Yeast, 1994 May, 10(5), 613 - 24 Molecular analysis of the malic enzyme gene (mae2) of Schizosaccharomyces pombe; Viljoen M et al.; Sequence analysis of a 4.6-kb HindIII fragment containing the malic enzyme gene (mae2) of Schizosaccharomyces pombe, revealed the presence of an open reading frame of 1695 nucleotides, coding for a 565 amino acid polypeptide . The mae2 gene is expressed constitutively and encodes a single mRNA transcript of 2.0 kb . The mae2 gene was mapped on chromosome III by chromoblotting . The coding region and inferred amino acid sequence showed significant homology with 12 malic enzyme genes and proteins from widely different origins . Eight highly homologous regions were found in these malic enzymes, suggesting that they contain functionally conserved amino acid sequences that are indispensable for activity of malic enzymes . Two of these regions have previously been reported to be NAD- and NADP-binding sites. Yeast, 1994 May, 10(5), 595 - 601 Chemical synthesis of the M-factor mating pheromone from Schizosaccharomyces pombe; Wang SH et al.; Conjugation in the fission yeast Schizosaccharomyces pombe is controlled by the reciprocal action of mating pheromones . We recently showed that M-factor, the pheromone released by cells of the cellular mating type Minus, is a nonapeptide in which the C-terminal cysteine residue is carboxyl-methylated and S-alkylated, probably with a farnesyl residue (Davey, 1992): Tyr-Thr-Pro-Lys-Val-Pro-Tyr-Met-Cys(S-farnesyl)- OCH3 . Here we describe the chemical synthesis of this modified peptide and show that it exhibits all of the properties of the native pheromone . These results confirm the structure of the M-factor while the production of relatively large amounts of pure pheromone will be invaluable for studying the mating response in this yeast. J Cell Sci, 1994 May, 107 ( Pt 5), 1197 - 204 The kinetics of H1 histone kinase activation during the cell cycle of wild-type and wee mutants of the fission yeast Schizosaccharomyces pombe; Creanor J et al.; H1 histone kinase activity has been followed in selection-synchronised cultures of fission yeast wild-type and wee1 mutant cells, and in induction-synchronised cells of the mutant cdc2-33 . The main conclusions are: (1) in all three cases, the peak of activity is near mitosis . (2) The rise in activity is relatively slow starting in wild type at 0.4 of the cycle before mitosis . It is proposed that the beginning of the rise is the first identified event in the mitotic control . (3) The rise is twice as fast in wee and starts nearer to mitosis . (4) In all cases the beginning of the rise is in G2 . (5) The fall in activity is also slow, lasting for 0.25 of the cycle, in wild type . Exit from mitosis happens well before activity has fallen to baseline . (6) In a range of size mutants, activity is roughly proportional to cell size . It is suggested that the kinase may have a cytoplasmic function . (7) Estimates have been made of the timing of mitosis in the mutants . In wee, mitosis is 0.14 of the cycle earlier than in wild type because the cells have a longer septated period at the end of the cycle . (8) A novel method has been developed for eliminating the effects of the partial asynchrony in synchronous cultures, without which the kinetic analysis would have been inaccurate. J Cell Sci, 1994 May, 107 ( Pt 5), 1131 - 6 Fission yeast protein kinase C gene homologues are required for protoplast regeneration: a functional link between cell wall formation and cell shape control; Kobori H et al.; Two novel protein kinase C (n PKC) gene homologues, pck1+ and pck2+ were isolated from the fission yeast Schizosaccharomyces pombe (Toda et al . (1993) EMBO J . 12, 1987) . We examined the functional differences of pck1+ and pck2+ in cell wall formation and actin organization of S . pombe . Regenerating protoplasts of a wild-type strain, single gene disruptants of pck1+ (delta pck1) and pck2+ (delta pck2) were used as a simple model to examine the functional links between PKC, cell wall formation and actin organization . Protoplasts of the wild-type strain and those of delta pck1 reverted to intact cells in osmotically stabilized liquid medium . A close spatial association between new cell wall formation and actin was observed in these two strains . In delta pck2, protoplasts did not revert to intact cells: (1) scarcely any new cell wall material was formed; (2) actin was not reorganized; and (3) nuclear division and an increase in the amount of cytoplasm were observed in the regenerating protoplasts . These findings demonstrate that the pck2+ gene has a function essential for protoplast regeneration but the pck1+ gene does not . Involvement of n PKCs in cell wall formation and actin organization was also clarified . The effect of staurosporine (a potent inhibitor of protein kinases) on regenerating protoplasts of the three strains confirmed the assumption that the pck2 protein is an in vivo target of staurosporine in the fission yeast. Mol Biol Cell, 1994 May, 5(5), 519 - 28 Localization of an alpha 1,2 galactosyltransferase activity to the Golgi apparatus of Schizosaccharomyces pombe; Chappell TG et al.; We have cloned a gene encoding an alpha 1,2 galactosyltransferase activity from Schizosaccharomyces pombe . The open reading frame of the gene (gma12 for galactomannan, alpha 1,2), combined with the previous protein purification (Chappell and Warren, 1989), predicts an O-linked glycoprotein with type II transmembrane topology . By homologous gene disruption, we have demonstrated that the gma12 gene product (gma12p) is nonessential . The deletion strain (gma12-D10::ura4) has a significantly reduced level of galactosyltransferase activity relative to the parental strain, but both in situ lectin binding and in vitro biochemical assays demonstrate the presence of further galactosyltransferase activity in addition to gma12p . Although gma12p is not the only galactosyltransferase in S . pombe, it produces a unique carbohydrate structure on the surface of the yeast cells . We have generated a polyclonal antiserum against this carbohydrate epitope and shown that gma12p is capable of synthesizing the epitope both in vitro and in vivo . Electron microscopic localization of the gma12+ specific epitope in gma12+ cells revealed that gma12p synthesizes the carbohydrate structure in the Golgi apparatus, and subsequent intracellular transport distributes the epitope to later stages of the secretory pathway . The immunolocalization studies confirm the presence of one or more galactosyltransferase activities in the Golgi apparatus in fission yeast. J Biol Chem, 1994 Apr 29, 269(17), 12527 - 35 Glycoprotein synthesis in yeast . Early events in N-linked oligosaccharide processing in Schizosaccharomyces pombe; Ziegler FD et al.; Oligosaccharide-lipid precursors and glycoprotein N-linked oligosaccharides isolated from the fission yeast, Schizosaccharomyces pombe, were compared with those from the budding yeast, Saccharomyces cerevisiae . Bio-Gel P-4 chromatography of oligosaccharide intermediates showed that Glc3Man9GlcNAc2-PP-dol synthesis, transfer of glycan to protein, and glucose removal to yield Man9GlcNAc2 proceeded in S . pombe as in S . cerevisiae . Two series of oligosaccharides were released from S . pombe glycoproteins by endo-beta-N-acetylglucosaminidase H; large "mannan-like" structures and smaller precursor or "core-filling" species . Unexpectedly, the smallest S . pombe N-linked glycan was Man9GlcNAc, confirmed by 500 MHz 1H NMR spectroscopy to be the lipid-linked isomer . No endoplasmic reticulum Man9-alpha 1,2-mannosidase activity was detected in S . pombe, thus identifying Man9GlcNAc as the minimum precursor for oligosaccharide elongation in contrast to the Man8GlcNAc2 intermediate identified in S . cerevisiae (Byrd, J . C., Tarentino, A . L., Maley, F., Atkinson, P . H., and Trimble, R . B . (1982) J . Biol . Chem . 257, 14657-14666) . S . pombe Hex10GlcNAc was at least four isomers by high pH anion-exchange chromatography with pulsed amperometric detection . Compositional analyses identified two of the major species as GalMan9GlcNAc and GlcMan9GlcNAc, the latter of which suggests that glycan trimming may be attenuated in the S . pombe endoplasmic reticulum . Hex13GlcNAc from S . pombe was homogeneous by mass spectrometry but yielded 12 species by high pH anion-exchange chromatography . Compositional analyses, alpha-galactosidase digestion, and lectin affinity chromatography on Griffonia simplicifolia lectin I-agarose indicated these to be a family of GalxMan13-xGlcNAc isomers (X = 1-4 residues) . The absence of Man9GlcNAc2 to Man8GlcNAc2 trimming in S . pombe and elongation of the lipid precursor of Man9GlcNAc with both Man and Gal to form "galactomannans" provides a novel system for N-linked glycoprotein processing studies. J Biol Chem, 1994 Apr 22, 269(16), 12074 - 9 p-nitrophenylphosphatase activity of plasma membrane H(+)-ATPase from yeast . Implications for the regulation of the catalytic cycle by H+; Ferreira-Pereira A et al.; The H(+)-ATPase from Schizosaccharomyces pombe belongs to the group of transport ATPases which displays two main conformational states, E1 and E2 (P-type ATPase) . In this report, we show that, as in the case of other P-type ATPase, the purified enzyme exhibits a p-nitrophenylphosphatase activity which can be completely inhibited by vanadate . In aqueous medium, p-nitrophenyl phosphate hydrolysis proceeds at only 0.5% of the rate of ATP hydrolysis, and both activities can be stimulated 3- to 4-fold by decreasing the pH from 7.5 to 6.5 . Addition of the organic solvent dimethyl sulfoxide (10-40%), which has been shown to favor the E2 conformation, stimulates the p-nitrophenylphosphatase activity but inhibits the ATPase activity . At pH 7.5, the Km for p-nitrophenyl phosphate decreases when dimethyl sulfoxide is present . In the presence of 30% (v/v) dimethyl sulfoxide, the phosphatase activity can be inhibited by ATP (K(i) 300 microM) or by P(i) (K(i) 1 mM) . The H(+)-ATPase incorporated into liposomes retains pNPPase activity, but it does not support H+ transport . Gel electrophoresis reveals that the pattern of H(+)-ATPase cleavage by trypsin changes when vanadate, Me2SO, or both compounds are present in the medium, regardless of the pH used during trypsinization . We propose that p-nitrophenyl phosphate is hydrolyzed by a H(+)-ATPase conformation distinct from that which hydrolyzes ATP, most probably an E2-like form . We also suggest that, in addition to the E1-E2 transition, the enzyme activity can be regulated by protons at another step of the catalytic cycle. J Biol Chem, 1994 Apr 22, 269(16), 12014 - 23 Cytoplasmic forms of fission yeast casein kinase-1 associate primarily with the particulate fraction of the cell; Wang PC et al.; Two novel casein kinase-1 homologs, cki1+ and cki2+, have been isolated from Schizosaccharomyces pombe and characterized . Both genes reside on chromosome II and encode approximately 50-kDa proteins that are related structurally and enzymatically to the YCK gene products of budding yeast . Subcellular fractionation experiments demonstrate that Cki1 and Cki2 are both cytoplasmic enzymes that do not overlap in subcellular distribution and that probably play distinct roles within the cell . Although gene disruption experiments show that neither cki1+ nor cki2+ is essential for cell viability, overexpression of cki2 leads to a severe growth defect and aberrant morphology . Cells become round or pear shaped and separate poorly following septation . These results suggest that of the four members of the casein kinase-1 family recognized in fission yeast, one member, Cki2, may contribute to the regulation of cell morphology. J Biol Chem, 1994 Apr 22, 269(16), 11921 - 6 ntf1+ encodes a 6-cysteine zinc finger-containing transcription factor that regulates the nmt1 promoter in fission yeast; Tang CS et al.; The nmt1+ gene of the fission yeast Schizosaccharomyces pombe is subject to transcriptional repression mediated by thiamine . The promoter of nmt1+ has been used to construct a series of vectors that are now commonly used for the regulated expression of genes in S . pombe . In this report, we described ntf1+, a gene involved in regulating nmt1+ expression . The ntf1+ gene was cloned in a high copy suppressor screen that utilized a construct, nmt1:wee1+, in which expression of the mitotic inhibitor Wee1 tyrosine kinase was placed under the control of the nmt1 promoter . ntf1+ encodes a 706-amino acid protein that shares substantial homology with a family of Cys6 zinc finger-containing transcription factors typified by GAL4 from Saccharomyces cerevisiae . Increased gene dosage of ntf1+ greatly increases both the repressed and derepressed activity of the nmt1 promoter . Cells having a disrupted version of ntf1+ are viable thiamine prototrophs, but basal expression from the nmt1 promoter is greatly reduced . These data demonstrate that Ntf1 plays an important role in regulating nmt1 expression. EMBO J, 1994 Apr 15, 13(8), 1881 - 7 A zinc finger protein controls the onset of premeiotic DNA synthesis of fission yeast in a Mei2-independent cascade; Sugiyama A et al.; In the fission yeast Schizosaccharomyces pombe, meiosis is initiated by the action of Mei2 in a complex cascade activated following conjugation . We have isolated a new gene named rep1+ that is required for the initiation of premeiotic DNA synthesis . rep1+ encodes a 53 kDa protein with one zinc finger motif that is essential for function, and effectively rescues a null mutant of the res1+ gene but only partially a temperature-sensitive mutant of the cdc10+ gene, both of which are required for the onset of mitotic, as well as premeiotic, S phase . Deletion of rep1+ has no apparent effects on the mitotic cell cycle or conjugation, but blocks the initiation of premeiotic DNA synthesis . However, this defect is partially suppressed when rapidly