Schizosaccharomyces

Nature, 1999 Jul 8, 400(6740), 181 - 4
Orientation of DNA replication establishes mating-type switching pattern in S . pombe; Dalgaard JZ et al.; The fission yeast Schizosaccharomyces pombe normally has haploid cells of two mating types, which differ at the chromosomal locus mat1 . After two consecutive asymmetric cell divisions, only one in four 'grand-daughter' cells undergoes a 'mating-type switch', in which genetic information is transferred to mat1 from the mat2-P or mat3-M donor loci . This switching pattern probably results from an imprinting event at mat1 that marks one sister chromatid in a strand-specific manner, and is related to a site-specific, double-stranded DNA break at mat1 . Here we show that the genetic imprint is a strand-specific, alkali-labile DNA modification at mat1 . The DNA break is an artefact, created from the imprint during DNA purification . We also propose and test the model that mat1 is preferentially replicated by a centromere-distal origin(s), so that the strand-specific imprint occurs only during lagging-strand synthesis . Altering the origin of replication, by inverting mat1 or introducing an origin of replication, affects the imprinting and switching efficiencies in predicted ways . Two-dimensional gel analysis confirmed that mat1 is preferentially replicated by a centromere-distal origin(s) . Thus, the DNA replication machinery may confer different developmental potential to sister cells.

Yeast, 1999 Jul, 15(10A), 893 - 901
DNA sequencing and analysis of a 67.4 kb region from the right arm of Schizosaccharomyces pombe chromosome II reveals 28 open reading frames including the genes his5, pol5, ppa2, rip1, rpb8 and skb1; Xiang Z et al.; 67 393 bp of contiguous DNA located between markers cdc18 and cdc14 on the right arm of fission yeast chromosome II has been sequenced as part of the European Union Schizosaccharomyces pombe genome sequencing project . The complete sequence, contained in cosmid clones c15C4 and c21H7, has been determined on both strands . Sequence analysis shows that it contains 28 open reading frames capable of coding for proteins, 16 split by one or more introns, but no tRNA, rRNA or transposon sequences . The gene density is one per 2 . 4 kb . Six genes have been previously described (his5, pol5, ppa2, rip1, rpb8 and skb1) and 22 are novel . Of the novel genes, 14 have significant similarity with proteins of known function, three have similarities with proteins of unknown function and five show no extensive similarities with known proteins . Sequence similarities suggest that three of the novel genes encode ATP-dependent RNA helicases, two encode transcription factor components and others encode a G-protein, a dehydrogenase, a Rab escort protein, an Abc1-like protein, a lipase, an ATP-binding transport protein, an amino acid permease, an acid phosphatase and a mannosyltransferase .

Yeast, 1999 Jul, 15(10A), 865 - 72
A systematic nomenclature for new translation initiation factor genes from S . pombe and other fungi; Linder P et al.; Eukaryotic translation initiation factors and their corresponding genes have been characterized using biochemical and genetic methods from a variety of different organisms . The designations of the factors relate to their apparent roles in the biochemical process . Many gene names indicate genetic interactions with other genes or the functional attributes used to identify them . On the other hand, progress in systematic sequencing of the genomes of organisms like Saccharomyces cerevisiae and Schizosaccharomyces pombe has revealed many genes homologous to known translation initiation factor genes . The genes defined by the systematic sequencing approach are assigned numerical designations completely unrelated to their biological function . So far there have been publications on only three genes encoding translation initiation factors from Schizosaccharomyces pombe . We therefore see this an an ideal opportunity to propose a systematic and logical nomenclature for genes encoding translation initiation factor genes that can be applied to all further genes of this type that are characterized in this fission yeast .

Yeast, 1999 Jul, 15(10A), 821 - 8
The topoisomerase I poison camptothecin generates a Chk1-dependent DNA damage checkpoint signal in fission yeast; Wan S et al.; The protein kinase Chk1 is essential for the DNA damage checkpoint . Cells lacking Chk1 are hypersensitive to DNA-damaging agents such as UV light and gamma-irradiation because they fail to arrest the cell cycle when DNA damage is generated . Phosphorylation of Chk1 occurs after DNA damage and is dependent on the integrity of the DNA damage checkpoint pathway . We have tested whether a topoisomerase I inhibitor, camptothecin (CPT), generates DNA damage in the fission yeast Schizosaccharomyces pombe that results in Chk1 phosphorylation . We demonstrate that Chk1 is phosphorylated in response to CPT treatment in a time- and dose-dependent manner and that phosphorylation is dependent on an intact DNA damage checkpoint pathway . Furthermore, we show that cells must be actively dividing in order for CPT to generate a Chk1-responsive DNA damage signal . This observation is consistent with a model whereby the cytotoxic event caused by CPT treatment is the production of a DNA double-strand break resulting from the collision of a DNA replication fork with a trapped CPT-topoisomerase I cleavable complex . Cells lacking Chk1 are hypersensitive to CPT treatment, suggesting that the DNA damage checkpoint pathway can be an important determinant for CPT sensitivity or resistance . Finally, as a well-characterized, soluble agent that specifically causes DNA damage, CPT will allow a biochemical analysis of the checkpoint pathway that responds to DNA damage .

Genome Res, 1999 Jun, 9(6), 550 - 7
The genomic tree as revealed from whole proteome comparisons; Tekaia F et al.; The availability of a number of complete cellular genome sequences allows the development of organisms' classification, taking into account their genome content, the loss or acquisition of genes, and overall gene similarities as signatures of common ancestry . On the basis of correspondence analysis and hierarchical classification methods, a methodological framework is introduced here for the classification of the available 20 completely sequenced genomes and partial information for Schizosaccharomyces pombe, Homo sapiens, and Mus musculus . The outcome of such an analysis leads to a classification of genomes that we call a genomic tree . Although these trees are phenograms, they carry with them strong phylogenetic signatures and are remarkably similar to 16S-like rRNA-based phylogenies . Our results suggest that duplication and deletion events that took place through evolutionary time were globally similar in related organisms . The genomic trees presented here place the Archaea in the proximity of the Bacteria when the whole gene content of each organism is considered, and when ancestral gene duplications are eliminated . Genomic trees represent an additional approach for the understanding of evolution at the genomic level and may contribute to the proper assessment of the evolutionary relationships between extant species.

Mol Biol Cell, 1999 Jul, 10(7), 2425 - 40
Functional characterization of the interaction of Ste50p with Ste11p MAPKKK in Saccharomyces cerevisiae; Wu C et al.; The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as the Schizosaccharomyces pombe Byr2p kinase . Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response . The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways . One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains . This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway . The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function . In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p . In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase . Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity.

Mol Biol Cell, 1999 Jul, 10(7), 2199 - 208
Effects of genome position and the DNA damage checkpoint on the structure and frequency of sod2 gene amplification in fission yeast; Patterson TE et al.; The Schizosaccharomyces pombe sod2 gene, located near the telomere on the long arm of chromosome I, encodes a Na+ (or Li+)/H+ antiporter . Amplification of sod2 has previously been shown to confer resistance to LiCl . We analyzed 20 independent LiCl-resistant strains and found that the only observed mechanism of resistance is amplification of sod2 . The amplicons are linear, extrachromosomal elements either 225 or 180 kb long, containing both sod2 and telomere sequences . To determine whether proximity to a telomere is necessary for sod2 amplification, a strain was constructed in which the gene was moved to the middle of the same chromosomal arm . Selection of LiCl-resistant strains in this genetic background also yielded amplifications of sod2, but in this case the amplified DNA was exclusively chromosomal . Thus, proximity to a telomere is not a prerequisite for gene amplification in S . pombe but does affect the mechanism . Relative to wild-type cells, mutants with defects in the DNA damage aspect of the rad checkpoint control pathway had an increased frequency of sod2 amplification, whereas mutants defective in the S-phase completion checkpoint did not . Two models for generating the amplified DNA are presented.

Mol Biol Cell, 1999 Jul, 10(7), 2175 - 90
Isolated mammalian and Schizosaccharomyces pombe ran-binding domains rescue S . pombe sbp1 (RanBP1) genomic mutants; Novoa I et al.; Mammalian Ran-binding protein-1 (RanBP1) and its fission yeast homologue, sbp1p, are cytosolic proteins that interact with the GTP-charged form of Ran GTPase through a conserved Ran-binding domain (RBD) . In vitro, this interaction can accelerate the Ran GTPase-activating protein-mediated hydrolysis of GTP on Ran and the turnover of nuclear import and export complexes . To analyze RanBP1 function in vivo, we expressed exogenous RanBP1, sbp1p, and the RBD of each in mammalian cells, in wild-type fission yeast, and in yeast whose endogenous sbp1 gene was disrupted . Mammalian cells and wild-type yeast expressing moderate levels of each protein were viable and displayed normal nuclear protein import . sbp1(-) yeast were inviable but could be rescued by all four exogenous proteins . Two RBDs of the mammalian nucleoporin RanBP2 also rescued sbp1(-) yeast . In mammalian cells, wild-type yeast, and rescued mutant yeast, exogenous full-length RanBP1 and sbp1p localized predominantly to the cytosol, whereas exogenous RBDs localized predominantly to the cell nucleus . These results suggest that only the RBD of sbp1p is required for its function in fission yeast, and that this function may not require confinement of the RBD to the cytosol . The results also indicate that the polar amino-terminal portion of sbp1p mediates cytosolic localization of the protein in both yeast and mammalian cells.

Biochim Biophys Acta, 1999 Jul 7, 1446(1-2), 93 - 101
Isolation of a novel gene, moc2, encoding a putative RNA helicase as a suppressor of sterile strains in Schizosaccharomyces pombe; Kawamukai M; A novel gene designated moc2, which encodes a putative RNA helicase, was isolated from Schizosaccharomyces pombe on the basis of its suppression of the sterility of two different mutant strains, one of which had elevated levels of cAMP and the other deregulated Ras functioning as a result of an ectopic expression of dominant negative RAS2 . Moc2 is highly homologous to the RNA helicase DED1 of Saccharomyces cerevisiae (58% identity) and PL10 of mouse (50% identity) . Disruption of the moc2 gene indicated that moc2 is essential for cell growth . The moc2 gene seems to have roles in both sexual differentiation and cell growth.

Gene, 1999 Jul 8, 234(2), 209 - 15
Molecular cloning of a cDNA encoding 14-3-3 protein from the protozoan Tetrahymena pyriformis and its mRNA expression during synchronous division; Zhao Y et al.; The 14-3-3 proteins have been identified in a wide variety of eukaryotic cells and diverse biochemical properties have been ascribed to them . Here we have cloned a cDNA encoding 14-3-3 protein from a cDNA library of Tetrahymena pyriformis . This cDNA (Tp14-3-3) encoded an open reading frame consisting of 244 amino acids with predicated molecular mass as 28.1kDa . The predicted protein shares 59.2%, 56.5% and 59.2% identity to Entamoeba histolytica 14-3-3-1, Schizosaccharomyces pombe Rad 24 and human 14-3-3 zeta/delta respectively . On the basis of comparison with other 14-3-3 proteins, two of the putative functional domains (dimerization domain and annexin-similarity domain) were found in Tp14-3-3 . In order to know the role of Tp14-3-3 in Tetrahymena, its mRNA levels during synchronous division were examined by Northern blot analysis . There was a marked increase in Tp14-3-3 mRNA level at 45min after the end of heat treatment, followed by a gradual decrease . These results suggest that the Tp14-3-3 mRNA level might vary during the cell cycle . The accumulation of Tp14-3-3 mRNA before cell division was assumed to be a prerequisite for the initiation of synchronous cell division.

Genomics, 1999 Jul 1, 59(1), 32 - 9
Mouse Hus1, a homolog of the Schizosaccharomyces pombe hus1+ cell cycle checkpoint gene; Weiss RS et al.; Cell cycle checkpoints are regulatory mechanisms that arrest the cell cycle or initiate programmed cell death when critical events such as DNA replication fail to be completed or when DNA or spindle damage occurs . In fission yeast, cell cycle checkpoint responses to DNA replication blocks and DNA damage require the hus1+ gene . Mammalian homologs of hus1+ were recently identified, and here we report a detailed analysis of mouse Hus1 . An approximately 4.2-kb full-length cDNA encoding the 32-kDa mouse Hus1 protein was isolated . The genomic structure and exon-intron boundary sequences of the gene were determined, and mouse Hus1 was found to consist of nine exons . Mouse Hus1 was mapped to the proximal end of chromosome 11 and is therefore a candidate gene for the mouse mutation germ cell deficient, which maps to the same genomic region . Finally, mouse Hus1 was found to be expressed in a variety of adult tissues and at several stages of embryonic development .

Mol Cell, 1999 Jun, 3(6), 781 - 91
The crystal structure of rna1p: a new fold for a GTPase-activating protein; Hillig RC et al.; rna1p is the Schizosaccharomyces pombe ortholog of the mammalian GTPase-activating protein (GAP) of Ran . Both proteins are essential for nuclear transport . Here, we report the crystal structure of rna1p at 2.66 A resolution . It contains 11 leucine-rich repeats that adopt the nonglobular shape of a crescent, bearing no resemblance to RhoGAP or RasGAP . The invariant residues of RanGAP form a contiguous surface, strongly indicating the Ran-binding interface . Alanine mutations identify Arg-74 as a critical residue for GTP hydrolysis . In contrast to RasGAP and RhoGAP, Arg-74 could be substituted by lysine and contributed significantly to the binding of Ran . Therefore, we suggest a GAP mechanism for rna1p, which constitutes a variation of the arginine finger mechanism found for Ras GAP and RhoGAP.

Mol Gen Genet, 1999 Jun, 261(4-5), 716 - 24
The function of the chicken p34CDC2 protein kinase in fission yeast is cold sensitive for cell cycle progression through the G1 phase and temperature sensitive for traversal of mitosis; Schmitz N; The protein kinase p34cdc2 is required at the onset of DNA replication and for entry into mitosis . The catalytic subunit and its regulatory proteins, notably the cyclins, are conserved from yeast to man . This suggests that the control mechanisms necessary for progression through the cell cycle in fission yeast are conserved throughout evolution . This work describes the characterization of a fission yeast strain that is dependent for cell cycle progression on the activity of the p34CDC2 protein kinase from chicken . The response of the chicken p34CDC2 protein kinase to cell cycle components of fission yeast was examined . Cells expressing the chicken p34CDC2 protein divide at reduced size at 31 degrees C . Cells are temperature sensitive at 35.5 degrees C and die as a result of mitotic catastrophe . This phenotype can be rescued by delaying cell cycle progression at the G1-S transition by adding low concentrations of hydroxyurea . Schizosaccharomyces pombe cells that are dependent on chicken p34CDC2 are cold sensitive . At 19 degrees C to 25 degrees C cells arrest in the G1 phase, while traversal of the G2-M transition is not blocked at low temperature . Expression of chicken p34CDC2 in the cold-sensitive G2-M mutant cdc2A21 suppresses the G1 arrest.

Yeast, 1999 Jun 15, 15(8), 669 - 86
Eleven novel sep genes of Schizosaccharomyces pombe required for efficient cell separation and sexual differentiation; Grallert A et al.; Genetic analysis of 20 sterile mutants prone to form hyphae revealed 11 novel ste genes (sep6 to sep16) of Schizosaccharomyces pombe . None of the mutants was completely mycelial . Most mutants formed branching hyphae and showed normal septation . Aberrant septal structures and actin distribution were seen only at 36 degrees C . sep9-307, sep14-576 and sep15-598 showed genetic interactions with sep1-1, a mutation in a forkhead transcription factor homologue . Additional genetic interactions were detected between sep6-194, sep15-598 and cdc16-116, a mutant allele of an anaphase modulator of p34cdc2 . sep9-307 and sep15-598 caused dikaryosis in wee1- background . In mating and sporulation tests, sep6-, sep7-, sep9-, sep10-, sep11- and sep15- proved to be defective in conjugation only, whereas sep8-, sep13- and sep16- were also defective in meiosis-sporulation . sep12- and sep14- were only partially sterile . All mutants could produce M-factor but sep8-, sep11-, sep15- and sep16- were defective in P-factor production . The mutations in sep8, sep11 and sep16 suppressed the pat1-114-driven meiosis . All mutants were sensitive to the presence of higher concentrations of chloride in the medium and to short heat shocks . The diversity of the mutant phenotypes and the pleiotropic effects of the mutations suggest that these sep genes might act in, or interact with, a multiple overlapping network of regulatory modules.

Yeast, 1999 Jun 15, 15(8), 639 - 45
Biotransformation of steroids by the fission yeast Schizosaccharomyces pombe; Pajic T et al.; The fungal biotransformation of steroids is of applied interest due to the economic importance of such stereo- and regiospecific reactions and also in the context of ergosterol pathway engineering to produce vitamin D and steroidal products . In Schizosaccharomyces pombe no steroid hydroxylation as is found in filamentous fungi was observed, but a cytosolic NAD(H)/NADP(H)-dependent hydroxysteroid dehydrogenase activity was identified . Progesterone was reduced at the delta 4 double bond (in vivo only) as well as at the C-3 and C-20 keto groups . Testosterone and 4-androstene-3,17-dione were interconverted and 5 alpha-pregnane-3,20-dione and 5 beta-pregnane-3,20-dione were reduced to 3-hydroxy products . The reactions were sometimes reversible and showed regio- and stereo specificity . In S . pombe more than one steroid dehydrogenase homologue is likely to occur, as has been observed in Saccharomyces cerevisiae . Our findings indicate that genes encoding soluble proteins should be examined as candidates for actual steroid dehydrogenase activity.

EMBO J, 1999 Jul 1, 18(13), 3746 - 56
The NES-Crm1p export pathway is not a major mRNA export route in Saccharomyces cerevisiae; Neville M et al.; Nuclear export signal (NES)-containing proteins are recognized by the NES receptor CRM1/Crm1p (also called exportin 1/Xpo1p) . In vertebrates and Schizosaccharomyces pombe, the toxin leptomycin B (LMB) inhibits CRM1-mediated export by interacting directly with CRM1 and disrupting the trimeric Ran-GTP-CRM1-NES export complex . In Saccharomyces cerevisiae, LMB is not toxic and is apparently unable to interact with Crm1p . A second difference between the systems is that LMB has no effect on mRNA export in vertebrate systems, whereas there is evidence that S.cerevisiae Crm1p plays a role in mRNA export . Here we show that a single amino acid change converts S . cerevisiae Crm1p from being LMB insensitive to fully LMB sensitive, indicating that Crm1p is the only relevant LMB target . This new strain has no phenotype, but LMB has a rapid and potent inhibitory effect on NES-mediated export . In situ hybridization assays show that LMB also causes nuclear accumulation of poly(A)+ RNA but with a significant delay compared with the effect on NES-mediated export . Biochemical assays indicate little or no LMB effect on cytoplasmic protein synthesis, indicating that the NES-Crm1p pathway is not a major mRNA export route in S.cerevisiae . We conclude that Crm1p structure and function is conserved from S.cerevisiae to man.

Nucleic Acids Res, 1999 Jul 15, 27(14), 2883 - 8
Terminator element mutations affect both the efficiency and position of RNA polymerase I termination in Schizosaccharomyces pombe; Shwed PS et al.; RNA polymerase I transcripts, purified from Schizosaccharomyces pombe cells, terminate at three sites that precede 'Sal box'-like termination element (TE) sequences . Essential features in these elements were investigated by the in vivo expression of targeted mutations . RNA analyses confirmed a functional significance for two of the elements (Boxes 1 and 3), but indicated that the third, less related, sequence (Box 2) does not function as a termination signal . The results further indicated that the most conserved residues in the two active TEs, as well as adjacent regions, are also most critical to function . Furthermore, some mutations in these elements or in immediately flanking sequences affect not only the efficiency of termination, but also alter the position of termination by as much as 35 nt . Since the element is able to influence the site of termination over a surprisingly long stretch of DNA sequence, these observations suggest that the TE does not act simply as a pause element by fixing the termination factor.

Nucleic Acids Res, 1999 Jul 15, 27(14), 2868 - 74
Removal of cyclobutane pyrimidine dimers by the UV damage repair and nucleotide excision repair pathways of Schizosaccharomyces pombe at nucleotide resolution; Lombaerts M et al.; In Schizosaccharomyces pombe two different repair mechanisms remove UV-induced lesions from DNA, i.e . nucleotide excision repair (NER) and UV damage repair (UVDR) . Here, the kinetics of removal of cyclobutane pyrimidine dimers (CPDs) by both pathways is determined at base resolution in the transcribed strand (TS) and the non-transcribed strand (NTS) of the sprpb2 +gene . UVDR does not remove lesions in a strand-specific manner, indicating that UVDR is neither stimulated nor inhibited by RNA polymerase II transcription . In contrast, in a UVDR-deficient strain the TS is repaired preferentially . This strong strand bias suggests that in S.pombe, as in other species, NER is coupled to transcription . In repair-proficient S.pombe the TS is repaired very rapidly, as a consequence of two efficiently operating pathways, while the NTS is repaired more slowly, mainly by UVDR . Furthermore, we demonstrate that UVDR is not always faster than NER.

Antimicrob Agents Chemother, 1999 Jul, 43(7), 1725 - 8
Purification, reconstitution, and inhibition of cytochrome P-450 sterol delta22-desaturase from the pathogenic fungus Candida glabrata; Lamb DC et al.; Sterol delta22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14alpha-demethylase (cytochrome P-45051; CYP51) . The purified cytochrome P-450 exhibited sterol delta22-desaturase activity in a reconstituted system with NADPH-cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 microM and a Vmax of 0 . 59 nmol of this substrate metabolized/min/nmol of P-450 . This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects . Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme . The azole antifungal compounds inhibited reconstituted sterol delta22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added . These results reveal the potential for sterol delta22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell.

Genetics, 1999 Jul, 152(3), 895 - 908
Identification and characterization of Schizosaccharomyces pombe asp1(+), a gene that interacts with mutations in the Arp2/3 complex and actin; Feoktistova A et al.; The Arp2/3 complex is an essential component of the actin cytoskeleton in yeast and is required for the movement of actin patches . In an attempt to identify proteins that interact with this complex in the fission yeast Schizosaccharomyces pombe, we sought high-copy suppressors of the S . pombe arp3-c1 mutant, and have identified one, which we have termed asp1(+) . The asp1(+) open reading frame (ORF) predicts a highly conserved protein of 921 amino acids with a molecular mass of 106 kD that does not contain motifs of known function . Neither asp1(+) nor its apparent Saccharomyces cerevisiae ortholog, VIP1, are essential genes . However, disruption of asp1(+) leads to altered morphology and growth properties at elevated temperatures and defects in polarized growth . The asp1 disruption strain also is hypersensitive to Ca+ ions and to low pH conditions . Although Asp1p is not stably associated with the Arp2/3 complex nor localized in any discrete structure within the cytoplasm, the asp1 disruption mutant was synthetically lethal with mutations in components of the Arp2/3 complex, arp3-c1 and sop2-1, as well as with a mutation in actin, act1-48 . Moreover, the vip1 disruption strain showed a negative genetic interaction with a las17Delta strain . We conclude that Asp1p/Vip1p is important for the function of the cortical actin cytoskeleton.

Genetics, 1999 Jul, 152(3), 869 - 80
Characterization of the ptr6(+) gene in fission yeast: a possible involvement of a transcriptional coactivator TAF in nucleocytoplasmic transport of mRNA; Shibuya T et al.; Transport of mRNA from the nucleus to the cytoplasm is one of the important steps in gene expression in eukaryotic cells . To elucidate a mechanism of mRNA export, we identified a novel ptr {poly(A)+ RNA transport} mutation, ptr6, which causes accumulation of mRNA in the nucleus and inhibition of growth at the nonpermissive temperature . The ptr6(+) gene was found to encode an essential protein of 393 amino acids, which shares significant homology in amino acid sequence with yTAFII67 of budding yeast Saccharomyces cerevisiae and human hTAFII55, a subunit of the general transcription factor complex TFIID . A Ptr6p-GFP fusion protein is localized in the nucleus, suggesting that Ptr6p functions there . Northern blot analysis using probes for 10 distinct mRNAs showed that the amount of tbp+ mRNA encoding the TATA-binding protein is increased five- to sixfold, whereas amounts of others are rapidly decreased at the nonpermissive temperature in ptr6-1 . ptr6 has no defects in nuclear import of an NLS-GFP fusion protein . These results suggest that Ptr6p required for mRNA transport is a Schizosaccharomyces pombe homologue of yTAFII67 and hTAFII55 . This is the first report suggesting that a TAF is involved in the nucleocytoplasmic transport of mRNA in addition to the transcription of the protein-coding genes.

Genetics, 1999 Jul, 152(3), 839 - 51
Rereplication phenomenon in fission yeast requires MCM proteins and other S phase genes; Snaith HA et al.; The fission yeast Schizosaccharomyces pombe can be induced to perform multiple rounds of DNA replication without intervening mitoses by manipulating the activity of the cyclin-dependent kinase p34(cdc2) . We have examined the role in this abnormal rereplication of a large panel of genes known to be involved in normal S phase . The genes analyzed can be grouped into four classes: (1) those that have no effect on rereplication, (2) others that delay DNA accumulation, (3) several that allow a gradual increase in DNA content but not in genome equivalents, and finally, (4) mutations that completely block rereplication . The rereplication induced by overexpression of the CDK inhibitor Rum1p or depletion of the Cdc13p cyclin is essentially the same and requires the activity of two minor B-type cyclins, cig1(+) and cig2(+) . In particular, the level, composition, and localization of the MCM protein complex does not alter during rereplication . Thus rereplication in fission yeast mimics the DNA synthesis of normal S phase, and the inability to rereplicate provides an excellent assay for novel S-phase mutants.

Genetics, 1999 Jul, 152(3), 827 - 38
Regulation of mRNA export by nutritional status in fission yeast; Whalen WA et al.; We have isolated a mutation in nup184(nup184-1) that is synthetically lethal with the mRNA export defective rae1-167 mutation in Schizosaccharomyces pombe . The consequence of the synthetic lethality is a defect in mRNA export . The predicted Nup184p is similar to Nup188p of Saccharomyces cerevisiae, and a Nup184p-GFP fusion localizes to the nuclear periphery in a punctate pattern . The Deltanup184 null mutant is viable and also is synthetically lethal with rae1-167 . In a rae1(+) background, both the nup184-1 and Deltanup184 mutations confer sensitivity to growth in nutrient-rich medium (YES) that is accompanied by nuclear poly(A)+ RNA accumulation . Removal of the cAMP-dependent protein kinase, Pka1p, relieved the growth and mRNA export defects of nup184 mutants when grown in nutrient-rich medium . The activation of Pka1p is necessary, but not sufficient, to cause the severe poly(A)+ RNA export defects when nup184 mutant cells are incubated in YES, suggesting nutritional status can also regulate poly(A)+ RNA export . Our results suggest that the regulation of poly(A)+ RNA export by Pka1p kinase appears to be indirect, via a translation-dependent step, but post-translationally in response to YES.

FEBS Lett, 1999 Jun 11, 452(3), 305 - 8
The Drosophila gene 2A5 complements the defect in mitochondrial F1-ATPase assembly in yeast lacking the molecular chaperone Atp11p; Wang ZG et al.; Assembly of mitochondrial F1-ATPase in Saccharomyces cerevisiae requires the molecular chaperone, Atp11p . Database searches have identified protein sequences from Schizosaccharomyces pombe and two species of Drosophila that are homologous to S . cerevisiae Atp11p . A cDNA encoding the putative Atp11p from Drosophila yakuba was shown to complement the respiratory deficient phenotype of yeast harboring an atp11::HIS3 disruption allele . Furthermore, the product of this Drosophila gene was shown to interact with the S . cerevisiae F1 beta subunit in the yeast two-hybrid assay . These results indicate that Atp11p function is conserved in higher eukaryotes.

Chromosoma, 1999 May, 108(2), 103 - 13
Functional compartmentalization of the nucleus in the budding yeast Saccharomyces cerevisiae; Leger-Silvestre I et al.; By combining cryofixation and cryosubstitution in a structural and functional analysis of the nucleus of Saccharomyces cerevisiae, we identified morphological subcompartments in the nucleolus . These were similar to those of nucleoli of higher eukaryotes, such as the fibrillar centre (FC), the dense fibrillar component (DFC) and the granular component (GC) . In situ hybridization and immunocytochemistry revealed RNA polymerase I and proteins involved in early steps of ribosomal maturation along the DFC, while the ribosomal genes were detected at the FCs . Our results also suggest that ribosomal transcripts are distributed along a nucleolar network that might include both DFC and GC . We also show that pre-ribosomal subunits may be exported along tracks to the cytoplasm . Export takes place through all the pores of the nuclear envelope, not just those in contact with the nucleolus . Moreover, comparison of the nucleolar organization in S . cerevisiae and in Schizosaccharomyces pombe demonstrated than the distribution of the 5S genes with respect to the 35S transcription unit does not modify the organization of the nucleolus . We also report, for the first time, the ultrastructural localization of RNA polymerase II in yeast . The distribution of RNA polymerase II and morphological details that could be observed in the extra-nucleolar region of cryofixed cells provided cytological evidence of a peripheral region extending along the nuclear envelope that could correspond to heterochromatin in higher eukaryotes.

J Cell Sci, 1999 Jul, 112 ( Pt 14), 2381 - 90
Functionally homologous DNA replication genes in fission and budding yeast; Sanchez M et al.; The cdc18(+) gene of the fission yeast Schizosaccharomyces pombe is involved in the initiation of DNA replication as well as in coupling the S phase to mitosis . In this work, we show that the Saccharomyces cerevisiae CDC6 gene complements cdc18-K46 ts and cdc18 deletion mutant S . pombe strains . The budding yeast gene suppresses both the initiation and the checkpoint defects associated with the lack of cdc18(+) . The Cdc6 protein interacts in vivo with Cdc2 kinase complexes . Interestingly, Cdc6 is an in vitro substrate for Cdc13/Cdc2 and Cig1/Cdc2, but not for Cig2/Cdc2-associated kinases . Overexpression of Cdc6 in fission yeast induces multiple rounds of S-phase in the absence of mitosis and cell division . This CDC6-dependent continuous DNA synthesis phenotype is independent of the presence of a functional cdc18(+) gene product and, significantly, requires only Cig2/Cdc2-associated kinase activity . Finally, these S . pombe over-replicating cells do not require any protein synthesis other than that of Cdc6 . Our data strongly suggest that CDC6 and cdc18(+) are functional homologues and also support the idea that controls restricting genome duplication diverge in fission and budding yeast.

J Cell Sci, 1999 Jul, 112 ( Pt 14), 2313 - 21
Asymmetry of the spindle pole bodies and spg1p GAP segregation during mitosis in fission yeast; Cerutti L et al.; In the fission yeast Schizosaccharomyces pombe, the onset of septum formation is induced by a signal transduction network involving several protein kinases and a GTPase switch . One of the roles of the spg1p GTPase is to localise the cdc7p protein kinase to the poles of the mitotic spindle, from where the onset of septation is thought to be signalled at the end of mitosis . Immunofluorescence studies have shown that cdc7p is located on both spindle pole bodies early in mitosis, but only on one during the later stages of anaphase . This is mediated by inactivation of spg1p on one pole before the other . The GAP for spg1p is a complex of two proteins, cdc16p and byr4p . Localisation of cdc16p and byr4p by indirect immunofluorescence during the mitotic cell cycle showed that both proteins are present on the spindle pole body in interphase cells . During mitosis, byr4p is seen first on both poles of the spindle, then on only one . This occurs prior to cdc7p becoming asymmetric . In contrast, the signal due to cdc16p decreases to a low level during early mitosis, before being seen strongly on the same pole as byr4p . Double staining indicates that this is the opposite pole to that which retains cdc7p in late anaphase . Examination of the effect of inactivating cdc16p at various stages of the cell cycle suggests that cdc16p, together with cdc2p plays a role in restraining septum formation during interphase . The asymmetric inactivation of spg1p is mediated by recruitment of the cdc16p-byr4p GAP to one of the poles of the spindle before the other, and the asymmetry of the spindle pole bodies may be established early during mitosis . Moreover, the spindle pole bodies appear to be non-equivalent even after division has been completed.

Can J Microbiol, 1999 Feb, 45(2), 125 - 9
The role of catalase in hydrogen peroxide resistance in fission yeast Schizosaccharomyces pombe; Mutoh N et al.; The role of catalase in hydrogen peroxide resistance in Schizosaccharomyces pombe was investigated . A catalase gene disruptant completely lacking catalase activity is more sensitive to hydrogen peroxide than the parent strain . The mutant does not acquire hydrogen peroxide resistance by osmotic stress, a treatment that induces catalase activity in the wild-type cells . The growth rate of the disruptant is not different from that of the parent strain . Additionally, transformed cells that overexpress the catalase activity are more resistant to hydrogen peroxide than wildtype cells with normal catalase activity . These results indicate that the catalase of S . pombe plays an important role in resistance to high concentrations of hydrogen peroxide but offers little in the way of protection from the hydrogen peroxide generated in small amounts under normal growth conditions.

Acta Microbiol Immunol Hung, 1999, 46(2-3), 297 - 302
Genetics, physiology and cytology of yeast-mycelial dimorphism in fission yeasts; Sipiczki M et al.; The order Schizosaccharomycetales contains a dimorphic and two yeast species . Sch . japonicus can form both yeast cells and mycelium, depending on the substrate and the culturing conditions . Sch . pombe is a strictly unicellular organism, but it can be forced to form mycelial cell chains by inactivating members of the sep gene family . The mutations in most of the sep genes confer pleitropic phenotypes indicating functional involvement in MAP-kinase-mediated signalling pathways . Two of them were found to encode transcription factor homologues of other eukaryotes.

RNA, 1999 Jun, 5(6), 739 - 50
C-terminal interaction of translational release factors eRF1 and eRF3 of fission yeast: G-domain uncoupled binding and the role of conserved amino acids; Ebihara K et al.; Translation termination in eukaryotes requires a stop codon-responsive (class-I) release factor, eRF1, and a guanine nucleotide-responsive (class-II) release factor, eRF3 . Schizosaccharomyces pombe eRF3 has an N-terminal polypeptide similar in size to the prion-like domain of Saccharomyces cerevisiae eRF3 in addition to the EF-1alpha-like catalytic domain . By in vivo two-hybrid assay as well as by an in vitro pull-down analysis using purified proteins of S . pombe as well as of S . cerevisiae, eRF1 bound to the C-terminal one-third domain of eRF3, named eRF3C, but not to the N-terminal two-thirds, which was inconsistent with the previous report by Paushkin et al . (1997, Mol Cell Biol 17:2798-2805) . The activity of S . pombe eRF3 in eRF1 binding was affected by Ala substitutions for the C-terminal residues conserved not only in eRF3s but also in elongation factors EF-Tu and EF-1alpha . These single mutational defects in the eRF1-eRF3 interaction became evident when either truncated protein eRF3C or C-terminally altered eRF1 proteins were used for the authentic protein, providing further support for the presence of a C-terminal interaction . Given that eRF3 is an EF-Tu/EF-1alpha homolog required for translation termination, the apparent dispensability of the N-terminal domain of eRF3 for binding to eRF1 is in contrast to importance, direct or indirect, in EF-Tu/EF-1alpha for binding to aminoacyl-tRNA, although both eRF3 and EF-Tu/EF-1alpha share some common amino acids for binding to eRF1 and aminoacyl-tRNA, respectively . These differences probably reflect the independence of eRF1 binding in relation to the G-domain function of eRF3 (i.e., probably uncoupled with GTP hydrolysis), whereas aminoacyl-tRNA binding depends on that of EF-Tu/EF-1alpha(i.e., coupled with GTP hydrolysis), which sheds some light on the mechanism of eRF3 function.

Nucleic Acids Res, 1999 Jul 1, 27(13), 2655 - 61
The role of Schizosaccharomyces pombe Rad32, the Mre11 homologue, and other DNA damage response proteins in non-homologous end joining and telomere length maintenance; Wilson S et al.; The Schizosaccharomyces pombe homologue of Mre11, Rad32, is required for repair of UV- and ionising radiation-induced DNA damage and meiotic recombination . In this study we have investigated the role of Rad32 and other DNA damage response proteins in non-homologous end joining (NHEJ) and telomere length maintenance in S.pombe . We show that NHEJ in S.pombe occurs by an error-prone mechanism, in contrast to the accurate repair observed in Saccharomyces cerevisiae . Deletion of the rad32 gene results in a modest reduction in NHEJ activity and the remaining repair events that occur are accurate . Mutations in two of the phosphoesterase motifs in Rad32 have no effect on the efficiency or accuracy of end joining, suggesting that the role of Rad32 protein may be to recruit another nuclease(s) for processing during the end joining reaction . We also analysed NHEJ in other DNA damage response mutants and showed that the checkpoint mutant rad3-d and two recombination mutants defective in rhp51 and rhp54 (homologues of S.cerevisiae RAD51 and RAD54, respectively) are not affected . However disruption of rad22, rqh1 and rhp9 / crb2 (homologues of the S.cerevisiae RAD52, SGS1 and RAD9 genes) resulted in increased NHEJ activity . Telomere lengths in the rad32, rhp9 and rqh1 null alleles were reduced to varying extents intermediate between the lengths observed in wild-type and rad3 null cells.

Mol Cell Biol, 1999 Jul, 19(7), 4703 - 10
A Uve1p-mediated mismatch repair pathway in Schizosaccharomyces pombe; Kaur B et al.; UV damage endonuclease (Uve1p) from Schizosaccharomyces pombe was initially described as a DNA repair enzyme specific for the repair of UV light-induced photoproducts and proposed as the initial step in an alternative excision repair pathway . Here we present biochemical and genetic evidence demonstrating that Uve1p is also a mismatch repair endonuclease which recognizes and cleaves DNA 5' to the mispaired base in a strand-specific manner . The biochemical properties of the Uve1p-mediated mismatch endonuclease activity are similar to those of the Uve1p-mediated UV photoproduct endonuclease . Mutants lacking Uve1p display a spontaneous mutator phenotype, further confirming the notion that Uve1p plays a role in mismatch repair . These results suggest that Uve1p has a surprisingly broad substrate specificity and may function as a general type of DNA repair protein with the capacity to initiate mismatch repair in certain organisms.

Mol Cell Biol, 1999 Jul, 19(7), 4633 - 42
Interaction between the product of the breast cancer susceptibility gene BRCA2 and DSS1, a protein functionally conserved from yeast to mammals; Marston NJ et al.; Germ line mutations in the breast cancer susceptibility gene BRCA2 predispose to early-onset breast cancer, but the function of the nuclear protein encoded by the gene is ill defined . Using the yeast two-hybrid system with fragments of human BRCA2, we identified an interaction with the human DSS1 (deleted in split hand/split foot) gene . Yeast and mammalian two-hybrid assays showed that DSS1 can associate with BRCA2 in the region of amino acids 2472 to 2957 in the C terminus of the protein . Using coimmunoprecipitation of epitope-tagged BRCA2 and DSS1 cDNA constructs transiently expressed in COS cells, we were able to demonstrate an association . Furthermore, endogenous BRCA2 could be coimmunoprecipitated with endogenous DSS1 in MCF7 cells, demonstrating an in vivo association . Apparent orthologues of the mammalian DSS1 gene were identified in the genome of the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae . Yeast strains in which these DSS1-like genes were deleted showed a temperature-sensitive growth phenotype, which was analyzed by flow cytometry . This provides evidence for a link between the BRCA2 tumor suppressor gene and a gene required for completion of the cell cycle.

FEBS Lett, 1999 May 28, 451(3), 321 - 6
Identification of cold-sensitive mutations in the Schizosaccharomyces pombe actin locus; McCollum D et al.; In recent years, the actin cytoskeleton in Schizosaccharomyces pombe has become the subject of intense scrutiny . However, to date, only a single actin mutation has been identified . Described here is the isolation and characterization of four new cold-sensitive actin mutations . Sequence analysis of the mutant actin genes indicated that each of these mutations caused alterations in single amino acids that are conserved in all actin sequences . These mutants differ in their phenotypes . One of these mutations (act1-48) was identified as an extragenic suppressor of a mutation in the cdc4 gene, which is required for actin ring formation and cytokinesis . Interestingly, when act1-48 mutant cells were shifted to the restrictive temperature, actin patches were not detected but the actin ring formation and stability was unaffected . The three other mutations, act1-16, act1-32 and act1-67, primarily affected the actin ring formation or stability while F-actin patches did not seem to be substantially different in appearance . Given that the ultrastructural architectures of F-actin patches and the F-actin ring are presently unclear, these mutations, which affect one structure or the other, should be useful for future studies on the role of actin itself in the function of these F-actin-containing structures in S . pombe.

