J Mol Endocrinol, 2000 Apr, 24(2), 183 - 91 Transcriptional activity of Drosophila melanogaster ecdysone receptor isoforms and ultraspiracle in Saccharomyces cerevisiae; Dela Cruz FE et al.; The Drosophila melanogaster ecdysone receptor (EcR) is produced in three isoforms, which mediate developmental processes such as metamorphosis . These isoforms were expressed in Saccharomyces cerevisiae to elucidate aspects of receptor transcription activity in a highly defined genetic model system . All three EcR isoforms showed ligand-independent transcriptional activation of an ecdysone reporter gene and the amount of activation correlated with the size of the N-terminal A/B (transactivation) domain present in the isoform: EcR-B1>EcR-A>>EcR-B2 . Upon co-expression with ultraspiracle (Usp), transcriptional activation was further increased with EcR-B1 or EcR-A, but was unchanged with EcR-B2 or a truncated EcR lacking the A/B N-terminal domain (EcRDeltaA/B) . Thus, the enhanced activity from Usp may depend on the presence of an N-terminal domain of EcR . Co-expression with Usp of several chimeric receptors of the EcR and the mouse androgen receptor (mAR) identified one chimera, composed of the mAR N-terminus and the remainder from EcR (mAR inverted question markEcR-CDEF) that was transcriptionally silent and inducible by Usp . In contrast, the vertebrate homologue, human retinoic acid receptor (RXRalpha), showed ligand-independent transcription when co-expressed with EcRDeltaA/B but not mAR inverted question mark EcR-CDEF . Therefore, RXRalpha does not require its partner to possess an N-terminal domain, yet is intolerant of a heterologous N-terminus . Similarly, the human vitamin D receptor, which has a short N-terminal region, showed greater ligand-independent transcription in the presence of RXRalpha than in the presence of Usp . These results reveal a mechanistic basis for the differential activities among the EcR isoforms, and between Usp and RXRalpha . Furthermore, they provided the foundation for a genetic screen to identify potential insecticides as well as accessory proteins for Usp and EcR. J Biol Chem, 2000 Jun 9, 275(23), 17611 - 8 Saccharomyces cerevisiae GATA sequences function as TATA elements during nitrogen catabolite repression and when Gln3p is excluded from the nucleus by overproduction of Ure2p; Cox KH et al.; Saccharomyces cerevisiae selectively uses good nitrogen sources (glutamine) in preference to poor ones (proline) by repressing GATA factor-dependent transcription of the genes needed to transport and catabolize poor nitrogen sources, a physiological process designated nitrogen catabolite repression (NCR) . We show that some NCR-sensitive genes (CAN1, DAL5, DUR1,2, and DUR3) produce two transcripts of slightly different sizes . Synthesis of the shorter transcript is NCR-sensitive and that of the longer transcript is not . The longer transcript also predominates in gln3Delta mutants irrespective of the nitrogen source provided . We demonstrate that the longer mRNA species arises through the use of an alternative transcription start site generated by Gln3p-binding sites (GATAAs) being able to act as surrogate TATA elements . The ability of GATAAs to serve as surrogate TATAs, i.e . when synthesis of the shorter, NCR-sensitive transcripts are inhibited, correlates with sequestration of enhanced green fluorescent protein (EGFP)-Gln3p in the cytoplasm in a way that is indistinguishable from that seen with EGFP-Ure2p . However, when the shorter, NCR-sensitive DAL5 transcript predominates, EGFP-Gln3p is nuclear . These data suggest that the mechanism underlying NCR involves the cytoplasmic association of Ure2p with Gln3p, an interaction that prevents Gln3p from reaching it is binding sites upstream of NCR-sensitive genes. Biochemistry, 2000 Apr 11, 39(14), 4096 - 104 Studies with recombinant Saccharomyces cerevisiae CaaX prenyl protease Rce1p; Dolence JM et al.; Eukaryotic proteins with carboxyl-terminal CaaX motifs undergo three post-translational processing reactions-protein prenylation, endoproteolysis, and carboxymethylation . Two genes in yeast encoding CaaX endoproteases, AFC1 and RCE1, have been identified . Rce1p is solely responsible for proteolysis of yeast Ras proteins . When proteolysis is blocked, plasma membrane localization of Ras2p is impaired . The mislocalization of undermodified Ras in the cell suggests that Rce1p is an attractive target for cancer therapeutics . Homologous expression of plasmid-encoded Saccharomyces cerevisiae RCE1 under the control of the GAL1 promoter gave a 370-fold increase in endoprotease activity over an uninduced control . Yeast Rce1p was detected by Western blotting with a yRce1p antibody or with an anti-myc antibody to Rce1p bearing a C-terminal myc-epitope . Membrane preparations were examined for their sensitivity to a variety of protease inhibitors, metal ion chelators, and heavy metals . The enzyme was sensitive to cysteine protease inhibitors, Zn(2+), and Ni(2+) . The substrate selectivity of yRce1p was determined for a variety of prenylated CaaX peptides including farnesylated and geranylgeranylated forms of human Ha-Ras, Ki-Ras, N-Ras, and yeast Ras2p, a-mating factor, and Rho2p . Six site-directed mutants of conserved polar and ionic amino acids in yRce1p were prepared . Four of the mutants, H194A, E156A, C251A, and H248A, were inactive . Results from the protease inhibition studies and the site-directed mutagenesis suggest that Rce1p is a cysteine protease. J Cell Biol, 2000 Apr 3, 149(1), 125 - 40 Dynamic localization of protein phosphatase type 1 in the mitotic cell cycle of Saccharomyces cerevisiae; Bloecher A et al.; Protein phosphatase type I (PP1), encoded by the single essential gene GLC7 in Saccharomyces cerevisiae, functions in diverse cellular processes . To identify in vivo subcellular location(s) where these processes take place, we used a functional green fluorescent protein (GFP)-Glc7p fusion protein . Time-lapse fluorescence microscopy revealed GFP-Glc7p localizes predominantly in the nucleus throughout the mitotic cell cycle, with the highest concentrations in the nucleolus . GFP-Glc7p was also observed in a ring at the bud neck, which was dependent upon functional septins . Supporting a role for Glc7p in bud site selection, a glc7-129 mutant displayed a random budding pattern . In alpha-factor treated cells, GFP-Glc7p was located at the base of mating projections, again in a septin-dependent manner . At the start of anaphase, GFP-Glc7p accumulated at the spindle pole bodies and remained there until cytokinesis . After anaphase, GFP-Glc7p became concentrated in a ring that colocalized with the actomyosin ring . A GFP-Glc7-129 fusion was defective in localizing to the bud neck and SPBs . Together, these results identify sites of Glc7p function and suggest Glc7p activity is regulated through dynamic changes in its location. Genetics, 2000 Apr, 154(4), 1549 - 59 Genetic analysis of the relationship between activation loop phosphorylation and cyclin binding in the activation of the Saccharomyces cerevisiae Cdc28p cyclin-dependent kinase; Cross FR et al.; We showed recently that a screen for mutant CDC28 with improved binding to a defective Cln2p G1 cyclin yielded a spectrum of mutations similar to those yielded by a screen for intragenic suppressors of the requirement for activation loop phosphorylation (T169E suppressors) . Recombination among these mutations yielded CDC28 mutants that bypassed the G1 cyclin requirement . Here we analyze further the interrelationship between T169E suppression, interaction with defective cyclin, and G1 cyclin bypass . DNA shuffling of mutations from the various screens and recombination onto a T169E-encoding 3' end yielded CDC28 mutants with strong T169E suppression . Some of the strongest T169E suppressors could suppress the defective Cln2p G1 cyclin even while retaining T169E . The strong T169E suppressors did not exhibit bypass of the G1 cyclin requirement but did so when T169E was reverted to T . These results suggested that for these mutants, activation loop phosphorylation and cyclin binding might be alternative means of activation rather than independent requirements for activation (as with wild type) . These results suggest mechanistic overlap between the conformational shift induced by cyclin binding and that induced by activation loop phosphorylation . This conclusion was supported by analysis of suppressors of a mutation in the Cdk phosphothreonine-binding pocket created by cyclin binding. Genetics, 2000 Apr, 154(4), 1523 - 32 Involvement of the PP2C-like phosphatase Ptc2p in the DNA checkpoint pathways of Saccharomyces cerevisiae; Marsolier MC et al.; RAD53 encodes a conserved protein kinase that acts as a central transducer in the DNA damage and the DNA replication checkpoint pathways in Saccharomyces cerevisiae . To identify new elements of these pathways acting with or downstream of RAD53, we searched for genes whose overexpression suppressed the toxicity of a dominant-lethal form of RAD53 and identified PTC2, which encodes a protein phosphatase of the PP2C family . PTC2 overexpression induces hypersensitivity to genotoxic agents in wild-type cells and is lethal to rad53, mec1, and dun1 mutants with low ribonucleotide reductase activity . Deleting PTC2 specifically suppresses the hydroxyurea hypersensitivity of mec1 mutants and the lethality of mec1Delta . PTC2 is thus implicated in one or several functions related to RAD53, MEC1, and the DNA checkpoint pathways. Genetics, 2000 Apr, 154(4), 1509 - 21 Glucose and ras activity influence the ubiquitin ligases APC/C and SCF in Saccharomyces cerevisiae; Irniger S et al.; In budding yeast, the Ras/cAMP pathway is involved in the coordination of cell growth and cell division . Glucose-rich medium stimulates Ras/cAMP signaling, which causes an increase in the critical cell size for cell cycle entry . Here we show that glucose and activated Ras proteins also influence the function of the anaphase-promoting complex (APC/C), a ubiquitin-protein ligase required for sister chromatid separation and mitotic exit . We found that apc10-22 and other mutants defective in the APC/C are suppressed by reduced Ras signaling activity, by a deletion of the RAS2 gene, by a cdc25 mutation, by elevated levels of PDE2, or by growth without glucose . Viability of these mutants is also enhanced by decreased Cdk1 activity . In contrast, a constitutively activated RAS2(Val19) allele or shifts to glucose medium are deleterious to apc10-22 mutants . Remarkably, cdc34-2 mutants, which are impaired in SCF function, are differently affected with respect to Ras activity . Viability of cdc34-2 mutants at elevated temperatures is dependent on glucose and the RAS2 gene . We conclude that glucose and Ras proteins influence the APC/C and the SCF complex in an opposite manner . These ubiquitin ligases might represent novel targets for modulating cell division in response to growth conditions. EMBO J, 2000 Apr 3, 19(7), 1598 - 612 Histone H2A is required for normal centromere function in Saccharomyces cerevisiae; Pinto I et al.; Histones are structural and functional components of the eukaryotic chromosome, and their function is essential for normal cell cycle progression . In this work, we describe the characterization of two Saccharomyces cerevisiae cold-sensitive histone H2A mutants . Both mutants contain single amino acid replacements of residues predicted to be on the surface of the nucleosome and in close contact with DNA . We show that these H2A mutations cause an increase-in-ploidy phenotype, an increased rate of chromosome loss, and a defect in traversing the G(2)-M phase of the cell cycle . Moreover, these H2A mutations show genetic interactions with mutations in genes encoding kinetochore components . Finally, chromatin analysis of these H2A mutants has revealed an altered centromeric chromatin structure . Taken together, these results strongly suggest that histone H2A is required for proper centromere-kinetochore function during chromosome segregation. Biotechnol Bioeng, 2000 May 20, 68(4), 389 - 95 Glutathione excretion in response to heterologous protein secretion in Saccharomyces cerevisiae; Bannister SJ et al.; Glutathione is excreted in a dose-dependent, non-stoichiometric fashion from Saccharomyces cerevisiae cells expressing and secreting Bovine Pancreatic Trypsin Inhibitor (BPTI), a small, disulfide-bonded protein . Glutathione excretion commences 40 hours following induction of BPTI synthesis . Expression of several secretory proteins with varying disulfide and cysteine contents results in glutathione excretion with no apparent requirement for protein disulfide content . Glutathione excretion is also triggered by overexpression of Kar2p/BiP, a native ER-resident protein-folding chaperone, indicating that the response is a general one not restricted to overexpression of thiol-containing heterologous proteins . Functional vesicular transport is not required at the time of glutathione excretion, and glutathione excretion requires the presence of molecular oxygen . These data are consistent with a delayed oxidative stress response potentiated by earlier heterologous secretion, but are inconsistent with secretory transport of glutathione spent as oxidizing equivalents for disulfide-bond formation in the endoplasmic reticulum . Curr Opin Microbiol, 2000 Apr, 3(2), 132 - 7 The Sir proteins of Saccharomyces cerevisiae: mediators of transcriptional silencing and much more; Gartenberg MR; The Sir2, Sir3, and Sir4 proteins of the yeast Saccharomyces cerevisiae elicit transcriptional silencing by forming repressive chromatin structures that are confined to specific chromosomal domains . Recent discoveries establish new and unexpected roles for the proteins in seemingly unrelated arenas of chromosome biology, including double-strand break repair, structure and function of the nucleolus, aging, cell cycle regulation, and checkpoint control. Genet Res, 2000 Feb, 75(1), 1 - 12 Tetrad analysis shows that gene conversion is the major mechanism involved in mutation at the human minisatellite MS1 integrated in Saccharomyces cerevisiae; Berg I et al.; Minisatellites are arrays of tandemly repeated DNA sequences which occur at thousands of locations in the human genome . They are frequently hypervariable with respect to allele length as a result of high rates of complex and incompletely understood recombination-based germline mutation events that alter the repeat copy number . MS1 is one of the most variable minisatellites so far isolated from the human genome . We have integrated MS1, flanked by synthetic markers, in the vicinity of a hot spot for meiotic double-strand breaks upstream of the LEU2 locus in chromosome III of Saccharomyces cerevisiae . Here we present the first tetrad analysis of mutations at a human minisatellite locus . The data showed that mutant alleles occur as single mutants in one of the spores in a tetrad, also when the mutant structure was the result of a combination of intra- and inter-allelic rearrangements . The conversional transfer of repeat units from one allele to the other was associated with flanking marker conversion which always involved the same flank of the minisatellite . The results demonstrate that conversion is the predominant mechanism by which minisatellite alleles mutate to new lengths, and also support the assumption that cis-acting elements are involved in the regulation of the mutational process in humans. J Biochem (Tokyo), 2000 Apr, 127(4), 611 - 6 Expression of plant group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed functional divergence among LEA proteins; Zhang L et al.; To study functions of late embryogenesis abundant (LEA) proteins, which accumulate in plant cells under water deficit conditions, in vivo functional analyses were carried out using a yeast (Saccharomyces cerevisiae) heterologous expression system . Two lea genes, tomato le4 (group 2) and barley HVA1 (group 3), were expressed under the GAL1 promoter, and the gene products were detected using specific antisera . The growth of the transformants was scored and compared with a control strain to analyze the effect of these proteins on yeast cells under stress conditions . The yeast cells expressing HVA1 showed shorter lag period when transferred to a medium containing 1.2 M NaCl as compared to a control strain, while the cells expressing le4 did not show improved growth . Attenuated growth inhibition in a medium containing 1.2 M KCl was observed in the yeast cells expressing le4 and HVA1 . No obvious growth improvement was observed in a high sorbitol medium in the cells expressing either le4 or HVA1 . Increased freezing tolerance was observed in both lea-expressing cells, while no effect on heat tolerance was observed . These results support the hypothesis that different LEA proteins play a distinctive role in the protection against cellular dehydration. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 475 - 7 Cloning, expression and crystallization of VMA13p, an essential subunit of the vacuolar H+-ATPase of Saccharomyces cerevisiae; Sagermann M et al.; The expression and crystallization of the VMA13p subunit of the vacuolar proton-translocating ATPase (V-ATPase) of Saccharomyces cerevisiae is described . This 478 amino-acid subunit is essential for activity but not for the assembly of this multisubunit complex . The protein has been recombinantly overexpressed in Escherichia coli and purified . Diffraction-quality crystals have been obtained using the hanging-drop vapor-diffusion method with ammonium sulfate as precipitant . Several different crystal forms were obtained . The most suitable crystal form for crystallographic characterization belongs to space group P3(1)21 or its enantiomorph, with unit-cell parameters a = b = 118.8, c = 119.3 A . Using an in-house X-ray source, the crystals diffract to about 3.5 A resolution under rapidly frozen conditions. Biochemistry (Mosc), 2000 Mar, 65(3), 349 - 54 Dependence of inorganic polyphosphate chain length on the orthophosphate content in the culture medium of the yeast Saccharomyces cerevisiae; Vagabov VM et al.; The content of inorganic linear polyphosphate (polyP) and the polymeric degree (n) of these compounds were determined in the process of growth of the yeast Saccharomyces cerevisiae VKM Y-1173 in a medium, which contained varying Pi amount with the constant level of all the necessary components . For this purpose, a combination of chemical methods of polyP extraction and 31P-NMR spectroscopy studies of their chain length were used . After 7 h of phosphate starvation, the yeast was shown to use almost completely the phosphate reserve in the form of polyP localized in various cell compartments to support their vitality . The polyP drop was followed by a considerable shortening of the polymer chain length of acid-soluble (polyP1) and two alkali-soluble (polyP3 and polyP4) fractions . Under the same conditions, the content of a salt-soluble fraction (polyP2) decreased almost 20-fold followed by a simultaneous increase of the chain length nearly 2-fold . As a result, fraction chain length ranged up to n = 40-45 . Replacement of the yeast cells after phosphate starvation to a complete phosphate- and glucose-containing medium resulted in super-accumulation ("overcompensation") of polyP within 2 h mainly in polyP3 and, to a lesser degree, in polyP1, polyP2, and polyP5 fractions . In polyP4 fraction localized as polyP3 at the cell surface, the polyP super-accumulation was not detected . The increase of polyP amount in the fractions mentioned turned out not to be accompanied by simultaneous elongation of their chain length and occurred at the lowest level that is characteristic of a polymer level for each fraction . Further cultivation of the yeast on the complete medium during 2 h had little or no effect on polyP content in the cells but led to elongation of polyP chain length especially in the polyP3 and polyP4 fractions . A phenomenon of considerable elongation of polyP chain length against the background of their fixed content revealed in the yeast growing on the complete medium suggests that these organisms possess a previously unknown discrete way of polyP biosynthesis, which results first in the formation of comparatively low-molecular-mass chains followed by that of high-molecular-mass polymers. Genes Genet Syst, 1999 Oct, 74(5), 241 - 56 Las21 participates in extracellular/cell surface phenomena in Saccharomyces cerevisiae; Tohe A et al.; Las21 (Yj1062W) is a member of the major facilitator super family, possessing multimembrane spanning domains . The LAS21 gene was identified as a responsible gene for a Saccharomyces cerevisiae mutan which shows sensitivity to a local anestheticum, tetracaine . The null las21 mutant (las21 delta) is viable but shows temperature sensitive growth . We found, in addition to this phenotype, that the las21 delta strain shows a number of defects; mating deficiency, calcofluor resistance, and formation of Zymolyase sensitive spores . Temperature sensitive growth of the las21 delta mutant was found to be suppressed by 0.1 M MgSO4 . Two multicopy suppressors were obtained . They are ECM33 (YBR078W) and PIR2/HSP150 (YJR159W) both have some roles in an extracellular function . The common features of the suppressors, genetic and physiological, of the las21 delta mutation suggest that Las21 participates in a global activity of extracellular phenomena . The las 21 phenotypes are consistent with the idea that Las21/Gpi7 acts in metabolism of glycosylphosphatidylinositol. FEBS Lett, 2000 Mar 24, 470(2), 102 - 6 Sterol metabolism and ERG2 gene regulation in the yeast Saccharomyces cerevisiae; Soustre I et al.; Certain exogenously-supplied sterols, like ergost-8-enol, are efficiently converted into ergosterol in yeast . We have taken advantage of this property to study the regulation of the Delta8-Delta7-sterol isomerase-encoding ERG2 gene in an ergosterol auxotrophic mutant devoid of squalene-synthase activity . Ergosterol starvation leads to an 8-16-fold increase in ERG2 gene expression . Such an increase was also observed in wild-type cells either grown anaerobically or treated with SR31747A a sterol isomerase inhibitor . Exogenously-supplied zymosterol is entirely transformed into ergosterol, which represses ERG2 transcription . By contrast, exogenously-supplied ergosterol has little or no effect on ERG2 transcription. J Biol Chem, 2000 Mar 31, 275(13), 9734 - 41 The stability of the Cdc6 protein is regulated by cyclin-dependent kinase/cyclin B complexes in Saccharomyces cerevisiae; Calzada A et al.; The Saccharomyces cerevisiae Cdc6 protein is necessary for the formation of prereplicative complexes that are a prerequisite for firing origins during DNA replication in the S phase . In budding yeast, the presence of Cdc6 protein is normally restricted to the G(1) phase of the cell cycle, at least partly because of its proteolytic degradation in the late G(1)/early S phase . Here we show that a Cdc28-dependent mechanism targets p57(CDC6) for degradation in mitotic-arrested budding yeast cells . Consistent with this observation, Cdc6-7 and Cdc6-8 proteins, mutants lacking Cdc28 phosphorylation sites, are stabilized relative to wild-type Cdc6 . Our data also suggest a correlation between the absence of Cdc28/Clb kinase activity and Cdc6 protein stabilization, because a drop in Cdc28/Clb-associated kinase activity allows mitotic-arrested cells to accumulate Cdc6 protein . Finally, we also show that cdc28 temperature-sensitive G(1) mutants accumulate Cdc6 protein because of a post-transcriptional mechanism . Our data suggest that budding yeast cells target Cdc6 for degradation through a Cdc28-dependent mechanism in each cell cycle. Protein Expr Purif, 2000 Apr, 18(3), 303 - 9 High-throughput assay for inorganic pyrophosphatases using the cytosolic enzymes of Saccharomyces cerevisiae and human as an example; Rumsfeld J et al.; This paper describes the development of a new, malachite green based, enzymatic assay for the identification of specific inhibitors of inorganic pyrophosphatase (iPPase) from Saccharomyces cerevisiae for antifungal drug discovery . The human iPPase was used as counterscreen . The coding regions of both enzymes were amplified, cloned into a vector providing a His-tag at the C-terminus, expressed in Escherichia coli, and purified by metal chelate affinity chromatography . Since the complete human sequence had not been published previously, the human iPPase was cloned on the basis of expressed sequence tag data . The human sequence was confirmed and showed about 55% amino acid identity with the yeast enzyme and 95% identity with an already published bovine enzyme . Both recombinant iPPases were characterized with regard to their biochemical properties, showing that the His-tag did not influence the specific activity, pH optimum, inhibitor profile, or dimerization . The enzyme activity was determined by quantifying released phosphate by complex formation with malachite green . The resulting complex was quantified spectrophotometrically . The assay was adapted to a microtiter plate format . Thus, it is possible to screen a large compound pool for iPPase inhibitors in a short period of time . Nucleic Acids Res, 2000 Apr 15, 28(8), 1676 - 83 Characterization of the Saccharomyces cerevisiae cyclic nucleotide phosphodiesterase involved in the metabolism of ADP-ribose 1",2"-cyclic phosphate; Nasr F et al.; ADP-ribose 1",2"-cyclic phosphate (Appr>p) is produced in yeast and other eukaryotes as a consequence of tRNA splicing . This molecule is converted to ADP-ribose 1"-phosphate (Appr-1"p) by the action of the cyclic nucleotide phosphodiesterase (CPDase) . Comparison of the previously cloned CPDase from Arabidopsis with proteins having related cyclic phosphodiesterase or RNA ligase activities revealed two histidine-containing tetrapeptides conserved in these enzyme families . Using the consensus phosphodiesterase signature, we have identified the yeast Saccharomyces cerevisiae open reading frame YGR247w as encoding CPDase . The bacterially expressed yeast protein, named Cpd1p, is able to hydrolyze Appr>p to Appr-1"p . Moreover, as with the previously characterized Arabidopsis and wheat CPDases, Cpd1p hydrolyzes nucleosides 2',3'-cyclic phosphates (N>p) to nucleosides 2'-phosphates . Apparent K (m)values for Appr>p, A>p, U>p, C>p and G>p are 0.37, 4.97, 8.91, 12.18 and 14.29 mM, respectively . Site-directed mutagenesis of individual amino acids within the two conserved tetrapeptides showed that H(40)and H(150)residues are essential for CPDase activity . Deletion analysis has indicated that the CPD1 gene is not important for cellular viability . Likewise, overexpression of Cpd1p had no effect on yeast growth . These results do not implicate an important role for Appr>p or Appr-1"p in yeast cells grown under standard laboratory conditions. Mol Cell Biol, 2000 Apr, 20(8), 2794 - 802 Posttranslational phosphorylation and ubiquitination of the Saccharomyces cerevisiae Poly(A) polymerase at the S/G(2) stage of the cell cycle; Mizrahi N et al.; The poly(A) polymerase of the budding yeast Saccharomyces cerevisiae (Pap1) is a 64-kDa protein essential for the maturation of mRNA . We have found that a modified Pap1 of 90 kDa transiently appears in cells after release from alpha-factor-induced G(1) arrest or from a hydroxyurea-induced S-phase arrest . While a small amount of modification occurs in hydroxyurea-arrested cells, fluorescence-activated cell sorting analysis and microscopic examination of bud formation indicate that the majority of modified enzyme is found at late S/G(2) and disappears by the time cells have reached M phase . The reduction of the 90-kDa product upon phosphatase treatment indicates that the altered mobility is due to phosphorylation . A preparation containing primarily the phosphorylated Pap1 has no poly(A) addition activity, but this activity is restored by phosphatase treatment . A portion of Pap1 is also polyubiquitinated concurrent with phosphorylation . However, the bulk of the 64-kDa Pap1 is a stable protein with a half-life of 14 h . The timing, nature, and extent of Pap1 modification in comparison to the mitotic phosphorylation of mammalian poly(A) polymerase suggest an intriguing difference in the cell cycle regulation of this enzyme in yeast and mammalian systems. Mol Gen Genet, 2000 Feb, 263(1), 60 - 72 Mutations in CDC14 result in high sensitivity to cyclin gene dosage in Saccharomyces cerevisiae; Yuste-Rojas M et al.; We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae . Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14 . Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms . In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p . Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation . The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor . We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication . We also describe genetic interactions between CDC28 and CDC14. Biochimie, 2000 Feb, 82(2), 123 - 7 Expression of the Trypanosoma brucei phosphoenolpyruvate carboxykinase gene in Saccharomyces cerevisiae; Yevenes A et al.; Plasmid pTbp60B (Kueng et al., J . Biol . Chem . 264 (1989) 5203-5209) was employed to obtain, through the polymerase chain reaction, the Trypanosoma brucei gene coding for phosphoenolpyruvate (PEP) carboxykinase, and then cloned into the yeast expression plasmid pYES2 . The cloned gene was completely sequenced and the expression plasmid transformed into Saccharomyces cerevisiae PUK-3B (MATalpha pck1 ura3 ade1) competent cells . Gene expression took place upon induction with 2% galactose, and the recombinant T . brucei PEP carboxykinase was purified to near homogeneity . The basic molecular and catalytic characteristics of the recombinant enzyme were determined, and they showed to be essentially similar to those reported for wild type T . brucei PEP carboxykinase (Hunt and Kohler, Biochim . Biophys . Acta 1249 (1995) 15-22) . The expression system here described is a reliable non-pathogenic source of T . brucei PEP carboxykinase. Biochem J, 2000 Apr 1, 347 Pt 1, 115 - 22 Nucleotides of the tRNA D-stem that play an important role in nuclear-tRNA export in Saccharomyces cerevisiae; Cleary JD et al.; Nuclear export of tRNA in Saccharomyces cerevisiae involves Los1p and Arc1p . Los1p facilitates tRNA translocation across the nuclear pore complex whereas Arc1p plays a role in delivering some species of tRNA exiting the nucleus to their cognate aminoacyl-tRNA synthetases . Here, we show that mutations of C11 and G24 of the D-stem of the yeast tyrosine amber-suppressor tRNA have different effects on nuclear export of the tRNA . Changing G24 had no effect on export of the tRNA to the cytoplasm . In contrast, mutating C11 resulted in nuclear retention of the tRNA . Nuclear retention of the tRNA mutants was not due to lack of processing, since only the mature forms of the tRNA mutants were found . The fact that mutations of G24 did not affect export of the tRNA also indicates that the effect of mutating C11 is not due to gross alteration of the tertiary structure resulting from disruption of the C11/G24 base pair . Expression of Los1p and the mammalian tRNA export receptor exportin-t rescued nuclear export of the tRNA with changes at position 11 . The export-defective mutations of the tRNA mutants were suppressed by introducing the complementary nucleotides at position 24 . Taken together, these findings suggest that C11 is important for binding of the tRNA to the export receptor, and that this binding is influenced by the conformation of the base . Finally, the export-defective tRNA mutants described can be used as reporters to identify eukaryotic proteins involved in the nuclear-tRNA export process, and characterize the molecular interactions between known receptors and the tRNA substrate. Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3094 - 9 Accuracy of thymine-thymine dimer bypass by Saccharomyces cerevisiae DNA polymerase eta; Washington MT et al.; The Saccharomyces cerevisiae RAD30 gene functions in error-free replication of UV-damaged DNA . RAD30 encodes a DNA polymerase, Pol eta, which inserts two adenines opposite the two thymines of a cis-syn thymine-thymine (T-T) dimer . Here we use steady-state kinetics to determine the accuracy of DNA synthesis opposite the T-T dimer . Surprisingly, the accuracy of DNA synthesis opposite the damaged DNA is nearly indistinguishable from that opposite nondamaged DNA, with frequencies of misincorporation of about 10(-2) to 10(-3) . These studies support the hypothesis that unlike most DNA polymerases, Pol eta is able to tolerate distortions in DNA resulting from damage, which then enables the polymerase to utilize the intrinsic base pairing ability of the T-T dimer. Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3364 - 9 Whole-genome expression analysis of snf/swi mutants of Saccharomyces cerevisiae; Sudarsanam P et al.; The Saccharomyces cerevisiae Snf/Swi complex has been previously demonstrated to control transcription and chromatin structure of particular genes in vivo and to remodel nucleosomes in vitro . We have performed whole-genome expression analysis, using DNA microarrays, to study mutants deleted for a gene encoding one conserved (Snf2) or one unconserved (Swi1) Snf/Swi component . This analysis was performed on cells grown in both rich and minimal media . The microarray results, combined with Northern blot, computational, and genetic analyses, show that snf2Delta and swi1Delta mutations cause similar effects on mRNA levels, that Snf/Swi controls some genes differently in rich and minimal media, and that Snf/Swi control is exerted at the level of individual genes rather than over larger chromosomal domains . In addition, this work shows that Snf/Swi controls mRNA levels of MATalpha-specific genes, likely via controlling transcription of the regulators MATalpha1 and MCM1 . Finally, we provide evidence that Snf/Swi acts both as an activator and as a repressor of transcription, and that neither mode of control is an indirect effect of the other. J Cell Sci, 2000 Apr, 113 ( Pt 8), 1435 - 45 Regulation of cytokinesis by the Elm1 protein kinase in Saccharomyces cerevisiae; Bouquin N et al.; A Saccharomyces cerevisiae mutant unable to grow in a cdc28-1N background was isolated and shown to be affected in the ELM1 gene . Elm1 is a protein kinase, thought to be a negative regulator of pseudo-hyphal growth . We show that Cdc11, one of the septins, is delocalised in the mutant, indicating that septin localisation is partly controlled by Elm1 . Moreover, we show that cytokinesis is delayed in an elm1delta mutant . Elm1 levels peak at the end of the cell cycle and Elm1 is localised at the bud neck in a septin-dependent fashion from bud emergence until the completion of anaphase, at about the time of cell division . Genetic and biochemical evidence suggest that Elm1 and the three other septin-localised protein kinases, Hsl1, Gin4 and Kcc4, work in parallel pathways to regulate septin behaviour and cytokinesis . In addition, the elm1delta;) morphological defects can be suppressed by deletion of the SWE1 gene, but not the cytokinesis defect nor the septin mislocalisation . Our results indicate that cytokinesis in budding yeast is regulated by Elm1. Biochim Biophys Acta, 2000 Mar 17, 1496(1), 99 - 116 Mitotic motors in Saccharomyces cerevisiae; Hildebrandt ER et al.; The budding yeast Saccharomyces cerevisiae provides a unique opportunity for study of the microtubule-based motor proteins that participate in mitotic spindle function . The genome of Saccharomyces encodes a relatively small and genetically tractable set of microtubule-based motor proteins . The single cytoplasmic dynein and five of the six kinesin-related proteins encoded have been implicated in mitotic spindle function . Each motor protein is unique in amino acid sequence . On account of functional overlap, no single motor is uniquely required for cell viability, however . The ability to create and analyze multiple mutants has allowed experimental dissection of the roles performed by each mitotic motor . Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle (kinesin-related Cin8p, Kip1p, Kip3p and Kar3p) . Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell (dynein and kinesin-related Kip2p, Kip3p and Kar3p) . The six motors apparently contribute three fundamental activities to spindle function: motility, microtubule cross-linking and regulation of microtubule dynamics. FEBS Lett, 2000 Mar 17, 470(1), 83 - 7 Biochemical characterization and subcellular localization of the sterol C-24(28) reductase, erg4p, from the yeast saccharomyces cerevisiae; Zweytick D et al.; The yeast ERG4 gene encodes sterol C-24(28) reductase which catalyzes the final step in the biosynthesis of ergosterol . Deletion of ERG4 resulted in a complete lack of ergosterol and accumulation of the precursor ergosta-5,7,22,24(28)-tetraen-3beta-ol . An erg4 mutant strain exhibited pleiotropic defects such as hypersensitivity to divalent cations and a number of drugs such as cycloheximide, miconazole, 4-nitroquinoline, fluconazole, and sodium dodecyl sulfate . Similar to erg6 mutants, erg4 mutants are sensitive to the Golgi-destabilizing drug brefeldin A . Enzyme activity measurements with isolated subcellular fractions revealed that Erg4p is localized to the endoplasmic reticulum . This view was confirmed in vivo by fluorescence microscopy of a strain expressing a functional fusion of Erg4p to enhanced green fluorescent protein . We conclude that ergosterol biosynthesis is completed in the endoplasmic reticulum, and the final product is supplied from there to its membranous destinations. Biochem, Educ. . 2000 Mar 1, 28(2), 96 - 99 Experimental treatment of the laws of heterogeneous catalysis with immobilized yeast cells (Saccharomyces cerevisiae); Grunwald P; A series of simple, low-cost experiments is described in this paper that allows students to be introduced to some basic kinetic laws relating to heterogeneous catalysis . Immobilized yeast cells are used as the example and therefore simultaneously offer the opportunity to acquaint the students with the theoretical and practical background of an important branch of biotechnology. J Biol Chem, 2000 Mar 24, 275(12), 8290 - 300 The transcriptional response of Saccharomyces cerevisiae to osmotic shock . Hot1p and Msn2p/Msn4p are required for the induction of subsets of high osmolarity glycerol pathway-dependent genes; Rep M et al.; We have analyzed the transcriptional response to osmotic shock in the yeast Saccharomyces cerevisiae . The mRNA level of 186 genes increased at least 3-fold after a shift to NaCl or sorbitol, whereas that of more than 100 genes was at least 1.5-fold diminished . Many induced genes encode proteins that presumably contribute to protection against different types of damage or encode enzymes in glycerol, trehalose, and glycogen metabolism . Several genes, which encode poorly expressed isoforms of enzymes in carbohydrate metabolism, were induced . The high osmolarity glycerol (HOG) pathway is required for full induction of many but not all genes . The recently characterized Hot1p transcription factor is required for normal expression of a subset of the HOG pathway-dependent responses . Stimulated expression of the genes that required the general stress-response transcription factors Msn2p and Msn4p was also reduced in a hog1 mutant, suggesting that Msn2p/Msn4p might be regulated by the HOG pathway . The expression of genes that are known to be controlled by the mating pheromone response pathway was stimulated by osmotic shock specifically in a hog1 mutant . Inappropriate activation of the mating response may contribute to the growth defect of a hog1 mutant in high osmolarity medium. Biochem Biophys Res Commun, 2000 Mar 24, 269(3), 767 - 74 A genome-wide screening in Saccharomyces cerevisiae for genes that confer resistance to the anticancer agent cisplatin; Burger H et al.; Cisplatin is a potent DNA-damaging agent that has demonstrated anticancer activities against several tumors . However, manifestation of cellular resistance is a major obstacle in anticancer therapy that severely limits the curative potential of cisplatin . Therefore, understanding the molecular basis of cisplatin resistance could significantly improve the clinical efficacy of this anticancer agent . Here, we employed Saccharomyces cerevisiae as a model organism to study cisplatin resistance mechanisms and describe a one-step cisplatin selection to identify and characterize novel cisplatin resistance genes . Screening a multicopy yeast genomic library enabled us to isolate several yeast clones for which we could confirm that the cisplatin resistance phenotype was linked to the introduced fragment . In a first attempt, a number of open reading frames could be identified . Among these genes, PDE2 and ZDS2 were repeatedly identified as genes whose overexpression confers cellular resistance to cisplatin . PDE2, encoding cAMP-phosphodiesterase 