growing cells are induced to conjugate, indicating that the rep1+ function is at least partly substituted by those of the genes controlling the 'start' of the mitotic cell cycle . The rep1 null mutant fails to induce the res2+ gene, a newly identified res1+ homolog cooperating with Cdc10 and acting for the onset of mitotic and premeiotic DNA synthesis, as well as for meiotic division . The rep1+ gene itself is induced moderately during nitrogen starvation but highly during conjugation, and this induction is dependent on both ste11+ and mating pheromones but independent of mei2+ . Thus, rep1+ controls the initiation of premeiotic DNA synthesis via induction and/or activation of Res2 and some other essential factors in a cascade independent of Mei2. EMBO J, 1994 Apr 15, 13(8), 1873 - 80 res2+, a new member of the cdc10+/SWI4 family, controls the 'start' of mitotic and meiotic cycles in fission yeast; Miyamoto M et al.; In the fission yeast Schizosaccharomyces pombe, the cdc10+ and res1+ genes play a crucial role in the start of mitotic and meiotic cycles . They encode structurally related transcriptional complex proteins and regulate some S phase-specific genes . Here we report the identification of a new member of this family named as res2+ . res2+ has been isolated as a multicopy suppressor of a res1- null mutant and specifies a 73 kDa protein, which has two copies of the Swi/ankyrin motif and shares the highest sequence and structure similarity with the Res1 protein . res2+ is largely redundant in function with res1+ and is required for the initiation of mitotic and premeiotic DNA synthesis, but has an additional role in meiotic division . Unlike res1+, res2+ is highly induced during conjugation and strongly depends on cdc10+ for its activity . We conclude that the fission yeast contains two functionally overlapping parallel 'start' systems, Res1-Cdc10 and Res2-Cdc10, the former of which plays a major role in mitotic cycle whereas the latter in meiotic cycle. Genes Dev, 1994 Apr 15, 8(8), 885 - 98 pct1+, which encodes a new DNA-binding partner of p85cdc10, is required for meiosis in the fission yeast Schizosaccharomyces pombe; Zhu Y et al.; The transcriptional activation of genes at late G1 is an important regulatory step in the commitment to a new cell division cycle . In Schizosaccharomyces pombe, this regulation is mediated by MCB elements that serve as binding sites for the MBF/DSC-1 complex . The cdc10(+)-encoded protein is a component of this complex . We report the cloning of a new gene, pct1+, encoding a 73-kD protein that interacts with p85cdc10 to form an MCB-binding heteromer . Pct1+ is related to, but distinct from, the res1+/sct1+ gene that also encodes a p85cdc10 partner . p73pct1 has centrally located ankyrin repeats and a putative amino-terminal DNA-binding domain that has extensive sequence similarity to the DNA-binding domains of the Saccharomyces cerevisiae SWI4 and MBP1 proteins . The p73pct1/p85cdc10 complex binds both in vitro and in vivo to MCB but not SCB or E2F sites . Overexpression of pct1+ is sufficient to rescue the growth of the cdc10-129 temperature-sensitive mutant at the restrictive temperature, although it is unable to rescue a cdc10 null mutation . A deletion of pct1+ is not lethal but does result in a severe meiotic defect . Our results indicate that there are two cdc10-containing heteromeric complexes that bind to MCB elements and play differential roles in mitotic division and meiosis. EMBO J, 1994 Apr 15, 13(8), 1863 - 72 A B-type cyclin negatively regulates conjugation via interacting with cell cycle 'start' genes in fission yeast; Obara-Ishihara T et al.; In the fission yeast Schizosaccharomyces pombe, the cdc10+/SWI family members constitute the cell cycle 'start' genes . res1+ and res2+ are the newly identified members of this family and encode putative association partners of the Cdc10 protein . The Pat1 kinase plays a pivotal role in switching between vegetative growth and sexual development, and its inactivation in haploid cells induces unconditional growth arrest and subsequent meiosis . We have identified as an extragenic suppressor of a temperature sensitive pat1-114 mutant, a new B-type cyclin that negatively regulates conjugation by interacting with these 'start' genes . This cyclin, named Cyc17, is highly homologous with Cdc13, but has no detectable activity as a mitotic cyclin . Deletion of cyc17+ markedly enhances conjugation, despite the presence of nitrogen source, and accelerates growth arrest in G1 upon nitrogen starvation . Conversely, overexpression of the cyc17+ gene strongly inhibits conjugation . The cyc17+ gene is transcribed into 3.2 kb poly(A)+ and 3.0 kb poly(A)- RNAs . Only the poly(A)+ species is expressed during vegetative growth and periodically with a peak in the G1 and S phases of the cell cycle . On the other hand, the poly(A)- transcript is highly induced during conjugation . This induction is lost in res2- cells, whereas the poly(A)+ transcript is significantly reduced in res1- cells . However, the mating inhibition as well as the ability to rescue the pat1 mutation by overexpression of res1+ and res2+ are totally abolished in cyc17- cells . Thus, in S.pombe, a B-type cyclin, regulated by the newly identified cell cycle 'start' genes, plays a crucial role in the control of sexual development. FEBS Lett, 1994 Apr 4, 342(2), 109 - 14 A comparison of demethoxyviridin and wortmannin as inhibitors of phosphatidylinositol 3-kinase; Woscholski R et al.; The mammalian Ptdlns 3-kinase is shown to be inhibited by low nanomolar concentrations of demethoxyviridin, an antifungal agent structurally related to wortmannin . The inhibitory potency of both compounds could be observed in purified Ptdlns 3-kinase whether or not the regulatory subunit (p85 alpha) was present, suggesting that the inhibitors bind to the catalytic subunit (p110) of the Ptdlns 3-kinase . These inhibitors also show similar potency against the intrinsic p85-phosphorylating activity of the p110-kinase . However, the structurally related Ptdlns 3-kinase from Saccharomyces cerevisiae (Vps34p) is not inhibited by either compound . Both inhibitors target the mammalian Ptdlns 3-kinase in vitro and in vivo, implying that these compounds should be useful in suppressing Ptdlns 3-kinase in mammalian systems . The inhibitors did not affect the mammalian Ptdlns 4-kinase, but they are able to inhibit a membrane-associated Ptdlns 4-kinase from Schizosaccharomyces pombe. Mol Gen Genet, 1994 Apr, 243(1), 1 - 8 Molecular analysis of the dhp1+ gene of Schizosaccharomyces pombe: an essential gene that has homology to the DST2 and RAT1 genes of Saccharomyces cerevisiae; Sugano S et al.; The DST2 gene of Saccharomyces cerevisiae encodes a DNA strand exchange protein, STP beta, which is required for homologous recombination in both mitotic or meiotic cells . We have cloned a DST2-related gene from the fission yeast Schizosaccharomyces pombe and designated it dhp1+ . The nucleotide sequence of dhp1+ revealed an open reading frame encoding a protein composed of 991 amino acids . The predicted amino acid sequence was significantly homologous to the S . cerevisiae STP beta, but lacked the carboxy-terminal sequence present in STP beta . Furthermore, dhp1+ shows greater homology to RAT1/HKE1, a gene which is involved in RNA trafficking and processing . Genetic experiments showed that dhp1+ on an S . cerevisiae expression vector could rescue both the defects of the S . cerevisiae DST2 disruptant, slow growth rate and a sporulation defect, and the lethality of the S . cerevisiae rat1ts mutation . This implies the functional similarity of dhp1+ to both DST2 and RAT1 . However unlike DST2, dhp1+ is an essential gene for cell growth in S . pombe, suggesting that dhp1+ is not the true homologue of DST2 but rather of RAT1 in S . pombe . The possible roles of dhp1+ in recombination and cell growth in S . pombe are discussed. EMBO J, 1994 Apr 1, 13(7), 1549 - 56 Cdc25A is a novel phosphatase functioning early in the cell cycle; Jinno S et al.; The cdc25+ tyrosine phosphatase is a key mitotic inducer of the fission yeast Schizosaccharomyces pombe, controlling the timing of the initiation of mitosis . Mammals contain at least three cdc25+ homologues called cdc25A, cdc25B and cdc25C . In this study we investigate the biological function of cdc25A . Although very potent in rescuing the S.pombe cdc25 mutant, cdc25A is less structurally related to the S.pombe enzyme . Northern and Western blotting detection reveals that unlike cdc25B, cdc25C and cdc2, cdc25A is predominantly expressed in late G1 . Moreover, immunodepletion of cdc25A in rat cells by microinjection of a specific antibody effectively blocks their cell cycle progression from G1 into the S phase, as determined by laser scanning single cell cytometry . These results indicate that cdc25A is not a mitotic regulator but a novel phosphatase that plays a crucial role in the start of the cell cycle . In view of its strong ability to activate cdc2 kinase and its specific expression in late G1, cdc2-related kinases functioning early in the cell cycle may be targets for this phosphatase. J Biol Chem, 1994 Apr 1, 269(13), 9632 - 7 Cloning of the pka1 gene encoding the catalytic subunit of the cAMP-dependent protein kinase in Schizosaccharomyces pombe; Maeda T et al.; We have isolated Schizosaccharomyces pombe genes that confer sterility to the fission yeast cell when expressed from a multicopy plasmid . One of these genes strongly hybridized to a probe carrying the open reading frame of Saccharomyces cerevisiae TPK1, which encodes a catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) . This S . pombe gene, named pka1, has a coding potential of 512 amino acids, and the deduced gene product is 60% identical with the S . cerevisiae Tpk1 protein in the C-terminal 320 amino acids . Disruption of pka1 slows cell growth but is not lethal . The resultant cells, however, are highly derepressed for sexual development, readily undergoing conjugation and sporulation in the absence of nitrogen starvation . They are, thus, phenotypically indistinguishable from the adenylyl cyclase-defective (cyr1-) cells previously characterized, except that the pka1- spores are retarded in germination, whereas the cyr1- spores are not . Disruption of pka1 is epistatic to a defect in cgs1, which encodes the regulatory subunit of protein kinase A . These results strongly suggest that the product of pka1 is a catalytic subunit of protein kinase A and, furthermore, that S . pombe has only one gene encoding it . This situation contrasts with the case of S . cerevisiae, in which three genes encode the catalytic subunits. J Cell Biochem, 1994 Apr, 54(4), 415 - 22 How does the G protein, Gi2, transduce mitogenic signals? Johnson GL, Gardner AM, Lange-Carter C, Qian NX, Russell M, Winitz S. Serpentine receptors coupled to the heterotrimeric G protein, Gi2, are capable of stimulating DNA synthesis in a variety of cell types . A common feature of the Gi2-coupled stimulation of DNA synthesis is the activation of the mitogen-activated protein kinases (MAPKs) . The regulation of MAPK activation by the Gi2-coupled thrombin and acetylcholine muscarinic M2 receptors occurs by a sequential activation of a network of protein kinases . The MAPK kinase (MEK) which phosphorylates and activates MAPK is also activated by phosphorylation . MEK is phosphorylated and activated by either Raf or MEK kinase (MEKK) . Thus, Raf and MEKK converge at MEK to regulate MAPK . Gi2-coupled receptors are capable of activating MEK and MAPK by Raf-dependent and Raf-independent mechanisms . Pertussis toxin catalyzed ADP-ribosylation of alpha i2 inhibits both the Raf-dependent and -independent pathways activated by Gi2-coupled receptors . The Raf-dependent pathway involves Ras activation, while the Raf-independent activation of MEK and MAPK does not involve Ras . The Raf-independent activation of MEK and MAPK most likely involves the activation of MEKK . The vertebrate MEKK is homologous to the Ste11 and Byr2 protein kinases in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively . The yeast Ste11 and Byr2 protein kinases are involved in signal transduction cascades initiated by pheromone receptors having a 7 membrane spanning serpentine structure coupled to G proteins . MEKK appears to be conserved in the regulation of G protein-coupled signal pathways in yeast and vertebrates . Raf represents a divergence in vertebrates from the yeast pheromone-responsive protein kinase system.