Yeast, 1992 Dec, 8(12), 1025 - 31 Transport of malic acid in the yeast Schizosaccharomyces pombe: evidence for a proton-dicarboxylate symport; Sousa MJ et al.; The transport system for malic acid present in Schizosaccharomyces pombe cells, growing in batch culture on several carbon sources, has been studied . It was found that the dicarboxylic acid carrier of S . pombe is a proton-dicarboxylate symporter that allows uphill transport and accumulation as a function of delta pH with the following kinetic parameters at pH 5.0: Vmax = 0.1 nmol of total malic acid s-1 mg (dry weight) of cells-1 and Km = 1.0 mM total malic acid . Malic acid uptake (pH 5.0) was accompanied by disappearance of extracellular protons, the uptake rates of which followed Michaelis-Menten kinetics as a function of the acid concentration . The Km values calculated as the concentrations either of anions or of undissociated acid, at various extracellular pH values, pointed to the monoanionic form as the transported species . Furthermore, accumulated free acid suffered rapid efflux after the addition of the protonophore carbonyl cyanid m-chlorophenyl hydrazone . These results suggested that the transport system was a dicarboxylate-proton symporter . Growth of cells in a medium with glucose (up to 14%, w/v) and malic acid (1.5%, w/v) also resulted in proton-dicarboxylate activity, suggesting that the system, besides being constitutive, was still active at high glucose concentrations . The following dicarboxylic acids acted as competitive inhibitors of malic acid transport at pH 5.0: D-malic acid, succinic acid, fumaric acid, oxaloacetic acid, alpha-ketoglutaric acid, maleic acid and malonic acid . In addition, all of these dicarboxylic acids induced proton movements that followed Michaelis-Menten kinetics.(ABSTRACT TRUNCATED AT 250 WORDS) Can J Microbiol, 1992 Dec, 38(12), 1313 - 9 Enzyme activities of D-glucose metabolism in the fission yeast Schizosaccharomyces pombe; Tsai CS et al.; The activities of key enzymes that are members of D-glucose metabolic pathways in Schizosaccharomyces pombe undergoing respirative, respirofermentative, and fermentative metabolisms are monitored . The steady-state activities of glycolytic enzymes, except phosphofructokinase, decrease with a reduced efficiency in D-glucose utilization by yeast continuous culture . On the other hand, the enzymic activities of pentose monophosphate pathway reach the maximum when the cell mass production of the cultures is optimum . Enzymes of tricarboxylate cycle exhibit the maximum activities at approximately the washout rate . The steady-state activity of pyruvate dehydrogenase complex increases rapidly when D-glucose is efficiently utilized . By comparison, the activity of pyruvate decarboxylase begins to increase only when ethanol production occurs . Depletion of dissolved oxygen suppresses the activity of pyruvate dehydrogenase complex but facilitates that of pyruvate decarboxylase . Acetate greatly enhances the acetyl CoA synthetase activity . Similarly, ethanol stimulates alcohol dehydrogenase and aldehyde dehydrogenase activities . Evidence for the existence of alcohol dehydrogenase isozymes in the fission yeast is presented. Nucleic Acids Res, 1992 Nov 25, 20(22), 5943 - 5 Detection and characterization of a ring chromosome in the fission yeast Schizosaccharomyces pombe; Fan JB et al.; NotI and SfiI genomic restriction maps were used to detect and characterize a ring chromosome II in a Schizosaccharomyces pombe strain with a meiotic defect on chromosome II . The ring chromosome was formed by an intrachromosomal fusion near, or at, the very ends of chromosome II. Gene, 1992 Nov 16, 121(2), 393 - 6 Cloning and sequencing of a human cDNA coding for dihydroorotate dehydrogenase by complementation of the corresponding yeast mutant; Minet M et al.; Dihydroorotate dehydrogenase (DHOdehase, EC 1.3.3.1) catalyses the fourth enzymatic step in de novo pyrimidine biosynthesis . A truncated human cDNA encoding this enzyme was isolated from a HeLa cell cDNA library by functional complementation of a corresponding deletion mutant from the yeast, Saccharomyces cerevisiae . The complementing clone contained a 1.5-kb poly(A)(+)-tailed insert with a 1191-bp open reading frame, hybridising with a unique human mRNA of 1.6 kb . The deduced amino acid sequence has 54%, 46% and 42% identity with Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli DHOdehases, respectively . In contrast, it has only 21% identity with the S . cerevisiae enzyme, which probably reflects the cytosolic location of the enzyme in the latter organism. J Biol Chem, 1992 Nov 15, 267(32), 23388 - 92 Purification and characterization of yeast RNA polymerase II general initiation factor g; Henry NL et al.; Yeast RNA polymerase II general initiation factor g was purified to near homogeneity on the basis of its function in a reconstituted transcription system . Polypeptides of 30, 54, and 105 kDa co-purified with transcriptional activity, forming a complex with a mass of 300 kDa as judged by gel filtration, but only 100 kDa based on sedimentation in glycerol gradients, suggesting an elongated shape . Transcription activity could be reconstituted after separation of the three polypeptides under denaturing conditions; the 54- and 105-kDa subunits were both essential, while the 30-kDa subunit was slightly stimulatory . Factor g was required for initiation at all promoters tested, including those from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and adenovirus . Factor g can stably associate with RNA polymerase II, as shown by cosedimentation in a glycerol gradient. Nucleic Acids Res, 1992 Nov 11, 20(21), 5571 - 7 Fission yeast cdc21+ belongs to a family of proteins involved in an early step of chromosome replication; Coxon A et al.; The cdc21+ gene of Schizosaccharomyces pombe was originally identified in a screen for cdc mutants affecting S phase and nuclear division . Here we show that the cdc21+ gene product belongs to a family of proteins implicated in DNA replication . These include the Saccharomyces cerevisiae MCM2 and MCM3 proteins, which are needed for the efficient function of certain replication origins, and S.cerevisiae CDC46, which is required for the initiation of chromosome replication . The cdc21 mutant is defective in the mitotic maintenance of some plasmids, like mcm2 and mcm3 . The mutant arrests with a single nucleus containing two genome equivalents of DNA, and maintains a cytoplasmic microtubular configuration . Activation of most, but not all, replication origins in the mutant may result in failure to replicate a small proportion of the genome, and this could explain the arrest phenotypes . Using the polymerase chain reaction technique, we have identified new cdc21(+)-related genes in S.cerevisiae, S.pombe and Xenopus laevis . Our results suggest that individual members of the cdc21(+)-related family are highly conserved in evolution. Nature, 1992 Nov 5, 360(6399), 84 - 7 A new tropomyosin essential for cytokinesis in the fission yeast S . pombe; Balasubramanian MK et al.; Mutations in the Schizosaccharomyces pombe cdc8 gene impair cytokinesis . Here we clone cdc8+ and find that it encodes a novel tropomyosin . Gene disruption results in lethal arrest of the cell cycle, but spore germination, cell growth, DNA replication and mitosis are all unaffected . Haploid cdc8 gene disruptants are rescued by expression of a fibroblast tropomyosin complementary DNA . Immunofluorescence microscopy of wild type and cdc8 gene disruptants indicates that cdc8 tropomyosin is present in two distinct cellular distributions: in dispersed patches, and during cytokinesis as a transient medial band . Collectively these results indicate that cdc8 tropomyosin has a specialized role which, we suggest, is to form part of the F-actin contractile ring at cytokinesis . These results establish the basis for further genetic studies of cytokinesis and of contractile protein function in S . pombe. Yeast, 1992 Nov, 8(11), 923 - 33 Schizosaccharomyces pombe mitochondria: morphological, respiratory and protein import characteristics; Moore AL et al.; A technique is described for the isolation and purification of intact, respiratory-competent mitochondria from Schizosaccharomyces pombe . The purified mitochondria are capable of oxidizing NADH and succinate as respiratory substrates, indicating the presence of succinate dehydrogenase and an NADH dehydrogenase located on the outer surface of the inner membrane . Mitochondria display good respiratory control with an ADP/O ratio of < 2 . Respiratory activity is linearly dependent upon the redox poise of the quinone pool, suggesting the presence of an unbranched respiratory pathway to molecular oxygen . Immunogold labelling using antisera raised against mitochondrial HSP70 proteins (SSP1, SSC1 and PHSP1) from three different species, namely S . pombe, Saccharomyces cerevisiae and the plant Pisum sativum respectively, has been used to investigate the presence and ultrastructure of the mitochondria isolated by this procedure . The immunocytochemistry was carried out using cells containing wild-type levels of SSP1 protein and cells over-expressing the protein . These results also demonstrate the capacity of mitochondria to import increased levels of protein in vivo . In vitro import experiments using COXIV-DHFR indicate that purified S . pombe mitochondria can efficiently import this precursor, and that protein translocation is dependent upon an oxidizable substrate and a membrane potential. Radiat Res, 1992 Nov, 132(2), 144 - 52 Radiation-induced mitotic delay: a genetic characterization in the fission yeast; Rowley R; Radiation-induced mitotic delay is under investigation in the fission yeast, Schizosaccharomyces pombe . A large range of cell cycle- and radiation-sensitive mutants of this yeast is available to facilitate this effort . Through an examination of such mutants it has been shown that the X-ray transition point and the p34cdc2 execution point are coincident; wee1- strains are not delayed by irradiation; and the radiation-sensitive mutants rad1-1, rad3-136, rad9-192, and rad17-W are not delayed by radiation or by inhibitors of DNA synthesis, including hydroxyurea . A model is proposed: Damaged DNA generates a signal to delay mitosis which is carried by the products of the rad genes to activate the tyrosine kinase p110wee1 . This in turn inactivates the serine/threonine kinase p34cdc2, thereby blocking entry to mitosis . Unreplicated DNA also initiates a signal to delay mitosis which is carried by these same rad genes but, as indicated in the literature, transmission to p34cdc2 does not require p110wee1 . The delay-deficient rad mutants may possess some properties of tumor suppressor genes, with implications for mutagenesis and oncogenesis. Oncogene, 1992 Nov, 7(11), 2249 - 58 PCTAIRE-1 and PCTAIRE-3, two members of a novel cdc2/CDC28-related protein kinase gene family; Okuda T et al.; We have isolated two murine cDNAs designated PCTAIRE-1 and -3 which encode putative serine/threonine-specific protein kinases . The predicted products of PCTAIRE-1 and -3 are 65% homologous and are organized into a core 295-residue kinase domain flanked by unique 161 and 117 amino acid N-terminal and 40 and 39 amino acid C-terminal domains respectively . The kinase domains are approximately 50-55% homologous to members of the cdc2/CDC28 kinase gene family, and each contains a cysteine-for-serine substitution within the conserved PSTAIRE motif . PCTAIRE-1 was ubiquitously expressed as a predominant 3.0-kb transcript and a minor 2.2-kb mRNA resulting from differential polyadenylation . In contrast, PCTAIRE-3 exhibited a more restricted pattern of expression with a single 3.0-kb mRNA detected in brain, kidney and intestine . The PCTAIRE-1 and -3 products produced by in vitro transcription-translation failed to bind to p13suc1 but were precipitated by antibodies directed to Schizosaccharomyces pombe p34cdc2 or to the human PSTAIRE motif . Thus, PCTAIRE-1 and -3 are members of a novel subfamily of cdc2/CDC28-related protein kinases. Genes Dev, 1992 Nov, 6(11), 2021 - 34 Cyclin-B homologs in Saccharomyces cerevisiae function in S phase and in G2; Richardson H et al.; We have cloned four cyclin-B homologs from Saccharomyces cerevisiae, CLB1-CLB4, using the polymerase chain reaction and low stringency hybridization approaches . These genes form two classes based on sequence relatedness: CLB1 and CLB2 show highest homology to the Schizosaccharomyces pombe cyclin-B homolog cdc13 involved in the initiation of mitosis, whereas CLB3 and CLB4 are more highly related to the S . pombe cyclin-B homolog cig1, which appears to play a role in G1 or S phase . CLB1 and CLB2 mRNA levels peak late in the cell cycle, whereas CLB3 and CLB4 are expressed earlier in the cell cycle but peak later than the G1-specific cyclin, CLN1 . Analysis of null mutations suggested that the CLB genes exhibit some degree of redundancy, but clb1,2 and clb2,3 cells were inviable . Using clb1,2,3,4 cells rescued by conditional overproduction of CLB1, we showed that the CLB genes perform an essential role at the G2/M-phase transition, and also a role in S phase . CLB genes also appear to share a role in the assembly and maintenance of the mitotic spindle . Taken together, these analyses suggest that CLB1 and CLB2 are crucial for mitotic induction, whereas CLB3 and CLB4 might participate additionally in DNA replication and spindle assembly. Mol Cell Biol, 1992 Nov, 12(11), 5033 - 40 Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins; Matviw H et al.; The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP . In S . cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway . Both yeast CAPs appear to be bifunctional proteins: the N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways . Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain . On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide . Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins . Expression of the human CAP protein in S . cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain . Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals. EMBO J, 1992 Nov, 11(11), 4205 - 11 Fission yeast and a plant have functional homologues of the Sar1 and Sec12 proteins involved in ER to Golgi traffic in budding yeast; d'Enfert C et al.; Sec12p and Sar1p are required for the formation of transport vesicles generated from the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae . Sec12p is an ER type II membrane protein that mediates the membrane attachment of the GTP-binding Sar1 protein . The SAR1 gene is a multi-copy suppressor of a thermosensitive sec12 mutation . In an attempt to identify functional homologues of Sec12p and Sar1p from other eukaryotic organisms, we screened cDNA expression libraries derived from the fission yeast Schizosaccharomyces pombe and from the plant Arabidopsis thaliana for complementation of the sec12ts mutation . Four individual cDNAs were isolated, two of which encode the S . pombe and A . thaliana homologues of Sar1p . The three Sar1 proteins are 67% identical on average . The two other cDNAs encode type II membrane proteins which were designated Stl1p for the S . pombe protein and Stl2p for the A . thaliana protein (Stl stands for Sec12p-like) . Both proteins have NH2-terminal cytoplasmic domains which resemble that of Sec12p: they are similar in size and present a significant degree of amino acid identity with the cytoplasmic domain of Sec12p . In contrast, the lumenal domains of Sec12p, Stl1p and Stl2p are very different in size and do not show any appreciable homology . That Stl1p and Stl2p are functional homologues of Sec12p was confirmed by showing that expression of either cloned gene complements a sec12 null mutation . Our results indicate that some of the mechanisms regulating vesicle formation at the ER are conserved not only in yeasts, but also in plants. Mol Gen Genet, 1992 Nov, 235(2-3), 413 - 21 The Ncypt1 gene from Neurospora crassa is located on chromosome 2: molecular cloning and structural analysis; Heintz K et al.; Small GTP-binding proteins are encoded by ras-like genes and play a central role in cell differentiation and membrane vesicle transport . By screening genomic and cDNA libraries of the Ascomycete fungus Neurospora crassa with Zmypt genes from Zea mays we have isolated a member of the ypt gene family, Ncypt1 . The gene resides on a 4 kb fragment of genomic DNA and contains four introns, which interrupt the coding sequence of a protein of 203 amino acid residues . The Ncytp1 gene was assigned to a single-copy gene encoding a transcript of 1.5 kb and a protein of 26,000 daltons . The gene maps on linkage group IIR between DB0001 and ccg-2 close to the Fsr-3 locus . Analysis of the nucleotide sequence and the deduced protein sequence revealed a striking homology to yeast, mouse and human genes encoding small GTP-binding proteins that are related to the ras supergene family . Homology was most significant to ypt1 from Schizosaccharomyces pombe, Mus musculus and Homo sapiens sharing 84.8%, 82.3%, and 82.3% identity, respectively . Common domains present in other small GTP-binding proteins were identified in the predicted sequence of the NCYPT1 protein, and the arrangement of peptide motifs sharing similarity with well characterized, small GTP-binding proteins suggests that the NCYPT1 protein is a GTPase . The C-terminal region extending from amino acid residues 175 to 199 shares only weak amino acid sequence similarity with other eukaryotic GTPases . Like other RAS proteins the NCYPT1 protein contains two conserved C-terminal cysteine residues, suggesting post-translational modification(s) by fatty acylation required for membrane anchoring . The high degree of homology between the NCYPT1 protein and eukaryotic YPT proteins suggests that NCYPT1 could be involved in the control of secretory processes. Science, 1992 Oct 16, 258(5081), 478 - 80 The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product; Crews CM et al.; Mitogen-activated protein (MAP) kinases, also known as extracellular signal-regulated kinases (ERKs), are thought to act at an integration point for multiple biochemical signals because they are activated by a wide variety of extracellular signals, rapidly phosphorylated on threonine and tyrosine, and highly conserved . A critical protein kinase lies upstream of MAP kinase and stimulates the enzymatic activity of MAP kinase . The structure of this protein kinase, denoted MEK1, for MAP kinase or ERK kinase, was elucidated from a complementary DNA sequence and shown to be a protein of 393 amino acids (43,500 daltons) that is related most closely in size and sequence to the product encoded by the Schizosaccharomyces pombe byr1 gene . The MEK gene was highly expressed in murine brain, and the product expressed in bacteria phosphorylated the ERK gene product. J Biolumin Chemilumin, 1992 Oct, 7(4), 245 - 53 Luminescence from the yeast Candida utilis and comparisons across three genera; Tilbury RN et al.; Weak luminescence was detected from oxygenated liquid cultures of the yeast Candida utilis during two stages of its growth cycle . The first period of emission occurred during the exponential phase of growth and comprised an ultraviolet band (270-390 nm; ca 19 photons s-1 cm-2 of culture surface) and a visible band (450-620 nm; ca 68 photons s-1 cm-2) . The second period of emission occurred late in the stationary phase of growth and was comprised almost entirely of a visible region band (450-620 nm; 6.8 x 10(2) photons s-1 cm-2) . No luminescence was observed when the yeast was grown anaerobically . These observations are compared with those previously obtained for two other yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe . The ratios of the intensities of the blue/red emissions in the stationary phase luminescences correlated with the ratio of the saturated/unsaturated lipid content for the three yeasts . This result provided further support for the claim that the stationary phase luminescence arises from the reactions associated with lipid peroxidation . A number of previously suggested sources of the exponential phase luminescence are discussed and rejected . Oxidative side reactions accompanying protein synthesis remain a possible source of that emission. Mol Gen Genet, 1992 Oct, 235(1), 122 - 30 Functional conservation between Schizosaccharomyces pombe ste8 and Saccharomyces cerevisiae STE11 protein kinases in yeast signal transduction; Styrkarsdottir U et al.; In fission yeast (Schizosaccharomyces pombe), the mat1-Pm gene, which is required for entry into meiosis, is expressed in response to a pheromone signal . Cells carrying a mutation in the ste8 gene are unable to induce transcription of mat1-Pm in response to pheromone, suggesting that the ste8 gene product functions in the signal transduction pathway . The ste8+ gene encodes a 659 amino acid putative protein kinase, which is identical to the previously identified byr2 suppressor of the ras1 defect . Furthermore, ste8+ is highly homologous to the Saccharomyces cerevisiae STE11 gene, which functions in signal transduction in budding yeast . Expression of the S . cerevisiae STE11 gene in S . pombe ste8 mutants restores the ability to transcribe mat1-Pm in response to pheromone . Also, such cells become capable of conjugation and sporulation . When mat1-Pm is artifically expressed from a heterologous promoter, ste8 mutant cells will enter meiosis . This demonstrates that the meiotic defect of ste8 mutants is due to the absence of the mat1-Pm gene product. Biochem J, 1992 Oct 1, 287 ( Pt 1), 195 - 200 Structurally and functionally conserved regions of cytochrome P-450 reductase as targets for DNA amplification by the polymerase chain reaction . Cloning and nucleotide sequence of the Schizosaccharomyces pombe cDNA; Miles JS; 1 . Alignments of the available cytochrome P-450 reductase amino acid sequences, and comparison with the crystal structure of ferredoxin-NADP reductase, indicate that two highly conserved regions are of functional importance . 2 . Degenerate oligonucleotide primers, based on these sequences, were used in the polymerase chain reaction to amplify a 309 bp fragment of the cytochrome P-450 reductase gene from Schizosaccharomyces pombe for use as an homologous probe . 3 . A 2.6 kb cDNA was cloned from a lambda library, and sequencing revealed an open-reading frame of 2034 bp encoding a protein of M(r) 76774 . This protein shares 38-41% identity with other eukaryotic cytochrome P-450 reductases, and 30% identity with that of Bacillus megaterium . 4 . Comparison of the N-terminal FMN-binding domain with flavodoxin, and the C-terminal FAD- and NADP-binding domain with ferredoxin-NADP reductase, indicates the presence of several functionally conserved regions . 5 . The Sc . pombe cytochrome P-450 reductase gene was shown to contain no introns. Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 8966 - 70 Divergent evolution of pyrimidine biosynthesis between anaerobic and aerobic yeasts; Nagy M et al.; A cDNA encoding the dihydroorotate dehydrogenase (DHOdehase; EC 1.3.3.1) of the yeast Schizosaccharomyces pombe was isolated by functional complementation in Saccharomyces cerevisiae . A divergent subcellular compartmentation of the DHOdehase of each yeast was shown . The DHOdehase from Sch . pombe was localized in the mitochondria whereas its homolog from S . cerevisiae was found to be cytosolic . The heterologous expression of the Sch . pombe enzyme in S . cerevisiae allowed us to demonstrate that the Sch . pombe DHOdehase activity requires the integrity of the mitochondrial electron transport chain . Indeed, the presence of a mutation inactivating cytochrome b abolished the complementation of a S . cerevisiae ura1 mutant by the corresponding Sch . pombe gene . By contrast, in vitro studies have revealed that the DHOdehase of S . cerevisiae uses fumarate as terminal electron acceptor . These results are discussed in relation to the anaerobic growth competence of the two yeasts and to the fermentative processes they use. Genes Dev, 1992 Oct, 6(10), 1914 - 26 Defects in mRNA 3'-end formation, transcription initiation, and mRNA transport associated with the yeast mutation prp20: possible coupling of mRNA processing and chromatin structure; Forrester W et al.; A temperature-sensitive lethal mutation in Saccharomyces cerevisiae, prp20-1, causes defects in several different steps in mRNA metabolism, including mRNA 3'-end formation, transcription initiation, and mRNA transport . Previous work has demonstrated that prp20 mutants are defective in actin pre-mRNA splicing . PRP20 is related, both in structure and function, to the RCC1 gene of mammals and the PIM1 gene of Schizosaccharomyces pombe, both of which appear to regulate entry into mitosis and chromosome condensation . In this report we demonstrate that, after a shift of prp20 mutants to the restrictive temperature, transcripts of several genes (CUP1, CYH2, and GAL10) are produced that extend 1-10 kb beyond their normal polyadenylation sites . The failure in 3'-end formation occurs within 1-2 min of the temperature shift . Transcription initiation also is disrupted, in that initiation sites upstream of the normal cap site are used . mRNA transport from nucleus to cytoplasm also is perturbed: In situ hybridization using an oligo(dT) probe demonstrates accumulation of poly(A) in the nucleus, consistent with the accumulation of longer bulk poly(A) (up to approximately 90-100 nucleotides) and with a failure to transport newly synthesized RNA to the cytoplasm . We demonstrate that prp20 and rna1 mutants are very similar, if not identical, with respect to each of these biochemical phenotypes . In light of the putative role of PRP20 in mitotic control, our results suggest a common step in that process and multiple steps in mRNA synthesis and maturation . We speculate that the perturbations in mRNA processing are the result of effects on the chromatin-nascent RNP-transcription complex or misregulation of a cell cycle component that modifies multiple mRNA-processing activities. Eur J Biochem, 1992 Oct 1, 209(1), 275 - 9 Altered plasma membrane H(+)-ATPase from the Dio-9-resistant pma1-2 mutant of Schizosaccharomyces pombe; Ghislain M et al.; The pma1-2 mutation affecting the plasma membrane H(+)-ATPase of Schizosaccharomyces pombe has been selected for resistance to the antibiotic Dio-9 . In membrane fractions purified from glucose-starved cells, the mutant ATPase activity is reduced by 96%, is insensitive to inhibition by vanadate and has a pH profile displaced in the acidic pH range when compared to the wild type . The maximum velocity of the H(+)-ATPase activity of plasma membranes from glucose-activated pma1-2 cells is activated 20-fold . This is in striking contrast with the wild-type ATPase activity, the maximal velocity of which is not affected by glucose . However, similar to the wild-type enzyme, glucose activation of the pma1-2 mutant H(+)-ATPase reduces the Km for MgATP 9-2 mM and shifts the optimal pH from 4.8 to 6.0-6.5 . The pma1-2 mutation modifies Lys250 to a threonine, which is highly conserved in fungal and plant H(+)-ATPases . These results, compared to those reported for mutations of neighbour residues in yeast or mammalian P-type ATPases, suggest that Lys250 could play a significant role, not only in phosphate binding and/or in the E1P-E2P conformational isomerisation, but also in glucose activation of the H(+)-ATPase. EMBO J, 1992 Oct, 11(10), 3787 - 96 Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae; Zhang S et al.; A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using {32P}poly(dG-m5dC) and {32P}oligo(dG-Br5dC)22 in the presence of B-DNA competitor . Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective . Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor . A Southwestern blot using {32P}poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa . The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast . Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites . The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus . A 60 amino acid segment of zuotin has similarity to several histone H1 sequences . Disruption of ZUO1 in yeast resulted in a slow growth phenotype. EMBO J, 1992 Oct, 11(10), 3491 - 9 Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter; Ortiz DF et al.; In response to heavy metal stress, plants and certain fungi, such as the fission yeast Schizosaccharomyces pombe, synthesize small metal-binding peptides known as phytochelatins . We have identified a cadmium sensitive S . pombe mutant deficient in the accumulation of a sulfide-containing phytochelatin-cadmium complex, and have isolated the gene, designated hmt1, that complements this mutant . The deduced protein sequence of the hmt1 gene product shares sequence identity with the family of ABC (ATP-binding cassette)-type transport proteins which includes the mammalian P-glycoproteins and CFTR, suggesting that the encoded product is an integral membrane protein . Analysis of fractionated fission yeast cell components indicates that the HMT1 polypeptide is associated with the vacuolar membrane . Additionally, fission yeast strains harboring an hmt1-expressing multicopy plasmid exhibit enhanced metal tolerance along with a higher intracellular level of cadmium, implying a relationship between HMT1 mediated transport and compartmentalization of heavy metals . This suggests that tissue-specific overproduction of a functional hmt1 product in transgenic plants might be a means to alter the tissue localization of these elements, such as for sequestering heavy metals away from consumable parts of crop plants. Curr Genet, 1992 Oct, 22(4), 289 - 92 Regulation of pho1-encoded acid phosphatase of Schizosaccharomyces pombe by adenine and phosphate; Schweingruber ME et al.; Expression of pho1-encoded acid phosphatase of Schizosaccharomyces pombe has been reported to be regulated by phosphate . In this communication we show that it is also regulated by adenine . Starving adenine auxotrophic strains for adenine leads to a drastic increase of the enzymatic activity while adenine represses this activity . Full repression by adenine only occurs when phosphate is not growth limiting and vice versa . Regulation occurs at the level of mRNA . We isolated adenine non-repressible mutants . They define four genes (anr1, anr2, anr3, and anr5) which are involved in adenine-dependent pho1 expression . All anr mutants are also phosphate non-repressible . These results indicate that the generation and/or transduction of the intracellular signal responsible for pho1 repression is simultaneously dependent on both adenine and phosphate. Plant Mol Biol, 1992 Oct, 20(2), 361 - 4 The tomato nia gene promoter functions in fission yeast but not in budding yeast; Truong HN et al.; A fragment comprising 1 kb of the 5' region and the 81 first nucleotides of the coding region of the tomato nitrate reductase nia gene was placed in translational fusion with the lacZ reporter gene . This construct was introduced in budding and in fission yeast using a derivative of the Saccharomyces cerevisiae/Schizosaccharomyces pombe autonomously replicating vector pUZL . Beta-galactosidase activity was detected in S . pombe but not in S . cerevisiae . Primer extension experiments show that in fission yeast transcripts are initiated at the same starting point as in tomato, indicating for the first time that a plant promoter can be correctly recognized in fission yeast. Virology, 1992 Oct, 190(2), 889 - 93 The BPV-1 E5 oncoprotein expressed in Schizosaccharomyces pombe exhibits normal biochemical properties and binds to the endogenous 16-kDa component of the vacuolar proton-ATPase; Goldstein DJ et al.; The 44-amino-acid E5 oncoprotein of bovine papillomavirus type 1 transforms immortalized murine fibroblast cell lines . This highly hydrophobic protein forms homodimers, localizes to intracellular membrane compartments (including the Golgi apparatus), and forms a complex with the 16-kDa membrane-embedded constituent (16k) of the vacuolar proton-ATPase . To develop a system for the genetic and biochemical analysis of the E5/16k interaction, the E5 gene was cloned into a new vector which was designed for expression in the fission yeast Schizosaccharomyces pombe . The E5 protein synthesized in this system dimerized normally and bound to endogenous and overexpressed S . pombe 16k protein . Comparison of the S . pombe and mammalian 16k proteins showed strong conservation in carboxyl-terminal amino acids but greater variation in the amino-terminal sequences, suggesting that E5 was interacting with the 16k carboxyl domains . Finally, a new protein epitope tag is described which permitted for the first time the coprecipitation of E5 with antibodies directed against the 16k protein. Biosci Biotechnol Biochem, 1992 Oct, 56(10), 1649 - 54 Inhibition of protein translocation in permeabilized cells of Schizosaccharomyces pombe by puromycin; Kambe-Honjoh H et al.; To investigate protein translocation in eukaryotes, we reconstituted a protein translocation system using the permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe . The precursor of a sex pheromone of Saccharomyces cerevisiae, prepro-alpha-factor, was translocated across the endoplasmic reticulum (ER) of S . pombe posttranslationally, and glycosylated to the same extent as in the ER of S . cerevisiae . This suggested that the size of N-linked core-oligosaccharide in the ER of S . pombe is similar to that in S . cerevisiae . This translocation into the ER of S . pombe was inhibited by puromycin, but the translocation in the P-cells of S . cerevisiae was not inhibited . This difference in sensitivity to puromycin was due to the membrane but not the cytosolic fraction . Our results suggested that the translocation machinery of S . pombe was sensitive to puromycin and different from that of S . cerevisiae. Biochem Cell Biol, 1992 Oct-Nov, 70(10-11), 954 - 71 Regulation of the G2-mitosis transition; Feilotter H et al.; The cell cycle is regulated by pathways composed of a dependent series of steps, by timers, and by checkpoint controls which ensure the completion of one event before the initiation of another . This review focuses on the regulation of the initiation of mitosis, with particular emphasis on the regulation of p34cdc2 activity at this point in the cell cycle . The review draws on data from various organisms, but strongly emphasizes the genetic framework as seen in the fission yeast Schizosaccharomyces pombe and the biology and biochemistry of maturation promoting factor in frog oocytes . An attempt is made to include all known genes and proteins where a link can be made to the initiation event . The nutritional size control and its major known controlling elements, the wee1/mik1 protein kinases, and cdc25 protein tyrosine phosphatase are considered in detail along with their regulation . In addition, the checkpoint control pathways which mediate G2 delay in response to failure of DNA replication or DNA damage are examined. Biochem Cell Biol, 1992 Oct-Nov, 70(10-11), 1088 - 96 Regulation of p105wee1 and p34cdc2 during meiosis in Schizosaccharomyces pombe; Daya-Makin M et al.; Temperature-sensitive pat1 mutants of the fission yeast Schizosaccharomyces pombe can be induced to undergo meiosis at the restrictive temperature, irrespective of the mat1 configuration and the nutritional conditions . Using a combination of exit from stationary phase and thermal inactivation of the 52-kilodalton protein kinase that is encoded by the pat1 (also called ran1) gene, highly synchronous meiotic cultures were obtained . Synthesis and tyrosyl phosphorylation of p34cdc2 was evident during meiotic G1 and S phases . During this period there was increased expression of p105wee1, a protein kinase implicated in the tyrosyl phosphorylation of p34cdc2 . Following a relatively brief G2 period, during which a reduction in the steady-state level of p105wee1 occurred, there was an approximately 19-fold increase in the histone H1 phosphotransferase activity of p34cdc2 . Only a single peak of histone H1 kinase activation was observed, which implies that unlike meiosis in amphibians and echinoderms, p34cdc2 is functional only during one of the meiotic divisions in S . pombe, presumably meiosis II . Stimulation of the kinase activity of p34cdc2 was associated with its tyrosyl dephosphorylation . This is analogous to mitotic M phase and suggests parallels in the mechanism of activation of p34cdc2 during mitosis and one of the meiotic divisions in S . pombe. Development, 1992 Oct, 116(2), 405 - 16 The Drosophila cdc25 homolog twine is required for meiosis; Courtot C et al.; We have identified a second cdc25 homolog in Drosophila . In contrast to string (the first homolog identified in Drosophila) this second homolog, twine, does not function in the mitotic cell cycle, but is specialized for meiosis . Expression of twine was observed exclusively in male and female gonads . twine transcripts are present in germ cells during meiosis, and appear only late during gametogenesis, well after the end of the mitotic germ cell divisions . The sterile Drosophila mutant, mat(2)synHB5, which had previously been isolated and mapped to the same genomic region as twine (35F), was found to carry a missense mutation in the twine gene . This missense mutation in twine abolished its ability to complement a mutation in Schizosaccharomyces pombe cdc25 . Phenotypic analysis of mat(2)synHB5 mutant flies revealed a complete block of meiosis in males and severe meiotic defects in females. Science, 1992 Sep 25, 257(5078), 1955 - 7 Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase; Parker LL et al.; Entry into mitosis in Schizosaccharomyces pombe is negatively regulated by the wee1+ gene, which encodes a protein kinase with serine-, theonine-, and tyrosine-phosphorylating activities . The wee1+ kinase negatively regulates mitosis by phosphorylating p34cdc2 on tyrosine 15, thereby inactivating the p34cdc2-cyclin B complex . The human homolog of the wee1+ gene (WEE1Hu) was overproduced in bacteria and assayed in an in vitro system . Unlike its fission yeast homolog, the product of the WEE1Hu gene encoded a tyrosine-specific protein kinase . The human WEE1 kinase phosphorylated the p34cdc2-cyclin B complex on tyrosine 15 but not on threonine 14 in vitro and inactivated the p34cdc2-cyclin B kinase . This inhibition was reversed by the human Cdc25C protein, which catalyzed the dephosphorylation of p34cdc2 . These results indicate that the product of the WEE1Hu gene directly regulates the p34cdc2-cyclin B complex in human cells and that a kinase other than that encoded by WEE1Hu phosphorylates p34cdc2 on threonine 14. Biochim Biophys Acta, 1992 Sep 24, 1132(2), 222 - 4 Functional and structural conservation of Schizosaccharomyces pombe dTMP kinase gene; Abaigar LT et al.; We describe the isolation and identification of the Schizosaccharomyces pombe dTMP kinase gene by the complementation of a Saccharomyces cerevisiae cell cycle mutant cell, cdc8 . The isolated cDNA contains an open reading frame which can encode a protein with the molecular weight of 24,151 . The deduced protein sequence is highly conserved among known dTMP kinase sequences from different organisms . The isolated gene should facilitate our study of its enzymatic activity, as well as nucleotide metabolism and cell cycle regulation in this organism. Gene, 1992 Sep 21, 119(1), 83 - 9 Isolation and characterization of the Schizosaccharomyces pombe rad3 gene, involved in the DNA damage and DNA synthesis checkpoints; Seaton BL et al.; We have cloned the Schizosaccharomyces pombe rad3 gene which is involved in G2 arrest following DNA damage, and in the dependence of mitosis on the completion of DNA replication . The gene was cloned by complementation of the sensitivity to UV light and gamma rays of the rad3-136 mutant with an Sz . pombe genomic library . Sublocalization of the complementing activity and sequencing of the clone identified an intronless 3210-bp open reading frame capable of encoding a 1070-amino acid protein with an M(r) of 121974 . The rad3 gene is a new gene with no homologs in existing sequence databases . The gene is poorly expressed, with a codon bias index of -0.01 . A disruption mutant affecting the coding region was only slightly more sensitive