|
|
|
|
|
| Mol Gen Genet, 1995 Dec 10, 249(4), 406 - 16 Structure and distribution of specific cis-elements for transcriptional regulation of PHO84 in Saccharomyces cerevisiae; Ogawa N et al.; Transcription of the PHO84 gene encoding a Pi transporter in Saccharomyces cerevisiae is regulated by the Pi concentration in the medium . The promoter region of PHO84 bears five copies of the motif 5'-CACGT(G/T)-3', a candidate for the upstream activation site (UAS) that binds the transcriptional activator protein of the phosphatase regulon, Pho4p . These motifs are found at nucleotides -880 (site A), -587 (B), -436 (C), -414 (D), and -262 (E) relative to the putative ATG codon of PHO84 . The Pho4p binds to all five 6-bp motifs with various affinities . Deletion analysis of the PHO84 promoter using a PHO84-lacZ fusion gene and base substitutions in the 6-bp motif revealed that two copies of the 6-bp motif, either C or D, and E, are necessary and sufficient for full regulation of the PHO84 gene . Results of expression studies with a CYC1-lacZ fusion gene with various 36-bp oligonucleotides including the 30-bp sequences around site D or E, or with modified sequences, inserted in the CYC1 promoter region indicated that the 6-bps motif flanked by a thymine nucleotide at its 5' end is much less effective as a UAS site for Pho4p in vivo than other versions . Thus, the consensus sequences for phosphatase regulation are 5'-GCACGTGGG-3' and 5'-GCACGTTTT-3' which differ from the binding sequences for the Cpflp protein required for transcription of the genes in methionine biosynthesis and for centromere function . However, Pho4p binding in vitro was unaffected by modification of the 5' or 3' flanking sites of the 6-bp motif, while modification inside the 6-bp motif affected it severely . The UAS function of the GCACGTTTT motif with respect to the Pi signal depends on its orientation in the promoter sequence. Oncogene, 1995 Dec 7, 11(11), 2267 - 71 Kir, a novel Ras-family G-protein, induces invasive pseudohyphal growth in Saccharomyces cerevisiae; Dorin D et al.; Kir belongs to a novel class of Ras-family G-proteins which includes Gem and Rad . These proteins are unique among Ras super-family G-proteins since their expression is under transcriptional regulation in mammalian cells . To gain insight into the function of Kir, we took advantage of the well-defined signal transduction pathways of yeast . When kir is expressed in Saccharomyces cerevisiae, the transformants form pseudohyphae and exhibit invasive properties characteristics of yeast cells undergoing a developmental transition induced by nitrogen starvation . Analysis of pseudohyphal signaling pathway mutants suggests that the Kir-induced pseudohyphae formation requires a MAP kinase cascade involving ste20, ste11, ste7 but not ste5 gene products . Furthermore, our results are consistent with the idea that Kir functions upstream of the STE20 kinase which plays a critical role in two distinct MAP kinase cascades. Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11539 - 43 14-3-3 proteins: potential roles in vesicular transport and Ras signaling in Saccharomyces cerevisiae; Gelperin D et al.; Deletion of the clathrin heavy-chain gene, CHC1, in the budding yeast Saccharomyces cerevisiae results in growth, morphological, and membrane trafficking defects, and in some strains chc1-delta is lethal . A previous study identified five genes which, in multicopy, rescue inviable strains of Chc- yeast . Now we report that one of the suppressor loci, BMH2/SCD3, encodes a protein of the 14-3-3 family . The 14-3-3 proteins are abundant acidic proteins of approximately 30 kDa with numerous isoforms and a diverse array of reported functions . The Bmh2 protein is > 70% identical to the mammalian epsilon-isoform and > 90% identical to a previously reported yeast 14-3-3 protein encoded by BMH1 . Single deletions of BMH1 or BMH2 have no discernable phenotypes, but deletion of both BMH1 and BMH2 is lethal . High-copy BMH1 also rescues inviable strains of Chc- yeast, although not as well as BMH2 . In addition, the slow growth of viable strains of Chc- yeast is further impaired when combined with single bmh mutations, often resulting in lethality . Overexpression of BMH genes also partially suppresses the temperature sensitivity of the cdc25-1 mutant, and high-copy TPK1, encoding a cAMP-dependent protein kinase, restores Bmh- yeast to viability . High-copy TPK1 did not rescue Chc- yeast . These genetic interactions suggest that budding-yeast 14-3-3 proteins are multifunctional and may play a role in both vesicular transport and Ras signaling pathways. Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11461 - 4 In vitro trans-splicing in Saccharomyces cerevisiae; Ghetti A et al.; The interactions established at the 5'-splice site during spliceosome assembly are likely to be important for both precise recognition of the upstream intron boundary and for positioning this site in the active center of the spliceosome . Definition of the RNA-RNA and the RNA-protein interactions at the 5' splice site would be facilitated by the use of a small substrate amenable to modification during chemical synthesis . We describe a trans-splicing reaction performed in Saccharomyces cerevisiae extracts in which the 5' splice site and the 3' splice site are on separate molecules . The RNA contributing the 5' splice site is only 20 nucleotides long and was synthesized chemically . The trans-splicing reaction is accurate and has the same sequence, ATP, and Mg2+ requirements as cis-splicing . We also report how deoxy substitutions around the 5'-splice site affect trans-splicing efficiency. EMBO J, 1995 Dec 1, 14(23), 5939 - 46 REG1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae; Tu J et al.; Protein phosphatase type 1 (PP1) is encoded by GLC7, an essential gene in Saccharomyces cerevisiae . The GLC7 phosphatase is required for glucose repression and appears to function antagonistically to the SNF1 protein kinase . Previously, we characterized a mutation, glc7-T152K, that relieves glucose repression but does not interfere with the function of GLC7 in glycogen metabolism . We proposed that the mutant GLC7T152K phosphatase is defective in its interaction with a regulatory subunit that directs participation of PP1 in the glucose repression mechanism . Here, we present evidence that REG1, a protein required for glucose repression, is one such regulatory subunit . We show that REG1 is physically associated with GLC7 . REG1 interacts with GLC7 strongly and specifically in the two-hybrid system, and REG1 and GLC7 fusion proteins co-immunoprecipitate from cell extracts . Moreover, overexpression of a REG1 fusion protein suppresses the glc7-T152K mutant defect in glucose repression . This and other genetic evidence indicate that the two proteins function together in regulating glucose repression . These results suggest that REG1 is a regulatory subunit of PP1 that targets its activity to proteins in the glucose repression regulatory pathway. EMBO J, 1995 Dec 1, 14(23), 5931 - 8 A downstream target of RHO1 small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae; Nonaka H et al.; The RHO1 gene in Saccharomyces cerevisiae encodes a homolog of the mammalian RhoA small GTP-binding protein, which is implicated in various actin cytoskeleton-dependent cell functions . In yeast, Rho1p is involved in bud formation . A yeast strain in which RHO1 is replaced with RhoA shows a recessive temperature-sensitive growth phenotype . A dominant suppressor mutant was isolated from this strain . Molecular cloning of the suppressor gene revealed that the mutation occurred at the pseuodosubstrate site of PKC1, a yeast homolog of mammalian protein kinase C . Two-hybrid analysis demonstrated that GTP-Rho1p, but not GDP-Rho1p, interacted with the region of Pkc1p containing the pseudosubstrate site and the C1 domain . MKK1 and MPK1 encode MAP kinase kinase and MAP kinase homologs, respectively, and function downstream of PKC1 . A dominant active MKK1-6 mutation or overexpression of MPK1 suppressed the temperature sensitivity of the RhoA mutant . The dominant activating mutation of PKC1 suppressed the temperature sensitivity of the RhoA mutant . The dominant activating mutation of PKC1 suppressed the temperature sensitivity of two effector mutants of RHO1, rho1(F44Y) and rho1(E451), but not that of rho1(V43T) . These results indicate that there are at least two signaling pathways regulated by Rho1p and that one of the downstream targets is Pkc1p, leading to the activation of the MAP kinase cascade. Yeast, 1995 Dec, 11(15), 1525 - 32 Sequencing of an 18.8 kb fragment from Saccharomyces cerevisiae chromosome VI; Naitou M et al.; The nucleotide sequence of lambda phage clone 4121, which contains the 18.8 kb fragment of Saccharomyces cerevisiae chromosome VI left arm, was determined . This sequence had seven open reading frames (ORFs), four of which were identical to known genes (ACT1, YPT1, TUB2 and RPO41) . Another three ORFs (4121orfR003, 4121orfR004 and 4121orfRN001) were highly homologous to FET3 multi-copper oxidase, glucose transport protein, and hypothetical protein of YIL106w on chromosome IX, respectively . 4121orfRN01 is suggested to contain an intron. Yeast, 1995 Dec, 11(15), 1513 - 8 Nucleotide sequence of the GDS1 gene of Saccharomyces cerevisiae; Konopinska A et al.; We have cloned and sequenced the GDS1 gene located on the right arm of chromosome XV of Saccharomyces cerevisiae . The gene codes for a 522 amino acid serine-rich protein with no obvious homology to proteins in the database . GDS1 gene was isolated as the multicopy suppressor of the glycerol-deficient phenotype caused by the nam9-1 mutation in the yeast nuclear gene encoding the mitochondrial ribosomal protein homologous to S4 proteins from various organisms . Disruption-deletion of the GDS1 open reading frame leads to a partial impairment of growth on medium containing glycerol as the carbon source, indicating mitochondrial function of the gene product. J Bioenerg Biomembr, 1995 Dec, 27(6), 597 - 603 Extracellular ascorbate stabilization as a result of transplasma electron transfer in Saccharomyces cerevisiae; Santos-Ocana C et al.; The presence of yeast cells in the incubation medium prevents the oxidation of ascrobate catalyzed by copper ions . Ethanol increases ascorbate retention . Pyrazole, an alcohol dehydrogenase inhibitor, prevents ascorbate stabilization by cells . Chelation of copper ions does not account for stabilization, since oxidation rates with broken or boiled cells or conditioned media are similar to control rates in the absence of cells . Protoplast integrity is needed to reach optimal values of stabilization . Chloroquine, a known inhibitor of plasma membrane redox systems, inhibits the ascorbate stabilization, the inhibition being partially reversed by coenzyme Q6 . Chloroquine does not inhibit ferricyanide reduction . Growth of yeast in iron-deficient media to increase ferric ion reductase activity also increases the stabilization . In conclusion, extracellular ascorbate stabilization by yeast cells can reflect a coenzyme Q dependent transplasmalemma electron transfer which uses NADH as electron donor . Iron deficiency increases the ascorbate stabilization but the transmembrane ferricyanide reduction system can act independently of ascorbate stabilization. J Biochem (Tokyo), 1995 Dec, 118(6), 1303 - 9 Localization and in vitro mutagenesis of the active site in the Saccharomyces cerevisiae mRNA capping enzyme; Shibagaki Y et al.; The yeast mRNA capping enzyme is composed of 52 (alpha) and 80 kDa (beta) polypeptides, which are responsible for its mRNA guanylyltransferase and RNA 5'-triphosphatase activities, respectively . We isolated the gene encoding the alpha subunit (CEG1) and showed that CEG1 is essential for yeast cell growth {Shibagaki et al., (1992) J . Biol . Chem . 267, 9521-9528} . In this study, CEG1 was expressed in Escherichia coli and the alpha subunit protein was purified to near homogeneity . A {32P}GMP-bound tryptic peptide derived from the recombinant enzyme-{32P}GMP covalent reaction intermediate was converted to a {32P}phosphoryl-peptide through periodate oxidation followed by beta-elimination . Hydrolysis of the {32P}phosphoryl-peptide with alkali resulted in {32P}N epsilon-phospholysine as the only phosphoamino acid, indicating that GMP in the enzyme-GMP complex is bound to a lysine residue via a phosphoamide linkage . Microsequencing of the {32P}GMP-peptide showed that the GMP binding site was located in the region between amino acids 60 and 75, which contained an internal trypsin-resistant lysine at position 70 . CEG1 was subjected to site-directed mutagenesis and the mutant proteins were expressed in E . coli . Substitution of His or Ile for Lys70 entirely abolished the enzyme-GMP formation activity, and this mutation was lethal to yeast in vivo, supporting the notion that the active site in the alpha subunit is located at Lys70 . Replacement of Lys70 with Arg reduced the ability to form the enzyme-GMP complex; however, yeast cells bearing this allele were not viable . A series of mutations, including 8 amino acid replacements and 3 insertions, near the active site (Lys70-Thr-Asp-Gly motif) were also introduced and the mutant polypeptides were examined for catalytic activity in vitro as well as yeast cell viability in vivo . There was a good correlation between the in vitro and in vivo functions of the mutant proteins, except when Asp72 was replaced with Glu, which allowed formation of the enzyme-GMP complex but failed to support cell growth . The results with Lys70 to Arg and Asp72 to Glu substitutions indicated that guanylyltransfer to RNA and/or additional roles besides cap formation per se are impaired in these mutant proteins. Yeast, 1995 Dec, 11(16), 1629 - 50 Processing of pre-ribosomal RNA in Saccharomyces cerevisiae; Venema J et al.; Post-transcriptional processing of precursor-ribosomal RNA comprises a complex pathway of endonucleolytic cleavages, exonucleolytic digestion and covalent modifications . The general order of the various processing steps is well conserved in eukaryotic cells, but the underlying mechanisms are largely unknown . Recent analysis of pre-rRNA processing, mainly in the yeast Saccharomyces cerevisiae, has significantly improved our understanding of this important cellular activity . Here we will review the data that have led to our current picture of yeast pre-rRNA processing. Yeast, 1995 Dec, 11(16), 1575 - 611 An overview of membrane transport proteins in Saccharomyces cerevisiae; Andre B; All eukaryotic cells contain a wide variety of proteins embedded in the plasma and internal membranes, which ensure transmembrane solute transport . It is now established that a large proportion of these transport proteins can be grouped into families apparently conserved throughout organisms . This article presents the data of an in silicio analysis aimed at establishing a preliminary classification of membrane transport proteins in Saccharomyces cerevisiae . This analysis was conducted at a time when about 65% of all yeast genes were available in public databases . In addition to approximately 60 transport proteins whose function was at least partially known, approximately 100 deduced protein sequences of unknown function display significant sequence similarity to membrane transport proteins characterized in yeast and/or other organisms . While some protein families have been well characterized by classical genetic experimental approaches, others have largely if not totally escaped characterization . The proteins revealed by this in silicio analysis also include a putative K+ channel, proteins similar to aquaporins of plant and animal origin, proteins similar to Na+-solute symporters, a protein very similar to electroneural cation-chloride cotransporters, and a putative Na+-H+ antiporter . A new research area is anticipated: the functional analysis of many transport proteins whose existence was revealed by genome sequencing. Yeast, 1995 Dec, 11(16), 1553 - 73 The chromosome ends of Saccharomyces cerevisiae; Louis EJ; Yeast chromosome ends are similar in structure and function to chromosome ends in most, if not all, eukaryotic organisms . There is a G-rich terminal repeat at the ends which is maintained by telomerase . In addition to the classical functions of protecting the end from degradation and end-to-end fusions, and completing replication, yeast telomeres have several interesting properties including: non-nucleosomal chromatin structure; transcriptional position effect variegation for genes with adjacent telomeres; nuclear peripheral localization; apparent physical clustering; non-random recombinational interactions . A number of genes have been identified that are involved in modifying one or more of these properties . These include genes involved in general DNA metabolism, chromatin structure and telomere maintenance . Adjacent to the terminal repeat is a mosaic of middle repetitive elements that exhibit a great deal of polymorphism both between individual strains and among different chromosome ends . Much of the sequence redundancy in the yeast genome is found in the sub-telomeric regions (within the last 25 kb of each end) . The sub-telomeric regions are generally low in gene density, low in transcription, low in recombination, and they are late replicating . The only element which appears to be shared by all chromosome ends is part of the previously defined X element containing an ARS consensus . Most of the 'core' X elements also contain an Abf1p binding site and a URS1-like element, which may have consequences for the chromatin structure, nuclear architecture and transcription of native telomeres . Possible functions of sub-telomeric repeats include: fillers for increasing chromosome size to some minimum threshold level necessary for chromosome stability; barrier against transcriptional silencing; a suitable region for adaptive amplification of genes; secondary mechanism of telomere maintenance via recombination when telomerase activity is absent. Biosci Biotechnol Biochem, 1995 Dec, 59(12), 2307 - 8 Effects of degraded schizophyllans on regeneration of protoplast cells of Saccharomyces cerevisiae; Hisamatsu M et al.; Schizophyllan was heated at 100 degrees C in 85% dimethyl sulfoxide (DMSO) containing 0.01 M H2SO4 for various times, and fractionated by gel-permeation chromatography . Molecular weights (M(r)) of the depolymerized products thus obtained were measured in water and DMSO by GPC-LALLS to estimate their conformations in water . The products with triple helical structure stimulated regeneration of Saccharomyces cerevisiae protoplast cells, while those of single chain conformation were totally inactive. Genetics, 1995 Dec, 141(4), 1275 - 85 Suppressors of a Saccharomyces cerevisiae pkc1 mutation identify alleles of the phosphatase gene PTC1 and of a novel gene encoding a putative basic leucine zipper protein; Huang KN et al.; The PKC1 gene product, protein kinase C, regulates a mitogen-activated protein kinase (MAPK) cascade, which is implicated in cell wall metabolism . Previously, we identified the pkc1-4 allele in a screen for mutants with increased rates of recombination, indicating that PKC1 may also regulate DNA metabolism . The pkc1-4 allele also conferred a temperature-sensitive (ts) growth defect . Extragenic suppressors were isolated that suppress both the ts and hyperrecombination phenotypes conferred by the pkc1-4 mutation . Eight of these suppressors for into two complementation groups, designated KCS1 and KCS2 . KCS1 was cloned and found to encode a novel protein with homology to the basic leucine zipper family of transcription factors . KCS2 is allelic with PTC1, a previously identified type 2C serine/threonine protein phosphatase . Although mutation of either KCS1 or PTC1 causes little apparent phenotype, the kcs1 delta ptc1 delta double mutant fails to grow at 30 degrees . Furthermore, the ptc1 deletion mutation is synthetically lethal in combination with a mutation in MPK1, which encodes a MAPK homologue proposed to act in the PKC1 pathway . Because PTC1 was initially isolated as a component of the Hog1p MAPK pathway, it appears that these two MAPK cascades share a common regulatory feature. Genetics, 1995 Dec, 141(4), 1263 - 74 Unigenic evolution: a novel genetic method localizes a putative leucine zipper that mediates dimerization of the Saccharomyces cerevisiae regulator Gcr1p; Deminoff SJ et al.; The GCR1 gene of Saccharomyces cerevisiae encodes a transcriptional activator that complexes with Rap1p and, through UASRPG elements (Rap1p DNA binding sites), stimulates efficient expression of glycolytic and translational component genes . To map the functionally important domains in Gcr1p, we combined multiple rounds of random mutagenesis in vitro with in vivo selection of functional genes to locate conserved, or hypomutable, regions . We name this method unigenic evolution, a statistical analysis of mutations in evolutionary variants of a single gene in an otherwise isogenic background . Examination of the distribution of 315 mutations in 24 variant alleles allowed the localization of four hypomutable regions in GCR1 (A, B, C, and D) . Dispensable N-terminal (intronic) and C-terminal portions of the evolved region of GCR1 were included in the analysis as controls and were, as expected, not hypomutable . The analysis of several insertion, deletion, and point mutations, combined with a comparison of the hypomutability and hydrophobicity plots of Gcr1p, suggested that some of the hypomutable regions may individually or in combination correspond to functionally important surface domains . In particular, we determined that region D contains a putative leucine zipper and is necessary and sufficient for Gcr1p homodimerization. Biokhimiia, 1995 Dec, 60(12), 2011 - 21 {Glycine amide ribonucleotide synthetase (EC 6.3.4.13)--is aminoimidazole ribonucleotide synthetase (EC 6.3.3.1) from Saccharomyces cerevisiae}; Tret'iakov OIu et al.; The bifunctional enzyme GAR-synthetase-AIR-synthetase (E2-E5) of the yeast Saccharomyces cerevisiae has been studied . The yeast strain with overproduction of E2-E5 has been obtained . The enzyme from this strain, E2-E5, has been purified and characterized . The protein is a dimer composed of two subunits with M(r) of 87 kDa . The pH and temperature optima, pH stability and thermostability for E2 and E5 have been determined . The kinetic constants for E2 and E5 have been estimated . E2 and E5 are active only in the presence of Mg2+ . E5 is a K(+)-dependent enzyme as is E5 from other sources . AMP is a competitive (to ATP) inhibitor for E5; hence, in yeast cells the purine nucleotide biosynthesis de novo is regulated at the first and fifth steps . Partial chymotryptic digestion of the purified protein gives rise to two fragments with M(r) of about 40 and 46 kDa; and E2 activity remains, while that of E5 disappears in the process. Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 451 - 8 Cloning of Corticium rolfsii glucoamylase cDNA and its expression in Saccharomyces cerevisiae; Nagasaka Y et al.; A cDNA coding for the glucoamylase of Corticium rolfsii AHU 9627 was cloned using synthetic oligonucleotide probes that code for inner amino acid sequences of the purified enzyme . This clone (CG 15) is 1900 base pairs long and contains the entire coding region for a polypeptide of 579 residues . Comparison with amino acid sequences of other fungal glucoamylases showed homologies of 35%-56%, and most homology with that of Aspergillus niger . The expression plasmid pACG 115 was constructed by introduction of the coding region of CG 15 into a yeast expression vector pAAH 5, containing the promoter and terminator of alcohol dehydrogenase (ADHI) . Saccharomyces cerevisiae AH 22, containing the recombinant plasmid pACG 115, acquired starch-saccharifying ability. Mol Biol Cell, 1995 Dec, 6(12), 1721 - 42 end5, end6, and end7: mutations that cause actin delocalization and block the internalization step of endocytosis in Saccharomyces cerevisiae; Munn AL et al.; Four mutants defective in endocytosis were isolated by screening a collection of temperature-sensitive yeast mutants . Three mutations define new END genes: end5-1, end6-1, and end7-1 . The fourth mutation is in END4, a gene identified previously . The end5-1, end6-1, and end7-1 mutations do not affect vacuolar protein localization, indicating that the defect in each mutant is specific for internalization at the plasma membrane . Interestingly, localization of actin patches on the plasma membrane is affected in each of the mutants . end5-1, end6-1, and end7-1 are allelic to VRP1, RVS161, and ACT1, respectively . VRP1 and RVS161 are required for correct actin localization and ACT1 encodes actin . To our surprise, the end6-1 mutation fails to complement the act1-1 mutation . Disruption of the RVS167 gene, which is homologous to END6/RVS161 and which is also required for correct actin localization, also blocks endocytosis . The end7-1 mutant allele has a glycine 48 to aspartic acid substitution in the DNase I-binding loop of actin . We propose that Vrp1p, Rvs161p, and Rvs167p are components of a cytoskeletal structure that contains actin and fimbrin and that is required for formation of endocytic vesicles at the plasma membrane. Arch Microbiol, 1995 Dec, 164(6), 435 - 43 Killer-toxin-resistant kre12 mutants of Saccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor; Schmitt MJ et al.; The Saccharomyces cerevisiae killer toxin K1 is a secreted alpha/beta-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process . The first step involves toxin binding to beta-1,6-D-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of the KRE genes whose products are involved in synthesis and/or assembly of cell wall beta-D-glucans . After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels . In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts . Classical yeast genetics and successive back-crossings to sensitive wild-type strains indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), rendering kre12 mutants incapable of binding significant amounts of toxin to the membrane . Since kre12 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from a kre12 mutant and from an isogenic Kre12(+) strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE . Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in the kre12 mutant . A model for K1 killer toxin action is presented in which the gene product of KRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation. Appl Microbiol Biotechnol, 1995 Dec, 44(1-2), 126 - 32 Effects of growth environment on recombinant plasmid stability in Saccharomyces cerevisiae grown in continuous culture; O'Kennedy R et al.; A recombinant strain of Saccharomyces cerevisiae, containing a 2-microns-fragment-based plasmid (pYE alpha a4) was grown under non-selective conditions in continuous culture . The decrease in the population carrying the plasmid-encoded auxotrophic marker, LEU2, was examined under different physiological conditions . The difference in growth rate (delta mu) between plasmid-free and plasmid-containing cells and the rate of plasmid segregation (R) were determined using a non-linear regression technique . Loss rates were greater in defined glucose-limited cultures than in complex glucose-limited cultures . Plasmid loss was delta mu-dominated in cultures grown on defined media whereas delta mu and R were co-dominant in cultures grown on complex medium . Loss rates increased with increasing dilution rate in complex glucose-limited cultures . The reverse was found in defined glucose-limited cultures . Plasmid retention and loss kinetic determined from defined magnesium-limited cultures were not significantly different from those observed in defined glucose-limited cultures . Although plasmid retention in defined phosphate-limited culture was not significantly different from that in defined glucose-limited culture, reduced R and increased delta mu indicated an alternative physiological effect of phosphate limitation on plasmid stability. Photochem Photobiol, 1995 Dec, 62(6), 997 - 1004 Photoreaction of 5-methoxypsoralen with thymidine and the thymine moiety of isolated and Saccharomyces cerevisiae DNA . Characterization and measurement of the two cis-syn furan-side monocycloadducts; Anselmino C et al.; The photoreaction of the furan-side moiety of 5-methoxypsoralen (5-MOP) with thymidine used as a DNA model compound was investigated in the dry state . Under these conditions, two main fluorescent photoadducts were formed and isolated by HPLC . The two modified nucleosides were characterized as the two cis-syn diastereoisomers of furan-side monoadducts of 5-MOP to thymidine on the basis of spectroscopic measurements including UV, fluorescence, 1H-NMR and circular dichroism analysis . The identification and quantification of the latter photoproducts within naked DNA exposed to photoexcited 5-MOP were achieved by enzymatic digestion completed by HPLC separation and fluorescence detection . Similarly, the two cis-syn furan-side monoadducts were found to be formed in the DNA of Saccharomyces cerevisiae cells after incubation with 5-MOP and subsequent exposure to 365 nm at an incident dose of 38.4 kJ m-2 . Under these conditions, the rate of induction of two diastereoisomeric photoadducts was as low as one modification per 10(6) and 2 x 10(5) bases, respectively. J Cell Biol, 1995 Dec, 131(6 Pt 2), 1677 - 97 Nuclear pore complex clustering and nuclear accumulation of poly(A)+ RNA associated with mutation of the Saccharomyces cerevisiae RAT2/NUP120 gene; Heath CV et al.; To identify genes involved in the export of messenger RNA from the nucleus to the cytoplasm, we used an in situ hybridization assay to screen temperature-sensitive strains of Saccharomyces cerevisiae . This identified those which accumulated poly(A)+ RNA in their nuclei when shifted to the non-permissive temperature of 37 degrees C . We describe here the properties of yeast strains carrying mutations in the RAT2 gene (RAT - ribonucleic acid trafficking) and the cloning of the RAT2 gene . Only a low percentage of cells carrying the rat2-1 allele showed nuclear accumulation of poly(A)+ RNA when cultured at 15 degrees or 23 degrees C, but within 4 h of a shift to the nonpermissive temperature of 37 degrees C, poly(A)+ RNA accumulated within the nuclei of approximately 80% of cells . No defect was seen in the nuclear import of a reporter protein bearing a nuclear localization signal . Nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope in wild-type cells . In cells carrying either the rat2-1 or rat2-2 allele, NPCs were clustered together into one or a few regions of the nuclear envelope . This clustering was a constitutive property of mutant cells . NPCs remained clustered in crude nuclei isolated from mutant cells, indicating that these clusters are not able to redistribute around the nuclear envelope when nuclei are separated from cytoplasmic components . Electron microscopy revealed that these clusters were frequently found in a protuberance of the nuclear envelope and were often located close to the spindle pole body . The RAT2 gene encodes a 120-kD protein without similarity to other known proteins . It was essential for growth only at 37 degrees C, but the growth defect at high temperature could be suppressed by growth of mutant cells in the presence of high osmolarity media containing 1.0 M sorbitol or 0.9 M NaCl . The phenotypes seen in cells carrying a disruption of the RAT2 gene were very similar to those seen with the rat2-1 and rat2-2 alleles . Epitope tagging was used to show that Rat2p is located at the nuclear periphery and co-localizes with yeast NPC proteins recognized by the RL1 monoclonal antibody . The rat2-1 allele was synthetically lethal with both the rat3-1/nup133-1 and rat7-1/nup159-1 alleles . These results indicate that the product of this gene is a nucleoporin which we refer to as Rat2p/Nup120p. Appl Environ Microbiol, 1995 Dec, 61(12), 4448 - 53 In situ 31P nuclear magnetic resonance for observation of polyphosphate and catabolite responses of chemostat-cultivated Saccharomyces cerevisiae after alkalinization; Castro CD et al.; The proposed pH buffering and phosphagenic functions of polyphosphate were investigated by subjecting chemostat-cultivated Saccharomyces cerevisiae to alkalinization (NaOH addition) and anaerobiosis . The subsequent changes in intracellular phosphate-containing species were observed in situ by nuclear magnetic resonance (NMR) spectroscopy by using the NMR cultivator we developed . For the alkalinization experiments, changes in catabolite secretion were also measured in parallel experiments . Additionally, a range of potential neutralization capacity was investigated: a dilute culture and concentrated cultures with low or high polyphosphate content . The concentrated cultures displayed increased cytosolic pH and rapid polyphosphate degradation to small chains . The pH changes and extent of polyphosphate degradation depended inversely on initial polyphosphate content . The dilute culture restored extracellular pH rapidly and secreted acetate . The concentrated culture with low polyphosphate reserves also secreted acetate . In contrast to the alkalinization-induced polyphosphate dynamics, anaerobiosis resulted in the complete hydrolysis of polyphosphate to P(i), as opposed to small chains, and reduced cytosolic pH . The results and calculations suggest that the bulk of NMR-observable polyphosphate (vacuolar) degradation to short polymers conceivably contributes to neutralizing added alkalinity . In other circumstances, such as anaerobiosis, degradation serves other functions, such as phosphorylation potential regulation. Gene, 1995 Dec 1, 166(1), 151 - 4 ROK1, a high-copy-number plasmid suppressor of kem1, encodes a putative ATP-dependent RNA helicase in Saccharomyces cerevisiae; Song Y et al.; The KEM1 gene is involved in nuclear fusion during conjugation, and chromosome transmission and spindle pole body duplication/or separation during mitotic cell division in the yeast Saccharomyces cerevisiae . KEM1 was also independently identified as DST2, SEP1, XRN1 and RAR5 on the basis of DNA strand transferase or exoribonuclease activity in vitro or mutations affecting plasmid stability . To understand the various functions suggested for KEM1 and to identify other genes with functions similar or related to those of KEM1, we have characterized the ROK1 gene which was isolated as a high-copy-number plasmid suppressor of the kem1 null mutation . Sequence analysis of the smallest subclone with the suppression activity revealed an open reading frame of 564 amino acids . The ROK1 aa sequence contains highly conserved domains found in the DEAD protein family of ATP-dependent RNA helicases . ROK1 is essential for viability and is closely linked to KEM1 on chromosome VII. Gene, 1995 Dec 1, 166(1), 145 - 9 The relationship between mRNA half-life and gene function in the yeast Saccharomyces cerevisiae; Moore J et al.; Saccharomyces cerevisiae (Sc) mRNAs have been described as falling into two major classes with respect to mRNA half-life {Santiago et al., Nucleic Acids Res . 14 (1986) 8347-8360} . We have used DNA sequence analysis to address the functional roles of eleven of the thirteen cDNAs upon which Santiago et al . based their conclusions . Eight had been described as copies of short half-life and five as copies of long-half-life mRNAs . We show here that five members of the short-half-life class encode known Sc cytosolic ribosomal proteins (rp) . One further short-half-life cDNA appears to encode a new Sc rp related to higher eukaryotic rp S12 . Among the long-half-life cDNAs, one encodes the glucose-inducible glycolytic enzyme enolase, while another is related to the mouse housekeeping gene MER5. Curr Microbiol, 1995 Dec, 31(6), 327 - 31 Decreased mitochondrial biogenesis in temperature-sensitive cell division cycle mutants of Saccharomyces cerevisiae; Genta HD et al.; The temperature-sensitive cell division cycle (cdc) G1 mutants cdc28 and cdc35 show decreased mitochondrial volumes with respect to the wild type strain A364A (WT) at the restrictive temperature . Of the three criteria of mitochondrial biogenesis studied, that is, number of mitochondria per cell, relative area of the cell occupied by mitochondria, or relative area of mitochondria occupied by inner membranes, only the second indicator was significantly lower in cdc mutants than in the WT . The mitochondrial inner membranes development did not compensate for the decrease in the organelles volume . Apparently, the reduced mitochondrial biogenesis was not due to the temperature shift because the relative area of the cell occupied by mitochondria was already significantly lower at 25 degrees C in cdc mutants . The specific fluxes of oxygen consumption confirmed that the respiratory capacity of cdc mutants is largely impaired in respect to the WT . Cdc28 and cdc35 mutants of Saccharomyces cerevisiae had been previously shown to exhibit high respiratory quotients (from 3 to 7) in respect to the WT (RQ approximately 1.0), which correlated with carbon and energy uncoupling probably the result of glucose-induced catabolite repression {Aon MA, Monaco ME, Cortassa S (1995) Exp Cell Res 217, 42-51; Monaco ME, Valdecantos PA, Aon MA (1995) Exp Cell Res 217, 52-56}. Mol Cell Biol, 1995 Dec, 15(12), 6987 - 98 Constitutive repression and nuclear factor I-dependent hormone activation of the mouse mammary tumor virus promoter in Saccharomyces cerevisiae; Chavez S et al.; To study the influence of various transactivators and the role of nucleosomal structure in gene regulation by steroid hormones, we have introduced mouse mammary tumor virus (MMTV) promoter sequences along with expression vectors for the glucocorticoid receptor (GR) and nuclear factor I (NFI) in Saccharomyces cerevisiae, an organism amenable to genetic manipulation . Both in the context of an episomal multicopy vector and in a centromeric single-copy plasmid, the MMTV promoter was virtually silent in the absence of inducer, even in yeast strains expressing GR and NFI . Induction was optimal with deacylcortivazol and required both GR and NFI . The transactivation function AF1 in the N-terminal half of GR is required for ligand-dependent induction and acts constitutively in truncated GR lacking the ligand binding domain . A piece of the MMTV long terminal repeat extending from -236 to +111 is sufficient to position a nucleosome, B, over the regulatory region of the promoter from -45 to -190 and another nucleosome over the transcription start region . The rotational orientation of the DNA on the surface of nucleosome B is the same as that previously found in animal cells and in reconstitution experiments . This orientation is compatible with binding of GR to two sites, while it should preclude binding of NFI and hence be responsible for constitutive repression . Upon ligand induction, there is no major chromatin rearrangement, but the proximal linker DNA, including the TATA box, becomes hypersensitive to nucleases . The transcriptional behavior of the MMTV promoter was unaffected by deletions of the genes for zuotin or SIN1/SPT2, two proteins which have been claimed to assume some of the functions of linker histones . Thus, despite the lack of histone H1, yeast cells could be a suitable system to study the contribution of nucleosomal organization to the regulated expression of the MMTV promoter. Mol Cell Biol, 1995 Dec, 15(12), 6979 - 86 Effects of mutations in the Saccharomyces cerevisiae RNA14, RNA15, and PAP1 genes on polyadenylation in vivo; Mandart E et al.; The RNA14 and RNA15 gene products have been implicated in a variety of cellular processes . Mutations in these genes lead to faster decay of some mRNAs and yield extracts that are deficient in cleavage and polyadenylation in vitro . These results suggest that the RNA14 and RNA15 gene products may be involved in both adenylation and deadenylation in vivo . To explore the roles of these gene products in vivo, we examined the site of adenylation and the rate of deadenylation for individual mRNAs in rna14 and rna15 mutant strains . We observed that the rates of deadenylation are not affected by lesions in either the RNA14 or the RNA15 gene . This result suggests that the proteins encoded by these genes are not involved in regulation of the deadenylation rate . In contrast, we observed that the site of adenylation for the ACT1 transcript can be altered in these mutants . Interestingly, we also observed that mutation of the poly(A) polymerase gene altered the site of ACT1 polyadenylation . These observations suggest that the RNA14, RNA15, and PAP1 proteins are involved in poly(A) site choice . This alteration in poly(A) site choice in the rna14 mutant can be corrected by the ssm4 suppressor, indicating that this suppression acts at the level of polyadenylation and not by slowing mRNA degradation. Mol Cell Biol, 1995 Dec, 15(12), 6838 - 44 Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae; Wang Y et al.; Inhibition of mitosis by antimitotic drugs is thought to occur by destruction of microtubules, causing cells to arrest through the action of one or more mitotic checkpoints . We have patterned experiments in the yeast Saccharomyces cerevisiae after recent studies in mammalian cells that demonstrate the effectiveness of antimitotic drugs at concentrations that maintain spindle structure . We show that low concentrations of nocodazole delay cell division under the control of the previously identified mitotic checkpoint genes BUB1, BUB3, MAD1, and MAD2 and independently of BUB2 . The same genes mediate the cell cycle delay induced in ctf13 mutants, limited for an essential kinetochore component . Our data suggest that a low concentration of nocodazole induces a cell cycle delay through checkpoint control that is sensitive to impaired kinetochore function . The BUB2 gene may be part of a separate checkpoint that responds to abnormal spindle structure. Mol Cell Biol, 1995 Dec, 15(12), 6754 - 69 A bipartite operator interacts with a heat shock element to mediate early meiotic induction of Saccharomyces cerevisiae HSP82; Szent-Gyorgyi C; Although key genetic regulators of early meiotic transcription in Saccharomyces cerevisiae have been well characterized, the activation of meiotic genes is still poorly understood in terms of cis-acting DNA elements and their associated factors . I report here that induction of HSP82 is regulated by the early meiotic IME1-IME2 transcriptional cascade . Vegetative repression and meiotic induction depend on interactions of the promoter-proximal heat shock element (HSE) with a nearby bipartite repression element, composed of the ubiquitous early meiotic motif, URS1 (upstream repression sequence 1), and a novel ancillary repression element . The ancillary repression element is required for efficient vegetative repression, is spatially separable from URS1, and continues to facilitate repression during sporulation . In contrast, URS1 also functions as a vegetative repression element but is converted early in meiosis into an HSE-dependent activation element . An early step in this transformation may be the antagonism of URS1-mediated repression by IME1 . The HSE also nonspecifically supports a second major mode of meiotic activation that does not require URS1 but does require expression of IME2 and concurrent starvation . Interestingly, increased rather than decreased URS1-mediated vegetative transcription can be artificially achieved by introducing rare point mutations into URS1 or by deleting the UME6 gene . These lesions offer insight into mechanisms of URS-dependent repression and activation . Experiments suggest that URS1-bound factors functionally modulate heat shock factor during vegetative transcription and early meiotic induction but not during heat shock . The loss of repression and activation observed when the IME2 activation element, T4C, is substituted for the HSE suggests specific requirements for URS1-upstream activation sequence interactions. Mol Cell Biol, 1995 Dec, 15(12), 6632 - 40 Requirement of the self-glucosylating initiator proteins Glg1p and Glg2p for glycogen accumulation in Saccharomyces cerevisiae; Cheng C et al.; Glycogen, a branched polymer of glucose, is a storage molecule whose accumulation is under rigorous nutritional control in many cells . We report the identification of two Saccharomyces cerevisiae genes, GLG1 and GLG2, whose products are implicated in the biogenesis of glycogen . These genes encode self-glucosylating proteins that in vitro can act as primers for the elongation reaction catalyzed by glycogen synthase . Over a region of 258 residues, the Glg proteins have 55% sequence identify to each other and approximately 33% identity to glycogenin, a mammalian protein postulated to have a role in the initiation of glycogen biosynthesis . Yeast cells defective in either GLG1 or GLG2 are similar to the wild type in their ability to accumulate glycogen . Disruption of both genes results in the inability of the cells to synthesize glycogen despite normal levels of glycogen synthase . These results suggest that a self-glucosylating protein is required for glycogen biosynthesis in a eukaryotic cell . The activation state of glycogen synthase in glg1 glg2 cells is suppressed, suggesting that the Glg proteins may additionally influence the phosphorylation state of glycogen synthase. Mol Cell Biol, 1995 Dec, 15(12), 6572 - 81 NDT80, a meiosis-specific gene required for exit from pachytene in Saccharomyces cerevisiae; Xu L et al.; We describe the identification of a new meiosis-specific gene of Saccharomyces cerevisiae, NDT80 . The ndt80 null and point mutants arrest at the pachytene stage of meiosis, with homologs connected by full-length synaptonemal complexes and spindle pole bodies duplicated but unseparated . Meiotic recombination in an ndt80 delta mutant is relatively normal, although commitment to heteroallelic recombination is elevated two- to threefold and crossing over is decreased twofold compared with those of the wild type . ndt80 arrest is not alleviated by mutations in early recombination genes, e.g., SPO11 or RAD50, and thus cannot be attributed to an intermediate block in prophase chromosome metabolism like that observed in several other mutants . The ndt80 mutant phenotype during meiosis most closely resembles that of a cdc28 mutant, which contains a thermolabile p34, the catalytic subunit of maturation-promoting factor . Cloning and molecular analysis reveal that the NDT80 gene maps on the right arm of chromosome VIII between EPT1 and a Phe-tRNA gene, encodes a 627-amino-acid protein which exhibits no significant homology to other known proteins, and is transcribed specifically during middle meiotic prophase . The NDT80 gene product could be a component of the cell cycle regulatory machinery involved in the transition out of pachytene, a participant in an unknown aspect of meiosis sensed by a pachytene checkpoint, or a SPO11- and RAD50-independent component of meiotic chromosomes that is the target of cell cycle signaling. J Cell Biol, 1995 Dec, 131(5), 1163 - 71 Interaction between BiP and Sec63p is required for the completion of protein translocation into the ER of Saccharomyces cerevisiae; Lyman SK et al.; To clarify the roles of Kar2p (BiP) and Sec63p in translocation across the ER membrane in Saccharomyces cerevisiae, we have utilized mutant alleles of the essential genes that encode these proteins: kar2-203 and sec63-1 . Sanders et al . (Sanders, S . L., K . M . Whitfield, J . P . Vogel, M . D . Rose, and R . W . Schekman . 