Schizosaccharomyces

EMBO J, 1991 Jan, 10(1), 221 - 6
S . pombe pac1+, whose overexpression inhibits sexual development, encodes a ribonuclease III-like RNase; Iino Y et al.; The Schizosaccharomyces pombe pac1 gene is a multicopy suppressor of the pat1 temperature-sensitive mutation, which directs uncontrolled meiosis at the restrictive temperature . Overexpression of the pac1 gene had no apparent effect on vegetative growth but inhibited mating and sporulation in wild type S . pombe cells . In such cells, expression of certain genes required for mating or meiosis was inhibited . The pac1 gene is essential for vegetative cell growth . The deduced pac1 gene product has 363 amino acids . Its C-terminal 230 residues revealed 25% amino acid identity with ribonuclease III, an enzyme that digests double-stranded RNA and is involved in processing ribosomal RNA precursors and certain mRNAs in Escherichia coli . The pac1 gene product could degrade double-stranded RNA in vitro . These observations establish the presence of a RNase III homolog in eukaryotic cells . The pac1 gene product probably inhibits mating and meiosis by degrading a specific mRNA(s) required for sexual development . It is likely that mRNA processing is involved in the regulation of sexual development in fission yeast.

EMBO J, 1991 Jan, 10(1), 123 - 32
Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box; van de Wetering M et al.; CD3-epsilon expression is controlled by a downstream T lymphocyte-specific enhancer element . We report the identification of a T cell-specific transcription factor, TCF-1, binding to this element . The multimerized recognition motif of TCF-1 constituted a T cell-specific enhancer . Subsequent cloning of TCF-1 identified three splice alternatives . TCF-1 contained a single DNA-binding HMG box most closely related to similar boxes in the putative mammalian sex-determining gene SRY and in the Schizosaccharomyces pombe Mc mating type gene . TCF-1 mRNA was expressed uniquely in T lymphocytes . Upon cotransfection into non-T cells, TCF-1 could transactivate through its cognate motif . These results identify TCF-1 as a T cell-specific transcription factor, which might play a role in the establishment of the mature T cell phenotype.

J Cell Biol, 1991 Jan, 112(2), 191 - 201
The chromatin structure of centromeres from fission yeast: differentiation of the central core that correlates with function; Polizzi C et al.; We have examined the chromatin structure of centromere regions from the fission yeast Schizosaccharomyces pombe . The large and complex centromere regions of the S . pombe chromosomes encompass many kilobase pairs of DNA and contain several classes of tandemly repeated DNA sequences . The repeated sequences are further organized into a large inverted repeat flanking a central core, a conserved structural feature among all three centromeres in S . pombe . The nucleosomal configuration of the centromere regions is nonuniform and highly varied . Most of the centromere-specific repeated DNA sequences are packaged into nucleosomes typical of bulk chromatin . However, the central core and core-associated repeated sequences from the centromere regions of chromosomes I (cen1) and II (cen2), when present in S . pombe, show an altered chromatin structure, with little or no evidence of regular nucleosomal packaging . The atypical chromatin organization of the cen2 central core is not due to transcription, as no transcripts from this region were detected . These same DNA sequences, however, are packaged into nucleosomes typical of bulk chromatin when present in a nonfunctional environment on a minichromosome in the budding yeast Saccharomyces cerevisiae . Because the cen2 central core sequences themselves do not preclude regular nucleosomal packaging, we speculate that in S . pombe they constitute a specialized site of kinetochore protein assembly . The atypical nucleosomal pattern of the cen2 central core remains constant during the cell cycle, with only minor differences observed for some sequences . We propose that the unusual chromatin organization of the core region forms the basis of a higher order structural differentiation that distinguishes the centromere from the chromosome arms and specifies the essential structure for centromere function.

Mol Cell Biol, 1991 Jan, 11(1), 289 - 98
The strong ADH1 promoter stimulates mitotic and meiotic recombination at the ADE6 gene of Schizosaccharomyces pombe; Grimm C et al.; The effect of the strong promoter from the alcohol dehydrogenase gene on mitotic and meiotic intragenic recombination has been studied at the ade6 locus of the fission yeast Schizosaccharomyces pombe . A 700-bp fragment containing the functional adh1 promoter was used to replace the weak wild-type promoter of the ade6 gene . Analysis of mRNA showed that strains with this ade6::adh1 fusion construct had strongly elevated ade6-specific mRNA levels during vegetative growth as well as in meiosis . These increased levels of mRNA correlated with a 20- to 25-fold stimulation of intragenic recombination in meiosis and a 7-fold increased prototroph formation during vegetative growth . Analysis of flanking marker configurations of prototrophic recombinants indicated that simple conversions as well as conversions associated with crossing over were stimulated in meiosis . The strongest stimulation of recombination was observed when the adh1 promoter was homozygous . Studies with heterologous promoter configurations revealed that the highly transcribed allele was the preferred acceptor of genetic information . The effect of the recombinational hot spot mutation ade6-M26 was also investigated in this system . Its effect was only partly additive to the elevated recombination rate generated by the ade6::adh1 fusion construct.

Mol Cell Biol, 1991 Jan, 11(1), 281 - 8
Regulation of heat shock factor in Schizosaccharomyces pombe more closely resembles regulation in mammals than in Saccharomyces cerevisiae; Gallo GJ et al.; The heat shock response appears to be universal . All eucaryotes studied encode a protein, heat shock factor (HSF), that is believed to regulate transcription of heat shock genes . This protein binds to a regulatory sequence, the heat shock element, that is absolutely conserved among eucaryotes . We report here the identification of HSF in the fission yeast Schizosaccharomyces pombe . HSF binding was not observed in extracts from normally growing S . pombe (28 degrees C) but was detected in increasing amounts as the temperature of heat shock increased between 39 and 45 degrees C . This regulation is in contrast to that observed in Saccharomyces cerevisiae, in which HSF binding is detectable at both normal and heat shock temperatures . The S . pombe factor bound specifically to the heat shock element, as judged by methylation interference and DNase I protection analysis . The induction of S . pombe HSF was not inhibited by cycloheximide, suggesting that induction occurs posttranslationally, and the induced factor was shown to be phosphorylated . S . pombe HSF was purified to near homogeneity and was shown to have an apparent mobility of approximately 108 kDa . Since heat-induced DNA binding by HSF had previously been demonstrated only in metazoans, the conservation of heat-induced DNA binding by HSF among S . pombe and metazoans suggests that this mode of regulation is evolutionarily ancient.

J Basic Microbiol, 1991, 31(2), 149 - 56
Use of alpha-aminoadipate and lysine as sole nitrogen source by Schizosaccharomyces pombe and selected pathogenic fungi; Ye ZH et al.; alpha-Aminodipate, an intermediate of the lysine biosynthetic pathway of fungi, or lysine when used as the sole nitrogen source in the medium was growth inhibitory and toxic to Saccharomyces cerevisiae . The fission yeast Schizosaccharomyces pombe and pathogenic fungi Candida albicans, Filobasidiella neoformans and Aspergillus fumigatus grew in the medium containing alpha-aminoadipate as the sole nitrogen source . C . albicans, A . fumigatus, and one of the strains of F . neoformans also grew in the medium containing lysine as the sole nitrogen source . When grown in the alpha-aminoadipate medium, only S . pombe accumulated a significant amount of alpha-ketoadipate in the culture supernatant . Also, 14C-alpha-aminoadipate was converted to 14C-alpha-ketoadipate in vivo . In the ammonium sulfate medium, S . pombe cells converted 14C-alpha-aminoadipate to lysine . The levels of glutamate-alpha-ketoadipate transaminase, an enzyme responsible for the conversion of alpha-aminoadipate to alpha-ketoadipate, and alpha-aminoadipate reductase, an enzyme required for the conversion of alpha-aminoadipate to lysine, were similar in S . pombe cells grown in the alpha-aminoadipate or ammonium sulfate medium . However, the level of homoisocitrate dehydrogenase, an enzyme before the alpha-ketoadipate step, was twelvefold lower in S . pombe cells grown in the alpha-aminoadipate medium compared to the level in cells grown in the ammonium sulfate medium . Pathogenic fungi used in this study did not accumulate alpha-ketoadipate and alpha-aminoadipate-delta-semialdehyde when grown in medium containing alpha-aminoadipate and lysine, respectively, as sole nitrogen source . However, only pathogenic fungi used both lysine and alpha-aminoadipate as sole nitrogen source . This unique metabolic property could be useful for the identification of these pathogens.

Genes Dev, 1991 Jan, 5(1), 60 - 73
Fission yeast genes that confer resistance to staurosporine encode an AP-1-like transcription factor and a protein kinase related to the mammalian ERK1/MAP2 and budding yeast FUS3 and KSS1 kinases; Toda T et al.; Staurosporine, a potent inhibitor of protein kinase C, arrests fission yeast cell elongation specifically at a stage immediately after cell division . We isolated two genes, which, when carried on multicopy plasmids, confer drug resistance in fission yeast . One, spk1+, encodes a protein kinase highly similar (54% identity) to those encoded by the mammalian ERK1/MAP2 kinase and the budding yeast KSS1 and FUS3 genes . It is not essential for vegetative growth of Schizosaccharomyces pombe cells but is required for conjugation . The spk1+ gene product is a 45-kD protein enriched in the nucleus, and its level increases 10-fold after addition of staurosporine . The other gene pap1+ encodes an AP-1-like transcription factor that contains a region rich in basic amino acids followed by a "leucine zipper" motif . The pap1+ gene is required for spk1(+)-conferred staurosporine resistance . These two genes appear to function as a part of the fission yeast growth control pathway.

Princess Takamatsu Symp, 1991, 22, 231 - 8
Mammalian G2 regulatory genes and their possible involvement in genetic instability in cancer cells; Okayama H et al.; In the fission yeast Schizosaccharomyces pombe, mitosis is initiated following the activation of the cdc2+/cyclin B kinase . The cdc2+/cyclin B kinase is positively regulated by cdc25+ tyrosine phosphatase and negatively regulated by wee1+/mik1+ tyrosine kinases . This regulatory system is evolutionarily conserved throughout higher eukaryotes . Drosophila and humans contain a cdc25+ gene homolog called String and CDC25Hs (hereafter referred to as CDC25Hul), respectively . We recently cloned a wee1+ homolog (WEE1Hu) and two additional cdc25+ homologs (CDC25Hu2 and CDC25Hu3) from human cells . Consequently, human cells contain at least one wee1+ and three cdc25+ homologs . Both CDC25Hu1 and CDC25Hu2 resemble the cdc25+ gene not only in structure and function but also in the mode of expression . They are expressed mostly in G2 . On the other hand, CDC25Hu3 is expressed mostly in early S, indicating that it has some novel function in the early phase of the cell cycle . In all the cell lines examined, CDC25Hu2 is expressed to a greater extent than CDC25Hu1 or CDC25Hu3 . The expression of CDC25Hu2 is particularly high in various cancer cells including those transformed by SV40 or human papilloma virus type 16 E6, E7, both of which are well known for their ability to induce genomic instability . In addition, there is a noticeable correlation between the extent of aneuploidy and the level of CDC25Hu2 expression in the cancer cells examined . In view of the fact that overexpression of cdc25+ under certain conditions induces genomic instability in the fission yeast, overexpression of CDC25Hu2 associated with many cancer cells might play at least a role in the induction of their chromosomal abnormalities.

Folia Microbiol (Praha), 1991, 36(2), 153 - 7
Formation and reversion of Schizosaccharomyces pombe cells with apical protoplast protuberances; Zemanova Z et al.; Lytic enzymes from the hepatopancreas of Helix pomatia do not induce a uniform digestion of the cell wall of Schizosaccharomyces pombe over the entire cell surface . Perforations are formed in growth zones through which a protoplast can locally protrude . Conditions were found under which the frequency of formation of apical protoplast protuberances is higher than 90% cells with such protuberances can reverse to normally multiplying cells.

Cold Spring Harb Symp Quant Biol, 1991, 56, 605 - 11
New elements in the mitotic control of the fission yeast Schizosaccharomyces pombe; Fantes PA et al.; The p107wee1 protein kinase plays a central role in regulating the cell cycle of fission yeast . It mediates transmission of signal(s) related to the nutritional status of the cell to the p34cdc2 protein kinase, which is an active component of the MPF complex driving cells into mitosis . p107wee1 is itself subject to control by the products of other genes such as nim1+/cdr1+, win1+, and perhaps wis1+ and other wis+ genes . At present, the relationships between these genes and their possible roles in the mitotic control are unclear and must await further analysis (Fig . 5) . It is likely that some of the gene products are concerned with the sensing and/or transmission of nutritional signals . p107wee1 negatively regulates the activity of p34cdc2, probably by direct tyrosine phosphorylation, and also appears to regulate the activities of the cdc1+ and cdc27+ gene products . The effects of nitrogen starvation and of wee1 mutations on conditional lethal mutations at the cdc1, cdc2, and cdc27 loci, taken together, support the largely speculative model shown in Figure 5 . During the normal cycle, the balance between phosphorylated and dephosphorylated p34cdc2 changes such that at the appropriate time, p34cdc2 is activated and the cell enters mitosis . We suggest that the cdc1+ and cdc27+ products may be regulated in a similar way . Such a mechanism would ensure coordinated activation of these and perhaps other proteins required for the G2/M transition . There are, of course, many uncertainties, and these must await elucidation by biochemical and genetic analysis.

Cold Spring Harb Symp Quant Biol, 1991, 56, 599 - 604
stf1: a new suppressor of the mitotic control gene, cdc25, in Schizosaccharomyces pombe; Hudson JD et al.; A novel element in the mitotic control, stf1, has been identified genetically by its ability to rescue cdc25-22 as well as a gene disruption of cdc25 . This is the first phenotypically non-wee mutation shown to do so . stf1-1 functions additively with cdc2-1w, cdc2-3w, or wee1-6 to rescue cdc25 . The available data are consistent with the wild-type gene product operating either on the same pathway as cdc25 or to stimulate cdc2 by a pathway independent of cdc25 or wee1 . The stf1 gene has been cloned and sequenced and encodes a putative protein of 50-65 kD, depending on whether a potential intron is present . It is a novel protein with no homology detected in the current data bases . When challenged with hydroxyurea, stf1-1 acts additively with cdc2-3w in rescuing cdc25 mutants and in allowing mitosis to occur without DNA synthesis . It does not appear to play a role in the nutritional sensing pathway nor in the pathway mediating radiation-induced G2 delay.

Oncogene, 1991 Jan, 6(1), 3 - 9
Identification and characterization of a human homolog of the Schizosaccharomyces pombe ras-like gene YPT-3; Drivas GT et al.; The Polymerase Chain Reaction was used to amplify ras and ras-like sequences from two human cDNA libraries . Members corresponding to each of the three major ras-subfamilies (ras, rho, and rab/YPT) were identified . The one homologous to rab/YPT, referred to here as YL8, appears to be the human homolog of the recently reported Schizosaccharomyces pombe YPT3 gene . The YL8 gene could encode a guanine nucleotide binding protein of 216 amino acids with about 70% amino acid sequence identity to S . pombe YPT3, and is transcriptionally active in a variety of human cell lines.

Cell Motil Cytoskeleton, 1991, 18(2), 86 - 93
Microtubules in the fission yeast Schizosaccharomyces pombe contain only the tyrosinated form of alpha-tubulin; Alfa CE et al.; The state of tubulin tyrosination in the fission yeast Schizosaccharomyces pombe was investigated using a combination of indirect immunofluorescence microscopy and Western blotting . Antibodies specific for the tyrosinated form of alpha-tubulin stained all microtubule arrays in wild type cells and recognised the two alpha-tubulin polypeptides in Western blots of cell extracts enriched for tubulin by DEAE-Sephadex chromatography . Antisera that specifically recognised the detyrosinated, glu, form, on the other hand, gave consistently negative results, both in cells undergoing rapid exponential growth and in those allowed to accumulate in stationary phase . Neither the "ageing" of microtubules, by arresting cells at different points (late G1 or G2/M) in the cell division cycle, nor stabilising them, using D2O, lead to any detectable tubulin detryrosination . These results suggest that S . pombe lacks the carboxypeptidase that carries out the tubulin detyrosination reaction . This is the first report of an organism that possesses the correct C-terminal alpha-tubulin sequence yet fails to carry out this post-translational modification . The implication of this novel finding for the biological role of these events is discussed.

Princess Takamatsu Symp, 1991, 22, 137 - 44
Protein phosphatases in cell division: how vital are they?
Yanagida M, Yamano H, Stone EM, Kinoshita N, Yoshida T, Shiozaki K.
In contrast to the wealth of information on cellular function of protein kinases, many of which are known to be the products of proto-oncogenes, little is known about how protein dephosphorylation is involved in growth control of normal and malignant cells . In the present study, roles of protein phosphatases in cell division cycle control were examined by molecular genetic approaches using a lower eukaryote, the fission yeast Schizosaccharomyces pombe . Nine protein phosphatase genes have been so far identified and characterized in this organism . Each of two (dis2+, sds21+, and ppa1+, ppa2+) gene products is highly similar to mammalian type 1 and 2A ser/thr phosphatases, respectively . The ppx1+ product is an intermediate of type 1 and 2A, while the ppb1+ product is similar to Ca(2+)-dependent type 2B . At least two protein tyrosine phosphatase genes (pyp1+ and pyp2+) exist . The cdc25 protein is now established to be a tyrosine phosphatase that activates cdc2 kinase . Some of these phosphatase genes are interrelated but have distinct, essential functions in cell cycle control . Missense mutations, deletions or high dosage expression of these phosphatase genes affect entry into and exit from mitosis, mitotic chromosome disjunction, cell size and cell shape . They seem to interact with the main regulators of mitosis, cdc2, cdc13/cyclin, cdc25 and weel, or with mitotic structural components, such as condensed chromosomes or the spindle apparatus . We show that the product of an essential gene, sds22+, is an important, positive factor in controlling the expression and modulating the activity of dis2 phosphatase.

Cell Motil Cytoskeleton, 1991, 20(1), 47 - 54
Segregation of the nucleolus during mitosis in budding and fission yeast; Granot D et al.; The segregation of the nucleolus during mitosis was examined in Saccharomyces cerevisiae and Schizosaccharomyces pombe by indirect immunofluorescence using antibodies directed to highly conserved anti-nucleolus antigens . In mitotic S . pombe cells, the nucleolus appears to trail the bulk of the DNA . In wild-type cells of S . cerevisiae, the nucleolus segregates alongside the bulk of the genomic DNA . Based on its distance from the centromere, we would expect the rDNA in both organisms to segregate behind the majority of the genomic DNA, if telomeric regions trail centromeric regions as in other eukaryotes . We therefore suggest that in S . cerevisiae the nucleolus is attached to other parts of the nucleus which enable it to segregate along with the bulk of the DNA . The segregation of the nucleolus in topoisomerase mutants and nuclear division mutants of S . cerevisiae was also investigated . In cdc14 mutants which arrest at late anaphase, the vast majority of the DNA is separated, but the nucleolar antigens remain extended between the mother and daughter cells . Thus, the CDC14 gene of S . cerevisiae appears to be important for the separation of the nucleolus at mitosis.

Verh K Acad Geneeskd Belg, 1991, 53(4), 365 - 85
{From ovocyte to biochemistry of the cell cycle}; Ozon R; The cell division cycle in eukaryotes contains up to three major transition points; the conversion of quiescent cells to a stage of active proliferation, the initiation of DNA synthesis (S phase) and the induction of mitosis in cells with newly replicated genome (M phase) . Within the past years two strategies, have converged to identify, genetically and biochemically a key protein kinase p34 cdc2 that governs the entry into mitosis . In the fission yeast Schizosaccharomyces pombe a number of mutants in the mitotic regulatory circuit have been isolated . A central gene in the network is cdc2 which is essential for the proper execution of mitosis . The cdc2 gene interacts with a number of other genes for correct mitotic control . The Amphibian oocyte, the oocyte from Xenopus laevis particularly, is arrested at the G2 phase of the first meiotic division; when it enters M phase, it contains a dominant regulatory factor known as MPF (M-phase or maturation promoting factor) . Purified MPF is an heterodimer formed of two polypeptides p34cdc2 an homologue of the product of the gene cdc2 and p45cdc13 or cyclin an homologue of the product of the gene cdc13 . Biochemical studies have revealed that p34cdc2 is a phosphotyrosine protein during the G2 phase of the cell cycle, both mitotic and meiotic . The tyrosine phosphorylation of p34cdc2 is regulated by the gradual accumulation of cyclin . At the onset of M phase, the complex p34cdc2/cyclin is activated as an histone H1 kinase, and p34cdc2 is tyrosine dephosphorylated . The mechanism of activation of p34cdc2 is negatively regulated by a form of protein phosphatase 2A . Ovulated vertebrate oocytes are arrested at metaphase of the second meiotic division (M II) under the control of the proto-oncogene c-mos a protein kinase . The exit of M II phase and the initiation of early embryonic mitotic cell cycles are physiologically induced by the spermatozoa at the time of fertilization . They requires the degradation of c-mos by a Ca2+ dependent proteolytic enzyme and the destruction of cyclin by an ubiquitin dependent pathway . The Xenopus oocyte has led to the molecular elucidation of MPF and identified links between cell cycle control, protein phosphorylation and proto-oncogenes . Despite the impresive progess of recent years, there is still much to be learned about the control of meiosis in Xenopus oocytes.

Science, 1990 Dec 14, 250(4987), 1573 - 6
Complementation of the mitotic activator, p80cdc25, by a human protein-tyrosine phosphatase; Gould KL et al.; The onset of M phase requires the activation of the pp34 protein kinase in all eukaryotes thus far examined . In Schizosaccharomyces pombe, pp34 is phosphorylated on Tyr15, and dephosphorylation of this residue regulates the initiation of mitosis . In this study, it is shown that dephosphorylation of Tyr15 triggered activation of the pp34-cyclin complex from fission yeast, that a human protein-tyrosine phosphatase can catalyze this event both in vitro and in vivo, and that activation of fission yeast pp34 does not require threonine dephosphorylation . The complementary DNA that encoded the tyrosine phosphatase replaced the mitotic activator p80cdc25, closely associating the cdc25(+)-activating pathway with tyrosine dephosphorylation of pp34.

Genes Dev, 1990 Dec, 4(12A), 2146 - 56
Evidence for base-pairing between mammalian U2 and U6 small nuclear ribonucleoprotein particles; Hausner TP et al.; Intramolecular and intermolecular snRNA cross-links were generated by irradiating HeLa nuclear extract with 365 nm light in the presence of the psoralen derivative AMT . After deproteinization, cross-linked RNAs were resolved by gel electrophoresis and identified as anomalously migrating species by Northern blotting . In addition to the U4/U6 snRNA cross-link, we detected an intermolecular U2/U6 cross-link, as well as several apparently intramolecular U1, U2, and U5 cross-links . Photoreversal of the U2/U6 cross-link with 254 nm irradiation released stoichiometric amounts of U2 and U6 snRNA . To localize the U2/U6 cross-link, the 3' end of U2 in the purified U2/U6 complex was labeled selectively using a novel oligonucleotide "splint" technique . The labeled U2/U6 complex was then subjected to rapid enzymatic RNA sequencing or to targeted digestion of the U2 and U6 components of the complex by RNase H and a panel of complementary oligonucleotides . The U2/U6 cross-link is located upstream of nucleotide 15 in U2 and downstream of nucleotide 85 in U6, suggesting that the phylogenetically conserved base-pairing between these regions (6 consecutive base pairs in human, Drosophila melanogaster, and Caenorhabditis elegans, 7 in Schizosaccharomyces pombe and Trypanasoma brucei, 8 in Pisum sativum, 11 in Saccharomyces cerevisiae) is significant.

Mol Cell Biol, 1990 Dec, 10(12), 6791 - 8
Two related families of retrotransposons from Schizosaccharomyces pombe; Levin HL et al.; Two related families of transposons were isolated from schizosaccharomyces pombe, an organism which has been the object of extensive genetic studies which had previously produced no evidence for the existence of such elements . These two classes of repeated DNAs, dubbed Tf1 (transposon of fission yeast 1) and Tf2 have many properties of retrotransposons . Tf1 and Tf2 both possess long terminal repeats and predicted protein sequences that resemble the protease, reverse transcriptase, and integrase domains of retroviruses . The chromosomal locations and total numbers of Tf1 and Tf2 differ greatly in various isolates of S . pombe . The Tf elements are expressed in the form of 4.5-kb mRNAs . The complete sequence of Tf1 was determined and suggests that a novel mechanism for regulating its gene expression may be used.

EMBO J, 1990 Dec, 9(12), 4007 - 16
Nuclear scaffold attachment stimulates, but is not essential for ARS activity in Saccharomyces cerevisiae: analysis of the Drosophila ftz SAR; Amati B et al.; Nuclei isolated from eukaryotic cells can be depleted of histones and most soluble nuclear proteins to isolate a structural framework called the nuclear scaffold . This structure maintains specific interactions with genomic DNA at sites known as scaffold attached regions (SARs), which are thought to be the bases of DNA loops . In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, genomic ARS elements are recovered as SARs . In addition, SARs from Drosophila melanogaster bind to yeast nuclear scaffolds in vitro and a subclass of these promotes autonomous replication of plasmids in yeast . In the present report, we present fine mapping studies of the Drosophila ftz SAR, which has both SAR and ARS activities in yeast . The data establish a close relationship between the sequences involved in ARS activity and scaffold binding: ARS elements that can bind the nuclear scaffold in vitro promote more efficient plasmid replication in vivo, but scaffold association is not a strict prerequisite for ARS function . Efficient interaction with nuclear scaffolds from both yeast and Drosophila requires a minimal length of SAR DNA that contains reiteration of a narrow minor groove structure of the double helix.

Biochem Cell Biol, 1990 Dec, 68(12), 1297 - 330
Protein kinase cascades in meiotic and mitotic cell cycle control; Pelech SL et al.; Eukaryotic cell cycle progression during meiosis and mitosis is extensively regulated by reversible protein phosphorylation . Many cell surface receptors for mitogens are ligand-stimulated protein-tyrosine kinases that control the activation of a network of cytoplasmic and nuclear protein-serine (threonine) kinases . Over 30 plasma membrane associated protein-tyrosine kinases are encoded by proto-oncogenes, i.e., genes that have the potential to facilitate cancer when disregulated . Proteins such as ribosomal protein S6, microtubule-associated protein-2, myelin basic protein, and casein have been used to detect intracellular protein-serine (threonine) kinases that are activated further downstream in growth factor signalling transduction cascades . Genetic analysis of yeast cell division control (cdc) mutants has revealed another 20 or so protein-serine (threonine) kinases . One of these, specified by the cdc-2 gene in Schizosaccharomyces pombe, has homologs that are stimulated during M phase in maturing sea star and frog oocytes and mammalian somatic cells . Furthermore, during meiotic maturation in these echinoderm and amphibian oocytes, this is followed by activation of many of the same protein-serine (threonine) kinases that are stimulated when quiescent mammalian somatic cells are prompted with mitogens to traverse from G0 to G1 phase . These findings imply that a similar protein kinase cascade may oversee progression at multiple points in the cell cycle.

Curr Genet, 1990 Dec, 18(6), 501 - 9
A mutated swi4 gene causes duplications in the mating-type region of Schizosaccharomyces pombe; Fleck O et al.; Efficient mating-type (MT) switching in homothallic strains of Schizosaccharomyces pombe is significantly reduced if they have a mutation in any of the eleven known swi genes . The swi4 mutation causes heterothallic as well as homothallic segregants, both of which have duplications in the MT region . In contrast to homothallic strains, h+ swi4 strains yield only a few duplications . The duplications originate in the process of MT switching, presumably by mistakes in the resolution of DNA intermediates . They always consist of one cassette and one of the intervening sequences, L and K respectively . Strains with up to seven cassettes in the MT region were found . The possible modes of their origins are discussed.

Curr Genet, 1990 Dec, 18(6), 511 - 6
Use of the Tn903 neomycin-resistance gene for promoter analysis in the fission yeast Schizosaccharomyces pombe; Lang-Hinrichs C et al.; The bacterial neo gene from transposon Tn903 (Tn601) was used for dominant transformation of the fission yeast Schizosaccharomyces pombe . It was found that high transformation efficiency was dependent on a high level of promoter activity, mediated by the strong promoter of the Schizosaccharomyces pombe alcohol dehydrogenase gene (adh1), as shown by comparing the efficiency of transformation to G418-resistance, the resistance levels of transformed cells, and the in vitro amino-glycoside phosphotransferase activity . On the other hand, the heterologous promoter of the Saccharomyces cerevisiae alcohol dehydrogenase I gene (adc1) is shown to be a weak promoter in Schizosaccharomyces pombe, though its activity is significantly enhanced in cells grown on glycerol as a carbon source . This system for selection and detection of promoter-active sequences may provide a useful basis for the analysis of promoter elements in fission yeast.

Nucleic Acids Res, 1990 Nov 25, 18(22), 6485 - 9
High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe; Okazaki K et al.; We describe a highly efficient alkali cation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of fission yeast Schizosaccharomyces pombe mutants . cDNA libraries constructed with the pcD or pcD2 vector are transduced into yeast by cotransfection with a linearized vector, which allows an enhanced homologous recombination between the yeast vector and the library plasmid leading to the efficient formation of concatemers containing pcD molecules . The transformation frequencies obtained by the method are 10(6) colonies per 10(8) cells transfected with 2 micrograms of library and 1 microgram of vector, 50-60% of which contain pcD molecules . The high-efficiency alkali cation method circumvents many of the shortcomings of the spheroplast method generally used for Schiz . pombe transfection . The vectors are maximized for the efficiency of library transduction and minimized for the rearrangements of pcD molecules during propagation in yeast . This system allows rapid screening of multi-million cDNA clone libraries for rare cDNAs in a routine scale of experiments . Using this system, various mammalian cDNAs that are extremely difficult, time-consuming, or unclonable to clone by other methods have been cloned.

Gene, 1990 Nov 15, 95(2), 179 - 86
Expression and characterization of infectious bursal disease virus polyprotein in yeast; Jagadish MN et al.; Various expression vectors containing a cDNA fragment encoding all but the first five amino acids (aa) of the large polyprotein (N-VP2-VP4-VP3-C) of infectious bursal disease virus were transformed into yeasts . In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, co- or post-translational processing of the unfused large polyprotein occurred, generating a stable C-terminal product (VP3) or correct size, but without any detectable N-terminal product (VP2) . Furthermore, when the processing of the polyprotein was interrupted, because of an engineered in-frame site-specific insertion of 4 aa, even VP3 (as part of the unprocessed polyprotein) was undetected . VP2 was detected in S . cerevisiae only when fused to yeast pre-sequences at the N terminus, suggesting that in yeast, VP2 or the unprocessed polyprotein, in the absence of its native N terminus or proper protection of its N-terminal aa residues is susceptible to proteolytic degradation . The first 8 aa of a modified pre-sequence of the CUP1 gene product and the pre-pro sequence of MF alpha 1 gene product have been used for stable intra- and extra-cellular production of VP2, respectively.

Science, 1990 Nov 9, 250(4982), 786 - 91
Cell cycle control of DNA replication by a homologue from human cells of the p34cdc2 protein kinase; D'Urso G et al.; The regulation of DNA replication during the eukaryotic cell cycle was studied in a system where cell free replication of simian virus 40 (SV40) DNA was used as a model for chromosome replication . A factor, RF-S, was partially purified from human S phase cells based on its ability to activate DNA replication in extracts from G1 cells . RF-S contained a human homologue of the Schizosaccharomyces pombe p34cdc2 kinase, and this kinase was necessary for RF-S activity . The limiting step in activation of the p34 kinase at the G1 to S transition may be its association with a cyclin since addition of cyclin A to a G1 extract was sufficient to start DNA replication . These observations suggest that the role of p34cdc2 in controlling the start of DNA synthesis has been conserved in evolution.

J Biol Chem, 1990 Nov 5, 265(31), 19351 - 5
A guanosine diphosphatase enriched in Golgi vesicles of Saccharomyces cerevisiae . Purification and characterization; Yanagisawa K et al.; We have recently described a luminal guanosine diphosphatase activity in Golgi-like vesicles of Saccharomyces cerevisiae (Abeijon, C., Orlean, P., Robbins, P . W., and Hirschberg, C . B . (1989) Proc . Natl . Acad . Sci . U . S . A . 86, 6935-6939) . The presumed in vivo role of this enzyme is to convert GDP into GMP . GDP is a reaction product following outer-chain mannosylation of luminal proteins and a known inhibitor of mannosyltransferases . It is hypothesized that GMP then returns to the cytosol . We have purified this enzyme to apparent homogeneity . Following solubilization from a membrane pellet using a buffer containing Triton X-100, the enzyme was purified on a concanavalin A-Sepharose column followed by Mono Q fast protein liquid chromatography (FPLC) and Superose-12 FPLC columns . After treatment with endoglycosidase H, the deglycosylated active enzyme was applied to a second Mono Q FPLC column and a phenyl-Superose FPLC column . The final enzyme activity was enriched 6500-fold over that of the Triton X-100 extract . The apparant molecular mass of the deglycosylated enzyme is 47 kDa . The purified enzyme is highly specific for guanosine diphosphate, requires Ca2+ for maximal activity, and has a broad pH optimum between 7.4 and 8.2 . The apparent Km for GDP is 0.1 mM; the Vmax is 4.9 mmol/min/mg of protein . An enzyme activity with similar substrate specificity has also been detected in membranes of Schizosaccharomyces pombe.

Mol Gen Genet, 1990 Nov, 224(2), 264 - 8
An electrophoretic karyotype of Aspergillus niger; Debets AJ et al.; An electrophoretic karyotype of Aspergillus niger was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis . Chromosome-sized DNA was separated into four bands . Seven of the eight linkage groups could be correlated with specific chromosomal bands . For this purpose DNA preparations from seven transformant strains of A . niger each carrying the heterologous amdS gene of Aspergillus nidulans on a different chromosome were analysed . Some of the assignments were confirmed with linkage group-specific A . niger probes . The estimated sizes of the A . niger chromosome range from 3.5 to 6.6 Mb, based on gel migration relative to the chromosomes of Schizosaccharomyces pombe strains, Saccharomyces cerevisiae and A . nidulans . The total genome size of A . niger significantly exceeds that of A . nidulans and is estimated to be about 35.5-38.5 Mb . Electrophoretic karyotyping was used to allocate non-mutant rRNA genes and to estimate the number of plasmids integrated in a high copy number transformant.