FEBS Lett, 1999 May 28, 451(3), 295 - 8
Schizosaccharomyces pombe UDP-galactose transporter: identification of its functional form through cDNA cloning and expression in mammalian cells; Segawa H et al.; The Schizosaccharomyces pombe UDP-galactose transporter cDNA (SpUGT cDNA), encoding the product of the gms1+ gene which consists of two exon sequences separated by a 173-bp intron, was cloned by RT-PCR . Its product, a hydrophobic protein of 353 amino acid residues resembling its human counterpart, was expressed in the Golgi membranes of UDP-galactose transporter-deficient Lec8 cells, and complemented the genetic defect of the mutant cells . This indicated that SpUGT cDNA encodes the functional S . pombe UDP-galactose transporter . The product of an ORF found in the second exon, which was previously assumed to be the S . pombe UDP-galactose transporter, thus represents an inactive, truncated form of the SpUGT protein.

J Mol Biol, 1999 Jun 18, 289(4), 691 - 9
Polyanionic inhibitors of phosphoglycerate mutase: combined structural and biochemical analysis; Rigden DJ et al.; The effects that the inhibitors inositol hexakisphosphate and benzene tri-, tetra- and hexacarboxylates have on the phosphoglycerate mutases from Saccharomyces cerevisiae and Schizosaccharomyces pombe have been determined . Their Kivalues have been calculated, and the ability of the inhibitors to protect the enzymes against limited proteolysis investigated . These biochemical data have been placed in a structural context by the solution of the crystal structures of S . cerevisiae phosphoglycerate mutase soaked with inositol hexakisphosphate or benzene hexacarboxylate . These large polyanionic compounds bind to the enzyme so as to block the entrance to the active-site cleft . They form multiple interactions with the enzyme, consistent with their low Kivalues, and afford good protection against limited proteolysis of the C-terminal region by thermolysin . The inositol compound is more efficacious because of its greater number of negative charges . The S . pombe phosphoglycerate mutase that is inherently lacking a comparable C-terminal region has higher Kivalues for the compounds tested . Moreover, the S . pombe enzyme is less sensititive to proteolysis, and the presence or absence of the inhibitor molecules has little effect on susceptibility to proteolysis .

EMBO J, 1999 Jun 15, 18(12), 3325 - 33
Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast; Clemens S et al.; Phytochelatins play major roles in metal detoxification in plants and fungi . However, genes encoding phytochelatin synthases have not yet been identified . By screening for plant genes mediating metal tolerance we identified a wheat cDNA, TaPCS1, whose expression in Saccharomyces cerevisiae results in a dramatic increase in cadmium tolerance . TaPCS1 encodes a protein of approximately 55 kDa with no similarity to proteins of known function . We identified homologs of this new gene family from Arabidopsis thaliana, Schizosaccharomyces pombe, and interestingly also Caenorhabditis elegans . The Arabidopsis and S.pombe genes were also demonstrated to confer substantial increases in metal tolerance in yeast . PCS-expressing cells accumulate more Cd2+ than controls . PCS expression mediates Cd2+ tolerance even in yeast mutants that are either deficient in vacuolar acidification or impaired in vacuolar biogenesis . PCS-induced metal resistance is lost upon exposure to an inhibitor of glutathione biosynthesis, a process necessary for phytochelatin formation . Schizosaccharomyces pombe cells disrupted in the PCS gene exhibit hypersensitivity to Cd2+ and Cu2+ and are unable to synthesize phytochelatins upon Cd2+ exposure as determined by HPLC analysis . Saccharomyces cerevisiae cells expressing PCS produce phytochelatins . Moreover, the recombinant purified S.pombe PCS protein displays phytochelatin synthase activity . These data demonstrate that PCS genes encode phytochelatin synthases and mediate metal detoxification in eukaryotes.

Plant Cell, 1999 Jun, 11(6), 1153 - 64
Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe; Ha SB et al.; Phytochelatins (PCs), a family of heavy metal-inducible peptides important in the detoxification of heavy metals, have been identified in plants and some microorganisms, including Schizosaccharomyces pombe, but not in animals . PCs are synthesized enzymatically from glutathione (GSH) by PC synthase in the presence of heavy metal ions . In Arabidopsis, the CAD1 gene, identified by using Cd-sensitive, PC-deficient cad1 mutants, has been proposed to encode PC synthase . Using a positional cloning strategy, we have isolated the CAD1 gene . Database searches identified a homologous gene in S . pombe, and a mutant with a targeted deletion of this gene was also Cd sensitive and PC deficient . Extracts of Escherichia coli cells expressing a CAD1 cDNA or the S . pombe gene catalyzing GSH-dependent, heavy metal-activated synthesis of PCs in vitro demonstrated that both genes encode PC synthase activity . Both enzymes were activated by a range of metal ions . In contrast, reverse transcription-polymerase chain reaction experiments showed that expression of the CAD1 mRNA is not influenced by the presence of Cd . A comparison of the two predicted amino acid sequences revealed a highly conserved N-terminal region, which is presumed to be the catalytic domain, and a variable C-terminal region containing multiple Cys residues, which is proposed to be involved in activation of the enzyme by metal ions . Interestingly, a similar gene was identified in the nematode, Caenorhabditis elegans, suggesting that PCs may also be expressed in some animal species.

J Biol Chem, 1999 Jun 18, 274(25), 17691 - 5
Phosphorylation of the myosin-II light chain does not regulate the timing of cytokinesis in fission yeast; McCollum D et al.; Proper coordination of cytokinesis with chromosome separation during mitosis is crucial to ensure that each daughter cell inherits an equivalent set of chromosomes . It has been proposed that one mechanism by which this is achieved is through temporally regulated myosin regulatory light chain (RLC) phosphorylation (Satterwhite, L . L., and Pollard, T . D . (1992) Curr . Opin . Cell Biol . 4, 43-52) . A variety of evidence is consistent with this model . A direct test of the importance of RLC phosphorylation in vivo has been done only in Dictyostelium and Drosophila; phosphorylation of the RLC is essential in Drosophila (Jordan, P., and Karess, R . (1997) J . Cell Biol . 139, 1805-1819) but not essential in Dictyostelium (Ostrow, B . D., Chen, P., and Chisholm, R . L . (1994) J . Cell Biol . 127, 1945-1955) . The Schizosaccharomyces pombe myosin light chain Cdc4p is essential for cytokinesis, but it was unknown whether phosphorylation played a role in its regulation . Here we show that the S . pombe myosin light chain Cdc4p is phosphorylated in vivo on either serine 2 or 6 but not both . Mutation of either or both of these sites to alanine did not effect the ability of Cdc4p to bind the type II myosin Myo2p, and cells expressing only these mutated versions of Cdc4p grew and divided normally . Similarly, mutation of Ser-2, Ser-6, or both residues to aspartic acid did not affect growth or division of cells . Thus we conclude that phosphorylation of Cdc4p is not essential in vivo for the function of the protein.

FEMS Microbiol Lett, 1999 Jun 1, 175(1), 143 - 8
Candida albicans exoglucanase as a reporter gene in Schizosaccharomyces pombe; Molero G et al.; The Candida albicans XOG1 gene, previously shown to be a good reporter gene in Saccharomyces cerevisiae and C . albicans, was tested in Schizosaccharomyces pombe . Unlike the budding yeast, S . pombe does not produce exoglucanase activity and hence this system would be applicable to any given strain of this organism . The XOG1 gene was located under the control of the nmt1 promoter and its functionality could be demonstrated even at high temperatures (37 degrees C) . The exoglucanase activity can be measured both in vivo and in vitro by either a simple biochemical reaction (on cells or media) or by flow cytometry, because the cells remain viable after the assay.

Biochem J, 1999 Jun 15, 340 ( Pt 3), 813 - 9
Interaction of mammalian neprilysin with binding protein and calnexin in Schizosaccharomyces pombe; Beaulieu H et al.; Neutral endopeptidase (neprilysin or NEP, EC 3.4.24.11) is a zinc metallo-endopeptidase expressed in many eukaryotic cell types and displaying several important physiological roles . In the brain (and central nervous system), this enzyme is involved in the molecular mechanism of pain by its action in the degradation of enkephalin molecules . In the kidney, NEP is implicated in the degradation of regulatory factors involved in the control of arterial pressure, including atrial natriuretic peptide and bradykinin . In this study we assessed the potential of the fission yeast Schizosaccharomyces pombe to overproduce rabbit NEP and secreted NEP (sNEP, a soluble derivative of this integral membrane protein) . Both recombinant NEP and sNEP were produced at high levels (5 mg/l) in this system . Enzymic studies revealed that these recombinant proteins were fully active and exhibit kinetic parameters similar to those of the bona fide enzyme . Immunofluorescence microscopy and enzymic assays demonstrated that recombinant NEP is correctly targeted to the cell membrane . Furthermore, co-immunoprecipitation studies showed that folding intermediates of NEP and sNEP, produced in S . pombe, interact in the endoplasmic reticulum (ER) with binding protein (BiP) and calnexin (Cnx1p) . The amount of sNEP coprecipitated with both BiP and Cnx1p augmented when cells were subjected to various stresses causing the accumulation of unfolded proteins in the ER . The interactions of NEP with BiP and Cnx1p were, however, more refractive to the same stresses.

Mol Biol Cell, 1999 Jun, 10(6), 1733 - 44
Cloning and characterization of peter pan, a novel Drosophila gene required for larval growth; Migeon JC et al.; We identified a new Drosophila gene, peter pan (ppan), in a screen for larval growth-defective mutants . ppan mutant larvae do not grow and show minimal DNA replication but can survive until well after their heterozygotic siblings have pupariated . We cloned the ppan gene by P-element plasmid rescue . ppan belongs to a highly conserved gene family that includes Saccharomyces cerevisiae SSF1 and SSF2, as well as Schizosaccharomyces pombe, Arabidopsis, Caenorhabditis elegans, mouse, and human homologues . Deletion of both SSF1 and SSF2 in yeast is lethal, and depletion of the gene products causes cell division arrest . Mosaic analysis of ppan mutant clones in Drosophila imaginal disks and ovaries demonstrates that ppan is cell autonomous and required for normal mitotic growth but is not absolutely required for general biosynthesis or DNA replication . Overexpression of the wild-type gene causes cell death and disrupts the normal development of adult structures . The ppan gene family appears to have an essential and evolutionarily conserved role in cell growth.

Genetics, 1999 Jun, 152(2), 495 - 508
Position effect variegation at the mating-type locus of fission yeast: a cis-acting element inhibits covariegated expression of genes in the silent and expressed domains; Ayoub N et al.; Schizosaccharomyces pombe switches its mating type by transposing a copy of unexpressed genes from the respective mat2 or mat3 cassettes to mat1 . The donor cassettes are located in a silent domain that is separated from the expressed mat1 cassette by the L region . We monitored the expression of ade6 from sites in the L region and examined the relationship between the expression state at these sites and at sites within the silent domain . Results indicate that: (1) the silent domain extends into the L region, but repression is gradually alleviated with increasing distance from mat2, and overexpression of swi6 enhances PEV in the L region; (2) a transcriptionally active chromatin state, associated with reporter gene expression in the L region, spreads toward the silent domain; (3) a cis-acting element, located at the junction between the L region and mat2-P, ensures repression in the silent domain, regardless of the expression state in the L region; and (4) repression in mat1-P cells is less stringently controlled than in mat1-M cells . We discuss the functional organization of the mat region and genetic elements that ensure separation between repressed and derepressed domains.

J Biochem (Tokyo), 1999 Jun, 125(6), 1061 - 6
A fission yeast gene (prr1(+)) that encodes a response regulator implicated in oxidative stress response; Ohmiya R et al.; An inspection of the Schizosaccharomyces pombe genome database revealed that this eukaryotic microorganism possesses a gene that may encode a bacterial type of histidine-to-aspartate (His-Asp) phosphorelay component, namely, a response regulator . The predicted gene, named prr1(+) (S . pombe response regulator), encodes a protein that contains a typical phospho-accepting receiver domain, preceded by a mammalian heat shock factor (HSF)-like DNA-binding domain . Inactivation of this prr1(+) gene resulted in mutant cells defective in some aspects of stress responses, including sensitivity to oxidative stress, cold-temperature, and heavy metal toxicity . It was also demonstrated that Prr1 is required for the transcription of some genes (e.g., trr1(+), ctt1(+)), which are induced by oxidative stress . These results suggest that a His-Asp phosphorelay system may be involved in a stress-activated signaling pathway in S . pombe.

J Biol Chem, 1999 Jun 4, 274(23), 16553 - 62
Characterization of human, Schizosaccharomyces pombe, and Candida albicans mRNA cap methyltransferases and complete replacement of the yeast capping apparatus by mammalian enzymes; Saha N et al.; Human and fission yeast cDNAs encoding mRNA (guanine-N7) methyltransferase were identified based on similarity of the human (Hcm1p; 476 amino acids) and Schizosaccharomyces pombe (Pcm1p; 389 amino acids) polypeptides to the cap methyltransferase of Saccharomyces cerevisiae (Abd1p) . Expression of PCM1 or HCM1 in S . cerevisiae complemented the lethal phenotype resulting from deletion of the ABD1 gene, as did expression of the NH2-terminal deletion mutants PCM1(94-389) and HCM1(121-476) . The CCM1 gene encoding Candida albicans cap methyltransferase (Ccm1p; 474 amino acids) was isolated from a C . albicans genomic library by selection for complementation of the conditional growth phenotype of S . cerevisiae abd1-ts mutants . Human cap methyltransferase was expressed in bacteria, purified, and characterized . Recombinant Hcm1p catalyzed quantitative S-adenosylmethionine-dependent conversion of GpppA-capped poly(A) to m7GpppA-capped poly(A) . We identified by alanine-scanning mutagenesis eight amino acids (Asp-203, Gly-207, Asp-211, Asp-227, Arg-239, Tyr-289, Phe-291, and Phe-354) that are essential for human cap methyltransferase function in vivo . All eight residues are conserved in other cellular cap methyltransferases . Five of the mutant human proteins (D203A, R239A, Y289A, F291A, and F354A) were expressed in bacteria and found to be defective in cap methylation in vitro . Concordance of mutational effects on Hcm1p, Abd1p, and vaccinia capping enzyme underscores a conserved structural basis for cap methylation in DNA viruses, yeast, and metazoans . This is in contrast to the structural and mechanistic divergence of the RNA triphosphatase components of the yeast and metazoan capping systems . Nevertheless, we demonstrate that the entire three-component yeast capping apparatus, consisting of RNA 5'-triphosphatase (Cet1p), RNA guanylyltransferase (Ceg1p), and Abd1p could be replaced in vivo by the two-component mammalian apparatus consisting of a bifunctional triphosphatase-guanylyltransferase Mce1p and the methyltransferase Hcm1(121-476)p . Isogenic yeast strains with fungal versus mammalian capping systems should facilitate rational screens for antifungal drugs that target cap formation in vivo.

J Cell Sci, 1999 Jun, 112 ( Pt 12), 1979 - 88
Sto1p, a fission yeast protein similar to tubulin folding cofactor E, plays an essential role in mitotic microtubule assembly; Grishchuk EL et al.; The proper functioning of microtubules depends crucially on the availability of polymerizable alpha/beta tubulin dimers . Their production occurs concomitant with the folding of the tubulin polypeptides and is accomplished in part by proteins known as Cofactors A through E . In the fission yeast, Schizosaccharomyces pombe, this tubulin folding pathway is essential . We have taken advantage of the excellent cytology available in S . pombe to examine the phenotypic consequences of a deletion of sto1(+), a gene that encodes a protein similar to Cofactor E, which is required for the folding of alpha-tubulin . The interphase microtubule cytoskeleton in sto1-delta cells is severely disrupted, and as cells enter mitosis their spindles fail to form . After a transient arrest with condensed chromosomes, the cells exit mitosis and resume DNA synthesis, whereupon they septate abnormally and die . Overexpression of Spo1p is toxic to cells carrying a cold-sensitive allele of the alpha- but not the beta-tubulin gene, consistent with the suggestion that this protein plays a role like that of Cofactor E . Unlike its presumptive partner Cofactor D (Alp1p), however, Sto1p does not localize to microtubules but is found throughout the cell . Overexpression of Sto1p has no toxic effects in wild-type cells, suggesting that it is unable to disrupt alpha/beta tubulin dimers in vivo.

Gene, 1999 May 17, 232(1), 53 - 8
A new inducible protein expression system in fission yeast based on the glucose-repressed inv1 promoter; Iacovoni JS et al.; Studies of the fission yeast Schizosaccharomyces pombe have made major contributions towards understanding cell-cycle control and many other important aspects of cell biology . A series of pREP expression vectors that utilize the thiamine-repressible nmt1 promoter are used routinely to manipulate the expression of genes in fission yeast . A shortcoming of the nmt1 promoter is that it is very slowly induced following removal of thiamine from the growth medium, requiring approx . 16h for full induction . Invertase, an enzyme responsible for sucrose metabolism, is regulated transcriptionally by glucose derepression in S . pombe . Using the inv1 promoter, we have developed the pINV1 set of inducible protein expression vectors . A shift from glucose to sucrose-based culture medium leads to a very rapid induction of the inv1 promoter . Genes that are regulated by the inv1 promoter are fully induced within 1h of the shift to sucrose-based medium . The pINV1 vectors utilize a simple induction protocol and enable studies in fission yeast requiring tight and rapid regulation of protein synthesis.

J Mol Biol, 1999 May 21, 288(5), 867 - 82
Influence of a replication enhancer on the hierarchy of origin efficiencies within a cluster of DNA replication origins; Kim SM et al.; DNA replication origins in animal cells sometimes occur in clusters . Often one of the multiple origins within these clusters fires more frequently than the others . The reason for this hierarchy remains unknown . Similar origin clusters occur in the fission yeast, Schizosaccharomyces pombe . One such cluster is located near the ura4 gene on chromosome III and contains three origins: ars3002, ars3003, and ars3004 . In their natural chromosomal context (ars3003 is about 2.5 kb upstream of ars3002 and ars3004 is adjacent to ars3002 on the downstream side) their initiation frequencies display a striking hierarchy: ars3002 >> ars3003 >> ars3004 . Here, we describe experiments that reveal a 400 bp replication enhancer within ars3004, adjacent to ars3002 . The enhancer is essential for ars3004 origin function in a plasmid, but even with the enhancer ars3004 is an inefficient origin . The enhancer is not essential for ars3002 plasmid origin activity, but dramatically stimulates this activity, converting ars3002 from an inefficient plasmid origin to a very efficient one . It also stimulates the plasmid origin activity of ars3001 and ars3003 at all tested positions and orientations on both sides of each autonomously replicating sequence (ARS) element . If ars3002 is redefined to include the enhancer, then the relative activities of the three ARS elements as single origins within separate plasmids or as origins when all three ARS elements are present in a single plasmid is the same as the chromosomal hierarchy . Thus, this replication enhancer defines the relative activities of the three origins in the ura4 origin region . Similar enhancers may affect relative activities in the origin clusters of animal cells .

Nucleic Acids Res, 1999 Jun 1, 27(11), 2256 - 64
Substrate specificity of ultraviolet DNA endonuclease (UVDE/Uve1p) from Schizosaccharomyces pombe; Avery AM et al.; Schizosaccharomyces pombe ultraviolet DNA endonuclease (UVDE or Uve1p) has been shown to cleave 5' to UV light-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP) . This endonuclease is believed to function in the initial step in an alternative excision repair pathway for the removal of DNA damage caused by exposure to UV light . An active truncated form of this protein, Delta228-Uve1p, has been successfully overexpressed, affinity purified and partially characterized . In the present study we present data from a detailed substrate specificity trial . We have determined that the substrate range of Uve1p is much greater than was originally believed . We demonstrate that this DNA damage repair protein is capable of recognizing an array of UV-induced DNA photoproducts (cis-syn-, trans-syn I- and trans-syn II CPDs, 6-4PP and Dewar isomers) that cause varying degrees of distortion in a duplex DNA molecule . We also demonstrate that Uve1p recognizes non-UV-induced DNA damage, such as platinum-DNA GG diadducts, uracil, dihydrouracil and abasic sites . This is the first time that a single DNA repair endonuclease with the ability to recognize such a diverse range of lesions has been described . This study suggests that Uve1p and the alternative excision repair pathway may participate broadly in the repair of DNA damage.

Nat Genet, 1999 May, 22(1), 82 - 4
Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome; Kitao S et al.; Rothmund-Thomson syndrome (RTS; also known as poikiloderma congenitale) is a rare, autosomal recessive genetic disorder characterized by abnormalities in skin and skeleton, juvenile cataracts, premature ageing and a predisposition to neoplasia . Cytogenetic studies indicate that cells from affected patients show genomic instability often associated with chromosomal rearrangements causing an acquired somatic mosaicism . The gene(s) responsible for RTS remains unknown . The genes responsible for Werner and Bloom syndromes (WRN and BLM, respectively) have been identified as homologues of Escherichia coli RecQ, which encodes a DNA helicase that unwinds double-stranded DNA into single-stranded DNAs . Other eukaryotic homologues thus far identified are human RECQL, Saccharomyces cerevisiae SGS1 and Schizosaccharomyces pombe rqh1 . We recently cloned two new human helicase genes, RECQL4 at 8q24.3 and RECQL5 at 17q25, which encode members of the RecQ helicase family . Here, we report that three RTS patients carried two types of compound heterozygous mutations in RECQL4 . The fact that the mutated alleles were inherited from the parents in one affected family and were not found in ethnically matched controls suggests that mutation of RECQL4 at human chromosome 8q24.3 is responsible for at least some cases of RTS.

Yeast, 1999 Apr, 15(6), 497 - 505
Schizosaccharomyces pombe stt3+ is a functional homologue of Saccharomyces cerevisiae STT3 which regulates oligosaccharyltransferase activity; Yoshida S et al.; The Saccharomyces cerevisiae STT3 (ScSTT3) gene encodes a protein which is involved in protein glycosylation via the regulation of oligosaccharyltransferase activity . We have cloned and isolated the Schizosaccharomyces pombe STT3 homologous gene (Spstt3+) . The Spstt3+ gene encodes a protein consisting of 749 amino acid residues which has significant homology with ScStt3p and the mouse Stt3p-homologue Itm1p . Disruption of the Spstt3+ gene shows that this gene is essential for growth . Like Itm1, Spstt3+ partially suppressed the temperature sensitivity of the stt3-1 mutation of S . cerevisiae, indicating that Spstt3+ is a functional and structural homologue of the ScSTT3 gene.

Mol Biol Cell, 1999 May, 10(5), 1495 - 510
Ssp1 promotes actin depolymerization and is involved in stress response and new end take-off control in fission yeast; Rupes I et al.; The ssp1 gene encodes a protein kinase involved in alteration of cell polarity in Schizosaccharomyces pombe . ssp1 deletion causes stress sensitivity, reminiscent of defects in the stress-activated MAP kinase, Spc1; however, the two protein kinases do not act through the same pathway . Ssp1 is localized mainly in the cytoplasm, but after a rise in external osmolarity it is rapidly recruited to the plasma membrane, preferentially to active growth zones and septa . Loss of Ssp1 function inhibits actin relocalization during osmotic stress, in cdc3 and cdc8 mutant backgrounds, and in the presence of latrunculin A, implicating Ssp1 in promotion of actin depolymerization . We propose a model in which Ssp1 can be activated independently of Spc1 and can partially compensate for its loss . The ssp1 deletion mutant exhibited monopolar actin distribution, but new end take-off (NETO) could be induced in these cells by exposure to KCl or to latrunculin A pulse treatment . This treatment induced NETO in cdc10 cells arrested in G1 but not in tea1 cells . This suggests that cells that contain intact cell end markers are competent to undergo NETO throughout interphase, and Ssp1 is involved in generating the NETO stimulus by enlarging the actin monomer pool.

Mol Biol Cell, 1999 May, 10(5), 1395 - 407
Active nucleocytoplasmic shuttling required for function and regulation of stress-activated kinase Spc1/StyI in fission yeast; Gaits F et al.; Transcriptional induction of many stress-response genes is dependent on stress-induced nuclear accumulation of stress-activated protein kinases (SAPKs) . In the fission yeast Schizosaccharomyces pombe, nuclear accumulation of the SAPK Spc1 (also known as StyI) requires activating phosphorylation catalyzed by the SAPK kinase Wis1; however, it is unknown whether the localization of Spc1 is regulated by nuclear transport factors . Herein are reported studies that show that Spc1 localization is regulated by active transport mechanisms during osmotic stress . Nuclear import of Spc1 requires Pim1, a homologue of the guanine nucleotide exchange factor RCC1 that is essential for nucleocytoplasmic shuttling of proteins . Nuclear export of Spc1 is regulated by the export factor Crm1 . An Spc1-Crm1 complex forms as Spc1 is exported from the nucleus . Wis1 and the tyrosine phosphatases Pyp1 and Pyp2 that inactivate Spc1 are excluded from the nucleus by a Crm1-independent mechanism; hence the nuclear import of Spc1 leads to transient isolation from its regulatory proteins . Thus, active nucleocytoplasmic shuttling is required for both the function and regulation of Spc1 during the osmotic shock response.

Cancer Res, 1999 May 1, 59(9), 2023 - 8
HRad17, a human homologue of the Schizosaccharomyces pombe checkpoint gene rad17, is overexpressed in colon carcinoma; Bao S et al.; Using the palindromic PCR-cDNA display method, we have cloned a novel gene overexpressed by human colon carcinoma relative to normal colon . Among normal tissues examined, only testis expresses it at a high level . Sequence analysis revealed its extensive homology with checkpoint genes rad17 of Schizosaccharomyces pombe and RAD24 of Saccharomyces cerevisiae . This novel gene designated as hRad17 is localized to chromosome 5q12,13.1, a region known to be deleted in a variety of human cancers . Promoter region and one pseudogene of hRad17 have been identified . Whereas the increased expression of hRad17 by human colon carcinomas may be related to the known resistance of these cells to DNA-damaging agents during therapy, the deletion of hRad17 in a variety of cancers may predispose them to increased rate of mutation and heightened sensitivity to DNA-damaging agents, including radiation and anticancer drugs.

Biochem Pharmacol, 1999 Jun 1, 57(11), 1297 - 303
Human CYP2C-mediated stereoselective phenytoin hydroxylation in Japanese: difference in chiral preference of CYP2C9 and CYP2C19; Yasumori T et al.; Regio- and stereoselective hydroxylation of phenytoin was determined in liver microsomes of nine extensive (EM) and three poor metabolizers (PM) of mephenytoin . Hydroxyphenytoins (HPPH) were isolated and quantified after separation into four regio- and stereoisomers . The total rates of microsomal phenytoin 4'- hydroxylation were approximately 3-fold higher than those of 3'-hydroxylation, and not significantly different in EM and PM . Formation of 4'-(R)-HPPH was 4.4-fold higher in EM than in PM, whereas no clear differences between EM and PM were detected in the formation of 4'-(S)-, 3'-(R)-, and 3'-(S)-HPPH . Cytochrome P450 (CYP)2C9, expressed in a fission yeast, Schizosaccharomyces pombe, catalyzed the formation of 4'-(R)- and 4'-(S)-HPPH stereoselectively, as observed with EM, in which predominantly 4'-(S)-HPPH was formed . Recombinant CYP2C19 was more stereoselective for 4'-(R)-HPPH formation . These results, in addition to inhibition experiments with anti-human CYP2C antibody, indicate that phenytoin hydroxylation is mainly catalyzed by CYP2C9 . Furthermore, CYP2C19 showed limited contribution to phenytoin 4'-hydroxylation with a different chiral preference from CYP2C9.

FEMS Microbiol Lett, 1999 Apr 15, 173(2), 373 - 8
Identification of the catalase gene promoter region involved in superinduction in Schizosaccharomyces pombe caused by cycloheximide and hydrogen peroxide; Nakagawa CW et al.; Superinduction of the catalase gene was observed in Schizosaccharomyces pombe cells treated with cycloheximide and hydrogen peroxide . The promoter analysis of the catalase gene revealed that element A (the region from -111 to -90, numbered with the transcription start site as +1), involved in the induction of the gene under oxidative stress, was required for superinduction by hydrogen peroxide and cycloheximide . Although Atf1 is a transcription factor responsible for the induction of the catalase gene by several stresses, a disruptant of atf1 exhibited superinduction . Moreover, in a deletion mutant that lacks element A but has an Atf1 binding site, the cells treated with hydrogen peroxide and cycloheximide expressed as much catalase mRNA as those treated with hydrogen peroxide alone . This suggests that cycloheximide does not stabilize the catalase mRNA but enhances the transcription via element A . Staurosporine, a strong inhibitor of protein phosphorylation, did not inhibit superinduction.

Curr Biol, 1999 Apr 22, 9(8), 441 - 4
Cdc2 activation in fission yeast depends on Mcs6 and Csk1, two partially redundant Cdk-activating kinases (CAKs); Lee KM et al.; Cyclin-dependent kinases (Cdks) are fully active only when phosphorylated by a Cdk-activating kinase (CAK) {1} . Metazoan CAK is itself a Cdk, Cdk7, whereas the CAK of Saccharomyces cerevisiae is a distinct enzyme unrelated to Cdks {1} . The Mcs6-Mcs2 complex of Schizosaccharomyces pombe is a putative CAK related to the metazoan enzyme {2} {3} . Although the loss of Mcs6 is lethal, it results in a phenotype that is inconsistent with a failure to activate Cdc2, the major Cdk in S . pombe {3} . We therefore tested the ability of Csk1, a putative regulator of Mcs6 {4}, to activate Cdk-cyclin complexes in vitro . Csk1 activated both the monomeric and the Mcs2-bound forms of Mcs6 . Surprisingly, Csk1 also activated Cdc2 in complexes with either Cdc13 or Cig2 cyclins . When a double mutant carrying a csk1 deletion and a temperature-sensitive mcs6 allele was incubated at the restrictive temperature, Cdc2 was not activated and the cells underwent a cell division arrest prior to mitosis . Cdc2-cyclin complexes isolated from the arrested cells could be activated in vitro by recombinant CAK, whereas complexes from wild-type cells or either of the single mutants were refractory to activation . Thus, fission yeast contains two partially redundant CAKs: the Mcs6-Mcs2 complex and Csk1 . Inactivation of both CAKs is necessary and sufficient to prevent Cdc2 activation and cause a cell-cycle arrest . Mcs6, which is essential, may therefore have required functions other than Cdk activation.

Curr Biol, 1999 Apr 22, 9(8), 425 - 8
The APC-associated protein EB1 associates with components of the dynactin complex and cytoplasmic dynein intermediate chain; Berrueta L et al.; Human EB1 is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein {1}, a domain of APC that is commonly deleted in colorectal neoplasia {2} . EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3 {3} and Saccharomyces cerevisiae Bim1p {4} . Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex {5} . Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle {6}, in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis {7} {8} {9} {10} . Here, we demonstrate that human EB1 can be coprecipitated with p150(Glued), a member of the dynactin protein complex . EB1 was also found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sedimented at approximately 5S in a sucrose density gradient . The association of EB1 with members of the dynactin complex was independent of APC and was preserved in the absence of an intact microtubule cytoskeleton . The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes.

Genetics, 1999 May, 152(1), 61 - 71
Caffeine-mediated override of checkpoint controls . A requirement for rhp6 (Schizosaccharomyces pombe); Rowley R et al.; Cells exposed to inhibitors of DNA synthesis or suffering DNA damage are arrested or delayed in interphase through the action of checkpoint controls . If the arrested cell is exposed to caffeine, relatively normal cell cycle progression is resumed and, as observed in checkpoint control mutants, loss of checkpoint control activity is associated with a reduction in cell viability . To address the mechanism of caffeine's action on cell progression, fission yeast mutants that take up caffeine but are not sensitized to hydroxyurea (HU) by caffeine were selected . Mutants 788 and 1176 are point mutants of rhp6, the fission yeast homolog of the budding yeast RAD6 gene . Mutant rhp6-788 is slightly HU sensitive, radiosensitive, and exhibits normal checkpoint responses to HU, radiation, or inactivation of DNA ligase . However, the addition of caffeine does not override the associated cell cycle blocks . Both point and deletion mutations show synthetic lethality at room temperature with temperature-sensitive mutations in cyclin B (cdc13-117) or the phosphatase cdc25 (cdc25-22) . These observations suggest that the rhp6 gene product, a ubiquitin-conjugating enzyme required for DNA damage repair, promotes entry to mitosis in response to caffeine treatment.

Appl Environ Microbiol, 1999 May, 65(5), 2020 - 4
Accumulation of trehalose by overexpression of tps1, coding for trehalose-6-phosphate synthase, causes increased resistance to multiple stresses in the fission yeast schizosaccharomyces pombe
Soto T, Fernandez J, Vicente-Soler J, Cansado J, Gacto M.
Recent studies have shown that heat shock proteins and trehalose synthesis are important factors in the thermotolerance of the fission yeast Schizosaccharomyces pombe . We examined the effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on resistance to several stresses in cells of S . pombe transformed with a plasmid bearing the tps1 gene, which codes for trehalose-6P synthase, under the control of the strong thiamine-repressible promoter . Upon induction of trehalose-6P synthase, the elevated levels of intracellular trehalose correlated not only with increased tolerance to heat shock but also with resistance to freezing and thawing, dehydration, osmostress, and toxic levels of ethanol, indicating that trehalose may be the stress metabolite underlying the overlap in induced tolerance to these stresses . Among the isogenic strains transformed with this construct, one in which the gene coding for the trehalose-hydrolyzing enzyme, neutral trehalase, was disrupted accumulated trehalose to a greater extent and was more resistant to the above stresses . Increased trehalose concentration is thus a major determinant of the general stress protection response in S . pombe.

J Biol Chem, 1999 May 7, 274(19), 13250 - 7
A fission yeast gene for mitochondrial sulfide oxidation; Vande Weghe JG et al.; A cadmium-hypersensitive mutant of the fission yeast Schizosaccharomyces pombe was found to accumulate abnormally high levels of sulfide . The gene required for normal regulation of sulfide levels, hmt2(+), was cloned by complementation of the cadmium-hypersensitive phenotype of the mutant . Cell fractionation and immunocytochemistry indicated that HMT2 protein is localized to mitochondria . Sequence analysis revealed homology between HMT2 and sulfide dehydrogenases from photosynthetic bacteria . HMT2 protein, produced in and purified from Escherichia coli, was soluble, bound FAD, and catalyzed the reduction of quinone (coenzyme Q2) by sulfide . HMT2 activity was also detected in isolated fission yeast mitochondria . We propose that HMT2 functions as a sulfide:quinone oxidoreductase . Homologous enzymes may be widespread in higher organisms, as sulfide-oxidizing activities have been described previously in animal mitochondria, and genes of unknown function, but with similarity to hmt2(+), are present in the genomes of flies, worms, rats, mice, and humans.

Yeast, 1999 Mar 30, 15(5), 419 - 26
DNA sequencing and analysis of a 40 kb region from the right arm of chromosome II from Schizosaccharomyces pombe; Sanchez M et al.; We have determined the complete nucleotide sequence of a 39,648 bp segment, contained in cosmid c32F12, derived from the right arm of chromosome II from the fission yeast Schizosaccharomyces pombe . Computer analysis of the sequence revealed the presence of 15 non-overlapping open reading-frames (ORFs) longer than 300 bp and one tRNA-Thr gene . Six ORFs correspond to the previously known rec14+, tug1+, rum1+, pch1+, gpd1+ and cyr1+ genes . Five ORFs code for putative proteins with significant homology to proteins from other organisms . SPBC32F12.01c shows considerable similarity to human neutral sphingomyelinase, whereas SPBC32F12.03c, SPBC32F12.10 and SPBC32F12.14 exhibit strong homology to glutathione peroxidase, phosphoglucomutase and ubiquitin protein ligase E-3 components from various organisms, respectively . The four remaining ORFs identified show weak or non-significant homology to previously sequenced genes . The nucleotide sequence has been submitted to the EMBL database under Accession Number AL023796.

Yeast, 1999 Mar 30, 15(5), 385 - 96
Biosynthesis of phytochelatins in the fission yeast . Phytochelatin synthesis: a second role for the glutathione synthetase gene of Schizosaccharomyces pombe; Al-Lahham A et al.; By complementation screening of a cadmium-sensitive Schizosaccharomyces pombe mutant deficient in phytochelatin synthesis, but with 44% of the wild-type glutathione content, we cloned a DNA fragment involved in phytochelatin synthesis . Sequence analysis revealed that it encodes the second enzyme involved in glutathione (GSH) biosynthesis, glutathione synthetase (GSH2) (E.C.6.3.2.3, Wang and Oliver, 1997) . The mutant allele shows a single base-pair exchange at the 3' end of the reading frame leading to a single amino acid change from glycine to aspartate . This mutation leads to a significant reduction of phytochelatin synthesis, whereas glutathione synthesis is impaired to a far lesser extent . Complementation with the Arabidopsis thaliana GSH2 cDNA led to a partial restoration of phytochelatin synthesis . These data strongly suggest that the GSH2 gene encodes a bifunctional enzyme that is able to catalyse both the synthesis of GSH by adding glycine to the dipeptide (gammaGlu-Cys) and the synthesis of phytochelatins . The sequence has been submitted to EMBL, Accession No . Y08414.

Nucleic Acids Res, 1999 May 15, 27(10), 2108 - 14
Isolation and identification of the third subunit of mammalian DNA polymerase delta by PCNA-affinity chromatography of mouse FM3A cell extracts; Hughes P et al.; Using proliferating cell nuclear antigen affinity chroma-tography and glycerol gradient centrifugation of partially purified fractions from mouse FM3A cells we have been able to isolate novel complexes of DNA polymerase delta and DNA ligase 1 containing clearly defined subunit compositions . In addition to the well known catalytic subunit of 125 kDa and accessory subunit of 48 kDa, the DNA polymerase delta complex contained three supplementary components, one of which reacted with antibodies directed against the p40 and p37 subunits of RF-C . Of the two remaining components, one termed p66 turned out to be coded by a gene whose putative C-terminal domain displayed significant homology with that of the Cdc27 subunit of Schizosaccharomyces pombe polymerase delta . On the basis of these and other observations, we propose p66 to be the missing third subunit of mammalian DNA polymerase delta . The DNA ligase 1 complex was made up of three novel components in addition to the 125 kDa catalytic subunit, two of which, p48 and p66, were common to DNA polymerase delta . We discuss the implications of our findings within the current framework of our understanding of DNA replication.

Biochimie, 1999 Jan-Feb, 81(1-2), 173 - 81
DNA structure checkpoint pathways in Schizosaccharomyces pombe; Caspari T et al.; The response to DNA damage includes a delay to progression through the cell cycle to aid DNA repair . Incorrectly replicated chromosomes (replication checkpoint) or DNA damage (DNA damage checkpoint) delay the onset of mitosis . These checkpoint pathways detect DNA perturbations and generate a signal . The signal is amplified and transmitted to the cell cycle machinery . Since the checkpoint pathways are essential for genome stability, the related proteins which are found in all eukaryotes (from yeast to mammals) are expected to have similar functions to the yeast progenitors . This review article focuses on the function of checkpoint proteins in the model system Schizosaccharomyces pombe . Checkpoint controls in Saccharomyces cerevisiae and mammalian cells are mentioned briefly to underscore common or diverse features.