to UV light than the original rad3-136 mutant . The rad3 gene was mapped to NotI fragment C on chromosome II. Biochem Biophys Res Commun, 1992 Sep 16, 187(2), 722 - 9 Binding selectivity of rhizoxin, phomopsin A, vinblastine, and ansamitocin P-3 to fungal tubulins: differential interactions of these antimitotic agents with brain and fungal tubulins; Li Y et al.; The binding of four potent antimitotic agents, rhizoxin (RZX), phomopsin A (PMS-A), ansamitocin P-3 (ASMP-3), and vinblastine (VLB), to tubulins from RZX-sensitive and -resistant strains of Aspergillus nidulans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae was investigated . Mycelial extracts to which RZX could bind contained beta-tubulin with Asn as the 100th amino acid residue (Asn-100) in all cases, and those without affinity for RZX contained beta-tubulins with either Ile-100 or Val-100 . Though PMS-A shares the same binding site as RZX and ASMP-3 on porcine brain tubulin (Asn-100), only ASMP-3 bound Asn-100 fungal tubulins in a competitive manner with respect to RZX . PMS-A and VLB, which strongly bind to porcine brain tubulin, did not bind to any of the fungal mycelial extracts examined . The results indicate differential interactions of these antimitotic agents with brain and fungal tubulins. Yeast, 1992 Sep, 8(9), 791 - 9 Sequence of the genes encoding subunits A and B of the vacuolar H(+)-ATPase of Schizosaccharomyces pombe; Ghislain M et al.; The genes encoding subunits A (vma1) and B (vma2) of the vacuolar H(+)-ATPase from Schizosaccharomyces pombe were cloned by hybridization to cDNAs of the homologous genes in Neurospora crassa . Both genes are interrupted by introns, two in vma1 and four in vma2 . Positions of introns do not appear to be conserved when compared to those of N . crassa . The subunit A gene encodes a single product of 619 amino acids and is not interrupted by the coding sequence for a second product as found for Saccharomyces cerevisiae (Kane, P . K., Yamashiro, C . T., Wolczyk, D . F., Neff, N., Goebl, M., and Stevens, T . H . (1990) . Science 250, 651-657). Mol Gen Genet, 1992 Sep, 234(3), 449 - 56 Mutations in the cdc10 start gene of Schizosaccharomyces pombe implicate the region of homology between cdc10 and SWI6 as important for p85cdc10 function; Reymond A et al.; The cdc10 gene of the fission yeast Schizosaccharomyces pombe is required for traverse of start and commitment to the mitotic cell division cycle rather than other fates . The product of the gene, p85cdc10, is a component of a factor that is thought to be involved in regulating the transcription of genes that are required for DNA synthesis . In order to define regions of the p85cdc10 protein that are important for its function a fine structure genetic map of the cdc10 gene was derived and the sequences of 13 cdc10ts mutants determined . The 13 mutants tested define eight alleles . Eleven of the mutants are located in the region that contains the two copies of the cdc10/SWI6 repeat motif, implicating it as important for p85cdc10 function. Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 8205 - 9 Purification of a murine protein-tyrosine/threonine kinase that phosphorylates and activates the Erk-1 gene product: relationship to the fission yeast byr1 gene product; Crews CM et al.; We report the purification to near homogeneity of a 45-kDa phorbol ester-stimulated protein kinase that phosphorylates and activates the Erk-1 gene product . This kinase, which we provisionally denote MEK for MAPK/Erk kinase, phosphorylated kinase-inactive Erk-1 protein primarily on a tyrosine residue and, to a lesser extent, on a threonine . We extend our previous results and show that two forms of purified MEK activated the myelin basic protein kinase encoded by Erk-1 . MEK was inactivated by the serine/threonine phosphatase 2A but not by the protein-tyrosine phosphatase 1B . Sequence analysis of peptides generated by trypsin digestion of MEK revealed similarity to the proteins encoded by the Schizosaccharomyces pombe byr1 and Saccharomyces cerevisiae STE7 genes . These data are discussed with regard to a possible signal transduction mechanism. Genetics, 1992 Sep, 132(1), 75 - 85 Meiotically induced rec7 and rec8 genes of Schizosaccharomyces pombe; Lin Y et al.; The Schizosaccharomyces pombe rec7 and rec8 genes, which are required for meiotic intragenic recombination but not for mitotic recombination, have been cloned and their DNA sequences determined . Genetic and physical analyses demonstrated that the cloned fragments contained the rec genes rather than rec mutation suppressors . A 1.6-kb DNA fragment contained a functional rec7 gene, and a 2.1-kb fragment contained a functional rec8 gene . The nucleotide sequences of these fragments revealed open reading frames predicting 249 amino acids for the rec7 gene product and 393 amino acids for the rec8 gene product . Northern hybridization analysis showed that both rec gene mRNAs were detectable only at 2-3 hr after induction of meiosis . The absence of these mRNAs in mitosis and their disappearance at 4 hr and later in meiosis suggest that the rec7 and rec8 gene products may be involved primarily in the early steps of meiotic recombination in S . pombe. J Bacteriol, 1992 Sep, 174(17), 5711 - 8 Regulation of CDP-diacylglycerol synthesis and utilization by inositol and choline in Schizosaccharomyces pombe; Gaynor PM et al.; CDP-diacylglycerol (CDP-DG) is an important branchpoint intermediate in eucaryotic phospholipid biosynthesis and could be a key regulatory site in phospholipid metabolism . Therefore, we examined the effects of growth phase, phospholipid precursors, and the disruption of phosphatidylcholine (PC) synthesis on the membrane-associated phospholipid biosynthetic enzymes CDP-DG synthase, phosphatidylglycerolphosphate (PGP) synthase, phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase in cell extracts of the fission yeast Schizosaccharomyces pombe . In complete synthetic medium containing inositol, maximal expression of CDP-DG synthase, PGP synthase, PI synthase, and PS synthase in wild-type cells occurred in the exponential phase of growth and decreased two- to fourfold in the stationary phase of growth . In cells starved for inositol, this decrease in PGP synthase, PI synthase, and PS synthase expression was not observed . Starvation for inositol resulted in a twofold derepression of PGP synthase and PS synthase expression, while PI synthase expression decreased initially and then remained constant . Upon the addition of inositol to inositol-starved cells, there was a rapid and continued increase in PI synthase expression . We examined expression of these enzymes in cho2 and cho1 mutants, which are blocked in the methylation pathway for synthesis of PC . Choline starvation resulted in a decrease in PS synthase and CDP-DG synthase expression in cho1 but not cho2 cells . Expression of PGP synthase and PI synthase was not affected by choline starvation . Inositol starvation resulted in a 1.7-fold derepression of PGP synthase expression in cho2 but not cho1 cells when PC was synthesized . PS synthase expression was not depressed, while CDP-DG synthase and PI synthase expression decreased in cho2 and cho1 cells in the absence of inositol . These results demonstrate that (i) CDP-DG synthase, PGP synthase, PI synthase, and PS synthase are similarly regulated by growth phase; (ii) inositol affects the expression of PGP synthase, PI synthase, and PS synthase; (iii) disruption of the methylation pathway results in aberrant patterns of regulation of growth phase and phospholipid precursors . Important differences between S . pombe and Saccharomyces cerevisiae with regard to regulation of these enzymes are discussed. J Mol Biol, 1992 Aug 20, 226(4), 1009 - 25 Chromatin structure of Schizosaccharomyces pombe . A nucleosome repeat length that is shorter than the chromatosomal DNA length; Godde JS et al.; We have used new methods for chromatin isolation, together with conventional methods for measuring the nucleosome repeat length, to determine the repeat length of Schizosaccharomyces pombe chromatin . We obtain a result of 156(+/- 2) bp . Equivalent results are obtained using a psoralen crosslinking method for measuring the repeat length in viable spheroplasts . That result, together with other control experiments, rules out many possible artifacts . The measured value of 156(+/- 2) bp is smaller than the length of DNA found in the chromatosome . Thus, the chromatosome cannot be the fundamental unit of chromatin structure in all eukaryotes . The crossed linker model of chromatin higher order structure is incompatible with a nucleosome repeat length of 156 bp, and thus cannot apply to all eukaryotes . The solenoid model of higher order structure is compatible with this repeat length only if the solenoid is right-handed . We note two other properties of this chromatin . (1) Early in digestion, the DNA length of mononucleosomes from S . pombe and Aspergillus nidulans exceeds the nucleosome repeat length . (2) Many methods for isolating chromatin from S . pombe yield an apparent nucleosome repeat length of less than or equal to 140 bp; this result is found to be an artifactual consequence of nucleosome sliding. Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7659 - 63 Simple derivation of TFIID-dependent RNA polymerase II transcription systems from Schizosaccharomyces pombe and other organisms, and factors required for transcriptional activation; Flanagan PM et al.; Resolution of whole cell extract through two chromatographic steps yields a single protein fraction requiring only the addition of TFIID for the initiation of transcription at RNA polymerase II promoters . This approach allows the convenient generation of RNA polymerase II transcription systems from Saccharomyces cerevisiae, human lymphocytes, and Schizosaccharomyces pombe . TFIIDs from all three organisms are interchangeable among all three systems . The S . cerevisiae and Sch . pombe systems support effects of acidic activator proteins, provided a further protein fraction from S . cerevisiae is supplied . This further fraction is distinct from the mediator of transcriptional activation described previously and represents a second component in addition to general initiation factors that may facilitate a response to acidic activators. Nature, 1992 Aug 13, 358(6387), 593 - 7 Anatomy of a transcription factor important for the start of the cell cycle in Saccharomyces cerevisiae; Primig M et al.; Entry of yeast cells into the mitotic cell cycle (Start) involves a form of the CDC28 kinase that associates with G1-specific cyclins encoded by CLN1 and CLN2 (ref . 1) . The onset of Start may be triggered by the activation of CLN1 and CLN2 transcription in late G1 (ref . 2) . SWI4 and SWI6 are components of a factor (SBF) that binds the CACGAAAA (SCB) promoter elements responsible for activation in late G1 of the HO endonuclease, CLN1 and CLN2 genes . A related factor (MBF) containing SWI6 and a 120K protein binds to the ACGCGTNA (MCB) promoter elements responsible for late G1-specific transcription of DNA replication genes . Nothing is known about how these heteromeric proteins bind DNA . We show here that SWI4 contains a novel DNA-binding domain at its N terminus that alone binds specifically to SCBs and a C-terminal domain that binds to SWI6 . SWI4's DNA-binding domain is similar to an N-terminal domain of the cdc10 protein that is a component of an MBF-like factor from Schizosaccharomyces pombe and is required for Start . An involvement of this kind of DNA-binding domain in transcriptional controls at Start may therefore be a conserved feature of eukaryotic cells. Gene, 1992 Aug 1, 117(1), 67 - 72 Identification and analysis of a DNA fragment from Saccharomyces kluyveri that can complement the loss of CDC25 function in Saccharomyces cerevisiae; Prigozy T et al.; In the budding yeast, Saccharomyces cerevisiae, the function of wild-type Ras proteins is dependent on the CDC25 protein, which promotes the exchange of guanine nucleotides bound to Ras . To facilitate the identification of proteins which similarly regulate Ras function in higher eukaryotes, we have identified the CDC25 gene from another budding yeast, Saccharomyces kluyveri, by low-stringency hybridization to an S . cerevisiae CDC25 restriction fragment . This protein, SKCDC25, shares significant amino acid homology with CDC25, SCD25, and Ste6 of Schizosaccharomyces pombe in the C-terminal portion of the protein . The expression of SKCDC25 in a temperature-sensitive cdc25 strain of S . cerevisiae complements the loss of endogenous CDC25 activity . The identification of the highly conserved C-terminal sequences, which direct bona fide CDC25 activity within these proteins, will aid in the isolation of CDC25 genes from higher eukaryotes. Gene, 1992 Aug 1, 117(1), 141 - 3 Cloning and regulation of Schizosaccharomyces pombe thi2, a gene involved in thiamine biosynthesis; Zurlinden A et al.; Biosyntheses of the pyrimidine and thiazole moieties of the thiamine molecule occur by separate pathways . In Schizosaccharomyces pombe, a gene, thi2, is responsible for thiazole synthesis {Schweingruber et al., Curr . Genet . 19 (1991) 249-254} . We have cloned a 3.1-kb genomic S . pombe fragment which can functionally complement a thi2 mutant . The fragment maps genetically at the thi2 site, indicating that it carries thi2 . As shown by Northern hybridization analysis, the appearance of thi2 mRNA levels is repressed when cells are grown in the presence of thiamine and 5-(2-hydroxyethyl)-4-methylthiazole . The thi3 gene involved in the biosynthesis of the pyrimidine moiety, is also regulated by thiamine {Maundrell, J . Biol . Chem . 