Schizosaccharomyces

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 expressed in vegetative and ectomycorrhizal hyphae, and SbCdc42p was detected in ectomycorrhiza-forming hyphae if growth and differentiation of the symbiotic hyphae took place . Cdc42p and actin were localized at the tips of S . bovinus vegetative hyphae . Similar to yeast, in filamentous fungi Cdc42p may be necessary to maintain the actin cytoskeleton at hyphal tips, making the polarized growth of the hyphae possible . In developing ectomycorrhiza, Cdc42p and actin were visualized in association with plasma membrane in swollen cells typical to the symbiotic hyphae . The role of Cdc42p and actin in regulation of the growth pattern and morphogenesis of ectomycorrhizal hyphae is discussed.

Arch Microbiol, 2000 Dec, 174(6), 386 - 92
The use of morphomutants to investigate septum formation and cell separation in Schizosaccharomyces pombe; Sipiczki M et al.; Cytokinesis in the fission yeast Schizosaccharomyces begins by formation of a medially placed actomyosin ring, continues by progressive development of a septum, and is completed by cleavage of the mature septum to bring about cell separation . The cytological analysis of the Schizosaccharomyces pombe morphomutants sph2-3 and sep1-1 presented here demonstrates that the medial actomyosin ring colocalizes to the leading edge of the centripetally growing septum, and cleavage of the septum is triggered by rupture of the mother cell wall at the septal basis.

Biosci Biotechnol Biochem, 2000 Nov, 64(11), 2493 - 6
Identification and characterization of a novel gene, hos2+, the function of which is necessary for growth under high osmotic stress in fission yeast; Nakamichi N et al.; hos2 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth . An S . pombe strain carrying the hos2-M10 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously . In this study, the hos2+ gene was identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases . The hos2-M10 mutation resulted in Gln-62 to TAG-termination codon . A Hos2-defective (hos2delta) strain, which we then constructed, showed the phenotype of high osmolarity sensitivity, as in the case of the original hos2-M10 mutant . For this hos2delta mutant, three multicopy suppressor genes were isolated and one of which was identified as the pgk1+ gene, encoding a phosphoglycerate kinase.

Curr Genet, 2000 Dec, 38(5), 227 - 32
The Schizosaccharomyces pombe sep15+ gene encodes a protein homologous to the Med8 subunit of the Saccharomyces cerevisiae transcriptional mediator complex; Zilahi E et al.; We previously described the isolation of mutants defective in cell separation and the identification of 16 sep genes with complex functions . Here we report on the cloning and analysis of sep15+ . The deduced amino acid sequence of the Sep15 protein shows significant homology to Med8, a component of the Saccharomyces cerevisiae transcription mediator complex . The mutation sep15-598 confers hyphal morphology and causes temperature-sensitive lethality . Disruption of sep15+ is lethal, indicating that Sep15 exerts an essential function and its role in cell separation is indirect.

Genome Biol . 2000;1(2):REVIEWS1011 . Epub 2000 Aug 04.
Where does fission yeast sit on the tree of life?
Sipiczki M.
The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are as different from each other as either is from animals: their ancestors separated about 420 to 330 million years ago . Now that S . pombe is poised to join the post-genome era, its evolutionary position should become much clearer.

Yeast, 2001 Feb, 18(3), 229 - 38
Hyperthermotolerant fission yeast mutations, sow1 and sow2, suppress the cell cycle defect and stress sensitivity of MAP kinase kinase wis1Delta; Prochnik S et al.; Wis1 is a mitogen-activated protein kinase kinase (MAPKK) that regulates mitosis and mediates stress responses in the fission yeast, Schizosaccharomyces pombe . wis1Delta strains are viable but stress-sensitive and show a mitotic delay . At high temperatures, wis1Delta cells cease division but cellular growth continues . Mutations that suppress the heat sensitivity of a wis1Delta strain were isolated and map to two apparently novel loci, sow1 (for suppressor of wis1Delta) and sow2 . In addition to suppressing wis1Delta heat sensitivity, sow1 and sow2 can suppress wis1Delta osmosensitivity and cell cycle defects . sow1 and sow2 mutants in a wis1+ background were able to grow at higher temperatures than wild-type and sow1 showed a mitotic advance . The sow genes may therefore define a novel connection between stress tolerance and cell cycle control.

Yeast, 2001 Feb, 18(3), 207 - 17
The cyclic AMP/PKA signal pathway is required for initiation of spore germination in Schizosaccharomyces pombe; Hatanaka M et al.; Spore germination, a transition from the quiescent G0 phase to the proliferation cycle, is triggered by glucose in Schizosaccharomyces pombe . The role of cAMP/protein kinase A (PKA) signalling in germination is investigated . Gene disruption of cyr1+, pka1+ and gpa2+ encoding adenylate cyclase, PKA and the alpha-subunit of a trimeric GTP-binding protein, respectively, reduced the colony-forming efficiency of spores in minimal medium . Isolated spores of these null mutants did not germinate in minimal medium for up to 12 h, at which time wild-type spores had completed germination and formed germ projections . In wild-type spores, cortical actin patches randomly distributed in the early stage of outgrowth and then localized to one side of spores before the formation of projections . In contrast, the mutant spores exhibited no actin patches, but the cell surface was predominantly stained, like ungerminated spores of wild-type . Flow fluorocytometric analysis of propidium iodide-stained spores revealed a distinct 1C DNA peak after germination was completed . The fluorescent profile of the mutant spores, however, did not change during 12 h incubation in the minimal medium . These observations indicate that spores harbouring either cyr1Delta, pka1Delta or gpa2Delta are hardly triggered to germination . When wild-type spores were exposed to glucose, the intracellular cAMP level transiently increased in a few minutes, but gpa2Delta spores did not respond to glucose . We conclude that S . pombe spores initiate germination in response to glucose through the cyclic AMP-PKA pathway.

Gene, 2001 Jan 10, 262(1-2), 43 - 50
Cloning and sequence analysis of the basidiomycete Lentinus edodes ribonucleotide reductase small subunit cDNA and expression of a corresponding gene in L . edodes; Kaneko S et al.; We have previously isolated the uck1 gene encoding UMP-CMP kinase from the basidiomycete Lentinus edodes (Kaneko et al., 1998) . It was shown to be most actively transcribed in hymenophores of mature fruiting bodies of L . edodes . The reduction of NDPs produced by the nucleoside monophosphate kinase to dNDPs has been known to be catalyzed by ribonucleotide reductase (RNR) which consists of a heterodimer of large and small subunits . So we attempted to isolate the L . edodes cDNA(s) of RNR and study the expression in L . edodes of the corresponding gene(s), resulting in an isolation of the small subunit cDNA from a mature fruiting-body cDNA library of the fungus . This cDNA, named Le.rnr2c, was shown to encode a 418 amino acids (aa) protein, named Le.RNR2, of which the deduced aa sequence shows an overall identity of 71.9% to that of Schizosaccharomyces pombe RNR small subunit . The Le.rnr2 gene was found to be most actively transcribed in hymenophores of mature fruiting body of L . edodes . The in situ RNA-RNA hybridization analysis showed the presence of markedly large amount of the Le.rnr2 transcript in both hymenium and outer region of trama in the hymenophore . The same experiment was done for the uck1 gene, obtaining a similar result . The hymenium contains many basidia in which fusion of two nuclei, meiosis, replication, etc . essential for production of basidiospores occur . The outer region of trama is the region branching out into subhymenium . These imply that Le.rnr2 gene (and uck1 gene) play a role mainly in the nucleotide biosynthesis essential both for production of basidiospores and for divergence of trama cells into subhymenium cells in the hymenophore.

Mol Biol Cell, 2001 Feb, 12(2), 407 - 19
Peroxide sensors for the fission yeast stress-activated mitogen-activated protein kinase pathway; Buck V et al.; The Schizosaccharomyces pombe stress-activated Sty1p/Spc1p mitogen-activated protein (MAP) kinase regulates gene expression through the Atf1p and Pap1p transcription factors, homologs of human ATF2 and c-Jun, respectively . Mcs4p, a response regulator protein, acts upstream of Sty1p by binding the Wak1p/Wis4p MAP kinase kinase kinase . We show that phosphorylation of Mcs4p on a conserved aspartic acid residue is required for activation of Sty1p only in response to peroxide stress . Mcs4p acts in a conserved phospho-relay system initiated by two PAS/PAC domain-containing histidine kinases, Mak2p and Mak3p . In the absence of Mak2p or Mak3p, Sty1p fails to phosphorylate the Atf1p transcription factor or induce Atf1p-dependent gene expression . As a consequence, cells lacking Mak2p and Mak3p are sensitive to peroxide attack in the absence of Prr1p, a distinct response regulator protein that functions in association with Pap1p . The Mak1p histidine kinase, which also contains PAS/PAC repeats, does not regulate Sty1p or Atf1p but is partially required for Pap1p- and Prr1p-dependent transcription . We conclude that the transcriptional response to free radical attack is initiated by at least two distinct phospho-relay pathways in fission yeast.

BMC Mol Biol . 2001;2(1):1 . Epub 2001 Jan 18.
Multiple redundant sequence elements within the fission yeast ura4 replication origin enhancer; Kim SM et al.; BACKGROUND: Some origins in eukaryotic chromosomes fire more frequently than others . In the fission yeast, Schizosaccharomyces pombe, the relative firing frequencies of the three origins clustered 4-8 kbp upstream of the ura4 gene are controlled by a replication enhancer - an element that stimulates nearby origins in a relatively position-and orientation-independent fashion . The important sequence motifs within this enhancer were not previously localized . RESULTS: Systematic deletion of consecutive segments of approximately 50, approximately 100 or approximately 150 bp within the enhancer and its adjacent core origin (ars3002) revealed that several of the approximately 50-bp stretches within the enhancer contribute to its function in partially redundant fashion . Other stretches within the enhancer are inhibitory . Some of the stretches within the enhancer proved to be redundant with sequences within core ars3002 . Consequently the collection of sequences important for core origin function was found to depend on whether the core origin is assayed in the presence or absence of the enhancer . Some of the important sequences in the core origin and enhancer co-localize with short runs of adenines or thymines, which may serve as binding sites for the fission yeast Origin Recognition Complex (ORC) . Others co-localize with matches to consensus sequences commonly found in fission yeast replication origins . CONCLUSIONS: The enhancer within the ura4 origin cluster in fission yeast contains multiple sequence motifs . Many of these stimulate origin function in partially redundant fashion . Some of them resemble motifs also found in core origins . The next step is to identify the proteins that bind to these stimulatory sequences.

Yeast, 2001 Jan 30, 18(2), 115 - 24
Functional analysis of the Neurospora crassa PZL-1 protein phosphatase by expression in budding and fission yeast; Vissi E et al.; The gene pzl-1 from the filamentous fungus Neurospora crassa encodes a putative Ser/Thr protein phosphatase that is reminiscent of the Ppz1/Ppz2 and Pzh1 phosphatases from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively . The entire PZL-1 protein, as well as its carboxyl-terminal domain, have been expressed in Escherichia coli as active protein phosphatases . To characterize its cellular role, PZL-1 was also expressed in Sz . pombe and in S . cerevisiae . Expression of PZL-1 in S . cerevisiae from the PPZ1 promoter was able to rescue the altered sensitivity to caffeine and lithium ions of a ppz1 strain . Furthermore, high copy number expression of PZL-1 alleviated the lytic phenotype of a S . cerevisiae slt2/mpk1 mitogen-activated protein (MAP) kinase mutant, similarly to that described for PPZ1, and mimicked the effects of high levels of Ppz1 on cell growth . Expression of PZL-1 in fission yeast from a weak version of the nmt1 promoter fully rescued the growth defect of a pzh1Delta strain in high potassium, but only partially complemented the sodium-hypertolerant phenotype . Strong overexpression of the N . crassa phosphatase in Sz . pombe affected cell growth and morphology . Therefore, PZL-1 appears to fulfil every known function carried out by its S . cerevisiae counterpart, despite the marked divergence in sequence within their NH(2)-terminal moieties .

Genes Cells, 2001 Jan, 6(1), 25 - 36
Transcription organization and mRNA levels of the genes for all 12 subunits of the fission yeast RNA polymerase II; Sakurai H et al.; The RNA polymerase II (Pol II) of eukaryotes is composed of 12 subunits, of which five are shared among Pol I, Pol II and Pol III . At present, however, little is known about the regulation of synthesis and assembly of the 12 Pol II subunits . To obtain an insight into the regulation of synthesis of these 12 Pol II subunits, Rpb1 to Rpb12, in the fission yeast Schizosaccharomyces pombe, we analysed the transcriptional organization of the rpb genes by use of the oligo capping method, and determined mRNA levels by quantitative competitive PCR assay . The intracellular concentrations of the 12 Rpb subunits in growing S . pombe cells are different, within a range of 15-fold difference between the least abundant Rpb3 and the most abundant Rpb12 . The transcription of one group of genes including rpb3, rpb4, rpb5, rpb6, rpb7 and rpb10 is mainly initiated at a single site, while that of the other group of genes for rpb1, rpb2, rpb8, rpb9, rpb11 and rpb12 is initiated at multiple sites . The promoters of the first group of genes contain the TATA box sequence between -26 and -62, while the second group of genes carry TATA-less promoters . Several common sequence segments, tentatively designated 'Rpb motifs', were identified in the promoter regions of the rpb genes . Competitive PCR analysis indicated that mRNAs for Rpb1, Rpb3, Rpb7 and Rpb9 were among the group which had a low abundance, while the levels of Rpb6 and Rpb10 mRNAs were about fivefold, and that of Rpb2 mRNA was about 40-fold higher than the Rpb3 mRNA level . The levels of rpb mRNAs do not correlate with those of Rpb proteins . The protein-to-mRNA ratio or the translation efficiency is low for the rpb1, rpb2, rpb3 and rpb11 genes, encoding the homologues of subunits beta', beta, alpha and alpha, respectively, of the prokaryotic RNA polymerase core enzyme.

Eur J Biochem, 2001 Feb, 268(3), 612 - 9
Intracellular contents and assembly states of all 12 subunits of the RNA polymerase II in the fission yeast Schizosaccharomyces pombe; Kimura M et al.; The RNA polymerase II (Pol II) of the fission yeast Schizosaccharomyces pombe is composed of 12 different polypeptides, Rpb1 to Rpb12, of which five, Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12, are shared among three forms of the RNA polymerase . To get an insight into the control of synthesis and assembly of individual subunits, we have measured the intracellular concentrations of all 12 subunits in S . pombe by quantitative immunoblotting . Results indicate that the levels are low for the three large subunits, Rpb1, Rpb2 and Rpb3, which are the homologues of beta', beta and alpha subunits, respectively, of prokaryotic RNA polymerase . On the other hand, the levels of small-sized subunits were between 2- to 15-fold higher than these three core subunits . The levels of the five common subunits shared among RNA polymerases I, II and III are about 10 times greater than those of the Pol II-specific core subunits . The assembly state of the Rpb proteins was analyzed by glycerol gradient centrifugation of S . pombe whole cell extracts . The three core subunits are mostly assembled in Pol II, but some of the small subunits were detected in the slowly sedimenting fractions, indicating that at least some of the excess Rpb proteins exist in unassembled forms . Based on the intracellular concentration of the least abundant Rpb3 subunit, the total number of Pol II in a growing S . pombe cell was estimated to be about 10,000 molecules . The intracellular distribution of some Pol II subunits was also analyzed by microscopic observation of the green fluorescent protein (GFP)-fused Rpb proteins . In agreement with the biochemical analysis, the GFP-Rpb1 and GFP-Rpb3 fusions were present in the nuclei but the GFP-Rpb4 was detected in the cytoplasm as well as the nuclei.

Genomics, 2001 Jan 15, 71(2), 252 - 5
Isolation of a ubiquitin-like (UBL5) gene from a screen identifying highly expressed and conserved iris genes; Friedman JS et al.; We have screened a human adult iris cDNA library to identify genes that are highly expressed and conserved between humans and pigs . We identified human iris cDNAs that hybridized at high stringency to a porcine choroidal ring cDNA probe . Of 1568 human iris cDNAs examined, 176 were found to have high expression in porcine choroidal rings . One of the 176 clones was identified as a previously uncharacterized cDNA that we have named the Ubiquitin-like 5 gene (UBL5) . The UBL5 gene is located on chromosome 19p13.2, and its genomic structure has been examined . There is a UBL5 pseudogene on chromosome 17p11.2 . We have also found homologues to the UBL5 gene in Arabidopsis thaliana, Caenorhabditis elegans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae . Northern blot analysis of the Ubiquitin-like gene 5 revealed expression in every tissue tested, with the highest levels of RNA expression in heart, skeletal muscle, kidney, liver, iris, and lymphoblasts . Intracellular localization experiments in COS-7 cells showed that the recombinant UBL5 protein is cytoplasmic . Western analysis demonstrated that the recombinant UBL5 protein is approximately 9 kDa, as predicted from the cDNA . A comparison between UBL5 and its homologues with other Ubiquitin-like proteins and Ubiquitin, using the PROTDIST program, suggests that the UBL5 genes are a separate class of Ubiquitin-like genes . Further characterization of the UBL5 gene will determine the function of the encoded protein and whether it is a candidate for ocular disease .

Mol Biol Cell, 2001 Jan, 12(1), 115 - 28
Role of fission yeast primase catalytic subunit in the replication checkpoint; Griffiths DJ et al.; To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe, spp1(+) and spp2(+) (S . pombe primase 1 and 2) . spp1(+) encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Polalpha to synthesize the initiation DNA . Here, we reported the isolation of the fission yeast spp1(+) gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle . Spp1 is essential for cell viability, and thermosensitive mutants of spp1(+) exhibit an allele-specific abnormal mitotic phenotype . Mutations of spp1(+) reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Polalpha-primase complex . The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase . Mutational analysis of Polalpha has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Polalpha . Together, these have led us to propose that suboptimal cellular levels of polalpha-primase complex due to the allele-specific mutations of Spp1 might not allow Polalpha to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response . Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.

J Lipid Res, 2001 Jan, 42(1), 150 - 4
A novel gene conserved from yeast to humans is involved in sterol biosynthesis; Gachotte D et al.; The ERG28 gene was originally identified by microarray expression profiling as possibly involved in the Saccharomyces cerevisiae sterol pathway . Microarray analyses suggested that the transcription pattern of ERG28 closely followed that of genes involved in sterol synthesis . ERG28 was also found in Schizosaccharomyces pombe and Arabidopsis as well as humans, and in the latter was shown to be highly expressed in adult testis tissue . All four proteins contain potential transmembrane domain(s) . Gas chromatography-mass spectrometry analysis of an ERG28-deleted S . cerevisiae strain (which is slow growing but not auxotrophic for ergosterol) indicates a lesion in sterol C-4 demethylation . Sterol profiles indicate accumulation of 3-keto and carboxylic acid sterol intermediates, which are involved in removing the two C-4 methyl groups from the sterol A ring . Similar intermediates have previously been demonstrated to accumulate in erg26 (sterol dehydrogenase/decarboxylase) and erg27 (3-ketoreductase) mutants in yeast.We speculate that the role of the Erg28 protein (Erg28p) may be either to tether Erg26p and Erg27p to the endoplasmic reticulum or to facilitate interaction between these proteins.-Gachotte, D., J . Eckstein, R . Barbuch, T . Hughes, C . Roberts, and M . Bard . A novel gene conserved from yeast to humans is involved in sterol biosynthesis . J . Lipid Res . 2001 . 42: 150;-154.

Genetics, 2001 Feb, 157(2), 519 - 32
Characterization of rec7, an early meiotic recombination gene in Schizosaccharomyces pombe; Molnar M et al.; rec7 is involved in intra- and intergenic meiotic recombination in all tested regions of the genome of the fission yeast Schizosaccharomyces pombe . Segregational analysis in a rec7 gene disruption mutant revealed frequent occurrence of two-spored asci . Spores giving rise to diploid colonies were shown to derive from skipping of the second meiotic division . Nondisjunction of homologous chromosomes at the first meiotic division was also frequent . The cytological structures and processes, such as formation of linear elements, pairing of homologous chromosomes, and clustering of telomeres and centromeres, are regular in the mutant . Northern blot experiments revealed meiosis-specific expression of rec7 . Screening of a meiotic cDNA library also identified transcripts from the opposite strand in the rec7 region . A Rec7-GFP fusion protein was localized in the nucleus of whole cells before karyogamy, during prophase, and after meiosis I . On spreads of prophase nuclei approximately 50 foci of Rec7-GFP were counted . Some of the observed phenotypes of the disruption mutant and the N-terminal sequence homology suggest that Rec7p is a functional homolog of Rec114p of Saccharomyces cerevisiae . The observed phenotypes of the disruption and the appearance of Rec7-GFP in mating haploid cells and after meiosis I are consistent with Rec7p functions before, during, and after meiotic prophase.

J Biol Chem, 2000 Apr 14, 275(15), 11198 - 206
The Saccharomyces cerevisiae Rheb G-protein is involved in regulating canavanine resistance and arginine uptake; Urano J et al.; The new member of the Ras superfamily of G-proteins, Rheb, has been identified in rat and human, but its function has not been defined . We report here the identification of Rheb homologues in the budding yeast Saccharomyces cerevisiae (ScRheb) as well as in Schizosaccharomyces pombe, Drosophila melanogaster, zebrafish, and Ciona intestinalis . These proteins define a new class of G-proteins based on 1) their overall sequence similarity, 2) high conservation of their effector domain sequence, 3) presence of a unique arginine in their G1 box, and 4) presence of a conserved CAAX farnesylation motif . Characterization of an S . cerevisiae strain deficient in ScRheb showed that it is hypersensitive to growth inhibitory effects of canavanine and thialysine, which are analogues of arginine and lysine, respectively . Accordingly, the uptake of arginine and lysine was increased in the ScRheb-deficient strain . This increased arginine uptake requires the arginine-specific permease Can1p . The function of ScRheb is dependent on having an intact effector domain since mutations in the effector domain of ScRheb are incapable of complementing canavanine hypersensitivity of scrheb disruptant cells . Furthermore, the conserved arginine in the G1 box plays a role in the activity of ScRheb, as a mutation of this arginine to glycine significantly reduced the ability of ScRheb to complement canavanine hypersensitivity of ScRheb-deficient yeast . Finally, a mutation in the C-terminal CAAX farnesylation motif resulted in a loss of ScRheb function . This result, in combination with our finding that ScRheb is farnesylated, suggests that farnesylation plays a key role in ScRheb function . Our findings assign the regulation of arginine and lysine uptake as the first physiological function for this new farnesylated Ras superfamily G-protein.

Curr Biol, 2000 Apr 6, 10(7), 397 - 400
Fission yeast myosin-II isoforms assemble into contractile rings at distinct times during mitosis; Bezanilla M et al.; Myosin-II is required for cytokinesis in Schizosaccharomyces pombe {1-3}, but unlike other unicellular organisms, S . pombe has two structurally distinct myosin-IIs, Myo2p and Myp2p, which are required under different conditions {4} . Disruption of myo2(+) is lethal, whereas disruption of myp2(+) leads to defects in cytokinesis when nutrients are limiting and to cold-sensitivity in 1 M KCl . In dividing cells, both myosin-IIs localize to a ring in the center of the cell, which is thought to contract, separating the cytoplasms of the daughter cells . Using deconvolution microscopy, we obtained three-dimensional reconstructions of fission yeast cells expressing green fluorescent protein-labeled (GFP)-myosin-II, providing for the first time detailed images of GFP-myosin-II rings . By time-lapse microscopy, we observed ring assembly and contraction in three dimensions using GFP-tubulin as a cell cycle marker . We determined that the Myo2p ring forms in metaphase/anaphase A whereas the Myp2p ring forms much later, at the end of anaphase B . Myo2p initiates ring formation while Myp2p acts later to increase the efficiency of cytokinesis.

J Biol Chem, 2001 Mar 30, 276(13), 10056 - 62 Epub 2000 Dec 27.
Fission yeast homolog of murine Int-6 protein, encoded by mouse mammary tumor virus integration site, is associated with the conserved core subunits of eukaryotic translation initiation factor 3; Akiyoshi Y et al.; The murine int-6 locus, identified as a frequent integration site of mouse mammary tumor viruses, encodes the 48-kDa eIF3e subunit of translation initiation factor eIF3 . Previous studies indicated that the catalytically active core of budding yeast eIF3 consists of five subunits, all conserved in eukaryotes, but does not contain a protein closely related to eIF3e/Int-6 . Whereas the budding yeast genome does not encode a protein closely related to murine Int-6, fission yeast does encode an Int-6 ortholog, designated here Int6 . We found that fission yeast Int6/eIF3e is a cytoplasmic protein associated with 40 S ribosomes . FLAG epitope-tagged Tif35, a putative core eIF3g subunit, copurified with Int6 and all five orthologs of core eIF3 subunits . An int6 deletion (int6Delta) mutant was viable but grew slowly in minimal medium . This slow growth phenotype was accompanied by a reduction in the amount of polyribosomes engaged in translation and was complemented by expression of human Int-6 protein . These findings support the idea that human and Schizosaccharomyces pombe Int-6 homologs are involved in translation . Interestingly, haploid int6Delta cells showed unequal nuclear partitioning, possibly because of a defect in tubulin function, and diploid int6Delta cells formed abnormal spores . We propose that Int6 is not an essential subunit of eIF3 but might be involved in regulating the activity of eIF3 for translation of specific mRNAs in S . pombe.

Genes Dev, 2001 Jan 1, 15(1), 36 - 41
Multiple roles for the yeast SUB2/yUAP56 gene in splicing; Libri D et al.; The UAP56 gene has been shown to be required for prespliceosome assembly in mammals . We report here the isolation of the Schizosaccharomyces pombe ortholog of this gene by heterologous complementation of a combined PRP40HA(3)/nam8Delta defect in budding yeast . The Saccharomyces cerevisiae ortholog, YDL084w/SUB2, is also able to suppress this defect . We show that SUB2 is involved in splicing in vivo as well as in vitro . Sub2 defective extracts form a stalled intermediate that contains U2snRNP and can be chased into functional spliceosomes . Our experiments also suggest a role for this protein in events that precede prespliceosome formation . Data reported here as well as in the accompanying papers strongly implicate Sub2p in multiple steps of the spliceosome assembly process.

Appl Microbiol Biotechnol, 2000 Dec, 54(6), 792 - 8
Differential uptake of fumarate by Candida utilis and Schizosaccharomyces pombe; Saayman M et al.; The dicarboxylic acid fumarate is an important intermediate in cellular processes and also serves as a precursor for the commercial production of fine chemicals such as L-malate . Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon . In this study we have shown that the yeast Candida utilis effectively degraded extracellular fumarate and L-malate, but glucose or other assimilable carbon sources repressed the transport and degradation of these dicarboxylic acids . The transport of both dicarboxylic acids was shown to be strongly inducible by either fumarate or L-malate while kinetic studies suggest that the two dicarboxylic acids are transported by the same transporter protein . In contrast, Schizosaccharomyces pombe effectively degraded extracellular L-malate, but not fumarate, in the presence of glucose or other assimilable carbon sources . The Sch . pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of L-malate.

