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| 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 lethality . These gene products have been localized throughout the secretory pathway and at the cell surface, suggesting a possible biosynthetic route . Current structural and genetic data have therefore allowed the development of models to predict biosynthetic events . Based on knowledge of beta-1,3-glucan and chitin synthesis, it is likely that the bulk of beta-1,6-glucan polymer synthesis occurs at the cell surface, but requires key prior intracellular events . However, the activity of most of the identified gene products remain unknown, making it unclear to what extent and how directly they contribute to the synthesis of this polymer . With the recent availability of new tools, reagents and methods (including genomics), the field is poised for a convergence of biochemical and genetic methods to identify and characterize the biochemical steps in the synthesis of this polymer. Eur J Biochem, 2000 Feb, 267(4), 1075 - 82 Contribution of Are1p and Are2p to steryl ester synthesis in the yeast Saccharomyces cerevisiae; Zweytick D et al.; In the yeast Saccharomyces cerevisiae, two acyl-CoA:sterol acyltransferases (ASATs) that catalyze the synthesis of steryl esters have been identified, namely Are2p (Sat1p) and Are1p (Sat2p) . Deletion of either ARE1 or ARE2 has no effect on cell viability, and are1are2 double mutants grow in a similar manner to wild-type despite the complete lack of cellular ASAT activity and steryl ester formation {Yang, H., Bard, M., Bruner, D . A., Gleeson, A., Deckelbaum, R . J., Aljinovic, G., Pohl, T . M., Rothstein, R . & Sturley, S . L . (1996) Science 272, 1353-1356; Yu, C., Kennedy, J., Chang, C . C . Y . & Rothblatt, J . A . (1996) J . Biol . Chem . 271, 24157-24163} . Here we show that both Are2p and Are1p reside in the endoplasmic reticulum as demonstrated by measuring ASAT activity in subcellular fractions of are1 and are2 deletion strains . This localization was confirmed by fluorescence microscopy using hybrid proteins of Are2p and Are1p fused to green fluorescent protein (GFP) . Lipid analysis of are1 and are2 deletion strains revealed that Are2p and Are1p utilize sterol substrates in vivo with different efficiency; Are2p has a significant preference for ergosterol as a substrate, whereas Are1p esterifies sterol precursors, mainly lanosterol, as well as ergosterol . The specificity towards fatty acids is similar for both isoenzymes . The lack of steryl esters in are1are2 mutant cells is largely compensated by an increased level of free sterols . Nevertheless, terbinafine, an inhibitor of ergosterol biosynthesis, inhibits growth of are1are2 cells more efficiently than growth of wild-type . In a growth competition experiment are1are2 cells grow more slowly than wild-type after several rounds of cultivation, suggesting that Are1p and Are2p or steryl esters, the product formed by these two enzymes, are more important in the natural environment than under laboratory conditions. Yeast, 2000 Mar 15, 16(4), 377 - 86 Disruption of six novel genes from chromosome VII of Saccharomyces cerevisiae reveals one essential gene and one gene which affects the growth rate; Fiori A et al.; Six ORFs of unknown function located on chromosome VII of Saccharomyces cerevisiae were disrupted in two different genetic backgrounds, and the phenotype of the generated mutants was analysed . Disruptions of ORFs YGR256w, YGR272c, YGR273c, YGR275w and YGR276c were carried out using the disruption marker kanMX4 flanked by short homology regions, whereas ORF YGR255c was inactivated with a long flanking homology (LFH) disruption cassette (Wach et al., 1994) . Tetrad analysis of the heterozygous disruptants revealed that ORF YGR255c, previously identified as COQ6 and encoding a protein involved in the biosynthesis of coenzime Q (Tzagoloff and Dieckmann, 1990), is an essential gene . The same analysis also revealed that sporulation of the ygr272cDelta heterozygous diploid produced two small colonies per ascus that were also G418-resistant, indicating that the inactivation of ORF YGR272c could result in a slower growth rate . This result was confirmed by growth tests of the haploid disruptants and by complementation of the phenotype after transformation with a plasmid carrying the cognate gene . No phenotypes could be associated to the inactivation of ORFs YGR256w, YGR273c, YGR275w and YGR276c . Two of these genes have recently been further characterized: ORF YGR255w, renamed RTT102, encodes a regulator of the Ty1-element transposition, whereas ORF YGR276c was found to encode the 70 kDa RNase H activity and was renamed RNH70 (Frank et al., 1999) . Yeast, 2000 Mar 15, 16(4), 365 - 75 Disruption and phenotypic analysis of six open reading frames from the left arm of Saccharomyces cerevisiae chromosome VII; Lillo JA et al.; Six open reading frames (ORFs) from Saccharomyces cerevisiae chromosome VII were deleted using the kanMX4 module and the long-flanking homology-PCR replacement strategy in at least two different backgrounds . Among these ORFs, two of them (YGL100w and YGL094c) are now known genes which encode well-characterized proteins (Seh1p, a nuclear pore protein, and Pan2p, a component of Pab1p-stimulated poly(A) ribonuclease, respectively) . The other four ORFs (YGL101w, YGL099w, YGL098w and YGL096w) code for proteins of unknown function, although the protein encoded by YGL101w has a strong similarity to the hypothetical protein Ybr242p . Gene disruptions were performed in diploid cells using the KanMX4 cassette, and the geneticin (G418)-resistant transformants were checked by PCR . Tetrad analysis of heterozygous deletant strains revealed that YGL098w is an essential gene for vegetative growth in three backgrounds, whereas the other five genes are non-essential, although we have found some phenotypes in one of them . YGL099wDelta strain did not grow at all at 15 degrees C and showed a highly impaired sporulation and a significantly lower mating efficiency . The other three deletants did not reveal any significant differences with respect to their parental strains in our basic phenotypic tests . Yeast, 2000 Mar 15, 16(4), 353 - 63 Identification of functionally important regions of the Saccharomyces cerevisiae mitochondrial translational activator Cbs1p; Krause-Buchholz U et al.; Translation of cytochrome b mRNA in yeast mitochondria requires activation by the nuclear-encoded Cbs1p . According to the current model, Cbs1p tethers cytochrome b mRNA to the inner mitochondrial membrane via interaction with the 5'-untranslated leader . Cbs1p is predicted to be a hydrophilic protein with two hydrophobic segments near the carboxyl-terminal end, which are both too short to span the membrane . Nevertheless Cbs1p is tightly associated with the mitochondrial membrane, as shown by its behaviour in extraction experiments with taurodeoxycholate . In an attempt to define functionally important regions of Cbs1p, we created a number of mutant alleles by random and directed mutagenesis . We report that a Cbs1p mutant protein lacking the mitochondrial presequence is still able to complement a Deltacbs1 strain, suggesting that the presequence does not contain essential mitochondrial targeting information . Mutations in a cluster of positively charged amino acids at the extremeC-terminus have no effect on Cbs1p function, but removal of this segment severely impairs Cbs1p function . Truncation of 12 or more amino acids from the C-terminus results in a completely defective protein . We further show that both short hydrophobic regions are essential for Cbs1p function, although membrane association is observed even in the absence of these regions . Biol Cell, 1999 Dec, 91(9), 649 - 63 The Saccharomyces cerevisiae Cdc14 phosphatase is implicated in the structural organization of the nucleolus; de Almeida A et al.; Cdc14, a dual-specificity protein phosphatase, has been previously implicated in triggering exit from mitosis in the yeast Saccharomyces cerevisiae . Using immunofluorescence microscopy and immunogold labeling, we demonstrate that a functional HA-tagged version of the phosphatase Cdc14 localizes to the nucleolus . Moreover, Cdc14-HA co-localized with the nucleolar NOP2 and GAR1 proteins . By immunofluorescence, Cdc14-HA was found in the nucleolus during most of the mitotic cell cycle, except during anaphase-telophase when it redistributed along the mitotic spindle . While this work was in progress, the same pattern of Cdc14 localization was described by others (Visintin et al, Nature 398 (1999) 818) . Constitutive overexpression of CDC14 was toxic and led to cell cycle arrest of cells, mainly in G1 . This correlated with the appearance of abnormal nuclear structures . A genetic search for suppressors of the lethality associated with CDC14 overexpression identified YJL076W . Because overproduction of Yj1076w buffered the toxic effect of Cdc14 overproduction, this suggested that it might be a substrate of Cdc14 . This has indeed been found to be the case by others who recently described Yj1076w/Netl as a nucleolar protein that physically associates with Cdc14 (Shou et al, Cell 97 (1999) 233) . The present data confirm several recently uncovered aspects of the regulation of Cdc14 localization and activity and suggest that the level of expression of CDC14 influences the structural organization of the nucleolus. Cancer Res, 2000 Jan 15, 60(2), 328 - 33 Differential toxicities of anticancer agents among DNA repair and checkpoint mutants of Saccharomyces cerevisiae; Simon JA et al.; Most cytotoxic anticancer agents damage DNA directly, interfere with DNA metabolism or chromosome segregation, and are particularly toxic in dividing cells . Although a considerable amount of information on the mechanisms of action of these agents is available, the molecular bases for selective tumor cell killing by chemotherapy are largely unknown . Many genetic alterations found in sporadic and hereditary cancers affect functions in DNA repair and cell cycle control and result in sensitivity to DNA damaging agents . We have therefore set out to determine the effects of these cancer mutations on sensitivity or resistance to various chemotherapeutic agents . Because most of the affected genes are well conserved among eukaryotes, we have carried out a comprehensive analysis of a panel of isogenic yeast strains, each defective in a particular DNA repair or cell cycle checkpoint function, for sensitivity to the Food and Drug Administration-approved cytotoxic anticancer agents . Widely different toxicity profiles were observed for 23 agents and X-rays, indicating that the type of DNA repair and cell cycle checkpoint mutations in individual tumors could strongly influence the outcome of a particular chemotherapeutic regimen. Acta Crystallogr D Biol Crystallogr, 2000 Feb, 56 ( Pt 2), 229 - 31 Crystallization and preliminary X-ray diffraction analysis of the Saccharomyces cerevisiae ran-binding protein Mog1p; Baker RP et al.; Mog1p binds the Ras-family GTPase Ran/Gsp1p, which has a central role in nucleocytoplasmic transport and cell-cycle progression . Overexpression of MOG1 is able to suppress temperature-sensitive gsp1 mutants in yeast; Deltamog1 null mutants display temperature-sensitive defects in nuclear trafficking . Orthorhombic crystals of bacterially expressed Mog1p that diffract to beyond 2 A resolution using synchrotron radiation have been obtained . The crystals have P2(1)2(1)2(1) symmetry, with unit-cell parameters a = 39.67, b = 51.96, c = 112.23 A, a Matthews coefficient of 2.44 A(3) Da(-1), an estimated solvent content of 49.5% and one chain in the asymmetric unit. Nucleic Acids Res, 2000 Mar 1, 28(5), 1114 - 9 Excision repair at the level of the nucleotide in the upstream control region, the coding sequence and in the region where transcription terminates of the Saccharomyces cerevisiae MFA2 gene and the role of RAD26; Teng Y et al.; RAD26, the yeast homologue of human CSB, has an essential role in transcription-coupled repair (TCR) . We have mapped the requisite of Rad26 for nucleotide excision repair (NER) within the different regions of the yeast Saccharomyces cerevisiae MFA2 gene at nucleotide resolution . Our results show that Rad26 is dispensable for enhanced NER in both the MFA2 upstream promoter, except in the TATA box region, and for enhanced NER in both strands of the active gene at a site close to the transcription termination region . As expected, it is not needed for repair of regions downstream of where transcription terminates . However, it is required for TCR in the transcription initiation and elongation regions . Our data support the hypothesis that Rad26 is required for the interchange between holo-TFIIH and a putative repairosome containing core TFIIH and other NER proteins . Close to the end of transcription, hotspots for the repair of CPDs in both the transcribed strand and the non-transcribed strand occur . This enhanced repair is independent of Rad26 . Hence, TFIIH may take a form favourable for forming a repairosome without Rad26 assistance; here the organisation of the DNA during the termination of transcription may facilitate access of a repair complex to enable enhanced repair of both strands. Folia Microbiol (Praha), 1999, 44(3), 299 - 305 Development of papillae on colonies of two isopolyauxotrophic strains of Saccharomyces cerevisiae allelic in RAD6 during adenine starvation; Rojas Gil AP et al.; Papilla formation on colonies of two isopolyauxotrophic strains (ade2 his3 leu2 trp1 ura3) allelic in RAD6 was compared in order to find proper conditions for selecting mutants of Saccharomyces cerevisiae with altered starvation-induced mutability . The most promising for this purpose appeared to be culturing low numbers of colonies on suboptimal plates with a growth-limiting amount of adenine at 28 degrees C for 20 d . Inactivation of the RAD6 gene which suppresses the level of starvation-associated mutagenesis markedly enhanced papilla formation under these conditions . Formation of almost all papillae on 20-d-old colonies of BJC3 was caused by mutation . Most of the papillae (75%) were white Ade+ revertants . Three groups of these papillae were distinguished (Ade+, Ade+Rad6+ and Ade+Trp+) . Both, Ade+Rad6+ and Ade+Trp+ double reversions were very probably caused by a suppressor mutation . The less frequent red papillae had the same auxotrophic markers and UV sensitivity as BJC3 but their outgrowth in liquid media was greater . It appears that creation of these papillae is caused by mutation affecting the cell response to growth limitation by low concentrations of adenine. Folia Microbiol (Praha), 1999, 44(3), 289 - 94 Life-cycle-dependent changes of aspartate carbamoyltransferase localization in membranes of Saccharomyces cerevisiae--centrifugal elutriation and ultracytochemical study; Vorisek J et al.; Exponential culture of a Saccharomyces cerevisiae strain with overexpressed aspartate carbamoyltransferase activity (ACTase) was chilled in ice and fractionated by centrifugal elutriation to several cell populations of increasing cell mass . The enzyme activity which belongs to the pyrimidine biosynthesis pathway, was detected in situ by a specific ultracytochemical reaction: the ACTase byproduct, monophosphate, was precipitated by cerium ions to cerium phosphate . During the outgrowth of nonbudding daughter cells (zero cells) the label appeared first in membranes of nuclear envelope and of mitochondria . In larger zero cells, this label appeared also in the endoplasmic reticulum, microvesicles and plasmalemma . In budding mother cells, the label was conspicuous in the whole cell-membrane complex . In most aged cells the ACTase activity was not detectable . The presence of ACTase activity in membranes of compartments conveying glycoproteins via the secretory pathway remains to be explained . To confirm the in situ detection of ACTase activity in membranes, we assayed the enzyme activity in both the 10,000 g sediment and supernatant prepared from yeast homogenate precentrifuged at 3000 g . From 23 to 43% of ACTase activity was detected in the sediments including membranes of wild-type and ACTase-overexpressing strains. J Biol Chem, 2000 Feb 11, 275(6), 4267 - 77 The accumulation of Man(6)GlcNAc(2)-PP-dolichol in the Saccharomyces cerevisiae Deltaalg9 mutant reveals a regulatory role for the Alg3p alpha1,3-Man middle-arm addition in downstream oligosaccharide-lipid and glycoprotein glycan processing; Cipollo JF et al.; N-Glycans in nearly all eukaryotes are derived by transfer of a precursor Glc(3)Man(9)GlcNAc(2) from dolichol (Dol) to consensus Asn residues in nascent proteins in the endoplasmic reticulum . The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide-lipid properly, and the alg9 mutant, accumulates Man(6)GlcNAc(2)-PP-Dol . High-field (1)H NMR and methylation analyses of Man(6)GlcNAc(2) released with peptide-N-glycosidase F from invertase secreted by Deltaalg9 yeast showed its structure to be Manalpha1,2Manalpha1,2Manalpha1, 3(Manalpha1,3Manalpha1,6)-Manbeta1,4GlcNAcbeta1, 4GlcNAcalpha/beta, confirming the addition of the alpha1,3-linked Man to Man(5)GlcNAc(2)-PP-Dol prior to the addition of the final upper-arm alpha1,6-linked Man . This Man(6)GlcNAc(2) is the endoglycosidase H-sensitive product of the Alg3p step . The Deltaalg9 Hex(7-10)GlcNAc(2) elongation intermediates were released from invertase and similarly analyzed . When compared with alg3 sec18 and wild-type core mannans, Deltaalg9 N-glycans reveal a regulatory role for the Alg3p-dependent alpha1,3-linked Man in subsequent oligosaccharide-lipid and glycoprotein glycan maturation . The presence of this Man appears to provide structural information potentiating the downstream action of the endoplasmic reticulum glucosyltransferases Alg6p, Alg8p and Alg10p, glucosidases Gls1p and Gls2p, and the Golgi Och1p outerchain alpha1,6-Man branch-initiating mannosyltransferase. Mol Gen Genet, 2000 Jan, 262(6), 1113 - 22 Multiple copies of MRG19 suppress transcription of the GAL1 promoter in a GAL80-dependent manner in Saccharomyces cerevisiae; Kabir MA et al.; A plasmid clone that suppresses galactose toxicity in a gal7 yeast strain has been isolated from a multicopy genomic DNA library . Molecular analysis revealed that the region responsible for the suppression of galactose toxicity corresponds to the ORF YPR030w, which was named MRG19 . A CEN-based plasmid carrying the above ORF was unable to suppress the toxicity . Galactokinase activity was substantially reduced in cell extracts obtained from transformants bearing multiple copies of MRG19 . Multiple copies of MRG19 were also able to suppress galactokinase expression driven by the CYC1 promoter but not the TEF1 promoter . Multiple copies of MRG19 could not suppress GAL1-driven galactokinase expression in a gal80 strain . However, MRG19-mediated suppression of CYC1-driven galactokinase expression was independent of GAL80 function . These results imply that multiple copies of MRG19 suppress galactokinase expression probably at the level of transcription . In agreement with this idea, multiple copies of MRG19 also suppress beta-galactosidase expression driven by the GAL1 promoter in a GAL80-dependent manner . Disruption of MRG19 leads to an increase in the cell density at stationary phase in synthetic complete medium . MRG19 encodes a previously uncharacterised 124-kDa protein that shows no sequence homology to any known proteins. Mol Gen Genet, 2000 Jan, 262(6), 1081 - 92 Functional analysis of RRD1 (YIL153w) and RRD2 (YPL152w), which encode two putative activators of the phosphotyrosyl phosphatase activity of PP2A in Saccharomyces cerevisiae; Rempola B et al.; In the context of the cooperative project for functional analysis of novel genes uncovered during the systematic sequencing of the Saccharomyces cerevisiae genome, we deleted two paralogous ORFs: YIL153w and YPL152w . Based on the resulting phenotypes, the corresponding genes were named RRD1 and RRD2, respectively . Rrd proteins show significant similarity to the human phosphotyrosyl phosphatase activator (PTPA) . Both single mutants, rrd1delta and rrd2delta, were viable . Deletion of RRD1 caused pleiotropic phenotypes under a wide range of conditions, including sensitivity to Ca2+, vanadate, ketoconazole, cycloheximide and Calcofluor white, and resistance to caffeine and rapamycin . The only phenotypes found for rrd2delta - resistance to caffeine and rapamycin - were weaker than the corresponding phenotypes of rrd1delta . The double mutant rrd1,2delta was inviable on rich glucose medium, but could grow in the presence of an osmotic stabilizer . The rrd1,2delta mutant was partially rescued by inactivation of HOG1 or PBS2, suggesting an interaction between the RRD genes and the Hog1p signal transduction pathway . Introduction of slt2delta into the rrd1,2delta background improved the growth of rrd1,2delta on sorbitol-containing medium, indicating that the Rrd proteins also interact with the Slt2p/Mpk1p signaling pathway . Suppression of the lethal phenotype of the rrd1,2delta mutant by overexpression of PPH22 suggested that the products of the RRD genes function positively with catalytic subunits of PP2A . The synthetic lethality was also suppressed by the "viable" allele (SSD1-v1) of the SSD1 gene. Mol Gen Genet, 2000 Jan, 262(6), 1036 - 46 In vivo analysis of mutated initiation codons in the mitochondrial COX2 gene of Saccharomyces cerevisiae fused to the reporter gene ARG8m reveals lack of downstream reinitiation; Bonnefoy N et al.; To examine normal and aberrant translation initiation in Saccharomyces cerevisiae mitochondria, we fused the synthetic mitochondrial reporter gene ARG8m to codon 91 of the COX2 coding sequence and inserted the chimeric gene into mitochondrial DNA (mtDNA) . Translation of the cox2(1-91)::ARG8m mRNA yielded a fusion protein precursor that was processed to yield wild-type Arg8p . Thus mitochondrial translation could be monitored by the ability of mutant chimeric genes to complement a nuclear arg8 mutation . As expected, translation of the cox2(1-91)::ARG8m mRNA was dependent on the COX2 mRNA-specific activator PET111 . We tested the ability of six triplets to function as initiation codons in both the cox2(1-91)::ARG8m reporter mRNA and the otherwise wild-type COX2 mRNA . Substitution of AUC, CCC or AAA for the initiation codon abolished detectable translation of both mRNAs, even when PET111 activity was increased . The failure of these mutant cox2(1-91)::ARG8m genes to yield Arg8p demonstrates that initiation at downstream AUG codons, such as COX2 codon 14, does not occur even when normal initiation is blocked . Three mutant triplets at the site of the initiation codon supported detectable translation, with efficiencies decreasing in the order GUG, AUU, AUA . Increased PET111 activity enhanced initiation at AUU and AUA codons . Comparisons of expression, at the level of accumulated product, of cox2(1-91)::ARG8m and COX2 carrying these mutant initiation codons revealed that very low-efficiency translation can provide enough Cox2p to sustain significant respiratory growth, presumably because Cox2p is efficiently assembled into stable cytochrome oxidase complexes. J Mol Biol, 2000 Feb 11, 296(1), 7 - 17 Domain III of Saccharomyces cerevisiae 25 S ribosomal RNA: its role in binding of ribosomal protein L25 and 60 S subunit formation; van Beekvelt CA et al.; Domain III of Saccharomyces cerevisiae 25 S rRNA contains the recognition site for the primary rRNA-binding ribosomal protein L25, which belongs to the functionally conserved EL23/L25 family of ribosomal proteins . The EL23/L25 binding region is very complex, consisting of several irregular helices held together by long-distance secondary and tertiary interactions . Moreover, it contains the eukaryote-specific V9 (D7a) expansion segment . Functional characterisation of the structural elements of this site by a detailed in vitro and in vivo mutational analysis indicates the presence of two separate regions that are directly involved in L25 binding . In particular, mutation of either of two conserved nucleotides in the loop of helix 49 significantly reduces in vitro L25 binding, thus strongly supporting their role as attachment sites for the r-protein . Two other helices appear to be primarily required for the correct folding of the binding site . Mutations that abolish in vitro binding of L25 block accumulation of 25 S rRNA in vivo because they stall pre-rRNA processing at the level of its immediate precursor, the 27 S(B) pre-rRNA . Surprisingly, several mutations that do not significantly affect L25 binding in vitro cause the same lethal defect in 27 S(B) pre-rRNA processing . Deletion of the V9 expansion segment also leads to under-accumulation of mature 25 S rRNA and a twofold reduction in growth rate . We conclude that an intact domain III, including the V9 expansion segment, is essential for normal processing and assembly of 25 S rRNA . J Mol Biol, 2000 Jan 28, 295(4), 755 - 65 Selection of amino acid substitutions restoring activity of HIV-1 integrase mutated in its catalytic site using the yeast Saccharomyces cerevisiae; Parissi V et al.; The integration of proviral DNA into the genome of the host cell is an essential step in the replication of retroviruses . This reaction is catalyzed by a viral-encoded enzyme, the integrase (IN) . We have previously shown that human immunodeficiency virus type 1 (HIV-1) IN causes a lethal effect when expressed in yeast cells . This system, called yeast lethal assay, was used as a tool to study IN activity in a cellular context . The yeast lethal assay allowed the selection and characterization of mutations affecting both the lethal phenotype and the in vitro IN activities.IN mutants were produced by random PCR mutagenesis in an IN gene bearing the inactivating D116A mutation in the catalytic site . The corresponding D116A substituted IN does not lead to lethality in yeast . Subsequent selection of mutants able to restore the lethal effect of IN was carried out using the yeast lethal assay . We isolated three mutants presenting a restored phenotype . The mutated IN genes were sequenced and the corresponding proteins were purified to characterize their in vitro activities . The three mutants presented restoration of the in vitro strand transfer activity, while 3' processing was only partially restored.The three mutants differ from D116A IN by at least one amino acid substitution located in the N-terminal domain of the protein, outside of the active site . These new mutated HIV-1 INs may therefore allow a better understanding of the N-terminal domain function in the integration reaction . In addition, these results support our hypothesis that explains the lethal effect as a consequence of the nuclear damage caused by wild-type IN in yeast cells . These data also indicate that the yeast lethal assay can be used as a tool to study the retroviral integration mechanism in a cellular context and to select specific inhibitors . Mutat Res, 1999 Dec 16, 431(1), 155 - 65 REV3 is required for spontaneous but not methylation damage-induced mutagenesis of Saccharomyces cerevisiae cells lacking O6-methylguanine DNA methyltransferase; Xiao W et al.; O6-methylguanine (O6-MeG) DNA methyltransferase (MTase) removes the methyl group from a DNA lesion and directly restores DNA structure . It has been shown previously that bacterial and yeast cells lacking such MTase activity are not only sensitive to killing and mutagenesis by DNA methylating agents, but also exhibit an increased spontaneous mutation rate . In order to understand molecular mechanisms of endogenous DNA alkylation damage and its effects on mutagenesis, we determined the spontaneous mutational spectra of the SUP4-o gene in various Saccharomyces cerevisiae strains . To our surprise, the mgt1 mutant deficient in DNA repair MTase activity exhibited a significant increase in G:C-->C:G transversions instead of the expected G:C-->A:T transition . Its mutational distribution strongly resembles that of the rad52 mutant defective in DNA recombinational repair . The rad52 mutational spectrum has been shown to be dependent on a mutagenesis pathway mediated by REV3 . We demonstrate here that the mgt1 mutational spectrum is also REV3-dependent and that the rev3 deletion offsets the increase of the spontaneous mutation rate seen in the mgt1 strains . These results indicate that the eukaryotic mutagenesis pathway is directly involved in cellular processing of endogenous DNA alkylation damage possibly by the translesion bypass of lesions at the cost of G:C-->C:G transversion mutations . However, the rev3 deletion does not affect methylation damage-induced killing and mutagenesis of the mgt1 mutant, suggesting that endogenous alkyl lesions may be different from O6-MeG. Nat Struct Biol, 2000 Feb, 7(2), 113 - 7 The aspartic proteinase from Saccharomyces cerevisiae folds its own inhibitor into a helix; Li M et al.; Aspartic proteinase A from yeast is specifically and potently inhibited by a small protein called IA3 from Saccharomyces cerevisiae . Although this inhibitor consists of 68 residues, we show that the inhibitory activity resides within the N-terminal half of the molecule . Structures solved at 2.2 and 1.8 A, respectively, for complexes of proteinase A with full-length IA3 and with a truncated form consisting only of residues 2-34, reveal an unprecedented mode of inhibitor-enzyme interactions . Neither form of the free inhibitor has detectable intrinsic secondary structure in solution . However, upon contact with the enzyme, residues 2-32 become ordered and adopt a near-perfect alpha-helical conformation . Thus, the proteinase acts as a folding template, stabilizing the helical conformation in the inhibitor, which results in the potent and specific blockage of the proteolytic activity. Proc Natl Acad Sci U S A, 2000 Feb 1, 97(3), 1107 - 12 Pho86p, an endoplasmic reticulum (ER) resident protein in Saccharomyces cerevisiae, is required for ER exit of the high-affinity phosphate transporter Pho84p; Lau WT et al.; In the budding yeast Saccharomyces cerevisiae, PHO84 and PHO86 are among the genes that are most highly induced in response to phosphate starvation . They are essential for growth when phosphate is limiting, and they function in the high-affinity phosphate uptake system . PHO84 encodes a high-affinity phosphate transporter, and mutations in PHO86 cause many of the same phenotypes as mutations in PHO84, including a phosphate uptake defect and constitutive expression of the secreted acid phosphatase, Pho5p . Here, we show that the subcellular localization of Pho84p is regulated in response to extracellular phosphate levels; it is localized to the plasma membrane in low-phosphate medium but quickly endocytosed and transported to the vacuole upon addition of phosphate to the medium . Moreover, Pho84p is localized to the endoplasmic reticulum (ER) and fails to be targeted to the plasma membrane in the absence of Pho86p . Utilizing an in vitro vesicle budding assay, we demonstrate that Pho86p is required for packaging of Pho84p into COPII vesicles . Pho86p is an ER resident protein, which itself is not transported out of the ER . Interestingly, the requirement of Pho86p for ER exit is specific to Pho84p, because other members of the hexose transporter family to which Pho84 belongs are not mislocalized in the absence of Pho86p. Genetics, 2000 Feb, 154(2), 587 - 98 Telomere structure regulates the heritability of repressed subtelomeric chromatin in Saccharomyces cerevisiae; Park Y et al.; Telomeres, the protein-DNA structures present at the termini of linear chromosomes, are capable of conferring a reversible repression of Pol II- and Pol III-transcribed genes positioned in adjacent subtelomeric regions . This phenomenon, termed telomeric silencing, is likely to be the consequence of a more global telomere position effect at the level of chromatin structure . To understand the role of telomere structure in this position effect, we have developed an assay to distinguish between the heritability of transcriptionally repressed and derepressed states in yeast . We have previously demonstrated that an elongated telomeric tract leads to hyperrepression of telomere-adjacent genes . We show here that the predominant effect of elongated telomeres is to increase the inheritance of the repressed state in cis . Interestingly, the presence of elongated telomeres overcomes the partial requirement of yCAF-1 in silencing . We propose that the formation of a specific telomeric structure is necessary for the heritability of repressed subtelomeric chromatin. Genetics, 2000 Feb, 154(2), 573 - 86 Roles for the Saccharomyces cerevisiae SDS3, CBK1 and HYM1 genes in transcriptional repression by SIN3; Dorland S et al.; The Saccharomyces cerevisiae Sin3 transcriptional repressor is part of a large multiprotein complex that includes the Rpd3 histone deacetylase . A LexA-Sin3 fusion protein represses transcription of promoters with LexA binding sites . To identify genes involved in repression by Sin3, we conducted a screen for mutations that reduce repression by LexA-Sin3 . One of the mutations identified that reduces LexA-Sin3 repression is in the RPD3 gene, consistent with the known roles of Rpd3 in transcriptional repression . Mutations in CBK1 and HYM1 reduce repression by LexA-Sin3 and also cause defects in cell separation and altered colony morphology . cbk1 and hym1 mutations affect some but not all genes regulated by SIN3 and RPD3, but the effect on transcription is much weaker . Genetic analysis suggests that CBK1 and HYM1 function in the same pathway, but this genetic pathway is separable from that of SIN3 and RPD3 . The remaining gene from this screen described in this report is SDS3, previously identified in a screen for mutations that increase silencing at HML, HMR, and telomere-linked genes, a phenotype also seen in sin3 and rpd3 mutants . Genetic analysis demonstrates that SDS3 functions in the same genetic pathway as SIN3 and RPD3, and coimmunoprecipitation experiments show that Sds3 is physically present in the Sin3 complex. Genetics, 2000 Feb, 154(2), 543 - 56 The Saccharomyces cerevisiae DNA recombination and repair functions of the RAD52 epistasis group inhibit Ty1 transposition; Rattray AJ et al.; RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies . The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or delta) elements . Here we examine the role of the cellular homologous recombination functions on Ty1 transposition . We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes . The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins . However, unincorporated Ty1 cDNA levels are markedly elevated . These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA. J Biol Chem, 2000 Feb 4, 275(5), 3593 - 602 Pex8p, an intraperoxisomal peroxin of Saccharomyces cerevisiae required for protein transport into peroxisomes binds the PTS1 receptor pex5p; Rehling P et al.; We report the characterization of ScPex8p, which is essential for peroxisomal biogenesis in Saccharomyces cerevisiae . Cells lacking Pex8p are characterized by the presence of peroxisomal membrane ghosts and mislocalization of peroxisomal matrix proteins of the PTS1 and PTS2 variety to the cytosol . Pex8p is tightly associated with the lumenal face of the peroxisomal membrane . Consistent with its intraperoxisomal localization, Pex8p contains a peroxisomal targeting signal 1, and it interacts with the PTS1 receptor Pex5p . However, the Pex5p/Pex8p association is also observed upon deletion of the PTS1 of Pex8p, suggesting that Pex8p contains a second binding site for Pex5p . The pex8Delta mutant phenotype and the observed PTS1-independent interaction with the PTS1 receptor suggest that Pex8p is involved in protein import into the peroxisomal matrix . In pex8Delta cells, the PTS1 and PTS2 receptor still associate with membrane bound components of the protein import machinery, supporting the assumption that the Pex8p function in protein translocation follows the docking event. Mol Cell Biol, 2000 Feb, 20(4), 1311 - 20 Identification of a novel element required for processing of intron-encoded box C/D small nucleolar RNAs in Saccharomyces cerevisiae; Villa T et al.; Processing of intron-encoded box C/D small nucleolar RNAs (snoRNAs) in metazoans through both the splicing-dependent and -independent pathways requires the conserved core motif formed by boxes C and D and the adjoining 5'-3'-terminal stem . By comparative analysis, we found that five out of six intron-encoded box C/D snoRNAs in yeast do not possess a canonical terminal stem . Instead, complementary regions within the flanking host intron sequences have been identified in all these cases . Here we show that these sequences are essential for processing of U18 and snR38 snoRNAs and that they compensate for the lack of a canonical terminal stem . We also show that the Rnt1p endonuclease, previously shown to be required for the processing of many snoRNAs encoded by monocistronic or polycistronic transcriptional units, is not required for U18 processing . Our results suggest a role of the complementary sequences in the early recognition of intronic snoRNA substrates and point out the importance of base pairing in favoring the communication between boxes C and D at the level of pre-snoRNA molecules for efficient assembly with snoRNP-specific factors. J Bacteriol, 2000 Feb, 182(4), 1167 - 71 Increase of external osmolarity reduces morphogenetic defects and accumulation of chitin in a gas1 mutant of Saccharomyces cerevisiae; Turchini A et al.; We have performed a physiological analysis of the effects of high-osmolarity media on gas1Delta cells . The reductions in the duplication time, number of pluribudded cells, hypersensitivity to Calcofluor and sodium dodecyl sulfate, and chitin level indicate a partial suppression of the mutant phenotype . GAS1 deletion was found to be lethal in the absence of the Bck1 and Slt2 (Mpk1) proteins of the cell integrity pathway. Yeast, 2000 Feb, 16(3), 277 - 88 Functional analysis of six novel ORFs on the left arm of chromosome XII in Saccharomyces cerevisiae reveals two essential genes, one of which is under cell-cycle control; El-Moghazy AN et al.; Six novel Open Reading Frames (ORFs) located on the left arm of chromosome XII (YLL044w, YLL042c, YLL040c, YLL038c, YLL035w and YLL034c) have been analysed using short-flanking homology (SFH) gene replacement . Sporulation and tetrad analysis showed that YLL035w and YLL034c are essential for cell growth; yll035w spores arrested after two or three cell divisions, while the majority of yll034c spores stopped growth within two cell cycles after germination . Complementation of the yll035w deletion with its cognate clone, and a promoter-substitution experiment, indicated that the promoter of YLL035w may lie within the adjacent ORF, YLL036c . Transcriptional analysis demonstrated that YLL035w is under cell-cycle regulation . Bioinformatic analyses produced significant matches between YLL034c and mammalian valosin and many other ATPases . The standard EUROFAN growth tests failed to reveal obvious phenotypes resulting from deletion of any of the four non-essential ORFs . Replacement cassettes, comprising the kanMX marker flanked by each ORF's promoter and terminator regions, were cloned into pUG7 . All the cognate clones, except for YLL040c, were generated using direct PCR products amplified from genomic DNA or using gap-repair . All clones and strains produced have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt) . Yeast, 2000 Feb, 16(3), 241 - 53 Analysis of deletion phenotypes and GFP fusions of 21 novel Saccharomyces cerevisiae open reading frames; Brachat A et al.; As part of EUROFAN (European Functional Analysis Network), we investigated 21 novel yeast open reading frames (ORFs) by growth and sporulation tests of deletion mutants . Two genes (YNL026w and YNL075w) are essential for mitotic growth and three deletion strains (ynl080c, ynl081c and ynl225c) grew with reduced rates . Two genes (YNL223w and YNL225c) were identified to be required for sporulation . In addition we also performed green fluorescent protein (GFP) tagging for localization studies . GFP labelling indicated the spindle pole body (Ynl225c-GFP) and the nucleus (Ynl075w-GFP) as the sites of action of two proteins . Ynl080c-GFP and Ynl081c-GFP fluorescence was visible in dot-shaped and elongated structures, whereas the Ynl022c-GFP signal was always found as one spot per cell, usually in the vicinity of nuclear DNA . The remaining C-terminal GFP fusions did not produce a clearly identifiable fluorescence signal . For 10 ORFs we constructed 5'-GFP fusions that were expressed from the regulatable GAL1 promoter . In all cases we observed GFP fluorescence upon induction but the localization of the fusion proteins remained difficult to determine . GFP-Ynl020c and GFP-Ynl034w strains grew only poorly on galactose, indicating a toxic effect of the overexpressed fusion proteins . In summary, we obtained a discernible GFP localization pattern in five of 20 strains investigated (25%) . A deletion phenotype was observed in seven of 21 (33%) and an overexpression phenotype in two of 10 (20%) cases . Yeast, 2000 Feb, 16(3), 207 - 17 Asparagine as a nitrogen source for improving the secretion of mouse alpha-amylase in Saccharomyces cerevisiae protease A-deficient strains; Chen DC et al.; A modified chemically defined medium was achieved by using asparagine as a nitrogen source to increase the production of secreted mouse alpha-amylase in several Saccharomyces cerevisiae protease A-deficient (pep4) strains . The specific productivity (quantity) and the 53 kDa non-glycosylated active form (quality) of mouse salivary alpha-amylase in liquid medium containing asparagine was remarkably improved compared to media containing other nitrogen sources, including ammonium sulphate, glutamic acid, arginine, casamino acids, yeast extract and peptone . Similar improvement was also observed on starch solid agar regarding the clarity and size of the halo zone formed by alpha-amylase activity . Compared with ammonium sulphate, advantages of using asparagine as the nitrogen source in liquid or solid medium included increasing the cell mass of test strains, recovering the viability of protease-deficient strains to levels similar to the wild-type strain, and increasing the copy number of the mouse alpha-amylase expression vector in test strains . In turn, these advantages apparently contributed to the increase of secretion of mouse alpha-amylase in several test strains and especially in the protease A-deficient strains . In addition to demonstrating the use of modified chemically defined medium to improve the quality and quantity of secreted mouse alpha-amylase, this study also provides a new strategy to improve the secretion of heterologous proteins in protease A deficient strains . Yeast, 2000 Feb, 16(3), 197 - 206 Cloning and characterization of the Yarrowia lipolytica squalene synthase (SQS1) gene and functional complementation of the Saccharomyces cerevisiae erg9 mutation; Merkulov S et al.; The squalene synthase (SQS) gene encodes a key regulatory enzyme, farnesyl-diphosphate farnesyltransferase (EC 2.5.1.21), in sterol biosynthesis . The SQS1 gene was isolated from a subgenomic library of the industrially important yeast Yarrowia lipolytica, using PCR-generated probes . Probes were based on conserved regions of homologues from different organisms . The complete nucleotide sequence of the coding region and the corresponding amino acid sequence were determined . The sequences showed extensive homologies with squalene synthase genes and enzymes from a number of other organisms and extreme amino acid conservation within the binding and catalytic domains . Direct cloning of a 4.3 kb genomic Y . lipolytica fragment, also comprising its own promoter and terminator sequences, into autonomously replicating plasmid YEp352 and subsequent transformation of a Saccharomyces cerevisiae mutant strain with relevant erg9: ura3-1 markers, resulted in functional complementation of these deficiencies, although Northern blot analyses did not reveal a unique full-length messenger . The availability of the Y . lipolytica SQS1 gene (GenBank Accession No . AF092497) offers prospects for metabolic engineering of the isoprenoid and sterol biosynthetic pathways . Nucleic Acids Res, 2000 Feb 15, 28(4), 974 - 81 Stp1p, Stp2p and Abf1p are involved in regulation of expression of the amino acid transporter gene BAP3 of Saccharomyces cerevisiae; de Boer M et al.; Expression of the BAP3 gene of Saccharomyces cerevisiae, encoding a branched chain amino acid permease, is induced in response to the availability of several naturally occurring amino acids in the medium . This induction is mediated via an upstream activating sequence (called UAS(aa)) in the BAP3 promoter, and dependent on Stp1p, a nuclear protein with zinc finger domains, suggesting that Stp1p is a transcription factor involved in BAP3 expression . In this paper, we show that Stp2p, a protein with considerable similarity to Stp1p, is also involved in the induction of BAP3 expression . To gain more insight into the roles of STP1 and STP2, we have overexpressed both Stp1p and Stp2p in yeast cells . Gel shift assays with the UAS(aa)as a probe show that the UAS(aa)can form two major complexes . One complex is dependent on Stp2p overexpression and the other is formed independently of STP1 or STP2, suggesting that the UAS(aa)is also bound by another factor . Here we show that the other factor is Abf1p, which binds specifically to the UAS(aa)of BAP3. Appl Microbiol Biotechnol, 1999 Dec, 53(1), 30 - 5 Enhanced sterol-acyl transferase activity promotes sterol accumulation in Saccharomyces cerevisiae; Polakowski T et al.; The sterol-acyl transferase encoded by the gene ARE2 was transcriptionally deregulated in the yeast Saccharomyces cerevisiae to understand its role in sterol storage and sterol enrichment . Our results show that sterols can indeed be enriched in yeast by enhancing the capacity of the cells to esterify sterols . ARE2 overexpression had no impact on the accumulation of the early sterols such as lanosterol, but influenced the later intermediates and the end product ergosterol . Thus an enhanced conversion of free sterols to their esterified counterparts may provide a tool to increase the overall sterol content of the yeast cell . We have previously shown that the overexpression of a truncated version of the key enzyme of the early sterol pathway, HMG-CoA reductase (HMG1), leads to an increase in the early sterols such as lanosterol and zymosterol . The simultaneous deregulation of both genes in one strain produces a cumulative effect in that both early and late sterols are enhanced . Karmellae-like structures can be detected when Are2p is overexpressed . Are2p therefore constitutes a new member of the karmellae-inducing protein family. Yeast, 2000 Jan 30, 16(2), 121 - 37 The level of cAMP-dependent protein kinase A activity strongly affects osmotolerance and osmo-instigated gene expression changes in Saccharomyces cerevisiae; Norbeck J et al.; The influence of cAMP-dependent protein kinase (PKA) on protein expression during exponential growth under osmotic stress was studied by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) . The responses of isogenic strains (tpk2Deltatpk3Delta) with either constitutively low (tpk1(w1)), regulated (TPK1) or constitutively high (TPK1bcy1Delta) PKA activity were compared . The activity of cAMP-dependent protein kinase (PKA) was shown to be a major determinant of osmotic shock tolerance . Proteins with increased expression during growth under sodium chloride stress could be grouped into three classes with respect to PKA activity, with the glycerol metabolic proteins GPD1, GPP2 and DAK1 standing out as independent of PKA . The other osmotically induced proteins displayed a variable dependence on PKA activity; fully PKA-dependent genes were TPS1 and GCY1, partly PKA-dependent genes were ENO1, TDH1, ALD3 and CTT1 . The proteins repressed by osmotic stress also fell into distinct classes of PKA-dependency . Ymr116c was PKA-independent, while Pgi1p, Sam1p, Gdh1p and Vma1p were fully PKA-dependent . Hxk2p, Pdc1p, Ssb1p, Met6p, Atp2p and Hsp60p displayed a partially PKA-dependent repression . The promotors of all induced PKA-dependent genes have STRE sites in their promotors suggestive of a mechanism acting via Msn2/4p . The mechanisms governing the expression of the other classes are unknown . From the protein expression data we conclude that a low PKA activity causes a protein expression resembling that of osmotically stressed cells, and furthermore makes cells tolerant to this type of stress . Yeast, 2000 Jan 30, 16(2), 99 - 110 A mannose-binding protein from the cell surface of flocculent Saccharomyces cerevisiae (NCIM 3528): its role in flocculation; Javadekar VS et al.; A cell surface lectin was isolated and purified to homogeneity from the cell walls of a highly flocculent strain of Saccharomyces cerevisiae (NCIM 3528) by chromatography on DEAE-cellulose, phenyl Sepharose and Sephacryl S-300 . It showed a molecular mass of 40 kDa on SDS-PAGE . It is an acidic protein with a pI of 4.0 and contains 44% hydrophobic amino acids . The N-terminal sequence up to 10 amino acid residues showed at least 70% homology with the predicted N-terminal sequence of the putative FLO1 as well as FLO5 gene products . The mannose-binding nature of the lectin was indicated by its high affinity and specificity towards the branched trisaccharide of mannose, a ligand which also inhibits the flocculation of yeast cells . Immunofluorescence studies confirmed the presence of lectin on the yeast cell surface and lectin-specific IgGs prevented flocculation of the cells . This cell surface mannose-specific lectin probably plays an important role in flocculation, with the branched trimannoside on the cell wall being the apparent carbohydrate receptor . The N-terminal sequence data gives a primary indication that the lectin could be a product of one of the FLO genes . FEMS Microbiol Rev, 2000 Jan, 24(1), 67 - 83 The role of ammonia metabolism in nitrogen catabolite repression in Saccharomyces cerevisiae; ter Schure EG et al.; Saccharomyces cerevisiae is able to use a wide variety of nitrogen sources for growth . Not all nitrogen sources support growth equally well . In order to select the best out of a large diversity of available nitrogen sources, the yeast has developed molecular mechanisms . These mechanisms consist of a sensing mechanism and a regulatory mechanism which includes induction of needed systems, and repression of systems that are not beneficial . The first step in use of most nitrogen sources is its uptake via more or less specific permeases . Hence the first level of regulation is encountered at this level . The next step is the degradation of the nitrogen source to useful building blocks via the nitrogen metabolic pathways . These pathways can be divided into routes that lead to the degradation of the nitrogen source to ammonia and glutamate, and routes that lead to the synthesis of nitrogen containing compounds in which glutamate and glutamine are used as nitrogen donor . Glutamine is synthesized out of ammonia and glutamate . The expression of the specific degradation routes is also regulated depending on the availability of a particular nitrogen source . Ammonia plays a central role as intermediate between degradative and biosynthetic pathways . It not only functions as a metabolite in metabolic reactions but is also involved in regulation of metabolic pathways at several levels . This review describes the central role of ammonia in nitrogen metabolism . This role is illustrated at the level of enzyme activity, translation and transcription. J Gen Virol, 2000 Jan, 81(Pt 1), 279 - 86 Expression of a plant virus non-structural protein in Saccharomyces cerevisiae causes membrane proliferation and altered mitochondrial morphology; Rubino L et al.; Carnation Italian ringspot tombusvirus encodes a protein, referred to as 36K, that possesses a mitochondrial targeting signal and two transmembrane segments which are thought to anchor this protein to the outer membrane of the mitochondrial envelope of infected plant cells . To determine the topology of the virus protein inserted in the cell membrane, as well as the sequence requirements for targeting and insertion, an in vivo system was set up in which this could be analysed in the absence of productive virus infection . The 36K protein was expressed in the yeast Saccharomyces cerevisiae in native form or fused to the green fluorescent protein . Using a fluorescence microscope, large green-fluorescing cytoplasmic aggregates were visible which stained red when cells were treated with the vital stain MitoTracker, which is specific for mitochondria . These aggregates were shown by electron microscopy to be composed of either mitochondria or membranes . The latter type was particularly abundant for the construct in which the green fluorescent protein was fused at the N terminus of the 36K protein . Immunoelectron microscopy demonstrated that the viral protein is present in the anomalous aggregates and Western blot analysis of protein extracts showed 36K to be resistant to alkaline, urea or salt extraction, a property of integral membrane proteins. J Cell Sci, 2000 Feb, 113 ( Pt 3), 533 - 44 Evidence for a novel pathway for the targeting of a Saccharomyces cerevisiae peroxisomal protein belonging to the isomerase/hydratase family; Karpichev IV et al.; We, and others, have identified a novel Saccharomyces cerevisiae peroxisomal protein that belongs to the isomerase/hydratase family . The protein, named Dci1p, shares 50% identity with Eci1p, a delta(3)-cis-delta(2)-trans-enoyl-CoA isomerase that acts as an auxiliary enzyme in the beta-oxidation of unsaturated fatty acids . Both of these proteins are localized to peroxisomes, and both contain motifs at their amino- and carboxyl termini that resemble peroxisome targeting signals (PTS) 1 and 2 . However, we demonstrate that the putative type 1 signaling motif is not required for the peroxisomal localization of either of these proteins . Furthermore, the correct targeting of Eci1p and Dci1p occurs in the absence of the receptors for the type 1 or type 2 peroxisome targeting pathway . Together, these data suggest a novel mechanism for the intracellular targeting of these peroxisomal proteins. J Cell Sci, 2000 Feb, 113 ( Pt 3), 521 - 32 Saccharomyces cerevisiae Arc35p works through two genetically separable calmodulin functions to regulate the actin and tubulin cytoskeletons; Schaerer-Brodbeck C et al.; Analysis of the arc35-1 mutant has revealed previously that this component of the Arp2/3 complex is involved in organization of the actin cytoskeleton . Further characterization uncovered a cell division cycle phenotype with arrest as large-budded cells . Cells with correctly positioned metaphase spindles accumulated at the restrictive temperature . The observed metaphase arrest most likely occurs by activation of the spindle assembly checkpoint, because arc35-1 was synthetically lethal with a deletion of BUB2 . Arc35p activity is required late in G(1) for its cell cycle function . Both the actin and microtubule defects of arc35-1 can be suppressed by overexpression of calmodulin . Analysis of a collection of ts cmd1 mutants for their ability to suppress the actin and/or microtubule defect revealed that the two defects observed in arc35-1 are genetically separable . These data suggest that the actin defect is probably not the cause of the microtubule defect. J Cell Sci, 2000 Feb, 113 ( Pt 3), 507 - 20 Type 1 protein phosphatase is required for maintenance of cell wall integrity, morphogenesis and cell cycle progression in Saccharomyces cerevisiae; Andrews PD et al.; GLC7 encodes the catalytic subunit of type 1 protein serine/threonine phosphatase (PP1) in the yeast Saccharomyces cerevisiae . Here we have characterized the temperature-sensitive glc7-10 allele, which displays aberrant bud morphology and an abnormal actin cytoskeleton at the restrictive temperature . At 37 degrees C glc7-10 strains accumulated a high proportion of budded cells with an unmigrated nucleus, duplicated spindle pole bodies, a short spindle, delocalized cortical actin and 2C DNA content, indicating a cell cycle block prior to the metaphase to anaphase transition . glc7-10 was suppressed by growth on high osmolarity medium and exhibited temperature-sensitive cell lysis upon hypo-osmotic stress . Pkc1p, the yeast protein kinase C homolog which is thought to regulate the Mpk1p MAP kinase pathway involved in cell wall remodelling and polarized cell growth, was found to act as a dosage suppressor of glc7-10 . Although neither activation of BCK1 (MEKK) by the dominant BCK1-20 mutation nor increased dosage of MKK1 (MEK) or MPK1 (MAP kinase) mimicked PKC1 as a glc7-10 dosage suppressor, extra copies of genes encoding upstream components of the Pkc1p pathway such as ROM2, RHO2, HCS77/WSC1/SLG1 and MID2 also suppressed glc7-10 effectively . Conversely, mpk1delta glc7-10 and bck1delta glc7-10 double mutants displayed a synthetic cell lysis defect compared with each single mutant and glc7-10 was hypersensitive to reduced PKC1 function, displaying highly aberrant morphologies and inviability even at the normally permissive temperature of 26 degrees C . Dephosphorylation by PP1 therefore functions positively to promote cell integrity, bud morphology and polarization of the actin cytoskeleton and glc7-10 cells require higher levels of Pkc1p activity to sustain these functions. EMBO J, 2000 Jan 17, 19(2), 223 - 33 Saccharomyces cerevisiae pex3p and pex19p are required for proper localization and stability of peroxisomal membrane proteins; Hettema EH et al.; The mechanisms by which peroxisomal membrane proteins (PMPs) are targeted to and inserted into membranes are unknown, as are the required components . We show that among a collection of 16 Saccharomyces cerevisiae peroxisome biogenesis (pex) mutants, two mutants, pex3Delta and pex19Delta, completely lack detectable peroxisomal membrane structures and mislocalize their PMPs to the cytosol where they are rapidly degraded . The other pexDelta mutants contain membrane structures that are properly inherited during vegetative growth and that house multiple PMPs . Even Pex15p requires Pex3p and Pex19p for localization to peroxisomal membranes . This PMP was previously hypothesized to travel via the endoplasmic reticulum (ER) to peroxisomes . We provide evidence that ER-accumulated Pex15p is not a sorting intermediate on its way to peroxisomes . Our results show that Pex3p and Pex19p are required for the proper localization of all PMPs tested, including Pex15p, whereas the other Pex proteins might only be required for targeting/import of matrix proteins. Neurobiol Aging, 1999 Sep-Oct, 20(5), 471 - 8 The RAS genes: a homeostatic device in Saccharomyces cerevisiae longevity; Jazwinski SM; The genetic analysis of the yeast replicative life span has revealed the importance of metabolic control and resistance to stress . It has also illuminated the pivotal role in determining longevity that the RAS genes play by the maintenance of homeostasis . This role