Drug Chem Toxicol, 2003 May, 26(2), 75 - 85 Toxicity of metal ions used in dental alloys: a study in the yeast Saccharomyces cerevisiae; Yang HC et al.; Metal ions are released from dental alloys into the oral environment, which can cause biological responses over short and extended periods . Since most toxic metal ions are capable of inducing oxidative stress on cells through the mitochondrial respiratory chain, mitochondria may contribute to and be a target of metal toxicity . In this study, we investigated the effect of metal ions on growth of the budding yeast, Saccharomyces cerevisiae, and on the morphology and function of yeast mitochondria . Moreover, we tested whether mitochondrial respiratory activity contributes to metal toxicity . Metal ions affected yeast cell growth . The toxicity of metal ions to yeast cells, ranked in decreasing order are as follows: Hg > Ag > Au > Cu, Ni, Co, Zn . This result mostly correlates with the degree of toxicity of those metal ions to growth of human cells . The MIC90 of Hg, Ag and Au ions in synthetic complete media are 0.325, 5 and 320 microM, respectively . None of the toxic metal ions resulted in loss of mitochondrial respiratory activity . However, respiration-deficient rho0 cells appeared to be resistant to Ag ion, but not to Hg and Au ions . Furthermore, at high concentrations, Ag ion caused morphological changes in mitochondria . These studies indicate that yeast may be used as a model system to screen for toxic effect of metals ions from dental alloys, and that oxidation activity in mitochondria may play a role in acute toxicity of silver ion. Cell Mol Biol Lett, 2003, 8(2), 261 - 8 The genetic characteristics Saccharomyces cerevisiae aci(+) mutants; Grochowalska R et al.; A series of 30 Saccharomyces cerevisiae aci(+) mutants (characterized as acidifying Ogur's glucose medium containing bromocresol purple) were isolated after EMS mutagenesis . All the mutants excreted acid metabolites to the medium after 24 or 48 hours of incubation . The character of the aci(+) mutations was defined using classical genetic techniques . Three of the aci(+) mutants were studied by molecular genetics techniques. Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7460 - 4 Epub 2003 Jun 16. Determination of the membrane topology of Ost4p and its subunit interactions in the oligosaccharyltransferase complex in Saccharomyces cerevisiae; Kim H et al.; Ost4p is a minimembrane protein containing only 36 amino acids and is a subunit of oligosaccharyltransferase (OT) in Saccharomyces cerevisiae . It was found previously when amino acid residues 18-25 of Ost4p were mutated to ionizable amino acids and defects were observed in the interaction between Ost4p and either Stt3p or Ost3p, two other components of OT . The transmembrane segment of Ost4p is likely to extend from residues 10-25 . This is consistent with the finding that alpha-helicity is estimated to be 36% by CD analysis of synthetic Ost4p in liposomes . This value is in reasonable agreement with the assumption that amino acids 10-25 (16 of 36 or 44%) are transmembrane . Therefore, the mutation-sensitive region (residues 18-25) is localized to only one half of the putative transmembrane domain of Ost4p . To learn where this region of Ost4p is situated in relation to the faces of endoplasmic reticulum (ER) membrane, we determined the membrane topology of Ost4p using an in vivo method and established that it is an Nlumen-Ccyto, type I membrane protein . These results indicate that the mutation-sensitive region of Ost4p is localized in the cytoplasmic leaflet of the ER membrane . In the current study, we also observed a loss of direct interaction between Ost3p and Stt3p in the presence of ost4 temperature-sensitive mutants, which indicates Ost4p, via interactions with amino acid residues in the cytosolic leaflet of the ER membrane, functions to bind these two proteins together in a subcomplex of OT. RNA, 2003 Jul, 9(7), 871 - 80 RNA-binding activity of translation initiation factor eIF4G1 from Saccharomyces cerevisiae; Berset C et al.; We identified and mapped RNA-binding sites of yeast Saccharomyces cerevisiae translation initiation factor eIF4G1 and examined their importance for eIF4G1 function in vitro and in vivo . Yeast eIF4G1 binds to single-stranded RNA with three different sites, the regions of amino acids 1-82 (N terminus), 492-539 (middle), and 883-952 (C terminus) . The middle and C-terminal RNA-binding sites represent RS (arginine and serine)-rich domains; the N-terminal site is asparagine-, glutamine- and glycine-rich . The three RNA-binding sites have similar affinity for single-stranded RNA, whereas the affinity for single-stranded RNA full-length eIF4G1 is about 100-fold higher (approximate K(d) of 5 x 10(-8) M) . Replacement of the arginine residues in the middle RS site by alanine residues abolishes its RNA-binding activity . Deletion of individual RNA-binding sites shows that eIF4G1 molecules lacking one binding site are still active in supporting growth of yeast cells and translation in vitro, whereas eIF4G1 molecules lacking two or all three RNA-binding sites are strongly impaired or inactive . These data suggest that RNA-binding activity is required for eIF4G1 function. Mol Biol Cell, 2003 Jun, 14(6), 2237 - 49 Epub 2003 Feb 06. She4p/Dim1p interacts with the motor domain of unconventional myosins in the budding yeast, Saccharomyces cerevisiae; Toi H et al.; She4p/Dim1p, a member of the UNC-45/CRO1/She4p (UCS) domain-containing protein family, is required for endocytosis, polarization of actin cytoskeleton, and polarization of ASH1 mRNA in Saccharomyces cerevisiae . We show herein that She4p/Dim1p is involved in endocytosis and actin polarization through interactions with the type I myosins Myo3p and Myo5p . Two-hybrid and biochemical experiments showed that She4p/Dim1p interacts with the motor domain of Myo3/5p through its UCS domain . She4p/Dim1p was required for Myo5p localization to cortical patch-like structures . Using random mutagenesis of the motor region of MYO5, we identified four independent dominant point mutations that suppress the temperature-sensitive growth phenotype of the she4/dim1 null mutant . All of the amino acid substitutions caused by these mutations, V164I, N168I, N209S, and K377M, could suppress the defects of endocytosis and actin polarization of the she4/dim1 mutant as well . She4p/Dim1p also showed two-hybrid interactions with the motor domain of a type II myosin Myo1p and type V myosins Myo2p and Myo4p, and was required for proper localization of Myo4p, which regulates polarization of ASH1 mRNA . Our results suggest that She4p/Dim1p is required for structural integrity or regulation of the motor domain of unconventional myosins. Mol Biol Cell, 2003 Jun, 14(6), 2226 - 36 Epub 2003 Mar 07. Pex15p of Saccharomyces cerevisiae provides a molecular basis for recruitment of the AAA peroxin Pex6p to peroxisomal membranes; Birschmann I et al.; The gene products (peroxins) of at least 29 PEX genes are known to be necessary for peroxisome biogenesis but for most of them their precise function remains to be established . Here we show that Pex15p, an integral peroxisomal membrane protein, in vivo and in vitro binds the AAA peroxin Pex6p . This interaction functionally interconnects these two hitherto unrelated peroxins . Pex15p provides the mechanistic basis for the reversible targeting of Pex6p to peroxisomal membranes . We could demonstrate that the N-terminal part of Pex6p contains the binding site for Pex15p and that the two AAA cassettes D1 and D2 of Pex6p have opposite effects on this interaction . A point mutation in the Walker A motif of D1 (K489A) decreased the binding of Pex6p to Pex15p indicating that the interaction of Pex6p with Pex15p required binding of ATP . Mutations in Walker A (K778A) and B (D831Q) motifs of D2 abolished growth on oleate and led to a considerable larger fraction of peroxisome bound Pex6p . The nature of these mutations suggested that ATP-hydrolysis is required to disconnect Pex6p from Pex15p . On the basis of these results, we propose that Pex6p exerts at least part of its function by an ATP-dependent cycle of recruitment and release to and from Pex15p. Genetics, 2003 Jun, 164(2), 443 - 55 Delineating the requirements for spontaneous DNA damage resistance pathways in genome maintenance and viability in Saccharomyces cerevisiae; Morey NJ et al.; Cellular metabolic processes constantly generate reactive species that damage DNA . To counteract this relentless assault, cells have developed multiple pathways to resist damage . The base excision repair (BER) and nucleotide excision repair (NER) pathways remove damage whereas the recombination (REC) and postreplication repair (PRR) pathways bypass the damage, allowing deferred removal . Genetic studies in yeast indicate that these pathways can process a common spontaneous lesion(s), with mutational inactivation of any pathway increasing the burden on the remaining pathways . In this study, we examine the consequences of simultaneously compromising three or more of these pathways . Although the presence of a functional BER pathway alone is able to support haploid growth, retention of the NER, REC, or PRR pathway alone is not, indicating that BER is the key damage resistance pathway in yeast and may be responsible for the removal of the majority of either spontaneous DNA damage or specifically those lesions that are potentially lethal . In the diploid state, functional BER, NER, or REC alone can support growth, while PRR alone is insufficient for growth . In diploids, the presence of PRR alone may confer a lethal mutation load or, alternatively, PRR alone may be insufficient to deal with potentially lethal, replication-blocking lesions. J Med Chem, 2003 Jun 19, 46(13), 2631 - 40 Discovery and structural modification of inhibitors of methionine aminopeptidases from Escherichia coli and Saccharomyces cerevisiae; Luo QL et al.; A series of pyridine-2-carboxylic acid derivatives were synthesized according to the leads from the screening, and potent inhibitors have been obtained by structural modification . They have shown submicromolar inhibition of the enzymes (for example, for 9n, IC(50) = 130 nM for EcMetAP1 and IC(50) = 380 nM for ScMetAP1) . They represent small-molecule MetAP inhibitors with novel structures different from alkylating fumagillin derivatives and peptidic bestatin-based MetAP inhibitor. Folia Microbiol (Praha), 2003, 48(2), 193 - 8 Trehalose, glycogen and ethanol metabolism in the gcr1 mutant of Saccharomyces cerevisiae; Seker T et al.; Since Gcr1p is pivotal in controlling the transcription of glycolytic enzymes and trehalose metabolism seems to be one of the control points of glycolysis, we examined trehalose and glycogen synthesis in response to 2% glucose pulse during batch growth in gcr1 (glucose regulation-1) mutant lacking fully functional glycolytic pathway and in the wild-type strain . An increase in both trehalose and glycogen stores was observed 1 and 2 h after the pulse followed by a steady decrease in both the wild-type and the gcr1 mutant . The accumulation was faster while the following degradation was slower in gcr1 cells compared to wild-type ones . Although there was no distinct glucose consumption in the mutant cells it seemed that the glucose repression mechanism is similar in gcr1 mutant and in wild-type strain at least with respect to trehalose and glycogen metabolism. Genome Res, 2003 Jun, 13(6A), 1146 - 54 Multimeric threading-based prediction of protein-protein interactions on a genomic scale: application to the Saccharomyces cerevisiae proteome; Lu L et al.; MULTIPROSPECTOR, a multimeric threading algorithm for the prediction of protein-protein interactions, is applied to the genome of Saccharomyces cerevisiae . Each possible pairwise interaction among more than 6000 encoded proteins is evaluated against a dimer database of 768 complex structures by using a confidence estimate of the fold assignment and the magnitude of the statistical interfacial potentials . In total, 7321 interactions between pairs of different proteins are predicted, based on 304 complex structures . Quality estimation based on the coincidence of subcellular localizations and biological functions of the predicted interactors shows that our approach ranks third when compared with all other large-scale methods . Unlike other in silico methods, MULTIPROSPECTOR is able to identify the residues that participate directly in the interaction . Three hundred seventy-four of our predictions can be found by at least one of the other studies, which is compatible with the overlap between two different other methods . From the analysis of the mRNA abundance data, our method does not bias towards proteins with high abundance . Finally, several relevant predictions involved in various functions are presented . In summary, we provide a novel approach to predict protein-protein interactions on a genomic scale that is a useful complement to experimental methods. Exp Cell Res, 2003 Jul 1, 287(1), 10 - 5 Generation and maintenance of synchrony in Saccharomyces cerevisiae continuous culture; Murray DB et al.; Cultures of Saccharomyces cerevisiae grown continuously produce an autonomous oscillation in many metabolic outputs . The most conveniently measured variable, i.e., dissolved oxygen concentration, oscillates with a period of 40-55 min . Previously we have identified two compounds capable of resetting phase, acetaldehyde and hydrogen sulfide . The phase-response curves constructed for acetaldehyde show a strong (Type 0) response at 3.0 mM and a weak (Type 1) response at 1.0 mM . Ammonium sulfide phase-response curves (pulse