Nucleic Acids Res, 2001 Jan 15, 29(2), 439 - 48
Structure of human DNMT2, an enigmatic DNA methyltransferase homolog that displays denaturant-resistant binding to DNA; Dong A et al.; DNMT2 is a human protein that displays strong sequence similarities to DNA (cytosine-5)-methyltransferases (m(5)C MTases) of both prokaryotes and eukaryotes . DNMT2 contains all 10 sequence motifs that are conserved among m(5)C MTases, including the consensus S:-adenosyl-L-methionine-binding motifs and the active site ProCys dipeptide . DNMT2 has close homologs in plants, insects and Schizosaccharomyces pombe, but no related sequence can be found in the genomes of Saccharomyces cerevisiae or Caenorhabditis elegans . The crystal structure of a deletion mutant of DNMT2 complexed with S-adenosyl-L-homocysteine (AdoHcy) has been determined at 1.8 A resolution . The structure of the large domain that contains the sequence motifs involved in catalysis is remarkably similar to that of M.HHAI, a confirmed bacterial m(5)C MTase, and the smaller target recognition domains of DNMT2 and M.HHAI are also closely related in overall structure . The small domain of DNMT2 contains three short helices that are not present in M.HHAI . DNMT2 binds AdoHcy in the same conformation as confirmed m(5)C MTases and, while DNMT2 shares all sequence and structural features with m(5)C MTases, it has failed to demonstrate detectable transmethylase activity . We show here that homologs of DNMT2, which are present in some organisms that are not known to methylate their genomes, contain a specific target-recognizing sequence motif including an invariant CysPheThr tripeptide . DNMT2 binds DNA to form a denaturant-resistant complex in vitro . While the biological function of DNMT2 is not yet known, the strong binding to DNA suggests that DNMT2 may mark specific sequences in the genome by binding to DNA through the specific target-recognizing motif.

Nucleic Acids Res, 2001 Jan 15, 29(2), 387 - 96
Characterization of Schizosaccharomyces pombe RNA triphosphatase; Pei Y et al.; RNA triphosphatase catalyzes the first step in mRNA cap formation which entails the cleavage of the beta-gamma phosphoanhydride bond of triphosphate-terminated RNA to yield a diphosphate end that is then capped with GMP by RNA guanylyltransferase . Here we characterize a 303 amino acid RNA triphosphatase (Pct1p) encoded by the fission yeast SCHIZOSACCHAROMYCES: pombe . Pct1p hydrolyzes the gamma phosphate of triphosphate-terminated poly(A) in the presence of magnesium . Pct1p also hydrolyzes ATP to ADP and P(i) in the presence of manganese or cobalt (K(m) = 19 microM ATP; k(cat) = 67 s(-1)) . Hydrolysis of 1 mM ATP is inhibited with increasing potency by inorganic phosphate (I(0.5) = 1 mM), pyrophosphate (I(0.5) = 0.4 mM) and tripolyphosphate (I(0.5) = 30 microM) . Velocity sedimentation indicates that Pct1p is a homodimer . Pct1p is biochemically and structurally similar to the catalytic domain of Saccharomyces cerevisiae RNA triphosphatase Cet1p . Mechanistic conservation between Pct1p and Cet1p is underscored by a mutational analysis of the putative metal-binding site of Pct1p . Pct1p is functional in vivo in S.cerevisiae in lieu of Cet1p, provided that it is coexpressed with the S.pombe guanylyltransferase . Pct1p and other yeast RNA triphosphatases are completely unrelated, mechanistically and structurally, to the metazoan RNA triphosphatases, suggesting an abrupt evolutionary divergence of the capping apparatus during the transition from fungal to metazoan species.

Genetics, 2001 Jan, 157(1), 63 - 77
Crossing over is rarely associated with mitotic intragenic recombination in Schizosaccharomyces pombe; Virgin JB et al.; Chromosomal rearrangements can result from crossing over during ectopic homologous recombination between dispersed repetitive DNA . We have previously shown that meiotic ectopic recombination between artificially dispersed ade6 heteroalleles in the fission yeast Schizosaccharomyces pombe frequently results in chromosomal rearrangements . The same recombination substrates have been studied in mitotic recombination . Ectopic recombination rates in haploids were approximately 1-4 x 10(-6) recombinants per cell generation, similar to allelic recombination rates in diploids . In contrast, ectopic recombination rates in heterozygous diploids were 2.5-70 times lower than allelic recombination or ectopic recombination in haploids . These results suggest that diploid-specific factors inhibit ectopic recombination . Very few crossovers occurred in ade6 mitotic recombination, either allelic or ectopic . Allelic intragenic recombination was associated with 2% crossing over, and ectopic recombination between multiple different pairing partners showed 1-7% crossing over . These results contrast sharply with the 35-65% crossovers associated with meiotic ade6 recombination and suggest either differential control of resolution of recombination intermediates or alternative pathways of recombination in mitosis and meiosis.

Mol Microbiol, 2001 Jan, 39(2), 445 - 54
Functional characterization of the alpha-glucoside transporter Sut1p from Schizosaccharomyces pombe, the first fungal homologue of plant sucrose transporters; Reinders A et al.; Disaccharide transporters have not previously been identified in Schizosaccharomyces pombe . This is in contrast to Saccharomyces cerevisiae in which several maltose permeases belonging to the sugar porter (SP) family have been characterized . Here we report that a novel S . pombe gene, sut1+, encodes a proton-coupled disaccharide uptake transporter in the glycoside-pentoside-hexuronide (GPH):cation symporter family . Previously, members of the GPH family were restricted to bacteria and plants . The closest homologues of sut1+ are the sucrose uptake transporters (SUTs) from higher plants that transport sucrose with a higher affinity than maltose . The transport function of Sut1p was analysed by expression in S . cerevisiae . Sut1p was found to transport maltose with a Km of 6.5 +/- 0.4 mM and sucrose with a Km of 36.3 +/- 9.7 mM . Therefore, the substrate specificity of Sut1p from S . pombe is different from that of its plant homologues . Glucose repression of sut1+ at the transcriptional level is also consistent with a physiological function for Sut1p in maltose uptake . These results indicate that, unlike S . cerevisiae, S . pombe utilizes maltose transporters derived from a common ancestor with the plant SUTs.

Mol Gen Genet, 2000 Nov, 264(4), 492 - 505
A novel member of the Swi6p family of fission yeast chromo domain-containing proteins associates with the centromere in vivo and affects chromosome segregation; Halverson D et al.; We previously used a genetic approach to identify a new class of Schizosaccharomyces pombe genes (chromosome loss when overexpressed; clo genes) that, when present in elevated dosage, cause the loss of an otherwise stable cen1 linear minichromosome at high rates . Here we report the identities of two clo genes; one encodes histone H3.3 and the other, designated clo2, encodes a novel protein with significant homology to fission yeast Swi6p, human and Drosophila HP1 heterochromatin proteins, and other chromo domain-containing proteins . Members of this group have been shown to localize to heterochromatic DNA, including centromeres, and to play roles in chromatin formation and organization . The S . pombe Clo2 protein localizes to centromere DNA in vivo, and overexpression of clo2 leads to a dramatic increase in the rate of mitotic loss of an artificial chromosome . Clo2p is not essential for mitotic growth, however, even in cells that also lack Swi6p . Thus, fission yeast appears to utilize multiple, functionally redundant, HP1-related proteins for heterochromatin-associated activities at centromeres and perhaps elsewhere in the genome.

Mol Gen Genet, 2000 Nov, 264(4), 441 - 51
The Prr1 response regulator is essential for transcription of ste11+ and for sexual development in fission yeast; Ohmiya R et al.; Schizosaccharomyces pombe expresses a putative transcription factor, named Prr1, which is intriguing in the sense that it contains a bacterial type of phospho-accepting receiver domain, preceded by a mammalian heat shock factor (HSF2)-like DNA-binding domain . The receiver domain is most probably involved in an as yet unidentified histidine-to-aspartate (His-to-Asp) phosphorelay pathway in S . pombe . In this study, the structure, function, and cellular localization of Prr1 were assessed in the context of oxidative stress and His-to-Asp phosphorelay . As the most intriguing result of this study, we found that Prr1 is essential not only for the expression of genes induced by oxidative stress (e.g., ctt1+ and trr1+), but also for the expression of ste11+, which in turn is responsible for the expression of a variety of genes required for sexual development . Accordingly, Prr1-deficient cells are not only hypersensitive to oxidative stress, but also severely defective in conjugation and/or spore formation . These results suggested that the transcription factor Prr1 plays a pivotal role in an as yet unknown signal transduction pathway that is implicated in sexual differentiation . These findings are discussed with special reference to the well-characterized transcription factors Pap1 and Atf1 of S . pombe.

Mol Gen Genet, 2000 Nov, 264(4), 392 - 401
A Neurospora double-strand-break repair gene, mus-11, encodes a RAD52 homologue and is inducible by mutagens; Sakuraba Y et al.; We isolated a Neurospora crassa cDNA that encodes a Rad52 homologue (ncRAD52) by PCR, using degenerate primers . RFLP mapping demonstrated that the cloned gene is located close to the ro-4 locus on the right arm of linkage group V (LGVR) . In a second experiment, we used sib selection to identify a cosmid clone containing the mus-11 gene in a N . crassa genomic library . Fine-scale mapping of the mus-11 mutant showed the gene order on LGVR near ro-4 to be: ad-7 - (9.5 mu) - pab-2 (7.8 mu) - mus-11 - (3.7 mu) - inv . The nucleotide sequence of the mus-11 gene matched that of the ncRAD52 cDNA . Thus, the mus-11 gene encodes the Rad52 homologue . The deduced amino acid sequence of the MUS11 protein shows 32.0% and 27.5% overall identity to the Schizosaccharomyces pombe Rad22 protein and the human hRad52 protein, respectively, and a higher level of identity (55-66%) within the conserved N-terminal region (141 residues) . The MUS11 protein shows homology to Rad52 from budding yeast only within the N-terminal region (53.2% identity over 141 amino acids) which is conserved among Rad52 homologues . Yeast two-hybrid analysis reveals that the MUS11 protein binds to both the MEI-3 protein, a Rad51 homologue, and to itself in vivo . An ncRAD52 mutant obtained by the RIPping procedure showed the same sensitivity as the original mus-11 mutant to the following mutagens and chemicals: UV light, 4NQO (4-nitroquinoline 1-oxide), MMS (methyl methanesulfonate), EMS (ethyl methanesulfonate), MNNG (N-methyl-N'-nitro-N-nitrosoguanidine), TBHP (tert-butyl hydroperoxide), HU (hydroxyurea) and histidine . Unlike the RAD52 transcript in Saccharomyces cerevisiae, the mus-11 transcript could not be detected in mycelium under normal growth conditions, but expression of the gene was induced by UV irradiation or treatment with MMS.

J Mol Biol, 2001 Jan 12, 305(2), 203 - 17
Only centromeres can supply the partition system required for ARS function in the yeast Yarrowia lipolytica; Vernis L et al.; Autonomously replicating sequences (ARSs) in the yeast Yarrowia lipolytica require two components: an origin of replication (ORI) and centromere (CEN) DNA, both of which are necessary for extrachromosomal maintenance . To investigate this cooperation in more detail, we performed a screen for genomic sequences able to confer high frequency of transformation to a plasmid-borne ORI . Our results confirm a cooperation between ORI and CEN sequences to form an ARS, since all sequences identified in this screen displayed features of centromeric DNA and included the previously characterized CEN1-1, CEN3-1 and CEN5-1 fragments . Two new centromeric DNAs were identified as they are unique, map to different chromosomes (II and IV) and induce chromosome breakage after genomic integration . A third sequence, which is adjacent to, but distinct from the previously characterized CEN1-1 region was isolated from chromosome I . Although these CEN sequences do not share significant sequence similarities, they display a complex pattern of short repeats, including conserved blocks of 9 to 14 bp and regions of dyad symmetry . Consistent with their A+T-richness and strong negative roll angle, Y . lipolytica CEN-derived sequences, but not ORIs, were capable of binding isolated Drosophila nuclear scaffolds . However, a Drosophila scaffold attachment region that functions as an ARS in other yeasts was unable to confer autonomous replication to an ORI-containing plasmid . Deletion analysis of CEN1-1 showed that the sequences responsible for the induction of chromosome breakage could be eliminated without compromising extrachromosomal maintenance . We propose that, while Y . lipolytica CEN DNA is essential for plasmid maintenance, this function can be supplied by several sub-fragments which, together, form the active chromosomal centromere . This complex organization of Y . lipolytica centromeres is reminiscent of the regional structures described in the yeast Schizosaccharomyces pombe or in multicellular eukaryotes .

Yeast, 2001 Jan 15, 18(1), 33 - 9
The Schizosaccharomyces pombe GPI8 gene complements a Saccharomyces cerevisiae GPI8 anchoring mutant; Shams-Eldin H et al.; The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction, in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides . The Saccharomyces cerevisiae GPI8 gene (ScGPI8) encodes a protein which is involved in the GPI transamidation reaction . We have cloned and isolated the Schizosaccharomyces pombe GPI8 homologous gene (SpGPI8) . The SpGPI8 gene encodes a protein of 411 amino acids with a calculated molecular weight of about 47 kDa . It shows 53.5% identity with the ScGPI8 and complements a S . cerevisiae GPI8 anchoring mutant.

Curr Opin Microbiol, 2000 Dec, 3(6), 631 - 6
Cell cycle regulation in Schizosaccharomyces pombe; Moser BA et al.; Cdc2, a cyclin-dependent kinase, controls cell cycle progression in fission yeast . New details of Cdc2 regulation and function have been uncovered in recent studies . These studies involve cyclins that associate with Cdc2 in G1-phase and the proteins that regulate inhibitory phosphorylation of Cdc2 during S-phase and G2-phase . Recent investigations have also provided a better understanding of proteins that regulate DNA replication and that are directly or indirectly controlled by Cdc2.

Pharmacol Rev, 2000 Dec, 52(4), 477 - 92
Yeast mutants as a model system for identification of determinants of chemosensitivity; Perego P et al.; The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae have become valuable tools for the study of basic cellular functions of eukaryotic cells, including DNA repair mechanisms and cell cycle control . Since the major signaling pathways and cellular processes involved in cellular response to cytotoxic agents are conserved between yeasts and mammalian cells, these simple eukaryotic systems could be excellent models for the identification of molecular/cellular mechanisms of sensitivity to antitumor drugs . We describe relevant biological features of yeast cells and potential applications derived by their genetic manipulation . In particular, we have outlined the role of genes involved in repair processes and in checkpoint control, with specific reference to genes regulating radiation-sensitivity . Specific examples are provided concerning the use of both yeasts in understanding the mechanism of action of platinum compounds and topoisomerase inhibitors . The availability of the genomic sequence of these organisms as well as of new technologies (microarrays, proteomics) is expected to allow the identification of potential drug targets, since the drug discovery process is moving toward a genomic orientation . Among eukaryotic organisms, yeasts are suitable for easy genetic manipulations, and specific genetic alterations are exploitable for assessing the effects of chemotherapeutic agents with different mechanism of action . Although still at an early stage, this fast-moving field shows promise as a novel and potentially useful method for development of target-specific therapeutic approaches.

Biochim Biophys Acta, 2000 Dec 15, 1517(1), 171 - 5
Cloning, expression and regulation of Schizosaccharomyces pombe gene encoding thioltransferase; Cho YW et al.; The genomic DNA encoding thioltransferase was isolated from Schizosaccharomyces pombe using the polymerase chain reaction . The amplified DNA fragment was confirmed by Southern hybridization, completely digested with HindIII and BamHI, and then ligated into the yeast-Escherichia coli shuttle vector pRS316, which resulted in plasmid pEH1 . The insert of plasmid pEH1 was transferred into the multi-copy vector YEp357 to generate plasmid pYEH1 . The determined nucleotide sequence harbors an open reading frame consisting of four exons and three introns, which encodes a polypeptide of 101 amino acids with a molecular mass of 11261 Da . Thioltransferase activity was increased 1.6-fold in Saccharomyces cerevisiae containing plasmid pYEH1, and 1.8- and 2.7-fold in S . pombe containing plasmid pEH1 and pYEH1, respectively . The upstream sequence and the region encoding the N-terminal six amino acids were fused into promoterless beta-galactosidase gene of the shuttle vector YEp357R to generate the fusion plasmid pYEHR1 . Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by zinc and NO-generating S-nitroso-N-acetylpenicillamine.

Mol Microbiol, 2000 Nov, 38(4), 839 - 53
Identification of proteases with shared functions to the proprotein processing protease Krp1 in the fission yeast Schizosaccharomyces pombe; Ladds G et al.; Many secretory proteins are synthesized as inactive proproteins that undergo proteolytic activation as they travel through the eukaryotic secretory pathway . The best characterized family of processing enzymes are the prohormone convertases or kexins, and these are responsible for the processing of a wide variety of prohormones and other precursors . Recent work has identified other proteases that appear to be involved in proprotein processing, but characterization of these enzymes is at an early stage . Krp1 is the only kexin identified in the fission yeast Schizosaccharomyces pombe, in which it is essential for cell viability . We have used a genetic screen to identify four proteases with specificities that overlap Krp1 . Two are serine proteases, one is a zinc metalloprotease (glycoprotease) and one is an aspartyl protease that belongs to the recently described yapsin family of processing enzymes . All four proteases support the growth of a yeast strain lacking Krp1, and each is able to process the P-factor precursor, the only substrate currently known to be processed by Krp1.

Yeast, 2000 Dec, 16(16), 1519 - 26
Sequence analysis of two cosmids from Schizosaccharomyces pombe chromosome III; Lucas M et al.; We report the complete sequence of two cosmids, SPCC895 (38457 bp insert, EMBL Accession No . AL035247) and SPCC1322 (42068 bp insert, EMBL Accession No . AL035259), localized on chromosome III of the Schizosaccharomyces pombe genome . Fourteen Coding DNA sequences (CDSs) were identified in SPCC895 and 17 in SPCC1322 . Two known genes were found in each cosmid: map2 and gms1 on SPCC895, encoding the mating type P-factor precursor and an UDP-galactose transporter, respectively, and bub1 and ade6 in SPCC1322, encoding a protein kinase and a phosphoribosylaminoimidazole carboxylase, respectively . The fission yeast K RNA gene has been localized to SPCC895 . Three ribosomal proteins have been predicted among these two cosmids . Nine CDSs similar to known proteins were found on SPCC895, and seven on SPCC1322 . They include putative genes for an uridylate kinase, a proteasome catalytic component, an ion transporter, a checkpoint protein, a translation initiation protein, a SNARE complex protein, a protein involved in cytoskeletal organization, a spindle pole body-associating protein, pre-mRNA splicing factor RNA helicase, a 3'-5' exonuclease for RNA 3' ss-tail, an UTP-glucose-1-phosphate uridylyltransferase, a leukotriene A(4) hydrolase, a member of the RanBP7-importin beta-Cse1p superfamily, a Ca(++)-calmodulin-dependent serine/threonine protein kinase and a prohibitin antiproliferative protein . One CDS is predicted to be an integral membrane protein . One CDS from SPCC895 is similar to a CDS of unknown function from Saccharomyces cerevisiae and three from SPCC1322 are similar to CDSs of unknown function from Candida albicans, S . cerevisiae and Sz . pombe, respectively . Finally, one CDS of SPCC895 and three of SPCC1322 correspond to orphan genes .

J Cell Sci, 2001 Jan, 114(Pt 1), 69 - 79
Two type V myosins with non-overlapping functions in the fission yeast Schizosaccharomyces pombe: Myo52 is concerned with growth polarity and cytokinesis, Myo51 is a component of the cytokinetic actin ring; Win TZ et al.; The fission yeast genome project has identified five myosin genes: one type I myosin, myo1(+), two type II myosins, myo2(+) and myp2(+), and two type V myosins, myo51(+) and myo52(+) . Cells deleted for myo51(+) show normal morphology and growth rates whereas deletion of myo52(+) results in a partial loss of cell polarity, slow growth and cytokinetic defects . Combining both deletions in a single strain is phenotypically non-additive, myo52(delta) being epistatic to myo51(delta) . Overproduction of Myo51 gives rise to elongated cells which fail to form functional septa whereas overproduction of Myo52 results in branched cells with aberrant septa that fail to cleave . Myo52 localises to the poles of growing cells but during cell division it relocalises to the cell equator as a bar that is bisected by the cytokinetic septum . Myo51 shows no obvious localisation during interphase but at cytokinesis it is associated with the contractile cytokinetic actin ring (CAR) . Both myosins are dependent upon an intact actin cytoskeleton for localisation . Myo52 partially colocalises with the (alpha)-glucan synthase Mok1 at the cell tips and to a lesser extent at the septum . Mok1 is delocalised and upregulated in myo52(delta) and myo52(delta) cell walls are resistant to digestion by the cell wall degrading enzyme zymolyase . Thus myo52(+) appears to be involved in the local delivery or positioning of vesicles containing cell wall precursors at the cell tips and has a role in the maturation or cleavage of the septum . Myo51 has a non-essential role in cytokinesis as a component of the cytokinetic actin ring.

Genomics, 2000 Dec 1, 70(2), 253 - 7
Cloning and characterization of human VPS35 and mouse Vps35 and mapping of VPS35 to human chromosome 16q13-q21; Zhang P et al.; Maintenance of different organelles in eukaryotic cells depends on sorting proteins, which ensure the proper delivery of organelle-specific proteins . The studies on yeast (Saccharomyces cerevisiae) VPS35, a hydrophilic membrane protein having a direct role in the retrieval of cargo proteins, suggest a mechanism underlying a possible lysosomal protein-sorting pathway in mammalian cells . Here, we report the isolation of human and mouse VPS35 cDNAs, which are 3208 and 3186 bp in length, respectively . The deduced proteins of the two cDNAs, which are both composed of 796 amino acids and share 99% identity, show homology to yeast VPS35 and other VPS35 homologues of various sources ranging from Schizosaccharomyces pombe to Drosophila melanogaster (31-56% identity and 49-71% similarity), especially in their amino- and carboxyl-termini . The conservation of VPS35 suggests that the function of this class of protein is important . The results of Northern hybridization of human VPS35 in 16 tissues showed that one transcript of 3.6 kb was highly expressed in brain, heart, testis, ovary, small intestine, spleen, skeletal muscle, and placenta and expressed at moderate or low levels in other tissues . Another transcript of 3.0 kb was also expressed with proportionally lower levels than the 3.6-kb transcript in all the tissues except that the 3.0-kb transcript was not detected in brain . Mouse Vps35 was widely expressed as a 3.4-kb transcript . In addition, human VPS35 was assigned to human chromosome 16q13-q21 by radiation hybrid mapping.

Eur J Biochem, 2000 Dec, 267(24), 7065 - 74
Characterization of active-site mutants of Schizosaccharomyces pombe phosphoglycerate mutase . Elucidation of the roles of amino acids involved in substrate binding and catalysis; Nairn J et al.; The roles of a number of amino acids present at the active site of the monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe have been explored by site-directed mutagenesis . The amino acids examined could be divided broadly into those presumed from previous related structural studies to be important in the catalytic process (R14, S62 and E93) and those thought to be important in substrate binding (R94, R120 and R121) . Most of these residues have not previously been studied by site-directed mutagenesis . All the mutants except R14 were expressed in an engineered null strain of Saccharomyces cerevisiae (S150-gpm:HIS) in good yield . The R14Q mutant was expressed in good yield in the transformed AH22 strain of S . cerevisiae . The S62A mutant was markedly unstable, preventing purification . The various mutants were purified to homogeneity and characterized in terms of kinetic parameters, CD and fluorescence spectra, stability towards denaturation by guanidinium chloride, and stability of phosphorylated enzyme intermediate . In addition, the binding of substrate (3-phosphoglycerate) to wild-type, E93D and R120,121Q enzymes was measured by isothermal titration calorimetry . The results provide evidence for the proposed roles of each of these amino acids in the catalytic cycle and in substrate binding, and will support the current investigation of the structure and dynamics of the enzyme using multidimensional NMR techniques.

Mutat Res, 2001 Jan 5, 461(4), 311 - 23
Characterization of RAD52 homologs in the fission yeast Schizosaccharomyces pombe; van den Bosch M et al.; The RAD52 gene of Saccharomyces cerevisiae is essential for repair of DNA double-strand breaks (DSBs) by homologous recombination . Inactivation of this gene confers hypersensitivity to DSB-inducing agents and defects in most forms of recombination . The rad22+ gene in Schizosaccharomyces pombe (here referred to as rad22A+) has been characterized as a homolog of RAD52 in fission yeast . Here, we report the identification of a second RAD52 homolog in Schizosaccharomyces pombe, called rad22B+ . The amino acid sequences of Rad22A and Rad22B show significant conservation (38% identity) . Deletion mutants of respectively, rad22A and rad22B, show different phenotypes with respect to sensitivity to X-rays and the ability to perform homologous recombination as measured by the integration of plasmid DNA . Inactivation of rad22A+ leads to a severe sensitivity to X-rays and a strong decrease in recombination (13-fold), while the rad22B mutation does not result in a decrease in homologous recombination or a change in radiation sensitivity . In a rad22A-rad22B double mutant the radiation sensitivity is further enhanced in comparison with the rad22A single mutant . Overexpression of the rad22B+ gene results in partial suppression of the DNA repair defects of the rad22A mutant strain . Meiotic recombination and spore viability are only slightly affected in either single mutant, but outgrowth of viable spores is almost 31-fold reduced in the rad22A-rad22B double mutant . The results obtained imply a crucial role for rad22A+ in repair and recombination in vegetative cells just like RAD52 in S . cerevisiae . The rad22B+ gene presumably has an auxiliary role in the repair of DSBs . The drastic reduced spore viability in the double mutant suggests that meiosis in S . pombe is dependent on the presence of either rad22A+ or rad22B+.

Mol Biol Cell, 2000 Dec, 11(12), 4393 - 401
Schizosaccharomyces pombe rho2p GTPase regulates cell wall alpha-glucan biosynthesis through the protein kinase pck2p; Calonge TM et al.; Schizosaccharomyces pombe rho1(+) and rho2(+) genes are involved in the control of cell morphogenesis, cell integrity, and polarization of the actin cytoskeleton . Although both GTPases interact with each of the two S . pombe protein kinase C homologues, Pck1p and Pck2p, their functions are distinct from each other . It is known that Rho1p regulates (1,3)beta-D-glucan synthesis both directly and through Pck2p . In this paper, we have investigated Rho2p signaling and show that pck2 delta and rho2 delta strains display similar defects with regard to cell wall integrity, indicating that they might be in the same signaling pathway . We also show that Rho2 GTPase regulates the synthesis of alpha-D-glucan, the other main structural polymer of the S . pombe cell wall, primarily through Pck2p . Although overexpression of rho2(+) in wild-type or pck1 delta cells is lethal and causes morphological alterations, actin depolarization, and an increase in alpha-D-glucan biosynthesis, all of these effects are suppressed in a pck2 delta strain . In addition, genetic interactions suggest that Rho2p and Pck2p are important for the regulation of Mok1p, the major (1-3)alpha-D-glucan synthase . Thus, a rho2 delta mutation, like pck2 delta, is synthetically lethal with mok1-664, and the mutant partially fails to localize Mok1p to the growing areas . Moreover, overexpression of mok1(+) in rho2 delta cells causes a lethal phenotype that is completely different from that of mok1(+) overexpression in wild-type cells, and the increase in alpha-glucan is considerably lower . Taken together, all of these results indicate the presence of a signaling pathway regulating alpha-glucan biosynthesis in which the Rho2p GTPase activates Pck2p, and this kinase in turn controls Mok1p.

Mol Biol Cell, 2000 Dec, 11(12), 4067 - 77
A conserved interaction between Moe1 and Mal3 is important for proper spindle formation in Schizosaccharomyces pombe; Chen CR et al.; Moe1 is a conserved fission yeast protein that negatively affects microtubule stability/assembly . We conducted a two-hybrid screen to search for Moe1-binding proteins and isolated Mal3, a homologue of human EB1 . We show that Moe1 and Mal3 expressed in bacteria form a complex and that Moe1 and Mal3 expressed in fission yeast cosediment with microtubules . Deletion of either moe1 or mal3 does not result in lethality; however, deletion of both moe1 and mal3 leads to cell death in the cold . The resulting cells appear to die of chromosome missegregation, which correlates with the presence of abnormal spindles . We investigated the cause for the formation of monopolar spindles and found that only one of the two spindle pole bodies (SPBs) contains gamma-tubulin, although both SPBs appear to be equal in size and properly inserted in the nuclear membrane . Moreover, the moe1 mal3 double null mutant in the cold contains abnormally short and abundant interphase microtubule bundles . These data suggest that Moe1 and Mal3 play a role in maintaining proper microtubule dynamics/integrity and distribution of gamma-tubulin to the SPBs during mitosis . Finally, we show that human Moe1 and EB1 can each rescue the phenotype of the moe1 mal3 double null mutant and form a complex, suggesting that these proteins are part of a well-conserved mechanism for regulating spindle functioning.

