S. cerevisiae

Cell Mol Life Sci, 1999 Mar, 55(3), 437 - 49
Roles of the DNA mismatch repair and nucleotide excision repair proteins during meiosis; Kirkpatrick DT; Numerous proteins are involved in the nucleotide excision repair (NER) and DNA mismatch repair (MMR) pathways . The function and specificity of these proteins during the mitotic cell cycle has been actively investigated, in large part due to the involvement of these systems in human diseases . In contrast, comparatively little is known about their functioning during meiosis . At least three repair pathways operate during meiosis in the yeast Saccharomyces cerevisiae to repair mismatches that occur as a consequence of heteroduplex formation in recombination . The first pathway is similar to the one acting during postreplicative mismatch repair in mitotically dividing cells, while two pathways are responsible for the repair of large loops during meiosis, using proteins from MMR and NER systems . Some MMR proteins also help prevent recombination between diverged sequences during meiosis, and act late in recombination to affect the resolution of crossovers . This review will discuss the current status of DNA mismatch repair and nucleotide excision repair proteins during meiosis, especially in the yeast S . cerevisiae.

EMBO J, 1999 May 4, 18(9), 2648 - 58
Sister chromatid-based DNA repair is mediated by RAD54, not by DMC1 or TID1; Arbel A et al.; In the mitotic cell cycle of the yeast Saccharomyces cerevisiae, the sister chromatid is preferred over the homologous chromosome (non-sister chromatid) as a substrate for DNA double-strand break repair . However, no genes have yet been shown to be preferentially involved in sister chromatid-mediated repair . We developed a novel method to identify genes that are required for repair by the sister chromatid, using a haploid strain that can embark on meiosis . We show that the recombinational repair gene RAD54 is required primarily for sister chromatid-based repair, whereas TID1, a yeast RAD54 homologue, and the meiotic gene DMC1, are dispensable for this type of repair . Our observations suggest that the sister chromatid repair pathway, which involves RAD54, and the homologous chromosome repair pathway, which involves DMC1, can substitute for one another under some circumstances . Deletion of RAD54 in S.cerevisiae results in a phenotype similar to that found in mammalian cells, namely impaired DNA repair and reduced recombination during mitotic growth, with no apparent effect on meiosis . The principal role of RAD54 in sister chromatid-based repair may also be shared by mammalian and yeast cells.

Genetics, 1999 May, 152(1), 179 - 90
Interorganelle signaling is a determinant of longevity in Saccharomyces cerevisiae; Kirchman PA et al.; Replicative capacity, which is the number of times an individual cell divides, is the measure of longevity in the yeast Saccharomyces cerevisiae . In this study, a process that involves signaling from the mitochondrion to the nucleus, called retrograde regulation, is shown to determine yeast longevity, and its induction resulted in postponed senescence . Activation of retrograde regulation, by genetic and environmental means, correlated with increased replicative capacity in four different S . cerevisiae strains . Deletion of a gene required for the retrograde response, RTG2, eliminated the increased replicative capacity . RAS2, a gene previously shown to influence longevity in yeast, interacts with retrograde regulation in setting yeast longevity . The molecular mechanism of aging elucidated here parallels the results of genetic studies of aging in nematodes and fruit flies, as well as the caloric restriction paradigm in mammals, and it underscores the importance of metabolic regulation in aging, suggesting a general applicability.

J Biol Chem, 1999 May 7, 274(19), 12979 - 83
SEC14-dependent secretion in Saccharomyces cerevisiae . Nondependence on sphingolipid synthesis-coupled diacylglycerol production; Stock SD et al.; The SEC14 gene in Saccharomyces cerevisiae encodes a phosphatidylinositol transfer protein required for secretory protein movement from the Golgi . Mutation of SAC1, a gene of unknown function, restores secretory flow in sec14-1(ts) strains . The existing model for the bypass of the sec14-1(ts) defect by sac1-22 involves stimulation of sphingolipid biosynthesis and, in particular, the synthesis of mannosyl-diinositolphosphoryl-ceramide with concomitant increases in Golgi diacylglycerol levels . To test this model, we disrupted IPT1, the mannosyl-diinositolphosphoryl-ceramide synthase of S . cerevisiae . Disruption of the IPT1 gene had no effect on the ability of sac1-22 to bypass sec14-1(ts) . Furthermore, sphingolipid analysis of sec14-1(ts) and sec14-1(ts) sac1-22 strains showed that mannosyl-diinositolphosphoryl-ceramide synthesis was not stimulated in the bypass mutant . However, the sec14-1(ts) strain had elevated mannosyl-monoinositolphosphoryl-ceramide levels, and the sec14-1(ts) sac1-22 strain showed an 8-fold increase in phosphatidylinositol 4-phosphate along with a decrease in phosphatidylinositol 4,5-bisphosphate . Cellular diacylglycerol levels, measured by {14C}acetate incorporation, did not differ between the sec14-1(ts) and the sec14-1 sac1-22 bypass strains, although disruption of IPT1 in the bypass strain resulted in reduced levels . These data indicate that phosphatidylinositol 4-phosphate, rather than mannosyl-diinositolphosphoryl-ceramide, accumulates in the sec14-1(ts) sac1-22 bypass strain, and that Golgi diacylglycerol accumulation is not required for bypass of the sec14-1(ts) growth and secretory phenotypes.

Appl Environ Microbiol, 1999 May, 65(5), 1915 - 8
Expression of an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae; Han Y et al.; Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste . Our objectives were to express an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae and to determine the effects of glycosylation on the phytase's activity and thermostability . A 1.4-kb DNA fragment containing the coding region of the phyA gene was inserted into the expression vector pYES2 and was expressed in S . cerevisiae as an active, extracellular phytase . The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition . The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60 degrees C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme . Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme . Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability . The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro . In conclusion, the phyA gene was expressed as an active, extracellular phytase in S . cerevisiae, and its thermostability was affected by glycosylation.

FEMS Microbiol Lett, 1999 Apr 1, 173(1), 63 - 8
Cell-cell recognition and pheromone response of the yeast Saccharomyces globosus; Fujimura H; Sexual agglutination and pheromone interaction between cells of two mating types, a and alpha, in the yeast Saccharomyces globosus were studied . S . globosus was shown to produce mating-type-specific factors analogs to a- and alpha-mating pheromones of Saccharomyces cerevisiae and to undergo the sexual agglutination reaction between cells of two mating types . While the sexual agglutination of cells of different species was not observed, mating type a cells of each species were shown to respond to alpha-factors produced by the other species . Thus, the mating response of S . globosus was shown to be identical to what has been observed in two other species of the same genera: S . cerevisiae and Saccharomyces kluyveri.

Proc Natl Acad Sci U S A, 1999 Apr 27, 96(9), 4780 - 5
Progress toward the evolution of an organism with an expanded genetic code; Liu DR et al.; Several significant steps have been completed toward a general method for the site-specific incorporation of unnatural amino acids into proteins in vivo . An "orthogonal" suppressor tRNA was derived from Saccharomyces cerevisiae tRNA2Gln . This yeast orthogonal tRNA is not a substrate in vitro or in vivo for any Escherichia coli aminoacyl-tRNA synthetase, including E . coli glutaminyl-tRNA synthetase (GlnRS), yet functions with the E . coli translational machinery . Importantly, S . cerevisiae GlnRS aminoacylates the yeast orthogonal tRNA in vitro and in E . coli, but does not charge E . coli tRNAGln . This yeast-derived suppressor tRNA together with yeast GlnRS thus represents a completely orthogonal tRNA/synthetase pair in E . coli suitable for the delivery of unnatural amino acids into proteins in vivo . A general method was developed to select for mutant aminoacyl-tRNA synthetases capable of charging any ribosomally accepted molecule onto an orthogonal suppressor tRNA . Finally, a rapid nonradioactive screen for unnatural amino acid uptake was developed and applied to a collection of 138 amino acids . The majority of glutamine and glutamic acid analogs under examination were found to be uptaken by E . coli . Implications of these results are discussed.

Nucleic Acids Res, 1999 May 15, 27(10), 2181 - 8
A Saccharomyces cerevisiae RNA 5'-triphosphatase related to mRNA capping enzyme; Rodriguez CR et al.; The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: the RNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1) . Using computer homology searching, a S . cerevisiae gene was identified that encodes a protein resembling the C-terminal region of Cet1 . Accordingly, we designated this gene CTL1 (capping enzyme RNAtriphosphatase-like 1) . CTL1 is not essential for cell viability and no genetic or physical interactions with the capping enzyme genes were observed . The protein is found in both the nucleus and cytoplasm . Recombinant Ctl1 protein releases gamma-phosphate from the 5'-end of RNA to produce a diphosphate terminus . The enzyme is specific for polynucleotide RNA in the presence of magnesium, but becomes specific for nucleotide triphosphates in the presence of manganese . Ctl1 is the second member of the yeast RNA triphosphatase family, but is probably involved in an RNA processing event other than mRNA capping.

FEBS Lett, 1999 Mar 19, 447(1), 65 - 70
Human members of the SCO1 gene family: complementation analysis in yeast and intracellular localization; Paret C et al.; Cytochrome c oxidase is a multiprotein complex in the mitochondrial membrane whose biogenesis requires a number of proteins besides the structural subunits . Several yeast proteins as well as a human disease-related protein have been reported which are involved in cytochrome c oxidase assembly . The S . cerevisiae Sco1p protein has been implicated in the transfer of copper to cytochrome c oxidase subunits Cox1p and/or Cox2p . Here we report on the complementation behavior in yeast of two recently identified ScSco1p homologs of chromosome 17 and chromosome 22 from human . When allotropically expressed in yeast, both genes fail to complement the lack of the ScSCO1 gene . However, a chimera of the N-terminal half of ScSco1p and the C-terminal half of the chromosome 17 homolog does substitute for the ScSco1p function . Interestingly, the respective chimera with the human homolog of chromosome 22 is not able to complement . Expression of EGFP fusions in HeLa cells shows that both human ScSco1p homologs are located in the mitochondria of human cells.

Eur J Biochem, 1999 May, 261(3), 812 - 20
Identification of Delta12-fatty acid desaturase from arachidonic acid-producing mortierella fungus by heterologous expression in the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae; Sakuradani E et al.; Based on the sequence information for the omega3-desaturase genes (from Brassica napus and Caenorhabditis elegans), which are involved in the desaturation of linoleic acid (Delta9, Delta12-18 : 2) to alpha-linolenic acid (Delta9, Delta12, Delta15-18 : 3), a cDNA was cloned from the filamentous fungal strain, Mortierella alpina 1S-4, which is used industrially to produce arachidonic acid . Homology analysis with protein databases revealed that the amino acid sequence showed 43.7% identity as the highest match with the microsomal omega6-desaturase (from Glycine max, soybean), whereas it exhibited 38.9% identity with the microsomal omega3-desaturase (from soybean) . The evolutionary implications of these enzymes will be discussed . The cloned cDNA was confirmed to encode a Delta12-desaturase, which was involved in the desaturation of oleic acid (Delta9-18 : 1) to linoleic acid, by its expression in both the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae . Analysis of the fatty acid composition of yeast and fungus transformants demonstrated that linoleic acid (which was not contained in the control strain of S . cerevisiae) was accumulated in the yeast transformant and that the fungal transformant contained a large amount of linoleic acid (71.9%) . Genomic Southern blot analysis of the transformants with the Mortierella Delta12-desaturase gene as a probe confirmed integration of this gene into the genome of A . oryzae . The M . alpina 1S-4 Delta12-desaturase is the first example of a cloned nonplant Delta12-desaturase.

J Cell Biol, 1999 Apr 19, 145(2), 291 - 304
Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA; Meeusen S et al.; Maintenance of mitochondrial DNA (mtDNA) during cell division is required for progeny to be respiratory competent . Maintenance involves the replication, repair, assembly, segregation, and partitioning of the mitochondrial nucleoid . MGM101 has been identified as a gene essential for mtDNA maintenance in S . cerevisiae, but its role is unknown . Using liquid chromatography coupled with tandem mass spectrometry, we identified Mgm101p as a component of highly enriched nucleoids, suggesting that it plays a nucleoid-specific role in maintenance . Subcellular fractionation, indirect immunofluorescence and GFP tagging show that Mgm101p is exclusively associated with the mitochondrial nucleoid structure in cells . Furthermore, DNA affinity chromatography of nucleoid extracts indicates that Mgm101p binds to DNA, suggesting that its nucleoid localization is in part due to this activity . Phenotypic analysis of cells containing a temperature sensitive mgm101 allele suggests that Mgm101p is not involved in mtDNA packaging, segregation, partitioning or required for ongoing mtDNA replication . We examined Mgm101p's role in mtDNA repair . As compared with wild-type cells, mgm101 cells were more sensitive to mtDNA damage induced by UV irradiation and were hypersensitive to mtDNA damage induced by gamma rays and H2O2 treatment . Thus, we propose that Mgm101p performs an essential function in the repair of oxidatively damaged mtDNA that is required for the maintenance of the mitochondrial genome.

Biochem Biophys Res Commun, 1999 Apr 21, 257(3), 835 - 8
Involvement of Spt7p in vacuolar polyphosphate level of Saccharomyces cerevisiae; Nishimura K et al.; Saccharomyces cerevisiae became less sensitive to nickel by a defect of the SPT7 gene encoding a transcription factor . Initial rate of nickel uptake by whole cells of a SPT7-negative mutant FY963 was nearly equal to that of the parent strain FY61, and FY963 accumulated nickel about 1.7-fold of the value of FY61 when cultured in medium containing 0.1 mM NiCl2; most of which was sequestered into vacuoles . The pH gradient-driven nickel uptake by vacuolar membrane vesicles was not altered in FY963, but the amount of polyphosphate in vacuoles was highly elevated . Involvement of Spt7p in nickel detoxification through regulation of vacuolar polyphosphate level in S . cerevisiae was discussed .

Biochem Soc Symp, 1999, 64, 13 - 27
Roles of the AMP-activated/SNF1 protein kinase family in the response to cellular stress; Hardie DG; The AMP-activated protein kinase (AMPK) in mammals, and its homologue in Saccharomyces cerevisiae, are activated by cellular stresses associated with ATP depletion . AMPK is a heterotrimer comprising a catalytic alpha subunit with associated beta and gamma subunits, these being homologous with the products of the SNF1, SIP1/SIP2/GAL83 and SNF4 genes in S . cerevisiae . The alpha subunit has at least two isoforms (alpha 1 and alpha 2), which differ in their AMP-dependence and subcellular localization, with alpha 2 complexes being partly nuclear . AMPK is activated allosterically by 5'-AMP, which also promotes phosphorylation and activation by an upstream kinase, and inhibits dephosphorylation and inactivation . Elevation of AMP always accompanies depletion of ATP due to the action of adenylate kinase . Since high ATP antagonizes the activating effects of AMP, the system behaves like a cellular 'fuel gauge' . It is activated by various types of stress associated with ATP depletion, such as hypoxia, heat shock, metabolic poisoning and, in muscle, exercise . AMPK phosphorylates multiple targets which switch off anabolic pathways and switch on alternative catabolic pathways . The yeast SNF1 complex is switched on by glucose starvation, and its targets include transcription factors that repress transcription of genes required for catabolism of alternative carbon sources.

Yeast, 1999 Mar 15, 15(4), 295 - 309
Identification and characterization of individual cyclin-dependent kinase complexes from Saccharomyces cerevisiae; Shellman YG et al.; In S . cerevisiae, regulation of cell cycle progression is known to be carried out by a single cyclin-dependent kinase homologue, Cdc28p, acting at different stages of the cell cycle in association with various cyclins and other regulatory subunits . However, a still unsolved problem is the identification of the physiologically relevant substrates of the different Cdc28p kinase complexes which participate in this regulation . Purification and characterization of the subunit composition and enzymological properties of these Cdc28p complexes would therefore contribute substantially to our understanding of the molecular mechanisms controlling the cell cycle . We have used a combination of ammonium sulphate fractionation, nickel nitrilotriacetate affinity purification, ATP Sepharose affinity chromatography and Resource Q ion exchange chromatography to purify two different Cdc28p kinase complexes . Using specific clb deletion mutants and plasmid or genomic HA epitope-tagged CLBs, we show that one of these complexes is composed almost exclusively (93% or greater) of Clb2p-Cdc28p, whereas the other is mainly (75% or greater) Clb3p-Cdc28p . These procedures provide the basis for the analysis of regulatory, enzymatic and functional properties of individual Cdc28p kinase complexes.

Mol Cell Biol, 1999 May, 19(5), 3580 - 7
Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70; Robinson NG et al.; The Rho3 protein plays a critical role in the budding yeast Saccharomyces cerevisiae by directing proper cell growth . Rho3 appears to influence cell growth by regulating polarized secretion and the actin cytoskeleton, since rho3 mutants exhibit large rounded cells with an aberrant actin cytoskeleton . To gain insights into how Rho3 influences these events, we have carried out a yeast two-hybrid screen using an S . cerevisiae cDNA library to identify proteins interacting with Rho3 . Two proteins, Exo70 and Myo2, were identified in this screen . Interactions with these two proteins are greatly reduced or abolished when mutations are introduced into the Rho3 effector domain . In addition, a type of mutation known to produce dominant negative mutants of Rho proteins abolished the interaction with both of these proteins . In contrast, Rho3 did not interact with protein kinase C (Pkc1), an effector of another Rho family protein, Rho1, nor did Rho1 interact with Exo70 or Myo2 . Rho3 did interact with Bni1, another effector of Rho1, but less efficiently than with Rho1 . The interaction between Rho3 and Exo70 and between Rho3 and Myo2 was also demonstrated with purified proteins . The interaction between Exo70 and Rho3 in vitro was dependent on the presence of GTP, since Rho3 complexed with guanosine 5'-O-(3-thiotriphosphate) interacted more efficiently with Exo70 than Rho3 complexed with guanosine 5'-O-(3-thiodiphosphate) . Overlapping subcellular localization of the Rho3 and Exo70 proteins was demonstrated by indirect immunofluorescence . In addition, patterns of localization of both Exo70 and Rho3 were altered when a dominant active allele of RHO3, RHO3(E129,A131), which causes a morphological abnormality, was expressed . These results provide a direct molecular basis for the action of Rho3 on exocytosis and the actin cytoskeleton.

EMBO J, 1999 Apr 1, 18(7), 1974 - 81
Translation termination efficiency can be regulated in Saccharomyces cerevisiae by environmental stress through a prion-mediated mechanism; Eaglestone SS et al.; {PSI+} is a protein-based heritable phenotype of the yeast Saccharomyces cerevisiae which reflects the prion-like behaviour of the endogenous Sup35p protein release factor . {PSI+} strains exhibit a marked decrease in translation termination efficiency, which permits decoding of translation termination signals and, presumably, the production of abnormally extended polypeptides . We have examined whether the {PSI+}-induced expression of such an altered proteome might confer some selective growth advantage over {psi-} strains . Although otherwise isogenic {PSI+} and {psi-} strains show no difference in growth rates under normal laboratory conditions, we demonstrate that {PSI+} strains do exhibit enhanced tolerance to heat and chemical stress, compared with {psi-} strains . Moreover, we also show that the prion-like determinant {PSI+} is able to regulate translation termination efficiency in response to environmental stress, since growth in the presence of ethanol results in a transient increase in the efficiency of translation termination and a loss of the {PSI+} phenotype . We present a model to describe the prion-mediated regulation of translation termination efficiency and discuss its implications in relation to the potential physiological role of prions in S.cerevisiae and other fungi.

Cell Death Differ, 1999 Feb, 6(2), 124 - 9
Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein; Missotten M et al.; ALG-2 is a EF hand calcium binding protein with sequence homologies to calmodulin . Vito et al have shown that ALG-2 expression is required for apoptosis following a number of death stimuli,1 although nothing is known about the effectors which underlie ALG-2 function . Here we have used ALG-2 as bait in a yeast two hybrid screen of a mouse brain cDNA library . We found that ALG-2 binds to itself and to a novel protein that we call ALG-2 interacting protein X, Alix . Using co-immunoprecipitation experiments, we confirmed ALG-2/ALG-2 binding and demonstrated that this interaction is calcium independent . ALG-2/Alix interaction was also validated by co-immunoprecipitation, but in this case, the binding was found to be strictly calcium dependent . Alix seems highly conserved throughout evolution since it shows significant homologies to a putative C . elegans protein (YNK-1) and to proteins of A . nidulans (PalA) and S . cerevisiae (BRO1) . Alix is a potential regulator or downstream effector of ALG-2 action.

Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4390 - 5
RNA polymerase switch in transcription of yeast rDNA: role of transcription factor UAF (upstream activation factor) in silencing rDNA transcription by RNA polymerase II; Vu L et al.; Transcription factor UAF (upstream activation factor) is required for a high level of transcription, but not for basal transcription, of rDNA by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae . RRN9 encodes one of the UAF subunits . We have found that rrn9 deletion mutants grow extremely slowly but give rise to faster growing variants that can grow without intact Pol I, synthesizing rRNA by using RNA polymerase II (Pol II) . This change is reversible and does not involve a simple mutation . The two alternative states, one suitable for rDNA transcription by Pol I and the other favoring rDNA transcription by Pol II, are heritable not only in mitosis, but also in meiosis . Thus, S . cerevisiae has an inherent ability to transcribe rDNA by Pol II, but this transcription activity is silenced in normal cells, and UAF plays a key role in this silencing by stabilizing the first state.

J Bacteriol, 1999 Apr, 181(8), 2620 - 3
The lactate-proton symport of Saccharomyces cerevisiae is encoded by JEN1; Casal M et al.; A mutant of Saccharomyces cerevisiae deficient in the lactate-proton symport was isolated . Transformation of the mutant with a yeast genomic library allowed the isolation of the gene JEN1 that restored lactate transport . Disruption of JEN1 abolished uptake of lactate . The results indicate that, under the experimental conditions tested, no other monocarboxylate permease is able to efficiently transport lactate in S . cerevisiae.

J Biol Chem, 1999 Apr 16, 274(16), 11296 - 302
Disruption of YHC8, a member of the TSR1 gene family, reveals its direct involvement in yeast protein translocation; Mamoun CB et al.; Genetic studies of Saccharomyces cerevisiae have identified many components acting to deliver specific proteins to their cellular locations . Genome analysis, however, has indicated that additional genes may also participate in such protein trafficking . The product of the yeast Yarrowia lipolytica TSR1 gene promotes the signal recognition particle-dependent translocation of secretory proteins through the endoplasmic reticulum . Here we describe the identification of a new gene family of proteins that is well conserved among different yeast species . The TSR1 genes encode polypeptides that share the same protein domain distribution and, like Tsr1p, may play an important role in the early steps of the signal recognition particle-dependent translocation pathway . We have identified five homologues of the TSR1 gene, four of them from the yeast Saccharomyces cerevisiae and the other from Hansenula polymorpha . We generated a null mutation in the S . cerevisiae YHC8 gene, the closest homologue to Y . lipolytica TSR1, and used different soluble (carboxypeptidase Y, alpha-factor, invertase) and membrane (dipeptidyl-aminopeptidase) secretory proteins to study its phenotype . A large accumulation of soluble protein precursors was detected in the mutant strain . Immunofluorescence experiments show that Yhc8p is localized in the endoplasmic reticulum . We propose that the YHC8 gene is a new and important component of the S . cerevisiae endoplasmic reticulum membrane and that it functions in protein translocation/insertion of secretory proteins through or into this compartment.

Biochem J, 1999 Apr 15, 339 ( Pt 2), 291 - 8
Cloning and expression of a human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine and phosphatidylethanolamine; Henneberry AL et al.; Cholinephosphotransferase catalyses the final step in the synthesis of phosphatidylcholine (PtdCho) via the Kennedy pathway by the transfer of phosphocholine from CDP-choline to diacylglycerol . Ethanolaminephosphotransferase catalyses an analogous reaction with CDP-ethanolamine as the phosphobase donor for the synthesis of phosphatidylethanolamine (PtdEtn) . Together these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PtdCho and PtdEtn synthesized de novo . A human choline/ethanolaminephosphotransferase cDNA (hCEPT1) was cloned, expressed and characterized . Northern blot analysis revealed one hCEPT1 2.3 kb transcript that was ubiquitous and not enriched, with respect to actin, in any particular cell type . The open reading frame predicts a protein (hCEPT1p) of 416 amino acid residues with a molecular mass of 46550 Da containing seven membrane-spanning domains . A predicted amphipathic helix resides within the active site of the enzyme with the final two aspartic residues of the CDP-alcohol phosphotransferase motif, DG(X)2AR(X)8G(X)3D(X)3D, positioned within this helix . hCEPT1p was successfully expressed in a full-length, active form in Saccharomyces cerevisiae cells devoid of endogenous cholinephosphotransferase or ethanolaminephosphotransferase activities (HJ091, cpt1::LEU2 ept1-) . In vitro, hCEPT1p displayed broad substrate specificity, utilizing both CDP-choline and CDP-ethanolamine as phosphobase donors to a broad range of diacylglycerols, resulting in the synthesis of both PtdCho and PtdEtn . In vivo, S . cerevisiae cells (HJ091, cpt1::LEU2 ept1-) expressing hCEPT1 efficiently incorporated both radiolabelled choline and ethanolamine into phospholipids, demonstrating that hCEPT1p has the ability to synthesize both choline- and ethanolamine- containing phospholipids in vitro and in vivo.

Syst Appl Microbiol, 1999 Feb, 22(1), 145 - 55
Characterization of Saccharomyces cerevisiae CBS 7764 isolated from rainbow trout intestine; Andlid T et al.; A wild-type Saccharomyces cerevisiae, strain CBS 7764, isolated from the intestine of rainbow trout, was analyzed with respect to general growth parameters and global protein expression . Characterization of this strain was of interest since previous data show non-typical S . cerevisiae cell surface properties and because data suggest a probiotic potential of CBS 7764 . The heat production rate (dQ/dt), monitored by microcalorimetry, showed that the typical growth phases resulting from diauxic growth on glucose were present in the fish isolate . However, CBS 7764 differentiated from a reference strain by becoming limited in the respiratory phase as demonstrated by a plateau in the dQ/dt signal . The global protein expression, as studied by two-dimensional gel electrophoresis (2D-PAGE), revealed a large degree of resemblance of the fish isolate to the reference strain, however, also clear qualitative and quantitative expression differences were detected; e.g . 14% of the proteins differed in expression level by a factor of at least 2 . In addition, the fish isolate expressed 12 unique proteins . The heat shock proteins, which for other organisms have been identified as important in mucosal colonization, were generally expressed to a higher level in CBS 7764.

Eur J Biochem, 1999 Apr, 261(1), 317 - 24
Pregnenolone esterification in Saccharomyces cerevisiae . A potential detoxification mechanism; Cauet G et al.; While studying the effect of steroids on the growth of the yeast Saccharomyces cerevisiae, we found that pregnenolone was converted into the acetate ester . This reaction was identified as a transfer of the acetyl group from acetyl-CoA to the 3beta-hydroxyl group of pregnenolone . The corresponding enzyme, acetyl-CoA:pregnenolone acetyltransferase (APAT) is specific for Delta5- or Delta4-3beta-hydroxysteroids and short-chain acyl-CoAs . The apparent Km for pregnenolone is approximately 0.5 microm . The protein associated with APAT activity was partially purified and finally isolated from an SDS/polyacrylamide gel . Tryptic peptides were generated and N-terminally sequenced . Two peptide sequences allowed the identification of an open reading frame (YGR177c, in the S . cerevisiae genome database) translating into a 62-kDa protein of hitherto unknown function . This protein encoded by a gene known as ATF2 displays 37% identity with an alcohol acetyltransferase encoded by the yeast gene ATF1 . Disruption of ATF2 led to the complete elimination of APAT activity and consequently abolished the esterification of pregnenolone . In addition, a toxic effect of pregnenolone linked to the disruption of ATF2 was observed . Pregnenolone toxicity is more pronounced when the atf2-Delta mutation is introduced in a yeast strain devoid of the ATP-binding cassette transporters, PDR5 and SNQ2 . Our results suggest that Atf2p (APAT) plays an active role in the detoxification of 3beta-hydroxysteroids in association with the efflux pumps Pdr5p and Snq2p.

Genetics, 1999 Apr, 151(4), 1341 - 51
Tempo and mode of Ty element evolution in Saccharomyces cerevisiae; Jordan IK et al.; The Saccharomyces cerevisiae genome contains five families of long terminal repeat (LTR) retrotransposons, Ty1-Ty5 . The sequencing of the S . cerevisiae genome provides an unprecedented opportunity to examine the patterns of molecular variation existing among the entire genomic complement of Ty retrotransposons . We report the results of an analysis of the nucleotide and amino acid sequence variation within and between the five Ty element families of the S . cerevisiae genome . Our results indicate that individual Ty element families tend to be highly homogenous in both sequence and size variation . Comparisons of within-element 5' and 3' LTR sequences indicate that the vast majority of Ty elements have recently transposed . Furthermore, intrafamily Ty sequence comparisons reveal the action of negative selection on Ty element coding sequences . These results taken together suggest that there is a high level of genomic turnover of S . cerevisiae Ty elements, which is presumably in response to selective pressure to escape host-mediated repression and elimination mechanisms.

Biotechnol Bioeng, 1999 May 5, 63(3), 290 - 7
Virus-like particle analysis in yeast homogenate using a laser light-scattering assay; Tsoka S et al.; Virus-like particles (VLPs) expressed intracellularly by the yeast S . cerevisiae have helped set the framework of a wide range of biologicals, particularly as carriers for viral antigens . This article investigates the use of dynamic light scattering (DLS) for the rapid evaluation of the concentration and purity of VLPs to aid the complex purification strategy . Development of the assay was performed in a high background process stream (yeast homogenate) and involved a change in the signal proportional to the VLP concentration by addition of antibodies that bind on the VLP surface and detection of that size change by DLS . Overall, the assay was found to provide a significant improvement of rapid monitoring alternatives for VLPs, exhibiting good sensitivity and speed of measurement . Data are given for the use of the DLS-based assay for optimization of VLP release during a yeast cell disruption treatment .

Biotechnol Bioeng, 1998 Sep 20, 59(6), 754 - 61
Optimization of polyphosphate degradation and phosphate secretion using hybrid metabolic pathways and engineered host strains
Van Dien SJ, Keasling JD.
Polyphosphate degradation and phosphate secretion were optimized in Escherichia coli strains overexpressing the E . coli polyphosphate kinase gene (ppk) and either the E . coli polyphosphatase gene (ppx) or the Saccharomyces cerevisiae polyphosphatase gene (scPPX1) from different inducible promoters on medium- and high-copy plasmids . The use of a host strain without functional ppk or ppx genes on the chromosome yielded the highest levels of polyphosphate, as well as the fastest degradation of polyphosphate when the gene for polyphosphatase was induced . The introduction of a hybrid metabolic pathway consisting of the E . coli ppk gene and the S . cerevisiae polyphosphatase gene resulted in lower polyphosphate concentrations than when using both the ppk and ppx genes from E . coli, and did not significantly improve the degradation rate . It was also found that the rate of polyphosphate degradation was highest when ppx was induced late in growth, most likely due to the high intracellular polyphosphate concentration . The phosphate released from polyphosphate allowed the growth of phosphate-starved cells; excess phosphate was secreted into the medium, leading to a down-regulation of the phosphate-starvation (Pho) response . The production of alkaline phosphatase, an indicator of the Pho response, can be precisely controlled by manipulating the degree of ppx induction .

Mol Cell Biochem, 1999 Jan, 191(1-2), 161 - 7
Transcriptional regulation of the S . cerevisiae ENA1 gene by casein kinase II; Tenney KA et al.; The regulatory subunit of S . cerevisiae casein kinase II (CKII) is encoded of two genes, CKB1 and CKB2 . Strains harboring deletions of either or both genes exhibit specific sensitivity to high concentrations of Na+ or Li+ . Na+ tolerance in S . cerevisiae is mediated primarily by transcriptional induction of ENA1, which encodes the plasma membrane sodium pump, and by conversion of the potassium uptake system to a higher affinity form that discriminates more efficiently against Na+ . To determine whether reduced ENA1 expression plays a role in the salt sensitivity of ckb mutants, we integrated an ENA1-lacZ reporter gene into isogenic wild-type, ckb1, ckb2, and ckb1 ckb2 strains and monitored beta-galactosidase activity at different salt concentrations . In all three mutants transcription from the ENA1 promoter remained salt-inducible, but both basal and salt-induced expression was depressed approximately 3- to 4-fold . The degree of reduction in ENA1 expression was comparable to that observed in an isogenic strain carrying a null mutation in protein phosphatase 2B (calcineurin), which is also required for salt tolerance . These results suggest that reduced expression ofENA1 contributes to the salt sensitivity of ckb strains . Consistent with this conclusion, overexpression of ENA1 from a heterologous promoter (GAL1) completely suppressed the salt sensitivity of ckb mutants . Induction of ENA1 expression by alkaline pH is also depressed in ckb mutants, but unlike calcineurin mutants, ckb strains are not growth inhibited by alkaline pH.

Biochem Biophys Res Commun, 1999 Apr 2, 257(1), 139 - 44
Molecular and functional study of AQY1 from Saccharomyces cerevisiae: role of the C-terminal domain; Laize V et al.; The yeast YPR192w gene, which encodes a protein (Aqy1p) with strong homology to aquaporins (AQPs), was cloned from nine S . cerevisiae strains . The osmotic water permeability coefficient (Pf) of X . laevis oocytes expressing the gene cloned from the Sigma1278b strain (AQY1-1) was 5.7 times higher than the Pf of oocytes expressing the gene cloned from other strains (AQY1-2) . Aqy1-1p, initially cloned without its C-terminus (Aqy1-1DeltaCp), mediated an approximately 3 times higher water permeability than the full-length protein . This corresponds to a 3-fold higher protein density in the oocyte plasma membrane, as shown by freeze-fracture electron microscopy . Pf measurements in yeast spheroplasts confirmed the presence of functional water channels in Sigma1278b and a pharmacological study indicated that this strain contains at least a second functional aquaporin .

Biosystems, 1999 Jan, 49(1), 63 - 70
Fractal properties of DNA walks; Abramson G et al.; We describe two dimensional DNA walks, and analyze their fractal properties . We show results for the complete genome of S . cerevisiae . We find that the mean square deviation of the walks is superdifussive, corresponding to a fractal structure of dimension lower than two . Furthermore, the coding part of the genome seems to have smaller fractal dimension, and longer correlations, than noncoding parts.

J Cell Biol, 1999 Mar 8, 144(5), 823 - 37
The beta4 integrin interactor p27(BBP/eIF6) is an essential nuclear matrix protein involved in 60S ribosomal subunit assembly; Sanvito F et al.; p27(BBP/eIF6) is an evolutionarily conserved protein that was originally identified as p27(BBP), an interactor of the cytoplasmic domain of integrin beta4 and, independently, as the putative translation initiation factor eIF6 . To establish the in vivo function of p27(BBP/eIF6), its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene was studied in the budding yeast, Saccharomyces cerevisiae . In epithelial cells containing beta4 integrin, p27(BBP/eIF6) is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of beta4 integrin . Surprisingly, in the absence and in the presence of the beta4 integrin subunit, p27(BBP/eIF6) is in the nucleolus and associated with the nuclear matrix . Deletion of the IIH S . cerevisiae gene, encoding the yeast p27(BBP/eIF6) homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level of free ribosomal 60S subunit . Furthermore, human p27(BBP/eIF6) can rescue the lethal effect of the iihDelta yeast mutation . The data obtained in vivo suggest an evolutionarily conserved function of p27(BBP/eIF6) in ribosome biogenesis or assembly rather than in translation . A further function related to the beta4 integrin subunit may have evolved specifically in higher eukaryotic cells.

Biochem J, 1999 Apr 1, 339 ( Pt 1), 185 - 92
Mammalian PIG-L and its yeast homologue Gpi12p are N-acetylglucosaminylphosphatidylinositol de-N-acetylases essential in glycosylphosphatidylinositol biosynthesis; Watanabe R et al.; Glycosylphosphatidylinositol (GPI) is used as a membrane anchor by many eukaryotic cell-surface proteins . The second step of GPI biosynthesis is de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) . We have previously cloned the rat PIG-L gene by expression cloning that complemented a mutant Chinese hamster ovary cell line defective in this step . Here we show that recombinant rat PIG-L protein purified from Escherichia coli as a complex with GroEL has GlcNAc-PI de-N-acetylase activity in vitro . The activity was not enhanced by GTP, which is known to enhance the de-N-acetylase activity of mammalian cell microsomes . As with other de-N-acetylases that act on the GlcNAc moiety, metal ions, in particular Mn2+ and Ni2+, enhanced the enzyme activity of PIG-L . The Saccharomyces cerevisiae YMR281W open reading frame encodes a protein (termed Gpi12p) with 24% amino acid identity with rat PIG-L . On transfection into mammalian PIG-L-deficient cells, this gene, GPI12, restored the cell-surface expression of GPI-anchored proteins and GlcNAc-PI de-N-acetylase activity . The disruption of the gene caused lethality in S . cerevisiae . These results indicate that GlcNAc-PI de-N-acetylase is conserved between mammals and yeasts and that the de-N-acetylation step is also indispensable in yeasts.

J Biol Chem, 1999 Mar 26, 274(13), 8850 - 7
Mitochondrial telomere-binding protein from Candida parapsilosis suggests an evolutionary adaptation of a nonspecific single-stranded DNA-binding protein; Nosek J et al.; The mitochondrial genome in a number of organisms is represented by linear DNA molecules with defined terminal structures . The telomeres of linear mitochondrial DNA (mtDNA) of yeast Candida parapsilosis consist of tandem arrays of large repetitive units possessing single-stranded 5' extension of about 110 nucleotides . Recently we identified the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of C . parapsilosis linear mtDNA and protects it from attack by various DNA-modifying enzymes (Tomaska, L'., Nosek, J., and Fukuhara, H . (1997) J . Biol . Chem . 272, 3049-3059) . Here we report the isolation of MTP1, the gene encoding mtTBP of C . parapsilosis . Sequence analysis revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded DNA-binding proteins that nonspecifically bind to single-stranded DNA with high affinity . Recombinant mtTBP displays a preference for the telomeric 5' overhang of C . parapsilosis mtDNA . The heterologous expression of a mtTBP-GFP fusion protein resulted in its localization to the mitochondria but was unable to functionally substitute for the loss of the S . cerevisiae homologue Rimlp . Analysis of the MTP1 gene and its translation product mtTBP may provide an insight into the evolutionary origin of linear mitochondrial genomes and the role it plays in their replication and maintenance.

J Biol Chem, 1999 Mar 26, 274(13), 8604 - 10
The Saccharomyces cerevisiae YOR163w gene encodes a diadenosine 5', 5"'-P1,P6-hexaphosphate (Ap6A) hydrolase member of the MutT motif (Nudix hydrolase) family; Cartwright JL et al.; The YOR163w open reading frame on chromosome XV of the Saccharomyces cerevisiae genome encodes a member of the MutT motif (nudix hydrolase) family of enzymes of Mr 21,443 . By cloning and expressing this gene in Escherichia coli and S . cerevisiae, we have shown the product to be a (di)adenosine polyphosphate hydrolase with a previously undescribed substrate specificity . Diadenosine 5',5"'-P1, P6-hexaphosphate is the preferred substrate, and hydrolysis in H218O shows that ADP and adenosine 5'-tetraphosphate are produced by attack at Pbeta and AMP and adenosine 5'-pentaphosphate are produced by attack at Palpha with a Km of 56 microM and kcat of 0.4 s-1 . Diadenosine 5',5"'-P1,P5-pentaphosphate, adenosine 5'-pentaphosphate, and adenosine 5'-tetraphosphate are also substrates, but not diadenosine 5',5"'-P1,P4-tetraphosphate or other dinucleotides, mononucleotides, nucleotide sugars, or nucleotide alcohols . The enzyme, which was shown to be expressed in log phase yeast cells by immunoblotting, displays optimal activity at pH 6.9, 50 degrees C, and 4-10 mM Mg2+ (or 200 microM Mn2+) . It has an absolute requirement for a reducing agent, such as dithiothreitol (1 mM), and is inhibited by Ca2+ with an IC50 of 3.3 mM and F- (noncompetitively) with a Ki of 80 microM . Its function may be to eliminate potentially toxic dinucleoside polyphosphates during sporulation.