appears to be performed by the coordination of a variety of cellular processes . Metabolic control seems to occupy a central position among these cellular processes that include stress resistance . Some of the features of metabolic control in yeast resemble the effects of the daf pathway for adult longevity in Caenorhabditis elegans and the metabolic consequences of selection for extended longevity in Drosophila melanogaster, as well as some of the features of caloric restriction in mammals . The distinction between dividing and nondividing cells is proposed to be less important for the aging process than generally believed because these cell types are part of a metabolic continuum in which the total metabolic capacity determines life span . As a consequence, the study of yeast aging may be helpful in understanding processes occurring in the aging brain. Nucleic Acids Res, 2000 Feb 1, 28(3), 736 - 43 The ribosomal P-proteins of the medfly Ceratitis capitata form a heterogeneous stalk structure interacting with the endogenous P-proteins, in conditional P0-null strains of the yeast Saccharomyces cerevisiae; Gagou ME et al.; The genes encoding the ribosomal P-proteins CcP0, CcP1 and CcP2 of Ceratitis capitata were expressed in the conditional P0-null strains W303dGP0 and D67dGP0 of Saccharomyces cerevisiae, the ribosomes of which contain either standard amounts or are totally deprived of the P1/P2 proteins, respectively . The presence of the CcP0 protein restored cell viability but reduced the growth rate . In the W303CcP0 strain, all four acidic yeast proteins were found on the ribosomes, but in notably less quantity, while a preferable binding of the YP1alpha/YP2betapair was established . In the absence of the endogenous P1/P2 proteins in the D67CcP0 strain, the complementation capacity of the CcP0 protein was considerably reduced . The simultaneous expression of the three medfly genes resulted in alterations of the stalk composition: both the CcP1 and CcP2 proteins were found on the particles substituting the YP1alphaand YP2alpha proteins, respectively, but their presence did not alter the growth rate, except in the case of the YP1alpha/betadefective strain, where a helping effect on the binding of the YP2alphaand YP2betaproteins on the ribo-somes was confirmed . Therefore, the medfly ribosomal P-proteins complement the yeast P-protein deficient strains forming an heterogeneous ribosomal stalk, which, however, is not functionally equivalent to the endogenous one. Mol Biol Cell, 2000 Jan, 11(1), 183 - 99 Characterization of alcohol-induced filamentous growth in Saccharomyces cerevisiae; Lorenz MC et al.; Diploid cells of the budding yeast Saccharomyces cerevisiae starved for nitrogen differentiate into a filamentous growth form . Poor carbon sources such as starches can also stimulate filamentation, whereas haploid cells undergo a similar invasive growth response in rich medium . Previous work has demonstrated a role for various alcohols, by-products of amino acid metabolism, in altering cellular morphology . We found that several alcohols, notably isoamyl alcohol and 1-butanol, stimulate filamentous growth in haploid cells in which this differentiation is normally repressed . Butanol also induces cell elongation and changes in budding pattern, leading to a pseudohyphal morphology, even in liquid medium . The filamentous colony morphology and cell elongation require elements of the pheromone-responsive MAPK cascade and TEC1, whereas components of the nutrient-sensing machinery, such as MEP2, GPA2, and GPR1, do not affect this phenomenon . A screen for 1-butanol-insensitive mutants identified additional proteins that regulate polarized growth (BUD8, BEM1, BEM4, and FIG1), mitochondrial function (MSM1, MRP21, and HMI1), and a transcriptional regulator (CHD1) . Furthermore, we have also found that ethanol stimulates hyperfilamentation in diploid cells, again in a MAPK-dependent manner . Together, these results suggest that yeast may sense a combination of nutrient limitation and metabolic by-products to regulate differentiation. Biosci Biotechnol Biochem, 1999 Nov, 63(11), 1970 - 6 Interaction among the subunits of Golgi membrane mannosyltransferase complexes of the yeast Saccharomyces cerevisiae; Kojima H et al.; Saccharomyces cerevisiae Mnn9 protein is a type II Golgi membrane protein which concerns in protein mannosylation . When solubilized by Triton X-100, it was recovered in two distinct complexes both having mannosyltransferase activity; one with Van1 protein (V-complex) and the other with Anp1, Hoc1, Mnn10, and Mnn11 proteins (A-complex) . Characterization of the null mutants suggested that A-complex is also concerned in protein O-glycosylation . A-complex was more resistant than V-complex to dissociating conditions . Interaction between the lumenal domains of Van1 and Mnn9 was detected by a two-hybrid experiment . The anchor domain of Mnn9 protein could be replaced with other membrane anchors without losing the ability to form complexes similar to V- and A-complexes . Thus the lumenal domains are important to assemble these distinct complexes. Biosci Biotechnol Biochem, 1999 Nov, 63(11), 1871 - 81 Involvement of thioredoxin peroxidase type II (Ahp1p) of Saccharomyces cerevisiae in Mn2+ homeostasis; Farcasanu IC et al.; To identify new proteins involved in Mn2+ homeostasis, we isolated Mn(2+)-resistant mutants of Saccharomyces cerevisiae starting from a calcineurin-deficient, Mn2+ hypersensitive strain (delta cmp1 delta cmp2) . The mutations were found to lie in the PMR1 gene, known to encode a "P-type" Ca(2+)-ATPase that transports Ca2+ and Mn2+ from the cytosol to the Golgi apparatus . A second gene, AHP1, was cloned as a suppressor of the Mn2+ tolerance of a delta cmp1 delta cmp2 pmr1 mutant . Ahp1p was recently described as a thioredoxin peroxidase type II, an antioxidant protein with alkyl hydroperoxide defense properties in yeast . AHP1 disruption in strain W303 decreased tolerance to Mn2+ and H2O2 . We found that a GFP-Ahp1p fusion construct was in the cytosol when cells were grown in glucose, and in the mitochondria when cells were grown in oleate . Based on Mn2+ transport data, we concluded that Ahp1p is involved in cellular Mn2+ homeostasis in trafficking of Mn2+ from cytosol to mitochondria and from cytosol for export across the plasma membrane. Mol Microbiol, 2000 Jan, 35(1), 161 - 72 Mth1 receives the signal given by the glucose sensors Snf3 and Rgt2 in Saccharomyces cerevisiae; Lafuente MJ et al.; We have determined that the mutant genes DGT1-1 and BPC1-1, which impair glucose transport and catabolite repression in Saccharomyces cerevisiae, are allelic forms of MTH1 . Deletion of MTH1 had only slight effects on the expression of HXT1 or SNF3, but increased expression of HXT2 in the absence of glucose . A two-hybrid screen revealed that the Mth1 protein interacts with the cytoplasmic tails of the glucose sensors Snf3 and Rgt2 . This interaction was affected by mutations in Mth1 and by the concentration of glucose in the medium . A double mutant, snf3 rgt2, recovered sensitivity to glucose when MTH1 was deleted, thus showing that glucose signalling may occur independently of Snf3 and Rgt2 . A model for the possible mode of action of Snf3 and Rgt2 is presented. Mol Cell Biochem, 1999 Nov, 201(1-2), 17 - 24 Direct observation of oxidative stress on the cell wall of Saccharomyces cerevisiae strains with atomic force microscopy; de Souza Pereira R et al.; We imaged pores on the surface of the cell wall of three different industrial strains of Saccharomyces cerevisiae using atomic force microscopy . The pores could be enlarged using 10 mM diamide, an SH residue oxidant that attacks surface proteins . We found that two strains showed signs of oxidative damage via changes in density and diameter of the surface pores . We found that the German strain was resistant to diamide induced oxidative damage, even when the concentration of the oxidant was increased to 50 mM . The normal pore size found on the cell walls of American strains had diameters of about 200 nm . Under conditions of oxidative stress the diameters changed to 400 nm . This method may prove to be a useful rapid screening process (45-60 min) to determine which strains are oxidative resistant, as well as being able to screen for groups of yeast that are sensitive to oxidative stress . This rapid screening tool may have direct applications in molecular biology (transference of the genes to inside of living cells) and biotechnology (biotransformations reactions to produce chiral synthons in organic chemistry. Genetics, 2000 Jan, 154(1), 109 - 20 Genetic analysis of transcription-associated mutation in Saccharomyces cerevisiae; Morey NJ et al.; High levels of transcription are associated with elevated mutation rates in yeast, a phenomenon referred to as transcription-associated mutation (TAM) . The transcription-associated increase in mutation rates was previously shown to be partially dependent on the Rev3p translesion bypass pathway, thus implicating DNA damage in TAM . In this study, we use reversion of a pGAL-driven lys2DeltaBgl allele to further examine the genetic requirements of TAM . We find that TAM is increased by disruption of the nucleotide excision repair or recombination pathways . In contrast, elimination of base excision repair components has only modest effects on TAM . In addition to the genetic studies, the lys2DeltaBgl reversion spectra of repair-proficient low and high transcription strains were obtained . In the low transcription spectrum, most of the frameshift events correspond to deletions of AT base pairs whereas in the high transcription strain, deletions of GC base pairs predominate . These results are discussed in terms of transcription and its role in DNA damage and repair. Genetics, 2000 Jan, 154(1), 99 - 107 Sip5 interacts with both the Reg1/Glc7 protein phosphatase and the Snf1 protein kinase of Saccharomyces cerevisiae; Sanz P et al.; The Snf1 protein kinase is an essential component of the glucose starvation signalling pathway in Saccharomyces cerevisiae . We have used the two-hybrid system to identify a new protein, Sip5, that interacts with the Snf1 kinase complex in response to glucose limitation . Coimmunoprecipitation studies confirmed the association of Sip5 and Snf1 in cell extracts . We found that Sip5 also interacts strongly with Reg1, the regulatory subunit of the Reg1/Glc7 protein phosphatase 1 complex, in both two-hybrid and coimmunoprecipitation assays . Previous work showed that Reg1/Glc7 interacts with the Snf1 kinase under glucose-limiting conditions and negatively regulates its activity . Sip5 is the first protein that has been shown to interact with both Snf1 and Reg1/Glc7 . Genetic analysis showed that the two-hybrid interaction between Reg1 and Snf1 is reduced threefold in a sip5Delta mutant . These findings suggest that Sip5 facilitates the interaction between the Reg1/Glc7 phosphatase and the Snf1 kinase. Genetics, 2000 Jan, 154(1), 83 - 97 Synthetic genetic interactions with temperature-sensitive clathrin in Saccharomyces cerevisiae . Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network; Bensen ES et al.; Clathrin is involved in selective protein transport at the Golgi apparatus and the plasma membrane . To further understand the molecular mechanisms underlying clathrin-mediated protein transport pathways, we initiated a genetic screen for mutations that display synthetic growth defects when combined with a temperature-sensitive allele of the clathrin heavy chain gene (chc1-521) in Saccharomyces cerevisiae . Mutations, when present in cells with wild-type clathrin, were analyzed for effects on mating pheromone alpha-factor precursor maturation and sorting of the vacuolar protein carboxypeptidase Y as measures of protein sorting at the yeast trans-Golgi network (TGN) compartment . By these criteria, two classes of mutants were obtained, those with and those without defects in protein sorting at the TGN . One mutant with unaltered protein sorting at the TGN contains a mutation in PTC1, a type 2c serine/threonine phosphatase with widespread influences . The collection of mutants displaying TGN sorting defects includes members with mutations in previously identified vacuolar protein sorting genes (VPS), including the dynamin family member VPS1 . Striking genetic interactions were observed by combining temperature-sensitive alleles of CHC1 and VPS1, supporting the model that Vps1p is involved in clathrin-mediated vesicle formation at the TGN . Also in the spectrum of mutants with TGN sorting defects are isolates with mutations in the following: RIC1, encoding a product originally proposed to participate in ribosome biogenesis; LUV1, encoding a product potentially involved in vacuole and microtubule organization; and INP53, encoding a synaptojanin-like inositol polyphosphate 5-phosphatase . Disruption of INP53, but not the related INP51 and INP52 genes, resulted in alpha-factor maturation defects and exacerbated alpha-factor maturation defects when combined with chc1-521 . Our findings implicate a wide variety of proteins in clathrin-dependent processes and provide evidence for the selective involvement of Inp53p in clathrin-mediated protein sorting at the TGN. Mol Gen Genet, 1999 Dec, 262(4-5), 683 - 702 Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach; Entian KD et al.; In a systematic approach to the study of Saccharomyces cerevisiae genes of unknown function, 150 deletion mutants were constructed (1 double, 149 single mutants) and phenotypically analysed . Twenty percent of all genes examined were essential . The viable deletion mutants were subjected to 20 different test systems, ranging from high throughput to highly specific test systems . Phenotypes were obtained for two-thirds of the mutants tested . During the course of this investigation, mutants for 26 of the genes were described by others . For 18 of these the reported data were in accordance with our results . Surprisingly, for seven genes, additional, unexpected phenotypes were found in our tests . This suggests that the type of analysis presented here provides a more complete description of gene function. J Biol Chem, 2000 Jan 14, 275(2), 1511 - 9 Regulatory mechanisms for modulation of signaling through the cell integrity Slt2-mediated pathway in Saccharomyces cerevisiae; Martin H et al.; Signal transduction mediated by the mitogen-activated protein kinase (MAPK) Slt2 pathway is essential to maintain the cell wall integrity in Saccharomyces cerevisiae . Stimulation of MAPK pathways results in activation by phosphorylation of conserved threonine and tyrosine residues of MAPKs . We have used an antibody that specifically recognizes dually phosphorylated Slt2 to gain insight into the activation and modulation of signaling through the cell integrity pathway . We show that caffeine and vanadate activate this pathway in the absence of osmotic stabilization . The lack of the putative cell surface sensor Mid2 prevents vanadate- but not caffeine-induced Slt2 phosphorylation . Disruption of the Rho1-GTPase-activating protein genes SAC7 and BEM2 leads to constitutive Slt2 activation, indicating their involvement as negative regulators of the pathway . MAPK kinases also seem to participate in signaling regulation, Mkk1 playing a greater role than Mkk2 in signal transmission to Slt2 . Additionally, one of the phosphatases involved in Slt2 dephosphorylation is likely to be the dual specificity phosphatase Msg5, since overexpression of MSG5 in a sac7Delta mutant eliminates the high Slt2 phosphorylation, and disruption of MSG5 in wild type cells results in increased phospho-Slt2 levels . These data present the first evidence for a negative regulation of the cell integrity pathway. Biochemistry, 1999 Nov 30, 38(48), 16010 - 5 Studies of cytochrome c oxidase-driven H(+)-coupled phosphate transport catalyzed by the Saccharomyces cerevisiae Pho84 permease in coreconstituted vesicles; Fristedt U et al.; The proton-coupled Pho84 phosphate permease of Saccharomyces cerevisiae, overexpressed as a histidine-tagged chimera in Escherichia coli, was detergent-solubilized, purified, and reconstituted into proteoliposomes . Proteoliposomes containing the Pho84 protein were fused with proteoliposomes containing purified cytochrome c oxidase from beef heart mitochondria . Both components of the coreconstituted system were functionally incorporated in tightly sealed membrane vesicles in which the cytochrome c oxidase-generated electrochemical proton gradient could drive phosphate transport via the proton-coupled Pho84 permease . The metal dependency of transport indicates that a metal-phosphate complex is the translocated substrate. Biochem Biophys Res Commun, 2000 Jan 7, 267(1), 473 - 7 The regulation of activity of main mevalonic acid pathway enzymes: farnesyl diphosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and squalene synthase in yeast Saccharomyces cerevisiae; Szkopinska A et al.; The co-regulation of the main mevalonic acid pathway enzymes was investigated in the yeast Saccharomyces cerevisiae . It was found that a 6-fold increase in FPPS activity compared with that of the wild-type strain FL100 did not cause significant changes in HMG-CoA reductase activity, while the amounts of synthesized dolichols and ergosterol increased by 80 and 32%, respectively . The disruption of the SQS gene in the strain grown in the presence of ergosterol repressed the activities of both FPP synthase and HMG-CoA reductase to a comparable degree, whereas in the same strain starved for ergosterol the activity of FPPS was 10-fold higher and HMG-CoA reductase activity was practically unchanged . We show that FPPS is the enzyme that regulates the flow rate of synthesized mevalonic acid pathway products independent of HMG-CoA reductase and SQS . Nat Cell Biol, 2000 Jan, 2(1), 36 - 41 The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae; Maddox PS et al.; Microtubule assembly in Saccharomyces cerevisiae is initiated from sites within spindle pole bodies (SPBs) in the nuclear envelope . Microtubule plus ends are thought to be organized distal to the SPBs, while minus ends are proximal . Several hypotheses for the function of microtubule motor proteins in force generation and regulation of microtubule assembly propose that assembly and disassembly occur at minus ends as well as at plus ends . Here we analyse microtubule assembly relative to the SPBs in haploid yeast cells expressing green fluorescent protein fused to alpha-tubulin, a microtubule subunit . Throughout the cell cycle, analysis of fluorescent speckle marks on cytoplasmic astral microtubules reveals that there is no detectable assembly or disassembly at minus ends . After laser-photobleaching, metaphase spindles recover about 63% of the bleached fluorescence, with a half-life of about 1 minute . After anaphase onset, photobleached marks in the interpolar spindle are persistent and do not move relative to the SPBs . In late anaphase, the elongated spindles disassemble at the microtubule plus ends . These results show for astral and anaphase interpolar spindle microtubules, and possibly for metaphase spindle microtubules, that microtubule assembly and disassembly occur at plus, and not minus, ends. Yeast, 2000 Jan 15, 16(1), 35 - 47 Saccharomyces cerevisiae gene ISW2 encodes a microtubule-interacting protein required for premeiotic DNA replication; Trachtulcova P et al.; A molecular genetic characterization of the ORF YOR304W (ISW2), identified in a screen of a yeast lambdagt11 library using a monoclonal antibody that reacts with a 210 kDa mammalian microtubule-interacting protein, is presented in this paper . The protein encoded by the ORF YOR304W is 50% identical to the Drosophila nucleosome remodelling factor ISWI and is therefore a new member of the SNF2 protein family and has been recently entered into SDG as ISW2 . Although not essential for vegetative growth, we found that the ISW2 gene is required for early stages in sporulation . The isw2 homozygous deletant diploid strain was blocked in the G(1) phase of the cell cycle, unable to execute the premeiotic DNA replication and progress through the nuclear meiotic division cycle . ISW2 expression from a multicopy plasmid had the same effect as deletion, but ISW2 expression from a centromeric plasmid rescued the deletion phenotype . In vegetatively growing diploid cells, the Isw2 protein was preferentially found in the cytoplasm, co-localizing with microtubules . An accumulation of the Isw2 protein within the nucleus was observed in cells entering sporulation . Together with data published very recently by Tsukiyama et al . (1999), we propose a role for the Isw2 protein in facilitating chromatin accessibility for transcriptional factor(s) that positively regulate meiosis/sporulation-specific genes . FEBS Lett, 1999 Dec 31, 464(3), 123 - 8 Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae; Wieczorke R et al.; The hexose transporter family of Saccharomyces cerevisiae comprises 18 proteins (Hxt1-17, Gal2) . Here, we demonstrate that all these proteins, except Hxt12, and additionally three members of the maltose transporter family (Agt1, Ydl247, Yjr160) are able to transport hexoses . In a yeast strain deleted for HXT1-17, GAL2, AGT1, YDL247w and YJR160c, glucose consumption and transport activity were completely abolished . However, as additional deletion of the glucose sensor gene SNF3 partially restored growth on hexoses, our data indicate the existence of even more proteins able to transport hexoses in yeast. Proc Natl Acad Sci U S A, 2000 Jan 4, 97(1), 250 - 5 Analysis of microsatellite mutations in the mitochondrial DNA of Saccharomyces cerevisiae; Sia EA et al.; In the nuclear genome of Saccharomyces cerevisiae, simple, repetitive DNA sequences (microsatellites) mutate at rates much higher than nonrepetitive sequences . Most of these mutations are deletions or additions of repeat units . The yeast mitochondrial genome also contains many microsatellites . To examine the stability of these sequences, we constructed a reporter gene (arg8(m)) containing out-of-frame insertions of either poly(AT) or poly(GT) tracts within the coding sequence . Yeast strains with this reporter gene inserted within the mitochondrial genome were constructed . Using these strains, we showed that poly(GT) tracts were considerably less stable than poly(AT) tracts and that alterations usually involved deletions rather than additions of repeat units . In contrast, in the nuclear genome, poly(GT) and poly(AT) tracts had similar stabilities, and alterations usually involved additions rather than deletions . Poly(GT) tracts were more stable in the mitochondria of diploid cells than in haploids . In addition, an msh1 mutation destabilized poly(GT) tracts in the mitochondrial genome. Proc Natl Acad Sci U S A, 2000 Jan 4, 97(1), 240 - 4 Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant {PSI(+)} of Saccharomyces cerevisiae; Eaglestone SS et al.; The cytoplasmic heritable determinant {PSI(+)} of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p . This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3 . In a {PSI(+)} background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination . We have investigated the process by which the {PSI(+)} determinant can be efficiently eliminated from strains, by growth in the presence of the protein denaturant guanidine hydrochloride (GuHCl) . Strains are "cured" of {PSI(+)} by millimolar concentrations of GuHCl, well below that normally required for protein denaturation . Here we provide evidence indicating that the elimination of the {PSI(+)} determinant is not derived from the direct dissolution of self-replicating {PSI(+)} seeds by GuHCl . Although GuHCl does elicit a moderate stress response, the elimination of {PSI(+)} is not enhanced by stress, and furthermore, exhibits an absolute requirement for continued cell division . We propose that GuHCl inhibits a critical event in the propagation of the prion conformer and demonstrate that the kinetics of curing by GuHCl fit a random segregation model whereby the heritable {PSI(+)} element is diluted from a culture, after the total inhibition of prion replication by GuHCl. Nat Genet, 2000 Jan, 24(1), 53 - 6 Novel dominant mutations in Saccharomyces cerevisiae MSH6; Das Gupta R et al.; Inherited mutations in the mismatch repair (MMR) genes MSH2 and MLH1 are found in most hereditary nonpolyposis colon cancer (HNPCC) patients studied . Eukaryotic MMR uses two partially redundant mispair-recognition complexes, Msh2p-Msh6p and Msh2p-Msh3p (ref.2) Inactivation of MSH2 causes high rates of accumulation of both base-substitution and frameshift mutations . Mutations in MSH6 or MSH3 cause partial defects in MMR, with inactivation of MSH6 resulting in high rates of base-substitution mutations and low rates of frameshift mutations; inactivation of MSH3 results in low rates of frameshift mutations . These different mutator phenotypes provide an explanation for the observation that MSH2 mutations are common in HNPCC families, whereas mutations in MSH3 and MSH6 are rare . We have identified novel missense mutations in Saccharomyces cerevisiae MSH6 that appear to inactivate both Msh2p-Msh6p- and Msh2p-Msh3p-dependent MMR . Our work suggests that such mutations may underlie some cases of inherited cancer susceptibility similar to those caused by MSH2 mutations. J Bacteriol, 2000 Jan, 182(1), 241 - 3 Monoubiquitination is sufficient to signal internalization of the maltose transporter in Saccharomyces cerevisiae; Lucero P et al.; Monoubiquitination of the 12-transmembrane segment (12-TMS) Saccharomyces cerevisiae maltose transporter promoted the maximal internalization rate of this protein . This modification is similar to that of the 7-TMS alpha-factor receptor but different from that of the 12-TMS uracil and general amino acid permeases . This result shows that binding of ubiquitin-Lys63 chains is not required for maximal internalization of all 12-TMS-containing proteins. FEMS Microbiol Lett, 2000 Jan 1, 182(1), 137 - 41 The SUN family of Saccharomyces cerevisiae: the double knock-out of UTH1 and SIM1 promotes defects in nucleus migration and increased drug sensitivity; Mouassite M et al.; UTH1 and SIM1 are two of four 'SUN' genes (SIM1, UTH1, NCA3 and SUN4/SCW3) whose products are involved in different cellular processes such as DNA replication, lifespan, mitochondrial biogenesis or cell septation . UTH1 or SIM1 inactivation did not affect cell growth, shape or nuclear migration, whereas the double null mutant presented phenotypes of numerous binucleate cells and benomyl sensitivity, suggesting that microtubule function could be altered; the uth1Deltasim1Delta strain also presented defects which could be related to the Ras/cAMP pathway: pet phenotype, heat shock sensitivity, inability to store glycogen, sensitivity to starvation and failure of spores to germinate . These observations suggested that Uth1p could be involved as a connection step between pathways controlling growth and those controlling division. FEMS Microbiol Lett, 2000 Jan 1, 182(1), 1 - 8 Metabolic surprises in Saccharomyces cerevisiae during adaptation to saline conditions: questions, some answers and a model; Blomberg A; This review describes the metabolic alterations and adaptations of yeast cells in response to osmotic stress . The basic theme of the cellular response is known to be exclusion of the extracellular stress agent salt and intracellular accumulation of the compatible solute glycerol . Molecular details of these basic processes are currently rather well known . However, analysis of expression changes during adaptation to salt has revealed a number of metabolic surprises . These include the induced expression of genes involved in glycerol dissimilation as well as trehalose turnover . The physiological rationale for these responses to osmotic stress is discussed . A model is presented in which it is hypothesised that the two pathways function as glycolytic safety valves during adaptation to stress. Virology, 2000 Jan 5, 266(1), 211 - 24 Creation and repair of specific DNA double-strand breaks in vivo following infection with adenovirus vectors expressing Saccharomyces cerevisiae HO endonuclease; Nicolas AL et al.; To study DNA double-strand break (DSB) repair in mammalian cells, the Saccharomyces cerevisiae HO endonuclease gene, or its recognition site, was cloned into the adenovirus E3 or E1 regions . Analysis of DNA from human A549 cells coinfected with the E3::HO gene and site viruses showed that HO endonuclease was active and that broken viral genomes were detectable 12 h postinfection, increasing with time up to approximately 30% of the available HO site genomes . Leftward fragments of approximately 30 kbp, which contain the packaging signal, but not rightward fragments of approximately 6 kbp, were incorporated into virions, suggesting that broken genomes were not held together tightly after cleavage . There was no evidence for DSB repair in E3::HO virus coinfections . In contrast, such evidence was obtained in E1::HO virus coinfections of nonpermissive cells, suggesting that adenovirus proteins expressed in the permissive E3::HO coinfection can inhibit mammalian DSB repair . To test the inhibitory role of E4 proteins, known to suppress genome concatemer formation late in infection (Weiden and Ginsberg, 1994), A549 cells were coinfected with E3::HO viruses lacking the E4 region . The results strongly suggest that the E4 protein(s) inhibits DSB repair . Mol Cell Biol, 2000 Jan, 20(2), 542 - 55 Distinct subcellular localization patterns contribute to functional specificity of the Cln2 and Cln3 cyclins of