(ABSTRACT TRUNCATED AT 250 WORDS) Biotechnology (N Y), 1994 Apr, 12(4), 400 - 4 High-level expression of human lipocortin I in the fission yeast Schizosaccharomyces pombe using a novel expression vector; Giga-Hama Y et al.; We have developed a novel expression system that allows the fission yeast, Schizosaccharomyces pombe, to be used for the efficient overproduction of heterologous proteins . As an example of the utility of this system, human lipocortin I was expressed to 50 percent of soluble protein, and 150 mg of highly purified material was obtained from 10 grams of wet cell paste . Expression of lipocortin I was driven by the human cytomegalovirus (hCMV) promoter in a vector that also contains a neomycin resistance gene (neo) under the control of the SV40 early promoter, permitting selection for increasing copy-number with increasing concentrations of the antibiotic G418 . The purified protein was equivalent to its native counterpart with respect to antigenicity and biochemical properties such as phospholipase A2 inhibition, actin binding and N-terminal acetylation . We have also used this system to produce comparable amounts of other proteins including rat arginase, rat NDP-kinase and human interleukin-6. Biotechnology (N Y), 1994 Apr, 12(4), 381 - 4 Protein disulfide isomerase overexpression increases secretion of foreign proteins in Saccharomyces cerevisiae; Robinson AS et al.; Overexpression of protein disulfide isomerase (PDI) from a single chromosomally integrated copy in Saccharomyces cerevisiae results in ten-fold higher levels of secretion of human platelet derived growth factor B homodimer, and a four-fold increase in secretion of Schizosaccharomyces pombe acid phosphatase . This result provides evidence that inefficient protein folding limits the secretion of some heterologous proteins, and that manipulation of the endoplasmic reticulum lumenal environment can help overcome this limitation. PCR Methods Appl, 1994 Apr, 3(5), 272 - 7 RNA associated with a heterodimeric protein that activates a meiotic homologous recombination hot spot: RL/RT/PCR strategy for cloning any unknown RNA or DNA; Wahls WP; The ade6-M26 mutation in the fission yeast Schizosaccharomyces pombe creates a meiotic homologous recombination hot spot . We have achieved 40,000-fold purification of a heterodimeric DNA-binding protein, Mts1/Mts2, that activates the recombination hot spot . Physical studies suggested the presence of a third subunit . It is demonstrated here that RNA molecules of approximately 210 nucleotides copurified with the heterodimer . To characterize the RNA component, it was necessary to develop a new strategy for cloning of the unknown, low-abundance, partially degraded RNAs that were present in purified Mts1/Mts2 protein preparations . The strategy uses RNA ligase to add DNA oligonucleotide priming sites to the RNA for subsequent reverse transcription and PCR (RNA ligase, reverse transcription-PCR, or RL/RT/PCR) . This cloning procedure could be applied to the cloning of any unknown RNA or DNA molecules . Because the cDNA clones obtained from Mts1/Mts2 were largely heterogeneous, it seems likely that the RNAs copurified as a result of tight but nonspecific interactions with the heterodimeric protein. Experientia, 1994 Mar 15, 50(3), 295 - 306 Homologous recombination in fission yeast: absence of crossover interference and synaptonemal complex; Kohli J et al.; The study of homologous recombination in the fission yeast Schizosaccharomyces pombe has recently been extended to the cytological analysis of meiotic prophase . Unlike in most eukaryotes no tripartite SC structure is detectable, but linear elements resembling axial cores of other eukaryotes are retained . They may be indispensable for meiotic recombination and proper chromosome segregation in meiosis I . In addition fission yeast shows interesting features of chromosome organization in vegetative and meiotic cells: Centromeres and telomeres cluster and associate with the spindle pole body . The special properties of fission yeast meiosis correlate with the absence of crossover interference in meiotic recombination . These findings are discussed . In addition homologous recombination in fission yeast is reviewed briefly. Experientia, 1994 Mar 15, 50(3), 234 - 41 Hotspots of homologous recombination; Smith GR; Homologous recombination occurs at higher than average frequency at and near hotspots . Hotspots are special nucleotide sequences recognized by proteins that promote, directly or indirectly, a rate limiting step of recombination . This review focuses on two well-studied examples, the Chi sites of the bacterium Escherichia coli and the M26 site of the fission yeast Schizosaccharomyces pombe . Chi, 5' G-C-T-G-G-T-G-G 3', is recognized by the RecBCD enzyme, which nicks the DNA near Chi and produces a 3'-ended single-stranded DNA 'tail'; this tail is a potent substrate for homologous pairing by RecA and single-stranded DNA binding proteins . M26, 5' A-T-G-A-C-G-T 3', is recognized by a heterodimeric protein and stimulates, by an as-yet-unknown mechanism, meiotic recombination at and near the ade6 gene . Additional hotspots in bacteria, fungi, and mammals enhance recombination directly or indirectly via a variety of mechanisms . Although hotspots are widespread among organisms, the biological role of their localized enhancement of recombination remains a matter of speculation. Experientia, 1994 Mar 15, 50(3), 223 - 33 The search for the right partner: homologous pairing and DNA strand exchange proteins in eukaryotes; Heyer WD; Finding the right partner is a central problem in homologous recombination . Common to all models for general recombination is a homologous pairing and DNA strand exchange step . In prokaryotes this process has mainly been studied with the RecA protein of Escherichia coli . Two approaches have been used to find homologous pairing and DNA strand exchange proteins in eukaryotes . A biochemical approach has resulted in numerous proteins from various organisms . Almost all of these proteins are biochemically fundamentally different from RecA . The in vivo role of these proteins is largely not understood . A molecular-genetical approach has identified structural homologs to the E . coli RecA protein in the yeast Saccharomyces cerevisiae and subsequently in other organisms including other fungi, mammals, birds, and plants . The biochemistry of the eukaryotic RecA homologs is largely unsolved . For the fungal RecA homologs (S . cerevisiae RAD51, RAD55, RAD57, DMC1; Schizosaccharomyces pombe rad51; Neurospora crassa mei3) a role in homologous recombination and recombinational repair is evident . Besides recombination, homologous pairing proteins might be involved in other cellular processes like chromosome pairing or gene inactivation. J Biol Chem, 1994 Mar 11, 269(10), 7304 - 9 Expression, purification, crystallization, and preliminary x-ray analysis of casein kinase-1 from Schizosaccharomyces pombe; Carmel G et al.; The catalytic domain of Schizosaccharomyces pombe casein kinase-1 (the product of the cki1 gene) has been overexpressed in Escherichia coli, purified by chromatographic methods, characterized in vitro, and crystallized in the presence and absence of nucleotide substrate . The best crystals belong to the trigonal space group P3(1)21 or its enantiomorph, have unit cell parameters a = b = 79 A, c = 121 A, and diffract x-rays to 2.0-A resolution . Kinetic characterization of the purified catalytic domain and other C-terminal deletion mutants of Cki1 suggests that it is subject to two forms of regulation . One mechanism involves autophosphorylation, and results in a 4-fold decrease in the affinity for protein substrate . In contrast, truncation of intact Cki1 results in a 3-fold activation in its catalytic rate . This activation may arise from the removal of an inhibitory domain present in the intact enzyme. Gene, 1994 Mar 11, 140(1), 51 - 7 The Histoplasma capsulatum cdc2 gene is transcriptionally regulated during the morphologic transition; Di Lallo G et al.; To understand the molecular mechanisms that control the reversible morphologic transition from mycelia to yeast in dimorphic fungi, we have isolated and characterized a cdc2 gene from Histoplasma capsulatum . This organism is a dimorphic pathogenic fungus that grows as a filamentous saprobic mold in soil and as a unicellular pathogenic yeast in human tissue . The cloned gene, whose protein product has a high degree of homology with other members of the cdc2 family, is split into four exons and three introns of 95, 52 and 85 nucleotides . Analyses of cDNA clones confirm the presence of the eukaryotic splice donor (GT) and acceptor (AG) sites . The spliced gene codes for a protein of 324 amino acids (aa) with a predicted molecular mass of 36.9 kDa . The H . capsulatum cdc2 product has 71% aa identity with Saccharomyces cerevisiae and 70% with Schizosaccharomyces pombe . The deduced protein contains the sequence, PSTAIRE, that is normally found in most p34cdc2 proteins . H . capsulatum cdc2 is transcriptionally regulated during the morphologic mycelium<==>yeast transitions and is more actively transcribed in the yeast than in the mycelial phase. Gene, 1994 Mar 11, 140(1), 41 - 9 The identification of a gene family in the Saccharomyces cerevisiae ergosterol biosynthesis pathway; Lai MH et al.; The Saccharomyces cerevisiae ERG24 gene, encoding sterol delta 14 reductase (Erg24p), was cloned by selecting strains carrying sequences on a 2 mu-based vector for resistance to the morpholine fungicide, fenpropimorph (Fp) . Four distinct plasmid inserts which conferred Fp resistance (FpR) were recovered (plasmids pML99, pML100, pML101 and pM103) . Although Fp is reported to inhibit activity of Erg24p and sterol delta 8-delta 7 isomerase (Erg2p; encoded by ERG2), none of the inserts had restriction maps resembling ERG2 . In addition, a 2 mu plasmid overexpression of the ERG2 sequence did not produce FpR . Characterization studies were focused on plasmid pML100, because it was the only plasmid to confer FpR consistently when tested in a number of different genetic backgrounds . Tests with a panel of fungicides indicated that pML100 conferred significant resistance only to compounds (Fp, tridemorph, fenpropidin and azasterol) which have a shared site of action, Erg24p . An insertional disruption of pML100 resulted in an obligate anaerobic phenotype, indicating a lesion in sterol biosynthesis . Sterol analysis of the disrupted mutant demonstrated the accumulation of ignosterol, indicating a loss of Erg24p activity . A SphI-XbaI fragment of pML100 was sequenced, revealing the presence of an ORF encoding a 438-amino-acid protein, which is highly similar to those encoded by two previously reported yeast drug sensitivity genes, sts1+ (Schizosaccharomyces pombe) and YGL022 (S . cerevisiae) . Analyses of these genes demonstrated that strains carrying disruptions of sts1+ or YGL022 have ergosterol biosynthesis defects in the enzyme, sterol C-24(28) reductase (Erg4p; encoded by ERG4).(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1994 Mar 4, 269(9), 6320 - 4 Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression; Nishi K et al.; The molecular action of leptomycin B (LMB), an agent inducing arrest of the eukaryotic cell cycle at G1 and G2 phases, was investigated by analyzing an LMB resistance gene of Schizosaccharomyces pombe . A genomic library of an LMB-resistant mutant was screened for LMB resistance, and a DNA fragment containing an open reading frame (ORF) of 1078 amino acids was cloned on a multicopy vector . The plasmid was found to confer drug resistance specifically to LMB . Nucleotide sequencing revealed that the ORF was a mutant gene for the essential nuclear protein crm1, which had been reported to complement a cold-sensitive mutation causing deformed nuclear morphology . The gene product named crm1-N1 had two amino acid replacements (Gly-503 to Asp and Met-546 to Ile) . Two allelic mutants of crm1 (crm1-809 and crm1-119) were found to be hypersensitive and resistant, respectively, to LMB . Nuclear morphology of the cold-sensitive crm1-809 mutant at the restrictive temperature was almost the same as that of the wild-type cells treated with LMB . Furthermore, a low concentration of LMB induced the intracellular accumulation of a 25-kDa protein in the wild-type cells, which was immunologically identical to the protein accumulating in the crm1-809 mutant cells . These results strongly suggest that LMB primarily inhibits the function of the crm1 gene which is required for maintaining higher order chromosome structures, correct gene expression, and cell growth in the fission yeast. Mol Cell Biol, 1994 Mar, 14(3), 2058 - 65 Sap1, a protein that binds to sequences required for mating-type switching, is essential for viability in Schizosaccharomyces pombe; Arcangioli B et al.; The pattern of mating-type switching in cell pedigrees of the fission yeast Schizosaccharomyces pombe is dictated by the inheritance of specific DNA chains at the mating-type locus (mat1) . The recombination event essential for switching is initiated by a site-specific double-strand break at mat1 . The switch-activating protein, Sap1, binds in vitro to a mat1 cis-acting site that was shown earlier to be essential for efficient mating-type switching . We isolated the sap1 gene by using oligonucleotides corresponding to the amino acid sequence of purified Sap1 protein . The sequence of that gene predicted a 30-kDa protein with no significant homology to other canonical DNA-binding protein motifs . To facilitate its biochemical characterization, Sap1 was expressed in Escherichia coli . The protein expressed in bacteria displayed the same DNA-binding specificities as the protein purified from S . pombe . Interestingly, analysis of a sap1 null mutation showed that the gene is essential for growth even in a strain in which mating-type switching is prohibited because of a defect in generation of the double-strand break . Thus, the sap1 gene product implicated in mating-type switching is shown to be essential for cell viability. Mol Cell Biol, 1994 Mar, 14(3), 2029 - 40 The rad16 gene of Schizosaccharomyces pombe: a homolog of the RAD1 gene of Saccharomyces cerevisiae; Carr AM et al.; The rad10, rad16, rad20, and swi9 mutants of the fission yeast Schizosaccharomyces pombe, isolated by their radiation sensitivity or abnormal mating-type switching, have been shown previously to be allelic . We have cloned DNA correcting the UV sensitivity or mating-type switching phenotype of these mutants and shown that the correcting DNA is encompassed in a single open reading frame . The gene, which we will refer to as rad16, is approximately 3 kb in length, contains seven introns, and encodes a protein of 892 amino acids . It is not essential for viability of S . pombe . The predicted protein is the homolog of the Saccharomyces cerevisiae RAD1 protein, which is involved in an early step in excision-repair of UV damage from DNA . The approximately 30% sequence identity between the predicted proteins from the two yeasts is distributed throughout the protein . Two-hybrid experiments indicate a strong protein-protein interaction between the products of the rad16 and swi10 genes of S . pombe, which mirrors that reported for RAD1 and