injected at 1.0 microM and 3.0 microM) revealed that sulfide is only an effective perturbation agent when endogenous sulfide concentrations are at a maximum . Also only Type 1 phase responses were observed . When the phase-response curve for sulfite (at 3.0 M) was constructed, phase responses were at a maximum at 60 degrees, indicating the possible involvement of sulfite in cell synchronization . It is concluded that endogenously produced acetaldehyde and sulfite tune the oscillation of mitochondrial energization state whereas sulfide mediates population synchrony. Eukaryot Cell, 2003 Jun, 2(3), 651 - 3 Evidence that synthesis of the Saccharomyces cerevisiae mitochondrially encoded ribosomal protein Var1p may be membrane localized; Fiori A et al.; The 5'-untranslated leaders of mitochondrial mRNAs appear to localize translation within the organelle . VAR1 is the only yeast mitochondrial gene encoding a major soluble protein . A chimeric mRNA bearing the VAR1 untranslated regions and the coding sequence for pre-Cox2p appears to be translated at the inner membrane surface . We propose that translation of the ribosomal protein Var1p is also likely to occur in close proximity to the inner membrane. Eukaryot Cell, 2003 Jun, 2(3), 599 - 608 Identification of in vivo enzyme activities in the cometabolism of glucose and acetate by Saccharomyces cerevisiae by using 13C-labeled substrates; dos Santos MM et al.; A detailed characterization of the central metabolic network of Saccharomyces cerevisiae CEN.PK 113-7D was carried out during cometabolism of different mixtures of glucose and acetate, using aerobic C-limited chemostats in which one of these two substrates was labeled with (13)C . To confirm the role of malic enzyme, an isogenic strain with the corresponding gene deleted was grown under the same conditions . The labeling patterns of proteinogenic amino acids were analyzed and used to estimate metabolic fluxes and/or make inferences about the in vivo activities of enzymes of the central carbon metabolism and amino acid biosynthesis . Malic enzyme flux increased linearly with increasing acetate fraction . During growth on a very-high-acetate fraction, the activity of malic enzyme satisfied the biosynthetic needs of pyruvate in the mitochondria, while in the cytosol pyruvate was supplied via pyruvate kinase . In several cases enzyme activities were unexpectedly detected, e.g., the glyoxylate shunt for a very-low-acetate fraction, phosphoenolpyruvate carboxykinase for an acetate fraction of 0.46 C-mol of acetate/C-mol of substrate, and glucose catabolism to CO(2) via the tricarboxylic acid cycle for a very-high-acetate fraction . Cytoplasmic alanine aminotransferase activity was detected, and evidence was found that alpha-isopropylmalate synthase has two active forms in vivo, one mitochondrial and the other a short cytoplasmic form. Eukaryot Cell, 2003 Jun, 2(3), 552 - 9 Rapamycin treatment results in GATA factor-independent hyperphosphorylation of the proline utilization pathway activator in Saccharomyces cerevisiae; Saxena D et al.; Treatment of Saccharomyces cerevisiae cells with the immunosuppressive drug rapamycin results in a variety of cellular changes in response to perceived nutrient deprivation . Among other effects, rapamycin treatment results in the nuclear localization of the global nitrogen activators Gln3p and Nil1p/Gat1p, which leads to expression of nitrogen assimilation genes . The proline utilization (Put) pathway genes were shown to be among the genes induced by rapamycin . Having previously shown that the Put pathway activator Put3p is differentially phosphorylated in response to the quality of the nitrogen source, we examined the phosphorylation status of Put3p after rapamycin treatment . Treatment with rapamycin resulted in the hyperphosphorylation of Put3p, which was independent of Gln3p, Nil1p, and Ure2p . The relative contributions of global nitrogen (Gln3p and Nil1p) and pathway-specific (Put3p) activators to rapamycin-induced expression of the target gene PUT1 were also examined . We found that Nil1p and Put3p, but not Gln3p, play major roles in rapamycin-induced PUT1 expression . Our findings show that perceived nitrogen deprivation triggered by rapamycin treatment and steady-state growth in nitrogen-derepressing conditions are associated with hyperphosphorylation of Put3p and increased PUT1 expression . Rapamycin treatment and nitrogen derepression may share some, but not all, regulatory elements, since Gln3p and Nil1p do not participate identically in both processes and are not required for hyperphosphorylation . A complex relationship exists among the global and pathway-specific regulators, depending on the nature and quality of the nitrogen source. Eukaryot Cell, 2003 Jun, 2(3), 534 - 41 A Saccharomyces cerevisiae mutant unable to convert glucose to glucose-6-phosphate accumulates excessive glucose in the endoplasmic reticulum due to core oligosaccharide trimming; Miseta A et al.; D-Glucose is the preferred carbon and energy source for most eukaryotic cells . Immediately following its uptake, glucose is rapidly phosphorylated to glucose-6-phosphate (Glc-6-P) . The yeast Saccharomyces cerevisiae has three enzymes (Hxk1p, Hxk2p, and Glk1p) that convert glucose to Glc-6-P . In the present study, we found that yeast mutants lacking any two of these enzymes retain the ability to efficiently convert glucose to Glc-6-P and thus maintain a low level of cellular glucose . However, a mutant strain lacking all three glucose-phosphorylating enzymes contained up to 225-fold more intracellular glucose than normal . Drugs that inhibit the synthesis or the trimming of the lipid-linked core oligosaccharide Glu(3)Man(9)GlcNac(2) effectively reduced the accumulation of glucose . Similarly, mutations that block the addition of glucose residues to the core oligosaccharide moiety, such as alg5Delta or alg6Delta, also diminished glucose accumulation . These results indicate that the intracellular glucose accumulation observed in the glucose phosphorylation mutant results primarily from the trimming of glucose residues from core oligosaccharide chains within the endoplasmic reticulum (ER) . Consistent with this conclusion, both {(14)C}glucose exchange and subcellular fractionation experiments indicate that much of the accumulated glucose is retained within an intracellular compartment, suggesting that the efficient transport of glucose from the ER to the cytosol in yeast may be coupled to its rephosphorylation to Glc-6-P . The high level of cellular glucose was associated with an increased level of protein glycation and the release of glucose into the culture medium via its transit through the secretory pathway . Finally, we also found that the accumulation of glucose may lead to a subtle alteration in ion homeostasis, particularly Ca(2+) uptake . This suggests that this mutant strain may serve as a useful model to study the consequences of excessive glucose accumulation and protein glycation. Eukaryot Cell, 2003 Jun, 2(3), 431 - 45 Ady4p and Spo74p are components of the meiotic spindle pole body that promote growth of the prospore membrane in Saccharomyces cerevisiae; Nickas ME et al.; Spore formation in Saccharomyces cerevisiae occurs via the de novo synthesis of the prospore membrane during the second meiotic division . Prospore membrane formation is triggered by assembly of a membrane-organizing center, the meiotic outer plaque (MOP), on the cytoplasmic face of the spindle pole body (SPB) during meiosis . We report here the identification of two new components of the MOP, Ady4p and Spo74p . Ady4p and Spo74p interact with known proteins of the MOP and are localized to the outer plaque of the SPB during meiosis II . MOP assembly and prospore membrane formation are abolished in spo74Delta/spo74Delta cells and occur aberrantly in ady4Delta/ady4Delta cells . Spo74p and the MOP component Mpc70p are mutually dependent for recruitment to SPBs during meiosis . In contrast, both Ady4p and Spo74p are present at SPBs, albeit at reduced levels, in cells that lack the MOP component Mpc54p . Our findings suggest a model for the assembled MOP in which Mpc54p, Mpc70p, and Spo74p make up a core structural unit of the scaffold that initiates synthesis of the prospore membrane, and Ady4p is an auxiliary component that stabilizes the plaque. Yeast, 2003 Jun, 20(8), 739 - 46 Construction of a set of Saccharomyces cerevisiae vectors designed for recombinational cloning; Van Mullem V et al.; The Gateway technology is becoming an increasingly popular method for cloning ORFs by recombination . It allows the transfer of any ORF flanked by specific recombination sites into any vectors harbouring the corresponding sites . Here we describe the construction of a set of 20 Saccharomyces cerevisiae Gateway compatible vectors . These plasmids bear an URA3 or TRP1 selection marker . They are designed for expression without tag sequence or for C- or N-terminal protein tagging with 3HA (haemagglutinin), 13MYC, 4TAP (tandem affinity purification) or GST (glutathione S-transferase) epitopes . The centromeric vectors allow expression of DNA sequence in yeast under tetracycline-regulatable promoters, while expression from the high copy vectors is driven by PGK promoter . To test their applicability, the genes encoding the RNA polymerase I subunit Rpa12p or the TFIIS transcription factor were cloned in these vectors . Their expression was demonstrated using Western blotting or complementation assays . J Med Virol, 2003 Aug, 70(4), 513 - 9 Saccharomyces cerevisiae-derived HBsAg preparations differ in their attachment to monocytes, immune-suppressive potential, and T-cell immunogenicity; Vanlandschoot P et al.; Expression of the hepatitis B virus S protein results in the formation of a lipoprotein particle, the hepatitis B surface antigen (HBsAg) . Such particles, produced in Saccharomyces cerevisiae, bind to the cell surface of monocytes through interaction with the lipopolysaccharide binding protein and the lipopolysaccharide receptor, CD14 . This attachment is suggested to depend on the presence of charged phospholipids in the particles . In addition, such particles interfere with the lipopolysaccharide and interleukin-2-induced activation of monocytes . In the present study, it is reported that of three Saccharomyces cerevisiae-derived HBsAg preparations, two have a reduced capacity to bind to monocytes . A correlation with a reduced potential to inhibit the lipopolysaccharide-induced activation of monocytes and an increased potential to stimulate HBsAg-specific T-cell proliferation is observed . Surprisingly, differences in phospholipid content that might explain these observations, were not detected . Int J Oncol, 2003 Jul, 23(1), 229 - 33 WD-40 repeat containing rat lethal giant larvae recessive oncogene, but not m-tomosyn, restores the salt sensitivity in Saccharomyces cerevisiae; Kim YS et al.; Recently, we cloned rgl-1, a putative rat homologue of the Drosophila tumor suppressor oncogene lethal (2) giant larvae (l(2)gl), from the rat brain . Sequence analysis revealed that rgl-1 encodes a 1,036 amino acid polypeptide containing a domain characteristic of WD-40 repeat sequence . It has been suggested that m-tomosyn is expressed in the rat brain, encodes a 1,116 amino acid polypeptide, contains WD-40 repeat sequence, and is homologous to the Drosophila l(2)gl and mouse mgl-1 genes . The comparison of amino acid sequence for m-tomosyn with l(2)gl and mgl-1 proteins using the clustal method showed only 17.8% and 20% amino acid identity, respectively . However, amino acid identity of rgl-1 protein to l(2)gl and mgl-1 proteins is much higher (30.6% and 96.8%, respectively) . Therefore, we investigated their biological function in the absence of Saccharomyces cerevisiae SOP1 and SOP2, the yeast homologues of the Drosophila l(2)gl . Functional analysis showed that the expression of the rgl-1 cDNA, but not m-tomosyn, restored partially salt sensitivity in yeast, indicating that rgl-1 is a homologue of lgl family members. J Biol Chem, 2003 Aug 29, 278(35), 32692 - 701 Epub 2003 Jun 04. Functional interactions between Hsp90 and the co-chaperones Cns1 and Cpr7 in Saccharomyces cerevisiae; Tesic M et al.; Hsp90 complexes contain a class of co-chaperones characterized by a tetratricopeptide repeat (TPR) domain, which mediates binding to a carboxyl-terminal EEVD region in Hsp90 . Among Hsp90 TPR co-chaperones in Saccharomyces cerevisiae, only Cns1 is essential . The amino terminus of Cns1, which harbors the TPR domain, is sufficient for viability when overexpressed . In a screen for temperature-sensitive alleles of CNS1, we identified mutations resulting in substitutions of conserved residues in the TPR domain . Mutations in CNS1 disrupt in vitro and in vivo interaction with Hsp90 and reduce Hsp90 function, indicating that Cns1 is a bona fide co-chaperone . Genetic interactions between CNS1 and another Hsp90 co-chaperone, CPR7, suggest that the two co-chaperones share an essential role in the cell . Although both the TPR and the isomerase domains of the cyclophilin Cpr7 are required for viability of cns1 mutant cells, this requirement does not depend on the catalytic function of the isomerase domain . Instead, hydrophilic residues on the surface of this domain appear to be important for the common Cns1.Cpr7 function . Although both co-chaperones interact with Hsp90 primarily through the carboxyl terminus (EEVD), Cns1 and Cpr7 are mostly found in complexes distinct from Hsp90 . EEVD is required for normal growth in cns1 mutant cells, demonstrating for the first time in vivo requirement for this conserved region of Hsp90 . Overall, our findings reveal