S. cerevisiae

Mol Cell, 2000 Sep, 6(3), 593 - 603
Checkpoint-dependent activation of mutagenic repair in Saccharomyces cerevisiae pol3-01 mutants; Datta A et al.; The Saccharomyces cerevisiae DNA polymerase delta proofreading exonuclease-defective mutation pol3-01 is known to cause high rates of accumulating mutations . The pol3-01 mutant was found to have abnormal cell cycle progression due to activation of the S phase checkpoint . Inactivation of the S phase checkpoint suppressed both the pol3-01 cell cycle progression defect and mutator phenotype, indicating that the pol3-01 mutator phenotype was dependent on the S phase damage checkpoint pathway . Epistasis analysis suggested that a portion of the pol3-01 mutator phenotype involves members of the RAD6 epistasis group that function in both error-free and error-prone repair . These results indicate that activation of a checkpoint in response to certain types of replicative defects can result in the accumulation of mutations.

J Cell Biochem, 2000 Sep 18, 80(1), 73 - 84
Initiation of DNA replication in Saccharomyces cerevisiae G1-phase nuclei by Xenopus egg extract; Wu JR et al.; Xenopus egg extracts initiate replication at specific origin sites within mammalian G1-phase nuclei . Similarly, S-phase extracts from Saccharomyces cerevisiae initiate DNA replication within yeast nuclei at specific yeast origin sequences . Here we show that Xenopus egg extracts can initiate DNA replication within G1-phase yeast nuclei but do not recognize yeast origin sequences . When G1-phase yeast nuclei were introduced into Xenopus egg extract, semiconservative, aphidicolin-sensitive DNA synthesis was induced after a brief lag period and was restricted to a single round of replication . The specificity of initiation within the yeast 2 microm plasmid as well as in the vicinity of the chromosomal origin ARS1 was evaluated by neutral two-dimensional gel electrophoresis of replication intermediates . At both locations, replication was found to initiate outside of the ARS element . Manipulation of both cis- and trans-acting elements in the yeast genome before introduction of nuclei into Xenopus egg extract may provide a system with which to elucidate the requirements for vertebrate origin recognition .

Cell Mol Life Sci, 2000 Aug, 57(8-9), 1161 - 71
Molecular basis of nutrient-controlled gene expression in Saccharomyces cerevisiae; Reece RJ; The ability of a unicellular organism to alter patterns of gene expression in response to nutrient availability is essential to its survival in a changing environment . How is the cell able to identify individual metabolites amongst a myriad of other similar molecules, and convert the information of its presence into a concerted change in the transcription of the genes required for the response to that metabolite? There is increasing evidence that the activity of transcription factors can be influenced directly by interaction with metabolites . A variety of mechanisms have been identified by which this type of gene regulation by small molecules can occur.

Mutat Res, 2000 Oct 31, 470(2), 125 - 32
Analysis of deletion mutations of the rpsL gene in the yeast Saccharomyces cerevisiae detected after long-term flight on the Russian space station Mir; Fukuda T et al.; Using the yeast Saccharomyces cerevisiae on board the Russian space station Mir, we studied the effects of long-term space flight on mutation of the bacterial ribosomal protein L gene (rpsL) cloned in a yeast-Escherichia coli shuttle vector . The mutation frequencies of the cloned rpsL gene on the Mir and the ground (control) yeast samples were estimated by transformation of E . coli with the plasmid DNAs recovered from yeast and by assessment of the conversion of the rpsL wild-type phenotype (Sm(S)) to its mutant phenotype (Sm(R)) . After a 40-day space flight, some part of space samples gave mutation frequencies two to three times higher than those of the ground samples . Nucleotide sequence analysis showed no apparent difference in point mutation rates between the space and the ground mutant samples . However, the greater part of the Mir mutant samples were found to have a total or large deletion in the rpsL sequence, suggesting that space radiation containing high-linear energy transfer (LET) might have caused deletion-type mutations.

