Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 452 - 5 {Studies on synthesis of glutathione by E . coli BL21 (pTrc-gsh) coupled with Saccharomyces cerevisiae}; Shen LX et al.; During synthesis of GSH by the engineered strain E . coli BL21(pTrc-gsh) coupled with Saccharomyces cerevisiae producing ATP from adenosin, the inconsistency of two systems in the concentration of phosphate buffer was solved by decreasing concentration to 250 mmol/L . The conditions under 250 mmol/L phosphate buffer were optimized and the yield of GSH was 1.6 g/L, which was higher than that of summation by two systems under the same conditions respectively . Addition of glycine later after glutamate and cysteine weakened the inhibition of GSH to GSHI . It made the yield of GSH reach to 2.13 g/L which was 30.7% higher than the control. Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 410 - 3 {Expression and characterization of human pulmonary surfactant-associated protein A1 in Saccharomyces cerevisiae}; Lan HK et al.; The cDNA encoding pulmonary surfactant-associated protein A1 (SP-A1) derived from healthy adult's lung was cloned into the pVT102U/alpha, expression vector of Saccharomyces cerevisiae, which contains the yeast alpha-factor signal sequence, leading to the secretion of expressed protein, and then transformed into Saccharomyces cerevisiae S-78 (leu2, ura3, rep4) by electroporation . After 2-3 days culture in adequate pH, the expressed SP-A1 accumulated up to 400 mg/L in supernatant . The pure proteins were obtained by Sephadex G-25, G-75, Sepharose 4B . The expressed recombinant products, 62 kD and 32 kD, reacted to specific antibody using ELISA and Western blot . The SP-A1 protein expressed in Saccharomyces cerevisiae was efficient in enhancing the phagocytosis of E . coli J5 by alveolar macrophages. Science, 2001 Dec 21, 294(5551), 2552 - 6 Epub 2001 Nov 08. A DNA microarray-based genetic screen for nonhomologous end-joining mutants in Saccharomyces cerevisiae; Ooi SL et al.; We describe a microarray-based screen performed by imposing different genetic selections on thousands of yeast mutants in parallel, representing most genes in the yeast genome . The presence or absence of mutants was detected by oligonucleotide arrays that hybridize to 20-nucleotide "barcodes." We used this method to screen for components of the nonhomologous end-joining (NHEJ) pathway . Known components of the pathway were identified, as well as a gene not previously known to be involved in NHEJ, NEJ1 . Nej1 protein interacts with the amino terminus of LIF1/XRCC4, a recently recognized "guardian of the genome" against cancer. Anal Biochem, 2001 Nov 15, 298(2), 283 - 92 High-performance liquid chromatography analysis of molecular species of sphingolipid-related long chain bases and long chain base phosphates in Saccharomyces cerevisiae after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; Lester RL et al.; The molecular species of dihydrosphingosines and phytosphingosines and their 1-phosphates with carbon chain lengths from 16 to 20 have been tagged with the fluorescent amino group reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate . All these derivatives could be resolved by reversed phase HPLC on a C18 column . A convenient one-pot method is described whereby lipid extracts from strains of Saccharomyces cerevisiae containing carbon chain length homologs of sphingolipid long chain bases and their phosphorylated derivatives were directly reacted with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, ester lipids were deacylated, and the reaction mixtures were subjected to liquid chromatography . Five molecular species of both sphingolipid long chain bases and their phosphorylated derivatives are for the first time separated and analyzed . The procedure is quite sensitive, requiring only approximately 10(8) wild-type cells . BMC Mol Biol . 2001;2(1):11 . Epub 2001 Oct 08. Poly purine.pyrimidine sequences upstream of the beta-galactosidase gene affect gene expression in Saccharomyces cerevisiae; Maiti AK et al.; BACKGROUND: Poly purine.pyrimidine sequences have the potential to adopt intramolecular triplex structures and are overrepresented upstream of genes in eukaryotes . These sequences may regulate gene expression by modulating the interaction of transcription factors with DNA sequences upstream of genes . RESULTS: A poly purine.pyrimidine sequence with the potential to adopt an intramolecular triplex DNA structure was designed . The sequence was inserted within a nucleosome positioned upstream of the beta-galactosidase gene in yeast, Saccharomyces cerevisiae, between the cycl promoter and gal 10 Upstream Activating Sequences (UASg) . Upon derepression with galactose, beta-galactosidase gene expression is reduced 12-fold in cells carrying single copy poly purine.pyrimidine sequences . This reduction in expression is correlated with reduced transcription . Furthermore, we show that plasmids carrying a poly purine.pyrimidine sequence are not specifically lost from yeast cells . CONCLUSION: We propose that a poly purine.pyrimidine sequence upstream of a gene affects transcription . Plasmids carrying this sequence are not specifically lost from cells and thus no additional effort is needed for the replication of these sequences in eukaryotic cells. Biochemistry, 2001 Nov 13, 40(45), 13699 - 709 Identification of the gene and characterization of the activity of the trans-aconitate methyltransferase from Saccharomyces cerevisiae; Cai H et al.; We have identified the yeast open reading frame YER175c as the gene encoding the trans-aconitate methyltransferase of Saccharomyces cerevisiae . Extracts of a yeast strain with a disrupted YER175c gene demonstrate a complete loss of activity toward the methyl-accepting substrates trans-aconitate, cis-aconitate, DL-isocitrate, and citrate . Reintroduction of the YER175c gene on a plasmid results in an overexpression of the activity toward each of these methyl-accepting substrates . We now designate this gene TMT1 for trans-aconitate methyltransferase . We examined the methyl-accepting substrate specificity of this enzyme in extracts from overproducing cells . We found that trans-aconitate was the best substrate with a Km of 0.66 mM . Other substrates were recognized much more poorly, including cis-aconitate with a Km of 74 mM and the decarboxylation product itaconate with a Km of 44 mM . The ratio of the maximal velocity to the Km of these substrates was only 0.24% and 0.9% that of trans-aconitate; for other substrates including citrate and other tricarboxylate and dicarboxylate derivatives, this ratio ranged from 0.0003% to 0.062% that of trans-aconitate . We then asked if any of these compounds were present endogenously in yeast extracts . We were able to identify trans-aconitate 5-methyl ester as well as additional unidentified radiolabeled products when S-adenosyl-L-{methyl-3H}methionine was mixed with TMT1+ extracts (but not with tmt1- extracts), suggesting that there may be additional substrates for this enzyme . We showed that the product 5-methyl ester of trans-aconitate is not readily metabolized in yeast extracts . Finally, we demonstrated that the activity of the yeast trans-aconitate methyltransferase is localized in the cytosol and increases markedly as cells undergo the metabolic transition at the diauxic shift. Nat Genet, 2001 Dec, 29(4), 482 - 6 Correlation between transcriptome and interactome mapping data from Saccharomyces cerevisiae; Ge H et al.; Genomic and proteomic approaches can provide hypotheses concerning function for the large number of genes predicted from genome sequences . Because of the artificial nature of the assays, however, the information from these high-throughput approaches should be considered with caution . Although it is possible that more meaningful hypotheses could be formulated by integrating the data from various functional genomic and proteomic projects, it has yet to be seen to what extent the data can be correlated and how such integration can be achieved . We developed a 'transcriptome-interactome correlation mapping' strategy to compare the interactions between proteins encoded by genes that belong to common expression-profiling clusters with those between proteins encoded by genes that belong to different clusters . Using this strategy with currently available data sets for Saccharomyces cerevisiae, we provide the first global evidence that genes with similar expression profiles are more likely to encode interacting proteins . We show how this correlation between transcriptome and interactome data can be used to improve the quality of hypotheses based on the information from both approaches . The strategy described here may help to integrate other functional genomic and proteomic data, both in yeast and in higher organisms. Mol Biol Cell, 2001 Nov, 12(11), 3428 - 38 The target of rapamycin signaling pathway regulates mRNA turnover in the yeast Saccharomyces cerevisiae; Albig AR et al.; The target of rapamycin (TOR) signaling pathway is an important mechanism by which cell growth is regulated by nutrient availability in eukaryotes . We provide evidence that the TOR signaling pathway controls mRNA turnover in Saccharomyces cerevisiae . During nutrient limitation (diauxic shift) or after treatment with rapamycin (a specific inhibitor of TOR), multiple mRNAs were destabilized, whereas the decay of other mRNAs was unaffected . Our findings suggest that the regulation of mRNA decay by the TOR pathway may play a significant role in controlling gene expression in response to nutrient depletion . The inhibition of the TOR pathway accelerated the major mRNA decay mechanism in yeast, the deadenylation-dependent decapping pathway . Of the destabilized mRNAs, two different responses to rapamycin were observed . Some mRNAs were destabilized rapidly, while others were affected only after prolonged exposure . Our data suggest that the mRNAs that respond rapidly are destabilized because they have short poly(A) tails prematurely either as a result of rapid deadenylation or reduced polyadenylation . In contrast, the mRNAs that respond slowly are destabilized by rapid decapping . In summary, the control of mRNA turnover by the TOR pathway is complex in that it specifically regulates the decay of some mRNAs and not others and that it appears to control decay by multiple mechanisms. Mol Biol Cell, 2001 Nov, 12(11), 3402 - 16 Cell cycle-dependent degradation of the Saccharomyces cerevisiae spindle motor Cin8p requires APC(Cdh1) and a bipartite destruction sequence; Hildebrandt ER et al.; Saccharomyces cerevisiae Cin8p belongs to the BimC family of kinesin-related motor proteins that are essential for spindle assembly . Cin8p levels were found to oscillate in the cell cycle due in part to a high rate of degradation imposed from the end of mitosis through the G1 phase . Cin8p degradation required the anaphase-promoting complex ubiquitin ligase and its late mitosis regulator Cdh1p but not the early mitosis regulator Cdc20p . Cin8p lacks a functional destruction box sequence that is found in the majority of anaphase-promoting complex substrates . We carried out an extensive mutagenesis study to define the cis-acting sequence required for Cin8p degradation in vivo . The C terminus of Cin8p contains two elements required for its degradation: 1) a bipartite destruction sequence composed of a KEN-box plus essential residues within the downstream 22 amino acids and 2) a nuclear localization signal . The bipartite destruction sequence appears in other BimC kinesins as well . Expression of nondegradable Cin8p showed very mild phenotypic effects, with an increase in the fraction of mitotic cells with broken spindles. Pharmacogenetics, 2001 Nov, 11(8), 709 - 18 Functional evaluation of cytochrome P450 2D6 with Gly42Arg substitution expressed in Saccharomyces cerevisiae; Tsuzuki D et al.; A single amino acid-substituted mutant protein, CYP2D6 (G42R) was expressed in Saccharomyces cerevisiae and its enzymatic properties were compared with those of other single (P34S, R296C and S486T) and double amino acid-substituted mutant proteins (P34S/S486T and R296C/S486T) expressed in yeast cells, all of which were known to occur in the CYP2D6 gene as single nucleotide polymorphisms . The protein levels of G42R, P34S and P34S/S486T in microsomal fractions and their oxidation capacities towards debrisoquine as a prototypic substrate and bunitrolol as a chiral substrate were different from those of wild-type CYP2D6, while the R296C, S486T and R296C/S486T behaved similarly to the wild-type in these indices . The CYP contents both in yeast microsomal and in whole cell fractions indicated that some part of G42R protein was localized in the endoplasmic reticulum membrane fraction, whereas most of G42R protein was in some subcellular fractions other than endoplasmic reticulum . In kinetic analysis, the G42R substitution increased apparent Km and decreased Vmax for debrisoquine 4-hydroxylation, while it increased both Km and Vmax for bunitrolol 4-hydroxylation . The P34S substitution did not drastically change Km but decreased Vmax for debrisoquine 4-hydroxylation, whereas Km was increased and Vmax unchanged or decreased for bunitrolol 4-hydroxylation by P34S substitution . These results suggest that the G42R substitution causes a change in the CYP2D6 conformation, which may be different from the change produced by the P34S substitution. Curr Genet, 2001 Sep, 40(2), 91 - 109 Sensors of extracellular nutrients in Saccharomyces cerevisiae; Forsberg H et al.; It has been known for a long time that yeast are capable of making rapid metabolic adjustments in response to changing extracellular nutrient conditions . Until recently it was thought that yeast, in contrast to mammalian cells, primarily monitored nutrient availability through the activity of