a considerable degree of complexity in the interactions not only between Hsp90 and its co-chaperones, but also among the co-chaperones themselves. Biochem Biophys Res Commun, 2003 Jun 20, 306(1), 5 - 9 Genome-wide expression analysis of NAP1 in Saccharomyces cerevisiae; Ohkuni K et al.; Nap1 is a nucleosome assembly protein which is necessary to keep proper nucleosome structures in transcription and replication in vitro . In Saccharomyces cerevisiae, additional functions have been ascribed to Nap1, as it has been shown to interact with Clb2 (B type cyclin) and Gin4 (septum formation) . In this study, we investigate genome-wide expression in the Deltanap1 cells using DNA microarrays . About 10% of all yeast open reading frames changed the transcription level more than 2-fold in the Deltanap1 strain, compared with wild-type strain, in one experiment . Interestingly, these genes, whose expressions are up- or down-regulated in the Deltanap1 cells, are clustered . This result suggests that yeast NAP1 is required for the maintenance of cumulative nucleosome formation in vivo and the loss of Nap1 leads to a change in the gene expression level in a cluster. Mutat Res, 2003 Jun 19, 527(1-2), 37 - 48 Validation of a novel assay for checkpoint responses: characterization of camptothecin derivatives in Saccharomyces cerevisiae; Zhang H et al.; The evolutionary conservation of pathways preserving genetic stability supports the use of a lower eukaryote such as the yeast Saccharomyces cerevisiae in screening for novel anti-neoplastic agents . Yeast is already established as a model system to characterize the cellular effects of the topoisomerase inhibitor and anti-cancer agent camptothecin (CPT) . Here, we demonstrate that a recently developed two-hybrid based plate assay that visualizes the DNA damage-induced homomeric complex formation of the yeast checkpoint protein Rad17 correctly predicts the biological activity of the tested camptothecin derivatives . The used criteria for biological activity include lethality, cell cycle arrest and Rad53p phosphorylation, an essential signaling event during checkpoint activation . Surprisingly, although responsive to camptothecin and not without influence on drug sensitivity, Rad17p appears to be dispensable for cell cycle arrest and for Rad53p phosphorylation following treatment with camptothecin . Such a role is only uncovered if double-strand break repair is compromised. Biotechnol Bioeng, 2003 Aug 5, 83(3), 335 - 43 Multicriteria optimization of biochemical systems by linear programming: application to production of ethanol by Saccharomyces cerevisiae; Vera J et al.; In this study we present a method for simultaneous optimization of several metabolic responses of biochemical pathways . The method, based on the use of the power law formalism to obtain a linear system in logarithmic coordinates, is applied to ethanol production by Saccharomyces cerevisiae . Starting from an experimentally based kinetic model, we translated it to its power law equivalent . With this new model representation, we then applied the multiobjective optimization method . Our intent was to maximize ethanol production and minimize each of the internal metabolite concentrations . To ensure cell viability, all optimizations were carried out under imposed constraints . The different solutions obtained, which correspond to alternative patterns of enzyme overexpression, were implemented in the original model . We discovered few discrepancies between the S-system-optimized steady state and the corresponding optimized state in the original kinetic model, thus demonstrating the suitability of the S-system representation as the basis for the optimization procedure . In all optimized solutions, the ATP level reached its maximum and any increase in its activity positively affected the optimization process . This work illustrates that in any optimization study no single criteria is of general application being the multiobjective and constrained task the proper way to address it . It is concluded that the proposed multiobjective method can serve to carry out, in a single study, the general pattern of behavior of a given metabolic system with regard to its control and optimization . FEBS Lett, 2003 Jun 5, 544(1-3), 160 - 4 Saccharomyces cerevisiae 14-3-3 proteins Bmh1 and Bmh2 participate in the process of catabolite inactivation of maltose permease; Mayordomo I et al.; In this study we show that Reg1, the regulatory subunit of the Reg1/Glc7 protein phosphatase (PP1) complex, interacts physically with the two yeast members of the 14-3-3 protein family, Bmh1 and Bmh2 . By using different fragments of the Reg1 protein we mapped the interaction domain at the N-terminal part of the protein . We also show that Reg1 and yeast 14-3-3 proteins participate actively in the regulation of the glucose-induced degradation of maltose permease (Mal61). FEBS Lett, 2003 Jun 5, 544(1-3), 148 - 52 Mitochondrial and cytosolic expression of human peroxiredoxin 5 in Saccharomyces cerevisiae protect yeast cells from oxidative stress induced by paraquat; Tien Nguyen-nhu N et al.; Human peroxiredoxin 5 is a recently discovered mitochondrial, peroxisomal and cytosolic thioredoxin peroxidase able to reduce hydrogen peroxide and alkyl hydroperoxides . To gain insight into peroxiredoxin 5 antioxidant role in cell protection, we investigated the resistance of yeast cells expressing human peroxiredoxin 5 in mitochondria or in the cytosol against oxidative stress induced by paraquat . The herbicide paraquat is a redox active drug known to generate superoxide anions in mitochondria and the cytosol of yeast and mammalian cells leading to the formation of several reactive oxygen species . Here, we report that mitochondrial and cytosolic human peroxiredoxin 5 protect yeast cells from cytotoxicity and lipid peroxidation induced by paraquat. Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1093 - 5 Epub 2003 May 23. Crystallization and X-ray diffraction data analysis of leukotriene A4 hydrolase from Saccharomyces cerevisiae; Andersson B et al.; The Saccharomyces cerevisiae leukotriene A4 (LTA4) hydrolase (scLTA4 hydrolase) has been crystallized in order to study the two activities of LTA4 hydrolase in an evolutionary perspective . Single well diffracting crystals are obtained after switching from the hanging-drop method to liquid-liquid diffusion in capillaries using PEG 8000 as precipitant . These crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.8, b = 98.1, c = 99.2 A . Intensity data to 2.3 A resolution were collected from a native scLTA4 hydrolase crystal using synchrotron radiation . A molecular-replacement solution was obtained using the human LTA4 hydrolase structure and the program BEAST. Mol Cell Biol, 2003 Jun, 23(12), 4207 - 18 Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II; Krogan NJ et al.; Set2 methylates Lys36 of histone H3 . We show here that yeast Set2 copurifies with RNA polymerase II (RNAPII) . Chromatin immunoprecipitation analyses demonstrated that Set2 and histone H3 Lys36 methylation are associated with the coding regions of several genes that were tested and correlate with active transcription . Both depend, as well, on the Paf1 elongation factor complex . The C terminus of Set2, which contains a WW domain, is also required for effective Lys36 methylation . Deletion of CTK1, encoding an RNAPII CTD kinase, prevents Lys36 methylation and Set2 recruitment, suggesting that methylation may be triggered by contact of the WW domain or C terminus of Set2 with Ser2-phosphorylated CTD . A set2 deletion results in slight sensitivity to 6-azauracil and much less beta-galactosidase produced by a reporter plasmid, resulting from a defect in transcription . In synthetic genetic array (SGA) analysis, synthetic growth defects were obtained when a set2 deletion was combined with deletions of all five components of the Paf1 complex, the chromodomain elongation factor Chd1, the putative elongation factor Soh1, the Bre1 or Lge1 components of the histone H2B ubiquitination complex, or the histone H2A variant Htz1 . SET2 also interacts genetically with components of the Set1 and Set3 complexes, suggesting that Set1, Set2, and Set3 similarly affect transcription by RNAPII. Biophys J, 2003 Jun, 84(6), 3917 - 23 15N-1H Residual dipolar coupling analysis of native and alkaline-K79A Saccharomyces cerevisiae cytochrome c; Assfalg M et al.; Residual dipolar couplings (RDCs) and pseudocontact shifts are experimentally accessible properties in nuclear magnetic resonance that are related to structural parameters and to the magnetic susceptibility anisotropy . We have determined RDCs due to field-induced orientation of oxidized-K79A and reduced cytochrome c at pH 7.0 and oxidized-K79A cytochrome c at pH 11.1 through measurements of amide (15)N-(1)H (1)J couplings at 800 and 500 MHz . The pH 7.0 RDCs for Fe(III)- and Fe(II)-cytochrome c together with available nuclear Overhauser effects were used to recalculate solution structures that were consistent with both sets of constraints . Molecular magnetic susceptibility anisotropy values were calculated for both redox states of the protein . By subtracting the residual dipolar couplings (RDCs) of the reduced form from those of the oxidized form measured at the same magnetic field (800 MHz), we found the RDC contribution of the paramagnetic metal ion in the oxidized protein . The magnetic susceptibility anisotropy, which was calculated from the structure, was found to be the same as that of the paramagnetic metal ion obtained independently from pseudocontact shifts, thereby indicating that the elements of secondary structure either are rigid or display the same mobility in both oxidation states . The residual dipolar coupling values of the alkaline-K79A form are small with respect to those of oxidized native cytochrome, whereas the pseudocontact shifts are essentially of the same magnitude, indicating local mobility . Importantly, this is the first time that mobility has been found through comparison of RDCs with pseudocontact shifts. J Biol Chem, 2003 Aug 8, 278(32), 30051 - 6 Epub 2003 May 23. Cloning and characterization of hCTF18, hCTF8, and hDCC1 . Human homologs of a Saccharomyces cerevisiae complex involved in sister chromatid cohesion establishment; Merkle CJ et al.; A growing body of evidence suggests that establishment of sister chromatid cohesion is dependent on replication fork passage over a precohesion area . In Saccharomyces cerevisiae, this process involves an alternative replication factor C (RFC) complex that contains the four small RFC subunits as well as CTF18, CTF8, and DCC1 . Here, we show that an evolutionarily conserved homologous complex exists in the nucleus of human cells . We demonstrate that hCTF18, hCTF8, and hDCC1 interact with each other as well as with the p38 subunit of RFC . This alternative RFC-containing complex interacts with proliferating cell nuclear antigen but not with the Rad9/Rad1/Hus1 complex, a proliferating cell nuclear antigen-like clamp involved in the DNA damage response . hCTF18 preferentially binds chromatin during S phase, suggesting a role during replication . Our data provide evidence for the existence of an alternative RFC complex with a probable role in mammalian sister chromatid cohesion establishment. Biochimie, 2003 Jan-Feb, 85(1-2), 133 - 43 Study on agglutinating factors from flocculent Saccharomyces cerevisiae strains; Ngondi-Ekome J et al.; The lectin-like theory suggest that yeast flocculation is mediated by an aggregating lectinic factor . In this study we isolated an agglutinating factor, which corresponds to lectin, from whole cells by treating the flocculent wild-type Saccharomyces cerevisiae NCYC 625 strain and its weakly flocculent mutant {rho degrees } with EDTA and two non-ionic surfactants (Hecameg and HTAC) . The dialysed crude extracts obtained in this way agglutinated erythrocytes and this hemagglutination was specifically inhibited by mannose and mannose derivatives . However, SDS-PAGE profiles showed that the three reagents had different effects on the yeast cells . The non-ionic surfactants appeared to be the most efficient, as their extracts possessed the highest specific agglutinating activity . The products released by the wild-type strain presented a higher specific agglutinating activity than those released by the {rho degrees } mutant . Purification of the agglutinating factor from extracts of both strains by affinity chromatography revealed two active bands of relative mass of 26 and 47 kDa on SDS-PAGE . Mass spectrometry analysis by MALDI-TOF, identified a 26 kDa band as the triose phosphate isomerase (TPI) whereas a 47 kDa band was identical to enolase . Edman degradation showed that the N-terminal sequences of these proteins were similar to TPI and enolase, respectively . The difference in the flocculation behaviour of the two strains is due to changes in the protein composition of the cell wall and in the protein structure involved in cell-cell recognition. Biotechniques, 2003 May, 34(5), 1024 - 8, 1033 Specific targeted integration of kanamycin resistance-associated nonselectable DNA in the genome of the yeast Saccharomyces cerevisiae; Waghmare SK et al.; Sophisticated genome manipulation requires the possibility to modify any intergenic or intragenic DNA sequence at will, without leaving large amounts of undesired vector DNA at the site of alteration . To this end, a series of vectors was developed from a previous gene knockout plasmid system to integrate nonselectable foreign DNA at any desired genomic location in yeast, with a minimum amount of residual plasmid DNA . These vectors have two mutated Flp recognition