Schizosaccharomyces

Mol Cell Biol, 1995 Jul, 15(7), 3697 - 707
Characterization of fus1 of Schizosaccharomyces pombe: a developmentally controlled function needed for conjugation; Petersen J et al.; In Schizosaccharomyces pombe, the fus1 mutation blocks conjugation at a point after cell contact and agglutination . The cell walls separating the mating partners are not degraded, which prevents cytoplasmic fusion . In order to investigate the molecular mechanism of conjugation, we cloned the fus1 gene and found that it is capable of encoding a 1,372-amino-acid protein with no significant similarities to other known proteins . Expression of the fus1 gene is regulated by the developmental state of the cells . Transcription is induced by nitrogen starvation and requires a pheromone signal in both P and M cell types . Consequently, mutants defective in the pheromone response pathway fail to induce fus1 expression . The ste11 gene, which encodes a transcription factor controlling expression of many genes involved in sexual differentiation, is also required for transcription of fus1 . Furthermore, deletion of two potential Ste11 recognition sites in the fus1 promoter region abolished transcription, and expression could be restored when we inserted a different Ste11 site from the mat1-P promoter . Since this element was inverted relative to the fus1 element, we conclude that activation of transcription by Ste11 is independent of orientation . Although the fus1 mutant has a phenotype very similar to that of Saccharomyces cerevisiae fus1 mutants, the two proteins appear to have different roles in the process of cell fusion . Budding yeast Fus1 is a typical membrane protein and contains an SH3 domain . Fission yeast Fus1 has no features of a membrane protein, yet it appears to localize to the projection tip . A characteristic proline-rich potential SH3 binding site may mediate interaction with other proteins.

Eur J Biochem, 1995 Jul 1, 231(1), 45 - 9
Inter-species DNA polymerase delta chimeras are functional in Saccharomyces cerevisiae; Moussy G et al.; The catalytic subunits of DNA polymerase delta of Schizosaccharomyces pombe and Saccharomyces cerevisiae share over 50% identity . The capability of S . pombe DNA polymerase delta to complement two thermosensitive mutants of S . cerevisiae was studied in vivo and it was determined that complementation was allele dependent . However, DNA polymerase delta from S . pombe did not restore growth of a S . cerevisiae strain containing a disrupted chromosomal copy of the POL3 gene that encodes DNA polymerase delta . To identify the regions of DNA polymerase delta responsible for species-specific interactions, we constructed different chimeras with S . cerevisiae and S . pombe DNA polymerase delta genes . The growth of a S . cerevisiae strain with a disrupted chromosomal POL3 gene was studied after transformation with plasmids expressing different chimeras . A 1254-bp region located in the 3' region of the S . cerevisiae POL3 gene is responsible for species-specific functions.

Differentiation, 1995 Jul, 59(1), 51 - 60
Liver fructose-1,6-bisphosphatase cDNA: trans-complementation of fission yeast and characterization of two human transcripts; Bertolotti R et al.; The SV40 early promoter is active both in mammalian cells and in the fission yeast Schizosaccharomyces pombe, and is used to drive full-length cDNA in polyvalent pcD-libraries . Two such liver libraries, of human and rat origin, were used to trans-complement a S . pombe mutant deficient in fructose-1,6-bisphosphatase (Fru-1,6-Pase) activity, a key gluconeogenic enzyme restricted to liver, kidney and intestine in mammals . A rat liver Fru-1,6-Pase cDNA was readily cloned and sequenced . Complementary PCR experiments revealed full-length Fru-1,6-Pase cDNA also present in the human liver library, however at a low abundance . Two human liver transcripts were thus characterized . Contrary to expectation, they were not differentially spliced products . They both encoded the same protein and were generated by a polyadenylation choice mechanism . The longest transcript comprised two polyadenylation signals and a consensus GT-rich element for the 3' processing of the upstream site . Rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) analysis of 3' ends from hepatic, renal and intestinal mRNA disclosed that both Fru-1,6-Pase transcripts are expressed in the three main gluconeogenic cell types and are subject to insulin differential modulation . On the other hand, overcoming liver cell heterogeneity problems, sequence analysis of 16 independent clones of 3' end-cDNA demonstrated that, in addition to a monocytic type corresponding to a previously described lambda gt11 clone, human liver does not contain a hepatic type Fru-1,6-Pase comprising a liver-specific carboxyl-terminal extension like its rat counterpart . This liver-specific extension is involved in enzyme up-regulation and appears to give a conclusive advantage to the rat hepatic enzyme over the human one when trans-complementing mutant yeast.(ABSTRACT TRUNCATED AT 250 WORDS)

Yeast, 1995 Jul, 11(9), 829 - 38
Characterization of Na+/H(+)-antiporter gene closely related to the salt-tolerance of yeast Zygosaccharomyces rouxii; Watanabe Y et al.; In order to clarify the relationship between salt-tolerance of Zygosaccharomyces rouxii and the function of Na+/H(+)-antiporter, a gene was isolated from Z . rouxii which exhibited homology to the Na+/H(+)-antiporter gene (sod2) from Schizosaccharomyces pombe . This newly isolated gene (Z-SOD2) encoded a product of 791 amino acids, which was larger than the product encoded by its Sz . pombe homologue . The predicted amino-acid sequence of Z-Sod2p was highly homologous to that of the Sz . pombe protein, but included an extra-hydrophilic stretch in the C-terminal region . The expression of Z-SOD2 was constitutive and independent of NaCl-shock . Z-SOD2-disruptants of Z . rouxii did not grow in media supplemented with 3 M-NaCl, but grew well in the presence of 50% sorbitol, indicating that the function of Z-SOD2 was closely related to the salt-tolerance of Z . rouxii . Several genes are also compared and discussed in relation to the salt-tolerance of Z . rouxii.

Yeast, 1995 Jul, 11(9), 801 - 8
Biochemical similarity of Schizosaccharomyces pombe ras1 protein with RAS2 protein of Saccharomyces cervisiae; Onozawa T et al.; Schizosaccharomyces pombe contains single ras oncogene homologue, ras1, that functions in the signal transduction pathway conducting the cell's mating processes . To understand the biochemical basis of yeast ras proteins, we have purified the ras1 protein and compared the major biochemical constants with those of RAS2 protein from Saccharomyces cerevisiae and mammalian ras proteins . The purified ras1 protein showed a remarkably high Kd value for GDP binding (178 nM) and for binding with ATP . In contrast, the Kd value for GTP binding and the rate of GTPase activity were 64 nM and 77 x 10(-6) s-1 at 37 degrees C, respectively; both were higher than normal p21ras protein, but at the same level as the RAS2 protein . We directly measured rate of GTP binding and GDP binding which were 3.9 x 10(-3) s-1 and 1.8 x 10(-3) s-1 at 30 degrees C, respectively . On the other hand, exchange rates between bound and free nucleotides remained almost constant throughout the tested combination of GTP and GDP, and were several-fold lower than the binding rate . These results suggest that the release of the guanine nucleotide is the rate-limiting step in the ras-GTP/GDP cycle . As a whole, the biochemical properties of the ras1 protein are close to those of the RAS2 protein, although these two proteins function differently in the signal transduction pathway in the cells.

Mol Gen Genet, 1995 Jun 25, 247(6), 698 - 708
Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease; Rotondo G et al.; The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P . We report here the isolation of the pac1+ gene as a multi-copy suppressor of snm1 . The pac1+ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed . The pac1+ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli . To investigate the essential structural features of the Pac1 RNase, we altered the pac1+ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility . These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347 . These amino acids are conserved in all RNase III-like proteins . The glycine and glutamic acid residues were previously identified as essential for E . coli RNase III activity . The valines are conserved in an element found in a family of double-stranded RNA binding proteins . Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.

Proc Natl Acad Sci U S A, 1995 Jun 20, 92(13), 6180 - 4
Shk1, a homolog of the Saccharomyces cerevisiae Ste20 and mammalian p65PAK protein kinases, is a component of a Ras/Cdc42 signaling module in the fission yeast Schizosaccharomyces pombe; Marcus S et al.; We describe a protein kinase, Shk1, from the fission yeast Schizosaccharomyces pombe, which is structurally related to the Saccharomyces cerevisiae Ste20 and mammalian p65PAK protein kinases . We provide genetic evidence for physical and functional interaction between Shk1 and the Cdc42 GTP-binding protein required for normal cell morphology and mating in S . pombe . We further show that expression of the STE20 gene complements the shk1 null mutation and that Shk1 is capable of signaling to the pheromone-responsive mitogen-activated protein kinase cascade in S . cerevisiae . Our results lead us to propose that signaling modules composed of small GTP-binding proteins and protein kinases related to Shk1, Ste20, and p65PAK, are highly conserved in evolution and participate in both cytoskeletal functions and mitogen-activated protein kinase signaling pathways.

EMBO J, 1995 Jun 15, 14(12), 2760 - 71
A pre-start checkpoint preventing mitosis in fission yeast acts independently of p34cdc2 tyrosine phosphorylation; Hayles J et al.; We have monitored the tyrosine (Y15) phosphorylated and dephosphorylated forms of p34cdc2 from Schizosaccharomyces pombe as cells proceed through the cell cycle . Y15 is dephosphorylated in G1 before start and becomes phosphorylated only after cells pass start and enter late G1 . This transition is associated with a switch from one checkpoint which restrains mitosis in pre-start G1, by a mechanism independent from Y15 phosphorylation, to a second checkpoint acting post-start during late G1 and S phase operating through Y15 phosphorylation . The pre-start checkpoint may act by preventing formation of the p34cdc2/p56cdc13 complex . The complex between Y15-phosphorylated p34cdc2 and p56cdc13 accumulates during S phase and G2, but the level generated is not solely dependent on the amount of p34cdc2 and p56cdc13 present in the cell . The extent of p56cdc13 breakdown at the end of mitosis may be determined by the amount complexed with p34cdc2 . We have also shown that an insoluble form of p34cdc2 is associated with the progression of the cell through late G1 into S phase.

EMBO J, 1995 Jun 15, 14(12), 2745 - 59
The role of Saccharomyces cerevisiae type 2A phosphatase in the actin cytoskeleton and in entry into mitosis; Lin FC et al.; We have prepared a temperature-sensitive Saccharomyces cerevisiae type 2A phosphatase (PP2A) mutant, pph21-102 . At the restrictive temperature, the pph21-102 cells arrested predominantly with small or aberrant buds, and their actin cytoskeleton and chitin deposition were abnormal . The involvement of PP2A in bud growth may be due to the role of PP2A in actin distribution during the cell cycle . Moreover, after a shift to the non-permissive temperature, the pph21-102 cells were blocked in G2 and had low activity of Clb2-Cdc28 kinase . Expression of Clb2 from the S.cerevisiae ADH promoter in pph21-102 cells was able to partially bypass the G2 arrest in the first cell cycle, but was not able to stimulate passage through a second mitosis . These cells had higher total amounts of Clb2-Cdc28 kinase activity, but the Clb2-normalized specific activity was lower in the pph21-102 cells compared with wild-type cells . Unlike wild-type strains, a PP2A-deficient strain was sensitive to the loss of MIH1, which is a homolog of the Schizosaccharomyces pombe mitotic inducer cdc25+ . Furthermore, the cdc28F19 mutation cured the synthetic defects of a PP2A-deficient strain containing a deletion of MIH1 . These results suggest that PP2A is required during G2 for the activation of Clb-Cdc28 kinase complexes for progression into mitosis.

Yeast, 1995 Jun 15, 11(7), 681 - 9
Sequence analysis of a 33.1 kb fragment from the left arm of Saccharomyces cerevisiae chromosome X, including putative proteins with leucine zippers, a fungal Zn(II)2-Cys6 binuclear cluster domain and a putative alpha 2-SCB-alpha 2 binding site; Miosga T et al.; In the framework of the European BIOTECH project for sequencing the Saccharomyces cerevisiae genome, we have determined the nucleotide sequence of the left part of the cosmid clone 232 and the cosmid clone 233 provided by F . Galibert (Rennes Cedex, France) . We present here 33,099 base pairs of sequence derived from the left arm of chromosome X of strain S288C . This sequence reveals 17 open reading frames (ORFs) with more than 299 base pairs, including the published sequences for ARG3, LIGTR/LIG1, ORF2, ACT3 and SCP160 . Two other ORFs showed similarity with S . cerevisiae genes: one with the CAN1 gene coding for an arginine permease, and one with genes encoding the family of transcriptional activators containing a fungal Zn(II)2-Cys6 binuclear cluster domain like that found in Ppr1p or Ga14p . Both putative proteins contain a leucine zipper motif, the Can1p homologue has 12 putative membrane-spanning domains and a putative alpha 2-SCB-alpha 2 binding site . In a diploid disruption mutant of ORF J0922 coding for the transcriptional activator homologue, no colonies appeared before 10 days after transformation and then grew slowly . In contrast, haploid disruption mutants showed a growth phenotype like wild-type cells . One ORF showed weak similarity to the rad4 gene product of Schizosaccharomyces pombe and is essential for yeast growth . Five ORFs showed similarity to putative genes on the right arm of chromosome XI of S . cerevisiae . Two of them have similarity to each other and belong to a family of extracellular proteins that groups mammalian SCP/Tpx-1, insects Ag3/Ag5, plants PR-1 and fungi Sc7/Sc14.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1995 Jun 11, 23(11), 1923 - 7
Site-specific cleavage of chromosomes in vitro through Cre-lox recombination; Qin M et al.; Site-specific recombination systems are useful tools for chromosome engineering in vivo and site-specific DNA cleavage methods have applications in genome analysis and gene isolation . Here, we report a new method to fragment chromosomes in vitro using the Cre-lox site-specific recombination system . Two lox sites were targeted into the 5.7 Mb chromosomes I of Schizosaccharomyces pombe . In vitro recombination between chromosomal lox sites and exogenously provided lox oligonucleotides 'cleaved' the chromosome at the defined lox sequences . Site-specific cleavage of lox sites in the tobacco genome was also demonstrated . This recombination-based cleavage method provides a novel approach for structural and functional analyses of eukaryotic chromosomes as it allows direct isolation of chromosome regions that correspond to phenotypes revealed through Cre-lox mediated chromosome rearrangements in vivo . Moreover, recombination with end-labeled lox oligonucleotides would permit the specific end-labeling of chromosome segments to facilitate the long range mapping of chromosomes.

Nucleic Acids Res, 1995 Jun 11, 23(11), 1912 - 8
gar2 is a nucleolar protein from Schizosaccharomyces pombe required for 18S rRNA and 40S ribosomal subunit accumulation; Gulli MP et al.; Several nucleolar proteins, such as nucleolin, NOP1/fibrillarin, SSB1, NSR1 and GAR1 share a common glycine and arginine rich structural motif called the GAR domain . To identify novel nucleolar proteins from fission yeast we screened Schizosaccharomyces pombe genomic DNA libraries with a probe encompassing the GAR structural motif . Here we report the identification and characterization of a S.pombe gene coding for a novel nucleolar protein, designated gar2 . The structure of the fission yeast gar2 is reminiscent of that of nucleolin from vertebrates and NSR1 from Saccharomyces cerevisiae . In addition, like these proteins, gar2 has a nucleolar localisation . The disruption of the gar2+ gene affects normal cell growth, leads to an accumulation of 35S pre-rRNA and a decrease of mature 18S rRNA steady state levels . Moreover, ribosomal profiles of the mutant show an increase of free 60S ribosomal subunits and an absence of free 40S ribosomal subunits . gar2 is able to rescue a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSR1 . We propose that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA.

Curr Genet, 1995 Jun, 28(1), 32 - 8
Schizosaccharomyces pombe pac2+ controls the onset of sexual development via a pathway independent of the cAMP cascade; Kunitomo H et al.; The Schizosaccharomyces pombe pac2 gene encodes a protein of 235 amino acids not similar to any protein of known function . Cells over-expressing pac2 were poor in mating and sporulation . Expression of ste11, which encodes a key transcription factor for sexual development, was not inducible by nitrogen starvation in these cells . Cells defective in pac2 could express ste11 and enter sexual development under incomplete starvation conditions . Although expression of ste11 is regulated primarily by the cAMP cascade, genetic analysis indicated that this cascade and pac2 can partially compensate for each other in the regulation of sexual development, and that neither of them is epistatic over the other . Thus, Pac2 appears to control ste11 expression via a signaling pathway independent of the cAMP cascade.

Curr Microbiol, 1995 Jun, 30(6), 367 - 72
Glutamine synthetase/glutamate synthase ammonium-assimilating pathway in Schizosaccharomyces pombe; Perysinakis A et al.; Kinetic parameters of glutamine synthetase (GS) and glutamate synthase (glutamine-oxoglutarate aminotransferase) (GOGAT) activities, including initial velocity, pH, and temperature optima, as well as Km values, were estimated in Schizosaccharomyces pombe crude cell-free extracts . Five glutamine auxotrophic mutants of S . pombe were isolated following MNNG treatment . These were designated gln1-1,2,3,4,5, and their growth could be repaired only by glutamine . Mutants gln1-1,2,3,4,5 were found to lack GS activity, but retained wild-type levels of NADP-glutamate dehydrogenase (GDH), NAD-GDH, and GOGAT . One further glutamine auxotrophic mutant, gln1-6, was isolated and found to lack both GS and GOGAT but retained wild-type levels of NADP-GDH and NAD-GDH activities . Fortuitously, this isolate was found to harbor an unlinked second mutation (designated gog1-1), which resulted in complete loss of GOGAT activity but retained wild-type GS activity . The growth phenotype of mutant gog1-1 (in the absence of the gln1-6 mutation) was found to be indistinguishable from the wild type on various nitrogen sources, including ammonium as a sole nitrogen source . Double-mutant strains containing gog1-1 and gdh1-1 or gdh2-1 (mutations that result specifically in the abolition of NADP-GDH activity) result in a complete lack of growth on ammonium as sole nitrogen source in contrast to gdh or gog mutants alone.

Mol Cell Biol, 1995 Jun, 15(6), 3310 - 7
A novel mechanism of self-primed reverse transcription defines a new family of retroelements; Levin HL; Retroviruses and long terminal repeat (LTR)-containing retrotransposons initiate reverse transcription by using a specific tRNA primer than anneals to the primer-binding site of the retroelement transcript . Sequences from a large number of retroviruses and LTR-containing retrotransposons had indicated that the role of tRNAs in priming reverse transcription is universal among these LTR-containing retroelements . Data presented here strongly support the surprising conclusion that Tf1, a highly active LTR-containing retrotransposon isolated from Schizosaccharomyces pombe, undergoes a novel self-priming process that requires hybridization between the primer-binding site and the first 11 bases of the Tf1 transcript . Single-base mutations in these regions block transposition and reverse transcription, while compensatory mutations that reestablish complementarily rescue both defects . In addition, the sequence of the minus-strand RNA primer of reverse transcription was consistent with its being derived from the 5' end of the Tf1 transcript . Evidence that this mechanism defines a new family of retroelements is presented.

Genetics, 1995 Jun, 140(2), 469 - 78
Hot spots of recombination in fission yeast: inactivation of the M26 hot spot by deletion of the ade6 promoter and the novel hotspot ura4-aim; Zahn-Zabal M et al.; The M26 mutation in the ade6 gene of Schizosaccharomyces pombe creates a hot spot of meiotic recombination . A single base substitution, the M26 mutation is situated within the open reading frame, near the 5' end . It has previously been shown that the heptanucleotide sequence 5' ATGACGT 3', which includes the M26 mutation, is required for hot spot activity . The 510-bp ade6-delXB deletion encompasses the promoter and the first 23 bp of the open reading frame, ending 112 bp upstream of M26 . Deletion of the promoter in cis to M26 abolishes hot spot activity, while deletion in trans to M26 has no effect . Homozygous deletion of the promoter also eliminates M26 hot spot activity, indicating that the heterology created through deletion of the promoter per se is not responsible for the loss of hot spot activity . Thus, DNA sequences other than the heptanucleotide 5' ATGACGT 3', which must be located at the 5' end of the ade6 gene, appear to be required for hot spot activity . While the M26 hotspot stimulates crossovers associated with M26 conversion, it does not affect the crossover frequency in the intervals adjacent to ade6 . The flanking marker ura4-aim, a heterology created by insertion of the ura4+ gene upstream of ade6, turned out to be a hot spot itself . It shows disparity of conversion with preferential loss of the insertion . The frequency of conversion at ura4-aim is reduced when the M26 hot spot is active 15 kb away, indicating competition for recombination factors by hot spots in close proximity.

Genetics, 1995 Jun, 140(2), 457 - 67
sck1, a high copy number suppressor of defects in the cAMP-dependent protein kinase pathway in fission yeast, encodes a protein homologous to the Saccharomyces cerevisiae SCH9 kinase; Jin M et al.; Schizosaccharomyces pombe regulates intracellular cAMP levels, and thus cAMP-dependent protein kinase (PKA) activity, in response to changes in nutrient conditions . Mutations in any of eight git genes inhibit glucose repression of fbp1 transcription, alter the cell morphology, and cause a reduction in the growth rate . The eight git genes encode components of an adenylate cyclase activation pathway, adenylate cyclase itself, and the catalytic subunit of PKA . Three of these genes have been identified in other studies as regulators of meiosis . Here we show that the sck1 gene, cloned as a high copy number suppressor of a mutation in git3, is able to suppress the defects conferred by a mutation in any of these git genes . Sequence analysis suggests that sck1 encodes a protein most closely related to the Saccharomyces cerevisiae SCH9 protein kinase that had previously been identified as a high copy number suppressor of mutations in S . cerevisiae that reduce or eliminate PKA activity . Disruption of the sck1 gene causes a significant delay in exit from stationary phase when combined with a disruption of the pka1 (git6) gene encoding the catalytic subunit of PKA . However, the sck1 disruption by itself has little or no effect upon fbp1 transcription, meiosis, or exit from stationary phase, and does not enhance the constitutive fbp1 transcription observed in a pka1 mutant . Therefore, sck1 appears to function in a redundant fashion to pka1, but to varying degrees, in the pathways regulated by pka1.

J Biol Chem, 1995 May 19, 270(20), 11860 - 5
RNA1 encodes a GTPase-activating protein specific for Gsp1p, the Ran/TC4 homologue of Saccharomyces cerevisiae; Becker J et al.; Ran/TC4 is a ras-related GTP-binding protein predominantly located in the nucleus . Ran/TC4 is essential for nuclear transport and is involved in mitotic control . In Saccharomyces cerevisiae a gene highly homologous to Ran/TC4 has been identified and named GSP1 . Like all ras-related GTP-binding proteins, Gsp1p undergoes cycles of GTP hydrolysis and GDP/GTP exchange . The switching between the two different nucleotide bound states regulates the function of these GTP-binding proteins . Here we identify the product of the yeast RNA1 gene as the GTPase-activating protein (GAP) of Gsp1p . RNA1 belongs to a group of genes which are conserved in a variety of different organisms . We have expressed and purified recombinant Gsp1p and Rna1p from Escherichia coli . The GTPase activity of Gsp1p is stimulated 10(7)-fold by Rna1p . In addition, we find that the previously identified human RanGAP1 and rna1p from Schizosaccharomyces pombe are also able to induce GTPase activity of Gsp1p . The GTP hydrolysis of Ran is induced by RanGAP1 and rna1p but not by Rna1p . Implications for the suggested functions of Ran/TC4/Gsp1p in nuclear transport and mitotic control are discussed.

Biochim Biophys Acta, 1995 May 17, 1262(1), 87 - 90
A cDNA of Schizosaccharomyces pombe encoding a homologue of DnaJ-like protein; Park SK et al.; A Schizosaccharomyces pombe homologue, Psi, was cloned from Schizosaccharomyces pombe cDNA library . Deduced amino acid sequence of the cDNA has 55% sequence homology with the Saccharomyces cerevisiae Sis1 protein and contains the structural features of a family of DnaJ proteins . This homology suggests Psi protein may be implicated in the initiation of translation as like Sis1 function of Saccharomyces cerevisiae.

Biochem Pharmacol, 1995 May 17, 49(10), 1395 - 401
Rescue of Schizosaccharomyces pombe from camptothecin-mediated death by a DNA topoisomerase I inhibitor, TAN-1518 A; Horiguchi T et al.; TAN-1518 A is a cytotoxic agent with suppressive activity against Meth A fibrosarcoma in vivo . This compound inhibits calf thymus DNA topoisomerase I (Topo I) but does not stimulate cleavable complex formation in the nuclei of Chinese hamster ovary (CHO)-K1 cells, suggesting that it inhibits Topo I in a manner different from that of camptothecin (CPT) . To clarify the mode of action of TAN-1518 A, we examined its effects on the eukaryotic microorganism Schizosaccharomyces pombe (S . pombe), which does not require Topo I as an essential factor for growth . TAN-1518 A inhibited purified S . pombe Topo I as potently as did CPT . TAN-1518 A, unlike CPT, did not stimulate Topo I-induced DNA cleavage; instead, it inhibited CPT-induced cleavable complex formation . We constructed a S . pombe strain, IR9, that produced excess Topo I . IR9 was hypersensitive to CPT, but its growth was not affected by TAN-1518 A . The CPT-mediated death of IR9 cells was reduced dramatically in the presence of TAN-1518 A . These findings clearly demonstrate that TAN-1518 A is a specific inhibitor of Topo I in eukaryotic cells and also suggest that this agent inhibits some earlier step(s) that occurs before the formation of cleavable complex on DNA strands in the catalytic cycle of this enzyme.

J Biol Chem, 1995 May 12, 270(19), 11298 - 303
Isolation of Schizosaccharomyces pombe isopentenyl diphosphate isomerase cDNA clones by complementation and synthesis of the enzyme in Escherichia coli; Hahn FM et al.; Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprene biosynthetic pathway . The Saccharomyces cerevisiae gene for IPP isomerase, IDI1, was recently isolated and characterized (Anderson, M . S., Muehlbacher, M., Street, I . P., Proffitt, J., and Poulter, C . D . (1989) J . Biol . Chem . 264, 19169-19175), and the wild-type gene, IDI1, was disrupted with a LEU2 marker to create a diploid yeast strain heterozygous for the idi1::leu2 disruption, which revealed that IDI1 was an essential single-copy gene (Mayer, M.P., Hahn, F . M., Stillman, D . J., and Poulter, C . D . (1992) Yeast 8, 743-748) . We now report the isolation of a cDNA clone from Schizosaccharomyces pombe by a plasmid shuffle-mediated complementation of the LEU2 disrupted yeast gene . The S . pombe clone encoded a 26,864-dalton polypeptide of 227 amino acids with a high degree of similarity to the S . cerevisiae IDI1 enzyme . S . pombe IPP isomerase contained the essential Cys and Glu catalytic residues identified in yeast isomerase (Street, I . P., Coffman, H . R., Baker, J., and Poulter, C . (1994) Biochemistry 33, 4212-4217) but was significantly smaller than the S . cerevisiae enzyme . The plasmid shuffle technique is an excellent procedure for screening expression libraries for IPP isomerase activity by complementation of the idi1 mutation.

Biochim Biophys Acta, 1995 May 10, 1229(3), 386 - 8
cDNA sequence of subunit VIII of ubiquinol-cytochrome-c oxidoreductase from Schizosaccharomyces pombe; Boumans H et al.; We have cloned a cDNA coding for subunit VIII of the ubiquinol-cytochrome-c oxidoreductase of Schizosaccharomyces pombe by functional complementation of the null mutant in the QCR8 gene of Saccharomyces cerevisiae . DNA sequence analysis reveals an open-reading frame of 276 bp encoding a 10.5 kDa protein with 51% amino acid sequence identity to its counterpart in S . cerevisiae.

DNA Cell Biol, 1995 May, 14(5), 359 - 71
Schizosaccharomyces pombe: a model for molecular studies of eukaryotic genes; Zhao Y et al.; Several features of the fission yeast Schizosaccharomyces pombe make it exceptionally well suited for the study of eukaryotic genes . It is a relatively simple eukaryote that can be readily grown and manipulated in the laboratory, using a variety of highly developed and sophisticated methodologies . Schizosaccharomyces pombe cells share many molecular, genetic, and biochemical features with cells from multicellular organisms, making it a particularly useful model to study the structure, function, and regulation of genes from more complex species . For examples, this yeast divides by binary fission, has many genes that contain introns, is capable of using mammalian gene promoters and polyadenylation signals, and has been used to clone mammalian genes by functional complementation of mutants . We present a summary of the biology of S . pombe, useful features that make it amenable to laboratory studies, and molecular techniques available to manipulate the genome of this organism as well as other eukaryotic genes within the fission yeast cellular environment.

Genes Dev, 1995 May 1, 9(9), 1059 - 73
The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells; Ohkura H et al.; We have identified a Schizosaccharomyces pombe gene with homology to the budding yeast gene CDC5, the Drosophila gene polo, and the mammalian family of genes encoding polo-like kinases . Disruption of this gene, plo1+, indicates that it is essential . Loss of plo1+ function leads to a mitotic arrest in which condensed chromosomes are associated with a monopolar spindle or to the failure of septation following the completion of nuclear division . In the latter case, cells show a failure both in the formation of an F-actin ring and in the deposition of septal material, suggesting that plo1+ function is required high in the regulatory cascade that controls septation . The overexpression of plo1+ in wild-type cells also results in the formation of monopolar spindles but also induces the formation of multiple septa without nuclear division . Septation can also be induced in the absence of mitotic commitment and concomitant spindle formation by the overexpression of plo1+ in cdc25-22 or cdc2-33 cells arrested in G2; in G1 cells arrested at Start by the cdc10-V50 mutation, or in cells lacking the cyclin B homolog cdc13 that undergo repeated S phases in the absence of mitosis.

Mol Cell Biol, 1995 May, 15(5), 2589 - 99
The Schizosaccharomyces pombe MBF complex requires heterodimerization for entry into S phase; Ayte J et al.; In Schizosaccharomyces pombe, MBF is a DNA-binding complex suspected to activate the transcription of genes necessary for entry into S phase . The MBF complex contains both p85cdc10 and p72res1/sct1 . To obtain a better understanding of how the MBF complex regulates gene expression at the G1/S transition, we have performed a genetic analysis of p72res1 . We determined that p72res1 can bind specifically to the cdc22 promoter, when analyzed by gel mobility shift assay, and that the N-terminal 157 amino acids of p72res1 are sufficient for this specific binding . When overexpressed in vivo, a fragment of p72res1 containing this DNA-binding domain could rescue a strain carrying a temperature-sensitive cdc10 allele at the restrictive temperature as well as a strain with a cdc10 null allele . We also determined that the C-terminal region of p72res1 is necessary and sufficient for binding to p85cdc10 . Overexpression of the cdc10-binding domain of p72res1 leads to a G1 arrest with a cdc phenotype and a decrease on MBF activity . Overexpression of full-length p72res1 also leads to a growth arrest that can be rescued by overexpression of p85cdc10 . These results imply that the MBF activity in vivo is dependent on the interaction of p85cdc10 with p72res1.

Mol Biol Cell, 1995 May, 6(5), 485 - 96
Isolation of protein glycosylation mutants in the fission yeast Schizosaccharomyces pombe; Huang KM et al.; We have isolated mutants in the fission yeast Schizosaccharomyces pombe that are defective in protein glycosylation . A collection of osmotically sensitive mutants was prepared and screened for glycosylation defects using lectin staining as an assay . Mutants singly defective in four glycoprotein synthesis genes (gps1-4) were isolated, all of which bind less galactose-specific lectin . Acid phosphatase and other glycoproteins from the gps mutants have increased electrophoretic mobility, suggesting that these mutants make glycans of reduced size . N-linked glycan analysis revealed that terminal oligosaccharide modification is defective in the gps1 and gps2 mutants . Both mutants synthesize the Man9GlcNAc2 core glycan but have reduced amounts of larger structures . Modified core glycans from gps1 cells have normal amounts of galactose (Gal) residues, but reduced amounts of Man, consistent with a defect in a Golgi mannosyltransferase in this mutant . In contrast, N-linked oligosaccharides from gps2 mutants have much less Gal than wild type, because of reduced levels of the Gal donor, UDP-Gal . This reduction is caused by decreased activity of UDP-glucose 4-epimerase, which synthesizes UDP-Gal . Neither the gps1 or gps2 mutations are lethal, although the cells grow at reduced rates . These findings suggest that S . pombe cells can survive with incompletely glycosylated cell wall glycoproteins . In particular, these results suggest that Gal, which comprises approximately 30% by weight of cell wall glycoprotein glycans, is not crucial for cell growth or survival.

Int Arch Allergy Immunol, 1995 May-Jun, 107(1-3), 298 - 300
Identification of the allergen Psi c 2 from the basidiomycete Psilocybe cubensis as a fungal cyclophilin; Horner WE et al.; Basidiospores are a prevalent and frequent cause of respiratory allergies, yet their allergens remain poorly defined; thus, we have attempted a molecular characterization of representative basidiomycete allergens . A Psilocybe cubensis mycelial cDNA library was immunoscreened with patient serum . A clone was isolated that expressed a 23-kD recombinant allergen as a fusion protein and inhibited a 16-kD band (Psi c 2) in immunoprints of P . cubenis extract, indicating antigenic identity . Sequence (cDNA) analysis of the clone indicates homology with cyclophilin and the deduced amino acid sequence of Psi c 2 showed 78% identity and 4% similarity with the amino acid sequence of Schizosaccharomyces pombe cyclophilin . This recombinant allergen is a useful model for epitope analysis of basidiospore allergens and fungal allergen cross-reactivity, and may provide an improved reagent for basidiospore allergy diagnosis and treatment.

FEMS Microbiol Lett, 1995 May 1, 128(2), 201 - 6
A rapid permeabilization procedure for accurate quantitative determination of beta-galactosidase activity in yeast cells; Kippert F; A procedure is described which allows the rapid permeabilization of yeast cells, Schizosaccharomyces pombe and Saccharomyces cerevisiae, for quantitative in situ assays of beta-galactosidase activity . Yeast cells are permeabilized by incubation in buffer containing 0.2% of the detergent sodium lauroyl sarcosinate without any need for washing or vortexing . This procedure is equally applicable to fresh and frozen samples . It is compared to earlier reported methods and found to be superior by being more accurate and less time-consuming.

Nature, 1995 Apr 27, 374(6525), 817 - 9
A kinase from fission yeast responsible for blocking mitosis in S phase; Murakami H et al.; In virtually all eukaryotes, mitosis starts after the completion of DNA synthesis . This orderly process is ensured by the checkpoint mechanism that blocks the onset of mitosis while DNA is being synthesized or is damaged . In the fission yeast Schizosaccharomyces pombe, this mechanism involves some rad+ and hus+ genes . However, it is not known how the checkpoint system monitors these events . Recently a multicopy suppressor of a temperature-sensitive DNA polymerase-alpha mutant was isolated . This gene, named cds1+ (checking DNA synthesis), encodes a typical protein kinase . Here we report that this protein kinase is a key component of the DNA replication-monitoring S/G2 checkpoint system . Our data suggest that its primary role is to monitor DNA synthesis by interacting with DNA polymerase alpha and send a signal to block the onset of mitosis while DNA synthesis is in progress.

Biochim Biophys Acta, 1995 Apr 26, 1229(2), 233 - 8
Functional expression of the ENA1(PMR2)-ATPase of Saccharomyces cerevisiae in Schizosaccharomyces pombe; Banuelos MA et al.; Na+ efflux and Na+ tolerance depend on a putative P-type ATPase encoded by the gene ENA1(PMR2) in Saccharomyces cerevisiae and on a putative Na+/H+ antiporter encoded by the gene sod2 in Schizosaccharomyces pombe . This report shows that a sod2::ura4 mutant of S . pombe transformed with the ENA1 gene of S . cerevisiae expressed the ENA1 protein, and recovered Na+ efflux and Na+ tolerance . The efflux of Na+ in the wild strain of S . pombe was sensitive to the transmembrane Na+ and H+ gradients, whereas in the sod2::ura4 mutant transformed with ENA1 it was independent of these gradients . The data give further support to the notion that ENA1 and sod2 encode Na+ transporters and not regulators of the process of Na+ export; they show also the physiological consequences of exporting Na+ through an Na(+)-ATPase or an Na+/H+ antiporter.