Genetics, 2000 Dec, 156(4), 1549 - 57
Decreased meiotic intergenic recombination and increased meiosis I nondisjunction in exo1 mutants of Saccharomyces cerevisiae; Kirkpatrick DT et al.; Exonuclease I was originally identified as a 5' --> 3' deoxyribonuclease present in fractionated extracts of Schizosaccharomyces pombe and Saccharomyces cerevisiae . Genetic analysis of exo1 mutants of both yeasts revealed no major defect in meiosis, suggesting that exonuclease I is unlikely to be the primary activity that processes meiosis-specific double-strand breaks (DSBs) . We report here that exo1 mutants of S . cerevisiae exhibit subtle but complex defects in meiosis . Diploids containing a homozygous deletion of EXO1 show decreased spore viability associated with an increase in meiosis I nondisjunction, while intergenic recombination is reduced about twofold . Exo1p functions in the same pathway as Msh5p for intergenic recombination . The length of heteroduplex tracts within the HIS4 gene is unaffected by the exo1 mutation . These results suggest that Exo1p is unlikely to play a major role in processing DSBs to form single-stranded tails at HIS4, but instead appears to promote crossing over to ensure disjunction of homologous chromosomes . In addition, our data indicate that exonuclease I may have a minor role in the correction of large DNA mismatches that occur in heteroduplex DNA during meiotic recombination at the HIS4 locus.

Front Biosci, 2000 Dec 01, 5, D905 - 16
Yeast perspectives on HIV-1 VPR; Zhao Y et al.; Increasing evidence suggests that HIV-1 viral protein R (Vpr) plays an important role in viral pathogenesis, as its functions are being linked to viral activation, suppression of human immune functions and depletion of human CD4 lymphocytes, which are the major clinical manifestation of AIDS . In vitro, Vpr shows multiple activities both in mammalian and yeast cells, which include nuclear transport, induction of cell cycle G2 arrest, morphological changes and cell death . The occurrence of these activities in yeast indicates that Vpr interacts with highly conserved cellular processes to cause these effects and allows Vpr activities to be studied in these genetically well characterized organisms . Studies of Vpr in fission yeast (Schizosaccharomyces pombe) and budding yeast (Saccharomyces cerevisiae) have helped to establish these major conclusions . 1) Vpr induces G2 arrest through inhibitory phosphorylation of the cyclin-dependent kinase by a pathway in which protein phosphatase 2A plays an important role . 2) Vpr fulfills its essential role in the nuclear transport of the viral pre-integration complex by binding to a novel site on importin ? . 3) Vpr induces apoptosis by directly permeabilizing the mitochondrial membrane . 4) Vpr also appears to kill cells by mitochondrial-independent mechanisms . 5) G2 arrest and cell death induced by Vpr are two independent functions, and 6) amino acid residues of Vpr at position 29, 33 and 71 are important sites for maintaining the overall structure of Vpr . Future studies of Vpr in yeast are expected to make additional contributions to understanding the mechanisms of Vpr activities and may also help address the importance of these activities during the course of a HIV-1 infection.

EMBO J, 2000 Dec 1, 19(23), 6569 - 81
Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry; Ajuh P et al.; Recently, we identified proteins that co-purify with the human spliceosome using mass spectrometry . One of the identified proteins, CDC5L, corresponds to the human homologue of the Schizosaccharomyces pombe CDC5(+) gene product . Here we show that CDC5L is part of a larger multiprotein complex in HeLa nuclear extract that incorporates into the spliceosome in an ATP-dependent step . We also show that this complex is required for the second catalytic step of pre-mRNA splicing . Immunodepletion of the CDC5L complex from HeLa nuclear extract inhibits the formation of pre-mRNA splicing products in vitro but does not prevent spliceosome assembly . The first catalytic step of pre-mRNA splicing is less affected by immunodepleting the complex . The purified CDC5L complex in HeLa nuclear extract restores pre-mRNA splicing activity when added to extracts that have been immunodepleted using anti-CDC5L antibodies . Using mass spectrometry and database searches, the major protein components of the CDC5L complex have been identified . This work reports a first purification and characterization of a functional, human non-snRNA spliceosome subunit containing CDC5L and at least five additional protein factors.

Nucleic Acids Res, 2000 Dec 1, 28(23), 4769 - 77
The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase; Izumi M et al.; Mcm10 (Dna43), first identified in Saccharomyces cerevisiae, is an essential protein which functions in the initiation of DNA synthesis . Mcm10 is a nuclear protein that is localized to replication origins and mediates the interaction of the Mcm2-7 complex with replication origins . We identified and cloned a human cDNA whose product was structurally homologous to the yeast Mcm10 protein . Human Mcm10 (HsMcm10) is a 98-kDa protein of 874 amino acids which shows 23 and 21% overall similarity to Schizosaccharomyces pombe Cdc23 and S . cerevisiae Mcm10, respectively . The messenger RNA level of HsMcm10 increased at the G(1)/S-boundary when quiescent human NB1-RGB cells were induced to proliferate as is the case of many replication factors . HsMcm10 associated with nuclease-resistant nuclear structures throughout S phase and dissociated from it in G(2) phase . HsMcm10 associated with human Orc2 protein when overexpressed in COS-1 cells . HsMcm10 also interacted with Orc2, Mcm2 and Mcm6 proteins in the yeast two-hybrid system . These results suggest that HsMcm10 may function in DNA replication through the interaction with Orc and Mcm2-7 complexes.

Nucleic Acids Res, 2000 Dec 1, 28(23), 4742 - 9
Species specificity of human RPA in simian virus 40 DNA replication lies in T-antigen-dependent RNA primer synthesis; Wang M et al.; Replication protein A (RPA) is a three-subunit protein complex with multiple functions in DNA replication . Previous study indicated that human RPA (h-RPA) could not be replaced by Schizosaccharomyces pombe RPA (sp-RPA) in simian virus 40 (SV40) replication, suggesting that h-RPA may have a specific function in SV40 DNA replication . To understand the specificity of h-RPA in replication, we prepared heterologous RPAs containing the mixture of human and S.pombe subunits and compared these preparations for various enzymatic activities . Heterologous RPAs containing two human subunits supported SV40 DNA replication, whereas those containing only one human subunit poorly supported DNA replication, suggesting that RPA complex requires at least two human subunits to support its function in SV40 DNA replication . All heterologous RPAs effectively supported single-stranded (ss)DNA binding activity and an elongation of a primed DNA template catalyzed by DNA polymerase (pol) alpha and delta . A strong correlation between SV40 DNA replication activity and large tumor antigen (T-ag)-dependent RNA primer synthesis by pol alpha-primase complex was observed among the heterologous RPAs . Furthermore, T-ag showed a strong interaction with 70- and 34-kDa subunits from human, but poorly interacted with their S.pombe counterparts, indicating that the specificity of h-RPA is due to its role in RNA primer synthesis . In the SV40 replication reaction, the addition of increasing amounts of sp-RPA in the presence of fixed amount of h-RPA significantly reduced overall DNA synthesis, but increased the size of lagging strand, supporting a specific role for h-RPA in RNA primer synthesis . Together, these results suggest that the specificity of h-RPA in SV40 replication lies in T-ag-dependent RNA primer synthesis.

Nucleic Acids Res, 2000 Dec 1, 28(23), 4709 - 16
Fission yeast retrotransposon Tf1 integration is targeted to 5' ends of open reading frames; Behrens R et al.; Target site selection of transposable elements is usually not random but involves some specificity for a DNA sequence or a DNA binding host factor . We have investigated the target site selection of the long terminal repeat-containing retrotransposon Tf1 from the fission yeast Schizosaccharomyces pombe . By monitoring induced transposition events we found that Tf1 integration sites were distributed throughout the genome . Mapping these insertions revealed that Tf1 did not integrate into open reading frames, but occurred preferentially in longer intergenic regions with integration biased towards a region 100-420 bp upstream of the translation start site . Northern blot analysis showed that transcription of genes adjacent to Tf1 insertions was not significantly changed.

Nucleic Acids Res, 2000 Dec 1, 28(23), 4604 - 10
Cloning and characterization of the Schizosaccharomyces pombe tRNA:pseudouridine synthase Pus1p; Hellmuth K et al.; Saccharomyces cerevisiae cells that carry deletions in both the LOS1 (a tRNA export receptor) and the PUS1 (a tRNA:pseudouridine synthase) genes exhibit a thermosensitive growth defect . A Schizosaccharomyces pombe gene, named spPUS1, was cloned from a cDNA library by complementation of this conditional lethal phenotype . The corresponding protein, spPus1p, shows sequence similarity to S . cerevisiae and murine Pus1p as well as other known members of the pseudouridine synthase family . Accordingly, recombinant spPus1p can catalyze in vitro the formation of pseudouridines at positions 27, 28, 34, 35 and 36 of yeast tRNA transcripts . The sequence and functional conservation of the Pus1p proteins in fungi and mammalian species and their notable absence from prokaryotes suggest that this family of pseudouridine synthases is required for a eukaryote-specific step of tRNA biogenesis, such as nuclear export.

Plant Mol Biol, 2000 Sep, 44(1), 53 - 60
Expression of a yeast RNase III gene in transgenic tobacco silences host nitrite reductase genes; Berthome R et al.; When a gene encoding the Schizosaccharomyces pombe dsRNA-specific RNase III, pac1, was expressed in transgenic tobacco plants, six out of thirteen transformed plants gave progeny among which were individuals displaying a distinctive chlorotic phenotype . These chlorotic plants strongly resemble those transformed with a 35S-Nii (nitrite reductase) transgene, in which both Nii host genes and the 35S-Nii transgene are silenced by co-suppression . RNA blots showed that the host Nii genes were silenced in chlorotic 35S-pac1 plants but not in individuals with a normal green phenotype . Neither the transcript levels of the other cellular genes tested nor the transcription of Nii genes was significantly affected by the expression of pac1 . This is the first observation of post-transcriptional silencing of host genes by a transgene with no apparent sequence similarity to the target gene.

FEMS Microbiol Lett, 2000 Dec 1, 193(1), 117 - 21
Role for trehalase during germination of spores in the fission yeast Schizosaccharomyces pombe; Beltran FF et al.; Spores from Schizosaccharomyces pombe contain neutral and acid trehalases . When spores from strains disrupted for ntp1(+), which encodes neutral trehalase, were induced to germinate, the onset of the process was markedly delayed as compared to wild-type spores . Further outgrowth was also reduced . Dormant spores lacking neutral trehalase contained twice the amount of trehalose present in wild-type spores and mobilised the intracellular pool of trehalose at a slower rate during germination . Inhibition by phloridzin of the sporulation-specific acid trehalase in ntp1-disrupted spores arrested germination completely while prompting no effect on wild-type spores . These results suggest that the two trehalase enzymes may support the utilisation of trehalose during germination but neutral trehalase is required for a more rapid and efficient process.

J Biol Chem, 2001 Mar 9, 276(10), 7027 - 32 Epub 2000 Nov 28.
The fission yeast TOR homolog, tor1+, is required for the response to starvation and other stresses via a conserved serine; Weisman R et al.; Targets of rapamycin (TORs) are conserved phosphatidylinositol kinase-related kinases that are involved in the coordination between nutritional or mitogenic signals and cell growth . Here we report the initial characterization of two Schizosaccharomyces pombe TOR homologs, tor1(+) and tor2(+) . tor2(+) is an essential gene, whereas tor1(+) is required only under starvation and other stress conditions . Specifically, Deltator1 cells fail to enter stationary phase or undergo sexual development and are sensitive to cold, osmotic stress, and oxidative stress . In complex with the prolyl isomerase FKBP12, the drug rapamycin binds a conserved domain in TORs, FRB, thus inhibiting some of the functions of TORs . Mutations at a conserved serine within the FRB domain of Saccharomyces cerevisiae TOR proteins led to rapamycin resistance but did not otherwise affect the functions of the proteins . The S . pombe tor1(+) exhibits different features; substitution of the conserved serine residue, Ser(1834), with arginine compromises its functions and has no effect on the inhibition that rapamycin exerts on sexual development in S . pombe.

J Bacteriol, 2000 Dec, 182(24), 6933 - 9
Phenotypes of fission yeast defective in ubiquinone production due to disruption of the gene for p-hydroxybenzoate polyprenyl diphosphate transferase; Uchida N et al.; Ubiquinone is an essential component of the electron transfer system in both prokaryotes and eukaryotes and is synthesized from chorismate and polyprenyl diphosphate by eight steps . p-Hydroxybenzoate (PHB) polyprenyl diphosphate transferase catalyzes the condensation of PHB and polyprenyl diphosphate in ubiquinone biosynthesis . We isolated the gene (designated ppt1) encoding PHB polyprenyl diphosphate transferase from Schizosaccharomyces pombe and constructed a strain with a disrupted ppt1 gene . This strain could not grow on minimal medium supplemented with glucose . Expression of COQ2 from Saccharomyces cerevisiae in the defective S . pombe strain restored growth and enabled the cells to produce ubiquinone-10, indicating that COQ2 and ppt1 are functional homologs . The ppt1-deficient strain required supplementation with antioxidants, such as cysteine, glutathione, and alpha-tocopherol, to grow on minimal medium . This suggests that ubiquinone can act as an antioxidant, a premise supported by our observation that the ppt1-deficient strain is sensitive to H(2)O(2) and Cu(2+) . Interestingly, we also found that the ppt1-deficient strain produced a significant amount of H(2)S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in S . pombe . Ppt1-green fluorescent protein fusion proteins localized to the mitochondria, indicating that ubiquinone biosynthesis occurs in the mitochondria in S . pombe . Thus, analysis of the phenotypes of S . pombe strains deficient in ubiquinone production clearly demonstrates that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.

J Biol Chem, 2001 Feb 23, 276(8), 5943 - 51 Epub 2000 Nov 21.
Structure of Cdc4p, a contractile ring protein essential for cytokinesis in Schizosaccharomyces pombe; Slupsky CM et al.; The Schizosaccharomyces pombe Cdc4 protein is required for the formation and function of the contractile ring, presumably acting as a myosin light chain . By using NMR spectroscopy, we demonstrate that purified Cdc4p is a monomeric protein with two structurally independent domains, each exhibiting a fold reminiscent of the EF-hand class of calcium-binding proteins . Although Cdc4p has one potentially functional calcium-binding site, it does not bind calcium in vitro . Three variants of Cdc4p containing single point mutations responsible for temperature-sensitive arrest of the cell cycle at cytokinesis (Gly-19 to Glu, Gly-82 to Asp, and Gly-107 to Ser) were also characterized by NMR and circular dichroism spectroscopy . In each case, the amino acid substitution only leads to small perturbations in the conformation of the protein . Furthermore, thermal unfolding studies indicate that, like wild-type Cdc4p, the three mutant forms are all extremely stable, remaining completely folded at temperatures significantly above those causing failure of cytokinesis in intact cells . Therefore, the altered phenotype must arise directly from a disruption of the function of Cdc4p rather than indirectly through a disruption of its overall structure . Several mutant alleles of Cdc4p also show interallelic complementation in diploid cells . This phenomenon can be explained if Cdcp4 has more than one essential function or, alternatively, if two mutant proteins assemble to form a functional complex . Based on the structure of Cdc4p, possible models for interallelic complementation including interactions with partner proteins and the formation of a myosin complex with Cdc4p fulfilling the role of both an essential and regulatory light chain are proposed.

J Biol Chem, 2001 Feb 23, 276(8), 5932 - 42 Epub 2000 Nov 21.
Cdc4p, a contractile ring protein essential for cytokinesis in Schizosaccharomyces pombe, interacts with a phosphatidylinositol 4-kinase; Desautels M et al.; The proposed function of Cdc4p, an essential contractile ring protein in Schizosaccharomyces pombe, is that of a myosin essential light chain . However, five conditionally lethal cdc4 alleles exhibit complementation in diploids . Such interallelic complementation is not readily explained if the sole function of Cdc4p is that of a myosin essential light chain . Complementation of cdc4 alleles could occur only if different mutant forms can assemble into an active oligomeric complex or if Cdc4p has more than one essential function . To search for other proteins that may interact with Cdc4p, we performed a two-hybrid screen and identified two such candidates: one similar to Saccharomyces cerevisiae Vps27p and the other a putative phosphatidylinositol (PI) 4-kinase . Binding of Cdc4p to the latter and to myosin heavy chain (Myo2p) was confirmed by immunosorbent assays . Deletion studies demonstrated interaction between the Cdc4p C-terminal domain and the PI 4-kinase C-terminal domain . Furthermore, interaction was abolished by the Cdc4p C-terminal domain point mutation, Gly107 to Ser . This allele also causes failure of cytokinesis . Ectopic expression of the PI 4-kinase C-terminal domain caused cytokinesis defects that were most extreme in cells carrying the G107S allele . We suggest that Cdc4p plays multiple roles in cytokinesis and that interaction with a PI 4-kinase may be important for contractile ring assembly and/or function.

J Cell Biol, 2000 Nov 27, 151(5), 1101 - 11
The fission yeast ran GTPase is required for microtubule integrity; Fleig U et al.; The microtubule cytoskeleton plays a pivotal role in cytoplasmic organization, cell division, and the correct transmission of genetic information . In a screen designed to identify fission yeast genes required for chromosome segregation, we identified a strain that carries a point mutation in the SpRan GTPase . Ran is an evolutionarily conserved eukaryotic GTPase that directly participates in nucleocytoplasmic transport and whose loss affects many biological processes . Recently a transport-independent effect of Ran on spindle formation in vitro was demonstrated, but the in vivo relevance of these findings was unclear . Here, we report the characterization of a Schizosaccharomyces pombe Ran GTPase partial loss of function mutant in which nucleocytoplasmic protein transport is normal, but the microtubule cytoskeleton is defective, resulting in chromosome missegregation and abnormal cell shape . These abnormalities are exacerbated by microtubule destabilizing drugs, by loss of the spindle checkpoint protein Mph1p, and by mutations in the spindle pole body component Cut11p, indicating that SpRan influences microtubule integrity . As the SpRan mutant phenotype can be partially suppressed by the presence of extra Mal3p, we suggest that SpRan plays a role in microtubule stability.

J Cell Sci, 2000 Dec, 113 Pt 24, 4557 - 62
Identification of an alpha-tubulin mutant of fission yeast from gamma-tubulin-interacting protein screening: genetic evidence for alpha-/gamma-tubulin interaction; Takeoka A et al.; gamma-Tubulin has been determined to be a central element of microtubule nucleation and, thus, indispensable for cellular organization of the microtubule . Utilizing the fact that human gamma-tubulin can function in the fission yeast Schizosaccharomyces pombe, we have generated a unique mutant screening procedure which can specifically select mutants of genes encoding gamma-tubulin-interacting proteins . One of the isolated mutants, cs76, turned out to carry a mutation in the alpha 1-tubulin gene (nda2(+)) . This result suggests a direct interaction between the alpha- and gamma-tubulins . We located the mutation site in the nda2 gene and characterized the mutant phenotype . Our results demonstrate the importance of the alpha-/gamma-tubulin interaction in microtubule nucleation and should complement previous knowledge.

Int J Oncol, 2000 Dec, 17(6), 1251 - 8
An additional transcript of the cdc25C gene from A431 cells encodes a functional protein; Bureik M et al.; Human p53 protein was found to be functional in fission yeast in terms of growth repression and checkpoint control . Expression of wild-type p53 or the hot spot mutant p53His273 results in dramatic morphological changes and loss of viability of recipient yeast cells . Overexpression of cdc25C phosphatase, the mitotic activator of cdc2, results in suppression of a p53-induced growth arrest . In order to understand the interplay between p53 and cdc25C in mammalian cells we isolated and sequenced cdc25C cDNA from the epidermoid carcinoma cell line A431, which is known to carry the p53His273 mutation . Two different transcripts of the human cdc25C gene were detected by RT-PCR analysis - one full-length transcript and a shortened version (cdc25Cdm) that carries two deletions in the 5'-region of the gene . In normal human skin fibroblasts only one full-length cdc25C transcript was detected . The two different transcripts code for proteins with a molecular weight of 55 kDa and 46 kDa, respectively . Both cdc25C cDNAs from A431 cells were found to complement a conditional lethal cdc25.22 mutant strain as well as a cdc25 deletion strain of Schizosaccharomyces pombe indicating that functional proteins were translated . Expression of cdc25Cdm variant leads to a stronger uncoupling of DNA replication from mitosis than expression of cdc25C suggesting that the deletion within the amino-terminus of cdc25C leads to a protein which might contribute some potential for oncogenic transformation . As with cdc25C, uncoupling of the DNA synthesis checkpoint by cdc25Cdm was reversed by coexpression of wild-type p53.

Mol Cell Biol, 2000 Dec, 20(23), 8958 - 68
Rdp1, a novel zinc finger protein, regulates the DNA damage response of rhp51(+) from Schizosaccharomyces pombe; Shim YS et al.; The Schizosaccharomyces pombe DNA repair gene rhp51(+) encodes a RecA-like protein with the DNA-dependent ATPase activity required for homologous recombination . The level of the rhp51(+) transcript is increased by a variety of DNA-damaging agents . Its promoter has two cis-acting DNA damage-responsive elements (DREs) responsible for DNA damage inducibility . Here we report identification of Rdp1, which regulates rhp51(+) expression through the DRE of rhp51(+) . The protein contains a zinc finger and a polyalanine tract similar to ones previously implicated in DNA binding and transactivation or repression, respectively . In vitro footprinting and competitive binding assays indicate that the core consensus sequences (NGG/TTG/A) of DRE are crucial for the binding of Rdp1 . Mutations of both DRE1 and DRE2 affected the damage-induced expression of rhp51(+), indicating that both DREs are required for transcriptional activation . In addition, mutations in the DREs significantly reduced survival rates after exposure to DNA-damaging agents, demonstrating that the damage response of rhp51(+) enhances the cellular repair capacity . Surprisingly, haploid cells containing a complete rdp1 deletion could not be recovered, indicating that rdp1(+) is essential for cell viability and implying the existence of other target genes . Furthermore, the DNA damage-dependent expression of rhp51(+) was significantly reduced in checkpoint mutants, raising the possibility that Rdp1 may mediate damage checkpoint-dependent transcription of rhp51(+).

Mol Cell Biol, 2000 Dec, 20(23), 8944 - 57
Novel Upf2p orthologues suggest a functional link between translation initiation and nonsense surveillance complexes; Mendell JT et al.; Transcripts harboring premature signals for translation termination are recognized and rapidly degraded by eukaryotic cells through a pathway known as nonsense-mediated mRNA decay (NMD) . In addition to protecting cells by preventing the translation of potentially deleterious truncated peptides, studies have suggested that NMD plays a broader role in the regulation of the steady-state levels of physiologic transcripts . In Saccharomyces cerevisiae, three trans-acting factors (Upf1p to Upf3p) are required for NMD . Orthologues of Upf1p have been identified in numerous species, showing that the NMD machinery, at least in part, is conserved through evolution . In this study, we demonstrate additional functional conservation of the NMD pathway through the identification of Upf2p homologues in Schizosaccharomyces pombe and humans (rent2) . Disruption of S . pombe UPF2 established that this gene is required for NMD in fission yeast . rent2 was demonstrated to interact directly with rent1, a known trans-effector of NMD in mammalian cells . Additionally, fragments of rent2 were shown to possess nuclear targeting activity, although the native protein localizes to the cytoplasmic compartment . Finally, novel functional domains of Upf2p and rent2 with homology to eukaryotic initiation factor 4G (eIF4G) and other translational regulatory proteins were identified . Directed mutations within these so-called eIF4G homology (4GH) domains were sufficient to abolish the function of S . pombe Upf2p . Furthermore, using the two-hybrid system, we obtained evidence for direct interaction between rent2 and human eIF4AI and Sui1, both components of the translation initiation complex . Based on these findings, a novel model in which Upf2p and rent2 effects decreased translation and accelerated decay of nonsense transcripts through competitive interactions with eIF4G-binding partners is proposed.

Mol Cell Biol, 2000 Dec, 20(23), 8767 - 82
Mex67p of Schizosaccharomyces pombe interacts with Rae1p in mediating mRNA export; Yoon JH et al.; We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 ts mutation . spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap . In contrast to scMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA . However, an spmex67 null mutation (Deltamex67) is synthetically lethal with the rae1-167 mutation and accumulates poly(A)(+) RNA in the nucleus . We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of the rae1-167 Deltamex67 synthetic lethality . This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions . The (149-505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell . Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149-505)-GFP fusion to the nucleus and the nuclear periphery . These results suggest that spMex67p competes for essential mRNA export factor(s) . Finally, we propose that the 149-505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.

Mol Cell Biol, 2000 Dec, 20(23), 8758 - 66
Damage tolerance protein Mus81 associates with the FHA1 domain of checkpoint kinase Cds1; Boddy MN et al.; Cds1, a serine/threonine kinase, enforces the S-M checkpoint in the fission yeast Schizosaccharomyces pombe . Cds1 is required for survival of replicational stress caused by agents that stall replication forks, but how Cds1 performs these functions is largely unknown . Here we report that the forkhead-associated-1 (FHA1) protein-docking domain of Cds1 interacts with Mus81, an evolutionarily conserved damage tolerance protein . Mus81 has an endonuclease homology domain found in the XPF nucleotide excision repair protein . Inactivation of mus81 reveals a unique spectrum of phenotypes . Mus81 enables survival of deoxynucleotide triphosphate starvation, UV radiation, and DNA polymerase impairment . Mus81 is essential in the absence of Bloom's syndrome Rqh1 helicase and is required for productive meiosis . Genetic epistasis studies suggest that Mus81 works with recombination enzymes to properly replicate damaged DNA . Inactivation of Mus81 triggers a checkpoint-dependent delay of mitosis . We propose that Mus81 is involved in the recruitment of Cds1 to aberrant DNA structures where Cds1 modulates the activity of damage tolerance enzymes.

J Bacteriol, 2000 Dec, 182(23), 6815 - 8
Hexokinase regulates kinetics of glucose transport and expression of genes encoding hexose transporters in Saccharomyces cerevisiae; Petit T et al.; Glucose transport kinetics and mRNA levels of different glucose transporters were determined in Saccharomyces cerevisiae strains expressing different sugar kinases . During exponential growth on glucose, a hxk2 null strain exhibited high-affinity hexose transport associated with an elevated transcription of the genes HXT2 and HXT7, encoding high-affinity transporters, and a diminished expression of the HXT1 and HXT3 genes, encoding low-affinity transporters . Deletion of HXT7 revealed that the high-affinity component is mostly due to HXT7; however, a previously unidentified very-high-affinity component (K(m) = 0.19 mM) appeared to be due to other factors . Expression of genes encoding hexokinases from Schizosaccharomyces pombe or Yarrowia lipolytica in a hxk1 hxk2 glk1 strain prevented derepression of the high-affinity transport system at high concentrations of glucose.

Mol Biol Cell, 2000 Nov, 11(11), 4005 - 18
Fission yeast Int6 is not essential for global translation initiation, but deletion of int6(+) causes hypersensitivity to caffeine and affects spore formation; Bandyopadhyay A et al.; Mammalian INT6 protein has been considered to be a subunit of the eukaryotic translation initiation factor, eIF3 . The Int6 locus is also known as a common integration site of mouse mammary tumor virus (MMTV) . However, the function of Int6 in translation initiation and the mechanism of Int6-mediated tumor induction are yet to be explored . In this study, the fission yeast, Schizosaccharomyces pombe, int6(+), which is 43% identical to the mammalian counterpart, was deleted . Despite the evidence that the majority of Int6 protein was associated with 40S particles in this organism, strains lacking int6(+) (Deltaint6) were viable and showed only moderate inhibition in the rate of in vivo global protein synthesis . Polysome profile analysis showed no apparent defects in translation initiation . Deltaint6 exhibited a hypersensitivity to caffeine, which could be suppressed by the addition of sorbitol to the growth medium . This and other phenotypes would imply that int6(+) is required for the integrity of cell membrane . In meiosis, Deltaint6 produced incomplete tetrads frequently . High dosage expression of a truncated mutant of int6(+) conferred a hypersensitivity to caffeine, but did not cause the defect in meiosis . A possible link between the function of int6(+) and the Deltaint6-phenotypes is discussed.