targets (FRT) sequences flanking the KanMX4 gene and multiple sites for subcloning the DNA fragment to be integrated . The selectable marker can be recycled by Flp site-specific excision between the identical FRTs, thereby allowing the integration of further DNA fragments . With this system, the NLS-tetR-GFP and DsRed genes were successfully integrated at the thr1 locus, and the RVB1 gene was tagged at the C-terminus with the V5-epitope-6-histidine tag . This plasmid system provides for a new molecular tool to integrate any DNA fragment at any genome location in {cir+} yeast strains . Moreover, the system can be extrapolated to other eukaryotic cells in which the FLP/FRT system functions efficiently. Curr Genet, 2003 Aug, 43(5), 327 - 36 Epub 2003 May 23. Suppressor mutations define two regions in the Cbp1 protein important for mitochondrial cytochrome b mRNA stability in Saccharomyces cerevisiae; Islas-Osuna MA et al.; Nuclear-encoded Cbp1 stabilizes and promotes translation of mitochondrial cytochrome b (COB) mRNA . A CCG triplet within the 5'UTR of COB mRNA is essential for Cbp1-dependent stability . Like cbp1 mutations, mutation of any nucleotide in CCG results in degradation of COB transcripts . In this study, CBP1-linked pseudorevertants of the temperature-sensitive CCU strain were isolated . The suppressors are missense mutations within a central cluster or a carboxyl cluster in the linear sequence of Cbp1 . Strains with mutations in the carboxyl half of the central cluster or the carboxyl cluster respire better than those with mutations in the amino half of the central cluster . COB mRNA levels in the suppressor strains were increased compared with that in the CCU strain and were positively correlated with respiratory capability . This correlation supports a model in which the primary role of Cbp1 is to protect COB mRNAs and deliver them to the mitochondrial translational apparatus. Antimicrob Agents Chemother, 2003 Jun, 47(6), 2012 - 4 Comparative roles of the cell wall and cell membrane in limiting uptake of xenobiotic molecules by Saccharomyces cerevisiae; Aouida M et al.; Using reversible electropermeabilization of cells and spheroplasts, we show that the cell wall and plasma membrane partly account for bleomycin resistance by acting as two independent barriers . We also report on the presence of a membrane protein that may be responsible for bleomycin internalization and toxicity in Saccharomyces cerevisiae. Curr Genet, 2003 Aug, 43(5), 337 - 50 Epub 2003 May 21. Characterization of mutations that are synthetic lethal with pol3-13, a mutated allele of DNA polymerase delta in Saccharomyces cerevisiae; Chanet R et al.; The pol3-13 mutation is located in the C-terminal end of POL3, the gene encoding the catalytic subunit of polymerase delta, and confers thermosensitivity onto the Saccharomyces cerevisiae mutant strain . To get insight about DNA replication control, we performed a genetic screen to identify genes that are synthetic lethal with pol3-13 . Mutations in genes encoding the two other subunits of DNA polymerase delta (HYS2, POL32) were identified . Mutations in two recombination genes (RAD50, RAD51) were also identified, confirming that homologous recombination is necessary for pol3-13 mutant strain survival . Other mutations were identified in genes involved in repair and genome stability (MET18/ MMS19), in the control of origin-firing and/or transcription (ABF1, SRB7), in the S/G2 checkpoint (RAD53), in the Ras-cAMP signal transduction pathway (MKS1), in nuclear pore metabolism (SEH1), in protein degradation (DOC1) and in folding (YDJ1) . Finally, mutations in three genes of unknown function were isolated (NBP35, DRE2, TAH18) . Synthetic lethality between pol3-13 and each of the three mutants pol32, mms19 and doc1 could be suppressed by a rad18 deletion, suggesting an important role of ubiquitination in DNA replication control . We propose that the pol3-13 mutant generates replicative problems that need both homologous recombination and an intact checkpoint machinery to be overcome. Biochem J, 2003 Sep 1, 374(Pt 2), 513 - 9 Protein S-thiolation targets glycolysis and protein synthesis in response to oxidative stress in the yeast Saccharomyces cerevisiae; Shenton D et al.; The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, a process by which protein SH groups form mixed disulphides with low-molecular-mass thiols such as glutathione . We report here the target proteins which are modified in yeast cells in response to H(2)O(2) . In particular, a range of glycolytic and related enzymes (Tdh3, Eno2, Adh1, Tpi1, Ald6 and Fba1), as well as translation factors (Tef2, Tef5, Nip1 and Rps5) are identified . The oxidative stress conditions used to induce S-thiolation are shown to inhibit GAPDH (glyceraldehyde-3-phosphate dehydrogenase), enolase and alcohol dehydrogenase activities, whereas they have no effect on aldolase, triose phosphate isomerase or aldehyde dehydrogenase activities . The inhibition of GAPDH, enolase and alcohol dehydrogenase is readily reversible once the oxidant is removed . In addition, we show that peroxide stress has little or no effect on glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, the enzymes that catalyse NADPH production via the pentose phosphate pathway . Thus the inhibition of glycolytic flux is proposed to result in glucose equivalents entering the pentose phosphate pathway for the generation of NADPH . Radiolabelling is used to confirm that peroxide stress results in a rapid and reversible inhibition of protein synthesis . Furthermore, we show that glycolytic enzyme activities and protein synthesis are irreversibly inhibited in a mutant that lacks glutathione, and hence cannot modify proteins by S-thiolation . In summary, protein S-thiolation appears to serve an adaptive function during exposure to an oxidative stress by reprogramming metabolism and protecting protein synthesis against irreversible oxidation. Mol Microbiol, 2003 May, 48(4), 1119 - 30 TFIIB and subunits of the SAGA complex are involved in transcriptional activation of phospholipid biosynthetic genes by the regulatory protein Ino2 in the yeast Saccharomyces cerevisiae; Dietz M et al.; In the yeast Saccharomyces cerevisiae, genes involved in phospholipid biosynthesis are activated by ICRE (inositol/choline-responsive element) up-stream motifs and the corresponding heterodimeric binding factor, Ino2 + Ino4 . Both Ino2 and Ino4 contain basic helix-loop-helix (bHLH) domains required for ICRE binding, whereas transcriptional activation is mediated exclusively by Ino2 . In this work, we describe a molecular analysis of functional minimal domains responsible for specific DNA recognition and transcriptional activation (TAD1 and TAD2) . We also define the importance of individual amino acids within the more important activation domain TAD1 . Random mutagenesis at five amino acid positions showed the importance of acidic as well as hydrophobic residues within this minimal TAD . We also investigated the contribution of known general transcription factors and co-activators for Ino2-dependent gene activation . Although an ada5 single mutant and a gal11 paf1 double mutant were severely affected, a partial reduction in activation was found for gcn5 and srb2 . Ino2 interacts physically with the basal transcription factor Sua7 (TFIIB of yeast) . Interestingly, interaction is mediated by the HLH dimerization domain of Ino2 and by two non-overlapping domains within Sua7 . Thus, Sua7 may compete with Ino4 for binding to the Ino2 activator, creating the possibility of positive and negative influence of Sua7 on ICRE-dependent gene expression. Proc Natl Acad Sci U S A, 2003 May 27, 100(11), 6640 - 5 Epub 2003 May 15. Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability; Myung K et al.; Some spontaneous gross chromosomal rearrangements (GCRs) seem to result from DNA-replication errors . The chromatin-assembly factor I (CAF-I) and replication-coupling assembly factor (RCAF) complexes function in chromatin assembly during DNA replication and repair and could play a role in maintaining genome stability . Inactivation of CAF-I or RCAF increased the rate of accumulating different types of GCRs including translocations and deletion of chromosome arms with associated de novo telomere addition . Inactivation of CAF-I seems to cause damage that activates the DNA-damage checkpoints, whereas inactivation of RCAF seems to cause damage that activates the DNA-damage and replication checkpoints . Both defects result in increased genome instability that is normally suppressed by these checkpoints, RAD52-dependent recombination, and PIF1-dependent inhibition of de novo telomere addition . Treatment of CAF-I- or RCAF-defective cells with methyl methanesulfonate increased the induction of GCRs compared with that seen for a wild-type strain . These results indicate that coupling of chromatin assembly to DNA replication and DNA repair is critical to maintaining genome stability. J Biol Chem, 2003 Jul 25, 278(30), 27372 - 81 Epub 2003 May 15. Evidence for a role of MCM (mini-chromosome maintenance)5 in transcriptional repression of sub-telomeric and Ty-proximal genes in Saccharomyces cerevisiae; Dziak R et al.; The MCM (mini-chromosome maintenance) genes have a well established role in the initiation of DNA replication and in the elongation of replication forks in Saccharomyces cerevisiae . In this study we demonstrate elevated expression of sub-telomeric and Ty retrotransposon-proximal genes in two mcm5 strains . This pattern of up-regulated genes resembles the genome-wide association of MCM proteins to chromatin that was reported earlier . We link the altered gene expression in mcm5 strains to a reversal of telomere position effect (TPE) and to remodeling of sub-telomeric and Ty chromatin . We also show a suppression of the Ts phenotype of a mcm5 strain by the high copy expression of the TRA1 component of the chromatin-remodeling SAGA/ADA (SPT-ADA-GCN5 acetylase/ADAptor) . We propose that MCM proteins mediate the establishment of silent chromatin domains around telomeres and Ty retrotransposons. Genetics, 2003 May, 164(1), 65 - 79 Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae; Galli A et al.; The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions . We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold . The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p . However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved . UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination . In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway . Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability. Genetics, 2003 May, 164(1), 47 - 64 Multiple roles for Saccharomyces cerevisiae histone H2A in telomere position effect, Spt phenotypes and double-strand-break repair; Wyatt HR et al.; Telomere position effects on transcription (TPE, or telomeric silencing) are nucleated by association of nonhistone silencing factors with the telomere and propagated in subtelomeric regions through association of silencing factors with the specifically modified histones H3 and H4 . However, the function of histone H2A in TPE is unknown . We found that deletion of either the amino or the carboxyltails of H2A substantially reduces TPE . We identified four H2A modification sites necessary for wild-type efficiency of TPE . These "hta1tpe" alleles also act as suppressors of a delta insertion allele of LYS2, suggesting shared elements of chromatin structure at both loci . Interestingly, we observed combinatorial effects of allele pairs, suggesting both interdependent acetylation and deacetylation events in the amino-terminal tail and a regulatory circuit between multiple phosphorylated residues in the carboxyl-terminal tail . Decreases in silencing and viability are observed in most hta1tpe alleles after treatment with low and high concentrations, respectively, of bleomycin, which forms double-strand breaks (DSBs) . In the absence of the DSB and telomere-binding protein yKu70, the bleomycin sensitivity of hta1tpe alleles is further enhanced . We also provide data suggesting the presence of a yKu-dependent histone H2A function in TPE . These data indicate that the amino- and carboxyl-terminal tails of H2A are essential for wild-type levels of yKu-mediated TPE and DSB repair. Proteomics, 2003 May, 3(5), 752 - 63 Fragmentation of dihydroxyacetone kinase 1 from Saccharomyces cerevisiae indicates a two-domain structure; Molin M et al.; Global protein expression in Saccharomyces cerevisiae strains either deleted for both yeast dihydroxyacetone kinases (DAK1 and DAK2) or overexpressing DAK1, was characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) . We found protein expression in the double deletion strain to be highly similar to wild-type . In the strain overexpressing Dak1p, nine spots representing fragments of the Dak1p protein in the size range 40-20 kDa and amounting to approximately 30% of total Dak1p, were discovered (native size Dak1p migrates at roughly 60 kDa) . Fragments were characterized by matrix-assisted laser desorption/ionization mass spectrometry and electrospray mass spectrometry analyses to represent either the N- or the C-terminal part of the DAK1 protein . Cleavage points, predicted from mass spectrometry and 2-D PAGE data, mapped almost exclusively in the middle region showing low sequence conservation between Dak1p and its closest homologues . We hypothesize that observed Dak1p fragments represent stable structural domains shielded from access by native endoproteases . Furthermore, overexpressing Dak1p with the non-native N-terminus (M)A-, resulted in native size Dak1p and N-terminal Dak1p