1992 . Cell . 69:353-365) showed that the translocation defect of the kar2-203 mutant lies in the inability of the precursor protein to complete its transit across the membrane, suggesting that the lumenal hsp70 homologue Kar2p (BiP) binds the transiting polypeptide in order to facilitate its passage through the pore . We now show that mutation of a conserved residue (A181-->T) (Nelson, M . K., T . Kurihara, and P . Silver . 1993 . Genetics . 134:159-173) in the lumenal DnaJ box of Sec63p (sec63-1) results in an in vitro phenotype that mimics the precursor stalling defect of kar2-203 . We demonstrate by several criteria that this phenotype results specifically from a defect in the lumenal interaction between Sec63p and BiP: Neither a sec62-1 mutant nor a mutation in the cytosolically exposed domain of Sec63p causes precursor stalling, and interaction of the sec63-1 mutant with the membranebound components of the translocation apparatus is unimpaired . Additionally, dominant KAR2 suppressors of sec63-1 partially relieve the stalling defect . Thus, proper interaction between BiP and Sec63p is necessary to allow the precursor polypeptide to complete its transit across the membrane. J Bacteriol, 1995 Dec, 177(23), 6836 - 43 Regulation of nuclear genes encoding mitochondrial proteins in Saccharomyces cerevisiae; Brown TA et al.; Selection for mutants which release glucose repression of the CYB2 gene was used to identify genes which regulate repression of mitochondrial biogenesis . We have identified two of these as the previously described GRR1/CAT80 and ROX3 genes . Mutations in these genes not only release glucose repression of CYB2 but also generally release respiration of the mutants from glucose repression . In addition, both mutants are partially defective in CYB2 expression when grown on nonfermentable carbon sources, indicating a positive regulatory role as well . ROX3 was cloned by complementation of a glucose-inducible flocculating phenotype of an amber mutant and has been mapped as a new leftmost marker on chromosome 2 . The ROX3 mutant has only a modest defect in glucose repression of GAL1 but is substantially compromised in galactose induction of GAL1 expression . This mutant also has increased SUC2 expression on nonrepressing carbon sources . We have also characterized the regulation of CYB2 in strains carrying null mutation in two other glucose repression genes, HXK2 and SSN6, and show that HXK2 is a negative regulator of CYB2, whereas SSN6 appears to be a positive effector of CYB2 expression. J Bacteriol, 1995 Dec, 177(23), 6761 - 5 Activation of the Ras/cyclic AMP pathway in the yeast Saccharomyces cerevisiae does not prevent G1 arrest in response to nitrogen starvation; Markwardt DD et al.; Cells carrying mutations that activate the Ras/cyclic AMP (Ras/cAMP) pathway fail to accumulate in G1 as unbudded cells and lose viability in response to nitrogen starvation . This observation has led to the idea that cells carrying this type of mutation are sensitive to nitrogen starvation because they are unable to appropriately arrest in G1 . In this study, we tested predictions made by this model . We found that cells with activating Ras/cAMP pathway mutations do not continue to divide after nitrogen starvation, show a normal decrease in steady state levels of START-specific transcripts, and are not rescued by removal of cAMP during nitrogen starvation . These findings are inconsistent with the idea that activation of the Ras/cAMP pathway prevents growth arrest in cells starved for nitrogen . Our finding that cells with an active Ras/cAMP pathway have dramatically reduced amino acid stores suggests an alternative model . We propose that cells at high cAMP levels are unable to store sufficient nutrients to allow return to the G1 phase of the cell cycle when they are suddenly deprived of nitrogen . It is this inability to return to G1, rather than a failure to arrest, which leaves cells at different points in the cell cycle following nitrogen starvation. J Biol Chem, 1995 Dec 1, 270(48), 28723 - 32 Binding sites for abundant nuclear factors modulate RNA polymerase I-dependent enhancer function in Saccharomyces cerevisiae; Kang JJ et al.; The 190-base pair (bp) rDNA enhancer within the intergenic spacer sequences of Saccharomyces cerevisiae rRNA cistrons activates synthesis of the 35S-rRNA precursor about 20-fold in vivo (Mestel,, R., Yip, M., Holland, J . P., Wang, E., Kang, J., and Holland, M . J . (1989) Mol . Cell . Biol . 9, 1243-1254) . We now report identification and analysis of transcriptional activities mediated by three cis-acting sites within a 90-bp portion of the rDNA enhancer designated the modulator region . In vivo, these sequences mediated termination of transcription by RNA polymerase I and potentiated the activity of the rDNA enhancer element . Two trans-acting factors, REB1 and REB2, bind independently to sites within the modulator region (Morrow, B . E., Johnson, S . P., and Warner, J . R . (1989) J . Biol . Chem . 264, 9061-9068) . We show that REB2 is identical to the ABF1 protien . Site-directed mutagenesis of REB1 and ABF1 binding sites demonstrated uncoupling of RNA polymerase I-dependent termination from transcriptional activation in vivo . We conclude that REB1 and ABF1 are required for RNA polymerase I-dependent termination and enhancer function, respectively, Since REB1 and ABF1 proteins also regulate expression of class II genes and other nuclear functions, our results suggest further similarities between RNA polymerase I and II regulatory mechanisms . Two rDNA enhancers flanking a rDNA minigene stimulated RNA polymerase I transcription in a "multiplicative" fashion . Deletion mapping analysis showed that similar cis-acting sequences were required for enhancer function when positioned upstream or downstream from a rDNA minigene. Genes Dev, 1995 Dec 1, 9(23), 2949 - 63 Mutation of RGA1, which encodes a putative GTPase-activating protein for the polarity-establishment protein Cdc42p, activates the pheromone-response pathway in the yeast Saccharomyces cerevisiae; Stevenson BJ et al.; We have selected yeast mutants that exhibit a constitutively active pheromone-response pathway in the absence of the beta subunit of the trimeric G protein . Genetic analysis of one such mutant revealed that it contained recessive mutations in two distinct genes, both of which contributed to the constitutive phenotype . One mutation identifies the RGA1 locus (Rho GTPase activating protein), which encodes a protein with homology to GAP domains and to LIM domains . Deletion of RGA1 is sufficient to activate the pathway in strains lacking the G beta subunit . Moreover, in wild-type strains, deletion of RGA1 increases signaling in the pheromone pathway, whereas over-expression of RGA1 dampens signaling, demonstrating that Rga1p functions as a negative regulator of the pheromone response pathway . The second mutation present in the original mutant proved to be an allele of a known gene, PBS2, which encodes a putative protein kinase that functions in the high osmolarity stress pathway . The pbs2 mutation enhanced the rga1 mutant phenotype, but by itself did not activate the pheromone pathway . Genetic and two-hybrid analyses indicate that an important target of Rga1p is Cdc42p, a p21 GTPase required for polarity establishment and bud emergence . This finding coupled with recent experiments with mammalian and yeast cells indicating that Cdc42p can interact with and activate Ste20p, a protein kinase that operates in the pheromone pathway, leads us to suggest that Rga1p controls the activity of Cdc42p, which in turn controls the magnitude of signaling in the pheromone pathway via Ste20p. Biochim Biophys Acta, 1995 Nov 30, 1269(3), 275 - 80 BAP2, a gene encoding a permease for branched-chain amino acids in Saccharomyces cerevisiae; Grauslund M et al.; To select the gene coding for an isoleucine permease, an isoleucine dependent strain (ilv1 cha1) was transformed with a yeast genomic multicopy library, and colonies growing at a low isoleucine concentration were selected . Partial sequencing of the responsible plasmid insert revealed the presence of a previously sequenced 609 codon open reading frame of chromosome II with homology to known permeases . Deletion, extra dosage and C-terminal truncation of this gene were constructed in a strain lacking the general amino acid permease, and amino acid uptake was measured during growth in synthetic complete medium . The following observations prompted us to name the gene BAP2 (branched-chain amino acid permease) . Deletion of BAP2 reduced uptake of leucine, isoleucine and valine by 25-50%, while the uptake of 8 other L-alpha-amino acids was unaltered or slightly increased . Introduction of BAP2 on a centromere-based vector, leading to a gene dosage of two or slightly more, caused a 50% increase in leucine uptake and a smaller increase for isoleucine and valine . However, when the 29 C-terminal codons of the plasmid-borne copy of BAP2 were substituted, the cells more than doubled the uptake of leucine, isoleucine and valine, while no or little increase in uptake was observed for the other 8 amino acids. FEBS Lett, 1995 Nov 27, 376(1-2), 120 - 4 Identification of regulatory proteins that might be involved in carbon catabolite repression of the aminopeptidase I gene of the yeast Saccharomyces cerevisiae; Bordallo J et al.; Transcription of the vacuolar aminopeptidase yscI (APE1) gene in Saccharomyces cerevisiae has previously been suggested to require the participation of a cis upstream activation sequence (UAS) involved in carbon catabolite repression that responds to glucose . To determine the structure of the APE1 UAS element, we used the 18-bp sequence 5'-ATGAATTAGTCAGCTTCT-3' as the DNA-binding site . Using gel mobility shift assays, we have identified a 78 kDa protein from yeast that binds specifically to both single and double-stranded forms of the UAS DNA-binding site . We have also identified a 48 kDa heterodimer from yeast that binds specifically to the single-stranded form of the UAS and whose DNA binding activity is remarkably heat stable . Even though the APE1 UAS contains a consensus sequence for the binding of the yeast activator protein yAP1, the two DNA-protein complexes could still be detected in a strain bearing a deletion in the YAP1 gene. Mol Gen Genet, 1995 Nov 27, 249(3), 309 - 16 The effects of a ring chromosome on the meiotic segregation of other chromosomes in Saccharomyces cerevisiae; Flatters M et al.; Meiotic chromosome segregation must occur with high fidelity in order to prevent the generation of aneuploid cells . We have previously described the identification and genetic characterization of a yeast mutant with defects in meiotic sister-chromatid segregation . We attributed the phenotype in this mutant to a dominant allele, which we referred to as SID1-1 . These mutants appeared to exhibit high levels of non-disjunction and precocious separation of sister-chromatids of chromosome III, as well as precocious separation of sister chromatids of chromosome VIII and a univalent artificial chromosome . We show here that the unusual meiotic behavior of chromosome III in these strains is due to the presence of a ring III chromosome, rather than a mutant gene . Additional experiments demonstrate that a ring III/rod III pair alters the meiotic segregation of a univalent artificial chromosome. Mol Gen Genet, 1995 Nov 27, 249(3), 289 - 96 A C-terminal region of the Saccharomyces cerevisiae transcription factor ADR1 plays an important role in the regulation of peroxisome proliferation by fatty acids; Simon MM et al.; The Saccharomyces cerevisiae transcriptional activator ADR1, which controls ADH2 gene expression, was shown to be involved in the regulation of peroxisome proliferation . To study the mode of action of ADR1, we compared strains carrying the adr1-1 mutation, high or low copy numbers of the ADR1 gene, the constitutive allele ADR1-5c, and 3'-deletions of ADR1 . High ADR1 gene dosage increased the transcription of genes encoding peroxisomal proteins as compared to one copy of the ADR1 gene . Furthermore, overexpression of ADR1 under ethanol growth conditions induced the proliferation of peroxisomal structures . The organelles were observed to be localized in clusters, a typical feature of peroxisomes induced by oleic acid . In contrast, the ADR1-5c allele, which induces ADH2 expression to a level comparable to that of high ADR1 gene dosage was found to have only a small effect . An analysis of functional domains of the ADR1 protein revealed that the N-terminal 220 amino acids of ADR1 were sufficient for wild-type levels of transcription of the FOX2, FOX3, and PAS1 genes, but the entire ADR1 protein was required for complete induction of the CTA1 gene and for growth oleic acid medium . Our data suggest that a functional domain of the ADR1 protein localized between residues 643 and 1323 is required for the induction of peroxisomal structures and for the utilization of oleic acid. Mol Gen Genet, 1995 Nov 27, 249(3), 257 - 64 Adaptation to high-salt stress in Saccharomyces cerevisiae is regulated by Ca2+/calmodulin-dependent phosphoprotein phosphatase (calcineurin) and cAMP-dependent protein kinase; Hirata D et al.; Ca2+/calmodulin-dependent phosphoprotein phosphatase (calcineurin, PP2B) of Saccharomyces cerevisiae is implicated in adaptation to high-salt conditions . Calcineurin mediates high salt-induced expression of the ENA1/PMR2 gene encoding the P-type ATPase, which is suggested to be involved in Na+ efflux . We identified the PDE1 gene encoding the low-affinity cAMP phosphodiesterase as a multicopy suppressor of the Li(+)- and Na(+)-sensitive calcineurin null mutant, suggesting that cAMP is a negative regulator of adaptation to high-salt stress . Genetic analysis indicated that calcineurin and cAMP act antagonistically in a common pathway for adaptation . The bcy1 disruption, which leads to constitutive cAMP-dependent protein kinase (PKA) activity inhibited high NaCl-induced expression of the ENA1/PMR2 gene, caused an elevation of the intracellular Na+ level and a growth defect in high-NaCl medium, all of which were analogous to the defects of a calcineurin mutant . A reduced cAMP level resulting from multiple copies of the PDE1 gene caused increased expression of the ENA1/PMR2 gene in response to high NaCl . We propose a model for the regulation of cation homeostasis, in which calcineurin antagonizes PKA to activate transcription of the ENA1/PMR2 gene in response to high-salt conditions. Nucleic Acids Res, 1995 Nov 25, 23(22), 4616 - 9 Cloning and characterisation of the gene encoding the ribosomal protein S5 (also known as rp14, S2, YS8) of Saccharomyces cerevisiae; Ignatovich O et al.; The protein sequence derived from a cloned yeast gene and partial cDNA has high sequence identity to 40S ribosomal subunit S5 proteins of higher eukaryotic origin . The open reading frame of the gene is flanked by consensus sequence motifs characteristic of ribosomal protein genes and the pattern of transcription of the gene in yeast cells subjected to nutritional shift or temperature shock is also typical of a ribosomal protein gene . The gene is single copy and essential for viability . The predicted sequence of the N-terminus of the protein identifies it as a phosphorylated ribosomal protein variously known as rp14, S2 or YS8, the least basic of the non-acidic ribosomal proteins of Saccharomyces cerevisiae. Biochem Biophys Res Commun, 1995 Nov 22, 216(3), 993 - 8 Glucose-6-phosphate dehydrogenase from Saccharomyces cerevisiae is a glycoprotein; Reilly KE et al.; A commercial preparation of glucose-6-phosphate dehydrogenase (G6PD) purified from Saccharomyces cerevisiae was subjected to PAGE analysis under both nondenaturing and denaturing conditions . The enzyme, identified by both activity staining and anti-yeast G6PD antibody immunoblotting, was shown to contain carbohydrate using the highly specific periodate-digoxigenin antidigoxigenin method which is diagnostic for glycoproteins. Biochem Biophys Res Commun, 1995 Nov 22, 216(3), 985 - 92 Cloning of the gene encoding a putative serine/threonine protein kinase which enhances spermine uptake in Saccharomyces cerevisiae; Kakinuma Y et al.; Polyamine uptake in Saccharomyces cerevisiae was modulated by extracellular magnesium; in a magnesium-limited medium, polyamine, especially spermine, was overaccumulated into the interior, whose level was then toxic for the growth of this organism (Maruyama, T., Masuda, N., Kakinuma, Y., and Igarashi K . (1994) Biochim . Biophys . Acta 1194, 289-295) . Here we isolated a mutant (strain YTM22-8) whose growth was tolerant to spermine in magnesium-limited medium . This mutant was defective in polyamine uptake and did not overaccumulate spermine . From a yeast genome library we cloned a gene (POT1) which restored the spermine uptake of this mutant and the sensitivity of the growth to spermine . The nucleotide sequence of the POT1 gene indicated that it encodes a putative serine/threonine protein kinase and is located on chromosome XI . The results suggest that spermine uptake by this organism is probably regulated by phosphorylation and dephosphorylation. Biochem Biophys Res Commun, 1995 Nov 22, 216(3), 1041 - 7 Induction of heat-shock proteins and accumulation of trehalose by TPN in Saccharomyces cerevisiae; Fujita K et al.; TPN {Tetrachloroisophthalonitrile}, a kind of disinfectant, affected growth in Saccharomyces cerevisiae . Exposed to TPN under no lethal conditions, 70-, 90-kDa protein and hsp104 were induced . Each of them was not uniformly induced; namely, the 70-kDa protein was more sensitive to TPN among other proteins . Trehalose was also accumulated depending on the concentration . The degree of thermotolerance in yeast cells pretreated with 1.0 mg/l TPN was about 100-fold greater than in the control . Under this condition, TPN-inducible proteins were synthesized but trehalose was not accumulated . Although TPN-inducible proteins and trehalose were definitely synthesized with 10.0 or 100.0mg/l TPN treatment, thermotolerance was not acquired. Biochim Biophys Acta, 1995 Nov 15, 1253(1), 25 - 32 The ATPase activity of purified CDC48p from Saccharomyces cerevisiae shows complex dependence on ATP-, ADP-, and NADH-concentrations and is completely inhibited by NEM; Frohlich KU et al.; The cell cycle protein CDC48p from Saccharomyces cerevisiae is a member of a protein superfamily (AAA superfamily) characterized by a common region of approximately 200 amino-acid residues including an ATP binding consensus . CDC48p purified to homogeneity showed considerable ATPase activity which could be completely abolished by preincubation with NEM in the absence of ATP . ATP protects the protein from NEM and stabilizes the otherwise labile enzyme . The ATPase activity is reversibly inhibited by NADH and shows cooperativity with its substrate ATP . The application of the in vitro ATPase activity to the identification of physiologically interacting molecules is discussed . By electron microscopy, the enzyme was shown to consist of hexameric ring structures similar to its vertebrate homologue. Biochim Biophys Acta, 1995 Nov 15, 1253(1), 13 - 5 The topology of CuA in relation to the other metal centres in cytochrome-c oxidase of Saccharomyces cerevisiae as determined by analysis of second-site reversions; Meunier B et al.; Second-site revertants were selected from a respiratory-deficient mutant carrying the mutation D369N located in a loop between helices IX and X close to H376 and H378, the proposed ligands of haem a3 and haem a, respectively . A reversion was observed in subunit II, in the vicinity of the CuA ligands . This same reversion compensates the subunit I deficiency mutation, S140L, assumed to be near H62, the second putative histidine ligand to haem a . These data enable us to propose a three-dimensional topology in which CuA in subunit II is located on top of the Positive-side of subunit I and in proximity to all three of its metal centres. Biochem J, 1995 Nov 15, 312 ( Pt 1), 83 - 90 A new model for disruption of the ornithine decarboxylase gene, SPE1, in Saccharomyces cerevisiae exhibits growth arrest and genetic instability at the MAT locus; Schwartz B et al.; Ornithine decarboxylase (ODC) is a rate-determining enzyme of the polyamine-biosynthetic pathway . We sought to produce cells with impaired ODC function in order to study the biological functions of polyamines . Saccharomyces cerevisiae strains were obtained by one-step gene replacement of a 900 bp fragment of the yeast ODC gene (SPE1) with the yeast URA3 gene . Spores derived from SPE1/spe1 cells germinated at reduced efficiency relative to SPE1/SPE1 . Sustained growth of spe1 haploid mutants in polyamine-free medium led to intracellular polyamine depletion, reduction in budding index, G1 arrest and cessation of growth, and cells that were large and misshapen . All of these effects were completely reversed by adding polyamines to the medium, even after 5 days of polyamine starvation . A diploid yeast strain bearing two copies of disrupted spe1 lost heterozygosity at the mating-type locus more often when grown in the absence of polyamines than when grown in their presence, indicating that polyamine deficiency leads to either chromosome loss or to mitotic recombination. Biochem Biophys Res Commun, 1995 Nov 13, 216(2), 676 - 85 Oxygen regulation of the cytochrome c oxidase subunit VI gene, COX6, in Saccharomyces cerevisiae; Wright RM et al.; Cytochrome c oxidase (CcO) in Saccharomyces cerevisiae is subject to intricate physiological control . Growth phase, carbon source, and oxygen level are three well recognized modulators of CcO expression . We focused on the subunit VI encoding gene, COX6, and detected unexpectedly complex oxygen regulation . We found that COX6 transcription possessed a critical threshold oxygen regulation between 0 and 2% . COX6 transcription was superinduced by elevated oxygen level up to 45%; however, superinduction was lost at 60% oxygen and above . The COX6 upstream activation region, UAS6, contains both glucose and heme responsive regions, and COX6 oxygen regulation was transduced through UAS6 by heme, as has been described for other oxygen regulated genes in yeast . We found that binding of the UAS6-domain 1 protein, BAF1, was unaltered by oxygen regulation . Nor were the alternative BAF1 complexes observed by growth in different glucose conditions formed by growth at different oxygen levels. Biochem Biophys Res Commun, 1995 Nov 13, 216(2), 458 - 66 Identification of a low specificity, oxygen, heme, and growth phase regulated DNA binding activity in Saccharomyces cerevisiae; Wright RM et al.; Cytochrome c oxidase from Saccharomyces cerevisiae is subject to intricate control at the level of transcription of the various genes encoding its subunits . Expression of the subunit VI encoding gene, COX6, is glucose repressed, growth phase induced, and dependent on oxygen and heme availability . An upstream activation region for COX6, UAS6, was found to contain a glucose responsive region, a heme dependent region (HDS1), and a binding site for the transcription factor, BAF1 . BAF1 was the only factor observed to form a protein complex with UAS6 in vitro . However, we found that binding of BAF1 was unaffected by oxygen or heme regulation . In the present communication, we have identified a DNA binding activity that was growth phase induced and dependent on oxygen and heme availability . This highly regulated activity was detected by its discrete binding to the heme responsive site, HDS1, in UAS6 . Nonetheless, it appears to have weak DNA binding specificity. Nucleic Acids Res, 1995 Nov 11, 23(21), 4320 - 8 Promoter elements of the PHR1 gene of Saccharomyces cerevisiae and their roles in the response to DNA damage; Sancar GB et al.; The PHR1 gene of Saccharomyces cerevisiae encodes the apoenzyme for the DNA repair enzyme photolyase . PHR1 transcription is induced in response to 254 nm radiation and a variety of chemical damaging agents . We report here the identification of promoter elements required for PHR1 expression . Transcription is regulated primarily through three sequence elements clustered within a 120 bp region immediately upstream of the translational start site . A 20 bp interrupted palindrome comprises UASPHR1 and is responsible for 80-90% of basal and induced expression . UASPHR1 alone can activate transcription of a CYC1 minimal promoter but does not confer damage responsiveness . In the intact PHR1 promoter UAS function is dependent upon an upstream essential sequence (UES) . URSPHR1 contains a binding site for the damage-responsive repressor Prp; consistent with this role, deletion or specific mutations of the URS increase basal level expression and decrease the induction ratio . Deletion of URSPHR1 also eliminates the requirement for UESPHR1 for promoter activation, indicating that the UES attenuates Prp-mediated repression . Sequences within UASPHR1 are similar to regulatory sequences found upstream of both damage responsive and nonresponsive genes involved in DNA repair and metabolism. J Biol Chem, 1995 Nov 10, 270(45), 27045 - 50 Regulation of profilin localization in Saccharomyces cerevisiae by phosphoinositide metabolism; Ostrander DB et al.; Profilin is an actin- and phosphatidylinositol 4,5-bisphosphate-binding protein that plays a role in the organization of the cytoskeleton and may be involved in growth factor signaling pathways . The subcellular localization of profilin was examined in the yeast Saccharomyces cerevisiae . Immunoblot analysis showed that profilin was localized in both the plasma membrane and cytosolic fractions of the cell . Actin was bound to the profilin localized in the cytosol . The association of profilin with the membrane was peripheral and mediated through interaction with phospholipid . The phospholipid dependence of profilin for membrane binding was examined in vitro using pure profilin and defined unilamellar phospholipid vesicles . The presence of phosphatidylinositol 4,5-bisphosphate in phospholipid vesicles was required for maximum profilin binding . Moreover, the binding of profilin to phospholipid vesicles was dependent on the surface concentration of phosphatidylinositol 4,5-bisphosphate . The subcellular localization of profilin was examined in vivo under growth conditions (i.e . inositol starvation of ino1 cells and glucose starvation of respiratory deficient cells) where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate were depleted . Depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate levels resulted in a translocation of profilin from the plasma membrane to the cytosolic fraction . Profilin translocated back to the membrane fraction from the cytosol under growth conditions where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate were replenished . These results suggested that phosphoinositide metabolism played a role in the localization of profilin. Biochem Pharmacol, 1995 Nov 9, 50(10), 1695 - 9 Induction in the gene RNR3 in Saccharomyces cerevisiae upon exposure to different agents related to carcinogenesis; Endo-Ichikawa Y et al.; The induction of the gene RNR3 was investigated in yeast Saccharomyces cerevisiae using RNR31 lacZ fusion . Gene induction was monitored by measuring beta-galactosidase activity . Various drugs that cause DNA damage effectively induced RNR3 expression; alkylating agents (cisplatin, mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine), a radical producer (bleomycin), and an intercalator (actinomycin D) induced RNR3 . When yeast expressing rat CYP1A1 was exposed to 2-aminofluorene, a concentration-dependent induction of RNR3 was observed . Aflatoxin B1 also induced the expression of RNR3 in the same yeast strain concomitant with inhibition of cell growth . In control yeast, no induction of RNR3 was observed upon exposure to 2-aminofluorene or aflatoxin B1 . Exposure to 2-acetylaminofluorene or benzo{a}pyrene did not lead to induction of RNR3 in yeast expressing CYP1A1 . These results indicate that DNA damage by chemicals related to carcinogenesis induces RNR3, and that activation of these procarcinogens was required for DNA damage-dependent induction of RNR3. Gene, 1995 Nov 7, 165(1), 97 - 102 Isolation and characterization of the Saccharomyces cerevisiae SUT1 gene involved in sterol uptake; Bourot S et al.; A new gene (SUT1) of Saccharomyces cerevisiae, implicated in sterol uptake, was isolated from a yeast genomic library constructed in a high-copy-number vector by virtue of conferring resistance to fenpropimorph in medium supplemented with ergosterol . The high expression of SUT1 in sterol auxotrophic mutant strains alleviates the requirement for accessory mutations affecting heme biosynthesis and allows sterol uptake in aerobiosis . Measurements of {14C}cholesterol uptake confirmed that SUT1 is involved in sterol absorption . Within the 4.1-kb insert isolated, the functional gene was localised on a 1.7-kb DNA fragment . The nucleotide sequence encodes a predicted protein of 299 amino acids . Northern blot analysis revealed that SUT1 is a new member of the hypoxic gene family . Gene disruption showed that SUT1 is not essential for aerobic or anaerobic yeast growth. Gene, 1995 Nov 7, 165(1), 25 - 9 Cloning and characterization of the Saccharomyces cerevisiae SVS1 gene which encodes a serine- and threonine-rich protein required for vanadate resistance; Nakamura T et al.; A novel Saccharomyces cerevisiae (Sc) SVS1 gene was cloned as a multicopy suppressor of vanadate (Vn) sensitivity (VnS) due to a calcineurin (CaN) null mutation . SVS1 encoded a 260-amino-acid protein abundant in Ser and Thr residues, with a putative signal sequence at the N terminus . Deletion of SVS1 resulted in increased sensitivity to Vn, but not to other metallic ions or drugs . Northern analysis of the SVS1 mRNA indicated that the induction of the gene occurred specifically in the response to Vn . These results suggested that Sc has a mechanism to enhance the tolerance to Vn by increasing the expression of SVS1 . The results of genetic experiments suggested that CaN and the Svs1 proteins act in separate pathways to enhance the tolerance to Vn. Proc Natl Acad Sci U S A, 1995 Nov 7, 92(23), 10595 - 8 Location of the active site of allosteric chorismate mutase from Saccharomyces cerevisiae, and comments on the catalytic and regulatory mechanisms; Xue Y et al.; The active site of the allosteric chorismate mutase (chorismate pyruvatemutase, EC 5.4.99.5) from yeast Saccharomyces cerevisiae (YCM) was located by comparison with the mutase domain (ECM) of chorismate mutase/prephenate dehydratase {prephenate hydro-lyase (decarboxylating), EC 4.2.1.51} (the P protein) from Escherichia coli . Active site domains of these two enzymes show very similar four-helix bundles, each of 94 residues which superimpose with a rms deviation of 1.06 A . Of the seven active site residues, four are conserved: the two arginines, which bind to the inhibitor's two carboxylates; the lysine, which binds to the ether oxygen; and the glutamate, which binds to the inhibitor's hydroxyl group in ECM and presumably in YCM . The other three residues in YCM (ECM) are Thr-242 (Ser-84), Asn-194 (Asp-48), and Glu-246 (Gln-88) . This Glu-246, modeled close to the ether oxygen of chorismate in YCM, may function as a polarizing or ionizable group, which provides another facet to the catalytic mechanism. J Biol Chem, 1995 Nov 3, 270(44), 26715 - 20 Structure-function relationships in the Saccharomyces cerevisiae poly(A) polymerase . Identification of a novel RNA binding site and a domain that interacts with specificity factor(s); Zhelkovsky AM et al.; We have constructed deletions in the nonconserved regions at the amino and carboxyl ends of the poly(A) polymerase (PAP) of Saccharomyces cerevisiae and examined the effects of these truncations on function of the enzyme . PAP synthesizes a poly(A) tail onto the 3'-end of RNA without any primer specificity but, in the presence of cellular factors, is directed specifically to the cleaved ends of mRNA precursors . The last 31 amino acids of PAP are dispensable for both nonspecific and specific activities . Removal of the next 36 amino acids affects an RNA binding domain, which is essential for the activity of the enzyme and for cell viability . This novel RNA binding site was further localized using additional deletions, cyanogen bromide cleavage of PAP cross-linked with RNA or 8-azido-ATP, and a monoclonal antibody against a COOH-terminal PAP epitope . A deletion that partially disrupts this domain has reduced nonspecific activity but functions in specific polyadenylation . In contrast, deletion of the first 18 amino acids of PAP has no effect on nonspecific polyadenylation but completely eliminates specific activity . This region is essential for enzyme function in vivo and is probably involved in the interaction of PAP with other protein(s) of the polyadenylation machinery. J Biol Chem, 1995 Nov 3, 270(44), 26505 - 10 Characterization of the in vivo phosphorylation sites of the mRNA.cap-binding complex proteins eukaryotic initiation factor-4E and p20 in Saccharomyces cerevisiae; Zanchin NI et al.; Eukaryotic translation is believed to be regulated via the phosphorylation of specific eukaryotic initiation factors (eIFs), including one of the cap-binding complex proteins, eIF-4E . We show that in the yeast Saccharomyces cerevisiae, both eIF-4E and another cap-binding complex protein, p20, are phosphoproteins . The major sites of phosphorylation of yeast eIF-4E are found to be located in the N-terminal region of its sequence (Ser2 and Ser15) and are thus in a different part of the protein from the main phosphorylation sites (Ser53 and Ser209) proposed previously for mammalian eIF-4E . The most likely sites of p20 phosphorylation are at Ser91 and/or Ser154 . All of the major sites in the two yeast proteins are phosphorylated by casein kinase II in vitro . Casein kinase II phosphorylation of cap-complex proteins should therefore be considered as potentially involved in the control of yeast protein synthesis . Mutagenesis experiments revealed that yeast eIF-4E activity is not dependent on the presence of Ser2 or Ser15 . On the other hand, we observed variations in the amount of (phosphorylated) p20 associated with the cap-binding complex as a function of cell growth conditions . Our results suggest that interactions of yeast eIF-4E with other phosphorylatable proteins, such as p20, could play a pivotal role in translational control. J Biol Chem, 1995 Nov 3, 270(44), 26446 - 50 Catabolite inactivation of fructose-1,6-bisphosphatase of Saccharomyces cerevisiae . Degradation occurs via the ubiquitin pathway; Schork SM et al.; Catabolite inactivation of fructose-1,6-bisphosphatase (FBPase), a key enzyme in gluconeogenesis, is due to phosphorylation and subsequent degradation in the yeast Saccharomyces cerevisiae . The degradation process of the enzyme had been shown to depend on the action of the proteasome . Here we report that components of the ubiquitin pathway target FBPase to proteolysis . Upon glucose addition to yeast cells cultured on nonfermentable carbon sources FBPase is ubiquitinated in vivo . A multiubiquitin chain containing isopeptide linkages at Lys48 of ubiquitin is attached to FBPase . Formation of a multiubiquitin chain is a prerequisite for the degradation of FBPase . Catabolite degradation of FBPase is dependent on the ubiquitin-conjugating enzymes Ubc1, Ubc4, and Ubc5 . The 26 S proteasome is involved in the degradation process. J Biol Chem, 1995 Nov 3, 270(44), 26168 - 77 Structure of Saccharomyces cerevisiae alpha-agglutinin . Evidence for a yeast cell wall protein with multiple immunoglobulin-like domains with atypical disulfides; Chen MH et al.; alpha-Agglutinin of Saccharomyces cerevisiae is a cell wall-associated protein that mediates cell interaction in mating . Although the mature protein includes about 610 residues, the NH2-terminal half of the protein is sufficient for binding to its ligand a-agglutinin . alpha-Agglutinin20-351, a fully active fragment of the protein, has been purified and analyzed . Circular dichroism spectroscopy, together with sequence alignments, suggest that alpha-agglutinin20-351 consists of three immunoglobulin variable-like domains: domain I, residues 20-104; domain II, residues 105-199; and domain III, residues 200-326 . Peptide sequencing data established the arrangement of the disulfide bonds in alpha-agglutinin20-351 . Cys97 is disulfide-bonded to Cys114, forming an interdomain bond between domains I and II . Cys202 is bonded to Cys300, in an atypical intradomain disulfide bond between the A and F strands of domain III . Cys227 and Cys256 have free sulfhydryls . Sequencing also showed that at least two of three potential N-glycosylation sites with sequence Asn-Xaa-Thr are glycosylated . At least one of three Asn-Xaa-Ser sequences is not glycosylated . No residues NH2-terminal to Ser282 were O-glycosylated, whereas Ser282, and all hydroxy amino acid residues COOH-terminal to this position were