Biochim Biophys Acta, 2003 Dec 3, 1618(1), 17 - 24 Structure-function studies of yeast C-4 sphingolipid long chain base hydroxylase; Idkowiak-Baldys J et al.; The roles of putative active site residues of the Saccharomyces cerevisiae sphingolipid C-4 long chain base hydroxylase (Sur2p) were investigated by site-directed mutagenesis . The replacement of any one of conserved His residues of three histidine-rich motifs with an alanine eliminated hydroxylase activity in vivo and in vitro, indicating that they are all essential elements of the active site . An additional conserved His residue (His 249) outside of the histidine-rich cluster region was also found to be crucial for activity . Additional mutants altered in residues in close proximity to the histidine-rich cluster were generated . In order to determine their roles in hydroxylase vs . desaturase activities, residues were replaced with conserved residues from the yeast Delta7-sterol-C5(6)-desaturase, Erg3p . Residues Phe 174, Asn 182, Ser 191, Leu 196, Pro 199, Asn 266, Tyr 269, Asp 271 and Gln 275 appear to be additionally important elements of the active site but their conversion into corresponding Erg3p residues did not lead to a gain in desaturase activity . It is concluded that Sur2p is a membrane-bound hydroxylase that belongs to the diiron family of eight-histidine motif enzymes. J Theor Biol, 2004 Feb 7, 226(3), 265 - 91 Integration of kinetic information on yeast sphingolipid metabolism in dynamical pathway models; Alvarez-Vasquez F et al.; For the first time, kinetic information from the literature was collected and used to construct integrative dynamical mathematical models of sphingolipid metabolism . One model was designed primarily with kinetic equations in the tradition of Michaelis and Menten whereas the other two models were designed as alternative power-law models within the framework of Biochemical Systems Theory . Each model contains about 50 variables, about a quarter of which are dependent (state) variables, while the others are independent inputs and enzyme activities that are considered constant . The models account for known regulatory signals that exert control over the pathway . Standard mathematical testing, repeated revisiting of the literature, and numerous rounds of amendments and refinements resulted in models that are stable and rather insensitive to perturbations in inputs or parameter values . The models also appear to be compatible with the modest amount of experimental experience that lends itself to direct comparisons . Even though the three models are based on different mathematical representations, they show dynamic responses to a variety of perturbations and changes in conditions that are essentially equivalent for small perturbations and similar for large perturbations . The kinetic information used for model construction and the models themselves can serve as a starting point for future analyses and refinements. Mutat Res, 2003 Nov 27, 532(1-2), 157 - 72 Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability; Weitao T et al.; We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae . Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis . We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB . Both pausing and breakage are elevated in the hypomorphic dna2-2 helicase mutant . Deletion of FOB1 suppresses the elevated pausing and DSB formation . Our current work shows that mutation inactivating Sgs1, the yeast RecQ helicase ortholog, also causes accumulation of stalled replication forks and DSBs at the rDNA RFB . Either deletion of FOB1, which suppresses fork blocking and certain types of rDNA recombination, or an increase in SIR2 gene dosage, which suppresses rDNA recombination, reduces the number of forks persisting at the RFB . Although dna2-2 sgs1Delta double mutants are conditionally lethal, they do not show enhanced rDNA defects compared to sgs1Delta alone . However, surprisingly, the dna2-2 sgs1Delta lethality is suppressed by deletion of FOB1 . On the other hand, the dna2-2 sgs1Delta lethality is only partially suppressed by deletion of rad51Delta . We propose that the replication-associated defects that we document in the rDNA are characteristic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones. Comput Biol Chem, 2003 Oct, 27(4-5), 497 - 506 Detection of potential positive regulatory motifs of transcription in yeast introns by comparative analysis of oligonucleotide frequencies; Zhang J et al.; We conducted a comparative statistical analysis of tetra- through hexanucleotide frequencies in two sets of introns of yeast genes . The first set consisted of introns of genes that have transcription rates higher than 30 mRNAs/h while the second set contained introns of genes whose transcription rates were lower than or equal to 10 mRNAs/h . Some oligonucleotides whose occurrence frequencies in the first set of introns are significantly higher than those in the second set of introns were detected . The frequencies of occurrence of most of these detected oligonucleotides are also significantly higher than those in the exons flanking the introns of the first set . Interestingly some of these detected oligonucleotides are the same as well known "signature" sequences of transcriptional regulatory elements . This could imply the existence of potential positive regulatory motifs of transcription in yeast introns. DNA Repair (Amst), 2003 Dec 9, 2(12), 1321 - 35 MEC1-dependent phosphorylation of yeast RPA1 in vitro; Kim HS et al.; Replication protein A (RPA) is a conserved single-stranded DNA (ssDNA) binding protein with well-characterized roles in DNA metabolism . RPA is phosphorylated in response to genotoxic stress and is required for efficient checkpoint function, although these aspects of RPA function are not well understood . We have investigated the association between RPA and the checkpoint kinase Mec1 in yeast . RPA and Mec1 were found to be physically associated during unperturbed cell growth and in response to DNA damage . Using a Mec1 immunoprecipitate (IP)-kinase assay, we show that the two large subunits, RPA1 and RPA2, are good substrates for Mec1 kinase . The major phosphorylation site of RPA1 was further investigated as it was found to be localized to its amino terminus (RPA1N), which is a non-ssDNA binding domain implicated in regulatory function . This phosphorylation site mapped to serine 178 and phosphorylation-defective mutant protein, expressed from rfa1-S178A, showed reduced physical interaction with Mec1 . Phenotypic analysis in vivo revealed that the rfa1-S178A mutation affected the kinetics of RPA1 and Rad53 phosphorylation but did not otherwise affect the checkpoint response . We suggest that phosphorylation of RPA1N by Mec1 may function together with other checkpoint events to regulate the checkpoint response. FEMS Immunol Med Microbiol, 2003 Dec 5, 39(3), 279 - 86 Extensive MHC class I-restricted CD8 T lymphocyte responses against various yeast genera in humans; Heintel T et al.; The human cellular immune response against 14 distantly related yeast species was analyzed by intracellular cytokine staining of lymphocytes after ex vivo stimulation of whole blood . While the CD4 T cell response was marginal, extensive MHC class I-restricted CD8 T cell responses were detected against a number of species including spoiling, environmental and human pathogenic yeasts . The yeast-specific CD8 T cells expressed interferon-gamma but lacked expression of CD27 and CCR7, indicating that they were end-differentiated effector memory cells . Mainly intact yeast cells rather than spheroplasts were able to induce cytokine expression in T cells demonstrating that the dominant immunogens were located in the yeast cell wall . Together these data underline the importance of the cellular immune response in protecting humans against yeast and fungal infections . And, from another perspective, recombinant yeast suggests itself as a potential vaccine candidate to efficiently induce antigen-specific CD8 T cell responses. Biochem Soc Trans, 2003 Dec, 31(Pt 6), 1409 - 12 Anti-glycation defences in yeast; Ponces Freire A et al.; Saccharomyces cerevisiae is an outstanding cellular model for metabolic studies in glycation . Due to its high glycolytic activity, it produces methylglyoxal, a highly reactive intracellular glycation agent, at a rate of approx . 0.1% of the glycolytic flux . We investigated methylglyoxal metabolism in Saccharomyces cerevisiae cells, using haploid null mutants . Growth studies showed that the most sensitive strains to 2-oxoaldehydes were the null mutants for GSH1 and GLO1, coding for glutathione synthase I and glyoxalase I respectively . The GRE3 null mutant, lacking aldose reductase activity, is as sensitive as the control strain . Kinetic modelling and computer simulation of this type of experiment were also performed, and we concluded that the most important parameters for controlling the intracellular concentration of methylglyoxal are the activity of glyoxalase I and the GSH concentration . Moreover, our model predicts an intracellular steady-state concentration of methylglyoxal of approx . 2 microM . Our results show that the glyoxalase pathway is the main detoxification pathway for 2-oxoaldehydes in yeast, and is likely to be the key enzymatic anti-glycation agent in these cells. Cytometry, 1996 Jan 1, 23(1), 28 - 38 Discrimination of respiratory dysfunction in yeast mutants by confocal microscopy, image, and flow cytometry; Petit P et al.; Living yeast cells can be selectively stained with the lipophilic cationic cyanine dye DiOC6(3) in a mitochondrial membrane potential-dependent manner . Our study extends the use of flow cytometric analysis and sorting to DiOC6(3)-stained yeast cells . Experimental conditions were developed that prevented the toxic side effect of the probe and gave a quantitative correlation between fluorescence and mitochondrial membrane potential, without any staining of other membranes . The localization of the fluorochrome was checked by confocal microscopy and image cytometry . The mitochondrial membrane alterations were also tested through cardiolipin staining with nonyl acridine orange . Differences in light scattering and in fluorescence were detected in mutants (rho-, rho degrees, mit-, or pet-) and wild-type (rho+mit+) populations of yeast . The dye uptake of respiratory-deficient yeast strains was significantly reduced as compared to that of the wild-type . Application of an uncoupler (mCICCP), which collapsed the mitochondrial membrane potential (alphapsi(m)), led to a drastic reduction of the dye uptake . It was observed that a decrease in deltapsi(m), was usually correlated with a decrease in cardiolipin stainability by nonyl acridine orange (NAO) . Quantitative flow cytometry is a fast and reproducible technique for rapid screening of yeast strains that might be suspected of respiratory dysfunction and/or mitochondrial structural changes . We give evidence that it is an adequate method to characterize and isolate respiratory mutants through sorting procedure, with selective enrichment of the population studied in respiring or non-respiring yeast cells . Confocal microscopy and image cytometry corroborate the flow cytometry results. Mol Genet Genomics, 2004 Apr, 271(3), 257 - 66 Epub 2003 Nov 27. Nuclear import of yeast Gcn4p requires karyopherins Srp1p and Kap95p; Pries R et al.; The yeast transcription factor Gcn4p contains two stretches of amino acid residues, NLS1 and NLS2, which are independently able to relocate the cytoplasmic protein chorismate mutase into the nucleus . Only NLS2 is conserved among fungi . A truncated version of CPCA (the counterpart of Gcn4p in Aspergillus nidulans), which lacks the conserved NLS, accumulates in the cytoplasm instead of the nucleus . Nuclear uptake mediated by the NLS1 of Gcn4p is impaired by defects in genes for several different karyopherins, whereas NLS2-dependent nuclear import specifically requires the alpha-importin Srp1p and the beta-importin Kap95p . Yeast strains that are defective in either of these two karyopherins are unable to respond to amino acid starvation . We have thus identified Gcn4p as a substrate for the Srp1p/Kap95p transport complex . Our data suggest that NLS2 is the essential and specific nuclear transport signal; NLS1 may play only an unspecific or accessory role. Planta, 2004 Mar, 218(5), 814 - 23 Epub 2003 Nov 25. Functional expression in yeast of an N-deleted form of At-ACA8, a plasma membrane Ca(2+)-ATPase of Arabidopsis thaliana, and characterization of a hyperactive mutant; Bonza MC et al.; A constitutively active form of At-ACA8, a plasma membrane Ca(2+)-ATPase from Arabidopsis thaliana (L.) Heynh., from which the first 74 amino acids containing the calmodulin-binding domain (delta74- At-ACA8) had been deleted, was expressed in Saccharomyces cerevisiae strain K616, which lacks the main endogenous active Ca(2+) transport systems . Delta74- At-ACA8 complemented the K616 phenotype, making it able to grow in a calcium-depleted medium . Delta74- At-ACA8 protein, which co-migrated with the endoplasmic reticulum marker BiP in a sucrose-density gradient, catalyzed MgATP-dependent Ca(2+) uptake and Ca(2+)-dependent MgATP hydrolysis, and retained the biochemical characteristics of the native plant plasma membrane Ca(2+)-ATPase (low specificity for nucleoside triphosphate, high sensitivity to inhibition by the fluorescein