2, is of particular interest because the overexpression of this yeast gene is known to induce cisplatin resistance in mammalian cells as well, providing proof of the principle of our experimental approach . In addition, the identification of PDE2 shows that our yeast screening system can directly be informative for drug resistance in mammalian cells . Gene, 2000 Jan 25, 242(1-2), 133 - 40 The growth of mdp1/rsp5 mutants of Saccharomyces cerevisiae is affected by mutations in the ATP-binding domain of the plasma membrane H+ -ATPase; Kaminska J et al.; Mutations in the PMA1 gene, encoding plasma membrane H+ -ATPase, were isolated that are able to suppress the temperature sensitivity (ts) phenotype of mdp1 mutations located in RSP5, the ubiquitin-protein ligase gene . The mdp1 mutants were previously found to change the mitochondrial/cytosolic distribution of Mod5p-I, the tRNA modifying enzyme, and to affect fluid phase endocytosis . The data presented reveal that mdp1 mutants are also pH sensitive, and hypersensitive to hygromycin B and paromomycin . The ts phenotype, hygromycin B and paromomycin sensitivity are suppressed by pmal-t, but the pH sensitivity, the effect of mdp1 on Mod5p-I cytoplasmic/mitochondrial localization and endocytosis are not . Characterization of pmal-t revealed the substitution of amino acid G(653)V in the ATP-binding domain of the H+ -ATPase . Our results indicate that Rsp5 ubiquitin-protein ligase may also influence, in addition to protein distribution, the functioning of plasma membrane H+ -ATPase and the response of cells to stress. Biochimie, 2000 Jan, 82(1), 59 - 64 Expression of the Fpg protein of Escherichia coli in Saccharomyces cerevisiae: effects on spontaneous mutagenesis and sensitivity to oxidative DNA damage; Guibourt N et al.; The biological relevance of oxidative DNA damage has been unveiled by the identification of genes such as fpg of E . coli or OGG1 of Saccharomyces cerevisiae . Both Fpg and Ogg1 proteins are DNA glycosylases/AP lyases that excise 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Me-FapyG) from damaged DNA . Although similar, the enzymatic and biological properties of Fpg and Ogg1 proteins are not identical . Furthermore, the Fpg and Ogg1 proteins do not show significant sequence homologies . In this study, we investigated the ability of the Fpg protein of E . coli to complement phenotypes thought to be due to oxidative DNA damage in Saccharomyces cerevisiae . To express Fpg in yeast, the coding sequence of the fpg gene was placed under the control of a strong yeast promoter in the expression vector pCM190 to generate the pFPG240 plasmid . The Ogg1-deficient yeast strain CD138, ogg1::TRP1, was transformed with pFPG240 and the expression of Fpg was measured . Expression of Fpg in yeast harboring pFPG240 was revealed by efficient release of Me-FapyG and cleavage of 8-OxoG-containing duplexes by cell free protein extracts . The production of the Fpg protein in yeast cells was further demonstrated by immunoblotting analysis using anti-Fpg antibodies . Fpg expression suppresses the spontaneous mutator phenotype of ogg1- yeast for the production of canavanin resistant mutants (CanR) and Lys+ revertants . Fpg expression also restores the capacity of plasmid DNA treated with methylene blue plus visible light (MB-light) to transform the yeast ogg1- rad1- double mutant. FEMS Microbiol Lett, 2000 Mar 15, 184(2), 219 - 24 Constitutive expression of the UGA4 gene in Saccharomyces cerevisiae depends on two positive-acting proteins, Uga3p and Uga35p; Garcia SC et al.; The first specific precursor of porphyrin biosynthesis is delta-aminolevulinic acid . delta-Aminolevulinic acid enters Saccharomyces cerevisiae cells through the gamma-aminobutyric acid specific permease Uga4p . It was described that this permease is inducible by gamma-aminobutyric acid and its regulation involves several specific and pleiotropic transcriptional factors . However, some studies showed that under certain growth conditions the synthesis of Uga4p was not dependent on the presence of gamma-aminobutyric acid . To study the effect of the trans-acting factors Uga43p, Uga3p, Uga35p, Ure2p and Gln3p on the expression of UGA4, we measured gamma-aminobutyric acid and delta-aminolevulinic acid uptake in yeast mutant cells, lacking one of these regulatory factors, grown under different conditions . Experiments analyzing the UGA4 promoter using a fusion construction UGA4::lacZ were also carried out . The results show that the constitutive expression of the UGA4 gene found in cells under certain growth conditions depends on the presence of Uga3p and Uga35p . In contrast, Gln3p and Ure2p do not seem to have any effect on this constitutive mechanism. FEBS Lett, 2000 Mar 10, 469(2-3), 151 - 4 Proteins in the early golgi compartment of Saccharomyces cerevisiae immunoisolated by Sed5p; Cho JH et al.; The yeast tSNARE Sed5p is considered to mainly reside in the early Golgi compartment at the steady state of its intracellular cycling . To better understand this compartment, we immunoisolated a membrane subfraction having Sed5p on the surface (the Sed5 vesicles) . Immunoblot studies showed that considerable portions (20-30%) of the Golgi mannosyltransferases (Mnt1p, Van1p, and Mnn9p) were simultaneously recovered while the late Golgi (Kex2p) or endoplasmic reticulum (Sec71p) proteins were almost excluded . The N-terminal sequences of the polypeptides detectable by Coomassie blue staining indicated that the prominent components of the Sed5 vesicles include Anp1p, Emp24p, Erv25p, Erp1p, Ypt52p, and a putative membrane protein of unknown function (Yml067c). Mol Cell Biol, 2000 Apr, 20(7), 2378 - 84 Involvement of the checkpoint protein Mec1p in silencing of gene expression at telomeres in Saccharomyces cerevisiae; Craven RJ et al.; Yeast strains with a mutation in the MEC1 gene are deficient in the cellular checkpoint response to DNA-damaging agents and have short telomeres (K . B . Ritchie, J . C . Mallory, and T . D . Petes, Mol . Cell . Biol . 19:6065-6075, 1999; T . A . Weinert, G . L . Kiser, and L . H . Hartwell, Genes Dev . 8:652-665, 1994) . In wild-type yeast cells, genes inserted near the telomeres are transcriptionally silenced (D . E . Gottschling, O . M . Aparichio, B . L . Billington, and V . A . Zakian, Cell 63:751-762, 1990) . We show that mec1 strains have reduced ability to silence gene expression near the telomere . This deficiency was alleviated by the sml1 mutation . Overexpression of Mec1p also resulted in a silencing defect, although this overexpression did not affect the checkpoint function of Mec1p . Telomeric silencing was not affected by mutations in several other genes in the Mec1p checkpoint pathway (null mutations in RAD9 and CHK1 or in several hypomorphic rad53 alleles) but was reduced by a null mutation of DUN1 . In addition, the loss of telomeric silencing in mec1 strains was not a consequence of the slightly shortened telomeres observed in these strains. Plant Physiol, 2000 Mar, 122(3), 715 - 20 Characterization of the Brassica napus extraplastidial linoleate desaturase by expression in Saccharomyces cerevisiae; Reed DW et al.; The substrate specificity and regioselectivity of the Brassica napus extraplastidial linoleate desaturase (FAD3) was investigated in vivo in a heterologous expression system . A strain of the yeast Saccharomyces cerevisiae producing the plant enzyme was constructed and cultured in media containing a variety of fatty acids . The products of desaturation of these potential substrates were determined by gas chromatographic and mass spectrometric analysis of the yeast cultures . The results indicate that the enzyme has: (a) omega-3, as opposed to Delta-15 or double-bond-related regioselectivity, (b) the ability to desaturate substrates in the 16 to 22 carbon range, (c) a preference for substrates with omega-6 double bonds, but the ability to desaturate substrates with omega-6 hydroxyl groups or omega-9 or omega-5 double bonds, and (d) a relative insensitivity to double bonds proximal to the carboxyl end of the substrate. Mol Biol Cell, 2000 Mar, 11(3), 983 - 98 Sec24p and Iss1p function interchangeably in transport vesicle formation from the endoplasmic reticulum in Saccharomyces cerevisiae; Kurihara T et al.; The Sec23p/Sec24p complex functions as a component of the COPII coat in vesicle transport from the endoplasmic reticulum . Here we characterize Saccharomyces cerevisiae SEC24, which encodes a protein of 926 amino acids (YIL109C), and a close homologue, ISS1 (YNL049C), which is 55% identical to SEC24 . SEC24 is essential for vesicular transport in vivo because depletion of Sec24p is lethal, causing exaggeration of the endoplasmic reticulum and a block in the maturation of carboxypeptidase Y . Overproduction of Sec24p suppressed the temperature sensitivity of sec23-2, and overproduction of both Sec24p and Sec23p suppressed the temperature sensitivity of sec16-2 . SEC24 gene disruption could be complemented by overexpression of ISS1, indicating functional redundancy between the two homologous proteins . Deletion of ISS1 had no significant effect on growth or secretion; however, iss1Delta mutants were found to be synthetically lethal with mutations in the v-SNARE genes SEC22 and BET1 . Moreover, overexpression of ISS1 could suppress mutations in SEC22 . These genetic interactions suggest that Iss1p may be specialized for the packaging or the function of COPII v-SNAREs . Iss1p tagged with His(6) at its C terminus copurified with Sec23p . Pure Sec23p/Iss1p could replace Sec23p/Sec24p in the packaging of a soluble cargo molecule (alpha-factor) and v-SNAREs (Sec22p and Bet1p) into COPII vesicles . Abundant proteins in the purified vesicles produced with Sec23p/Iss1p were indistinguishable from those in the regular COPII vesicles produced with Sec23p/Sec24p. Mol Biol Cell, 2000 Mar, 11(3), 957 - 68 Dual lipid modification motifs in G(alpha) and G(gamma) subunits are required for full activity of the pheromone response pathway in Saccharomyces cerevisiae; Manahan CL et al.; To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide-binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae . The yeast G protein gamma subunit (Ste18p) is unusual among G(gamma) subunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106 . Substitution of either cysteine results in a strong signaling defect . In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107 . Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation . However, G protein activation released prenylation- or thioacylation-defective Ste18p into the cytoplasm . Hence, lipid modifications of the G(gamma) subunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of G(betagamma) after receptor-stimulated release from G(alpha) . The G protein alpha subunit (Gpa1p) is tandemly modified at its N terminus with amide- and thioester-linked fatty acids . Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate . Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone . Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway. Mol Biol Cell, 2000 Mar, 11(3), 863 - 72 Dynein-dependent movements of the mitotic spindle in Saccharomyces cerevisiae Do not require filamentous actin; Heil-Chapdelaine RA et al.; In budding yeast, the mitotic spindle is positioned in the neck between the mother and the bud so that both cells inherit one nucleus . The movement of the mitotic spindle into the neck can be divided into two phases: (1) Kip3p-dependent movement of the nucleus to the neck and alignment of the short spindle, followed by (2) dynein-dependent movement of the spindle into the neck and oscillation of the elongating spindle within the neck . Actin has been hypothesized to be involved in all these movements . To test this hypothesis, we disrupted the actin cytoskeleton with the use of mutations and latrunculin A (latrunculin) . We assayed nuclear segregation in synchronized cell populations and observed spindle movements in individual living cells . In synchronized cell populations, no actin cytoskeletal mutant segregated nuclei as poorly as cells lacking dynein function . Furthermore, nuclei segregated efficiently in latrunculin-treated cells . Individual living cell analysis revealed that the preanaphase spindle was mispositioned and misaligned in latrunculin-treated cells and that astral microtubules were misoriented, confirming a role for filamentous actin in the early, Kip3p-dependent phase of spindle positioning . Surprisingly, mispositioned and misaligned mitotic spindles moved into the neck in the absence of filamentous actin, albeit less efficiently . Finally, dynein-dependent sliding of astral microtubules along the cortex and oscillation of the elongating mitotic spindle in the neck occurred in the absence of filamentous actin. Protein Eng, 2000 Feb, 13(2), 73 - 6 Lethal effect of the expression of a killer gene SMK1 in Saccharomyces cerevisiae; Suzuki C et al.; Expression of the SMK1 gene which encodes the yeast killer toxin SMKT is lethal in Saccharomyces cerevisiae . Effects of deletion and site-directed mutagenesis of SMK1 on the lethality and the secretion of the gene products were examined . Deletion of the interstitial gamma peptide or the C-terminal loop from Ala208 to the C-terminal Asp222 had no effect on the lethality . Those SMK1 products that lacked either the gamma peptide or the C-terminal loop were expressed in the cells but were not secreted into the culture medium, suggesting that these peptides may have a role in secretion or in protein stability . On the other hand, deletion of the signal sequence resulted in complete loss of the lethal activity . Entering the secretory pathway may be critical