fragments appearing in two major 2-D PAGE forms of approximately equal size and abundance, but with slightly different isoelectric points . However, when overexpressing Dak1p with the native N-terminus (M)S-, only the more acidic 2-D PAGE form appeared . In the N-terminal acetyltransferase mutant nat1delta, (M)A-Dak1p species were converted into the basic form, arguing twin spots to represent forms with acetylated and deacetylated N-termini . Data thus indicated that (M)A-N-termini, in the Dak1p context, were NatA substrates recognized with 50% lower efficiency than (M)S-N-termini. Mol Cell Biol, 2003 Jun, 23(11), 3721 - 34 The Rad51 pathway of telomerase-independent maintenance of telomeres can amplify TG1-3 sequences in yku and cdc13 mutants of Saccharomyces cerevisiae; Grandin N et al.; In the yeast Saccharomyces cerevisiae, Cdc13, Yku, and telomerase define three parallel pathways for telomere end protection that prevent chromosome instability and death by senescence . We report here that cdc13-1 yku70delta mutants generated telomere deprotection-resistant cells that, in contrast with telomerase-negative senescent cells, did not display classical crisis events . cdc13-1 yku70delta cells survived telomere deprotection by exclusively amplifying TG(1-3) repeats (type II recombination) . In a background lacking telomerase (tlc1delta), this process predominated over type I recombination (amplification of subtelomeric Y' sequences) . Strikingly, inactivation of the Rad50/Rad59 pathway (which is normally required for type II recombination) in cdc13-1 yku70delta or yku70delta tlc1delta mutants, but also in cdc13-1 YKU70(+) tlc1delta mutants, still permitted type II recombination, but this process was now entirely dependent on the Rad51 pathway . In addition, delayed senescence was observed in cdc13-1 yku70delta rad51delta and cdc13-1 tlc1delta rad51delta cells . These results demonstrate that in wild-type cells, masking by Cdc13 and Yku prevents the Rad51 pathway from amplifying telomeric TG(1-3) sequences . They also suggest that Rad51 is more efficient than Rad50 in amplifying the sequences left uncovered by the absence of Cdc13 or Yku70. J Biol Chem, 2003 Jul 25, 278(30), 27605 - 11 Epub 2003 May 14. Saccharomyces cerevisiae PIP2 mediating oleic acid induction and peroxisome proliferation is regulated by Adr1p and Pip2p-Oaf1p; Rottensteiner H et al.; Saccharomyces cerevisiae genes involved in fatty acid degradation contain in their promoters oleate response elements (OREs) and type 1 upstream activation sequences (UAS1s) that bind Pip2p-Oaf1p and Adr1p, respectively . The promoter of the PIP2 gene was found to contain a potential UAS1 that consists of a tandem array of CYCCRR half-sites in an overlapping arrangement with a previously characterized ORE . Electrophoretic mobility shift analysis demonstrated that Adr1p bound to UAS1PIP2, and Northern analysis in combination with a lacZ reporter gene confirmed that Adr1p influenced the transcription of PIP2 . Immunoprecipitation showed that, in adr1delta mutant cells grown on oleic acid, Pip2p was less abundant compared with the corresponding wild-type . In addition, the amount of Pip2p-Oaf1p that bound to a target ORE in vitro was reduced in mutant extracts compared with the wild-type . Transcription of the oleic acid-inducible genes SPS19 and CTA1, which rely on both Pip2p-Oaf1p and Adr1p for their regulation, was reduced in adr1delta mutant cells . However, by ectopically restoring levels of Pip2p in adr1delta cells grown on oleic acid medium, transcription of both genes increased 2-fold compared with the control . This partial suppression of the adr1delta mutant phenotype was additionally manifested by moderate utilization of oleic acid . Hence, both the expression as well as the action of the two transcription factors, Adr1p and Pip2p-Oaf1p, are interconnected, which allows for an elaborate control of fatty acid-inducible genes. J Cell Biol, 2003 May 12, 161(3), 483 - 8 Spindle orientation in Saccharomyces cerevisiae depends on the transport of microtubule ends along polarized actin cables; Hwang E et al.; Microtubules and actin filaments interact and cooperate in many processes in eukaryotic cells, but the functional implications of such interactions are not well understood . In the yeast Saccharomyces cerevisiae, both cytoplasmic microtubules and actin filaments are needed for spindle orientation . In addition, this process requires the type V myosin protein Myo2, the microtubule end-binding protein Bim1, and Kar9 . Here, we show that fusing Bim1 to the tail of the Myo2 is sufficient to orient spindles in the absence of Kar9, suggesting that the role of Kar9 is to link Myo2 to Bim1 . In addition, we show that Myo2 localizes to the plus ends of cytoplasmic microtubules, and that the rate of movement of these cytoplasmic microtubules to the bud neck depends on the intrinsic velocity of Myo2 along actin filaments . These results support a model for spindle orientation in which a Myo2-Kar9-Bim1 complex transports microtubule ends along polarized actin cables . We also present data suggesting that a similar process plays a role in orienting cytoplasmic microtubules in mating yeast cells. Nature, 2003 May 8, 423(6936), 181 - 5 Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae; Anderson RM et al.; Calorie restriction extends lifespan in a broad range of organisms, from yeasts to mammals . Numerous hypotheses have been proposed to explain this phenomenon, including decreased oxidative damage and altered energy metabolism . In Saccharomyces cerevisiae, lifespan extension by calorie restriction requires the NAD+-dependent histone deacetylase, Sir2 (ref . 1) . We have recently shown that Sir2 and its closest human homologue SIRT1, a p53 deacetylase, are strongly inhibited by the vitamin B3 precursor nicotinamide . Here we show that increased expression of PNC1 (pyrazinamidase/nicotinamidase 1), which encodes an enzyme that deaminates nicotinamide, is both necessary and sufficient for lifespan extension by calorie restriction and low-intensity stress . We also identify PNC1 as a longevity gene that is responsive to all stimuli that extend lifespan . We provide evidence that nicotinamide depletion is sufficient to activate Sir2 and that this is the mechanism by which PNC1 regulates longevity . We conclude that yeast lifespan extension by calorie restriction is the consequence of an active cellular response to a low-intensity stress and speculate that nicotinamide might regulate critical cellular processes in higher organisms. J Mol Microbiol Biotechnol, 2003, 5(2), 78 - 81 Maize Activator (AC) transposase (TPase) is expressed in Saccharomyces cerevisiae from a genomic clone . detection via Elisa, and proposed use in complementation studies; MacRae AF; The maize Activator (Ac) transposase (TPase) was expressed as a Histidine (His)-tagged protein in Saccharomyces cerevisiae from a full length genomic clone . Expression was demonstrated via the highly specific nickel-coated Elisa plate method, using an anti-His antibody and 2 separate anti-Ac TPase antibodies, to Ac residues 103-465 and 189-807 . AC TPase expression in Saccharomyces is important for two reasons: (a) because the expression from a genomic clone herein permits the future study of RNA splicing mechanisms in common between maize and yeast systems, and (b) because a yeast system can easily be used for demonstrating complementation of function . Thus, such transformed yeast systems could be used in future, to experimentally test whether Ac TPase could complement various yeast mutations . Specifically, Ac TPase may be able to complement (i.e . provide the same function) to yeast transcription factor mutants or to genes mutated in other essential yeast functions . If confirmed, this would lend support to Barbara McClintock's hypothesis that transposable elements can serve as 'controlling elements' within the genome, by their ability to supplement other essential genes' functions, as needed . Work herein is contrasted with existing studies on Ac in yeast . Nucleic Acids Res, 2003 May 15, 31(10), 2576 - 85 Multiple recombination pathways for sister chromatid exchange in Saccharomyces cerevisiae: role of RAD1 and the RAD52 epistasis group genes; Dong Z et al.; Sister chromatid exchange (SCE) can occur by several recombination mechanisms, including those directly initiated by double-strand breaks (DSBs), such as gap repair and break-induced replication (BIR), and those initiated when DNA polymerases stall, such as template switching . To elucidate SCE recombination mechanisms, we determined whether spontaneous and DNA damage-associated SCE requires specific genes within the RAD52 and RAD3 epistasis groups in Saccharomyces cerevisiae strains containing two his3 fragments, his3-Delta5' and his3-Delta3'::HOcs . SCE frequencies were measured after cells were exposed to UV, X-rays, 4-nitroquinoline 1-oxide (4-NQO) and methyl methanesulfonate (MMS), or when an HO endonuclease-induced DSB was introduced at his3-Delta3'::HOcs . Our data indicate that genes involved in gap repair, such as RAD55, RAD57 and RAD54, are required for DNA damage-associated SCE but not for spontaneous SCE . RAD50 and RAD59, genes required for BIR, are required for X-ray-associated SCE but not for SCE stimulated by HO-induced DSBs . In comparison with wild type, rates of spontaneous SCE are 10-fold lower in rad51 rad1 but not in either rad51 rad50 or rad51 rad59 double mutants . We propose that gap repair mechanisms are important in DNA damage-associated recombination, whereas alternative pathways, including a template switch pathway, play a role in spontaneous SCE. Nucleic Acids Res, 2003 May 15, 31(10), 2524 - 33 RRP20, a component of the 90S preribosome, is required for pre-18S rRNA processing in Saccharomyces cerevisiae; Senapin S et al.; A strain of Saccharomyces cerevisiae, defective in small subunit ribosomal RNA processing, has a mutation in YOR145c ORF that converts Gly235 to Asp . Yor145c is a nucleolar protein required for cell viability and has been reported recently to be present in 90S pre-ribosomal particles . The Gly235Asp mutation in YOR145c is found in a KH-type RNA-binding domain and causes a marked deficiency in 18S rRNA production . Detailed studies by northern blotting and primer extension analyses show that the mutant strain impairs the early pre-rRNA processing cleavage essentially at sites A1 and A2, leading to accumulation of a 22S dead-end processing product that is found in only a few rRNA processing mutants . Furthermore, U3, U14, snR10 and snR30 snoRNAs, involved in early pre-rRNA cleavages, are not destabilized by the YOR145c mutation . As the protein encoded by YOR145c is found in pre-ribosomal particles and the mutant strain is defective in ribosomal RNA processing, we have renamed it as RRP20. Curr Genet, 2003 Jul, 43(4), 263 - 72 Epub 2003 May 07. Rsf1p, a protein required for respiratory growth of Saccharomyces cerevisiae; Lu L et al.; A central problem in our understanding of mitochondrial (mt) function remains the question of how coordinate transcriptional control is accomplished between nucleus and mitochondria . Here, we report the initial characterization of a protein of previously unknown function, the product of the YMR030 W gene, that appears to mediate such coordinate gene expression . Expression of YMR030 W is glucose-repressible; a deletion mutant for this gene shows a severe growth defect on glycerol-, but not glucose- or ethanol-based medium . In that mutant, transcript levels from GUT1 and GUT2 are highly attenuated compared with those of the wild-type parent when both are grown on glycerol-based medium . Under the same growth conditions, transcripts from the mt OLI1 gene, which has one copy of a mt upstream activating sequence (UAS) in its 5'-flanking region, are attenuated in the DeltaYMR030 W mutant, but mRNA from the mt COX3 ( OXI2) gene, which lacks the mt UAS, are not . Some nuclear genes encoding mt-related proteins also show low transcript levels in the DeltaYMR030 W mutant in comparison with those of the wild-type parent strain during glycerol-based growth . Localization of the protein, via its expression fused to green fluorescent protein, indicates that it is present in both nucleus and mitochondria, supporting a respiration-related transcriptional role for this gene product in both cellular genetic compartments . Because of its role in both respiratory growth and mt function, we designate the YMR030 W coding sequence RSF1 (respiration factor 1). Mol Genet Genomics, 2003 Jun, 269(3), 370 - 80 Epub 2003 May 06. Merging of multiple signals regulating delta9 fatty acid desaturase gene transcription in Saccharomyces cerevisiae; Nakagawa Y et al.; Fatty acid desaturation, which requires molecular oxygen (O2) as an electron acceptor, is catalyzed by delta9 fatty acid desaturase, which is encoded by OLE1 in Saccharomyces cerevisiae . Transcription of the OLE1 gene is repressed by unsaturated fatty acids (UFAs) and activated by hypoxia and low temperatures via the endoplasmic reticulum membrane protein Mga2p . We previously reported the isolation of the nfo3-1 (negative factor for OLE1) mutant, which exhibits enhanced expression of OLE1 in the presence of UFA and under aerobic conditions . In this work, we demonstrated that the NFO3 gene is identical to OLE1 and that the nfo3-1 mutation (renamed ole1-101) alters arginine-346, in the vicinity of the conserved histidine-rich motif essential for the catalytic function of the Ole1 protein, to lysine . The ratio of UFAs to total fatty acids in the ole1-101 mutant was 60%, compared to 75% in the wild type, suggesting that the reduction in relative levels