RAD10 in S . cerevisiae . We have identified the mutations in the four alleles of rad16 . They mapped to the N-terminal (rad10), central (rad20), and C-terminal (rad16 and swi9) regions . The rad10 and rad20 mutations are in the splice donor sequences of introns 2 and 4, respectively . The plasmid correcting the UV sensitivity of the rad20 mutation was missing the sequence corresponding to the 335 N-terminal amino acids of the predicted protein . Neither smaller nor larger truncations were, however, able to correct its UV sensitivity. Mol Cell Biol, 1994 Mar, 14(3), 1796 - 805 Specific initiation at an origin of replication from Schizosaccharomyces pombe; Caddle MS et al.; Using a genetic assay for efficient autonomous replication, we have isolated from Schizosaccharomyces pombe a 6.2-kb fragment which shows the properties expected of an origin of DNA replication in S . pombe . A 2.8-kb subclone of the fragment has the same replication properties . Two-dimensional gel analysis of replication intermediates throughout plasmids carrying the 6.2- or 2.8-kb fragments shows that replication initiates only in a specific region, which can be localized to within several hundred base pairs, in the fragments . This region is also a site of replication initiation in the S . pombe chromosome where the fragments normally reside . These results provide strong evidence that initiation of replication in S . pombe is localized and mediated by specific DNA sequence signals. Mol Biol Cell, 1994 Mar, 5(3), 273 - 82 Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14; Kornbluth S et al.; The key regulator of entry into mitosis is the serine/threonine kinase p34cdc2 . This kinase is regulated both by association with cyclins and by phosphorylation at several sites . Phosphorylation at Tyr 15 and Thr 14 are believed to inhibit the kinase activity of cdc2 . In Schizosaccharomyces pombe, the wee1 (and possibly mik1) protein kinase catalyzes phosphorylation of Tyr 15 . It is not clear whether these or other, as yet unidentified, protein kinases phosphorylate Thr 14 . In this report we show, using extracts of Xenopus eggs, that the Thr 14-directed kinase is tightly membrane associated . Specifically, we have shown that a purified membrane fraction, in the absence of cytoplasm, can promote phosphorylation of cdc2 on both Thr 14 and Tyr 15 . In contrast, the cytoplasm can phosphorylate cdc2 only on Tyr 15, suggesting the existence of at least two distinctly localized subpopulations of cdc2 Tyr 15-directed kinases . The membrane-associated Tyr 15 and Thr 14 kinase activities behaved similarly during salt or detergent extraction and were similarly regulated during the cell cycle and by the checkpoint machinery that delays mitosis while DNA is being replicated . This suggests the possibility that a dual-specificity membrane-associated protein kinase may catalyze phosphorylation of both Tyr 15 and Thr 14. J Cell Sci, 1994 Mar, 107 ( Pt 3), 615 - 23 Study of the higher eukaryotic gene function CDK2 using fission yeast; Paris J et al.; In the fission yeast Schizosaccharomyces pombe, cdc2 function is required both in G1 to enter the cell cycle and in G2 to initiate mitosis . In higher eukaryotes, these functions appeared to be shared between several cdc2-like genes including CDK2 . Temperature-sensitive mutations in S . pombe cdc2 that arrest the cell cycle in both G1 and G2 phases are not complemented by CDK2 . We have used S . pombe to investigate what functions CDK2 can perform . We found that overexpression of the human homologue (HsCDK2) caused cell cycle arrest in G2/M showing that HsCDK2 interfered with mitotic events . Xenopus CDK2 (XlCDK2) overexpression did not cause cell cycle arrest and could rescue the G1 block but not the G2 block of a cdc2-M26 ts strain . A mutant XlCDK2-R33, which is inactive as a kinase, failed to rescue the G1 block, suggesting that the protein kinase activity of CDK2 is required to enter the cell cycle in these circumstances . We designed screens to select mutants that would require XlCDK2 expression for viability, hoping to isolate new gene functions interacting with, or that could be replaced by, XlCDK2 in G1, or new cdc2 mutants altered solely in their G1 role . From these screens several cell cycle mutants were selected that were XlCDK2-dependent . These were all cdc2 mutants altered only in their G2/M function . Therefore XlCDK2 can influence both the G1/S and G2/M transition points of cdc2 in S . pombe. J Cell Sci, 1994 Mar, 107 ( Pt 3), 601 - 13 Analysis of the Schizosaccharomyces pombe cyclin puc1: evidence for a role in cell cycle exit; Forsburg SL et al.; The puc1+ gene, encoding a G1-type cyclin from the fission yeast Schizosaccharomyces pombe, was originally isolated by complementation in the budding yeast Saccharomyces cerevisiae . Here, we report the molecular characterization of this gene and analyse its role in S . pombe . We fail to identify any function of this cyclin at the mitotic G1/S transition in S . pombe, but demonstrate that it does function in exit from the mitotic cycle . Expression of the puc1+ gene is increased during nitrogen starvation, and puc1 affects the timing of sexual development in response to starvation . Overexpression of the puc1 protein blocks sexual development, and rescues pat1ts cells, which would otherwise undergo a lethal meiosis . We conclude that puc1 contributes to negative regulation of the timing of sexual development in fission yeast, and functions at the transition between cycling and non-cycling cells. J Cell Sci, 1994 Mar, 107 ( Pt 3), 469 - 86 Unusual chromosome structure of fission yeast DNA in mouse cells; McManus J et al.; Chromosomes from the fission yeast Schizosaccharomyces pombe have been introduced into mouse cells by protoplast fusion . In most cell lines the yeast DNA integrates into a single site within a mouse chromosome and results in striking chromosome morphology at metaphase . Both light and electron microscopy show that the yeast chromosome region is narrower than the flanking mouse DNA . Regions of the yeast insert stain less intensely with propidium iodide than surrounding DNA and bear a morphological resemblance to fragile sites . We investigate the composition of the yeast transgenomes and the modification and chromatin structure of this yeast DNA in mouse cells . We suggest that the underlying basis for the structure we see lies above the level of DNA modification and nucleosome assembly, and may reflect the attachment of the yeast DNA to the rodent cell nucleoskeleton . The yeast integrant replicates late in S phase at a time when G bands of the mouse chromosomes are being replicated, and participates in sister chromatid exchanges at a high frequency . We discuss the implications of these studies to the understanding of how chromatin folding relates to metaphase chromosome morphology and how large stretches of foreign DNA behave when introduced into mammalian cells. Genetics, 1994 Mar, 136(3), 849 - 56 Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe; Keeney JB et al.; Homologous integration into the fission yeast Schizosaccharomyces pombe has not been well characterized . In this study, we have examined integration of plasmids carrying the leu1+ and ura4+ genes into their chromosomal loci . Genomic DNA blot analysis demonstrated that the majority of transformants have one or more copies of the plasmid vector integrated via homologous recombination with a much smaller fraction of gene conversion to leu1+ or ura4+ . Non-homologous recombination events were not observed for either gene . We describe the construction of generally useful leu1+ and ura4+ plasmids for targeted integration at the leu1-32 and ura4-294 loci of S . pombe. Genetics, 1994 Mar, 136(3), 769 - 79 Transient, meiosis-induced expression of the rec6 and rec12 genes of Schizosaccharomyces pombe; Lin Y et al.; Two meiotic recombination genes, rec6 and rec12, from Schizosaccharomyces pombe have been cloned by genetic complementation and their DNA sequences determined . Gene replacements demonstrated that the cloned fragments contained the rec6 and rec12 genes . Further analysis showed that the functional rec6 gene was within a 1.3-kb fragment and rec12 within a 1.7-kb fragment . The nucleotide sequences of these fragments revealed open reading frames (ORFs) predicting 143 amino acids for the rec6 gene product and 139 amino acids for the rec12 gene product . After pat1-114 temperature-induced meiosis, the transcripts of rec6 and rec12 were induced to maximal levels at 2-3 hr, at about the time of premeiotic DNA synthesis, but were present at much lower levels before and after this time . The transient induction of the transcripts and the phenotypes of the mutants suggest that the rec6 and rec12 gene products are involved primarily in the early steps of meiotic recombination in S . pombe . Near the rec6 gene are two additional ORFs potentially encoding proteins with homology to ribosomal protein S7 of Saccharomyces cerevisiae (ORF137) and to the homeodomain of developmental regulatory proteins (ORF201) . The roles of these S . pombe ORFs remain to be determined. FEBS Lett, 1994 Feb 28, 340(1-2), 145 - 50 A phorbol ester-responsive PKC-zeta generated by fusion with the regulatory domain of PKC-delta; Goode NT et al.; A hybrid molecule generated by fusing the regulatory domain of PKC-delta with the catalytic domain of PKC-zeta is, like PKC-delta but unlike PKC-zeta, a phorbol ester-dependent enzyme . However, the substrate specificity of this hybrid resembles that of PKC-zeta . Expression of mammalian PKC-delta, but not PKC-zeta, in the fission yeast Schizosaccharomyces pombe causes growth retardation and phorbol esters amplify the PKC-delta phenotype without affecting that of PKC-zeta (Goode et al., submitted) . The chimaeric molecule also inhibited growth and this effect was phorbol-ester dependent . Both the hybrid and PKC-delta holoenzyme, in contrast to PKC-zeta, down-regulate upon prolonged exposure to phorbol esters in vivo . Thus, this hybrid retains the regulatory properties conferred by PKC-delta but the catalytic properties of PKC-zeta . This regulatable chimaeric molecule will be useful in assessing the function of PKC-zeta. Nucleic Acids Res, 1994 Feb 25, 22(4), 686 - 93 Inhibition of protein synthesis by an efficiently expressed mutation in the yeast 5.8S ribosomal RNA; Abou Elela S et al.; Recent studies on the inhibition of protein synthesis by specific anti 5.8S rRNA oligonucleotides strongly suggested that this RNA plays an important role in eukaryotic ribosome function . To evaluate this possibility further, a ribosomal DNA transcription unit from Schizosaccharomyces pombe was cloned into yeast shuttle vectors with copy numbers ranging from 2 to approximately 90 per cell; to allow direct detection of expressed RNA and to disrupt the function of the 5.8S rRNA molecule, a five base insertion was made in a universally conserved GAAC sequence . The altered mobility of the mutant RNA was readily detected by gel electrophoresis and analyses indicated that mutant RNA transcription reflected the ratio of plasmid to endogenous rDNA . The highest copy number plasmid resulted in about 40-50% mutant RNA . This mutant RNA was readily integrated into the ribosome structure resulting in an in vivo ribosome population which was also about 40-50% mutant; the rates of growth and protein synthesis were equally reduced by approximately 40% . A comparable level of inhibition in protein synthesis was demonstrated in vitro and polyribosomal profiles revealed a consistent increase in size . Subsequent RNA analyses indicated a normal distribution of mutant RNA in both monoribosomes and polyribosomes, but elevated tRNA levels in mutant polyribosomes . Additional mutations in alternate GAAC sequences revealed similar but cumulative effects on both protein synthesis and polyribosome profiles . Taken together, these results suggest little or no effect on initiation but provide in vivo evidence of a functional role for the 5.8S rRNA in protein elongation. Science, 1994 Feb 11, 263(5148), 805 - 7 RNA polymerase II initiation factor interactions and transcription start site selection; Li Y et al.; An RNA polymerase II transcription system was resolved and reconstituted from extracts of Schizosaccharomyces pombe . Exchange with components of a Saccharomyces cerevisiae system was undertaken to reveal the factor or factors responsible for the difference in location of the transcription start site, about 30 base pairs and 40 to 120 base pairs downstream of the TATA box in S . pombe and S . cerevisiae, respectively . Two components, counterparts of human transcription factor IIF (TFIIF) and TFIIH, could be exchanged individually between systems without effect on the start site . Three components, counterparts of human TFIIB, TFIIE, and RNA polymerase II, could not be exchanged individually but could be swapped in the pairs TFIIE-TFIIH and TFIIB-RNA polymerase II, which demonstrates that there are functional interactions between these components . Moreover, exchange of the latter pair shifted the starting position, which shows that TFIIB and RNA polymerase II are solely responsible for determining the start site of transcription. J Biol Chem, 1994 Feb 4, 269(5), 3498 - 502 Structure/function relationship of the Chlorella glucose/H+ symporter; Caspari T et al.; The Clorella kessleri HUP 1 gene coding for a hexose/H+ symporter has been expressed in a glucose uptake-deficient mutant of Schizosaccharomyces pombe . The transformants are able to grow on glucose and to accumulate 3-O-methylglucose 100-fold . This system has been used to test the activity of specifically mutated HUP 1 cDNAs . All three histidyl residues were exchanged with arginine (H73R, H170R, and H495R) without a major effect on transport activity . When Asp-44 within the first transmembrane helix was replaced by Asn, the transporter was