Proc Natl Acad Sci U S A, 1990 Nov, 87(22), 8711 - 5
Leucine-rich repeats and carboxyl terminus are required for interaction of yeast adenylate cyclase with RAS proteins; Suzuki N et al.; A Saccharomyces cerevisiae gene encoding adenylate cyclase has been analyzed by deletion and insertion mutagenesis to localize regions required for activation by the Sa . cerevisiae RAS2 protein . The NH2-terminal 657 amino acids were found to be dispensable for the activation . However, almost all 2-amino acid insertions in the middle 600 residues comprising leucine-rich repeats and deletions in the COOH-terminal 66 residues completely abolished activation by the RAS2 protein, whereas insertion mutations in the other regions generally had no effect . Chimeric adenylate cyclases were constructed by swapping the upstream and downstream portions surrounding the catalytic domains between the Sa . cerevisiae and Schizosaccharomyces pombe adenylate cyclases and examined for activation by the RAS2 protein . We found that the fusion containing both the NH2-terminal 1600 residues and the COOH-terminal 66 residues of the Sa . cerevisiae cyclase rendered the catalytic domain of the Sc . pombe cyclase, which otherwise did not respond to RAS proteins, activatable by the RAS2 protein . Thus the leucine-rich repeats and the COOH terminus of the Sa . cerevisiae adenylate cyclase appear to be required for interaction with RAS proteins.

Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8272 - 6
A putative protein kinase gene (kin1+) is important for growth polarity in Schizosaccharomyces pombe; Levin DE et al.; Mixed synthetic oligonucleotides encoding a sequence conserved among tyrosine-specific protein kinases were used to probe the genome of the fission yeast Schizosaccharomyces pombe . A single gene (kin1+) was isolated that encodes a putative protein kinase closely related to the KIN1- and KIN2-encoded serine/threonine-specific protein kinases of Saccharomyces cerevisiae . kin1+ is transcribed into a 3.5-kilobase mRNA that contains an uninterrupted open reading frame encoding a polypeptide of 98 kDa . In contrast to results obtained with kin mutants of S . cerevisiae, disruption of the Sc . pombe kin1+ gene resulted in recessive morphological and growth defects . kin1-disrupted cells grew slowly on enriched medium and grew as spheres, in contrast to wild-type Sc . pombe cells, which grow as rods . Relative to kin1+ cells, kin1-disrupted cells were differentially sensitive to lysis by treatment with alpha- and beta-glucanases, suggesting an alteration in either the composition or the organization of their cell walls.

J Gen Microbiol, 1990 Nov, 136 ( Pt 11), 2251 - 9
Glycoprotein molecules in the walls of Schizosaccharomyces pombe wild-type cells and a morphologically altered mutant resistant to papulacandin B; de Mora JF et al.; Schizosaccharomyces pombe cell walls contain two major glycoprotein species, I and II, with molecular masses of 2 x 10(6) and 5 x 10(5) Da respectively, as determined by gel filtration chromatography and PAGE . The ratio of sugar to protein is higher in species I than in species II . Much of the sugar in both glycoproteins (about 85% in wild-type cells) is O-linked to the peptide moiety . The morphological sph1 mutant is resistant to papulacandin B, and its cell wall contains less glycoprotein II (but not less glycoprotein I) than the parental wild-type strain, although glycoprotein II is still synthesized and released into the growth medium . Papulacandin B largely reverses the morphological alteration of the mutant, and returns the ratio between species I and II to about that found in the parental strain, although the absolute amount of species II is still lower in the mutant . The results point to the importance of the relative amounts of the different wall polymers in determining cell morphology.

EMBO J, 1990 Nov, 9(11), 3573 - 81
Drosophila cdc2 homologs: a functional homolog is coexpressed with a cognate variant; Lehner CF et al.; Using probes obtained by PCR amplification, we have cloned Drosophila cDNAs encoding structural homologs of the p34cdc2 cell cycle kinase . Southern blot experiments and in situ hybridization to polytene chromosomes demonstrated that the isolated cDNAs, were derived from two distinct genes, Dm cdc2 (31E) and Dm cdc2c (92F) . Northern blot and in situ hybridization experiments revealed that these two genes are coexpressed during embryogenesis and that expression is correlated with cell proliferation . However, despite the similarity in structure and expression, the two gene products differed in functional assays in yeasts . Expression of Dm cdc2 in Schizosaccharomyces pombe and Saccharomyces cerevisiae rescued cell cycle arrest caused by mutations in cdc2+ and CDC28, the genes encoding the p34cdc2 kinase homologs of these yeasts . In contrast, the Dm cdc2c gene product did not restore cell cycle progression . Thus, in addition to the identification of a functional homolog in Drosophila, our results indicate the presence of a closely related cognate of the p34cdc2 cell cycle kinase.

EMBO J, 1990 Nov, 9(11), 3565 - 71
Complementation of fission yeast cdc2ts and cdc25ts mutants identifies two cell cycle genes from Drosophila: a cdc2 homologue and string; Jimenez J et al.; We have exploited the universality of the molecular mechanisms that control entry into mitosis to clone the Drosophila melanogaster homologues of fission yeast Schizosaccharomyces pombe cell division control (cdc) genes by the complementation of temperature sensitive mutations . The Drosophila genes were expressed in S.pombe as cDNAs from the SP6 promoter . Successful recovery of complementing plasmids required that we first 'adapt' pooled plasmids from a Drosophila embryonic cDNA library for propagation in fission yeast by introducing an ars1-LEU2 DNA fragment into the vector . This library was introduced into S.pombe cdc2 and cdc25 mutants, and plasmids isolated carrying cDNAs that complement these mutations . The gene that encodes the Drosophila cdc2 homologue maps to a single locus in the Drosophila genome at 31E on chromosome 2 . It is expressed maternally to provide mRNA in syncytial embryos, and appears to be zygotically expressed in mitotically active regions of the cellularized embryo . We have isolated two different cDNAs that complement cdc25-22 . One corresponds to a transcript of string, previously described as the Drosophila homologue of cdc25, and the other to a gene that has not been previously characterized.

J Gen Microbiol, 1990 Nov, 136 ( Pt 11), 2261 - 5
The occurrence of glucosaminoglycan in the wall of Schizosaccharomyces pombe; Sietsma JH et al.; The major part of the wall of Schizosaccharomyces pombe consists of (1----3)-alpha-glucan and (1----3)-beta-glucan with some (1----6)-beta-linkages . Although in hydrolysed samples only a minute amount of glucosamine could be detected, this amino sugar may play an essential role as an integral part of a glucosaminoglycan/glucan complex . Treatment of the wall with either nitrous acid or chitinase changed the solubility properties of the beta-glucan, which suggests that the glucosaminoglycan/glucan complex is essentially similar to that found in walls of other fungi . An enzyme with properties similar to that of chitin synthase of other fungi, and probably responsible for the synthesis of the glucosaminoglycan, was detected in a mixed-membrane fraction.

J Cell Sci, 1990 Nov, 97 ( Pt 3), 509 - 16
Mitochondrial growth and DNA synthesis occur in the absence of nuclear DNA replication in fission yeast; Sazer S et al.; Cell growth and division require the doubling of cellular constituents followed by their equal distribution to the two daughter cells . Within a growing population, the ratio of mitochondrial to cellular volume is maintained, as is the number of mitochondrial genomes per cell . The mechanisms responsible for coordinating nuclear and mitochondrial DNA synthesis, and for balancing increases in cell and mitochondrial size are not well understood . In studies of the fission yeast Schizosaccharomyces pombe we quantified cellular and mitochondrial DNA content by both Southern blot analysis and flow cytometry of cells stained with a variety of DNA-binding fluorochromes, which we show are able to detect nuclear and mitochondrial DNA with different efficiencies . In the conditional cell division cycle mutant cdc10, which is unable to initiate nuclear DNA synthesis, we found that there was an increase in the mitochondrial DNA content in the absence of nuclear DNA replication . This demonstrates that mitochondrial and nuclear DNA synthesis are not obligately linked . We also show that mitochondrial DNA replication is not required for the increase in mitochondrial size that occurs as cells elongate, although this results in a decrease in the ratio of mitochondrial DNA to mitochondrial volume.

Gene, 1990 Oct 30, 95(1), 91 - 8
The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors; Stotz A et al.; We have determined the sequence of a DNA fragment encoding the ADE2 gene from Saccharomyces cerevisiae . A DNA fragment of 2241 bp capable of complementing ade2 mutations was modified so it is available as a single BglII fragment for use in yeast vectors or for gene disruptions . The minimal fragment codes for a putative protein which is highly similar to the protein encoded by the ADE6 gene from Schizosaccharomyces pombe and to the proteins encoded by the purEK operon of Escherichia coli.

Gene, 1990 Oct 30, 95(1), 105 - 10
Isolation of a gene encoding a mitochondrial HSP70 protein from Schizosaccharomyces pombe; Powell MJ et al.; We have isolated cDNA and genomic clones encoding a mitochondrial HSP70 protein from Schizosaccharomyces pombe . Nucleotide sequence analysis indicates that the encoded protein is homologous to the HSP70s of other organisms . The highest degree of amino acid conservation is with the proteins encoded by the Escherichia coli dnaK gene, the SSC1 gene of Saccharomyces cerevisiae and the MTP70 gene of Trypanosoma cruzi, the latter two having recently been shown to be located in the mitochondria . Western-blot analysis with immunoglobulin G raised against a peptide corresponding to the C terminus of the SSP1 protein indicates a 70-kDa protein which is associated with the mitochondria.

FEBS Lett, 1990 Oct 29, 273(1-2), 107 - 10
Heat shock induces enzymes of trehalose metabolism, trehalose accumulation, and thermotolerance in Schizosaccharomyces pombe, even in the presence of cycloheximide; De Virgilio C et al.; Exponentially growing cells of the fission yeast, Schizosaccharomyces pombe, contained virtually no trehalose at 27 degrees C but rapidly accumulated large quantities during heat shock at 40 degrees C . Activities of trehalose-6-phosphate synthase and trehalase also increased upon heat shock . Thermotolerance of the cells, measured as survival at 52 degrees C, increased in parallel to trehalose accumulation and decreased in parallel to the trehalose levels when cells were shifted back to 27 degrees C . Trehalose levels, activities of enzymes of trehalose metabolism and thermotolerance strongly increased upon heat shock even in the presence of cycloheximide, indicating that none of these effects requires protein synthesis . The data support the hypothesis that trehalose acts as a thermoprotectant in Schizosaccharomyces pombe.

J Biol Chem, 1990 Oct 25, 265(30), 18400 - 7
Calcium homeostasis and transport are affected by disruption of cta3, a novel gene encoding Ca2(+)-ATPase in Schizosaccharomyces pombe; Ghislain M et al.; A new P-type ATPase gene, cta3, has been identified in Schizosaccharomyces pombe . The deduced amino acid sequence presents a 45% identity with the Saccharomyces cerevisiae putative Ca2(+)-ATPase encoded by the PMR2 gene . The cta3 protein contains 7 out of the 8 amino acid residues involved in high affinity Ca2+ binding in the sarcoplasmic reticulum Ca2(+)-ATPase from muscles . It also contains a region similar to the phospholamban-binding domain that characterizes this Ca2+ pump . A null mutation of cta3 leads to higher levels of cytosolic free Ca2+ and to lower amounts of sequestered and bound Ca2+ . Cellular Ca2+ efflux and rates of uptake into intracellular compartments are reduced by the loss of cta3 function . The sequence analysis and the physiological results strongly support the conclusion that the cta3 gene encodes a Ca2(+)-ATPase, probably located in intracellular membranes.

Nature, 1990 Oct 18, 347(6294), 680 - 2
Distinct nuclear and spindle pole body population of cyclin-cdc2 in fission yeast; Alfa CE et al.; Cyclins, as subunits of the protein kinase encoded by the cdc2 gene are major controlling elements of the eukaryotic cell cycle . The fission yeast Schizosaccharomyces pombe has a B-type cyclin, which is a nuclear protein encoded by the cdc13 gene . Here we demonstrate the presence of two spatially distinct cdc13 cyclin populations in the nucleus of S . pombe, one of which is associated with the mitotic spindle poles . Both populations colocalize with the product of the cdc2 gene (p34cdc2) . Treatment of cells with the antimicrotubule drug thiabendazole prevents cyclin degradation and blocks the tyrosine dephosphorylation and activation of cdc2 . These results suggest a key regulatory role of the cdc2-cyclin complex in the initiation of mitotic spindle formation and also that mitotic microtubule function is required for cdc2 activation.

J Biol Chem, 1990 Oct 5, 265(28), 16856 - 62
Protein transport into mitochondria is conserved between plant and yeast species; Chaumont F et al.; Protein targeting into plant mitochondria was investigated by in vitro translocation experiments . The precursor of the mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia was synthesized in vitro, translocated to, processed, and assembled in purified Vicia faba mitochondria . Transport (but not binding) required a membrane potential and external nucleotides and was conserved among plant species . beta subunit precursors from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe were imported and correctly processed in plant mitochondria . This translocation used protease-sensitive components of the outer membrane . Conversely, the N . plumbaginifolia beta subunit precursor was efficiently translocated and cleaved in yeast mitochondria . However, a precursor for a chloroplast protein was not targeted to plant or yeast mitochondria . We conclude that the machinery for protein import into mitochondria is specific and conserved in plant and yeast organisms . These results are discussed in the context of a poly- or monophyletic origin of mitochondria.

Mol Cell Biol, 1990 Oct, 10(10), 5548 - 52
Evolutionary origin of the U6 small nuclear RNA intron; Reich C et al.; U6 is the most conserved of the five small nuclear RNAs known to participate in pre-mRNA splicing . In the fission yeast Schizosaccharomyces pombe, the single-copy gene encoding this RNA is itself interrupted by an intron (T . Tani and Y . Ohshima, Nature (London) 337:87-90, 1989) . Here we report analysis of the U6 genes from all four Schizosaccharomyces species, revealing that each is interrupted at an identical position by a homologous intron; in other groups, including ascomycete and basidiomycete fungi, as well as more distantly related organisms, the U6 gene is colinear with the RNA . The most parsimonious interpretation of our data is that the ancestral U6 gene did not contain an intron, but rather, it was acquired via a single relatively recent insertional event.

J Appl Bacteriol, 1990 Oct, 69(4), 569 - 77
Growth inhibitory and biocidal activity of some isothiazolone biocides; Collier PJ et al.; Similar patterns of growth inhibition were observed for the three biocides, benzisothiazol-3-one (BIT), 5-chloro-N-methylisothiazol-3-one (CMIT) and N-methylisothiazol-3-one (MIT) against Escherichia coli ATCC 8739 and Schizosaccharomyces pombe NCYC 1354 . After periods of induced stasis, proportional to biocide concentration, growth proceeded at an inhibited rate . Extrapolation of the static periods and inhibited growth rates against biocide concentration gave minimum growth inhibitory concentration estimates of 0.1-0.5 micrograms/ml for CMIT, 15-20 micrograms/ml for BIT and 40-250 micrograms/ml for MIT . Patterns of growth inhibition by CMIT and induced morphological changes in inhibited cultures suggested this compound to also inhibit initiation of DNA replication . Growth inhibitory activity was rapidly quenched by the addition of thiol-containing materials such as glutathione and cysteine . The activity of CMIT was additionally quenched by the presence of the non-thiol amino acids valine and/or histidine . These results suggest that the chlorinated isothiazolones can react with amines as well as with essential thiol groups.

Curr Genet, 1990 Oct, 18(3), 269 - 72
The structural gene coding for thiamin-repressible acid phosphatase in Schizosaccharomyces pombe; Yang JW et al.; The pho4 gene of the fission yeast Schizosaccharomyces pombe is regulated by thiamin . The nucleotide sequence of this gene is given here and it is shown that it matches the amino acid sequence of thiamin-repressible acid phosphatase, corroborating genetic evidence that pho4 represents the structural gene of this enzyme . The gene codes for a protein of 463 amino acids in length and shows regions of strong similarity with the phosphate-repressible acid phosphatase of Schizosaccharomyces pombe . The enzyme has a cleavable signal sequence 18 amino acids long and carries nine potential N-glycosylation sites.

Curr Genet, 1990 Oct, 18(3), 193 - 7
The recombinational hot spot mutation ade6-M26 of Schizosaccharomyces pombe stimulates recombination at sites in a nearby interval; Grimm C et al.; With the help of in vitro constructed intragenic double mutants, we investigated the influence of the recombinational hot spot mutation ade6-M26 on meiotic recombination between two additional ade6 mutations proximal to it . Recombination was stimulated four-fold when M26 was present in a heterozygous condition and ten-fold when homozygous . M26 itself remained unaffected in a substantial number of these events . This indicates that the stimulation can not only be due to a preferred conversion of M26 to wild-type with co-conversion of the second mutation in cis . A model is proposed in which M26 acts as an "entry site" for recombinational enzymes.

Genetics, 1990 Oct, 126(2), 309 - 15
stf1: non-wee mutations epistatic to cdc25 in the fission yeast Schizosaccharomyces pombe; Hudson JD et al.; In Schizosaccharomyces pombe, cdc25 is a cell cycle regulated inducer of mitosis . wee1 and phenotypically wee alleles of cdc2 are epistatic to cdc25 . Mutant alleles of a new locus, stf1 (suppressor of twenty-five), identified in a reversion analysis of conditionally lethal cdr1-76 cdc25-22 and cdr2-96 cdc25-22 double mutant strains, also suppress both temperature-sensitive and gene disruption alleles of cdc25 . These mutants, by themselves, are phenotypically indistinguishable from wild type strains; hence they represent the first known mutations that are epistatic to cdc25 and do not display a wee phenotype . stf1 genetically interacts with other elements of mitotic control in S . pombe . stf1-1 is additive with wee1-50, cdc2-1w and cdc2-3w for suppression of cdc25-22 . Also, like wee1- and cdc2-w, stf1- suppression of cdc25 is reversed by overexpression of the putative type 1 protein phosphatase bws1+/dis2+ . Interaction with various mutants and plasmid overexpression experiments suggest that stf1 does not operate either upstream or downstream of wee1 . Similarly, it does not operate through cdc25 since it rescues the disruption . stf1 appears to encode an important new element of mitotic control.

FEBS Lett, 1990 Oct 1, 271(1-2), 189 - 93
The RNA component of RNase P in Schizosaccharomyces species; Zimmerly S et al.; In the fission yeast Schizosaccharomyces pombe, the enzyme RNAse P copurifies with two RNAs, K1- and K2-RNA, which are identical except for their termini {1} and which are encoded by a single gene {2} . We have undertaken the cloning of the K-RNA genes in related organisms in order to gain comparative structural information . Because the K-RNA sequence is poorly conserved across species, we have cloned the K-RNA genes in the Schizosaccharomyces species S . malidevorans, S . japonicus, S . versatilis, and S . octosporus . Of the 4 species, only S . octosporus contains a K-RNA gene different from that in S . pombe; the gene diverges by 20% . Based on the two sequences, nuclease protection data and computer analysis, we have proposed a secondary structure model for the K-RNA . Northern analysis shows the K-RNA genes in all four Schizosaccharomyces species to be expressed as two RNAs, as in S . pombe.

EMBO J, 1990 Oct, 9(10), 3045 - 50
Primary structure, genomic organization and heterologous expression of a glucose transporter from Arabidopsis thaliana; Sauer N et al.; Both genomic and full length cDNA clones of an Arabidopsis thaliana sugar carrier, STP1, have been obtained using a cDNA clone of the H+/hexose cotransporter from the green alga Chlorella kessleri as hybridization probe . The peptide predicted from these sequences in 522 amino acids long and has a molecular weight of 57,518 kd . This higher plant sugar carrier contains 12 putative transmembrane segments and is highly homologous to the H+/hexose cotransporter from Chlorella, with an overall identity in the amino acid sequence of 47.1% . It is also homologous to the human HepG2 glucose transporter (28.4%), and other sugar carriers from man, rat, yeast and Escherichia coli . The definite proof for the function of the STP1 protein as a hexose transporter and data on its substrate specificity were obtained by heterologous expression in the fission yeast Schizosaccharomyces pombe . Transformed yeast cells transport D-glucose with a 100-fold lower KM value than control cells . Moreover only the transformed cells were able to accumulate the non-metabolizable D-glucose analogue 3-O-methyl-D-glucose, indicating that the Arabidopsis carrier catalyses an energy dependent, active uptake of hexoses . Expression of STP1 mRNA is low in heterotrophic tissues like roots or flowers . High levels of expression are found in leaves.

Proc Natl Acad Sci U S A, 1990 Oct, 87(20), 7814 - 8
Adenylyl cyclase is dispensable for vegetative cell growth in the fission yeast Schizosaccharomyces pombe; Maeda T et al.; Disruption of the cyr1 gene of Schizosaccharomyces pombe, which encodes adenylyl cyclase, did not confer lethality to fission yeast cells, although they grew 40% slower than wild-type strains in complete medium . These cells contained no measurable amount of cAMP and no adenylyl cyclase activity . When h+ and h- cyr1 disruptants were mixed, they underwent mating even in rich medium . Propagation of homothallic cyr1 disruptants was difficult, probably because such cells readily mate and produce asci and thus stop growing . A greater than 10-fold increase in the amount of cyr1 mRNA was observed when cloned cyr1+ was introduced into Sch . pombe cells on a multicopy plasmid . The total adenylyl cyclase activity was similarly high in these transformants . However, the level of intracellular cAMP was hardly affected . Evidence suggests that this was not due to increased phosphodiesterase activity . Thus, cAMP level in growing fission yeast cells appears to be regulated not by the amount of adenylyl cyclase protein but by a feedback mechanism at the enzyme level . The cAMP level fell by approximately 50% under nitrogen starvation, which triggers sexual development in Sch . pombe . We suggest that fission yeast controls the level of intracellular cAMP primarily to regulate sexual development rather than to drive or arrest the cell cycle.

Mol Cell Biol, 1990 Oct, 10(10), 5442 - 54
Drosophila scaffold-attached regions bind nuclear scaffolds and can function as ARS elements in both budding and fission yeasts; Amati B et al.; Histone-depleted nuclei maintain sequence-specific interactions with genomic DNA at sites known as scaffold attachment regions (SARs) or matrix attachment regions . We have previously shown that in Saccharomyces cerevisiae, autonomously replicating sequence elements bind the nuclear scaffold . Here, we extend these observations to the fission yeast Schizosaccharomyces pombe . In addition, we show that four SARs previously mapped in the genomic DNA of Drosophila melanogaster bind in vitro to nuclear scaffolds from both yeast species . In view of these results, we have assayed the ability of the Drosophila SARs to promote autonomous replication of plasmids in the two yeast species . Two of the Drosophila SARs have autonomously replicating sequence activity in budding yeast, and three function in fission yeast, while four flanking non-SAR sequences are totally inactive in both.

Bioessays, 1990 Oct, 12(10), 457 - 63
Yeast as a model system for understanding the control of DNA replication in Eukaryotes; Bartlett R et al.; In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the initiation of DNA replication is controlled at a point called START . At this point, the cellular environment is assessed; only if conditions are appropriate do cells traverse START, thus becoming committed to initiate DNA replication and complete the remainder of the cell cycle . The cdc2+/CDC28+ gene, encoding the protein kinase p34, is a key element in this complex control . The identification of structural and functional homologues of p34 suggests that it has a role in the control of DNA replication in all eukaryotes . The WHI1+, CLN1+ and CLN2+ gene products, identified in S . cerevisiae, are positive regulators that function at START and may interact with p34 . Determining how passing the START control point leads to the initiation of DNA replication is a major outstanding challenge in cell cycle studies.

J Biol Chem, 1990 Sep 15, 265(26), 15503 - 5
Effects of phosphate and hydrophobic molecules on two mutations in the beta-strand sector of the H(+)-ATPase from the yeast plasma membrane; Goffeau A et al.; At concentrations from 10 to 100 mM, inorganic phosphate and sulfate stimulate the activity of the H(+)-ATPase purified from the wild type Schizosaccharomyces pombe plasma membranes . Compared to the wild type ATPase, the stimulation by phosphate is more pronounced in the mutant pma1-1 (Gly-268----Asp) and is much reduced in the mutant pma1-2 (Lys-250----Thr) enzymes . In contrast, the inhibition by trifluoperazine is less pronounced in the pma1-1 mutant than in the wild type or pma1-2 mutant . The mutant pma1-2 ATPase activity is markedly stimulated by 10-20% dimethyl sulfoxide, which has a limited effect on the wild type and pma1-1 enzymes . These data indicate that the protein domain located in the beta-strand sector, including Lys-250 and Gly-268, is located in the active site and that its hydrophobic character influences the interactions of the yeast H(+)-ATPase with inorganic phosphate, as well as with the hydrophobic inhibitor trifluoperazine or the hydrophobic solvent dimethyl sulfoxide.

Gene, 1990 Sep 14, 93(2), 265 - 70
Synthesis, phosphorylation, and nuclear localization of human papillomavirus E7 protein in Schizosaccharomyces pombe; Tommasino M et al.; The complete E7 protein-encoding open reading frame of human papillomavirus type 16 (HPV-16) was expressed in the fission yeast Schizosaccharomyces pombe, under the control of a cloned yeast promoter . The HPV-16 E7 protein synthesized in S . pombe is a 17-kDa phosphoprotein which is recognized by anti-E7 antibodies (raised in rabbits against E7 fusion protein produced in Escherichia coli) . The mobility during sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of native E7 phosphoprotein synthesized in S . pombe is identical to that of the E7 phosphoprotein immunoprecipitated from human CaSki cells . Immunofluorescence staining showed that HPV-16 E7 phosphoprotein is localized in the nuclei of transformed S . pombe . These results indicate that E7 protein synthesized by S . pombe is apparently indistinguishable from HPV-16 E7 protein synthesized in higher eukaryotic cells expressing genes of HPV-16, and also that the phosphorylated, nuclear HPV-16 E7 protein is synthesized in S . pombe in a form compatible with its biological activity.

Nucleic Acids Res, 1990 Sep 11, 18(17), 5207 - 12
Immunoprecipitation distinguishes non-overlapping groups of snRNPs in Schizosaccharomyces pombe; Tollervey D et al.; The large number of snRNAs in the fission yeast Schizosaccharomyces pombe can be divided into four non-overlapping groups by immunoprecipitation with antibodies directed against mammalian snRNP proteins . 1) Of the abundant snRNAs, anti-Sm sera precipitate only the spliceosomal snRNAs U1, U2, U4, U5 and U6 . Surprisingly, three Sm-sera tested distinguish between U2, U4 and U5 and U1 from S.pombe; one precipitating only U1 and two precipitating U2, U4 and U5 but not U1 . 2) A group of 11 moderately abundant snRNAs are not detectably precipitated by human anti-Sm sera, but are specifically precipitated by monoclonal antibody H57 specific for the human B/B' polypeptides . From Aspergillus nidulans this antibody also precipitates at least 12 snRNAs . 3) Anti-(U3)RNP sera do not precipitate the above snRNAs, but precipitate at least 6 further snRNAs, including the homologues of U3 . Both the anti-(U3)RNP sera and H57 also efficiently precipitate a number of discrete non-capped RNAs . 4) A small number of additional snRNAs are not detectably precipitated by any anti-serum tested to date, further analysis may identify antisera specific for these snRNPs . Western blots of purified snRNP proteins were used to identify the S.pombe proteins responsible for these immunoprecipitations . Several Sm-sera decorate a 16.3kD protein which may be a D protein homologue, monoclonal H57 decorates a further protein of 16kD and an anti-(U3)RNP serum decorates the homologue of the 36kD U3-specific protein, fibrillarin.

Mol Gen Genet, 1990 Sep, 223(3), 407 - 16
DNA sequence elements required for transcription initiation of the Schizosaccharomyces pombe ADH gene in Saccharomyces cerevisiae; Furter-Graves EM et al.; The roles of the TATA element and sequences near the mRNA initiation site in specifying the location of initiation sites in Saccharomyces cerevisiae were examined, using the Schizosaccharomyces pombe ADH gene . The importance of spacing was demonstrated by analysis of a series of deletions that removed from 8-50 bp between the TATA element and ATG translation initiation site of this gene . Primer extension mapping showed that increasing deletion length is associated with a progressive shift downstream in the location of the initiation sites . The distance of a given site from the promoter affected the relative ability of the site to be utilized for initiation . For this gene, a permissive region for transcription initiation exists between 55 and 125 bases downstream of the TATA element, and a zone of 75-115 bases allows maximal usage of an initiation site . The presence of a TATA sequence was shown to be necessary in S . cerevisiae for maintaining the location of this "window" of initiation . The TATA sequence is essential for function of the gene in S . pombe . This gene, as well as the majority of the 63 S . cerevisiae genes surveyed, uses initiation sites which fit a PyAA/T(Pu) consensus . Cis-acting mutations were recovered which restored ADH activity to a deletion allele that initiates its mRNAs downstream of the ATG . DNA sequence and transcript analysis with these mutants confirmed the requirement of proper spacing and conformity of initiation sites to the PyAA/T(Pu) consensus for efficient transcript initiation.

Mutat Res, 1990 Sep-Nov, 236(2-3), 277 - 87
Eukaryotic DNA ligases; Lasko DD et al.; Recent studies on eukaryotic DNA ligases are briefly reviewed . The two distinguishable enzymes from mammalian cells, DNA ligase I and DNA ligase II, have been purified to homogeneity and characterized biochemically . Two distinct DNA ligases have also been identified in Drosophila melanogaster embryos . The genes encoding DNA ligases from Schizosaccharomyces pombe, Saccharomyces cerevisiae and vaccinia virus have been cloned and sequenced . These 3 proteins exhibit about 30% amino acid sequence identity; the 2 yeast enzymes share 53% amino acid sequence identity or conserved changes . Altered DNA ligase I activity has been found in cell lines from patients with Bloom's syndrome, although a causal link between the enzyme deficiency and the disease has not yet been proven.

Proc Natl Acad Sci U S A, 1990 Sep, 87(17), 6679 - 83
Human DNA ligase I cDNA: cloning and functional expression in Saccharomyces cerevisiae; Barnes DE et al.; Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods . In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I . In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomyces cerevisiae . The sequence of an apparently full-length cDNA encodes a 102-kDa protein, indistinguishable in size from authentic human DNA ligase I . The deduced amino acid sequence of the human DNA ligase I cDNA is 40% homologous to the smaller DNA ligases of S . cerevisiae and Schizosaccharomyces pombe, homology being confined to the carboxyl-terminal regions of the respective proteins . Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is transcribed from a single-copy gene on chromosome 19.

Cell Regul, 1990 Sep, 1(10), 763 - 9
Biological activity of the mammalian RAP genes in yeast; Xu HP et al.; We have screened expression libraries for mammalian cDNAs capable of suppressing defects in ras1- Schizosaccharomyces pombe . Both the RAP1A and RAP1B genes were identified in this manner . They suppress defects in cell morphology and sporulation, although not conjugation . In contrast, RAP genes do not suppress phenotypes in the yeast Saccharomyces cerevisiae that are deficient in RAS . Indeed, expression of RAP1A appears to antagonize the activated S . cerevisiae RAS2val19 gene . These results indicate that RAP proteins can interact with RAS targets, sometimes productively, sometimes nonproductively.

Genes Dev, 1990 Aug, 4(8), 1332 - 44
Human cDNAs encoding homologs of the small p34Cdc28/Cdc2-associated protein of Saccharomyces cerevisiae and Schizosaccharomyces pombe; Richardson HE et al.; The Cks1 protein is a component of the Cdc28 protein kinase in the budding yeast Saccharomyces cerevisiae . This paper reports the cloning of two homologs of the S . cerevisiae CKS1 gene from human cells . These homologs, CKShs1 and CKShs2, both encode proteins of 79 amino acids that share considerable homology at the amino acid level with the products of CKS1 from S . cerevisiae and suc1+ from the fission yeast Schizosaccharomyces pombe . Both human homologs are capable of rescuing a null mutation of the S . cerevisiae CKS1 gene when expressed from the S . cerevisiae GAL1 promoter . S . pombe suc1+ expressed from the GAL1 promoter is also capable of rescuing a S . cerevisiae cks1 null mutation . Ckshs1 or Ckshs2 protein linked to Sepharose beads can bind the Cdc28/Cdc2 protein kinase from both S . cerevisiae and human cells . The CKShs1 and CKShs2 mRNAs are expressed in different patterns through the cell cycle in HeLa cells, which may reflect specialized roles for the encoded proteins.

Proc Natl Acad Sci U S A, 1990 Aug, 87(15), 5697 - 701
Mitotic role for the Cdc28 protein kinase of Saccharomyces cerevisiae; Reed SI et al.; The Cdc28 protein kinase functions in the G1 to S phase transition of the cell cycle of the budding yeast Saccharomyces cerevisiae . This is in contrast with observations of the homologous protein kinase from a variety of metazoans, where activity and function are associated with the G2 to M phase transition . We present evidence that the Cdc28 protein kinase is also required for mitosis and that this function is executed in the G2 interval of the cell cycle . We show, in addition, that the protein kinase is highly active during this phase of the cell cycle . The dual role of the Cdc28 protein kinase in the S . cerevisiae cell cycle thus parallels that demonstrated for the cdc2 protein kinase of the fission yeast Schizosaccharomyces pombe.

J Cell Sci, 1990 Aug, 96 ( Pt 4), 683 - 9
Cell cycle regulation of p34cdc2 kinase activity in Physarum polycephalum; Ducommun B et al.; The regulation of the mitotic histone H1 kinase activity has been analyzed during the naturally synchronous cell cycle of Physarum polycephalum plasmodia . The universal binding property of the p13suc1 Schizosaccharomyces pombe gene product was used to precipitate and assay the cdc2 histone H1 kinase activity . The kinase activity peaks at the beginning of metaphase and its decline, which requires protein synthesis, appears to be an early event during the metaphase process . Microtubular poisons, temperature shifts and DNA synthesis inhibitors were used to perturb cell cycle regulatory pathways and characterize their effects on cdc2 kinase activation . Our results suggest that the full activation of the mitotic kinase requires at least two successive triggering signals involving microtubular components and DNA synthesis.

Mol Gen Mikrobiol Virusol, 1990 Aug, (8), 5 - 8
{Phenotypic manifestations of reverse transcriptase activity in yeast cells}; Reznik NL et al.; The yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe transformed by plasmids containing retrotransposon from yeast or Drosophila under the control of a strong promoter show the remarkable reverse transcriptase activity . The activity results in the impaired yeast growth and decreased mitotic stability of the plasmids . The phenotypic expression of the reverse transcriptase activity is observed within 30 days.