J Cell Sci, 2000 Dec, 113 Pt 23, 4351 - 62
The SHR3 homologue from S . pombe demonstrates a conserved function of ER packaging chaperones; Martinez P et al.; In Saccharomyces cerevisiae cells lacking SHR3, amino acid permeases do not enter into COPII transport vesicles and specifically accumulate in the membrane of the endoplasmic reticulum . Shr3p functions as a packaging chaperone to prime transport vesicle formation in the proximity of amino acid permeases . A genetic screen was developed that enabled the Schizosaccharomyces pombe SHR3 functional homologue, designated psh3(+) (pombe SHR3), to be cloned . The psh3(+) gene encodes a protein of 215 amino acids, which shares a high degree of structural and functional similarity with Shr3p . The heterologous expression of psh3(+) complements many, but not all, shr3 null mutant phenotypes in S . cerevisiae in a temperature-dependent manner . Psh3p is localised to the endoplasmic reticulum of S . pombe cells, and strains lacking the psh3(+ )gene exhibit decreased rates of amino acid uptake due to reduced levels of functional permeases in the plasma membrane . No packaging chaperones, or proteins exhibiting homology with packaging chaperones, have so far been identified in other eukayotic organisms . The findings reported here are the first to establish that specific packaging chaperones exist in divergent organisms, and demonstrate a conserved function of packaging chaperones in facilitating the export of large polytopic membrane proteins from the endoplasmic reticulum.

J Cell Sci, 2000 Dec, 113 Pt 23, 4341 - 50
The Spd1p S phase inhibitor can activate the DNA replication checkpoint pathway in fission yeast; Borgne A et al.; Spd1p (for S phase delayed) is a cell cycle inhibitor in Schizosaccharomyces pombe . Spd1p overexpression blocks the onset of both S phase and mitosis . In this study, we have investigated the mechanisms by which Spd1p overexpression blocks cell cycle progression, focussing on the block over mitotic onset . High levels of Spd1p lead to an increase in Y15 phosphorylation of Cdc2p and we show that the block over G(2) requires the Wee1p kinase and is dependent on the rad and chk1/cds1 checkpoint genes . We propose that high levels of Spd1p in G(2) cells activate the DNA replication checkpoint control, which leads to a Wee1p-dependent increase of Cdc2p Y15 phosphorylation blocking onset of mitosis . The Spd1p block at S phase onset may act by interfering directly with DNA replication, and also activates the G(2 )rad/hus checkpoint pathway to block mitosis.

J Cell Sci, 2000 Dec, 113 Pt 23, 4177 - 91
Live analysis of lagging chromosomes during anaphase and their effect on spindle elongation rate in fission yeast; Pidoux AL et al.; The fission yeast Schizosaccharomyces pombe is widely used as a model system for studies of the cell cycle and chromosome biology . To enhance these studies we have fused GFP to the chromodomain protein Swi6p, thus allowing nuclear and chromosome behaviour to be followed in living cells using time-lapse fluorescence microscopy . Like endogenous Swi6p, GFP-Swi6p localises to the nucleus and is concentrated at the heterochromatic centromeres and telomeres . The nucleus is highly dynamic during interphase: the clustered centromeres, in particular, are highly mobile . By expressing GFP-(&agr;)2-tubulin and GFP-Swi6p in the same cells we observe that the clustered centromeres move in concert with the cytoplasmic microtubules, which is likely to reflect their association with the spindle pole body . Drug treatment indicates that this movement is dependent on intact cytoplasmic microtubules . We have also used GFP-Swi6p to investigate the properties of lagging chromosomes observed in mutants with defects in chromosome segregation . Lagging chromosomes display a variety of behaviours on anaphase spindles, most surprisingly, chromosomes appear to initiate microtubule interactions and move to the poles late in anaphase B . Interestingly, in cells displaying lagging chromosomes, the rate of spindle elongation is slowed by a factor of two . This suggests that cells are able to sense the presence of a lagging chromosome and slow anaphase B in order to allow it extra time to reach the pole . However, this mechanism is not dependent on the spindle checkpoint proteins Bub1p or Dma1p, raising the possibility that a novel checkpoint mechanism operates to retard spindle elongation if lagging chromosomes are detected . An alternative model is also discussed in which single defective kinetochores on lagging chromatids are able to interact simultaneously with microtubules emanating from both poles and affect spindle dynamics by counteracting the spindle elongation force.

J Cell Sci, 2000 Dec, 113 Pt 23, 4157 - 63
The S . pombe rlc1 gene encodes a putative myosin regulatory light chain that binds the type II myosins myo3p and myo2p; Le Goff X et al.; In order to identify additional components important for cell division in the fission yeast Schizosaccharomyces pombe we have screened a bank of conditional cold-sensitive mutants for cytokinesis defects . One of these mutants showed a delay in cell cleavage, and strong genetic interactions with other genes implicated in medial ring formation . Cloning of the corresponding gene indicates that it encodes a protein with significant homology to the regulatory light chain of non-muscle myosins . We have named the gene rlc1 (regulatory light chain 1) . The gene is not essential for division, but null mutants display a cell cleavage defect and form an aberrant F-actin ring . Two myosin-II heavy chains have been identified in fission yeast: Co-immunoprecipitation experiments indicate that rlc1p associates more strongly with myo3p than myo2p.

Mol Microbiol, 2000 Oct, 38(2), 308 - 21
bgs2+, a sporulation-specific glucan synthase homologue is required for proper ascospore wall maturation in fission yeast; Martin V et al.; The formation of the ascospore cell wall of Schizosaccharomyces pombe requires the co-ordinated activity of enzymes involved in the biosynthesis of its components, such as glucans . We have cloned the bgs2+ gene . bgs2+ belongs to the glucan synthase family of S . pombe and is homologous to the Saccharomyces cerevisiae FKS1 and FKS2 genes . Deletion or overexpression of this gene does not lead to any apparent defect during vegetative growth, but homozygous bgs2Delta diploids do show a sporulation defect . Although meiosis takes place normally, ascospores are unable to mature, and their wall differs from that of wild-type ascospores . Moreover, bgs2Delta zygotes were not able to release ascospores spontaneously, and the ascospores were unable to germinate . We show that expression of bgs2+ is restricted to sporulation and that a bgs2-green fluorescent protein (GFP) fusion protein localizes to the ascospore envelope . The glucan synthase activity in sporulating diploids bearing a bgs2 deletion was diminished in comparison with that of the wild-type diploids, a fact that underscores the importance of the bgs2+ gene and glucan synthesis for the proper formation and maturation of the ascospore wall.

Genetics, 2000 Nov, 156(3), 995 - 1004
Fission yeast Ras1 effector Scd1 interacts with the spindle and affects its proper formation; Li YC et al.; Ras1 GTPase is the Schizosaccharomyces pombe homolog of the mammalian Ha-Ras proto-oncoprotein . Ras1 interacts with Scd1 (aka Ral1), a presumptive guanine nucleotide exchange factor for Cdc42sp, to control organization of the cytoskeleton . In this study, we demonstrated that the scd1 deletion (scd1Delta) induced hypersensitivity to microtubule destabilizing drugs and instability of the minichromosome . Overexpression of scd1 induced formation of abnormal spindles and chromosome missegregation . The scd1 deletion worsened the defects of spindle formation in tubulin mutants; by contrast, it did not induce lethality in mutants defective in the spindle pole bodies . These genetic data suggest that Scd1 can interact with tubulin with substantial specificity to affect proper spindle formation and chromosome segregation . Subcellular localization data further illustrated that a GFP-Scd1 fusion protein can associate with the spindle . Finally, we showed that unlike ras1Delta and scd1Delta, byr2Delta (affecting the Ras1 effector for mating) is not synthetically lethal with the tubulin mutations . These data collectively suggest that the Ras1 pathway can impinge upon microtubules through Scd1, but not Byr2, to affect proper spindle formation and chromosome segregation.

Genetics, 2000 Nov, 156(3), 983 - 94
A fission yeast repression element cooperates with centromere-like sequences and defines a mat silent domain boundary; Ayoub N et al.; REII is a Schizosaccharomyces pombe repression element located at the centromere-proximal end of the mat silent domain . Here we show that inversion of REII enhances silencing on its centromere-proximal side while suppressing silencing on its centromere-distal side . Transplacement of REII to a position 2.5 kb from its native locus extends the region of stringent repression to the new REII site . These results suggest that REII defines a mat silent domain boundary by acting preferentially toward its centromere-distal side . To investigate cooperation between REII and a K-region sequence that shares homology with the centromeric dg dh repeats (cen2 homology), we targeted combinations of these elements to an ectopic site and monitored expression of an adjacent reporter gene . Centromeric dh-like sequences conferred low-level silencing on the adjacent reporter gene, and REII, which did not display silencing activity on its own, enhanced cen2 homology-mediated silencing . Cooperation was also apparent at the mat locus, where deletion of REII impaired repression stability . We propose that REII and the cen2 homology play different yet complementary roles in silencing establishment and inheritance at the mat locus.

EMBO J, 2000 Nov 1, 19(21), 5801 - 12
LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae; Rouse J et al.; We identified YDR499W as a Saccharomyces cerevisiae open reading frame with homology to several checkpoint proteins, including S . cerevisiae Rfc5p and Schizosaccharomyces pombe Rad26 . Disruption of YDR499W (termed LCD1) results in lethality that is rescued by increasing cellular deoxyribonucleotide levels . Cells lacking LCD1 are very sensitive to a range of DNA-damaging agents, including UV irradiation, and to the inhibition of DNA replication . LCD1 is necessary for the phosphorylation and activation of Rad53p in response to DNA damage or DNA replication blocks, and for Chk1p activation in response to DNA damage . LCD1 is also required for efficient DNA damage-induced phosphorylation of Rad9p and for the association of Rad9p with the FHA2 domain of Rad53p after DNA damage . In addition, cells lacking LCD1 are completely defective in the G(1)/S and G(2)/M DNA damage checkpoints . Finally, we reveal that endogenous Mec1p co-immunoprecipitates with Lcd1p both before and after treatment with DNA-damaging agents . These results indicate that Lcd1p is a pivotal checkpoint regulator, involved in both the essential and checkpoint functions of the Mec1p pathway.

Nucleic Acids Res, 2000 Nov 1, 28(21), 4340 - 9
Mutant alleles of Schizosaccharomyces pombe rad9(+) alter hydroxyurea resistance, radioresistance and checkpoint control; Hang H et al.; Schizosaccharomyces pombe rad9 mutations can render cells sensitive to hydroxyurea (HU), gamma-rays and UV light and eliminate associated checkpoint controls . In vitro mutagenesis was performed on S.pombe rad9 and altered alleles were transplaced into the genome to ascertain the functional significance of five groups of evolutionarily conserved amino acids . Most targeted regions were changed to alanines, whereas rad9-S3 encodes a protein devoid of 22 amino acids normally present in yeast but absent from mammalian Rad9 proteins . We examined whether these rad9 alleles confer radiation and HU sensitivity and whether the sensitivities correlate with checkpoint control deficiencies . One rad9 mutant allele was fully active, whereas four others demonstrated partial loss of function . rad9-S1, which contains alterations in a BH3-like domain, conferred HU resistance but increased sensitivity to gamma-rays and UV light, without affecting checkpoint controls . rad9-S2 reduced gamma-ray sensitivity marginally, without altering other phenotypes . Two alleles, rad9-S4 and rad9-S5, reduced HU sensitivity, radiosensitivity and caused aberrant checkpoint function . HU-induced checkpoint control could not be uncoupled from drug resistance . These results establish unique as well as overlapping functional domains within Rad9p and provide evidence that requirements of the protein for promoting resistance to radiation and HU are not identical.

Curr Genet, 2000 Oct, 38(3), 113 - 25
The genetic control of spontaneous and UV-induced mitotic intrachromosomal recombination in the fission yeast Schizosaccharomyces pombe; Osman F et al.; An artificially created non-tandem hetero-allelic duplication was constructed to assay mitotic intrachromosomal recombination in Schizosaccharomyces pombe . Two classes of recombinants could be distinguished: deletion-types, in which one copy of the duplicated sequence and the intervening sequence were lost, and conversion-types which retained the duplication . For spontaneous recombination, compared to wild-type cells, a rad22 mutant (corresponding to a Saccharomyces cerevisiae rad52 mutant) had wild-type levels of deletion-types, but was hypo-recombinant for conversion-types; rad16 (S . cerevisiae rad1), rad22 rad16 (S . cerevisiae rad52 rad1) and swi10 (S . cerevisiae rad10) mutants were hyper-recombinant for both types; rad22 swi10 (S . cerevisiae rad52 rad10) mutants were hypo-recombinant for both types; rhp51 (S . cerevisiae rad51) and rhp54 (S . cerevisiae rad54) mutants were hyper-recombinant for deletion-types, but almost completely lacked conversion-types . For wild-type cells, UV-irradiation induced both types of recombinant, but mainly conversion-types . All of the mutants lacked UV-induced recombination.

Biotechniques, 2000 Oct, 29(4), 892 - 7
Use of high specific activity StarFire oligonucleotide probes to visualize low-abundance pre-mRNA splicing intermediates in S . pombe; Behlke MA et al.; An oligonucleotide labeling system was developed that can produce radiolabeled hybridization probes with tenfold or more higher specific activity than is obtained by traditional 5'-end-labeling with polynucleotide kinase . Yet the system is as rapid and simple as kinase labeling . The reaction uses the Klenow fragment of E . coli DNA polymerase to add alpha-32P-dA residues to the 3'-end of an oligonucleotide in a primer-extension reaction . Unlike other methods of radioactive tailing (e.g., terminal transferase), a single species is produced of both known length and known specific activity . The reaction is efficient, and over 90% of probe molecules are routinely labeled . Using this method of labeling, an oligonucleotide was shown to be tenfold more sensitive in detecting target DNA sequences in a dot blot hybridization assay, compared to the same oligonucleotide labeled using polynucleotide kinase . Northern blots of Schizosaccharomyces pombe RNA were probed with an oligonucleotide specific for intron 1 of the tf2d gene, a TATA-box binding transcription factor . Kinase-labeled tf2d probe detected only unspliced RNA, while the same oligonucleotide labeled using the new method detected both unspliced tf2d RNA and rare pre-mRNA splicing intermediates.

Nat Cell Biol, 2000 Nov, 2(11), 855 - 8
Type II myosin regulatory light chain relieves auto-inhibition of myosin-heavy-chain function; Naqvi NI et al.; The F-actin based motor protein myosin II has a key role in cytokinesis . Here we show that the Schizosaccharomyces pombe regulatory light chain (RLC) protein Rlc1p binds to Myo2p in manner that is dependent on the IQ sequence motif (the RLC-binding site), and that Rlc1p is a component of the actomyosin ring . Rlc1p is important for cytokinesis at all growth temperatures and is essential for this process at lower temperatures . Interestingly, all deleterious phenotypes associated with the loss of Rlc1p function are suppressed by deletion of the RLC binding site on Myo2p . We conclude that the sole essential function of RLCs in fission yeast is to relieve the auto-inhibition of myosin II function, which is mediated by the RLC-binding site, on the myosin heavy chain (MHC).

Yeast, 2000 Nov, 16(15), 1405 - 11
Analysis of 114 kb of DNA sequence from fission yeast chromosome 2 immediately centromere-distal to his5; Xiang Z et al.; One hundred and fourteen kilobase pairs (kb) of contiguous genomic sequence have been determined immediately distal to the his5 genetic marker located about 0.9 Mb from the centromere on the long arm of Schizosaccharomyces pombe chromosome 2 . The sequence is contained in overlapping cosmid clones c16H5, c12D12, c24C6 and c19G7, of which 20 kb are identical to previously reported sequence from clone c21H7 . The remaining 93 781 bp of sequence contains 10 known genes (cdc14, cdm1, cps1, gpa1, msh2, pck2, rip1, rps30-2, sad1 and ubl1), 32 open reading frames (ORFs) capable of coding for proteins of at least 100 amino acid residues in length, one 5S rRNA gene, one tRNA(Pro) gene, one lone Tf1-type long terminal repeat (LTR) and one lone Tf2-type LTR . There is a density of one protein-coding gene per 2.2 kb and 22 of the 42 ORFs (52%) incorporate one or more introns . Twenty-one of the novel ORFs show sequence similarities which suggest functions of their products, including a cyclin C, a MADS box transcription factor, mad2-like protein, telomere binding protein, topoisomerase II-associated protein, ATP-dependent DEAH box RNA helicase, G10 protein, ubiquitin-activating e1-like enzyme, nucleoporin, prolyl-tRNA synthetase, peptidylprolyl isomerase, delta-1-pyrroline-5-carboxylate dehydrogenase, protein transport protein, coatomer epsilon, TCP-1 chaperonin, beta-subunit of 6-phosphofructokinase, aminodeoxychorismate lyase, a phosphate transport protein and a thioredoxin.

J Biol Chem, 2001 Feb 2, 276(5), 3004 - 9 Epub 2000 Oct 19.
The Cdc42p GTPase and its regulators Nrf1p and Scd1p are involved in endocytic trafficking in the fission yeast Schizosaccharomyces pombe; Murray JM et al.; Nrf1p was first identified in a screen for negative regulators of the Cdc42p GTPase . Overexpression of Nrf1p resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology and abnormal vacuolar phenotypes including an increase in vacuolar fusion . Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to vacuolar membranes and GFP-Nrf1p vacuolar localization depended on Scd1p, the Schizosaccharomyces pombe homolog of the Cdc24p guanine nucleotide exchange factor . In this study, site-directed mutagenesis was conducted on Nrf1p to determine its functional domains . Mutations in the three putative transmembrane domains resulted in mislocalization of GFP-Nrf1p and an inability to induce lethality, suggesting a loss of function . Mutations in the second extramembranous loop of Nrf1p also resulted in a loss of function and altered the ability of GFP-Nrf1p to localize to vacuolar membranes . Analysis of Deltanrf1 and Deltascd1 mutants revealed defects in endocytosis . In addition, overexpression of constitutively active Cdc42(G12V)p resulted in an increase in endocytosis and an ability to rescue the endocytic defects in Deltanrf1 and Deltascd1 cells . These data are consistent with Nrf1p and Scd1p being necessary for efficient endocytosis, possibly through the regulation of Cdc42p.

Bioorg Khim, 2000 Aug, 26(8), 623 - 30
{Chromosomal localization of rpb9+ and tfa1+ genes, coding for components of the mRNA synthesis apparatus of Schizosaccharomyces pombe}; Shpakovskii GV et al.; Using DNA hybridization on cosmid filters of high density, we established chromosomal localization of the rpb9+ gene encoding one of the specific subunits of RNA polymerase II of Schizosaccharomyces pombe and thus filled in the last gap in the mapping of the genes encoding components of RNA polymerase II of the fission yeast . The primary structure of three extended regions of the Sz . pombe chromosome I was elucidated and, as a result, genes neighboring on rpb9+ were identified . One of them proved to be the tfa1+ gene, encoding the large (alpha) subunit of the general factor of transcription initiation TFIIE.

Glycobiology, 2000 Oct, 10(10), 983 - 91
Dolichol phosphate mannose synthase from the filamentous fungus Trichoderma reesei belongs to the human and Schizosaccharomyces pombe class of the enzyme; Kruszewska JS et al.; Dolichol phosphate mannose (DPM) synthase activity, which is required in N:-glycosylation, O-mannosylation, and glycosylphosphatidylinositol membrane anchoring of protein, has been postulated to regulate the Trichoderma reesei secretory pathway . We have cloned a T.reesei cDNA that encodes a 243 amino acid protein whose amino acid sequence shows 67% and 65% identity, respectively, to the Schizosaccharomyces pombe and human DPM synthases, and which lacks the COOH-terminal hydrophobic domain characteristic of the Saccharomyces cerevisiae class of synthase . The Trichoderma dpm1 (Trdpm1) gene complements a lethal null mutation in the S.pombe dpm1(+) gene, but neither restores viability of a S.cerevisiae dpm1-disruptant nor complements the temperature-sensitivity of the S . cerevisiae dpm1-6 mutant . The T.reesei DPM synthase is therefore a member of the "human" class of enzyme . Overexpression of Trdpm1 in a dpm1(+)::his7/dpm1(+) S.pombe diploid resulted in a 4-fold increase in specific DPM synthase activity . However, neither the wild type T . reesei DPM synthase, nor a chimera consisting of this protein and the hydrophobic COOH terminus of the S.cerevisiae DPM synthase, complemented an S.cerevisiae dpm1 null mutant or gave active enzyme when expressed in E.coli . The level of the Trdpm1 mRNA in T.reesei QM9414 strain was dependent on the composition of the culture medium . Expression levels of Trdpm1 were directly correlated with the protein secretory capacity of the fungus.

Mol Cell, 2000 Sep, 6(3), 637 - 48
Structure and function of Cdc6/Cdc18: implications for origin recognition and checkpoint control; Liu J et al.; Cdc6/Cdc18 is a conserved and essential component of prereplication complexes . The 2.0 A crystal structure of an archaeal Cdc6 ortholog, in conjunction with a mutational analysis of the homologous Cdc18 protein from Schizosaccharomyces pombe, reveals novel aspects of Cdc6/Cdc18 function . Two domains of Cdc6 form an AAA+-type nucleotide binding fold that is observed bound to Mg.ADP . A third domain adopts a winged-helix fold similar to known DNA binding modules . Sequence comparisons show that the winged-helix domain is conserved in Orc1, and mutagenesis data demonstrate that this region of Cdc6/Cdc18 is required for function in vivo . Additional mutational analyses suggest that nucleotide binding and/or hydrolysis by Cdc6/Cdc18 is required not only for progression through S phase, but also for maintenance of checkpoint control during S phase.

Plant J, 2000 Oct, 24(1), 11 - 20
Activation of CDK-activating kinase is dependent on interaction with H-type cyclins in plants; Yamaguchi M et al.; cDNAs encoding cyclin H homologs were isolated from poplar (Populus tremula X tremuloides) and rice (Oryza sativa) plants, and were designated Pt;cycH;1 and Os;cycH;1, respectively . The deduced amino-acid sequences showed 40-60% similarity to human cyclin H and Schizosaccharomyces pombe Mcs2, with higher similarity in the cyclin box region . While Pt;cycH;1 and Os;cycH;1 were expressed in all tissues examined, the transcripts accumulated abundantly in dividing cells . Expression of Os;cycH;1 was abundant in the S-phase in partially synchronized suspension cells, and was induced by submergence in internodes of deepwater rice . A yeast two-hybrid assay demonstrated that both Pt;CycH;1 and Os;CycH;1 were able to interact with rice R2 kinase, which is structurally and functionally similar to cyclin-dependent kinase (CDK)-activating kinase (CAK) of vertebrates . Moreover, an in vitro pull-down assay showed that Os;CycH;1 specifically bound to R2 but not to other rice CDKs . When R2 was expressed in budding yeast CAK mutant, the suppression activity in terms of temperature-sensitivity was enhanced by co-expression with Os;cycH;1 . Furthermore, in vitro kinase assay indicated that the kinase activities of R2 on CDKs and the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II were markedly elevated by binding to Os;CycH;1 . Our results suggest that cyclin H is a regulatory subunit of CAK, which positively controls CDK- and CTD-kinase activities in plant cells.

J Biol Chem, 2001 Jan 5, 276(1), 835 - 43
Differential regulation of growth and checkpoint control mediated by a Cdc25 mitotic phosphatase from Pneumocystis carinii; Gustafson MP et al.; Pneumocystis carinii is an opportunistic fungal pathogen phylogenetically related to the fission yeast Schizosaccharomyces pombe . P . carinii causes severe pneumonia in immunocompromised patients with AIDS and malignancies . Although the life cycle of P . carinii remains poorly characterized, morphologic studies of infected lung tissue indicate that P . carinii alternates between numerous small trophic forms and fewer large cystic forms . To understand further the molecular mechanisms that regulate progression of the cell cycle of P . carinii, we have sought to identify and characterize genes in P . carinii that are important regulators of eukaryotic cell cycle progression . In this study, we have isolated a cDNA from P . carinii that exhibits significant homology, but unique functional characteristics, to the mitotic phosphatase Cdc25 found in S . pombe . P . carinii Cdc25 was shown to rescue growth of the temperature-sensitive S . pombe cdc25-22 strain and thus provides an additional tool to investigate the unique P . carinii life cycle . Although P . carinii Cdc25 could also restore the DNA damage checkpoint in cdc25-22 cells, it was unable to restore fully the DNA replication checkpoint . The dissociation of checkpoint control at the level of Cdc25 indicates that Cdc25 may be under distinct regulatory control in mediating checkpoint signaling.

Mol Biol Cell, 2000 Oct, 11(10), 3265 - 75
Protection of telomeres by the Ku protein in fission yeast; Baumann P et al.; Schizosaccharomyces pombe cells survive loss of telomeres by a unique pathway of chromosome circularization . Factors potentially involved in this survival mechanism include the heterodimeric Ku protein and ligase IV, both of which are involved in the repair of DNA double-strand breaks in mammalian cells . Furthermore, Ku plays a role in telomere maintenance as well as in DNA double-strand break repair in Saccharomyces cerevisiae . We have identified Ku and ligase IV homologues in S . pombe and analyzed their functions during normal growth and in cells undergoing senescence . In the absence of either a Ku subunit (pku70(+)) or ligase IV (lig4(+)), nonhomologous DNA end-joining was severely reduced . Lack of functional Ku led to shorter but stable telomeres and caused striking rearrangements of telomere-associated sequences, indicating a function for Ku in inhibiting recombinational activities near chromosome ends . In contrast to S . cerevisiae, concurrent deletion of pku70(+) and the gene for the catalytic subunit of telomerase (trt1(+)) was not lethal, allowing for the first time the dissection of the roles of Ku during senescence . Our results support a model in which Ku protects chromosome termini from nucleolytic and recombinational activities but is not involved in the formation of chromosome end fusions during senescence . The conclusion that nonhomologous end-joining is not required for chromosome circularization was further supported by analysis of survivors in strains lacking the genes for both trt1(+) and lig4(+).

Mol Cell Biol, 2000 Nov, 20(21), 7955 - 70
Evidence for splice site pairing via intron definition in Schizosaccharomyces pombe; Romfo CM et al.; Schizosaccharomyces pombe pre-mRNAs are generally multi-intronic and share certain features with pre-mRNAs from Drosophila melanogaster, in which initial splice site pairing can occur via either exon or intron definition . Here, we present three lines of evidence suggesting that, despite these similarities, fission yeast splicing is most likely restricted to intron definition . First, mutating either or both splice sites flanking an internal exon in the S . pombe cdc2 gene produced almost exclusively intron retention, in contrast to the exon skipping observed in vertebrates . Second, we were unable to induce skipping of the internal microexon in fission yeast cgs2, whereas the default splicing pathway excludes extremely small exons in mammals . Because nearly quantitative removal of the downstream intron in cgs2 could be achieved by expanding the microexon, we propose that its retention is due to steric occlusion . Third, several cryptic 5' junctions in the second intron of fission yeast cdc2 are located within the intron, in contrast to their generally exonic locations in metazoa . The effects of expanding and contracting this intron are as predicted by intron definition; in fact, even highly deviant 5' junctions can compete effectively with the standard 5' splice site if they are closer to the 3' splicing signals . Taken together, our data suggest that pairing of splice sites in S . pombe most likely occurs exclusively across introns in a manner that favors excision of the smallest segment possible.