derivatives erythrosin B and eosin Y), thus confirming that it is correctly folded and functional . Substitution of the (794)HE residues (numbers refer to full-length At-ACA8) following the highly conserved TGDG(TV)NDP(AS)L motif in the cytoplasmic headpiece with two lysine residues generated an hyperactive protein, with a catalytic activity 2-fold higher than that of delta74- At-ACA8 . The (794)HE-->KK mutant was also about 6-fold more sensitive than delta74- At-ACA8 to inhibition by vanadate, indicating that the mutation determines an increase in the proportion of enzyme in the E(2) state during the catalytic cycle. Curr Genet, 2004 Mar, 45(3), 183 - 6 Epub 2003 Nov 26. High-efficiency transformation of the pathogenic yeast Candida parapsilosis; Zemanova J et al.; A recently developed transformation system for the pathogenic yeast Candida parapsilosis opened a venue for studying the biological phenomena of this species at the molecular level . However, the standard chemical method yielded only about 1x10(3) transformants/microg of DNA, which is insufficient for certain types of experiment . With the aim of increasing the transformation efficiency, we employed two alternative methods for the introduction of plasmids into the recipient cells . Whereas biolistics resulted in about 5x10(2) transformants/microg of plasmid DNA, electroporation was an order of magnitude more efficient than the chemical method . Pretreatment of cells with 100 mM lithium acetate or 10 mM dithiothreitol resulted in a 5-fold (5x10(4)) or a 10-fold (1x10(5)) increase in transformation efficiency, respectively . This high-efficiency transformation method should be suitable for experiments such as the screening of DNA libraries. Biochem J, 2004 Mar 15, 378(Pt 3), 899 - 908 Lipid dynamics in yeast under haem-induced unsaturated fatty acid and/or sterol depletion; Ferreira T et al.; In the yeast Saccharomyces cerevisiae, UFA (unsaturated fatty acids) and ergosterol syntheses are aerobic processes that require haem . We took advantage of a strain affected in haem synthesis ( hem1 Delta) to starve specifically for one or the other of these essential lipids in order to examine the consequences on the overall lipid composition . Our results demonstrate that reserve lipids (i.e . triacylglycerols and steryl esters) are depleted independently of haem availability and that their UFA and sterol content is not crucial to sustain residual growth under lipid depletion . In parallel to UFA starvation, a net accumulation of SFA (saturated fatty acids) is observed as a consequence of haem biosynthesis preclusion . Interestingly, the excess SFA are not mainly stored within triacylglycerols and steryl esters but rather within specific phospholipid species, with a marked preference for PtdIns . This results in an increase in the cellular PtdIns content . However, neutral lipid homoeostasis is perturbed under haem starvation . The contribution of two lipid particle-associated proteins (namely Tgl1p and Dga1p) to this process is described. Biochemistry, 2003 Dec 9, 42(48), 14139 - 49 Characterization of the yeast peroxiredoxin Ahp1 in its reduced active and overoxidized inactive forms using NMR; Trivelli X et al.; Peroxiredoxins (Prx's) are a superfamily of thiol-specific antioxidant proteins present in all organisms and involved in the hydroperoxide detoxification of the cell . The catalytic cysteine of Prx's reduces hydroperoxides and is transformed into a transient sulfenic acid (Cys-SOH) . At high hydroperoxide concentration, the sulfenic acid can be overoxidized into a sulfinate, or even a sulfonate . We present here the first peroxiredoxin characterization by solution NMR of the Saccharomyces cerevisiae alkylhydroperoxide reductase (Ahp1) in its reduced and in vitro overoxidized forms . NMR (15)N relaxation data and ultracentrifugation experiments indicate that the protein behaves principally as a homodimer (2 x 19 kDa) in solution, regardless of the redox state . In vitro treatment of Ahp1 by a large excess of tBuOOH leads to an inactive form, with the catalytic cysteine overoxidized into sulfonate, as demonstrated by (13)C NMR . Depending on the amino acid sequence of their active site, Prx's are classified into five different families . In this classification, Ahp1 is a member of the scarcely studied D-type Prx's . Ahp1 is unique among the D-type Prx's in its ability to form an intermolecular disulfide . The peptidic sequence of Ahp1 was analyzed and compared to other D-type Prx sequences. J Bioenerg Biomembr, 2003 Aug, 35(4), 313 - 21 Assembly and regulation of the yeast vacuolar H+-ATPase; Kane PM et al.; The yeast vacuolar proton-translocating ATPase (V-ATPase) is an excellent model for V-ATPases in all eukaryotic cells . Activity of the yeast V-ATPase is reversibly down-regulated by disassembly of the peripheral (V1) sector, which contains the ATP-binding sites, from the membrane (V0) sector, which contains the proton pore . A similar regulatory mechanism has been found in Manduca sexta and is believed to operate in other eukaryotes . We are interested in the mechanism of reversible disassembly and its implications for V-ATPase structure . In this review, we focus on (1) characterization of the yeast V-ATPase stalk subunits, which form the interface between V1 and V0, (2) potential mechanisms of silencing ATP hydrolytic activity in disassembled V1 sectors, and (3) the structure and function of RAVE, a recently discovered complex that regulates V-ATPase assembly. J Bioenerg Biomembr, 2003 Aug, 35(4), 301 - 12 Structure and assembly of the yeast V-ATPase; Graham LA et al.; The yeast V-ATPase belongs to a family of V-type ATPases present in all eucaryotic organisms . In Saccharomyces cerevisiae the V-ATPase is localized to the membrane of the vacuole as well as the Golgi complex and endosomes . The V-ATPase brings about the acidification of these organelles by the transport of protons coupled to the hydrolysis of ATP . In yeast, the V-ATPase is composed of 13 subunits consisting of a catalytic V1 domain of peripherally associated proteins and a proton-translocating V0 domain of integral membrane proteins . The regulatory subunit, Vma13p, was the first V-ATPase subunit to have its crystal structure determined . In addition to proteins forming the functional V-ATPase complex, three ER-localized proteins facilitate the assembly of the V0 subunits following their translation and insertion into the membrane of the ER . Homologues of the Vma21p assembly factor have been identified in many higher eukaryotes supporting a ubiquitous assembly pathway for this important enzyme complex. J Bioenerg Biomembr, 2003 Aug, 35(4), 291 - 9 Subunit structure, function, and arrangement in the yeast and coated vesicle V-ATPases; Inoue T et al.; The vacuolar (H+)-ATPases (or V-ATPases) are ATP-dependent proton pumps that function both to acidify intracellular compartments and to transport protons across the plasma membrane . Acidification of intracellular compartments is important for such processes as receptor-mediated endocytosis, intracellular trafficking, protein processing, and coupled transport . Plasma membrane V-ATPases function in renal acidification, bone resorption, pH homeostasis, and, possibly, tumor metastasis . This review will focus on work from our laboratories on the V-ATPases from mammalian clathrin-coated vesicles and from yeast . The V-ATPases are composed of two domains . The peripheral V1 domain has a molecular mass of 640 kDa and is composed of eight different subunits (subunits A-H) of molecular mass 70-13 kDa . The integral V0 domain, which has a molecular mass of 260 kDa, is composed of five different subunits (subunits a, d, c, c', and c'') of molecular mass 100-17 kDa . The V1 domain is responsible for ATP hydrolysis whereas the V0 domain is responsible for proton transport . Using a variety of techniques, including cysteine-mediated crosslinking and electron microscopy, we have defined both the overall shape of the V-ATPase and the V0 domain as well as the location of various subunits within the complex . We have employed site-directed and random mutagenesis to identify subunits and residues involved in nucleotide binding and hydrolysis, proton translocation, and the coupling of these two processes . We have also investigated the mechanism of regulation of the V-ATPase by reversible dissociation and the role of different subunits in this process. J Bioenerg Biomembr, 2003 Aug, 35(4), 281 - 9 A journey from mammals to yeast with vacuolar H+-ATPase (V-ATPase); Nelson N; The vacuolar H+-ATPase (V-ATPase) is one of the most fundamental enzymes in nature . It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes . V-ATPase has a structure and mechanism of action similar to F-ATPase and several of their subunits probably evolved from common ancestors . In eukaryotic cells, F-ATPase is confined to the semiautonomous organelles, chloroplasts and mitochondria, which contain their own genes that encode some of the F-ATPase subunits . In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the protonmotive force (pmf), V-ATPases function exclusively as ATP-dependent proton pumps . The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes . It was the survival of the yeast mutant without the active enzyme and yeast genetics that allowed the identification of genuine subunits of the V-ATPase . It also revealed special properties of individual subunits, factors that are involved in the enzyme's biogenesis and assembly, as well as the involvement of V-ATPase in the secretory pathway, endocytosis, and respiration . It may be the insect V-ATPase that unconventionally resides in the plasma membrane of their midgut, that will give the first structure resolution of this complex. Nat Rev Genet, 2003 Nov, 4(11), 916 - 23 Clues to catastrophic telomere loss in mammals from yeast telomere rapid deletion; Lustig AJ; Catastrophic losses of telomeric sequences have recently been described during apoptosis, senescence and tumorigenesis in murine and human cells, in ataxia telangiectasia patients and in immortalized cells in which telomerase is inactive . A mechanism that underlies a single-step non-reciprocal telomere deletion called telomere rapid deletion in Saccharomyces cerevisiae might provide clues for future studies of catastrophic telomere loss in higher eukaryotes. Genes Dev, 2003 Dec 1, 17(23), 2902 - 21 Epub 2003 Nov 21. Hierarchical assembly of the budding yeast kinetochore from multiple subcomplexes; De Wulf P et al.; Kinetochores are multiprotein complexes that assemble on centromeric DNA and attach chromosomes to spindle microtubules . Over the past six years, the number of proteins known to localize to the Saccharomyces cerevisiae kinetochore has increased from around 10 to over 60 . However, relatively little is known about the protein-protein interactions that mediate kinetochore assembly or about the overall structure of microtubule-attachment sites . Here we used biophysical techniques, affinity purification, mass spectrometry, and in vivo assays to examine the state of association of 31 centromere-binding proteins, including six proteins newly identified as kinetochore subunits . We found that yeast kinetochores resemble transcriptional enhancers in being composed of at least 17 discrete subcomplexes that assemble on DNA to form a very large structure with a mass in excess of 5 MD . Critical to kinetochore assembly are proteins that bridge subunits in direct contact with DNA and subunits bound to microtubules . We show that two newly identified kinetochore complexes, COMA (Ctf19p-Okp1p-Mcm21p-Ame1p) and MIND (Mtw1p including Nnf1p-Nsl1p-Dsn1p) function as bridges . COMA, MIND, and the previously described Ndc80 complex constitute three independent and essential platforms onto which outer kinetochore proteins assemble . In addition, we propose that the three complexes have different functions with respect to force generation and MT attachment. Am J Pathol, 2003 Dec, 163(6), 2201 - 9 Reconstructed beta-catenin/TCF4 signaling in yeast applicable to functional evaluation of APC mutations; Yamada H et al.; In human genetics and molecular oncology, mutation research is necessary not only to identify mutations in nucleic acid sequences, but also to analyze the loss of function caused by mutant proteins . We reconstructed a protein-protein network system of human beta-catenin and TCF4, in Saccharomyces cerevisiae . beta-Catenin and TCF4 proteins form a complex and transactivate reporter genes . Co-expressed wild-type APC with beta-catenin and TCF4 inhibit the transcriptional activity of the beta-catenin/TCF4 complex in yeast, as well as in mammals . This unique method in which the beta-catenin/TCF4 signaling pathway is reconstructed in vivo may prove useful for the functional evaluation of APC mutants, including a type of APC truncated and missense mutants influenced to the ability of binding to beta-catenin. Proc Natl Acad Sci U S A, 2003 Dec 9, 100(25), 14994 - 9 Epub 2003 Nov 20. Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast; Storici F et al.; The repair of chromosomal double-strand breaks (DSBs) can be accomplished through homologous recombination in most organisms . We report here that exogenous oligonucleotides can efficiently target for repair a single DSB induced in a chromosome of yeast . The efficiency of recombinational targeting leading to a desired DNA change can be as high as 20% of cells . The DSB was generated either by a regulatable I-SceI endonuclease just before transformation or appeared spontaneously at the site of a long inverted repeat composed of human Alu sequences . The approach used features of our previously described delitto perfetto system for selecting transformants with integrative recombinant oligonucleotides . The DSB repair mediated by pairs of complementary integrative recombinant oligonucleotides was efficient for targeting to homologous sequences that were close to or distant from the DSB and in the presence of a competing homologous chromosome in diploid cells . We also demonstrate that a DSB can strongly stimulate recombination with single-stranded DNA, without strand bias . These findings expand current models of DSB repair . In addition, we establish a high-throughput system for rapid genome-wide modification with oligonucleotides. Exp Diabesity Res, 2003 Apr-Jun, 4(2), 125 - 32 Hyperglycemia-induced protein kinase C activation inhibits phagocytosis of C3b- and immunoglobulin g-opsonized yeast particles in normal human neutrophils; Saiepour D et al.; The aim of this study was to investigate the effects of elevated glucose concentrations on complement receptor- and Fcgamma receptor-mediated phagocytosis in normal human neutrophils . D-Glucose at 15 or 25 mM dose-dependently inhibited both complement receptor- and Fcgamma receptor-mediated phagocytosis, as compared to that at a normal physiological glucose concentration . The protein kinase C (PKC) inhibitors GF109203X and Go6976 both dose-dependently and completely reversed the inhibitory effect of 25 mM D-glucose on phagocytosis . Complement receptor-mediated phagocytosis was dose-dependently inhibited by the cell permeable diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol (DAG), an effect that was abolished by PKC inhibitors . Furthermore, suboptimal inhibitory concentrations of DAG and glucose showed an additive inhibitory effect on complement receptor-mediated phagocytosis . The authors conclude that elevated glucose concentrations can inhibit complement receptor and Fcgamma receptor-mediated phagocytosis in normal human neutrophils by activating PKCalpha and/or PKCbeta, an effect possibly mediated by DAG. J Microsc, 2003 Dec, 212(Pt 3), 239 - 43 Encapsulated yeast cells inside Paramecium primaurelia: a model system for protection capability of polyelectrolyte shells; Krol S et al.; One of the most promising applications of encapsulated living cells is their use as protected transplanted tissue into the human body . A suitable system for the protection of living cells is the use of nano- or microcapsules of polyelectrolytes . These shells can be deposited easily on top of the cells by means of a layer-by-layer technique . An interesting feature of the capsules is the possibility to control their properties on a nanometre level, tuning their wall texture via the preparation conditions . Here we introduce a model system to test the protection ability of polyelectrolyte capsules . Common bakery yeast cells were encapsulated . They were coated with a fluorescently labelled shell at conditions known to guarantee cell survival, and the cell interior was stained with DAPI . The protozoan Paramecium primaurelia was incubated with this double-stained living yeast and visualized by means of two-photon excitation fluorescence microscopy . Cross-sections of the dye-stained material as well as autofluorescence of the fixed protozoan allowed us to follow the digestion of the coated yeast with time . Our investigation reveals that capsules prepared under these deposition conditions are permeable to lysosomal enzymes, leading to degradation of the yeast inside the intact capsules . Our preliminary results indicate the suitability of the introduced model as a test system of this permeability. Biochem J, 2004 Mar 1, 378(Pt 2), 599 - 607 Role of YHM1, encoding a mitochondrial carrier protein, in iron distribution of yeast; Lesuisse E et al.; Mitochondrial carrier proteins are a large protein family, consisting of 35 members in Saccharomyces cerevisiae . Members of this protein family have been shown to transport varied substrates from cytoplasm to mitochondria or mitochondria to cytoplasm, although many family members do not have assigned substrates . We speculated whether one or more of these transporters will play a role in iron metabolism . Haploid yeast strains each deleted for a single mitochondrial carrier protein were analysed for alterations in iron homoeostasis . The strain deleted for YHM1 was characterized by increased and misregulated surface ferric reductase and high-affinity ferrous transport activities . Siderophore uptake from different sources was also increased, and these effects were dependent on the AFT1 iron sensor regulator . Mutants of YHM1 converted into rho degrees, consistent with secondary mitochondrial DNA damage from mitochondrial iron accumulation . In fact, in the Delta yhm1 mutant, iron was found to accumulate in mitochondria . The accumulated iron showed decreased availability for haem synthesis, measured in isolated mitochondria using endogenously available metals and added porphyrins . The phenotypes of Delta yhm1 mutants indicate a role for this mitochondrial transporter in cellular iron homoeostasis. Eur J Cell Biol, 2003 Oct, 82(10), 531 - 8 The spindle pole body of the pathogenic yeast Exophiala dermatitidis: variation in morphology and positional relationship to the nucleolus and the bud in interphase cells; Yamaguchi M et al.; The spindle pole body (SPB) in the interphase cell of the pathogenic yeast Exophiala dermatitidis was studied in detail . The SPB was located on the outer nuclear envelope and was 342 +/- 86 nm long in a haploid strain . It consisted of two disk elements that measured 151 +/- 43 nm in diameter and 103 +/- 17 nm in thickness, connected by a rod-shaped midpiece that measured 56 +/- 20 nm in length and 37 +/- 9 nm in diameter . There were considerable variations in size and morphology of interphase SPB . Some disk elements appeared spherical but others were more flattened, and there was variation in electron density . A few SPBs did not have the midpiece . The SPB of a diploid strain was 486 +/- 118 nm long, thus significantly bigger than that of the haploid strain . The SPB tended to be localized away from the nucleolus (110 +/- 48 degrees), but close to the bud (78 +/- 45 degrees) . The present study highlights the necessity of observing a large number of micrographs in three-dimensions to describe accurately the ultrastructure of the SPB in yeast. Nucleic Acids Res, 2003 Dec 1, 31(23), 7024 - 31 Identifying cooperativity among transcription factors controlling the cell cycle in yeast; Banerjee N et al.; Transcription regulation in eukaryotes is known to occur through the coordinated action of multiple transcription factors (TFs) . Recently, a few genome-wide transcription studies have begun to explore the combinatorial nature of TF interactions . We propose a novel approach that reveals how multiple TFs cooperate to regulate transcription in the yeast cell cycle . Our method integrates genome-wide gene expression data and chromatin immunoprecipitation (ChIP-chip) data to discover more biologically relevant synergistic interactions between different TFs and their target genes than previous studies . Given any pair of TFs A and B, we define a novel measure of cooperativity between the two TFs based on the expression patterns of sets of target genes of only A, only B, and both A and B . If the cooperativity measure is significant then there is reason to postulate that the presence of both TFs is needed to influence gene expression . Our results indicate that many cooperative TFs that were previously characterized experimentally indeed have high values of cooperativity measures in our analysis . In addition, we propose several novel, experimentally testable predictions of cooperative TFs that play a role in the cell cycle and other biological processes . Many of them hold interesting clues for cross talk between the cell cycle and other processes including metabolism, stress response and pseudohyphal differentiation . Finally, we have created a web tool where researchers can explore the exhaustive list of cooperative TFs and survey the graphical representation of the target genes' expression profiles . The interface includes a tool to dynamically draw a TF cooperativity network of 113 TFs with user-defined significance levels . This study is an example of how systematic combination of diverse data types along with new functional genomic approaches can provide a rigorous platform to map TF interactions more efficiently. Nucleic Acids Res, 2003 Dec 1, 31(23), 6788 - 97 A complex pathway for 3' processing of the yeast U3 snoRNA; Kufel J et al.; Mature U3 snoRNA in yeast is generated from the 3'-extended precursors by endonucleolytic cleavage followed by exonucleolytic trimming . These precursors terminate in poly(U) tracts and are normally stabilised by binding of the yeast La homologue, Lhp1p . We report that normal 3' processing of U3 requires the nuclear Lsm proteins . On depletion of any of the five essential proteins, Lsm2-5p or Lsm8p, the normal 3'-extended precursors to the U3 snoRNA were lost . Truncated fragments of both mature and pre-U3 accumulated in the Lsm-depleted strains, consistent with substantial RNA degradation . Pre-U3 species were co-precipitated with TAP-tagged Lsm3p, but the association with spliced pre-U3 was lost in strains lacking Lhp1p . The association of Lhp1p with pre-U3 was also reduced on depletion of Lsm3p or Lsm5p, indicating that binding of Lhp1p and the Lsm proteins is interdependent . In contrast, a tagged Sm-protein detectably co-precipitated spliced pre-U3 species only in strains lacking Lhp1p . We propose that the Lsm2-8p complex functions as a chaperone in conjunction with Lhp1p to stabilise pre-U3 RNA species during 3' processing . The Sm complex may function as a back-up to stabilise 3' ends that are not protected by Lhp1p. Cell Stress Chaperones, 2003 Summer, 8(2), 120 - 4 Protection against oxidation during dehydration of yeast; Pereira Ede J et al.; Based on the well-documented notion that oxygen affects the stability of dried cells, the role of the cytosolic and mitochondrial forms of superoxide dismutase (Sod) in the capacity of cells to resist dehydration was examined . Both enzymes are important for improving survival, and the absence of only 1 isoform did not impair tolerance against dehydration . In addition, sod strains showed the same Sod activity as the control strain, indicating that the deficiency in either cytoplasmic Cu/Zn or mitochondrial Mn was overcome by an increase in activity of the remaining Sod . To measure the level of intracellular oxidation produced by dehydration, a fluorescent probe, 2',7'-dichlorofluorescein, was used . Dry cells exhibited a high increase in fluorescence: both control and sod mutant strains became almost 10-fold more oxidized after dehydration . Furthermore, the disaccharide trehalose was shown to protect dry cells against oxidation. Proc Natl Acad Sci U S A, 1979 Oct, 76(10), 5264 - 8 Control of yeast cell types by mobile genes: a test; Kushner PJ et al.; The yeast Saccharomyces cerevisiae changes cell types by switching the alleles of the mating type locus (MAT) from a to alpha and vice versa . In the cassette model, these switches, --e.g, from a to alpha--occur when a replica of silent alpha information (an alpha "cassette") replaces the resident a cassette at the mating type locus and is thereby expressed . We have identified a mutation in the locus postulated to be the silent alpha information (HMLalpha) and find that a mutation is introduced into the mating type locus as a result of interconversion: HMLalpha(-) MATalpha cells switch to MATa and then to MATalpha(-) . The MATalpha(-) mutation leads to defective mating and behaves like some previously identified MATalpha(-) mutations . These observations satisfy the prediction of the cassette and controlling element models that genetic information is transmitted from HMLalpha to the mating type locus. Biotechnol Lett, 2003 Oct, 25(20), 1775 - 81 Calcium alginate entrapment of the yeast Rhodosporidium toruloides for the kinetic resolution of 1,2-epoxyoctane; Maritz J et al.; Resting cells of the yeast Rhodosporidium toruloides (UOFS Y-0471) were immobilised in calcium alginate beads for the enantioselective kinetic resolution of racemic-1,2-epoxyoctane . The initial activity exhibited by immobilised cells was almost 50% lower than that of the free counterpart but was extremely stable when compared to the free cells . The concentration of the immobilised biomass had no effect on apparent enzyme activity but did lead to a decrease in single cell activity . An increase in both the alginate and CaCl2 concentrations used for bead preparation led to a decrease in enzyme stability . An increase in the alginate concentration led to an increase in bead diameter . The stoichiometric equation for cross-linking of alginate was only obeyed when CaCl2 concentrations higher than 0.4 M were utilised for bead preparation. Biochem Biophys Res Commun, 2003 Nov 28, 311(4), 1019 - 25 Yeast two-hybrid screens implicate DISC1 in brain development and function; Millar JK et al.; DISC1 is a candidate gene for involvement in the aetiology of major psychiatric illnesses including schizophrenia . We report