FEBS Lett, 1990 Jul 30, 268(1), 217 - 21
Human chorionic gonadotropin alpha and human cytomegalovirus promoters are extremely active in the fission yeast Schizosaccharomyces pombe; Toyama R et al.; We have investigated the transcriptional activity of human cytomegalovirus, herpes thymidine kinase, human chorionic gonadotropin alpha, somatostatin, immunoglobulin kappa chain, alpha crystallin, albumin and interferon-beta promoters in the fission yeast Schizosaccharomyces pombe . Among these, the human cytomegalovirus, human chorionic gonadotropin alpha, and somatostatin promoters were found to be very active, approximately 11-, 9-, and 0.9-fold as active as the SV40 early promoter, respectively . The remainder of the promoters studied were weak, having only 10-20% of the SV40 promoter activity . Primer extension analysis showed that the strong promoters initiated transcription in S . pombe at the same sites as in mammalian cells, indicating the high similarity between both transcriptional systems.

Nature, 1990 Jul 19, 346(6281), 291 - 4
Striking conservation of TFIID in Schizosaccharomyces pombe and Saccharomyces cerevisiae; Fikes JD et al.; Eukaryotic promoters contain binding sites for basic transcription factors and gene-specific activator proteins . The transcription factors interact at the TATA box, which lies close to the position of transcription initiation . Activators typically bind to distant sites that can lie kilobases away from the initiation site . The factor TFIID binds specifically to the TATA box to initiate an ordered pathway of assembly of the basic transcription factors . Biochemical analyses have shown that human and Saccharomyces cerevisiae TFIID are functionally interchangeable in vitro . To study further the functional conservation of this critical factor, we are surveying proteins from divergent organisms that can substitute in vivo for the S . cerevisiae TFIID . We report here the isolation of a unique gene from Schizosaccharomyces pombe that fully complements a null mutation in SPT15, the gene that encodes TFIID in S . cerevisiae . The Schiz . pombe gene encodes a protein 93% identical (166/178) to S . cerevisiae TFIID in a region consisting of a direct repeat.

Nature, 1990 Jul 19, 346(6281), 240 - 4
A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif; Sinclair AH et al.; A search of a 35-kilobase region of the human Y chromosome necessary for male sex determination has resulted in the identification of a new gene . This gene is conserved and Y-specific among a wide range of mammals, and encodes a testis-specific transcript . It shares homology with the mating-type protein, Mc, from the fission yeast Schizosaccharomyces pombe and a conserved DNA-binding motif present in the nuclear high-mobility-group proteins HMG1 and HMG2 . This gene has been termed SRY (for sex-determining region Y) and proposed to be a candidate for the elusive testis-determining gene, TDF.

Mol Cell Biol, 1990 Jul, 10(7), 3750 - 60
Cloning and analysis of a gene involved in DNA repair and recombination, the rad1 gene of Schizosaccharomyces pombe; Sunnerhagen P et al.; We have cloned the rad1 gene of Schizosaccharomyces pombe by complementation of the rad1-1 mutant, which is deficient in DNA repair and recombination . The coding region of the gene is 582 base pairs long and contains no introns . The predicted product is a strongly acidic, 22-kilodalton protein containing 194 amino acid residues . This gene does not exhibit significant homology to any other known repair gene . The major transcription start site is at 27 base pairs upstream of the putative start codon . Insertion mutagenesis revealed that besides the coding region, at least 151 base pairs of 5'-flanking sequence are required for full complementing activity . A strain carrying a null allele of rad1 was constructed and found to have a phenotype closely similar to that of the rad1-1 mutant . Expression in Escherichia coli of the coding region yielded a protein product of a size close to that predicted from the DNA sequence . This product reacted with antibodies raised against a synthetic peptide with a sequence from that predicted for the protein product . We have localized the rad1 gene to NotI fragment E of the S . pombe genome.

Mol Gen Genet, 1990 Jul, 222(2-3), 169 - 75
Molecular basis for determining the sensitivity of eucaryotes to the antimitotic drug rhizoxin; Takahashi M et al.; Rhizoxin, an antibiotic, exhibits potent anti-mitotic activity against most eucaryotic cells including those of higher vertebrates, plants and fungi by binding to beta-tubulin . The benA gene of three independently isolated rhizoxin-resistant (Rhir) mutants of Aspergillus nidulans was cloned, sequenced and compared with that of the wild-type, rhizoxin-sensitive (Rhis) strain . In all three Rhir mutants, the AAC codon for Asn-100 of the benA beta-tubulin gene was altered to ATC, coding for Ile . Sequence displacement experiments confirmed that the substitution of Ile for Asn-100 confers resistance to rhizoxin in this organism . The amino acid sequences of beta-tubulin surrounding the 100th amino acid residue from the N-terminus including Asn-100 are highly conserved with a few exceptions . The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are naturally occurring Rhir organisms whose beta-tubulin genes encode Ile and Val respectively at the 100th amino acid residue . The Ile-100 of S . pombe and the Val-100 of S . cerevisiae were altered to Asn using site-directed mutagenesis and gene displacement techniques . The resultant haploid strains of these two yeasts uniquely expressing beta-tubulin (Asn-100) instead of beta-tubulin (Ile-100 or Val-100) were found to be Rhis . Haploid yeast expressing beta-tubulin (Asn-100) is normal except for its sensitivity to rhizoxin . These results suggest that rhizoxin resistance has a common basis in both naturally occurring species and experimentally selected mutants in the substitution of Ile or Val for Asn-100 in beta-tubulin.

J Gen Microbiol, 1990 Jul, 136 ( Pt 7), 1271 - 7
Effect of ethanol on the phospholipid and fatty acid content of Schizosaccharomyces pombe membranes; Koukou AI et al.; Ethanol at concentrations up to 5% (v/v) had no effect on the growth of Schizosaccharomyces pombe, whereas concentrations over 7.5% were inhibitory . The major membrane phospholipids in S . pombe cells growing aerobically in the absence of added ethanol were phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine . Oleic acid (18:1) was the main fatty acid . When ethanol (7.5%) was added to aerobically growing cultures, the phosphatidylinositol content increased, whereas the 18:1 content decreased . Similar changes were observed in the membrane phospholipids of cells grown anaerobically without ethanol . However, the presence of ethanol in anaerobically growing cultures had an opposite effect on fatty acids, as the 18:1 content increased . The results support the idea that ethanol tolerance in S . pombe may be connected with a high content of 18:1 fatty acids, and with the ability to maintain a high rate of phospholipid biosynthesis.

J Cell Sci, 1990 Jul, 96 ( Pt 3), 435 - 8
Continued DNA synthesis after a mitotic block in the double mutant cut1 cdc11 of the fission yeast Schizosaccharomyces pombe; Creanor J et al.; DNA synthesis is normally dependent on a cell having previously gone through mitosis . Hirano et al . (1986), however, found that DNA synthesis continued at the restrictive temperature in the double mutant cut1 cdc11 of Schizosaccharomyces pombe even though mitosis was blocked in some of the cells . We have confirmed this result with bulk DNA assays of asynchronous cultures . Synchronous cultures of a diploid double mutant at the restrictive temperature showed two peaks of incorporation with an interval between them that was approximately the same as the doubling time in cell length . Flow cytometry showed that the cells had increased their DNA content from 4C (the diploid value) to about 16C after 7h . The cytological appearance at this time was mixed, with uninucleate, binucleate and dead cells, but fluorescence measurements on single cells indicated that about half the population had single nuclei with about the 16C value and had therefore gone through two rounds of DNA synthesis without mitosis.

Genes Dev, 1990 Jul, 4(7), 1141 - 8
Cloning of the Schizosaccharomyces pombe TFIID gene reveals a strong conservation of functional domains present in Saccharomyces cerevisiae TFIID; Hoffmann A et al.; The gene encoding the Schizosaccharomyces pombe TATA box-binding factor (TFIID) was cloned and sequenced . The gene contains three introns and codes for a polypeptide of 231 amino acids . The cDNA-expressed protein showed both TATA box-binding and basal transcription activities . The carboxy-terminal three-quarters of S . pombe TFIID shares an extraordinary degree of amino acid sequence homology with a corresponding region of Saccharomyces cerevisiae TFIID that has been shown to be necessary and sufficient for TATA box-binding and basal transcription activities . In contrast, the amino-terminal regions of the S . pombe and S . cerevisiae TFIIDs differ markedly in amino acid sequence and composition . Structure and function relationships of TFIID are discussed in light of these data.

Mol Cell Biol, 1990 Jul, 10(7), 3524 - 34
Differential distribution of factors involved in pre-mRNA processing in the yeast cell nucleus; Potashkin JA et al.; The yeast cell nucleus has previously been shown to be divided into two regions by a variety of microscopic approaches . We used antibodies specific for the 2,2,7-trimethylguanosine cap structure of small nuclear ribonucleic acids (snRNAs) and for a protein component of small nuclear ribonucleoprotein particles to identify the distribution of small nuclear ribonucleoprotein particles within the yeast cell nucleus . These studies were performed with the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae . By using immunofluorescence microscopy and immunoelectron microscopy, most of the abundant snRNAs were localized to the portion of the nucleus which has heretofore been referred to as the nucleolus . This distribution of snRNAs is different from that found in mammalian cells and suggests that the nucleolar portion of the yeast nucleus contains functional domains in addition to those associated with RNA polymerase I activity.

Proc Natl Acad Sci U S A, 1990 Jul, 87(13), 4917 - 21
Neurospora crassa a mating-type region; Staben C et al.; The a mating-type region of Neurospora crassa controls several major events in both the sexual and asexual phases of the fungal life cycle . This 3235-base-pair DNA segment is not homologous to the comparable genetic region of the A mating type . The unique a and A regions are bordered by nearly identical DNA sequences . The a genetic region contains at least two functional segments . One segment encodes a perithecium maturation function that is dependent on the second segment for phenotypic expression . This second a segment encodes a spliced mRNA that specifies the mt a-1 polypeptide . This polypeptide appears to be responsible for vegetative incompatibility, mating identity, and perithecium induction . The a-1 transcript is produced vegetatively and under conditions that induce sexual differentiation . The amino-terminal half of the mt a-1 polypeptide is homologous to the shorter Schizosaccharomyces pombe mat-Mc polypeptide . This homology and the properties of mt a-1 mutants suggest that the a-1 polypeptide segment that is homologous to the mat-Mc polypeptide may be primarily responsible for mating functions, while the distal segment is required for vegetative incompatibility.

J Cell Sci, 1990 Jul, 96 ( Pt 3), 429 - 33
Changes in the rate of oxygen consumption in synchronous cultures of the fission yeast Schizosaccharomyces pombe; Novak B et al.; Oxygen consumption was measured with an oxygen electrode in synchronous cultures of S . pombe . There were changes during the cell cycle in the rate of oxygen uptake, which are most clearly shown as oscillations in acceleration curves (rate of the rate of uptake) . Under various conditions of selection and induction synchrony the acceleration curves are similar to those found earlier for CO2 production . As with CO2 production, the oscillations continued after a block to the DNA-division cycle . There were, however, two differences between oxygen uptake and CO2 production . The oxygen oscillations were more marked and also were out of phase by half a cycle . The respiratory coefficient therefore changes through the cycle.

Can J Microbiol, 1990 Jun, 36(6), 390 - 4
Pattern of end growth of the fission yeast Schizosaccharomyces pombe; Miyata H et al.; The patterns of end growth of individual cells of Schizosaccharomyces pombe, wild-type cells (strain 972 h-), cells exposed to 8 mM hydroxyurea, and cdc mutants (cdc11-123 and cdc2-33), were investigated by time-lapse photomicrography . It was reconfirmed that there are three patterns of end growth: cells growing at the old end, at the new end, and at both ends from the beginning of the cell cycle . Cells that initiated growth at the old (new) end increased their growth rate at the new (old) end and became constant in their growth rate at the old (new) end when cells had their growth rate higher than a critical value: 0.08, 0.09, 0.08, and 0.11 microns/min in wild-type cells, cells exposed to hydroxyurea, cdc11-123 cells, and cdc2-33 cells, respectively . The critical value is proportional to the doubling time in length.

Proc Natl Acad Sci U S A, 1990 Jun, 87(11), 4043 - 7
Introduction of large linear minichromosomes into Schizosaccharomyces pombe by an improved transformation procedure; Allshire RC; The efficiency of transformation of Schizosaccharomyces pombe has been increased 10- to 50-fold over previously reported methods . By using 1 microgram of plasmid, 7.0 x 10(5) transformants are regularly obtained . This increased transformation efficiency is mainly due to the inclusion of the cationic liposome-forming reagent Lipofectin in the protocol . Various parameters affecting transformation of Sc . pombe in the presence of Lipofectin have been examined . Lipofectin can also be used to increase transformation efficiency in Saccharomyces cerevisiae . It is also demonstrated that by using this improved transformation procedure, linear minichromosomes of greater than 500 kilobases can be introduced into Sc . pombe with relative ease . These minichromosomes can replicate as stable linear molecules upon reintroduction into Sc . pombe, demonstrating that Sc . pombe telomeres retain function when reintroduced as naked DNA . The ability of Sc . pombe to admit large DNA molecules indicates that it should be feasible to clone large DNA from other organisms in Sc . pombe.

EMBO J, 1990 Jun, 9(6), 1929 - 37
Substrate recognition by RNase P and by the catalytic M1 RNA: identification of possible contact points in pre-tRNAs; Kahle D et al.; Modified bases were introduced into pre-tRNAs during in vitro RNA synthesis or by chemical modification . These RNAs were used as substrates for the catalytic M1 RNA and the RNase P holoenzyme from Schizosaccharomyces pombe . The synthetic approach permitted the insertion of 100% m7GTP into pre-tRNAs and this resulted in complete inhibition of the specific 5' processing reactions . Partially modified RNAs were obtained by chemical modifications of purines and uridines in the pre-tRNAs . This allowed detailed analyses of specific bases excluded in the products . With pre-tRNA(Ser) and initiator pre-tRNA(Met), strong effects were observed in the T arm and weaker effects in the anticodon stem . Only minor base exclusions were detected in the acceptor stem of pre-tRNA(Ser) and in the D arm of pre-tRNA(Met).

Curr Genet, 1990 Jun, 17(6), 473 - 9
Evolutionary conservation of transcriptional machinery between yeast and plants as shown by the efficient expression from the CaMV 35S promoter and 35S terminator; Hirt H et al.; Complementation of fission yeast mutants by plant genomic libraries could be a promising method for the isolation of novel plant genes . One important prerequisite is the functioning of plant promoters and terminators in Schizosaccharomyces pombe and Saccharomyces cerevisiae . Therefore, we studied the expression of the bacterial beta-glucuronidase (GUS) reporter gene under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and 35S terminator . We show here that S . pombe initiates transcription at exactly the same start site as was reported for tobacco . The 35S CaMV terminator is appropriately recognized leading to a polyadenylated mRNA of the same size as obtained in plant cells transformed with the same construct . Furthermore, the GUS-mRNA is translated into fully functional GUS protein, as determined by an enzymatic assay . Interestingly, expression of the 35S promoter in the budding yeast S . cerevisiae was found to be only moderate and about hundredfold lower than in S . pombe . To investigate whether different transcript stabilities are responsible for this enormous expression difference in the two yeasts, the 35S promoter was substituted by the ADH (alcohol dehydrogenase) promoter from fission yeast . In contrast to the differential expression pattern of the 35S promoter, the ADH promoter resulted in equally high expression rates in both fission and budding yeast, comparable to the 35S promoter in S . pombe . Since the copy number of the 35S-GUS constructs differs only by a factor of two in the two yeasts, it appears that differential recognition of the 35S promoter is responsible for the different transcription rates.

Mol Cell Biol, 1990 Jun, 10(6), 2874 - 81
U1 small nuclear RNA from Schizosaccharomyces pombe has unique and conserved features and is encoded by an essential single-copy gene; Porter G et al.; We have cloned, sequenced, and disrupted the gene encoding U1 small nuclear RNA (snRNA) in the fission yeast Schizosaccharomyces pombe . This RNA is close in size and exhibits a high degree of secondary structure homology to human U1 RNA . There exist two regions of extended primary sequence identity between S . pombe and human U1 RNAs; the first comprises nucleotides involved in hydrogen bonding to 5' splice junctions, and the second is a single-stranded region which, in the human snRNA, forms part of the A protein binding site . S . pombe U1 lacks two nucleotides just following the 5' cap structure which are present in all other U1 homologs examined to date, and the region which corresponds to the binding site for the human 70K protein (molecular weight of 55,000) is more divergent than in other organisms . A putative upstream transcription signal is conserved in sequence and location among all loci encoding spliceosomal snRNAs in S . pombe with the exception of U6 . Disruption of the single-copy U1 gene, designated snu1, reveals that this RNA is indispensable for viability.

EMBO J, 1990 Jun, 9(6), 1957 - 62
Structural and functional analysis of ypt2, an essential ras-related gene in the fission yeast Schizosaccharomyces pombe encoding a Sec4 protein homologue; Haubruck H et al.; Using the cloned Saccharomyces cerevisiae YPT1 gene as hybridization probe, a gene, designated ypt2, was isolated from the fission yeast Schizosaccharomyces pombe and found to encode a 200 amino acid long protein most closely related to the ypt branch of the ras superfamily . Disruption of the ypt2 gene is lethal . The bacterially produced ypt2 gene product is shown to bind GTP . A region of the ypt2 protein corresponding to but different from the 'effector region' of ras proteins is also different from that of ypt1 proteins of different species but identical to the 'effector loop' of the S.cerevisiae SEC4 gene product, a protein known to be required for vesicular protein transport . The S.pombe ypt2 gene under control of the S.cerevisiae GAL10 promoter is able to suppress the temperature-sensitive phenotype of a S . cerevisiae sec4 mutant, indicating a functional similarity of these GTP-binding proteins from the two very distantly related yeasts.

EMBO J, 1990 Jun, 9(6), 1949 - 55
The ryh1 gene in the fission yeast Schizosaccharomyces pombe encoding a GTP-binding protein related to ras, rho and ypt: structure, expression and identification of its human homologue; Hengst L et al.; A gene, ryh1, of the fission yeast Schizosaccharomyces pombe encoding a GTP-binding protein of 201 amino acids and belonging to the ras superfamily was isolated using the protein-coding region of the cloned Saccharomyces cerevisiae YPT1 gene as hybridization probe . The ryh1 gene is interrupted by three introns . ryh1 null mutants are viable but unable to grow at temperatures greater than 35.5 degrees C . Invertase of ryh1- cells is properly secreted but has a faster electrophoretic mobility compared to that of wild-type cells . The temperature-sensitive phenotype of ryh1 null mutants is complemented by the human rab6 cDNA expressed either under transcriptional control of the S.pombe adh or the SV40 early promoter.

Anal Biochem, 1990 May 15, 187(1), 94 - 7
A versatile microtiter assay for the universal cdc2 cell cycle regulator; Ducommun B et al.; A microassay for p34cdc2 based on the high affinity association between cdc2 and Schizosaccharomyces pombe p13suc1 has been developed . p13 purified from Escherichia coli was immobilized on microtiter plates and cellular lysate was incubated in the wells to allow the binding of cdc2 and its associated proteins . p34cdc2 was assayed either as a histone kinase or by immunological methods . The method was optimized for S . pombe cell extracts but can also be applied to other organisms such as Xenopus oocytes or HeLa cells . This rapid assay allows the specific determination of p34cdc2 histone H1 kinase activity in a very large number of samples.

Mol Gen Genet, 1990 May, 221(3), 403 - 10
Effects of seven different mutations in the pho1 gene on enzymatic activity, glycosylation and secretion of acid phosphatase in Schizosaccharomyces pombe; Schwaninger R et al.; Structural gene mutants of the cell-surface glycoprotein acid phosphatase of Schizosaccharomyces pombe were analysed to define structural determinants that are responsible for enzymatic activity, N-glycosylation and secretion . All seven defined mutations cause a single amino acid substitution in the mature acid phosphatase protein and destroy the enzymatic activity . The mutational lesions are distributed throughout the pho1 gene . A ser to phe substitution at position 349 abolishes enzymatic activity only and does not affect glycosylation and secretion . Two mutations create a new N-glycosylation site by substitution of pro at position 56 by phe and ser, respectively . This new site is apparently used in the mutants . Their core-glycosylated acid phosphatase is slightly larger than that of the wild type . Overglycosylation seems not to affect secretion . Four different mutations (a gly to asp substitution at position 281 and ser to phe substitutions at positions 150, 271 and 277) cause intracellular accumulation of enzymatically inactive core-glycosylated acid phosphatase precursor . These mutational lesions apparently block transport of acid phosphatase from the endoplasmic reticulum to the Golgi apparatus.

EMBO J, 1990 May, 9(5), 1417 - 22
Identification of ras-related, YPT family genes in Schizosaccharomyces pombe; Miyake S et al.; Screening for genes homologous to ras in Schizosaccharomyces pombe resulted in the isolation of a homolog of Saccharomyces cerevisiae YPT1 . This S . pombe gene, named ypt3, has a coding capacity of 214 amino acids interrupted by two introns, and is essential for cell growth . Two more YPT1 homologs were isolated from S . pombe using a part of the ypt3 gene as the probe . One of them, named ypt1, is highly homologous to S . cerevisiae YPT1 and mouse ypt1 and is essential for cell growth . This gene has four introns and encodes 203 amino acids . Its cDNA placed downstream of the S . cerevisiae GAL7 promoter could complement S . cerevisiae ypt1-, indicating that Sp ypt1 and Sc YPT1 are functionally homologous . The other isolate, named ryh1, and a fourth homolog, ypt2, have been characterized by Gallwitz and co-workers . The ypt1, ypt2 and ypt3 genes, but not ryh1, constitute a family, their products having double cysteine as their C terminus and serine in place of a glycine residue highly conserved in ras proteins (mammalian Gly-12 or S . pombe Gly-17) . The physiological roles of these genes appear to be distinct because each of them is indispensable for cell growth.

EMBO J, 1990 May, 9(5), 1407 - 15
The developmental fate of fission yeast cells is determined by the pattern of inheritance of parental and grandparental DNA strands; Klar AJ; A key feature for development consists of producing sister cells that differ in their potential for cellular differentiation . Following two cell divisions, a haploid Schizosaccharomyces pombe cell produces one cell in four 'granddaughters' with a changed mating cell type, implying nonequivalence of sister cells in each of two consecutive cell divisions . The observed pattern of switching is analogous to the mammalian 'stem cell' lineage by which a cell produces one daughter like itself while the other daughter is advanced in its developmental program . It is tested here whether sisters differ because of unequal distribution of cytoplasmic and/or nuclear components to them or due to inheriting a specific parental DNA chain at the mating type locus . Only the DNA strand-segregation model predicts that those cells engineered to contain an inverted tandem duplication of the mating type locus should produce equivalent sisters . Consequently, two 'cousins' in four related granddaughter cells should switch . The results verified the prediction, thus establishing that all cells otherwise fully possess the potential to switch . Therefore, the program of cell type change in S.pombe cell lineages is determined by the pattern of DNA strand inheritance at the mating type locus . A specific DNA sequence present at the mating type locus is postulated to be the cause of developmental asymmetry between sister cells . A general model for cellular differentiation is proposed in which the act of DNA replication itself is hypothesized to produce developmentally nonequivalent sister genomes.

Mol Cell Biol, 1990 May, 10(5), 2341 - 8
A gene family for acidic ribosomal proteins in Schizosaccharomyces pombe: two essential and two nonessential genes; Beltrame M et al.; We have cloned the genes for small acidic ribosomal proteins (A-proteins) of the fission yeast Schizosaccharomyces pombe . S . pombe contains four transcribed genes for small A-proteins per haploid genome, as is the case for Saccharomyces cerevisiae . In contrast, multicellular eucaryotes contain two transcribed genes per haploid genome . The four proteins of S . pombe, besides sharing a high overall similarity, form two couples of nearly identical sequences . Their corresponding genes have a very conserved structure and are transcribed to a similar level . Surprisingly, of each couple of genes coding for nearly identical proteins, one is essential for cell growth, whereas the other is not . We suggest that the unequal importance of the four small A-proteins for cell survival is related to their physical organization in 60S ribosomal subunits.

Mol Cell Biol, 1990 May, 10(5), 1863 - 72
Functional analysis of a centromere from fission yeast: a role for centromere-specific repeated DNA sequences; Clarke L et al.; A circular minichromosome carrying functional centromere sequences (cen2) from Schizosaccharomyces pombe chromosome II behaves as a stable, independent genetic linkage group in S . pombe . The cen2 region was found to be organized into four large tandemly repeated sequence units which span over 80 kilobase pairs (kb) of untranscribed DNA . Two of these units occurred in a 31-kb inverted repeat that flanked a 7-kb central core of nonhomology . The inverted repeat region had centromere function, but neither the central core alone nor one arm of the inverted repeat was functional . Deletion of a portion of the repeated sequences that flank the central core had no effect on mitotic segregation functions or on meiotic segregation of a minichromosome to two of the four haploid progeny, but drastically impaired centromere-mediated maintenance of sister chromatid attachment in meiosis I . This requirement for centromere-specific repeated sequences could not be satisfied by introduction of random DNA sequences . These observations suggest a function for the heterochromatic repeated DNA sequences found in the centromere regions of higher eucaryotes.

J Cell Sci, 1990 May, 96 ( Pt 1), 71 - 7
Distribution of tubulin and actin through the cell division cycle of the fission yeast Schizosaccharomyces japonicus var . versatilis: a comparison with Schizosaccharomyces pombe; Alfa CE et al.; Changes in the distribution of microtubules and F-actin through the cell division cycle of the fission yeast Schizosaccharomyces japonicus var . versatilis were investigated by fluorescence microscopy . The fluorescence images obtained with S . japonicus were markedly superior to those previously reported for S . pombe and revealed new details of cytoskeletal organization in this important genus . As in S . pombe, F-actin in S . japonicus was present as a concentration of 'dots' at the growing poles of interphase cells and as a filamentous equatorial ring directing the deposition of the cytokinetic septum . The transition between these two states occurred at late anaphase, in contrast to the situation in S . pombe where the appearance of the equatorial actin ring is tightly coupled to the early events of mitosis . During the course of cytokinesis in S . japonicus the actin ring constricted and broadened, suggesting that it is contractile . Microtubule organization in S . japonicus also revealed interesting differences from S . pombe . Whereas in S . pombe cytoplasmic microtubules are reinitiated from a pair of microtubule organizing centres (MTOCs) at the cell equator, in S . japonicus they arise by extensive microtubule growth from the spindle poles . Western blots of cell extracts enriched for tubulin by DEAE-Sephadex chromatography showed that, like S . pombe, S . japonicus contains two alpha-tubulins and a single beta-tubulin . Whilst the alpha 1- and beta-tubulins from the two species comigrated on one-dimensional polyacrylamide gels, the alpha 2 species were electrophoretically distinct . Although fundamental differences clearly exist between the two species, S . japonicus could prove to be a useful tool in basic studies of fission yeast cell biology.

Trends Genet, 1990 May, 6(5), 150 - 4
Centromeres of budding and fission yeasts; Clarke L; Centromeres of the budding yeast Saccharomyces cerevisiae are structurally relatively simple, are specified by only about 125 base pairs of DNA, and contain no repeated DNA sequences . The centromere regions in the fission yeast Schizosaccharomyces pombe span many kilobase pairs of DNA and contain repeated DNA sequences that appear to be necessary for full centromere function . A portion of the repeated sequences is organized into a large inverted repeated structure in the centromere region of each S . pombe chromosome . Fission yeast provides an excellent model system for studying the role of repeated DNA sequences in centromere function.

Biochem J, 1990 May 1, 267(3), 697 - 702
Purification and characterization of the invertase from Schizosaccharomyces pombe . A comparative analysis with the invertase from Saccharomyces cerevisiae; Moreno S et al.; Invertase (EC 3.2.1.26) was purified to homogeneity from exponentially growing cells of Schizosaccharomyces pombe fully de-repressed for synthesis of the enzyme, and was shown to be a high-molecular-mass glycoprotein that can be dissociated in the presence of 8 M-urea/1% SDS into identical subunits with an apparent molecular mass of 205 kDa . The carbohydrate moiety, accounting for 67% of the total mass, is composed of equimolar amounts of mannose and galactose . There is a small amount of glucosamine, which is probably involved in the linkage to the protein moiety, since the enzyme is sensitive to treatment with endoglycosidase H . The composition of the carbohydrate moiety resembles that found in higher-eukaryotic glycoproteins and differs from glycoproteins found in Saccharomyces cerevisiae . The protein portion of each subunit is a polypeptide of molecular mass 60 kDa, very similar to the invertase of Sacch . cerevisiae . Both proteins cross-react with antibodies raised against the protein fractions of the other, indicating that the two enzymes are similar.

J Cell Biol, 1990 May, 110(5), 1617 - 21
Intramitotic controls in the fission yeast Schizosaccharomyces pombe: the effect of cell size on spindle length and the timing of mitotic events; Hagan IM et al.; We have used a new cinemicroscopy technique in combination with antitubulin immunofluorescence microscopy to investigate the timing of mitotic events in cells of the fission yeast Schizosaccharomyces pombe having lengths at division between 7 and 60 microns . Wild-type fission yeast cells divide at a length of 14 microns . Separation of daughter nuclei (anaphase B) proceeds at a rate of 1.6 +/- 0.2 microns min-1, until the spindle extends the length of the cell . Coincident with spindle depolymerization, the nuclei reverse direction and take up positions that will become the center of the two daughter cells . This post-mitotic nuclear migration occurs at a rate of 1.4 +/- 0.5 microns-1 . In cells in which the weel+ gene is overexpressed fivefold and that have an average length at mitosis of 28 microns, the rate of nuclear separation was only slightly reduced but, as spindles in these cells measure 20-22 microns, the duration of anaphase B was extended by approximately 40% . By contrast, in the mutant weel.50, which divides at 7 microns, both the rate and duration of anaphase B were indistinguishable from wild type . Nuclei reach the ends of these cells earlier but remain there until a point corresponding to the time of postmitotic nuclear migration in wild type . Thus, the events of mitosis can be extended but not abbreviated . These results are discussed in terms of a mitotic termination control that monitors many different events, one of which is spindle elongation.

EMBO J, 1990 May, 9(5), 1423 - 30
The rhp6+ gene of Schizosaccharomyces pombe: a structural and functional homolog of the RAD6 gene from the distantly related yeast Saccharomyces cerevisiae; Reynolds P et al.; The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin conjugating enzyme and is required for DNA repair, DNA-damage-induced mutagenesis and sporulation . Here, we show that RAD6 and the rhp6+ gene from the distantly related yeast Schizosaccharomyces pombe share a high degree of structural and functional homology . The predominantly acidic carboxyl-terminal 21 amino acids present in the RAD6 protein are absent in the rhp6(+)-encoded protein; otherwise, the two proteins are very similar, with 77% identical residues . Like rad6, null mutations of the rhp6+ gene confer a defect in DNA repair, UV mutagenesis and sporulation, and the RAD6 and rhp6+ genes can functionally substitute for one another . These observations suggest that functional interactions between RAD6 (rhp6+) protein and other components of the DNA repair complex have been conserved among eukaryotes.

Mol Cell Biol, 1990 May, 10(5), 2261 - 8
A novel mammalian protein kinase gene (mak) is highly expressed in testicular germ cells at and after meiosis; Matsushime H et al.; We isolated a novel gene designated mak (male germ cell-associated kinase) by using weak cross-hybridization with a tyrosine kinase gene (v-ros) . Sequence analysis of the cDNA corresponding to the 2.6-kilobase transcript revealed that the predicted product of rat mak consisted of 622 amino acids and contained protein kinase consensus motifs in its amino-terminal region . Comparison of the deduced amino acid sequence of mak in the kinase domain with those of other protein kinase genes demonstrated that mak was approximately 40% identical to the cdc2-CDC28 gene family in Schizosaccharomyces pombe, Saccharomyces cerevisiae, and humans but less identical to most other protein kinase gene products . Expression of mak was highly tissue specific, and its transcripts were detected almost exclusively in testicular cells entering and after meiosis but hardly detectable in ovarian cells including oocytes, after the dictyotene stage . These results suggest that the mak gene plays an important role in spermatogenesis.

J Cell Sci, 1990 May, 96 ( Pt 1), 79 - 91
CO2 production after induction synchrony of the fission yeast Schizosaccharomyces pombe: the origin and nature of entrainment; Novak B et al.; Earlier work has shown that there is a periodic change in the rate of production of CO2 during the cell cycle of fission yeast and that this periodicity persists after a block to the DNA-division cycle and also after a block to protein synthesis . It appears that there is a periodic control or 'oscillator' affecting CO2 production that is normally closely entrained to the cell cycle, but which can 'free-run' after a block . In this paper, we examine what events in the DNA-division cycle can generate the entrainment signals and what is the nature of such signals . In the first set of experiments, CO2 production was measured by manometry during induction synchrony produced by blocking the DNA-division cycle in an asynchronous culture for a period and then releasing the block . Synchronous cell division occurs after the release with cell cycles shorter than normal . After release from a block imposed by shifting up the mutant cdc2.33 to the restrictive temperature, oscillations in CO2 production started rapidly and remained closely entrained to the division cycles (with slightly different patterns and timing from those after selection synchrony) . This showed that there was an entrainment signal but did not show whether it came from start, the S period or mitosis . A similar experiment with cdc10.129 showed that an early signal came from either start or the S period, as did an experiment with release from N-starvation . The results with cdc25.22 were similar to those with cdc2.33 . After a block with hydroxyurea, there was entrainment but with no signs of the early signal that occurred with cdc10 . This showed that the early signal came from start and not from the S period . In a second set of double-block experiments, the first block was followed by a second different block . With cdc25.22 followed by MBC (an inhibitor of nuclear division) the cells passed through a narrow window of the cell cycle between the transition point of cdc25.22 and mitosis . This was sufficient to start the oscillations, showing that an entrainment signal could be generated at about the time of mitosis . The results from using hydroxyurea followed by cdc2.33 showed no genuine oscillations, confirming the conclusion from the single hydroxyurea block . The results from using hydroxyurea followed by cdc10.129 confirmed the existence of a mitotic signal.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochim Biophys Acta, 1990 Apr 30, 1023(3), 380 - 2
Phenylmethylsulfonyl fluoride protects L-lysine transport in Schizosaccharomyces pombe against inactivation by ammonium ions; Horak J et al.; Ammonium ions inactivate the basic amino acid transport system in Schizosaccharomyces pombe in an irreversible manner . The inactivation is accompanied by a 4-fold decrease of KT of L-lysine transport, leaving its Jmax unchanged; phenylmethylsulfonyl fluoride protects the system against inactivation . In contrast, two basic amino acid transport systems in a gap1 mutant of Saccharomyces cerevisiae are influenced by NH4+ ions in such a way that only the Jmax decreases while the KT of L-lysine transport is unchanged . Phenylmethylsulfonyl fluoride does not act here as a protective agent.

