J Biol Chem, 2004 Sep 10, 279(37), 38495 - 502 Epub 2004 Jul 13. The Ca2+ homeostasis defects in a pgm2Delta strain of Saccharomyces cerevisiae are caused by excessive vacuolar Ca2+ uptake mediated by the Ca2+-ATPase Pmc1p; Aiello DP et al.; Loss of the major isoform of phosphoglucomutase (PGM) causes an accumulation of glucose 1-phosphate when yeast cells are grown with galactose as the carbon and energy source . Remarkably, the pgm2Delta strain also exhibits a severe imbalance in intracellular Ca(2+) homeostasis when grown under these conditions . In the present study, we examined how the pgm2Delta mutation alters yeast Ca(2+) homeostasis in greater detail . We found that a shift from glucose to galactose as the carbon source resulted in a 2-fold increase in the rate of cellular Ca(2+) uptake in wild-type cells, whereas Ca(2+) uptake increased 8-fold in the pgm2Delta mutant . Disruption of the PMC1 gene, which encodes the vacuolar Ca(2+)-ATPase Pmc1p, suppressed the Ca(2+)-related phenotypes observed in the pgm2Delta strain . This suggests that excessive vacuolar Ca(2+) uptake is tightly coupled to these defects in Ca(2+) homeostasis . An in vitro assay designed to measure Ca(2+) sequestration into intracellular compartments confirmed that the pgm2Delta mutant contained a higher level of Pmc1p-dependent Ca(2+) transport activity than the wild-type strain . We found that this increased rate of vacuolar Ca(2+) uptake also coincided with a large induction of the unfolded protein response in the pgm2Delta mutant, suggesting that Ca(2+) uptake into the endoplasmic reticulum compartment was reduced . These results indicate that the excessive Ca(2+) uptake and accumulation previously shown to be associated with the pgm2Delta mutation are due to a severe imbalance in the distribution of cellular Ca(2+) into different intracellular compartments. FEBS Lett, 2004 Jul 16, 570(1-3), 155 - 60 P-domain and lectin site are involved in the chaperone function of Saccharomyces cerevisiae calnexin homologue; Xu X et al.; Cne1p, a calnexin homologue from Saccharomyces cerevisiae, has been shown to possess a conserved P-domain and lectin site as mammalian calnexin . The effect of P-domain and lectin site on the function of Cne1p was investigated in vitro using recombinant P-domain, P-domain deletion mutant of Cne1p, and lectin site mutant of Cne1ps (E181A and E398A) The binding of monoglucosylated oligosaccharide (G1M9) with Cne1p was clearly demonstrated using lectin site mutants . The P-domain deletion mutant and the letcin site mutants partially decreased the ability to suppress the aggregation of citrate synthase (CS) and chicken egg yolk immunoglobulin at levels different from Cne1p . Furthermore, the P-domain deletion mutant and the lectin site mutants decreased the ability to enhance the refolding of CS . These results suggest that the cooperation between the P-domain and the lectin site are important for the complete function of Cne1p . Thus, we conclude that P-domain in cooperation with the lectin site of Cne1p functions as a chaperone. Proc Natl Acad Sci U S A, 2004 Jul 20, 101(29), 10655 - 60 Epub 2004 Jul 12. Bipolar orientation of chromosomes in Saccharomyces cerevisiae is monitored by Mad1 and Mad2, but not by Mad3; Lee MS et al.; The spindle checkpoint governs the timing of anaphase separation of sister chromatids . In budding yeast, Mad1, Mad2, and Mad3 proteins are equally required for arrest in the presence of damage induced by antimicrotubule drugs or catastrophic loss of spindle structure . We find that the MAD genes are not equally required for robust growth in the presence of more subtle kinetochore and microtubule damage . A mad1Delta synthetic lethal screen identified 16 genes whose deletion in cells lacking MAD1 results in death or slow growth . Eleven of these mad1Delta genetic interaction partners encode proteins at the kinetochore-microtubule interface . Analysis of the entire panel revealed similar phenotypes in combination with mad2Delta . In contrast, 13 panel mutants exhibited a less severe phenotype in combination with mad3Delta . Checkpoint arrest in the absence of bipolar orientation and tension (induced by replication block in a cdc6 mutant) was lacking in cells without MAD1 or MAD2 . Cells without MAD3 were checkpoint-proficient . We conclude that Mad1 and Mad2 are required to detect bipolar orientation and/or tension at kinetochores, whereas Mad3 is not. Biochem J, 2004 Nov 15, 384(Pt 1), 47 - 58 Characterization of a transport and detoxification pathway for the antitumour drug bleomycin in Saccharomyces cerevisiae; Aouida M et al.; BLM (bleomycin) is effective in combination therapy against various cancers including testicular cancer . However, several other cancers such as colon cancer are refractory to BLM treatment . The exact mechanism for this differential response of cancer cells to the drug is not known . In the present study, we created fluorescently labelled BLM-A5, which retained nearly full genotoxic potential, and used this molecule to conduct the first study to understand the transport pathway of the drug in Saccharomyces cerevisiae . Uptake studies revealed that fluoro-BLM-A5 is transported into the cell in a concentration-dependent manner . Transport of a non-saturating concentration of fluoro-BLM-A5 was modest for the first 90 min, but thereafter it was sharply induced until 300 min . The inducible transport was completely abolished by the addition of cycloheximide, suggesting that BLM-A5 uptake into the cell is dependent on new protein synthesis . Interestingly, transport of fluoro-BLM-A5 was blocked if the cells were preincubated with increasing concentrations of spermine . Moreover, a mutant lacking the Ptk2 kinase, necessary for positively regulating polyamine transport, was defective in fluoro-BLM-A5 uptake and exhibited extreme resistance to the drug . A simple interpretation of these results is that BLM-A5 may enter the cell through the polyamine transport system . We showed further that after the uptake, fluoro-BLM-A5 accumulated into the vacuole of the parent, but localized to the cytoplasm of mutants disrupted for the END3 gene required for an early step of the endocytotic pathway . In general, mutants with a defect in the endocytic pathway to the vacuole were hypersensitive to BLM-A5 . We suggest that BLM-A5 is transported across the yeast plasma membrane and sequestered into the vacuole for detoxification. J Biol Chem, 2004 Sep 3, 279(36), 37512 - 7 Epub 2004 Jul 09. NPR1 kinase and RSP5-BUL1/2 ubiquitin ligase control GLN3-dependent transcription in Saccharomyces cerevisiae; Crespo JL et al.; The GATA transcription factors GLN3 and GAT1 activate nitrogen-regulated genes in Saccharomyces cerevisiae . NPR1 is a protein kinase that controls post-Golgi sorting of amino acid permeases . In the presence of a good nitrogen source, TOR (target of rapamycin) maintains GLN3 and NPR1 phosphorylated and inactive by inhibiting the type 2A-related phosphatase SIT4 . We identified NPR1 as a regulator of GLN3 . Specifically, loss of NPR1 causes nuclear translocation and activation of GLN3, but not GAT1, in nitrogen-rich conditions . NPR1-mediated inhibition of GLN3 is independent of the phosphatase SIT4 . We also demonstrate that the E3/E4 ubiquitin-protein ligase proteins RSP5 and BUL1/2 are required for GLN3 activation under poor nitrogen conditions . Thus, NPR1 and BUL1/2 antagonistically control GLN3-dependent transcription, suggesting a role for regulated ubiquitination in the control of nutrient-responsive transcription. Mol Biol Cell, 2004 Sep, 15(9), 4191 - 202 Epub 2004 Jul 07. Promoter-dependent roles for the Srb10 cyclin-dependent kinase and the Hda1 deacetylase in Tup1-mediated repression in Saccharomyces cerevisiae; Green SR et al.; The Tup1-Ssn6 complex has been well characterized as a Saccharomyces cerevisiae general transcriptional repressor with functionally conserved homologues in metazoans . These homologues are essential for cell differentiation and many other developmental processes . The mechanism of repression of all of these proteins remains poorly understood . Srb10 (a cyclin-dependent kinase associated with the Mediator complex) and Hda1 (a class I histone deacetylase) have each been implicated in Tup1-mediated repression . We present a statistically based genome-wide analysis that reveals that Hda1 partially represses roughly 30% of Tup1-repressed genes, whereas Srb10 kinase activity contributes to the repression of approximately 15% of Tup1-repressed genes . These effects only partially overlap, suggesting that different Tup1-repression mechanisms predominate at different promoters . We also demonstrate a distinction between histone deacetylation and transcriptional repression . In an HDA1 deletion, many Tup1-repressed genes are hyperacetylated at lysine 18 of histone H3, yet are not derepressed, indicating deacetylation alone is not sufficient to repress most Tup1-controlled genes . In a strain lacking both Srb10 and Hda1 functions, more than half of the Tup1-repressed genes are still repressed, suggesting that Tup1-mediated repression occurs by multiple, partially overlapping mechanisms, at least one of which is unknown. Genetics, 2004 Jun, 167(2), 607 - 17 Sal1p, a calcium-dependent carrier protein that suppresses an essential cellular function associated With the Aac2 isoform of ADP/ATP translocase in Saccharomyces cerevisiae; Chen XJ; Adenine nucleotide translocase (Ant) catalyzes ADP/ATP exchange between the cytosol and the mitochondrial matrix . It is also proposed to form or regulate the mitochondrial permeability transition pore, a megachannel of high conductancy on the mitochondrial membranes . Eukaryotic genomes generally contain multiple isoforms of Ant . In this study, it is shown that the Ant isoforms are functionally differentiated in Saccharomyces cerevisiae . Although the three yeast Ant proteins can equally support respiration (the R function), Aac2p and Aac3p, but not Aac1p, have an additional physiological function essential for cell viability (the V function) . The loss of V function in aac2 mutants leads to a lethal phenotype under both aerobic and anaerobic conditions . The lethality is suppressed by a strain-polymorphic locus, named SAL1 (for Suppressor of aac2 lethality) . SAL1 was identified to encode an evolutionarily conserved protein of the mitochondrial carrier family . Notably, the Sal1 protein was shown to bind calcium through two EF-hand motifs located on its amino terminus . Calcium binding is essential for the suppressor activity . Finally, Sal1p is not required for oxidative phosphorylation and its overexpression does not complement the R(-) phenotype of aac2 mutants . On the basis of these observations, it is proposed that Aac2p and Sal1p may define two parallel pathways that transport a nucleotide substrate in an operational mode distinct from ADP/ATP exchange. Genetics, 2004 Jun, 167(2), 579 - 91 The origin recognition complex links replication, sister chromatid cohesion and transcriptional silencing in Saccharomyces cerevisiae; Suter B et al.; Mutations in genes encoding the origin recognition complex (ORC) of Saccharomyces cerevisiae affect initiation of DNA replication and transcriptional repression at the silent mating-type loci . To explore the function of ORC in more detail, a screen for genetic interactions was undertaken using large-scale synthetic lethal analysis . Combination of orc2-1 and orc5-1 alleles with the complete set of haploid deletion mutants revealed synthetic lethal/sick phenotypes with genes involved in DNA replication, chromatin structure, checkpoints, DNA repair and recombination, and other genes that were unexpected on the basis of previous studies of ORC . Many of these genetic interactions are shared with other genes that are involved in initiation of DNA replication . Strong synthetic interactions were demonstrated with null mutations in genes that contribute to sister chromatid cohesion . A genetic interaction between orc5-1 and the cohesin mutant scc1-73 suggested that ORC function contributes to sister chromatid cohesion . Thus, comprehensive screening for genetic interactions with a replication gene revealed a connection between initiation of DNA replication and sister chromatid cohesion . Further experiments linked sister chromatid cohesion genes to silencing at mating-type loci and telomeres. Biochemistry, 2004 Jul 13, 43(27), 8878 - 84 Common chromatin architecture, common chromatin remodeling, and common transcription kinetics of Adr1-dependent genes in Saccharomyces cerevisiae; Agricola E et al.; The chromatin structure of several Saccharomyces cerevisiae ADR1-dependent genes was comparatively analyzed in vivo in order to evaluate the role of promoter architecture in transcriptional control . In repressing conditions (high glucose) a nucleosome particle always obstructs the TATA box, immediately adjacent to an upstream-located nucleosome-free region containing a cluster of Adr1 binding sites . Upon derepression the TATA box-containing nucleosome is destabilized according to a mechanism shared by all of the genes studied . The transcription factor Adr1 is always required for the observed chromatin remodeling . mRNA accumulation of all of the genes analyzed is strongly delayed in the absence of the acetyltransferase Gcn5 and is decreased in the presence of a temperature-sensitive Esa1 mutant . The results suggest that a defined promoter chromatin structure, controlled by DNA conformational features, is relevant for the activation of coregulated genes. Genome Res, 2004 Jul, 14(7), 1291 - 7 Recovery of a function involving gene duplication by retroposition in Saccharomyces cerevisiae; Schacherer J et al.; The duplication of DNA sequences contributes to genomic plasticity and is known to be one of the key factors responsible for evolution . The mechanisms underlying these rare events, which have been