Biochim Biophys Acta, 1999 Apr 1, 1449(3), 239 - 53
Ibd1p, a possible spindle pole body associated protein, regulates nuclear division and bud separation in Saccharomyces cerevisiae; Lee J et al.; The proper spatial and temporal coordination of mitosis and cytokinesis is essential for maintaining genomic integrity . We describe the identification and characterization of the Saccharomyces cerevisiae IBD1 gene, which encodes a novel protein that regulates the proper nuclear division and bud separation . IBD1 was identified by the limited homology to byr4, a dosage-dependent regulator of cytokinesis in Schizosaccharomyces pombe . IBD1 is not an essential gene, and the knock-out cells show no growth defects except for the reduced mating efficiency {1} . However, upon ectopic expression from an inducible promoter, IBD1 is lethal to the cell and leads to abnormal nuclear division and bud separation . In detail, approximately 90% of the IBD1 overexpressing cells arrest at large bud stages with dividing or divided nuclei . In some IBD1 overexpressing cells, spindle elongation and chromosome separation occur within the mother cell, leading to anucleated and binucleate daughter cells . The anucleated cell can not bud, but the binucleate cell proceeds through another cell cycle(s) to produce a cell with multiple nuclei and multiple buds . Observations of the F-actin and chitin rings in the IBD1 overexpressing cells reveal that these cells lose the polarity for bud site selection and growth or attain the hyper-polarity for growth . Consistent with the phenotypes, the IBD1 overexpressing cells contain a broad range of DNA content, from 2 to 4 N or more . A functional Ibd1p-GFP fusion protein localizes to a single dot at the nuclear DNA boundary in the divided nuclei or to double dots in dividing nuclei, suggesting its localization on the spindle pole body (SPB) . The cross-species expressions of IBD1 in S . pombe and byr4 in S . cerevisiae cause defects in shape, implicating the presence of a conserved mechanism for the control of cytokinesis in eukaryotes . We propose that Ibd1p is an SPB associated protein that links proper nuclear division to cytokinesis and bud separation.

Oncogene, 1999 Mar 4, 18(9), 1689 - 99
hRAD17, a structural homolog of the Schizosaccharomyces pombe RAD17 cell cycle checkpoint gene, stimulates p53 accumulation; Li L et al.; The RAD17 gene product of S . Pombe is an essential component of the checkpoint control pathway which responds to both DNA damage and disruption of replication . We have identified a human cDNA that encodes a polypeptide which is structurally conserved with the S . Pombe Rad17 protein . The human gene, designated hRAD17, predicts an encoded protein of 590 amino acids and a molecular weight of 69 kD . Amino acid sequence alignment revealed that hRadl7 has 28.3% and 52.5% similarity with the S . Pombe Rad17 protein, and 21.8% identity and 45.8% similarity to the budding yeast cell cycle checkpoint protein, Rad 24 . When introduced into the S . Pombe rad17 mutant, hRAD17 was able to partially revert its hydroxyurea and ionizing radiation hypersensitivity, but not its UV hypersensitivity . Permanent overexpression of the hRAD17 gene in human fibrosarcoma cells resulted in p53 activation and a significant reduction of S- and G2/M-phase cells accompanied by an accumulation of the G1-phase population, suggesting that hRAD17 may have a role in cell cycle checkpoint control . Immunostaining of HT-1080 cells transiently transfected with a hRAD17 construct confirmed the nuclear accumulation of p53, which mimics the induction caused by DNA damage . Using FISH analysis, we have mapped the hRAD17 locus to human chromosome 5q11.2.

Glycobiology, 1999 May, 9(5), 507 - 15
Schizosaccharomyces pombe produces novel Gal0-2Man1-3 O-linked oligosaccharides; Gemmill TR et al.; Schizosaccharomyces pombe whole-cell glycoproteins, previously depleted of N-linked glycans by sequential treatment with endo-ss-N-acetylglucosaminidase H and peptide-N4-asparagine amidohydrolase F, were ss-eliminated with 0.1 M NaOH/1 M NaBH4 to release the O-linked oligosaccharides . The saccharide-alditols were separated by gel-exclusion chromatography into pools from Hexitol to Hex4Hexitol in size . Analysis of the Hexitol pool indicated Man to be the only sugar linked to Ser or Thr residues . The Hex1Hexitol pool contained two components, Galalpha1,2Man-ol (2A) and Manalpha1, 2Man-ol (2B) . The Hex2Hexitol pool contained two components, Galalpha1,2Manalpha1,2Man-ol (3A) and Manalpha1,2Manalpha1,2Man-ol (3B) . The two Hex3Hexitol components were Galalpha1,2(Galalpha1, 3)Manalpha1,2Man-ol (4A) and Manalpha1,2(Galalpha1,3)Manalpha1, 2Man-ol (4B) . The Hex4Hexitol component was found to be a single isomer with the composition of Galalpha1,2(Galalpha1,3)Manalpha1, 2Manalpha1,2Man-ol (5AB) . Surprisingly, galactobiose was not detected in any of these oligosaccharides . The gma12 (T . G . Chappell and G . Warren (1989) J . Cell Biol., 109, 2693-2707) and gth1 (T . G . Chappell personal communication) alpha1, 2-galactosyltransferase-deficient mutants and the gma12/gth1 double mutant S.pombe strains were similarly examined . The results indicated that gma12p is solely responsible for the addition of terminal alpha1,2-linked Gal in compound 2A, while one or both of gma12p and gth1p are required for the alpha1,2-linked Gal in 4A . Both transferases are largely responsible for terminal Gal in isomer 5AB . Neither gma12 nor gth1 had any discernible effect on the structure of the large N-linked galactomannans as determined by 1H NMR spectroscopy . Thus, while gth1p and gma12p appear responsible for adding alpha1,2-linked Gal to terminal Man, neither adds galactose side chains to the N-linked poly alpha1,6-Man outerchain, nor the O-linked branch-forming alpha1,3-linked Gal . Furthermore, the presence of Hexalpha1,2(Galalpha1,3)Manalpha1,2- structures in the O-linked glycans implies the presence of a novel branch-forming alpha1,3-galactosyltransferase in S.pombe.

Mol Cell Biol, 1999 May, 19(5), 3829 - 41
Isolation of a mammalian homologue of a fission yeast differentiation regulator; Yamamoto H et al.; In the fission yeast Schizosaccharomyces pombe the nrd1(+) gene encoding an RNA binding protein negatively regulates the onset of differentiation . Its biological role is to block differentiation by repressing a subset of the Ste11-regulated genes essential for conjugation and meiosis until the cells reach a critical level of nutrient starvation . By using the phenotypic suppression of the S . pombe temperature-sensitive pat1 mutant that commits lethal haploid meiosis at the restrictive temperature, we have cloned ROD1, a functional homologue of nrd1(+), from rat and human cDNA libraries . Like nrd1(+), ROD1 encodes a protein with four repeats of typical RNA binding domains, though its amino acid homology to Nrd1 is limited . When expressed in the fission yeast, ROD1 behaves in a way that is functionally similar to nrd1(+), being able to repress Ste11-regulated genes and to inhibit conjugation upon overexpression . ROD1 is predominantly expressed in hematopoietic cells or organs of adult and embryonic rat . Like nrd1(+) for fission yeast differentiation, overexpressed ROD1 effectively blocks both 12-O-tetradecanoyl phorbol-13-acetate-induced megakaryocytic and sodium butyrate-induced erythroid differentiation of the K562 human leukemia cells without affecting their proliferative ability . These results suggest a role for ROD1 in differentiation control in mammalian cells . We discuss the possibility that a differentiation control system found in the fission yeast might well be conserved in more complex organisms, including mammals.

Mol Cell Biol, 1999 May, 19(5), 3515 - 28
Rec8p, a meiotic recombination and sister chromatid cohesion phosphoprotein of the Rad21p family conserved from fission yeast to humans; Parisi S et al.; Our work and that of others defined mitosis-specific (Rad21 subfamily) and meiosis-specific (Rec8 subfamily) proteins involved in sister chromatid cohesion in several eukaryotes, including humans . Mutation of the fission yeast Schizosaccharomyces pombe rec8 gene was previously shown to confer a number of meiotic phenotypes, including strong reduction of recombination frequencies in the central region of chromosome III, absence of linear element polymerization, reduced pairing of homologous chromosomes, reduced sister chromatid cohesion, aberrant chromosome segregation, defects in spore formation, and reduced spore viability . Here we extend the description of recombination reduction to the central regions of chromosomes I and II . We show at the protein level that expression of rec8 is meiosis specific and that Rec8p localizes to approximately 100 foci per prophase nucleus . Rec8p was present in an unphosphorylated form early in meiotic prophase but was phosphorylated prior to meiosis I, as demonstrated by analysis of the mei4 mutant blocked before meiosis I . Evidence for the persistence of Rec8p beyond meiosis I was obtained by analysis of the mutant mes1 blocked before meiosis II . A human gene, which we designate hrec8, showed significant primary sequence similarity to rec8 and was mapped to chromosome 14 . High mRNA expression of mouse and human rec8 genes was found only in germ line cells, specifically in testes and, interestingly, in spermatids . hrec8 was also expressed at a low level in the thymus . Sequence similarity and testis-specific expression indicate evolutionarily conserved functions of Rec8p in meiosis . Possible roles of Rec8p in the integration of different meiotic events are discussed.

EMBO J, 1999 Apr 1, 18(7), 1772 - 82
Characterization of GAPCenA, a GTPase activating protein for Rab6, part of which associates with the centrosome; Cuif MH et al.; The Rab6 GTPase regulates intracellular transport at the level of the Golgi apparatus, probably in a retrograde direction . Here, we report the identification and characterization of a novel human Rab6-interacting protein named human GAPCenA (for 'GAP and centrosome-associated') . Primary sequence analysis indicates that GAPCenA displays similarities, within a central 200 amino acids domain, to both the yeast Rab GTPase activating proteins (GAPs) and to the spindle checkpoint proteins Saccharomyces cerevisiae Bub2p and Schizosaccharomyces pombe Cdc16p . We demonstrate that GAPCenA is indeed a GAP, specifically active in vitro on Rab6 and, to a lesser extent, on Rab4 and Rab2 proteins . Immunofluorescence and cell fractionation experiments showed that GAPCenA is mainly cytosolic but that a minor pool is associated with the centrosome . Moreover, GAPCenA was found to form complexes with cytosolic gamma-tubulin and to play a role in microtubule nucleation . Therefore, GAPCenA may be involved in the coordination of microtubule and Golgi dynamics during the cell cycle.

Biochemistry, 1999 Apr 13, 38(15), 4809 - 17
Processing of UV damage in vitro by FEN-1 proteins as part of an alternative DNA excision repair pathway; Yoon JH et al.; Ultraviolet (UV) irradiation induces predominantly cyclobutane and (6-4) pyrimidine dimer photoproducts in DNA . Several mechanisms for repairing these mutagenic UV-induced DNA lesions have been identified . Nucleotide excision repair is a major pathway, but mechanisms involving photolyases and DNA glycosylases have also been characterized . Recently, a novel UV damage endonuclease (UVDE) was identified that initiates an excision repair pathway different from previously established repair mechanisms . Homologues of UVDE have been found in eukaryotes as well as in bacteria . In this report, we have used oligonucleotide substrates containing site-specific cyclobutane pyrimidine dimers and (6-4) photoproducts for the characterization of this UV damage repair pathway . After introduction of single-strand breaks at the 5' sides of the photolesions by UVDE, these intermediates became substrates for cleavage by flap endonucleases (FEN-1 proteins) . FEN-1 homologues from humans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe all cleaved the UVDE-nicked substrates at similar positions 3' to the photolesions . T4 endonuclease V-incised DNA was processed in the same way . Both nicked and flapped DNA substrates with photolesions (the latter may be intermediates in DNA polymerase-catalyzed strand displacement synthesis) were cleaved by FEN-1 . The data suggest that the two enzymatic activities, UVDE and FEN-1, are part of an alternative excision repair pathway for repair of UV photoproducts.

Chromosoma, 1999 Apr, 108(1), 26 - 31
Identification and characterization of mouse homologue to yeast Cdc7 protein and chromosomal localization of the cognate mouse gene Cdc7l; Faul T et al.; The Cdc7 kinase is required for the G1/S-phase transition during the cell cycle and plays a direct role in the activation of individual origins of replication in Saccharomyces cerevisiae . Here, we report the identification of a mouse cDNA, MmCdc7, whose product is closely related in sequence to Saccharomyces cerevisiae Cdc7 as well as their human, Xenopus and Schizosaccharomyces pombe homologues . The MmCdc7p contains the conserved subdomains common to all protein-serine/threonine kinases and three kinase inserts that are characteristic of members of the Cdc7 protein family . We have mapped the locus of the MmCdc7 gene to chromosome 5, band 5E . Conservation of structures among members of the Cdc7-related proteins suggests that these proteins play a key role in the regulation of DNA replication during the cell cycle in all eukaryotes.

Mol Biol Cell, 1999 Apr, 10(4), 1061 - 75
Sla1p is a functionally modular component of the yeast cortical actin cytoskeleton required for correct localization of both Rho1p-GTPase and Sla2p, a protein with talin homology; Ayscough KR et al.; SLA1 was identified previously in budding yeast in a genetic screen for mutations that caused a requirement for the actin-binding protein Abp1p and was shown to be required for normal cortical actin patch structure and organization . Here, we show that Sla1p, like Abp1p, localizes to cortical actin patches . Furthermore, Sla1p is required for the correct localization of Sla2p, an actin-binding protein with homology to talin implicated in endocytosis, and the Rho1p-GTPase, which is associated with the cell wall biosynthesis enzyme beta-1,3-glucan synthase . Mislocalization of Rho1p in sla1 null cells is consistent with our observation that these cells possess aberrantly thick cell walls . Expression of mutant forms of Sla1p in which specific domains were deleted showed that the phenotypes associated with the full deletion are functionally separable . In particular, a region of Sla1p encompassing the third SH3 domain is important for growth at high temperatures, for the organization of cortical actin patches, and for nucleated actin assembly in a permeabilized yeast cell assay . The apparent redundancy between Sla1p and Abp1p resides in the C-terminal repeat region of Sla1p . A homologue of SLA1 was identified in Schizosaccharomyces pombe . Despite relatively low overall sequence homology, this gene was able to rescue the temperature sensitivity associated with a deletion of SLA1 in Saccharomyces cerevisiae.

Mol Biol Cell, 1999 Apr, 10(4), 1031 - 41
Cloning and sequencing of a protein involved in phagosomal membrane fusion in Paramecium; Yamauchi K et al.; An mAb was raised to the C5 phagosomal antigen in Paramecium multimicronucleatum . To determine its function, the cDNA and genomic DNA encoding C5 were cloned . This antigen consisted of 315 amino acid residues with a predicted molecular weight of 36,594, a value similar to that determined by SDS-PAGE . Sequence comparisons uncovered a low but significant homology with a Schizosaccharomyces pombe protein and the C-terminal half of the beta-fructofuranosidase protein of Zymomonas mobilis . Lacking an obvious transmembrane domain or a possible signal sequence at the N terminus, C5 was predicted to be a soluble protein, whereas immunofluorescence data showed that it was present on the membranes of vesicles and digestive vacuoles (DVs) . In cells that were minimally permeabilized but with intact DVs, C5 was found to be located on the cytosolic surface of the DV membranes . Immunoblotting of proteins from the purified and KCl-washed DVs showed that C5 was tightly bound to the DV membranes . Cryoelectron microscopy also confirmed that C5 was on the cytosolic surface of the discoidal vesicles, acidosomes, and lysosomes, organelles known to fuse with the membranes of the cytopharynx, the DVs of stages I (DV-I) and II (DV-II), respectively . Although C5 was concentrated more on the mature than on the young DV membranes, the striking observation was that the cytopharyngeal membrane that is derived from the discoidal vesicles was almost devoid of C5 . Approximately 80% of the C5 was lost from the discoidal vesicle-derived membrane after this membrane fused with the cytopharyngeal membrane . Microinjection of the mAb to C5 greatly inhibited the fusion of the discoidal vesicles with the cytopharyngeal membrane and thus the incorporation of the discoidal vesicle membranes into the DV membranes . Taken together, these results suggest that C5 is a membrane protein that is involved in binding and/or fusion of the discoidal vesicles with the cytopharyngeal membrane that leads to DV formation.

Mol Biol Cell, 1999 Apr, 10(4), 1019 - 30
Uridine diphosphate-glucose transport into the endoplasmic reticulum of Saccharomyces cerevisiae: in vivo and in vitro evidence; Castro O et al.; It has been proposed that synthesis of beta-1,6-glucan, one of Saccharomyces cerevisiae cell wall components, is initiated by a uridine diphosphate (UDP)-glucose-dependent reaction in the lumen of the endoplasmic reticulum (ER) . Because this sugar nucleotide is not synthesized in the lumen of the ER, we have examined whether or not UDP-glucose can be transported across the ER membrane . We have detected transport of this sugar nucleotide into the ER in vivo and into ER-containing microsomes in vitro . Experiments with ER-containing microsomes showed that transport of UDP-glucose was temperature dependent and saturable with an apparent Km of 46 microM and a Vmax of 200 pmol/mg protein/3 min . Transport was substrate specific because UDP-N-acetylglucosamine did not enter these vesicles . Demonstration of UDP-glucose transport into the ER lumen in vivo was accomplished by functional expression of Schizosaccharomyces pombe UDP-glucose:glycoprotein glucosyltransferase (GT) in S . cerevisiae, which is devoid of this activity . Monoglucosylated protein-linked oligosaccharides were detected in alg6 or alg5 mutant cells, which transfer Man9GlcNAc2 to protein; glucosylation was dependent on the inhibition of glucosidase II or the disruption of the gene encoding this enzyme . Although S . cerevisiae lacks GT, it contains Kre5p, a protein with significant homology and the same size and subcellular location as GT . Deletion mutants, kre5Delta, lack cell wall beta-1,6 glucan and grow very slowly . Expression of S . pombe GT in kre5Delta mutants did not complement the slow-growth phenotype, indicating that both proteins have different functions in spite of their similarities.

Mol Biol Cell, 1999 Apr, 10(4), 833 - 45
Cdc25 inhibited in vivo and in vitro by checkpoint kinases Cds1 and Chk1; Furnari B et al.; In the fission yeast Schizosaccharomyces pombe, the protein kinase Cds1 is activated by the S-M replication checkpoint that prevents mitosis when DNA is incompletely replicated . Cds1 is proposed to regulate Wee1 and Mik1, two tyrosine kinases that inhibit the mitotic kinase Cdc2 . Here, we present evidence from in vivo and in vitro studies, which indicates that Cds1 also inhibits Cdc25, the phosphatase that activates Cdc2 . In an in vivo assay that measures the rate at which Cdc25 catalyzes mitosis, Cds1 contributed to a mitotic delay imposed by the S-M replication checkpoint . Cds1 also inhibited Cdc25-dependent activation of Cdc2 in vitro . Chk1, a protein kinase that is required for the G2-M damage checkpoint that prevents mitosis while DNA is being repaired, also inhibited Cdc25 in the in vitro assay . In vitro, Cds1 and Chk1 phosphorylated Cdc25 predominantly on serine-99 . The Cdc25 alanine-99 mutation partially impaired the S-M replication and G2-M damage checkpoints in vivo . Thus, Cds1 and Chk1 seem to act in different checkpoint responses to regulate Cdc25 by similar mechanisms.

J Biol Chem, 1999 Apr 16, 274(16), 11339 - 43
Regions of Byr4, a regulator of septation in fission yeast, that bind Spg1 or Cdc16 and form a two-component GTPase-activating protein with Cdc16; Furge KA et al.; In the fission yeast Schizosaccharomyces pombe, septation and constriction of the actomyosin ring for cell division are positively regulated by the Spg1 GTPase, a member of the Ras superfamily . Spg1 is negatively regulated by Byr4 and Cdc16, which together form a two-component GTPase-activating protein for the Spg1 GTPase . To better understand how Byr4 regulates septation, Byr4 mutants were tested for in vitro functions . This analysis revealed that Byr4 contained one Cdc16-binding site and four Spg1-binding sites (SBS), designated SBS1-SBS4 . Although mutants with a single SBS bound Spg1 and inhibited GTP dissociation, the equilibrium binding affinity of these mutants was 28-280-fold weaker than Byr4 . Because some Byr4 mutants with multiple SBSs bound Spg1 tighter than the corresponding mutants with a single SBS, multiple SBSs probably interact to cause the high affinity binding of Byr4 to Spg1 . A region of Byr4 that bound Spg1, SBS4, and the region that bound Cdc16, Cdc16-binding site, was necessary and sufficient to form Cdc16-dependent Spg1GAP activity that was similar to that of wild-type Byr4 with Cdc16.

Methods, 1999 Apr, 17(4), 292 - 304
Methods of assaying Bcl-2 and Bax family proteins in yeast; Xu Q et al.; Bcl-2 family proteins play an evolutionarily conserved role in regulating the life and death of the cell . Certain proapoptotic members of the Bcl-2 family, Bax and Bak, have intrinsic cytotoxic activities in that they not only induce or sensitize mammalian cells to undergo apoptosis but also display a lethal phenotype when ectopically expressed in two yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe . Furthermore, the antiapoptotic Bcl-2 and Bcl-XL proteins can protect yeast against Bax-mediated lethality, suggesting that the death-regulatory functions of these Bcl-2 family proteins are well preserved in yeast . These observations provide the opportunity to study the function of Bcl-2 family proteins in genetically tractable yeast and to apply classical yeast genetics and functional cloning approaches to the dissection of programmed cell death pathway regulated by Bcl-2 family proteins . We describe here methods used in our laboratory to express and to study the functions of Bcl-2 family proteins in both the budding yeast S . cerevisiae and the fission yeast S . pombe .

J Biol Chem, 1999 Apr 9, 274(15), 9969 - 75
Transcriptional regulation of the Schizosaccharomyces pombe malic enzyme gene, mae2; Viljoen M et al.; The NAD-dependent malic enzyme from Schizosaccharomyces pombe catalyzes the oxidative decarboxylation of L-malate to pyruvate and CO2 . Transcription of the S . pombe malic enzyme gene, mae2, was studied to elucidate the regulatory mechanisms involved in the expression of the gene . No evidence for substrate-induced expression of mae2 was observed in the presence of 0.2% L-malate . However, transcription of mae2 was induced when cells were grown in high concentrations of glucose or under anaerobic conditions . The increased levels of malic enzyme may provide additional pyruvate or assist in maintaining the redox potential under fermentative conditions . Deletion and mutation analyses of the 5'-flanking region of the mae2 gene revealed the presence of three novel negative cis-acting elements, URS1, URS2, and URS3, that seem to function cooperatively to repress transcription of the mae2 gene . URS1 and URS2 are also present in the promoter region of the S . pombe malate transporter gene, suggesting co-regulation of their expression . Furthermore, two positive cis-acting elements in the mae2 promoter, UAS1 and UAS2, show homology with the DNA recognition sites of the cAMP-dependent transcription factors ADR1, AP-2, and ATF (activating transcription factor)/CREB (cAMP response element binding).

Mol Gen Genet, 1999 Mar, 261(2), 364 - 73
The fission yeast rpa17+ gene encodes a functional homolog of AC19, a subunit of RNA polymerases I and III of Saccharomyces cerevisiae; Imai K et al.; Eukaryotic RNA polymerases I and III consist of multiple subunits . Each of these enzymes includes two distinct and evolutionarily conserved subunits called alpha-related subunits which are shared only by polymerases I and III . The alpha-related subunits show limited homology with the alpha-subunit of prokaryotic RNA polymerase . To gain further insight into the structure and function of alpha-related subunits, we cloned and characterized a gene from Schizosaccharomyces pombe that encodes a protein of 17 kDa which can functionally replace AC19 - an alpha-related subunit of RNA polymerases I and III of Saccharomyces cerevisiae - and was thus named rpa17+ . RPA17 has 125 amino acids and shows 63% identity to AC19 over a 108-residue stretch, whereas the N-terminal regions of the two proteins are highly divergent . Disruption of rpa17+ shows that the gene is essential for cell growth . Sequence comparison with other alpha-related subunits from different species showed that RPA17 contains an 81-amino acid block that is evolutionarily conserved . Deletion analysis of the N- and C-terminal regions of RPA17 and AC19 confirms that the 81-amino acid block is important for the function of the alpha-related subunits.

Mol Gen Genet, 1999 Mar, 261(2), 297 - 306
Isolation of multicopy suppressors of the calcium sensitivity of a mutant lacking the bZIP transcription factor Atf1 in fission yeast; Ohmiya R et al.; In Schizosaccharomyces pombe, recent studies have uncovered a set of putative transcription factors of the basic leucine zipper (bZIP) type (e.g., Atf1, Pcr1, Pap1), which function downstream of the Sty1 mitogen-activated protein kinase (MAPK) cascade which is involved in stress-activated signal transduction . Accordingly, a delta atf1 mutant is known to exhibit osmosensitivity for growth, since one of the targets of Atf1 is the gpd1+ gene, which is responsible for the osmoadaptive glycerol production mediated by the Sty1 MAPK cascade . During the course of our studies on the osmotic response in S . pombe, we found that growth of a delta atf1 mutant is highly sensitive to the level of Ca2+ ions in the medium (but less sensitive to Mg2+ and Na+ ions) . This phenotype seemed to be relevant to the osmosensitivity, because an delta gpd1 mutant showed a similar phenotype . An attempt was therefore made to isolate multicopy suppressors of the calcium sensitivity exhibited by the delta atf1 cells . Among such suppressors were several bZIP factors, including two known proteins (Atf21 and Pcr1), and two new ones (named Atf31 and Zip1) . These factors were characterized further, in comparison to Atf1, with special reference to the Sty1 MAPK signaling pathway.

Mol Gen Genet, 1999 Mar, 261(2), 290 - 6
An aureobasidin A resistance gene isolated from Aspergillus is a homolog of yeast AUR1, a gene responsible for inositol phosphorylceramide (IPC) synthase activity; Kuroda M et al.; The AUR1 gene of Saccharomyces cerevisiae, mutations in which confer resistance to the antibiotic aureobasidin A, is necessary for inositol phosphorylceramide (IPC) synthase activity . We report the molecular cloning and characterization of the Aspergillus nidulans aurA gene, which is homologous to AUR1 . A single point mutation in the aurA gene of A . nidulans confers a high level of resistance to aureobasidin A . The A . nidulans aurA gene was used to identify its homologs in other Aspergillus species, including A . fumigatus, A . niger, and A . oryzae . The deduced amino acid sequence of an aurA homolog from the pathogenic fungus A . fumigatus showed 87% identity to that of A . nidulans . The AurA proteins of A . nidulans and A . fumigatus shared common characteristics in primary structure, including sequence, hydropathy profile, and N-glycosylation sites, with their S . cerevisiae, Schizosaccharomyces pombe, and Candida albicans counterparts . These results suggest that the aureobasidin resistance gene is conserved evolutionarily in various fungi.

Mol Gen Genet, 1999 Mar, 261(2), 281 - 9
The ukc1 gene encodes a protein kinase involved in morphogenesis, pathogenicity and pigment formation in Ustilago maydis; Durrenberger F et al.; The fungal phytopathogen Ustilago maydis alternates between budding and filamentous growth during its life cycle . This dimorphic transition, which is influenced by environmental factors and mating, is regulated in part by cAMP-dependent protein kinase (PKA) . We have recently identified a related protein kinase, encoded by the ukc1 gene, that also plays a role in determining cell shape . The ukc1 gene is homologous to several other protein kinase-encoding genes including the cot-I gene of Neurospora crassa, the TB3 gene of Colletotrichum trifolii, the orb6 gene of Schizosaccharomyces pombe, the warts tumor suppressor gene of Drosophila melanogaster and the myotonic dystrophy kinase gene in humans . Disruption of the ukc1 gene in U . maydis resulted in cells that were highly distorted in their morphology, incapable of generating aerial filaments during mating in culture and defective in their ability to cause disease on corn seedlings . In addition, the cells of ukc1 mutants became highly pigmented and resembled the chlamydospore-like cells that have been described for U . maydis . Overall, these results demonstrate an important role for the ukc1-encoded protein kinase in the morphogenesis, pathogenesis and pigmentation of U . maydis.

Mol Gen Genet, 1999 Mar, 261(2), 242 - 50
Genetic interactions between Hsp90 and the Cdc2 mitotic machinery in the fission yeast Schizosaccharomyces pombe; Munoz MJ et al.; In Schizosaccharomyces pombe, wee1 encodes a tyrosine kinase that inhibits entry into mitosis by phophorylating Cdc2, the universal cyclin-dependent kinase (Cdk) that regulates the G2/M transition in all eukaryotic cells . A search for suppressors of the G2 arrest caused by overexpression of weel led to the isolation of a new allele of swo1 (named swo1-w1), the gene coding for chaperone Hsp90, which is required to stabilise Weel . The swo1-w1 allele carries a glycine to aspartic acid substitution at amino acid 155 that results in a partial loss of Hsp90 function . Cells bearing the swo1-w1 mutation in combination with the point mutation cdc2-33 or cdc2-M26 showed severe mitotic defects . Genetic interactions were not observed in combination with point mutations in other cdc genes, suggesting that Cdc2 specifically interacts with Hsp90 . This synthetic lethal swo1-w1 cdc2-33 (or cdc2-M26) strain had normal levels of Cdc2 protein and histone H1 phosphorylation activity, indicating that Hsp90 is required to enable Cdc2 to interact with its mitotic substrates or regulators, rather than for its proper folding or stabilisation . In a wild-type background, swo1-w1 mutant cells were sensitive to temperature as well as to other stress agents, such as KCI, ethanol and formamide . Under these stressful growth conditions, the swo1-w1 cells displayed anaphase B arrest and aberrant septation patterns, indicating that a subset of proteins involved in mitosis and cytokinesis is highly dependent on chaperone Hsp90 for function.

FEBS Lett, 1999 Mar 5, 446(1), 182 - 8
Mutational analysis of two Arabidopsis thaliana cyclin-dependent kinases in fission yeast; Porceddu A et al.; We have analyzed five mutant alleles of two cyclin-dependent kinases from Arabidopsis thaliana, CDC2aAt and CDC2bAt, in Schizosaccharomyces pombe . Two of the five mutant alleles produced similar phenotypes for both cyclin-dependent kinases . The other three mutants caused phenotypes dependent on the particular cyclin-dependent kinase . Of all the mutant alleles, only two were found to possess a detectable kinase activity . Our mutational analysis lends further support for CDC2aAt being the true orthologue of the yeast cdc2 . CDC2bAt, even though quite divergent from S . pombe cdc2, still retains the ability to interact with at least some essential cell cycle regulators, suggesting some functional homology with the yeast protein . Additionally, we demonstrated that the three amino acid deletion in the DL50 mutants results in the loss of the ability to interact with the suc1/CKS1 proteins.

Biochem Cell Biol, 1998, 76(4), 645 - 8
Regulation of D-glucose-6-phosphate dehydrogenase from Schizosaccharomyces pombe; Tsai CS et al.; D-Glucose-6-phosphate dehydrogenase is a regulatory enzyme of the oxidative pentose phosphate pathway in Schizasaccharomyces pombe . The enzyme is subject to negative cooperative regulation by D-glucose-6-phosphate as characterized by the Hill coefficient of 0.68 +/- 0.04 . D-Glyceraldehyde-3-phosphate and D-ribulose-5-phosphate rectify the negative cooperativity as evidenced from a change in the Hill coefficients to 0.98 +/- 0.05 and 1.02 +/- 0.05, respectively . These pentose phosphate pathway intermediates also inhibit the enzyme competitively with respect to D-glucose-6-phosphate . Thus, D-glucose-6-phosphate dehydrogenase provides an avenue for regulating the partitioning of D-glucose between the redundant branches of the oxidative phosphate pathway in S . pombe.

Biochem Cell Biol, 1998, 76(4), 637 - 44
Purification and kinetic characterization of 6-phosphogluconate dehydrogenase from Schizosaccharomyces pombe; Tsai CS et al.; 6-Phosphogluconate dehydrogenase is the pivotal enzyme that links the gluconate route and the oxidative phase of the pentose phosphate pathway in Schizosaccharomyces pombe . The enzyme differs from the known 6-phosphogluconate dehydrogenases of other sources in that the Schizosaccharomyces enzyme is tetrameric having a subunit mass of 38 kDa, that it requires NADP+ obligatorily for activity, and that it can be activated by divalent metal ions such as Co2+ and Mn2+ . Steady-state kinetic studies were undertaken . Initial rate and product inhibition results suggest that 6-phosphogluconate dehydrogenase from Schizosaccharomyces pombe catalyzes NADP(+)-linked oxidative decarboxylation of 6-phosphogluconate by an equilibrium random mechanism with two independent binding sites, namely one site for the nicotinamide coenzyme, NADP+/NADPH, and another site for 6-phosphogluconate-D-ribulose-5-phosphate and for CO2 . Studies of pH dependence implicated a basic residue with a pK value of 7.4 in the binding of 6-phosphogluconate and an acidic residue with a pK value of 6.7 in the cation-mediated interaction of NADP+ with the enzyme.

Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 4180 - 5
Characterization of maize (Zea mays L.) Wee1 and its activity in developing endosperm; Sun Y et al.; We report the characterization of a maize Wee1 homologue and its expression in developing endosperm . Using a 0.8-kb cDNA from an expressed sequence tag project, we isolated a 1.6-kb cDNA (ZmWee1), which encodes a protein of 403 aa with a calculated molecular size of 45.6 kDa . The deduced amino acid sequence shows 50% identity to the protein kinase domain of human Wee1 . Overexpression of ZmWee1 in Schizosaccharomyces pombe inhibited cell division and caused the cells to enlarge significantly . Recombinant ZmWee1 obtained from Escherichia coli is able to inhibit the activity of p13(suc1)-adsorbed cyclin-dependent kinase from maize . ZmWee1 is encoded by a single gene at a locus on the long arm of chromosome 4 . RNA gel blots showed the ZmWee1 transcript is about 2.4 kb in length and that its abundance reaches a maximum 15 days after pollination in endosperm tissue . High levels of expression of ZmWee1 at this stage of endosperm development imply that ZmWee1 plays a role in endoreduplication . Our results show that control of cyclin-dependent kinase activity by Wee1 is conserved among eukaryotes, from fungi to animals and plants.

Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3824 - 9
DRC1, DNA replication and checkpoint protein 1, functions with DPB11 to control DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae; Wang H et al.; In addition to DNA polymerase complexes, DNA replication requires the coordinate action of a series of proteins, including regulators Cdc28/Clb and Dbf4/Cdc7 kinases, Orcs, Mcms, Cdc6, Cdc45, and Dpb11 . Of these, Dpb11, an essential BRCT repeat protein, has remained particularly enigmatic . The Schizosaccharomyces pombe homolog of DPB11, cut5, has been implicated in the DNA replication checkpoint as has the POL2 gene with which DPB11 genetically interacts . Here we describe a gene, DRC1, isolated as a dosage suppressor of dpb11-1 . DRC1 is an essential cell cycle-regulated gene required for DNA replication . We show that both Dpb11 and Drc1 are required for the S-phase checkpoint, including the proper activation of the Rad53 kinase in response to DNA damage and replication blocks . Dpb11 is the second BRCT-repeat protein shown to control Rad53 function, possibly indicating a general function for this class of proteins . DRC1 and DPB11 show synthetic lethality and reciprocal dosage suppression . The Drc1 and Dpb11 proteins physically associate and function together to coordinate DNA replication and the cell cycle.

Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3745 - 50
A human Cds1-related kinase that functions downstream of ATM protein in the cellular response to DNA damage; Brown AL et al.; Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer . Much of our current understanding of checkpoints comes from genetic studies conducted in yeast . In the fission yeast Schizosaccharomyces pombe (Sp), SpRad3 is an essential component of both the DNA damage and DNA replication checkpoints . The SpChk1 and SpCds1 protein kinases function downstream of SpRad3 . SpChk1 is an effector of the DNA damage checkpoint and, in the absence of SpCds1, serves an essential function in the DNA replication checkpoint . SpCds1 functions in the DNA replication checkpoint and in the S phase DNA damage checkpoint . Human homologs of both SpRad3 and SpChk1 but not SpCds1 have been identified . Here we report the identification of a human cDNA encoding a protein (designated HuCds1) that shares sequence, structural, and functional similarity to SpCds1 . HuCds1 was modified by phosphorylation and activated in response to ionizing radiation . It was also modified in response to hydroxyurea treatment . Functional ATM protein was required for HuCds1 modification after ionizing radiation but not after hydroxyurea treatment . Like its fission yeast counterpart, human Cds1 phosphorylated Cdc25C to promote the binding of 14-3-3 proteins . These findings suggest that the checkpoint function of HuCds1 is conserved in yeast and mammals.

Eur J Biochem, 1999 Feb, 260(1), 31 - 7
The Schizosaccharomyces pombe Pzh1 protein phosphatase regulates Na+ ion influx in a Trk1-independent fashion; Balcells L et al.; We have previously shown that fission yeast encodes a PPZ-like phosphatase, designated Pzhl, which is an important determinant of cation homeostasis . pzh1 delta mutants display increased tolerance to Na+ ions, but they are hypersensitive to KC1 {Balcells, L., Gomez, N., Casamayor, A., Clotet, J . & Arino, J . (1997) Eur . J . Biochem . 250, 476-483} . We have immunodetected Pzh1 in yeast extracts and found that this phosphatase is largely associated with particulate fractions . Cells defective in Pzh1 do not show altered efflux of Na+ or Li+ ions, but they accumulate these cations more slowly than wild-type cells . K+ ion content of pzh1 delta cells is about twice that of wild-type cells, and this can be explained by decreased efflux of K+ . Therefore, Pzh1 may regulate both Na+ influx and K+ efflux in fission yeast . To test the possible relationship between K+ uptake, Na+ tolerance and Pzh1 function, we deleted the trk1+ gene, which encodes a putative high-affinity transporter of K+ ions . trkl delta mutants grew well even at relatively low concentrations of KCl and did not show significantly altered content or influx of K+ ions . However, they showed a Na(+)-sensitive phenotype which was greatly intensified by deletion of the sod2+ gene (which encodes the major determinant for efflux of Na+ ions), and clearly ameliorated by deletion of the pzh1 phosphatase, as well as by moderate concentrations of KCl in the medium . These results suggest that Trk1 does not mediate the effect of Pzh1 on NaCl tolerance and that fission yeast contains efficient systems, other than Trk1, for uptake of K+ ions.

Biochemistry, 1999 Mar 23, 38(12), 3649 - 55
Schizosaccharomyces pombe Aps1, a diadenosine 5',5' "-P1, P6-hexaphosphate hydrolase that is a member of the nudix (MutT) family of hydrolases: cloning of the gene and characterization of the purified enzyme; Ingram SW et al.; The fission yeast Schizosaccharomyces pombe contains a gene on chromosome I that encodes a hypothetical nudix hydrolase, YA9E . The gene, designated aps1, has been cloned and the protein has been purified from Escherichia coli with a yield of 10 mg of Aps1/L of culture . Aps1, composed of 210 amino acids with a calculated molecular mass of 23 724 Da, behaves as a monomer with a sedimentation coefficient of 1.92 S as determined by analytical ultracentrifugation . The effective hydrodynamic radius is about 29 A as determined by both analytical ultracentrifugation and gel-filtration chromatography . Aps1, whose expression was detected in S . pombe by Western blotting, is an enzyme that catalyzes the hydrolysis of dinucleoside oligophosphates, with Ap6A and Ap5A being the preferred substrates . The major reaction products are ADP and p4A from Ap6A and ADP and ATP from Ap5A . Values of Km for Ap6A and Ap5A are 19 microM and 22 microM, respectively, and the corresponding values of kcat are 2.0 s-1 and 1.7 s-1, respectively . The enzyme has limited activity on Ap4A and negligible activity on Ap3A, ADP-ribose, and NADH . Aps1 catalyzes the hydrolysis of mononucleotides with decreasing activity in order from p5A to AMP . Optimal activity with Ap6A as substrate is observed at pH 7.6 and in the presence of 0.1-1 mM MnCl2 . Aps1 is the first nudix hydrolase isolated from S . pombe, and it is the first enzyme identified with this specific substrate specificity and reaction products.