Saccharomyces cerevisiae; Miller ME et al.; The G(1) cyclins of budding yeast drive cell cycle initiation by different mechanisms, but the molecular basis of their specificity is unknown . Here we test the hypothesis that the functional specificity of G(1) cyclins is due to differential subcellular localization . As shown by indirect immunofluorescence and biochemical fractionation, Cln3p localization appears to be primarily nuclear, with the most obvious accumulation of Cln3p to the nuclei of large budded cells . In contrast, Cln2p localizes to the cytoplasm . We were able to shift localization patterns of truncated Cln3p by the addition of nuclear localization and nuclear export signals, and we found that nuclear localization drives a Cln3p-like functional profile, while cytoplasmic localization leads to a partial shift to a Cln2p-like functional profile . Therefore, forcing Cln3p into a Cln2p-like cytoplasmic localization pattern partially alters the functional specificity of Cln3p toward that of Cln2p . These results suggest that there are CLN-dependent cytoplasmic and nuclear events important for cell cycle initiation . This is the first indication of a cytoplasmic function for a cyclin-dependent kinase . The data presented here support the idea that cyclin function is regulated at the level of subcellular localization and that subcellular localization contributes to the functional specificity of Cln2p and Cln3p. FEBS Lett, 1999 Dec 17, 463(3), 307 - 11 The E-box DNA binding protein Sgc1p suppresses the gcr2 mutation, which is involved in transcriptional activation of glycolytic genes in Saccharomyces cerevisiae; Sato T et al.; Glycolytic gene expression is mediated by the Gcr1p-Gcr2p transcriptional activation complex . A screen for multicopy suppressors of gcr2 yielded SGC1 . SGC1's suppression activity was specific to gcr2, it did not extend to gcr1 . Disruption of SGC1 moderately affected glycolytic enzyme activities, although no growth defect was evident . Sgc1p exhibits a bHLH motif which is characteristic of E-box DNA-binding proteins . DNA footprinting experiments demonstrated Sgc1p's ability to bind at an E-box . However, its binding specificity was less than 10-fold, which is also characteristic of E-box binding proteins . LexA fusion experiments demonstrated that Sgc1p has weak intrinsic activating activity independent of GCR1 and GCR2 . We propose that Sgc1p binds at E-boxes of glycolytic genes and contributes to their activation. J Biol Chem, 1999 Dec 24, 274(52), 36835 - 8 Fidelity and processivity of Saccharomyces cerevisiae DNA polymerase eta; Washington MT et al.; The yeast RAD30 gene functions in error-free replication of UV-damaged DNA, and RAD30 encodes a DNA polymerase, pol eta, that has the ability to efficiently and correctly replicate past a cis-syn-thymine-thymine dimer in template DNA . To better understand the role of pol eta in damage bypass, we examined its fidelity and processivity on nondamaged DNA templates . Steady-state kinetic analyses of deoxynucleotide incorporation indicate that pol eta has a low fidelity, misincorporating deoxynucleotides with a frequency of about 10(-2) to 10(-3) . Also pol eta has a low processivity, incorporating only a few nucleotides before dissociating . We suggest that pol eta's low fidelity reflects a flexibility in its active site rendering it more tolerant of DNA damage, while its low processivity limits its activity to reduce errors. J Bacteriol, 1999 Dec, 181(24), 7618 - 20 CDC64 encodes cytoplasmic alanyl-tRNA synthetase, Ala1p, of Saccharomyces cerevisiae; Wrobel C et al.; The cdc64-1 mutation causes G(1) arrest in Saccharomyces cerevisiae corresponding to a type II Start phenotype . We report that CDC64 encodes Ala1p, an alanyl-tRNA synthetase . Thus, cdc64-1 might affect charging of tRNA(Ala) and thereby initiation of cell division. J Bacteriol, 1999 Dec, 181(24), 7414 - 20 Localization of synthesis of beta1,6-glucan in Saccharomyces cerevisiae; Montijn RC et al.; Beta1,6-Glucan is a key component of the yeast cell wall, interconnecting cell wall proteins, beta1,3-glucan, and chitin . It has been postulated that the synthesis of beta1,6-glucan begins in the endoplasmic reticulum with the formation of protein-bound primer structures and that these primer structures are extended in the Golgi complex by two putative glucosyltransferases that are functionally redundant, Kre6 and Skn1 . This is followed by maturation steps at the cell surface and by coupling to other cell wall macromolecules . We have reinvestigated the role of Kre6 and Skn1 in the biogenesis of beta1,6-glucan . Using hydrophobic cluster analysis, we found that Kre6 and Skn1 show significant similarities to family 16 glycoside hydrolases but not to nucleotide diphospho-sugar glycosyltransferases, indicating that they are glucosyl hydrolases or transglucosylases instead of genuine glucosyltransferases . Next, using immunogold labeling, we tried to visualize intracellular beta1,6-glucan in cryofixed sec1-1 cells which had accumulated secretory vesicles at the restrictive temperature . No intracellular labeling was observed, but the cell surface was heavily labeled . Consistent with this, we could detect substantial amounts of beta1,6-glucan in isolated plasma membrane-derived microsomes but not in post-Golgi secretory vesicles . Taken together, our data indicate that the synthesis of beta1, 6-glucan takes place largely at the cell surface . An alternative function for Kre6 and Skn1 is discussed. J Bacteriol, 1999 Dec, 181(24), 7409 - 13 Genome-wide transcriptional analysis of aerobic and anaerobic chemostat cultures of Saccharomyces cerevisiae; ter Linde JJ et al.; The yeast Saccharomyces cerevisiae is unique among eukaryotes in exhibiting fast growth in both the presence and the complete absence of oxygen . Genome-wide transcriptional adaptation to aerobiosis and anaerobiosis was studied in assays using DNA microarrays . This technique was combined with chemostat cultivation, which allows controlled variation of a single growth parameter under defined conditions and at a fixed specific growth rate . Of the 6,171 open reading frames investigated, 5,738 (93%) yielded detectable transcript levels under either aerobic or anaerobic conditions; 140 genes showed a >3-fold-higher transcription level under anaerobic conditions . Under aerobic conditions, transcript levels of 219 genes were >3-fold higher than under anaerobic conditions. Protein Expr Purif, 1999 Dec, 17(3), 477 - 84 Purification of virus-like particles of recombinant human papillomavirus type 11 major capsid protein L1 from Saccharomyces cerevisiae; Cook JC et al.; Recombinant major capsid protein, L1 (M(r) = 55,000), of human papillomavirus type 11 was expressed intracellularly at high levels in a galactose-inducible Saccharomyces cerevisiae expression system by an HPV6/11 hybrid gene . The capsid protein self-assembled into virus-like particles (VLPs) and accounted for 15% of the total soluble protein . A purification process was developed that consisted of two main steps: microfiltration and cation-exchange chromatography . The purified VLPs were 98% homogeneous, and the overall purification yield was 10% . The final product was characterized by several analytical methods and was highly immunogenic in mice . Protein Expr Purif, 1999 Dec, 17(3), 456 - 64 Heterologous expression, purification, and kinetic comparison of the cytoplasmic and mitochondrial glyoxalase II enzymes, Glo2p and Glo4p, from Saccharomyces cerevisiae; Bito A et al.; The aims of the present study are (i) to purify a mitochondrial glyoxalase II to homogeneity for the first time from any organism and (ii) to compare its kinetic properties with those of the cytoplasmic enzyme . Both the cytoplasmic and the mitochondrial glyoxalases II from Saccharomyces cerevisiae, which are the products of two distinct genes, GLO2 and GLO4, were purified from yeast and in recombinant form from Escherichia coli . To obtain a higher protein yield (compared to wild-type expression) in yeast, the genes were placed under the control of the strong GAL1 promoter on a multicopy plasmid . Amino-terminal sequencing and molecular mass determination by MALDI-TOF mass spectrometry of the mitochondrial Glo4 protein revealed Met-11 of the primary translation product of the gene as the N-terminal amino acid . Judged by enzyme kinetic properties the recombinant and natural proteins were equivalent . The cytoplasmic and the mitochondrial enzyme differed in the pH dependence of the kinetic parameters for the main substrate, S-d-lactoylglutathione . Whereas the cytoplasmic protein showed a pronounced peak of enzyme activity between pH 7-8 and a continuous up to fivefold increase of the K(M) value with increasing pH (from 5 . 5-9.0), the mitochondrial protein had a nearly constant K(M) value and an activity maximum over a broad pH range (6.5-9.0) . The kinetic parameters (at pH 7.5) of both the cytoplasmic and the mitochondrial enzyme for S-D-lactoylglutathione were of the same order of magnitude as reported recently for the human and Arabidopsis thaliana enzymes which are presumably of cytoplasmic origin . However, both yeast enzymes showed a severalfold lower preference for the more hydrophobic substrate, S-d-mandeloylglutathione . Protein Expr Purif, 1999 Dec, 17(3), 414 - 20 Functional expression and characterization of the myrosinase MYR1 from Brassica napus in Saccharomyces cerevisiae; Chen S et al.; Myrosinases are thioglucosidases that hydrolyze the natural plant products glucosinolates . We have expressed the myrosinase MYR1 from Brassica napus in Saccharomyces cerevisiae . The recombinant myrosinase was enzymatically active which shows that the MYR1, which in the plant is complex bound with myrosinase-binding proteins and myrosinase-associated proteins, is functional in its free form . Characterization of the recombinant MYR1 with respect to pH optimum, substrate specificity, activation by ascorbic acid, and inhibitors showed similar characteristics as previously observed for other plant myrosinases . The indolizidine alkaloid castanospermine, an inhibitor of O-glycosidases, inhibited the hydrolysis of p-hydroxybenzylglucosinolate with a K(i) value of 0.3 microM and 2-deoxy-2-fluoroglucotropaeolin, a specific inhibitor of thioglucosidases, inhibited the enzyme with a K(i) value of 1 mM . The expression of the myrosinase in yeast was transient and the growth of the yeast cells was significantly reduced during the period of expression of the myrosinase . Immunoblot analysis showed that the highest level of expression of MYR1 was obtained 24 h after induction with galactose . The amount of myrosinase protein correlated with the level of enzyme activity . The transient expression of myrosinase indicates that myrosinase is toxic to the cells . This is the first report on successful heterologous expression of a myrosinase and provides an important tool for, e.g., further characterization of myrosinase by site-directed mutagenesis and for studying the interaction between myrosinase and myrosinase-binding proteins, myrosinase-associated proteins, and epithiospecifier proteins . Arch Biochem Biophys, 1999 Dec 15, 372(2), 360 - 6 Lysine 219 participates in NADPH specificity in a flavin-containing monooxygenase from Saccharomyces cerevisiae; Suh JK et al.; The flavin-containing monooxygenase from Saccharomyces cerevisiae (yFMO) uses NADPH and O(2) to oxidize thiol containing substrates such as GSH and thereby generates the oxidizing potential for the ER . The enzyme uses NADPH 12 times more efficiently than NADH . Amino acid sequence analysis suggests that Lys 219 and/or Lys 227 may act as counterions to the 2' phosphate of NADPH and to help determine the preference for pyridine nucleotides . Site directed mutations show that Lys 219 makes the greater contribution to cosubstrate recognition . Conversion of Lys 219 to Ala reduces NADPH dependent activity 90-fold, but has no effect on NADH-dependent activity . Conversion of Lys 227 to Ala reduces NADPH-dependent activity fivefold and NADH-dependent activity threefold . Dissociation constants for NADP(+) to oxidized yFMO were measured spectroscopically . K(d) is 12 microM for the wild-type enzyme and 243 microM for the K219A mutant, consistent with the role of Lys 219 in pyridine nucleotide binding . Arch Biochem Biophys, 1999 Dec 15, 372(2), 300 - 8 Saccharomyces cerevisiae expresses two genes encoding isozymes of methylenetetrahydrofolate reductase; Raymond RK et al.; The identification, expression, and assay of two Saccharomyces cerevisiae genes encoding methylenetetrahydrofolate reductases (MTHFR) is described . MTHFR catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, used to methylate homocysteine in methionine synthesis . The MET12 gene is located on chromosome XVI and encodes a protein of 657 amino acids . The MET13 gene is located on chromosome VII and encodes a protein of 599 amino acids . The deduced amino acid sequences of these two genes are 34% identical to each other and 32-37% identical to the human MTHFR . A phenotype for the single disruption of MET12 was not observed, however, single disruption of MET13 resulted in methionine auxotrophy . Double disruption of both MET12 and MET13 also resulted in methionine auxotrophy . Growth of the methionine auxotrophs was supported by both methionine and S-adenosylmethionine . Transcripts of both MET12 and MET13 were detected in total RNA from wild type cells grown in the presence or absence of methionine . The methionine requirement of the met12 met13 double disruptant was complemented by plasmid-borne MET13, but not MET12 even when a multicopy plasmid was used . Furthermore, overexpression of the human MTHFR in the met12 met13 double disruptant complemented the methionine auxotrophy of this strain . In contrast, overexpression of the Escherichia coli metF gene did not complement the methionine requirement of met12 met13 cells . Assays for MTHFR in crude extracts and expression of the yeast proteins in Escherichia coli verified that both MET12 and MET13 encode functional MTHFR isozymes . Biochemistry, 1999 Dec 14, 38(50), 16671 - 7 Characterization of a novel dUTP-dependent activity of CTP synthetase from Saccharomyces cerevisiae; Pappas A et al.; CTP synthetase {EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)} from the yeast Saccharomyces cerevisiae catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP . In this work, we demonstrated that CTP synthetase utilized dUTP as a substrate to synthesize dCTP . The dUTP-dependent activity was linear with time and with enzyme concentration . Maximum dUTP-dependent activity was dependent on MgCl(2) (4 mM) and GTP (K(a) = 14 microM) at a pH optimum of 8.0 . The apparent K(m) values for dUTP, ATP, and glutamine were 0.18, 0.25, and 0.41 mM, respectively . dUTP promoted the tetramerization of CTP synthetase, and the extent of enzyme tetramerization correlated with dUTP-dependent activity . dCTP was a poor inhibitor of dUTP-dependent activity, whereas CTP was a potent inhibitor of this activity . The enzyme catalyzed the synthesis of dCTP and CTP when dUTP and UTP were used as substrates together . CTP was the major product synthesized when dUTP and UTP were present at saturating concentrations . When dUTP and UTP were present at concentrations near their K(m) values, the synthesis of dCTP increased relative to that of CTP . The synthesis of dCTP was favored over the synthesis of CTP when UTP was present at a concentration near its K(m) value and dUTP was varied from subsaturating to saturating concentrations . These data suggested that the dUTP-dependent synthesis of dCTP by CTP synthetase activity may be physiologically relevant. Biol Chem, 1999 Oct, 380(10), 1167 - 73 Mitochondrial iron metabolism in the yeast Saccharomyces cerevisiae; Craig EA et al.; Iron is fundamental to many biological processes, but is also detrimental as it fosters the synthesis of destructive oxygen radicals . Recent experiments have increased our knowledge of the critical process of regulation of mitochondrial iron metabolism . A number of genes directly involved in iron homeostasis in this organelle have been identified . Intriguingly, a minor Hsp70 molecular chaperone of the mitochondrial matrix has been implicated as a player in this process as well. Mol Cell Biol, 2000 Jan, 20(1), 224 - 32 Rsp5, a ubiquitin-protein ligase, is involved in degradation of the single-stranded-DNA binding protein rfa1 in Saccharomyces cerevisiae; Erdeniz N et al.; In Saccharomyces cerevisiae, RAD1 and RAD52 are required for alternate pathways of mitotic recombination . Double-mutant strains exhibit a synergistic interaction that decreases direct repeat recombination rates dramatically . A mutation in RFA1, the largest subunit of a single-stranded DNA-binding protein complex (RP-A), suppresses the recombination deficiency of rad1 rad52 strains (J . Smith and R . Rothstein, Mol . Cell . Biol . 15:1632-1641, 1995) . Previously, we hypothesized that this mutation, rfa1-D228Y, causes an increase in recombinogenic lesions as well as the activation of a RAD52-independent recombination pathway . To identify gene(s) acting in this pathway, temperature-sensitive (ts) mutations were screened for those that decrease recombination levels in a rad1 rad52 rfa1-D228Y strain . Three mutants were isolated . Each segregates as a single recessive gene . Two are allelic to RSP5, which encodes an essential ubiquitin-protein ligase . One allele, rsp5-25, contains two mutations within its open reading frame . The first mutation does not alter the amino acid sequence of Rsp5, but it decreases the amount of full-length protein in vivo . The second mutation results in the substitution of a tryptophan with a leucine residue in the ubiquitination domain . In rsp5-25 mutants, the UV sensitivity of rfa1-D228Y is suppressed to the same level as in strains overexpressing Rfa1-D228Y . Measurement of the relative rate of protein turnover demonstrated that the half-life of Rfa1-D228Y in rsp5-25 mutants was extended to 65 min compared to a 35-min half-life in wild-type strains . We propose that Rsp5 is involved in the degradation of Rfa1 linking ubiquitination with the replication-recombination machinery. Mol Cell Biol, 2000 Jan, 20(1), 173 - 80 Stabilizing effects of interruptions on trinucleotide repeat expansions in Saccharomyces cerevisiae; Rolfsmeier ML et al.; In most trinucleotide repeat (TNR) diseases, the primary factor determining the likelihood of expansions is the length of the TNR . In some diseases, however, stable alleles contain one to three base pair substitutions that interrupt the TNR tract . The unexpected stability of these alleles compared to the frequent expansions of perfect TNRs suggested that interruptions somehow block expansions and that expansions occur only upon loss of at least one interruption . The work in this study uses a yeast genetic assay to examine the mechanism of stabilization conferred by two interruptions of a 25-repeat tract . Expansion rates are reduced up to 90-fold compared to an uninterrupted allele . Stabilization is greatest when the interruption is replicated early on the lagging strand, relative to the rest of the TNR . Although expansions are infrequent, they are often polar, gaining new DNA within the largest available stretch of perfect repeats . Surprisingly, interruptions are always retained and sometimes even duplicated, suggesting that expansion in yeast cells can proceed without loss of the interruption . These findings support a stabilization model in which interruptions contribute in cis to reduce hairpin formation during TNR replication and thus inhibit expansion rates. Curr Genet, 1999 Oct, 36(4), 195 - 200 A 'natural' mutation in Saccharomyces cerevisiae strains derived from S288c affects the complex regulatory gene HAP1 (CYP1); Gaisne M et al.; The HAP1 gene encodes a complex transcriptional regulator of many genes involved in electron-transfer reactions and is essential in anaerobic or heme-depleted conditions . We show here that strains derived from S288c carry a defective Ty1 element inserted in the 3' region of the HAP1 ORF . This mutant allele acts as a HAP1 null allele in terms of cytochrome c expression and CYC1 UAS1-dependent transcription, but is able to sustain limited growth in heme-depleted conditions. Mol Microbiol, 1999 Oct, 34(1), 124 - 33 Distinct roles for Rev1p and Rev7p during translesion synthesis in Saccharomyces cerevisiae; Baynton K et al.; Translesion synthesis (TLS) in Saccharomyces cerevisiae requires at least Rev1p and polymerase zeta (Pol zeta), a complex of the Rev3 polymerase and its accessory factor Rev7p . Although their precise role(s) are poorly characterized, in vitro studies suggest that each protein contributes to TLS in a manner dependent on the particular lesion and surrounding DNA sequence . In the present study, strand segregation analysis is used to attempt to identify the role(s) of the Rev1 and Rev7 proteins during TLS . This assay uses double-stranded plasmids containing a genetic marker opposite to a replication blocking lesion (N-2-acetylaminofluorene; AAF) to measure TLS quantitatively and qualitatively in vivo . The AAF adduct is localized within a repetitive sequence in a manner that allows the formation of misaligned primer-template replication intermediates . Elongation from a misaligned intermediate fixes a frameshift mutation (slipped TLS), while extension of the correctly aligned lesion terminus yields error-free (non-slipped) TLS . The results indicate that there is a strong requirement for Rev7p during Pol zeta-mediated TLS measured in vivo . Furthermore, Rev1p is needed only for non-slipped TLS; slipped TLS remains efficient in its absence, revealing a previously uncharacterized Rev1p activity similar to Escherichia coli UmuDC function . Specifically, this activity is required for elongation from a correctly aligned lesion terminus. Nucleic Acids Res, 2000 Jan 1, 28(1), 81 - 4 TRIPLES: a database of gene function in Saccharomyces cerevisiae; Kumar A et al.; Using a novel multipurpose mini-transposon, we have generated a collection of defined mutant alleles for the analysis of disruption phenotypes, protein localization, and gene expression in Saccharomyces cerevisiae . To catalog this unique data set, we have developed TRIPLES, a Web-accessible database of TRansposon-Insertion Phenotypes, Localization and Expression in Saccharomyces . Encompassing over 250 000 data points, TRIPLES provides convenient access to information from nearly 7800 transposon-mutagenized yeast strains; within TRIPLES, complete data reports of each strain may be viewed in table format, or if desired, downloaded as tab-delimited text files . Each report contains external links to corresponding entries within the Saccharomyces Genome Database and International Nucleic Acid Sequence Data Library (GenBank) . Unlike other yeast databases, TRIPLES also provides on-line order forms linked to each clone report; users may immediately request any desired strain free-of-charge by submitting a completed form . In addition to presenting a wealth of information for over 2300 open reading frames, TRIPLES constitutes an important medium for the distribution of useful reagents throughout the yeast scientific community . Maintained by the Yale Genome Analysis Center, TRIPLES may be accessed at http://ycmi.med.yale.edu/ygac/triples.htm Curr Genet, 1999 Nov, 36(5), 256 - 61 Analysis of the genes activated by the FLO8 gene in Saccharomyces cerevisiae; Kobayashi O et al.; It is thought that the FLO8 gene encodes a transcriptional activator of the dominant flocculation gene FLO1 in Saccharomyces cerevisiae . To determine other genes which are regulated by FLO8, a detailed comparison of the transcripts from the FLO8 and Deltaflo8 strains was carried out . In addition to the FLO1 gene, it was found that transcription of the FLO11 and STA1 genes is positively regulated by FLO8 . In flo8 strains, not only transcripts of the FLO11, STA1, and FLO1 genes but also invasive growth, extracellular glucoamylase production, and flocculation were undetected . From these results, it is suggested that FLO8 regulates these characteristics via the transcriptional regulation of the FLO11, STA1, and FLO1 genes. Curr Genet, 1999 Nov, 36(5), 249 - 55 Analysis of strains of Saccharomyces cerevisiae with amino acid substitutions in the Cu(A)-binding region of subunit II of cytochrome c oxidase; Mazourek M et al.; Cytochrome c oxidase accepts electrons from cytochrome c and transfers them to oxygen to form water . Electrons enter the complex through the Cu(A) site, formed by two copper atoms bound to mitochondrially encoded subunit II . The effect of amino-acid alterations in one of the Cu(A) ligands and in an amino acid adjacent to another of the ligands in the yeast enzyme is examined . Substitution of tyrosine for the Cu(A) ligand, cysteine 221, completely abolishes enzyme activity . In addition, 19 independent revertants of this mutant yeast strain recover function by restoring the cysteine codon . Replacement of a non-conserved glycine at position 228 by valine, adjacent to the Cu(A)-ligand histidine 229, virtually blocks enzyme activity . Activity is restored by inserting alanine or phenylalanine at position 228 or by amino-acid substitutions at nearby codons . Our results demonstrate that the Cu(A) ligand appears to be essential for enzyme function while other residues in the copper-binding region are less functionally constrained. Mutat Res, 1999 Nov 29, 430(1), 99 - 107 Methionine reduces spontaneous and alkylation-induced mutagenesis in Saccharomyces cerevisiae cells deficient in O6-methylguanine-DNA methyltransferase; Bawa S et al.; The exposure of DNA to reactive intracellular metabolites is thought to be a major cause of spontaneous mutagenesis . DNA alkylation is implicated in the above process by the fact that bacterial and yeast cells lacking DNA alkylation-specific repair genes exhibit elevated spontaneous mutation rates . The origin of the intracellular alkylating molecules is not clear; however, S-adenosylmethionine (SAM) has been proposed as one source because it has a reactive methyl group known to methylate proteins and DNA . We supplemented yeast cultures with excess methionine and examined the effects of increased endogenous SAM concentration on spontaneous and alkylation-induced mutagenesis in the absence of various DNA repair pathways . Our results show that either the excess methionine, or the increased SAM produced as a result of this treatment, is able to protect yeast cells from mutagenesis, and that this effect is alkylation-damage-specific . The protective effect was observed only in the mgt1 mutant deficient in the O(6)-methylguanine-DNA repair methyltransferase, but not in the wild type or other DNA repair-deficient strains, indicating that the protection is specific for O-methyl lesions . Thus, our results may lend support to the recently reported chemopreventive effect of SAM in rodents and further suggest that the observed tumor prevention by SAM may be, in part, due to its suppression of spontaneous mutagenesis in mammals . Given that a strong correlation has been established between O(6)-methylguanine and carcinogenicity, this study may offer a novel approach to preventing carcinogenesis. Yeast, 1999 Dec, 15(16), 1769 - 74 The trp1- delta FA designer deletion for PCR-based gene functional analysis in Saccharomyces cerevisiae; Horecka J et al.; PCR-based gene deletion and modification are now common techniques for rapid gene manipulation in the yeast Saccharomyces cerevisiae . The techniques work best when the host strain lacks sequence homology to the PCR-amplified selectable markers . One of the most versatile sets of PCR deletion/modification vectors is the pFA system described by Longtine et al.