frequently mentioned by authors performing genomic analysis, have not yet been completely elucidated . These mechanisms were approached here in the yeast Saccharomyces cerevisiae, using a positive selection screen based on a particular mutated allele of the URA2 gene . Spontaneous revertants containing a duplication of the terminal part of the URA2 gene were selected and analyzed . Some important features of the duplicated regions, such as their chromosome location, size, and insertion sites, were characterized . The events selected correspond to a single inter- or intrachromosomal gene duplication process . The duplicated ATCase sequence is generally punctuated by a poly(A) tract and is always located in Ty1 sequences . In addition, the activation of a Ty1 transcription process increased the frequency of the duplication events . All in all, these data suggest that the duplication mechanism involves the reverse transcription of mRNA and the subsequent integration of the cDNA into a Ty1 area . The Ty1 elements and the retrotransposon-encoded function are key factors contributing to chromosomal reshaping . The genomic rearrangements described constitute experimental evidence for the recovery of a function involving duplication by retroposition . Mol Microbiol, 2004 Jul, 53(2), 469 - 83 Dissection of septin actin interactions using actin overexpression in Saccharomyces cerevisiae; Norden C et al.; Although many proteins can be overexpressed several fold without much effect on cell viability and morphology, some become toxic upon a slight increase in their intracellular level . This is particularly true for cytoskeletal proteins and has proven useful in the past for studying the cytoskeleton . In yeast, actin and tubulin are examples of proteins that cannot be overexpressed without affecting cell viability . Here, we have analysed the effect of actin overexpression in Saccharomyces cerevisiae . We show that actin overexpression interferes differently with distinct aspects of actin function . For example, two- to fourfold overexpression of actin did not affect the establishment of actin polarity, whereas it abrogated its maintenance . Also, actin structures that are barely visible in wild-type cells could be observed upon actin overexpression . This allowed us to identify a new ring-like actin structure genetically distinguishable from the actomyosin contractile ring . Formation of this actin structure upon actin overexpression was dependent on the septin cytoskeleton, the poorly understood cytokinetic protein Hof1 and the Arp2/3 complex . In contrast to the actomyosin ring, the ring formed upon actin overexpression required neither Myo1 nor formins for assembly . Therefore, we propose that Hof1 acts as a linker between actin and septins . Furthermore, we found that, in the absence of actin overexpression, a novel, Hof1-dependent actin belt is formed at the bud neck of anaphase cells . The physiological role of this belt might be related to that of the similar structure observed in dividing fission yeast. Biochem J, 2004 Oct 1, 383(Pt 1), 139 - 47 Absolute transcript levels of thioredoxin- and glutathione-dependent redox systems in Saccharomyces cerevisiae: response to stress and modulation with growth; Monje-Casas F et al.; We report the co-ordinated fine-tune of mRNA molecules that takes place in yeast (Saccharomyces cerevisiae) in response to diverse environmental stimuli . We performed a systematic and refined quantification of the absolute expression patterns of 16 genes coding for thioredoxin- and glutathione-dependent redox system components . Quantifications were performed to examine the response to oxidants, to sudden temperature upshifts and in association with metabolic changes accompanying culture growth and to explore the contribution of mRNA decay rates to the differences observed in basal expression levels . Collectively, these quantifications show (i) vast differences in the steady-state amounts of the investigated transcripts, cTPxI being largely overexpressed compared with GPX1 during the exponential phase and GPX2 beyond this growth stage; (ii) drastic changes in the relative abundance of the transcripts in response to oxidants and heat shock; and (iii) a unique temporal expression profile for each transcript as cells proceed from exponential to stationary growth phase, yet with some general trends such as maximal or near-maximal basal amounts of most mRNA species at early growth stages when glucose concentration is high and cells are actively growing . Moreover, the results indicate that (i) the half-lives of the investigated transcripts are longer and distributed within a narrower range than previously reported global mRNA half-lives and (ii) transcriptional initiation may play an important role in modulating the significant alterations that most mRNAs exhibit in their steady-state levels along with culture growth. Biochemistry, 2004 Jul 6, 43(26), 8439 - 46 Direct evidence for the interaction of stigmatellin with a protonated acidic group in the bc(1) complex from Saccharomyces cerevisiae as monitored by FTIR difference spectroscopy and 13C specific labeling; Ritter M et al.; In this study a combined electrochemical and FTIR spectroscopic approach was applied to monitor the binding of stigmatellin, a Q(o) site inhibitor of the cytochrome bc(1) complex from Saccharomyces cerevisiae . Natural stigmatellin A induced clear shifts in the redox-induced FTIR difference spectra . For data interpretation a stigmatellin derivative (UST) with the conjugated trienes replaced by an aliphatic tail was synthesized, and the carbonyl group shown in crystal structures to interact with His181, the {2Fe-2S} ligand of the Rieske, was specifically (13)C labeled . Electrochemically induced FTIR difference spectra of the inhibitors in CH(3)OD were obtained and revealed signals characteristic for the oxidized and reduced forms of the labeled and unlabeled compounds . On the basis of signals from the inhibitors alone, the binding of the inhibitor to the bc(1) complex was monitored . Direct evidence for the interaction of the carbonyl group with the protein was provided by the observed shift of the nu(C=O) vibrational mode of about 10 cm(-1) . In addition, redox-dependent reorganizations of the protein were identified, including protonation changes of acidic residues at 1746 and 1734 cm(-1) . The conformational changes observed upon inhibitor binding are discussed with respect to the crystal structures and proposed mechanistic models {Hunte, C., Koepke, J., Lange, C., Rossmanith, T., and Michel, H . (2000) Structure 8, 669-684; Palsdottir, H., Lojero, C . G., Trumpower, B . L., and Hunte, C . (2003) J . Biol . Chem . 278, 31303-31311}. Mol Genet Genomics, 2004 Jul, 271(6), 697 - 708 Epub 2004 Jun 22. Combinatorial control by the protein kinases PKA, PHO85 and SNF1 of transcriptional induction of the Saccharomyces cerevisiae GSY2 gene at the diauxic shift; Enjalbert B et al.; Genes involved in storage carbohydrate metabolism are coordinately induced when yeast cells are subjected to conditions of stress, or when they exit the exponential growth phase on glucose . We show that the STress Responsive Elements (STREs) present in the promoter of GSY2 are essential for gene activation under conditions of stress, but dispensable for gene induction and glycogen accumulation at the diauxic shift on glucose . Using serial promoter deletion, we found that the latter induction could not be attributed to a single cis -regulatory sequence, and present evidence that this mechanism depends on combinatorial transcriptional control by signalling pathways involving the protein kinases Pho85, Snf1 and PKA . Two contiguous regions upstream of the GSY2 coding region are necessary for negative control by the cyclin-dependent protein kinase Pho85, one of which is a 14-bp G/C-rich sequence . Positive control by Snf1 is mediated by Mig1p, which acts indirectly on the distal part of the GSY2 promoter . The PKA pathway has the most pronounced effect on GSY2, since transcription of this gene is almost completely abolished in an ira1ira2 mutant strain in which PKA is hyperactive . The potent negative effect of PKA is dependent upon a branched pathway involving the transcription factors Msn2/Msn4p and Sok2p . The SOK2 branch was found to be effective only under conditions of high PKA activity, as in a ira1ira2 mutant, and this effect was independent of Msn2/4p . The Msn2/4p branch, on the other hand, positively controls GSY2 expression directly through the STREs, and indirectly via a factor that still remains to be discovered . In summary, this study shows that the transcription of GSY2 is regulated by several different signalling pathways which reflect the numerous factors that influence glycogen synthesis in yeast, and suggests that the PKA pathway must be deactivated to allow gene induction at the diauxic shift. J Biol Chem, 2004 Sep 10, 279(37), 39165 - 74 Epub 2004 Jun 25. The Reg1-interacting proteins, Bmh1, Bmh2, Ssb1, and Ssb2, have roles in maintaining glucose repression in Saccharomyces cerevisiae; Dombek KM et al.; In Saccharomyces cerevisiae, a type 1 protein phosphatase complex composed of the Glc7 catalytic subunit and the Reg1 regulatory subunit represses expression of many glucose-regulated genes . Here we show that the Reg1-interacting proteins Bmh1, Bmh2, Ssb1, and Ssb2 have roles in glucose repression . Deleting both BMH genes causes partially constitutive ADH2 expression without significantly increasing the level of Adr1 protein, the major activator of ADH2 expression . Adr1 and Bcy1, the regulatory subunit of cAMP-dependent protein kinase, are both required for this effect indicating that constitutive expression in Deltabmh1Deltabmh2 cells uses the same activation pathway that operates in Deltareg1 cells . Deletion of both BMH genes and REG1 causes a synergistic relief from repression, suggesting that Bmh proteins also act independently of Reg1 during glucose repression . A two-hybrid interaction with the Bmh proteins was mapped to amino acids 187-232, a region of Reg1 that is conserved in different classes of fungi . Deleting this region partially releases SUC2 from glucose repression . This indicates a role for the Reg1-Bmh interaction in glucose repression and also suggests a broad role for Bmh proteins in this process . An in vivo Reg1-Bmh interaction was confirmed by copurification of Bmh proteins with HA(3)-TAP-tagged Reg1 . The nonconventional heat shock proteins Ssb1 and Ssb2 are also copurified with HA(3)-TAP-tagged Reg1 . Deletion of both SSB genes modestly decreases repression of ADH2 expression in the presence of glucose, suggesting that Ssb proteins, perhaps through their interaction with Reg1, play a minor role in glucose repression. J Biol Chem, 2004 Aug 27, 279(35), 36906 - 15 Epub 2004 Jun 25. Functional characterization of the eukaryotic cysteine desulfurase Nfs1p from Saccharomyces cerevisiae; Muhlenhoff U et al.; Previous studies have indicated that the essential protein Nfs1 performs a crucial role in cellular iron-sulfur (Fe/S) protein maturation . The protein is located predominantly in mitochondria, yet low amounts are present in cytosol and nucleus . Here we examined several aspects concerning the molecular function of yeast Nfs1p as a model protein . First, we demonstrated that purified Nfs1p facilitates the in vitro assembly of Fe/S proteins by using cysteine as its specific substrate . Thus, eukaryotic Nfs1 is a functional orthologue of the bacterial cysteine desulfurase IscS . Second, we showed that only the mitochondrial version but not the extramitochondrial version of Nfs1p is functional in generating cytosolic and nuclear Fe/S proteins . Mutation of the nuclear targeting signal of Nfs1p did not affect the maturation of cytosolic and nuclear Fe/S proteins, despite a severe growth defect under this condition . Nfs1p could not assemble an Fe/S cluster on the Isu scaffold proteins when they were located in the yeast cytosol . The lack of function of these central Fe/S cluster assembly components suggests that the maturation of extramitochondrial Fe/S protein does not involve functional copies of the mitochondrial Fe/S cluster assembly machinery in the yeast cytosol . Third, the extramitochondrial version of Nfs1p was shown to play a direct role in the thiomodification of tRNAs . Finally, we identified a highly conserved N-terminal beta-sheet of Nfs1p as a functionally essential part of the protein . The implication of these findings for the structural stability of Nfs1p and for its targeting mechanism to mitochondria and cytosol/nucleus will be discussed. Mol Biol Cell, 2004 Sep, 15(9), 3977 - 85 Epub 2004 Jun 23. Pxl1p, a paxillin-like protein in Saccharomyces cerevisiae, may coordinate Cdc42p and Rho1p functions during polarized growth; Gao XD et al.; Rho-family GTPases Cdc42p and Rho1p play critical roles in the budding process of the yeast Saccharomyces cerevisiae . However, it is not clear how the functions of these GTPases are coordinated temporally and spatially during this process . Based on its ability to suppress cdc42-Ts mutants when overexpressed, a novel gene PXL1 was identified . Pxl1p resembles mammalian paxillin, which is involved in integrating various signaling events at focal adhesion . Both proteins share amino acid sequence homology and structural organization . When expressed in yeast, chicken paxillin localizes to the sites of polarized growth as Pxl1p does . In addition, the LIM domains in both proteins are the primary determinant for targeting the proteins to the cortical sites in their native cells . These data strongly suggest that Pxl1p is the "ancient paxillin" in yeast . Deletion of PXL1 does not produce any obvious phenotype . However, Pxl1p directly binds to Rho1p-GDP in vitro, and inhibits the growth of rho1-2 and rho1-3 mutants in a dosage-dependent manner . The opposite effects of overexpressed