J Biol Chem, 1995 Apr 21, 270(16), 9178 - 84
Cloning and expression of a yeast gene encoding a protein with ATPase activity and high identity to the subunit 4 of the human 26 S protease; Lucero HA et al.; The cloning, expression, and biochemical characterization of an essential gene of Saccharomyces cerevisiae that encodes for a new member of the TBP1-like subfamily of putative ATPases are described . The protein is 72% identical at the amino acid level to subunit four (S4) of the human 26 S protease and 73% identical to Schizosaccharomyces pombe MTS2 gene product . The purified, recombinant protein, designated Yhs4p, has an estimated molecular mass of 49 kDa and exhibits a Mg(2+)-dependent ATPase activity with nucleotide specificity and Km for ATP similar to those exhibited by the human 26 S protease . The observed ATPase activity was reduced by 73% upon the introduction of point mutation K229Q in the "P-loop" domain of the ATP-binding site relative to the nonmutated form of the protein . This is the first direct biochemical evidence supporting the putative ATPase activity of a member of the TBP1-like subfamily . Furthermore, the experimental results demonstrate a regulatory function for the amino-terminal region of the molecule . The amino-terminal truncated form of Yhs4p lacking two clusters of positively charged amino acids exhibits a greater ATPase activity . The ATPase activity of both the truncated and complete forms of Yhs4p is stimulated by polyanions . Polylysine partially inhibits the ATPase activity of the amino-terminal truncated form having no observable effect on the complete protein . N-Ethylmaleimide inhibits the ATPase activity of both forms of Yhs4p . We propose that Yhs4p ATPase may play an essential role in the regulatory function of the proteolytic activity of the yeast 26 S protease.

Mol Gen Genet, 1995 Apr 20, 247(2), 247 - 54
Characterisation of Saccharomyces cerevisiae genes encoding ribosomal protein YL6; Moore J et al.; We have characterised a Saccharomyces cerevisiae cDNA (cDNA13), originally isolated on the basis of the short half-life of the corresponding mRNA . We show here that its sequence is closely related to that of the genes encoding ribosomal proteins K37, KD4 and K5 of Schizosaccharomyces pombe . 'mRNA13' also behaves like other mRNAs encoding ribosomal proteins, in that its abundance increases sharply when glucose is added to cells grown on ethanol (nutrient-upshift), and declines when cells are subjected to a mild heat-shock . Unspliced mRNA13 accumulates when cells bearing a temperature-sensitive splicing mutation are grown at the restrictive temperature . The gene(s) corresponding to cDNA13, like other ribosomal protein genes of S . cerevisiae, thus contain an intron . Southern blot analysis indicates the presence of two separate loci related to cDNA13 in the S . cerevisiae genome . From the sequence of one of these, a complete polypeptide sequence was deduced . The first 40 amino acids are identical to those of YL6, a S . cerevisiae ribosomal protein characterised only by N-terminal protein sequence analysis . There is clear evidence within the genomic sequence for the predicted intron, and for elements similar to those that regulate expression of other S . cerevisiae ribosomal protein genes.

Structure, 1995 Apr 15, 3(4), 321 - 5
The cell cycle and suc1: from structure to function?
Endicott JA, Nurse P.
Structures have recently been determined for the yeast Schizosaccharomyces pombe cell cycle regulatory protein, CKS/suc1, and its human equivalent . The structures provide some long-awaited clues about the role of CKS/suc1 in cell cycle control.

Biochim Biophys Acta, 1995 Apr 4, 1261(2), 319 - 20
Nucleotide sequence of a Aspergillus parasiticus gene strongly repressed by thiamine; Cary JW et al.; A cDNA clone demonstrating a high degree of homology to the thiamine repressed nmt1 gene of Schizosaccharomyces pombe was isolated from the aflatoxigenic fungus, Aspergillus parasiticus . The deduced polypeptide of a cDNA clone from A . parasiticus had an amino acid sequence identity of 60% with that of the nmt1 gene of S . pombe . Transcription of the nmt1 gene homolog in the fungus was strongly inhibited by concentrations of thiamine of 2.0 microM or higher.

Mol Cell Biol, 1995 Apr, 15(4), 2028 - 36
p13suc1 of Schizosaccharomyces pombe regulates two distinct forms of the mitotic cdc2 kinase; Basi G et al.; suc1 is an essential gene initially identified for its ability to rescue certain temperature-sensitive alleles of cdc2 in Schizosaccharomyces pombe . The role of suc1 in the regulation of the cdc2 kinase is not well understood . In our study, we have characterized the biochemical effect of loss of suc1 function on specific cdc2-cyclin complexes . We show that the cig1 cyclin is associated with cdc2 and that the cdc2-cig1 kinase is activated at mitosis, with kinetics similar to those of the cdc2-cdc13 kinase . We provide evidence that loss of suc1 function affects the kinase activity of the two distinct mitotic forms of the cdc2 kinase . We also show that a dramatic increase in the level of the cdc13 protein is associated with loss of suc1 . These results suggest that mitosis cannot be properly completed in the absence of suc1, possibly because of an increase in the level of cdc2-cdc13 complex, and support the idea of a role for suc1 in the regulation of multiple forms of the cdc2 kinase.

Microbiology, 1995 Apr, 141 ( Pt 4), 883 - 90
A temperature-compensated ultradian clock ticks in Schizosaccharomyces pombe; Kippert F et al.; An ultradian oscillation is described for Schizosaccharomyces pombe which meets the criteria for a cellular clock, i.e . timekeeping device . The rhythm can be induced by transfer from circadian conditions (stationary phase or very slow growth) to ultradian conditions (rapid growth) . It can also be synchronized by ultradian temperature cycles of 6 degrees C difference . Released to constant temperature, the rhythm persists for 20 h without damping . The period of the free-running rhythm is temperature-compensated and in no experiment did period length fall outside the narrow range between 40 and 44 min . The parameter observed is the septum index, i.e . the percentage of cells occupying the last stage of the cell cycle in wild-type cells before final division . The results suggest control of the cell division processes by the ultradian clock.

Biosci Biotechnol Biochem, 1995 Apr, 59(4), 678 - 82
Phosphatidylinositol-3 kinase in fission yeast: a possible role in stress responses; Kimura K et al.; A DNA fragment coding for a part of a putative phosphatidylinositol 3 kinase was cloned from Schizosaccharomyces pombe by cross-hybridization with Saccharomyces cerevisiae VPS34 gene, a yeast homologue of mammalian PI-3 kinase . The clone contained an open reading frame of 797 amino acids but lacked the initiation codon, ATG . The predicted amino acid sequence was homologous to those of S . cerevisiae VPS34 and mammalian PI-3 kinase genes . Disruption of the gene resulted in extremely low levels of PI-3-P and higher levels of PI-4-P, supporting the idea that the gene codes for the PI-3 kinase of S . pombe . The disruptants harbored large vacuoles and were sensitive to stresses such as high temperature or high concentration of monovalent and divalent cations.

Mol Biol Cell, 1995 Apr, 6(4), 371 - 85
The Wee1 protein kinase regulates T14 phosphorylation of fission yeast Cdc2; Den Haese GJ et al.; The Cdc2 protein kinase is a key regulator of the G1-S and G2-M cell cycle transitions in the fission yeast Schizosaccharomyces pombe . The activation of Cdc2 at the G2-M transition is triggered by dephosphorylation at a conserved tyrosine residue Y15 . The level of Y15 phosphorylation is controlled by the Wee1 and Mik1 protein kinases acting in opposition to the Cdc25 protein phosphatase . Here, we demonstrate that Wee1 overexpression leads to a high stoichiometry of phosphorylation at a previously undetected site in S . pombe Cdc2, T14 . T14 phosphorylation was also detected in certain cell cycle mutants blocked in progression through S phase, indicating that T14 phosphorylation might normally occur at low stoichiometry during DNA replication or early G2 . Strains in which the chromosomal copy of cdc2 was replaced with either a T14A or a T14S mutant allele were generated and the phenotypes of these strains are consistent with T14 phosphorylation playing an inhibitory role in the activation of Cdc2 as it does in higher eukaryotes . We have also obtained evidence that Wee1 but not Mik1 or Chk1 is required for phosphorylation at this site, that the Mik1 and Chk1 protein kinases are unable to drive T14 phosphorylation in vivo, that residue 14 phosphorylation requires previous phosphorylation at Y15, and that the T14A mutant, unlike Y15F, is recessive to wild-type Cdc2 activity . Finally, the normal duration of G2 delay after irradiation or hydroxyurea treatment in a T14A mutant strain indicates that T14 phosphorylation is not required for the DNA damage or replication checkpoint controls.

Curr Opin Genet Dev, 1995 Apr, 5(2), 162 - 7
Eukaryotic replicators and associated protein complexes; Bell SP; In the past year, genetic studies have provided a detailed understanding of the DNA sequence elements that constitute Saccharomyces cerevisiae origins of DNA replication and have identified larger DNA domains that direct DNA replication in both Schizosaccharomyces pombe and human cells . In vivo studies of the proteins associated with S . cerevisiae origins of DNA replication indicate that there are dynamic changes in origin chromatin structure during the cell cycle and suggest that the Cdc7 protein kinase is among the associated proteins.

Curr Genet, 1995 Apr, 27(5), 447 - 50
Electrophoretic karyotype of the astaxanthin-producing yeast Phaffia rhodozyma; Adrio JL et al.; The electrophoretic karyotype of three different strains of Phaffia rhodozyma was determined by contour-clamped homogeneous electric field (CHEF)-gel electrophoresis . Significant differences in electrophoretic karyotyping patterns were found among the three strains studied . Between nine and 17 bands were observed . The size of these bands, based on their migration relative to the chromosomal DNA of Schizosaccharomyces pombe, Hansenula wingei was estimated to be between 0.48 and 3.1 Mb.

Curr Genet, 1995 Apr, 27(5), 440 - 6
Molecular cloning of the meiosis-induced rec10 gene of Schizosaccharomyces pombe; Lin Y et al.; The meiotic recombination gene rec10, which encodes a region-specific activator of recombination in Schizosaccharomyces pombe, has been cloned by genetic complementation and its nucleotide sequence determined . The rec10 gene was identified in a 5.6-kb cloned fragment by partial-deletion and insertion experiments . The nucleotide sequence of 3.5 kb of this clone revealed an open reading frame (ORF) encoding a 791 amino-acid polypeptide for the rec10 gene product . During meiosis, thermally induced in a temperature-sensitive pat1-114 mutant, the transcript of rec10 was induced to a maximal level at 2-3 h but was present at much lower levels before and after this time . The transient induction of the rec10 transcript and the rec10 mutant phenotype suggest that the rec10 gene product is involved primarily in the early steps of meiotic recombination localized to chromosome III in S . pombe.

Semin Cell Biol, 1995 Apr, 6(2), 95 - 104
Pheromone communication in the fission yeast Schizosaccharomyces pombe; Nielsen O et al.; Conjugation between two haploid yeast cells is generally controlled by the reciprocal action of diffusible mating pheromones, cells of each mating type releasing pheromones that induce mating-specific changes in cells of the opposite type . Recent studies into pheromone signalling in the fission yeast Schizosaccharomyces pombe have revealed significant parallels with processes in higher eukaryotes and could provide the opportunity for investigating communication in an organism that is amenable to both biochemical and genetic manipulation.

Semin Cell Biol, 1995 Apr, 6(2), 79 - 87
The control of septum formation and cytokinesis in fission yeast; Simanis V; Our understanding of the control of cytokinesis is limited in comparison with our knowledge of the controls over the initiation of S phase or mitosis . Study of genetically tractable systems such as Schizosaccharomyces pombe are a useful way to address this problem, since mutants defective in regulation of cytokinesis have been identified . Cloning and analysis of the proteins they encode has begun to shed light upon how formation of the division septum is initiated and directed to the correct place in the cell . Some of these mutants may also be implicated in coordinating mitosis and cytokinesis.

Semin Cell Biol, 1995 Apr, 6(2), 73 - 8
Cyclins of the fission yeast Schizosaccharomyces pombe; Fisher D et al.; Five cyclin-like genes, cig1, cig2/cyc17, mcs2, puc1 and cdc13, have been discovered in S . pombe to date . It is not yet clear what their functions are or even whether they are all involved with control of the cell cycle . Conflicting data for cig1 and cig2/cyc17 have obscured analysis of their function and cig1 remains largely uncharacterized, although clues to the role of cig2/cyc17 have emerged . There is genetic data available for the more distant cyclin homologue mcs2, which has an essential although as yet unspecified role . Puc1 may be involved in regulation of exit from the cell cycle . The first cyclin to be discovered, and the best understood, is cdc13 which with cdc2 promotes mitosis . Studies of the roles of cdc2 and cdc13 in the overall ordering of the cell cycle suggest that cdc13 and probably other cyclins are key regulators, maintaining the order of S phase and mitosis during the cell cycle.

J Biol Chem, 1995 Mar 31, 270(13), 7703 - 11
The rad21 gene product of Schizosaccharomyces pombe is a nuclear, cell cycle-regulated phosphoprotein; Birkenbihl RP et al.; The rad21 gene of Schizosaccharomyces pombe is involved in the repair of double-strand breaks in DNA and is essential for mitotic growth (Birkenbihl, R . P., and Subramani, S . (1992) Nucleic Acids Res . 20, 6605-6611) . We show that the Rad21 protein migrates with an aberrantly slow mobility, has a thrombin cleavage site, and is multiply phosphorylated mainly at serine residues . The expression of the rad21 mRNA and the Rad21 protein is cell cycle-regulated, with the peak of mRNA and protein expression occurring near the G1 to S transition . Following translation of the protein, hypophosphorylated forms of the protein appear . However, the most phosphorylated form of Rad21 appears only later in the cell cycle (in S to G2) . Analysis of the radiosensitive mutant rad21-45 revealed that the mutant protein is permanently hypophosphorylated . The Rad21 protein is nuclear during the cell cycle . The nuclear localization signal was identified in the C-terminal third of the protein . Upon repression of the Rad21 protein expressed from the repressible nmt1 promoter, the unphosphorylated and hypophosphorylated forms of Rad21 disappeared first . When the concentration of the most highly phosphorylated form of Rad21 sank under a critical level, the cells underwent aberrant mitoses . They exhibited loss of proper nuclear organization and abnormal septation.

J Biol Chem, 1995 Mar 31, 270(13), 7411 - 9
A mutation in the Schizosaccharomyces pombe rae1 gene causes defects in poly(A)+ RNA export and in the cytoskeleton; Brown JA et al.; A collection of fission yeast Schizosaccharomyces pombe conditional mutants was screened for defective nucleocytoplasmic transport of poly(A)+ RNA by fluorescence in situ hybridization . We identified a temperature-sensitive mutant that accumulated poly(A)+ RNA in the nucleus and have named it rae1-1, for ribonucleic acid export . All rae1-1 cells exhibit the defect in poly(A)+ RNA export within 30 min following a shift to the non-permissive temperature . In addition, in the rae1-1 mutant, actin and tubulin become disorganized, and cells undergo an irreversible cycle arrest . Results from experiments in which rae1-1 cells were arrested in various phases of the cell division cycle and then shifted to nonpermissive temperature suggest that cells are particularly vulnerable to loss of rae1 function during G2/M . However, the inability to export RNA from the nucleus to the cytoplasm was not limited to a particular phase of the cell division cycle . The rae1 gene was isolated by complementation and encodes a predicted protein of 352 amino acids with four beta-transducin/WD40 repeats.

Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 2790 - 4
Schizosaccharomyces pombe mutants that are defective in glycoprotein galactosylation; Ballou L et al.; Several mutants of Schizosaccharomyces pombe were obtained that are defective in protein glycosylation . One of the mutants, strain Sp550, makes galactomannoproteins with about half of the wild-type amount of galactose, whereas another strain, Sp137, makes glycoproteins that are almost devoid of galactose . Nondenaturing gel electrophoresis of cell extracts of both mutants revealed that they make invertases with a greatly increased mobility relative to the wild type . Additional study showed that Sp137 invertase has a subunit molecular mass that is about half that reported for the wild-type enzyme, owing to a reduction in carbohydrate content, whereas the native multimeric state appears unaltered . Structural studies on bulk cell-wall glycoprotein from Sp137 showed that the N-linked carbohydrate chains consist of a typical branched core oligosaccharide to which is attached an unsubstituted alpha 1-->6-polymannose outer chain . Consequently, the cells are agglutinated by antibodies against alpha 1-->6-linked mannose and have N-linked carbohydrate chains that are structurally analogous to the mnn2 mutant of Saccharomyces cerevisiae.

Gene, 1995 Mar 21, 155(1), 139 - 40
Cloning and sequence analysis of an ERG24 homolog from Schizosaccharomyces pombe; Smith S; The Schizosaccharomyces pombe (Sp) erg24 cDNA, encoding C-14 sterol reductase (erg24p), has been cloned and sequenced . The nucleotide sequence of Sp erg24 contains an open reading frame encoding a 424-amino-acid protein . The deduced aa sequence of Sp erg24 shows significant homology with Saccharomyces cerevisiae (Sc) Erg24p, as well as with other members of a larger gene family that includes yeast C-24(28) sterol reductase (Erg4p) and a vertebrate inner nuclear membrane protein, the lamin B receptor (LBR).

Gene, 1995 Mar 21, 155(1), 137 - 8
Schizosaccharomyces pombe and Candida albicans cDNA homologues of the Saccharomyces cerevisiae UBC4 gene; Damagnez V et al.; cDNA homologues of the Saccharomyces cerevisiae UBC4 and UBC5 genes, encoding putative ubiquitin conjugating enzymes, were isolated and characterized from the fission yeast Schizosaccharomyces pombe and from the pathogenic dimorphic yeast Candida albicans . The Sz . pombe and C . albicans deduced amino-acid sequences are 82.3 and 90.5% similar to the Sa . cerevisiae UBC4 gene product, respectively.

Gene, 1995 Mar 21, 155(1), 119 - 22
Isolation and sequencing of two cDNA clones encoding Rho proteins from the fission yeast Schizosaccharomyces pombe; Nakano K et al.; The rho genes encode a group of low-molecular-weight GTP-binding proteins that show about 30% identity in the amino-acid sequence to the ras gene product . Two cDNA clones, both of which are similar to the rho genes, were isolated from a cDNA library of the fission yeast Schizosaccharomyces pombe, using the human rhoA cDNA as a probe . These genes were called rho1+ and rho2+ . The encoded Rho1 protein showed 72.7% identity to the budding yeast RHO1 and 66.8% to human RhoA, and the encoded Rho2 protein showed 53.2% identity to the budding yeast RHO2 and RhoA.

Gene, 1995 Mar 21, 155(1), 113 - 7
Cdc54 belongs to the Cdc46/Mcm3 family of proteins which are essential for initiation of eukaryotic DNA replication; Whitebread LA et al.; CDC54 is a gene essential for initiation of DNA replication in Saccharomyces cerevisiae, and which is known to genetically interact with other regulators of the S-phase, including CDC46 . We describe the isolation and sequencing of CDC54 and show that it encodes a protein structurally related to Cdc46p, Mcm2p and Mcm3p by the presence of a conserved domain of 145 amino acids which is internal to each polypeptide . This conserved domain resembles the DEAD box of RNA helicases and is similar to the conserved domain associated with a group of transcription and replication factors with known or assumed DNA-dependent ATPase activity, suggesting it may be involved in nucleic-acid recognition . Comparison of Cdc54p to related proteins from other species revealed that it closely resembles cdc21p from Schizosaccharomyces pombe.

Mol Gen Genet, 1995 Mar 20, 246(6), 671 - 9
A large circular minichromosome of Schizosaccharomyces pombe requires a high dose of type II DNA topoisomerase for its stabilization; Murakami S et al.; We have constructed circular minichromosomes, ranging in size from 36 to 110 kb, containing the centromeric repeats of Schizosaccharomyces pombe cen3 . Comparison of their mitotic stability showed that the circular minichromosomes became more unstable with increasing in size, however, a linear cen3 minichromosome, which is almost the same size as the largest circular one tested, does not show such instability . High levels of expression of the top2+ (type II DNA topoisomerase; topo II) but not top1+ gene (type I DNA topoisomerase) suppressed the instability of the largest circular minichromosome, whereas partial inactivation of topo II dramatically destabilized the minichromosome . A mutant topo II, defective in nuclear localization but still retaining its in vitro relaxation activity, did not stabilize the circular minichromosome . These results indicate that endogenous type II DNA topoisomerase is insufficient for accurate segregation of the circular minichromosome . In addition, the replication of the minichromosomal DNA appears to proceed normally, because the presence of the unstable minichromosome did not cause G2 delay . A likely cause of the instability is intertwining of the minichromosome DNA possibly occurring after DNA replication . An interaction between topo II and the centromeric repeats is implied by the finding that multiple copies of the centromeric repeat, dg-dh, affect stability of the minichromosome similarly to top2+ gene dosage.

Mol Gen Genet, 1995 Mar 20, 246(6), 663 - 70
Characterization of uvi15+, a stress-inducible gene from Schizosaccharomyces pombe; Lee JK et al.; The uvi15+ gene of Schizosaccharomyces pombe is a member of a group of stress-inducible genes transcription levels of which increase in response to DNA-damaging agents or heat shock . It encodes a polypeptide of calculated molecular mass 11641 Da, with no significant sequence similarity to other known heat shock proteins . The steady-state level of the uvi15+ gene product of about 12 kDa was increased by heat shock and canavanine, an amino acid analog . This gene also showed a transient increase in expression as cells moved into diauxic shift phase . Although deletion of the uvi15+ gene did not affect the mitotic growth or thermotolerance of cells, the mutant cells rapidly lost viability in stationary phase and under starvation conditions . These cells also showed a defect in sporulation ability . These results suggest that the uvi15+ gene encodes a stress response protein involved in the maintenance of cell viability during entry into stationary phase or under starvation conditions.

Eur J Biochem, 1995 Mar 15, 228(3), 976 - 80
Partial purification and characterization of RNase P from Dictyostelium discoideum; Stathopoulos C et al.; Ribonuclease P (RNase P) from Dictyostelium discoideum has been purified 470-fold . D . discoideum RNase P cleaves the precursor to Schizosaccharomyces pombe suppressor tRNA(Ser) at the same site as S . pombe RNase P, producing the mature 5' end of tRNA(Ser) . pH and temperature optima for enzyme activity are 7.6 and 37 degrees C, respectively . The enzyme shows optimal activity in the presence of 5 mM MgCl2 and 10 mM NH4Cl or 5 mM KCl . The apparent Km for the S . pombe tRNA precursor derived from the supS1 tRNA(Ser) gene is 240 nM, and the apparent Vmax is 3.6 pmol/min . Inhibition of D . discoideum RNase P by proteinase K and micrococcal nuclease strongly indicates that the activity requires both protein and RNA components . In cesium sulfate density gradients, the enzyme has a buoyant density of 1.23 g/ml, indicating a low RNA/protein ratio for the holoenzyme.

Arch Biochem Biophys, 1995 Mar 10, 317(2), 487 - 96
Asparagine-linked glycosylation in Schizosaccharomyces pombe: functional conservation of the first step in oligosaccharide-lipid assembly; Zou J et al.; The gene gpt encoding uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetylglucosaminylphosphoryltransferase (L-G1PT) was isolated by screening a Schizosaccharomyces pombe genomic DNA library in lambda phage under low-stringency hybridization using the Saccharomyces cerevisiae gene ALG7 as probe . Sequencing 2.4 kb of S . pombe DNA revealed a 1338-bp open reading frame (ORF) encoding a hydrophobic protein of 446 amino acids with a predicted molecular weight of 49,852 . The S . pombe protein was 50% identical to the S . cerevisiae protein and 43% identical to the protein from Chinese hamster ovary (CHO) cells . Overexpression of the gpt gene in S . pombe cells increased resistance to tunicamycin 25-fold and increased the specific activity of the enzyme in isolated cell membranes 13-fold . This was accompanied by a 50-fold increase in poly(A)+ RNA hybridizing to the gpt probe . Northern analysis indicated a single 1.8-kb message is transcribed from the gpt gene . The gpt gene is essential for viability of S . pombe . Cells containing a disrupted ORF could be rescued by an expression plasmid containing either the intact S . pombe gpt ORF or the CHO L-G1PT cDNA . The S . pombe gpt gene was mapped to chromosome 2 near top1 and ade1.

Mol Gen Genet, 1995 Mar 10, 246(5), 561 - 9
DNA polymerase delta is required for the replication feedback control of cell cycle progression in Schizosaccharomyces pombe; Francesconi S et al.; DNA replication and DNA repair are essential cell cycle steps ensuring correct transmission of the genome . The feedback replication control system links mitosis to completion of DNA replication and partially overlaps the radiation checkpoint control . Deletion of the chk1/rad27 gene abolishes the radiation but not the replication feedback control . Thermosensitive mutations in the DNA polymerase delta, cdc18 or cdc20 genes lead cells to arrest in the S phase of the cell cycle . We show that strains carrying any of these mutations enter lethal mitosis in the absence of the radiation checkpoint chk1/rad27 . We interpret these data as an indication that an assembled replisome is essential for replication dependent control of mitosis and we propose that the arrest of the cell cycle in the thermosensitive mutants is due to the chk1+/rad27+ pathway, which monitors directly DNA for signs of damage.

FEBS Lett, 1995 Mar 6, 360(3), 235 - 41
An actin-related protein from Dictyostelium discoideum is developmentally regulated and associated with mitochondria; Murgia I et al.; An actin-related protein (ACLA) has been identified in the cellular slime mould Dictyostelium discoideum . The complete cDNA sequence indicates that within the actin superfamily it belongs to the ARP3 family of actin-related proteins together with Arp66B from Drosophila melanogaster, Actin2 from Bos taurus, act2 from Schizosaccharomyces pombe and possibly act2 from Caenorhabditis elegans . The ACLA mRNA is regulated during development, showing a maximum between 2 and 4 h after starvation . The protein has been expressed in E . coli and antibodies raised against it . Immunofluorescence microscopy shows that ACLA protein co-localises with mitochondria; the protein copurifies with Dictyostelium mitochondria.

J Biol Chem, 1995 Mar 3, 270(9), 4845 - 53
The Schizosaccharomyces pombe homologue of the chaperone calnexin is essential for viability; Jannatipour M et al.; We have cloned a Schizosaccharomyces pombe gene, here designated cnx1, encoding the homologue of the endoplasmic reticulum molecular chaperone calnexin . Disruption of the cnx1 gene was lethal, demonstrating that it has an essential cellular function . Transcription of cnx1 mRNA is initiated at multiple sites, and it can be induced by various stress treatments that lead to the accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum . The encoded Cnx1p protein more closely resembles its plant and animal calnexin homologues than that of Saccharomyces cerevisiae . Cnx1p is acidic and migrates aberrantly on SDS-polyacrylamide gel electrophoresis, similar to its mammalian counterparts . Cnx1p contains the hallmark KPEDWD motifs that are found in all members of the calnexin/calreticulin family of proteins . Using an in vitro translation-processing system, we have shown that Cnx1p has the characteristic type I topology of calnexin proteins . Unlike its higher eukaryotic homologues, Cnx1p has a site for N-glycosylation that was modified in an in vitro translation-processing assay.

J Biol Chem, 1995 Mar 3, 270(9), 4721 - 8
Transport of metal-binding peptides by HMT1, a fission yeast ABC-type vacuolar membrane protein; Ortiz DF et al.; The Schizosaccharomyces pombe hmt1 gene encodes an ABC (ATP-binding cassette)-type protein essential for Cd2+ tolerance . Immunoblot analysis of subcellular fractions indicates that the native HMT1 polypeptide is associated with the vacuolar membrane . Vacuolar membrane vesicles were purified from strains that hyperproduce, or are deficient in, the HMT1 protein . In vitro transport of radiolabeled substrates by these vesicles indicates that HMT1 is an ATP-dependent transporter of phytochelatins, the metal-chelating peptides involved in heavy metal tolerance of plants and certain fungi . Vacuolar vesicles containing HMT1 are capable of taking up both apo-phytochelatins and phytochelatin-Cd2+ complexes . HMT1 activity is sensitive to antibodies directed against this protein and to vanadate, but not to inhibitors affecting the vacuolar proton ATPase or ionophores that abolish the pH gradient across the vacuolar membrane . Vacuolar uptake of Cd2+ and of a glutathione conjugate were also observed, but are not attributable to HMT1 . These studies highlight the importance of the yeast vacuole in detoxification of xenobiotics.

EMBO J, 1995 Mar 1, 14(5), 1015 - 23
Crystal structure of casein kinase-1, a phosphate-directed protein kinase; Xu RM et al.; The structure of a truncated variant of casein kinase-1 from Schizosaccharomyces pombe, has been determined in complex with MgATP at 2.0 A resolution . The model resembles the 'closed', ATP-bound conformations of the cyclin-dependent kinase 2 and the cAMP-dependent protein kinase, with clear differences in the structure of surface loops that impart unique features to casein kinase-1 . The structure is of unphosphorylated, active conformation of casein kinase-1 and the peptide-binding site is fully accessible to substrate.

J Bacteriol, 1995 Mar, 177(6), 1536 - 43
bfr1+, a novel gene of Schizosaccharomyces pombe which confers brefeldin A resistance, is structurally related to the ATP-binding cassette superfamily; Nagao K et al.; We have isolated a Schizosaccharomyces pombe gene, bfr1+, which on a multicopy plasmid vector, pDB248', confers resistance to brefeldin A (BFA), an inhibitor of intracellular protein transport . This gene encodes a novel protein of 1,531 amino acids with an intramolecular duplicated structure, each half containing a single ATP-binding consensus sequence and a set of six transmembrane sequences . This structural characteristic of bfr1+ protein resembles that of mammalian P-glycoprotein, which, by exporting a variety of anticancer drugs, has been shown to be responsible for multidrug resistance in tumor cells . Consistent with this is that S . pombe cells harboring bfr1+ on pDB248' are resistant to actinomycin D, cerulenin, and cytochalasin B, as well as to BFA . The relative positions of the ATP-binding sequences and the clusters of transmembrane sequences within the bfr1+ protein are, however, transposed in comparison with those in P-glycoprotein; the bfr1+ protein has N-terminal ATP-binding sequence followed by transmembrane segments in each half of the molecule . The bfr1+ protein exhibited significant homology in primary and secondary structures with two recently identified multidrug resistance gene products of Saccharomyces cerevisiae, Snq2 and Sts1/Pdr5/Ydr1 . The bfr1+ gene is not essential for cell growth or mating, but a delta bfr1 mutant exhibited hypersensitivity to BFA . We propose that the bfr1+ protein is another member of the ATP-binding cassette superfamily and serves as an efflux pump of various antibiotics.

Mol Cell Biol, 1995 Mar, 15(3), 1479 - 88
The sak1+ gene of Schizosaccharomyces pombe encodes an RFX family DNA-binding protein that positively regulates cyclic AMP-dependent protein kinase-mediated exit from the mitotic cell cycle; Wu SY et al.; In Schizosaccharomyces pombe, meiosis is initiated by conditions of nutrient deprivation . Mutations in genes encoding elements of the cyclic AMP-dependent protein kinase (cAPK) pathway interfere with meiosis . Loss-of-function alleles of genes that stimulate the activity of cAPK allow cells to bypass the normal requirement of starvation for conjugation and meiosis . Alternatively, loss-of-function alleles of genes that inhibit cAPK lead to the inability to undergo sexual differentiation . The cgs1+ gene encodes the regulatory subunit of cAPK, and the cgs2+ gene encodes a cyclic AMP phosphodiesterase . Thus, both genes encode proteins which negatively regulate the activity of cAPK . Loss of either cgs1 or cgs2 prevents haploid cells from conjugating and diploid cells from undergoing meiosis . In addition to these defects, cells are unable to enter stationary phase . We describe a novel gene, sak1+, which when present on a plasmid overcomes the aberrant phenotypes associated with unregulated cAPK activity . Genetic analysis of sak1+ (suppressor of A-kinase) reveals that it functions downstream of cyclic AMP-dependent protein kinase to allow cells to exist the mitotic cycle and enter either stationary phase or the pathway leading to sexual differentiation . The sak1+ gene is essential for cell viability, and a null allele causes multiple defects in cell morphology and nuclear division . Thus, sak1+ is an important regulatory element in the life cycle of S . pombe . Sequence analysis shows that the predicted product of the sak1+ gene is an 87-kDa protein which shares homology to the RFX family of DNA-binding proteins identified in humans and mice . One member of this family, RFX1, is a transcription factor for a variety of viral and cellular genes.

Mol Cell Biol, 1995 Mar, 15(3), 1431 - 8
Activation of phospholipase C gamma in Schizosaccharomyces pombe by coexpression of receptor or nonreceptor tyrosine kinases; Arkinstall S et al.; The fission yeast Schizosaccharomyces pombe has no detectable endogenous receptor tyrosine kinases or associated signalling apparatus, and we have used this cell system to reconstitute mammalian platelet-derived growth factor beta (PDGF beta) receptor-linked activation of phospholipase C gamma 2 (PLC gamma 2) . The PDGF beta receptor migrates as a glycosylated protein of 165 kDa associated exclusively with membrane fractions . No tyrosine autophosphorylation was detected when PDGF beta was expressed alone . PLC gamma 2 appears as a 140-kDa protein distributed between particulate and soluble fractions which exhibits characteristic selectivity for phosphatidylinositol 4,5-bisphosphate and is sensitive to powerful activation by Ca2+ . When coexpressed, both PDGF beta and PLC gamma 2 undergo tyrosine phosphorylation, and this is accompanied by a > 26-fold increase in {3H}inositol 4,5-biphosphate ({3H}IP2) and {3H}inositol 1,4,5-triphosphate {3H}IP3 production . Treatment with the tyrosine phosphatase inhibitor pervanadate further increased PLC gamma 2 tyrosine phosphorylation as well as {3H}IP2 and {3H}IP3 generation . Phosphorylated PLC gamma 2 was found predominantly in membrane fractions . To test a nonreceptor tyrosine kinase, we then expressed the human proto-oncogene c-src together with its negative regulator Csk . These were immunodetectable as bands at 60 kDa (c-Src) and 50 kDa (Csk) and distributed between membrane and cytosolic fractions . When yeast coexpressing c-Src, Csk, and PLC gamma 2 was incubated with pervanadate, PLC gamma 2 was tyrosine phosphorylated and {3H}IP2 and {3H}IP3 production increased 11.0- and 7.0-fold, respectively . Csk expressed alone with PLC gamma 2 was ineffective . Similar PLC gamma 2 activation was observed upon in vitro mixing with the extracts expressing either c-Src or the PDGF beta receptor . In summary, this is the first report of a reconstitution of mammalian tyrosine kinase-linked effector activation in yeast cells and also the first demonstration of direct PLC gamma 2 activation by the proto-oncogene c-src . These observations indicate that S . pombe provides a powerful cell system in which to study critical molecular interactions and activities underlying receptor and nonreceptor tyrosine kinase-dependent cell signaling.

Yeast, 1995 Mar, 11(3), 271 - 82
Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated, sorted and matured in the fission yeast Schizosaccharomyces pombe; Simeon A et al.; Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPYsc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 mu derived plasmid . Immunoblot analysis revealed that CPYsc produced in the fission yeast has a higher molecular mass than mature CPYsc produced by the budding yeast . CPYsc is glycosylated when expressed in S . pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion . Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPYsc produced by S . cerevisiae . CPYsc isolated from S . pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation . Subcellular fractionation experiments showed a cofractionation of CPYsc with the S . pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.

Yeast, 1995 Mar, 11(3), 225 - 31
Effects of phleomycin-induced DNA damage on the fission yeast Schizosaccharomyces pombe cell cycle; Belenguer P et al.; The effect of phleomycin, a bleomycin-like antibiotic, has been investigated in the fission yeast, Schizosaccharomyces pombe . We report that in response to phleomycin-induced DNA damage, growth was inhibited and S . pombe cells arrested in the G2-phase of the cell cycle . DNA repair mutants rad9 and rad17 did not arrest and were hypersensitive to phleomycin . Cell cycle mutants that entered mitosis without monitoring the completion of DNA replication also displayed an increased sensitivity to this DNA-damaging agent . Thus, phleomycin could be used as a tool in the fission yeast S . pombe model system for the study of DNA damage and cell cycle checkpoints, or as a new selective agent.

Can J Microbiol, 1995 Mar, 41(3), 273 - 7
Pattern of polar extension of the cell wall in the fission yeast Schizosaccharomyces pombe; May JW et al.; The indirect fluorescent-antibody technique has been used to establish the pattern of polar extension in the fission yeast Schizosaccharomyces pombe 160 over a complete cell cycle in liquid medium, thus avoiding the possibility of perturbations being introduced by growth on an agar pad, which is the technique used in most other investigations . Nearly all of the cells (about 98%) showed more growth at the old end than at the new end that was formed by cleavage of the septum at the previous division . Importantly, there was no evidence of the abnormal growth pattern (i.e., the significant contribution of new ends to extension) in cells of S . pombe growing on agar pads reported by Miyata et al . (H . Miyata, M . Miyata, and B.F . Johnson . 1986 . Can . J . Microbiol . 32: 528-530 and 1990 . Can . J . Microbiol . 36: 390-394) . In addition, extension over the cycle was inversely related to birth length (cells shorter than the mean at birth tended to produce daughter cells longer than themselves and vice versa), there was a small but significant asymmetry in the position of the septum, and the time of initiation of extension at the new end was estimated at about 0.24 of the cycle.

