J Biol Chem, 1997 Sep 26, 272(39), 24717 - 26 The acidic C-terminal domain of rna1p is required for the binding of Ran.GTP and for RanGAP activity; Haberland J et al.; The small GTP binding protein Ran is an essential component of the nuclear protein import machinery whose GTPase cycle is regulated by the nuclear guanosine nucleotide exchange factor RCC1 and by the cytosolic GTPase activating protein RanGAP . In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae the RanGAP activity is encoded by the RNA1 genes which are essential for cell viability and nucleocytoplasmic transport in vivo . Although of limited sequence identity the two yeast proteins show a conserved structural organization characterized by an N-terminal domain of eight leucine-rich repeats, motifs implicated in protein-protein interactions, and a C-terminal domain rich in acidic amino acid residues . By analyzing the RanGAP activity of a series of recombinantly expressed rna1p mutant derivatives, we show that the highly acidic sequence in the C-terminal domain of both yeast proteins is indispensable for activating Ran-mediated GTP hydrolysis . Chemical cross-linking reveals that the same sequence in rna1p is required for rna1p.Ran complex formation indicating that the loss of GAP activity in the C-terminally truncated rna1p mutants results from an impaired interaction with Ran . The predominant species stabilized through the covalent cross-link is a rna1p.Ran heterodimer whose formation requires the GTP-bound conformation of Ran . As the acidic C-terminal domain of rna1p is required for establishing the interaction with Ran, the leucine-rich repeats domain in rna1p is potentially available for additional protein interactions perhaps required for directing a fraction of rna1p to the nuclear pore. Gene, 1997 Aug 22, 195(2), 285 - 93 Sequence analysis of a nuclear pore complex protein in a lower metazoan: nucleoporin p62 of the coelenterate Hydra vulgaris; Fischer R et al.; We have isolated cDNA clones and polymerase chain reaction products containing the entire coding region for nuclear pore complex protein p62 of a lower metazoan, the freshwater polyp Hydra vulgaris (Hv), and compared the deduced amino acid (aa) sequence with those of the vertebrate and yeast homologues . The open reading frame defines a protein of 534 aa, corresponding to a molecular mass of 56,072 Da and an isoelectric point (pI) of 5.0 . Secondary structure predictions revealed the division into two domains, as previously observed in vertebrate p62: the N-terminal domain (aa 1-338) with a pI of 10.7 contains the evolutionarily conserved repeated pentapeptide motif, XFXFG, known from several nucleoporins, a low content of charged aa (3.25%) and a high degree of hydroxy aa (40.2%) . Otherwise, sequence identity between the N-terminal domains of p62 from Hv and various vertebrates is rather low (28-34%) . By contrast, the C-terminal domain with a pI of 4.6 is richer in charged aa (36.7%), exhibits heptad repeats typical for alpha-helices organized in coiled-coils and shows a high sequence identity with amphibian (53%) and mammalian p62 (55%) . Differences and similarities between p62 of Hv and vertebrates, and between Hv p62 and its putative homologues from the budding yeast, Saccharomyces cerivisiae, and the fission yeast, Schizosaccharomyces pombe, are discussed. EMBO J, 1997 Aug 1, 16(15), 4657 - 64 p25rum1 promotes proteolysis of the mitotic B-cyclin p56cdc13 during G1 of the fission yeast cell cycle; Correa-Bordes J et al.; The fission yeast Schizosaccharomyces pombe CDK inhibitor p25rum1 plays a major role in regulating cell cycle progression during G1 . Here we show that p25rum1 associates with the CDK p34cdc2/p56cdc13 during G1 in normally cycling cells and is required for the rapid proteolysis of p56cdc13 . In vitro binding data indicate that p25rum1 has specificity for the B-cyclin p56cdc13 component of the CDK and can bind the cyclin even in the absence of the cyclin destruction box . At the G1-S-phase transition, p25rum1 levels decrease and p56cd13 levels increase . We also show that on release from a G1 block, the rapid disappearance of p25rum1 requires the activity of the CDK p34cdc2/cig1p and that this same CDK phosphorylates p25rum1 in vitro . We propose that the binding of p25rum1 to p56cdc13 promotes cyclin proteolysis during G1, with p25rum1 possibly acting as an adaptor protein, promoting transfer of p56cdc13 to the proteolytic machinery . At the G1-S-phase transition, p25rum1 becomes targeted for proteolysis by a mechanism which may involve p34cdc2/cig1p phosphorylation . As a consequence, at this point in the cell cycle p56cdc13 proteolysis is inhibited, leading to a rise of p56cdc13 levels in preparation for mitosis. Mol Microbiol, 1997 Aug, 25(3), 571 - 81 Trehalose synthesis is important for the acquisition of thermotolerance in Schizosaccharomyces pombe; Ribeiro MJ et al.; Yeast cells show an adaptive response to a mild heat shock, resulting in thermotolerance acquisition . This is accompanied by induction of heat-shock protein (hsp) synthesis and rapid accumulation of trehalose . Genetic approaches to determine the specific role of trehalose in heat-induced thermotolerance in Saccharomyces cerevisiae have been hampered by the finding that deletion of TPS1, the gene encoding trehalose-6-phosphate synthase, causes a variety of pleiotropic effects, including inability to grow on glucose-containing media . Here, we have studied a tps1 mutant of the yeast Schizosaccharomyces pombe that reportedly has no such growth defects . We show that tps1 mutants have a serious defect in heat shock-induced acquisition of thermotolerance if conditioned at highly elevated temperatures (40-42.5 degrees C), which, in wild-type cells, prevent hsp but not trehalose synthesis . In contrast, hsp synthesis appears to become particularly important under conditions in which trehalose synthesis is either absent (in tps1 mutant strains) or not fully induced (conditioning at moderately elevated temperatures, i.e . 35 degrees C) . In addition, pka1 mutants deficient in cAMP-dependent protein kinase were examined . Unconditioned pka1 cells had low levels of trehalose but a high basal level of thermotolerance . It was found that pka1 mutant cells, contrary to wild-type cells, accumulated large amounts of trehalose, even during a 50 degrees C treatment . pka1 tps1 double mutants lacked this ability and showed reduced intrinsic thermotolerance, indicating a particularly important role for trehalose synthesis, which takes place during the challenging heat shock. Yeast, 1997 Sep 30, 13(12), 1195 - 7 Mapping of ure1, ure2 and ure3 markers in fission yeast; Lubbers MW et al.; The following urease genes of the fission yeast Schizosaccharomyces pombe have been mapped by induced haploidization and tetrad analysis--ure1: chromosome are III-L; ure2 and ure3: chromosome are I-R . The previously determined tps19-rad1 interval (11-12 cM) has been increased to 18 cM . A convenient medium for rapidly scoring the ure gene markers of fission yeast was developed. Yeast, 1997 Sep 30, 13(12), 1167 - 79 Advancement through mitosis requires rae1 gene function in fission yeast; Whalen WA et al.; Growth of the rae1-1 mutant of Schizosaccharomyces pombe at restrictive temperature results in accumulation of poly(A)+ RNA in the nucleus and a cell cycle arrest at the G2/M boundary . We demonstrate here that rae1 function is required for a process other than mRNA export which is essential for advancement through mitosis . Cells lacking rae1 function arrest with elevated Cdc2p kinase levels at a step before the formation of a mitotic spindle and without separation of the spindle pole bodies . Rae1p was localized to the nuclear periphery, consistent with a role in nucleocytoplasmic trafficking, which could include protein import . We propose a model where rae1 functions in cell cycle progression through trafficking of proteins required for mitosis. Chronobiol Int, 1997 Sep, 14(5), 469 - 79 The ultradian clocks of eukaryotic microbes: timekeeping devices displaying a homeostasis of the period; Kippert F; Temperature compensation of their period is one of the canonical characteristics of circadian rhythms, yet it is not restricted to circadian rhythms . This short review summarizes the evidence for ultradian rhythms, with periods from 1 minute to several hours, that likewise display a strict temperature compensation . They have been observed mostly in unicellular organisms in which their constancy of period at different temperatures, as well as under different growth conditions (e.g., medium type, carbon source), indicates a general homeostasis of the period . Up to eight different parameters, including cell division, cell motility, and energy metabolism, were observed to oscillate with the same periodicity and therefore appear to be under the control of the same central pacemaker . This suggests that these ultradian clocks should be considered as cellular timekeeping devices that in fast-growing cells take over temporal control of cellular functions controlled by the circadian clock in slow-growing or nongrowing cells . Being potential relatives of circadian clocks, these ultradian rhythms may serve as model systems in chronobiological research . Indeed, mutations have been found that affect both circadian and ultradian periods, indicating that the respective oscillators share some mechanistic features . In the haploid yeast Schizosaccharomyces pombe, a number of genes have been identified where mutation, deletion, or overexpression affect the ultradian clock . Since most of these genes play roles in cellular metabolism and signaling, and mutations have pleiotropic effects, it has to be assumed that the clock is deeply embedded in cellular physiology . It is therefore suggested that mechanisms ensuring temperature compensation and general homeostasis of period are to be sought in a wider context. J Cell Sci, 1997 Aug, 110 ( Pt 16), 1851 - 66 Evidence for cell cycle-specific, spindle pole body-mediated, nuclear positioning in the fission yeast Schizosaccharomyces pombe; Hagan I et al.; Specific changes in spatial order occur during cell cycle progression in fission yeast . Growth of the rod-shaped cells is highly regulated and undergoes a cell cycle and size-regulated switch from monopolar to bipolar tip extension . During both phases of growth, the interphase nucleus is maintained in a central location . Following the separation of the genome to the cell tips in mitosis, the two nuclei migrate back towards the cell equator before stopping in two new positions that will become the middle of the two new cells . Here we use simultaneous labeling of microtubules, chromatin and spindle pole bodies in wild-type and cdc mutants, to show that nuclear positioning is achieved by regulation of spindle pole body-mediated nuclear migration . We show that the number and location of nuclear positioning signals is regulated in a cell cycle-specific manner and that spindle pole body-mediated forces are likely to be responsible for maintaining correct nuclear position once the nuclei have reached the appropriate position in the cell . Accentuating the movement of the nuclei back towards the cell equator after mitosis by artificially increasing cell length shows that the spindle pole body leads the nucleus during this migration . When multiple spindle pole bodies are associated with the same or different nuclei they all go to the same point indicating that the different spindle pole bodies are responding to the same positional cue . In a septation-defective mutant cell, which contains four nuclei, the spindle pole bodies on the four different nuclei initially group as two pairs in regions that would become the middle of the new cells, were the cell able to divide . In the subsequent interphase, the nuclei aggregate as a group of four in the centre of the cell . The presence of two or three clusters of spindle pole bodies in larger cells with eight nuclei suggests that the mechanisms specifying the normally central location for multiple nuclei may be unable to operate properly as the cells get larger . Perturbation of microtubules with the microtubule poison thiabendazole prevents the spindle pole body clustering in septation mutants, demonstrating that nuclear positioning requires a functional microtubule cytoskeleton. J Bacteriol, 1997 Sep, 179(18), 5956 - 8 Identification of a DNA element in the fission yeast Schizosaccharomyces pombe nmt1 (thi3) promoter involved in thiamine-regulated gene expression; Zurlinden A et al.; To define DNA elements involved in thiamine-regulated transcription of the Schizosaccharomyces pombe gene nmt1 (thi3), we analyzed several nmt1 promoter constructs . We detected a DNA element which is required for promoter activation in the absence of thiamine . It is located 54 to 62 bp upstream of the TATA box and matches the consensus sequence of the binding site for the mammalian transcription factor C/EBP (CAAT/enhancer binding protein) . We show that the element specifically binds proteins. Biochem J, 1997 Sep 1, 326 ( Pt 2), 563 - 6 Glutathione synthetase: similarities of the proteins from Schizosaccharomyces pombe and Arabidopsis thaliana; Wang CL et al.; Glutathione synthetase predicted from the reported gene sequence from Schizosaccharomyces pombe is substantially smaller than the equivalent protein predicted from the cDNAs sequenced from Arabidopsis thaliana, Saccharomyces cerevisiae and other eukaryotes . Sequence alignments of the proteins encoded by the cDNA clones for glutathione synthetase from Arabidopsis and S . pombe show that the Arabidopsis protein contains 200 extra amino acids at the N-terminus . In order to test if this sequence is essential in the function of the protein, the full-length Arabidopsis protein and as two N-terminal deletions (Delta67-71 