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<