(1998), which is based on both heterologous (kanMX6 and HIS3MX6) and homologous (TRP1) markers . Here we describe the trp1-DeltaFA designer deletion allele that removes precisely from the genome TRP1 sequences carried in the pFA vectors . The trp1-DeltaFA allele can be introduced easily into TRP1 and most trp1 starting strains, and its use increases the frequency of correct integrants when using the pFA system's TRP1-based constructs . Unlike trp1-Delta1, trp1-DeltaFA does not remove neighbouring GAL3 upstream activating sequences and therefore does not interfere with GAL gene induction . Yeast, 1999 Dec, 15(16), 1733 - 45 Selection of genes repressed by cAMP that are induced by nutritional limitation in Saccharomyces cerevisiae; Tadi D et al.; DNA-lacZ fusion libraries of yeast Saccharomyces cerevisiae were used to select genes coordinately regulated by the Ras-cAMP-cAPK signalling pathway . Sixteen new genes (AGP1, APE2, APE3, FPS1, GUT2, MDH2, PLB2, PYK2, RNR3, SUR1, UGA1, YHR033w, YBR006w, YHR143w, YMR086w and YOR173w) were found to be repressed by cAMP . Most of these genes encode for metabolic enzymes and are induced by nutritional limitations . These common properties suggest a role of this pathway in the metabolic adjustment of the cell to nutritional variations . The induction of 10 of these genes is reduced in the msn2,msn4 double mutant, which emphasizes the role of the Msn2/4p transcriptional activators in mediating the Ras-cAMP-cAPK signalling pathway . The Msn2p/Msn4p-independent expression of the six other genes suggests the existence of other regulatory systems under the control of this pathway . Yeast, 1999 Dec, 15(16), 1719 - 31 RGD1 genetically interacts with MID2 and SLG1, encoding two putative sensors for cell integrity signalling in Saccharomyces cerevisiae; de Bettignies G et al.; The RGD1 gene was identified during systematic genome sequencing of Saccharomyces cerevisiae . To further understand Rgd1p function, we set up a synthetic lethal screen for genes interacting with RGD1 . Study of one lethal mutant made it possible to identify the SLG1 and MID2 genes . The gene SLG1/HCS77/WSC1 was mutated in the original synthetic lethal strain, whereas MID2/SMS1 acted as a monocopy suppressor . The SLG1 gene has been described to be an upstream component in the yeast PKC pathway and encodes a putative cell surface sensor for the activation of cell integrity signalling . First identified by viability loss of shmoos after pheromone exposure, and since found in different genetic screens, MID2 was recently reported as also encoding an upstream activator of the PKC pathway . The RGD1 gene showed genetic interactions with both sensors of cell integrity pathway . The rgd1 slg1 synthetic lethality was rescued by osmotic stabilization, as expected for mutants altered in cell wall integrity . The slight viability defect of rgd1 in minimal medium, which was exacerbated by mid2, was not osmoremediated . As for mutants altered in PKC pathway, the accumulation of small-budded dead cells in slg1, rgd1 and mid2 after heat shock was prevented by 1 M sorbitol . In addition, the rgd1 strain also displayed dead shmoos after pheromone treatment, like mid2 . Taken together, the present results indicate close functional links between RGD1, MID2 and SLG1 and suggest that RGD1 and MID2 interact in a cell integrity signalling functionally linked to the PKC pathway . Yeast, 1999 Dec, 15(16), 1711 - 7 Transcription of some PHO genes in Saccharomyces cerevisiae is regulated by spt7p; Nishimura K et al.; Spt7p is a new global transcription factor in Saccharomyces cerevisiae(Gansheroff et al., 1995) . We report here that the activities of high affinity phosphate transport and acid phosphatase in particular were decreased in a spt7 null mutant . Northern blot experiments revealed that transcription of the PHO84 and PHO5 genes was impaired in this mutant; expression of the PHO regulatory genes, PHO4 and PHO2, was normal . Spt7p is thus linked with expression of several structural genes of the PHO regulon in yeast . Hum Mol Genet, 2000 Jan 1, 9(1), 93 - 100 CGG/CCG repeats exhibit orientation-dependent instability and orientation-independent fragility in Saccharomyces cerevisiae; Balakumaran BS et al.; An expansion to >200 CGG/CCG repeats (hereafter called CGG) in the 5' region of the FMR1 gene causes fragile X syndrome, and this locus becomes a folate-sensitive fragile site . We used Saccharomyces cerevisiae as a model system to study the stability and fragility of CGG repeats . Tracts of (CGG)(81)and (CGG)(160)were integrated onto a yeast chromosome in both orientations relative to the nearest replication origin . Tracts of this length are pre-mutation alleles in humans, with a high probability of expansion in future generations . The CGG tracts in yeast colonies showed a length-dependent instability with longer tracts being more prone to contraction than shorter tracts . In addition, there was an orientation bias for tract stability with tracts having fewer contractions when the CCG strand was the template for lagging strand synthesis . Expansions of the CGG tracts also occurred in an orientation-dependent manner, although at a lower frequency than contractions . To determine whether CGG tracts are fragile sites in yeast, the CGG tracts were flanked by direct repeats, and the rate of recombination between the repeats determined . Strains carrying the (CGG)(160)tract in either orientation had a large increase in their rate of recombination compared with a no-tract control strain . Because this increase was dependent on genes involved in double-strand break repair, recombination was likely to be initiated by CGG tract-induced breakage between the direct repeats . The observation of orientation-dependent instability and orientation-independent fragility suggests that at least some aspects of their underlying mechanisms are different. Mol Gen Genet, 1999 Oct, 262(3), 481 - 92 A novel element in the promoter of the Saccharomyces cerevisiae gene SPS19 enhances ORE-dependent up-regulation in oleic acid and is essential for de-repression; Gurvitz A et al.; In Saccharomyces cerevisiae cells grown on oleic acid, genes encoding enzymes of beta-oxidation are induced by the interaction of a transcription factor composed of Pip2p and Oaflp with an oleate response element (ORE) in their promoters . The SPS19 gene, which encodes peroxisomal 2,4-dienoyl-CoA reductase, an auxiliary beta-oxidation enzyme, has been shown previously to be up-regulated by a canonical ORE . To determine whether additional elements contribute to this transcriptional upregulation, deletion analysis of the SPS19 promoter was conducted using SPS19-lacZ reporter genes . In a reporter construct containing a deletion adjacent to the ORE, transcriptional activation of SPS19 in oleic acid medium was impaired . Together with an additional segment that overlaps a portion of the canonical ORE, this region forms a continuous element (termed UAS(SPS19)) that is essential for de-repression of SPS19 when glucose levels are low . The potentially bi-partite UAS(SPS19) element was able to initiate bi-directional transcription from a promoterless CYC1-lacZ reporter construct under de-repression conditions, whereas the canonical ORE was not . In oleic acid-containing medium, UAS(SPS19) stimulated transcription of the reporter gene 2.4-fold compared to the intact SPS19 ORE, but did so only in the presence of Pip2p and Oaf1p . UAS(SPS19), which is similar to a transcriptional enhancer in the promoter of the sporulation-specific gene SPS4, was shown specifically to bind several proteins, including Pip2p and Oaflp . We propose that UAS(SPS19) and other sequences like it are required to enhance the transcriptional effects mediated by more specific response elements. Mol Gen Genet, 1999 Oct, 262(3), 437 - 47 The mitochondrial cytochrome c peroxidase Ccp1 of Saccharomyces cerevisiae is involved in conveying an oxidative stress signal to the transcription factor Pos9 (Skn7); Charizanis C et al.; In Saccharomyces cerevisiae two transcription factors, Pos9 (Skn7) and Yap1, are involved in the response to oxidative stress . Fusion of the Pos9 response-regulator domain to the Gal4 DNA-binding domain results in a transcription factor which renders the expression of a GAL1-lacZ reporter gene dependent on oxidative stress . To identify genes which are involved in the oxygen-dependent activation of the Gal4-Pos9 hybrid protein we screened for mutants that failed to induce the heterologous test system upon oxidative stress (fap mutants for factors activating Pos9) . We isolated several respiration-deficient and some respiration-competent mutants by this means . We selected for further characterization only those mutants which also displayed an oxidative-stress-sensitive phenotype . One of the respiration-deficient mutants (complementation groupfap6) could be complemented by the ISM1 gene, which encodes mitochondrial isoleucyl tRNA synthetase, suggesting that respiration competence was important for signalling of oxidative stress . In accordance with this notion a rho0 strain and a wild-type strain in which respiration had been blocked (by treatment with antimycin A or with cyanide) also failed to activate Gal4-Pos9 upon imposition of oxidative stress . Another mutant, fap24, which was respiration-competent, could be complemented by CCP1, which encodes the mitochondrial cytochrome c peroxidase . Mitochondrial cytochrome c peroxidase degrades reactive oxygen species within the mitochondria . This suggested a possible sensor function for the enzyme in the oxidative stress response . To test this we used the previously described point mutant ccp1 W191F, which is characterized by a 10(4)-fold decrease in electron flux between cytochrome c and cytochrome c peroxidase . The Ccp1W191F mutant was still capable of activating the Pos9 transcriptional activation domain, suggesting that the signalling function of Ccp1 is independent of electron flux rates. J Mol Biol, 1999 Dec 10, 294(4), 897 - 907 Saccharomyces cerevisiae ISU1 and ISU2: members of a well-conserved gene family for iron-sulfur cluster assembly; Garland SA et al.; Recent studies in bacteria and eukaryotes have led to the identification of several new genes implicated in the biogenesis of iron-sulfur (Fe/S) cluster-containing proteins . This report focuses on two genes of bakers yeast Saccharomyces cerevisiae, ISU1 and ISU2, which encode homologues to bacterial IscU and NifU, potential iron-binding or cluster-assembly proteins . As with other yeast genes implicated in Fe/S protein assembly, deletion of either ISU1 or ISU2 results in increased accumulation of iron within the mitochondria, loss of activity of the {4Fe-4S} aconitase enzyme, and suppression of oxidative damage in cells lacking cytosolic copper/zinc superoxide dismutase . Both genes are induced in strains expressing an activated allele of Aft1p, the iron-sensing transcription factor, suggesting that they are regulated by the iron status of the cell . Immunoblotting studies using an antibody directed against Escherichia coli IscU reveal that both Isu1p and Isu2p are localized primarily in the mitochondria and that Isu1p is the predominant form expressed under all growth conditions tested . The possible role of the Isu proteins in the assembly and/or repair of Fe/S clusters is discussed . Proc Natl Acad Sci U S A, 1999 Dec 7, 96(25), 14366 - 71 Nuclear tRNA aminoacylation and its role in nuclear export of endogenous tRNAs in Saccharomyces cerevisiae; Sarkar S et al.; Nuclear tRNA aminoacylation was proposed to provide a proofreading step in Xenopus oocytes, ensuring nuclear export of functional tRNAs {Lund, E . & Dahlberg, J . E . (1998) Science 282, 2082-2085} . Herein, it is documented that tRNA aminoacylation also occurs in yeast nuclei and is important for tRNA export . We propose that tRNA aminoacylation functions in one of at least two parallel paths of tRNA export in yeast . Alteration of one aminoacyl-tRNA synthetase affects export of only cognate tRNA, whereas alterations of two other aminoacyl-tRNA synthetases affect export of both cognate and noncognate tRNAs . Saturation of tRNA export pathway is a possible explanation of this phenomenon. Proc Natl Acad Sci U S A, 1999 Dec 7, 96(25), 14360 - 5 The polo-box-dependent induction of ectopic septal structures by a mammalian polo kinase, plk, in Saccharomyces cerevisiae; Lee KS et al.; Members of the polo subfamily of protein kinases play pivotal roles in cell-cycle control and proliferation . In addition to a high degree of sequence similarity in the kinase domain, polo kinases contain a strikingly conserved motif termed "polo-box" in the noncatalytic C-terminal domain . We have previously shown that the mammalian polo-like kinase Plk is a functional homolog of Saccharomyces cerevisiae Cdc5 . Here, we show that, in a polo-box- and kinase activity-dependent manner, ectopic expression of Plk in budding yeast can induce a class of cells with abnormally elongated buds . In addition to localization at spindle poles and cytokinetic neck filaments, Plk induces and localizes to ectopic septin ring structures within the elongated buds . In contrast, mutations in the polo-box abolish both localization to, and induction of, septal structures . Consistent with the polo-box-dependent subcellular localization, the C-terminal domain of Plk, but not its polo-box mutant, is sufficient for subcellular localization . Our data suggest that Plk may contribute a signal to initiate or promote cytokinetic event(s) and that an intact polo-box is required for regulation of these cellular processes. Biochemistry, 1999 Dec 7, 38(49), 16195 - 204 Metabolic effects of mislocalized mitochondrial and peroxisomal citrate synthases in yeast Saccharomyces cerevisiae; Velot C et al.; Genes CIT1 and CIT2 from Saccharomyces cerevisiae encode mitochondrial and peroxisomal citrate synthases involved in the Krebs tricarboxylic acid (TCA) cycle and glyoxylate pathway, respectively . A Deltacit1 mutant does not grow on acetate, despite the presence of Cit2p that could, in principle, bypass the resulting block in the TCA cycle . To elucidate this absence of cross-complementation, we have examined the ability of Cit1p to function in the cytosol, and that of Cit2p to function in mitochondria . A cytosolically localized form of Cit1p was also incompetent for restoration of growth of a Deltacit1 strain on acetate, suggesting that mitochondrial localization of Cit1p is essential for its function in the TCA cycle . Cit2p was able, when mislocalized in mitochondria, to restore a wild-type phenotype in a strain lacking Cit1p . We have purified these two isoenzymes as well as mitochondrial malate dehydrogenase, Mdh1p, and have shown that Cit2p was also able to mimic Cit1p in its in vitro interaction with Mdh1p . Models of Cit1p and Cit2p structures generated on the basis of that of pig citrate synthase indicate very high structural and electrostatic surface potential similarities between the two yeast isozymes . Altogether, these data indicate that metabolic functions may require structural as well as catalytic roles for the enzymes. Philos Trans R Soc Lond B Biol Sci, 1999 Sep 29, 354(1389), 1577 - 80; discussion 1580-1 Molecular mechanism of autophagy in yeast, Saccharomyces cerevisiae; Ohsumi Y; Bulk degradation of cytosol and organelles is important for cellular homeostasis under nutrient limitation, cell differentiation and development . This process occurs in a lytic compartment, and autophagy is the major route to the lysosome and/or vacuole . We found that yeast, Saccharomyces cerevisiae, induces autophagy under various starvation conditions . The whole process is essentially the same as macroautophagy in higher eukaryotic cells . However, little is known about the mechanism of autophagy at a molecular level . To elucidate the molecules involved, a genetic approach was carried out and a total of 16 autophagy-defective mutants (apg) were isolated . So far, 14 APG genes have been cloned . Among them we recently found a unique protein conjugation system essential for autophagy . The C-terminal glycine residue of a novel modifier protein Apg12p, a 186-amino-acid protein, is conjugated to a lysine residue of Apg5p, a 294-amino-acid protein, via an isopeptide bond . We also found that apg7 and apg10 mutants were unable to form an Apg12p-Apg5p conjugate . The conjugation reaction is mediated via Apg7p, E1-like activating enzyme and Apg10p, indicating that it is a ubiquitination-like system . These APG genes have mammalian homologues, suggesting that the Apg12 system is conserved from yeast to human . Further molecular and cell biological analyses of APG gene products will give us crucial clues to uncover the mechanism and regulation of autophagy. Philos Trans R Soc Lond B Biol Sci, 1999 Sep 29, 354(1389), 1513 - 22 The Saccharomyces cerevisiae ubiquitin-proteasome system; Hochstrasser M et al.; Our studies of the yeast ubiquitin-proteasome pathway have uncovered a number of general principles that govern substrate selectivity and proteolysis in this complex system . Much of the work has focused on the destruction of a yeast transcription factor, MAT alpha 2 . The alpha 2 protein is polyubiquitinated and rapidly degraded in alpha-haploid cells . One pathway of proteolytic targeting, which depends on two distinct endoplasmic reticulum-localized ubiquitin-conjugating enzymes, recognizes the hydrophobic face of an amphipathic helix in alpha 2 . Interestingly, degradation of alpha 2 is blocked in a/alpha-diploid cells by heterodimer formation between the alpha 2 and a1 homeodomain proteins . The data suggest that degradation signals may overlap protein-protein interaction surfaces, allowing a straightforward steric mechanism for regulated degradation . Analysis of alpha 2 degradation led to the identification of both 20S and 26S proteasome subunits, and several key features of proteasome assembly and active-site formation were subsequently uncovered . Finally, it has become clear that protein (poly) ubiquitination is highly dynamic in vivo, and our studies of yeast de-ubiquitinating enzymes illustrate how such enzymes can facilitate the proteolysis of diverse substrates. Appl Environ Microbiol, 1999 Dec, 65(12), 5398 - 402 Mitochondrial function in cell wall glycoprotein synthesis in Saccharomyces cerevisiae NCYC 625 (Wild type) and {rho(0)} mutants; Iung AR et al.; We studied phosphopeptidomannans (PPMs) of two Saccharomyces cerevisiae NCYC 625 strains (S . diastaticus): a wild type strain grown aerobically, anaerobically, and in the presence of antimycin and a {rho(0)} mutant grown aerobically and anaerobically . The aerobic wild-type cultures were highly flocculent, but all others were weakly flocculent . Ligands implicated in flocculation of mutants or antimycin-treated cells were not aggregated as much by concanavalin A as were those of the wild type . The {rho(0)} mutants and antimycin-treated cells differ from the wild type in PPM composition and invertase, acid phosphatase, and glucoamylase activities . PPMs extracted from different cells differ in the protein but not in the glycosidic moiety . The PPMs were less stable in mitochondrion-deficient cells than in wild-type cells grown aerobically, and this difference may be attributable to defective mitochondrial function during cell wall synthesis . The reduced flocculation of cells grown in the presence of antimycin, under anaerobiosis, or carrying a {rho(0)} mutation may be the consequence of alterations of PPM structures which are the ligands of lectins, both involved in this cell-cell recognition phenomenon . These respiratory chain alterations also affect peripheral, biologically active glycoproteins such as extracellular enzymes and peripheral PPMs. Appl Environ Microbiol, 1999 Dec, 65(12), 5303 - 6 Transformation of Escherichia coli with DNA from Saccharomyces cerevisiae cell lysates; Adam AC et al.; We developed a system to monitor the transfer of heterologous DNA from a genetically manipulated strain of Saccharomyces cerevisiae to Escherichia coli . This system is based on a yeast strain that carries multiple integrated copies of a pUC-derived plasmid . The bacterial sequences are maintained in the yeast genome by selectable markers for lactose utilization . Lysates of the yeast strain were used to transform E . coli . Transfer of DNA was measured by determining the number of ampicillin-resistant E . coli clones . Our results show that transmission of the Amp(r) gene to E . coli by genetic transformation, caused by DNA released from the yeast, occurs at a very low frequency (about 50 transformants per microg of DNA) under optimal conditions (a highly competent host strain and a highly efficient transformation procedure) . These results suggest that under natural conditions, spontaneous transmission of chromosomal genes from genetically modified organisms is likely to be rare. Genetics, 1999 Dec, 153(4), 1583 - 90 Frequent meiotic recombination between the ends of truncated chromosome fragments of Saccharomyces cerevisiae; Arbel T et al.; A single truncated chromosome fragment (TCF) in diploid cells undergoes frequent ectopic recombination during meiosis between markers located near the ends of the fragment . Tetrads produced by diploids with a single TCF show frequent loss of one of the two markers . This marker loss could result either from recombination of the TCF with one of the two copies of the chromosome from which it was derived or from ectopic recombination between the ends of the TCF . The former would result in shortening of a normal chromosome and lethality in one of the four spores . The high frequency of marker loss in tetrads with four viable spores supports recombination between the TCF ends as the main source of marker loss . Most of the spore colonies that display TCF marker loss contained a TCF with the same marker on both ends . Deletion of most of the pBR322 sequences distal to the marker at one of the subtelomeric regions of the TCF did not reduce the overall frequency of recombination between the ends, but affected the loss of one marker significantly more than the other . We suggest that the mechanism by which the duplication of one end marker and loss of the other occurs is based on association and recombination between the ends of the TCF. Genetics, 1999 Dec, 153(4), 1547 - 60 The role of centromere alignment in meiosis I segregation of homologous chromosomes in Saccharomyces cerevisiae; Guerra CE et al.; During meiosis, homologous chromosomes pair and then segregate from each other at the first meiotic division . Homologous centromeres appear to be aligned when chromosomes are paired . The role of centromere alignment in meiotic chromosome segregation was investigated in Saccharomyces cerevisiae diploids that contained one intact copy of chromosome I and one copy bisected into two functional centromere-containing fragments . The centromere on one fragment was aligned with the centromere on the intact chromosome while the centromere on the other fragment was either aligned or misaligned . Fragments containing aligned centromeres segregated efficiently from the intact chromosome, while fragments containing misaligned centromeres segregated much less efficiently from the intact chromosome . Less efficient segregation was correlated with crossing over in the region between the misaligned centromeres . Models that suggest that these crossovers impede proper segregation by preventing either a segregation-promoting chromosome alignment on the meiotic spindle or some physical interaction between homologous centromeres are proposed. Genetics, 1999 Dec, 153(4), 1535 - 46 Saccharomyces cerevisiae G1 cyclins are differentially involved in invasive and pseudohyphal growth independent of the filamentation mitogen-activated protein kinase pathway; Loeb JD et al.; Several lines of evidence suggest that the morphogenetic transition from the yeast form to pseudohyphae in Saccharomyces cerevisiae may be regulated by the cyclin-dependent kinase (Cdk) . To examine this hypothesis, we mutated all of the G1 cyclin genes in strains competent to form pseudohyphae . Interestingly, mutation of each G1 cyclin results in a different filamentation phenotype, varying from a significant defect in cln1/cln1 strains to enhancement of filament production in cln3/cln3 strains . cln1 cln2 double mutants are more defective in pseudohyphal development and haploid invasive growth than cln1 strains . FLO11 transcription, which correlates with the level of invasive growth, is low in cln1 cln2 mutants and high in grr1 cells (defective in proteolysis of Cln1,2), suggesting that Cln1,2/Cdks regulate the pseudohyphal transcriptional program . Epistasis analysis reveals that Cln1,2/Cdk and the filamentation MAP kinase pathway function in parallel in regulating filamentous and invasive growth . Cln1 and Cln2, but not Ste20 or Ste12, are responsible for most of the elevated FLO11 transcription in grr1 strains . Furthermore, phenotypic comparison of various filamentation mutants illustrates that cell elongation and invasion/cell-cell adhesion during filamentation are separable processes controlled by the pseudohyphal transcriptional program . Potential targets for G1 cyclin/Cdks during filamentous growth are discussed. Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 190 - 5 Difference in strength of autonomously replicating sequences among repeats in the rDNA region of Saccharomyces cerevisiae; Reppe S et al.; The rDNA region of Saccharomyces cerevisiae contains 100-200 tandemly repeated copies of a 9 kb unit, each with a potential replication origin . In the present studies of cloned fragments from the region involved in the regulation of replication of rDNA, we detected differences in autonomously replicating sequence (ARS) activity for clones from the same yeast strain . One clone, which showed very low ARS activity, carried a point mutation, a C instead of T, in position 9 of the essential 11 bp consensus ARS as compared to clones carrying the normal 10-of-11-bp match to the consensus . The mutation could be traced back to genomic rDNA where it represents about one-third of the rDNA units in that strain . Differences in ARS activity have implications for understanding the regulation of replication of rDNA, and the ratio of active to inactive ARS in the rDNA region may be important for potential generation of extrachromosomal copies . Mech Ageing Dev, 1999 Oct 1, 110(1-2), 119 - 29 The DLP1 mutant of the yeast Saccharomyces cerevisiae with an increased copy number of the 2micron plasmid shows a shortened lifespan; Xu Z et al.; We isolated and characterized a recessive mutant, named dlp1, which shows the Dlp phenotype (delayed loss of proliferation activity) during the autophagic death of cdc28 . The dip1 mutant was found to consist of two subtypes of cells based on colony morphology . One subtype with the Dlp phenotype, named dlp1-1, became large, red, and nibbled during the incubation, suggesting that the cells on the surface of the colonies were dying . The other without the Dlp phenotype, named dlp1-s, retained small, white colonies even after a prolonged incubation and was found to be a petite mutant . The change from dlp1-1 to dlp1-s (petite) occurred much more frequently (about 15%) than that from the wild-type to petite mutant (less than 1%) . The lifespan of both subtypes of cells was severely shortened . The copy number of the endogenous 2micron plasmid of dlp1-1 was 68-fold that of the original cdc28, and decreased by half after the conversion to dlp1-s (petite) . A 4.0-kbp fragment of the 2micron plasmid containing REP2 decreased the copy number of the endogenous 2micron plasmid to 8-fold that of the original cdc28 cells and partially rescued the shortened lifespan, in addition to resulting in the complete complementation of the Dlp and nibbled-colony phenotypes . These results suggest that DLP1 is a chromosomal gene that regulates the copy number of the 2micron plasmid, and that the shortening of the lifespan and other effects of the dlp1 mutation are likely caused by the increased copy number of the endogenous 2micron plasmid. FEBS Lett, 1999 Nov 26, 462(1-2), 37 - 42 Intracellular localization of an active green fluorescent protein-tagged Pho84 phosphate permease in Saccharomyces cerevisiae; Petersson J et al.; Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the carboxy-terminus of the Pho84 phosphate permease of Saccharomyces cerevisiae . Both components of the fusion protein displayed their native functions and revealed a cellular localization and degradation of the Pho84-GFP chimera consistent with the behavior of the wild-type Pho84 protein . The GFP-tagged chimera allowed for a detection of conditions under which the Pho84 transporter is localized to its functional environment, i.e . the plasma membrane, and conditions linked to relocation of the protein to the vacuole for degradation . By use of the methodology described, GFP should be useful in studies of localization and degradation also of other membrane proteins in vivo. FEBS Lett, 1999 Sep 24, 458(3), 309 - 12 Molecular characterization of Vig4/Vrg4 GDP-mannose transporter of the yeast Saccharomyces cerevisiae; Abe M et al.; Saccharomyces cerevisiae Vig4/Vrg4 protein is a Golgi membrane protein which has multiple transmembrane domains and is essential for transport of GDP-mannose across the Golgi membrane . By immunoprecipitation of detergent-solubilized tagged protein, we found that this protein exists as oligomer . Two mutants vig4-1 and vig4-2 had amino acid substitutions in the C-terminal region, Ala286Val and Ser278Cys, respectively . In accord with these mutations, trimming of the C-terminal hydrophobic part close to the region impaired the function and traffic of the proteins from the endoplasmic reticulum to the Golgi compartments. J Nutr, 1999 Dec, 129(12), 2236 - 8 Boron stimulates yeast (Saccharomyces cerevisiae) growth; Bennett A et al.; Boron is required for the growth of vascular plants and embryonic development in fish . The molecular basis of boron's essentiality, however, remains unknown for both . The objective of this study was to determine whether yeast (Saccharomyces cerevisiae) could be used as a model for the evaluation of intracellular boron trafficking . Three experiments were conducted to assess the effect of boron supplementation on yeast growth . Cultures were grown in low boron media containing 0.04 micromol B/L . After 24 h, a new flask was inoculated with this culture; it was allowed to reach early log phase growth (9 h) and was then divided between two flasks . One flask was supplemented with ultrapure boric acid to achieve a concentration of 185 micromol B/L (+B); the other was supplemented with an equivalent volume of ultrapure water (NB) . Boron significantly stimulated cell growth rate into the stationary phase of growth . Yeast cell boron concentrations decreased in both treatments over the course of the experiment, but analysis by inductively coupled plasma-mass spectrometry (ICPMS) did not detect differences in cellular concentration between the boron supplemented (B) and nonsupplemented (NB) groups . Ethanol concentrations did not differ between the two treatments, demonstrating that boron-stimulated growth was not a secondary effect of alcohol dehydrogenase inhibition . The demonstration of boron-dependent growth stimulation in yeast suggests that Saccharomyces cerevisiae can be used as a model system for the study of intracellular boron trafficking. Biochim Biophys