Nucleic Acids Res, 1990 Apr 25, 18(8), 2025 - 32
Schizosaccharomyces U6 genes have a sequence within their introns that matches the B box consensus of tRNA internal promoters; Frendewey D et al.; The gene for the U6 small nuclear RNA (snRNA) in the fission yeast Schizosaccharomyces pombe is interrupted by an intron whose structure is similar to those found in messenger RNA precursors (pre-mRNAs) (1) . This is the only known example of a split snRNA gene from any organism--animal, plant, or yeast . To address the uniqueness of the S . pombe U6 gene, we have investigated the structures of the U6 genes from five Schizosaccharomyces strains and three other fungi . A fragment of the U6 coding sequence was amplified from the genomic DNA of each strain by the polymerase chain reaction (PCR) . The sizes of the PCR products indicated that all of the fission yeast strains possess intron-containing U6 genes; whereas, the U6 genes from the other fungi appeared to be uninterrupted . The sequences of the Schizosaccharomyces U6 gene fragments revealed that each had an intron of approximately 50 base pairs in precisely the same position . In addition to the splice sites and putative branch point regions, a sequence immediately upstream of the branch point consensus was found to be conserved in all of the Schizosaccharomyces U6 genes . This sequence matches the consensus for the B box of eukaryotic tRNA promoters . These results raise the interesting possibility that synthesis of U6 RNA in fission yeast might involve the use of internal promoter elements similar to those found in other genes transcribed by RNA polymerase III.

Nature, 1990 Apr 5, 344(6266), 549 - 52
Regulation of mitosis by cyclic accumulation of p80cdc25 mitotic inducer in fission yeast; Moreno S et al.; The coordination of somatic cell division with cell size must be accomplished by the accumulation of mitotic inducers or the dilution, in the course of cell growth, of mitotic inhibitors . In fission yeast (Schizosaccharomyces pombe), cell size at mitosis is determined by expression of the cdc25+ and nim1+ inducer genes and of the inhibitor gene wee1+, which between them regulate the M-phase protein kinase p34cdc2 . We now report that both the phosphoprotein product of cdc25+ (p80cdc25, with apparent relative molecular mass 80,000) and the corresponding messenger RNA increase in concentration as cells proceed through interphase, peaking at mitosis . We propose that the cell-cycle timing of mitosis in somatic cells is regulated by the cyclic accumulation of the cdc25 mitotic inducer, which on reaching a critical level results in activation of p34cdc2 protein kinase . Accumulation of this inducer could play a part in coordinating cell division with growth.

Mol Cell Biol, 1990 Apr, 10(4), 1432 - 8
Identification of Schizosaccharomyces pombe transcription factor PGA4, which binds cooperatively to Saccharomyces cerevisiae GAL4-binding sites; Ruden DM; When the DNA-binding site for the Saccharomyces cerevisiae transcription activator GAL4 is placed upstream of the Schizosaccharomyces pombe ADH1 TATA box, transcription of the ADH1 gene is activated in S . pombe in vivo by an endogenous transcription factor . In vitro studies show that this S . pombe protein, PGA4, binds specifically to DNA containing a GAL4 site and that when two GAL4 sites are present, this protein binds cooperatively . Cooperating binding of PGA4 to DNA is favored if the GAL4 sites are separated by an integral number of turns of the DNA helix.

Genetics, 1990 Apr, 124(4), 807 - 16
Isolation and characterization of mutants constitutive for expression of the fbp1 gene of Schizosaccharomyces pombe; Hoffman CS et al.; Transcription of the fbp1 gene of Schizosaccharomyces pombe, encoding fructose-1,6-bisphosphatase, is glucose repressible . We have constructed two hybrid genes, containing the fbp1 promoter, that allow selection for mutations that alter transcriptional regulation of fbp1 . Strains carrying fbp1-ura4 and fbp1-lacZ fusions are phenotypically Ura-, resistant to 5-fluoro-orotic acid, and express a low level of beta-galactosidase activity when grown under repressing conditions (8% glucose) . By selecting for Ura+ strains grown under repressing conditions, we have isolated 187 independent mutants that constitutively express the fbp1-ura4 fusion . These mutants identify ten complementation groups that represent ten unlinked git (glucose insensitive transcription) genes . The git gene products are required in trans for glucose repression of expression from the fbp1 promoter since these mutations also alter expression of the fbp1-lacZ fusion . We have shown that transcription of the wild type fbp1 gene in most git mutants is elevated to a level consistent with the increased expression of the fbp1-lacZ hybrid gene . Mutations in some git genes confer additional phenotypes such as slow growth, temperature-sensitive lethality and reduced spore viability . Therefore, some of these genes are likely to encode factors that are of general importance for S . pombe transcription.

Nature, 1990 Mar 22, 344(6264), 355 - 7
Homologous activators of ras in fission and budding yeast; Hughes DA et al.; The ras proto-oncogene products are plasma membrane-bound, guanine nucleotide-binding proteins implicated in signal transduction across the plasma membrane . But the signal(s) that activates the ras pathway(s) is not known . In the budding yeast Saccharomyces cerevisiae, the CDC25 gene product acts upstream of Ras proteins, but it has not been clear whether CDC25 function is unique to the S . cerevisiae ras pathway . Here we report that the ste6 gene of fission yeast Schizosaccharomyces pombe is a homologue of CDC25: the ste6 gene product and the CDC25 gene product have significant amino-acid similarity in their C-terminal regions . Like the S . pombe ras1 gene, ste6 is essential for mating . Epistatic interactions indicate that the ste6 gene functions upstream of ras1 . We propose that ste6 and CDC25 activate Ras protein through a common mechanism, perhaps by promoting GDP-GTP exchange, even though it seems that the function of Ras protein in budding yeast differs from that in fission yeast . Homologues of ste6 and CDC25 could regulate ras activity in other eukaryotic cells.

Exp Cell Res, 1990 Mar, 187(1), 150 - 6
Effects of leptomycin B on the cell cycle of fibroblasts and fission yeast cells; Yoshida M et al.; An antifungal antibiotic, leptomycin B (LMB), which induced cell elongation of fission yeast, Schizosaccharomyces pombe, was found to be a unique inhibitor of the cell cycle of mammalian and fission yeast cells . Proliferation of rat 3Y1 fibroblasts was reversibly blocked by LMB in both the G1 and G2 phases and the treated cells were presumably introduced into the resting state (GO) . After removal of LMB, proliferative tetraploid cells were produced from the cells which had been arrested by LMB at the G2 phase, as a result of DNA replication without passage through the M phase . LMB also inhibited the proliferation of S . pombe in both the G1 and G2 phases . These results suggest that the molecular target of LMB is one of the components necessary for progression of both G1 and G2 in the eukaryotic cell cycle.

FEBS Lett, 1990 Feb 26, 261(2), 413 - 8
Adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is not regulated by guanyl nucleotides; Engelberg D et al.; The adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is localized to the plasma membrane of the cell . The enzyme utilizes Mn2+/ATP as substrate and free Mn2+ ions as an effector . Unlike the baker yeast Saccharomyces cerevisiae, S . pombe adenylyl cyclase does not utilize Mg2+/ATP as substrate and the activity is not stimulated by guanyl nucleotides . The optimal pH for the S . pombe adenylyl cyclase activity is 6.0 . The activity dependence on ATP is cooperative with a Hill coefficient of 1.68 +/- 0.14.

J Mol Biol, 1990 Feb 20, 211(4), 699 - 712
Structural analysis of the internal transcribed spacer 2 of the precursor ribosomal RNA from Saccharomyces cerevisiae; Yeh LC et al.; Full-length precursor ribosomal RNA molecules (6440 bases) were produced in vitro using a plasmid containing the yeast 35 S pre-rRNA operon under the control of phage T7 promoter . The higher-order structure of the internal transcribed spacer 2 (ITS-2) region (between the 5.8 S and 25 S rRNA sequence) in the pre-rRNA molecule was investigated using a combination of enzymatic and chemical structural probes . The data were used to evaluate several structural models predicted by a minimum free-energy calculation . The results supported a model in which the 3' end of the 5.8 S rRNA and the 5' end of the 25 S rRNA are hydrogen-bonded better than the one in which the ends are not . The model contains a high degree of secondary structure with several stable hairpins . Similar structural models for the ITS-2 regions of Schizosaccharomyces pombe, Saccharomyces carlsbergensis, mung bean and Xenopus laevis were derived . Certain common folding features appear to be conserved, in spite of extensive sequence divergence . The yeast model should be useful as a prototype in future investigations of the structure, function and processing of pre-rRNA.

Gene, 1990 Feb 14, 86(2), 257 - 61
An inducible expression vector for both fission and budding yeast; Picard D et al.; We have developed a vector system for inducible gene expression in both fission yeast (Schizosaccharomyces pombe) and budding yeast (Saccharomyces cerevisiae) . The autonomously replicating expression vector contains multiple glucocorticoid response elements, rendering a linked promoter inducible 20-70-fold by glucocorticoid hormones in the presence of the mammalian glucocorticoid receptor . A polylinker with several unique cloning sites allows insertion of cDNAs of interest . Glucocorticoids are gratuitous signalling molecules in yeast, exerting little or no effect on the expression of genes other than those fused to the regulated promoter.

EMBO J, 1990 Feb, 9(2), 525 - 34
A mutation in a single gene of Schizosaccharomyces pombe affects the expression of several snRNAs and causes defects in RNA processing; Potashkin J et al.; A bank of temperature sensitive (ts-) mutants of Schizosaccharomyces pombe was screened for snRNA expression mutants using an oligodeoxynucleotide that recognizes U2 RNA . One mutant with a novel phenotype was identified that has reduced steady-state levels of the spliceosomal snRNAs U1, U2, U4, U5 and U6 . In addition, the mutant exhibits a temperature-dependent accumulation of aberrant U2 and U4 transcripts elongated at their 3' end . The steady-state concentration of the RNA component of RNase P is also reduced in the mutant, whereas the amount of U3 RNA, 7SL RNA, tRNA, rRNA and mRNA are the same as wild-type . Pre-mRNA, pre-tRNA and U6 RNA precursor processing are impaired in the mutant . Genetic analysis demonstrates that the snRNA defects are tightly linked to the ts- growth defect and are recessive . We have named this mutant snm1 to indicate a defect in snRNA maintenance . The data on snm1 suggest that a single trans-acting factor is essential for the maintenance of steady-state levels of several snRNAs and for proper 3' end formation of U2 and U4 RNAs.

J Cell Biol, 1990 Feb, 110(2), 417 - 25
In vitro reactivation of spindle elongation in fission yeast nuc2 mutant cells; Masuda H et al.; To investigate the mechanisms of spindle elongation and chromosome separation in the fission yeast Schizosaccharomyces pombe, we have developed an in vitro assay using a temperature-sensitive mutant strain, nuc2 . At the restrictive temperature, nuc2 cells are arrested at a metaphase-like stage with short spindles and condensed chromosomes . After permeabilization of spheroplasts of the arrested cells, spindle elongation was reactivated by addition of ATP and neurotubulin both at the restrictive and the permissive temperatures, but chromosome separation was not . This suggests that the nuc2 cells are impaired in function at a stage before sister chromatid disjunction . Spindle elongation required both ATP and exogenous tubulin and was inhibited by adenylyl imidodiphosphate (AMPPNP) or vanadate . The ends of yeast half-spindle microtubules pulse-labeled with biotinylated tubulin moved past each other during spindle elongation and a gap formed between the original half-spindles . These results suggest that the primary mechanochemical event responsible for spindle elongation is the sliding apart of antiparallel microtubules of the two half-spindles.

Mol Cell Biol, 1990 Feb, 10(2), 549 - 60
Schizosaccharomyces pombe ras1 and byr1 are functionally related genes of the ste family that affect starvation-induced transcription of mating-type genes; Nadin-Davis SA et al.; We have further investigated the function of the ras1 and byr1 genes, which were previously shown to be critical for sexual differentiation in fission yeast cells . Several physiological similarities between strains containing null alleles of these genes supports the idea that ras1 and byr1 are functionally closely related . Furthermore, we have found that byr1 is allelic to ste1, one of at least 10 genes which when mutated can cause sterility . Since ras1 had previously been found to be allelic to ste5, both ras and byr genes are now clearly shown to be a part of the ste gene family, thus confirming their close functional relationship . The observation that the mating-type loci could overcome the sporulation block of ras1 and byr1 mutant strains prompted investigation of the role of the ras-byr pathway in the induction of the mating-type gene transcripts upon nitrogen starvation . By Northern analysis of RNA preparations from strains carrying wild-type or mutant ras1 alleles and grown to different stages of the growth cycle, we have shown that ras1 plays an important role in inducing the Pi transcript of the mating-type loci and the mei3 gene transcript . These observations provide a molecular basis for the role of the ste gene family, including ras1 and byr1, in meiosis and indicate that further characterization of other ste genes would be very useful for elucidating the mechanism of ras1 function in fission yeast cells.

Mol Biochem Parasitol, 1990 Jan 15, 38(2), 169 - 73
A molecular karyotype of Eimeria tenella as revealed by contour-clamped homogeneous electric field gel electrophoresis; Shirley MW et al.; DNA from sporozoites of Eimeria tenella was resolved by pulsed field gel electrophoresis into nine chromosomal bands . Some bands of this molecular karyotype contained more than one chromosome as determined by the relative intensity of both staining with ethidium bromide and hybridisation to an E . tenella telomeric probe . Haploid forms of the parasite must be presumed to contain at least 12 chromosomes . The two smallest chromosomes were about 1.1 and 1.4 megabases . Most chromosomes were in excess of 3 Mb with the largest over 5 Mb as determined by comparison with the co-migration of chromosomes from Schizosaccharomyces pombe . A 5S ribosomal gene probe hybridised to a single chromosomal band.

Biochem Biophys Res Commun, 1990 Jan 15, 166(1), 113 - 8
Electrofusion of oriented Schizosaccharomyces pombe cells through apical protoplast-protuberances; Vondrejs V et al.; The electrofusion of oriented Schizosaccharomyces pombe cells through apical protoplast-protuberances was demonstrated . The protuberances arose after an exposure of early-exponential phase cells to digestive enzymes from hepatopancreas of Helix pomatia . The orientation of cylindric cells within pearl chains was produced by the application of inhomogenous alternating electric fields.

J Mol Biol, 1990 Jan 5, 211(1), 7 - 9
An intron-containing Schizosaccharomyces pombe U6 RNA gene can be transcribed by human RNA polymerase III; Kleinschmidt AM et al.; A Schizosaccharomyces pombe U6 small nuclear RNA gene containing an intron has been described . We find that the S . pombe U6 gene is transcribed in a human (HeLa) cell S100 extract with an alpha-amanitin sensitivity characteristic of RNA polymerase III . The S . pombe U6 gene is also transcribed after transfection into human cells . The transcription of vertebrate U6 RNA genes by RNA polymerase III does not require intragenic control elements . The intron of the S . pombe U6 gene disrupts a "box A"-like intragenic sequence that is typically an RNA polymerase III transcription control element . This, together with the transcription of the S . pombe U6 gene by human RNA polymerase III, suggests that it is recognized by human U6 gene-specific transcription machinery.

Mol Gen Genet, 1990 Jan, 220(2), 314 - 6
Expression of the beta-glucuronidase gene under the control of the CaMV 35s promoter in Schizosaccharomyces pombe; Pobjecky N et al.; We have transformed Schizosaccharomyces pombe with the beta-glucuronidase (GUS) gene from Escherichia coli under the control of the plant cauliflower mosaic virus (CaMV) 35S promoter element . Efficient expression of GUS enzyme was observed . Moreover, transcription initiated at a unique site identical to that used in plant cells.

Curr Genet, 1990 Jan, 17(1), 13 - 9
Construction of an h+S strain of Schizosaccharomyces pombe; Heim L; By spontaneous in vivo integration of a mat2:1 degrees plasmid, containing a Plus (P) cassette, into an h-L MT region of Schizosaccharomyces pombe an h+ strain was obtained which neither mutates to h- nor to h90 . Southern blotting showed that it possesses the same mating-type (MT) configuration as h-S except that P information resides in both cassettes . Therefore the strain was called h+S . By crossing h+S with the h- strain LK42 of Engelke et al . (1987) it was possible to obtain h- recombinants with the MT configuration mat1:1(M)smt-o-L-mat2:3(P) . Because of the totally defective smt signal (smt-o) in these recombinants no MT switching occurs, so that M information is conserved in mat1:1; furthermore the cassette mat2:3(P) is not expressed like in strains with a K region . This proves that the K region does not cause the silencing of mat2:3(P).

Ciba Found Symp, 1990, 150, 168 - 77; discussion 177-83
Controls of cell proliferation in yeast and animals; Norbury C et al.; Genetic studies using fission yeast (Schizosaccharomyces pombe) have identified a gene, cdc2, whose product (p34cdc2) is a protein kinase required for traversal of both the G1 and G2 cell cycle control points . Genetic complementation has been used to demonstrate that p34cdc2 homologues are functionally and structurally conserved in distantly related eukaryotes, and p34cdc2-related proteins are components of both maturation-promoting factor (MPF) and the M phase (growth-associated) histone H1 kinase . The p34cdc2 homologues of multicellular eukaryotes undergo potentially regulatory phosphorylation changes through the cell cycle . Phosphorylation on serine during late G1 is accompanied by a significant increase in p34cdc2 kinase activity which, by analogy with fission yeast, may betray a function related to control over entry into S phase . Phosphorylation on threonine and tyrosine in G2 precedes dephosphorylation of these residues during kinase hyperactivation and entry into mitosis . In addition, long-term control of expression of mammalian p34cdc2 homologues is likely to be exerted at the transcriptional level . These observations provide the framework of a universal model for the control of eukaryotic cell proliferation, in which the p34cdc2 protein kinase integrates multiple cues to signal the initiation of S phase and, subsequently, mitosis.

Ciba Found Symp, 1990, 150, 262 - 71; discussion 271-8
Cellular proteins that are targets for transformation by DNA tumour viruses; Buchkovich K et al.; Small DNA tumour viruses produce proteins that redirect cellular gene expression and growth control . The E1A polypeptides of adenovirus perform the functions of transcriptional activation and cellular transformation . These two functions are carried out by different domains within the E1A protein . The E1A protein associates with several cellular proteins, including the product of the retinoblastoma gene, pRb-1 . Mutational analysis correlates transformation with the sites required for binding pRb and two other cellular proteins, p107 and a 300 kDa polypeptide . This correlation suggests that these proteins are targets for E1A-mediated transformation . Transforming proteins from other small DNA tumour viruses interact with pRb, raising the possibility that a common event in viral transformation is the inactivation of proteins that inhibit cellular proliferation . The role of the E1A-associated 60 kDa protein, p60, in transformation is being investigated . In the absence of E1A, p60 binds to the human homologue of the Schizosaccharomyces pombe cdc2 gene product, p34, to form a complex that has kinase activity that oscillates during the cell cycle . Ongoing studies of the effect of adenovirus infection, and specifically E1A expression, on this cellular kinase may provide clues to how E1A overcomes cell cycle controls and transforms cells.

Dev Suppl . 1990;:3-8.
Regulation of fission yeast mating-type interconversion by chromosome imprinting; Klar AJ; Mating types of the fission yeast Schizosaccharomyces pombe interchange nonrandomly in a cell lineage so that only one cell among four granddaughters of a cell ever switches, and the sister of the newly switched cell switches efficiently in consecutive cell divisions, thereby producing chains of recurrent switching . The programme of cellular differentiation is mediated by inheritance of parental DNA chains at the mating type locus (mat1) by progeny cells . This review summarizes recent results suggesting that two types of imprinting events at the mat1 locus are required to generate the specific pattern of switching in a cell lineage . One of those is a site- and strand- specific event that is required before the mat1 locus can be cleaved in vivo . The other is a double-stranded break at mat1 that is healed by gene conversion in the progeny cells resulting in switching the mat1 locus.

New Biol, 1990 Jan, 2(1), 10 - 9
Centromere structure and function in budding and fission yeasts; Carbon J et al.; Functional centromeric DNAs have now been isolated and characterized from both budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeasts . Artificial chromosomes containing these centromere DNA sequences segregate faithfully in both mitotic and meiotic cell divisions, but only in the parent organism . Structure-function analyses have revealed surprising fundamental differences between these two centromere classes . In the budding yeast centromeres, a 125-bp consensus DNA sequence contains all the information needed in cis to provide proper chromosome segregation . In contrast, the fission yeast centromeres each contain a long run (40 to 100 kb) of untranscribed repetitive DNA sequences arranged into a large inverted repeat, most of which is required for full centromere function . The fission yeast centromere-kinetochore appears to be a highly relevant experimental model for analysis of the mechanism of chromosome segregation in higher eukaryotes, in which the centromere regions often contain megabases of transcriptionally silent repetitive DNA sequences of unknown function.

EMBO J, 1989 Dec 20, 8(13), 4281 - 8
A selection for mutants of the RNA polymerase III transcription apparatus: PCF1 stimulates transcription of tRNA and 5S RNA genes; Willis I et al.; A genetic approach has been developed to study transcription by RNA polymerase III . A pair of Schizosaccharomyces pombe nonsense suppressor tRNA genes were arranged in tandem such that expression of the downstream (supS1) tRNA suppressor was dependent upon transcription initiated by the internal promoter of the upstream (sup9-e) gene . Dominant mutant strains of Saccharomyces cerevisiae were isolated that suppress in trans the effect of an A block promoter mutation (A19) in the sup9-e gene and restore supS1 suppressor activity . Fifteen mutant strains, eight of which were independently isolated, all have elevated steady-state levels of sup9-e A19 RNA consistent with an increase in gene transcription . Extracts of a strain carrying the dominant mutant gene, PCF1, show a general 6-fold stimulation in transcription of mutant (A19) and wild-type tRNA genes and increase 5S gene transcription 4-fold compared with extracts from a wild-type strain . A transcription factor exclusion assay was used to show that the PCF1 mutation affects two distinct stages in transcription: one prior to and one after stable complex formation; and that these effects are mediated by a component of the stable complex . Further evidence of an effect during complex assembly was obtained in a time-course experiment that showed a shortened lag phase in the PCF1 extract . The results indicate that PCF1 is either a component of the stable complex or a positive regulator of its activity.

Nature, 1989 Dec 14, 342(6251), 830 - 3
The yeast SWI4 protein contains a motif present in developmental regulators and is part of a complex involved in cell-cycle-dependent transcription; Andrews BJ et al.; Transcription of the HO gene of Saccharomyces cerevisiae, which encodes a site-specific endonuclease that initiates cell-type switching (reviewed in refs . 1,2), is restricted to a short window of the cell cycle in late G1 (refs 3,4) . A repeated element in the upstream region of HO (the cell-cycle box, CCB) and two regulatory proteins, SWI4 and SWI6, are required for cell-cycle-dependent expression of HO . Biochemical experiments have identified a factor, CCBF (cell-cycle box factor), that binds to the CCB elements and that presumably plays a key part in cell-cycle regulation of HO . The SWI4 and SWI6 genes are required for formation of the CCBF-DNA complex . Here we report the nucleotide sequence of the SWI4 gene and show that it contains two copies of the conserved SWI6-cdc10 motif observed in SWI6 of budding yeast, the Schizosaccharomyces pombe cdc10 gene required for progression through G19, the Drosophila Notch gene, and in the Caenorhabditis elegans lin-12 and glp-1 genes . We demonstrate by using antibodies to the SWI4 protein in gel-shift assays that the protein is present in the CCBF-DNA complex.

Gene, 1989 Dec 14, 84(2), 473 - 9
A transcriptionally regulated expression vector for the fission yeast Schizosaccharomyces pombe; Hoffman CS et al.; An expression vector for the fission yeast Schizosaccharomyces pombe is described . The vector is designed to facilitate the construction of transcriptional fusions to the promoter of the S . pombe fructose bisphosphatase gene . Transcription from this promoter is regulated by glucose repression over a range of greater than 100-fold . The tight regulation by this promoter should allow for the maintenance of genes whose products are lethal to S . pombe and for the high level production of their protein or RNA products . Intermediate levels of expression can also be achieved by growth on different carbon sources.

Proc Natl Acad Sci U S A, 1989 Dec, 86(24), 9737 - 41
Substitution at position 116 of Schizosaccharomyces pombe calmodulin decreases its stability under nitrogen starvation and results in a sporulation-deficient phenotype; Takeda T et al.; We constructed Schizosaccharomyces pombe strains that carry phenylalanine, instead of arginine, as residue 116 of calmodulin by site-directed mutagenesis of the cam1 gene . Whereas haploid strains carrying the mutant allele, designated cam1-F116, exhibit no defects in growth and mating, diploid strains homozygous for cam1-F116 are deficient in sporulation . The four nuclei generated by the two serial meiotic divisions are not encapsulated in these diploids . The mutation is recessive . Semiquantitative analysis using polyclonal antibodies showed that vegetatively growing cam1-F116 cells have a smaller amount of calmodulin than wild-type cells . The quantitative difference becomes more remarkable if the cells are starved for nitrogen, which is a condition for induction of sporulation . In addition to this in vivo observation, we showed in vitro that the mutant protein is susceptible to a proteolytic activity induced by nitrogen starvation that hardly affects the wild-type calmodulin . Thus, the sporulation deficiency of the cam1-F116 mutant may be ascribed to shortage of calmodulin due to proteolysis of the mutant molecules under nitrogen starvation . Two other mutations at position 116 resulted in similar but leakier Spo- phenotypes.

J Cell Sci, 1989 Dec, 94 ( Pt 4), 657 - 62
The first transition point of the mutant cdc2.33 in the fission yeast Schizosaccharomyces pombe; Novak B et al.; We show that the first of the two transition points of cdc2.33, a mutant of Schizosaccharomyces pombe, exists in exponential phase cells . Using flow cytometry and a double-block experiment, we have measured the position of this transition point both in the single mutant and in the double mutant cdc2.33 wee1.6 . In the single mutant, this point is in early G1 . In the double mutant, however, this point is only delayed slightly, if at all, despite much larger delays in the S period and in the transition point of cdc10, another 'start' mutant . There is therefore a significant dissociation in the timing of what are thought to be two start events, and the first one appears not to be subject to a size control and to be associated with the completion of mitosis rather than the G1/S boundary.

J Cell Sci, 1989 Dec, 94 ( Pt 4), 647 - 56
Dynamics of cytoplasmic organelles in the cell cycle of the fission yeast Schizosaccharomyces pombe: three-dimensional reconstruction from serial sections; Kanbe T et al.; Changes in the ultrastructure of the fission yeast Schizosaccharomyces pombe during the cell division cycle were analyzed by three-dimensional reconstruction of serial section electron micrographs of freeze-substituted cells . Cytoplasmic vesicles were found at the cell ends during interphase and at the equatorial zone of cells undergoing cytokinesis . Filasomes behaved in a similar but temporally retarded way to vesicles . Microfilament(mf)-associated granules were found attached to the plasma membrane at the growing ends . Microfilaments were identified against the plasma membrane and adjacent to developing septa . From these observations it is suggested that mf-associated structures such as filasomes constitute dense knots of actin network that function in localized cell wall growth by controlling the deposition of cytoplasmic vesicles . Dictyosomes occur as tubular and fenestrated cisternae with associated cytoplasmic vesicles . They were distributed uniformly in the cytoplasm and did not change significantly during the cell cycle . Changes in the three-dimensional localization of cytoplasmic microtubules and mitochondria are also described.

J Cell Sci, 1989 Dec, 94 ( Pt 4), 635 - 46
Actin is associated with the formation of the cell wall in reverting protoplasts of the fission yeast Schizosaccharomyces pombe; Kobori H et al.; To clarify the involvement of actin in the formation of the yeast cell wall, reverting protoplasts of Schizosaccharomyces pombe were used as a simple model system . Actin of reverting protoplasts was labeled with rhodamine-conjugated phalloidin and observed by conventional fluorescence microscopy and laser scanning confocal microscopy . A close spatial as well as temporal relationship between actin and cell wall formation was observed in protoplast reversion . That is, the site of actin 'dots' in the reverting protoplasts coincided with the site of new wall formation and the timing of rearrangement of actin coincided with the initiation of cell wall formation and with the timing of cell wall expansion . Treatment of reverting protoplasts with cytochalasin D (CD) further clarified the close relationship between actin and cell wall organization . The effect of CD was dose dependent . A high dose of CD caused the absence of actin as well as the complete inhibition of cell wall formation . A low dose of CD caused weakly stained unlocalized actin, which induced grossly aberrant cell wall deposition as well as substantial changes in the morphology of the reverting protoplasts . These results demonstrated that actin is associated with initiation of cell wall formation, the proper deposition of cell wall materials, and maintaining the normal morphology of reverting protoplasts . Scanning electron microscopy revealed the presence of a fibrillar net structure on the surface of non-treated control reverting protoplasts . However, the absence of a fibrillar network on the surface of reverting protoplasts was observed in the presence of a high concentration of CD . Lack of localization of microfibrils as well as poor development of the fibrillar network were also observed in the presence of a low concentration of CD . Recovery experiments confirmed the close relationship between actin and cell wall formation.

Curr Genet, 1989 Dec, 16(5-6), 361 - 7
A ribosomal protein gene family from Schizosaccharomyces pombe consisting of three active members; Gatermann KB et al.; Recently, we have reported the isolation and characterization of a ribosomal protein gene from the fission yeast Schizosaccharomyces pombe . This gene was called K37 . Here we describe the isolation of two genes which are related to the K37 gene . Sequence analysis of these genes revealed open reading frames encoding proteins which are almost identical to the ribosomal protein K37 . Furthermore, all three genes are functional as determined by Northern analysis using transformed and wild type cells . The results indicate that S . pombe contains a ribosomal protein gene family, designated the K-family, consisting of three active members . The promoter regions of the three members are compared and several common motifes are identified which might serve as transcriptional activators in these genes.

Mol Cell Biol, 1989 Dec, 9(12), 5617 - 22
Characterization of the Schizosaccharomyces pombe ral2 gene implicated in activation of the ras1 gene product; Fukui Y et al.; Mutations in the Schizosaccharomyces pombe ral2 gene cause a phenotype indistinguishable from that of the ras1-defective mutant . Using cloned ral2 DNA, we disrupted the chromosomal gene . The disruptants showed the same phenotype as the original ral2 isolates, i.e., they had spherical cells, had no detectable mating activity, and exhibited no response to the mating pheromone, but their vegetative growth was apparently normal . Sequence analysis of the ral2 gene suggests that it encodes a polypeptide of 611 amino acid residues whose predicted amino acid sequence shows no strong homology to any known protein . Either multiple copies or even a single copy of the ras1Val-17 allele, which is an activated form of ras1, restored rodlike cell morphology and ability to respond to the mating factor to ral2 mutants . These results suggest that the ral2 and ras1 gene products interact intimately and that the ral2 gene product is involved in activation of the ras1 protein in S . pombe.

Mol Gen Genet, 1989 Dec, 220(1), 95 - 101
Cauliflower mosaic virus promoters direct efficient expression of a bacterial G418 resistance gene in Schizosaccharomyces pombe; Gmunder H et al.; A system is presented for transformation of the fission yeast Schizosaccharomyces pombe to resistance against the antibiotic G418 . The bacterial resistance gene of the transposon Tn5 is expressed under the control of promoters and transcription terminators from cauliflower mosaic virus (CaMV) . The promoter of the S . pombe alcohol dehydrogenase gene has also been used . Transformants can be selected directly on medium containing G418 (up to 1 mg/ml) due to inactivation of G418 by the Tn5 gene product, the aminoglycoside 3'-phosphotransferase (II) . The plant viral promoter 35S confers higher resistance to G418 than the 19S promoter . This corresponds to the relative strengths of these promoters in plant cells . The strong plant promoter 35S yields resistance comparable to that obtained with the strong S . pombe promoter from the alcohol dehydrogenase gene . The constructions with the two plant promoters have been used on multicopy shuttle plasmids that replicate autonomously in S . pombe and Escherichia coli . In addition the 35S and the 19S constructions have been inserted into the S . pombe genome where they confer G418 resistance as single copy genes . Since vector sequences are excluded in this case, all the necessary signals for expression of G418 resistance are contained within the DNA fragments containing the plant promoters, the resistance gene and the plant terminators . This transformation system is independent of S . pombe mutants . It may be useful for the transformation of other lower eukaryotes . The activity of the CaMV promoters in S . pombe may be exploited for the expression of plant genes in fission yeast.

J Cell Biol, 1989 Dec, 109(6 Pt 2), 3223 - 30
Saccharomyces cerevisiae and Schizosaccharomyces pombe contain a homologue to the 54-kD subunit of the signal recognition particle that in S . cerevisiae is essential for growth; Hann BC et al.; We have isolated and sequenced genes from Saccharomyces cerevisiae (SRP54SC) and Schizosaccharomyces pombe (SRP54sp) encoding proteins homologous to both the 54-kD protein subunit (SRP54mam) of the mammalian signal recognition particle (SRP) and the product of a gene of unknown function in Escherichia coli, ffh (Romisch, K., J . Webb, J . Herz, S . Prehn, R . Frank, M . Vingron, and B . Dobberstein . 1989 . Nature (Lond.) . 340:478-482; Bernstein H . D., M . A . Poritz, K . Strub, P . J . Hoben, S . Brenner, P . Walter . 1989 . Nature (Lond.) . 340:482-486) . To accomplish this we took advantage of short stretches of conserved sequence between ffh and SRP54mam and used the polymerase chain reaction (PCR) to amplify fragments of the homologous yeast genes . The DNA sequences predict proteins for SRP54sc and SRP54sp that are 47% and 52% identical to SRP54mam, respectively . Like SRP54mam and ffh, both predicted yeast proteins contain a GTP binding consensus sequence in their NH2-terminal half (G-domain), and methionine-rich sequences in their COOH-terminal half (M-domain) . In contrast to SRP54mam and ffh the yeast proteins contain additional Met-rich sequences inserted at the COOH-terminal portion of the M-domain . SRP54sp contains a 480-nucleotide intron located 78 nucleotides from the 5' end of the open reading frame . Although the function of the yeast homologues is unknown, gene disruption experiments in S . cerevisiae show that the gene is essential for growth . The identification of SRP54sc and SRP54sp provides the first evidence for SRP related proteins in yeast.