Mol Cell Biol, 1999 Apr, 19(4), 2535 - 46
The schizosaccharomyces pombe dim1(+) gene interacts with the anaphase-promoting complex or cyclosome (APC/C) component lid1(+) and is required for APC/C function; Berry LD et al.; The Schizosaccharomyces pombe dim1(+) gene is required for entry into mitosis and for chromosome segregation during mitosis . To further understand dim1p function, we undertook a synthetic lethal screen with the temperature-sensitive dim1-35 mutant and isolated lid (for lethal in dim1-35) mutants . Here, we describe the temperature-sensitive lid1-6 mutant . At the restrictive temperature of 36 degrees C, lid1-6 mutant cells arrest with a "cut" phenotype similar to that of cut4 and cut9 mutants . An epitope-tagged version of lid1p is a component of a multiprotein approximately 20S complex; the presence of lid1p in this complex depends upon functional cut9(+) . lid1p-myc coimmunoprecipitates with several other proteins, including cut9p and nuc2p, and the presence of cut9p in a 20S complex depends upon the activity of lid1(+) . Further, lid1(+) function is required for the multiubiquitination of cut2p, an anaphase-promoting complex or cyclosome (APC/C) target . Thus, lid1p is a component of the S . pombe APC/C . In dim1 mutants, the abundances of lid1p and the APC/C complex decline significantly, and the ubiquitination of an APC/C target is abolished . These data suggest that at least one role of dim1p is to maintain or establish the steady-state level of the APC/C.

Nat Genet, 1999 Mar, 21(3), 314 - 7
Involvement of nucleotide-excision repair in msh2 pms1-independent mismatch repair; Fleck O et al.; Nucleotide-excision repair (NER) and mismatch repair (MMR) are prominent examples of highly conserved DNA repair systems which recognize and replace damaged and/or mispaired nucleotides in DNA . In humans, inheritable defects in components of the NER system are associated with severe diseases such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS), whereas inactivation of MMR is accompanied by predisposition to certain types of cancer . In Schizosaccharomyces pombe, the msh2- and pms1-dependent long-patch MMR system efficiently corrects small insertion/deletion loops and all base-base mismatches, except C/C . Up to 70% of C/C mismatches generated in recombination intermediates, and to a lesser extent also other base-base mismatches, are thought to undergo correction by a minor, short-patch excision repair system . We identify here the NER genes rhpl4, swi10 and rad16 as components of this repair pathway and show that they act independently of msh2 and pms1.

Bioorg Khim, 1998 Dec, 24(12), 933 - 7
{Molecular cloning and characteristics of rpc19+ and rpc40+ Schizosaccharomyces pombe genes, coding for common subunits of nuclear RNA polymerase I and III}; Shpakovskii GV et al.; Full-length copies of cDNAs of the rpc19+ and rpc40+ genes encoding the common subunits of nuclear RNA polymerases I and III and the corresponding fragments of chromosomes were isolated from genomic and cDNA libraries of Schizosaccharomyces pombe and characterized . It was established that the cloned genes are located on chromosomes III and II of the fission yeast, respectively . The rpc40+ gene lacks introns, and the rpc19+ gene contains two intervening sequences . The comparison of subunits Rpc19 (125 aa; M 13 722 Da; pI 4.51) and Rpc40 (348 aa; M 39 141 Da; pI 5.40) of Sz . pombe, whose characteristics were deduced from the sequences of their cDNAs, with the orthologous components of other eukaryotes allowed the most conserved structure-functional domains of these proteins to be identified.

Bioorg Khim, 1998 Nov, 24(11), 877 - 80
{Molecular identification and characteristics of hRPC11, the smallest specific subunit of human RNA polymerase III}; Spakovskii GV et al.; Full-length copies of cDNA of the hRPC11 gene encoding the smallest specific subunit of nuclear RNA polymerase III were identified among human transcripts with the use of the RT-PCR technique . The cloning of the first orthologue of the subunit RPC11 from a multicellular organism and the comparison of subunit hRPC11 of Homo sapiens (108 aa; M(r), 12.3 kDa; pI 8.05) deduced from the cDNA primary structure with the homologous components of RNA polymerase III from Saccharomyces cerevisiae and Schizosaccharomyces pombe revealed the most important functional domains: a Zn-binding motif of the classic type (CxxCx16-17CxxC) at the N-terminal region, and two extended regions of homology (KEVDDVLGG and RSADEPM) in the central and C-terminal parts of the molecule, respectively . The C-terminus of the RPC11 subunits is highly homologous to the unique zinc ribbon of the elongation factor TFIIS, which suggests a role for this subunit in the elongation or termination of RNA synthesis.

Yeast, 1999 Feb, 15(3), 255 - 62
Analysis of TFIIH subunit through isolation of the gene from Schizosaccharomyces pombe corresponding to that of Saccharomyces cerevisiae SSL1, reveals the presence of conserved structural motifs; Adachi N et al.; We isolated a Schizosaccharomyces pombe (Sz . pombe) gene encoding the counterpart of the TFIIH subunit Homo sapiens (H . sapiens) p44 and Saccharomyces cerevisiae (S . cerevisiae) SSL1, and we named this gene product p47 . Contrary to the case of SSL1, which is an essential gene of S . cerevisiae, p47 is not essential for the viability of Sz . pombe . The deduced amino acid sequence revealed that this TFIIH subunit is highly conserved during evolution . Comparison of the primary structures revealed differences in the predicted positions of introns in the Caenorhabditis elegans (C . elegans) gene encoding the p47 counterpart found during the genome project . A charged cluster in the N-terminal region is present in the two yeasts . Two putative zinc-binding motifs, an extended C2H2 zinc finger with a 'C8 motif' and a second putative zinc-binding motif common to the two TFIIH subunits, were also found, the former being completely conserved . The latter motif consists of five cysteine residues and is also present in hp44, SSL1 and another TFIIH subunit, human p34 (hp34) . Since one zinc atom can bind to four ligands in zinc-binding motifs, the conservation of cysteine residues was given attention . This motif is completely conserved in p47 homologues derived from the four species . As one cysteine residue is not conserved among the homologues of hp34, the consensus of this motif is concluded to be Cys X2-Cys-X(10,12)-Cys-X2-Cys . This nucleotide sequence has been deposited in the GenBank Data Library under Accession Number AF017646.

Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 2656 - 61
The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks; Chuang RY et al.; The origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae as a protein that specifically binds to origins of DNA replication . Although ORC appears to play an essential role in the initiation of DNA replication in the cells of all eukaryotes, its interactions with DNA have not been defined in species other than budding yeast . We have characterized a Schizosaccharomyces pombe homologue of the ORC subunit, Orc4p . The homologue (Orp4p) consists of two distinct functional domains . The C-terminal domain shows strong sequence similarity to human, frog, and yeast Orc4 proteins, including conserved ATP-binding motifs . The N-terminal domain contains nine copies of the AT-hook motif found in a number of DNA-binding proteins, including the members of the HMG-I(Y) family of chromatin proteins . AT-hook motifs are known from biochemical and structural studies to mediate binding to the minor groove of AT-tracts in DNA . Orp4p is essential for viability of Sc . pombe and is expressed throughout the cell cycle . The Orp4 protein (and its isolated N-terminal domain) binds to the Sc . pombe replication origin, ars1 . The DNA binding properties of Orp4p provide a plausible explanation for the characteristic features of Sc . pombe origins of replication, which differ significantly from those of Sa . cerevisiae.

Plant Cell, 1999 Mar, 11(3), 417 - 30
Isolation and characterization of SYN1, a RAD21-like gene essential for meiosis in Arabidopsis; Bai X et al.; The proper pairing, recombination, and segregation of chromosomes are central to meiosis and sexual reproduction . The syn1 mutation was previously identified as a synaptic mutant in a T-DNA-tagged population of plants . SYN1 has been isolated and found to exhibit similarity to Schizosaccharomyces pombe RAD21 and RAD21-like proteins, which are required for chromosome condensation and sister chromatid cohesion during mitosis . Plants homozygous for syn1 are male and female sterile and show defects in chromosome condensation and pairing beginning at leptonema of meiosis I . Fragmentation of the chromosomes was observed at metaphase I . Alternative promoters produced two SYN1 transcripts . One transcript was expressed at low levels in most tissues, whereas the other was expressed only in prebolting buds . DNA blot analyses suggest that Arabidopsis contains a small RAD21 gene family . Consistent with the DNA blot data, a second Arabidopsis RAD21-like gene has been identified . These results suggest that different RAD21-like proteins play essential roles in chromosome condensation and pairing during both meiosis and mitosis.

Mol Gen Genet, 1999 Feb, 261(1), 177 - 83
A Schizosaccharomyces pombe gene, ksg1, that shows structural homology to the human phosphoinositide-dependent protein kinase PDK1, is essential for growth, mating and sporulation; Niederberger C et al.; Fission yeast (Schizosaccharomyces pombe) requires inositol for growth, mating and sporulation . To define putative genes that are involved in the processing and transduction of the inositol signal, mutants that are temperature sensitive for growth and sporulation were selected on a medium containing non-limiting amounts of inositol . Two such mutants (ksg1-208 and ksg1-358) were analyzed, which are impaired in mating and sporulation at 30 degrees C and undergo growth arrest in the G2 phase of the cell cycle at 35 degrees C . The ksg1 gene was isolated by functional complementation . It maps on the left arm of chromosome II and encodes a putative 592-amino acid protein which exhibits good structural homology to a human 3-phosphoinositide-dependent protein kinase (PDK1) and its rat and Drosophila homologues . The two mutants have the same substitution at amino acid position 159: a glycine residue is replaced by glutamic acid . Deletion of the gene is lethal for haploid cells . We propose that ksg1 is involved in one or several phosphoinositide signalling processes that are responsible for control of the life cycle.

Mol Gen Genet, 1999 Feb, 261(1), 161 - 9
Mutations in the Schizosaccharomyces pombe heat shock factor that differentially affect responses to heat and cadmium stress; Saltsman KA et al.; Heat shock factor (hsf) is the transcriptional activator that governs the transcriptional response of eukaryotic cells to stressful conditions . The structure and regulation of hsf is highly conserved . We describe deletion mutations in hsf+ that alter the ability of Schizosaccharomyces pombe to respond to different stressful conditions . One mutation causes increased sensitivity to cadmium while maintaining near normal sensitivity to heat stress, while another mutation confers increased sensitivity to heat stress but retains normal sensitivity to cadmium . Despite the differential sensitivity of these two strains to cadmium and heat stress, the mutant hsf proteins in each strain were activated by both cadmium and heat . However, we found that these mutations differentially affected the ability of hsf to activate different promoters: one mutated hsf activated the ssp1+ gene better than the wis2+ gene following either stress, while the other mutated hsf activated wis2+ better than ssp1+ . We propose that the differential ability of strains that contain these mutant hsfs to survive cadmium and heat stress is not caused by differences in activation of hsf, but is caused instead by differential abilities of the mutant hsfs to activate the appropriate sets of genes needed for survival.

J Biochem (Tokyo), 1999 Mar, 125(3), 574 - 85
Disruption of the YRB2 gene retards nuclear protein export, causing a profound mitotic delay, and can be rescued by overexpression of XPO1/CRM1; Noguchi E et al.; Disruption of the YRB2 gene encoding a nuclear Ran-binding protein homologous to Yrb1p/RanBP1 makes Saccharomyces cerevisiae cold sensitive for colony-formation, but not for growth in liquid medium . Schizosaccharomyces pombe Hba1p, which is homologous to Saccharomyces cerevisiae Yrb2p, rescued the cold sensitivity of Deltayrb2 cells . When released from an alpha factor block, Deltayrb2 cells underwent a prolonged delay at the short spindle stage of mitosis with a normal level of Clb/p34(CDC28) kinase activity, but there was no chromosome loss, this being consistent with the finding that Deltayrb2 was synthetic lethal with neither Deltamad1 nor Deltamad3 . The cold sensitive colony-formation of Deltayrb2 cells was rescued by both XPO1/CRM1 and GSP1, but not CDC5, carried on a multicopy vector . XPO1/CRM1 rescued Deltayrb2 even in a single copy . Consistent with such a tight functional interaction, Xpo1p/Crm1p directly bound to Yrb2p, but not Yrb1p, and Deltayrb2 cells were found to have a defect in nuclear export signal (NES)-dependent nuclear protein export . From these results together, the ability of Xpo1/Crm1p to export NES-proteins is suggested to be enhanced by both Yrb2p and Gsp1p, and thereby disruption of YRB2 retards nuclear protein export, resulting in the mitotic delay.

Genetics, 1999 Mar, 151(3), 1027 - 39
Evolution of the RECQ family of helicases: A drosophila homolog, Dmblm, is similar to the human bloom syndrome gene; Kusano K et al.; Several eukaryotic homologs of the Escherichia coli RecQ DNA helicase have been found . These include the human BLM gene, whose mutation results in Bloom syndrome, and the human WRN gene, whose mutation leads to Werner syndrome resembling premature aging . We cloned a Drosophila melanogaster homolog of the RECQ helicase family, Dmblm (Drosophila melanogaster Bloom), which encodes a putative 1487-amino-acid protein . Phylogenetic and dot plot analyses for the RECQ family, including 10 eukaryotic and 3 prokaryotic genes, indicate Dmblm is most closely related to the Homo sapiens BLM gene, suggesting functional similarity . Also, we found that Dmblm cDNA partially rescued the sensitivity to methyl methanesulfonate of Saccharomyces cerevisiae sgs1 mutant, demonstrating the presence of a functional similarity between Dmblm and SGS1 . Our analyses identify four possible subfamilies in the RECQ family: (1) the BLM subgroup (H . sapiens Bloom, D . melanogaster Dmblm, and Caenorhabditis elegans T04A11.6); (2) the yeast RECQ subgroup (S . cerevisiae SGS1 and Schizosaccharomyces pombe rqh1/rad12); (3) the RECQL/Q1 subgroup (H . sapiens RECQL/Q1 and C . elegans K02F3.1); and (4) the WRN subgroup (H . sapiens Werner and C . elegans F18C5.2) . This result may indicate that metazoans hold at least three RECQ genes, each of which may have a different function, and that multiple RECQ genes diverged with the generation of multicellular organisms . We propose that invertebrates such as nematodes and insects are useful as model systems of human genetic diseases.

Genetics, 1999 Mar, 151(3), 945 - 63
Localization and properties of a silencing element near the mat3-M mating-type cassette of Schizosaccharomyces pombe; Thon G et al.; Transcription is repressed in a segment of Schizosaccharomyces pombe chromosome II that encompasses the mat2-P and mat3-M mating-type cassettes . Chromosomal deletion analysis revealed the presence of a repressor element within 500 bp of mat3-M . This element acted in synergy with the trans-acting factors Swi6, Clr1, Clr2, Clr3, and Clr4 and had several properties characteristic of silencers: it did not display promoter specificity, being able to silence not only the M mating-type genes but also the S . pombe ura4 and ade6 genes placed on the centromere-distal side of the mat3-M cassette; it could repress a gene when placed further than 2.6 kb from the promoter and it acted in both orientations, although with different efficiencies, the natural orientation repressing more stringently than the reverse . Following deletion of this element, two semistable states of expression of the mat3-M region were observed and these two states could interconvert . The deletion did not affect gene expression in the vicinity of the mat2-P cassette, 11 kb away from mat3-M . Conversely, deleting 1.5 kb on the centromere-proximal side of the mat2-P cassette, which was previously shown to partially derepress transcription around mat2-P, had no effect on gene expression near mat3-M . A double deletion removing the mat2-P and mat3-M repressor elements had the same effect as the single deletions on their respective cassettes when assayed in cells of the M mating type . These observations allow us to refine a model proposing that redundant pathways silence the mating type region of S . pombe.

J Biol Chem, 1999 Mar 5, 274(10), 6212 - 8
Functional expression of the plant alternative oxidase affects growth of the yeast Schizosaccharomyces pombe; Affourtit C et al.; We have investigated the extent to which functional expression of the plant alternative oxidase (from Sauromatum guttatum) in Schizosaccharomyces pombe affects yeast growth . When cells are cultured on glycerol, the maximum specific growth rate is decreased from 0.13 to 0.11 h-1 while growth yield is lowered by 20% (from 1 . 14 x 10(8) to 9.12 x 10(7) cells ml-1) . Kinetic studies suggest that the effect on growth is mitochondrial in origin . In isolated mitochondria we found that the alternative oxidase actively competes with the cytochrome pathway for reducing equivalents and contributes up to 24% to the overall respiratory activity . Metabolic control analysis reveals that the alternative oxidase exerts a considerable degree of control (22%) on total electron flux . Furthermore, the negative control exerted by the alternative oxidase on the flux ratio of electrons through the cytochrome and alternative pathways is comparable with the positive control exerted on this flux-ratio by the cytochrome pathway . To our knowledge, this is the first paper to report a phenotypic effect because of plant alternative oxidase expression . We suggest that the effect on growth is the result of high engagement of the non-protonmotive alternative oxidase in yeast respiration that, consequently, lowers the efficiency of energy conservation and hence growth.

J Cell Sci, 1999 Mar, 112 ( Pt 6), 939 - 46
Regulation of the start of DNA replication in Schizosaccharomyces pombe; Carlson CR et al.; Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources under steady-state conditions, with doubling times ranging from 2.5 to 14 hours . Flow cytometry and fluorescence microscopy confirmed earlier findings that at rapid growth rates, the G1 phase was short and cell separation occurred at the end of S phase . For some nitrogen sources, the growth rate was greatly decreased, the G1 phase occupied 30-50% of the cell cycle, and cell separation occurred in early G1 . In contrast, other nitrogen sources supported low growth rates without any significant increase in G1 duration . The method described allows manipulation of the length of G1 and the relative cell cycle position of S phase in wild-type cells . Cell mass was measured by flow cytometry as scattered light and as protein-associated fluorescence . The extensions of G1 were not related to cell mass at entry into S phase . Our data do not support the hypothesis that the cells must reach a certain fixed, critical mass before entry into S . We suggest that cell mass at the G1/S transition point is variable and determined by a set of molecular parameters . In the present experiments, these parameters were influenced by the different nitrogen sources in a way that was independent of the actual growth rate.

J Cell Sci, 1999 Mar, 112 ( Pt 6), 927 - 37
Caffeine can override the S-M checkpoint in fission yeast; Wang SW et al.; The replication checkpoint (or 'S-M checkpoint') control prevents progression into mitosis when DNA replication is incomplete . Caffeine has been known for some time to have the capacity to override the S-M checkpoint in animal cells . We show here that caffeine also disrupts the S-M checkpoint in the fission yeast Schizosaccharomyces pombe . By contrast, no comparable effects of caffeine on the S . pombe DNA damage checkpoint were seen . S . pombe cells arrested in early S phase and then exposed to caffeine lost viability rapidly as they attempted to enter mitosis, which was accompanied by tyrosine dephosphorylation of Cdc2 . Despite this, the caffeine-induced loss of viability was not blocked in a temperature-sensitive cdc2 mutant incubated at the restrictive temperature, although catastrophic mitosis was prevented under these conditions . This suggests that, in addition to S-M checkpoint control, a caffeine-sensitive function may be important for maintenance of cell viability during S phase arrest . The lethality of a combination of caffeine with the DNA replication inhibitor hydroxyurea was suppressed by overexpression of Cds1 or Chk1, protein kinases previously implicated in S-M checkpoint control and recovery from S phase arrest . In addition, the same combination of drugs was specifically tolerated in cells overexpressing either of two novel S . pombe genes isolated in a cDNA library screen . These findings should allow further molecular investigation of the regulation of S phase arrest, and may provide a useful system with which to identify novel drugs that specifically abrogate the checkpoint control.

Mol Microbiol, 1999 Jan, 31(2), 511 - 20
Cloning of two genes encoding potassium transporters in Neurospora crassa and expression of the corresponding cDNAs in Saccharomyces cerevisiae; Haro R et al.; Two Neurospora crassa genes, trk-1 and hak-1, encode K+ transporters that show sequence similarities to the TRK transporters described in Saccharomyces cerevisiae and Schizosaccharomyces pombe, and to the HAK transporters described in Schwanniomyces occidentalis and barley . The N . crassa TRK1 and HAK1 transporters expressed by the corresponding cDNAs in a trk1 delta trk2 delta mutant of S . cerevisiae exhibited a high affinity for Rb+ and K+ . Northern blot analysis and comparison of the kinetic characteristics of the two transporters in the trk1 delta trk2 delta mutant with the kinetic characteristics of K+ uptake in N . crassa cells allowed TRK1 to be identified as the dominant K+ transporter and HAK1 as a transporter that is only expressed when the cells are K+ starved . The HAK1 transporter showed a high concentrative capacity and is identified as the K(+)-H+ symporter described in N . crassa, whereas TRK1 might be a K+ uniporter . Although the co-existence of K+ transporters of the TRK and HAK types in the same species had not been reported formerly, we discuss whether this co-existence may be the normal situation in soil fungi.

FEBS Lett, 1999 Jan 29, 443(3), 256 - 60
Phosphorylation of yeast TBP by protein kinase CK2 reduces its specific binding to DNA; Maldonado E et al.; Protein kinase CK2 is a ubiquitous Ser/Thr kinase which phosphorylates a large number of proteins including several transcription factors . Recombinant Xenopus laevis CK2 phosphorylates both recombinant Saccharomyces cerevisiae and Schizosaccharomyces pombe TATA binding protein (TBP) . The phosphorylation of TBP by CK2 reduces its binding activity to the TATA box . CK2 copurifies with the transcription factor IID (TFIID) complex from HeLa cell extracts and phosphorylates several of the TBP-associated factors within TFIID . Taken together these findings argue for a role of CK2 in the control of transcription by RNA polymerase II through the modulation of the binding activity of TBP to the TATA box.

J Mol Biol, 1999 Feb 26, 286(3), 695 - 708
Essential structural features in the Schizosaccharomyces pombe pre-rRNA 5' external transcribed spacer; Intine RV et al.; The proximal region in the 5' external transcribed spacer (5'ETS) of the genes encoding ribosomal RNAs in Schizosaccharomyces pombe was examined with respect to structural features which underlie rRNA maturation . Computer analyses and partial digestion with nuclease probes indicate a crucifix-like structure composed primarily of three extended hairpins which are more highly ordered than previously proposed in Saccharomyces cerevisiae . A re-evaluation of the same region in S . cerevisiae indicates a conserved core structure, including the U3 snoRNA binding site within this higher-order structure . The sequences encoding the individual hairpins were deleted by PCR-mediated mutagenesis and the mutant rDNAs were expressed in vivo to determine the effect of these features on rRNA maturation . Quantitative hybridization analyses indicate that the first hairpin only has modest effects on 18 S rRNA maturation, but the other two regions are critical and no mature 18 S rRNA was observed . When smaller changes were systematically introduced into the critical regions, strong correlations were observed with known or putative events in rRNA maturation . Changes associated with an intermediate cleavage site in helix II and with the putative U3 snoRNA binding site were again critical to 18 S rRNA production . In each case, the effects were sequence dependent and not simply the result of disrupted structure . Further analyses of the 5.8 S rRNA indicate that the large ribosomal subunit RNA can be properly processed in each case but the efficiency is reduced by as much as 60 %, an observation which provides new evidence of interdependency in the maturation process . The results illustrate that rRNA processing is more critically dependent on the 5'ETS than previously believed .

Mol Cell Biol, 1999 Mar, 19(3), 2351 - 65
A new member of the Sin3 family of corepressors is essential for cell viability and required for retroelement propagation in fission yeast; Dang VD et al.; Tf1 is a long terminal repeat (LTR)-containing retrotransposon that propagates within the fission yeast Schizosaccharomyces pombe . LTR-retrotransposons possess significant similarity to retroviruses and therefore serve as retrovirus models . To determine what features of the host cell are important for the proliferation of this class of retroelements, we screened for mutations in host genes that reduced the transposition activity of Tf1 . We report here the isolation and characterization of pst1(+), a gene required for Tf1 transposition . The predicted amino acid sequence of Pst1p possessed high sequence homology with the Sin3 family of proteins, known for their interaction with histone deacetylases . However, unlike the SIN3 gene of Saccharomyces cerevisiae, pst1(+) is essential for cell viability . Immunofluorescence microscopy indicated that Pst1p was localized in the nucleus . Consistent with the critical role previously reported for Sin3 proteins in the histone acetylation process, we found that the growth of the strain with the pst1-1 allele was supersensitive to the specific histone deacetylase inhibitor trichostatin A . However, our analysis of strains with the pst1-1 mutation was unable to detect any changes in the acetylation of specific lysines of histones H3 and H4 as measured in bulk chromatin . Interestingly, the pst1-1 mutant strain produced wild-type levels of Tf1-encoded proteins and cDNA, indicating that the defect in transposition occurred after reverse transcription . The results of immunofluorescence microscopy showed that the nuclear localization of the Tf1 capsid protein was disrupted in the strain with the pst1-1 mutation, indicating an important role of pst1(+) in modulating the nuclear import of Tf1 virus-like particles.

Mol Cell Biol, 1999 Mar, 19(3), 1928 - 37
Control of growth and differentiation by Drosophila RasGAP, a homolog of p120 Ras-GTPase-activating protein; Feldmann P et al.; Mammalian Ras GTPase-activating protein (GAP), p120 Ras-GAP, has been implicated as both a downregulator and effector of Ras proteins, but its precise role in Ras-mediated signal transduction pathways is unclear . To begin a genetic analysis of the role of p120 Ras-GAP we identified a homolog from the fruit fly Drosophila melanogaster through its ability to complement the sterility of a Schizosaccharomyces pombe (fission yeast) gap1 mutant strain . Like its mammalian homolog, Drosophila RasGAP stimulated the intrinsic GTPase activity of normal mammalian H-Ras but not that of the oncogenic Val12 mutant . RasGAP was tyrosine phosphorylated in embryos and its Src homology 2 (SH2) domains could bind in vitro to a small number of tyrosine-phosphorylated proteins expressed at various developmental stages . Ectopic expression of RasGAP in the wing imaginal disc reduced the size of the adult wing by up to 45% and suppressed ectopic wing vein formation caused by expression of activated forms of Breathless and Heartless, two Drosophila receptor tyrosine kinases of the fibroblast growth factor receptor family . The in vivo effects of RasGAP overexpression required intact SH2 domains, indicating that intracellular localization of RasGAP through SH2-phosphotyrosine interactions is important for its activity . These results show that RasGAP can function as an inhibitor of signaling pathways mediated by Ras and receptor tyrosine kinases in vivo . Genetic interactions, however, suggested a Ras-independent role for RasGAP in the regulation of growth . The system described here should enable genetic screens to be performed to identify regulators and effectors of p120 Ras-GAP.

Mol Cell Biol, 1999 Mar, 19(3), 1800 - 9
The Saccharomyces cerevisiae ETH1 gene, an inducible homolog of exonuclease III that provides resistance to DNA-damaging agents and limits spontaneous mutagenesis; Bennett RA; The recently sequenced Saccharomyces cerevisiae genome was searched for a gene with homology to the gene encoding the major human AP endonuclease, a component of the highly conserved DNA base excision repair pathway . An open reading frame was found to encode a putative protein (34% identical to the Schizosaccharomyces pombe eth1(+) {open reading frame SPBC3D6.10} gene product) with a 347-residue segment homologous to the exonuclease III family of AP endonucleases . Synthesis of mRNA from ETH1 in wild-type cells was induced sixfold relative to that in untreated cells after exposure to the alkylating agent methyl methanesulfonate (MMS) . To investigate the function of ETH1, deletions of the open reading frame were made in a wild-type strain and a strain deficient in the known yeast AP endonuclease encoded by APN1 . eth1 strains were not more sensitive to killing by MMS, hydrogen peroxide, or phleomycin D1, whereas apn1 strains were approximately 3-fold more sensitive to MMS and approximately 10-fold more sensitive to hydrogen peroxide than was the wild type . Double-mutant strains (apn1 eth1) were approximately 15-fold more sensitive to MMS and approximately 2- to 3-fold more sensitive to hydrogen peroxide and phleomycin D1 than were apn1 strains . Elimination of ETH1 in apn1 strains also increased spontaneous mutation rates 9- or 31-fold compared to the wild type as determined by reversion to adenine or lysine prototrophy, respectively . Transformation of apn1 eth1 cells with an expression vector containing ETH1 reversed the hypersensitivity to MMS and limited the rate of spontaneous mutagenesis . Expression of ETH1 in a dut-1 xthA3 Escherichia coli strain demonstrated that the gene product functionally complements the missing AP endonuclease activity . Thus, in apn1 cells where the major AP endonuclease activity is missing, ETH1 offers an alternate capacity for repair of spontaneous or induced damage to DNA that is normally repaired by Apn1 protein.

EMBO J, 1999 Feb 15, 18(4), 854 - 62
Evidence for F-actin-dependent and -independent mechanisms involved in assembly and stability of the medial actomyosin ring in fission yeast; Naqvi NI et al.; Cell division in a number of eukaryotes, including the fission yeast Schizosaccharomyces pombe, is achieved through a medially placed actomyosin-based contractile ring . Although several components of the actomyosin ring have been identified, the mechanisms regulating ring assembly are still not understood . Here, we show by biochemical and mutational studies that the S.pombe actomyosin ring component Cdc4p is a light chain associated with Myo2p, a myosin II heavy chain . Localization of Myo2p to the medial ring depended on Cdc4p function, whereas localization of Cdc4p at the division site was independent of Myo2p . Interestingly, the actin-binding and motor domains of Myo2p are not required for its accumulation at the division site although the motor activity of Myo2p is essential for assembly of a normal actomyosin ring . The initial assembly of Myo2p and Cdc4p at the division site requires a functional F-actin cytoskeleton . Once established, however, F-actin is not required for the maintenance of Cdc4p and Myo2p medial rings, suggesting that the attachment of Cdc4p and Myo2p to the division site involves proteins other than actin itself.

J Biochem (Tokyo), 1999 Feb, 125(2), 230 - 5
Identification and characterization of a Drosophila homologue of the yeast UBC9 and hus5 genes; Ohsako S et al.; The yeast UBC9 and hus5 gene products have been identified as putative E2 members of the ubiquitin-conjugating enzyme (UBC) family and have been shown to play an essential role in cell cycle progression . We have identified a Drosophila Ubc9/Hus5 homologue (termed dUBC9) in an attempt to identify proteins that interact with the amino-terminal transcriptional repression domain of the Groucho corepressor by use of the yeast two-hybrid system . The predicted dUBC9 protein consists of 159 amino acids and shows 85, 68, and 54% amino acid sequence identities with human UBC9 homologue, Schizosaccharomyces pombe Hus5, and Saccharomyces cerevisiae Ubc9 proteins, respectively . Expression of dUBC9 cDNA complements a temperature-sensitive ubc9-1 mutation of S . cerevisiae to fully restore normal growth, indicating that the dUBC9 protein can act as a substitute for the yeast Ubc9 protein . The dUBC9 transcripts were about 1.2 kb and were detected at all stages of Drosophila development and in ovaries and Schneider cells . However, an increased level was observed in early embryos and ovaries . The dUBC9 gene is present as a single copy in the genome and localized in segment 21C-D on the left arm of the second chromosome.

J Biol Chem, 1999 Feb 19, 274(8), 5104 - 13
Functional organization of two large subunits of the fission yeast Schizosaccharomyces pombe RNA polymerase II . Location of the catalytic sites; Wlassoff WA et al.; The catalytically competent transcription complex of RNA polymerase II from the fission yeast Schizosaccharomyces pombe was affinity labeled with photoreactive nucleotide analogues incorporated at 3' termini of nascent RNA chains . To locate the catalytic site for RNA polymerization, the labeled subunits were separated by SDS-polyacrylamide gel electrophoresis and subjected to partial proteolysis . After microsequencing of proteolytic fragments, a complex multidomain organization was indicated for both of the two large subunits, Rpb1 and Rpb2, with the most available sites of proteolysis in junctions between the conserved sequences among RNA polymerase from both prokaryotes and eukaryotes . The cross-linking studies indicate the following: (i) the 3' termini of growing RNA chains are most extensively cross-linked to the second largest subunit Rpb2 between amino acids 825 and 994; (ii) the regions 298-535 of Rpb2 and 614-917 of Rpb1 are cross-linked to less extents, suggesting that these regions are situated in the vicinity of the catalytic site . All these regions include the conserved sequences of RNA polymerases, and the catalytic site of Rpb2 belongs to an NH2-terminal part of its conserved sequence H.

Fungal Genet Biol, 1998 Nov, 25(2), 79 - 86
Enhancement of neutral trehalase activity by oxidative stress in the fission yeast Schizosaccharomyces pombe; Fernandez J et al.; Addition of hydrogen peroxide, menadione, or plumbagin to growing cultures of the fission yeast Schizosaccharomyces pombe increased trehalase activity . The effect was inhibited only slightly in the presence of cycloheximide, indicating that the stimulation of trehalase triggered by oxidative stress is mostly due to posttranscriptional activation . Northern blot analysis of trehalase mRNA level revealed that oxidative stress also induces a moderate rise in transcription of trehalase . Mutants disrupted in genes encoding elements of the mitogen-activated protein kinase (MAPK) cascade showed a reduced increase in trehalase activity upon oxidative challenge, which was coincident with a block in transcription of trehalase . Taken together, the results support the idea that the enhancement of trehalase by oxidative stress is due to enzyme activation (via the Pka1/Sck1 phosphorylation pathway) and induction of trehalase mRNA (via the MAPK signaling pathway) . In spite of the trehalase increase, a net accumulation of trehalose was noticed during the oxidative stress.

J Bacteriol, 1999 Feb, 181(4), 1356 - 9
Cell surface galactosylation is essential for nonsexual flocculation in Schizosaccharomyces pombe; Tanaka N et al.; We have isolated fission yeast mutants that constitutively flocculate upon growth in liquid media . One of these mutants, the gsf1 mutant, was found to cause dominant, nonsexual, and calcium-dependent aggregation of cells into flocs . Its flocculation was inhibited by the addition of galactose but was not affected by the addition of mannose or glucose, unlike Saccharomyces cerevisiae FLO mutants . The gsf1 mutant coflocculated with Schizosaccharomyces pombe wild-type cells, while no coflocculation was found with galactose-deficient (gms1Delta) cells . Moreover, flocculation of the gsf1 mutant was also inhibited by addition of cell wall galactomannan from wild-type cells but not from gms1Delta cells . These results suggested that galactose residues in the cell wall glycoproteins may be receptors of gsf1-mediated flocculation, and therefore cell surface galactosylation is required for nonsexual flocculation in S . pombe.

Mol Biol Cell, 1999 Feb, 10(2), 245 - 57
Liz1p, a novel fission yeast membrane protein, is required for normal cell division when ribonucleotide reductase is inhibited; Moynihan EB et al.; Ribonucleotide reductase activity is required for generating deoxyribonucleotides for DNA replication . Schizosaccharomyces pombe cells lacking ribonucleotide reductase activity arrest during S phase of the cell cycle . In a screen for hydroxyurea-sensitive mutants in S . pombe, we have identified a gene, liz1(+), which when mutated reveals an additional, previously undescribed role for ribonucleotide reductase activity during mitosis . Inactivation of ribonucleotide reductase, by either hydroxyurea or a cdc22-M45 mutation, causes liz1(-) cells in G2 to undergo an aberrant mitosis, resulting in chromosome missegregation and late mitotic arrest . liz1(+) encodes a 514-amino acid protein with strong similarity to a family of transmembrane transporters, and localizes to the plasma membrane of the cell . These results reveal an unexpected G2/M function of ribonucleotide reductase and establish that defects in a transmembrane protein can affect cell cycle progression.

Glycobiology, 1999 Feb, 9(2), 133 - 41
Coexpression of alpha1,2 galactosyltransferase and UDP-galactose transporter efficiently galactosylates N- and O-glycans in Saccharomyces cerevisiae; Kainuma M et al.; We have studied in vivo neo-galactosylation in Saccharomyces cerevisiae and analyzed the critical factors involved in this system . Two heterologous genes, gma12(+) encoding alpha1, 2-galactosyltransferase (alpha1,2 GalT) from Schizosaccharomyces pombe and UGT2 encoding UDP-galactose (UDP-Gal) transporter from human, were functionally expressed to examine the intracellular conditions required for galactosylation . Detection by fluorescence labeled alpha-galactose specific lectin revealed that 50% of the cells incorporated galactose to cell surface mannoproteins only when the gma12(+) and hUGT2 genes were coexpressed in galactose media . Integration of both genes in the Delta mnn1 background cells increased galactosylation to 80% of the cells . Correlation between cell surface galactosylation and UDP-galactose transport activity indicated that an exogenous supply of UDP-Gal transporter rather than alpha1,2 GalT played a key role for efficient galactosylation in S.cerevisiae . In addition, this heterologous system enabled us to study the in vivo function of S . pombe alpha1,2 GalT to prove that it transfers galactose to both N - and O -linked oligosaccharides . Structural analysis indicated that this enzyme transfers galactose to O -mannosyl residue attached to polypeptides and produces Galalpha1,2-Man1-O-Ser/Thr structure . Thus, we have successfully generated a system for efficient galactose incorporation which is originally absent in S . cerevisiae, suggesting further possibilities for in vivo glycan remodeling toward therapeutically useful galactose containing heterologous proteins in S . cerevisiae.

Genomics, 1999 Jan 15, 55(2), 219 - 28
Human and mouse homologs of the Schizosaccharomyces pombe rad17+ cell cycle checkpoint control gene; Bluyssen HA et al.; The Schizosaccharomyces pombe rad17+ cell cycle checkpoint control gene is required for S-phase and G2/M arrest in response to both DNA damage and incomplete DNA replication . We isolated and characterized the putative human (RAD17Sp) and mouse (mRAD17Sp) homologs of the S . pombe Rad17 (Rad17Sp) protein . The human RAD17Sp open reading frame (ORF) encodes a protein of 681 amino acids; the mRAD17Sp ORF codes for a protein of 688 amino acids . The mRAD17Sp messenger is highly expressed in the testis as a single 3-kb mRNA species . The human RAD17Sp and mRAD17Sp proteins are 24% identical and 46% similar to the S.pombe Rad17Sp protein . Sequence homology was also noted with the Saccharomyces cerevisiae Rad24Sc (which is the structural counterpart of S.pombe Rad17Sp) and structurally related polypeptides from Caenorhabditis elegans, Arabidopsis thaliana, Pyrococcus horikoshii, and Drosophila melanogaster . The degree of conservation between the mammalian RAD17Sp proteins and those of the other species is consistent with the evolutionary distance between the species, indicating that these proteins are most likely true counterparts . In addition, homology was found between the Rad17Sp homologs and proteins identified as components of mammalian replication factor C (RF-C)/activator 1, especially in several highly conserved RF-C-like domains including a "Walker A" motif . Using FISH and analysis of a panel of rodent-human cell hybrids, the human RAD17Sp gene (HGMW-approved symbol RAD17 could be localized on human chromosome 5q13-q14, a region implicated in the etiology of small cell lung carcinoma, non-small-cell lung carcinoma, duodenal adenocarcinoma, and head and neck squamous cell carcinoma . Our results suggest that the structure and function of the checkpoint "rad" genes in the G2/M checkpoint pathway are evolutionary conserved between yeast and higher eukaryotes .

Prog Nucleic Acid Res Mol Biol, 1999, 62, 369 - 95
DNA damage and replication checkpoints in the fission yeast, Schizosaccharomyces pombe; Huberman JA; Eukaryotic organisms have developed an array of mechanisms for minimizing the consequences of damage to their DNA molecules and the consequences of interference with their DNA replication . Among these mechanisms are the DNA damage and replication checkpoints, which inhibit passage from one cell cycle stage to the next when DNA is damaged or replication is incomplete . Studies of these checkpoints in the fission yeast, Schizosaccharomyces pombe, complement studies in other organisms and provide valuable insight into the nature of the proteins responsible for these checkpoints and how such proteins may function.