modified . Therefore O-glycosylated Ser and Thr residues cluster in the COOH-terminal region of domain III, and the O-glycosylation continues into a Ser/Thr-rich sequence that extends from domain III to the COOH-terminal of the full-length protein. Plasmid, 1995 Nov, 34(3), 184 - 97 The CDC6 gene is required for centromeric, episomal, and 2-microns plasmid stability in the yeast Saccharomyces cerevisiae; Storici F et al.; Temperature-sensitive Saccharomyces cerevisiae cdc6 mutants, under restrictive conditions, show an increase in recombination frequency, as well as chromosome and circular minichromosome loss . The role of the essential CDC6 gene was tested in trans and in cis to study circular plasmid stability . It was possible to demonstrate that the product of the CDC6 gene, acting in trans, is important for centromeric, episomal, and also 2-microns plasmid maintenance, while the gene sequence itself has no effect in cis on the stability of the plasmids tested . A high percentage of phenotypic revertants for the cdc6 mutation loses 2 microns upon shifting to the restrictive temperature and, under semipermissive conditions, the endogenous plasmid becomes very unstable, favoring a more efficient curing procedure . A positive correlation between centromeric plasmid size and stability was demonstrated even for small circular plasmids. Mol Microbiol, 1995 Nov, 18(4), 605 - 14 Organization of the Saccharomyces cerevisiae actin gene UAS: functional significance of reiterated REB1 binding sites and AT-rich elements; McLean M et al.; The upstream activation sequence (UAS) in the Saccharomyces cerevisiae actin gene promoter contains three different motifs, specifically two AT-rich tracts, two binding sites for the yeast protein REB1, and an Mlul site . Synthetic UAS elements containing individual motifs, or combinations of them, were inserted in place of the natural UAS, and assayed using a lacZ reporter gene . The REB1 binding sites were found to be essential for, and sufficient to restore partial, UAS activity . AT-rich tracts alone were inactive . Multimerization of a REB1 binding site created a UAS that in galactose is more active, but in glucose less active, than a UAS having a single REB1 site with one AT-rich tract . In general, transcription during growth in galactose or glycerol/lactate responds more to multimerization of motifs . The results suggest that the natural actin promoter UAS retains activity on these alternative carbon sources because of reiteration of sequence elements within it; the additional elements appear to be redundant when cells are grown on glucose . The Mlul site, which is present upstream of a number of yeast genes involved in DNA synthesis and confers cell cycle periodicity to those genes, contributes to the activity of the synthetic UAS elements, but not in a cell-cycle-dependent manner. Eur J Cell Biol, 1995 Nov, 68(3), 275 - 87 Autophagic death after cell cycle arrest at the restrictive temperature in temperature-sensitive cell division cycle and secretory mutants of the yeast Saccharomyces cerevisiae; Motizuki M et al.; When virgin temperature-sensitive mutant cdc28 cells of the yeast Saccharomyces cerevisiae were incubated at restrictive temperatures, they continued to grow, reaching a maximum of 3.3-fold the original size after 24 h . The protein and RNA levels increased during the first 24 h, then gradually decreased . The cells that reached the maximal size lost proliferative activity and synthesized less protein . After a 72-h incubation, cellular components, protein, RNA and DNA, were progressively degraded, resulting in extensive fragmentation within 7 days . Light and electron microscopic observation revealed that cdc28 cells incubated at the restrictive temperature for 24 h were enriched with double-unit membranes in the cytoplasm, and the vacuoles were filled with autophagic body-like structures . After 7 days most cellular contents were lost, and the membrane systems were fragmented . The protein synthesis inhibitor cycloheximide added at 24 h inhibited degradation of protein for at least 7 days suggesting that protein synthesis was involved in the activation of autophagic death . All other temperature-sensitive cdc and secretory (sec) mutants tested showed similar morphological changes when arrested in the cell division cycle at the restrictive temperature . In contrast, the temperature-insensitive wild-type cells grew normally at 38 degrees C and only a few percent of them underwent autolysis 7 days after transfer to the stationary phase . These results suggested that the yeast cells underwent autophagic death when arrested at any stage of the cell division cycle, whereas those arrested at the stationary phase rarely underwent autophagic death. Mutagenesis, 1995 Nov, 10(6), 543 - 8 Preferential repair in Saccharomyces cerevisiae rad mutants after induction of interstrand cross-links by 8-methoxypsoralen plus UVA; Meniel V et al.; The gene specific induction and the incision step of the removal of 8-methoxypsoralen (8-MOP) plus UVA-induced interstrand cross-links (ICL) was measured in repair mutants of Saccharomyces cerevisiae . Events were examined at the MAT alpha and HML alpha loci in mutants deficient in the repair of ICL, namely rad1, rad2 delta, rad52, pso2 and the rad16 mutant which is impaired in the removal of UV-induced pyrimidine dimers from the silent HML alpha locus . Previously, we observed in a wild-type strain (K107) preferential repair concerning the incision of 8-MOP photo-induced ICL . The present study indicates that the two mutants rad1 and rad2 delta show no repair in either locus, due presumably to their deficiency in the incision step of ICL repair . The rad52 mutant which is defective in recombination, is proficient in the preferential incision of ICL at the MAT alpha locus versus the HML alpha locus . The same is true for the pso2 mutant which also lacks the ability to perform complete repair of ICL . The rad16 mutant is unable to repair ICL in the silent locus HML alpha but is proficient in repair (i.e . the incision of ICL) in the transcriptionally active MAT alpha locus. Curr Genet, 1995 Nov, 28(6), 521 - 5 Effects of the CDC2 gene on adaptive mutation in the yeast Saccharomyces cerevisiae; Baranowska H et al.; We have studied the influence of a temperature-sensitive cdc2-1 mutation in DNA polymerase delta on the selection-induced mutation occurring at the LYS-2 locus in the yeast Saccharomyces cerevisiae . It was found that in cells plated on synthetic complete medium lacking only lysine, the numbers of Lys+ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number . When cdc2-1 mutant cells, after selective plating, were incubated at the restrictive temperature of 37 degrees C for 5 h daily for 7 days, the frequency of an adaptive reversion of lys(-)-->Lys+ was significantly higher than the frequency in cells incubated only at the permissive temperature, or in wild-type cells incubated either at 23 degrees C or 37 degrees C . Therefore, when the proofreading activity of DNA polymerase delta is impaired under restrictive conditions, the frequency of adaptive mutations is markedly enhanced. Curr Genet, 1995 Nov, 28(6), 517 - 20 Nature of abortive transformation in Saccharomyces cerevisiae; Yap WY et al.; Disruption mutagenesis by homologous recombination in Saccharomyces cerevisiae is carried out by transforming-DNA fragments containing the target gene disrupted by a selectable marker . A large number of transient (abortive) transformants are often formed that may hinder the isolation of integrants containing the gene disruption . We show that abortive transformants result from re-circularization of the linear transforming-DNA in vivo . Their number was greatly reduced when the cut DNA could not readily re-ligate, either by digestions that gave non-compatible or blunt ends, or by de-phosphorylation . In addition, true integrants could be readily distinguished from abortive transformants through replica plating onto selective media . Enhanced disruption-mutagenesis was also observed when non-compatible ends were generated in an ARS-containing insertion vector. Curr Genet, 1995 Nov, 28(6), 512 - 6 Identification and characterization of CEN12 in the budding yeast Saccharomyces cerevisiae; Gammie AE et al.; In this paper we report the cloning, sequencing and functional characterization of CEN12 and an associated autonomously replicating sequence (ARS) from the budding yeast Saccharomyces cerevisiae . In the course of studying a dynamin-related gene, DNM1, we previously physically mapped the gene to chromosome 12 . Genetic mapping showed that the gene was tightly linked (0.35 cM) to the centromere . Subcloning experiments revealed that a centromere-like activity was included in a small segment of DNA immediately downstream from the DNM1 gene . Mitotic centromere activity was discerned by the ability of the region to de-stabilize a centromere-containing plasmid, and to stabilize an ARS-containing plasmid . Meiotic centromere activity was determined by the first-division segregation in crosses of ARS plasmids containing this region . The DNA sequence of this region revealed a sequence with strong homology to the consensus for yeast centromeres. Curr Genet, 1995 Nov, 28(6), 503 - 11 Positive and negative elements involved in the differential regulation by heme and oxygen of the HEM13 gene (coproporphyrinogen oxidase) in Saccharomyces cerevisiae; Amillet JM et al.; The Saccharomyces cerevisiae HEM13 gene codes for coproporphyrinogen oxidase (CPO), an oxygen-requiring enzyme catalysing the sixth step of heme biosynthesis . Its transcription is increased 40-50-fold in response to oxygen- or heme-deficiency . We have analyzed CPO activity and HEM13 mRNA levels in a set of isogenic strains carrying single or double deletions of the CYP1 (HAP1), ROX1, SSN6, or TUP1 genes . The cells were grown in the presence or absence of oxygen and under heme-deficiency (hem1 delta background) . Both Rox1p and Cyp1p partially repressed HEM13 in aerobic heme-sufficient cells, probably in an independent manner . In the absence of heme, Cyp1p activated HEM13 and strongly repressed ROX1, allowing de-repression of HEM13 . Cyp1p had no effect on HEM13 expression in anaerobic cells . Deletions of SSN6 or TUP1 dramatically de-repressed HEM13 in aerobic cells . A series of deletions in the HEM13 promoter identified at least four regulatory regions that are required for HEM13 regulation . Two regions, containing motifs similar to the Rox1p consensus sequences, act as repression sites under aerobic growth . The two other sites act as activation sequences required for full induction under oxygen- or heme-deficiency . Taken together, these results suggest that induction of HEM13 occurs in part through relief of repression exerted by Rox1p and Cyp1p, and in part by activation mediated partly by Cyp1p under heme-deficiency and by unknown factors under oxygen-deficiency. Appl Microbiol Biotechnol, 1995 Nov, 43(6), 1050 - 5 Characterization of rice alpha-amylase isozymes expressed by Saccharomyces cerevisiae; Terashima M et al.; Two rice alpha-amylase isozymes, AmylA and Amy3D, were produced by secretion from genetically engineered strains of Saccharomyces cerevisiae . They have distinct differences in enzymatic characteristics that can be related to the physiology of the germinating rice seed . The rice isozymes were purified with immunoaffinity chromatography . The pH optima for Amy3D (pH optimum 5.5) and Amy1A (pH optimum 4.2) correlate with the pH of the endosperm tissue at the times in rice seedling development when these isozymes are produced . Amy3D showed 10-14 times higher reactivity to oligosaccharides than Amy1A . Amy1A, on the other hand, showed higher reactivity to soluble starch and starch granules than Amy3D . These results suggest that the isozyme Amy3D, which is expressed at an early stage of germination, produces sugars from soluble starch during the early stage of seed germination and that the isozyme Amy1A works to initiate hydrolysis of the starch granules. Yeast, 1995 Nov, 11(14), 1421 - 7 The sequence of a 27 kb segment on the right arm of chromosome VII from Saccharomyces cerevisiae reveals MOL1, NAT2, RPL30B, RSR1, CYS4, PEM1/CHO2, NSR1 genes and ten new open reading frames; Skala J et al.; The DNA sequence of a 26,677 bp fragment from the right arm of chromosome VII from Saccharomyces cerevisiae reveals 18 open reading frames (ORFs) longer than 300 bp . Eight ORFs correspond to previously characterized genes . G6620 is the 3' end of the MOL1 gene coding for a polypeptide similar to stress-inducible proteins from Fusarium; G6630 is the NAT2 gene which encodes a methionine N-acetyltransferase; G6635 is the RPL30B gene coding for the ribosomal protein L30; G6658 is RSR1 encoding a ras-related protein; G6667 is CYS4, the gene for cystathionine beta-synthase; G6670 is identical to ORF2 located close to CYS4; G6673 is PEM1/CHO2 encoding a phosphatidylethanolamine methyltransferase; G7001 is the NSR1 gene coding for a nuclear signal recognition protein . G6664 shares significant homology with the ORF YKR076w from chromosome XI . The other nine ORFs show no significant homology to any protein sequence presently available in the public data bases. Yeast, 1995 Nov, 11(14), 1393 - 8 The low-affinity component of the glucose transport system in Saccharomyces cerevisiae is not due to passive diffusion; Gamo FJ et al.; It has been claimed that the low-affinity component of glucose transport in Saccharomyces cerevisiae is due to passive diffusion of the sugar across the plasma membrane . We have investigated this possibility . For this purpose we have measured the permeability coefficient of hexoses in this organism . We have found that this coefficient is at least two to three orders of magnitude lower than required to account for the low-affinity component of glucose transport, and have concluded that this component is not due to passive diffusion. Yeast, 1995 Nov, 11(14), 1367 - 80 Further definition of the sequence and position requirements of the arginine control element that mediates repression and induction by arginine in Saccharomyces cerevisiae; Crabeel M et al.; Repression or induction of the genes involved in arginine biosynthesis or catabolism, respectively, both require participation of the ArgRp/Mcm1p regulatory complex . Our previous work showed that those opposite effects were mediated by a similar arginine-responsive element of 23 nucleotides (that we now call ARC, for ARginine Control) situated close to the start of transcription in the repressed promoters and far upstream of the TATA-element in the induced promoters . To define more precisely the sequence and position requirements of the ARC element, we have now characterized by mutagenesis the promoter elements of the arginine-repressible ARG1 and ARG8 genes . We also identify a functional ARC in the CPA1 promoter, thereby confirming, in agreement with our previous mRNA pulse-labelling data, the participation of a transcriptional component in the arginine regulation of that gene otherwise submitted to a translational regulation . From the 12 ARC elements now characterized, we have derived a consensus sequence and show that such a synthetic element is able to mediate ArgRp/Mcm1p-dependent arginine regulation . An important new finding illustrated by ARG1 and CPA1, is that contrary to what all the previous data suggested, repression can be mediated by ARC elements located far upstream of the TATA-box . The new data suggest that the arginine repressor might inhibit transcription in an active process. Genetics, 1995 Nov, 141(3), 889 - 902 Roles of ABF1, NPL3, and YCL54 in silencing in Saccharomyces cerevisiae; Loo S et al.; A sensitized genetic screen was carried out to identify essential genes involved in silencing in Saccharomyces cerevisiae . This screen identified temperature-sensitive alleles of ORC2 and ORC5, as described elsewhere, and ABF1, NPL3, and YCL54, as described here . Alleles of ABF1 that caused silencing defects provided the genetic proof of Abflp's role in silencing . The roles of Npl3p and Ycl54p are less clear . These proteins did not act exclusively through any one of the three protein binding sites of the HMR-E silencer . Unlike the orc2, orc5, and abf1 mutations that were isolated in the same (or a similar) screen for silencing mutants, neither temperature-sensitive mutation in NPL3 or YCL54 caused overt replication defects. Genetics, 1995 Nov, 141(3), 833 - 44 vph6 mutants of Saccharomyces cerevisiae require calcineurin for growth and are defective in vacuolar H(+)-ATPase assembly; Hemenway CS et al.; We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B) . A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype . The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain . We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase) . The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins . We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification . In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1 . Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis. Mol Gen Genet, 1995 Nov 1, 249(1), 51 - 7 Artificial antisense RNA regulation of YBR1012 (YBR136w), an essential gene from Saccharomyces cerevisiae which is important for progression through G1/S; Nasr F et al.; YBR1012 (YBR136w) is an essential gene from Saccharomyces cerevisiae identified during the systematic sequencing of part of the right arm of chromosome II . We previously constructed a conditional allele of YBR1012 based on antisense RNA, by inserting a small fragment of this gene downstream from the inducible UASGAL10-CYC1 promoter . Several other antisense RNA constructions have since been made and their activity tested . The response of the system appears to be very delicate, as the presence or absence of 13 nucleotides of polylinker in the 300 nucleotide antisense transcript can dramatically modify its effectiveness . The most effective antisense RNA construction was used in flow cytometry studies to investigate the role of ybr1012p . The results show that during the antisense RNA block some 80% of the cells are arrested with their DNA unreplicated, suggesting that Ybr1012p is needed for progression through G1 or early S phase. Mol Gen Genet, 1995 Nov 1, 249(1), 1 - 7 Overexpression of the RNR1 gene rescues Saccharomyces cerevisiae mutants in the mitochondrial DNA polymerase-encoding MIP1 gene; Lecrenier N et al.; A multicopy suppressor gene which rescues the temperature-sensitive growth defect of Saccharomyces cerevisiae mutants in the mitochondrial DNA (mtDNA) polymerase-encoding MIP1 gene has been isolated and identified as the RNR1 gene . This gene, whose transcript is cell cycle-regulated and mainly expressed at the G1 to S phase transition, encodes the large subunit of ribonucleotide reductase . This enzyme catalyses a limiting step in the production of deoxynucleotides needed for DNA synthesis . The presence of a high copy number of the RNR1 gene also decreases the accumulation of rho- mutants observed in diploids that harbour a single copy of the MIP1 gene . In cell cycle-synchronised cells, the presence of a high copy number of RNR1 does not modify its cell cycle transcription regulation and increases its transcript level by a factor of 10 throughout the cell cycle . Our results show that an increased supply of dNTPs in mitochondria can stimulate the mtDNA polymerase activity and indicate that the dNTP concentration may be rate limiting for the replication of mtDNA. J Cell Biol, 1995 Nov, 131(3), 603 - 17 VPS27 controls vacuolar and endocytic traffic through a prevacuolar compartment in Saccharomyces cerevisiae; Piper RC et al.; Newly synthesized vacuolar hydrolases such as carboxypeptidase Y (CPY) are sorted from the secretory pathway in the late-Golgi compartment and reach the vacuole after a distinct set of membrane-trafficking steps . Endocytosed proteins are also delivered to the vacuole . It has been proposed that these pathways converge at a "prevacuolar" step before delivery to the vacuole . One group of genes has been described that appears to control both of these pathways . Cells carrying mutations in any one of the class E VPS (vacuolar protein sorting) genes accumulate vacuolar, Golgi, and endocytosed proteins in a novel compartment adjacent to the vacuole termed the "class E" compartment, which may represent an exaggerated version of the physiological prevacuolar compartment . We have characterized one of the class E VPS genes, VPS27, in detail to address this question . Using a temperature-sensitive allele of VPS27, we find that upon rapid inactivation of Vps27p function, the Golgi protein Vps10p (the CPY-sorting receptor) and endocytosed Ste3p rapidly accumulate in a class E compartment . Upon restoration of Vps27p function, the Vps10p that had accumulated in the class E compartment could return to the Golgi apparatus and restore correct sorting of CPY . Likewise, Ste3p that had accumulated in the class E compartment en route to the vacuole could progress to the vacuole upon restoration of Vps27p function indicating that the class E compartment can act as a functional intermediate . Because both recycling Golgi proteins and endocytosed proteins rapidly accumulate in a class E compartment upon inactivation of Vps27p, we propose that Vps27p controls membrane traffic through the prevacuolar/endosomal compartment in wild-type cells. J Bacteriol, 1995 Nov, 177(22), 6672 - 5 The concentration of ammonia regulates nitrogen metabolism in Saccharomyces cerevisiae; ter Schure EG et al.; Saccharomyces cerevisiae was grown in a continuous culture at a single dilution rate with input ammonia concentrations whose effects ranged from nitrogen limitation to nitrogen excess and glucose limitation . The rate of ammonia assimilation (in millimoles per gram of cells per hour) was approximately constant . Increased extracellular ammonia concentrations are correlated with increased intracellular glutamate and glutamine concentrations, increases in levels of NAD-dependent glutamate dehydrogenase activity and its mRNA (gene GDH2), and decreases in levels of NADPH-dependent glutamate dehydrogenase activity and its mRNA (gene GDH1), as well as decreases in the levels of mRNA for the amino acid permease-encoding genes GAP1 and PUT4 . The governing factor of nitrogen metabolism might be the concentration of ammonia rather than its flux. Mol Cell Biol, 1995 Nov, 15(11), 6232 - 45 The Saccharomyces cerevisiae HSP12 gene is activated by the high-osmolarity glycerol pathway and negatively regulated by protein kinase A; Varela JC et al.; The HSP12 gene encodes one of the two major small heat shock proteins of Saccharomyces cerevisiae . Hsp12 accumulates massively in yeast cells exposed to heat shock, osmostress, oxidative stress, and high concentrations of alcohol as well as in early-stationary-phase cells . We have cloned an extended 5'-flanking region of the HSP12 gene in order to identify cis-acting elements involved in regulation of this highly expressed stress gene . A detailed analysis of the HSP12 promoter region revealed that five repeats of the stress-responsive CCCCT motif (stress-responsive element {STRE}) are essential to confer wild-type induced levels on a reporter gene upon osmostress, heat shock, and entry into stationary phase . Disruption of the HOG1 and PBS2 genes leads to a dramatic decrease of the HSP12 inducibility in osmostressed cells, whereas overproduction of Hog1 produces a fivefold increase in wild-type induced levels upon a shift to a high salt concentration . On the other hand, mutations resulting in high protein kinase A (PKA) activity reduce or abolish the accumulation of the HSP12 mRNA in stressed cells . Conversely, mutants containing defective PKA catalytic subunits exhibit high basal levels of HSP12 mRNA . Taken together, these results suggest that HSP12 is a target of the high-osmolarity glycerol (HOG) response pathway under negative control of the Ras-PKA pathway . Furthermore, they confirm earlier observations that STRE-like sequences are responsive to a broad range of stresses and that the HOG and Ras-PKA pathways have antagonistic effects upon CCCCT-driven transcription. Mol Cell Biol, 1995 Nov, 15(11), 6109 - 17 Mutational analysis of Rox1, a DNA-bending repressor of hypoxic genes in Saccharomyces cerevisiae; Deckert J et al.; Rox1 is a repressor of the hypoxic genes of Saccharomyces cerevisiae . It binds to a specific hypoxic consensus sequence in the upstream region of these genes and represses transcription in conjunction with the general repression complex Tup1-Ssn6 . In this study, we demonstrated that the first 100 amino acids comprising the HMG domain of Rox1 were responsible for DNA binding and that when bound, Rox1 bent DNA at an angle of 90 degrees . A mutational analysis resulted in the isolation of seven missense mutations, all located within the HMG domain, that caused loss of DNA binding . The effect of these mutations on the structure of Rox1 was evaluated on the basis of the homology between Rox1 and the human male sex-determining protein SRY, for which a structural model is available . The failure to isolate missense mutations in the carboxy-terminal three-quarters of the protein prompted a deletion analysis of this region . The results suggested that this region was responsible for the repression function of Rox1 and that the repression information was redundant . This hypothesis was confirmed by using a set of fusions between sequences encoding the GAL4 DNA-binding domain and portions of ROX1 . Those fusions containing either the entire carboxy-terminal region or either half of it were capable of repression . Repression by selected fusions was demonstrated to be dependent on Ssn6. Mol Cell Biol, 1995 Nov, 15(11), 5917 - 28 Mcm1 is required to coordinate G2-specific transcription in Saccharomyces cerevisiae; Althoefer H et al.; In the budding yeast Saccharomyces cerevisiae, MCM1 encodes an essential DNA-binding protein that regulates transcription of many genes in cooperation with different associated factors . With the help of a conditional expression system, we show that Mcm1 depletion has a distinct effect on cell cycle progression by preventing cells from undergoing mitosis . Genes that normally exhibit a G2-to-M-phase-specific expression pattern, such as CLB1, CLB2, CDC5, SWI5, and ACE2, remain uninduced in the absence of functional Mcm1 . In vivo footprinting experiments show that Mcm1, in conjunction with an Mcm1-recruited factor, binds to the promoter regions of SWI5 and CLB2 at sites shown to be involved in cell cycle regulation . However, promoter occupation at these sites is cell cycle independent, and therefore the regulatory system seems to operate on constitutively bound Mcm1 complexes . A gene fusion that provides Mcm1 with a strong transcriptional activation domain causes transcription of SWI5, CLB1, CLB2, and CDC5 at inappropriate times of the cell cycle . Thus, Mcm1 and a cooperating, cell cycle-regulated activation partner are directly involved in the coordinated expression of multiple G2-regulated genes . The arrest phenotype of Mcm1-depleted cells is consistent with low levels of Clb1 and Clb2 kinase . However, constitutive CLB2 expression does not suppress the mitotic defect, and therefore other essential activities required for the G2-to-M transition must also depend on Mcm1 function. J Cell Biol, 1995 Nov, 131(4), 913 - 27 Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment; Lussier M et al.; The yeast Kre2p/Mnt1p alpha 1,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane-spanning region, and a large catalytic luminal domain containing one N-glycosylation site . Anti-Kre2p/Mnt1p antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNN1-dependent manner . Kre2p/Mnt1p is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnn1p, and distinct from that including the late Golgi protein Kex1p . To determine which regions of Kre2p/Mnt1p are required for Golgi localization, Kre2p/Mnt1p mutant proteins were assembled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p . Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence . The results demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable . However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36-amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi. EMBO J, 1995 Nov 1, 14(21), 5258 - 70 The GTPase Ypt7p of Saccharomyces cerevisiae is required on both partner vacuoles for the homotypic fusion step of vacuole inheritance; Haas A et al.; In the budding yeast Saccharomyces cerevisiae, vacuoles are inherited by the projection of vesicles and tubules from the mother-cell vacuole into the growing daughter cell during the S phase . These vesicles then fuse and form the daughter-cell organelle . We have described previously in vitro reactions of the formation of vacuole-derived segregation structures and of vacuole-vacuole fusion . Homotypic vacuole fusion requires cytosol, ATP and a physiological temperature, and is sensitive to GTPase inhibitors . These reactions are divisible into early stages which require ATP and cytosol, and late stages which require neither . Here, we report that Ypt7p, a ras-like GTPase implicated previously in endocytosis in yeast, is largely localized to the vacuole and is required on both partners during the in vitro vacuole fusion reaction . The in vitro fusion reaction is inhibited either by Gdi1p, which extracts the GDP-bound form of ras-like GTPases from membranes, or by antibodies specific for Ypt7p . The presence of anti-Ypt7p during the early stages of the reaction inhibits the development of cytosol- and ATP-independent intermediates . Although cytosol and ATP are no longer needed for the late stage of vacuole inheritance in vitro, the inhibition of this late stage by anti-Ypt7p or Gdi1p requires the continued presence of ATP and cytosol . Ypt7p is the first GTPase for which a direct role in organelle inheritance has been established. J Biol Chem, 1995 Oct 27, 270(43), 25905 - 14 Casein kinase II is required for cell cycle progression during G1 and G2/M in Saccharomyces cerevisiae; Hanna DE et al.; The catalytic subunit of Saccharomyces cerevisiae casein kinase II (Sc CKII) is encoded by the CKA1 and CKA2 genes, which together are essential for viability . Five independent temperature-sensitive alleles of the CKA2 gene were isolated and used to analyze the function of CKII during the cell cycle . Following a shift to the nonpermissive temperature, cka2ts strains arrested within a single cell cycle and exhibited a dual arrest phenotype consisting of 50% unbudded and 50% large-budded cells . The unbudded half of the arrested population contained a single nucleus and a single focus of microtubule staining, consistent with arrest in G1 . Most of the large-budded fraction contained segregated chromatin and an extended spindle, indicative of arrest in anaphase, though a fraction contained an undivided nucleus with a short thick intranuclear spindle, indicative of arrest in G2 and/or metaphase . Flow cytometry of pheromone-synchronized cells confirmed that CKII is required in G1, at a point which must lie at or beyond Start but prior to DNA synthesis . Similar analysis of hydroxyurea-synchronized cells indicated that CKII is not required for completion of previously initiated DNA replication but confirmed that the enzyme is again required for cell cycle progression in G2 and/or mitosis . These results establish a role for CKII in regulation and/or execution of the eukaryotic cell cycle. Biochem Biophys Res Commun, 1995 Oct 24, 215(3), 961 - 7 High level production and localization of bovine rod cGMP-gated cation channel subunit 1 in baculovirus-infected insect cells and Saccharomyces cerevisiae; Marheineke K et al.; The cGMP-gated cation channel is responsible for the last step in the vertebrate phototransduction cascade which couples light activation of rhodopsin to a change in membrane permeability . Two different expression systems, baculovirus-infected Spodoptera frugiperda (Sf9) insect cells and Saccharomyces cerevisiae, were used for the overproduction of the cGMP-gated cation channel subunit 1 of bovine rod cells . Presence of recombinant channel protein was monitored by SDS-PAGE and Western-blot analysis . Through immunogold labeling, the heterologously expressed cation channel was found to be localized predominantly in the inner compartments of the infected insect cells and in the vacuole of recombinant yeast cells. Proc Natl Acad Sci U S A, 1995 Oct 24, 92(22), 10418 - 21 Mutations in the MSH3 gene preferentially lead to deletions within tracts of simple repetitive DNA in Saccharomyces cerevisiae; Strand M et al.; Eukaryotic genomes contain tracts of DNA in which a single base or a small number of bases are repeated (microsatellites) . Mutations in the yeast DNA mismatch repair genes MSH2, PMS1, and MLH1 increase the frequency of mutations for normal DNA sequences and destabilize microsatellites . Mutations of human homologs of MSH2, PMS1, and MLH1 also cause microsatellite instability and result in certain types of cancer . We find that a mutation in the yeast gene MSH3 that does not substantially affect the rate of spontaneous mutations at several loci increases microsatellite instability about 40-fold, preferentially causing deletions . We suggest that MSH3 has different substrate specificities than the other mismatch repair proteins and that the human MSH3 homolog (MRP1) may be mutated in some tumors with microsatellite instability. J Biol Chem, 1995 Oct 20, 270(42), 24982 - 8 Differential biochemical regulation of the URA7- and URA8-encoded CTP synthetases from Saccharomyces cerevisiae; Nadkarni AK et al.; The URA7- and URA8-encoded CTP synthetases (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming) are functionally overlapping enzymes responsible for the biosynthesis of CTP in the yeast Saccharomyces cerevisiae . URA8-encoded CTP synthetase was purified to apparent homogeneity by ammonium sulfate fractionation of the cytosolic fraction followed by chromatography with Q-Sepharose, Affi-Gel Blue, Mono Q, and Superose 6 . The subunit molecular mass (67 kDa) of purified URA8-encoded CTP synthetase was in good agreement with the predicted size of the URA8 gene product . Antibodies raised against a fusion protein constructed from the coding sequences of the URA8 gene and expressed in Escherichia coli reacted with purified URA8-encoded CTP synthetase . Native URA8-encoded CTP synthetase existed as a dimer which oligomerized to a tetramer in the presence of its substrates UTP and ATP . Maximum URA8-encoded CTP synthetase activity was dependent on Mg2+ ions (Ka = 2.4 mM) and 2-mercaptoethanol at the pH optimum of 7.5 . The enzyme followed saturation kinetics toward UTP (Km = 74 microM), ATP (Km = 22 microM), and glutamine (Km = 0.14 mM) . GTP stimulated (Ka = 26 microM) URA8-encoded CTP synthetase activity 12-fold . CTP potently inhibited (IC50 = 85 microM) URA8-encoded CTP synthetase activity and, in addition, caused the dependence of activity toward UTP to become cooperative . The URA8-encoded CTP synthetase and the previously purified URA7-encoded CTP synthetase differed significantly with respect to several biochemical properties including turnover number, pH optimum, substrate dependences, and sensitivity to inhibition by CTP . The URA7-encoded CTP synthetase mRNA was 2-fold more abundant when compared with URA8-encoded CTP synthetase mRNA . Both CTP synthetase isoforms were maximally expressed in the exponential phase of growth. J Biol Chem, 1995 Oct 20, 270(42), 24831 - 8 Myristoylation of calcineurin B is not required for function or interaction with immunophilin-immunosuppressant complexes in the yeast Saccharomyces cerevisiae; Zhu D et al.; Calcineurin is a heterodimeric Ca2+/calmodulin-dependent protein phosphatase that regulates signal transduction and is the target of immunophilin-immunosuppressive drug complexes in T-lymphocytes and in yeast . Calcineurin is composed of a catalytic A subunit and a regulatory B subunit that is myristoylated at its amino terminus . We employed genetic and biochemical approaches to investigate the functional roles of myristoylation of calcineurin B (CNB1) in Saccharomyces cerevisiae . A calcineurin B mutant in which glycine 2 was substituted by alanine (CNB1-G2A) did not incorporate {3H}myristate when expressed in yeast . Both wild-type calcineurin B and the CNB1-G2A mutant protein are partially associated with membranes and cytoskeletal structures; hence, myristoylation is not required for these associations . In several independent genetic assays of calcineurin functions (recovery from alpha-factor arrest, survival during cation stress, and viability of a calcineurin-dependent strain), the nonmyristoylated CNB1-G2A mutant protein exhibited full biological activity . In vitro, both wild-type and CNB1-G2A mutant proteins formed complexes with both cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 that contained calcineurin A . Interestingly, expression of the nonmyristoylated CNB1-G2A mutant protein rendered yeast cells partially resistant to the immunosuppressant CsA, but not to FK506 . This study demonstrates that calcineurin B myristoylation is not required for function, but may participate in inhibition by the cyclophilin A-CsA complex. Oncogene, 1995 Oct 19, 11(8), 1623 - 30 Cyclic AMP signaling is required for function of the N-terminal and CR1 domains of adenovirus E1A in Saccharomyces cerevisiae; Miller ME et al.; We have constructed yeast vectors in which derivatives of the adenovirus E1A gene are expressed from the GAL1 promoter . Cells expressing E1A289 grow poorly and accumulate cells with a 1C DNA content . Using a series of E1A deletion mutants, we have identified three regions within the E1A protein that are necessary for the G1 growth phenotype; each deletion partially relieves the growth defect . These deletions span residues 4-25, 38-60 and 140-186, which fall within the N-terminal, CR1 and CR3 domains of E1A respectively . Expression of the first 82 residues of E1A, spanning just the N-terminal and CR1 domains, strongly inhibits yeast cell growth in G1 showing that these domains can function independently of other domains of E1A . Using this strong growth inhibition, we isolated a yeast mutant in the net1 gene that conferred resistance to the expression of E1A1-82 . The mutant was insensitive to expression of both E1A1-82 and full length E1A, but remained sensitive to the toxicity caused by over-expression of a Gal4p-VP16 fusion . Finally, we found that the function of E1A in yeast depends on the cyclic AMP signaling pathway, providing a striking parallel with the action of E1A at the c-fos promoter in mammalian cells . These results suggest that a genetic analysis of the yeast model system will provide relevant new insights into mechanisms of gene regulation by E1A proteins. Biochim Biophys Acta, 1995 Oct 17, 1264(1), 79 - 86 Expression of genes encoding peroxisomal proteins in Saccharomyces cerevisiae is regulated by different circuits of transcriptional control; Kos W et al.; In Saccharomyces cerevisiae induction of the FOX3 gene, encoding peroxisomal 3-oxoacyl-CoA thiolase, by growth on oleate as sole carbon source, is exerted via the cis-acting DNA element designated oleate response element (ORE) (Einerhand et al . (1991) Eur . J . Biochem . 