265 (1990) 10857-10864; Schweingruber et al., Curr . Genet . 19 (1991) 249-254} . We previously identified and analyzed four regulatory genes (tnr1, tnr2, tnr3, and thi1) that are responsible for the regulation of thi3 {Schweingruber et al., Genetics (1992) in press} . Mutants defective in these regulatory genes affect expression of thi2 in a similar way to thi3 . This indicates that biosynthesis of the pyrimidine and thiazole moieties are under common genetic control in S . pombe. Plant Mol Biol, 1992 Aug, 19(5), 847 - 57 Molecular characterization of tobacco cDNAs encoding two small GTP-binding proteins; Dallmann G et al.; We have isolated two cDNAs encoding small GTP-binding proteins from leaf cDNA libraries . These cDNAs encode distinct proteins which show considerable homology to members of the ras superfamily . Np-ypt3, a 1044 bp long Nicotiana plumbaginifolia cDNA, encodes a 24.4 kDa protein which shows 65% amino acid sequence similarity to the Schizosaccharomyces pombe ypt3 protein . The N-ypt3 gene is differentially expressed in mature flowering plants . Expression of this gene is weak in leaves, higher in stems and roots, but highest in petals, stigmas and stamens . Nt-rab5, a 712 bp long Nicotiana tabacum SR1 cDNA, encodes a 21.9 kDa protein which displays 65% amino acid sequence similarity to mammalian rab5 proteins . The expression pattern of the Nt-rab5 gene is very similar to that of the Np-ypt3 gene . The Nt-rab5 gene is virtually not expressed in leaves, higher in stems and roots, and highest in flowers . Both the Nt-rab5 and Np-ypt3 proteins were expressed in Escherichia coli and shown to bind GTP. Yeast, 1992 Aug, 8(8), 647 - 53 Mutational analysis of Schizosaccharomyces pombe U4 snRNA by plasmid exchange; Dandekar T et al.; We have developed a system for testing mutations by plasmid exchange in the fission yeast Schizosaccharomyces pombe . This system has been used to test the requirement for different regions of the small nuclear RNA U4 in S . pombe . Surprisingly, five of seven deletion and substitution mutations tested in different regions of U4 prevent the accumulation of the mutant RNA . Substitution of the U4 sequence in stem 1 of the U4/U6 interaction domain allows accumulation of the mutant U4, but does not support viability . Two sequences with homology to the Sm binding site are found in the 3' region of S . pombe U4; substitution of the 3' sequence of the two does not interfere with accumulation or function of U4, indicating that the 5' sequence is the functional Sm-binding site. Semin Cancer Biol, 1992 Aug, 3(4), 209 - 18 Genetic analysis of ras homologs in yeasts; Powers S; Ras proteins with extensive structural homology to mammalian p21ras have been studied in the two yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe . Comparative analysis of these two yeasts has revealed significant differences in the biochemical and physiological functions that are controlled by this subgroup of eukaryotic GTP-binding regulatory proteins . Despite such divergence of cellular functions, proteins and mechanisms involved in the regulation and modification of Ras proteins are highly conserved in yeasts and other eukaryotes . Genetic analysis of the function of yeast proteins that regulate or modify Ras proteins has provided important information about Ras proteins in general. EMBO J, 1992 Aug, 11(8), 2869 - 75 A mechanosensitive ion channel in Schizosaccharomyces pombe; Zhou XL et al.; Protoplast protuberances (blebs) of Schizosaccharomyces pombe were examined using the patch-clamp technique . In addition to several voltage-gated ion channels, we encountered the activities of a mechanosensitive ion channel with a conductance of 180 pS . Microscopic currents of one or two units were observed in some excised patches and ensemble currents of several tens of units were observed in all blebs examined in whole-bleb configuration . This channel opens at pressures of cm Hg applied to whole blebs and it passes cations, including Ca2+ . It is inactivated by membrane depolarizations and blocked by Gd3+ . We discuss the possible functions of such a channel, including its activation upon cell cycle dependent cytoskeletal reorganizations. Eur J Biochem, 1992 Aug 1, 207(3), 1003 - 8 An intracellular ATP-dependent calcium pump within the yeast Schizosaccharomyces pombe, encoded by the gene cta3; Halachmi D et al.; We have permeabilized the plasma membranes of Schizosaccharomyces pombe cell with nystatin and measured ATP-dependent Ca2+ uptake in the presence of KNO3 and a protonophore in order to inhibit Ca2+ uptake into the vacuole . ATP-dependent Ca2+ accumulation into non-vacuolar Ca(2+)-storing organelles was detected . This Ca2+ uptake activity was maximal at pH 6 and inhibited by vanadate, the inhibitor of P-type ATPases . The null mutation of cta3, a putative Ca2+ gene, {Ghislain, M., Goffeau, A., Halachmi, D . and Eilam, Y . (1990) J . Biol . Chem . 265, 18400-18407} strongly reduced the level of ATP-dependent Ca2+ uptake into non-vacuolar intracellular storing organelles . This result suggests that cta3 encodes an intracellular ATP-dependent Ca2+ pump . The residual ATP-dependent Ca2+ uptake in the mutant strain indicated the presence of a second nonvacuolar, intracellular Ca(2+)-ATPase encoded by a different gene. Biochem Biophys Res Commun, 1992 Jul 31, 186(2), 838 - 45 Protein transport in the permeabilized cell of Schizosaccharomyces pombe; Kambe-Honjoh H et al.; We reconstituted a protein translocation-transport system composed of permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe and the precursor of alpha sex pheromone, prepro-alpha-factor of the budding yeast Saccharomyces cerevisiae . We found that P-cells prepared from the spheroplasts formed in 0.7M KCl as an osmotic stabilizer had the activity to transport pro-alpha-factor to the Golgi apparatus . Electron microscopic observations showed that membranes were preserved more intact in the P-cells prepared from the spheroplasts formed in 0.7M KCl than in 0.7M sorbitol . A glycoprotein of S . pombe contains galactose residues, and we detected incorporation of radiolabeled galactose residues into the anti-prepro-alpha-factor immunoprecipitable fractions in this S . pombe system, but not in the S . cerevisiae system . This paper reports that a heterologous system of in vitro protein transport was performed, and prepro-alpha-factor has the signals necessary for early steps of the transport in S . pombe. Biochemistry, 1992 Jul 28, 31(29), 6769 - 73 A single-stranded DNA exonuclease from Schizosaccharomyces pombe; Szankasi P et al.; We have purified to near homogeneity a DNA exonuclease from meiotic cells of Schizosaccharomyces pombe . The enzyme, designated exonuclease II (ExoII), had an apparent molecular weight of 134,000 and was abundant in the cell . It specifically degraded single-stranded DNA in the 5'----3' direction with an apparent Km for 5' DNA ends of 3.6 x 10(-11) M and produced 5' deoxynucleoside monophosphates . Its mode of degradation is similar to that of the RecJ protein from Escherichia coli; ExoII may, therefore, be involved in genetic recombination and DNA damage repair. Nucleic Acids Res, 1992 Jul 25, 20(14), 3679 - 84 Phenol-treatment and a homologous pairing-assay; Arai N et al.; Homologous pairing is a key step in homologous genetic recombination . In the early stage of trials for the identification of homologous pairing-promoting proteins from a fission yeast, Schizosaccharomyces pombe, we treated DNA products with phenol in the presence of a salt for the removal of tightly bound proteins from DNA before the assay, but we found that this treatment caused very efficient protein-independent double-strand formation from complementary single-stranded DNAs . Using an assay including the phenol treatment, we detected another species of apparent homologous pairing-promoting proteins in the nuclei, in addition to a homologous pairing-promoting protein consisting of three components which we reported previously . However, studies involving the use of an assay without the phenol-treatments revealed that the second one was not really a homologous pairing-protein . Thus, the protein-independent double-strand formation by phenol-treatment in the presence of a salt could cause the erroneous identification of homologous pairing-promoting proteins. Biochem Biophys Res Commun, 1992 Jul 15, 186(1), 122 - 8 Computer modeling of two inorganic pyrophosphatases; Vihinen M et al.; The yeast Saccharomyces cerevisiae has two inorganic pyrophosphatases that are structurally related . One, PPA1, is a cytoplasmic enzyme . The other, PPA2, is located in the mitochondria and appears to be energy-linked . The sequence similarity of PPA1 and PPA2 is about 66% and the identity is about 50% . All amino acids known to be important for catalysis are conserved, except one glutamate which is substituted by an aspartate in PPA2 . The structures of PPA2 and the cytoplasmic PPase from Schizosaccharomyces pombe were modeled based on the three dimensional structure of PPA1 . Two cysteines in PPA2 and one in the S . pombe enzyme are located at the catalytic cleft . Four residues form an unique insertion near the entrance of the catalytic cleft in the mitochondrial enzyme. Nucleic Acids Res, 1992 Jul 11, 20(13), 3383 - 90 Mammalian promoter element function in the fission yeast Schizosaccharomyces pombe; Prentice HL et al.; We have analyzed the function of several mammalian promoter elements in the fission yeast, Schizosaccharomyces pombe . Mutants of the human HSP70 promoter were introduced into S . pombe as single copy integrants at a specific location . Transcription initiation sites utilized in S . pombe with the HSP70 TATA element were similar to those used in mammalian cells . Of three mammalian TATA elements tested, only the HSP70 TATA element functioned in S . pombe . The adenovirus Ella TATA element had little or no activity in S . pombe, indicating that S . pombe is deficient in the factor(s) necessary for recognition of this element . Of upstream promoter elements tested, the CCAAT, Sp1 binding, ATF binding and heat shock elements were functional in S . pombe . Strains containing mutant promoters fused to the ble gene were used to demonstrate that phleomycin can be used as a graded selection in S . pombe . These data demonstrate that S . pombe should provide a useful system in which to characterize and isolate mammalian factors involved in initiation site determination and transcriptional regulation. J Immunol, 1992 Jul 1, 149(1), 17 - 23 Expression of the murine homologue of the cell cycle control protein p34cdc2 in T lymphocytes; Kim YH et al.; The mammalian homologue of the cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a p34cdc2 cyclin-dependent kinase that regulates the cell cycle of a wide variety of cell types . Resting murine T lymphocytes contained no detectable p34cdc2 protein, histone kinase activity, or specific mRNA for the cdc2 gene . Activation of the T cells by immobilized anti-CD3 resulted in the expression of specific mRNA late in the G1 phase of the cell cycle, and p34cdc2 protein was detectable at or near G1/S . At this point in the cell cycle, the protein was phosphorylated at tyrosine and displayed no H1 histone kinase activity . As the cells progressed through the cycle, the amount of specific mRNA and p34cdc2 increased, and H1 histone kinase activity was detectable when the cells were blocked at G2/M by nocodazole . The activation of T cells by phorbol dibutyrate induced the expression of IL-2R but failed to induce the synthesis of IL-2 or the expression of cdc2-specific mRNA . Under these conditions, the activated cells failed to enter the S phase of the cell cycle . Because the presence of IL-2 added exogenously during activation by phorbol dibutyrate resulted in the expression of cdc2-specific mRNA and progression through the cell cycle, either IL-2 or the interaction with IL-2R may be involved in the expression of cdc2 and regulation of the G1/S transition. Eur J Biochem, 1992 Jul 1, 207(1), 201 - 5 Spectral characteristics of cadmium-containing phytochelatin complexes isolated from Schizosaccharomyces pombe; Plocke DJ et al.; Phytochelatins, heavy-metal-containing peptides with structures (gamma EC)nG, where n = 2-8, have been isolated from higher plants and the fission yeast Schizosaccharomyces pombe . The present work describes the isolation and characterization of several naturally occurring mixed complexes of these peptides from S . pombe exposed to 1 mM CdCl2 . A lower-molecular-mass fraction from Sephadex G-50 chromatography yielded three distinct species on further fractionation . HPLC chromatography revealed the presence of peptides with n = 1-4 in varying amounts in these three complexes, referred to as complexes I, II and III . Stoichiometries are proposed for these complexes, based on {Cd}, {SH}, {S2-} and the amino acid content . Ultraviolet absorption and magnetic circular dichroism spectra of complexes II and III are similar, whereas the CD spectra of these two complexes are strikingly different . Compared to both complexes II and III, the CD bands of complex I are relatively weak . Ultraviolet absorption, CD and magnetic circular dichroism spectra provide