of intracellular UFAs activates OLE1 transcription . However, in ole1-101 cells grown in the presence of oleic acid, the level of OLE1 expression remained high, although the relative amount of UFAs in the ole1-101 mutant cells was almost the same as that in wild-type cells growing under the same conditions . By contrast, when cells were grown with linoleic acid, which has a lower melting point than oleic acid, the elevation of the OLE1 expression level due to the ole1-101 mutation was almost completely suppressed . These observations suggest that the ole1-101 cells activate OLE1 transcription by sensing not only the intracellular UFA level, but also membrane fluidity or the nature of the UFA species itself . Furthermore, we found that not only the fatty acid- regulated (FAR) element but also the O2- regulated (O2R) element in the OLE1 promoter was involved in the activation of OLE1 transcription by the ole1-101 mutation, and that the effects of the low-oxygen signal and the ole1-101-generated signal on OLE1 expression were not additive . Taken together, these findings suggest that signals associated with hypoxia, low temperatures and intracellular UFA depletion activate OLE1 transcription by a common pathway. J Cell Biol, 2003 May 12, 161(3), 497 - 505 Epub 2003 May 05. Oscillatory nucleocytoplasmic shuttling of the general stress response transcriptional activators Msn2 and Msn4 in Saccharomyces cerevisiae; Jacquet M et al.; Msn2 and Msn4 are two related transcriptional activators that mediate a general response to stress in yeast Saccharomyces cerevisiae by eliciting the expression of specific sets of genes . In response to stress or nutritional limitation, Msn2 and Msn4 migrate from the cytoplasm to the nucleus . Using GFP-tagged constructs and high-resolution time-lapse video microscopy on single cells, we show that light emitted by the microscope also triggers this migration . Unexpectedly, the population of Msn2 or Msn4 molecules shuttles repetitively into and out of the nucleus with a periodicity of a few minutes . A large heterogeneity in the oscillatory response to stress is observed between individual cells . This periodic behavior, which can be induced by various types of stress, at intermediate stress levels, is not dependent upon protein synthesis and persists when the DNA-binding domain of Msn2 is removed . The cAMP-PKA pathway controls the sensitivity of the oscillatory nucleocytoplasmic shuttling . In the absence of PKA, Msn4 continues to oscillate while Msn2 is maintained in the nucleus . We show that a computational model based on the possibility that Msn2 and Msn4 participate in autoregulatory loops controlling their subcellular localization can account for the oscillatory behavior of the two transcription factors. Electrophoresis, 2003 May, 24(9), 1458 - 70 Proteome analysis of Saccharomyces cerevisiae under metal stress by two-dimensional differential gel electrophoresis; Hu Y et al.; The defense mechanism by which cells combat metal stress remains poorly understood . By utilizing a newly developed technique - the differential gel electrophoresis (DIGE) - we evaluated the biological alterations of metal stress on Saccharomyces cerevisiae at its translational level . By simultaneously comparing the differential expression profiles of thousands of proteins as results of 15 different metal treatments, we were able to closely examine the response of a large number of proteins within the yeast proteome towards individual metals, as well as the response of the same proteins towards different metals . This, to our knowledge, is the first case which demonstrates the potential of DIGE as a high-throughput tool for large-scale proteome analysis . From our studies, where yeast cells were exhaustively treated with exogenous metals, 20-30% of all proteins detected showed statistically significant changes . According to different effects (up-/downregulation) of protein expression levels observed, we were able to tentatively divide the 15 metals into three groups . By mass spectrometric analysis, more than 50 protein spots were positively identified, both quantitatively and qualitatively . One of the proteins was identified to be Cu/Zn superoxide dismutase (SOD1), and its expression levels as a result of 15 different metal treatments was further examined in greater details . Significant changes in SOD1 expression were observed throughout all 15 DIGE gels. Int Microbiol, 2003 Mar, 6(1), 49 - 55 Epub 2003 Jan 25. Intracellular transport of a heterologous membrane protein, the human transferrin receptor, in Saccharomyces cerevisiae; Prinz B et al.; We have analyzed the intracellular behavior of the human transferrin receptor (TfR) in Saccharomyces cerevisiae . The major part of the heterologously expressed TfR, which has previously been used as a model for heterologous expression of membrane proteins in yeast, is localized in the endoplasmic reticulum (ER) membranes; a minor fraction is present in the plasma membrane (PM) . The stability of the TfR depends on vacuolar proteases, implying that it is degraded in the vacuolar compartment . Degradation is further dependent on favorable transport conditions to this compartment . The main bottleneck of transport seems to be the transition from the ER to the PM . The chaperone Cne1p, which is involved in quality control in the ER, plays a role in regulating the amount of heterologous TfR, as deletion of CNE1 leads to significant accumulation of the protein . This is the first demonstration of the involvement of CNE1 in regulating the level of heterologous membrane proteins. J Nutr, 2003 May, 133(5 Suppl 1), 1532S - 5S Multiple regulatory mechanisms maintain zinc homeostasis in Saccharomyces cerevisiae; Eide DJ; Zinc is an essential nutrient, yet it is toxic if it accumulates in excess amounts within cells; therefore the intracellular labile zinc content of cells is tightly controlled . In Saccharomyces cerevisiae, zinc homeostasis is regulated by the controlled activity of zinc uptake transporters in the plasma membrane and transporters responsible for intracellular zinc compartmentalization . The activity of these transporters is regulated at both transcriptional and posttranscriptional levels in response to zinc . These different mechanisms work together to precisely balance zinc uptake and its storage and utilization. J Biol Chem, 2003 Jul 4, 278(27), 24636 - 43 Epub 2003 May 02. Nuclear thiol peroxidase as a functional alkyl-hydroperoxide reductase necessary for stationary phase growth of Saccharomyces cerevisiae; Cha MK et al.; Yeast nucleus-localized thiol peroxidase (nTPx) was characterized as a functional peroxidase . There are two cysteine residues in nTPx . Replacement of Cys-106 or Cys-111 with serine resulted in a complete loss of thioredoxin-linked peroxidase activity . However, when their activities were measured in terms of the ability to inhibit oxidation of glutamine synthetase, C111S showed the same antioxidant activity as the wild type protein . SDS-PAGE gel analysis revealed that only C111S existed as the dimer form . In addition to the identification of Cys-106 as the primary catalytic site, these data suggest the formation of the intradisulfide bond as a part of the catalytic cycle between nTPx and thioredoxin . nTPx preferentially reduced alkyl-hydroperoxides rather than H2O2 . Furthermore, a nTPx mutant strain showed higher sensitivity toward alkyl-hydroperoxide than hydrogen peroxide . Also, reduction of the viability of nTPx mutant strain against various oxidants supports an in vivo antioxidant role for nTPx . nTPx transcriptional activity was not significantly detectable in log phase yeast, but the activity was exponentially increased after the diauxic shift . The transcriptional activity was highly induced even in the log phase yeast grown in nonfermentable carbon source . Deletion of Tor1p, Ras1p, and Ras2p resulted in considerable induction when compared with their parent strains, demonstrating the activation of the transcription of nTPx gene at the diauxic shift . Transcription of nTPx gene was induced in response to oxidative stress . Viability of a stationary phase nTPx mutant was considerably reduced compared with the isogenic strain . Collectively, these data demonstrate that nTPx is a thiol peroxidase family acting as alkyl-hydroperoxide reductase in the nucleus during post-diauxic growth. Biosci Biotechnol Biochem, 2003 Feb, 67(2), 448 - 50 Cooperation of Sly1/SM-family protein and sec18/NSF of Saccharomyces cerevisiae in disassembly of cis-SNARE membrane-protein complexes; Kosodo Y et al.; Assembly and disassembly of the SNARE membrane-protein complexes plays a key role in vesicular trafficking . The SM-family Slyl protein binds to the tSNARE Sed5 protein and stimulates its assembly into a trans-SNARE complex . Disassembly of the resulting cis-SNARE complex containing Sed5 was retarded in a temperature-sensitive yeast mutant of Slyl protein with a defect in binding to Sed5 . A temperature-sensitive mutation (sec18-1) of Sec18/NSF disassembly ATPase showed synthetic lethality with the sly1(ts) mutation . These results suggest that Slyl and Sec18 proteins work cooperatively and that the binding of Slyl to Sed5 stimulates the disassembly of the cis-SNARE complex by Sec18 ATPase. FEMS Microbiol Lett, 2003 Apr 25, 221(2), 249 - 55 Optimisation of interdelta analysis for Saccharomyces cerevisiae strain characterisation; Legras JL et al.; A new primer pair (delta12-delta21) for polymerase chain reaction-based yeast typing was designed using the yeast genome sequence . The specificity of this primer pair was checked by the comparison of the electrophoresis pattern with a virtual profile calculated from Blast data . The analysis of 53 commercial and laboratory Saccharomyces cerevisiae yeast strains showed a clear improvement of interdelta analysis using the newly designed primers. FEMS Microbiol Lett, 2003 Apr 25, 221(2), 151 - 4 Human TSG101 does not replace Saccharomyces cerevisiae VPS23 role in the quality control of plasma membrane proteins; Blanco S et al.; The Saccharomyces cerevisiae VPS23 (STP22) gene is implicated in the control of vesicle movement and quality of plasma membrane proteins . VPS23 mutants have defects either in removing defective membrane proteins such as alpha-mating factor receptor and arginine permease . The human ortholog TSG101 and its variants, isolated from tumor cells, do not substitute VPS23 in its ability to rescue the phenotype of defective plasma membrane proteins. Mol Cell Biol, 2003 May, 23(10), 3681 - 91 Telomeric position effect variegation in Saccharomyces cerevisiae by Caenorhabditis elegans linker histones suggests a mechanistic connection between germ line and telomeric silencing; Jedrusik MA et al.; Linker histones are nonessential for the life of single-celled eukaryotes . Linker histones, however, can be important components of specific developmental programs in multicellular animals and plants . For Caenorhabditis elegans a single linker histone variant (H1.1) is essential in a chromatin silencing process which is crucial for the proliferation and differentiation of the hermaphrodite germ line . In this study we analyzed the whole linker histone complement of C . elegans by telomeric position effect variegation in budding yeast . In this assay an indicator gene (URA3) placed close to the repressive telomeric chromatin structure is subject to epigenetically inherited gene inactivation . Just one out of seven C . elegans linker histones (H1.1) was able to enhance the telomeric position effect in budding yeast . Since these results reflect the biological function of H1.1 in C . elegans, we suggest that chromatin silencing in C . elegans is governed by molecular mechanisms related to the telomere-dependent silencing in budding yeast . We confirmed this hypothesis by testing C . elegans homologs of three yeast genes which are established modifiers of the yeast telomeric chromatin structure (SIR2, SET1, and RAD17) for their influence on repeat-dependent transgene silencing for C . elegans. Mol Cell Biol, 2003 May, 23(10), 3405 - 16 Rad53 checkpoint kinase phosphorylation site preference identified in the Swi6 protein of Saccharomyces cerevisiae; Sidorova JM et al.; Rad53 of Saccharomyces cerevisiae is a checkpoint kinase whose structure and function are conserved among eukaryotes . When a cell detects damaged DNA, Rad53 activity is dramatically increased, which ultimately leads to changes in DNA replication, repair, and cell division . Despite its central role in checkpoint signaling, little is known about Rad53 substrates or substrate specificity . A number of proteins are implicated as Rad53 substrates; however, the evidence remains indirect . Previously, we have provided evidence that Swi6, a subunit of the Swi4/Swi6 late-G(1)-specific transcriptional activator, is a substrate of Rad53 in the G(1)/S DNA damage checkpoint . In the present study we identify Rad53 phosphorylation sites in Swi6 in vitro and demonstrate that at least one of them is targeted by Rad53 in vivo . Mutations in these phosphorylation sites in Swi6 shorten but do not eliminate the Rad53-dependent delay of the G(1)-to-S transition after DNA damage . We derive a consensus for Rad53 site preference at positions -2 and +2 (-2/+2) and identify its potential substrates in the yeast proteome . Finally, we present evidence that one of these candidates, the cohesin complex subunit Scc1 undergoes DNA damage-dependent phosphorylation, which is in part dependent on Rad53. Microbiology, 2003 May, 149(Pt 5), 1205 - 16 Synergistic inhibition of APC/C by glucose and activated Ras proteins can be mediated by each of the Tpk1-3 proteins