Acta, 1999 Oct 18, 1472(1-2), 395 - 8 Sensitivity of antioxidant-deficient yeast Saccharomyces cerevisiae to peroxynitrite and nitric oxide; Jakubowski W et al.; Sensitivity of Saccharomyces cerevisiae strains deficient in superoxide dismutases and catalases and of decreased level of glutathione to peroxynitrite and a nitric oxide donor, S-nitrosoglutathione was compared . Moderate but significant differences observed point to increased sensitivity to both agents of yeast deficient in antioxidant defense, the superoxide dismutase-deficient strain showing the highest sensitivity, The sequence of sensitivity of various strains to peroxynitrite and nitric oxide was the same . The results are compatible with the view that cytotoxic effects of peroxynitrite involve formation of secondary reactive oxygen species. Yeast, 1999 Nov, 15(15), 1691 - 701 Disruption and phenotypic analysis of six novel genes from chromosome IV of Saccharomyces cerevisiae reveal YDL060w as an essential gene for vegetative growth; Casalone E et al.; The disruption of six novel genes (YDL059c, YDL060w, YDL063c, YDL065c, YDL070w and YDL110c), localized on the left arm of chromosome IV in Saccharomyces cerevisiae, is reported . A PCR-based strategy was used to construct disruption cassettes in which the kanMX4 dominant marker was introduced between two long flanking homology regions, homologous to the promoter and terminator sequences of the target gene (Wach et al., 1994) . The disruption cassettes were used to generate homologous recombinants in two diploid strains with different genetic backgrounds (FY1679 and CEN . PK2), selecting for geneticin (G418) resistance conferred by the presence of the dominant marker kanMX4 . The correctness of the cassette integration was tested by PCR . After sporulation and tetrad analysis of the heterozygous deletant diploids, geneticin-resistant haploids carrying the disrupted allele were isolated . YDL060w was shown to be an essential gene for vegetative growth . A more detailed phenotypic analysis of the non-lethal haploid deletant strains was performed, looking at cell and colony morphology, growth capability on different media at different temperatures, and ability to conjugate . Homozygous deletant diploids were also constructed and tested for sporulation . Only minor differences between parental and mutant strains were found for some deletant haploids . Yeast, 1999 Nov, 15(15), 1619 - 29 A potential role of the cytoskeleton of Saccharomyces cerevisiae in a functional organization of glycolytic enzymes; Gotz R et al.; Numerous individual enzymes participate in a given synthetic or degradative pathway in which the product of one reaction becomes the substrate for the subsequent enzyme . This raises the question of whether the product of one 'soluble' enzyme diffuses freely through the available cell volume, where it accidentally collides with the subsequent 'soluble' enzyme . Alternatively, enzymes acting in a given pathway may be organized in ordered structures, metabolons . Certain glycolytic enzymes have been shown to co-localize with the cytoskeleton in mammalian cells . We deleted genes coding for proteins associated with the cytoskeleton of Saccharomyces cerevisiae: TPM1 coding for tropomyosin, SAC6 for fimbrin and CIN1 for a microtubule-associated protein . Single deletions or deletions of two such genes had no effect on the specific activities of glycolytic enzymes, or on the rates of glucose consumption and ethanol production . However, the concentrations of glycolytic metabolites during a switch from a gluconeogenic mode of metabolism, growth on an ethanol medium, to glycolysis after glucose addition showed transient deviations from the normal change in metabolite concentrations, as observed in wild type cells . However, all metabolites in mutant strains reached wild-type levels within 2-4 h after the shift . Only ATP levels remained low in all but the tmp1-Delta-sac6-Delta double mutant strains . These observations can be interpreted to mean that metabolic reorganization from a gluconeogenic to a glycolytic metabolism is facilitated by an intact cytoskeleton in yeast . Nucleic Acids Res, 1999 Dec 15, 27(24), 4751 - 8 Different positioning elements select poly(A) sites at the 3'-end of GCN4 mRNA in the yeast Saccharomyces cerevisiae; Duvel K et al.; Cleavage and polyadenylation of eukaryotic mRNA requires efficiency and positioning elements in the 3'-untranslated region (3'-UTR) of the mRNA . Specific point mutations were introduced into the yeast GCN4 3'-UTR to detect sequence motifs which are involved in the positioning of the poly(A) site . 3'-End proces-sing activities of different GCN4 3'-UTR alleles were measured in an in vivo test system . Point mutations in an AAGAA motif defocussed selection of the poly(A) sites of the GCN4 3'-UTR to various additional poly(A) sites instead of the single site of the wild-type GCN4 3'-UTR . A strain with an intact wild-type GCN4 3'-UTR but impaired in RNA15 encoding an RNA-binding processing factor showed a similar defocussed pattern of poly(A) site selection . Remarkably, two additional sequence motifs upstream of the AAGAA motif which resemble yeast efficiency motifs independently affected poly(A) site positioning but not efficiency of 3'-end processing . Mutations in one motif resulted in an additional upstream poly(A) site . Alterations of the other motif shifted the poly(A) sites exclusively to two downstream poly(A) sites . These data suggest several contact points between the precursor mRNA and the polyadenylation machinery in yeast. Infect Immun, 1999 Dec, 67(12), 6619 - 30 WdChs4p, a homolog of chitin synthase 3 in Saccharomyces cerevisiae, alone cannot support growth of Wangiella (Exophiala) dermatitidis at the temperature of infection; Wang Z et al.; By using improved transformation methods for Wangiella dermatitidis, and a cloned fragment of its chitin synthase 4 structural gene (WdCHS4) as a marking sequence, the full-length gene was rescued from the genome of this human pathogenic fungus . The encoded chitin synthase product (WdChs4p) showed high homology with Chs3p of Saccharomyces cerevisiae and other class IV chitin synthases, and Northern blotting showed that WdCHS4 was expressed at constitutive levels under all conditions tested . Reduced chitin content, abnormal yeast clumpiness and budding kinetics, and increased melanin secretion resulted from the disruption of WdCHS4 suggesting that WdChs4p influences cell wall structure, cellular reproduction, and melanin deposition, respectively . However, no significant loss of virulence was detected when the wdchs4Delta strain was tested in an acute mouse model . Using a wdchs1Delta wdchs2Delta wdchs3Delta triple mutant of W . dermatitidis, which grew poorly but adequately at 25 degrees C, we assayed WdChs4p activity in the absence of activities contributed by its three other WdChs proteins . Maximal activity required trypsin activation, suggesting a zymogenic nature . The activity also had a pH optimum of 7.5, was most stimulated by Mg(2+), and was more inhibited by polyoxin D than by nikkomycin Z . Although the WdChs4p activity had a broad temperature optimum between 30 to 45 degrees C in vitro, this activity alone did not support the growth of the wdchs1Delta wdchs2Delta wdchs3Delta triple mutant at 37 degrees C, a temperature commensurate with infection. FEBS Lett, 1999 Nov 19, 461(3), 173 - 7 The effect of iron limitation on glycerol production and expression of the isogenes for NAD(+)-dependent glycerol 3-phosphate dehydrogenase in Saccharomyces cerevisiae; Ansell R et al.; When deprived of iron, Saccharomyces cerevisiae rearranges its metabolic flux towards increased glycerol production . This work examines the role and regulation of GPD1 and GPD2, encoding two isoforms of glycerol 3-phosphate dehydrogenase, in glycerol production during iron starvation . The two genes respond differently on transfer of cells to iron-limited conditions . Whereas the expression of GPD2 increases about 3-fold, that of GPD1 does not exhibit significant changes . Deletion of either GPD1 or GPD2 alters the capacity for glycerol production during iron-limited as well as iron sufficient conditions . However, loss of function of either gene does not seem to provoke compensatory flux via the other gene product . As judged from the glycerol production, the amount produced by each single mutant adds approximately up to the level produced by the parental strain . In agreement with the pattern of expression of GPD2, this gene product was estimated to account for the bulk of the glycerol production (about 60%) during iron-limited conditions . The strong growth inhibition caused by iron starvation was reversed by the addition of iron also for a gpd1Deltagpd2Delta double deletion mutant, which is unable to produce any detectable glycerol. Mol Cell Biol, 1999 Dec, 19(12), 8180 - 90 Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae; Rodriguez-Manzaneque MT et al.; Glutaredoxins are members of a superfamily of thiol disulfide oxidoreductases involved in maintaining the redox state of target proteins . In Saccharomyces cerevisiae, two glutaredoxins (Grx1 and Grx2) containing a cysteine pair at the active site had been characterized as protecting yeast cells against oxidative damage . In this work, another subfamily of yeast glutaredoxins (Grx3, Grx4, and Grx5) that differs from the first in containing a single cysteine residue at the putative active site is described . This trait is also characteristic for a number of glutaredoxins from bacteria to humans, with which the Grx3/4/5 group has extensive homology over two regions . Mutants lacking Grx5 are partially deficient in growth in rich and minimal media and also highly sensitive to oxidative damage caused by menadione and hydrogen peroxide . A significant increase in total protein carbonyl content is constitutively observed in grx5 cells, and a number of specific proteins, including transketolase, appear to be highly oxidized in this mutant . The synthetic lethality of the grx5 and grx2 mutations on one hand and of grx5 with the grx3 grx4 combination on the other points to a complex functional relationship among yeast glutaredoxins, with Grx5 playing a specially important role in protection against oxidative stress both during ordinary growth conditions and after externally induced damage . Grx5-deficient mutants are also sensitive to osmotic stress, which indicates a relationship between the two types of stress in yeast cells. Mol Cell Biol, 1999 Dec, 19(12), 8083 - 93 Telomere-telomere recombination is an efficient bypass pathway for telomere maintenance in Saccharomyces cerevisiae; Teng SC et al.; Many Saccharomyces telomeres bear one or more copies of the repetitive Y' element followed by approximately 350 bp of telomerase-generated C(1-3)A/TG(1-3) repeats . Although most cells lacking a gene required for the telomerase pathway die after 50 to 100 cell divisions, survivors arise spontaneously in such cultures . These survivors have one of two distinct patterns of telomeric DNA (V . Lundblad and E . H . Blackburn, Cell 73:347-360, 1993) . The more common of the two patterns, seen in type I survivors, is tandem amplification of Y' followed by very short tracts of C(1-3)A/TG(1-3) DNA . By determining the structure of singly tagged telomeres, chromosomes in type II survivors were shown to end in very long and heterogeneous-length tracts of C(1-3)A/TG(1-3) DNA, with some telomeres having 12 kb or more of C(1-3)A/TG(1-3) repeats . Maintenance of these long telomeres required the continuous presence of Rad52p . Whereas type I survivors often converted to the type II structure of telomeric DNA, the type II pattern was maintained for at least 250 cell divisions . However, during outgrowth, the structure of type II telomeres was dynamic, displaying gradual shortening as well as other structural changes that could be explained by continuous gene conversion events with other telomeres . Although most type II survivors had a growth rate similar to that of telomerase-proficient cells, their telomeres slowly returned to wild-type lengths when telomerase was reintroduced . The very long and heterogeneous-length telomeres characteristic of type II survivors in Saccharomyces are reminiscent of the telomeres in immortal human cell lines and tumors that maintain telomeric DNA in the absence of telomerase. J Biol Chem, 1999 Nov 26, 274(48), 34294 - 300 Direct involvement of phosphatidylinositol 4-phosphate in secretion in the yeast Saccharomyces cerevisiae; Hama H et al.; The SEC14 gene encodes an essential phosphatidylinositol (PtdIns) transfer protein required for formation of Golgi-derived secretory vesicles in yeast . Suppressor mutations that rescue temperature-sensitive sec14 mutants provide an approach for determining the role of Sec14p in secretion . One suppressor, sac1-22, causes accumulation of PtdIns(4)P . SAC1 encodes a phosphatase that can hydrolyze PtdIns(4)P and certain other phosphoinositides . These findings suggest that PtdIns(4)P is limiting in sec14 cells and that elevation of PtdIns(4)P production can suppress the secretory defect . Correspondingly, we found that PtdIns(4)P levels were decreased significantly in sec14-3 mutants shifted to 37 degrees C and that sec14-3 cells could grow at an otherwise nonpermissive temperature (34 degrees C) when carrying a plasmid overexpressing PIK1, encoding one of two essential PtdIns 4-kinases . This effect is specific because overexpression of the other PtdIns 4-kinase gene (STT4) or a PtdIns 3-kinase gene (VPS34) did not rescue sec14-3 cells . To further address Pik1p function in secretion, two different pik1(ts) mutants were examined . Upon shift to restrictive temperature (37 degrees C), the PtdIns(4)P levels dropped by about 60% in both pik1(ts) strains within 1 h . During the same period, cells displayed a reduction (40-50%) in release of a secreted enzyme (invertase) . However, similar treatment did not effect maturation of a vacuolar enzyme (carboxypeptidase Y) . These findings indicate that, first, PtdIns(4)P limitation is a major contributing factor to the secretory defect in sec14 cells; second, Sec14p function is coupled to the action of Pik1p, and; third, PtdIns(4)P has an important role in the Golgi-to-plasma membrane stage of secretion. J Biol Chem, 1999 Nov 26, 274(48), 33843 - 6 Generation of the amyloid-beta peptide N terminus in Saccharomyces cerevisiae expressing human Alzheimer's amyloid-beta precursor protein; Greenfield JP et al.; The Alzheimer's amyloid-beta precursor protein (betaAPP) is a type 1 membrane-spanning protein from which the Alzheimer's disease amyloid-beta peptide (Abeta) is proteolytically derived . To date, attempts to identify the enzymes responsible for Abeta generation have failed . Here we report the accumulation of Abeta-immunoreactive peptides in yeast expressing human betaAPP . Characterization of these peptides by metabolic labeling, immunoprecipitation with Abeta-specific antibodies, and N-terminal radiosequencing indicates that these peptides include the Abeta peptide at their N termini . The Abeta-like peptides generated in yeast were recovered predominantly as 8- and 12-14-kDa species . A 4-kDa species was recovered either when a protease-deficient strain was used to prevent breakdown or when the 8- and 12-14-kDa species were treated with disaggregating agents . The likely existence in yeast of enzymes generating the Abeta N terminus indicates that the molecular identification of yeast beta-secretase-like enzymes may be accomplished using genetic screens or empirical approaches based upon the sequenced genome of Saccharomyces cerevisiae. FEMS Microbiol Lett, 1999 Dec 1, 181(1), 177 - 85 Variations in mRNA transcript levels of cell wall-associated genes of Saccharomyces cerevisiae following spheroplasting; Braley R et al.; mRNA transcript levels of 38 genes from Saccharomyces cerevisiae were investigated during attempted spheroplast regeneration . Many of the genes selected are involved in cell wall biosynthesis . Spheroplasts did not regenerate into osmotically competent cells during the experiment . However, at a mRNA level, the quantities of transcripts were altered between the experimental and control populations . KRE11, EGT2 and MSS10 had their transcript levels increased by more than 10-fold during attempted spheroplast regeneration . A further six genes, FLO1, TIR1, SED1, HKR1, YGR189 and MUC1, showed transcript level increases of at least 5-fold . Five genes showed a change in transcript levels from an undetectable level to a detectable level: SKT5, KRE1, KRE5, SEC53 and DHS1 . PMT2 showed a rapid decrease in mRNA levels followed by an increase to the basal level . Thus, cell stress genes, biosynthetic genes and some glycosylphosphatidylinositol-anchored cell wall proteins have their transcript levels increased in regenerating spheroplasts, but their transcription was not sufficient to initiate the replacement of the cell wall in liquid medium. Biochim Biophys Acta, 1999 Nov 16, 1472(3), 587 - 94 Saccharomyces cerevisiae cultured under aerobic and anaerobic conditions: air-level oxygen stress and protection against stress; Ohmori S et al.; Cells of Saccharomyces cerevisiae were grown aerobically and anaerobically, and levels of the protective compounds, cysteine and glutathione, and activities of defensive enzymes, catalase and superoxide dismutase, against an oxygen stress were determined and compared in both cells . Aerobiosis increased both the compounds and enzyme activities . The elevated synthesis of glutathione could be associated with the increased levels of cysteine which in its turn was found to be controlled by the oxygen-dependent activation of cystathionine beta-synthase. Mol Microbiol, 1999 Nov, 34(4), 701 - 13 Contribution of N- and C-terminal domains to the function of Hsp90 in Saccharomyces cerevisiae; Scheibel T et al.; The molecular chaperone Hsp90 is a regulatory component of some key signalling proteins in the cytosol of eukaryotic cells . For some of these functions, its interaction with co-chaperones is required . Limited proteolysis defined stable folded units of Hsp90 . Both an N-terminal (N210) and a C-terminal (262C) fragment interact with non-native substrate proteins in vitro, but with different specificity and ATP dependence . Here, we analysed the functional properties of these Hsp90 fragments in vivo and in vitro . We determined their influence on the general viability and cell growth of Saccharomyces cerevisiae . Expression of N210 or 262C resulted in a dominant-negative phenotype in several yeast strains tested . Their expression was not toxic, but inhibited cell growth . Further, both were unable to restore viability to Hsp90-depleted cells . In addition, N210 and 262C influence the maturation of Hsp90 substrates, such as the glucocorticoid receptor and pp60v-Src kinase . Specifically, 262C forms partially active chaperone complexes, leading to an arrest of the chaperoned substrate at a certain stage of its maturation cycle . This demonstrates the requirement of a sophisticated and cofactor-regulated interplay between N- and C-terminal activities for Hsp90 function in vivo. Tsitologiia, 1999, 41(8), 685 - 92 {C-terminal domain of saccharomyces cerevisiae protein ChI4 binds to centromere DNA fragment of yeast chromosome III}; Shmelev AV et al.; We have expressed in Escherichia coli a recombinant protein consisting of N-terminal peptide omega 10s10 (11 aa) fused with part (aa 135-458) of yeast protein Chl4 involved in the chromosome segregation in Saccharomyces cerevisiae . Mice were immunized with the antigen purified from inclusion bodies, and a polyclonal serum against yeast protein Chl4 was raised . MW of the detected yeast protein Chl4 was approximately 54 kDa, corresponding to the full length ORF translation . C-terminal portion of Chl4 (aa 376-458), containing Helin-Turn-Helix (HTH) motif of DNA-binding, was fused in frame after E . coli maltose binding protein MalE . The soluble fusion was affinity purified using an alternative procedure on the preswollen amylose column . This protein and a 32P labelled 620 bp fragment of yeast CEN3 DNA were used in the DNA-mobility shift assay in polyacrylamide and agarose gels . The binding was detected in the presence and absence of Zn2+ ions . The data obtained could support participation of Chl4 in a direct binding to the yeast centromere in the CBF complex . The result is in a certain agreement with the data on photocrosslinking proteins of the CBF3 complex with the centromere DNA, where the minor protein with a molecular weight of 55-55 kDa was also detected (Espelin C . W . et al., 1997 . J . Cell Biol . 139: 1383-1396). EMBO J, 1999 Nov 15, 18(22), 6448 - 54 Sum1 and Hst1 repress middle sporulation-specific gene expression during mitosis in Saccharomyces cerevisiae; Xie J et al.; Meiotic development in yeast is characterized by the sequential induction of temporally distinct classes of genes . Genes that are induced at the middle stages of the pathway share a promoter element, termed the middle sporulation element (MSE), which interacts with the Ndt80 transcriptional activator . We have found that a subclass of MSEs are strong repressor sites during mitosis . SUM1 and HST1, genes previously associated with transcriptional silencing, are required for MSE-mediated repression . Sum1 binds specifically in vitro to MSEs that function as strong repressor sites in vivo . Repression by Sum1 is gene specific and does not extend to neighboring genes . These results suggest that mechanisms used to silence large regions of chromatin may also be used to regulate the expression of specific genes during development . NDT80 is regulated during mitosis by both the Sum1 and Ume6 repressors . These results suggest that progression through sporulation may be controlled by the regulated competition between the Sum1 repressor and Ndt80 activator at key MSEs. J Cell Biol, 1999 Nov 15, 147(4), 845 - 56 PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae; Holly SP et al.; During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and cell surface growth are polarized, mediating bud emergence, bud growth, and cytokinesis . We have determined whether p21-activated kinase (PAK)-family kinases regulate cell and actin polarization at one or several points during the yeast cell cycle . Inactivation of the PAK homologues Ste20 and Cla4 at various points in the cell cycle resulted in loss of cell and actin cytoskeletal polarity, but not in depolymerization of F-actin . Loss of PAK function in G1 depolarized the cortical actin cytoskeleton and blocked bud emergence, but allowed isotropic growth and led to defects in septin assembly, indicating that PAKs are effectors of the Rho-guanosine triphosphatase Cdc42 . PAK inactivation in S/G2 resulted in depolarized growth of the mother and bud and a loss of actin polarity . Loss of PAK function in mitosis caused a defect in cytokinesis and a failure to polarize the cortical actin cytoskeleton to the mother-bud neck . Cla4-green fluorescent protein localized to sites where the cortical actin cytoskeleton and cell surface growth are polarized, independently of an intact actin cytoskeleton . Thus, PAK family kinases are primary regulators of cell and actin cytoskeletal polarity throughout most or all of the yeast cell cycle . PAK-family kinases in higher organisms may have similar functions. Mol Microbiol, 1999 Oct, 34(2), 247 - 56 A constitutive role for GPI anchors in Saccharomyces cerevisiae: cell wall targeting; De Sampaio G et al.; GPI anchors are widely represented among organisms and have several cellular functions . It has been proposed that in yeast there are two groups of GPI proteins: plasma membrane-resident proteins, such as Gas1p or Yap3p, and cell wall-targeted proteins, such as Tir1p or alpha-agglutinin . A model has been proposed for the plasma membrane retention of proteins from the first group because of a dibasic motif located just upstream of the GPI-anchoring signal . The results we report here are not in agreement with such a model as we show that constructs containing the C-terminal parts of Gas1p and Yap3p are also targeted to the cell wall . We also detect the genuine Gas1p after cell wall treatment with Quantazyme or Glucanex glycanases . In addition, we show that the GPI-anchoring signal from the human placental alkaline phosphatase (PLAP) is not compatible with the yeast machinery unless the human transamidase hGpi8p is co-expressed . In this condition, this human signal is able to target a protein to the cell wall . Moreover, TIR1 proved to be a multicopy suppressor of Deltagas1 mutation . The present findings suggest a constitutive role for GPI anchors in yeast: the cell wall targeting of proteins. Biochim Biophys Acta, 1999 Sep 21, 1421(1), 64 - 76 Transbilayer movement of phosphatidylcholine in the mitochondrial outer membrane of Saccharomyces cerevisiae is rapid and bidirectional; Janssen MJ et al.; The process of transmembrane movement of phosphatidylcholine (PC) across the outer membrane of mitochondria was investigated in vitro in mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces cerevisiae . Phosphatidylcholine-transfer protein (PC-TP) was used to extract radiolabeled PC from OMV, with small unilamellar vesicles serving as acceptor system . Endogenously radiolabeled PC synthesized either via the CDP-choline pathway or via methylation of phosphatidylethanolamine can be extracted completely from the OMV with a t(1/2) of 1 min or less at 30 degrees C . The size of the pool of PC in OMV available for exchange by PC-TP is not affected by pretreatment of the OMV with proteinase K or sulfhydryl reagents . In the reverse experiment where radiolabeled PC was introduced into the OMV, similar characteristics for the exchange were found . The accessibility of labeled PC to externally added phospholipase A(2) was used as a measure for its transmembrane distribution . It was found that PC is not exclusively located in the outer leaflet of the OMV . Only 30-35% can be degraded in intact OMV by phospholipase A(2), irrespective of whether the PC is introduced by PC-TP or endogenously synthesized via either of the pathways of biosynthesis . The results demonstrate the occurrence of rapid bidirectional transbilayer movement of both endogenous and in vitro introduced PC in OMV . Furthermore, there appears to be no preference for mitochondrial import of PC synthesized by either of the pathways in vivo. J Bacteriol, 1999 Nov, 181(22), 7052 - 64 Synergistic operation of the CAR2 (Ornithine transaminase) promoter elements in Saccharomyces cerevisiae; Park HD et al.; Dal82p binds to the UIS(ALL) sites of allophanate-induced genes of the allantoin-degradative pathway and functions synergistically with the GATA family Gln3p and Gat1p transcriptional activators that are responsible for nitrogen catabolite repression-sensitive gene expression . CAR2, which encodes the arginine-degradative enzyme ornithine transaminase, is not nitrogen catabolite repression sensitive, but its expression can be modestly induced by the allantoin pathway inducer . The dominant activators of CAR2 transcription have been thought to be the ArgR and Mcm1 factors, which mediate arginine-dependent induction . These observations prompted us to investigate the structure of the CAR2 promoter with the objectives of determining whether other transcription factors were required for CAR2 expression and, if so, of ascertaining their relative contributions to CAR2's expression and control . We show that Rap1p binds upstream of CAR2 and plays a central role in its induced expression irrespective of whether the inducer is arginine or the allantoin pathway inducer analogue oxalurate (OXLU) . Our data also explain the early report that ornithine transaminase production is induced when cells are grown with urea . OXLU induction derives from the Dal82p binding site, which is immediately downstream of the Rap1p site, and Dal82p functions synergistically with Rap1p . This synergism is unlike all other known instances of Dal82p synergism, namely, that with the GATA family transcription activators Gln3p and Gat1p, which occurs only in the presence of an inducer . The observations reported suggest that CAR2 gene expression results from strong constitutive transcriptional activation mediated by Rap1p and Dal82p being balanced by the down regulation of an equally strong transcriptional repressor, Ume6p . This balance is then tipped in the direction of expression by the presence of the inducer . The formal structure of the CAR2 promoter and its operation closely follow the model proposed for CAR1. Mutat Res, 1999 Oct 22, 435(2), 129 - 39 Transient expression of Saccharomyces cerevisiae endo-exonuclease NUD1 gene increases the frequency of extrachromosomal homologous recombination in mouse Ltk- fibroblasts; Semionov A et al.; Endo-exonucleases (EEs) are nucleolytic enzymes which have been shown to participate in the processes of DNA repair and recombination in eukaryotes . Recently, we have demonstrated that transient expression of Saccharomyces cerevisiae EE NUD1 gene in HeLa cells increased the resistance of the latter to ionizing radiation and cisplatin, suggesting the involvement of the NUD1 gene product in the recombination repair of double-strand breaks (DSB) . Here, we report that transient expression of NUD1 results in up to 62% increase in the frequency of homologous recombination between two co-transfected linear plasmids in mouse Ltk- cells. FEBS Lett, 1999 Nov 5, 460(3), 411 - 6 Dominant-negative alleles of 14-3-3 proteins cause defects in actin organization and vesicle targeting in the yeast