a basis for the discussion of structural differences in these complexes. Mol Biol Cell, 1992 Jul, 3(7), 721 - 34 A gene encoding a protein with seven zinc finger domains acts on the sexual differentiation pathways of Schizosaccharomyces pombe; Xu HP et al.; Byr3 was selected as a multicopy suppressor of the sporulation defects of diploid Schizosaccharomyces pombe cells that lack ras1 . Like cells mutant at byr1 and byr2, two genes that encode putative protein kinases and that in multiple copies are also suppressors of the sporulation defects of ras1 null diploid cells, cells mutant at byr3 are viable but defective in conjugation . Nucleic acid sequence indicates byr3 has the capacity to encode a protein with seven zinc finger binding domains, similar in structure to the cellular nucleic acid binding protein (CNBP), a human protein that was identified on the basis of its ability to bind DNA . Expression of CNBP in yeast can partially suppress conjugation defects of cells lacking byr3. Nat Genet, 1992 Jul, 1(4), 273 - 7 Complete coverage of the Schizosaccharomyces pombe genome in yeast artificial chromosomes; Maier E et al.; The genome of the fission yeast, Schizosaccharomyces pombe, consists of some 14 million base pairs of DNA contained in three chromosomes . On account of its excellent genetics we used it as a test system for a strategy designed to map mammalian chromosomes and genomes . Data obtained from hybridization fingerprinting established an ordered library of 1,248 yeast artificial chromosome clones with an average size of 535 kilobases . The clones fall into three contigs completely representing the three chromosomes of the organism . This work provides a high resolution physical and clone map of the genome, which has been related to available genetic and physical map information. Biochemistry, 1992 Jun 30, 31(25), 5791 - 8 Differential nucleotide binding to catalytic and noncatalytic sites and related conformational changes involving alpha/beta-subunit interactions as monitored by sensitive intrinsic fluorescence in Schizosaccharomyces pombe mitochondrial F1; Divita G et al.; Mitochondrial F1 from the yeast Schizosaccharomyces pombe exhibits an intrinsic tryptophan fluorescence sensitive to adenine nucleotides and inorganic phosphate {Divita, G., Di Pietro, A., Deleage, G., Roux, B., & Gautheron, D.C . (1991) Biochemistry 30, 3256-3262} . The present results indicate that the intrinsic fluorescence is differentially modified by nucleotide binding to either catalytic or noncatalytic sites . Guanine or hypoxanthine nucleotides, which selectively bind to the catalytic site, produce a hyperbolic saturation monitored by fluorescence quenching at 332 nm, the maximal emission wavelength . On the contrary, adenine nucleotides, which bind to both catalytic and noncatalytic sites, exhibit a biphasic saturation . High-affinity ATP binding produces a marked quenching as opposed to the lower-affinity one . In contrast, ADP exhibits a sigmoidal saturation, with high-affinity binding producing no quenching but responsible for positive cooperativity of binding to the lower-affinity site . The catalytic-site affinity for GDP is almost 20-fold higher at pH 5.0 as compared to pH 9.0, and the high sensitivity of the method allows detection of the 10-fold lower-affinity GMP binding . In contrast, high-affinity binding of ADP, or AMP, is not pH-dependent . The selective catalytic-site saturation induces a F1 conformational change decreasing the Stern-Volmer constant for acrylamide and the tryptophan fraction accessible to iodide . ATP saturation of both catalytic and noncatalytic sites produces an additional reduction of the accessible fraction to acrylamide. Cell, 1992 Jun 26, 69(7), 1159 - 69 Mutations at the 3' splice site can be suppressed by compensatory base changes in U1 snRNA in fission yeast; Reich CI et al.; U1 snRNA is an essential splicing factor known to base pair with 5' splice sites of premessenger RNAs . We demonstrate that pairing between the universally conserved CU just downstream from the 5' junction interaction region and the 3' splice site AG contributes to efficient splicing of Schizosaccharomyces pombe introns that typify the AG-dependent class described in mammals . Strains carrying mutations in the 3' AG of an artificial intron accumulate linear precursor, indicative of a first step block . Lariat formation is partially restored in these mutants by compensatory changes in nucleotides C7 and U8 of U1 snRNA . Consistent with a general role in fission yeast splicing, mutations at C7 are lethal, while U8 mutants are growth impaired and accumulate linear, unspliced precursor to U6 snRNA . U1 RNA-mediated recognition of the 3' splice site may have origins in analogous intramolecular interactions in an ancestral self-splicing RNA. Nature, 1992 Jun 11, 357(6378), 505 - 8 SWI6 protein is required for transcription of the periodically expressed DNA synthesis genes in budding yeast; Lowndes NF et al.; In budding yeast many genes are expressed under cell-cycle control in late G1 . These include a large group of DNA synthesis genes, the HO gene involved in mating-type switching, CTS1 (chitinase) and also CLN1 and CLN2 (ref . 4) encoding G1 cyclins . Two factors, encoded by the SWI4 and SWI6 genes, are required for HO (ref . 5), CLN (refs 6, 7) and CTS1 (ref . 3) gene expression and, at least in the HO promoter, bind to CACGA4 upstream sequences (CCBs) . This motif is not found upstream of the DNA synthesis genes, which instead have a hexamer element, ACGCGT1 (MCB), an MluI restriction site, that is recognized by a cell-cycle regulated transcription complex DSC1 (ref . 1) . This MluI-activation system consisting of the MCBs and DSC1 is conserved in fission yeast where a DSC1-like complex controls the cdc22+ ribonucleotide reductase gene . The Schizosaccharomyces pombe cdc10+ gene encodes a component of DSC1 (ref . 10) and, significantly, this has homology with both the Swi4 and Swi6 proteins . Here we show that Swi6 is an essential component of DSC1 and that deletion of SWI6 impairs the cell-cycle regulation of the DNA synthesis genes, as well as CLN1 and CLN2 . Thus Swi6 is the common factor in regulation of all the above genes and may therefore be responsible for the timing of their expression in late G1. Nucleic Acids Res, 1992 Jun 11, 20(11), 2673 - 8 Cloning and characterisation of the S . pombe rad15 gene, a homologue to the S . cerevisiae RAD3 and human ERCC2 genes; Murray JM et al.; The RAD3 gene of Saccharomyces cerevisiae encodes an ATP-dependent 5'-3' DNA helicase, which is involved in excision repair of ultraviolet radiation damage . By hybridisation of a Schizosaccharomyces pombe genomic library with a RAD3 gene probe we have isolated the S . pombe homologue of RAD3 . We have also cloned the rad15 gene of S . pombe by complementation of radiation-sensitive phenotype of the rad15 mutant . Comparison of the restriction map and DNA sequence, shows that the S . pombe rad15 gene is identical to the gene homologous to S . cerevisiae RAD3, identified by hybridisation . The S . pombe rad15.P mutant is highly sensitive to UV radiation, but only slightly sensitive to ionising radiation, as expected for a mutant defective in excision repair . DNA sequence analysis of the rad15 gene indicates an open reading frame of 772 amino acids, and this is consistent with a transcript size of 2.6 kb as detected by Northern analysis . The predicted rad15 protein has 65% identity to RAD3 and 55% identity to the human homologue ERCC2 . This homology is particularly striking in the regions identified as being conserved in a group of DNA helicases . Gene deletion experiments indicate that, like the S . cerevisiae RAD3 gene, the S . pombe rad15 gene is essential for viability, suggesting that the protein product has a role in cell proliferation and not solely in DNA repair. FEBS Lett, 1992 Jun 8, 304(1), 73 - 7 Exon-intron organization of the Arabidopsis thaliana protein kinase genes CDC2a and CDC2b; Imajuku Y et al.; We have previously shown by cDNA cloning that a higher plant, Arabidopsis thaliana, possesses at least two CDC2 genes (CDC2a and CDC2b) similar to the cell-cycle-controlling cdc2 gene of Schizosaccharomyces pombe . To understand the exon-intron organization of these genes, genomic clones were isolated and their nucleotide sequences determined . The coding and 5'-untranslated regions of CDC2a were interrupted by seven and one introns, respectively, whilst CDC2b contained three introns within the coding portion . These intron positions partly overlapped with each other and with those of the yeast cdc2 gene, nevertheless the lengths and sequences of the corresponding introns were diverse. J Biol Chem, 1992 Jun 5, 267(16), 11329 - 36 Post-translational processing of Schizosaccharomyces pombe YPT proteins; Newman CM et al.; ras proteins are post-translationally processed at their carboxyl-terminal CAAX motif by a triplet of modifications: prenylation of C with farnesyl, proteolytic trimming of AAX, and carboxyl-methylation . These modifications co-operate with palmitoylation of nearby sites or a polybasic region to target plasma membrane localization . The related YPT/rab proteins in contrast are localized to compartments of the endo-membrane system and may be involved in directing membrane traffic . These proteins end in XCC or CXC motifs . We have analyzed the processing of members of this subfamily form the fission yeast Schizosaccharomyces pombe . We find using in vitro translation in reticulocyte lysates that YPT1, -3, and -5 are prenylated with geranylgeranyl and that they incorporate label from {3H}mevalonic acid when expressed in transfected COS cells in vivo . Furthermore, prenylation was necessary for membrane binding in vivo . The CXC protein YPT5, but neither of the two XCC proteins YPT1 and YPT3, was carboxyl-methylated in S . pombe and in COS cells in vivo . However, YPT5 was not carboxyl-methylated in vitro in lysates which were able to methylate ras protein . YPT3 was detectably palmitoylated when expressed in COS cells, though at a much lower level than ras. Genetics, 1992 Jun, 131(2), 255 - 60 Nuclear mutations in the petite-negative yeast Schizosaccharomyces pombe allow growth of cells lacking mitochondrial DNA; Haffter P et al.; The fission yeast Schizosaccharomyces pombe has never been found to give rise to viable cells totally lacking mitochondrial DNA (rho(o)) . This paper describes the isolation of rho(o) strains of S . pombe by very long term incubation of cells in liquid medium containing glucose, potassium acetate and ethidium bromide . Once isolated, the rho(o) strains did not require potassium acetate or any other novel growth factors . These nonrespiring strains contained no mitochondrial DNA (mtDNA) detectable either by gel-blot hybridization using as probe a clone containing the entire S . pombe mtDNA, or by 1',6-diamidino-2-phenylindole staining of whole cells . Induction of rho(o) derivatives of standard laboratory strains was not reproducible from culture to culture . The cause of this irreproducibility appears to be that growth of the rho(o) strains of S . pombe depended on nuclear mutations that occurred in some, but not all, of the initial cultures . Two independent rho(o) isolates contained mutations in unlinked genes, termed ptp1-1 and ptp2-1 . These mutations allowed reproducible ethidium bromide induction of viable rho(o) strains . No other phenotypes were associated with ptp mutations in rho+ strains. Mol Gen Genet, 1992 Jun, 233(3), 436 - 42 The switching gene swi6 affects recombination and gene expression in the mating-type region of Schizosaccharomyces pombe; Lorentz A et al.; The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in homothallic (h90) strains of Schizosaccharomyces pombe . The MT region of h90 comprises three cassette genes: the expression site mat1:1 and two silent loci, mat2:2 and mat3:3 . Besides reducing MT switching, the swi6 mutation leads to deletions in the MT region caused by intrachromosomal cross-overs between two paired cassettes . These deletions only arise if DNA double-strand breaks are present at mat1:1, which initiate MT switching . Furthermore, swi6 allows meiotic recombination in the K region, a region of 16 kb between mat2:2 and mat3:3; in wild-type strains no recombination occurs in K . swi6 also allows the simultaneous expression of two different cassettes in the same haploid cell . Thus swi6 may have an influence on the general chromatin structure in the MT region. J Bacteriol, 1992 Jun, 174(12), 4078 - 85 Mutant enrichment of Schizosaccharomyces pombe by inositol-less death; Minskoff SA et al.; Enrichment procedures, such as those utilizing inositol-less death, have proven to be extremely powerful for increasing the efficiency of identification of spontaneous mutants in a variety of procaryotic and eucaryotic organisms . We characterized inositol-less death in several widely used strains of the inositol-requiring yeast Schizosaccharomyces pombe and determined conditions under which this phenomenon can be used to enrich for mutants . Conflicting reports in the literature on the effects of inositol starvation upon viability of S . pombe had cast doubt on the suitability of using inositol-less death in a mutant enrichment procedure for this organism . We determined that inositol-less death was strain dependent, with differences in viability of up to 5 orders of magnitude observed between the most-sensitive strain, 