J Invertebr Pathol, 1995 Mar, 65(2), 118 - 24
Electrophoretic karyotype of intracellular yeast-like symbiotes in rice planthoppers and anobiid beetles; Noda H et al.; Chromosomal DNA molecules of intracellular yeast-like symbiotes (YLS) of three species of rice planthoppers and two species of anobiid beetles have been separated by pulsed-field gel electrophoresis . Probable chromosome numbers of Nilaparvata lugens, Sogatella furcifera, and Laodelphax striatellus, were 4, 4, and 5, respectively, and tentative genome sizes were 17.3, 17.6, and 20.1 Mbp, respectively, based upon migration of individual chromosome-sized DNA relative to the size standards of Schizosaccharomyces pombe, Hansenula wingei, and Saccharomyces cerevisiae chromosomes . Chromosome numbers of Lasioderma serricorne and Stegobium paniceum were 11 and 15, respectively, and total genome sizes were 20.9 and 15.1 Mbp, respectively . The chromosomes carrying ribosomal RNA genes were identified by Southern blot analysis . Chromosomal organization and the genome size of YLS were similar to those of nonsymbiotic yeasts and fungi.

Curr Genet, 1995 Mar, 27(4), 293 - 7
Construction of a marker gene cassette which is repeatedly usable for gene disruption in yeast; Toh-e A; A disruption cassette has been constructed containing the LEU2 gene flanked by directly repeated site-specific recombination sites of the yeast plasmid, pSB3, which resembles the 2 microns DNA of Saccharomyces cerevisiae . A disruption constructed by inserting this DNA fragment acquires a Leu+ phenotype, which can be easily removed by expressing the FLP-PSB3 gene encoding the site-specific recombinase of pSB3 . A test was made using a Schizosaccharomyces pombe host . The ura4+ gene of S . pombe was replaced with the ura4::LEU2 gene constructed by inserting the disruption cassette into the ura4+ gene . Then, the FLP-pSB3 gene driven by the nmt1+ promoter was introduced into this disruptant . Upon de-repression of the nmt1 promoter by removing thiamine from the medium, the rate of appearance of Leu- was increased . As expected the ura4+ locus underwent a structural change . Thus, the FLP-pSB3 protein and its target site can function adequately in S . pombe.

J Struct Biol, 1995 Mar-Apr, 114(2), 140 - 52
Ultrastructure of the cell wall of Schizosaccharomyces pombe following treatment with various glucanases; Kopecka M et al.; The ultrastructure of isolated cell walls of Schizosaccharomyces pombe was studied by electron microscopy after treatment with the following purified enzymes: endo-beta-(1-->3)-glucanase, endo-beta-(-->6)-glucanase, and endo-alpha-(1-->3)-glucanase produced by Bacillus circulans; exo-beta-(1-->3)-glucanase and endo-beta-(1-->3)-glucanase produced by Schizosaccharomyces japonicus var . versatilis . The exo-beta-(1-->3)-glucanase had no detectable effect on the walls, but amorphous wall material was removed by action of the endo-beta-(1-->3)- and endo-beta-(1-->6)-glucanases of B . circulans to reveal a wall component consisting of densely interwoven microfibrils . The fibrils were hydrolyzed by treatment with the Schiz . japonicus endo-beta-(1-->3)-glucanase followed by B . circulans endo-alpha-(1-->3)-glucanase--suggesting that they were composed of -beta-(1-->3)-linked glucan and alpha-(1-->3)-linked glucan . The presence of a fibrillar component in untreated walls was evident after negative staining.

Biotechnol Prog, 1995 Mar-Apr, 11(2), 171 - 7
Constitutive overexpression of secreted heterologous proteins decreases extractable BiP and protein disulfide isomerase levels in Saccharomyces cerevisiae; Robinson AS et al.; High-level gene expression does not always lead to corresponding high-level secretion of heterologous proteins in yeast . The rate-limiting step in many cases has been shown to exit from the endoplasmic reticulum (ER) . Within the ER, the correct folding of secreted proteins is required for export competence; hence, the cellular proteins involved in these events are likely to be important for efficient secretion . We have found that the extractable levels of two ER-resident proteins involved in folding--heavy chain binding protein (BiP) and protein disulfide isomerase (PDI)--are significantly reduced by prolonged constitutive overexpression of human granulocyte colony stimulating factor (GCSF), human erythropoietin, or Schizosaccharomyces pombe acid phosphatase . However, the rate of BiP synthesis measured in pulse--chase radiolabeling experiments is not reduced by GCSF overexpression, and galactose-directed transcription of the BiP gene does not restore normal BiP protein levels once they have been depleted . The observed loss of lumenal resident proteins, either by proteolysis or irreversible aggregation, is expected to contribute significantly to the inefficiency of foreign protein secretion in yeast.

Proc Natl Acad Sci U S A, 1995 Feb 28, 92(5), 1749 - 53
Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p involved in mRNA processing and transport; Bischoff FR et al.; RanGAP1 is the GTPase activator for the nuclear Ras-related regulatory protein Ran, converting it to the putatively inactive GDP-bound state . Here, we report the amino acid sequence of RanGAP1, derived from cDNA and peptide sequences . We found it to be homologous to murine Fug1, implicated in early embryonic development, and to Rna1p from Saccharomyces cerevisiae and Schizosaccharomyces pombe . Mutations of budding yeast RNA1 are known to result in defects in RNA processing and nucleocytoplasmic mRNA transport . Concurrently, we have isolated Rna1p as the major RanGAP activity from Sc . pombe . Both this protein and recombinant Rna1p were found to stimulate RanGTPase activity to an extent almost identical to that of human RanGAP1, indicating the functional significance of the sequence homology . The Ran-specific guanine nucleotide exchange factor RCC1 and its yeast homologues are restricted to the nucleus, while Rna1p is reported to be localized to the cytoplasm . We suggest a model in which both activities, nuclear GDP-to-GTP exchange on Ran and cytoplasmic hydrolysis of Ran-bound GTP, are essential for shuttling of Ran between the two cellular compartments . Thus, a defect in either of the two antagonistic regulators of Ran would result in a shutdown of Ran-dependent transport processes, in agreement with the almost identical phenotypes described for such defects in budding yeast.

Proc Natl Acad Sci U S A, 1995 Feb 28, 92(5), 1436 - 40
A mutation in the RCC1-related protein pim1 results in nuclear envelope fragmentation in fission yeast; Demeter J et al.; Members of the RCC1 protein family are chromatin-associated guanine nucleotide exchange factors that have been implicated in diverse cellular processes in various organisms, yet no consensus has been reached as to their primary biological role . The fission yeast Schizosaccharomyces pombe, a single-celled eukaryote, provides an in vivo system in which to study the RCC1/Ran switch by using a temperature-sensitive mutant in the RCC1-related protein pim1 . Mitotic entry in the pim1-d1ts mutant is normal, but mitotic exit leads to the accumulation of cells arrested with a medial septum and condensed chromosomes . Although the yeast nuclear envelope normally remains intact throughout the cell cycle, we found a striking fragmentation of the nuclear envelope in the pim1-d1ts mutant following mitosis . This resulted in chromatin that was no longer compartmentalized and an accumulation of pore-containing membranes in the cytoplasm . The development of this terminal phenotype was dependent on the passage of cells through mitosis and was coincident with the loss of viability . We propose that pim1 is required for the reestablishment of nuclear structure following mitosis in fission yeast.

Biochemistry, 1995 Feb 28, 34(8), 2621 - 7
The 2-oxoglutarate/malate translocator of chloroplast envelope membranes: molecular cloning of a transporter containing a 12-helix motif and expression of the functional protein in yeast cells; Weber A et al.; The 2-oxoglutarate/malate translocator of spinach chloroplasts transports carbon skeletons into chloroplasts for net glutamate synthesis . A sequence of a endoprotease Lys-C peptide derived from the purified protein allowed the design of an oligonucleotide which was then used for a hybridization screening of a cDNA library . A 1945 bp insert of 1 of the isolated clones codes for the entire 569 amino acid residues of the precursor protein corresponding to a molecular mass of 60,288 Da . There was no significant homology to the mitochondrial 2-oxoglutarate/malate carrier from bovine heart or to any other known protein . The translocator protein is composed of a hydrophilic N-terminal region (the transit peptide) with a length of about 90-100 amino acid residues which shows, in contrast to presequences of other known envelope membrane proteins, typical features of higher plant chloroplast transit sequences . The mature protein contains 12 putative transmembrane segments in alpha-helical conformation . It is suggested that this translocator, in contrast to other known transporters of organellar origin which are all homodimers with a 6 + 6 helix folding pattern, may function as a monomer . The in vitro synthesized precursor protein is directed to chloroplasts where it is inserted into the chloroplast envelope membrane in a protease-resistant manner . The cDNA coding for the precursor protein was cloned into the yeast expression vector pEVP11, and this construct was used to transform cells from the fission yeast Schizosaccharomyces pombe . The 2-oxoglutarate/malate translocator could be functionally expressed in the transformed yeast cells, and the recombinant protein showed substrate specificities identical to those of the authentic chloroplast protein.

Gene, 1995 Feb 27, 154(1), 109 - 13
A cDNA from Schizosaccharomyces pombe encoding a putative enolase; Jackson JC et al.; Here we report the isolation of an enolase (Eno)-encoding cDNA clone from Schizosaccharomyces pombe . The deduced amino acid (aa) sequence of the 1.4-kb cDNA shares identifies with a number of Eno from Escherichia coli to humans . The highest degree of similarity is to the known Eno from Saccharomyces cerevisiae and an Eno from Candida albicans . Northern blot analysis identified a single transcript of approx . 1.4 kb, which was most abundant when cells were grown in media with glucose as the carbon source, as opposed to glycerol/lactate or ethanol.

Science, 1995 Feb 24, 267(5201), 1166 - 9
A role for exonuclease I from S . pombe in mutation avoidance and mismatch correction; Szankasi P et al.; Exonuclease I (Exo I) from Schizosaccharomyces pombe, a 5'-->3' double-stranded DNA exonuclease, is induced during meiotic prophase I . The exo1 gene is a member of a family of related DNA repair genes, including RAD2/rad13/xpgc and YKL510/rad2, conserved from yeast to humans . An exo1 mutant displays a mutator phenotype and alters activity of the ade6-M387 marker effect . These results suggest that Exo I acts in a pathway that corrects mismatched base pairs.

FEMS Microbiol Lett, 1995 Feb 15, 126(2), 197 - 202
Effects of ethanol and acetic acid on the transport of malic acid and glucose in the yeast Schizosaccharomyces pombe: implications in wine deacidification; Sousa MJ et al.; Ethanol and acetic acid, at concentrations which may occur during wine-making, inhibited the transport of L-malic acid in Schizosaccharomyces pombe . The inhibition was non-competitive, the decrease of the maximum initial velocity following exponential kinetics . Glucose transport was not significantly affected either by ethanol (up to 13%, w/v) or by acetic acid (up to 1.5%, w/v) . The uptake of labelled acetic acid followed simple diffusion kinetics, indicating that a carrier was not involved in its transport . Therefore, the undissociated acid appears to be the only form that enters the cells and is probably responsible for the toxic effects . Accordingly, deacidification by Ss . pombe during wine fermentation should take place before, rather than after, the main alcoholic fermentation by Saccharomyces cerevisiae.

Biochemistry, 1995 Feb 14, 34(6), 1902 - 11
In vitro activation of purified human heat shock factor by heat; Larson JS et al.; A major regulatory step in the heat-induced transcription of heat shock protein (hsp) genes in eukaryotes is the activation of heat shock factor (HSF) . In metazoans and Schizosaccharomyces pombe, HSF is present in unstressed cells but is unable to bind to its target DNA sequence element, the heat shock element (HSE) . Heat induction of the DNA binding activity of HSF is a critical component required for activation of heat shock genes . Inactive HSF in extracts of non-heat shocked human cells can be heated in vitro to activate HSF, suggesting the factors required to sense temperature and activate HSF are soluble factors {Larson, J . S., Schuetz, T . J., & Kingston, R . E . (1988) Nature 335, 372-375} . We utilized the ability to purify human HSF in the active form to characterize further the in vitro activation of HSF . Here we have developed a procedure to deactivate the DNA binding ability of HSF . When purified and deactivated HSF is heated, the DNA binding ability of HSF is activated . This activation occurs most efficiently at 43 degrees C (heat shock temperature), but, in contrast to activation in the crude system, some activation of HSF is observed at 37 degrees C (non-heat shock temperature) . We show that purified and deactivated HSF is similar to natural inactive HSF in both size and shape . Thus, the monomer to trimer transition that activates HSF can occur in a temperature-dependent fashion in the absence of other proteins . It is possible that these biochemical properties of HSF contribute to the ability of HSF to respond to heat in vivo.

FEBS Lett, 1995 Feb 13, 359(2-3), 192 - 4
The use of mass spectrometry to examine the formation and hydrolysis of the phosphorylated form of phosphoglycerate mutase; Nairn J et al.; Electrospray mass spectrometry has been used to study the formation and hydrolysis of the phosphorylated forms of two phosphoglycerate mutases . The half-life of the enzyme from Saccharomyces cerevisiae was 35 min at 20 degrees C in 10 mM ammonium bicarbonate, pH 8.0 . Addition of 1 mM 2-phosphoglycollate reduced this value by at least 100-fold . The phosphorylated form of the enzyme from Schizosaccharomyces pombe was much less stable with a half-life of less than 1 min . The results are discussed in terms of the kinetic properties of the enzymes . Mass spectrometry would appear to be a powerful method to study the formation and breakdown of phosphorylated proteins, processes which are of widespread significance in regulatory mechanisms.

Nucleic Acids Res, 1995 Feb 11, 23(3), 383 - 8
Cloning and characterisation of the Schizosaccharomyces pombe rad32 gene: a gene required for repair of double strand breaks and recombination; Tavassoli M et al.; A new Schizosaccharomyces pombe mutant (rad32) which is sensitive to gamma and UV irradiation is described . Pulsed field gel electrophoresis of DNA from irradiated cells indicates that the rad32 mutant, in comparison to wild type cells, has decreased ability to repair DNA double strand breaks . The mutant also undergoes decreased meiotic recombination and displays reduced stability of minichromosomes . The rad32 gene has been cloned by complementation of the UV sensitive phenotype . The gene, which is not essential for cell viability and is expressed at a moderate level in mitotically dividing cells, has significant homology to the meiotic recombination gene MRE11 of Saccharomyces cerevisiae . Epistasis analysis indicates that rad32 functions in a pathway which includes the rhp51 gene (the S.pombe homologue to S.cerevisiae RAD51) and that cells deleted for the rad32 gene in conjunction with either the rad3 deletion (a G2 checkpoint mutation) or the rad2 deletion (a chromosome stability and potential nucleotide excision repair mutation) are not viable.

Nucleic Acids Res, 1995 Feb 11, 23(3), 357 - 61
Characterization of cDNA encoding mouse homolog of fission yeast dhp1+ gene: structural and functional conservation; Shobuike T et al.; The dhp1+ gene of Schizosaccharomyces pombe is a homolog of Saccharomyces cerevisiae HKE1/RAT1/TAP1 gene that is involved in RNA metabolism such as RNA trafficking and RNA synthesis . dhp1+ is also related to S . cerevisiae DST2 (SEP1) that encodes a DNA strand exchange protein required for sporulation and homologous recombination in S.cerevisiae . We isolated several clones of Dhm1, a mouse homolog of dhp1+, from mouse spermatocyte cDNA library and determined its nucleotide sequence . The Dhm1 gene consists of an open reading frame predicting a protein with 947 amino acids and molecular weight of 107,955 . Northern blot analysis revealed that Dhm1 is transcribed at high level in testis, liver and kidney . The predicted product of Dhm1 (Dhm1p) has a significant homology with Dhp1p, Hke1p/Rat1p/Tap1p and Dst2p . In particular, Dhm1p, Dhp1p and Hke1p/Rat1p/Tap1p share strong similarity at the two regions of their N- and C-terminal parts . The Dhm1 gene on a multicopy plasmid rescued the temperature-sensitivity of dhp1ts and lethality of dhp1 null mutation, suggesting that Dhm1 is a mouse homolog of S.pombe dhp1+ and functions similarly in mouse as dhp1+.

J Biol Chem, 1995 Feb 10, 270(6), 2669 - 73
Protein kinase C (PKC)-induced PKC down-regulation . Association with up-regulation of vesicle traffic; Goode NT et al.; Phorbol esters cause long term activation of protein kinase C (PKC) and frequently the down-regulation of PKC protein levels in mammalian cells . Mammalian PKC-gamma, -delta, and -eta down-regulated in response to phorbol esters when expressed in Schizosaccharomyces pombe . However, PKC-epsilon does not down-regulate in S . pombe, in contrast to the behavior of this isotype in mammalian cells . Co-expression of PKC-gamma or -delta with PKC-epsilon in S . pombe renders PKC-epsilon susceptible to down-regulation . A protein kinase defective form of PKC-delta does not down-regulate efficiently in S . pombe but, like PKC-epsilon, is susceptible when co-expressed with PKC-gamma or full-length PKC-delta . Thus, down-regulation is a consequence of the catalytic function of certain PKC isotypes with other isotypes being affected in trans . PKC down-regulation parallels a striking accumulation of vesicles in S . pombe, suggesting a direct relationship between these events.

Mol Gen Genet, 1995 Feb 6, 246(3), 316 - 26
The RCK1 and RCK2 protein kinase genes from Saccharomyces cerevisiae suppress cell cycle checkpoint mutations in Schizosaccharomyces pombe; Dahlkvist A et al.; The protein kinase-encoding genes RCK1 and RCK2 from Saccharomyces cerevisiae have been identified as suppressors of Schizosaccharomyces pombe cell cycle checkpoint mutations . Upon expression of these genes, radiation resistance is partially restored in S . pombe mutants with checkpoint deficiencies, but not in mutants with DNA repair defects . Some checkpoint mutants are sensitive to the DNA synthesis inhibitor hydroxyurea, and this sensitivity is also suppressed by RCK1 and RCK2 . The degree of suppression can be modulated by varying expression levels . Expression of RCK1 or RCK2 in S . pombe causes cell elongation and decelerated growth . Cells expressing these genes have a single nucleus and a 2n DNA content . We conclude that these genes act in S . pombe to prolong the G2 phase of the cell cycle.

J Biol Chem, 1995 Feb 3, 270(5), 1979 - 82
Protein kinase Byr2 is a target of Ras1 in the fission yeast Schizosaccharomyces pombe; Masuda T et al.; Conservation of the structure and function of Ras proteins has been observed in a variety of eukaryotic organisms . However, the nature of their downstream effectors appears to be quite divergent; adenylyl cyclase and a protein kinase Raf-1, which do not share any structural homology with each other, are effectors of Ras in the budding yeast and in higher organisms, respectively . We show here that a protein kinase Byr2, which has been known to act downstream of Ras1 in a mating pheromone signal transduction system of Schizosaccharomyces pombe, binds directly to Ras proteins in a GTP-dependent manner . The region of Byr2 responsible for the Ras binding was mapped by a gene deletion analysis to its N-terminal segment of 206 amino acid residues, which does not possess any significant homology with the other effectors of Ras . The affinity of the Byr2 N terminus for Saccharomyces cerevisiae Ras2 was determined by measuring its activity to competitively inhibit Ras-dependent adenylyl cyclase activity and found to be comparable with those of yeast adenylyl cyclase and human Raf-1, with a dissociation constant (Kd) of about 1 nM . Furthermore, Byr2 inhibited a Ras GTPase-activating activity of Ira2, a S . cerevisiae homologue of neurofibromin . These results indicate that Byr2 is an immediate downstream target of Ras1 in S . pombe.

J Cell Biol, 1995 Feb, 128(4), 445 - 54
A novel cis-acting centromeric DNA element affects S . pombe centromeric chromatin structure at a distance; Marschall LG et al.; The chromatin structure of the central core region of Schizosaccharomyces pombe centromeric DNA is unusual . This distinctive chromatin structure is associated only with central core sequences in a functional context and is modulated by a novel cis-acting DNA element (centromere enhancer) within the functionally critical K centromeric repeat, which is found in multiple copies in all three S . pombe centromeres . The centromere enhancer alters central core chromatin structure from a distance and in an orientation-independent manner without altering the nucleosomal packaging of sequences between the enhancer and the central core . These findings suggest a functionally relevant structural interaction between the enhancer and the centromeric central core brought about by DNA looping.

EMBO J, 1995 Feb 1, 14(3), 492 - 502
Counteractive roles of protein phosphatase 2C (PP2C) and a MAP kinase kinase homolog in the osmoregulation of fission yeast; Shiozaki K et al.; With the goal of discovering the cellular functions of type 2C protein phosphatases, we have cloned and analyzed two ptc (phosphatase two C) genes, ptc2+ and ptc3+, from the fission yeast Schizosaccharomyces pombe . Together with the previously identified ptc1+ gene, the enzymes encoded by these genes account for approximately 90% of the measurable PP2C activity in fission yeast cells . No obvious growth defects result from individual disruptions of ptc genes, but a delta ptc1 delta ptc3 double mutant displays aberrant cell morphology and temperature-sensitive cell lysis that is further accentuated in a delta ptc1 delta ptc2 delta ptc3 triple mutant . These phenotypes are almost completely suppressed by the presence of osmotic stabilizers, strongly indicating that PP2C has an important role in osmoregulation . Genetic suppression of delta ptc1 delta ptc3 lethality identified two loci, mutations of which render cells hypersensitive to high-osmolarity media . One locus is identical to wis1+, encoding a MAP kinase kinase (MEK) homolog . The Wis1 sequence is most closely related to the Saccharomyces cerevisiae MEK encoded by PBS2, which is required for osmoregulation . These data indicate that divergent yeasts have functionally conserved MAP kinase pathways, which are required to increase intracellular osmotic concentrations in response to osmotic stress . Moreover, our observations implicate PP2C enzymes as also having an important role in signal transduction processes involved in osmoregulation, probably acting to negatively regulate the osmosensing signal that is transmitted through Wis1 MAP kinase kinase.

Glycobiology, 1995 Feb, 5(1), 129 - 36
Genomic organization and expression of hamster UDP-N-acetylglucosamine:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase; Scocca JR et al.; The hamster gene for uridine diphosphate N-acetyl-D-glucosamine:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase (L-G1PT) was found to extend over 6.5 kb and to contain nine exons . The exons ranged in size from 63 to 214 bp, encoding the 408 amino acid protein . The introns ranged from 85 bp to 1.4 kb . Upstream 5' sequences included two possible TATA boxes, one possible CCAAT box and at least two potential GC boxes . Heterologous expression was successful in Schizosaccharomyces pombe, and resulted in cells that were tunicamycin resistant and had 12-fold more L-G1PT activity than wild-type cells . Antiserum prepared to a hydrophilic peptide (residues 300-320) of the L-G1PT protein reacted with a 35-36 kDa protein in membrane samples from Chinese hamster ovary (CHO) cells and S . pombe cells that had increased levels of L-G1PT activity . In both cases, antigenic peptide competed with the 35-36 kDa protein detected by the antiserum.

J Cell Sci, 1995 Feb, 108 ( Pt 2), 475 - 86
The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis; al-Khodairy F et al.; Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired . Controls called 'checkpoints', mediate cell cycle arrest in response to unreplicated or damaged DNA . Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation . We have cloned and sequenced the hus5+ gene . It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs) . To understand the role of hus5+ in cell cycle control we have characterized the phenotypes of the hus5 mutants and the hus5 gene disruption . We find that, whilst the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest . Thus, the hus5+ gene product is not directly involved in checkpoint control . However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants . In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation . We conclude that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation.

Cell Biophys, 1995 Feb, 26(1), 57 - 75
Smashed fission yeast walls . Structural discontinuities related to wall growth; Johnson BF et al.; Twenty-three samples of fission yeast cells (Schizosaccharomyces pombe) were smashed by shaking them with glass beads . The samples represented all phases of the culture cycle, with the lag and log phases emphasized . Ruptured walls of the smashed cells were observed by phase-contrast and electron microscopy . Ruptures were tabulated with respect to their magnitudes and locations . Ruptures occurred not at random, nor at sites directed by geometry, but predominated in certain definable wall regions . These discontinuities were correlated with morphogenetic activities of the cell . Thus, the extensile end was found to be most fragile through most of the culture cycle . Also fragile was the nonextensile end, its edge more than its middle . Further, the data were applied to the testing of predictions from extant models (Johnson endohydrolytic softening model and Wessels presoftened-posthardened and crosslinking model) for hyphal tip extension . The frequency of rupture at the extensile (old) end of the cell was qualitatively predicted by both models; the frequency at the nonextensile (new) end was not predictable by either . Rupture frequencies and characteristics at other regions conformed to predictions by one or the other model, but rarely by both.

Curr Opin Genet Dev, 1995 Feb, 5(1), 12 - 6
Checkpoints in the cell cycle of fission yeast; D'Urso G et al.; When cell cycle progression in fission yeast is disrupted, checkpoint controls ensure that the normal sequence of cell cycle events is maintained . Activation of a checkpoint relies on monitoring signals that might involve assembly of macromolecular structures essential for specific cell cycle processes . The past year has seen further elucidation of two new checkpoints operating during the cell cycle of Schizosaccharomyces pombe . One involves the product of the rum1 gene and prevents cells from entering mitosis from the pre-Start G1 interval . The second checkpoint operates during the later stages of the cell cycle and is essential for coupling the events of mitosis and cell division.

Yeast, 1995 Feb, 11(2), 179 - 85
Molecular cloning of the plc1+ gene of Schizosaccharomyces pombe, which encodes a putative phosphoinositide-specific phospholipase C; Andoh T et al.; Exploiting the polymerase chain reaction, we have isolated a gene that encodes a putative phosphoinositide-specific phospholipase C (PLC) of the fission yeast Schizosaccharomyces pombe . Inspection of the nucleotide sequence of the gene revealed an open reading frame that can encode a polypeptide of 899 amino acid residues with a calculated molecular mass of 102 kDa . This putative polypeptide contains both the X and Y regions that are conserved among three classes of mammalian PLC, and also contains a presumptive Ca(2+)-binding site (an E-F hand motif) . The structure of the putative protein is most similar to that of the delta class of PLC isozymes . To investigate the role of this gene, designated plc1+, gene disruption was carried out by interrupting the coding region with the ura4+ marker . Growth of plc1 cells was temperature-sensitive in rich medium, and cells could not grow in synthetic medium . Expression of the PLC1 gene of Saccharomyces cerevisiae suppressed the growth defect phenotype of plc1- cells, a strong suggestion that the plc1+ gene encodes PLC.

Yeast, 1995 Feb, 11(2), 157 - 67
Characterization of Schizosaccharomyces pombe his1 and his5 cDNAs; Erickson FL et al.; We have isolated Schizosaccharomyces pombe cDNAs corresponding to the genes his1+ and his5+ . The his1 cDNA was isolated by functional complementation of the His- phenotype in a his1-29 gcn3 Saccharomyces cerevisiae strain, while the his5 cDNA was isolated as a suppressor of the 3-amino-1,2, 4-triazole (3-AT) sensitivity in a gcn3 S . cerevisiae strain . his1 and his5 are each present in single copy in haploid S . pombe . As is the case with S . cerevisiae, we have found that the growth of wild-type strains of S . pombe is sensitive to 3-AT, an inhibitor of imidazoleglycerol-phosphate dehydratase . This enzyme is encoded by the HIS3 gene in S . cerevisiae and the his5+ gene in S . pombe . Treatment of S . pombe cells with 3-AT leads to a small increase in the level of the his5 transcript, but no effect is seen on the level of the his1 transcript . This is in contrast to larger increases in transcription of amino acid biosynthetic genes, regulated by the general amino acid control, seen previously in similarly treated cultures of S . cerevisiae . These results suggest that there are likely to be some differences in the regulation of amino acid biosynthesis between these two yeasts.

J Biochem (Tokyo), 1995 Feb, 117(2), 283 - 8
Molecular cloning and nucleotide sequencing of the gamma-glutamylcysteine synthetase gene of the fission yeast Schizosaccharomyces pombe; Mutoh N et al.; A DNA fragment encoding gamma-glutamylcysteine synthetase {EC 6.3.2.2} of Schizosaccharomyces pombe was cloned by complementation of the cadmium hypersensitivity of a S . pombe mutant deficient in the enzyme . Sequence analysis of the cloned DNA revealed that the enzyme was consisted of 669 amino acid residues and was homologous to the enzymes of human liver, rat kidney, and Saccharomyces cerevisiae . The deduced amino acid sequence coincides with the amino acid sequences of the proteolytic peptides obtained from the purified enzyme . A cysteine residue was deduced to be important for catalytic activity by comparing the amino acid sequences of the enzymes of the four species . The gene contains one intron and the splicing point was confirmed by sequencing a cDNA amplified by PCR . Northern blot analysis showed an RNA of 2,200 bases hybridized with the cloned gene.

Philos Trans R Soc Lond B Biol Sci, 1995 Jan 30, 347(1319), 49 - 56
Regulation and mechanisms of gene amplification; Smith KA et al.; Amplification in rodent cells usually involves bridge-breakage-fusion (BBF) cycles initiated either by end-to-end fusion of sister chromatids, or by chromosome breakage . In contrast, in human cells, resistance to the antimetabolite N-(phosphonacetyl)-L-aspartate (PALA) can be mediated by several different mechanisms that lead to overexpression of the target enzyme carbamyl-P synthetase, aspartate transcarbamylase, dihydro-orotase (CAD) . Mechanisms involving BBF cycles account for only a minority of CAD amplification events in the human fibrosarcoma cell line HT 1080 . Here, formation of a 2p isochromosome and overexpression of CAD by other types of amplification events (and even without amplification) are much more prevalent . Broken DNA is recognized by mammalian cells with intact damage-recognition pathways, as a signal to arrest or to die . Loss of these pathways by, for example, loss of p53 or pRb tumour suppressor function, or by increased expression of ras and myc oncogenes, causes non-permissive rat and human cells to become permissive both for amplification and for other manifestations of DNA damage . In cells that are already permissive, amplification can be stimulated by overexpressing oncogenes such as c-myc or ras, or by damaging DNA in a variety of ways . To supplement genetic analysis of amplification in mammalian cells, an amplification selection has been established in Schizosaccharomyces pombe . Selection with LiCl yields cells with amplified sod2 genes in structures related to those observed in mammalian cells . The effect on amplification in S . pombe can now be tested for any mutation in a gene involved in repair of damaged DNA or in normal cellular responses to DNA damage.

Gene, 1995 Jan 23, 152(2), 209 - 13
Structures of genes encoding TATA box-binding proteins from Trimeresurus gramineus and T . flavoviridis snakes; Nakashima K et al.; A cDNA encoding the Trimeresurus gramineus (Tg; green habu snake) TATA-box-binding protein (TgTBP) was cloned and sequenced . The cDNA encodes a 33-kDa protein with an extensive sequence similarity to those derived from other organisms, except for the N-terminal domain . Genes encoding TgTBP and Trimeresurus flavoviridis (Tf; habu snake) TBP (TfTBP) were isolated using a TgTBP cDNA and their nt sequences were determined . They are the first TBP genes entirely sequenced in higher animals . Both genes span over 15 kb and are constructed from eight exons and seven introns . Comparison of the loci of introns on the aligned amino-acid sequences of TBP from six organisms (Tg, Tf, mouse, Arabidopsis thaliana, Schizosaccharomyces pombe and Acanthamoeba castellanii) indicated that there are three highly conserved loci in the C-terminal domain.

Gene, 1995 Jan 11, 152(1), 117 - 20
Cloning and sequence of a gene encoding the pyruvate dehydrogenase E1 beta subunit of Schizosaccharomyces pombe; Cavan G et al.; A 4906-bp DNA fragment, which complemented Schizosaccharomyces pombe strains partly defective in pyruvate dehydrogenase (PDH), was cloned and sequenced . The fragment contained an open reading frame (ORF) of 366 amino acids (aa), which showed 62% identity with the Saccharomyces cerevisiae gene encoding the PDH E1 beta subunit, and significant similarity to subunits from a number of other 2-oxo-acid dehydrogenase complexes from mammals, Gram+ and some Gram- bacteria . The clone hybridised to a 1.6-1.7-kb mRNA from wild type, and the 5' ends of the mRNA were mapped 73-83 bp upstream from the AUG start codon of the ORF . No other ORFs were found in this 4.9-kb segment of the Sz . pombe genome . Plasmids containing the ORF complemented the PDH-defective strains of Sz . pombe both for growth and for enzyme activity.

Arch Biochem Biophys, 1995 Jan 10, 316(1), 163 - 8
Kinetic studies of gluconate pathway enzymes from Schizosaccharomyces pombe; Tsai CS et al.; Glucose dehydrogenase and gluconate kinase which catalyze two-step reactions of the gluconate pathway have been purified from Schizosaccharomyces pombe . Their steady-state kinetic studies were undertaken . The yeast glucose dehydrogenase requires NADP+ as an obligatory coenzyme and mediates the oxidation of D-glucose to D-gluconate via an ordered Bi Bi mechanism with NADP+ as the leading substrate . Kinetic constants for the dehydrogenase reactions have been measured . The yeast gluconate kinase requires Mg2+ as an activator . The phosphorylation catalyzed by the fission yeast gluconate kinase has been studied kinetically at a fixed concentration of Mg2+ . The initial velocity and product inhibition results are consistent with a rapid equilibrium random Bi Bi mechanism with the formation of an abortive enzyme-ADP-gluconate complex . Dissociation constants of the two substrates, ATP and D-gluconate from various binary and ternary enzymic complexes, have been determined.

Arch Biochem Biophys, 1995 Jan 10, 316(1), 155 - 62
Carbon-13 NMR studies and purification of gluconate pathway enzymes from Schizosaccharomyces pombe; Tsai CS et al.; Evidence is presented to show that D-glucose in Schizosaccharomyces pombe can be metabolized via a new alternative route (gluconate pathway) in addition to the regular D-glucose 6-phosphate route . This gluconate pathway consists of two steps: oxidation of D-glucose to D-gluconate by NADP(+)-dependent glucose dehydrogenase and phosphorylation of D-gluconate to 6-phosphogluconate by gluconate kinase . The formation of D-gluconate and 6-phosphogluconate from D-glucose was monitored by 13C nuclear magnetic resonance spectroscopy using D-{1-13C}glucose and D-{U-13C}glucose . The operation of the gluconate pathway was further substantiated by the purification of its two member enzymes, glucose dehydrogenase and gluconate kinase, from the cell-free extract of the fission yeast . Glucose dehydrogenase has been purified (580-fold) to homogeneity by the combined procedures of ammonium sulfate fractionation, Sephadex gel filtration, cation-exchange chromatography, matrex gel chromatography, and agarose-NADP+ affinity chromatography . The purified enzyme is monomeric with a relative molecular weight of 6.65 x 10(4) Da . Gluconate kinase has been purified (410-fold) to near homogeneity by a combination of chromatographic procedures using Bio-gels, matrex gel, and agarose gels . The purified enzyme is monomeric with a relative molecular weight of 2.4 x 10(4) Da . The gluconate pathway presented here provides an alternative route for the D-glucose metabolism in Sch . pombe . Meanwhile, this paper documents another metabolic difference between the fission and budding yeasts.

Virology, 1995 Jan 10, 206(1), 126 - 35
Synthesis and assembly of virus-like particles of human papillomaviruses type 6 and type 16 in fission yeast Schizosaccharomyces pombe; Sasagawa T et al.; We have synthesized capsid proteins of human papillomavirus types 6 (HPV 6) and 16 (HPV 16) in fission yeast Schizosaccharomyces pombe and produced virus-like particles (VLP) . The capsid proteins were localized in the nucleus by indirect immunofluorescence and cell fractionation analyses . The VLP were produced in both yeast clones synthesizing L1 alone and L/L2 and purified by sulfato-cellulofine chromatography . Electron microscopic examination showed that these VLP were similar in structure to native HPV particles . Two HPV 16 L1 variants (16 B27L1 and 16 T3L1), isolated from benign cervical samples, produced many more (68- and 14-fold) VLP than the prototype L1 (16 PL1) derived from cervical carcinoma . Coexpression of the HPV 6 L2 protein with 6 L1 and 16 B27L1 proteins increased the production level of the VLP four- and twofold, respectively . The L2 was not detected in the VLP purified with sulfato-cellulofine column, although the L2 was purified in the same fraction containing HPV 6 and 16 B27-VLP by size-fractionation using Sepharose column . Interaction between 6 L2 and 6/16 L1 proteins was not detected by the coimmunoprecipitation assays with either L1 or L2 antibodies . These results suggest that the L2 is not incorporated into the VLP synthesized in yeast.