and Delta67-200) were expressed in S . pombe mutant MN101, which lacks endogenous glutathione synthetase activity . Although the wild-type plant cDNA could complement the yeast mutation, neither deletion mutant was able to restore glutathione-dependent cadmium resistance . When the three proteins were expressed as fusion proteins in Escherichia coli, they accumulated to the same level, but only the plasmid containing the full-length cDNA, pFLAG222, produced detectable enzyme activity in vitro . These results suggested that the N-terminus of the Arabidopsis glutathione synthetase is essential for its function and opened up the possibility that there was a sequencing error in the reported S . pombe sequence . Therefore the gsh2 sequence from wild-type S . pombe and the mutant strain MN101 were determined . The wild-type S . pombe gsh2 encodes a protein that is about the same length as that found in Arabidopsis, and the MN101 mutation involves a frameshift mutation early in the glutathione synthetase reading frame. Fungal Genet Biol, 1997 Jun, 21(3), 388 - 405 Evolution of a fungal regulatory gene family: the Zn(II)2Cys6 binuclear cluster DNA binding motif; Todd RB et al.; The coevolution of DNA binding proteins and their cognate binding sites is essential for the maintenance of function . As a result, comparison of DNA binding proteins of unknown function in one species with characterized DNA binding proteins in another can identify potential targets and functions . The Zn(II)2Cys6 (or C6 zinc) binuclear cluster DNA binding domain has thus far been identified exclusively in fungal proteins, generally transcriptional regulators, and there are more than 80 known or predicted proteins which contain this motif, the best characterized of which are GAL4, PPR1, LEU3, HAP1, LAC9, and PUT3 . Here we review all known proteins containing the Zn(II)2Cys6 motif, along with their function, DNA binding, dimerization, and zinc(II) coordination properties and DNA binding sites . In addition, we have identified all of the Zn(II)2Cys6 motif-containing proteins in the sequence databases, including a large number with unknown function from the completed Saccharomyces cerevisiae and ongoing Schizosaccharomyces pombe genome projects, and examined the phylogenetic relationships of all the Zn(II)2Cys6 motifs from these proteins . Based on these relationships, we have assigned potential functions to a number of these unknown proteins. Fungal Genet Biol, 1997 Jun, 21(3), 373 - 87 Autonomous plasmid replication in Aspergillus nidulans: AMA1 and MATE elements; Aleksenko A et al.; With few exceptions, in eukaryotic organisms the presence of a chromosomal replicator on a circular vector molecule is not sufficient to confer on it the ability to persist and replicate extrachromosomally . However, it is possible to isolate from genomes of some filamentous fungi DNA fragments which can provide extrachromosomal maintenance of plasmids . In Aspergillus nidulans, two functional classes of such sequences can be distinguished: effective plasmid replicators (e.g., AMA1) and transformation enhancers (e.g., ANS1 or MATEs), which apparently are able to initiate aberrant replication, leading to vector rearrangement and multimerization and eventually resulting in chromosomal integration . We discuss the similarity of these events to DNA amplification in other eukaryotes . A model is suggested which accounts for the formation of effective replicating plasmids as a result of sequence amplification . The model is based on the observation that in some organisms, including A . nidulans and Schizosaccharomyces pombe, duplication of an inefficient replicator enhances its efficiency dramatically . Some structural traits of transformation enhancers in A . nidulans imply a role for topoisomerases in amplification and replication of circular DNA molecules . We discuss practical applications of replicative vectors for gene cloning and expression studies. J Biol Chem, 1997 Sep 12, 272(37), 23042 - 9 Isolation, expression, and regulation of the pgr1(+) gene encoding glutathione reductase absolutely required for the growth of Schizosaccharomyces pombe; Lee J et al.; The pgr1(+) gene encoding glutathione reductase (GR, EC 1.6.4.2) was isolated from Schizosaccharomyces pombe using a polymerase chain reaction fragment as a probe . The gene consists of two exons and an intron of 55 nucleotides, encoding a polypeptide of 465 amino acids (50,238 Da) with conserved residues characteristic of GR . The transcriptional start site was localized at 239 nucleotides upstream from the ATG initiation codon . The level of transcript as well as the GR enzyme activity increased more than 11-fold when the cloned pgr1(+) gene was expressed on a multicopy plasmid . This overexpression conferred on S . pombe cells more resistance against menadione, a redox cycling agent, but not against H2O2 . The level of pgr1(+) transcripts increased by treatment with oxidants such as menadione, cumene hydroperoxide, and diamide . It also increased by treatment with high osmolarity, heat shock, or at the stationary growth phase . The deletion of the pap1(+) gene encoding an AP-1 homolog in S . pombe caused reduction in the pgr1(+) gene expression . Furthermore, Deltapap1 cells lost the inducibility of pgr1(+) gene expression by the above stresses, implying that Pap1 is involved in general stress-inducible gene expression . When the pgr1(+) gene was disrupted, the haploid spores were not viable . Repression of nmt1 promoter-driven pgr1(+) expression by thiamine caused cessation of growth, which was rescued by the episomal pgr1(+) gene . These results indicate that GR activity, which efficiently reduces GSSG, is essentially required for the growth of S . pombe, unlike in Saccharomyces cerevisiae or Escherichia coli. Science, 1997 Sep 5, 277(5331), 1497 - 501 Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25; Sanchez Y et al.; In response to DNA damage, mammalian cells prevent cell cycle progression through the control of critical cell cycle regulators . A human gene was identified that encodes the protein Chk1, a homolog of the Schizosaccharomyces pombe Chk1 protein kinase, which is required for the DNA damage checkpoint . Human Chk1 protein was modified in response to DNA damage . In vitro Chk1 bound to and phosphorylated the dual-specificity protein phosphatases Cdc25A, Cdc25B, and Cdc25C, which control cell cycle transitions by dephosphorylating cyclin-dependent kinases . Chk1 phosphorylates Cdc25C on serine-216 . As shown in an accompanying paper by Peng et al . in this issue, serine-216 phosphorylation creates a binding site for 14-3-3 protein and inhibits function of the phosphatase . These results suggest a model whereby in response to DNA damage, Chk1 phosphorylates and inhibits Cdc25C, thus preventing activation of the Cdc2-cyclin B complex and mitotic entry. Genetics, 1997 Sep, 147(1), 101 - 15 The prp1+ gene required for pre-mRNA splicing in Schizosaccharomyces pombe encodes a protein that contains TPR motifs and is similar to Prp6p of budding yeast; Urushiyama S et al.; The prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe have a defect in pre-mRNA splicing and accumulate mRNA precursors at a restrictive temperature . One of the prp mutants, prp1-4, also has a defect in poly(A)+ RNA transport . The prp1+ gene encodes a protein of 906 amino acid residues that contains 19 repeats of 34 amino acids termed tetratrico peptide repeat (TPR) motifs, which were proposed to mediate protein-protein interactions . The amino acid sequence of Prp1p shares 29.6% identity and 50.6% similarity with that of the PRP6 protein of Saccharomyces cerevisiae, which is a component of the U4/U6 snRNP required for spliceosome assembly . No functional complementation was observed between S . pombe prp1+ and S . cerevisiae PRP6 . We examined synthetic lethality of prp1-4 with the other known prp mutations in S . pombe . The results suggest that Prp1p interacts either physically or functionally with Prp4p, Prp6p and Prp13p . Interestingly, the prp1+ gene was found to be identical with the zer1+ gene that functions in cell cycle control . These results suggest that Prp1p/Zer1p is either directly or indirectly involved in cell cycle progression and/or poly(A)+ RNA nuclear export, in addition to pre-mRNA splicing. Nucleic Acids Res, 1997 Sep 1, 25(17), 3433 - 9 Sce3, a suppressor of the Schizosaccharomyces pombe septation mutant cdc11, encodes a putative RNA-binding protein; Schmidt S et al.; In the fission yeast Schizosaccharomyces pombe, the cdc11 gene is required for the initiation of septum formation at the end of mitosis . The sce3 gene was cloned as a multi-copy suppressor of the heat-sensitive mutant cdc11-136 . When over-expressed, it rescues all mutants of cdc11 and also a heat-sensitive allele of cdc14, but not the cdc14 null mutant . Deletion shows that sce3 is not essential for cell proliferation . It encodes a putative RNA-binding protein which shows homology to human eIF4B . Immunolocalisation indicates that Sce3p is located predominantly in the cytoplasm . Elevated expression of sce3 increases the steady-state level of cdc14 mRNA . Possible mechanisms of its action are discussed. J Cell Biol, 1997 Aug 25, 138(4), 845 - 60 PSTPIP: a tyrosine phosphorylated cleavage furrow-associated protein that is a substrate for a PEST tyrosine phosphatase; Spencer S et al.; We have investigated proteins which interact with the PEST-type protein tyrosine phosphatase, PTP hematopoietic stem cell fraction (HSCF), using the yeast two-hybrid system . This resulted in the identification of proline, serine, threonine phosphatase interacting protein (PSTPIP), a novel member of the actin- associated protein family that is homologous to Schizosaccharomyces pombe CDC15p, a phosphorylated protein involved with the assembly of the actin ring in the cytokinetic cleavage furrow . The binding of PTP HSCF to PSTPIP was induced by a novel interaction between the putative coiled-coil region of PSTPIP and the COOH-terminal, proline-rich region of the phosphatase . PSTPIP is tyrosine phosphorylated both endogenously and in v-Src transfected COS cells, and cotransfection of dominant-negative PTP HSCF results in hyperphosphorylation of PSTPIP . This dominant-negative effect is dependent upon the inclusion of the COOH-terminal, proline-rich PSTPIP-binding region of the phosphatase . Confocal microscopy analysis of endogenous PSTPIP revealed colocalization with the cortical actin cytoskeleton, lamellipodia, and actin-rich cytokinetic cleavage furrow . Overexpression of PSTPIP in 3T3 cells resulted in the formation of extended filopodia, consistent with a role for this protein in actin reorganization . Finally, overexpression of mammalian PSTPIP in exponentially growing S . pombe results in a dominant-negative inhibition of cytokinesis . PSTPIP is therefore a novel actin-associated protein, potentially involved with cytokinesis, whose tyrosine phosphorylation is regulated by PTP HSCF. Chromosoma, 1997 Sep, 106(4), 254 - 65 Integrated map of the Schizosaccharomyces pombe genome; Garkavtsev I et al.; A restriction map of the entire Schizosaccharomyces pombe genome was constructed using two restriction enzymes (BamHI and PstI) that recognize 6 bp . The restriction map contains 420 minimally overlapping clones (miniset) and has 22 gaps . We located 126 genes, marker fragments of DNA (NotI and SfiI linking clones), and 36 transposable elements by hybridization to unique restriction fragments. Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9147 - 52 Modeling the control of DNA replication in fission yeast; Novak B et al.; A central event in the eukaryotic cell cycle is the decision to commence DNA replication (S phase) . Strict controls normally operate to prevent repeated rounds of DNA replication without intervening mitoses ("endoreplication") or initiation of mitosis before DNA is fully replicated ("mitotic catastrophe") . Some of the genetic interactions involved in these controls have recently been identified in yeast . From this evidence we propose a molecular mechanism of "Start" control in Schizosaccharomyces pombe . Using established principles of biochemical kinetics, we compare the properties of this model in detail with the observed behavior of various mutant strains of fission yeast: wee1(-) (size control at Start), cdc13Delta and rum1(OP) (endoreplication), and wee1(-) rum1Delta (rapid division cycles of diminishing cell size) . We discuss essential features of the mechanism that are responsible for characteristic properties of Start control in fission yeast, to expose our proposal to crucial experimental tests. Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9119 - 24 mRNA binding protein mrnp 41 localizes to both nucleus and cytoplasm; Kraemer D et al.; We have identified and molecularly characterized a human protein with a Mr of 40,880 Da . After UV irradiation of HeLa cells, this protein was cross-linked to poly(A)-containing mRNA and was therefore designated mrnp 41 (for mRNA binding protein of 41 kDa) . Cell fractionation and immunoblotting showed mrnp 41 in both the cytoplasm and the nucleus and particularly in the nuclear envelope . Immunofluorescence microscopy localized mrnp 41 to distinct foci in the nucleoplasm, to the nuclear rim, and to meshwork-like structures throughout the cytoplasm . The cytoplasmic meshwork staining was disrupted by prior treatment of cells with the actin filament- or microtubule-disrupting drugs cytochalasin or nocodazole, respectively, suggesting association of mrnp 41 with the cytoskeleton . Double immunofluorescence with antibodies against mrnp 41 and the cytoplasmic poly(A) binding protein showed colocalization to the cytoplasmic meshwork . Immunogold electronmicroscopy confirmed mrnp 41's cytoplasmic and nucleoplasmic localization and revealed a striking labeling of nuclear pore complexes . Together these data suggest that mrnp 41 may function in nuclear export of mRNPs and/or in cytoplasmic transport on, or attachment to, the cytoskeleton . Consistent with a role of mrnp 41 in nuclear export are previous reports that mutations in homologs of mrnp 41 in Schizosaccharomyces pombe, designated Rae1p, or in Saccharomyces cerevisiae, designated Gle2p, result in mRNA accumulation in the nucleus although it is presently not known whether these homologs are mRNA binding proteins as well. Science, 1997 Aug 15, 277(5328), 955 - 9 Telomerase catalytic subunit homologs from fission yeast and human; Nakamura TM et al.; Catalytic protein subunits of telomerase from the ciliate Euplotes aediculatus and the yeast Saccharomyces cerevisiae contain reverse transcriptase motifs . Here the homologous genes from the fission yeast Schizosaccharomyces pombe and human are identified . Disruption of the S . pombe gene resulted in telomere shortening and senescence, and expression of mRNA from the human gene correlated with telomerase activity in cell lines . Sequence comparisons placed the telomerase proteins in the reverse transcriptase family but revealed hallmarks that distinguish them from retroviral and retrotransposon relatives . Thus, the proposed telomerase catalytic subunits are phylogenetically conserved and represent a deep branch in the evolution of reverse transcriptases. FEBS Lett, 1997 Aug 11, 413(1), 16 - 20 Functional cloning of a cDNA encoding Mei2-like protein from Arabidopsis thaliana using a fission yeast pheromone receptor deficient mutant; Hirayama T et al.; To isolate Arabidopsis cDNAs that encode signal transducers and components involved in the regulation of meiosis, a trans-complementation analysis was performed using a Schizosaccharomyces pombe meiosis-defective mutant in which the genes for pheromone receptors were disabled . One cDNA obtained in this screening encodes a polypeptide, named AML1, that shows significant similarity to S . pombe Mei2 protein and has three putative RNA-recognition motifs like as Mei2 . Mei2 is involved in the regulation of meiosis in fission yeast . Northern blot analysis showed that the AML1 gene is expressed in each organ . The possible functions of AML1 are discussed. J Biol Chem, 1997 Aug 8, 272(32), 19993 - 20002 A novel protein, Psp1, essential for cell cycle progression of Schizosaccharomyces pombe is phosphorylated by Cdc2-Cdc13 upon entry into G0-like stationary phase of cell growth; Jang YJ et al.; A novel gene, psp1(+), which functionally complements a temperature-sensitive mutant defective in cell cycle progression both in G1/S and G2/M has been isolated from the genomic and cDNA libraries of Schizosaccharomyces pombe . Disruption of this gene is lethal for cell growth at 30 degrees C indicating that it is an essential gene for vegetative cell growth . Western analysis of the protein by polyclonal antibody made from glutathione S-transferase-Psp1 fusion protein indicated that the Psp1 protein exists in two different molecular weight forms depending on the growth state of the cell . In vitro experiments with a phosphatase showed that this difference is due to phosphorylation . The dephosphorylated form of the protein is dominant in actively growing cells whereas the phosphorylated form becomes the major species when cells enter the stationary phase . The Cdc2-Cdc13 complex is shown to phosphorylate the GST-Psp1 fusion protein in vitro, and site-directed mutagenesis and phosphoamino acid analysis indicated that the serine residue at position 333 in the carboxyl-terminal region is required for phosphorylation . In situ fluorescein isothiocyanate-conjugated antibody staining showed that this protein tends to be localized to both ends of the cell upon entry into the stationary phase of cell growth . However, overexpression of the novel protein Psp1 in actively growing cells inhibits cell growth causing accumulation of DNA (4n or 8n) . Thus we speculate that Psp1 can function at both G1/S and G2/M phases complementing the defect of the new mutant we have isolated . It is likely that Psp1 is required both for proper DNA replication and for the process of mitosis. Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8427 - 32 Purification and characterization of a CENP-B homologue protein that binds to the centromeric K-type repeat DNA of Schizosaccharomyces pombe; Lee JK et al.; We have purified and characterized a novel 60-kDa protein that binds to centromeric K-type repeat DNA from Schizosaccharomyces pombe . This protein was initially purified by its ability to bind to the autonomously replicating sequence 3002 DNA . Cloning of the gene encoding this protein revealed that it possesses significant homology to the mammalian centromere DNA-binding protein CENP-B and S . pombe Abp1, and this gene was designated as cbh+ (CENP-B homologue) . Cbh protein specifically interacts in vitro with the K-type repeat DNA, which is essential for centromere function . The Cbh-binding consensus sequence was determined by DNase I footprinting assays as PyPuATATPyPuTA, featuring an inverted repeat of the first four nucleotides . Based on its binding activity to centromeric DNA and homology to centromere proteins, we suggest that this protein may be a functional homologue of the mammalian CENP-B in S . pombe. FEBS Lett, 1997 Aug 4, 412(3), 415 - 9 PP2C gamma: a human protein phosphatase with a unique acidic domain; Travis SM et al.; We have cloned a novel cDNA from human skeletal muscle which encodes a protein phosphatase with a unique acidic domain . It is 34% identical to mammalian PP2C alpha and PP2C beta and we call it PP2C gamma . It more closely resembles PP2Cs from Paramecium tetraurelia and Schizosaccharomyces pombe than mammalian PP2Cs . Northern blot analysis shows that PP2C gamma is widely expressed, and is most abundant in testis, skeletal muscle, and heart . Like known PP2Cs, recombinant PP2C gamma requires Mg2+ or Mn2+ for activity . Unlike any other known phosphatase, PP2C gamma has a highly acidic domain: 75% of the 54 residues are glutamate or aspartate. Mol Biol Cell, 1997 Aug, 8(8), 1461 - 79 The spindle pole body of Schizosaccharomyces pombe enters and leaves the nuclear envelope as the cell cycle proceeds; Ding R et al.; The cycle of spindle pole body (SPB) duplication, differentiation, and segregation in Schizosaccharomyces pombe is different from that in some other yeasts . Like the centrosome of vertebrate cells, the SPB of S . pombe spends most of interphase in the cytoplasm, immediately next to the nuclear envelope . Some gamma-tubulin is localized on the SPB, suggesting that it plays a role in the organization of interphase microtubules (MTs), and serial sections demonstrate that some interphase MTs end on or very near to the SPB . gamma-Tubulin is also found on osmiophilic material that lies near the inner surface of the nuclear envelope, immediately adjacent to the SPB, even though there are no MTs in the interphase nucleus . Apparently, the MT initiation activities of gamma-tubulin in S . pombe are regulated . The SPB duplicates in the cytoplasm during late G2 phase, and the two resulting structures are connected by a darkly staining bridge until the mitotic spindle forms . As the cell enters mitosis, the nuclear envelope invaginates beside the SPB, forming a pocket of cytoplasm that accumulates dark amorphous material . The nuclear envelope then opens to form a fenestra, and the duplicated SPB settles into it . Each part of the SPB initiates intranuclear MTs, and then the two structures separate to lie in distinct fenestrae as a bipolar spindle forms . Through metaphase, the SPBs remain in their fenestrae, bound to the polar ends of spindle MTs; at about this time, a small bundle of cytoplasmic MTs forms in association with each SPB . These MTs are situated with one end near to, but not on, the SPBs, and they project into the cytoplasm at an orientation that is oblique to the simple axis . As anaphase proceeds, the nuclear fenestrae close, and the SPBs are extruded back into the cytoplasm . These observations define new fields of enquiry about the control of SPB duplication and the dynamics of the nuclear envelope. Yeast, 1997 Aug, 13(10), 985 - 90 Phylogenetic relationships of fungal cytochromes c; Janbon G et al.; The CYC1 gene encoding cytochrome c in the yeast Candida albicans was cloned by complementation of a cytochrome c-deficient mutant of Saccharomyces cerevisiae, and its DNA sequence was determined . The analysis of the amino acid sequences of cytochrome c from 14 fungal species and two isoforms from S . cerevisiae revealed sequences unique to fungi, and revealed a phylogenetic relationship with a pronounced divergence between Schizosaccharomyces pombe and other ascomycetous budding yeast. J Cell Sci, 1997 Aug, 110 ( Pt 15), 1805 - 12 Calmodulin localizes to the spindle pole body of Schizosaccharomyces pombe and performs an essential function in chromosome segregation; Moser MJ et al.; The essential calmodulin genes in both Saccharomyces cerevisiae and Schizosaccharomyces pombe were precisely replaced with genes encoding fusions between calmodulin and the green fluorescent protein (GFP) . In living budding yeast the GFP-calmodulin fusion protein (GFP-Cmd1p) localized simultaneously to sites of cell growth and to the spindle pole body (SPB), the yeast analog of the centrosome . Having demonstrated proper localization of GFP-calmodulin in budding yeast, we examined the localization of a fusion between GFP and calmodulin (GFP-Camlp) in fission yeast, where calmodulin had not been localized by any method . We find GFP-Camlp also localizes both to sites of polarized cell growth and to the fission yeast SPB . The localization of calmodulin to the SPB by GFP fusion was confirmed by indirect immunofluorescence . Antiserum to S . pombe calmodulin labeled the ends of the mitotic spindle stained with anti-tubulin antiserum . This pattern was identical to that seen using antiserum to Sad1p, a known SPB component . We then characterized the defects in a temperature-sensitive S . pombe calmodulin mutant . Mutant cam1-E14 cells synchronized in S phase completed DNA synthesis, but lost viability during transit of mitosis . Severe defects in chromosome segregation, including hypercondensation, fragmentation, and unequal allocation of chromosomal material were observed . Immunofluorescence analysis of tubulin revealed a population of cells containing either broken or mislocalized mitotic spindles, which were never observed in wild-type cells . Taken together with the subcellular localization of calmodulin, the observed spindle and chromosome segregation defects suggest that calmodulin performs an essential role during mitosis at the fission yeast SPB. Genetics, 1997 Aug, 146(4), 1275 - 86 Mismatch repair in Schizosaccharomyces pombe requires the mutL homologous gene pms1: molecular cloning and functional analysis; Schar P et al.; Homologues of the bacterial mutS and mutL genes involved in DNA mismatch repair have been found in organisms from bacteria to humans . Here, we describe the structure and function of a newly identified Schizosaccharomyces pombe that encodes a predicted amino acid sequence of 794 residues with a high degree of homology to MutL related proteins . On the basis of its closer relationship to the eukaryotic "PMS" genes than to the "MLH" genes, we have designated the S . pombe homologue pms1 . Disruption of the pms1 gene causes a significant increase of spontaneous mutagenesis as documented by reversion rate measurements . Tetrad analyses of crosses homozygous for the pms1 mutation reveal a reduction of spore viability from > 92% to 80% associated with a low proportion (approximately 50%) of meioses producing four viable spores and a significant, allele-dependent increase of the level of post-meiotic segregation of genetic marker allele pairs . The mutant phenotypes are consistent with a general function of pms1 in correction of mismatched base pairs arising as a consequence of DNA polymerase errors during DNA synthesis, or of hybrid DNA formation between homologous but not perfectly complementary DNA strands during meiotic recombination. Genetics, 1997 Aug, 146(4), 1253 - 64 A WD repeat protein, Rec14, essential for meiotic recombination in Schizosaccharomyces pombe; Evans DH et al.; Mutations in the Schizosaccharomyces pombe rec14 gene reduce meiotic recombination by as much as a factor of 1000 in the three intervals tested on chromosomes I and III . A DNA clone complementing the rec14 mutation was shown by genetic and physical analysis to contain the rec14 gene, which was functional in plasmid-borne inserts as small as 1.4 kb . The rec14 gene contains two exons separated by a 53-bp intron, which was confirmed by analysis of rec14 transcripts . The spliced transcript encodes a protein product of 302 amino acids, which contains six WD repeat motifs found in the G-beta transducin family of proteins and other proteins, including the Saccharomyces cerevisiae Ski8 (Rec103) protein . Although the rec14 transcripts were present in mitotically dividing cells, rec14 mutations had no detectable effect on mitotic recombination . The pattern of expression of rec14 differes from that of previously analyzed S . pombe rec genes . Based upon mutant phenotypes and amino acid sequence similarities, we propose that S . pombe Rec14 is a functional homologue of S . cerevisiae Rec103. Genetics, 1997 Aug, 146(4), 1221 - 38 A recombinationally repressed region between mat2 and mat3 loci shares homology to centromeric repeats and regulates directionality of mating-type switching in fission yeast; Grewal SI et al.; Cells of the fission yeast Schizosaccharomyces pombe switch mating type by replacing genetic information at the transcriptionally active mat1 locus with