Mol Cell Biol, 1999 Apr, 19(4), 2967 - 76
An activator binding module of yeast RNA polymerase II holoenzyme; Lee YC et al.; The Mediator complex of Saccharomyces cerevisiae is required for both general and regulated transcription of RNA polymerase II (PolII) and is composed of two stable subcomplexes (Srb4 and Rgr1 subcomplexes) . To decipher the function of each Mediator subcomplex and to delineate the functional relationship between the subcomplexes, we characterized the compositions and biochemical activities of PolII-Mediator complexes (holoenzymes) prepared from several Mediator mutant strains of S . cerevisiae . We found that holoenzymes devoid of a functional Gal11 module were defective for activated but not basal transcription in a reconstituted in vitro system . This activation-specific defect was correlated with a crippled physical interaction to transcriptional activator proteins, which could be bypassed by artificial recruitment of a mutant holoenzyme to a promoter . Consistent with this observation, a direct interaction between Gal11 and gene-specific transcriptional activator proteins was detected by far-Western analyses and column binding assays . In contrast, the srb5 deletion mutant holoenzyme was defective for both basal and activated transcription, despite its capacity for activator binding that is comparable to that of the wild-type holoenzyme . These results demonstrate that the Gal11 module of the Rgr1 subcomplex is required for the efficient recruitment of PolII holoenzyme to a promoter via activator-specific interactions, while the Srb4 subcomplex functions in the modulation of general polymerase activity.

Mol Cell Biol, 1999 Apr, 19(4), 2929 - 35
Overlapping specificities of base excision repair, nucleotide excision repair, recombination, and translesion synthesis pathways for DNA base damage in Saccharomyces cerevisiae; Swanson RL et al.; The removal of oxidative damage from Saccharomyces cerevisiae DNA is thought to be conducted primarily through the base excision repair pathway . The Escherichia coli endonuclease III homologs Ntg1p and Ntg2p are S . cerevisiae N-glycosylase-associated apurinic/apyrimidinic (AP) lyases that recognize a wide variety of damaged pyrimidines (H . J . You, R . L . Swanson, and P . W . Doetsch, Biochemistry 37:6033-6040, 1998) . The biological relevance of the N-glycosylase-associated AP lyase activity in the repair of abasic sites is not well understood, and the majority of AP sites in vivo are thought to be processed by Apn1p, the major AP endonuclease in yeast . We have found that yeast cells simultaneously lacking Ntg1p, Ntg2p, and Apn1p are hyperrecombinogenic (hyper-rec) and exhibit a mutator phenotype but are not sensitive to the oxidizing agents H2O2 and menadione . The additional disruption of the RAD52 gene in the ntg1 ntg2 apn1 triple mutant confers a high degree of sensitivity to these agents . The hyper-rec and mutator phenotypes of the ntg1 ntg2 apn1 triple mutant are further enhanced by the elimination of the nucleotide excision repair pathway . In addition, removal of either the lesion bypass (Rev3p-dependent) or recombination (Rad52p-dependent) pathway specifically enhances the hyper-rec or mutator phenotype, respectively . These data suggest that multiple pathways with overlapping specificities are involved in the removal of, or tolerance to, spontaneous DNA damage in S . cerevisiae . In addition, the fact that these responses to induced and spontaneous damage depend upon the simultaneous loss of Ntg1p, Ntg2p, and Apn1p suggests a physiological role for the AP lyase activity of Ntg1p and Ntg2p in vivo.

Adv Exp Med Biol, 1999, 448, 247 - 54
Intracellular pathways of copper trafficking in yeast and humans; Culotta VC et al.; In the bakers yeast S . cerevisiae, there at least four intracellular targets requiring copper ions-1) Ccc2p and Fet3p in the secretory pathway (homologues to Menkes/Wilson proteins and ceruloplasmin); 2) cytochrome oxidase in the mitochondria; 3) copper transcription factors in the nucleus; and 4) Cu/Zn superoxide dismutase (SOD1) in the cytosol . We have discovered a small soluble copper carrier that specifically delivers copper ions to the secretory pathway . This 8.2 kDa factor known as Atx1p, exhibits striking homology to the MERp mercury carrier of bacteria and contains a single MTCXXC metal binding site also found in the Menkes/Wilson family of copper transporting ATPases . Our studies show that Atx1p is cytosolic and facilitates the delivery of copper ions from the cell surface copper transporter to Ccc2p and Fet3p in the secretory pathway; furthermore, it is not involved in the delivery of copper ions to the mitochondria, the nucleus or cytosolic SOD1, implicating specific signals directing Atx1p to the secretory pathway . Homologues to Atx1p have been found in invertebrates, plants and humans, and the human gene is abundantly expressed in all tissues . In addition to Atx1p, we have recently uncovered an additional metal trafficking protein that appears to specifically deliver copper ions to SOD1 . Mutants in the corresponding gene (lys7) are defective for SOD1 activity, and are unable to incorporate copper into SOD1, while there is no obvious impairment in copper delivery to cytochrome oxidase of Fet3p . The encoded 27 kDa protein contains a single MHCXXC consensus copper binding sequence and close homologues have been identified in a wide array of eukaryotic species including humans.

Curr Genet, 1999 Mar, 35(2), 103 - 8
Mitochondrial copper metabolism in yeast: mutational analysis of Sco1p involved in the biogenesis of cytochrome c oxidase; Rentzsch A et al.; Saccharomyces cerevisiae Sco1p is believed to be involved in the transfer of copper from the carrier Cox17p to the mitochondrial cytochrome c oxidase subunits 1 and 2 . We here report on the results of a mutational analysis of Sco1p . The two cysteine residues of a potential metal-binding motif (CxxxC) are essential for protein function as shown by their substitution by alanines . Chimeras consisting of Sco1p and its homolog S . cerevisiae Sco2p restrict the specificity of Sco1p function to the N-terminal half of the protein . A candidate region for conferring specificity on Sco1p is a stretch of hydrophobic amino acids, which act as a membrane anchor . In line with this suggestion is the result that alterations of individual amino acids within this region impair Sco1p function.

Mol Gen Genet, 1999 Feb, 261(1), 11 - 20
Functional analysis of multiple AUG codons in the transcripts of the STA2 glucoamylase gene from Saccharomyces cerevisiae; Vivier MA et al.; A scanning ribosome will usually initiate translation as soon as it encounters the first favourable AUG codon and only a few eukaryotic transcripts have more complex arrangements . These relatively few complex transcripts are normally characterized by structural features such as multiple AUGs and significant secondary structure . However, the functional relevance of these features has rarely been established . We present here a study of the functional significance of the multiple AUGs in the leader of STA2 transcripts of the budding yeast Saccharomyces cerevisiae, and extrapolate, where applicable, these results to a co-regulated gene, MUC1 . The STA2 gene (a representative member of the polymorphic STA1-3 gene family), encodes an extracellular glucoamylase, and is evolutionarily linked to, and transcriptionally co-regulated with, the MUC1 gene, which encodes a mucin-like protein essential for pseudohyphal/invasive growth and cell-adhesion in S . cerevisiae . Each of these genes contains a putative upstream ORF, while STA2 has two additional in-frame AUG codons 5' to the major cistron . We show that utilization of the alternative translational start-sites of STA2 results in glucoamylases that differ at their N-termini, which are associated with differences in their localization patterns . Analysis of mutants revealed the presence of a putative secretion-enhancing signal that might prove to be relevant to the alternative targeting mechanism recently uncovered in S . cerevisiae . We show that a short up-stream ORF present in the leaders of STA1-3 and MUC1 is probably bypassed by a process of leaky scanning.

Mol Biol Cell, 1999 Mar, 10(3), 581 - 96
GCS1, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, is required for normal actin cytoskeletal organization in vivo and stimulates actin polymerization in vitro; Blader IJ et al.; Recent cloning of a rat brain phosphatidylinositol 3,4, 5-trisphosphate binding protein, centaurin alpha, identified a novel gene family based on homology to an amino-terminal zinc-binding domain . In Saccharomyces cerevisiae, the protein with the highest homology to centaurin alpha is Gcs1p, the product of the GCS1 gene . GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth . Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects . Here, we have shown that similar to centaurin alpha, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity . A novel GCS1 disruption strain (gcs1Delta) exhibited morphological defects, as well as mislocalization of cortical actin patches . gcs1Delta was hypersensitive to the actin monomer-sequestering drug, latrunculin-B . Synthetic lethality was observed between null alleles of GCS1 and SLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton . In addition, synthetic growth defects were observed between null alleles of GCS1 and SAC6, the gene encoding the yeast fimbrin homologue . Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro . These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S . cerevisiae.

J Mol Biol, 1999 Mar 12, 286(5), 1507 - 17
Sulphate ions observed in the 2.12 A structure of a new crystal form of S . cerevisiae phosphoglycerate mutase provide insights into understanding the catalytic mechanism; Rigden DJ et al.; The structure of a new crystal form of Saccharomyces cerevisiae phosphoglycerate mutase has been solved and refined to 2.12 A with working and free R-factors of 19.7 and 22.9 %, respectively . Higher-resolution data and greater non-crystallographic symmetry have produced a more accurate protein structure than previously . Prominent among the differences from the previous structure is the presence of two sulphate ions within each active site cleft . The separation of the sulphates suggests that they may occupy the same sites as phospho groups of the bisphosphate ligands of the enzyme . Plausible binding modes for 2,3-bisphosphoglycerate and 1, 3-bisphosphoglycerate are thereby suggested . These results support previous conclusions from mutant studies, highlight interesting new targets for mutagenesis and suggest a possible mechanism of enzyme phosphorylation .

Biochemistry, 1999 Mar 2, 38(9), 2628 - 35
DNA ligation during excision repair in yeast cell-free extracts is specifically catalyzed by the CDC9 gene product; Wu X et al.; Excision repair of DNA is an important cellular response to DNA damage induced by radiation and many chemicals . In eukaryotes, base excision repair (BER) and nucleotide excision repair (NER) are two major excision repair pathways which are completed by a DNA ligation step . Using a cell-free system, we have determined the DNA ligase requirement during BER and NER of the yeast S . cerevisiae . Under nonpermissive conditions in extracts of the cdc9-2 temperature-sensitive mutant, DNA ligation in both BER and NER pathways was defective, and the repair patches were enlarged . At the permissive temperature (23 degrees C), DNA ligation during excision repair was only partially functional in the mutant extracts . In contrast, deleting the DNA ligase IV gene did not affect DNA ligation of BER or NER . Defective DNA ligation of BER and NER in cdc9-2 mutant extracts was complemented in vitro by purified yeast Cdc9 protein, but not by DNA ligase IV even when overexpressed . These results demonstrate that the ligation step of excision repair in yeast cell-free extracts is catalyzed specifically by the Cdc9 protein, the homologue of mammalian DNA ligase I.

Appl Environ Microbiol, 1999 Mar, 65(3), 1191 - 7
Stimulation of strontium accumulation in linoleate-enriched Saccharomyces cerevisiae is a result of reduced Sr2+ efflux; Avery SV et al.; The influence of modified plasma membrane fatty acid composition on cellular strontium accumulation in Saccharomyces cerevisiae was investigated . Growth of S . cerevisiae in the presence of 1 mM linoleate (18:2) (which results in 18:2 incorporation to approximately 70% of total cellular and plasma membrane fatty acids, with no effect on growth rate) yielded cells that accumulated Sr2+ intracellularly at approximately twice the rate of S . cerevisiae grown without a fatty acid supplement . This effect was evident over a wide range of external Sr2+ concentrations (25 microM to 5 mM) and increased with the extent of cellular 18:2 incorporation . Stimulation of Sr2+ accumulation was not evident following enrichment of S . cerevisiae with either palmitoleate (16:1), linolenate (18:3) (n-3 and n-6 isomers), or eicosadienoate (20:2) (n-6 and n-9 isomers) . Competition experiments revealed that Ca2+- and Mg2+-induced inhibition of Sr2+ accumulation did not differ between unsupplemented and 18:2-supplemented cells . Treatment with trifluoperazine (TFP) (which can act as a calmodulin antagonist and Ca2+-ATPase inhibitor), at a low concentration that precluded nonspecific K+ efflux, increased intracellular Sr2+ accumulation by approximately 3.6- and 1.4-fold in unsupplemented and 18:2-supplemented cells, respectively . Thus, TFP abolished the enhanced Sr2+ accumulation ability of 18:2-supplemented cells . Moreover, the rate of Sr2+ release from Sr2+-loaded fatty acid-unsupplemented cells was found to be at least twice as great as that from Sr2+-loaded 18:2-enriched cells . The influence of enrichment with other fatty acids on Sr2+ efflux was variable . The results reveal an enhanced Sr2+ accumulation ability of S . cerevisiae following 18:2-enrichment, which is attributed to diminished Sr2+ efflux activity in these cells.

J Cell Physiol, 1999 Feb, 178(2), 216 - 26
Carbon source-dependent regulation of cell growth by murine protein kinase C epsilon expression in Saccharomyces cerevisiae; Parissenti AM et al.; Protein kinase C is known to play a role in cell cycle regulation in both lower and higher eucaryotic cells . Since mutations in yeast proteins involved in cell cycle regulation can often be rescued by the mammalian homolog and since significant conservation exists between PKC-signalling pathways in yeast and mammalian cells, cell cycle regulation by mammalian PKC isoforms may be effectively studied in a simpler genetically-accessible model system such as Saccharomyces cerevisiae . With this objective in mind, we transfected S . cerevisiae cells with a plasmid (pYECepsilon) coding for the expression of murine protein kinase C epsilon (PKCepsilon) under the control of a galactose-inducible promoter . Unlike mock-transfected cells, yeast cells transformed with pYECepsilon expressed, in a galactose-dependent manner, an 89 kDa protein that was recognized by a human PKCepsilon antibody . Extracts from these pYECepsilon-transfected cells could phosphorylate a PKCepsilon substrate peptide in a phospholipid/phorbol ester-dependent manner . Moreover, this catalytic activity could be inhibited by a fusion protein in which the regulatory domain of murine PKCepsilon was fused in frame with GST (GST-Repsilon), further confirming the successful expression of murine PKCepsilon . Induction of PKCepsilon expression by galactose in cells transformed with pYECepsilon increased Ca++ uptake by the cells approximately 5-fold and resulted in a dramatic inhibition of cell growth in glycerol . However, when glucose was used as the carbon source, PKCepsilon expression had no effect on cell growth . This was in contrast to what was observed upon bovine PKCalpha or PKCbeta-I expression in yeast, where expression of these PKC isoforms strongly and moderately inhibited growth in glucose, respectively . Visualization of the cells by phase contrast microscopy indicated that murine PKCepsilon expression in the presence of glycerol resulted in a significant increase in the number of yeast cells exhibiting very small buds . Since overall growth of the cells was dramatically decreased, the data suggests that PKCepsilon expression potently inhibits the progression of yeast cells through the cell cycle after the initiation of budding . In addition, a small amount of the PKCepsilon-expressing yeast cells (1-2%) exhibited gross alterations in cell morphology and defects in both chromosome segregation and septum formation . This suggests that for those cells which do complete DNA synthesis, murine PKCepsilon expression may nevertheless inhibit yeast cell growth by retarding and/or imparing cell division . Taken together, the data suggests murine PKCepsilon expression potently reduces the growth of yeast cells in a carbon source-dependent fashion by affecting progression through multiple points within the cell cycle . This murine PKCepsilon-expressing yeast strain may serve as a very useful tool in the elucidation of mechanism(s) by which external environmental signals (possibly through specific PKC isoforms) regulate cell cycle progression in both yeast and mammalian cells.

Cell Signal, 1999 Feb, 11(2), 127 - 35
The SH3 domain of the S . cerevisiae Cdc25p binds adenylyl cyclase and facilitates Ras regulation of cAMP signalling; Mintzer KA et al.; Cdc25 and Ras are two proteins required for cAMP signalling in the budding yeast Saccharomyces cerevisiae . Cdc25 is the guanine nucleotide exchange protein that activates Ras . Ras, in turn, activates adenylyl cyclase . Cdc25 has a Src homology 3 (SH3) domain near the N-terminus and a catalytic domain in the C-terminal region . We find that a point mutation in the SH3 domain attenuates cAMP signalling in response to glucose feeding . Furthermore, we demonstrate, by using recombinant adenylyl cyclase and Cdc25, that the SH3 domain of Cdc25 can bind directly to adenylyl cyclase . Binding was specific, because the SH3 domain of Abp1p (actin-binding protein 1), which binds the 70,000 Mr subunit of adenylyl cyclase, CAP/Srv2, failed to bind adenylyl cyclase . A binding site for Cdc25-SH3 localised to the C-terminal catalytic region of adenylyl cyclase . Finally, pre-incubation with Ras enhanced the SH3-bound adenylyl cyclase activity . These studies suggest that a direct interaction between Cdc25 and adenylyl cyclase promotes efficient assembly of the adenylyl cyclase complex.

J Biotechnol, 1999 Feb 5, 68(1), 15 - 28
Non-linear optimization of biotechnological processes by stochastic algorithms: application to the maximization of the production rate of ethanol, glycerol and carbohydrates by Saccharomyces cerevisiae; Rodriguez-Acosta F et al.; A non-linear optimization, based on an stochastic multi-start search algorithm, has been applied to the maximization of the production rates of ethanol, glycerol and carbohydrates by Saccharomyces cerevisiae . This optimization is applied to two alternative (non-linear) model representations of the same system, namely the Michaelis-Menten and the generalized mass action forms . We find a complete agreement between the results obtained using both representations . This is, maximization of the ethanol production rate requires modulation of up to six enzymes, while modification of only one enzyme is sufficient to obtain a significant improvement in the production rate of glycerol and carbohydrates . When the results are compared with those previously obtained using an indirect linear optimization method (Torres, N.V., Voit, E.O., Gonzalez-Alcon, C., Rodriguez, F . 1997 . An integrated optimization method for biochemical systems . Description of method and application to ethanol, glycerol and carbohydrate production in S . cerevisiae . Biotechnol . Bioeng . 55(5), 758-772.), we find close agreement between both optimization techniques . Qualitatively, both optimization approaches render the same profile of enzymes to be modulated, while quantitatively, discrepancies arise when the objective function is the maximization of the ethanol production rate . Reasons for such discrepancies and an evaluation of the advantages of each method (linear vs non-linear) are given.

Curr Genet, 1999 Feb, 35(1), 51 - 7
Transformation of Candida oleophila and survival of a transformant on orange fruit under field conditions; Chand-Goyal T et al.; Histidine auxotrophs of wild-type strain I-182 of Candida oleophila, produced using ethyl methanesulfonate, were transformed with plasmids containing the HIS3, HIS4 and HIS5 genes of Saccharomyces cerevisiae . Histidine auxotrophy was complemented by the HIS5 gene of S . cerevisiae . Stability of the transformants under non-selective conditions and DNA gel-blot analysis suggested that the transforming DNA had integrated into the C . oleophila genome . There were no detectable physiological differences between the wild-type and the transformants . The biological control ability of C . oleophila was not affected by the transformation . A genetically marked transformant (with a beta-glucuronidase gene) colonized wounds on oranges, and its population increased under field conditions . The identity of the genetically marked transformant was established by PCR-amplification of a portion of the beta-glucuronidase gene.

Mol Cell Biol, 1999 Mar, 19(3), 2142 - 54
Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA; Massenet S et al.; Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method . In contrast to vertebrate UsnRNAs, S . cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions . At these positions, UsnRNAs are universally modified . When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs . Indeed, Psi44 formation in U2 snRNA was abolished . By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S . cerevisiae U2 snRNA . Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA . As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S . cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.

Genes Cells, 1998 Nov, 3(11), 737 - 49
Cell cycle dependent topological changes of chromosomal replication origins in Saccharomyces cerevisiae; Fujita M et al.; BACKGROUND: The ORC (Origin Recognition Complex) of Saccharomyces cerevisiae is a protein complex for the initiation of replication which interacts with a cis-element, ACS (ARS Consensus Sequence), essential for DNA replication . The protein-DNA complex detected by the DNase I genomic footprinting method has been shown to vary depending on cell cycle progression . Further studies on topological changes of replication origin in vivo caused by ORC association are crucial for an understanding of chromosomal DNA replication in S . cerevisiae . RESULTS: Topological changes in the replication origins of the S . cerevisiae chromosome were studied by an in vivo UV photofootprinting method which is capable of detecting the change in the flexibility of DNA caused by protein binding . The footprinting method detected the inhibition and enhancement of UV-induced pyrimidine dimer formation in A and B1 elements of a chromosomal origin, ARS1, depending on the activity of native ORC subunits . Furthermore, footprint patterns were reproduced in vitro with purified ORC . The inhibition regarding the A element was stronger during the S to late M phase than that during the progression through the G1 phase . Functional CDC6 and MCM5 were required for maintaining the weaker inhibition state in G1-arrested cells . CONCLUSION: The application of in vivo UV photofootprinting in studies of topological changes of S . cerevisiae replication origins revealed the presence of two modes of topological ORC-ACS interaction . The weaker footprint in the G1 phase represents a specific topology of ACS, resulting from an alteration of the ORC-ACS interaction aided by CDC6 and MCM5, and this topological change may make the replication origin competent for initiating DNA replication.

Eur J Biochem, 1998 Dec 15, 258(3), 941 - 7
Mutational analysis of chitin synthase 2 of Saccharomyces cerevisiae . Identification of additional amino acid residues involved in its catalytic activity; Yabe T et al.; Saccharomyces cerevisiae harbors three chitin synthases termed Chs1p, Chs2p and Chs3p . Previously, we demonstrated that con1, a region that is highly conserved among all chitin synthases, contains amino acids essential for the catalytic activity of the enzyme and that Asp562, Gln601, Arg604, and Trp605 found in con1 together with Asp441 were probable catalytic sites of the enzyme . Here we report that another region, con2, in the C-terminal half of Chs2p is also conserved exclusively in chitin synthases that resemble S . cerevisiae Chs1p and Chs2p . Alanine substitutions for the conserved amino acids in con2 identified five amino acids, Asn797, His799, Asp800, Trp803, and Thr805, the mutation of which severely diminished enzymatic activity and the enzyme's ability to rescue the yeast chs2 delta chs3 delta null mutant strain . Although the activities of some of the mutant enzymes were too low to measure enzyme kinetics, most of the alanine mutations in con2 affected the kcat values rather than the K(m) values . Whereas a conservative mutation of Asn797 restored the activity, those of His799, Asp800, Trp803, and Thr805 did not . A fine alignment of the amino acid sequences of con2 and Chs3p revealed that Asp800, Trp803 and Thr805 are completely conserved near the C-terminal ends of Chs3p and its homologs in other fungi . On the basis of these findings, we propose that Asp800, Trp803, and Thr805 in con2 are additional residues involved in catalysis, and hypothesise that Asp800 together with the previously identified Asp441 and Asp562 serve as polar residues necessary for the acid-based catalytic reaction of chitin synthase.

J Biol Chem, 1999 Feb 19, 274(8), 4869 - 75
Mutations in ribosomal protein L10e confer resistance to the fungal-specific eukaryotic elongation factor 2 inhibitor sordarin; Justice MC et al.; The natural product sordarin, a tetracyclic diterpene glycoside, selectively inhibits fungal protein synthesis by impairing the function of eukaryotic elongation factor 2 (eEF2) . Sordarin and its derivatives bind to the eEF2-ribosome-nucleotide complex in sensitive fungi, stabilizing the post-translocational GDP form . We have previously described a class of Saccharomyces cerevisiae mutants that exhibit resistance to varying levels of sordarin and have identified amino acid substitutions in yeast eEF2 that confer sordarin resistance . We now report on a second class of sordarin-resistant mutants . Biochemical and molecular genetic analysis of these mutants demonstrates that sordarin resistance is dependent on the essential large ribosomal subunit protein L10e in S . cerevisiae . Five unique L10e alleles were characterized and sequenced, and several nucleotide changes that differ from the wild-type sequence were identified . Changes that result in the resistance phenotype map to 4 amino acid substitutions and 1 amino acid deletion clustered in a conserved 10-amino acid region of L10e . Like the previously identified eEF2 mutations, the mutant ribosomes show reduced sordarin-conferred stabilization of the eEF2-nucleotide-ribosome complex . To our knowledge, this report provides the first description of ribosomal protein mutations affecting translocation . These results and our previous observations with eEF2 suggest a functional linkage between L10e and eEF2.

Mol Microbiol, 1999 Jan, 31(1), 103 - 16
Msn1p/Mss10p, Mss11p and Muc1p/Flo11p are part of a signal transduction pathway downstream of Mep2p regulating invasive growth and pseudohyphal differentiation in Saccharomyces cerevisiae; Gagiano M et al.; In Saccharomyces cerevisiae, a network of signal transduction pathways governs the switch from yeast-type growth to pseudohyphal and invasive growth that occurs in response to nutrient limitation . Important elements of this network have been identified, including nutrient signal receptors, GTP-binding proteins, components of the pheromone-dependent MAP kinase cascade and several transcription factors . However, the structural and functional mapping of these pathways is far from complete . Here, we present data regarding three genes, MSN1/MSS10, MSS11 and MUC1/FLO11, which form an essential part of the signal transduction network establishing invasive growth . Both MSN1 and MSS11 are involved in the co-regulation of starch degradation and invasive growth . Msn1p and Mss11p act downstream of Mep2p and Ras2p and regulate the transcription of both STA2 and MUC1 . We show that MUC1 mediates the effect of Msn1p and Mss11p on invasive growth . In addition, our results suggest that the activity of Msn1p is independent of the invasive growth MAP kinase cascade, but the Mss11p is required for the activation of pseudohyphal and invasive growth by Ste12p . We also show that starch metabolism in S . cerevisiae is subject to regulation by components of the MAP kinase cascade.

Biosci Biotechnol Biochem, 1998 Dec, 62(12), 2425 - 7
Cloning of genomic DNA of Rhizopus niveus lipase and expression in the yeast Saccharomyces cerevisiae; Kohno M et al.; Genomic DNA encoding Lipase I was cloned from Rhizopus niveus strain IFO4759 . For expression of this gene in S . cerevisiae, DBY746 was transformed with YEp352PLipS, which had the cloned lipase gene under the control of a PGK promoter . This strain secreted the lipase at a high level (350 U/ml) . The strain ND-12B was produced by a mating of DBY746, harboring YEp352PLipS, and NA74-3A, and dissection of asci . This new strain secreted the lipase up to 530 U/ml . Moreover, the lipase was produced most effectively in a medium containing Bacto-yeast extract, soy-peptide, and sucrose.

Biosci Biotechnol Biochem, 1998 Nov, 62(11), 2280 - 2
Identification of the orotidine-5'-phosphate decarboxylase gene and development of a transformation system in the yeast Saccharomyces exiguus Yp74L-3; Hisatomi T et al.; To investigate the uracil biosynthetic pathway of the yeast Saccharomyces exiguus Yp74L-3, uracil auxotrophic mutants were isolated . Using conventional genetic techniques, four mutant genes concerned in uracil biosynthesis were identified and denoted as ura1, ura2, ura3, and ura4 . Mutations in the URA3 and URA4 genes were specifically selected with 5-fluoroorotic acid (5-FOA) . Vector plasmids containing the URA3 gene and an autonomously replicating sequence (ARS) of S . cerevisiae produced sufficient amounts of Ura+ transformants from the ura4 mutant of S . exiguus . This fact indicates that the S . exiguus URA4 gene encodes orotidine-5'-phosphate decarboxylase (OMP decarboxylase) and demonstrates that vector plasmids for S . cerevisiae are also usable in S . exiguus.

Lipids, 1998 Dec, 33(12), 1229 - 34
Lysophosphatidylcholine acyltransferase activity in Saccharomyces cerevisiae: regulation by a high-affinity Zn2+ binding site; Richard MG et al.; Saccharomyces cerevisiae cells were demonstrated to contain lysophosphatidylcholine (lysoPtdCho) acyltransferase (E.C . 2.3.1.23) activity . The enzyme displayed Km(app) of 69 microM for lysoPtdCho and 152 microM for oleoyl CoA . Enzyme activity was not affected by the addition of 1 mM Mg2+, Mn2+, Ca2+, or 200 mM EDTA . However, Zn2+ inhibited lysoPtdCho acyltransferase activity to 33% control values at 0.1 mM and to 7% at 1.0 mM Zn2+ . To further explore the possibility that lysoPtdCho acyltransferase may contain a high-affinity Zn2+ binding site, we tested the strong Zn2+ chelator o-phenanthroline for its ability to inhibit enzyme activity . LysoPtdCho acyltransferase activity was inhibited to 18 and 27%, respectively, those of control values in the presence of 2 and 1 mM o-phenanthroline, implying that a high-affinity Zn2+ binding site exists in lysoPtdCho acyltransferase or in an accessory protein that is essential for protein stability and/or activity . Saccharomyces cerevisiae lysoPtdCho acyltransferase activity displayed a broad lysoPtdCho fatty acyl chain substrate specificity utilizing lysoPtdCho molecules ranging in length from C10-C20 (the entire range tested) . In addition, the enzyme was capable of using the ether-linked analog of lysoPtdCho, 1-O-alkyl-2-hydroxy-sn-3-glycerophosphocholine, as a substrate . The ability of S . cerevisiae to incorporate radiolabeled 1-O-alkyl-2-hydroxy-sn-3-glycerophosphocholine into phosphatidylcholine in vitro was exploited to demonstrate a direct precursor-product relationship between lysoPtdCho molecules and their incorporation into phosphatidylcholine in vivo . Identical labeling results were obtained in S . cerevisiae cells disrupted for their major transacylase activity, PLB1, demonstrating that the incorporation of lysolipid was via acyltransferase, and not transacylase, activity.

J Mol Evol, 1999 Feb, 48(2), 133 - 41
Relationships between transcriptional and translational control of gene expression in Saccharomyces cerevisiae: a multiple regression analysis; Pavesi A; Natural selection for an increased translation efficiency has been proposed as the main determinant for the bias in codon usage observed in many genes of Saccharomyces cerevisiae . Recently, the efficiency of transcription of a large number of yeast genes has been determined, based on the cellular content of the respective mRNAs: this provides an additional dimension to the study of the multisep process of gene expression . Using a representative set of yeast genes with a known level of transcription, the relationship between transcriptional and translational steps was evaluated by a multiple linear regression model . This analysis demonstrated a positive correlation between the amount of transcript, given as the number of mRNA copies per cell for each individual gene, and indices evaluating the effects of translational selection on the corresponding codon usage pattern . This finding suggests a close association of the cellular mRNA content, regulated also at the transcriptional level, to its efficiency of translation, mediated by a fine-tuning of codon usage strategy . Moreover, multiple regression analysis demonstrated that the transcription level of a gene can be approximately predicted using indices of bias deriving from its nucleotide sequence . This allowed for an extensive investigation of uncharacterized regions of the complete genome sequence of S . cerevisiae, to detect new potential short protein coding genes that were not considered by previous searching procedures . Several small open reading frames exhibiting a statistically significant coding potential were thus identified as good candidates for functional analysis.

Mol Gen Genet, 1999 Jan, 260(6), 510 - 21
Molecular characterization of HymA, an evolutionarily highly conserved and highly expressed protein of Aspergillus nidulans; Karos M et al.; Aspergillus nidulans reproduces asexually via uninucleate, haploid spores, which are produced on morphologically differentiated aerial structures, called conidiophores . These consist of four distinct cell types, a foot with a terminally swollen stalk, metulae, phialides and conidiospores . The molecular mechanisms underlying the morphological changes that occur during conidiophore development have been studied by mutant analysis . We have isolated the hym A mutant, in which conidiophore development is affected at the metula stage . In the mutant metulae do not differentiate properly but come to resemble hyphae (hym = hypha-like metulae) . In this paper we have analyzed the corresponding gene . It encodes a highly expressed 44 kDa protein which resides in the cytoplasm and has homologues in yeast, plants, fly, worm, fish, mice and man . We constructed hym deletion strains of Saccharomyces cerevisiae and of A . nidulans and found that the gene is essential in S . cerevisiae but is dispensable in the filamentous fungus . A cellular function for the Hym protein has not yet been defined in any organism . To demonstrate functional conservation we constructed a chimeric protein comprised of the N-terminal half of the A . nidulans and the C-terminal half of the mouse homologue MO25 . This hybrid protein could fully substitute for HymA function in A . nidulans . In addition, the mouse protein itself partially rescued the hym A mutation in the fungus . HymA is thus highly conserved in evolution and probably serves similar functions . The fact that hym A is required for conidiophore development in A . nidulans suggests that homologous genes in other organisms might also be involved in morphogenesis.

Biochim Biophys Acta, 1998 Dec 8, 1429(1), 93 - 101
Interaction of adenosine nucleotide analogs with Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase; Carrasco L et al.; The substrate characteristics and interactions of different adenosine nucleotide analogs with Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase were investigated by steady-state kinetic analysis and calculations of interaction energies . Comparison of Vmax/Km values showed that analogs substituted at C8 in the adenine ring (8-Br-ATP, 8-N3-ATP, 8-N3-ADP) gave almost the same kinetic values as ATP and ADP, whereas those substituted in the ribose hydroxyls (3'(2')-O-(N-methylanthraniloyl)-ATP (MANT-ATP), 3'(2')-O-(N-methylanthraniloyl)-ADP (MANT-ADP), 2'(3')-O-(2,4,6-trinitrophenyl)-ADP (TNP-ADP), 2'(3')-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP)) showed 1-8% the value for the corresponding physiological substrate . A comparison between the experimental results and molecular mechanics calculations was performed, employing a model for the S . cerevisiae PEP carboxykinase-ATP-Mn2+ complex . The calculated interaction energies of S . cerevisiae PEP carboxykinase with ATP, MANT-ATP, TNP-ATP, 8-Br-ATP, and 8-N3-ATP were linearly related (correlation coefficient 0.92) with -ln(Vmax/Km) . This good correlation supports the proposal that the interaction of the substituent with the enzyme affects the interaction of the common region of ATP with the active site, thus leading to effects in Vmax.

Biochim Biophys Acta, 1998 Dec 8, 1429(1), 29 - 39
D-arabinose dehydrogenase and its gene from Saccharomyces cerevisiae; Kim ST et al.; D-Arabinose dehydrogenase was purified 843-fold from the cytosolic fraction of Saccharomyces cerevisiae with a recovery of 9% . The purified enzyme gave two bands with a molecular mass of 40 and 39 kDa on SDS-PAGE . The native enzyme had a molecular mass of 74 kDa as estimated by Sephacryl S-200 chromatography . Therefore, this enzyme was considered to be a heterodimer . The purified enzyme exhibited maximum activity at pH 10.0 and around 30 degrees C . The enzyme catalysed the oxidation of D-arabinose, L-xylose, L-fucose and L-galactose in the presence of NADP+ . The apparent Km values at pH 10.0 with 50 microM NADP+ for D-arabinose, L-xylose, L-fucose, and L-galactose were 161, 24, 98 and 180 mM, respectively . The pH profile of Vmax and kcat/Km showed one ionisable groups around pH 8.3 . D-Erythroascorbic acid was formed in vitro from D-arabinose by D-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase . The N-terminal amino acid sequence of the heavy subunit was Ser-Thr-Glu-Asn-Ile-Val-Glu-Asn-Met-Leu-His-Pro-Lys-Thr- . The N-terminus of the light subunit was blocked . The obtained peptide sequence was identical to the translational product of an unknown open reading frame, YBR149W, in chromosome II of S . cerevisiae . When compared with the translational product of this open reading frame, the peptide sequence was identical to the amino acid sequences of residues 7 to 20 . The first six amino acids of this open reading frame were lost in protein sequence, which may be modified post-translationally . The heavy subunit was composed of 344 amino acid residues and its deduced amino acid sequence contained the motifs I, II, and III of aldo-keto reductase and also leucine zipper motif . This enzyme is the first heterodimeric protein of aldo-keto reductase family . In the deletion mutant of this gene, D-arabinose dehydrogenase activity and D-erythroascorbic acid were not detected.

Biochem Biophys Res Commun, 1999 Jan 19, 254(2), 345 - 50
Med8, a subunit of the mediator CTD complex of RNA polymerase II, directly binds to regulatory elements of SUC2 and HXK2 genes; Chaves RS et al.; In a search to identify new factors required for expression of SUC2 gene in Saccharomyces cerevisiae, we have partially purified a 27 kDa protein (p27) that bound both the DRSs of the HXK2 gene and the UASs of SUC2 gene . The amino terminal sequence of p27 identified the MED8 gene (open reading frame YBR193C), located in chromosome II of S . cerevisiae, as the gene coding for the protein . Disruption of this gene has demonstrated that is an essential gene for yeast growth . To determine whether the p27 protein represents the Med8 product, we expressed MED8 gene in E . coli and demonstrated that the heterologous synthesized protein specifically binds to both UASSUC2 and DRS2HXK2 . This observation suggests that Med8 may be important for the coupling of the glucose repression pathway of SUC2 gene to the HXK2 gene expression . Med8 has been described as a mediator protein interacting with the CTD of the RNA polymerase II . Thus, the role of Med8 could be to act as coupling factor by linking activating and repressing transcription complexes to the RNA polymerase II holoenzyme transcriptional machinery .

J Biol Chem, 1999 Jan 29, 274(5), 2609 - 12
A built-in arginine finger triggers the self-stimulatory GTPase-activating activity of rho family GTPases; Zhang B et al.; Signal transduction through the Rho family GTPases requires regulated cycling of the GTPases between the active GTP-bound state and the inactive GDP-bound state . Rho family members containing an arginine residue at position 186 in the C-terminal polybasic region were found to possess a self-stimulatory GTPase-activating protein (GAP) activity through homophilic interaction, resulting in significantly enhanced intrinsic GTPase activities . This arginine residue functions effectively as an "arginine finger" in the GTPase activating reaction to confer the catalytic GAP activity but is not essential for the homophilic binding interactions of Rho family proteins . The arginine 186-mediated negative regulation seems to be absent from Cdc42, a Rho family member important for cell-division cycle regulation, of lower eukaryotes, yet appears to be a part of the turn-off machinery of Cdc42 from higher eukaryotes . Introduction of the arginine 186 mutation into S . cerevisiae CDC42 led to phenotypes consistent with down-regulated CDC42 function . Thus, specific Rho family GTPases may utilize a built-in arginine finger, in addition to RhoGAPs, for negative regulation.

Eur J Biochem, 1999 Jan, 259(1-2), 244 - 52
Analysis of a conditional degradation signal in yeast and mammalian cells; Levy F et al.; The N-end rule pathway is a ubiquitin-dependent proteolytic system, the targets of which include proteins that bear destabilizing N-terminal residues . The latter are a part of the degradation signal called the N-degron . Arg-DHFRts, an engineered N-end rule substrate, bears N-terminal arginine (a destabilizing residue) and DHFRts {a temperature-sensitive mouse dihydrofolate reductase (DHFR) moiety} . Previous work has shown that Arg-DHFRts is long-lived at 23 degreesC but short-lived at 37 degreesC in the yeast Saccharomyces cerevisiae . In the present work, we extended this analysis, and found that the degradation of Arg-DHFRts can be nearly completely inhibited in vivo by methotrexate (MTX), a low-Mr ligand of DHFR . In S . cerevisiae, Arg-DHFRts is degraded at 37 degreesC exclusively by the N-end rule pathway, whereas in mouse cells the same protein at the same temperature is also targeted by another proteolytic system, through a degron in the conformationally perturbed DHFRts moiety . In mouse cells, MTX completely inhibits the degradation of Arg-DHFRts through its degron within the DHFRts moiety, but only partially inhibits degradation through the N-degron . When the N-terminus of Arg-DHFRts was extended with a 42-residue lysine-lacking extension, termed eDeltaK, the resulting Arg-eDeltaK-DHFRts was rapidly degraded at both 23 degreesC and 37 degreesC . Moreover, the degradation of Arg-eDeltaK-DHFRts, in contrast with that of Arg-DHFRts, could not be inhibited by MTX, suggesting that the metabolic stability of Arg-DHFRts at 23 degreesC results, at least in part, from steric inaccessibility of its N-terminal arginine . The N-degron of Arg-DHFRts is the first example of a portable degradation signal the activity of which can be modulated in vivo by a cell-penetrating compound . We discuss implications of this advance and the mechanics of targeting by the ubiquitin system.

Mol Cell Biol, 1999 Feb, 19(2), 1369 - 80
Control of Saccharomyces cerevisiae filamentous growth by cyclin-dependent kinase Cdc28; Edgington NP et al.; The ascomycete Saccharomyces cerevisiae exhibits alternative vegetative growth states referred to as the yeast form and the filamentous form, and it switches between the two morphologies depending on specific environmental signals . To identify molecules involved in control of morphologic differentiation, this study characterized mutant S . cerevisiae strains that exhibit filamentous growth in the absence of the normal external signals . A specific amino acid substitution in the cyclin-dependent protein kinase Cdc28 was found to cause constitutive expression of most filamentous growth characteristics . These effects include specifically modified cell polarity characteristics in addition to the defined shape and division cycle alterations typical of the filamentous form . Several other mutations affecting Cdc28 function also had specific effects on filamentous growth . Constitutive filamentous growth resulting from deletion of the protein kinase Elm1 was prevented by modification of Cdc28 such that it could not be phosphorylated on tyrosine residue 19 . In addition, various mutations affecting Hsl1 or Swe1, known or presumed components of a protein kinase cascade that mediates Cdc28 phosphorylation on Y19, either prevented or enhanced filamentous growth . The data suggest that a protein kinase cascade involving Elm1, Hsl1, and Swe1 can modulate Cdc28 activity and that Cdc28 in turn exerts global effects that cause filamentous growth.