intracellular sensors . Recent advances in our understanding of nutrient sensing indicate that yeast cells possess several nutrient-sensing systems localized in the plasma membrane that transduce information regarding the presence of extracellular amino acids, ammonium . and glucose . Strikingly, the transmembrane components of several of these sensors, Ssylp, Mep2p, Snf3p . and Rgt2p, are unique members of nutrient-transport protein families . Perhaps with the exception of Mep2p, the ability of these transporter homologues to transduce nutrient-(ligand)-induced signals across the plasma membrane appears to be independent of nutrient uptake; and thus these sensor components may function analogously to traditional ligand-dependent receptors . Additionally, the G protein-coupled receptor Gpr1p has been shown to exhibit properties consistent with it being a sensor . These recent advances indicate that yeast cells obtain information regarding their growth environments using sensing systems that are more similar to those present in mammalian cells than previously thought . The fact that yeast plasma membrane nutrient sensors have only recently been discovered reveals how little is understood regarding the molecular signals that enable eukaryotic cells to adapt to changing environments. Mol Aspects Med, 2001 Aug-Oct, 22(4-5), 217 - 46 Oxidative stress and signal transduction in Saccharomyces cerevisiae: insights into ageing, apoptosis and diseases; Costa V et al.; In yeast, as in higher eukaryotes, reactive oxygen species are produced as normal by-products of cellular metabolism . Under physiological conditions, the cell defence mechanisms are able to avoid molecular damages . This balance is disturbed when yeast cells are exposed to diverse environmental stress conditions, such as the presence of oxidants, heat shock, ethanol and metal ions . The increased production of reactive oxygen species is sensed by the cell, leading to the induction of defence mechanisms - the oxidative stress response . The present review discusses the mechanisms by which reactive oxygen species are sensed and the signalling pathways that are coupled with changes in genomic expression programs . Yeast has been used as an eukaryotic cell system to characterise the molecular mechanisms underlying the oxidative stress response . Furthermore, yeast has been utilised to elucidate the role of oxidative stress in ageing, apoptosis, and diseases, such as familial amyotrophic lateral sclerosis and Friedreich's ataxia. Gene, 2001 Aug 22, 274(1-2), 151 - 6 A genomic screen identifies AUT8 as a novel gene essential for autophagy in the yeast Saccharomyces cerevisiae; Barth H et al.; Autophagy is a starvation-induced transport pathway delivering parts of the cytosol into the lysosome (vacuole) for degradation . Autophagy significantly differs from other transport pathways by using double membrane layered transport intermediates . Based on the identification of autophagy genes in Saccharomyces cerevisiae, which served as a pacemaker for higher cells, our mechanistic knowledge of autophagy notably increased over the past few years . We here identify AUT8 as a novel gene essential for autophagy by screening a collection of approximately 5000 yeast deletion strains, each containing a defined deletion in an individual gene . This collection is a result of the world-wide Saccharomyces deletion project and covers the non-essential genes of the whole yeast genome . Homozygous aut8 Delta cells are impaired in maturation of proaminopeptidase I, and they fail to undergo the cell differentiation process of sporulation . The essential function of AUT8 for autophagy is further demonstrated by the lack of accumulation of autophagic vesicles in the vacuoles of aut8 Delta cells starved of nitrogen in the presence of the proteinase B inhibitor phenylmethylsulfonyl fluoride. Gene, 2001 Aug 22, 274(1-2), 15 - 25 Nonsense-mediated mRNA decay in Saccharomyces cerevisiae; Gonzalez CI et al.; Cell survival depends on the precise and correct production of polypeptides . Eukaryotic cells have evolved conserved proofreading mechanisms to get rid of incomplete and potentially deleterious proteins . The nonsense-mediated mRNA decay (NMD) pathway is an example of a surveillance mechanism that monitors premature translation termination and promotes degradation of aberrant transcripts that code for nonfunctional or even harmful proteins . In this review we will describe our current knowledge of the NMD pathway, analyzing primarily the results obtained from the yeast Saccharomyces cerevisiae, but establishing functional comparisons with those obtained in higher eukaryotes . Based on these observations, we present two related working models to explain how this surveillance pathway recognizes and selectively degrades aberrant mRNAs. J Biol Chem, 2001 Dec 28, 276(52), 49244 - 50 Epub 2001 Oct 22. Three cell wall mannoproteins facilitate the uptake of iron in Saccharomyces cerevisiae; Protchenko O et al.; Analysis of iron-regulated gene expression in Saccharomyces cerevisiae using cDNA microarrays has identified three putative cell wall proteins that are directly regulated by Aft1p, the major iron-dependent transcription factor in yeast . FIT1, FIT2, and FIT3 (for facilitator of iron transport) were more highly expressed in strains grown in low concentrations of iron and in strains in which AFT1-1(up), a constitutively active allele of AFT1, was expressed . Northern blot analysis confirmed that FIT1, FIT2, and FIT3 mRNA transcript levels were increased 60-230-fold in response to iron deprivation in an Aft1p-dependent manner . Fit1p was localized exclusively to the cell wall by indirect immunofluorescence . Deletion of the FIT genes, individually or in combination, resulted in diminished uptake of iron bound to the siderophores ferrioxamine B and ferrichrome, without diminishing the uptake of ferric iron salts, or the siderophores triacetylfusarinine C and enterobactin . FIT-deletion strains exhibited increased expression of Aft1p target genes as measured by a FET3-lacZ reporter gene or by Arn1p Western blotting, indicating that cells respond to the absence of FIT genes by up-regulating systems of iron uptake . Aft1p activation in FIT-deleted strains occurred when either ferrichrome or ferric salts were used as sources of iron during growth, suggesting that the FIT genes enhance uptake of iron from both sources . Enzymatic digestion of the cell wall resulted in the release of significant amounts of iron from cells, and the relative quantity of iron released was reduced in FIT-deletion strains . Fit1p, Fit2p, and Fit3p may function by increasing the amount of iron associated with the cell wall and periplasmic space. Biochem J, 2001 Nov 1, 359(Pt 3), 631 - 7 gamma-Glutamyl transpeptidase in the yeast Saccharomyces cerevisiae and its role in the vacuolar transport and metabolism of glutathione; Mehdi K et al.; In the yeast Saccharomyces cerevisiae, the enzyme gamma-glutamyl transpeptidase (gamma-GT; EC 2.3.2.2) is a glycoprotein that is bound to the vacuolar membrane . The kinetic parameters of GSH transport into isolated vacuoles were measured using intact vacuoles isolated from the wild-type yeast strain Sigma 1278b, under conditions of gamma-GT synthesis (nitrogen starvation) and repression (growth in the presence of ammonium ions) . Vacuoles devoid of gamma-GT displayed a K(m) (app) of 18+/-2 mM and a V(max) (app) of 48.5+/-5 nmol of GSH/min per mg of protein . Vacuoles containing gamma-GT displayed practically the same K(m), but a higher V(max) (app) (150+/-12 nmol of GSH/min per mg of protein) . Vacuoles prepared from a disruptant lacking gamma-GT showed no increase in V(max) (app) with nitrogen starvation . From a comparison of the transport data obtained for vacuoles isolated from various reference and mutant strains, it appears that the yeast cadmium factor 1 (YCF1) transport system accounts for approx . 70% of the GSH transport capacity of the vacuoles, the remaining 30% being due to a vacuolar (H(+)) ATPase-coupled system . The V(max) (app)-increasing effect of gamma-GT concerns only the YCF1 system . gamma-GT in the vacuolar membrane activates the Ycf1p transporter, either directly or indirectly . Moreover, GSH accumulating in the vacuolar space may exert a feedback effect on its own entry . Excretion of glutamate from radiolabelled GSH in isolated vacuoles containing gamma-GT was also measured . It is proposed that gamma-GT and a L-Cys-Gly dipeptidase catalyse the complete hydrolysis of GSH stored in the central vacuole of the yeast cell, prior to release of its constitutive amino acids L-glutamate, L-cysteine and glycine into the cytoplasm . Yeast appears to be a useful model for studying gamma-GT physiology and GSH metabolism. Biotechnol Bioeng, 2001 Nov, 76(3), 259 - 68 Cell cycle-dependent protein secretion by Saccharomyces cerevisiae; Frykman S et al.; Synchronized Saccharomyces cerevisiae cell populations were used to examine secretion rates of a heterologous protein as a function of cell cycle position . The synchronization procedure had a profound effect on the type and quality of data obtained . When cell synchrony was induced by cell cycle-arresting drugs, a significant physiological perturbation of cells was observed that obscured representative secretion data . In contrast, synchronization with centrifugal elutriation resulted in synchronized first-generation daughter cells with undetectable perturbation of the physiological state . The synchronized cells did not secrete significant amounts of protein until they reached cell division, suggesting that the secretion process in these cells is strongly cell cycle dependent . However, the maximum secretion rate of the synchronized culture (7-14 molecules/cell/second) was significantly lower than that of an asynchronous culture (29-51 molecules/cell/second) . This result indicates that young daughter cells isolated in the synchronization process exhibit different protein secretion behavior than older mother cells that are absent in the synchronized cell population but present in the asynchronous culture . Genetika, 2001 Sep, 37(9), 1213 - 24 {New mutation in Saccharomyces cerevisiae SRM genes and some features of their phenotypic effects}; Koltovaia NA et al.; The effects of the previously identified mutations in nuclear genes SRM8, SRM12, SRM15, and SRM17 on the maintenance of chromosomes and recombinant plasmids in Saccharomyces cerevisiae cells and on cell sensitivity to ionizing radiation were studied . The srm8 mutation caused instability of chromosome maintenance in diploid cells . In yeast cells with the intact mitochondrial genome, all examined srm mutations decreased the mitotic stability of a centromeric recombinant plasmid with the chromosomal ARS element . Mutations srm12, srm15, and srm17 also decreased the mitotic stability of a centromereless plasmid containing the same ARS element, whereas the srm8 mutation did not markedly affect the maintenance of this plasmid . Mutations srm8, srm12, and srm17 were shown to increase cell sensitivity to gamma-ray irradiation . The SRM8 gene was mapped, cloned, and found to correspond to the open reading frame YJLO76w in chromosome X. Proc Natl Acad Sci U S A, 2001 Oct 23, 98(22), 12608 - 13 Epub 2001 Oct 16. A genome-wide screen in Saccharomyces cerevisiae for genes affecting UV radiation sensitivity; Birrell GW et al.; The recent completion of the deletion of essentially all of the ORFs in yeast is an important new resource for identifying the phenotypes of unknown genes . Each ORF is replaced with a cassette containing unique tag sequences that allow rapid parallel analysis of strains in a pool by using hybridization to a high-density oligonucleotide array . We examined the utility of this system to identify genes conferring resistance to UV irradiation by using a pool of 4,627 individual homozygous deletion strains (representing deletions of all nonessential genes) . We identified most of the nonessential genes previously shown to be involved in nucleotide excision repair, in cell cycle checkpoints, in homologous recombination, and in postreplication repair after UV damage . We also identified and individually confirmed, by replacing the genes, three new genes, to our knowledge not previously reported to confer UV sensitivity when deleted . Two of these newly identified genes have human orthologs associated with cancer, demonstrating the potential of this system to uncover human genes affecting sensitivity to DNA-damaging agents and genes potentially involved in cancer formation. Genetics, 2001 Oct, 159(2), 487 - 97 Genetic interactions of Spt4-Spt5 and TFIIS with the RNA polymerase II CTD and CTD modifying enzymes in Saccharomyces cerevisiae; Lindstrom DL et al.; Genetic and biochemical studies have identified many factors thought to be important for transcription elongation . We investigated relationships between three classes of these factors: (1) transcription elongation factors Spt4-Spt5, TFIIS, and Spt16; (2) the C-terminal heptapeptide repeat domain (CTD) of RNA polymerase II; and (3) protein kinases that phosphorylate the CTD and a phosphatase that dephosphorylates it . We observe that spt4 and spt5 mutations cause strong synthetic phenotypes in combination with mutations that shorten or alter the composition of the CTD; affect the Kin28, Bur1, or Ctk1 CTD kinases; and affect the CTD phosphatase Fcp1 . We show that Spt5 co-immunoprecipitates with RNA polymerase II that has either a hyper- or a hypophosphorylated CTD . Furthermore, mutation of the CTD or of CTD modifying enzymes does not affect the ability of Spt5 to bind RNA polymerase II . We find a similar set of genetic interactions between the CTD, CTD modifying enzymes, and TFIIS . In contrast, an spt16 mutation did not show these interactions . These results