here the results of DISC1 yeast two-hybrid screens using human foetal and adult brain libraries . Twenty-one proteins from a variety of subcellular locations were identified, consistent with observations that DISC1 occupies multiple subcellular compartments . The cellular roles of the proteins identified implicate DISC1 in several aspects of central nervous system development and function, including gene transcription, mitochondrial function, modulation of the actin cytoskeleton, neuronal migration, glutamate transmission, and signal transduction . Intriguingly, mutations in one of the proteins identified, WKL1, have been previously suggested to underlie the aetiology of catatonic schizophrenia. FEBS Lett, 2003 Nov 20, 554(3), 422 - 6 Cuprous oxidase activity of yeast Fet3p and human ceruloplasmin: implication for function; Stoj C et al.; The Fet3 protein in Saccharomyces cerevisiae and mammalian ceruloplasmin are multicopper oxidases (MCO) that are required for iron homeostasis via their catalysis of the ferroxidase reaction, 4Fe(2+)+O(2)+4H(+)-->4Fe(3+)+2H(2)O . The enzymes may play an essential role in copper homeostasis since they exhibit a strikingly similar kinetic activity towards Cu(1+) as substrate . In contrast, laccase, an MCO that exhibits weak activity towards Fe(2+), exhibits a similarly weak activity towards Cu(1+) . Kinetic analyses of the Fet3p reaction demonstrate that the ferroxidase and cuprous oxidase activities are due to the same electron transfer site on the enzyme . These two ferroxidases are fully competent kinetically to play a major role in maintaining the cuprous-cupric redox balance in aerobic organisms. Cell, 2003 Nov 14, 115(4), 401 - 11 Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast; Ira G et al.; Very few gene conversions in mitotic cells are associated with crossovers, suggesting that these events are regulated . This may be important for the maintenance of genetic stability . We have analyzed the relationship between homologous recombination and crossing-over in haploid budding yeast and identified factors involved in the regulation of crossover outcomes . Gene conversions unaccompanied by a crossover appear 30 min before conversions accompanied by exchange, indicating that there are two different repair mechanisms in mitotic cells . Crossovers are rare (5%), but deleting the BLM/WRN homolog, SGS1, or the SRS2 helicase increases crossovers 2- to 3-fold . Overexpressing SRS2 nearly eliminates crossovers, whereas overexpression of RAD51 in srs2Delta cells almost completely eliminates the noncrossover recombination pathway . We suggest Sgs1 and its associated topoisomerase Top3 remove double Holliday junction intermediates from a crossover-producing repair pathway, thereby reducing crossovers . Srs2 promotes the noncrossover synthesis-dependent strand-annealing (SDSA) pathway, apparently by regulating Rad51 binding during strand exchange. Eur J Biochem, 2003 Dec, 270(23), 4689 - 95 Sensitivity to Hsp90-targeting drugs can arise with mutation to the Hsp90 chaperone, cochaperones and plasma membrane ATP binding cassette transporters of yeast; Piper PW et al.; The Hsp90 molecular chaperone catalyses the final activation step of many of the most important regulatory proteins of eukaryotic cells . The antibiotics geldanamycin and radicicol act as highly selective inhibitors of in vivo Hsp90 function through their ability to bind within the ADP/ATP binding pocket of the chaperone . Drugs based on these compounds are now being developed as anticancer agents, their administration having the potential to inactivate simultaneously several of the targets critical for counteracting multistep carcinogenesis . This investigation used yeast to show that cells can be rendered hypersensitive to Hsp90 inhibitors by mutation to Hsp90 itself (within the Hsp82 isoform of yeast Hsp90, the point mutations T101I and A587T); with certain cochaperone defects and through the loss of specific plasma membrane ATP binding cassette transporters (Pdr5p, and to a lesser extent, Snq2p) . The T101I hsp82 and A587T hsp82 mutations do not cause higher drug affinity for purified Hsp90 but may render the in vivo chaperone cycle more sensitive to drug inhibition . It is shown that these mutations render at least one Hsp90-dependent process (deactivation of heat-induced heat shock factor activity) more sensitive to drug inhibition in vivo. AAPS PharmSciTech . 2003;4(3):E41. Acid-treated yeast cell wall as a binder displaying function of disintegrant; Ozeki T et al.; This investigation examined the application of acid-treated yeast cell wall (AYC) as a binder functioning as a disintegrant . Acetylsalicylic acid (ASA) was granulated with AYC, hydroxypropylcellulose (HPC), polyvinylpyrrolidone (PVP), or pullulan (PUL) and compressed into a tablet in the absence of disintegrant . Particle size and angle of repose of the granules, tensile strength, disintegration time, and water absorption behavior of the tablets and ASA release profiles from the tablets were measured . The surface of AYC-granules was observed with a scanning electron microscope . As was the case with the granules of HPC, PVP, or PUL, D50 of the granules of AYC increased with increasing AYC addition percentage, indicating that it is possible to granulate ASA with AYC . Tablets incorporating HPC, PVP, and PUL failed to disintegrate within 30 minutes at all percentages of binder addition because in the case of the HPC, PVP, or PUL tablets in the dissolution medium, water scarcely penetrated into the inner region of the tablet, causing no disintegration . In the case of the AYC tablets, disintegration was not detected at 3% or less of AYC . When AYC was equal to or greater than 5%, AYC tablets disintegrated in approximately 4 minutes and rapid ASA release from the tablets was observed . These results may have been caused by the following . In the case of the AYC 3% granules, ungranulated aspirin powder remained, but in the case of the AYC 5% granules, ASA powder was granulated and covered with AYC . Water absorption was observed initially; however, a plateau was reached in the case of the AYC 3%-tablet . In contrast, in the cases of the AYC 5% and more tablets, water absorption was greater and increased with time . The angle of repose of the AYC 5% granules was 25.7 degrees, which represented high fluidity . The tablets produced by compressing the granules demonstrated sufficient tensile strength greater than 0.8 MPa . The tablets rapidly disintegrated and rapid ASA release was obtained . AYC functioned as a binder at granulation; additionally, AYC served as a disintegrant in the dissolution of drug from the tablets . These results indicate that AYC affords high utility as a unique pharmaceutical additive possessing contrary functions such as binding and disintegration. Yeast, 2003 Nov, 20(15), 1255 - 62 An improved tetO promoter replacement system for regulating the expression of yeast genes; Yen K et al.; Regulatable promoters are commonly used to control the expression of, especially, essential genes in a conditional manner . Integration of such promoters upstream of an ORF using one-step PCR-mediated homologous recombination should be particularly efficient . However, integration of the original KanMX4-tetO promoter cassette (Belli et al., 1998a) into the relatively short upstream regions of many yeast genes is often problematic, presumably due to the size (3.9 kb) of the replacement cassette . We have created a new, shorter, KanMX4-tetO cassette by removing the transactivator (tTA) sequence from the original cassette . The transactivator (tTA) has been integrated into the yeast genome to create a new strain for use with the new system, which has a greatly increased efficiency of promoter substitution . With it, we have been able to create strains that could not be made with the original cassette . To increase the throughput of promoter substitutions, we have developed a new assay for testing doxycycline sensitivity, based on liquid culture using microtitre trays . Altogether, the components of this new 'tool kit' greatly increase the efficiency of systematic promoter substitutions . Cell Mol Life Sci, 2003 Oct, 60(10), 2115 - 30 Yeast as a sensor of factors affecting the accuracy of protein synthesis; Valente L et al.; The cell monitors and maintains the fidelity of translation during the three stages of protein synthesis: initiation, elongation and termination . Errors can arise by multiple mechanisms, such as altered start site selection, reading frame shifts, misincorporation or nonsense codon suppression . All of these events produce incorrect protein products . Translational accuracy is affected by both cis- and trans-acting elements that insure the proper peptide is synthesized by the protein synthetic machinery . Many cellular components are involved in the accuracy of translation, including RNAs (transfer RNAs, messenger RNAs and ribosomal RNAs) and proteins (ribosomal proteins and translation factors) . The yeast Saccharomyces cerevisiae has proven an ideal system to study translational fidelity by integrating genetic approaches with biochemical analysis . This review focuses on the ways studies in yeast have contributed to our understanding of the roles translation factors and the ribosome play in assuring the accuracy of protein synthesis. EMBO Rep, 2003 Dec, 4(12), 1150 - 5 Epub 2003 Nov 14. X-ray structure and activity of the yeast Pop2 protein: a nuclease subunit of the mRNA deadenylase complex; Thore S et al.; In Saccharomyces cerevisiae, a large complex, known as the Ccr4-Not complex, containing two nucleases, is responsible for mRNA deadenylation . One of these nucleases is called Pop2 and has been identified by similarity with PARN, a human poly(A) nuclease . Here, we present the crystal structure of the nuclease domain of Pop2 at 2.3 A resolution . The domain has the fold of the DnaQ family and represents the first structure of an RNase from the DEDD superfamily . Despite the presence of two non-canonical residues in the active site, the domain displays RNase activity on a broad range of RNA substrates . Site-directed mutagenesis of active-site residues demonstrates the intrinsic ability of the Pop2 RNase D domain to digest RNA . This first structure of a nuclease involved in the 3'-5' deadenylation of mRNA in yeast provides information for the understanding of the mechanism by which the Ccr4-Not complex achieves its functions. Annu Rev Genet, 2003, 37, 435 - 60 Yeast vacuole inheritance and dynamics; Weisman LS; The vacuole/lysosome of the budding yeast Saccharomyces cerevisiae is actively divided between mother and daughter cells . Vacuole inheritance initiates early in the cell cycle and ends in G2, just prior to nuclear migration . The process begins with a portion of the vacuole extending into the emerging bud . This tubular-vesicular entity, the segregation structure, enables continued exchange of vacuole contents between mother and daughter vacuoles . Genetic, biochemical, and cytological analyses of vacuole inheritance have provided insight into the molecular basis of membrane movement, the spatial and temporal control of organelle transport, and the molecular basis of membrane fusion and fission. BioDrugs, 2003, 17(6), 413 - 24 Yeast genetic methods for the detection of membrane protein interactions: potential use in drug discovery; Fetchko M et al.; Due to the pivotal role of membrane proteins in many cellular processes, their direct link to human disease and their often extracellular accessibility towards drugs, an understanding of membrane protein function is desirable . However, the hydrophobic nature of membrane proteins often results in insoluble proteins which makes protein isolation difficult and therefore hinders the determination of protein complex composition and protein function . Recently, several yeast genetic techniques have made the characterisation of interactions among membrane proteins more feasible . Techniques such as the guanine-nucleotide binding protein fusion assay, the reverse Ras recruitment system and the split-ubiquitin system have been fruitful in monitoring known protein interactions and uncovering novel interactions . Since many disease states have altered membrane protein function, one can use these systems to recreate interactions involving disease causing membrane proteins . Once established, screens for small molecules, peptides and/or single chain antibodies which disrupt such interactions can provide insight into the biology of the interaction and thus help guide therapeutical research . In this review, we speculate on the feasibility of using inhibitors of protein interactions as drugs and the adaptation of these techniques to select for inhibitors of defined protein interactions. J Vet Sci, 2002 Mar, 3(1), 1 - 5 Additive estrogenic activities of the binary mixtures of four estrogenic chemicals in recombinant yeast expressing human estrogen receptor; Kang KS et al.; To evaluate the estrogenic activities of several chemicals such as 17beta-estradiol (E2), rho-nonylphenol, bisphenol A, butylparaben, and combinations of these chemicals, we used recombinant yeasts containing the human estrogen receptor {Saccharomyces cerevisiae ER + LYS 8127} . We evaluated E2 was most active in the recombinant yeast assay, followed by rho-nonylphenol, bisphenol A, butylparaben . The combinations of some concentrations of 17-estradiol as a strong