Pxl1p on cdc42 and rho1 mutants suggest that the functions of Cdc42p and Rho1p may be coordinately regulated during budding and that Pxl1p may be involved in this coordination. Cell Mol Biol Lett, 2004, 9(2), 271 - 86 Alpha-Ketoglutarate dehydrogenase and lipoic acid synthase are important for the functioning of peroxisomes of Saccharomyces cerevisiae; Smaczynska-de Rooij I et al.; A method was devised to search for yeast mutants impaired in peroxisome functioning, indicating cross-talk between metabolic pathways . Two mutants were isolated; they are impaired in oleate utilisation and carry mutations in the KGD1 and LIP5 genes encoding the E1 component of the mitochondrial alpha-ketoglutarate dehydrogenase complex and lipoic acid synthase, respectively . The results presented indicate that the Kgd1 and Lip5 proteins are important for the expression of genes encoding peroxisomal matrix proteins, although they are not necessary for the biogenesis of this cellular compartment. J Biol Chem, 2004 Aug 20, 279(34), 35469 - 78 Epub 2004 Jun 21. Gle2p is essential to induce adaptation of the export of bulk poly(A)+ mRNA to heat shock in Saccharomyces cerevisiae; Izawa S et al.; The export of bulk poly(A)(+) mRNA is blocked under heat-shocked (42 degrees C) conditions in Saccharomyces cerevisiae . We found that an mRNA export factor Gle2p rapidly dissociated from the nuclear envelope and diffused into the cytoplasm at 42 degrees C . However, in exponential phase cells pretreated with mild heat stress (37 degrees C for 1 h), Gle2p did not dissociate at 42 degrees C, and the export of bulk poly(A)(+) mRNA continued . Cells in stationary phase also continued with the export of bulk poly(A)(+) mRNA at 42 degrees C without the dissociation of Gle2p from the nuclear envelope . The dissociation of Gle2p was caused by increased membrane fluidity and correlated closely with blocking of the export of bulk poly(A)(+) mRNA . Furthermore, the mutants gle2Delta and rip1Delta could not induce such an adaptation of the export of bulk poly(A)(+) mRNA to heat shock . Our findings indicate that Gle2p plays a crucial role in mRNA export especially under heat-shocked conditions . Our findings also indicate that the nuclear pore complexes that Gle2p constitutes need to be stabilized for the adaptation and that the increased membrane integrity caused by treatment with mild heat stress or by survival in stationary phase is likely to contribute to the stabilization of the association between Gle2p and the nuclear pore complexes. Acta Pharmacol Sin, 2004 Jul, 25(7), 907 - 14 Type I methionine aminopeptidase from Saccharomyces cerevisiae is a potential target for antifungal drug screening; Chen LL et al.; AIM: To screen antifungal drug candidates using in vitro and in vivo assays based on type I methionine aminopeptidase from Saccharomyces cerevisiae (ScMetAP1) . METHODS: A colorimetric assay suitable for high throughput screening (HTS) using recombinant ScMetAP1 protein expressed in Escherichia coli was established for antifungal lead discovery . A series of pyridine-2-carboxylic acid derivatives were characterized and a chemical library of 12,800 pure organic compounds was screened with the in vitro ScMetAP1 assay . Active compounds from the in vitro assay were further evaluated by a growth inhibition assay on yeast strain with deletion of ScMetAP1 gene map1 in comparison with the wild-type yeast strain and the yeast strain with deletion of type II enzyme (ScMetAP2) gene map2 . RESULTS: Active ScMetAP1 inhibitors were identified from HTS . Some of the pyridine-2-carboxylic acid derivatives (compound 2 and 3) had selective inhibition of the growth of map2 deletion yeast and weak inhibition on wild-type yeast growth, while no inhibition on map1 deletion yeast . CONCLUSION: ScMetAP1 is a novel potential target for developing antifungal drugs . The in vitro and in vivo ScMetAP1 assays can serve as tools in discovering antifungal drug candidates. Biochemistry, 2004 Jun 29, 43(25), 8107 - 15 Saccharomyces cerevisiae Hsp104 enhances the chaperone capacity of human cells and inhibits heat stress-induced proapoptotic signaling; Mosser DD et al.; Hsp104, the most potent thermotolerance factor in Saccharomyces cerevisiae, is an unusual molecular chaperone that is associated with the dispersal of aggregated, non-native proteins in vivo and in vitro . The close cooperation between Hsp100 oligomeric disaggregases and specific Hsp70 chaperone/cochaperone systems to refold and reactivate heat-damaged proteins has been dubbed a "bichaperone network" . Interestingly, animal genomes do not encode a Hsp104 ortholog . To investigate the biochemical and biological consequences of introducing into human cells a stress tolerance factor that has protein refolding capabilities distinct from those already present, Hsp104 was expressed as a transgene in a human leukemic T-cell line (PEER) . Hsp104 inhibited heat-shock-induced loss of viability in PEER cells, and this action correlated with reduced procaspase-3 cleavage but not with reduced c-Jun N-terminal kinase phosphorylation . Hsp104 cooperated with endogenous human Hsp70 and Hsc70 molecular chaperones and their J-domain-containing cochaperones Hdj1 and Hdj2 to produce a functional hybrid bichaperone network capable of refolding aggregated luciferase . We also established that Hsp104 shuttles across the nuclear envelope and enhances the chaperoning capacity of both the cytoplasm and nucleoplasm of intact cells . Our results establish the fundamental properties of protein disaggregase function in human cells with implications for the use of Hsp104 or related proteins as therapeutic agents in diseases associated with protein aggregation. RNA, 2004 Jul, 10(7), 1073 - 83 Npa1p is an essential trans-acting factor required for an early step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae; Rosado IV et al.; Ribosome biogenesis requires >100 nonribosomal proteins, which are associated with different preribosomal particles . The substrates, the interacting partners, and the timing of action of most of these proteins are largely unknown . To elucidate the functional environment of the putative ATP-dependent RNA helicase Dbp6p from Saccharomyces cerevisiae, which is required for 60S ribosomal subunit assembly, we have previously performed a synthetic lethal screen and thereby revealed a genetic interaction network between Dbp6p, Rpl3p, Nop8p, and the novel Rsa3p . In this report, we extended the characterization of this functional network by performing a synthetic lethal screen with the rsa3 null allele . This screen identified the so far uncharacterized Npa1p (YKL014C) . Polysome profile analysis indicates that there is a deficit of 60S ribosomal subunits and an accumulation of halfmer polysomes in the slowly growing npa1-1 mutant . Northern blotting and primer extension analysis shows that the npa1-1 mutation negatively affects processing of all 27S pre-rRNAs and the normal accumulation of both mature 25S and 5.8S rRNAs . In addition, 27SA(2) pre-rRNA is prematurely cleaved at site C(2) . Moreover, GFP-tagged Npa1p localizes predominantly to the nucleolus and sediments with large complexes in sucrose gradients, which most likely correspond to pre-60S ribosomal particles . We conclude that Npa1p is required for ribosome biogenesis and operates in the same functional environment of Rsa3p and Dbp6p during early maturation of 60S ribosomal subunits. Res Microbiol, 2004 Jun, 155(5), 301 - 10 Experimental studies of deleterious mutation in Saccharomyces cerevisiae; Korona R; Yeast has proven to be a very useful model organism for studying eukaryotic cell functions . Its applicability for population and quantitative genetics is less well known . Among its advantages is the ease of screening for mutants . The present paper reviews experiments aimed at estimating the parameters of spontaneous mutations deleterious to fitness . The rate of deleterious mutation was found to be moderately high . A large fraction of detectable mutants were lethal; among the non-lethal mutants, the least harmful ones dominated . Deleterious mutations, and especially the lethal ones, were generally very well masked by wild-type alleles when in heterozygous loci . The negative effects of mutations were much stronger under stressful than under benign conditions . Interactions between loci with deleterious mutations did alter their fitness, but no strong overall effect of synergism or antagonisms was observed. J Biol Chem, 2004 Aug 27, 279(35), 36354 - 62 Epub 2004 Jun 16. Mapping subunit location on the Saccharomyces cerevisiae origin recognition complex free and bound to DNA using a novel nanoscale biopointer; Chastain PD 2nd et al.; The Saccharomyces cerevisiae origin recognition complex (ORC) is composed of six subunits and is an essential component in the assembly of the replication apparatus . To probe the organization of this multiprotein complex by electron microscopy, each subunit was tagged on either its C or N terminus with biotin and assembled into a complex with the five other unmodified subunits . A nanoscale biopointer consisting of a short DNA duplex with streptavidin at one end was used to map the location of the N and C termini of each subunit . These observations were made using ORC free in solution and bound to the ARS1 origin of replication . This mapping confirms and extends previous studies mapping the sites of subunit interaction with origin DNA . In particular, we provide new information concerning the stoichiometry of the ORC-ARS1 complex and the changes in conformation that are associated with DNA binding by ORC . This versatile, new approach to mapping protein structure has potential for many applications. J Antimicrob Chemother, 2004 Jul, 54(1), 46 - 55 Epub 2004 Jun 16. Response of Saccharomyces cerevisiae to a monoterpene: evaluation of antifungal potential by DNA microarray analysis; Parveen M et al.; Plant-derived essential oils with monoterpenoids have been used as antifungal drugs since ancient times, but the mode of action of these natural hydrocarbons at the molecular level is not understood . In order to understand the mechanisms of toxicity of alpha-terpinene (a cyclic monoterpene), a culture of Saccharomyces cerevisiae was exposed to 0.02% alpha-terpinene for 2 h and transcript profiles were obtained using yeast DNA arrays . These profiles, when compared with transcript profiles of untreated cultures, revealed that the expression of 793 genes was affected . For 435 genes, mRNA levels in treated cells compared with control cells differed by more than two-fold, whereas for 358 genes, it was <0.5-fold . Northern blots were performed for selected genes to verify the microarray results . Functional analysis of the up-regulated genes indicates that, similar to commonly used antifungal drugs, alpha-terpinene exposure affected genes involved in ergosterol biosynthesis and sterol uptake . In addition, transcriptional induction of genes related to lipid metabolism, cell wall structure and function, detoxification and cellular transport was observed in response to terpinene toxicity . Notably, the functions of 192 up-regulated genes are still unknown, but their characterization will probably shed light on the mechanisms of drug resistance and sensitivity . Taken together, this study showed that alpha-terpinene has strong antifungal activities and its modes of action resemble those of presently used antifungal drugs. J Mol Biol, 2004 Jul 2, 340(2), 195 - 202 Mcm10 and Cdc45 cooperate in origin activation in Saccharomyces cerevisiae; Sawyer SL et al.; Mcm10 has recently been found to play a crucial role in multiple steps of the DNA replication initiation process in eukaryotes . Here, we have examined the role of Mcm10 in assembling initiation factors at a well-characterized yeast replication origin, ARS1 . We find that the pre-replication complex (pre-RC) components Cdc6 and Mcm7 associate with ARS1 in the mcm10-1 mutant, suggesting that establishment of the pre-RC is not compromised in this mutant . Association of Cdc45 with ARS1 is reduced in the mcm10-1 mutant, suggesting that Mcm10 is involved in recruiting Cdc45 to the pre-RC . We find that overexpression of either Mcm10-1 or Cdc45 suppresses the growth defect of mcm10-1, and that a physical interaction between Cdc45 and Mcm10 is disrupted in the mcm10-1 mutant . Our results show that interaction between the Mcm10 and Cdc45 proteins facilitates the recruitment of Cdc45 onto the ARS1 origin. Genes Dev, 2004 Jun 15, 18(12), 1439 - 51 Spindle checkpoint regulates Cdc20p stability in Saccharomyces cerevisiae; Pan J et al.; The spindle checkpoint arrests cells at the metaphase-to-anaphase transition until all chromosomes have properly attached to the mitotic spindle . Checkpoint proteins Mad2p and Mad3p/BubR1p bind and inhibit Cdc20p, an activator for the anaphase-promoting complex (APC) . We find that upon spindle checkpoint activation by microtubule inhibitors benomyl or nocodazole, wild-type Saccharomyces cerevisiae contains less Cdc20p than spindle checkpoint mutants do, whereas their CDC20 mRNA levels are similar . The difference in Cdc20p levels correlates with their difference in the half-lives of Cdc20p, indicating that the spindle checkpoint destabilizes Cdc20p . This process requires the association between Cdc20p and Mad2p, and functional APC, but is independent of the known destruction boxes in Cdc20p and the other APC activator Cdh1p . Importantly, destabilization of Cdc20p is important for the spindle checkpoint, because a modest overexpression of Cdc20p causes benomyl sensitivity and premature Pds1p degradation in cells treated with nocodazole . Our study suggests that the spindle checkpoint reduces Cdc20p to below a certain threshold level to ensure a complete inhibition of Cdc20p before anaphase. Yeast, 2004 Jun, 21(8), 661 - 70 Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae; Sheff