J Cell Sci, 1999 Feb, 112 ( Pt 3), 339 - 47
Antagonistic effects of NES and NLS motifs determine S . cerevisiae Rna1p subcellular distribution; Feng W et al.; Nucleus/cytosol exchange requires a GTPase, Ran . In yeast Rna1p is the GTPase activating protein for Ran (RanGAP) and Prp20p is the Ran GDP/GTP exchange factor (GEF) . RanGAP is primarily cytosolic and GEF is nuclear . Their subcellular distributions led to the prediction that Ran-GTP hydrolysis takes place solely in the cytosol and GDP/GTP exchange solely in the nucleus . Current models propose that the Ran-GTP/Ran-GDP gradient across the nuclear membrane determines the direction of exchange . We provide three lines of evidence that Rna1p enters and leaves the nuclear interior . (1) Rna1p possesses leucine-rich nuclear export sequences (NES) that are able to relocate a passenger karyophilic protein to the cytosol; alterations of consensus residues re-establish nuclear location . (2) Rna1p possesses other sequences that function as a novel nuclear localization sequence able to deliver a passenger cytosolic protein to the nucleus . (3) Endogenous Rna1p location is dependent upon Xpo1p/Crm1p, the yeast exportin for leucine-rich NES-containing proteins . The data support the hypothesis that Rna1p exists on both sides of the nuclear membrane, perhaps regulating the Ran-GTP/Ran-GDP gradient, participating in a complete RanGTPase nuclear cycle or serving a novel function.

Yeast, 1998 Dec, 14(16), 1471 - 510
Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae; Daum G et al.; The yeast Saccharomyces cerevisiae is a powerful experimental system to study biochemical, cell biological and molecular biological aspects of lipid synthesis . Most but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of this unicellular eukaryote have been cloned, and many gene products have been functionally characterized . Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes, turnover and degradation of complex lipids, regulation of lipid biosynthesis, and linkage of lipid metabolism to other cellular processes . Here we summarize current knowledge about lipid biosynthetic pathways in S . cerevisiae and describe the characteristic features of the gene products involved . We focus on recent discoveries in these fields and address questions on the regulation of lipid synthesis, subcellular localization of lipid biosynthetic steps, cross-talk between organelles during lipid synthesis and subcellular distribution of lipids . Finally, we discuss distinct functions of certain key lipids and their possible roles in cellular processes.

Cell Signal, 1998 Nov, 10(10), 699 - 711
Casein kinase I: spatial organization and positioning of a multifunctional protein kinase family; Gross SD et al.; The casein kinase I family of serine/threonine protein kinases is highly conserved from yeast to humans . Until only recently, both the function and regulation of these enzymes remained poorly uncharacterised in that they appeared to be constitutively active and were capable of phosphorylating an untold number of other proteins . While relatively little was known regarding the exact function of the higher eukaryotic isoforms, the casein kinase I (CKI) isoforms from yeast have been genetically linked to vesicular trafficking, DNA repair, cell cycle progression and cytokinesis . All five S . cerevisiae isoforms are known to be associated with discrete cellular compartments and this localization has been shown to be absolutely essential for their respective functions . New evidence now suggests that the CKI isoforms in more complex systems also exhibit non-homogeneous subcellular distributions that may prove vital to defining the function and regulation of these enzymes . In particular, CKIalpha, the most-characterized vertebrate isoform, is associated with cytosolic vesicles, the mitotic spindle and structures within the nucleus . Functions associated with these localizations coincide with those previously reported in yeast, suggesting a conservation of function . Other reports have indicated that each of the remaining CKI isoforms have the capacity to make associations with components of several signal transduction pathways, thereby channeling CKI function toward specific regulatory events . This review will examine what is now known about the higher eukaryotic CKI family members from the perspective localization as a means of gaining a better understanding of the function and regulation of these kinases.

J Biol Chem, 1999 Jan 15, 274(3), 1783 - 90
Red1p, a MEK1-dependent phosphoprotein that physically interacts with Hop1p during meiosis in yeast; de los Santos T et al.; The synaptonemal complex (SC) is a proteinaceous structure formed between pairs of homologous chromosomes during prophase I of meiosis . The proper assembly of axial elements (AEs), lateral components of the SC, during meiosis in the yeast, Saccharomyces cerevisiae, is essential for wild-type levels of recombination and for the accurate segregation of chromosomes at the first meiotic division . Genetic experiments have indicated that the stoichiometry between two meiosis-specific components of AEs in S . cerevisiae, HOP1 and RED1, is critical for proper assembly and function of the SC . A third meiosis-specific gene, MEK1, which encodes a putative serine/threonine protein kinase, is also important for proper AE function, suggesting that AE formation is regulated by phosphorylation . In this paper, we demonstrate that Mek1p is a functional kinase in vitro and that catalytic activity is an essential part of the meiotic function of Mek1 in vivo . Immunoblot analysis revealed that Red1p is a MEK1-dependent phosphoprotein . Co-immunoprecipitation experiments demonstrated that the interaction between Hop1p and Red1p is enhanced by the presence of MEK1 . Thus, MEK1-dependent phosphorylation of Red1p facilitates the formation of Hop1p/Red1p hetero-oligomers, thereby enabling the formation of functional AEs.

Biochim Biophys Acta, 1999 Jan 6, 1426(2), 385 - 400
The Gas1 glycoprotein, a putative wall polymer cross-linker; Popolo L et al.; The yeast cell wall, which for years has been regarded as a static cellular component, has been revealed to be dynamic in its structure and composition and complex in its enzymatic activity . The S . cerevisiae cell wall is composed of beta-1,3/beta-1,6-glucans, mannoproteins, and chitin, which are assembled into an extracellular matrix essential for maintenance of cell integrity . Gas1p, a glycoprotein anchored to the outer leaflet of the plasma membrane through a glycosylphosphatidylinositol, plays a key role in cell wall assembly . Loss of Gas1p leads to several morphogenetic defects and to a decrease in the amount of cross-links between the cell wall glucans . These defects in turn trigger a compensatory response that guarantees cell viability . Several Gas1p homologs have been isolated from Candida species and S . pombe . The Gas1p family also includes two plant proteins with endo-beta-1,3-glucanase activity . Sequence comparisons reveal that Gas1p family proteins have a modular organization of domains . The genetic and molecular analyses reviewed here suggest that Gas1p could play a role as a polymer cross-linker, presumably by catalyzing a transglycosylation reaction.

Biochim Biophys Acta, 1999 Jan 6, 1426(2), 335 - 45
Mannosylphosphate transfer to yeast mannan; Jigami Y et al.; Mannoproteins located in the outermost layer of yeast cell wall determine the wall's porosity and thereby regulate leakage of proteins from the periplasmic space and entrance of macromolecules from the environment . In several yeasts, including Saccharomyces cerevisiae, the glycan portion of mannoproteins is composed not only of neutral oligosaccharides containing mannose and N-acetylglucosamine, but also of acidic oligosaccharides containing mannosylphosphate . The mannosylphosphate residues confer a net negative charge on the cell wall, and so change the properties and environment of the cell surface . Progress on mannosylphosphorylation and its regulation in S . cerevisiae is summarized . Two genes required for mannosylphosphate transfer, MNN4 and MNN6, have been cloned, and a functional analysis of these genes suggests a mechanism for mannosylphosphate transfer . Possible functions for mannosylphosphate transfer in yeast are also discussed . These include supply of GMP for sugar nucleotide transport in the Golgi, cross-linking of mannoproteins to beta-glucan, and a cellular stress response to environmental changes . Glycans in pathogenic yeast and protozoa are also modified with mannosylphosphate, and the potential contribution of this modification to the pathogenicity of these organisms is evaluated.

Eur J Biochem, 1998 Dec 1, 258(2), 402 - 10
Isolation of a cDNA from Arabidopsis thaliana that complements the sec14 mutant of yeast; Jouannic N et al.; The SEC14 gene of Saccharomyces cerevisiae codes for a phosphatidylinositol-transfer protein (Sec14p(sc)) which is capable of transferring both phosphatidylinositol and phosphatidylcholine between membranes in vitro . Genetic and biochemical studies conducted in S . cerevisiae have shown that this protein acts as an inhibitor of phosphatidylcholine biosynthesis via the so-called Kennedy pathway only . This inhibition is controlled by the binding of phospholipids to the Sec14p(sc) protein . Here we describe the isolation of a cDNA from Arabidopsis thaliana by functional complementation of a sec14(ts) mutant of S . cerevisiae . This cDNA, designated AtSEC14, is capable of restoring the growth of the sec14(ts) mutant at the restrictive temperature of 37 degrees C . Extracellular invertase measurements indicated that the cDNA can partly restore protein secretion . In addition, the phosphatidylinositol-transfer activity measured in protein extracts is greatly enhanced in the complemented mutant strain when compared with the sec14(ts) mutant . The best sequence similarity at the amino acid level is found with the Sec14p protein of S . cerevisiae (36.5% similarity), and most of the amino acids that are thought to be involved in the binding of phospholipids in the yeast protein are conserved in the AtSEC14 gene product . Southern analysis suggests the presence of a single gene in the Arabidopsis genome, although the existence of distantly related sequences cannot be excluded . This gene is expressed in roots, leaves, flowers and siliques of Arabidopsis.

Curr Microbiol, 1999 Feb, 38(2), 122 - 5
The autonomously replicating sequence (ARS) of the yeast Saccharomyces exiguus Yp74L-3; Hisatomi T et al.; Fragments containing ARSes were cloned from the genomic DNA of the yeast Saccharomyces exiguus Yp74L-3, and the essential regions for ARSes were restricted for these fragments . Mapping studies of ARS-acting sequences in one of these fragments suggested that S . exiguus recognizes a sequence as an ARS that is different from that recognized by Saccharomyces cerevisiae . Two ARS essential regions of S . exiguus were sequenced, and an ARS core consensus sequence of S . exiguus was deduced to be MATTAMWAWWTK . This sequence differs significantly from that of S . cerevisiae in two positions, suggesting that these nucleotide substitutions cause the difference in the ARS-recognition modes between S . exiguus and S . cerevisiae.

Glycoconj J, 1998 Aug, 15(8), 815 - 22
Characterization of oligosaccharides from an antigenic mannan of Saccharomyces cerevisiae; Young M et al.; Mannans of the yeast Saccharomyces cerevisiae have been implicated as containing the allergens to which bakers and brewers are sensitive and also the antigen recognized by patients with Crohn's disease . A fraction of S . cerevisiae mannan, Sc500, having high affinity for antibodies in Crohn's patients has been characterized by NMR spectroscopy followed by fragmentation using alkaline elimination, partial acid hydrolysis and acetolysis . The released oligosaccharides were separated by gel filtration on a Biogel P4 column and analyzed by fluorescence labeling, HPLC and methylation analysis . The relationship between structure and antigen activity was measured by competitive ELISA . The antigenic activity of the original high molecular weight mannan could be ascribed to terminal Manalpha1-->3Manalpha1-->2 sequences which are rarely found in human glycoproteins but were over-represented in Sc500 compared to other yeast mannans.

J Eukaryot Microbiol, 1998 Nov-Dec, 45(6), 589 - 99
The HSP70 gene family in Pneumocystis carinii: molecular and phylogenetic characterization of cytoplasmic members; Stedman TT et al.; Pneumocystis carinii, a major opportunistic lung pathogen of AIDS patients, is found in a number of mammals and is proposed to be a member of the fungi . In this work, several members of the highly conserved HSP70 multigene family were characterized from rat-derived P . carinii . Previously, we reported characterization of the ER resident HSP70 homolog known as BiP from prototype (P.c . carinii) and variant (P . c . rattus) strains of the organism . We report here, from P . c . carinii, characterization of Pcsa1, an HSP70 homolog that encodes a cognate/stress-induced HSP70 homolog of the SSA subfamily in Saccharomyces cerevisiae . We also identify, from both rat strains and from a human isolate of P . carinii (P.c . hominis), a third set of HSP70 homologs that apparently encode a ribosome-associated cytoplasmic HSP70 homologous to the S . cerevisiae SSB subfamily . Our data indicate that Pcsal mRNA, like Pcbip mRNA, bears an intron in the 5' untranslated region, is induced by heat shock, and suggest that this gene undergoes alternative transcription and splicing . The SSB homologs display significant sequence heterogeneity between P . carinii source strains, supporting the genetic divergence and likely speciation of P . carinii isolates within and between host species . Phylogenetic analysis with the PcSA1 protein supports inclusion of P . carinii among the higher fungi.

J Cell Biol, 1998 Dec 28, 143(7), 1859 - 69
Pex18p and Pex21p, a novel pair of related peroxins essential for peroxisomal targeting by the PTS2 pathway; Purdue PE et al.; We have identified ScPex18p and ScPex21p, two novel S . cerevisiae peroxins required for protein targeting via the PTS2 branch of peroxisomal biogenesis . Targeting by this pathway is known to involve the interaction of oligopeptide PTS2 signals with Pex7p, the PTS2 receptor . Pex7p function is conserved between yeasts and humans, with defects in the human protein causing rhizomelic chondrodysplasia punctata (RCDP), a severe, lethal peroxisome biogenesis disorder characterized by aberrant targeting of several PTS2 peroxisomal proteins, but uncertainty remains about the subcellular localization of this receptor . Previously, we have reported that ScPex7p resides predominantly in the peroxisomal matrix, suggesting that it may function as a highly unusual intraorganellar import receptor, and the data presented in this paper identify Pex18p and Pex21p as key components in the targeting of Pex7p to peroxisomes . They each interact specifically with Pex7p both in two-hybrid analyses and in vitro . In cells lacking both Pex18p and Pex21p, Pex7p remains cytosolic and PTS2 targeting is completely abolished . Pex18p and Pex21p are weakly homologous to each other and display partial functional redundancy, indicating that they constitute a two-member peroxin family specifically required for Pex7p and PTS2 targeting.

J Bacteriol, 1999 Jan, 181(1), 347 - 52
APT1, but not APT2, codes for a functional adenine phosphoribosyltransferase in Saccharomyces cerevisiae; Alfonzo JD et al.; The yeast Saccharomyces cerevisiae has two separate genes (APT1 and APT2) that encode two potentially different forms of adenine phosphoribosyltransferase (APRT) . However, genetic analysis indicated that only APT1 could code for a complementing activity . Cloning and expression of both the APT1 and APT2 genes in Escherichia coli showed that although discrete proteins (APRT1 and APRT2) were made by these genes, only APRT1 had detectable APRT activity . Northern and Western blot analyses demonstrated that only APT1 was transcribed and translated under normal physiological conditions in yeast . Phylogenetic analysis revealed that APRT1 and APRT2 are evolutionary closely related and that they arise from a gene duplication event . We conclude that APT1 is the functional gene in S . cerevisiae and that APT2 is a pseudogene.

Mol Gen Genet, 1998 Nov, 260(2-3), 232 - 41
Efficient initiation of S-phase in yeast requires Cdc40p, a protein involved in pre-mRNA splicing; Boger-Nadjar E et al.; The S . cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is essential only at elevated temperatures . Cells carrying mutations in this gene arrest with a large bud and a single nucleus with duplicated DNA content . Cdc40p is also required for spindle establishment or maintenance . Sequence analysis reveals that CDC40 is identical to PRP17, a gene involved in pre-mRNA splicing . In this paper, we show that Cdc40p is required at all temperatures for efficient entry into S-phase and that cell cycle arrest associated with cdc40 mutations is independent of all the known checkpoint mechanisms . Using immunofluorescence, we show that Cdc40p is localized to the nuclear membrane, weakly associated with the nuclear pore . Our results point to a link between cell cycle progression, pre-mRNA splicing, and mRNA export.

Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15388 - 93
A protein required for nuclear-protein import, Mog1p, directly interacts with GTP-Gsp1p, the Saccharomyces cerevisiae ran homologue; Oki M et al.; We previously isolated 25 temperature-sensitive gsp1 alleles of Saccharomyces cerevisiae Ran homologue, each of which possesses amino acid changes that differ from each other . We report here isolation of three multicopy suppressors-PDE2, NTF2, and a gene designated MOG1-all of which rescued a growth defect of these gsp1 strains . The gsp1 suppression occurred even in the absence of GSP2, another S . cerevisiae GSP1-like gene . Previously, NTF2 was reported to suppress gsp1 but not PDE2 . Mog1p, with a calculated molecular mass of 24 kDa, was found to be encoded by the yeast ORF YJR074W . Both MOG1 and NTF2 suppressed a series of gsp1 alleles with similar efficiency, and both suppressed gsp1 even with a single gene dose . Consistent with the high efficiency of gsp1 suppression, Mog1p directly bound to GTP, but not to GDP-Gsp1p . The disruption of MOG1 made yeast temperature-sensitive for growth . Deltamog1, which was suppressed by overexpression of NTF2, was found to have a defect in both classic and nonclassic nuclear localization signal-dependent nuclear-protein imports, but not in mRNA export . Thus, Mog1p, which was localized in the nucleus, is a Gsp1p-binding protein involved in nuclear-protein import and that functionally interacts with Ntf2p . Furthermore, the finding that PDE2 suppressed both gsp1 and rna1-1 indicates that the Ran GTPase cycle is regulated by the Ras-cAMP pathway.

Gene, 1998 Nov 26, 223(1-2), 67 - 76
Cre/loxP-mediated in vivo excision of large segments from yeast genome and their amplification based on the 2microm plasmid-derived system; Yoon YG et al.; In vivo excision and amplification of pre-determined, large genomic segments, directly from the genome of a natural host, provides an alternative to conventional cloning in foreign vectors . Using this approach, we have devised an in vivo procedure for excising large segments of Saccharomyces cerevisiae genome using Cre/loxP system of bacteriophage P1, followed by amplification of excised circles, as based on the yeast 2microm plasmid-derived ori and Flp/FRT machinery . To provide the excision and replication enzymes, trans-acting genes cre and FLP, which were under a very tight control of GAL1 and GAL10 promoters, respectively, were inserted by homologous recombination into the URA3 gene on chromosome V . Two parallel loxP sequences, which serve as the recognition sites for the Cre recombinase, were also integrated into the genome at pre-determined sites that are 50-100kb apart . Moreover, 2microm ori, REP3 and two inverted FRTs, which serve as a conditional replication system, were also integrated between the loxP sites . The strain carrying all these inserted elements was perfectly stable . Only after the induction by galactose of the Cre excision function, the genomic segment flanked by two loxP sites was excised and circularized . Applying this procedure, the 50-kb LEU2-YCR011c and 100-kb LEU2-YCR035c regions of chromosome III were successfully excised from the S . cerevisiae genome, whereas the 2microm ori, as aided by FRT/Flp, provided the amplification function . Such excised and amplified genomic segments can be used for the sequencing and functional analysis of any yeast genes.

Yeast, 1998 Nov, 14(15), 1417 - 27
Quantitative analysis of yeast gene function using competition experiments in continuous culture; Baganz F et al.; One possible route to the evaluation of gene function is a quantitative approach based on the concepts of metabolic control analysis (MCA) . An important first step in such an analysis is to determine the effect of deleting individual genes on the growth rate (or fitness) of S . cerevisiae . Since the specific growth-rate effects of most genes are likely to be small, we employed competition experiments in chemostat culture to measure the proportion of deletion mutants relative to that of a standard strain by using a quantitative PCR method . In this paper, we show that both densitometry and GeneScan analysis can be used with similar accuracy and reproducibility to determine the proportions of (at least) two strains simultaneously, in the range 10-90% of the total cell population . Furthermore, we report on a model competition experiment between two diploid nuclear petite mutants, homozygous for deletions in the cox5a or pet191 genes, and the standard strain (ho::kanMX4/ho::kanMX4) in chemostat cultures under six different physiological conditions . The results indicate that competition experiments is continuous culture are a suitable method to distinguish quantitatively between deletion mutants that qualitatively exhibit the same phenotype.

Yeast, 1998 Nov, 14(15), 1373 - 86
Cloning and characterization of the peroxisomal acyl CoA oxidase ACO3 gene from the alkane-utilizing yeast Yarrowia lipolytica; Wang H et al.; The ACO3 gene, which encodes one of the acyl-CoA oxidase isoenzymes, was isolated from the alkane-utilizing yeast Yarrowia lipolytica as a 10 kb genomic fragment . It was sequenced and found to encode a 701-amino acid protein very similar to other ACOs, 67.5% identical to Y . lipolytica Aco1p and about 40% identical to S . cerevisiae Pox1p . Haploid strains with a disrupted allele were able to grow on fatty acids . The levels of acyl-CoA oxidase activity in the ACO3 deleted strain, in an ACO1 deleted strain and in the wild-type strain, suggested that ACO3 encodes a short chain acyl-CoA oxidase isoenzyme . This narrow substrate spectrum was confirmed by expression of Aco3p in E . coli.

J Cell Biol, 1998 Dec 14, 143(6), 1471 - 84
Interaction of ZPR1 with translation elongation factor-1alpha in proliferating cells; Gangwani L et al.; The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF) . Homologues of ZPR1 were identified in yeast and mammals . These ZPR1 proteins bind to eukaryotic translation elongation factor-1alpha (eEF-1alpha) . Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1alpha and the redistribution of both proteins to the nucleus . In the yeast Saccharomyces cerevisiae, genetic analysis demonstrated that ZPR1 is an essential gene . Deletion analysis demonstrated that the NH2-terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S . cerevisiae . The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation . Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth . Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth . We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1alpha contributes to normal cellular proliferation.

J Bacteriol, 1998 Dec, 180(24), 6736 - 42
Disruption of PMR1, encoding a Ca2+-ATPase homolog in Yarrowia lipolytica, affects secretion and processing of homologous and heterologous proteins; Sohn YS et al.; The Yarrowia lipolytica PMR1 gene (YlPMR1) is a Saccharomyces cerevisiae PMR1 homolog which encodes a putative secretory pathway Ca2+-ATPase . In this study, we investigated the effects of a YlPMR1 disruption on the processing and secretion of native and foreign proteins in Y . lipolytica and found variable responses by the YlPMR1-disrupted mutant depending on the protein . The secretion of 32-kDa mature alkaline extracellular protease (AEP) was dramatically decreased, and incompletely processed precursors were observed in the YlPMR1-disrupted mutant . A 36- and a 52-kDa premature AEP were secreted, and an intracellular 52-kDa premature AEP was also detected . The acid extracellular protease activity of the YlPMR1-disrupted mutant was increased by 60% compared to that of the wild-type strain . The inhibitory effect of mutations in secretory pathway Ca2+-ATPase genes on the secretion of rice alpha-amylase was also observed in the Y . lipolytica and S . cerevisiae PMR1-disrupted mutants . Unlike rice alpha-amylase, the secretion of Trichoderma reesei endoglucanase I (EGI) was not influenced by the YlPMR1 disruption . However, the secreted EGI from the YlPMR1-disrupted mutant had different characteristics than that of the control . While wild-type cells secreted the hyperglycosylated form of EGI, hyperglycosylation was completely absent in the YlPMR1-disrupted mutant . Our results indicate that the effects of the YlPMR1 disruption as manifested by the phenotypic response depend on the characteristics of the reporter protein in the recombinant yeast strain evaluated.

Nucleic Acids Res, 1999 Jan 1, 27(1), 161 - 4
A comprehensive database for the small nucleolar RNAs from Saccharomyces cerevisiae; Samarsky DA et al.; Small nucleolar RNAs (snoRNAs) are involved in cleavage of rRNA, modification of rRNA nucleotides and, perhaps, other aspects of ribosome biogenesis in eukaryotic cells . Scores of snoRNAs have been discovered in recent years from various eukaryotes, and the total number is predicted to be up to 200 different snoRNA species per individual organism . We have created a comprehensive database for snoRNAs from the yeast Saccharomyces cerevisiae which allows easy access to detailed information about each species known (almost 70 snoRNAs are featured) . The database consists of three major parts: (i) a utilities section; (ii) a master table; and (iii) a collection of tables for the individual snoRNAs . The utilities section provides an introduction to the database . The master table lists all known S . cerevisiae snoRNAs and their major properties . Information in the individual tables includes: alternate names, size, family classification, genomic organization, sequences (with major features identified), GenBank accession numbers, occurrence of homologues, gene disruption phenotypes, functional properties and associated RNAs and proteins . All information is accompanied with appropriate literature references . The database is available on the World Wide Web , and should be useful for a wide range of snoRNA studies.

Cell, 1998 Nov 25, 95(5), 705 - 16
Complex formation and functional versatility of Mre11 of budding yeast in recombination; Usui T et al.; Meiotic recombination of S . cerevisiae contains two temporally coupled processes, formation and processing of double-strand breaks (DSBs) . Mre11 forms a complex with Rad50 and Xrs2, acting as the binding core, and participates in DSB processing . Although these proteins are also involved in DSB formation, Mre11 is not necessarily holding them . The C-terminal region of Mre11 is required only for DSB formation and binds to some meiotic proteins . The N-terminal half specifies nuclease activities that are collectively required for DSB processing . Mre11 has a DNA-binding site for DSB formation and another site for DSB processing . It has two regions to bind to Rad50 . Mre11 repairs methyl methanesulfonate-induced DSBs by reactions that require the nuclease activities and those that do not.

Mol Cell, 1998 Nov, 2(5), 571 - 80
Ubiquitination and degradation of the substrate recognition subunits of SCF ubiquitin-protein ligases; Zhou P et al.; The S . cerevisiae SCFCdc4p ubiquitin-protein ligase complex promotes cell cycle transitions through degradation of cell cycle regulators . To investigate SCFCdc4p regulation in vivo, we examined the stability of individual SCFCdc4p components . Whereas Cdc53p and Skp1p were stable, Cdc4p, the F box-containing component responsible for substrate recognition, was short lived and subject to SCF-mediated ubiquitination . Grr1p, another F box component of SCF complexes, was also ubiquitinated . A stable truncated Cdc4pF-beta-gal hybrid protein capable of binding Skp1p and entering into an SCF complex interfered with proteolysis of SCF targets and inhibited cell proliferation . The finding that the F box-containing SCF components are unstable suggests a mechanism of regulating SCF function through ubiquitination and proteolysis of F box components.

Mol Cell Probes, 1998 Dec, 12(6), 397 - 405
Sequence analysis of DNA randomly amplified from the Saccharomyces cerevisiae genome; McGrath A et al.; Despite its widespread use, the molecular basis of random amplification is poorly understood . Here the basis of random amplification has been investigated by cloning and sequencing the products of a random amplification of polymorphic DNA (RAPD) amplification from Saccharomyces cerevisiae DNA . The genomic origin of the amplified products was determined by sequence comparison with the S . cerevisiae Genome Database (SGD) . This allowed analysis of the degree of identity between the random primer and the primer binding sites on the genome . There was no relationship between RAPD size, GC content and relative abundance . The degree of matching between the primer and the primer binding sites increased towards the 3; end of the primer and decreased towards the 5; end . The maximum number of mismatches observed between primer and primer binding sites was never more than one between positions 1-7 of the primer . Nucleotide compositional biases were also observed upstream and downstream of the primer binding site with a marked preference for AT richness upstream of the primer binding sites and for a GC preference directly following the 3; end of the primer . These findings have important ramifications for primer design for multiplex, low stringency and degenerate polymerase chain reaction (PCR) .

Biochemistry, 1998 Nov 24, 37(47), 16620 - 6
Phosphorylation of ribosomal protein P0 is not essential for ribosome function but can affect translation; Rodriguez-Gabriel MA et al.; Protein P0, an essential component of the eukaryotic ribosomal stalk, is found phosphorylated in the ribosome . Substitution of serine 302 in the amino acid sequence of the Saccharomyces cerevisiae P0 by either aspartic acid or cysteine abolishes in vitro and in vivo phosphorylation of the protein . On the contrary, the replacement of this serine by a threonine results in an increase in the protein phosphorylation under both sets of conditions . Therefore, this serine residue, which is part of a consensus casein kinase II modification site, SDDD, seems to be the phosphorylation site in protein P0 . The effect of the mutations on the protein activity has been tested in S . cerevisiae W303dGP0 and D67dGP0, both of which carry a genomic P0 gene under the control of the GAL1 promoter . Transformation of the mutated genes in S . cerevisiae W303dGP0 allows cell growth at 30 degreesC in glucose-to repress the wild-type P0 expression-at the same rate as controls, and the ribosomes contain a normal amount of the other stalk components . A similar absence of effect of the mutations on growth was found in strain D67dGP0, which has ribosomes deprived of the P1 and P2 proteins . Therefore, P0 phosphorylation is not a requirement for ribosome activity in standard growth conditions either in the presence or in the absence of the other stalk proteins . However, a phenotypic effect is detected in the case of strain D67 transformed with the overphosphorylated threonine containing P0, which contrary to the wild-type and the other mutated proteins is unable to support cell growth at 37 degreesC in the presence of either 0.3 M NaCl or 0.8 M sorbitol . In vitro polymerizing tests indicate that this effect is not due to the thermosensitivity of the mutated protein . The results indicate that although P0 phosphorylation is not required for the overall ribosome activity, it may affect the expression of specific proteins involved in metabolic processes such as osmoregulation.

Acta Histochem, 1998 Nov, 100(4), 419 - 38
Cytochemical images of secretion in Saccharomyces cerevisiae and animal cells are different; Vorisek J; Like in animal cells, the major secretory pathway of the ascomycetous budding yeast Saccharomyces (s.) cerevisiae consists of membrane-bound compartments which transport soluble and membrane (glyco)peptides to lysosomal vacuoles, cell wall, or out of the cell . The established model of the cellular machinery of the yeast secretory pathway was deduced largerly from molecular ex situ analyses and for budding yeast cells it was assumed to be identical with that of secretory animal cells . Interphase yeast cells were never considered . Glycosylation of peptides was detected in the endoplasmic reticulum (ER) and the putative Golgi cisternae . Coated membrane vesicles were assumed to transport intermediates into and within the Golgi cascade . Proteolytic trimming would occur in the last Golgi compartment . Golgi-derived membrane vesicles would serve for exocytosis or fuse with lysosomal vacuoles . In contrast to this notion, yeast cytologists showed specific features of secretion in S . cerevisiae and other Ascomycetes . Cytochemical observations in situ of both dividing and interphase yeast showed direct communication between nuclear envelope, ER and segregated Golgi cisternae . A new class of constitutive conveyors, coated protein globules smaller than membrane vesicles, was shown to exist throughout the cell cycle . The function of Golgi-derived membrane vesicles was constrained to promotion of exocytosis in budding yeast . Some of the Golgi apparatus functions were detected in both these classes of exocytotic conveyors . Uptake (phagocytosis) of transport conveyors and lipoprotein condensates has been shown to deliver enzymes and secretory compounds into vacuoles . This simplified machinery of secretion, postulated for S . cerevisiae, does not include the Golgi cascade.

J Biol Chem, 1998 Dec 11, 273(50), 33635 - 43
The difference in recognition of terminal tripeptides as peroxisomal targeting signal 1 between yeast and human is due to different affinities of their receptor Pex5p to the cognate signal and to residues adjacent to it; Lametschwandtner G et al.; Pex5p is the receptor for the peroxisomal targeting signal 1 (PTS1) that consists of a C-terminal tripeptide (consensus (S/A/C)(K/R/H)(L/M)) . Hexadecapeptides recognized by Pex5p from Homo sapiens and Saccharomyces cerevisiae were identified by screening a two-hybrid peptide library, and the targeting ability of the peptides was demonstrated using the green fluorescent protein as reporter . The PTS1 receptors recognized in a species-specific manner a broad range of C-terminal tripeptides, and these are reported herein . In addition, residues upstream of the tripeptide influenced the strength of the interaction in the two-hybrid system as well as in an in vitro competition assay . In peptides interacting with the human protein, hydrophobic residues were found with high frequency especially at positions -2 and -5, whereas peptides interacting with S . cerevisiae Pex5p were more hydrophilic and frequently contained arginine at position -2 . In instances where the terminal tripeptide deviated from the consensus, upstream residues exerted a greater influence on the ability of the hexadecapeptides to bind Pex5p.

J Biol Chem, 1998 Dec 11, 273(50), 33184 - 91
Molecular characterization of Saccharomyces cerevisiae Delta3, Delta2-enoyl-CoA isomerase; Geisbrecht BV et al.; We report here the identification of the Saccharomyces cerevisiae peroxisomal Delta3,Delta2-enoyl-CoA isomerase, an enzyme that is essential for the beta-oxidation of unsaturated fatty acids . The yeast gene YLR284C was identified in an in silico screen for genes that contain an oleate response element, a transcription factor-binding site common to most fatty acid-induced genes . Growth on oleic acid resulted in a significant increase in YLR284C mRNA, demonstrating that it is indeed an oleate-induced gene . The deduced product of YLR284C contains a type 1 peroxisomal targeting signal-like sequence at its C terminus and localizes to the peroxisome in a PEX8-dependent manner . Removal of YLR284C from the S . cerevisiae genome eliminated growth on oleic acid, but had no effect on peroxisome biogenesis, indicating a role for YLR284C in fatty acid metabolism . Cells lacking YLR284C had no detectable Delta3,Delta2-enoyl-CoA isomerase activity, and a bacterially expressed form of this protein catalyzed the isomerization of 3-cis-octenoyl-CoA to 2-trans-octenoyl-CoA with a specific activity of 16 units/mg . We conclude that YLR284C encodes the yeast peroxisomal Delta3,Delta2-enoyl-CoA isomerase and propose a new name, ECI1, to reflect its enoyl-CoA isomerase activity.

Biosci Biotechnol Biochem, 1998 Oct, 62(10), 1956 - 61
Hemiacetal dehydrogenation activity of alcohol dehydrogenases in Saccharomyces cerevisiae; Kusano M et al.; Some methylotrophic yeasts produce methyl formate from methanol and formaldehyde via hemiacetal formation . We investigated Saccharomyces cerevisiae to find whether this yeast has a carboxylate ester producing pathway that proceeds via hemiacetal dehydrogenation . We confirmed that the purified alcohol dehydrogenase (Adh) protein from S . cerevisiae can catalyze the production of esters . High specific activities were observed toward the hemiacetals corresponding to the primary alcohols when ether groups were substituted for methylene groups, resulting in the formation of formate esters . Both ADH and methyl formate synthesizing activities were sharply reduced in the delta adh1 delta adh2 mutant . The ADH1 and ADH2 genes encode the major Adh proteins in S . cerevisiae . Thus, it was concluded that the S . cerevisiae Adh protein catalyzes activities for the production of certain carboxylate esters.

Biosci Biotechnol Biochem, 1998 Oct, 62(10), 1852 - 7
Cloning and nucleotide sequence of the alcohol acetyltransferase II gene (ATF2) from Saccharomyces cerevisiae Kyokai No . 7; Nagasawa N et al.; The ATF2 gene, which encodes alcohol acetyltransferase II (AATase II), was cloned from Saccharomyces cerevisiae Kyokai No . 7 (sake yeast) . The ATF2 gene coded for a protein of 535 amino acid residues with a calculated molecular mass of 61,909 daltons . The deduced amino acid sequences of the ATF2 showed 36.9% similarity with that of ATF1, which encodes AATase I . The hydrophobicity profiles for the Atf2 protein and Atf1 protein were similar . A transformant carrying multiple copies of the ATF2 gene had 2.5-fold greater AATase activity than the control, and this activity was not significantly inhibited by linoleic acid . A Southern analysis of the yeast genomes in which the ATF2 gene was used as a probe showed that S . cerevisiae and brewery larger yeast have one ATF2 gene, while S . bayanus has no similar gene.

Appl Environ Microbiol, 1998 Dec, 64(12), 4857 - 61
Assimilation of cellooligosaccharides by a cell surface-engineered yeast expressing beta-glucosidase and carboxymethylcellulase from aspergillus aculeatus
Murai T, Ueda M, Kawaguchi T, Arai M, Tanaka A.
Since Saccharomyces cerevisiae lacks the cellulase complexes that hydrolyze cellulosic materials, which are abundant in the world, two types of hydrolytic enzymes involved in the degradation of cellulosic materials to glucose were genetically co-immobilized on its cell surface for direct utilization of cellulosic materials, one of the final goals of our studies . The genes encoding FI-carboxymethylcellulase (CMCase) and beta-glucosidase from the fungus Aspergillus aculeatus were individually fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin and introduced into S . cerevisiae . The delivery of CMCase and beta-glucosidase to the cell surface was carried out by the secretion signal sequence of the native signal sequence of CMCase and by the secretion signal sequence of glucoamylase from Rhizopus oryzae for beta-glucosidase, respectively . The genes were expressed by the glyceraldehyde-3-phosphate dehydrogenase promoter from S . cerevisiae . The CMCase and beta-glucosidase activities were detected in the cell pellet fraction, not in the culture supernatant . The display of CMCase and beta-glucosidase proteins on the cell surface was confirmed by immunofluorescence microscopy . The cells displaying these cellulases could grow on cellobiose or water-soluble cellooligosaccharides as the sole carbon source . The degradation and assimilation of cellooligosaccharides were confirmed by thin-layer chromatography . This result showed that the cell surface-engineered yeast with these enzymes can be endowed with the ability to assimilate cellooligosaccharides . This is the first step in the assimilation of cellulosic materials by S . cerevisiae expressing heterologous cellulase genes.

J Biochem (Tokyo), 1998 Dec 1, 124(6), 1124 - 9
C-terminal peptide of fusarium heterosporum lipase is necessary for its increasing thermostability; Nagao T et al.; Saccharomyces cerevisiae bearing a lipase cDNA from Fusarium heterosporum produced two lipases, A and B . Lipase B was significantly more stable to temperature than lipase A, but their optimum temperatures were similar . Lipase B was composed of one polypeptide (301 amino acids), and lipase A was composed of two polypeptides (275 and 26 amino acids) generated by the cleavage between Arg275 and Asp276 with a trypsin-like protease . It was suggested that the C-terminal peptide (26 amino acids) tightened the lipase structure when bound to the catalytic domain (275 amino acids) through a peptide bond . The tight structure was loosened by cleavage of the C-terminal peptide, even though the peptide interacted noncovalently with the catalytic domain, possibly through charged amino acids, in which it is rich . Deletion of the C-terminal peptide greatly decreased the lipase production by the recombinant S . cerevisiae, although its transcriptional level was the same as that of cells carrying the wild-type gene . These facts suggested that the C-terminal peptide affected the lipase production in the post-transcriptional step.

J Biol Chem, 1998 Dec 4, 273(49), 32771 - 5
Functional conservation of the human homolog of the yeast pre-mRNA splicing factor Prp17p; Lindsey LA et al.; Splicing of pre-mRNAs involves two sequential transesterification reactions commonly referred to as the first and second steps . In Saccharomyces cerevisiae, four proteins, Prp16p, Prp17p, Prp18p, and Slu7p are exclusively required for the second step of splicing . The human homologs of Prp16p, Prp17p, and Prp18p have been identified, and the human proteins hPrp16 and hPrp18 have been shown to be required for the second step of splicing in vitro . Here we provide further evidence for the functional conservation of the second step factors between yeast and humans . Human hPrp17, which is 35% identical to the S . cerevisiae protein, is able to partially rescue the temperature-sensitive phenotype in a yeast strain where PRP17 has been knocked out, suggesting that the human and yeast proteins are functionally conserved . Overexpression of hPrp17 in the knockout yeast strain partially rescues the splicing defect seen in vitro and in vivo . In HeLa cells, hPrp17 is highly concentrated in the nuclear speckles, as is SC35 and many other splicing factors, thus providing further support that this protein also functions as a splicing factor in humans.

J Bacteriol, 1998 Dec, 180(23), 6404 - 7
PRS5, the fifth member of the phosphoribosyl pyrophosphate synthetase gene family in Saccharomyces cerevisiae, is essential for cell viability in the absence of either PRS1 or PRS3; Hernando Y et al.; In Saccharomyces cerevisiae, an open reading frame, YOL061w, encodes a polypeptide with sequence similarity to the four known 5-phosphoribosyl-1(alpha)-pyrophosphate synthetase (PRS) genes since it contains a divalent cation binding site and a phosphoribosyl pyrophosphate binding site . We regard YOL061w as the fifth member of the PRS gene family, PRS5 . Loss of Prs5p has a significant impact on PRS enzyme activity, causing it to be reduced by 84% . On the other hand, Deltaprs5 strains are not affected in growth or in the size of their nucleotide pools . However, simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 rendered the strains inviable, which implies that PRS5 plays an important role in the maintenance of PRS function in S . cerevisiae.