200, 113-122) . The transcription factor(s) binding to this upstream activation site (UAS) are still unknown, however . Induction of another peroxisomal enzyme, citrate synthase (CIT2) is dependent on the products of two genes called RTG1 and RTG2 (Liao and Butow (1993) Cell 72, 61-71) . In the present study we have investigated whether RTG1 controls other genes coding for peroxisomal proteins, and whether such control takes place via the ORE . A number of genes coding for a variety of peroxisomal proteins such as: thiolase and catalase (peroxisomal matrix proteins), PAS3p (a peroxisomal membrane protein) and PAS10p (a protein involved in the import of peroxisomal proteins) were studied in their response to RTG1 . Although the RTG1 and 2 products proved to be required for the increase in number and volume of peroxisomes upon induction by oleate, the single promoter output of the chosen set of genes remained practically unchanged in a rtg1 mutant strain . In addition gel retardation experiments indicated that RTG1 does not bind to the ORE . The behavior of genes coding for the various proteins also varied during repression, derepression and induction, indicating that probably a number of proteins are involved in tuning the output of each gene to cellular demand. FEMS Microbiol Lett, 1995 Oct 15, 132(3), 253 - 8 Ultrastructural effects of pressure stress to the nucleus in Saccharomyces cerevisiae: a study by immunoelectron microscopy using frozen thin sections; Kobori H et al.; The effects of hydrostatic pressure on subcellular structures, particularly the nucleus, of Saccharomyces cerevisiae were investigated by immunoelectron microscopy . Cells were treated with hydrostatic pressure from 0.1 to 400 MPa for 10 min at room temperature . Frozen thin sections of the cells revealed that spindle pole bodies disappeared at 100 MPa . At 150 MPa, the deposition of gold particles for anti alpha-tubulin was noticed in the nucleus, although the filamentous structure of microtubules was lost . At 200 MPa, fewer gold particles were scattered in the nucleus and the nuclear membrane in several portions was also observed to be open at 300 MPa . These results show that elements of the nuclear division apparatus were susceptible to pressure stress, particularly spindle pole bodies and microtubules . The damage to spindle pole bodies, microtubules, and nuclear membrane caused by pressure stress was followed by the inhibition of nuclear division . After the release of pressure, the spindle pole bodies and microtubules of pressurized cells at below 200 MPa regained their normal appearance at 24 h. Eur J Biochem, 1995 Oct 15, 233(2), 584 - 92 Differential functional activities of rainbow trout and human estrogen receptors expressed in the yeast Saccharomyces cerevisiae; Petit F et al.; The cDNA of rainbow trout estrogen receptor (rtER), highly and stably expressed in yeast, Saccharomyces cerevisiae, was used to analyse the biological activity of the receptor . The rtER mRNA encoded a 65-kDa protein which was immunorevealed by a specific antibody and migrated with the authentic rtER major protein form detected in trout liver . Yeast rtER bound estradiol with high affinity and the dissociation constant (Kd = 1.35 nM) was very similar to the value measured from trout liver extracts but 3-5-fold higher than the Kd found for human estrogen receptor (hER) . This indicates therefore that the rtER has a lower estradiol affinity compared to the human receptor . While the hER Kd remained unchanged at both 4 degrees C or 22 degrees C, it was slightly modified at 30 degrees C . The Kd measured for rtER at 22 degrees C and 30 degrees C were about 2-fold, and 12-fold higher, respectively, than the Kd obtained at 4 degrees C suggesting an alteration of the rtER affinity for its ligand at elevated temperature . To examine the estrogen-receptor-mediated activation of transcription in yeast, reporter plasmids integrated or not in the yeast genome were used . The reporter genes consist of one, two, or three copies of estrogen-responsive elements (ERE) upstream of the yeast proximal CYC1 or URA3 promoters fused to the lacZ gene of Escherichia coli coding for beta-galactosidase . The induction of beta-galactosidase activity for all reporter genes was strictly dependent on the presence of rtER and estrogens . The activation of transcription mediated by rtER responded in an estradiol-dose-dependent manner as in animal cells . However, compared to hER, the estradiol concentration necessary to achieve maximal activation was 10-fold higher . This is probably a consequence of the lower estradiol-affinity for rtER compared to hER . The levels of induction of the reporter genes containing two or three ERE were strongly enhanced compared to the one ERE construct . This is in agreement with the synergistic effect previously described for multiple ERE . The magnitudes of transcriptional induction mediated by rtER and hER were similar when the reporter gene containing three ERE was used but changed when the one ERE construct was used . In this case transcriptional activation indicated by rtER was 10-20 fold lower . This suggests that rtER requires protein/protein interaction for its stabilization on DNA . Antiestrogens were able to bind rtER and promote gene transcription . However, to produce effects comparable to those obtained with estrogens, much higher concentrations were required . This may imply nonetheless that antihormones were capable of provoking efficient interactions of rtER with the transcriptional machinery. Eur J Biochem, 1995 Oct 15, 233(2), 520 - 30 Genetic and biochemical characterization of the UGP1 gene encoding the UDP-glucose pyrophosphorylase from Saccharomyces cerevisiae; Daran JM et al.; We report here that the open reading frame YKL248, previously identified during the systematic sequencing of yeast chromosome XI {Purnelle B., Skala, J., Van Dijck, L . & Goffeau, A . (1992) Yeast 8, 977-986} encodes UDP-glucose pyrophosphorylase (UGPase), the enzyme which catalyses the reversible formation of UDP-Glc from glucose 1-phosphate and UTP . Proof for this function come from sequence alignment of the YKL248 product with UGPase of other species, from complementation studies of an Escherichia coli galU mutant deficient in UGPase activity, and from overexpression studies . In particular, the amino acid sequence motifs involved in the binding of glucose 1-phosphate and UDP-Glc are entirely conserved between the yeast, bovine, human and potato tuber UGPases, and multi-copy expression of YKL248 resulted in a 40-fold increase in UGPase activity . This gene was, therefore, renamed UGP1 . Gene disruption at the UGP1 locus in a diploid strain, followed by tetrad analysis, showed that UGPase is essential for cell viability . Functional analysis of UGP1 was, therefore, carried out by generating strains in which UGPase could be either overexpressed or depleted . This was done by generating haploid strains carrying either UGP1 on a multicopy vector or the chromosomal deletion of UGP1, and rescued by a vector bearing the wild-type gene under the control of the glucose-repressible galactose-inducible promoter . The effects of overproducing UGPase on the cell metabolism and morphology were carbon-source dependent . On glucose medium, the 40-fold increase of UGPase activity was restricted to a twofold increase in the concentration of glycogen and UDP-Glc, with no significant effect on growth . In contrast, on galactose, the 40-fold increase in UGPase activity was accompanied by several effects, including a threefold reduction of the growth rate, a 3-5-fold increase in the concentrations of UDP-Glc, UDP-Gal and galactose 1-phosphate, a higher sensitivity to calcofluor white and an increase in the degree of protein glycosylation . Depletion of UGPase activity was performed by transferring the mutant strains from galactose to glucose medium . Unexpectedly, growth of these mutants on glucose was as efficient as that of the control, although the mutants contained only 5-10% wild-type UGPase activity, and a growth defect could never been obtained, even after serial transfers of the mutants to a 10% glucose medium . However, the 10-fold reduction of UGPase activity induced a multi-budding pattern, a higher resistance to zymolyase, a slight increase in the calcofluor sensitivity and a decrease in the cell-wall beta-glucan content . All these alterations, induced by manipulating the UGP1 gene, are discussed in the context of the strategic position of UDP-Glc in yeast metabolism. J Biol Chem, 1995 Oct 13, 270(41), 24252 - 7 Gene RPA43 in Saccharomyces cerevisiae encodes an essential subunit of RNA polymerase I; Thuriaux P et al.; Yeast RNA polymerase I contains 14 distinct polypeptides, including A43, a component of about 43 kDa . The corresponding gene, RPA43, encodes a 326-amino acid polypeptide matching the peptidic sequence of two tryptic fragments isolated from A43 . Gene inactivation leads to a lethal phenotype that is rescued by a plasmid containing the 35S ribosomal RNA gene fused to the GAL7 promoter, which allows the synthesis of 35S rRNA by RNA polymerase II in the presence of galactose . A screening for mutants rescued by the presence of GAL7-35SrDNA identified a nonsense rpa43 allele truncating the protein at amino acid position 217 . {3H}Uridine pulse labeling showed that this mutation abolishes 35S rRNA synthesis without significant effects on the synthesis of 5 S RNA and tRNAs . These properties establish that A43 is an essential component of RNA polymerase I . This highly hydrophilic phosphoprotein has a strongly acidic carboxyl-terminal domain, and shows no homology to entries in current sequence data banks, including all the genetically identified components of the other two yeast RNA polymerases . RPA43 mapped next to RPA190, encoding the largest subunit of polymerase I . These genes are divergently transcribed and may thus share upstream regulatory elements ensuring their co-regulation. Biochem Biophys Res Commun, 1995 Oct 13, 215(2), 578 - 90 A single base-pair change (ATG-->ATC) nullifies the activity of cytosolic fumarase in Saccharomyces cerevisiae; Wu M et al.; A respiratory defective pet mutant (W303 delta FUM1) of Saccharomyces cerevisiae deficient in fumarase was transformed with the plasmid construct pG5/ST7 . This plasmid contains the entire FUM1 gene with only a single base pair change (ATG-->ATC) confined to the putative second inframe translation initiation codon . While transformation of the fumarase deficient mutant with pG5/ST7 resulted in an elevation of fumarase activity in the mitochondria of the transformed strain, fumarase activity in the cytosol remained negligible his result indicated that the cytosolic fumarase isoenzyme is synthesized exclusively from the second translation initiation codon of FUM1 . Results of RACE-PCR of the 5' ends of FUM1 transcripts confirmed that the FUM1 gene synthesizes two distinct species of transcripts . These data provide strong evidence for the synthesis and targeting of two fumarase isomers. Proc Natl Acad Sci U S A, 1995 Oct 10, 92(21), 9786 - 90 Cloning, expression, and function of TFC5, the gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIB; Kassavetis GA et al.; TFC5, the unique and essential gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been cloned . It encodes a 594-amino acid protein (67,688 Da) . Escherichia coli-produced B" has been used to reconstitute entirely recombinant TFIIIB that is fully functional for TFIIIC-directed, as well as TATA box-dependent, DNA binding and transcription . The DNase I footprints of entirely recombinant TFIIIB, composed of B", the 67-kDa Brf, and TATA box-binding protein, and TFIIIB reconstituted with natural B" are indistinguishable . A truncated form of B" lacking 39 N-terminal and 107 C-terminal amino acids is also functional for transcription. Proc Natl Acad Sci U S A, 1995 Oct 10, 92(21), 9450 - 4 Role of the GATA factors Gln3p and Nil1p of Saccharomyces cerevisiae in the expression of nitrogen-regulated genes; Stanbrough M et al.; We have isolated the NIL1 gene, whose product is an activator of the transcription of nitrogen-regulated genes, by virtue of the homology of its zinc-finger domain to that of the previously identified activator, the product of GLN3 . Disruption of the chromosomal NIL1 gene enabled us to compare the effects of Gln3p and of Nil1p on the expression of the nitrogen-regulated genes GLN1, GDH2, and GAP1, coding respectively for glutamine synthetase, NAD-linked glutamate dehydrogenase, and general amino acid permease . Our results show that the nature of GATAAG sequence that serve as the upstream activation sequence elements for these genes determines their abilities to respond to Gln3p and Nil1p . The results further indicate that Gln3p is inactivated by an increase in the intracellular concentration of glutamine and that Nil1p is inactivated by an increase in intracellular glutamate. FEBS Lett, 1995 Oct 2, 373(1), 66 - 70 Mutations in RCA1 and AFG3 inhibit F1-ATPase assembly in Saccharomyces cerevisiae; Paul MF et al.; The RCA1 (YTA12) and AFG3 (YTA10) genes of Saccharomyces cerevisiae code for homologous mitochondrial proteins that belong to the recently described AAA protein-family {Kunau et al . (1993) Biochimie 75,209-224} . Mutations in either gene have been shown to induce a respiratory defect . In the case of rca1 mutants this phenotype has been ascribed to defective assembly of cytochrome oxidase and ubiquinol-cytochrome c reductase . In the present study we show that the respiratory defect of afg3 mutants, like that of rca1 mutants, is also caused by an arrest in assembly of cytochrome oxidase and ubiquinol-cytochrome c reductase . In addition to the absence of the respiratory complexes, rca1 and afg3 mutants exhibit reduced mitochondrial ATPase activity . As a first step to an understanding of the biochemical basis for the ATPase defect we have examined the assembly of the F1 and F0 constituents of the ATPase complex . We present evidence that the ATPase lesion stems at least in part from the failure of rca1 and afg3 mutants to assemble F1 . Although the mutants also display lower steady-state concentrations of some F0 subunits, this could be a secondary effect of defective F1 assembly. EMBO J, 1995 Oct 2, 14(19), 4803 - 13 Roles and regulation of Cln-Cdc28 kinases at the start of the cell cycle of Saccharomyces cerevisiae; Dirick L et al.; In budding yeast G1 cells increase in cell mass until they reach a critical cell size, at which point (called Start) they enter S phase, bud and duplicate their spindle pole bodies . Activation of the Cdc28 protein kinase by G1-specific cyclins Cln1, Cln2 or Cln3 is necessary for all three Start events . Transcriptional activation of CLN1 and CLN2 by SBF and MBF transcription factors also requires an active Cln-Cdc28 kinase and it has therefore been proposed that the sudden accumulation of CLN1 and CLN2 transcripts during late G1 occurs via a positive feedback loop . We report that whereas Cln1 and Cln2 are required for the punctual execution of most, if not all, other Start-related events, they are not required for the punctual activation of SBF- or MBF-driven transcription . Cln3, on the other hand, is essential . By turning off cyclin B proteolysis and turning on proteolysis of the cyclin B-Cdc28 inhibitor p40SIC1, Cln1 and Cln2 kinases activate cyclin B-Cdc28 kinases and thereby trigger S phase . Thus the accumulation of Cln1 and Cln2 kinases which starts the yeast cell cycle is set in motion by prior activation of SBF- and MBF-mediated transcription by Cln3-Cdc28 kinase . This dissection of regulatory events during late G1 demands a rethinking of Start as a single process that causes cells to be committed to the mitotic cell cycle. Genetics, 1995 Oct, 141(2), 465 - 79 Isolation of mutants of Saccharomyces cerevisiae requiring DNA topoisomerase I; Sadoff BU et al.; Despite evidence that DNA topoisomerase I is required to relieve torsional stress during DNA replication and transcription, yeast strains with a top1 null mutation are viable and display no gross defects in DNA or RNA synthesis, possibly because other proteins provide overlapping functions . We isolated mutants whose inviablility or growth defect is relieved when TOP1 is expressed {trf mutants (topoisomerase one-requiring function)} . The TRF genes define at least four complementation groups . TRF3 is allelic to TOP2 . TRF1 is allelic to HPR1, previously shown to be homologous to TOP1 over two short regions . TRF4 encodes a novel 584-amino acid protein with homology to the N-terminus of Saccharomyces cerevisiae topo I . Like top1 mutants, trf4 mutants have elevated rDNA recombination and fail to shut off RNA polymerase II transcription in stationary phase . trf4 null mutants are cs for viability, display reduced expression of GAL1 and Cell Cycle Box UAS::LacZ fusions, and are inviable in combination with trfI null mutants, indicating that both proteins may share a common function with DNA topoisomerase I . The existence of multiple TRF complementation groups suggests that not all biological functions of topo I can be carried out by topo II. Curr Genet, 1995 Oct, 28(5), 467 - 73 Cloning and expression of an Aspergillus kawachii endo-1,4-beta-xylanase gene in Saccharomyces cerevisiae; Crous JM et al.; First-strand cDNA was prepared from mRNA isolated from Aspergillus kawachii IFO4308 and the beta-xylanase gene (xynC) amplified by using the polymerase chain reaction (PCR) technique . This gene was inserted between the yeast phosphoglycerate kinase (PGK1) gene promoter (PGK1p) and terminator (PGK1T) sequences . The PGK1p-xynC-PGK1T construct (designated XYN3) was cloned into a multicopy episomal plasmid and the XYN3 gene was expressed in Saccharomyces cerevisiae . Functional beta-xylanase (Xyn3) was produced and secreted by the recombinant yeast . Xyn3 was stable between 30 and 50 degrees C, and the optimum temperature and pH were shown to be at 60 degrees C and lower than pH 3, respectively . An autoselective furl::LEU2 XYN3 recombinant strain was developed that allowed beta-xylanase production at a level of 300 nkat/ml in a non-selective complex medium. Curr Genet, 1995 Oct, 28(5), 441 - 6 A two-reporter gene system for the analysis of bi-directional transcription from the divergent MAL6T-MAL6S promoter in Saccharomyces cerevisiae; Bell PJ et al.; Many sets of genes in Saccharomyces cerevisiae are divergently transcribed, but at present there are no vectors generally available for the simultaneous analysis of divergent transcription from these promoters . In the present study MEL1 and lacZ were used to construct a vector capable of measuring the divergent expression initiated by the MAL6T-MAL6S bi-directional promoter . Our observations demonstrate that the expression of both reporter genes was regulated in a similar fashion to the native MAL6T and MAL6S genes, and that induction was dependent upon the presence of a functional MALR activator gene . The results confirmed that the MAL6T-MAL6S promoter was co-ordinately regulated, repressed by glucose, induced by maltose, and that basal expression was more active in the MAL6S direction than in the MAL6T direction. Mol Biol Cell, 1995 Oct, 6(10), 1381 - 96 Organelle-cytoskeletal interactions: actin mutations inhibit meiosis-dependent mitochondrial rearrangement in the budding yeast Saccharomyces cerevisiae; Smith MG et al.; During early stages of meiosis I, yeast mitochondria fuse to form a single continuous thread . Thereafter, portions of the mitochondrial thread are equally distributed to daughter cells . Using time-lapse fluorescence microscopy and a membrane potential sensing dye, mitochondria are resolved as small particles at the cell periphery in pre-meiotic, living yeast . These organelles display low levels of movement . During meiosis I, we observed a threefold increase in mitochondrial motility . Mitochondrial movements were linear, occurred at a maximum velocity of 25 +/- 6.7 nm/s, and resulted in organelle collision and fusion to form elongated tubular structures . Mitochondria do not co-localize with microtubules . Destabilization of microtubules by nocodazole treatment has no significant effect on the rate and extent of thread formation . In contrast, yeast bearing temperature-sensitive mutations in the actin-encoding ACT1 gene (act1-3 and act1-133) exhibit abnormal mitochondrial aggregation, fragmentation, and enlargement as well as loss of mitochondrial motility . In act1-3 cells, mitochondrial defects and actin delocalization occur only at restrictive temperatures . The act1-133 mutation, which perturbs the myosin-binding site of actin without significantly affecting actin cytoskeletal structure in meiotic yeast, results in mitochondrial morphology and motility defects at restrictive and permissive temperatures . These studies support a role for the actin cytoskeleton in the control of mitochondrial position and movements in meiotic yeast. Mol Biol Cell, 1995 Oct, 6(10), 1263 - 70 The actin-related protein Act3p of Saccharomyces cerevisiae is located in the nucleus; Weber V et al.; Actin-related proteins, a group of protein families that exhibit about 50% sequence identity among each other and to conventional actin, have been found in a variety of eukaryotic organisms . In the budding yeast Saccharomyces cerevisiae, genes for one conventional actin (ACT1) and for three actin-related proteins (ACT2, ACT3, and ACT5) are known . ACT3, which we recently discovered, is an essential gene coding for a polypeptide of 489 amino acids (Act3p), with a calculated molecular mass of 54.8 kDa . Besides its homology to conventional actin, Act3p possesses a domain exhibiting weak similarity to the chromosomal protein HMG-14 as well as a potential nuclear localization signal . An antiserum prepared against a specific segment of the ACT3 gene product recognizes a polypeptide band of approximately 55 kDa in yeast extract . Indirect immunofluorescence experiments with this antiserum revealed that Act3p is located in the nucleus . Nuclear staining was observed in all cells regardless of the stage of the cell cycle . Independently, immunoblotting experiments with subcellular fractions showed that Act3p is indeed highly enriched in the nuclear fraction . We suggest that Act3p is an essential constituent of yeast chromatin. Antonie Van Leeuwenhoek, 1995 Oct, 68(3), 231 - 5 High molecular weight precursors of glucans in Saccharomyces cerevisiae; Ruiz-Herrera J et al.; Nascent beta-1,3 glucan synthesized by mixed membrane fractions from Saccharomyces cerevisiae was solubilized by extraction with hot SDS or urea . Nature of the material was analyzed by electrophoresis and gel filtration . As determined by gel filtration, Mr of synthesized glucans exceeded 1,500 kDa, but was below 20,000 kDa . This nascent material served as an acceptor for further glucose transfer reactions, giving rise to glucan molecules over 20,000 kDa . It is suggested that the high Mr precursor components represent protein-bound glucan molecules in transit to the cell surface. Plant Cell Physiol, 1995 Oct, 36(7), 1179 - 88 Characterization of DNA-binding proteins involved in the assembly of mitochondrial nucleoids in the yeast Saccharomyces cerevisiae; Miyakawa I et al.; Mitochondrial nucleoids (mt-nucleoids) isolated from the yeast Saccharomyces cerevisiae were analyzed to identify the protein components that are involved in the compact packaging of mtDNA . The isolated mt-nucleoids were disassembled by the addition of 2 M NaCl and the disassembled mt-nucleoids were reassembled once again into compact structures by dialysis against a buffer that contained NaCl at concentrations below 0.1 M, as monitored by staining of the DNA with 4',6-diamidino-2-phenylindole . DNA-binding proteins with molecular masses of 67 kDa, 52 kDa, 50 kDa, 38 kDa, 30 kDa and 20 kDa were separated from isolated mt-nucleoids by column chromatography on DNA-cellulose after digestion of mt-nucleoids by DNase I in the presence or absence of 2 M NaCl . Purified mtDNA was compactly packaged into nucleoid-like structures upon the addition of fractions that contained DNA-binding proteins and subsequent dialysis to reduce the concentration of NaCl . Five proteins, with molecular masses of 67 kDa, 52 kDa, 50 kDa, 38 kDa and 30 kDa, respectively, had lower affinity for double-stranded DNA than that of the 20-kDa protein . The fraction that contained the five DNA-binding proteins other than the 20-kDa protein was also able to fold mtDNA compactly into nucleoid-like structures . By contrast, the combination of the 20-kDa protein and mtDNA resulted in formation of less tightly packed, string-of-bead structures . These results suggest that at least six different DNA-binding proteins are involved in the organization of the mt-nucleoids. Yeast, 1995 Oct, 11(13), 1275 - 80 Precise gene disruption in Saccharomyces cerevisiae by double fusion polymerase chain reaction; Amberg DC et al.; We adapted a fusion polymerase chain reaction (PCR) strategy to synthesize gene disruption alleles of any sequenced yeast gene of interest . The first step of the construction is to amplify sequences flanking the reading frame we want to disrupt and to amplify the selectable marker sequence . Then we fuse the upstream fragment to the marker sequence by fusion PCR, isolate this product and fuse it to the downstream sequence in a second fusion PCR reaction . The final PCR product can then be transformed directly into yeast . This method is rapid, relatively inexpensive, offers the freedom to choose from among a variety of selectable markers and allows one to construct precise disruptions of any sequenced open reading frame in Saccharomyces cerevisiae. Yeast, 1995 Oct, 11(13), 1265 - 74 Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae; Schneider BL et al.; Epitope tagging is the insertion of a short stretch of amino acids constituting an epitope into another protein . Tagged proteins can be identified by Western, immunoprecipitation and immunofluorescence assays using pre-existing antibodies . We have designed vectors containing the URA3 gene flanked by direct repeats of epitope tags . We use the polymerase chain reaction (PCR) to amplify the tag-URA3-tag cassette such that the ends of the PCR fragments possess homology to the gene of interest . In vivo recombination is then used to direct integration of the fragment to the location of interest, and transformants are selected by their Ura+ phenotype . Finally, selection for Ura- cells on 5-fluoro-orotic acid plates yields cells where recombination between the repeated epitopes has 'popped out' the URA3 gene, leaving a single copy of the epitope at the desired location . PCR epitope tagging (PET) provides a rapid and direct technique for tagging that does not require any cloning steps . We have used PET to tag three Saccharomyces cerevisiae proteins, Cln1, Sic1 and Est1. Yeast, 1995 Oct, 11(13), 1233 - 40 DOGR1 and DOGR2: two genes from Saccharomyces cerevisiae that confer 2-deoxyglucose resistance when overexpressed; Randez-Gil F et al.; Saccharomyces cerevisiae contains two genes (DOGR1 and DOGR2) that are able to confer 2-deoxyglucose resistance when they are overexpressed . These genes are very similar, sharing 92% identity at the protein level . They code for two isoenzymes with 2-deoxyglucose-6 phosphate (2-DOG-6P) phosphatase activity . These enzymes have been purified and characterized . DogR1p shows an optimum pH of 6, an optimum temperature of 30 degrees C and a KM on 2-DOG-6P of 17 mM . DogR2p shows a similar optimum pH, but the optimum temperature is 40 degrees C and it exhibits a KM on 2-DOG-6P of 41 mM . Both enzymes require 10 mM-MgCl2 for maximal activity and they are inhibited by inorganic phosphate. Protein Sci, 1995 Oct, 4(10), 2168 - 78 Homology modeling of an immunoglobulin-like domain in the Saccharomyces cerevisiae adhesion protein alpha-agglutinin; Lipke PN et al.; The Saccharomyces cerevisiae adhesion protein alpha-agglutinin is expressed by cells of alpha mating type . On the basis of sequence similarities, alpha-agglutinin has been proposed to contain variable-type immunoglobulin-like (IgV) domains . The low level of sequence similarity to IgV domains of known structure made homology modeling using standard sequence-based alignment algorithms impossible . We have therefore developed a secondary structure-based method that allowed homology modeling of alpha-aggulutinin domain III, the domain most similar to IgV domains . The model was assessed and where necessary refined to accommodate information obtained by biochemical and molecular genetic approaches, including the positions of a disulfide bond, glycosylation sites, and proteolytic sites . The model successfully predicted surface exposure of glycosylation and proteolytic sites, as well as identifying residues essential for binding activity . One side of the domain was predicted to be covered by carbohydrate residues . Surface accessibility and volume packing analyses showed that the regions of the model that have greatest sequence dissimilarity from the IgV consensus sequence are poorly structured in the biophysical sense . Nonetheless, the utility of the model suggests that these alignment and testing techniques should be of general use for building and testing of models of proteins that share limited sequence similarity with known structures. Biosci Biotechnol Biochem, 1995 Oct, 59(10), 1869 - 74 Cloning and nucleotide sequence of the ribonuclease T1 gene (rntA) from Aspergillus oryzae and its expression in Saccharomyces cerevisiae and Aspergillus oryzae; Fujii T et al.; A genomic DNA encoding ribonuclease (RNase) T1 from Aspergillus oryzae was cloned using a synthetic oligonucleotide probe . The cloned gene (designated rntA) encoded functional RNase T1, since an A . oryzae transformant with multiple copies of the rntA gene showed higher RNase T1 activity (over 200 times) than a transformant with a vector . A cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) with primers corresponding to the 5' terminus and 3' terminus of the reading frame of the rntA gene . Nucleotide sequencing analysis of both DNAs found that RNase T1 had a prepro-sequence consisting of 26 amino acids and the rntA gene had only one intron (114 bp) in the region encoding the signal sequence . The A . oryzae transformant with cDNA controlled by the amyB promoter also showed higher activity (over 300 times), indicating that the cloned cDNA encoded functional RNase T1 . On the other hand, the Saccharomyces cerevisiae transformant with cDNA controlled by the GAL1 promoter could not grow on a medium containing galactose . These results suggests that A . oryzae may have a protection mechanism from RNase T1. Eur J Biochem, 1995 Oct 1, 233(1), 200 - 8 Regulation of mitochondrial biogenesis in Saccharomyces cerevisiae . Intricate interplay between general and specific transcription factors in the promoter of the QCR8 gene; De Winde JH et al.; Transcription of the QCR8 gene, encoding subunit VIII of the Saccharomyces cerevisiae mitochondrial ubiquinol-cytochrome c oxidoreductase (QCR), is controlled by the carbon-source-dependent heme-activator protein complex HAP2/3/4 and the general transcriptional regulators autonomous replication-site-binding factor ABF1 and centromere-binding and promoter-binding factor CPF1 . In this study, we investigate and dissect the relative contributions and mutual interactions of these regulators in transcriptional control . Transcription was analyzed both under steady-state conditions and during nutritional shifts, in hap delta mutants and after site-specific mutagenesis of the various binding sites in the chromosomal context of the QCR8 gene . We present evidence for both direct and indirect interactions between ABF1 and HAP2/3/4, and show that HAP2/3/4 is essential for a rapid transcriptional induction during transition from repressed to derepressed conditions . However, the activator is not the only determinant for carbon-source-dependent regulation, and we observe a functional difference between HAP2/3/4 and the HAP2/3 subcomplex . ABF1 is required for maintainance of basal repressed and derepressed transcription in the steady state of growth . The repressive action of the negative modulator CPF1 during escape from glucose repression is overcome through the cooperative action of ABF1 and HAP2/3/4 . The implications of the intricate interactions of these DNA-binding regulators for control of expression of mitochondrial protein genes are discussed. Microbiology, 1995 Oct, 141 ( Pt 10), 2463 - 9 Analysis of lysine-dependent yeast sporulation: a decrease in cyclic AMP is not required for initiation of meiosis and sporulation in Saccharomyces cerevisiae; Suizu T et al.; Cells of the yeast Saccharomyces cerevisiae sporulated in nutrient-rich medium containing L-lysine . Sporulation was specific to the presence of L-lysine and was initiated when the cellular content of this basic amino acid reached approximately 0.2-0.5 mmol (g cells)-1, at early stationary phase . The formation of asci was most efficient at pH 7.0 and 50-100 mM L-lysine; in these optimum conditions, the sporulation frequency reached about 60% after 5 d incubation . The L-lysine-dependent sporulation system in nutrient-rich conditions was distinct from the currently used potassium-acetate-dependent system in nutrient-deficient conditions . Analysis of the L-lysine-dependent system indicated that, prior to entrance into meiosis and/or sporulation processes, the yeast cells change in shape, their pool sizes for L-cysteine and glutathione alter, and they synthesize a protein with a molecular mass of 15 kDa . A low level of cAMP was not required for the entrance into meiosis and/or sporulation. Appl Microbiol Biotechnol, 1995 Oct, 43(5), 877 - 9 CEN14 sequences cause slower proliferation, reduced cell size and asporogeny in Saccharomyces cerevisiae; Dickinson JR et al.; The introduction of CEN14-based plasmids into haploid strains of Saccharomyces cerevisiae resulted in reduced proliferation rates and significantly smaller (20%) cell size than in untransformed control cells . This could be useful to those yeast biotechnology processes that require high levels of gene expression but little or no yeast growth and proliferation . In diploids similar plasmids caused asporogeny, which was possibly a consequence of the reduced cell size. Mol Cell Biol, 1995 Oct, 15(10), 5607 - 17 Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes; Tran HT et al.; Small direct repeats, which are frequent in all genomes, are a potential source of genome instability . To study the occurrence and genetic control of repeat-associated deletions, we developed a system in the yeast Saccharomyces cerevisiae that was based on small direct repeats separated by either random sequences or inverted repeats . Deletions were examined in the LYS2 gene, using a set of 31- to 156-bp inserts that included inserts with no apparent potential for secondary structure as well as two quasipalindromes . All inserts were flanked by 6- to 9-bp direct repeats of LYS2 sequence, providing an opportunity for Lys+ reversion via precise excision . Reversions could arise by extended deletions involving either direct repeats or random sequences and by -1-or +2-bp frameshift mutations . The deletion breakpoints were always associated with short (3- to 9-bp) perfect or imperfect direct repeats . Compared with the POL+ strain, deletions between small direct repeats were increased as much as 100-fold, and the spectrum was changed in a temperature-sensitive DNA polymerase delta pol3-t mutant, suggesting a role for replication . The type of deletion depended on orientation relative to the origin of replication . On the basis of these results, we propose (i) that extended deletions between small repeats arise by replication slippage and (ii) that the deletions occur primarily in either the leading or lagging strand . The RAD50 and RAD52 genes, which are required for the recombinational repair of many kinds of DNA double-strand breaks, appeared to be required also for the production of up to 90% of the deletions arising between separated repeats in the pol3-t mutant, suggesting a newly identified role for these genes in genome stability and possibly replication. Mol Cell Biol, 1995 Oct, 15(10), 5246 - 57 Pheromone signalling in Saccharomyces cerevisiae requires the small GTP-binding protein Cdc42p and its activator CDC24; Zhao ZS et al.; Pheromone signalling in Saccharomyces cerevisiae is mediated by the STE4-STE18 G-protein beta gamma subunits . A possible target for the subunits is Ste20p, whose structural homolog, the serine/threonine kinase PAK, is activated by GTP-binding p21s Cdc42 and Rac1 . The putative Cdc42p-binding domain of Ste20p, expressed as a fusion protein, binds human and yeast GTP-binding Cdc42p . Cdc42p is required for alpha-factor-induced activation of FUS1.cdc24ts strains defective for Cdc42p GDP/GTP exchange show no pheromone induction at restrictive temperatures but are partially rescued by overexpression of Cdc42p, which is potentiated by Cdc42p12V mutants . Epistatic analysis indicates that CDC24 and CDC42 lie between STE4 and STE20 in the pathway . The two-hybrid system revealed that Ste4p interacts with Cdc24p . We propose that Cdc42p plays a pivotal role both in polarization of the cytoskeleton and in pheromone signalling. Yeast, 1995 Sep 30, 11(12), 1187 - 94 The sequence of an 11.1 kb fragment on the left arm of Saccharomyces cerevisiae chromosome VII reveals six open reading frames including NSP49, KEM1 and four putative new genes; Bertani I et al.; We report the sequence of an 11.1 kb fragment located on the left arm of chromosome VII of Saccharomyces cerevisiae . By sequence analysis we have detected six open reading frames (ORFs) longer that 300 bp, which cover 87% of the entire sequence . ORF G1645 is 100% identical to the KEM1 gene, also identified as DST2, XRN1, SEP1 and RAR5, while G1648 is 100% identical to the NSP49 or NUP49 gene . ORF G1642 shares some identity with a hypothetical protein of Caenorhabditis elegans, while the other four ORFs show no significant homology to known proteins. Yeast, 1995 Sep 30, 11(12), 1157 - 69 A double flow cytometric tag allows tracking of the dynamics of cell cycle progression of newborn Saccharomyces cerevisiae cells during balanced exponential growth; Porro D et al.; Studies on the dynamics of growth of single eukaryotic cells and their relationships with cell cycle regulations are generally carried out following cell synchronization procedures or, on a relatively low number of cells, by time-lapse studies . Establishment of both time-lapse studies and synchronous cell populations usually requires elaborate experimental efforts and is prone to perturb the physiological state of the cell . In this paper we use a new flow cytometric approach which allows, in asynchronous growing Saccharomyces cerevisiae populations, tagging of both the cell age and the cell protein content of a cohort of daughter cells at the different cell cycle set points . Since the cell protein content is a good estimation of the cell size, it is possible to follow the kinetics of the cell size increase during cell cycle progression . The experimental findings obtained indicate an exponential increase of the cell size during growth, that the daughter and the parent subpopulations grow with the same specific growth rate, that the average cell size increase rate of each individual cell is almost identical to the specific growth rate of the overall population and provide the opportunity to estimate the cell cycle length for the daughter cell population as well as the identification of the complex structure of asynchronously growing yeast populations. Nucleic Acids Res, 1995 Sep 25, 23(18), 3750 - 5 Escherichia coli, Saccharomyces cerevisiae, rat and human 3-methyladenine DNA glycosylases repair 1,N6-ethenoadenine when present in DNA; Saparbaev M et al.; The human carcinogen vinyl chloride is metabolized in the liver to reactive intermediates which generate various ethenobases in DNA . It has been reported that 1,N6-ethenoadenine (epsilon A) is excised by a DNA glycosylase present in human cell extracts, whereas protein extracts from Escherichia coli and yeast were devoid of such an activity . We confirm that the human 3-methyladenine-DNA glycosylase (ANPG protein) excises epsilon A residues . This finding was extended to the rat (ADPG protein) . We show, at variance with the previous report, that pure E.coli 3-methyladenine-DNA glycosylase II (AlkA protein) as well as its yeast counterpart, the MAG protein, excise epsilon A from double stranded oligodeoxynucleotides that contain a single epsilon A . Both enzymes act as DNA glycosylases . The full length and the truncated human (ANPG 70 and 40 proteins, respectively) and the rat (ADPG protein) 3-methyladenine-DNA glycosylases activities towards epsilon A are 2-3 orders of magnitude more efficient than the E.coli or yeast enzyme for the removal of epsilon A . The Km of the various proteins were measured . They are 24, 200 and 800 nM for the ANPG, MAG and AlkA proteins respectively . These three proteins efficiently cleave duplex oligonucleotides containing epsilon A positioned opposite T, G, C or epsilon A . However the MAG protein excises A opposite cytosine much faster than opposite thymine, guanine or adenine. Genomics, 1995 Sep 20, 29(2), 546 - 50 Cloning and chromosomal localization of a mouse cDNA with homology to the Saccharomyces cerevisiae gene zuotin; Hughes R et al.; The eukaryotic DnaJ homologs form a family of proteins with diverse functions . One member of the family, the Saccharomyces cerevisiae gene zuotin, was isolated for its ability to bind Z-DNA . Here, we have isolated a mouse cDNA called ZRF1 (for zuotin-related factor1) with significant homology to zuotin . The DnaJ domain and candidate phosphorylation sites of zuotin and ZRF1 are highly conserved . ZRF1 gene is localized on chromosome 5 . The structural similarity of zuotin and ZRF1 suggests conservation of function of this DnaJ subfamily. Anal Biochem, 1995 Sep 20, 230(2), 308 - 14 Purification of functionally sealed cytoplasmic side-out plasma membrane vesicles from Saccharomyces cerevisiae; Menendez A et al.; Highly purified plasma membrane vesicles were prepared from yeast protoplasts by a combination of osmotic lysis, differential centrifugation, and separation in an aqueous dextran/polyethylene glycol two-phase system . The vesicles were predominantly (85-90%) of cytoplasmic side-out orientation and displayed large ATP-dependent proton pumping activity which was inhibited by vanadate (100 microM) but not by bafilomycin or nitrate . The preparation presented a distinct polypeptide profile with respect to the total membrane fraction and was enriched in the 110-kDa polypeptide corresponding to the plasma membrane H(+)-ATPase . This preparation of native plasma membranes vesicles is especially suitable for functional studies in vitro. Biochim Biophys Acta, 1995 Sep 19, 1263(3), 285 - 8 A high dose of the STM1 gene suppresses the temperature sensitivity of the tom1 and htr1 mutants in Saccharomyces cerevisiae; Utsugi T et al.; A new gene (STM1; suppressor of tom1) of Saccharomyces cerevisiae was isolated by the ability to suppress the temperature sensitivity of a tom1 mutant, by increasing its gene dosage . The gene could also suppress the temperature sensitivity of the htr1 disruptant (Kikuchi et al . (1994) Mol . Gen . Genet . 