Saccharomyces cerevisiae; Roth D et al.; 14-3-3 Proteins are thought to function as adapters in signaling complexes {1,2}, thereby participating in cellular processes including vesicle trafficking and exocytosis {3,4} . To delineate further the function of 14-3-3 proteins during vesicle trafficking, we generated dominant-negative alleles of the two 14-3-3 homologues, Bmh1p and Bmh2p, in budding yeast and analyzed their phenotype in respect to exocytosis . Cells overexpressing the carboxy-terminal region of Bmh2p failed to polarize vesicular transport although bulk exocytosis remained unaffected and showed a disrupted actin cytoskeleton . Our data suggest that 14-3-3 proteins may act primarily on the actin cytoskeleton to regulate vesicle targeting. Nucleic Acids Res, 1999 Dec 1, 27(23), 4598 - 608 Spb1p is a putative methyltransferase required for 60S ribosomal subunit biogenesis in Saccharomyces cerevisiae; Kressler D et al.; Several mutants ( spb1 - spb7 ) have been previously identified as cold-sensitive extragenic suppressors of loss-of-function mutations in the poly(A)(+)-binding protein 1 of Saccharomyces cerevisiae . Cloning, sequence and disruption analyses revealed that SPB1 (YCL054W) encodes an essential putative S -adenosylmethionine-dependent methyltransferase . Polysome analyses showed an under-accumulation of 60S ribosomal subunits in the spb1-1 mutant and in a strain genetically depleted of Spb1p . Northern and primer extension analyses indicated that this was due to inhibition of processing of the 27SB precursors, which results in depletion of the mature 25S and 5.8S rRNAs . At later time points of Spb1p depletion, the stability of 40S ribosomal subunits is also affected . These results suggest that Spb1p is involved in 60S ribosomal subunit biogenesis and associates early with the pre-ribosomes . Consistent with this, hemagglutinin epitope-tagged Spb1p localizes to the nucleus with nucleolar enrichment . Despite the expected methyltransferase activity of Spb1p, global methylation of pre-rRNA is not affected upon Spb1p depletion . We propose that Spb1p is required for proper assembly of pre-ribosomal particles during the biogenesis of 60S ribosomal subunits. Nucleic Acids Res, 1999 Dec 1, 27(23), 4547 - 52 A sequence immediately upstream of the plus-strand primer is essential for plus-strand DNA synthesis of the Saccharomyces cerevisiae Ty1 retrotransposon; Wilhelm M et al.; Priming of plus-strand DNA is a critical step in reverse transcription of retroviruses and retrotransposons . All retroelements use an RNase H-resistant oligoribonucleotide spanning a purine-rich sequence (the polypurine tract or PPT) to prime plus-strand DNA synthesis . Plus-strand DNA synthesis of the yeast Saccharomyces cerevisiae Ty1-H3 retrotransposon is initiated at two sites, PPT1 and PPT2, located at the upstream boundary of the 3'-long terminal repeat and near the middle of the pol gene in the integrase coding region . The two plus-strand primers have the same purine-rich sequence GGGTGGTA . This sequence is not sufficient by itself to generate a plus-strand origin since two identical sequences located upstream of PPT2 in the integrase coding region are not used efficiently as primers for plus-strand DNA synthesis . Thus, other factors must be involved in the formation of a specific plus-strand DNA primer . We show here that mutations upstream of the PPT in a highly conserved T-rich region severely alters plus-strand DNA priming of Ty1 . Our results demonstrate the importance of sequences or structural elements upstream of the PPT for initiation of plus-strand DNA synthesis. Mol Biotechnol, 1999 Aug, 12(1), 35 - 73 Nitrogen catabolite repression in Saccharomyces cerevisiae; Hofman-Bang J; In Saccharomyces cerevisiae the expression of all known nitrogen catabolite pathways are regulated by four regulators known as Gln3, Gat1, Dal80, and Deh1 . This is known as nitrogen catabolite repression (NCR) . They bind to motifs in the promoter region to the consensus sequence 5'GATAA 3' . Gln3 and Gat1 act positively on gene expression whereas Dal80 and Deh1 act negatively . Expression of nitrogen catabolite pathway genes known to be regulated by these four regulators are glutamine, glutamate, proline, urea, arginine . GABA, and allantonie . In addition, the expression of the genes encoding the general amino acid permease and the ammonium permease are also regulated by these four regulatory proteins . Another group of genes whose expression is also regulated by Gln3, Gat1, Dal80, and Deh1 are some proteases, CPS1, PRB1, LAP1, and PEP4, responsible for the degradation of proteins into amino acids thereby providing a nitrogen source to the cell . In this review, all known promoter sequences related to expression of nitrogen catabolite pathways are discussed as well as other regulatory proteins . Overview of metabolic pathways and promotors are presented. J Biol Chem, 1999 Nov 12, 274(46), 32613 - 8 Functional characterization of human methylenetetrahydrofolate reductase in Saccharomyces cerevisiae; Shan X et al.; Human methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate . 5-Methyltetrahydrofolate is a major methyl donor in the remethylation of homocysteine to methionine . Impaired MTHFR can cause high levels of homocysteine in plasma, which is an independent risk factor for vascular disease and neural tube defects . We have functionally characterized wild-type and several mutant alleles of human MTHFR in yeast, Saccharomyces cerevisiae . We have shown that yeast MET11 is a functional homologue of human MTHFR . Expression of the human MTHFR cDNA in a yeast strain deleted for MET11 can restore the strain's MTHFR activity in vitro and complement its methionine auxotrophic phenotype in vivo . To understand the domain structure of human MTHFR, we have truncated the C terminus (50%) of the protein and demonstrated that expressing an N-terminal human MTHFR in met11(-) yeast cells rescues the growth phenotype, indicating that this region contains the catalytic domain of the enzyme . However, the truncation leads to the reduced protein levels, suggesting that the C terminus may be important for protein stabilization . We have also functionally characterized four missense mutations identified from patients with severe MTHFR deficiency and two common missense polymorphisms found at high frequency in the general population . Three of the four missense mutations are unable to complement the auxotrophic phenotype of met11(-) yeast cells and show less than 7% enzyme activity of the wild type in vitro . Both of the two common polymorphisms are able to complement the growth phenotype, although one exhibited thermolabile enzyme activity in vitro . These results shall be useful for the functional characterization of MTHFR mutations and analysis structure/function relationship of the enzyme. J Biol Chem, 1999 Nov 12, 274(46), 32847 - 54 Biotin protein ligase from Saccharomyces cerevisiae . The N-terminal domain is required for complete activity; Polyak SW et al.; Catalytically active biotin protein ligase from Saccharomyces cerevisiae (EC 6.3.4.15) was overexpressed in Escherichia coli and purified to near homogeneity in three steps . Kinetic analysis demonstrated that the substrates ATP, biotin, and the biotin-accepting protein bind in an ordered manner in the reaction mechanism . Treatment with any of three proteases of differing specificity in vitro revealed that the sequence between residues 240 and 260 was extremely sensitive to proteolysis, suggesting that it forms an exposed linker between an N-terminal 27-kDa domain and the C-terminal 50-kDa domain containing the active site . The protease susceptibility of this linker region was considerably reduced in the presence of ATP and biotin . A second protease-sensitive sequence, located in the presumptive catalytic site, was protected against digestion by the substrates . Expression of N-terminally truncated variants of the yeast enzyme failed to complement E . coli strains defective in biotin protein ligase activity . In vitro assays performed with purified N-terminally truncated enzyme revealed that removal of the N-terminal domain reduced BPL activity by greater than 3500-fold . Our data indicate that both the N-terminal domain and the C-terminal domain containing the active site are necessary for complete catalytic function. J Cell Sci, 1999 Nov, 112 ( Pt 22), 4079 - 87 Peroxisome degradation in Saccharomyces cerevisiae is dependent on machinery of macroautophagy and the Cvt pathway; Hutchins MU et al.; Organelle biogenesis and turnover are necessary to maintain biochemical processes that are appropriate to the needs of the eukaryotic cell . Specific degradation of organelles in response to changing environmental cues is one aspect of achieving proper metabolic function . For example, the yeast Saccharomyces cerevisiae adjusts the level of peroxisomes in response to differing nutritional sources . When cells are grown on oleic acid as the sole carbon source, peroxisome biogenesis is induced . Conversely, a subsequent shift to glucose-rich or nitrogen-limiting conditions results in peroxisome degradation . The degradation process, pexophagy, requires the activity of vacuolar hydrolases . In addition, peroxisome degradation is specific . Analyses of cellular marker proteins indicate that peroxisome degradation under these conditions occurs more rapidly and to a greater extent than mitochondrial, Golgi, or cytosolic protein delivery to the vacuole by the non-selective autophagy pathway . To elucidate the molecular mechanism of selective peroxisome degradation, we examined pexophagy in mutants that are defective in autophagy (apg) and the selective targeting of aminopeptidase I to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway . Inhibition of peroxisome degradation in cvt and apg mutants indicates that these pathways overlap and that peroxisomes are delivered to the vacuole by a mechanism that utilizes protein components of the Cvt/autophagy pathways. Acta Biochim Pol, 1999, 46(2), 289 - 98 The essential DNA polymerases delta and epsilon are involved in repair of UV-damaged DNA in the yeast Saccharomyces cerevisiae; Halas A et al.; We have studied the ability of yeast DNA polymerases to carry out repair of lesions caused by UV irradiation in Saccharomyces cerevisiae . By the analysis of postirradiation relative molecular mass changes in cellular DNA of different DNA polymerases mutant strains, it was established that mutations in DNA polymerases delta and epsilon showed accumulation of single-strand breaks indicating defective repair . Mutations in other DNA polymerase genes exhibited no defects in DNA repair . Thus, the data obtained suggest that DNA polymerases delta and epsilon are both necessary for DNA replication and for repair of lesions caused by UV irradiation . The results are discussed in the light of current concepts concerning the specificity of DNA polymerases in DNA repair. Hum Mol Genet, 1999 Nov, 8(12), 2263 - 73 Functional analysis of human FEN1 in Saccharomyces cerevisiae and its role in genome stability; Greene AL et al.; The flap endonuclease, FEN1, is an evolutionarily conserved component of DNA replication from archaebacteria to humans . Based on in vitro results, it processes Okazaki fragments during replication and is involved in base excision repair . FEN1 removes the last primer ribonucleotide on the lagging strand and it cleaves a 5' flap that may result from strand displacement during replication or during base excision repair . Its biological importance has been revealed largely through studies in the yeast Saccharomyces cerevisiae where deletion of the homologous gene RAD27 results in genome instability and mutagen sensitivity . While the in vivo function of Rad27 has been well characterized through genetic and biochemical approaches, little is understood about the in vivo functions of human FEN1 . Guided by our recent results with yeast RAD27, we explored the function of human FEN1 in yeast . We found that the human FEN1 protein complements a yeast rad27 null mutant for a variety of defects including mutagen sensitivity, genetic instability and the synthetic lethal interactions of a rad27 rad51 and a rad27 pol3-01 mutant . Furthermore, a mutant form of FEN1 lacking nuclease function exhibits dominant-negative effects on cell growth and genome instability similar to those seen with the homologous yeast rad27 mutation . This genetic impact is stronger when the human and yeast PCNA-binding domains are exchanged . These data indicate that the human FEN1 and yeast Rad27 proteins act on the same substrate in vivo . Our study defines a sensitive yeast system for the identification and characterization of mutations in FEN1. Genetics, 1999 Nov, 153(3), 1219 - 31 The Saccharomyces cerevisiae RanGTP-binding protein msn5p is involved in different signal transduction pathways; Alepuz PM et al.; In eukaryotes, control of transcription by extracellular signals involves the translocation to the nucleus of at least one component of the signal transduction pathway . Transport through the nuclear envelope requires the activity of an import or export receptor that interacts with the small GTPase Ran . We have cloned the MSN5 gene of the yeast Saccharomyces cerevisiae that is postulated to encode one of these receptors . Msn5p belongs to a family of proteins with a conserved N-terminal sequence that acts as a RanGTP-binding domain . The results presented here provide genetic data supporting Msn5p involvement in several different signal transduction pathways . All of these pathways include changes in gene expression, and regulated nucleocytoplasmic redistribution of a component in response to external conditions has already been described in some of them . We have cloned MSN5 following two different strategies . Msn5p was constitutively localized in the nucleus . Phenotypic analysis of the msn5 mutant demonstrated that this protein participates in processes such as catabolite repression, calcium signaling, mating, and cell proliferation, as well as being involved in previously characterized phosphate utilization . Therefore, Msn5p could be a receptor for several proteins involved in different signaling pathways. Genetics, 1999 Nov, 153(3), 1205 - 18 Multiple functions of Saccharomyces cerevisiae splicing protein Prp24 in U6 RNA structural rearrangements; Vidaver RM et al.; U6 spliceosomal RNA has a complex secondary structure that includes a highly conserved stemloop near the 3' end . The 3' stem is unwound when U6 RNA base-pairs with U4 RNA during spliceosome assembly, but likely reforms when U4 RNA leaves the spliceosome prior to the catalysis of splicing . A mutation in yeast U6 RNA that hyperstabilizes the 3' stem confers cold sensitivity and inhibits U4/U6 assembly as well as a later step in splicing . Here we show that extragenic suppressors of the 3' stem mutation map to the gene coding for splicing factor Prp24 . The suppressor mutations are located in the second and third of three RNA-recognition motifs (RRMs) in Prp24 and are predicted to disrupt RNA binding . Mutations in U6 RNA predicted to destabilize a novel helix adjacent to the 3' stem also suppress the 3' stem mutation and enhance the growth defect of a suppressor mutation in RRM2 of Prp24 . Both phenotypes are reverted by a compensatory mutation that restores pairing in the novel helix . These results are best explained by a model in which RRMs 2 and 3 of Prp24 stabilize an extended intramolecular structure in U6 RNA that competes with the U4/U6 RNA interaction, and thus influence both association and dissociation of U4 and U6 RNAs during the splicing cycle. Genetics, 1999 Nov, 153(3), 1171 - 82 A role for the replication proteins PCNA, RF-C, polymerase epsilon and Cdc45 in transcriptional silencing in Saccharomyces cerevisiae; Ehrenhofer-Murray AE et al.; Transcriptional silencing in the budding yeast Saccharomyces cerevisiae may be linked to DNA replication and cell cycle progression . In this study, we have surveyed the effect of 41 mutations in genes with a role in replication, the cell cycle, and DNA repair on silencing at HMR . Mutations in PCNA (POL30), RF-C (CDC44), polymerase epsilon (POL2, DPB2, DPB11), and CDC45 were found to restore silencing at a mutant HMR silencer allele that was still a chromosomal origin of replication . Replication timing experiments indicated that the mutant HMR locus was replicated late in S-phase, at the same time as wild-type HMR . Restoration of silencing by PCNA and CDC45 mutations required the origin recognition complex binding site of the HMR-E silencer . Several models for the precise role of these replication proteins in silencing are discussed. Genetics, 1999 Nov, 153(3), 1131 - 43 Genetic analysis of the shared role of CLN3 and BCK2 at the G(1)-S transition in Saccharomyces cerevisiae; Wijnen H et al.; The transcription complexes SBF and MBF mediate the G(1)-S transition in the cell cycle of Saccharomyces cerevisiae . In late G(1), SBF and MBF induce a burst of transcription in a number of genes, including G(1)- and S-phase cyclins . Activation of SBF and MBF depends on the G(1) cyclin Cln3 and a largely uncharacterized protein called Bck2 . We show here that the induction of SBF/MBF target genes by Bck2 depends partly, but not wholly, on SBF and MBF . Unlike Cln3, Bck2 is capable of inducing its transcriptional targets in the absence of functional Cdc28 . Our results revealed promoter-specific mechanisms of regulation by Cln3, Bck2, SBF, and MBF . We isolated high-copy suppressors of the cln3 bck2 growth defect; all of these had the ability to increase CLN2 expression . One of these suppressors was the negative regulator of meiosis RME1 . Rme1 induces CLN2, and we show that it has a haploid-specific role in regulating cell size and pheromone sensitivity . Genetic analysis of the cln3 bck2 defect showed that CLN1, CLN2, and other SBF/MBF target genes have an essential role in addition to the degradation of Sic1. Genetics, 1999 Nov, 153(3), 1105 - 15 A mutation in a methionine tRNA gene suppresses the prp2-1 Ts mutation and causes a pre-mRNA splicing defect in Saccharomyces cerevisiae; Kim DH et al.; The PRP2 gene in Saccharomyces cerevisiae encodes an RNA-dependent ATPase that activates spliceosomes for the first transesterification reaction in pre-mRNA splicing . We have identified a mutation in the elongation methionine tRNA gene EMT1 as a dominant, allele-specific suppressor of the temperature-sensitive prp2-1 mutation . The EMT1-201 mutant suppressed prp2-1 by relieving the splicing block at high temperature . Furthermore, EMT1-201 single mutant cells displayed pre-mRNA splicing and cold-sensitive growth defects at 18 degrees . The mutation in EMT1-201 is located in the anticodon, changing CAT to CAG, which presumably allowed EMT1-201 suppressor tRNA to recognize CUG leucine codons instead of AUG methionine codons . Interestingly, the prp2-1 allele contains a point mutation that changes glycine to aspartate, indicating that EMT1-201 does not act by classical missense suppression . Extra copies of the tRNA(Leu)(UAG) gene rescued the cold sensitivity and in vitro splicing defect of EMT1-201 . This study provides the first example in which a mutation in a tRNA gene confers a pre-mRNA processing (prp) phenotype. Genetics, 1999 Nov, 153(3), 1091 - 103 Activation of the Saccharomyces cerevisiae filamentation/invasion pathway by osmotic stress in high-osmolarity glycogen pathway mutants; Davenport KD et al.; Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes . Of the five MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress . We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media . Three high-copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1 . Msg5p is a dual-specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast . Deletions of the putative MAPK targets of Msg5p revealed that kss1delta could suppress the osmosensitivity of pbs2-3 . Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5 . Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion-pathway-dependent manner . Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7 . These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype. Carcinogenesis, 1999 Nov, 20(11), 2143 - 52 Heterocyclic aromatic amines efficiently induce mitotic recombination in metabolically competent Saccharomyces cerevisiae strains; Paladino G et al.; Heterocyclic aromatic amines (HAs) represent a class of potent bacterial mutagens and rodent carcinogens which gain their biological activity upon metabolic conversion by phase I and phase II enzymes . Subsequent to cytochrome P450 (CYP)-dependent hydroxylation, mainly catalyzed by CYP1A2, acetylation mediated by the activity of N-acetyltransferase, NAT2, produces the ultimate electrophilic product that may react with DNA . In addition to point mutations observed in HA-exposed cells as genotoxic endpoint in vitro, loss of heterozygosity (LOH) has often been identified in HA-related rodent tumors as another endpoint in vivo . LOH may reflect a chromosomal deletion, a chromosome loss or a previous mitotic recombination event and it represents a prominent mechanism for the inactivation of tumor suppressor alleles . In this study we have investigated whether LOH observed in several HA-induced rodent tumors is related to a recombinogenic activity of HA compounds, and to address this question we have studied the genotoxic activity of several HAs in metabolically competent Saccharomyces cerevisiae strains . For this purpose expression vectors have been constructed providing simultaneous expression of three human enzymes, CYP1A2, NADPH-cytochrome P450 oxidoreductase and NAT2 in different genotoxicity tester strains . Evidence for functional expression of all three enzymes has been obtained . One strain allowed us to monitor HA-induced gene conversion, another one HA-induced chromosomal translocation . A third strain allowed us to study HA-induced forward mutations in the endogenous URA3 gene . It was found that 2-amino-3-methylimidazo-{4,5-f}quinoline and 2-amino-3, 8-dimethylimidazo-{4,5-f}quinoxaline produced a strong recombinogenic response in either recombination tester strain . The recombinogenic activity was comparable with the mutagenic activity of the compounds . The other HAs, 2-amino-3, 4-dimethyl-imidazo-{4, 5-f}quinoline, 2-amino-6-methyldipyrido-{1,2-a:3',2'-d}imidazole, 2-aminodipyrido-{1,2-a:3', 2'-d}imidazole, 3-amino-1-methyl-5H pyrido-{4,3-b}indole and 2-amino-1-methyl-6-phenyl-imidazo-{4, 5-b}pyridine, produced weak or no increases in the genotoxic endpoints of interest . The described strains may provide a suitable tool to characterize the genotoxic potential of HAs in more detail. Anal Biochem, 1999 Nov 1, 275(1), 109 - 15 Characterization of novel peptide agonists of the alpha mating factor of Saccharomyces cerevisiae; Siegel EG et al.; Alpha-factor {WHWLQLKPGQPMY}, a secreted tridecapeptide pheromone, is required for mating between the a- and alpha-haploid mating types of Saccharomyces cerevisiae (MATa, MATalpha) . New analogues of alpha-factor were synthesized and evaluated by morphogenesis assays and receptor binding studies . The Y(0)Nle(12)F(13) analogue {YWHWLQLKPGQPNleF} (MFN5) caused growth arrest and morphological alteration in MATa cells in a fashion identical to that of the native pheromone . Binding of (125)I-labeled MFN5 was saturable, and reversible as shown by equipotent label displacement by MFN5 and native alpha-mating factor . Scatchard analysis of equilibrium binding data on plasma membranes and intact cells indicated the existence of a single high-affinity binding site (K(d) = 6.4 x 10(-8)) . Specific binding of (125)I-labeled MFN5 was significantly reduced by guanosine nucleotides . Affinity cross-linking of (125)I-labeled MFN5 to MATa cell membranes identified a specifically labeled 49-kDa protein . The novel synthetic alpha-factor analogue MFN5 can be easily iodinated and used as a probe for the alpha-factor receptor . EMBO J, 1999 Nov 1, 18(21), 6155 - 68 Identification and specificities of N-terminal acetyltransferases from Saccharomyces cerevisiae; Polevoda B et al.; N-terminal acetylation can occur cotranslationally on the initiator methionine residue or on the penultimate residue if the methionine is cleaved . We investigated the three N-terminal acetyltransferases (NATs), Ard1p/Nat1p, Nat3p and Mak3p . Ard1p and Mak3p are significantly related to each other by amino acid sequence, as is Nat3p, which was uncovered in this study using programming alignment procedures . Mutants deleted in any one of these NAT genes were viable, but some exhibited diminished mating efficiency and reduced growth at 37 degrees C, and on glycerol and NaCl-containing media . The three NATs had the following substrate specificities as determined in vivo by examining acetylation of 14 altered forms of iso-1-cytochrome c and 55 abundant normal proteins in each of the deleted strains: Ard1p/Nat1p, subclasses with Ser-, Ala-, Gly- and Thr-termini; Nat3p, Met-Glu- and Met-Asp- and a subclass of Met-Asn-termini; and Mak3p subclasses with Met-Ile- and Met-Leu-termini . In addition, a special subclass of substrates with Ser-Glu- Phe-, Ala-Glu-Phe- and Gly-Glu-Phe-termini required all three NATs for acetylation. EMBO J, 1999 Nov 1, 18(21), 5843 - 52 Molecular characterization of carnitine-dependent transport of acetyl-CoA from peroxisomes to mitochondria in Saccharomyces cerevisiae and identification of a plasma membrane carnitine transporter, Agp2p; van Roermund CW et al.; In Saccharomyces cerevisiae, beta-oxidation of fatty acids is confined to peroxisomes . The acetyl-CoA produced has to be transported from the peroxisomes via the cytoplasm to the mitochondrial matrix in order to be degraded to CO(2) and H(2)O . Two pathways for the transport of acetyl-CoA to the mitochondria have been proposed . The first involves peroxisomal conversion of acetyl-CoA into glyoxylate cycle intermediates followed by transport of these intermediates to the mitochondria . The second pathway involves peroxisomal conversion of acetyl-CoA into acetylcarnitine, which is subsequently transported to the mitochondria . Using a selective screen, we have isolated several mutants that are specifically affected in the second pathway, the carnitine-dependent acetyl-CoA transport from the peroxisomes to the mitochondria, and assigned these CDAT mutants to three different complementation groups . The corresponding genes were identified using functional complementation of the mutants with a genomic DNA library . In addition to the previously reported carnitine acetyl-CoA transferase (CAT2), we identified the genes for the yeast orthologue of the human mitochondrial carnitine acylcarnitine translocase (YOR100C or CAC) and for a transport protein (AGP2) required for carnitine transport across the plasma membrane. Microbiology, 1999 Oct, 145 ( Pt 10), 2739 - 45 Involvement of glutathione in the regulation of respiratory oscillation during a continuous culture of Saccharomyces cerevisiae; Murray DB et al.; Respiratory oscillation occurred during aerobic continuous culture of Saccharomyces cerevisiae . During oscillation, phase-related changes in NAD(P)H and GSH levels occur . Perturbation of oscillation and inhibition of respiration occurred when GSH or GSSG was injected; however, there was a phase delay in perturbation in the case of an injection during high respiration . The perturbation phase delay was not apparent when a combination of DL-buthionine-(S,R)-sulphoximine, GSH and 5-nitro-2-furaldehyde was injected . Perturbation by GSH injection caused the intracellular GSH concentration to increase, the GSSG concentration to decrease and the cessation of ethanol uptake . NAD(P)H during perturbation was inversely related to dissolved oxygen . Perturbation by calcium pantothenate and pyridoxal-HCl caused a period of enhanced respiration before oscillation returned . These results suggest that the NAD+/NADH redox is not directly involved in oscillation control and regulation involves glutathione metabolism . Possible regulation points include alcohol dehydrogenase inhibition and/or respiratory-chain inhibition. Proc Natl Acad Sci U S A, 1999 Oct 26, 96(22), 12655 - 60 Characterization of the Saccharomyces cerevisiae ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation; Gachotte D et al.; The last unidentified gene encoding an enzyme involved in ergosterol biosynthesis in Saccharomyces cerevisiae has been cloned . This gene, designated ERG27, encodes the 3-keto sterol reductase, which, in concert with the C-4 sterol methyloxidase (ERG25) and the C-3 sterol dehydrogenase (ERG26), catalyzes the sequential removal of the two methyl groups at the sterol C-4 position . We developed a strategy to isolate a mutant deficient in converting 3-keto to 3-hydroxy-sterols . An ergosterol auxotroph unable to synthesize sterol or grow without sterol supplementation was mutagenized . Colonies were then selected that were nystatin-resistant in the presence of 3-ketoergostadiene and cholesterol . A new ergosterol auxotroph unable to grow on 3-ketosterols without the addition of cholesterol was isolated . The gene (YLR100w) was identified by complementation . Segregants containing the YLR100w disruption failed to grow on various types of 3-keto sterol substrates . Surprisingly, when erg27 was grown on cholesterol- or ergosterol-supplemented media, the endogenous compounds that accumulated were noncyclic sterol intermediates (squalene, squalene epoxide, and squalene dioxide), and there was little or no accumulation of lanosterol or 3-ketosterols . Feeding experiments in which erg27 strains were supplemented with