Microbiol Mol Biol Rev, 2001 Jun, 65(2), 319 - 33 ; third page, table of contents Cytokinesis in prokaryotes and eukaryotes: common principles and different solutions; Nanninga N; Cytokinesis requires duplication of cellular structures followed by bipolarization of the predivisional cell . As a common principle, this applies to prokaryotes as well as eukaryotes . With respect to eukaryotes, the discussion has focused mainly on Saccharomyces cerevisiae and on Schizosaccharomyces pombe . Escherichia coli and to a lesser extent Bacillus subtilis have been used as prokaryotic examples . To establish a bipolar cell, duplication of a eukaryotic origin of DNA replication as well as its genome is not sufficient . Duplication of the microtubule-organizing center is required as a prelude to mitosis, and it is here that the dynamic cytoskeleton with all its associated proteins comes to the fore . In prokaryotes, a cytoskeleton that pervades the cytoplasm appears to be absent . DNA replication and the concomitant DNA segregation seem to occur without help from extensive cytosolic supramacromolecular assemblies but with help from the elongating cellular envelope . Prokaryotic cytokinesis proceeds through a contracting ring, which has a roughly 100-fold-smaller circumference than its eukaryotic counterpart . Although the ring contains proteins that can be considered as predecessors of actin, tubulin, and microtubule-associated proteins, its macromolecular composition is essentially different. Yeast, 2001 Jun, 18(8), 745 - 57 Functional characterization of Gms1p/UDP-galactose transporter in Schizosaccharomyces pombe; Tanaka N et al.; Galactosylation of glycoproteins in the fission yeast Schizosaccharomyces pombe requires the transport of UDP-galactose as substrate for the galactosyltransferase into the lumen of the Golgi apparatus, which is achieved by the UDP-galactose transporter . We isolated a mutant (gms1) that is deficient in galactosylation of cell surface glycoproteins in Sz.pombe, and found that the gms1(+) gene encodes a UDP-galactose transporter . In the prediction of secondary structure of the Gms1 protein, an eight-membrane-spanning structure was obtained . Fluorescent microscopy revealed the functional Gms1-GFP fusion protein to be stably localized at the Golgi membrane . Sequencing analysis of the coding region of Gms1p derived from galactosylation-defective mutants identified a single amino acid mutation (A102T or A258E) located within the putative transmembrane region, helix 2 or helix 7, respectively . The mutagenized Gms1(A102T or A258E)p exhibited loss of UDP-galactose transport activity but no change in the localization to the Golgi membrane . The C-terminal truncated Gms1p mutants demonstrated that the C-terminal hydrophilic region was dispensable for targeting and function as UDP-galactose transporter at the Golgi membrane.We suggest that the putative eighth (the most C-terminus-proximal) transmembrane helix of Gms1p is critical to targeting from ER to the Golgi membrane . Nucleic Acids Res, 2001 Jun 1, 29(11), 2327 - 37 Comprehensive isolation of meiosis-specific genes identifies novel proteins and unusual non-coding transcripts in Schizosaccharomyces pombe; Watanabe T et al.; In order to isolate meiosis-specific genes in Schizosaccharomyces pombe, we have constructed a subtracted cDNA library enriched in clones whose expression is enhanced during meiosis induced by nitrogen starvation . Using northern blot analysis, we isolated 31 kinds of clones whose expression was induced in a meiosis/sporulation-specific manner . We comprehensively named them meu after meiotic expression upregulated . The transcription of 20 meu genes was found to be dependent on the mei4(+) gene, which encodes a transcription factor required for the progression of meiosis . DNA sequencing indicated that most of the meu genes encode novel proteins . Notably, five of the meu genes harbor no apparent protein coding sequences, and the transcripts form stable hairpin structures, suggesting that they may generate non-coding RNAs or antisense RNAS: The results presented here imply that RNAs are also important for the comprehensive characterization of genomic expression. Nucleic Acids Res, 2001 May 15, 29(10), 2003 - 11 Transcription of the Schizosaccharomyces pombe U2 gene in vivo and in vitro is directed by two essential promoter elements; Zhou D et al.; As compared to the metazoan small nuclear RNAs (snRNAs), relatively little is known about snRNA synthesis in unicellular organisms . We have analyzed the transcription of the Schizosaccharomyces pombe U2 snRNA gene in vivo and in the homologous in vitro system . Deletion and linker-scanning analyses show that the S.pombe U2 promoter contains at least two elements: the spUSE centered at -55, which functions as an activator, and a TATA box at -26, which is essential for basal transcription . These data point to a similar architecture among S.pombe, plant and invertebrate snRNA promoters . Factors recognizing the spUSE can be detected in whole cell extracts by DNase I footprinting and competition studies show that the binding of these factors correlates with transcriptional activity . Electrophoretic mobility shift assays and gel-filtration chromatography revealed a native molecular mass of approximately 200 kDa for the spUSE binding activity . Two polypeptides of molecular masses 25 and 65 kDa were purified by virtue of their ability to specifically bind the spUSE. Mol Biol Cell, 2001 May, 12(5), 1367 - 80 Identification of two type V myosins in fission yeast, one of which functions in polarized cell growth and moves rapidly in the cell; Motegi F et al.; We characterized the novel Schizosaccharomyces pombe genes myo4(+) and myo5(+), both of which encode myosin-V heavy chains . Disruption of myo4 caused a defect in cell growth and led to an abnormal accumulation of secretory vesicles throughout the cytoplasm . The mutant cells were rounder than normal, although the sites for cell polarization were still established . Elongation of the cell ends and completion of septation required more time than in wild-type cells, indicating that Myo4 functions in polarized growth both at the cell ends and during septation . Consistent with this conclusion, Myo4 was localized around the growing cell ends, the medial F-actin ring, and the septum as a cluster of dot structures . In living cells, the dots of green fluorescent protein-tagged Myo4 moved rapidly around these regions . The localization and movement of Myo4 were dependent on both F-actin cables and its motor activity but seemed to be independent of microtubules . Moreover, the motor activity of Myo4 was essential for its function . These results suggest that Myo4 is involved in polarized cell growth by moving with a secretory vesicle along the F-actin cables around the sites for polarization . In contrast, the phenotype of myo5 null cells was indistinguishable from that of wild-type cells . This and other data suggest that Myo5 has a role distinct from that of Myo4. Mol Biol Cell, 2001 May, 12(5), 1275 - 91 Fission yeast Aip3p (spAip3p) is required for an alternative actin-directed polarity program; Jin H et al.; Aip3p is an actin-interacting protein that regulates cell polarity in budding yeast . The Schizosaccharomyces pombe-sequencing project recently led to the identification of a homologue of Aip3p that we have named spAip3p . Our results confirm that spAip3p is a true functional homologue of Aip3p . When expressed in budding yeast, spAip3p localizes similarly to Aip3p during the cell cycle and complements the cell polarity defects of an aip3Delta strain . Two-hybrid analysis shows that spAip3p interacts with actin similarly to Aip3p . In fission yeast, spAip3p localizes to both cell ends during interphase and later organizes into two rings at the site of cytokinesis . spAip3p localization to cell ends is dependent on microtubule cytoskeleton, its localization to the cell middle is dependent on actin cytoskeleton, and both patterns of localization require an operative secretory pathway . Overexpression of spAip3p disrupts the actin cytoskeleton and cell polarity, leading to morphologically aberrant cells . Fission yeast, which normally rely on the microtubule cytoskeleton to establish their polarity axis, can use the actin cytoskeleton in the absence of microtubule function to establish a new polarity axis, leading to the formation of branched cells . spAip3p localizes to, and is required for, branch formation, confirming its role in actin-directed polarized cell growth in both Schizosaccharomyces pombe and Saccharomyces cerevisiae. J Biol Chem, 2001 Jul 20, 276(29), 27731 - 9 Epub 2001 May 16. The essential Smp3 protein is required for addition of the side-branching fourth mannose during assembly of yeast glycosylphosphatidylinositols; Grimme SJ et al.; The major glycosylphosphatidylinositols (GPIs) transferred to protein in mammals and trypanosomes contain three mannoses . In Saccharomyces cerevisiae, however, the GPI transferred to protein bears a fourth, alpha1,2-linked Man on the alpha1,2-Man that receives the phosphoethanolamine (EthN-P) moiety through which GPIs become linked to protein . We report that temperature-sensitive smp3 mutants accumulate a GPI containing three mannoses and that smp3 is epistatic to the gpi11, gpi13, and gaa1 mutations, which normally result in the accumulation of Man(4)-GPIs, including the presumed substrate for the yeast GPI transamidase . The Smp3 protein, which is encoded by an essential gene, is therefore required for addition of the fourth Man to yeast GPI precursors . The finding that smp3 prevents the formation of the Man(4)-GPI that accumulates when addition of EthN-P to Man-3 is blocked in a gpi13 mutant suggests that the presence of the fourth Man is important for transfer of EthN-P to Man-3 of yeast GPIs . The Man(3)-GPI that accumulates in smp3 is a mixture of two dominant isoforms, one bearing a single EthN-P side branch on Man-1, the other with EthN-P on Man-2, and these isoforms can be placed in separate arms of a branched GPI assembly pathway . Smp3-related proteins are encoded in the genomes of Schizosaccharomyces pombe, Candida albicans, Drosophila melanogaster, and Homo sapiens and form a subgroup of a family of proteins, the other groups of which are defined by the Pig-B(Gpi10) protein, which adds the third GPI mannose, and by the Alg9 and Alg12 proteins, which act in the dolichol pathway for N-glycosylation . Because Man(4)-containing GPI precursors are normally formed in yeast and Plasmodium falciparum, whereas addition of a fourth Man during assembly of mammalian GPIs is rare and not required for GPI transfer to protein, Smp3p-dependent addition of a fourth Man represents a target for antifungal and antimalarial drugs. Mol Cells, 2001 Apr 30, 11(2), 198 - 203 Cloning, expression, and complementation test of the RNA lariat debranching enzyme cDNA from mouse; Kim HC et al.; The RNA lariat debranching enzyme of mouse (mDBR1) was cloned by screening a NIH/3T3 cDNA library . The sequence of full-length mDBR1 cDNA contained a single 515 amino acid open reading frame of 58 kDa protein . Comparison of the amino acid sequence of mDBR1 to other DBR proteins showed 40%, 44%, 43%, 42%, and 80% identity to Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, and human debranching enzymes, respectively . The mDBR1 cDNA was shown to be functional in an interspecies specific complementation experiment, and an in vitro debranching enzyme assay . Mouse DBR1 could complement the intron accumulation phenotype of a S . cerevisiae dbrl null mutant strain . However, the level of complementation depended on the copy number of the mDBR1 cDNA . The integration of the mDBR1 cDNA in the chromosome of S . pombe also complemented both intron accumulation and slow growth phenotypes of the S . pombe dbr1 knock-out mutant strain. Environ Toxicol Chem, 2001 Jan, 20(1), 198 - 204 Potential multidrug resistance gene POHL: an ecologically relevant indicator in marine sponges; Krasko A et al.; Sponges are sessile filter feeders found in all aquatic habitats from the tropics to the arctic . Against potential environmental hazards, they are provided with efficient defense systems, e.g., protecting chaperones and/or the P-170/multidrug resistance pump system . Here we report on a further multidrug resistance pathway that is related to the pad one homologue (POH1) mechanism recently identified in humans . It is suggested that proteolysis is involved in the inactivation of xenobiotics by the POH1 system . Two cDNAs were cloned, one from the demosponge Geodia cydonium and a second from the hexactinellid sponge Aphrocallistes vastus . The cDNA from G . cydonium, termed GCPOHL, encodes a deduced polypeptide with a size of 34,591 Da and that from A . vastus, AVPOHL, a protein of a calculated M(r) of 34,282 . The two sponge cDNAs are highly similar to each other as well as to the known sequences from fungi (Schizosaccharomyces pombe and Saccharomyces cerevisiae) and other Metazoa (from Schistosoma mansoni to humans) . Under controlled laboratory conditions, the expression of the potential multidrug resistance gene POHL is, in G . cydonium, strongly upregulated in response to the toxins staurosporin (20 microM) or taxol (50 microM); the first detectable transcripts appear after 1 d and reach a maximum after 3 to 5 d of incubation . The relevance of the expression pattern of the G . cydonium gene POHL for the assessment of pollution in the field was determined at differently polluted sites in the area around Rovinj (Croatia; Mediterranean Sea, Adriatic Sea) . The load of the selected sites was assessed by measuring the potency of