245, 107-116) and was physically mapped in the region near PEP3 on chromosome XII R . The predicted gene product (29,999 Da) is basic and partially homologous to various histone H1 . The level of the gene expression increased 2-fold when exposed to mating pheromone. Biochim Biophys Acta, 1995 Sep 19, 1263(3), 261 - 5 Characterization of a new gene family developing pleiotropic phenotypes upon mutation in Saccharomyces cerevisiae; Revardel E et al.; The aim of this paper is to gather and complete data about four members of a new gene family . Mutation in SUR4 gene was originally selected as a suppressor of defects caused by mutations in RVS161 or RVS167 genes . Cloning and sequencing of the SUR4 gene were performed . The deduced protein contains six putative transmembrane domains . Sequence comparison revealed that two yeast genes, FEN1 and JO343, shared significant similarities with SUR4 . Mutants for SUR4 and FEN1 have the same pleiotropic phenotype, including bud localization defects, resistance to an immunosuppressor and resistance to ergosterol biosynthesis inhibitors . The double inactivation of SUR4 and FEN1 genes is lethal . These data and other aspects implicating SUR4 in glucose metabolism, suggest an involvement of these genes in the dynamics of cortical actin cytoskeleton in response to nutrient availability . Moreover, the existence of a fourth homologous gene in C . elegans extends the family to pluricellular organisms. FEBS Lett, 1995 Sep 18, 372(1), 29 - 32 Import of sterols into mitochondria of the yeast Saccharomyces cerevisiae; Tuller G et al.; An in vitro assay was designed to study the import of radiolabeled ergosterol and cholesterol from unilamellar vesicles into isolated mitochondria of the yeast Saccharomyces cerevisiae . Supply of ergosterol to the mitochondrial surface was enhanced by a cytosolic fraction containing a lipid transfer protein, whereas no such additive to the assay was required for cholesterol transport . Both sterols reached the inner mitochondrial membrane . During import, they were detected in contact sites between the outer and the inner mitochondrial membrane supporting the idea, that these zones are sites of intramitochondrial lipid translocation . Transport of ergosterol between the outer and the inner mitochondrial membrane was not affected by addition of ATP, depletion of ATP caused by treatment of mitochondria with apyrase and oligomycin, and incubation with the uncoupler CCCP, indicating that this process is energy-independent. J Biol Chem, 1995 Sep 15, 270(37), 21793 - 9 BTS1 encodes a geranylgeranyl diphosphate synthase in Saccharomyces cerevisiae; Jiang Y et al.; Protein prenylation utilizes different types of isoprenoids groups, namely farnesyl and geranylgeranyl, to modify proteins . These lipophilic moieties attach to carboxyl-terminal cysteine residues to promote the association of soluble proteins to membranes . Most prenylated proteins are geranylgeranylated . Geranylgeranylation is catalyzed by two different prenyltransferases, the type I and type II geranylgeranyl transferases, both of which utilize geranylgeranyl diphosphate as a lipid donor . In the yeast Saccharomyces cerevisiae, the BET2 gene encodes the beta-subunit of the type II geranylgeranyl transferase . Mutations in this gene cause a defect in the geranylgeranylation of small GTP-binding proteins that mediate vesicular traffic . In an attempt to analyze those genes whose products may interact with Bet2, we isolated a suppressor of the bet2-1 mutant . This suppressor gene, called BTS1, encodes the yeast geranylgeranyl diphosphate synthase . BTS1 is not essential for the vegetative growth of cells; however, disrupting it impedes the geranylgeranylation of many cellular proteins and renders cells cold sensitive for growth . Our findings imply that BTS1 suppresses the bet2-1 mutant by increasing the intracellular pool of geranylgeranyl diphosphate. J Biol Chem, 1995 Sep 15, 270(37), 21453 - 6 ATP-dependent processivity of a telomerase activity from Saccharomyces cerevisiae; Lue NF et al.; Extracts of Saccharomyces cerevisiae were shown to support the elongation of oligodeoxynucleotides with telomere-like sequences . The primer sequence specificity of this elongation activity, its incorporation of dG and dT but not dA or dC from the corresponding triphosphates, and its sensitivity to RNase A and RNase H are all consistent with it being a telomerase . In contrast to the reported properties of other telomerases, the presence of ATP enhances the efficiency of initiation of the yeast enzyme and improves its processivity . Hydrolysis of ATP appears to be unnecessary for the observed effects, as the beta,gamma-imido or the gamma-thio derivative of ATP is nearly as effective. Eur J Biochem, 1995 Sep 15, 232(3), 712 - 7 Protein phosphatase 2B of Saccharomyces cerevisiae is required for tolerance to manganese, in blocking the entry of ions into the cells; Farcasanu IC et al.; The role of protein phosphatase 2B (PP2B/calcineurin) of Saccharomyces cerevisiae in the tolerance to divalent cations was investigated . PP2B-deficient mutants were found to be sensitive to MnCl2, but not to ZnCl2, CuCl2, NiCl2 and CoCl2 . By measuring both manganese uptake and its efflux, it was found that the sensitivity of the mutant cells was due to an increase in manganese uptake and that the wild-type cells were able to prevent manganese entry into the cells, rather than export it in a more efficient manner . In the presence of the immunosuppressant FK506, the behavior of wild-type cells became similar to that of PP2B mutants . Out of various divalent cations tested, externally added magnesium ions were able to block manganese uptake in both wild-type and PP2B mutant strains. EMBO J, 1995 Sep 15, 14(18), 4589 - 98 The nucleotide mapping of DNA double-strand breaks at the CYS3 initiation site of meiotic recombination in Saccharomyces cerevisiae; de Massy B et al.; Initiation of meiotic recombination in the yeast Saccharomyces cerevisiae occurs by localized DNA double-strand breaks (DSBs) at several locations in the genome, corresponding to hot spots for meiotic gene conversion and crossing over . The meiotic DSBs occur in regions of chromatin that are hypersensitive to nucleases . To gain insight into the molecular mechanism involved in the formation of these DSBs, we have determined their positions at the nucleotide level at the CYS3 hot spot of gene conversion on chromosome I . We found four major new features of these DSBs: (i) sites of DSBs are multiple with varying intensities and spacing within the promoter region of the CYS3 gene; (ii) no consensus sequence can be found at these sites, indicating that the activity involved in DSB formation has little or no sequence specificity; (iii) the breaks are generated by blunt cleavages; and (iv) the 5' ends are modified in rad50S mutant strains, where the processing of these ends is known to be prevented . We present a model for the initiation of meiotic recombination taking into account the implications of these results. Yeast, 1995 Sep 15, 11(11), 1103 - 12 Sequence analysis of a 78.6 kb segment of the left end of Saccharomyces cerevisiae chromosome II; Obermaier B et al.; We report the sequence analysis of a 78,601 bp DNA segment on the left arm of chromosome II of Saccharomyces cerevisiae . This 78.6 kb segment spans the region from the start of a subtelomeric Y' element up to the ILS1 gene . It contains 49 open reading frames (ORFs) with more than 100 amino acids length including 14 internal and five overlapping ORFs . The gene density, excluding the internal ORFs, was calculated as one ORF per 2.2 kb . Eight ORFs (PKC1, TyA, TyB, ATP1, ROX3, RPL17a, PET112 and ILS1) correspond to previously characterized genes . ORF YBL0718 was identified as CDC27; YBL0706 as TEL1 . Four other ORFs show strong similarities to already known genes . The gene product of YBL0838 is 60% identical to the ribosomal protein RPL32 from rat, mouse and man . YBL0701 encodes a protein with significant similarity to the initiation factor eIF2 associated p67 glycoprotein from rat . Eight ORFs were disrupted and the resulting yeast strains analysed with respect to their phenotype. Yeast, 1995 Sep 15, 11(11), 1087 - 91 The complete sequence of a 9000 bp fragment of the right arm of Saccharomyces cerevisiae chromosome VII contains four previously unknown open reading frames; Guerreiro P et al.; We report the sequence of a 9000 bp fragment from the right arm of Saccharomyces cerevisiae chromosome VII . Analysis of the sequence revealed four complete previously unknown open reading frames, which were named G7587, G7589, G7591 and G7594 following standard rules for provisional nomenclature . Outstanding features of some of these proteins were the homology of the putative protein coded by G7589 with proteins involved in transcription regulation and the transmembrane domains predicted in the putative protein coded by G7591. Yeast, 1995 Sep 15, 11(11), 1001 - 13 Review: the dominant flocculation genes of Saccharomyces cerevisiae constitute a new subtelomeric gene family; Teunissen AW et al.; The quality of brewing strains is, in large part, determined by their flocculation properties . By classical genetics, several dominant, semidominant and recessive flocculation genes have been recognized . Recent results of experiments to localize the flocculation genes FLO5 and FLO8, combined with the in silicio analysis of the available sequence data of the yeast genome, have revealed that the flocculation genes belong to a family which comprises at least four genes and three pseudogenes . All members of this gene family are located near the end of chromosomes, just like the SUC, MEL and MAL genes, which are also important for good quality baking or brewing strains . Transcription of the flocculation genes is repressed by several regulatory genes . In addition, a number of genes have been found which cause cell aggregation upon disruption or overexpression in an as yet unknown manner . In total, 33 genes have been reported that are involved in flocculation or cell aggregation. Biochem Biophys Res Commun, 1995 Sep 14, 214(2), 723 - 9 On the relationship of the ATP-independent, mitochondrial associated DNA topoisomerase of Saccharomyces cerevisiae to the nuclear topoisomerase I; Wang J et al.; A mitochondrial type I-like, ATP-independent, DNA topoisomerase, isolated from highly purified yeast mitochondria, is genetically related to nuclear topoisomerase I . We found that the mitochondrial topoisomerase activity cannot be detected in yeast mitochondrial extracts prepared from strains in which the topoisomerase I gene (TOP1) is disrupted . Thus, the topoisomerase activity associated with mitochondria is dependent upon the expression of the nuclear topoisomerase I gene. Biochem Biophys Res Commun, 1995 Sep 14, 214(2), 709 - 15 Characterization of SFP2, a putative sulfate permease gene of Saccharomyces cerevisiae; Jin YH et al.; The SFP2 gene of Saccharomyces cerevisiae has been characterized . The deduced amino acid sequence contained twelve highly hydrophobic domains and showed 50, 47, 44 and 48% homologies to Neurospora crassa sulfate permease II (CYS14), soybean GMAK170 nodulin, human colon mucosa protein (DRA) and a putative open reading frame (ORF) downstream of Escherichia coli prs (phosphoribosyl pyrophosphatate synthetase) gene, respectively, in the aligned regions . Cells lacking SFP2 were viable and displayed no obvious decrease in their growth rate . Southern blot analysis revealed that SFP2 exists as a single copy in haploid genome . Northern blot analysis showed that SFP2 produced a 2.8-kb transcript which was highly expressed under sulfur derepressing condition . SFP2 mRNA was found to turn over with a half-life of approximately 15 min, which may contribute to the regulation of sulfate permease function, and reached its maximal level in about 22 h after depression. Proc Natl Acad Sci U S A, 1995 Sep 12, 92(19), 8921 - 5 Functional complementation of the ste6 gene of Saccharomyces cerevisiae with the pfmdr1 gene of Plasmodium falciparum; Volkman SK et al.; The pfmdr1 gene has been associated with a drug-resistant phenotype in Plasmodium falciparum, and overexpression of pfmdr1 has been associated with mefloquine- and halofantrine-resistant parasites, but little is known about the functional role of pfmdr1 in this process . Here, we demonstrate that the pfmdr1 gene expressed in a heterologous yeast system functions as a transport molecule and complements a mutation in ste6, a gene which encodes a mating pheromone a-factor export molecule . In addition, the pfmdr1 gene containing two mutations which are associated with naturally occurring chloroquine resistance abolishes this mating phenotype, suggesting that these genetic polymorphisms alter this transport function . Our results support the functional role of pfmdr1 as a transport molecule in the mediation of drug resistance and provide an assay system to address the nature of this transport function. Proc Natl Acad Sci U S A, 1995 Sep 12, 92(19), 8911 - 5 Expression of a plant viral polycistronic mRNA in yeast, Saccharomyces cerevisiae, mediated by a plant virus translational transactivator; Sha Y et al.; We demonstrate that the cauliflower mosaic virus (CaMV) gene VI product can transactivate the expression of a reporter gene in bakers' yeast, Saccharomyces cerevisiae . The gene VI coding sequence was placed under the control of the galactose-inducible promoter GAL1, which is presented in the yeast shuttle vector pYES2, to create plasmid JS169 . We also created a chloramphenicol acetyltransferase (CAT) reporter plasmid, JS161, by inserting the CAT reporter gene in-frame into CaMV gene II and subsequently cloning the entire CaMV genome into the yeast vector pRS314 . When JS161 was transformed into yeast and subsequently assayed for CAT activity, only a very low level of CAT activity was detected in cellular extracts . To investigate whether the CaMV gene VI product would mediate an increase in CAT activity, we cotransformed yeast with JS169 and JS161 . Upon induction with galactose, we found that CAT activity in yeast transformed with JS161 and JS169 was about 19 times higher than the level in the transformants that contained only JS161 . CAT activity was dependent on the presence of the gene VI protein, because essentially no CAT activity was detected in yeast cells grown in the presence of glucose, which represses expression from the GAL1 promoter . RNase protection assays showed that the gene VI product had no effect on transcription from the 35S RNA promoter, demonstrating that regulation was occurring at the translation level . This yeast system will prove useful for understanding how the gene VI product of CaMV mediates the translation of genes present on a eukaryotic polycistronic mRNA. Biochemistry, 1995 Sep 12, 34(36), 11385 - 98 Three-dimensional solution structure of the cyanide adduct of a Met80Ala variant of Saccharomyces cerevisiae iso-1-cytochrome c . Identification of ligand-residue interactions in the distal heme cavity; Banci L et al.; The 1H NMR spectrum of the the cyanide adduct of a triply mutated Saccharomyces cerevisiae iso-1-cytochrome c (His39Gln/Met80Ala/Cys102Ser) in the oxidized form has been assigned through 1D NOE and 2D COSY, TOCSY, NOESY, and NOE-NOESY experiments; 562 protons out of a total of 683 have been assigned . The solution structure, the first of a paramagnetic heme protein, was determined using 1426 meaningful NOE constraints out of a total of 1842 measured NOEs . The RMSD values at the stage of restrained energy minimization of 17 structures obtained from distance geometry calculations are 0.68 +/- 0.11 and 1.32 +/- 0.14 A for the backbone and all heavy atoms, respectively . The quality, in terms of RMSD, of the present structure is the same as that obtained for the solution structure of the diamagnetic horse heart ferrocytochrome c {Qi, P . X., et al . (1994) Biochemistry 33, 6408-6419} . The secondary structure elements and the overall folding in the variant are observed to be the same as those of the wild-type protein for which the X-ray structure is available . However, the replacement of the methionine axial ligand with an alanine residue creates a ligand-binding "distal cavity" . The properties of the distal cavity seen in this solution structure are compared to those of other heme proteins. Nucleic Acids Res, 1995 Sep 11, 23(17), 3493 - 500 HYS2, an essential gene required for DNA replication in Saccharomyces cerevisiae; Sugimoto K et al.; To investigate cell cycle regulation at the S or G2 phase in Saccharomyces cerevisiae, we have isolated mutants displaying supersensitivity to hydroxyurea (HU), a chemical that inhibits DNA replication . Such mutants, which we have named hydroxyurea sensitive (hys), defined four linkage groups and we characterized the hys2 mutation in this study . The hys2-1 mutant displays temperature sensitive growth and a constellation of phenotypes indicating defective DNA metabolism . At the restrictive temperature, hys2-1 cells arrest as large budded cells with a single nucleus at the neck of the bud and a short spindle . The hys2-1 mutant exhibits increased rates of chromosome loss and recombination . Additionally, hys2-1 appears to accumulate incompletely replicated DNA that can be detected by a pulse field electrophoresis assay . Finally, deletion of RAD9 in a hys2-1 strain decreases the percentage of arrested cells, suggesting that an intact RAD9-checkpoint is required for the cell cycle arrest in hys2-1 cells . HYS2 encodes a 55 kDa protein that is essential for viability at all temperatures . Taken together, these data suggest that Hys2 plays a role in DNA replication. Gene, 1995 Sep 11, 162(2), 239 - 43 Stress- and sequence-dependent release into the culture medium of HIV-1 Nef produced in Saccharomyces cerevisiae; Macreadie IG et al.; We have produced human immunodeficiency virus type 1 (HIV-1) Nef (a myristylated 206-amino-acid protein) in Saccharomyces cerevisaie and shown that, while Nef is normally found as a predominantly intracellular protein, amounts up to 40 micrograms/ml of Nef are also released into the extracellular medium during stress . By electrophoretic (SDS-PAGE) analysis the extracellular Nef is indistinguishable from intracellular Nef . Conditions of stress that lead to the release of Nef include elevated levels of copper or magnesium ions or growth at elevated temperatures . This release appears to be dependent upon the N-terminal sequences of Nef, including the presence of a myristylation site . Our observations concerning Nef release in yeast suggest new ways in which the behaviour of Nef should be examined in order to gain further insights into the development of AIDS . If the release of Nef is important in the development of AIDS, our work reveals that Nef-associated symptoms may be reduced or delayed by reducing stresses, such as fevers. J Biol Chem, 1995 Sep 8, 270(36), 21028 - 34 High level expression, partial purification, and functional reconstitution of the human AE1 anion exchanger in Saccharomyces cerevisiae; Sekler I et al.; Human erythroid anion exchanger AE1 (Band 3) was expressed in the yeast Saccharomyces cerevisiae under the control of the constitutive promoter and transcriptional terminator of the yeast phosphoglycerate kinase gene . AE1 expression in stable yeast transformants was estimated to be approximately 0.7 mg AE1 per liter . Density gradient sedimentation analysis indicated that the AE1 protein was associated with a membrane fraction distinct from plasma membrane, most likely the endoplasmic reticulum . AE1 protein was solubilized from yeast membranes with lysophosphatidyl choline, and the protein, tagged with six histidines at its amino terminus, was purified to 35% homogeneity by metal chelation affinity chromatography . Size-exclusion chromatography in the presence of octaethylene glycol monododecyl ether indicated that the solubilized yeast-expressed AE1, like endogenous erythroid AE1, eluted at a stokes radius of 77 A, consistent with a dimeric oligomeric state . Binding of partially purified yeast-expressed AE1 to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate resin was competitive with the transportable substrate chloride but not the nontransported anion citrate, suggesting that the structure of the anion binding site is preserved . The specific activity of sulfate transport by partially purified yeast AE1 was determined in proteoliposomes to be similar to that of authentic AE1 purified from erythrocyte membranes . These data show that this expression system has the capacity to produce functional mammalian plasma membrane anion exchangers at levels sufficient for biochemical and biophysical analysis. J Biol Chem, 1995 Sep 8, 270(36), 20997 - 1002 Mutations that perturb cyclophilin A ligand binding pocket confer cyclosporin A resistance in Saccharomyces cerevisiae; Cardenas ME et al.; In complex with the peptidyl-prolyl isomerase cyclophilin A, the immunosuppressive antifungal drug cyclosporin A (CsA) inhibits a Ca2+/calmodulin-dependent protein phosphatase, calcineurin, which regulates signal transduction . We isolated and characterized cyclophilin A mutations that confer CsA resistance in a Saccharomyces cerevisiae strain whose growth is CsA-sensitive . Three mutations (G70S, H90Y, and G102A) alter single amino acids conserved between yeast and human cyclophilin A, which structural analyses implicate in CsA binding to human cyclophilin A . By Western analysis, all three mutant proteins are expressed in yeast . In vitro, two purified mutant cyclophilins (G70S, G102A) retain prolyl isomerase activity and have moderately reduced affinity for CsA and calcineurin but, when bound to CsA, do bind and inhibit calcineurin phosphatase activity . In contrast, the purified H90Y mutant cyclophilin is dramatically decreased in prolyl isomerase activity, CsA affinity, and calcineurin binding and inhibition . These studies identify conserved cyclophilin A residues that participate in CsA binding and catalysis. J Biochem (Tokyo), 1995 Sep, 118(3), 607 - 13 Three ATP1 genes are present on chromosome II in Saccharomyces cerevisiae; Takeda M et al.; Chromosome fragmentation, ATP1 disruption, and Southern blot analyses of total DNAs and prime clones of chromosome II showed that three identical ATP1s are present, directing from the telomere to the centromere on the 35-55 kb far from the left telomere sequence of chromosome II . That is, the coding and 5'-, 3'-non-coding regions of ATP1 are repeated 3 times at approximately 7 kb intervals . These three ATP1s are expressed, and one and two ATP1s-disrupted strains, respectively, showed ca.70 and 40% decreases in their ATPase activities and alpha subunit contents, compared to those of the wild type, DC-5 or W303-1A strain, but could grow on glycerol. Z Naturforsch {C}, 1995 Sep-Oct, 50(9-10), 732 - 4 Effect of platinum(II) complexes of benzoic and 3-methoxybenzoic acid hydrazides on Saccharomyces cerevisiae; Tabakova S et al.; The inhibitory effect of benzoic acid hydrazide (bah) and 3-methoxybenzoic acid hydrazide (mbah) on Saccharomyces cerevisiae strains has been compared to that of their platinum(II) complexes: cis-{Pt(mbah)2X2}, cis-{Pt(NH3)(mbah)Cl2}.0.5 H2O, cis-{Pt(mbah)2X2} and cis{Pt(NH3)(mbah)Cl2} (mean = Cl, Br = I), and cis-{Pt(NH3)2Cl2} . The minimal inhibitory concentrations for bah and mbah were 5000-20,000 microM whereas for their Pt(II) complexes they were much less (25-800 microM) and did not exceed these of cisplatin (100-800 microM) . The activity of the Pt(II) complexes of bah and mbah varied in wide ranges for the different yeast strains tested . Osmotically unstable mutants were found to be more susceptible . The most active complexes were {Pt(NH3)(bah)Cl2}.0.5 H2O and {Pt(NH3)(mbah)Cl2}. Glycobiology, 1995 Sep, 5(6), 633 - 42 Proliferation-dependent differential regulation of the dolichol pathway genes in Saccharomyces cerevisiae; Lennon K et al.; The dolichol pathway serves in the synthesis of the dolichol-linked oligosaccharide precursor for protein N-glycosylation . Recently, we reported that mRNAs of genes that function at the early steps in the dolichol pathway in yeast, ALG7, ALG1 and ALG2, were co-ordinately induced following growth stimulation of G0-arrested cells in a manner similar to that of the transcripts of the early growth response genes (Kukuruzinska, M.A . and Lennon, K . Glycobiology, 4, 437-443, 1994) . To determine whether the entire dolichol pathway was co-ordinately regulated with growth, we examined the expression of genes functioning late in the pathway, including two genes encoding oligosaccharyltransferase subunits, at two critical control points in the G1 phase of cell cycle: G0/G1 and START . We show that early in G1, at the G0/G1 transition point, the late ALG genes and the two oligosaccharyltransferase-encoding genes examined were regulated co-ordinately with the early ALG genes: they were downregulated upon exit from the mitotic cell cycle into G0, and they were induced following growth stimulation in the absence of de novo protein synthesis . All the dolichol pathway genes produced transcripts with short half-lives that were rapidly stabilized in the presence of cycloheximide . In contrast, cell division arrest late in G1, at START, was accompanied by a selective downregulation of only the first dolichol pathway gene, ALG7, and not of the genes functioning later in the pathway . These results indicate that, depending on their position in G1, cells either co-ordinately or differentially regulate the dolichol pathway genes. Cell Mol Biol (Noisy-le-grand), 1995 Sep, 41(6), 843 - 9 GABA uptake in a Saccharomyces cerevisiae strain; Bermudez Moretti M et al.; The aim of this work was to characterize the 4-aminobutyric acid (GABA) transport in the Saccharomyces cerevisiae D27 strain, followed by the study of the relationship between 5-aminolevulinic acid (ALA) and GABA transport systems . It was found that the general amino acid permease (GAP) is not active in D27 strain, suggesting that GABA incorporation should be mediated by PUT4 and UGA4 permeases . However, after kinetic studies only one system was detected . It was also shown that GABA uptake is competitively inhibited by ALA . GABA incorporation is regulated by the carbon source but not by the nitrogen source . When cells were grown in the presence of GABA, its entrance was very low. Cell Mol Biol (Noisy-le-grand), 1995 Sep, 41(6), 763 - 9 The correlative evidence suggesting that trehalose stabilizes membrane structure in the yeast Saccharomyces cerevisiae; Iwahashi H et al.; The contribution of trehalose and hsp104 to membrane fluidity and the mobility of non-freezing cell water were examined on the basis of whole cell NMR analysis of the yeast Saccharomyces cerevisiae . Membrane fluidity was dependent on the accumulation of trehalose not hsp104 and non-freezing cell water was dependent on the accumulation of hsp104 not trehalose . Thus, the correlative evidence suggesting that trehalose protects yeast cells from temperature extremes by stabilizing the membrane structure was observed in vivo. Mol Biol Cell, 1995 Sep, 6(9), 1125 - 43 Glucose-dependent turnover of the mRNAs encoding succinate dehydrogenase peptides in Saccharomyces cerevisiae: sequence elements in the 5' untranslated region of the Ip mRNA play a dominant role; Cereghino GP et al.; We have demonstrated previously that glucose repression of mitochondrial biogenesis in Saccharomyces cerevisiae involves the control of the turnover of mRNAs for the iron protein (Ip) and flavoprotein (Fp) subunits of succinate dehydrogenase (SDH) . Their half-lives are > 60 min in the presence of a nonfermentable carbon source (YPG medium) and < 5 min in glucose (YPD medium) . This is a rare example in yeast in which the half-lives are > 60 min in the presence of a nonfermentable carbon source (YPG medium) and < 5 min in glucose (YPD medium) . This is a rare example in yeast in which the half-life of an mRNA can be controlled by manipulating external conditions . In our current studies, a series of Ip transcripts with internal deletions as well as chimeric transcripts with heterologous sequences (internally or at the ends) have been examined, and we established that the 5'-untranslated region (5' UTR) of the Ip mRNA contains a major determinant controlling its differential turnover in YPG and YPD . Furthermore, the 5' exonuclease encoded by the XRN1 gene is required for the rapid degradation of the Ip and Fp mRNAs upon the addition of glucose . In the presence of cycloheximide the nucleolytic degradation of the Ip mRNA can be slowed down by stalled ribosomes to allow the identification of intermediates . Such intermediates have lost their 5' ends but still retain their 3' UTRs . If protein synthesis is inhibited at an early initiation step by the use of a prt1 mutation (affecting the initiation factor eIF3), the Ip and Fp mRNAs are very rapidly degraded even in YPG . Significantly, the arrest of translation by the introduction of a stable hairpin loop just upstream of the initiation codon does not alter the differential stability of the transcript in YPG and YPD . These observations suggest that a signaling pathway exists in which the external carbon source can control the turnover of mRNAs of specific mitochondrial proteins . Factors must be present that control either the activity or more likely the access of a nuclease to the select mRNAs . As a result, we propose that a competition between initiation of translation and nuclease action at the 5' end of the transcript determines the half-life of the Ip mRNA. Mol Biol Cell, 1995 Sep, 6(9), 1103 - 10 Mutations in nucleolar proteins lead to nucleolar accumulation of polyA+ RNA in Saccharomyces cerevisiae; Kadowaki T et al.; Synthesis of mRNA and rRNA occur in the chromatin-rich nucleoplasm and the nucleolus, respectively . Nevertheless, we here report that a Saccharomyces cerevisiae gene, MTR3, previously implicated in mRNA transport, codes for a novel essential 28-kDa nucleolar protein . Moreover, in mtr3-1 the accumulated polyA+ RNA actually colocalizes with nucleolar antigens, the nucleolus becomes somewhat disorganized, and rRNA synthesis and processing are inhibited . A strain with a ts conditional mutation in RNA polymerase I also shows nucleolar accumulation of polyA+ RNA, whereas strains with mutations in the nucleolar protein Nop1p do not . Thus, in several mutant backgrounds, when mRNA cannot be exported i concentrates in the nucleolus . mRNA may normally encounter nucleolar components before export and proteins such as Mtr3p may be critical for export of both mRNA and ribosomal subunits. Protein Sci, 1995 Sep, 4(9), 1832 - 43 UME6, a negative regulator of meiosis in Saccharomyces cerevisiae, contains a C-terminal Zn2Cys6 binuclear cluster that binds the URS1 DNA sequence in a zinc-dependent manner; Anderson SF et al.; UME6 is a protein of 836 amino acids from Saccharomyces cerevisiae that acts as a repressor and activator of several early meiotic genes . UME6 contains, near the C-terminus, the amino acid sequence-771C-X2-C-X6-C-X6-C-X2-C-X6-C-, in which the spacings of the six Cys residues are identical to those found in 39 N-terminal Cys-rich DNA binding subdomains of fungal transcription factors . This sequence has been shown in GAL4 and other proteins to form a zinc binuclear cluster . In spite of the different location, the C-rich sequence, cloned and over-produced within the last 111 amino acid residues of UME6, UME6(111), forms a binuclear cluster and exhibits a Zn-dependent binding to the URS1 DNA sequence . The latter, TAGCCGCCGA, is required for the repression or activation of meiosis-specific genes by UME6 . UME6(111) contains 1.8 +/- 0.4 mol Zn/mol protein and the Zn can be exchanged for Cd to yield a protein containing 1.9 +/- 0.1 mol Cd/mol protein . At 5 degrees C, 113Cd2UME6(111) shows two 113Cd NMR signals, with chemical shifts of 699 and 689 ppm, similar to those observed for 113Cd2GAL4(149) . The magnitude of these chemical shifts suggests that each 113Cd nucleus is coordinated to four -S- ligands, compatible with a 113Cd2 cluster structure in which two thiolates from bridging ligands . The entire UME6 gene has been cloned and overexpressed and binds more tightly to the URS1 sequence than the zinc binuclear cluster domain alone . DNase I footprints of UME6 on URS1-containing DNA show that the protein protects the phosphodiesters of the 5'-CCGCCG-3' region within the URS1 sequence. C R Acad Sci III, 1995 Sep, 318(9), 919 - 26 In vitro yeast (Saccharomyces cerevisiae) presqualene and squalene synthesis related to substrate and cofactor availability; Socaciu C et al.; Squalene synthase catalyses the synthesis of squalene from trans-farnesyl diphosphate in 2 separate steps requiring NAD(P)H . The kinetics of this enzyme in different fractions extracted from a wild-type Saccharomyces cerevisiae were studied . Although this protein is known to be a membrane-bound enzyme, we have found a cytosolic squalene synthase activity besides the microsomal enzyme . A spectrophotometric enzyme assay, not involving isotopic labelling, was established . The relative synthesis of presqualene and squalene was evaluated by using different substrate and cofactor concentrations during the incubation . The involvement of a single catalytic site promoting the 2 reactions of squalene synthesis is suggested. Biosci Biotechnol Biochem, 1995 Sep, 59(9), 1694 - 8 Chromosome bisection in the yeast Saccharomyces cerevisiae facilitated by yeast artificial chromosomes bearing a site-specific recombination system; Kawasaki H et al.; A chromosome bisection method was constructed using yeast artificial chromosomes (YAC) and a GAL1-promoted site-specific recombination system . This method was applied to bisect chromosome IV into the left and right parts of the centromere region . The bisection occurred at frequencies of about 10% when the recombination site DNAs were integrated onto YAC and chromosome IV in the same direction, but were less than 10(-3) when they were in opposite directions . Reconstruction of the original chromosome IV from the bisected chromosomes was also induced by galactose at high frequencies . Loss of the left part chromosome was found at the frequencies of 0.9 x 10(-3) after hybrid cells between the chromosome-bisected strain and a normal haploid were subcultivated in a complete medium . The bisection and reconstruction of chromosome IV and deletion of the left part chromosome were demonstrated by electrophoretical karyotyping. Mutat Res, 1995 Sep, 331(1), 149 - 59 Analysis of spontaneous frameshift mutations in REV1 and rev1-1 strains of Saccharomyces cerevisiae; Kalinowski DP et al.; Frameshift mutations occur by a number of mechanisms . To better understand the nature of these mechanisms, we determined the DNA sequence changes of 232 independent, spontaneous frameshift mutations in the HIS4 gene of REV1 and rev1-1 strains of Saccharomyces cerevisiae . All frameshift mutants were selected based on their ability to revert the +1 frameshift mutation his4-38 . DNA sequence information was recovered using two approaches-the double-strand gap repair of plasmid pMP4, and the polymerase chain reaction (PCR) . Using these techniques, saturated mutation spectra for the spontaneous reversion of his4-38 were generated . The most frequently occurring mutational events in both strains were -1 frameshifts, but +2 frameshifts, larger deletions, larger insertions and more complex mutations were also observed . Between the REV1 and rev1-1 strains, we noticed a significant difference in the distribution of -1 frameshift mutations . In addition, while for -1 frameshift events there was no significant difference between the reversion spectra determined by double-strand gap repair or PCR, there was a surprisingly significant difference between the types of frameshift mutations recovered by double-strand gap repair (only -1 frameshifts and one +2 frameshift), and those recovered using PCR (-1 frameshifts, +2 frameshifts, larger deletions and insertions, and more complex mutations) . This difference may reflect a selectional mechanism inherent in double-strand break repair that avoids chromosomal sequences which include complex alterations. Curr Microbiol, 1995 Sep, 31(3), 163 - 8 Transcriptional control of the Saccharomyces cerevisiae ADH1 gene by autonomously replicating sequence binding factor 1; Yoo HY et al.; Autonomously replicating sequence (ARS)-binding factor 1 (ABF1) is a multifunctional protein involved in transcriptional activation and repression, as well as DNA replication, in yeast . The ADH1 gene, encoding alcohol dehydrogenase 1, contains two ABF1 consensus binding sites in the promoter and the coding regions . To examine the effect of ABF1 on expression of the ADH1 gene, we constructed an ADH1-lacZ fusion plasmid . Both ABF1 binding sites appeared to be transcriptional activators because deletions and mutations of these sites decreased transcriptional activity . The ABF1 binding sites also acted in an orientation-independent manner when a synthetic ABF1 binding site was inserted into the yeast CYC1 gene lacking its transcriptional activation region . A gel mobility shift assay showed that ABF1 bound in vitro to both ABF1 binding sites in the promoter and coding regions . In a glycerol medium the degree of activation by ABF1 was higher than in a glucose medium . The expression of ADH1 was activated synergistically by both ABF1 binding sites . These observations suggest that ABF1 transactivates the ADH1 gene through its binding sequences in both the promoter and coding regions. J Biol Chem, 1995 Sep 1, 270(35), 20742 - 7 The cellular content of Cdc25p, the Ras exchange factor in Saccharomyces cerevisiae, is regulated by destabilization through a cyclin destruction box; Kaplon T et al.; The Cdc25p and Sdc25p proteins were the first members of the family of guanine nucleotide exchange factors to be identified . These proteins promote the formation of active Ras-GTP complex from inactive Ras-GDP complex by exchange of GDP for GTP . Therefore Cdc25p which is the main positive regulator of Ras, regulates through Ras the activity of adenylate cyclase in Saccharomyces cerevisiae . The amino-terminal part of Cdc25p has a sequence similar to the cyclin destruction box (CDB) of mitotic cyclins . This sequence has been reported to be required for ubiquitin-dependent proteolysis . In this study we show that Cdc25p is an unstable polypeptide with a half-life of 15-20 min . Its instability depends upon the presence of the CDB which can also confer instability to other proteins . Degradation of Cdc25p and CDB containing beta-galactosidase was found to be independent of various cell cycle arrest points . The fast degradation of Cdc25p opens the possibility that Ras and the cAMP cascade in yeast are directly modulated by the cellular content of the guanine nucleotide exchange factor rather than variation in activity or localization control. Mol Cell Biol, 1995 Sep, 15(9), 5030 - 42 Negative regulation of G1 and G2 by S-phase cyclins of Saccharomyces cerevisiae; Basco RD et al.; Cell cycle progression in the budding yeast Saccharomyces cerevisiae is controlled by the Cdc28 protein kinase, which is sequentially activated by different sets of cyclins . Previous genetic analysis has revealed that two B-type cyclins, Clb5 and Clb6, have a positive role in DNA replication . In the present study, we show, in addition, that these cyclins negatively regulate G1- and G2-specific functions . The consequences of this negative regulation were most apparent in clb6 mutants, which had a shorter pre-Start G1 phase as well as a shorter G2 phase than congenic wild-type cells . As a consequence, clb6 mutants grew and proliferated more rapidly than wild-type cells . It was more difficult to assess the role of Clb5 in G1 and G2 by genetic analysis because of the extreme prolongation of S phase in clb5 mutants . Nevertheless, both Clb5 and Clb6 were shown to be responsible for down-regulation of the protein kinase activities associated with Cln2, a G1 cyclin, and Clb2, a mitotic cyclin, in vivo . These observations are consistent with the observed cell cycle phase accelerations associated with the clb6 mutant and are suggestive of similar functions for Clb5 . Genetic evidence suggested that the inhibition of mitotic cyclin-dependent kinase activities was dependent on and possibly mediated through the CDC6 gene product . Thus, Clb5 and Clb6 may stabilize S phase by promoting DNA replication while inhibiting other cell cycle activities. Mol Cell Biol, 1995 Sep, 15(9), 4754 - 62 Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae; Remacha M et al.; Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained . Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins . All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C . Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins . Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes . However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract . In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes . These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently. Int J Lepr Other Mycobact Dis, 1995 Sep, 63(3), 381 - 90 Immunogenic properties of the M . leprae recombinant 18-kDa antigen purified from Saccharomyces cerevisiae; enhancement of delayed-type hypersensitivity after gamma-irradiation; Pinho JR et al.; In this paper we report the purification and study of the immunogenic properties of the Mycobacterium leprae 18-kDa protein antigen produced and secreted by the yeast Saccharomyces cerevisiae, using an expression system we recently described {Biotech . Lett . 16 (1994) 1241-1246} . The 18-kDa protein was purified from the yeast culture media by precipitation, ion exchange chromatography (MonoQ) and exclusion size chromatography (Sephacryl S-100) . The biological properties of the recombinant protein, previously irradiated with gamma rays, were assayed by immunization of mice . Humoral and cellular responses, monitored by antibody production and delayed-type hypersensitivity, respectively, were obtained . Furthermore, gamma-irradiation of the recombinant protein prior to the administration was shown to significantly potentiate the T-cell response . The data suggest that this irradiated recombinant antigen could be used in a more sensitive standardized skin test to monitor M . leprae infection. Microbiol Rev, 1995 Sep, 59(3), 345 - 86 Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae; Cid VJ et al.; In fungi and many other organisms, a thick outer cell wall is responsible for determining the shape of the cell and for maintaining its integrity . The budding yeast Saccharomyces cerevisiae has been a useful model organism for the study of cell wall synthesis, and over the past few decades, many aspects of the composition, structure, and enzymology of the cell wall have been elucidated . The cell wall of budding yeasts is a complex and dynamic structure; its arrangement alters as the cell grows, and its composition changes in response to different environmental conditions and at different times during the yeast life cycle . In the past few years, we have witnessed a profilic genetic and molecular characterization of some key aspects of cell wall polymer synthesis and hydrolysis in the budding yeast . Furthermore, this organism has been the target of numerous recent studies on the topic of morphogenesis, which have had an enormous impact on our understanding of the intracellular events that participate in directed cell wall synthesis . A number of components that direct polarized secretion, including those involved in assembly and organization of the actin cytoskeleton, secretory pathways, and a series of novel signal transduction systems and regulatory components have been identified . Analysis of these different components has suggested pathways by which polarized secretion is directed and controlled . Our aim is to offer an overall view of the current understanding of cell wall dynamics and of the complex network that controls polarized growth at particular stages of the budding yeast cell cycle and life cycle. J Cell Biol, 1995 Sep, 130(6), 1333 - 44 Identification of six complementation classes involved in the biosynthesis of glycosylphosphatidylinositol anchors in Saccharomyces cerevisiae; Benghezal M et al.; Glycosylphosphatidylinositol (GPI)-anchored membrane proteins are synthesized by the posttranslational attachment of a preformed glycolipid to newly made glycoproteins . alpha-Agglutinin is a GPI-anchored glycoprotein that gets expressed at the cell surface of MAT alpha cells after induction with type a mating factor . Mutants affecting the biosynthesis of GPI anchors were obtained by selecting for the absence of alpha-agglutinin from the cell wall after induction with a-factor at 37 degrees C . 