suggest that the CTD plays a key role in modulating elongation in vivo and that at least a subset of elongation factors are dependent upon the CTD for their normal function. Genetics, 2001 Oct, 159(2), 453 - 70 Genes involved in sister chromatid separation and segregation in the budding yeast Saccharomyces cerevisiae; Biggins S et al.; Accurate chromosome segregation requires the precise coordination of events during the cell cycle . Replicated sister chromatids are held together while they are properly attached to and aligned by the mitotic spindle at metaphase . At anaphase, the links between sisters must be promptly dissolved to allow the mitotic spindle to rapidly separate them to opposite poles . To isolate genes involved in chromosome behavior during mitosis, we microscopically screened a temperature-sensitive collection of budding yeast mutants that contain a GFP-marked chromosome . Nine LOC (loss of cohesion) complementation groups that do not segregate sister chromatids at anaphase were identified . We cloned the corresponding genes and performed secondary tests to determine their function in chromosome behavior . We determined that three LOC genes, PDS1, ESP1, and YCS4, are required for sister chromatid separation and three other LOC genes, CSE4, IPL1, and SMT3, are required for chromosome segregation . We isolated alleles of two genes involved in splicing, PRP16 and PRP19, which impair alpha-tubulin synthesis thus preventing spindle assembly, as well as an allele of CDC7 that is defective in DNA replication . We also report an initial characterization of phenotypes associated with the SMT3/SUMO gene and the isolation of WSS1, a high-copy smt3 suppressor. Genetics, 2001 Oct, 159(2), 441 - 52 Direct estimate of the mutation rate and the distribution of fitness effects in the yeast Saccharomyces cerevisiae; Wloch DM et al.; Estimates of the rate and frequency distribution of deleterious effects were obtained for the first time by direct scoring and characterization of individual mutations . This was achieved by applying tetrad analysis to a large number of yeast clones . The genomic rate of spontaneous mutation deleterious to a basic fitness-related trait, that of growth rate, was U = 1.1 x 10(-3) per diploid cell division . Extrapolated to the fruit fly and humans, the per generation rate would be 0.074 and 0.92, respectively . This is likely to be an underestimate because single mutations with selection coefficients s < 0.01 could not be detected . The distribution of s > or = 0.01 was studied both for spontaneous and induced mutations . The latter were induced by ethyl methanesulfonate (EMS) or resulted from defective mismatch repair . Lethal changes accounted for approximately 30-40% of the scored mutations . The mean s of nonlethal mutations was fairly high, but most frequently its value was between 0.01 and 0.05 . Although the rate and distribution of very small effects could not be determined, the joint share of such mutations in decreasing average fitness was probably no larger than approximately 1%. EMBO Rep, 2001 Nov, 2(11), 1035 - 9 Epub 2001 Oct 17. Yarrowia lipolytica Pex20p, Saccharomyces cerevisiae Pex18p/Pex21p and mammalian Pex5pL fulfil a common function in the early steps of the peroxisomal PTS2 import pathway; Einwachter H et al.; Import of peroxisomal matrix proteins is essential for peroxisome biogenesis . Genetic and biochemical studies using a variety of different model systems have led to the discovery of 23 PEX genes required for this process . Although it is generally believed that, in contrast to mitochondria and chloroplasts, translocation of proteins into peroxisomes involves a receptor cycle, there are reported differences of an evolutionary conservation of this cycle either with respect to the components or the steps involved in different organisms . We show here that the early steps of protein import into peroxisomes exhibit a greater similarity than was thought previously to be the case . Pex20p of Yarrowia lipolytica, Pex18p and Pex21p of Saccharomyces cerevisiae and mammalian Pex5pL fulfil a common function in the PTS2 pathway of their respective organisms . These non-orthologous proteins possess a conserved sequence region that most likely represents a common PTS2-receptor binding site and di-aromatic pentapeptide motifs that could be involved in binding of the putative docking proteins . We propose that not necessarily the same proteins but functional modules of them are conserved in the early steps of peroxisomal protein import. Int J Biochem Cell Biol, 2001 Dec, 33(12), 1202 - 7 UGA4 gene encoding the gamma-aminobutyric acid permease in Saccharomyces cerevisiae is an acid-expressed gene; Moretti MB et al.; BACKGROUND AND AIMS: biological processes in all organisms are controlled by environmental conditions, however, information concerning the molecular responses to external pH is scarce . In this work we studied the pH response of UGA4 gene encoding delta-aminolevulinic acid and gamma-aminobutyric acid permease in Saccharomyces cerevisiae . METHODS: we analyzed the effect of pH on the expression of UGA4 gene measuring beta-galactosidase activity in cells carrying a UGA4::lacZ fusion gene . RESULTS: results indicate that UGA4 expression is higher at acidic pH . The expression of UGA3 and UGA35 genes, which encode two positive transcription factors, is not regulated by external pH, while the expression of UGA43 gene encoding a repressor of UGA4 transcription is dependent on pH . Using a strain lacking Uga43p we clearly showed that the effect of ambient pH on UGA4 expression is not a secondary effect of the pH regulation on UGA43 . We have also demonstrated that the effect of pH can only be detected when UGA4 gene is not subject to a strong repression by Uga43p nor to GABA induction . CONCLUSION: here, we demonstrate that UGA4 is an acid-expressed gene . This regulation is probably mediated by Rim101p through the consensus site 5'-GCCARG-3' at 237 bp preceding the UGA4 coding sequence (201). Res Microbiol, 2001 Sep, 152(7), 605 - 12 Protein-based inheritance in Saccharomyces cerevisiae: {URE3} as a prion form of the nitrogen regulatory protein Ure2; Edskes HK; The {URE3} element of the yeast Saccharomyces cerevisiae results from the presence of an altered form of the nitrogen regulatory protein Ure2 . This altered form acts as an infectious protein (prion) . Genes affecting {URE3} initiation and propagation should give valuable information about prion diseases as well as other conformational diseases. Prikl Biokhim Mikrobiol, 2001 Sep-Oct, 37(5), 578 - 81 {Effect of culture media on the composition of free amino acids in Saccharomyces cerevisiae yeast}; Khalilova EA et al.; The quantitative and qualitative compositions of free amino acids of the yeast Saccharomyces cerevisiae Y-503 cultivated in different nutrient media were studied by liquid chromatography . The yeast grown in the medium containing geothermal water was shown to accumulate more amino acids . During lyophilization, the stabilization of the physiological activity of the yeast in this nutrient medium was observed . The increased biological value of dry yeast was shown to depend on the content of free amino acids, including essential amino acids: arginine, histidine, leucine, isoleucine, lysine, threonine, serine and phenylalanine. Mol Cell Biol, 2001 Nov, 21(22), 7663 - 72 Peripheral mitochondrial inner membrane protein, Mss2p, required for export of the mitochondrially coded Cox2p C tail in Saccharomyces cerevisiae; Broadley SA et al.; Cytochrome oxidase subunit 2 (Cox2p) is synthesized on the matrix side of the mitochondrial inner membrane, and its N- and C-terminal domains are exported across the inner membrane by distinct mechanisms . The Saccharomyces cerevisiae nuclear gene MSS2 was previously shown to be necessary for Cox2p accumulation . We have used pulse-labeling studies and the expression of the ARG8(m) reporter at the COX2 locus in an mss2 mutant to demonstrate that Mss2p is not required for Cox2p synthesis but rather for its accumulation . Mutational inactivation of the proteolytic function of the matrix-localized Yta10p (Afg3p) AAA-protease partially stabilizes Cox2p in an mss2 mutant but does not restore assembly of cytochrome oxidase . In the absence of Mss2p, the Cox2p N terminus is exported, but Cox2p C-terminal export and assembly of Cox2p into cytochrome oxidase is blocked . Epitope-tagged Mss2p is tightly, but peripherally, associated with the inner membrane and protected by it from externally added proteases . Taken together, these data indicate that Mss2p plays a role in recognizing the Cox2p C tail in the matrix and promoting its export. Mol Cell Biol, 2001 Nov, 21(22), 7576 - 86 Overproduction of PDR3 suppresses mitochondrial import defects associated with a TOM70 null mutation by increasing the expression of TOM72 in Saccharomyces cerevisiae; Koh JY et al.; Most mitochondrial proteins are synthesized with cleavable amino-terminal targeting signals that interact with the mitochondrial import machinery to facilitate their import from the cytosol . We previously reported that the presequence of the F(1)-ATPase beta subunit precursor (pre-F(1)beta) acts as an intramolecular chaperone that maintains the precursor in an import-competent conformation prior to import (P . Hajek, J . Y . Koh, L . Jones, and D . M . Bedwell, Mol . Cell . Biol . 17:7169-7177, 1997) . We also found that a mutant form of pre-F(1)beta with a minimal targeting signal (Delta 1,2 pre-F(1)beta) is inefficiently imported into mitochondria because it rapidly folds into an import-incompetent conformation . We have now analyzed the consequences of reducing the pre-F(1)beta targeting signal to a minimal unit in more detail . We found that Delta 1,2 pre-F(1)beta is more dependent upon the Tom70p receptor for import than WT pre-F(1)beta is, resulting in a growth defect on a nonfermentable carbon source at 15 degrees C . Experiments using an in vitro mitochondrial protein import system suggest that Tom70p functions to maintain a precursor containing the Delta 1,2 pre-F(1)beta import signal in an import-competent conformation . We also identified PDR3, a transcriptional regulator of the pleiotropic drug resistance network, as a multicopy suppressor of the mitochondrial import defects associated with Delta 1,2 pre-F(1)beta in a tom70 Delta strain . The overproduction of PDR3 mediated this effect by increasing the import of Delta 1,2 pre-F(1)beta into mitochondria . This increased the mitochondrial ATP synthase activity to the extent that growth of the mutant strain was restored under the selective conditions . Analysis of the transcription patterns of components of the mitochondrial outer membrane import machinery demonstrated that PDR3 overproduction increased the expression of TOM72, a little studied TOM70 homologue . These results suggest that Tom72p possesses overlapping functions with Tom70p and that the pleiotropic drug resistance network plays a previously unappreciated role in mitochondrial biogenesis. J Biol Chem, 2001 Dec 21, 276(51), 48539 - 48 Epub 2001 Oct 15. Phosphatidylethanolamine has an essential role in Saccharomyces cerevisiae that is independent of its ability to form hexagonal phase structures; Storey MK et al.; Two yeast enzymes, Psd1p and Psd2p, catalyze the decarboxylation of phosphatidylserine to produce phosphatidylethanolamine (PtdEtn) . Mitochondrial Psd1p provides approximately 90% of total cellular phosphatidylserine decarboxylase activity . When the PSD1 gene is deleted, the resultant strain (psd1Delta) grows normally at 30 degrees C in glucose and in the absence of exogenous choline or ethanolamine . However, at elevated temperature (37 degrees C) or on the nonfermentable carbon source lactate, the growth of psd1Delta strains is minimal without ethanolamine supplementation . The reduced growth and viability correlate with a PtdEtn content below 4% of total phospholipid . These results suggest that there is a critical level of PtdEtn required to support growth . This theory is supported by growth data revealing that a psd1Delta psd2Delta dpl1Delta strain can only grow in the presence of ethanolamine . In contrast, a psd1Delta psd2Delta strain, which makes low levels of PtdEtn from sphingolipid breakdown, can be rescued by ethanolamine, choline, or the ethanolamine analogue propanolamine . psd1Delta psd2Delta cells grown in 2 mm propanolamine accumulate a novel lipid, which was determined by mass spectrometry to be phosphatidylpropanolamine (PtdPrn) . PtdPrn can comprise up to 40% of the total phospholipid content in supplemented cells at the expense of phosphatidylcholine and PtdEtn . The absolute level of PtdEtn required for growth when PtdPrn is present appears to be 1% of the total phospholipid content . The essential function of the PtdEtn in the presence of propanolamine does not appear to be the formation of hexagonal phase lipid, insofar as PtdPrn readily forms hexagonal phase structures detectable by (31)P NMR. J Biol Chem, 2001 Dec 14, 276(50), 47556 - 62 Epub 2001 Oct 15. Manganese superoxide dismutase in Saccharomyces cerevisiae acquires its metal co-factor through a pathway involving the Nramp metal transporter, Smf2p; Luk EE et al.; Eukaryotes express both copper/zinc (SOD1)- and manganese (SOD2)-requiring superoxide dismutase enzymes that guard against oxidative damage . Although SOD1 acquires its copper through a specific copper trafficking pathway, nothing is known regarding the intracellular manganese trafficking pathway for SOD2 . We demonstrate here that in Saccharomyces cerevisiae cells delivery of manganese to SOD2 in the mitochondria requires the Nramp metal transporter, Smf2p . SOD2 activity is greatly diminished in smf2Delta mutants, even though the mature SOD2 polypeptide accumulates to normal levels in mitochondria . Treating smf2Delta cells with manganese supplements corrected the SOD2 defect, as did elevating intracellular manganese through mutations in PMR1 . Hence, manganese appears to be inaccessible to mitochondrial SOD2 in smf2 mutants . Cells lacking SMF2 also exhibited defects in manganese-dependent steps in protein glycosylation and showed an overall decrease in steady-state levels of accumulated manganese . By comparison, mutations in the cell surface Nramp transporter, Smf1p, had very little impact on manganese accumulation and trafficking . Smf2p resides in intracellular vesicles and shows no evidence of plasma membrane localization, even in an end4 mutant blocked for endocytosis . We propose a model in which Smf2p-containing vesicles play a central role in manganese trafficking to the mitochondria and other cellular sites as well. Nucleic Acids Res, 2001 Oct 15, 29(20), 4134 - 43 Efficient repair of large DNA loops in Saccharomyces cerevisiae; Corrette-Bennett SE et al.; Small looped mispairs are efficiently corrected by mismatch repair . The situation with larger loops is less clear . Repair activity on large loops has been reported as anywhere from very low to quite efficient . There is also uncertainty about how many loop repair activities exist and whether any are conserved . To help address these issues, we studied large loop repair in Saccharomyces cerevisiae using in vivo and in vitro assays . Transformation of heteroduplexes containing 1, 16 or 38 nt loops led to >90% repair for all three substrates . Repair of the 38 base loop occurred independently of mutations in key genes for mismatch repair (MR) and nucleotide excision repair (NER), unlike other reported loop repair functions in yeast . Correction of the 16 base loop was mostly independent of MR, indicating that large loop repair predominates for this size heterology . Similarities between mammalian and yeast large loop repair were suggested by the inhibitory effects of loop secondary structure and by the role of defined nicks on the relative proportions of loop removal and loop retention products . These observations indicate a robust large loop repair pathway in yeast, distinct from MR and NER, and conserved in mammals. J Radiat Res (Tokyo), 2001 Jun, 42(2), 217 - 27 Effect of a hypoxic radiosensitizer, AK 2123 (Sanazole), on yeast Saccharomyces cerevisiae; Pasupathy K et al.; Sanazole/DNA repair/Hypoxic radiosensitization/DNA polymerases/Saccharomyces cerevisiae Yeast Saccharomyces cerevisiae can exist in two physiological states, namely anaerobic and aerobic . They differ in their response to gamma- radiation and radiomodification . We report hereon our results concerning radiosensitization by Sanazole (AK-2123), a well-known hypoxic radio sensitizer, whose mechanism of action has been studied extensively . The results have revealed that Sanazole (1 mM) when present during irradiation could specifically sensitize wild-type anaerobic yeast cells with a DMF of 2.4 . In a radiation-sensitive mutant which lacks a DNA repair pathway specific for the recovery from gamma-radiation induced DNA damage, the extent of sensitization was considerably lower and the DMF was only 1.3 . Studies on the liquid holding recovery of cells of both wild- type and rad52 yeast cells exposed to radiation in presence of Sanazole revealed that sensitization by Sanazole is due to a preferential increase in the DNA damage, and not by impairing DNA repair . This system thus holds promise for screening potential hypoxic chemical radiosensitizers. Mol Biol Cell, 2001 Oct, 12(10), 3295 - 306 The GPI transamidase complex of Saccharomyces cerevisiae contains Gaa1p, Gpi8p, and Gpi16p; Fraering P et al.; Gpi8p and Gaa1p are essential components of the GPI transamidase that adds glycosylphosphatidylinositols (GPIs) to newly synthesized proteins . After solubilization in 1.5% digitonin and separation by blue native PAGE, Gpi8p is found in 430-650-kDa protein complexes . These complexes can be affinity purified and are shown to consist of Gaa1p, Gpi8p, and Gpi16p (YHR188c) . Gpi16p is an essential N-glycosylated transmembrane glycoprotein . Its bulk resides on the lumenal side of the ER, and it has a single C-terminal transmembrane domain and a small C-terminal, cytosolic extension with an ER retrieval motif . Depletion of Gpi16p results in the accumulation of the complete GPI lipid CP2 and of unprocessed GPI precursor proteins . Gpi8p and Gpi16p are unstable if either of them is removed by depletion . Similarly, when Gpi8p is overexpressed, it largely remains outside the 430-650-kDa transamidase complex and is unstable . Overexpression of Gpi8p cannot compensate for the lack of Gpi16p . Homologues of Gpi16p are found in all eucaryotes . The transamidase complex is not associated with the Sec61p complex and oligosaccharyltransferase complex required for ER insertion and N-glycosylation of GPI proteins, respectively . When GPI precursor proteins or GPI lipids are depleted, the transamidase complex remains intact. Environ Toxicol Chem, 2001 Oct, 20(10), 2353 - 60 Bioassay of cadmium using a DNA microarray: genome-wide expression patterns of Saccharomyces cerevisiae response to cadmium; Momose Y et al.; DNA microarray technology enables genome-wide detection of cell response at the transcriptional level . We are planning to make bioassay systems that can detect environmental chemicals to screen for potential bioreactive agents . To develop a DNA microarray for our purposes, the changes in gene expression underlying the yeast stress response to cadmium were analyzed by a microarray of total mRNA . Cadmium is a potent cell poison known to cause oxidative stress by changing intracellular glutathione levels . We report here that not only the glutathione synthesis gene (GSH1) but also almost all transcripts of the enzymes involved in the sulfur amino acid metabolism, especially MET14 and MET17, were greatly induced after exposure to cadmium . While several common stress-responsive genes, such as HSP26, GRE1, HSP12, and DDR48, were up-regulated more than almost fourfold by cadmium, there were also 42 other genes up-regulated more than fourfold . Based on these results, we concluded that DNA microarrays are very useful instruments for creating new bioassay systems and finding genetic promoters of stress indicators. Physiol Genomics, 2001 Oct 10, 7(1), 27 - 34 Two-hybrid analysis of the Saccharomyces cerevisiae 26S proteasome; Cagney G et al.; A two-hybrid screen against an activation domain array of Saccharomyces cerevisiae proteins was carried out for 31 yeast proteasome proteins . Fifty-five putative interactions were identified: 21 between components of the proteasome complex and 34 between proteasome proteins and other proteins . Many of these latter interactions involved either proteins of the ubiquitin pathway, cell cycle proteins, protein kinases or a translation initiation factor subunit . The role of eleven proteins associated with proteasome function by these screens was analyzed by examining the corresponding deletion strains for temperature sensitivity and canavanine sensitivity and for the stability of a ubiquitin-beta-galactosidase fusion protein . These assays additionally implicated three proteins, Bim1, Ump1, and YKL171W, in proteasome function . This study demonstrates the utility of genome-wide two-hybrid assays as an entry point for the further analysis of a large protein complex. FEBS Lett, 2001 Oct 5, 506(2), 149 - 56 Functional characterization of the Bag7, Lrg1 and Rgd2 RhoGAP proteins from Saccharomyces cerevisiae; Roumanie O et al.; Rho proteins are down-regulated in vivo by specific GTPase activating proteins (RhoGAP) . We have functionally studied three Saccharomyces cerevisiae putative RhoGAP . By first identifying Rho partners with a systematic two-hybrid approach and then using an in vitro assay, we have demonstrated that the Bag7 protein stimulated the GTPase activity of the Rho1 protein, Lrg1p acted on the Cdc42 and Rho2 GTPases and we showed that Rgd2p has a GAP activity on both Cdc42p and Rho5p . In addition, we brought the first evidence for the existence of a sixth functional Rho in yeast, the Cdc42/Rac-like GTPase Rho5. Biotechnol Bioeng, 2001 Nov 5, 75(3), 334 - 44 Statistical reconciliation of the elemental and molecular biomass composition of Saccharomyces cerevisiae; Lange HC et al.; A systematic mathematical procedure capable of detecting the presence of a gross error in the measurements and of reconciling connected data sets by using the maximum likelihood principle is applied to the biomass composition data of yeast . The biomass composition of Saccharomyces cerevisiae grown in a chemostat under glucose limitation was analyzed for its elemental and for its molecular composition . Both descriptions initially resulted in conflicting results concerning the elemental composition, molecular weight, and degrees of reduction . The application of the statistical reconciliation method, based on elemental balances and equality relations, is used to obtain a consistent biomass composition . Simultaneously, the error margins of the data sets are significantly reduced in the reconciliation process . On the basis of statistical analysis it was found that inclusion of about 4% water in the list of biomass constituents is essential to adequately describe the dry biomass and match both set of measurements . The reconciled carbon content of the biomass varied 4% from the ones obtained from the molecular analysis . The proposed method increases the accuracy of biomass composition data of its elements and its molecules by providing a best estimate based on all available data and thus provides an improved and consistent basis for metabolic flux analysis as well as black box modeling approaches . Eur J Biochem, 2001 Oct, 268(19), 5209 - 14 The AUG start codon of the Saccharomyces cerevisiae NFS1 gene can be substituted for by UUG without increased initiation of translation at downstream codons; Nett JH et al.; The selection of the site for initiation of translation for the Saccharomyces cerevisiae NFS1 gene was examined using mutated AUG1, AUG2 and AUG3 codons . When AUG1 of the yeast NFS1 gene was mutated to UUG and the resulting mRNA was translated in vitro using a reticulocyte system, initiation from the mutated codon was abolished and occurred instead at downstream codons at increased rates . When the same mRNA was translated using a yeast extract, translation initiated at the mutated codon, albeit at a reduced rate, and there was no increased translation at downstream AUG codons . The NFS1 gene in which AUG1 was replaced by UUG was also able to substitute for the wild-type gene in vivo in yeast . Western blots confirmed that the encoded protein was the same size as that encoded by the wild-type gene and that both the wild-type and mutated proteins localized to mitochondria . This is apparently the first example of a yeast protein where mutagenesis of AUG1 does not lead to alternate use of a downstream AUG. Eur J Biochem, 2001 Oct, 268(19), 5057 - 65 Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae; Boubekeur S et al.; This work was undertaken to clarify the role of acetaldehyde dehydrogenases in Saccharomyces cerevisiae metabolism during growth on respiratory substrates . Until now, there has been little agreement concerning the ability of mutants deleted in gene ALD4, encoding mitochondrial acetaldehyde dehydrogenase, to grow on ethanol . Therefore we constructed mutants in two parental strains (YPH499 and W303-1a) . Some differences appeared in the growth characteristics of mutants obtained from these two parental strains . For these experiments we used ethanol, pyruvate or lactate as substrates . Mitochondria can oxidize lactate into pyruvate using an ATP synthesis-coupled pathway . The ald4Delta mutant derived from the YPH499 strain failed to grow on ethanol, but growth was possible for the ald4Delta mutant derived from the W303-1a strain . The co-disruption of ALD4 and PDA1 (encoding subunit E1alpha of pyruvate dehydrogenase) prevented the growth on pyruvate for both strains but prevented growth on lactate only in the double mutant derived from the YPH499 strain, indicating that the mutation effects are strain-dependent . To understand these differences, we measured the enzyme content of these different strains . We found the following: (a) the activity of cytosolic acetaldehyde dehydrogenase in YPH499 was relatively low compared to the W303-1a strain; (b) it was possible to restore the growth of the mutant derived from YPH499 either by addition of acetate in the media or by introduction into this mutant of a multicopy plasmid carrying the ALD6 gene encoding cytosolic acetaldehyde dehydrogenase . Therefore, the lack of growth of the mutant derived from the YPH499 strain seemed to be related to the low activity of acetaldehyde oxidation . Therefore, when cultured on ethanol, the cytosolic acetaldehyde dehydrogenase can partially compensate for the lack of mitochondrial acetaldehyde dehydrogenase only when the activity of the cytosolic enzyme is sufficient . However, when cultured on pyruvate and in the absence of pyruvate dehydrogenase, the cytosolic acetaldehyde dehydrogenase cannot compensate for the lack of the mitochondrial enzyme because the mitochondrial form produces intramitochondrial NADH and consequently ATP through oxidative phosphorylation. Mol Genet Genomics, 2001 Sep, 266(1), 20 - 7 Overexpression of Bud5p can suppress mutations in the Gsp1p guanine nucleotide exchange factor Prp20p in Saccharomyces cerevisiae; Clement M et al.; The gene product Prp20p, which is located in the nucleus, serves as the nucleotide exchange factor (GEF) for the small nuclear G protein Gsp1p in Saccharomyces cerevisiae, and catalyses the replacement of Gsp1-bound GDP by GTP . These proteins are involved in numerous