MA et al.; Green fluorescent protein (GFP) has become an increasingly popular protein tag for determining protein localization and abundance . With the availability of GFP variants with altered fluorescence spectra, as well as GFP homologues from other organisms, multi-colour fluorescence with protein tags is now possible, as is measuring protein interactions using fluorescence resonance energy transfer (FRET) . We have created a set of yeast tagging vectors containing codon-optimized variants of GFP, CFP (cyan), YFP (yellow), and Sapphire (a UV-excitable GFP) . These codon-optimized tags are twice as detectable as unoptimized tags . We have also created a tagging vector containing the monomeric DsRed construct tdimer2, which is up to 15-fold more detectable than tags currently in use . These tags significantly improve the detection limits for live-cell fluorescence imaging in yeast, and provide sufficient distinguishable fluorophores for four-colour imaging . Genome Res, 2004 Jul, 14(7), 1298 - 309 Epub 2004 Jun 14. Reconstruction and validation of Saccharomyces cerevisiae iND750, a fully compartmentalized genome-scale metabolic model; Duarte NC et al.; A fully compartmentalized genome-scale metabolic model of Saccharomyces cerevisiae that accounts for 750 genes and their associated transcripts, proteins, and reactions has been reconstructed and validated . All of the 1149 reactions included in this in silico model are both elementally and charge balanced and have been assigned to one of eight cellular locations (extracellular space, cytosol, mitochondrion, peroxisome, nucleus, endoplasmic reticulum, Golgi apparatus, or vacuole) . When in silico predictions of 4154 growth phenotypes were compared to two published large-scale gene deletion studies, an 83% agreement was found between iND750's predictions and the experimental studies . Analysis of the failure modes showed that false predictions were primarily caused by iND750's limited inclusion of cellular processes outside of metabolism . This study systematically identified inconsistencies in our knowledge of yeast metabolism that require specific further experimental investigation . J Biol Chem, 2004 Aug 13, 279(33), 34421 - 30 Epub 2004 Jun 10. Functional characterization of the Saccharomyces cerevisiae VHS3 gene: a regulatory subunit of the Ppz1 protein phosphatase with novel, phosphatase-unrelated functions; Ruiz A et al.; The yeast gene VHS3 (YOR054c) has been recently identified as a multicopy suppressor of the G(1)/S cell cycle blockade of a conditional sit4 and hal3 mutant . Vhs3 is structurally related to Hal3, a negative regulatory subunit of the Ser/Thr protein phosphatase Ppz1 important for cell integrity, salt tolerance, and cell cycle control . Phenotypic analyses using vhs3 mutants and overexpressing strains clearly show that Vhs3 has functions reminiscent to those of Hal3 and contrary to those of Ppz1 . Mutation of Vhs3 His(459), equivalent to the supposedly functionally relevant His(90) in the plant homolog AtHal3a, did not affect Vhs3 functions mentioned above . Similarly to Hal3, Vhs3 binds in vivo to the C-terminal catalytic moiety of Ppz1 and inhibits in vitro its phosphatase activity . Therefore, our results indicate that Vhs3 plays a role as an inhibitory subunit of Ppz1 . We have found that the vhs3 and hal3 mutations are synthetically lethal . Remarkably, lethality is not suppressed by deletion of PPZ1, PPZ2, or both phosphatase genes, indicating that it is not because of an excess of Ppz phosphatase activity . Furthermore, a Vhs3 version carrying the H459A mutation did not rescue the synthetically lethal phenotype . A conditional vhs3 tetO:HAL3 double mutant displays, in the presence of doxycycline, a flocculation phenotype that is dependent on the presence of Flo8 and Flo11 . These results indicate that, besides its role as Ppz1 inhibitory subunit, Vhs3 (and probably Hal3) might have important Ppz-independent functions. J Biol Chem, 2004 Aug 13, 279(33), 34865 - 72 Epub 2004 Jun 10. Genome-wide analysis of ARS (autonomously replicating sequence) binding factor 1 (Abf1p)-mediated transcriptional regulation in Saccharomyces cerevisiae; Miyake T et al.; Autonomously replicating sequence-binding factor-1 (Abf1p) is an essential sequence-specific transcription factor in Saccharomyces cerevisiae that participates in multiple nuclear events including DNA replication, transcription activation, and gene silencing . Numerous gene-specific analyses have implicated Abf1p in the transcriptional control of genes involved in a diverse range of cellular functions, leading to the notion that Abf1p acts as a global transcriptional regulator . Here we report findings from a genome-wide comparison of the gene expression profiles in the wild-type and abf1-1 temperature-sensitive mutant . The study identifies a total of 86 Abf1p-regulated genes (1.4% of the genome) of which 50 are activated and 36 are repressed by Abf1p . Interestingly, Abf1p binds to its own promoter in vivo and strongly represses its own transcription, suggesting a potential negative regulatory loop in Abf1p-mediated gene regulation . A comparison of our microarray data with the available databases reveals a significant overlap of genes regulated by Abf1p and those by several general transcription factors such as Mot1p and TAFs (TATA-binding protein-associated factors) . Different mutant alleles of abf1 affect Abf1p-mediated transcription in a gene-dependent manner . Furthermore, Abf1p in vivo is associated with the promoter region of most Abf1p-activated but not with that of most Abf1p-repressed genes . Taken together, these results strongly suggest distinct underlying mechanisms by which Abf1p regulates gene expression. J Biol Chem, 2004 Aug 27, 279(35), 36720 - 31 Epub 2004 Jun 09. Genetic, biochemical, and transcriptional responses of Saccharomyces cerevisiae to the novel immunomodulator FTY720 largely mimic those of the natural sphingolipid phytosphingosine; Welsch CA et al.; Sphingolipids are signaling molecules that influence diverse cellular functions from control of the cell cycle to degradation of plasma membrane proteins . The synthetic sphingolipid-like compound FTY720 is an immunomodulating agent in clinical trials for transplant graft maintenance . In this report, we compare the effects of the natural yeast sphingolipid phytosphingosine with FTY720 in Saccharomyces cerevisiae . We show that the multicopy suppressor genes that induce growth resistance to FTY720 also confer resistance to growth-inhibitory concentrations of phytosphingosine . In addition, mutants for ubiquitination pathway proteins are shown to be resistant to the growth-inhibiting effect of both FTY720 and phytosphingosine . We observe fewer similarities between sphingosine and FTY720 than between FTY720 and phytosphingosine as revealed by genetic studies . Yeast cells lacking the specific sphingosine kinase LCB4 are sensitive to phytosphingosine and FTY720 but resistant to sphingosine, suggesting that FTY720 and phytosphingosine have a more related mechanism of action . Gene expression profile comparisons of sensitive and resistant yeast cells exposed to FTY720 and phytosphingosine highlight a number of similarities . In response to treatment with these compounds, approximately 77% of the genes that are regulated >2-fold by FTY720 also respond to phytosphingosine in the same direction in the parent strain . In addition, a close inspection of TAT1 and TAT2 transporters following exposure to phytosphingosine indicates that TAT1 protein is degraded in a similar fashion upon treatment with FTY720 and phytosphingosine . There were differences, however, with respect to the TAT2 protein level and the expression profiles of a subset of genes . The genetic, transcriptional, and biochemical data together indicate that FTY720 and phytosphingosine influence similar pathways in yeast cells . These findings offer further insights into the physiological pathways influenced by these compounds in all eukaryotic cells and help us to understand the therapeutic consequences of FTY720 in humans. Eukaryot Cell, 2004 Jun, 3(3), 695 - 704 Hph1p and Hph2p, novel components of calcineurin-mediated stress responses in Saccharomyces cerevisiae; Heath VL et al.; Calcineurin is a Ca2+- and calmodulin-dependent protein phosphatase that plays a key role in animal and yeast physiology . In the yeast Saccharomyces cerevisiae, calcineurin is required for survival during several environmental stresses, including high concentrations of Na+, Li+, and Mn2+ ions and alkaline pH . One role of calcineurin under these conditions is to activate gene expression through its regulation of the Crz1p transcription factor . We have identified Hph1p as a novel substrate of calcineurin . HPH1 (YOR324C) and its homolog HPH2 (YAL028W) encode tail-anchored integral membrane proteins that interact with each other in the yeast two-hybrid assay and colocalize to the endoplasmic reticulum . Hph1p and Hph2p serve redundant roles in promoting growth under conditions of high salinity, alkaline pH, and cell wall stress . Calcineurin modifies the distribution of Hph1p within the endoplasmic reticulum and is required for full Hph1p activity in vivo . Furthermore, calcineurin directly dephosphorylates Hph1p and interacts with it through a sequence motif in Hph1p, PVIAVN . This motif is related to calcineurin docking sites in other substrates, such as NFAT and Crz1p, and is required for regulation of Hph1p by calcineurin . In contrast, Hph2p neither interacts with nor is dephosphorylated by calcineurin . Ca2+-induced Crz1p-mediated transcription is unaffected in hph1delta hph2delta mutants, and genetic analyses indicate that HPH1/HPH2 and CRZ1 act in distinct pathways downstream of calcineurin . Thus, Hph1p and Hph2p are components of a novel Ca2+- and calcineurin-regulated response required to promote growth under conditions of high Na+, alkaline pH, and cell wall stress . Eukaryot Cell, 2004 Jun, 3(3), 620 - 31 SYM1 is the stress-induced Saccharomyces cerevisiae ortholog of the mammalian kidney disease gene Mpv17 and is required for ethanol metabolism and tolerance during heat shock; Trott A et al.; Organisms rapidly adapt to severe environmental stress by inducing the expression of a wide array of heat shock proteins as part of a larger cellular response program . We have used a genomics approach to identify novel heat shock-induced genes in Saccharomyces cerevisiae . The uncharacterized open reading frame (ORF) YLR251W was found to be required for both metabolism and tolerance of ethanol during heat shock . YLR251W has significant homology to the mammalian peroxisomal membrane protein Mpv17, and Mpv17(-/-) mice exhibit age-onset glomerulosclerosis, deafness, hypertension, and, ultimately, death by renal failure . Expression of Mpv17 in ylr251wdelta cells complements the 37 degrees C ethanol growth defect, suggesting that these proteins are functional orthologs . We have therefore renamed ORF YLR251W as SYM1 (for "stress-inducible yeast Mpv17") . In contrast to the peroxisomal localization of Mpv17, we find that Sym1 is an integral membrane protein of the inner mitochondrial membrane . In addition, transcriptional profiling of sym1delta cells uncovered changes in gene expression, including dysregulation of a number of ethanol-repressed genes, exclusively at 37 degrees C relative to wild-type results . Together, these data suggest an important metabolic role for Sym1 in mitochondrial function during heat shock . Furthermore, this study establishes Sym1 as a potential model for understanding the role of Mpv17 in kidney disease and cardiovascular biology . Methods Mol Biol, 2004, 280, 291 - 306 Purification and analysis of checkpoint protein complexes from Saccharomyces cerevisiae; Green CM et al.; The DNA damage-dependent checkpoint of Saccharomyces cerevisiae is a paradigm for eukaryotic checkpoint pathways that regulate cell cycle progression in the presence of insults to the genetic material . In order to better understand this pathway, we undertook a biochemical study of the proteins implicated in its functioning . Analysis of the hydrodynamic properties of a protein in a crude mixture can give insights into possible tertiary organization such as participation in high-molecular-mass protein complexes . We here describe the determination of Stokes radius and sedimentation coefficients for the Rad24 protein, which enabled us to predict that this protein was a component of a protein complex in crude yeast extracts . This led us to develop a protocol to purify this complex to homogeneity in order to determine the component proteins . The methods described here should be applicable to the hydrodynamic analysis and subsequent purification of any soluble protein from organisms amenable to genetic manipulation, such as yeast, as long as the function of that protein is not perturbed by the addition of an epitope tag. Methods Mol Biol, 2004, 280, 275 - 90 Assaying the spindle checkpoint in the budding yeast Saccharomyces cerevisiae; Yellman CM et al.; The spindle checkpoint is assayed in Saccharomyces cerevisiae using several criteria . Sensitivity to benzimidazole drugs is assayed in cells grown in liquid medium and cells grown on solid medium on petri plates . Cell cycle delays are measured using cells synchronized by treatment with mating pheromone alpha-factor, and the population is monitored by flow cytometry measuring DNA content in cells . There are two different transitions that are monitored, and cytological assays for individual cells and biochemical assays for populations of cells are presented . The metaphase to anaphase transition is assayed by monitoring sister chromatid separation using GFP-tagged chromosomes, Pds1 stability using immunofluorescence, and Mcd1/Scc1 association with chromatin using chromosome spreads . Pds1 and Mcd1/Scc1 stability is measured in populations