FEBS Lett, 1995 Jan 3, 357(2), 173 - 7
Recombinant expression and domain structure of the Rna1 protein from Schizosaccharomyces pombe; Haberland J et al.; The amino acid sequence of Rna1p, a yeast protein implicated in the maturation and/or nucleocytoplasmic transport of RNA, is characterised by the presence of eight leucine-rich repeats (LLRs) as well as two intervening repeats of a different type and a highly acidic C-terminal region . Limited proteolysis of purified Rna1p expressed recombinantly in bacteria reveals that the C-terminal extension but not the region containing the two types of repeats is highly accessible to proteolytic attack and that the C-terminal region most likely harbours (a) low affinity Ca(2+)-binding site(s) . These results are indicative of the domain structure of the Rna1p molecule, with the repeats and the C-terminal region being accessible for different interactions.

Cell Mol Biol Res, 1995, 41(5), 333 - 46
Temporal and spatial distributions of yeast nucleoside diphosphate kinase activities and its association with the Cdc8p; Zhang SQ et al.; Nucleoside diphosphate kinase (E.C . 2.7.4.6.) is a broad substrate-specific enzyme that catalyzes the phosphorylation of nucleoside diphosphates to the corresponding triphosphates in nucleic acid biosynthesis . In this report, we investigate its spatial and temporal distributions in yeast to understand how the enzyme exerts its gene function(s) . Our results show that the enzyme is predominantly cytoplasmic . A substantial amount of enzyme activity (40-50%) may be associated with the cell membrane . Less than 1% of total activity was detected in the nuclear fraction . Approximately 3% was found in the mitochondrial fraction . When yeast cultures were synchronized, we found that Saccharomyces cerevisiae nucleoside diphosphate kinase did not show cell cycle periodicity, as Schizosaccharomyces pombe enzyme did . To explore its link with DNA synthesis, we investigated its relationship with the Cdc8p (dTMP kinase) . We demonstrated a physical interaction between these proteins in vitro, as evidenced that the GST:Cdc8p protein affinity column could retain a subpopulation of nucleoside diphosphate kinase activity from yeast crude extract . Furthermore, when GST:Cdc8p protein was expressed in yeast, the protein could bind to the glutathione-agarose, along with nucleoside diphosphate kinase, suggesting that there is an interaction between GST:Cdc8p and nucleoside diphosphate kinase in vivo . Our results provide evidence for at least a two-enzyme complex that may well facilitate nucleotide channeling in the cell.

Folia Microbiol (Praha), 1995, 40(5), 519 - 27
Changes in cell wall composition of deformed ras1- cells of Schizosaccharomyces pombe; Harmouch N et al.; Disruption of the Schizosaccharomyces pombe ras1 gene results in a morphological transformation to large spheres, in contrast to wild-type cells which grow as rods . Chemical analysis of isolated cell walls showed no significant changes in saccharide content but an increase in protein and phosphate contents in ras1- walls relative to parent walls . Polymers tightly bound to the cell wall were solubilized by SDS treatment . Several compounds with molar mass ranging from 22 to 130 kDa and more were resolved by gel filtration and SDS-PAGE . Among low-molar-mass species, a component moving as a band at 31 kDa was conspicuous in ras1- cell walls . It was solubilized by heating in Tris-HCl buffer and shown to have a beta-1,3-glucanase activity against laminarin . The level of the enzyme was by 30% higher in the ras1- cell wall than in the wild-type cell wall . This enzyme may participate in the remodelling of the rigid glucan network and account (at least partially) for the aberrant cell shape . The ras1- cell wall contained a high level of charged polymers, especially phosphoproteins, raising the appealing possibility that ras1- is involved in a putative kinase cascade required to sense and respond to external stimuli destined for the cell wall . Although the present study shows that ras1 loss of function and altered cell wall composition are closely linked defects, it has still to be shown that the ras1 protein is directly involved in alterations found in the mutant cell walls.

Antisense Res Dev, 1995 Winter, 5(4), 295 - 305
The ade6 gene of the fission yeast as a target for antisense and ribozyme RNA-mediated suppression; Atkins D et al.; A genetic system for the analysis of antisense and ribozyme mechanisms is a much needed experimental tool, and yeast represent a favorable organism on which to base such a system . We have shown previously that the fission yeast Schizosaccharomyces pombe has potential to satisfy the requirements of such a system . This report describes experiments designed to determine if antisense and ribozyme RNA-mediated gene suppression will be generally applicable to other genes in S . pombe . Antisense and ribozyme RNAs designed to suppress the ade6 gene were expressed at high levels from episomal expression vectors . The ade6 gene was chosen as a target as mutations within the gene confer adenine auxotrophy and a red colony phenotype, and it was expected that antisense or ribozyme RNA-mediated mutant phenocopies would exhibit the same readily detectable phenotype . No phenotypic indication of ade6 suppression was detected in transformed yeast, and ade6 target mRNA was analyzed by primer extension and Northern analysis . Initially, conflicting results were obtained from these techniques, which were determined to be due to duplex formation between antisense and target RNA in vitro . No detectable reduction in the ade6 mRNA levels was found, and it was concluded that the gene was not suppressed by the antisense or ribozyme RNAs tested . These results confirm that in S . pombe as with other organisms, the susceptibility of genes to RNA-mediated suppression may be gene specific and that design of antisense and ribozyme genes will be an empirical process.

Biochimie, 1995, 77(4), 279 - 87
Effect of phenylarsine oxide on the fission yeast Schizosaccharomyces pombe cell cycle; Oustrin ML et al.; Phosphotyrosyl turnover is an essential regulatory mechanism for many biological processes, and the balance between tyrosine kinases and phosphatases plays a major role in the control of cell proliferation . Phenylarsine oxide (PAO), a potent inhibitor of tyrosine phosphatases (PTPase), was used to investigate the involvement of PTPase in the growth and control of the cell cycle of the fission yeast Schizosaccharomyces pombe . Cell proliferation was arrested by treatment with PAO, which was found to inhibit cdc25 PTPase in vitro but appeared not to act in vivo on this mitosis inducer . The PAO-treated cells displayed a mono- or binucleated phenotype and a DNA content that was either 2C or 4C, indicating a cell cycle arrest with a failure to complete cytokinesis . Entry into the cell division cycle from the G0 quiescent stage was also delayed by treatment with PAO . These results suggest that a number of key events in the mitotic cell cycle are regulated by as yet unidentified PTPases.

Chin J Biotechnol, 1995, 11(2), 125 - 30
A study of growth kinetics of the flocs from Schizosaccharomyces pombe; Bai F et al.; Inducing yeast cells to self-flocculate could be considered a cell immobilization method . The growth kinetics of the flocs from Schizosaccharomyces pombe was studied in an experimental suspended-bed bioreactor with starch hydrolysate obtained by two-stage enzymatic hydrolysis . It was discovered that a limited oxygen supply was necessary during continuous ethanol fermentation with yeast flocs . The oxygen supplied was a kind of limited substrate effecting on the floc growth . Further, a kinetic model describing this floc growth was proposed.

Hum Mol Genet, 1995, 4 Spec No, 1765 - 77
Epigenetic regulation of gene expression: the effect of altered chromatin structure from yeast to mammals; Hendrich BD et al.; Epigenetic gene regulation refers to different states of phenotypic expression caused by differential effects of chromosome or chromatin packaging rather than by differences in DNA sequence . Examples of epigenetic regulation can be found in organisms as diverse as the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and mammals . Three major types of epigenetic regulation are considered in this review: dosage compensation, imprinting and position effect variegation . While the specific details and mechanisms of each is quite different, they all involve either local or extensive alterations in chromatin structure . A number of genes implicated in epigenetic regulation have been isolated and their products identified as proteins or RNA molecules involved at various levels in DNA, chromatin or chromosome binding . While in general our understanding of mammalian epigenetic phenomena is not as advanced as that in model systems, the detailed molecular and genetic understanding of processes responsible for conditional gene silencing in invertebrate systems provides strong models for consideration of such effects in human and mouse genetics.

Plant Physiol, 1995 Jan, 107(1), 33 - 41
Subcellular localization of the inducible Chlorella HUP1 monosaccharide-H+ symporter and cloning of a Co-induced galactose-H+ symporter; Stadler R et al.; The unicellular green alga Chlorella kessleri can induce monosaccharide-H+ symport catalyzing the energy-dependent transport of D-glucose (D-Glc) and several other pentoses and hexoses across the plasmalemma . The gene coding for the inducible HUP1 monosaccharide-H+ symporter has been cloned and the protein has been characterized previously . The data presented in this paper demonstrate that the presence of the HUP1 gene product alone is not sufficient to cover the broad substrate specificity of monosaccharide transport in induced Chlorella cells . Two other HUP genes are shown to be co-induced in Chlorella in response to D-Glc in the medium . The cloning of HUP2 and HUP3 cDNA and genomic sequences is described, both being very homologous to HUP1 . Modification of the 5' untranslated sequences of full-length cDNA clones of HUP2 and HUP3 allowed the functional expression of both transporters in Schizosaccharomyces pombe . HUP2 was shown to be a galactose-H+ symporter, whereas the substrate specificity of the HUP3 gene product is very similar to that of the HUP1 protein . However, HUP3 does not seem to be induced to high levels in Glc-treated Chlorella cells . Results are also presented proving that the product of the HUP1 gene is localized in the plasmalemma of D-Glc-induced Chlorella cells and is absent in plasma membranes of noninduced cells . Incubation of thin sections of Chlorella cells with anti-HUP1 antibodies and a fluorescence-labeled, second antibody yielded a ring of fluorescence on the surface of Glc-induced Chlorella cells.

DNA Res, 1995, 2(1), 45 - 9
Cloning and characterization of rat cellular nucleic acid binding protein (CNBP) cDNA; Yasuda J et al.; We cloned and sequenced the cDNAs which code for rat cellular nucleic acid binding protein (CNBP) . In-frame insertion/deletion differences were found among the clones at two sites in the open reading frame, suggesting alternative splicing of the message or the presence of multiple genes which code for this protein . The deduced amino acid sequence revealed that one rat CNBP sequence was completely identical to its human counterpart . This striking conservation, together with the fact that homologous genes have been found in various organisms including Schizosaccharomyces pombe, suggests that CNBP plays a basic biological role in eukaryotic cells . The recombinant GST-CNBP fusion protein produced in Escherichia coli bound to a G-rich single-stranded RNA and DNA in a sequence-specific manner.

J Eukaryot Microbiol, 1995 Jan-Feb, 42(1), 12 - 9
Transcription factor genes from rat Pneumocystis carinii; Sunkin SM et al.; Genes encoding the TFIID TATA-box binding protein (TBP) from two probable species of rat Pneumocystis carinii (prototype and variant) were sequenced . The two P . carinii TBP gene sequences were 91% identical to each other, and 65-77% identical to TBP genes from other species . A cDNA from one of the two P . carinii TBP genes was sequenced, which showed that four small introns resided in identical positions within the TBP genes from the prototype and variant rat P . carinii . Conservation of the 180 amino acids that constitute the conserved core of TBP was 97% between the P . carinii TBP, which were 95% and 97% identical to conserved core sequences of TBP from Saccharomyces cerevisiae and Schizosaccharomyces pombe respectively.

Dev Comp Immunol, 1995 Jan-Feb, 19(1), 13 - 9
Antifungal activity of Aplysianin E, a cytotoxic protein of sea hare (Aplysia kurodai) eggs; Iijima R et al.; We observed for the first time that antifungal activity was exhibited by Aplysianin E (AKE), an antineoplastic and antibacterial glycoprotein purified from the eggs of Aplysia kurodai . AKE completely suppressed growth of the yeast form fungi, Saccharomyces cerevisiae A 5 8 1 A, Schizosaccharomyces pombe JY 1 and Candida albicans ATCC 36232 at a concentration of over 16 micrograms/mL . The colony-forming abilities of the fungi were also significantly decreased after contact with AKE . These results indicate that AKE has an antifungal property and that its mode of action is fungicidal.

Gene, 1994 Dec 30, 151(1-2), 243 - 6
A Drosophila melanogaster homolog of the TIS11 family of immediate early genes that can rescue a cdr1 cdc25 mutant strain of fission yeast; Warbrick E et al.; A Drosophila melanogaster (Dm) embryonic cDNA library was screened for genes capable of inhibiting wee1+/mik1+ protein kinase (Pk) function . We expected to identify homologs of the Schizosaccharomyces pombe gene nim1+ . This gene encodes a Pk capable of phosphorylating and so inhibiting the wee1+ Pk that in turn inhibits p34cdc2 . Dm cDNAs capable of complementing the temperature-sensitive phenotype of a nim1/cdr1 cdc25 double mutant strain were identified and found to fall into two classes . One class encodes the Dm Cdc2 protein . The second cDNA class encodes a novel protein containing a central motif consisting of two tandem repeats of a putative Zn(2+)-finger motif . This region is highly conserved in the TIS11 family of immediate early genes, which in mammalian cells are rapidly and transiently induced in response to 12-O-tetradecanoyl phorbol-13-acetate (TPA) and to mitogens such as epidermal growth factor and fibroblast growth factor.

Gene, 1994 Dec 30, 151(1-2), 215 - 20
The Schizosaccharomyces pombe pka1 gene, encoding a homolog of cAMP-dependent protein kinase; Yu G et al.; We have isolated 16 independent Schizosaccharomyces pombe cDNA clones that suppress the temperature-sensitive (ts) phenotype of a Saccharomyces cerevisiae strain containing the dominant-negative RAS2val19ala22 allele . Fourteen of these cDNAs encode Sz . pombe Ras1 . The other two clones encode the C-terminal region of a protein we have named Pka1 . We have cloned the pka1 gene from a Sz . pombe genomic library . It contains an uninterrupted open reading frame encoding a 512-amino-acid (aa) protein . The C-terminal region (aa 200-512) of Pka1 is 51-63% identical to cAMP-dependent protein kinase (Pka) catalytic subunits from other eukaryotes . Production of Pka1 suppresses the ts phenotypes exhibited by Sa . cerevisiae ras1-ras2ts or cyr1ts strains . Furthermore, overproduction of Pka1 in Sz . pombe results in a sterile phenotype and an abnormal morphology similar to that exhibited by cells in which the cAMP pathway is constitutively activated . These observations suggest that pka1 encodes the Sz . pombe Pka catalytic subunit.

Proc Natl Acad Sci U S A, 1994 Dec 20, 91(26), 12832 - 6
Comparisons of eukaryotic genomic sequences; Karlin S et al.; A method for assessing genomic similarity based on relative abundances of short oligonucleotides in large DNA samples is introduced . The method requires neither homologous sequences nor prior sequence alignments . The analysis centers on (i) dinucleotide (and tri- and tetra-) relative abundance extremes in genomic sequences, (ii) distances between sequences based on all dinucleotide relative abundance values, and (iii) a multidimensional partial ordering protocol . The emphasis in this paper is on assessments of general relatedness of genomes as distinguished from phylogenetic reconstructions . Our methods demonstrate that the relative abundance distances almost always differ more for genomic interspecific sequence comparisons than for genomic intraspecific sequence comparisons, indicating congruence over different genome sequence samples . The genomic comparisons are generally concordant with accepted phylogenies among vertebrate and among fungal species sequences . Several unexpected relationships between the major groups of metazoa, fungal, and protist DNA emerge, including the following . (i) Schizosaccharomyces pombe and Saccharomyces cerevisiae in dinucleotide relative abundance distances are as similar to each other as human is to bovine . (ii) S . cerevisiae, although substantially far from, is significantly closer to the vertebrates than are the invertebrates (Drosophila melanogaster, Bombyx mori, and Caenorhabditis elegans) . This phenomenon may suggest variable evolutionary rates during the metazoan radiations and slower changes in the fungal divergences, and/or a polyphyletic origin of metazoa . (iii) The genomic sequences of D . melanogaster and Trypanosoma brucei are strikingly similar . This DNA similarity might be explained by some molecular adaptation of the parasite to its dipteran (tsetse fly) host, a host-parasite gene transfer hypothesis . Robustness of the methods may be due to a genomic signature of dinucleotide relative abundance values reflecting DNA structures related to dinucleotide stacking energies, constraints of DNA curvature, and mechanisms attendant to replication, repair, and recombination.

FEMS Microbiol Lett, 1994 Dec 15, 124(3), 361 - 5
Mutations which reduce levels of pyruvate dehydrogenase in Schizosaccharomyces pombe cause a requirement for arginine or glutamine; Cavan G et al.; Forty-four mutants of Schizosaccharomyces pombe were isolated which required supplementation with arginine or glutamine . These mutants appear to define three genes, provisionally named agg1, agg2 and agg3 (arginine, glutamine requiring) . Mutants in all three genes were found to have reduced levels of pyruvate dehydrogenase compared to wild-type.

Gene, 1994 Dec 15, 150(2), 281 - 6
Schizosaccharomyces malidevorans and Sz . octosporus homologues of Sz . pombe rad9, a gene that mediates radioresistance and cell-cycle progression; Lieberman HB et al.; The rad9 gene of Schizosaccharomyces pombe is involved in promoting resistance to ionizing radiation and UV light, as well as regulating cell cycle progression after irradiation . We have isolated functional rad9 cognates from two other fission yeasts, Sz . malidevorans and Sz . octosporus, that can restore radioresistance and the radiation-induced G2 delay response to Sz . pombe rad9::ura4 cells . The Sz . pombe and Sz . malidevorans genes are identical at the nucleotide sequence level, which reflects their close evolutionary relationship . Each bears three introns and codes for a 47 464-Da protein that contain 426 amino acids (aa) . In contrast, Sz . octosporus rad9 contains five introns and codes for a 48 210-Da protein that is 432-aa long . The Sz . pombe rad9 product is only 65% identical and 80% similar to the corresponding Sz . octosporus gene product . All of the strains synthesize a rad9 RNA of approx . 1.6 kb . The presence of a rad9-like gene in these yeasts suggests that the cellular process(es) mediated by rad9, and used by these organisms to increase survival and transiently delay cycling in G2 after irradiation, are conserved . The isolation, analyses and comparison of rad9 genes from different organisms should aid in elucidating the specific biological role of the corresponding protein and especially help pinpoint regions important for function.

Gene, 1994 Dec 15, 150(2), 275 - 80
A copy-number-controlled expression vector for the fission yeast Schizosaccharomyces pombe; Tohda H et al.; A novel expression vector for the fission yeast Schizosaccharomyces pombe carries the neomycin-resistance-encoding gene regulated by the SV40 early promoter, and its copy number is controlled by the level of Geneticin (G418) . Foreign gene expression is driven by the human cytomegalovirus (hCMV) promoter which is transcriptionally active in S . pombe . Moreover, the vector expresses foreign genes at high levels, due to the 5'-untranslated region (5'-UTR) containing an A + T-rich sequence of about 50 nucleotides located between the TATA box of the hCMV promoter and the start codon . Recombinant human lipocortin I was produced at levels of up to 50% of the total soluble protein in the presence of 100-200 micrograms/ml of G418 in the media . Southern and Northern blotting showed that this high level of expression was due to an increase in copy number induced by G418, the high transcriptional activity of the hCMV promoter and the high translational efficiency of the 5'-UTR . We modified the vector into an 'ATG vector', named pTL2M, that maintains the 5'-UTR optimized for gene expression and into which any foreign gene, whose exact sequence is known, can be easily inserted.

EMBO J, 1994 Dec 15, 13(24), 5910 - 21
Isolation and characterization of krp, a dibasic endopeptidase required for cell viability in the fission yeast Schizosaccharomyces pombe; Davey J et al.; The activation of pro-hormones and many precursor proteins involves cleavage by endopeptidases belonging to the subtilisin-like family of enzymes . Here we describe the isolation and characterization of the first member of this family from the fission yeast Schizosaccharomyces pombe . The enzyme, which has been named krp for KEX2-related protease, is a type I membrane-bound endopeptidase that cleaves substrates after pairs of dibasic residues . It appears to be synthesized as a pre-pro-protein that is likely to undergo processing following translocation into the endoplasmic reticulum . Processing has been characterized in a cell-free translation/translocation system prepared from Xenopus eggs . Krp is N-glycosylated on all five of its potential sites and both the pre-sequence and the pro-sequence are quickly removed following translocation, the latter probably by autocatalytic cleavage . The inhibitor profile of krp broadly reflects the known properties of the eukaryotic subtilisin proteases, while its pH and Ca2+ dependence are consistent with it being active within the secretory pathway . One of its physiological substrates is likely to be the pheromone precursor pro-P-factor, which it is shown to process in an in vitro system, but identification of other substrates is complicated because, unlike other members of this family, krp is essential for cell viability.

Nucleic Acids Res, 1994 Dec 11, 22(24), 5296 - 301
Cloning of cDNAs from Arabidopsis thaliana that encode putative protein phosphatase 2C and a human Dr1-like protein by transformation of a fission yeast mutant; Kuromori T et al.; We characterized three Arabidopsis thaliana cDNA clones that could rescue the sterile phenotype of the Schizosaccharomyces pombe pde1 mutant, which is defective in cAMP phosphodiesterase . The first clone had a coding capacity of 399 amino acids that is 35% identical with rat protein phosphatase 2C (PP2C) . The second had a coding capacity of 159 amino acids that is 41% identical with human Dr1 . Dr1 has been shown to interact with TATA-binding protein (TBP) and block its ability to activate transcription . The third encoded Arabidopsis TBP itself . Saccharomyces cerevisiae TBP also could suppress the sterile phenotype if expressed in S.pombe pde1 cells, but overexpression of S.pombe TBP could do so very poorly . These observations suggest preliminarily that PP2C may counteract cAMP-dependent protein kinase in fission yeast cells, and that the heterologous TBPs and Dr1 may interfere with the general transcription factors of S.pombe so that the gene expression in the host cell becomes affirmative of sexual development . Furthermore, the identification of a Dr1-like protein in A.thaliana strongly argues for the ubiquity of this protein among eukaryotic genera and for a conserved mechanism to regulate transcription initiation that involves Dr1.

Nucleic Acids Res, 1994 Dec 11, 22(24), 5289 - 95
Identification of two mismatch-binding activities in protein extracts of Schizosaccharomyces pombe; Fleck O et al.; We have performed band-shift assays to identify mismatch-binding proteins in cell extracts of Schizosaccharomyces pombe . By testing heteroduplex DNA containing either a T/G or a C/C mismatch, two distinct band shifts were produced in the gels . A low mobility complex was observed with the T/G substrate, while a high mobility complex was present with C/C . Further analysis of the mismatch-binding specificities revealed that the T/G binding activity also binds to T/C, C/T, T/T, T/-, A/-, C/-, G/-, G/G, A/A, A/C, A/G, G/T, G/A, and C/A substrates with varying efficiencies, but not binds to C/C . The C/C binding activity efficiently binds to C/C, T/C, C/T, C/A, A/C, C/-, and weakly also to T/T, while all other mispairs are not recognized . Protein extracts of a mutant strain, defective in the mutS homologue swi4, displayed both mismatch-binding activities . Thus, swi4 does not encode for either one of the mismatch-binding proteins.

Nucleic Acids Res, 1994 Dec 11, 22(24), 5279 - 88
Hac1: a novel yeast bZIP protein binding to the CRE motif is a multicopy suppressor for cdc10 mutant of Schizosaccharomyces pombe; Nojima H et al.; We cloned by phenotypic complementation a novel Saccharomyces cerevisiae's multicopy suppressor of the Schizosaccharomyces pombe cdc10-129 mutant which we call HAC1, an acronym of 'homologous to ATF/CREB 1' . It encodes a bZIP (basic-leucine zipper) protein of 230 amino acids with close homology to the mammalian ATF/CREB transcription factor and gel-retardation assays showed that it binds specifically to the CRE motif . HAC1 is not essential for viability . However, the hac1 disruptant becomes caffeine sensitive, which is suppressed by multicopy expression of the yeast PDE2 (Phosphodiesterase 2) gene . Although the mRNA level of HAC1 is almost constitutive throughout the cell cycle, it fluctuates during meiosis . The upstream region of the HAC1 gene contains a T4C site, a URS (upstream repression sequence) and a TR (T-rich) box-like sequence, which reside upstream of many meiotic genes . These results suggest that HAC1 may also be one of the meiotic genes.

Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 12046 - 50
Covalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases; Shuman S et al.; Formation of the 5' cap structure of eukaryotic mRNAs occurs via transfer of GMP from GTP to the 5' terminus of the primary transcript . RNA guanylyltransferase, the enzyme that catalyzes this reaction, has been isolated from many viral and cellular sources . Though differing in molecular weight and subunit structure, the various guanylyltransferases employ a common catalytic mechanism involving a covalent enzyme-(Lys-GMP) intermediate . Saccharomyces cerevisiae CEG1 is the sole example of a cellular capping enzyme gene . In this report, we describe the identification and characterization of the PCE1 gene encoding the capping enzyme from Schizosaccharomyces pombe . PCE1 was isolated from a cDNA library by functional complementation in Sa . cerevisiae . Induced expression of PCE1 in bacteria and in yeast confirmed that the 47-kDa Sc . pombe protein was enzymatically active . The amino acid sequence of PCE1 is 38% identical (152 of 402 residues) to the 52-kDa capping enzyme from Sa . cerevisiae . Comparison of the two cellular capping enzymes with guanylyltransferases encoded by DNA viruses revealed local sequence similarity at the enzyme's active site and at four additional collinear motifs . Mutational analysis of yeast CEG1 demonstrated that four of the five conserved motifs are essential for capping enzyme function in vivo . Remarkably, the same motifs are conserved in the polynucleotide ligase family of enzymes that employ an enzyme-(Lys-AMP) intermediate . These findings illuminate a shared structural basis for covalent catalysis in nucleotidyl transfer and suggest a common evolutionary origin for capping enzymes and ligases.

J Biol Chem, 1994 Dec 2, 269(48), 30701 - 6
Purification to homogeneity of UDP-glucose:glycoprotein glucosyltransferase from Schizosaccharomyces pombe and apparent absence of the enzyme fro Saccharomyces cerevisiae; Fernandez FS et al.; The UDP-Glc:glycoprotein glucosyltransferase was purified to homogeneity from the fission yeast Schizosaccharomyces pombe . The enzyme has been recently suggested to be involved in the mechanism by which unfolded, partially folded, or misfolded glycoproteins are retained in the endoplasmic reticulum . The pure yeast glucosyltransferase formed protein-linked Glc1-Man9GlcNAc2,Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2 when incubated with UDP-Glc and denatured thyroglobulin . The same compounds were formed upon glucosylation of endogenous acceptors by crude microsomes . The enzyme was a soluble microsomal protein that required Ca2+ for activity, used UDP-Glc and not TDP-Glc, ADP-Glc, or UDP-Gal as sugar donor, had an almost neutral optimum pH value, and as the glucosyl-transferase obtained from rat liver, glucosylated denatured but not native glycoproteins or glycopeptides . A similar enzymatic activity could not be detected in Saccharomyces cerevisiae microsomes and transient glucosylation of glycoproteins (addition of a single glucose unit to glucose-free oligosaccharides by the glucosyltransferase followed by its removal by glucosidase II) could not be detected in intact S . cerevisiae cells . These are the only eukaryotic cells described so far in which these processing reactions of the endoplasmic reticulum do not occur . Availability of the pure S . pombe enzyme will eventually allow testing the possible involvement of the glucosyltransferase in sensing glycoprotein tertiary structures in the endoplasmic reticulum.

J Biol Chem, 1994 Dec 2, 269(48), 30530 - 7
mik1+ encodes a tyrosine kinase that phosphorylates p34cdc2 on tyrosine 15; Lee MS et al.; mik1+ and wee1+ function to regulate the tyrosine phosphorylation of p34cdc2 in Schizosaccharomyces pombe (Lundgren, K., Walworth, N., Booher, R., Dembski, M., Kirschner, M., and Beach, D . (1991) Cell 64, 1111-1122) . wee1+ encodes a tyrosine kinase that directly phosphorylates p34cdc2 on tyrosine 15, resulting in the inactivation of the cyclin B/p34cdc2 complex . We have overproduced the mik1+ gene product in insect cells and in S . pombe in order to characterize it biochemically . Immunoprecipitates of Mik1 from both sources catalyzed the phosphorylation of p34cdc2 on tyrosine 15 whereas immunoprecipitates of a kinase-deficient mutant of Mik1 were negative in this assay . Mik1 overproduced in insect cells was partially purified by column chromatography, and column fractions were assayed for their ability to phosphorylate p34cdc2 on tyrosine 15 . Two major peaks of Mik1 protein were detected by gel filtration chromatography . One peak eluted in the void volume, and a second peak eluted with an apparent molecular mass expected for monomeric Mik1 (approximately 68 kDa) . The tyrosine 15 kinase activity co-eluted with the 68 kDa form of Mik1 . These results indicate that mik1+ encodes a tyrosine kinase that directly phosphorylates p34cdc2 on tyrosine 15.

Mol Cell Biol, 1994 Dec, 14(12), 7839 - 54
The Srp54 GTPase is essential for protein export in the fission yeast Schizosaccharomyces pombe; Althoff SM et al.; Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein required for targeting a subset of presecretory proteins to the endoplasmic reticulum (ER) membrane . Here we report the results of a series of experiments to define the function of the Schizosaccharomyces pombe homolog of the 54-kDa subunit of mammalian SRP . One-step gene disruption reveals that the Srp54 protein, like SRP RNA, is essential for viability in S . pombe . Precursor to the secretory protein acid phosphatase accumulates in cells in which Srp54 synthesis has been repressed under the control of a regulated promoter, indicating that S . pombe SRP functions in protein targeting . In common with other Srp54 homologs, the S . pombe protein has a modular structure consisting of an amino-terminal G (GTPase) domain and a carboxyl-terminal M (methionine-rich) domain . We have analyzed the effects of 17 site-specific mutations designed to alter the function of each of the four GTPase consensus motifs individually . Several alleles, including some with relatively conservative amino acid substitutions, confer lethal or conditional phenotypes, indicating that GTP binding and hydrolysis are critical to the in vivo role of the protein . Two mutations (R to L at position 194 {R194L} and R194H) which were designed, by analogy to oncogenic mutations in rats, to dramatically decrease the catalytic rate and one (T248N) predicted to alter nucleotide binding specificity produce proteins that are unable to support growth at 18 degrees C . Consistent with its design, the R194L mutant hydrolyzes GTP at a reduced rate relative to wild-type Srp54 in enzymatic assays on immunoprecipitated proteins . In strains that also contain wild-type srp54, this mutant protein, as well as others designed to be locked in a GTP-bound conformation, exhibits temperature-dependent dominant inhibitory effects on growth, while a mutant predicted to be GDP locked does not interfere with the function of the wild-type protein . These results form the basis of a simple model for the role of GTP hydrolysis by Srp54 during the SRP cycle.

Jpn J Genet, 1994 Dec, 69(6), 671 - 8
Molecular cloning and characterization of a fission yeast gene responsible for supersensitivity to the spindle poison, isopropyl N-3-chlorophenyl carbamate; Ishiguro J et al.; The cps3 gene of the fission yeast, Schizosaccharomyces pombe, was previously identified as a mutation conferring supersensitivity to the spindle poison, isopropyl N-3-chlorophenyl carbamate (CIPC) . A 3.2 kb DNA fragment that complements the mutant phenotype was cloned from a S . pombe genomic library . The base sequence analysis showed that the fragment contains a maximum 1086 nucleotide open reading frame and that the putative product consists of 362 amino acids, having a molecular weight of 39.3 KDa . No significant homology of the potential product with known proteins could be found by database searches . A disruptant of the gene, produced by insertion of a ura4+ fragment was able to germinate, but not to undergo cell division, suggesting that the gene to be essential for the cell cycle progression . The disruption experiment suggests that the gene is an extragenic suppressor of cps3 mutation.

Mol Gen Genet, 1994 Dec 1, 245(5), 654 - 7
A Drosophila gene encoding a DEAD box RNA helicase can suppress loss of wee1/mik1 function in Schizosaccharomyces pombe; Warbrick E et al.; We describe a screen to isolate cDNAs encoding Drosophila mitosis inhibitors capable of suppressing the mitotic catastrophe phenotype resulting in Schizosaccharomyces pombe from the combination of the wee1-50 mutation with either a deletion allele of mik1, or with overexpression of cdc25+ . One plasmid was isolated which could suppress the temperature sensitive lethality of both these strains . The cDNA in this plasmid encodes a protein highly homologous to the DEAD-box family of ATP-dependent RNA helicases, rather than to protein kinases as might be expected . It is possible that the RNA helicase described here may regulate entry into mitosis by down regulating the expression of other genes whose activity may be rate-limiting for entry into mitosis.

Mol Gen Genet, 1994 Dec 1, 245(5), 628 - 35
Analysis of a histone H2A variant from fission yeast: evidence for a role in chromosome stability; Carr AM et al.; We have isolated and characterised the pht1 gene from the fission yeast Schizosaccharomyces pombe . The sequence of the predicted translation product has revealed a striking similarity to the family of H2A.F/Z histone variant proteins, which have been found in a variety of different organisms . Cells deleted for the pht1 gene locus grow slowly, exhibit an altered colony morphology, increased resistance to heat shock and show a significant decrease in the fidelity of segregation of an S . pombe minichromosome . We propose that the histone H2A variant encoded by the pht1 gene is important for chromosomal structure and function, possibly including a role in controlling the fidelity of chromosomal segregation during mitosis.

Protein Sci, 1994 Dec, 3(12), 2185 - 93
Six new candidate members of the alpha/beta twisted open-sheet family detected by sequence similarity to flavodoxin; Grandori R et al.; Strong sequence similarity has been reported among WrbA (the Trp repressor-binding protein of Escherichia coli); Ycp4, a protein of unknown function from the budding yeast Saccharomyces cerevisiae; P25, the pap1-dependent protein of the fission yeast Schizosaccharomyces pombe; and the translation product of a partial cDNA sequence from rice seedling root (Oryza sativa, locus Ricr02421a; here referred to as RicR) . Further homology search with the profile method indicates that all the above sequences are related to the flavodoxin family and, in turn, allows detection of the recently proposed flavodoxin-like proteins from E . coli, MioC and the hypothetical protein YihB . We discuss sequence conservation with reference to the known 3-dimensional structures of flavodoxins . Conserved sequence and hydrophobicity patterns, as well as residue-pair interaction potentials, strongly support the hypothesis that these proteins share the alpha/beta twisted open-sheet fold typical of flavodoxins, with an additional alpha/beta unit in the WrbA family . On the basis of the proposed structural homology, we discuss the details of the putative FMN-binding sites . Our analysis also suggests that the helix-turn-helix motif we identified previously in the C-terminal region of the WrbA family is unlikely to reflect a DNA-binding function of this new protein family.

Yeast, 1994 Dec, 10(12), 1657 - 62
Sequence analysis of a 40.2 kb DNA fragment located near the left telomere of yeast chromosome X; Vandenbol M et al.; We have sequenced on both strands a 40,257 bp fragment located near the left telomere of chromosome X of Saccharomyces cerevisiae . The sequenced segment contains 21 open reading frames (ORFs) at least 100 amino acids long . Five of the ORFs correspond to known amino acid sequences: two hypothetical proteins in the subtelomeric Y' repeat region of 65.4 and 12.8 KDa, the cytochrome B pre-mRNA processing CBP1 protein, the mitochondrial nuclease NUC1 and the CRT1 protein . Of the 16 remaining ORFs, eight show highest homologies with the S . cerevisiae hexose transporters family (two ORFs), the yeast alpha-glucosidase (two ORFs), the yeast PEP1 precursor, the Escherichia coli galactoside O-acetyltransferase, the S . cerevisiae 137.7 KDa protein located in the Y' region and a protein of unknown function of Schizosaccharomyces pombe . Finally, eight of the ORFs exhibit no significant similarity with any amino acid sequences described in data banks . DNA sequence comparison has revealed the presence of different repeated elements characteristic of yeast chromosome ends . Disruption studies have been performed on two ORFs encoding putative proteins of unknown function.