XAD-7 concentrates of water samples taken from those places to induce the level of benzo{a}pyrene monooxygenase (BaPMO) in fish and to impair the multidrug resistance (MDR)/P-170 extrusion pump in clams . These field experiments revealed that the levels of inducible BaPMO activity in fish and of the MDR potential by the water concentrates are highly correlated with the level of expression of the potential multidrug resistance gene POHL in G . cydonium . This report demonstrates that the detoxification POH pathway, here mediated by the G . cydonium GCPOHL gene, is an additional marker for the assessment of the environmental load in a given marine area. Biochem Biophys Res Commun, 2001 May 18, 283(4), 908 - 14 Characterization of the manganese-containing superoxide dismutase and its gene regulation in stress response of Schizosaccharomyces pombe; Jeong JH et al.; Fission yeast Schizosaccharomyces pombe contains two superoxide dismutases (SODs), one in the cytosol and the other in mitochondria . The sod2+ gene encoding putative mitochondrial superoxide dismutase containing manganese (MnSOD) has been isolated . Purification and analysis of the sod2+ gene product revealed that it contained only manganese as a cofactor, thus verified to be a genuine MnSOD . It was localized in mitochondria as expected . Its N-terminal amino acid sequence indicated that the mitochondrial targeting sequence of 21 amino acids was removed . The native form consisted of two identical subunits . The sod2+ expression was induced by external stresses, such as treatments with superoxide generators, high osmolarity, and heat . The induction by these stress treatments depended on Wis1-Spc1 MAPK signal transduction pathway being independent of transcription factors Atf1 or Pap1 . The sod2 disruption rendered cells sensitive to various superoxide-generators, heat, and high osmolarity, suggesting that the mitochondrial MnSOD acts as a general defense agent against multiple stresses . RNA, 2001 May, 7(5), 671 - 81 Mutation in the prp12+ gene encoding a homolog of SAP130/SF3b130 causes differential inhibition of pre-mRNA splicing and arrest of cell-cycle progression in Schizosaccharomyces pombe; Habara Y et al.; prp12-1 is one of the mutants defective in pre-mRNA splicing at a nonpermissive temperature in Schizosaccharomyces pombe . We found that the prp12+ gene encodes a protein highly homologous with a human splicing factor, SAP130/SF3b130, a subunit of a U2 snRNP-associated complex SF3b . Prp12p was shown to interact genetically with Prp10p that is a homolog of SAP155/SF3b155, another subunit in SF3b, suggesting that Prp12p is a functional homolog of human SAP130/SF3b130 . Prp12p tagged with GFP is uniformly localized in the nuclear DNA region . In addition to pre-mRNA splicing defects, the prp12-1 mutant produced elongated cells, a typical phenotype of cell division cycle (cdc) mutants, suggesting a possible link between pre-mRNA splicing and cell-cycle progression . We examined kinetics of splicing defects in prp12-1 and several other prp mutants using northern blot hybridization and found that, among all the tested pre-mRNAs, only Tflld pre-mRNA with low splicing efficiency showed detectable splicing defects at the nonpermissive temperature in prp12-1 . In addition, we found that other prp mutants with the cdc phenotype also showed differential splicing defects in tested pre-mRNAs at the nonpermissive temperature . On the other hand, prp mutants that do not exhibit the cdc phenotype showed a rapid and complete block of pre-mRNA splicing in all the tested pre-mRNAs at the nonpermissive temperature, indicating that prp mutants with weak splicing defects have a tendency to exhibit the cdc phenotype . These results suggest that the cdc phenotype in prp12-1 is caused by a selective reduction of spliced transcripts encoding a protein (or proteins) required for G2/M transition. Pharmacogenetics, 2001 Apr, 11(3), 207 - 15 Functional characterization of human N-acetyltransferase 2 (NAT2) single nucleotide polymorphisms; Fretland AJ et al.; N-Acetyltransferase 2 (NAT2) catalyses the activation and/or deactivation of a variety of aromatic amine drugs and carcinogens . Polymorphisms in the N-acetyltransferase 2 (NAT2) gene have been associated with a variety of drug-induced toxicities, as well as cancer in various tissues . Eleven single nucleotide polymorphisms (SNPs) have been identified in the NAT2 coding region, but the specific effects of each of these SNPs on expression of NAT2 protein and N-acetyltransferase enzymatic activity are poorly understood . To investigate the functional consequences of SNPs in the NAT2 coding region, reference NAT2*4 and NAT2 variant alleles possessing one of the 11 SNPs in the NAT2 coding region were cloned and expressed in yeast (Schizosaccharomyces pombe) . Reductions in catalytic activity for the N-acetylation of a sulfonamide drug (sulfamethazine) and an aromatic amine carcinogen (2-aminofluorene) were observed for NAT2 variants possessing G191A (R64Q), T341C (I114T), A434C (E145P), G590A (R197Q), A845C (K282T) or G857A (G286T) . Reductions in expression of NAT2 immunoreactive protein were observed for NAT2 variants possessing T341C, A434C or G590A . Reductions in protein stability were noted for NAT2 variants possessing G191A, A845C, G857A or, to some extent, G590A . No significant differences in mRNA expression or transformation efficiency were observed among any of the NAT2 alleles . These results suggest two mechanisms for slow acetylator phenotype(s) and more clearly define the effects of individual SNPs on human NAT2 expression, stability and catalytic activity. Science, 2001 May 18, 292(5520), 1382 - 5 Epub 2001 May 03. Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome; Lyapina S et al.; SCF ubiquitin ligases control various processes by marking regulatory proteins for ubiquitin-dependent proteolysis . To illuminate how SCF complexes are regulated, we sought proteins that interact with the human SCF component CUL1 . The COP9 signalosome (CSN), a suppressor of plant photomorphogenesis, associated with multiple cullins and promoted cleavage of the ubiquitin-like protein NEDD8 from Schizosaccharomyces pombe CUL1 in vivo and in vitro . Multiple NEDD8-modified proteins uniquely accumulated in CSN-deficient S . pombe cells . We propose that the broad spectrum of activities previously attributed to CSN subunits--including repression of photomorphogenesis, activation of JUN, and activation of p27 nuclear export--underscores the importance of dynamic cycles of NEDD8 attachment and removal in biological regulation. J Biol Chem, 2001 Jul 6, 276(27), 24736 - 42 Epub 2001 May 02. Rapamycin blocks sexual development in fission yeast through inhibition of the cellular function of an FKBP12 homolog; Weisman R et al.; FKBP12 is a ubiquitous and a highly conserved prolyl isomerase that binds the immunosuppressive drugs FK506 and rapamycin . Members of the FKBP12 family have been implicated in many processes that include intracellular protein folding, transport, and assembly . In the budding yeast Saccharomyces cerevisiae and in human T cells, rapamycin forms a complex with FKBP12 that inhibits cell cycle progression by inhibition of the TOR kinases . We reported previously that rapamycin does not inhibit the vegetative growth of the fission yeast Schizosaccharomyces pombe; however, it specifically inhibits its sexual development . Here we show that disruption of the S . pombe FKBP12 homolog, fkh1(+), at its chromosomal locus results in a mating-deficient phenotype that is highly similar to that obtained by treatment of wild type cells with rapamycin . A screen for fkh1 mutants that can confer rapamycin resistance identified five amino acids in Fkh1 that are critical for the effect of rapamycin in S . pombe . All five amino acids are located in the putative rapamycin binding pocket . Together, our findings indicate that Fkh1 has an important role in sexual development and serves as the target for rapamycin action in S . pombe. Trends Genet, 2001 May, 17(5), 273 - 8 Establishment of a cellular axis in fission yeast; Chang F; Recent studies in fission yeast Schizosaccharomyces pombe reveal how cells establish a cellular axis that specifies domains as the functional 'ends' and 'middle' of the cell . During interphase, dynamic microtubules position the nucleus at the middle of the cell and orientate microtubule 'plus' ends towards the ends of the cell . At the cell ends, the microtubule plus ends might establish a zone of polarized cell growth and actin assembly by depositing factors such as Tea1p . At the cell middle, the nucleus might specify the position of the actin contractile ring and the future cell division site by positioning cytokinesis factors such as Mid1p. Genetics, 2001 May, 158(1), 77 - 85 Control of GT repeat stability in Schizosaccharomyces pombe by mismatch repair factors; Mansour AA et al.; The mismatch repair (MMR) system ensures genome integrity by removing mispaired and unpaired bases that originate during replication . A major source of mutational changes is strand slippage in repetitive DNA sequences without concomitant repair . We established a genetic assay that allows measuring the stability of GT repeats in the ade6 gene of Schizosaccharomyces pombe . In repair-proficient strains most of the repeat variations were insertions, with addition of two nucleotides being the most frequent event . GT repeats were highly destabilized in strains defective in msh2 or pms1 . In these backgrounds, mainly 2-bp insertions and 2-bp deletions occurred . Surprisingly, essentially the same high mutation rate was found with mutants defective in msh6 . In contrast, a defect in swi4 (a homologue of Msh3) caused only slight effects, and instability was not further increased in msh6 swi4 double mutants . Also inactivation of exo1, which encodes an exonuclease that has an MMR-dependent function in repair of base-base mismatches, caused only slightly increased repeat instability . We conclude that Msh2, Msh6, and Pms1 have an important role in preventing tract length variations in dinucleotide repeats . Exo1 and Swi4 have a minor function, which is at least partially independent of MMR. Genetics, 2001 May, 158(1), 65 - 75 Requirement for Msh6, but not for Swi4 (Msh3), in Msh2-dependent repair of base-base mismatches and mononucleotide loops in Schizosaccharomyces pombe; Tornier C et al.; The msh6 mismatch repair gene of Schizosaccharomyces pombe was cloned, sequenced, and inactivated . Strains bearing all combinations of inactivated msh6, msh2, and swi4 (the S . pombe MSH3 ortholog) alleles were tested for their defects in mitotic and meiotic mismatch repair . Mitotic mutation rates were similarly increased in msh6 and msh2 mutants, both for reversion of a base-base substitution as well as of an insertion of one nucleotide in a mononucleotide run . Tetrad analysis and intragenic two-factor crosses revealed that meiotic mismatch repair was affected in msh6 to the same extent as in msh2 background . In contrast, loss of Swi4 likely did not cause a defect in mismatch repair, but rather resulted in reduced recombination frequency . Consistently, a mutated swi4 caused a two- to threefold reduction of recombinants in intergenic crosses, while msh2 and msh6 mutants were not significantly different from wild type . In summary, our study showed that Msh6 plays the same important role as Msh2 in the major mismatch repair pathway of S . pombe, while Swi4 rather functions in recombination. RNA, 2001 Mar, 7(3), 342 - 50 Activation of a cryptic 5' splice site by U1 snRNA; Alvarez CJ et al.; In the course of analyzing 5' splice site mutations in the second intron of Schizosaccharomyces pombe cdc2, we identified a cryptic 5' junction containing a nonconsensus nucleotide at position +2 . An even more unusual feature of this cryptic 5' junction was its pattern of activation . By analyzing the profile of splicing products for an extensive series of cdc2 mutants in the presence and absence of compensatory U1 alleles, we have obtained evidence that the natural 5' splice site participates in activation of the cryptic 5' splice site, and that it does so via base pairing to U1 snRNA . Furthermore, the results of follow-up experiments strongly suggest that base pairing between U1 snRNA and the cryptic 5' junction itself plays a dominant role in its activation . Most remarkably, a mutant U1 can activate the cryptic 5' splice site even in the presence of a wild-type sequence at the natural 5' junction, providing unambiguous evidence that this snRNA redirects splicing via base pairing . Although previous work has demonstrated that U5 and U6 snRNAs can activate cryptic 5' splice sites through base pairing interactions, this is the first example in which U1 snRNA has been implicated in the final selection of a cryptic 5' junction. Yeast, 2001 May, 18(7), 657 - 62 Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe; Tasto JJ et al.; We describe the construction of tagging cassettes and plasmids for tandem affinity purification (TAP) of proteins in Schizosaccharomyces pombe . The tagging cassettes are designed for either carboxy- or amino-terminal tagging of proteins . The carboxyl terminal tags differ in that they contain either two or four repeats of IgG binding units . For tagging endogenous loci, the cassettes contain the kan MX6 module to allow for selection of G418-resistant cells . The amino-terminal tagging vectors allow for the regulated expression of proteins . Sz . pombe Cdc2p was chosen to test these new affinity tags . Several known binding proteins co-purified with both Cdc2p-CTAP and N-TAP-Cdc2p, indicating the usefulness of these tags