by Western blots . The exit from mitosis is monitored by Cdc14 immunofluorescence and Clb2 Western blots. J Appl Microbiol, 2004, 97(1), 57 - 67 Comprehensive gene expression analysis of the response to straight-chain alcohols in Saccharomyces cerevisiae using cDNA microarray; Fujita K et al.; AIMS: The purpose of this study was to examine the gene expression profiles of yeast Saccharomyces cerevisiae subjected to straight-chain alcohols . METHODS AND RESULTS: Lipophilic alcohols with high log Pow values were more toxic to yeast than those with low log Pow values . Morphological changes after exposure to ethanol, 1-pentanol, 1-octanol were observed, whereas n-pentane as a model hydrocarbon affected the surface of the outer membrane, with little change in organelles . Using cDNA microarrays, quite a few up-regulated gene categories were classified into the category 'cell rescue, defence and virulence' by ethanol, and the category 'energy' and 'metabolism' by 1-pentanol . Meanwhile, the characteristic genes up-regulated by n-pentane were not observed, and the expression profile was distantly related to ethanol, 1-pentanol and 1-octanol . CONCLUSIONS: This study suggests that gene expression profiles at the whole genome level were intimately associated with the cell growth inhibition and morphological changes by straight-chain alcohols with differing log Pow values . SIGNIFICANCE AND IMPACT OF THE STUDY: The study of comprehensive gene expression profiles by cDNA microarrays elucidates the straight-chain alcohol adaptation mechanisms. Mol Microbiol, 2004 Jun, 52(6), 1653 - 63 In UV-irradiated Saccharomyces cerevisiae, overexpression of Swi2/Snf2 family member Rad26 increases transcription-coupled repair and repair of the non-transcribed strand; Bucheli M et al.; Nucleotide excision repair (NER) in eukaryotes is a pathway conserved from yeast to humans that removes many bulky chemical adducts and UV-induced photoproducts from DNA in a relatively error-free manner . In addition to the recognition and excision of DNA damage throughout the genome (GGR), there exists a mechanism, transcription-coupled nucleotide excision repair (TCR), for recognizing some types of DNA damage in the transcribed strand of genes in Escherichia coli, yeast and mammalian cells . An obstacle in the repair of the transcribed strand of active genes is the RNA polymerase complex stalled at sites of DNA damage . The stalled RNA polymerase complex may then mediate recruitment of repair proteins to damage in the transcribed strand . Proteins enabling TCR are the Cockayne syndrome B (CSB) protein in humans and its yeast homologue Rad26 . Both CSB and Rad26 belong to the Swi2/Snf2 family of DNA-dependent ATPases, which change DNA accessibility to proteins by altering chromatin structure . To address how Rad26 functions in yeast repair, we used the genetic approach of overexpressing Rad26 and examined phenotypic changes, i.e . changes in NER . We found that repair of both the transcribed and the non-transcribed strands is increased . In addition, overexpression of Rad26 partially bypasses the requirement for Rad7 in GGR, specifically in the repair of non-transcribed sequences . As TCR takes place in very localized regions of DNA (i.e . within genes) in wild-type cells, we propose that overexpression of recombinant Rad26 increases accessibility of the damaged DNA in chromatin for interaction with repair proteins. Dig Dis Sci, 2004 Apr, 49(4), 611 - 8 Frequency and significance of antibodies to Saccharomyces cerevisiae in autoimmune hepatitis; Czaja AJ et al.; Our aims were to determine the frequency of antibodies to Saccharomyces cerevisiae in autoimmune hepatitis and assess associations with concurrent mucosal diseases, genetic factors, and corticosteroid response . Seropositivity was determined by enzyme immunoassay in 385 samples obtained from 178 patients . Antibodies to Saccharomyces cerevisiae were detected in 49 patients (28%), and serum levels of immunoglobulin A were higher in seropositive patients (410 +/- 35 versus 321 +/- 20 mg/dL; P = 0.02) . Individuals with and without antibodies were not otherwise distinguished by concurrent mucosal diseases, laboratory findings, or outcomes . Antibodies to tissue transglutaminase occurred more commonly in seropositive patients (16 versus 4%; P = 0.008), but this association was lost when corroborating serological criteria for celiac disease were sought . We conclude that antibodies to Saccharomyces cerevisiae are common in autoimmune hepatitis . They may be associated with non-disease-specific immune responses, but they do not define individuals with a distinctive clinical phenotype, associated mucosal diseases, or treatment outcome. Proc Natl Acad Sci U S A, 2004 Jun 15, 101(24), 9039 - 44 Epub 2004 Jun 07. Mutator genes for suppression of gross chromosomal rearrangements identified by a genome-wide screening in Saccharomyces cerevisiae; Smith S et al.; Different types of gross chromosomal rearrangements (GCRs), including translocations, interstitial deletions, terminal deletions with de novo telomere additions, and chromosome fusions, are observed in many cancers . Multiple pathways, such as S-phase checkpoints, DNA replication, recombination, chromatin remodeling, and telomere maintenance that suppress GCRs have been identified . To experimentally expand our knowledge of other pathway(s) that suppress GCRs, we developed a generally applicable genome-wide screening method . In this screen, we identified 10 genes (ALO1, CDC50, CSM2, ELG1, ESC1, MMS4, RAD5, RAD18, TSA1, and UFO1) that encode proteins functioning in the suppression of GCRs . Moreover, the breakpoint junctions of GCRs from these GCR mutator mutants were determined with modified breakpoint-mapping methods . We also identified nine genes (AKR1, BFR1, HTZ1, IES6, NPL6, RPL13B, RPL27A, RPL35A, and SHU2) whose mutations generated growth defects with the pif1Delta mutation . In addition, we found that some of these mutations changed the telomere size. J Clin Microbiol, 2004 Jun, 42(6), 2840 - 2 Use of paraffin-embedded tissue for identification of Saccharomyces cerevisiae in a baker's lung nodule by fungal PCR and nucleotide sequencing; Ren P et al.; A 40-year-old healthy male employed in a bakery presented with a single lung nodule and underwent investigations to rule out pulmonary carcinoma . Biopsy was positive for yeast cells, which did not match common fungal pathogens . PCR assay of paraffin-embedded tissue and nucleotide sequencing with ribosomal ITS1-ITS2 universal primers revealed the presence of Saccharomyces cerevisiae. Appl Environ Microbiol, 2004 Jun, 70(6), 3766 - 8 Immobilization of Saccharomyces cerevisiae cystathionine gamma-lyase and application of the product to cystathionine synthesis; Yamagata S et al.; Cystathionine gamma-lyase of Saccharomyces cerevisiae was immobilized to aminohexyl-Sepharose through the cofactor pyridoxal 5'-phosphate and was characterized with respect to its cystathionine gamma-synthase activity . The immobilized product was so stable that it repeatedly catalyzed as many as five cycles of the reaction without losing activity. J Biol Chem, 2004 Aug 6, 279(32), 33057 - 62 Epub 2004 Jun 04. Effectors of lysine 4 methylation of histone H3 in Saccharomyces cerevisiae are negative regulators of PHO5 and GAL1-10; Carvin CD et al.; Post-translational modifications of histone amino-terminal tails are a key determinant in gene expression . Histone methylation plays a dual role in gene regulation . Methylation of lysine 9 of histone H3 in higher eukaryotes is associated with transcriptionally inactive heterochromatin, whereas H3 lysine 4 methylation correlates with active chromatin . Methylation of lysine 4 of H3 via Set1, a component of the Saccharomyces cerevisiae COMPASS complex, is regulated by the transcriptional elongation Paf1-Rtf1 and histone ubiquitination Rad6-Bre1 complexes, which are required for the expression of a subset of genes . This suggests that lysine 4 methylation of histone H3 may play an activating role in transcription; however, the mechanism of Set1 function remains unclear . We show here that H3 lysine 4 methylation also negatively regulated gene expression, as strains without Set1 showed enhanced expression of PHO5, wherein chromatin structure plays an important transcriptional regulatory role . Di- and trimethylation of H3 lysine 4 was detected at the PHO5 promoter, and a strain expressing a mutant version of histone H3 with lysine 4 changed to arginine, (which cannot be methylated) exhibited PHO5 derepression . Moreover, PHO5 was derepressed in strains that lacked components of either the Paf1-Rtf1 elongation or Rad6-Bre1 histone ubiquitination complexes . Lastly, PHO84 and GAL1-10 transcription was also increased in set1Delta cells . These results suggest that H3 methylation at lysine 4, in conjunction with transcriptional elongation, may function in a negative feedback pathway for basal transcription of some genes, although being a positive effector at others. Parasitol Res, 2004 Jul, 93(4), 318 - 21 Epub 2004 Jun 04. Targeting a DBL3gamma domain of the Plasmodium falciparum erythrocyte membrane protein 1 to the surface of Saccharomyces cerevisiae; Schieck E et al.; The availability of the full genomes of the malarial parasite Plasmodium falciparum and its two hosts, man and Anopheles gambiae, has dramatically increased the demand for protein display systems to study host/parasite interactions at the molecular level . Here, we explored the potential of a Saccharomyces cerevisiae expression and display system that allows proteins of interest to be targeted to the yeast surface . As proof of this principle, we used a P . falciparum erythrocyte membrane protein 1 DBL3gamma domain which mediates the binding of P . falciparum-infected erythrocytes to chondroitin-4-sulfate, a host receptor involved in parasite sequestration in the placenta . Our data revealed localization of the DBL3gamma domain to the yeast surface, demonstrating the value of the yeast system as a tool for displaying P . falciparum protein fragments . However, binding of the respective yeast strains to chondroitin-4-sulfate could not be demonstrated . Biochem Biophys Res Commun, 2004 Jun 25, 319(2), 349 - 57 Functional and physical interactions of Faf1p, a Saccharomyces cerevisiae nucleolar protein; Karkusiewicz I et al.; We report the discovery and characterisation of a novel nucleolar protein of Saccharomyces cerevisiae . We identified this protein encoded by ORF YIL019w, designated in SGD base as Faf1p, in a two hybrid interaction screen using the known nucleolar protein Krr1 as bait . The presented data indicate that depletion of the Faf1 protein has an impact on the 40S ribosomal subunit biogenesis resulting from a decrease in the production of 18S rRNA . The primary defect is apparently due to inefficient processing of 35S rRNA at the A(0), A(1), and A(2) cleavage sites. Genetika, 2004 Apr, 40(4), 437 - 44 {Induction of synthesis of Hsp104 of Saccharomyces cerevisiae in heat shock is controlled by mitochondria}; Rikhvanov EG et al.; Heat shock protein Hsp104 of Saccharomyces cerevisiae functions as a protector of cells against heat stress . When yeast are grown in media containing nonfermentable carbon sources, the constitutive level of this protein increases, which suggests an association between the expression of Hsp104 and yeast energy metabolism . In this work, it is shown that distortions in the function of mitochondria appearing as a result of mutation petite or after exposure of cells to the mitochondrial inhibitor sodium azide reduce the induction of Hsp104 synthesis during heat shock . Since the addition of sodium azide suppressed the formation of induced thermotolerance in the parent type and in mutant hsp104, the expression of gene HSP104 and other stress genes during heat shock is apparently regulated by mitochondria. Proc Natl Acad Sci U S A, 2004 Jun 8, 101(23), 8581 - 6 Epub 2004 Jun 01. Polyadenylation of rRNA in Saccharomyces cerevisiae; Kuai L et al.; In contrast to mRNAs, rRNAs are transcribed by RNA polymerase I or III and are not believed to be polyadenylated . Here we show that in Saccharomyces cerevisiae, at least a small fraction of rRNAs do have a poly(A) tail . The levels of polyadenylated rRNAs are dramatically increased in strains lacking the degradation function of Rrp6p, a component of the nuclear exosome . Pap1p, the poly(A) polymerase, is responsible for adenylating the rRNAs despite the fact that the rRNAs do not have a canonical polyadenylation signal . Polyadenylated rRNAs reside mainly within the nucleus and are in turn degraded . For at least one rRNA type, the polyadenylation preferentially occurs on the precursor rather than the mature product . The existence of polyadenylated rRNAs may reflect a quality-control mechanism of rRNA biogenesis. J Biochem (Tokyo), 2004 May, 135(5), 615 - 8 Expression and characterization of Saccharomyces cerevisiae Cne1p, a calnexin homologue; Xu X et al.; The calnexin homologue (Cne1p) of Saccharomyces cerevisiae was expressed in Escherichia coli to evaluate its chaperone function . The chaperone function was examined as to the effects on the suppression of thermal denaturation and the enhancement of refolding, using citrate synthase (CS) as a nonspecific chaperone substrate . Cne1p effectively suppressed the thermal denaturation of CS and enhanced the refolding of thermally or chemically denatured CS in a concentration-dependent manner . In addition, the chaperone function of Cne1p was greatly affected in the presence of monoglucosylated oligosaccharides (G1M9) that specifically bind to the lectin site . These results indicated that Cne1p functions as a molecular chaperone in Saccharomyces cerevisiae. Biochemistry (Mosc), 2004 Apr, 69(4), 387 - 93 Purification and properties of exopolyphosphatase from the cytosol of Saccharomyces cerevisiae not encoded by the