Mol Cell Biol, 2000 Nov, 20(21), 7922 - 32
Schizosaccharomyces pombe Hsk1p is a potential cds1p target required for genome integrity; Snaith HA et al.; The fission yeast Hsk1p kinase is an essential activator of DNA replication . Here we report the isolation and characterization of a novel mutant allele of the gene . Consistent with its role in the initiation of DNA synthesis, hsk1(ts) genetically interacts with several S-phase mutants . At the restrictive temperature, hsk1(ts) cells suffer abnormal S phase and loss of nuclear integrity and are sensitive to both DNA-damaging agents and replication arrest . Interestingly, hsk1(ts) mutants released to the restrictive temperature after early S-phase arrest in hydroxyurea (HU) are able to complete bulk DNA synthesis but they nevertheless undergo an abnormal mitosis . These findings indicate a second role for hsk1 subsequent to HU arrest . Consistent with a later S-phase role, hsk1(ts) is synthetically lethal with Deltarqh1 (RecQ helicase) or rad21ts (cohesin) mutants and suppressed by Deltacds1 (RAD53 kinase) mutants . We demonstrate that Hsk1p undergoes Cds1p-dependent phosphorylation in response to HU and that it is a direct substrate of purified Cds1p kinase in vitro . These results indicate that the Hsk1p kinase is a potential target of Cds1p regulation and that its activity is required after replication initiation for normal mitosis.

Mol Cell Biol, 2000 Nov, 20(21), 7853 - 66
Analysis of fission yeast primase defines the checkpoint responses to aberrant S phase initiation; Tan S et al.; To investigate the checkpoint response to aberrant initiation, we analyzed the cell cycle checkpoint response induced by mutations of Schizosaccharomyces pombe DNA primase . DNA primase has two subunits, Spp1 and Spp2 (S . pombe primases 1 and 2) . Spp1 is the catalytic subunit that synthesizes the RNA primer, which is then extended by DNA polymerase alpha (Polalpha) to synthesize an initiation DNA structure, and this catalytic function of Polalpha is a prerequisite for generating the S-M phase checkpoint . Here we show that Spp2 is required for coupling the function of Spp1 to Polalpha . Thermosensitive mutations of spp2(+) destabilize the Polalpha-primase complex, resulting in an allele-specific S phase checkpoint defect . The mutant exhibiting a more severe checkpoint defect also has a higher extent of Polalpha-primase complex instability and deficiency in the hydroxyurea-induced Cds1-mediated intra-S phase checkpoint response . However, this mutant is able to activate the Cds1 response to S phase arrest induced by temperature . These findings suggest that the Cds1 response to the S-phase arrest signal(s) induced by a initiation mutant is different from that induced by hydroxyurea . Interestingly, a polalphats mutant with a defective S-M phase checkpoint and an spp2 mutant with an intact checkpoint have a similar Polalpha-primase complex stability, and the Cds1 response induced by hydroxyurea or by the mutant arrests at the restrictive temperature . Thus, the Cds1-mediated intra-S phase checkpoint response induced by hydroxyurea can also be distinguished from the S-M phase checkpoint response that requires the initiation DNA synthesis by Polalpha.

J Cell Biol, 2000 Oct 2, 151(1), 15 - 28
Tea2p is a kinesin-like protein required to generate polarized growth in fission yeast; Browning H et al.; Cytoplasmic microtubules are critical for establishing and maintaining cell shape and polarity . Our investigations of kinesin-like proteins (klps) and morphological mutants in the fission yeast Schizosaccharomyces pombe have identified a kinesin-like gene, tea2(+), that is required for cells to generate proper polarized growth . Cells deleted for this gene are often bent during exponential growth and initiate growth from improper sites as they exit stationary phase . They have a reduced cytoplasmic microtubule network and display severe morphological defects in genetic backgrounds that produce long cells . The tip-specific marker, Tea1p, is mislocalized in both tea2-1 and tea2Delta cells, indicating that Tea2p function is necessary for proper localization of Tea1p . Tea2p is localized to the tips of the cell and in a punctate pattern within the cell, often coincident with the ends of cytoplasmic microtubules . These results suggest that this kinesin promotes microtubule growth, possibly through interactions with the microtubule end, and that it is important for establishing and maintaining polarized growth along the long axis of the cell.

Mol Gen Genet, 2000 Sep, 264(1-2), 173 - 83
Schizosaccharomyces pombe ehs1p is involved in maintaining cell wall integrity and in calcium uptake; Carnero E et al.; The Schizosaccharomyces pombe mutant ehs1-1 mutant was isolated on the basis of its hypersensitivity to Echinocandin and Calcofluor White, which inhibit cell wall synthesis . The mutant shows a thermosensitive growth phenotype that is suppressed in the presence of an osmotic stabiliser . The mutant also showed other cell wall-associated phenotypes, such as enhanced sensitivity to enzymatic cell wall degradation and an imbalance in polysaccharide synthesis . The ehs1 + gene encodes a predicted integral membrane protein that is 30% identical to Saccharomyces cerevisiae Mid1p, a protein that has been proposed to form part of a calcium channel . As expected for such a function, we found that ehs1+ is involved in intracellular Ca2+ accumulation . High external Ca2+ concentrations suppressed all phenotypes associated with the ehs1 null mutation, suggesting that the cell integrity defects of ehs1 mutants result from inadequate levels of calcium in the cell . We observed a genetic relationship between ehs1+ and the protein kinase C homologue pck2+ . pck2+ suppressed all phenotypes of ehs1-1 mutant cells . Overproduction of pck2p is deleterious to wild-type cells, increasing 1,3-beta-D-glucan synthase activity and promoting accumulation of extremely high levels of Ca2+ . The lethality associated with pck2p, the increase in 1,3-beta-D-glucan synthase production and the strong Ca2+ accumulation are all dependent on the presence of ehs1p . Our results suggest that in fission yeast ehs1p forms part of a calcium channel that is involved in the cell wall integrity pathway that includes the kinase pck2p.

Yeast, 2000 Oct, 16(14), 1261 - 71
Schizosaccharomyces pombe gmd3(+)/alg11(+) is a functional homologue of Saccharomyces cerevisiae ALG11 which is involved in N-linked oligosaccharide synthesis; Umeda K et al.; The oligosaccharide of glycoproteins in the fission yeast Schizosaccharomyces pombe is unique in containing galactose . We isolated four mutants that had reduced amounts of galactose residues on their cell surface glycoproteins by fluorescence-activated cell sorter . The isolated four recessive mutants, gmd1 to gmd4, showed a defect in glycosylation of acid phosphatase, a cell surface glycoprotein . In gmd3 mutant cells, the amounts of both mannose and galactose residues were decreased on the cell surface galactomannoproteins, suggesting an underglycosylation of galactomannoproteins . The gmd3(+) gene encodes a protein that has significant similarity with Saccharomyces cerevisiae Alg11p and is likely to be involved in N-linked core oligosaccharide synthesis . ALG11 suppressed the gmd3 mutation, indicating that gmd3(+) gene is a functional homologue of the ALG11 gene . We therefore designated gmd3(+) as alg11(+) .

Genetics, 2000 Oct, 156(2), 513 - 21
Glucose monitoring in fission yeast via the Gpa2 galpha, the git5 Gbeta and the git3 putative glucose receptor; Welton RM et al.; The fission yeast Schizosaccharomyces pombe responds to environmental glucose by activating adenylate cyclase . The resulting cAMP signal activates protein kinase A (PKA) . PKA inhibits glucose starvation-induced processes, such as conjugation and meiosis, and the transcription of the fbp1 gene that encodes the gluconeogenic enzyme fructose-1,6-bisphosphatase . We previously identified a collection of git genes required for glucose repression of fbp1 transcription, including pka1/git6, encoding the PKA catalytic subunit, git2/cyr1, encoding adenylate cyclase, and six "upstream" genes required for adenylate cyclase activation . The git8 gene, identical to gpa2, encodes the alpha subunit of a heterotrimeric guanine-nucleotide binding protein (Galpha) while git5 encodes a Gbeta subunit . Multicopy suppression studies with gpa2(+) previously indicated that S . pombe adenylate cyclase activation may resemble that of the mammalian type II enzyme with sequential activation by Galpha followed by Gbetagamma . We show here that an activated allele of gpa2 (gpa2(R176H), carrying a mutation in the coding region for the GTPase domain) fully suppresses mutations in git3 and git5, leading to a refinement in our model . We describe the cloning of git3 and show that it encodes a putative seven-transmembrane G protein-coupled receptor . A git3 deletion confers the same phenotypes as deletions of other components of the PKA pathway, including a germination delay, constitutive fbp1 transcription, and starvation-independent conjugation . Since the git3 deletion is fully suppressed by the gpa2(R176H) allele with respect to fbp1 transcription, git3 appears to encode a G protein-coupled glucose receptor responsible for adenylate cyclase activation in S . pombe.

Pharmacol Ther, 2000 Aug-Sep, 87(2-3), 117 - 39
Specific and nonspecific enzymes involved in the catabolism of mononucleoside and dinucleoside polyphosphates; Guranowski A; This review concerns enzymes that can degrade nucleoside 5'-tetra- and pentaphosphates (p(4)N and p(5)N) and those that can degrade various dinucleoside polyphosphates (Np(3-6)N') . Most of these enzymes are hydrolases, and they occur in all types of organisms . Certain fungi and protozoa also possess specific Np(n)N' phosphorylases . Specific p(4)N hydrolases have been demonstrated in mammals and in plants . In yeast, p(4)N and p(5)N are hydrolyzed by exopolyphosphatases . Among other hydrolases that can degrade these minor mononucleotides are phosphatases, apyrase, and (asymmetrical) Np(4)N' hydrolase, as well as the nonspecific adenylate deaminase . Np(n)N's are good substrates for Type I phosphodiesterases and nucleotide pyrophosphatases, and diadenosine polyphosphates are easily deaminated to diinosine polyphosphates by nonspecific adenylate deaminases . Specific Np(3)N' hydrolases occur in both prokaryotes and eukaryotes . Interestingly, the human fragile histidine triad (Fhit) tumor suppressor protein appears to be a typical Np(3)N' hydrolase . Among the specific Np(4)N' hydrolases are asymmetrically cleaving ones, which are typical of higher eukaryotes, and symmetrically cleaving enzymes found in Physarum polycephalum and in many bacteria . An enzyme that hydrolyzes both diadenosine tetraphosphate and diadenosine triphosphate has been found in the fission yeast Schizosaccharomyces pombe . Its amino acid sequence is similar to that of the human Fhit/Np(3)N' hydrolase . Very recently, a typical (asymmetrical) Np(4)N' hydrolase has been demonstrated for the first time in a bacterium-the pathogenic Bartonella bacilliformis . Another novelty is the discovery of diadenosine 5', 5"'-P(1),P 6-hexaphosphate hydrolases in budding and fission yeasts and in mammalian cells . These enzymes and the (asymmetrical) Np(4)N' hydrolases have the amino acid motif typical of the MutT (or Nudix hydrolase) family . In contrast, the Schizosaccharomyces pombe Ap(4)A/Ap(3)A hydrolase, the human Fhit protein, and the yeast Np(n)N' phosphorylases belong to a superfamily GAFH, which includes the histidine triad proteins.

J Bacteriol, 2000 Oct, 182(20), 5880 - 4
Characterization of tpp1(+) as encoding a main trehalose-6P phosphatase in the fission yeast Schizosaccharomyces pombe; Franco A et al.; We have characterized an open reading frame of 2,454 bp on chromosome I of Schizosaccharomyces pombe as the gene encoding trehalose-6P phosphatase (tpp1(+)) . Disruption of tpp1(+) caused in vivo accumulation of trehalose-6P upon heat shock and prevented cell growth at 37 to 40 degrees C . Accumulation of trehalose-6P in cells bearing a chromosomal disruption of the tpp1(+) gene and containing a plasmid with tpp1(+) under the control of the thiamine-repressible promotor correlated with tpp1(+) repression . The level of tpp1(+) mRNA rose upon heat shock, osmostress, or oxidative stress and was negatively controlled by cyclic AMP-dependent protein kinase activity . Expression of tpp1(+) during oxidative or osmotic stress, but not during heat shock, was under positive control by the wis1-sty1 (equivalent to phh1 and spc1) mitogen-activated protein kinase pathway . Analysis of Tpp1 protein levels suggests that the synthesis of trehalose-6P phosphatase may also be subjected to translational or posttranslational control.

Mol Cell Biol, 2000 Oct, 20(20), 7798 - 812
Nuclear import of the retrotransposon Tf1 is governed by a nuclear localization signal that possesses a unique requirement for the FXFG nuclear pore factor Nup124p; Dang VD et al.; Retroviruses, such as human immunodeficiency virus, that infect nondividing cells generate integration precursors that must cross the nuclear envelope to reach the host genome . As a model for retroviruses, we investigated the nuclear entry of Tf1, a long-terminal-repeat-containing retrotransposon of the fission yeast Schizosaccharomyces pombe . Because the nuclear envelope of yeasts remains intact throughout the cell cycle, components of Tf1 must be transported through the envelope before integration can occur . The nuclear localization of the Gag protein of Tf1 is different from that of other proteins tested in that it has a specific requirement for the FXFG nuclear pore factor, Nup124p . Using extensive mutagenesis, we found that Gag contained three nuclear localization signals (NLSs) which, when included individually in a heterologous protein, were sufficient to direct nuclear import . In the context of the intact transposon, mutations in the NLS that mapped to the first 10 amino acid residues of Gag significantly impaired Tf1 retrotransposition and abolished nuclear localization of Gag . Interestingly, this NLS activity in the heterologous protein was specifically dependent upon the presence of Nup124p . Deletion analysis of heterologous proteins revealed the surprising result that the residues in Gag with the NLS activity were independent from the residues that conveyed the requirement for Nup124p . In fact, a fragment of Gag that lacked NLS activity, residues 10 to 30, when fused to a heterologous protein, was sufficient to cause the classical NLS of simian virus 40 to require Nup124p for nuclear import . Within the context of the current understanding of nuclear import, these results represent the novel case of a short amino acid sequence that specifies the need for a particular nuclear pore complex protein.

Hum Mol Genet, 2000 Sep 22, 9(15), 2223 - 9
Myotubularin, a phosphatase deficient in myotubular myopathy, acts on phosphatidylinositol 3-kinase and phosphatidylinositol 3-phosphate pathway; Blondeau F et al.; Myotubular myopathy (MTM1) is an X-linked disease, characterized by severe neonatal hypotonia and generalized muscle weakness, with pathological features suggesting an impairment in maturation of muscle fibres . The MTM1 gene encodes a protein (myotubularin) with a phosphotyrosine phosphatase consensus . It defines a family of at least nine genes in man, including the antiphosphatase hMTMR5/Sbf1 and hMTMR2, recently found mutated in a recessive form of Charcot-Marie-Tooth disease . Myotubularin shows a dual specificity protein phosphatase activity in vitro . We have performed an in vivo test of tyrosine phosphatase activity in Schizosaccharomyces pombe, indicating that myotubularin does not have a broad specificity tyrosine phosphatase activity . Expression of active human myotubularin inhibited growth of S.pombe and induced a vacuolar phenotype similar to that of mutants of the vacuolar protein sorting (VPS) pathway and notably of mutants of VPS34, a phosphatidylinositol 3-kinase (PI3K) . In S.pombe cells deleted for the endogenous MTM homologous gene, expression of human myotubularin decreased the level of phosphatidylinositol 3-phosphate (PI3P) . We have created a substrate trap mutant which shows relocalization to plasma membrane projections (spikes) in HeLa cells and was inactive in the S.pombe assay . This mutant, but not the wild-type or a phosphatase site mutant, was able to immunoprecipitate a VPS34 kinase activity . Wild-type myotubularin was also able to directly dephosphorylate PI3P and PI4P in vitro . Myotubularin may thus decrease PI3P levels by down-regulating PI3K activity and by directly degrading PI3P.

Mol Microbiol, 2000 Sep, 37(6), 1480 - 93
FAP1, a homologue of human transcription factor NF-X1, competes with rapamycin for binding to FKBP12 in yeast; Kunz J et al.; The immunosuppressive drug rapamycin binds to the peptidyl-prolyl cis-trans isomerase FKBP12, and this complex arrests growth of yeast cells and activated T lymphocytes in the G1 phase of the cell cycle . In yeast, loss-of-function mutations in FPR1, the gene encoding FKBP12, or dominant gain-of-function mutations in TOR1 and TOR2, the genes encoding the physical targets of the FKBP12-rapamycin complex, confer rapamycin resistance . Here, we report the cloning and characterization of a novel gene, termed FAP1, which confers resistance to rapamycin by competing with the drug for binding to FKBP12 . FAP1 encodes a member of an evolutionarily conserved family of putative transcription factors that includes human NF-X1, Drosophila melanogaster shuttle craft and previously undescribed homologues in Caenorhabditis elegans, Arabidopsis thaliana and Schizosaccharomyces pombe . We provide genetic and biochemical evidence that FAP1 interacts physically with FKBP12 in vivo and in vitro, and that it competes with rapamycin for interaction . Furthermore, mutations in the FKBP12 drug binding/active site or surface residues abolish binding to FAP1 . Our results suggest that FAP1 is a physiological ligand for FKBP12 that is highly conserved from yeast to man . Furthermore, prolyl isomerases may commonly bind and regulate transcription factors.

FEBS Lett, 2000 Sep 15, 481(2), 122 - 6
Schizosaccharomyces pombe Rad9 contains a BH3-like region and interacts with the anti-apoptotic protein Bcl-2; Komatsu K et al.; Here we report that the Schizosaccharomyces pombe Rad9 (SpRad9) protein contains a group of amino acids with similarity to the Bcl-2 homology 3 death domain, which is required for SpRad9 interaction with human Bcl-2 and apoptosis induction in human cells . Overexpression of Bcl-2 in S . pombe inhibits cell growth independently of rad9, but enhances resistance of rad9-null cells to methyl methanesulfonate, ultraviolet and ionizing radiation . These observations suggest that SpRad9 may represent the first member of the Bcl-2 protein family identified in yeast, though the cell death pathways in S . pombe may differ from those found in mammals.

FEBS Lett, 2000 Sep 15, 481(2), 105 - 8
The S . pombe sep1 gene encodes a nuclear protein that is required for periodic expression of the cdc15 gene; Zilahi E et al.; The Schizosaccharomyces pombe sep1 gene encodes a putative transcription factor that is required for cell separation . Among the genes required for septum formation and cytokinesis in fission yeast examined to date, the only one whose mRNA fluctuates significantly during the cell cycle is cdc15 . In this study we have examined cdc15 mRNA levels in sep1 mutant and null backgrounds and have found that sep1p function is required for periodic accumulation of cdc15 mRNA . We have also localised sep1p and find that it is a nuclear protein, consistent with its proposed role as a transcription factor.

J Mol Biol, 2000 Sep 29, 302(4), 751 - 9
Periodic accumulation of cdc15 mRNA is not necessary for septation in Schizosaccharomyces pombe; Utzig S et al.; Analysis of Schizosaccharomyces pombe mutants that are defective in septum formation and cytokinesis has identified the product of the cdc15 gene as a key element in formation of a division septum . S . pombe cells lacking cdc15p function cannot assemble a functional medial ring, and do not make a division septum . cdc15 mRNA accumulates periodically during the cell cycle, peaking after entry into mitosis, and increased expression of the gene in G2-arrested cells can promote F-actin ring formation . Here, we have investigated the effects of mutations that block cell division upon the expression of cdc15 in synchronised cell populations, and analysed the expression of cdc15 when septum formation is induced by ectopic activation of the septation signalling network . We concluded the following: (i) the septation signalling network genes are not required for periodic accumulation of cdc15 mRNA; (ii) induction of septum formation in G2-arrested cells by activation of the septation signalling network does not result in accumulation of cdc15 mRNA, which is therefore not a prerequisite for septum formation; (iii) failure to turn off septum formation at the end of mitosis results in continued expression of cdc15; and (iv) periodic accumulation of cdc15 mRNA is mediated by a 97 bp region 5' to the mRNA start site .

Folia Microbiol (Praha), 1999, 44(4), 401 - 5
Development of the primer set for the detection of the hsp60 gene in Trichophyton mentagrophytes cDNA; Kopecek P et al.; Three sequences of hsp60 from Saccharomyces cerevisiae, Schizosaccharomyces pombe and Histoplasma capsulatum were compared . Local multiple alignment of these sequences allowed the selection of two oligonucleotides suitable as primers for the polymerase chain reaction . This primer set was used for the amplification of a part of the hsp60 gene from cDNA of Trichophyton mentagrophytes and S . cerevisiae . Similar fragments detected in both PCR's imply the possible future use of the developed primer set for the detection of the hsp60 gene in other fungal species.

Nucleic Acids Res, 2000 Sep 15, 28(18), 3666 - 73
Human RNA lariat debranching enzyme cDNA complements the phenotypes of Saccharomyces cerevisiae dbr1 and Schizosaccharomyces pombe dbr1 mutants; Kim JW et al.; The cDNA encoding the human RNA lariat debranching enzyme (hDBR1) was identified and cloned by searching the Expressed Sequence Tag (EST) database and screening a HeLa cDNA library, based on predicted amino acid sequence homologies with the Saccharomyces cerevisiae, Schizosaccharomyces pombe and Caenorhabditis elegans debranching enzymes . The hDBR1 cDNA expressed in Escherichia coli showed debranching activity in vitro and was also shown to be functional in an interspecies specific complementation experiment . hDBR1 cDNA in a S . cerevisiae expression vector complemented the intron accumulation phenotype of a S . cerevisiae dbr1 null mutant . Integration of the cDNA for hDBR1 into the ura4 locus of S . pombe also complemented both the intron accumulation and slow growth phenotypes of a S . pombe dbr1 null mutant strain . Comparison of the amino acid sequence of hDBR1 with the other DBR protein sequences showed several conserved regions, with 40, 44 and 43% identity to the S . cerevisiae, S . pombe and C . elegans debranching enzymes, respectively.

Mol Biol Cell, 2000 Sep, 11(9), 3205 - 17
A zinc-finger protein, Rst2p, regulates transcription of the fission yeast ste11(+) gene, which encodes a pivotal transcription factor for sexual development; Kunitomo H et al.; Schizosaccharomyces pombe ste11 encodes a high-mobility group family transcriptional activator that is pivotal in sexual development . Transcription of ste11 is induced by starvation of nutrients via a decrease of the cAMP-dependent protein kinase (PKA) activity . Here we report the identification of a novel transcription factor, Rst2p, that directly regulates ste11 expression . Cells in which the rst2 gene was disrupted expressed ste11 poorly and were sterile, and this sterility could be suppressed by artificial expression of ste11 . Disruption of rst2 suppressed hypermating and hypersporulation in the PKA-null mutant, whereas overexpression of rst2 induced sexual development in the PKA-activated mutant . Cloning analysis indicated that Rst2p was a Cys(2)His(2) zinc-finger protein carrying 567 amino acid residues . Rst2p could bind specifically to a stress response element-like cis element located in the ste11 promoter region, which was important for ste11 expression . Meanwhile, transcription of ste11 was reduced significantly by a defective mutation in itself . An artificial supply of functional Ste11p circumvented this reduction . A complete Ste11p-binding motif (TR box) found in the promoter region was necessary for the full expression of ste11, suggesting that Ste11p is involved in the activation of ste11 . We conclude that transcription of ste11 is under autoregulation in addition to control through the PKA-Rst2p pathway.

Mol Biol Cell, 2000 Sep, 11(9), 3177 - 90
Neuronal cell shape and neurite initiation are regulated by the Ndr kinase SAX-1, a member of the Orb6/COT-1/warts serine/threonine kinase family; Zallen JA et al.; The Caenorhabditis elegans sax-1 gene regulates several aspects of neuronal cell shape . sax-1 mutants have expanded cell bodies and ectopic neurites in many classes of neurons, suggesting that SAX-1 functions to restrict cell and neurite growth . The ectopic neurites in sensory neurons of sax-1 mutants resemble the defects caused by decreased sensory activity . However, the activity-dependent pathway, mediated in part by the UNC-43 calcium/calmodulin-dependent kinase II, functions in parallel with SAX-1 to suppress neurite initiation . sax-1 encodes a serine/threonine kinase in the Ndr family that is related to the Orb6 (Schizosaccharomyces pombe), Warts/Lats (Drosophila), and COT-1 (Neurospora) kinases that function in cell shape regulation . These kinases have similarity to Rho kinases but lack consensus Rho-binding domains . Dominant negative mutations in the C . elegans RhoA GTPase cause neuronal cell shape defects similar to those of sax-1 mutants, and genetic interactions between rhoA and sax-1 suggest shared functions . These results suggest that SAX-1/Ndr kinases are endogenous inhibitors of neurite initiation and cell spreading.

Mol Biol Cell, 2000 Sep, 11(9), 2845 - 62
A pcl-like cyclin activates the Res2p-Cdc10p cell cycle "start" transcriptional factor complex in fission yeast; Tanaka K et al.; In the fission yeast Schizosaccharomyces pombe, the "start" of the cell cycle is controlled by the two functionally redundant transcriptional regulator complexes, Res1p-Cdc10p and Res2p-Cdc10p, that activate genes essential for the onset and progression of S phase . The activity of the Res2p-Cdc10p complex is regulated at least by the availability of the Rep2 trans-activator subunit in the mitotic cell cycle . We have recently isolated the pas1(+) gene as a multicopy suppressor of the res1 null mutant . This gene encodes a novel cyclin that shares homology with the Pho85 kinase-associated cyclins of the budding yeast Saccharomyces cerevisiae . Genetic analysis reveals that Pas1 cyclin is unrelated to phosphate metabolism and stimulates the G(1)-S transition by specifically activating the Res2p-Cdc10p complex independently of Rep2p . Pas1 cyclin also controls mating pheromone signaling . Cells lacking pas1(+) are highly sensitive to mating pheromone, responding with facilitated G(1) arrest and premature commitment to conjugation . Pas1 cyclin associates in vivo with both Cdc2 and Pef1 kinases, the latter of which is a fission yeast counterpart of the budding yeast Pho85 kinase, but genetic analysis indicates that the Pef1p-associated Pas1p is responsible for the activation of Res2p-Cdc10p during the G(1)-S transition.

Genetics, 2000 Sep, 156(1), 59 - 68
A family of cAMP-response-element-related DNA sequences with meiotic recombination hotspot activity in Schizosaccharomyces pombe; Fox ME et al.; The heptamer sequence ATGACGT is essential for activity of the M26 meiotic recombination hotspot in the ade6 gene of Schizosaccharomyces pombe . Hotspot activity is associated with binding of the heterodimeric transcription factor Atf1.Pcr1 to M26 . We have found that the sequences (C/T/G) TGACGT also bound Atf1.Pcr1 and acted as meiotic hotspots, but unlike M26 they must be followed by A or C for Atf1.Pcr1 binding and hotspot activity . The basis of the hotspot activity of CTGACGTA (ade6-3013) appears to be identical to that of M26: hotspot activity of both sequences was abolished in cells mutant for atf1, pcr1, spc1, or wis1 and was undetectable in mitotic recombination and in meiotic recombination when located on a plasmid . Both hotspot sequences were sites of micrococcal nuclease hypersensitivity in meiotic chromatin, suggesting that they create an open chromatin structure during meiosis at the site of the hotspots . The newly identified hotspot sequences (C/T/G)TGACGT(A/C) and M26 are closely related to the cAMP response element (CRE) consensus sequence for binding of cAMP-responsive transcription factors such as Atf1.Pcr1, suggesting a link between transcription and meiotic recombination . These results significantly expand the list of identified sequences with meiotic recombination hotspot activity in S . pombe from a single sequence to a family of CRE-related sequences.