for the rapid purification of stable protein complexes from Sz . pombe . Curr Genet, 2001 Feb, 39(1), 2 - 9 Sequence of Crm1/exportin 1 mutant alleles reveals critical sites associated with multidrug resistance; Carobbio S et al.; We have previously shown that genes involved in a novel pathway of multidrug resistance (MDR) in the fission yeast Schizosaccharomyces pombe are functionally conserved in human cells (V . Spataro et al . (1997) J Biol Chem 272: 30470-30475) . The human homologue of one of these genes, hCRM1, has recently been identified and found to function in nucleocytoplasmic export, a process which controls the subcellular localization and hence activity of a number of key cell cycle regulators and transcription factors . Several mutant alleles of crm1 confer a phenotype of MDR in S . pombe, through the nuclear accumulation of the AP-1 transcription factor Pap1 . We therefore sequenced mutations of crm1 in fission yeast in order to guide the search for analogous hCRM1 mutations which could play a role in tumour-drug resistance . Fifteen yeast crm1 mutants were assessed by PCR and DNA sequencing . Four mis-sense mutations were identified in the open reading frame, three of which (G to A transitions at nucleotide positions 385, 895 and 1,288) were capable of conferring the MDR phenotype alone . For three of the four mutations found, the corresponding amino acid changes affect residues which are conserved in the human homologue hCRM1 and lie in highly conserved regions of the CRM1 protein . We analysed the corresponding hCRM1 coding regions by RT-PCR and sequencing in a panel of ten tumour cell lines, including three ovarian lines resistant either to cisplatin or paclitaxel, or to both and one MDR breast cancer cell line with nuclear accumulation of the transcription factor YB-1 . No hCRM1 mutations were found in the three cDNA fragments examined in this panel of tumour cell lines . However, the identification of amino acid residues within the CRM1 protein that are critical for the export of the MDR-associated transcription factor Pap1 in fission yeast can guide further analysis of hCRM1 mutations in tumours with a MDR phenotype. Exp Parasitol, 2001 Mar, 97(3), 119 - 28 Primary structure of the Plasmodium vivax crk2 gene and interference of the yeast cell cycle upon its conditional expression; Speranca MA et al.; The cdc2 gene product, a 34-kDa protein kinase, plays a universal role in the M phase of the eukaryotic cell cycle . To study the cell cycle regulation in malarial parasites, we have characterized a cdc2-related gene from the most widely distributed human malaria, Plasmodium vivax (Pvcrk2) . The full-length Pvcrk2 revealed 90--99% homology with Crk2 proteins from other Plasmodium species and approximately 60% homology with p34(cdc2) proteins from higher eukaryotes . We used the temperature-sensitive Schizosaccharomyces pombe cdc2 mutant (cdc2-33(ts)) for gene complementation studies . Expression of the full-length 33-kDa PvCrk2 protein, a truncated 27-kDa version, and two chimeric proteins in which we exchanged the N- and C-terminal regions of PvCrk2 with their S . pombe counterparts at the restrictive temperature in the mutant cdc2-33(ts) did not complement the cell cycle defect . However, conditional expression of the Pvcrk2 genes or the chimera containing the C terminus from Spcdc2 in mutant cdc2-33(ts) cells produced cell-cycle-arrested phenotypes only in the induced state and at the permissive temperature . Our results thus provide the first compelling genetic evidence that the plasmodial Crk2 gene product(s) is capable of interfering with the well-conserved eukaryotic cell cycle machinery . J Cell Biol, 2001 Apr 16, 153(2), 397 - 411 A mechanism for nuclear positioning in fission yeast based on microtubule pushing; Tran PT et al.; The correct positioning of the nucleus is often important in defining the spatial organization of the cell, for example, in determining the cell division plane . In interphase Schizosaccharomyces pombe cells, the nucleus is positioned in the middle of the cylindrical cell in an active microtubule (MT)-dependent process . Here, we used green fluorescent protein markers to examine the dynamics of MTs, spindle pole body, and the nuclear envelope in living cells . We find that interphase MTs are organized in three to four antiparallel MT bundles arranged along the long axis of the cell, with MT plus ends facing both the cell tips and minus ends near the middle of the cell . The MT bundles are organized from medial MT-organizing centers that may function as nuclear attachment sites . When MTs grow to the cell tips, they exert transient forces produced by plus end MT polymerization that push the nucleus . After an average of 1.5 min of growth at the cell tip, MT plus ends exhibit catastrophe and shrink back to the nuclear region before growing back to the cell tip . Computer modeling suggests that a balance of these pushing MT forces can provide a mechanism to position the nucleus at the middle of the cell. Cell, 2001 Apr 6, 105(1), 115 - 26 Dynamic coupling between the SH2 and SH3 domains of c-Src and Hck underlies their inactivation by C-terminal tyrosine phosphorylation; Young MA et al.; The effect of C-terminal tyrosine phosphorylation on molecular motions in the Src kinases Hck and c-Src is investigated by molecular dynamics simulations . The SH2 and SH3 domains of the inactive kinases are seen to be tightly coupled by the connector between them, impeding activation . Dephosphorylation of the tail reduces the coupling between the SH2 and SH3 domains in the simulations, as does replacement of connector residues with glycine . A mutational analysis of c-Src expressed in Schizosaccharomyces pombe demonstrates that replacement of residues in the SH2-SH3 connector with glycine activates c-Src . The SH2-SH3 connector appears to be an inducible "snap lock" that clamps the SH2 and SH3 domains upon tail phosphorylation, but which allows flexibility when the tail is released. Eur J Biochem, 2001 Apr, 268(8), 2281 - 9 Hsp90 chaperone complexes are required for the activity and stability of yeast protein kinases Mik1, Wee1 and Swe1; Goes FS et al.; The Wee1 protein kinase negatively regulates entry into mitosis by mediating the inhibitory tyrosine phosphorylation of Cdc2-cyclin B kinase . The stability and activity of Wee1 from the fission yeast Schizosaccharomyces pombe is critically dependent on functional Hsp90 chaperones . Here we identify two related tyrosine protein kinases, Mik1 from fission yeast and its Saccharomyces cerevisiae homolog Swe1, as Hsp90 substrates and show that the kinase domain is sufficient to mediate this interaction . Morphological and biochemical defects arising from overexpression of the kinases in fission yeast are suppressed in the conditional Hsp90 mutant swo1-26 . A subset of all three kinases is associated with the Hsp90 cochaperones cyclophilin 40 and p23 . Under conditions of impaired chaperone function or treatment with the Hsp90 inhibitory drug geldanamycin, intracellular levels of the kinases are reduced and the proteins become rapidly degraded by the proteasome machinery, indicating that Wee1, Mik1 and Swe1 require Hsp90 heterocomplexes for their stability and maintenance of function. Eur J Biochem, 2001 Apr, 268(8), 2270 - 80 Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of alpha-glucosidase from Schizosaccharomyces pombe; Okuyama M et al.; cDNA encoding Schizosaccharomyces pombe alpha-glucosidase was cloned from a library constructed from mRNA of the fission yeast, and expressed in Saccharomyces cerevisiae . The cDNA, 4176 bp in length, included a single ORF composed of 2910 bp encoding a polypeptide of 969 amino-acid residues with M(r) 106 138 . The deduced amino-acid sequence showed a high homology to those of alpha-glucosidases from molds, plants and mammals . Therefore, the enzyme was categorized into the alpha-glucosidase family II . By site-directed mutagenesis, Asp481, Glu484 and Asp647 residues were confirmed to be essential in the catalytic reaction . The carboxyl group (-COOH) of the Asp647 residue was for the first time shown to be the most likely proton donor acting as the acid catalyst in the alpha-glucosidase of family II . Studies with the chemical modifier conduritol B epoxide suggested that the carboxylate group (-COO-) of the Asp481 residue was the catalytic nucleophile, although the role of the Glu484 residue remains obscure. Mol Biol Cell, 2001 Apr, 12(4), 1161 - 75 Profilin binding to poly-L-proline and actin monomers along with ability to catalyze actin nucleotide exchange is required for viability of fission yeast; Lu J et al.; We tested the ability of 87 profilin point mutations to complement temperature-sensitive and null mutations of the single profilin gene of the fission yeast Schizosaccharomyces pombe . We compared the biochemical properties of 13 stable noncomplementing profilins with an equal number of complementing profilin mutants . A large quantitative database revealed the following: 1) in a profilin null background fission yeast grow normally with profilin mutations having >10% of wild-type affinity for actin or poly-L-proline, but lower affinity for either ligand is incompatible with life; 2) in the cdc3-124 profilin ts background, fission yeast function with profilin having only 2-5% wild-type affinity for actin or poly-L-proline; and 3) special mutations show that the ability of profilin to catalyze nucleotide exchange by actin is an essential function . Thus, poly-L-proline binding, actin binding, and actin nucleotide exchange are each independent requirements for profilin function in fission yeast. Mol Biol Cell, 2001 Apr, 12(4), 1061 - 77 Roles of a fimbrin and an alpha-actinin-like protein in fission yeast cell polarization and cytokinesis; Wu JQ et al.; Eukaryotic cells contain many actin-interacting proteins, including the alpha-actinins and the fimbrins, both of which have actin cross-linking activity in vitro . We report here the identification and characterization of both an alpha-actinin-like protein (Ain1p) and a fimbrin (Fim1p) in the fission yeast Schizosaccharomyces pombe . Ain1p localizes to the actomyosin-containing medial ring in an F-actin-dependent manner, and the Ain1p ring contracts during cytokinesis . ain1 deletion cells have no obvious defects under normal growth conditions but display severe cytokinesis defects, associated with defects in medial-ring and septum formation, under certain stress conditions . Overexpression of Ain1p also causes cytokinesis defects, and the ain1 deletion shows synthetic effects with other mutations known to affect medial-ring positioning and/or organization . Fim1p localizes both to the cortical actin patches and to the medial ring in an F-actin-dependent manner, and several lines of evidence suggest that Fim1p is involved in polarization of the actin cytoskeleton . Although a fim1 deletion strain has no detectable defect in cytokinesis, overexpression of Fim1p causes a lethal cytokinesis defect associated with a failure to form the medial ring and concentrate actin patches at the cell middle . Moreover, an ain1 fim1 double mutant has a synthetical-lethal defect in medial-ring assembly and cell division . Thus, Ain1p and Fim1p appear to have an overlapping and essential function in fission yeast cytokinesis . In addition, protein-localization and mutant-phenotype data suggest that Fim1p, but not Ain1p, plays important roles in mating and in spore formation. Mol Biol Cell, 2001 Apr, 12(4), 901 - 17 The Schizosaccharomyces pombe spo20(+) gene encoding a homologue of Saccharomyces cerevisiae Sec14 plays an important role in forespore membrane formation; Nakase Y et al.; The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth . Herein, we report that S . pombe, spo20(+) is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle . We also demonstrate that the spo20(+) gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast . This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other . Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein . That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures . However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation . Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells . We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus . This nuclear localization persists during conjugation and meiosis . On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells . In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus . Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function. Mol Biol Cell, 2001 Apr, 12(4), 780 - 94 A millennial myosin census; Berg JS et al.; The past decade has seen a remarkable explosion in our knowledge of the size and diversity of the myosin superfamily . Since these actin-based motors are candidates to provide the molecular basis for many cellular movements, it is essential that motility researchers be aware of the complete set of myosins in a given organism . The availability of cDNA and/or draft genomic sequences from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Dictyostelium discoideum has allowed us to tentatively define and compare the sets of myosin genes in these organisms . This analysis has also led to the identification of several putative myosin genes that may be of general interest . In humans, for example, we find a total of 40 known or predicted myosin genes including two new myosins-I, three new class II (conventional) myosins, a second member of the class III/ninaC myosins, a gene similar to the class XV deafness myosin, and a novel myosin sharing at most 33% identity with other members of the superfamily . These myosins are in addition to the recently discovered class XVI myosin with N-terminal ankyrin repeats and two human genes with similarity to the class XVIII PDZ-myosin from mouse . We briefly describe these newly recognized myosins and extend our previous phylogenetic analysis of the myosin superfamily to include a comparison of the complete or nearly complete inventories of myosin genes from several experimentally important organisms. Nucleic Acids Res, 2001 Apr 15, 29(8), 1724 - 32 Gain- and loss-of-function of Rhp51, a Rad51 homolog in fission yeast, reveals dissimilarities in chromosome integrity; Kim WJ et al.; Rad51 is crucial not only in homologous recombination and recombinational repair but also in normal cellular growth . To address the role of Rad51 in normal cell growth we investigated morphological changes of cells after overexpression of wild-type and a dominant negative form of Rad51 in fission yeast . Rhp51, a Rad51 homolog in Schizosaccharomyces pombe, has a highly conserved ATP-binding motif . Rhp51 K155A, which has a single substitution in this motif, failed to rescue hypersensitivity of a rhp51 mutant to methyl methanesulfonate (MMS) and UV, whereas it binds normally to Rhp51 and Rad22, a Rad52 homolog . Two distinct cellular phenotypes were observed when Rhp51 or Rhp51 K155A was overexpressed in normal cells . Overexpression of Rhp51 caused lethality in the absence of DNA-damaging agents, with acquisition of a cell cycle mutant phenotype and accumulation of a 1C DNA population . On the other hand, overexpression of Rhp51 K155A led to a delay in G(2) with decondensed nuclei, which resembled the phenotype of rhp51 . The latter also exhibited MMS and UV sensitivity, indicating that Rhp51 K155A has a dominant negative effect . These results suggest an association between DNA replication and Rad51 function. Genetics, 2001 Apr, 157(4), 1513 - 22 Fission yeast Mog1p homologue, which interacts with the small GTPase Ran, is required for mitosis-to-interphase transition and poly(A)(+) RNA metabolism; Tatebayashi K et al.; We have cloned and characterized the Schizosaccharomyces pombe gene mog1(+), which encodes a protein with homology to the Saccharomyces cerevisiae Mog1p participating in the Ran-GTPase system . The S . pombe Mog1p is predominantly localized in the nucleus . In contrast to the S . cerevisiae MOG1 gene, the S . pombe mog1(+) gene is essential for cell viability . mog1(+) is required for the mitosis-to-interphase transition, as the mog1-1 mutant arrests at restrictive temperatures as septated, binucleated cells with highly condensed chromosomes and an aberrant nuclear envelope . FACS analysis showed that these cells do not undergo a subsequent round of DNA replication . Surprisingly, also unlike the Delta mog1 mutation in S . cerevisiae, the mog1-1 mutation causes nucleolar accumulation of poly(A)(+) RNA at the restrictive temperature in S . pombe, but the signals do not overlap with the fibrillarin-rich region of the nucleolus . Thus, we found that mog1(+) is required for the mitosis-to-interphase transition and a class of RNA metabolism . In our attempt to identify suppressors of mog1-1, we isolated the spi1(+) gene, which encodes the fission yeast homologue of Ran . We found that overexpression of Spi1p rescues the S . pombe Delta mog1 cells from death . On the basis of these results, we conclude that mog1(+) is involved in the Ran-GTPase system. Yeast, 2001 Apr, 18(6), 543 - 54 Hut1 proteins identified in Saccharomyces cerevisiae and Schizosaccharomyces pombe are functional homologues involved in the protein-folding process at the endoplasmic reticulum; Nakanishi H et al.; The Saccharomyces cerevisiae HUT1 gene (scHUT1) and the Schizosaccharomyces pombe hut1(+) gene (sphut1(+)) encode hydrophobic proteins with approximately 30% identity to a human UDP-galactose transporter-related gene (UGTrel1) product . These proteins show a significant similarity to the nucleotide sugar transporter and are conserved in many eukaryotic species, but their physiological functions are not known . Both scHUT1 and sphut1(+) genes are non-essential for cell growth under normal conditions, and their disruptants show no defects in the modification of O- and N-linked oligosaccharides, but are sensitive to a membrane-permeable reducing agent, dithiothreitol (DTT) . Consistent with this phenotype, scHUT1 has genetic interaction with ERO1, which plays an essential role in the oxidation of secretory proteins at the endoplasmic reticulum (ER) . Overexpression of the MPD1 or MPD2 genes, which were isolated as multicopy suppressors of protein disulphide isomerase (PDI) depletion, could not replace the essential function of PDI in Delta hut1 S . cerevisiae cells . Our results indicate that scHut1p and spHut1p are functional homologues, and their physiological function is to maintain the optimal environment for the folding of secretory pathway proteins in the ER . Yeast, 2001 Apr, 18(6), 533 - 41 Overexpression of HUT1 gene stimulates in vivo galactosylation by enhancing UDP-galactose transport activity in Saccharomyces cerevisiae; Kainuma M et al.; Transfer of activated sugar-nucleotides from the cytoplasm to the lumen of the Golgi is an essential requirement for glycosylation of glycoproteins, proteoglycans and glycosphingolipids . Although mannosylation is the major modification in the yeast Saccharomyces cerevisiae, several reports suggest the presence of galactose residues on yeast proteins and sphingolipids . We have detected alpha-galactosylated O-linked chitinase by lectin blotting from cells that functionally express the gma12(+) gene, encoding alpha 1,2-galactosyltransferase from Schizosaccharomyces pombe . This result implies the presence of a UDP-galactose transporter in S . cerevisiae . A conserved gene, HUT1, which encodes a putative multi-transmembrane protein, was cloned and characterized for its possible involvement in galactosylation . The HUT1 gene is not essential and is expressed at a relatively low level under the physiological conditions we examined . The disruption of this gene did not show any apparent impairments in glycosylation . However, a temperature- and concentration-dependent increase in UDP--galactose transport activity was detected from cells overexpressing HUT1 in the presence of gma12(+) . The surface of these cells was confirmed to carry galactose residues by staining with FITC-conjugated alpha-galactose-specific lectin . These results suggest a role for Hut1p in the transport of UDP--galactose from the cytosol into the Golgi lumen in S . cerevisiae . J Exp Bot, 2001 Feb, 52(355), 193 - 202 Origins and complexes: the initiation of DNA replication; Bryant JA et al.; Eukaryotic DNA is organized for replication as multiple replicons . DNA synthesis in each replicon is initiated at an origin of replication . In both budding yeast, Saccharomyces cerevisiae and fission yeast, Schizosaccharomyces pombe, origins contain specific sequences that are essential for initiation, although these differ significantly between the two yeasts with those of S . pombe being more complex then those of S . cerevisiae . However, it is not yet clear whether the replication origins of plants contain specific essential sequences or whether origin sites are determined by features of chromatin structure . In all eukaryotes there are several biochemical events that must take place before initiation can occur . These are the marking of the origins by the origin recognition complex (ORC), the loading onto the origins, in a series of steps, of origin activation factors including the MCM proteins, and the initial denaturation of the double helix to form a replication "bubble" . Only then can the enzymes that actually initiate replication, primase and DNA polymerase-alpha, gain access to the template . In many cells this complex series of events occurs only once per cell cycle, ensuring that DNA is not re-replicated within one cycle . However, regulated re-replication of DNA within one cell cycle (DNA endoreduplication) is relatively common in plants, indicating that the "once-per-cycle" controls can be overridden. J Biol Chem, 2001 Jun 15, 276(24), 21235 - 41 Epub 2001 Mar 30. Antizyme regulates the degradation of ornithine decarboxylase in fission yeast Schizosaccharomyces pombe . Study in the spe2 knockout strains; Chattopadhyay MK et al.; The mechanism of the regulatory degradation of ornithine decarboxylase (ODC) by polyamines was studied in fission yeast, Schizosaccharomyces pombe . To regulate cellular spermidine experimentally, we cloned and disrupted S-adenosylmethionine decarboxylase gene (spe2) in S . pombe . The null mutant of spe2 was devoid of spermidine and spermine, accumulated putrescine, and contained a high level of ODC . Addition of spermidine to the culture medium resulted in rapid decrease in the ODC activity caused by the acceleration of ODC degradation, which was dependent on de novo protein synthesis . A fraction of ODC forming an inactive complex concomitantly increased . The accelerated ODC degradation was prevented either by knockout of antizyme gene or by selective inhibitors of proteasome . Thus, unlike budding yeast, mammalian type antizyme-mediated ODC degradation by proteasome is operating in S . pombe. J Biol Chem, 2001 May 18, 276(20), 16655 - 9 Epub 2001 Feb 13. A chaperone for ribosome maturation; Lalev AI et al.; The nascent pre-rRNA of eukaryotic ribosomes is fully transcribed and assembled into an 80-90 S nucleolar particle before being cleaved into mature ribosomal RNA . The interdependence of steps in the processing of this precursor RNA indicates that RNA processing, at least in part, acts as a quality control mechanism that helps ensure that only functional RNA is incorporated into mature ribosomes . In search of structural components that underlie this interdependence using the Schizosaccharomyces pombe internal transcribed spacer 1 (ITS) as a ligand for affinity chromatography of ITS1-specific proteins, we have isolated a large spliceosome-like protein complex, a ribosome assembly chaperone (RAC) of 20 or more polypeptides (Lalev, A . I., Abeyrathne, P . D., and Nazar, R . N . (2000) J . Mol . Biol . 302, 65-77) . When the ITS2 spacer was used in the present study to isolate ITS2-specific proteins, the same proteins were identified consistent with a complex containing multiple specific binding sites . Subsequent competition binding studies indicated that the protein complex actually contains independent binding sites for all four of the transcribed spacers in the pre-rRNA . Because disruption of protein-binding sites in these spacer RNAs is known to severely affect rRNA processing, taken together these results suggest that the RAC complex is a chaperone for ribosome maturation acting as a "rack" on which critical structure is organized. J Biol Chem, 2001 May 18, 276(20), 17117 - 24 Epub 2001 Mar 09. Two WD repeat-containing TATA-binding protein-associated factors in fission yeast that suppress defects in the anaphase-promoting complex; Mitsuzawa H et al.; The general transcription factor IID consists of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs) . Here we report the isolation of two related TAF genes from the fission yeast Schizosaccharomyces pombe as multicopy suppressors of a temperature-sensitive mutation in the ubiquitin-conjugating enzyme gene ubcP4(+) . The ubcP4(ts) mutation causes cell cycle arrest in mitosis, probably due to defects in ubiquitination mediated by the anaphase-promoting complex/cyclosome . One multicopy suppressor is the previously reported gene taf72(+), whereas the other is a previously unidentified gene named taf73(+) . We show that the taf73(+) gene, like taf72(+), is essential for cell viability . The taf72(+) and taf73(+) genes encode proteins homologous to WD repeat-containing TAFs such as human TAF100, Drosophila TAF80/85, and Saccharomyces cerevisiae TAF90 . We demonstrate that TAF72 and TAF73 proteins are present in the same complex with TBP and other TAFs and that TAF72, but not TAF73, is associated with the putative histone acetylase Gcn5 . We also show that overexpression of TAF72 or TAF73 suppresses the cell cycle arrest in mitosis caused by a mutation in the anaphase-promoting complex/cyclosome subunit gene cut9(+) . These results suggest that TAF72 and TAF73 may regulate the expression of genes involved in ubiquitin-dependent proteolysis during mitosis . Our study thus provides evidence for a possible role of WD repeat-containing TAFs in the expression of genes involved in progression through the M phase of the cell cycle. J Biol Chem, 2001 May 4, 276(18), 15472 - 80 Epub 2001 Jan 26. The fission yeast copper-sensing transcription factor Cuf1 regulates the copper transporter gene expression through an Ace1/Amt1-like recognition sequence; Beaudoin J et al.; Transcriptional regulation of genes encoding critical components of copper transport is essential for copper homeostasis and growth in yeast . Analysis of regulatory regions in the promoter of the ctr4(+) copper transporter gene in fission yeast Schizosaccharomyces pombe reveals the identity of a conserved copper-signaling element (CuSE), which is recognized by the transcription factor Cuf1 . We demonstrate that CuSE is necessary for transcriptional activation in response to copper deprivation conditions . Interestingly, the CuSE element bears a strong sequence similarity to the recognition site, denoted MRE (metal regulatory element), which is recognized by a distinct class of copper sensors required for copper detoxification, including Ace1 from Saccharomyces cerevisiae and Amt1 from Candida glabrata . When a consensus MRE from S . cerevisiae is introduced into S . pombe, transcription is induced by copper deprivation in a Cuf1-dependent manner, similar to regulation by Mac1, the nuclear sensor for regulating the expression of genes encoding components involved in copper transport in S . cerevisiae . UV-cross-linking experiments show that the Cuf1 protein directly binds the CuSE . These results demonstrate that the Cuf1 nutritional copper-sensing factor possesses a module that functions similarly to domains found in the Ace1/Amt1 class of metalloregulatory factors, which allows the protein to act through a closely related MRE-like sequence to regulate copper transport gene expression in S . pombe. J Biol Chem, 2001 May 11, 276(19), 15861 - 7 Epub 2001 Feb 14. Identification of 12 new yeast mitochondrial ribosomal proteins including 6 that have no prokaryotic homologues; Saveanu C et al.; Mitochondrial ribosomal proteins were studied best in yeast, where the small subunit was shown to contain about 35 proteins . Yet, genetic and biochemical studies identified only 14 proteins, half of which were predictable by sequence homology with prokaryotic ribosomal components of the small subunit . Using a recently described affinity purification technique and tagged versions of yeast Ykl155c and Mrp1, we isolated this mitochondrial ribosomal subunit and identified a total of 20 proteins, of which 12 are new . For a subset of the newly described ribosomal proteins, we showed that they are localized in mitochondria and are required for the respiratory competency of the yeast cells . This brings to 26 the total number of proteins described as components of the mitochondrial small ribosomal subunit . Remarkably, almost half of the previously and newly identified mitochondrial ribosomal components showed no similarity to any known ribosomal protein . Homologues could be found, however, in predicted protein sequences from Schizosaccharomyces pombe . In more distant species, putative homologues were detected for Ykl155c, which shares conserved motifs with uncharacterized proteins of higher eukaryotes including humans . Another newly identified ribosomal protein, Ygl129c, was previously shown to be a member of the DAP-3 family of mitochondrial apoptosis mediators. J Biol Chem, 2001 May 11, 276(19), 16580 - 6 Epub 2001 Feb 06. ATM-dependent phosphorylation of human Rad9 is required for ionizing radiation-induced checkpoint activation; Chen MJ et al.; ATM (ataxia-telangiectasia-mutated) is a Ser/Thr kinase involved in cell cycle checkpoints and DNA repair . Human Rad9 (hRad9) is the homologue of Schizosaccharomyces pombe Rad9 protein that plays a critical role in cell cycle checkpoint control . To examine the potential signaling pathway linking ATM and hRad9, we investigated the modification of hRad9 in response to DNA damage . Here we show that hRad9 protein is constitutively phosphorylated in undamaged cells and undergoes hyperphosphorylation upon treatment with ionizing radiation (IR), ultraviolet light (UV), and hydroxyurea (HU) . Interestingly, hyperphosphorylation of hRad9 induced by IR is dependent on ATM . Ser(272) of hRad9 is phosphorylated directly by ATM in vitro . Furthermore, hRad9 is phosphorylated on Ser(272) in response to IR in vivo, and this modification is delayed in ATM-deficient cells . Expression of hRad9 S272A mutant protein in human lung fibroblast VA13 cells disturbs IR-induced G(1)/S checkpoint activation and increased cellular sensitivity to IR . Together, our results suggest that the ATM-mediated phosphorylation of hRad9 is required for IR-induced checkpoint activation. J Biol Chem, 2001 May 4, 276(18), 14549 - 52 Epub 2001 Mar 09. The highly conserved protein methyltransferase, Skb1, is a mediator of hyperosmotic stress response in the fission yeast Schizosaccharomyces pombe; Bao S et al.; The p21-activated kinase, Shk1, is required for cell viability, establishment and maintenance of cell polarity, and proper mating response in the fission yeast, Schizosaccharomyces pombe . Previous genetic studies suggested that a presumptive protein methyltransferase, Skb1, functions as a positive modulator of Shk1 . However, unlike Shk1, Skb1 is not required for viability or mating of S . pombe cells and contributes only modestly to the regulation of cell morphology under normal growth conditions . Here we demonstrate that Skb1 plays a more significant role in regulating cell growth and polarity under conditions of hyperosmotic stress . We provide evidence that the inability of skb1Delta cells to properly maintain cell polarity in hyperosmotic conditions results from inefficient subcellular targeting of F-actin . We show that Skb1 localizes to cell ends, sites of septation, and nuclei of S . pombe cells . Hyperosmotic shock results in substantial delocalization of Skb1 from cell ends and nuclei, as well as stimulation of Skb1 protein methyltransferase activity . Taken together, our results demonstrate a new role for Skb1 as a mediator of hyperosmotic stress response in fission yeast . We show that the protein methyltransferase activity of the human Skb1 homolog, Skb1Hs, is also stimulated by hyperosmotic stress in fission yeast, providing evidence for evolutionary conservation of a role for Skb1-related proteins as mediators of hyperosmotic stress response, as well as mechanisms involved in regulating this novel class of protein methyltransferases. J Biol Chem, 2001 Jun 8, 276(23), 20529 - 35 Epub 2001 Mar 26. Identification of a novel high affinity copper transport complex in the fission yeast Schizosaccharomyces pombe; Zhou H et al.; Copper is an essential nutrient that serves as a co-factor for enzymes involved in critical cellular processes including energy generation, peptide hormone maturation, oxidative stress protection, and iron homeostasis . Although genes have been identified from yeast and mammals encoding a homologous subunit of a plasma membrane high affinity copper transporter, the presence of additional subunits that function as part of a copper transport complex has not been reported . We observed that ctr4(+), a previously identified copper transport protein from the fission yeast Schizosaccharomyces pombe, fails to complement bakers' yeast cells defective in high affinity copper transport and fails to be targeted to the plasma membrane . However, selection for S . pombe genes, which, when co-expressed with Ctr4, confer high affinity copper transport to S . cerevisiae cells resulted in the identification of ctr5(+) . Both Ctr4 and Ctr5 are integral membrane proteins, are co-regulated by copper levels and the copper-sensing transcription factor Cuf1, physically associate in vivo, are interdependent for secretion to the plasma membrane, and are each essential for high affinity copper transport . These studies in S . pombe identify Ctr4 and Ctr5 as components of a novel eukaryotic heteromeric plasma membrane complex that is essential for high affinity copper transport. J Biol Chem, 2001 Jun 15, 276(24), 21670 - 7 Epub 2001 Mar 23. Human BIN3 complements the F-actin localization defects caused by loss of Hob3p, the fission yeast homolog of Rvs161p; Routhier EL et al.; The BAR adaptor proteins encoded by the RVS167 and RVS161 genes from Saccharomyces cerevisiae form a complex that regulates actin, endocytosis, and viability following starvation or osmotic stress . In this study, we identified a human homolog of RVS161, termed BIN3 (bridging integrator-3), and a Schizosaccharomyces pombe homolog of RVS161, termed hob3+ (homolog of Bin3) . In human tissues, the BIN3 gene was expressed ubiquitously except for brain . S . pombe cells lacking Hob3p were often multinucleate and characterized by increased amounts of calcofluor-stained material and mislocalized F-actin . For example, while wild-type cells localized F-actin to cell ends during interphase, hob3Delta mutants had F-actin patches distributed randomly around the cell . In addition, medial F-actin rings were rarely found in hob3Delta mutants . Notably, in contrast to S . cerevisiae rvs161Delta mutants, hob3Delta mutants showed no measurable defects in endocytosis or response to osmotic stress, yet hob3+ complemented the osmosensitivity of a rvs161Delta mutant . BIN3 failed to rescue the osmosensitivity of rvs161Delta, but the actin localization defects of hob3Delta mutants were completely rescued by BIN3 and partially rescued by RVS161 . These findings suggest that hob3+ and BIN3 regulate F-actin localization, like RVS161, but that other roles for this gene have diverged somewhat during evolution. J Mol Biol, 2001 Mar 30, 307(3), 861 - 70 Solution structure, domain features, and structural implications of mutants of the chromo domain from the fission yeast histone methyltransferase Clr4; Horita DA et al.; The encapsulation of otherwise transcribable loci within transcriptionally inactive heterochromatin is rapidly gaining recognition as an important mechanism of epigenetic gene regulation . In the fission yeast Schizosaccharomyces pombe, heterochromatinization of the mat2/mat3 loci silences the mating-type information encoded within these loci . Here, we present the solution structure of the chromo domain from the cryptic loci regulator protein Clr4 . Clr4 is known to regulate silencing and switching at the mating-type loci and to affect chromatin structure at centromeres . Clr4 and its human and Drosophila homologs have been identified as histone H3-specific methyltransferases, further implicating this family of proteins in chromatin remodeling . Our structure highlights a conserved surface that may be involved in chromo domain-ligand interactions . We have also analyzed two chromo domain mutants (W31G and W41G) that previously were shown to affect silencing and switching in full-length Clr4 . Both mutants are significantly destabilized relative to wild-type . Arch Microbiol, 2001 Jan, 175(1), 62 - 9 Bypass of the requirement for cdc16p GAP function in Schizosaccharomyces pombe by mutation of the septation initiation network genes; Fournier N et al.; The onset of septum formation in the fission yeast Schizosaccharomyces pombe is signaled via the spglp GTPase-switch, which is part of the septation initiation network . This is negatively regulated by the two-component GTPase-activating protein (GAP) comprised of the products of the cdc16 and byr4 genes . Loss-of-function mutations in either of these genes result in multiple rounds of septum formation without cell cleavage . In this work, we demonstrate that attenuation of the protein kinase cdc7p can rescue the lethality of a null allele of cdc16 . This observation provides the basis for selection of chromosomal mutations and multicopy suppressors that attenuate the signaling of septation . Using this screen, mutations in all the previously described septation initiation network genes were obtained, with the exception of byr4, sid4 and plo1 . We also demonstrate that increased expression of the dma1 gene can rescue the lethality of a null allele of cdc16 . The implications for the regulation of septum formation in fission yeast are discussed. Curr Genet, 2001 Jan, 38(6), 307 - 13 Schizosaccharomyces pombe taf1+ is required for nitrogen starvation-induced sexual development and for entering the dormant GO state; Ueno M et al.; Environmental change, such as nutritional starvation, induces physiological and morphological alterations that enable fission yeast cells to survive . We isolated a novel gene, taf1+, required for the response to nitrogen starvation in the fission yeast Schizosaccharomyces pombe . taf1 disruptants could not mate upon nitrogen starvation, but could upon carbon starvation . taf1 disruptants had a defect in inducing stell+ expression under nitrogen starvation conditions . Furthermore, they lost viability quickly in nitrogen-depleted medium . Unlike wild-type cells, starved taf1-cells had nuclear chromatin that were flat and adhered to the cell periphery . These results indicate that tqf1+ is required for nitrogen starvation-induced sexual development and entering the dormant G0 state. Curr Genet, 2001 Jan, 38(6), 299 - 306 A novel genetic screen identifies checkpoint-defective alleles of Schizosaccharomyces pombe chk1; Wan S et al.; The protein kinase Chk1 is required in the fission yeast Schizosaccharomyces pombe for delaying cell cycle progression in response to DNA damage . Chk1 becomes phosphorylated when DNA is damaged by a variety of agents, including the anti-tumor drug camptothecin . To further characterize the behavior of Chk1 in response to DNA damage, we used PCR-based mutagenesis of the chk1 gene coupled with in vivo gap repair to generate mutant alleles . Of 44 chk1 mutants recovered, six encode full-length proteins that confer a DNA damage-sensitive phenotype . All of the alleles render cells checkpoint-defective, but confer subtle differences in sensitivity to camptothecin or UV light . Mutant alleles were sequenced and served to identify regions of the protein that are critical for checkpoint function. Biochim Biophys Acta, 2001 Mar 19, 1518(1-2), 194 - 9 Characterization and regulation of Schizosaccharomyces pombe gene encoding thioredoxin; Cho Y et al.; A cDNA coding thioredoxin (TRX) was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization . The 438 bp EcoRI fragment, which was detected by Southern hybridization, reveals an open reading frame which encodes a protein of 103 amino acids . The genomic DNA encoding TRX was also isolated from S . pombe chromosomal DNA using PCR . The cloned sequence contains 1795 bp and encodes a protein of 103 amino acids . However, the C-terminal region obtained from the cDNA clone is -Val-Arg-Leu-Asn-Arg-Ser-Leu, whereas the C-terminal region deduced from the genomic DNA appears to contain -Ala-Ser-Ile-Lys-Ala-Asn-Leu . This indicates that S . pombe cells contain two kinds of TRX genes which have dissimilar amino acid sequences only at the C-terminal regions . The heterologous TRX 1C produced from the cDNA clone could be used as a subunit of T7 DNA polymerase, while the TRX 1G from the genomic DNA could not . The upstream sequence and the region encoding the N-terminal 18 amino acids of the genomic DNA were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357 to generate the fusion plasmid pYKT24 . Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by hydrogen peroxide, menadione and aluminum chloride . It indicates that the expression of the cloned TRX gene is induced by oxidative stress. J Cell Biol, 2001 Jan 22, 152(2), 349 - 60 The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation; Wigge PA et al.; We have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p . Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation . In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle . Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere . Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere . A human homologue of Nuf2p was identified in the expressed sequence tag database . Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells . Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates. EMBO Rep, 2000 Aug, 1(2), 145 - 50 Fate of mat1 DNA strands during mating-type switching in fission yeast; Arcangioli B; The mating-type switching of the fission yeast, Schizosaccharomyces pombe, is highly regulated . Two consecutive asymmetric divisions are required to produce one mating-type switched cell among the four progeny . Using DNA density-gradient centrifugation we demonstrate that one-fourth of the mat1 DNA is not replicated by the conventional semi-conservative mode, but instead both DNA strands are synthesized de novo . Our data are consistent with a gene conversion event, initiated by a site- and strand-specific DNA break (SSB) . We further demonstrate that the virgin switched mat1-containing chromatid no longer contained the nick, while it is reintroduced during the lagging strand synthesis of the mat1 locus on the sister chromatid . This finding establishes at the molecular level a firm experimental link between the phenotype and genotype in the process of asymmetric mating-type switching during mitotic divisions. Biochem Biophys Res Commun, 2001 Mar 23, 282(1), 10 - 5 Rkp1/Cpc2, a fission yeast RACK1 homolog, is involved in actin cytoskeleton organization through protein kinase C, Pck2, signaling; Won M et al.; The Rkp1/Cpc2, a fission yeast RACK1 homolog, interacted with Pck2, one of the known PKC homologs, in vivo and in vitro . The rkp1-deletion mutants (Deltarkp1) are elongated and the pck2-deletion mutant (Deltapck2) showed abnormal morphology . The double-deletion mutant (Deltarkp1Deltapck2) showed more aberrant cell shapes and was sensitive to high salt concentration . Both Deltarkp1 and Deltapck2 cells were sensitive to latrunculin B (Lat B) which inhibits actin polymerization . The cells expressing the human RACK1 homolog complemented the latrunculin B sensitivity of Deltarkp1 indicating that human RACK1 is a functional homolog of Rkp1/Cpc2 . We propose that Rkp1/Cpc2 may function as a receptor for Pck2 in the regulation of actin cytoskeleton organization during cell wall synthesis and morphogenesis of Schizosaccharomyces pombe . Traffic, 2001 Mar, 2(3), 189 - 201 Vrp1p functions in both actomyosin ring-dependent and Hof1p-dependent pathways of cytokinesis; Naqvi SN et al.; Vrp1p/verprolin/End5p is a Saccharomyces cerevisiae proline-rich protein, structurally and functionally related to human Wiskott-Aldrich syndrome protein-interacting protein . Vrp1p is required for viability at 37 degrees C, but not 24 degrees C . Here, we show that loss of Vrp1p (vrp1Delta) leads to a 3-4-fold delay in cytokinesis, wide bud necks, abnormal actomyosin rings, and aberrant septa even at 24 degrees C . Like other mutations affecting the actomyosin ring, vrp1Delta is synthetic lethal with deletion of HOF1 (or CYK2), which encodes a protein related to mammalian proline serine threonine phosphatase-interacting protein and Schizosaccharomyces pombe Cdc15p required for an actomyosin ring-independent pathway of cytokinesis in S . cerevisiae . At 37 degrees C, vrp1Delta cells rapidly cease dividing and exhibit a novel terminal phenotype: a single large bud, two well-separated nuclei, and an interphase microtubule array . The arrested cells have a persistent ring containing both actin and myosin at the bud neck . Many also exhibit some polarisation of cortical actin patches to the bud neck . Vrp1p binds an SH3-domain-containing fragment of Hof1p in vitro . Vrp1p is required in vivo for Hof1p relocalisation to a single ring at the bud neck prior to cytokinesis at 37 degrees C, but not at 24 degrees C . Vrp1p thus acts in both actomyosin ring formation and function, as well as in Hof1p localisation during cytokinesis. Mol Cell Biol, 2001 Apr, 21(7), 2449 - 62 The Cbk1p pathway is important for polarized cell growth and cell separation in Saccharomyces cerevisiae; Bidlingmaier S et al.; During the early stages of budding, cell wall remodeling and polarized secretion are concentrated at the bud tip (apical growth) . The CBK1 gene, encoding a putative serine/threonine protein kinase, was identified in a screen designed to isolate mutations that affect apical growth . Analysis of cbk1Delta cells reveals that Cbk1p is required for efficient apical growth, proper mating projection morphology, bipolar bud site selection in diploid cells, and cell separation . Epitope-tagged Cbk1p localizes to both sides of the bud neck in late anaphase, just prior to cell separation . CBK1 and another gene, HYM1, were previously identified in a screen for genes involved in transcriptional repression and proposed to function in the same pathway . Deletion of HYM1 causes phenotypes similar to those observed in cbk1Delta cells and disrupts the bud neck localization of Cbk1p . Whole-genome transcriptional analysis of cbk1Delta suggests that the kinase regulates the expression of a number of genes with cell wall-related functions, including two genes required for efficient cell separation: the chitinase-encoding gene CTS1 and the glucanase-encoding gene SCW11 . The Ace2p transcription factor is required for expression of CTS1 and has been shown to physically interact with Cbk1p . Analysis of ace2Delta cells reveals that Ace2p is required for cell separation but not for polarized growth . Our results suggest that Cbk1p and Hym1p function to regulate two distinct cell morphogenesis pathways: an ACE2-independent pathway that is required for efficient apical growth and mating projection formation and an ACE2-dependent pathway that is required for efficient cell separation following cytokinesis . Cbk1p is most closely related to the Neurospora crassa Cot-1; Schizosaccharomyces pombe Orb6; Caenorhabditis elegans, Drosophila, and human Ndr; and Drosophila and mammalian WARTS/LATS kinases . Many Cbk1-related kinases have been shown to regulate cellular morphology. Electrophoresis, 2001 Feb, 22(3), 576 - 85 Expression of the small tyrosine phosphatase (Stp1) in Saccharomyces cerevisiae: a study on protein tyrosine phosphorylation; Modesti A et al.; Small tyrosine phoshatase 1 (Stp1) is a Schizosaccharomyces pombe low-molecular-mass phosphotyrosine-phosphatase 50% identical to Saccharomyces cerevisiae Ltp1 . In order to investigate the role of Stp1 in yeast, a mutant was generated having the characteristic of a dominant negative molecule . Changes in protein tyrosine phosphorylation in S . cerevisiae proteome in response to Stp1 or its dominant negative mutant expression were analyzed by high-resolution two-dimensional (2-D) electrophoresis . The most remarkable result is the modification by phosphorylation on tyrosine of several proteins involved in carbohydrate metabolism . Twelve proteins were identified on the basis of their positions in the anti-phosphotyrosine immunoblot of the 2-D electrophoresis . Ten of these present tyrosyl residues that are within the consensus sequence for protein kinase CK2 (casein kinase-2) . These data open the possibility for the identification of Stp1 substrates in yeast and provide hints about the nature of tyrosine phosphorylating agents in yeast and in other organisms where bona fide tyrosine kinases are lacking. Yeast, 2001 Mar 30, 18(5), 463 - 8 Construction of FLAG and histidine tagging vectors for Schizosaccharomyces pombe; Huang Y et al.; Schizosaccharomyces pombe is becoming an increasingly popular model system for investigating important cellular processes . To facilitate detection, purification and functional studies of Sz . pombe gene products, we constructed two tagging expression vectors for use in Sz . pombe . These vectors allow proteins to be expressed ectopically as fusion proteins with a FLAG epitope and six histidine residue tags attached to their N-terminus or C-terminus . The function and applicability of these vectors were examined and the results are shown using the N-terminal tagging vector encoding Sfc6p, a subunit of the Sz . pombe RNA polymerase III general transcription factor, TFIIIC . Yeast, 2001 Mar 30, 18(5), 433 - 44 In situ localization of beta-glucans in the cell wall of Schizosaccharomyces pombe; Humbel BM et al.; The chemical composition of the cell wall of Sz . pombe is known as beta-1,3-glucan, beta-1,6-glucan, alpha-1,3-glucan and alpha-galactomannan; however, the three-dimensional interactions of those macromolecules have not yet been clarified . Transmission electron microscopy reveals a three-layered structure: the outer layer is electron-dense, the adjacent layer is less dense, and the third layer bordering the cell membrane is dense . In intact cells of Sz . pombe, the high-resolution scanning electron microscope reveals a surface completely filled with alpha-galactomannan particles . To better understand the organization of the cell wall and to complement our previous studies, we set out to locate the three different types of beta-glucan by immuno-electron microscopy . Our results suggest that the less dense layer of the cell wall contains mainly beta-1,6-branched beta-1,3-glucan . Occasionally a line of gold particles can be seen, labelling fine filaments radiating from the cell membrane to the alpha-galactomannan layer, suggesting that some of the radial filaments contain beta-1,6-branched beta-1,3-glucan . beta-1,6-glucan is preferentially located underneath the alpha-galactomannan layer . Linear beta-1,3-glucan is exclusively located in the primary septum of dividing cells . beta-1,6-glucan only labels the secondary septum and does not co-localize with linear beta-1,3-glucan, while beta-1,6-branched beta-1,3-glucan is present in both septa . Linear beta-1,3-glucan is present from early stages of septum formation and persists until the septum is completely formed; then just before cell division the label disappears . From these results we suggest that linear beta-1,3-glucan is involved in septum formation and perhaps the separation of the two daughter cells . In addition, we frequently found beta-1,6-glucan label on the Golgi apparatus, on small vesicles and underneath the cell membrane . These results give fresh evidence for the hypothesis that beta-1,6-glucan is synthesized in the endoplasmic reticulum-Golgi system and exported to the cell membrane . Mamm Genome, 2001 Mar, 12(3), 227 - 31 Synteny of orthologous genes conserved in mammals, snake, fly, nematode, and fission yeast; Trachtulec Z et al.; Four mouse genes, programmed cell death 2 (Pdcd2 or Rp8), brain protein 44-like (Brp441), bystin-like (Bysl), and uncoordinated-93-like (Unc931) genes were mapped to Chromosome (Chr) 17 . The orthologs of these and other mouse Chr 17 genes are localized on Chr III of Caenorhabditis elegans, thus defining a syntenic group conserved between vertebrates and nonvertebrates . In human, mouse, and snake, the PDCD2-, and TATA-binding protein (TBP)-encoding genes are adjacent tail-to-tail . The TBP- and PDCD2-encoding genes are linked also in Drosophila, and, together with proteasomal subunit C5 gene, they are syntenic in human, mouse, C . elegans, and Schizosaccharomyces pombe . The orthologs of tightly linked C . elegans genes, coding for BRP44L and PDCD2, map to about 2-Mb interval on human region 6q27 and on mouse Chr 17 . Hitherto, 13 members of synteny conserved between C . elegans and vertebrates have been detected, of which six are located on Drosophila Chr X . Such a distribution of transcription units is nonrandom and could indicate a long-range cis-acting relationship among the genes within the conserved syntenic group. Mamm Genome, 2001 Mar, 12(3), 192 - 8 Molecular cloning, genetic mapping, and expression of the mouse Sf3b1 (SAP155) gene for the U2 snRNP component of spliceosome; Isono K et al.; SAP155, a subunit of the U2 snRNP, is essential for prespliceosome assembly and splicing catalysis of the major spliceosome . Moreover, the protein has been identified in the minor (U12-dependent) spliceosome . These facts strongly suggest that SAP155 is shared by two distinct complexes owing to its importance in the removal of any type of intron . Here we have isolated a cDNA encoding the 146-kDa mouse homolog, designated Sf3b1 . The amino acid sequence of Sf3b1 is very highly conserved among homologs from Schizosaccharomyces pombe (52.4% identity) to human (99.6%), and the C-terminal 825 residues of these Sf3b1 homologs show even higher identities . This C-terminal region shows significant similarity to the PR65 subunit of protein phosphatase 2A, which is composed of 15 tandem repeats of a 39 amino acid sequence . Mouse genome analyses showed Sf3bh1 to be a single-copy gene mapping to the central part of Chromosome (Chr) 1 . Northern blot analysis and whole mount in situ hybridization revealed Sf3b1 to be ubiquitously expressed in a variety of adult tissues and mid-gestation embryos. EMBO J, 2001 Mar 15, 20(6), 1259 - 70 The role of Plo1 kinase in mitotic commitment and septation in Schizosaccharomyces pombe; Tanaka K et al.; Plo1-associated casein kinase activity peaked during mitosis before septation . Phosphatase treatment abolished this activity . Mitotic Plo1 activation had a requirement for prior activation of M-phase promoting factor (MPF), suggesting that Plo1 does not act as a mitotic trigger kinase to initiate MPF activation during mitotic commitment . A link between Plo1 and the septum initiating network (SIN) has been suggested by the inability of plo1 Delta cells to septate and the prolific septation following plo1(+) overexpression . Interphase activation of Spg1, the G protein that modulates SIN activity, induced septation but did not stimulate Plo1-associated kinase activity . Conversely, SIN inactivation did not affect the mitotic stimulation of Plo1-associated kinase activity . plo1.ts4 cells formed a misshapen actin ring, but rarely septated at 36 degrees C . Forced activation of Spg1 enabled plo1.ts4 mutant cells, but not cells with defects in the SIN component Sid2, to convert the actin ring to a septum . The ability of plo1(+) overexpression to induce septation was severely compromised by SIN inactivation . We propose that Plo1 acts before the SIN to control septation. FEBS Lett, 2001 Mar 9, 492(1-2), 133 - 8 Involvement of mitochondrial ferredoxin and Cox15p in hydroxylation of heme O; Barros MH et al.; Cox15p is essential for the biogenesis of cytochrome oxidase {Glerum et al., J . Biol . Chem . 