972, and the least-sensitive strain, SP837 . Inositol-less death was also dependent upon the cell concentration at the time of initiation of starvation . While inositol-less death occurred at all four temperatures tested, the kinetics of death was slower at 16 degrees C than at 23, 30, or 37 degrees C . Inositol-less death was observed during growth in fermentable and nonfermentable carbon sources, although loss of viability in glycerol-ethanol was significantly slower than that in glucose, sucrose, or raffinose . The feasibility of exploiting inositol-less death to enrich for spontaneous mutants was demonstrated by the identification of amino acid auxotrophs, nucleotide auxotrophs, carbon source utilization mutants, and temperature-sensitive mutants . By varying starvation conditions, some mutants were recovered at frequencies as high as 5.7 x 10(-2), orders of magnitude higher than the spontaneous mutation rate. Proc Natl Acad Sci U S A, 1992 Jun 1, 89(11), 4952 - 6 The rad3+ gene of Schizosaccharomyces pombe is involved in multiple checkpoint functions and in DNA repair; Jimenez G et al.; A number of important molecular checkpoints are believed to control the orderly progression of cell cycle events . We have found that the radiation-sensitive Schizosaccharomyces pombe mutant rad3-136 is deficient in two molecular checkpoint functions . Unlike wild-type cells, the mutant cells are unable to arrest in the G2 phase of the cell cycle after DNA damage by gamma-irradiation and are also incapable of maintaining the dependence of mitosis upon the completion of DNA synthesis . An S . pombe genomic clone that complements the UV sensitivity of the rad3-136 mutant completely restores the missing checkpoint functions . The rad3+ gene is also likely to play a role in DNA repair. J Cell Biol, 1992 Jun, 117(5), 1055 - 66 In vitro microtubule-nucleating activity of spindle pole bodies in fission yeast Schizosaccharomyces pombe: cell cycle-dependent activation in xenopus cell-free extracts; Masuda H et al.; The spindle pole body (SPB) is the equivalent of the centrosome in fission yeast . In vivo it nucleates microtubules (MTs) during mitosis, but, unlike animal centrosomes, does not act as a microtubule organizing center (MTOC) during interphase . We have studied the MT-nucleating activity of SPBs in vitro and have found that SPBs in permeabilized cells retain in vivo characteristics . SPBs in cells permeabilized during mitosis can nucleate MTs, and are recognized by two antibodies: anti-gamma-tubulin and MPM-2 which recognizes phosphoepitopes . SPBs in cells permeabilized during interphase cannot nucleate MTs and are only recognized by anti-gamma-tubulin . Interphase SPBs which cannot nucleate can be converted to a nucleation competent state by incubation in cytostatic factor (CSF)-arrested Xenopus egg extracts . After incubation, they are recognized by MPM-2, and can nucleate MTs . The conversion does not occur in Xenopus interphase extract, but occurs in Xenopus interphase extract driven into mitosis by preincubation with exogenous cyclin B . The conversion is ATP dependent and inhibited by protein kinase inhibitors and alkaline phosphatase . Purified, active, cdc2 kinase/cyclin B complex in itself is not effective for activation of MT nucleation, although some interphase SPBs are now stained with MPM-2 . These results suggest that the ability of SPBs in vitro to nucleate MTs after exposure to CSF-arrested extracts is activated through a downstream pathway which is regulated by cdc2 kinase. Cell Calcium, 1992 Jun-Jul, 13(6-7), 435 - 44 Mutational analysis of calmodulin in Saccharomyces cerevisiae; Davis TN; Calmodulin is well characterized as an intracellular Ca2+ receptor in nonproliferating tissues such as muscle and brain . Several observations indicate that calmodulin is also required for cellular growth and division . Deletion of the calmodulin gene is a lethal mutation in Saccharomyces cerevisiae, Schizosaccharomyces pombe and Aspergillus nidulans . Expression of calmodulin antisense RNA in mouse C127 cells causes a transient arrest at G1 and metaphase . Although these results indicate calmodulin plays a critical function during proliferation, they do not reveal the function . S . cerevisiae offers an excellent system for identifying calmodulin functions . Because calmodulin mutants can be readily constructed by gene replacement the consequences of mutations in calmodulin can be directly examined in vivo without interference from wild-type calmodulin . The available wealth of information concerning all aspects of the yeast life cycle provides a large framework for interpretation of new results . The recent dissection of cell cycle regulation is just the latest example of the important insights provided by analyzing basic cellular processes in yeast . Whether studies of calmodulin in yeast will reveal a universal function is unknown . One encouraging result is that yeast cells relying on vertebrate calmodulin as their only source of calmodulin survive and grow well, even if the amount of vertebrate calmodulin is equivalent to the normal steady state levels of yeast calmodulin . This review discusses the varied techniques we are using to identify the functions of calmodulin in yeast . As part of the analysis, we are defining the essential elements of calmodulin structure. Mutat Res, 1992 Jun, 267(2), 193 - 200 Antimutagenicity in yeast; Bronzetti G et al.; In recent years there has been increasing interest in antimutagenesis, and studies have been done using both prokaryotic and eukaryotic systems . In eukaryotic systems the first studies were performed with different strains of Schizosaccharomyces pombe . In particular, caffeine and L-methionine were investigated . Different strains of Saccharomyces cerevisiae were employed in studies of a wide variety of compounds, including acridine, saccharin, salts, tumor promoters and co-carcinogens . Strain D7 was widely employed and antimutagenic activity of spermine, chlorophyllin, cobaltous chloride and fermented milk is reported. J Bioenerg Biomembr, 1992 Jun, 24(3), 309 - 17 An alignment of 17 deduced protein sequences from plant, fungi, and ciliate H(+)-ATPase genes; Wach A et al.; Seventeen protein sequences of H(+)-ATPases from plants (Arabidopsis thaliana, Nicotiana plumbaginifolia, Lycopersicum esculentum), fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Zygosaccharomyces rouxii, Neuropora crassa, Candida albicans), and a parasitic ciliate (Leishmania donovani) have been aligned . Twenty sequence fragments were identified which were conserved in H(+)-, Na+/K(+)-, and Ca++ plasma membrane-ATPases . In addition, a total of 118 residues not located in these fragments were found to be conserved in all H(+)-ATPases . Among those, 38 amino acid residues were screened out as being priority targets for site-directed mutagenesis experiments aimed at the identification of the amino acid residues specifically involved in cation specificity. Nucleic Acids Res, 1992 May 25, 20(10), 2591 - 6 Transcriptional activity of the human immunodeficiency virus-1 LTR promoter in fission yeast Schizosaccharomyces pombe; Toyama R et al.; We have analyzed the transcriptional activity of the human immunodeficiency virus type I (HIV-1) LTR promoter in the fission yeast Schizosaccharomyces pombe (S.pombe) . The ability of a series of 5'-deleted forms of the HIV-1 LTR promoter to direct transcription of the chloramphenicol acetyltransferase reporter gene was studied . We found that the HIV-1 promoter is functional in S.pombe and that deletion of sequences upstream of the NF-kB binding site previously identified to contain the negative regulatory element (NRE) in mammalian cells, resulted in about thirty-fold increase in transcriptional activity . Sequences in the HIV-1 promoter that bind NF-kB were found to be essential for transcriptional activation in S.pombe . In mammalian cells, transactivation of the HIV-1 LTR requires TAR sequences and the viral Tat protein . In fission yeast, Tat failed to transactivate the HIV-1 LTR, suggesting that S.pombe may lack a cellular factor(s) required for the Tat transactivation process. J Biol Chem, 1992 May 15, 267(14), 10024 - 30 Isolation and characterization of a second protein tyrosine phosphatase gene, PTP2, from Saccharomyces cerevisiae; Guan K et al.; A putative protein tyrosine phosphatase (PTPase) gene, PTP2, was cloned from Saccharomyces cerevisiae . The complete yeast PTP2 gene encodes a 750-amino acid residue protein with a predicted mass of 86 kDa . The conserved PTPase domain was localized in the C-terminal half of the protein . Amino acid sequence alignment of the yeast PTPase domain with other phosphatases indicated approximately 20-25% sequence identity with the mammalian PTPase and a similar degree of identity with the PTPase encoded by the yeast PTP1 gene . The PTP2 gene is closely linked to the yeast RET1 and STE4 genes and is localized on the right arm of chromosome 15 . Gene disruption experiments demonstrated that neither PTP2 alone nor PTP2 in combination with PTP1 was essential for growth under the conditions tested . The ability of PTP2 to complement the cdc25-22 mutant of Schizosaccharomyces pombe was also examined, and unlike the human T-cell PTPase, which was able to complement the cdc25-22 mutant, the S . cerevisiae PTP2 was unable to complement the cdc25-22 mutant of S . pombe. Nucleic Acids Res, 1992 May 11, 20(9), 2327 - 34 The Schizosaccharomyces pombe rhp3+ gene required for DNA repair and cell viability is functionally interchangeable with the RAD3 gene of Saccharomyces cerevisiae; Reynolds PR et al.; The RAD3 gene of Saccharomyces cerevisiae is required for excision repair and is essential for cell viability . RAD3 encoded protein possesses a single stranded DNA-dependent ATPase and DNA and DNA.RNA helicase activities . Mutational studies have indicated a requirement for the RAD3 helicase activities in excision repair . To examine the extent of conservation of structure and function of RAD3 during eukaryotic evolution, we have cloned the RAD3 homolog, rhp3+, from the distantly related yeast Schizosaccharomyces pombe . RAD3 and rhp3+ encoded proteins are highly similar, sharing 67% identical amino acids . We show that like RAD3, rhp3+ is indispensable for excision repair and cell viability, and our studies indicate a requirement of the putative rhp3+ DNA helicase activity in DNA repair . We find that the RAD3 and rhp3+ genes can functionally substitute for one another . The level of complementation provided by the rhp3+ gene in S.cerevisiae rad3 mutants or by the RAD3 gene in S.pombe rhp3 mutants is remarkable in that both the excision repair and viability defects in both yeasts are restored to wild type levels . These observations suggest a parallel evolutionary conservation of other protein components with which RAD3 interacts in mediating its DNA repair and viability functions. Gene, 1992 May 1, 114(1), 59 - 66 Versatile shuttle vectors and genomic libraries for use with Schizosaccharomyces pombe; Barbet N et al.; We have constructed a variety of pUC-based vectors designed for maintenance in Schizosaccharomyces pombe . These can be used for both gene bank construction and subcloning . Plasmids pUR18 and pUR19 are modifications of pUC vectors containing the Sc . pombe ars1 and ura4 sequences and retaining the lacZ XGal blue-white selection system for screening for DNA inserts . These vectors have been used to construct representative Sc . pombe and Saccharomyces cerevisiae genomic libraries . To assist in the creation of gene deletions, we have constructed another two plasmids . Combined with the technique of partially filling-in 5' overhangs created with restriction enzymes, these plasmids simplify the replacement of all or part of an open reading frame by a functional ura4 gene . Furthermore, such constructs can be excised with SfiI as a linear fragment for use in Sc . pombe transformations . When integrated into the Sc . pombe genome, the site of integration can be easily mapped by pulsed-field gel electrophoresis using the presence of a novel NotI site. Gene, 1992 May 1, 114(1), 153 - 4 A homologue of the ras-related CDC42 gene from Schizosaccharomyces pombe; Fawell E et al.; A cDNA was isolated from the fission yeast, Schizosaccharomyces pombe, using mixed oligodeoxyribonucleotides encoding part of the GTP-binding site of the ras superfamily . The encoded protein is the homologue of the budding yeast CDC42 gene product and the human proteins, CDC42Hs and G25K. Proc Natl Acad Sci U S A, 1992 May 1, 89(9), 3671 - 5 A heat shock gene from Saccharomyces cerevisiae encoding a secretory glycoprotein; Russo P et al.; We report the finding of a secretory heat shock protein, HSP150, of Saccharomyces cerevisiae, and the characterization of the gene coding for it . HSP150 is constitutively expressed, extensively O-glycosylated, and secreted efficiently to the growth medium . When cells grown at 25 degrees C were shifted to 37 degrees C, a 7-fold increase in the level of HSP150 was observed within 1 hr . The HSP150 gene encodes a primary translation product of 412 amino acids . Direct amino acid sequencing of the mature secreted protein showed that an N-terminal sequence of 18 amino acids is removed, and a KEX2 protease-specific site is cleaved to yield two subunits of 53 and 341 amino acids, which remain noncovalently associated during secretion . The larger subunit is highly repetitive, containing 11 tandem repeats