Eur J Cell Biol, 1998 Dec, 77(4), 284 - 93
Effects of the myosin inhibitor 2,3-butanedione monoxime on the physiology of fission yeast; Steinberg G et al.; F-actin and associated myosins are thought to take part in a wide range of cellular processes, like motility and contraction, polarized growth, and secretion . The reagent 2,3-butanedione monoxime (BDM) is a well characterized inhibitor of the contraction of vertebrate muscle that reversibly affects myosin function and influences the intracellular concentration of Ca2+ . Here we describe the influence of BDM on growth and division of the fission yeast Schizosaccharomyces pombe . At concentrations from 1-30 mM, BDM gradually inhibited formation and growth of S . pombe colonies on agar plates, with a lethal effect at > or = 15 mM . In strains of S . pombe that were blocked by elevated temperature from entry into mitosis, drug treatment reversibly decreased microtubule-independent tip growth and septation, with an IC50 value around 12 mM; nuclear division, on the other hand, was essentially unaffected by up to 15 mM BDM . At 30 mM BDM the secretion of invertase, which required both F-actin and microtubules, was decreased to the same extent as that seen when cytochalasin D was used to disrupt F-actin . However, the actin cytoskeleton was insensitive to up to 10 mM BDM, while the actin patches lost their polar distribution at 20-30 mM BDM . Cells treated with 5-20 mM BDM for 3 hours and then high pressure frozen did not show an accumulation of secretory vesicles . However, 10 mM BDM treatment disorganized the fungal cell wall, resulting in some unusually thick parts lying next to regions were the wall was almost absent . These defects could be rescued by incubating the cells in inhibitors of glucanases . Osmolytic stabilization with sorbitol rescued the effect of 15 mM BDM on colony survival, indicating that the secretion of wall components and/or wall-modifying enzymes may be the principal reason for cell death caused by BDM . Our results are consistent with the hypothesis that BDM influences actin-dependent processes in fission yeast and that actomyosin-dependent motility contributes to the secretory process of tip growth.

Nucleic Acids Res, 1999 Feb 15, 27(4), 1047 - 55
Sequence divergence of the RNA polymerase shared subunit ABC14.5 (Rpb8) selectively affects RNA polymerase III assembly in Saccharomyces cerevisiae; Voutsina A et al.; ABC14.5 (Rpb8) is a eukaryotic subunit common to all three nuclear RNA polymerases . In Saccharomyces cerevisiae, ABC14.5 (Rpb8) is essential for cell viability, however its function remains unknown . We have cloned and characterised the Schizosaccharomyces pombe rpb8(+) cDNA . We found that S.pombe rpb8, unlike the similarly diverged human orthologue, cannot substitute for S.cerevisiae ABC14 . 5 in vivo . To obtain information on the function of this RNA polymerase shared subunit we have used S.pombe rpb8 as a naturally altered molecule in heterologous expression assays in S.cerevisiae . Amino acid residue differences within the 67 N-terminal residues contribute to the functional distinction of the two yeast orthologues in S.cerevisiae . Overexpression of the S.cerevisiae largest subunit of RNA polymerase III C160 (Rpc1) allows S.pombe rpb8 to functionally replace ABC14.5 in S.cerevisiae, suggesting a specific genetic interaction between the S.cerevisiae ABC14.5 (Rpb8) and C160 subunits . We provide further molecular and biochemical evidence showing that the heterologously expressed S.pombe rpb8 molecule selectively affects RNApolymerase III but not RNA polymerase I complex assembly . We also report the identification of a S.cerevisiae ABC14.5-G120D mutant which affects RNA polymerase III.

RNA, 1999 Jan, 5(1), 49 - 65
Molecular genetic analysis of U2AF59 in Schizosaccharomyces pombe: differential sensitivity of introns to mutational inactivation; Romfo CM et al.; The large subunit of the mammalian U2AF heterodimer (U2AF65) is essential for splicing in vitro . To expand our understanding of how this protein functions in vivo, we have created a null allele of the gene encoding the Schizosaccharomyces pombe ortholog, U2AF59, and employed it in a variety of genetic complementation assays . First, analysis of an extensive series of double amino acid substitutions indicates that this splicing factor is surprisingly refractory to mutations . Second, despite extensive structural conservation, we find that metazoan large subunit orthologs cannot substitute in vivo for fission yeast U2AF59 . Third, because the activity of U2AF65 in vitro involves binding to the 3' polypyrimidine tract, we examined the splicing of introns containing or lacking this feature in a U2AF59 mutant described here as well as a previously isolated temperature-sensitive mutant (Potashkin et al., 1993, Science 262:573-575) . Our data indicate that all four introns tested, including two that lack extensive runs of pyrimidines between the branchpoint and 3' splice site, show splicing defects upon shifting to the nonpermissive condition . In all cases, splicing is blocked prior to the first transesterification reaction in the mutants, consistent with the role inferred for human U2AF65 based on in vitro experiments.

Mol Gen Genet, 1998 Dec, 260(5), 434 - 43
Cloning of caf1+, caf2+ and caf4+ from Schizosaccharomyces pombe: their involvement in multidrug resistance, UV and pH sensitivity; Benko Z et al.; We previously identified four nuclear genes (caf1+ to caf4+) in Schizosaccharomyces pombe, mutations in which can confer caffeine resistance . Here we report the cloning and sequencing of caf1+, caf2+ and caf4+ . All three genes are allelic to genes (hba1+, crm1+ and trr1+, respectively) involved in multidrug resistance mechanisms or in stress response systems . In agreement with this the caffeine-resistant mutants caf1(hba1)-21, caf2(crm1)-3 and caf4(trr1)-83 are also resistant to brefeldin . Disruption of caf1(hba1)+ and caf4(trr1)+ makes cells sensitive to high pH . The overlapping ranges of pleiotropic effects and the genetic interaction detected between caf1(hba1)+ and caf2(crm1)+ suggest that the three genes function in interlinked systems.

Proc Natl Acad Sci U S A, 1999 Jan 19, 96(2), 517 - 22
Moe1, a conserved protein in Schizosaccharomyces pombe, interacts with a Ras effector, Scd1, to affect proper spindle formation; Chen CR et al.; In fission yeast, Scd1/Ral1 is a putative guanine nucleotide exchange factor for Cdc42sp and also acts as a Ras1 effector necessary for the regulation of cytoskeleton organization . In this study, we have characterized a protein, Moe1, that binds directly to Scd1 . A moe1 null (Delta) mutant exhibits numerous phenotypes indicative of abnormal microtubule functioning, including an abnormality in the spindle . moe1Delta mutants are resistant to microtubule destabilizing agents; moreover, moe1Delta rescued the growth defects of tubulin mutants containing unstable microtubules . These results suggest that Moe1 induces instability in microtubules . Biochemical and subcellular localization studies suggest that Moe1 and Scd1 colocalize in the nucleus . Furthermore, loss of function in Scd1 or Ras1 also induced abnormality in the spindle and is synthetically lethal with moe1Delta producing cells that lack a detectable spindle . These data demonstrate that Moe1 is a component of the Ras1 pathway necessary for proper spindle formation in the nucleus . Human and nematode Moe1 both can substitute for yeast Moe1, indicating that the function of Moe1 in spindle formation has been conserved substantially during evolution.

Mol Cell Biol, 1999 Feb, 19(2), 1251 - 61
Definition of transcriptional pause elements in fission yeast; Aranda A et al.; Downstream elements (DSEs) with transcriptional pausing activity play an important role in transcription termination of RNA polymerase II . We have defined two such DSEs in Schizosaccharomyces pombe, one for the ura4 gene and a new one in the 3'-end region of the nmt2 gene . Although these DSEs do not have sequence homology, both are orientation specific and are composed of multiple and redundant sequence elements that work together to achieve full pausing activity . Previous studies on the nmt1 and nmt2 genes revealed that transcription extends several kilobases past the genes' poly(A) sites . We show that the insertion of either DSE immediately downstream of the nmt1 poly(A) site induces more immediate termination . nmt2 termination efficiency can be increased by moving the DSE closer to the poly(A) site . These results suggest that DSEs may be a common feature in yeast genes.

Mol Cell Biol, 1999 Feb, 19(2), 1126 - 35
Mutator phenotype induced by aberrant replication; Liu VF et al.; We have identified thermosensitive mutants of five Schizosaccharomyces pombe replication proteins that have a mutator phenotype at their semipermissive temperatures . Allele-specific mutants of DNA polymerase delta (poldelta) and mutants of Polalpha, two Poldelta subunits, and ligase exhibited increased rates of deletion of sequences flanked by short direct repeats . Deletion of rad2(+), which encodes a nuclease involved in processing Okazaki fragments, caused an increased rate of duplication of sequences flanked by short direct repeats . The deletion mutation rates of all the thermosensitive replication mutators decreased in a rad2Delta background, suggesting that deletion formation requires Rad2 function . The duplication mutation rate of rad2Delta was also reduced in a thermosensitive polymerase background, but not in a ligase mutator background, which suggests that formation of duplication mutations requires normal DNA polymerization . Thus, although the deletion and duplication mutator phenotypes are distinct, their mutational mechanisms are interdependent . The deletion and duplication replication mutators all exhibited decreased viability in combination with deletion of a checkpoint Rad protein, Rad26 . Interestingly, deletion of Cds1, a protein kinase functioning in a checkpoint Rad-mediated reversible S-phase arrest pathway, decreased the viability and exacerbated the mutation rate only in the thermosensitive deletion replication mutators but had no effect on rad2Delta . These findings suggest that aberrant replication caused by allele-specific mutations of these replication proteins can accumulate potentially mutagenic DNA structures . The checkpoint Rad-mediated pathways monitor and signal the aberrant replication in both the deletion and duplication mutators, while Cds1 mediates recovery from aberrant replication and prevents formation of deletion mutations specifically in the thermosensitive deletion replication mutators.

Biochemistry, 1999 Jan 5, 38(1), 243 - 6
Excision of products of oxidative DNA base damage by human NTH1 protein; Dizdaroglu M et al.; A functional human homologue of Escherichia coli endonuclease III (Nth-Eco protein) has recently been cloned and characterized {Aspinwall, R., Rothwell, D . G., Roldan-Arjona, T., Anselmino, C., Ward, C . J., Cheadle, J . P., Sampson, J . R., Lindahl, T., Harris, P . C., and Hickson, I . D . (1997) Proc . Natl . Acad . Sci . U.S.A., 94, 109-114} . This enzyme, designated hNTH1 protein, shares an extensive sequence similarity with Nth-Eco protein and a related enzyme from Schizosaccharomyces pombe (Nth-Spo protein) . We investigated the substrate specificity of this human enzyme for oxidative DNA base damage, using the technique of gas chromatography/isotope-dilution mass spectrometry . Four different DNA substrates damaged by various free radical-generating systems were used . 5-Hydroxycytosine, thymine glycol, 5-hydroxy-6-hydrothymine, 5,6-dihydroxycytosine, and 5-hydroxyuracil were substrates of hNTH1 protein among 17 lesions found in DNA substrates . The substrate specificity and excision kinetics of the human enzyme were found to be significantly different from those of Nth-Spo and Nth-Eco proteins.

Microbiology, 1998 Dec, 144 ( Pt 12), 3475 - 85
Growth polarity transitions in a dimorphic fission yeast; Sipiczki M et al.; Fission yeast cells grow by extension at the ends (poles) and divide by transverse fission . It has previously been reported that Schizosaccharomyces japonicus var . japonicus can switch to unipolar, filamentous growth . Here it is shown that the yeast-to-mycelium transition is a gradual process involving a changeover to unipolar growth associated with asymmetric divisions, the development of large polarly located vacuoles, the modifications of the actin and microtubular cytoskeleton and the repression of cell separation after division . High concentrations of glucose in the medium or supplementation of the medium with caffeine or cAMP support the bipolar yeast phase, inhibit the transition to the mycelial phase and induce the conversion of hyphae to yeasts . These effects suggest that cAMP may be involved in the regulation of dimorphism . Temperatures below 18 degrees C or over 35 degrees C are restrictive for the mycelial phase and provoke a return to yeast phase.

Genes Genet Syst, 1998 Aug, 73(4), 181 - 91
Genetic control of fission yeast cell wall synthesis: the genes involved in wall biogenesis and their interactions in Schizosaccharomyces pombe; Ishiguro J; The fungal cell wall is an essential structure which protects cells from various environmental stresses such as hyper- or hypo-osmosis, and endows them with specific morphology in response to their life or cell division cycle . In addition, the cell wall has a variety of enzymatic activities per se, which are required for nutritional uptake, secretion, and cell adhesion including mating processes . In addition to these cytological interests, clinical demands to clarify the regulatory mechanisms of cell wall synthesis have been increasing, since the cell wall is a unique and effective target of antifungal agents . However, the molecular mechanisms are poorly understood at present, although the role of several signal transduction pathways have recently been implicated in regulation . In this review, the author focuses on genes and their interactions which are involved in fission yeast cell wall biogenesis.

Mol Biol Cell, 1999 Jan, 10(1), 91 - 104
The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling; Ma P et al.; The yeast Saccharomyces cerevisiae contains two genes, PDE1 and PDE2, which respectively encode a low-affinity and a high-affinity cAMP phosphodiesterase . The physiological function of the low-affinity enzyme Pde1 is unclear . We show that deletion of PDE1, but not PDE2, results in a much higher cAMP accumulation upon addition of glucose or upon intracellular acidification . Overexpression of PDE1, but not PDE2, abolished the agonist-induced cAMP increases . These results indicate a specific role for Pde1 in controlling glucose and intracellular acidification-induced cAMP signaling . Elimination of a putative protein kinase A (PKA) phosphorylation site by mutagenesis of serine252 into alanine resulted in a Pde1(ala252) allele that apparently had reduced activity in vivo . Its presence in a wild-type strain partially enhanced the agonist-induced cAMP increases compared with pde1Delta . The difference between the Pde1(ala252) allele and wild-type Pde1 was strongly dependent on PKA activity . In a RAS2(val19) pde2Delta background, the Pde1(ala252) allele caused nearly the same hyperaccumulation of cAMP as pde1Delta, while its expression in a PKA-attenuated strain caused the same reduction in cAMP hyperaccumulation as wild-type Pde1 . These results suggest that serine252 might be the first target site for feedback inhibition of cAMP accumulation by PKA . We show that Pde1 is rapidly phosphorylated in vivo upon addition of glucose to glycerol-grown cells, and this activation is absent in the Pde1(ala252) mutant . Pde1 belongs to a separate class of phosphodiesterases and is the first member shown to be phosphorylated . However, in vitro the Pde1(ala252) enzyme had the same catalytic activity as wild-type Pde1, both in crude extracts and after extensive purification . This indicates that the effects of the S252A mutation are not caused by simple inactivation of the enzyme . In vitro phosphorylation of Pde1 resulted in a modest and variable increase in activity, but only in crude extracts . This was absent in Pde1(ala252), and phosphate incorporation was strongly reduced . Apparently, phosphorylation of Pde1 does not change its intrinsic activity or affinity for cAMP but appears to be important in vivo for protein-protein interaction or for targeting Pde1 to a specific subcellular location . The PKA recognition site is conserved in the corresponding region of the Schizosaccharomyces pombe and Candida albicans Pde1 homologues, possibly indicating a similar control by phosphorylation.

Biochim Biophys Acta, 1999 Jan 6, 1426(2), 287 - 95
Reglucosylation of glycoproteins and quality control of glycoprotein folding in the endoplasmic reticulum of yeast cells; Parodi AJ; Proteins entering the secretory pathway may be glycosylated upon transfer of an oligosaccharide (Glc3Man9GlcNAc2) from a dolichol-P-P derivative to nascent polypeptide chains in the lumen of the endoplasmic reticulum (ER) . Oligosaccharides are then deglucosylated by glucosidases I and II (GII) . Also in the ER, glycoproteins acquire their final tertiary structures, and species that fail to fold properly are retained and eventually degraded in the proteasome . It has been proposed that in mammalian cells the monoglucosylated oligosaccharides generated either by partial deglucosylation of the transferred compound or by reglucosylation of glucose-free oligosaccharides by the UDP-Glc:glycoprotein glucosyltransferase (GT) are recognized by ER resident lectins (calnexin and/or calreticulin) . GT is a sensor of glycoprotein conformation as it only glucosylates misfolded species . The lectin-monoglucosylated oligosaccharide interaction would retain glycoproteins in the ER until correctly folded, and also facilitate their acquisition of proper tertiary structures by preventing aggregation . GII would liberate glycoproteins from the calnexin/calreticulin anchor, but species not properly folded would be reglucosylated by GT, and so continue to be retained by the lectins . Only when the protein becomes properly folded would it cease to be retained by the lectins . This review presents evidence suggesting that a similar quality control mechanism of glycoprotein folding is operative in Schizosaccharomyces pombe and that the mechanism in Saccharomyces cerevisiae probably differs substantially from that occurring in mammalian and Sch . pombe cells.

J Mol Biol, 1998 Dec 18, 284(5), 1341 - 51
Conserved core structure in the internal transcribed spacer 1 of the Schizosaccharomyces pombe precursor ribosomal RNA; Lalev AI et al.; The structure of the internal transcribed spacer 1 (ITS1) in Schizosaccharomyces pombe was examined with respect to phylogenetically conserved features in yeasts as well as the binding of transacting factors that potentially play a role in ribosomal maturation . Computer analyses and probes for nuclease protection indicate a compact, more highly organized structure than previously proposed in Saccharomyces cerevisiae, with distinct structural features which can be recognized in S . cerevisiae . These include a central extended hairpin structure as well as smaller hairpins immediately adjacent to the maturing termini . Comparisons with ITS sequences in more diverse organisms indicate that the same features also can be recognized . This is especially clear in organisms which contain very short sequences in which the putative structures are much less ambiguous . Again nuclease protection analyses in one of these, Verticillium albo-atrum, confirm a central hairpin with additional hairpins linked to the maturing termini . Protein binding and gel retardation studies with the S . pombe ITS1 further indicate that, as observed in the 3' external transcripted spacer (ETS) region, the extended hairpin is not only the site of intermediate RNA cleavage during rRNA processing, but also a site for specific interactions with one or more soluble factors . Taken together with other analyses on transcribed spacer regions, the present data provide evidence that the spacer regions act not only to organize the maturing terminal sequences but also may serve to organize specific soluble factors, possibly acting in a manner which is analogous with that of the free small nucleolar ribonucleo protein particles (snoRNPs) .

Genomics, 1998 Dec 15, 54(3), 424 - 36
cDNA cloning and gene mapping of human homologs for Schizosaccharomyces pombe rad17, rad1, and hus1 and cloning of homologs from mouse, Caenorhabditis elegans, and Drosophila melanogaster; Dean FB et al.; Mutations in DNA repair/cell cycle checkpoint genes can lead to the development of cancer . The cloning of human homologs of yeast DNA repair/cell cycle checkpoint genes should yield candidates for human tumor suppressor genes as well as identifying potential targets for cancer therapy . The Schizosaccharomyces pombe genes rad17, rad1, and hus1 have been identified as playing roles in DNA repair and cell cycle checkpoint control pathways . We have cloned the cDNA for the human homolog of S . pombe rad17, RAD17, which localizes to chromosomal location 5q13 by fluorescence in situ hybridization and radiation hybrid mapping; the cDNA for the human homolog of S . pombe rad1, RAD1, which maps to 5p14-p13.2; and the cDNA for the human homolog of S . pombe hus1, HUS1, which maps to 7p13-p12 . The human gene loci have previously been identified as regions containing tumor suppressor genes . In addition, we report the cloning of the cDNAs for genes related to S . pombe rad17, rad9, rad1, and hus1 from mouse, Caenorhabditis elegans, and Drosophila melanogaster . These include Rad17 and Rad9 from D . melanogaster, hpr-17 and hpr-1 from C . elegans, and RAD1 and HUS1 from mouse . The identification of homologs of the S . pombe rad checkpoint genes from mammals, arthropods, and nematodes indicates that this cell cycle checkpoint pathway is conserved throughout eukaryotes .

J Biol Chem, 1999 Jan 8, 274(2), 1147 - 55
Regulation of phosphatidylinositol 4-phosphate 5-kinase from Schizosaccharomyces pombe by casein kinase I; Vancurova I et al.; Phosphatidylinositol ()P 5-kinase (PtdIns(4)P 5-kinase) catalyzes the last step in the synthesis of phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) . PtdIns(4,5)P2 is a precursor of diacylglycerol and inositol 1,4,5-trisphosphate and is also involved in regulation of actin cytoskeleton remodeling and membrane traffic . To satisfy such varied demands in several aspects of cell physiology, synthesis of PtdIns(4,5)P2 must be stringently regulated . In this paper we describe extraction, purification, and characterization of PtdIns(4)P 5-kinase from the plasma membranes of Schizosaccharomyces pombe . We also provide evidence that PtdIns(4)P 5-kinase is phosphorylated and inactivated by Cki1, the S . pombe homolog of casein kinase I . Phosphorylation by Cki1 in vitro decreases the activity of PtdIns(4)P 5-kinase . In addition, and most importantly, overexpression of Cki1 in S . pombe results in a reduced synthesis of PtdIns(4,5)P2 and in a lower activity of PtdIns(4)P 5-kinase associated with the plasma membrane . These results suggest that PtdIns(4)P 5-kinase is a target of Cki1 in S . pombe and that Cki1 is involved in regulation of PtdIns(4, 5)P2 synthesis by phosphorylating and inactivating PtdIns(4)P 5-kinase.

J Biol Chem, 1999 Jan 8, 274(2), 781 - 6
An evolutionarily conserved family of Hsp70/Hsc70 molecular chaperone regulators; Takayama S et al.; Heat Shock Protein 70 kDa (Hsp70) family molecular chaperones play critical roles in protein folding and trafficking in all eukaryotic cells . The mechanisms by which Hsp70 family chaperones are regulated, however, are only partly understood . BAG-1 binds the ATPase domains of Hsp70 and Hsc70, modulating their chaperone activity and functioning as a competitive antagonist of the co-chaperone Hip . We describe the identification of a family of BAG-1-related proteins from humans (BAG-2, BAG-3, BAG-4, BAG-5), the invertebrate Caenorhabditis elegans (BAG-1, BAG-2), and the fission yeast Schizosaccharomyces pombe (BAG-1A, BAG-1B) . These proteins all contain a conserved approximately 45-amino acid region near their C termini (the BAG domain) that binds Hsc70/Hsp70, but they differ widely in their N-terminal domains . The human BAG-1, BAG-2, and BAG-3 proteins bind with high affinity (KD congruent with 1-10 nM) to the ATPase domain of Hsc70 and inhibit its chaperone activity in a Hip-repressible manner . The findings suggest opportunities for specification and diversification of Hsp70/Hsc70 chaperone functions through interactions with various BAG-family proteins.

J Biol Chem, 1999 Jan 8, 274(2), 567 - 70
Human homologs of Schizosaccharomyces pombe rad1, hus1, and rad9 form a DNA damage-responsive protein complex; Volkmer E et al.; DNA damage activates cell cycle checkpoints in yeast and human cells . In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe checkpoint-deficient mutants have been characterized, and the corresponding genes have been cloned . Searches for human homologs of S . pombe rad1, rad9, and hus1 genes identified the potential human homologs hRad1, hRad9, and hHus1; however, little is known about the roles of these proteins in human cells . The present studies demonstrate that hRad1 and hHus1 associate in a complex that interacts with a highly modified form of hRad9, but hHus1 and hRad1 do not associate with hRad17 . In addition to being a key participant in complex formation, hRad9 is phosphorylated in response to DNA damage . Together, these results suggest that hRad9, hRad1, and hHus1 are central components of a DNA damage-responsive protein complex in human cells.

FEBS Lett, 1998 Dec 4, 440(3), 430 - 3
Repression of enzymes of the pentose phosphate pathway by glucose in fission yeast; Mehta S et al.; We examine here the effect of carbon sources on the synthesis of the shunt pathway enzymes in the fission yeast Schizosaccharomyces pombe growing on a mixture of ethanol and glycerol . Delta-gluconolactone induces practically every one of these enzymes . Glucose in contrast tends to attenuate the synthesis of the majority of them . RNA analysis confirms that their induction and repression reflect changes in the levels of their transcripts.

Curr Genet, 1998 Dec, 34(5), 343 - 50
Schizosaccharomyces pombe exo1 is involved in the same mismatch repair pathway as msh2 and pms1; Rudolph C et al.; Besides the MutLS-like system, Schizosaccharomyces pombe has an additional pathway of mismatch repair . This minor pathway, producing short excision tracts, repairs C/C and, with lower efficiency, other mismatches also . We investigated the involvement of the exo1+, msh2+ and pms1+ genes in the two pathways . The exo1+ gene encodes a 5' to 3' exonuclease, while msh2+ and pms1+ are homologs of Escherichia coli mutS and mutL, respectively . Intragenic two-factor crosses showed that exo1+, msh2+ and pms1+ are involved in the major, but not in the C/C-correcting, pathway . Post-meiotic segregation frequencies and mitotic mutation rates in single and double mutants supported this finding . Furthermore, msh2 delta was epistatic over exo1 delta, and the ExoI enzyme is likely to be redundant with other exonucleases.

Mol Gen Genet, 1998 Nov, 260(4), 319 - 34
Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe; Osman F et al.; Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells . In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S . pombe that was absent in the budding yeast Saccharomyces cerevisiae . Studies with radiation-sensitive S . pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair . The alternative pathway was thought to be recombinational and to operate in the G2 phase of the cell cycle . However, in this study we show that cells held in G1 of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair . We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect . Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair . The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G2 following DNA damage or incomplete replication . The gene products may also have an additional role in a DNA repair or damage tolerance pathway . The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway . Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G2 DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA . In contrast, caffeine has no effect on the DNA replication S phase checkpoint in response to inhibition of DNA synthesis by hydroxyurea.

Genes Dev, 1998 Dec 15, 12(24), 3857 - 71
The RNA cleavage activity of RNA polymerase III is mediated by an essential TFIIS-like subunit and is important for transcription termination; Chedin S et al.; Budding yeast RNA polymerase III (Pol III) contains a small, essential subunit, named C11, that is conserved in humans and shows a strong homology to TFIIS . A mutant Pol III, heterocomplemented with Schizosaccharomyces pombe C11, was affected in transcription termination in vivo . A purified form of the enzyme (Pol III Delta), deprived of C11 subunit, initiated properly but ignored pause sites and was defective in termination . Remarkably, Pol III Delta lacked the intrinsic RNA cleavage activity of complete Pol III . In vitro reconstitution experiments demonstrated that Pol III RNA cleavage activity is mediated by C11 . Mutagenesis in C11 of two conserved residues, which are critical for the TFIIS-dependent cleavage activity of Pol II, is lethal . Immunoelectron microscopy data suggested that C11 is localized on the mobile thumb-like stalk of the polymerase . We propose that C11 allows the enzyme to switch between an RNA elongation and RNA cleavage mode and that the essential role of the Pol III RNA cleavage activity is to remove the kinetic barriers to the termination process . The integration of TFIIS function into a specific Pol III subunit may stem from the opposite requirements of Pol III and Pol II in terms of transcript length and termination efficiency.

Cell Growth Differ, 1998 Dec, 9(12), 961 - 7
The mammalian Rad24 homologous to yeast Saccharomyces cerevisiae Rad24 and Schizosaccharomyces pombe Rad17 is involved in DNA damage checkpoint; Bao S et al.; Cell cycle checkpoint proteins play critical roles in maintaining genomic stability and integrity to prevent the development of cancer and hereditary diseases . Here we report the isolation of a novel mouse gene encoding the protein MmRad24 {MmRad24 is the mouse homologue of HRad17, which was described recently by A . E . Parker et al . (J . Biol . Chem., 273: 18340-18346, 1998)}, which shares significant sequence and structural homology with the budding yeast Rad24 and its fission yeast counterpart Rad17, both of which are required for DNA damage checkpoints . Confocal microscopy revealed that the green fluorescent protein-tagged MmRad24 protein is localized to the nucleus in living cells . Fluorescence-activated cell-sorting analysis showed that overexpression of the wild-type MmRad24 in diploid fibroblast WI-38 cells caused a significant G2 arrest of the cell cycle, whereas overexpression of a mutant MmRad24 (mutated on the nucleotide-binding site) that likely functions as a dominant-negative protein resulted in a defect in cell cycle arrest after DNA damage treatment as measured by bromodeoxyuridine pulse-chase labeling experiments . Taken together, these results suggest that the mammalian Rad24 protein may function as a critical gatekeeper in DNA damage checkpoint control.

J Biol Chem, 1999 Jan 1, 274(1), 36 - 40
ATP synthase of yeast mitochondria . Isolation of subunit j and disruption of the ATP18 gene; Arnold I et al.; The subunit composition of the mitochondrial ATP synthase from Saccharomyces cerevisiae was analyzed using blue native gel electrophoresis and high resolution SDS-polyacrylamide gel electrophoresis . We report here the identification of a novel subunit of molecular mass of 6,687 Da, termed subunit j (Su j) . An open reading frame of 127 base pairs (ATP18), which encodes for Su j, was identified on chromosome XIII . Su j does not display sequence similarity to ATP synthase subunits from other organisms . Data base searches, however, identified a potential homolog from Schizosaccharomyces pombe with 51% identity to Su j of S . cerevisiae . Su j, a small protein of 59 amino acid residues, has the characteristics of an integral inner membrane protein with a single transmembrane segment . Deletion of the ATP18 gene encoding Su j led to a strain (Deltasu j) completely deficient in oligomycin-sensitive ATPase activity and unable to grow on nonfermentable carbon sources . The presence of Su j is required for the stable expression of subunits 6 and f of the F0 membrane sector . In the absence of Su j, spontaneously arising rho- cells were observed that lacked also ubiquinol-cytochrome c reductase and cytochrome c oxidase activities . We conclude that Su j is a novel and essential subunit of yeast ATP synthase.

Nucleic Acids Res, 1999 Jan 15, 27(2), 462 - 9
A key role for replication factor C in DNA replication checkpoint function in fission yeast; Reynolds N et al.; Replication factor C (RF-C) is a five subunit DNA polymerase (Pol) delta/straightepsilon accessory factor required at the replication fork for loading the essential processivity factor PCNA onto the 3'-ends of nascent DNA strands . Here we describe the genetic analysis of the rfc2 +gene of the fission yeast Schizosaccharomyces pombe encoding a structural homologue of the budding yeast Rfc2p and human hRFC37 proteins . Deletion of the rfc2 + gene from the chromosome is lethal but does not result in the checkpoint-dependent cell cycle arrest seen in cells deleted for the gene encoding PCNA or for those genes encoding subunits of either Pol delta or Pol straightepsilon . Instead, rfc2 Delta cells proceed into mitosis with incompletely replicated DNA, indicating that the DNA replication checkpoint is inactive under these conditions . Taken together with recent results, these observations suggest a simple model in which assembly of the RF-C complex onto the 3'-end of the nascent RNA-DNA primer is the last step required for the establishment of a checkpoint-competent state.

Biochemistry, 1998 Dec 8, 37(49), 17253 - 61
Calnexin and BiP interact with acid phosphatase independently of glucose trimming and reglucosylation in Schizosaccharomyces pombe; Jannatipour M et al.; The association of newly synthesized glycoproteins with the ER molecular chaperones calnexin and immunoglobulin binding protein (BiP) has been well documented in a variety of higher eukaryotes . Here we report that Cnx1p, the calnexin homologue in Schizosaccharomyces pombe, associates with newly synthesized molecules of the secreted glycoprotein acid phosphatase . Unlike ligand binding to mammalian calnexin, glucose trimming and reglucosylation of acid phosphatase by UDP-Glc:glycoprotein glucosyltransferase were shown to be dispensable for its binding to Cnx1p . Thus, despite the essentiality of Cnx1p for S . pombe viability, the glucose trimming and reglucosylation cycle does not appear to be required for protein folding in the fission yeast . The association of core-glycosylated acid phosphatase with Cnx1p after exposure of cells to heat shock or to DTT was shown to be reversible . However, Cnx1p stably associated with unglycosylated acid phosphatase after treatment with the core-glycosylation inhibitor tunicamycin . BiP was found to coprecipitate with Cnx1p, under normal and stress conditions, and following inhibition of protein synthesis by cycloheximide . We postulate that Cnx1p and BiP are part of a complex that is involved in the folding of both core-glycosylated trimmed ligands and unglycosylated proteins.

Mol Cell Biol, 1999 Jan, 19(1), 602 - 11
Genetic evidence for Pak1 autoinhibition and its release by Cdc42; Tu H et al.; Pak1 protein kinase of Schizosaccharomyces pombe, a member of the p21-GTPase-activated protein kinase (PAK) family, participates in signaling pathways including sexual differentiation and morphogenesis . The regulatory domain of PAK proteins is thought to inhibit the kinase catalytic domain, as truncation of this region renders kinases more active . Here we report the detection in the two-hybrid system of the interaction between Pak1 regulatory domain and the kinase catalytic domain . Pak1 catalytic domain binds to the same highly conserved region on the regulatory domain that binds Cdc42, a GTPase protein capable of activating Pak1 . Two-hybrid, mutant, and genetic analyses indicated that this intramolecular interaction rendered the kinase in a closed and inactive configuration . We show that Cdc42 can induce an open configuration of Pak1 . We propose that Cdc42 interaction disrupts the intramolecular interactions of Pak1, thereby releasing the kinase from autoinhibition.

Mol Cell Biol, 1999 Jan, 19(1), 241 - 50
The msh2 gene of Schizosaccharomyces pombe is involved in mismatch repair, mating-type switching, and meiotic chromosome organization; Rudolph C et al.; We have identified in the fission yeast Schizosaccharomyces pombe a MutS homolog that shows highest homology to the Msh2 subgroup . msh2 disruption gives rise to increased mitotic mutation rates and increased levels of postmeiotic segregation of genetic markers . In bandshift assays performed with msh2Delta cell extracts, a general mismatch-binding activity is absent . By complementation assays, we showed that S . pombe msh2 is allelic with the previously identified swi8 and mut3 genes, which are involved in mating-type switching . The swi8-137 mutant has a mutation in the msh2 gene which causes a truncated Msh2 peptide lacking a putative DNA-binding domain . Cytological analysis revealed that during meiotic prophase of msh2-defective cells, chromosomal structures were frequently formed; such structures are rarely found in the wild type . Our data show that besides having a function in mismatch repair, S . pombe msh2 is required for correct termination of copy synthesis during mating-type switching as well as for proper organization of chromosomes during meiosis.

Mol Cell Biol, 1999 Jan, 19(1), 99 - 106
AnCF, the CCAAT binding complex of Aspergillus nidulans, contains products of the hapB, hapC, and hapE genes and is required for activation by the pathway-specific regulatory gene amdR; Steidl S et al.; CCAAT binding factors (CBFs) positively regulating the expression of the amdS gene (encoding acetamidase) and two penicillin biosynthesis genes (ipnA and aatA) have been previously found in Aspergillus nidulans . The factors were called AnCF and PENR1, respectively . Deletion of the hapC gene, encoding a protein with significant similarity to Hap3p of Saccharomyces cerevisiae, eliminated both AnCF and PENR1 binding activities . We now report the isolation of the genes hapB and hapE, which encode proteins with central regions of high similarity to Hap2p and Hap5p of S . cerevisiae and to the CBF-B and CBF-C proteins of mammals . An additional fungus-specific domain present in HapE was revealed by comparisons with the homologs from S . cerevisiae, Neurospora crassa, and Schizosaccharomyces pombe . The HapB, HapC, and HapE proteins have been shown to be necessary and sufficient for the formation of a CCAAT binding complex in vitro . Strains with deletions of each of the hapB, hapC, and hapE genes have identical phenotypes of slow growth, poor conidiation, and reduced expression of amdS . Furthermore, induction of amdS by omega amino acids, which is mediated by the AmdR pathway-specific activator, is abolished in the hap deletion mutants, as is growth on gamma-aminobutyric acid as a sole nitrogen or carbon source . AmdR and AnCF bind to overlapping sites in the promoters of the amdS and gatA genes . It is known that AnCF can bind independently of AmdR . We suggest that AnCF binding is required for AmdR binding in vivo.

EMBO J, 1998 Dec 15, 17(24), 7320 - 36
A cdc15-like adaptor protein (CD2BP1) interacts with the CD2 cytoplasmic domain and regulates CD2-triggered adhesion; Li J et al.; A human CD2 cytoplasmic tail-binding protein, termed CD2BP1, was identified by an interaction trap cloning method . Expression of CD2BP1 is restricted to hematopoietic tissue, being prominent in T and natural killer (NK) cells, with long (CD2BP1L) and short (CD2BP1S) variants arising by alternative RNA splicing . Both CD2BP1 molecules are homologous to Schizosaccharomyces pombe cdc15, and include a helical domain, variable length intervening PEST sequence and C-terminal SH3 domain . Although the CD2BP1 SH3 domain binds directly to the CD2 sequence, KGPPLPRPRV (amino acids 300-309), its association is augmented markedly by the CD2BP1 N-terminal segment . Upon ligand-induced clustering of surface CD2 molecules, CD2BP1 redistributes from a cytosolic to a surface membrane compartment, co-localizing with CD2 . In turn, CD2-stimulated adhesion is downregulated by CD2BP1, apparently through coupling of the protein tyrosine phosphatase (PTP)-PEST to CD2 . These findings offer the first molecular view into the control processes for T cell adhesion.

EMBO J, 1998 Dec 15, 17(24), 7239 - 49
Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses; Martinho RG et al.; Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways . In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage . During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks . The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1) . We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase . We have also isolated and characterized a temperature-sensitive allele of rad3 . Rad3 functions differently depending on which checkpoint pathway is activated . Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response . When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response . We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro . Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses . Keywords: ATM/ATR/cell-cycle checkpoints/Chk1/Rad3

EMBO J, 1998 Dec 15, 17(24), 7230 - 8
Fission yeast Csk1 is a CAK-activating kinase (CAKAK); Hermand D et al.; Cell cycle progression is dependent on the sequential activity of cyclin-dependent kinases (CDKs) . For full activity, CDKs require an activating phosphorylation of a conserved residue (corresponding to Thr160 in human CDK2) carried out by the CDK-activating kinase (CAK) . Two distinct CAK kinases have been described: in budding yeast Saccharomyces cerevisiae, the Cak1/Civ1 kinase is responsible for CAK activity . In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7-cyclin H (Mcs6-Mcs2 in fission yeast) . Here we identify the fission yeast Csk1 kinase as an in vivo activating kinase of the Mcs6-Mcs2 CAK defining Csk1 as a CAK-activating kinase (CAKAK).

J Biol Chem, 1998 Dec 25, 273(52), 35063 - 73
Substrate specificity of the SpCCE1 holliday junction resolvase of Schizosaccharomyces pombe; Whitby MC et al.; SpCCE1 from Schizosaccharomyces pombe is an endonuclease that resolves Holliday junctions in vitro . SpCCE1 also binds and cleaves a range of other DNAs (Y-junction; flap; and flayed, nicked, and partial duplexes) with varying efficiency . Cleavage sites are always 3' of thymine nucleotides positioned at or close to the branch point or strand interruption . SpCCE1's favored substrate is the X-junction . Up to two dimers of SpCCE1 can bind concurrently to the same X-junction at its crossover point . From mixing experiments of SpCCE1 and the Escherichia coli RuvA protein, we show that each dimer of SpCCE1 binds to a different face of the X-junction and that both are seemingly competent for strand cleavage . We propose that this provides a mechanism whereby SpCCE1 can scrutinize all four junction strands simultaneously for cleavable thymine nucleotides . SpCCE1 appears to resolve X-junctions by a nick and counter-nick mechanism . Therefore, to ensure a high probability of bilateral strand cleavage, SpCCE1 has a relatively long lifetime on X-junctions . This mechanism has the drawback of limiting dissociation from noncleavable junctions . We discuss why this might not be a problem in vivo.

Biochem J, 1999 Jan 1, 337 ( Pt 1), 89 - 95
The role of the C-terminal region in phosphoglycerate mutase; Walter RA et al.; Removal of the C-terminal seven residues from phosphoglycerate mutase from Saccharomyces cerevisiae by limited proteolysis is associated with loss of mutase activity, but no change in phosphatase activity . The presence of the cofactor 2, 3-bisphosphoglycerate, or of the cofactor and substrate 3-phosphoglycerate together, confers protection against proteolysis . The substrate alone offers no protection . Replacement of either or both of the two lysines at the C-terminus by glycines has only limited effects on the kinetic properties of phosphoglycerate mutase, indicating that these residues are unlikely to be involved in crucial electrostatic interactions with the substrate, intermediate or product in the reaction . However, the double-mutant form of the enzyme is more sensitive to proteolysis and is no longer protected against proteolysis by the presence of cofactor . The proteolysed wild-type and two of the mutated forms of the enzyme show a reduced response to 2-phosphoglycollate, which enhances the instability of the phospho form of the native enzyme . The phosphoglycerate mutase from Schizosaccharomyces pombe, which lacks the analogous C-terminal tail, has an inherently lower mutase activity and is also less responsive to stimulation by 2-phosphoglycollate . It is proposed that the C-terminal region of phosphoglycerate mutase helps to maintain the enzyme in its active phosphorylated form and assists in the retention of the bisphosphoglycerate intermediate at the active site . However, its role seems not to be to contribute directly to ligand binding, but rather to exert indirect effects on the transfer of the phospho group between substrate, enzyme, intermediate and product.