Curr Genet, 2000 Aug, 38(2), 71 - 7
Glutathione depletion leads to delayed growth stasis in Saccharomyces cerevisiae: evidence of a partially overlapping role for thioredoxin; Sharma KG et al.; Disruption of the first enzyme of glutathione biosynthesis in both Saccharomyces cerevisiae and Schizosaccharomyces pombe leads to a glutathione auxotrophy phenotype on plates . However, growth experiments in liquid medium revealed that the cessation of growth resulting from glutathione depletion in these yeasts is very delayed in S . cerevisiae compared to S . pombe . Glutathione metabolism was investigated to understand this delayed growth stasis in S . cerevisiae . The assimilation of reduced and oxidized glutathione, the intracellular storage pools of glutathione and the turnover of this compound were investigated and found to be similar in both yeasts . A possible overlapping role of intracellular thioredoxin in causing delayed stasis was studied . Yeast thioredoxin was overexpressed in S . cerevisiae and was found to partially relieve the dependence of S . cerevisiae glutathione auxotrophs on extracellular glutathione in glucose-grown cultures, as well as in glycerol-grown cultures where conditions of increased glutathione requirements exists in the cell . By partially, but not completely, compensating for glutathione deficiency in this yeast, thioredoxin thus appeared to be the major factor that was causing the delayed growth stasis following glutathione depletion in this yeast.

Biochem J, 2000 Sep 15, 350 Pt 3, 663 - 9
Disruption and overexpression of the Schizosaccharomyces pombe aph1 gene and the effects on intracellular diadenosine 5',5'''-P1, P4-tetraphosphate (Ap4A), ATP and ADP concentrations; Ingram SW et al.; Diadenosine oligophosphates are ubiquitous compounds that were discovered over 30 years ago . Diadenosine 5',5"'-P(1), P(4)-tetraphosphate (Ap(4)A) is the most studied member of this family, and its function in yeast is unknown . To investigate possible functions, we changed the intracellular Ap(4)A concentration in Schizosaccharomyces pombe via disruption and overexpression of the aph1 gene, which encodes an Ap(4)A hydrolase (Aph1) . S . pombe Aph1 is 52% identical with a human tumour suppressor protein, Fhit, in a core region of 109 amino acids . Disruption of aph1 resulted in an 85% decrease in Ap(4)A hydrolase activity and a 290-fold increase in the intracellular Ap(4)A concentration . The disruption and subsequent increase in intracellular Ap(4)A concentration had no significant effect on the growth of S . pombe . Overexpression of the S . pombe aph1 gene, resulting in 17- and 84-fold increases in Ap(4)A hydrolase activity above wild-type levels, resulted in 60 and 80% decreases respectively in the intracellular Ap(4)A concentration . This represents the first report of a decrease in the intracellular Ap(4)A concentration in response to overexpression of a degradative enzyme in any eukaryotic organism . We describe a new S . pombe expression plasmid, pPOX, which was used to achieve the largest increase in expression of aph1 . Overexpression of aph1 at the highest level resulted in a 46% increase in generation time in comparison with the control strain . Neither overexpression nor disruption had any effect on the intracellular ATP or ADP concentrations . This is the first report of ADP and ATP concentrations in S . pombe . These data also indicate that Aph1 functions in vivo to degrade Ap(4)A, and that high-level overexpression of this enzyme reduces the growth rate.

Mamm Genome, 2000 Sep, 11(9), 786 - 8
The genomic structure of c14orf1 is conserved across eukarya; Ottolenghi C et al.; We have recently cloned the gene C14orf1, which is strongly expressed in normal testis and in several cancer cell lines and tumors . This gene maps to 14q24.3 and is interrupted by four introns . Two of them are also represented in the open reading frame of Schizosaccharomyces pombe in the same phase . In Arabidopsis taliana only the first of the two introns was found, in the same phase as the corresponding ones in S . pombe and human . Disruption of the ortholog in Saccharomyces cerevisiae (Yer044c) led to a severe growth defect, and C14orf1 failed to complement mutant yeast when put under the control of the natural Yer044c promoter . Further studies are needed to understand the causes underlying the high degree of conservation of the C14orf1 genomic structure.

Int Rev Cytol, 2000, 200, 101 - 41
The Chlorella hexose/H(+)-symporters; Tanner W; The physiology, molecular biology, and biochemistry of the inducible hexose uptake protein of Chlorella kessleri is reviewed . The protein encoded by the HUP1 gene is the most intensively studied membrane transporter of plants . Responsible for substrate accumulation up to 1500-fold, it translocates one proton together with one hexose, and the cell invests 1 ATP per sugar transported . Kinetics suggest that substrate accumulation is mainly brought about by a large delta Km (Kminside >> Kmoutside) . The HUP1 protein (534aa) consists of 12 transmembrane helices of which at least helices I, V, VII, and XI interact with the sugar during translocation and participate in lining the transport path through the membrane . The helix packing might very well be identical to the one suggested for the E . coli lac permease, although the mechanism for transport and proton coupling that has been suggested for lac permease (Kaback, 1997) certainly does not hold for the Chlorella symporter; both are distantly related members, however, of the MFS-family of transporters . HUP1 has been functionally expressed in Schizosaccharomyces pombe, Saccharomyces cerevisiae, Escherichia coli, Volvox carteri, and in Xenopus oocytes.

J Mol Biol, 2000 Sep 8, 302(1), 65 - 77
Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1; Lalev AI et al.; The interdependency of steps in the processing of pre-rRNA in Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes . To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins . When incubated with protein extract, the spacer formed a specific large RNP . This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresis . Modification exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers . Mutagenic analyses confirmed an interaction with this sequence and indicated that this domain is critical to the efficient maturation of the precursor RNA . The protein constituents, purified by affinity chromatography using the ITS1 sequence, retained an ability to form stable RNP . Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicated at least 20 protein components ranging in size from 20-200 kDa . Peptide mapping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA binding proteins which included four different RNA-binding motifs . Another protein was putatively identified as a pseudouridylate synthase . Additional RNA constituents were not detected . The significance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed .

Eur J Cell Biol, 2000 Jul, 79(7), 469 - 77
The Cdc42p GTPase is targeted to the site of cell division in the fission yeast Schizosaccharomyces pombe; Merla A et al.; The Rho-family GTPase Cdc42p regulates many aspects of cell polarity and growth in eukaryotic cells, including the organization of the actin cytoskeleton . To further examine Cdc42p function in the fission yeast Schizosaccharomyces pombe, a functional green fluorescent protein (GFP)-Cdc42p fusion protein was generated . GFP-Cdc42p was observed at the medial region of the cell at the cell-division site early in cytokinesis and remained there through cell separation, and was also localized to the periphery of the cell and to internal membranes . Unexpectedly, treatment with the actin-depolymerizing drug latrunculin-A disrupted the medial region targeting pattern, and cells deficient in the actin-binding proteins tropomyosin and profilin also did not exhibit medial GFP-Cdc42p staining . In addition, medial GFP-Cdc42p localization was eliminated in a number of cytokinesis mutants, including strains defective in assembling the medial actinomyosin ring, medial ring contraction, and septum assembly . GFP-Cdc42p targeting was less affected in mutants that formed misplaced or multiple septa . These results suggest that the localization of Cdc42p at the cell-division site was dependent upon the actin cytoskeleton and that Cdc42p may function in the interdependent processes of cytokinesis and septation.

J Biol Chem, 2000 Nov 24, 275(47), 36575 - 83
Biochemical and functional characterization of inositol 1,3,4,5, 6-pentakisphosphate 2-kinases; Ives EB et al.; Synthesis of inositol 1,2,3,4,5,6-hexakisphosphate (IP(6)), also known as phytate, is integral to cellular function in all eukaryotes . Production of IP(6) predominately occurs through phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (IP(5)) by a 2-kinase . Recent cloning of the gene encoding this kinase from Saccharomyces cerevisiae, designated scIpk1, has identified a cellular role for IP(6) production in the regulation of mRNA export from the nucleus . In this report, we characterize the biochemical and functional parameters of recombinant scIpk1 . Purified recombinant scIpk1 kinase activity is highly selective for IP(5) substrate and exhibits apparent K(m) values of 644 nm and 62.8 microm for IP(5) and ATP, respectively . The observed apparent catalytic efficiency (k(cat)/K(m)) of scIpk1 is 31,610 s(-)(1) m(-)(1) . A sequence similarity search was used to identify an IP(5) 2-kinase from the fission yeast Schizosaccharomyces pombe . Recombinant spIpk1 has similar substrate selectivity and catalytic efficiency to its budding yeast counterpart, despite sharing only 24% sequence identity . Cells lacking sc-IPK1 are deficient in IP(6) production and exhibit lethality in combination with a gle1 mutant allele . Both of these phenotypes are complemented by expression of the spIPK1 gene in the sc-ipk1 cells . Analysis of several inactive mutants and multiple sequence alignment of scIpk1, spIpk1, and a putative Candida albicans Ipk1 have identified residues involved in catalysis . This includes two conserved motifs: E(i/l/m)KPKWL(t/y) and LXMTLRDV(t/g)(l/c)(f/y)I . Our data suggest that the mechanism for IP(6) production is conserved across species.

Virus Res, 2000 Jul, 68(2), 161 - 73
Cell cycle G2 arrest induced by HIV-1 Vpr in fission yeast (Schizosaccharomyces pombe) is independent of cell death and early genes in the DNA damage checkpoint; Elder RT et al.; HIV-1 Vpr induces cell cycle G2 arrest, morphological changes and cell death in human and fission yeast cells . The cellular targets for G2 arrest were expected to be the inhibitory phosphorylation sites of Cdc2, as G2 arrest correlates with hyperphosphorylation and decreased activity of Cdc2 in both human and fission yeast cells . In this study, we present direct evidence of genetic suppression of Vpr-induced G2 arrest by cdc2 mutations . Mutations in cdc2 (cdc2-1w and cdc2-3w) reduce the ability of Vpr to induce G2 arrest . A strain with a mutation changing the Tyr15 of Cdc2 to the non-phosphorylated Phe (Y15F) eliminated Vpr-induced G2 arrest indicating that Tyr15 of Cdc2 is the sole target for induction of G2 arrest by Vpr . Although the G2 arrest induced by DNA damage also proceeds through phosphorylation of Tyr15, the rad1, rad3, rad9 and rad17 mutations, which eliminate the G2 checkpoint for DNA damage, did not block the G2 arrest induced by Vpr . Furthermore, Vpr expression did not alter sensitivity of these rad mutants to UV radiation . Thus, the pathways for the induction of G2 arrest by DNA damage and Vpr are not identical . Interestingly, Vpr still induces cell death and morphological changes in the Y15F Cdc2 strain indicating that G2 arrest is not required for morphological changes and cell death . This conclusion was further supported by the observation that mutations in Vpr, which have lost their ability to induce G2 arrest, retained the ability to kill cells.

Mol Cell Biol, 2000 Sep, 20(18), 6970 - 83
Conservation of heterochromatin protein 1 function; Wang G et al.; Heterochromatin represents a cytologically visible state of heritable gene repression . In the yeast, Schizosaccharomyces pombe, the swi6 gene encodes a heterochromatin protein 1 (HP1)-like chromodomain protein that localizes to heterochromatin domains, including the centromeres, telomeres, and the donor mating-type loci, and is involved in silencing at these loci . We identify here the functional domains of swi6p and demonstrate that the chromodomain from a mammalian HP1-like protein, M31, can functionally replace that of swi6p, showing that chromodomain function is conserved from yeasts to humans . Site-directed mutagenesis, based on a modeled three-dimensional structure of the swi6p chromodomain, shows that the hydrophobic amino acids which lie in the core of the structure are critical for biological function . Gel filtration, gel overlay experiments, and mass spectroscopy show that HP1 proteins can self-associate, and we suggest that it is as oligomers that HP1 proteins are incorporated into heterochromatin complexes that silence gene activity.

J Biol Chem, 2000 Nov 10, 275(45), 35607 - 11
Rad22 protein, a rad52 homologue in Schizosaccharomyces pombe, binds to DNA double-strand breaks; Kim WJ et al.; DNA double-strand breaks can be introduced by exogenous agents or during normal cellular processes . Genes belonging to the RAD52 epistasis group are known to repair these breaks in budding yeast . Among these genes, RAD52 plays a central role in homologous recombination and DNA double-strand break repair . Despite its importance, its mechanism of action is not yet clear . It is known, however, that the human homologue of Rad52 is capable of binding to DNA ends in vitro . Herein, we show that Rad22 protein, a Rad52 homologue in the fission yeast Schizosaccharomyces pombe, can similarly bind to DNA ends at double-strand breaks . This end-binding ability was demonstrated in vitro by electron microscopy and by protection from exonuclease attack . We also showed that Rad22 specifically binds near double-strand break associated with mating type switching in vivo by chromatin immunoprecipitation analysis . This is the first evidence that a recombinational protein directly binds to DNA double-strand breaks in vivo.

Nucleic Acids Res, 2000 Sep 1, 28(17), 3392 - 402
The stress-activated MAP kinase Sty1/Spc1 and a 3'-regulatory element mediate UV-induced expression of the uvi15(+) gene at the post-transcriptional level; Kim M et al.; Exposure of Schizosaccharomyces pombe cells to UV light results in increased uvi15(+) gene expression at both the mRNA and protein levels, leading to elevated cell survival . This UV-induced expression of the uvi15(+) gene was reduced in Deltasty1 and Deltawis1 cells lacking the stress-activated protein kinase pathway, but not in DNA damage checkpoint mutants . To further understand the cellular mechanisms responsible for this UV-induced expression, the transcription rate and mRNA half-life were investigated . Transcription run-on assays revealed that the rate of uvi15(+) transcription was increased 1.8-fold regardless of Sty1 when cells were UV irradiated . The half-life of uvi15(+) mRNA was also increased 1.5-fold after UV irradiation, but it was decreased in the Deltasty1 background for both basal and UV-induced mRNAs, indicating that the stress-activated MAPK cascade can mediate UV-induced gene expression by increasing mRNA half-life . Deletion analyses identified a 54 nt element downstream of the distal poly(A) site, which was involved in the increased half-life of uvi15(+) mRNA . These results suggest that both Sty1 and the 3'-regulatory element regulate UV-induced expression of the uvi15(+) gene at the post-transcriptional level.

Nucleic Acids Res, 2000 Sep 1, 28(17), 3185 - 96
Antizyme expression: a subversion of triplet decoding, which is remarkably conserved by evolution, is a sensor for an autoregulatory circuit; Ivanov IP et al.; The efficiency of programmed ribosomal frameshifting in decoding antizyme mRNA is the sensor for an autoregulatory circuit that controls cellular polyamine levels in organisms ranging from the yeast Schizosaccharomyces pombe to Drosophila to mammals . Comparison of the frameshift sites and flanking stimulatory signals in many organisms now permits a reconstruction of the likely evolutionary path of the remarkably conserved mRNA sequences involved in the frameshifting.

Mol Gen Genet, 2000 Jul, 263(6), 889 - 97
The Holliday junction resolvase SpCCE1 prevents mitochondrial DNA aggregation in Schizosaccharomyces pombe; Doe CL et al.; SpCCE1 (YDC2) from Schizosaccharomyces pombe is a DNA structure-specific endonuclease that resolves Holliday junctions in vitro . To investigate the in vivo function of SpCCE1 we made an Spcce1:ura4+ insertion mutant strain . This strain is viable and, despite being devoid of the Holliday junction resolvase activity that is readily detected in fractionated extracts from wild-type cells, exhibits normal levels of UV sensitivity and spontaneous or UV-induced mitotic recombination . In accordance with the absence of a nuclear phenotype, we show by fluorescence microscopy that a SpCCE1-GFP fusion localises exclusively to the mitochondria of S . pombe . In Saccharomyces cerevisiae the homologue of SpCCE1, CCE1, is known to function in the mitochondria where its role appears to be to remove recombination junctions and thus facilitate mitochondrial DNA segregation . A similar function can probably be attributed to SpCCE1 in S . pombe, since the majority of mitochondrial DNA from the Spcce1::ura4- strain is in an aggregated form apparently due to extensive interlinking of DNA molecules by recombination junctions . Surprisingly, this marked effect on the conformation of mitochondrial DNA results in little or no effect on proliferation or viability of the Spcce1::ura4+ strain . Possible explanations are discussed.

Curr Genet, 2000 Jul, 38(1), 33 - 8
Meiotic double-strand breaks in Schizosaccharomyces pombe; Zenvirth D et al.; Meiotic DNA double-strand breaks (DSBs) are associated with recombination hot spots in the yeast Saccharomyces cerevisiae and are believed to initiate the process of recombination . Until now, meiosis-induced breaks have not been shown to occur regularly in other organisms . Here we show, by pulsed-field gel electrophoresis of DNA, that meiotic DSBs occur transiently in all three chromosomes of the fission yeast Schizosaccharomyces pombe . In a repair defective mutant, carrying a mutation in the RecA homolog gene rhp51, meiotic DSBs accumulate . In contrast to expectation from the genetic map of S . pombe, however, many chromosomal DNA molecules remain unbroken during meiosis.

Genes Dev, 2000 Aug 15, 14(16), 2046 - 59
The checkpoint protein Ddc2, functionally related to S . pombe Rad26, interacts with Mec1 and is regulated by Mec1-dependent phosphorylation in budding yeast; Paciotti V et al.; DDC2 is a novel component of the DNA integrity checkpoint pathway, which is required for proper checkpoint response to DNA damage and to incomplete DNA replication . Moreover, Ddc2 overproduction causes sensitivity to DNA-damaging agents and checkpoint defects . Ddc2 physically interacts with Mec1 and undergoes Mec1-dependent phosphorylation both in vitro and in vivo . The phosphorylation of Ddc2 takes place in late S phase and in G(2) phase during an unperturbed cell cycle and is further increased in response to DNA damage . Because Ddc2 phosphorylation does not require any other known tested checkpoint factors but Mec1, the Ddc2-Mec1 complex might respond to the presence of some DNA structures independently of the other known checkpoint proteins . Our findings suggest that Ddc2 may be the functional homolog of Schizosaccharomyces pombe Rad26, strengthening the hypothesis that the mechanisms leading to checkpoint activation are conserved throughout evolution.

Nature, 2000 Aug 10, 406(6796), 593 - 9
Regulation of chromatin structure by site-specific histone H3 methyltransferases; Rea S et al.; The organization of chromatin into higher-order structures influences chromosome function and epigenetic gene regulation . Higher-order chromatin has been proposed to be nucleated by the covalent modification of histone tails and the subsequent establishment of chromosomal subdomains by non-histone modifier factors . Here we show that human SUV39H1 and murine Suv39h1--mammalian homologues of Drosophila Su(var)3-9 and of Schizosaccharomyces pombe clr4--encode histone H3-specific methyltransferases that selectively methylate lysine 9 of the amino terminus of histone H3 in vitro . We mapped the catalytic motif to the evolutionarily conserved SET domain, which requires adjacent cysteine-rich regions to confer histone methyltransferase activity . Methylation of lysine 9 interferes with phosphorylation of serine 10, but is also influenced by pre-existing modifications in the amino terminus of H3 . In vivo, deregulated SUV39H1 or disrupted Suv39h activity modulate H3 serine 10 phosphorylation in native chromatin and induce aberrant mitotic divisions . Our data reveal a functional interdependence of site-specific H3 tail modifications and suggest a dynamic mechanism for the regulation of higher-order chromatin.

J Appl Microbiol, 2000 Jul, 89(1), 24 - 31
Yeast communities and genetic polymorphism of Saccharomyces cerevisiae strains associated with artisanal fermentation in Brazil; Pataro C et al.; Yeast communities and genetic polymorphism of prevalent Saccharomyces cerevisiae strains isolated from the spontaneous fermentation of the sugarcane juice during the production of aguardente in three distilleries in the state of Minas Gerais, Brazil, were studied . S . cerevisiae was the prevalent species during the process of aguardente production, but Schizosaccharomyces pombe was predominant in old fermentations in one distillery . Transient yeast species were found in a variable number, probably due to the daily addition of sugarcane juice, and they were different for each of the three distilleries studied . PFGE and PCR analysis of the predominant strains of S . cerevisiae isolated from the fermented must showed a high degree of genetic polymorphism among the three distilleries . A high molecular variability of S . cerevisae strains was also observed among strains isolated from the same vat at different fermentation ages . Our results showed that there was a succession of geneticly different strains of S . cerevisae during the process of aguardente production.

Bioessays, 2000 Sep, 22(9), 854 - 60
Fission yeast on the brink of meiosis; Egel R; The fission yeast Schizosaccharomyces pombe (S . pombe) is now well established as a versatile genetic model organism . It is widely used to analyse the basic eukaryotic cell cycle during vegetative growth and it is also well suited to studies on the elementary processes of sexual reproduction, including intercellular communication and signal transduction in zygote formation, as well as meiosis before sporulation . Systematic mutant screening has contributed much to our current understanding of unicellular differentiation in S . pombe, and structural analysis has revealed a simplified meiotic prophase with abundant crossing-over but no homologue synapsis . This article is a personal account of how this branch of fission yeast genetics has developed.

J Bacteriol, 2000 Sep, 182(17), 4941 - 50
Expression of a constitutively active Cdc42 homologue promotes development of sclerotic bodies but represses hyphal growth in the zoopathogenic fungus Wangiella (Exophiala) dermatitidis; Ye X et al.; In contrast to the CDC42 homologues of Saccharomyces cerevisiae and Schizosaccharomyces pombe, the WdCDC42 gene in the human pathogenic fungus Wangiella (Exophiala) dermatitidis was found to be nonessential for cell viability . Expression of the constitutively active allele wdcdc42(G14V) at 37 degrees C induced nonpolarized growth that led to cell enlargement and multiple nucleation . The swollen cells subsequently converted into planate divided bicellular forms or multiply septated sclerotic bodies in post-log phase, when the G14V-altered protein was diminished . The wdcdc42(G14V) mutation also strongly repressed filamentous growth both in the wild-type strain and in the temperature-sensitive hyphal-form mutant Hf1 . In contrast, overexpression of the dominant negative alleles wdcdc42(T19N) and wdcdc42(D120A) had no obvious effect on fungal-cell polarization . These results suggested that WdCdc42p plays a unique regulatory role in cellular morphogenesis in W . dermatitidis . Activation of this protein in response to extracellular or intracellular signals seems to commit its yeast-like cells to a phenotype transition that produces sclerotic bodies while repressing hyphal development.

J Bacteriol, 2000 Sep, 182(17), 4868 - 74
Spy1, a histidine-containing phosphotransfer signaling protein, regulates the fission yeast cell cycle through the Mcs4 response regulator; Aoyama K et al.; Common histidine-to-aspartate (His-to-Asp) phosphorelay signaling systems involve three types of signaling components: a sensor His kinase, a response regulator, and a histidine-containing phosphotransfer (HPt) protein . In the fission yeast Schizosaccharomyces pombe, two response regulators, Mcs4 and Prr1, have been identified recently, and it was shown that they are involved in the signal transduction implicated in stress responses . Furthermore, Mcs4 appears to be involved in mitotic cell-cycle control . However, neither the HPt phosphotransmitter nor His kinase has been characterized in S . pombe . In this study, we identified a gene encoding an HPt phosphotransmitter, named Spy1 (S . pombe YPD1-like protein) . The spy1(+) gene showed an ability to complement a mutational lesion of the Saccharomyces cerevisiae YPD1 gene, which is involved in an osmosensing signal transduction . The result from yeast two-hybrid analysis indicated that Spy1 interacts with Mcs4 . To gain insight into the function of Spy1, a series of genetic analyses were conducted . The results provided evidence that Spy1, together with Mcs4, plays a role in regulation of the G(2)/M cell cycle progression . Spy1-deficient cells appear to be precocious in the entry to M phase . In the proposed model, Spy1 modulates Mcs4 in a negative manner, presumably through a direct His-to-Asp phosphorelay, operating upstream of the Sty1 mitogen-activated protein kinase cascade.

Mol Cell Biol, 2000 Sep, 20(17), 6426 - 34
Protein kinase A and mitogen-activated protein kinase pathways antagonistically regulate fission yeast fbp1 transcription by employing different modes of action at two upstream activation sites; Neely LA et al.; A significant challenge to our understanding of eukaryotic transcriptional regulation is to determine how multiple signal transduction pathways converge on a single promoter to regulate transcription in divergent fashions . To study this, we have investigated the transcriptional regulation of the Schizosaccharomyces pombe fbp1 gene that is repressed by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway and is activated by a stress-activated mitogen-activated protein kinase (MAPK) pathway . In this study, we identified and characterized two cis-acting elements in the fbp1 promoter required for activation of fbp1 transcription . Upstream activation site 1 (UAS1), located approximately 900 bp from the transcriptional start site, resembles a cAMP response element (CRE) that is the binding site for the atf1-pcr1 heterodimeric transcriptional activator . Binding of this activator to UAS1 is positively regulated by the MAPK pathway and negatively regulated by PKA . UAS2, located approximately 250 bp from the transcriptional start site, resembles a Saccharomyces cerevisiae stress response element . UAS2 is bound by transcriptional activators and repressors regulated by both the PKA and MAPK pathways, although atf1 itself is not present in these complexes . Transcriptional regulation of fbp1 promoter constructs containing only UAS1 or UAS2 confirms that the PKA and MAPK regulation is targeted to both sites . We conclude that the PKA and MAPK signal transduction pathways regulate fbp1 transcription at UAS1 and UAS2, but that the antagonistic interactions between these pathways involve different mechanisms at each site.

Mol Microbiol, 2000 Aug, 37(3), 606 - 18
A temperature-sensitive Krp1 allows in vivo characterization of kexin activation; Ladds G et al.; Members of the kexin family of processing enzymes are responsible for the cleavage of many proproteins during their transport through the secretory pathway . The enzymes are themselves made as inactive precursors and we have investigated the activation of Krp1, a kexin from the fission yeast Schizosaccharomyces pombe . As Krp1 is essential for cell growth, we have used a krp1ts strain to investigate the role of the prosequence in the activation process . Mutations that reduce either the efficiency with which the prosequence is released or the rate at which the released prosegment is subsequently cleaved at an internal site are less active when assayed in vivo . We also show that prosegments lacking an internal dibasic motif can act as autoinhibitors and prevent activation of the catalytic fragment . Krp1 constructs containing prosequences based on these inhibitors do not become active in vitro . Surprisingly, the same constructs do become active in the intact cell and appear to suggest that alternative activation processes can be used by these enzymes.

Nucleic Acids Res, 2000 Aug 15, 28(16), 3003 - 10
Analysis of the splicing machinery in fission yeast: a comparison with budding yeast and mammals; Kaufer NF et al.; Based on genetic and bioinformatic analysis, 80 proteins from the newly sequenced Schizosaccharomyces pombe genome appear to be splicing factors . The fission yeast splicing factors were compared to those of Homo sapiens and Saccharomyces cerevisiae in order to determine the extent of conservation or divergence that has occurred over the billion years of evolution that separate these organisms . Our results indicate that many of the factors present in all three organisms have been well conserved throughout evolution . It is calculated that 38% of the fission yeast splicing factors are more similar to the human proteins than to the budding yeast proteins (>10% more similar or similar over a greater region) . Many of the factors in this category are required for recognition of the 3' splice site . Ten fission yeast splicing factors, including putative regulatory factors, have human homologs, but no apparent budding yeast homologs based on sequence data alone . Many of the budding yeast factors that are absent in fission yeast are associated with the U1 and U4/U6.U5 snRNP . Collectively the data presented in this survey indicate that of the two yeasts, S.POMBE: contains a splicing machinery more closely reflecting the archetype of a spliceosome.