of a 19-amino acid sequence . Northern blot hybridization analysis showed a substantial increase in HSP150 mRNA level after heat shock . The upstream flanking region of the gene contains several heat shock element-like sequences . Disruption of HSP150 did not lead to inviability or significant effects on growth rate, mating, or thermotolerance . However, heat-regulated antigenic homologs of HSP150 were found in divergent yeasts such as Schizosaccharomyces pombe. Mol Cell Biol, 1992 May, 12(5), 1997 - 2009 Yeast telomere repeat sequence (TRS) improves circular plasmid segregation, and TRS plasmid segregation involves the RAP1 gene product; Longtine MS et al.; Telomere repeat sequences (TRSs) can dramatically improve the segregation of unstable circular autonomously replicating sequence (ARS) plasmids in Saccharomyces cerevisiae . Deletion analysis demonstrated that yeast TRSs, which conform to the general sequence (C(1-3)A)n, are able to stabilize circular ARS plasmids . A number of TRS clones of different primary sequence and C(1-3)A tract length confer the plasmid stabilization phenotype . TRS sequences do not appear to improve plasmid replication efficiency, as determined by plasmid copy number analysis and functional assays for ARS activity . Pedigree analysis confirms that TRS-containing plasmids are missegregated at low frequency and that missegregated TRS-containing plasmids, like ARS plasmids, are preferentially retained by the mother cell . Plasmids stabilized by TRSs have properties that distinguish them from centromere-containing plasmids and 2 microns-based recombinant plasmids . Linear ARS plasmids, which include two TRS tracts at their termini, segregate inefficiently, while circular plasmids with one or two TRS tracts segregate efficiently, suggesting that plasmid topology or TRS accessibility interferes with TRS segregation function on linear plasmids . In strains carrying the temperature-sensitive mutant alleles rap1grc4 and rap1-5, TRS plasmids are not stable at the semipermissive temperature, suggesting that RAP1 protein is involved in TRS plasmid stability . In Schizosaccharomyces pombe, an ARS plasmid was stabilized by the addition of S . pombe telomere sequence, suggesting that the ability to improve the segregation of ARS plasmids is a general property of telomere repeats. J Cell Sci, 1992 May, 102 ( Pt 1), 43 - 53 Vertebrate p34cdc2 phosphorylation site mutants: effects upon cell cycle progression in the fission yeast Schizosaccharomyces pombe; Krek W et al.; We have used the fission yeast Schizosaccharomyces pombe to analyse the effects of in vitro mutagenesis of the four known phosphorylation sites in the chicken p34(cdc2) protein, Thr 14, Tyr 15, Thr 161 and Ser 277, upon cell cycle progression . We have studied both the effect of overexpression of mutant proteins in a cdc2+ background and assayed their ability to rescue null and temperature-sensitive alleles of cdc2 . Mutations of Thr 14 and Tyr 15 within the ATP binding domain of p34(cdc2) that mimic constitutive phosphorylation cause dominant negative cell cycle arrest when overexpressed . In contrast, some substitutions that simulate permanent dephosphorylation of the corresponding sites advance dephosphorylation of the corresponding sites advance mitosis . These data confirm the model that p34(cdc2) function is negatively regulated by phosphorylation of residues in the ATP binding site . Mutagenesis of the conserved residue Thr 161 functionally inactivates p34(cdc2), and our data suggest that both phosphorylation and dephosphorylation events at Thr 161 are required for progression through the cell cycle . Mutations at the fourth site of phosphorylation . Ser 277, lead to cold-sensitive cell cycle arrest, in minimal but not rich growth medium, suggesting that this site is involved in monitoring the nutritional status of the cell. Mol Gen Genet, 1992 May, 233(1-2), 17 - 24 Reduction in the intracellular cAMP level triggers initiation of sexual development in fission yeast; Mochizuki N et al.; Schizosaccharomyces pombe initiates sexual development in response to nutritional starvation . The level of cAMP in S . pombe cells changed during the transition from exponential growth to stationary phase . It also changed in response to a shift from nitrogen-rich medium to nitrogen-free medium . A decrease of approximately 50% was observed in either case, suggesting that S . pombe cells contain less cAMP when they initiate sexual development . S . pombe cells that expressed the catalytic domain of Saccharomyces cerevisiae adenylyl cyclase from the S . pombe adh1 promoter contained 5 times as much cAMP as the wild type and could not initiate mating and meiosis . These observations, together with previous findings that exogenously added cAMP inhibits mating and meiosis and that cells with little cAMP are highly derepressed for sexual development, strongly suggest that cAMP functions as a key regulator of sexual development in S . pombe . The pde1 gene, which encodes a protein homologous to S . cerevisiae cAMP phosphodiesterase I, was isolated as a multicopy suppressor of the sterility caused by a high cAMP level . Disruption of pde1 made S . pombe cells partially sterile and meiosis-deficient, indicating that this cAMP phosphodiesterase plays an important role in balancing the cAMP level in vivo. Mol Cell Biol, 1992 May, 12(5), 2331 - 8 Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases; Kulkosky J et al.; Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region . The same constellation is found in the transposases of a number of bacterial insertion sequences . The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin . We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro . Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets . The severity of the defects depended upon the site and the nature of the amino acid substitution(s) . All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions.(ABSTRACT TRUNCATED AT 250 WORDS) Hum Mol Genet, 1992 May, 1(2), 91 - 6 Isolation and characterization of a novel gene with differential expression in benign and malignant human breast tumours; Adams SM et al.; We report the identification of a novel cDNA representing an mRNA showing significantly higher levels of expression in benign breast lesions than in carcinomas . This cDNA was identified by differential screening of a cDNA library generated from a breast carcinoma, and shows consistently higher expression in fibroadenomas than in carcinomas . The expression in both benign and malignant tissues is highest in epithelial cells as determined by in situ hybridization to tissue sections . The nucleotide sequence of the full-length cDNA has been determined, and the deduced protein is highly basic with no signal or transmembrane sequence, but two potential nuclear localization signals . Neither the DNA nor the protein sequence show any significant homology to sequences in current databases . The cDNA hybridizes to multiple sequences within both human and other mammalian genomes, but to single genomic sequences in Drosophila, Physarum and Schizosaccharomyces pombe . This cDNA therefore represents a highly conserved gene sequence . We have identified only one major transcript in human cells, and it seems likely that there are several pseudogenes within the human genome. FEMS Microbiol Lett, 1992 Apr 15, 71(2), 151 - 6 Synthesis and degradation of polyphosphate in the fission yeast Schizosaccharomyces pombe: mutations in phosphatase genes do not affect polyphosphate metabolism; Muller J et al.; The fission yeast Schizosaccharomyces pombe was found to accumulate large amounts of polyphosphate, particularly when grown on arginine as the nitrogen source . Upon transfer to a medium without phosphate, polyphosphate was degraded and served as an endogenous phosphate reserve . When phosphate was added again after a prolonged period of phosphate starvation, fission yeast cells synthesized more polyphosphate than they had contained before starvation, a phenomenon known as over-compensation . Strains carrying mutated structural genes for three different phosphatases, pho1, pho2 or pho3, degraded polyphosphate at the same rate as the wild-type strain during phosphate starvation and showed the same type of over-compensation when phosphate was added again. Nucleic Acids Res, 1992 Apr 11, 20(7), 1607 - 15 Random mutagenesis of Schizosaccharomyces pombe SRP RNA: lethal and conditional lesions cluster in presumptive protein binding sites; Liao X et al.; Signal recognition particle (SRP), a ribonucleoprotein composed of six polypeptides and one RNA subunit, serves as an adaptor between the cytoplasmic protein synthetic machinery and the translocation apparatus of the endoplasmic reticulum . To begin constructing a functional map of the 7SL RNA component of SRP, we extensively mutagenized the Schizosaccharomyces pombe SRP7 gene . Phenotypes are reported for fifty-two mutant alleles derived from random point mutagenesis, seven alleles created by site-directed mutagenesis to introduce restriction sites into the SRP7 gene, nine alleles designed to pinpoint conditional lesions, and three alleles with extra nucleotides inserted at position 84 . Our data indicate that virtually all single nucleotide changes as well as many multiple substitutions in this highly structured RNA are phenotypically silent . Six lethal alleles and eleven which result in sensitivity to the combination of high temperature and elevated osmotic strength were identified . These mutations cluster in conserved regions which, in the mammalian RNA, are protected from nucleolytic agents by SRP proteins . The effects of mutations in the presumptive binding site for a fission yeast SRP 9/14 homolog indicate that both the identity of a conserved residue and the secondary structure within which it is embedded are functionally important . The phenotypes of mutations in Domain IV suggest particular residues as base-specific contacts for the fission yeast SRP54 protein . A single allele which confers temperature-sensitivity in the absence of osmotic perturbants was identified in this study; the growth properties of the mutant strain suggest that the encoded RNA is somewhat defective even at the permissive temperature, and is most likely unable to correctly assemble with SRP proteins at the nonpermissive temperature. J Chromatogr, 1992 Apr 3, 596(1), 141 - 9 DNA electrophoresis in uncross-linked polyacrylamide solution, studied by epifluorescence microscopy; Rampino NJ et al.; Electrophoresis of human DNA fragments (approximately 1 x 10(5) to 1 x 10(7) bases in size) was conducted in a solution of uncross-linked polyacrylamide contained in a horizontally mounted 1 mm diameter glass tube and monitored by epifluorescence microscopy . In presence of the polymer, molecular conformations described as a "trailing network" of DNA and a globular "head" were observed . The migration velocity varies between species differing in the size of the "head", and in the ratio between the size of the "head" and that of the trailing "network" . By contrast, in pure buffer, lambda phage DNA migrates in a globular form at a mobility consistent with known macroscopic data . When electrophoresis in the polymer solution of an agarose plug preparation of Schizosaccharomyces pombe DNA was carried out after melting at 70 degrees C, a migrating DNA-agarose complex was observed . The complex was not fully dissociated by an agarose-hydrolyzing enzyme (Gelase). Mol Gen Genet, 1992 Apr, 232(3), 440 - 6 Five novel elements involved in the regulation of mitosis in fission yeast; Warbrick E et al.; Five new elements of the mitotic control in the fission yeast Schizosaccharomyces pombe were isolated from gene libraries as multicopy suppressors of the conditional lethal phenotype of win1-1 wee1ts cdc25ts triple mutant strains . These genes were designated wis1(+)-wis5+ for win suppressing, and do not correspond to win1+ or any of the previously characterised mitotic control genes . None of the wis genes is capable of suppressing the cdc phenotype of cdc25ts strains, suggesting that their effect is not simply to reverse the effect of loss of cdc25 function . wis1+ has been previously reported to encode a putative serine/threonine protein kinase that acts as a dosage-dependent inducer of mitosis . wis4+ appears to be a specific suppressor of the win1-1 mutation . wis2+ and wis3+ are capable of suppressing a wide range of cdc phenotypes arising from the combination of various mutations with wee1ts and cdc25ts, suggesting that the wis2+ and wis3+ products may interact with elements central to the mitotic control. Mol Gen Genet, 1992 Apr, 232(3), 367 - 76 Molecular cloning and analysis of Schizosaccharomyces pombe rad9, a gene involved in DNA repair and mutagenesis; Lieberman HB et al.; The mutant allele rad9-192 renders Schizosaccharomyces pombe cells sensitive to ionizing radiation and UV light . We have isolated from a S . pombe genomic DNA library a unique recombinant plasmid that is capable of restoring wild-type levels of radioresistance to a rad9-192-containing cell population . Plasmid integration studies using the cloned DNA, coupled with mating and tetrad analyses, indicate that this isolated DNA contains the wild-type rad9 gene . We inactivated the repair function of the cloned fragment by a single insertion of the S . pombe ura4 gene . This nonfunctional fragment was used to create a viable disruption mutant, thus demonstrating