Mol Gen Genet, 1998 Oct, 260(1), 120 - 30
Function of hybrid human-yeast cyclin-dependent kinases in Saccharomyces cerevisiae; Bitter GA; It is now well established that progression through the eukaryotic cell cycle is controlled by oscillations in the activity of cyclin- dependent kinases (CDKs) . In many cases, however, the physiological substrate(s) of CDKs are unknown . The Saccharomyces cerevisiae PHO5 gene encodes a secreted acid phosphatase which is induced in response to phosphate starvation . The PHO5 gene is activated by the Pho4p transcription factor, which itself is negatively regulated through phosphorylation by the products of PHO80 and PHO85 . Pho80p and Pho85p are homologous to cyclins and CDKs, respectively, and the Pho80p/Pho85p heterodimer satisfies the biochemical definition of a cyclin/CDK . In the present study, several reporter genes were expressed in S . cerevisiae from promoters which are activated by the transcription factor Pho4p, thereby generating yeast strains which exhibit quantifiable phenotypes that reflect the activity of a specific cyclin/CDK . Positive genetic selections for inhibition of cyclin/CDK function were characterized using the E . coli neo and yeast LEU2 genes . Chromosomal disruptions of the yeast PHO80 and PHO85 genes were constructed and conditions for complementation by plasmid-borne genes were defined . Complementation is achieved at very low levels of expression of both Pho80p and Pho85p . High-level expression of Pho80p results in aberrant PHO5 promoter regulation, characterized by failure to derepress in low-phosphate medium . Genes encoding hybrid CDKs in which regions of Pho85p were replaced with the homologous region of human Cdk2 were constructed, and tested for function in S . cerevisiae by complementation of the pho85 chromosomal gene disruption . Hybrid proteins in which more than two-thirds of the molecule were derived from human Cdk2 retained Pho85p function with respect to high-phosphate repression of the PHO5 promoter . The hybrid proteins require the PHO80 gene product for this function . A hybrid human-yeast CDK in which a single amino acid is deleted, within a nonapeptide sequence which is perfectly conserved in Pho85p and human Cdk2, retains full function . These results demonstrate that, within the context of the conserved structure of CDKs, considerable primary sequence variability can be introduced without loss of the cyclin-dependent function of the CDK.

Cytokines Cell Mol Ther, 1998 Sep, 4(3), 147 - 51
Expression of the human GM-CSF receptor alpha subunit in Saccharomyces cerevisiae; Tu J et al.; The alpha subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 45 kDa membrane protein with a higher apparent molecular weight of 50-85 kDa due to glycosylation . Previously, we had demonstrated that N-glycosylation plays a critical role in the GM-CSF receptor-ligand interaction . To assess the activity of the alpha subunit of the human GM-CSF receptor (GMRalpha) in a lower eukaryote, we expressed GMRalpha in the yeast S . cerevisiae and found that the protein has a lower apparent molecular weight compared with that expressed in mammalian cells . Using indirect immunofluorescence microscopy, we showed that GMRalpha protein expressed in yeast localizes to the plasma membrane . Although the yeast-expressed GMRalpha is able to interact with anti-GMRalpha antibody, the heterologously expressed receptor does not bind GM-CSF . Our results indicate that specific sites and/or forms of glycosylation of the GM-CSF receptor are crucial for ligand binding.

Cancer Res, 1998 Nov 15, 58(22), 5027 - 31
Identification of a human gene encoding a homologue of Saccharomyces cerevisiae EXO1, an exonuclease implicated in mismatch repair and recombination; Tishkoff DX et al.; The EXO1 gene was identified in Saccharomyces cerevisiae as a gene encoding an exonuclease that interacts with MSH2 and functions in mismatch repair and genetic recombination . To understand the role of EXO1 in higher eukaryotes, we identified the human EXO1 gene . The hEXO1 predicted amino acid sequence shares 26.6% identity with the S . cerevisiae EXO1 amino acid sequence . The human and S . cerevisiae proteins showed a similar ability to complement the mutator phenotype of S . cerevisiae rad27 mutants indicating that the two proteins are functionally similar . There appear to be two forms of hEXO1 that differ by the COOH-terminal 1 and 44 amino acids, respectively, and these appear to result from alternative RNA splicing . The hEXO1 gene consists of 14 exons and is transcribed to yield a 3-kb mRNA . Radiation hybrid and fluorescence in situ hybridization mapping studies indicate that the human gene is located at 1q42.2-qter . Northern blot analysis demonstrates that hEXO1 is expressed in high levels in testis; elevated expression was also observed in thymus and colon and to a lesser extent in small intestine, placenta, spleen, and ovary.

Mol Microbiol, 1998 Nov, 30(3), 603 - 13
Regulation of expression of the amino acid transporter gene BAP3 in Saccharomyces cerevisiae; De Boer M et al.; The BAP3 gene of Saccharomyces cerevisiae encodes a protein with a high similarity to the BAP2 gene product, a high-affinity permease for branched-chain amino acids . In this paper, we show that, like BAP2, the expression of the BAP3 gene in S . cerevisiae is induced by the addition of branched-chain amino acids to the medium . Unexpectedly, most other naturally occurring L-amino acids found in proteins (with the exception of proline, lysine, arginine and histidine) have the same effect on the expression of BAP3 . The induction of BAP3 expression appears to be dependent on Stp1p, a nuclear protein, previously shown to be involved in pre-tRNA maturation and also required for the expression of BAP2, as induction is no longer observed in an stp1 - mutant . The transcriptional regulator Leu3p is not involved in the induction of BAP3 expression, but may act as a repressor of BAP3 expression in the absence of leucine, as can be inferred from a transcriptional analysis in a Deltaleu3 mutant . By extensive deletion analysis of the BAP3 promoter fused to a GUS reporter, as well as by fusions of different parts of the BAP3 promoter to a LacZ reporter, we have found that a portion of the BAP3 promoter from - 418 to - 392 relative to the ATG start codon is both necessary and sufficient for the Stp1p-dependent induction of BAP3 expression by (most) amino acids . We have therefore named this sequence UASaa (amino acid-dependent upstream activator sequence) . Neither Stp1p nor Leu3p appear to bind to the UASaa, at least in vitro, as judged from gel retardation assays . Sequences similar to the UASaa can be found in the promoters of BAP2, PTR2 and TAT1; genes that, like BAP3, encode permeases inducible by amino acids, suggesting that amino acid induction of all these genes is exerted via a common mechanism.

Curr Biol, 1998 Nov 19, 8(23), 1281 - 4
The role of Saccharomyces cerevisiae coronin in the actin and microtubule cytoskeletons; Heil-Chapdelaine RA et al.; Coronin was originally identified as a cortical protein associated with the actin cytoskeleton in Dictyostelium {1} . More recent studies have revealed that coronin is involved in actin-based motility, cytokinesis and phagocytosis {2,3} . Here, we describe the identification of a single homolog of coronin in Saccharomyces cerevisiae, which we show localizes to cortical actin patches in an actin-dependent manner . Unlike Dictyostelium mutants that lack coronin, yeast strains lacking coronin had no detectable defects in actin-based processes . This may reflect differences in the functions of the actin cytoskeleton in these two organisms . Previous studies have shown that cortical actin may mediate astral microtubule-based movements of the mitotic spindle in S . cerevisiae {4,5} and that, during mitosis in Dictyostelium, the regions of the cell cortex that overlap with astral microtubules become enriched in actin and coronin {6} . We therefore examined whether yeast lacking coronin had defects in the microtubule cytoskeleton . The mutant strains had increased sensitivity to the microtubule-destabilizing drug benomyl and an increased number of large-budded cells with short spindles . Further examination of microtubule-related processes, including spindle formation, migration of the mitotic spindle to the bud neck, spindle elongation, and translocation of the elongating spindle through the bud neck, failed to reveal any defects in the coronin mutant . Taken together, these results suggest that S . cerevisiae coronin is a component of the actin cytoskeleton that may interact with the microtubule cytoskeleton.

FEBS Lett, 1998 Oct 30, 438(1-2), 61 - 5
Mammalian Bax triggers apoptotic changes in yeast; Ligr M et al.; Apoptosis is co-regulated by the conserved family of Bcl-2-related proteins, which includes both its agonists (Bax) and antagonists (Bcl-X(L)) . A mutant strain of the yeast Saccharomyces cerevisiae has been shown to express all morphological signs of apoptosis . Overexpression of Bax is lethal in S . cerevisiae, whereas simultaneous overexpression of Bcl-X(L) rescues the cells . We report that overexpression of mammalian Bax in a S . cerevisiae wild type strain triggers morphological changes similar to those of apoptotic metazoan cells: the loss of asymmetric distribution of plasma membrane phosphatidylserine, plasma membrane blebbing, chromatin condensation and margination, and DNA fragmentation . Simultaneous overexpression of Bcl-X(L) prevents these changes . We demonstrate that Bax triggers phenotypic alterations in yeast strongly resembling those it causes in metazoan apoptotic cells.

Curr Opin Biotechnol, 1998 Oct, 9(5), 510 - 7
Production of G-protein-coupled receptors in yeast; Reilander H et al.; Yeasts combine the advantages of fast and easy handling with the potential to perform eukaryotic post-translational modifications and are for this reason interesting hosts for heterologous production of G-protein-coupled receptors . The possibility to connect foreign receptors to a yeast internal MAP kinase pathway was used to establish yeast-based systems for high-throughput screening of compound libraries . In addition, yeasts have the potential for high level production of G-protein-coupled receptors . In this field, non-Saccharomyces yeasts seems to be interesting alternatives to S . cerevisiae, as well as to systems based on higher eukaryotic cells.

J Gen Virol, 1998 Nov, 79 ( Pt 11), 2651 - 60
Co-expression of human eIF-4G and poliovirus 2Apro in Saccharomyces cerevisiae: effects on gene expression; Barco A et al.; The poliovirus 5' untranslated region (5' UTR) confers on mRNAs the capacity to be translated by internal initiation . The functionality of this RNA motif has been tested in yeast cells (Saccharomyces cerevisiae) using luciferase (luc) as a reporter gene . Although some luciferase is synthesized from luc mRNA containing the poliovirus 5' UTR (Leader-luc mRNA), much more luciferase is synthesized in cells that express luc mRNA devoid of the poliovirus 5' UTR . Since poliovirus 2Apro enhances the translation of Leader-luc mRNAs after eIF-4G cleavage in mammalian cells, yeast cells were produced that synthesize three heterologous proteins, luciferase, poliovirus 2Apro and human eIF-4G . Initially, S . cerevisiae cells constitutively expressing human eIF-4G were isolated . The human eIF-4G gene does not complement yeast cells defective in the initiation factor counterpart, p150, indicating that the human and yeast eIF-4G are not interchangeable . Expression of poliovirus 2Apro in an inducible manner does not affect p150, but led to the efficient cleavage of human eIF-4G in yeast cells . Induction of 2Apro was detrimental to luciferase synthesis either from luc mRNA or Leader-luc mRNA irrespective of the presence or absence of human eIF-4G . 2Apro blocked luciferase expression at the transcriptional level . Finally, the effects of 16 point mutations of poliovirus 2Apro on luciferase expression and human eIF-4G cleavage were analysed . Only those 2Apro variants that generate viable polioviruses actively cleave eIF-4G in yeast.

Mol Cell Biol, 1998 Dec, 18(12), 7353 - 9
Cns1 is an essential protein associated with the hsp90 chaperone complex in Saccharomyces cerevisiae that can restore cyclophilin 40-dependent functions in cpr7Delta cells; Marsh JA et al.; Saccharomyces cerevisiae harbors two cyclophilin 40-type enzymes, Cpr6 and Cpr7, which are components of the Hsp90 molecular chaperone machinery . Cpr7 is required for normal growth and is required for maximal activity of heterologous Hsp90-dependent substrates, including glucocorticoid receptor (GR) and the oncogenic tyrosine kinase pp60(v-src) . In addition, it has recently been shown that Cpr7 plays a major role in negative regulation of the S . cerevisiae heat shock transcription factor (HSF) . To better understand functions associated with Cpr7, a search was undertaken for multicopy suppressors of the cpr7Delta slow-growth phenotype . The screen identified a single gene, designated CNS1 (for cyclophilin seven suppressor), capable of suppressing the cpr7Delta growth defect . Overexpression of CNS1 in cpr7Delta cells also largely restored GR activity and negative regulation of HSF . In vitro protein retention experiments in which Hsp90 heterocomplexes were precipitated resulted in coprecipitation of Cns1 . Interaction between Cns1 and the carboxy terminus of Hsp90 was also shown by two-hybrid analysis . The functional consequences of CNS1 overexpression and its physical association with the Hsp90 machinery indicate that Cns1 is a previously unidentified component of molecular chaperone complexes . Thus far, Cns1 is the only tetratricopeptide repeat-containing component of Hsp90 heterocomplexes found to be essential for cell viability under all conditions tested.

Yeast, 1998 Jun 30, 14(9), 793 - 803
A novel esterase from Saccharomyces carlsbergensis, a possible function for the yeast TIP1 gene; Horsted MW et al.; An extracellular esterase was isolated from the brewer's yeast, Saccharomyces carlsbergensis . Inhibition by diisopropyl fluorophosphate shows that the enzyme has a serine active site . By mass spectrometry, the molecular weight of the enzyme was 16.9 kDa . The optimal pH for activity was in the range of four to five . Esterase activity was found in beer before pasteurization, and a low level of activity was still present after pasteurization . Caprylic acid, which is present in beer, competitively inhibited the esterase . The substrate preference towards esters of p-nitrophenol indicated that the enzyme prefers esters of fatty acids from four to 16 carbon atoms . The esterase has lipolytical activity; olive oil (C-18:1), which is a classical substrate for lipase, was hydrolysed . N-terminal sequence analysis of the esterase yielded a sequence which was identical to the deduced amino acid sequence of the S . cerevisiae TIP1 gene . The esterase preparation did not appear to contain significant amounts of other proteins than Tip1p, indicating that the TIP1 gene is the structural gene for the esterase.

Gene, 1998 Nov 5, 222(1), 69 - 75
Overlapping 3'-end formation signals and ARS elements: tightly linked but functionally separable; Magrath C et al.; 3'-End formation signals are closely associated with autonomous replicating sequences (ARSs) in Saccharomyces cerevisiae in that ARSs frequently contain signals that direct 3'-end formation (Chen et al., 1996) . Mutationally-inactivated ARSs that co-reside with 3'-end formation sequences do not disrupt 3'-end formation, thus demonstrating that replication function does not affect termination function . To test the corollary possibility that 3'-end formation is important for replication function, we made point mutations in ARS305 that increase readthrough of the 3'-end formation signals and determined plasmid replication efficiency . Replication efficiency, as assessed by plasmid stability assays, was not altered by mutations affecting 3'-end formation when transcription through the ARS was either absent or highly-induced . Under conditions of high-level transcription through the ARS, the rate of plasmid loss in both wild-type and mutated terminators increased over five-fold from rates observed during transcriptionally repressed conditions . This result indicates that the native 3'-end formation signal is incapable of protecting the replication function when high levels of transcription are directed into the ARS . Thus, the compact nature of the S . cerevisiae genome, rather than a functional inter-dependence, may account for close association of transcription terminators and ARSs.

Genes Cells, 1998 Sep, 3(9), 587 - 601
Meiotic behaviours of chromosomes and microtubules in budding yeast: relocalization of centromeres and telomeres during meiotic prophase; Hayashi A et al.; BACKGROUND: Meiosis is a process of universal importance in eukaryotic organisms, generating variation in the heritable haploid genome by recombination and re-assortment of chromosomes . The intranuclear movement of chromosomes is expected to achieve pairing and recombination of homologous chromosomes during meiosis . Meiosis in the budding yeast Saccharomyces cerevisiae has been extensively studied, both genetically and by molecular biology; here we report cytological observations of meiotic chromosomal events in this organism . RESULTS: Using fluorescence microscopy, we have examined the behaviour of chromosomes and microtubules during meiosis in S . cerevisiae . We first observed the dynamic behaviour of nuclei in living cells using jellyfish green fluorescent protein (GFP) fused with nucleoplasmin, a Xenopus oocyte nuclear protein . The characterization of nuclear movement in living cells was extended by an analysis of chromosomes and microtubules in fixed specimens . In addition, the nuclear localization of centromeres and telomeres was determined by indirect immunofluorescence microscopy in synchronous populations of meiotic cells . While telomeres remain in clusters of 5-8 throughout meiosis, centromeres change their nuclear localization dramatically during the progression of meiosis: centromeres are first clustered at a single site near the spindle-pole body before the induction of meiosis, and become scattered during the meiotic prophase . CONCLUSIONS: Our observations have demonstrated that nuclear and cytoskeletal reorganization take place with meiosis in S . cerevisiae . In particular, the distinct relocalization of centromeres during meiosis indicates a considerable movement of chromosomes within the meiotic prophase nucleus.

J Cell Biol, 1998 Nov 2, 143(3), 751 - 65
The yeast centrin, cdc31p, and the interacting protein kinase, Kic1p, are required for cell integrity; Sullivan DS et al.; Cdc31p is the yeast homologue of centrin, a highly conserved calcium-binding protein of the calmodulin superfamily . Previously centrins have been implicated only in microtubule-based processes . To elucidate the functions of yeast centrin, we carried out a two-hybrid screen for Cdc31p-interacting proteins and identified a novel essential protein kinase of 1,080 residues, Kic1p (kinase that interacts with Cdc31p) . Kic1p is closely related to S . cerevisiae Ste20p and the p-21- activated kinases (PAKs) found in a wide variety of eukaryotic organisms . Cdc31p physically interacts with Kic1p by two criteria; Cdc31p coprecipitated with GST-Kic1p and it bound to GST-Kic1p in gel overlay assays . Furthermore, GST-Kic1p exhibited in vitro kinase activity that was CDC31-dependent . Although kic1 mutants were not defective for spindle pole body duplication, they exhibited a variety of mutant phenotypes demonstrating that Kic1p is required for cell integrity . We also found that cdc31 mutants, previously identified as defective for spindle pole body duplication, exhibited lysis and morphological defects . The cdc31 kic1 double mutants exhibited a drastic reduction in the range of permissive temperature, resulting in a severe lysis defect . We conclude that Kic1p function is dependent upon Cdc31p both in vivo and in vitro . We postulate that Cdc31p is required both for SPB duplication and for cell integrity/morphogenesis, and that the integrity/morphogenesis function is mediated through the Kic1p protein kinase.

J Biol Chem, 1998 Nov 20, 273(47), 31138 - 44
Suppressors of superoxide dismutase (SOD1) deficiency in Saccharomyces cerevisiae . Identification of proteins predicted to mediate iron-sulfur cluster assembly; Strain J et al.; Yeast deficient in the cytosolic copper/zinc superoxide dismutase (SOD1) exhibit metabolic defects indicative of oxidative damage even under non-stress conditions . To help identify the endogenous sources of this oxidative damage, we isolated mutant strains of S . cerevisiae that suppressed metabolic defects associated with loss of SOD1 . Six complementation groups were isolated and three of the corresponding genes have been identified . One sod1Delta suppressor represents SSQ1 which encodes a hsp70-type molecular chaperone found in the mitochondria . A second sod1Delta suppressor gene, designated JAC1, represents a new member of the 20-kDa J-protein family of co-chaperones . Jac1p contains a mitochondrial targeting consensus sequence and may serve as the partner for Ssq1p . Homologues of Ssq1p and Jac1p are found in bacteria in close association with genes proposed to be involved in iron-sulfur protein biosynthesis . The third suppressor gene identified was NFS1 . Nfs1p is homologous to cysteine desulfurase enzymes that function in iron-sulfur cluster assembly and is also predicted to be mitochondrial . Each of the suppressor mutants identified exhibited diminished rates of respiratory oxygen consumption and was found to have reduced mitochondrial aconitase and succinate dehydrogenase activities . Taken together these results suggest a role for Ssq1p, Jac1p, and Nfs1p in assembly/maturation of mitochondrial iron-sulfur proteins and that one or more of the target Fe/S proteins contribute to oxidative damage in cells lacking copper/zinc SOD.

Curr Biol, 1998 Nov 5, 8(22), 1219 - 22
The stress-activated phosphatidylinositol 3-phosphate 5-kinase Fab1p is essential for vacuole function in S . cerevisiae; Cooke FT et al.; Polyphosphoinositides have many roles in cell signalling and vesicle trafficking {1-3} . Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a recently discovered PIP2 isomer, is ubiquitous in eukaryotic cells and rapidly accumulates in hyperosmotically stressed yeast . PI(3,5)P2 is synthesised from PI(3)P in both yeast and mammalian cells {4,5} . A search of the Saccharomyces cerevisiae genome database identified FAB1, a gene encoding a PIP kinase homologue and potential PI(3)P 5-kinase . Fab1p shows PI(3)P 5-kinase activity both in vivo and in vitro . A yeast strain in which FAB1 had been deleted was unable to synthesise PI(3,5)P2, either in the presence or absence of osmotic shock . A loss of PI(3,5)P2 was observed also in a temperature-sensitive FAB1 strain at the non-permissive temperature . A recombinant glutathione-S-transferase (GST)-Fab1p fusion protein was shown to have selective PI(3)P 5-kinase activity in vitro . Thus, we have demonstrated that Fab1p is a PI(3)P-specific 5-kinase and represents a third class of PIP kinase activity, which we have termed type III . Deletion of the FAB1 gene produces a loss of vacuolar morphology {6}; it is therefore concluded that PI(3,5)P2, the lipid product of Fab1p, is required for normal vacuolar function.

Biochimie, 1998 Jul, 80(7), 579 - 90
Purification and properties of cysteinyl-tRNA synthetase from rabbit liver; Motorin Y et al.; Cysteinyl-tRNA synthetase (CRS) from rabbit liver was purified 8300-fold to a constant specific activity . SDS-PAGE revealed the presence of two polypeptides of 86 kDa and 92 kDa, in the proportions of 60% and 40% respectively . The SDS-electrophoretic migration of the major 86 kDa component was indistinguishable from that of the single polypeptide previously found in CRS from S . cerevisiae . The two polypeptides from rabbit CRS were inaccessible to Edman degradation, but internal peptides generated from each by in-gel proteolysis after SDS-electrophoretic separation, yielded sequences found in the deduced protein sequence of human CRS . Moreover, subjecting the two polypeptides separated by SDS-PAGE to a renaturation treatment showed that CRS activity was associated with both . The structure of the native enzyme was probed by limited proteolysis with elastase . The strikingly simple degradation pattern observed supported a model according to which the two polypeptides derive from the same gene, differing only by a approximately 6 kDa extension located at the C-terminal extremity of the 92 kDa component . Moreover, the finding that notwithstanding the presence of the two polypeptides, the behaviour of rabbit CRS upon gel-filtration or chemical cross-linking was indistinguishable from that of homodimeric yeast CRS, indicated that the 6 kDa C-terminal extension on the 92 kDa polypeptide does not impede dimerisation . The origin of the two components of rabbit CRS is discussed in light of the deduced protein sequence of human CRS derived from the published cDNA sequence and the recently released genomic sequence of the human enzyme.

FEMS Microbiol Lett, 1998 Oct 15, 167(2), 321 - 6
Enrichment with a polyunsaturated fatty acid enhances the survival of Saccharomyces cerevisiae in the presence of tributyltin; Masia A et al.; The toxicity of inorganic metal species towards Saccharomyces cerevisiae has been shown to be markedly dependent on cellular fatty acid composition . In this investigation, the influence of fatty acid supplementation on the toxicity of the lipophilic organometal, tributyltin was investigated . Growth of S . cerevisiae was increasingly inhibited when the tributyltin concentration was increased from 0 to 10 microM . However, the inhibitory effect was partly alleviated by supplementation of the medium with 1 mM linoleate (18:2), a treatment that leads to large-scale incorporation of this polyunsaturated fatty acid (to > 60% of total fatty acids) in yeast membrane lipids . Cells that were previously enriched with 18:2 also showed reduced loss of vitality compared to cells grown in the absence of a fatty acid supplement, when exposed to tributyltin . For example, addition of tributyltin to a concentration of 0.1 microM was associated with an approximate 10% reduction in the H+ efflux activity of 18:2-enriched cells, but a 70% reduction in that of fatty acid-unsupplemented cells . Despite the increased tributyltin resistance of 18:2-enriched S . cerevisiae, the level of cell-associated tributyltin was found to be approximately two-fold higher in these organisms than in fatty acid-unsupplemented cells . These results demonstrate an increased resistance of 18:2-enriched membranes to the direct toxic action(s) of tributyltin . This is in contrast to the previously reported effect of 18:2 enrichment on sensitivity of S . cerevisiae to inorganic metal cations.

Curr Microbiol, 1998 Dec, 37(6), 426 - 30
A short region from the LEU2 gene of Saccharomyces cerevisiae functions as an ARS in the yeast Saccharomyces exiguus Yp74L-3; Hisatomi T et al.; We examined the autonomously replicating sequence (ARS) activity of some fragments derived from the LEU2 region of Saccharomyces cerevisiae onto Saccharomyces exiguus Yp74L-3 . A DNA fragment functioning as an ARS in S . exiguus, but not in S . cerevisiae, was shown to exist . The ARS activity for S . exiguus was reduced by the 2-microm plasmid origin of S . cerevisiae when both elements coexisted on a single circular plasmid . Analysis of ARS activity with the PCR products from the fragment revealed that the ARS-acting sequence was located in the 3'-terminal area of the transcribed region of the LEU2 gene of S . cerevisiae . It is suggested that the ARS recognition system in S . exiguus is significantly different from that of S . cerevisiae.

Radiat Res, 1998 Nov, 150(5 Suppl), S80 - 91
Identification of genes involved in repair of DNA double-strand breaks in mammalian cells; Jeggo PA; At least two mechanisms of DNA double-strand break (DSB) repair operate in mammalian cells . Homologous recombination, which plays a major role in lower organisms, plays a less significant role in higher organisms . In contrast, the majority of DSBs in mammalian cells are rejoined by a mechanism, termed non-homologous end joining (NHEJ), that does not depend upon extensive regions of homology . This process is also used to rejoin site-specific DSBs introduced during V(D)J recombination . From the analysis of defective rodent mutants, four proteins (Ku70, Ku80, DNA-PKcs and Xrcc4) that function in this process in mammalian cells have been identified . DNA ligase IV is also strongly implicated since it associates strongly with XRCC4, and since DNA ligase IV-deficient yeast are defective in their ability to carry out NHEJ . In S . cerevisiae, Sir2p, Sir3p and Sir4p, three proteins required for transcriptional silencing, are also required for NHEJ . Additionally, the yeast mutants, xrs2, rad50 and mre11, which are defective in meiotic recombination, are also defective in NHEJ . Here I review the evidence implicating these proteins as functioning in NHEJ and discuss their properties and role in other pathways . The significance of DSB repair to clinical radiosensitivity and human disorders is also evaluated.

Yeast, 1998 Oct, 14(14), 1249 - 55
Stabilization of two families of critical targets for hyperthermic cell killing and acquired thermotolerance of yeast cells; Obuchi K et al.; Hyperthermic cell killing profiles of Saccharomyces cerevisiae cells were biphasic and a shoulder (phase 1) was followed by an exponential killing (phase 2) . Assuming that (i) the rate of thermal damage in particular macromolecules or their assemblies limits the rate of hyperthermic cell killing (the critical target model), and (ii) the damages of two families of targets are lethal independently, we built a 'dual critical target model' in order to interpret the biphasic cell killing . Time-courses of temperature-programmed fractional survival were traced for S . cerevisiae cells in exponentially growing phase, heat shocked, and in stationary phase . Non-linear curve-fitting of the time-courses by using the dual critical target model provided the Arrhenius parameters of denaturation of the two families of targets . The cells were killed more slowly in phase 1 than in phase 2 . Arrest in stationary phase, not heat shock, stabilizes the family of targets that is critical to phase 1 death . On the other hand, both heat-shock response and arrest in stationary phase stabilizes the other family of targets that, in addition to the previous one, is responsible for phase 2 death.

Free Radic Biol Med, 1998 Oct, 25(6), 682 - 7
Treatment of the budding yeast Saccharomyces cerevisiae with the lipid peroxidation product 4-HNE provokes a temporary cell cycle arrest in G1 phase; Wonisch W et al.; The effects of 4-hydroxy-2-nonenal (HNE) on the cell division cycle were investigated in the yeast Saccharomyces cerevisiae . A short treatment with HNE at a concentration in the range of the IC50 value in S . cerevisiae SP-4 cells induced a significant increase in the proportion of G0/G1 cells at the expense of S-phase cells . A similar delay in cell cycle progression upon treatment with HNE has recently been shown for HL-60 neoplastic cells . Long-term exposure in a synchronized yeast culture resulted in a pronounced dose-dependent block between G0G1- and S-phase, probably at or close to a point in the cell cycle that has been designated as "START." Incorporation of radioactively labeled precursors of macromolecules revealed that DNA synthesis was most susceptible to HNE in comparison to RNA and protein synthesis . Production of glutathione appeared to be required for the continuation of the cell cycle . HNE-treated yeast cells reentered the cell cycle when their glutathione content exceeded about twice the level of control cells . The release from the cell division cycle delay was followed by an enhanced growth to an extent that HNE-treated cells exceeded the number of control cells . These results indicate that HNE causes a biphasic modulation of cell proliferation . It was concluded that this effect was conserved during evolution from yeast to mammalian cells, emphasizing once more the usefulness of this unicellular organism as a model system for the investigation of the effects of free radical-derived products on the proliferation of eukaryotes.

Appl Environ Microbiol, 1998 Nov, 64(11), 4283 - 90
Threonine aldolase overexpression plus threonine supplementation enhanced riboflavin production in Ashbya gossypii; Monschau N et al.; Riboflavin production in the filamentous fungus Ashbya gossypii is limited by glycine, an early precursor required for purine synthesis . We report an improvement of riboflavin production in this fungus by overexpression of the glycine biosynthetic enzyme threonine aldolase . The GLY1 gene encoding the threonine aldolase of A . gossypii was isolated by heterologous complementation of the glycine-auxotrophic Saccharomyces cerevisiae strain YM13 with a genomic library from A . gossypii . The deduced amino acid sequence of GLY1 showed 88% similarity to threonine aldolase from S . cerevisiae . In the presence of the GLY1 gene, 25 mU of threonine aldolase specific activity mg-1 was detectable in crude extracts of S . cerevisiae YM13 . Disruption of GLY1 led to a complete loss of threonine aldolase activity in A . gossypii crude extracts, but growth of and riboflavin production by the knockout mutant were not affected . This indicated a minor role of the enzyme in glycine biosynthesis of A . gossypii . However, overexpression of GLY1 under the control of the constitutive TEF promoter and terminator led to a 10-fold increase of threonine aldolase specific activity in crude extracts along with a 9-fold increase of riboflavin production when the medium was supplemented with threonine . This strong enhancement, which could not be achieved by supplementation with glycine alone, was attributed to an almost quantitative uptake of threonine and its intracellular conversion into glycine . This became evident by a subsequent partial efflux of the glycine formed.

Gene, 1998 Oct 23, 221(2), 179 - 84
Characterization of alg2 encoding a mannosyltransferase in the zygomycete fungus Rhizomucor pusillus; Yamazaki H et al.; ALG2 of Saccharomyces cerevisiae encodes the glycosyltransferase that mannosylates Man2GlcNAc2-dolichol diphosphate (PP-Dol) and Man1GlcNAc2-PP-Dol to form Man3GlcNAc2-PP-Dol . The genomic DNA and cDNA encoding an ALG2 homologue were cloned from the zygomycete fungus, Rhizomucor pusillus, and their nucleotide sequences were determined . The cloned cDNA under the control of the yeast GAL1 promoter complemented the temperature-sensitive (ts) growth of the alg2-1 mutant of S . cerevisiae, indicating that it represented a functional ALG2 homologue of R . pusillus . Five introns intervened the R . pusillus alg2 encoding a 455-amino-acid (aa) protein that showed end-to-end similarity in aa sequence to yeast Alg2 and contained a dolichol-binding consensus sequence (Val/Ile-x-Phe-x-x-Ile, where x is any aa) very near its C-terminus . The yeast alg2-1 gene had two mutation points at 377Gly to Arg and 386Gln to Lys . alg2-2 also contained two mutations at 54Glu to Lys and 377Gly to Arg . Site-directed mutagenesis of the fungal Alg2 and determination of their phenotypes in the yeast alg2-1 mutant showed that a mutation at 368Gly (equivalent to 377Gly of yeast Alg2) to Arg resulted in generation of a ts enzyme . The fungal Alg2 containing a mutation at the position corresponding to 54Glu or 386Gln of yeast Alg2 still complemented the ts growth of yeast alg2-1.

J Biol Chem, 1998 Nov 6, 273(45), 29360 - 6
Identification of Delta5-desaturase from Mortierella alpina by heterologous expression in Bakers' yeast and canola; Knutzon DS et al.; A DNA fragment with homology to Delta6-desaturases from borage and cyanobacteria was isolated after polymerase chain reaction amplification of Mortierella alpina cDNA with oligonucleotide primers corresponding to the conserved regions of known Delta6-desaturase genes . This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 446 amino acids from a M . alpina library . Expression of this open reading frame from an inducible promoter in Saccharomyces cerevisiae in the presence of various substrates revealed that the recombinant product had Delta5-desaturase activity . The effects of growth and induction conditions as well as host strain on activity of the recombinant Delta5-desaturase in S . cerevisiae were evaluated . Expression of the M . alpina Delta5-desaturase cDNA in transgenic canola seeds resulted in the production of taxoleic acid (Delta5,9-18:2) and pinolenic acid (Delta5,9,12-18:3), which are the Delta5-desaturation products of oleic and linoleic acids, respectively.

Yeast, 1998 Sep 30, 14(13), 1233 - 40
Cloning and characterization of the Hansenula polymorpha homologue of the Saccharomyces cerevisiae PMR1 gene; Kang HA et al.; A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha . The partial DNA fragment of the H . polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids . The putative gene product contains all ten of the conserved regions observed in P-type ATPase . The cloned gene product exhibits 60.3% amino acid identity to the S . cerevisiae PMR1 gene product and complemented the growth defect of a S . cerevisiae pmr1 null mutant in the EGTA-containing medium . The results demonstrate that the H . polymorpha gene encodes the functional homologue of the S . cerevesiae PMR1 gene product, a P-type Ca(2+)-ATPase.

Yeast, 1998 Sep 30, 14(13), 1223 - 32
Cloning and sequence analysis of the gene encoding invertase (INV1) from the yeast Candida utilis; Chavez FP et al.; The gene INV1 encoding invertase from the yeast Candida utilis has been cloned using a homologous PCR hybridization probe, amplified with two sets of degenerate primers designed considering sequence comparisons between yeast invertases . The cloned gene was sequenced and found to encode a polypeptide of 533 amino acids that contain a 26 amino-acid signal peptide and 12 potential N-glycosylation sites . The nucleotide sequences of the 5' and 3' non-coding regions were found to contain motifs probably involved in initiation, regulation and termination of gene transcription . The amino-acid sequence shows significant identity with other yeast, bacterial and plant beta-fructofuranosidases . The INV1 gene from C . utilis was able to complement functionally the suc2 mutation of S . cerevisiae.

Genomics, 1998 Oct 15, 53(2), 184 - 90
Human CDC23: cDNA cloning, mapping to 5q31, genomic structure, and evaluation as a candidate tumor suppressor gene in myeloid leukemias; Zhao N et al.; The transition from metaphase to anaphase and exit from mitosis involve the degradation of active cyclin B-CDC2 complexes by ubiquitin-mediated proteolysis . The anaphase-promoting complex (APC) catalyzes the formation of cyclin B-ubiquitin conjugates, thereby targeting cyclin B for degradation . The APC is composed of eight proteins, including four members of a family characterized by multiple tetratricopeptide repeats (TPR) . We mapped two overlapping expressed sequence tag clones within a genomic contig on human chromosome 5, band q31 . A search revealed high homology to Saccharomyces cerevisiae CDC23, a TPR protein component of the APC . We have isolated the human CDC23 cDNA containing the full-length predicted open reading frame . The approximately 3.3-kb message is ubiquitously expressed and encodes a protein with 591 amino acids (MW = 68,293 Da) and 9 TPR units . The protein has 30% identity and 51% similarity to the S . cerevisiae protein . The human CDC23 gene contains 16 exons and spans approximately 31 kb . CDC23 maps within the smallest commonly deleted segment in myeloid leukemias characterized by a deletion of 5q; however, we detected no mutations of CDC23 in leukemia cells with loss of 5q . Thus, CDC23 is unlikely to be involved in the pathogenesis of myeloid leukemias characterized by abnormalities of chromosome 5 .

Genomics, 1998 Oct 1, 53(1), 69 - 80
Cloning and characterization of the human and Caenorhabditis elegans homologs of the Saccharomyces cerevisiae MSH5 gene; Winand NJ et al.; In Saccharomyces cerevisiae the MSH5 gene encoding a MutS homolog was identified as a gene required for meiotic crossing over . To understand the role of MSH5 in higher eukaryotes, we have identified both the human and the Caenorhabditis elegans MSH5 genes . The human and C . elegans MSH5 predicted amino acid sequences share, respectively, 25.3 and 22.0% identity with the S . cerevisiae MSH5 amino acid sequence . The human MSH5 gene consists of 25 exons and spans at least 12 kb of genomic DNA, while the C . elegans gene comprises 17 exons distributed over at least 5.8 kb . Radiation hybrid mapping studies indicate that the human gene is located at 6p22.3-p21.3 . Northern blot analysis demonstrates that human MSH5 is expressed to some extent in all tissues, but that particularly high levels of expression occur in testis, thymus, and other tissues of the immune system . Two-hybrid interaction analysis demonstrates that the human MSH4 and MSH5 proteins interact as observed for S . cerevisiae MSH4 and MSH5 .

Oncogene, 1998 Sep 17, 17(11 Reviews), 1395 - 413
Increasing complexity of Ras signaling; Campbell SL et al.; The initial discovery that ras genes endowed retroviruses with potent carcinogenic properties and the subsequent determination that mutated ras genes were present in a wide variety of human cancers, prompted a strong suspicion that the growth-promoting actions of mutated Ras proteins contribute to their aberrant regulation of growth stimulatory signaling pathways . In 1993, a remarkable convergence of experimental observations from genetic analyses of Drosophila, S . cerevisiae and C . elegans as well as biochemical and biological studies in mammalian cells came together to define a clear role for Ras in signal transduction . What emerged was an elegant linear signaling pathway where Ras functions as a relay switch that is positioned downstream of cell surface receptor tyrosine kinases and upstream of a cytoplasmic cascade of kinases that included the mitogen-activated protein kinases (MAPKs) . Activated MAPKs in turn regulated the activities of nuclear transcription factors . Thus, a signaling cascade where every component between the cell surface and the nucleus was defined and conserved in worms, flies and man . This was a remarkable achievement in our efforts to appreciate how the aberrant function of Ras proteins may contribute to the malignant growth properties of the cancer cell . However, the identification of this pathway has proven to be just the beginning, rather than the culmination, of our understanding of Ras in signal transduction . Instead, we now appreciate that this simple linear pathway represents but a minor component of a very complex signaling circuitry . Ras signaling has emerged to involve a complex array of signaling pathways, where cross-talk, feedback loops, branch points and multi-component signaling complexes are recurring themes . The simplest concept of a signaling cascade, where each component simply relays the same message to the next, is clearly not the case . In this review, we summarize our current understanding of Ras signal transduction with an emphasis on new complexities associated with the recognition and/or activation of cellular effectors, and the diverse array of signaling pathways mediated by interaction between Ras and Ras-subfamily proteins with multiple effectors.

Development, 1998 Nov, 125(22), 4391 - 402
A highly conserved centrosomal kinase, AIR-1, is required for accurate cell cycle progression and segregation of developmental factors in Caenorhabditis elegans embryos; Schumacher JM et al.; S . cerevisiae Ipl1, Drosophila Aurora, and the mammalian centrosomal protein IAK-1 define a new subfamily of serine/threonine kinases that regulate chromosome segregation and mitotic spindle dynamics . Mutations in ipl1 and aurora result in the generation of severely aneuploid cells and, in the case of aurora, monopolar spindles arising from a failure in centrosome separation . Here we show that a related, essential protein from C . elegans, AIR-1 (Aurora/Ipl1 related), is localized to mitotic centrosomes . Disruption of AIR-1 protein expression in C . elegans embryos results in severe aneuploidy and embryonic lethality . Unlike aurora mutants, this aneuploidy does not arise from a failure in centrosome separation . Bipolar spindles are formed in the absence of AIR-1, but they appear to be disorganized and are nucleated by abnormal-looking centrosomes . In addition to its requirement during mitosis, AIR-1 may regulate microtubule-based developmental processes as well . Our data suggests AIR-1 plays a role in P-granule segregation and the association of the germline factor PIE-1 with centrosomes.

Mol Cell Biol, 1998 Nov, 18(11), 6560 - 70
Global regulatory functions of Oaf1p and Pip2p (Oaf2p), transcription factors that regulate genes encoding peroxisomal proteins in Saccharomyces cerevisiae; Karpichev IV et al.; Two transcription factors, Oaf1p and Pip2p (Oaf2p), are key components in the pathway by which several Saccharomyces cerevisiae genes encoding peroxisomal proteins are activated in the presence of a fatty acid such as oleate . By searching the S . cerevisiae genomic database for the consensus sequence that acts as a target for these transcription factors, we identified 40 genes that contain a putative Oaf1p-Pip2p binding site in their promoter region . Quantitative Northern analysis confirmed that the expression of 22 of the genes identified is induced by oleate and that either one or both of these transcription factors are required for the activation . In addition to known peroxisomal proteins, the regulated genes encode novel peroxisomal proteins, a mitochondrial protein, and proteins of unknown location and function . We demonstrate that Oaf1p regulates certain genes in the absence of Pip2p and that both of these transcription factors play a role in maintaining the glucose-repressed state of one gene . Furthermore, we provide evidence that the defined consensus binding site is not required for the regulation of certain oleate-responsive genes.