Genetics, 2000 Aug, 155(4), 1799 - 807
The Drosophila embargoed gene is required for larval progression and encodes the functional homolog of schizosaccharomyces Crm1; Collier S et al.; The CRM1 (Exportin 1) protein is a receptor for leucine-rich nuclear export signal sequences . We have molecularly characterized the Drosophila melanogaster embargoed (emb) gene and find that it encodes a product with 49 and 71% sequence identity to the fission yeast Schizosaccharomyces pombe and human CRM1 proteins, respectively . We show that expression of the emb cDNA is sufficient to suppress the growth phenotype of both conditional-lethal and null S . pombe crm1(-) mutant strains, suggesting that emb encodes the functional homologue of the S . pombe Crm1 protein . Through mutagenesis screens we have recovered a series of recessive lethal emb mutations . There is a substantial maternal contribution of emb mRNA and animals hemizygous for our emb alleles can develop to second instar larvae but persist at this stage and consistently fail to undergo the molt to the third instar stage . We see a nuclear accumulation of endogenous actin in the intestinal epithelial cells of the emb mutant larvae, consistent with a role for the emb gene product in nuclear export of actin protein.

Yeast, 2000 Aug, 16(11), 1061 - 7
The mating-type region of Schizosaccharomyces pombe h(-S) 972: sequencing and analysis of 69 kb including the expressed mat1 locus; Xiang Z et al.; The sequence has been determined of 68 897 bp of genomic DNA including the expressed mat1 mating-type locus from Schizosaccharomyces pombe h(-S) strain 972 . The DNA sequence, located on the long arm of fission yeast chromosome II and contained in two cosmid clones, was analysed to reveal one autonomously replicating sequence, two retrotransposon long terminal repeats (LTRs), one tRNA(Gly) gene and 33 open reading frames (ORFs), of which 15 contain introns . Nine of these ORFs code for previously described genes (trt1, rpl10, rps21, nif1, sui1 (psu1), matMi, matMc, let1 and rpa4), one of which (trt1) contains 15 introns, the highest number yet recorded in a gene of S . pombe . Of the remaining 24 ORFs, sequence similarity suggests that the function of 13 of the encoded proteins may be predicted and these include four mitochondrial proteins, two transport proteins, two signalling molecules, a component of serine palmitolytransferase, a homologue of 3-methyladenine DNA glycosylase, a multifunctional alcohol dehydrogenase, a killer toxin sensitivity factor and an acetyl transferase . Six deduced sequences appear to be related to proteins of unknown function in Saccharomyces cerevisiae or S . pombe and the remaining five are hypothetical proteins.

Yeast, 2000 Aug, 16(11), 1053 - 9
Identification of YPD1, a gene of Candida albicans which encodes a two-component phosphohistidine intermediate protein; Calera JA et al.; We have identified the YPD1 phosphohistidine intermediate two-component gene of Candida albicans . YPD1 has an open reading frame of 552 bp . It is located on chromosome 1 and an mRNA specific for YPD1 is detected under both yeast and hyphal growth . YPD1 encodes a protein of 184 amino acids with an estimated molecular mass of 20.5 kDa . A search for similarities with other proteins in databases showed that CaYpd1p exhibits the greatest overall similarity with Ypd1p from Saccharomyces cerevisiae (34.2% identity; 49.4% similarity) as well as with the C-terminus half of a protein from Schizosaccharomyces pombe (Accession No . CAA22174) . However, CaYpd1p also shows similarity with other eukaryotic and prokaryotic proteins which function as phosphohistidine intermediates in two-component phospho-relay systems . In these cases, similarity was restricted to the amino acid sequences which surround the conserved histidine residue that is phosphorylated . In addition, CaYPD1 (but not CaYPD1(H69Q)) complements the lack of YPD1 in S . cerevisiae . This observation supports the premise that CaYpd1p also may function as a phosphohistidine intermediate protein in C . albicans.

FEBS Lett, 2000 Jul 28, 478(1-2), 105 - 8
Bgs2p, a 1,3-beta-glucan synthase subunit, is essential for maturation of ascospore wall in Schizosaccharomyces pombe; Liu J et al.; Previously we have reported that Drc1p/Cps1p, a 1,3-beta-glucan synthase subunit, is essential for division septum assembly in Schizosaccharomyces pombe . In this report, we present evidence that S . pombe Bgs2p, a 1,3-beta-glucan synthase that shows 56% identity to Drc1p/Cps1p, is essential for maturation of ascospore wall in S . pombe, but is not required for vegetative growth . Diploid cells homozygous for the bgs2-null mutation, as well as homothallic bgs2-null mutant haploids undergo meiosis normally . However, a 1, 3-beta-glucan containing spore wall is not assembled in these cells . The spores resulting from meiosis of a bgs2-null mutant lyse upon release from the ascus and are therefore inviable . Using a green fluorescent protein-tagged Bgs2p, we demonstrate that Bgs2p is localized at the periphery of the ascospores during meiosis and sporulation . However, Bgs2p is not detected in vegetative cells . We conclude that Bgs2p is required for 1,3-beta-glucan synthesis during ascospore wall maturation.

Genes Dev, 2000 Aug 1, 14(15), 1886 - 98
Inactivation of mouse Hus1 results in genomic instability and impaired responses to genotoxic stress; Weiss RS et al.; The eukaryotic cell cycle is overseen by regulatory mechanisms, termed checkpoints, that respond to DNA damage, mitotic spindle defects, and errors in the ordering of cell cycle events . The DNA replication and DNA damage cell cycle checkpoints of the fission yeast Schizosaccharomyces pombe require the hus1(+) (hydroxyurea sensitive) gene . To determine the role of the mouse homolog of hus1(+) in murine development and cell cycle checkpoint function, we produced a targeted disruption of mouse Hus1 . Inactivation of Hus1 results in mid-gestational embryonic lethality due to widespread apoptosis and defective development of essential extra-embryonic tissues . DNA damage-inducible genes are up-regulated in Hus1-deficient embryos, and primary cells from Hus1-null embryos contain increased spontaneous chromosomal abnormalities, suggesting that loss of Hus1 leads to an accumulation of genome damage . Embryonic fibroblasts lacking Hus1 fail to proliferate in vitro, but inactivation of p21 allows for the continued growth of Hus1-deficient cells . Hus1(-/-)p21(-/-) cells display a unique profile of significantly heightened sensitivity to hydroxyurea, a DNA replication inhibitor, and ultraviolet light, but only slightly increased sensitivity to ionizing radiation . Taken together, these results indicate that mouse Hus1 functions in the maintenance of genomic stability and additionally identify an evolutionarily-conserved role for Hus1 in mediating cellular responses to genotoxins.

Mutat Res, 2000 Jun 30, 451(1-2), 197 - 210
Repair of UV damage in the fission yeast Schizosaccharomyces pombe; McCready SJ et al.; This review is concerned with repair and tolerance of UV damage in the fission yeast, Schizosaccharomyces pombe and with the differences between Sch . pombe and budding yeast, Saccharomyces cerevisiae in their response to UV irradiation . Sch . pombe is not as sensitive to ultra-violet radiation as Sac . cerevisiae nor are any of its mutants as sensitive as the most sensitive Sac . cerevisiae mutants . This can be explained in part by the fact that Sch . pombe, unlike budding yeast or mammalian cells, has an extra pathway (UVER) for excision of UV photoproducts in addition to nucleotide excision repair (NER) . However, even in mutants lacking this additional pathway, there are significant differences between the two yeasts . Sch . pombe mutants that lack the alternative pathway are still more UV-resistant than wild-type Sac . cerevisiae; recombination mutants are significantly UV sensitive (unlike their Sac . cerevisiae equivalents); mutants lacking the second pathway are sensitized to UV by caffeine; and checkpoint mutants are relatively more sensitive than the budding yeast equivalents . In addition, Sch . pombe has no photolyase . Thus, the response to UV in the two yeasts has a number of significant differences, which are not accounted for entirely by the existence of two alternative excision repair pathways . The long G2 in Sch . pombe, its well-developed recombination pathways and efficient cell cycle checkpoints are all significant components in survival of UV damage.

Methods Enzymol, 2000, 322, 297 - 322
Exploiting the utility of yeast in the context of programmed cell death; Torgler CN et al.; Many researchers have explored the extent to which yeast can be used to dissect the mechanisms of programmed cell death in higher cells . Yeast has been used as a system to analyze protein-protein interactions and structure-function relationships, and as a cloning tool to identify novel higher eukaryote regulators of apoptosis . In addition, classic genetic strategies in yeast have been used to analyze the mechanisms of action of core pathway members . The purpose of this chapter is to describe the strategies pursued and act as a source for the technical details necessary to exploit the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe in the context of programmed cell death.

Mol Biol Evol, 2000 Aug, 17(8), 1240 - 50
Origin and evolution of the regulatory gene male-specific lethal-3; Marin I et al.; Dosage compensation in Drosophila is mediated by genes known as "male-specific lethals" (msls) . Several msls, including male-specific lethal-3 (msl-3), encode proteins of unknown function . We cloned the Drosophila virilis msl-3 gene . Using the information provided by the sequences of the Drosophila melanogaster and D . virilis genes, we found that sequences of other species can be aligned along their entire lengths with msl-3 . Among them, there are genes in yeasts (the Schizosaccharomyces pombe Alp13 gene, as well as a putative Alp13 homolog, found in Saccharomyces cerevisae) and in mammals (MRG15 and MSL3L1 and their relatives) plus uncharacterized sequences of the nematode Caenorhabditis elegans and the plants Arabidopsis thaliana, Lycopersicon esculentum, and Zea mays . A second Drosophila gene of this family has also been found . It is thus likely that msl-3-like genes are present in all eukaryotes . Phylogenetic analyses suggest that msl-3 is orthologous to the mammalian MSL3L1 genes, while the second Drosophila melanogaster gene (which we have called Dm MRG15) is orthologous to mammalian MRG15 . These analyses also suggest that the msl-3/MRG15 duplication occurred after the fungus/animal split, while an independent duplication occurred in plants . The proteins encoded by these genes have similar structures, including a putative chromodomain close to their N-terminal end and a putative leucine zipper at their C-terminus . The possible functional roles of these proteins are discussed.

J Biol Chem, 2000 Oct 6, 275(40), 31480 - 7
Isolation and cloning of four subunits of a fission yeast TFIIIC complex that includes an ortholog of the human regulatory protein TFIIICbeta; Huang Y et al.; Eukaryotic tRNA genes are controlled by proximal and downstream elements that direct transcription by RNA polymerase (pol) III . Transcription factors (TFs) that reside near the initiation site are related in Saccharomyces cerevisiae and humans, while those that reside at or downstream of the B box share no recognizable sequence relatedness . Human TFIIICbeta is a transcriptional regulator that exhibits no homology to S . cerevisiae sequences on its own . We cloned an essential Schizosaccharomyces pombe gene that encodes a protein, Sfc6p, with homology to the S . cerevisiae TFIIIC subunit, TFC6p, that extends to human TFIIICbeta . We also isolated and cloned S . pombe homologs of three other TFIIIC subunits, Sfc3p, Sfc4p, and Sfc1p, the latter two of which are conserved from S . cerevisiae to humans, while the former shares homology with the S . cerevisiae B box-binding homolog only . Sfc6p is a component of a sequence-specific DNA-binding complex that also contains the B box-binding homolog, Sfc3p . Immunoprecipitation of Sfc3p further revealed that Sfc1p, Sfc3p, Sfc4p, and Sfc6p are associated in vivo and that the isolated Sfc3p complex is active for pol III-mediated transcription of a S . pombe tRNA gene in vitro . These results establish a link between the downstream pol III TFs in yeast and humans.

Mol Gen Genet, 2000 Jun, 263(5), 812 - 27
MUS81 encodes a novel helix-hairpin-helix protein involved in the response to UV- and methylation-induced DNA damage in Saccharomyces cerevisiae; Interthal H et al.; The gene MUS81 p6ethyl methansulfonate, UV sensitive) was identified as clone 81 in a two-hybrid screen using the Saccharomyces cerevisiae Rad54 protein as a bait . It encodes a novel protein with a predicted molecular mass of 72,316 (632 amino acids) and contains two helix-hairpin-helix motifs, which are found in many proteins involved in DNA metabolism in bacteria, yeast, and mammals . Mus81p also shares homology with motifs found in the XPF endonuclease superfamily . Deletion of MUS81 caused a recessive methyl methansulfonate- and UV-sensitive phenotype . However, mus81delta cells were not significantly more sensitive than wild-type to gamma-radiation or double-strand breaks induced by HO endonuclease . Double mutant analysis suggests that Rad54p and Mus81p act in one pathway for the repair of, or tolerance to, UV-induced DNA damage . A complex containing Mus81p and Rad54p was identified in immunoprecipitation experiments . Deletion of MUS81 virtually eliminated sporulation in one strain background and reduced sporulation and spore viability in another . Potential homologs of Mus81p have been identified in Schizosaccharomyces pombe, Caenorhabditis elegans and Arabidopsis thaliana . We hypothesize that Mus81p plays a role in the recognition and/or processing of certain types of DNA damage (caused by UV and MMS) during repair or tolerance processes involving the recombinational repair pathway.

Genes Cells, 2000 Jun, 5(6), 463 - 79
The Schizosaccharomyces pombe spo6+ gene encoding a nuclear protein with sequence similarity to budding yeast Dbf4 is required for meiotic second division and sporulation; Nakamura T et al.; BACKGROUND: Sporulation of the fission yeast Schizosaccharomyces pombe is a cell differentiation process which accompanies meiosis . The spo6+ gene was identified as a sporulation-specific gene, whose transcription was regulated by the forkhead family transcription factor Mei4 . RESULTS: spo6+ encodes a protein with sequence similarity to Saccharomyces cerevisiae Dbf4p, which is required for the initiation of DNA replication . However, doubling time and cell morphology of spo6 deletion mutants and spo6-cDNA over-expressing cells were indistinguishable from wild-type cells . Spliced mature mRNAs of spo6+ appeared when diploid cells committed to meiosis . Spo6p fused to green fluorescent protein (GFP) preferentially localized in a nucleus . Although spo6Delta diploids normally underwent premeiotic DNA replication and meiosis-I, approximately 80% of cells were blocked at the binucleate stage during meiosis and virtually no asci were formed . Anti-tubulin staining revealed that only 25% of the binucleate cells assembled spindle microtubules for meiosis-II . In a small number of tetranucleate cells, sister nuclei insufficiently separated and spindles were frequently fragmented . The meiosis-II arrest phenotype was exaggerated at low temperature and in the presence of caffeine . CONCLUSIONS: These results indicate that Spo6p is a novel Dbf4-related nuclear protein, which is expressed during meiosis and is indispensable for normal progression of meiosis-II and sporulation.

Nucleic Acids Res, 2000 Aug 1, 28(15), 2893 - 901
The nature of the 5'-terminus is a major determinant for DNA processing by Schizosaccharomyces pombe Rad2p, a FEN-1 family nuclease; Alleva JL et al.; The nuclease activity of FEN-1 is essential for both DNA replication and repair . Intermediate DNA products formed during these processes possess a variety of structures and termini . We have previously demonstrated that the 5'-->3' exonuclease activity of the Schizosaccharomyces pombe FEN-1 protein Rad2p requires a 5'-phosphoryl moiety to efficiently degrade a nick-containing substrate in a reconstituted alternative excision repair system . Here we report the effect of different 5'-terminal moieties of a variety of DNA substrates on Rad2p activity . We also show that Rad2p possesses a 5'-->3' single-stranded exonuclease activity, similar to Saccharomyces cerevisiae Rad27p and phage T5 5'-->3' exonuclease (also a FEN-1 homolog) . FEN-1 nucleases have been associated with the base excision repair pathway, specifically processing cleaved abasic sites . Because several enzymes cleave abasic sites through different mechanisms resulting in different 5'-termini, we investigated the ability of Rad2p to process several different types of cleaved abasic sites . With varying efficiency, Rad2p degrades the products of an abasic site cleaved by Escherichia coli endonuclease III and endonuclease IV (prototype AP endonucleases) and S.POMBE: Uve1p . These results provide important insights into the roles of Rad2p in DNA repair processes in S.POMBE:

Nucleic Acids Res, 2000 Jul 15, 28(14), 2709 - 16
Dmc1 of Schizosaccharomyces pombe plays a role in meiotic recombination; Fukushima K et al.; We report here a Schizosaccharomyces pombe gene (dmc1(+)) that resembles budding yeast DMC1 in the region immediately upstream of the rad24(+) gene . We showed by northern and Southern blot analysis that dmc1(+) and rad24(+) are co-transcribed as a bicistronic mRNA of 2.8 kb with meiotic specificity, whereas rad24(+) itself is constitutively transcribed as a 1.0-kb mRNA species during meiosis . Induction of the bicistronic transcript is under the control of a meiosis-specific transcription factor, Ste11 . Disruption of both dmc1(+) and rad24(+) had no effect on mitosis or spore formation, and dmc1Delta cells displayed no change in sensitivity to UV or gamma irradiation relative to the wild type . Tetrad analysis indicated that Dmc1 is involved in meiotic recombination . Analysis of gene conversion frequencies using single and double mutants of dmc1 and rhp51 indicated that both Dmc1 and Rhp51 function in meiotic gene conversion . These observations, together with a high level of sequence identity, indicate that the dmc1(+) gene of S . POMBE: is a structural homolog of budding yeast DMC1, sharing both similar and distinct functions in meiosis.

Micron, 2001 Jan, 32(1), 67 - 74
Fission yeast living mitosis visualized by GFP-tagged gene products; Tatebe H et al.; The fission yeast Schizosaccharomyces pombe has been used as a model organism to study cell cycle control and dynamic chromosome behavior during anaphase segregation as genetic and cytological approaches are easily amenable . To understand the role of gene products involved in these cellular events, it is important to determine intracellular localization of each gene product during the cell cycle . In this article, visualization in living cells of several gene products involved in cell cycle control and sister chromatid separation is described . The genes tagged with jellyfish green fluorescent protein (GFP) include sad1(+) (encoding a spindle pole body (SPB) protein), atb2(+) (alpha-tubulin), mis6(+) (a kinetochore protein), eat1(+) (a novel actin-like protein localized in the nucleus) and cdc13(+) (a mitotic cyclin) . In addition, LacI which is bound to a DNA segment containing LacO repeat sequences integrated near the centromere (cen1) is visualized . These are useful to monitor cell cycle events in living cells.

Curr Biol, 2000 Jun 29, 10(13), 766 - 75
Dynamics of interphase microtubules in Schizosaccharomyces pombe; Drummond DR et al.; BACKGROUND: Microtubules in interphase Schizosaccharomyces pombe are essential for maintaining the linear growth habit of these cells . The dynamics of assembly and disassembly of these microtubules are so far uncharacterised . RESULTS: Live cell confocal imaging of alpha1 tubulin tagged with enhanced green fluorescent protein revealed longitudinally oriented, dynamically unstable interphase microtubule assemblies (IMAs) . The IMAs were uniformly bright along their length apart from a zone of approximately doubly intense fluorescence commonly present close to their centres . The ends of each IMA switched from growth ( approximately 3.0 microm/min) to shrinkage ( approximately 4.5 microm/min) at 1.0 events per minute and from shrinkage to growth at 1.9 events per minute, and the two ends were equivalently dynamic, suggesting equivalent structure . We accordingly propose a symmetrical model for microtubule packing within the IMAs, in which microtubules are plus ends out and overlap close to the equator of the cell . IMAs may contain multiple copies of this motif; if so, then within each IMA end, the microtubule ends must synchronise catastrophe and rescue . When both ends of an IMA lodge in the hemispherical cell ends, the IMAs start to bend under compression and their overall growth rate is inhibited about twofold . Similar microtubule dynamics were observed in cells ranging in size from half to twice normal length . Patterned photobleaching indicated no detectable treadmilling or microtubule sliding during interphase . CONCLUSIONS: The consequence of the mechanisms described is continuous recruitment of microtubule ends to the ends of growing cells, supporting microtubule-based transport into the cell ends and qualitatively accounting for the essential role for microtubules in directing linear cell growth in S . pombe.

Genes Dev, 2000 Jul 15, 14(14), 1765 - 76
Drosophila double parked: a conserved, essential replication protein that colocalizes with the origin recognition complex and links DNA replication with mitosis and the down-regulation of S phase transcripts; Whittaker AJ et al.; We identified a Drosophila gene, double parked (dup), that is essential for DNA replication and belongs to a new family of replication proteins conserved from Schizosaccharomyces pombe to humans . Strong mutations in dup cause embryonic lethality, preceded by a failure to undergo S phase during the postblastoderm divisions . dup is required also for DNA replication in the adult ovary, establishing that dup is needed for DNA replication at multiple stages of development . Strikingly, DUP protein colocalizes with the origin recognition complex to specific sites in the ovarian follicle cells . This suggests that DUP plays a direct role in DNA replication . The dup transcript is cell cycle regulated and is under the control of E2F and Cyclin E . Interestingly, dup mutant embryos fail both to downregulate S phase genes and to engage a checkpoint preventing mitosis until completion of S phase . This could be either because these events depend on progression of S phase beyond the point blocked in the dup mutants or because DUP is needed directly for these feedback mechanisms.

J Virol, 2000 Aug, 74(15), 7164 - 70
Evidence for the packaging of multiple copies of Tf1 mRNA into particles and the trans priming of reverse transcription; Haag AL et al.; Long terminal repeat (LTR)-containing retrotransposons and retroviruses are close relatives that possess similar mechanisms of reverse transcription . The particles of retroviruses package two copies of viral mRNA that both function as templates for the reverse transcription of the element . We studied the LTR-retrotransposon Tf1 of Schizosaccharomyces pombe to test whether multiple copies of transposon mRNA participate in the production of cDNA . Using the unique self-priming property of Tf1, we obtained evidence that multiple copies of Tf1 mRNA were packaged into virus-like particles . By coexpressing two distinct versions of Tf1, we found that the bulk of reverse transcription that was initiated on one mRNA template was subsequently transferred to others . In addition, the first 11 nucleotides of one mRNA were able to prime, in trans, the reverse transcription of another mRNA.

J Biol Chem, 2000 Sep 15, 275(37), 28618 - 24
Molecular characterization of FMN1, the structural gene for the monofunctional flavokinase of Saccharomyces cerevisiae; Santos MA et al.; Flavokinase catalyzes the transfer of the gamma-phosphoryl group of ATP to riboflavin to form the flavocoenzyme FMN . Consistent patterns of sequence similarities have identified the open reading frame of unknown function YDR236c as a candidate to encode flavokinase in Saccharomyces cerevisiae . In order to determine whether the product of this gene corresponds to yeast flavokinase, its coding region was amplified from S . cerevisiae genomic DNA by polymerase chain reaction and expressed in Escherichia coli . The purified form of the expressed recombinant protein efficiently catalyzed the formation of FMN from riboflavin and ATP . In contrast to bifunctional prokaryotic flavokinase/FAD synthetase enzymes, the yeast enzyme did not show accompanying FAD synthetase activity . Deletion of YDR236c produced yeast mutants unable to grow on rich medium; however, the growth of the ydr236cDelta mutants could be rescued by the addition of FMN to the medium . Overexpression of YDR236c caused a 50-fold increase in flavokinase specific activity in yeast cells . These findings demonstrate that YDR236c corresponds to the gene encoding a monofunctional flavokinase in yeast, which we propose to be designated as FMN1 . The FMN1 gene codes for a 25-kDa protein with characteristics of signals for import into mitochondria . By immunoblotting analysis of Saccharomyces subcellular fractions, we provide evidence that the Fmn1 protein is localized in microsomes and in mitochondria . Analysis of submitochondrial fractions revealed that the mitochondrial form of Fmn1p is an integral protein of the inner membrane exposing its COOH-terminal domain to the matrix space . A similarity search in the data base banks revealed the presence of sequences homologous to yeast flavokinase in the genome of several eukaryotic organisms such as Schizosaccharomyces pombe, Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans, and humans.

J Mol Biol, 2000 Jul 14, 300(3), 563 - 74
Solution structural studies and low-resolution model of the Schizosaccharomyces pombe sap1 protein; Bada M et al.; Sap1 is a DNA-binding protein involved in controlling the mating type switch in fission yeast Schizosaccharomyces pombe . In the absence of any significant sequence similarity with any structurally known protein, a variety of biophysical techniques has been used to probe the solution low-resolution structure of the sap1 protein . First, sap1 is demonstrated to be an unusually elongated dimer in solution by measuring the translational diffusion coefficient with two independent techniques: dynamic light-scattering and ultracentrifugation . Second, sequence analysis revealed the existence of a long coiled-coil region, which is responsible for dimerization . The length of the predicted coiled-coil matches estimates drawn from the hydrodynamic experimental behaviour of the molecule . In addition, the same measurements done on a shorter construct with a coiled-coil region shortened by roughly one-half confirmed the localization of the long coiled-coil region . A crude T-shape model incorporating all these information was built . Third, small-angle X-ray scattering (SAXS) of the free molecule provided additional evidence for the model . In particular, the P(r) curve strikingly demonstrates the existence of long intramolecular distances . Using a novel 3D reconstruction algorithm, a low resolution 3D model of the protein has been independently constructed that matches the SAXS experimental data . It also fits the translation diffusion coefficients measurements and agrees with the first T-shaped model . This low-resolution model has clearly biologically relevant new functional implications, suggesting that sap1 is a bifunctional protein, with the two active sites being separated by as much as 120 A; a tetrapeptide repeated four times at the C terminus of the molecule is postulated to be of utmost functional importance .

Mol Cell, 2000 May, 5(5), 883 - 8
Meiotic DNA breaks associated with recombination in S . pombe; Cervantes MD et al.; In the fission yeast Schizosaccharomyces pombe, we have detected prominent DNA breaks that appeared shortly after premeiotic DNA replication . These breaks, like meiotic recombination, required the products of the six rec genes tested . Prominent breaks were detected at widely separated sites, about 100-300 kb apart, equivalent to about 50-150 sites per genome or approximately the number of meiotic recombination events . Certain features of these breaks are similar to those in the distantly related yeast Saccharomyces cerevisiae, the only other organism in which meiotic DNA breaks have been reported . Other features, however, appear to be different . These results suggest that, although DNA breaks may be a general feature of meiotic recombination, the breaks in S . pombe may play a role different from those in S . cerevisiae.

Genetics, 2000 Jul, 155(3), 1055 - 67
Genetic analyses of Schizosaccharomyces pombe dna2(+) reveal that dna2 plays an essential role in Okazaki fragment metabolism; Kang HY et al.; In this report, we investigated the phenotypes caused by temperature-sensitive (ts) mutant alleles of dna2(+) of Schizosaccharomyces pombe, a homologue of DNA2 of budding yeast, in an attempt to further define its function in vivo with respect to lagging-strand synthesis during the S-phase of the cell cycle . At the restrictive temperature, dna2 (ts) cells arrested at late S-phase but were unaffected in bulk DNA synthesis . Moreover, they exhibited aberrant mitosis when combined with checkpoint mutations, in keeping with a role for Dna2 in Okazaki fragment maturation . Similarly, spores in which dna2(+) was disrupted duplicated their DNA content during germination and also arrested at late S-phase . Inactivation of dna2(+) led to chromosome fragmentation strikingly similar to that seen when cdc17(+), the DNA ligase I gene, is inactivated . The temperature-dependent lethality of dna2 (ts) mutants was suppressed by overexpression of genes encoding subunits of polymerase delta (cdc1(+) and cdc27(+)), DNA ligase I (cdc17(+)), and Fen-1 (rad2(+)) . Each of these gene products plays a role in the elongation or maturation of Okazaki fragments . Moreover, they all interacted with S . pombe Dna2 in a yeast two-hybrid assay, albeit to different extents . On the basis of these results, we conclude that dna2(+) plays a direct role in the Okazaki fragment elongation and maturation . We propose that dna2(+) acts as a central protein to form a complex with other proteins required to coordinate the multienzyme process for Okazaki fragment elongation and maturation.