inactive; replacement by Glu (D44E) resulted in a loss of activity by 90% and a 15-fold increased Km value . Glutamine residues conserved in all glucose transporters sequenced so far were exchanged: Q179N (in helix 5), Q298G and Q299N (both in helix 7) . Whereas Q298G only resulted in a small Km change, both Q179N and Q299N showed an increase in Km by a factor of 10 . Inserting 4 additional amino acids each into the two largest loops (1 and 6) reduced the activity dramatically; only in the latter case this was due to decreased protein synthesis or stability . Two COOH-terminal deletions (-27 and -43 amino acids) were also tested . The 27 COOH-terminal amino acids, but not the 43 COOH-terminal amino acids, could be removed without affecting transporter activity. Genes Dev, 1994 Feb 1, 8(3), 328 - 38 The fission yeast mating pheromone P-factor: its molecular structure, gene structure, and ability to induce gene expression and G1 arrest in the mating partner; Imai Y et al.; Schizosaccharomyces pombe h+ cells secrete a diffusable mating pheromone called P-factor . Here we show that the map2 gene, a defect of which confers h(+)-specific sterility, encodes the precursor of P-factor . We purified P-factor from cells overexpressing map2 and determined its amino acid sequence . P-factor is a peptide of 23 residues, with the sequence Thr-Tyr-Ala-Asp-Phe-Leu-Arg-Ala-Tyr-Gln-Ser- Trp-Asn-Thr-Phe-Val-Asn-Pro-Asp-Arg-Pro-Asn-Leu . A synthetic peptide of this sequence gave the same specific activity and chromatographic profile as the purified P-factor, suggesting that P-factor is unmodified . h- cells starved for nutrition showed a morphological response to P-factor . Transcription of the sxa2 gene, which encodes a protease thought to degrade P-factor, was activated in these cells . The cry1 null mutant, which lacks adenylyl cyclase and has little intracellular cAMP, was susceptible to P-factor even in the presence of nutrients . Combination of the cyr1 and sxa2 mutations enhanced this susceptibility . P-factor induced not only responses toward mating but also arrest of the cell cycle at the G1 phase in h- cyr1 sxa2 cells . This proves that the S . pombe mating pheromone has the ability to arrest cell cycle progression, which has previously been obscured by the usual requirement for mating of nutritional starvation and subsequent growth arrest. EMBO J, 1994 Feb 1, 13(3), 606 - 15 A fission yeast RCC1-related protein is required for the mitosis to interphase transition; Sazer S et al.; The isolation and characterization of the mutant dcdts (defect in chromatin decondensation) has led to the identification of two conserved proteins required for the re-establishment of the interphase state following the completion of mitosis . The gene that rescues the dcdts mutant encodes a protein similar to the human chromatin binding protein, RCC1 . A suppressor of dcdts encodes a protein nearly identical to the human GTP-binding protein, RAN, encoded by the TC4 gene . These results indicate that completion of mitosis is regulated at least in part by a GTPase molecular switch . The gene and suppressor of dcdts are identical to the previously described Schizosaccharomyces pombe genes pim1 (premature initiation of mitosis) and spi1 (suppressor of pim), but the dcdts mutant does not enter mitosis prematurely, a phenotype that has been reported for the pim1-46ts mutant . Based on our studies we propose that the pim1 gene product is required for regulating chromatin condensation with a primary role at the end of mitosis and pleiotropic effects on other aspects of cell behavior. Mol Cell Biol, 1994 Feb, 14(2), 1075 - 83 Cdc42p GTPase is involved in controlling polarized cell growth in Schizosaccharomyces pombe; Miller PJ et al.; Cdc42p is a highly conserved low-molecular-weight GTPase that is involved in controlling cellular morphogenesis . We have isolated the Cdc42p homolog from the fission yeast Schizosaccharomyces pombe by its ability to complement the Saccharomyces cerevisiae cdc42-1ts mutation . S . pombe Cdc42p is 85% identical in predicted amino acid sequence to S . cerevisiae Cdc42p and 83% identical to the human Cdc42p homolog . The Cdc42p protein fractionates to both soluble and particulate fractions, suggesting that it exists in two cellular pools . We have disrupted the cdc42+ gene and shown that it is essential for growth . The cdc42 null phenotype is an arrest as small, round, dense cells . In addition, we have generated three site-specific mutations, G12V, Q61L, and D118A, in the Cdc42p GTP-binding domains that correspond to dominant-lethal mutations in S . cerevisiae CDC42 . In contrast to the S . cerevisiae cdc42 mutations, the S . pombe cdc42 mutant alleles were not lethal when overexpressed . However, the cdc42 mutants did exhibit an abnormal morphological phenotype of large, misshapen cells, suggesting that S . pombe Cdc42p is involved in controlling polarized cell growth. Yeast, 1994 Feb, 10(2), 173 - 83 Structural modification of spindle pole bodies during meiosis II is essential for the normal formation of ascospores in Schizosaccharomyces pombe: ultrastructural analysis of spo mutants; Hirata A et al.; In order to characterize the morphological steps defined by sporulation (spo) genes during the formation of ascospores in the fission yeast Schizosaccharomyces pombe, we performed an electron microscopic study of the ultrastructure of the spindle pole body (SPB) and of the development of the forespore membrane during the second meiotic division (meiosis II) in sporulation-deficient (spo) mutants (spo4, spo5, spo14 and spo18) . No difference was found in terms of the function and the structure of the SPB during the first meiotic division (meiosis I) between the four mutants and wild-type cells . However, during meiosis II, the spo4 and spo18 mutants underwent nuclear division but in neither case were the SPBs modified nor were forespore membranes formed . The SPBs of the spo18 mutant diminished in size after meiosis II and eventually disappeared after 18 h in sporulation medium . By contrast, the SPBs of the spo4 mutant remained unchanged even after an 18-h incubation . The outer plaques of SPBs of spo5 and spo14 mutants were sufficiently modified to allow them to initiate development of the forespore membrane, but the membrane had an abnormally expanded lumen and did not enclose the nuclei during meiosis II . The spo5 mutant produced anucleate spore-like bodies while the spo14 mutant formed unorganized structures with irregular peripheries which, presumably, contained spore-wall precursors, instead of anucleate spore-like bodies . We conclude that the modification of the SPB is essential for the formation of ascospores and at least two genes (spo5 and spo14) participate in the development of the forespore membrane . The defective phenotypes define discrete steps in the development of ascospores, which proceeds via steps defined by the mutant spo4, spo18, spo14 and spo5 genes respectively . Our observations provide further substantial evidence that the SPB plays a pivotal role in the normal development of ascospores in yeasts. Yeast, 1994 Feb, 10(2), 159 - 72 A homologous cell-free system for studying protein translocation across the endoplasmic reticulum membrane in fission yeast; Brennwald P et al.; We report the development of a homologous in vitro assay system for analysing translocation of proteins across the endoplasmic reticulum (ER) membrane of the fission yeast Schizosaccharomyces pombe . Our protocol for preparing an S . pombe extract capable of translating natural messenger RNAs was modified from a procedure previously used for Saccharomyces cerevisiae, in which cells are lysed in a bead-beater . However, we were unable to prepare fission yeast microsomes active in protein translocation using existing budding yeast protocols . Instead, our most efficient preparations were isolated by fractionating spheroplasts, followed by extensive washing and size exclusion chromatography of the crude membranes . Translocation of two ER-targeted proteins, pre-acid phosphatase from S . pombe and prepro-alpha-factor from S . cerevisiae, was monitored using two distinct assays . First, evidence that a fraction of both proteins was sequestered within membrane-enclosed vesicles was provided by resistance to exogenously added protease . Second, the protected fraction of each protein was converted to a higher molecular weight, glycosylated form; attachment of carbohydrate to the translocated proteins was confirmed by their ability to bind Concanavalin A-Sepharose . Finally, we examined whether proteins could be translocated across fission yeast microsomal membranes after their synthesis was complete . Our results indicate that S . cerevisiae prepro-alpha-factor can be post-translationally imported into the fission yeast ER, while S . pombe pre-acid phosphatase crosses the membrane only by a co-translational mechanism. Curr Opin Genet Dev, 1994 Feb, 4(1), 82 - 9 MAP kinase kinase kinase, MAP kinase kinase and MAP kinase; Marshall CJ; Signal transduction pathways that respond to external signals through the MAP kinase family of protein kinases are involved in diverse responses in eukaryotic cells . MAP kinases are one element in a series of kinases that serve to connect the plasma membrane with cytoplasmic and nuclear events . MAP kinases have the unusual feature that their activation requires threonine and tyrosine phosphorylation carried out by a dual specificity protein kinase . Recent advances have shown that in two MAP kinase pathways (the mating response pathway in the fission yeast Schizosaccharomyces pombe, and receptor tyrosine kinase signalling), the small GTP binding protein ras p21 links membrane events to kinase pathway activation. Curr Opin Cell Biol, 1994 Feb, 6(1), 50 - 4 Genetic and biochemical approaches to spindle function and chromosome segregation in eukaryotic microorganisms; Kilmartin JV; The past year saw the molecular characterization of components of the Saccharomyces cerevisiae kinetochore and spindle pole body . In Schizosaccharomyces pombe, new cytological methods have been described for detection of centromeric DNA by light microscopy and probable kinetochores by electron microscopy. Curr Opin Cell Biol, 1994 Feb, 6(1), 110 - 9 The yeast actin cytoskeleton; Welch MD et al.; Budding and fission yeast present significant advantages for studies of the actin cytoskeleton . The application of classical and molecular genetic techniques provides a facile route for the analysis of structure/function relationships, for the isolation of novel proteins involved in cytoskeletal function, and for deciphering the signals that regulate actin assembly in vivo . This review focuses on the budding yeast Saccharomyces cerevisiae and also identifies some recent advances from studies on the fission yeast Schizosaccharomyces pombe, for which studies on the actin cytoskeleton are still in their infancy. Eur J Biochem, 1994 Feb 1, 219(3), 775 - 80 Mammalian phosphatidylinositol 3'-kinase induces a lethal phenotype on expression in Schizosaccharomyces pombe; comparison with the VPS34 gene product; Kodaki T et al.; The 110-kDa catalytic subunit of the phosphatidylinositol 3'-kinase (p110) is shown to be expressed in Schizosaccharomyces pombe as a functional protein, as judged by the accumulation of 3'-phosphorylated lipids in vivo and the extraction of 3'-kinase activity in vitro . On expression of p110, the cells fail to grow and lose viability . In contrast, while the Saccharomyces cerevisiae protein Vps34p can be expressed in S . pombe as a functional, extractable, phosphatidylinositol 3-kinase, expression of this protein fails to increase substantially 3'-phosphorylated lipids in vivo and does not induce a phenotype equivalent to that induced by p110 . The results indicate that (over-)-accumulation of 3'-phosphorylated inositol lipids in S . pombe causes loss of viability. Mol Biol Cell, 1994 Feb, 5(2), 147 - 60 Identification and characterization of new elements involved in checkpoint and feedback controls in fission yeast; al-Khodairy F et al.; To investigate the mechanisms that ensure the dependency relationships between cell cycle events and to investigate the checkpoints that prevent progression through the cell cycle after DNA damage, we have isolated mutants defective in the checkpoint and feedback control pathways . We report the isolation and characterization of 11 new loci that define distinct classes of mutants defective in one or more of the checkpoint and feedback control pathways . Two mutants, rad26.T12 and rad27.T15, were selected for molecular analysis . The null allele of the rad26 gene (rad26.d) shares the phenotype reported for the "checkpoint rad" mutants rad1, rad3, rad9, rad17, and hus1, which are defective in the radiation checkpoint and in the feedback controls that ensure the order of cell cycle events . The null allele of the rad27 gene (rad27.d) defines a new class of Schizosaccharomyces pombe mutant . The rad27 complementing gene codes for a putative protein kinase that is required for cell cycle arrest after DNA damage but not for the feedback control that links mitosis to the completion of prior DNA synthesis (the same gene has recently been described by Walworth et al . (1993) as chk1) . These properties are similar to those of the rad9 gene of Saccharomyces cerevisiae . A comparative analysis of the radiation responses in rad26.d, rad26.T12, and rad27.d cells has revealed the existence of two separable responses to DNA damage controlled by the "checkpoint rad" genes . The first, G2 arrest, is defective in rad27.d and rad26.d but is unaffected in rad26.T12 cells . The second response is not associated with G2 arrest after DNA damage and is defective in rad26.d and rad26.T12 but not rad27.d cells . A study of the radiation sensitivity of these mutants through the cell cycle suggests that this second response is associated with S phase and that the checkpoint rad mutants, in addition to an inability to arrest mitosis after radiation, are defective in an S phase radiation checkpoint. Biochem Biophys Res Commun, 1994 Jan 28, 198(2), 770 - 9 UV induction of excision repair enzymes detected in protein extracts from Schizosaccharomyces pombe; Jaeg JP et al.; Induction of genes and proteins after DNA damaging treatment is well documented in various biological systems . In order to monitor repair activity in Schizosacchromyces pombe, we adapted the biochemical assay that allowed specific quantification of excision repair in mammalian cells (Wood et al . 