sequences copied from one of two closely linked silent loci, mat2-P or mat3-M . By a process referred to as directionality of switching, cells predominantly switch to the opposite mat1 allele; the mat1-P allele preferentially recombines with mat3, while mat1-M selects the mat2 . In contrast to efficient recombination at mat1, recombination within the adjoining mat2-mat3 interval is undetectable . We defined the role of sequences between mat2 and mat3, designated the K-region, in directionality as well as recombinational suppression . Cloning and sequencing analysis revealed that a part of the K-region is homologous to repeat sequences present at centromeres, which also display transcriptional and recombinational suppression . Replacement of 7.5 kb of the K-region with the ura4+ gene affected directionality in a variegated manner . Analysis of the swi6-mod locus, which was previously shown to affect directionality, in K delta::ura4+ strains suggested the existence of at least two overlapping directionality mechanisms . Our work furthers the model that directionality is regulated by cell-type-specific organization of the heterochromatin-like structure in the mating-type region and provides evidence that the K-region contributes to silencing of the mat2-mat3 interval. J Biol Chem, 1997 Jul 25, 272(30), 19088 - 94 COX15 codes for a mitochondrial protein essential for the assembly of yeast cytochrome oxidase; Glerum DM et al.; The respiratory defect of Saccharomyces cerevisiae mutants assigned to complementation group G4 of a pet strain collection stems from their failure to synthesize cytochrome oxidase . The mutations do not affect expression of either the mitochondrially or nuclearly encoded subunits of the enzyme . The cytochrome oxidase deficiency also does not appear to be related to mitochondrial copper metabolism or heme a biosynthesis . These data suggest that the mutants are likely to be impaired in assembly of the enzyme . A gene designated COX15 has been cloned by transformation of mutants from complementation group G4 . This gene is identical to reading frame YER141w on chromosome 5 . To facilitate further studies, Cox15p has been expressed as a biotinylated protein . Biotinylated Cox15p fully restores cytochrome oxidase in cox15 mutants, indicating that the carboxyl-terminal sequence with biotin does not affect its function . Cox15p is a constituent of the mitochondrial inner membrane and, because of its resistance to proteolysis, probably is largely embedded in the phospholipid bilayer of the membrane . The present studies further emphasize the complexity of cytochrome oxidase assembly and report a new constituent of mitochondria involved in this process . The existence of COX15 homologs in Schizosaccharomyces pombe and Caenorhabditis elegans suggests that it may be widely distributed in eucaryotic organisms. J Biol Chem, 1997 Jul 25, 272(30), 18910 - 9 A DNA helicase from Schizosaccharomyces pombe stimulated by single-stranded DNA-binding protein at low ATP concentration; Park JS et al.; A DNA helicase named DNA helicase I was isolated from cell-free extracts of the fission yeast Schizosaccharomyces pombe . Both DNA helicase and single-stranded DNA-dependent ATPase activities copurified with a polypeptide of 95 kDa on an SDS-polyacrylamide gel . The helicase possessed a sedimentation coefficient of 6.0 S and a Stokes radius of 44.8 A determined by glycerol gradient centrifugation and gel filtration analysis, respectively . From these data the native molecular mass was calculated to be 110 kDa, indicating that the active enzyme is a monomer . The DNA-unwinding and ATP hydrolysis activities associated with DNA helicase I have been examined . One notable property of the enzyme was its relatively high rate of ATP turnover (35-50 molecules of ATP hydrolyzed/s/enzyme molecule) that may contribute to its inefficient unwinding activity at low concentrations of ATP (<0.2 mM) . Addition of an ATP-regenerating system to the reaction mixture restored the DNA-unwinding activity of the enzyme . S . pombe single-stranded DNA-binding protein (SpSSB, also called SpRPA) stimulated the DNA helicase activity significantly at low levels of ATP (0.025-0.2 mM) even in the absence of an ATP-regenerating system . In contrast, SpRPA had no effect on ATP hydrolysis at any ATP concentration examined . These observations suggest that the stimulation of DNA unwinding by SpRPA is not simply a result of suppression of nonproductive ATP hydrolysis . Rather, the role of SpRPA is to lower the Km for ATP in the unwinding reaction, allowing the helicase to function efficiently at low ATP concentrations. Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 7965 - 70 The Schizosaccharomyces pombe spindle checkpoint protein mad2p blocks anaphase and genetically interacts with the anaphase-promoting complex; He X et al.; The spindle checkpoint monitors mitotic spindle integrity and the attachment of kinetochores to the spindle . Upon sensing a defect the checkpoint blocks cell cycle progression and thereby prevents chromosome missegregation . Previous studies in budding yeast show that the activated spindle checkpoint inhibits the onset of anaphase by an unknown mechanism . One possible target of the spindle checkpoint is anaphase promoting complex (APC), which controls all postmetaphase events that are blocked by spindle checkpoint activation . We have isolated mad2, a spindle checkpoint component in fission yeast, and shown that mad2 overexpression activates the checkpoint and causes a cell cycle arrest at the metaphase-to-anaphase transition . In addition to the observation that mad2-induced arrest can be partially relieved by mitosis-promoting factor inactivation, we present genetic evidence consistent with the hypothesis that the spindle checkpoint imposes a cell cycle arrest by inhibiting APC-dependent proteolysis. Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 7873 - 8 Human and Saccharomyces cerevisiae dolichol phosphate mannose synthases represent two classes of the enzyme, but both function in Schizosaccharomyces pombe; Colussi PA et al.; Dolichol phosphate mannose (Dol-P-Man), formed upon transfer of Man from GDPMan to Dol-P, is a mannosyl donor in pathways leading to N-glycosylation, glycosyl phosphatidylinositol membrane anchoring, and O-mannosylation of protein . Dol-P-Man synthase is an essential protein in Saccharomyces cerevisiae . We have cloned cDNAs encoding human and Schizosaccharomyces pombe proteins that resemble S . cerevisiae Dol-P-Man synthase . Disruption of the gene for the S . pombe Dol-P-Man synthase homolog, dpm1(+), is lethal . The known Dol-P-Man synthase sequences can be divided into two classes . One contains the S . cerevisiae, Ustilago maydis, and Trypanosoma brucei enzymes, which have a COOH-terminal hydrophobic domain, and the other contains the human, S . pombe, and Caenorhabditis synthases, which lack a hydrophobic COOH-terminal domain . The two classes of synthase are functionally equivalent, because S . cerevisiae DPM1 and its human counterpart both complement the lethal null mutation in S . pombe dpm1(+) . The findings that Dol-P-Man synthase is essential in yeast and that the Ustilago and Trypanosoma synthases are in a different class from the human enzyme raise the possibility that Dol-P-Man synthase could be exploited as a target for inhibitors of pathogenic eukaryotic microbes. EMBO J, 1997 Jul 16, 16(14), 4340 - 51 Human and Xenopus cDNAs encoding budding yeast Cdc7-related kinases: in vitro phosphorylation of MCM subunits by a putative human homologue of Cdc7; Sato N et al.; Saccharomyces cerevisiae Cdc7 kinase is essential for initiation of DNA replication, and Hsk1, a related kinase of Schizosaccharomyces pombe, is also required for DNA replication of fission yeast cells . We report here cDNAs encoding Cdc7-related kinases from human and Xenopus (huCdc7 and xeCdc7, respectively) . The cloned cDNA for huCdc7 contains an open reading frame consisting of 574 amino acids with a predicted molecular weight of 63,847 that possesses overall amino acid identity of 32% (54% including similar residues) to Cdc7 and Hsk1 . huCDC7 is transcribed in the various tissues examined, but most abundantly in testis . Three transcripts of 4.4, 3.5 and 2.4 kb in length are detected . The 3.5 kb transcript is the most predominant and is expressed in all the tissues examined . A cDNA containing a 91 nucleotide insertion at the N-terminal region of huCDC7 is also detected, suggesting the presence of multiple splicing variants . The huCdc7 protein is expressed at a constant level during the mitotic cell cycle and is localized primarily in nuclei in interphase and distributed diffusibly in cytoplasm in the mitotic phase . The wild-type huCdc7 protein expressed in COS7 cells phosphorylates MCM2 and MCM3 proteins in vitro, suggesting that huCdc7 may regulate processes of DNA replication by modulating MCM functions. Cancer Lett, 1997 Jul 15, 117(1), 23 - 8 The ribosomal 5.8S RNA as a target site for p53 protein in cell differentiation and oncogenesis; Abou Elela S et al.; Previous studies have shown that the ribosomal 5.8S rRNA of human cells is partially methylated in a tissue-specific fashion, a modification which occurs largely or entirely in the cytoplasm . More recent studies have shown that the 5.8S rRNA forms a covalent linkage with tumor suppressor p53 protein and have suggested that this RNA plays a functional role in protein elongation . We now show that the expression of p53 protein in Schizosaccharomyces pombe results in cells which: morphologically resemble transformed cells expressing mutant 5.8S rRNA; are equally compromised in their ability to sustain protein synthesis, in vitro; and contain polyribosome profiles which strongly resemble the elevated profiles which also are observed with mutated 5.8S rRNA . Taken together, these results provide new physiological evidence of the possibility that the 5.8S rRNA is an important target in the control of ribosome function during cell differentiation and oncogenesis. Nucleic Acids Res, 1997 Jul 15, 25(14), 2823 - 7 Complementation of the DNA repair-deficient swi10 mutant of fission yeast by the human ERCC1 gene; Rodel C et al.; In human cells DNA damage caused by UV light is mainly repaired by the nucleotide excision repair pathway . This mechanism involves dual incisions on both sides of the damage catalyzed by two nucleases . In mammalian cells XPG cleaves 3' of the DNA lesion while the ERCC1-XPF complex makes the 5' incision . The amino acid sequence of the human excision repair protein ERCC1 is homologous with the fission yeast Swi10 protein . In order to test whether these proteins are functional homologues, we overexpressed the human gene in a Schizosaccharomyces pombe swi10 mutant . A swi10 mutation has a pleiotropic effect: it reduces the frequency of mating type switching (a mitotic transposition event from a silent cassette into the expression site) and causes increased UV sensitivity . We found that the full-length ERCC1 gene only complements the transposition defect of the fission yeast mutant, while a C-terminal truncated ERCC1 protein also restores the DNA repair capacity of the yeast cells . Using the two-hybrid system of Saccharomyces cerevisiae we show that only the truncated human ERCC1 protein is able to interact with the S . pombe Rad16 protein, which is the fission yeast homologue of human XPF . This is the first example yet known that a human gene can correct a yeast mutation in nucleotide excision repair. J Biol Chem, 1997 Jul 11, 272(28), 17873 - 9 Protein phosphatase 2C acts independently of stress-activated kinase cascade to regulate the stress response in fission yeast; Gaits F et al.; Stress-activated signal transduction pathways, which are largely conserved among a broad spectrum of eukaryotic species, have a crucial role in the survival of many forms of stress . It is therefore important to discover how these pathways are both positively and negatively regulated . Recent genetic studies have implicated protein phosphatase 2C (PP2C) as a novel negative regulator of stress response pathways in both budding and fission yeasts . Moreover, it was hypothesized that PP2C dephosphorylates one or more components of protein kinase cascades that are at the core of stress-activated signal transduction pathways . Herein we present genetic and biochemical studies of the fission yeast Schizosaccharomyces pombe that disprove this hypothesis and indicate that PP2C instead negatively regulates a downstream element of the pathway . First, high expression of PP2C produces phenotypes that are inconsistent with negative regulation of the Wik1-Wis1-Spc1 stress-activated kinase cascade . Second, high expression of PP2C leads to sustained activating tyrosine phosphorylation of Spc1 . Third, Spc1-dependent phosphorylation of Atf1, a transcription factor substrate of Spc1, is unaffected by high expression of PP2C . Fourth, high expression of PP2C suppresses Atf1-dependent transcription of a stress-response gene . These studies strongly suggest that PP2C acts downstream of Spc1 kinase in the stress-activated signal transduction pathway. Gene, 1997 Jul 9, 193(2), 203 - 10 Cloning and characterization of the S . pombe gene efc25+, a new putative guanine nucleotide exchange factor; Tratner I et al.; We report the cloning and characterization of a new S . pombe gene, efc25+, for 'exchange factor Cdc25-like' . The C-terminal region of the predicted product of this gene displays high sequence homology with a number of guanine nucleotide exchange factors for Ras . These include Cdc25 of Saccharomyces cerevisiae, Cdc25 of Saccharomyces kluyveri, Csc25 of Candida albicans, Sdc25 of S . cerevisiae and Ste6 of Schizosaccharomyces pombe . Disruption of efc25+ resulted in cells with a spherical shape reminiscent of the abnormal morphological phenotype of ras1 deletion mutants . However, unlike ras1 null mutants, strains deleted for efc25+ were proficient for mating and sporulation . This differs from the only other Ras1 exchange factor characterized so far in S . pombe, the Ste6 protein, whose deletion results in defects in mating and sporulation but not in cell shape . We hypothesize that Efc25 is an exchange factor for Ras1 and that it is involved in a signaling pathway different from that involving Ste6. Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7487 - 92 Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2; Tishkoff DX et al.; A two-hybrid screen was used to identify Saccharomyces cerevisiae genes encoding proteins that interact with MSH2 . One gene was found to encode a homologue of Schizosaccharomyces pombe EXO1, a double-stranded DNA-specific 5'-3' exonuclease . S . cerevisiae EXO1 interacted with both S . cerevisiae and human MSH2 in two-hybrid and coimmunoprecipitation experiments . exo1 mutants showed a mutator phenotype, and epistasis analysis was consistent with EXO1 functioning in the MSH2-dependent mismatch repair pathway . exo1 mutations were lethal in combination with rad27 mutations, and overexpression of EXO1 suppressed both the temperature sensitive and mutator phenotypes of rad27 mutants. Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7446 - 51 Position- and orientation-independent activity of the Schizosaccharomyces pombe meiotic recombination hot spot M26; Fox ME et al.; The activity of the M26 meiotic recombination hot spot of Schizosaccharomyces pombe depends on the presence of the heptamer 5'-ATGACGT-3' . Transplacement of DNA fragments containing the ade6-M26 gene to other chromosomal loci has previously demonstrated that the heptamer functions in some, but not all, transplacements, suggesting that hot spot activity depends on chromosomal context . In this study, hot spot activity was tested in the absence of gross DNA changes by using site-directed mutagenesis to create the heptamer sequence at novel locations in the genome . When created by mutagenesis of 1-4 bp in the ade6 and ura4 genes, the heptamer was active as a recombination hot spot, in an orientation-independent manner, at all locations tested . Thus, the heptamer sequence can create an active hot spot in other chromosomal contexts, provided that the gross chromosomal structure is not altered; this result is consistent with the hypothesis that a specific higher-order chromatin structure is required for M26 hot spot activity. Mol Microbiol, 1997 Jul, 25(2), 261 - 73 Functional analysis of chimerical plasma membrane H+-ATPases from Saccharomyces cerevisiae and Schizosaccharomyces pombe; de Kerchove d'Exaerde A et al.; The plasma membrane H+-ATPase from the fission yeast Schizosaccharomyces pombe does not support growth of H+-ATPase-depleted cells of the budding yeast Saccharomyces cerevisiae, even after deletion of the enzyme's carboxy terminus . Functional chimerical H+-ATPase proteins in which appropriate regions of the S . pombe enzyme were replaced with their S . cerevisiae counterparts were generated by in vivo gene recombination . Site-directed mutagenesis of the H+-ATPase chimeras showed that a single amino acid replacement, tyrosine residue 596 by alanine, resulted in functional expression of the S . pombe H+-ATPase . The reverse Ala-598-->Tyr substitution was introduced into the S . cerevisiae enzyme to better understand the role of this alanine residue . However, no obvious effect on ATPase activity could be detected . The S . cerevisiae cells expressing the S . pombe H+-ATPase substituted with alanine were enlarged and grew more slowly than wild-type cells . ATPase activity showed a more alkaline pH optimum, lower K(m) values for MgATP and decreased Vmax compared with wild-type S . cerevisiae activity . None of these kinetic parameters was found to be modified in glucose-starved cells, indicating that the S . pombe H+-ATPase remained fully active . Interestingly, regulation of ATPase activity by glucose was restored to a chimera in which the S . cerevisiae sequence spans most of the catalytic site. Mol Gen Genet, 1997 Jul, 255(3), 332 - 40 Characterisation of the Schizosaccharomyces pombe rad4/cut5 mutant phenotypes: dissection of DNA replication and G2 checkpoint control function; McFarlane RJ et al.; Mutation of the essential Schizosaccharomyces pombe rad4/cut5 gene causes sensitivity to UV and ionising radiation at the permissive temperature whilst at the restrictive temperature cells fail to undergo DNA replication but still attempt mitosis owing to a defective S-phase checkpoint response . Many mutations in genes encoding DNA replication proteins also abolish checkpoint responses, possibly because the replication machinery is a pre-requisite for the generation of the signal . We demonstrate here that rad4/cut5 cells fail to arrest cell division when treated with the replication inhibitor hydroxyurea at the semi-permissive temperature 32 degrees C, but retain essentially normal replicative capacity . This demonstrates that the replication and checkpoint function of the rad4/cut5 gene product can be separated and that the Rad4 protein differs from other replication proteins in being directly involved in generating the S-phase checkpoint signal . Furthermore, we have investigated the checkpoint response or rad4/cut5-deficient cells to gamma-irradiation and UV-mimetic drugs . We find that, at the restrictive temperature, the rad4-/cut5- cells fail to delay mitosis in response to gamma-irradiation whilst retaining a normal checkpoint response to the UV-mimetic drug 4-nitroquinoline-1-oxide . The lack of the gamma-irradiation checkpoint is reminiscent of the deficiency associated with mutation of the human ATM locus, the causative deficiency of the heritable disorder ataxia telangiectasia . The implications of our results for the organisation of distinct checkpoint-response pathways in both fission yeast and mammalian cells are discussed . Moreover the data are consistent with a model in which the generation of the S-Phase checkpoint signal is DNA polymerase epsilon dependent. Eur J Biochem, 1997 Jul 1, 247(1), 314 - 21 The ribosomal-RNA-processing pathway in Schizosaccharomyces pombe; Good L et al.; In all cells, a long precursor RNA is processed into mature rRNAs for ribosome biogenesis . In eukaryotes, the complexity and speed of the overall process often has made it difficult to establish finer details of the maturation pathway . Since phylogenetic comparisons can provide evidence for critical events, the major rRNA processing pathway for the yeast Schizosaccharomyces pombe was determined using primer extension, nuclease protection and Northern-hybridisational analyses . Transcript mapping of the 5' external transcribed spacer revealed six cleavage sites which occur upstream of the mature 18S termini . Two of these sites as well as a site adjacent to the 18S termini are complementary to conserved Box sequences in the S . pombe U3 small nucleolar RNA . Transcript mapping of the internal transcribed spacers (1 and 2) suggest similar maturation schemes for the two spacers, in which an initial endonuclease cleavage is followed by processing to the mature termini . The mature 5' termini of 25S rRNA appear to be heterogeneous in S . pombe, as has been demonstrated for 5.8S rRNA, suggesting an essential limiting structure in the ribosome-integrated mature RNA . Together with our previous analysis of the 3' external spacer region, the results reveal the major processing pathway for S . pombe and further support a maturation process which acts as a quality assurance mechanism. Microbiology, 1997 Jul, 143 ( Pt 7), 2457 - 63 Protein kinase Sck1 is involved in trehalase activation by glucose and nitrogen source in the fission yeast Schizosaccharomyces pombe; Soto T et al.; Trehalase activity is markedly enhanced upon addition of glucose and a nitrogen source to cells of the fission yeast Schizosaccharomyces pombe . This increase corresponds to a post-translational activation of the enzyme, which is controlled by cAMP-dependent and cAMP-independent pathways . Recent work has shown that overexpression of SCK1 in Schiz . pombe is able to suppress mutations that result in reduced Pka1 (cAMP-dependent protein kinase A activity, suggesting that Sck1 (suppressor of loss of cAMP-dependent protein kinase) might be a functional analogue of Pka1 in the fission yeast . Here, an analysis of the possible role of Sck1 in the activation of trehalase triggered by glucose and a nitrogen source is reported in cells that were deficient in either Pka1, Sck1 or both protein kinases . The results showed that, except in repressed cells, Sck1 probably mediates a cAMP-independent activation of trehalase following the signal(s) triggered by glucose and the nitrogen source . The absence of functional Sck1 in depressed cells renders trehalase insensitive to activation by glucose and the nitrogen source even in the presence of Pka1, indicating that the Sck1-dependent, cAMP-independent pathway is the main signalling pathway controlling trehalase activation under derepression conditions . It is proposed that, during the activation of trehalase induced by glucose or a nitrogen source, the cAMP-Pka1 activation pathway previously characterized is to some extent parallel to this newly described one which includes Sck1 as phosphorylating enzyme . Neither of these two pathways, however, plays a key role in the heat-induced increase in trehalase activity. EMBO J, 1997 Jul 1, 16(13), 4021 - 33 Cell differentiation by interaction of two HMG-box proteins: Mat1-Mc activates M cell-specific genes in S.pombe by recruiting the ubiquitous transcription factor Ste11 to weak binding sites; Kjaerulff S et al.; The Schizosaccharomyces pombe mfm1 gene is expressed in an M cell-specific fashion . This regulation requires two HMG-box proteins: the ubiquitous Ste11 transcription factor and the M cell-controlling protein Mat1-Mc . Here we report that the mfm1 promoter contains a single, weak Stell-binding site (a so-called TR-box) that can confer M-specificity on a heterologous promoter when present in eight copies . In vitro, both Mat1-Mc and Ste11 can bind this box with approximately the same affinity . The Mat1-Mc protein caused a dramatic increase in the DNA-binding of Ste11 to this box, under conditions where we could not detect Mat1-Mc in the resulting protein-DNA complex . When we changed a single base in the mfm1 TR-box, such that it resembled those boxes found in ubiquitously expressed genes, Ste11 binding was enhanced, and in vivo the mfm1 gene also became expressed in P cells where Mat1-Mc is absent . These findings suggest that M-specificity results from Mat1-Mc-mediated Ste11 binding to weak TR-boxes . We have also defined a novel motif (termed M-box), adjacent to the mfm1 TR-box, to which Mat1-Mc binds strongly . A DNA fragment containing both the TR- and the M-box allowed the formation of a complex containing both Ste11 and Mat1-Mc . A single copy of this fragment was sufficient to activate a heterologous promoter in an M-specific fashion, suggesting that these two boxes act in a synergistic manner. Protein Expr Purif, 1997 Jul, 10(2), 192 - 201 A general purification protocol for E7 proteins from "high- and low-risk" human papillomavirus types expressed in the yeast Schizosaccharomyces pombe; Braspenning J et al.; A purification protocol was developed to obtain human papillomavirus (HPV) type 16 E7 protein expressed in the yeast Schizosaccharomyces pombe . Only three chromatographic steps were necessary to purify the unfused HPV 16 E7 protein to homogeneity (95-99%) as shown by silver staining after polyacrylamide gel electrophoresis . Approximately 0.8 mg of highly purified E7 was obtained from 5 x 10(10) yeast cells . The purified HPV 16 E7 phosphoprotein (Ser 31/32) was refolded and assayed for functionality . Binding to the proteins Rb1 and p107 in vitro and induction of DNA synthesis after microinjection into serum-deprived NIH 3T3 cells suggest that the E7 protein retains some of its biological activities . Most importantly, the purification strategy is also applicable for different HPV 16 E7 mutants and for E7 proteins from other HPV types such as HPV 18 and 11. J Bacteriol, 1997 Jul, 179(13), 4179 - 89 Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe; Tabuchi M et al.; PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe . The cloned S . pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site . It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles . The C-terminal mature region is highly conserved in other serine carboxypeptidases . In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues . The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each . Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions . Like S . cerevisiae CPY, S . pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery . To investigate the vacuolar sorting signal of S . pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S . pombe Cpy1p fused to the secreted enzyme S . cerevisiae invertase . The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole . These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal. Mol Cell Biol, 1997 Jul, 17(7), 3508 - 19 Mkh1, a MEK kinase required for cell wall integrity and proper response to osmotic and temperature stress in Schizosaccharomyces pombe; Sengar AS et al.; We have identified a Schizosaccharomyces pombe gene, mkh1, that encodes a MEK kinase (MEKK) homolog . The coding region of mkh1 is contained within a single exon encoding a 1,116-amino-acid protein . The putative catalytic domain of Mkh1 is 54% identical to the catalytic domain of S . cerevisiae Bck1, the most closely related protein . Deletion of mkh1 did not significantly affect cell growth or division under standard conditions . However, mkh1delta cell growth was inhibited by high KCl or NaCl concentrations . mkh1delta cells required a longer time to reenter the cell cycle after prolonged stationary-phase arrest . Also, mkh1delta cells exhibited a round cell shape, while overexpression of Mkh1 resulted in an elongated cell shape . mkh1delta cells exhibited a more dramatic phenotype when grown in nutrient-limiting conditions at high temperature or in hyperosmotic medium . In such conditions, completion of cytokinesis was inhibited, resulting in the growth of pseudohyphal filaments with multiple septa and nuclei . Also, mkh1delta cells were hypersensitive to beta-glucanase treatment . Together these results suggest that Mkh1 regulates cell morphology, cell wall integrity, salt resistance, cell cycle reentry from stationary-phase arrest, and filamentous growth in response to stress . These phenotypes are essentially identical to those exhibited by cells lacking Pmk1/Spm1, a recently identified mitogen-activated protein kinase . Our evidence suggests that Pmk1/Spm1 acts downstream from Mkh1 in a common pathway . Our results also suggest that Mkh1 and Pck2 act independently to maintain cell wall integrity, cell morphology, and salt resistance but act in opposition to regulate filamentous growth. J Cell Biol, 1997 Jun 16, 137(6), 1337 - 54 Fission yeast dim1(+) encodes a functionally conserved polypeptide essential for mitosis; Berry LD et al.; In a screen for second site mutations capable of reducing the restrictive temperature of the fission yeast mutant cdc2-D217N, we have isolated a novel temperature-sensitive mutant, dim1-35 . When shifted to restrictive temperature, dim1-35 mutant cells arrest before entry into mitosis or proceed through mitosis in the absence of nuclear division, demonstrating an uncoupling of proper DNA segregation from other cell cycle events . Deletion of dim1 from the Schizosaccharomyces pombe genome produces a lethal G2 arrest phenotype . Lethality is rescued by overexpression of the mouse dim1 homolog, mdim1 . Likewise, deletion of the Saccharomyces cerevisiae dim1 homolog, CDH1, is lethal . Both mdim1 and dim1(+) are capable of rescuing lethality in the cdh1::HIS3 mutant . Although dim1-35 displays no striking genetic interactions with various other G2/M or mitotic mutants, dim1-35 cells incubated at restrictive temperature arrest with low histone H1 kinase activity . Morevoer, dim1-35 displays sensitivity to the microtubule destabilizing drug, thiabendazole (TBZ) . We conclude that Dim1p plays a fundamental, evolutionarily conserved role as a protein essential for entry into mitosis as well as for chromosome segregation during mitosis . Based on TBZ sensitivity and failed chromosome segregation in dim1-35, we further speculate that Dim1p may play a role in mitotic spindle formation and/or function. J Cell Biol, 1997 Jun 16, 137(6), 1309 - 19 Type II myosin heavy chain encoded by the myo2 gene composes the contractile ring during cytokinesis in Schizosaccharomyces pombe; Kitayama C et al.; We cloned the myo2 gene of Schizosaccharomyces pombe, which encodes a type II myosin heavy chain, by virtue of its ability to promote diploidization in fission yeast cells . The myo2 gene encodes 1,526 amino acids in a single open reading frame . Myo2p shows homology to the head domains and the coiledcoil tail of the conventional type II myosin heavy chain and carries putative binding sites for ATP and actin . It also carries the IQ motif, which is a presumed binding site for the myosin light chain . However, Myo2p apparently carries only one IQ motif, while its counterparts in other species have two . There are nine proline residues, which should break alpha-helix, in the COOH-terminal coiled-coil region of Myo2p . Thus, Myo2p is rather unusual as a type II myosin heavy chain . Disruption of myo2 inhibited cell proliferation . myo2Delta cells showed normal punctate distribution of interphase actin, but they produced irregular actin rings and septa and were impaired in cell separation . Overproduction of Myo2p was also lethal, apparently blocking actin relocation . Nuclear division proceeded without actin ring formation and cytokinesis in cells overexpressing Myo2p, giving rise to multinucleated cells with dumbbell morphology . Analysis using tagged Myo2p revealed that Myo2p colocalizes with actin in the contractile ring, suggesting that Myo2p is a component of the ring and responsible for its contraction . Furthermore, genetic evidence suggested that the acto-myosin system may interact with the Ras pathway, which regulates mating and the maintenance of cell morphology in S . pombe. Genes Dev, 1997 Jun 15, 11(12), 1519 - 34 The Spg1p GTPase is an essential, dosage-dependent inducer of septum formation in Schizosaccharomyces pombe; Schmidt S et al.; The spg1 gene (septum-promoting GTPase) was cloned as a multicopy suppressor of a dominant-negative mutant of the Cdc7p kinase . It encodes a small GTPase of the Ras superfamily . spg1 is an essential gene . Null or heat-sensitive alleles do not make a division septum, but growth, S-phase, and mitosis continue in the absence of cell division, producing elongated, multinucleate cells . Increased expression of Spg1p induces septum formation in G2, S-phase, and pre-Start G1-arrested cells . This requires the activity of Cdc7p kinase, but not p34(cdc2) . Increased expression of Cdc7p bypasses the requirement for Spg1p . Spg1p and Cdc7p can be coimmunoprecipitated from cell extracts, and interact in the two-hybrid system . These data indicate that Spg1p is a key element in controlling the onset of septum formation in Schizosaccharomyces pombe, and that it acts through the Cdc7p kinase. Biochim Biophys Acta, 1997 Jun 5, 1357(1), 41 - 8 Osmo-stress-induced changes in neutral trehalase activity of the fission yeast Schizosaccharomyces pombe; Fernandez J et al.; Exposure of repressed growing cultures of Schizosaccharomyces pombe to various extracellular concentrations of NaCl, sorbitol or glycerol resulted in a reversible increase in neutral trehalase activity which was maintained while the cells were in the presence of high environmental osmolarity . Treatment of osmo-stress-induced trehalase by phosphatase lead to a decreased activity indicating that the active enzyme is phosphorylated . The stress response following the osmotic shock required protein synthesis and was independent of the cAMP-dependent protein kinase pathway . Cells disrupted for wis} or phh1 (identical to sty1 and spc1), which encode members of the mitogen-activated protein kinase (MAPK) cascade, showed that the osmo-stress-induced increase in trehalase markedly diminished . In contrast, the heat shock-induced increase in trehalase remained unchanged in these cells . Taken together, the data suggest that the elevation of trehalase activity in Schiz . pombe under conditions of high osmolarity is due to de novo synthesis of the enzyme and that this process is modulated through a MAPK signal transduction pathway as part of the physiological response to the osmotic stress . The wisl-phhl MAPK cascade, however, does not appear to form part of the mechanism underlaying the increase in trehalase after heat stress. Gene, 1997 Jun 3, 191(2), 191 - 5 General purpose tagging vectors for fission yeast; Forsburg SL et al.; We have designed a series of vectors for use in the fission yeast Schizosaccharomyces pombe that allow fusion of any protein of interest to a triple HA epitope or a GST domain . The HA epitope may be placed at the N terminus or the C terminus under three different versions of the nmt1 promoter, to allow varying levels of gene expression . The GST tag may be placed at the N terminus or C terminus under control of a fully active nmt1 promoter . This family of vectors has compatible restriction sites and modular design, so that the protein under study may be exchanged easily between different plasmids . Using the Cdc19p protein as a test case, we have demonstrated that these plasmids can express functional tagged proteins in the fission yeast cell. Genes Cells, 1997 Jun, 2(6), 381 - 99 HsMCM6: a new member of the human MCM/P1 family encodes a protein homologous to fission yeast Mis5; Tsuruga H et al.; BACKGROUND: The tight regulatory mechanism that prevents more than one round of chromosomal DNA replication per cell cycle is thought to require the function of Mcm/P1 proteins . We report here the structural and functional analyses of HsMcm6, a human homologue of the Mis5 of Schizosaccharomyces pombe . RESULTS: We demonstrate here that the transcription of the HsMCM6 gene was repressed in quiescent cells but was rapidly induced at the G1/S phase by growth factor stimulation . The 5' regulatory region of the HsMCM6 gene was found to harbour four putative E2F binding motifs, and these were responsible for the promoter activity . The HsMcm6 protein level oscillated during the cell cycle, with a peak at the G1/S phase . We also showed that the cell-cycle dependent change of subcellular localization of HsMcm6 resembles those of other Mcm/P1 proteins . HsMcm6 consists of two forms, a form extractable by Nonidet P-40 and the nucleus-bound form . A demonstration of the association of HsMcm6 with HsMcm2 and HsMcm7 in vivo supports the idea that they behave as a heteromeric complex . We mapped the HsMCM6 gene at 2q12-14 . CONCLUSION: The results indicate that the behaviour of HsMcm6 is reminiscent of replication licensing factor like other Mcm/P1 family members. Mol Gen Genet, 1997 Jun, 255(2), 226 - 36 The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secretion of M-factor; Christensen PU et al.; In the fission yeast Schizosaccharomyces pombe, cells of opposite mating type communicate via diffusible peptide pheromones prior to mating . We have cloned the S . pombe mam1 gene, which encodes a 1336-amino acid protein belonging to the ATP-binding cassette (ABC) superfamily . The mam1 gene is only expressed in M cells and the gene product is responsible for the secretion of the mating pheromone . M-factor, a nonapeptide that is S-farnesylated and carboxy-methylated on its C-terminal cysteine residue . The predicted Mam1 protein is highly homologous to mammalian multiple drug-resistance proteins and to the Saccharomyces cerevisiae STE6 gene product, which mediates export of a-factor mating pheromone . We show that STE6 can also mediate secretion of M-factor in S . pombe. EMBO J, 1997 Jun, 16(12), 3633 - 43 Transcriptional termination signals for RNA polymerase II in fission yeast; Birse CE et al.; Transcription 'run-on' (TRO) analysis using permeabilized yeast cells indicates that transcription terminates between 180 and 380 bp downstream of the poly(A) site of the Schizosaccharomyces pombe ura4 gene . Two signals direct RNA polymerase II (pol II) to stop transcription: the previously identified 3' end formation signals located close to the poly(A) site and an additional downstream element (DSE) located at the region of termination . The downstream signal (135 bp) appears to act by pausing the elongating polymerase . TRO analysis indicates that elevated levels of transcribing polymerases accumulate over the DSE and that removal of this signal leads to transcription proceeding beyond the normal termination region . Furthermore, when inserted between two competing polyadenylation signals, this DSE increases the utilization of upstream poly(A) sites in vivo . We show that polymerase pausing over an extended region of template ensures termination of pol II transcription close to the poly(A) site. Chromosoma, 1997 Jun, 105(7-8), 542 - 52 Ultrastructural changes in the Schizosaccharomyces pombe nucleolus following the disruption of the gar2+ gene, which encodes a nucleolar protein structurally related to nucleolin; Leger-Silvestre I et al.; The nucleolar protein gar2, from the fission yeast Schizosaccharomyces pombe, is the functional homolog of NSR1 from Saccharomyces cerevisiae, and is structurally related to nucleolin from vertebrates . By immunocytochemistry at the electron microscope level, we show that gar2 co-localizes with RNA polymerase I and the gar1 protein along the dense fibrillar component of the nucleolus in a wild-type strain of S . pombe, suggesting that gar2 is involved in the transcription and/or in the early steps of maturation of the ribosomal RNAs . Since the effects of disruption of the gar2+ gene might also shed light on the role of the gar2 protein, we analyzed the ultrastructure of the nucleolus of a gar2-disruption mutant . The nucleolus of the gar2- mutant is dramatically reorganized when compared with that of the wild-type gar2+ strain: a truncated protein containing the NH2-terminus of the gar2 protein is accumulated in an unusual nucleolar "dense body" . Our results also suggest that the NH2-terminus might be sufficient for nucleolar localization via interaction with specific nucleolar components and support the hypothesis that gar2 in wild-type S . pombe interacts with nascent pre-rRNA via its two RNA-binding domains in combination with the glycine/arginine-rich domain . We also report that disruption of the gar2+ gene results in a mutant that is defective in cytokinesis and nuclear division. Chromosoma, 1997 Jun, 105(7-8), 532 - 41 Mitosis-specific phosphorylation of gar2, a fission yeast nucleolar protein structurally related to nucleolin; Gulli MP et al.; The nucleolar protein gar2 of fission yeast is structurally related to the multifunctional nucleolar protein nucleolin from vertebrates and has been shown to be implicated in production of 18S rRNA . gar2 contains several potential casein kinase 2 (CK2) phosphorylation sites and a single putative p34(cdc2 )phosphorylation site in the consensus S50PKK . Here, we show that, like nucleolin, gar2 is phosphorylated in vitro by both highly purified CK2 from CHO cells and p34(cdc2 )from starfish