for the lethality . Further, deletion of the region from the C-terminus to Leu207 resulted in loss of the lethal activity . Leu207 is located at the C-terminus of the central strand of the beta-sheet structure of SMKT and its side chain is thrust into a hydrophobic environment between the beta-sheet and the alpha-helices . The result obtained upon substitutions of Ala, Ser or Glu for Leu207 suggested that the side chain of Leu207 stabilizes the hydrophobic environment that contributes to the overall structure of the SMK1 product. Nucleic Acids Res, 2000 Apr 1, 28(7), 1604 - 17 Computational identification of cis-acting elements affecting post-transcriptional control of gene expression in Saccharomyces cerevisiae; Jacobs Anderson JS et al.; Understanding the regulation of gene expression requires the identification of cis -acting control elements that modulate gene function . The recent availability of complete genome sequences and profiles of mRNA expression has facilitated the development and utilization of computational methods to identify discrete regulatory elements . We have developed an oligomer counting method that identifies sequences that occur significantly more often in a group of interest relative to other genes in the genome . The use of a second parameter, which measures the frequency of oligomers within the group of interest, allows the detection of false positive signals caused by very infrequent oligomers that would otherwise appear as significant . Applying this method to gene groups that have a common expression pattern or shared function should identify oligomers that comprise cis -acting control elements . As a test of this method, we applied this approach to a set of intron-containing yeast genes, where we easily identified the known splicing signals as control elements . We have used this training set to examine how this method is affected by the length of the oligomer examined, as well as the size and composition of the gene group . These simulations allowed us to identify rules for selecting groups of genes to analyze . Finally, application of this method to nuclear genes encoding proteins targeted to the mitochondria identified a new putative cis -acting sequence in the 3'-untranslated region of this family of genes, which may play a role in mRNA localization or the regulation of mRNA stability or translation. Nucleic Acids Res, 2000 Apr 1, 28(7), 1499 - 505 SURVEY AND SUMMARY: Saccharomyces cerevisiae basic helix-loop-helix proteins regulate diverse biological processes; Robinson KA et al.; Basic helix-loop-helix (bHLH) proteins are among the most well studied and functionally important regulatory proteins in all eukaryotes . The HLH domain dictates dimerization to create homo- and heterodimers . Dimerization juxtaposes the basic regions of the two monomers to create a DNA interaction surface that recognizes the consensus sequence called the E-box, 5'-CANNTG-3' . Several bHLH proteins have been identified in the yeast Saccharomyces cerevisiae using traditional genetic methodologies . These proteins regulate diverse biological pathways . The completed sequence of the yeast genome, combined with novel methodologies allowing whole-genome expression studies, now offers a unique opportunity to study the function of these bHLH proteins . It is the purpose of this review to summarize the current knowledge of bHLH protein function in yeast. Microbiology, 2000 Feb, 146 ( Pt 2), 377 - 84 Long-chain alkyl ester of AMP acts as an antagonist of glucose-induced signal transduction that mediates activation of plasma membrane proton pump in Saccharomyces cerevisiae; Tanaka T et al.; One of the long-chain alkyl esters of AMP, adenosine 5'-hexadecylphosphate (AMPC16), exhibited a cytotoxic growth inhibitory effect on cells of various yeast strains . The growth inhibitory effect of AMPC16 on Saccharomyces cerevisiae cells was observed only in medium containing Mg2+, which accelerated cellular uptake of the nucleotide analogue . In the presence of Mg2+, AMPC16 completely inhibited glucose-induced extracellular acidification by the intact cells and also interfered with activation of the plasma membrane ATPase, but did not directly inhibit the ATPase activity itself . AMPC16 treatment prevented cells from increasing their intracellular sn-1,2-diacylglycerol (DAG) level in response to glucose, whereas the inhibition of proton extrusion by the cells could be largely reversed by the coaddition of a membrane-permeable DAG analogue . The DAG analogue, a physiological activator of protein kinase C (PKC), was not protective against the inhibition of glucose-induced proton extrusion by staurosporine, which is capable of directly interfering with the action of PKC . These results implied that AMPC16 caused a Mg(2+)-dependent cytotoxic effect on Sac . cerevisiae cells by interfering with a phosphatidylinositol type of signal that mediates activation of the plasma membrane proton pump. Biol Pharm Bull, 2000 Jan, 23(1), 108 - 11 Biosynthesis of thiamin under anaerobic conditions in Saccharomyces cerevisiae; Tanaka K et al.; We studied the biosynthetic route of thiamin in Saccharomyces cerevisiae to see whether the route differed under aerobic and anaerobic conditions . Histidine and pyridoxine are the precursors of the pyrimidine moiety of thiamin under aerobic conditions . Formate is incorporated into the pyrimidine via histidine . The incorporation of {13C}formate and {5'-(2)H2}pyridoxine into the pyrimidine was examined under anaerobic conditions . The labels from {13C}formate and {5'-(2)H2}pyridoxine were not incorporated into the pyrimidine under anaerobic conditions, indicating that the biosynthetic pathway of the pyrimidine differed from that under aerobic conditions . On the other hand, {15N}glycine was incorporated into the thiazole under both anaerobic and aerobic conditions . The biosynthetic pathway of the thiazole was therefore unaltered by the O2 concentration. Mol Cell Biochem, 1999 Dec, 202(1-2), 109 - 18 Characterization of Saccharomyces cerevisiae strains expressing ira1 mutant alleles modeled after disease-causing mutations in NF1; Gil R et al.; The 2818 amino acids of neurofibromin, the product of the human NF1 gene, include a 230 amino acid Ras-GAP related domain (GRD) . Functions which may be associated with the rest of the protein remain unknown . However, many NF1 mutations in neurofibromatosis 1 patients are found downstream of the GRD, suggesting that the C-terminal region of the protein is also functionally important . Since the C-terminal region of neurofibromin encompassing these mutations is homologous with the corresponding regions in the two Saccharomyces cerevisiae Ras-GAPs, Ira1p and Ira2p, we chose yeast as a model system for functional exploration of this region (Ira-C region) . Three missense mutations that affect the Ira-C region of NF1 were used as a model for the mutagenesis of IRA1 . The yeast phenotypes of heat shock sensitivity, iodine staining, sporulation efficiency, pseudohyphae formation, and GAP activity were scored . Even though none of the mutations directly affected the Ira1p-GRD, mutations at two of the three sites resulted in a decrease in the GAP activity present in ira1 cells . The third mutation appeared to disassociate the phenotypes of sporulation ability and GAP activity . This and other evidence suggest an effector function for Ira1p. Yeast, 2000 Mar 30, 16(5), 439 - 49 YHP1 encodes a new homeoprotein that binds to the IME1 promoter in Saccharomyces cerevisiae; Kunoh T et al.; The IME1 gene is essential for initiation of meiosis in the yeast Saccharomyces cerevisiae . Transcription of IME1 is detected under conditions of starvation for nitrogen and glucose, and in the presence of the MATa1 and MATalpha2 gene products . In our previous work, we have shown that there are two elements acting as TUP1-dependent upstream repression sequence (URS) and tup1 mutation-dependent upstream activation sequence (UAS) between nt -915 and -621 of the IME1 promoter under nutritional conditions . The region from -915 to -621 has also been reported to harbour meiotic URS and UAS when a/alpha cells were transferred to sporulation conditions . To identify proteins that are able to bind to the region, we screened a cDNA library fused with the Gal4-activation domain by means of the one-hybrid system . We identified a previously unknown gene (YDR451c), which we designated YHP1, encoding a homeodomain protein of the Drosophila antennapedia type . The region for binding of Yhp1 was delimited to the 28 bp region between nt -702 and -675 of the IME1 promoter in vivo and in vitro, and the 28 bp region harboured a URS activity in a Yhp1-dependent manner under nutrient growth conditions . Although a yhp1 single-disruption mutation did not give rise to a scorable phenotype under nutritional and sporulation conditions, the level of the YHP1 transcript was significantly lower in the cells grown in acetate medium (presporulation medium) and sporulation medium than those grown in glucose medium, and the reduction of YHP1 transcription in acetate medium coincided with an increment of the IME1 transcript . We suggest that the homeoprotein Yhp1 that binds directly to the 28 bp region of the IME1 promoter is a new repressor acting under glucose growth conditions . Yeast, 2000 Mar 30, 16(5), 387 - 400 The GTP hydrolysis defect of the Saccharomyces cerevisiae mutant G-protein Gpa1(G50V); Kallal L et al.; The Saccharomyces cerevisiae haploid cell response to pheromone involves two seven-transmembrane-domain pheromone receptors that couple to a heterotrimeric G protein . The G50V mutation in the G protein alpha subunit (G(alpha)), Gpa1p, is analogous to the p21(ras) transforming mutation Gly-->Val 12, and has been extensively examined for the phenotypes it produces in yeast cells . Here we have characterized the Gpa1(G50V) mutant protein in vitro by examining GTPgammaS binding, GDP exchange, GTP occupancy and guanosine triphosphatase (GTPase) activity . Compared to wild-type (WT) Gpa1p, Gpa1(G50V)p was found to have a moderately reduced GTPase activity and increased GTP occupancy, while GTPgammaS binding and GDP exchange were not significantly altered . The yeast regulator of G protein Signalling (RGS) protein, Sst2p, was also expressed and purified, and found to have a significantly reduced ability to stimulate the initial rate of GTP hydrolysis of Gpa1(G50V)p compared to its effect on WT Gpa1p . Probing conformational transitions by a protease sensitivity assay suggested that Gpa1(G50V)p did not bind the transition state mimetic GDP/AlF(4)(-) as efficiently as the WT Gpa1p . These biochemical results can explain many of the known gpa1(G50V) yeast cell phenotypes . J Cell Sci, 2000 Apr, 113 ( Pt 7), 1199 - 211 The Saccharomyces cerevisiae SDA1 gene is required for actin cytoskeleton organization and cell cycle progression; Buscemi G et al.; The organization of the actin cytoskeleton is essential for several cellular processes . Here we report the characterization of a Saccharomyces cerevisiae novel gene, SDA1, encoding a highly conserved protein, which is essential for cell viability and is localized in the nucleus . Depletion or inactivation of Sda1 cause cell cycle arrest in G(1) by blocking both budding and DNA replication, without loss of viability . Furthermore, sda1-1 temperature-sensitive mutant cells arrest at the non-permissive temperature mostly without detectable structures of polymerized actin, although a normal actin protein level is maintained, indicating that Sda1 is required for proper organization of the actin cytoskeleton . To our knowledge, this is the first mutation shown to cause such a phenotype . Recovery of Sda1 activity restores proper assembly of actin structures, as well as budding and DNA replication . Furthermore we show that direct actin perturbation, either in sda1-1 or in cdc28-13 cells released from G(1) block, prevents recovery of budding and DNA replication . We also show that the block in G(1) caused by loss of Sda1 function is independent of Swe1 . Altogether our results suggest that disruption of F-actin structure can block cell cycle progression in G(1) and that Sda1 is involved in the control of the actin cytoskeleton. Cell Motil Cytoskeleton, 2000 Mar, 45(3), 235 - 46 Rpg1p, the subunit of the Saccharomyces cerevisiae eIF3 core complex, is a microtubule-interacting protein; Hasek J et al.; The essential gene RPG1/TIF32 of Saccharomyces cerevisiae encodes the 110-kDa subunit of the translation initiation factor 3 (eIF3) core complex . In this study, the Rpg1p-specific monoclonal antibody PK1/1 was used to analyse the cellular distribution of Rpg1p by epifluorescence and confocal laser scanning microscopy (CLSM) . In budded cells, a portion of Rpg1p was obviously co-localised with microtubules . In addition, CLSM revealed an accumulation of Rpg1p in a patch at the very end of cytoplasmic microtubules reaching the bud tip . A punctate fluorescence pattern was typical for separated unbudded cells . Distribution of Rpg1p was confirmed using a strain expressing exclusively a hemaglutinin-tagged version of Rpg1p . In nocodazole-treated cells, the pattern of the PK1/1 staining was disturbed . No staining was observed in Rpg1p-depleted cells . In vitro experiments revealed that Rpg1p was specifically co-immunoprecipitated with alpha-tubulin from the yeast cell free extract and this observation was further supported by showing that Rpg1p co-sedimented with hog brain microtubules . We conclude that Rpg1p is a microtubule-interacting protein that indicates an interesting connection between the translation initiation machinery and cytoskeleton in yeast Saccharomyces cerevisiae . Antonie Van Leeuwenhoek, 2000 Jan, 77(1), 65 - 9 Molecular cloning of Saccharomyces cerevisiae MGC1/YDR473c gene which is essential for cell growth; Fujimura H; Saccharomyces cerevisiae haploid cells undergo morphological changes in response to mating pheromones, a- and alpha-factors, during sexual conjugation . As a first step to elucidate the mechanism, I had previously identified the mgc1 mutation which affected the morphogenesis induced by mating pheromones . The mutation had been designated mgc1 for morphogenesis control . In the present study I cloned the MGC1 gene . Sequencing analysis indicates that the MGC1 gene corresponds to the YDR473c gene . The MGC1 gene was shown to be essential for cell growth and required for the transition from the G1 to S phase of cell cycle . Protein-protein interaction of Mgc1 protein was shown by using yeast two-hybrid system . Mgc1 protein was also proposed to be localized in the nucleus in yeast cells. J Biol Chem, 2000 Mar 10, 275(10), 7198 - 204 Functional domain mapping and subcellular distribution of Dal82p in Saccharomyces cerevisiae; Scott S et al.; Previous studies have shown that (i) Dal81p and Dal82p are required for allophanate-induced gene expression in Saccharomyces cerevisiae; (ii) the cis-acting element mediating the induced transcriptional response to allophanate is a dodecanucleotide, UIS(ALL); and (iii) Dal82p binds specifically to UIS(ALL) . Here we show that Dal82p is localized to the nucleus and parallels movement of the DNA through the cell cycle . Deletion analysis of DAL82 identified and localized three functional domains . Electrophoretic mobility shift assays identified a peptide (consisting of Dal82p amino acids 1-85) that is sufficient to bind a DNA fragment containing UIS(ALL) . LexA-tethering experiments demonstrated that Dal82p is capable of mediating transcriptional activation . The activation domain consists of two parts: (i) an absolutely required core region (amino acids 66-99) and (ii) less well defined regions flanking residues 66-99 that are required for full wild-type levels of activation . The Dal82p C terminus contains a predicted coiled-coil motif that down-regulates Dal82p-mediated transcriptional activation. J Biol Chem, 2000 Mar 10, 275(10), 6876 - 84 Cloning of an alkaline ceramidase from Saccharomyces cerevisiae . An enzyme with reverse (CoA-independent) ceramide synthase activity; Mao C et al.; Ceramide is not only a core intermediate of sphingolipids but also an important modulator of many cellular events including apoptosis, cell cycle arrest, senescence, differentiation, and stress responses . Its turnover may be tightly regulated . However, little is known about the regulation of its metabolism because most enzymes responsible for its synthesis and breakdown have yet to be cloned . Here we report the cloning and characterization of the yeast gene YPC1 (YBR183w) by screening Saccharomyces cerevisiae genes whose overexpression bestows resistance to fumonisin B1 . We demonstrate that the yeast gene YPC1 encodes an alkaline ceramidase activity responsible for the breakdown of dihydroceramide and phytoceramide but not unsaturated ceramide . YPC1 ceramidase activity was confirmed by in vitro studies using an Escherichia coli expression system . Importantly, YPC1p also has reverse activity, catalyzing synthesis of phytoceramide from palmitic acid and phytosphingosine . This ceramide synthase activity is CoA-independent and is resistant to fumonisin B1, thus explaining why YPC1 was cloned as a fumonisin B1-resistant gene. J Mol Biol, 2000 Mar 10, 296(5), 1205 - 14 Computational identification of cis-regulatory elements associated with groups of functionally related genes in Saccharomyces cerevisiae; Hughes JD et al.; AlignACE is a Gibbs sampling algorithm for identifying motifs that are over-represented in a set of DNA sequences . When used to search upstream of apparently coregulated genes, AlignACE finds motifs that often correspond to the DNA binding preferences of transcription factors . We previously used AlignACE to analyze whole genome mRNA expression data . Here, we present a more detailed study of its effectiveness as applied to a variety of groups of genes in the Saccharomyces cerevisiae genome . Published functional catalogs of genes and sets of genes grouped by common name provided 248 groups, resulting in 3311 motifs . In conjunction with this analysis, we present measures for gauging the tendency of a motif to target a given set of genes relative to all other genes in the genome and for gauging the degree to which a motif is preferentially located in a certain distance range upstream of translational start sites . We demonstrate improved methods for comparing and clustering sequence motifs . Many previously identified cis-regulatory elements were found . We also describe previously unidentified motifs, one of which has been verified by experiments in our laboratory . An extensive set of AlignACE runs on randomly selected sets of genes and on sets of genes whose upstream regions contain known transcription factor binding sites serve as controls . FEBS Lett, 2000 Feb 25, 468(2-3), 142 - 8 Two different modes of cyclin clb2 proteolysis during mitosis in Saccharomyces cerevisiae; Baumer M et al.; Sister chromatid separation and mitotic exit are triggered by the anaphase-promoting complex (APC/C) which is a multi-subunit ubiquitin ligase required for proteolytic degradation of various target proteins . Cdc20 and Cdh1 are substrate-specific activators of the APC/C . It was previously proposed that Cdh1 is essential for proteolysis of the yeast mitotic cyclin Clb2 . We show that Clb2 proteolysis is triggered by two different modes during mitosis . A fraction of Clb2 is degraded during anaphase in the absence of Cdh1 . However, a second fraction of Clb2 remains stable during anaphase and is degraded in a Cdh1-dependent manner as cells exit from mitosis . Most of cyclin Clb3 is degraded independently of Cdh1 . Our data imply that degradation of mitotic cyclins is initiated by a Cdh1-independent mechanism. Mol Microbiol, 2000 Feb, 35(4), 936 - 48 The essential protein fap7 is involved in the oxidative stress response of Saccharomyces cerevisiae; Juhnke H et al.; Pos9 (Skn7) is an important transcription factor that, together with Yap1, induces the expression of oxidative stress target genes in Saccharomyces cerevisiae . The activation of Pos9 upon an oxidative stress signal occurs post-translationally . In a mutant screen for factors involved in the activation of a Pos9-dependent reporter gene upon oxidative stress, we identified the mutant fap7-1 (for factor activating Pos9) . This point mutant failed to activate a Gal4-Pos9 hybrid transcription factor, assayed by hydrogen peroxide-induced GAL1-lacZ reporter gene activities . Additionally, the fap7-1 mutant strain was sensitive to oxidative stress and revealed slow growth on glucose compared with the wild type . The fap7-1 mutation also affected the induction of the Pos9 target gene TPX1 and of a synthetic promoter previously identified to be regulated in a Yap1- and Pos9-dependent manner . This lack of induction was specific as the fap7-1 mutant response to other stresses such as sodium chloride or co-application of both hydrogen peroxide and sodium chloride was not affected, as tested with the Pos9-independent expression pattern of a TPS2-lacZ reporter system . We identified the gene YDL166c to be allelic to the FAP7 gene and to be essential . Fluorescence microscopy of Fap7-GFP fusion proteins indicated a nuclear localization of the Fap7 protein . Our data suggest that Fap7 is a nuclear factor important for Pos9-dependent target gene transcription upon oxidative stress. Annu Rev Genet, 1999, 33, 261 - 311 Ribosome synthesis in Saccharomyces cerevisiae; Venema J et al.; The synthesis of ribosomes is one of the major metabolic pathways in all cells . In addition to around 75 individual ribosomal proteins and 4 ribosomal RNAs, synthesis of a functional eukaryotic ribosome requires a remarkable number of trans-acting factors . Here, we will discuss the recent, and often surprising, advances in our understanding of ribosome synthesis in the yeast Saccharomyces cerevisiae . These will underscore the unexpected complexity of eukaryotic ribosome synthesis. FEMS Microbiol Lett, 2000 Mar 1, 184(1), 103 - 8 Proline accumulation by mutation or disruption of the proline oxidase gene improves resistance to freezing and desiccation stresses in Saccharomyces cerevisiae; Takagi H et al.; We examined the role of intracellular proline under freezing and desiccation stress conditions in Saccharomyces cerevisiae . When cultured in liquid minimal medium, the proline-nonutilizing mutant containing the put1 mutation (proline oxidase-deficient) produced more intracellular proline, and increased the cell survival rate as compared to the wild-type strain after freezing and desiccation . We also constructed two PUT1 gene disruptants . PUT1-disrupted mutants in minimal medium supplemented with external proline at 0.1% accumulated higher proline levels than those of the control strains (17-22-fold) . These disruptants also had a 2-5-fold increase in cell viability compared to the control strains after freezing and desiccation stresses . These results indicate that proline has a stress-protective function in yeast. FEMS Microbiol Lett, 2000 Mar 1, 184(1), 69 - 72 Energetics of the effect of acetic acid on growth of Saccharomyces cerevisiae; Pampulha ME et al.; In batch cultures of a respiratory deficient mutant of Saccharomyces cerevisiae the maximum specific growth rate and the yield coefficient decreased, but the specific glucose consumption rate increased, in the presence of acetic acid . The ATP yield decreased from approximately 14 to 4 g biomass (mol ATP)(-1) when the concentration of acetic acid increased from 0 to 170 mM . Intracellular acidification was much weaker than previously reported for non-adapted cells . A linear relation was obtained between the ATP specific production rate and the uptake rate of acetic acid, suggesting that about 1 mol ATP is consumed per mol of acetic acid diffusing into the cells. Mol Biol Cell, 2000 Feb, 11(2), 663 - 76 Essential functions of protein tyrosine phosphatases PTP2 and PTP3 and RIM11 tyrosine phosphorylation in Saccharomyces cerevisiae meiosis and sporulation; Zhan XL et al.; Tyrosine phosphorylation plays a central role in eukaryotic signal transduction . In yeast, MAP kinase pathways are regulated by tyrosine phosphorylation, and it has been speculated that other biochemical processes may also be regulated by tyrosine phosphorylation . Previous genetic and biochemical studies demonstrate that protein tyrosine phosphatases (PTPases) negatively regulate yeast MAP kinases . Here we report that deletion of PTP2 and PTP3 results in a sporulation defect, suggesting that tyrosine phosphorylation is involved in regulation of meiosis and sporulation . Deletion of PTP2 and PTP3 blocks cells at an early stage of sporulation before premeiotic DNA synthesis and induction of meiotic-specific genes . We observed that tyrosine phosphorylation of several proteins, including 52-, 43-, and 42-kDa proteins, was changed in ptp2Deltaptp3Delta homozygous deletion cells under sporulation conditions . The 42-kDa tyrosine-phosphorylated protein was identified as Mck1, which is a member of the GSK3 family of protein kinases and previously known to be phosphorylated on tyrosine . Mutation of MCK1 decreases sporulation efficiency, whereas mutation of RIM11, another GSK3 member, specifically abolishes sporulation; therefore, we investigated regulation of Rim11 by Tyr phosphorylation during sporulation . We demonstrated that Rim11 is phosphorylated on Tyr-199, and the Tyr phosphorylation is essential for its in vivo function, although Rim11 appears not to be directly regulated by Ptp2 and Ptp3 . Biochemical characterizations indicate that tyrosine phosphorylation of Rim11 is essential for the activity of Rim11 to phosphorylate substrates . Our data demonstrate important roles of protein tyrosine phosphorylation in meiosis and sporulation Mol Biol Cell, 2000 Feb, 11(2), 593 - 611 Roles of Hof1p, Bni1p, Bnr1p, and myo1p in cytokinesis in Saccharomyces cerevisiae; Vallen EA et al.; Cytokinesis in Saccharomyces cerevisiae occurs by the concerted action of the actomyosin system and septum formation . Here we report on the roles of HOF1, BNI1, and BNR1 in cytokinesis, focusing on Hof1p . Deletion of HOF1 causes a temperature-sensitive defect in septum formation . A Hof1p ring forms on the mother side of the bud neck in G2/M, followed by the formation of a daughter-side ring . Around telophase, Hof1p is phosphorylated and the double rings merge into a single ring that contracts slightly and may colocalize with the actomyosin structure . Upon septum formation, Hof1p splits into two rings, disappearing upon cell separation . Hof1p localization is dependent on septins but not Myo1p . Synthetic lethality suggests that Bni1p and Myo1p belong to one functional pathway, whereas Hof1p and Bnr1p belong to another . These results suggest that Hof1p may function as an adapter linking the primary septum synthesis machinery to the actomyosin system . The formation of the actomyosin ring is not affected by bni1Delta, hof1Delta, or bnr1Delta . However, Myo1p contraction is affected by bni1Delta but not by hof1Delta or bnr1Delta . In bni1Delta cells that lack the actomyosin contraction, septum formation is often slow and asymmetric, suggesting that actomyosin contraction may provide directionality for efficient septum formation. Biochemistry, 2000 Feb 22, 39(7), 1716 - 24 Catalytic and DNA binding properties