cellular processes, including nucleocytoplasmic trafficking of macromolecules, cell cycle progression, DNA replication and maintenance of chromosome structure/stability . It is believed that in order to complete a full GDP/GTP cycle, Gsp1p has to shuttle between the nucleus and the cytoplasm, where its GTPase Activating Protein (GAP) Rna1p is located . Here, we report on the ability of Bud5p, the exchange factor for Rsr1p, to suppress conditional prp20 mutants when an extra copy of GSP1 is present . This suppression by BUD5 can be reversed by simultaneous overexpression of RNA1, and is not Rsr1p-dependent, nor allele-specific . We also show that Bud5p can physically interact with Gsplp, both in vitro and in vivo . These,findings raise the possibility that Bud5p could act as a cytoplasmic exchange factor for Gsp1p and, therefore, that a complete GDP/GTP cycle could take place in the cytoplasm. Mol Genet Genomics, 2001 Sep, 266(1), 142 - 55 Mutations in WSC genes for putative stress receptors result in sensitivity to multiple stress conditions and impairment of Rlm1-dependent gene expression in Saccharomyces cerevisiae; Zu T et al.; Intracellular signaling by mitogen-activated protein (MAP) kinase cascades plays an essential role in the cellular response to environmental stress . In the yeast Saccharomyces cerevisiae, the PKC1-regulated, stress-activated MAP kinase pathway, the MPK1 cascade, is activated by heat and by a decrease in osmolarity . The genes WSC1, WSC2 and WSC3 encode putative receptors that maintain cell wall integrity under conditions of heat stress . Genetic studies place the function of the WSC genes upstream of the MPK1 kinase cascade . To further define the role of the WSC family in the stress response we determined whether: (1) the wscdelta mutants are sensitive to other environmental stress conditions, in addition to heat shock; (2) expression from four transcriptional control elements, known to be activated by stress, is impaired in wscdelta mutants; and (3) Wsc4, a Wsc homolog, has functions that overlap with those of the other Wsc family members . We report here that deletion of WSC and PKC1 causes hypersensitivity to ethanol, hydrogen peroxide and DNA-damaging drugs . In wscdelta mutants expression of beta-galactosidase from the AP-1 response element (ARE), the heat shock response element (HSE) or the stress response element (STRE) is not reduced . In contrast, expression of a reporter gene placed under the control of the Rlm1 (transcription factor)-dependent response element is significantly reduced in wscdelta mutants . This suggests that the lysis defect of wscdelta mutants is at least in part caused by a defect in transcriptional regulation by Rlm1 . Phenotypic analysis of the effect of deleting WSC4 in a wsc1delta mutant show that, unlike WSC2 or WSC3, deletion of WSC4 does not exacerbate the lysis defect of a wsc1delta strain . In contrast, deletion of WSC4 enhances the sensitivity of the wsc1delta mutant to heat shock, ethanol, and a DNA-damaging drug, suggesting that WSC4 plays a role in the response to environmental stress but that its function may differ from those of the other WSC family members. Mol Genet Genomics, 2001 Sep, 266(1), 115 - 25 Role of the kinetochore protein Ndc10 in mitotic checkpoint activation in Saccharomyces cerevisiae; Fraschini R et al.; Mitotic checkpoints delay cell cycle progression in response to alterations in the mitotic apparatus, thus ensuring correct chromosome segregation . While improper spindle orientation activates the Bub2/Bfa1-dependent checkpoint in budding yeast, delaying exit from mitosis, lack of bipolar kinetochore-microtubule attachment activates a signal transduction cascade that prevents both anaphase onset and exit from mitosis by inhibiting the Cdc20/APC (Anaphase Promoting Complex)-mediated proteolysis of securin and inactivation of mitotic cyclin-dependent kinases (CDKs), respectively . Proteolysis of the securin Pdsl is necessary to liberate the separase Esp1, which then triggers sister chromatid separation, whereas inactivation of mitotic CDKs is a prerequisite for exit from mitosis and for starting a new round of DNA replication in the next cell cycle . In budding yeast, this latter checkpoint response involves the proteins Mad1, 2, 3, Bub1 and Bub3, whose vertebrate counterparts localize to unattached kinetochores . Mutations that alter other kinetochore proteins result in mitotic checkpoint activation, while the ndc10-1 mutation not only impairs kinetochore function, but also disrupts the checkpoint response, indicating a role for Ndc10 in this process . Here we present evidence that Ndc10 is not part of the Bub2/Bfa1-dependent pathway, and its role in the checkpoint response might also be different from that of the other Mad and Bub proteins . Indeed, Ndc10, unlike other mitotic checkpoint proteins, is not required for the mitotic block induced by overexpression of the Mpsl protein kinase, which is implicated in mitotic checkpoint control . Furthermore, the delay in mitotic exit caused by non-degradable Pds1, which does not require Mad and Bub proteins, depends on Ndc10 function . We propose that a pathway involving Ndc10 might monitor defects in the mitotic apparatus independently of the Mad and Bub proteins . Since the Espl separase is required for exit from mitosis in both ndc10-1 and nocodazole-treated mad2delta cells, the two signal transduction cascades might ultimately converge on the inactivation of Esp1. Methods Enzymol, 2001, 342, 159 - 67 Purification and characterization of Saccharomyces cerevisiae Rnt1p nuclease; Lamontagne B et al.; In this article, we have described methods used to purify Rnt1p and study its biochemical properties . Rnt1p can be easily purified from bacteria as N-terminal His6-tagged protein and its activity may be monitored in vitro . Rnt1p cleaves the RNA by binding to a cleavage site followed by hydrolysis and product release . The kinetic parameters of Rnt1p are similar to those of other nucleases, including bacterial RNase III . The ability of Rnt1p to bind substrate without cleaving it in the absence of divalent metal ions provides a convenient means to study RNA recognition and binding independent of catalysis . The gel mobility shift and in-the-gel cleavage assays described here reveal the formation of two Rnt1p-RNA complexes with different cleavage activities, suggesting that the protein may bind the substrate in two different forms or through a two-step binding reaction. J Biol Chem, 2001 Dec 14, 276(50), 47671 - 4 Epub 2001 Oct 03. Binding and partial denaturing of G-quartet DNA by Cdc13p of Saccharomyces cerevisiae; Lin YC et al.; The protein Cdc13p binds telomeres in vivo and is essential for the maintenance of the telomeres of Saccharomyces cerevisiae . In addition, Cdc13p is known to bind single-stranded TG(1-3) DNA in vitro . Here we have shown that Cdc13p also binds DNA quadruplex, G-quartet, formed by TG(1-3) DNA . Moreover, the binding of Cdc13p causes a partial denaturing of the G-quartet DNA . Formation of DNA quadruplexes may involve the intermolecular association of TG(1-3) DNA and inhibit the extension of telomeres by telomerase . Thus, our finding suggests that Cdc13p may disrupt telomere association and facilitate telomere replication. Cancer Res, 2001 Oct 1, 61(19), 6982 - 6 SKY1 is involved in cisplatin-induced cell kill in Saccharomyces cerevisiae, and inactivation of its human homologue, SRPK1, induces cisplatin resistance in a human ovarian carcinoma cell line; Schenk PW et al.; The therapeutic potential of cisplatin, one of the most active and widely used anticancer drugs, is severely limited by the occurrence of cellular resistance . In this study, using budding yeast Saccharomyces cerevisiae as a model organism to identify novel drug resistance genes, we found that disruption of the yeast gene SKY1 (serine/arginine-rich protein-specific kinase from budding yeast) by either transposon insertion or one-step gene replacement conferred cellular resistance to cisplatin . Heterologous expression of the human SKY1 homologue SRPK1 (serine/arginine-rich protein-specific kinase) in SKY1 deletion mutant yeast cells restored cisplatin sensitivity, suggesting that SRPK1 is a cisplatin sensitivity gene, the inactivation of which could lead to cisplatin resistance . Subsequently, we investigated the role of SRPK1 in cisplatin sensitivity and resistance in human ovarian carcinoma A2780 cells using antisense oligodeoxynucleotides . Treatment of A2780 cells with antisense oligodeoxynucleotides directed against the translation initiation site of SRPK1 led to down-regulation of SRPK1 protein and conferred a 4-fold resistance to cisplatin . The human SRPK1 gene has not been associated with drug resistance before . Our new findings strongly suggest that SRPK1 is involved in cisplatin-induced cell kill and indicate that SRPK1 might potentially be of importance for studying clinical drug resistance. FEMS Microbiol Lett, 2001 Sep 25, 203(2), 223 - 7 Saccharomyces cerevisiae pyruvate kinase Pyk1 is PKA phosphorylation substrate in vitro; Cytrynska M et al.; Fractionation of Saccharomyces cerevisiae postribosomal extract on DEAE-cellulose revealed two fractions of cAMP-dependent protein kinase (PKA-1 and PKA-2) . The presence of PKA in both fractions was confirmed by immunoblotting with anti-Bcy1 antibodies . Yeast pyruvate kinase Pyk1 identified by amino acid microsequencing analysis and immunoblotting with anti-Pyk1 antibodies copurified with the PKA-1 but not the -2 fraction . Pyk1 can be phosphorylated by yeast PKA in vitro in the presence of cAMP and cGMP . Two-dimensional gel electrophoretic analysis revealed four phosphorylated forms of Pyk1 modified by PKA . In phosphorylation of Pyk1 mainly the Tpk2 catalytic subunit of yeast PKA was involved. Mol Microbiol, 2001 Sep, 41(6), 1311 - 26 Lre1 affects chitinase expression, trehalose accumulation and heat resistance through inhibition of the Cbk1 protein kinase in Saccharomyces cerevisiae; Versele M et al.; The addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae triggers activation of the cAMP pathway with a rapid drop in stress resistance as a consequence . We have isolated the LRE1 gene as a multicopy suppressor of glucose-induced loss of heat resistance . Overexpression of LRE1 in a wild-type strain causes the same phenotype as observed in strains with reduced activity of the cAMP-PKA pathway: higher heat resistance and enhanced trehalose levels . Deletion of LRE1 results in the opposite phenotypes . Epistasis analysis indicates that these effects are independent of cAMP and PKA, of the protein kinases Yak1, Sch9 and Rim15 and of the transcription factors Msn2 and Msn4 . Lre1 has recently been isolated in a two-hybrid screen using the conserved protein kinase Cbk1 as a bait . Cbk1 controls the expression of CTS1 (encoding chitinase) through the transcription factor Ace2 . We demonstrate here that overexpression of LRE1 represses CTS1 whereas deletion of LRE1 induces the expression of CTS1 . Repression of CTS1 results in deficient cell separation as a result of inefficient degradation of the chitin ring after cytokinesis . Neither deletion nor overexpression of LRE1 has any effect on CTS1 expression in a cbk1Delta mutant, indicating that Lre1 inhibits Cbk1 . In addition, we show that increased trehalose accumulation and increased heat resistance caused by overexpression of LRE1 are also the result of inhibition of Cbk1, revealing a novel control pathway for certain targets affected by PKA . The yeast genome contains a homologue of LRE1, YDR528w, which we have called HLR1 (for homologue of Lre1) . Deletion and overexpression of HLR1 causes similar but less pronounced effects compared with LRE1. Microbiology, 2001 Oct, 147(Pt 10), 2849 - 55 New aspects of the glucose activation of the H(+)-ATPase in the yeast Saccharomyces cerevisiae; Souza MA et al.; The glucose-induced activation of plasma membrane ATPase from Saccharomyces cerevisiae was first described by Serrano in 1983 . Many aspects of this signal transduction pathway are still obscure . In this paper, evidence is presented for the involvement of Snf3p as the glucose sensor related to this activation process . It is shown that, in addition to glucose detection by Snf3p, sugar transport is also necessary for activation of the ATPase . The participation of the G protein, Gpa2p, in transducing the internal signal (phosphorylated sugars) is also demonstrated . Moreover, the involvement of protein kinase C in the regulation of ATPase activity is confirmed . Finally, a model pathway is presented for sensing and transmission of the glucose activation signal of the yeast H(+)-ATPase. Nucleic Acids Res, 2001 Oct 1, 29(19), 4043 - 51 Specific distribution of the Saccharomyces cerevisiae linker histone homolog HHO1p in the chromatin; Freidkin I et al.; In virtually all eukaryotic organisms, linker DNA between nucleosomes is associated with a histone termed linker histone or histone H1 . In Saccharomyces cerevisiae, HHO1 encodes a putative linker histone with very significant homology to histone H1 . The encoded protein is expressed in the nucleus, but has not been shown to affect global chromatin structure, nor has its deletion shown any detectable phenotype . In vitro chromatin assembly experiments with recombinant HHO1p have shown that it is able to complex with dinuncleosomes in a similar manner to histone H1 . Here we report that while disruption of HHO1 has little affect on RNA levels of most cellular transcripts, there are numerous exceptions . Measurement of HHO1p concentration in the wild-type cell showed a stoichiometry of about one HHO1p molecule per 37 nucleosomes . Localization of HHO1p in the chromatin, using an immunoprecipitation technique, showed preferential HHO1p binding to rDNA sequences . These results