Mol Cell Biol, 1998 Nov, 18(11), 6430 - 5
Homologous recombination, but not DNA repair, is reduced in vertebrate cells deficient in RAD52; Yamaguchi-Iwai Y et al.; Rad52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination . Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and Rad52 act cooperatively . To define the role of Rad52 in vertebrates, we generated RAD52(-/-) mutants of the chicken B-cell line DT40 . Surprisingly, RAD52(-/-) cells were not hypersensitive to DNA damages induced by gamma-irradiation, methyl methanesulfonate, or cis-platinum(II)diammine dichloride (cisplatin) . Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52(-/-) cells as in wild-type cells . Targeted integration frequencies, however, were consistently reduced in RAD52(-/-) cells, showing a clear role for Rad52 in genetic recombination . These findings reveal striking differences between S . cerevisiae and vertebrates in the functions of RAD51 and RAD52.

Mol Cell Biol, 1998 Nov, 18(11), 6423 - 9
Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation; Rijkers T et al.; The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation . In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway . Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination . Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination . Unlike the yeast Scrad52 mutant, MmRAD52(-/-) ES cells were not hypersensitive to agents that induce DSBs . MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system . These results show that, as in S . cerevisiae, MmRAD52 is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others . The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.

J Biol Chem, 1998 Oct 23, 273(43), 28219 - 28
Identification of limiting steps for efficient trans-activation of HIV-1 promoter by Tat in Saccharomyces cerevisiae; Daviet L et al.; Cellular context is an important determinant for the activity of Tat, the trans-activator of human immunodeficiency virus (HIV) . We have investigated HIV-1 promoter expression and trans-activation in Saccharomyces cerevisiae to provide clues about the limiting steps for Tat activity in this organism . A minimal 43-nucleotide HIV promoter (HIV43) has the activity of a weak yeast promoter in the presence or absence of various enhancer binding sites (bs), whereas the entire long terminal repeat is not expressed . None of these constructs could be trans-activated by Tat . Fusion proteins Gal4 binding domain (BD)-Tat48 and Gal4BD-Tat72 are active with different efficiencies on various yeast promoters that have Gal4 bs . They have 70 and 50% of Gal4 wild type activity on hybrid HIV promoters fused to Gal4 bs only in the presence of AP1 bs . This study shows that trans-activation of the HIV-1 promoter by Tat occurs in yeast when Tat is targeted to the promoter and a functional enhancer activity is present . Sp1 function and Tat transfer from the RNA to the promoter are two major elements for in vivo trans-activation of HIV-1 that are defective in S . cerevisiae but can be replaced by functional equivalents.

Carcinogenesis, 1998 Sep, 19(9), 1673 - 8
Rearrangements in minisatellite sequences induced by aflatoxin B1 in a metabolically competent strain of Saccharomyces cerevisiae; Kaplanski C et al.; The role of aflatoxin B1 (AFB1) in the induction of rearrangements affecting minisatellite sequences was studied in an in vitro yeast model . The Saccharomyces cerevisiae strain used expresses human cytochrome P450 1A2 and NADPH-cytochrome P450 oxidoreductase and has previously been used to study genetic recombination events induced by AFB1 . DNA multilocus fingerprinting was performed using probe M13 core hybridizing to a set of hypervariable minisatellite sequences in S . cerevisiae . Frequent spontaneous genomic alterations that affect the minisatellite fingerprint pattern were observed . Control cultures showed 15.8% rearrangements in minisatellites, and this frequency increased to 40.0% in cultures exposed to AFB1 (80 microg/ml) . A total of approximately 29 minisatellite loci were visualized for each culture . Given the number of cultures examined (40 AFB1-treated and 38 controls) the rearrangement frequency per detectable minisatellite was 2.59% in the AFB1-treated group and 0.73% in the control group, which represents a statistically significant (P = 0.001) difference . Thus, our data strongly suggest that AFB1 can promote the genetic events responsible for minisatellite rearrangements in the yeast genome . Such genetic rearrangements may be important events during the etiology of liver carcinogenesis in people chronically exposed to dietary aflatoxins.

RNA, 1998 Oct, 4(10), 1304 - 12
Identification and functional analysis of hPRP17, the human homologue of the PRP17/CDC40 yeast gene involved in splicing and cell cycle control; Ben Yehuda S et al.; The PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that participates in the second step of the splicing reaction . It was found recently that the yeast PRP17 gene is identical to the cell division cycle CDC40 gene . The PRP17/CDC40 gene codes for a protein with several copies of the WD repeat, a motif found in a large family of proteins that play important roles in signal transduction, cell cycle progression, splicing, transcription, and development . In this report, we describe the identification of human, nematode, and fission yeast homologues of the PRP17/CDC40 gene of S . cerevisiae . The newly identified proteins share homology with the budding yeast protein throughout their entire sequence, with the similarity being greatest in the C-terminal two thirds that includes the conserved WD repeats . We show that a yeast-human chimera, carrying the C-terminal two thirds of the hPRP17 protein, is able to complement the cell cycle and splicing defects of a yeast prp17 mutant . Moreover, the yeast and yeast-human chimeric proteins co-precipitate the intron-exon 2 lariat intermediate and the intron lariat product, providing evidence that these proteins are spliceosome-associated . These results show the functional conservation of the Prp17 proteins in evolution and suggest that the second step of splicing takes place by a similar mechanism throughout eukaryotes.

Res Microbiol, 1997 Mar-Apr, 148(3), 205 - 15
Identification of different daughter and parent subpopulations in an asynchronously growing Saccharomyces cerevisiae population; Porro D et al.; Under all growth conditions, a growing Saccharomyces cerevisiae yeast population is extremely heterogeneous, since individual cells differ in their cell size; this is due to their position in the cell division cycle and their genealogical age . To gain insight into the structure of a growing yeast population, we used a recently developed flow cytometric approach which enables, in asynchronously growing S . cerevisiae populations, tagging of both the cell age and the protein content of individual cells . This approach enabled the identification of daughter cells belonging to different cell cycle positions (i.e . newborn, G1, S + G2 + M + G1*, and dividing), thus yielding information about the relative fraction in the whole population, cell size and variability . More limited information could be obtained for the parent subpopulation; however, we were able to identify and characterize the dividing parent cells . The coefficient of variation (CV) of the protein content distribution for the dividing parents (27) was much higher than the CV of dividing daughters (18) . Further findings obtained indicated a large overlap between the cell protein content distributions of daughter and parent cells as well as between the protein content of cells of the same subpopulation but belonging to different stages of the cell division cycle . The analysis of these differences enables a better understanding of the complex structure of an asynchronously growing yeast population.

Bioseparation, 1998, 7(2), 107 - 15
Ribose-5-phosphate isomerase from Saccharomyces cerevisiae: purification and molecular analysis of the enzyme; Reuter R et al.; Purification and molecular analysis of ribose-5-phosphate isomerase (EC 5.3.1.6) from Saccharomyces cerevisiae is described first time . The enzyme was enriched from a haploid deletion mutant containing the wild-type gene on a multicopy plasmid elaborating the following steps: ammonium sulphate precipitation, interfacial salting out on Sepharose 6B, high performance liquid chromatography on Fractogel EMD DEAE and on Resource Phenyl . The enzyme activity was found to be rather unstable possibly caused by removal of stabilizing cofactors or proteins during the purification procedure . The purified enzyme showed a hyperbolic dependence on the substrate ribose-5-phosphate with a K(m)-value of 1.6 +/- 0.3 mmol/l . For the native enzyme a molecular mass of 115 +/- 10 kDa was determined as found by saccharose density gradient centrifugation, sedimentation equilibrium analysis, size exclusion chromatography and polyacrylamide gel electrophoresis . Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blotting revealed one band with a molecular mass of 31 +/- 2 kDa . Thus, the native enzyme is composed of four subunits of identical size . The molecular mass of the subunit and the identified N-terminal sequence of 33 amino acids fits well the 258 amino acid protein encoded by the S . cerevisiae RKI open reading frame, which was characterized previously only by increasing specific activities of ribose-5-phosphate isomerase in cells after cloning the gene . On the basis of the conserved amino acids an alignment of the amino acid sequence of ribose-5-phosphate isomerase from yeast with those of the enzyme from mouse, spinach and Escherichia coli is presented.

Acta Crystallogr D Biol Crystallogr, 1998 Jul 1, 54 ( Pt 4), 659 - 61
Crystallization and preliminary X-ray diffraction analysis of aspartate aminotransferase from Saccharomyces cerevisiae; Jeffery CJ et al.; Diffraction-quality crystals of S . cerevisiae cytoplasmic aspartate aminotransferase have been obtained by the hanging-drop vapor-diffusion method in the presence of pyridoxal phosphate and maleic acid, sodium acetate, ammonium acetate and polyethylene glycol . The crystals have the symmetry of the orthorhombic space groups P212121 or P21212 with unit-cell dimensions a = 130.2, b = 134.6 and c = 98.7 A . Square rod-shaped crystals with dimensions of approximately 0.2 x 0.2 x 0.5 mm diffract to spacings of 2 A . The calculated value of the Matthews coefficient, Vm = 2.4 A3 Da-1, is consistent with four subunits of aspartate aminotransferase per asymmetric unit.

Annu Rev Biochem, 1998, 67, 27 - 48
Sphingolipid functions in Saccharomyces cerevisiae: comparison to mammals; Dickson RC; Many roles for sphingolipids have been identified in mammals . Available data suggest that sphingolipids and their intermediates also have diverse roles in Saccharomyces cerevisiae . These roles include signal transduction during the heat stress response, regulation of calcium homeostasis or components in calcium-mediated signaling pathways, regulation of the cell cycle, and functions as components in trafficking of secretory vesicles from the endoplasmic reticulum to the Golgi apparatus and as the lipid moiety in many glycosylphosphatidylinositol-anchored proteins . S . cerevisiae is likely to be the first organism in which all genes involved in sphingolipid metabolism are identified . This information will provide an unprecedented opportunity to determine, for the first time in any organism, how sphingolipid synthesis is regulated . Through the use of both genetic and biochemical techniques, the identification of the complete array of processes regulated by sphingolipid signals is likely to be possible, as is the quantification of the physiological contribution of each.

J Biol Chem, 1998 Oct 9, 273(41), 26875 - 9
Transcriptional factor mutations reveal regulatory complexities of heat shock and newly identified stress genes in Saccharomyces cerevisiae; Treger JM et al.; A computer-aided pattern search of the entire yeast genome was designed and used to identify 186 putative stress response element-regulated genes in Saccharomyces cerevisiae . Transcript levels of eight of these candidate genes were examined, and three (37%) were shown to be heat shock- and DNA damage-inducible and to require the Msn2p and Msn4p transcriptional activators for stress regulation . Significantly, several heat shock protein (HSP) genes were identified in this computer search . Using a series of single and multiple regulatory mutants, we demonstrate unexpected regulatory complexities among the HSP genes from S . cerevisiae following heat shock.

J Biol Chem, 1998 Oct 9, 273(41), 26836 - 43
Identification of the MNN2 and MNN5 mannosyltransferases required for forming and extending the mannose branches of the outer chain mannans of Saccharomyces cerevisiae; Rayner JC et al.; The mannan structure found on the N-linked glycans of the yeast Saccharomyces cerevisiae is composed of a long backbone of alpha-1, 6-linked mannose to which are attached branches consisting of two alpha-1,2-linked mannoses followed by an alpha-1,3-linked mannose . In the mutants mnn2 and mnn5, the addition of the first and second of these two mannoses, respectively, is defective . In this paper, we report the identification of the genes corresponding to these mutations . The two genes encode closely related proteins with distant homology to the known Mnn1p alpha-1,3-mannosyltransferase . We show that these proteins are localized in an early compartment of the yeast Golgi and that they are not physically associated with each other or with the two protein complexes known to be involved in synthesizing the alpha-1,6-linked backbone . The identification of Mnn2p and Mnn5p allows us to assign Golgi proteins to all of the catalytic steps in S . cerevisiae mannan synthesis.

J Biol Chem, 1998 Oct 9, 273(41), 26298 - 304
Production of human compatible high mannose-type (Man5GlcNAc2) sugar chains in Saccharomyces cerevisiae; Chiba Y et al.; A yeast mutant capable of producing Man5GlcNAc2 human compatible sugar chains on glycoproteins was constructed . An expression vector for alpha-1,2-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae . An in vitro alpha-1,2-mannosidase assay and Western blot analysis showed that it was successfully localized in the endoplasmic reticulum . A triple mutant yeast lacking three glycosyltransferase activities was then transformed with an alpha-1, 2-mannosidase expression vector . The oligosaccharide structures of carboxypeptidase Y as well as cell surface glycoproteins were analyzed, and the recombinant yeast was shown to produce a series of high mannose-type sugar chains including Man5GlcNAc2 . This is the first report of a recombinant S . cerevisiae able to produce Man5GlcNAc2-oligosaccharides, the intermediate for hybrid-type and complex-type sugar chains.

J Biochem (Tokyo), 1998 Oct, 124(4), 756 - 63
Deletion analysis of protein kinase Calpha reveals a novel regulatory segment; Rotenberg SA et al.; Using a combined pharmacological and genetic approach, we have identified aa 260-280 in the C2 region as a critical factor in the catalytic function of protein kinase Calpha (PKCalpha) . Progressive truncations from the N-terminus as well as selected internal deletion mutants were expressed in Saccharomyces cerevisiae and tested for altered sensitivity to dequalinium, a PKC inhibitor whose target site was previously mapped to the catalytic domain . PKC mutants representing truncations of up to 158 amino acid residues (aa) from the N-terminus (ND84 and ND158) displayed 60-63% inhibition of kinase activity by 50 microM dequalinium, somewhat more sensitive than the wild-type PKCalpha enzyme (45% inhibition) . Mutant ND262, lacking N-terminal aa 1-262, was inhibited by almost 72% with 50 microM dequalinium, but mutant ND278, which lacked an additional 16 aa, was inhibited by only 9% of total activity . This result suggests that a C-terminal segment of the C2 region (aa 263-278) influences inhibition by dequalinium at low micromolar concentrations . An internal deletion mutant (D260-280) which retains the entire primary structure of PKCalpha except for aa 260-280, was similarly inhibited by only 4% with 50 microM dequalinium . In the absence of dequalinium and despite the presence of a nearly complete regulatory domain, this mutant exhibited constitutive activity (both in vitro and in a phenotypic assay with S . cerevisiae) that could not be further stimulated even by the potent activator TPA . Taken together, our findings suggest that, in the native structure of PKCalpha, the segment described by aa 260-280 regulates PKCalpha activity and influences the sensitivity of PKCalpha to dequalinium.

J Bacteriol, 1998 Oct, 180(19), 5020 - 9
Isolation of Candida glabrata homologs of the Saccharomyces cerevisiae KRE9 and KNH1 genes and their involvement in cell wall beta-1,6-glucan synthesis; Nagahashi S et al.; The Candida glabrata KRE9 (CgKRE9) and KNH1 (CgKNH1) genes have been isolated as multicopy suppressors of the tetracycline-sensitive growth of a Saccharomyces cerevisiae mutant with the disrupted KNH1 locus and the KRE9 gene placed under the control of a tetracycline-responsive promoter . Although a cgknh1Delta mutant showed no phenotype beyond slightly increased sensitivity to the K1 killer toxin, disruption of CgKRE9 resulted in several phenotypes similar to those of the S . cerevisiae kre9Delta null mutant: a severe growth defect on glucose medium, resistance to the K1 killer toxin, a 50% reduction of beta-1,6-glucan, and the presence of aggregates of cells with abnormal morphology on glucose medium . Replacement in C . glabrata of the cognate CgKRE9 promoter with the tetracycline-responsive promoter in a cgknh1Delta background rendered cell growth tetracycline sensitive on media containing glucose or galactose . cgkre9Delta cells were shown to be sensitive to calcofluor white specifically on glucose medium . In cgkre9 mutants grown on glucose medium, cellular chitin levels were massively increased.

Genes Dev, 1998 Sep 15, 12(18), 2932 - 42
mei-W68 in Drosophila melanogaster encodes a Spo11 homolog: evidence that the mechanism for initiating meiotic recombination is conserved; McKim KS et al.; Meiotic recombination requires the action of several gene products in both Saccharomyces cerevisiae and Drosophila melanogaster . Genetic studies in D . melanogaster have shown that the mei-W68 gene is required for all meiotic gene conversion and crossing-over . We cloned mei-W68 using a new genetic mapping method in which P elements are used to promote crossing-over at their insertion sites . This resulted in the high-resolution mapping of mei-W68 to a <18-kb region that contains a homolog of the S . cerevisiae spo11 gene . Molecular analysis of several mutants confirmed that mei-W68 encodes an spo11 homolog . Spo11 and MEI-W68 are members of a family of proteins similar to a novel type II topoisomerase . On the basis of this and other lines of evidence, Spo11 has been proposed to be the enzymatic activity that creates the double-strand breaks needed to initiate meiotic recombination . This raises the possibility that recombination in Drosophila is also initiated by double-strand breaks . Although these homologous genes are required absolutely for recombination in both species, their roles differ in other respects . In contrast to spo11, mei-W68 is not required for synaptonemal complex formation and does have a mitotic role.

Cell, 1998 Sep 4, 94(5), 607 - 13
Cse4p is a component of the core centromere of Saccharomyces cerevisiae; Meluh PB et al.; Histones are fundamental structural components of chromatin and are expected to play important roles in chromosome dynamics . Here, we present direct evidence that Cse4p, a histone H3 variant, is a structural component of the core centromere of S . cerevisiae . In histone H4 and Cse4p mutants, the core centromere chromatin structure is disrupted at restrictive temperature . Overexpression of Cse4p suppresses this defect in the H4 mutant, implying that the two proteins act together in centromere structure . We show by chromatin immunoprecipitation experiments that Cse4p is specifically cross-linked to centromeric DNA . Furthermore, by immunofluorescence microscopy, Cse4p is found in discrete foci consistent with that expected for centromeres . These results suggest the kinetochore is assembled on a specialized centromeric nucleosome containing Cse4p.

Genomics, 1998 Aug 15, 52(1), 50 - 61
Cloning, structural characterization, and chromosomal localization of the human orthologue of Saccharomyces cerevisiae MSH5 gene; Her C et al.; We have cloned and characterized the human orthologue of the Saccharomyces cerevisiae MutS homologue 5 (MSH5) cDNA, as well as the human gene that encodes the MSH5 cDNA, as a step toward understanding the molecular genetic mechanisms involved in the biological function of this novel human protein . The identified cDNA contains a 2505-bp open reading frame (ORF) that encodes an 834-amino-acid polypeptide with a predicted molecular mass of 92.9 kDa . The amino acid sequence encoded by this cDNA includes sequence motifs that are conserved in all known MutS homologues existing in bacteria to humans . The cDNA appears, on the basis of amino acid sequence analysis, to be a member of the MutS family and shares 30% sequence identity with that of S . cerevisiae MSH5, a yeast gene that plays a critical role in facilitating crossover during meiosis . Northern blot analysis demonstrated the presence of a 2.9-kb human MSH5 mRNA species in all human tissues tested, but the highest expression was in human testis, an organ containing cells that undergo constant DNA synthesis and meiosis . The expression pattern of human MSH5 resembled that of the previously identified human MutS homologues MSH2, MSH3, and MSH6-genes that are involved in the pathogenesis of hereditary nonpolyposis colorectal cancer (HNPCC) . In an effort to expedite the search for potential disease association with this new human MutS homologue, we have also determined the chromosomal location and structure of the human MSH5 locus . Sequence and structural characterization demonstrated that MSH5 spans approximately 25 kb and contains 26 exons that range in length from 36 bp for exon 8 to 254 bp for exon 25 . MSH5 has been mapped to human chromosome band 6p21.3 by fluorescence in situ hybridization . Knowledge of the sequence and gene structure of MSH5 will now enable studies of the possible roles MSH5 may play in meiosis and/or DNA replicative mismatch repair .

Nahrung, 1998 Aug, 42(3-4), 141 - 4
Construction of poly-met DNA yeast hybrids for increased methionine content: technofunctional properties of the hybrid yeasts; Halasz A et al.; Methionine is a limiting essential amino acid in human nutrition, to overcome the possible overdosage and improve bioavailability methionine supply should be in protein bound form . So insertion a poly-met encoding DNA sequence results a more efficient solution in increasing methionine content of yeast . Poly-met DNA yeast hybrids were constructed of Saccharomyces cerevisiae CB89, an auxotrophic mutant strain . Synthetic DNA sequence encoding methionine polypeptide was inserted into the polylinker region of pVT-U 100 vector with 2 mu plasmid replicon . After transformation of E . coli HB101 cells the efficiency of the ligation and transformation was checked by digesting the minipreps . S . cerevisiae CB89 was transformed with vector-poly-met insert and with the plasmid vector only as well . Hybrid yeasts were selected on uracilless medium . PVT-U 100 can be used as vector for the expression of DNA sequence in S . cerevisiae . The vector harbours the promoter of ADC1 gene immediately downstream from the promoter lies a polylinker sequence comprising unique restriction enzyme sites for BamHI, HindIII, PvuII, SacI, Xhol . The polylinker sequence is followed by the transcriptional stop site and polyadenylation signal of ADC1 gene . Plasmid pVT-U 100 has selection markers for S . cerevisiae (URA3) and for E . coli (amp, per F) . Results show that the poly-met DNA hybrids methionine content is influenced by the length of the insert . Fusion hybrids containing 600 bp oligo insert showed the best values . Distribution of methionine content in the protein subfractions of polymet DNA hybrid and parent strain CB89 was determined in dependence of glucose concentration and aeration intensity . The increase in synthesized methionine appeared in fractions 1 + 2 and residue . Technofunctional properties of parent strains and hybrids were compared for whole cells and cell wall (residue) . Results demonstrate that enrichment in methionine in the cell wall fraction resulted improvement of emulsifying ability . Bioavailability of methionine content was better in DNA hybrid yeast than in parent strain and was the best when propagated in whey medium.

Eur J Biochem, 1998 Aug 1, 255(3), 544 - 51
Stability of neutral trehalase during heat stress in Saccharomyces cerevisiae is dependent on the activity of the catalytic subunits of cAMP-dependent protein kinase, Tpk1 and Tpk2; Zahringer H et al.; In Saccharomyces cerevisiae cAMP-dependent protein kinase (cAPK) is involved in nutrient sensing and growth regulation via the Ras/cAMP pathway . Target enzymes, e.g . neutral trehalase, are activated or inactivated rapidly by cAPK-mediated phosphorylation . In addition, stress-induced transcription of genes of the general stress-response, e.g . HSP12, is negatively regulated via cAPK . We have investigated the effect of low cAPK activity on the stress-induced expression of neutral trehalase Nth1p . For this purpose we used mutants (tpk1tpk2TPK3, tpk1TPK2tpk3 and TPK1tpk2tpk3) with double knockouts of the three TPK genes encoding catalytic subunits of cAPK . It is shown that the tpk1tpk2TPK3 mutant, which has very low cAPK activity, exhibits a heat-stress-induced inactivation of neutral trehalase that is not observed in tpk1TPK2tpk3, TPK1tpk2tpk3 mutants and wild-type cells . However, heat stress induces an increase in NTH1 mRNA in the tpk1tpk2TPK3 mutant . Introduction of a plasmid carrying the TPK1 or TPK2 gene into tpk1tpk2TPK3 cells restores the heat-induced increase of neutral trehalase activity . In vitro and in vivo results suggest that the heat induced inactivation of neutral trehalase is due to a reversible inactivation of Nth1p . Our data indicate that a certain level of phosphorylation is essential for maintenance of neutral trehalase activity during heat shock in S . cerevisiae . Two identical putative cAPK phosphorylation sites have been found in the sequence predicted for the Nth1p . Stabilization and activation of neutral trehalase may be regulated by these sites . Furthermore, our data suggest that the heat-stress-induced transcription of the NTH1 gene is not negatively regulated by cAPK, that the TPK genes have no effect on the glucose repression of the NTH1 gene, and that non-detectable neutral trehalase activity in derepressed tpk1tpk2TPK3 cells is correlated with the reduced thermotolerance observed in this strain, similar to the heat-shock-recovery defect reported for the nth1delta mutant.

Mol Gen Genet, 1998 Jul, 259(1), 29 - 38
Ste50p is involved in regulating filamentous growth in the yeast Saccharomyces cerevisiae and associates with Ste11p; Ramezani Rad M et al.; STE50 is required to sustain pheromone-induced signal transduction in S . cerevisiae . Here we report that Ste50p is involved in regulating pseudohyphal development . Both of these processes are also dependent on Ste11p . Deletion of STE50 leads to defects in filamentous growth, which can be suppressed by overproduction of Ste11p . Overexpression of STE11 also suppresses the mating defects of ste50 mutants . We have analysed the physical association between Ste50p and Ste11p in extracts of cells harvested under various conditions . A Ste11p-Ste50p complex can be isolated from extracts of cells in which the pheromone response has been activated, as well as from normally growing cells . Formation of the Ste50p-Ste11p complex does not require G(alpha), G(beta), Ste20p or Ste5p . Oligomerisation of Ste11p is shown to be independent of activation of the pheromone response pathway, and occurs in the absence of Ste50p . We conclude that Ste50p is necessary for Ste11p activity in at least two differentiation programmes: mating and filamentous growth.

FEBS Lett, 1998 Aug 28, 434(1-2), 71 - 6
The hexokinase 2 protein participates in regulatory DNA-protein complexes necessary for glucose repression of the SUC2 gene in Saccharomyces cerevisiae; Herrero P et al.; The HXK2 gene plays an important role in glucose repression in the yeast Saccharomyces cerevisiae . Recently we have described that the HXK2 gene product, isoenzyme 2 of hexokinase, is located both in the nucleus and in the cytoplasm of S . cerevisiae cells . In this work we used deletion analysis to identify the essential part of the protein-mediating nuclear localisation . Determinations of fructose-kinase activity and immunoblot analysis using anti-Hxk2 antibodies in isolated nuclei, together with observations of the fluorescence distribution of Hxk2-GFP fusion protein in cells transformed with an HXK2::gfp mutant gene, indicated that the decapeptide KKPQARKGSM, located between amino acid residues 7 and 16 of hexokinase 2, is important for nuclear localisation of the protein . Further experimental evidence, measuring invertase activity in wild-type and mutant cells expressing a truncated version of the Hxk2 protein unable to enter the nucleus, shows that a nuclear localisation of Hxk2 is necessary for glucose repression signalling of the SUC2 gene . Furthermore, we demonstrate using gel mobility shift analysis that Hxk2 participates in DNA-protein complexes with cis-acting regulatory elements of the SUC2 gene promoter.

Mol Cell, 1998 Aug, 2(2), 173 - 82
CLB5-dependent activation of late replication origins in S . cerevisiae; Donaldson AD et al.; Replication origins in chromosomes are activated at specific times during the S phase . We show that the B-type cyclins are required for proper execution of this temporal program . clb5 cells activate early origins but not late origins, explaining the previously described long clb5 S phase . Origin firing appears normal in cIb6 mutants . In clb5 clb6 double mutant cells, the late origin firing defect is suppressed, accounting for the normal duration of the phase despite its delayed onset . Therefore, Clb5p promotes the timely activation of early and late origins, but Clb6p can activate only early origins . In clb5 clb6 mutants, the other B-type cyclins (Clb1-4p) promote an S phase during which both early and late replication origins fire.

J Biol Chem, 1998 Sep 18, 273(38), 24529 - 34
The Saccharomyces cerevisiae NDE1 and NDE2 genes encode separate mitochondrial NADH dehydrogenases catalyzing the oxidation of cytosolic NADH; Luttik MA et al.; In Saccharomyces cerevisiae, the NDI1 gene encodes a mitochondrial NADH dehydrogenase, the catalytic side of which projects to the matrix side of the inner mitochondrial membrane . In addition to this NADH dehydrogenase, S . cerevisiae exhibits another mitochondrial NADH-dehydrogenase activity, which oxidizes NADH at the cytosolic side of the inner membrane . To investigate whether open reading frames YMR145c/NDE1 and YDL 085w/NDE2, which exhibit sequence similarity with NDI1, encode the latter enzyme, NADH-dependent mitochondrial respiration was assayed in wild-type S . cerevisiae and nde deletion mutants . Mitochondria were isolated from aerobic, glucose-limited chemostat cultures grown at a dilution rate (D) of 0 . 10 h-1, in which reoxidation of cytosolic NADH by wild-type cells occurred exclusively by respiration . Compared with the wild type, rates of mitochondrial NADH oxidation were about 3-fold reduced in an nde1Delta mutant and unaffected in an nde2Delta mutant . NADH-dependent mitochondrial respiration was completely abolished in an nde1Delta nde2Delta double mutant . Mitochondrial respiration of substrates other than NADH was not affected in nde mutants . In shake flasks, an nde1Delta nde2Delta mutant exhibited reduced specific growth rates on ethanol and galactose but not on glucose . Glucose metabolism in aerobic, glucose-limited chemostat cultures (D = 0.10 h-1) of an nde1Delta nde2Delta mutant was essentially respiratory . Apparently, under these conditions alternative systems for reoxidation of cytosolic NADH could replace the role of Nde1p and Nde2p in S . cerevisiae.

J Mol Biol, 1998 Sep 11, 282(1), 13 - 24
Missense translation errors in Saccharomyces cerevisiae; Stansfield I et al.; We describe the development of a novel plasmid-based assay for measuring the in vivo frequency of misincorporation of amino acids into polypeptide chains in the yeast Saccharomyces cerevisiae . The assay is based upon the measurement of the catalytic activity of an active site mutant of type III chloramphenicol acetyl transferase (CATIII) expressed in S . cerevisiae . A His195(CAC)-->Tyr195(UAC) mutant of CATIII is completely inactive, but catalytic activity can be restored by misincorporation of histidine at the mutant UAC codon . The average error frequency of misincorporation of histidine at this tyrosine UAC codon in wild-type yeast strains was measured as 0 . 5x10(-5) and this frequency was increased some 50-fold by growth in the presence of paromomycin, a known translational-error-inducing antibiotic . A detectable frequency of misincorporation of histidine at a mutant Ala195 GCU codon was also measured as 2x10(-5), but in contrast to the Tyr195-->His195 misincorporation event, the frequency of histidine misincorporation at Ala195 GCU was not increased by paromomycin, inferring that this error did not result from miscognate codon-anticodon interaction . The His195 to Tyr195 missense error assay was used to demonstrate increased frequencies of missense error at codon 195 in SUP44 and SUP46 mutants . These two mutants have previously been shown to exhibit a translation termination error phenotype and the sup44+ and sup46+ genes encode the yeast ribosomal proteins S4 and S9, respectively . These data represent the first accurate in vivo measurement of a specific mistranslation event in a eukaryotic cell and directly confirm that the eukaryotic ribosome plays an important role in controlling missense errors arising from non-cognate codon-anticodon interactions .

J Cell Biol, 1998 Sep 7, 142(5), 1301 - 12
Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis; Bi E et al.; In Saccharomyces cerevisiae, the mother cell and bud are connected by a narrow neck . The mechanism by which this neck is closed during cytokinesis has been unclear . Here we report on the role of a contractile actomyosin ring in this process . Myo1p (the only type II myosin in S . cerevisiae) forms a ring at the presumptive bud site shortly before bud emergence . Myo1p ring formation depends on the septins but not on F-actin, and preexisting Myo1p rings are stable when F-actin is depolymerized . The Myo1p ring remains in the mother-bud neck until the end of anaphase, when a ring of F-actin forms in association with it . The actomyosin ring then contracts to a point and disappears . In the absence of F-actin, the Myo1p ring does not contract . After ring contraction, cortical actin patches congregate at the mother-bud neck, and septum formation and cell separation rapidly ensue . Strains deleted for MYO1 are viable; they fail to form the actin ring but show apparently normal congregation of actin patches at the neck . Some myo1Delta strains divide nearly as efficiently as wild type; other myo1Delta strains divide less efficiently, but it is unclear whether the primary defect is in cytokinesis, septum formation, or cell separation . Even cells lacking F-actin can divide, although in this case division is considerably delayed . Thus, the contractile actomyosin ring is not essential for cytokinesis in S . cerevisiae . In its absence, cytokinesis can still be completed by a process (possibly localized cell-wall synthesis leading to septum formation) that appears to require septin function and to be facilitated by F-actin.

J Cell Biol, 1998 Sep 7, 142(5), 1223 - 33
Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure; Jakob CA et al.; In Saccharomyces cerevisiae, transfer of N-linked oligosaccharides is immediately followed by trimming of ER-localized glycosidases . We analyzed the influence of specific oligosaccharide structures for degradation of misfolded carboxypeptidase Y (CPY) . By studying the trimming reactions in vivo, we found that removal of the terminal alpha1,2 glucose and the first alpha1,3 glucose by glucosidase I and glucosidase II respectively, occurred rapidly, whereas mannose cleavage by mannosidase I was slow . Transport and maturation of correctly folded CPY was not dependent on oligosaccharide structure . However, degradation of misfolded CPY was dependent on specific trimming steps . Degradation of misfolded CPY with N-linked oligosaccharides containing glucose residues was less efficient compared with misfolded CPY bearing the correctly trimmed Man8GlcNAc2 oligosaccharide . Reduced rate of degradation was mainly observed for misfolded CPY bearing Man6GlcNAc2, Man7GlcNAc2 and Man9GlcNAc2 oligosaccharides, whereas Man8GlcNAc2 and, to a lesser extent, Man5GlcNAc2 oligosaccharides supported degradation . These results suggest a role for the Man8GlcNAc2 oligosaccharide in the degradation process . They may indicate the presence of a Man8GlcNAc2-binding lectin involved in targeting of misfolded glycoproteins to degradation in S . cerevisiae.

J Biol Chem, 1998 Sep 11, 273(37), 23716 - 21
Metalloregulation of FRE1 and FRE2 homologs in Saccharomyces cerevisiae; Martins LJ et al.; The high affinity uptake systems for iron and copper ions in Saccharomyces cerevisiae involve metal-specific permeases and two known cell surface Cu(II) and Fe(III) metalloreductases, Fre1 and Fre2 . Five novel genes found in the S . cerevisiae genome exhibit marked sequence similarity to Fre1 and Fre2, suggesting that the homologs are part of a family of proteins related to Fre1 and Fre2 . The homologs are expressed genes in S . cerevisiae, and their expression is metalloregulated as is true with FRE1 and FRE2 . Four of the homologs (FRE3-FRE6) are specifically iron-regulated through the Aft1 transcription factor . These genes are expressed either in cells limited for iron ion uptake by treatment with a chelator or in cells lacking the high affinity iron uptake system . Expression of FRE3-FRE6 is elevated in AFT1-1 cells and attenuated in aft1 null cells, showing that iron modulation occurs through the Aft1 transcriptional activator . The fifth homolog FRE7 is specifically copper-metalloregulated . FRE7 is expressed in cells limited in copper ion uptake by a Cu(I)-specific chelator or in cells lacking the high affinity Cu(I) permeases . The constitutive expression of FRE7 in MAC1 cells and the lack of expression in mac1-1 cells are consistent with Mac1 being the critical transcriptional activator of FRE7 expression . The 5' promoter sequence of FRE7 contains three copper-responsive promoter elements . Two elements are critical for Mac1-dependent FRE7 expression . Combinations of either the distal and central elements or the central and proximal elements result in copper-regulated FRE7 expression . Spacing between Mac1-responsive sites is important as shown by the attenuated expression of FRE7 and CTR1 when two elements are separated by over 100 base pairs . From the three Mac1-responsive elements in FRE7, a new consensus sequence for Mac1 binding can be established as TTTGC(T/G)C(A/G).

J Biol Chem, 1998 Sep 4, 273(36), 23248 - 57
Growth regulation of the expression of mouse cDNA and gene encoding a serine/threonine kinase related to Saccharomyces cerevisiae CDC7 essential for G1/S transition . Structure, chromosomal localization, and expression of mouse gene for s . cerevisiae Cdc7-related kinase; Kim JM et al.; Saccharomyces cerevisiae CDC7 encodes a serine/threonine kinase required for G1/S transition of the yeast cells . We previously reported human and Xenopus cDNAs encoding CDC7-related kinases and suggested the possibility that chromosomal replication of higher eukaryotes may be regulated through conserved mechanisms involving Cdc7-related kinases . Here we report a murine cDNA and gene (muCdc7) encoding a serine/threonine kinase related to CDC7 . The predicted coding frame for the longest cDNA for muCdc7 consists of 564 amino acids, which shares 46, 77, and 93% identity, respectively, with those of budding yeast, Xenopus, and human in kinase conserved domains . The chromosomal gene for muCdc7, located at the band 5E5 on the mouse chromosome 5, consists of 12 exons, and its exon/intron organization shares some similarity with that of other protein kinases including Cdk and cAMP-dependent kinase . Transcription of muCdc7, initiated at multiple sites over the 370-base pair promoter region, is repressed in the resting state and is induced at the G1/S boundary after growth factor stimulation in a growth factor-dependent cell line . Transient transfection assays indicated that a 231-base pair segment of the muCdc7 promoter containing three putative E2F binding sites and one Sp1 site but lacking TATA sequence is sufficient for response to growth stimulation.

J Biol Chem, 1998 Sep 4, 273(36), 22873 - 6
Glutamine-rich domains activate transcription in yeast Saccharomyces cerevisiae; Xiao H et al.; Activation domains of eukaryotic transcription factors can be classified into at least three distinct types based on their amino acid composition: acidic, proline-rich, and glutamine-rich . Acidic activators, such as yeast GAL4 and GCN4 and herpes simplex virus VP16, have been shown to stimulate transcription in various higher and lower eukaryotic cells . Similarly, proline-rich activators also function in both mammalian and yeast cells . These activators are regarded to possess "universal" activating potentials . By contrast, several studies have suggested that glutamine-rich activators such as human Sp1 are active in higher (mammalian) but not lower (yeast) eukaryotic cells . One interpretation is that lower eukaryotic cells lack a critical co-factor necessary for a glutamine-rich domain . This reasoning is counter-intuitive because many native yeast activator proteins contain glutamine-rich domains . Here, we have investigated the activity of a glutamine-rich GAL4-Sp1 domain A (Sp1A) hybrid protein in yeast Saccharomyces cerevisiae . We show that GAL4-Sp1A activated a GAL1-lacZ reporter by more than 200-fold over basal when the reporter was carried on a 2mu vector . The generality of the Sp1A results is supported by our finding that yeast glutamine-rich domains from HAP2 and MCM1 are also transcriptionally active in S . cerevisiae . Interestingly, we found that glutamine-rich domains are considerably less potent when responsive promoters (i.e . GAL1-lacZ) are integrated into yeast chromosome . Thus our results segregate the inherent transcriptional activity of a glutamine-rich domain in yeast S . cerevisiae from its apparent lack of activity when assayed on chromosomally embedded promoters.

FEMS Microbiol Lett, 1998 Aug 1, 165(1), 15 - 20
Overproduction of acetyl-coenzyme A synthetase isoenzymes in respiring Saccharomyces cerevisiae cells does not reduce acetate production after exposure to glucose excess; de Jong-Gubbels P et al.; To investigate whether the production of acetate which occurs after exposure of respiring Saccharomyces cerevisiae cells to excess glucose can be reduced by overproduction of acetyl-CoA synthetase (ACS, EC 6.2.1.1), the ACS1 and ACS2 genes were introduced on multi-copy plasmids . For each isoenzyme, the level in glucose-limited chemostat cultures was increased by 3-6-fold, relative to an isogenic reference strain . However, ACS overproduction did not result in a reduced production of acetate after a glucose pulse (100 mmol l-1) to these cultures . This indicates that a limited capacity of ACS is not the sole cause of acetate accumulation in S . cerevisiae.

Mol Cell Biol, 1998 Sep, 18(9), 5425 - 34
A role for SRp54 during intron bridging of small introns with pyrimidine tracts upstream of the branch point; Kennedy CF et al.; One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3' splice site region . U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S . cerevisiae), thereby participating in bridging either exons or introns . In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3' splice site . Many small introns, especially those in nonvertebrates, lack a classical 3' pyrimidine tract . Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5' splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5' and 3' splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor . The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1 . Addition of a strong 3' pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory . We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.