estrogen and bisphenol A or butylparaben as a weak estrogen showed additive estrogenic effects . Also, the combinations of some concentrations of nonlyphenol and butylparaben and combination of butylparaben and bisphenol A showed additive effects in the estrogenic activity . Therefore, the estrogenic activities of the combinations of two chemicals were additive, not synergistic. Genome Res, 2003 Dec, 13(12), 2691 - 9 Epub 2003 Nov 12. A strategy for constructing large protein interaction maps using the yeast two-hybrid system: regulated expression arrays and two-phase mating; Zhong J et al.; Maps representing the binary interactions among proteins have become valuable tools for understanding how proteins work together to mediate biological processes . One of the most effective methods for detecting biologically important protein interactions has been the yeast two-hybrid system . Here we present an efficient two-hybrid strategy to facilitate construction of protein interaction maps on a genome-wide scale . The strategy begins with two arrays of yeast expressing known proteins fused to either a DNA binding domain (BD), or a transcription activation domain (AD) . The fusion proteins are conditionally expressed using regulated promoters that can be repressed during construction and amplification of the yeast arrays . Interaction assays are conducted in two phases . In the first phase, small pools of AD strains are mated with the array of BD strains . In the second phase, individual BD strains are mated with appropriate subsets of the AD array corresponding to positive pools in the first phase . This strategy has several advantages over previously described approaches, including the ability to detect interactions with proteins that inhibit yeast growth or that activate transcription as BD fusions . Moreover, by minimizing the number of mating operations and sequencing reactions needed to test large sets of binary interactions, this strategy is more efficient than either matrix or library screening approaches . We also present a three-dimensional pooling scheme to further increase the efficiency of large-scale two-hybrid analyses. FEMS Yeast Res, 2003 Nov, 4(2), 149 - 55 The role of nitrate reductase in the regulation of the nitrate assimilation pathway in the yeast Hansenula polymorpha; Navarro FJ et al.; The role of nitrate reductase (NR) in the regulation of the nitrate assimilation pathway was evaluated in the yeast Hansenula polymorpha . Posttranscriptional regulation of NR in response to reduced nitrogen sources and the effect of a heterologous NR on the transcriptional regulation of nitrate-assimilatory gene expression was examined . The strain bearing YNR1 (nitrate reductase gene) under the control of the methanol-induced MOX (methanol oxidase) promoter showed that NR is active in the presence of reduced nitrogen sources . In cells incubated with glutamine plus nitrate, rapamycin abolished nitrogen catabolite repression, NR activity being very similar to that in cells induced by nitrate alone . This reveals the involvement of the Tor-signalling pathway in the transcriptional regulation of H . polymorpha nitrate assimilation genes . To assess the role of NR in nitrate-assimilatory gene expression, different strains lacking YNR1, or both YNR1 and YNT1 (high-affinity nitrate transporter) genes, or expressing the tobacco NR under the YNR1 promoter, were used . Tobacco NR abolished the constitutive nitrate-assimilatory gene induction shown by an NR gene disruptant strain . Moreover, in strains lacking the high-affinity nitrate transporter and NR this deregulation disappeared . These facts discard the role of NR protein in the transcriptional induction of the nitrate-assimilatory genes and point out the involvement of the high-affinity nitrate transporter as a part of the nitrate-signalling pathway. Mol Cell Biol, 2003 Dec, 23(23), 8913 - 23 Yeast Rad52 and Rad51 recombination proteins define a second pathway of DNA damage assessment in response to a single double-strand break; Lee SE et al.; Saccharomyces cells with a single unrepaired double-strand break adapt after checkpoint-mediated G(2)/M arrest . We have found that both Rad51 and Rad52 recombination proteins play key roles in adaptation . Cells lacking Rad51p fail to adapt, but deleting RAD52 suppresses rad51Delta . rad52Delta also suppresses adaptation defects of srs2Delta mutants but not those of yku70Delta or tid1Delta mutants . Neither rad54Delta nor rad55Delta affects adaptation . A Rad51 mutant that fails to interact with Rad52p is adaptation defective; conversely, a C-terminal truncation mutant of Rad52p, impaired in interaction with Rad51p, is also adaptation defective . In contrast, rad51-K191A, a mutation that abolishes recombination and results in a protein that does not bind to single-stranded DNA (ssDNA), supports adaptation, as do Rad51 mutants impaired in interaction with Rad54p or Rad55p . An rfa1-t11 mutation in the ssDNA binding complex RPA partially restores adaptation in rad51Delta mutants and fully restores adaptation in yku70Delta and tid1Delta mutants . Surprisingly, although neither rfa1-t11 nor rad52Delta mutants are adaptation defective, the rad52Delta rfa1-t11 double mutant fails to adapt and exhibits the persistent hyperphosphorylation of the DNA damage checkpoint protein Rad53 after HO induction . We suggest that monitoring of the extent of DNA damage depends on independent binding of RPA and Rad52p to ssDNA, with Rad52p's activity modulated by Rad51p whereas RPA's action depends on Tid1p. Mol Cell Biol, 2003 Dec, 23(23), 8820 - 8 Yeast Mre11 and Rad1 proteins define a Ku-independent mechanism to repair double-strand breaks lacking overlapping end sequences; Ma JL et al.; End joining of double-strand breaks (DSBs) requires Ku proteins and frequently involves base pairing between complementary terminal sequences . To define the role of terminal base pairing in end joining, two oppositely oriented HO endonuclease cleavage sites separated by 2.0 kb were integrated into yeast chromosome III, where constitutive expression of HO endonuclease creates two simultaneous DSBs with no complementary end sequence . Lack of complementary sequence in their 3' single-strand overhangs facilitates efficient repair events distinctly different from when the 3' ends have a 4-bp sequence base paired in various ways to create 2- to 3-bp insertions . Repair of noncomplementary ends results in a set of nonrandom deletions of up to 302 bp, annealed by imperfect microhomology of about 8 to 10 bp at the junctions . This microhomology-mediated end joining (MMEJ) is Ku independent, but strongly dependent on Mre11, Rad50, and Rad1 proteins and partially dependent on Dnl4 protein . The MMEJ also occurs when Rad52 is absent, but the extent of deletions becomes more limited . The increased gamma ray sensitivity of rad1Delta rad52Delta yku70Delta strains compared to rad52Delta yku70Delta strains suggests that MMEJ also contributes to the repair of DSBs induced by ionizing radiation. Mol Cell Biol, 2003 Dec, 23(23), 8809 - 19 The DIVa maturase binding site in the yeast group II intron aI2 is essential for intron homing but not for in vivo splicing; Huang HR et al.; Splicing of the Saccharomyces cerevisiae mitochondrial DNA group II intron aI2 depends on the intron-encoded 62-kDa reverse transcriptase-maturase protein (p62) . In wild-type strains, p62 remains associated with the excised intron lariat RNA in ribonucleoprotein (RNP) particles that are essential for intron homing . Studies of a bacterial group II intron showed that the DIVa substructure of intron domain IV is a high-affinity binding site for its maturase . Here we first present in vitro evidence extending that conclusion to aI2 . Then, experiments with aI2 DIVa mutant strains show that the binding of p62 to DIVa is not essential for aI2 splicing in vivo but is essential for homing . Because aI2 splicing in the DIVa mutant strains remains maturase dependent, splicing must rely on other RNA-protein contacts . The p62 that accumulates in the mutant strains has reverse transcriptase activity, but fractionation experiments at high and low salt concentrations show that it associates more weakly than the wild-type protein with endogenous mitochondrial RNAs, and that phenotype probably explains the homing defect . Replacing the DIVa of aI2 with that of the closely related intron aI1 improves in vivo splicing but not homing, indicating that DIVa contributes to the specificity of the maturase-RNA interaction needed for homing. Hepatobiliary Pancreat Dis Int, 2002 May, 1(2), 238 - 42 Cloning and expression of the preS1 gene of hepatitis B virus in yeast cells; Lu YY et al.; OBJECTIVE: To investigate the complex functions of HBV preS1 protein, we constructed HBV preS1 gene expression vector and expressed it in yeast cells . METHODS: Polymerase chain reaction (PCR) was performed to amplify the gene of HBV preS1 from the plasmid pCP10 containing the whole DNA fragment of HBV ayw subtype as template and the PCR product was cloned into the pGEM-T vector for sequencing . After being identified, the HBV preS1 gene was cut from the pGEM-T vector by EcoR I and Pst I restriction enzymes, and cloned into yeast expressive plasmid pGBKT7 to construct pGBKT7-preS1 recombinant expressive plasmid . This plasmid was transformed into yeast cell AH109 and expressed in it . The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting . RESULTS: The HBV preS1 gene was amplified successfully and identified by DNA sequencing . The PCR products were coincided completely with the reported sequence . The digested fragments were cloned into the pGBKT7 vector and transformed into yeast cell AH109 . The results of SDS-PAGE and Western blotting assay showed: (1) The HBV preS1 protein was expressed and existed in yeast cells; (2) The molecular weight of the expression product was about 30 000 D . CONCLUSION: The HBV preS1 gene was successfully cloned and expressed in yeast cells. FEMS Microbiol Lett, 2003 Nov 7, 228(1), 111 - 3 Use of novel assays to indicate that O-esters and S-esters are produced by the same enzyme in brewing yeast; Bamforth CW et al.; Ethanol- and methanethiol-dependent removal of acetyl-CoA by crude extracts of ale yeast has been monitored using a decrease in OD232 . Activity has also been detected in these extracts after fractionation on polyacrylamide gels, in this case using a novel assay in which the coenzyme A produced in the reaction is linked via DCPIP reduction to color formation from nitroblue tetrazolium . Ethanol- and methanethiol-dependent activities migrate identically on such gels, and only one band of color formation was observed . Furthermore they displayed closely similar sensitivity to heating at 40 degrees C and 60 degrees C and pH optima, with activity maximal at pH 7.5 . It is likely that a single enzyme is responsible for the formation of O-esters and S-esters in yeast . Initial kinetic studies indicate that methanethiol has higher affinity for the enzyme than has ethanol and a higher maximum velocity . However, the enzyme has a much lower Km for acetyl-CoA, suggesting that the alcohol or thiol substrate is the more likely substrate to be limiting. Genome Biol . 2003;4(11):233 . Epub 2003 Oct 15. Multi-level response of the yeast genome to glucose; Gelade R et al.; The yeast Saccharomyces cerevisiae shows a great variety of cellular responses to glucose via several glucose-sensing and signaling pathways . Recent microarray analysis has revealed multiple levels of genomic sensitivity to glucose and highlighted the power of genome-wide analysis to detect cellular responses to minute environmental changes. J Biol Chem, 2004 Feb 6, 279(6), 4498 - 506 Epub 2003 Nov 10. Inhibition of sodium/proton exchange by a Rab-GTPase-activating protein regulates endosomal traffic in yeast; Ali R et al.; Endosomal Na+/H+ exchangers are important for salt and osmotolerance, vacuolar pH regulation, and endosomal trafficking . We show that the C terminus of yeast Nhx1 interacts with Gyp6, a GTPase-activating protein for the Ypt/Rab family of GTPases, and that Gyp6 colocalizes with Nhx1 in the endosomal/prevacuolar compartment (PVC) . The gyp6 null mutant exhibits novel phenotypes consistent with loss of negative regulation of Nhx1, including increased tolerance to hygromycin, increased vacuolar pH, and decreased plasma membrane potential . In contrast, overexpression of Gyp6 increases sensitivity to hygromycin, decreases vacuolar pH, and results in a slight missorting of vacuolar carboxypeptidase Y to the cell surface . We conclude that Gyp6 is a negative regulator of Nhx1-dependent trafficking out of the PVC . Taken together with its GTPase-activating protein-dependent role as a negative regulator of Ypt6-mediated retrograde traffic to the Golgi, we propose that Gyp6 coordinates upstream and downstream events in the PVC to Golgi pathway . Our findings provide a possible molecular link between intraendosomal pH and regulation of vesicular trafficking. Nahrung, 2003 Oct, 47(5), 312 - 9 Structural and farinographic changes during mixing of a yeast sweet dough; Calderon-Dominguez G et al.; Sweet dough requires longer mixing time than salty or white pan bread doughs to reach a developed stage . Although many studies have dealt with the effect of mixing time on dough, few have referred to yeast sweet doughs . The aim of this study was to evaluate if the changes in dough