suggest that HHO1p may play a similar role to linker histones, but at restricted locations in the chromatin. Biochem Biophys Res Commun, 2001 Oct 5, 287(4), 837 - 42 Properties of the cysteine-less Pho84 phosphate transporter of Saccharomyces cerevisiae; Berhe A et al.; The derepressible Pho84 high-affinity phosphate permease of Saccharomyces cerevisiae, encoded by the PHO84 gene belongs to a family of phosphate:proton symporters (PHS) . The protein contains 12 native cysteine residues of which five are predicted to be located in putative transmembrane regions III, VI, VIII, IX, and X, and the remaining seven in the hydrophilic domains of the protein.Here we report on the construction of a Pho84 transporter devoid of cysteine residues (C-less) in which all 12 native residues were replaced with serines using PCR mutagenesis and the functional consequences of this . Our results clearly demonstrate that the C-less Pho84 variant is able to support growth of yeast cells to the same extent as the wild-type Pho84 and is stably expressed under derepressible conditions and is fully active in proton-coupled phosphate transport across the yeast plasma membrane . Yeast, 2001 Oct, 18(14), 1317 - 30 The HTL1 gene (YCR020W-b) of Saccharomyces cerevisiae is necessary for growth at 37 degrees C, and for the conservation of chromosome stability and fertility; Lanzuolo C et al.; A small 78 codon ORF, named HTL1 (Chen et al., unpublished results), situated between loci MAK31 and HSP30 on chromosome III of Saccharomyces cerevisiae, is required for growth at 37 degrees C . In this communication, we characterize the ORF and show that disruption of HTL1, besides preventing growth at 37 degrees C, causes genetic and/or epigenetic instability at 26 degrees C: ploidy increases in about 10% of cells grown from individual disruptants and a fraction of disruptant clones are predestined to a rapid and progressive loss of fertility during growth at 26 degrees C . Yeast, 2001 Oct, 18(14), 1309 - 16 Human pancreatic glucokinase (GlkB) complements the glucose signalling defect of Saccharomyces cerevisiae hxk2 mutants; Mayordomo I et al.; Human pancreatic glucokinase (GlkB, hexokinase IV) has been expressed in Saccharomyces cerevisiae . The recombinant protein showed similar enzyme kinetics to those described for the original enzyme . When expressed in hxk2 yeast mutants, GlkB complemented both the glucose induction and the glucose repression defects present in the mutant . It was also functional in regulating the activity of the Snf1 kinase complex in response to glucose, participating in the regulation of the Reg1/Glc7 phosphatase complex, as its yeast counterpart . BMC Mol Biol . 2001;2(1):9 . Epub 2001 Aug 24. Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae; Urakov VN et al.; BACKGROUND: Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3 . eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl - dependent manner . Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough . RESULTS: We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state . This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes . Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons . Itt1p interacts in vitro with both eRFl and eRF3 . Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p . CONCLUSIONS: The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast . This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation. J Biol Chem, 2001 Nov 23, 276(47), 43824 - 8 Epub 2001 Sep 21. Structure of DNA polymerase delta from Saccharomyces cerevisiae; Johansson E et al.; DNA polymerase delta (Pol delta) from Saccharomyces cerevisiae consists of three subunits, Pol3 (125 kDa), Pol31 (55 kDa), and Pol32 (40 kDa), present at a 1:1:1 stoichiometry in purified preparations . Previously, based on gel filtration studies of Pol delta, we suggested that the enzyme may be a dimer of catalytic cores, with dimerization mediated by the Pol32 subunit (Burgers, P . M., and Gerik, K . J . (1998) J . Biol . Chem . 273, 19756-19762) . We now report on extensive gel filtration, glycerol gradient sedimentation, and analytical equilibrium centrifugation studies of Pol delta and of several subassemblies of Pol delta . The hydrodynamic parameters of these assemblies indicate that (i) Pol32 is a rod-shaped protein with a frictional ratio f/f(0) = 2.22; (ii) any complex containing Pol32 also has an extremely asymmetric shape; (iii) the results of these studies are independent of concentration (varied between 0.1-20 microm); (iv) all complexes are monomeric under the conditions studied (up to 20 microm) . Moreover, a two-hybrid analysis of the Pol32 subunit did not detect a Pol32-Pol32 interaction in vivo . Therefore, we conclude that the assembly structure of Pol delta is that of a monomer. Biomol Eng, 2001 Oct 15, 18(3), 135 - 42 The major exoglucanase secreted by Saccharomyces cerevisiae as a model to study protein glycosylation; Larriba G et al.; The major yeast exoglucanase (ExgIb) consists of a 408 amino acid polypeptide carrying two short N-linked oligosaccharides attached to asparagines 165 (Asn(165)) and 325 (Asn(325)) . These oligosaccharides are very similar, in both length and composition, to those present in the vacuolar protease carboxypeptidase Y . Minor glycoforms of exoglucanase arise by underglycosylation of the protein precursor (Exg(165) and Exg(325)) or by elongation of the second oligosaccharide (ExgIa) . The fact that these glycoforms can be readily separated and identified by HPLC and/or Western blots converts ExgI in an excellent model to study the role of the several components or branches of the precursor oligosaccharide in the efficiency and selectivity of the oligosaccharidyl transferase in vivo . We have found that the presence of a single glucose attached to Dol-PP-GlcNAc(2)-Man(9) increases the efficiency of transfer of that oligosaccharide to the protein acceptor . Also, the glucotriose unit appears to be involved in the selection of the sequons to be occupied, in such a way that its absence results in a bias towards the glycosylation of a particular sequon . Finally, we have shown the transfer of GlcNAc(2) from Dol-PP-GlcNAc(2) to exoglucanase, an indication that this intermediate is able to translocate from the cytoplasmic to the lumenal face of the endoplasmic reticulum membrane. Biochim Biophys Acta, 2001 Sep 28, 1533(2), 81 - 5 Degradation of conjugated linoleic acid isomers in the yeast Saccharomyces cerevisiae; Gurvitz A et al.; Propagation of Saccharomyces cerevisiae cells in conjugated linoleic acid (CLA) medium resulted in activation of the transcriptional machinery that responds to fatty acids . Cells utilized efficiently trans-10,cis-12 CLA, but not the corresponding cis-9,trans-11 isomer, probably due to the formation of cis-3,trans-5-dienoyl-CoA intermediates that are recalcitrant to beta-oxidation. Electrophoresis, 2001 Aug, 22(14), 2955 - 68 Identification of peroxisomal membrane proteins of Saccharomyces cerevisiae by mass spectrometry; Schafer H et al.; The identification of peroxisomal membrane proteins is very important to understand the import mechanisms of substrates and proteins into these organelles and the pathogenesis of human peroxisomal disorders like the Zellweger Syndrom . Peroxisomal membrane proteins were identified after separation by gel electrophoresis, tryptic digestion and mass spectrometric analysis . Using matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and nanoliquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), it was possible to identify 45 proteins of isolated yeast peroxisomal membranes. Biochem J, 2001 Oct 1, 359(Pt 1), 193 - 201 Regulatory elements in the FBP1 promoter respond differently to glucose-dependent signals in Saccharomyces cerevisiae; Zaragoza O et al.; In Saccharomyces cerevisiae expression of the fructose-1,6-bisphosphatase-encoding gene, FBP1, is controlled by glucose through the upstream activating sequences UAS1 and UAS2 and the upstream repressing sequence URS1 in its promoter . We have studied the regulation of the proteins that could bind to these elements . We have investigated the role of the putative transcription factors Cat8 and Sip4 in the formation of specific DNA-protein complexes with UAS1 and UAS2, and in the expression of UAS1-lacZ and UAS2-lacZ . The expression of CAT8-lacZ and SIP4-lacZ has been also measured in mig1, tup1 or hxk2 mutants, partially refractory to catabolite repression . We conclude that there is no strict correlation between Cat8 and Sip4 expression or in vitro formation of DNA-protein complexes and expression of UAS1-lacZ and UAS2-lacZ . The URS1 element binds the regulatory protein Mig1, which blocks transcription by recruiting the proteins Cyc8 and Tup1 . The pattern of complexes of URS1 with nuclear extracts was dependent on the carbon source and on Cyc8, but not on Tup1; it was also affected by the protein kinase Snf1 and by the exportin Msn5 . The repression caused by URS1 in a fusion gene was dependent on Mig1, Cyc8 and Tup1, and on the carbon source in the medium; in a snf1 strain the repression observed was independent of the carbon source . Expression of Mig1 could occur in the absence of Snf1 and was moderately sensitive to glucose . We present data showing that different elements of the regulatory system controlling FBP1 responded differently to the concentration of glucose in the medium. Mol Cells, 2001 Aug 31, 12(1), 77 - 83 Characterization of the CDC10 product and the timing of events of the budding site of Saccharomyces cerevisiae; Jeong JW et al.; Budding cells of the yeast Saccharomyces cerevisiae possess a ring of septin filaments of unknown biochemical nature that lies under the inner surface of the plasma membrane in the neck that connects the mother cell to its bud . Mutants, defective in any of the four genes (CDC3, CDC10, CDC11, CDC12), lack these septin filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis . The cloned CDC10 was fused to bacterial genes to generate antibodies specific for the CDC10 product was a constituent of the septin filaments . Cdc10p-specific antibodies for septin staining and actin-specific rhodamine-phalloidine were used to investigate the timing of the localization of septin and actin at the budding site using the immunofluorescence microscopic technique . In wild-type cells, the timing of the appearance and disappearance of these proteins was indistinguishable . In addition, the cdc10 mutant did not prevent actin localization at the budding site . The mutant that was blocked in the actin function also did not prevent the septin localization of the Cdc10p . This result may suggest an organizational independence between these proteins in the bud formation . Finally, the localization of septin and actin in the cdc24 mutant cell was examined . It was found that the CDC24 function was necessary for the organization of septin and actin at the budding site. Yeast, 2001 Sep 30, 18(13), 1207 - 16 A PBS2 homologue from Debaryomyces hansenii shows a differential effect on calcofluor and polymyxin B sensitivity in Saccharomyces cerevisiae; Bansal PK et al.; The PBS2 gene encodes a MAP kinase kinase that plays a pivotal role in osmosensing signal-transduction pathway in the yeast Saccharomyces cerevisiae . Mutation in the PBS2 gene has a pleotropic effect . Besides being osmosensitive, pbs2 mutants show altered sensitivity to polymyxin B and calcofluor . Recent studies revealed that Pbs2p plays a different role in osmoadaptation and calcofluor sensitivity . We have isolated a gene homologous to PBS2 from the highly salt-tolerant yeast Debaryomyces hansenii by phenotypic complementation . DNA sequencing of the clone revealed that the gene encoded a protein of 683 amino acid residues . Like Pbs2p, this protein also has a proline-rich motif . Further characterization revealed that this gene could complement polymyxin B sensitivity but did not affect calcofluor sensitivity . Thus, it appeared that Pbs2p also has an independent role in these two physiological processes . The GenBank Accession No . of this sequence is AF371315 . Genetics, 2001 Sep, 159(1), 77 - 89 The Ras/PKA signaling pathway of Saccharomyces cerevisiae exhibits a functional interaction with the Sin4p complex of the RNA polymerase II holoenzyme; Howard SC et al.; Saccharomyces cerevisiae cells enter into the G(0)-like resting state, stationary phase, in response to specific types of nutrient limitation . We have initiated a genetic analysis of this resting state and have identified a collection of rye mutants that exhibit a defective transcriptional response to nutrient deprivation . These transcriptional defects appear to disrupt the control of normal growth because the rye mutants are unable to enter into a normal stationary phase upon nutrient deprivation . In this study, we examined the mutants in the rye1 complementation group and found that rye1 mutants were also defective for stationary phase entry . Interestingly, the RYE1 gene was found to be identical to SIN4, a gene that encodes a component of the yeast Mediator complex within the RNA polymerase II holoenzyme . Moreover, mutations that affected proteins within the Sin4p module of the Mediator exhibited specific genetic interactions with the Ras protein signaling pathway . For example, mutations that elevated the levels of Ras signaling, like RAS2(val19), were synthetic lethal with sin4 . In all, our data suggest that specific proteins within the RNA polymerase II holoenzyme might be targets of signal transduction pathways that are responsible for coordinating gene expression with cell growth. Genetics, 2001 Sep, 159(1), 65 - 75 Spontaneous frameshift mutations in Saccharomyces cerevisiae: accumulation during DNA replication and removal by proofreading and mismatch repair activities; Greene CN et al.; The accumulation of frameshift