Cell, 1998 Aug 7, 94(3), 375 - 86
The meiosis-specific Hop2 protein of S . cerevisiae ensures synapsis between homologous chromosomes; Leu JY et al.; The hop2 mutant of S . cerevisiae displays a novel phenotype: meiotic chromosomes form nearly wild-type amounts of synaptonemal complex, but most chromosomes are engaged in synapsis with nonhomologous partners . The meiosis-specific Hop2 protein localizes to chromosomes prior to and during synapsis and in the absence of the double-strand breaks that initiate recombination . hop2 strains sustain a wild-type level of meiotic double-strand breaks, but these breaks remain unrepaired . The hop2 mutant arrests at the pachytene stage of meiotic prophase with the RecA-like protein Dmc1 located at numerous sites along synapsed chromosomes . We propose that the Hop2 protein functions to prevent synapsis between nonhomologous chromosomes.

Proc Natl Acad Sci U S A, 1998 Aug 18, 95(17), 9997 - 10002
Generation of a strong mutator phenotype in yeast by imbalanced base excision repair; Glassner BJ et al.; Increased spontaneous mutation is associated with increased cancer risk . Here, by using a model system, we show that spontaneous mutation can be increased several hundred-fold by a simple imbalance between the first two enzymes involved in DNA base excision repair . The Saccharomyces cerevisiae MAG1 3-methyladenine (3MeA) DNA glycosylase, when expressed at high levels relative to the apurinic/apyrimidinic endonuclease, increases spontaneous mutation by up to approximately 600-fold in S . cerevisiae and approximately 200-fold in Escherichia coli . Genetic evidence suggests that, in yeast, the increased spontaneous mutation requires the generation of abasic sites and the processing of these sites by the REV1/REV3/REV7 lesion bypass pathway . Comparison of the mutator activity produced by Mag1, which has a broad substrate range, with that produced by the E . coli Tag 3MeA DNA glycosylase, which has a narrow substrate range, indicates that the removal of endogenously produced 3MeA is unlikely to be responsible for the mutator effect of Mag1 . Finally, the human AAG 3-MeA DNA glycosylase also can produce a small (approximately 2-fold) but statistically significant increase in spontaneous mutation, a result which could have important implications for carcinogenesis.

EMBO J, 1998 Aug 17, 17(16), 4809 - 17
Zuotin, a ribosome-associated DnaJ molecular chaperone; Yan W et al.; Correct folding of newly synthesized polypeptides is thought to be facilitated by Hsp70 molecular chaperones in conjunction with DnaJ cohort proteins . In Saccharomyces cerevisiae, SSB proteins are ribosome-associated Hsp70s which interact with the newly synthesized nascent polypeptide chain . Here we report that the phenotypes of an S.cerevisiae strain lacking the DnaJ-related protein Zuotin (Zuo1) are very similar to those of a strain lacking Ssb, including sensitivities to low temperatures, certain protein synthesis inhibitors and high osmolarity . Zuo1, which has been shown previously to be a nucleic acid-binding protein, is also a ribosome-associated protein localized predominantly in the cytosol . Analysis of zuo1 deletion and truncation mutants revealed a positive correlation between the ribosome association of Zuo1 and its ability to bind RNA . We propose that Zuo1 binds to ribosomes, in part, by interaction with ribosomal RNA and that Zuo1 functions with Ssb as a chaperone on the ribosome.

Annu Rev Nutr, 1998, 18, 441 - 69
The molecular biology of metal ion transport in Saccharomyces cerevisiae; Eide DJ; Transition metals such as iron, copper, manganese, and zinc are essential nutrients . The yeast Saccharomyces cerevisiae is an ideal organism for deciphering the mechanism and regulation of metal ion transport . Recent studies of yeast have shown that accumulation of any single metal ion is mediated by two or more substrate-specific transport systems . High-affinity systems are active in metal-limited cells, whereas low-affinity systems play the predominant roles when the substrate is more abundant . Metal ion uptake systems of cells are tightly controlled, and both transcriptional and posttranscriptional regulatory mechanisms have been identified . Most importantly, studies of S . cerevisiae have identified a large number of genes that function in metal ion transport and have illuminated the existence of importance of gene families that play related roles in these processes in mammals.

J Clin Microbiol, 1998 Sep, 36(9), 2613 - 7
Species identification and virulence attributes of Saccharomyces boulardii (nom . inval.); McCullough MJ et al.; Saccharomyces boulardii (nom . inval.) has been used for the treatment of several types of diarrhea . Recent studies have confirmed that S . boulardii is effective in the treatment of diarrhea, in particular chronic or recurrent diarrhea, and furthermore that it is a safe and well-tolerated treatment . The aim of the present study was to identify strains of S . boulardii to the species level and assess their virulence in established murine models . Three strains of S . boulardii were obtained from commercially available products in France and Italy . The three S . boulardii strains did not form spores upon repeated testing . Therefore, classical methods used for the identification of Saccharomyces spp . could not be undertaken . Typing by using the restriction fragment length polymorphisms (RFLPs) of the PCR-amplified intergenic transcribed spacer regions (including the 5 . 8S ribosomal DNA) showed that the three isolates of S . boulardii were not separable from authentic isolates of Saccharomyces cerevisiae with any of the 10 restriction endonucleases assessed, whereas 9 of the 10 recognized species of Saccharomyces could be differentiated . RFLP analysis of cellular DNA with EcoRI showed that all three strains of S . boulardii had identical patterns and were similar to other authentic S . cerevisiae isolates tested . Therefore, the commercial strains of S . boulardii available to us cannot be genotypically distinguished from S . cerevisiae . Two S . boulardii strains were tested in CD-1 and DBA/2N mouse models of systemic disease and showed intermediate virulence compared with virulent and avirulent strains of S . cerevisiae . The results of the present study show that these S . boulardii strains are asporogenous strains of the species S . cerevisiae, not representatives of a distinct and separate species, and possess moderate virulence in murine models of systemic infection . Therefore, caution should be advised in the clinical use of these strains in immunocompromised patients until further study is undertaken.

Mol Cell, 1998 Jul, 2(1), 135 - 40
Circularization of mRNA by eukaryotic translation initiation factors; Wells SE et al.; Communication between the 5' cap structure and 3' poly(A) tail of eukaryotic mRNA results in the synergistic enhancement of translation . The cap and poly(A) tail binding proteins, eIF4E and Pab1p, mediate this effect in the yeast S . cerevisiae through their interactions with different parts of the translation factor eIF4G . Here, we demonstrate the reconstitution of an eIF4E/eIF4G/Pab1p complex with recombinant proteins, and show by atomic force microscopy that the complex can circularize capped, polyadenylated RNA . Our results suggest that formation of circular mRNA by translation factors could contribute to the control of mRNA expression in the eukaryotic cell.

Mol Cell, 1998 Jul, 2(1), 65 - 73
A genome-wide transcriptional analysis of the mitotic cell cycle; Cho RJ et al.; Progression through the eukaryotic cell cycle is known to be both regulated and accompanied by periodic fluctuation in the expression levels of numerous genes . We report here the genome-wide characterization of mRNA transcript levels during the cell cycle of the budding yeast S . cerevisiae . Cell cycle-dependent periodicity was found for 416 of the 6220 monitored transcripts . More than 25% of the 416 genes were found directly adjacent to other genes in the genome that displayed induction in the same cell cycle phase, suggesting a mechanism for local chromosomal organization in global mRNA regulation . More than 60% of the characterized genes that displayed mRNA fluctuation have already been implicated in cell cycle period-specific biological roles . Because more than 20% of human proteins display significant homology to yeast proteins, these results also link a range of human genes to cell cycle period-specific biological functions.

Mol Cell, 1998 Jul, 2(1), 9 - 22
Chromosomal rearrangements occur in S . cerevisiae rfa1 mutator mutants due to mutagenic lesions processed by double-strand-break repair; Chen C et al.; Three temperature-sensitive S . cerevisiae RFA1 alleles were found to cause elevated mutation rates . These mutator phenotypes resulted from the accumulation of base substitutions, frameshifts, gross deletions (8 bp-18 kb), and nonreciprocal translocations . A representative rfa1 mutation exhibited a growth defect in conjunction with rad51, rad52, or rad10 mutations, suggesting an accumulation of double-strand breaks . rad10 and rad52 mutations eliminated deletion and translocation formation, whereas a rad51 mutation increased the frequency of these events and revealed a new class of genetic rearrangements--loss of a portion of a chromosome arm combined with telomere addition . The breakpoints of the translocations and deletions were flanked by imperfect direct repeats of 2-20 bp, similar to the breakpoint structures observed at translocations and gross deletions, including LOH events, underlying human cancer and other hereditary diseases.

Biochemistry, 1998 Aug 11, 37(32), 11323 - 31
Characterization of recombinant Saccharomyces cerevisiae manganese-containing superoxide dismutase and its H30A and K170R mutants expressed in Escherichia coli; Borders CL Jr et al.; All known Mn-containing superoxide dismutases (MnSODs) have a highly conserved histidine (His-30 in Escherichia coli FeSOD) in the active-site channel, and nearly all have an active-site arginine (Arg-170) that has been proposed to play a combined structural and functional role {Chan et al., Arch . Biochem . Biophys . 279, 195-201 (1990)} . In Saccharomyces cerevisiae MnSOD, the active-site arginine is replaced by a lysine . The S . cerevisiae MnSOD gene has been cloned and expressed in E . coli, and H30A and K170R site-specific mutants have been prepared . The purified recombinant native (RN) and mutant enzymes were compared to one another and to the native enzyme purified from S . cerevisiae (SC) in terms of activity, temperature stability, and sensitivity to 2,4,6-trinitrobenzenesulfonate (TNBS) and phenylglyoxal (PG) . All enzymes had high specific activities (SC = 5000, RN = 5600, H30A = 4500, K170R = 4600) (U/mg, using the pyrogallol assay) . SC, RN, and H30A were very stable at 75 degreesC (pH 8.0), with half-lives of 4.7, 2.8, and 2.7 h, respectively, while K170R had a much greater temperature lability, with a half-life of 0.36 h under these conditions . TNBS (0.5 mM, pH 9.0, 25 degreesC) rapidly inactivated SC, RN, and H30A, with half-lives of 3 . 5, 5.1, and 5.5 min, respectively, but only slowly inactivated K170R, with a half-life of 101 min . PG (20 mM, pH 9.0, 25 degreesC) caused very slow inactivation of SC, RN, and H30A by biphasic kinetics, and each enzyme retained >/=25% activity after 3 h of modification . K170R, on the other hand, was completely inactivated by PG under these conditions by first-order kinetics, with a half-life of 7.0 min . The data suggest that His-30, a residue highly conserved in the active-site channel of MnSODs and FeSODs, does not play a crucial role in catalysis or stability . In addition, Lys-170, a residue that is almost always arginine in the numerous other MnSODs and FeSODs sequenced to date, can be replaced by arginine with no loss of catalytic activity, but K170R is less stable and Arg-170 in this mutant is more exposed than the corresponding arginine in other SODs . RN and SC showed some surprising differences . Thus, while the specific activities of RN and SC are very similar, SC is more stable to inactivation at 75 degreesC, and less susceptible to inactivation by phenylglyoxal, than RN . These data suggest that there may be slight differences in the tertiary structures of SC, the native enzyme expressed in S . cerevisiae, and RN, the recombinant native enzyme expressed in E . coli.

Biochim Biophys Acta, 1998 Jun 10, 1365(1-2), 125 - 34
Mechanism of ubiquinol oxidation by the cytochrome bc1 complex: pre-steady-state kinetics of cytochrome bc1 complexes containing site-directed mutants of the Rieske iron-sulfur protein; Snyder C et al.; To facilitate characterization of mutated cytochrome bc1 complexes in S . cerevisiae we have developed a new approach using a rapid scanning monochromator to examine pre-steady-state reduction of the enzyme with menaquinol . The RSM records optical spectra of cytochromes b and c1 at 1-ms intervals after a dead time of 2 ms, and menaquinol fully reduces both cytochromes bH and c1 and a portion of cytochrome bL . The rapid-mixing, rapid-scanning monochromator methodology obviates limitations inherent in previous rapid kinetics methods and permits measurements of rates exceeding 200 s-1 . To document the validity of this methodology we have examined the reduction kinetics of the cytochrome bc1 complexes from wild-type yeast and yeast that lack ubiquinone . The results establish that menaquinol reacts via the Q cycle pathway both in the presence and absence of ubiquinone . From analyzing bc1 complexes containing Rieske proteins in which the midpoint potential of the iron-sulfur cluster has been altered from +280 to +105 mV, we propose a mechanism in which the protonated quinol displaces a proton from the imidazole nitrogen of one of the histidines that is a ligand to the iron-sulfur cluster and forms a quinol-imidazolate complex that is the electron donor to the redox active iron.

Genetics, 1998 Aug, 149(4), 1763 - 75
Identification of the genes encoding the cytosolic translation release factors from Podospora anserina and analysis of their role during the life cycle; Gagny B et al.; In an attempt to decipher their role in the life history and senescence process of the filamentous fungus Podospora anserina, we have cloned the su1 and su2 genes, previously identified as implicated in cytosolic translation fidelity . We show that these genes are the equivalents of the SUP35 and SUP45 genes of Saccharomyces cerevisiae, which encode the cytosolic translation termination factors eRF3 and eRF1, respectively . Mutations in these genes that suppress nonsense mutations may lead to drastic mycelium morphology changes and sexual impairment but have little effect on life span . Deletion of su1, coding for the P . anserina eRF3, is lethal . Diminution of its expression leads to a nonsense suppressor phenotype whereas its overexpression leads to an antisuppressor phenotype . P . anserina eRF3 presents an N-terminal region structurally related to the yeast eRF3 one . Deletion of the N-terminal region of P . anserina eRF3 does not cause any vegetative alteration; especially life span is not changed . However, it promotes a reproductive impairment . Contrary to what happens in S . cerevisiae, deletion of the N terminus of the protein promotes a nonsense suppressor phenotype . Genetic analysis suggests that this domain of eRF3 acts in P . anserina as a cis-activator of the C-terminal portion and is required for proper reproduction.

Genetics, 1998 Aug, 149(4), 1707 - 15
GIT1, a gene encoding a novel transporter for glycerophosphoinositol in Saccharomyces cerevisiae; Patton-Vogt JL et al.; Phosphatidylinositol catabolism in Saccharomyces cerevisiae cells cultured in media containing inositol results in the release of glycerophosphoinositol (GroPIns) into the medium . As the extracellular concentration of inositol decreases with growth, the released GroPIns is transported back into the cell . Exploiting the ability of the inositol auxotroph, ino1, to use exogenous GroPIns as an inositol source, we have isolated mutants (Git-) defective in the uptake and metabolism of GroPIns . One mutant was found to be affected in the gene encoding the transcription factor, SPT7 . Mutants of the positive regulatory gene INO2, but not of its partner, INO4, also have the Git- phenotype . Another mutant was complemented by a single open reading frame (ORF) termed GIT1 (glycerophosphoinositol) . This ORF consists of 1556 bp predicted to encode a polypeptide of 518 amino acids and 57.3 kD . The predicted Git1p has similarity to a variety of S . cerevisiae transporters, including a phosphate transporter (Pho84p), and both inositol transporters (Itr1p and Itr2p) . Furthermore, Git1p contains a sugar transport motif and 12 potential membrane-spanning domains . Transport assays performed on a git1 mutant together with the above evidence indicate that the GIT1 gene encodes a permease involved in the uptake of GroPIns.

Proc Natl Acad Sci U S A, 1998 Aug 4, 95(16), 9167 - 71
Molecular remedy of complex I defects: rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria restores the NADH oxidase activity of complex I-deficient mammalian cells; Seo BB et al.; The NDI1 gene encoding rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria was cotransfected into the complex I-deficient Chinese hamster CCL16-B2 cells . Stable NDI1-transfected cells were obtained by screening with antibiotic G418 . The NDI1 gene was shown to be expressed in the transfected cells . The expressed Ndi1 enzyme was recognized to be localized to mitochondria by immunoblotting and confocal immunofluorescence microscopic analyses . Using digitonin-permeabilized cells, it was shown that the transfected cells, but not nontransfected control cells, exhibited the electron transfer activities with glutamate/malate as the respiratory substrate . The activities were inhibited by flavone, antimycin A, and KCN but not by rotenone . Added NADH did not serve as the substrate, suggesting that the expressed Ndi1 enzyme was located on the matrix side of the inner mitochondrial membranes . Furthermore, although nontransfected cells could not survive in a medium low in glucose (0.6 mM), which is a substrate of glycolysis, the NDI1-transfected cells were able to grow in the absence of added glucose . When glycolysis is slow, either at low glucose concentrations or in the presence of galactose, respiration is required for cells to survive . The mutant cells do not survive at low glucose or in galactose, but they can be rescued by Ndi1 . These results indicated that the S . cerevisiae Ndi1 was expressed functionally in CCL16-B2 cells and catalyzed electron transfer from NADH in the matrix to ubiquinone-10 in the inner mitochondrial membranes . It is concluded that the NDI1 gene provides a potentially useful tool for gene therapy of mitochondrial diseases caused by complex I deficiency.

Appl Environ Microbiol, 1998 Aug, 64(8), 2794 - 9
Expression and secretion of defined cutinase variants by Aspergillus awamori; van Gemeren IA et al.; Several cutinase variants derived by molecular modelling and site-directed mutagenesis of a cutinase gene from Fusarium solani pisi are poorly secreted by Saccharomyces cerevisiae . The majority of these variants are successfully produced by the filamentous fungus Aspergillus awamori . However, the L51S and T179Y mutations caused reductions in the levels of extracellular production of two cutinase variants by A . awamori . Metabolic labelling studies were performed to analyze the bottleneck in enzyme production by the fungus in detail . These studies showed that because of the single L51S substitution, rapid extracellular degradation of cutinase occurred . The T179Y substitution did not result in enhanced sensitivity towards extracellular proteases . Presumably, the delay in the extracellular accumulation of this cutinase variant is caused by the enhanced hydrophobicity of the molecule . Overexpression of the A . awamori gene encoding the chaperone BiP in the cutinase-producing A . awamori strains had no significant effect on the secretion efficiency of the cutinases . A cutinase variant with the amino acid changes G28A, A85F, V184I, A185L, and L189F that was known to aggregate in the endoplasmic reticulum of S . cerevisiae, resulting in low extracellular protein levels, was successfully produced by A . awamori . An initial bottleneck in secretion occurred before or during translocation into the endoplasmic reticulum but was rapidly overcome by the fungus.

Appl Microbiol Biotechnol, 1998 Jun, 49(6), 751 - 7
Manganese toxicity towards Saccharomyces cerevisiae: dependence on intracellular and extracellular magnesium concentrations
Blackwell KJ, Tobin JM, Avery SV.
Inhibition of the growth of Saccharomyces cerevisiae was evident at concentrations of 0.5 mM Mn2+ or higher, but a tolerance to lower Mn2+ concentrations was observed . The inhibitory effects of 2.0 mM Mn2+ were eliminated by supplementing the medium with excess Mg2+ (10 mM), whereas addition of excess Ca2+ and K+ had negligible effect on Mn2+ toxicity . Growth inhibition by Mn2+, in the absence of a Mg2+ supplement, was attributed to Mn2+ accumulation to toxic intracellular levels . Mn levels in S . cerevisiae grown in Mg(2+)-supplemented medium were severalfold lower than those of cells growing in unsupplemented medium . Mn2+ toxicity was also influenced by intracellular Mg, as Mn2+ toxicity was found to be more closely correlated with the cellular Mg:Mn ratio than with cellular Mn levels alone . Cells with low intracellular levels of Mg were more susceptible to Mn2+ toxicity than cells with high cellular Mg, even when sequestered Mn2+ levels were similar . A critical Mg:Mn ratio of 2.0 was identified below which Mn2+ toxicity became acute . The results demonstrate the importance of intracellular and extracellular competitive interactions in determining the toxicity of Mn2+.

Arch Microbiol, 1998 Aug, 170(2), 99 - 105
Physiological and genetic characterisation of osmosensitive mutants of Saccharomyes cerevisiae; Bruning AR et al.; The screening of 20,000 Saccharomyces cerevisiae random mutants to identify genes involved in the osmotic stress response yielded 14 mutants whose growth was poor in the presence of elevated concentrations of NaCl and glucose . Most of the mutant strains were more sensitive to NaCl than to glucose at the equivalent water activity (aw) and were classified as salt-sensitive rather than osmosensitive . These mutants fell into 11 genetic complementation groups and were designated osr1-osr11 (osmotic stress response) . All mutations were recessive and showed a clear 2(+) : 2(-) segregation of the salt-stress phenotype upon tetrad analysis when crossed to a wild-type strain . The complementation groups osr1, osr5 and osr11 were allelic to the genes PBS2, GPD1 and KAR3, respectively . Whereas intracellular and extracellular levels of glycerol increased in the wild-type strains when exposed to NaCl, all mutants demonstrated some increase in extracellular glycerol production upon salt stress, but a number of the mutants showed little or no increase in intracellular glycerol concentrations . The mutants had levels of glycerol-3-phosphate dehydrogenase, an enzyme induced by osmotic stress, either lower than or similar to those of the parent wild-type strain in the absence of osmotic stress . In the presence of NaCl, the increase in glycerol-3-phosphate dehydrogenase activity in the mutants did not match that of the parent wild-type strain . None of the mutants had defective ATPases or were sensitive to heat stress . It is evident from this study and from others that a wide spectrum of genes is involved in the osmotic stress response in S . cerevisiae.

J Bacteriol, 1998 Aug, 180(15), 3864 - 72
Control by nutrients of growth and cell cycle progression in budding yeast, analyzed by double-tag flow cytometry; Alberghina L et al.; To gain insight on the interrelationships of the cellular environment, the properties of growth, and cell cycle progression, we analyzed the dynamic reactions of individual Saccharomyces cerevisiae cells to changes and manipulations of their surroundings . We used a new flow cytometric approach which allows, in asynchronous growing S . cerevisiae populations, tagging of both the cell age and the cell protein content of cells belonging to the different cell cycle set points . Since the cell protein content is a good estimation of the cell size, it is possible to follow the kinetics of the cell size increase during cell cycle progression . The analysis of the findings obtained indicates that both during a nutritional shift-up (from ethanol to glucose) and following the addition of cyclic AMP (cAMP), two important delays are induced . The preexisting cells that at the moment of the nutritional shift-up were cycling before the Start phase delay their entrance into S phase, while cells that were cycling after Start are delayed in their exit from the cycle . The combined effects of the two delays allow the cellular population that preexisted the shift-up to quickly adjust to the new growth condition . The effects of a nutritional shift-down were also determined.

FEMS Microbiol Lett, 1998 Jul 15, 164(2), 249 - 55
Cloning, molecular characterization, and expression of an endo-polygalacturonase-encoding gene from Saccharomyces cerevisiae IM1-8b; Blanco P et al.; A structural polygalacturonase-encoding gene (PGU1) from Saccharomyces cerevisiae IM1-8b was cloned and sequenced . The predicted protein comprises 361 amino acids, with a signal peptide between residues 1 and 18 and two potential glycosylation points in residues 318 and 330 . The putative active site is a conserved histidine in position 222 . This polygalacturonase showed 54% homology with the fungal ones and only 24% homology with their plant and bacterial counterparts . The gene is present in a single gene copy per haploid genome and it is detected in all strains, regardless of their phenotype . The expression of PGU1 gene in several strains of S . cerevisiae revealed that the polygalacturonase activity depended on the plasmid used and also on the genetic background of each strain but in all cases the enzymatic activity increased.

Methods Mol Biol, 1998, 103, 209 - 25
Expression of EGF and HIV envelope glycoprotein; Clare J et al.; 1 . The S . cerevisiae alpha-factor prepro leader is functional and is correctly processed in P . pastoris . 2 . P . pastoris has a high secretory capacity, but yields can be severely reduced by extracellular proteases . This problem can be reduced by altering the medium composition, e.g., adjusting the pH or by adding casamino acids . 3 . A rapid DNA dot-blot technique can be used for mass screening of transformants to obtain high-copy-number, high-expressing strains . 4 . For mEGF, which is an efficiently secreted protein, there was a good correlation between gene dosage and yield, and maximum levels were obtained at high copy number . 5 . Vectors conferring resistance to G418 have been developed for the selection of high-copy-number transformants . These vectors can also be used to isolate a series of transformants with increasing copy number of optimizing the expression of genes where high copy number may be detrimental . 6 . The HIV-1 ENV gene was not expressed in P . pastoris owing to fortuitous termination of transcription within AT-rich regions . This is a species-specific phenomenon, since full-length HIV-1 ENV transcripts are produced in S . cerevisiae . The problem was overcome by synthesizing the relevent portion of the gene with increased GC content . 7 . ENV was hyperglycosylated and immunologically inactive when secreted by P . pastoris . The yield was reduced by extracellular proteases, but like mEGF, this could be significantly improved by altering the pH of the culture medium and by adding casamino acids . 8 . In single-copy integrants, transcripts from the semisynthetic HIV-1 ENV gene were almost as abundant as endogenous AOX1 . Transcript levels increased progressively with increasing copy number, showing that the AOX1 promoter is not greatly limited by the level of trans-activating factors.

Cell Mol Biol (Noisy-le-grand), 1998 Jun, 44(4), 585 - 90
UGA4 gene expression in Saccharomyces cerevisiae depends on cell growth conditions; Bermudez Moretti M et al.; In the yeast Saccharomyces cerevisiae, gamma-aminobutyric acid (GABA) transport is mediated by three permeases: the general amino acid permease GAP1, the proline permease PUT4 and the specific GABA permease UGA4 . Expression of UGA4 gene is induced in the presence of GABA . We started a comparative study about UGA4 gene induction in cells grown on different culture media . Results presented here indicate that under certain growth conditions UGA4 permease is constitutive . Therefore, we demonstrate that in S . cerevisiae the UGA4 gene expression depends on cell growth conditions and that its synthesis not always depends on the presence of GABA.

J Biol Chem, 1998 Jul 31, 273(31), 19437 - 42
The LCB4 (YOR171c) and LCB5 (YLR260w) genes of Saccharomyces encode sphingoid long chain base kinases; Nagiec MM et al.; Sphingolipid long chain bases (LCBs) and phosphorylated derivatives, particularly sphingosine 1-phosphate, are putative signaling molecules . To help elucidate the physiological roles of LCB phosphates, we identified two Saccharomyces cerevisiae genes, LCB4 (YOR171c) and LCB5 (YLR260w), which encode LCB kinase activity . This conclusion is based upon the synthesis of LCB kinase activity in Escherichia coli expressing either LCB gene . LCB4 encodes most (97%) Saccharomyces LCB kinase activity, with the remainder requiring LCB5 . Log phase lcb4-deleted yeast cells make no LCB phosphates, showing that the Lcb4 kinase synthesizes all detectable LCB phosphates under these growth conditions . The Lcb4 and Lcb5 proteins are paralogs with 53% amino acid identity but are not related to any known protein, thus revealing a new class of lipid kinase . Two-thirds of the Lcb4 and one-third of the Lcb5 kinase activity are in the membrane fraction of yeast cells, a puzzling finding in that neither protein contains a membrane-localization signal . Both enzymes can use phytosphingosine, dihydrosphingosine, or sphingosine as substrate . LCB4 and LCB5 should be useful for probing the functions of LCB phosphates in S . cerevisiae . Potential mammalian cDNA homologs of the LCB kinase genes may prove useful in helping to understand the function of sphingosine 1-phosphate in mammals.

J Biol Chem, 1998 Jul 31, 273(31), 19383 - 90
Cloning and characterization of the two enzymes responsible for trypanothione biosynthesis in Crithidia fasciculata; Tetaud E et al.; Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (gamma-Glu-Cys-Gly) and spermidine to form trypanothione (N1,N8-bis(glutathionyl)spermidine), which is involved in maintaining intracellular thiol redox and in defense against oxidants . In this study, the genes from Crithidia fasciculata, Cf-GSS and Cf-TRS, which encode, respectively, glutathionylspermidine synthetase (EC 6.3.1.8) and trypanothione synthetase (EC 6.3.1.9) have been cloned and expressed . The deduced amino acid sequence of both Cf-GSS and Cf-TRS share 50% sequence similarity with the Escherichia coli glutathionylspermidine synthetase/amidase . Both genes are present as single copies in the C . fasciculata genome . When expressed in E . coli and Saccharomyces cerevisiae, neither protein was present in an active soluble form . However, thiol analysis of S . cerevisiae demonstrated that cells transformed with the Cf-GSS gene contained substantial amounts of glutathionylspermidine, whereas cells expressing both the Cf-GSS and Cf-TRS genes contained glutathionylspermidine and trypanothione, confirming that these genes encode the functional glutathionylspermidine and trypanothione synthetases from C . fasciculata . The translation products of Cf-GSS and Cf-TRS show significant homology to the amidase domain present in E . coli glutathionylspermidine synthetase, which can catalyze both synthesis and degradation of glutathionylspermidine . Glutathionylspermidine synthetase isolated from C . fasciculata was found to possess a similar amidase activity.

Yeast, 1998 Jun 15, 14(8), 723 - 31
Comparison of the structural characteristics of chromosome VI in Saccharomyces sensu stricto: the divergence, species-dependent features and uniqueness of saké yeasts; Nakazato A et al.; Previous studies have revealed that chromosome VI of sake yeasts is much larger than that of the other strains of Saccharomyces cerevisiae . Southern analysis using segments of chromosome VI of a laboratory strain as probes suggested that the nucleotide sequence of a major portion of this chromosome is conserved, but considerable diversity was found in the distal parts in the other strains . Physical maps also indicated that differences in length of chromosome VI were mainly due to differences in its ends . NotI was found to generate 9 kb and/or 16 kb fragments from the left telomere of chromosome VI in most sake yeasts, but no fragment in the case of AB972 . SfiI produced one or two 30-50 kb fragments from the right end of this chromosome in all sake yeasts tested, but produced a 20 kb fragment in the case of AB972 . All S . cerevisiae strains not employed in sake brewing were the same as AB972 in these respects . S . paradoxus had one NotI site in chromosome VI, while S . bayanus had two, one of which is possibly common to both species . The SfiI site mentioned above was present in chromosome VI of all species, while that of S . bayanus and S . paradoxus each had a second site distinct from the other . Chromosome VI of S . pastorianus was not distinguishable from that of S . bayanus.

FEMS Microbiol Lett, 1998 Jul 1, 164(1), 29 - 34
Identification and disruption of the gene encoding the K(+)-activated acetaldehyde dehydrogenase of Saccharomyces cerevisiae; Tessier WD et al.; The identity of the gene encoding the mitochondrial K(+)-activated acetaldehyde dehydrogenase (K(+)-ACDH) of Saccharomyces cerevisiae has been confirmed . The gene is situated on the right arm of chromosome XV, bears the systematic name YOR374w and the deduced product shows significant homology to other members of the S . cerevisiae aldehyde dehydrogenase (ALDH) family . YOR374w has now been assigned the gene name ALD7 . The N-terminal amino acid sequences of K(+)-ACDHs purified from several diverse strains of S . cerevisiae were determined, and found to have 81-100% identity in alignments with the product of ALD7 . Haploid mutants containing a deletion of ALD7 were constructed and, in these strains, the K(+)-ACDH was not detectable under any growth conditions examined . The activity of the Mg(2+)-activated acetaldehyde dehydrogenase (Mg(2+)-ACDH), encoded by ALD6, remained at wild-type levels in the mutants . Growth on glucose was not affected in the mutants lacking ALD7 (in contrast to the behaviour of ald6 mutants), whereas growth on ethanol was severely impaired . This observation, together with previous work by our group, shows that both the Mg(2+)- and K(+)-ACDHs are required for growth on ethanol, whilst only the former plays a role during growth on glucose.

Biochem Biophys Res Commun, 1998 Jul 9, 248(1), 87 - 92
Disruption of a gene encoding phosphatidic acid phosphatase causes abnormal phenotypes in cell growth and abnormal cytokinesis in Saccharomyces cerevisiae; Katagiri T et al.; Phosphatidic acid phosphatase (PAP) is an enzyme involved in lipid metabolism . Diacylglycerol (DG) and phosphatidic acid (PA) are a substrate and a product of PAP, respectively, and function as second messengers in several signal transduction pathways in animals . To investigate the function of PAP in Saccharomyces cerevisiae, we analyzed changes in cellular phenotypes of a mutant that has a disrupted PAP gene . Two putative genes for PAP (ScPAP1 and ScPAP2) are in the S . cerevisiae genome . We generated a ScPAP1-null mutant and observed its cellular phenotypic changes . The ScPAP1-null mutant cells aggregated in liquid culture, and microscopical analyses showed that these mutant cells have an abnormal cell shape and abnormal cytokinesis during cell division . The ScPAP1 is possibly involved in cell growth and cytokinesis in S . cerevisiae . Yeast phosphatidic acid phosphatase is possibly involved in cell growth and cytokinesis.

Proc Natl Acad Sci U S A, 1998 Jul 21, 95(15), 8733 - 8
Bloom's and Werner's syndrome genes suppress hyperrecombination in yeast sgs1 mutant: implication for genomic instability in human diseases; Yamagata K et al.; Bloom's syndrome (BS) and Werner's syndrome (WS) are genetic disorders in which an increased rate of chromosomal aberration is detected . The genes responsible for these diseases, BLM and WRN, have been found to be homologs of Escherichia coli recQ and Saccharomyces cerevisiae SGS1 genes . Here we show that yeast Sgs1 helicase acts as a suppressor of illegitimate recombination through homologous recombination and that human BLM and WRN helicases can suppress the increased homologous and illegitimate recombinations in the S . cerevisiae sgs1 mutant . The results imply a role of BLM and WRN helicases to control genomic stability in human cells . Similar to Sgs1 helicase, BLM helicase suppressed the cell growth in the top3 sgs1 mutation background and restored the increased sensitivity of the sgs1 mutant to hydroxyurea, but the WRN helicase did not . We discussed differential roles of BLM and WRN helicases in human cells . BLM- and WRN-bearing yeasts provide new useful models to investigate human BS and WS diseases.

Mol Cell Biol, 1998 Aug, 18(8), 4935 - 46
Identification of a translation initiation factor 3 (eIF3) core complex, conserved in yeast and mammals, that interacts with eIF5; Phan L et al.; Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3 . We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni2+ affinity and gel filtration chromatography and obtained a complex of approximately 600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p . All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S . cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p . The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3 . The purified complex could rescue Met-tRNAiMet binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3 . These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5 . Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays . Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon . Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.

Mol Gen Genet, 1998 Jun, 258(6), 628 - 38
Identification, cloning and characterization of a derepressible Na+-coupled phosphate transporter in Saccharomyces cerevisiae; Martinez P et al.; Based on the high sequence homology between the yeast ORF YBR296c (accession number P38361 in the SWISS-PROT database) and the PHO4 gene of Neurospora crassa, which codes for a Na+/Pi cotransporter with twelve putative transmembrane domains, the YBR296c ORF was considered to be a promising candidate gene for a plasma membrane-bound phosphate transporter in Saccharomyces cerevisiae . Therefore, this gene, here designated PHO89, was cloned and a set of deletion mutants was constructed . We then studied their Pi uptake activity under different conditions . We show here that a transport activity displayed by PHO89 strains under alkaline conditions and in the presence of Na+ is absent in pho89 null mutants . Moreover, when the pH was lowered to pH 4.5 or when Na+ was omitted, this activity decreased significantly, reaching values close to those exhibited by the deltapho89 mutant . Studies of the acid phosphatase activity of these strains, as well as promoter sequence analysis, suggest that expression of the PHO89 gene is under the control of the PHO regulatory system . Northern analysis shows that this gene is only transcribed under conditions of Pi limitation . This is, to our knowledge, the first demonstration that the PHO89 gene codes for the Na+/Pi cotransporter previously characterized by kinetic studies, and represents the only Na+-coupled secondary anion transport system so far identified in S . cerevisiae . Pho89p has been shown to have an apparent Km of 0.5 microM and a pH optimum of 9.5, and is highly specific for Na+; activation of transport is maximal at a Na+ concentration of 25 mM.

EMBO J, 1998 Jul 15, 17(14), 4188 - 98
Saccharomyces cerevisiae LIF1: a function involved in DNA double-strand break repair related to mammalian XRCC4; Herrmann G et al.; Saccharomyces cerevisiae DNA ligase IV (LIG4) has been shown previously to be involved in non-homologous DNA end joining and meiosis . The homologous mammalian DNA ligase IV interacts with XRCC4, a protein implicated in V(D)J recombination and double-strand break repair . Here, we report the discovery of LIF1, a S.cerevisiae protein that strongly interacts with the C-terminal BRCT domain of yeast LIG4 . LIG4 and LIF1 apparently occur as a heterodimer in vivo . LIF1 shares limited sequence homology with mammalian XRCC4 . Disruption of the LIF1 gene abolishes the capacity of cells to recircularize transformed linearized plasmids correctly by non-homologous DNA end joining . Loss of LIF1 is also associated with conditional hypersensitivity of cells to ionizing irradiation and with reduced sporulation efficiency . Thus, with respect to their phenotype, lif1 strains are similar to the previously described lig4 mutants . One function of LIF1 is the stabilization of the LIG4 enzyme . The finding of a XRCC4 homologue in S.cerevisiae now allows for mutational analyses of structure-function relationships in XRCC4-like proteins to define their role in DNA double-strand break repair.

J Biol Chem, 1998 Jul 24, 273(30), 18974 - 8
Requirement for Hsp90 and a CyP-40-type cyclophilin in negative regulation of the heat shock response; Duina AA et al.; The heat shock response is a highly conserved mechanism that allows cells to withstand a variety of stress conditions . Activation of this response is characterized by increased synthesis of heat shock proteins (HSPs), which protect cellular proteins from stress-induced denaturation . Heat shock transcription factors (HSFs) are required for increased expression of HSPs during stress conditions and can be found in complexes containing components of the Hsp90 molecular chaperone machinery, raising the possibility that Hsp90 is involved in regulation of the heat shock response . To test this, we have assessed the effects of mutations that impair activity of the Hsp90 machinery on heat shock related events in Saccharomyces cerevisiae . Mutations that either reduce the level of Hsp90 protein or eliminate Cpr7, a CyP-40-type cyclophilin required for full Hsp90 function, resulted in increased HSF-dependent activities . Genetic tests also revealed that Hsp90 and Cpr7 function synergistically to repress gene expression from HSF-dependent promoters . Conditional loss of Hsp90 activity resulted in both increased HSF-dependent gene expression and acquisition of a thermotolerant phenotype . Our results reveal that Hsp90 and Cpr7 are required for negative regulation of the heat shock response under both stress and nonstress conditions and establish a specific endogenous role for the Hsp90 machinery in S . cerevisiae.

Mol Cells, 1998 Jun 30, 8(3), 251 - 8
Small-subunit ribosomal DNA of an ectomycorrhizal fungus tricholoma matsutake: sequence, structure and phylogenetic analysis; Hwang SK et al.; A 2861 bp nucleotide sequence containing Tricholoma matsutake SSU rRNA gene and its flanking regions was determined and analyzed . Comparison with known SSU rDNA sequences and primer extension analysis revealed that the SSU rRNA coding region and intergenic spacer 2 (IGS2) are 1805 bp and 1043 bp in length, respectively . The IGS2 has an imperfect direct repeat (type 1) homologous to the region downstream to the 5S rRNA gene and the three imperfect tandem direct repeats (type 2) upstream to the SSU rRNA-encoding sequence . Structural analysis by a comparative method showed that the overall secondary structure of the SSU rRNA is generally similar to that of S . cerevisiae, whereas the secondary structures of the V4 regions predicted by the thermodynamics-based method showed different configurations between distantly related taxa, implying that these structural differences can provide phylogenetically informative features . Phylogenetic trees based on both the aligned SSU rRNA sequences with almost full-length sequences and V4 sequences revealed that T . matsutake is very closely related to other basidiomycetes belonging to Agaricales . Thus we propose that the V4 region is also a good source for the inference of phylogeny to support the SSU rRNA phylogeny.

J Mol Evol, 1998 Jul, 47(1), 14 - 20
Evidence for the role of recombination in the regulatory evolution of Saccharomyces cerevisiae Ty elements; Jordan IK et al.; The recent completion of the sequencing of the Saccharomyces cerevisiae genome provides a unique opportunity to analyze the evolutionary relationships existing among the entire complement of retrotransposons residing within a single genome . In this article we report the results of such an analysis of two closely related families of yeast long terminal repeat (LTR) retrotransposons, Ty1 and Ty2 . In our study, we analyzed the molecular variation existing among the 32 Ty1 and 13 Ty2 elements present within the S . cerevisiae genome recently sequenced within the context of the yeast genome project . Our results indicate that while the Ty1 family is most likely ancestral to Ty2 elements, both families of elements are relatively recent components of the S . cerevisiae genome . Our results also indicate that both families of elements have been subject to purifying selection within their protein coding regions . Finally, and perhaps most interestingly, our results indicate that a relatively recent recombination event has occurred between Ty2 and a subclass of Ty1 elements involving the LTR regulatory region . We discuss the possible biological significance of these findings and, in particular, how they contribute to a better overall understanding of LTR retrotransposon evolution.