lanosterol (an upstream intermediate of the C-4 demethylation process) and cholesterol (an end-product sterol) demonstrated accumulation of four types of 3-keto sterols identified by GC/MS and chromatographic properties: 4-methyl-zymosterone, zymosterone, 4-methyl-fecosterone, and ergosta-7,24 (28)-dien-3-one . In addition, a fifth intermediate was isolated and identified by (1)H NMR as a 4-methyl-24, 25-epoxy-cholesta-7-en-3-one . Implications of these results are discussed. Genomics, 1999 Oct 15, 61(2), 170 - 82 A mouse homolog of the Saccharomyces cerevisiae meiotic recombination DNA transesterase Spo11p; Keeney S et al.; The Saccharomyces cerevisiae Spo11 protein is thought to catalyze formation of the DNA double-strand breaks that initiate meiotic recombination . We have cloned cDNA and genomic DNA for a mouse gene encoding a protein with significant sequence similarity to conserved domains found in proteins of the Spo11p family . This putative mouse Spo11 gene maps to the distal region of chromosome 2 (homologous to human chromosome 20q13.2-q13.3) and comprises at least 12 exons, spanning approximately 15-18 kb . Strong expression of the Spo11 message is seen in juvenile and adult testis by RNA in situ hybridization, RT-PCR, and Northern blot, with much weaker expression in thymus and brain . In situ hybridization detects expression in oocytes of embryonic ovary, but not of adult ovary . RT-PCR and in situ hybridization analyses of a time course of juvenile testis development indicate that Spo11 expression begins in early meiotic Prophase I, prior to the pachytene stage, with increasing accumulation of mRNA through the pachytene stage . Taken together, these results strongly suggest that this gene encodes the functional homolog of yeast Spo11p, which in turn suggests that the mechanism of meiotic recombination initiation is conserved between yeast and mammals . Int J Biochem Cell Biol, 1999 Sep, 31(9), 903 - 14 Identification of the essential EPE1 gene involved in retention of secreted proteins on the cell surface of Saccharomyces cerevisiae cells; Alexieva KI et al.; Saccharomyces cerevisiae yeast cells secrete extracellularly low amounts of a few proteins . The reasons for retardation of secreted proteins on the cell surface remain obscure . We describe here a mutant able to export enhanced amount of proteins . Classical genetic methods, nucleic acids manipulations and cloning procedures were used to isolate and characterize the mutant and to clone and sequence the corresponding wild type gene . The isolated Saccharomyces cerevisiae mutant MW11, is temperature sensitive and exports on average twenty-fold more proteins at 37 degrees C than parental wild type strain (80 micrograms of proteins/1 x 10(8) mutant cells, SEM +/- 5, n22; versus 3 micrograms of proteins/1 x 10(8) parental cells, SEM +/- 1, n22) . Protein overexport in the mutant requires a functional SEC1 pathway and is independent of cell lysis . Cloning and sequencing of the corresponding wild type gene identified an open reading frame of 786 bp coding for a hydrophilic protein with predicted molecular mass of 30 kDa and cytosolic localization . The newly identified gene, designated EPE1, is an essential gene . Its DNA and amino acids sequence showed no homology with other yeast genes and proteins . It is concluded that the function of unknown yet genes, such as EPE1 is needed for retention of secreted proteins on the surface of Saccharomyces cerevisiae cells. J Biol Chem, 1999 Oct 29, 274(44), 31359 - 65 Expression of hepatitis B virus polymerase in Ty1-his3AI retroelement of Saccharomyces cerevisiae; Qadri I et al.; Hepatitis B virus (HBV), although a DNA virus, replicates using reverse transcriptase encoded by the HBV polymerase (pol) gene . The biochemical dissection of HBV pol has been hampered by failure to liberate enzymatically active protein from nucleocapsids . Here, we have employed a yeast-based genetic approach to express the HBV reverse transcriptase . In this strategy, the reverse transcriptase of yeast retrotransposon Ty1 element is replaced with the HBV pol gene to produce the hybrid Ty1/HBV element . Additionally, the indicator gene his3AI is combined in an antisense orientation to the transcripts of the hybrid Ty1/HBVRT element . The splicing of his3AI, cDNA synthesis of the Ty1/HBVRT RNA and subsequent integration relies on the reverse transcriptase activity . The production of histidine prototrophs results from the successful reverse transcription of Ty1/HBVRThis3AI transcripts followed by either homologous recombination or integrase-mediated insertion and subsequent expression of HIS3 gene . Using this approach we successfully detected the reverse transcriptase activity of HBV in yeast strains defective in endogenous Ty1 expression . Consistent with the unique priming activity associated with HBV pol, the minus strand DNA synthesis was protein-primed . Deletion of HBV reverse transcriptase (RT) or RNase H domains resulted in a dramatic drop in histidine prototrophs . The addition of HBV encoded HBx protein in virus-like particles during in vitro RT reaction stimulated the RT reaction by severalfold . Furthermore, in the presence of 3TC, a known inhibitor of HBV reverse transcriptase, yeast His(+) growth of His protrophs was not observed . Thus, this approach, which is based on genetic selection in yeast, is safe, economic, and a reliable strategy with a potential for large scale screening of cofactors and inhibitors of HBV polymerase functions. Biochem J, 1999 Nov 1, 343 Pt 3, 621 - 6 Opposite roles of trehalase activity in heat-shock recovery and heat-shock survival in Saccharomyces cerevisiae; Wera S et al.; A variety of results has been obtained consistent with activation of neutral trehalase in Saccharomyces cerevisiae through direct phosphorylation by cAMP-dependent protein kinase (PKA) . A series of neutral trehalase mutant alleles, in which all evolutionarily conserved putative phosphorylation sites were changed into alanine, was tested for activation in vitro (by PKA) and in vivo (by glucose addition) . None of the mutations alone affected the activation ratio, whereas all mutations combined resulted in an inactive enzyme . All mutant alleles were expressed to similar levels, as shown by Western blotting . Several of the point mutations significantly lowered the specific activity . Using this series of mutants with different activity levels we show an inverse relationship between trehalase activity and heat-shock survival during glucose-induced trehalose mobilization . This is consistent with a stress-protective function of trehalose . On the other hand, reduction of trehalase activity below a certain threshold level impaired recovery from a sublethal heat shock . This suggests that trehalose breakdown is required for efficient recovery from heat shock, and that the presence of trehalase protein alone is not sufficient for efficient heat-stress recovery. Mutat Res, 1999 Sep 13, 435(1), 1 - 11 Genetic interactions between error-prone and error-free postreplication repair pathways in Saccharomyces cerevisiae; Xiao W et al.; Evidence obtained from recent studies supports the existence of an error-free postreplication repair (PRR) and a mutagenesis pathway within the Saccharomyces cerevisiae RAD6 DNA repair group . The MMS2 gene is the only known yeast gene involved in error-free PRR that, when mutated, significantly increases the spontaneous mutation rate . In this study, the mutational spectrum of the mms2 mutator was determined and compared to the wild type strain . In addition, mutagenenic effects and genetic interactions of the mms2 mutator and rev3 anti-mutator were examined with respect to forward mutations, frameshift reversions as well as amber and ochre suppressions . It was concluded from these results that the mms2 mutator phenotype is largely dependent on the functional REV3 gene . The synergistic effects of mms2 and rev3 mutations towards killing by a variety of DNA-damaging agents ruled out the possibility that MMS2 simply acts to suppress REV3 activity and favored the hypothesis that MMS2 and REV3 form two alternative subpathways within the RAD6 DNA repair pathway . Taken together, we propose that two pathways represented by MMS2 and REV3 deal with a similar range of endogenous and environmental DNA damage but with different biological consequences, namely, error-free repair and mutagenesis, respectively. FEBS Lett, 1999 Oct 15, 459(3), 427 - 32 Analysis by atomic force microscopy of Med8 binding to cis-acting regulatory elements of the SUC2 and HXK2 genes of saccharomyces cerevisiae; Moreno-Herrero F et al.; Med8 protein is a regulator that specifically binds to upstream activating sequences (UASs) of SUC2 promoter, to downstream repressing sequences (DRSs) of the HXK2 gene and to the carboxy-terminal domain of the RNA polymerase II . Atomic force microscopy has allowed for direct visualization of Med8 interactions with a 305 bp fragment of SUC2 promoter and with a 676 bp fragment of HXK2 gene, containing respectively the UASs and DRSs regulatory regions . This approach has provided complementary information about the position and the structure of the DNA-protein complexes . Med8 binding to DNA results in total covering of one of the two existing 7 bp motives (consensus, (A/C)(A/G)GAAAT) in the studied DNA fragments . No preference for binding either of the two UASs of SUC2 promoter as well as for the two DRSs of HXK2 gene has been found . We also discuss whether this protein works as dimer or as a monomer. J Cell Biol, 1999 Oct 18, 147(2), 335 - 50 Novel roles for saccharomyces cerevisiae mitotic spindle motors; Cottingham FR et al.; The single cytoplasmic dynein and five of the six kinesin-related proteins encoded by Saccharomyces cerevisiae participate in mitotic spindle function . Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle . Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell . This study reveals that kinesin-related Kar3p and Kip3p are unique in that they perform roles both inside and outside the nucleus . Kar3p, like Kip3p, was found to be required for spindle positioning in the absence of dynein . The spindle positioning role of Kar3p is performed in concert with the Cik1p accessory factor, but not the homologous Vik1p . Kar3p and Kip3p were also found to overlap for a function essential for the structural integrity of the bipolar spindle . The cytoplasmic and nuclear roles of both these motors could be partially substituted for by the microtubule-destabilizing agent benomyl, suggesting that these motors perform an essential microtubule-destabilizing function . In addition, we found that yeast cell viability could be supported by as few as two microtubule-based motors: the BimC-type kinesin Cin8p, required for spindle structure, paired with either Kar3p or Kip3p, required for both spindle structure and positioning. Mol Cell Biol, 1999 Nov, 19(11), 7801 - 15 Saccharomyces cerevisiae pol30 (proliferating cell nuclear antigen) mutations impair replication fidelity and mismatch repair; Chen C et al.; To understand the role of POL30 in mutation suppression, 11 Saccharomyces cerevisiae pol30 mutator mutants were characterized . These mutants were grouped based on their mutagenic defects . Many pol30 mutants harbor multiple mutagenic defects and were placed in more than one group . Group A mutations (pol30-52, -104, -108, and -126) caused defects in mismatch repair (MMR) . These mutants exhibited mutation rates and spectra reminiscent of MMR-defective mutants and were defective in an in vivo MMR assay . The mutation rates of group A mutants were enhanced by a msh2 or a msh6 mutation, indicating that MMR deficiency is not the only mutagenic defect present . Group B mutants (pol30-45, -103, -105, -126, and -114) exhibited increased accumulation of either deletions alone or a combination of deletions and duplications (4 to 60 bp) . All deletion and duplication breakpoints were flanked by 3 to 7 bp of imperfect direct repeats . Genetic analysis of one representative group B mutant, pol30-126, suggested polymerase slippage as the likely mutagenic mechanism . Group C mutants (pol30-100, -103, -105, -108, and -114) accumulated base substitutions and exhibited synergistic increases in mutation rate when combined with msh6 mutations, suggesting increased DNA polymerase misincorporation as a mutagenic defect . The synthetic lethality between a group A mutant, pol30-104, and rad52 was almost completely suppressed by the inactivation of MSH2 . Moreover, pol30-104 caused a hyperrecombination phenotype that was partially suppressed by a msh2 mutation . These results suggest that pol30-104 strains accumulate DNA breaks in a MSH2-dependent manner. Mol Cell Biol, 1999 Nov, 19(11), 7558 - 67 Separation-of-function mutations in Saccharomyces cerevisiae MSH2 that confer mismatch repair defects but do not affect nonhomologous-tail removal during recombination; Studamire B et al.; Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication . In addition to their role in mismatch repair (MMR), the MSH2 and MSH3 gene products are required to remove 3' nonhomologous DNA tails during genetic recombination . The mismatch repair genes MSH6, MLH1, and PMS1, whose products interact with Msh2p, are not required in this process . We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair . Twenty-four msh2 mutations that conferred a dominant negative phenotype for mismatch repair were isolated . A subset of these mutations mapped to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds . Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the removal of nonhomologous DNA tails during genetic recombination . The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein complexes that are thought to play different roles during DNA repair and genetic recombination. Mol Cell Biol, 1999 Nov, 19(11), 7428 - 35 Context-dependent modulation of replication activity of Saccharomyces cerevisiae autonomously replicating sequences by transcription factors; Kohzaki H et al.; Evidence for transcription factor involvement in the initiation of DNA replication at certain replication origins in Saccharomyces cerevisiae mainly comes from an indirect assay which measures the mitotic stability of plasmids containing an autonomously replicating sequence (ARS), a selectable marker gene, and a centromere . In order to eliminate the effect of transcription factor binding to the selectable marker gene or centromere in such assays, we have adapted the DpnI assay to directly measure ARS replication activity in vivo by using ARS plasmids devoid of extraneous transcription elements . Using this assay, we found that the B3 element of ARS1, which serves as a binding site for the transcription factor Abf1p, does not stimulate ARS activity on plasmids lacking a centromere and a selectable marker gene . We also found with such plasmids that exogenous expression of the strong transcriptional activators Gal4 and Gal4-VP16 inhibited the replication activity of ARS1 when B3 was replaced by the Gal4 binding site, although these activators had previously been shown to stimulate replication activity in the stability assay . Moreover, a chromosomally inactive ARS, ARS301, which was active by itself on a plasmid, was inactivated by placing an Abf1p binding site in its vicinity . These results indicate that the sequences surrounding the ARS as well as properties of the ARS element itself determine its response to transcription factors. Mol Cell Biol, 1999 Nov, 19(11), 7369 - 76 Saccharomyces cerevisiae RNA polymerase I terminates transcription at the Reb1 terminator in vivo; Reeder RH et al.; We have mapped transcription termination sites for RNA polymerase I in the yeast Saccharomyces cerevisiae . S1 nuclease mapping shows that the primary terminator is the Reb1p terminator located at +93 downstream of the 3' end of 25S rRNA . Reverse transcription coupled with quantitative PCR shows that approximately 90% of all transcripts terminate at this site . Transcripts which read through the +93 site quantitatively terminate at a fail-safe terminator located further downstream at +250 . Inactivation of Rnt1p (an RNase III involved in processing the 3' end of 25S rRNA) greatly stabilizes transcripts extending to both sites and increases readthrough at the +93 site . In vivo assay of mutants of the Reb1p terminator shows that this site operates in vivo by the same mechanism as has previously been delineated through in vitro studies. EMBO J, 1999 Oct 15, 18(20), 5714 - 23 The Saccharomyces cerevisiae MER3 gene, encoding a novel helicase-like protein, is required for crossover control in meiosis; Nakagawa T et al.; The MER3 gene is identified as a novel meiosis-specific gene, whose transcript is spliced in an MRE2/MER1-dependent manner . The predicted Mer3 protein contains the seven motifs characteristic of the DExH-box type of helicases as well as a putative zinc finger . Double strand breaks (DSBs), the initial changes of DNA in meiotic recombination, do not disappear completely and are hyperresected late in mer3 meiosis, indicating that MER3 is required for the transition of DSBs to later intermediates . A mer3 mutation reduces crossover frequencies, and the remaining crossovers show random distribution along a chromosome, resulting in a high incidence of non-disjunction of homologous chromosomes at the first meiotic division . MER3 appears to be very important for both the DSB transition and crossover control. Biochemistry (Mosc), 1999 Sep, 64(9), 990 - 3 Hydrolysis of tripolyphosphate by purified exopolyphosphatase from Saccharomyces cerevisiae cytosol: kinetic model; Kulakovskaya TV et al.; The kinetics of hydrolysis of tripolyphosphate by purified exopolyphosphatase from Saccharomyces cerevisiae cytosol has been studied in the presence of Mg2+ . Two kinetic models suggesting the formation of complexes of tripolyphosphate and the enzyme with Mg2+ are compared . Both models suggest that only enzyme--substrate complexes containing Mg2+ and tripolyphosphate simultaneously are able to hydrolyze the tripolyphosphate . The first model suggests that the enzyme is able to bind to Mg2+ independently from substrate binding . The second model does not consider this possibility, but suggests that both complexes containing tripolyphosphate and Mg2+ in proportion 1:1 and 1:2 can serve as the reaction substrates . The description of the experimental data by both models is essentially the same . The complex containing tripolyphosphate and Mg2+ in proportion 1:1 is optimal for the enzyme activity, the complex containing tripolyphosphate and Mg2+ in proportion 1:2 being hydrolyzed at a lower rate. Glycobiology, 1999 Oct, 9(10), 1045 - 51 Mnt2p and Mnt3p of Saccharomyces cerevisiae are members of the Mnn1p family of alpha-1,3-mannosyltransferases responsible for adding the terminal mannose residues of O-linked oligosaccharides; Romero PA et al.; The genome of Saccharomyces cerevisiae contains five genes that encode type II transmembrane proteins with significant amino acid similarity to the alpha-1,3-mannosyltransferase Mnn1p . The roles of the three genes most closely related to MNN1 were examined in mutants carrying single and multiple combinations of the disrupted genes . Paper chromatographic analysis of {2-3H}mannose-labeled O-linked oligosaccharides released by beta-elimination showed that the MNT2 (YGL257c) and MNT3 (YIL014w) genes in combination with MNN1 have overlapping roles in the addition of the fourth and fifth alpha-1,3-linked mannose residues to form Man4 and Man5 oligosaccharides whereas MNT4 (YNR059w) does not appear to be required for O-glycan synthesis. Genes Dev, 1999 Oct 1, 13(19), 2570 - 80 The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms; Kaeberlein M et al.; The SIR genes are determinants of life span in yeast mother cells . Here we show that life span regulation by the Sir proteins is independent of their role in nonhomologous end joining . The short life span of a sir3 or sir4 mutant is due to the simultaneous expression of a and alpha mating-type information, which indirectly causes an increase in rDNA recombination and likely increases the production of extrachromosomal rDNA circles . The short life span of a sir2 mutant also reveals a direct failure to repress recombination generated by the Fob1p-mediated replication block in the rDNA . Sir2p is a limiting component in promoting yeast longevity, and increasing the gene dosage extends the life span in wild-type cells . A possible role of the conserved SIR2 in mammalian aging is discussed. Biochemistry, 1999 Oct 12, 38(41), 13551 - 9 Functional expression and characterization of the wild-type mammalian renal cortex sodium/phosphate cotransporter and an 215R mutant in Saccharomyces cerevisiae; Bernhardt F et al.; The wild-type and an R215E mutant of the rat renal cortex sodium/phosphate cotransporter type 2 (NaPi-2) were functionally expressed in the yeast Saccharomyces cerevisiae strain MB192, a cell line lacking the high-affinity endogenous H+/P(i) cotransporter . The expression of the mRNA molecules and corresponding proteins was confirmed by Northern and Western blot analysis, respectively . As detected by indirect immunofluorescence and antibody capture assay, both wild-type and mutant NaPi-2 proteins are expressed in the yeast plasma membrane in comparable amounts . In the presence of 5 microM phosphate, Na+ promotes phosphate uptake into yeast cells expressing the wild-type NaPi-2 with a K(0.5) of 5.6 +/- 1.1 mM . The maximum uptake of phosphate (649 +/- 30 pmol/10 min) is approximately 8-fold higher than the uptake obtained with nontransformed cells (76.8 +/- 8 pmol/10 min) . Yeast cells expressing the R215E mutant of NaPi-2 accumulate 213 +/- 9 pmol of phosphate/10 min under the same conditions . The K(0.5) for the stimulation of phosphate uptake by Na+ is 4.2 +/- 0.8 mM for the R215E mutant and thus not significantly different from the value obtained with cells expressing the wild-type cotransporter . The reduced level of accumulation of phosphate in yeast cells expressing the R215E mutant is probably due to a reduction of the first-order rate constant k for phosphate uptake: while cells expressing wild-type NaPi-2 accumulate phosphate with a k of 0.06 min(-1), the rate for phosphate uptake into cells expressing the R215E mutant (k) is 0.016 min(-1) and therefore about 4-fold lower . In comparison, the rate for phosphate uptake into nontransformed cells (k) is 0.0075 min(-1) . Phosphate uptake into yeast cells that express the wild-type NaPi-2 in the presence of 150 mM NaCl is promoted by extracellular phosphate with a K(0.5) of 45 +/- 4 microM . A phosphate-dependent phosphate accumulation is also observed with cells expressing the R215E mutant, but the K(0.5) is twice as high (86 +/- 5 microM) as that obtained with the wild-type cotransporter . We conclude that the yeast expression system is a useful tool for the investigation of structure-function relationships of the renal sodium/phosphate cotransporter and that (215)R, although not involved in Na+ recognition, is a part of the structure involved in phosphate recognition and considerably influences the rate of phosphate uptake by the NaPi-2 cotransporter. Genetika, 1999 Jul, 35(7), 1012 - 5 {Cloning and characterization of the homologue of the Saccharomyces cerevisiae gene in Drosophila melanogaster}; Nabirochkina EN et al.; RAD23 is an evolutionary conserved protein, which is essential for DNA excision repair . It is believed that this protein is present in all eukaryotic organisms from yeast to mammals . In this work, molecular cloning of the Drosophila melanogaster RAD23 gene and an analysis of the encoded protein are reported. FEBS Lett, 1999 Sep 10, 458(1), 1 - 5 Characterization of purified and unidirectionally reconstituted Pho84 phosphate permease of Saccharomyces cerevisiae; Fristedt U et al.; Hydropathy analysis of the amino acid sequence of the Pho84 phosphate permease of Saccharomyces cerevisiae suggests that the protein consists of 12 transmembrane domains connected by hydrophilic loops . The Pho84 protein has been modified by a gene fusion approach, yielding two different N-terminal His-tagged chimeras which can be expressed in Escherichia coli, purified and functionally reconstituted into defined proteoliposomes . The continuous epitopes in the N- and C-terminal sequences of the Pho84 chimeras were shown to be accessible in proteoliposomes containing the purified active Pho84 proteins . Site-specific proteolysis of the immunoreactive N-terminal sequence in the reconstituted protein suggests a unidirectional insertion into liposomes. FEMS Microbiol Lett, 1999 Oct 15, 179(2), 327 - 32 The yeast Saccharomyces cerevisiae does not sequester chloride but can express a functional mammalian chloride channel; Coury LA et al.; Chloride uptake into yeast was measured as a function of pH . A small amount of uptake was seen at pH values of 3.0 and 4.0; at pH 6.0 chloride uptake was substantially less than the uptake of phosphate and rubidium . Because chloride uptake is inefficient, we expressed the putative mammalian chloride channel, pI(Cln), in yeast and observed a chloride-selective current when total membrane protein was reconstituted into lipid bilayers . The current was inhibited by a specific chloride channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoic acid . These results suggest that yeast may serve as a means to characterize chloride channels from other organisms. Mol Gen Genet, 1999 Sep, 262(2), 332 - 41 The Saccharomyces cerevisiae LEP1/SAC3 gene is associated with leucine transport; Stella CA et al.; Leucine uptake by Saccharomyces cerevisiae is mediated by three transport systems, the general amino acid transport system (GAP), encoded by GAP1, and two group-specific systems (S1 and S2), which also transport isoleucine and valine . A new mutant defective in both group-specific transport activities was isolated by employing a gap1 leu4 strain and selecting for trifluoroleucine-resistant mutants which also showed greatly reduced ability to utilize L-leucine as sole nitrogen source and very low levels of {14C}L-leucine uptake . A multicopy plasmid containing a DNA fragment which complemented the leucine transport defect was isolated by selecting for transformants that grew normally on minimal medium containing leucine as nitrogen source and subsequently assaying {14C}L-leucine uptake . Transformation of one such mutant, lep1, restored sensitivity to trifluoroleucine . The complementing gene, designated LEP1, was subcloned and sequenced . The LEP1 ORF encodes a large protein that lacks characteristics of a transporter or permease (i.e., lacks hydrophobic domains necessary for membrane association) . Instead, Lep1p is a very basic protein (pI of 9.2) that contains a putative bipartite signal sequence for targeting to the nucleus, suggesting that it might be a DNA-binding protein . A database search revealed that LEP1 encodes a polypeptide that is identical to Sac3p except for an N-terminal truncation . The original identification of SAC3 was based on the isolation of a mutant allele, sac3-1, that suppresses the temperature-sensitive growth defect of an actin mutant containing the allele act1-1 . Sac3p has been previously shown to be localized in the nucleus . When a lep1 mutant was crossed with a sac3 deletion mutant, no complementation was observed, indicating that the two mutations are functionally allelic. Mol Gen Genet, 1999 Sep, 262(2), 275 - 82 Regulated nuclear localisation of the yeast transcription factor Ace2p controls expression of chitinase (CTS1) in Saccharomyces cerevisiae; O'Conallain C et al.; The yeast transcription factor Ace2p regulates expression of the chitinase gene CTS1 in a cell cycle-dependent manner . Nuclear localisation of Ace2p is restricted to late M and early G phases of the mitotic cell cycle . We show here that this nuclear localisation is directly associated with regulation of CTS1 expression . Using a version of Ace2p tagged with a c-myc epitope, we show that the protein is excluded from the nucleus of cells during most phases of the mitotic cell cycle . A mutant derivative in which one threonine and two serine residues, which are candidate phosphorylation sites, were replaced by alanine (to mimic constitutive dephosphorylation) is localised in the nucleus throughout the cell cycle . The mechanism of localisation of Ace2p therefore involves regulation of its phosphorylation state, and closely resembles that used by the homologous transcription factor Swi5p . The wild-type Ace2 protein associates with Cdc28p in vivo, suggesting this may be the kinase that mediates the phosphorylation event . The stability of the protein is greatly reduced in a mutant that is constitutively localised to the nucleus, but is restored in a deletion derivative which remains in the cytoplasm . Ace2p is therefore controlled throughout the cell cycle at three levels: transcription, nuclear localisation, and proteolysis. J Bacteriol, 1999 Oct, 181(20), 6441 - 8 Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae; Athenstaedt K et al.; Lipid particles of the yeast Saccharomyces cerevisiae were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced . In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions . All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism . Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation . To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis . Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids . Other deletion mutants were viable under standard conditions . Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism.
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