mutations during DNA synthesis is determined by the rate at which frameshift intermediates are generated during DNA polymerization and the efficiency with which frameshift intermediates are removed by DNA polymerase-associated exonucleolytic proofreading activity and/or the postreplicative mismatch repair machinery . To examine the relative contributions of these factors to replication fidelity in Saccharomyces cerevisiae, we determined the reversion rates and spectra of the lys2 Delta Bgl +1 frameshift allele . Wild-type and homozygous mutant diploid strains with all possible combinations of defects in the exonuclease activities of DNA polymerases delta and epsilon (conferred by the pol3-01 and pol2-4 alleles, respectively) and in mismatch repair (deletion of MSH2) were analyzed . Although there was no direct correlation between homopolymer run length and frameshift accumulation in the wild-type strain, such a correlation was evident in the triple mutant strain lacking all repair capacity . Furthermore, examination of strains defective in one or two repair activities revealed distinct biases in the removal of the corresponding frameshift intermediates by exonucleolytic proofreading and/or mismatch repair . Finally, these analyses suggest that the mismatch repair machinery may be important for generating some classes of frameshift mutations in yeast. Genetics, 2001 Sep, 159(1), 35 - 45 Roles for internal and flanking sequences in regulating the activity of mating-type-silencer-associated replication origins in Saccharomyces cerevisiae; Sharma K et al.; ARS301 and ARS302 are inactive replication origins located at the left end of budding yeast (Saccharomyces cerevisiae) chromosome III, where they are associated with the HML-E and -I silencers of the HML mating type cassette . Although they function as replication origins in plasmids, they do not serve as origins in their normal chromosomal locations, because they are programmed to fire so late in S phase that they are passively replicated by the replication fork from neighboring early-firing ARS305 before they have a chance to fire on their own . We asked whether the nucleotide sequences required for plasmid origin function of these silencer-associated chromosomally inactive origins differ from the sequences needed for plasmid origin function by nonsilencer-associated chromosomally active origins . We could not detect consistent differences in sequence requirements for the two types of origins . Next, we asked whether sequences within or flanking these origins are responsible for their chromosomal inactivity . Our results demonstrate that both flanking and internal sequences contribute to chromosomal inactivity, presumably by programming these origins to fire late in S phase . In ARS301, the function of the internal sequences determining chromosomal inactivity is dependent on the checkpoint proteins Mec1p and Rad53p. Genetics, 2001 Sep, 159(1), 17 - 33 Mutations in SID2, a novel gene in Saccharomyces cerevisiae, cause synthetic lethality with sic1 deletion and may cause a defect during S phase; Jacobson MD et al.; SIC1 encodes a nonessential B-type cyclin/CDK inhibitor that functions at the G1/S transition and the exit from mitosis . To understand more completely the regulation of these transitions, mutations causing synthetic lethality with sic1 Delta were isolated . In this screen, we identified a novel gene, SID2, which encodes an essential protein that appears to be required for DNA replication or repair . sid2-1 sic1 Delta strains and sid2-21 temperature-sensitive strains arrest preanaphase as large-budded cells with a single nucleus, a short spindle, and an approximately 2C DNA content . RAD9, which is necessary for the DNA damage checkpoint, is required for the preanaphase arrest of sid2-1 sic1 Delta cells . Analysis of chromosomes in mutant sid2-21 cells by field inversion gel electrophoresis suggests the presence of replication forks and bubbles at the arrest . Deleting the two S phase cyclins, CLB5 and CLB6, substantially suppresses the sid2-1 sic1 Delta inviability, while stabilizing Clb5 protein exacerbates the defects of sid2-1 sic1 Delta cells . In synchronized sid2-1 mutant strains, the onset of replication appears normal, but completion of DNA synthesis is delayed . sid2-1 mutants are sensitive to hydroxyurea indicating that sid2-1 cells may suffer DNA damage that, when combined with additional insult, leads to a decrease in viability . Consistent with this hypothesis, sid2-1 rad9 cells are dead or very slow growing even when SIC1 is expressed. Eur J Biochem, 2001 Sep, 268(18), 4860 - 7 Neurotensin induces mating in Saccharomyces cerevisiae cells that express human neurotensin receptor type 1 in place of the endogenous pheromone receptor; Leplatois P et al.; Heterologous expression of the human neurotensin receptor type I (hNT1-R) has been achieved in the yeast Saccharomyces cerevisiae . Immunoanalysis of membranes prepared from cells expressing a c-myc-tagged version of hNT1-R revealed multiple c-myc cross-reacting polypeptides of high molecular mass, suggesting that hNT1-R was glycosylated in yeast . High-affinity binding sites for 125I-labeled-{monoiodo-Tyr3}neurotensin ({125I-Tyr3}NT) were detected on hNT1-R-expressing cells with Kd and Bmax values of 3.2 nM and of 500 receptors per cell, respectively . Competition binding studies of neurotensin with SR142948 and SR48692, two nonpeptidic antagonists of hNT1-R, indicated that the yeast-produced recombinant receptor displayed the same pharmacological properties as hNT1-R expressed in mammalian cells . Interestingly, neurotensin activated the pheromone pathway in hNT1-R-expressing cells in a dose-dependent fashion, as revealed by a beta-galactosidase activity assay with a pheromone-responsive Fus1:lacZ construct . Mutational inactivation of the SST2 and STE2 genes increased the level of beta-galactosidase activity in response to neurotensin by twofold . Recombinant hNT1-R-producing cells, which lacked the endogenous G-protein-coupled receptor for the alpha pheromone, mated with wild-type MATalpha haploid cells in response to neurotensin, leading to bona fide diploid zygote formation . This is the first report of a mammalian receptor that can replace the endogenous pheromone receptor when produced in yeast, by signaling a fully effective, agonist-induced, mating process. Mikrobiologiia, 2001 Jul-Aug, 70(4), 531 - 5 {Heat shock-induced changes in the respiration of the yeast Saccharomyces cerevisiae}; Rikhvanov EG et al.; The incubation of Saccharomyces cerevisiae at elevated temperature (45 degrees C) stimulated the respiration of yeast cells and decreased their survival rate . The respiration-deficient mutant of this yeast was found to be more tolerant to the elevated temperature than the wild-type strain . At the same time, the cultivation of the wild-type strain in an ethanol-containing medium enhanced the respiration, catalase activity, and thermotolerance of yeast cells, as compared with their growth in a glucose-containing medium . It is suggested that the enhanced respiration of yeast cells at 45 degrees C leads to an intense accumulation of reactive oxygen species, which may be one of the reasons for the heat shock-induced cell death. J Biol Chem, 2001 Nov 23, 276(47), 43548 - 56 Epub 2001 Sep 13. The membrane proteins, Spt23p and Mga2p, play distinct roles in the activation of Saccharomyces cerevisiae OLE1 gene expression . Fatty acid-mediated regulation of Mga2p activity is independent of its proteolytic processing into a soluble transcription activator; Chellappa R et al.; The Saccharomyces OLE1 gene encodes the Delta-9 fatty acid desaturase, an enzyme that converts saturated fatty acyl-CoAs into cis-Delta-9 unsaturated fatty acids . OLE1 gene expression is regulated by unsaturated fatty acids, which repress transcription and destabilize the OLE1 mRNA . Expression of OLE1 is activated by N-terminal proteolytic fragments of two homologous endoplasmic reticulum membrane proteins, Spt23p and Mga2p . Disruption of either gene does not significantly affect cell growth or fatty acid metabolism; cells that contain null alleles of both genes, however, are unsaturated fatty acid auxotrophs . An analysis of spt23Delta and mga2Delta strains shows that Spt23p and Mga2p differentially activate and regulate OLE1 transcription . In glucose-grown cells, both genes activate transcription to similar levels of activity . Expressed alone, Mga2p induces high levels of OLE1 transcription in cells exposed to cobalt or grown in glycerol-containing medium . Spt23p expressed alone activates OLE1 transcription to levels similar to those in wild type cells . OLE1 expression is strongly repressed by unsaturated fatty acids in spt23Delta or mga2Delta cells, under all growth conditions . To test if OLE1 expression is controlled by fatty acids at the level of membrane proteolysis, soluble N-terminal fragments of Spt23p and Mga2p that lack their membrane-spanning regions (Deltatm) were expressed under the control of their native promoters in spt23Delta;mga2Delta cells . Under those conditions, Mga2pDeltatm acts as a powerful transcription activator that is strongly repressed by unsaturated fatty acids . By comparison, the Spt23pDeltatm polypeptide weakly activates transcription and shows little regulation by unsaturated fatty acids . Co-expression of the two soluble fragments results in activation to levels observed with the Mga2pDeltatm protein alone . The fatty acid repression of transcription under those conditions is attenuated by Spt23Deltatm, however, suggesting that the two proteins may interact to modulate OLE1 gene expression. Arch Biochem Biophys, 2001 Sep 15, 393(2), 207 - 14 The functional role of conserved acidic residues of the Qcr7 protein of the cytochrome bc(1) complex in Saccharomyces cerevisiae; Lee SY et al.; The 14-kDa Qcr7 protein represents one of the 10 subunits that are components of a functional cytochrome bc(1) complex in Sacharomyces cerevisiae . Previous studies have shown that the N-terminus of the Qcr7 protein may be involved in the assembly of the cytochrome bc(1) complex and its C-terminus by interacting with cytochrome b and QCR8 proteins . It has also been suggested that Qcr7 protein may be involved in proton pumping . The coding sequence for two highly conserved aspartate residues, D46 and D47, in the QCR7 gene was altered by site-directed mutagenesis and the mutated genes expressed in cells lacking a functional QCR7 gene . Mutants D46E, D46G, D46N, and D47E were comparable to wild type in growth phenotype on nonfermentable carbon sources . Mutants D47G and D47N were respiratory deficient and analysis of complex components by immunoblotting and spectral analysis of cytochrome b suggests defective assembly . Despite being respiratory competent and having normal electron transport rates in broken mitochondria, the mutant D46G had markedly reduced ATP synthesis from electron transport reactions catalyzed by complexes II plus III of the respiratory chain . This suggests that the geometry of proton uptake by the bc(1) complex is disturbed by the mutation in D46 . Hum Mol Genet, 2001 Sep 1, 10(18), 1889 - 900 Functional analysis of human MLH1 and MSH2 missense variants and hybrid human-yeast MLH1 proteins in Saccharomyces cerevisiae; Ellison AR et al.; Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant inherited disease caused by defects in the process of DNA mismatch repair (MMR), and mutations in the hMLH1 or hMSH2 genes are responsible for the majority of HNPCC . In addition to clear loss-of-function mutations conferred by nonsense or frameshift alterations in the coding sequence or by splice variants, genetic screening has revealed a large number of missense codons with less obvious functional consequences . The ability to discriminate between a loss-of-function mutation and a silent polymorphism is important for genetic testing for inherited diseases like HNPCC where the opportunity exists for early diagnosis and preventive intervention . In this study, quantitative in vivo DNA MMR assays in the yeast Saccharomyces cerevisiae were performed to determine the functional significance of amino acid replacements observed in the human population . Missense codons previously observed in human genes were introduced at the homologous residue in the yeast MLH1 or MSH2 genes . This study also demonstrated feasibility of constructing genes that encode functional hybrid human-yeast MLH1 proteins . Three classes of missense codons were found: (i) complete loss of function, i.e . mutations; (ii) variants indistinguishable from wild-type protein, i.e . silent polymorphisms; and (iii) functional variants which support MMR at reduced efficiency, i.e . efficiency polymorphisms . There was a good correlation between the functional results in yeast and available human clinical data regarding penetrance of the missense codon . The results reported here raise the intriguing possibility that differences in the efficiency of DNA MMR exist between individuals in the human population due to common polymorphisms. Genes Cells, 2001 Sep, 6(9), 775 - 88 PKC1, a protein kinase C homologue of Saccharomyces cerevisiae, participates in microtubule function through the yeast EB1 homologue, BIM1; Hosotani T et al.; BACKGROUND: RSC is a chromatin-remodelling complex of Saccharomyces cerevisiae and essential for growth . Its catalytic subunit is encoded by the NPS1/STH1 gene . At the present time, little is known regarding the cellular function of RSC . RESULTS: To identify genes with functions related to NPS1, we screened high-copy suppressor genes for the temperature- and thiabendazole (TBZ)-sensitive mutant allele of NPS1, nps1-105 . Amongst the suppressors we cloned PKC1/STT1 and BIM1 that encoded a homologue of mammalian protein kinase C and a conserved microtubule