Yeast, 1994 Dec, 10(12), 1631 - 8
Characterization of an active GST-human Cdc2 fusion protein kinase expressed in the fission yeast Schizosaccharomyces pombe: a new approach to the study of cell cycle control proteins; Leroy D et al.; Characterization of cdk (cyclin dependent kinases) substrates and studies of their regulation require purified enzymatic complexes of cdc2-related catalytic and cyclin regulatory subunits . We produced human Cdc2 kinase in the fission yeast Schizosaccharomyces pombe as a fusion protein with glutathione S-transferase (GST) . The GST-human Cdc2p fusion protein was active in vivo since it rescued a temperature-sensitive allele of cdc2 . The fusion protein was purified using a one-step chromatography procedure with glutathione-Sepharose and exhibited a catalytic activity in vitro . Yeast cyclin B and suc1 were found in association with GST-Cdc2 . A 17-fold stimulation of GST-Cdc2 kinase activity was obtained by incubation of recombinant human cyclin A with the S . pombe cellular extract prior to affinity purification . This indicates that cyclin concentration is limiting in this overexpression system . These findings describe a fast and easy production of active recombinant human Cdc2 kinase in yeast that can be used for biochemical studies.

Eur J Cell Biol, 1994 Dec, 65(2), 298 - 304
Cytosolic factors block antibody binding to the C-terminal cytoplasmic tail of the KDEL receptor; Tang BL et al.; The mammalian KDEL receptor is an extremely hydrophobic membrane protein . One of the longest stretches of hydrophilic sequence resides at the C-terminus . Various antibodies against a synthetic peptide corresponding to this region confirmed that the C-terminus is exposed to the cytoplasm . It was observed that antibody binding to the C-terminus of the KDEL receptor was diminished during immunofluorescence microscopy procedures which involved fixation prior to permeabilization as compared to when cells were permeabilized before fixation . Binding of both polyclonal and monoclonal antibodies, as assessed by indirect immunofluorescence microscopy in digitonin permeabilized cells, was inhibited by preincubation with rat liver cytosol . This inhibition was not observed with antibody against another membrane protein (p28) with a cytoplasmically exposed epitope also residing in the Golgi/intermediate compartment . Rabbit reticulocyte lysate had a similar effect while Schizosaccharomyces pombe cytosol inhibited binding to a greater degree than Saccharomyces cerevisiae cytosol . This inhibition by cytosol was prevented by coincubation with the antibody and was dose-dependent on the cytosol . Inhibition did not occur on ice or at 15 degrees C, or when the cytosol was energy-depleted by apyrase treatment . Interestingly, pretreatment of permeabilized cells with N-ethylmaleimide or its addition into the incubation mixture abolished inhibition . N-ethylmaleimide-treated cytosol, however, remained inhibitory . The findings suggest the existence of cytosolic factor (s) which interacts specifically with the cytoplasmic C-terminus of the KDEL receptor, which are likely to be components of the KDEL protein retrieval machinery.

J Cell Sci, 1994 Dec, 107 ( Pt 12), 3635 - 42
Transcriptional regulation of a Ras nucleotide-exchange factor gene by extracellular signals in fission yeast; Hughes DA et al.; The ste6 gene of Schizosaccharomyces pombe encodes a putative GDP-GTP exchange factor for the ras1 gene product . Genetic analysis of the ste6 and ras1 genes has shown that they are required for mating and for the response to mating pheromones . In this study we show that expression of the ste6-encoded mRNA is induced by nitrogen starvation, the physiological signal that triggers mating and sexual differentiation . Exposure to mating pheromones enhances the induction of ste6 expression upon nitrogen starvation . Pheromone-induced expression requires not only the function of components of the pheromone-signalling pathway, but also ras1 function . Furthermore, mutants in which the Ras1 protein is activated have higher basal and induced levels of ste6 gene expression than wild-type cells . These observations indicate the existence of a positive-feedback loop through which Ras1 stimulates the expression of its own activator . Since Ste6 is likely to promote the exchange of guanine nucleotides on Ras1 protein, our results suggest an important role for GDP-GTP exchange in the regulation of Ras1 activity during the mating process in S . pombe.

Biochem Pharmacol, 1994 Nov 29, 48(11), 2139 - 41
Dnacin A1 and dnacin B1 are antitumor antibiotics that inhibit cdc25B phosphatase activity; Horiguchi T et al.; The p80cdc25 protein is a protein phosphatase directly involved in p34cdc2 protein kinase activation by dephosphorylation . The cdc25B gene is one of three human cdc25 homologs which can complement the temperature-sensitive cdc25 mutation of Schizosaccharomyces pombe, and is expressed a high levels in human cell lines, particularly in some cancer cells . A fusion protein of glutathione-S-transferase (GST) and the catalytic domain of cdc25B protein was constructed and found to retain phosphatase activity in the manner of a p80cdc25 phosphatase by using a chromogenic substrate, p-nitrophenylphosphate . Two benzoquinoid antitumor compounds, dnacin A1 and dnacin B1, inhibited phosphatase activity in a non-competitive manner.

J Biol Chem, 1994 Nov 18, 269(46), 28535 - 8
Structural and functional homology between mammalian DNase IV and the 5'-nuclease domain of Escherichia coli DNA polymerase I; Robins P et al.; A nuclear 42-kDa 5'-->3'-exonuclease, DNase IV, was found previously in animal tissues . The enzyme has been purified from HeLa cells and shown to possess two catalytic properties characteristic of the 5'-nuclease function of Escherichia coli DNA polymerase I,-DNase IV removes single-stranded 5' regions from splayed-arm DNA structures by endonucleolytic incision at the bifurcation point and possesses RNase H activity . Determination of the molecular masses of tryptic and V8 peptides of DNase IV by mass spectrometry identified the enzyme as the human homolog of the Schizosaccharomyces pombe Rad2 protein . The protein sequence retains conserved residues and shows significant homology to the sequences of the 5'-nuclease domain of E . coli DNA polymerase I and related microbial enzymes.

J Biol Chem, 1994 Nov 11, 269(45), 27996 - 9
Low molecular weight protein-tyrosine phosphatases are highly conserved between fission yeast and man; Mondesert O et al.; Cdc25 protein phosphatase dephosphorylates tyrosine 15 of Cdc2, thereby activating Cdc2/cyclin B kinase, which then brings about mitosis . A fission yeast (Schizosaccharomyces pombe) cDNA expression library was screened for clones that rescue cdc25-22 . In addition to the cdc25+ and pyp3+ protein-tyrosine phosphatase genes, a third gene was discovered . This gene, named stp1+ (small tyrosine phosphatase), encodes a approximately 17.5-kDa protein that is approximately 42% identical to members of an unusual class of small (approximately 18 kDa) cytosolic phosphatases previously known to exist only in mammalian species . The biological functions of these proteins are unknown, but they have vigorous protein-tyrosine phosphatase activity in vitro and have a sequence motif, Cys-X5-Arg, that is present at the active sites of all known types of protein-tyrosine phosphatases . Sequence homology between S . pombe Stp1 and its mammalian homologs is particularly high in the active site region of the proteins . Rescue of cdc25-22 by overproduction of Stp1 protein is probably due to an ability of Stp1 to dephosphorylate tyrosine 15 of Cdc2 . Disruption of stp1+ causes no obvious phenotype . The fact that Stp1 homologs are highly conserved between yeast and man suggests that they have important functions.

J Bacteriol, 1994 Nov, 176(21), 6631 - 5
Cloning, nucleotide sequence, and regulation of Schizosaccharomyces pombe thi4, a thiamine biosynthetic gene; Zurlinden A et al.; thi4 mutants of Schizosaccharomyces pombe exhibit defective thiamine biosynthesis, and thi4 mutations define a gene which is believed to be involved in the phosphorylation of 4-amino-5-hydroxymethyl-2-methylpyrimidine or 5-(2-hydroxyethyl)-4-methylthiazole and/or in the coupling of the two phosphorylated precursors to thiamine monophosphate (A . M . Schweingruber, J . Dlugonski, E . Edenharter, and M . E . Schweingruber, Curr . Genet . 19:249-254, 1991) . The thi4 gene was cloned by functional complementation of a thi4 mutant and physically mapped on the left arm of chromosome I close to the genetic marker gln1 . The thi4-carrying DNA fragment shows an open reading frame encoding a protein of 518 amino acids and a calculated molecular mass of 55.6 kDa . The appearance of thi4 mRNA is strongly repressed by thiamine and to a lesser extent by 5-(2-hydroxyethyl)-4-methylthiazole . thi4 mRNA production is under the control of the thi1 gene-encoded transcription factor and of the negative regulators encoded by genes tnr1, tnr2, and tnr3 . thi4 is expressed and regulated in manners similar to those of other S . pombe genes involved in thiamine metabolism, including thi2, thi3, and pho4.

EMBO J, 1994 Nov 1, 13(21), 5212 - 9
Preferential strand transfer and hybrid DNA formation at the recombination hotspot ade6-M26 of Schizosaccharomyces pombe; Schar P et al.; The ade6-M26 mutation of Schizosaccharomyces pombe stimulates intragenic and intergenic meiotic recombination . M26 is a single base pair change creating a specific heptanucleotide sequence that is crucial for recombination hotspot activity . This sequence is recognized by proteins that may facilitate rate-limiting steps of recombination at the ade6 locus . To start the elucidation of the intermediate DNA structures formed during M26 recombination, we have analyzed the aberrant segregation patterns of two G to C transversion mutations flanking the heptanucleotide sequence in crosses homozygous for M26 . At both sites the level of post-meiotic segregation is typical for G to C transversion mutations in S . pombe in general . Quantitative treatment of the data provides strong evidence for heteroduplex DNA being the major recombination intermediate at the M26 site . We can now exclude a double-strand gap repair mechanism to account for gene conversion across the recombination hotspot . Furthermore, the vast majority (> 95%) of the heteroduplexes covering either of the G to C transversion sites are produced by transfer of the transcribed DNA strand . These results are consistent with ade6-M26 creating an initiation site for gene conversion by the introduction of a single-strand or a double-strand break in its vicinity, followed by transfer of the transcribed DNA strands for heteroduplex DNA formation.

Mol Endocrinol, 1994 Nov, 8(11), 1455 - 64
Reconstitution of thyroid hormone receptor and retinoic acid receptor function in the fission yeast Schizosaccharomyces pombe; Sande S et al.; We report here a characterization of the thyroid hormone receptors (T3Rs), retinoic acid receptors (RARs), and retinoid X receptors (RXRs) by reconstituting their actions in the fission yeast Schizosaccharomyces pombe . S . pombe provide a well defined and readily manipulated genetic background devoid of known endogenous nuclear hormone receptors . All the receptors tested, when introduced exogenously into S . pombe, induced high levels of reporter gene activation in response to physiological concentrations of hormone ligand . In these properties, the S . pombe system exhibits significant advantages over the previously employed Saccharomyces cerevisiae system . Use of the S . pombe system permitted the elucidation of previously undescribed differences in the DNA sequence recognition properties of different isoforms of the RXR and RARs, and the identification of apparently novel forms of response element for RXRs and RARs . Intriguingly, the v-erb A allele of T3R, a transcriptional repressor in vertebrate cells, acts as a transcriptional activator both in S . cerevisiae and in the evolutionarily highly divergent S . pombe, underscoring the importance of cellular factors in the regulation of receptor transcriptional activity.

Curr Genet, 1994 Nov-Dec, 26(5-6), 553 - 6
Physical mapping of the Schizosaccharomyces pombe histone genes; Lind M et al.; The histone-encoding genes in Schizosaccharomyces pombe were physically mapped by hybridisation to filters containing cosmid and P1 genomic libraries . The H2A.2 gene and the H2A.1-H2B.1 gene pair mapped between the ade6 and rikI genes on chromosome III . The three H4-H3 gene pairs were mapped to three different regions by a H4.1 probe . Southern analysis of clones from each region revealed the positions of the three H4-H3 gene pairs . H4.1-H3.1 was localised to chromosome I between the mei2 and rad1 genes; H4.2-H3.2 mapped between rad3 and cdc2 on chromosome II; H4.3-H3.3 was localised to a region between the nuc1 and puc1 genes on chromosome II.

Genetics, 1994 Nov, 138(3), 621 - 32
The mutator gene swi8 effects specific mutations in the mating-type region of Schizosaccharomyces pombe; Fleck O et al.; The swi8+ gene of Schizosaccharomyces pombe appears to be involved in the termination step of copy synthesis during mating-type (MT) switching . Mutations in swi8 confer a general mutator phenotype and, in particular, generate specific mutations in the MT region . Sequencing of the MT cassettes of the h90 swi8-137 mutant revealed three altered sites . One is situated at the switching (smt) signal adjacent to the H1 homology box of the expression locus mat1:1 . It reduces the rate of MT switching . The alteration at the smt signal arose frequently in other h90 swi8 strains and is probably caused by gene conversion in which the sequence adjacent to the H1 box of mat2:2 is used as template . This change might be generated during the process of MT switching when hybrid DNA formation is anomalously extended into the more heterologous region flanking the H1 homology box . In addition to the gene conversion at mat1:1, two mutations were found in the H3 homology boxes of the silent cassettes mat2:2 and mat3:3.

J Exp Biol, 1994 Nov, 196, 483 - 91
Hexose/H+ symporters in lower and higher plants; Caspari T et al.; A well-studied transporter of plant cells is the hexose/H+ symporter of the unicellular alga Chlorella kessleri . Its properties, studied in vivo, are briefly summarized . In part, they are atypical and it has been suggested that this porter acts in an asymmetric way . Three genes coding for Chlorella hexose transport activity have been identified (HUP1, HUP2 and HUP3) . HUP1 cDNA expressed in a mutant of Schizosaccharomyces pombe not transporting any D-glucose has been studied in detail . Several mutants with changed Km values for substrate were obtained, some by random polymerase chain reaction mutation and selection for decreased sensitivity towards the toxic sugar 2-deoxyglucose, some by site-directed mutagenesis . The amino acids affected clustered in the centre of the putative transmembrane helices V, VII and XI . Large families of hexose transporter genes are found in higher plants (Arabidopsis, Chenopodium, Ricinus) . Their functional role is discussed . Finally, the progress made in studying plant transporters in a vesicle system energized by cytochrome c oxidase is summarized.

Insect Mol Biol, 1994 Nov, 3(4), 195 - 20
cDNA structure and characterization of a kinesin-like protein from the silkworm Bombyx mori; Okano K et al.; We have isolated a 1224 bp cDNA clone from a Bombyx mori embryonic cDNA library which contains sequences homologous to the kinesin-like protein gene, ncd, which is required for distribution of chromosomes at meiosis in Drosophila melanogaster females . This clone includes both a microtubule motor and the ATP-binding domains found in kinesin-like proteins . The motor domain is classified in the group of the BimC and cut7, which have a role in spindle formation during mitosis of Aspergillus nidulans and Schizosaccharomyces pombe, respectively . However, the location of the domain at the carboxy terminus is not common in this family, except for ncd and KAR3.

Mutat Res, 1994 Nov, 315(3), 295 - 305
Cloning of human and mouse genes homologous to RAD52, a yeast gene involved in DNA repair and recombination; Muris DF et al.; The RAD52 gene of Saccharomyces cerevisiae is required for recombinational repair of double-strand breaks . Using degenerate oligonucleotides based on conserved amino acid sequences of RAD52 and rad22, its counterpart from Schizosaccharomyces pombe, RAD52 homologs from man and mouse were cloned by the polymerase chain reaction . DNA sequence analysis revealed an open reading frame of 418 amino acids for the human RAD52 homolog and of 420 amino acid residues for the mouse counterpart . The identity between the two proteins is 69% and the overall similarity 80% . The homology of the mammalian proteins with their counterparts from yeast is primarily concentrated in the N-terminal region . Low amounts of RAD52 RNA were observed in adult mouse tissues . A relatively high level of gene expression was observed in testis and thymus, suggesting that the mammalian RAD52 protein, like its homolog from yeast, plays a role in recombination . The mouse RAD52 gene is located near the tip of chromosome 6 in region G3 . The human equivalent maps to region p13.3 of chromosome 12 . Until now, this human chromosome has not been implicated in any of the rodent mutants with a defect in the repair of double-strand breaks.

Mutat Res, 1994 Nov 1, 311(1), 111 - 23
A new shuttle vector system for the identification of spontaneous and radiation-induced mutations in the fission yeast Schizosaccharomyces pombe; Zhao Y et al.; A shuttle vector, pCRR1, has been constructed for the detection of spontaneous and radiation-induced mutations in the fission yeast Schizosaccharomyces pombe . This vector contains an Escherichia coli supF suppressor tRNA gene as the target for mutagenesis and bacterial pMB1 and yeast ars1 replication origins, which can be used to propagate the plasmid in bacterial and fission yeast cells, respectively . supF mutations can be detected after plasmid transformation into S . pombe and recovery in a bacterial indicator system, KS40/pKY241, by selecting for nalidixic acid resistance and/or by screening for lacZ- cells . We found that UV light or gamma-rays induced mutations in a dose-dependent manner in this system . Treatment of ultraviolet light (UV)-irradiated DNA with E . coli photolyase, which monomerizes cyclobutane pyrimidine dimers, before introduction into S . pombe reduced mutation frequencies to nearly background levels, indicating that this type of lesion is the major source of mutations . Comparison of spontaneous and UV-induced mutation frequencies in rad+, rad8-190 and rad13-A cells revealed no significant difference in background levels or induced levels after exposure to 100 J/m2 of UV . However, when plasmid DNA was UV-irradiated with 500 J/m2, the rad8-190 cells generated only 38% as many induced supF mutations as the rad+ strain, whereas the rad13-A cells produced more than a 6-fold increase in mutability relative to the level observed for the wild-type strain . These mutability patterns are consistent with previous studies that characterized rad8-190 cells as hypomutable and rad13-A cells as hypermutable by UV light at chromosomal loci . Thus, this shuttle vector system provides a useful and sensitive tool to assess mutability in S . pombe.

J Mol Biol, 1994 Oct 21, 243(2), 157 - 66
Transposable group II introns in fission and budding yeast . Site-specific genomic instabilities and formation of group II IVS plDNAs; Schmidt WM et al.; The recent report on RNA-mediated group II intron (IVS, intervening sequence) transposition in mitochondria (mt) of Saccharomyces cerevisiae and Podospora anserina and the demonstration of reverse transcriptase (RT) activity encoded by the mobile S . cerevisiae intron cox1-aI1 suggests that group II introns constitute a new class of site-specific retro-like (retroid) elements . This is supported by the finding that the mitochondrial cob1-bI1 intron from the fission yeast Schizosaccharomyces pombe, encoding an RT-like open reading frame, is transposed in mtDNA populations . In agreement with the involvement of an RNA-intermediate in IVS transposition: First, the insertion sites were preceded by at least an IBS1-like (intron binding site) motif, which corresponds to the upstream exon and suffices to form the IBS1/EBS1 (EBS: exon binding site) base-pairing interactions . Second, intron transposition was conservative with respect to sequences flanking the insertion sites . We formulated the hypothesis that transient IVS insertion at non-allelic sites followed by recombination can be viewed as a general molecular mechanism, applicable equally well to site-specific genomic instabilities involving splice-site borders of group II introns and to the formation of extra-genomic IVS plasmid DNAs (plDNAs) . We used polymerase chain reaction (PCR) techniques to detect infrequent rearrangements in mtDNA and report here on duplicative IVS transposition, twintron formation (e.g . bI1 insertion into another bI1 intron), and IVS insertions at canonical 5' exon-intron borders in S . pombe (cob1-bI1) and in S . cerevisiae (cox1-aI1) . These data substantiate the concept that group II intron homing, IVS transposition and circular IVS plDNA formation involve a common RNA-mediated mechanism . Finally, the findings suggest that extra-genomic group II IVS copies are not restricted to senescence mycelia of P . anserina, but constitute natural components of group II IVS-containing genomes.

Mol Gen Genet, 1994 Oct 17, 245(1), 86 - 95
Ethanol-hypersensitive and ethanol-dependent cdc- mutants in Schizosaccharomyces pombe; Jimenez J et al.; Ethanol-hypersensitive strains (ets mutants), unable to grow on media containing 6% ethanol, were isolated from a sample of mutagenized Schizosaccharomyces pombe wild-type cells . Genetic analysis of these ets strains demonstrated that the ets phenotype is associated with mutations in a large set of genes, including cell division cycle (cdc) genes, largely non-overlapping with the set represented by the temperature conditional method; accordingly, we isolated some ets non-ts cdc- mutants, which may identify novel essential genes required for regulation of the S . pombe cell cycle . Conversely, seven well characterized ts cdc- mutants were tested for their ethanol sensitivity; among them, cdc1-7 and cdc13-117 exhibited a tight ets phenotype . Ethanol sensitivity was also tested in strains bearing different alleles of the cdc2 gene, and we found that some of them were ets, but others were non-ets; thus, ethanol hypersensitivity is an allele-specific phenotype . Based on the single base changes found in each particular allele of the cdc2 gene, it is shown that a single amino acid substitution in the p34cdc2 gene product can produce this ets phenotype, and that ethanol hypersensitivity is probably due to the influence of this alcohol on the secondary and/or tertiary structure of the target protein . Ethanol-dependent (etd) mutants were also identified as mutants that can only be propagated on ethanol-containing media . This novel type of conditional phenotype also covers many unrelated genes . One of these etd mutants, etd1-1, was further characterized because of the lethal cdc- phenotype of the mutant cells under restrictive conditions (absence of ethanol) . The isolation of extragenic suppressors of etd1-1, and the complementation cloning of a DNA fragment encompassing the etd1+ wild-type gene (or an extragenic multicopy suppressor) demonstrate that current genetic techniques may be applied to mutants isolated by using ethanol as a selective agent.

J Biol Chem, 1994 Oct 14, 269(41), 25447 - 53
A novel GTP-binding protein which is selectively repressed in SV40 transformed fibroblasts; Schenker T et al.; We have used a subtractive hybridization procedure to isolate cDNA clones for proteins that are produced by human fibroblasts, but not by their SV40-transformed counterparts . With this technique we found, in addition to fibronectin and collagen VI, a novel GTP-binding protein . Sequencing of overlapping cDNA clones demonstrated that this protein is composed of 364 amino acids with a molecular mass of 41 kDa and a calculated isoelectric point of 9.4 . It contains the five sequence motifs G1-G5 that are conserved in all GTP-binding proteins . Apart from these characteristic motifs the amino acid sequence differs substantially from those of the well characterized G-proteins, but it is similar to those of some recently identified proteins from Caenorhabditis elegans, from Schizosaccharomyces pombe, and from an archaebacterium, suggesting the existence of a new subfamily within the superfamily of the GTP-binding proteins . The striking conservation of the primary structure between distantly related species indicates a fundamental function of the new protein . Since it is produced in normal, but not in virally transformed fibroblasts, it may play a role in the expression of the transformed phenotype or in growth control.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 10163 - 7
Km mutants of the Chlorella monosaccharide/H+ cotransporter randomly generated by PCR; Will A et al.; The HUP1 gene codes for the monosaccharide/H+ cotransporter protein of Chlorella kessleri . The gene is functionally expressed in Schizosaccharomyces pombe . This heterologous system has been used to screen for Km mutants of the Chlorella symporter . Since S . pombe transformed with HUP1 cDNA showed a 1000-fold increase in sensitivity toward the toxic sugar analogue 2-deoxyglucose, we screened for transformants with a decreased 2-deoxyglucose sensitivity . The transformants were produced with HUP1 cDNA randomly mutagenized by PCR . From 73 transformants with decreased 2-deoxyglucose sensitivity, four mutants with increased Km values for D-glucose were obtained . The amino acid exchanges responsible for the increased Km values are located in the center of the putative transmembrane helices V (Q179E), VII (Q298R), and XI (V433L/N436Y) . Q179N and Q299N had previously been shown by directed mutagenesis to affect the Km value of the transporter for D-glucose . The drastic mutational changes Q298R and N436Y gave rise to very high Km values; however, the corresponding conservative amino acid changes Q298N or N436Q obtained by directed mutagenesis also result in Km values increased by a factor of 10 or 20, respectively . The data therefore support the proposal that at least helices V, VII, and XI may line the sugar translocation path and determine its specificity . These results are discussed in relation to other sugar transporters and to the interaction of the yeast hexokinase B with D-glucose as known from published crystal structures.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 10084 - 8
Negative regulation of mitosis in fission yeast by catalytically inactive pyp1 and pyp2 mutants; Hannig G et al.; The Schizosaccharomyces pombe genes pyp1+ and pyp2+ encode protein tyrosine phosphatases (PTPases) that act as negative regulators of mitosis upstream of the wee1+/mik1+ pathway . Here we provide evidence that pyp1+ and pyp2+ function independently of cdr1+(nim1+) in the inhibition of mitosis and that the wee1 kinase is not a direct substrate of either PTPase . In a pyp1::ura4 cdc25-22 genetic background, overexpression of either the N-terminal domain of pyp1+ or a catalytically inactive mutant, pyp1C470S, causes cell cycle arrest . This phenotype reverses the suppression of a cdc25 temperature-sensitive mutation at 35 degrees C caused by a pyp1 disruption . Furthermore, pyp1C470S and a catalytically inactive mutant of pyp2, pyp2C630S, induce mitotic delay as do their wild-type counterparts . Analysis of pyp1+ and pyp2+ further reveals that in vitro PTPase activity of pyp1 and pyp2, as well as their biological activity, is dependent on the presence of N-terminal sequences that are not normally considered part of PTPase catalytic domains.

Gene, 1994 Oct 11, 148(1), 155 - 9
The Schizosaccharomyces pombe rad1 gene consists of three exons and the cDNA sequence is partially homologous to the Ustilago maydis REC1 cDNA; Long KE et al.; We show that the rad1 gene of Schizosaccharomyces pombe is comprised of three exons and encodes a protein of 37 kDa . A cDNA clone containing these three exons complements the sensitivity of the rad1-1 mutant to ultraviolet and gamma-radiation and to hydroxyurea . The newly identified ORF of the rad1 gene was found to exhibit partial homology to the REC1 gene of Ustilago maydis . These two genes share putative functional similarities in their respective organisms.

Microbiology, 1994 Oct, 140 ( Pt 10), 2617 - 23
Glucose-transport-deficient mutants of Schizosaccharomyces pombe: phenotype, genetics and use for genetic complementation; Milbradt B et al.; Glucose-transport-deficient mutants of Schizosaccharomyces pombe were obtained by treatment of wild-type cells (972h-) with N-methyl-N'-nitro-N- nitrosoguanidine, and by selection of resulting mutants on gluconate medium containing 0.05% 2-deoxy-D-glucose (2DG) . One mutant, designated YGS-B22, was unable to grow on D-glucose and/or D-fructose as a carbon source (Glc/Fru-), and was resistant to 2DG; hence, none of the three sugars was taken up by the mutant cells . The hexokinase activity in the wild-type and the mutant cells was equal . Genetic purification of YGS-B22 by back-crossing with a leucine-auxotrophic mutant and the wild-type resulted in two strains: YGS-4, with reduced 2DG resistance, and YGS-5, which had lost 2DG-resistance . YGS-5 grew in D-glucose-containing media, albeit very slowly . No measurable sugar uptake was detectable in either of the two mutants within the 1 h test interval . Tetrad analyses proved a Mendelian segregation of growth on D-glucose and leucine auxotrophy . However, 2DG resistance did not co-segregate with the Glc/Fru- phenotype, indicating that the transport deficiency and 2DG resistance characters are not encoded on the same genomic locus . Using a genomic bank of Sch . pombe, two transformants, YGS-5-G7 and YGS-5-G12, were found which had regained the wild-type growth and transport phenotype by complementation . Correspondingly, both D-glucose uptake and 2DG accumulation were restored in the transformed strains.(ABSTRACT TRUNCATED AT 250 WORDS)

J Cell Biol, 1994 Oct, 127(2), 273 - 85
Dynamics of chromosome organization and pairing during meiotic prophase in fission yeast; Scherthan H et al.; Interactions between homologous chromosomes (pairing, recombination) are of central importance for meiosis . We studied entire chromosomes and defined chromosomal subregions in synchronous meiotic cultures of Schizosaccharomyces pombe by fluorescence in situ hybridization . Probes of different complexity were applied to spread nuclei, to delineate whole chromosomes, to visualize repeated sequences of centromeres, telomeres, and ribosomal DNA, and to study unique sequences of different chromosomal regions . In diploid nuclei, homologous chromosomes share a joint territory even before entry into meiosis . The centromeres of all chromosomes are clustered in vegetative and meiotic prophase cells, whereas the telomeres cluster near the nucleolus early in meiosis and maintain this configuration throughout meiotic prophase . Telomeres and centromeres appear to play crucial roles for chromosome organization and pairing, both in vegetative cells and during meiosis . Homologous pairing of unique sequences shows regional differences and is most frequent near centromeres and telomeres . Multiple homologous interactions are formed independently of each other . Pairing increases during meiosis, but not all chromosomal regions become closely paired in every meiosis . There is no detectable axial compaction of chromosomes in meiotic prophase . S . pombe does not form mature synaptonemal complexes, but axial element-like structures (linear elements), which were analyzed in parallel . Their appearance coincides with pairing of interstitial chromosomal regions . Axial elements may define minimal structures required for efficient pairing and recombination of meiotic chromosomes.

Yeast, 1994 Oct, 10(10), 1383 - 7
Sequence of a 10.27 kb segment on the left arm of chromosome XV from Saccharomyces cerevisiae includes part of the IRA2 gene and a putative new gene; Zumstein E et al.; A 10,270 bp fragment from the left arm of chromosome XV of Saccharomyces cerevisiae was sequenced and analysed . The sequence reveals the presence of two open reading frames (ORFs), one of them is the larger part of the previously sequenced gene IRA2 (YOL0951) . The other ORF, YOL0950, has a length of 1245 nucleotides and exhibits no significant homology with any known gene, although there is some similarity of its upstream region to the corresponding region of the Schizosaccharomyces pombe cdr1/nim1 gene which is involved in the control of mitotic cell size.

Yeast, 1994 Oct, 10(10), 1347 - 54
Assessment of pheromone production and response in fission yeast by a halo test of induced sporulation; Egel R et al.; We describe a rapid, sensitive and semi-quantitative plate assay for monitoring pheromone activity in the fission yeast Schizosaccharomyces pombe . It is based on the observation that meiosis requires stimulation by pheromone and exploits diploid strains that will only sporulate after addition of exogenous pheromone . The tester strains are heterozygous for mating type, are non-switching, and are mutated in one of the early subfunctions (either mat1-Mc or mat1-Pc), so that meiosis is only induced after exposure to exogenous pheromone (M-factor or P-factor, respectively) . Pheromone activity is assessed as an iodine-positive halo of sporulation surrounding the pheromone source, and the width of the halo is related to the amount of pheromone being produced . The assay is sufficiently sensitive to monitor the low amount of M-factor produced by an M mam1 strain, and its sensitivity towards P-factor is greatly increased by using a hyper-sensitive tester strain lacking the Sxa2 protease that is believed to degrade this pheromone . We also demonstrate that the production of P-factor is very much stimulated by exposure of P cells to M-factor.

Biol Chem Hoppe Seyler, 1994 Oct, 375(10), 715 - 9
Two-chain bacteriorhodopsin synthesized by Schizosaccharomyces pombe; Hansen OK et al.; Bacteriorhodopsin (BR), the light-driven proton pump of Halobacterium salinarium purple membrane, was produced in functional form as a two-chain protein by simultaneous expression in the fission yeast Schizosaccharomyces pombe of two separate structural genes, one coding for an aminoterminal BR fragment encompassing the first two transmembrane helices of BR, the other coding for the remainder of the protein . The fragments assemble spontaneously in vivo to yield functional BR which can be purified by immobilized metal ion affinity chromatography.

Curr Genet, 1994 Oct, 26(4), 315 - 20
Biochemical and genetical studies of NADP-specific glutamate dehydrogenase in the fission yeast Schizosaccharomyces pombe; Perysinakis A et al.; The initial velocity, pH and temperature optima, and Km values of Schizosaccharomyces pombe NADP-glutamate dehydrogenase (NADP-GDH:EC 1.4.1.4) have been determined . NADP-GDH was found to be specific for the substrates used in the reaction mixtures . NADP-GDH activity showed a sigmoidal response to changes in alpha-ketoglutarate concentrations, following Hill kinetics with a coefficient nH = 2 . A two-fold and a three-fold increase in activity was found in extracts of cells grown on a medium containing cytosine or histidine as a sole nitrogen source, respectively, relative to the activity found in cells grown on other sole nitrogen sources including ammonium, adenine, arginine, aspartate, asparagine, glutamate, glutamine, leucine, lysine, proline, uridine and urea . Five NADP-GDH-defective mutants were isolated on the basis of no growth on ammonium plus allantoin as sole nitrogen sources . The mutants also failed to grow on allantoin alone but, in contrast, they were phenotypically indistinguishable from the wild-type growing on solid minimal medium with ammonium . Additionally, the mutants were found to grow as wild-type on minimal medium with alanine, arginine, asparagine, aspartate, glutamate, glutamine, leucine, ornithine and proline in the absence or presence of allantoin . In liquid minimal medium with ammonium as sole nitrogen source they had a slower growth than the wild-type . Normal growth was observed in cells grown on alanine, arginine, asparagine, aspartate, glutamate, glutamine, leucine, ornithine and proline . The mutants had undetectable levels of NADP-GDH activity, but retained wild-type levels of NAD-GDH, glutame synthase (GOGAT) and glutamine synthetase (GS).(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosoma, 1994 Oct, 103(6), 414 - 22
Comparison of the two major ARS elements of the ura4 replication origin region with other ARS elements in the fission yeast, Schizosaccharomyces pombe; Zhu J et al.; We have previously reported that the replication origin region located near the ura4 gene on chromosome III of the fission yeast, Schizosaccharomyces pombe, contains three closely spaced origins, each associated with an autonomously replicating sequence (ARS) element . Here we report the nucleotide sequences of two of these ARS elements, ars3002 and ars3003 . The two ARS elements are located on either side of a transcribed 1.5 kb open reading frame . Like 11 other S . pombe ARS elements whose sequences have previously been determined in other laboratories, the 2 new ARS elements are unusually A+T-rich . All 13 ARS elements contain easily unwound stretches of DNA . Each of the ARS elements contains numerous copies, at a higher than expected frequency, of short stretches of A+T-rich DNA in which most of the Ts are on one strand and most of the As are on the complementary strand . We discuss the potential significance for ARS function of these multiple asymmetric A+T-rich sequences.

Chromosoma, 1994 Oct, 103(6), 369 - 80
The spindle pole body of yeast; Snyder M; Microtubule organizing centers play an essential cellular role in nucleating microtubule assembly and establishing the microtubule array . The microtubule organizing center of yeast, the spindle pole body (SPB), shares many functions and properties with those other organisms . In recent years considerable new information has been generated concerning components associated with the SPB, and the mechanism by which it duplicates . This article reviews our current view of the cytology and molecular composition of the SPB of the budding yeast, Saccharomyces cerevisiae, and the fission yeast, Schizosaccharomyces pombe . Genetic studies in these organisms has revealed information about how the SPB duplicates and separates, and its roles during vegetative growth, mating and meiosis.

Genomics, 1994 Oct, 23(3), 592 - 9
Cloning and molecular characterization of the Chinese hamster ERCC2 nucleotide excision repair gene; Kirchner JM et al.; The Chinese hamster ERCC2 nucleotide excision repair gene, encoding a presumed ATP-dependent DNA helicase, was cloned from the V79 cell line, and its nucleotide sequence was determined . The approximately 15-kb gene comprises 23 exons with a 2283-base open reading frame . The predicted 760-amino-acid protein is 98% identical to the human ERCC2/XPD (760 amino acids), 51% identical to the Saccharomyces cerevisiae RAD3 (778 amino acids), and 54% identical to the Schizosaccharomyces pombe rad15 (772 amino acids) proteins . The promoter region of the hamster ERCC2 gene contains a pyrimidine-rich stretch (42 nucleotides, 88% C+T) similar to sequences found in the promoter regions of two other nucleotide excision repair genes, a GC box, a putative alpha-Pal transcription factor binding site, and two CAAT boxes . There is no apparent TAATA box . No consensus polyadenylation sequence (AATAAA or its variants) was found within 663 bases 3' of the translation termination codon.