1988, Cell, 53, 97-106) to yeast-free extracts . Repair synthesis determined on UV-damaged plasmid DNA with S . pombe total protein extract relied on base excision repair and not nucleotide excision repair . Under conditions that allowed optimal repair activity, an enhanced repair synthesis was found with extract from yeast previously irradiated with UV light (254 nm) . A 4-fold induction factor was obtained with 70 J/m2 irradiation dose after 40 min incubation post-irradiation . This base excision repair activity on UV photoproducts was transiently induced since it returned to the level of untreated yeast after about 2 hours post-irradiation. Nucleic Acids Res, 1994 Jan 25, 22(2), 200 - 7 Replacement of the Saccharomyces cerevisiae RPR1 gene with heterologous RNase P RNA genes; Pagan-Ramos E et al.; Phylogenetic studies of yeast nuclear RNase P RNA genes have shown a striking conservation of secondary structure for the Saccharomyces and Schizosaccharomyces RNase P RNAs, yet much of the primary sequence and many substructures vary among the RNAs examined . To investigate which sequences and structural features can be varied and still allow function in a heterologous organism, RNase P genes from several yeast species were tested for the ability to substitute for the Saccharomyces cerevisiae RNA . The RNase P genes from Saccharomyces carlsbergensis and Saccharomyces kluyveri could act as the sole source of RNase P RNA within S . cerevisiae cells, whereas the genes from Saccharomyces globosus and Schizosaccharomyces pombe could not . Although heterologous RNase P RNAs were synthesized by the cells in all cases, the RNAs that complemented tended to be processed from longer precursor transcripts into mature-sized RNase P RNA, while the RNAs that did not complement tended to accumulate as the longer precursor form . The results identified sequences and structures in the RNA that are not essential for interaction with species-specific proteins, processing or localization, and suggested other positions that may be candidates for such processes. EMBO J, 1994 Jan 15, 13(2), 471 - 83 The Schizosaccharomyces pombe cdc5+ gene encodes an essential protein with homology to c-Myb; Ohi R et al.; The Schizosaccharomyces pombe cdc5+ gene was identified in the first screen for cell division cycle mutants in this yeast . The cdc5+ gene was reported to be required for nuclear division but because of its modest elongation and leaky nature at the non-permissive temperature, it was not investigated further . Here, we report the characterization of the single allele of this gene, cdc5-120, in more detail . The mutant arrests with a 2N DNA content and a single interphase nucleus . Further genetic analyses suggest that cdc5+ gene function is essential in the G2 phase of the cell cycle . We have cloned and sequenced the cdc5+ gene . The deduced protein sequence predicts that Cdc5 is an 87 kDa protein and contains a region sharing significant homology with the DNA binding domain of the Myb family of transcription factors . Deletion mapping of the cdc5+ gene has shown that the N-terminal 232 amino acids of the protein, which contain the Myb-related region, are sufficient to complement the cdc5ts strain . A cdc5 null mutant was generated by homologous recombination . Haploid cells lacking cdc5+ are inviable, indicating that cdc5+ is an essential gene . A fusion protein consisting of bacterial glutathione S-transferase joined in-frame to the N-terminal 127 amino acids of the Cdc5 protein is able to bind to DNA cellulose at low salt concentrations . This evidence suggests that cdc5+ might encode a transcription factor whose activity is required for cell cycle progression and growth during G2. Cell, 1994 Jan 14, 76(1), 157 - 69 Position effect variegation at fission yeast centromeres; Allshire RC et al.; Chromatin structure at Schizosaccharomyces pombe centromeres is unusual . The insertion of the ura4 gene within these centromeres resulted in genetically identical cells mosaic for its expression . Placement of the ade6 gene within cen1 or cen3 resulted in red-white sectored colonies, demonstrating the instability of gene expression . The occurrence of pink colonies implied that intermediate levels of repression were established . Repression of both genes within centromeres was temperature sensitive . The chromatin structure of the ura4 gene at centromeres was altered, suggesting that the unusual chromatin encroaches into the gene and inhibits normal expression . These repressive effects at S . pombe centromeres resemble the classical phenomenon of position effect variegation imposed by Drosophila heterochromatin on nearby genes . However, since the epigenetic states can be set at intermediate levels of expression, a purely euchromatin-heterochromatin dichotomy does not apply . A model for the epigenetic regulation of genes placed within S . pombe centromeres is presented. Biochim Biophys Acta, 1994 Jan 4, 1183(3), 550 - 2 Molecular cloning and nucleotide sequencing of Schizosaccharomyces pombe homologue of the class II fructose-1,6-bisphosphate aldolase gene; Mutoh N et al.; DNA fragment containing Schizosaccharomyces pombe homologue of the class II fructose-1,6-bisphosphate aldolase gene was cloned and sequenced . A long open reading frame, which encodes a polypeptide of 358 amino acid residues, was found in the sequence . Amino acid sequence deduced from the nucleotide sequence is 63% homologous to the amino acid sequence of the enzyme of Saccharomyces cerevisiae . Northern blot analysis revealed that 1.3 kb poly(A)+ RNA is transcribed from this DNA sequence. Genes Dev, 1994 Jan, 8(2), 203 - 10 Region-specific activators of meiotic recombination in Schizosaccharomyces pombe; DeVeaux LC et al.; Schizosaccharomyces pombe rec mutants were previously isolated on the basis of their deficiency in meiotic recombination at the ade6 locus . We surveyed their meiotic recombination deficiencies at and between other loci . In rec10 mutants recombinant frequencies in the approximately 2-Mb region surrounding the ade6 locus were reduced 10- to 100-fold, but recombinant frequencies at or between nine other unlinked loci were reduced < 3-fold . The rec10 mutations are recessive and are on chromosome I; the ade6 region is on chromosome III . These results indicate that the rec10 gene product is required for activation of meiotic recombination in the approximately 2-Mb region surrounding ade6 but not in the other regions surveyed . Similar ade6 regional specificities were observed for rec8 and rec11 . We infer that there are multiple activators of meiotic recombination, each specific for a limited set of loci, and we discuss how these regional activators may work. J Bacteriol, 1994 Jan, 176(1), 232 - 9 Coexpression of eukaryotic tRNASer and yeast seryl-tRNA synthetase leads to functional amber suppression in Escherichia coli; Weygand-Durasevic I et al.; In order to gain insight into the conservation of determinants for tRNA identity between organisms, Schizosaccharomyces pombe and human amber suppressor serine tRNA genes have been examined for functional expression in Escherichia coli . The primary transcripts, which originated from E . coli plasmid promoters, were processed into mature tRNAs, but they were poorly aminoacylated in E . coli and thus were nonfunctional as suppressors in vivo . However, coexpression of cloned Saccharomyces cerevisiae seryl-tRNA synthetase led to efficient suppression in E . coli . This shows that some, but not all, determinants specifying the tRNASer identity are conserved in evolution. Mol Cell Biol, 1994 Jan, 14(1), 768 - 76 Interaction between the Cig1 and Cig2 B-type cyclins in the fission yeast cell cycle; Connolly T et al.; In this report, we describe the cloning and characterization of a B-type cyclin, Cig2 from the fission yeast Schizosaccharomyces pombe . The cig2 gene encodes a 45-kDa protein that is most similar to a previously identified B-type cyclin in S . pombe, Cdc13 . Deletion of cig2 had no observable effect on cell viability or progression through the cell cycle . Strains carrying the cig2 null allele do, however, exhibit an enhanced ability to undergo conjugation relative to a wild-type strain . The cig2 transcript was found to undergo periodic oscillation during the cell cycle, peaking at the G1/S-phase boundary . We have investigated the relationship between Cig2 and the other B-type cyclins, Cig1 and Cdc13, in the fission yeast . We found that cells carrying disruptions of both the cig1 and cig2 genes contain multiple nuclei with a 1C DNA content, suggesting that they are delayed in progression through the G1 phase of the cell cycle . The phenotype of this double mutant suggests that there is a delay in septum formation, possibly as a result of defective nuclear separation. Mol Cell Biol, 1994 Jan, 14(1), 576 - 86 The Schizosaccharomyces pombe casein kinase II alpha and beta subunits: evolutionary conservation and positive role of the beta subunit; Roussou I et al.; Casein kinase II is a key regulatory enzyme involved in many cellular processes, including the control of growth and cell division . We report the molecular cloning and sequencing of cDNAs encoding the alpha and the beta subunits of casein kinase II of Schizosaccharomyces pombe . The deduced amino acid sequence of Cka1, the alpha catalytic subunit, shows high sequence similarity to alpha subunits identified in other species . The amino acid sequence of Ckb1, the S . pombe beta subunit, is 57% identical to that of the human beta subunit . Cka1 overexpression results in no detectable phenotype . In contrast, Ckb1 overexpression inhibits cell growth and cytokinesis, with formation of multiseptated cells . Disruption of the ckb1+ gene causes a cold-sensitive phenotype and abnormalities in cell shape . In these cells, the casein kinase II activity is reduced to undetectable levels, demonstrating that Ckb1 is required for enzyme activity in vivo . In agreement with this, the activity measured in a strain expressing high levels of Cka1 is enhanced only when the Ckb1 protein is coexpressed . Altogether, our data suggest that Ckb1 is a positive regulator of the enzyme activity, and that it plays a role in mediating the interaction of casein kinase II with downstream targets and/or with additional regulators. Mol Cell Biol, 1994 Jan, 14(1), 50 - 8 Concerted action of RAS and G proteins in the sexual response pathways of Schizosaccharomyces pombe; Xu HP et al.; We have shown that the expression of mam2, the gene encoding the Schizosaccharomyces pombe P-factor pheromone receptor, is dependent upon components of the pheromone signal transduction pathway, including Ras1, Gpa1, Byr1 and Byr2, each of which is required for both conjugation and sporulation . Studies of the expression of mam2 in mutant S . pombe cells confirm previous conclusions, based on the ability of cells to sporulate, that the Byr1 protein kinase acts downstream of the Byr2 protein kinase and that both act downstream of Ras1, the S . pombe RAS homolog, and Gpa1, the G alpha component that mediates the occupancy of the mam2 receptor . In addition, our present studies show that Ras1 and Gpa1 each act downstream from the other and hence act in concert . The Spk1 kinase, which is required for conjugation and sporulation and which is a structural and functional homolog of the vertebrate MAP kinases, is not required for mam2 expression. Yeast, 1994 Jan, 10(1), 1 - 11 Inhibition of protein synthesis disrupts the Golgi apparatus in the fission yeast, Schizosaccharomyces pombe; Ayscough K et al.; Schizosaccharomyces pombe was treated with either cycloheximide or anisomycin at levels sufficient to inhibit > 95% of protein synthesis for periods upon to 3 h, equivalent to one cell cycle . Treatment for as little as 1 h caused significant loss of the Golgi apparatus by both immunofluorescence and electron microscopy . The loss was quantitated by stereology on electron micrographs . Nearly 90% of the stacked Golgi was lost over a 3 h period . No other intracellular membrane compartment seemed to be affected . Measurement of enzyme activities confirmed these observations . The activity of a resident of the Golgi apparatus, alpha-1,2 galactosyltransferase, was reduced over this time, whereas the endoplasmic reticulum marker, BiP, and the cytoplasmic enzyme, hexokinase, were unaffected . The morphological changes associated with cycloheximide addition were reversed on its removal, though there was a lag before cells recommenced growth or secretion of the enzyme, acid phosphatase. Radiat Environ Biophys, 1994, 33(1), 9 - 21 Randomly distributed DNA double-strand breaks as measured by pulsed field gel electrophoresis: a series of explanatory calculations; Cedervall B et al.; The aim of this article is to characterize expressions of relevance to the interpretation of pulsed field gel electrophoresis (PFGE) experiments where randomly distributed double-strand breaks (DSBs) are detected as smears of DNA fragments . Specifically, equations for conversion of percentages of fragments in defined size ranges to DSBs were derived . Several models have been used, one of which is based on theoretically fragmented DNA from the fission yeast Schizosaccharomyces pombe, which has three PFGE separable chromosomes. J Cell Sci, 