J Cell Biol, 1989 Dec, 109(6 Pt 1), 2693 - 702
A galactosyltransferase from the fission yeast Schizosaccharomyces pombe; Chappell TG et al.; A membrane-associated galactosyltransferase has been purified to homogeneity from the fission yeast, Schizosaccharomyces pombe . The enzyme has a molecular weight of 61,000 and is capable of transfering galactose from UDP-galactose (UDP-Gal) to a variety of mannose-based acceptors to form an alpha-1,2 galactosyl mannoside linkage . Immunofluorescence localization of the protein is consistent with the presence of the enzyme in the Golgi apparatus of S . pombe . This, together with the presence of terminal, alpha-linked galactose on the N-linked oligosaccharides of S . pombe secretory proteins, suggests that the galactosyltransferase is an enzyme involved in the processing of glycoproteins transported through the Golgi apparatus in fission yeast.

J Biol Chem, 1989 Nov 25, 264(33), 19577 - 82
Biochemical characterization of the p34cdc2 protein kinase component of purified maturation-promoting factor from Xenopus eggs; Erikson E et al.; Genetic studies in the fission yeast Schizosaccharomyces pombe and biochemical data in oocytes and eggs of Xenopus laevis have implicated the product of the cdc2+ gene as critical for the G2 to M transition in the cell cycle . The product of the cdc2+ gene is a 34-kDa serine/threonine protein kinase, designated p34cdc2, that is a component of purified maturation-promoting factor (MPF) and also of purified mammalian growth-associated histone H1 kinase . The biochemical properties of p34cdc2 H1 kinase activity in the MPF complex were studied . Phosphorylation of the p45cyclin component in the MPF complex by p34cdc2 exhibited kinetics consistent with an intramolecular reaction . On glycerol gradient centrifugation, MPF kinase against several substrates sedimented with an apparent Mr = 45,000-55,000 . p34cdc2 was found to utilize ATP, GTP, and adenosine 5'-O-(3-thiotriphosphate) with apparent Km values of 75, 700, and 250 microM, respectively . The kinase activity was inhibited by beta-glycerophosphate, NaF, and zinc, whereas p-nitrophenyl phosphate was slightly stimulatory . The relative rates of phosphorylation of various substrates by MPF and growth-associated H1 kinase were similar . These findings should prove useful in further work on the regulation of MPF kinase activity and characterization of its substrates.

J Biol Chem, 1989 Nov 15, 264(32), 19221 - 7
Interaction of yeast transcription factor IIIC with dimeric Schizosaccharomyces pombe tRNA(Ser)-tRNA(Met) genes; Johnson DL et al.; A unique tRNA(Ser)-tRNA(Met) tandem gene arrangement was characterized previously from Schizosaccharomyces pombe . Three alleles exist in which a tRNA(Ser) gene is separated by 7 base pairs from an initiator tRNA(Met) gene . Promotion of transcription occurs only within the tRNA(Ser) gene, yielding a dimeric precursor transcript . Using nuclease protection and gel retardation assays, we have analyzed how the Saccharomyces cerevisiae RNA polymerase III transcription factor C (TFIIIC) interacts with this dimeric gene template . The primary interaction site of TFIIIC with the tRNA(Ser) gene is at the 3'-internal control region (ICR), which can be distinguished kinetically from its weaker interaction with the 5'-ICR of the gene . We examined a variety of point mutations and double mutations within the tRNA(Ser) gene which reduce transcription . We found that changes in highly conserved nucleotides within the ICRs reduce TFIIIC binding up to 7-fold compared with the parent suppressor gene . The interaction of TFIIIC with the tRNA(Ser) gene does not sterically prevent stable binding of TFIIIC to the 3'-ICR of the tRNA(Met) gene . However, the affinity of binding of TFIIIC to the dimeric template is 7-fold higher than to the tRNA(Met) gene, alone, demonstrating that the tRNA(Met) gene contains intrinsically weak promoter elements . This may contribute to the inability of the tRNA(Met) gene to independently direct transcription from its ICR elements.

Nature, 1989 Nov 2, 342(6245), 39 - 45
Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis; Gould KL et al.; The cdc2+ protein kinase (pp34) is found to be phosphorylated on tyrosine as well as serine and threonine residues in exponentially growing Schizosaccharomyces pombe . At mitosis, the level of pp34 phosphorylation on both threonine and tyrosine residues decreases . The single detectable site of tyrosine phosphorylation in pp34 has been mapped to Tyr 15, a residue within the presumptive ATP-binding domain . Substitution of this tyrosine by phenylalanine advances cells prematurely into mitosis, establishing that tyrosine phosphorylation/dephosphorylation directly regulates pp34 function.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1989 Nov, 22(4), 229 - 41
Maloalcoholic fermentation by immobilized Schizosaccharomyces pombe; Guo LY et al.; Cells of Schizosaccharomyces pombe TMB 1138, which are capable of metabolizing-malate, was immobilized in calcium alginate gel to carry out maloalcoholic fermentation . Four milliliters of cell suspension containing about 2.0 X 10(7) cells were entrapped in 16 ml of sodium alginate solution in order to prepare 2% Na-alginate (w/v) gel bead . After activation by incubating at 28 degrees C for 24 h in grape juice, 300 beads of immobilized cells were inoculated into the fermentation medium . After fermentation was proceeded at 25 or 28 degrees C for 24 h by shaking, it could metabolize L-malate completely and the total acidity was also reduced . Under the same condition for batch fermentation, it was found that the utilization of L-malic acid was over 97% for the first 7 days in fermentation medium, 85% for the first 4 days in grape juice and 87% for the first 4 days in wine . Furthermore, for the continuous fermentation in wine, the conversion of L-malic acid reached 92% in 24 h and could be maintained at 75% in the following 9 days.

J Biol Chem, 1989 Oct 15, 264(29), 17084 - 90
Multiple mutations of the first gene of a dimeric tRNA gene abolish in vitro tRNA gene transcription; Nichols M et al.; Eukaryotic tRNA expression initiates with transcription by RNA polymerase III and requires two additional protein factors and two regions within the tRNA gene (the 5'-internal control region (ICR) or A-box and the 3'-ICR or B-box) . Using a reconstituted Saccharomyces cerevisiae RNA polymerase III system, the transcription of various 5'-ICR, 3'-ICR, and double mutation alleles of the Schizosaccharomyces pombe sup3-e dimeric tRNA gene were studied . The sup3-e tRNA locus consists of an upstream serine tRNA gene and a downstream initiator methionine tRNA gene which are transcribed as a dimeric precursor and processed to give two tRNAs . Only the ICRs of the tRNA(Ser) gene are active in directing dimeric gene transcription . Mutations in the 3'-ICR of the tRNA(Ser) gene reduce transcription of the dimer more than those in the 5'-ICR . Mutations in the 5'-ICR were found which greatly increased or decreased transcription of the dimer, while base changes in the 3'-ICR were only found to decrease transcription . This suggests a modulatory role for the 5'-ICR in transcription regulation . Mutation of the methionine tRNA gene ICR has little effect on sup3-e transcription, and no detectable transcripts initiate from the methionine tRNA gene when the tRNA(Ser) gene promoter is inactivated by mutation . Comparison with transcription studies of other mutant tRNA genes suggests that nucleotides sites within the ICRs, such as nucleotides 8, 10, 13, 18, and 19 in the 5'-ICR and 48, 53, 56, 57, and 58 in the 3'-ICR, appear to have evolved universal importance for RNA polymerase III transcription in eukaryotes . Thus these ICR sequences may play a critical role in regulation of tRNA expression.

Nucleic Acids Res, 1989 Oct 11, 17(19), 7821 - 31
Splicing of the U6 RNA precursor is impaired in fission yeast pre-mRNA splicing mutants; Potashkin J et al.; U6 RNA is a member of a class of small abundant stable nuclear RNAs that are essential for splicing . In all species examined so far, the U6 RNA is a RNA polymerase III transcript . The U6 gene of the fission yeast Schizosaccharomyces pombe is unusual in that it is interrupted by an intron whose structure is similar to those found in pre-mRNAs . As part of our previous analysis of three S . pombe temperature sensitive pre-mRNA splicing mutants we examined their spliceosomal snRNA content . In contrast to the other snRNAs, the amount of U6 RNA is reduced at the restrictive temperature in all three of the mutants compared to the wild type . To investigate the cause of this reduction we have analyzed the efficiency of splicing of the U6 RNA precursor (U6 pre-RNA) in the pre-mRNA splicing mutants . At the restrictive temperature the ratio of unspliced U6 precursor to mature RNA is elevated in the mutants compared to the wild type grown under identical conditions, indicating a defect in U6 pre-RNA splicing . In this regard, the U6 RNA precursor behaves similarly to pre-mRNAs . Unspliced U6 pre-RNA was also detected in wild type cells under certain growth conditions.

FEMS Microbiol Lett, 1989 Oct 1, 52(1-2), 231 - 6
Interconversion of polyamines in wild-type strains and mutants of yeasts and the effects of polyamines on their growth; Hamana K et al.; Yeasts of wild-type strains, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans were shown to have the ability to form aminopropylcadaverine and aminopropylhomospermidine from cadaverine and homospermidine, respectively . A polyamine autotroph S . cerevisiae 179-5, which lacks ornithine decarboxylase, produced both aminopropylcadaverine and aminopropylhomospermidine, while another mutant S . cerevisiae Y 260 A, which lacks spermine synthase, formed only aminopropylcadaverine . Naturally-occurring triamines and tetraamines except norspermidine and norspermine stimulated the growth of S . cerevisiae 179-5 . All the six aliphatic diamines with carbon chain length ranging from one to six were effective in activating the growth of S . cerevisiae 179-5, though all of them were not converted to either triamines or tetraamines.

Proc Natl Acad Sci U S A, 1989 Oct, 86(20), 7989 - 93
The adenylyl cyclase gene from Schizosaccharomyces pombe; Young D et al.; We cloned the adenylyl cyclase gene from the fission yeast Schizosaccharomyces pombe using low-stringency hybridization to the Saccharomyces cerevisiae adenylyl cyclase gene . The Sc . pombe gene encodes a 1692-amino acid-residue protein . The identity of this gene was confirmed by studies of its expression in Sa . cerevisiae . Expression of the carboxyl-terminal region of the Sc . pombe adenylyl cyclase protein will suppress a temperature-sensitive mutation in the Sa . cerevisiae adenylyl cyclase gene . Furthermore, Sa . cerevisiae that lack their endogenous adenylyl cyclase gene and express the carboxyl-terminal region of the Sc . pombe adenylyl cyclase protein have measurable adenylyl cyclase activity . The carboxyl-terminal region of this protein has strong homology with the catalytic domain of the Sa . cerevisiae adenylyl cyclase . Also, Sc . pombe adenylyl cyclase, like Sa . cerevisiae adenylyl cyclase, contains a tandemly repeated motif rich in leucine . Neither yeast protein is particularly homologous to the recently cloned Gs-responsive mammalian adenylyl cyclase {Krupinski, J., Coussen, F., Bakalyar, H . A., Tang, W.-J., Feinstein, P . G., Orth, K., Slaughter, C., Reed, R . R . & Gilman, A . G . (1989) Science 244, 1558-1564}.

Exp Cell Res, 1989 Oct, 184(2), 273 - 86
A review of mitosis in the fission yeast Schizosaccharomyces pombe; Hayles J et al.; Mitosis and cell division are the final events of the cell cycle, resulting in the precise segregation of chromosomes into two daughter cells . A highly controlled and accurate segregation of the chromosomes is required to ensure that each daughter cell receives a complete genome and remains viable . The fission yeast, Schizosaccharomyces pombe, is a unicellular eukaryotic organism which is particularly convenient for investigating these problems . It is very amenable to genetic analysis and its predominantly haploid life cycle has allowed the isolation of recessive temperature-sensitive mutants unable to complete the cell cycle . Classical genetic analysis of these mutants has been used to identify over 40 gene functions that are required for cell cycle progress in S . pombe . Many of these genes have now been cloned and sequenced and in some cases the encoded gene product has been identified . This approach, coupling classical and molecular genetics, allows identification of the molecules important in the mitotic processes and provides a means for establishing what functional roles they may play.

EMBO J, 1989 Oct, 8(10), 3045 - 52
Characterization of Schizosaccharomyces pombe minichromosome deletion derivatives and a functional allocation of their centromere; Niwa O et al.; A 530 kb long Schizosaccharomyces pombe linear minichromosome, Ch16, containing a centric region of chromosome III, has previously been made . In the present study, we constructed a number of deletions in the right and/or left arms of Ch16, and compared their structure and behaviour with Ch16 . The functional centromere, cen3, is allocated within a 120 kb long region which is covered by the shortest derivative, Ch10, and is comprised mostly of centromeric repeating sequences . The shortest minichromosome is stable in mitosis and the copy number control is apparently precise . In monosomic meiosis it segregates normally . In disomic meioses, however, the frequency of non-disjunction is very high, suggesting that it may not form a pair . The mitotic loss rate of one of the left-arm deletions, ChR32, which lacks a part of the centromeric repeating sequence, is the highest of all the deletions . This deletion also exhibits the highest precocious sister chromatid separation in meiosis I, suggesting that sister chromatid association might become weakened in ChR32 . Our results indicate that the proper meiotic segregation of S.pombe minichromosomes is dependent upon the formation of a bivalent . S.pombe may not have the 'distributive segregation' found with Saccharomyces cerevisiae minichromosomes.

Gene, 1989 Sep 30, 81(2), 227 - 35
Cloning of Schizosaccharomyces pombe genes encoding the U1, U2, U3 and U4 snRNAs; Dandekar T et al.; Schizosaccharomyces pombe contains a group of five relatively abundant small nuclear RNAs (snRNAs) which are immunoprecipitated by human autoimmune antibodies of Sm serotype . The S . pombe RNAs hybridise to probes specific for human U1, U2, U4, U5 and U6 and in each case are similar in size to the human species . A further group of snRNAs from S . pombe are precipitated by antibodies against U3 containing ribonucleoprotein; the most abundant of these species hybridises to a probe specific for human U3 . We have cloned the genes encoding U1, U2, U3 and U4 from S . pombe, together with that encoding another abundant snRNA, previously designated SPU43 . U2 and U4 are encoded by single-copy genes, while two genes encode U3 . The latter are not clustered, since a chromosomal Southern transfer shows them to lie on different chromosomes.

Genetics, 1989 Sep, 123(1), 45 - 54
Meiotic recombination-deficient mutants of Schizosaccharomyces pombe; Ponticelli AS et al.; A mutant screen employing the ade6-M26 recombination hotspot was developed and used to isolate Schizosaccharomyces pombe mutants deficient in meiotic recombination . Nine rec mutations were recessive, defining six complementation groups, and reduced ade6 meiotic recombination 3-fold to greater than or equal to 300-fold when homozygous . Three recessive rec mutations analyzed further also reduced meiotic intragenic recombination at ura4 on chromosome III and intergenic recombination between pro2 and arg3 on chromosome I . The observed non-co-ordinate reductions of the recombinant frequencies in the three test intervals suggest a degree of locus (or intragenic vs . intergenic) specificity of the corresponding rec+ gene products . None of the mutations specifically inactivated the ade6-M26 hotspot . Additional rec genes may be identified with these methods.

Mol Gen Genet, 1989 Sep, 218(3), 554 - 8
Isolation and initial characterization of a Schizosaccharomyces pombe mutant exhibiting temperature-dependent radiation sensitivity due to a mutation in a previously unidentified rad locus; Lieberman HB et al.; We have isolated a mutant of the yeast Schizosaccharomyces pombe which exhibits sensitivity to UV light when grown at either 30 degrees or 37 degrees C, as compared to the parental wild-type strain . This increased sensitivity is more pronounced when cells are grown at 37 degrees C . The mutant is also sensitive to 18 MeV electrons at the high temperature . Tetrad analysis of spores generated by crossing the mutant and a Rad+ strain revealed that sensitivity to both types of radiation cosegregate 2:2, relative to wild-type resistance, indicating that a single altered chromosomal locus is responsible for the radiation sensitivities observed . In addition, analysis of spores resulting from crosses between the mutant and all other known S . pombe rad mutants indicates that the temperature-dependent sensitivity described in this report is mediated by a mutation in a previously unidentified rad locus.

Mol Cell Biol, 1989 Sep, 9(9), 3860 - 8
Mammalian growth-associated H1 histone kinase: a homolog of cdc2+/CDC28 protein kinases controlling mitotic entry in yeast and frog cells; Langan TA et al.; Mammalian growth-associated H1 histone kinase, an enzyme whose activity is sharply elevated at mitosis, is similar to cdc2+ protein kinase from Schizosaccharomyces pombe and CDC28 protein kinase from Saccharomyces cerevisiae with respect to immunoreactivity, molecular size, and specificity for phosphorylation sites in H1 histone . Phosphorylation of specific growth-associated sites in H1 histone is catalyzed by yeast cdc2+/CDC28 kinase, as shown by the in vitro thermal lability of this activity in extracts prepared from temperature-sensitive mutants . In addition, highly purified Xenopus maturation-promoting factor catalyzes phosphorylation of the same sites in H1 as do the mammalian and yeast kinases . The data indicate that growth-associated H1 kinase is encoded by a mammalian homolog of cdc2+/CDC28 protein kinase, which controls entry into mitosis in yeast and frog cells . Since H1 histone is known to be an in vivo substrate of the mammalian kinase, this suggests that phosphorylation of H1 histone or an H1 histone counterpart is an important component of the mechanism for entry of cells into mitosis.

J Biol Chem, 1989 Aug 15, 264(23), 13373 - 6
Mapping of the active site tyrosine of eukaryotic DNA topoisomerase I; Eng WK et al.; DNA topoisomerase I from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe was overproduced using the cloned genes . Extracts from cells overproducing DNA topoisomerase I were prepared and incubated with 32P-labeled DNA . Alkali was used to trap the topoisomerase I-DNA covalent intermediate . Most of the DNA was digested with nuclease, and the resultant 32P-labeled topoisomerase I was subjected to cleavage with cyanogen bromide or formic acid . From the molecular weights of the resultant labeled peptides and by comparison of the amino acid sequences derived from the cloned genes, we were able to deduce that the active site tyrosine of eukaryotic DNA topoisomerase I is very near the carboxyl terminus, at amino acid 771 for S . pombe and 727 for S . cerevisiae . Site-directed mutagenesis was used to change tyrosine 727 of S . cerevisiae topoisomerase I to a phenylalanine . The resulting mutant topoisomerase I protein lost all DNA relaxation activity and rendered cells resistant to the topoisomerase I inhibitor, camptothecin . The amino acid sequence of human topoisomerase I has significant similarity to the two yeast topoisomerase I sequences . Based on this similarity, we infer that tyrosine 723 is the active site tyrosine of human enzyme.

J Mol Biol, 1989 Aug 5, 208(3), 371 - 9
Schizosaccharomyces pombe U4 small nuclear RNA closely resembles vertebrate U4 and is required for growth; Dandekar T et al.; The single-copy gene snu4, which encodes the small nuclear RNA (snRNA) U4, has been cloned and sequenced . Schizosaccharomyces pombe U4 is 128 nucleotides in length, similar in size to vertebrate U4 and shows substantial primary and secondary structure homology . The gene lacks sequences closely resembling vertebrate snRNA transcription signals, but has a TATA box at -33 to -30; TATA sequences flanked by several additional conserved nucleotides are found in the same position in the 5' regions of other snRNA genes from Schiz . pombe . The cloned snu4 gene was disrupted by transposon mutagenesis and used to replace one chromosomal copy of snu4 in a diploid strain . On sporulation snu4- haploid strains could not be recovered, demonstrating that U4 is required, at least for spore germination . Haploid snu4- strains are viable if they also carry snu4+ on a replicating plasmid but are unable to loose the plasmid under non-selective growth, demonstrating a continuous requirement for U4 for viability.

Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6260 - 3
Electrophoretic karyotype of Aspergillus nidulans; Brody H et al.; An electrophoretic karyotype of Aspergillus nidulans has been obtained using contour-clamped homogeneous electric field gel electrophoresis . Six chromosomal bands were separated, with two of the bands migrating as doublets . Using the Schizosaccharomyces pombe and Saccharomyces cerevisiae chromosomes as size standards, we estimate the sizes of the chromosomes to be between 2.9 and 5.0 megabase pairs (mb) with a total genome size of approximately 31 mb . Four of the eight genetic linkage groups were assigned to chromosomal bands by hybridization of contour-clamped homogeneous electric field gel blots with various radiolabeled probes each specific to a particular linkage group . Contour-clamped homogeneous electric field gel analysis of reciprocal translocation strains gave chromosomal assignments for the four remaining linkage groups . In order of decreasing size, the A . nidulans chromosomes are: VIII (5.0 mb), VII (4.5 mb), II (4.2 mb), I and V (3.8 mb), III and VI (3.5 mb), and IV (2.9 mb).

Proc Natl Acad Sci U S A, 1989 Aug, 86(15), 5693 - 7
Adenylate cyclases in yeast: a comparison of the genes from Schizosaccharomyces pombe and Saccharomyces cerevisiae; Yamawaki-Kataoka Y et al.; A Schizosaccharomyces pombe gene encoding adenylate cyclase has been cloned by cross-hybridization with the Saccharomyces cerevisiae adenylate cyclase gene . The protein encoded consists of 1692 amino acids and has adenylate cyclase activity that cannot be activated by the Sa . cerevisiae RAS2 protein . Sc . pombe cyclase has a high degree of homology (approximately 60%) with the catalytic domain of Sa . cerevisiae cyclase precisely mapped by a gene-deletion analysis . A 25-40% identity is observed throughout the middle segments of approximately 1000 residues of both cyclases, large parts of which are composed of repetitions of a 23-amino acid motif similar to those found in human glycoproteins, Drosophila chaoptin, and Toll gene product . However, a segment corresponding to the NH2-terminal 620 residues of Sa . cerevisiae cyclase appears lost from Sc . pombe cyclase, and the COOH-terminal 140 residues are not well conserved between the two yeast species . Deletions involving the COOH-terminal residues of Sa . cerevisiae cyclase cause loss of activation by the RAS2 protein . These results suggest that Sc . pombe cyclase may have lost the ability to interact with RAS proteins by the loss of a regulatory site.

Curr Genet, 1989 Aug, 16(2), 89 - 94
Some of the swi genes of Schizosaccharomyces pombe also have a function in the repair of radiation damage; Schmidt H et al.; In Schizosaccharomyces pombe the frequency of mating-type (MT) switching is reduced by mutations in the swi genes . The ten hitherto known swi genes can be subdivided into three classes: Ia, Ib and II . Strains having swi5 (class Ib), swi9 (class II) and swi10 (class II) mutations do not only show reduced MT switching, but also exhibit an increased sensitivity to UV- and gamma-rays . For that reason, 19 previously described rad genes were tested for their effect on MT switching . We found that swi9, "rad10", "rad16" and "rad20" are allelic with each other indicating that the former allocation of these rad mutations to three different genes must have been erroneous . Among the remaining 16 rad genes examined, rad22 seems to be a new class II swi gene . The double mutants swi5 swi9 and swi5 swi10, but not swi9 swi10, are much more sensitive to radiation than the respective single mutants . Thus a cumulative increase in sensitivity occurs only if the mutants belong to different classes; previously the same correlation was found with regard to cumulative effects in MT switching.

Biochem Cell Biol, 1989 Aug, 67(8), 464 - 7
Acetate assimilation by the fission yeast, Schizosaccharomyces pombe; Tsai CS et al.; The fission yeast Schizosaccharomyces pombe utilizes acetate at subinhibitory concentrations in the presence of D-glucose . The nonionized form of acetate is preferentially utilized, oxidized to 14CO2, and assimilated into lipids and proteins . Acetyl CoA synthetase activity greatly increases in the yeast cells grown in media containing acetate . However, glyoxylate cycle enzymes are not detectable in Schizosaccharomyces pombe . {1-14C}Acetate is incorporated into stereols, sterol esters, neutral lipids, and phospholipids . Assimilation of {1-14C}acetate into the peptide structure of proteins was confirmed by a proteolytic digestion experiment.

J Bacteriol, 1989 Aug, 171(8), 4525 - 9
Nucleotide sequences of fic and fic-1 genes involved in cell filamentation induced by cyclic AMP in Escherichia coli; Kawamukai M et al.; The nucleotide sequences of fic-1 involved in the cell filamentation induced by cyclic AMP in Escherichia coli and its normal counterpart fic were analyzed . The open reading frame of both fic-1 and fic coded for 200 amino acids . The Gly at position 55 in the Fic protein was changed to Arg in the Fic-1 protein . The promoter activity of fic was confirmed by fusing fic and lacZ . The gene downstream from fic was found to be pabA (p-aminobenzoate) . There is an open reading frame (ORF190) coding for 190 amino acids upstream from the fic gene . Computer-assisted analysis showed that Fic has sequence similarity with part of CDC28 of Saccharomyces cerevisiae, CDC2 of Schizosaccharomyces pombe, and FtsA of E . coli . In addition, ORF190 has sequence similarity with the cyclosporin A-binding protein cyclophilin.

Biochimie, 1989 Aug, 71(8), 931 - 40
Structure-function relationships of mitochondrial ATPase-ATPsynthase using Schizosaccharomyces pombe yeast mutants with altered F1 subunits; Di Pietro A et al.; Phenotypic revertants have been selected from mutants of the yeast Schizosaccharomyces pombe devoid of either alpha or beta subunits of mitochondrial ATPase-ATPsynthase . In contrast to parental mutants, phenotypic revertants are able to grow on glycerol respiratory medium and show immunodetectable alpha and beta subunits . However, growth and cellular respiration are only partially restored as compared to the wild strain, indicating that the recovered subunits are mutated . ATPase activity of revertant submitochondrial particles shows markedly different parameters: more acidic optimal pH, absence of bicarbonate activation and decreased sensitivity to azide inhibition in the alpha subunit-modified R3.51 . Opposite differences are observed in the beta subunit-modified R4.3: more alkaline optimal pH, much higher bicarbonate activation, and increased sensitivity to azide . The ITPase activity of R4.3 submitochondrial particles is also more sensitive to azide as compared to the wild strain . ATPase activity of purified F1 also exhibits marked differences: loss of bicarbonate-sensitive negative cooperativity, decreased sensitivity to both ADP and azide inhibitions in the R3.51 revertant . On the contrary, increased negative cooperativity and increased sensitivity to both ADP and azide inhibitions are observed for the R4.3 revertant enzyme which in addition exhibits a much lower maximal rate . The beta subunit-mutation of R4.3 also increases the sensitivity of ITPase activity to tripolyphosphate inhibition, whereas the alpha subunit-mutation of R3.51 is without any effect . Soluble F1 with beta subunit-mutation is very sensitive to high ammonium sulfate concentrations required for enzyme precipitation and concentration and known to partially deplete the enzyme from its endogenous nucleotides . On the contrary, poly(ethylene)glycol is very efficient for preparing from any strain a pure and very stable enzyme retain-ing high amounts of endogenous nucleotides . The R4.3 revertant F1 retains even more nucleotides than the wild-strain F1 and is much less sensitive to high iodide concentrations which favor enzyme dissociation and precipitation . The tryptophan intrinsic fluorescence of F1 is modified by both mutations that increase the maximal emission intensity . The most important effect is produced by beta subunit-mutation which decreases the quenchable fraction, one-third to one-half tryptophans being no longer accessible to iodide . The overall results suggest that both mutations modify enzyme-nucleotide interactions: the alpha subunit-mutation of R3.51 would favor ADP release by lowering interactions with the adenine moiety, whereas the beta subunit-mutation of R4.3 would lower ADP release by strengthening interactions with the phosphate chain moiety.

FEBS Lett, 1989 Jul 17, 251(1-2), 84 - 8
Substrate structural requirements of Schizosaccharomyces pombe RNase P; Drainas D et al.; RNase P from Schizosaccharomyces pombe has been purified over 2000-fold . The apparent Km for two S . pombe tRNA precursors derived from the supS1 and sup3-e tRNA(Ser) genes is 20 nM; the apparent Vmax is 2.5 nM/min (supS1) and 1.1 nM/min (sup3-e) . Processing studies with precursors of other mutants show that the structures of the acceptor stem and anticodon/intron loop of tRNA are crucial for S . pombe RNase P action.

FEMS Microbiol Lett, 1989 Jul 1, 51(1), 45 - 8
A mating-type-specific sterility gene map1 is required for transcription of a mating-type gene mat1-Pi in the fission yeast Schizosaccharomyces pombe; Fujioka H et al.; Transcriptional activation of the mating-type gene mat1-Pi essential for meiosis in the fission yeast Schizosaccharomyces pombe was studied . A homozygous diploid harboring the mutation at the h+-specific sterility gene, map1, was arrested before premeiotic DNA synthesis in the nitrogen-free sporulation medium . Transcription of mat1-Pi was totally absent in the map1 mutant . The mei2 gene encoding a positive regulator for meiosis was normally transcribed in the map1 mutant, suggesting that the map1 function was specific to mat1-Pi.

Mol Gen Genet, 1989 Jul, 218(1), 41 - 9
Molecular cloning and sequence analysis of mutant alleles of the fission yeast cdc2 protein kinase gene: implications for cdc2+ protein structure and function; Carr AM et al.; The cdc2+ gene function plays a central role in the control of the mitotic cell cycle of the fission yeast Schizosaccharomyces pombe . Recessive temperature-sensitive mutations in the cdc2 gene cause cell cycle arrest when shifted to the restrictive temperature, while a second class of mutations within the cdc2 gene causes a premature advancement into mitosis . Previously the cdc2+ gene has been cloned and has been shown to encode a 34 kDa phosphoprotein with in vitro protein kinase activity . Here we describe the cloning of 11 mutant alleles of the cdc2 gene using two simple methods, one of which is presented here for the first time . We have sequenced these alleles and find a variety of single amino acid substitutions mapping throughout the cdc2 protein . Analysis of these mutations has identified a number of regions within the cdc2 protein that are important for cdc2+ activity and regulation . These include regions which may be involved in the interaction of the cdc2+ gene product with the proteins encoded by the wee1+, cdc13+ and suc1+ genes.

Bioessays, 1989 Jul, 11(1), 9 - 14
Regulation of meiosis: from DNA binding protein to protein kinase; McLeod M; The transition from mitotic cell division to meiosis in yeast is governed by both the mating-type genes and signals from the environment . Analysis of mutants that are unable to regulate entry into meiosis has identified many genes that function in this process and in some cases, the biochemical activity of their protein products has been described . At least two of the the mating-type genes of Saccharomyces cerevisiae encode DNA binding proteins that regulate transcription of unlinked genes required for entry into meiosis . Meiotic development of the distantly related yeast, Schizosaccharomyces pombe, is also controlled by the mating-type genes but in this yeast, their role is to regulate expression of a protein that acts as an inhibitor of a protein kinase . The ability to use the powerful tool of genetics in yeast has provided us with many new insights into the problem of meiotic development.

Biochim Biophys Acta, 1989 Jun 23, 975(1), 119 - 26
Purification from a yeast mutant of mitochondrial F1 with modified beta-subunit . High affinity for nucleotides and high negative cooperativity of ATPase activity; Falson P et al.; Mitochondrial F1 containing genetically modified beta-subunit was purified for the first time from a mutant of the yeast Schizosaccharomyces pombe . Precipitation by poly(ethylene glycol) allowed us to obtain a very stable and pure enzyme from either mutant or wild-type strain . In the presence of EDTA, purified F1 retained high amounts of endogenous nucleotides: 4.6 mol/mol and 3.7 mol/mol for mutant and wild-type F1, respectively . The additional nucleotide in mutant F1 was ATP; it was lost in the presence of Mg2+, which led to a total of 3.4 mol of nucleotides/mol whereas wild-type F1 retained all its nucleotides . Mutant F1 bound more exogenous ADP than wild-type F1 and the same total nucleotide amount was reached with both enzymes . Kinetics of ATPase activity revealed a much higher negative cooperativity for mutant than for wild-type F1 . Bicarbonate abolished this negative cooperativity, but higher concentrations were required for mutant F1 . The mutant enzyme was more sensitive than the wild-type one to azide inhibition and ADP competitive inhibition; this indicated stronger interactions between nucleotide and F1 in the mutant enzyme . The latter also showed increased sensitivity to N,N'-dicyclohexylcarbodiimide irreversible inhibition.

Nature, 1989 Jun 22, 339(6226), 626 - 9
Dephosphorylation and activation of Xenopus p34cdc2 protein kinase during the cell cycle; Gautier J et al.; Genetic studies in the fission yeast Schizosaccharomyces pombe have established that a critical element required for the G2----M-phase transition in the cell cycle is encoded by the cdc2+ gene . The product of this gene is a serine/threonine protein kinase, designated p34cdc, that is highly conserved functionally from yeast to man2 and has a relative molecular mass of 34,000 (34 K) . Purified maturation-promoting factor (MPF) is a complex of p34cdc2 and a 45K substrate that appears in late G2 phase and is sufficient to drive cells into mitosis . This factor has been identified in all eukaryotic cells, and in vitro histone H1 is the preferred substrate for phosphorylation . The increase in the activity of H1 kinase in M-phase is associated with a large increase in total cell protein phosphorylation which is believed to be a consequence of MPF activation . We show here that the H1 kinase activity of p34cdc2 oscillates during the cell cycle in Xenopus, and maximal activity correlates with the dephosphorylated state of p34cdc2 . Direct inactivation of MPF in vitro is accompanied by phosphorylation of p34cdc2 and reduction of its protein kinase activity.

Proc Natl Acad Sci U S A, 1989 Jun, 86(11), 4137 - 41
Genetic analysis of Schizosaccharomyces pombe 7SL RNA: a structural motif that includes a conserved tetranucleotide loop is important for function; Liao XB et al.; We have studied the effects of mutations in a 6-base segment of Schizosaccharomyces pombe 7SL RNA, which lies within a 35-nucleotide domain whose sequence and secondary structure are conserved in RNAs from many divergent organisms, including the 7SL component of human signal recognition particle (SRP) . Surprisingly, many changes in this region can be tolerated under normal growth conditions . An exception is the lethality of several mutations at positions 159 and 160, 2 nucleotides previously shown to be protected from RNase digestion by the 19-kDa canine SRP protein . Nucleotide 160 is, in addition, the most highly conserved base in a consensus sequence for the most common tetranucleotide loop in ribosomal RNAs . Mutations that are likely to affect the stability and/or conformation of the RNA give rise to a conditional phenotype: when osmolarity of the medium is raised, the RNAs become partially or completely defective in function at high temperature.