oocytes . Moreover, the substitution of alanine for the N-terminal serine 50 abolishes phosphorylation by p34(cdc2 )in vitro . We also provide evidence that gar2 is phosphorylated in vitro by a p13(suc1)-Sepharose-bound kinase from Schizosaccharomyces pombe extracts that displays cell cycle-regulated activity similar to that of the p34(cdc2(kinase . In vivo 32P labeling of cells indicates that gar2 is a phosphoprotein and that incorporation of phosphate on residue 50 occurs specifically at mitosis . Taken together, these results lead us to propose that gar2 is likely to be an in vivo substrate for the mitotic p34(cdc2 )kinase . However, this posttranslational modification of the gar2 protein does not appear to be essential for normal production of 18S rRNA. Mol Biol Cell, 1997 Jun, 8(6), 1117 - 28 Ran1 functions to control the Cdc10/Sct1 complex through Puc1; Caligiuri M et al.; We have undertaken a biochemical analysis of the regulation of the G1/S-phase transition and commitment to the cell cycle in the fission yeast Schizosaccharomyces pombe . The execution of Start requires the activity of the Cdc2 protein kinase and the Sct1/Cdc10 transcription complex . Progression through G1 also requires the Ran1 protein kinase whose inactivation leads to activation of the meiotic pathway under conditions normally inhibitory to this process . We have found that in addition to Cdc2, Sct1/Cdc10 complex formation requires Ran1 . We demonstrate that the Puc1 cyclin associates with Ran1 and Cdc10 in vivo and that the Ran1 protein kinase functions to control the association between Puc1 and Cdc10 . In addition, we present evidence that the phosphorylation state of Cdc10 is altered upon inactivation of Ran1 . These results provide biochemical evidence that demonstrate one mechanism by which the Ran1 protein kinase serves to control cell fate through Cdc10 and Puc1. Mol Biol Cell, 1997 Jun, 8(6), 1105 - 15 The Cdc2 protein kinase controls Cdc10/Sct1 complex formation; Connolly T et al.; In the fission yeast Schizosaccharomyces pombe, the execution of Start requires the activity of the Cdc2 protein kinase and the Cdc10/Sct1 transcription complex . The loss of any of these genes leads to G1 arrest and activation of the mating pathway under appropriate conditions . We have undertaken a genetic and biochemical analysis of these genes and their protein products to elucidate the molecular mechanism that governs the regulation of Start . We demonstrate that serine-196 of Cdc10 is phosphorylated in vivo and provide evidence that suggests that phosphorylation of this residue is required for Cdc10 function . Substitution of serine-196 of Cdc10 with alanine (Cdc10 S196A) leads to inactivation of Cdc10 . We show that Cdc10 S196A is incapable of associating with Sct1 to form a heteromeric complex, whereas substitution of this serine with aspartic acid (S196D) restores DNA-binding activity by allowing Cdc10 to associate with Sct1 . Furthermore, we demonstrate that Cdc2 activity is required for the formation of the heteromeric Sct1/Cdc10 transcription complex and that the Cdc10 S196D mutation alleviates this requirement . We thus provide biochemical evidence to demonstrate one mechanism by which the Cdc2 protein kinase may regulate Start in the fission yeast cell cycle. Curr Biol, 1997 Jun 1, 7(6), 418 - 26 The Drosophila grapes gene is related to checkpoint gene chk1/rad27 and is required for late syncytial division fidelity; Fogarty P et al.; BACKGROUND: Cell cycle checkpoints maintain the fidelity of the somatic cell cycle by ensuring that one step in the cell cycle is not initiated until a previous step has been completed . The extent to which cell cycle checkpoints play a role in the initial rapid embryonic divisions of higher eukaryotes is unclear . The initial syncytial divisions of Drosophila embryogenesis provide an excellent opportunity to address this issue as they are amenable to both genetic and cellular analysis . In order to study the relevance of cell cycle checkpoints in early Drosophila embryogenesis, we have characterized the maternal-effect grapes (grp) mutation, which may affect feedback control during early syncytial divisions . RESULTS: The Drosophila grp gene encodes a predicted serine/threonine kinase and has significant homology to chk1/rad27, a gene required for a DNA damage checkpoint in Schizosaccharomyces pombe . Relative to normal embryos, embryos derived from grp-mutant mothers exhibit elevated levels of DNA damage . During nuclear cycles 12 and 13, alignment of the chromosomes on the metaphase plate was disrupted in grp-derived embryos, and the embryos underwent a progression of cytological events that were indistinguishable from those observed in normal syncytial embryos exposed to X-irradiation . The mutant embryos also failed to progress through a regulatory transition in Cdc2 activity that normally occurs during interphase of nuclear cycle 14 . CONCLUSION: We propose that the primary defect in grp-derived embryos is a failure to replicate or repair DNA completely before mitotic entry during the late syncytial divisions . This suggests that wild-type grp functions in a developmentally regulated DNA replication/damage checkpoint operating during the late syncytial divisions . These results are discussed with respect to the proposed function of the chk1/rad27 gene. Biotechniques, 1997 Jun, 22(6), 1134 - 9 Cassette for the generation of sequential gene disruptions in the yeast Schizosaccharomyces pombe; McNabb DS et al.; The ability to conveniently construct gene disruptions is an important methodology for genetic analysis of the fission yeast Schizosaccharomyces pombe . Because of the limited number of selectable markers available for generating gene disruptions in fission yeast, the construction of strains that contain multiple gene disruptions can be quite difficult . This becomes a particular problem when episomal plasmids carrying selectable markers are also required within the same strains . To alleviate these difficulties, we have constructed a hisG-ura(4+)-hisG cassette that can be used repeatedly for constructing gene disruptions in S . pombe . This cassette allows the recycling of the ura4+ marker, thereby permitting the disruption of an indefinite number of genes sequentially within the same strain and/or for subsequently introducing a ura(4+)-marked plasmid. Mol Cell Biol, 1997 Jun, 17(6), 3408 - 17 Plk is a functional homolog of Saccharomyces cerevisiae Cdc5, and elevated Plk activity induces multiple septation structures; Lee KS et al.; Plk is a mammalian serine/threonine protein kinase whose activity peaks at the onset of M phase . It is closely related to other mammalian kinases, Snk, Fnk, and Prk, as well as to Xenopus laevis Plx1, Drosophila melanogaster polo, Schizosaccharomyces pombe Plo1, and Saccharomyces cerevisiae Cdc5 . The M phase of the cell cycle is a highly coordinated process which insures the equipartition of genetic and cellular materials during cell division . To enable understanding of the function of Plk during M phase progression, various Plk mutants were generated and expressed in Sf9 cells and budding yeast . In vitro kinase assays with Plk immunoprecipitates prepared from Sf9 cells indicate that Glu206 and Thr210 play equally important roles for Plk activity and that replacement of Thr210 with a negatively charged residue elevates Plk specific activity . Ectopic expression of wild-type Plk (Plk WT) complements the cell division defect associated with the cdc5-1 mutation in S . cerevisiae . The degree of complementation correlates closely with the Plk activity measured in vitro, as it is enhanced by a mutationally activated Plk, T210D, but is not observed with the inactive forms K82M, D194N, and D194R . In a CDC5 wild-type background, expression of Plk WT or T210D, but not of inactive forms, induced a sharp accumulation of cells in G1 . Consistent with elevated Plk activity, this phenomenon was enhanced by the C-terminally deleted forms WT deltaC and T210D deltaC . Expression of T210D also induced a class of cells with unusually elongated buds which developed multiple septal structures . This was not observed with the C-terminally deleted form T210D deltaC, however . It appears that the C terminus of Plk is not required for the observed cell cycle influence but may be important for polarized cell growth and septal structure formation. Mol Cell Biol, 1997 Jun, 17(6), 3356 - 63 Discrete roles of the Spc1 kinase and the Atf1 transcription factor in the UV response of Schizosaccharomyces pombe; Degols G et al.; Exposure of mammalian cells to UV irradiation or alkylating agents leads to the activation of the c-Jun N-terminal kinase and p38 stress-activated protein kinase cascades, phosphorylation of c-Jun and ATF-2 bZIP transcription factors, and finally to selective induction of gene expression . This UV response is believed to be crucially important for cell survival, although conclusive evidence is lacking . Here, we address this issue by investigating a homologous UV response pathway in the fission yeast Schizosaccharomyces pombe . In fission yeast cells, UV irradiation induces activation of Spc1 stress-activated protein kinase, which in turn phosphorylates the Atf1 bZIP transcription factor . spc1 mutants are hypersensitive to killing by UV at a level equivalent to some checkpoint rad mutants . Whereas checkpoint rad mutants fail to arrest division in response to DNA damage, spc1 mutants are defective at resuming cell division after UV exposure . Levels of basal and UV-induced transcription of ctt1+, which encodes a catalase believed important for combating oxidative stress caused by UV, are extremely low in spc1 mutants . Atf1 is required for UV-induced transcription of ctt1+, but atf1 mutants are not hypersensitive to killing by UV . This surprising finding is explained by the observation that ctt1+ basal expression is unaffected in atf1 single mutant and spc1 atf1 double mutant cells, suggesting that unphosphorylated Atf1 represses ctt1+ expression in spc1 cells . In fact, the level of UV sensitivity of spc1 atf1 double mutant cells is intermediate between those of the wild type and spc1 mutants . These findings suggest the following . (i) Key properties of UV response mechanisms are remarkably similar in mammals and S . pombe . (ii) Activation of Spc1 kinase greatly enhances survival of UV-irradiated cells . (iii) Induction of gene expression by activation of Atf1 may not be the most important mechanism by which stress-activated kinases function in the UV response. Mol Cell Biol, 1997 Jun, 17(6), 3305 - 14 The centromere enhancer mediates centromere activation in Schizosaccharomyces pombe; Ngan VK et al.; The centromere enhancer is a functionally important DNA region within the Schizosaccharomyces pombe centromeric K-type repeat . We have previously shown that addition of the enhancer and cen2 centromeric central core to a circular minichromosome is sufficient to impart appreciable centromere function . A more detailed analysis of the enhancer shows that it is dispensable for centromere function in a cen1-derived minichromosome containing the central core and the remainder of the K-type repeat, indicating that the critical centromeric K-type repeat, like the central core, is characterized by functional redundancy . The centromeric enhancer is required, however, for a central core-carrying minichromosome to exhibit immediate centromere activity when the circular DNA is introduced via transformation into S . pombe . This immediate activation is probably a consequence of a centromere-targeted epigenetic system that governs the chromatin architecture of the region . Moreover, our studies show that two entirely different DNA sequences, consisting of elements derived from two native centromeres, can display centromere function . An S . pombe CENP-B-like protein, Abp1p/Cbp1p, which is required for proper chromosome segregation in vivo, binds in vitro to sites within and adjacent to the modular centromere enhancer, as well as within the centromeric central cores . These results provide direct evidence in fission yeast of a model, similar to one proposed for mammalian systems, whereby no specific sequence is necessary for centromere function but certain classes of sequences are competent to build the appropriate chromatin foundation upon which the centromere/kinetochore can be formed and activated. Mol Cell Biol, 1997 Jun, 17(6), 3103 - 15 A novel mutant allele of Schizosaccharomyces pombe rad26 defective in monitoring S-phase progression to prevent premature mitosis; Uchiyama M et al.; A semipermissive growth condition was defined for a Schizosaccharomyces pombe strain carrying a thermosensitive allele of DNA polymerase delta (pol delta ts03) . Under this condition, DNA polymerase delta is semidisabled and causes a delay in S-phase progression . Using a genetic strategy, we have isolated a panel of mutants that enter premature mitosis when DNA replication is incomplete but which are not defective for arrest in G2/M following DNA damage . We characterized the aya14 mutant, which enters premature mitosis when S phase is arrested by genetic or chemical means . However, this mutant is sensitive to neither UV nor gamma irradiation . Two genomic clones, rad26+ and cds1+, were found to suppress the hydroxyurea sensitivity of the aya14 mutant . Genetic analysis indicates that aya14 is a novel allele of the cell cycle checkpoint gene rad26+, which we have named rad26.a14 . cds1+ is a suppressor which suppresses the S-phase feedback control defect of rad26.a14 when S phase is inhibited by either hydroxyurea or cdc22, but it does not suppress the defect when S phase is arrested by a mutant DNA polymerase . Analyses of rad26.a14 in a variety of cdc mutant backgrounds indicate that strains containing rad26.a14 bypass S-phase arrest but not G1 or