PPX1 gene; Andreeva NA et al.; A novel exopolyphosphatase has been isolated from the cytosol of Saccharomyces cerevisiae grown to the stationary phase after its transfer from phosphate-deficient to complete medium . The PPX1 gene responsible for 40-kD exopolyphosphatase of the cytosol does not encode it . Specific activity of the preparation is 150 U/mg, purification degree is 319, and the yield is 16.9% . The minimal molecular mass of the active but unstable enzyme complex is approximately 125 kD . A stable enzyme complex with a molecular mass of approximately 500 kD is composed of two polypeptides of approximately 32 and 35 kD and apparently polyphosphates (polyP) . Unlike the enzyme encoded by PPX1, the high-molecular-mass exopolyphosphatase is slightly active with polyP3, not inhibited by antibodies suppressing the activity of 40-kD exopolyphosphatase, inhibited by EDTA, and stimulated by divalent cations to a lesser extent . The high-molecular-mass exopolyphosphatase hydrolyzes polyP with an average chain length of 208 to 15 phosphate residues to the same extent, but is inactive with ATP, PPi, and p-nitrophenyl phosphate . The activity with polyP3 is 13% of that with polyP208 . The Km values for polyP208, polyP15, and polyP3 hydrolysis are 3.5, 75, and 1100 microM, respectively . The enzyme is most active at pH approximately 7 . Co2+ at the optimal concentration of 0.1 mM stimulates the activity 6-fold, while Mg2+ at the optimal concentration of 1 mM enhances it 2-fold . The enzyme under study is similar in some properties to an exopolyphosphatase purified earlier from yeast vacuoles. Mol Cell Biol, 2004 Jun, 24(12), 5290 - 303 Mutations in a partitioning protein and altered chromatin structure at the partitioning locus prevent cohesin recruitment by the Saccharomyces cerevisiae plasmid and cause plasmid missegregation; Yang XM et al.; The 2 microm circle is a highly persistent "selfish" DNA element resident in the Saccharomyces cerevisiae nucleus whose stability approaches that of the chromosomes . The plasmid partitioning system, consisting of two plasmid-encoded proteins, Rep1p and Rep2p, and a cis-acting locus, STB, apparently feeds into the chromosome segregation pathway . The Rep proteins assist the recruitment of the yeast cohesin complex to STB during the S phase, presumably to apportion the replicated plasmid molecules equally to daughter cells . The DNA-protein and protein-protein interactions of the partitioning system, as well as the chromatin organization at STB, are important for cohesin recruitment . Rep1p variants that are incompetent in binding to Rep2p, STB, or both fail to assist the assembly of the cohesin complex at STB and are nonfunctional in plasmid maintenance . Preventing the cohesin-STB association without impeding Rep1p-Rep2p-STB interactions also causes plasmid missegregation . During the yeast cell cycle, the Rep1p and Rep2p proteins are expelled from STB during a short interval between the late G(1) and early S phases . This dissociation and reassociation event ensures that cohesin loading at STB is replication dependent and is coordinated with chromosomal cohesin recruitment . In an rsc2 Delta yeast strain lacking a specific chromatin remodeling complex and exhibiting a high degree of plasmid loss, neither Rep1p nor the cohesin complex can be recruited to STB . The phenotypes of the Rep1p mutations and of the rsc2 Delta mutant are consistent with the role of cohesin in plasmid partitioning being analogous to that in chromosome partitioning. Mol Cell Biol, 2004 Jun, 24(12), 5130 - 43 A new Saccharomyces cerevisiae strain with a mutant Smt3-deconjugating Ulp1 protein is affected in DNA replication and requires Srs2 and homologous recombination for its viability; Soustelle C et al.; The Saccharomyces cerevisiae Srs2 protein is involved in DNA repair and recombination . In order to gain better insight into the roles of Srs2, we performed a screen to identify mutations that are synthetically lethal with an srs2 deletion . One of them is a mutated allele of the ULP1 gene that encodes a protease specifically cleaving Smt3-protein conjugates . This allele, ulp1-I615N, is responsible for an accumulation of Smt3-conjugated proteins . The mutant is unable to grow at 37 degrees C . At permissive temperatures, it still shows severe growth defects together with a strong hyperrecombination phenotype and is impaired in meiosis . Genetic interactions between ulp1 and mutations that affect different repair pathways indicated that the RAD51-dependent homologous recombination mechanism, but not excision resynthesis, translesion synthesis, or nonhomologous end-joining processes, is required for the viability of the mutant . Thus, both Srs2, believed to negatively control homologous recombination, and the process of recombination per se are essential for the viability of the ulp1 mutant . Upon replication, mutant cells accumulate single-stranded DNA interruptions . These structures are believed to generate different recombination intermediates . Some of them are fixed by recombination, and others require Srs2 to be reversed and fixed by an alternate pathway. Curr Genet, 2004 Aug, 46(2), 67 - 71 Epub 2004 May 27. Intragenic suppressors that restore the activity of the maturase encoded by the second intron of the Saccharomyces cerevisiae cyt b gene; Maciaszczyk E et al.; The protein encoded by the second intron (bi2) of the mitochondrial cyt b gene from Saccharomyces cerevisiae functions as a maturase promoting intron splicing . This protein belongs to a large family characterized by the presence of two conserved motifs: LAGLIDADG (or P1 and P2) . We have isolated and characterized spontaneous revertants from two mis-sense mutations, G85D and H92P (localized in the P1 motif of the bi2-maturase), that have a detrimental effect on intron splicing . All analyzed revertants are intragenic and resulted from monosubstitutions in the mutated codons . Only true back-mutations that restor the initial glycine 85 and a pseudoreversion that replaces the deleterious aspartic acid 85 by alanine were found in revertants of the mutant G85D . In contrast, all possible monosubstitutions in the mutated codon H92P were identified among the revertants of this mutant . The maturase activity of all novel forms of the protein is similar to the wild-type protein. J Biol Chem, 2004 Jul 23, 279(30), 31983 - 9 Epub 2004 May 27. Oxidative damage to specific proteins in replicative and chronological-aged Saccharomyces cerevisiae: common targets and prevention by calorie restriction; Reverter-Branchat G et al.; Oxidative modifications of cellular components have been described as one of the main contributions to aged phenotype . In Saccharomyces cerevisiae, two distinct life spans can be considered, replicative and chronological . The relationship between both aging models is still not clear despite suggestions that these phenomena may be related . In this work, we show that replicative and chronological-aged yeast cells are affected by an oxidative stress situation demonstrated by increased protein carbonylation when compared with young cells . The data on the identification of these oxidatively modified proteins gives clues to better understand cellular dysfunction that occurs during aging . Strikingly, although in both aging models metabolic differences are important, major targets are almost the same . Common targets include stress resistance proteins (Hsp60 and Hsp70) and enzymes involved in glucose metabolism such as enolase, glyceraldehydes-3-P dehydrogenase, fructose-1,6-biphosphate aldolase, pyruvate decarboxylase, and alcohol dehydrogenase . In both aging models, calorie restriction results in decreased damage to these proteins . In addition, chronological-aged cells grown under glucose restriction displayed lowered levels of lipid peroxidation product lipofuscin . Intracellular iron concentration is kept almost unchanged, whereas in non-restricted cells, the values increase up 4-5 times . The pro-oxidant effects of such increased iron concentration would account for the damage observed . Also, calorie-restricted cells show undamaged catalase, which clearly appears carbonylated in cells grown at a high glucose concentration . These results may explain lengthening of the viability of chronological-aged cells and could have an important role in replicative life span extension by calorie restriction. J Biol Chem, 2004 Jul 30, 279(31), 32262 - 8 Epub 2004 May 27. Global analyses of sumoylated proteins in Saccharomyces cerevisiae . Induction of protein sumoylation by cellular stresses; Zhou W et al.; We have undertaken a global analysis of sumoylated proteins in Saccharomyces cerevisiae by tandem mass spectrometry . Exposure of cells to oxidative and ethanol stresses caused large increases in protein sumoylation . A large number of new sumoylated proteins were identified in untreated, hydrogen peroxide-treated, and ethanol-treated cells . These proteins are known to be involved in diverse cellular processes, including gene transcription, protein translation, DNA replication, chromosome segregation, metabolic processes, and stress responses . Additionally, the known enzymes, including E1, E2, and E3 of the sumoylation cascade were found to be auto-sumoylated . Taken together, these results show that protein sumoylation is broadly involved in many cellular functions and this mass spectrometry-based proteomic approach is useful in studying the regulation of protein sumoylation in the cells. Genetics, 2004 May, 167(1), 93 - 105 Genetic interactions with C-terminal domain (CTD) kinases and the CTD of RNA Pol II suggest a role for ESS1 in transcription initiation and elongation in Saccharomyces cerevisiae; Wilcox CB et al.; Ess1 is an essential prolyl isomerase that binds the C-terminal domain (CTD) of Rpb1, the large subunit of RNA polymerase II . Ess1 is proposed to control transcription by isomerizing phospho-Ser-Pro peptide bonds within the CTD repeat . To determine which step(s) in the transcription cycle might require Ess1, we examined genetic interactions between ESS1 and genes encoding the known CTD kinases (KIN28, CTK1, BUR1, and SRB10) . Although genetic interactions were identified between ESS1 and all four kinases, the clearest interactions were with CTK1 and SRB10 . Reduced dosage of CTK1 rescued the growth defect of ess1(ts) mutants, while overexpression of CTK1 enhanced the growth defects of ess1(ts) mutants . Deletion of SRB10 suppressed ess1(ts) and ess1Delta mutants . The interactions suggest that Ess1 opposes the functions of these kinases, which are thought to function in preinitiation and elongation . Using a series of CTD substitution alleles, we also identified Ser5-Pro6 as a potential target for Ess1 isomerization within the first "half" of the CTD repeats . On the basis of the results, we suggest a model in which Ess1-directed conformational changes promote dephosphorylation of Ser5 to stimulate preinitiation complex formation and, later, to inhibit elongation. Genetics, 2004 May, 167(1), 35 - 49 Analysis of beta-1,3-glucan assembly in Saccharomyces cerevisiae using a synthetic interaction network and altered sensitivity to caspofungin; Lesage G et al.; Large-scale screening of genetic and chemical-genetic interactions was used to examine the assembly and regulation of beta-1,3-glucan in Saccharomyces cerevisiae . Using the set of deletion mutants in approximately 4600 nonessential genes, we scored synthetic interactions with genes encoding subunits of the beta-1,3-glucan synthase (FKS1, FKS2), the glucan synthesis regulator (SMI1/KNR4), and a beta-1,3-glucanosyltransferase (GAS1) . In the resulting network, FKS1, FKS2, GAS1, and SMI1 are connected to 135 genes in 195 interactions, with 26 of these genes also interacting with CHS3 encoding chitin synthase III . A network core of 51 genes is multiply connected with 112 interactions . Thirty-two of these core genes are known to be involved in cell wall assembly and polarized growth, and 8 genes of unknown function are candidates for involvement in these processes . In parallel, we screened the yeast deletion mutant collection for altered sensitivity to the glucan synthase inhibitor, caspofungin . Deletions in 52 genes led to caspofungin hypersensitivity and those in 39 genes to resistance . Integration of the glucan interaction network with the caspofungin data indicates an overlapping set of genes involved in FKS2 regulation, compensatory chitin synthesis, protein mannosylation, and the PKC1-dependent cell integrity pathway. Genetics, 2004 May, 167(1), 21 - 33 An essential role for the Saccharomyces cerevisiae DEAD-box helicase DHH1 in G1/S DNA-damage checkpoint recovery; Bergkessel M et al.; The eukaryotic cell cycle displays a degree of plasticity in its regulation; cell cycle progression can be transiently arrested in response to environmental stresses . While the signaling pathways leading to cell cycle arrest are beginning to be well understood, the regulation of the release from arrest has not been well characterized . Here we show that DHH1, encoding a DEAD-box RNA helicase orthologous to the human putative proto-oncogene p54/RCK, is important in release from DNA-damage-induced cell cycle arrest at the G1/S checkpoint . DHH1 mutants are not defective for DNA repair and recover normally from the G2/M and replication checkpoints, suggesting a specific function for Dhh1p in recovery from G1/S checkpoint arrest . Dhh1p has been suggested to play a role in partitioning mRNAs between translatable and nontranslatable pools, and our results implicate this modulation of mRNA metabolism in the recovery from G1/S cell cycle arrest following DNA damage . Furthermore, the high degree of conservation between DHH1 and its human ortholog suggests that this mechanism is conserved among all eukaryotes and potentially important in human disease. Mol Microbiol, 2004 Jun, 52(5), 1413 - 25 