J Mol Biol, 1994 Sep 30, 242(4), 389 - 96
On the identification of group II introns in nucleotide sequence data; Knoop V et al.; Four different consensus sequences (GTI, group II identifiers) have been derived from domains V of known group II introns and are used as query input sequences for sensitive database screenings with the FASTA and LFASTA programs . The set of four GTI sequences can identify all domains V of the 96 known group II introns in the completely sequenced chloroplast genomes of Marchantia polymorpha, Epifagus virginiana, Oryza sativa, Nicotiana tabacum and the completely sequenced mitochondrial genomes of Saccharomyces cerevisiae, Podospora anserina, Schizosaccharomyces pombe and Marchantia polymorpha . Seven moderately high-scoring hits can easily be rejected as false-positives since they do not fulfil secondary structure requirements . Large FASTA outputs obtained after screening the entire nucleotide sequence database are evaluated in a second step by a program (D5SCAN) that allows the assignment of variable selection criteria for potential domain V secondary structures . Database searches with these routines yield evidence for several group II intron sequences previously unrecognized . These include novel intron structures in the cyanobacterium Synechocystis and in the mitochondrial genomes of Marchantia, soybean, pea, broad bean, sugar beet and a heterobasidiomycete . Potential intron remnants are found contributing to the secondary structure of rRNAs in several trypanosome species . At a given sensitivity of 95% positively identified true domains V, the search routine produces one false positive hit per 10,000 kb.

J Biol Chem, 1994 Sep 30, 269(39), 24229 - 36
Brefeldin A sensitivity and resistance in Schizosaccharomyces pombe . Isolation of multiple genes conferring resistance; Turi TG et al.; The fungal metabolite brefeldin A (BFA) causes the inhibition of protein secretion and the disruption of the structure and function of the Golgi complex in mammalian cells . Here we show that BFA has identical effects in the fission yeast Schizosaccharomyces pombe which normally contains a Golgi complex of stacked cisternae similar to the Golgi complexes in animal cells . After treatment with BFA, secretion was inhibited, Golgi complexes disappeared, and there was an accumulation of endoplasmic reticulum . These results indicate that the effects of BFA in fungi are very similar to those in mammalian cells and provide direct evidence for an effect of BFA on Golgi morphology in fungi . Five spontaneous BFA-resistant mutants were isolated . Genetic analysis showed that the mutations conferring BFA resistance were dominant and in two separate linkage groups . One of the BFA-resistant mutations was found to be allelic to crm1, a gene affecting chromatin structure . All BFA-resistant mutants overexpressed a 20-kDa protein, and the corresponding gene obr1 was isolated and sequenced . However, obr1 overexpression was not sufficient to confer BFA resistance . Plasmids capable of conferring BFA resistance to wild type cells were isolated from libraries constructed from the two BFA-resistant mutants . These plasmids contain six different genes capable of conferring resistance when present in high copy . One of these genes encoded the transcription factor pap1, a homolog of the mammalian AP1 protein . The overexpression of pap1 probably confers BFA resistance indirectly by inducing expression of one or more other proteins . The isolation of several genes conferring BFA resistance suggests several mechanisms are involved.

Proc Natl Acad Sci U S A, 1994 Sep 27, 91(20), 9367 - 71
Photoactive mitochondria: in vivo transfer of a light-driven proton pump into the inner mitochondrial membrane of Schizosaccharomyces pombe; Hoffmann A et al.; The light-driven proton pump bacteriorhodopsin (bR) from Halobacterium salinarium has been genetically transferred into the inner mitochondrial membrane (IM) of the eukaryotic cell Schizosaccharomyces pombe, where the archaebacterial proton pump replaces or increases the proton gradient usually formed by the respiratory chain . For targeting and integration, as well as for the correct orientation of bR in the IM, the bacterioopsin gene (bop) was fused to signal sequences of IM proteins . Northern and Western blot analysis proved that all hybrid gene constructs containing the bop gene and a mitochondrial signal sequence were expressed and processed to mature bR . Fast transient absorption spectroscopy showed photocycle activity of bR integrated in the IM by formation of the M intermediate . Experiments with the pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein revealed bR-mediated proton pumping from the mitochondrial matrix into the intermembrane space . Glucose uptake measurements under anaerobic conditions showed that yeast cells containing photoactive mitochondria need less sugar under illumination . In summary, our experiments demonstrate the functional genetic transfer of a light energy converter to a naturally nonphotoactive eukaryotic organism.

Proc Natl Acad Sci U S A, 1994 Sep 27, 91(20), 9327 - 31
Schizosaccharomyces pombe glycosylation mutant with altered cell surface properties; Ballou CE et al.; Mutagenesis of Schizosaccharomyces pombe cells yielded a strain that made reduced amounts of invertase . A comparison of the O- and N-linked carbohydrate chains of the wild-type and mutant glycoproteins revealed that a single type of alpha 1-->2-linked mannose was missing in the mutant . Analysis of the wild-type galactomannoprotein showed that it contained a heterogeneous small "core" oligosaccharide fraction linked to asparagine with sugar compositions that ranged from Man9(GlcNAc)2- to Gal4Man10(GlcNAc)2- . The galactose units are in terminal positions of a Man10(GlcNAc)2- unit that is similar to the mannoprotein core of Saccharomyces cerevisiae . Attached to this core in a larger oligosaccharide fraction is an alpha 1-->6-linked polymannose chain that is substituted at position 2 with alpha-linked mannose and galactose . The O-linked sugars consist of mannose, alpha 1-->2-linked mannosylmannose and alpha 1-->2-linked galactosylmannose, along with small amounts of tri- and tetrasaccharides . The glycosylation mutant lacks alpha 1-->2-linked mannose on both the O-linked chains and the outer chain of the large N-linked chains, suggesting that it may be defective in regulation of an alpha 1,2-mannosyltransferase that adds mannose to glycoproteins in the Golgi.

Gene, 1994 Sep 15, 147(1), 63 - 9
The fission yeast Schizosaccharomyces pombe rpb6 gene encodes the common phosphorylated subunit of RNA polymerase and complements a mutation in the corresponding gene of Saccharomyces cerevisiae; Shpakovski GV; A single-copy gene, homologous to the RPB6 gene from Saccharomyces cerevisiae, encoding a small phosphorylated subunit common to all three forms of nuclear DNA-dependent RNA polymerase was isolated from the fission yeast Schizosaccharomyces pombe . Its cDNA copy consists of an open reading frame of 142 codons and encodes an acidic protein (predicted pI 4.1) with a M(r) of 15,730 . The genomic copy of Sz . pombe rpb6 contains an intron (219 nucleotides) located at codon 92, a position which does not correspond to the single intron of the S . cerevisiae gene . The sequencing of both genomic and cDNA copies of rpb6 allowed us to determine the probable positions of the start and stop of rpb6 transcription and to identify a putative TATA box . The primary structures of the Sz . pombe and S . cerevisiae Rpb6 proteins have 60.7% identity, with the same general organization: a highly acidic N-terminal region followed by a short basic region and a C terminus featuring a putative heptad Leu repeat . The C-terminal half of the sequence is particularly well conserved and, therefore, probably contains the most important functional domain . Moreover, a heterospecific complementation test showed that rpb6 from Sz . pombe fully complements a complete deletion of its S . cerevisiae homologue.

Gene, 1994 Sep 15, 147(1), 141 - 4
Thiamine-repressible genes in Schizosaccharomyces pombe are regulated by a Cys6 zinc-finger motif-containing protein; Fankhauser H et al.; Our previous genetic data indicate that the product of the Schizosaccharomyces pombe thi1 gene acts as an activator of several thiamine-repressible genes which are involved in the control of thiamine metabolism {Schweingruber et al., Genetics 130 (1992) 445-449; Zurlinden and Schweingruber, Gene 117 (1992) 141-143} . In this communication, we report the cloning and sequencing of thi1 and show that it carries an open reading frame which translates into a 775-amino-acid protein with the characteristics of a Cys6 zinc-finger-motif-containing transcription factor, as typified by Saccharomyces cerevisae GAL4 . We, therefore, suggest that the thi1-encoded protein binds to upstream activator sequences of thiamine-repressible genes.

Gene, 1994 Sep 15, 147(1), 131 - 5
The isolation and characterization of the gene (dfr1) encoding dihydrofolate reductase (DHFR) in Schizosaccharomyces pombe; Bertani LE et al.; A sequence encoding dihydrofolate reductase (DHFR) was isolated from a Schizosaccharomyces pombe cDNA library by selecting for trimethoprim resistance in Escherichia coli . The sequence was found to be functional in both Saccharomyces cerevisiae and Sz . pombe . When present on a multicopy plasmid, it confers increased resistance to concentrations of the drug methotrexate that are otherwise inhibitory for the standard yeast strains . The sequence was mapped by DNA hybridizations between genes adh1 and ade5 on chromosome III of Sz . pombe . The 1.6-kb insert contains a 1.5-kb open reading frame (ORF) with strong sequence similarity to other described DHFR-encoding genes . The similarity, however, is limited to a 678-bp sequence, occupying the 3'-half of the ORF . No similarity to other described DNA sequences or proteins could be found for the 5'-half . Southern and Northern blots indicate that the entire insert is present intact in the Sz . pombe genome and produces a 1.7-kb RNA transcript.

J Cell Biol, 1994 Sep, 126(6), 1465 - 73
Human gamma-tubulin functions in fission yeast; Horio T et al.; gamma-Tubulin is a phylogenetically conserved component of microtubule-organizing centers that is essential for viability and microtubule function . To examine the functional conservation of gamma-tubulin, we have tested the ability of human gamma-tubulin to function in the fission yeast Schizosaccharomyces pombe . We have found that expression of a human gamma-tubulin cDNA restores viability and a near-normal growth rate to cells of S . pombe lacking endogenous gamma-tubulin . Immunofluorescence microscopy showed that these cells contained normal mitotic spindles and interphase microtubule arrays, and that human gamma-tubulin, like S . pombe gamma-tubulin, localized to spindle pole bodies, the fungal microtubule-organizing centers . These results demonstrate that human gamma-tubulin functions in fission yeast, and they suggest that in spite of the great morphological differences between the microtubule-organizing centers of humans and fission yeasts, gamma-tubulin is likely to perform the same tasks in both . They suggest, moreover, that the proteins that interact with gamma-tubulin, including, most obviously, microtubule-organizing center proteins, must also be conserved . We have also found that a fivefold overexpression of S . pombe gamma-tubulin causes no reduction in growth rates or alteration of microtubule organization . We hypothesize that the excess gamma-tubulin is maintained in the cytoplasm in a form incapable of nucleating microtubule assembly . Finally, we have found that expression of human gamma-tubulin or overexpression of S . pombe gamma-tubulin causes no significant alteration of resistance to the antimicrotubule agents benomyl, thiabendazole and nocodazole.

Mol Gen Genet, 1994 Sep 1, 244(5), 456 - 64
The ste13+ gene encoding a putative RNA helicase is essential for nitrogen starvation-induced G1 arrest and initiation of sexual development in the fission yeast Schizosaccharomyces pombe; Maekawa H et al.; When the fission yeast Schizosaccharomyces pombe is starved for nitrogen, the cells are arrested in the G1 phase, enter the G0 phase and initiate sexual development . The ste13 mutant, however, fails to undergo a G1 arrest when starved for nitrogen and since this mutant phenotype is not suppressed by a mutation in adenylyl cyclase (cyr1), it would appear that ste13+ either acts independently of the decrease in the cellular cAMP level induced by starvation for nitrogen, or functions downstream of this controlling event . We have used functional complementation to clone the ste13+ gene from an S . pombe genomic library and show that its disruption is not lethal, indicating that, while the gene is required for sexual development, it is not essential for cell growth . Nucleotide sequencing predicts that ste13+ should encode a protein of 485 amino acids in which the consensus motifs of ATP-dependent RNA helicases of the DEAD box family are completely conserved . Point mutations introduced into these consensus motifs abolished the ste13+ functions . The predicted Ste13 protein is 72% identical to the Drosophila melanogaster Me31B protein over a stretch of 391 amino acids . ME31B is a developmentally regulated gene that is expressed preferentially in the female germline and may be required for oogenesis . Expression of ME31B cDNA in S . pombe suppresses the ste13 mutation . These two evolutionarily conserved genes encoding putative RNA helicases may play a pivotal role in sexual development.

Mol Cell Biol, 1994 Sep, 14(9), 6361 - 71
Identification of cut8+ and cek1+, a novel protein kinase gene, which complement a fission yeast mutation that blocks anaphase; Samejima I et al.; The fission yeast Schizosaccharomyces pombe {corrected} temperature sensitivity cut8-563 mutation causes chromosome overcondensation and short spindle formation in the absence of sister chromatid separation . The cut8-563 mutation allows cytokinesis before the completion of anaphase, thus producing cells with a cut phenotype . The cut8+ gene product may be required for normal progression of anaphase . Diploidization occurs at the restrictive temperature, and 60 to 70% of the cells surviving after two generations are diploid . These phenotypes are reminiscent of those of budding yeast (Saccharomyces cerevisiae) ctf13 and ctf14 (ndc10) mutations . The cut8+ gene, isolated by complementation of the mutant, predicts a 262-amino-acid protein; the amino and carboxy domains are hydrophilic, while the central domain contains several hydrophobic stretches . It has a weak overall similarity to the budding yeast DBF8 gene product . DBF8 is an essential gene whose mutations result in delay in mitotic progression and chromosome instability . Anti-cut8 antibodies detect a 33-kDa polypeptide . Two multicopy suppressor genes for cut8-563 are identified . They are the cut1+ gene essential for nuclear division, and a new gene (designated cek1+) which encodes a novel protein kinase . The cek1+ gene product is unusually large (1,309 amino acids) and has a 112-amino-acid additional sequence in the kinase domain . The cek1+ gene is not an essential gene . Protein phosphorylation by cek1 may facilitate the progression of anaphase through direct or indirect interaction with the cut8 protein.

Genetics, 1994 Sep, 138(1), 39 - 45
Glucose repression of fbp1 transcription of Schizosaccharomyces pombe is partially regulated by adenylate cyclase activation by a G protein alpha subunit encoded by gpa2 (git8); Nocero M et al.; In the fission yeast Schizosaccharomyces pombe, genetic studies have identified genes that are required for glucose repression of fbp1 transcription . The git2 gene, also known as cyr1, encodes adenylate cyclase . Adenylate cyclase converts ATP into the second messenger cAMP as part of many eukaryotic signal transduction pathways . The git1, git3, git5, git7, git8 and git10 genes act upstream of adenylate cyclase, presumably encoding an adenylate cyclase activation pathway . In mammalian cells, adenylate cyclase enzymatic activity is regulated by heterotrimeric guanine nucleotide-binding proteins (G proteins) . In the budding yeast Saccharomyces cerevisiae, adenylate cyclase enzymatic activity is regulated by monomeric, guanine nucleotide-binding Ras proteins . We show here that git8 is identical to the gpa2 gene that encodes a protein homologous to the alpha subunit of a G protein . Mutations in two additional genes, git3 and git5 are suppressed by gpa2+ in high copy number . Furthermore, a mutation in either git3 or git5 has an additive effect in strains deleted for gpa2 (git8), as it significantly increases expression of an fbp1-lacZ reporter gene . Therefore, git3 and git5 appear to act either in concert with or independently from gpa2 (git8) to regulate adenylate cyclase activity.

Genetics, 1994 Sep, 138(1), 29 - 38
Three additional linkage groups that repress transcription and meiotic recombination in the mating-type region of Schizosaccharomyces pombe; Thon G et al.; The mating-type genes of Schizosaccharomyces pombe are found at three locations in the same chromosomal region . These genes are in an active configuration at the mat1 locus and in an inactive configuration at the mat2 and mat3 loci . The mechanism that represses transcription of mat2 and mat3 also inactivates other promoters introduced nearby and is accompanied by a block to meiotic recombination in the mat2-mat3 interval, suggesting that this mechanism involves a particular chromatin structure . We present evidence that the transcription and recombination blocks require three newly defined trans-acting loci, clr2, clr3 and clr4, in addition to the previously identified clr1, rik1 and swi6 loci . We also investigated the role of mat2 cis-acting sequences in silencing . Four cis-acting elements that repress mat2 in a plasmid context were previously identified . Deletion of two of these elements proved to have little effect in a chromosomal context . However, when combined with mutations in trans-acting genes, deletion of the same two elements greatly enhanced mat2 expression . The observed cumulative effects suggest a redundancy in the silencing mechanism.

Exp Parasitol, 1994 Sep, 79(2), 137 - 47
Plasmodium falciparum: the pfmdr2 protein is not overexpressed in chloroquine-resistant isolates of the malaria parasite; Rubio JP et al.; We have isolated and sequenced a full-length gene (pfmdr2) that has homology to the ABC (ATP-binding cassette)-type transport proteins which includes the mammalian P-glycoproteins, CFTR, and the protein product of the Plasmodium falciparum pfmdr1 gene . The protein encoded by the pfmdr2 gene has 10 hydrophobic domains followed by a region homologous to the nucleotide binding fold of the ABC transport proteins . The pfmdr2 protein also shows homology outside the nucleotide binding fold and some structural similarity to HMT1, a protein involved in heavy metal tolerance in Schizosaccharomyces pombe . Antibodies raised to the pfmdr2 protein react with a 110-kDa band and localization by immunofluorescence suggests that protein is expressed over the whole parasite and may be located on the plasma membrane of the parasite . Comparison of the level of expression of the pfmdr2 protein in chloroquine-resistant and -sensitive parasites show that it is present at approximately equal levels which is in contrast to previous results that determined the level of the pfmdr2 transcript . These results support the evidence that pfmdr2 is not involved in the chloroquine resistance phenotype.

Curr Biol, 1994 Sep 1, 4(9), 798 - 806
The activation of phosphatidylinositol 3-kinase by Ras; Kodaki T et al.; BACKGROUND: Activation of the mammalian phosphatidylinositol 3-kinase complex can play a critical role in transducing growth factor responses . The lipid kinase complex, which is made up of p85 alpha and p110 alpha regulatory and catalytic subunits, becomes associated with a number of activated receptor protein tyrosine kinases, but the mechanism of its activation has not yet been defined . Recent evidence indicates that Ras can bind to the p85 alpha/p110 alpha complex . We describe here the functional regulation of the mammalian phosphatidylinositol 3-kinase complex by Ras . RESULTS: Expression of p110 alpha, the catalytic subunit of phosphatidylinositol 3-kinase, in the fission yeast, Schizosaccharomyces pombe, has been used to demonstrate an inhibitory effect of p85 alpha on p110 alpha activity in intact cells; inhibition did not result from a decrease in p110 alpha expression . In this cellular context, we have investigated the effect of a constitutively active mutant of Ras, v-Ras, either on p85 alpha or p110 alpha-alone, or on the p85 alpha/p110 alpha complex . In the presence of the p85 alpha/p110 alpha complex, v-Ras suppressed cell growth, but an effector-domain mutant of v-Ras did not . The growth-suppressive effect of v-Ras was not seen for any other combination of expressed proteins . The phenotype induced by v-Ras was consistent with activation of the p85 alpha/p110 alpha complex: it was sensitive to the phosphatidylinositol 3-kinase inhibitor, wortmannin, and the cells accumulated 3-phosphorylated polyphosphoinositides . Activation of purified p85 alpha/p110 alpha by purified recombinant Ras in vitro was also demonstrated . CONCLUSIONS: The phosphatidylinositol 3-kinase complex, p85 alpha/p110 alpha, shows a suppressed catalytic function in vivo when compared with free p110 alpha . This complex can, however, be activated by Ras . We suggest that the phosphatidylinositol 3-kinase p85 alpha/p110 alpha complex is a downstream effector of Ras.

Mutat Res, 1994 Sep, 322(3), 161 - 7
Mutagenic and carcinogenic effects of waste oil of frying bean cake on Saccharomyces cerevisiae and Schizosaccharomyces pombe; Zaied KA; The present investigation was conducted to study the genotoxic effects of waste oil of frying bean cake (Taamiah oil) using Saccharomyces cerevisiae and Schizosaccharomyces pombe as test organisms . The results showed that the different concentrations of Taamiah oil exert different toxicity on yeast cells . the induced toxicity in both organisms was gradually increased with rising the concentration of Taamiah oil While, the differences between cellular survival and respiratory deficient mutants in treated and untreated samples were significant . Though Taamiah oil induced a concentration-dependent toxicity, it did not exert an induction of recombination . Thus, it seems likely that there was a cytotoxic effect and a weak effect on the induction of cytoplasmic petite mutations in yeast . The results suggest that Taamiah oil does not seem to induce lesions in DNA that are subject to excision repair . However, in Schizosaccharomyces pombe some point mutations seem to be induced in addition to toxicity . The conclusion is straight forward that waste oil of frying bean cake is mutagenic and may be carcinogenic in humans.

Biochem Biophys Res Commun, 1994 Aug 30, 203(1), 231 - 6
Molecular cloning and sequence analysis of two novel fission yeast casein kinase-1 isoforms; Kearney PH et al.; The cDNAs for two casein kinase-1 homologs, hhp1 and hhp2, have been isolated from Schizosaccharomyces pombe and characterized . Their corresponding genes reside on chromosomes II and I, respectively, and encode approximately 42-46 kDa proteins that are related structurally to the HRR25 gene product of budding yeast . On the basis of multiple sequence alignment, the CK1 family appears to consist of three main branches . We predict that the branch containing the hhp genes encodes nuclear kinases involved in the regulation of DNA metabolism.

Nucleic Acids Res, 1994 Aug 25, 22(16), 3365 - 72
Identification of a novel HIV-1 TAR RNA bulge binding protein; Baker B et al.; The Tat protein binds to TAR RNA to stimulate the expression of the human immunodeficiency virus type 1 (HIV-1) genome . Tat is an 86 amino acid protein that contains a short region of basic residues (aa49-aa57) that are required for RNA binding and TAR is a 59 nucleotide stem-loop with a tripyrimidine bulge in the upper stem . TAR is located at the 5' end of all viral RNAs . In vitro, Tat specifically interacts with TAR by recognising the sequence of the bulge and upper stem, with no requirement for the loop . However, in vivo the loop sequence is critical for activation, implying a requirement for accessory cellular TAR RNA binding factors . A number of TAR binding cellular factors have been identified in cell extracts and various models for the function of these factors have been suggested, including roles as coactivators and inhibitors . We have now identified a novel 38 kD cellular factor that has little general, single-stranded or double-stranded RNA binding activity, but that specifically recognises the bulge and upper stem region of TAR . The protein, referred to as BBP (bulge binding protein), is conserved in mammalian and amphibian cells and in Schizosaccharomyces pombe but is not found in Saccharomyces cerevisiae . BBP is an effective competitive inhibitor of Tat binding to TAR in vitro . Our data suggest that the bulge-stem recognition motif in TAR is used to mediate cellular factor/RNA interactions and indicates that Tat action might be inhibited by such competing reactions in vivo.

Biochim Biophys Acta, 1994 Aug 24, 1194(1), 149 - 54
The HUP1 gene product of Chlorella kessleri: H+/glucose symport studied in vitro; Opekarova M et al.; An in vitro system was established to measure secondary active transport mediated by plant H+ symporters . For this purpose plasma membranes of Schizosaccharomyces pombe cells transformed with the HUP1 gene coding for the H+/hexose symporter of Chlorella kessleri were fused with cytochrome-c oxidase containing proteoliposomes . After energization with ascorbate/TMPD/cytochrome c these vesicles built up a protonmotive force of > 130 mV consisting mainly of a membrane potential of > 100 mV (inside negative) . Energized vesicles accumulated D-glucose in a pH-dependent way up to 30-fold which was not the case with control vesicles prepared from cells transformed with the plasmid not containing the HUP1 gene . The Km value for D-glucose uptake was 5 x 10(-5) M . The pH-dependence of accumulation was not due to a difference in protonmotive force, but reflected the pH-dependence of the carrier activity, i.e., the accumulation was determined by kinetic and by thermodynamic parameters . In the system both components of protonmotive force delta psi and delta pH can be manipulated individually, which allows to evaluate to what extent they contribute to sugar accumulation . The results indicate that under certain conditions the internal pH may be a limiting factor for D-glucose accumulation.

J Biol Chem, 1994 Aug 19, 269(33), 21010 - 5
Dominance of metallothionein in metal ion buffering in yeast capable of synthesis of (gamma EC)nG isopeptides; Yu W et al.; The relationship of yeast metallothionein (MT) and (gamma EC)nG isopeptides (phytochelatins) in metal ion buffering was assessed . The effect of constitutive expression of yeast metallothionein (MT) genes on accumulation of metal-(gamma EC)nG isopeptide (phytochelatin) complexes was analyzed in Candida glabrata and Schizosaccharomyces pombe cultures incubated in the presence of cadmium salts . Constitutive expression of the Saccharomyces cerevisiae MT (CUP1) gene inhibited the accumulation of metal-phytochelatin complexes in both C . glabrata and S . pombe . Intracellular Cd(II) sequestration occurred by formation of CdMT complexes . Phytochelatin (gamma EC)nG complexes appear to function in metal buffering in cells when MT genes are not present or expressed . A third condition in which metal-(gamma EC)nG complexes are observed is when constitutively expressed MT does not accumulate . We observed that C . glabrata lacking the AMT1 gene necessary for copper induction of the MT genes expressed MTII constitutively, but this expression does not lead to CdMTII accumulation . Only Cd-(gamma EC)nG complexes accumulate . Likewise, metal exposed cultures of S . cerevisiae (cup1) transformed with C . glabrata MTII under the constitutive ADH1 promoter resulted in constitutive expression of MTII and accumulation of CuMTII complexes but no CdMTII complexes . The inability of constitutively expressed C . glabrata MTII to buffer Cd(II) ions may arise in part from an inherent kinetic lability of CdMTII complexes . Incubation of ZnMTII with a metallochromic chelator, 4-(2-pyridylazo)resorcinol resulted in greater Zn(II) loss than Zn(II) complexes with CUP1 MT and C . glabrata MTI . C . glabrata MTII appears to be the first MT described which forms an unstable Cd(II) complex.

EMBO J, 1994 Aug 15, 13(16), 3801 - 11
Telomere-associated chromosome breakage in fission yeast results in variegated expression of adjacent genes; Nimmo ER et al.; The sequence requirements for in vivo telomere function in the fission yeast, Schizosaccharomyces pombe, have been investigated . A 258 bp tract of previously characterized cloned fission yeast terminal repeats adjacent to 800 bp of telomere-associated sequences is sufficient to seed new telomeres onto linearized ars-containing plasmids when introduced into cells . The resulting transformants contain unrearranged, acentric, linear episomes . Cloned telomeres, with and without telomere-associated sequences adjacent to the 258 bp terminal repeats, were utilized to introduce chromosome breaks at specific sites in a non-essential minichromosome . Truncated minichromosome derivatives were recovered containing the ura4 or ade6 gene adjacent to a newly formed telomere . These telomeres exert reversible position effects on the expression of the adjacent ura4 or ade6 genes.

FEMS Microbiol Lett, 1994 Aug 15, 121(2), 223 - 7
Use of Yarrowia lipolytica hexokinase for the quantitative determination of trehalose 6-phosphate; Blazquez MA et al.; This paper describes a procedure for the quantitative determination of trehalose 6-phosphate (T6P) based on its ability to inhibit hexokinase from Yarrowia lipolytica . The assay is linear between 1 nmol and at least 8 nmol . The concentration of T6P in wild-type Saccharomyces cerevisiae (0.15 mM) and in ras2 mutants (0.25 mM) remained unchanged in the exponential or stationary phase of growth or after heat shock . A tps1 mutant affected in T6P synthase did not show detectable T6P . Heat shock increased the concentration of T6P in Schizosaccharomyces pombe from 0.43 to 0.75 mM.

J Biol Chem, 1994 Aug 12, 269(32), 20517 - 21
Identification of a human rasGAP-related protein containing calmodulin-binding motifs; Weissbach L et al.; Conversion of active GTP-bound Ras to its inactive GDP-bound form is catalyzed by GTPase-activating proteins (GAPs) . Two mammalian Ras-specific GAPs, p120GAP and neurofibromin, the product of the NF1 tumor suppressor gene, have been previously described . We report here the identification of a new human cDNA clone, IQGAP1, which predicts a 1657-amino acid protein that displays extensive sequence similarity to the catalytic domain of all previously reported RasGAPs . IQGAP1 is most closely related to the Schizosaccharomyces pombe RasGAP-like protein, Sar1 . Sequence similarity to IQGAP1 is seen throughout the entire Sar1 protein . The N-terminal half of IQGAP1, which does not overlap with Sar1, contains six copies of a unique amino acid motif, as well as four so-called IQ motifs . The latter motifs are found in several proteins, including conventional and unconventional myosins, and mediate the interaction with calmodulin and calmodulin-related proteins . Thus, IQGAP1 appears to represent a novel RasGAP-like protein that may link Ras signaling to some calmodulin-mediated process.

Nucleic Acids Res, 1994 Aug 11, 22(15), 3104 - 12
An essential gene, ESR1, is required for mitotic cell growth, DNA repair and meiotic recombination in Saccharomyces cerevisiae; Kato R et al.; A new mutant, which was sensitive to both methyl-methanesulfonate (MMS) and ultra-violet light (UV) and defective in meiotic recombination, was isolated from Saccharomyces cerevisiae . The gene, ESR1, was cloned by complementation of the MMS sensitivity of the mutant and found to be essential for cell growth, as the deleted haploid strain was lethal . The ESR1 gene was adjacent to the CKS1 gene on chromosome II and encoded a putative 2368-amino acid protein with a molecular weight of 273 k . The ESR1 transcript was 8.0 kb long and was induced during meiosis . The predicted Esr1 protein had a mosaic structure composed of homologous regions and showed amino acid sequence similarities to Schizosaccharomyces pombe rad3+ protein, which monitors completion of DNA repair synthesis, and cut1+ protein, which is required for spindle pole body (SPB) duplication . The Esr1 protein was also similar to phosphatidylinositol (PI) 3-kinases, including Saccharomyces cerevisiae TOR2 (and DRR1), which are involved in G1 progression . These results suggest that ESR1 is multi-functional throughout mitosis and meiosis.

Nucleic Acids Res, 1994 Aug 11, 22(15), 3026 - 32
A new ATP-independent DNA endonuclease from Schizosaccharomyces pombe that recognizes cyclobutane pyrimidine dimers and 6-4 photoproducts; Bowman KK et al.; We have discovered a new DNA endonuclease in the fission yeast Schizosaccharomyces pombe which recognizes cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts . S . pombe DNA endonuclease (SPDE) catalyzes a single ATP-independent incision immediately 5' to the UV photoproduct and generates termini containing 3' hydroxyl and 5' phosphoryl groups . Based on these properties, we propose that SPDE may function in a DNA repair capacity, representing the initial recognition/cleavage step of a DNA excision repair pathway.

Nucleic Acids Res, 1994 Aug 11, 22(15), 2930 - 7
Identification of the DNA-binding domains of the switch-activating-protein Sap1 from S.pombe by random point mutations screening in E.coli; Arcangioli B et al.; Mating type switching in fission yeast, Schizosaccharomyces pombe, is initiated by a site-specific double-strand break (DSB) at the mat1 locus . The DSB is controlled from a distance by cis- and trans-acting elements . The switch-activating protein, Sap1 binds to the SAS1 cis-acting element which controls the frequency of the DSB at the mat1 locus and, consequently the efficiency of mating type switching . We developed a general method for screening randomly mutagenized expression libraries of DNA-binding protein in E.coli . Sap1 gene was mutagenized by PCR under conditions of reduced Taq polymerase fidelity . The mutated DNA was expressed in E.coli and screened for SAS1-recognition . This method was used to isolated 16 point mutations that abolished SAS1 interaction together with 18 mutations that did not affect binding . The position of these point mutations allowed the identification of three protein domains located in the N-terminal part of Sap1 that are essential for DNA-binding . Deletions and biochemical analysis showed that Sap1 is a dimer both in solution and when bound to SAS1 sequence . The dimerization domain was localized C-terminally to the three domains described above and when used in exess it inhibited DNA binding.

Gene, 1994 Aug 5, 145(2), 205 - 10
The mating-type region of Schizosaccharomyces pombe contains an essential gene encoding a protein homologous to human modulators of HIV transactivation; Michael H et al.; In Schizosaccharomyces pombe, an intrachromosomal crossover between the mating type (MT) expression locus and one of the silent donor cassettes is lethal due to the loss of the intervening L region . The region contains one essential gene, let1 . This gene was cloned and sequenced . The deduced amino acid (aa) sequence of let1 shows extensive homologies with SUG1 from Saccharomyces cerevisiae . Significant homologies were also found with the human HIV transactivation modulators, MSS1 and TBP-1, as well as with subunit 4 of the mammalian 26 S protease . The data indicate that let1 is a member of a recently defined multigene family of ATPases.

EMBO J, 1994 Aug 1, 13(15), 3638 - 47
Three ARS elements contribute to the ura4 replication origin region in the fission yeast, Schizosaccharomyces pombe; Dubey DD et al.; The ura4 replication origin region, which is located near the ura4 gene on chromosome III of the fission yeast, Schizosaccharomyces pombe, contains multiple initiation sites . We have used 2D gel electrophoretic replicon mapping methods to study the distribution of these initiation sites, and have found that they are concentrated near three ARS elements (stretches of DNA which permit autonomous plasmid replication) . To determine the roles of these ARS elements in the function of the ura4 origin region, we deleted either one or two of them from the chromosome and then assessed the consequences of the deletions by 2D gel electrophoresis . The results suggest that each of the three ARS elements is responsible for the initiation events in its vicinity and that the ARS elements interfere with each other in a hierarchical fashion . It is possible that the large initiation zones of animal cells are similarly composed of multiple mutually interfering origins.

Mutat Res, 1994 Aug, 324(4), 147 - 52
Molecular analysis of CXPD mutations in the repair-deficient hamster mutants UV5 and UVL-13; Weber CA et al.; The cDNA sequence of the Chinese hamster xeroderma pigmentosum group D (CXPD) nucleotide excision repair gene was analyzed from three Chinese hamster ovary (CHO) cell lines: repair proficient strain AA8 and repair deficient, UV complementation group 2 strains UV5 and UVL-13 . CXPD encodes a presumed ATP-dependent DNA helicase and is single copy in CHO lines due to the hemizygosity of chromosome 9 . Comparison of the deduced wild-type AA8 CXPD protein sequence with that of the Chinese hamster V79 lung-derived cell line revealed two amino acid polymorphisms . Position 285 is glutamine in AA8 and arginine in V79, and position 298 is alanine in AA8 and threonine in V79 . Comparison with the human XPD, Saccharomyces cerevisiae RAD3, and Schizosaccharomyces pombe rad15 homologs shows variability at these positions . Analysis of the CXPD sequence in the repair deficient CHO lines UV5 and UVL-13 revealed, in each case, a single base substitution resulting in an amino acid substitution . Position 116 is tyrosine in UV5 and cysteine in AA8, and the corresponding positions of XPD, RAD3, and rad15 are cysteine . Position 615 is glutamic acid in UVL-13 and glycine in AA8, and the corresponding positions of XPD, RAD3, and rad15 are glycine . In both UV5 and UVL-13, positions 285 and 298 are glutamine and alanine, respectively, as seen in AA8 . These results suggest that cysteine 116 and glycine 615 are critical to the repair function of CXPD.

Infect Immun, 1994 Aug, 62(8), 3572 - 5
Cloning of Entamoeba genes encoding proteolipids of putative vacuolar proton-translocating ATPases; Descoteaux S et al.; Molecular cloning techniques were used to identify genes encoding the proteolipids of putative vacuolar proton-transporting ATPases (V-ATPases; EC 3.6.1.35) of Entamoeba histolytica (Ehvma3) and Entamoeba dispar . The Ehvma3 gene encoded a 177-amino-acid peptide, with an M(r) of 18,110, which showed extensive positional identities with peptides of E . dispar (92%), Schizosaccharomyces pombe (58%), and humans (56%).

Curr Genet, 1994 Aug, 26(2), 187 - 9
Mapping of additional markers in fission yeast, especially fus1 and three mfm genes; Egel R; The following genes of the fission yeast Schizosaccharomyces pombe have been mapped by tetrad analysis--chromosome arm I-L: mfm2, rad24, rad25; I-R: abc1, fus1, mfm1; II-L: mfm3; II-R: mam1, rad13 . A hot-spot of meiotic recombination although not quite so active as suggested by previous maps, may be located between rad25 and aro5 on I-L.

Curr Genet, 1994 Aug, 26(2), 105 - 12
Mutations in cyr1 and pat1 reveal pheromone-induced G1 arrest in the fission yeast Schizosaccharomyces pombe; Davey J et al.; Investigations into sexual differentiation and pheromone response in the fission yeast Schizosaccharomyces pombe are complicated by the need to first starve the cells of nitrogen . Most mating-related experiments are therefore performed on non-dividing cells . Here we overcome this problem by using two mutants that bypass the nutritional requirements and respond to the M-factor mating pheromone in rich medium . The first mutant lacks the cyr1 gene which encodes adenylate cyclase and these cells contain no measurable amounts of cAMP . When M-factor is added to a growing h+ cyr1- strain it causes a transient G1 arrest of cell division, transcription of mat1-Pm, and elongation of the cells to form shmoos . The second mutant contains the temperature-sensitive pat1-114 allele . At 30 degrees C this mutant was previously shown not only to bypass the nutritional signal but also to stop growing in a state derepressed for pheromone-controlled functions . We now report that an h+ pat1-114 strain growing mitotically at 23 degrees C responds to M-factor . This shows that the pat1 protein kinase can be tuned to derepress nutritional signalling while repressing the other stages in the differentiation process.