binding protein homologous to human EB1, respectively . Both the temperature sensitive mutation of PKC1, stt1, and the bim1 null mutation caused synthetic growth defects with nps1-105 . A genetic analysis of the functional relationships between these genes revealed that PKC1 suppressed the defect of nps1-105 through the BIM1 function but not by the activation of the MPK1/MAPK pathway . The stt1 mutation alone showed TBZ sensitivity and delayed the G2-phase progression at semi-permissive temperatures . Both of these stt1 phenotypes were suppressed by the over-expression of BIM1 . In addition, stt1 as well as nps1-105, mis-segregated a mini-chromosome at frequencies higher than the wild-type at a permissive temperature . The mis-segregation was enhanced in the nps1-105 stt1 double mutant . CONCLUSION: These results suggest that Pkc1p plays a role which is relevant to microtubule functions and that this role is mediated by a hitherto unknown PKC signalling pathway and by Bim1p Radiat Res, 2001 Oct, 156(4), 379 - 87 Cell cycle arrest determines the intensity of the global transcriptional response of Saccharomyces cerevisiae to ionizing radiation; De Sanctis V et al.; Whole-genome analysis was performed using DNA microarrays to define the changes in the gene expression patterns occurring in Saccharomyces cerevisiae cells exposed to ionizing radiation . The effects of sublethal dose on wild-type, rad53 (enhanced sensitivity to radiation and impaired in a cell cycle damage checkpoint), and rad6 (enhanced sensitivity to radiation and functional cell cycle block by radiation) mutant backgrounds and of a higher dose on the wild-type and G(2)-phase-arrested cells were analyzed . Several gene pathways were identified as being implicated in the response to radiation . In particular, the cell cycle blockage that occurred in the wild-type strain after a high radiation dose and in the rad6 mutant after a lower dose entailed modifications of defined gene expression patterns, which are described here and are compared with the gene modulation patterns observed in the rad53 strain in the absence of efficient blockage . Loss of the RAD53 function caused a major increase in the number of genes modulated by radiation . Given that Rad53-Sad1p, the protein encoded by RAD53, has functions other than those directly connected to cell cycle arrest, we determined the gene patterns that were modulated upon irradiation of rad53 cells that had been forced to arrest in G(2) phase by nocodazole treatment . These differential whole-genome analyses shed light on the multiplicity of functions of the pivotal Rad53-Sad1p protein . The results obtained describe how the cells respond to different irradiation conditions by modulating important gene classes, including those associated with stress defense, ribosomal proteins, histones, ergosterol and GCR1-controlled sugar metabolism. Mol Biol Cell, 2001 Sep, 12(9), 2646 - 59 ADY1, a novel gene required for prospore membrane formation at selected spindle poles in Saccharomyces cerevisiae; Deng C et al.; ADY1 is identified in a genetic screen for genes on chromosome VIII of Saccharomyces cerevisiae that are required for sporulation . ADY1 is not required for meiotic recombination or meiotic chromosome segregation, but it is required for the formation of four spores inside an ascus . In the absence of ADY1, prospore formation is restricted to mainly one or two spindle poles per cell . Moreover, the two spores in the dyads of the ady1 mutant are predominantly nonsisters, suggesting that the proficiency to form prospores is not randomly distributed to the four spindle poles in the ady1 mutant . Interestingly, the meiosis-specific spindle pole body component Mpc54p, which is known to be required for prospore membrane formation, is localized predominantly to only one or two spindle poles per cell in the ady1 mutant . A partially functional Myc-Pfs1p is localized to the nucleus of mononucleate meiotic cells but not to the spindle pole body or prospore membrane . These results suggest that Pfs1p is specifically required for prospore formation at selected spindle poles, most likely by ensuring the functionality of all four spindle pole bodies of a cell during meiosis II. Mol Biol Cell, 2001 Sep, 12(9), 2614 - 28 Aux1p/Swa2p is required for cortical endoplasmic reticulum inheritance in Saccharomyces cerevisiae; Du Y et al.; In the yeast Saccharomyces cerevisiae, the endoplasmic reticulum (ER) is found at the periphery of the cell and around the nucleus . The segregation of ER through the mother-bud neck may occur by more than one mechanism because perinuclear, but not peripheral ER, requires microtubules for this event . To identify genes whose products are required for cortical ER inheritance, we have used a Tn3-based transposon library to mutagenize cells expressing a green fluorescent protein-tagged ER marker protein (Hmg1p) . This approach has revealed that AUX1/SWA2 plays a role in ER inheritance . The COOH terminus of Aux1p/Swa2p contains a J-domain that is highly related to the J-domain of auxilin, which stimulates the uncoating of clathrin-coated vesicles . Deletion of the J-domain of Aux1p/Swa2p leads to vacuole fragmentation and membrane accumulation but does not affect the migration of peripheral ER into daughter cells . These findings suggest that Aux1p/Swa2p may be a bifunctional protein with roles in membrane traffic and cortical ER inheritance . In support of this hypothesis, we find that Aux1p/Swa2p localizes to ER membranes. Curr Biol, 2001 Sep 4, 11(17), 1328 - 35 The role of the Mre11-Rad50-Xrs2 complex in telomerase- mediated lengthening of Saccharomyces cerevisiae telomeres; Tsukamoto Y et al.; BACKGROUND: The Saccharomyces Mre11p, Rad50p, and Xrs2p proteins form a complex, called the MRX complex, that is required to maintain telomere length . Cells lacking any one of the three MRX proteins and Mec1p, an ATM-like protein kinase, undergo telomere shortening and ultimately die, phenotypes characteristic of cells lacking telomerase . The other ATM-like yeast kinase, Tel1p, appears to act in the same pathway as MRX: mec1 tel1 cells have telomere phenotypes similar to those of telomerase-deficient cells, whereas the phenotypes of tel1 cells are not exacerbated by the loss of a MRX protein . RESULTS: The nuclease activity of Mre11p was found to be dispensable for the telomerase-promoting activity of the MRX complex . The association of the single-stranded TG1-3 DNA binding protein Cdc13p with yeast telomeres occurred efficiently in the absence of Tel1p, Mre11p, Rad50p, or Xrs2p . Targeting of catalytically active telomerase to the telomere suppressed the senescence phenotype of mec1 mrx or mec1 tel1 cells . Moreover, when telomerase was targeted to telomeres, telomere lengthening was robust in mec1 mrx and mec1 tel1 cells . CONCLUSIONS: These data rule out models in which the MRX complex is necessary for Cdc13p binding to telomeres or in which the MRX complex is necessary for the catalytic activity of telomerase . Rather, the data suggest that the MRX complex is involved in recruiting telomerase activity to yeast telomeres. J Biol Chem, 2001 Nov 9, 276(45), 41906 - 12 Epub 2001 Sep 10. Saccharomyces cerevisiae Dmc1 protein promotes renaturation of single-strand DNA (ssDNA) and assimilation of ssDNA into homologous super-coiled duplex DNA; Hong EL et al.; Dmc1 and Rad51 are eukaryotic RecA homologues that are involved in meiotic recombination . The expression of Dmc1 is limited to meiosis, whereas Rad51 is expressed in mitosis and meiosis . Dmc1 and Rad51 have unique and overlapping functions during meiotic recombination . Here we report the purification of the Dmc1 protein from the budding yeast Saccharomyces cerevisiae and present basic characterization of its biochemical activity . The protein has a weak DNA-dependent ATPase activity and binds both single-strand DNA (ssDNA) and double-strand DNA . Electrophoretic mobility shift assays suggest that DNA binding by Dmc1 is cooperative . Dmc1 renatures linearized plasmid DNA with first order reaction kinetics and without requiring added nucleotide cofactor . In addition, Dmc1 catalyzes strand assimilation of ssDNA oligonucleotides into homologous supercoiled duplex DNA in a reaction promoted by ATP or the non-hydrolyzable ATP analogue AMP-PNP. Epidemiol Mikrobiol Imunol, 2001 Aug, 50(3), 131 - 5 {Initial experience with detection of Saccharomyces cerevisiae antibodies in patients with primary nonspecific inflammatory bowel disease}; Malickova K et al.; INTRODUCTION: Inflammatory bowel diseases (IBD), with Crohn's disease (CD) and ulcerative colitis (UC) as the two main disorders, is a heterogeneous group of diseases of unknown etiology . Actually we have no ideal disease marker, to identify people at risk of the disease, which can differentiate CD from UC, be highly specific for CD or UC and easily applicable in routine laboratory praxis . AIMS: Determine the clinical significance of serological testing p-ANCA and ASCA in patients with IBD . METHODS: P-ANCA in IgG isotype were detected by indirect fluorescence assay on human ethanol-fixed granulocytes, ASCA antibodies in IgG and IgA isotypes were determined by ELISA with mannan as a target antigen . RESULTS: P-ANCA and ASCA were studied in a group of 86 patients (38 CD, 26 UC, 3 non-inflammatory gastrointestinal disorder, 19 health controls) . P-ANCA was associated with UC in 46% . ASCA was associated with CD in 76% . Specificity of ANCA for UC compared to healthy controls was 100%, specificity of ASCA for CD compared to healthy controls was 89.5% . CONCLUSION: Although the sensitivity of ASCA and p-ANCA is low, their specificity is high, especially when combining these two markers . We think that combined assay for ASCA and p-ANCA is more useful in IBD. J Biomed Sci, 2001 Sep, 8(5), 416 - 20 Functional correlation between the nuclear localization of Fht1p and its flocculation and heat tolerance activities in budding yeast Saccharomyces cerevisiae; Iha H et al.; Fht1p is involved in the flocculation and heat tolerance machinery of budding yeast Saccharomyces cerevisiae . Despite knowledge of its involvement in those phenotypes, a precise mechanism has yet to be discovered . To this end, we monitored the relationship between subcellular localization of Fht1p and its flocculation or heat tolerance function using newly developed expression vectors with a recombinant green fluorescent protein (GFP; S65T/S147P) of Aequorea victoria added at both the N- and C-terminus of Fht1p . The main fluorescent signal of the GFP tagged with either a wild-type Fht1p or mutants which preserve their flocculation function was detected in the nucleus, whereas signals of functionless mutants were dispersed to the cytoplasm . Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 513 - 6 Coupling effects of osmotic pressure and temperature on the viability of Saccharomyces cerevisiae; Beney L et al.; The osmotic tolerance of cells of Saccharomyces cerevisiae as a function of glycerol concentration and temperature has been investigated . Results show that under isothermal conditions (25 degrees C) cells are resistant (94% viability) to hyperosmotic treatment at 49.2 MPa . A thigher osmotic pressure, cell viability decreases to 25% at 99 MPa . Yeast resistance to high osmotic stress (99 Mpa) is enhanced at low temperatures (5-11 degrees C) . Therefore, the temperature at which hyperosmotic pressure is achieved greatly affects cell viability . These results suggest that temperature control is a suitable means of enhancing cell survival in response to osmotic dehydration. J Biol Chem, 2001 Nov 9, 276(45), 41769 - 81 Epub 2001 Sep 06. Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p; Dodt G et al.; PEX5 functions as an import receptor for proteins with the type-1 peroxisomal targeting signal (PTS1) . Although PEX5 is not involved in the import of PTS2-targeted proteins in yeast, it is essential for PTS2 protein import in mammalian cells . Human cells generate two isoforms of PEX5 through alternative splicing, PEX5S and PEX5L, and PEX5L contains an additional insert 37 amino acids long . Only one isoform, PEX5L, is involved in PTS2 protein import, and PEX5L physically interacts with PEX7, the import receptor for PTS2-containing proteins . In this report we map the regions of human PEX5L involved in PTS2 protein import, PEX7 interaction, and targeting to peroxisomes . These studies revealed that amino acids 1-230 of PEX5L are required for PTS2 protein import, amino acids 191-222 are sufficient for PEX7 interaction, and amino acids 1-214 are sufficient for targeting to peroxisomes . We also identified a 21-amino acid-long peptide motif of PEX5L, amino acids 209-229, that overlaps the regions sufficient for full PTS2 rescue activity and PEX7 interaction and is shared by Saccharomyces cerevisiae Pex18p and Pex21p, two yeast peroxins that act only in PTS2 protein import in yeast . A mutation in PEX5 that changes a conserved serine of this motif abrogates PTS2 protein import in mammalian cells and reduces the interaction of PEX5L and PEX7 in vitro . This peptide motif also lies within regions of Pex18p and Pex21p that interact with yeast PEX7 . Based on these and other results, we propose that mammalian PEX5L may have acquired some of the functions that yeast Pex18p and/or Pex21p perform in PTS2 protein import . This hypothesis may explain the essential role of PEX5L in PTS2 protein import in mammalian cells and its lack of importance for PTS2 protein import in yeast. Int J Food Microbiol, 2001 Aug 15, 68(1-2), 155 - 60 Release of nitrogen compounds to the extracellular medium by three strains of Saccharomyces cerevisiae during induced autolysis in a model wine system; Martinez-Rodriguez AJ et al.; To detect differences among three strains of Saccharomyces cerevisiae used in the man