microstructure during different stages of mixing were the same between sweet pan bread and white pan bread, using as control a flour water system . Scanning electronic microscopy (SEM) and some bread characteristics were used as evaluation parameters . Doughs were prepared in a Brabender Farinograph instrument . Different mixing times were used for each formulation, which correspond to common farinographic parameters such as: arrival time, peak time, departure time, etc . Farinographic consistency was evaluated at those times . Results showed that sweet dough farinogram was quite different from those obtained from the other two samples; it starts with a low consistency value (260 BU), and after 8 min of mixing it began to increase until almost reaching the 500 BU line; then the graphic follows the classical curve . Larger products were obtained from arrival time to departure time for both formulations . SEM showed that as mixing proceeds the dough structure opens, changing its appearance from a compact structure at the beginning to a very open one at the end of the mixing process . SEM also showed that the process of mixing is the same for the two samples and control; however, the time needed to reach each stage was different among samples . Farinogram can be used to get information about mixing behavior of yeast sweet doughs . The study of mixing can be easier using sweet dough formulations because it was possible to get more points between the onset of mixing and full dough development, and the process was very similar no matter the formulation. Mol Genet Genomics, 2003 Dec, 270(5), 442 - 7 Epub 2003 Nov 08. Interrelations between the efficiency of translation start sites and other sequence features of yeast mRNAs; Kochetov AV et al.; The translation start site (TSS) plays an important role in the control of the translational efficiency and cytoplasmic stability of eukaryotic mRNAs . The efficiency of TSS recognition is known to be influenced by sequence context, and mRNAs with "weak" TSSs are relatively abundant . We analyzed a sample of 4113 yeast genes in a search for features that might serve to compensate for the inefficient recognition of "weak" TSSs by initiating ribosomes . The first feature found to correlate with variations in TSS strength is differences in the stability of secondary structure upstream and downstream of the start AUG codon . The second feature concerns the characteristics of AUG triplets found at the beginning of the coding sequence, i.e., downstream of the predicted TSS . In particular, the proximal downstream AUG lies in frame with the CDS significantly more often if the TSS itself is located in a "weak" context . The accuracy of TSS annotation, the possibility of polypeptide heterogeneity due to the use of alternative downstream AUGs, and the influence of related features of mRNA sequences are discussed. J Biochem (Tokyo), 2003 Oct, 134(4), 615 - 23 Patch clamp analysis of a H+ pump heterologously expressed in giant yeast vacuoles; Nakanishi Y et al.; Despite the usefulness of the patch-clamp technique, its application to ion pumps and transporters in biomembranes is limited . We developed a novel method for determining the activity of a proton-pumping pyrophosphatase (H(+)-PPase) made of a single protein . We heterologously highly expressed the enzyme in Saccharomyces cerevisiae, prepared giant vacuoles from the cells, and measured a PPi-dependent electrical current of 18 pA (10.5 fA/ micro m(2)) using the patch-clamp technique in the whole-vacuole recording mode . We determined the inhibitor sensitivity and affinity for substrate (K(m), 4.6 micro M) . The enzyme number in a giant vacuole (4.2 x 10(6)) and the molecular activity of the expressed H(+)-PPase (14 s(-1)) were determined . An uncoupling-type H(+)-PPase mutant, of which the 263rd glutamate residue was replaced by aspartate, and of which H(+) pump activity was not detected with the fluorescence quenching method, showed a weak current with a high K(m) . The high accuracy, effectiveness and applicability of the method for exogenously expressed ion transporters were also discussed. Hepatobiliary Pancreat Dis Int, 2003 Feb, 2(1), 81 - 4 Screening of augmenter of liver regeneration-binding proteins by yeast-two hybrid technique; Cheng J et al.; OBJECTIVE: To investigate the biological function of augmenter of liver regeneration (ALR), we used yeast-two hybrid technique to detect proteins in hepatocytes interacting with ALR . METHODS: ALR bait plasmid was constructed by using yeast-two hybrid system 3, then transformed into yeast AH109 . The transformed yeast was mated with yeast Y187 containing liver cDNA library plasmid in a 2XYPDA medium . Diploid yeast was plated on a synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selection and screening . After extracting and sequencing of the plasmid from blue colonies . Analysis was performed by bioinformatics . RESULTS: Of 36 colonies sequenced, 14 are metallothionein, 12 albumin, and 3 selenoprotein P . One colony is a new gene with unknown function . CONCLUSION: The successful cloning of gene of ALR interacting protein has paved the way for studying the physiological function of ALR and associated proteins. Hepatobiliary Pancreat Dis Int, 2002 Feb, 1(1), 87 - 91 Cloning and expression of the gene of augmenter of liver regeneration in yeast cells; Cheng J et al.; OBJECTIVE: To study the function of augmenter of liver regeneration (ALR) as a regulatory factor that specifically stimulates hepatic cell regeneration, we constructed yeast expressive vector of ALR and expressed it in yeast cells . METHODS: Total RNA was extracted from HepG2 cells, and reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the coding region of ALR . The products were cloned into PGEM-T vector and sequenced, then cloned into PGBK T7 vector . The recombinant plasmid PGBK T7-ALR was transformed into yeast AH109 . The yeast protein was extracted and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization technique . RESULTS: DNA sequencing results confirmed that the coding region of ALR was correctly inserted into the yeast expression vector, and Western blotting assay showed that recombinant ALR was successfully expressed in yeast . Its molecular weight was identical to the theoretical value of 15 000 Da; the protein was found inside the yeast cells . CONCLUSION: The successful expression of ALR in yeast cells makes it possible to study further on its biological function. Cell Signal, 2004 Jan, 16(1), 43 - 9 Galpha protein dependent and independent effects of human RGS1 expression in yeast; Li XY et al.; Regulators of G-protein Signalling (RGS) regulate the functional lifetime of G-Protein Coupled Receptor (GPCR)-activated heterotrimeric G-protein by serving as GTPase Accelerating Proteins (GAPs) for the G(alpha) subunit . A number of mammalian RGSs can functionally replace the yeast RGS containing SST2 gene and inhibit GPCR signalling . Using yeast strains harbouring a G(betagamma)-responsive FUS1-LacZ reporter gene, we demonstrate that heterologously expressed mammalian RGS1 also serves to decrease basal signalling in the absence of agonist . Although this effect was dependent on the expression of a GPA1-encoded functional G(alpha) protein, the GPCR itself was nevertheless not required . Using the GAL1 inducible promoter to express RGS1, we further demonstrate that in addition to serving as a GAP for Gpa1p in yeast, RGS1 is a dosage-dependent inhibitor of growth . This effect is specific to RGS1 since growth is not altered in cells expressing either mammalian RGS2 or RGS5 . We further demonstrate that neither of the two yeast G(alpha) proteins is responsible for mediating this growth inhibitory effect of RGS1 . Taken together, our results indicate that RGS1 can function in both G-protein-dependent and -independent manners in yeast. Structure (Camb), 2003 Nov, 11(11), 1403 - 11 Structure of the yeast Hst2 protein deacetylase in ternary complex with 2'-O-acetyl ADP ribose and histone peptide; Zhao K et al.; Sir2 proteins are NAD(+)-dependant protein deactylases that have been implicated in playing roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology . To define the mechanism of Sir2 activity, we report the 1.5 A crystal structure of the yeast Hst2 (yHst2) Sir2 protein in ternary complex with 2'-O-acetyl ADP ribose and an acetylated histone H4 peptide . The structure captures both ligands meeting within an enclosed tunnel between the small and large domains of the catalytic protein core and permits the assignment of a detailed catalytic mechanism for the Sir2 proteins that is consistent with solution and enzymatic studies . Comparison of the ternary complex with the yHst2/NAD(+) complex, also reported here, and nascent yHst2 structure also reveals that NAD(+) binding accompanies intramolecular loop rearrangement for more stable NAD(+) and acetyl-lysine binding, and that acetyl-lysine peptide binding induces a trimer-monomer protein transition involving nonconserved Sir2 residues. Sci Aging Knowledge Environ . 2001 Oct 03;2001(1):pe1. Using yeast to discover the fountain of youth; Kaeberlein M et al.; The budding yeast Saccharomyces cerevisiae has long served as a model organism for the study of basic cellular processes . Its short generation time, well-established molecular genetics, and fully sequenced genome have made this organism a favorite of researchers in diverse fields . Much of the information obtained has been shown to apply to higher eukaryotes, including humans . Recently, researchers have begun using yeast to tackle one of the outstanding questions in science: How and why do organisms age? The identification of individual genes in yeast that can affect the aging process itself has elevated this single-celled fungus to full contender status in the aging field . In this Perspective, we present two fundamentally different measures of aging in yeast: replicative life-span and stationary phase survival (chronological life-span) . We describe the benefits and limitations of each and present models that attempt to explain these "aging" phenomena . Finally, we present compelling evidence that the use of yeast as a model system will ultimately prove beneficial to the study of human aging. Dev Cell, 2003 Nov, 5(5), 723 - 34 Spatial and temporal pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis; Wu JQ et al.; Microscopy of fluorescent fusion proteins and genetic dependencies show that fission yeast assemble and constrict a cytokinetic contractile ring in a precisely timed, sequential order . More than 90 min prior to separation of the spindle pole bodies (SPB), the anillin-like protein (Mid1p) migrates from the nucleus and specifies a broad band of cortex around the equator as the division site . Between 10 min before and 2 min after SPB separation, conventional myosin-II (Myo2p), IQGAP (Rng2p), PCH protein (Cdc15p), and formin (Cdc12p) join the broad band independent of actin filaments . Over the subsequent 10 min prior to anaphase B, this broad band of proteins condenses into a contractile ring including actin, tropomyosin (Cdc8p), and alpha-actinin (Ain1p) . During anaphase B, unconventional myosin-II (Myp2p) joins the ring followed by the septin (Spn1p) . Ring contraction and disassembly begin 37 min after SPB separation . This spatial and temporal hierarchy provides the framework for analysis of molecular mechanisms. Biometrics, 2003 Sep, 59(3), 562 - 9 Models for yeast prions; Morgan BJ et al.; The cytoplasmic heritable determinant {PSI+} of the yeast Saccharomyces cerevisiae exhibits prion-like properties . The properties of yeast prions are studied in the hope that this will enhance the understanding of mammalian prions, which cause mad-cow, Creutzfeldt-Jakob, and related neurodegenerative diseases . When host cells divide, the yeast prions distribute themselves without loss over the daughter cells . Experimental data provide information on how the proportion of cells with prions decreases over time when priori replication is inhibited . One feature of scientific interest is the unknown mean number, n0, of prions assumed to be present in the cells at the start of the experiment . We develop several stochastic models and by fitting them to the data, we obtain substantially larger estimates of n0 compared with a previous analysis . An interesting feature of a model with constant cell generation times is that the predicted proportion of cells with prions varies over time as a sequence of linked hyperbolic curves . Avenues for future research are outlined, which relax simplifying assumptions made in the models . We make several recommendations for the design of future experiments. Hepatobiliary Pancreat Dis Int, 2003 May, 2(2), 247 - 51 Transcriptional activation function of hepatitis B virus Pre S1 protein in yeast; Xiao SX et al.; OBJECTIVE: To explore the feasibility of cloning of the hepatocyte receptor interacting with the Pre S1 protein of HBV by two-hybrid system . METHODS: Yeast expression plasmids encoding fusion proteins of full length or portions of Pre S1 of HBV and DNA binding domain of yeast protein GAL4 were constructed and used to transform yeast reporter strain SFY526 . Reporter gene product beta-galactosidase activity was assayed as a measure of transcriptional activation in yeast . Mammalian expression plasmid encoding fusion proteins of full length Pre S1 and DNA binding domain of GAL4 was constructed and used