Gene, 1998 Oct 9, 221(1), 59 - 68
Vectors for the expression of tagged proteins in Schizosaccharomyces pombe; Craven RA et al.; A series of vectors is described which enables the episomal expression of proteins fused to different tag sequences in Schizosaccharomyces pombe . Proteins can be expressed with their amino termini fused to GFP/EGFP, three copies of the HA or Pk epitopes or a combined tag which contains two copies of the myc epitope and six histidine residues (MH) . Fusion of the carboxyl terminus of a protein to a tag is possible with GFP/EGFP or Pk . Expression of the fusion proteins is controlled by the medium strength mutant version of the regulatable nmt1 promoter.

Gene, 1998 Oct 9, 221(1), 11 - 6
Identification of the gene and the protein of RNA polymerase II subunit 9 (Rpb9) from the fission yeast Schizosacharomyces pombe; Sakurai H et al.; Both the rpb9 gene and its cDNA encoding the subunit 9 of RNA polymerase II were cloned from the fission yeast Schizosaccharomyces pombe . From the DNA sequences, Rpb9 was predicted to consist of 113 amino acid residues with a molecular mass of 13,175 . S . pombe Rpb9 is 47, 40 and 36% identical in amino acid sequence to the corresponding subunits from Saccharomyces cerevisiae, human and Drosophila melanogaster, respectively . Previously, we failed to detect Rpb9 in the purified RNA polymerase II by amino-terminal micro-sequencing of proteolytic fragments of subunits separated by SDS-gel electrophoresis . After Western blot analysis using antibodies raised against the protein product of the newly isolated rpb9 gene, we found that the purified RNA polymerase II contains Rpb9.

J Cell Biol, 1998 Dec 14, 143(6), 1603 - 16
Role of polo kinase and Mid1p in determining the site of cell division in fission yeast; Bahler J et al.; The fission yeast Schizosaccharomyces pombe divides symmetrically using a medial F-actin- based contractile ring to produce equal-sized daughter cells . Mutants defective in two previously described genes, mid1 and pom1, frequently divide asymmetrically . Here we present the identification of three new temperature-sensitive mutants defective in localization of the division plane . All three mutants have mutations in the polo kinase gene, plo1, and show defects very similar to those of mid1 mutants in both the placement and organization of the medial ring . In both cases, ring formation is frequently initiated near the cell poles, indicating that Mid1p and Plo1p function in recruiting medial ring components to the cell center . It has been reported previously that during mitosis Mid1p becomes hyperphosphorylated and relocates from the nucleus to a medial ring . Here we show that Mid1p first forms a diffuse cortical band during spindle formation and then coalesces into a ring before anaphase . Plo1p is required for Mid1p to exit the nucleus and form a ring, and Pom1p is required for proper placement of the Mid1p ring . Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells . Genetic and two-hybrid analyses suggest that Plo1p and Mid1p act in a common pathway distinct from that involving Pom1p . Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation . Taken together, the data indicate that Plo1p plays a role in the positioning of division sites by regulating Mid1p . Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.

J Biol Chem, 1998 Dec 18, 273(51), 34603 - 10
Multiple forms of the U2 small nuclear ribonucleoprotein auxiliary factor U2AF subunits expressed in higher plants; Domon C et al.; Requirements for intron recognition during pre-mRNA splicing in plants differ from those in vertebrates and yeast . Plant introns contain neither conserved branch points nor distinct 3' splice site-proximal polypyrimidine tracts characteristic of the yeast and vertebrate introns, respectively . However, they are strongly enriched in U residues throughout the intron, property essential for splicing . To understand the roles of different sequence elements in splicing, we are characterizing proteins involved in intron recognition in plants . In this work we show that Nicotiana plumbaginifolia, a dicotyledonous plant, contains two genes encoding different homologs of the large 50-65-kDa subunit of the polypyrimidine tract binding factor U2AF, characterized previously in animals and Schizosaccharomyces pombe . Both plant U2AF65 isoforms, referred to as NpU2AF65a and NpU2AF65b, support splicing of an adenovirus pre-mRNA in HeLa cell nuclear extracts depleted of the endogenous U2AF factor . Both proteins interact with RNA fragments containing plant introns and show affinity for poly(U) and, to a lesser extend, poly(C) and poly(G) . The branch point or the 3' splice site regions do not contribute significantly to intron recognition by NpU2AF65 . The existence of multiple isoforms of U2AF may be quite general in plants because two genes expressing U2AF65 have been identified in Arabidopsis, and different isoforms of the U2AF small subunit are expressed in rice.

Mol Biochem Parasitol, 1998 Oct 30, 96(1-2), 139 - 50
Stage-specific activity of the Leishmania major CRK3 kinase and functional rescue of a Schizosaccharomyces pombe cdc2 mutant; Wang Y et al.; Cell cycle control by cdc2-related kinases (CRKs) is essential to the regulation of cell proliferation and developmental processes in many organisms . Alternating phases of growth, arrest, and differentiation are characteristics of the infectious cycle of many trypanosomatid parasites, raising the possibility that members of the trypanosomatid CRK gene family participate in the regulation of these essential processes . Here we describe properties of the CRK3 gene from Leishmania major, which encodes a 36 kDa protein kinase showing 60% amino acid sequence identity with human CDK2, including several conserved sites implicated in regulation of kinase activity . CRK3 mRNA was constitutively expressed throughout the parasite life cycle, but histone H1 kinase activity of an epitope tagged CRK3 protein was greater in log-phase than in stationary-phase promastigotes . When integrated into the genome and expressed at the optimal level, CRK3 was able to rescue the growth defect of a Schizosaccharomyces pombe cdc2 mutant (cdc2-33(ts)), indicating that CRK3 is a functional homolog of cdc2 . Mutants of CRK3 at several key regulatory residues showed the expected dominant negative effects on the S . pombe mutant . This is the first example of functional expression of a trypanosomatid CRK in yeast, opening the way for further genetic studies within this amenable organism.

Microbiology, 1998 Nov, 144 ( Pt 11), 2941 - 50
Candida albicans SSD1 can suppress multiple mutations in Saccharomyces cerevisiae; Chen CY et al.; The SSD1 gene of Saccharomyces encodes a 160 kDa cytoplasmic protein that can suppress mutations in a number of other genes . A functional homologue of SSD1 from the human pathogen Candida albicans was isolated on the basis of its ability to restore viability at the restrictive temperature in a Saccharomyces cerevisiae swi4 ssd1-d strain . The C . albicans gene, designated CaSSD1, encodes a 1262 aa protein which has 47% identity overall to S . cerevisiae SSD1 as well as significant identity to Schizosaccharomyces pombe dis3 and sts5 products . It is shown that CaSSD1 expression is constitutive through the mitotic cell cycle, which is consistent with a role for the protein in cell growth . CaSSD1 rescues the swi4ts defect in an ssd1-d background when expressed from its own promoter on a single-copy plasmid and under the same conditions can rescue mutations in genes encoding protein phosphatase type 2A catalytic subunits . These data suggest that CaSSD1, like its S . cerevisiae homologue, can limit the effect of mutations on a variety of cellular processes.

Proc Natl Acad Sci U S A, 1998 Dec 8, 95(25), 14781 - 6
Negative regulation of mitosis in fission yeast by the shk1 interacting protein skb1 and its human homolog, Skb1Hs; Gilbreth M et al.; We previously provided evidence that the protein encoded by the highly conserved skb1 gene is a putative regulator of Shk1, a p21(Cdc42/Rac)-activated kinase (PAK) homolog in the fission yeast Schizosaccharomyces pombe . skb1 null mutants are viable and competent for mating but less elongate than wild-type S . pombe cells, whereas cells that overexpress skb1 are hyperelongated . These phenotypes suggest a possible role for Skb1 as a mitotic inhibitor . Here we show genetic interactions of both skb1 and shk1 with genes encoding key mitotic regulators in S . pombe . Our results indicate that Skb1 negatively regulates mitosis by a mechanism that is independent of the Cdc2-activating phosphatase Cdc25 but that is at least partially dependent on Shk1 and the Cdc2 inhibitory kinase Wee1 . We provide biochemical evidence for association of Skb1 and Shk1 with Cdc2 in S . pombe, suggesting that Skb1 and Shk1 inhibit mitosis through interaction with the Cdc2 complex, rather than by an indirect mechanism . These results provide evidence of a previously undescribed role for PAK-related protein kinases as mitotic inhibitors . We also describe the cloning of a human homolog of skb1, SKB1Hs, and show that it can functionally replace skb1 in S . pombe . Thus, the molecular functions of Skb1-related proteins have likely been substantially conserved through evolution.

Mol Biol Cell, 1998 Dec, 9(12), 3399 - 415
The cdr2(+) gene encodes a regulator of G2/M progression and cytokinesis in Schizosaccharomyces pombe; Breeding CS et al.; Schizosaccharomyces pombe cells respond to nutrient deprivation by altering G2/M cell size control . The G2/M transition is controlled by activation of the cyclin-dependent kinase Cdc2p . Cdc2p activation is regulated both positively and negatively . cdr2(+) was identified in a screen for regulators of mitotic control during nutrient deprivation . We have cloned cdr2(+) and have found that it encodes a putative serine-threonine protein kinase that is related to Saccharomyces cerevisiae Gin4p and S . pombe Cdr1p/Nim1p . cdr2(+) is not essential for viability, but cells lacking cdr2(+) are elongated relative to wild-type cells, spending a longer period of time in G2 . Because of this property, upon nitrogen deprivation cdr2(+) mutants do not arrest in G1, but rather undergo another round of S phase and arrest in G2 from which they are able to enter a state of quiescence . Genetic evidence suggests that cdr2(+) acts as a mitotic inducer, functioning through wee1(+), and is also important for the completion of cytokinesis at 36 degrees C . Defects in cytokinesis are also generated by the overproduction of Cdr2p, but these defects are independent of wee1(+), suggesting that cdr2(+) encodes a second activity involved in cytokinesis.

Mol Biol Cell, 1998 Dec, 9(12), 3321 - 34
The protein kinase Cdr2, related to Nim1/Cdr1 mitotic inducer, regulates the onset of mitosis in fission yeast; Kanoh J et al.; Cdc2-Cyclin B, the protein kinase that catalyzes the onset of mitosis, is subject to multiple forms of regulation . In the fission yeast Schizosaccharomyces pombe and most other species, a key mode of Cdc2-Cyclin B regulation is the inhibitory phosphorylation of Cdc2 on tyrosine-15 . This phosphorylation is catalyzed by the protein kinases Wee1 and Mik1 and removed by the phosphatase Cdc25 . These proteins are also regulated, a notable example being the inhibition of Wee1 by the protein kinase Nim1/Cdr1 . The temperature-sensitive mutation cdc25-22 is synthetic lethal with nim1/cdr1 mutations, suggesting that a synthetic lethal genetic screen could be used to identify novel mitotic regulators . Here we describe that such a screen has identified cdr2(+), a gene that has an important role in the mitotic control . Cdr2 is a 775 amino acid protein kinase that is closely related to Nim1 and mitotic control proteins in budding yeast . Deletion of cdr2 causes a G2-M delay that is more severe than that caused by nim1/cdr1 mutations . Genetic studies are consistent with a model in which Cdr2 negatively regulates Wee1 . This model is supported by experiments showing that Cdr2 associates with the N-terminal regulatory domain of Wee1 in cell lysates and phosphorylates Wee1 in vitro . Thus, Cdr2 is a novel mitotic control protein that appears to regulate Wee1.

Eur J Biochem, 1998 Nov 1, 257(3), 630 - 7
Differences in in vivo acceptor specificity of two galactosyltransferases, the gmh3+ and gma12+ gene products from Schizosaccharomyces pombe; Yoko-o T et al.; In the fission yeast Schizosaccharomyces pombe, gmh1+, gmh2+ and gmh3+ genes encode alpha-1,2-galactosyltransferase homologues . In an in vitro galactosyltransferase assay, the gmh3+ gene product showed galactose transfer activity toward a-methyl-D-mannoside as an acceptor . The disruption of gma12+, the major galactosyltransferase gene {Chappell, T . G., Hajibagheri, M . A . N., Ayscough, K., Pierce, M . & Warren, G . (1994) Mol . Biol . Cell 5, 519-528}, and of gmh3+ in S . pombe caused decreases in the total remaining galactosyltransferase activity and cell surface galactose content . Disruption of gma12+ and gmh3+ also caused an increase in electrophoretic mobility of acid phosphatase, indicating their involvement in the galactosylation of cell surface glycoproteins . The gmh3delta gma12delta double mutant cells had more severe galactose-less phenotypes than single gene mutant cells . HPLC analysis of O-linked mannoprotein oligosaccharides from wild-type and disrupted strains revealed that the gma12+ gene product is responsible for the galactosylation of O-linked oligosaccharide, whereas gmh3+ has no involvement in the process . In contrast, both the gmh3+ and gma12+ gene products are involved in the galactosylation of the N-linked core oligosaccharide Man9GlcNAc2 . From these results, it is evident that there are some functional differences between the enzymes in the process of galactosylation . It appears that the gmh3+ gene product transfers galactose to N-linked oligosaccharide, while the gma12+ gene product transfers galactose to both N-linked and O-linked oligosaccharides.

Biochim Biophys Acta, 1998 Nov 26, 1443(1-2), 225 - 9
Analysis of the ntp1+ gene, encoding neutral trehalase in the fission yeast Schizosaccharomyces pombe; Soto T et al.; We have cloned and sequenced the ntp1+ gene that codes for neutral trehalase in the fission yeast Schizosaccharomyces pombe . The ntp1+ gene product (Ntp1p) showed a 45-55% identity with neutral trehalases from other yeasts at the amino acid sequence level . However, in clear contrast to other neutral yeast trehalases so far characterized (which show two cAMP phospho-sites), only one consensus site for cAMP-dependent protein phosphorylation was found in Ntp1p . Northern blot hybridization experiments demonstrated that the Wis-Phh1/Sty1 MAP kinase cascade regulates ntp1+ expression during osmostress.

Nucleic Acids Res, 1998 Dec 15, 26(24), 5662 - 9
The fission yeast prp10(+) gene involved in pre-mRNA splicing encodes a homologue of highly conserved splicing factor, SAP155; Habara Y et al.; In the fission yeast Schizosaccharomyces pombe, 14 prp (pre-mRNAprocessing) mutants have been isolated to date . We cloned the prp10(+) gene by complementation of the temperature-sensitive growth of prp10 . Five types of transcripts were found that were alternatively spliced with respect to two possible introns located in the 5'-terminal region . Three of them are probably functional and code for putative proteins of approximately 1200 amino acids . Proteins highly homologous to Prp10p are present in other organisms, one of which is a human spliceosome-associated protein SAP155, a subunit of the splicing factor complex SF3 . The C-terminal two-thirds of Prp10p is highly conserved among species, and contains consensus repeats for the regulatory subunit A of protein phosphatase PP2A . A gene disruption experiment indicated that the prp10(+) gene is essential for viability in S.pombe . Prp10p tagged with GFP is predominantly localized in the nuclear DNA region . A series of deletions showed that the less conserved N-terminal region of approximately 300 amino acids in Prp10p is dispensable, although the corresponding region was thought to play important roles in the mammalian splicing system.

Nucleic Acids Res, 1998 Dec 15, 26(24), 5609 - 16
Interaction of the resolving enzyme YDC2 with the four-way DNA junction; White MF et al.; Holliday junctions (four-way DNA junctions), formed during homologous recombination, are bound and resolved by junction-specific endonucleases to yield recombinant duplex DNA products . The junction-resolving enzymes are a structurally diverse class of proteins that nevertheless have many properties in common; in particular a high structure specificity for binding and metal-dependent, (frequently) sequence-specific cleavage activity . In Saccharomyces cerevisiae, the enzyme CCE1 is necessary for the resolution of recombining mitochondrial genomes, and in Schizosaccharomyces pombe the homologous protein YDC2 is thought to have a similar function . We have generated an inactive mutant of YDC2, D226N, that retains structure-specific junction binding and have analysed the interaction of this protein with the four-way DNA junction . YDC2 binds the four-way junction in two specific complexes (I and II), unfolding the stacked X-structure into a conformation where the arms extend to the four corners of a square . This structure is reminiscent of that of the free junction in the absence of metal ions and of the structures imposed on the Holliday junction by CCE1 and RuvA . DNase I probing reveals footprints specific for complexes I and II which extend from the junction centre on all four arms . No protection is observed with the small, hydrophobic probe DMS.

Science, 1998 Dec 4, 282(5395), 1893 - 7
Linkage of ATM to cell cycle regulation by the Chk2 protein kinase; Matsuoka S et al.; In response to DNA damage and replication blocks, cells prevent cell cycle progression through the control of critical cell cycle regulators . We identified Chk2, the mammalian homolog of the Saccharomyces cerevisiae Rad53 and Schizosaccharomyces pombe Cds1 protein kinases required for the DNA damage and replication checkpoints . Chk2 was rapidly phosphorylated and activated in response to replication blocks and DNA damage; the response to DNA damage occurred in an ataxia telangiectasia mutated (ATM)-dependent manner . In vitro, Chk2 phosphorylated Cdc25C on serine-216, a site known to be involved in negative regulation of Cdc25C . This is the same site phosphorylated by the protein kinase Chk1, which suggests that, in response to DNA damage and DNA replicational stress, Chk1 and Chk2 may phosphorylate Cdc25C to prevent entry into mitosis.

Plant Cell, 1998 Dec, 10(12), 2087 - 101
Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato; Schiebel W et al.; A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato . The putative protein encoded by this ORF does not share homology with any characterized proteins . Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein . The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and poly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns . DNA gel blot analysis revealed a single copy of the RdRP gene in tomato . RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction . A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans . The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA . The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed.

Genetics, 1998 Dec, 150(4), 1361 - 75
Suppressors of cdc25p overexpression identify two pathways that influence the G2/M checkpoint in fission yeast; Forbes KC et al.; Checkpoints maintain the order of cell-cycle events . At G2/M, a checkpoint blocks mitosis in response to damaged or unreplicated DNA . There are significant differences in the checkpoint responses to damaged DNA and unreplicated DNA, although many of the same genes are involved in both responses . To identify new genes that function specifically in the DNA replication checkpoint pathway, we searched for high-copy suppressors of overproducer of Cdc25p (OPcdc25(+)), which lacks a DNA replication checkpoint . Two classes of suppressors were isolated . One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled mitosis (sum3(+)) . This gene negatively regulates the cell-cycle response to stress when overexpressed and restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation . The second class includes chk1(+) and the two Schizosaccharomyces pombe 14-3-3 genes, rad24(+) and rad25(+), which appear to suppress the checkpoint defect by inhibiting Cdc25p . We show that rad24Delta mutants are defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37 degrees and that cds1Delta rad24Delta mutants, like cds1Delta chk1Delta mutants, are entirely checkpoint deficient at 29 degrees . These results suggest that chk1(+) and rad24(+) may function redundantly with cds1(+) in the checkpoint response to unreplicated DNA.

Genes Dev, 1998 Nov 15, 12(22), 3541 - 50
The molecular mechanism of mitotic inhibition of TFIIH is mediated by phosphorylation of CDK7; Akoulitchev S et al.; TFIIH is a multisubunit complex, containing ATPase, helicases, and kinase activities . Functionally, TFIIH has been implicated in transcription by RNA polymerase II (RNAPII) and in nucleotide excision repair . A member of the cyclin-dependent kinase family, CDK7, is the kinase subunit of TFIIH . Genetically, CDK7 homologues have been implicated in transcription in Saccharomyces cerevisiae, and in mitotic regulation in Schizosaccharomyces pombe . Here we show that in mitosis the CDK7 subunit of TFIIH and the largest subunit of RNAPII become hyperphosphorylated . MPF-induced phosphorylation of CDK7 results in inhibition of the TFIIH-associated kinase and transcription activities . Negative and positive regulation of TFIIH requires phosphorylation within the T-loop of CDK7 . Our data establishes TFIIH and its subunit CDK7 as a direct link between the regulation of transcription and the cell cycle.

Cell Motil Cytoskeleton, 1998, 41(3), 247 - 53
Isolation and characterization of alpha-tubulin genes from Septoria tritici and Rhynchosporium secalis, and comparative analysis of fungal alpha-tubulin sequences; Rohel EA et al.; The alpha-tubulin genes from Septoria tritici and Rhynchosporium secalis have been cloned and sequenced . The predicted amino acid sequence and intron structure showed strong homology with other known filamentous fungal alpha-tubulins . Comparison of sixteen fungal alpha-tubulin sequences based on amino acid sequence homology and intron structure identified five groups of proteins . Group 1 consists of filamentous fungi, including S . tritici and R . secalis, the dimorphic fungus Histoplasma capsulatum, and Pneumocystis carinii . Group 2 includes two divergent isoforms from Neurospora crassa and Aspergillus nidulans . Group 3 includes the yeast Saccharomyces cerevisiae and the dimorphic fungus Candida albicans . Group 4 contains the single yeast Schizosaccharomyces pombe . Group 5 includes the only Basidiomycete, Schizophyllum commune . This analysis supports the classification of P carinii as a primitive Ascomycete . The presence of an additional glycine residue between the second and third amino acid found only in Group 2 proteins may indicate a functionally distinct fungal isotype . Implications in terms of structure-function relationships for alpha-tubulin molecules are discussed.

J Bacteriol, 1998 Dec, 180(23), 6338 - 41
Methionine induces sexual development in the fission yeast Schizosaccharomyces pombe via an ste11-dependent signalling pathway; Schweingruber AM et al.; Methionine added to minimal medium overcomes the repressing effects of ammonium and cyclic AMP (cAMP) on sexual development and efficiently induces mating and sporulation in homothallic strains of Schizosaccharomyces pombe . In heterothallic strains it induces G1 arrest when cells enter stationary phase . We show that methionine reduces the intracellular cAMP pool and induces the expression of at least two cAMP-repressible genes, including fbp1 and ste11 . The easiest interpretation of the results is that methionine induces sexual development via a cAMP-dependent ste11 signalling pathway.

Genomics, 1998 Dec 1, 54(2), 344 - 7
RAD1, a human structural homolog of the Schizosaccharomyces pombe RAD1 cell cycle checkpoint gene; Marathi UK et al.; Cell cycle checkpoints are gating mechanisms that govern cell cycle progression in the presence of DNA damage and incomplete DNA replication . The Schizosaccharomyces pombe Rad1 protein is an essential component of cell cycle checkpoints activated by both types of genomic stress . In this study, we report the isolation of a human homolog of the S . pombe RAD1 gene . The hRAD1 protein is also similar to the Saccharomyces cerevisiae cell cycle checkpoint protein Rad17 and the Ustilago maydis 3' --> 5' exonuclease, Rec1 . We show that human RAD1 partially complements the hydroxyurea and ionizing radiation hypersensitivities of a S . pombe rad1 mutant, suggesting phylogenetic conservation of the DNA damage and replication checkpoints . The human RAD1 locus was mapped to human chromosome 5p13.2, a locus frequently altered in non-small-cell lung cancer and bladder cancer .

Genomics, 1998 Dec 1, 54(2), 331 - 7
A human and mouse homolog of the Schizosaccharomyces pombe rad1+ cell cycle checkpoint control gene; Bluyssen HA et al.; The Schizosaccharomyces pombe rad1+ cell cycle checkpoint control gene is required for S-phase and G2/M arrest in response to both DNA damage and incomplete DNA replication . We isolated and characterized the putative human RAD1 (hRAD1) and mouse RAD1 (mRAD1) homologs of the S . pombe Rad1 (Rad1) protein . The human RAD1 open reading frame (ORF) encodes a protein of 282 amino acids; the mRAD1 ORF codes for a protein of 280 amino acids . The human RAD1 and mRAD1 messengers are highly expressed in the testis as different mRNA species (varying from 1.0, 1.4, 1.5, to 3.0 kb) . The hRAD1 and mRAD1 proteins are 30% identical and 56% similar to the S . pombe Rad1 protein . Sequence homology was also noted with the Saccharomyces cerevisiae Rad17p, the putative 3'-5' exonuclease Rec1 from Ustilago maydis, and the structurally related polypeptides from Arabidopsis thaliana and Caenorhabditis elegans . The degree of conservation between the mammalian RAD1 proteins and those of the other species is consistent with the evolutionary distance between the species, implicating that these proteins are most likely true counterparts . Together, this suggests that the structure and function of the checkpoint "rad" genes in the G2/M checkpoint pathway are evolutionarily conserved between yeasts and higher eukaryotes . The human RAD1 gene could be localized on human chromosome 5p13, a region that has been implicated in the etiology of small cell lung carcinomas, squamous cell carcinomas, adenocarcinomas, and bladder cancer .

Nucleic Acids Res, 1998 Dec 1, 26(23), 5270 - 6
Substrate specificities of the ntg1 and ntg2 proteins of Saccharomyces cerevisiae for oxidized DNA bases are not identical; Senturker S et al.; Two genes of Saccharomyces cerevisiae, NTG1 and NTG2, encode proteins with a significant sequence homology to the endonuclease III of Escherichia coli . The Ntg1 and Ntg2 proteins were overexpressed in E.coli and purified to apparent homogeneity . The substrate specificity of Ntg1 and Ntg2 proteins for modified bases in oxidatively damaged DNA was investigated using gas chromatography/isotope-dilution mass spectrometry . The substrate used was calf-thymus DNA exposed to gamma-radiation in N2O-saturated aqueous solution . The results reveal excision by Ntg1 and Ntg2 proteins of six pyrimidine-derived lesions, 5-hydroxy-6-hydrothymine, 5-hydroxy-6-hydrouracil, 5-hydroxy-5-methylhydantoin, 5-hydroxyuracil, 5-hydroxycytosine and thymine glycol, and two purine-derived lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine from gamma-irradiated DNA . In contrast, Ntg1 and Ntg2 proteins do not release 8-hydroxyguanine or 8-hydroxyadenine from gamma-irradiated DNA . The Ntg1 and Ntg2 proteins also release 2, 6-diamino-4-hydroxy-5-N-methylformamido-pyrimidine from damaged poly(dG-dC).poly(dG-dC) . Excision was measured as a function of enzyme concentration and time . Furthermore, kinetic parameters were determined for each lesion . The results show that kinetic constants varied among the different lesions for the same enzyme . We also investigated the capacity of the Ntg1 and Ntg2 proteins to cleave 34mer DNA duplexes containing a single 8-OH-Gua residue mispaired with each of the four DNA bases . The results show that the Ntg1 protein preferentially cleaves a DNA duplex containing 8-OH-Gua mispaired with a guanine . Moreover, the Ntg1 protein releases free 8-OH-Gua from 8-OH-Gua/Gua duplex but not from duplexes containing 8-OH-Gua mispaired with adenine, thymine or cytosine . In contrast, the Ntg2 protein does not incise duplexes containing 8-OH-Gua mispaired with any of the four DNA bases . These results demonstrate that substrate specificities of the Ntg1 and Ntg2 proteins are similar but not identical and clearly different from that of the endonuclease III of E.coli and its homologues in Schizosaccharomyces pombe or human cells.

Nucleic Acids Res, 1998 Dec 1, 26(23), 5261 - 9
Schizosaccharomyces pombe rad32 protein: a phosphoprotein with an essential phosphoesterase motif required for repair of DNA double strand breaks; Wilson S et al.; The Schizosaccharomyces pombe Rad32 protein is required for repair of DNA double strand breaks, minichromosome stability and meiotic recombination . We show here that the Rad32 protein is phosphorylated in a cell cycle-dependent manner and during meiosis . The phosphorylation is not dependent on the checkpoint protein Rad3 . Analysis of a partially purified protein preparation indicates that Rad32 is likely to act in a complex . Characterisation of the rad32-1 mutation and site-directed mutagenesis indicate that three aspartate residues in the conserved phosphoesterase motifs are important for both mitotic and meiotic functions, namely response to UV and ionising radiation and spore viability.

EMBO J, 1998 Nov 16, 17(22), 6465 - 76
Localization of the 26S proteasome during mitosis and meiosis in fission yeast; Wilkinson CR et al.; The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins . We have investigated the localization of this complex in the fission yeast, Schizosaccharomyces pombe . Immunofluorescence microscopy shows a striking localization pattern whereby the proteasome is found predominantly at the nuclear periphery, both in interphase and throughout mitosis . Electron microscopic analysis revealed a concentration of label near the inner side of the nuclear envelope . The localization of green fluorescent protein (GFP)-tagged 26S proteasomes was analyzed in live cells during mitosis and meiosis . Throughout mitosis the proteasome remained predominantly at the nuclear periphery . During meiosis the proteasome was found to undergo dramatic changes in its localization . Throughout the first meiotic division, the signal is more dispersed over the nucleus . During meiosis II, there was a dramatic re-localization, and the signal became restricted to the area between the separating DNA until the end of meiosis when the signal dispersed before returning to the nuclear periphery during spore formation . These findings strongly imply that the nuclear periphery is a major site of protein degradation in fission yeast both in interphase and throughout mitosis . Furthermore they raise interesting questions as to the spatial organization of protein degradation during meiosis.

Folia Microbiol (Praha), 1998, 43(4), 369 - 72
Stability and refractoriness of the high catalase activity in the oxidative-stress-resistant fission yeast Schizosaccharomyces pombe; Sigler K et al.; Effect of oxygen and metabolic substrates (glucose, ethanol) on the catalase activity of anaerobically grown Schizosaccharomyces pombe cells was assessed and compared with that of Saccharomyces cerevisiae in order to determine the catalase activity regulation in S . pombe . In contrast to S . cerevisiae, the total catalase activity of permeabilized S . pombe anaerobically grown cells is higher than that found in aerobically grown cells, is stable and constant under all circumstances (i.e . it is not induced by oxygen and/or substrates), and only a negligible part (3-5%) of it is contributed by de novo protein synthesis during aeration with or without substrates . The patent catalase activity of intact cells rises 2-fold during 6-h aeration without substrate and 7-8-fold in the presence of glucose or ethanol . The increase is not inhibited by cycloheximide and is thus not due to de novo catalase synthesis, but may reflect enhanced transport of catalase to the cell surface or a permeabilization of the plasma membrane during the aeration.

Folia Microbiol (Praha), 1998, 43(4), 361 - 7
Inactivation of the plasma membrane ATPase of Schizosaccharomyces pombe by hydrogen peroxide and by the Fenton reagent (Fe2+/H2O2): nonradical vs . radical-induced oxidation; Sigler K et al.; In the absence of added Fe2+, the ATPase activity of isolated Schizosaccharomyces pombe plasma membranes (5-7 mumol P(i) per mg protein per min) is moderately inhibited by H2O2 in a concentration-dependent manner . Sizable inactivation occurs only at 50-80 mmol/L H2O2 . The process, probably a direct oxidative action of H2O2 on the enzyme, is not induced by the indigenous membrane-bound iron (19.3 nmol/mg membrane protein), is not affected by the radical scavengers mannitol and Tris, and involves a decrease of both the K(m) of the enzyme for ATP and the V of ATP splitting . On exposing the membranes to the Fenton reagent (50 mumol/L Fe2+ + 20 mmol/L H2O2), which causes a fast production of HO . radicals, the ATPase is 50-60% inactivated and 90% of added Fe2+ is oxidized to Fe3+ within 1 min . The inactivation occurs only when Fe2+ is added before H2O2 and can thus bind to the membranes . The lack of effect of radical scavengers (mannitol, Tris) indicates that HO . radicals produced in the bulk phase play no role in inactivation . Blockage of the inactivation by the iron chelator deferrioxamine implies that the process requires the presence of Fe2+ ions bound to binding sites on the enzyme molecules . Added catalase, which competes with Fe2+ for H2O2, slows down the inactivation but in some cases increases its total extent, probably due to the formation of the superoxide radical that gives rise to delayed HO . production.

Mol Cell Biol, 1998 Dec, 18(12), 7575 - 83
Regulation of the Mts1-Mts2-dependent ade6-M26 meiotic recombination hot spot and developmental decisions by the Spc1 mitogen-activated protein kinase of fission yeast; Kon N et al.; The M26 meiotic recombination hot spot in the ade6 gene of Schizosaccharomyces pombe is activated by the heterodimeric M26 binding protein Mts1-Mts2 . The individual Mts1 (Atf1, Gad7) and Mts2 (Pcr1) proteins are also transcription factors involved in developmental decisions . We report that the Mts proteins are key effectors of at least two distinct classes of developmental decisions regulated by the mitogen-activated protein (MAP) kinase cascade . The first class (osmoregulation, spore viability, and spore quiescence) requires the Spc1 MAP kinase and the Mts1 protein but does not require the Mts2 protein . The second class (mating, meiosis, and recombination hot spot activation) requires the Spc1 kinase and the Mts1-Mts2 heterodimer . Northern and Western blotting eliminated any significant role for the Spc1 kinase in regulating the expression levels of the Mts proteins . Gel mobility shift experiments indicated that the Mts1-Mts2 heterodimer does not need to be phosphorylated to bind to ade6-M26 DNA in vitro . However, in vivo dimethyl sulfate footprinting demonstrated that protein-DNA interaction within cells is dependent upon the Spc1 MAP kinase, which phosphorylates the Mts1 protein . Thus, the Spc1 kinase helps regulate the effector activities of the Mts1-Mts2 heterodimer in part by modulating its ability to occupy the M26 DNA site in vivo . Meiotic recombination hot spot function is likely the result of DNA conformational changes imparted by binding of the Mts1-Mts2 meiotic transcription factor.

Mol Cell Biol, 1998 Dec, 18(12), 7317 - 26
The mating-type proteins of fission yeast induce meiosis by directly activating mei3 transcription; Van Heeckeren WJ et al.; Cell type control of meiotic gene regulation in the budding yeast Saccharomyces cerevisiae is mediated by a cascade of transcriptional repressors, a1-alpha2 and Rme1 . Here, we investigate the analogous regulatory pathway in the fission yeast Schizosaccharomyces pombe by analyzing the promoter of mei3, the single gene whose expression is sufficient to trigger meiosis . The mei3 promoter does not appear to contain a negative regulatory element that represses transcription in haploid cells . Instead, correct regulation of mei3 transcription depends on a complex promoter that contains at least five positive elements upstream of the TATA sequence . These elements synergistically activate mei3 transcription, thereby constituting an on-off switch for the meiosis pathway . Element C is a large region containing multiple sequences that resemble binding sites for Mc, an HMG domain protein encoded by the mating-type locus . The function of element C is extremely sensitive to spacing changes but not to linker-scanning mutations, suggesting the possibility that Mc functions as an architectural transcription factor . Altered-specificity experiments indicate that element D interacts with Pm, a homeodomain protein encoded by the mating-type locus . This indicates that Pm functions as a direct activator of the meiosis pathway, whereas the homologous mating-type protein in S . cerevisiae (alpha2) functions as a repressor . Thus, despite the strong similarities between the mating-type loci of S . cerevisiae and S . pombe, the regulatory logic that governs the tight control of the key meiosis-inducing genes in these organisms is completely different.

Mol Cell Biol, 1998 Dec, 18(12), 7294 - 303
Multiple orientation-dependent, synergistically interacting, similar domains in the ribosomal DNA replication origin of the fission yeast, Schizosaccharomyces pombe; Kim SM et al.; Previous investigations have shown that the fission yeast, Schizosaccharomyces pombe, has DNA replication origins (500 to 1500 bp) that are larger than those in the budding yeast, Saccharomyces cerevisiae (100 to 150 bp) . Deletion and linker substitution analyses of two fission yeast origins revealed that they contain multiple important regions with AT-rich asymmetric (abundant A residues in one strand and T residues in the complementary strand) sequence motifs . In this work we present the characterization of a third fission yeast replication origin, ars3001, which is relatively small ( approximately 570 bp) and responsible for replication of ribosomal DNA . Like previously studied fission yeast origins, ars3001 contains multiple important regions . The three most important of these regions resemble each other in several ways: each region is essential for origin function and is at least partially orientation dependent, each region contains similar clusters of A+T-rich asymmetric sequences, and the regions can partially substitute for each other . These observations suggest that ars3001 function requires synergistic interactions between domains binding similar proteins . It is likely that this requirement extends to other fission yeast origins, explaining why such origins are larger than those of budding yeast.

J Cell Sci, 1998 Dec 18, 111 ( Pt 24), 3609 - 19
The novel murine calmodulin-binding protein Sha1 disrupts mitotic spindle and replication checkpoint functions in fission yeast; Craig R et al.; Entry into mitosis is normally blocked in eukaryotic cells that have not completed replicative DNA synthesis; this 'S-M' checkpoint control is fundamental to the maintenance of genomic integrity . Mutants of the fission yeast Schizosaccharomyces pombe defective in the S-M checkpoint fail to arrest the cell cycle when DNA replication is inhibited and hence attempt mitosis and cell division with unreplicated chromosomes, resulting in the 'cut' phenotype . In an attempt to identify conserved molecules involved in the S-M checkpoint we have screened a regulatable murine cDNA library in S . pombe and have identified cDNAs that induce the cut phenotype in cells arrested in S phase by hydroxyurea . One such cDNA encodes a novel protein with multiple calmodulin-binding motifs that, in addition to its effects on the S-M checkpoint, perturbed mitotic spindle functions, although spindle pole duplication was apparently normal . Both aspects of the phenotype induced by this cDNA product, which we term Sha1 (for spindle and hydroxyurea checkpoint abnormal), were suppressed by simultaneous overexpression of calmodulin . Sha1 is structurally related to the product of the Drosophila gene abnormal spindle (asp) . These data suggest that calmodulin-binding protein(s) are important in the co-ordination of mitotic spindle functions with mitotic entry in fission yeast, and probably also in multicellular eukaryotes.

Hum Mol Genet, 1998 Dec, 7(13), 2149 - 56
Characterization of a gene encoding survival motor neuron (SMN)-related protein, a constituent of the spliceosome complex; Talbot K et al.; Mutations in the gene encoding the Survival Motor Neuron (SMN) protein are responsible for autosomal recessive proximal spinal muscular atrophy (SMA) . SMN orthologues have been identified in the nematode worm Caenorhabditis elegans and the yeast Schizosaccharomyces pombe but, to date, no human paralogues have been described . Here we describe identification and characterization of an SMN-related protein (SMNrp) gene that encodes a novel protein of 239 amino acids, which has recently been identified as a constituent of the spliceosome complex and designated SPF30 . Significant similarity to the SMN protein is apparent only within a central region of SMNrp that represents a tudor domain . The SMNrp/SPF30 gene has been mapped to chromosome 10q23 . It is differentially expressed, with abundant levels in skeletal muscle . An exclusively nuclear localization for SMNrp in cultured cells and muscle sections was revealed using GFP fusion constructs and thereafter confirmed with a polyclonal antibody raised against SMNrp . Overexpression of SMNrp as a fusion protein in HeLa cells in culture induced dose-dependent apoptosis with positive TUNEL staining . In addition to a possible role for this protein as a pro-apoptotic factor, SMN and its related protein share significant similarities in sequence and cellular function.

J Cell Biol, 1998 Nov 16, 143(4), 887 - 99
Nuclear import and the evolution of a multifunctional RNA-binding protein; Rosenblum JS et al.; La (SS-B) is a highly expressed protein that is able to bind 3'-oligouridylate and other common RNA sequence/structural motifs . By virtue of these interactions, La is present in a myriad of nuclear and cytoplasmic ribonucleoprotein complexes in vivo where it may function as an RNA-folding protein or RNA chaperone . We have recently characterized the nuclear import pathway of the S . cerevisiae La, Lhp1p . The soluble transport factor, or karyopherin, that mediates the import of Lhp1p is Kap108p/Sxm1p . We have now determined a 113-amino acid domain of Lhp1p that is brought to the nucleus by Kap108p . Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La . Furthermore, when expressed in Saccharomyces cerevisiae, the nuclear localization of Schizosaccharomyces pombe, Drosophila, and human La proteins are independent of Kap108p . We have been able to reconstitute the nuclear import of human La into permeabilized HeLa cells using the recombinant human factors karyopherin alpha2, karyopherin beta1, Ran, and p10 . As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins . Our results are consistent with an intermingling of the nuclear import and evolution of La.