Mol Cell, 1998 May, 1(6), 807 - 17
Osmotin, a plant antifungal protein, subverts signal transduction to enhance fungal cell susceptibility; Yun DJ et al.; The plant pathogenesis-related protein osmotin is an antifungal cytotoxic agent that causes rapid cell death in the yeast S . cerevisiae . We show here that osmotin uses a signal transduction pathway to weaken defensive cell wall barriers and increase its cytotoxic efficacy . The pathway activated by osmotin includes the regulatory elements of the mating pheromone response STE4, STE18, STE20, STE5, STE11, STE7, FUS3, KSS1, and STE12 . Neither the pheromone receptor nor its associated G protein alpha subunit GPA1 are required for osmotin action . However, mutation of SST2, a negative regulator of G alpha proteins, resulted in supersensitivity to osmotin . Phosphorylation of STE7 was rapidly stimulated by osmotin preceding any changes in cell vitality or morphology . These results demonstrate that osmotin subverts target cell signal transduction as part of its mechanism of action.

Mol Cell, 1998 Feb, 1(3), 327 - 36
The Mitochondrial F0F1-ATPase proton pump is required for function of the proapoptotic protein Bax in yeast and mammalian cells; Matsuyama S et al.; The proapoptotic mammalian protein Bax associates with mitochondrial membranes and confers a lethal phenotype when expressed in yeast . By generating Bax-resistant mutant yeast and using classical complementation cloning methods, subunits of the mitochondrial F0F1-ATPase proton pump were determined to be critical for Bax-mediated killing in S . cerevisiae . A pharmacological inhibitor of the proton pump, oligomycin, also partially abrogated the cytotoxic actions of Bax in yeast . In mammalian cells, oligomycin also inhibited Bax-induced apoptosis and activation of cell death proteases . The findings imply that an intact F0F1-ATPase in the inner membrane of mitochondria is necessary for optimal function of Bax in both yeast and mammalian cells.

Mol Cell, 1998 Jan, 1(2), 193 - 202
A function for monoubiquitination in the internalization of a G protein-coupled receptor; Terrell J et al.; Modification of an S . cerevisiae G protein-coupled receptor with ubiquitin is required for its ligand-stimulated internalization . We now demonstrate that monoubiquitination on a single lysine residue is sufficient to signal receptor internalization, a modification distinct from that required for proteasome recognition . Formation of a polyubiquitin chain is not necessary, as demonstrated by the ability of mutant ubiquitins that lack lysine residues to serve as efficient internalization signals . Fusion of ubiquitin in-frame to a receptor that lacks cytoplasmic tail lysines also promotes rapid receptor internalization, indicating that ubiquitin itself and not a specific type of linkage to the receptor acts as an internalization signal . Thus, we have defined a cellular function for monoubiquitination in alpha-factor receptor endocytosis.

J Bacteriol, 1998 Jul, 180(14), 3592 - 7
Identification of a gene product induced by hard-surface contact of Colletotrichum gloeosporioides conidia as a ubiquitin-conjugating enzyme by yeast complementation; Liu ZM et al.; The germinating conidia of many phytopathogenic fungi on hosts must differentiate into an infection structure called the appressorium in order to penetrate their hosts . Chemical signals, such as the host's surface wax or fruit ripening hormone, ethylene, trigger germination and appressorium formation of the avocado pathogen Colletotrichum gloeosporioides only after the conidia are in contact with a hard surface . What role this contact plays is unknown . Here, we describe isolation of genes expressed during the early stage of hard-surface treatment by a differential-display method and report characterization of one of these cloned genes, chip1 (Colletotrichum hard-surface induced protein 1 gene), which encodes a ubiquitin-conjugating enzyme . RNA blots clearly showed that it is induced by hard-surface contact and that ethylene treatment enhanced this induction . The predicted open reading frame (ubc1Cg) would encode a 16.2-kDa ubiquitin-conjugating enzyme, which shows 82% identity to the Saccharomyces cerevisiae UBC4-UBC5 E2 enzyme, comprising a major part of total ubiquitin-conjugating activity in stressed yeast cells . UBC1Cg can complement the proteolysis deficiency of the S . cerevisiae ubc4 ubc5 mutant, indicating that ubiquitin-dependent protein degradation is involved in conidial germination and appressorial differentiation.

Biochim Biophys Acta, 1998 Jun 29, 1385(2), 201 - 19
Thiamin metabolism and thiamin diphosphate-dependent enzymes in the yeast Saccharomyces cerevisiae: genetic regulation; Hohmann S et al.; The yeast Saccharomyces cerevisiae utilises external thiamin for the production of thiamin diphosphate (ThDP) or can synthesise the cofactor itself . Prior to uptake into the cell thiamin phosphates are first hydrolysed and thiamin is taken up as free vitamin which is then pyrophosphorylated by a pyrophosphokinase . Synthesis of ThDP starts with the production of hydroxyethylthiazole and hydroxymethylpyrimidine . Those are linked to yield thiamin phosphate which is hydrolysed to thiamin and subsequently pyrophosphorylated . The THI genes encoding the enzymes of these final steps of ThDP production and of thiamin utilisation have been identified . Their expression is controlled by the level of thiamin and a number of regulatory proteins involved in regulated expression of the THI genes are known . However, the molecular details of the regulatory circuits need to be deciphered . Since the nucleotide sequence of the entire yeast genome is known we can predict the number of ThDP-dependent enzymes in S . cerevisiae . Eleven such proteins have been found: pyruvate decarboxylase (Pdc, three isoforms), acetolactate synthase, a putative alpha-ketoisocaproate decarboxylase with a regulatory role in ThDP synthesis and two proteins of unknown function form the group of Pdc related enzymes . In addition there are two isoforms for transketolase as well as the E1 subunits of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase . Expression of most of these genes is either induced or repressed by glucose . Surprisingly, it has been found recently that expression of one of the genes for Pdc is repressed by thiamin . In addition, the regulatory protein Pdc2p was shown to be required for high level expression of both the THI and the PDC genes . Apparently, the production of ThDP and of the enzymes using this cofactor is coordinately regulated . Future research will focus on the elucidation of the molecular mechanisms of this novel type of regulation.

DNA Cell Biol, 1998 Jun, 17(6), 511 - 8
Production of recombinant hydroxylated human type III collagen fragment in Saccharomyces cerevisiae; Vaughn PR et al.; A recombinant hydroxylated fragment of human type III collagen has been produced in Saccharomyces cerevisiae by coordinated coexpression of a collagen gene fragment together with both the alpha- and beta-subunit genes for prolyl-4-hydroxylase (EC 1.14.11.2) . The collagen fragment consisted of 255 residues of the helical domain and the complete C-telopeptide and C-propeptide domains . It was inserted under the control of the ethanol-inducible ADH2 promoter in a multicopy, TRP1-selectable, yeast expression vector, YEpFlag1 . The prolyihydroxylase subunit genes were cloned on either side of a bidirectional galactose-inducible promoter in a low-copy minichromosome yeast expression vector, pYEUra3, which is URA3 selectable . Coordinated expression of the three different gene products after cotransformation into S . cerevisiae was detected by immunoblotting . Amino acid analysis of an immunoreactive collagen fraction demonstrated the presence of hydroxyproline, while the presence of a triple-helical domain in the collagen fragment was demonstrated by its resistance to pepsin proteolysis.

Eur J Biochem, 1998 May 15, 254(1), 202 - 6
Role of the actin cytoskeleton in regulating the outer phosphatidylethanolamine levels in yeast plasma membrane; Dixit BL et al.; Transbilayer phosphatidylethanolamine (PtdEtn) movements in the plasma membrane of Saccharomyces cerevisiae are regulated by an ATP-dependent, protein-mediated process(es) . To examine whether this process is influenced by the actin cytoskeleton, we have studied the PtdEtn translocation in S . cerevisiae cells after treatment with microfilament disrupting and microtubule-disrupting agents . PtdEtn translocation was studied by measuring the external PtdEtn levels, using fluorescamine as the external membrane probe, in the ATP-depleted, ATP-depleted and repleted, and N-ethylmaleimide-treated cells . The microfilaments and microtubules were disrupted by treatment with various cytochalasins and colchicine (or benomyl) respectively PtdEtn translocation became abnormal in the cytochalasin-treated cells but not in cells that were treated with microtubule-disrupting agents, such as colchicine or benomyl . These results have been interpreted to suggest that the actin cytoskeleton is involved in regulating the PtdEtn translocase activity in the yeast cell plasma membrane.

FEBS Lett, 1998 Jun 12, 429(2), 194 - 6
Purification and properties of exopolyphosphatase isolated from Saccharomyces cerevisiae vacuoles; Andreeva NA et al.; An exopolyphosphatase (polyPase) with a specific activity of 60 U/mg protein has been purified from the vacuolar sap of Saccharomyces cerevisiae . The molecular mass of the intact enzyme was found to be 245 kDa . It is highly specific towards high-molecular polyphosphates (polyP) . The activity with polyP9 is 24% of that with polyP208 . The apparent Km for polyP15 and polyP208 hydrolysis is 93 and 2.4 microM, respectively . The enzyme is slightly active with polyP3 and adenosine-5'-tetraphosphate, but does not hydrolyze pyrophosphate, ATP, GTP and p-nitrophenylphosphate . It is stimulated by divalent metal cations . Co2+, the best activator, stimulates it 6-fold . Antibodies that inhibit the cell envelope and cytosol polyPases of S . cerevisiae have no effect on the vacuolar polyPase . The vacuolar polyPase differs from other yeast polyPases in molecular mass, substrate specificity and effects of activators.

FEBS Lett, 1998 Jun 12, 429(2), 173 - 8
Topological and functional analysis of the rat liver carnitine palmitoyltransferase 1 expressed in Saccharomyces cerevisiae; Prip-Buus C et al.; The rat liver carnitine palmitoyltransferase 1 (L-CPT 1) expressed in Saccharomyces cerevisiae was correctly inserted into the outer mitochondrial membrane and shared the same folded conformation as the native enzyme found in rat liver mitochondria . Comparison of the biochemical properties of the yeast-expressed L-CPT 1 with those of the native protein revealed the same detergent lability and similar sensitivity to malonyl-CoA inhibition and affinity for carnitine . Normal Michaelis-Menten kinetics towards palmitoyl-CoA were observed when careful experimental conditions were used for the CPT assay . Thus, the expression in S . cerevisiae is a valid model to study the structure-function relationships of L-CPT 1.

Biochem Biophys Res Commun, 1998 Jun 29, 247(3), 827 - 32
Alpha 4 associates with protein phosphatases 2A, 4, and 6; Chen J et al.; Protein phosphatases participate in the regulation of a variety of cellular processes . Control of their enzymatic activity and specificity is made possible largely by an array of regulatory subunits . Novel serine/threonine phosphatases--PP4 and PP6 in human cells--have been discovered recently, for which regulatory subunits are yet to be identified . We report here the identification of a potential regulatory subunit of these phosphatases . Using the yeast two-hybrid system, we have found that alpha 4, a previously identified phosphoprotein, associates constitutively with the catalytic subunits of PP4, PP6, and both isoforms of PP2A . These interactions have been confirmed by direct binding and do not require phosphorylation of alpha 4, although it is unclear whether alpha 4 phosphorylation has any effect on its association with the phosphatases . The binding activity appears to reside in the N-terminal 50 amino acids of the phosphatases, consistent with a previous observation that the first 55 residues of PPV, a Drosophila homolog of PP6, may harbor the element for regulation . alpha 4 shares 37% sequence homology with Tap42, an S . cerevisiae protein that has been reported to associate with PP2A and Sit4 (yeast homolog of PP6) and comprises a regulatory component in the rapamycin-sensitive Tor signalling pathway . By analogy, alpha 4 and its associated phosphatases may participate in the mammalian rapamycin-sensitive pathway mediated by FRAP.

Curr Genet, 1998 Jun, 33(6), 412 - 9
Isolation of UBP3, encoding a de-ubiquitinating enzyme, as a multicopy suppressor of a heat-shock mutant strain of S . cerevisiae; Baxter BK et al.; Yeast strains lacking functional copies of the two genes SSA1 and SSA2, which encode cytosolic molecular chaperones, are temperature-sensitive . In this report, we describe the isolation of a high-copy suppressor of this temperature sensitivity, UBP3, which encodes a de-ubiquitinating enzyme . We show that ubp3 mutant yeast strains have a mild slow-growth phenotype and accumulate ubiquitin-protein conjugates . We propose a model in which Ubp3p acts in vivo to reverse the ubiquitination of substrate proteins, allowing temporarily misfolded proteins an opportunity to fold correctly.

Biochem Biophys Res Commun, 1998 Jun 18, 247(2), 204 - 6
A second-step splicing activity is conserved from yeast to human; Cicarelli RM et al.; We describe a defective HeLa nuclear extract which is particularly deficient in step 2 of splicing reaction . With this extract we have studied the conservation of a second-step activity from yeast to human cells . We detected a S . cerevisiae second-step splicing activity that allows restoration of step 2 of the defective HeLa nuclear extract, which indicates that yeast purified fraction has a second-step activity that is conserved from yeast to human cells . The activity is a yeast UsnRNP protein(s) since it is purified with anti-trimethylguanosine by immunoaffinity columns .

Microbiology, 1998 Jun, 144 ( Pt 6), 1683 - 90
Distribution of 14C-labelled carbon from glucose and glutamate during anaerobic growth of Saccharomyces cerevisiae; Albers E et al.; The distribution of carbon from glucose and glutamate was studied using anaerobically grown Saccharomyces cerevisiae . The yeast was grown on glucose (20 g l-1) as the carbon/energy source and glutamic acid (3.5 g l-1) as additional carbon and sole nitrogen source . The products formed were identified using labelled {U-14C}glucose or {U-14C}glutamic acid . A seldom-reported metabolite in S . cerevisiae, 2-hydroxyglutarate, was found in significant amounts . It is suggested that 2-hydroxyglutarate is formed from the reduction of 2-oxoglutarate in a reaction catalysed by a dehydrogenase . Succinate, 2-oxoglutarate and 2-hydroxyglutarate were found to be derived exclusively from glutamate . Based on radioactivity measurements, 55%, 17% and 14% of the labelled glutamate was converted to 2-oxoglutarate, succinate and 2-hydroxyglutarate, respectively, and 55%, 9% and 3% of the labelled glucose was converted to ethanol, glycerol and pyruvate, respectively . No labelled glucose was converted to 2-oxoglutarate, succinate or 2-hydroxyglutarate . Furthermore, very little of the evolved CO2 was derived from glutamate . Separation of the amino acids from biomass by paper chromatography revealed that the glutamate family of amino acids (glutamic acid, glutamine, proline, arginine and lysine) originated almost exclusively from the carbon skeleton of glutamic acid . It can be concluded that the carbon flow follows two separate paths, and that the only major reactions utilized in the tricarboxylic acid (TCA) cycle are those reactions involved in the conversion of 2-oxoglutarate to succinate.

Biochem J, 1998 Jul 1, 333 ( Pt 1), 117 - 20
Re-design of Saccharomyces cerevisiae flavocytochrome b2: introduction of L-mandelate dehydrogenase activity; Sinclair R et al.; Flavocytochrome b2 from Saccharomyces cerevisiae is an l-lactate dehydrogenase which exhibits only barely detectable activity levels towards another 2-hydroxyacid, l-mandelate . Using protein engineering methods we have altered the active site of flavocytochrome b2 and successfully introduced substantial mandelate dehydrogenase activity into the enzyme . Changes to Ala-198 and Leu-230 have significant effects on the ability of the enzyme to utilize l-mandelate as a substrate . The double mutation of Ala-198-->Gly and Leu-230-->Ala results in an enzyme with a kcat value (25 degrees C) with L-mandelate of 8.5 s-1, which represents an increase of greater than 400-fold over the wild-type enzyme . Perhaps more significantly, the mutant enzyme has a catalytic efficiency (as judged by kcat/Km values) that is 6-fold higher with l-mandelate than it is with L-lactate . Closer examination of the X-ray structure of S . cerevisiae flavocytochrome b2 led us to conclude that one of the haem propionate groups might interfere with the binding of L-mandelate at the active site of the enzyme . To test this idea, the activity with l-mandelate of the independently expressed flavodehydrogenase domain (FDH), was examined and found to be higher than that seen with the wild-type enzyme . In addition, the double mutation of Ala-198-->Gly and Leu-230-->Ala introduced into FDH produced the greatest mandelate dehydrogenase activity increase, with a kcat value more than 700-fold greater than that seen with the wild-type holoenzyme . In addition, the enzyme efficiency (kcat/Km) of this mutant enzyme was more than 20-fold greater with L-mandelate than with l-lactate . We have therefore succeeded in constructing an enzyme which is now a better mandelate dehydrogenase than a lactate dehydrogenase.

Yeast, 1998 May, 14(7), 617 - 22
An extracellular meiosis-promoting factor in Saccharomyces cerevisiae; Hayashi M et al.; Meiosis and sporulation in the yeast Saccharomyces cerevisiae has been classically viewed as an example of unicellular, eukaryotic differentiation that occurs in response to nutritional starvation . We present evidence that S . cerevisiae produces an extracellular factor(s), called meiosis-promoting factor (MEP), that is required, in addition to starvation conditions, for efficient meiosis and sporulation . This factor is secreted and accumulates in a cell density-dependent fashion such that cells at a low density sporulate poorly under conditions in which cells at a high density sporulate efficiently . Conditioned medium from sporulating cells at a high density contains a small anionic molecule that has cytostatic activity and stimulates sporulation of cells at low density under a normal starvation condition . These results indicate that MEP-mediated social communication between cells is required for meiosis and sporulation.

Cell, 1998 May 29, 93(5), 863 - 73
Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin; Vainberg IE et al.; We describe the discovery of a heterohexameric chaperone protein, prefoldin, based on its ability to capture unfolded actin . Prefoldin binds specifically to cytosolic chaperonin (c-cpn) and transfers target proteins to it . Deletion of the gene encoding a prefoldin subunit in S . cerevisiae results in a phenotype similar to those found when c-cpn is mutated, namely impaired functions of the actin and tubulin-based cytoskeleton . Consistent with prefoldin having a general role in chaperonin-mediated folding, we identify homologs in archaea, which have a class II chaperonin but contain neither actin nor tubulin . We show that by directing target proteins to chaperonin, prefoldin promotes folding in an environment in which there are many competing pathways for nonnative proteins.

Gene, 1998 May 12, 211(2), 395 - 400
A high-copy-number ADE2-bearing plasmid for transformation of Candida glabrata; Hanic-Joyce PJ et al.; The Candida glabrata ADE2 gene encoding aminoimidazole ribonucleotide (AIR) carboxylase (EC 4.1.1.21) was isolated by complementation of the ade2-1 mutation in Saccharomyces cerevisiae . The predicted amino acid (aa) sequence is 75% identical to that of S . cerevisiae . Integrative transformation was used to produce a C . glabrata strain bearing a deletion of ADE2 coding sequences . A high-copy-number shuttle vector bearing the ADE2 gene was constructed and contains a fragment of S . cerevisiae mitochondrial (mt) DNA that confers the ability to replicate autonomously in C . glabrata.

Gene, 1998 May 12, 211(2), 323 - 9
Isolation of the Aspergillus nidulans sudD gene and its human homologue; Anaya P et al.; We have been studying the heat-sensitive bimD6 mutation of Aspergillus nidulans . At a restrictive temperature, the chromosomes of bimD6 mutant strains fail to attach properly to the spindle microtubules, and the mutant also displays a high rate of chromosome loss . We previously cloned the sudA gene, an extragenic suppressor of the heat-sensitive bimD6 mutation and showed that it coded for a DA-box or SMC protein . SMC proteins have been demonstrated to function in chromosome condensation, segregation and global gene regulation . We have now cloned the sudD gene, another of the extragenic suppressor genes of the bimD6 mutation . The predicted SUDD protein is the founding member of a widely expressed protein family . Similar proteins are found in sequence databases for Saccharomyces cerevisiae, Caenorhabditis elegans, mammals and four species of archaebacteria . We have also cloned and sequenced a human cDNA that encodes the human homologue of SUDD and mapped the gene to 18q11.2 . The predicted SUDD proteins from A . nidulans, Homo sapiens and S . cerevisiae all share a variety of features . The predicted proteins are approximately 60000Da in mass and have a serine-plus-threonine content of about 11% . The evolutionary conservation of the proteins suggests an ancient origin and conserved function for these proteins.

Gene, 1998 May 12, 211(2), 259 - 66
Cloning, sequence analysis and expression of the basidiomycete Lentinus edodes gene uck1, encoding UMP-CMP kinase, the homologue of Saccharomyces cerevisae URA6 gene; Kaneko S et al.; Sequence analysis of the downstream region of the basidiomycete Lentinus edodes priB gene encoding a protein with a 'Zn(II)2Cys6 zinc cluster' DNA-binding motif (Endo, H., Kajiwara, S., Tunoka, O., Shishido, K., 1994 . A novel cDNA, priBc, encoding a protein with a Zn(II)2Cys6 zinc cluster DNA-binding motif, derived from the basidiomycete Lentinus edodes . Gene 139, 117-121) suggested the presence of a Saccharomyces cerevisiae URA6 gene homologue encoding UMP kinase . We isolated a corresponding cDNA from a mature fruiting-body cDNA library of L . edodes . The nucleotide sequence of this was determined and compared with that of the genomic DNA, revealing that the URA6 gene homologue encodes 227 amino acids (aa) and is interrupted by four small introns . The deduced aa sequence showed an overall identity of 51.1% to that of the S . cerevisiae URA6 gene product . The URA6 homologue protein produced in Escherichia coli using the glutathione S-transferase gene fusion system was found to catalyze the phosphoryl transfer from ATP to UMP and CMP efficiently and also to AMP and dCMP with lower efficiencies . Thus, the URA6 gene homologue was designated uck1 and its product UMP-CMP kinase . Northern-blot analysis showed that the uck1 is actively transcribed in the gill tissue of mature fruiting bodies of L . edodes, implying that uck1 may play a role during the formation of basidiospores occurs in the gill tissue.

J Eukaryot Microbiol, 1998 May-Jun, 45(3), 233 - 9
Quantitation of absolute Pneumocystis carinii nuclear DNA content . Trophic and cystic forms isolated from infected rat lungs are haploid organisms; Wyder MA et al.; The Pneumocystis carinii carinii DNA content in nuclei of trophic forms and cysts (spore cases) containing 2, 4, or 8 intracystic bodies, were compared using quantitative fluorescence image analysis . The nuclear DNA content was found to be lower than the theoretical limits of Feulgen cytophotometry . Several fluorescent DNA dyes provide brighter staining, but these techniques suffer from nonspecific binding to other cellular components, such as RNA . It was demonstrated that the thick glycocalyx surfaces of trophic forms and the cyst walls of P . carinii organisms, as well as the cell wall of S . cerevisiae, bound all fluorescent dyes tested to varying degrees . Hence in this study, measurements were performed on cells in which the outer surfaces of organisms were first removed with lyticase . Two stains that appeared most specific for DNA, DB181 and 4',6-diamidino-2-phenylindole (DAPI), were used for quantitations; lower deviations of fluorescence intensities were observed with DB181 . Haploid wild type Saccharomyces cerevisiae and cdc-28 temperature-sensitive mutant cells, accumulated at the restrictive temperature (37 degrees C), were used as quantitative internal standards for estimating the absolute nuclear DNA content of P . carinii . Haploid wild type and mutant nuclei stained with DAPI had the same relative fluorescence intensities . The P . carinii nuclear DNA content of trophic forms and individual intracystic bodies (spores), regardless of life cycle stage, were not different . The mean values obtained were 6.9 and 6.7 fg DNA/nucleus with DB181 and DAPI, respectively (approximately 9.26 and 8.99 Mbp nucleotides, respectively) . Since these would include 2C (G-2 phase) and S-phase nuclei, a 1C population of nuclei was selected by histogram distributions of DB181-stained nuclei . Almost all nuclei analyzed in all life cycle stages fell within this population . The 1C mean of 6.55 fg DNA/nucleus (median, 6.62 fg DNA/nucleus) was estimated as representing 8.79 Mbp nucleotides, assuming only A-T binding of the dye and taking into account the G + C content of S . cerevisiae and P . carinii . A 4C (G-2-phase diploid nuclei) population was not detected in histograms of DB181- or DAPI-stained nuclei . The P . carinii nuclear DNA content values obtained in this study were similar to those independently obtained by calculating the total DNA in the organism's chromosomes resolved by electrophoretic techniques . Together, the data on total chromosome numbers and the estimated DNA content of those chromosomes, with our quantitation of nuclear DNA content of different life-cycle stages demonstrate that P . carini carinii isolated from infected rat lungs are haploid organisms.

Anat Rec, 1998 Jun, 251(2), 256 - 64
Role of endoplasmic reticulum-derived vesicles in the formation of Golgi elements in sec23 and sec18 Saccharomyces Cerevisiae mutants; Morin-Ganet MN et al.; BACKGROUND: In the yeast Saccharomyces cerevisiae, the Golgi apparatus consists of individual networks of membranous tubules interspersed throughout the cytoplasm . When sec23 and sec18 mutants are shifted from the permissive (20 degrees C) to the restrictive (37 degrees C) temperature, the secretory pathway is blocked between endoplasmic reticulum (ER) and Golgi elements . When examined with an electron microscope, sec23 displays an excess of ER membranes, whereas sec18 accumulates small vesicles . The present investigation describes the kinetics of the ultrastructural modifications of the Golgi and vesicular elements when sec23 and sec18 mutants are shifted for 10 min to restrictive temperature and then returned to permissive temperature for various time intervals . METHODS: S . cerevisiae sec23 and sec18 mutants from exponentially growing cultures at 20 degrees C were maintained for 10 min at the restrictive temperature of 37 degrees C and returned to the permissive temperature of 20 degrees C for different time intervals . Following fixation in glutaraldehyde and postfixation in potassium ferrocyanide reduce osmium, 80- to 200-nm-thick sections were prepared from Epon-embedded yeast cells . Using the thicker sections, stereopairs of electron microscopy photographs were prepared and used to visualize the three-dimensional configuration of the organelles . To follow the modifications of cell organelles, cell sections were selected at random in thinner sections and cell organelles were scored . RESULTS: At permissive temperature (20 degrees C), the Golgi apparatus consisted of individual networks of tubules dispersed in the cytoplasm, as in the wild type strain . When both mutants were shifted for 10 min at the restrictive temperature (37 degrees C), the main structural feature was the disappearance of all Golgi networks . In sec23 mutant cells, there was an increase in number of tubular, nonnodular networks corresponding to terminal portions of the endoplasmic reticulum; in sec18 cells, small 20- to 50-nm tubules and vesicles accumulated in the cytoplasm . Within minutes after the return of sec23 cells to permissive temperature (20 degrees C), small vesicles and tubules started to accumulate to reach a number similar or greater than that noted in sec18 cells observed under the same conditions . At later time intervals and in both mutants, the small tubules and vesicles decreased in number . This decrease was concomitant with the reappearance of fine nodular networks, followed later on by the reconstruction of networks of larger caliber and the formation of secretion granules . CONCLUSIONS: It is concluded that a block of the secretory pathway upstream of the Golgi compartment leads to the disappearance of Golgi networks . The small vesicles and tubules that accumulate during the block in sec18 cells and within minutes after the shift at 20 degrees C in sec23 cells appear to fuse together to form new Golgi networks . Thus, the small vesicles would not fuse with a preexisting Golgi apparatus but would rather fuse together to produce new Golgi networks . Such networks appear as transitory structures continuously undergoing renewal.

Biochemistry, 1998 May 5, 37(18), 6295 - 302
The strongly conserved lysine 256 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase is essential for phosphoryl transfer; Krautwurst H et al.; Lysine 256, a conserved amino acid of Saccharomycescerevisiae phosphoenolpyruvate (PEP) carboxykinase located in the consensus kinase 1a sequence of the enzyme, was changed to alanine, arginine, or glutamine by site-directed mutagenesis . These substitutions did not result in gross changes in the protein structure, as indicated by circular dichroism, tryptophan fluorescence spectroscopy, and gel-exclusion chromatography . The three variant enzymes showed almost unaltered Km for MnADP but about a 20 000-fold decrease in Vmax for the PEP carboxylation reaction, as compared to wild-type PEP carboxykinase . The variant enzymes presented oxaloacetate decarboxylase activity at levels similar to those of the native protein; however, they lacked pyruvate kinase-like activity . The dissociation constant for the enzyme-MnATP complex was 1.3 +/- 0.3 microM for wild-type S . cerevisiae PEP carboxykinase, and the corresponding values for the Lys256Arg, Lys256Gln, and Lys256Ala mutants were 2.0 +/- 0.6 microM, 17 +/- 2 microM, and 20 +/- 6 microM, respectively . These results collectively show that a positively charged residue is required for proper binding of MnATP and that Lys256 plays an essential role in transition state stabilization during phosphoryl transfer for S . cerevisiae PEP carboxykinase.

J Biol Chem, 1998 Jun 5, 273(23), 14331 - 8
Isolation and characterization of the Saccharomyces cerevisiae LPP1 gene encoding a Mg2+-independent phosphatidate phosphatase; Toke DA et al.; The DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase enzyme accounts for half of the Mg2+-independent phosphatidate (PA) phosphatase activity in Saccharomyces cerevisiae . The LPP1 (lipid phosphate phosphatase) gene encodes a protein that contains a novel phosphatase sequence motif found in DGPP phosphatase and in the mouse Mg2+-independent PA phosphatase . A genomic copy of the S . cerevisiae LPP1 gene was isolated and was used to construct lpp1Delta and lpp1Delta dpp1Delta mutants . A multicopy plasmid containing the LPP1 gene directed a 12.9-fold overexpression of Mg2+-independent PA phosphatase activity in the S . cerevisiae lpp1Delta dpp1Delta double mutant . The heterologous expression of the S . cerevisiae LPP1 gene in Sf-9 insect cells resulted in a 715-fold overexpression of Mg2+-independent PA phosphatase activity relative to control insect cells . The Mg2+-independent PA phosphatase activity encoded by the LPP1 gene was associated with the membrane fraction of the cell . The LPP1 gene product also exhibited lyso-PA phosphatase and DGPP phosphatase activities . The order of substrate preference was PA > lyso-PA > DGPP . Like the dpp1Delta mutant, the lpp1Delta mutant and the lpp1Delta dpp1Delta double mutant were viable and did not exhibit obvious growth defects . Biochemical analyses of lpp1Delta, dpp1Delta, and lpp1Delta dpp1Delta mutants showed that the LPP1 and DPP1 gene products encoded nearly all of the Mg2+-independent PA phosphatase and lyso-PA phosphatase activities and all of the DGPP phosphatase activity in S . cerevisiae . Moreover, the analyses of the mutants showed that the LPP1 and DPP1 gene products played a role in the regulation of phospholipid metabolism and the cellular levels of phosphatidylinositol and PA.

Curr Genet, 1998 May, 33(5), 345 - 51
Reciprocal translocation at duplicated RPL2 loci might cause speciation of Saccharomyces bayanus and Saccharomyces cerevisiae; Ryu SL et al.; By a genomic comparison of two sibling yeasts, Saccharomyces bayanus and S . cerevisiae, we previously demonstrated that chromosomes II and IV of S . cerevisiae were rearranged into chromosomes 12 and 14 of S . bayanus or vice versa . In the present study we have delimited the translocation break sites in chromosomes II and IV by Southern hybridization using DNA fragments of S . cerevisiae cosmid clones as probes . The results suggest that the reciprocal translocation of chromosomes II and IV had occurred at duplicated RPL2 loci . Furthermore, the translocation sites in S . bayanus were confirmed by the cloning and sequence analysis of the regions flanking RPL2 loci . Several genes in the regions flanking the RPL2 loci were present in the order expected for a translocation at these loci between the two species . These results indicated that the reciprocal translocation between chromosomes II and IV was generated by homologous recombination at duplicated RPL2 loci on the two chromosomes . Therefore, we propose that duplicated genes or duplicated regions play an important role in altering genomic organization during the speciation of S . bayanus and S . cerevisiae.

J Bacteriol, 1998 Jun, 180(11), 2875 - 82
Identification and characterization of MAE1, the Saccharomyces cerevisiae structural gene encoding mitochondrial malic enzyme; Boles E et al.; Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase . Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis . A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate . Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells . Conversely, overexpression of YKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity . Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol . Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures . Disruption of MAE1 alone did not result in a clear phenotype . Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring beta-galactosidase activities in a strain carrying a MAE1::lacZ fusion . Both in shake flask cultures and in carbon-limited chemostat cultures, MAE1 was constitutively expressed . A three- to fourfold induction was observed during anaerobic growth on glucose . Subcellular fractionation experiments indicated that malic enzyme in S . cerevisiae is a mitochondrial enzyme . Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions . However, since null mutants could still grow anaerobically, this function is apparently not essential.

Mol Gen Genet, 1998 Apr, 258(1-2), 60 - 8
The novel function of the Saccharomyces cerevisiae CBP2 gene as a splicing factor essential to excision of the Saccharomyces douglasii LSU intron in vivo; Tian GL et al.; In the yeast Saccharomyces cerevisiae, the product of the nuclear gene CBP2 is required exclusively for the splicing of the terminal intron of the mitochondrial cytochrome b gene . The homologous gene from the related yeast, Saccharomyces douglasii, has been shown to be essential for respiratory growth in the presence of a wild-type S . douglasii mitochondrial genome and dispensable in the presence of an intronless mitochondrial genome . The two CBP2 genes are functionally interchangeable although the target intron of the S . cerevisiae CBP2 gene is absent from the S . douglasii mitochondrial genome . To determine the function of the CBP2 gene in S . douglasii mitochondrial pre-RNA processing we have constructed and analyzed interspecific hybrid strains between the nuclear genome of S . cerevisiae carrying an inactive CBP2 gene and S . douglasii mitochondrial genomes with different intron contents . We have demonstrated that inactivation of the S . cerevisiae CBP2 gene affects the maturation of the S . douglasii LSU pre-RNA, leading to a respiratory-deficient phenotype in the hybrid strains . We have shown that the CBP2 gene is essential for excision of the S . douglasii LSU intron in vivo and that the gene is dispensable when this intron is deleted or replaced by the S . cerevisiae LSU intron.

Gene, 1998 Jun 8, 212(2), 323 - 32
Isolation and characterization of a new gene encoding a member of the HIRA family of proteins from Drosophila melanogaster; Kirov N et al.; The HIRA family of genes (named after yeast HIR genes; HIR is an acronym for 'histone regulator') includes the yeast HIR1 and HIR2 repressors of histone gene transcription in S . cerevisiae, human TUPLE-1/HIRA, chicken HIRA, and mouse HIRA . Here, we describe a new member of the HIRA family, Dhh, for the Drosophila homolog of HIRA . Northern analysis with poly (A)+ mRNA isolated from different developmental stages of Drosophila melanogaster shows hybridization with a single Dhh transcript of 4.1kb . Hybridization is strong in female adults, unfertilized eggs and 0-3-h-old embryos, then diminishes, but is still detectable, during later stages of development and in adult males . More specifically, in-situ hybridization shows that Dhh transcripts, which are initially detected in nurse cells during mid-oogenesis, become localized to the developing oocyte at high levels . Transcripts persist strongly during early blastoderm stages then fade dramatically by 3h of development . The Dhh cDNA encodes an open reading frame of 1061 amino acids with high similarity scores to the HIRA polypeptides, as well as two hypothetical polypeptides from C . elegans and S . pombe, in a protein database search . They all share three highly homologous regions: a WD-repeat cluster, a small domain with clustered positively charged amino acids, and a domain comprising two repeats with close resemblance to WD repeats plus a region with no homology outside of the family . The conservation of these homologous regions in HIRA-encoded proteins from evolutionary distant organisms suggests that they are important for the activity of the members of the family.

J Biol Chem, 1998 May 29, 273(22), 13482 - 7
Scanning alanine mutagenesis of the CDP-alcohol phosphotransferase motif of Saccharomyces cerevisiae cholinephosphotransferase; Williams JG et al.; Cholinephosphotransferase (EC 2.7.8.2) catalyzes the formation of a phosphoester bond via the transfer of a phosphocholine moiety from CDP-choline to diacylglycerol forming phosphatidylcholine and releasing CMP . A motif, Asp113-Gly114-(X)2-Ala117-Arg118-(X)8-Gly127+ ++-(X)3-Asp131-(X)3-Asp135, located within the CDP-choline binding region of Saccharomyces cerevisiae cholinephosphotransferase (CPT1 ?/Author: Please confirm that a gene is meant here.) is also found in several other phospholipid synthesizing enzymes that catalyze the formation of a phosphoester bond utilizing a CDP-alcohol and a second alcohol as substrates . To determine if this motif is diagnostic of the above reaction type scanning alanine mutagenesis of the conserved residues within S . cerevisiae cholinephosphotransferase was performed . Enzyme activity was assessed in vitro using a mixed micelle enzyme assay and in vivo by determining the ability of the mutant enzymes to restore phosphatidylcholine synthesis from radiolabeled choline in an S . cerevisiae strain devoid of endogenous cholinephosphotransferase activity . Alanine mutants of Gly114, Gly127, Asp131, and Asp135 were inactive; mutants, Ala117 and Arg118 displayed reduced enzyme activity, and Asp113 displayed wild type activity . The analysis described is the first molecular characterization of a CDP-alcohol phosphotransferase motif and results predict a catalytic role utilizing a general base reaction proceeding through Asp131 or Asp135 via a direct nucleophilic attack of the hydroxyl of diacylglyerol on the phosphoester bond of CDP-choline that does not proceed via an enzyme bound intermediate . Residues Ala117 and Arg118 do not participate directly in catalysis but are likely involved in substrate binding or positioning with Arg118 predicted to associate with a phosphate moiety of CDP-choline.

Microbiologia, 1997 Dec, 13(4), 481 - 8
Distribution of the trehalase activation response and the regulatory trehalase gene among yeast species; Soto T et al.; In Saccharomyces cerevisiae and other yeasts the activity of regulatory trehalases increases in response to the addition of glucose and to thermal changes in the extracellular medium . We have performed an screening on the extent of this response among different representative yeast species and the results show that this ability is displayed only by a few members of the Saccharomycetaceae family . However, all yeasts examined contain a gene related to that coding for regulatory trehalase in S . cerevisiae . This finding reveals that the operational distinction between regulatory and nonregulatory trehalase in yeasts is not a property of the enzyme by itself but relays on the expression of accompanying mechanisms able to modulate trehalase activity.

Arch Biochem Biophys, 1998 May 15, 353(2), 297 - 311
Overexpression, purification, and stereochemical studies of the recombinant (S)-adenosyl-L-methionine: delta 24(25)- to delta 24(28)-sterol methyl transferase enzyme from Saccharomyces cerevisiae; Nes WD et al.; The ERG6 gene that encodes (S)-adenosyl-L-methionine: delta 24(25)-to delta 24(28)-sterol methyl transferase (SMT) enzyme from Saccharomyces cerevisiae was introduced into plasmid pET23a(+) and the resulting native protein was overexpressed in BL21 (DE3) host cells under control of a T7 promoter . This enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, anion exchange, and hydrophobic interaction chromatography . N-Terminal sequence analysis of the first 10 amino acids of the purified SMT protein confirmed the identity of the start triplet and expected primary structure . The enzyme exhibited a turnover number of 0.01/s and an isoelectric point of 5.95 . A combination of Superose 6 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified SMT enzyme possessed a native molecular weight of 172,000 and was tetrameric . The purified SMT enzyme generated kinetics in which velocity versus substrate curves relative to zymosterol (preferred sterol acceptor molecule) and AdoMet were sigmoidal rather than hyperbolic, indicating enzyme cooperativity among the subunits . Studies on product formation using {27-13C}zymosterol and {2H3-methyl}AdoMet incubated with the pure SMT enzyme confirmed the reaction mechanism of sterol methylation to involve a 1,2-hydride shift of H-24 to C-25 from the Re-face of the original 24,25- double bond . Deduced amino acid sequence comparisons of the SMT polypeptide from S . cerevisiae with related sterol methyl transferase enzymes of plant and fungal origin indicate that there is a significant degree of similarity between these enzymes . Specifically, there is a conserved sequence (in yeast from amino acids ca . 79 to 92 which contains an YEXGWG motif; referred to as Region I) that is not present in other AdoMet-dependent methyl transferase enzymes.

Yeast, 1998 Apr 30, 14(6), 527 - 33
Identification of YHR019 in Saccharomyces cerevisiae chromosome VIII as the gene for the cytosolic asparaginyl-tRNA synthetase; Landrieu I et al.; Exploiting the asparagine auxotrophy of the Saccharomyces cerevisiae mutant strain 8556a, we have isolated the gene for the cytosolic asparaginyl-tRNA synthetase (AsnRS) of S . cerevisiae, by functional complementation of the mutation affecting this strain . The isolated gene could be identified to the open reading frame YHR019, called DED81, located on chromosome VIII . The mutant gene from the 8556a strain, asnrs-1, was amplified from genomic DNA by PCR . This gene contains a point mutation, leading to the replacement of a glycine residue by a serine in a region of the protein probably important for the asparaginyl-adenylate recognition . The protein encoded by YHR019 is very similar to cytosolic AsnRS from other eukaryotic sources . In a phylogenetic analysis based on AsnRS sequences from various organisms, the eukaryotic sequences were clustered . Expression of YHR019 in Escherichia coli demonstrated that a yeast AsnRS activity was produced . The recombinant enzyme was purified to homogeneity in three chromatography steps . We showed that the recombinant S . cerevisiae AsnRS was able to charge unfractionated yeast tRNA, but not E . coli tRNA, with asparagine.