in Saccharomyces cerevisiae; Bolte M et al.; Proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for the progression through mitosis . APC/C is a highly conserved ubiquitin ligase whose activity is regulated during the cell cycle by various factors, including spindle checkpoint components and protein kinases . The cAMP-dependent protein kinase (PKA) was identified as negative regulator of APC/C in yeast and mammalian cells . In the yeast Saccharomyces cerevisiae, PKA activity is induced upon glucose addition or by activated Ras proteins . This study shows that glucose and the activated Ras2(Val19) protein synergistically inhibit APC/C function via the cAMP/PKA pathway in yeast . Remarkably, Ras2 proteins defective in the interaction with adenylate cyclase fail to influence APC/C, implying that its function is regulated exclusively by PKA, but not by alternative Ras pathways . Furthermore, it is shown that the three PKAs in yeast, Tpk1, Tpk2 and Tpk3, have redundant functions in regulating APC/C in response to glucose medium . Single or double deletions of TPK genes did not prevent inhibition of APC/C, suggesting that each of the Tpk proteins can take over this function . However, Tpk2 seems to inhibit APC/C function more efficiently than Tpk1 and Tpk3 . Finally, evidence is provided that Cdc20 is involved in APC/C regulation by the cAMP/PKA pathway. Microbiology, 2003 May, 149(Pt 5), 1193 - 204 Response to high osmotic conditions and elevated temperature in Saccharomyces cerevisiae is controlled by intracellular glycerol and involves coordinate activity of MAP kinase pathways; Wojda I et al.; In the yeast Saccharomyces cerevisiae, response to an increase in external osmolarity is mediated by the HOG (high osmolarity glycerol) MAP kinase pathway . HOG pathway mutant strains display osmosensitive phenotypes . Recently evidence has been obtained that the osmosensitivity of HOG pathway mutants is reduced during growth at elevated temperature (37 degrees C) . A notable exception is the ste11ssk2ssk22 mutant, which displays hypersensitivity to osmotic stress at 37 degrees C . This paper reports that overexpression of FPS1 or GPD1 (encoding the glycerol transport facilitator and glycerol-3-phosphate dehydrogenase, respectively, and both affecting intracellular glycerol levels) reduces the hypersensitivity to osmotic stress of ste11ssk2ssk22 at 37 degrees C . Although in this particular HOG pathway mutant a correlation between suppression of the phenotype and glycerol content could be demonstrated, the absolute level of intracellular glycerol per se does not determine whether a strain is osmosensitive or not . Rather, evidence was obtained that the glycerol level may have an indirect effect, viz . by influencing signalling through the PKC (protein kinase C) MAP kinase pathway, which plays an important role in maintenance of cellular integrity . In order to validate the data obtained with a HOG pathway mutant strain for wild-type yeast cells, MAP kinase signalling under different growth conditions was examined in wild-type strains . PKC pathway signalling, which is manifest at elevated growth temperature by phosphorylation of MAP kinase Mpk1p, is rapidly lost when cells are shifted to high external osmolarity conditions . Expression of bck1-20 or overexpression of WSC3 in wild-type cells resulted in restoration of PKC signalling . Both PKC and HOG signalling, cell wall phenotypes and high osmotic stress responses in wild-type cells were found to be influenced by the growth temperature . The data taken together indicate the intricate interdependence of growth temperature, intracellular glycerol, cell wall structure and MAP kinase signalling in the hyperosmotic stress response of yeast. Biosci Biotechnol Biochem, 2003 Mar, 67(3), 517 - 24 Characterization of a Saccharomyces cerevisiae mutant with pseudohyphae and cloning of a gene complementing the mutation; Maneesri J et al.; Screening for morphological mutants of a haploid strain of Saccharomyces cerevisiae was done on the basis of their cell-shape on a solid medium containing isoamyl alcohol, which causes cell elongation, to obtain information on the morphogenesis . Mutant J19, which had pseudohyphae in liquid medium even in the absence of isoamyl alcohol, had many elongated cells . Few reports exist of haploid cells growing as pseudohyphae in liquid culture . Cell-wall analysis showed that J19 had ordinary amounts of alkali-insoluble glucan and chitin, but that isoamyl alcohol in the medium caused structural changes in the cell wall . Addition of a DNA fragment that included the wild-type SCL1 gene to J19 complemented its morphological phenotype . Sequencing of J19 SCL1 showed that the glycine at position 226 in the Scl1 protein had been replaced by asparatic acid, suggesting that this mutation in the protein, a subunit of proteasomes, may be involved in the morphological change. J Biol Chem, 2003 Jul 11, 278(28), 25308 - 16 Epub 2003 Apr 29. The Saccharomyces cerevisiae COQ6 gene encodes a mitochondrial flavin-dependent monooxygenase required for coenzyme Q biosynthesis; Gin P et al.; Coenzyme Q (Q) is a lipid that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes . There are eight complementation groups of Q-deficient Saccharomyces cerevisiae mutants, designated coq1-coq8 . Here we have isolated the COQ6 gene by functional complementation and, in contrast to a previous report, find it is not an essential gene . coq6 mutants are unable to grow on nonfermentable carbon sources and do not synthesize Q but instead accumulate the Q biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid . The Coq6 polypeptide is imported into the mitochondria in a membrane potential-dependent manner . Coq6p is a peripheral membrane protein that localizes to the matrix side of the inner mitochondrial membrane . Based on sequence homology to known proteins, we suggest that COQ6 encodes a flavin-dependent monooxygenase required for one or more steps in Q biosynthesis. Protein Sci, 2003 May, 12(5), 1136 - 40 A novel member of the split betaalphabeta fold: Solution structure of the hypothetical protein YML108W from Saccharomyces cerevisiae; Pineda-Lucena A et al.; As part of the Northeast Structural Genomics Consortium pilot project focused on small eukaryotic proteins and protein domains, we have determined the NMR structure of the protein encoded by ORF YML108W from Saccharomyces cerevisiae . YML108W belongs to one of the numerous structural proteomics targets whose biological function is unknown . Moreover, this protein does not have sequence similarity to any other protein . The NMR structure of YML108W consists of a four-stranded beta-sheet with strand order 2143 and two alpha-helices, with an overall topology of betabetaalphabetabetaalpha . Strand beta1 runs parallel to beta4, and beta2:beta1 and beta4:beta3 pairs are arranged in an antiparallel fashion . Although this fold belongs to the split betaalphabeta family, it appears to be unique among this family; it is a novel arrangement of secondary structure, thereby expanding the universe of protein folds. Mol Genet Genomics, 2003 Apr, 269(1), 78 - 89 Epub 2003 Feb 12. Disruption of the Saccharomyces cerevisiae cell-wall pathway gene SLG1 causes hypersensitivity to the antitumor drug bleomycin; Leduc A et al.; Bleomycin is an antitumor drug that damages DNA via a free radical-dependent mechanism, and yeast mutants defective in DNA repair are hypersensitive to the drug . To identify possible pathways that may contribute to bleomycin resistance in yeast, we characterized a panel of bleomycin-sensitive mutants that were previously isolated by insertion mutagenesis using the transposon miniTn3::Leu2::LacZ::AMP( R) . One of these mutants harbored a single insertion in the SLG1 gene, which encodes a cell membrane protein that senses cell wall stress, and functions to maintain cell wall function by activating the protein kinase C signaling pathway . Deletion of the SLG1 gene in parental strains caused hypersensitivity to bleomycin, and this correlated with an accumulation of damaged DNA . A plasmid that expresses the native SLG1 gene or that increases PKC1 gene dosage restored bleomycin resistance to the slg1Delta mutant . Two-dimensional gel electrophoresis revealed that exposure to bleomycin triggered the expression of certain proteins, presumably to maintain cell wall function, in a Slg1-dependent manner . In addition, mutants lacking cell wall function were found to be hypersensitive to bleomycin . We conclude that mutants deficient in proteins that maintain cell wall function are severely compromised in their ability to limit bleomycin entry into the cell . Therefore, these mutants are burdened with increased genotoxicity upon exposure to bleomycin in the medium . Our results show that major mechanisms other than DNA repair are operating in yeast to mediate bleomycin resistance. Nucleic Acids Res, 2003 May 1, 31(9), 2289 - 96 Identification of stop codon readthrough genes in Saccharomyces cerevisiae; Namy O et al.; We specifically sought genes within the yeast genome controlled by a non-conventional translation mechanism involving the stop codon . For this reason, we designed a computer program using the yeast database genomic regions, and seeking two adjacent open reading frames separated only by a unique stop codon (called SORFs) . Among the 58 SORFs identified, eight displayed a stop codon bypass level ranging from 3 to 25% . For each of the eight sequences, we demonstrated the presence of a poly(A) mRNA . Using isogenic {PSI(+)} and {psi(-)} yeast strains, we showed that for two of the sequences the mechanism used is a bona fide readthrough . However, the six remaining sequences were not sensitive to the PSI state, indicating either a translation termination process independent of eRF3 or a new stop codon bypass mechanism . Our results demonstrate that the presence of a stop codon in a large ORF may not always correspond to a sequencing error, or a pseudogene, but can be a recoding signal in a functional gene . This emphasizes that genome annotation should take into account the fact that recoding signals could be more frequently used than previously expected. Fish Shellfish Immunol, 2003 May, 14(5), 375 - 88 A mannose-receptor is possibly involved in the phagocytosis of Saccharomyces cerevisiae by seabream (Sparus aurata L.) leucocytes; Rodriguez A et al.; In this paper the possible involvement of the mannose-receptor on the non-specific recognition and phagocytosis of heat killed yeast cells (Saccharomyces cerevisiae) by gilthead seabream (Sparus aurata L.) head-kidney leucocytes was established by studying the ability of different sugars to inhibit the uptake of the yeast cells by leucocytes . Leucocytes were preincubated for 30min with different concentrations of sugar (alpha-mannan, d-mannose, d-fucose, l-fucose, d-glucose, d-glucosamine and n-acetyl-glucosamine, all of them described as specific ligands of the vertebrate mannose-receptor) and afterwards incubated with FITC-labelled yeast cells for phagocytosis assays . The phagocytic ability (percentage of cells with one or more ingested yeast cells within the total cell population) and capacity (number of ingested yeast cells per cell) of leucocytes was analysed by flow cytometry . The results demonstrate the potential existence of a specific receptor-sugar or receptor-yeast cell binding process, which was saturable, specific and dose-dependent . More specifically, when leucocytes were preincubated with appropriate doses of d-mannose, d- or l-fucose, d-glucose or n-acetyl-glucosamine the phagocytosis of yeast cells by head-kidney leucocytes was partially blocked . Seabream leucocytes were also preincubated with chloroquine, a lysosomotropic drug which downregulates (in a nonspecific manner) the expression of mannose-receptors in mammals, before phagocytosis assays were performed . The results demonstrated that the phagocytosis of yeast was completely blocked by this substance . The overall results seem to corroborate the presence of the mannose-receptor in seabream phagocytes, which is involved in the non-specific binding and phagocytosis of yeast cells by head-kidney leucocytes. J Biol Chem, 2003 Jun 27, 278(26), 23610 - 6 Epub 2003 Apr 22. Phosphorylation of Saccharomyces cerevisiae CTP synthetase at Ser424 by protein kinases A and C regulates phosphatidylcholine synthesis by the CDP-choline pathway; Choi MG et al.; The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinases A and C . Previous studies have revealed that Ser424 is the target site for protein kinase A . Using a purified S424A mutant CTP synthetase enzyme, we examined the effect of Ser424 phosphorylation on protein kinase C phosphorylation . The S424A mutation in CTP synthetase caused a 50% decrease in the phosphorylation of the enzyme by protein kinase C and an 80% decrease in the stimulatory effect on CTP synthetase activity by protein kinase C . The S424A mutation caused increases in the apparent Km values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction . The effect of the S424A mutation on the phosphorylation reaction was dependent on time and protein kinase C concentration . A CTP synthetase synthetic peptide (SLGRKDSHSA) containing Ser424 was a substrate for protein kinase C . Comparison of phosphopeptide maps of the wild type and S424A mutant CTP synthetase enzymes phosphorylated by protein kinases A and C indicated that Ser424 was also a target site for protein kinase C . Phosphorylation of Ser424 accounted for 10% of the total phosphorylation of CTP synthetase by protein kinase C . The incorporation of {methyl-3H}choline into phosphocholine, CDP-choline, and phosphatidylcholine