to cotransfect hepatoma cell line Huh-7 together with CAT reporter plasmid . Cell extracts were assayed for CAT activity by thin-layer chromatography . RESULTS: The fusion proteins of full length Pre S1 protein and GAL4 DNA binding domain presented transcriptional activation function in yeast . The transcription activating sequence was localized to the 21 to 47 amino acids of Pre S1 protein . Fusion proteins of full length Pre S1 and GAL4 DNA binding domain did not show transcriptional activation function in mammalian cells . CONCLUSIONS: The transcription activating sequence of HBV Pre S1 protein in yeast overlaps the hepatocyte receptor binding site . The transcriptional activation function of HBV Pre S1 protein in yeast may prevent researchers from using yeast two-hybrid system to clone HBV receptor interacting with Pre S1 protein . However, the Pre S1 protein does not show transcriptional activation function in mammalian cells . Mammalian two-hybrid system may be a practical method to clone the HBV hepatocyte receptor interacting with Pre S1 protein. Toxicol In Vitro, 2003 Oct-Dec, 17(5-6), 753 - 9 Protective effect of lipoic acid against hydrogen peroxide in yeast cells; Della Croce C et al.; Lipoic acid (LA) is found in all kinds of cells, it is widely used in medicine and as a dietary supplement, and it is involved in different physiological functions . Even if there are many papers regarding therapeutic effects of LA, medical research does not always support its effectiveness and little is known about LA metabolism in eukaryotic cells . In this work the probable protective effect of LA was investigated employing five strains of yeast Saccharomyces cerevisiae through short term assays . In particular LA behaviour in oxidative stress conditions was studied . For this purpose hydrogen peroxide was used as oxidant . In D7 strain, LA showed antimutagenic effects against hydrogen peroxide and decreased significantly cytochrome P450 . To better elucidate the effect of LA the following yeast strains carrying deletions in superoxide dismutase genes (SOD) were employed: EG-103 (wild type), EG-110 strain (without mytochondrial SOD), EG-118 (without cytoplasmatic SOD) and EG-133 (without both enzymes) . LA increased the number of mitotic divisions in EG-103, EG-110 and EG-133 and in growing cells (EG-103, EG-110, EG-118) it increased survival percentage with respect to hydrogen peroxide . The positive action was evident in D7 and in EG strains and it showed that LA can be protective and antimutagenic against oxidants in yeast cells, via its antioxidant activity. Toxicol In Vitro, 2003 Oct-Dec, 17(5-6), 709 - 16 A yeast-based method for the detection of cyto and genotoxicity; Lichtenberg-Frate H et al.; A miniaturized short-term in vivo genotoxicity screening assay based on genetically modified yeast (Saccharomyces cerevisiae) cells was performed to explore the capacity of this eukaryotic organism to detect the presence of genotoxic compounds . An increased general sensitivity of yeast cells to toxic compounds was obtained by using a strain being deleted in the prominent pleiotropic drug resistance mediating efflux transporters PDR5, SNQ2 and YOR1 . In order to detect genotoxic effects, a yeast optimized version of the green fluorescent protein (GFP) was fused to the RAD54 promoter that is activated upon DNA damage . Various model substances including the oxygenated fuel additive methyl tertiary-butyl ether (MTBE) and the direct acting genotoxins methyl-N-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline-1-oxide (4-NQO) were tested . All model substances were in parallel examined for chronic cytotoxicity . The results point out the sufficiency of both the sensitivity of the yeast cells to detect chronic cytotoxicity and the intensity of the fluorescence signal for the assessment of genotoxic effects . Thus, the test enables simultaneous detection of cytotoxic and genotoxic effects . By partial automation and implementation of the test in the microtitre scale this bioassay allows parallel sensitive pre-screening of numerous samples. Genes Dev, 2003 Nov 1, 17(21), 2698 - 708 Negative regulation of calcineurin signaling by Hrr25p, a yeast homolog of casein kinase I; Kafadar KA et al.; Calcineurin is a Ca2+/calmodulin-regulated protein phosphatase required for Saccharomyces cerevisiae to respond to a variety of environmental stresses . Calcineurin promotes cell survival during stress by dephosphorylating and activating the Zn-finger transcription factor Crz1p/Tcn1p . Using a high-throughput assay, we screened 119 yeast kinases for their ability to phosphorylate Crz1p in vitro and identified the casein kinase I homolog Hrr25p . Here we show that Hrr25p negatively regulates Crz1p activity and nuclear localization in vivo . Hrr25p binds to and phosphorylates Crz1p in vitro and in vivo . Overexpression of Hrr25p decreases Crz1p-dependent transcription and antagonizes its Ca2+-induced nuclear accumulation . In the absence of Hrr25p, activation of Crz1p by Ca2+/calcineurin is potentiated . These findings represent the first identification of a negative regulator for Crz1p, and establish a novel physiological role for Hrr25p in antagonizing calcineurin signaling. Gene, 2003 Nov 27, 320, 137 - 44 Human wild-type SEDL protein functionally complements yeast Trs20p but some naturally occurring SEDL mutants do not; Gecz J et al.; X-linked spondyloepiphyseal dysplasia tarda (SEDT, or SEDL) is a primary skeletal dysplasia affecting mostly spinal vertebral bodies and epiphyses . Previously, we have identified the SEDL gene and determined the spectrum of 21 different SEDL causing mutations . The SEDL gene is a highly conserved gene with an as yet unknown function . The yeast SEDL protein ortholog, Trs20p, has been isolated as a member of a large multi-protein complex ( approximately 10 proteins) called transport protein particle (TRAPP), which is involved in endoplasmic reticulum (ER)-to-Golgi transport . While the SEDL gene mutations cause a tissue-specific (epiphyses) and relatively mild phenotype, the Trs20p function is essential for the yeast cell . We now provide evidence that recombinant human SEDL protein is able to functionally complement the Saccharomyces cerevisiae TRS20 (TRAPP subunit 20 gene) knockout mutant . This finding strongly supports the speculated conserved nature of the SEDL/Trs20p function . To shed further light on the SEDL/Trs20p protein function, five different naturally occurring SEDL gene mutations have been tested in complementation studies . While two truncation mutations (157delAT and C271T) and one missense mutation (G139T) were unable to rescue the trs20Delta lethal phenotype, two other missense mutations (C218T and T389A) did complement trs20Delta . Interestingly, there is no obvious correlation between the nature and position of the SEDL mutation and the clinical severity of the disorder among the human SEDL patients . Although the identification of complementing SEDL gene mutations may suggest the existence of subtle phenotypic differences among SEDL patients, it might also point towards the identification of SEDL protein residues/domains specific for normal, vertebrate bone growth. Biochemistry, 2003 Nov 11, 42(44), 12813 - 20 Novel destabilization of nucleotide binding by the gamma phosphate of ATP in the yeast SR protein kinase Sky1p; Aubol BE et al.; SR protein kinases (SRPKs) regulate the temporal and cell-specific selection of alternative splice sites . These enzymes are highly unique members of the protein kinase family . SRPKs contain a large domain insert (approximately 200 residues) within the kinase core, do not require phosphorylation for regulation, have an extended helix insert near the nucleotide pocket, and possess unusual substrate specificity determinants . The yeast SRPK, Sky1p, rapidly phosphorylates its natural substrate Npl3 but binds ATP with a high K(m), suggesting that some of these distinctive structural features may be correlated with nucleotide binding {Aubol et al . (2002) Biochemistry 41, 10002-10009} . To address this issue, the nucleotide binding properties of Sky1p were studied using fluorescence spectroscopy . The affinities of several nucleotides (ATP, ADP, AMP, adenosine, and AMPPNP) to Sky1p and the prototype kinase, cAMP-dependent protein kinase, were compared in the absence and presence of the metal activator, Mg(2+), using a fluorescence-based displacement assay . The data indicate that Sky1p, unlike cAMP-dependent protein kinase, potently destabilizes the gamma phosphate of ATP . This novel finding suggests that rapid phosphoryl transfer may be facilitated by unique mechanisms in both protein kinases. Mol Biol Cell, 2004 Jan, 15(1), 11 - 23 Epub 2003 Oct 31. Mek1 kinase activity functions downstream of RED1 in the regulation of meiotic double strand break repair in budding yeast; Wan L et al.; Three meiosis-specific chromosomal components in budding yeast, Mek1, Red1, and Hop1, are required for recombination, proper segregation of homologs, and the meiotic recombination checkpoint . Mek1 is a protein kinase . Mutations that increase the size of the ATP binding pocket of Mek1 (mek1-as1) sensitize the kinase to specific small molecule inhibitors . Experiments using mek1-as1 demonstrate that the requirement for Mek1 kinase activity coincides with the formation of double strand breaks (DSBs) and that this activity is necessary after DSB formation to prevent repair by DMC1-independent pathways . Contrary to previous reports, Red1 is not a substrate for Mek1 . Instead, RED1 is required for wild-type levels of Mek1 kinase activity . In addition, activation of Mek1 requires HOP1, the formation of Red1/Hop1 complexes and a functional Mek1 FHA domain . The requirement for RED1 to produce active kinase can be bypassed by a mek1 mutation that creates a constitutively active Mek1 kinase . We propose that Red1 is phosphorylated by a kinase other than MEK1 and that phosphothreonines on Red1 then interact with the Mek1 FHA domain to recruit the kinase to sites of DSBs where Mek1 is activated to prevent DMC1-independent DSB repair. Mol Biol Cell, 2004 Feb, 15(2), 946 - 56 Epub 2003 Oct 31. Tor pathway regulates Rrn3p-dependent recruitment of yeast RNA polymerase I to the promoter but does not participate in alteration of the number of active genes; Claypool JA et al.; Yeast cells entering into stationary phase decrease rRNA synthesis rate by decreasing both the number of active genes and the transcription rate of individual active genes . Using chromatin immunoprecipitation assays, we found that the association of RNA polymerase I with the promoter and the coding region of rDNA is decreased in stationary phase, but association of transcription factor UAF with the promoter is unchanged . Similar changes were also observed when growing cells were treated with rapamycin, which is known to inhibit the Tor signaling system . Rapamycin treatment also caused a decrease in the amount of Rrn3p-polymerase I complex, similar to stationary phase . Because recruitment of Pol I to the rDNA promoter is Rrn3p-dependent as shown in this work, these data suggest that the decrease in the transcription rate of individual active genes in stationary phase is achieved by the Tor signaling system acting at the Rrn3p-dependent polymerase recruitment step . Miller chromatin spreads of cells treated with rapamycin and cells in post-log phase confirm this conclusion and demonstrate that the Tor system does not participate in alteration of the number of active genes observed for cells entering into stationary phase. J Biol Chem, 2004 Feb 6, 279(6), 4322 - 9 Epub 2003 Nov 21. Genome-wide analysis of iron-dependent growth reveals a novel yeast gene required for vacuolar acidification; Davis-Kaplan SR et al.; We conducted a genome-wide screen in the budding yeast Saccharomyces cerevisiae of 4,792 homozygous diploid deletions to identify genes that function in iron metabolism . Strains unable to grow on iron-restricted medium contained deletions of genes that encode the structural components of the high affinity iron transport system (FET3, FTR1), the iron-sensing transcription factor AFT1 or genes required for the assembly of the transport system . We also identified genes that were not previously known to play a role in iron metabolism . Deletion of the gene CWH36 resulted in a severe growth defect on iron-limited medium, as well as increased sensitivity to Congo red and calcofluor white . Iron transport studies demonstrated that Deltacwh36 cells have an inability to copper load apoFet3p . Furthermore, Deltacwh36 cells demonstrated additional phenotypes including distorted vacuole morphology and altered kinetics of FM4-64 trafficking . We show that Deltacwh36 cells have a defect in vacuolar acidification through the use of the pH-sensitive dye LysoSensor Green DND-189 . In Deltacwh36 cells, the vacuolar H+-ATPase is not assembled and there are reduced levels of at least one subunit of the V0 complex . The open reading frame responsible for the Deltacwh36 phenotypes is YCL005W-A . This gene contains two introns, has homologues in other Saccharomyces strains, and shows weak homology to a component of the vacuolar H+-ATPase found in organisms as diverse as insect and cow. Appl Microbiol Biotechnol, 2004 May, 64(4), 537 - 42 Epub 2003 Oct 31. A screening system for antioxidants using thioredoxin-deficient yeast: discovery of thermostable antioxidant