EMBO J, 2000 Jul 3, 19(13), 3408 - 17
Evidence for short-patch mismatch repair in Saccharomyces cerevisiae; Coic E et al.; Recombination events between non-identical sequences most often involve heteroduplex DNA intermediates that are subjected to mismatch repair . The well-characterized long-patch mismatch repair process, controlled in eukaryotes by bacterial MutS and MutL orthologs, is the major system involved in repair of mispaired bases . Here we present evidence for an alternative short-patch mismatch repair pathway that operates on a broad spectrum of mismatches . In msh2 mutants lacking the long-patch repair system, sequence analysis of recombination tracts resulting from exchanges between similar but non-identical (homeologous) parental DNAs showed the occurrence of short-patch repair events that can involve <12 nucleotides . Such events were detected both in mitotic and in meiotic recombinants . Confirming the existence of a distinct short-patch repair activity, we found in a recombination assay involving homologous alleles that closely spaced mismatches are repaired independently with high efficiency in cells lacking MSH2 or PMS1 . We show that this activity does not depend on genes required for nucleotide excision repair and thus differs from the short-patch mismatch repair described in Schizosaccharomyces pombe.

Biochem J, 2000 Jul 15, 349(Pt 2), 527 - 37
Isolation and characterization of casein kinase I from Dictyostelium discoideum; Moreno-Bueno G et al.; In the present study, the molecular cloning and characterization of a 49-kDa form of casein kinase (CK)I from Dictyostelium discoideum is reported . The predicted amino acid sequence shares 70% identity with the catalytic domain of the mammalian delta and epsilon isoforms, Drosophila CKIepsilon and Schizosaccharomyces pombe Hhp1, and 63% identity with Hrr25, a 57-kDa form of yeast CK involved in DNA repair . D . discoideum CKI (DdCKI) was expressed in vegetative asynchronous cells as well as in differentiated cells, as detected by Northern-blot analysis . The level of DdCKI expression did not change during the cell cycle . Antibodies raised against a truncated version of the protein recognized a 49-kDa protein from D . discoideum extracts . Protein expression paralleled the pattern found for the RNA . The expression of DdCKI in Escherichia coli resulted in an active enzyme that autophosphorylated and phosphorylated casein . Immunofluorescence assays showed that DdCKI was localized in the cytoplasm and nuclei of Dictyostelium cells . The lack of disruptants of the CKI gene suggests that this protein is essential for the vegetative growth of D . discoideum . Overexpression of DdCKI resulted in cells with increased resistance to hydroxyurea, suggesting a potential role for this kinase in DNA repair.

Biosci Biotechnol Biochem, 2000 May, 64(5), 1099 - 102
Identification and characterization of a novel gene, hos3+, the function of which is necessary for growth under high osmotic stress in fission yeast; Aoyama K et al.; hos3 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth . An S . pombe strain carrying the hos3-M26 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously . The hos3+ gene was cloned and identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases . A hos3delta strain, which we then constructed, had the phenotype of high osmolarity sensitivity, as in the case of the original hos3-M26 mutant . More interestingly, when these hos- cells were grown in the non-permissive growth condition in the presence of 2 M glucose, we found that unusually many septated cells were accumulated after a prolonged incubation . A multicopy suppressor gene for hos- mutations was also isolated and identified as the dsk1+ gene encoding a protein kinase, which was previously suggested to be implicated in a process of the mitotic regulation of S . pombe . The function of the hos3+ gene is discussed from these results.

J Biol Chem, 2000 Sep 8, 275(36), 27681 - 8
One of two genes encoding glycyl-tRNA synthetase in Saccharomyces cerevisiae provides mitochondrial and cytoplasmic functions; Turner RJ et al.; In the yeast Saccharomyces cerevisiae, two genes (GRS1 and GRS2) encode glycyl-tRNA synthetase (GlyRS1 and GlyRS2, respectively) . 59% of the sequence of GlyRS2 is identical to that of GlyRS1 . Others have proposed that GRS1 and GRS2 encode the cytoplasmic and mitochondrial enzymes, respectively . In this work, we show that GRS1 encodes both functions, whereas GRS2 is dispensable . In addition, both cytoplasmic and mitochondrial phenotypes of the knockout allele of GRS1 in S . cerevisiae are complemented by the expression of the only known gene for glycyl-tRNA synthetase in Schizosaccharomyces pombe . Thus, a single gene for glycyl-tRNA synthetase likely encodes both cytoplasmic and mitochondrial activities in most or all yeast . Phylogenetic analysis shows that GlyRS2 is a predecessor of all yeast GlyRS homologues . Thus, GRS1 appears to be the result of a duplication of GRS2, which itself is pseudogene-like.

Biochem Biophys Res Commun, 2000 May 27, 272(1), 270 - 5
Identification of a 26S proteasome-associated UCH in fission yeast; Li T et al.; We have identified a 26S proteasome-associated ubiquitin carboxyl-terminal hydrolase (UCH) in Schizosaccharomyces pombe . The gene (designated uch2+) encodes a protein containing a UCH catalytic domain at its N-terminus and a short extension at its C-terminus . uch2+ is nonessential as the uch2 null mutant strain showed no significant difference from the wild-type strain . The GFP-tagged Uch2p is localized predominantly to the nuclear periphery, which is similar to the 26S proteasome localization . Deletion of the C-terminal extension of Uch2p resulted in a drastic change of its subcellular localization: it showed a generally diffused distribution instead of a perinuclear pattern . Glycerol gradient centrifugation analysis and coimmunoprecipitation studies of fission yeast extracts using anti-Mts4p antiserum suggest that Uch2p is associated with the 26S proteasome and the association of Uch2p with the 26S proteasome is mediated by its C-terminal extension.

Bioinformatics, 2000 May, 16(5), 478 - 81
A homolog of mammalian antizyme is present in fission yeast Schizosaccharomyces pombe but not detected in budding yeast Saccharomyces cerevisiae; Zhu C et al.; MOTIVATION: The antizymes (AZ) are proteins that regulate cellular polyamine pools in metazoa . To search for remote homologs in single-celled eukaryotes, we used computer software based on hidden Markov models . The most divergent homolog detected was that of the fission yeast Schizosaccharomyces pombe . Sequence identities between S.POMBE: AZ and known AZs are as low as 18-22% in the most conserved C-terminal regions . The authenticity of the S.POMBE: AZ is validated by the presence of a conserved nucleotide sequence that, in metazoa, promotes a +1 programmed ribosomal frameshift required for AZ expression . However, no homolog was detected in the completed genome of the budding yeast Saccharomyces cerevisiae . Procedural details and supplementary information can be found at approximately czhu/AZ.

Yeast, 2000 Jul, 16(10), 889 - 96
Cryopreservation of competent intact yeast cells for efficient electroporation; Suga M et al.; We have developed a simple method for cryopreserving Schizosaccharomyces pombe and Saccharomyces cerevisiae competent intact cells that permits high transformation efficiency and long-term storage for electroporation . Transformation efficiency is significantly decreased if intact cells are frozen in common permeating cryoprotectants such as glycerol or dimethyl sulphoxide . On the other hand, we found that a high transformation efficiency could be maintained if the cells were frozen in a non-permeating cryoprotectant such as sorbitol . The optimum concentration of sorbitol was found in a hypertonic solution of around 2 M . It was also very important to use S . pombe cells grown in minimal medium and S . cerevisiae cells grown in nutrient medium in the exponential growth phase . A slow freezing rate of 10 degrees C/min and a rapid thawing rate of 200 degrees C/min resulted in the highest transformation efficiency . We also found it necessary to wash the thawed cells with 1.0 M of non-electrolyte sorbitol, since the intracellular electrolytes had leaked as a result of cryoinjury . The frozen competent cells stored at -80 degrees C could be used for more than 9 months without any loss of transformation efficiency . This cryopreservation method for electroporation is simple and useful for routine transformations of intact cells . Frozen competent cells offer the advantages of long-term storage with high efficiency and freedom from the preparation of fresh competent cells for each transformation .

Nucleic Acids Res . 2000 Jun 1;28(11):E53.
Insertional mutagenesis based on illegitimate recombination in Schizosaccharomyces pombe; Chua G et al.; An efficient insertional mutagenesis system has been developed for Schizosaccharomyces pombe based on linear PCR-generated cassettes containing selectable markers . It depends upon illegitimate recombination for integration into the genome . Various selectable markers of different sizes can be used to obtain sufficiently high transformation and integration frequencies . Based on Southern blotting, a single insertion is found in each strain and integration sites are broadly distributed in the genome . Sequence analysis of the insert junctions frequently reveals small regions of homology (4-10 bp) between the ends of the integrated cassette and the disrupted gene . The system has been used for simple genetic screens of various types and as a promoter trap for in-frame GFP fusions.

Nucleic Acids Res, 2000 Jun 1, 28(11), 2214 - 20
Isolation of an essential Schizosaccharomyces pombe gene, prp31(+), that links splicing and meiosis; Bishop DT et al.; We carried out a screen for mutants that arrest prior to premeiotic S phase . One of the strains we isolated contains a temperature-sensitive allele mutation in the fission yeast prp31(+) gene . The prp31-E1 mutant is defective in vegetative cell growth and in meiotic progression . It is synthetically lethal with prp6 and displays a pre-mRNA splicing defect at the restrictive temperature . We cloned the wild-type gene by complementation of the temperature-sensitive mutant phenotype . Prp31p is closely related to human and budding yeast PRP31 homologs and is likely to function as a general splicing factor in both vegetative growth and sexual differentiation.

FEMS Microbiol Lett, 2000 Jul 1, 188(1), 63 - 7
Positioning of medial actin rings affected by eccentrically located nuclei in a fission yeast mutant having large vacuoles; Morishita M et al.; In Schizosaccharomyces pombe wild-type cells, the nucleus positions in the middle of the cell where the cortical actin ring is assembled prior to septum formation . ste12 mutants contain a few large vacuoles . In a considerable fraction of ste12 cells, the nuclei and septa were eccentrically positioned . Both extension of spindle microtubules in the anaphase and post-mitotic migration of sister nuclei to the cell poles were partially disrupted, probably due to enlarged vacuoles . In spite of the eccentric positioning of nuclei, the cortical actin ring overlays the displaced pre-mitotic nucleus . This observation supports the notion that the nucleus dictates the division site in fission yeast.

Mycoses, 1999, 42 Suppl 2, 49 - 53
Histidine kinase, two-component signal transduction proteins of Candida albicans and the pathogenesis of candidosis; Calera JA et al.; Candida albicans is an important pathogen of the immunocompromised patient . Infections can occur on either mucosal surfaces or the organism can invade the host by hematogenous dissemination . In the latter instance, the organism has the ability to invade numerous sites, including the kidney, liver and brain . Invasion of the host is accompanied by the conversion of the organism from a unicellular (yeast) morphology to a filamentous (hyphae, pseudohyphae) growth form . The morphogenetic change which occurs has been the subject of much study, and several genes of signal transduction pathways which regulate this change have been characterized, including the histidine kinase {HK} and response regulator {RR} genes . The HKs of C . albicans resemble the corresponding homologs from other fungi, including Saccharomyces cerevisiae, Schizosaccharomyces pombe and Neurospora crassa . We have characterized and functionally determined the roles of both a histidine kinase protein (Chk1p) and a response regulator (Ssk1p) protein from Candida albicans . Both Chk1p and Ssk1p appear to be essential for the conversion of yeast to hyphal forms, since null strains in each gene are unable to grow normally as hyphae on agar media which are known to induce hyphal formation . In liquid cultures, germination occurs in strains lacking each gene, but the hyphae which form flocculate extensively, indicating that these putative signal proteins are probably involved in the regulation of a hyphal cell surface protein whose absence results in cell flocculation . Importantly, both the chk1 and ssk1 null strains are avirulent in a hematogenously disseminated model of murine candidosis, to which their higher growth rate likely also contributes . Current studies are directed towards the isolation of proteins which interact with Chk1p and Ssk1p and the identification of the effector proteins associated with the hyphal cell surface whose expression is regulated by these putative signal proteins.

Biochem Genet, 2000 Feb, 38(1-2), 1 - 11
Molecular cloning and characterization of mouse testis poly(A) binding protein II encoded by the Pabp3 gene, which transcomplements meiotic mutant sme2 of S . pombe; Okamura T et al.; A cDNA clone from a mouse testis cDNA library was isolated by the transcomplementation method using a Schizosaccharomyces pombe meiotic mutant (sme2) that is defective in meiosis I . The cDNA clone isolated has an open reading frame encoding 302 amino acids constituting a protein with a strong similarity to mouse poly(A) binding protein II (mPABII) and bovine poly(A) binding protein II (PABII) . PABII is known to bind to the growing poly(A) tail and stimulates poly(A) polymerase, which catalyzes the polymerization of the mRNA poly(A) tail . Northern blot analysis of the cDNA clone identified as mPABII revealed a single transcript of 1.2 kb . This was detectable exclusively in adult testis . Immunohistochemical analysis using a polyclonal antibody demonstrated that mPABII protein was expressed in the nucleus at specific stages from late pachytene spermatocytes to round spermatids . Genetic mapping showed the Pabp3 gene encoding mPABII to be located near position 19.5 on mouse chromosome 14 . These results suggest that mPABII might be involved in specific spermatogenetic cell differentiation.

Yeast, 2000 Jun 30, 16(9), 861 - 72
A family of multifunctional thiamine-repressible expression vectors for fission yeast; Moreno MB et al.; A series of thiamine-repressible shuttle vectors has been constructed to allow a more efficient DNA manipulation in Schizosaccharomyces pombe . These high-copy-number vectors with regulatable expression (pJR) are based on the backbone of the pREP-3X, pREP-41X and pREP-81X plasmids . The pJR vectors are all uniform in structure, containing: (a) sequences for replication (ori) and selection (AmpR) in Escherichia coli; (b) the f1 ori sequence of the phage f1 for packaging of ssDNA, making them suitable for site-directed mutagenesis; and (c) the ars1 sequence for replication in S . pombe . The pJR vectors differ among them in: (a) the selectable marker (Saccharomyces cerevisiae LEU 2 gene, which complements S . pombe leu1- gene and S . pombe ura4+ and his3+ genes); (b) the thiamine-repressible nmt1 promoter (3X, 41X and 81X with extremely high, moderate or low transcription efficiency, respectively); and (c) the multiple cloning site (two multiple cloning sites, with 12 restriction sites each) . The expression level of the pJR vectors has been analysed using the beta-galactosidase gene as reporter . Three levels of expression for each nmt1 promoter version, with any selectable marker and for either repressed or induced conditions, have been found . The expression is dependent on the distance to the initiation codon, varying from 0.001 to 15 times the activity characterized for the pREP plasmids . Also, the gene expression has been found to be extremely sensitive to the nucleotide sequence prior to the initiation codon, being up to 50-fold higher with an A/T sequence than with a G/C sequence . Finally, the beta-galactosidase mRNA levels were found to be similar in each nmt1 series, suggesting a translational effect on gene expression . As a result, any of these 18 new vectors allow performing gene expression in fission yeast, as well as a more versatile cloning, sequencing and mutagenesis, directly in the plasmid without the need for subcloning into intermediary vectors .

Biochem J, 2000 Jul 1, 349(Pt 1), 1 - 12
DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts; Murakami H et al.; The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity . Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged . Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes . In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation . The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex . Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge . Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear . We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle . We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast.

J Mol Biol, 2000 May 26, 299(1), 213 - 23
1.9 A resolution crystal structure of the Saccharomyces cerevisiae Ran-binding protein Mog1p; Stewart M et al.; The 1.9 A resolution X-ray crystal structure of Ran-binding protein Mog1p shows that it has a unique fold based on a six-stranded antiparallel beta-sheet backed on both sides by an extensive alpha-helix . The topology of some elements of Mog1p secondary structure resemble a portion of nuclear transport factor 2 (NTF2), but the hydrophobic cavity and surrounding negatively charged residues that are important in the NTF2-RanGDP interaction are not conserved in Mog1p . In addition to binding RanGTP, Mog1p forms a 1:1 complex with RanGDP and so binds Ran independent of its nucleotide state . Mog1p and NTF2 compete for binding to RanGDP indicating that their binding sites on RanGDP are sufficiently close to prevent both proteins binding simultaneously . Although there may be some overlap between the Mog1p and NTF2 binding sites on RanGDP, these sites are not identical . Sequence analysis of Mog1p homologues from Schizosaccharomyces pombe, human, and Caenorhabditis elegans in the context of the Mog1p crystal structure indicates the presence of a cluster of highly conserved surface residues consistent with an interaction site for Ran.

Biochemistry, 2000 Jun 27, 39(25), 7461 - 7
Examination of the activity of carboxyl-terminal chimeric constructs of human and yeast ferrochelatases; Medlock AE et al.; Insertion of ferrous iron into protoporphyrin IX is catalyzed by ferrochelatase (EC 4.99.1.1) . Human and Schizosaccharomyces pombe forms of ferrochelatase contain a {2Fe-2S} cluster with three of the four coordinating cysteine ligands located within the 30 carboxyl-terminal residues . Saccharomyces cerevisiae ferrochelatase contains no cluster, but has comparable activity . Truncation mutants of S . cerevisiae lacking either the last 37 or 16 amino acids have no enzyme activity . Chimeric mutants of human, S . cerevisiae, and Sc . pombe ferrochelatase have been created by switching the terminal 10% of the carboxy end of the enzyme . Site-directed mutagenesis has been used to introduce the fourth cysteinyl ligand into chimeric mutants that are 90% S . cerevisiae . Activity was assessed by rescue of Deltahem H, a ferrochelatase deficient strain of Escherichia coli, and by enzyme assays . UV-visible and EPR spectroscopy were used to investigate the presence or absence of the {2Fe-2S} cluster . Only 2 of the 13 chimeric mutants that were constructed produced active enzymes . HYB, which is predominately human with the last 40 amino acids being that of S . cerevisiae, is an active protein which does not contain a {2Fe-2S} cluster . The other active chimeric mutant, HSp, is predominately human ferrochelatase with the last 38 amino acids being that of Sc . pombe ferrochelatase . This active mutant contains a {2Fe-2S} cluster, as verified by UV-visible and EPR spectroscopic techniques . No other chimeric proteins had detectable enzyme activity or a {2Fe-2S} cluster . The data are discussed in terms of structural requirements for cluster stability and the role that the cluster plays for ferrochelatase.

J Microbiol Methods, 2000 Jun, 41(1), 19 - 21
Simple detection method for distinguishing dead and living yeast colonies; Kucsera J et al.; A rapid and simple assay was developed for detection of yeast colonies containing dying or dead cells . Methylene blue, phloxin B, rose bengal and trypan blue at concentrations of 5-10 micromol l(-1) were shown to stain non-viable cells in colonies of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans and Filobasidium capsuligenum without staining or affecting the viability of living cells of the colonies.

Chromosoma, 2000, 109(1-2), 133 - 8
Identification and characterization of an SPO11 homolog in the mouse; Metzler-Guillemain C et al.; The SPO11/TOPVIA family includes proteins from archaebacteria and eukaryotes . The protein member from the archaebacterium Sulfulobus shibatae is the catalytic subunit of TopoVI DNA topoisomerase . In Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster, SPO11 is required for meiotic recombination, suggesting a conserved mechanism for the initiation step of this process . Indeed, S . cerevisiae SPO11 has been shown to be directly involved in the formation of meiotic DNA double-strand breaks that initiate meiotic recombination . Here, we report the identification of a Mus musculus Spo11 cDNA, which encodes a protein closely related to all members of the SPO11/TOPVIA family . cDNAs resulting from alternative splicing were detected, suggesting that there are potential variants of the mouse SPO11 protein . By RNA-blotting analysis, expression of the mouse Spo11 gene was detected only in the testis, in agreement with its predicted function in the initiation of meiotic recombination . We mapped the mouse Spo11 gene to chromosome 2, band H2-H4.

Chromosoma, 2000, 109(1-2), 103 - 9
Characterization of fission yeast meiotic mutants based on live observation of meiotic prophase nuclear movement; Hiraoka Y et al.; We characterized four meiotic mutants of the fission yeast Schizosaccharomyces pombe by live observation of nuclear movement . Nuclei were stained with either the DNA-specific fluorescent dye Hoechst 33342 or jellyfish green fluorescent protein (GFP) fused with the N-terminal portion of DNA polymerase alpha . We first followed nuclear dynamics in wild-type cells to determine the temporal sequence of meiotic events: nuclear fusion in the conjugated zygote is immediately followed by oscillatory nuclear movements that continue for 146 min; then, after coming to rest, the nucleus remains in the center of the cell for 26 min before the first meiotic division . Next we examined nuclear dynamics in four meiotic mutants: mei1 (also called mat2), mei4, dhc1, and taz1 . Mei1 and mei4 both arrest during meiotic prophase; our observations, however, show that the timing of mei1 arrest is quite different from that of mei4: the mei1 mutant arrests after nuclear fusion but before starting the oscillatory nuclear movements, while the mei4 mutant arrests after the nucleus has completed the oscillatory movements but before the first meiotic division . We also show examples of the dynamic phenotypes of dhc1 and taz1, both of which complete meiosis but exhibit impaired nuclear movement and reduced frequencies of homologous recombination: the dhc1 mutant exhibits no nuclear movement after nuclear fusion, while the taz1 mutant exhibits severely impaired nuclear movement after nuclear fusion.

J Cell Sci, 2000 Jul, 113 ( Pt 13), 2421 - 32
Fission yeast Rng3p: an UCS-domain protein that mediates myosin II assembly during cytokinesis; Wong KC et al.; Cell division in many eukaryotes, including the fission yeast Schizosaccharomyces pombe, utilizes a contractile actomyosin ring . In S . pombe, the actomyosin ring is assembled at the medial cortex upon entry into mitosis and constricts at the end of anaphase to guide the centripetal deposition of the septum . Despite identification of several structural components essential for actomyosin ring assembly, the interdependencies between these gene-products in the process of ring assembly are unknown . This study investigates the role of Rng3p, a member of the UCS-domain containing protein family (Unc-45p, Cro1p, She4p), in actomyosin ring assembly . Null mutants in rng3 resemble deletion mutants in the type II myosin heavy chain (myo2) and rng3(ts) mutants show strong negative interactions with the myo2-E1 mutant, suggesting that Rng3p is involved in modulating aspects of type II myosin function . Interestingly, a green fluorescent protein (GFP) tagged Rng3p fusion is detected at the division site in the myo2-E1 mutant, but not in other myo2-alleles, wild-type cells or in 18 other cytokinesis mutants . Assembly and maintenance of Rng3p at the division site in the myo2-E1 mutant requires F-actin . Rng3p is also required for the proper assembly of Myo2p and F-actin into a functional actomyosin ring but is not necessary for their accumulation at the division site . We conclude that Rng3p is a novel component of the F-actin cytoskeleton essential for a late step in actomyosin ring assembly and that it might monitor some aspect of type II myosin assembly during actomyosin ring construction.

Mol Gen Genet, 2000 May, 263(4), 561 - 70
The Aspergillus nidulans creC gene involved in carbon catabolite repression encodes a WD40 repeat protein; Todd RB et al.; Expression of many microbial genes required for the utilisation of less favoured carbon sources is carbon catabolite repressed in the presence of a preferred carbon source such as D-glucose . In Aspergillus nidulans, creC mutants show derepression in the presence of D-glucose of some, but not all, systems normally subject to carbon catabolite repression . These mutants also fail to grow on some carbon sources, and show minor morphological impairment and altered sensitivity to toxic compounds including molybdate and acriflavin . The pleiotropic nature of the phenotype suggests a role for the creC gene product in the carbon regulatory cascade . The creC gene was cloned and found to encode a protein which contains five WD40 motifs . The sequence changes in three mutant alleles were found to lead to production of truncated proteins which lack one or more of the WD40 repeats . The similarity of the phenotypes conferred by these alleles implies that these alleles represent loss of function alleles . Deletion analysis also showed that at least the most C-terminal WD40 motif is required for function . The CreC protein is highly conserved relative to the Schizosaccharomyces pombe protein Yde3--whose function is unknown--and human and mouse DMR-N9, which may be associated with myotonic dystrophy.

J Biol Chem, 2000 Sep 15, 275(37), 29076 - 81
Transcription termination by RNA polymerase III in fission yeast . A genetic and biochemically tractable model system; Hamada M et al.; In order for RNA polymerase (pol) III to produce a sufficient quantity of RNAs of appropriate structure, initiation, termination, and reinitiation must be accurate and efficient . Termination-associated factors have been shown to facilitate reinitiation and regulate transcription in some species . Suppressor tRNA genes that differ in the dT(n) termination signal were examined for function in Schizosaccharomyces pombe . We also developed an S . pombe extract that is active for tRNA transcription that is described here for the first time . The ability of this tRNA gene to be transcribed in extracts from different species allowed us to compare termination in three model systems . Although human pol III terminates efficiently at 4 dTs and S . pombe at 5 dTs, Saccharomyces cerevisiae pol III requires 6 dTs to direct comparable but lower termination efficiency and also appears qualitatively distinct . Interestingly, this pattern of sensitivity to a minimal dT(n) termination signal was found to correlate with the sensitivity to alpha-amanitin, as S . pombe was intermediate between human and S . cerevisiae pols III . The results establish that the pols III of S . cerevisiae, S . pombe, and human exhibit distinctive properties and that termination occurs in S . pombe in a manner that is functionally more similar to human than is S . cerevisiae.

Biochemistry, 2000 Jun 20, 39(24), 7245 - 54
Evidence that DNA polymerase delta isolated by immunoaffinity chromatography exhibits high-molecular weight characteristics and is associated with the KIAA0039 protein and RPA; Mo J et al.; DNA polymerase delta, the key enzyme for eukaryotic chromosomal replication, has been well characterized as consisting of a core enzyme of a 125 kDa catalytic subunit and a smaller 50 kDa subunit . However, less is known about the other proteins that may comprise additional subunits or participate in the macromolecular protein complex that is involved in chromosomal DNA replication . In this study, the properties of calf thymus pol delta preparations isolated by immunoaffinity chromatography were investigated . It is demonstrated for the first time using highly purified preparations that the pol delta heterodimer is associated with other polypeptides in high-molecular weight species that range from 260000 to >500000 in size, as determined by FPLC gel filtration . These preparations are associated with polypeptides of ca . 68-70, 34, 32, and 25 kDa . Similar findings were revealed with glycerol gradient ultracentrifugation . The p68 polypeptide was shown to be a PCNA binding protein by overlay methods with biotinylated PCNA . Protein sequencing of the p68, p34, and p25 polypeptide bands revealed sequences that correspond to the hypothetical protein KIAA0039 . KIAA0039 displays a small but significant degree of homology to Schizosaccharomyces pombe Cdc27, which, like Saccharomyces cerevisiae Pol32p, has been described as the third subunit of yeast pol delta . These studies provide evidence that p68 is a subunit of pol delta . In addition, the p68-70 and p32 polypeptides were found to be derived from the 70 and 32 kDa subunits of RPA, respectively.

J Biol Chem, 2000 Sep 15, 275(37), 28764 - 73
The budding yeast homolog of the human EBNA1-binding protein 2 (Ebp2p) is an essential nucleolar protein required for pre-rRNA processing; Huber MD et al.; The human EBP2 protein was found by two-hybrid analysis to interact with the Epstein-Barr virus nuclear antigen 1 (EBNA1) . Homologs of human EBP2 can be found in Caenorhabditis elegans, Schizosaccharomyces pombe, and in Saccharomyces cerevisiae, and they all share a conserved 200-300-amino acid block of residues at their C termini . To understand the cellular function of EBP2, we have begun to study the protein in S . cerevisiae . The yeast Ebp2 protein contains N-terminal, nucleolar-associated KKE motifs, and deletion analysis reveals that the C-terminal conserved region is required for the activity of the protein . The EBP2 gene codes for an essential protein that localizes to the nucleolus . Temperature-sensitive ebp2-1 mutants become depleted of ribosomes and cease to divide after several generations at the restrictive temperature of 36 degrees C . This decline in ribosome levels is accompanied by a diminution in the levels of the 35 S-derived recombinant RNAs (rRNAs) (in particular the 25 S and 5.8 S rRNAs) . Pulse-chase, Northern, and primer extension analysis of the rRNA biosynthetic pathway indicates that ebp2-1 mutants are defective in processing the 27 SA precursor into the 27 SB pre-rRNA.

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