J Cell Biol, 1998 Nov 2, 143(3), 719 - 36
Role of the yeast Gin4p protein kinase in septin assembly and the relationship between septin assembly and septin function; Longtine MS et al.; To identify septin-interacting proteins in Saccharomyces cerevisiae, we screened for mutations that are synthetically lethal with a cdc12 septin mutation . One of the genes identified was GIN4, which encodes a protein kinase related to Hsl1p/Nik1p and Ycl024Wp in S . cerevisiae and to Nim1p/Cdr1p and Cdr2p in Schizosaccharomyces pombe . The Gin4p kinase domain displayed a two-hybrid interaction with the COOH-terminal portion of the Cdc3p septin, and Gin4p colocalized with the septins at the mother-bud neck . This localization depended on the septins and on the COOH-terminal (nonkinase) region of Gin4p, and overproduction of this COOH-terminal region led to a loss of septin organization and associated morphogenetic defects . We detected no effect of deleting YCL024W, either alone or in combination with deletion of GIN4 . Deletion of GIN4 was not lethal but led to a striking reorganization of the septins accompanied by morphogenetic abnormalities and a defect in cell separation; however, remarkably, cytokinesis appeared to occur efficiently . Two other proteins that localize to the neck in a septin-dependent manner showed similar reorganizations and also appeared to remain largely functional . The septin organization observed in gin4Delta vegetative cells resembles that seen normally in cells responding to mating pheromone, and no Gin4p was detected in association with the septins in such cells . The organization of the septins observed in gin4Delta cells and in cells responding to pheromone appears to support some aspects of the model for septin organization suggested previously by Field et al . (Field, C.M., O . Al-Awar, J . Rosenblatt, M.L . Wong, B . Alberts, and T.J . Mitchison . 1996 . J . Cell Biol . 133:605-616).

J Cell Biol, 1998 Nov 2, 143(3), 625 - 35
The UDP-Glc:Glycoprotein glucosyltransferase is essential for Schizosaccharomyces pombe viability under conditions of extreme endoplasmic reticulum stress; Fanchiotti S et al.; Interaction of monoglucosylated oligosaccharides with ER lectins (calnexin and/or calreticulin) facilitates glycoprotein folding but this interaction is not essential for cell viability under normal conditions . We obtained two distinct single Schizosaccharomyces pombe mutants deficient in either one of the two pathways leading to the formation of monoglucosylated oligosaccharides . The alg6 mutant does not glucosy- late lipid-linked oligosaccharides and transfers Man9GlcNAc2 to nascent polypeptide chains and the gpt1 mutant lacks UDP-Glc:glycoprotein glucosyltransferase (GT) . Both single mutants grew normally at 28 degreesC . On the other hand, gpt1/alg6 double-mutant cells grew very slowly and with a rounded morphology at 28 degreesC and did not grow at 37 degreesC . The wild-type phenotype was restored by transfection of the double mutant with a GT-encoding expression vector or by addition of 1 M sorbitol to the medium, indicating that the double mutant is affected in cell wall formation . It is suggested that facilitation of glycoprotein folding mediated by the interaction of monoglucosylated oligosaccharides with calnexin is essential for cell viability under conditions of extreme ER stress such as underglycosylation of proteins caused by the alg6 mutation and high temperature . In contrast, gls2/alg6 double-mutant cells that transfer Man9GlcNAc2 and that are unable to remove the glucose units added by GT as they lack glucosidase II (GII), grew at 37 degreesC and had, when grown at 28 degreesC, a phenotype of growth and morphology almost identical to that of wild-type cells . These results indicate that facilitation of glycoprotein folding mediated by the interaction of calnexin and monoglucosylated oligosaccharides does not necessarily require cycles of reglucosylation-deglucosylation catalyzed by GT and GII.

Biochimie, 1998 Jul, 80(7), 621 - 5
Functional analysis of domains in the Byr2 kinase; Bauman P et al.; The activation of mitogen-activated protein (MAP) kinase cascades by the Ras GTPase is an evolutionarily conserved signal transduction mechanism . To better understand the interaction between Ras and its target kinase, we study the yeast Schizosaccharomyces pombe where the Ras1 GTPase activates the Byr2 kinase . The Byr2 kinase contains an N-terminal regulatory region and a C-terminal kinase region . The regulatory region can be divided into a sterile-alpha motif (SAM) that binds Ste4, a Ras1-binding domain (RBD) that binds activated Ras1, and a catalytic binding domain (CBD) that interacts with the Byr2 kinase domain . To analyze the importance of functional domains of the Byr2 kinase, a biological assay was used that exploited the ability of Byr2 to partially bypass the need for Ras1 in sporulation . Analysis of mutants using this assay showed that SAM and RBD were very important for Ras1-stimulated sporulation . Three activating mutations were identified within the N-terminal lobe of the Byr2 kinase domain that partially bypassed the need for Ras1 for sporulation . These activating mutations may identify a region of the Byr2 kinase domain that interacts with the CBD since mutations in the CBD which disrupt binding to the kinase domain also increase Byr2 function.

Trends Biochem Sci, 1998 Oct, 23(10), 399 - 402
Checkpoints on the road to mitosis; Russell P; Eukaryotic organisms use cell-cycle checkpoints to ensure that nuclear division is restrained while DNA is undergoing replication or repair . Recent studies of the fission yeast Schizosaccharomyces pombe have illuminated these checkpoint mechanisms . These investigations have connected checkpoint proteins with central elements of the mitotic-control machinery.

FEMS Microbiol Lett, 1998 Oct 15, 167(2), 157 - 62
Mutation of Gly-444 inactivates the S . pombe malic enzyme; Viljoen M et al.; A mutant malic enzyme gene, mae2-, was cloned from a strain of Schizosaccharomyces pombe that displayed almost no malic enzyme activity . Sequence analysis revealed only one codon-altering mutation, a guanine to adenine at nucleotide 1331, changing the glycine residue at position 444 to an aspartate residue . Gly-444 is located in Region H, previously identified as one of eight highly conserved regions in malic enzymes . We found that Gly-444 is absolutely conserved in 27 malic enzymes from various prokaryotic and eukaryotic sources, as well as in three bacterial malolactic enzymes investigated . The evolutionary conservation of Gly-444 suggests that this residue is important for enzymatic function.

Biochim Biophys Acta, 1998 Nov 8, 1442(2-3), 361 - 8
Cloning and characterization of two genes encoding dihydroxyacetone kinase from Schizosaccharomyces pombe IFO 0354; Kimura T et al.; We report the cloning and characterization of two genes encoding dihydroxyacetone kinase (EC 2.7.1.29), SpDAK1 and SpDAK2, from Schizosaccharomyces pombe IFO 0354 . The open reading frames of both genes encode 591 amino acids and have Mrs of 62158 and 62170, respectively . Both predicted amino acid sequences exhibited a high identity to each other (99.8%) and relatively high identities (30% to 76%) to other putative dihydroxyacetone kinase gene products . A Western blot analysis showed that these enzymes are induced by glycerol and repressed by glucose . A genomic Southern blot analysis indicated the presence of SpDAK1 and the absence of SpDAK2 in a standard laboratory strain, S . pombe 972h-.

J Biol Chem, 1998 Nov 13, 273(46), 30398 - 405
The high mobility group domain protein Cmb1 of Schizosaccharomyces pombe binds to cytosines in base mismatches and opposite chemically altered guanines; Fleck O et al.; The mismatch-binding activity Cmb1 of Schizosaccharomyces pombe was enriched from wild type cells, and N-terminal sequencing enabled cloning of the respective gene . The deduced amino acid sequence of cmb1(+) contains a high mobility group domain, a motif that is common to a heterogeneous family of DNA-binding proteins . In crude protein extracts of a cmb1 gene-disruption strain, specific binding to C/T, C/A, and C/Delta was abolished . Weak binding to C/C revealed the presence of a second mismatch-binding activity, Cmb2 . Cmb1, enriched from S . pombe and purified from Escherichia coli, bound specifically to C/C, C/T, C/A, T/T, and C/Delta but showed little or no affinity to other mismatches and small loops . Cmb1 recognizes 1,2 GpG intrastrand cross-links, produced by the chemotherapeutic drug cisplatin, when two cytosines are opposite the cross-linked guanines but not when other bases are present . Consistently, O6-methylguanine:C but not O6-methylguanine/T lesions were bound . Thus, cytosines in mismatches and opposite chemically modified guanines are the preferred target of Cmb1 recognition . cmb1 mutant cells are more sensitive to cisplatin than wild type cells, indicating a role of Cmb1 in repair of cisplatin-induced DNA damage.

J Biol Chem, 1998 Nov 13, 273(46), 30301 - 5
A highly conserved glutamate residue (Glu-270) is essential for plant alternative oxidase activity; Albury MS et al.; We have previously demonstrated that expression of a Sauromatum guttatum alternative oxidase in Schizosaccharomyces pombe confers cyanide-resistant respiratory activity on these cells (Albury, M . S., Dudley, P., Watts, F . Z., and Moore, A . L . (1996) J . Biol . Chem . 271, 17062-17066) . Using this functional expression system we have investigated the active site of the plant alternative oxidase, which has been postulated to comprise a non-heme binuclear iron center . Mutation of a conserved glutamate (Glu-270), previously postulated to be a bridging ligand within the active site, to asparagine abolishes catalytic activity because mitochondria containing the E270N mutant protein do not exhibit antimycin A-resistant respiration . Western blot analysis, using antibodies specific for the alternative oxidase, revealed that the E270N mutant protein was targeted to and processed by S . pombe mitochondria in a manner similar to that of the wild-type protein . It is possible that lack of antimycin A-insensitive respiration observed in mitochondria containing the E270N mutant protein is due to incorrect insertion of the mutant alternative oxidase into the inner mitochondrial membrane . However, Western blot analysis of subfractionated mitochondria shows that both wild-type and E270N alternative oxidase are specifically located in the inner mitochondrial membrane, suggesting that misfolding or lack of insertion is unlikely . These results provide the first experimental evidence to support the structural model in which the active site of the alternative oxidase contains a coupled binuclear iron center.

Yeast, 1998 Oct, 14(14), 1307 - 10
Molecular cloning and sequence analysis of the Schizosaccharomyces pombe ade10+ gene; Liedtke C et al.; We have cloned and sequenced the Schizosaccharomyces pombe ade10 gene encoding 5-phosphoribosyl-4-carboxamide 5-aminoimidazole transformylase inosine monophosphate cyclohydrolase . The sequence has an uninterrupted open reading frame of 1755 nucleotides corresponding to 585 amino acid residues . The deduced amino acid sequence shows a high degree of similarity to the purH gene product of many species, including Saccharomyces cerevisiae, human, chicken and Escherichia coli . Moreover our data indicate that intrachromosomal recombination in Schiz . pombe is enhanced if the ade10 gene product is defective.

Yeast, 1998 Oct, 14(14), 1297 - 306
A new method for quantitative determination of polysaccharides in the yeast cell wall . Application to the cell wall defective mutants of Saccharomyces cerevisiae; Dallies N et al.; A reliable acid hydrolysis method for quantitative determination of the proportion of beta-glucan, mannan and chitin in Saccharomyces cerevisiae cell wall is reported together with a simple extraction procedure to quantify within a standard error of less than 2% the proportion of the wall per gram of cell dry mass . This method is an optimized version of Saeman's procedure based on sulfuric acid hydrolysis of complex polysaccharides . It resulted in an almost complete release of glucose, mannose and glucosamine residues from cell wall polysaccharides . After complete removal of sulfate ions by precipitation with barium hydroxide, the liberated monosaccharides were separated and quantified by high performance anion-exchange chromatography with pulsed amperometric detection . The superiority of this method over the hydrolysis in either trifluoroacetic or hydrochloric acid resides in its higher efficiency regarding the release of glucose from beta 1,6-glucan and of glucosamine from chitin . The sulfuric acid method was successfully applied to determine the beta-glucan, mannan and chitin contents in cell walls of genetically well-characterized yeast mutants defective in cell wall biosynthesis, and in Schizosaccharomyces pombe cell walls . The simplicity and reliability of this procedure make it the method of choice for the characterization of cell walls from S . cerevisiae mutants generated in the EUROFAN programme, as well as for other pharmacological and biotechnological applications.

Mol Biol Cell, 1998 Nov, 9(11), 3211 - 25
Dynamics of centromeres during metaphase-anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle; Nabeshima K et al.; In higher eukaryotic cells, the spindle forms along with chromosome condensation in mitotic prophase . In metaphase, chromosomes are aligned on the spindle with sister kinetochores facing toward the opposite poles . In anaphase A, sister chromatids separate from each other without spindle extension, whereas spindle elongation takes place during anaphase B . We have critically examined whether such mitotic stages also occur in a lower eukaryote, Schizosaccharomyces pombe . Using the green fluorescent protein tagging technique, early mitotic to late anaphase events were observed in living fission yeast cells . S . pombe has three phases in spindle dynamics, spindle formation (phase 1), constant spindle length (phase 2), and spindle extension (phase 3) . Sister centromere separation (anaphase A) rapidly occurred at the end of phase 2 . The centromere showed dynamic movements throughout phase 2 as it moved back and forth and was transiently split in two before its separation, suggesting that the centromere was positioned in a bioriented manner toward the poles at metaphase . Microtubule-associating Dis1 was required for the occurrence of constant spindle length and centromere movement in phase 2 . Normal transition from phase 2 to 3 needed DNA topoisomerase II and Cut1 but not Cut14 . The duration of each phase was highly dependent on temperature.

Nucleic Acids Res, 1998 Nov 15, 26(22), 5223 - 4
A novel expression cloning method to isolate mammalian transcription factors in Schizosaccharomyces pombe; Remacle JE et al.; We describe a novel expression cloning strategy in the fission yeast for the isolation of mammalian transcription factors using a mammalian promoter as target . This strategy is possible because of the conservation between mammalian cells and Schizosaccharomyces pombe of the mechanism that leads to the selection of the transcription start site . It also opens new perspectives to investigate the transcriptional regulation of genes for which detailed promoter analysis is difficult.

Nucleic Acids Res, 1998 Nov 15, 26(22), 5052 - 60
A Schizosaccharomyces pombe artificial chromosome large DNA cloning system; Young DJ et al.; The feasibility of using the fission yeast, Schizosaccharomyces pombe , as a host for the propagation of cloned large fragments of human DNA has been investigated . Two acentric vector arms were utilized; these carry autonomously replicating sequences ( ars elements), selectable markers ( ura4(+) or LEU2 ) and 250 bp of S . pombe terminal telomeric repeats . All cloning was performed between the unique sites in both vector arms for the restriction endonuclease Not I . Initially the system was tested by converting six previously characterized cosmids from human chromosome 11p13 into a form that could be propagated in S.pombe as linear episomal elements of 50-60 kb in length . In all transformants analysed these cosmids were maintained intact . To test if larger fragments of human DNA could also be propagated total human DNA was digested with Not I and size fractionated by pulsed field gel electrophoresis (PFGE) . Fractions of 100-1000 kb were ligated to Not I-digested vector arms and transformed into S.pombe protoplasts in the presence of lipofectin . Prototrophic ura+leu+transformants were obtained which upon examination by PFGE were found to contain additional linear chromosomes migrating at between 100 and 500 kb with a copy number of 5-10 copies/cell . Hybridization analyses revealed that these additional bands contained human DNA . Fluorescent in situ hybridization (FISH) analyses of several independent clones indicated that the inserts were derived from single loci within the human genome . These analyses clearly demonstrate that it is possible to clone large fragments of heterologous DNA in fission yeast using this S.p ombe artificial chromosome system which we have called SPARC . This vector-host system will complement the various other systems for cloning large DNA fragments.

Curr Genet, 1998 Oct, 34(4), 250 - 8
Cdm1, the smallest subunit of DNA polymerase d in the fission yeast Schizosaccharomyces pombe, is non-essential for growth and division; Reynolds N et al.; Highly purified DNA polymerase delta from the fission yeast Schizosaccharomyces pombe is a complex of at least four distinct subunits . Genes encoding three of these (pol3+/cdc6+, cdc1+ and cdc27+) have been characterised previously . Here we describe the isolation and characterisation of cdm1+, the gene encoding the smallest (22kDa) subunit of the Pol delta complex . Over-expression of cdm1+, which encodes a 160 amino-acid protein with no significant sequence similarity to proteins in current databases, is able to rescue cells carrying temperature-sensitive mutations in either pol3+/cdc6+, cdc1+ or cdc27+ . Cells deleted for cdm1+ are viable, indicating that cdm1+ is non-essential for mitotic growth, and are no more sensitive to a variety of DNA replication inhibitors and DNA damaging agents than are wild-type cells . In addition, over-expression of cdm1+ suppresses the temperature-sensitive cdc24-M38 mutant suggesting that cdc24+ may also have a role in DNA polymerase delta function.

Genetics, 1998 Nov, 150(3), 1007 - 18
Characterization of functional regions in the Schizosaccharomyces pombe mei3 developmental activator; Wang W et al.; The Schizosaccharomyces pombe mei3(+) gene is expressed only in diploid cells undergoing meiosis . Ectopic expression of mei3(+) in haploid cells causes meiotic catastrophe . Mei3 is an inhibitor of Ran1/Pat1 kinase and contains a nine-amino-acid motif, Mei3-RKDIII, that resembles two regions in the Ste11 substrate for Ran1/Pat1 . Substitution of serine for Arg-81 within Mei3-RKDIII transforms the inhibitor into a substrate for Ran1/Pat1 . Thus, it is likely that Mei3-RKDIII defines a pseudosubstrate sequence . In this study, we constructed a series of mei3 deletion mutations and assayed each for activity . This analysis indicates that the carboxy-terminal domain of Mei3 is sufficient for function in vivo . Alanine-scanning mutagenesis identifies critical residues within the inhibitory domain . Two mutations, SM1 and SM8, fail to cause meiotic catastrophe . The SM1 mutation contains alterations of amino acid residues in Mei3-RKDIII . Recombinant SM1 protein exhibits reduced ability to inhibit Ran1/Pat1 kinase in vitro and interacts inefficiently with the kinase in a two-hybrid assay . The SM8 protein binds to Ran1/Pat1 in a two-hybrid assay but fails to inhibit Ran1/Pat1 substrate phosphorylation in vitro . These findings provide evidence that Mei3-RKDIII defines a Ran1/Pat1-binding site that is necessary but not sufficient for inhibition of the kinase . Using fusions to green fluorescent protein, the cellular localization of Ran1 and Mei3 was examined in living cells . Ran1 is concentrated in the nucleus . Mei3 is also enriched in the nucleus and, consistent with the genetic and biochemical results, the inhibitory domain of Mei3 is sufficient for nuclear localization.

Biochem J, 1998 Nov 1, 335 ( Pt 3), 671 - 9
Inositol hexakisphosphate in Schizosaccharomyces pombe: synthesis from Ins(1,4,5)P3 and osmotic regulation; Ongusaha PP et al.; Schizosaccharomyces pombe extracts synthesize InsP6 (myo-inositol hexaphosphate) from Ins(1,4,5)P3 plus ATP . An S . pombe soluble fraction converts Ins(1,4,5)P3 into Ins(1,4,5,6)P4 and Ins(1,3,4, 5)P4, in a constant ratio of approximately 5:1, and thence to Ins(1, 3,4,5,6)P5 and InsP6 . We have purified a soluble Mg2+-dependent kinase of molecular mass approximately 41 kDa that makes Ins(1,4,5, 6)P4 and Ins(1,3,4,5)P4 in the same ratio and also converts Ins(1,4, 5,6)P4 or Ins(1,3,4,5)P4 into Ins(1,3,4,5,6)P5 and InsP6 . Of InsP3 isomers other than Ins(1,4,5)P3, only the non-biological molecule Ins(1,4,6)P3 potently 'competed' with all steps in conversion of Ins(1,4,5)P3 into InsP6 . Examination of molecular graphics representations allowed us to draw tentative conclusions about the environment needed for an hydroxyl group to be phosphorylated by this kinase and to predict successfully that the purified kinase would phosphorylate the 5-hydroxyl of Ins(1,4,6)P3 . S . pombe that have been cultured with {3H}inositol contains a variety of 3H-labelled inositol polyphosphates, with Ins(1,4,5)P3 and InsP6 the most prominent, and the InsP6 concentration quickly increases in hyper-osmotically stressed S . pombe . This yeast therefore contains InsP6 and Ins(1,4,5)P3 as normal constituents, makes more InsP6 when hyper-osmotically stressed and contains a versatile inositol polyphosphate kinase that synthesizes InsP6 from Ins(1,4,5)P3.

Biochem J, 1998 Nov 1, 335 ( Pt 3), 581 - 8
Mutant DNA polymerase delta from thermosensitive Schizosaccharomyces pombe strains display reduced stimulation by proliferating cell nuclear antigen; Perderiset M et al.; We have isolated and characterized DNA polymerase delta (pol delta) from two thermosensitive Schizosaccharomyces pombe strains, poldeltats1 and poldeltats3, mutated in two different evolutionarily conserved domains of the catalytic subunit . At the restrictive temperature of 37 degreesC poldeltats1 and poldeltats3 mutant strains arrest growth in the S phase of the cell cycle . We show that at low levels of primer ends, in vitro stimulation by proliferating cell nuclear antigen (PCNA) of mutant enzymes is lower than stimulation of wild-type pol delta . Affinity for primer (3'-OH) ends and processivity of mutant enzymes do not appear different from wild-type pol delta . In contrast, Vmax values are lower than the wild-type value . The major in vitro defect appears to be decreased stimulation of mutant enzymes by PCNA, resulting in reduced velocity of DNA synthesis . In addition, ts1 pol delta is not stimulated by low PCNA concentration at 37 degreesC, although low concentrations stimulate activity at 25 degreesC, suggesting that this thermolability at low levels of primer ends could be its critical defect in vivo . Thus, both ts1 and ts3 pol delta mutations are located in regions of the catalytic subunit that seem necessary, directly or indirectly, for its efficient interaction with PCNA.

Yeast, 1998 Sep 30, 14(13), 1167 - 74
Characterization of a second gene (ZSOD22) of Na+/H+ antiporter from salt-tolerant yeast Zygosaccharomyces rouxii and functional expression of ZSOD2 and ZSOD22 in Saccharomyces cerevisiae; Iwaki T et al.; We reported in our previous paper on the characterization of the Na+/H(+)-antiporter gene (ZSOD2) closely related to the salt-tolerance of yeast Zygosaccharomyces rouxii . In the present paper, we have cloned a second gene (ZSOD22) of Na+/H+ antiporter from Z . rouxii . The deduced amino acid sequence of Zsod22p was highly homologous to that of Zsod2p, Sod2p from Schizosaccharomyces pombe, and Nhalp from Saccharomyces cerevisiae . The open reading frames (ORFs) from ZSOD2 or ZSOD22 were inserted into a yeast expression vector pYES2, and their constructs (pZSOD2 and pZSOD22) were used to transform the salt-sensitive S . cerevisiae . pZSOD2- or pZSOD22-harboring-recombinant S . cerevisiae cells showed increases in salt tolerance . However, the Z . rouxii disruptant of ZSOD22 did not show any phenotypes related to salt tolerance or osmotolerance, unlike that of ZSOD2 . The transcriptional expression of ZSOD22 was not observed by Northern blot analysis even in Z . rouxii cells subjected to NaCl-shock . From these results we conclude that although Z . rouxii includes at least two copies of the Na+/H(+)-antiporter gene (ZSOD2 and ZSOD22), ZSOD2 encodes a functional product as an antiporter and ZSOD22 is poorly transcribed, if at all.

Biochem Biophys Res Commun, 1998 Oct 29, 251(3), 720 - 6
Identification of a fission yeast dynamin-related protein involved in mitochondrial DNA maintenance; Pelloquin L et al.; Members of the dynamin-related proteins family have been identified in a wide range of organisms, however their precise functions remain elusive . We have identified a new member of that GTPase family in the fission yeast Schizosaccharomyces pombe . We show that Msp1+ is an essential nuclear gene encoding a 101 kDa protein whose closest homologue is the S . cerevisiae MGM1 gene product . We also report that msp1 conditional loss of function affects the maintenance of mitochondrial DNA and leads to growth arrest associated with respiratory deficiency .

Biochem Biophys Res Commun, 1998 Oct 29, 251(3), 714 - 9
A mutation Ser213/Asn in the hexokinase 1 from Schizosaccharomyces pombe increases its affinity for glucose; Petit T et al.; Alignment of amino acids of the region implicated in glucose binding from a series of hexokinases showed that Schizosaccharomyces pombe hexokinase 1 had a Ser residue in a place where all other kinases had an Asn . We changed an AGT codon to AAT to place an Asn in the Ser213 position . This mutation decreased Km for glucose from 9.4 mM to 1.6 mM and the ratio Vmax (Fructose)/Vmax (Glucose) from 5 to 2.5 . Also the Km for 2-deoxyglucose decreased from 2.7 mM to 0.8 mM . A mutation in the similar position of S . pombe hexokinase 2 (Asn196/Ser) increased the Km for glucose from 0.16 mM to 0.56 mM . Fermentation of glucose is not detectable in a S . pombe mutant with only hexokinase 1 activity but expression of the hxk1(S213/N) gene conferred ability to ferment the sugar . While the mutated hexokinase 1 partially mimicked S . cerevisiae hexokinase II in catabolite repression of invertase, the wild type one could not substitute for it .

Protein Expr Purif, 1998 Nov, 14(2), 247 - 53
Purification and characterization of pyruvate kinase from Schizosaccharomyces pombe: evidence for an unusual quaternary structure; Nairn J et al.; Earlier attempts to purify and characterize nonrecombinant pyruvate kinase from Schizosaccharomyces pombe proved difficult due to problems associated with the instability of the protein . The enzyme has been overexpressed in Saccharomyces cerevisiae strain AH22, permitting studies to determine the conditions required to stabilize the enzyme during purification . Recombinant S . pombe pyruvate kinase was purified by a combination of ion-exchange chromatography and gel filtration . The purified enzyme showed sigmoidal kinetics with respect to PEP; in the presence of FBP, the kinetics were restored to Michaelis-Menten behavior . With respect to ADP, the Hill coefficient was not affected by FBP . Determination of the molecular mass of the purified enzyme by ultracentrifugation showed that it behaved as a dimer-tetramer system with a Kd of approximately 1 microM .

Mol Gen Genet, 1998 Sep, 259(4), 437 - 48
A phosphorylation site mutant of Schizosaccharomyces pombe cdc2p fails to promote the metaphase to anaphase transition; Gould KL et al.; The protein kinase cdc2p is a key regulator of the G1-S and G2-M cell cycle transitions in the yeast Schizosaccharomyces pombe . Activation of cdc2p is regulated by its phosphorylation state and by interaction with other proteins . We have analyzed the consequences for cell cycle progression of altering the conserved threonine phosphorylation site, within the activation loop of cdc2p, to glutamic acid . This mutant, T167 E, promotes entry into mitosis, as judged by the accumulation of mitotic spindles and condensed chromosomes, despite the fact that it lacks demonstrable kinase activity both in vitro and in vivo . However, T167 E cannot promote the metaphase-anaphase transition . Since a component of the anaphase-promoting complex (APC) in S . pombe, cut9p, remains hypophosphorylated at the T167 E arrest point, the cell cycle block might be due to the inability of T167 E to activate the APC . T167 E is lethal when overexpressed, and overproduction also causes a mitotic arrest . Multicopy suppressors of the dominant negative phenotype were isolated, and identified as cdc13+ and suc1+ . Overexpression of suc1+ suppresses the effects of T167 E overproduction by restoring sufficient amounts of suc1p to the cell to allow passage through mitosis.

Mol Gen Genet, 1998 Sep, 259(5), 549 - 56
Characterization of the lys2 gene of Penicillium chrysogenum encoding alpha-aminoadipic acid reductase; Casqueiro J et al.; A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78 . It encodes a protein of 1409 amino acids (Mr 154859) with strong similarity to the S . cerevisiae (49.9% identity) Schizosaccharomyces pombe (51.3% identity) and Candida albicans (48.12% identity) alpha-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the alpha-aminoadipate-activating domain of the alpha-aminoadipyl-cysteinyl-valine synthetase of P . chrysogenum (12.4% identical amino acids) . The lys2 gene contained one intron in the 5'-region and other in the 3'-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA . The lys2 gene was localized on chromosome III (7.5 Mb) in P . chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains . The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region.

Mol Gen Genet, 1998 Sep, 259(5), 449 - 56
Growth arrest of Schizosaccharomyces pombe following overexpression of mouse type 1 protein phosphatases; Sangrador A et al.; Three mouse genes encoding type 1 protein phosphatase isotypes alpha, gamma and delta were expressed in Schizosaccharomyces pombe cells under the control of the regulatable promoter nmt1 . In the repressed state, basal expression of mouse genes was able to rescue the S . pombe mutant dis2-11 . An integrated mouse gene could not, however, genetically complement S . pombe double mutants carrying disrupted dis2 and sds21 protein phosphatase genes . Overexpression of any of the three mouse protein phosphatase1 isotypes produced growth arrest and loss of viability in wild-type S . pombe cells . Overexpressing cells showed defects in chromosome distribution and in the formation of septa . These defects are characteristic of the expression of the mammalian protein phosphatases in S . pombe, since they are not observed after overexpression of endogenous S . pombe type 1 protein phosphatases . Overexpression of a truncated version of isotype delta, which lacked protein phosphatase activity, reproduced most of the characteristics of the lethal phenotype . We conclude that the lethal effect may be due to interactions with essential cell cycle proteins.

Cell Motil Cytoskeleton, 1998, 41(2), 117 - 25
The myosin ATPase inhibitor 2,3-butanedione-2-monoxime (BDM) inhibits tip growth and cytokinesis in the fission yeast, Schizosaccharomyces pombe; May KM et al.; The growth of fission yeast cultures was reversibly inhibited by exposure to the myosin-ATPase inhibitor 2,3-butanedione-2-monoxime (BDM) . Wild-type cells treated with 20 mM BDM for approximately two generation times were smaller than untreated controls and had a septation index approximately twice that seen in the absence of the inhibitor . The organization of actin at the cell poles was somewhat disorganized in the presence of BDM; however, cells formed a cytokinetic actin ring . When nitrogen-starved stationary-phase cells were reinoculated into fresh medium in the presence of BDM, the time taken to repolarize the actin cytoskeleton and to resume the characteristic vegetative cell shape before initiation of the first cell division were both substantially delayed . BDM significantly inhibited the increase in cell length of cdc25.22 cells arrested for cell cycle progress by incubation at the restrictive temperature and substantially delayed the initiation of both mitosis and cytokinesis in arrested cdc25.22 cells after release of the temperature block . These results suggest that tip growth and cytokinesis--processes in fission yeast that involve the actin cytoskeleton--also require myosin activity.

J Cell Biol, 1998 Oct 19, 143(2), 415 - 27
imp2, a new component of the actin ring in the fission yeast Schizosaccharomyces pombe; Demeter J et al.; Cytokinesis is the part of the cell cycle in which the cell is cleaved to form two daughter cells . The unicellular yeast, Schizosaccharomyces pombe is an excellent model organism in which to study cell division, since it shows the general features of eukaryotic cell division and is amenable to genetic analysis . In this manuscript we describe the isolation and characterization of a new protein, imp2, which is required for normal septation in fission yeast . imp2, which colocalizes with the medial ring during septation, is structurally similar to a group of proteins including the S . pombe cdc15 and the mouse PSTPIP that are localized to, and thought to be involved in actin ring organization . Cells in which the imp2 gene is deleted or overexpressed have septation and cell separation defects . An analysis of the actin cytoskeleton shows the lack of a medial ring in septating cells that overexpress imp2, and the appearance of abnormal medial ring structures in septated cells that lack imp2 . These observations suggest that imp2 destabilizes the medial ring during septation . imp2 also shows genetic interactions with several, previously characterized septation genes, strengthening the conclusion that it plays a role in normal fission yeast septation.

J Biol Chem, 1998 Oct 30, 273(44), 28912 - 20
Mak21p of Saccharomyces cerevisiae, a homolog of human CAATT-binding protein, is essential for 60 S ribosomal subunit biogenesis; Edskes HK et al.; Mak21-1 mutants are unable to propagate M1 double-stranded RNA, a satellite of the L-A double-stranded RNA virus, encoding a secreted protein toxin lethal to yeast strains that do not carry M1 . We cloned MAK21 using its map location and found that Mak21p is homologous to a human and mouse CAATT-binding protein and open reading frames in Schizosaccharomyces pombe and Caenorhabditis elegans . Although the human protein regulates Hsp70 production, Mak21p is essential for growth and necessary for 60 S ribosomal subunit biogenesis . mak21-1 mutants have decreased levels of L-A coat protein and L-A double-stranded RNA . Electroporation with reporter mRNAs shows that mak21-1 cells cannot optimally express mRNAs which, like L-A viral mRNA, lack 3'-poly(A) or 5'-cap structures but can normally express mRNA with both cap and poly(A) . The virus propagation phenotype of mak21-1 is suppressed by ski2 or ski6 mutations, each of which derepresses translation of non-poly(A) mRNA.

Biochem Pharmacol, 1998 Sep 1, 56(5), 577 - 82
Pharmacological characterisation of the D2 dopamine receptor expressed in the yeast Schizosaccharomyces pombe; Presland J et al.; The rat D2(long) dopamine receptor has been expressed in the fission yeast Schizosaccharomyces pombe at levels of about 1 pmol/mg of protein . The recombinant receptor, analysed in ligand binding experiments, exhibits properties typical of a D2 dopamine receptor and the affinities of antagonists agree with values obtained for the receptor expressed in mammalian systems although the affinities of some antagonists are lower . Substituted benzamide antagonists show lower affinities in the absence of sodium ions whereas clozapine and classical antagonists mostly show higher affinities . Agonist binding is insensitive to the effects of GTP indicating lack of a stable interaction with G-proteins.

Mol Microbiol, 1998 Sep, 29(6), 1357 - 67
Characterization of the geranylgeranyl transferase type I from Schizosaccharomyces pombe; Arellano M et al.; The Schizosaccharomyces pombe cwg2+ gene encodes the beta-subunit of geranylgeranyl transferase I (GGTase I), which participates in the post-translational C-terminal modification of several small GTPases, allowing their targeting to the membrane . Using the two-hybrid system, we have identified the cwp1+ gene that encodes the alpha-subunit of the GGTase I . cwp1p interaction with cwg2p was mapped to amino acids 1-244 or 137-294 but was not restricted to amino acids 137-244 . The genomic cwp1+ was isolated and sequenced . It has two putative open reading frames of 677 and 218 bp, separated by a 51 bp intron . The predicted amino acid sequence shows significant similarity to GGTase I alpha-subunits from different species . However, complementation of Saccharomyces cerevisiae ram2-1 mutant by overexpressing the cwp1+ gene was not possible . Expression of both cwg2+ and cwp1+ in Escherichia coli allowed 'in vitro' reconstitution of the GGTase I activity . S . pombe cells expressing the mutant enzyme containing the cwg2-1 mutation do not grow at 37 degrees C, but the growth defect can be suppressed by the addition of sorbitol . Actin immunostaining of the cwg2-1 mutant strain grown at 37 degrees C showed an abnormal distribution of actin patches . The cwg2-1 mutation was identified as a guanine to adenine substitution at nucleotide 604 of the coding region, originating the change A202T in the cwg2p . Deletion of the cwg2 gene is lethal; delta cwg2 spores can divide two or three times before losing viability . Most cells have aberrant morphology and septation defects . Overexpression of the rho1G15VC199R double-mutant allele in S . pombe caused loss of polarity but was not lethal and did not render the (1-3)beta-D-glucan synthase activity independent of GTP . Therefore, geranylgeranylation of rho1p is required for the appropriate function of this GTPase.

FEBS Lett, 1998 Oct 2, 436(2), 193 - 6
phd1+, a histone deacetylase gene of Schizosaccharomyces pombe, is required for the meiotic cell cycle and resistance to trichostatin A; Kim YB et al.; A gene named phd1+ encoding a protein highly homologous to the yeast and human histone deacetylases, such as Saccharomyces cerevisiae Rpd3p and human HDAC1, was cloned from Schizosaccharomyces pombe . The immune complex isolated from S . pombe cells expressing Phd1 fused to the FLAG epitope showed histone deacetylase activity, which was inhibited by trichostatin A (TSA), a specific inhibitor of histone deacetylase . The null mutation of phd1+ resulted in a marked decrease in the total cellular histone deacetylase activity and an increase in the sensitivity to TSA . Although the phd1 disruptant showed no obvious defect in the mitotic cell cycle or mating, both homothallic haploid and heterothallic diploid cells failed to form spores in the absence of phd1+ . These results indicate that phd1+ encodes a histone deacetylase, which is involved in the meiotic cell cycle in S . pombe.

Mol Cell Biol, 1998 Nov, 18(11), 6859 - 69
Reverse transcription of a self-primed retrotransposon requires an RNA structure similar to the U5-IR stem-loop of retroviruses; Lin JH et al.; An inverted repeat (IR) within the U5 region of the Rous sarcoma virus (RSV) mRNA forms a structure composed of a 7-bp stem and a 5-nucleotide (nt) loop . This U5-IR structure has been shown to be required for the initiation of reverse transcription . The mRNA of Tf1, long terminal repeat-containing retrotransposon from fission yeast (Schizosaccharomyces pombe) contains nucleotides with the potential to form a U5-IR stem-loop that is strikingly similar to that of RSV . The putative U5-IR stem-loop of Tf1 consists of a 7-bp stem and a 25-nt loop . Results from mutagenesis studies indicate that the U5-IR stem-loop in the mRNA of Tf1 does form and that it is required for Tf1 transposition . Although the loop is required for transposition, we were surprised that the specific sequence of the nucleotides within the loop was unimportant for function . Additional investigation indicates that the loss of transposition activity due to a reduction in the loop size to 6 nt could be rescued by increasing the GC content of the stem . This result indicates that the large loop in the Tf1 mRNA relative to that of the RSV allows the formation of the relatively weak U5-IR stem . The levels of Tf1 proteins expressed and the amounts of Tf1 RNA packaged into the virus-like particles were not affected by mutations in the U5-IR structure . However, all of the mutations in the U5-IR structure that caused defects in transposition produced low amounts of reverse transcripts . A unique feature in the initiation of Tf1 reverse transcription is that, instead of a tRNA, the first 11 nt of the Tf1 mRNA serve as the minus-strand primer . Analysis of the 5' end of Tf1 mRNA revealed that the mutations in the U5-IR stem-loop that resulted in defects in reverse transcription caused a reduction in the cleavage activity required to generate the Tf1 primer . Our results indicate that the U5-IR stems of Tf1 and RSV are conserved in size, position, and function.

Mol Cell Biol, 1998 Nov, 18(11), 6839 - 52
Schizosaccharomyces pombe retrotransposon Tf2 mobilizes primarily through homologous cDNA recombination; Hoff EF et al.; The Tf2 retrotransposon, found in the fission yeast Schizosaccharomyces pombe, is nearly identical to its sister element, Tf1, in its reverse transcriptase-RNase H and integrase domains but is very divergent in the gag domain, the protease, the 5' untranslated region, and the U3 domain of the long terminal repeats . It has now been demonstrated that a neo-marked copy of Tf2 overexpressed from a heterologous promoter can mobilize into the S . pombe genome and produce true transposition events . However, the Tf2-neo mobilization frequency is 10- to 20-fold lower than that of Tf1-neo, and 70% of the Tf2-neo events are homologous recombination events generated independently of a functional Tf2 integrase . Thus, the Tf2 element is primarily dependent on homologous recombination with preexisting copies of Tf2 for its propagation . Finally, production of Tf2-neo proteins and cDNA was also analyzed; surprisingly, Tf2 was found to produce its reverse transcriptase as a single species in which it is fused to protease, unlike all other retroviruses and retrotransposons.