Yeast, 1994 Aug, 10(8), 1075 - 82
nmt2 of fission yeast: a second thiamine-repressible gene co-ordinately regulated with nmt1; Manetti AG et al.; We previously described a screen for thiamine-repressible genes in Schizosaccharomyces pombe and reported on one such gene, nmt1, required for thiamine biosynthesis . Here we describe a second gene, nmt2, recovered in the same screen . Disruption of nmt2 also resulted in thiamine auxotrophy, indicating a role for the nmt2 gene product in thiamine biosynthesis . Both genes are highly transcribed in minimal medium and repressed in medium containing thiamine, and nuclear 'run-on' experiments confirm that expression in both cases is controlled by the rate of transcription initiation . The virtually identical kinetics of induction and repression suggest that the two genes are co-ordinately regulated . Sequence comparison of the two promoters reveals a canonical TATA box, downstream of which is a perfectly conserved 11 bp element . Transcript mapping experiments show that transcription initiation of both genes is centred on this element.

Yeast, 1994 Aug, 10(8), 1003 - 17
Functional expression of human poly(ADP-ribose) polymerase in Schizosaccharomyces pombe results in mitotic delay at G1, increased mutation rate, and sensitization to radiation; Avila MA et al.; The activity of poly(ADP-ribose) polymerase (PADPRP), a chromatin-associated enzyme present in most eukaryotic cells, is stimulated by DNA strand breaks, suggesting a role for the enzyme in the cellular response to DNA damage . However, the primary function of PADPRP remains unknown . We have selected Schizosaccharomyces pombe as a simple eukaryotic system in which to study PADPRP function because this fission yeast shares with mammalian cells important cellular features possibly associated with poly-(ADP-ribos)ylation pathways . We investigated the existence of an endogenous yeast PADPRP by DNA and RNA hybridization to mammalian probes under low-stringency conditions and by PADPRP activity assays . Our data indicate that fission yeasts are naturally devoid of PADPRP . We therefore isolated S . pombe strains expressing PADPRP by transformation with a human full-length PADPRP cDNA under the control of the SV40 early promoter . The human PADPRP construct was transcribed and translated in S . pombe, generating a major transcript of the same size (3.7 kb) as that detected in mammalian cells and a 113-kDa polypeptide, identical in size to the native human PADPRP protein . Yeast recombinant PADPRP was enzymatically active and was recognized by antibodies to human PADPRP . S . pombe cells expressing PADPRP (SPT strains) showed a stable phenotype that was characterized by: (i) cell cycle retardation as a result of a specific delay at the G1 phase, (ii) decreased cell viability in stationary cultures, (iii) enhanced rates of spontaneous and radiation-induced ade6-ade7 mutations, and (iv) increased sensitivity to radiation . SPT strains may prove efficient tools with which to investigate PADPRP functions in eukaryotic cells.

FEMS Microbiol Rev, 1994 Aug, 14(4), 303 - 8
The role of glutathione biosynthesis in heavy metal resistance in the fission yeast Schizosaccharomyces pombe; Coblenz A et al.; Plants and the fission yeast Schizosaccharomyces pombe synthesize small cadmium-binding peptides, called phytochelatins, in response to cadmium . Derived from glutathione (GSH: gamma-Glu-Cys-Gly), they have the general structure (gamma-Glu-Cys)nGly, where n is 2-11 . In order to study the biosynthesis of phytochelatins, we used the mutagen N-methyl-N'-nitro-N nitrosoguanidine (MNNG) to select mutants with a lowered GSH content . GSH-deficient mutants show a Cd-sensitive phenotype, whereas resistance to Cu is only slightly influenced . These Cd-sensitive mutants contain 2-15% of the wild-type GSH level . For three mutants a lowered activity of gamma-glutamylcysteine synthetase was measured . One of the mutants was transformed to Cd-resistance and the complementing fragment was analyzed further . The complementing fragment hybridized with chromosome III . In the transformants, GSH content was restored up to wild-type levels, whereas the activity of gamma-glutamylcysteine synthetase was significantly increased compared with the wild-type . Possible mechanisms for Cd-resistance in the transformants are discussed.

J Bioenerg Biomembr, 1994 Aug, 26(4), 447 - 56
Preparation of highly phosphorylating mitochondria from the yeast Schizosaccharomyces pombe; Jault JM et al.; Schizosaccharomyces pombe yeast cells grown on either fermentable or respiratory media were efficiently converted to stable spheroplasts by the alpha-(1-->3)-glucanase Novozym 234 in the presence of 1.2 M sorbitol . Lysis of spheroplasts by gentle homogenization in dilute sorbitol resulted in the preparation of mitochondria with a structure similar to that observed within the starting yeast cells . The isolated mitochondria exhibited high oxidation rates with various respiratory substrates, NADH being the most efficient . The mitochondria appeared well coupled since the second State 4 rate observed after ADP consumption was identical to the initial one . The State 3 rate in the presence of ADP was completely inhibited by low oligomycin concentrations, similarly to the concomitant ATP synthesis of 900 nmol/min x mg protein . These NADH oxidation and dependent ATP-synthesis activities are much higher than those previously described for mitochondria isolated from Schizosaccharomyces pombe, and similar to the highest values reported for Saccharomyces cerevisiae.

Cell Biol Int, 1994 Aug, 18(8), 813 - 7
Both glucose-type monosaccharides and one of their metabolites are required for activation of yeast plasma membrane H(+)-ATPase; Kotyk A et al.; Saccharomyces cerevisiae and Schizosaccharomyces pombe cells were grown on D-glucose, D-galactose, D-fructose, D-mannose, maltose, trehalose and ethanol . All these substrates were separately added to cells thus grown and the onset and rate of acidification mediated by the plasma membrane H(+)-ATPase were determined . Irrespective of the growth substrate, the best triggers of acidification in both species were fructose, mannose and glucose (with average rates of 5.2, 5.0 and 4.8 nmol H+ per min per mg dry weight, respectively, for S . cerevisiae, and 4.5, 6.8 and 5.8 for S . pombe) . These were followed in S . cerevisiae by galactose in Gal-, Man- and Tre-grown cells (about 0.40 nmol H+) and by maltose in Mal- and Tre-grown cells (about 0.15 nmol H+) . Trehalose elicited some response in only ethanol-grown cells while ethanol itself was completely ineffective in activating the H(+)-ATPase . In S . pombe, however, maltose caused an acidification rate of 3.6 nmol H+ per min per mg dry wt., followed by EtOH (().38), Gal (0.13) and Tre (0.05) . 6-Deoxy-D-glucose and 2-deoxy-D-glucose, not metabolized or improperly metabolized analogues of glucose, had no effect whatsoever . It appears that the sensor triggering the ATPase-activating pathway is a complex responding both to a glucose-type sugar (Glc, Man, Fru) and possibly identical with one of the glucose carriers, and to one of its metabolites, most probably fructose-6-phosphate.

Biochem Mol Biol Int, 1994 Aug, 33(6), 1145 - 9
Univalent-cation-elicited acidification by yeasts; Kotyk A et al.; Addition of univalent cations to sugar-metabolizing Saccharomyces cerevisiae, Schizosaccharomyces pombe and Lodderomyces elongisporus brought about a powerful acidification of the external medium with rates up to nearly 20 nmol H+ per min per mg dry wt . in S . cerevisiae, over 15 nmol in S . pombe, and 4.7 nmol in L . elongisporus . These rates were as much as 20 times, 5.5 times and 10.3 times, respectively . higher than in the absence of K+ . Use of galactose-induced cells, of H(+)-ATPase-deficient mutants and observations over the entire growth curve indicated that the K+ effect on H+ extrusion is not connected with the H(+)-ATPase function as such but rather depends on metabolic reactions producing ATP . The effect has apparently nothing to do with the electrical potential across the plasma membrane.

Mol Biol Cell, 1994 Aug, 5(8), 907 - 20
Expression of mammalian protein kinase C in Schizosaccharomyces pombe: isotype-specific induction of growth arrest, vesicle formation, and endocytosis; Goode NT et al.; Mammalian protein kinase C (PKC) isotypes elicit a number of effects on expression in Schizosaccharomyces pombe . A small decrease in growth rate results from PKC-gamma expression, and treatment of these cells with phorbol esters leads to marked growth inhibition and vesicle formation . PKC-delta and -eta expression causes growth inhibition and vesiculation, and the magnitude of both of these effects is increased by phorbol esters . In contrast, PKC-epsilon expression produces growth inhibition but no vesicle accumulation, and this effect is not responsive to phorbol ester . Finally, PKC-zeta has no observable effect . Thus, isotype-specific biological effects are observed . The accumulation of vesicles correlates with phorbol ester-dependent growth inhibition and occurs only with expression of those isotypes that down-regulate in response to phorbol esters in these cells . Antibodies against mammalian clathrin light chain 1a identified clathrin-coated vesicles and up-regulation of clathrin expression in those cells where vesicles accumulate; the increased vesicular traffic includes an element of endocytosis . Thus expression of specific mammalian PKC isotypes up-regulates endocytosis in S . pombe, providing a likely explanation for PKC-mediated receptor internalization in higher eukaryotes.

Mol Biol Cell, 1994 Aug, 5(8), 877 - 86
Budding and fission yeast casein kinase I isoforms have dual-specificity protein kinase activity; Hoekstra MF et al.; We have examined the activity and substrate specificity of the Saccharomyces cerevisiae Hrr25p and the Schizosaccharomyces pombe Hhp1, Hhp2, and Cki1 protein kinase isoforms . These four gene products are isotypes of casein kinase I (CKI), and the sequence of these protein kinases predicts that they are protein serine/threonine kinases . However, each of these four protein kinases, when expressed in Escherichia coli in an active form, was recognized by anti-phosphotyrosine antibodies . Phosphoamino acid analysis of 32P-labeled proteins showed phosphorylation on serine, threonine, and tyrosine residues . The E . coli produced forms of Hhp1, Hhp2, and Cki1 were autophosphorylated on tyrosine, and both Hhp1 and Hhp2 were capable of phosphorylating the tyrosine-protein kinase synthetic peptide substrate polymer poly-E4Y1 . Immune complex protein kinases assays from S . pombe cells showed that Hhp1-containing precipitates were associated with a protein-tyrosine kinase activity, and the Hhp1 present in these immunoprecipitates was phosphorylated on tyrosine residues . Although dephosphorylation of Hhp1 and Hhp2 by Ser/Thr phosphatase had little effect on the specific activity, tyrosine dephosphorylation of Hhp1 and Hhp2 caused a 1.8-to 3.1-fold increase in the Km for poly-E4Y1 and casein . These data demonstrate that four different CKI isoforms from two different yeasts are capable of protein-tyrosine kinase activity and encode dual-specificity protein kinases.

Mol Biol Cell, 1994 Aug, 5(8), 839 - 49
Physical mapping of origins of replication in the fission yeast Schizosaccharomyces pombe; Wohlgemuth JG et al.; We isolated four fragments from the Schizosaccharomyces pombe genome that mediate autonomous replication . A two-dimensional gel analysis revealed that in each case initiation could be mapped to within the S . pombe sequences . In three of the fragments, initiation could be mapped to one discrete location . In the fourth fragment, subcloning and two-dimensional gel analysis suggested that two discrete origins of replication were located within 3 kb of each other . When in proximity, usually only one of these origins fired, suggesting origin interference . Two-dimensional gel analysis of the four origin fragments at their genomic locations demonstrated that each is used in the chromosomes, but in only a subset of cells or cell divisions . The S . pombe genome appears to contain many discrete origins, not all of which fire in any given cell and some of which are closely spaced . Not I/Sfi I mapping of the five origins from this and a previous study indicates that they are randomly distributed throughout the genome and appear to be representative of chromosomal origins of replication in this organism . We compare the features of S . pombe replication origins with those of S . cerevisiae and animal cells.

Biosci Biotechnol Biochem, 1994 Aug, 58(8), 1467 - 70
Electrophoretic karyotype and gene assignment to chromosomes of Aspergillus oryzae; Kitamoto K et al.; An electrophoretic karyotype of Aspergillus oryzae was obtained by transverse altering-field electrophoresis . Seven chromosomal bands were found . With Schizosaccharomyces pombe chromosomes as size standards, we estimated the sizes of the chromosomes to be 7.0, 5.2, 5.0, 4.5, 4.0, 3.7, and 2.8 megabase pairs (Mbp) . The chromosomal DNA bands were identified with use of 13 cloned genes including ribosomal DNA . The intensity of ethidium staining and the results of Southern blotting with 100 random clones isolated from A . oryzae suggested that the smallest band migrated as doublet and that the total genome size was approximately 35 Mbp.

Biochem Biophys Res Commun, 1994 Jul 29, 202(2), 1113 - 9
Isolation of UV-inducible transcripts from Schizosaccharomyces pombe; Lee JK et al.; Four UV-inducible cDNA clones, UVI15, UVI18, UVI22 and UVI31, were isolated from Schizosaccharomyces pombe by subtraction hybridization . All transcripts of these clones were rapidly induced about 5 to 10 fold within 1 hour after UV-irradiation and the nucleotide sequences of these clones did not showed any significant sequence homology to the known genes in the data bases . Transcripts of UVI18 and UVI31 were induced only by UV-irradiation and those of UVI22 were also induced by alkylating agents, suggesting that inductions of these transcripts are specific responses to DNA damages . However, transcript levels of UVI15 were also increased by other cytotoxic agents including heat shock . These results indicate that UVI15 might be a stress responsive gene.

Mol Gen Genet, 1994 Jul 25, 244(2), 183 - 8
The archaebacterial membrane protein bacterio-opsin is expressed and N-terminally processed in the yeast Saccharomyces cerevisiae; Lang-Hinrichs C et al.; The bop gene codes for the membrane protein bacterio-opsin (BO), which on binding all-trans-retinal, constitutes the light-driven proton pump bacteriorhodopsin (BR) in the archaebacterium Halobacterium salinarium . This gene was cloned in a yeast multi-copy vector and expressed in Saccharomyces cerevisiae under the control of the constitutive ADH1 promoter . Both the authentic gene and a modified form lacking the precursor sequence were expressed in yeast . Both proteins are incorporated into the membrane in S . cerevisiae . The presequence is thus not required for membrane targeting and insertion of the archaebacterial protein in budding yeast, or in the fission yeast Schizosaccharomyces pombe, as has been shown previously . However, in contrast to S . pombe transformants, which take on a reddish colour when all-trans-retinal is added to the culture medium as a result of the in vivo regeneration of the pigment, S . cerevisiae cells expressing BO do not take on a red colour . The precursor of BO is processed to a protein identical in size to the mature BO found in the purple membrane of Halobacterium . The efficiency of processing in S . cerevisiae is dependent on growth phase, as well as on the composition of the medium and on the strain used . The efficiency of processing of BR is reduced in S . pombe and in a retinal-deficient strain of H . salinarium, when retinal is present in the medium.

Mol Gen Genet, 1994 Jul 25, 244(2), 111 - 9
Structural analyses of DNA fragments integrated by illegitimate recombination in Schizosaccharomyces pombe; Tatebayashi K et al.; In order to elucidate the mechanisms of illegitimate recombination in eukaryotes, we have studied the structure of DNA fragments integrated by illegitimate recombination into the genome of fission yeast . Nonhomologous recombination was rarely identified when a long region of homology with the chromosomal leu1+ gene was present in the introduced leu1::ura4+ DNA fragment; but a decrease in length of homology leads to an increase in the ratio of non-homologous to homologous recombination events . The introduced DNA fragments were integrated into different sites in the chromosomes by nonhomologous recombination . The results suggested that there are multiple modes of integration; most events simply involve both ends of the fragments, while in other cases, fragments were integrated in a more complicated manner, probably via circularization or multimerization . To analyze the mechanism of the major type of integration, DNA fragments containing the recombination junctions of three recombinants were amplified by inverted polymerase chain reaction (IPCR) and their nucleotide sequences were determined . There was no obvious homology between introduced DNA and chromosomal DNA at these recombination sites . Furthermore it was found that each terminal region of the introduced DNA was deleted, but that there were no or very small deletions in the target sites of chromosomal DNA . Two models are proposed to explain the mechanism of nonhomologous integration.

Mol Gen Genet, 1994 Jul 25, 244(2), 176 - 82
Isolation and characterization of phosphoprotein phosphatase 1 from alfalfa; Pay A et al.; Protein phosphatases are central regulatory components of diverse processes in eukaryotes and are among the most highly conserved proteins known . In this paper, we report the cloning and sequencing of a type 1 protein phosphatase (pp1Ms) cDNA from alfalfa . Southern analysis indicates the presence of a gene family of PP1 proteins in alfalfa . The pp1Ms open reading frame is very similar to one of five predicted Arabidopsis type 1 protein phosphatases, indicating that different subtypes are individually conserved . Expression of the alfalfa pp1Ms in a temperature-sensitive Schizosaccharomyces pombe PP1 mutant, dis2-11, revealed no complementation, suggesting that PP1Ms is not involved in mitotic regulation . In different plant organs, different pp1Ms transcript levels were observed; in contrast, mRNA levels remained constant in all phases of the cell cycle and in logarithmically growing cells . However, when cells entered stationary phase pp1Ms transcript levels decreased considerably.

Gene, 1994 Jul 22, 145(1), 155 - 6
Identification of a Xenopus cDNA that prevents mitotic catastrophe in the fission yeast Schizosaccharomyces pombe; Su JY et al.; A Xenopus total ovary cDNA library was constructed in a fission yeast expression vector . Using a genetic functional complementation method, we have identified a Xenopus cDNA clone that can rescue several different yeast mitotic catastrophe mutants defective in Wee1 kinase function at the restrictive temperature . The 3.0-kb cDNA clone contains an open reading frame (ORF) of 2226 nucleotides, encoding a predicted 82-kDa protein . The deduced amino acid (aa) sequence shows seven almost identical 30-aa tandem repeats, each of which contains a phosphorylation site meeting the consensus for both Cdc2 kinase and MAP kinase.

Science, 1994 Jul 22, 265(5171), 533 - 5
14-3-3 protein homologs required for the DNA damage checkpoint in fission yeast; Ford JC et al.; During the cell cycle, DNA is replicated and segregated equally into two daughter cells . The DNA damage checkpoint ensures that DNA damage is repaired before mitosis is attempted . Genetic studies of the fission yeast Schizosaccharomyces pombe have identified two genes, rad24 and rad25, that are required for this checkpoint . These genes encode 14-3-3 protein homologs that together provide a function that is essential for cell proliferation . In addition, S . pombe rad24 null mutants, and to a lesser extent rad25 null mutants, enter mitosis prematurely, which indicates that 14-3-3 proteins have a role in determining the timing of mitosis.

Genes Dev, 1994 Jul 15, 8(14), 1693 - 702
A heteromeric protein that binds to a meiotic homologous recombination hot spot: correlation of binding and hot spot activity; Wahls WP et al.; Homologous recombination hot spots are DNA sites that increase the frequency of recombination in their vicinity . The M26 allele of the ade6 gene in Schizosaccharomyces pombe is the first meiotic hot spot with an identified unique nucleotide sequence . We have purified 40,000-fold a heteromeric protein, containing polypeptides Mts1 (70 kD) and Mts2 (28 kD), that binds to the M26 site . Binding in vitro strictly correlates with hot spot activity in vivo for numerous single base pair substitutions in the vicinity of the M26 site, indicating that Mts1/Mts2 activates the M26 site and promotes a rate-limiting step of meiotic recombination . These and other data suggest that homologous recombination may be regulated primarily by discrete DNA sites and proteins that interact with those sites.

Genomics, 1994 Jul 15, 22(2), 482 - 6
An algorithm to detect chimeric clones and random noise in genomic mapping; Grigoriev A et al.; Experimental noise and noncontiguous clone inserts can pose serious problems in reconstructing genomic maps from hybridization data . We describe an algorithm that easily identifies false positive signals and clones containing chimeric inserts/internal deletions . The algorithm "dechimerizes" clones, splitting them into independent contiguous components and cleaning the initial library into a more consistent data set for further ordering . The effectiveness of the algorithm is demonstrated on both simulated data and the real YAC map of the whole genome of the fission yeast Schizosaccharomyces pombe.

Nucleic Acids Res, 1994 Jul 11, 22(13), 2687 - 93
SCR: novel human suppressors of cdc2/cdc13 mutants of Schizosaccharomyces pombe harbour motifs for RNA binding proteins; Kanaoka Y et al.; By phenotypic complementation of the cdc2 and the cdc13 mutants of the fission yeast Schizosaccharomyces pombe, we have cloned two novel multicopy suppressors from a cDNA library of the human fibroblast . They encode homologous proteins containing two regions that are highly conserved among RNA binding proteins . We named them scr2 and scr3, the acronyms of the suppressor of cdc2 (cdc13) with RNA binding motif . They encode proteins of 403 (Scr2) and 407 (Scr3) amino acids . Western blot analysis showed that the amount of Cdc2 increased when either rat kidney fibroblasscr2 or scr3 was introduced into the cdc2-L7 and cdc13-117 mutant cells of S.pombe . No conspicuous alteration in the transcript level was detected as judged by Northern analysis . Considering that the cdc2+ suppresses the cdc13 mutant and vice versa, one of the possible interpretations of these result is that these genes suppress the mutants through the induction of the translation of Cdc2.

Nucleic Acids Res, 1994 Jul 11, 22(13), 2557 - 67
Genetic and biochemical analysis of the fission yeast ribonucleoprotein particle containing a homolog of Srp54p; Selinger D et al.; Mammalian signal recognition particle (SRP), a complex of six polypeptides and one 7SL RNA molecule, is required for targeting nascent presecretory proteins to the endoplasmic reticulum (ER) . Earlier work identified a Schizosaccharomyces pombe homolog of human SRP RNA and showed that it is a component of a particle similar in size and biochemical properties to mammalian SRP . The recent cloning of the gene encoding a fission yeast protein homologous to Srp54p has made possible further characterization of the subunit structure, subcellular distribution, and assembly of fission yeast SRP . S . pombe SRP RNA and Srp54p co-sediment on a sucrose velocity gradient and coimmunoprecipitate, indicating that they reside in the same complex . In vitro assays demonstrate that fission yeast Srp54p binds under stringent conditions to E . coli SRP RNA, which consists essentially of domain IV, but not to the full-length cognate RNA nor to an RNA in which domain III has been deleted in an effort to mirror the structure of bacterial homologs . Moreover, the association of S . pombe Srp54p with SRP RNA in vivo is disrupted by conditional mutations not only in domain IV, which contains its binding site, but in domains I and III, suggesting that the particle may assemble cooperatively . The growth defects conferred by mutations throughout SRP RNA can be suppressed by overexpression of Srp54p, and the degree to which growth is restored correlates inversely with the severity of the reduction in protein binding . Conditional mutations in SRP RNA also reduce its sedimentation with the ribosome/membrane pellet during cell fractionation . Finally, immunoprecipitation under native conditions of an SRP-enriched fraction from {35S}-labeled fission yeast cells suggests that five additional polypeptides are complexed with Srp54p; each of these proteins is similar in size to a constituent of mammalian SRP, implying that the subunit structure of this ribonucleoprotein is conserved over vast evolutionary distances.

Genetics, 1994 Jul, 137(3), 701 - 7
An analysis of interference in the fission yeast Schizosaccharomyces pombe; Munz P; The evaluation of three-point crosses at the tetrad and random spore level leads to the conclusion that both chiasma and chromatid interference are absent in the fission yeast Schizosaccharomyces pombe.

J Eukaryot Microbiol, 1994 Jul-Aug, 41(4), 381 - 7
A cdc2-like kinase associated with commitment to division in Paramecium tetraurelia; Tang L et al.; Cell division in higher eukaryotes is mainly controlled by p34cdc2 or related kinases and by other components of these kinase complexes . We present evidence that cdc2-like kinases also occur in Paramecium . Two polypeptides reacted with an antibody directed against the perfectly conserved PSTAIR region found in cdc2 kinases in other eukaryotes . Only the less abundant peptide bound to p13suc1 from Schizosaccharomyces pombe . Using centrifugal elutriation to select cells on the basis of size, we isolated highly synchronous Paramecium G1 cells . With this procedure, we demonstrated that the p13suc1-associated cdc2-like histone H1 kinase was activated before cell division at the point of commitment to division in Paramecium . Further, we show that Paramecium cdc2-like proteins occurred principally as monomers and that these monomers were active as histone H1 kinases in vitro.

FEMS Microbiol Lett, 1994 Jul 1, 120(1-2), 211 - 6
Dipeptidyl aminopeptidase yspI mutants of Schizosaccharomyces pombe: genetic mapping of dpa1+ on chromosome III; Villa L et al.; A mutant strain of Schizosaccharomyces pombe lacking dipeptidyl aminopeptidase yspI was isolated from a strain already defective in aminopeptidase activity by means of a staining technique with the chromogenic substrate ala-pro-4-methoxy-beta-naph-thylamide to screen colonies for the absence of the enzyme . The defect segregated 2+:2- in meiotic tetrads, indicating a single chromosomal gene mutation, which was shown to be recessive . Gene dosage experiments indicated that the mutation resides in the structural gene of dipeptidyl aminopeptidase yspI, dpa1+ . The dpa1+ gene was located on chromosome III by using m-fluorophenylalanine-induced haploidization and mitotic analysis . dpa1 mutants did not show any obvious phenotype under a variety of conditions tested.

EMBO J, 1994 Jul 1, 13(13), 3011 - 9
The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast; Fankhauser C et al.; Mutation of the Schizosaccharomyces pombe cdc7 gene prevents formation of the division septum and cytokinesis . We have cloned the cdc7 gene and show that it encodes a protein kinase which is essential for cell division . In the absence of cdc7 function, spore germination, DNA synthesis and mitosis are unaffected, but cells are unable to initiate formation of the division septum . Overexpression of p120cdc7 causes cell cycle arrest; cells complete mitosis and then undergo multiple rounds of septum formation without cell cleavage . This phenotype, which is similar to that resulting from inactivation of cdc16 protein, requires the kinase activity of p120cdc7 . Mutations inactivating the early septation gene, cdc11, suppress the formation of multiple septa and allow cells to proliferate normally . If formation of the division septum is prevented by inactivation of either cdc14 or cdc15, p120cdc7 overproduction does not interfere with other events in the mitotic cell cycle . Septation is not induced by overexpression of p120cdc7 in G2 arrested cells, indicating that it does not bypass the normal dependency of septation upon initiation of mitosis . These findings indicate that the p120cdc7 protein kinase plays a key role in initiation of septum formation and cytokinesis in fission yeast and suggest that p120cdc7 interacts with the cdc11 protein in the control of septation.

J Bacteriol, 1994 Jul, 176(13), 3895 - 902
Trehalose-6-P synthase is dispensable for growth on glucose but not for spore germination in Schizosaccharomyces pombe; Blazquez MA et al.; Trehalose-6-P inhibits hexokinases in Saccharomyces cerevisiae (M . A . Blazquez, R . Lagunas, C . Gancedo, and J . M . Gancedo, FEBS Lett . 329:51-54, 1993), and disruption of the TPS1 gene (formerly named CIF1 or FDP1) encoding trehalose-6-P synthase prevents growth in glucose . We have found that the hexokinase from Schizosaccharomyces pombe is not inhibited by trehalose-6-P even at a concentration of 3 mM . The highest internal concentration of trehalose-6-P that we measured in S . pombe was 0.75 mM after heat shock . We have isolated from S . pombe the tps1+ gene, which is homologous to the Saccharomyces cerevisiae TPS1 gene . The DNA sequence from tps1+ predicts a protein of 479 amino acids with 65% identity with the protein of S . cerevisiae . The tps1+ gene expressed from its own promoter could complement the lack of trehalose-6-P synthase in S . cerevisiae tps1 mutants . The TPS1 gene from S . cerevisiae could also restore trehalose synthesis in S . pombe tps1 mutants . A chromosomal disruption of the tps1+ gene in S . pombe did not have a noticeable effect on growth in glucose, in contrast with the disruption of TPS1 in S . cerevisiae . However, the disruption prevented germination of spores carrying it . The level of an RNA hybridizing with an internal probe of the tps1+ gene reached a maximum after 20 min of heat shock treatment . The results presented support the idea that trehalose-6-P plays a role in the control of glycolysis in S . cerevisiae but not in S . pombe and show that the trehalose pathway has different roles in the two yeast species.

Mol Cell Biol, 1994 Jul, 14(7), 4878 - 88
Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage; Murray JM et al.; The rad2 mutant of Schizosaccharomyces pombe is sensitive to UV irradiation and deficient in the repair of UV damage . In addition, it has a very high degree of chromosome loss and/or nondisjunction . We have cloned the rad2 gene and have shown it to be a member of the Saccharomyces cerevisiae RAD2/S . pombe rad13/human XPG family . Using degenerate PCR, we have cloned the human homolog of the rad2 gene . Human cDNA has 55% amino acid sequence identity to the rad2 gene and is able to complement the UV sensitivity of the rad2 null mutant . We have thus isolated a novel human gene which is likely to be involved both in controlling the fidelity of chromosome segregation and in the repair of UV-induced DNA damage . Its involvement in two fundamental processes for maintaining chromosomal integrity suggests that it is likely to be an important component of cancer avoidance mechanisms.

Yeast, 1994 Jul, 10(7), 923 - 33
Cloning and sequencing of Schizosaccharomyces pombe car1 gene encoding arginase . Expression of the arginine anabolic and catabolic genes in response to arginine and related metabolites; Van Huffel C et al.; We report here the cloning and sequencing of the gene encoding arginase (car1) from Schizosaccharomyces pombe . Since no arginase-less strain exists in this organism, we cloned the gene by functional complementation of a car1 mutant strain from Saccharomyces cerevisiae . The S . pombe car1 gene encodes a 323 amino acids polypeptide sharing identity with arginases from different organisms . Measurements of arg3, arg11 and car1 mRNA under different growth conditions confirm the very weak repression by arginine of the two anabolic genes and show that the induction of arginase synthesis operates at a transcriptional level . The promoter of S . pombe car1 gene does not contain the 'arginine boxes' defined as the target of the ARGR-MCM1 proteins in the promoters of the arginine co-regulated genes in S . cerevisiae . The heterologous expression of S . pombe car1 gene in S . cerevisiae is independent of the ARGRII gene product (ArgRIIp/Arg81p) . Determination of arginine, ornithine and citrulline intracellular concentrations shows the efficiency of the different controls operating in S . cerevisiae, and also indicates that in S . pombe enzyme compartmentation is not always sufficient to control the arginine metabolic flux.

Yeast, 1994 Jul, 10(7), 883 - 94
A fission yeast gene encoding a protein that preferentially associates with curved DNA; Yamada H et al.; We searched for fission yeast (Schizosaccharomyces pombe) proteins that preferentially bind to a synthetic curved DNA sequence, by means of a DNA-binding gel shift assay in the presence of an excess amount of a non-curved DNA sequence as a competitor . We identified such a protein in S . pombe . The protein, thus purified, has an apparent molecular weight of 42,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . It was suggested that this protein (42 K-protein) recognizes and binds to a curved DNA structure in a given nucleotide sequence, although it also binds to a non-curved DNA sequence with lower affinity . As its putative coding sequence, a 1.9-kilobase genomic DNA from S . pombe was cloned and sequenced . Sequencing of a cDNA clone also revealed the existence of an open reading frame, with no intron, encoding a 381-amino-acid protein with a calculated molecular mass, 41,597 . This protein appears to be located in the nucleus . The predicted protein sequence revealed that the 42 K-protein exhibits no significant similarity to any other known proteins, except to a hypothetical protein of Caenorhabditis elegans.

J Cell Sci, 1994 Jul, 107 ( Pt 7), 1725 - 35
A calcineurin-like gene ppb1+ in fission yeast: mutant defects in cytokinesis, cell polarity, mating and spindle pole body positioning; Yoshida T et al.; A calcineurin (type 2B)-like protein phosphatase gene designated ppb1+ was isolated from the fission yeast Schizosaccharomyces pombe . The predicted amino acid sequence was 57% identical to rat PP2B alpha . ppb1 null mutant could form colonies at 33 degrees C but the size of the colonies was small at 22 degrees C . Cytokinesis was greatly delayed at 22 degrees C, and a large number of multi-septate cells were produced . The cell polarity control was impaired, causing branched cells . ppb1 null was virtually sterile . These phenotypes were rescued by a plasmid carrying the ppb1+ gene . Multi-septate cells were also produced in wild type at 22 degrees C by cyclosporin A, an inhibitor of calcineurin . This drug effect was enhanced in stst1 null mutant, which was hypersensitive to various drugs and cations . ppb1 null was not affected by cyclosporin A, consistent with the hypothesis that ppb1 is its target . Double-mutant analysis indicated that ppb1 had a function related to that of two other phosphatases, type 1-like dis2 and 2A-like ppa2.ppb1 null-sts1 null showed the severe multi-septate phenotype in the absence of cyclosporin A . ppb1+ and sts1+ gene functions are related . The double mutant ppb1-sts5 was lethal, indicating that the ppb1+ gene shared an essential function with the sts5+ gene . Overexpression of ppb1+ caused anomalies in cell and nuclear shape, microtubule arrays and spindle pole body positioning in interphase cells . Thus the ppb1+ gene appears to be involved in cytokinesis, mating, transport, nuclear and spindle pole body positioning, and cell shape.

Curr Genet, 1994 Jul, 26(1), 31 - 7
Identification and characterization of genes induced during sexual differentiation in Schizosaccharomyces pombe; Sato S et al.; Five cDNA clones, harboring genetic messages preferentially expressed during the sexual differentiation process, were isolated from a cDNA library of Schizosaccharomyces pombe by subtractive screening . Transcription of the corresponding genes, termed isp3, 4, 5, 6, and 7, was dependent on nitrogen starvation and their induction occurred at several stages of spore formation . Analysis of the cDNA primary structures revealed a capacity for the coding of polypeptides of 19.2 kDa, 88.3 kDa, 60.1 kDa, 49.7 kDa, and 43.8 kDa, respectively . The translated amino-acid sequences of isp5 and isp6 were found to show significant similarities to those of amino-acid permeases and proteinase B of Saccharomyces cerevisiae, respectively . Disruption of isp6 arrested the cell cycle prior to conjugation and caused a drastic blocking effect on spore formation.

Curr Biol, 1994 Jul 1, 4(7), 596 - 603
A molecular evolutionary framework for eukaryotic model organisms; Sidow A et al.; BACKGROUND: Implicit in the characterization of a model organism is the hope that insights into its biology can be extended to other species . For this hope to be fulfilled, the phylogenetic position of the model organism within a larger evolutionary framework must be known . We focus here on major model organisms of developmental genetics and cell biology . We first consider the positions of the nematode Caenorhabditis elegans and the arthropod Drosophila melanogaster within a phylogeny of the major advanced metazoan groups . Then we consider the evolutionary relationships between fungi (represented by Saccharomyces cerevisiae and Schizosaccharomyces pombe), plants, and animals . RESULTS: We show, by a direct comparison with small subunit ribosomal RNA (18 S rRNA), that RNA polymerase II is an appropriate molecule for addressing the phylogenetic branchings in the early evolution of eukaryotes . The results from the analyses of newly determined and previously published sequences of the two largest subunits of RNA polymerase II suggest the following . Firstly, that plants and animals share a last common ancestor that excludes fungi, the lineage of which originated earlier . Secondly, that the lineage leading to the nematode Caenorhabditis elegans diverged earlier from the Metazoa than the lineages of arthropods, deuterostomes, annelids and molluscs . Finally, that deuterostomes arose from within protostomes . CONCLUSIONS: RNA polymerase II is well-suited for the elucidation of the evolutionary relationships among eukaryotes . We emphasize the implications of our results for other biological disciplines in addition to molecular evolution, as a phylogenetic framework allows predictions and inferences to be made about the existence of fundamental biological mechanisms elucidated in model organisms.