Sed1p and Srl1p are required to compensate for cell wall instability in Saccharomyces cerevisiae mutants defective in multiple GPI-anchored mannoproteins; Hagen I et al.; The covalently linked cell wall protein Ccw12p of Saccharomyces cerevisiae is a GPI-anchored protein (V . Mrsa et al., 1999, J Bacteriol 181: 3076-3086) . Although only 121 amino acids long, the haemagglutinin-tagged protein released by laminarinase from the cell wall possesses an apparent molecular mass of > 300 kDa . A membrane-bound form with an apparent molecular mass of 58 kDa is highly O- and N-glycosylated and contains the GPI anchor . With a half-life of 2 min, the membrane form is transformed to the > 300 kDa form . The deletion mutant ccw12Delta grows slower than the wild type, is highly sensitive to Calcofluor white and contains 2.5 times more chitin . Further, compared with wild-type yeast, significantly more proteins are released from intact cells when treated with dithiothreitol . Interestingly, these defects become less pronounced when further GPI-anchored cell wall proteins are deleted . Mutant DeltaGPI (simultaneous deletion of CCW12, CCW13/DAN1, CCW14, TIP1 and CWP1) is similar in many respects to wild-type yeast . To find out how the cell wall is stabilized in mutant DeltaGPI, a genome-wide transcription analysis was performed . Of 159 significantly regulated genes, 14 encode either known or suspected cell wall-associated proteins . Analysis of genes affected in transcription revealed that SED1 and SRL1 in particular are required to reconstruct cell wall stability in the absence of multiple GPI-anchored mannoproteins. J Environ Pathol Toxicol Oncol, 2004, 23(2), 145 - 51 Evaluation of radioprotective action of compounds using Saccharomyces cerevisiae; Nemavarkar P et al.; The yeast Saccharomyces cerevisiae serves as a model eukaryotic system to screen radioprotectors that can be used primarily in radiotherapy as well as in occupational workers in nuclear and allied industries . A number of antioxidants are suggested to be radioprotectors by virtue of their ability to quench reactive oxygen species, but their radioprotective action has not been investigated so far . In this study, a number of antioxidants were tested for their efficacy in radioprotection using yeast cells . Sulfhydryl compounds (disulfiram at 100 and 200 microM) and reduced glutathione (10 and 100 mM), purified compounds of plant origin, such as curcumin (1 mM, 10 mM, and 100 mM), quercetin (100 and 500 microM), rutin (100 and 200 microM), ellagic acid (100,200, and 500 microM) and gallic acid (100 and 500 microM)-were studied . The results revealed that all compounds, except gallic acid, specifically protected normal yeast cells from gamma-radiation damage . Using rad 52 mutants, which lack recombinational DNA repair pathway, it has been found that protection was solely brought about by reducing DNA damage rather than by interfering with DNA repair . Results with DNA repair polymerase further substantiated this contention . We conclude that simple eukaryotic yeast cells can serve as a test system not only for rapid screening of radiomodifiers but also to study their mode of action. J Environ Pathol Toxicol Oncol, 2004, 23(2), 139 - 44 Radioprotective effect of podophyllotoxin in Saccharomyces cerevisiae; Bala M et al.; Recent reports showed that whole extract of Podophyllum hexandrum was radioprotective in mice . Podophyllotoxin is one of the major constituents of the whole extract of Podophyllum . In this study we report on the radioprotective action of podophyllotoxin in Saccharomyces cerevisiae yeast . Proliferating yeast cells pretreated with podophyllotoxin (2.5-5.0 microg/mL) for > or =3 hours showed a higher surviving fraction after (60)Co-gamma-irradiation (200-600 Gy) than did the irradiated cells not pretreated with podophyllotoxin . The maximum increase (2.0 times) in surviving fraction was observed in cells treated with 2.5 microg/mL podophyllotoxin, 5 hours before (60)Co-gamma-irradiation (400 Gy) . Podophyllotoxin was not mutagenic or recombinogenic at radioprotective doses (2.5 microg/mL) . A post-irradiation decrease in revertants and gene convertants was observed in cells treated with podophyllotoxin (2.5 microg/mL podophyllotoxin, -5 hours, 400 Gy) . This study indicates that podophyllotoxin is radioprotective in yeast, and its radioprotective effects in higher eukaryotes would be worth investigating. Proc Natl Acad Sci U S A, 2004 Jun 8, 101(23), 8658 - 63 Epub 2004 May 25. A genome-wide screen for Saccharomyces cerevisiae deletion mutants that affect telomere length; Askree SH et al.; Telomeres are nucleoprotein structures present at the ends of eukaryotic chromosomes that play a central role in guarding the integrity of the genome by protecting chromosome ends from degradation and fusion . Length regulation is central to telomere function . To broaden our knowledge about the mechanisms that control telomere length, we have carried out a systematic examination of approximately 4,800 haploid deletion mutants of Saccharomyces cerevisiae for telomere-length alterations . By using this screen, we have identified >150 candidate genes not previously known to affect telomere length . In two-thirds of the identified mutants, short telomeres were observed; whereas in one-third, telomeres were lengthened . The genes identified are very diverse in their functions, but certain categories, including DNA and RNA metabolism, chromatin modification, and vacuolar traffic, are overrepresented . Our results greatly enlarge the number of known genes that affect telomere metabolism and will provide insights into how telomere function is linked to many other cellular processes. J Am Chem Soc, 2004 Jun 2, 126(21), 6579 - 89 Ferrous binding to the multicopper oxidases Saccharomyces cerevisiae Fet3p and human ceruloplasmin: contributions to ferroxidase activity; Quintanar L et al.; The multicopper oxidases are a family of enzymes that couple the reduction of O(2) to H(2)O with the oxidation of a range of substrates . Saccharomyces cerevisiae Fet3p and human ceruloplasmin (hCp) are members of this family that exhibit ferroxidase activity . Their high specificity for Fe(II) has been attributed to the existence of a binding site for iron . In this study, mutations at the E185 and Y354 residues, which are putative ligands for iron in Fet3p, have been generated and characterized . The effects of these mutations on the electronic structure of the T1 Cu site have been assessed, and the reactivities of this site toward 1,4-hydroquinone (a weak binding substrate) and Fe(II) have been evaluated and interpreted in terms of the semiclassical Marcus theory for electron transfer . The electronic and geometric structure of the Fe(II) substrate bound to Fet3p and hCp has been studied for the first time, using variable-temperature variable field magnetic circular dichroism (VTVH MCD) spectroscopy . The iron binding sites in Fet3p and hCp appear to be very similar in nature, and their contributions to the ferroxidase activity of these proteins have been analyzed . It is found that these iron binding sites play a major role in tuning the reduction potential of iron to provide a large driving force for the ferroxidase reaction, while still supporting the delivery of the Fe(III) product to the acceptor protein . Finally, the analysis of possible electron-transfer (ET) pathways from the protein-bound Fe(II) to the T1 Cu site indicates that the E185 residue not only plays a role in iron binding, but also provides the dominant ET pathway to the T1 Cu site. J Agric Food Chem, 2004 Jun 2, 52(11), 3666 - 73 Alkali extraction of beta-d-glucans from Saccharomyces cerevisiae cell wall and study of their adsorptive properties toward zearalenone; Yiannikouris A et al.; The isolated cell wall of Saccharomyces cerevisiae has some capacity to adsorb zearalenone (affinity near 30%) and reduce the bioavailability of toxins in the digestive tract . The adsorption process was quantified in vitro, and the data obtained when plotted with Hill's equation indicated a cooperative process . The model showed that the adsorption capacity was related to the yeast cell wall composition . This work focused on the role of various beta-d-glucan types in the efficacy of zearalenone adsorption by yeast cell wall and sought to elucidate some of the adsorption mechanisms . Zearalenone was mixed at 37 degrees C with a constant quantity of alkali-soluble or alkali-insoluble beta-d-glucans isolated from yeast cell walls, and the amount of adsorbed zearalenone was measured . Given that the alkali solubility of beta-d-glucans is a determining factor for their three-dimensional conformation and that the alkali-insoluble fraction had a greater affinity (up to 50%) than the alkali-soluble fraction ( approximately 16%), it was concluded that the three-dimensional structure strongly influences the adsorption process . The alkali insolubility of beta-d-glucans led to the formation of single and/or triple helices, which have been identified as the most favorable structures for zearalenone adsorption efficacy . The beta(1,3)-d-glucan and beta(1,6)-d-glucan compositions of the two alkali-extracted fractions and their involvement in the adsorption process are discussed. Biochim Biophys Acta, 2004 May 27, 1663(1-2), 9 - 13 Targeting of proteins involved in sterol biosynthesis to lipid particles of the yeast Saccharomyces cerevisiae; Mullner H et al.; In the yeast Saccharomyces cerevisiae, three enzymes of the sterol biosynthetic pathway, namely Erg1p, Erg6p and Erg7p, are located in lipid particles . Whereas Erg1p (squalene epoxidase) is also present in the endoplasmic reticulum (ER) to a significant amount, only traces of Erg6p (sterol C-24 methyltransferase) and Erg7p (lanosterol synthase) are found in the ER . We have chosen these three Erg-proteins as typical representatives of lipid particle proteins to study targeting to their destination . Lipid particle proteins do not contain obvious targeting motifs, but the only common structural feature is the presence of one or two hydrophobic domains near the C-termini . We constructed truncated versions of Erg1p, Erg6p and Erg7p to test the role of these hydrophobic domains in subcellular distribution . Our results demonstrate that lack of the hydrophobic domains prevents at least in part the association of the proteins with lipid particles and causes their retention to the ER . This result strongly supports the view that ER and lipid particles are related organelles. Eur J Biochem, 2004 Jun, 271(11), 2264 - 71 Modeling the Qo site of crop pathogens in Saccharomyces cerevisiae cytochrome b; Fisher N et al.; Saccharomyces cerevisiae has been used as a model system to characterize the effect of cytochrome b mutations found in fungal and oomycete plant pathogens resistant to Q(o) inhibitors (QoIs), including the strobilurins, now widely employed in agriculture to control such diseases . Specific residues in the Q(o) site of yeast cytochrome b were modified to obtain four new forms mimicking the Q(o) binding site of Erysiphe graminis, Venturia inaequalis, Sphaerotheca fuliginea and Phytophthora megasperma . These modified versions of cytochrome b were then used to study the impact of the introduction of the G143A mutation on bc(1) complex activity . In addition, the effects of two other mutations F129L and L275F, which also confer levels of QoI insensitivity, were also studied . The G143A mutation caused a high level of resistance to QoI compounds such as myxothiazol, axoxystrobin and pyraclostrobin, but not to stigmatellin . The pattern of resistance conferred by F129L and L275F was different . Interestingly G143A had a slightly deleterious effect on the bc(1) function in V . inaequalis, S . fuliginea and P . megasperma Q(o) site mimics but not in that for E . graminis . Thus small variations in the Q(o) site seem to affect the impact of the G143A mutation on bc(1) activity . Based on this observation in the yeast model, it might be anticipated that the G143A mutation might affect the fitness of pathogens differentially . If so, this could contribute to observed differences in the rates of evolution of QoI resistance in fungal and oomycete pathogens. FEBS Lett, 2004 May 21, 566(1-3), 218 - 22 Complementation of Saccharomyces cerevisiae ccc2 mutant by a putative P1B-ATPase from Brassica napus supports a copper-transporting function; Southron JL et al.; Copper transport across membranes plays an important role in plant growth and survival . P(1B)-type ATPases participate in transmembrane transport of copper in various organisms . A Brassica napus cDNA (BnRAN1) encoding a putative Cu(2+)-ATPase was cloned in this study . A complementation assay demonstrated that the protein encoded by this cDNA could functionally replace Ccc2p, a Saccharomyces cerevisiae Cu(2+)-ATPase, rescuing growth of ccc2 mutant under iron-limited conditions . Our results suggest that this rescue likely resulted from restoration of copper delivery, mediated by BnRAN1, to Fet3p . This study is amongst the first to demonstrate that a putative plant P(1B)-ATPase is functional and to examine its substrate specificity. FEBS Lett, 2004 May 21, 566(1-3), 141 - 6 Yap8p activation in Saccharomyces cerevisiae under arsenic conditions; Menezes RA et al.; Yap8p, a member of the Saccharomyces cerevisiae Yap family, is activated in response to arsenic . Both the mechanisms by which this activation takes place and its regulation have not yet been identified . In this report, we show that Yap8p is not activated at the transcriptional level but, rather, its nuclear transport is actively regulated and dependent on the exportin chromosome region maintenance protein . In addition, it is shown that Cys(132), Cys(137)and Cys(274) are essential for Yap8p localization and transactivation function both of which are required for its biological activity. J Biol Chem, 2004 