of the ogg1 protein of Saccharomyces cerevisiae: comparison between the wild type and the K241R and K241Q active-site mutant proteins; Guibourt N et al.; The Ogg1 protein of Saccharomyces cerevisiae belongs to a family of DNA glycosylases and apurinic/apyrimidinic site (AP) lyases, the signature of which is the alpha-helix-hairpin-alpha-helix-Gly/Pro-Asp (HhH-GPD) active site motif together with a conserved catalytic lysine residue, to which we refer as the HhH-GPD/K family . In the yeast Ogg1 protein, yOgg1, the HhH-GPD/K motif spans residues 225-260 and the conserved lysine is K241 . In this study, we have purified the K241R and K241Q mutant proteins and compared their catalytic and DNA binding properties to that of the wild-type yOgg1 . The results show that the K241R mutation greatly impairs both the DNA glycosylase and the AP lyase activities of yOgg1 . Specificity constants for cleavage of a 34mer oligodeoxyribonucleotide containing a 7,8-dihydro-8-oxoguanine (8-OxoG) paired with a cytosine, {8-OxoG.C}, are 56 x 10(-)(3) and 5 x 10(-)(3) min(-)(1) nM(-)(1) for the wild-type and the K241R protein, respectively . On the other hand, the K241Q mutation abolishes the DNA glycosylase and AP lyase activities of yOgg1 . In contrast, the K241R and K241Q proteins have conserved wild-type DNA binding properties . K(dapp) values for binding of {8-OxoG.C} are 6.9, 7.4, and 4.8 nM for the wild-type, K241R, and K241Q proteins, respectively . The results also show that AP site analogues such as 1, 3-propanediol (Pr), tetrahydrofuran (F), or cyclopentanol (Cy) are not substrates but constitute good inhibitors of the wild-type yOgg1 . Therefore, we have used a 59mer {Pr.C} duplex to further analyze the DNA binding properties of the wild-type, K241R, and K241Q proteins . Hydroxyl radical footprints of the wild-type yOgg1 show strong protection of six nucleotides centered around the Pr lesion in the damaged strand . On the complementary strand, only the cytosine placed opposite Pr was strongly protected . The same footprints were observed with the K241R and K241Q proteins, confirming their wild-type DNA binding properties . These results indicate that the K241Q mutant protein can be used to study interactions between yOgg1 and DNA containing metabolizable substrates such as 8-OxoG or an AP site. Mol Cell Biol, 2000 Mar, 20(6), 2087 - 97 ArgRII, a component of the ArgR-Mcm1 complex involved in the control of arginine metabolism in Saccharomyces cerevisiae, is the sensor of arginine; Amar N et al.; Repression of arginine anabolic genes and induction of arginine catabolic genes are mediated by a three-component protein complex, interacting with specific DNA sequences in the presence of arginine . Although ArgRI and Mcm1, two MADS-box proteins, and ArgRII, a zinc cluster protein, contain putative DNA binding domains, alone they are unable to bind the arginine boxes in vitro . Using purified glutathione S-transferase fusion proteins, we demonstrate that ArgRI and ArgRII1-180 or Mcm1 and ArgRII1-180 are able to reconstitute an arginine-dependent binding activity in mobility shift analysis . Binding efficiency is enhanced when the three recombinant proteins are present simultaneously . At physiological concentration, the full-length ArgRII is required to fulfill its functions; however, when ArgRII is overexpressed, the first 180 amino acids are sufficient to interact with ArgRI, Mcm1, and arginine, leading to the formation of an ArgR-Mcm1-DNA complex . Several lines of evidence indicate that ArgRII is the sensor of the effector arginine and that the binding site of arginine would be the region downstream from the zinc cluster, sharing some identity with the arginine binding domain of bacterial arginine repressors. Mol Cell Biol, 2000 Mar, 20(6), 2066 - 74 RRS1, a conserved essential gene, encodes a novel regulatory protein required for ribosome biogenesis in Saccharomyces cerevisiae; Tsuno A et al.; A secretory defect causes specific and significant transcriptional repression of both ribosomal protein and rRNA genes (K . Mizuta and J . R . Warner, Mol . Cell . Biol . 14:2493-2502, 1994), suggesting the coupling of plasma membrane and ribosome syntheses . In order to elucidate the molecular mechanism of the signaling pathway, we isolated a cold-sensitive mutant with a mutation in a gene termed RRS1 (regulator of ribosome synthesis), which appeared to be defective in the signaling pathway . The rrs1-1 mutation greatly reduced transcriptional repression of both rRNA and ribosomal protein genes that is caused by a secretory defect . RRS1 is a novel, essential gene encoding a nuclear protein of 203 amino acid residues that is conserved in eukaryotes . A conditional rrs1-null mutant was constructed by placing RRS1 under the control of the GAL1 promoter . Rrs1p depletion caused defects in processing of pre-rRNA and assembly of ribosomal subunits. Protein Expr Purif, 2000 Mar, 18(2), 202 - 12 Vectors allowing amplified expression of the Saccharomyces cerevisiae Gal3p-Gal80p-Gal4p transcription switch: applications to galactose-regulated high-level production of proteins; Sil AK et al.; The Gal4, Gal80, and Gal3 proteins of Saccharomyces cerevisiae constitute a galactose-responsive regulatory switch for GAL gene promoters . The low cellular levels of these proteins have hampered mechanistic studies and limit the utility of the GAL gene promoters for high-yield production of endogenous and exogenous proteins . We have constructed two new vectors, pMEGA2 and pMEGA2-DeltaURA3, that increase the level of the Gal4p-Gal80p-Gal3p switch proteins under conditions that preserve the Gal3p-Gal80p-Gal4p stoichiometries required for normal switch function . Cells carrying pMEGA2 show 15- to 20-fold more Gal4p and 30- to 40-fold more Gal3p and Gal80p than cells lacking pMEGA2 . These high levels of Gal4p, Gal80p, and Gal3p do not perturb the integrity of galactose-inducible regulation . Cells that carry pMEGA2 exhibit normal galactose-induction kinetics for the chromosomal MEL1 gene expression and normal, albeit slower, log-phase growth . Insertion of the MEL1 gene into pMEGA2 provides a 24- to 30-fold increase in the Mel1 protein . Cells carrying a 2-microm-based URA3-selectable plasmid containing a GAL1pro:lacZ reporter gene and a second plasmid, pMEGA2-DeltaURA3, produce 12-fold more beta-galactosidase than cells carrying only the GAL1pro:lacZ reporter plasmid . The performance of the MEGA plasmids in providing amplified production of the Gal3, Gal80, and Gal4 proteins should prove useful in investigations of the mechanistic aspects of these transcription switch proteins and in work aimed at achieving high-level, galactose-regulatable production of proteins in yeast . Biochemistry, 2000 Feb 29, 39(8), 1903 - 14 Crystal structures of a low-molecular weight protein tyrosine phosphatase from Saccharomyces cerevisiae and its complex with the substrate p-nitrophenyl phosphate; Wang S et al.; Low-molecular weight protein tyrosine phosphatases are virtually ubiquitous, which implies that they have important cellular functions . We present here the 2.2 A resolution X-ray crystallographic structure of wild-type LTP1, a low-molecular weight protein tyrosine phosphatase from Saccharomyces cerevisiae . We also present the structure of an inactive mutant substrate complex of LTP1 with p-nitrophenyl phosphate (pNPP) at a resolution of 1.7 A . The crystal structures of the wild-type protein and of the inactive mutant both have two molecules per asymmetric unit . The wild-type protein crystal was grown in HEPES buffer, a sulfonate anion that resembles the phosphate substrate, and a HEPES molecule was found with nearly full occupancy in the active site . Although the fold of LTP1 resembles that of its bovine counterpart BPTP, there are significant changes around the active site that explain differences in their kinetic behavior . In the crystal of the inactive mutant of LTP1, one molecule has a pNPP in the active site, while the other has a phosphate ion . The aromatic residues lining the walls of the active site cavity exhibit large relative movements between the two molecules . The phosphate groups present in the structures of the mutant protein bind more deeply in the active site (that is, closer to the position of nucleophilic cysteine side chain) than does the sulfonate group of the HEPES molecule in the wild-type structure . This further confirms the important role of the phosphate-binding loop in stabilizing the deep binding position of the phosphate group, thus helping to bring the phosphate close to the thiolate anion of nucleophilic cysteine, and facilitating the formation of the phosphoenzyme intermediate. Nucleic Acids Res, 2000 Mar 15, 28(6), 1332 - 9 Interactions of the human, rat, Saccharomyces cerevisiae and Escherichia coli 3-methyladenine-DNA glycosylases with DNA containing dIMP residues; Saparbaev M et al.; In DNA, the deamination of dAMP generates 2'-deoxy-inosine 5'-monophosphate (dIMP) . Hypoxanthine (HX) residues are mutagenic since they give rise to A.T-->G.C transition . They are excised, although with different efficiencies, by an activity of the 3-methyl-adenine (3-meAde)-DNA glycosylases from Escherichia coli (AlkA protein), human cells (ANPG protein), rat cells (APDG protein) and yeast (MAG protein) . Comparison of the kinetic constants for the excision of HX residues by the four enzymes shows that the E.coli and yeast enzymes are quite inefficient, whereas for the ANPG and the APDG proteins they repair the HX residues with an efficiency comparable to that of alkylated bases, which are believed to be the primary substrates of these DNA glycosylases . Since the use of various substrates to monitor the activity of HX-DNA glycosylases has generated conflicting results, the efficacy of the four 3-meAde-DNA glycosylases of different origin was compared using three different substrates . Moreover, using oligo-nucleotides containing a single dIMP residue, we investigated a putative sequence specificity of the enzymes involving the bases next to the HX residue . We found up to 2-5-fold difference in the rates of HX excision between the various sequences of the oligonucleotides studied . When the dIMP residue was placed opposite to each of the four bases, a preferential recognition of dI:T over dI:dG, dI:dC and dI:dA mismatches was observed for both human (ANPG) and E.coli (AlkA) proteins . At variance, the yeast MAG protein removed more efficiently HX from a dI:dG over dI:dC, dI:T and dI:dA mismatches. FEBS Lett, 2000 Feb 18, 468(1), 28 - 32 Inositol polyphosphate multikinase (ArgRIII) determines nuclear mRNA export in Saccharomyces cerevisiae; Saiardi A et al.; The ARGRIII gene of Saccharomyces cerevisiae encodes a transcriptional regulator that also has inositol polyphosphate multikinase (ipmk) activity {Saiardi et al . (1999) Curr . Biol . 9, 1323-1326} . To investigate how inositol phosphates regulate gene expression, we disrupted the ARGRIII gene . This mutation impaired nuclear mRNA export, slowed cell growth, increased cellular {InsP(3)} 170-fold and decreased {InsP(6)} 100-fold, indicating reduced phosphorylation of InsP(3) to InsP(6) . Levels of diphosphoinositol polyphosphates were decreased much less dramatically than was InsP(6) . Low levels of InsP(6), and considerable quantities of Ins(1,3,4,5)P(4), were synthesized by an ipmk-independent route . Transcriptional control by ipmk reflects that it is a pivotal regulator of nuclear mRNA export via inositol phosphate metabolism. Mol Cell Biol Res Commun, 2000 Jan, 3(1), 20 - 5 Increased chitin synthesis in response to type II myosin deficiency in Saccharomyces cerevisiae; Cruz JA et al.; We reported previously that the chitin content in cell walls of type II myosin-deficient Saccharomyces cerevisiae strains is increased relative to wild-type cells suggesting that increased chitin synthesis is induced in these strains . In the present study, we have performed enzyme activity assays for chitin synthases 1, 2, and 3 to determine the enzyme isoform(s) involved . To determine if transcriptional regulation is involved, we conducted quantitative mRNA assays of the corresponding chitin synthase genes . We show that the enzyme activities of all three chitin synthases increase substantially over the wild-type strain while eight- and twofold increases in the mRNA levels for chitin synthases 1 and 3 were detected . Increases in enzyme activities and mRNA levels were not proportional . We conclude that the enzyme activities for all three chitin synthases are elevated in this strain and that this increase is mediated mainly by a posttranslational mechanism(s) . The heightened sensitivity to osmotic stress and the corresponding increase in cell wall chitin content reported in these strains are consistent with a compensatory "stress response" mechanism induced by abnormal cell wall assembly . Arch Biochem Biophys, 2000 Mar 1, 375(1), 154 - 60 The "SUN" family: UTH1, an ageing gene, is also involved in the regulation of mitochondria biogenesis in Saccharomyces cerevisiae; Camougrand NM et al.; Since it was shown in previous work that NCA3 (one of the four genes of the SUN family) is involved in mitochondrial protein synthesis regulation, the effect of the other members of this gene family was tested . UTH1 (but not SUN4 or SIM1) was also shown to interfere with mitochondria biogenesis . In Deltauth1 cells, cytochromes aa(3), c, and b were lowered by 25 and 15%, respectively . In the double-null mutant Deltauth1Deltanca3, only cytochrome aa(3) was lowered by 50% relative to the wild type . However, the ratio of cellular respiration to cytochrome oxidase was greatly enhanced in the double-null mutant . Measurements