C R Acad Sci III, 1994 Jul, 317(7), 607 - 13
YBR1012 an essential gene from S . cerevisiae: construction of an RNA antisense conditional allele and isolation of a multicopy suppressor; Nasr F et al.; The gene YBR1012 was identified during the systematic sequencing of chromosome II of the yeast Saccharomyces cerevisiae . We have inactivated the gene and shown that it is essential for cellular viability . Using antisense RNA technology we have constructed a conditional allele, expression of the antisense RNA strongly inhibits growth . To our knowledge this is the first successful use of antisense RNA technology in S . cerevisiae . Comparison of the deduced ybr1012p sequence with the data banks revealed the presence of a putative phosphatidylinositol kinase domain and a strong homology to the Schizosaccharomyces pombe rad3p . These results suggest that ybr1012p may be involved in signal transduction, possibly related to the control of replication and/or DNA damage repair . The link with DNA damage repair was reinforced by the isolation of the DUN1 gene as a multicopy suppressor of the YBR1012 deletion.

Mol Biol Cell, 1994 Jul, 5(7), 785 - 95
Inhibition of G2/M progression in Schizosaccharomyces pombe by a mutant calmodulin kinase II with constitutive activity; Rasmussen C et al.; Intracellular signaling by the second messenger Ca2+ through its receptor calmodulin (CaM) regulates cell function via the activation of CaM-dependent enzymes . Previous studies have shown that cell cycle progression at G1/S and G2/M is sensitive to intracellular CaM levels . However, little is known about the CaM-regulated enzymes involved . Protein phosphorylation has been shown to be important for cell-cycle regulation . Because CaM regulates several protein kinases, and at least one protein phosphatase, our studies are focusing on the roles of these enzymes within the cell cycle . As an initial approach to this problem, cDNAs encoding either normal or mutant calcium/calmodulin kinase II (CaMKII) have been expressed in Schizosaccharomyces pombe . The results show that overexpression of a constitutively active mutant CaMKII caused cell-cycle arrest in G2 . Arrest was associated with a failure to activate the p34/cdc2 protein kinase . Expression of the mutant CaMKII in strains of S . pombe with altered timing of mitosis revealed that this effect is not mediated either by cdc25+ or wee1+, suggesting that CaMKII may regulate G2/M progression by another mechanism.

Mol Biol Cell, 1994 Jul, 5(7), 747 - 61
The centromeric K-type repeat and the central core are together sufficient to establish a functional Schizosaccharomyces pombe centromere; Baum M et al.; The DNA requirements for centromere function in fission yeast have been investigated using a minichromosome assay system . Critical elements of Schizosaccharomyces pombe centromeric DNA are portions of the centromeric central core and sequences within a 2.1-kilobase segment found on all three chromosomes as part of the K-type (K/K"/dg) centromeric repeat . The S . pombe centromeric central core contains DNA sequences that appear functionally redundant, and the inverted repeat motif that flanks the central core in all native fission yeast centromeres is not essential for centromere function in circular minichromosomes . Tandem copies of centromeric repeat K", in conjunction with the central core, exert an additive effect on centromere function, increasing minichromosome mitotic stability with each additional copy . Centromeric repeats B and L, however, and parts of the central core and its core-associated repeat are dispensable and cannot substitute for K-type sequences . Several specific protein binding sites have been identified within the centromeric K-type repeat, consistent with a recently proposed model for centromere/kinetochore function in S . pombe.

Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 5863 - 7
A small nuclear GTP-binding protein from tomato suppresses a Schizosaccharomyces pombe cell-cycle mutant; Ach RA et al.; Ran is a 25-kDa Ras-related nuclear GTP-binding protein which is very highly conserved in humans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe . Ran has been found to form a stable, noncovalent complex with the chromatin-associated protein RCC1, a negative regulator of mitosis . In Sch . pombe, a temperature-sensitive mutation in the RCC1 homolog encoded by the pim1 gene causes premature induction of mitosis, and this mutation can be suppressed by overexpression of the Ran homolog encoded by spi1 . We report here the cloning of three Ran cDNAs from tomato . The Ran protein is very highly conserved among plants, animals, and fungi . In tomato, Ran mRNA is expressed in all tissues examined, even those with little or no cell division, indicating that Ran in plants may have functions other than just control of mitosis . We have found that the tomato Ran protein can direct a beta-glucuronidase reporter protein to the plant cell nucleus, confirming that Ran is a nuclear protein in plants . We show that the tomato Ran protein can suppress the Sch . pombe pim1 mutation, indicating that the tomato Ran protein and the Sch . pombe spi1 protein are functionally homologous.

Eur J Biochem, 1994 Jun 15, 222(3), 879 - 84
Catabolite inactivation of heterologous fructose-1,6-bisphosphatases and fructose-1,6-bisphosphatase-beta-galactosidase fusion proteins in Saccharomyces cerevisiae; Gamo FJ et al.; Fructose-1,6-bisphosphatase (FruP2ase) from Saccharomyces cerevisiae is rapidly inactivated upon addition of glucose to a culture growing on non-sugar carbon sources . Under the same conditions the FruP2ases from Schizosaccharomyces pombe or Escherichia coli expressed in S . cerevisiae were not affected . A chimaeric protein containing the first 178 amino acids from the N-terminal half of S . cerevisiae FruP2ase fused to E . coli beta-galactosidase was susceptible to catabolite inactivation . Elimination of a putative destruction box, RAELVNLVG .. . KK ... . K., beginning at amino acid 60 did not prevent catabolite inactivation . Similarly a change of the vacuole-targeting sequence QKKLD, amino acids 80-84, to QKNSD did not affect significantly the course of inactivation of beta-galactosidase . A fusion protein carrying only the first 138 amino acids from FruP2ase was inactivated at a higher rate than the one carrying the first 178, suggesting the existence of a protective region between amino acids 138 and 178 . A fusion protein carrying the first 81 amino acids from FruP2ase was inactivated by glucose at a similar rate to the one carrying the 178 amino acids, but one with only the first 18 amino acids was resistant to catabolite inactivation . Inactivation of FruP2ase in mutants ubr1 that lack a protein required for ubiquitin-dependent proteolysis, or pra1 that lack vacuolar protease A, proceeded as in a wild type . Our results suggest that at least two domains of FruP2ase may mark beta-galactosidase for catabolite inactivation and that FruP2ase can be inactivated by a mechanism independent of transfer to the vacuole.

EMBO J, 1994 Jun 15, 13(12), 2777 - 88
Characterization of two protein kinases from Schizosaccharomyces pombe involved in the regulation of DNA repair; Dhillon N et al.; We have identified two novel genes designated hhp1+ and hhp2+ in the fission yeast Schizosaccharomyces pombe . The hhp1+ and hhp2+ genes encode two closely related protein kinases that share significant sequence identities with Hrr25p from Saccharomyces cerevisiae . Characterization of strains harboring single and double mutations in the hhp+ genes reveals DNA repair defects in these cells . Schizosaccharomyces pombe strains lacking either or both Hhp activities reveal differences in their ability to withstand DNA lesions caused by either methyl methanesulfonate (MMS) or gamma-rays which correlate with their ability to repair DNA strand breaks caused by these agents . We suggest that Hhp1 and Hhp2 are involved in the regulation of distinct and overlapping DNA repair pathways in S . pombe.

Nucleic Acids Res, 1994 Jun 11, 22(11), 2094 - 101
Nonhomologous DNA end joining in Schizosaccharomyces pombe efficiently eliminates DNA double-strand-breaks from haploid sequences; Goedecke W et al.; Cells of higher eucaryotes are known to possess mechanisms of illegitimate recombination which promote the joining between nonhomologous ends of broken DNA and thus may serve as basic tools of double-strand-break (DSB) repair . Here we show that cells of the fission yeast Schizosaccharomyces pombe also contain activities of nonhomologous DNA end joining resembling the ones found in higher eucaryotes . Nonhomologous end joining activities were detected by transformation of linearized self-replicating plasmids in yeast cells employing a selection procedure which only propagates transformants carrying recircularized plasmid molecules . Linear plasmid substrates were generated by duplicate restriction cuts carrying either blunt ends or 3' or 5' protruding single strands (PSS) of 4 nt which were efficiently joined in any tested combination . Sequence analysis of joined products revealed that junctional sequences were shortened by 1 to 14 nt . Two mechanisms may account for junction formation (i) loss of terminal nucleotides from PSS tails to produce blunt ends which can be joined to abutting ends and (ii) interactions of DNA termini at patches of sequence homologies (1-4 bp) by formation of overlap intermediates which are subsequently processed . A general feature of the yeast joining system is that end joining can only be detected in the absence of sequence homology between the linear substrate and host genome . In the presence of homology, nonhomologous DNA end joining is efficiently competed by activities of homologous recombination.

J Mol Biol, 1994 Jun 3, 239(2), 170 - 80
Intragenic processing in yeast rRNA is dependent on the 3' external transcribed spacer; Melekhovets YF et al.; The nucleotide sequence of the 3' external transcribed spacer (3' ETS) region in Schizosaccharomyces pombe rDNA was determined to define structural features which mediate the termination of RNA transcription and subsequent rRNA maturation . S1 nuclease protection studies suggest three alternative termination sites and four cleavage sites in the processing of the 3' ETS sequence . Each of the termination sites precedes a "Sal box"-like sequence which has been demonstrated to mediate the termination of rRNA transcription in mammalian cells . A highly conserved extended hairpin structure in the ETS sequence was deleted by PCR-mediated mutagenesis and the mutant rDNA was expressed in vivo to determine its role in rRNA maturation . Despite an efficient expression of the mutant gene, mature 5.8 S or 25 S rRNA was not observed . Labelling kinetics and S1 nuclease protection analyses indicate that the deletion not only fully inhibits the removal of the 3' ETS but also fully inhibits the processive excision of the second internal transcribed spacer (ITS2) . Instead, a relatively stable 27 S nRNA precursor remains easily detectable in the whole cell RNA population . The results demonstrate a critical dependence of ITS processing on the 3' ETS raising the possibility that these sequences interact in a common processing domain.

J Cell Biol, 1994 Jun, 125(6), 1289 - 301
The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis; Balasubramanian MK et al.; The fission yeast Schizosaccharomyces pombe divides by medial fission and, like many higher eukaryotic cells, requires the function of an F-actin contractile ring for cytokinesis . In S . pombe, a class of cdc- mutants defective for cytokinesis, but not for DNA replication, mitosis, or septum synthesis, have been identified . In this paper, we present the characterization of one of these mutants, cdc3-124 . Temperature shift experiments reveal that mutants in cdc3 are incapable of forming an F-actin contractile ring . We have molecularly cloned cdc3 and used the cdc3+ genomic DNA to create a strain carrying a cdc3 null mutation by homologous recombination in vivo . Cells bearing a cdc3-null allele are inviable . They arrest the cell cycle at cytokinesis without forming a contractile ring . DNA sequence analysis of the cdc3+ gene reveals that it encodes profilin, an actin-monomer-binding protein . In light of recent studies with profilins, we propose that Cdc3-profilin plays an essential role in cytokinesis by catalyzing the formation of the F-actin contractile ring . Consistent with this proposal are our observations that Cdc3-profilin localizes to the medial region of the cell where the F-actin contractile ring forms, and that it is essential for F-actin ring formation . Cells overproducing Cdc3-profilin become elongated, dumbbell shaped, and arrest at cytokinesis without any detectable F-actin staining . This effect of Cdc3-profilin overproduction is relieved by introduction of a multicopy plasmid carrying the actin encoding gene, act1+ . We attribute these effects to potential sequestration of actin monomers by profilin, when present in excess.

Mol Cell Biol, 1994 Jun, 14(6), 3895 - 905
Analysis of the structural genes encoding M-factor in the fission yeast Schizosaccharomyces pombe: identification of a third gene, mfm3; Kjaerulff S et al.; We previously identified two genes, mfm1 and mfm2, with the potential to encode the M-factor mating pheromone of the fission yeast Schizosaccharomyces pombe (J . Davey, EMBO J . 11:951-960, 1992), but further analysis revealed that a mutant strain lacking both genes still produced active M-factor . Here we describe the isolation and characterization of a third M-factor gene, mfm3 . A mutant lacking all three genes fails to produce M-factor, indicating that all functional M-factor genes now have been identified . The triple mutant exhibits an absolute mating defect in M cells, a defect that is not rescued by addition of exogenous M-factor . A mutational analysis reveals that all three mfm genes contribute to the production of M-factor . Their transcription is limited to M cells and requires the mat1-Mc and ste11 gene products . Each gene is induced when the cells are starved of nitrogen and further induced by a pheromone signal . Additionally, the signal transduction machinery associated with the pheromone response is required for transcription of the mfm genes in both stimulated and unstimulated cells.

Mol Cell Biol, 1994 Jun, 14(6), 3742 - 51
Protein phosphatase 2C, encoded by ptc1+, is important in the heat shock response of Schizosaccharomyces pombe; Shiozaki K et al.; Protein phosphatase 2C (PP2C), an Mg(2+)-dependent enzyme that dephosphorylates serine and threonine residues, defines one of the three major families of structurally unrelated eukaryotic protein phosphatases . Members of the two other families of protein phosphatases are known to have important cellular roles, but very little is known about the biological functions of PP2C . In this report we describe a genetic investigation of a PP2C enzyme in the fission yeast Schizosaccharomyces pombe . We discovered ptc1+ (phosphatase two C) as a multicopy suppressor gene of swo1-26, a temperature-sensitive mutation of a gene encoding the heat shock protein hsp90 . The ptc1+ gene product is a 40-kDa protein with approximately 24% identity to a rat PP2C protein . Purified Ptc1 has Mg(2+)-dependent casein phosphatase activity, confirming that it is a PP2C enzyme . A ptc1 deletion mutant is viable and has approximately normal levels of PP2C activity, observations consistent with the fact that ptc1+ is a member of a multigene family . Although a ptc1 deletion mutant is viable, it has a greatly reduced ability to survive brief exposure to elevated temperature . Moreover, ptc1+ mRNA levels increase 5- to 10-fold during heat shock . These data, demonstrating that Ptc1 activity is important for survival of heat shock, provide one of the first genetic clues as to the biological functions of PP2C.

Mol Cell Biol, 1994 Jun, 14(6), 3707 - 18
Two types of RAS mutants that dominantly interfere with activators of RAS; Jung V et al.; In the fission yeast Schizosaccharomyces pombe, ras1 regulates both sexual development (conjugation and sporulation) and cellular morphology . Two types of dominant interfering mutants were isolated in a genetic screen for ras1 mutants that blocked sexual development . The first type of mutation, at Ser-22, analogous to the H-rasAsn-17 mutant (L . A . Feig and G . M . Cooper, Mol . Cell . Biol . 8:3235-3243, 1988), blocked only conjugation, whereas a second type of mutation, at Asp-62, interfered with conjugation, sporulation, and cellular morphology . Analogous mutations at position 64 of Saccharomyces cerevisiae RAS2 or position 57 of human H-ras also resulted in dominant interfering mutants that interfered specifically and more profoundly than mutants of the first type with RAS-associated pathways in both S . pombe or S . cerevisiae . Genetic evidence indicating that both types of interfering mutants function upstream of RAS is provided . Biochemical evidence showing that the mutants are altered in their interaction with the CDC25 class of exchange factors is presented . We show that both H-rasAsn-17 and H-rasTyr-57, compared with wild-type H-ras, are defective in their guanine nucleotide-dependent release from human cdc25 and that this defect is more severe for the H-rasTyr-57 mutant . Such a defect would allow the interfering mutants to remain bound to, thereby sequestering RAS exchange factors . The more severe interference phenotype of this novel interfering mutant suggests that it functions by titrating out other positive regulators of RAS besides those encoded by ste6 and CDC25.

J Cell Physiol, 1994 Jun, 159(3), 506 - 14
Cisplatin sensitivity correlates with its ability to cause cell cycle arrest via a wee1 kinase-dependent pathway in Schizosaccharomyces pombe; Thiebaut F et al.; Mutants of Schizosaccharomyces pombe were used to define genes involved in the cell cycle arrest produced by cisplatin (DDP), an agent that causes both DNA damage and inhibition of DNA synthesis . Previous work has demonstrated that strains with defective or absent wee1+ function fail to arrest in G2 when DNA is damaged, but do arrest when DNA synthesis is inhibited (Rowley et al., 1992a, Nature, 356:353-355) . Strains defective in wee1+ function, or in the ability of the wee1+ kinase to regulate cdc2, failed to arrest following DDP exposure, as did a rad1-1 mutant . All strains failing to arrest in G2 were hypersensitive to DDP . Thus, DNA damage rather than inhibition of DNA synthesis is causative of DDP-induced cell cycle arrest . In addition, this work shows that the wee1+ and rad1+ gene products are required for successful DDP-induced arrest, and suggests that the ability of S . pombe to arrest is a major determinant of sensitivity to DDP.

Curr Genet, 1994 Jun, 25(6), 497 - 503
Meiosis-dependent mRNA splicing of the fission yeast Schizosaccharomyces pombe mes1+ gene; Kishida M et al.; The mes1+ gene of the fission yeast Schizosaccharomyces pombe is essential for the second meiotic division . We have cloned a 1.1-kb HindIII fragment containing mes1+ by complementation from an S . pombe genomic library . Sequencing of the genomic and cDNA fragments indicates the existence of one small intron of 75 nucleotides, although both the 5'(G/GTTAGT) and 3'(CAG/T) intron-exon junctions deviate from the consensus sequences proposed for S . pombe . The putative translation product of the mature mes1+ mRNA is a 11-kDa protein of 101 amino acids which has no significant homology to any previously-reported proteins . Disruption of mes1 has no effect on cell growth but causes an arrest of meiosis before the second meiotic division . Northern-blot analysis revealed that mes1+ was preferentially transcribed under conditions of nitrogen starvation . When a h90 homothallic strain was shifted to a nitrogen-deficient medium, a pre-mRNA accumulated and then was gradually processed to generate a mature mRNA . This splicing did not occur in either a heterothallic haploid strain or in a homothallic mei2 mutant strain which was defective in the initiation of meiosis . Expression of the first exon alone was not able to suppress the mes1 null allele . These results indicate that mes1+ is required for the completion of meiosis, that splicing is required for the function of the mes1+ gene, and that this splicing requires the function of the mei2+ product.

Microbiology, 1994 Jun, 140 ( Pt 6), 1467 - 72
Cyclic AMP signalling pathway and trehalase activation in the fission yeast Schizosaccharomyces pombe; Carrillo D et al.; The response of derepressed cells of Schizosaccharomyces pombe to the addition of glucose included a marked and reversible activation of neutral trehalase that was not produced in repressed cells . The protein synthesis inhibitor cycloheximide, the protonophore 2,4-dinitrophenol or the uncoupler sodium azide also enhanced trehalase activity in derepressed cells provided glucose was present in the incubation assays . However, only 2,4-dinitrophenol or cycloheximide was able to induce trehalase activation in repressed cells . Stimulation of trehalase by these compounds was preceded in all cases by a rapid increase in adenosine 3'-5'-cyclic monophosphate (cAMP) content . Since exogenous cAMP can activate trehalase both in repressed and derepressed growing cells, the results provide evidence for the existence of an induced cAMP signalling pathway in the fission yeast with several entries for trehalase activation . The correlation between cAMP increase and trehalase activation was not maintained when the enzyme was heat-shock-activated, supporting the concept that trehalase activity can be also enhanced in cells by another mechanism in which cAMP does not act as second messenger.

Yeast, 1994 Jun, 10(6), 757 - 70
Mating pheromone-induced expression of the mat1-Pm gene of Schizosaccharomyces pombe: identification of signalling components and characterization of upstream controlling elements; Aono T et al.; Transcription of the mat1-Pm gene of Schizosaccharomyces pombe controlling entry into meiosis is stimulated by the mating pheromone, M-factor . We have studied its expression by monitoring beta-galactosidase activity in cells carrying a plasmid-borne mat1-Pm/lacZ fusion construct . Stimulation required the M-factor receptor (Map3) and other proteins (Gpa1, Byr1, Byr2 and Spk1) thought to be involved in propagating the pheromone signal within the cell . Mutational activation of gpa1 encoding an alpha subunit of the receptor-coupled heterotrimeric G protein causes full expression of mat1-Pm even in the absence of pheromone, suggesting that Gpa1 is a key signal transmitter . Furthermore, an activated ras1val17 mutant exhibited a much stronger level of induction than wild-type cells, though full expression needs M-factor treatment . Deletion analysis of the mat1-Pm promoter region identified a stretch of 21 bp that is shown to play a critical role in controlling expression . This region lies just upstream of a TATA-like box and contains a TR-box (TTCTTTGTTY) motif which is the recognition site of a putative transcription factor Ste11 . Point mutations in the TR-box motif abolished the expression of mat1-Pm/lacZ . Almost no expression of mat1-Pm was detected in a ste11 deletion mutant, whereas overproduction of Ste11 greatly increased the expression.

J Cell Sci, 1994 Jun, 107 ( Pt 6), 1671 - 8
Comparison of human CAP and CAP2, homologs of the yeast adenylyl cyclase-associated proteins; Yu G et al.; We previously reported the identification of human CAP, a protein that is related to the Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylyl cyclase-associated CAP proteins . The two yeast CAP proteins have similar functions: the N-terminal domains are required for the normal function of adenylyl cyclase, while loss of the C-terminal domains result in morphological and nutritional defects that are unrelated to the cAMP pathways . We have amplified and cloned cDNAs from a human glioblastoma library that encode a second CAP-related protein, CAP2 . The human CAP and CAP2 proteins are 64% identical . Expression of either human CAP or CAP2 in S . cerevisiae cap- strains suppresses phenotypes associated with deletion of the C-terminal domain of CAP, but does not restore hyper-activation of adenylyl cyclase by RAS2val19 . Similarly, expression of either human CAP or CAP2 in S . pombe cap- strains suppresses the morphological and temperature-sensitive phenotypes associated with deletion of the C-terminal domain of CAP in this yeast . In addition, expression of human CAP, but not CAP2, suppresses the propensity to sporulate due to deletion of the N-terminal domain of CAP in S . pombe . This latter observation suggests that human CAP restores normal adenylyl cyclase activity in S . pombe cap- cells . Thus, functional properties of both N-terminal and C-terminal domains are conserved between the human and S . pombe CAP proteins.

J Cell Sci, 1994 Jun, 107 ( Pt 6), 1519 - 28
A single p34cdc2 protein kinase (encoded by nimXcdc2) is required at G1 and G2 in Aspergillus nidulans; Osmani AH et al.; We have cloned and sequenced a homolog of cdc2 from Aspergillus nidulans that can complement the Schizosaccharomyces pombe cdc2-33 mutation . The gene was deleted and is required for continued nuclear DNA replication but not for mitochondrial DNA replication . Three different temperature-sensitive alleles were generated by reverse genetics . All of the mutations generate the nim phenotype of A . nidulans . The new gene was designated nimXcdc2 as it is not allelic to any of the other nim genes (nimA to nimW) of A . nidulans . Reciprocal shift experiments place an essential function for nimXcdc2 in G1 and G2 . Antipeptide antibodies were generated that detect NIMXcdc2, and antisera were also generated to detect NIMEcyclinB . The two p34cdc2 protein species previously detected in A . nidulans, p34 and p37, both precipitate using NIMXcdc2 C-terminus-specific antibodies but only p34 co-precipitates with NIMEcyclinB . Dephosphorylation of denatured p34 converts it to the p37 form, showing p37 to be the non-phosphorylated form of NIMXcdc2 . The phosphorylation of p34 is therefore associated with its interaction with NIMEcyclinB.

Chromosoma, 1994 Jun, 103(3), 162 - 70
Analysis of Schizosaccharomyces pombe mitochondrial DNA replication by two dimensional gel electrophoresis; Han Z et al.; The entire mitochondrial genome of Schizosaccharomyces pombe ura4-294h- was analyzed by the 2D pulsed field gel electrophoresis technique developed by Brewer and Fangman . The genome consists of multimers with an average size of 100 kb and analysis of the overlapping restriction fragments of the complete mitochondrial DNA (mtDNA) genome resulted in simple Y 2D gel patterns . Large single-stranded DNA molecules or double-stranded DNA molecules containing large or numerous single-stranded regions were found in the S . pombe mtDNA preparation . The replication of mtDNA monomers was found to occur in either direction . On the basis of these results, a replication mechanism for S . pombe mtDNA that is most consistent with a rolling circle model is suggested.

Gene, 1994 May 27, 143(1), 139 - 43
Switching gene swi6, involved in repression of silent mating-type loci in fission yeast, encodes a homologue of chromatin-associated proteins from Drosophila and mammals; Lorentz A et al.; The switching gene swi6 of Schizosaccharomyces pombe is involved in the repression of the silent mating-type loci mat2 and mat3 . We have cloned the gene by functional complementation of the switching defect of the swi6-115 mutation . DNA sequence analyses revealed an open reading frame of 984 bp coding for a putative protein of 328 amino acids (aa) . The isolation of a swi6 cDNA confirmed this result . Gene replacement showed that swi6 is not essential for viability . The Swi6 protein is very hydrophilic; it contains 41% charged aa . A region of 48 aa is homologous to a sequence motif found in the chromatin-associated proteins, HP1 and Polycomb (Drosophila melanogaster), M31, M32 and M33 (mouse), and the human HSM1 protein . This motif is called chromo domain (chromatin organization modifier) . Our results indicate that Swi6 is a structural component of chromatin . Swi6 may have the function to compact mat2 and mat3 into a heterochromatin-like conformation which represses the transcription of these silent cassettes.

Gene, 1994 May 16, 142(2), 207 - 11
Cloning and sequence analysis of rhp51+, a Schizosaccharomyces pombe homolog of the Saccharomyces cerevisiae RAD51 gene; Jang YK et al.; A homology (rhp51+) of the RAD51 gene in Schizosaccharomyces pombe was cloned by screening a Sz . pombe genomic library using the 3'-end of RAD51 from Saccharomyces cerevisiae as a probe . As in S . cerevisiae, the sequence of rhp51+ showed two MluI cell-cycle boxes and a putative DNA damage-responsive element in its upstream region . The open reading frame codes for a 365-amino-acid (aa) polypeptide with an estimated molecular mass of 40,555 Da . The deduced aa sequence shows 27, 66, 75 and 80% identity with Escherichia coli RecA, S . cerevisiae Rad51 and the Rad51 homologs from chicken and humans, respectively . The aa sequence encoded by rhp51+ contains A- and B-type nucleotide-binding consensus sequences, as found in other RAD51 homologs . Northern blot analysis showed that rhp51+ encodes a 1.7-kb transcript . Methyl methanesulfonate treatment increased the level of this transcript three- to fivefold . Southern hybridization analysis suggests that a single copy of rhp51+ exists in the Sz . pombe genome.

EMBO J, 1994 May 15, 13(10), 2441 - 51
RNA 3' end signals of the S.pombe ura4 gene comprise a site determining and efficiency element; Humphrey T et al.; We have defined sequences in the 3' non-coding region of the Schizosaccharomyces pombe ura4 gene that are required for efficient mRNA 3' end formation . Three separate sequence elements have been identified . Two of these are site determining elements which specify alternative sites of polyadenylation {the major poly(A) site and a minor downstream poly(A) site} . The third sequence, located downstream of both poly(A) sites, functions as an efficiency element that enhances utilization of either polyadenylation site . By employing sensitive RT-PCR analysis, we demonstrate that although low levels of transcripts are detected up to the efficiency element, none is detected beyond this point . The downstream site determining element and efficiency element have both been delineated to specific 16 nt sequences which we show are together sufficient for ura4 mRNA 3' end formation . We have further characterized the interaction between these two elements and show that the efficiency element behaves in a position-independent, orientation-dependent manner, but cannot form 3' ends independently of the site determining element . Surprisingly, we find that the efficiency element can be functionally replaced by a second copy of either site determining element . We present a model for the mechanism of RNA 3' end formation of the ura4 gene and note that this bipartite structure for a poly(A) signal in S.pombe may be related to the AAUAAA and downstream GU-rich sequences of poly(A) signals in mammalian genes.

J Biol Chem, 1994 May 13, 269(19), 14103 - 10
Schizosaccharomyces pombe fatty acid synthase mediates DNA strand exchange in vitro; Kaslan E et al.; During purification of a strand exchange activity from Schizosaccharomyces pombe using the three-strand reaction of double-stranded linear and circular single-stranded DNA, we identified p190/210 as an activity that stimulated the strand exchange activity of p140exo2 by about 10-fold . The accompanying report (Kaslin, E., and Heyer, W.-D . (1994) J . Biol . Chem . 269, 0000-0000) described the purification and characterization of p140exo2, likely to be the S . pombe homolog of the Saccharomyces cerevisiae strand exchange protein p175SEP1 . Here, we report the purification of p190/210 from S . pombe cells and its identification as fatty acid synthase (FAS) . S . pombe FAS (p190/210) binds to single-stranded and double-stranded DNA, leading to condensation of DNA into large aggregates . In addition, it is capable of renaturing complementary single-stranded DNA . Besides stimulating the strand exchange activity of p140exo2, FAS (p190/210) itself exhibits strand exchange activity provided the double-stranded substrate has single-stranded tails . We propose a probable mechanism for the action of FAS (p190/210) during DNA strand exchange in vitro . Since FAS (p190/210) is highly unlikely to have a role in homologous recombination in vivo, we discuss the implications of our data on the interpretation of other homologous pairing and strand exchange proteins purified from eukaryotes using this or similar assays.

J Biol Chem, 1994 May 13, 269(19), 14094 - 102
A multifunctional exonuclease from vegetative Schizosaccharomyces pombe cells exhibiting in vitro strand exchange activity; Kaslin E et al.; A 140-kDa polypeptide (p140) has been purified over 2000-fold from vegetative Schizosaccharomyces pombe cells using an assay of homologous pairing and strand exchange between linear double-stranded DNA (dsDNA) and circular single-stranded DNA (ssDNA) in vitro . Electron microscopic analysis of the reaction products showed displacement of one strand of the linear duplex DNA by the circular ssDNA molecule . In addition, the protein contained 5' to 3' exonuclease activity on ssDNA and dsDNA (with a 50-fold preference on the single-stranded substrate) as well as on single-stranded RNA . Furthermore, p140 was capable of renaturing complementary ssDNA as shown by S1 nuclease assays . p140 behaved like a monomer in solution under reaction conditions . Direct comparison of the biochemical properties, sequence analysis, and cross-reactivity to a monoclonal antibody suggests that p140 is probably identical with ExoII, purified from S . pombe meiotic cells as a ssDNA exonuclease (Szankasi, P., and Smith, G . R . (1992) Biochemistry 31, 6769-6773) . Given the diverse activities of p140, the protein might be involved in DNA and/or RNA metabolism in vivo.

Nucleic Acids Res, 1994 May 11, 22(9), 1750 - 9
Fission yeast gene structure and recognition; Zhang MQ et al.; A database of 210 Schizosaccharomyces pombe DNA sequences (524,794 bp) was extracted from GenBank (release number 81.0) and examined by a number of methods in order to characterize statistical features of these sequences that might serve as signals or constraints for messenger RNA splicing . The statistical information compiled includes splicing signal (donor, acceptor and branch site) profiles, translational initiation start profile, exon/intron length distributions, ORF distribution, CDS size distribution, codon usage table, and 6-tuple distribution . The information content of the various signals are also presented . A rule-based interactive computer program for finding introns called INTRON.PLOT has been developed and was used to successfully analyze 7 newly sequenced genes.

Proc Natl Acad Sci U S A, 1994 May 10, 91(10), 4589 - 93
Identification of Drosophila cytoskeletal proteins by induction of abnormal cell shape in fission yeast; Edwards KA et al.; To clone metazoan genes encoding regulators of cell shape, we have developed a functional assay for proteins that affect the morphology of a simple organism, the fission yeast Schizosaccharomyces pombe . A Drosophila melanogaster cDNA library was constructed in an inducible expression vector and transformed into S . pombe . When expression of the Drosophila sequences was induced, aberrant cell shapes were found in 0.2% of the transformed colonies . Four severe phenotypes representing defects in cytokinesis and/or cell shape maintenance were examined further . Each displayed drastic and specific reorganizations of the actin cytoskeleton . Three of the cDNAs responsible for these defects appear to encode cytoskeletal components: the actin binding proteins profilin and cofilin/actin depolymerizing factor and a membrane-cytoskeleton linker of the ezrin/merlin family . These results demonstrate that a yeast phenotypic screen efficiently identifies conserved genes from more complex organisms and sheds light on their potential in vivo functions.

Proc Natl Acad Sci U S A, 1994 May 10, 91(10), 4461 - 5
Top-down construction of an ordered Schizosaccharomyces pombe cosmid library; Grothues D et al.; A very rapid and efficient method for sorting and ordering large numbers of clones is presented . This top-down mapping approach divides the entire ordering problem into many smaller tasks and analyzes in parallel a gridded membrane array of clones by hybridization with probe pools . The strategy was tested on a 15-fold-coverage Schizosaccharomyces pombe cosmid library . About 1600 clones were assigned to chromosomes and to regions defined by the Not I and Sfi I restriction maps . Then, the clones were ordered into 20 contigs, which is consistent with statistical expectations for the degree of genome coverage used . The parallel ordering of clones and the computer-based analysis of digitized images make this approach very efficient; it is about 8-fold faster than existing methods . Only 61 hybridizations were needed to order 1600 clones.

FEBS Lett, 1994 May 9, 344(1), 41 - 6
Expression of the human D2S dopamine receptor in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe: a comparative study; Sander P et al.; The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe were tested for heterologous expression of the human D2S dopamine receptor . The cDNA coding for the dopamine receptor was cloned into high copy number plasmids with inducible promoters . After transformation into the yeasts recombinant clones were examined for the presence of functional receptor by radioligand binding using the antagonist {3H}spiperone . Subsequent Western blot analysis of positive recombinants with an antiserum raised against a peptide from the third intracellular domain of the receptor protein revealed the production of a protein with an apparent molecular mass of 40 kDa in both yeasts . Membranes harvested from recombinant yeast clones exhibited saturable binding of the dopaminergic antagonist {3H}spiperone with Kd values of 1.3 nM in S . cerevisiae and 0.25 nM in S . pombe . The rank order of potencies for several dopaminergic ligands to displace specific {3H}spiperone binding to membranes were the same in both yeasts, whereas the affinities for ligands differed significantly.

Gene, 1994 May 3, 142(1), 119 - 22
Two genes encoding ribosomal protein L3 of Schizosaccharomyces pombe and their proximal promoter regions; Liebich I et al.; We have cloned and sequenced two genes, rpl3-1 and rpl3-2, encoding the ribosomal protein L3 of Schizosaccharomyces pombe . The two genes contain an open reading frame encoding 388 amino acids (aa) with a M(r) of 43,808 . The aa sequences are identical, except at position 78, where Rpl3-1 displays a valine residue and Rpl3-2 contains isoleucine . The aa sequences show 75% identity to the RPL3 aa sequence from Saccharomyces cerevisiae . S1-nuclease protection analysis revealed that both genes are transcribed . The promoter sequences of the two rpl3 genes are significantly different, but both promoters contain the conserved homol-D element . Transcription starts between 40 and 50 nt downstream from this element.

EMBO J, 1994 May 1, 13(9), 2066 - 74
Genetic analysis of cell morphogenesis in fission yeast--a role for casein kinase II in the establishment of polarized growth; Snell V et al.; We have initiated a study to identify genes regulating cell morphogenesis in the fission yeast Schizosaccharomyces pombe . Five genes have been identified, orb1-orb5, whose mutation gives rise to spherical cells, indicative of an inability to polarize growth . Two further genes have been identified, tea1 and ban1, whose mutant alleles have disturbed patterns of tip growth, leading to T-shaped and curved cells . In fission yeast, sites of cell wall deposition are defined by actin localization, with actin distributions and therefore growth patterns undergoing cell cycle stage-specific reorganization . Studies of double mutants constructed between orb5-19 and various cdc mutants blocked before and after cell division show that orb5 is required for the re-establishment of polar growth following cytokinesis . This indicates that the mutant allele orb5-19 is defective in the reinitiation of polarized growth, even though actin reorganization to the cell tips occurs normally . orb5 encodes a fission yeast homologue of casein kinase II alpha . We propose that this kinase plays a role in the translation of cell polarity into polarized growth, but not in the establishment of polarity itself.

Curr Genet, 1994 May, 25(5), 465 - 8
The ade4 gene of Schizosaccharomyces pombe: cloning, sequence and regulation; Ludin KM et al.; We report the isolation and sequence of the Schizosaccharomyces pombe ade4 gene which encodes the glutamine phosphoribosylpyrophosphate amidotransferase, the first enzyme of the purine nucleotide de-novo biosynthetic pathway . The enzyme contains 533 amino acids and its sequence exhibits homologies to the corresponding enzymes of Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis, chicken, rat, and human . In contrast to the situation in S . cerevisiae, adenine does not repress ade4 expression at the mRNA level and also other nutritional signals seem not to affect its expression.

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