Mol Cell Biol, 1998 Nov, 18(11), 6679 - 97
Cell cycle-regulated expression of mammalian CDC6 is dependent on E2F; Hateboer G et al.; The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation . In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have functions similar to those of E2F proteins in higher eukaryotes, by regulating the timed expression of genes implicated in cell cycle progression and DNA synthesis . The CDC6 gene is a target for MBF and SBF-regulated transcription . S . cerevisiae Cdc6p induces the formation of the prereplication complex and is essential for initiation of DNA replication . Interestingly, the Cdc6p homolog in Schizosaccharomyces pombe, Cdc18p, is regulated by DSC1, the S . pombe homolog of MBF . By cloning the promoter for the human homolog of Cdc6p and Cdc18p, we demonstrate here that the cell cycle-regulated transcription of this gene is dependent on E2F . In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells enter S phase . Our data also demonstrate that the human CDC6 protein (hCDC6) is essential and limiting for DNA synthesis, since microinjection of an anti-CDC6 rabbit antiserum blocks DNA synthesis and CDC6 cooperates with cyclin E to induce entry into S phase in cotransfection experiments . Furthermore, E2F is sufficient to induce expression of the endogenous CDC6 gene even in the absence of de novo protein synthesis . In conclusion, our results provide a direct link between regulated progression through G1 controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication.

J Biol Chem, 1998 Oct 23, 273(43), 28341 - 5
Interaction of Bnr1p with a novel Src homology 3 domain-containing Hof1p . Implication in cytokinesis in Saccharomyces cerevisiae; Kamei T et al.; Proteins containing the formin homology (FH) domains FH1 and FH2 are involved in cytokinesis or establishment of cell polarity in a variety of organisms . We have shown that the FH proteins Bni1p and Bnr1p are potential targets of the Rho family small GTP-binding proteins and bind to an actin-binding protein, profilin, at their proline-rich FH1 domains to regulate reorganization of the actin cytoskeleton in the yeast Saccharomyces cerevisiae . We found here that a novel Src homology 3 (SH3) domain-containing protein, encoded by YMR032w, interacted with Bnr1p in a GTP-Rho4p-dependent manner through the FH1 domain of Bnr1p and the SH3 domain of Ymr032wp . Ymr032wp weakly bound to Bni1p . Ymr032wp was homologous to cdc15p, which is involved in cytokinesis in Schizosaccharomyces pombe, and we named this gene HOF1 (homolog of cdc 15) . Both Bnr1p and Hof1p were localized at the bud neck, and both the bnr1 and hof1 mutations showed synthetic lethal interactions with the bni1 mutation . The hof1 mutant cells showed phenotypes similar to those of the septin mutants, indicating that HOF1 is involved in cytokinesis . These results indicate that Bnr1p directly interacts with Hof1p as well as with profilin to regulate cytoskeletal functions in S . cerevisiae.

EMBO J, 1998 Oct 15, 17(20), 5877 - 86
A misfolded protein conformation is not a sufficient condition for in vivo glucosylation by the UDP-Glc:glycoprotein glucosyltransferase; Fernandez F et al.; A key element in the quality control of glycoprotein folding is the UDP-Glc:glycoprotein glucosyltransferase (GT), which in cell-free assays exclusively glucosylates misfolded glycoproteins . In order to test if such a protein conformation is a sufficient condition for in vivo glucosylation of all N-linked oligosaccharides by GT, a Schizosaccharomyces pombe double mutant (gls2/alg6) was constructed . With this mutant, Man9GlcNAc2 is transferred to proteins and no removal of glucose units added by GT occurs as it lacks glucosidase II . The same proportion of glucosylated (Glc1Man9GlcNAc2) and unglucosylated (Man9GlcNAc2 and Man8GlcNAc2) endoplasmic reticulum (ER)-specific compounds was produced when cells were pre-incubated for 10, 20 or 30 min and further incubated with {14C}glucose for 10 min at 28 degrees C with or without 5 mM dithiothreitol (DTT), thus indicating not only that DTT did not affect protein glucosylation but also that no increased glucosylation of glycoproteins occurred in the presence of the drug . Monitoring Golgi-specific modifications of oligosaccharides after pulse-chase experiments performed in the presence or absence of 5 mM DTT showed that exit of the bulk of glycoproteins synthesized from the ER and thence their proper folding had been prevented by the drug . Cells pulse-chase labeled at 37 degrees C in the absence of DTT also yielded glucosylated and unglucosylated protein-linked oligosaccharides without Golgi-specific modifications . It was concluded that a misfolded protein conformation is not a sufficient condition for in vivo glucosylation of all N-linked oligosaccharides by GT.

Science, 1998 Oct 16, 282(5388), 493 - 6
Two modes of survival of fission yeast without telomerase; Nakamura TM et al.; Deletion of the telomerase catalytic subunit gene trt1+ in Schizosaccharomyces pombe results in death for the majority of cells, but a subpopulation survives . Here it is shown that most survivors have circularized all of their chromosomes, whereas a smaller number maintain their telomeres presumably through recombination . When the telomeric DNA-binding gene taz1+ is also deleted, trt1- taz1- survivors use the recombinational mode more frequently . Moreover, the massive elongation of telomeres in taz1- cells is absent in the double mutant . Thus, Taz1p appears to regulate telomeric recombination as well as telomerase activity in fission yeast.

RNA, 1998 Oct, 4(10), 1230 - 8
Programmed frameshifting in the synthesis of mammalian antizyme is +1 in mammals, predominantly +1 in fission yeast, but -2 in budding yeast; Ivanov IP et al.; The coding sequence for mammalian ornithine decarboxylase antizyme is in two different partially overlapping reading frames with no independent ribosome entry to the second ORF . Immediately before the stop codon of the first ORF, a proportion of ribosomes undergo a quadruplet translocation event to shift to the +1 reading frame of the second and main ORF . The proportion that frameshifts is dependent on the polyamine level and, because the product antizyme is a negative regulator of intracellular polyamine levels, the frameshifting acts to complete an autoregulatory circuit by sensing polyamine levels . An mRNA element just 5' of the shift site and a 3' pseudoknot are important for efficient frameshifting . Previous work has shown that a cassette with the mammalian shift site and associated signals directs efficient shifting in the budding yeast Saccharomyces cerevisiae at the same codon to the correct frame, but that the shift is -2 instead of +1 . The product contains an extra amino acid corresponding to the shift site . The present work shows efficient frameshifting also occurs in the fission yeast, Schizosaccharomyces pombe . This frameshifting is 80% +1 and 20% -2 . The response of S . pombe translation apparatus to the mammalian antizyme recoding signals is more similar to that of the mammalian system than to that of S . cerevisiae . S . pombe provides a good model system for genetic studies on the mechanism of at least this type of programmed mammalian frameshifting.

J Cell Physiol, 1998 Nov, 177(2), 241 - 7
Molecular cloning and tissue-specific expression of Mrad9, a murine orthologue of the Schizosaccharomyces pombe rad9+ checkpoint control gene; Hang H et al.; We have isolated a murine cDNA, Mrad9, that is orthologous to the fission yeast rad9+ and human HRAD9 genes . Mrad9 encodes a 389 amino acid long, 42,032 Dalton protein that is 27% identical and 56% similar to Rad9p, and 82% identical and 88% similar to HRAD9, at the amino acid level . Expression of the Mrad9 cDNA in Schizosaccharomyces pombe rad9::ura4+ cells restores nearly wild-type levels of hydroxyurea resistance and early S phase checkpoint control to mutant fission yeast cell populations . However, UV resistance is only minimally restored, and mutant cells remain sensitive to gamma radiation . Mrad9 genomic DNA was isolated from a mouse 129/SvEv library . The Mrad9 gene was local ized to a 15-kbp genomic DNA fragment, and contains 10 exons separated by 9 introns . Northern blot analysis indicates that the gene is expressed in many different tissues of the adult mouse, but the mRNA is most abundant in the heart and present at very low levels in the liver . These studies demonstrate the existence of a murine orthologue of the fission yeast rad9+ gene and underscore at least the partial evolutionary conservation of rad9+-dependent checkpoint control mechanisms.

Gene, 1998 Oct 5, 220(1-2), 45 - 53
Molecular analysis of the split cox1 gene from the Basidiomycota Agrocybe aegerita: relationship of its introns with homologous Ascomycota introns and divergence levels from common ancestral copies; Gonzalez P et al.; The Basidiomycota Agrocybe aegerita (Aa) mitochondrial cox1 gene (6790 nucleotides), encoding a protein of 527aa (58377Da), is split by four large subgroup IB introns possessing site-specific endonucleases assumed to be involved in intron mobility . When compared to other fungal COX1 proteins, the Aa protein is closely related to the COX1 one of the Basidiomycota Schizophyllum commune (Sc) . This clade reveals a relationship with the studied Ascomycota ones, with the exception of Schizosaccharomyces pombe (Sp) which ranges in an out-group position compared with both higher fungi divisions . When comparison is extended to other kingdoms, fungal COX1 sequences are found to be more related to algae and plant ones (more than 57.5% aa similarity) than to animal sequences (53.6% aa similarity), contrasting with the previously established close relationship between fungi and animals, based on comparisons of nuclear genes . The four Aa cox1 introns are homologous to Ascomycota or algae cox1 introns sharing the same location within the exonic sequences . The percentages of identity of the intronic nucleotide sequences suggest a possible acquisition by lateral transfers of ancestral copies or of their derived sequences . These identities extend over the whole intronic sequences, arguing in favor of a transfer of the complete intron rather than a transfer limited to the encoded ORF . The intron i4 shares 74% of identity, at the nucleotidic level, with the Podospora anserina (Pa) intron i14, and up to 90.5% of aa similarity between the encoded proteins, i.e . the highest values reported to date between introns of two phylogenetically distant species . This low divergence argues for a recent lateral transfer between the two species . On the contrary, the low sequence identities (below 36%) observed between Aa i1 and the homologous Sp i1 or Prototheca wickeramii (Pw) i1 suggest a long evolution time after the separation of these sequences . The introns i2 and i3 possessed intermediate percentages of identity with their homologous Ascomycota introns . This is the first report of the complete nucleotide sequence and molecular organization of a mitochondrial cox1 gene of any member of the Basidiomycota division.

Plant Physiol, 1998 Oct, 118(2), 407 - 17
Arabidopsis Rho-related GTPases: differential gene expression in pollen and polar localization in fission yeast; Li H et al.; The Rho small GTP-binding proteins are versatile, conserved molecular switches in eukaryotic signal transduction . Plants contain a unique subfamily of Rho-GTPases called Rop (Rho-related GTPases from plants) . Our previous studies involving injection of antibodies indicated that the pea Rop GTPase Rop1Ps is critical for pollen tube growth . In this study we show that overexpression of an apparent Arabidopsis ortholog of Rop1Ps, Rop1At, induces isotropic cell growth in fission yeast (Schizosaccharomyces pombe) and that green fluorescence protein-tagged Rop1At displays polar localization to the site of growth in yeast . We found that Rop1At and two other Arabidopsis Rops, Rop3At and Rop5At, are all expressed in mature pollen . All three pollen Rops fall into the same subgroup as Rop1Ps and diverge from those Rops that are not expressed in mature pollen, suggesting a coupling of the structural conservation of Rop GTPases to their gene expression in pollen . However, pollen-specific transcript accumulation for Rop1At is much higher than that for Rop3At and Rop5At . Furthermore, Rop1At is specifically expressed in anthers, whereas Rop3At and Rop5At are also expressed in vegetative tissues . In transgenic plants containing the Rop1At promoter:GUS fusion gene, GUS is specifically expressed in mature pollen and pollen tubes . We propose that Rop1At may play a predominant role in the regulation of polarized cell growth in pollen, whereas its close relatives Rop3At and Rop5At may be functionally redundant to Rop1At in pollen.

J Virol, 1998 Nov, 72(11), 9318 - 22
A map of interactions between the proteins of a retrotransposon; Steele SJ et al.; The yeast two-hybrid system and in vitro binding assays were used to characterize 54 potential interactions between the proteins of Tf1, an LTR-retrotransposon found in Schizosaccharomyces pombe . The Tf1 integrase (IN) protein was found to interact strongly with itself and not with other control proteins . In addition, the IN core domain interacted strongly with itself and full-length IN . Interestingly, the two-hybrid analysis detected an interaction between the RNase H domain of reverse transcriptase and IN . The biological implications of these interactions are discussed.

Eur J Cell Biol, 1998 Aug, 76(4), 288 - 95
Subcellular localization and possible function of actin, tropomyosin and actin-related protein 3 (Arp3) in the fission yeast Schizosaccharomyces pombe; Arai R et al.; We investigated subcellular localizations and interactions of actin and two actin cytoskeleton-related proteins, Cdc8 tropomyosin and actin-related protein 3, Arp3, in the fission yeast Schizosaccharomyces pombe, using specific antibodies and by gene disruption . Actin was localized to the medial microfilamentous ring in the region of the septum during cytokinesis and to cortical patches by immunoelectron microscopy . F-actin cables were detected throughout the cell cycle by fluorescent staining with Bodipy-phallacidin . Cables were often linked to the patches and to the medial ring during its formation . Tropomyosin was localized to the medial ring and the cables . It was also distributed in the cell as patches, although co-localization with F-actin was not frequent . In cdc8ts mutant cells, F-actin cables were not observed although the F-actin patches were detected and cell polarity was maintained . These observations suggest that the F-actin cables may be involved in the formation of the medial ring, and that tropomyosin plays an important role in organizing both the ring and the cable, but is not involved in the F-actin patch formation or maintenance of cell polarity . Binding of Arp3 to actin was revealed by immunoprecipitation as well as by DNase I column chromatography . Arp3 seemed to form a complex with several proteins in the cell extracts, as previously reported for other organisms . Contrary to a previous report (McCollum et al., EMBO J . 15, 6438-6446, 1996), Arp3 was found to be concentrated in the medial region from early anaphase to late cytokinesis . Following arp3 gene disruption, F-actin patches were delocalized throughout the cell and cells did not undergo polarized growth, suggesting that Arp3 influences the proper localization of the actin patches in the cell and thereby controls the polarized growth of the cell.

Mol Biol Cell, 1998 Oct, 9(10), 2839 - 55
cut11(+): A gene required for cell cycle-dependent spindle pole body anchoring in the nuclear envelope and bipolar spindle formation in Schizosaccharomyces pombe; West RR et al.; The "cut" mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality . Analysis of temperature-sensitive alleles of cut11(+) suggests that this gene is required for the formation of a functional bipolar spindle . Defective spindle structure was revealed with fluorescent probes for tubulin and DNA . Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm . Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase . This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope . Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes . Cloning and sequencing showed that cut11(+) encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1 . These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis.

Mol Biol Cell, 1998 Oct, 9(10), 2739 - 50
The role of topoisomerase II in meiotic chromosome condensation and segregation in Schizosaccharomyces pombe; Hartsuiker E et al.; Topoisomerase II is able to break and rejoin double-strand DNA . It controls the topological state and forms and resolves knots and catenanes . Not much is known about the relation between the chromosome segregation and condensation defects as found in yeast top2 mutants and the role of topoisomerase II in meiosis . We studied meiosis in a heat-sensitive top2 mutant of Schizosaccharomyces pombe . Topoisomerase II is not required until shortly before meiosis I . The enzyme is necessary for condensation shortly before the first meiotic division but not for early meiotic prophase condensation . DNA replication, prophase morphology, and dynamics of the linear elements are normal in the top2 mutant . The top2 cells are not able to perform meiosis I . Arrested cells have four spindle pole bodies and two spindles but only one nucleus, suggesting that the arrest is nonregulatory . Finally, we show that the arrest is partly solved in a top2 rec7 double mutant, indicating that topoisomerase II functions in the segregation of recombined chromosomes . We suggest that the inability to decatenate the replicated DNA is the primary defect in top2 . This leads to a loss of chromatin condensation shortly before meiosis I, failure of sister chromatid separation, and a nonregulatory arrest.

FEBS Lett, 1998 Sep 18, 435(2-3), 241 - 4
The Pzh1 protein phosphatase and the Spm1 protein kinase are involved in the regulation of the plasma membrane H+-ATPase in fission yeast; Balcells L et al.; We have previously shown that the mutation of the Schizosaccharomyces pombe PPZ-like protein phosphatase encoded by the gene pzh1+ results in increased tolerance to sodium and in hypersensitivity to potassium ions . A similar phenotype has also been reported for deletants in the spm1/pmk1 gene, encoding a mitogen-activated protein (MAP) kinase . We have found that the sodium tolerance phenotype of pzh1 deletants is stronger than that of spm1 mutants, and both effects are additive . Therefore, most probably both gene products mediate different pathways on sodium tolerance . In our hands, mutation of the kinase does not alter the tolerance to potassium, but it yields cells more tolerant to magnesium ions . While in budding yeast the mutations are synthetically lethal, fission yeast cells lacking both the phosphatase and the kinase genes are viable . Interestingly, their ability to export H+ to the medium is greatly impaired (although not that of pzh1 or spm1 single mutants) . We have observed that, although the amount of the H+-ATPase in the plasma membrane is not altered, the activity of the enzyme is lower than normal and cannot be induced by glucose . These observations suggest that the activity of the H+-ATPase in fission yeast might be regulated by phospho-dephosphorylation mechanisms that might involve the pzh1+ and spm1+ gene products.

Appl Environ Microbiol, 1998 Oct, 64(10), 3983 - 8
A novel ATP-binding cassette transporter involved in multidrug resistance in the phytopathogenic fungus Penicillium digitatum; Nakaune R et al.; Demethylation inhibitor (DMI)-resistant strains of the plant pathogenic fungus Penicillium digitatum were shown to be simultaneously resistant to cycloheximide, 4-nitroquinoline-N-oxide (4NQO), and acriflavine . A PMR1 (Penicillium multidrug resistance) gene encoding an ATP-binding cassette (ABC) transporter (P-glycoprotein) was cloned from a genomic DNA library of a DMI-resistant strain (LC2) of Penicillium digitatum by heterologous hybridization with a DNA fragment containing an ABC-encoding region from Botrytis cinerea . Sequence analysis revealed significant amino acid homology to the primary structures of PMR1 (protein encoded by the PMR1 gene) and ABC transporters of Saccharomyces cerevisiae (PDR5 and SNQ2), Schizosaccharomyces pombe (HBA2), Candida albicans (CDR1), and Aspergillus nidulans (AtrA and AtrB) . Disruption of the PMR1 gene of P . digitatum DMI-resistant strain LC2 demonstrated that PMR1 was an important determinant of resistance to DMIs . The effective concentrations inhibiting radial growth by 50% (EC50s) and the MICs of fenarimol and bitertanol for the PMR1 disruptants (Deltapmr1 mutants) were equivalent to those for DMI-sensitive strains . Northern blot analysis indicated that severalfold more PMR1 transcript accumulated in the DMI-resistant strains compared with those in DMI-sensitive strains in the absence of fungicide . In both DMI-resistant and -sensitive strains, transcription of PMR1 was strongly enhanced within 10 min after treatment with the DMI fungicide triflumizole . These results suggested that the toxicant efflux system comprised of PMR1 participates directly in the DMI resistance of the fungus.

Genetics, 1998 Oct, 150(2), 553 - 62
The Schizosaccharomyces pombe cho1+ gene encodes a phospholipid methyltransferase; Kanipes MI et al.; The isolation of mutants of Schizosaccharomyces pombe defective in the synthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine is reported . These mutants are choline auxotrophs and fall into two unlinked complementation groups, cho1 and cho2 . We also report the analysis of the cho1+ gene, the first structural gene encoding a phospholipid biosynthetic enzyme from S . pombe to be cloned and characterized . The cho1+ gene disruption mutant (cho1Delta) is viable if choline is supplied and resembles the cho1 mutants isolated after mutagenesis . Sequence analysis of the cho1+ gene indicates that it encodes a protein closely related to phospholipid methyltransferases from Saccharomyces cerevisiae and rat . Phospholipid methyltransferases encoded by a rat liver cDNA and the S . cerevisiae OPI3 gene are both able to complement the choline auxotrophy of the S . pombe cho1 mutants . These results suggest that both the structure and function of the phospholipid N-methyltransferases are broadly conserved among eukaryotic organisms.

EMBO J, 1998 Oct 1, 17(19), 5670 - 8
The role of the destruction box and its neighbouring lysine residues in cyclin B for anaphase ubiquitin-dependent proteolysis in fission yeast: defining the D-box receptor; Yamano H et al.; Programmed proteolysis of proteins such as mitotic cyclins and Cut2/Pds1p requires a 9-residue conserved motif known as the destruction box (D-box) . Strong expression of protein fragments containing destruction boxes, such as the first 70 residues of Cdc13 (N70), inhibits the growth of Schizosaccharomyces pombe at metaphase . This inhibition can be overcome either by removal of all lysine residues from N70 using site-directed mutagenesis (K0-N70) or by raising the concentration of intracellular ubiquitin . Consistent with the idea that competition for ubiquitin accounts for some of its inhibitory effects, wild-type N70 not only stabilized D-box proteins, but also Rum1 and Cdc18, which are degraded by a different pathway . The K0-N70 construct was neither polyubiquitinated nor degraded in vitro, but it blocked the growth of strains of yeast in which anaphase-promoting complex/cyclosome (APC/C) function was compromised by mutation, and specifically inhibited proteolysis of APC/C substrates in vivo . Both K0-N70 and 20-residue D-box peptides blocked polyubiquitination of other D-box-containing substrates in a cell-free ubiquitination assay system . These data suggest the existence of a D-box receptor protein that recognizes D-boxes prior to ubiquitination.

Nucleic Acids Res, 1998 Oct 15, 26(20), 4783 - 4
Use of gap repair in fission yeast to obtain novel alleles of specific genes; Kostrub CF et al.; We have adapted a method for making libraries of mutations in any specific gene for use in the fission yeast Schizosaccharomyces pombe . This elegant and simple method consists of PCR amplification of the gene of interest, followed by co-transformation of fission yeast with the PCR fragment and a linearized plasmid vector prepared such that the ends of the vector share DNA sequence with the ends of the PCR fragment . Homologous recombination between the vector and the PCR fragment occurs at a high frequency and results in a collection of yeast transformants, most harboring a mutated allele of the original gene within the vector of choice . This library can then be screened or selected for phenotypes of interest.

Genes Cells, 1998 Jul, 3(7), 405 - 13
Meiotic telomeres: a matchmaker for homologous chromosomes; Hiraoka Y; Telomeres, with their special structures and special schemes of synthesis, are essential for protecting the ends of eukaryotic linear chromosomes during cell proliferation . In addition to this basic function, the meiosis-specific functions of telomeres have long been inferred from the cytological observations of characteristic chromosome configurations in meiotic prophase . Recent studies in the fission yeast Schizosaccharomyces pombe have provided deeper insights into the role of meiotic telomeres in the pairing of homologous chromosomes . Here I have summarized our current understanding of the meiotic behaviour of telomeres in S . pombe, and discuss the role of telomeres in meiosis.

Prog Nucleic Acid Res Mol Biol, 1998, 61, 345 - 78
Control of meiotic recombination in Schizosaccharomyces pombe; Fox ME et al.; Homologous recombination occurs at high frequency during meiosis and is essential for the proper segregation of chromosomes and the generation of genetic diversity . Meiotic recombination is controlled in numerous ways . In the fission yeast Schizosaccharomyces pombe nutritional starvation induces meiosis and high-level expression of many genes, including numerous recombination (rec) genes, whose products are required for recombination . Accompanying the two meiotic divisions are profound changes in nuclear and chromosomal structure and movement, which may play an important role in meiotic recombination . Although recombination occurs throughout the genome, it occurs at high frequency in some intervals (hotspots) and at low frequency in others (coldspots) . The well-characterized hotspot M26 is activated by the Mts1/Mts2 protein; this site and its binding proteins interact with the local chromosomal structure to enhance recombination . A coldspot between the silent mating-type loci is repressed by identified proteins, which may also alter local chromatin . We discuss in detail the rec genes and the possible functions of their products, some but not all of which share homology with other identified proteins . Although some of the rec gene products are required for recombination throughout the genome, others demonstrate regional specificity and are required in certain genomic regions but not in others . Throughout the review contrasts are made with meiotic recombination in the more thoroughly studied budding yeast Saccharomyces cerevisiae.

Glycobiology, 1998 Nov, 8(11), 1087 - 95
All pyruvylated galactose in Schizosaccharomyces pombe N-glycans is present in the terminal disaccharide, 4, 6-O-{(R)-(1-carboxyethylidine)}-Galbeta1,3Galalpha1-; Gemmill TR et al.; The large N-linked oligosaccharides released from Schizosaccharomyces pombe by endo-beta-N-acetylglucosaminidase H were examined to determine how the negatively chargedpyruvylated galactoses present (Gemmill,T.R., and Trimble,R.B., 1996, J . Biol . Chem ., 271, 25945-25949) were attached to the oligosaccharide chains . Binding of biotinylated human serum amyloid P and peanut agglutinin to native and depyruvylated S.pombe glycoproteins, respectively, indicated that the pyruvylated epitope was likely to be in the beta configuration . Examination by high-field 1H NMR of whole glycans and a disaccharide fragment released from them on partial acid hydrolysis showed that the pyruvylated galactose species was in fact beta1,3-linked to a second galactose, and this occurred an average of five to six times on nominal Gal57Man64GlcNAc N-glycans . The pyruvate-2,(4,6)Gal-beta1,3Gal epitope is chemically similar to acetaldehyde-Galbeta1,3Gal groups found on the glycoproteins from Paramyxovirus-infected bovine kidney cells (Prehm, P., Scheid,A . and Choppin,P.W . ,1979, J . Biol . Chem ., 254, 9669-9677) . The 1:1 stoichiometry between pyruvate and beta-linked galactose in these S.pombe glycans indicates that either pyruvate addition to terminal beta1,3Gal is highly efficient or that pyruvylated Gal is transferred en bloc to alpha1,2-linked Gal residues in theN-linked chains . In contradiction to many galactomannan-producing fungi, which add substantial amounts of Gal in the furanose form to their glycoproteins, all detectable Gal in the large S.pombe galactomannans is in the pyranose form, as found in higher eukaryotes . The current work shows that the S.pombe outer chain structure is a poly-alpha1,6Man backbone 2-O-substituted with either Gal or the pyruvylated galactobiose and contains little alpha1,2-linked or 2-O-substituted Man . This is in contrast to the S . cerevisiae outer chain, which is poly-alpha1,6Man substituted with alpha1,2-linked Man sidechains (Ballou,C.E . ,1990, Methods Enzymol , 185, 440-470).

Mol Cells, 1998 Aug 31, 8(4), 431 - 7
Thioltransferase from Schizosaccharomyces pombe: purification to homogeneity and some properties; Kim HG et al.; Two types of thioltransferase were identified in the cytosolic extract of Schizosaccharomyces pombe, a fission yeast . In the present study, the major one of them was purified to homogeneity using chromatography processes such as ion-exchange chromatography and gel filtration . Purification was monitored by the transhydrogenase activity of thioltransferase with 2-hydroxyethyl disulfide as a substrate . Its molecular weight was estimated to be about 14,000 on SDS-polyacrylamide gel electrophoresis . The purified enzyme catalyzes the reduction of various disulfide compounds such as S-sulfocysteine, L-cystine, and insulin . It was also found to contain the reducing activity on non-disulfide substrates such as dehydroascorbic acid and alloxan . Its activity was greatly activated by high concentrations of reduced glutathione . It was found to be very heat-stable as like other thioltransferases . It was characterized on other aspects such as kinetic parameters and optimal reaction conditions.

J Bacteriol, 1998 Oct, 180(19), 5038 - 43
Isolation and characterization of high-osmolarity-sensitive mutants of fission yeast; Aiba H et al.; For the fission yeast Schizosaccharomyces pombe, adaptation to high-osmolarity medium is mediated by a mitogen-activated protein (MAP) kinase cascade, involving the Wis1 MAP kinase kinase and the Sty1 MAP kinase . The MAP kinase pathway transduces an osmotic signal and accordingly regulates the expression of the downstream target gene (gpd1(+)) that encodes NADH-dependent glycerol-3-phosphate dehydrogenase, in order to adaptively accumulate glycerol inside the cells as an osmoprotectant . We previously characterized a set of high-osmolarity-sensitive S . pombe mutants, including wis1, sty1, and gpd1 . In this study, we attempted to further isolate novel osmolarity-sensitive mutants . For some of the mutants isolated, profiles of glycerol production in response to the osmolarity of the growth medium were indistinguishable from that of the wild-type cells, suggesting that they are novel types . They were classified into three distinct types genetically and, thus, were designated hos1, hos2, and hos3 (high osmolarity sensitive) mutants . One of them, the hos1 mutant, was characterized in detail . The hos1 mutant was demonstrated to have a mutational lesion in the known ryh1(+) gene, which encodes a small GTP-binding protein . Disruption of the ryh1(+) gene results not only in osmosensitivity but also in temperature sensitivity for growth . It was also found that the delta ryh1 mutant is severely sterile . These results are discussed with special reference to the osmoadaptation of S . pombe.

J Biol Chem, 1998 Oct 2, 273(40), 26078 - 86
Cloning and disruption of caPLB1, a phospholipase B gene involved in the pathogenicity of Candida albicans; Leidich SD et al.; The Candida albicans PLB1 gene was cloned using a polymerase chain reaction-based approach relying on degenerate oligonucleotide primers designed according to the amino acid sequences of two peptide fragments obtained from a purified candidal enzyme displaying phospholipase activity (Mirbod, F., Banno, Y., Ghannoum, M . A., Ibrahim, A . S., Nakashima, S., Yasuo, K., Cole, G . T., and Nozawa, Y . (1995) Biochim . Biophys . Acta 1257, 181-188) . Sequence analysis of a 6.7-kilobase pair EcoRI-ClaI genomic clone revealed a single open reading frame of 1818 base pairs that predicts for a pre-protein of 605 residues . Comparison of the putative candidal phospholipase with those of other proteins in data base revealed significant homology to known fungal phospholipase Bs from Saccharomyces cerevisiae (45%), Penicillium notatum (42%), Torulaspora delbrueckii (48%), and Schizosaccharomyces pombe (38%) . Thus, we have cloned the gene encoding a C . albicans phospholipase B homolog . This gene, designated caPLB1, was mapped to chromosome 6 . Disruption experiments revealed that the caplb1 null mutant is viable and displays no obvious phenotype . However, the virulence of strains deleted for caPLB1, as assessed in a murine model for hematogenously disseminated candidiasis, was significantly attenuated compared with the isogenic wild-type parental strain . Although deletion of caPLB1 did not produce any detectable effects on candidal adherence to human endothelial or epithelial cells, the ability of the caplb1 null mutant to penetrate host cells was dramatically reduced . Thus, phospholipase B may well contribute to the pathogenicity of C . albicans by abetting the fungus in damaging and traversing host cell membranes, processes which likely increase the rapidity of disseminated infection.

Mol Gen Genet, 1998 Aug, 259(2), 216 - 23
Molecular cloning and characterization of the gene encoding N'-{(5'-phosphoribosyl)-formimino}-5-aminoimidazole-4-carboxamide ribonucleotide (BBM II) isomerase from Arabidopsis thaliana; Fujimori K et al.; We have isolated an Arabidopsis BBM II isomerase cDNA from an Arabidopsis cDNA library, by means of functional complementation of the E . coli hisA mutant strain HfrG6 . The isolated cDNA encodes a polypeptide of 304 amino acids with a calculated molecular weight of 33,363 . Sequence comparison with the HIS6 proteins of yeasts revealed that Arabidopsis BBM II isomerase contains an N-terminal extension of approximately 40 amino acids that shows the general properties of chloroplast transit peptides . This finding is consistent with the localization of other histidine biosynthetic enzymes, such as imidazoleglycerolphosphate dehydratase and histidinol dehydrogenase, in the chloroplasts in higher plants . The primary structure of the mature protein was 50% and 42% identical, respectively, to the HIS6 proteins of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively, while no prominent sequence similarity to the bacterial BBM II isomerase was found . That the isolated Arabidopsis cDNA actually encodes a functionally active BBM II isomerase activity was confirmed in an in vitro enzyme assay using a crude extract prepared from strain HfrG6 transformed with the Arabidopsis BBM II isomerase cDNA.

Eur J Biochem, 1998 Aug 15, 256(1), 112 - 8
Purification, characterization and complete amino acid sequence of nuclease C1 from Cunninghamella echinulata var . echinulata; Ho HC et al.; It is known, from the zymogram method of nuclease activity assay, that the crude extracts of Cunninghamella echinulata var . echinulata contained at least three distinct extracellular nucleases . Among them, the major form was 30 kDa in molecular mass and termed nuclease C1 . In this report, nuclease C1 was purified to apparent homogeneity by chromatography on Cibacron blue-3GA, phenyl-Sepharose 4B and HiTrap Heparin . Nuclease C1 acquired enzymatic activity in the presence of Mn2+ or Mg2+ and was inhibited by EDTA . The activity was maximal at pH 7-8.5 . The primary structure of nuclease C1 was completely determined using enzymatic digestion and gene cloning . The N-terminal 49 residues of nuclease C1 were first elucidated from a tryptic digest . Two degenerate upstream primers were subsequently designed to amplify the cDNA encoding nuclease C1 . The resulting protein sequence of nuclease C1 was shown to be composed of 252 residues . It was intriguing to find that the protein sequence of nuclease C1 showed significant similarities with the sequences of the mitochondrial nucleases of Saccharomyces cerevisiae (44% identity) and Schizosaccharomyces pombe (42% identity) . Residue His87 of nuclease C1 was postulated to be located at the active site from sequence similarity with secreted nuclease from Serratia marcescens.

Curr Genet, 1998 Sep, 34(3), 172 - 82
ras1 and pat1 alleles interact to quantitatively and qualitatively alter conjugation in fission yeast; Mach KE et al.; To identify novel components of ras1+ signalling in Schizosaccharomyces pombe, extragenic suppressors of the mating defect of ras1 effector mutants were isolated . A novel allele of pat1, pat1-e1, was isolated that increases the mating of ras1-D43E mutants to near wild-type levels but does not suppress the mating defect of ras1-I41M, ras1-Y37F, or ras1-Y45I mutants . This allele-specific suppression is not a characteristic of all pat1 alleles since pat1-3 and pat1-114 partially and equally suppress ras1-D43E and ras1-I41M mutants . Analysis of mating cultures showed that ras1-D43E and pat1-e1 interact to qualitatively alter the mating response . While pat1-e1 ras1-D43E cells were delayed in agglutination, cell-cycle delay, and mat1-Pm transcription, they induce mat1-Mc at the same time and mate more rapidly than other mating cultures . These results suggest that pheromone signalling, but not nutritional signalling, is delayed in pat1-e1 ras1-D43E cells . We hypothesize that this delay causes an elevated pheromone response and thus suppression of the mating defect of the ras1-D43E mutant by pat1-e1.

Curr Genet, 1998 Sep, 34(3), 164 - 71
The essential schizosaccharomyces pombe cdc23 DNA replication gene shares structural and functional homology with the Saccharomyces cerevisiae DNA43 (MCM10) gene; Aves SJ et al.; The fission yeast cdc23 gene is required for correct DNA replication: cdc23 mutants show reduced rates of DNA synthesis and become elongated after cell-cycle arrest . We have cloned the Schizosaccharomyces pombe cdc23 gene by complementation of the temperature-sensitive phenotype of cdc23-M36 and confirmed the identity of the gene by integrative mapping . Analysis of the DNA sequence reveals that cdc23 can encode a protein of 593 amino acids (Mr=67 kDa) with 22% overall identity and many structural homologies with the product of the Saccharomyces cerevisiae DNA43 (MCM10) gene which is required for correct initiation of DNA synthesis at chromosomal origins of replication . Construction of a cdc23 null allele has established that the cdc23 gene is essential for viability, with cdc23 deletion mutant spores germinating but undergoing arrest with undivided nuclei in the first or second cell cycle . The S . pombe cdc23 gene on an expression plasmid is able to complement the S . cerevisiae dna43-1 mutant . These structural and functional homologies between two distantly related species suggest that cdc23 and DNA43 may represent genes for a conserved essential eukaryotic DNA replication function.

Curr Genet, 1998 Sep, 34(3), 153 - 63
Cell-division-cycle defects associated with fission yeast pre-mRNA splicing mutants; Potashkin J et al.; We have isolated six new pre-mRNA splicing mutants (prp) from a collection of temperature-sensitive (ts-) Schizosaccharomyces pombe strains . The prp mutants are defective in the splicing of both messenger RNA and U6 small nuclear RNA precursors . A single recessive mutation is responsible for both the ts- growth and the splicing phenotypes in each of the prp mutants . The six prp mutations are unlinked and fall into separate complementation groups . Two are allelic with the previously described prp3 and prp4 mutations; the remaining four define the new alleles prp5-1, prp6-1, prp7-1, and prp9-1 . The six mutants exhibit three splicing phenotypes: accumulation of unspliced precursor at the restrictive but not at the permissive temperature; accumulation of unspliced precursor at both the permissive and restrictive temperatures; and accumulation of unspliced precursor, the intron-exon lariat intermediate, and the intron lariat final product . In addition to their aberrant splicing phenotypes, the prp5-1 and prp6-1 mutants express classical cell-division-cycle defects, while prp7-1 exhibits an unusual hyphal morphology . These results suggest a connection between pre-mRNA splicing and the control of cell division in fission yeast.

Curr Biol, 1998 Aug 27, 8(17), 963 - 6
Conjugation in S . pombe: identification of a microtubule-organising centre, a requirement for microtubules and a role for Mad2; Petersen J et al.; During the G1 phase of the cell cycle, cells of the fission yeast Schizosaccharomyces pombe can be induced to mate by nitrogen starvation and the presence of mating pheromones . Polarised growth towards cells of the opposite mating type (P or M) leads to the formation of a projection tip and, upon contact, localised cell wall degradation results in conjugation and cell fusion {1} . Here, we have investigated the role of microtubules in this process . We describe a previously unidentified microtubule-organising centre (MTOC) that forms at projection tips upon cell-to-cell contact, before cells fuse . Treatment of mating cells with the microtubule-destabilising drug thiabendazole (TBZ) showed that microtubule integrity was required for mating at two distinct stages: during projection tip formation and cell fusion . Projection tip formation requires filamentous (F) actin function {2} and microtubules are required for the localisation of F actin to the projection tip . We also identify a role during mating for Mad2--a mitotic checkpoint protein that is required in all eukaryotes to maintain the mitotic state in response to microtubule depolymerisation {3} . S . pombe mad2 mutant cells were compromised in their ability to mate upon removal of TBZ, indicating that in fission yeast, in the absence of microtubules, Mad2 is also required to maintain mating competence.

Curr Biol, 1998 Aug 27, 8(17), 947 - 54
Byr4 and Cdc16 form a two-component GTPase-activating protein for the Spg1 GTPase that controls septation in fission yeast; Furge KA et al.; BACKGROUND: Spatial and temporal control of cytokinesis ensures the accurate transmission of genetic material and the correct development of multicellular organisms . An excellent model system in which to study cytokinesis is Schizosaccharomyces pombe because there are similarities between cytokinesis in S . pombe and mammals and because genes involved in S . pombe cytokinesis have been characterized . In particular, formation of the septum is positively regulated by the Spg1 GTPase and its effector, the Cdc7 kinase . Septation is negatively regulated by Cdc16, a protein similar to GTPase-activating proteins (GAPs) for Ypt GTPases, and by Byr4, a protein of unknown biochemical function . This study investigates the relationship between Byr4, Cdc16, and Spg1 . RESULTS: Genetic interactions were observed between byr4, cdc16, and spg1 mutants . Byr4 bound to Cdc16 and Spg1 in yeast two-hybrid assays and in coprecipitations in vitro and in yeast . Byr4 inhibited the dissociation and hydrolysis of GTP bound to Spg1, but when Byr4 and Cdc16 were combined together they displayed Spg1GAP activity in vitro; Cdc16 alone had no detectable GAP activity . The binding of Byr4 to Spg1 and the Byr4-Cdc16 Spg1GAP activity were specific because Byr4 and Cdc16 did not bind to or affect the GTPase activities of the seven known S pombe Ypt family GTPase . CONCLUSIONS: Byr4 and Cdc16 form a two-component GAP for the Spg1 GTPase . Byr4 and Cdc16 appear to negatively regulate septation in S . pombe by modulating the nucleotide state of Spg1 possibly in a spatially or temporally controlled manner.

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