1994 Jan, 107 ( Pt 1), 253 - 65 A human nuclear protein with sequence homology to a family of early S phase proteins is required for entry into S phase and for cell division; Todorov IT et al.; Molecular cloning and characterisation of a human nuclear protein designated BM28 is reported . On the amino acid level this 892 amino acid protein, migrating on SDS-gels as a 125 kDa polypeptide, shares areas of significant similarity with a recently defined family of early S phase proteins . The members of this family, the Saccharomyces cerevisiae Mcm2p, Mcm3p, Cdc46p/Mcm5p, the Schizosaccharomyces pombe Cdc21p and the mouse protein P1 are considered to be involved in the onset of DNA replication . The highest similarity was found with Mcm2p (42% identity over the whole length and higher than 75% over a conservative region of 215 amino acid residues), suggesting that BM28 could represent the human homologue of the S . cerevisiae MCM2 . Using antibodies raised against the recombinant BM28 the corresponding antigen was found to be localised in the nuclei of various mammalian cells . Microinjection of anti-BM28 antibody into synchronised mouse NIH3T3 or human HeLa cells presents evidence for the involvement of the protein in cell cycle progression . When injected in G1 phase the anti-BM28 antibody inhibits the onset of subsequent DNA synthesis as tested by the incorporation of bromodeoxyuridine . Microinjection during the S phase had no effect on DNA synthesis, but inhibits cell division . The data suggest that the nuclear protein BM28 is required for two events of the cell cycle, for the onset of DNA replication and for cell division. Mol Gen Genet, 1994 Jan, 242(2), 121 - 9 Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae; Kimura M et al.; Aspergillus terreus produces a unique enzyme, blasticidin S deaminase, which catalyzes the deamination of blasticidin S (BS), and in consequence confers high resistance to the antibiotic . A cDNA clone derived from the structural gene for BS deaminase (BSD) was isolated by transforming Escherichia coli with an Aspergillus cDNA expression library and directly selecting for the ability to grow in the presence of the antibiotic . The complete nucleotide sequence of BSD was determined and proved to contain an open reading frame of 393 bp, encoding a polypeptide of 130 amino acids . Comparison of its nucleotide sequence with that of bsr, the BS deaminase gene isolated from Bacillus cereus, indicated no homology and a large difference in codon usage . The activity of BSD expressed in E . coli was easily quantified by an assay based on spectrophotometric recording . The BSD gene was placed in a shuttle vector for Schizosaccharomyces pombe, downstream of the SV40 early region promoter, and this allowed direct selection with BS at high frequency, following transformation into the yeast . The BSD gene was also employed as a selectable marker for Pyricularia oryzae, which could not be transformed to BS resistance by bsr . These result promise that the BSD gene will be useful as a new dominant selectable marker for eukaryotes. Genetics, 1994 Jan, 136(1), 53 - 64 Mutations in rik1, clr2, clr3 and clr4 genes asymmetrically derepress the silent mating-type loci in fission yeast; Ekwall K et al.; In Schizosaccharomyces pombe the mating-type information is stored at two transcriptionally silent loci (mat2 and mat3) . The region between these sites (K region) is inert for meiotic crossing over . The mating-type genes (M or P) are expressed only when present at a third, active locus (mat1) . We have earlier shown that the positional regulation of P genes is based on repression at the silent site, caused by elements in the flanking DNA sequences . In this study we have mutagenized a sterile mat1 deleted strain and selected for cells that are able to conjugate . Recessive mutations of this type should define genes encoding trans-acting factors involved in repression of the silent mating-type loci . Before this work mutations in two genes, clr1 and swi6, had been shown to allow both expression of the silent loci and recombination in the K region . The sensitivity of the present selection is demonstrated by the isolation of new mutations that derepress one or both of the silent loci (M-mating or bi-mating) . The frequency of M-mating mutants was almost two orders of magnitude higher than that of bi-mating mutants and in all mutants analyzed mat3-M expression was significantly higher than mat2-P expression . The mutations define three new genes, clr2, clr3 and clr4 . In addition we show that the rik1 mutant previously known to allow recombination in the K region also depresses the silent loci. Curr Genet, 1994 Jan, 25(1), 80 - 3 Complete absence of mitochondrial DNA in the petite-negative yeast Schizosaccharomyces pombe leads to resistance towards the alkaloid lycorine; Massardo DR et al.; The petite-positive yeast Saccharomyces cerevisiae can be efficiently and completely converted to respiratory-deficient cytoplasmic petite mutants by intercalating drugs . Rho0 petites from Schizosaccharomyces pombe could only be obtained in strains carrying a nuclear mutation . In this paper we report the efficient isolation of rho0 mutants in a Sch . pombe strain containing a mitochondrial mutator mutation . We also show that the alkaloid lycorine is able to differentiate between cells containing defective mitochondrial DNA (mit-) and those lacking mitochondrial DNA completely (rho0) . Rho0 cells are resistant to the alkaloid whereas mit- and wild-type cells show the same sensitivity. Microbios, 1994, 79(319), 73 - 9 Sensitivity of fructose-1,6-bisphosphatase to glucose and cyclic AMP in the fission yeast Schizosaccharomyces pombe; Carrillo D et al.; Stationary phase cells from resting cultures of Schizosaccharomyces pombe, suspended in buffer, did not decrease fructose-1,6-bisphosphatase (FbPase) activity upon addition of glucose to the cell suspension . In contrast, the addition of glucose to cells from derepressed growing cultures resulted in a 4- to 5-fold reduction in FbPase activity within minutes after the addition of the sugar . This response was independent of protein synthesis, was accompanied by a rise in the cyclic AMP (cAMP) level and was concomitant with an increase in the activity of neutral trehalase . The addition of exogenous cAMP to these cells provoked a decrease in FbPase similar to that induced by glucose . The occurrence of a glucose-induced cAMP signal was not sufficient to trigger the decrease in FbPase activity, which appeared to require additional transduction elements not active in growth-arrested cells . In assays performed in vitro it was found that the enzyme activity was strongly inhibited by fructose-2,6-bisphosphate. Bioorg Med Chem, 1994 Jan, 2(1), 27 - 34 Synthesis of steroid intermediates via alkylation of dianion derived from acetoacetic ester; Wang KC et al.; A synthetic route for A-ring aromatic steroid intermediates starting from alkylation of dianion derived from acetoacetic ester with m-methoxyphenylethyl bromide to form benzene ring connected to a linear six-carbon fragment is described . This unit, after chemical modifications to 5, was condensed with 2-methylcyclopentan-1,3-dione (6a) to form prochiral trione, 7a, a key synthetic intermediate in A-ring aromatic steroid . Microbial reduction of 7a with Schizosaccharomyces pombe (NRRL Y-164) gave chiral (-)-11 in 65% yield . Starting from 2,2-dimethylsuccinic acid, 2,4,4-trimethylcyclopentan-1,3-dione (6a) was prepared, which was condensed subsequently with 5 to form racemic 7b trione intermediate . Asymmetric cyclization of 7b in the presence of L-(-)-phenylanlanine, followed by acidic cyclization led to regiospecific synthesis of 16,16-dimethyl tetracyclic steroid intermediate. Genetics, 1994 Jan, 136(1), 41 - 51 M26 recombinational hotspot and physical conversion tract analysis in the ade6 gene of Schizosaccharomyces pombe; Grimm C et al.; At the ade6 locus of Schizosaccharomyces pombe flanking markers have been introduced as well as five silent restriction site polymorphisms: four in the 5' upstream region and one in the middle of the gene . The mutations ade6-706, ade6-M26 (both at the 5' end) and ade6-51 (middle of the gene) were used as partners for crosses with the 3' mutation ade6-469 . From these three types of crosses, wild-type recombinants were selected and analyzed genetically to assess association with crossing-over and physically to determine conversion tract lengths . The introduced restriction site polymorphisms (five vs . only one) neither influenced the pattern of recombinant types nor the distribution of conversion tracts . The hotspot mutation M26 enhances crossing-over and conversion to the same proportion . M26 not only stimulates conversion at the 5' end, but does this also (to a lower extent) at the 3' end of ade6 at a distance of more than 1 kb . The majority of meiotic conversion tracts are continuous and postmeiotic segregation of polymorphic sites is rare . Conversion tracts are slightly shorter with M26 in comparison with its control 706 . The mean minimal length of tracts varies from 670 bp (M26) to 890 bp (706) to 1290 bp (51) . It is concluded that M26 acts as an initiation site of recombination or enhances initiation of recombination . M26 does not act by termination of conversion . A region of recombination initiation exists at the 5' end of the ade6 gene also in the absence of the ade6-M26 hotspot mutation. Cell Mol Biol Res, 1994, 40(3), 241 - 3 Cellular function of protein phosphatase 2C in yeast; Shiozaki K et al.; Protein phosphatase 2C (PP2C) is one of four major classes of protein serine/threonine-phosphatases, which requires Mg2+ for its activity . PP2C activity distributes ubiquitously among eukaryotes and enzymes purified from mammalian tissues were well characterized biochemically, however, very little is known about their biological function . We have been trying to identify cellular function of PP2C by genetic analyses in fission yeast Schizosaccharomyces pombe . So far, three PP2C homologs, ptc1+, ptc2+, and ptc3+, have been cloned and their activity were detected in the S . pombe cell lysates . Experiments using ptc mutants constructed by gene disruption technique have revealed the involvement of PP2C in the heat shock response and possibly osmoregulation in yeast cells. Symp Soc Exp Biol, 1994, 48, 155 - 65 Molecular biology of sugar transporters of the plant plasma membrane; Sauer N et al.; Plants possess highly efficient transport systems for organic substances such as monosaccharides and disaccharides . In many lower plants these transport systems can be used for heterotrophic growth in the absence of light . In higher plants which are a complex mosaic of autotrophic and heterotrophic cells, tissues and organs, they play an important role in the distribution of assimilated carbon amongst these various parts . Genes and cDNAs encoding transport proteins for mono-or disaccharides have been cloned recently . The relationship of these transporters to a large superfamily of uni-, sym-, and antiporters cloned from many other organisms is discussed . The paper describes strategies for cloning, heterologous expression (in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Xenopus oocytes and heterologous plant systems), and functional characterization of individual transporters . Data on the tissue-specific expression of two monosaccharide transporters from Arabidopsis thaliana will be presented and possible glycosylation of the proteins is discussed. Proc Int Conf Intell Syst Mol Biol, 1994, 2, 78 - 86 Genetic map construction with constraints; Clark DA et al.; A pilot program, CME, is described for generating a physical genetic map from hybridization fingerprinting data . CME is implemented in the parallel constraint logic programming language ElipSys . The features of constraint logic programming are used to enable the integration of pre-existing mapping information (partial probe orders from cytogenetic maps and local physical maps) into the global map generation process, while parallelism enables the search space to be traversed more efficiently . CME was tested using data from chromosome 2 of Schizosaccharomyces pombe and was found able to generate maps as well as (and sometimes better than) a more traditional method . This paper illustrates the practical benefits of using a symbolic logic programming language and shows that the features of constraint handling and parallel execution bring the development of practical systems based on AI programming technologies nearer to being a reality. Arch Virol, 1994, 137(3-4), 341 - 53 Antibodies against linear and conformational epitopes of the human papillomavirus (HPV) type 16 E6 and E7 oncoproteins in sera of cervical cancer patients; Nindl I et al.; Sera obtained from 137 cervical cancer patients were analysed for the presence of antibodies to the human papillomavirus (HPV) type 16 proteins E6 and E7 by the aid of different assays, i.e . ELISA using as antigen either synthetic peptides or the complete E7 protein and radio-immunoprecipitation (RIPA) which uses the viral protein made by in vitro transcription/translation . In agreement with previous reports, reactivity to the E7 protein was found more frequently than to the E6 protein (31.4% vs . 