10 recessive mutants were grouped into 6 complementation classes, gpi4 to gpi9 . Mutants are considered to be deficient in the biosynthesis of GPI anchors, since each mutant accumulates an abnormal, incomplete GPI glycolipid containing either zero, two, or four mannoses . One mutant accumulates a complete precursor glycolipid, suggesting that it might be deficient in the transfer of complete precursor lipids to proteins . When labeled with {2-3H}inositol, mutants accumulate reduced amounts of radiolabeled GPI-anchored proteins, and the export of the GPI-anchored Gas1p out of the ER is severely delayed in several mutant strains . On the other hand, invertase and acid phosphatase are secreted by all but one mutant . All mutants show an increased sensitivity to calcofluor white and hygromycin B . This suggests that GPI-anchored proteins are required for the integrity of the yeast cell wall. Microbiology, 1995 Sep, 141 ( Pt 9), 2201 - 9 IMP2, a gene involved in the expression of glucose-repressible genes in Saccharomyces cerevisiae; Lodi T et al.; Two mutants carrying different deletions of the IMP2 coding sequence of Saccharomyces cerevisiae, delta T1, which encodes a protein lacking the last 26 C-terminal amino acids, and delta T2, which completely lacks the coding region, were analysed for derepression of glucose-repressible maltose, galactose, raffinose and ethanol utilization pathways in response to glucose limitation . The role of the IMP2 gene product in the regulation of carbon catabolite repressible enzymes maltase, invertase, alcohol dehydrogenase, NAD-dependent glutamate dehydrogenase (NAD-GDH) and L-lactate:ferricytochrome-c oxidoreductase (L-LCR) was also analysed . The IMP2 gene product is required for the rapid glucose derepression of all above-mentioned carbon source utilization pathways and of all the enzymes except for L-LCR . NAD-GDH is regulated by IMP2 in the opposite way and, in fact, this enzyme was released at higher levels in both imp2 mutants than in the wild-type strain . Therefore, the product of IMP2 appears to be involved in positive and negative regulation . Both deletions result in growth and catalytic defects; in some cases partial modification of the gene product yielded more dramatic effects than its complete absence . Moreover, evidence is provided that the IMP2 gene product regulates galactose- and maltose-inducible genes at the transcriptional level and is a positive regulator of maltase, maltose permease and galactose permease gene expression. Gene, 1995 Aug 30, 162(1), 87 - 92 Cloning and characterization of the Saccharomyces cerevisiae LYS7 gene: evidence for function outside of lysine biosynthesis; Horecka J et al.; The Saccharomyces cerevisiae LYS7 gene has been cloned based on its genetic map position and complementation of a lys7 mutant . The 1453-bp sequence contains an open reading frame (ORF) that predicts a unique 249 amino acid (aa) protein . A Northern blot experiment demonstrated that LYS7 transcription was not regulated by lysine-specific or general aa control mechanisms . To investigate the effects of total loss of LYS7 function, we created a complete deletion of the gene and introduced this allele into wild-type yeast . The lys7 delta mutant requires lysine and simultaneously displays an array of phenotypes that include pH and temperature sensitivity . The pleiotropic phenotypes of the lys7 delta mutant and the constitutive transcription pattern are at odds with the hypothesis that Lys7p functions solely in the lysine biosynthesis pathway. Gene, 1995 Aug 30, 162(1), 81 - 5 The NAM9-1 suppressor mutation in a nuclear gene encoding ribosomal mitochondrial protein of Saccharomyces cerevisiae; Dmochowska A et al.; The nuclear gene NAM9 from Saccharomyces cerevisiae (Sc) codes for a protein which, on the basis of sequence homology, was previously postulated to be a mitochondrial (mt) equivalent of the Escherichia coli (Ec) S4 ribosomal protein (r-protein) {Boguta et al., Mol . Cell . Biol . 12 (1992) 402-412} . The mt-r character of the NAM9 product is now confirmed by cross-reaction with the antisera for the Sc mt r-proteins . The NAM9-1 mutation, characterized previously as the nuclear suppressor of some ochre mt mit- mutants, is found to be a single nucleotide substitution changing Ser82 to Leu within the part of NAM9 corresponding to the S4 region involved in interaction with the 16S rRNA . This indicates that the mechanism of NAM9-1 suppression could be analogous to the suppression due to ram (ribosomal ambiguity) mutations in the Ec structural gene encoding r-protein S4 . The NAM9-1 mutation leads also to defect in respiratory growth in the background of the wild-type mit+ genome. Proc Natl Acad Sci U S A, 1995 Aug 29, 92(18), 8224 - 8 Identification and characterization of a TFIID-like multiprotein complex from Saccharomyces cerevisiae; Poon D et al.; Although the mechanisms of transcriptional regulation by RNA polymerase II are apparently highly conserved from yeast to man, the identification of a yeast TATA-binding protein (TBP)-TBP-associated factor (TAFII) complex comparable to the metazoan TFIID component of the basal transcriptional machinery has remained elusive . Here, we report the isolation of a yeast TBP-TAFII complex which can mediate transcriptional activation by GAL4-VP16 in a highly purified yeast in vitro transcription system . We have cloned and sequenced the genes encoding four of the multiple yeast TAFII proteins comprising the TBP-TAFII multisubunit complex and find that they are similar at the amino acid level to both human and Drosophila TFIID subunits . Using epitope-tagging and immunoprecipitation experiments, we demonstrate that these genes encode bona fide TAF proteins and show that the yeast TBP-TAFII complex is minimally composed of TBP and seven distinct yTAFII proteins ranging in size from M(r) = 150,000 to M(r) = 25,000 . In addition, by constructing null alleles of the cloned TAF-encoding genes, we show that normal function of the TAF-encoding genes is essential for yeast cell viability. Nucleic Acids Res, 1995 Aug 25, 23(16), 3174 - 80 STD1 (MSN3) interacts directly with the TATA-binding protein and modulates transcription of the SUC2 gene of Saccharomyces cerevisiae; Tillman TS et al.; STD1 (MSN3) was isolated independently as a multicopy suppressor of mutations in the TATA-binding protein and in SNF4, suggesting that STD1 might couple the SNF1 kinase signaling pathway to the transcriptional machinery . We report here a direct physical interaction between STD1 and the TATA-binding protein (TBP), observed in vivo by the two-hybrid system and in vitro by binding studies . STD1 bound both native TBP in yeast cell-free extracts and purified recombinant TBP . This interaction was altered when TBP delta 57 was used, suggesting a role for the non-conserved N-terminal domain of TBP in mediating protein-protein interactions . We also show that perturbation of STD1-TBP stoichiometry alters SUC2 expression in vivo and that this effect is dependent on the N-terminal domain of TBP . The activation of SUC2 expression by increased copy number of STD1 occurs at the level of mRNA accumulation and it requires the same TATA element and uses the same transcription start site as does activation of SUC2 by glucose limitation . Taken together, these results suggest that STD1 modulates SUC2 transcription through direct interactions with TBP. J Mol Biol, 1995 Aug 18, 251(3), 334 - 45 The efficiency of translation termination is determined by a synergistic interplay between upstream and downstream sequences in Saccharomyces cerevisiae; Bonetti B et al.; In a recent study we found that the efficiency of translation termination could be decreased several hundred fold by altering the local sequence context surrounding stop codons in the yeast Saccharomyces cerevisiae . Suppression of termination was shown to be mediated by near-cognate tRNA mispairing with the termination codon . We have now examined in greater detail how the local sequence context affects the efficiency of translation termination in this organism . Our results indicate that the sequence immediately upstream of the termination codon plays a significant role in determining the efficiency of translation termination . An extended termination sequence (containing the stop codon and the following three nucleotides) was also found to be a major determinant of termination efficiency, with effects attributable to the fourth nucleotide being largely independent of the termination codon . For the UGA and UAA stop codons, the influence of the fourth position on termination efficiency (from most efficient to least efficient termination) was found to be G > U,A > C, while for the UAG codon it was U,A > C > G . These sequence-specific effects on the efficiency of translation termination suggest that polypeptide chain release factor (or another molecule that may play a role in translation termination, such as rRNA) recognizes an extended termination sequence in yeast . A previous study found a statistically significant bias toward certain tetranucleotide sequences (containing the stop codon and the first distal nucleotide) in several organisms . We found that tetranucleotide sequences most frequently used in yeast are among the most efficient at mediating translation termination, while rare tetranucleotide sequences mediate much less efficient termination . Taken together, our results indicate that upstream and downstream components of an extended sequence context act synergistically to determine the overall efficiency of translation termination in yeast. EMBO J, 1995 Aug 15, 14(16), 4031 - 43 The product of the nuclear gene PET309 is required for translation of mature mRNA and stability or production of intron-containing RNAs derived from the mitochondrial COX1 locus of Saccharomyces cerevisiae; Manthey GM et al.; Expression of the Saccharomyces cerevisiae mitochondrial COX1 locus, which contains several introns and is co-transcribed with the downstream genes AAP1, OLI2 and ENS2, is controlled by at least 18 nuclear-encoded proteins . The PET309 gene, encoding one of these proteins, was cloned, sequenced and shown to contain an open reading frame of 965 codons . Isonuclear PET309+ and delta pet309::URA3 strains carrying mitochondrial genomes that differ in the number of COX1 introns, were generated . Analysis of RNA species from these strains demonstrated an inverse relationship between the number of introns present in the precursor RNA and the amount of COX1 and AAP1/OLI2/ENS2 RNAs accumulated in a pet309 mutant . Hence, PET309 plays a role either in transcription of intron-containing primary transcripts from the COX1-AAP1-OLI2-ENS2 transcription unit or in stabilization of primary transcripts . PET309 is also required in translation of COX1 mRNA . A mitochondrial bypass suppressor of the pet309 deletion mutation was isolated, and shown to consist of a DNA rearrangement at the COX1 locus, such that the 5' untranslated leader region (UTR) of the COB gene was fused to COX1 at nucleotide -174 of its 5' UTR . This result suggests that Pet309p acts through the COX1 5' UTR to activate initiation of translation of the COX1 coding region. Biochem Biophys Res Commun, 1995 Aug 15, 213(2), 484 - 9 Detection and expression of the 70 kDa heat shock protein SSB1P at different temperatures in Saccharomyces cerevisiae; Iwahashi H et al.; Ssb1p and ssb2p are two members of the hsp70 family in yeast . Up to now there has been no evidence to indicate any differences between these two members of the hsp70 family, and it was suggested that ssb1p and ssb2p were 99% identical . Here we show that an antibody prepared against the C-terminal domain of human hsp71 recognizes specifically ssb1p out of the eight hsp(c)70s in Saccharomyces cerevisiae . An amino acid peptide sequence at the C-terminal end (VTATDKSTGK) is suggested to be the sequence which has high homology between ssb1p and hu-hsp71 and to be responsible for the specificity of recognition of this unique member of the hsp70 family . Using this antibody in immunoblot assays, we have determined the cellular content of ssb1p after heat shock and at different growth temperatures . Ssb1p is shown to be degraded during heat shock treatment while it shows a higher level of expression at low temperatures. Nucleic Acids Res, 1995 Aug 11, 23(15), 2980 - 7 Structure-function analysis of the DNA binding domain of Saccharomyces cerevisiae ABF1; Cho G et al.; To localize the DNA binding domain of the Saccharomyces cerevisiae Ars binding factor 1 (ABF1), a multifunctional DNA binding protein, plasmid constructs carrying point mutations and internal deletions in the ABF1 gene were generated and expressed in Escherichia coli . Normal and mutant ABF1 proteins were purified by affinity chromatography and their DNA binding activities were analyzed . The substitution of His61, Cys66 and His67 respectively, located in the zinc finger motif in the N-terminal region (amino acids 40-91), eliminated the DNA binding activity of ABF1 protein . Point mutations in the middle region of ABF1, specifically at Leu353, Leu399, Tyr403, Gly404, Phe410 and Lys434, also eliminated or reduced DNA binding activity . However, the DNA binding activity of point mutants of Ser307, Ser496 and Glu649 was the same as that of wild-type ABF1 protein and deletion mutants of amino acids 200-265, between the zinc finger region and the middle region (residues 323-496) retained DNA binding activity . As a result, we confirmed that the DNA binding domain of ABF1 appears to be bipartite and another DNA binding motif, other than the zinc finger motif, is situated between amino acid residues 323 and 496. J Mol Biol, 1995 Aug 11, 251(2), 229 - 36 Novel mutagenic properties of abasic sites in Saccharomyces cerevisiae; Gibbs PE et al.; Abasic sites are particularly important in mutation research because they are frequently the ultimate lesion in chemical mutagenesis, and because they are believed to be a paradigm for non-pairing lesions . Although preferential insertion of dAMP ("A-rule") opposite the lesion has been observed in almost all previous studies with other organisms, we find that in budding yeast, Saccharomyces cerevisiae, the preferred nucleotide is dCMP, suggesting that yeast has a "C-rule", at least with respect to the vector constructs used . These constructs contained a single abasic site specifically located within a 28 nucleotide single-stranded region in an otherwise duplex vector . Nucleotide insertions were determined by sequence analysis of replicated vectors taken from a random set of yeast transformants . In three different sequence contexts, the frequencies of dCMP and dAMP insertion were 83% and 13%, 62% and 31%, and 85% and 8%, respectively . A similar bias in favor of cytosine insertion was found using vectors that were entirely single-stranded . However, a preference for dAMP insertion was found when Escherichia coli, rather than yeast, was transfected with samples of the same gapped duplex vector DNA . Preferential insertion of dCMP is not likely to have arisen by previously proposed mechanisms, but is also unlikely to have occurred by a primer/template misalignment mechanism, in which a nearby template guanine directs the insertion of cytosine . Predominant dCMP insertion was observed even when template guanine bases were excluded from a region extending 19 nucleotides 5', and 13 nucleotides 3', to the abasic site. J Biol Chem, 1995 Aug 11, 270(32), 18774 - 80 Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by CTP; McDonough VM et al.; In the yeast Saccharomyces cerevisiae, the major membrane phospholipid phosphatidylcholine is synthesized by the CDP-diacylglycerol and CDP-choline pathways . We examined the regulation of phosphatidylcholine synthesis by CTP . The cellular concentration of CTP was elevated (2.4-fold) by overexpressing CTP synthetase, the enzyme responsible for the synthesis of CTP . The overexpression of CTP synthetase resulted in a 2-fold increase in the utilization of the CDP-choline pathway for phosphatidylcholine synthesis . The increase in CDP-choline pathway usage was not due to an increase in the expression of any of the enzymes in this pathway . CDP-choline, the product of the phosphocholine cytidylyltransferase reaction, was the limiting intermediate in the CDP-choline pathway . The apparent Km of CTP (1.4 mM) for phosphocholine cytidylyltransferase was 2-fold higher than the cellular concentration of CTP (0.7 mM) in control cells . This provided an explanation of why the overexpression of CTP synthetase caused an increase in the cellular concentration of CDP-choline . Phosphatidylserine synthase activity was reduced in cells overexpressing CTP synthetase . This was not due to a transcriptional repression mechanism . Instead, the decrease in phosphatidylserine synthase activity was due, at least in part, to a direct inhibition of activity by CTP . These results show that CTP plays a role in the regulation of the pathways by which phosphatidylcholine is synthesized . This regulation includes the supple of CTP for the phosphocholine cytidylyltransferase reaction in the CDP-choline pathway and the inhibition of the phosphatidylserine synthase reaction in the CDP-diacylglycerol pathway. Gene, 1995 Aug 8, 161(1), 81 - 5 Cloning and characterization of the adenine phosphoribosyltransferase-encoding gene (APT1) from Saccharomyces cerevisiae; Alfonzo JD et al.; We have cloned, sequenced and characterized the APT1 (adenine phosphoribosyltransferase) gene from Saccharomyces cerevisiae . The APT1 sequence includes an open reading frame encoding 221 amino acids and is contained within a 1322-bp insert that complements APRT-deficient mutants to wild-type levels of enzyme activity . Analysis by primer extension revealed multiple transcription start points (tsp) and a major tsp 21-bp upstream from the ATG start codon . A transcript initiated at the major tsp would yield a 700-nt mRNA which is in agreement with the size observed by Northern analysis . Sequence comparison indicates that the yeast enzyme shares strong similarities with other known APRT of bacterial, invertebrate, plant and mammalian origins. Mol Microbiol, 1995 Aug, 17(4), 653 - 62 A genomic locus in Saccharomyces cerevisiae with four genes up-regulated by osmotic stress; Miralles VJ et al.; A locust within chromosome XIII of Saccharomyces cerevisiae containing four genes upregulated by osmotic stress has been characterized . Two of the genes, but not their osmotic induction, were already described: the DNA damage-inducible gene DDR48 and the protease inhibitor gene PAI3 . The two novel genes encode a cytoplasmic aldehyde dehydrogenase (ALD2) and a peptide of unknown function (SIP18) . These genes form a cluster of two pairs of divergent promoters regulated by osmotic stress . The regulation of the divergent ALD2 and DDR48 genes, however, occurs by different mechanisms . ALD2 exhibits maximum induction with 0.3 M NaCl, negative regulation by protein kinase A and dependence on PBS2 and HOG1 protein kinases for osmotic induction . DDR48 requires 1 M NaCl for maximum induction and its expression in independent of PBS2 and HOG1 protein kinases and less sensitive to protein kinase A . PAI3 and SIP18 are as dependent on the above protein kinases as ALD2 . Deletion analysis indicates that most of the regulation of the ALD2 promoter is mediated by a negative element counteracted by osmotic stress. J Biochem (Tokyo), 1995 Aug, 118(2), 302 - 11 Molecular cloning of CWP1: a gene encoding a Saccharomyces cerevisiae cell wall protein solubilized with Rarobacter faecitabidus protease I; Shimoi H et al.; A yeast cell wall glycoprotein with a molecular weight of 40,000, named gp40, was solubilized from SDS-extracted cell wall of Saccharomyces cerevisiae by incubation with Rarobacter faecitabidus protease I, which is a yeast-lytic enzyme . Based on its amino acid sequence, we cloned and sequenced the gene encoding the precursor of gp40, named CWP1; cell wall protein gene . The DNA sequence of the CWP1 gene was identical to YKL443, an open reading frame identified in a genome sequencing program for yeast chromosome XI . This gene encoded a serine-rich protein of 239 amino acids with a molecular weight of 24,267 . The presence of hydrophobic sequences in the N- and C-termini of the CWP1 protein suggests that it is secreted as a glycosylphosphatidylinositol-anchored protein and is subsequently integrated into the cell wall . Since a gene disruption experiment showed no growth defect, the CWP1 gene is not essential for growth . Mutant CWP1 protein deficient in the C-terminal hydrophobic sequence was secreted into the culture medium, not anchored to the cell wall, thereby indicating that this hydrophobic sequence plays a crucial role in anchoring to the cell wall . Homology between the CWP1 protein and TIP1 family of cold shock proteins suggests that they belong to a new family of cell wall proteins. Biochem Mol Biol Int, 1995 Aug, 36(6), 1217 - 23 Effect of trehalose during stress in a heat-shock resistant mutant of Saccharomyces cerevisiae; Eleutherio EC et al.; Cells of a heat-shock resistant mutant were approximately 1000-times more resistant to lethal heat shock than those of the parental strain . We observed that exponentially growing cells of the mutant synthesized trehalose and showed increased osmotolerance, dehydration tolerance an ethanol tolerance, a fact not observed in wild type strains . The mutant synthesizes constitutively six proteins, among them two proteins of 56 and 63 kDa . Interestingly these molecular weights could correspond to the subunit of trehalose-6-phosphate synthase and to phosphoglucomutase II, respectively . Our results showed that glucose-growing cells of the hsr 1 mutant possessed high levels of activity of these enzymes when compared to the control strain. Yeast, 1995 Aug, 11(10), 961 - 6 New open reading frames, one of which is similar to the nifV gene of Azotobacter vinelandii, found on a 12.5 kbp fragment of chromosome IV of Saccharomyces cerevisiae; Verhasselt P et al.; The nucleotide sequence of a 12.5 kbp segment of the left arm of chromosome IV is described . Five open reading frames (ORFs) longer than 100 amino acids were detected, all of which are completely confined to the 12.5 kbp region . Two ORFs (D1271 and D1286) correspond to previously sequenced genes (PPH22 and VMA1 or TFP1, respectively) . ORF D1298 shows the characteristics of alpha-isopropylmalate and homocitrate synthase genes and is similar to the nifV gene of Azotobacter vinelandii . Two more ORFs have no apparent homologue in the data libraries . Conversely, two smaller ORFs of 25 and 85 amino acids encoding the ribosomal protein YL41A and an ATPase inhibitor, respectively, were detected . Although a substantial part of the 12.5 kbp fragment apparently lacks protein-coding characteristics, no other elements, such as tRNA genes or transposons, were found. Yeast, 1995 Aug, 11(10), 913 - 28 Targeting of heterologous membrane proteins into proliferated internal membranes in Saccharomyces cerevisiae; Wittekindt NE et al.; Overproduction of chimeric proteins containing the HMG2/1 peptide, which comprises the seven transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl-CoA reductase isozymes 1 and 2, has previously been observed to induce the proliferation of internal endoplasmic reticulum-like membranes . In order to exploit this amplified membrane surface area for the accommodation of heterologous microsomal proteins, we fused sequences coding for human cytochrome P4501A1 (CYP1A1) to sequences encoding the HMG2/1 peptide and expressed the hybrid genes in yeast . The heterologous hybrid proteins were targeted into strongly proliferated membranes, as shown by electron microscopic and immunofluorescent analysis . Fusion proteins comprising the whole CYP1A1 polypeptide (HMG2/1-CYP1A1) exhibited 7-ethoxyresorufin-O-deethylase activity, whereas fusion proteins lacking the N-terminal 56 amino acids of CYP1A1 (HMG2/1-delta CYP1A1) were inactive and appeared to be unable to incorporate protoheme . Similar amounts of heterologous protein were detected in cells expressing HMG2/1-CYP1A1, HMG2/1-delta CYP1A1 and CYP1A1, respectively . Replacement of the N-terminal membrane anchor domain of human NADPH-cytochrome P450 oxidoreductase by the HMG2/1 peptide also resulted in a functional fusion enzyme, which was able to interact with HMG2/1-CYP1A1 and the yeast endogenous P450 enzyme lanosterol-14 alpha-demethylase. Curr Genet, 1995 Aug, 28(3), 258 - 66 MIG1-dependent and MIG1-independent glucose regulation of MAL gene expression in Saccharomyces cerevisiae; Hu Z et al.; Glucose repression is a global regulatory system in Saccharomyces cerevisiae controlling carbon-source utilization, mitochondrial biogenesis, gluconeogenesis and other metabolic pathways . Mig1p, a zinc-finger class of DNA-binding protein, is a transcriptional repressor regulating GAL and SUC gene expression in response to glucose . This report demonstrates that Mig1 protein represses transcription of the MAL61 and MAL62 structural genes and also the MAL63 gene, which encodes the Mal-activator . Mig1p DNA-binding sites were identified upstream of all three MAL genes . Both of the Mig1p-binding sites found in the bidirectional MAL61-MAL62 promoter were shown to function in the Mig1p-dependent glucose repression . Studies using constitutive Mal-activator alleles suggest that glucose regulation of inducer availability is a second major contributing factor in glucose repression of MAL gene expression and is even stronger than the Mig1p-dependent component of repression . Moreover, our results also suggest the contribution of other minor mechanisms in glucose regulation of MAL gene expression. Protein Expr Purif, 1995 Aug, 6(4), 537 - 45 Multicopy overexpression of bovine pancreatic trypsin inhibitor saturates the protein folding and secretory capacity of Saccharomyces cerevisiae; Parekh R et al.; Bovine pancreatic trypsin inhibitor (BPTI) was expressed and secreted from a synthetic gene as a model system for the study of protein folding and secretion in Saccharomyces cerevisiae . The efficiency of different leader sequences in directing BPTI secretion was examined, and up to 11 micrograms/ml of active BPTI was secreted . In some fusion constructs, inefficient proteolytic processing by Kex2p, Ste13p, and signal peptidase were observed immediately adjacent to the BPTI N terminus . Insertion of dipeptide spacers improved endoproteolytic processing substantially but the level of secretion was unchanged . Overexpression from a 2-microns multicopy vector results in essentially unchanged BPTI secretion as compared to expression from a single copy centromere vector . BPTI expressed from a multicopy vector accumulates intracellularly in an unfolded form, indicating that available secretory chaperones and foldases can be saturated by increasing the rate of BPTI synthesis. FEMS Microbiol Lett, 1995 Aug 1, 130(2-3), 307 - 12 Hydrostatic pressure promotes the acidification of vacuoles in Saccharomyces cerevisiae; Abe F et al.; Application of hydrostatic pressure caused a delay or cessation of cell growth in Saccharomyces cerevisiae . The yeast vacuole is an acidic organelle involved in cellular ion homeostasis and degradation of proteins . Hydrostatic pressure promoted the acidification of the vacuoles in the strain IFO 2347 . A pressure of 40 to 60 MPa reduced the vacuolar pH, defined using 6-carboxyfluorescein, from 6.05 to 5.88, while a pressure of 20 MPa did not affect the pH . Similar results were obtained with the strain X2180 . Bafilomycin A1, a specific inhibitor of vacuolar H(+)-ATPase (V-H(+)-ATPase), caused a significant alkalization of vacuoles in the strain X2180 . The pHs rose to 7.34 and 6.84 at both atmospheric pressure and a pressure of 40 MPa, respectively . Meanwhile, vacuolar accumulation of the weak base quinacrine was increased by a pressure of 40 MPa, suggesting that uptake of the dye was induced by the increased pH gradient across the vacuolar membrane. Eur J Biochem, 1995 Aug 1, 231(3), 704 - 13 ACS2, a Saccharomyces cerevisiae gene encoding acetyl-coenzyme A synthetase, essential for growth on glucose; Van den Berg MA et al.; In Saccharomyces cerevisiae, the conversion of pyruvate to acetyl-coenzyme A may proceed directly via the pyruvate dehydrogenase complex (PDH) or indirectly via the so-called PDH bypass, which requires the sequential action of pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-coenzyme A synthetase . The relative contribution of both pathways to the rate of acetyl-coenzyme A synthesis varies in an unknown way with cultural conditions . To determine the possible role of acetyl-coenzyme A synthetase in this central part of metabolism, we have analyzed the genes encoding this enzyme . Disruption of the recently cloned ACS1 gene {De Virgilio, C., Burckert, N., Barth, G., Neuhaus, J., Boller, T . & Wiemken, A . (1992) Yeast 8, 1043-1051} did not cause an apparent phenotype, except for a prolonged lag-phase during growth on glucose or C2 compounds such as acetate and ethanol . In fact, a product from a different gene is responsible for acetyl-coenzyme A formation in the acs1 mutant . We cloned a second gene encoding acetyl-coenzyme A synthetase, which we called ACS2 . Inactivation of this gene caused inability to grow on media containing glucose, but not on media with acetate or ethanol as the sole carbon source . This indicates that ACS2 is essential for growth on glucose in batch cultures . The acs1-acs2 double mutant was not viable . The role of both genes in glucose metabolism and acetate or ethanol metabolism is discussed. Exp Cell Res, 1995 Aug, 219(2), 477 - 86 Expression of the first N-glycosylation gene in the dolichol pathway, ALG7, is regulated at two major control points in the G1 phase of the Saccharomyces cerevisiae cell cycle; Pretel R et al.; The Saccharomyces cerevisiae ALG7 gene, which functions by initiating the dolichol pathway of protein N-glycosylation, displays properties of an early growth-response gene . To initiate studies of the involvement of ALG7 in cellular proliferation, we have now more precisely analyzed ALG7 expression in the G1 phase of cell cycle . We show that the rapid rate of ALG7 mRNA accumulation following growth stimulation was attenuated soon thereafter and that ALG7 growth induction occurred irrespective of alpha-factor . ALG7 growth induction was observed in mutants conditionally defective for reentry into the cell cycle from the stationary phase, indicating that the induction occurred prior to the performance of START . In addition, the steady-state levels of ALG7 mRNAs declined four-fold in response to START-I cell division arrest brought about by alpha-factor treatment later in G1 . Importantly, deregulated expression of ALG7 resulted in an aberrant alpha-factor response . Our data not only indicate that ALG7 expression is regulated at two critical control points in G1 that determine the proliferative potential of cells, but also provide a link between ALG7 and START. EMBO J, 1995 Aug 1, 14(15), 3788 - 99 Cdc6 is an unstable protein whose de novo synthesis in G1 is important for the onset of S phase and for preventing a 'reductional' anaphase in the budding yeast Saccharomyces cerevisiae; Piatti S et al.; S phase entry depends on cyclin-dependent kinases whose activation during late G1 due partly to the synthesis of unstable cyclin subunits . We identify here a second type of unstable protein, Cdc6, whose synthesis during G1 is important for initiation of DNA replication . The CDC6 gene is normally transcribed at the end of mitosis, but in cells with a prolonged G1 phase there is a second burst of transcription in late G1 . The former is due to Swi5, while the latter is due to MBF or SBF transcription factors . Small G1 cells that cannot synthesize Cdc6 in late G1 progress through S phase very slowly . Cells that transcribe CDC6 neither at the end of mitosis nor in late G1 fail to replicate DNA but, despite this, undergo mitosis and produce daughter cells with fractional DNA contents . This 'reductional' anaphase occurs with almost wild-type kinetics and depends on the activity of G2 cyclins . Thus, cells that fail to duplicate chromosomes due to a cdc6 defect cannot prevent the onset of mitosis, unlike other mutants with replication defects . We show, by fluorescence in situ hybridization, that chromosomes which remain unduplicated due to a lack of Cdc6 synthesis are segregated intact to spindle poles during the 'reductional' anaphase. J Bacteriol, 1995 Aug, 177(15), 4517 - 9 Different internal metabolites trigger the induction of glycolytic gene expression in Saccharomyces cerevisiae; Muller S et al.; In the yeast Saccharomyces cerevisiae, the sugar-induced expression of various genes coding for glycolytic enzymes is triggered by increases in the concentrations of different internal metabolites . Here, we show that the induction of the glycolytic isoenzyme enolase 2 is strictly dependent on the abilities of different mutant strains to increase the level of glucose-6-phosphate after the addition of sugars . In contrast, the induction of alcohol dehydrogenase I is dependent on increasing concentrations of metabolites in the late stages of glycolysis. Mol Cell Biol, 1995 Aug, 15(8), 3998 - 4008 The essential helicase gene RAD3 suppresses short-sequence recombination in Saccharomyces cerevisiae; Bailis AM et al.; We have isolated an allele of the essential DNA repair and transcription gene RAD3 that relaxes the restriction against recombination between short DNA sequences in Saccharomyces cerevisiae . Double-strand break repair and gene replacement events requiring recombination between short identical or mismatched sequences were stimulated in the rad3-G595R mutant cells . We also observed an increase in the physical stability of double-strand breaks in the rad3-G595R mutant cells . These results suggest that the RAD3 gene suppresses recombination involving short homologous sequences by promoting the degradation of the ends of broken DNA molecules. J Cell Biol, 1995 Aug, 130(3), 687 - 700 Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae; Yeh E et al.; We have used time-lapse digital- and video-enhanced differential interference contrast (DE-DIC, VE-DIC) microscopy to study the role of dynein in spindle and nuclear dynamics in the yeast Saccharomyces cerevisiae . The real-time analysis reveals six stages in the spindle cycle . Anaphase B onset appears marked by a rapid phase of spindle elongation, simultaneous with nuclear migration into the daughter cell . The onset and kinetics of rapid spindle elongation are identical in wild type and dynein mutants . In the absence of dynein the nucleus does not migrate as close to the neck as in wild-type cells and initial spindle elongation is confined primarily to the mother cell . Rapid oscillations of the elongating spindle between the mother and bud are observed in wild-type cells, followed by a slower growth phase until the spindle reaches its maximal length . This stage is protracted in the dynein mutants and devoid of oscillatory motion . Thus dynein is required for rapid penetration of the nucleus into the bud and anaphase B spindle dynamics . Genetic analysis reveals that in the absence of a functional central spindle (ndcl), dynein is essential for chromosome movement into the bud . Immunofluorescent localization of dynein-beta-galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies and the cell cortex: with spindle pole body localization dependent on intact microtubules . A kinetic analysis of nuclear movement also revealed that cytokinesis is delayed until nuclear translocation is completed, indicative of a surveillance pathway monitoring nuclear transit into the bud. J Cell Biol, 1995 Aug, 130(3), 567 - 77 Functional characterization of Ost3p . Loss of the 34-kD subunit of the Saccharomyces cerevisiae oligosaccharyltransferase results in biased underglycosylation of acceptor substrates; Karaoglu D et al.; Within the lumen of the rough endoplasmic reticulum, oligosaccharyltransferase catalyzes the en bloc transfer of a high mannose oligosaccharide moiety from the lipid-linked oligosaccharide donor to asparagine acceptor sites in nascent polypeptides . The Saccharomyces cerevisiae oligosaccharyltransferase was purified as a heteroligomeric complex consisting of six subunits (alpha-zeta) having apparent molecular masses of 64 kD (Ost1p), 45 kD (Wbp1p), 34 kD, 30 kD (Swp1p), 16 kD, and 9 kD . Here we report a structural and functional characterization of Ost3p which corresponds to the 34-kD gamma-subunit of the oligosaccharyltransferase . Unlike Ost1p, Wbp1p, and Swp1p, expression of Ost3p is not essential for viability of yeast . Instead, ost3 null mutant yeast grow at wild-type rates on solid or in liquid media irrespective of culture temperature . Nonetheless, detergent extracts prepared from ost3 null mutant membranes are twofold less active than extracts prepared from wild-type membranes in an in vitro oligosaccharyltransferase assay . Furthermore, loss of Ost3p is accompanied by significant underglycosylation of soluble and membrane-bound glycoproteins in vivo . Compared to the previously characterized ost1-1 mutant in the oligosaccharyltransferase, and the alg5 mutant in the oligosaccharide assembly pathway, ost3 null mutant yeast appear to be selectively impaired in the glycosylation of several membrane glycoproteins . The latter observation suggests that Ost3p may enhance oligosaccharide transfer in vivo to a subset of acceptor substrates. Mutat Res, 1995 Aug, 347(3-4), 129 - 33 Low environmental radiation background impairs biological defence of the yeast Saccharomyces cerevisiae to chemical radiomimetic agents; Satta L et al.; Background radiation is likely to constitute one of the factors involved in biological evolution since radiations are able to affect biological processes . Therefore, it is possible to hypothesize that organisms are adapted to environmental background radiation and that this adaptation could increase their ability to respond to the harmful effects of ionizing radiations . In fact, adaptive responses to alkylating agents and to low doses of ionizing radiation have been found in many organisms . In order to test for effects of adaptation, cell susceptibility to treatments with high doses of radiomimetic chemical agents has been studied by growing them in a reduced environmental radiation background . The experiment has been performed by culturing yeast cells (Saccharomyces cerevisiae D7) in parallel in a standard background environment and in the underground Gran Sasso National Laboratory, with reduced environmental background radiation . After a conditioning period, yeast cells were exposed to recombinogenic doses of methyl methanesulfonate . The yeast cells grown in the Gran Sasso Laboratory showed a higher frequency of radiomimetic induced recombination as compared to those grown in the standard environment . This suggests that environmental radiation may act as a conditioning agent. EMBO J, 1995 Aug 1, 14(15), 3820 - 7 The Saccharomyces cerevisiae translation initiation factor Tif3 and its mammalian homologue, eIF-4B, have RNA annealing activity; Altmann M et al.; The Saccharomyces cerevisiae TIF3 gene encodes the yeast homologue of mammalian translation initiation factor eIF-4B . We have added six histidine residues to the C-terminus of Tif3 protein (Tif3-His6p) and purified the tagged protein by affinity chromatography . Tif3-His6p stimulates translation and mRNA binding to ribosomes in a Tif3-dependent in vitro system . Furthermore, it binds to single-stranded RNA and catalyses the annealing of partially complementary RNA strands in vitro . In parallel experiments, RNA annealing activity could also be demonstrated for mammalian eIF-4B . A role for Tif3/eIF-4B and RNA annealing activity in the scanning process is proposed. Genetics, 1995 Aug, 140(4), 1213 - 22 Genetics of the synthesis of serine from glycine and the utilization of glycine as sole nitrogen source by Saccharomyces cerevisiae; Sinclair DA et al.; Saccharomyces cerevisiae can grow on glycine as sole nitrogen source and can convert glycine to serine via the reaction catalyzed by the glycine decarboxylase multienzyme complex (GDC) . Yeast strains with mutations in the single gene for lipoamide dehydrogenase (lpd1) lack GDC activity, as well as the other three 2-oxoacid dehydrogenases dependent on this enzyme . The LPD1 gene product is also required for cells to utilize glycine as sole nitrogen source . The effect of mutations in LPD1 (L-subunit of GDC), SER1 (synthesis of serine from 3-phosphoglycerate), ADE3 (cytoplasmic synthesis of one-carbon units for the serine synthesis from glycine), and all combinations of each has been determined . The results were used to devise methods for isolating mutants affected either in the generation of one-carbon units from glycine (via GDC) or subsequent steps in serine biosynthesis . The mutants fell into six complementation groups (gsd1-6 for defects in conversion of glycine to serine) . Representatives from three complementation groups were also unable to grow on glycine as sole nitrogen source (gsd1-3) . Assays of the rate of glycine uptake and decarboxylation have provided insights into the nature of the mutations. Biochem Biophys Res Commun, 1995 Jul 26, 212(3), 854 - 60 A role for the Gal11 protein in pheromone-induced transcription in Saccharomyces cerevisiae; Dolan JW et al.; The product of the Saccharomyces cerevisiae GAL11 gene, Gal11p, is required for the proper expression of a wide range of genes including many mating-type-specific genes . In this study Gal11p is shown to have a role in the induction of transcription by pheromone . Basal transcription