Microbiol Rev, 1989 Jun, 53(2), 171 - 85
The ras oncogene--an important regulatory element in lower eucaryotic organisms; Gibbs JB et al.; The ras proto-oncogene in mammalian cells encodes a 21-kilodalton guanosine triphosphate (GTP)-binding protein . This gene is frequently activated in human cancer . As one approach toward understanding the mechanisms of cellular transformation by ras, the function of this gene in lower eucaryotic organisms has been studied . In the yeast Saccharomyces cerevisiae, the RAS gene products serve as essential function by regulating cyclic adenosine monophosphate metabolism . Stimulation of adenylyl cyclase is dependent not only on RAS protein complexed to GTP, but also on the CDC25 and IRA gene products, which appear to control the RAS GTP-guanosine diphosphate cycle . Although analysis of RAS biochemistry in S . cerevisiae has identified mechanisms central to RAS action, RAS regulation of adenylyl cyclase appears to be strictly limited to this particular organism . In Schizosaccharomyces pombe, Dictyostelium discoideum, and Drosophila melanogaster, ras-encoded proteins are not involved with regulation of adenylyl cyclase, similar to what is observed in mammalian cells . However, the ras gene product in these other lower eucaryotes is clearly required for appropriate responses to extracellular signals such as mating factors and chemoattractants and for normal growth and development of the organism . The identification of other GTP-binding proteins in S . cerevisiae with distinct yet essential functions underscores the fundamental importance of G-protein regulatory processes in normal cell physiology.

Mol Cell Biol, 1989 Jun, 9(6), 2536 - 43
Partial characterization of an RNA component that copurifies with Saccharomyces cerevisiae RNase P; Lee JY et al.; Saccharomyces cerevisiae cellular RNase P is composed of both protein and RNA components that are essential for activity . The isolated holoenzyme contains a highly structured RNA of 369 nucleotides that has extensive sequence similarities to the 286-nucleotide RNA associated with Schizosaccharomyces pombe RNase P but bears little resemblance to the analogous RNA sequences in procaryotes or S . cerevisiae mitochondria . Even so, the predicted secondary structure of S . cerevisiae RNA is strikingly similar to the bacterial phylogenetic consensus rather than to previously predicted structures of other eucaryotic RNase P RNAs.

FEBS Lett, 1989 May 8, 248(1-2), 105 - 10
New expression vectors for the fission yeast Schizosaccharomyces pombe; Broker M et al.; A general expression vector (pMB332) for the fission yeast Schizosaccharomyces pombe was constructed . The heterologous gene expression is driven by the S . pombe alcohol dehydrogenase (adh) promoter . Transcription termination signals were isolated from the S . pombe actin gene . The vectors carry the Saccharomyces cerevisiae Ura3 gene, which complements the S . pombe ura4 mutation . The plasmid stability is conferred by the S . pombe ars and stb elements isolated from pFL120 {(1983) Cell 32, 371-377} . An 'ATG' vector (pMB340) was created, which allows the expression of protein fragments fused to a translational start codon downstream of the adh promoter . The function of this vector system is shown by the production of the human blood coagulation protein factor XIIIa.

Mol Cell Biol, 1989 May, 9(5), 2034 - 41
The Saccharomyces cerevisiae CKS1 gene, a homolog of the Schizosaccharomyces pombe suc1+ gene, encodes a subunit of the Cdc28 protein kinase complex; Hadwiger JA et al.; The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for cell cycle initiation . In an attempt to identify genes encoding proteins that interact with the Cdc28 protein kinase, high-copy plasmid suppressors of a temperature-sensitive cdc28 mutation were isolated . One such suppressor, CKS1, was found to encode an 18-kilodalton protein that shared a high degree of homology with the suc1+ protein (p13) of Schizosaccharomyces pombe (67% amino acid sequence identity) . Disruption of the chromosomal CKS1 gene conferred a G1 arrest phenotype similar to that of cdc28 mutants . The presence of the 18-kilodalton Cks1 protein in yeast lysates was demonstrated by using Cks-1 specific antiserum . Furthermore, the Cks1 protein was shown to be physically associated with active forms of the Cdc28 protein kinase . These data suggest that Cks1 is an essential component of the Cdc28 protein kinase complex.

J Cell Sci, 1989 May, 93 ( Pt 1), 185 - 9
Nucleoside diphosphokinase, an enzyme with step changes in activity during the cell cycle of the fission yeast Schizosaccharomyces pombe . II . Dissociation of the steps from the DNA-division cycle after induction synchronization; Creanor J et al.; Synchrony was induced in cultures of the mitotic mutant cdc2.33 of Schizosaccharomyces pombe by shifting up an asynchronous culture to the restrictive temperature for a period of 3.5-4.5 h and then shifting down to the permissive temperature . The resulting synchronous divisions had short cycle times, down to 50% of the normal cycle . The oscillatory control of nucleoside diphosphokinase activity was also synchronized by the shift-down and the activity rose in a step pattern . Unlike the situation in the normal cycle, this step pattern was dissociated from the shortened cell cycle and had a longer period and different phase relations . It may be that the normal entrainment or coupling between the cell cycle and the activity control fails if the cell cycle is too short . The period of the activity control (equal to the protein doubling time at the restrictive temperature) appears to be temperature-compensated.

Proc Natl Acad Sci U S A, 1989 May, 86(10), 3559 - 63
Peptide sequencing and site-directed mutagenesis identify tyrosine-727 as the active site tyrosine of Saccharomyces cerevisiae DNA topoisomerase I; Lynn RM et al.; Extensive digestion of the covalent intermediate between DNA and Saccharomyces cerevisiae DNA topoisomerase I with trypsin yields a 7-amino acid peptide covalently linked to DNA . Direct sequencing of the DNA-linked peptide identifies Tyr-727 as the active site tyrosine that forms an O4-phosphotyrosine bond with DNA when the enzyme cleaves a DNA phosphodiester bond . Site-directed mutagenesis of the cloned yeast TOP1 gene encoding the enzyme confirms the essentiality of Tyr-727 for the relaxation of supercoiled DNA by the enzyme . From amino acid sequence homology, Tyr-771 and -773 are readily identified as the active site tyrosines of Schizosaccharomyces pombe and human DNA topoisomerase I, respectively . Sequence comparison and site-directed mutagenesis also implicate Tyr-274 of vaccinia virus DNA topoisomerase as the active site residue . There appears to be a 70-amino acid domain near the carboxyl terminus of eukaryotic DNA topoisomerase I and vaccinia topoisomerase, within which the active site tyrosine resides.

Nucleic Acids Res, 1989 Apr 25, 17(8), 3217 - 27
Improved separation of chromosome-sized DNA from Trypanosoma brucei, stock 427-60; Van der Ploeg LH et al.; Separation of chromosome-sized DNA from the parasitic protozoan Trypanosoma brucei had previously resulted in the fractionation of DNA molecules that ranged in size from 50 kb up to roughly 1.5 Mb . The number of larger chromosomes and their size, accounting for 80% of the DNA of T . brucei remained unclear . We have now size separated these larger DNA molecules by pulsed field gel electrophoresis (PFG) and resolve a total of 20 bands, accounting for roughly 120 chromosomes, ranging in size from 50 kb up to the size of the largest, 5.7 Mb chromosome of Schizosaccharomyces pombe . Three different VSG gene expression sites were located to chromosomes of 430 kb, 1.5 Mb and 3 Mb, respectively . We have not been able to identify additional, previously cryptic DNA rearrangements, that could explain the activation or inactivation of the expression sites.

FEBS Lett, 1989 Apr 24, 247(2), 242 - 6
The primary structure of rat ribosomal protein L18a; Aoyama Y et al.; The amino acid sequence of rat ribosomal protein L18a was deduced from the sequence of nucleotides in a recombinant cDNA . Ribosomal protein L18a contains 175 amino acids and has a molecular mass of 20,047 Da . Hybridization of the cDNA to digests of rat nuclear DNA and to a preparation of poly(A)+ mRNA suggests that there are 8-11 copies of the L18a gene and that the mRNA for the protein is about 700 nucleotides in length . Rat L18a is related to Schizosaccharomyces pombe L17 and perhaps to Halobacterium marismortui L19.

Nucleic Acids Res, 1989 Apr 11, 17(7), 2801 - 18
Construction of a Not I restriction map of the fission yeast Schizosaccharomyces pombe genome; Fan JB et al.; Pulsed field gel electrophoresis and large DNA technology were used to construct a Not I restriction map of the entire genome of the fission yeast Schizosaccharomyces pombe . There are 14 detectable Not I sites in S . pombe 972h: 9 sites on chromosome I and 5 sites on chromosome II, while no Not I sites were found on chromosome III . The 17 fragments (including intact chromosome III) generated by Not I digestion were resolved by PFG electrophoresis . These fragments ranged in size from 4.5 kb to approximately 3.5 Mb . Various strategies were applied in determining, efficiently, the order of the fragments on the chromosomes . The genomic size measured by adding all the fragments together is about 14 Mb and the sizes of the three chromosomes are I, 5.7 Mb, II, 4.6 to 4.7 Mb, and III, 3.5 Mb . These are generally somewhat smaller than estimated previously.

Yeast, 1989 Apr, 5 Spec No, S399 - 404
Group-specific differences in the secondary structure of the 28S ribosomal RNA of yeasts; Blanz PA et al.; Ribosomal RNA (rRNA) sequences represent a good taxonomic character . Even small but significant parts of rRNA molecules can be used as a basis for taxonomic conclusions . A highly variable region within the 28S ribosomal nucleotide sequences from various yeasts was analysed . Despite numerous nucleotide exchanges in this region the secondary structure is generally conserved as in helices 17.1 and 17.2 . However, among the organisms analysed the basidiomycetous yeasts show an insertion of 30 nucleotides which is missing in other yeasts . In this feature the Taphrinales and Schizosaccharomycetaceae resemble the 28S rRNA of Saccharomyces carlsbergensis rather than that of the basidiomycetous yeasts . Optimal sequence alignment and a good understanding of the secondary structure is an indispensible prerequisite for any evaluation of the taxonomic significance of differences in ribosomal RNA nucleotide sequences.

Anal Biochem, 1989 Apr, 178(1), 82 - 7
Isolation of DNA from yeasts; Mann W et al.; Methods are described that allow DNA to be prepared from widely different yeasts (Candida utilis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) . The methods are reliably reproducible, and the DNA obtained is of appropriate quality for the construction of gene libraries (upper limit of size range consistently 50-150 kbp) . In method A, yeast cells are converted into spheroplasts by treatment with a highly purified mixture of enzymes from Trichoderma harzianum, the spheroplasts are lysed in a lauroylsarcosinate/EDTA buffer, and the lysate is incubated with proteinase K and then directly centrifuged through a cesium trifluoroacetate gradient . DNA is recovered from the appropriate fractions by ethanol precipitation, and the redissolved precipitate is incubated with ribonuclease . For the rest of the isolation, two protocols are given, one avoiding and one including phenol/chloroform extraction . In this way, DNA up to about 150 kbp in size can be obtained . In method B, spheroplasts are not made . Yeast cells are broken by grinding under liquid nitrogen and are then worked up in a manner similar to method A, protocol 2 . Subsequent steps depend on the purpose for which the DNA is required . Traditional methods of sucrose or salt density gradient centrifugation or agarose gel electrophoresis are applicable for size selection . A sodium iodide/silica matrix technique allows fast and effective DNA recovery from agarose gels.

Mol Cell Biol, 1989 Apr, 9(4), 1526 - 35
Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe; Gatermann KB et al.; Insertion of a 36-base-pair (bp) synthetic oligonucleotide comprising the sequence 5'-GTAGGT(19N)CTAAT (4N)AG-3' into several different positions within the coding region of the naturally intronless ura4 gene of Schizosaccharomyces pombe leads to an efficiently spliced gene producing a functional product . This suggests that the proper signals within an intron are sufficient to initiate and complete a splicing event independent of the location of the intron in the gene . Point mutations in the 5' junction (5'-GTAGGT-3') and in the putative branch sequence (5'-CTAAT-3') affect splicing efficiency significantly . A G-to-A transition at the first nucleotide at the 5' splice junction (5'-ATAGGT-3') abolishes the use of the authentic splice junction and leads to the increased use of an alternative splice site . No functional product is produced from this transcript . An A-to-G transition of the second A in the putative branch sequence (5'-CTAGT-3') lowers the splicing efficiency drastically, but still results in a functional gene product . Furthermore, extension of the 36-bp intron to introns more than 180 bp in size abolishes splicing, suggesting that the splicing apparatus might be restricted to very short introns . We discuss the possibility that S . pombe introns represent a simple type of eucaryotic intron.

J Bacteriol, 1989 Apr, 171(4), 1893 - 7
Sexual reproduction as a response to H2O2 damage in Schizosaccharomyces pombe; Bernstein C et al.; Although sexual reproduction is widespread, its adaptive advantage over asexual reproduction is unclear . One major advantage of sex may be its promotion of recombinational repair of DNA damage during meiosis . This idea predicts that treatment of the asexual form of a facultatively sexual-asexual eucaryote with a DNA-damaging agent may cause it to enter the sexual cycle more frequently . Endogenous hydrogen peroxide is a major natural source of DNA damage . Thus, we treated vegetative cells of Schizosaccharomyces pombe with hydrogen peroxide to test if sexual reproduction increases . Among untreated stationary-phase S . pombe populations the sexual spores produced by meiosis represented about 1% of the total cells . However, treatment of late-exponential-phase vegetative cells with hydrogen peroxide increased the percentage of meiotic spores in the stationary phase by 4- to 18-fold . Oxidative damage therefore induces sexual reproduction in a facultatively sexual organism, a result expected by the hypothesis that sex promotes DNA repair.

Exp Parasitol, 1989 Apr, 68(3), 283 - 8
Blastocystis hominis: phylogenetic affinities determined by rRNA sequence comparison; Johnson AM et al.; In 1912 Blastocystis hominis was identified as a new species and classified as a yeast (Brumpt 1912) . In the early 1920s several groups confirmed its classification as a yeast, specifically a member of the genus Schizosaccharomyces (discussed by Zierdt et al . 1967) . Apart from an occasional case report, the classification of B . hominis and its role as a harmless intestinal yeast was not questioned for another 50 years . Then, Zierdt (1967) suggested that it should be classified in the phylum Protozoa, subphylum Sporozoa, and that it should be considered as a potential pathogen . The likely role of B . hominis as a human pathogen has recently become more firmly established (Garcia et al . 1984; Sheehan et al . 1986) and its classification has been changed . Although the classification of B . hominis as a protozoon was assumed widely, classification as a sporozoon was not accepted, and the most recent definitive classification of the Protozoa did not even list B . hominis (Lee et al . 1985) . Then, based essentially on a review of the known characteristics of the organism, it was recently reclassified into the subphylum Sarcodina (Zierdt 1988) . Clearly, the phylogeny of this emerging human pathogen needs definitive analysis (Mehlhorn 1988).

J Cell Biol, 1989 Apr, 108(4), 1195 - 207
Higher order chromosome structure is affected by cold-sensitive mutations in a Schizosaccharomyces pombe gene crm1+ which encodes a 115-kD protein preferentially localized in the nucleus and its periphery; Adachi Y et al.; We isolated a novel class of Schizosaccharomyces pombe cold-sensitive mutants with deformed nuclear chromosome domains consisting of thread- or rodlike condensed segments at restrictive temperature . Their mutations were mapped in a novel, identical locus designated crm1 (chromosomal region maintenance) . The crm1 mutants also show the following phenotypes . DNA, RNA, and protein syntheses diminish at restrictive temperature . At permissive temperature, the amount of one particular protein, p25, greatly increases . The mutant growth is hypersensitive to Ca2+ and resistant to protein kinase inhibitors . We cloned the 4.1-kb-long crm1+ gene that rescued the above phenotypes by transformation and determined its nucleotide sequence, which predicts a 1,077-residue protein . Affinity-purified antiserum raised against the crm1+ polypeptide expressed in Escherichia coli detected a 115-kD protein in S . pombe extracts . Genomic Southern hybridization and immunoblotting suggested that the crm1+ product might be highly conserved in distant organisms . Through immunofluorescence microscopy, the crm1+ protein appeared to be principally localized within the nucleus and also at its periphery . We speculate that the crm1+ protein might be one of those nuclear components that modify the chromosome structures or regulate the nuclear environment required for maintaining higher order chromosome structures.

Genes Dev, 1989 Apr, 3(4), 488 - 99
Identification and characterization of an RNA molecule that copurifies with RNase P activity from HeLa cells; Bartkiewicz M et al.; An RNA molecule, 340 nucleotides in length and designated H1 RNA, copurifies with RNase P activity from extracts of HeLa cells or isolated HeLa cell nuclei . When the genomic DNA of various organisms is probed with H1 cDNA in Southern hybridization assays, only mammalian DNA gives a positive signal . The gene coding for H1 RNA in human cells is present in one to three copies per cell . The nucleotide sequence of H1 RNA, which shows little homology to the known sequences of its analogs from prokaryotes and yeast, can be drawn as a two-dimensional, hydrogen-bonded structure that resembles similar structures proposed for the RNA subunit of RNase P from these other sources . Part of the hypothetical structure is virtually identical to structures that can be drawn for analogous RNAs from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and S . octosporus.

Mol Cell Biol, 1989 Mar, 9(3), 983 - 7
Electrophoretic analysis of Histoplasma capsulatum chromosomal DNA; Steele PE et al.; Seven chromosome-sized DNA molecules in the Downs strain of Histoplasma capsulatum were resolved by using chromosome-specific DNA probes in blot hybridizations of contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE) agarose gels . The sizes of the chromosomal DNA bands extended from that of the largest Saccharomyces cerevisiae chromosome to beyond that of the Schizosaccharomyces pombe chromosomes . Under our experimental conditions, the order of the five largest DNA bands was inverted in the FIGE gel relative to the CHEF gel, demonstrating a characteristic of FIGE whereby large DNA molecules may have greater rather than lesser mobility with increasing size . Comparison of the Downs strain with other H . capsulatum strains by CHEF and FIGE analysis revealed considerable variability in band mobility . The resolution of seven chromosome-sized DNA molecules in the Downs strain provides a minimum estimate of the chromosome number.

Microbiol Rev, 1989 Mar, 53(1), 148 - 70
Transformation in fungi; Fincham JR; Transformation with exogenous deoxyribonucleic acid (DNA) now appears to be possible with all fungal species, or at least all that can be grown in culture . This field of research is at present dominated by Saccharomyces cerevisiae and two filamentous members of the class Ascomycetes, Aspergillus nidulans and Neurospora crassa, with substantial contributions also from fission yeast (Schizosaccharomyces pombe) and another filamentous member of the class Ascomycetes, Podospora anserina . However, transformation has been demonstrated, and will no doubt be extensively used, in representatives of most of the main fungal classes, including Phycomycetes, Basidiomycetes (the order Agaricales and Ustilago species), and a number of the Fungi Imperfecti . The list includes a number of plant pathogens, and transformation is likely to become important in the analysis of the molecular basis of pathogenicity . Transformation may be maintained either by using an autonomously replicating plasmid as a vehicle for the transforming DNA or through integration of the DNA into the chromosomes . In S . cerevisiae and other yeasts, a variety of autonomously replicating plasmids have been used successfully, some of them designed for use as shuttle vectors for Escherichia coli as well as for yeast transformation . Suitable plasmids are not yet available for use in filamentous fungi, in which stable transformation is dependent on chromosomal integration . In Saccharomyces cerevisiae, integration of transforming DNA is virtually always by homology; in filamentous fungi, in contrast, it occurs just as frequently at nonhomologous (ectopic) chromosomal sites . The main importance of transformation in fungi at present is in connection with gene cloning and the analysis of gene function . The most advanced work is being done with S . cerevisiae, in which the virtual restriction of stable DNA integration to homologous chromosome loci enables gene disruption and gene replacement to be carried out with greater precision and efficiency than is possible in other species that show a high proportion of DNA integration events at nonhomologous (ectopic) sites . With a little more trouble, however, the methodology pioneered for S . cerevisiae can be applied to other fungi too . Transformation of fungi with DNA constructs designed for high gene expression and efficient secretion of gene products appears to have great commercial potential.

J Gen Microbiol, 1989 Mar, 135 ( Pt 3), 697 - 701
Purification and properties of glycerol:NADP+ 2-oxidoreductase from Schizosaccharomyces pombe; Marshall JH et al.; Glycerol:NADP+ 2-oxidoreductase (EC 1.1.1.156) was isolated from Schizosaccharomyces pombe, purified and characterized . It had an Mr of 57,000, and SDS-PAGE revealed two polypeptides, of Mr 25,000 and 30,000 . Its coenzyme requirement was satisfied exclusively by NADP . The pH optimum for glycerol oxidation was 9.5, for dihydroxyacetone reduction 6.0 . Rates of oxidation with some structurally related diols were three- to six-fold lower than for glycerol, while glyceraldehyde and other carbonyl compounds showed negligible rates of reduction . Neither monovalent nor divalent cations activated the enzyme . Apparent Km and Vmax values were determined . The enzyme is similar to glycerol dehydrogenases isolated from Mucor javanicus and from Dunaliella parva but differs considerably from the glycerol:NAD+ 2-oxidoreductase of S . pombe.

J Cell Sci, 1989 Mar, 92 ( Pt 3), 345 - 8
Observations on ultracentrifuging wild-type and mutant (cdc2.33) cells of Schizosaccharomyces pombe; Khar A et al.; Ultracentrifuging (400,000 g for 4-6 h at 4 degrees C) living wild-type cells of the fission yeast Schizosaccharomyces pombe moves the nucleus towards the ends of the cells but scarcely affects their viability . However, in the long cells produced by growing the mutant cdc2.33 for 4-6 h at the restrictive temperature (36.5 degrees C), ultracentrifuging (as above) gives an intense fluorescence with DAPI in about half of the cytoplasm in about 80% of the cells . This is probably nuclear DNA that has moved into the cytoplasm, both because of the DAPI stain and because it is removed by DNase treatment . These cells ultimately divide and are viable, and we suggest that the extended cytoplasmic DNA returns to the nucleus.

Proc Natl Acad Sci U S A, 1989 Mar, 86(6), 1934 - 8
Use of the DNA polymerase chain reaction for homology probing: isolation of partial cDNA or genomic clones encoding the iron-sulfur protein of succinate dehydrogenase from several species; Gould SJ et al.; The DNA polymerase chain reaction was developed for in vitro amplification of specific DNA sequences, and it has been used for a wide variety of purposes in several fields . We have developed an application of the polymerase chain reaction that is useful for the isolation of partial cDNA or genomic clones of conserved genes . We used this technique to clone the gene encoding the iron protein subunit (27 kDa) of succinate dehydrogenase (EC 1.3.5.1) from several species, including human, rat, Drosophila melanogaster, Arabidopsis thaliana, Schizosaccharomyces pombe, and Saccharomyces cerevisiae . Mixed oligonucleotide primers corresponding to two conserved regions of the protein were used in conjunction with genomic and cDNA templates in the reaction . The primers contained all possible nucleotide combinations that could encode the corresponding peptide sequences . These oligonucleotide mixtures contained 262,144 (2(18} and 8192 (2(13} unique sequences, respectively . Use of the polymerase chain reaction for homology probing allows one to utilize more complex mixtures of oligonucleotides as probes than is possible with filter hybridization screening techniques . In addition, the polymerase chain reaction offers the advantage of synthesizing the DNA product directly, in some cases obviating the need to construct cDNA or genomic libraries . This application of the polymerase chain reaction should be useful not only for the identification of conserved genes in a variety of species but also for the isolation of previously unknown members of gene families.

EMBO J, 1989 Feb, 8(2), 551 - 9
Pre-mRNA splicing mutants of Schizosaccharomyces pombe; Potashkin J et al.; A collection of temperature sensitive (ts-) mutants was prepared by chemical mutagenesis of a wild type Schizosaccharomyces pombe strain . To screen the ts- mutants for pre-mRNA splicing defects, an oligodeoxynucleotide that recognizes one of the introns of the beta-tubulin pre-mRNA was used as a probe in a Northern blot assay to detect accumulation of intron sequences . This screening procedure identified three pre-mRNA splicing mutants from 100 ts- strains . The three mutants are defective in an early step of the pre-mRNA splicing reaction; none accumulate intermediates . The precursors that accumulate at 37 degrees C are polyadenylated . Analysis of the splicing of another pre-mRNA showed that the mutations are not specific for beta-tubulin . The total RNA pattern in the three splicing mutants appears to be normal . In addition, the amounts of the spliceosomal snRNAs are not drastically changed compared to the wild type and splicing of pre-tRNAs is not blocked . Genetic analyses demonstrate that all three splicing mutations are tightly linked to the ts- growth defects and are recessive . Crosses among the mutants place them in three complementation groups . The mutants have been named prp1, prp2 and prp3.

J Cell Biol, 1989 Feb, 108(2), 243 - 53
Essential roles of the RNA polymerase I largest subunit and DNA topoisomerases in the formation of fission yeast nucleolus; Hirano T et al.; A temperature-sensitive lethal mutant nuc1-632 of Schizosaccharomyces pombe shows marked reduction in macromolecular synthesis and a defective nuclear phenotype with an aberrant nucleolus, indicating a structural role of the nuc1+ gene product in nucleolar organization . We cloned the nuc1+ gene by transformation and found that it appears to encode the largest subunit of RNA polymerase I . We raised antisera against nuc1+ fusion polypeptides and detected a polypeptide (approximately 190 kD and 2 x 10(4) copies/cell) in the S . pombe nuclear fraction . By immunofluorescence microscopy, anti-nuc1+ antibody revealed intense staining at a particular nuclear domain previously defined as the nucleolus . The nucleolar immunofluorescence by anti-nuc1+ was faded in nuc1-632 at restrictive temperature and dramatically diminished in the absence of DNA topoisomerases I and II . Thus active RNA polymerase I appears to be required for the formation of the nucleolus as its major component, and DNA topoisomerases appear to be required for the folding of rDNA and RNA polymerase I molecules into the functional organization of nucleolar genes.

Biochem Biophys Res Commun, 1989 Jan 31, 158(2), 392 - 9
A yeast strain with mutated beta-subunits of mitochondrial ATPase-ATPsynthase: high azide and bicarbonate sensitivity of the ATPase activity; Jault JM et al.; A phenotypic revertant with modified beta-subunits of mitochondrial ATPase-ATP synthase has been obtained for the first time by selection from a beta-less mutant of the yeast Schizosaccharomyces pombe . Contrary to the parental mutant, the phenotypic revertant grows on glycerol, has normal respiratory activity and shows immunodetectable beta-subunits . However the kinetic properties of its submitochondrial particles ATPase activity differ markedly from those of the wild strain . The optimal pH is increased by about one unit . The maximal rate of the revertant ATPase activity at pH 8.5 is 4 to 5-fold lower than that of the wild strain, but it can be greatly increased upon addition of bicarbonate whereas the wild strain is completely insensitive to this anion . Furthermore the revertant ATPase activity is much more sensitive to azide inhibition . The results suggest that ADP dissociation is the rate-limiting step of ATP hydrolysis by the revertant.

FEBS Lett, 1989 Jan 30, 243(2), 137 - 40
Genetic transfer of the pigment bacteriorhodopsin into the eukaryote Schizosaccharomyces pombe; Hildebrandt V et al.; The gene encoding for bacterio-opsin (bop gene) from Halobacterium halobium has been introduced in a yeast expression vector . After transformation in Schizosaccharomyces pombe, bacterio-opsin (BO) is expressed and was detected by antisera . The precursor protein of BO (pre-BO) is processed by cleavage of amino acids at the N-terminal end as in H . halobium . Addition of the chromophore, retinal, to the culture medium results in a slight purple colour of the yeast cells indicating the in vivo regeneration of BO to bacteriorhodopsin (BR) and its incorporation into membranes . Therefore, in contrast to the expression in E . coli, isolation of the membrane protein and reconstitution in lipid vesicles is not necessary for functional analysis . The kinetics of the ground state signal of the photocycle BR in protoplasts is demonstrated by flash spectroscopy and is comparable to that of the natural system . The present investigation shows for the first time the transfer of an energy converting protein from archaebacteria to eukaryotes by genetic techniques . This is a basis for further studies on membrane biogenesis, genetics, and bioenergetics by analysis of in vivo active mutants.

Biochim Biophys Acta, 1989 Jan 30, 978(2), 203 - 8
Transport of L-lysine in the fission yeast Schizosaccharomyces pombe; Sychrova H et al.; Systems of L-lysine transport in Schizosaccharomyces pombe are not constitutive, as at no phase of growth in a rich medium is lysine taken up . Transport activity appears only after preincubation of harvested cells with glucose or another suitable source of energy . If cycloheximide is added during this preincubation no transport systems are synthesized . After removal of glucose, the activity of the transport system decays with a half-time of 13 min . The transport of L-lysine into S . pombe cells from the stationary phase of growth preincubated for 60 min with 1% D-glucose is mediated by at least two systems, the high-affinity one with a Kt of 26 mumol/l and Jmax of 4.95 nmol/min per mg dry wt., the low-affinity one with a KT of 1.1 mmol/l and Jmax of 11.8 nmol/min per mg dry wt . The transport of lysine mediated by these two systems proceeds uphill . The high-affinity system has a pH optimum at 4.0-4.2, the accumulation ratio is highest at a cell density 2-5 mg dry wt . per ml and decreases with increasing lysine concentrations . Lysine accumulated by this system does not exit from cells . The only potent competitive inhibitors are L-arginine, L-histidine and D-lysine . The other amino acids tested do not behave as competitive inhibitors . Of the various metabolic inhibitors tested, the most potent were proton conductors and antimycin A.

Nature, 1989 Jan 5, 337(6202), 87 - 90
The gene for the U6 small nuclear RNA in fission yeast has an intron; Tani T et al.; The small nuclear RNAs (snRNAs) are a class of metabolically stable small RNAs present in the nuclei of eukaryotic cells . In mammalian cells, there are six major molecular species (U1 to U6 snRNA), which are complexed with proteins, forming small nuclear ribonucleoprotein particles, snRNPs . Of these, the U1, U2, U4, U5, U6 snRNPs are thought to participate in pre-mRNA splicing as part of the spliceosome . Here, we describe the characterization of the gene coding for the Schizosaccharomyces pombe U6 snRNA . Unexpectedly, the Schiz . pombe U6 RNA gene was found to contain an intron-like sequence of 50 base pairs . Northern blot analysis and RNA sequencing revealed that this intron-like sequence is precisely removed from the transcript . The mature U6 RNA of Schiz . pombe has 77% sequence homology with the mammalian U6 RNA . In Schiz . pombe, it is possible that U6 RNA is not only involved in pre-mRNA splicing, but is also a splicing substrate . This is the first report of an intron in a snRNA gene.

J Cell Sci, 1989 Jan, 92 ( Pt 1), 51 - 6
Characterization of the fission yeast cdc10+ protein that is required for commitment to the cell cycle; Simanis V et al.; We have used antiserum raised against a beta-galactosidase-cdc10+ fusion protein to identify the protein product of the cdc10+ start gene of Schizosaccharomyces pombe . This gene is required for progress through the G1 phase of the cell cycle and for activating processes such as the increase in histone mRNA level in preparation for S phase . The protein has an apparent molecular weight of 87,000 and is phosphorylated on multiple serine residues . The protein remains phosphorylated throughout the mitotic cell cycle and shows no significant steady-state changes in level . The antiserum has also detected a protein similar in size to p87cdc10 in human cells.

Curr Genet, 1989 Jan, 15(1), 71 - 4
An abnormal cell division cycle in an AIR carboxylase-deficient mutant of the fission yeast Schizosaccharomyces pombe; Ishiguro J; Adenine-requiring mutant strains of S . pombe enter the stationary phase after depleting a culture medium of adenine or its analogues . Stationary phase cells of six mutants defective at different stages of the purine nucleotide synthetic pathway were examined for cell volume and DNA content, and then compared in these respects with those of a prototrophic wild-type strain . The cell cycle of the wild-type strain was arrested in the G2 phase (2C state) in the nitrogen rich medium, as is evident from DNA content per cell (0.0425 pg) and cell volume (47.7 microns 3) . An AIR carboxylase-deficient (ade6) mutant strain was found to have an unusual cell volume (307.4 microns 3) and DNA content (0.1187 pg) . By DAPI fluorescence microscopy, each mutant cell was seen to contain only one enlarged nucleus, which indicates the absence of cell populations containing cells in the 4C state of the S phase following nuclear division . It then follows that in ade6 mutant cells, DNA synthesis occurs in the absence of a completed nuclear division . Thus in S . pombe cells, the completion of nuclear division is not necessarily required for the next cycle initiation of DNA synthesis under certain physiological conditions.

Arch Microbiol, 1989, 151(2), 177 - 9
Circadian control of heat tolerance in stationary phase cultures of Schizosaccharomyces pombe; Kippert F; The capacity of stationary phase cultures of Schizosaccharomyces pombe to survive a heat treatment at 55 degrees C is controlled by a circadian rhythm . In a synchronizing light-dark-cycle this rhythm shows a stable phase relationship to the onset of light . In continuous darkness it persists for several cycles without marked damping . The free-running period of about 27 h at 30 degrees C is only slightly longer at 20 degrees C, hence temperature-compensated . These results indicate that S . pombe is a suitable experimental organism for further research into both heat tolerance and circadian rhythms.

J Cell Sci Suppl, 1989, 12, 1 - 8
Regulation of the cell cycle timing of mitosis; Moreno S et al.; Considerable advances have been made recently in our understanding of how the cell cycle timing of mitosis is regulated . This has come about because links have been established between two independent areas of research, one based on a genetic approach using the fission yeast Schizosaccharomyces pombe and the second based on a biochemical approach using Xenopus and starfish oocytes . In this chapter we review work that has identified a number of the mitotic regulating genes in fission yeast and has established links with controls operative in multicellular eukaryotes.

Curr Genet, 1989 Jan, 15(1), 27 - 30
The Saccharomyces cerevisiae RAD2 gene complements a Schizosaccharomyces pombe repair mutation; McCready SJ et al.; Two Saccharomyces cerevisiae genes necessary for excision repair of UV damage in DNA, RAD1 and RAD2, were introduced individually, on a yeast shuttle vector, into seven Schizosaccharomyces pombe mutants - rads 1, 2, 5, 13, 15, 16 and 17 . The presence of the cloned RAD1 gene did not affect survival of any of the S . pombe mutants . The RAD2 gene increased survival of S . pombe rad13 to near the wild-type level after UV irradiation and had no effect on any of the other mutants tested . S . pombe rad13 mutants are somewhat defective in removal of pyrimidine dimers so complementation by the S . cerevisiae RAD2 gene suggests that the genes may code for equivalent proteins in the two yeasts.