Neoplasma, 1997, 44(6), 374 - 9
Searching for a functional analogy between yeast Pso4 and bacterial RecA proteins in induced mitotic recombination; Vlckova V et al.; The pso4-1 mutant of S . cerevisiae is phenotypically similar to the recA mutant of E . coli; it is sensitive to DNA cross-linking agents and defective in both recombination and mutagenesis . In this paper we have measured the effect of the recA gene expression on the frequency of mitotic crossing-over and mitotic gene conversion in response to DNA damage induced by photoactivated 8-methoxypsoralen (8-MOP + UVA), ultraviolet radiation (UV) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . The diploid pso4-1 mutant and the repair wild type strain were transformed with the multicopy plasmid carrying the recA gene placed under the control of the ADH1 promoter . The results showed that RecA is not able to restore block in induced mitotic recombination in pso4-1 cells after DNA damaging agents used . Thus RecA protein is not able to substitute Pso4 protein in homologous mitotic recombination indicating that they have probably different functions in this process.

J Cell Sci, 1998 Jun, 111 ( Pt 12), 1717 - 27
The S . cerevisiae CLU1 and D . discoideum cluA genes are functional homologues that influence mitochondrial morphology and distribution; Fields SD et al.; The cluA gene, encoding a novel 150 kDa protein, was recently characterized in Dictyostelium discoideum; disruption of cluA impaired cytokinesis and caused mitochondria to cluster at the cell center . The genome of Saccharomyces cerevisiae contains an open reading frame (CLU1) that encodes a protein that is 27% identical, 50% similar, to this Dictyostelium protein . Deletion of CLU1 from S . cerevisiae did not affect cell viability, growth properties, sporulation efficiency, or frequency of occurrence of cells lacking functional mitochondria . However, in clu1Delta cells the mitochondrial reticulum, which is normally highly branched, was condensed to one side of the cell . Transformation of cluA- Dictyostelium mutants with the yeast CLU1 gene yielded amoebae that divided normally and had dispersed mitochondria . The mitochondria in cluA- Dictyostelium cells complemented with CLU1 were not as widely scattered as in cluA+ Dictyostelium cells, but formed loose clusters throughout the cytoplasm . These results indicate that the products of the CLU1 and cluA genes, in spite of their limited homology, are functional homologues.

Genetics, 1998 May, 149(1), 73 - 85
Mutations synthetically lethal with cep1 target S . cerevisiae kinetochore components; Baker RE et al.; CP1 (encoded by CEP1) is a Saccharomyces cerevisiae chromatin protein that binds a DNA element conserved in centromeres and in the 5'-flanking DNA of methionine biosynthetic (MET) genes . Strains lacking CP1 are defective in chromosome segregation and MET gene transcription, leading to the hypothesis that CP1 plays a general role in assembling higher order chromatin structures at genomic sites where it is bound . A screen for mutations synthetically lethal with a cep1 null allele yielded five recessive csl (cep1 synthetic lethal) mutations, each defining a unique complementation group . Four of the five mutations synergistically increased the loss rate of marker chromosomes carrying a centromere lacking the CP1 binding site, suggesting that the cep1 synthetic lethality was due to chromosome segregation defects . Three of these four CSL genes were subsequently found to be known or imputed kinetochore genes: CEP3, NDC10, and CSE4 . The fourth, CSL4, corresponded to ORF YNL232w on chromosome XIV, and was found to be essential . A human cDNA was identified that encoded a protein homologous to Csl4 and that complemented the csl4-1 mutation . The results are consistent with the view that the major cellular role of CP1 is to safeguard the biochemical integrity of the kinetochore.

Glycobiology, 1998 Jan, 8(1), 17 - 33
Identification and analysis of a class 2 alpha-mannosidase from Aspergillus nidulans; Eades CJ et al.; A Class 2 alpha-mannosidase gene was cloned and sequenced from the filamentous fungus Aspergillus nidulans . A portion of the gene was amplified using degenerate oligonucleotide primers which were designed based on similarity between the Saccharomyces cerevisiae vacuolar and rat ER/cytosolic Class 2 protein sequences . The PCR amplification product was used to isolate the full length gene, and DNA sequencing revealed a 3383 bp coding region containing three introns . The predicted 1049 amino acid reading frame contained six potential N-glycosylation sites and encoded a protein of 118 kDa . The protein sequence did not appear to encode a typical fungal signal sequence or membrane spanning domain . Although the cellular location of the A.nidulans mannosidase was not determined, experimental evidence suggested that it was located within a subcellular organelle . The Matchbox sequence similarity matrix indicated that the A.nidulans protein sequence was more highly similar to the rat ER/cytosolic (Rij = 0.33) and S.cerevisiae vacuolar alpha-mannosidases (Rij = 0.43) than the rat and yeast sequences were to each other (Rij = 0.29) . These three enzymes were found to be distantly related to other Class 2 sequences, and compose a third subgroup of Class 2 alpha-mannosidases, as shown by ClustalW sequence alignment.

FEBS Lett, 1998 Apr 10, 426(1), 7 - 16
Updated catalogue of homologues to human disease-related proteins in the yeast genome; Andrade MA et al.; The recent availability of the full Saccharomyces cerevisiae genome offers a perfect opportunity for revising the number of homologues to human disease-related proteins . We carried out automatic analysis of the complete S . cerevisiae genome and of the set of human disease-related proteins as identified in the SwissProt sequence data base . We identified 285 yeast proteins similar to 155 human disease-related proteins, including 239 possible cases of human-yeast direct functional equivalence (orthology) . Of these, 40 cases are suggested as new, previously undiscovered relationships . Four of them are particularly interesting, since the yeast sequence is the most phylogenetically distant member of the protein family, including proteins related to diseases such as phenylketonuria, lupus erythematosus, Norum and fish eye disease and Wiskott-Aldrich syndrome.

FEMS Microbiol Lett, 1998 May 1, 162(1), 155 - 60
The YGR194c (XKS1) gene encodes the xylulokinase from the budding yeast Saccharomyces cerevisiae; Rodriguez-Pena JM et al.; We report the finding of a Saccharomyces cerevisiae gene necessary for growth in culture media with D-xylulose as the sole carbon source . This gene corresponds to the YGR194c open reading frame that we have previously described, and it is renamed now XKS1 . Data bank comparisons of the protein encoded by the XKS1 gene showed significant homology with different xylulokinases, indicating a possible role in xylulose phosphorylation . The wild-type gene in a centromeric plasmid complemented defective growth of xks1 S . cerevisiae mutant strains in xylulose . By contrast, overexpression negatively influenced cell growth in this carbon source.

FEMS Microbiol Lett, 1998 May 1, 162(1), 83 - 91
Ca(2+)-ATPases of Saccharomyces cerevisiae: diversity and possible role in protein sorting; Okorokov LA et al.; The PMR1 gene of Saccharomyces cerevisiae is thought to encode a putative Ca(2+)-ATPase {1} . Membranes isolated from wild-type cells and from pmr1 null mutant of S . cerevisiae were fractionated on sucrose density gradients . In the pmr1 mutant we found a decrease in activity of the P-type ATPase and of ATP-dependent, protonophore-insensitive Ca2+ transport in light membranes, that comigrate with the Golgi marker GDPase . We conclude that the product of the PMR1 gene (Pmr1p) is indeed a Ca(2+)-ATPase of the Golgi and Golgi-like membranes . Surprisingly, the pmr1 null mutation abolished Ca(2+)-ATPase activity in Golgi and/or Golgi-like membranes only to 50% under conditions where they are separated from vacuolar membranes . This indicates that an additional Ca(2+)-ATPase is localized in Golgi and/or Golgi-like membranes . Moreover, a third Ca(2+)-ATPase is found in the ER and ER-like membranes . The data are consistent with the assumption that these Ca(2+)-ATPases are encoded by gene(s) different from PMR1 . Disruption of PMR1 Ca(2+)-ATPase causes significant redistribution of enzyme activities and of total protein in compartments of the secretory pathway . A decrease in activity is observed for three integral membrane proteins: NADPH cytochrome c reductase, dolichyl phosphate mannose synthase, and Ca(2+)-ATPase, and also for total protein in Golgi, Golgi-like compartments and in vacuoles, whereas a corresponding increase of these activities is observed in endoplasmic reticulum and endoplasmic reticulum-like membranes . We assume that Ca(2+)-ATPases and sufficient Ca2+ gradients across the organellar membranes are important for the correct sorting of proteins to the various compartments of the secretory apparatus.

Antimicrob Agents Chemother, 1998 May, 42(5), 1034 - 41
Identification of Saccharomyces cerevisiae genes conferring resistance to quinoline ring-containing antimalarial drugs; Delling U et al.; To identify genes that can confer resistance to antimalarial drugs in yeast, we transformed the quinidine-sensitive strain CYX247-9A of Saccharomyces cerevisiae with a yeast genomic library and selected for transformants that grow in the presence of elevated levels of antimalarial drugs . Plasmids were rescued from such clones and were analyzed for the presence of individual open reading frames that can confer drug resistance . Using quinidine as the selective drug, we were able to identify three genes that can cause resistance to antimalarial drugs . Overexpression of the yeast genes CIN5 (a member of the family of bZIP transcription factors), STII (a Hsp90 cochaperone), and YOR273c (a member of the major facilitator superfamily of transmembrane transporters) conferred 3.9-, 7.0-, and 4.3-fold resistance to quinidine, respectively, over that of control yeast . Cross-resistance assays determined that STI1 also conferred resistance to mefloquine (3.4-fold), while CIN5 also conferred resistance to mefloquine (9.6-fold) and chloroquine (5.4-fold) . Using mefloquine as the selective drug, we determined that overexpression of YBR233w, a member of the hnRNPK family of nuclear RNA binding proteins, conferred resistance to mefloquine (13.5-fold) . Expression of the human hnRNPK homolog of YBR233w in S . cerevisiae also conferred mefloquine resistance, suggesting that homologs of the identified resistance genes may perform similar functions in species other than yeast . Our experiments have identified heretofore unknown pathways of resistance to quinoline ring-containing antimalarial drugs in S . cerevisiae.

Biotechniques, 1998 May, 24(5), 782 - 6, 788
Vectors for expressing T7 epitope- and His6 affinity-tagged fusion proteins in S . cerevisiae; Enomoto S et al.; We have constructed a series of vectors (YGALSETs) for the expression of epitope- and affinity-tagged fusion proteins in yeast cells using the regulated GAL10 promoter . Fusion proteins produced from YGALSET plasmids include a leader peptide at the N terminus that encodes both a T7 gene 10 epitope tag and a His6 affinity tag . The YGALSET vector series includes centromere plasmids for low-copy plasmid maintenance and 2 micron episomal plasmids for multicopy plasmid maintenance and four different selectable markers: TRP1, URA3, LEU2 and HIS3 . We also provide a convenient approach for transferring cloned genes from a bacterial expression vector into YGALSET vectors by in vivo recombination and a rapid method to screen directly for clones that express the fusion protein of interest.

Genome Res, 1998 May, 8(5), 464 - 78
Transposable elements and genome organization: a comprehensive survey of retrotransposons revealed by the complete Saccharomyces cerevisiae genome sequence; Kim JM et al.; We conducted a genome-wide survey of Saccharomyces cerevisiae retrotransposons and identified a total of 331 insertions, including 217 Ty1, 34 Ty2, 41 Ty3, 32 Ty4, and 7 Ty5 elements . Eighty-five percent of insertions were solo long terminal repeats (LTRs) or LTR fragments . Overall, retrotransposon sequences constitute >377 kb or 3.1% of the genome . Independent evolution of retrotransposon sequences was evidenced by the identification of a single-base pair insertion/deletion that distinguishes the highly similar Ty1 and Ty2 LTRs and the identification of a distinct Ty1 subfamily (Ty1') . Whereas Ty1, Ty2, and Ty5 LTRs displayed a broad range of sequence diversity (typically ranging from 70%-99% identity), Ty3 and Ty4 LTRs were highly similar within each element family (most sharing >96% nucleotide identity) . Therefore, Ty3 and Ty4 may be more recent additions to the S . cerevisiae genome and perhaps entered through horizontal transfer or past polyploidization events . Distribution of Ty elements is distinctly nonrandom: 90% of Ty1, 82% of Ty2, 95% of Ty3, and 88% of Ty4 insertions were found within 750 bases of tRNA genes or other genes transcribed by RNA polymerase III . tRNA genes are the principle determinant of retrotransposon distribution, and there is, on average, 1.2 insertions per tRNA gene . Evidence for recombination was found near many Ty elements, particularly those not associated with tRNA gene targets . For these insertions, 5'- and 3'-flanking sequences were often duplicated and rearranged among multiple chromosomes, indicating that recombination between retrotransposons can influence genome organization . S . cerevisiae offers the first opportunity to view organizational and evolutionary trends among retrotransposons at the genome level, and we hope our compiled data will serve as a starting point for further investigation and for comparison to other, more complex genomes.

Proc Natl Acad Sci U S A, 1998 May 12, 95(10), 5667 - 71
Humanizing the yeast telomerase template; Henning KA et al.; Saccharomyces cerevisiae contains an irregular telomere sequence (TG1-3)n, which differs from the regular repeat (TTAGGG)n found at the telomeres of higher organisms including humans . We have modified the entire 16-nt template region of the S . cerevisiae telomerase RNA gene (TLC1) to produce (TTAGGG)n repeats, the human telomere sequence . Haploid yeast strains with the tlc1-human allele are viable with no growth retardation and express the humanized gene at a level comparable to wild type . Southern hybridization demonstrates that (TTAGGG)n repeats are added onto the yeast chromosome ends in haploid strains with the tlc1-human allele, and sequencing of rescued yeast artificial chromosome ends has verified the addition of human telomeric repeats at the molecular level . These data suggest that the irregularity of the yeast telomere sequence is because of the template sequence of the yeast telomerase RNA . Haploid strains with the tlc1-human allele will provide an important tool for studying the function of telomerase and its regulation by telomere-binding proteins, and these strains will serve as good hosts for human artificial chromosome assembly and propagation.

Curr Biol, 1998 Apr 23, 8(9), 497 - 507
The Polo-related kinase Cdc5 activates and is destroyed by the mitotic cyclin destruction machinery in S . cerevisiae; Charles JF et al.; BACKGROUND: Following chromosome segregation in anaphase, ubiquitin-dependent degradation of mitotic cyclins contributes to the exit from mitosis . A key step in this process is catalyzed by a ubiquitin-protein ligase known as the anaphase-promoting complex (APC), the regulation of which is poorly understood . The Polo-related protein kinase Cdc5 in Saccharomyces cerevisiae might encode a regulator of the APC, because cdc5 mutant cells arrest with a late mitotic phenotype similar to that observed in cells with defective cyclin destruction . RESULTS: We investigated the role of Cdc5 in the regulation of mitotic cyclin degradation . In cdc5-1 mutant cells, we observed a defect in the destruction of cyclins and a reduction in the cyclin-ubiquitin ligase activity of the APC . Overexpression of CDC5 resulted in increased APC activity and mitotic cyclin destruction in asynchronous cells or in cells arrested in metaphase . CDC5 mutation or overexpression did not affect the degradation of the APC substrate Pds 1, which is normally degraded at the metaphase-to-anaphase transition . Cyclin-specific APC activity in cells overexpressing CDC5 was reduced in the absence of the APC regulatory proteins Hct 1 and Cdc20 . In G1, Cdc5 itself was degraded by an APC-dependent and Hct1-dependent mechanism . CONCLUSIONS: We conclude that Cdc5 is a positive regulator of cyclin-specific APC activity in late mitosis . Degradation of Cdc5 in G1 might provide a feedback mechanism by which the APC destroys its activator at the onset of the next cell cycle.

Development, 1998 Jun, 125(12), 2291 - 302
YakA, a protein kinase required for the transition from growth to development in Dictyostelium; Souza GM et al.; When Dictyostelium cells starve they arrest their growth and induce the expression of genes necessary for development . We have identified and characterized a protein kinase, YakA, that is essential for the proper regulation of both events . Amino acid sequence and functional similarities indicate that YakA is a homolog of Yak1p, a growth-regulating protein kinase in S . cerevisiae . Purified YakA expressed in E . coli is able to phosphorylate myelin basic protein . YakA-null cells are smaller and their cell cycle is accelerated relative to wild-type cells . When starved, YakA-null cells fail to decrease the expression of the growth-stage gene cprD, and do not induce the expression of genes required for the earliest stages of development . YakA mRNA levels increase during exponential growth and reach a maximum at the point of starvation, consistent with a role in mediating starvation responses . YakA mRNA also accumulates when cells are grown in medium conditioned by cells grown to high density, suggesting that yakA expression is under the control of an extracellular signal that accumulates during growth . Expression of yakA from a conditional promoter causes cell-cycle arrest in nutrient-rich medium and promotes developmental events, such as the expression of genes required for cAMP signaling . YakA appears to regulate the transition from growth to development in Dictyostelium.

Curr Biol, 1998 Mar 26, 8(7), 393 - 403
Repair of oxidatively damaged guanine in Saccharomyces cerevisiae by an alternative pathway; Bruner SD et al.; BACKGROUND: Transversion mutations are caused by 8-oxoguanine (OG), a DNA lesion produced by the spontaneous oxidation of guanine nucleotides, which mis-pairs with adenine during replication . Resistance to this mutagenic threat is mediated by the GO system, the components of which are functionally conserved in bacteria and mammals . To date, only one of three GO system components has been identified in the budding yeast Saccharomyces cerevisiae, namely the OG:C-specific glycosylase/lyase yOgg1 . Furthermore, S . cerevisiae has been reported to contain a unique glycosylase/lyase activity, yOgg2, which excises OG residues opposite adenines . Paradoxically, according to the currently accepted model, yOgg2 activity should increase the mutagenicity of OG lesions . Here we report the isolation of yOgg2 and the elucidation of its role in oxidative mutagenesis . RESULTS: Borohydride-dependent cross-linking using an OG-containing oligonucleotide substrate led to the isolation of yOgg1 and a second protein, Ntg1, which had previously been shown to process oxidized pyrimidines in DNA . We demonstrate that Ntg1 has OG-specific glycosylase/lyase activity indistinguishable from that of yOgg2 . Targeted disruption of the NTG1 gene resulted in complete loss of yOgg2 activity and yeast lacking NTG1 had an elevated rate of A:T to C:G transversions . CONCLUSIONS: The Ntg1 and yOgg2 activities are encoded by a single gene . We propose that yOgg2 has evolved to process OG:A mis-pairs that have arisen through mis-incorporation of 8-oxo-dGTP during replication . Thus, the GO system in S . cerevisiae is fundamentally distinct from that in bacteria and mammals.

Yi Chuan Xue Bao, 1997, 24(6), 561 - 8
{Construction of yeast ADH2-SUC2 hybrid promoter and its regulation in gene expression}; Li W et al.; By fusing the upstream activation sequence (UAS) of ADH2 gene to the 5' end of a series of deletions in the upstream region of SUC2 gene, 4 expression plasmids YFD110 delta 1, YFD110 delta 9, YFD110 delta 17 and YFD110 delta 11 were constructed . They contained different hybrid promoters for transcription of SUC2 gene . After transforming them together with two control plasmids YFD26 delta 1, YFD25 into yeast . S . cerevisiae Y33 respectively, the transformants were grown in the repression or derepression media and the invertase produced by each transformants were analyzed by colormetry and gel electrophoresis . The results were as follow is: (1) UASSUC2 and UASADH2 in the hybrid plasmid YFD110 delta 1 worked synergically under derepression condition . Under repression condition, Y33/YFD110 delta 1 produced very low level of glycosylation invertase . (2) Compared with three different derepression media, the medium containing low concentration glucose gave higher derepression efficiency of glycosylated invertase than the media containing glycerol and ethanol.

Gene, 1998 Apr 14, 210(2), 195 - 201
Spermine is not essential for growth of Saccharomyces cerevisiae: identification of the SPE4 gene (spermine synthase) and characterization of a spe4 deletion mutant; Hamasaki-Katagiri N et al.; Spermine, ubiquitously present in most organisms, is the final product of the biosynthetic pathway for polyamines and is synthesized from spermidine . In order to investigate the physiological roles of spermine, we identified the SPE4 gene, which codes for spermine synthase, on the right arm of chromosome XII of Saccharomyces cerevisiae and prepared a deletion mutant in this gene . This mutant has neither spermine nor spermine synthase activity . Using the spe4 deletion mutant, we show that S . cerevisiae does not require spermine for growth, even though spermine is normally present in the wild-type organism . This is in striking contrast to the absolute requirement of S . cerevisiae for spermidine for growth, which we had previously reported using a mutant lacking the SPE3 gene (spermidine synthase) {Hamasaki-Katagiri, N., Tabor, C . W., Tabor, H., 1997 . Spermidine biosynthesis in Saccharomyces cerevisiae: Polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase) . Gene 187, 35-43}.

J Bacteriol, 1998 May, 180(9), 2345 - 9
Control of 5',5'-dinucleoside triphosphate catabolism by APH1, a Saccharomyces cerevisiae analog of human FHIT; Chen J et al.; The putative human tumor suppressor gene FHIT (fragile histidine triad) (M . Ohta et al., Cell 84:587-597, 1996) encodes a protein behaving in vitro as a dinucleoside 5',5"'-P1,P3-triphosphate (Ap3A) hydrolase . In this report, we show that the Saccharomyces cerevisiae APH1 gene product, which resembles human Fhit protein, also hydrolyzes dinucleoside 5',5'-polyphosphates, with Ap3A being the preferred substrate . Accordingly, disruption of the APH1 gene produced viable S . cerevisiae cells containing reduced Ap3A-hydrolyzing activity and a 30-fold-elevated Ap3N concentration.

J Bacteriol, 1998 May, 180(9), 2292 - 7
The Pol beta-14 dominant negative rat DNA polymerase beta mutator mutant commits errors during the gap-filling step of base excision repair in Saccharomyces cerevisiae; Clairmont CA et al.; We demonstrated recently that dominant negative mutants of rat DNA polymerase beta (Pol beta) interfere with repair of alkylation damage in Saccharomyces cerevisiae . To identify the alkylation repair pathway that is disrupted by the Pol beta dominant negative mutants, we studied the epistatic relationship of the dominant negative Pol beta mutants to genes known to be involved in repair of DNA alkylation damage in S . cerevisiae . We demonstrate that the rat Pol beta mutants interfere with the base excision repair pathway in S . cerevisiae . In addition, expression of one of the Pol beta dominant negative mutants, Pol beta-14, increases the spontaneous mutation rate of S . cerevisiae whereas expression of another Pol beta dominant negative mutant, Pol beta-TR, does not . Expression of the Pol beta-14 mutant in cells lacking APN1 activity does not result in an increase in the spontaneous mutation rate . These results suggest that gaps are required for mutagenesis to occur in the presence of Pol beta-14 but that it is not merely the presence of a gap that results in mutagenesis . Our results suggest that mutagenesis can occur during the gap-filling step of base excision repair in vivo.

Biometals, 1998 Apr, 11(2), 101 - 6
Evidence for the involvement of vacuolar activity in metal(loid) tolerance: vacuolar-lacking and -defective mutants of Saccharomyces cerevisiae display higher sensitivity to chromate, tellurite and selenite; Gharieb MM et al.; The responses of Saccharomyces cerevisiae towards the oxyanions tellurite, selenite and chromate were investigated in order to establish the involvement of the yeast vacuole in their detoxification . Three mutants of S . cerevisiae with defective vacuolar morphology and function were used; mutant JSR180 delta 1 is devoid of any vacuolar-like structure while ScVatB and ScVatC are deficient in specific protein subunits of the vacuolar (V)-H(+)-ATPase . All the mutant strains showed increased sensitivity to tellurite and chromate compared to their parental strains . Such sensitivity of the mutants was associated with increased accumulation of tellurium and chromium . These results indicate that accumulation of both tellurium and chromium occurred mainly in the cytosolic compartment of the cell, with detoxification influenced by the presence of a functionally-active vacuole which may play a role in compartmentation as well as regulation of the cytosolic compartment for optimal expression of a detoxification mechanism, e.g . reduction . In contrast, the vacuolar-lacking mutant, JSR180 delta 1, and the defective V-H+ATPase mutant ScVatB displayed lower selenium accumulation than their parental strains . Additionally, the mutant strain ScVatB displayed a higher tolerance to selenite than the parental strain . This result suggests that accumulation of selenium occurs mainly in the vacuolar compartment of the cell with tolerance depending on the ability of the cytosolic component to reduce selenite to elemental selenium, which might, in turn, be related to activity of the V-H(+)-ATPase . These results are discussed in relation to vacuolar compartmentation and the significance of the vacuolar H(+)-ATPase in cytosolic homeostasis of H+ both of which may affect the accumulation, reduction, and tolerance to the tested metal(loids).

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3752 - 7
Parallel analysis of genetic selections using whole genome oligonucleotide arrays; Cho RJ et al.; Thousands of genes have recently been sequenced in organisms ranging from Escherichia coli to human . For the majority of these genes, however, available sequence does not define a biological role . Efficient functional characterization of these genes requires strategies for scaling genetic analyses to the whole genome level . Plasmid-based library selections are an established approach to the functional analysis of uncharacterized genes and can help elucidate biological function by identifying, for example, physical interactors for a gene and genetic enhancers and suppressors of mutant phenotypes . The application of these selections to every gene in a eukaryotic genome, however, is generally limited by the need to manipulate and sequence hundreds of DNA plasmids . We present an alternative approach in which identification of nucleic acids is accomplished by direct hybridization to high-density oligonucleotide arrays . Based on the complete sequence of Saccharomyces cerevisiae, high-density arrays containing oligonucleotides complementary to every gene in the yeast genome have been designed and synthesized . Two-hybrid protein-protein interaction screens were carried out for S . cerevisiae genes implicated in mRNA splicing and microtubule assembly . Hybridization of labeled DNA derived from positive clones is sufficient to characterize the results of a screen in a single experiment, allowing rapid determination of both established and previously unknown biological interactions . These results demonstrate the use of oligonucleotide arrays for the analysis of two-hybrid screens . This approach should be generally applicable to the analysis of a range of genetic selections.

Exp Physiol, 1998 Mar, 83(2), 259 - 65
Mitochondrial carrier proteins can reversibly change their transport mode: the cases of the aspartate/glutamate and the phosphate carrier; Kramer R; A number of mitochondrial carrier systems function both in homologous and in heterologous exchange mode, which, in the case of the phosphate carrier is a homologous Pi(-)-Pi- and a heterologous Pi(-)-OH- exchange . In addition, we showed that mitochondrial carriers, e.g . the aspartate/glutamate and the phosphate carrier, can undergo a functional shift from coupled antiport to uncoupled uniport after modification of cysteine residues . In this transport mode a mixture of carrier- and channel-type properties is observed . To address this question on the molecular level, the phosphate carrier from yeast (S . cerevisiae) mitochondria was expressed in E . coli, solubilized, purified and functionally reconstituted . From three cysteine residues present in the yeast phosphate carrier at positions 28, 134 and 300, only one single cysteine residue (C28) was found responsible for the functional switch from antiport to uniport . Upon replacement by a serine residue, this interconversion was blocked . After incorporation of the carrier into giant liposomes, electrophysiological methods (patch clamp) were applied . Under these conditions, a fourth transport mode of the phosphate carrier was observed, namely an action as anion-selective channel, which could be reversibly blocked by phosphate.

Mol Cell Biol, 1998 May, 18(5), 2559 - 70
Association of transcription factor IIA with TATA binding protein is required for transcriptional activation of a subset of promoters and cell cycle progression in Saccharomyces cerevisiae; Ozer J et al.; The general transcription factor IIA (TFIIA) interacts with the TATA binding protein (TBP) and promoter DNA to mediate transcription activation in vitro . To determine if this interaction is generally required for activation of all class II genes in vivo, we have constructed substitution mutations in yeast TFIIA which compromise its ability to bind TBP . Substitution mutations in the small subunit of TFIIA (Toa2) at residue Y69 or W76 significantly impaired the ability of TFIIA to stimulate TBP-promoter binding in vitro . Gene replacement of wild-type TOA2 with a W76E or Y69A/W76A mutant was lethal in Saccharomyces cerevisiae, while the Y69F/W76F mutant exhibited extremely slow growth at 30 degrees C . Both the Y69A and W76A mutants were conditionally lethal at higher temperatures . Light microscopy indicated that viable toa2 mutant strains accumulate as equal-size dumbbells and multibudded clumps . Transcription of the cell cycle-regulatory genes CLB1, CLB2, CLN1, and CTS1 was significantly reduced in the toa2 mutant strains, while the noncycling genes PMA1 and ENO2 were only modestly affected, suggesting that these toa2 mutant alleles disrupt cell cycle progression . The differential effect of these toa2 mutants on gene transcription was examined for a number of other genes . toa2 mutant strains supported high levels of CUP1, PHO5, TRP3, and GAL1 gene activation, but the constitutive expression of DED1 was significantly reduced . Activator-induced start site expression for HIS3, GAL80, URA1, and URA3 promoters was defective in toa2 mutant strains, suggesting that the TFIIA-TBP complex is important for promoters which require an activator-dependent start site selection from constitutive to regulated expression . We present evidence to indicate that transcription defects in toa2 mutants can be both activator and promoter dependent . These results suggest that the association of TFIIA with TBP regulates activator-induced start site selection and cell cycle progression in S . cerevisiae.

Mol Biochem Parasitol, 1998 Mar 15, 91(2), 281 - 93
Stage-specific expression of the 14-3-3 gene in Echinococcus multilocularis; Siles-Lucas M et al.; A cDNA expression library representing the metacestode developmental stage of the tapeworm Echinococcus multilocularis was immunoscreened with monospecific antibodies affinity purified following differential immunoblot analysis . Using this procedure, a metacestode-specific clone was isolated representing a 14-3-3 gene of the parasite, which is present as a single copy in the parasite genome . The identity of this clone was demonstrated by cross-reactivity of the recombinant E . multilocularis 14-3-3 protein with antibodies raised against a heterologous 14-3-3 protein from Saccharomyces cerevisiae . In addition, expression of the E . multilocularis 14-3-3 gene in the mutant S . cerevisiae strain, DS9-22, resulted in complementation of the phenotypic deficiency of this strain, thus demonstrating the functionality of the respective gene product . By reverse transcription-polymerase chain reaction (RT-PCR) we showed that the E . multilocularis 14-3-3 protein is about 10-fold overexpressed in the metacestode stage compared with the expression level in the adult stage . Immunolocalization of the 14-3-3 protein in E . multilocularis metacestodes revealed its predominant presence in the germinal layer of the parasite . The results of this study, taken together with the current knowledge on the 14-3-3 protein family, suggest that this parasite molecule may contribute to the promotion of the progressive, potentially unlimited growth behaviour of the E . multilocularis metacestode within the host tissue.

Int J Syst Bacteriol, 1998 Jan, 48 Pt 1, 295 - 303
Phylogenetic analysis of the Saccharomyces cerevisiae group based on polymorphisms of rDNA spacer sequences; Montrocher R et al.; The phylogenetic relationships between species of yeasts assigned to the Saccharomyces sensu stricto group, which includes Saccharomyces cerevisiae and Saccharomyces bayanus, were studied together with Saccharomyces pastorianus and Saccharomyces paradoxus . The experimental approaches used were RFLP analysis of the PCR-amplified rDNA internal transcribed spacer (ITS) and intergenic spacer, and total ITS sequence analysis . Both RFLP and sequence analyses gave fairly similar results . The gene trees generated with either of the two data sets showed the distribution of the yeasts into two major, well-separated, phylogenetic clusters called 'cerevisiae' and 'bayanus' . The 'cerevisiae' cluster included the S . cerevisiae type strain, together with most of the species (16 out of 23), whereas the 'bayanus' cluster included the remaining seven type strains . Therefore, analysis of rDNA sequences confirmed S . cerevisiae and S . bayanus as two well-defined taxa . However, S . pastorianus and S . paradoxus, the two other usually accepted taxa of the now-defined Saccharomyces sensu stricto complex, could not be clearly separated from S . bayanus and S . cerevisiae, respectively . However, in both PCR-RFLP and ITS sequence analyses, S . paradoxus had the outermost position in the 'cerevisiae' cluster . PCR-RFLP analysis of the ribosomal spacer sequences was also carried out on 26 Saccharomyces strains isolated in various wine-growing regions of France in an attempt to clarify their positions in the Saccharomyces phylogenetic tree . Compared to the diversity of the Saccharomyces type strains, less genetic diversity was detected among these yeasts and several of them exhibited identical RFLP patterns . Most of the wine yeast strains (16 out of 26) were closely related to each other and were found within the 'cerevisiae' cluster . The remaining 10 wine yeast strains branched within the 'bayanus' cluster . PCR-RFLP analysis of ribosomal spacer sequences thus appears to be a useful and appropriate method for the correct characterization of Saccharomyces yeast strains used in food processing.

Genetics, 1998 Apr, 148(4), 1715 - 29
Identification and characterization of an essential family of inositol polyphosphate 5-phosphatases (INP51, INP52 and INP53 gene products) in the yeast Saccharomyces cerevisiae; Stolz LE et al.; We recently demonstrated that the S . cerevisiae INP51 locus (YIL002c) encodes an inositol polyphosphate 5-phosphatase . Here we describe two related yeast loci, INP52 (YNL106c) and INP53 (YOR109w) . Like Inp51p, the primary structures of Inp52p and Inp53p resemble the mammalian synaptic vesicle-associated protein, synaptojanin, and contain a carboxy-terminal catalytic domain and an amino-terminal SAC1-like segment . Inp51p (108 kD), Inp52p (136 kD) and Inp53p (124 kD) are membrane-associated . Single null mutants (inp51, inp52, or inp53) are viable . Both inp51 inp52 and inp52 inp53 double mutants display compromised cell growth, whereas an inp51 inp53 double mutant does not . An inp51 inp52 inp53 triple mutant is inviable on standard medium, but can grow weakly on media supplemented with an osmotic stabilizer (1 M sorbitol) . An inp51 mutation, and to a lesser degree an inp52 mutation, confers cold-resistant growth in a strain background that cannot grow at temperatures below 15 degrees . Analysis of inositol metabolites in vivo showed measurable accumulation of phosphatidylinositol 4,5-bisphosphate in the inp51 mutant . Electron microscopy revealed plasma membrane invaginations and cell wall thickening in double mutants and the triple mutant grown in sorbitol-containing medium . A fluorescent dye that detects endocytic and vacuolar membranes suggests that the vacuole is highly fragmented in inp51 inp52 double mutants . Our observations indicate that Inp51p, Inp52p, and Inp53p have distinct functions and that substrates and/or products of inositol polyphosphate 5-phosphatases may have roles in vesicle trafficking, membrane structure, and/or cell wall formation.

Yeast, 1998 Mar 30, 14(5), 431 - 42
Application of mRNA differential display to investigate gene expression in thermotolerant cells of Saccharomyces cerevisiae; Gross C et al.; We have described the use of differential display of PCR-amplified reverse transcribed mRNA (DDRT-PCR) to survey changes in gene expression profiles induced by heat shock and carbon catabolite derepression in Saccharomyces cerevisiae . It is well established that either of these states elicits thermotolerant phenotypes . An initial analysis conducted on cells of an inherently thermosensitive strain (Ysen) indicated that approximately 10% of the total number of cDNAs detected were either up or down regulated following heat shock at 37 degrees C (30 min) in comparison to control cells (25 degrees C) . In addition, whereas 7% of all PCR products were preferentially expressed during derepressive growth, approximately 2% were found to be common to both heat-shocked and derepressed cells . A repeat analysis, performed on all three cell types of Ysen as well as cells of a relatively thermoresistant strain (Yres) yielded 30 differentially displayed cDNA fragments common to heat-shocked and derepressed cells of both strains . Eighteen of these generated signals on Northern blots, of which three were confirmed as regulated . Five amplicons, including one not detected by Northern analysis and another from the derepressed state, were cloned and sequenced . Three of these exhibited homology to S . cerevisiae genes with well-characterized protein products: HSP 90, HXK1 and STA1 . The remaining two applicons showed nucleotide identity to YTIS11, a homolog of the mammalian TIS11 and putative transcriptional activator, and an orphan gene encoding a hypothetical transmembrane protein belonging to the multi-drug resistance translocase family . Our novel application of DDRT-PCR has identified new and known genes that may be further evaluated as factors involved in stress regulation and has demonstrated the potential of the technique to systematically analyse gene expression in yeast.

Yeast, 1998 Mar 15, 14(4), 323 - 34
Assembly of phosphofructokinase-1 from Saccharomyces cerevisiae in extracts of single-deletion mutants; Klinder A et al.; Phosphofructokinase-1 from Saccharomyces cerevisiae is an octameric enzyme comprising two non-identical subunits, alpha and beta, which are encoded by the unlinked genes PFK1 and PFK2 . In this paper, assembly and reactivation of the enzyme have been studied in cell-free extracts of single-deletion mutants . In contrast to the previously described lack of phosphofructokinase-1 activity in cell-free extracts of these mutants, we could measure a temporary enzyme activity immediately after lysis of protoplasts . This result supports the assumption that each of the subunits forms an enzyme structure which is active in vivo but not stable after cell disruption . Upon mixing of separately prepared cell-free extracts of both deletion mutants very low activity could be measured . About 40% of the wild-type activity was regained when both mutants were mixed prior to disruption . The reactivation rate could be slightly increased by addition of ATP and fructose 6-phosphate and was found to be a function of the growth state, particularly of the beta-subunit-carrying cells . The individual subunits did not interact with Cibacron Blue F3G-A, a biomimetic ligand of phosphofructokinase-1 . After reassembly of both subunits in vitro a strong affinity of the reconstituted phosphofructokinase-1 to the dye-ligand was observed . The inability of the subunits to reconstitute under certain conditions seems to result from alterations of the intracellular environment following disruption . These changes give rise to induce an unproductive side reaction like self-aggregation of the subunits . Because reconstitution of phosphofructokinase-1 from S . cerevisiae behaves in a similar way to that of hemoglobin and luciferase, we would speculate a general mechanism for assembly of oligomeric proteins in vivo.

Antimicrob Agents Chemother, 1998 Apr, 42(4), 889 - 94
Use of a rapid throughput in vivo screen to investigate inhibitors of eukaryotic topoisomerase II enzymes; Hammonds TR et al.; Topoisomerase II catalyzes the passage of one DNA helix through another via a transient double-stranded break . The essential nature of this enzyme in cell proliferation and its mechanism of action make it an ideal target for cytotoxic agents . Saccharomyces cerevisiae topoisomerase II has been frequently used as a model for testing potential inhibitors of eukaryotic topoisomerase II as antitumor agents . The standard in vivo method of estimating the sensitivity of S . cerevisiae to the antitopoisomerase drugs is via inhibition or kill curves which rely on viable-cell counts and is labor intensive . We present an alternative to this, a high-throughput in vivo screen . This method makes use of a drug-permeable S . cerevisiae strain lacking endogenous topoisomerase II, which is modified to express either human topoisomerase IIalpha or IIbeta or S . cerevisiae topoisomerase II carried on plasmids . Each modified strain expresses a full-length topoisomerase II enzyme, as opposed to the more commonly used temperature-sensitive S . cerevisiae mutant expressing yeast or yeast/human hybrid enzymes . A comparison of this new method with a plating-and-counting method gave similar drug sensitivity results, with increased accuracy and reduced manual input for the new method . The information generated has highlighted the sensitivities of different topoisomerase II enzymes and isoenzymes to several different classes of topoisomerase II inhibitor.

Prog Cell Cycle Res, 1996, 2, 197 - 204
The cyclin C/Cdk8 kinase; Leclerc V et al.; Cyclin C was originally identified in a genetic screen for metazoan cDNAs that complement a triple knock-out of the CLN genes, involved in G1/S progression in S . cerevisiae . Unlike cyclin Ds and cyclin E, also identified in this screen, cyclin C has not been found to have a cell-cycle role in metazoa . Identified as the catalytic partner of cyclin C, Cdk8 is a novel protein-kinase of the Cdk family structurally related to the yeast Srb10 kinase . Cyclin C, Cdk8 and RNA polymerase II are found in a large multi-protein complex that shows structural as well as functional homologies with the yeast polymerase II holoenzyme . These observations and the sequence similarity to the kinase/cyclin pair Srb10/Srb11 in S . cerevisiae, suggest that cyclin C and Cdk8 control RNA polymerase II function.

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