of an a-specific gene is unaffected by loss of Gal11p but pheromone treatment fails to increase transcription . A haploid-specific gene is shown to retain pheromone-inducibility in the absence of Gal11p but the extent of induction is drastically reduced . Evidence is also presented that suggests the existence of strain differences that can alter the phenotype of gal11 mutants. FEBS Lett, 1995 Jul 24, 368(3), 505 - 8 The mature AEP2 gene product of Saccharomyces cerevisiae, required for the expression of subunit 9 of ATP synthase, is a 58 kDa mitochondrial protein; Finnegan PM et al.; The nucleotide sequence of the yeast nuclear AEP2 gene, required for the expression of the mitochondrial DNA-encoded subunit 9 of ATP synthase, predicts a primary translation product of 67.5 kDa . The ATP13 gene is allelic to AEP2 but was reported to encode a protein of about 42 kDa in size . We thus investigated genetically and biochemically the size of the AEP2 gene product . Genetic complementation assays using 3' truncated AEP2 genes, here shows that function is abolished by the removal of only 32 amino acids from the C-terminus of the predicted protein product . Cell-free translation of AEP2 produces a 64 kDa polypeptide (consistent with the AEP2 sequence) which is imported into mitochondria and processed to a 58 kDa product by the removal of a presequence of about 50 amino acids. Mol Gen Genet, 1995 Jul 22, 248(1), 59 - 68 Regulation of the Saccharomyces cerevisiae Srs2 helicase during the mitotic cell cycle, meiosis and after irradiation; Heude M et al.; The expression of the SRS2 gene, which encodes a DNA helicase involved in DNA repair in Saccharomyces cerevisiae, was studied using an SRS2-lacZ fusion integrated at the chromosomal SRS2 locus . It is shown here that this gene is expressed at a low level and is tightly regulated . It is cell-cycle regulated, with induction probably being coordinated with that of the DNA-synthesis genes, which are transcribed at the G1-S boundary . It is also induced by DNA-damaging agents, but only during the G2 phase of the cell cycle; this distinguishes it from a number of other repair genes, which are inducible throughout the cycle . During meiosis, the expression of SRS2 rises at a time nearly coincident with commitment to recombination . Since srs2 null mutants are radiation sensitive essentially when treated in G1, the mitotic regulation pattern described here leads us to postulate that either secondary regulatory events limit Srs2 activity of G1 cells or Srs2 functions in a repair mechanism associated with replication. J Biol Chem, 1995 Jul 21, 270(29), 17139 - 47 A mutation affecting signal peptidase inhibits degradation of an abnormal membrane protein in Saccharomyces cerevisiae; Mullins C et al.; Signal peptidase removes amino-terminal signal peptides from precursor proteins during or immediately following their translocation to the lumen of the endoplasmic reticulum (ER) and may participate in ER degradation, a poorly defined process whereby abnormal proteins are rapidly degraded early in the secretory pathway . Here, the involvement of signal peptidase in ER degradation is examined through the use of two chimeric membrane proteins that lack amino-terminal signal peptides: A189invHD, which contains sequences derived from arginine permease and histidinol dehydrogenase, and AHDK2, containing the ER-resident protein Kar2p fused to the carboxyl terminus of A189invHD . Degradation of approximately 95% of A189invHD is observed in yeast cells expressing enzymatically active signal peptidase, whereas only 60% undergoes rapid degradation in a sec11 mutant bearing a temperature-sensitive mutation in the gene encoding the 18-kDa subunit (Sec11p) of the signal peptidase complex . AHDK2 is proteolyzed in a reaction yielding at least two fragments in wild-type cells and in the sec11 mutant containing a plasmid bearing the SEC11 gene . The proteolytic reaction is catalyzed in a temperature-dependent manner in the sec11 mutant, with AHDK2 remaining stable at the nonpermissive temperature . Using conditional mutants defective in protein translocation into and out of the ER and in vitro protease protection studies, the site of degradation for AHDK2 is localized to the ER lumen . The data therefore indicate (i) A189invHD is degraded through both signal peptidase-dependent and independent processes; (ii) signal peptidase, specifically the Sec11p subunit, is required for the proteolysis of AHDK2; and (iii) the Kar2 fragment at the carboxyl terminus of AHDK2 permits detection of proteolytic intermediates. EMBO J, 1995 Jul 17, 14(14), 3472 - 9 Peroxisomal and mitochondrial carnitine acetyltransferases of Saccharomyces cerevisiae are encoded by a single gene; Elgersma Y et al.; Carnitine acetyltransferase (CAT) is present in mitochondria and peroxisomes of oleate-grown Saccharomyces cerevisiae . Both proteins are encoded by the same gene, YCAT, which encodes a protein with a mitochondrial targeting signal (MTS) at the N-terminus, and a peroxisomal targeting signal type 1 (PTS-1) at the C-terminus . Deletion of both motifs revealed the presence of an additional internal targeting sequence . Import of CAT via this internal signal was shown to be dependent on PAS10, a protein which is required for the import of PTS-1 containing proteins . An interaction of PAS10 with this internal targeting signal was demonstrated using the yeast two-hybrid technique . Expression of the YCAT gene behind a heterologous promoter resulted in loss of peroxisomal targeting, indicating that differential targeting is controlled at transcriptional or translational level . Determination of the 5'-ends of YCAT mRNAs revealed that YCAT transcripts initiating after the first AUG were present in oleate-grown cells . These transcripts were virtually absent in acetate- or glycerol-grown cells . We propose that in response to oleate, shorter transcripts are produced from which the peroxisomal form of CAT is translated, resulting in a CAT protein without a MTS, which can be targeted to peroxisomes. EMBO J, 1995 Jul 17, 14(14), 3349 - 64 A human virus protein, poliovirus protein 2BC, induces membrane proliferation and blocks the exocytic pathway in the yeast Saccharomyces cerevisiae; Barco A et al.; Inducible synthesis of poliovirus protein 2BC in Saccharomyces cerevisiae arrests cell growth in the G2 phase of the cell cycle, while no effects are observed upon expression of poliovirus genes 2B or 2C, either individually or in combination . Expression of 2BC induces a number of morphological modifications in yeast cells, one of the most striking being the proliferation of small membranous vesicles that fill most of the cytoplasm . These vesicles are morphologically similar to the cytopathic vacuoles that proliferate during the infection of human cells by poliovirus . The transport and processing of several yeast proteins, including vacuolar carboxypeptidase Y, aminopeptidase I or yeast alpha-mating factor, is hampered upon expression of poliovirus 2BC, suggesting that transport of proteins through the Golgi apparatus is impaired by this viral protein . Finally, a number of 2BC variants were generated and the effects of their expression on yeast growth, cellular morphology and protein processing were analyzed . 2BC variants defective in the NTPase activity were still able to interfere with yeast growth and the exocytic system, while deletion of 30 amino acids at the N-terminus of 2BC impairs its function . These findings lend support to the idea that 2BC, but not 2B or 2C, is the protein responsible for vesicle proliferation in poliovirus-infected cells . In addition, the activity of a human virus protein in yeast cells opens new avenues to investigate the exact location at which poliovirus 2BC interferes with the vesicular system and to test the action of other animal virus proteins potentially involved in modifying the vesicular system in mammalian cells. Genes Dev, 1995 Jul 15, 9(14), 1728 - 39 MSH5, a novel MutS homolog, facilitates meiotic reciprocal recombination between homologs in Saccharomyces cerevisiae but not mismatch repair; Hollingsworth NM et al.; Using a screen designed to identify yeast mutants specifically defective in recombination between homologous chromosomes during meiosis, we have obtained new alleles of the meiosis-specific genes, HOP1, RED1, and MEK1 . In addition, the screen identified a novel gene designated MSH5 (MutS Homolog 5) . Although Msh5p exhibits strong homology to the MutS family of proteins, it is not involved in DNA mismatch repair . Diploids lacking the MSH5 gene display decreased levels of spore viability, increased levels of meiosis I chromosome nondisjuction, and decreased levels of reciprocal exchange between, but not within, homologs . Gene conversion is not reduced . Msh5 mutants are phenotypically similar to mutants in the meiosis-specific gene MSH4 (Ross-Macdonald and Roeder 1994) . Double mutant analysis using msh4 msh5 diploids demonstrates that the two genes are in the same epistasis group and therefore are likely to function in a similar process--namely, the facilitation of interhomolog crossovers during meiosis. Eur J Biochem, 1995 Jul 15, 231(2), 329 - 36 Use of monoclonal antibodies in the functional characterization of the Saccharomyces cerevisiae Sep1 protein; Holler A et al.; The Saccharomyces cerevisiae strand-exchange protein 1 (Sep1 also known as Xrn1, Kem1, Rar5, Stp beta/DST2) has been demonstrated to mediate the formation of hybrid DNA from model substrates of linear double-stranded and circular single-stranded DNA in vitro . To delineate the mechanism by which Sep1 acts in the strand-exchange reaction, we analyzed mouse anti-Sep1 monoclonal antibodies for inhibition of the Sep1 in vitro activity . Of 12 class-G immunoglobulins tested, four were found to consistently inhibit the Sep1-mediated strand-exchange reaction . The inhibiting antibodies were tested for inhibition of a variety of Sep1-catalyzed DNA reactions including exonuclease activity on double-stranded and single-stranded DNA, renaturation of complementary single-stranded DNA and condensation of DNA into large aggregates . All four inhibiting antibodies had no effect on the exonuclease activity of Sep1 . Three antibodies specifically blocked DNA aggregation . In addition, one antibody inhibited renaturation of complementary single-stranded DNA . This inhibition pattern underlines the importance of condensation of DNA into large aggregates in conjunction with double-stranded DNA exonuclease activity for the in vitro homologous pairing activity of Sep1 . The implications of these data for the interpretation of proteins which promote homologous pairing of DNA are discussed, in particular in light of the reannealing activity of the p53 human tumor-suppressor protein. Nucleic Acids Res, 1995 Jul 11, 23(13), 2548 - 54 Characterization of three new snRNAs from Saccharomyces cerevisiae: snR34, snR35 and snR36; Samarsky DA et al.; Genes for three novel snRNAs of Saccharomyces cerevisiae have been isolated, sequenced and tested for essentiality . The RNAs encoded by these genes are designated snR34, snR35 and snR36 respectively and contain 203, 204 and 182 nucleotides . Each RNA is derived from a single copy gene and all three RNAs are believed to be nucleolar, i.e . snoRNAs, based on extraction properties and association with fibrillarin . SnR34 and snR35 contain a trimethylguanosine cap, but this feature is absent from snR36 . The novel RNAs lack elements conserved among several other snoRNAs, including box C, box D and long sequence complementarities with rRNA . Genetic disruption analyses showed each of the RNAs to be dispensable and a haploid strain lacking all three RNAs and a previously characterized fourth snoRNA (snR33) is also viable . No differences in the levels of precursors or mature rRNAs were apparent in the four gene knock-out strain . Possible roles for the new RNAs in ribosome biogenesis are discussed. Arch Biochem Biophys, 1995 Jul 10, 320(2), 305 - 14 3-Hexaprenyl-4-hydroxybenzoic acid forms a predominant intermediate pool in ubiquinone biosynthesis in Saccharomyces cerevisiae; Poon WW et al.; The biosynthesis of ubiquinone (coenzyme Q) was studied in Saccharomyces cerevisiae . Lipid extracts were prepared from wild-type yeast grown in the presence of p-{U-14C}- and p-{carboxy-14C}hydroxybenzoic acid . Ergosterol was removed by adsorption to digitonin-celite, and radiolabeled lipids were purified by sequential reverse-phase and normal-phase HPLC steps . Radiolabeled peaks were identified by comparison with synthetic standards using retention time and electron ionization mass spectrometric criteria . The recovery and identification of the unstable 3-hexaprenyl-4-hydroxybenzoic acid molecule were facilitated by treatment of the lipid extract with diazomethane under conditions that resulted in the formation of the stable derivatives methyl 3-hexaprenyl-4-hydroxybenzoate or methyl 3-hexaprenyl-4-methoxybenzoate . In stationary-phase yeast cultures, the major radioactive lipid products are coenzyme Q and 3-hexaprenyl-4-hydroxybenzoic acid, constituting 62 and 38% of the radioactive lipids, respectively . However, under log-phase growth conditions the biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid predominates (accounting for 81% of the radioactive lipids) . The data indicate that in wild-type yeast, 3-hexaprenyl-4-hydroxybenzoic acid forms a predominant intermediate pool in ubiquinone biosynthesis and that in log-phase growth this ubiquinone intermediate is present at fourfold higher abundance than the end product . The physiological rationale for this high concentration of a membrane-bound intermediate is unclear. J Biol Chem, 1995 Jul 7, 270(27), 16153 - 9 ABF1 Ser-720 is a predominant phosphorylation site for casein kinase II of Saccharomyces cerevisiae; Upton T et al.; ABF1 is a multifunctional phosphoprotein that binds specifically to yeast origins of replication and to transcriptional regulatory sites of a variety of genes . We isolated a protein kinase from extracts of Saccharomyces cerevisiae on the basis of its ability to specifically phosphorylate the ABF1 protein . Physical and biochemical properties of this kinase identify it as casein kinase II (CKII) . The purified kinase has a high affinity for the ABF1 substrate as indicated by a relatively low Km value . Furthermore, when incubated with ABF1 and anti-ABF1 antibodies, the kinase forms an immunocomplex active in the phosphorylation of ABF1 . Biochemical and genetic mapping localized a major site for phosphorylation at Ser-720 near the C terminus of the ABF1 protein . This serine is embedded within a domain enriched for acidic amino acid residues . A Ser-720 to Ala mutation abolishes phosphorylation by CKII in vitro . The same mutation also abolishes phosphorylation of this site in vivo, suggesting that CKII phosphorylates Ser-720 in vivo as well . Although three CKII enzymes, yeast, sea star, and recombinant human, utilize casein as a substrate with similar efficiencies, only the yeast enzyme efficiently phosphorylates the ABF1 protein . This suggests that ABF1 is a specific substrate of the yeast CKII and that this specificity may reside within one of the beta regulatory subunits of the enzyme . Thus, phosphorylation of ABF1 by yeast CKII may prove to be a useful system for studying targeting mechanisms of CKII to a physiological substrate. J Biol Chem, 1995 Jul 7, 270(27), 16063 - 9 5'-exonuclease-2 of Saccharomyces cerevisiae . Purification and features of ribonuclease activity with comparison to 5'-exonuclease-1; Stevens A et al.; 5'-Exonuclease-2 has been purified 17,000-fold from whole cell extracts of Saccharomyces cerevisiae . A 116-kDa polypeptide parallels the enzyme activity when the purified protein is examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate . Amino-terminal sequencing of the 116-kDa protein shows that the sequence agrees with that encoded by the HKE1 gene, previously reported to encode exonuclease-2 . A 45-kDa polypeptide also parallels the enzyme activity upon purification, and Sephacryl S-200 molecular sieve chromatography of the purified enzyme shows a parallel elution of most of the 116- and 45-kDa polypeptides, suggesting a close association of the two . Enzyme instability has precluded a more detailed analysis of their associative properties . The enzyme hydrolyzes RNA substrates to 5'-mononucleotides in a processive manner . Measurements of its substrate specificity and mode of action are compared with 5'-exonuclease-1 . Restriction cut single-stranded T7 DNA is hydrolyzed at approximately 5-7% of the rate of 18 S rRNA of yeast by both enzymes . That 5'-exonuclease-2 hydrolyzes in a processive manner and lacks endonuclease activity is shown by the finding that {5'-32P}GMP is the only product of its hydrolysis of {alpha-32P}GTP-labeled synthetic RNAs . That 5'-exonuclease-2 hydrolyzes by a 5'-->3' mode is shown by: 1) its poor hydrolysis of both 5'-capped and triphosphate-ended RNA substrates; 2) the products of its hydrolysis of {5'-32P,3H}(pA)4; and 3) the accumulation of 3'-stall fragments when a strong artificial RNA secondary structure is present in synthetic RNAs . 5'-Exonuclease-1 hydrolyzes the synthetic RNAs and (pA)4 in an identical manner. J Biol Chem, 1995 Jul 7, 270(27), 15984 - 92 Molecular characterization of Ste20p, a potential mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase kinase from Saccharomyces cerevisiae; Wu C et al.; The Ste20p protein kinase was immunopurified from yeast cells and analyzed in an in vitro assay system . Ste20p immune complexes exhibited autophosphorylating activity at serine and threonine residues and specifically phosphorylated a bacterially expressed glutathione S-transferase (GST) fusion of Ste11p (a mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase homologue) at serine and threonine residues . In contrast, GST fusions either of Ste7p (a MEK homologue) or the beta-subunit of the mating response G-protein and immunoprecipitated Ste5p were not phosphorylated by the Ste20p immune complexes . Myelin basic protein was identified as an excellent in vitro substrate, whereas histone H1 was only poorly phosphorylated . Evidence was obtained that autophosphorylation might play a regulatory role for the in vitro kinase activity . The in vitro activity was found to be Ca(2+)-independent . Both the in vivo and in vitro activities were abolished by mutational changes of either the conserved lysine residue 649 within the ATP binding site or threonine 777 between the catalytic subdomains VII and VIII . Wild-type Ste20p and the catalytically inactive T777A mutant were identified as phosphoproteins in vivo . The phosphorylation occurred at serine and threonine residues independent of pheromone stimulation . Based on the genetically determined significance of Ste20p in pheromone signal transduction and on our in vitro studies, we propose the model that Ste20p represents a yeast MEK kinase kinase whose function is to link G-protein-coupled receptors through G beta gamma to a mitogen-activated protein kinase module. EMBO J, 1995 Jul 3, 14(13), 3105 - 15 Nin1p, a regulatory subunit of the 26S proteasome, is necessary for activation of Cdc28p kinase of Saccharomyces cerevisiae; Kominami K et al.; The nin1-1 mutant of Saccharomyces cerevisiae cannot perform the G1/S and G2/M transitions at restrictive temperatures . At such temperatures, nin1-1 strains fail to activate histone H1 kinase after release from alpha factor-imposed G1 block and after release from hydroxyurea-imposed S block . The nin1-1 mutation shows synthetic lethality with certain cdc28 mutant alleles such as cdc28-IN . Two lines of evidence indicate that Nin1p is a component of the 26S proteasome complex: (i) Nin1p, as well as the known component of the 26S proteasome, shifted to the 26S proteasome peak in the glycerol density gradient after preincubation of crude extract with ATP-Mg2+, and (ii) nin1-1 cells accumulated polyubiquitinated proteins under restrictive conditions . These results suggest that activation of Cdc28p kinase requires proteolysis . We have cloned a human cDNA encoding a regulatory subunit of the 26S proteasome, p31, which was found to be a homolog of Nin1p. FEBS Lett, 1995 Jul 3, 367(3), 219 - 22 Glucose-induced inactivation of isocitrate lyase in Saccharomyces cerevisiae is mediated by an internal decapeptide sequence; Ordiz I et al.; In this work we have investigated the role of specific peptide sequences for glucose-inactivation of the yeast isocitrate lyase . Thus, different fragments of the ICL1 coding region were fused to the lacZ gene of E . coli to provide a reporter construction . Determinations of beta-galactosidase activities indicated that the decapeptide sequence KTKRNYSARD, located between amino acid residues 37 and 46 of isocitrate lyase, is important for glucose induced proteolytic inactivation . Further experimental evidence was provided by insertion of this sequence into a glucokinase-beta-galactosidase fusion protein, which is not sensitive to glucose regulation . The decapeptide inserted conferred glucose inactivation to this construct, confirming that it is both necessary and sufficient as a signal. J Biochem (Tokyo), 1995 Jul, 118(1), 39 - 45 Cloning and sequence of the SCS2 gene, which can suppress the defect of INO1 expression in an inositol auxotrophic mutant of Saccharomyces cerevisiae; Nikawa J et al.; Saccharomyces cerevisiae ire15 mutant has a defect in the expression of INO1, showing the inositol auxotrophic phenotype {Nikawa, J . (1994) Gene 149, 367-372} . We have isolated five yeast genes which suppress the ire15 mutation in multiple copies by genetic complementation . Among them, one gene, designated as SCS2, also suppressed the choline-sensitive dominant mutation, CSE1 {Hosaka, K . et al . (1992) J . Biochem . 111, 352-358} . The CSE1 mutation is not allelic to ire15 . Sequencing analysis revealed that the SCS2 gene encodes 244 amino acid residues with a calculated molecular mass of 26,866 . INO2/SCS1, which is another suppressor gene for CSE1 and is known to be a positive factor for INO1 expression, also suppressed the growth defect of the ire15 mutant . These results clearly indicate that the ire15 and CSE1 mutations genetically interact and the SCS2 and INO2/SCS1 genes are involved in the regulation of INO1 expression. J Biochem (Tokyo), 1995 Jul, 118(1), 168 - 77 Expression of rat cathepsin D cDNA in Saccharomyces cerevisiae: implications for intracellular targeting of cathepsin D to vacuoles; Nishimura Y et al.; To investigate the intracellular transport mechanisms of lysosomal cathepsin D in yeast cells, we produced cathepsin D in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene . Immunoblotting analysis by the use of an antibody specific for rat cathepsin D coding sequence produced an intermediate species which had a slightly higher molecular weight than that of the mature cathepsin D . Cell fractionation experiments demonstrated that the cathepsin D polypeptide was colocalized to the yeast vacuoles with the marker enzyme carboxypeptidase Y in a Ficoll step gradient . A biosynthesis study with pulse-chase kinetic analysis revealed that the precursor polypeptide was accurately sorted to the yeast vacuoles as determined by cell fractionation, and that N-linked carbohydrate modifications were not required for vacuolar sorting of this protein . To elucidate the role of the propeptide region of cathepsin D, which might function in the intracellular targeting to the vacuole, a deletion mutant of cathepsin D lacking the propeptide was prepared and its intracellular targeting was examined after transfection into yeast cells . Immunoblotting analysis demonstrated that the propeptide-deleted mutant protein was recovered in a low quantity as compared with that in the case of yeast cells expressing the wild-type protein in the isolated vacuolar fraction . Immunofluorescence analysis revealed that the deletion mutant protein appeared to be accumulated within the intracellular small vesicles but not in the carboxypeptidase Y-positive vacuoles . Overall, these results indicate that the rat cathepsin D precursor polypeptide is recognized by mechanisms similar to those involved in the intracellular sorting of vacuolar proteins through the ER/Golgi/vacuolar sorting pathway in yeast cells, and that the propeptide has an important function in translocation of the cathepsin D polypeptide to the vacuole. Mol Cell Biol, 1995 Jul, 15(7), 3934 - 44 A 15-base-pair element activates the SPS4 gene midway through sporulation in Saccharomyces cerevisiae; Hepworth SR et al.; Sporulation of the yeast Saccharomyces cerevisiae represents a simple developmental process in which the events of meiosis and spore wall formation are accompanied by the sequential activation of temporally distinct classes of genes . In this study, we have examined expression of the SPS4 gene, which belongs to a group of genes that is activated midway through sporulation . We mapped the upstream boundary of the regulatory region of SPS4 by monitoring the effect of sequential deletions of 5'-flanking sequence on expression of plasmid-borne versions of SPS4 introduced into a MATa/MAT alpha delta sps4/delta sps4 strain . This analysis indicated that the 5' boundary of the regulatory region was within 50 bp of the putative TATA box of the gene . By testing various oligonucleotides that spanned this boundary and the downstream sequence for their ability to activate expression of a heterologous promoter, we found that a 15-bp sequence sufficed to act as a sporulation-specific upstream activation sequence . This 15-bp fragment, designated UASSPS4, activated expression of a CYC1-lacZ reporter gene midway through sporulation and was equally active in both orientations . Extending the UAS fragment to include the adjacent 14-bp enhanced its activity 10-fold . We show that expression of SPS4 is regulated in a manner distinct from that of early meiotic genes: mutation of UME6 did not lead to vegetative expression of SPS4, and sporulation-specific expression was delayed by mutation of IME2 . In vivo and in vitro assays suggested that a factor present in vegetative cells bind to the UASSPS4 element . We speculate that during sporulation this factor is modified to serve as an activator of the SPS4 gene or, alternatively, that it recruits an activator to the promoter. Mol Cell Biol, 1995 Jul, 15(7), 3635 - 43 Inhibition of G-protein signaling by dominant gain-of-function mutations in Sst2p, a pheromone desensitization factor in Saccharomyces cerevisiae; Dohlman HG et al.; Genetic analysis of cell-cell signaling in Saccharomyces cerevisiae has led to the identification of a novel factor, known as Sst2p, that promotes recovery after pheromone-induced growth arrest (R . K . Chan and C . A . Otte, Mol . Cell . Biol . 2:11-20, 1982) . Loss-of-function mutations lead to increased pheromone sensitivity, but this phenotype is partially suppressed by overexpression of the G protein alpha subunit gene (GPA1) . Suppression is allele specific, however, suggesting that there is direct interaction between the two gene products . To test this model directly, we isolated and characterized several dominant gain-of-function mutants of SST2 . These mutations block the normal pheromone response, including a loss of pheromone-stimulated gene transcription, cell cycle growth arrest, and G protein myristoylation . Although the SST2 mutations confer a pheromone-resistant phenotype, they do not prevent downstream activation by overexpression of G beta (STE4), a constitutively active G beta mutation (STE4Hpl), or a disruption of GPA1 . None of the SST2 alleles affects the expression or stability of G alpha . These results point to the G protein alpha subunit as being the direct target of Sst2p action and underscore the importance of this novel desensitization factor in G-protein-mediated signaling. Mol Cell Biol, 1995 Jul, 15(7), 3608 - 17 Disturbance of normal cell cycle progression enhances the establishment of transcriptional silencing in Saccharomyces cerevisiae; Laman H et al.; Previous studies have indicated that mutation of RAP1 (rap1s) or of the HMR-E silencer ARS consensus element leads to metastable repression of HMR . A number of extragenic suppressor mutations (sds, suppressors of defective silencing) that increase the fraction of repressed cells in rap1s hmr delta A strains have been identified . Here we report the cloning of three SDS genes . SDS11 is identical to SWI6, a transcriptional regulator of genes required for DNA replication and of cyclin genes . SDS12 is identical to RNR1, which encodes a subunit of ribonucleotide reductase . SDS15 is identical to CIN8, whose product is required for spindle formation . We propose that mutations in these genes improve the establishment of silencing by interfering with normal cell cycle progression . In support of this idea, we show that exposure to hydroxyurea, which increases the length of S phase, also restores silencing in rap1s hmr delta A strains . Mutations in different cyclin genes (CLN3, CLB5, and CLB2) and two cell cycle transcriptional regulators (SWI4 and MBP1) also suppress the silencing defect at HMR . The effect of these cell cycle regulators is not specific to the rap1s or hmr delta A mutation, since swi6, swi4, and clb5 mutations also suppress mutations in SIR1, another gene implicated in the establishment of silencing . Several mutations also improve the efficiency of telomeric silencing in wild-type strains, further demonstrating that disturbance of the cell cycle has a general effect on position effect repression in Saccharomyces cerevisiae . We suggest several possible models to explain this phenomenon. Genetics, 1995 Jul, 140(3), 933 - 43 Sodium orthovanadate-resistant mutants of Saccharomyces cerevisiae show defects in Golgi-mediated protein glycosylation, sporulation and detergent resistance; Kanik-Ennulat C et al.; Orthovanadate is a small toxic molecule that competes with the biologically important oxyanion orthophosphate . Orthovanadate resistance arises spontaneously in Saccharomyces cerevisiae haploid cells by mutation in a number of genes . Mutations selected at 3 nM sodium orthovanadate have different degrees of vanadate resistance, hygromycin sensitivity, detergent sensitivity and sporulation defects . Recessive vanadate-resistant mutants belong to at least six genetic loci . Most mutants are defective in outer chain glycosylation of secreted invertase (van1, van2, van4, van5, van6, VAN7-116 and others), a phenotype found in some MNN or VRG mutants . The phenotypes of these vanadate-resistant mutants are consistent with an alteration in the permeability or specificity of the Golgi apparatus . The previously published VAN1 gene product has a 200 amino acid domain with 40% identity with the MNN9 gene product and 70% identity with the ANP1 gene product . Cells containing the van1-18, mnn9 (vrg6) or anp1 mutations have some phenotypic similarities . The VAN2 gene was isolated and its coding region is identified and reported . It is an essential gene on chromosome XV and its translated amino acid sequence predicts a unique 337 amino acid protein with multiple transmembrane domains. Nat Genet, 1995 Jul, 10(3), 261 - 8 Analysis of the nucleotide sequence of chromosome VI from Saccharomyces cerevisiae; Murakami Y et al.; The complete nucleotide sequence of Saccharomyces cerevisiae chromosome VI (270 kb) has revealed that it contains 129 predicted or known genes (300 bp or longer) . Thirty-seven (28%) of which have been identified previously . Among the 92 novel genes, 39 are highly homologous to previously identified genes . Local sequence motifs were compared to active ARS regions and inactive loci with perfect ARS core sequences to examine the relationship between these motifs and ARS activity . Additional ARS sequences were predominantly observed in 3' flanking sequences of active ARS loci. Genes Dev, 1995 Jul 1, 9(13), 1559 - 71 The protein kinase C-activated MAP kinase pathway of Saccharomyces cerevisiae mediates a novel aspect of the heat shock response; Kamada Y et al.; The PKC1 gene of budding yeast encodes a homolog of the alpha, beta, and gamma isoforms of mammalian PKC that is proposed to regulate a MAPK-activation pathway . Mutants in this pathway undergo cell lysis resulting from a deficiency in cell wall construction when they attempt to grow at elevated temperatures . We show that the PKC1-regulated pathway is important for induced thermotolerance and that the MPK1 protein kinase (the MAPK of this pathway) is strongly activated by mild heat shock . This activation is sustained during growth at high temperature and is dependent on the function of pathway components proposed to function upstream of MPK1, including PKC1 . Expression of genes under the control of known heat shock-inducible promoter elements (HSEs and STREs) was not compromised in PKC1 pathway mutants, indicating that this pathway mediates a novel aspect of the yeast heat shock response . We propose that the heat-induced signal for pathway activation is generated in response to weakness in the cell wall created during growth under thermal stress, perhaps as a result of increased membrane fluidity . Evidence is presented that the mechanism by which the cell detects this weakness is by measuring stretch of the plasma membrane. Mutat Res, 1995 Jul, 343(4), 185 - 99 Induction of chromosome loss in Saccharomyces cerevisiae strain D61.M by selected benzimidazole compounds; Goin CJ et al.; Twenty-two benzimidazole compounds were tested for induction of chromosome loss (CHRL) in the diploid yeast Saccharomyces cerevisiae strain D61.M . Six compounds tested positive for CHRL induction: mebendazole, albendazole, RS-9237-000, fenbendazole, 2-benzimidazolylacetonitrile, and thiabendazole . Mebendazole, albendazole, RS-9237-000, and fenbendazole were strongly positive only after modified testing media were used to enhance solubility . The compounds that tested negative for CHRL were 2-phenylbenzimidazole, 2-(2-pyridyl)benzimidazole, benzimidazole, 2-aminobenzimidazole, 2-amino-5,6-dimethylbenzimidazole, 2-(aminomethyl)benzimidazole dihydrochloride hydrate, 5,6-dimethylbenzimidazole, 2-guanidinobenzimidazole, 2-methylbenzimidazole, 2-(methylmercapto) benzimidazole, 1-methyl-2-phenylbenzimidazole, 2-benzimidazolylurea, RS-65255-000, oxibendazole, and RS-95005-000 . One chemical, cambendazole, tested negative or only marginally positive . Modified testing medium was also used to enhance the solubility of 2-phenylbenzimidazole, oxibendazole, and RS-95005-000 . Because no toxicity was observed with oxibendazole or RS-95005-000, the negative results obtained with these two compounds could not be considered definitive. Biochem J, 1995 Jul 1, 309 ( Pt 1), 321 - 4 Aminoacyl-tRNA synthetase complex in Saccharomyces cerevisiae; Harris CL et al.; The size distribution of aminoacyl-tRNA synthetase activity was investigated in cell extracts prepared from Saccharomyces cerevisiae . Bio-Gel A-5M chromatography of 105,000 g supernatants separated isoleucyl-tRNA synthetase activity into three peaks, with apparent molecular masses (Da) of about 100,000, 350,000 and 10(6) or greater . Similar results were obtained with synthetases specific for glutamic acid, serine and tyrosine . Sucrose-density-gradient centrifugation of yeast supernatants also provided evidence for the existence of synthetase complexes . These data provide the first evidence for the existence of a high-molecular-mass aminoacyl-tRNA synthetase complex in yeast, perhaps similar to those reported in higher eukaryotes. J Cell Biol, 1995 Jul, 130(2), 299 - 312 Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae; TerBush DR et al.; In the yeast Saccharomyces cerevisiae, the products of at least 14 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane . Two of these genes, SEC8 and SEC15, encode components of a 1-2-million D multi-subunit complex that is found in the cytoplasm and associated with the plasma membrane . In this study, oligonucleotide-directed mutagenesis is used to alter the COOH-terminal portion of Sec8 with a 6-histidine tag, a 9E10 c-myc epitope, or both, to allow the isolation of the Sec8/15 complex from yeast lysates either by immobilized metal affinity chromatography or by immunoprecipitation . Sec6 cofractionates with Sec8/15 by immobilized metal affinity chromatography, gel filtration chromatography, and by sucrose velocity centrifugation . Sec6 and Sec15 coimmunoprecipitate from lysates with c-myc-tagged Sec8 . These data indicate that the Sec8/15 complex contains Sec6 as a stable component . Additional proteins associated with Sec6/8/15 were identified by immunoprecipitations from radiolabeled lysates . The entire Sec6/8/15 complex contains at least eight polypeptides which range in molecular mass from 70 to 144 kD . Yeast strains containing temperature sensitive mutations in the SEC genes were also transformed with the SEC8-c-myc-6-histidine construct and analyzed by immunoprecipitation . The composition of the Sec6/8/15 complex is disrupted specifically in the sec3-2, sec5-24, and sec10-2 strain backgrounds . The c-myc-Sec8 protein is localized by immunofluorescence to small bud tips indicating that the Sec6/8/15 complex may function at sites of exocytosis. J Bacteriol, 1995 Jul, 177(14), 4190 - 3 Recognition of nitrogen-responsive upstream activation sequences of Saccharomyces cerevisiae by the product of the GLN3 gene; Blinder D et al.; We describe the purification of the product of the GLN3 gene of Saccharomyces cerevisiae and the demonstration that the purified product, Gln3p, binds specifically to the DNA sequences GATAAG and GATTAG, previously identified as nitrogen-responsive upstream activation sequences (UASN) . When Gln3p is overproduced, it is released from the cells in a highly aggregated form incapable of specific binding to UASN . We used Gln3p tagged with six histidine codons at the 5' terminus and equipped with a galactose-inducible promoter to overproduce histidine-tagged Gln3p . The material was denatured, adsorbed to an Ni-nitrilotriacetic acid (NTA)-agarose column, eluted with imidazole, and after renaturation further purified on a gel filtration column . We then demonstrated the specific binding of the more than 90% pure Gln3p to the UASN by gel shift and footprinting methods. Mol Biol Cell, 1995 Jul, 6(7), 889 - 909 Phosphorylation and localization of Kss1, a MAP kinase of the Saccharomyces cerevisiae pheromone response pathway; Ma D et al.; Kss1 protein kinase, and the homologous Fus3 kinase, are required for pheromone signal transduction in Saccharomyces cerevisiae . In MATa haploids exposed to alpha-factor, Kss1 was rapidly phosphorylated on both Thr183 and Tyr185, and both sites were required for Kss1 function in vivo . De novo protein synthesis was required for sustained pheromone-induced phosphorylation of Kss1 . Catalytically inactive Kss1 mutants displayed alpha-factor-induced phosphorylation on both residues, even in kss1 delta cells; hence, autophosphorylation is not obligatory for these modifications . In kss1 delta fus3 delta double mutants, Kss1 phosphorylation was elevated even in the absence of pheromone; thus, cross-phosphorylation by Fus3 is not responsible for Kss1 activation . In contrast, pheromone-induced Kss1 phosphorylation was eliminated in mutants deficient in two other protein kinases, Ste11 and Ste7 . A dominant hyperactive allele of STE11 caused a dramatic increase in the phosphorylation of Kss1, even in the absence of pheromone stimulation, but required Ste7 for this effect, suggesting an order of function: Ste11-->Ste7-->Kss1 . When overproduced, Kss1 stimulated recovery from pheromone-imposed G1 arrest . Catalytic activity was essential for Kss1 function in signal transmission, but not for its recovery-promoting activity . Kss1 was found almost exclusively in the particulate material and its subcellular fractionation was unaffected by pheromone treatment . Indirect immunofluorescence demonstrated that Kss1 is concentrated in the nucleus and that its distribution is not altered detectably during signaling. Mol Biol Cell, 1995 Jul, 6(7), 793 - 807 Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C; Meluh PB et al.; The MIF2 gene of Saccharomyces cerevisiae has been implicated in mitosis . Here we provide genetic evidence that MIF2 encodes a centromere protein . Specifically, we found that mutations in MIF2 stabilize dicentric minichromosomes and confer high instability (i.e., a synthetic acentric phenotype) to chromosomes that bear a cis-acting mutation in element I of the yeast centromeric DNA (CDEI) . Similarly, we observed synthetic phenotypes between mutations in MIF2 and trans-acting mutations in three known yeast centromere protein genes-CEP1/CBF1/CPF1, NDC10/CBF2, and CEP3/CBF3B . In addition, the mif2 temperature-sensitive phenotype can be partially rescued by increased dosage of CEP1 . Synthetic lethal interactions between a cep1 null mutation and mutations in either NDC10 or CEP3 were also detected . Taken together, these data suggest that the Mif2 protein interacts with Cep1p at the centromere and that the yeast centromere indeed exists as a higher order protein-DNA complex . The Mif2 and Cep1 proteins contain motifs of known transcription factors, suggesting that assembly of the yeast centromere is analogous to that of eukaryotic enhancers and origins of replication . We also show that the predicted Mif2 protein shares two short regions of homology with the mammalian centromere Ag CENP-C and that two temperature-sensitive mutations in MIF2 lie within these regions . These results provide evidence for structural conservation between yeast and mammalian centromeres. Microbiology, 1995 Jul, 141 ( Pt 7), 1559 - 66 Constitutive glucose-induced activation of the Ras-cAMP pathway and aberrant stationary-phase entry on a glucose-containing medium in the Saccharomyces cerevisiae glucose-repression mutant hex2; Dumortier F et al.; Addition of glucose to cells of the yeast Saccharomyces cerevisiae growing on a nonfermentable carbon source triggers a rapid, transient increase in the cAMP level . The occurrence of this cAMP spike appears to be correlated inversely with the glucose-repression state of the cells . This was also observed for the hex2 mutant, which is deficient in glucose repression and which displayed the cAMP signal constitutively . When cells of the hex2 mutant were starved for nitrogen on a glucose-containing medium, they rapidly lost viability, similarly to mutants with overactivation of the Ras-adenylate cyclase pathway . Flow cytometry measurements showed that G1 arrest of the hex2 mutant under such conditions was incomplete . Trehalose accumulation, a typical feature of cells entering the stationary phase G0, was very short-lived in the hex2 mutant under the same conditions . These results are in agreement with the presence of continuous glucose-triggered activation of cAMP synthesis in hex2 cells on a glucose-containing nitrogen-starvation medium . In the course of these experiments a spontaneous suppressor mutant, shx (for suppressor of hex2), was isolated which survived nitrogen starvation on a glucose-containing medium much better than the hex2 strain . It also showed normal G1 arrest and much longer accumulation of trehalose . The suppressor mutation also caused inability to grow on nonfermentable carbon sources and absence of invertase depression, and it was epistatic to hex2 for these characteristics also . The isolation of this epistatic depression mutation supports the idea that the defect in glucose repression of the hex2 mutant is the cause of its rapid loss of viability during nitrogen starvation on a glucose-containing medium.