272 (1997) 19088-19094} . We show here that cox15 mutants are blocked in heme A but not heme O biosynthesis . In Schizosaccharomyces pombe COX15 is fused to YAH1, the yeast gene for mitochondrial ferredoxin (adrenodoxin) . A fusion of Cox15p and Yah1p in Saccharomyces cerevisiae rescued both cox15 and yah1 null mutants . This suggests that Yah1p functions in concert with Cox15p . We propose that Cox15p functions together with Yah1p and its putative reductase (Arh1p) in the hydroxylation of heme O. J Bacteriol, 2001 Apr, 183(7), 2226 - 33 Functional characterization of alanine racemase from Schizosaccharomyces pombe: a eucaryotic counterpart to bacterial alanine racemase; Uo T et al.; Schizosaccharomyces pombe has an open reading frame, which we named alr1(+), encoding a putative protein similar to bacterial alanine racemase . We cloned the alr1(+) gene in Escherichia coli and purified the gene product (Alr1p), with an M(r) of 41,590, to homogeneity . Alr1p contains pyridoxal 5'-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent K(m) and V(max) values as follows: for L-alanine, 5.0 mM and 670 micromol/min/mg, respectively, and for D-alanine, 2.4 mM and 350 micromol/min/mg, respectively . The enzyme is almost specific to alanine, but L-serine and L-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of L-alanine, respectively . S . pombe uses D-alanine as a sole nitrogen source, but deletion of the alr1(+) gene resulted in retarded growth on the same medium . This indicates that S . pombe has catabolic pathways for both enantiomers of alanine and that the pathway for L-alanine coupled with racemization plays a major role in the catabolism of D-alanine . Saccharomyces cerevisiae differs markedly from S . pombe: S . cerevisiae uses L-alanine but not D-alanine as a sole nitrogen source . Moreover, D-alanine is toxic to S . cerevisiae . However, heterologous expression of the alr1(+) gene enabled S . cerevisiae to grow efficiently on D-alanine as a sole nitrogen source . The recombinant yeast was relieved from the toxicity of D-alanine. J Mol Biol, 2001 Mar 2, 306(4), 703 - 16 Fission yeast nascent polypeptide-associated complex binds to four-way DNA junctions; Whitby MC et al.; The four-way DNA junction (X-junction) is both a central intermediate of recombination reactions and, in some cases, a controlling element in transcription and the initiation of DNA replication . Many different proteins have been found to bind to X-junctions in a structure-specific manner . In some cases, this ability only reflects the proteins' general predilection for distorted DNAs but in others the interaction is highly specific and usually signifies that the X-junction is the real target for the protein in vivo . Here we identify the Schizosaccharomyces pombe (Sp) nascent polypeptide associated complex (NAC) as a potent binder of X-junction DNA . NAC is highly conserved in eukaryotes and has reported functions in transcription and the targeting of proteins within the cytosol . NAC is composed of alpha and beta subunits . Each SpNAC subunit has the capacity to bind X-junction DNA, but optimal binding depends on a heterodimer of subunits . Competition assays and binding comparisons using a range of different DNA substrates reveal that SpNAC is highly selective for the X-junction structure . By comparative gel electrophoresis we show that the X-junction is held in its open square conformation when bound by SpNAC . Junction binding is inhibited by concentrations of magnesium ions that are sufficient to "stack" the X-junction, suggesting that SpNAC recognises only the open junction structure . Finally, SpNAC can bind to X-junctions that are already bound by a tetramer of the Escherichia coli RuvA protein, indicating that it interacts with only one face of the junction . The possible biological significance of X-junction binding by SpNAC is discussed . DNA Cell Biol, 2001 Jan, 20(1), 53 - 65 Gene structure for adenosine kinase in Chinese hamster and human: high-frequency mutants of CHO cells involve deletions of several introns and exons; Singh B et al.; The structure for the adenosine kinase (AK) gene has been determined from Chinese hamster (CH) and human cells . The AK gene in CH is comprised of 11 exons ranging in length from 36 to 765 nt, with the majority <100 nt . The exact lengths of the intervening introns have not been determined, but most of them are indicated to be very large (>15 kb) . A 6.6-kb fragment from human cells was also sequenced, and it contained only a single exon corresponding to exon 10 in CH . The BLAST searches of the subsequently released draft human genome sequence have revealed that the AK gene structure in human is identical to that in CH . In the human genome, the AK exons are distributed over four genomic clones totaling 752 kb, providing direct evidence that the AK gene in mammalian species is unusually large . In contrast to CH and human, the AK genes from several other eukaryotic organisms whose complete genomes are now known are quite small (between 1.2 and 2.5 kb) and either contain no introns (Saccharomyces cerevisiae and Schizosaccharomyces pombe) or various numbers of introns (Drosophila melanogaster {2}, Caenorhabditis elegans {4}, Arabidopsis thaliana {10}) . Some of the intron-exon junctions in these species are in the same positions as in mammals . The AK gene in CH and human, as well as mouse, is linked upstream in a head-to-head fashion with the gene for the clathrin adaptor mu3 protein (or beta 3A subunit of the AP-3 protein complex), which is affected in type 2 Hermansky-Pudlak syndrome . These two genes are separated by <200 nt, and it is possible that they have a common or overlapping promoter(s) . We have also determined the nature of the genetic alterations in two of the class A AK(-) mutants of CHO cells, which are obtained at a very high spontaneous frequency (10(-3)-10(-4)) in this cell line . Both mutants contained large deletions within the AK gene and greatly shortened AK transcripts . The cloning and sequencing of the transcripts from these mutants showed that the deletion in one of them led to the loss of exons 5 through 8, whereas in the other, all exons from 2 through 8 are deleted . The endpoints of these deletions lie in the large introns within the AK gene. Nature, 2001 Mar 1, 410(6824), 120 - 4 Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain; Bannister AJ et al.; Heterochromatin protein 1 (HP1) is localized at heterochromatin sites where it mediates gene silencing . The chromo domain of HP1 is necessary for both targeting and transcriptional repression . In the fission yeast Schizosaccharomyces pombe, the correct localization of Swi6 (the HP1 equivalent) depends on Clr4, a homologue of the mammalian SUV39H1 histone methylase . Both Clr4 and SUV39H1 methylate specifically lysine 9 of histone H3 (ref . 6) . Here we show that HP1 can bind with high affinity to histone H3 methylated at lysine 9 but not at lysine 4 . The chromo domain of HP1 is identified as its methyl-lysine-binding domain . A point mutation in the chromo domain, which destroys the gene silencing activity of HP1 in Drosophila, abolishes methyl-lysine-binding activity . Genetic and biochemical analysis in S . pombe shows that the methylase activity of Clr4 is necessary for the correct localization of Swi6 at centromeric heterochromatin and for gene silencing . These results provide a stepwise model for the formation of a transcriptionally silent heterochromatin: SUV39H1 places a 'methyl marker' on histone H3, which is then recognized by HP1 through its chromo domain . This model may also explain the stable inheritance of the heterochromatic state. Nucleic Acids Res, 2001 Mar 15, 29(6), 1326 - 33 The dhp1(+) gene, encoding a putative nuclear 5'-->3' exoribonuclease, is required for proper chromosome segregation in fission yeast; Shobuike T et al.; The Schizosaccharomyces pombe dhp1(+) gene is an ortholog of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5'-->3' exoribonuclease, and is essential for cell viability . To clarify the cellular functions of the nuclear 5'-->3' exoribonuclease, we isolated and characterized a temperature-sensitive mutant of dhp1 (dhp1-1 mutant) . The dhp1-1 mutant showed nuclear accumulation of poly(A)(+) RNA at the restrictive temperature, as was already reported for the rat1 mutant . Interestingly, the dhp1-1 mutant exhibited aberrant chromosome segregation at the restrictive temperature . The dhp1-1 cells frequently contained condensed chromosomes, most of whose sister chromatids failed to separate during mitosis despite normal mitotic spindle elongation . Finally, chromosomes were displaced or unequally segregated . As similar mitotic defects were also observed in Dhp1p-depleted cells, we concluded that dhp1(+) is required for proper chromosome segregation as well as for poly(A)(+) RNA metabolism in fission yeast . Furthermore, we isolated a multicopy suppressor of the dhp1-1 mutant, referred to as din1(+) . We found that the gene product of dhp1-1 was unstable at high temperatures, but that reduced levels of Dhp1-1p could be suppressed by overexpressing Din1p at the restrictive temperature . Thus, Din1p may physically interact with Dhp1p and stabilize Dhp1p and/or restore its activity. Mol Cell Biol, 2001 Mar, 21(5), 1499 - 508 Roles of the mitotic inhibitors Wee1 and Mik1 in the G(2) DNA damage and replication checkpoints; Rhind N et al.; The G(2) DNA damage and DNA replication checkpoints in many organisms act through the inhibitory phosphorylation of Cdc2 on tyrosine-15 . This phosphorylation is catalyzed by the Wee1/Mik1 family of kinases . However, the in vivo role of these kinases in checkpoint regulation has been unclear . We show that, in the fission yeast Schizosaccharomyces pombe, Mik1 is a target of both checkpoints and that the regulation of Mik1 is, on its own, sufficient to delay mitosis in response to the checkpoints . Mik1 appears to have two roles in the DNA damage checkpoint; one in the establishment of the checkpoint and another in its maintenance . In contrast, Wee1 does not appear to be involved in the establishment of either checkpoint. Genetics, 2001 Mar, 157(3), 1205 - 15 Transcriptional regulators of the Schizosaccharomyces pombe fbp1 gene include two redundant Tup1p-like corepressors and the CCAAT binding factor activation complex; Janoo RT et al.; The Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK) . To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity . Two such clones express amino-terminally truncated forms of the S . pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor . These clones appear to act as dominant negative alleles . Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription . In strains lacking tup11 and tup12, the atf1-pcr1 transcriptional activator continues to play a central role in fbp1-lacZ expression; however, spc1 MAPK phosphorylation of atf1 is no longer essential for its activation . We discuss possible models for the role of tup11- and tup12-mediated repression with respect to signaling from the MAPK and PKA pathways . A third clone identified in our screen expresses the php5 protein subunit of the CCAAT-binding factor (CBF) . Deletion of php5 reduces fbp1 expression under both repressed and derepressed conditions . The CBF appears to act in parallel to atf1-pcr1, although it is unclear whether or not CBF activity is regulated by PKA. Genetics, 2001 Mar, 157(3), 1191 - 203 Functional redundancies, distinct localizations and interactions among three fission yeast homologs of centromere protein-B; Irelan JT et al.; Several members of protein families that are conserved in higher eukaryotes are known to play a role in centromere function in the fission yeast Schizosaccharomyces pombe, including two homologs of the mammalian centromere protein CENP-B, Abp1p and Cbh1p . Here we characterize a third S . pombe CENP-B homolog, Cbh2p (CENP-B homolog 2) . cbh2Delta strains exhibited a modest elevation in minichromosome loss, similar to cbh1Delta or abp1Delta strains . cbh2Delta cbh1Delta strains showed little difference in growth or minichromosome loss rate when compared to single deletion strains . In contrast, cbh2Delta abp1Delta strains displayed dramatic morphological and chromosome segregation defects, as well as enhancement of the slow-growth phenotype of abp1Delta strains, indicating partial functional redundancy between these proteins . Both cbh2Delta abp1Delta and cbh1Delta abp1Delta strains also showed strongly enhanced sensitivity to a microtubule-destabilizing drug, consistent with a mitotic function for these proteins . Cbh2p was localized to the central core and core-associated repeat regions of centromeric heterochromatin, but not at several other centromeric and arm locations tested . Thus, like its mammalian counterpart, Cbh2p appeared to be localized exclusively to a portion of centromeric heterochromatin . In contrast, Abp1p was detected in both centromeric heterochromatin and in chromatin at two of three replication origins tested . Cbh2p and Abp1p homodimerized in the budding yeast two-hybrid assay, but did not interact with each other . These results suggest that indirect cooperation between different CENP-B-like DNA binding proteins with partially overlapping chromatin distributions helps to establish a functional centromere. J Mol Biol, 2001 Feb 16, 306(2), 275 - 90 Solution structure and dynamics of an open beta-sheet, glycolytic enzyme, monomeric 23.7 kDa phosphoglycerate mutase from Schizosaccharomyces pombe; Uhrinova S et al.; The structure and backbone dynamics of a double labelled (15N,13C) monomeric, 23.7 kD phosphoglycerate mutase (PGAM) from Schizosaccharomyces pombe have been investigated in solution using NMR spectroscopy . A set of 3125 NOE-derived distance restraints, 148 restraints representing inferred hydrogen bonds and 149 values of (3)J(HNHalpha) were used in the structure calculation . The mean rmsd from the average structure for all backbone atoms from residues 6-205 in the best 21 calculated structures was 0.59 A . The core of the enzyme includes an open, twisted, six-stranded beta-sheet flanked by four alpha-helices and a short 3(10)-helix . An additional smaller domain contains two short antiparallel beta-strands and a further pair of alpha-helices . The C(alpha) atoms of the S . pombe PGAM may be superimposed on their equivalents in one of the four identical subunits of Saccharomyces cerevisiae PGAM with an rmsd of 1.34 A (0.92 A if only the beta-sheet is considered) . Small differences between the two structures are attributable partly to the deletion in the S . pombe sequence of a 25 residue loop involved in stabilising the S . cerevisiae tetramer . Analysis of 15N relaxation parameters indicates that PGAM tumbles isotropically with a rotational correlation time of 8.7 ns and displays a range of dynamic features . Of 178 residues analysed, only 77 could be fitted without invoking terms for fast internal motion or chemical exchange, and out of the remainder, 77 required a chemical exchange term . Significantly, 46 of the slowly exchanging (milli- to microsecond) residues lie in helices, and these account for two-thirds of all analysed helix residues . On the contrary, only one beta-sheet residue required an exchange term . In contrast to other analyses of backbone dynamics reported previously, residues in slow exchange appeared to correlate with architectural features of the enzyme rather than congregating close to ligand binding sites. Nat Cell Biol, 2001 Mar, 3(3), 235 - 44 Role of actin polymerization and actin cables in actin-patch movement in Schizosaccharomyces pombe; Pelham RJ Jr et al.; Factors that are involved in actin polymerization, such as the Arp2/3 complex, have been found to be packaged into discrete, motile, actin-rich foci . Here we investigate the mechanism of actin-patch motility in S . pombe using a fusion of green fluorescent protein (GFP) to a coronin homologue, Crn1p . Actin patches are associated with cables and move with rates of 0.32 microm s(-1) primarily in an undirected manner at cell tips and also in a directed manner along actin cables, often away from cell tips . Patches move more slowly or stop when actin polymerization is attenuated by Latrunculin A or in arp3 and cdc3 (profilin) mutants . In a cdc8 (tropomyosin) mutant, actin cables are absent, and patches move with similar speed but in a non-directed manner . Patches are sites of Arp3-dependent F-actin polymerization in vitro . Rapid F-actin turnover rates in vivo indicate that patches and cables are maintained continuously by actin polymerization . Our studies give rise to a model in which actin patches are centres for actin polymerization that drive their own movement on actin cables using Arp2/3-based actin polymerization. FEBS Lett, 2001 Jan 26, 489(1), 75 - 80 Schizosaccharomyces pombe och1(+) encodes alpha-1,6-mannosyltransferase that is involved in outer chain elongation of N-linked oligosaccharides; Yoko-o T et al.; The fission yeast Schizosaccharomyces pombe attaches an outer chain containing mannose and galactose to the N-linked oligosaccharides on many of its glycoproteins . We identified an S . pombe och1 mutant that did not synthesize the outer chains on acid phosphatase . The S . pombe och1(+) gene was a functional homolog of Saccharomyces cerevisiae OCH1, and its gene product (SpOch1p) incorporated alpha-1,6-linked mannose into pyridylaminated Man(9)GlcNAc(2), indicating that och1(+) encodes an alpha-1,6-mannosyltransferase . Our results indicate that SpOch1p is a key enzyme of outer chain elongation . The substrate specificity of SpOch1p was different from that of S . cerevisiae OCH1 gene product (ScOch1p), suggesting that SpOch1p may have a wider substrate specificity than that of ScOch1p. EMBO J, 2001 Mar 1, 20(5), 1064 - 73 Fission yeast Pom1p kinase activity is cell cycle regulated and essential for cellular symmetry during growth and division; Bahler J et al.; Schizosaccharomyces pombe cells grow from both ends during most of interphase and divide symmetrically into two daughter cells . The pom1 gene, encoding a member of the Dyrk family of protein kinases, has been identified through a mutant showing abnormal cellular morphogenesis . Here we show that Pom1p kinase activity is cell cycle regulated in correlation with the state of cellular symmetry: the activity is high during symmetrical growth and division, but lower when cells grow at just one end . Point mutations in the catalytic domain lead to asymmetry during both cell growth and division, whilst cells overexpressing Pom1p form additional growing ends . Manipulations of kinase activity indicate a negative role for Pom1p in microtubule growth at cell ends . Pom1p is present in a large protein complex and requires its non-catalytic domain to localize to the cell periphery and its kinase activity to localize to cell ends . These data establish that Pom1p kinase activity plays an important role in generating cellular symmetry and suggest that there may be related roles of homologous protein kinases ubiquitously present in all eukaryotes. Trends Genet, 2001 Mar, 17(3), 153 - 7 Does S . pombe exploit the intrinsic asymmetry of DNA synthesis to imprint daughter cells for mating-type switching? Dalgaard JZ, Klar AJ. Typically cell division is envisaged to be symmetrical, with both daughter cells being identical . However, during development and cellular differentiation, asymmetrical cell divisions have a crucial role . In this article, we describe a model of how Schizosaccharomyces pombe exploits the intrinsic asymmetry of DNA replication machinery--the difference between the replication of the leading strand and the lagging strand--to establish an asymmetrical mating-type switching pattern . This is the first system where the direction of DNA replication is involved in the formation of differentiated chromosomes . The discovery raises the possibility that DNA replication might be more generally involved in the establishment of asymmetric cellular differentiation. EMBO J, 2001 Jan 15, 20(1-2), 210 - 21 Novel functional requirements for non-homologous DNA end joining in Schizosaccharomyces pombe; Manolis KG et al.; DNA double strand break (DSB) repair by non-homologous end joining (NHEJ) in mammalian cells requires the Ku70-Ku80 heterodimer, the DNA-PK catalytic subunit DNA-PKcs, as well as DNA ligase IV and Xrcc4 . NHEJ of plasmid DSBs in Saccharomyces cerevisiae requires Ku, Xrcc4 and DNA ligase IV, as well as Mre11, Rad50, Xrs2 and DNA damage checkpoint proteins . Saccharomyces cerevisiae Ku is also required for telomere length maintenance and transcriptional silencing . We have characterized NHEJ in Schizosaccharomyces pombe using an extrachromosomal assay and find that, as anticipated, it is Ku70 and DNA ligase IV dependent . Unexpectedly, we find that Rad32, Rad50 (the S.pombe homologues of Mre11 and Rad50, respectively) and checkpoint proteins are not required for NHEJ . Furthermore, although S.pombe Ku70 is required for maintenance of telomere length, it is dispensable for transcriptional silencing at telomeres and is located throughout the nucleus rather than concentrated at the telomeres . Together, these results provide insight into the mechanism of NHEJ and contrast significantly with recent studies in S.cerevisiae. Yeast, 2001 Mar 15, 18(4), 355 - 61 Subtelomeric sequence from the right arm of Schizosaccharomyces pombe chromosome I contains seven permease genes; Hunt C et al.; The sequence has been determined of 80 888 bp of contiguous subtelomeric DNA, including the isp5 gene, from the right arm of chromosome I of Schizosaccharomyces pombe; 27 open reading frames (ORFs) longer than 100 codons are present, giving a density of one gene per 3.0 kb . Seven of the predicted proteins are members of the major facilitator superfamily (MFS) of transport proteins, including four amino acid permease homologues, bringing this family of amino acid permease sequences to 17 in Sz . pombe, and a phylogenetic analysis is presented . Also encoded is an allantoate permease homologue, a sulphate permease homologue and a probable urea active transporter . Predicted non-membrane proteins include a 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase), a class III aminotransferase, serine acetyltransferase, protein-L-isoaspartate O-methyltransferase, alpha-glucosidase, alpha-galactosidase, esterase/lipase, oxidoreductase of the short-chain dehydrogenase/reductase (SDR) family, aldehyde dehydrogenase, formamidase, amidase, flavohaemoprotein, a putative translation initiation inhibitor and a protein with similarity to a filamentous fungal conidiation-specific protein . The remaining six ORFs are likely to encode proteins, either because they have sequence similarity with hypothetical proteins or because they are known to be transcribed . Introns are scarce in the sequenced region: only three ORFs contain introns, with only one having multiple introns . The sequenced region also contains a single Tf1 transposon long terminal repeat (LTR) . The sequence is derived from cosmid clones c869, c922 and c1039 and has been submitted to the EMBL database under entries SPAC869 (Accession No . AL132779), SPAC922 (AL133522) and SPAC1039 (AL133521) . Biosci Biotechnol Biochem, 2000 Dec, 64(12), 2675 - 9 Efflux system for pyridoxine in Schizosaccharomyces pombe; Hirose K et al.; Pyridoxine-charged Schizosaccharomyces pombe released pyridoxine rapidly at 30 degrees C: very low amounts of three other B6 vitamers were also released . The rate of efflux was temperature-dependent . The initial rate of efflux was dependent on the concentration of pyridoxine in the cells: the rate was almost zero at lower than 0.02 mM and became saturated at higher than 0.2 mM . Na+, sodium azide, and dinitrophenol increased the rate in both the presence and absence of D-glucose . Mg++, thiamine, and menadione inhibited the efflux . The intracellular concentration of ATP did not significantly affect the efflux rate . The system may be dependent on a membrane potential of the yeast cells . It was found that the fission yeast cells have a gate or carrier system for efflux of pyridoxine, which was distinct from that in Saccharomyces cerevisiae. Mol Plant Microbe Interact, 2001 Feb, 14(2), 135 - 44 Characterization of small GTPases Cdc42 and Rac and the relationship between Cdc42 and actin cytoskeleton in vegetative and ectomycorrhizal hyphae of Suillus bovinus; Gorfer M et al.; This work reports the isolation and molecular characterization of CDC42 and RAC1 cDNAs from the ectomycorrhiza forming filamentous homobasidiomycete Suillus bovinus . Previously, no RAC gene was described from filamentous fungi and no CDC42 gene was described from homobasidiomycetes . Southern hybridization with SbCDC42 and SbRAC1 cDNAs indicated that the S . bovinus genome contains only one CDC42 and one RAC1 gene . The predicted amino acid sequence of SbRaclp is 77% identical with the Rac1B protein of chick, whereas SbCdc42p is most identical with Schizosaccharomyces pombe Cdc42p, showing 88% identity . In the predicted amino acid sequences of SbRaclp and SbCdc42p, the five guanine nucleotide binding regions, switch I and II, and the effector domain are highly identical to those known in other small GTPases . These domain structures suggest that in S . bovinus, SbRac1p and SbCdc42p function as molecular switches regulating the organization of actin cytoskeleton, similar to yeasts and mammals . SbRAC1 and SbCDC42 were e