activity from Agaricus blazei Murill; Izawa S et al.; Previously, we found that cytosolic thioredoxin is a negative regulator for an oxidative stress responsive transcription factor, Yap1p (yeast AP-1-like transcription factor), i.e., this transcription factor is constitutively concentrated in the nucleus in the thioredoxin-deficient mutant ( trx1delta trx2delta) due to an impairment of the reactive oxygen species-scavenging activity of this mutant {Izawa et al . (1999) J Biol Chem 274:28459-28465} . Based on these findings, we developed a screening method to discover substances that show antioxidant activity . With this method, antioxidant activity was evaluated by monitoring the subcellular localization of Yap1p . Since Yap1p is oxidized and accumulates in the nucleus in trx1delta trx2Delta cells, it is easy to identify antioxidant activity by observing the localization of green fluorescent protein (GFP)-tagged Yap1p . If exogenous substances taken in by trx1delta trx2Delta cells were able to function as antioxidants to reduce the oxidized form of Yap1p, GFP1-Yap1p would diffuse into the cytoplasm . We used this system to screen for antioxidant activity in mushrooms, and found that the edible mushroom Agaricus blazei Murill is an excellent source of antioxidants . Mol Biol Cell, 2003 Nov, 14(11), 4676 - 84 Receptor internalization in yeast requires the Tor2-Rho1 signaling pathway; deHart AK et al.; Efficient internalization of proteins from the cell surface is essential for regulating cell growth and differentiation . In a screen for yeast mutants defective in ligand-stimulated internalization of the alpha-factor receptor, we identified a mutant allele of TOR2, tor2G2128R . Tor proteins are known to function in translation initiation and nutrient sensing and are required for cell cycle progression through G1 . Yeast Tor2 has an additional role in regulating the integrity of the cell wall by activating the Rho1 guanine nucleotide exchange factor Rom2 . The endocytic defect in tor2G2128R cells is due to disruption of this Tor2 unique function . Other proteins important for cell integrity, Rom2 and the cell integrity sensor Wsc1, are also required for efficient endocytosis . A rho1 mutant specifically defective in activation of the glucan synthase Fks1/2 does not internalize alpha-factor efficiently, and fks1Delta cells exhibit a similar phenotype . Removal of the cell wall does not inhibit internalization, suggesting that the function of Rho1 and Fks1 in endocytosis is not through cell wall synthesis or structural integrity . These findings reveal a novel function for the Tor2-Rho1 pathway in controlling endocytosis in yeast, a function that is mediated in part through the plasma membrane protein Fks1. Biochem Biophys Res Commun, 2003 Nov 14, 311(2), 525 - 32 Small angle X-ray scattering study of the yeast prion Ure2p; Zhu L et al.; The GdmCl-induced equilibrium unfolding and dissociation of the dimeric yeast prion protein Ure2, and its prion domain deletion mutants Delta 15-42Ure2 and 90Ure2, was studied by small angle X-ray scattering (SAXS) using synchrotron radiation and by chemical cross-linking with dithiobis(succinimidyl propionate) (DTSP) . The native state is globular and predominantly dimeric prior to the onset of unfolding . R(g) values of 32 and 45A were obtained for the native and 5M GdmCl denatured states of Delta 15-42Ure2, respectively; the corresponding values for 90Ure2 were 2-3A lower . SAXS suggests residual structure in the 4M GdmCl denatured state and chemical cross-linking detects persistence of dimeric structure under these conditions . Hexamers consisting of globular subunits could be detected by SAXS at high protein concentration under partially denaturing conditions . The increased tendency of partially folded states to form small oligomers points to a mechanism for prion formation. Yeast, 2003 Oct 30, 20(14), 1189 - 211 Screening for new yeast mutants affected in mannosylphosphorylation of cell wall mannoproteins; Conde R et al.; We have carried out a screen of 622 deletion strains generated during the EUROFAN B0 project to identify non-essential genes related to the mannosylphosphate content of the cell wall . By examining the affinity of the deletants for the cationic dye alcian blue and the ion exchanger QAE-Sephadex, we have selected 50 strains . On the basis on their reactivity (blue colour intensity) in the alcian blue assay, mutants with a lower phosphate content than wild-type cells were then arranged in groups defined by previously characterized mutants, as follows: group I (mnn6), group II (between mnn6 and mnn9) and group III (mnn9) . Similarly, strains that behaved like mnn1 (i.e . a blue colour deeper than wild-type) were included in group VI . To confirm the association between the phenotype and a specific mutation, strains were complemented with clones or subjected to tetrad analysis . Selected strains were further tested for extracellular invertase and exoglucanase . Within groups I, II and III, we found some genes known to be involved in oligosaccharide biosynthesis (ALG9, ALG12, HOC1), secretion (BRE5, COD4/COG5, VPS53), transcription (YOL072w/THP1, ELP2, STB1, SNF11), cell polarity (SEP7, RDG1), mitochondrial function (YFH1), cell metabolism, as well as orphan genes . Within group VI, we found genes involved in environmentally regulated transduction pathways (PAL2 and RIM20) as well as others with miscellaneous or unknown functions . We conclude that mannosylphosphorylation is severely impaired in some deletants deficient in specific glycosylation/secretion processes, but many other different pathways may also modulate the amount of mannosylphosphate in the cell wall . Mol Cell Biol, 2003 Nov, 23(22), 8395 - 403 Replication checkpoint protein Mrc1 is regulated by Rad3 and Tel1 in fission yeast; Zhao H et al.; Fission yeast Mrc1 (mediator of replication checkpoint 1) is an adaptor checkpoint protein required for Rad3-dependent activation of the checkpoint kinase Cds1 in response to arrest of replication forks . Here we report studies on the regulation of Mrc1 by phosphorylation . Replication arrest induced by hydroxyurea (HU) induces Mrc1 phosphorylation that is detected by a change in Mrc1 electrophoretic mobility . Phosphorylation is maintained in cds1Delta, rad3Delta, and tel1Delta single mutants but eliminated in a rad3Delta tel1Delta double mutant . Mrc1 has two clusters of S/TQ motifs that are potential Rad3/Tel1 phosphorylation sites . Mutation of six S/TQ motifs in these two clusters strongly impairs Mrc1 phosphorylation . Two motifs located at S604 and T645 are vital for HU resistance . The T645A mutation strongly impairs a Cds1-Mrc1 yeast two-hybrid interaction that is dependent on a functional forkhead-associated (FHA) domain in Cds1, indicating that phosphorylation of T645 mediates Mrc1's association with Cds1 . Consistent with this model, the T645 region of Mrc1 effectively substitutes for the T11 region of Cds1 that is thought to be phosphorylated by Rad3 and to mediate FHA-dependent oligomerization of Cds1 . The S/TQ cluster that includes S604 is needed for Mrc1's increased association with chromatin in replication-arrested cells . These data indicate that Rad3 and Tel1 regulate Mrc1 through differential phosphorylation to control Cds1. Mol Cell Biol, 2003 Nov, 23(22), 8316 - 22 The mechanism of nucleotide incorporation by human DNA polymerase eta differs from that of the yeast enzyme; Washington MT et al.; DNA polymerase eta (Poleta) catalyzes the efficient and accurate synthesis of DNA opposite cyclobutane pyrimidine dimers, and inactivation of Poleta in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum . Pre-steady-state kinetic studies of yeast Poleta have indicated that the low level of fidelity of this enzyme results from a poorly discriminating induced-fit mechanism . Here we examine the mechanistic basis of the low level of fidelity of human Poleta . Because the human and yeast enzymes behave similarly under steady-state conditions, we expected these enzymes to utilize similar mechanisms of nucleotide incorporation . Surprisingly, however, we find that human Poleta differs from the yeast enzyme in several important respects . The human enzyme has a 50-fold-faster rate of nucleotide incorporation than the yeast enzyme but binds the nucleotide with an approximately 50-fold-lower level of affinity . This lower level of binding affinity might provide a means of regulation whereby the human enzyme remains relatively inactive except when the cellular deoxynucleoside triphosphate concentrations are high, as may occur during DNA damage, thereby avoiding the mutagenic consequences arising from the inadvertent action of this enzyme during normal DNA replication. J Biol Chem, 2004 Jan 9, 279(2), 1191 - 6 Epub 2003 Oct 29. Studies on substrate recognition by the budding yeast separase; Sullivan M et al.; Sister chromatid cohesion is resolved at anaphase onset when separase, a site-specific protease, cleaves the Scc1 subunit of the chromosomal cohesin complex that is responsible for holding sister chromatids together . This mechanism to initiate anaphase is conserved in eukaryotes from budding yeast to man . Budding yeast separase recognizes and cleaves two conserved peptide motifs within Scc1 . In addition, separase cleaves a similar motif in the kinetochore and spindle protein Slk19 . Separase may cleave further substrate proteins to orchestrate multiple cellular events that take place during anaphase . To investigate substrate recognition by budding yeast separase we analyzed the sequence requirements at one of the Scc1 cleavage site motifs by systematic mutagenesis . We derived a cleavage site consensus motif (not(FKRWY))(ACFHILMPVWY)(DE)X(AGSV)R/X . This motif is found in 1,139 of 5,889 predicted yeast proteins . We analyzed 28 candidate proteins containing this motif as well as 35 proteins that contain a core (DE)XXR motif . We could so far not confirm new separase substrates, but we have uncovered other forms of mitotic regulation of some of the proteins . We studied whether determinants other than the cleavage site motif mediate separase-substrate interaction . When the separase active site was occupied with a peptide inhibitor covering the cleavage site motif, separase still efficiently interacted with its substrate Scc1 . This suggests that separase recognizes both a cleavage site consensus sequence as well as features outside the cleavage site. J Cell Biol, 2003 Oct 27, 163(2), 215 - 22 Architecture of the budding yeast kinetochore reveals a conserved molecular core; Westermann S et al.; How kinetochore proteins are organized to connect chromosomes to spindle microtubules, and whether any structural and organizational themes are common to kinetochores from distantly related organisms, are key unanswered questions . Here, we used affinity chromatography and mass spectrometry to generate a map of kinetochore protein interactions . The budding yeast CENP-C homologue Mif2p specifically copurified with histones H2A, H2B, and H4, and with the histone H3-like CENP-A homologue Cse4p, strongly suggesting that Cse4p replaces histone H3 in a specialized centromeric nucleosome . A novel four-protein Mtw1 complex, the Nnf1p subunit of which has homology to the vertebrate kinetochore protein CENP-H, also copurified with Mif2p and a variety of central kinetochore proteins . We show that Mif2 is a critical in vivo target of the Aurora kinase Ipl1p . Chromatin immunoprecipitation studies demonstrated the biological relevance of these associations . We propose that a molecular core consisting of CENP-A, -C, -H, and Ndc80/HEC has been conserved from yeast to humans to link centromeres to spindle microtubules. Biophys J, 2003 Nov, 85(5), 3255 - 61 Predissociated dimers and molten globule monomers in the equilibrium unfolding of yeast glutathione reductase; Louzada PR et al.; The equilibrium unfolding of dimeric yeast glutathione reductase (GR) by guanidine hydrochloride (GdnHCl) was investigated . Unfolding was monitored by a variety of techniques, including intrinsic fluorescence emission, anisotropy and iodide quenching measurements, far-ultraviolet circular dichroism and thiol reactivity measurements . At 1 M GdnHCl, one thiol group of GR became accessible to modification with 5,5'-dithiobis-(2-nitrobenzoic) acid (DTNB), whereas no changes could be detected in the spectroscopic properties (fluorescence, circular dichroism) of the protein . Between 2 and 3 M GdnHCl, two partially folded intermediate states possessing flexible tertiary structures (revealed by fluorescence data) but compact secondary structures (as indicated by circular dichroism measurements) were identified . The quaternary structure of GR in the presence of GdnHCl was also investigated by size-exclusion liquid chromatography . These results indicated the presence of an expanded predissociated dimer at 2.5 M GdnHCl and partially folded monomers at 3 M GdnHCl . Taken together, these results suggest the existence of two molten-globule-like intermediate species (one dimeric and one monomeric) in the unfolding of GR . The results are discussed in terms of the mechanism of GR folding and dimerization. Cryobiology, 2003 Oct, 47(2), 155 - 64 Screening of genes that respond to cryopreservation stress using yeast DNA microarray; Odani M et al.; We studied the response of yeast cells after cryopreservation treatment using DNA microarray technology . G