(ABSTRACT TRUNCATED AT 250 WORDS) Electrophoresis, 1995 Jul, 16(7), 1170 - 4 A Saccharomyces cerevisiae Internet protein resource now available; Latter GI et al.; The QUEST Protein Database Center is now making available two Saccharomyces cerevisiae protein databases via the Internet . The yeast electrophoretic protein database (YEPD) is a database of approximately one hundred protein identifications on two-dimensional gels . The yeast protein database (YPD) is a database of gene names and properties of over 3500 yeast proteins of known sequence . These databases can be accessed via a World-Wide Web (WWW) server (URL YPD is available via public ftp (isis.cshl.org) as well, in a spreadsheet format, and in ASCII format . When accessed via WWW, both of these databases have hypertext links to other biological data, such as the SWISS-PROT protein sequence database and the Saccharomyces Genome Database (SacchDB), and to each other. Yeast, 1995 Jul, 11(9), 885 - 90 A new essential gene located on Saccharomyces cerevisiae chromosome IX; Kurlandzka A et al.; A new 1150 amino acids long open reading frame (ORF), coding for an essential protein of unknown function was found in Saccharomyces cerevisiae by sequencing 3754 bp of geonomic DNA . The clone was isolated in a search for a fatty acid-binding protein (FABP) and was localized on chromosome IX . The ORF bears no homology to FABP, but it shows weak similarity to Plasmodium vivax reticulocyte binding protein 1 and to aggregation-specific adenylate cyclase from Dictyostelium discoideum . The new gene is constitutively transcribed regardless of the carbon source used. Yeast, 1995 Jul, 11(9), 849 - 64 Expression and secretion of antifreeze peptides in the yeast Saccharomyces cerevisiae; Driedonks RA et al.; The antifreeze peptide AFP6 from the polar fish Pseudopleuronectus americanus has been expressed in and secreted by the yeast Saccharomyces cerevisiae as a biologically active molecule . The gene for the 37 amino acid long peptide has been chemically synthesized using yeast preferred codons . Subsequently, the gene has been cloned into an episomal expression vector as well as in a multicopy integration vector, which is mitotically more stable . The expression is under the control of the inducible GAL7 promoter . The enzyme alpha-galactosidase has been investigated as a carrier protein to facilitate expression and secretion of AFP . In order to reach increased expression levels, tandem repeats of the AFP gene (up to eight copies) have been cloned . In most cases the genes are efficiently expressed and the products secreted . The expression level amounts to approximately 100 mg/l in the culture medium . In a number of genetic constructs the genes are directly linked and expressed as AFP multimers . In other constructs linker regions have been inserted between the AFP gene copies, that allow the peptide to be processed by specific proteinases, either from the endogenous yeast proteolytic system or from a non-yeast source . The latter requires a separate processing step after yeast cultivation to obtain mature AFP . In all these cases proteolytic processing is incomplete, generating a heterogeneous mixture of mature AFP, carrier and chimeric protein, and/or a mixture of AFP-oligomers . The antifreeze activity has been demonstrated for such mixtures as well as for AFP multimers. Yeast, 1995 Jul, 11(9), 809 - 22 The Saccharomyces cerevisiae FLO1 flocculation gene encodes for a cell surface protein; Bidard F et al.; The sequencing of a 6619 bp region encoding for a flocculation gene previously cloned from a strain defined as FLO5 (Bidard et al., 1994) has revealed that it was a FLO1 gene . The FLO1 gene product has been localized at the cell surface of the yeast cell by immunofluorescent microscopy . The Flo1 protein contains four regions with repeated sequences which account for about 70% of the amino acids of this protein . A functional analysis of the major repeated region has revealed that it plays an important role in determining the flocculation level . A gene disruption experiment has shown that FLO5 strain STX 347-1D contains at least two flocculation genes of the FLO1 type but that they are supposed to be inactive and do not contribute to its flocculation . However, enzyme-linked immunosorbent assays performed on intact cells have revealed that a protein expressed at the cell surface of the FLO5 strain STX 347-1D is antigenically related to Flo1p . A deletion analysis of the 5' region of the FLO1 gene has shown that the expression is submitted to controls which depend on the genetic background of the strain. J Biol Chem, 1995 Jun 30, 270(26), 15821 - 6 Secretion and maturation study of endothiapepsin in Saccharomyces cerevisiae . A first step toward improving its substrate specificity; Valverde V et al.; The gene encoding endothiapepsin (EAP), an extracellular aspartic proteinase from the filamentous ascomycete Cryphonectria parasitica, was expressed into Saccharomyces cerevisiae . Efficient secretion of an active and correctly processed enzyme was achieved when expressing the entire cDNA encoding prepro-EAP under the control of the galactose-inducible GRAP1 yeast promoter . Since three independent, site-directed mutations of EAP, including the substitution of an aspartyl catalytic residue, resulted in the intracellular accumulation of zymogen forms, we assumed that the EAP propeptide was autocatalytically processed . As a prerequisite to further improve the specificity of EAP, we therefore attempted to bypass this self-processing step in three different ways: 1) introduction of a Kex2-like recognition site between the pro and the mature part, 2) deletion of the prosequence (pre-EAP), and 3) co-expression in trans of the pre-EAP with its preprosequence . No improvement in the secretion of mutant enzymes was obtained in any of these experiments . As an alternative, we finally replaced the EAP processing site by the chymosin cleavage sequence of kappa-casein . Such a modification remained efficient in directing the secretion of active EAP only when a putative alpha-helix structural motif was conserved at the C terminus of the pro region. Yeast, 1995 Jun 30, 11(8), 789 - 91 SSU71, encoding the largest subunit of TFIIF, is located on the right arm of chromosome VII in Saccharomyces cerevisiae; Sun ZW et al.; SSU71 (TFG1) is an essential nuclear gene encoding the largest subunit of the yeast general transcription factor TFIIF . The SSU71 gene was physically mapped to the right arm of chromosome VII, physically linked to QCR9, by hybridization of the cloned gene to CHEF and lambda clone grid blots . This assignment was confirmed by genetic mapping . A search of the nucleotide sequence databases revealed that SSU71 is immediately adjacent to the TYS1 gene, which encodes tRNA(Tyr) synthetase . TYS1 was reported previously to lie on chromosome XV based on sequence overlap with the adjacent UBR1 gene . The mapping data reported here established that TYS1 and UBR1 do not lie on chromosome XV; rather the SSU71-TYS1-UBR1 gene cluster lies on the right arm of chromosome VII, physically linked to QCR9 and genetically linked to ade3 and ser2. Yeast, 1995 Jun 30, 11(8), 761 - 6 Sequence, map position and genome organization of the RPL17B gene, encoding ribosomal protein L17b in Saccharomyces cerevisiae; Berroteran RW et al.; Sequence analysis of the newly defined SSU81 gene revealed an adjacent open reading frame (ORF) encoding a protein whose deduced amino acid sequence is identical to that of ribosomal protein L17 . The DNA sequence of this region is different from that of the RPL17A gene and therefore represents a duplicate gene encoding L17 . We have designated this gene RPL17B . The RPL17B coding region is split by an intron that occurs in the same position (codons 14/15) as the intron in RPL17A . The RPL17B promoter region includes two TATA boxes, a canonical UASRPG motif, and several pyrimidine-rich tracts . RPL17B was mapped by CHEF and lambda clone grid hybridization blots to the right arm of chromosome V, linked to the TRP2 and RAD51 genes . A partial ORF was identified adjacent to RPL17B and SSU81 that is homologous to an ORF (designated A509) physically linked to RPL17A . This observation, and the identical position of the introns within the RPL17 genes, suggest that one RPL17 locus arose by duplication and translocation of the other. FEBS Lett, 1995 Jun 26, 367(2), 201 - 4 Intracellular transport of inositol-containing sphingolipids in the yeast, Saccharomyces cerevisiae; Hechtberger P et al.; Organelles of the early protein secretion pathway (ER, Golgi) are involved in biosynthesis and intracellular migration of the yeast sphingolipids, inositolphosphorylceramide (IPC), mannosylinositolphosphorylceramide (MIPC), and mannosyldiinositolphosphorylceramide (M(IP)2C) . Cycloheximide and nocodazole neither block biosynthesis of sphingolipids, nor ER to Golgi transport of IPC . In contrast, treatment of yeast cells with brefeldin A, which affects integrity of the Golgi, decreases formation of IPC and MIPC . Interruption of late steps of protein secretion (Golgi to plasma membrane transport) in temperature-sensitive secretory mutants prevents sphingolipids from being transported to the cell periphery. Mol Gen Genet, 1995 Jun 25, 247(6), 716 - 25 Mutations causing high basal level transcription that is independent of transcriptional activators but dependent on chromosomal position in Saccharomyces cerevisiae; Harashima S et al.; Two single (bel2 and bel4) and two double (bel3 bel7 and bel5 be16) mutations causing enhanced transcription of a gene fusion, consisting of the open reading frame of PHO5 connected to the HIS5 promoter (HIS5p) integrated at the ura3 or leu2 locus, were isolated from a gcn4-disrupted mutant of Saccharomyces cerevisiae . The PHO5 gene, encoding repressible acid phosphatase, in the HIS5p-PHO5 construct was derepressed under amino acid starved conditions by the action of the transcriptional activator Gcn4p . The bel mutants showed temperature-sensitive cell growth and/or cell aggregation . All the mutants except bel4 also showed high levels of transcription of an intact PHO5 DNA integrated at the URA3 locus in the absence of the cognate transcriptional activator, Pho4p, and in the absence of upstream activating sequences of PHO5 . The HIS5 and PHO5 genes at their original chromosomal positions were, however, not affected by the bel2 mutation . The BEL2 gene was found to be identical with SIN4/TSF3, mutations in which cause high levels of transcription of the HO and GAL genes in the absence of their respective transcriptional activators, Swi5p and Gal4p . The effect of the bel2/sin4/tsf3 mutation on PHO5 transcription was additive with the Pho4p function . Thus the effect of the bel2/sin4/tsf3 mutation is dependent on the position of PHO5 in the chromosome and independent of Pho4p and Gen4p activation. J Biol Chem, 1995 Jun 23, 270(25), 15037 - 44 VMA8 encodes a 32-kDa V1 subunit of the Saccharomyces cerevisiae vacuolar H(+)-ATPase required for function and assembly of the enzyme complex; Graham LA et al.; The isolated Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) is composed of at least 10 subunits . We have identified VMA8, the gene encoding the 32-kDa subunit of the V-ATPase, by 100% match between the sequences of tryptic peptides and the predicted protein sequence of ORF11 . The VMA8 gene contains a 768-base pair open reading frame encoding a 256-amino acid protein with a predicted molecular mass of 29,176 Da . Disruption of VMA8 resulted in a mutant exhibiting pH-sensitive growth, slowed growth under all conditions, and an inability to grow on nonfermentable carbon sources . Vacuolar membranes isolated from vma8 delta yeast cells exhibited no V-ATPase activity . Immunoblot analysis of vma8 delta cells revealed normal levels of both V1 and Vo subunits . Whereas the V1 subunits failed to associated with the vacuolar membrane in vma8 delta cells, the Vo polypeptides were transported to and stable in the vacuolar membrane . Density gradient fractionation revealed that Vma8p associated only with the fully assembled V-ATPase and did not associate with a separate lower density Vo subcomplex fraction . Finally, Vma8p was unable to assemble onto the vascular membranes in the absence of other V1 subunits. J Biol Chem, 1995 Jun 23, 270(25), 14983 - 8 Phosphorylation of CTP synthetase from Saccharomyces cerevisiae by protein kinase C; Yang WL et al.; Phosphorylation of CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) from Saccharomyces cerevisiae protein kinase C was examined . Using pure CTP by synthetase as a substrate, protein kinase C activity was dose- and time-dependent and required calcium, diacylglycerol, and phosphatidylserine for full activation . Protein kinase C activity was also dependent on the concentration of CTP synthetase . Protein kinase C phosphorylated CTP synthetase on serine and threonine residues in vitro whereas the enzyme was primarily phosphorylated on serine residues in vivo . Phosphopeptide mapping analysis of CTP synthetase phosphorylated in vitro and in vivo indicated that the enzyme was phosphorylated on more than one site . Most of the phosphopeptides derived from CTP synthetase phosphorylated in vivo were the same as those derived from CTP synthetase phosphorylated by protein kinase C in vitro . The stoichiometry of the phosphorylation of native CTP synthetase was 0.4 mol of phosphate/mol of enzyme whereas the stoichiometry of the phosphorylation of alkaline phosphatase-treated CTP synthetase was 2.2 mol of phosphate/mol of enzyme . This indicated that CTP synthetase was purified in a phosphorylated state . Phosphorylation of CTP synthetase resulted in a 3-fold activation in enzyme activity whereas alkaline phosphatase treatment of CTP synthetase resulted in a 5-fold decrease in enzyme activity . Overall, the results reported here were consistent with the conclusion that CTP synthetase was regulated by protein kinase C phosphorylation. Biochem J, 1995 Jun 15, 308 ( Pt 3), 847 - 51 Replacement of Lys by Glu in a transmembrane segment strongly impairs the function of the uracil permease from Saccharomyces cerevisiae; Urban-Grimal D et al.; The co-transport of uracil and protons through the plasma membrane of the yeast Saccharomyces cerevisiae is mediated by a specific permease encoded by the FUR4 gene . The uracil permease is a multi-spanning membrane protein that follows the secretory pathway to the plasma membrane . Recent experimental data led to the proposal of a two-dimensional model of its topology . A spontaneous mutant corresponding to the substitution of Lys-272 by glutamic acid was obtained . The influence of this mutation was studied by comparing the wild-type and mutant permeases produced in a strain carrying a chromosomal deletion of the FUR4 gene . The mutant permease is correctly targeted to the plasma membrane and its stability is similar to that of the wild-type permease . The uptake parameters for the mutant permease were impaired and showed an approximately 65-fold increase of apparent K(m) and a decrease in apparent Vmax . Equilibrium binding measurements with enriched plasma membrane preparations showed an approximately 70-fold increase in apparent Kd in the mutant, whereas its Bmax . was similar to that of the wild type . Lys-272 is fully conserved in the uracil permease family and is predicted to lie in the fourth transmembrane segment of the protein . It seems to be essential for both efficient uracil binding and translocation. Eur J Biochem, 1995 Jun 15, 230(3), 1118 - 24 Characterization of mutations in the mitochondrial cytochrome b gene of Saccharomyces cerevisiae affecting the quinone reductase site (QN); Brasseur G et al.; The revertant {G33A}cytochrome b recently isolated from the {G33D}cytochrome b mutant {Coppee, J . Y., Tokutake, N., Marc, D., di Rago, J.-P., Miyoshi, H . & Colson, A.-M . (1994) FEBS Lett . 339, 1-6} exhibits cross resistance to center-N inhibitors 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) and funiculosin and a spectral shift in the cytochrome b562 heme . This indicates that the conserved G33 residue is in the vicinity of this heme, and thus agrees with the previous suggestion that glycine may play a role in the helix packing around the hemes . The {S206L}cytochrome b and {M221K}cytochrome b respiratory-growth-deficient mutants {Lemesle-Meunier, D., Brivet-Chevillotte, P., di Rago, J . P., Slonimski, P . P., Bruel, C., Tron, T . & Forget, N . (1993) J . Biol . Chem . 268, 15,626-15,632}, which synthesize cytochrome b and retain little or no bc1 complex activity, show no change in the reduction kinetics of cytochrome b via center P, which suggests that the oxidizing site is functional . Impairment of both the reduction and oxidation of heme b562 at the ubiquinone reduction center of the mitochondrial ubiquinone-cytochrome-c oxidoreductase site is, therefore, responsible for the deficient catalytic activity and respiratory growth in these strains. Yeast, 1995 Jun 15, 11(7), 697 - 700 Mapping of the ACC1/FAS3 gene to the right arm of chromosome XIV of Saccharomyces cerevisiae; Guerra CE et al.; The ACC1/FAS3 gene has been mapped to the right arm of chromosome XIV by both genetic and physical methods . The gene is closely linked to RNA2 and is allelic to the ABP2 gene of chromosome XIV. Yeast, 1995 Jun 15, 11(7), 659 - 66 High-resolution cosmid mapping of the left arm of Saccharomyces cerevisiae chromosome XII; a first step towards an ordered sequencing approach; Scholler P et al.; For the sequencing of the left arm of chromosome XII of Saccharomyces cerevisiae, we fine-mapped the entire 450 kb fragment between the ribosomal DNA (rDNA) and the left telomere . Total yeast DNA in agarose blocks was digested with I-PpoI, which exclusively cuts once in each repeat unit of the rDNA . The resulting fragment was isolated from pulsed-field gels, together with the equally sized chromosome IX . A cosmid library of some 30-fold chromosome coverage was generated from this material, with the cloning efficiency being around 20,000 clones per microgram genomic DNA . The chromosome XII and IX specific clones were identified by complementary hybridizations with the respective chromosomes . For the left arm of chromosome XII, a contiguous cosmid array (contig) with an average map resolution better than 9 kb was generated by clone hybridization procedures . The ordered library serves as a tool for the physical mapping of genetic markers . Also, a minimal set of 15 clones was selected that covers the entire fragment . This subset forms the basis for the generation of a template map of much higher resolution for a directed sequencing of the left arm of chromosome XII. Yeast, 1995 Jun 15, 11(7), 641 - 58 The maintenance of self-replicating plasmids in Saccharomyces cerevisiae: mathematical modelling, computer simulations and experimental tests; Van der Sand ST et al.; A distributive model has been constructed to describe the maintenance of the native 2 microns and 2 micron-based plasmids in the yeast Saccharomyces cerevisiae . This model includes elements which represent the influence of selection, segregation, replication and amplification on plasmid stability . A computer program has been written in TURBO PASCAL to implement the model and a number of simulation experiments have been carried out . These simulations permitted the choice of a form of the model which is compatible with the available experimental evidence . The form chosen involves an amplification system in which the RAF gene product binds to the Rep1/Rep2 dimer to prevent the latter acting to repress the activity of the FLP gene . At the same time an upper limit (or 'ceiling') was imposed on the number of plasmid molecules able to replicate . Maternal bias was accommodated by 'tagging' a small proportion of molecules for inheritance by the mother nucleus and these tags being removed (or 'cleared') by the Rep1/Rep2 dimers . This final form of the model makes specific predictions about the stability of 2 microns and YEp plasmids in yeast populations and about the distribution of plasmid copy number between cells in such populations . The predictions on stability have been subjected to experimental test and results provide good support for the model. Yeast, 1995 Jun 15, 11(7), 601 - 13 Two-dimensional protein map of Saccharomyces cerevisiae: construction of a gene-protein index; Boucherie H et al.; This publication marks the beginning of the construction of a gene-protein index that relates proteins which are resolved on the two-dimensional protein map of Saccharomyces cerevisiae with their corresponding genes . We report the identification of 36 novel polypeptide spots on the yeast protein map . They correspond to the products of 26 genes . Together with the polypeptide spots previously identified, this raises to 41 the number of genes whose products have been identified on the protein map . The proteins identified here are concerned with four major areas of yeast cellular physiology: carbon metabolism, heat shock, amino acid biosynthesis and purine biosynthesis . Given the molecular weight and isoelectric point of the identified proteins, and the codon-usage bias of the corresponding genes, it can be estimated that 25 to 35% of all the soluble yeast proteins are detectable under the labelling and running gel conditions used in this study. Anal Biochem, 1995 Jun 10, 228(1), 143 - 9 Determination of intracellular trehalose and glycogen in Saccharomyces cerevisiae; Schulze U et al.; A simple, sensitive and nonlaborious enzyme-based method has been developed for determination of both trehalose and glycogen in yeast cells . The method is based on extraction of trehalose and glycogen into a 40 mM acetate buffer (pH 4.8) by mechanical disintegration of the cells in a bead mill . Subsequently, trehalose and glycogen can be hydrolyzed to glucose by the enzymes trehalase and amyloglycosidase, respectively . The formed glucose is quantified by a flow injection analyzer based on the enzyme glucose oxidase . The method gives results comparable to traditional methods but the simplicity of the analysis results in a much lower relative standard deviation . The excellent sensitivity of the glucose analyzer means that as little as 1 microgram trehalose or glycogen can be determined which reduces the required sample volume . This makes the method ideal for physiological studies, e.g., of transients in continuous cultures of Saccharomyces cerevisiae . In addition, a consistent procedure has been derived for pretreatment and storage of samples. J Biol Chem, 1995 Jun 9, 270(23), 13726 - 32 The Saccharomyces cerevisiae VMA10 is an intron-containing gene encoding a novel 13-kDa subunit of vacuolar H(+)-ATPase; Supekova L et al.; The vacuolar H(+)-ATPase (V-ATPase) functions as a primary proton pump that generates an electrochemical gradient of protons across the membranes of several internal organelles . It is composed of distinct catalytic and membrane sectors, each containing several subunits . We identified a protein (M16) that copurifies with the V-ATPase complex from Saccharomyces cerevisiae and appears to be present at multiple copies/enzyme . Amino acid sequencing of its proteolytic products yielded three nonoverlapping peptide sequences matching an unidentified reading frame located on chromosome VIII . Sequence analysis of cDNA encoding M16 revealed that the gene encoding this protein (VMA10) is interrupted by a 162-nucleotide intron that begins after the ATG codon of the initiator methionine . The cDNA encodes an hydrophilic protein of 12,713 Da with a basic isoelectric point of pH 9 . A delta vma10::URA3 null mutant exhibited growth characteristics typical of other vma disruptant mutants in genes encoding subunits of V-ATPase . The null mutant does not grow on medium buffered at pH 7.5 . It fails to accumulate quinacrine into its vacuole, and subunits of the catalytic sector are not assembled onto the vacuolar membrane in the absence of M16 . A cold inactivation experiment demonstrated that M16 is a subunit of the membrane sector of V-ATPase . M16 exhibits a significant sequence homology with subunit b of F-ATPase membrane sector. J Biol Chem, 1995 Jun 2, 270(22), 13171 - 8 Regulation of lipid biosynthesis in Saccharomyces cerevisiae by fumonisin B1; Wu WI et al.; The regulation of lipid biosynthesis in the yeast Saccharomyces cerevisiae by fumonisin B1 was examined . Fumonisin B1 inhibited the growth of yeast cells . Cells supplemented with fumonisin B1 accumulated free sphinganine and phytosphingosine in a dose-dependent manner . The cellular concentration of ceramide was reduced in fumonisin B1-supplemented cells . Ceramide synthase activity was found in yeast cell membranes and was inhibited by fumonisin B1 . Fumonisin B1 inhibited the synthesis of the inositol-containing sphingolipids inositol phosphorylceramide, mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide . Fumonisin B1 also caused a decrease in the synthesis of the major phospholipids synthesized via the CDP-diacylglycerol-dependent pathway and the synthesis of neutral lipids . The effects of fumonisin B1 and sphingoid bases on the activities of enzymes in the pathways leading to the synthesis of sphingolipids, phospholipids, and neutral lipids were also examined . Other than ceramide synthase, fumonisin B1 did not affect the activities of any of the enzymes examined . However, sphinganine and phytosphingosine inhibited the activities of inositol phosphorylceramide synthase, phosphatidylserine synthase, and phosphatidate phosphatase . These are key enzymes responsible for the synthesis of lipids in yeast . The data reported here indicated that the biosynthesis of sphingolipids, phospholipids and neutral lipids was coordinately regulated by fumonisin B1 through the regulation of lipid biosynthetic enzymes by sphingoid bases. Curr Genet, 1995 Jun, 28(1), 1 - 11 Functional analysis of the ABF1-binding sites within the Ya regions of the MATa and HMRa loci of Saccharomyces cerevisiae; McBroom LD et al.; Cell type in the yeast Saccharomyces cerevisiae is determined by information present at the MAT locus . Cells can switch mating types when cell-type information located at a silent locus, HML or HMR, is transposed to the MAT locus . The HML and HMR loci are kept silent through the action of a number of proteins, one of which is the DNA-binding protein, ABF1 . We have identified a binding site for ABF1 within the Ya region of MATa and HMRa . In order to examine the function of this ABF1-binding site, we have constructed strains that lack the site in the MATa or HMRa loci . Consistent with the idea that ABF1 plays a redundant role in silencing, it was found that a triple deletion of the ABF1-binding sites at HMRE, Ya and I did not permit the expression of HMRa . We have also shown that chromosomal deletion of the binding site at MATYa had no effect on the level of cutting by the HO endonuclease nor on the amount of mating-type switching observed . Similarly, chromosomal deletion of all three ABF1-binding sites at HMRa had no effect on the directionality of mating-type switching. J Cell Biol, 1995 Jun, 129(6), 1601 - 15 Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle; Winey M et al.; The three dimensional organization of microtubules in mitotic spindles of the yeast Saccharomyces cerevisiae has been determined by computer-aided reconstruction from electron micrographs of serially cross-sectioned spindles . Fifteen spindles ranging in length from 0.6-9.4 microns have been analyzed . Ordered microtubule packing is absent in spindles up to 0.8 micron, but the total number of microtubules is sufficient to allow one microtubule per kinetochore with a few additional microtubules that may form an interpolar spindle . An obvious bundle of about eight interpolar microtubules was found in spindles 1.3-1.6 microns long, and we suggest that the approximately 32 remaining microtubules act as kinetochore fibers . The relative lengths of the microtubules in these spindles suggest that they may be in an early stage of anaphase, even though these spindles are all situated in the mother cell, not in the isthmus between mother and bud . None of the reconstructed spindles exhibited the uniform populations of kinetochore microtubules characteristic of metaphase . Long spindles (2.7-9.4 microns), presumably in anaphase B, contained short remnants of a few presumed kinetochore microtubules clustered near the poles and a few long microtubules extending from each pole toward the spindle midplane, where they interdigitated with their counterparts from the other pole . Interpretation of these reconstructed spindles offers some insights into the mechanisms of mitosis in this yeast. Lett Appl Microbiol, 1995 Jun, 20(6), 353 - 6 X-ray microanalysis of chlorhexidine-treated cells of Saccharomyces cerevisiae; Hiom SJ et al.; The use of energy dispersive analysis of X-rays (EDAX) to identify and quantify the presence of chlorhexidine diacetate (CHA) within Saccharomyces cerevisiae cells was examined . Chlorine was used as the elemental marker tag . Saccharomyces cerevisiae cells exposed to 1000 micrograms ml-1 CHA took up increasing amounts of CHA over a time period of 30 s to 30 min . Electron probe micro-analysis was employed to examine the specific accumulation of CHA across the treated cells . These results showed that CHA was distributed evenly between the cell wall, cytoplasm and vacuoles of cells pre-treated with this concentration of CHA for 30 min . The EDAX system therefore provides a useful tool for examining the qualitative and quantitative effects of chlorhexidine on yeast cells, although quantitative data must be interpreted with caution. Virology, 1995 Jun 1, 209(2), 506 - 18 Sequence determination of human papillomavirus type 6a and assembly of virus-like particles in Saccharomyces cerevisiae; Hofmann KJ et al.; Human papillomavirus 6a (HPV6a), the most abundant HPV6 subtype, was detected in a vulvar condyloma acuminatum . The complete genome of HPV6a was cloned, and its DNA sequence was shown to be over 97% identical to the HPV6b sequence . Of the eight open reading frames (ORFs) of HPV6a, only the imputed amino acid sequence of the major capsid protein L1 was identical to the corresponding HPV6b sequence; all other HPV6a ORFs showed amino acid changes compared to the HPV6b ORFs . The HPV6a L1 or the L1 + L2 ORFs were expressed in the yeast Saccharomyces cerevisiae . Self-assembly of the L1 capsid protein into virus-like particles (VLPs) was demonstrated both in the L1 as well as L1 + L2 coexpressing yeast strains . Copurification of the L1 and L2 proteins showed complex formation of the L1 and L2 proteins in the yeast-derived VLPs of coexpressing strains. J Bacteriol, 1995 Jun, 177(12), 3563 - 72 Global changes in protein synthesis during adaptation of the yeast Saccharomyces cerevisiae to 0.7 M NaCl; Blomberg A; Exponentially growing Saccharomyces cerevisiae was challenged to increased salinity by transfer to 0.7 M NaCl medium, and changes in protein synthesis were examined during the 1st h of adaptation by use of two-dimensional gel electrophoresis coupled to computerized quantification . An impressive number of proteins displayed changes in the relative rate of synthesis, with most differences from nonstressed cells being found at between 20 and 40 min . During this period, 18 proteins exhibited more than eightfold increases in their rates of synthesis and were classified as highly NaCl responsive . Only two proteins were repressed to the same level . Most of these highly NaCl-responsive proteins seemed to constitute gene products not earlier reported to respond to dehydration . Applying a selection criterion to subsequent samples of a twofold change in the relative rate of synthesis, 14 different regulatory patterns were discerned . Most identified glycolytic enzymes exhibited a delayed response, and their rates of synthesis did not change until the middle phase of adaptation, with only a minor decrease in the rate of production . A slight salt-stimulated response was observed for some members of the HSP70 gene family . Overall, the data presented indicate complex intracellular signalling as well as involvement of diverse regulatory mechanisms during the period of adaptation to NaCl. J Bacteriol, 1995 Jun, 177(11), 3251 - 8 Computer-assisted nonlinear regression analysis of the multicomponent glucose uptake kinetics of Saccharomyces cerevisiae; Coons DM et al.; The kinetics of glucose uptake in Saccharomyces cerevisiae are complex . An Eadie-Hofstee (rate of uptake versus rate of uptake over substrate concentration) plot of glucose uptake shows a nonlinear form typical of a multicomponent system . The nature of the constituent components is a subject of debate . It has recently been suggested that this nonlinearity is due to either a single saturable component together with free diffusion of glucose or a single constitutive component with a variable Km, rather than the action of multiple hexose transporters . Genetic data support the existence of a family of differentially regulated glucose transporters, encoded by the HXT genes . In this work, kinetic expressions and nonlinear regression analysis, based on an improved zero trans-influx assay, were used to address the nature of the components of the transport system . The results indicate that neither one component with free diffusion nor a single permease with a variable Km can explain the observed uptake rates . Results of uptake experiments, including the use of putative alternative substrates as inhibitory compounds, support the model derived from genetic analyses of a multicomponent system with at least two components, one a high-affinity carrier and the other a low-affinity carrier . This approach was extended to characterize the activity of the SNF3 protein and identify its role in the depression of high-affinity uptake . The kinetic data support a role of SNF3 as a regulatory protein that may not itself be a transporter. J Bacteriol, 1995 Jun, 177(11), 3227 - 34 Cloning and characterization of GNS1: a Saccharomyces cerevisiae gene involved in synthesis of 1,3-beta-glucan in vitro; el-Sherbeini M et al.; The GNS1 gene product is required for the synthesis of 1,3-beta-glucan in vitro, since mutations in this gene result in exhibit an 80 to 90% reduction in 1,3-beta-glucan synthase specific activity . gns1 mutant strains display a pleiotropic phenotype including resistance to a pneumocandin B0 analog (L-733,560), slow growth, and mating and sporulation defects . The gns1-1 mutation was genetically mapped to within 1.35 centimorgans from the MAT locus on chromosome III . The wild-type GNS1 gene was isolated by complementing the pneumocandin resistance phenotype of the gns1-1 mutation and by hybridization with a chromosome III-derived sequence being used as a probe . The nucleotide sequence of GNS1 was determined and compared with the homologous region of the chromosome . The genetic and nucleotide sequence analyzes revealed that GNS1 and the open reading frame, YCR34 {S . Oliver, Q . van der Aart, M . Agostoni-Carbone, and the Chromosome III Sequencing Group, Nature (London) 357:38-46, 1992}, represent identical loci in the genome . Cells deleted for GNS1 are viable but exhibit slow growth as well as the pleiotropic phenotype of the gns1 mutants . The putative protein product is predicted to be an integral membrane protein with five transmembrane helices displaying an exoplasmic orientation for the N terminus and a cytoplasmic orientation for the C terminus . This protein may be a subunit of 1,3-beta-glucan synthase. J Bacteriol, 1995 Jun, 177(11), 3104 - 10 Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae; van der Vaart JM et al.; Three glucanase-extractable cell wall proteins from Saccharomyces cerevisiae were purified, and their N-terminal amino acid sequences were determined . With this information, we were able to assign gene products to three known open reading frames (ORFs) . The N-terminal sequence of a 55-kDa mannoprotein corresponded with the product of ORF YKL096w, which we named CWP1 (cell wall protein 1) . A 80-kDa mannoprotein was identified as the product of the TIP1 gene, and a 180-kDa mannoprotein corresponded to the product of the ORF YKL444, which we named CWP2 . CWP1, TIP1, and CWP2 encode proteins of 239, 210, and 92 amino acids, respectively . The C-terminal regions of these proteins all consist for more than 40% of serine/threonine and contain putative glycosylphosphatidylinositol attachment signals . Furthermore, Cwp1p and Tip1p were shown to carry a beta 1,6-glucose-containing side chain . The cwp2 deletion mutant displayed an increased sensitivity to Congo red, calcofluor white, and Zymolyase . Electron microscopic analysis of the cwp2 deletion mutant showed a strongly reduced electron-dense layer on the outside of the cell wall . These results indicate that Cwp2p is a major constituent of the cell wall and plays an important role in stabilizing the cell wall . Depletion of Cwp1p or Tip1p also caused increased sensitivities to Congo red and calcofluor white, but the effects were less pronounced than for cwp2 delta . All three cell wall proteins show a substantial homology with Srp1p, which also appears to be localized in the cell wall . We conclude that these four proteins are small structurally related cell wall proteins. J Bacteriol, 1995 Jun, 177(11), 3087 - 94 The posttranslational modification of phosphoglucomutase is regulated by galactose induction and glucose repression in Saccharomyces cerevisiae; Fu L et al.; The enzyme phosphoglucomutase functions at a key point in carbohydrate metabolism . In this paper, we show that the synthesis of the major isoform of yeast phosphoglucomutase, encoded by the GAL5 (PGM2) gene, is regulated in a manner that is distinct from that previously described for other enzymes involved in galactose metabolism in the yeast Saccharomyces cerevisiae . Accumulation of this isoform increased four- to sixfold when the culture experienced either glucose depletion or heat shock . However, heat shock induction did not occur unless the cells were under glucose repression . This nonadditive increase in expression suggests that the regulatory mechanisms controlling the heat shock induction and glucose repression of the GAL5 gene are functionally related . We previously demonstrated that phosphoglucomutase is modified by a posttranslational Glc-phosphorylation reaction . We now show that this posttranslational modification, like phosphoglucomutase expression itself, is also regulated by galactose induction and glucose repression . Finally, no evidence was found to indicate that the Glc-phosphorylation of phosphoglucomutase alters its enzymatic activity under the conditions examined. Mol Cell Biol, 1995 Jun, 15(6), 3405 - 14 The upstream region of the FOX3 gene encoding peroxisomal 3-oxoacyl-coenzyme A thiolase in Saccharomyces cerevisiae contains ABF1- and replication protein A-binding sites that participate in its regulation by glucose repression; Einerhand AW et al.; Expression of the FOX3 gene, which encodes yeast peroxisomal 3-oxoacyl-coenzyme A thiolase, can be induced by oleate and repressed by glucose . Previously, we have shown that induction was mediated by an oleate response element . Just upstream of this element a negatively acting control region that mediated glucose repression was found . In order to study this negative control region, we carried out DNA-binding assays and analyzed phenotypes of mutations in this region and in the trans-acting factor CAR80, which is identical to UME6 . DNA-binding assays showed that two multifunctional yeast proteins, ABF1 and RP-A, interacted with the negative control element independently of the transcriptional activity of the FOX3 gene . ABF1 and RP-A, the latter being identical to BUF, were able to bind to DNA independently of one another but also simultaneously . The phenotypes of mutations in either DNA-binding sites of ABF1, RP-A, or both, which affected the DNA binding of these factors in vitro, indicated that these sites and the proteins that interact with them participate in glucose repression . The involvement of the RP-A site in glucose repression was further supported by our observation that the CAR80 gene product, which is required for repression mediated by the RP-A site, was essential for maintenance of glucose repression . In addition to the RP-A site in the FOX3 promoter, similar sequences were observed in other genes involved in peroxisomal function . RP-A proved to bind to all of these sequences, albeit with various affinities . From these results it is concluded that the ABF1 and RP-A sites are being required in concert to mediate glucose repression of the FOX3 gene . In addition, coordinated regulation of expression of genes involved in peroxisomal function in response to glucose is mediated by proteins associated with the RP-A site, probably RP-A and CAR80.
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