Mol Cell Biol, 1989 Jan, 9(1), 224 - 31
Ribosomal protein L7a is encoded by a gene (Surf-3) within the tightly clustered mouse surfeit locus; Giallongo A et al.; The mouse Surfeit locus, which contains a cluster of at least four genes (Surf-1 to Surf-4), is unusual in that adjacent genes are separated by no more than 73 base pairs (bp) . The heterogeneous 5' ends of Surf-1 and Surf-2 are separated by only 15 to 73 bp, the 3' ends of Surf-1 and Surf-3 are only 70 bp apart, and the 3' ends of Surf-2 and Surf-4 overlap by 133 bp . This very tight clustering suggests a cis interaction between adjacent Surfeit genes . The Surf-3 gene (which could code for a basic polypeptide of 266 amino acids) is a highly expressed member of a pseudogene-containing multigene family . By use of an anti-peptide serum (against the C-terminal nine amino acids of the putative Surf-3 protein) for immunofluorescence and immunoblotting of mouse cell components and by in vitro translation of Surf-3 cDNA hybrid-selected mRNA, the Surf-3 gene product was identified as a 32-kilodalton ribosomal protein located in the 60S ribosomal subunit . From its subunit location, gel migration, and homology with a limited rat ribosomal peptide sequence, the Surf-3 gene was shown to encode the mouse L7a ribosomal protein . The Surf-3 gene is highly conserved through evolution and was detected by nucleic acid hybridization as existing in multiple copies (multigene families) in other mammals and as one or a few copies in birds, Xenopus, Drosophila, and Schizosaccharomyces pombe . The Surf-3 C-terminal anti-peptide serum detects a 32-kilodalton protein in other mammals, birds, and Xenopus but not in Drosophila and S . pombe . The possible effect of interaction of the Surf-3 ribosomal protein gene with adjacent genes in the Surfeit locus at the transcriptional or posttranscriptional level or both levels is discussed.

Proc Natl Acad Sci U S A, 1989 Jan, 86(2), 577 - 81
Construction of functional artificial minichromosomes in the fission yeast Schizosaccharomyces pombe; Hahnenberger KM et al.; The centromere DNAs from chromosomes I and III of Schizosaccharomyces pombe have been cloned in an artificial chromosome vector in both budding and fission yeasts . In S . pombe, synthetic linear and circular minichromosomes containing an intact centromere are stable mitotically and behave as independent genetic linkage groups that segregate properly through meiosis . These experiments present a general strategy for the isolation of centromeres from other organisms.

Proc Natl Acad Sci U S A, 1989 Jan, 86(1), 90 - 3
Purification of tropomyosin from Saccharomyces cerevisiae and identification of related proteins in Schizosaccharomyces and Physarum; Liu HP et al.; Tropomyosin is a key component of the contractile systems found in muscle and nonmuscle cells of higher eukaryotes . Based on properties common to all tropomyosins, we have purified a protein from Saccharomyces cerevisiae that resembles tropomyosins from higher cells . The yeast protein remains soluble after heat treatment at 90 degrees C, has an apparent polypeptide molecular weight of 33,000, an isoelectric point of 4.5, a Stokes radius of 3.5 nm, and a sedimentation coefficient of 2.6 S . It binds F-actin in a Mg2+-dependent, KCl-modulated manner, up to a stoichiometry of about 1 polypeptide per 3.0 actin monomers . In all these properties it is very similar to tropomyosins from higher cells . Antigen-affinity-purified antibodies specifically recognize the Mr 33,000 polypeptide among total yeast proteins and crossreact with bovine brain tropomyosin . In addition, the antibodies specifically crossreact with heat-stable Mr 33,000 polypeptides in extracts of Schizosaccharomyces pombe and Physarum polycephalum . Our detection of tropomyosin in lower eukaryotes suggests that they might have contractile systems very similar to those found in higher organisms.

Folia Microbiol (Praha), 1989, 34(4), 279 - 85
Peculiarities of amino acid transport in Schizosaccharomyces pombe: effects of growth medium; Sychrova H et al.; Transport systems for amino acids in the wild-type strain of Schizosaccharomyces pombe are not constitutive . During growth on different media no transport of acidic, neutral and basic amino acids is detectable . To acquire the ability to transport amino acids, cells must be preincubated with a metabolic source of energy, such as glucose . The appearance of transport activity is associated with protein synthesis (suppression by cycloheximide) at all phases of culture growth . After such preincubation the initial rate of amino acid uptake depends on the phase of growth of the culture and on the amount of glucose in the growth medium but not on the nitrogen source used . L-Proline and 2-aminoisobutyric acid are practically not transported under any of the conditions tested.

J Cell Sci Suppl, 1989, 12, 99 - 116
Temporal regulation of cdc2 mitotic kinase activity and cyclin degradation in cell-free extracts of Xenopus eggs; Felix MA et al.; In cleaving Xenopus eggs, the cell division cycle is abbreviated to a rapid succession of S and M phases . During mitosis a number of proteins show increased phosphorylation due to the activation of a histone H1 kinase, the homologue of the cdc2+ gene product of the yeast Schizosaccharomyces pombe . We have studied the regulation of the activity of this enzyme in cell-free extracts of Xenopus eggs . In extracts of activated eggs incubated at 22 degrees C, histone H1 kinase activity shows two peaks of activation and disappearance . Activation occurs in two stages . The first stage requires protein synthesis, whereas the second does not . The second stage of activation involves post-translational activation of the kinase . Kinase activity rises to a peak and then abruptly disappears . Added sea urchin cyclin is degraded at the time of disappearance of kinase activity . The oscillation in kinase activity is then repeated, usually with lower amplitude . Post-translational activation of the kinase requires a membrane-containing particulate cellular component, whose role has yet to be defined . The kinase can still be activated in the presence of EDTA or in the presence of the ATP analogue, 6-dimethylaminopurine, which implies that phosphorylation of the kinase complex is not required for activation . Under these conditions, however, the kinase activity does not show its normal sudden disappearance, and added cyclin is perfectly stable . These observations are consistent with the idea that post-translational activation of the kinase involves protein phosphatase activity, whereas switching off the kinase requires an ATP-Mg2(+)-dependent reaction, perhaps due to protein phosphorylation

J Cell Sci Suppl, 1989, 12, 213 - 29
Gene products required for chromosome separation; Yanagida M; Gene products required for mitotic chromosome separation in the fission yeast Schizosaccharomyces pombe are described . They have been identified by two distinct strategies of mutant isolation, followed by gene cloning and immunochemical characterization of gene products . The roles of four representative genes, namely nda3+, nuc2+, top2+ and dis2+, encoding beta-tubulin, a nuclear scaffold-like protein, DNA topoisomerase II and type-1 protein phosphatase, respectively, are discussed in regard to the mechanisms and control of chromosome separation.

Adv Enzymol Relat Areas Mol Biol, 1989, 62, 227 - 313
The phosphoglycerate mutases; Fothergill-Gilmore LA et al.; The phosphoglycerate mutase family is generally very well documented with respect to structure, evolution, and mode of action . However, a few individuals in the family remain relatively poorly characterized and will clearly require more detailed study . Furthermore, certain aspects of the detailed behavior of these enzymes are, as yet, incompletely understood and require further investigation . Cofactor-dependent monophosphoglycerate mutase and bisphosphoglycerate mutase are undoubtedly very closely related . Their amino acid sequences are strongly similar, they can form active heterodimers, and they catalyze the same three reactions, albeit at substantially different relative rates . Both enzymes catalyze a ping-pong type of reaction with a phosphohistidine intermediate . The presence of an additional phospho ligand at the active site of monophosphoglycerate mutase helps to explain why this enzyme is better at retaining the 2,3-bisphosphoglycerate intermediate and why it is thus more efficient (by a factor of about 10(3)) at catalyzing the interconversion of 3- and 2-phosphoglycerates . The reason why 1,3-bisphosphoglycerate is a better substrate for bisphosphoglycerate mutase than for monophosphoglycerate mutase (by a factor of about 30) is not yet apparent but presumably relates to the relative positioning of the two phospho-binding sites . Both enzymes are equally good as phosphatases when the reaction is activated by 2-phosphoglycollate . Available evidence indicates that these mutases are similar in many respects to the much smaller, cofactor-dependent monophosphoglycerate mutase from Schizosaccharomyces pombe, but further information is required to define the relationship more precisely . Cofactor-independent monophosphoglycerate mutase belongs to a quite distinct branch of the phosphoglycerate mutase family . It is not known at present whether this branch is related divergently or convergently to the cofactor-dependent monophosphoglycerate mutase/bisphosphoglycerate mutase branch . Existing evidence can be argued both ways . For example, the kinetic evidence shows a ping-pong type of reaction and would be consistent with a phosphohistidine intermediate as encountered in the other mutases . Thus the cofactor-independent enzyme may also have arisen by gene duplication--but, in this case, yielding an enzyme of about twice the size, with slightly different residues at the active site and C-terminal tail . An alternative possibility, of course, is that the two branches of the phosphoglycerate mutase family are quite unrelated in a divergent sense and are little more similar structurally than is, for example, the catalytically similar enzyme phosphoglucomutase.(ABSTRACT TRUNCATED AT 400 WORDS)

J Cell Sci Suppl, 1989, 12, 9 - 19
Fission yeast cyclin: subcellular localisation and cell cycle regulation; Alfa CE et al.; Entry into mitosis in the fission yeast Schizosaccharomyces pombe involves the interaction of a number of genes with the major cell cycle control gene, cdc2+ . One of these, cdc13+, encodes a protein with homology to cyclin . By indirect immunofluorescence microscopy using antibodies to the appropriate bacterially-expressed protein, we have shown that both cdc13 and cdc2 are nuclear proteins in S . pombe . Both are localised to a nuclear domain distinct from that occupied by the DAPI-staining chromatin . The immunofluorescence signals of both proteins show a progressive increase during interphase but are undetectable at mitosis . Loss of cdc13 fluorescence at mitosis reflects the destruction of the protein . Thus, it behaves as a classic cyclin . This is not the case for cdc2, the level of which remains constant through the cell cycle . Cells carrying a disrupted copy of the cdc13+ gene fail to accumulate either cdc13 or cdc2 in the nucleus . Cells carrying a disrupted cdc2+ gene fail to accumulate cdc2 but reveal apparently normal levels of cdc13 . cdc13 therefore appears to be required to localise cdc2 to the nucleus but not vice versa . The destruction of cdc13 at mitosis may allow cdc2 to redistribute to the cytoplasm.

J Electron Microsc (Tokyo), 1989, 38(6), 457 - 68
Cell wall formation in regenerating protoplasts of Schizosaccharomyces pombe: study by high resolution, low voltage scanning electron microscopy; Osumi M et al.; The ultrastructure of regenerating cell wall in Schizosaccharomyces pombe protoplasts was studied with a high resolution, low voltage scanning electron microscope (LVSEM) . In contrast to the transmission electron microscopy, the LVSEM images give three-dimensional information on the cell wall regeneration in yeast protoplasts . We found that, after only a few minutes of incubation, the protoplasts began to show protuberances in a unipolar manner, and a fibrilar network was formed asymmetrically which covered the whole surface of the protoplasts after 5 hr . The network consisted of microfibrils about 8 to 10 nm wide, forming flat and wavy bundles of various widths and lengths, up to about 200 nm wide and 1 micron long, mainly made of yeast glucan . Free ends of microfibrils were seldom found . Interfibrillar spaces were progressively filled with granular particles and finally the complete cell wall was formed after 12 hr . The fibrillar network was destroyed by the digestion with beta (1----3)-glucanase . When protoplasts were regenerating in the presence of aculeacin A, the fibrillar networks were not formed, resulting in incomplete cell wall formation . These observations suggest that beta-glucan is the main component of the microfibrils and that it plays an important role in the formation of the cell wall in S . pombe.

Chin J Biotechnol, 1989, 5(1), 55 - 63
Continuous fermentation with yeast (Shizosaccharomyces pombe) floccules for ethanol production; Feng PS et al.; This paper describes an experimental study of continuous fermentation of saccharified starch solution to produce ethanol with yeast particles of a highly flocculent strain of Schizosaccharomyces pombe, which were treated as immobilized microbial cells . Observations were initially made on yeast floccule characteristics during fermentation . An aeration-type fluidized-bed bioreactor that retained virtually all the yeast particles was built and operated continuously for 3 months . Bioreactor operation was found to be quite favorable for this fermentation system . The yeast concentration exceeded 40 g (dry mass)/liter; delivery of a limited amount of supplementary oxygen enhanced throughput to give levels as high as 20-24 g/liter.hr . The kinetic equations were also determined.

Gene, 1988 Dec 30, 74(2), 503 - 15
Cloning and sequence determination of the gene encoding the largest subunit of the fission yeast Schizosaccharomyces pombe RNA polymerase I; Yamagishi M et al.; The gene encoding the largest subunit of RNA polymerase I (SPRPA190) was cloned from the fission yeast Schizosaccharomyces pombe by cross-hybridization with a probe containing part of the corresponding Saccharomyces cerevisiae gene RPA190 . The SPRPA190 gene is present in a single copy per haploid genome and is essential for cell growth . The polypeptide encoded by this gene, as deduced from the nucleotide sequence of the uninterrupted coding frame, consists of 1689 amino acids and its calculated Mr is 189,300 . The amino acid identity between the subunits of the two yeast species is 50% . Amino acid sequence conservation covers the regions previously suggested to be functionally important for the S . cerevisiae enzyme . In addition, two markedly hydrophilic regions recognized in the S . cerevisiae polypeptide can also be recognized in the S . pombe polypeptide in approximately the same positions, even though the amino acid sequences in these regions are diverged from each other . In the 5'-flanking region of the gene, several nucleotide sequence elements are detected which are also found in the two S . pombe ribosomal protein genes so far sequenced.

J Mol Biol, 1988 Dec 20, 204(4), 917 - 25
DNA sequence analysis of the ade6 gene of Schizosaccharomyces pombe . Wild-type and mutant alleles including the recombination host spot allele ade6-M26; Szankasi P et al.; The gene ade6 is located on chromosome III of the fission yeast Schizosaccharomyces pombe . It codes for the enzyme phosphoribosylaminoimidazole carboxylase involved in purine biosynthesis . A DNA fragment of 3043 nucleotides has been sequenced . It complements ade6 mutations when present on plasmids . An uninterrupted open reading frame of 552 amino acid residues was identified . A method for the cloning of chromosomal mutations by repair of gapped replication vectors in vivo has been developed . Twelve ade6 mutant alleles have been isolated . The sequence alterations of four mutant alleles have been determined . Among them are the ade6-M26 recombination hot spot mutation and the nearby ade6-M375 control mutation . Both are G to T base substitutions, converting adjacent glycine codons to TGA termination codons . They are suppressed by defined tRNA nonsense suppressors of the UGA type . The ade6-M26 mutation leads to a tenfold increase of the occurrence of conversion tetrads in comparison with other ade6 mutations . Possible explanations for the M26-induced increase of recombination frequency are discussed in relation to specific features of the nucleotide sequence identified in the region of the M26 mutation.

EMBO J, 1988 Dec 20, 7(13), 4335 - 46
The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog; Nash R et al.; WHI1-1 is a dominant mutation that reduces cell volume by allowing cells to commit to division at abnormally small sizes, shortening the G1 phase of the cell cycle . The gene was cloned, and dosage studies indicated that the normal gene activated commitment to division in a dose-dependent manner, and that the mutant gene had a hyperactive but qualitatively similar function . Mild over-expression of the mutant gene eliminated G1 phase, apparently entirely relaxing the normal G1 size control, but revealing hitherto cryptic controls . Sequence analysis showed that the hyperactivity of the mutant was caused by the loss of the C-terminal third of the wild-type protein . This portion of the protein contained PEST regions, which may be signals for protein degradation . The WHI1 protein had sequence similarity to clam cyclin A, to sea urchin cyclin and to Schizosaccharomyces pombe cdc13, a cyclin homolog . Since cyclins are inducers of mitosis, WHI1 may be a direct regulator of commitment to division . A probable accessory function of the WHI1 activator is to assist recovery from alpha factor arrest; WHI1-1 mutant cells could not be permanently arrested by pheromone, consistent with a hyperactivation of division.

Mol Cell Biol, 1988 Dec, 8(12), 5575 - 80
U2 small nuclear RNA is remarkably conserved between Schizosaccharomyces pombe and mammals; Brennwald P et al.; We report the molecular cloning and sequencing of the most abundant trimethylguanosine-capped small nuclear RNA from the fission yeast Schizosaccharomyces pombe, a highly conserved homolog of mammalian U2 small nuclear RNA . This RNA is 186 nucleotides in length, just 2 nucleotides shorter than its human counterpart; this is in contrast to Saccharomyces cerevisiae U2, which is 1,175 nucleotides long . Moreover, the secondary structure of Schizosaccharomyces pombe U2 is virtually identical to that of mammalian U2, including the 3' half of the RNA, which shows limited primary sequence identity . Northern (RNA) blot analysis revealed that the size of this RNA is conserved not only in fission yeasts but in many organisms, including other ascomycetes.

Curr Genet, 1988 Dec, 14(6), 573 - 82
Organization of ribosomal RNA genes in the fungus Cochliobolus heterostrophus; Garber RC et al.; The genes encoding the 17S, 5.8S and 25S ribosomal RNAs in the Ascomycete Cochliobolus heterostrophus were cloned and analyzed . They are arranged in tandemly repeated units (rDNA) either 9.0 or 9.15 kilobases in length, depending upon the strain . The 5S rRNA genes are not part of the tandemly repeated rDNA . Instead, many and perhaps all of the 5S genes are dispersed in the genome, as they are in the fungi Neurospora, Aspergillus and Schizosaccharomyces . Comparative restriction maps of the rDNA from C . heterostrophus and other filamentous fungi and yeasts are presented . A survey of rDNAs from twenty-three field isolates of C . heterostrophus collected worldwide demonstrated that each isolate has one or the other of two rDNA forms, which differ in length and in the presence or absence of at least three restriction enzyme sites . The differences are all located in spacer DNA outside the coding regions for the rRNA genes . Heterogeneity in the rDNA repeat was not observed within any single isolate . The copy number of the rRNA gene cluster in C . heterostrophus is approximately 130 per haploid genome.

Mol Gen Genet, 1988 Dec, 215(1), 87 - 93
Observations on integrative transformation in Schizosaccharomyces pombe; Grimm C et al.; Three different Schizosaccharomyces pombe strains have been transformed with a circular or linearized non-ars plasmid carrying the ura4+ gene as a selectable marker . The first strain shows full homology between the genomic ura4-294 gene (point mutation) and the marker gene on the plasmid . The second strain carries a 600 bp deletion (ura4-D6) that decreases homology between plasmid and chromosome . No homology remains in the third strain which has a complete deletion of the ura4 gene on the chromosome (ura4-D18) . When sequence homology exists between transforming DNA and the chromosomal ura4 region, gene conversion is strongly preferred over integration of the circular plasmid . Reduction of the length of homology leads to a decrease of transformation frequencies, and homology dependent as well as a minority of homology independent integrations are observed . In the complete absence of homology two rare types of transformants are encountered: either the circular plasmid replicates autonomously, although it is devoid of an ars sequence, or alternatively the plasmid integrates into the genome at various positions . Transformation with plasmid cut within the coding region of ura4 can lead to tandemly arranged multiple integrations, when no homology exists between the free ends and the chromosome . The integrations occur at the ura4 locus, when homology is retained between plasmid and chromosome, and at various sites in the genome of the strain with a complete deletion of the ura4 gene . The results suggest that homology dependent events (conversion, integration) are strongly preferred in transformation of S . pombe with non-ars plasmids . In addition low frequency integration by illegitimate recombination is observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1988 Dec, 215(1), 81 - 6
Genetic engineering of Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker; Grimm C et al.; A system is described for gene disruption and replacement in Schizosaccharomyces pombe based on the homologous selectable marker, ura4, the structural gene for orotidine-5'-phosphate decarboxylase . The presence of a single copy of the wild-type gene can rescue a ura4 auxotrophic mutant . Furthermore, ura4- cells can be selected for in the presence of 5-fluoroorotic acid (5-FOA) . This allows a convenient means of selecting for both forward and backward mutations . The sequence of a 1.8 kb HindIII fragment which contains the functional gene is reported . It encodes a single open reading frame of 264 amino acids which shows considerable conservation with the orotidine-5'-phosphate (OMP) decarboxylases from other organisms . The ura4 transcript is approximately 850 nucleotides long . It begins 51 bp upstream of the protein coding sequence and is unusual in that transcription termination occurs at or very close to the translational stop codon . To facilitate the use of ura4 in gene disruption experiments we have also constructed a novel strain of S . pombe called ura4-D18, in which the 1.8 kb HindIII fragment has been deleted from the chromosome . Using a combination of this strain and vectors containing ura4 as a selectable marker, we present a general method for targeting recombination events to the chromosomal locus under investigation.

Can J Microbiol, 1988 Dec, 34(12), 1338 - 43
Pseudo-exponential growth in length of the fission yeast, Schizosaccharomyces pombe; Miyata H et al.; The growth patterns of individual cells of the fission yeast (Schizosaccharomyces pombe wild-type cells, strain 972 h-; cells exposed to hydroxyurea; and cdc mutants, 11-123, 2-33) were investigated by time-lapse photomicrography . Wild-type cells showed one, two, or three linear-growth segments followed by a constant-length stage . Cells with two segments were most frequent . Hydroxyurea cells that divided as oversized cells (about three times the birth length) had three linear-growth segments in a cycle . Mutant cdc11-123 cells did not divide but had a constant-length stage separating the cycles; both the first and second cycles consisted of two linear-growth segments, and cells were oversized at the second constant-length stage (about 3.5 times the birth length) . Elongating cdc2-33 cells that did not divide and were oversized (about five times the birth length) while under observation, showed four linear-growth segments . Cells of all strains showed 30 to 40% increase in growth rate at the rate-change point and maintained approximate exponential (pseudo-exponential) growth . We conclude that the normal growth pattern of individual fission-yeast cells is the pseudo-exponential pattern.

J Mol Evol, 1988 Dec-1989 Feb, 28(1-2), 113 - 24
Phylogenetic calibration of the 5' terminal domain of large rRNA achieved by determining twenty eucaryotic sequences; Qu LH et al.; Due to their high information content and their particular mode of variation, large rRNA molecules potentially represent powerful indicators of phylogenetic relationships . Even partial sequences may suffice to generate reliable estimations, provided they correspond to well-chosen portions of the molecule . We have systematically analyzed a specific portion of the large rRNA (the region extending over nearly 400 nucleotides from the 5' end) as a general index of eucaryotic phylogeny . By means of fast and direct rRNA sequencing, we have determined the sequence of this region for 20 additional eucaryotes, including several representatives of each vertebrate class, an invertebrate metazoan (mussel), a fungus (Schizosaccharomyces pombe), and three higher plants . Comparative treatment of these new data and previously reported rRNA sequences shows that this region can serve as an indicator of eucaryotic phylogeny for evaluating both long-range and short-range relationships . Its conservative domains appear to possess a rather uniform rate of nucleotide changes in all the eucaryotic lineages analyzed and the phylogenetic tree we derived agrees with classical views.

J Bacteriol, 1988 Dec, 170(12), 5968 - 70
Lysine biosynthesis pathway and biochemical blocks of lysine auxotrophs of Schizosaccharomyces pombe; Ye ZH et al.; The alpha-aminoadipate (AA) pathway for the biosynthesis of lysine was investigated in the wild type and in lysine auxotrophs of the fission yeast Schizosaccharomyces pombe . Of the eight enzyme activities of the AA pathway that have been examined so far, six were present in the extract of wild-type S . pombe cells . Growth response to AA and accumulation studies indicated that three lysine auxotrophs, the lys2-97, lys4-95, and lys8-1 strains, were blocked before the AA step and that four lysine auxotrophs, the lys1-131, lys3-37, lys6-3, and lys7-2 strains, were blocked after the AA step . Among the mutants investigated, the lys2-97 mutant exhibited an enzyme lesion at the cis-homoaconitate hydratase step, the lys1-131 and lys7-2 mutants exhibited lesions at the AA reductase step, and lys3-37 exhibited a lesion at the saccharopine dehydrogenase step . These results demonstrated the basic similarity of the AA pathway in S . pombe and Saccharomyces cerevisiae.

Mol Gen Genet, 1988 Dec, 215(1), 26 - 31
Isolation and characterization of Schizosaccharomyces pombe mutants phenotypically similar to ras1-; Fukui Y et al.; We isolated mutants of Schizosaccharomyces pombe which have deformed cell morphology, are deficient in conjugation and poor in sporulation . This phenotype is characteristic of the ras1 defective mutant previously identified . Tests of the mutants for allelism using cell fusion showed that they define five complementation groups, one of which is ras1 itself . The others are named ral1 through ral4 (ras like) . Mutants in ral3 or ral4 conjugate at a very low frequency, while the others apparently do not conjugate at all . Plasmid clones complementing ral1, ral2 or ral3, which apparently carry the respective gene, were isolated from S . pombe genomic libraries . Multiple copies of either the ral2 or the ral3 gene could partially restore mating ability in ral1- strains . Multiple copies of the ras1 gene could partially restore mating ability in ral1- and ral2- strains . These results suggest that the ral1, ral2 and ras1 genes may function in a common pathway in that order . The ral3 gene may influence this pathway . Analysis of these gene products will aid identification of factors which interact with Ras proteins.

Mol Pharmacol, 1988 Dec, 34(6), 755 - 60
Evidence that DNA topoisomerase I is necessary for the cytotoxic effects of camptothecin; Eng WK et al.; The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are both sensitive to camptothecin, an inhibitor of DNA topoisomerase I . An S . cerevisiae DNA repair mutant, rad52, is hypersensitive to the drug . In both species, topoisomerase I mutants totally lacking the enzyme are completely resistant to the drug . A strain with a mutation leading to a temperature-sensitive topoisomerase I exhibits temperature dependence in its in vivo response to camptothecin . A strain carrying a plasmid that overproduces topoisomerase I is hypersensitive to the drug . The rad52 mutant is killed by overproduction of the enzyme, even in the absence of the drug . The response of several of these strains to camptothecin analogs, to DNA topoisomerase II inhibitors, and to other drugs is reported . The cytotoxic effects of camptothecin are discussed in terms of the drug extending the lifetime of a topoisomerase I-DNA covalent intermediate, which is recognized as DNA damage by a DNA repair system.

Nucleic Acids Res, 1988 Nov 11, 16(21), 10131 - 52
The sequence of U3 from Schizosaccharomyces pombe suggests structural divergence of this snRNA between metazoans and unicellular eukaryotes; Porter GL et al.; We have cloned and sequenced one of the two genes encoding a 255 nucleotide small nuclear RNA from the fission yeast Schizosaccharomyces pombe . Based on the presence of four regions of primary sequence conservation and a predicted secondary structure similar to that previously proposed for human U3, we conclude that this molecule is the fission yeast homologue of this mammalian snRNA . The 5' one-third of fission yeast U3 is, however, unable to form a single stable hairpin as proposed for this region of the human RNA, but rather folds into two stem-loop structures . By analogy to fission yeast U3, we propose revised secondary structures containing two hairpins for this portion of the U3-like snRNAs from Saccharomyces cerevisiae and Dictyostelium discoideum . Thus, our data suggest that the structure of U3 snRNA has diverged in lower and higher eukaryotes.

Nature, 1988 Oct 6, 335(6190), 547 - 50
The Caenorhabditis elegans lin-12 gene encodes a transmembrane protein with overall similarity to Drosophila Notch; Yochem J et al.; The lin-12 gene seems to control certain binary decisions during Caenorhabditis elegans development, from genetic and anatomical studies of lin-12 mutants that have either elevated or reduced levels of lin-12 activity . We report here the complete DNA sequence of lin-12: 13.5 kilobases (kb) derived from genomic clones and 4.5 kb from complementary DNA clones . It is of interest that the predicted product is a putative transmembrane protein, given that many of the decisions controlled by lin-12 activity require cell-cell interactions for the correct choice of cell fate . In addition, the predicted lin-12 product may be classified into several regions, based on amino acid sequence similarities to other proteins . These include extensive overall sequence similarity to the Drosophila Notch protein, which also is involved in cell-cell interactions that specify cell fate; a repeated motif found in proteins encoded by the yeast cell-cycle control genes cdc10 (Schizosaccharomyces pombe) and SWI6 (Saccharomyces cerevisiae); and a repeated motif exemplified by epidermal growth factor, found in many mammalian proteins.

Curr Genet, 1988 Oct, 14(4), 375 - 9
The primary structure of the leu1+ gene of Schizosaccharomyces pombe; Kikuchi Y et al.; A DNA fragment which carries the leu1 gene encoding beta-isopropylmalate dehydrogenase in Schizosaccharomyces pombe has been isolated by complementation of an E . coli leuB mutation . This 1.5 kb DNA fragment complements not only the S . pombe leu1 mutation, but also the S . cerevisiae leu2 mutation . The nucleotide sequence of the essential part of the leu1 gene and its flanking regions was determined . This sequence contains an open reading frame of 371 codons, from which a protein having a Mr = 39,732 can be predicted . The deduced amino acid sequence and its codon usage were compared with those of the S . cerevisiae LEU2 protein . The cloned DNA will be a useful marker when transforming S . pombe.

J Biol Chem, 1988 Sep 15, 263(26), 12832 - 5
Sulfide stabilization of the cadmium-gamma-glutamyl peptide complex of Schizosaccharomyces pombe; Reese RN et al.; Addition of cadmium salts to the growth medium of Schizosaccharomyces pombe leads to synthesis of a Cd.gamma-Glu peptide complex and an enhanced generation of sulfide ions . The gamma-Glu peptide complex functions in the detoxification of heavy metal ions . Native Cd.gamma-Glu peptide complexes contain acid-labile sulfide in the metal-thiolate cluster . Two forms of the complex exist differing primarily in their sulfide content . Sulfide concentrations up to 0.2 and 1.2 mol/mol of peptide were observed in native isolates of forms I and II, respectively . Addition of sulfide to the low sulfide form I converted it to a complex similar to form II . Properties of the Cd.gamma-Glu peptide complex were altered by the incorporation of sulfide ions . Sulfide-dependent electronic transitions in the ultraviolet were evident, and the absorbance maximum of the transition was related to the sulfide content and the bound metal ion . High sulfide forms of the Cd and Zn complexes exhibited absorbance peaks at 318 nm and 255 nm, respectively . Incorporation of sulfide into the Cd.gamma-Glu peptide complex imparted greater thermodynamic stability to the complex, an increased Stokes radius, and an enhanced Cd(II) binding capacity . Sulfide generation may be a cellular response in part to enhance the effectiveness of the gamma-Glu peptide system for Cd(II) detoxification.

FEBS Lett, 1988 Sep 12, 237(1-2), 31 - 4
Two changes of the same nucleotide confer resistance to diuron and antimycin in the mitochondrial cytochrome b gene of Schizosaccharomyces pombe; Weber S et al.; Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and antimycin, both inhibitors of mitochondrial respiration, block electron flow between cytochromes b and c1 . Mutants resistant to either drug have been selected using Schizosaccharomyces pombe strains with an extrachromosomally inherited mutator . In analogy to Saccharomyces cerevisiae these mutational sites were assumed to map in the cytochrome b gene . DNA sequence analysis showed that two changes in the same nucleotide are responsible for resistance to antimycin and diuron . Analysis of resistant and sensitive progeny of crosses between the mutants and the wild type confirmed the correlation between mutational alteration and resistant phenotype.

Arch Biochem Biophys, 1988 Sep, 265(2), 381 - 9
Cu(I) binding to the Schizosaccharomyces pombe gamma-glutamyl peptides varying in chain lengths; Mehra RK et al.; The metal-gamma-glutamyl peptide complex of Schizosaccharomyces pombe is an oligomer of peptides of the general structure (gamma-Glu-Cys)n-Gly with n defining the number of dipeptide repeats . The complexes induced with either cadmium or copper salts are heterogeneous with respect to the number of repeat units or n . Peptides isolated from two preparations of the Cd-gamma-Glu complex by reverse-phase HPLC at low pH were of an n range of 2 to 6 with n3 and n4 peptides being predominant . In addition to peptides of the mentioned structure, peptides of n3 and n4 without the terminal Gly were isolated . These n3 and n4 desGly peptides were present in an abundance of about 10-20% of the concentration of the parent peptide . Peptides of unique n were studied in Cu(I) reconstitution experiments in an attempt to understand the significance of the peptide length heterogeneity in the oligomeric metal-thiolate cluster . Cu-gamma-Glu complexes were formed with each peptide as determined by the characteristic 260-nm shoulder in the ultraviolet absorption spectrum and luminescence indicative of Cu(I)-thiolate coordination in a solvent-inaccessible environment . Cluster formation also occurs with desGly peptides, so the carboxyl-terminal Gly is not critical for cluster formation . Maximal Cu binding stoichiometry with n3 and n4 peptides was markedly less than the maximal Cu(I) stoichiometry of a peptide mixture or the native complex . Cu ions in complexes formed with unique n peptides were more reactive with bathocuproine than Cu ions in complexes with a peptide n mixture . The results suggest that metal-peptide complexes consisting of peptides differing in n probably exist and not all metal-peptide complexes have the same n peptide constituents.

Curr Genet, 1988 Sep, 14(3), 235 - 40
Multiple phosphorylated forms of the product of the fission yeast cell division cycle gene cdc2+; Potashkin JA et al.; The 34 kilodalton protein product (p34) of the cdc2+ cell cycle control gene of Schizosaccharomyces pombe was expressed in bacteria . Monoclonal antibodies raised against this protein are capable of immunoprecipitating p34cdc2 from yeast lysates . Immunoprecipitates of {35S}methionine- and {32P}orthophosphate-labeled p34cdc2 were analyzed by two-dimensional gel electrophoresis . The cdc2+ gene product is homogeneous in size but resolves into seven species of differing charge . At least four of these species are phosphorylated . Phosphoamino acid analysis reveals that phosphorylation occurs mainly on threonine residues . The pattern of p34 phosphorylation is unaltered at the nonpermissive temperature in strains carrying temperature sensitive alleles of weel-50 and ran1-114 or in a strain overproducing the ran1+ gene product.