in cells carrying the S424A mutant CTP synthetase enzyme was reduced by 48, 32, and 46%, respectively, when compared with control cells . These data indicated that phosphorylation of Ser424 by protein kinase A or by protein kinase C was required for maximum phosphorylation and stimulation of CTP synthetase and that the phosphorylation of this site played a role in the regulation of phosphatidylcholine synthesis by the CDP-choline pathway. Eur J Biochem, 2003 May, 270(9), 2013 - 22 Saccharomyces cerevisiae Pip2p-Oaf1p regulates PEX25 transcription through an adenine-less ORE; Rottensteiner H et al.; The role of the Saccharomyces cerevisiae Pip2p-Oaf1p transcription factor was examined in reference to the regulation of the peroxin gene PEX25 involved in peroxisome proliferation . The PEX25 promoter contains an oleate response element (ORE)-like sequence comprising a CGG palindrome lacking a canonical adenine, which is considered critical for element function and Pip2p-Oaf1p binding . Pex25p levels were higher in wild-type cells grown on oleic acid medium than in those grown on ethanol, but this induction was abolished in cells devoid of Pip2p-Oaf1p . Studies based on lacZ reporter genes and in vitro protein-DNA interactions revealed that the PEX25 ORE could bind Pip2p-Oaf1p and confer activation on a basal promoter . These findings reinforced the central role played by Pip2p-Oaf1p in regulating peroxisome proliferation . We also investigated whether Pip2p-Oaf1p is important for regulating genes encoding peroxins involved in protein import into the peroxisomal matrix . Pip2p-Oaf1p was able to bind efficiently to the PEX5 ORE but not to an ORE-like CGG palindrome in the PEX14 promoter . However, immunoblotting revealed that both Pex5p and Pex14p (as well as Pex7p and Pex13p) were not more abundant in cells grown on oleic acid medium compared with ethanol . These data on a functional, adenine-less, PEX25 ORE and a nonfunctional N13-spaced ORE-like sequence in the PEX14 promoter capable of binding Pip2p-Oaf1p prompts readjustment of the ORE consensus to comprise CGGN3TNA/(R)N8-12CCG. Chromosoma, 2003 Apr, 111(7), 417 - 28 Epub 2003 Mar 18. Analysis of the distribution of the kinetochore protein Ndc10p in Saccharomyces cerevisiae using 3-D modeling of mitotic spindles; Muller-Reichert T et al.; Ndc10p is one of the DNA-binding constituents of the kinetochore in Saccharomyces cerevisiae but light microscopy analysis suggests that Ndc10p is not limited to kinetochore regions . We examined the localization of Ndc10p using immunoelectron microscopy and showed that Ndc10p is associated with spindle microtubules from S-phase through anaphase . By serial section reconstruction of mitotic spindles combined with immunogold detection, we showed that Ndc10p interacts with microtubules laterally as well as terminally . About 50% of the gold label in serial section reconstructions of short mitotic spindles was associated with the walls of spindle microtubules . Interaction of kinetochore components with microtubule walls was also shown for kinetochore protein Ndc80p . Our data suggest that at least a subset of kinetochore-associated protein is dispersed throughout the mitotic spindle. Genetics, 2003 Apr, 163(4), 1287 - 98 Mode of selection and experimental evolution of antifungal drug resistance in Saccharomyces cerevisiae; Anderson JB et al.; We show that mode of selection, degree of dominance of mutations, and ploidy are determining factors in the evolution of resistance to the antifungal drug fluconazole in yeast . In experiment 1, yeast populations were subjected to a stepwise increase in fluconazole concentration over 400 generations . Under this regimen, two mutations in the same two chromosomal regions rose to high frequency in parallel in three replicate populations . These mutations were semidominant and additive in their effect on resistance . The first of these mutations mapped to PDR1 and resulted in the overexpression of the ABC transporter genes PDR5 and SNQ2 . These mutations had an unexpected pleiotropic effect of reducing the residual ability of the wild type to reproduce at the highest concentrations of fluconazole . In experiment 2, yeast populations were subjected to a single high concentration of fluconazole . Under this regimen, a single recessive mutation appeared in each of three replicate populations . In a genome-wide screen of approximately 4700 viable deletion strains, 13 were classified as resistant to fluconazole (ERG3, ERG6, YMR102C, YMR099C, YPL056C, ERG28, OSH1, SCS2, CKA2, SML1, YBR147W, YGR283C, and YLR407W) . The mutations in experiment 2 all mapped to ERG3 and resulted in the overexpression of the gene encoding the drug target ERG11, but not PDR5 and SNQ2 . Diploid hybrids from experiments 1 and 2 were less fit than the parents in the presence of fluconazole . In a variation of experiment 2, haploids showed a higher frequency of resistance than diploids, suggesting that degree of dominance and ploidy are important factors in the evolution of antifungal drug resistance. Genetics, 2003 Apr, 163(4), 1273 - 86 Crossover interference in Saccharomyces cerevisiae requires a TID1/RDH54- and DMC1-dependent pathway; Shinohara M et al.; Two RecA-like recombinases, Rad51 and Dmc1, function together during double-strand break (DSB)-mediated meiotic recombination to promote homologous strand invasion in the budding yeast Saccharomyces cerevisiae . Two partially redundant proteins, Rad54 and Tid1/Rdh54, act as recombinase accessory factors . Here, tetrad analysis shows that mutants lacking Tid1 form four-viable-spore tetrads with levels of interhomolog crossover (CO) and noncrossover recombination similar to, or slightly greater than, those in wild type . Importantly, tid1 mutants show a marked defect in crossover interference, a mechanism that distributes crossover events nonrandomly along chromosomes during meiosis . Previous work showed that dmc1Delta mutants are strongly defective in strand invasion and meiotic progression and that these defects can be partially suppressed by increasing the copy number of RAD54 . Tetrad analysis is used to show that meiotic recombination in RAD54-suppressed dmc1Delta cells is similar to that in tid1; the frequency of COs and gene conversions is near normal, but crossover interference is defective . These results support the proposal that crossover interference acts at the strand invasion stage of recombination. FEMS Yeast Res, 2001 Apr, 1(1), 57 - 65 The essential and ancillary role of glutathione in Saccharomyces cerevisiae analysed using a grande gsh1 disruptant strain; Lee JC et al.; A grande gsh1 disruptant mutant of Saccharomyces cerevisiae was generated by crossing a petite disruptant to a wild-type grande strain . This strain was relatively stable, but generated petites at an elevated frequency, illustrating the ancillary role of glutathione (GSH) in the maintenance of the genetic integrity of the mitochondrial genome . The availability of the grande gsh1 deletant enabled an evaluation of the role of GSH in the cellular response to hydrogen peroxide independent of the effects of a petite mutation . The mutant strain was more sensitive to hydrogen peroxide than the wild-type strain but was still capable of producing an adaptive stress response to this compound . GSH was found to be essential for growth and sporulation of the yeast, but the intracellular level needed to support growth was at least two orders of magnitude less than that normally present in wild-type cells . This surprising result indicates that there is an essential role for GSH but only very low amounts are needed for growth . This result was also found in anaerobic conditions, thus this essential function does not involve protection from oxidative stress . Suppressors of the gsh1 deletion mutation were isolated by ethylmethanesulfonate mutagenesis . These were the result of a single recessive mutation (sgr1, suppressor for glutathione requirement) that relieved the requirement for GSH for growth on minimal medium but did not affect the sensitivity to H(2)O(2) stress . Interestingly, the gsh1 sgr1 mutant generated petites at a lower rate than the gsh1 mutant . Thus, it is suggested that the essential role of GSH is involved in the maintenance of the mitochondrial genome. FEMS Yeast Res, 2001 Apr, 1(1), 47 - 55 The impact of GAL6, GAL80, and MIG1 on glucose control of the GAL system in Saccharomyces cerevisiae; Ostergaard S et al.; The role of the proteins encoded by the GAL80 gene, the MIG1 gene and the GAL6 gene in glucose control of galactose consumption by Saccharomyces cerevisiae was studied by physiological characterisation of various GAL mutant strains . Dynamic experiments with the CEN.PK 113-7D wild-type strain and a deltagal80deltamig1 double-mutant strain in aerobic nitrogen-limited continuous cultivations at a dilution rate of 0.1 h(-1), showed simultaneous glucose and galactose consumption by the deltagal80deltamig1 strain . The wild-type strain did not consume galactose in the presence of glucose . Aerobic batch cultivations on glucose-galactose mixtures with the wild-type strain and with recombinant strains with a de-regulated GAL system (the deltagal80deltamig1 strain, a deltagal6 deleted strain, a deltagal6deltagal80deltamig1 triple mutant, and a deltagal6deltagal80deltamig1 triple mutant harbouring a GAL4 high-copy vector) were carried out . Generally, a reduction of glucose control lowered the maximum specific growth rate on glucose and increased the ethanol yield obtained on galactose with more than 100% . In contrast to the wild-type strain, the deltagal6deltagal80deltamig1 triple mutant strain consumed glucose and galactose simultaneously, and this strain also showed the highest ethanol production with an overall ethanol yield of 0.35 g g-1 sugar, which is 17% higher than the yield on glucose obtained with the wild-type strain . GAL80 and MIG1 were demonstrated to be responsible for the majority of the glucose control on the GAL system, whereas GAL6 has a minor role in glucose control . Deletion of GAL6 was shown to have a major impact on biomass and ethanol formation when cells were grown on galactose, and from the data obtained we speculate that Gal6 may be involved in mRNA degradation of the GAL gene transcripts. FEMS Yeast Res, 2001 Apr, 1(1), 33 - 45 The role of hexose transport and phosphorylation in cAMP signalling in the yeast Saccharomyces cerevisiae; Rolland F et al.; Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation . The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose . Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling . Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis . Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling . This indicates that a regulatory function may be associated with the hexose kinases . Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase . This may indicate that intracellular acidification can boost a downstream effect that amplifies the residual signal transmitted via the hexose kinases when glucose uptake is too low. FEMS Yeast Res, 2003 Apr, 3(2), 201 - 9 Chronological and replicative lifespan of polyploid Saccharomyces cerevisiae (syn . S . pastorianus); Maskell DL et al.; Chronological lifespan may be defined as the result of accumulation of irreversible damage to intracellular components during extended stationary phase, compromising cellular integrity and leading to death and autolysis . In contrast, replicative lifespan relates to the number of divisions an individual cell has undertaken before entering a non-replicative state termed senescence, leading to cell death and autolysis . Both forms of lifespan have been considered to represent models of ageing in higher eukaryotes, yet the relation between chronologically and replicatively aged populations has not been investigated . In this study both forms of lifespan have been investigated in Saccharomyces cerevisiae (Syn . S . pastorianus) to establish the relationship between chronological and replicative ageing. FEMS Yeast Res, 2001 Dec, 1(3), 225 - 32 Phospholipase D1 is required for efficient mating projection formation in Saccharomyces cerevisiae; Hairfield ML et al.; Phospholipase D1 (PLD1) is an important enzyme involved in lipid signal transduction in eukaryotes . A role for PLD1 in signaling in Saccharomyces cerevisiae was examined . Pheromone response in yeast is controlled by a well-characterized protein kinase cascade . Loss of PLD1 activity was found to impair pheromone-induced changes in cellular morphology that result in formation of mating projections . The rate at which projections appeared following pheromone treatment was delayed, suggesting that PLD1 facilitates the execution of a rate-limiting step in morphogenesis . Mutants were found to be less sensitive to pheromone, again arguing that PLD1 is acting at a rate-limiting step . The fact that morphogenesis is most dramatically affected indicates that PLD1 functions primarily in the morphogenic branch of the pheromone response pathway. FEMS Yeast Res, 2001 Dec, 1(3), 169 - 75 The glutamate synthase (GOGAT) of Saccharomyces cerevisiae plays an important role in central nitrogen metabolism; Guillamon JM et al.; Central nitrogen metabolism contains two pathways for glutamate biosynthesis, glutaminases and glutamate synthase (GOGAT), using glutamine as the sole nitrogen source . GOGAT's importance for cellular metabolism is still unclear . For a further physiological characterisation of the GOGAT function in central nitrogen metabolism, a GOGAT-negative (Deltaglt1) mutant strain (VWk274 LEU(+)) was studied in glutamine-limited continuous cultures . As reference, we did the same exper