Jul 23, 279(30), 31937 - 42 Epub 2004 May 15. Glycerophosphoinositol, a novel phosphate source whose transport is regulated by multiple factors in Saccharomyces cerevisiae; Almaguer C et al.; Git1p mediates the transport of the phospholipid metabolite, glycerophosphoinositol, into Saccharomyces cerevisiae . We report that phosphate limitation and inositol limitation affect GIT1 expression and Git1p transport activity via distinct mechanisms that involve multiple transcription factors . GIT1 transcript levels and Git1p activity are greater in cells starved for phosphate, with or without inositol limitation, than in cells only limited for inositol . Furthermore, the kinetics of GIT1 transcript accumulation and Git1p activity upon transfer of cells to phosphate starvation media are different from those obtained upon transfer of cells to inositol-free media . Pho2p and Pho4p are required for GIT1 expression and for Git1p transport activity under all growth conditions tested . In contrast, Ino2p and Ino4p are required for full GIT1 expression when inositol is limiting, with or without phosphate limitation, but not when only phosphate is limiting . Greatly reduced transport activity was detected in ino2Delta and ino4Delta cells under all growth conditions . A 300-base pair region of the GIT1 promoter containing potential Pho4p binding sites was shown to be required for full GIT1 expression . Git1p appears to act as a H(+)-symporter, and neither inositol nor phosphate effectively compete with glycerophosphoinositol for transport by Git1p . Glycerophosphoinositol was shown previously to support the growth of an inositol auxotroph . Remarkably, we now report that glycerophosphoinositol can act as the sole source of phosphate for the cell, providing functional relevance for the regulation of Git1p transport activity by phosphate. Anal Chem, 2004 May 15, 76(10), 2852 - 8 Molecular-level description of proteins from saccharomyces cerevisiae using quadrupole FT hybrid mass spectrometry for top down proteomics; Meng F et al.; For improved detection of diverse posttranslational modifications (PTMs), direct fragmentation of protein ions by top down mass spectrometry holds promise but has yet to be achieved on a large scale . Using lysate from Saccharomyces cerevisiae, 117 gene products were identified with 100% sequence coverage revealing 26 acetylations, 1 N-terminal dimethylation, 1 phosphorylation, 18 duplicate genes, and 44 proteolytic fragments . The platform for this study combined continuous-elution gel electrophoresis, reversed-phase liquid chromatography, automated nanospray coupled with a quadrupole-FT hybrid mass spectrometer, and a new search engine for querying a custom database . The proteins identified required no manual validation, ranged from 5 to 39 kDa, had codon biases from 0.93 to 0.083, and were primarily associated with glycolysis and protein synthesis . Illustrations of gene-specific identifications, PTM detection and subsequent PTM localization (using either electron capture dissociation or known PTM data stored in a database) show how larger scale proteome projects incorporating top down may proceed in the future using commercial Q-FT instruments. Biochem J, 2004 Sep 15, 382(Pt 3), 867 - 75 Regulation of transcription by Saccharomyces cerevisiae 14-3-3 proteins; Bruckmann A et al.; 14-3-3 proteins form a family of highly conserved eukaryotic proteins involved in a wide variety of cellular processes, including signalling, apoptosis, cell-cycle control and transcriptional regulation . More than 150 binding partners have been found for these proteins . The yeast Saccharomyces cerevisiae has two genes encoding 14-3-3 proteins, BMH1 and BMH2 . A bmh1 bmh2 double mutant is unviable in most laboratory strains . Previously, we constructed a temperature-sensitive bmh2 mutant and showed that mutations in RTG3 and SIN4, both encoding transcriptional regulators, can suppress the temperature-sensitive phenotype of this mutant, suggesting an inhibitory role of the 14-3-3 proteins in Rtg3-dependent transcription {van Heusden and Steensma (2001) Yeast 18, 1479-1491} . In the present paper, we report a genome-wide transcription analysis of a temperature-sensitive bmh2 mutant . Steady-state mRNA levels of 60 open reading frames were increased more than 2.0-fold in the bmh2 mutant, whereas those of 78 open reading frames were decreased more than 2.0-fold . In agreement with our genetic experiments, six genes known to be regulated by Rtg3 showed elevated mRNA levels in the mutant . In addition, several genes with other cellular functions, including those involved in gluconeogenesis, ergosterol biosynthesis and stress response, had altered mRNA levels in the mutant . Our data show that the yeast 14-3-3 proteins negatively regulate Rtg3-dependent transcription, stimulate the transcription of genes involved in ergosterol metabolism and in stress response and are involved in transcription regulation of multiple other genes. FEMS Microbiol Lett, 2004 May 15, 234(2), 371 - 8 Swm1p, a subunit of the APC/cyclosome, is required to maintain cell wall integrity during growth at high temperature in Saccharomyces cerevisiae; Ufano S et al.; Swm1p, a subunit of the APC cyclosome, was originally identified for its role in the later stages of the sporulation process and is required for spore wall assembly . In addition, this protein is required to maintain cell wall integrity in vegetative cells during growth at high temperature . Electron microscopy analyses of mutant cells grown at the restrictive temperature in the absence of osmotic support show that the cell wall is clearly abnormal, with large number of discontinuities that may be responsible for the observed lysis . The mutant cells show a 7-fold reduction in glucan synthase activity during growth at 38 degrees C and a 3.5-fold increase in the chitin content of the cell wall . The chitin is deposited in a delocalized manner all over the cell wall, where it accumulates in patches in abnormal regions . The excess chitin is mainly synthesized by the action of chitin synthase III (Chs3p), since it disappears in the swm1 chs3 double-mutant . FEBS Lett, 2004 May 7, 565(1-3), 106 - 10 Rrs1p, a ribosomal protein L11-binding protein, is required for nuclear export of the 60S pre-ribosomal subunit in Saccharomyces cerevisiae; Miyoshi K et al.; Rrs1p is a ribosomal protein L11-binding protein in Saccharomyces cerevisiae . We have obtained temperature-sensitive rrs1 mutants by random PCR mutagenesis . {(3)H}Methionine pulse-chase analysis reveals that the rrs1 mutations cause a defect in maturation of 25S rRNA . Ribosomal protein L25-enhanced green fluorescent protein, a reporter of the 60S ribosomal subunit, concentrates in the nucleus with enrichment in the nucleolus when the rrs1 mutants are shifted to the restrictive temperature . These results suggest that Rrs1p stays on the pre-60S particle from the early stage to very late stage of the large-subunit maturation and is required for export of 60S subunits from the nucleolus to the cytoplasm. Mol Genet Genomics, 2004 Jun, 271(5), 603 - 15 Epub 2004 May 06. Genetic interactions between an RNA polymerase II phosphatase and centromeric elements in Saccharomyces cerevisiae; Pierstorff E et al.; The Saccharomyces cerevisiase protein phosphatase Fcp1 has been implicated in the regulation of transcription by RNA polymerase II, and is encoded by the essential gene FCP1 . A screen was carried out for multicopy suppressors of the temperature-sensitive phenotype of two phosphatase mutants, fcp1-2 and fcp1-4 . Only the wild-type FCP1 was found to suppress (complement) the fcp1-4 mutation . For fcp1-2 three second-site suppressors were identified . One contained the ORF for ZDS1 . The remaining two suppressors mapped to the centromere regions of chromosomes I and V . Suppression due to centromere DNA was found to be more dependent on the CDEIII region than on other regions of the centromere . The presence of a suppressor centromere affected the level of Fcp1 protein and the overall phosphorylation state of RNA polymerase II (RNAPII) in fcp1-2 cells, but not wild-type cells, grown at both permissive and non-permissive temperatures . In addition, genetic interactions were identified between this FCP1 mutant and the genes SKP1, CEP3 and CBF1, which code for centromere binding proteins . The mechanism of suppression and regulation of Fcp1-2 protein activity by centromeric DNA is discussed. Nucleic Acids Res, 2004 May 07, 32(8), 2529 - 40 Print 2004. The Saccharomyces cerevisiae gene CDC40/PRP17 controls cell cycle progression through splicing of the ANC1 gene; Dahan O et al.; The timing of events in the cell cycle is of crucial importance, as any error can lead to cell death or cancerous growth . This accurate timing is accomplished through the activation of specific CDC genes . Mutations in the CDC40/PRP17 gene cause cell cycle arrest at the G2/M stage . It was previously found that the CDC40 gene encodes a pre-mRNA splicing factor, which participates in the second step of the splicing reaction . In this paper we dissect the mechanism by which pre-mRNA splicing affects cell cycle progression . We identify ANC1 as the target of CDC40 regulation . Deletion of the ANC1 intron relieves the cell cycle arrest and temperature sensitivity of cdc40 mutants . Furthermore, we identify, through point mutation analysis, specific residues in the ANC1 intron that are important for its splicing dependency on Cdc40p . Our results demonstrate a novel mechanism of cell cycle regulation that relies on the differential splicing of a subset of introns by specific splicing factors. J Biol Chem, 2004 Jul 16, 279(29), 30298 - 306 Epub 2004 May 09. Cti6 is an Rpd3-Sin3 histone deacetylase-associated protein required for growth under iron-limiting conditions in Saccharomyces cerevisiae; Puig S et al.; Iron and copper are redox active metals essential for life . In the budding yeast Saccharomyces cerevisiae, expression of iron and copper genes involved in metal acquisition and utilization is tightly regulated at the transcriptional level . In addition iron and copper metabolism are inextricably linked because of the dependence on copper as a co-factor for iron uptake or mobilization . To further identify genes that function in iron and copper homeostasis, we screened for novel yeast mutants defective for iron limiting growth and thereby identified the CTI6 gene . Cti6 is a PHD finger-containing protein that has been shown to participate in the interaction of the Ssn6-Tup1 co-repressor with the Gcn5-containing SAGA chromatin-remodeling complex . In this report we show that CTI6 mRNA levels are increased under iron-limiting conditions, and that cti6 mutants display a growth defect under conditions of iron deprivation . Furthermore, we demonstrate that Cti6 is a nuclear protein that functionally associates with the Rpd3-Sin3 histone deacetylase complex involved in transcriptional repression . Cti6 demonstrates Rpd3-dependent transcriptional repression, and cti6 mutants exhibit an enhanced silencing of telomeric, rDNA and HMR loci, similar to mutants in genes encoding other Rpd3-Sin3-associated proteins . Microarray experiments with cti6 mutants grown under iron-limiting conditions show a down-regulation of telomeric genes and an up-regulation of Aft1 and Tup1 target genes involved in iron and oxygen regulation . Taken together, these data suggest a specific role for Cti6 in the regulation of gene expression under conditions of iron limitation. Appl Bioinformatics, 2003, 2(3 Suppl), S37 - 46 Complementary techniques of clustering and composite pattern analysis to Saccharomyces cerevisiae gene expression; Magusin A; We present the combined application of clustering and composite pattern analysis to Saccharomyces cerevisiae gene expression and sequence data . The aim is to devise a system for the efficient investigation of the upstream regions associated with regulating the coordinated transcriptional response of genes via the location of cis-acting elements . The clustering algorithm presented allows clusters to overlap in light of the fact that many genes are regulated by multiple factors and/or are associated with multiple functions in the organism . The sequence clusters are analysed for shared monad and dyad patterns . These motifs are commonly regarded as putative cis-acting regulatory elements . Computationally, dyad patterns may be difficult and expensive to discover and verify if the sequences are very large . This has highlighted the need to obtain maximally--with regards to the pattern in question--'enriched' sets of sequences to uncover these motifs . We present overlapping clusters as an ideal basis for the discovery of conserved motifs. Lett Appl Microbiol, 2004, 38(6), 454 - 8 Surface alteration of Saccharomyces cerevisiae induced by thymol and eugenol; Bennis S et al.; AIMS: This study aims to bring some information about the mechanism of the fungicidal action of thymol and eugenol; phenolic major components of thyme and clove essential oils respectively . Saccharomyces cerevisiae was used as yeast model . METHODS AND RESULTS: Treatment of yeast cells with these components led to their lysis as shown by the release of substances absorbing at 260 nm . In addition, scanning electron microscope observations revealed that the surface of the treated cells was significantly damaged . CONCLUSIONS: Antifungal activity of thymol and eugenol involve alteration of both membrane and cell wall of the yeast . SIGNIFICANCE AND IMPACT OF THE STUDY: This work is a preliminary contribution aiming to develop a new generation of efficient and natural antifungal agents. J Biol Chem, 2004 Jul 16, 279(29), 30274 - 8 Epub 2004 May 05. Comprehensive chimeric analysis of amino acid residues critical for high affinity glucose transport by Hxt2 of Saccharomyces cerevisiae; Kasahara T et al.; Chimeras of Hxt2 and Hxt1,