J Mol Biol, 1988 Aug 20, 202(4), 725 - 34
Two distinct mechanisms for deletion in mitochondrial DNA of Schizosaccharomyces pombe mutator strains . Slipped mispairing mediated by direct repeats and erroneous intron splicing; Ahne A et al.; Mutator strains of the fission yeast Schizosaccharomyces pombe produce mitochondrial respiratory deficient mutants at a high rate, and roughly 20% of these mutants carry deletions in the range of 50 to 1500 base-pairs . To elucidate the mechanism of deletion we have sequenced ten deletion mutants in the mosaic gene encoding apocytochrome b (cob) and three in the split gene coding for the first subunit of cytochrome c oxidase (cox1) . Of 13 deletions, ten are correlated with the presence of direct repeats, which could promote deletions by slipped mispairing during DNA replication . In some of these mutants, the termini are located in possible DNA secondary structures . In three independently isolated mutants with identical deletions in the cob gene, the 5' deletion endpoint coincides with the 3' splice point of the intron, whereas the 3' endpoint of the deletion exhibits pronounced homology with the 5' splice point of the intron . This result suggests that these deletions might be initiated by erroneous RNA splicing.

Mol Gen Genet, 1988 Aug, 213(2-3), 529 - 34
The role of sterility genes (ste and aff) in the initiation of sexual development in Schizosaccharomyces pombe; Sipiczki M; Haploid homothallic strains of Schizosaccharomyces pombe with mutations in any of nine "sterility genes" (ste) do not mate with wild-type fertile strains . Those defective in genes ste1 to ste4 and ste7 to ste9 are also deficient in meiosis and sporulation . I found that the ste1, ste3 and ste8 genes act very early in the sexual development, presumably before the pat1-controlled conjugation-specific event . ste5 and ste6 exert their function downstream of pat1 in the initiation of conjugation and do not play any role in the meiotic pathway . ste2, ste4, ste7 and ste9 are involved in both sexual pathways: they seem to act downstream of pat1 in conjugation but upstream of pat1 in the initiation of meiosis . A new gene, aff1, whose defective allele suppresses the pat1-114-provoked haploid sporulation and arrest of vegetative growth is also described . It is supposed that the aff1+ gene product participates in a cascade of regulatory events, as a factor antagonistic to pat1.

EMBO J, 1988 Aug, 7(8), 2321 - 7
Involvement of cdc13+ in mitotic control in Schizosaccharomyces pombe: possible interaction of the gene product with microtubules; Booher R et al.; Previous genetic studies have shown that the fission yeast cdc13+ gene product interacts closely with the cdc2+ protein kinase during mitosis . Here, we have cloned the cdc13+ gene from a S . pombe gene bank by complementation of the temperature-sensitive defect of a cdc13-117 mutant strain . The complementing activity was localized to a 1.9-kb XbaI-NsiI DNA fragment, and nucleotide sequencing revealed a 1446-bp open reading frame . The predicted amino acid sequence contained 482 residues and was not homologous to any protein in a protein database . The cdc13+ gene function was confirmed to be essential for cell division since cells carrying a cdc13 null allele arrested with a cdc phenotype . However, unlike any existing temperature-sensitive cdc13 mutants, cdc13 null mutants arrested in G2 without septa or condensed chromosomes indicating that cdc13+ gene function is required at or prior to the initiation of mitotis . cdc13-117 mutant strains were found to be hypersensitive to the tubulin inhibitor thiabendazole . This observation suggests that the cdc13+ gene product, which is required for mitotic initiation, may interact with microtubules.

FEBS Lett, 1988 Jul 4, 234(1), 95 - 9
Synthesis of Saccharomyces cerevisiae invertase by Schizosaccharomyces pombe; Sanchez Y et al.; In order to gain information on the ability of the glycosylation system of Schizosaccharomyces pombe to process heterologous glycoproteins, the expression of Saccharomyces cerevisiae invertase in the former yeast was studied . Sc . pombe cells are able to produce enzymatically active invertase from the S . cerevisiae SUC2 gene introduced by transformation and the enzyme is glycosylated and secreted into the cell wall . However, Sc . pombe transformants do not glycosylate the heterologous enzyme as their own invertase since it is not bound by the lectin from Bandeiraea simplicifolia seeds, which indicates the absence of terminal galactose residues . Moreover, the electrophoretic mobility of the heterologous invertase is similar to that of the large enzyme from S . cerevisiae, both in its native form and after being deglycosylated with Endo H . These results suggest that the polypeptide chain of S . cerevisiae invertase is the primary factor for the glycosylation in Sc . pombe cells.

Genetics, 1988 Jul, 119(3), 491 - 7
Genetic and physical analysis of the M26 recombination hotspot of Schizosaccharomyces pombe; Ponticelli AS et al.; The ade6-M26 mutation of Schizosaccharomyces pombe has previously been reported to stimulate ade6 intragenic meiotic recombination . We report here that the ade6-M26 mutation is a single G----T nucleotide change, that M26 stimulated recombination within ade6 but not at other distinct loci, and that M26 stimulated meiotic but not mitotic recombination . In addition, M26 stimulated recombination within ade6 when M26 is homozygous; this result demonstrates that a base-pair mismatch at the M26 site was not required for the stimulation . These results are consistent with the ade6-M26 mutation creating a meiotic recombination initiation site.

EMBO J, 1988 Jul, 7(7), 2203 - 9
Sequence analysis of ARS elements in fission yeast; Maundrell K et al.; Chromosomal DNA of Schizosaccharomyces pombe contains sequences with properties analogous to ARS elements of Saccharomyces cerevisiae . Following Sau3A fragmentation of the S . pombe genome we have recovered a number of such fragments in an M13-based shuttle vector, suitable for subsequent sequence analysis . The complete nucleotide sequence has been obtained for eight ARS+ inserts derived from the Sau3A cloning and for the ARS present in pFL20 isolated previously by Losson and Lacroute (Cell, 32, 371-377, 1983) . The Sau3A clones are single fragments between 0.8 and 1.8 kb . No ARS+ clones smaller than this were recovered even though the average size Sau3A fragment in S . pombe is approximately 200-300 bp . The sequence analysis revealed that all clones are AT-rich (69-75% A + T residues), and all contain a particularly AT-rich 11 bp core element represented by the consensus sequence 5' (A/T)PuTT-TATTTA(A/T) 3' . Deletion mapping indicates that the consensus in all cases is in the vicinity of a functional ARS domain . However precise excision of the consensus by in vitro mutagenesis has little effect on ARS activity as judged by the transformation assay . We argue that the association of the consensus with the ARS domain occurs too reproducibly to be explained by chance alone . We suggest that although it may not be essential for the extrachromosomal maintenance of plasmids in S . pombe, the consensus does have a function in situ in the chromosome and thus is always present as a cryptic sequence in the isolated ARS element.

Mol Cell Biol, 1988 Jul, 8(7), 2976 - 9
Invariant phosphorylation of the Saccharomyces cerevisiae Cdc28 protein kinase; Hadwiger JA et al.; The phosphorylation level of the Saccharomyces cerevisiae Cdc28 protein remained invariant under conditions that resulted in cell cycle arrest in the G1 phase and loss of Cdc28-specific protein kinase activity when the activity was assayed in vitro . These results are in contrast to the proposed regulation of the homologous Cdc2 protein kinase of Schizosaccharomyces pombe.

Science, 1988 Jun 10, 240(4858), 1439 - 43
Yeast: an experimental organism for modern biology; Botstein D et al.; The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have become popular and successful model systems for understanding eukaryotic biology at the cellular and molecular levels . The reasons for this success are experimental tractability, especially in applying classical and molecular genetic methods to associate genes with proteins and functions within the cell.

Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4315 - 9
Small ribonucleoproteins in Schizosaccharomyces pombe and Yarrowia lipolytica homologous to signal recognition particle; Poritz MA et al.; We have partially purified ribonucleoproteins (RNPs) from Schizosaccharomyces pombe and Yarrowia lipolytica with properties resembling those of mammalian signal recognition particle (SRP) . In both species of yeast we have identified a single major RNA species in the size range of SRP RNA (256 nucleotides in S . pombe and 270 nucleotides in Y . lipolytica) present in postribosomal salt extracts of the cytoplasm . The RNPs containing these RNAs sediment in sucrose gradients at 11 S and 10 S for S . pombe and Y . lipolytica, respectively . Analysis of genomic clones of these RNAs has revealed that (i) they are encoded by single copy genes; (ii) they share two short conserved sequences that match the A and B boxes defined for polymerase III promoters; (iii) they can be folded into secondary structures that closely match that defined by phylogenetic analysis of higher eukaryotic SRP RNAs; and (iv) they show primary sequence conservation in short regions predicted to be single stranded . Both of the yeast RNAs bind under stringent conditions to canine SRP proteins . Most importantly, RNase protection of the S . pombe RNA by the individual canine SRP proteins, p19 and p68/72, shows that the proteins recognize homologous elements of the mammalian and yeast RNA . Taken together these data suggest strongly that we have identified yeast SRP homologues.

J Biol Chem, 1988 May 5, 263(13), 6051 - 7
Characterization of the gene for fructose-1,6-bisphosphatase from Saccharomyces cerevisiae and Schizosaccharomyces pombe . Sequence, protein homology, and expression during growth on glucose; Rogers DT et al.; We have determined the nucleotide sequence of the gene for fructose-1,6-bisphosphatase from both Saccharomyces cerevisiae and Schizosaccharomyces pombe . The predicted protein sequence for fructose-1,6-bisphosphatase from S . cerevisiae contains 347 amino acids and has a molecular weight of 38,100; that from S . pombe, contains 346 amino acids and has a molecular weight of 38,380 . Comparison of these amino acid sequences with each other and that of pig kidney fructose-1,6-bisphosphatase shows several regions of strong homology separated by regions of divergence . These homologous regions are likely candidates for functional domains . A gene cassette was constructed for fructose-1,6-bisphosphatase from S . cerevisiae and the gene cassette expressed from the regulated PHO5 and GAL1 promoters of yeast . Yeast cells expressing fructose-1,6-bisphosphatase, while growing on glucose, accumulated large amounts of enzyme intracellularly, suggesting that glucose-regulated proteolytic inactivation does not operate efficiently under these conditions . Growth on glucose was not inhibited by the expression of fructose 1,6-bisphosphatase.

EMBO J, 1988 May, 7(5), 1465 - 73
Cold-sensitive and caffeine-supersensitive mutants of the Schizosaccharomyces pombe dis genes implicated in sister chromatid separation during mitosis; Ohkura H et al.; We isolated novel classes of Schizosaccharomyces pombe cold-sensitive dis mutants that block mitotic chromosome separation (nine mapped in the dis1 gene and one each in the dis2 and dis3 genes) . Defective phenotype at restrictive temperature is similar among the mutants; the chromosomes condense and anomalously move to the cell ends in the absence of their disjoining so that they are unequally distributed at the two cell ends . Synchronous culture analyses indicate that the cells can enter into mitosis at normal timing but become lethal during mitosis . In comparison with the wild-type mitosis, defects are found in the early spindle structure, the mitotic chromosome structure, the poleward chromosome movement by the spindle elongation and the telophase spindle degradation . The dis mutants lose at permissive temperature an artificial minichromosome at higher rates than occur in the wild type . We found that all the dis mutants isolated are supersensitive to caffeine at permissive temperature . Furthermore, the mutant cells in the presence of caffeine produce a phenotype similar to that obtained at restrictive temperature . We suggest that the dis genes are required for the sister chromatid separation at the time of mitosis and that caffeine might affect the dis gene expression . We cloned, in addition to the dis2+ and dis3+ genes, multicopy extragenic suppressor sequences which complement dis1 and dis2 mutations . A complex regulatory system may exist for the execution of the dis+ gene functions.

Nature, 1988 Apr 7, 332(6164), 509 - 14
A specific inhibitor of the ran1+ protein kinase regulates entry into meiosis in Schizosaccharomyces pombe; McLeod M et al.; In fission yeast, meiosis is initiated by transcriptional activation of the mei3+ gene, under the combined influence of the four mating-type genes . The product of the mei3+ gene acts as a critical meiotic inducer by binding non-covalently to a newly identified protein kinase encoded by the ran1+ gene and inhibiting its enzymatic activity . Inactivation of the ran1+ protein kinase is both necessary and sufficient to divert a vegetative cell from mitotic division to meiotic differentiation.

J Cell Biol, 1988 Apr, 106(4), 1171 - 83
A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase; Hirano T et al.; A temperature-sensitive mutant nuc2-663 of the fission yeast Schizosaccharomyces pombe specifically blocks mitotic spindle elongation at restrictive temperature so that nuclei in arrested cells contain a short uniform spindle (approximately 3-micron long), which runs through a metaphase plate-like structure consisting of three condensed chromosomes . In the wild-type or in the mutant cells at permissive temperature, the spindle is fully extended approximately 15-micron long in anaphase . The nuc2' gene was cloned in a 2.4-kb genomic DNA fragment by transformation, and its complete nucleotide sequence was determined . Its coding region predicts a 665-residues internally repeating protein (76.250 mol wt) . By immunoblots using anti-sera raised against lacZ-nuc2+ fused proteins, a polypeptide (designated p67; 67,000 mol wt) encoded by nuc2+ is detected in the wild-type S . pombe extracts; the amount of p67 is greatly increased when multi-copy or high-expression plasmids carrying the nuc2+ gene are introduced into the S . pombe cells . Cellular fractionation and Percoll gradient centrifugation combined with immunoblotting show that p67 cofractionates with nuclei and is enriched in resistant structure that is insoluble in 2 M NaCl, 25 mM lithium 3,5'-diiodosalicylate, and 1% Triton but is soluble in 8 M urea . In nuc2 mutant cells, however, soluble p76, perhaps an unprocessed precursor, accumulates in addition to insoluble p67 . The role of nuc2+ gene may be to interconnect nuclear and cytoskeletal functions in chromosome separation.

Genome, 1988 Apr, 30(2), 211 - 7
Transformation of Drosophila melanogaster with a suppressor tRNA gene (Sup3e tRNA(SerUGA)) from Schizosaccharomyces pombe; Molnar CM et al.; P-element mediated transformation was utilized to introduce a suppressor tRNA gene (Sup3e tRNA(UGASER)) from Schizosaccharomyces pombe into Drosophila melanogaster . Thirteen independently transformed lines were characterized as to the number of cytological locations of the transposons . It was ascertained that the suppressor tRNA gene of interest was introduced into each transformed strain . The helper P element used (p pi 25.1) allows further transposition to occur, and it was determined that from one to seven copies of the heterologous tRNA(UGASER) gene per strain were present among the respective transformed strains . The number of transposons per transformed line was established by in situ hybridization to salivary gland chromosomes as well as by Southern hybridization analyses and there was good agreement in the totals determined by these two techniques.

EMBO J, 1988 Apr, 7(4), 985 - 93
A gene which encodes a predicted protein kinase can restore some functions of the ras gene in fission yeast; Nadin-Davis SA et al.; The ras1- mutation of the fission yeast Schizosaccharomyces pombe interferes with sexual differentiation by preventing conjugation and causing inefficient sporulation . From a gene library, we have isolated a gene, byr1+, which when in high copy number restores efficient sporulation to ras1- strains . byr1+ encodes a putative 340-amino acid protein product, the sequence of which strongly suggests that it functions as a protein kinase . Gene disruption experiments show that loss of byr1+ function does not interfere with mitotic growth but it completely prevents both conjugation and sporulation . byr1 is thus another important gene in the sexual differentiation pathway and we believe that at least part of ras1 function is to act directly or indirectly through byr1 to modulate protein phosphorylation.

Mol Cell Biol, 1988 Apr, 8(4), 1469 - 73
An electrophoretic karyotype of Neurospora crassa; Orbach MJ et al.; A molecular karyotype of Neurospora crassa was obtained by using an alternating-field gel electrophoresis system which employs contour-clamped homogeneous electric fields . The migration of all seven N . crassa chromosomal DNAs was defined, and five of the seven molecules were separated from one another . The estimated sizes of these molecules, based on their migration relative to Schizosaccharomyces pombe chromosomal DNA molecules, are 4 to 12.6 megabases . The seven linkage groups were correlated with specific chromosomal DNA bands by hybridizing transfers of contour-clamped homogeneous electric field gels with radioactive probes specific to each linkage group . The mobilities of minichromosomal DNAs generated from translocation strains were also examined . The methods used for preparation of chromosomal DNA molecules and the conditions for their separation should be applicable to other filamentous fungi.

Hum Genet, 1988 Apr, 78(4), 333 - 7
Evolutionary conservation of the human homologue of the yeast cell cycle control gene cdc2 and assignment of Cd2 to chromosome 10; Spurr NK et al.; The human homologue of the fission yeast Schizosaccharomyces pombe cell cycle control gene cdc2 has been assigned to chromosome 10 . DNA hybridization reveals that this gene is highly conserved in vertebrates . The human CDC2 gene probe detects a simple two-allele polymorphism in Taq1-digested DNA.

Curr Genet, 1988 Apr, 13(4), 305 - 14
Deficiency in both type I and type II DNA topoisomerase activities differentially affect rRNA and ribosomal protein synthesis in Schizosaccharomyces pombe; Yamagishi M et al.; The synthesis of rRNA and r-proteins was studied in temperature-sensitive topoisomerase mutants of the fisson yeast Schizosaccharomyces pombe . To reduce the severity of heatshock response seen in the wild type strain, slow temperature shift-up of the cultures was used to inactivate the mutant topoisomerases . It was found that the temperature shift caused a large preferential reduction of rRNA synthesis in the top1top2 double mutant . In contrast, no preferential inhibition of rRNA synthesis was observed in top1 or top2 single mutants, although some reduction in the total RNA synthesis was observed in the top2 mutant . Thus, as observed with Saccharomyces cerevisiae (Brill et al . 1987), relaxation of supercoiled DNA structures by either topoisomerase I or II appears to be essential for efficient transcription of rRNA genes . Analysis of r-protein synthesis indicated that there were small decreases in the differential synthesis rates of r-proteins after temperature shift-up in the top1top2 mutant, but the observed negative effects on r-protein synthesis was much smaller than that on rRNA synthesis, and degradation of the newly synthesized r-proteins was observed . These observations indicate the apparent lack of tight coupling between rRNA and r-protein synthesis in S . pombe under these experimental conditions.

Mol Cell Biol, 1988 Apr, 8(4), 1580 - 90
Identification of an essential Schizosaccharomyces pombe RNA homologous to the 7SL component of signal recognition particle; Brennwald P et al.; We have cloned the gene encoding a novel small cytoplasmic RNA from the fission yeast Schizosaccharomyces pombe . Four lines of evidence support the idea that this RNA is a homolog of the 7SL RNA component of mammalian signal recognition particle (SRP), which targets presecretory proteins to the endoplasmic reticulum membrane . First, it shares limited but significant primary sequence homology with previously identified 7SL RNAs and can be folded into a similar secondary structure . Second, it possesses the 5' triphosphate characteristic of unprocessed RNA polymerase III transcripts, and moreover, it is the only fission yeast RNA in this size range with such a terminus . Third, its behavior in cell fractionation experiments suggests that it is part of a small ribonucleoprotein which forms salt-labile contacts with larger structures . Fourth, the particle containing S . pombe 7SL RNA resembles mammalian SRP in both size (11S) and affinity for DEAE-Sepharose . Disruption of the single-copy gene, designated slr1+, reveals that the RNA is indispensable for growth in fission yeast . This result is not surprising, since secretion is an essential cellular process.

J Biol Chem, 1988 Mar 25, 263(9), 4186 - 92
Studies on the gamma-glutamyl Cu-binding peptide from Schizosaccharomyces pombe; Reese RN et al.; The gamma-glutamyl peptide induced in Schizosaccharomyces pombe in response to metal stress has been purified following exposure of the organism to cadmium and copper salts . Induction of the peptide enables S . pombe to proliferate in media containing high concentrations of cadmium and copper . Two Cd-gamma-Glu peptide complexes are produced which differ in the content of acid-labile sulfur . One Cu-gamma-Glu peptide complex is induced, and it lacks acid-labile sulfur in the metal-binding cluster . The peptides are composed of repeating dipeptide units of gamma-Glu-Cys with a carboxyl-terminal glycine with heterogeneity observed in the repeat unit n . The number of repeats averages 3.2 and 3.8 for the Cd-peptides I and II and 3.6 for the Cu-peptide, in the case of the Cu-complex peptides with n values from 2 to 4 were separated by reverse phase high pressure liquid chromatography . The Cu-gamma-Glu peptide complex is oligomeric, but the exact number of peptide units per complex is not known . The copper binding stoichiometry averages 2.3 g atoms of Cu/mol of peptide, whereas Cd-peptides I and II average 1.8 and 2.7 mol eq of Cd(II)/peptide unit . The pH of half-dissociation of Cu ions from the gamma-Glu peptide is near 1.3, whereas pH values of 4 and 5.4 are sufficient for half-displacement of Cd ions from the sulfide-containing and -lacking peptides II and I, respectively . In the Cu-peptide complex copper is bound as Cu(I) as the complex exhibits luminescence characteristic of Cu(I)-S chelation . The luminescence emission peaks at 619 nm with a corrected excitation peak centered at 290 nm . The luminescence of the Cu-complex indicates the clustering of Cu(I) ions within a solvent-inaccessible complex . The complex is air-labile as the luminescence emission is gradually lost upon air exposure.

Curr Genet, 1988 Mar, 13(3), 235 - 9
Immunological homologies between yeast ribosomal protein L2 and rat liver ribosomal proteins L4 and L24; Kreutzfeldt C et al.; Polyclonal antibodies raised against ribosomal protein (r-protein) L2 of Schizosaccharomyces pombe were used to check for cross-reactions with total r-proteins of rat liver . Using this procedure, the rat liver r-proteins, L4 and L24, were identified as being immunologically related to yeast L2 . In additional, homologies between rat liver L4 and L24 were detected . The possible implications for the regulation of r-protein synthesis are discussed.

J Cell Biochem, 1988 Mar, 36(3), 261 - 73
Posttranslational modification of ras proteins: detection of a modification prior to fatty acid acylation and cloning of a gene responsible for the modification; Tamanoi F et al.; Products of ras genes are synthesized as precursors in the cytosol and transported to the plasma membrane by a process which involves posttraslational modification by fatty acid . In this paper, we present evidence for the occurrence in the cytosol of an intermediate modification of ras proteins prior to the fatty acid acylation . The modification is detected by a slight shift in the mobility of the protein on SDS polyacrylamide gel . The fatty acid acylation does not contribute to this mobility shift . This modification is affected by the dprl mutation which has recently been shown to affect the processing of yeast RAS proteins . To further characterize the nature of the modification event, we have cloned DPR1 gene from the DNA of Saccharomyces cerevisiae . The gene is actively transcribed in yeast cells producing mRNA of approximately 1.6 kb . Genes related to the DRP1 appear to be present in a distantly related yeast, Schizosaccharomyces pombe as well as in guinea pig and human cells.

Proc Natl Acad Sci U S A, 1988 Mar, 85(5), 1379 - 83
Yeast RNase P: catalytic activity and substrate binding are separate functions; Nichols M et al.; During tRNA biosynthesis the 5'-leader sequences in precursor tRNAs are removed by the ribonucleoprotein RNase P, an enzyme whose RNA moiety is required for activity . To clarify some aspects of the enzyme mechanism, we examined substrate binding and product formation with mutant precursor tRNAs . Mutations G-1----A or U-2----C in the Schizosaccharomyces pombe sup3-e tRNASer, which cause mispairing at or near the top of the acceptor stem, prevent the removal of the 5'-leader sequences by Saccharomyces cerevisiae RNase P . Equilibrium binding studies involving specific gel retardation of RNase P-precursor tRNA complexes showed that complexes with wild-type and A-1 and C-2 mutant precursor tRNAs had very similar dissociation constants (average Kd for sup3 = 1.5 +/- 0.2 nM) . Thus, the 5'-terminal nucleotides of mature tRNA, on the 3' proximal side of the RNase P cleavage site, affect the enzyme's catalytic function but not substrate binding . The catalytic integrity of the RNA component of RNase P is not essential for binding of tRNA precursors, as demonstrated by gel retardation of micrococcal nuclease-inactivated enzyme . This suggests a possible role for the protein component of the enzyme in substrate binding . Upon restoration of base pairing to the acceptor stem in the A-1 or C-2 mutants, we found that, in addition to a requirement for pairing at these positions, conservation of the wild-type first and second nucleotides of the tRNA was necessary to obtain maximal cleavage by RNase P . This indicates a distinct sequence preference of this enzyme.

J Biochem (Tokyo), 1988 Mar, 103(3), 508 - 21
Peptide elongation factor 1 from yeasts: purification and biochemical characterization of peptide elongation factors 1 alpha and 1 beta (gamma) from Saccharomyces carlsbergensis and Schizosaccharomyces pombe; Miyazaki M et al.; Cytoplasmic elongation factor 1 alpha (EF-1 alpha) {corrected} was purified to homogeneity in high yield from the two different yeasts Saccharomyces carlsbergensis (S . carls.) and Schizosaccharomyces pombe (S . pombe) . The purification was easily achieved by CM-Sephadex column chromatography of the breakthrough fractions from DEAE-Sephadex chromatography of cell-free extracts . The basic proteins have a molecular weight of 47,000 for the S . carls . factor and of 49,000 for the S . pombe factor . While the purified yeast EF-1 alpha s function analogously to other eukaryotic factors and the E . coli EF-Tu in Phe-tRNA binding and polyphenylalanine synthesis, the yeast factor unusually hydrolyzed GTP on yeast ribosomes upon addition of Phe-tRNA in the absence of poly(U) as mRNA . This novelty is probably owing to the yeast ribosomes, which are assumed to lack elongation factor 3-equivalent component(s) . Trypsin and chymotrypsin selectively cleaved the two yeast factors to generate resistant fragments with the same molecular weight of 43,000 (by trypsin) and of 44,000 (by chymotrypsin), respectively . Those cleavage sites were characteristically protected by the presence of several ligands bound to EF-1 alpha such as GDP, GTP, and aminoacyl-tRNA . Based on the sequence analysis of the fragments generated by the two proteases, the partial amino acid sequence of the S . carls . EF-1 alpha was deduced to be in accordance with the N-terminal region covering positions (1) to 94 and two Lys residues at the C-terminal end of the predicted total sequence of the Saccharomyces cerevisiae (S . cerev.) factor derived from DNA analysis, except for a few N-terminal residues, confirming the predicted S . cerev . sequence at the protein level . EF-1 beta and EF-1 beta gamma were isolated and highly purified as biologically active entities from the two yeasts . EF-1 beta s from the two yeasts have the same molecular weight of 27,000, whereas component gamma of the S . carls . EF-1 beta gamma showed a higher molecular weight (47,000) than that of the S . pombe factor (40,000) . It was also shown that a stoichiometric complex was formed between EF-1 alpha and EF-1 beta gamma from S . pombe . Furthermore, a considerable amount of Phe-tRNA binding activity was distributed in the EF-1H (probably EF-1 alpha beta gamma) fraction from freshly prepared cell-free extracts of yeast.

J Cell Sci, 1988 Mar, 89 ( Pt 3), 343 - 57
The use of cell division cycle mutants to investigate the control of microtubule distribution in the fission yeast Schizosaccharomyces pombe; Hagan IM et al.; We have characterized the changes in microtubule organization that occur through the cell division cycle of the fission yeast Schizosaccharomyces pombe by indirect immunofluorescence microscopy . During interphase, groups of cytoplasmic microtubules, independent of the spindle pole body (SPB), form an array extending between the cell tips . These microtubules are involved in positioning the nucleus at the cell equator and in the establishment of cell polarity . At mitosis, the interphase array disappears and is replaced by an intranuclear spindle extending between the now duplicated SPBs . Elongation of the spindle sees the appearance of astral microtubules emanating from the cytoplasmic face of the SPBs . These persist until the end of anaphase whereupon the spindle microtubules depolymerize and two microtubule organizing centres (MTOCs) at the cell equator re-establish the interphase array . We have used the unique properties of various cell division cycle mutants to investigate further the function of these different microtubule arrays and their temporal and positional control.

EMBO J, 1988 Mar, 7(3), 761 - 7
The S.pombe mei2 gene encoding a crucial molecule for commitment to meiosis is under the regulation of cAMP; Watanabe Y et al.; The complete nucleotide sequence of the mei2 gene of Schizosaccharomyces pombe, which is essential for initiation of meiosis, is presented and four transcriptional start sites assigned . Transcription of mei2 and other genes involved in life cycle control of S . pombe, which is inducible by nitrogen starvation, is inhibited by addition of cAMP, suggesting that cAMP can mediate the signal of nitrogen supply in S.pombe . mei2 is the furthest downstream among target genes regulated by cAMP and genetic or physiological factors so far shown to block uncontrolled meiosis in S.pombe, which is provoked by inactivation of the part1 gene product, are either mutations at the mei2 locus or inhibitors of its expression . Cooperation of two regulatory pathways, one leading to the inactivation of pat1 activity and the other to the supply of the mei2 product, appears to commit cells to meiosis in S.pombe.

J Biochem (Tokyo), 1988 Mar, 103(3), 522 - 30
Characterization of the ATPase and GTPase activities of elongation factor 3 (EF-3) purified from yeasts; Uritani M et al.; Three steps of chromatography of a post-ribosomal supernatant fraction have provided a highly purified preparation of peptide elongation factor 3 (EF-3) with a molecular weight of 125,000 from the typical budding yeast Saccharomyces carlsbergensis and of the factor with a molecular weight of 120,000 from the fission yeast Schizosaccharomyces pombe . Both of the proteins consist of a single peptide chain . The purified factors fulfilled the requirement for polyphenylalanine synthesis on yeast ribosomes and exhibited strong ATPase and GTPase activities dependent on yeast ribosomes . The activity profiles of the nucleotidases dependent on pH and salt concentration and the inhibition studies indicated that the ATPase and GTPase activities of EF-3 were displayed by the same active site with a wide substrate specificity, showing the highest activity with ATP . Those experiments also revealed that the ATPase and GTPase of EF-3 were characteristically different from the GTPases of EF-1 alpha and EF-2 . Both Km and kcat of EF-3 for ATP (Km = 0.12 mM and Kcat = 610 mol/mol/min) and GTP (Km = 0.20 mM and kcat = 390 mol/mol/min) are much higher than those of the GTPases of EF-1 alpha and EF-2 . Inactivation experiments and studies on the ATP effect led us to conclude that this ATPase activity was an essential requirement for the functional role of EF-3 and therefore, in addition to the GTPases of EF-1 alpha and EF-2, the third nucleoside triphosphate hydrolyzing step by the ATPase of EF-3 was necessary for the yeast peptide elongation cycle.

Biochem Biophys Res Commun, 1988 Feb 29, 151(1), 32 - 9
Isolation of mutants of Schizosaccharomyces pombe unable to synthesize cadystin, small cadmium-binding peptides; Mutoh N et al.; Schizosaccharomyces pombe synthesize small cadmium-binding peptides cadystin, structure of which is (gamma-Glu-Cys)n-Gly, in response to cadmium . Mutants unable to synthesize cadystin were found in the mutants hypersensitive to cadmium . Some of them lack activity of either gamma-glutamylcysteine synthetase (EC 6.3.2.2) or glutathione synthetase (EC 6.3.2.3), enzyme involved in glutathione biosynthesis . Some mutants have the same activity levels of these enzymes as wild type has . These results indicate that some steps of cadystin biosynthesis are catalyzed by the enzymes catalyzing glutathione biosynthesis.

FEBS Lett, 1988 Feb 29, 229(1), 30 - 4
Electroporation: high frequency of occurrence of a transient high-permeability state in erythrocytes and intact yeast; Weaver JC et al.; We present the first determinations of population distributions of macromolecule uptake due to electroporation, the percentage of cells which participate and, for the yeast, the subpopulation of cells whose membranes exhibit significant recovery following macromolecule uptake . Flow cytometry is used to measure the uptake of a first test molecule (green fluorescence, FITC-dextran; 70 kDa) and also, for the yeast, the subsequent uptake of a second, much smaller, test molecule (red fluorescence, propidium iodide; 660 Da), which provides a measure of membrane recovery . A dramatic 20% (erythrocytes) to 75% (intact Schizosaccharomyces pombe) of cells can take up the first test molecule within 5 min of a pulse.

J Biol Chem, 1988 Feb 25, 263(6), 2891 - 6
The primary structure of rat ribosomal protein S6; Chan YL et al.; The amino acid sequence of rat ribosomal protein S6, the major phosphoprotein in the organelle, was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed from the sequence of amino acids in portions of the protein . Ribosomal protein S6 contains 249 amino acids and has a molecular weight of 28,683 . The protein has 15 seryl residues; 7 are located in the carboxyl-terminal sequence of 15 amino acids and probably include most if not all of the residues that are phosphorylated . There are related repeated sequences of 10 amino acids each that occur at four separate positions in S6 and that are very basic . Rat S6 is homologous to Saccharomyces cerevisiae S10 (the extent of the identity is 75%) and most likely also to Schizosaccharomyces pombe S6.

Mol Cell Biol, 1988 Feb, 8(2), 754 - 63
Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe; Fishel B et al.; Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments . The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases {kb}, chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I) . Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions . On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat . Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function . The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin . Thus, S . pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.

FEBS Lett, 1988 Jan 4, 226(2), 250 - 4
Identification and isolation of core histones from Schizosaccharomyces pombe; Whiteside SG et al.; Core histones have been isolated from Schizosaccharomyces pombe and compared electrophoretically to core histones from Saccharomyces cerevisiae and rat liver . The molecular masses of all cognate histones examined were found to be very similar as determined by SDS gel electrophoresis . Histones H3, H2A and H2B from Sch . pombe migrated almost identically to their respective counterparts from S . cerevisiae as determined by acid/urea gel electrophoresis . Two-dimensional gel electrophoresis with a Triton X-100 acid/urea gel in the first dimension followed by an SDS gel in the second dimension was used to separate Sch . pombe histones from contaminating ribosomal proteins.

Curr Genet, 1988, 13(1), 57 - 63
Primary structure of the ribosomal protein gene S6 from Schizosaccharomyces pombe; Gross T et al.; We have determined the nucleotide sequence of a ribosomal protein gene which codes for the ribosomal protein S6 (rps6) . The sequence analysis revealed that the gene comprises 239 amino acids, giving rise to a basic protein with a molecular weight of 27,502 Da . The product of this gene is the equivalent of the ribosomal protein S10 from Saccharomyces cerevisiae . Northern analyses and S1 mapping of both the 5' and the 3' end of the transcripts of this gene show that it is transcribed into three distinct transcripts with different sizes and heterogeneous termini . In the DNA region flanking the coding sequence, several conserved elements are present that may be involved in the transcription initiation and termination.

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