late S/G2 arrest . A model of how Rad26 monitors S-phase progression to maintain the dependency of cell cycle events and coordinates with other rad/hus checkpoint gene products in responding to radiation damage is proposed. Nucleic Acids Res, 1997 Jun 1, 25(11), 2138 - 46 Isolation and characterization of the Schizosaccharomyces pombe rhp9 gene: a gene required for the DNA damage checkpoint but not the replication checkpoint; Willson J et al.; Checkpoint controls exist in eukaryotic cells to ensure that cells do not enter mitosis in the presence of DNA damage or unreplicated chromosomes . In Schizosaccharomyces pombe many of the checkpoint genes analysed to date are required for both the DNA damage and the replication checkpoints, an exception being chk1 . We report here on the characterization of nine new methylmethane sulphonate (MMS)-sensitive S.pombe mutants, one of which is defective in the DNA damage checkpoint but not the replication checkpoint . We have cloned and sequenced the corresponding gene . The predicted protein is most similar to the Saccharomyces cerevisiae Rad9 protein, having 46% similarity and 26% identity . The S.pombe protein, which we have named Rhp9 (Rad9 homologue in S . pombe) on the basis of structural and phenotypic similarity, also contains motifs present in BRCA1 and 53BP1 . Deletion of the gene is not lethal and results in a DNA damage checkpoint defect . Epistasis analysis with other S.pombe checkpoint mutants indicates that rhp9 acts in a process involving the checkpoint rad genes and that the rhp9 mutant is phenotypically very similar to chk1. Mol Gen Genet, 1997 May 20, 254(5), 520 - 8 Tolerance of low pH in Schizosaccharomyces pombe requires a functioning pub1 ubiquitin ligase; Saleki R et al.; A strain of Schizosaccharomyces pombe carrying a disrupted Na+/H+ antiporter gene (sod2::sup3-5), in addition to the common auxotrophic mutations, ade6-216, ura4-D18 and leu1-32, is highly sensitive to media adjusted to pH 6.9 . Reversion analysis of this strain yielded a group of revertants capable of growth at pH 6.9 . Two of the revertants elongated and failed to form colonies at pH 3.5 . Genetic characterization of one of the pH-sensitive elongated strains, J227, showed the presence of two independently segregating mutations . One, pub1 (protein ubiquitin ligase 1), has recently been reported as an E3 protein ubiquitin ligase involved in cdc25 turnover . The second has been named elp3-1 (elongated at low pH) . Genetic dissection of the original strain revealed that poor growth at high pH was due to the presence of the auxotrophic markers, suggesting a possible inhibitory effect of high pH on the function of permeases responsible for uptake of the necessary nutrients . Suppression of the high pH sensitivity required the presence of both the pub1-1 and elp3-1 mutations . While the pub1-1 mutation reduced the capacity of cells to tolerate relatively moderate concentrations of LiCl (3 mM) in liquid culture, it was capable of partially suppressing the extreme Li+ sensitivity caused by the sod2 disruption . Under these conditions, the growth of pub1-1 sod2::ura4 double mutant cells was improved over that of either pub1-1 or sod2::ura4 cells . The elp3-1 mutation had no effect on the Li+ tolerance in either wild-type or sod2::ura4 backgrounds . pub1-1 cells are elongated and incapable of colony formation at pH 3.5 . In contrast, elp3-1 cells are elongated at pH 3.5 and pH 5.5 (the normal pH of minimal medium) but can form colonies under both conditions . J227 cells are significantly longer than either single mutant at pH 3.5 and do not form colonies but are visually similar to elp3-1 cells at pH 5.5 . Complementation cloning in the J227 background yielded a genomic clone of pub1, allowing us to define the intron-exon structure of the gene . Sequences with high homology to the predicted amino acid sequence of pub1 have been identified in Saccharomyces cerevisiae (RSP5/NPI1), human (hRPF1), mouse (mNedd4), and rat (rNedd4) . Based on the nature of our mutant selection, the pH-sensitive phenotype of the strains selected, and the known involvement of RSP5/ NPI1 in membrane permease turnover in S . cerevisiae, we hypothesize a role for pub1, either directly or indirectly, in regulating membrane transport processes . This is further supported by the broad range of effects that the pub1-1 mutation exerts on overall performance of cells at high and low external pH, and in the presence of toxic levels of Li+. J Biol Chem, 1997 May 16, 272(20), 13320 - 5 Regulation of Schizosaccharomyces pombe Wee1 tyrosine kinase; Aligue R et al.; Wee1 tyrosine kinase regulates mitosis by carrying out the inhibitory tyrosine 15 phosphorylation of Cdc2 M-phase inducing kinase . Schizosaccharomyces pombe Wee1 is a large protein, consisting of a C-terminal catalytic domain of approximately 350 amino acids preceded by a N-terminal domain of approximately 550 residues . The functional properties of the Wee1 N-terminal domain were investigated by expressing truncated forms of Wee1 in S . pombe . Both positive and negative regulatory domains were identified . Sequences important for Wee1 function were mapped to a central region (residues 363-408) . This region is not required for kinase activity or nuclear localization, suggesting it may be involved in substrate recognition . The negative regulatory domain resides in the N-terminal third of Wee1, Wee1 constructs lacking this domain are more effective at delaying mitosis than wild-type Wee1 . The negative regulatory domain contains clusters of potential Cdc2 phosphorylation sites . Investigations to monitor the abundance of Wee1 mRNA and protein during the cell cycle were also carried out. J Biol Chem, 1997 May 16, 272(20), 13203 - 10 A 2'-phosphotransferase implicated in tRNA splicing is essential in Saccharomyces cerevisiae; Culver GM et al.; The last step of tRNA splicing in the yeast Saccharomyces cerevisiae is catalyzed by an NAD-dependent 2'-phosphotransferase, which transfers the splice junction 2'-phosphate from ligated tRNA to NAD to produce ADP-ribose 1"-2" cyclic phosphate . We have purified the phosphotransferase about 28,000-fold from yeast extracts and cloned its structural gene by reverse genetics . Expression of this gene (TPT1) in yeast or in Escherichia coli results in overproduction of 2'-phosphotransferase activity in extracts . Tpt1 protein is essential for vegetative growth in yeast, as demonstrated by gene disruption experiments . No obvious binding motifs are found within the protein . Several candidate homologs in other organisms are identified by searches of the data base, the strongest of which is in Schizosaccharomyces pombe. Gene, 1997 May 6, 190(2), 287 - 96 Poly(ADP-ribose) polymerase interacts with a novel human ubiquitin conjugating enzyme: hUbc9; Masson M et al.; Poly(ADP-ribose) polymerase (PARP) has been suggested to play a regulatory role in vivo, in DNA replication and/or DNA repair based mainly on its capacity to bind to DNA strand breaks . This interaction is modulated through auto poly(ADP-ribosylation) . However, the biological function of PARP may also involve interactions with proteins such as topoisomerase I or DNA polymerase alpha, which may or may not be themselves ADP-ribosylated . Using the yeast two-hybrid method search for other proteins interacting with PARP, we have isolated a full-length cDNA clone coding for a protein of 158 amino acid residues . This amino acid sequence is 66 and 56% identical to yeast ubiquitin-conjugating enzymes Hus5 and Ubc9 of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively . Moreover, we have demonstrated that the expressed protein complements a S . cerevisiae yeast strain deficient for Ubc9 . The protein encoded by the isolated cDNA is thus a new human counterpart of the ubiquitin-conjugating enzyme family and has been called hUbc9 . The hubc9 gene locus has been assigned to the chromosomal location 16p13.2-p13.3 . By means of two-hybrid analysis it was discovered that hUbc9 interacts with the automodification domain of PARP . This interaction was further confirmed using GST (glutathione-S-transferase) tagged fusion proteins: (i) in vivo, by transfecting cos7 cells with hUbc9 cloned in an eukaryotic expression vector, and (ii) in vitro, by mixing purified PARP with hUbc9 purified and expressed in bacteria . The possible significance and function of this interaction is discussed while taking into account the possible intracellular role of hUbc9. J Biol Chem, 1997 May 2, 272(18), 12100 - 6 Pch1(+), a second essential C-type cyclin gene in Schizosaccharomyces pombe; Furnari BA et al.; The Schizosaccharomyces pombe gene pch1(+) (pombe cyclin C homology) was isolated in a two-hybrid screen for proteins that interact with Cdc2 . The cyclin box region of Pch1 protein shares greatest sequence identity with mammalian and Drosophila C-type cyclins ( approximately 33% identity) . Pch1 is significantly less similar to Mcs2 (19% identity), a second member of the C-type cyclin family in S . pombe . Cdc2 co-precipitates with Pch1 in S . pombe cell lysates, although Cdc2 may not be the major catalytic partner of a Pch1 kinase in vivo . Purified Pch1-associated kinase phosphorylated myelin basic protein, histone H1, and a peptide corresponding to the carboxyl-terminal domain repeat of RNA polymerase II . The amount of pch1 mRNA does not oscillate during the cell cycle, as is the case for mRNA transcripts of other C-type cyclin genes . Deltapch1 cells are inviable, therefore S . pombe has two essential genes that encode members of the C-type cyclin family, pch1(+) and mcs2(+) . The Deltapch1 mutation causes pleiotropic morphological defects and an associated growth deficiency, but loss of Pch1 activity does not result in a cdc cell cycle-arrest phenotype. Bioorg Khim, 1997 May, 23(5), 441 - 8 {Molecular cloning and characterization of cDNA of the rpc10+ gene encoding the smallest subunit of nuclear RNA polymerases of Schizosaccharomyces pombe}; Shpakovskii GV et al.; The full-length cDNA of the rpc10+ gene encoding mini-subunit Rpc10, which is common for all three nuclear RNA polymerases of the fission yeast Schizosaccharomyces pombe, was cloned and sequenced . The Rpc10 subunit of Sz . pombe and its homologs from S . cerevisiae and H . sapiens are positively charged proteins with a highly conserved C-terminal region and an invariant zinc-binding domain (Zn-finger) of a typical amino acid composition: YxCx2Cx12RCx2CGxR . Functional tests of heterospecific complementation, using tetrad analysis or plasmid shuffling, showed that the Rpc10 subunit of Sz . pombe can successfully replace the homologous ABC10 alpha subunit in nuclear RNA polymerases I-III of S . cerevisiae. Yeast, 1997 May, 13(6), 561 - 72 Characterization of the Saccharomyces cerevisiae cdc42-1ts allele and new temperature-conditional-lethal cdc42 alleles; Miller PJ et al.; Cdc42p is a highly conserved GTPase involved in controlling cell polarity and polarizing the actin cytoskeleton . The CDC42 gene was first identified by the temperature-sensitive cell-division-cycle mutant cdc42-1ts in Saccharomyces cerevisiae . We have determined the DNA and predicted amino-acid sequence of the cdc42-1ts allele and identified multiple mutations in the coding region and 5' promoter region, thereby limiting its usefulness in genetic screens . Therefore, we generated additional temperature-conditional-lethal alleles in highly conserved amino-acid residues of both S . cerevisiae and Schizosaccharomyces pombe Cdc42p . The cdc42W97R temperature-sensitive allele in S . cerevisiae displayed the same cell-division-cycle arrest phenotype (large, round unbudded cells) as the cdc42-1ts mutant . However, it exhibited a bud-site selection defect and abnormal bud morphologies at the permissive temperature of 23 degrees C . These phenotypes suggest that Cdc42p functions in bud-site selection early in the morphogenetic process and also in polarizing growth patterns leading to proper bud morphogenesis later in the process . In S . pombe, the cdc42W97R mutant displayed a cold-sensitive, los-of-function phenotype when expressed from the thiamine-repressible nmt1 promoter under repressing conditions . In addition, cdc42T58A and cdc42S71P mutants showed a temperature-sensitive loss-of-function phenotype when expressed in S . pombe: these mutants did not display a conditional phenotype when expressed in S . cerevisiae . These new conditional-lethal cdc42 alleles will be important reagents for the further dissection of the cell polarity pathway in both yeasts. Yeast, 1997 May, 13(6), 499 - 513 Yarrowia lipolytica SRP54 homolog and translocation of Kar2p; Lee IH et al.; To investigate the role of Srp54p in protein translocation, the Yarrowia lipolytica SRP54 homolog was cloned . Sequencing revealed an open reading frame of 536 amino acids coding for a 57.2 kilodalton polypeptide with 55 to 57% sequence identity to Srp54ps of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and mouse . Like these Srp54ps, Y . lipolytica Srp54p has an N-terminal domain with a highly conserved GTP-binding site and a methionine-rich C-terminal domain . Differing results regarding the essentiality of SRP subunits were obtained . SRP54 is important but not essential for growth, but it was reconfirmed that at least one SRP RNA gene is essential . Cells with SRP54 deleted grow about six times more slowly than wild type; faster-growing colonies, still growing much slower than wild type, appeared quite frequently . In srp54 delta cells, no untranslocated alkaline extracellular protease precursor was detected . Therefore, to develop another reporter molecule the Y . lipolytica KAR2 homolog was cloned and Kar2p antibodies were produced . For Kar2p an untranslocated precursor was detected in srp54 delta but not in wild-type cells, suggesting that its transl