16.8%) when the sera were assayed by peptide-based ELISA . In contrast, when RIPA was employed, reactivity to either protein was obtained at similar frequency (38.7% vs 46.7%) . When the protein was denatured prior to immuno-precipitation the reactivity was lost in all sera tested for E6-specific antibodies but only in a few samples in the E7-RIPA . Therefore it was concluded that the increased sensitivity of the E6-RIPA as compared to the E6 peptide-ELISA is due to the detection of antibodies to conformational epitopes which are presented by the in vitro product but not by the synthetic peptides . Eighty-two sera from healthy donors were tested by HPV 16E6- and E7-RIPA and also by ELISA using the HPV 16E7 protein which was produced in the fission yeast Schizosaccharomyces pombe . One sample reacted each in the E6- and E7-RIPA indicating a high specificity of these assays . The E7 protein-ELISA proved to be less sensitive for the detection of antibodies in cervical cancer patients' sera (22.6% positive) as compared to peptide-based ELISA or RIPA. Nucleic Acids Res, 1993 Dec 25, 21(25), 5964 - 71 Cloning and characterisation of the Schizosaccharomyces pombe rad8 gene, a member of the SNF2 helicase family; Doe CL et al.; The Schizosaccharomyces pombe rad8 mutant is sensitive to both UV and gamma irradiation . We have cloned the rad8 gene by complementation of the UV sensitivity of a rad8.190 mutant strain . The gene comprises an open reading frame of 3.4 kb which does not contain any introns and is capable of encoding a 1133 amino acid protein of 129 kDa . Deletion of the gene indicates that it is not essential for cell viability . Recognisable motifs are present for a nuclear localisation signal, a RING finger and helicase domains . The predicted protein is a member of the SNF2 subfamily of proteins and shows particular homology to the Saccharomyces cerevisiae RAD5 protein . Double mutant analysis demonstrated that the rad8 mutant is not epistatic to mutants in the excision repair pathway (rad13) or checkpoint pathway (rad9) . Analysis of radiation sensitivity though the cell cycle indicates that, unlike most other rad mutants, rad8 is most sensitive to irradiation during the G1/S period. Nucleic Acids Res, 1993 Dec 25, 21(25), 5940 - 4 The fission yeast rad22 gene, having a function in mating-type switching and repair of DNA damages, encodes a protein homolog to Rad52 of Saccharomyces cerevisiae; Ostermann K et al.; The gene rad22 of the fission yeast Schizosaccharomyces pombe has a function in DNA repair and mating-type switching . We have cloned the rad22 gene from a genomic gene bank by functional complementation of the switching defect . An open reading frame coding for a putative protein of 469 amino acids was found by sequence analyses . The rad22 gene contains no intron . A region of 126 amino acids in the N-terminal half of the Rad22 protein has significant homologies (56% identity and 36% similarity) to the Rad52 protein of Saccharomyces cerevisiae . A rad22 disruption strain was constructed which seems to be inviable in a homothallic background . Southern blot analyses have shown that the rad22-67 mutant frequently gives rise to deletions in the mating-type region . These data indicate that the Rad22 protein has a function in the repair of DNA double-strand breaks. EMBO J, 1993 Dec 15, 12(13), 5245 - 54 The Schizosaccharomyces pombe cwg2+ gene codes for the beta subunit of a geranylgeranyltransferase type I required for beta-glucan synthesis; Diaz M et al.; The product of the Schizosaccharomyces pombe cwg2+ gene is involved in the biosynthesis of beta-D-glucan . When grown at the non-permissive temperature, cwg2-1 mutant cells lyse in the absence of an osmotic stabilizer and display a reduced (1-3) beta-D-glucan content and (1-3) beta-D-glucan synthase activity . The cwg2+ gene was cloned by the rescue of the cwg2-1 mutant phenotype using an S . pombe genomic library and subsequently verified by integration of the appropriate insert into the S . pombe genome . Determination of the nucleotide sequence of this gene revealed a putative open reading frame of 1065 bp encoding a polypeptide of 355 amino acids with a calculated M(r) of 40,019 . The cwg2+ DNA hybridizes to a main transcript, the 5' end of which maps to a position 469 bp upstream of the predicted start of translation . The sequence between the transcription and the translation start sites is unusually long and has several short open reading frames which suggest a translational control of the gene expression . Comparative analysis of the predicted amino acid sequence shows that it possesses significant similarity to three Saccharomyces cerevisiae proteins, encoded by the DPR1/RAM1, CDC43/CAL1 and ORF2/BET2 genes respectively, which are beta subunits of different prenyltransferases . When grown at 37 degrees C, cwg2-1 mutant extracts were specifically deficient in geranylgeranyltransferase type I activity, as measured in vitro . Multiple copies of the CDC43 gene can partially suppress the growth and (1-3) beta-D-glucan synthase defect of the cwg2-1 mutant at the restrictive temperature . In a similar manner, the cwg2+ gene can partially suppress the cdc43-2 growth defect . These results indicate that cwg2+ is the structural gene for the beta subunit of geranylgeranyltransferase type I in S . pombe and that this enzyme is required for (1-3) beta-D-glucan synthase activity . The functional homology of Cwg2 with Cdc43, which has been implicated in the control of cell polarity, suggests a link between two morphogenetic events such as establishment of cell polarity and cell wall biosynthesis. Gene, 1993 Dec 8, 134(2), 191 - 200 Cloning of a mouse cDNA encoding DNA polymerase delta: refinement of the homology boxes; Cullmann G et al.; A mouse DNA polymerase delta (Pol delta)-encoding cDNA (pol delta) was isolated by PCR amplification and cDNA library screening . The sequenced cDNA has a length of 3386 bp and the open reading frame (ORF) encodes a protein of 1105 amino acids (aa) with an M(r) of 123,743 . The aa identity to the proteins encoded by the corresponding cDNA from Bos taurus (93%) and Homo sapiens (92%) is very high . The identity to the Pol delta from Schizosaccharomyces pombe, Saccharomyces cerevisiae and Plasmodium falciparum is around 50% . An aa comparison between all available Pol delta sequences reveals several common structural motifs . Polyclonal antibodies raised against a 31-aa synthetic peptide deduced from the ORF specifically recognize Pol delta polymerases from human cells and calf thymus in an immunoblot. J Biol Chem, 1993 Dec 5, 268(34), 25463 - 8 Cloning of Chinese hamster DNA topoisomerase I cDNA and identification of a single point mutation responsible for camptothecin resistance; Tanizawa A et al.; A camptothecin-resistant (DC3F/C-10) Chinese hamster cell line that contains a catalytically altered and camptothecin (CPT)-resistant DNA topoisomerase I (top 1) (Tanizawa, A., and Pommier, Y . (1992) Cancer Res . 52, 1848-1854) and the parent cell line (DC3F) were used to compare top 1 mRNAs and cDNAs . Northern blot analysis showed a single 4.1-kilobase band without quantitative reduction between the two cell lines . We have cloned and sequenced top 1 cDNAs . DC3F and DC3F/C-10 top 1 c-DNA are 3591 and 3626 base pair long, respectively, and encode 767 amino acids . The homology of deduced amino acid sequences between Chinese hamster and mouse or human top 1 are 98.1 and 96.7, respectively . cDNAs from DC3F/C-10 and DC3F cells differ by a single base point mutation (G to A) which results in an amino acid change from Gly505 to Ser (Gly505-->Ser) . G505 corresponds to Gly503 of human top 1 cDNA and is located 220 amino acids away from the presumed catalytic Tyr725 . The point mutation in the Chinese hamster top 1 is located in a region that is highly conserved among all cloned top 1 cDNAs (plant ATH, vaccinia virus, Shope fibroma virus, Drosophila, Saccharomyces cerevisiae, Schizosaccharomyces pombe, mouse, and Human) . A mutation of Asp533 to Gly in this same region has been shown to confer CPT resistance for human top 1 . Chinese hamster top 1 protein with a Gly505-->Ser mutation that was expressed in bacteria was resistant to CPT, indicating that this single base mutation is involved in CPT resistance . Our results suggest that the highly conserved region around Gly505 plays an important role in the interactions among top 1, DNA, and CPT. Semin Cell Biol, 1993 Dec, 4(6), 433 - 42 Reversible tyrosine phosphorylation and cell cycle control; Atherton-Fessler S et al.; In eukaryotic organisms, reversible tyrosine phosphorylation has been established as an important element in the regulation of cell growth and more recently as an essential element in the regulation of the cell division cycle . The activity of p34cdc2, a protein kinase whose activity is required for the entry of cells into mitosis, is tightly controlled by reversible phosphorylation at tyrosine 15 . A complex network of interacting protein kinases and protein phosphatases regulate the state of p34cdc2 tyrosine phosphorylation and therefore the entry of cells into mitosis . In the fission yeast Schizosaccharomyces pombe, genes encoding several of these protein kinases and protein phosphatases have been obtained through genetic approaches . In this review, we will focus on the protein kinases encoded by wee1+, mik1+ and cdr1+/nim1+ and the protein phosphatases encoded by cdc25+ and pyp1+, pyp2+ and pyp3+ . Homologs of many of these regulators have been identified and characterized in higher eukaryotes underscoring the importance of reversible tyrosine phosphorylation as a universal mechanism for the regulation of the cell division cycle. Curr Genet, 1993 Dec, 24(6), 544 - 7 Isolation and structure of an acetolactate synthase gene from Schizosaccharomyces pombe and complementation of the ilv2 mutation in Saccharomyces cerevisiae; Bekkaoui F et al.; A gene encoding a functional acetolactate synthase (ALS) subunit has been isolated from the fission yeast Schizosaccharomyces pombe, and has been structurally and genetically characterized . The approximate 5-kbp cloned DNA segment was found to contain a 2007-bp open reading frame capable of encoding a 669 aminoacid polypeptide which exhibited 57.1% similarity to the corresponding ALS subunit from Saccharomyces cerevisiae . The putative ilv1 isolated from S . pombe was shown to encode a functional subunit of acetolactate synthase by complementation of an S . cerevisiae strain deleted for the ILV2 locus. Curr Genet, 1993 Dec, 24(6), 496 - 9 An electrophoretic karyotype of the cultivated mushroom--Agaricus bisporus; Lodder S et al.; Thirteen chromosomal-sized DNA bands of the cultivated mushroom Agaricus bisporus have been resolved using the method of clamped homogeneous electric field (CHEF) electrophoresis . Using chromosome size standards from Schizosaccharomyces pombe, Saccharomyces cerevisiae and Candida albicans, the estimated size of the chromosomal DNAs ranged from 3.5 to 1.2 megabase pairs (Mb) . By Southern hybridization with homologous gene probes, the chromosomal location of cellulase and laccase genes have been mapped . In addition, rDNA has been assigned to chromosomal bands using a heterologous gene probe . Genomic rearrangement is suggested in the commercial heterokaryon, as indicated by the presence of non-comigrating homologous chromosomes, identified by a number of probes for particular DNA sequences. Curr Genet, 1993 Dec, 24(6), 491 - 5 Cloning and manipulation of the Schizosaccharomyces pombe his7+ gene as a new selectable marker for molecular genetic studies; Apolinario E et al.; We have cloned the his7+ gene of the fission yeast Schizosaccharomyces pombe by complementation of the recessive mutant allele his7-366 . The his7+ gene is able to complement a mutation of the Escherichia coli hisI gene, suggesting that his7+ encodes a phosphoribosyl-AMP cyclohydrase . Subcloning experiments localize the gene to a 1.9-kb XbaI-BglII fragment . We describe the construction of plasmids to facilitate the use of his7+ as a selectable marker in S . pombe studies . Plasmid pEA2 carries his7+ cloned into the pUC18 polylinker . From either pEA2 or the original his7+ clone, pMN1, fragments carrying his7+ can be isolated using a variety of restriction enzymes for the construction of gene disruptions . Plasmid pEA500 is a cloning vector that carries his7+ and ars1, yet retains the ability to use the blue/white color screen to identify recombinants. Plant Physiol, 1993 Dec, 103(4), 1277 - 83 Barley pathogenesis-related proteins with fungal cell wall lytic activity inhibit the growth of yeasts; Grenier J et al.; Proteins from intercellular fluid extracts of chemically stressed barley (Hordeum vulgare L.) leaves were separated by native polyacrylamide gel electrophoresis at alkaline or acid pH . Polyacrylamide gels contained Saccharomyces cerevisiae (bakers' yeast) or Schizosaccharomyces pombe (fission yeast) crude cell walls for assaying yeast wall lysis . In parallel, gels were overlaid with a suspension of yeasts for assaying growth inhibition by pathogenesis-related proteins . The same assays were also performed with proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions . In alkaline native polyacrylamide gels, only one band corresponding to yeast cell wall lytic activity was found to be inhibitory to bakers' yeast growth, whereas in acidic native polyacrylamide gels one band inhibited the growth of both yeasts . Under denaturing nonreducing conditions, one band of 19 kD inhibited the growth of both fungi . The 19-kD band corresponded to a basic protein after two-dimensional gel analysis . The 19-kD protein with yeast cell wall lytic activity and inhibitory to both yeasts was found to be different from previously reported barley chitosanases that were lytic to fungal spores . It could be different from other previously reported lytic antifungal activities related to pathogenesis-related proteins. EMBO J, 1993 Dec, 12(12), 4885 - 95 Novel gene expression mechanism in a fission yeast retroelement: Tf1 proteins are derived from a single primary translation product; Levin HL et al.; In sharp contrast to the single ORF of the Schizosaccharomyces pombe retrotransposon Tf1, retroviruses and most retrotransposons employ two different ORFs to separately encode the Gag and Pol proteins . The different ORFs are thought to allow for overexpression of the Gag protein relative to Pol protein presumed necessary for the assembly of functional retrovirus par