on whole lysed cells showed that another mitochondrial enzyme, citrate synthase, was also lowered in Deltauth1 and Deltauth1Deltanca3 whereas hexokinase was not . Electron micrographs showed no difference in global mitochondria content in Deltauth1Deltanca3, but mitochondria appeared less dense to electrons compared to the wild type . Cardiolipin and mtDNA were equivalent in parental and mutant strains . Measurements on isolated mitochondria showed that the cyt aa(3)/cyt b ratio was also lowered in Deltauth1Deltanca3, but the control exerted by the oxidase on the respiratory flux was higher . The activity of other mitochondrial complexes versus oxidase was equivalent in mutants compared to the wild type . These results suggest that the protein equipment could be lowered in mitochondria from strains inactivated for UTH1 . Arch Biochem Biophys, 2000 Mar 1, 375(1), 83 - 9 Ribosomal stalk protein phosphorylating activities in Saccharomyces cerevisiae; Bou G et al.; With ribosomal P protein as a substrate, five peaks of protein kinase activity are eluted after chromatography of a Saccharomyces cerevisiae cellular extract on DEAE-cellulose . Two of them correspond to CK-II and the other three have been called RAP-1, RAP-II, and RAP-III . RAP-I was previously characterized . RAP-III is present in a very small amount, which hindered its purification . RAP-II was further purified on phosphocellulose, heparin-Sepharose, and P protein-Sepharose, studied in detail, and compared with other acidic protein kinases, including RAP-I, CK-II, and PK60 . RAP-II is shown by SDS-PAGE and centrifugation on glycerol linear density gradients to have a molecular mass of around 62 kDa and it is immunologically different from RAP-I and PK60 . RAP-II phosphorylates the P proteins in the last serine residue at the highly conserved carboxyl terminal domain as other P-protein kinases . The ribosome-bound stalk P proteins are not equally phosphorylated by the different kinases . Thus, RAP-II and PK60 mainly phosphorylate P1beta and P2alpha whereas RAP-I and CK-II modify all of them . A comparative study of the K(m) and V(max) of the phosphorylation reaction by the different kinases using individual purified acidic proteins suggests changes in the substrate susceptibility upon binding to the ribosome . All the data available reveal clear differences in the characteristics of the various P protein kinases and suggest that the cell may use them to differentially modify the stalk depending, perhaps, on metabolic requirements . Arch Biochem Biophys, 2000 Mar 1, 375(1), 78 - 82 A winged helix protein from yeast Saccharomyces cerevisiae recognizes centromere sequences; Myrich E et al.; The winged helix-turn-helix motif was initially identified in the mammalian hepatocyte-enriched transcription factor HNF-3 and the Drosophila forkhead homeotic protein . Proteins containing the winged helix motif have been shown to play important roles in tissue-specific developmental regulation . In this report, by using a genomic binding site selection method, we demonstrate that the winged helix protein YFKH-1 from the yeast Saccharomyces cerevisiae recognizes conserved sequence in yeast centromeres . Thus, our data suggest that the winged helix proteins of the yeast may be involved in centromeric functions of the yeast . J Biol Chem, 2000 Feb 25, 275(8), 5431 - 40 Loss of the major isoform of phosphoglucomutase results in altered calcium homeostasis in Saccharomyces cerevisiae; Fu L et al.; Phosphoglucomutase (PGM) is a key enzyme in glucose metabolism, where it catalyzes the interconversion of glucose 1-phosphate (Glc-1-P) and glucose 6-phosphate (Glc-6-P) . In this study, we make the novel observation that PGM is also involved in the regulation of cellular Ca(2+) homeostasis in Saccharomyces cerevisiae . When a strain lacking the major isoform of PGM (pgm2Delta) was grown on media containing galactose as sole carbon source, its rate of Ca(2+) uptake was 5-fold higher than an isogenic wild-type strain . This increased rate of Ca(2+) uptake resulted in a 9-fold increase in the steady-state total cellular Ca(2+) level . The fraction of cellular Ca(2+) located in the exchangeable pool in the pgm2Delta strain was found to be as large as the exchangeable fraction observed in wild-type cells, suggesting that the depletion of Golgi Ca(2+) stores is not responsible for the increased rate of Ca(2+) uptake . We also found that growth of the pgm2Delta strain on galactose media is inhibited by 10 microM cyclosporin A, suggesting that activation of the calmodulin/calcineurin signaling pathway is required to activate the Ca(2+) transporters that sequester the increased cytosolic Ca(2+) load caused by this high rate of Ca(2+) uptake . We propose that these Ca(2+)-related alterations are attributable to a reduced metabolic flux between Glc-1-P and Glc-6-P due to a limitation of PGM enzymatic activity in the pgm2Delta strain . Consistent with this hypothesis, we found that this "metabolic bottleneck" resulted in an 8-fold increase in the Glc-1-P level compared with the wild-type strain, while the Glc-6-P and ATP levels were normal . These results suggest that Glc-1-P (or a related metabolite) may participate in the control of Ca(2+) uptake from the environment. J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 295 - 9 Three-step chromatographic purification of Cpr6, a cyclophilin from Saccharomyces cerevisiae; Mayr C; Cyclophilins constitute a group of peptidyl-prolyl cis-trans isomerases (PPIs), known to be involved in protein folding . Because of their ability to bind the immunosuppresant drug Cyclosporin A (CsA), they are also called immunophilins . Immunophilins, which exhibit a relative molecular mass higher than 40 000, are further found in complex with Hsp90, a major cytosolic molecular chaperone . The present work describes a three-step chromatographic purification of recombinant Cpr6, a cyclophilin from Saccharomyces cerevisiae . The cDNA of Cpr6 was cloned into a pRSET A-plasmid with an N-terminal 6 x histidine-tag (his-tag) and transformed into the BL21{DE3}pLysS strain . After collection of the bacterial material and lysis of the cells the cell lysate was centrifuged and loaded onto a metal chelating column . After extensive washing the protein was eluted with a step gradient from 20 to 250 mM imidazol . The pooled protein was dialysed against ethylenedinitrilo tetraacetic acid (EDTA)-buffer, and loaded onto a strong anion-exchanger . Cpr6 containing fractions were then, in a last step, loaded onto a gel permeation chromatography column . The purity of the resulting protein was measured by silver stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and, additionally, as Cpr6 does not contain tryptophan residues by tryptophan residue titration . Based on a standard curve the content of contaminating tryptophan residues in the purified protein solution was determined . A typical yield of 1 mg pure protein per g of wet cells was achieved with the described procedure. Curr Opin Genet Dev, 2000 Feb, 10(1), 47 - 53 Cell-cycle checkpoints that ensure coordination between nuclear and cytoplasmic events in Saccharomyces cerevisiae; Lew DJ; Cytoskeletal organization is crucial for several aspects of cell-cycle progression but cytoskeletal elements are quite sensitive to environmental perturbations . Two novel checkpoint controls monitor the function of the actin and microtubule systems in budding yeast and operate to delay cell-cycle progression in response to cytoskeletal perturbations . In cells whose actin cytoskeleton has been perturbed, bud formation is frequently delayed and the morphogenesis checkpoint introduces a compensatory delay of nuclear division until a bud has been formed . In cells whose microtubule cytoskeleton has been perturbed, anaphase spindle elongation often occurs entirely within the mother cell, and the post-anaphase nuclear migration checkpoint introduces a compensatory delay of cytokinesis until one pole of the anaphase nucleus enters the bud . Recent studies indicate that regulators of entry into mitosis are localized to the daughter side of the mother-bud neck whereas regulators of exit from mitosis are localized to the spindle pole bodies . Thus, specific cell-cycle regulators are well-placed to monitor whether a cell has formed a bud and whether a daughter nucleus has been delivered accurately to the bud following mitosis. Biochim Biophys Acta, 2000 Feb 15, 1463(2), 477 - 82 Expression and subcellular localization of a membrane protein related to Hsp30p in Saccharomyces cerevisiae; Wu K et al.; The Saccharomyces cerevisiae YDR033w gene product is homologous to Hsp30p and Yro2p, both of which are induced during heat shock . To investigate the subcellular localization of the YDR033w gene product, hemagglutinin (HA) epitope-tagged protein was expressed, detected on immunoblots, and localized by immunofluorescence to cell membranes, primarily the plasma membrane . A punctuate immunofluorescence pattern was observed within cell buds . The nuclear envelope, but not the vacuole or mitochondrial membranes, were also immunostained . We refer to YDR033w as MRH1 to denote that it encodes a membrane protein related to Hsp30p. Biochim Biophys Acta, 2000 Feb 15, 1463(2), 267 - 78 The LEA-like protein HSP 12 in Saccharomyces cerevisiae has a plasma membrane location and protects membranes against desiccation and ethanol-induced stress; Sales K et al.; The LEA-like protein HSP 12 was identified as having a plasma membrane location in yeast . Gold particles, indicative of the presence of HSP 12, were observed on the external side of the plasma membrane when yeast grown to stationary phase were subjected to immunocytochemical analysis . Growth of yeast in the osmolyte mannitol resulted in an increased number of gold particles that were now observed to be present on both sides of the plasma membrane . No gold particles were observed using a mutant strain of the same yeast that did not express HSP 12 . A model liposome system encapsulating the fluorescent dye calcein was used to investigate the protection by HSP 12 of membranes during desiccation . HSP 12 was found to act in an analogous manner to trehalose and protect liposomal membrane integrity against desiccation . The interaction between HSP 12 and the liposomal membrane was judged to be electrostatic as membrane protection was only observed with positively charged liposomes and not with either neutral or negatively charged liposomes . The ability of the wild-type and mutant yeast to grow in media containing ethanol was compared . It was found that yeast not expressing the HSP 12 protein were less able to grow in media containing ethanol . HSP 12 was shown to confer increased integrity on the liposomal membrane in the presence of ethanol . Ethanol, like mannitol, was found to induce HSP 12 protein synthesis . However, yeast grown in both ethanol and mannitol showed a decreased HSP 12 response compared with yeast grown in the presence of either osmolyte alone. Gene, 2000 Feb 8, 243(1-2), 37 - 45 Nob1p, a new essential protein, associates with the 26S proteasome of growing saccharomyces cerevisiae cells; Tone Y et al.; Nob1p, which interacts with Nin1p/Rpn12, a subunit of the 19S regulatory particle (RP) of the yeast 26S proteasome, has been identified by two-hybrid screening . NOB1 was found to be an essential gene, encoding a protein of 459 amino acid residues . Nob1p was detected in growing cells but not in cells in the stationary phase . During the transition to the stationary phase, Nob1p was degraded, at least in part, by the 26S proteasome . Nob1p was found only in proteasomal fractions in a glycerol gradient centrifugation profile and immuno-coprecipitated with Rpt1, which is an ATPase component of the yeast proteasomes . These results suggest that association of Nob1p with the proteasomes is essential for the function of the proteasomes in growing cells. FEBS Lett, 2000 Feb 11, 467(2-3), 311 - 5 The Saccharomyces cerevisiae DNA damage checkpoint is required for efficient repair of double strand breaks by non-homologous end joining; de la Torre-Ruiz M et al.; In this work we report that the Saccharomyces cerevisiae RAD9, RAD24, RAD17, MEC1, MEC3 and RAD53 checkpoint genes are required for efficient non-homologous end joining (NHEJ) . RAD9 and RAD24 function additionally in this process . Defective NHEJ in rad9Delta-rad24Delta, but not yku80Delta cells, is only partially rescued by imposing G1 or G2/M delays . Thus, checkpoint functions other than transient cell cycle delays may be required for normal levels of NHEJ . Epistasis analysis also indicated that YKU80 and RAD9/RAD24 function in the same pathway for repair of lesions caused by MMS and gamma-irradiation . Unlike NHEJ, the checkpoint pathway is not required for efficient site-specific integration of plasmid DNA into the yeast genome, which is RAD52-dependent, but RAD51-independent. Mol Microbiol, 2000 Feb, 35(3), 477 - 89 beta-1,6-Glucan synthesis in Saccharomyces cerevisiae; Shahinian S et al.; beta-1,6-Glucan is an essential fungal-specific component of the Saccharomyces cerevisiae cell wall that interconnects all other wall components into a lattice . Considerable biochemical and genetic effort has been directed at the identification and characterization of the steps involved in its biosynthesis . Structural studies show that the polymer plays a central role in wall structure, attaching mannoproteins via their glycosylphosphatidylinositol (GPI) glycan remnant to beta-1,3-glucan and chitin . Genetic approaches have identified genes that upon disruption result in beta-1,6-glucan defects of varying severity, often with reduced growth or lethali