Biochem Biophys Res Commun, 2000 Oct 5, 276(3), 879 - 84
Expression of ZRC1 coding for suppressor of zinc toxicity is induced by zinc-starvation stress in Zap1-dependent fashion in Saccharomyces cerevisiae; Miyabe S et al.; The ZRC1 gene was cloned as a multicopy suppressor of zinc toxicity in Saccharomyces cerevisiae . Zrc1 belongs to CDF (cation diffusion facilitator) family . The transporters belonging to this family are thought to play an important role in metal detoxification . However, we found that cell growth of zrc1Delta mutant was lowered under the metal-limited conditions, which was restored by zinc . The Zap1 transcription factor is crucial for expression of several genes responsive to zinc-starvation stress . The expression of ZRC1 was induced in Zap1-dependent fashion when the intracellular zinc level was decreased and this induction was repressed by zinc . These results imply an important role of Zrc1 in the zinc-starvation stress .

Proc Natl Acad Sci U S A, 2000 Oct 10, 97(21), 11383 - 90
Inaugural article: global mapping of meiotic recombination hotspots and coldspots in the yeast Saccharomyces cerevisiae; Gerton JL et al.; In the yeast Saccharomyces cerevisiae, meiotic recombination is initiated by double-strand DNA breaks (DSBs) . Meiotic DSBs occur at relatively high frequencies in some genomic regions (hotspots) and relatively low frequencies in others (coldspots) . We used DNA microarrays to estimate variation in the level of nearby meiotic DSBs for all 6,200 yeast genes . Hotspots were nonrandomly associated with regions of high G + C base composition and certain transcriptional profiles . Coldspots were nonrandomly associated with the centromeres and telomeres.

Mol Cell Biol, 2000 Nov, 20(21), 8157 - 67
Regulatory networks revealed by transcriptional profiling of damaged Saccharomyces cerevisiae cells: Rpn4 links base excision repair with proteasomes; Jelinsky SA et al.; Exposure to carcinogenic alkylating agents, oxidizing agents, and ionizing radiation modulates transcript levels for over one third of Saccharomyces cerevisiae's 6,200 genes . Computational analysis delineates groups of coregulated genes whose upstream regions bear known and novel regulatory sequence motifs . One group of coregulated genes contain a number of DNA excision repair genes (including the MAG1 3-methyladenine DNA glycosylase gene) and a large selection of protein degradation genes . Moreover, transcription of these genes is modulated by the proteasome-associated protein Rpn4, most likely via its binding to MAG1 upstream repressor sequence 2-like elements, that turn out to be almost identical to the recently identified proteasome-associated control element (G . Mannhaupt, R . Schnall, V . Karpov, I . Vetter, and H . Feldmann, FEBS Lett . 450:27-34, 1999) . We have identified a large number of genes whose transcription is influenced by Rpn4p.

Mol Cell Biol, 2000 Nov, 20(21), 8093 - 102
Tryptophan permease gene TAT2 confers high-pressure growth in Saccharomyces cerevisiae; Abe F et al.; Hydrostatic pressure in the range of 15 to 25 MPa was found to cause arrest of the cell cycle in G(1) phase in an exponentially growing culture of Saccharomyces cerevisiae, whereas a pressure of 50 MPa did not . We found that a plasmid carrying the TAT2 gene, which encodes a high-affinity tryptophan permease, enabled the cells to grow under conditions of pressure in the range of 15 to 25 MPa . Additionally, cells expressing the Tat2 protein at high levels became endowed with the ability to grow under low-temperature conditions at 10 or 15 degrees C as well as at high pressure . Hydrostatic pressure significantly inhibited tryptophan uptake into the cells, and the Tat2 protein level was down-regulated by high pressure . The activation volume associated with tryptophan uptake was found to be a large positive value, 46.2 +/- 3.85 ml/mol, indicating that there was a net volume increase in a rate-limiting step in tryptophan import . The results showing cell cycle arrest in G(1) phase and down-regulation of the Tat2 protein seem to be similar to those observed upon treatment of cells with the immunosuppressive drug rapamycin . Although rapamycin treatment elicited the rapid dephosphorylation of Npr1 and induction of Gap1 expression, hydrostatic pressure did not affect the phosphorylation state of Npr1 and it decreased the level of Gap1 protein, suggesting that the pressure-sensing pathway may be independent of Npr1 function . Here we describe high-pressure sensing in yeast in comparison with the TOR-signaling pathway and discuss an important factor involved in adaptation of organisms to high-pressure environments.

Mol Cell Biol, 2000 Nov, 20(21), 8047 - 58
Identification of novel Saccharomyces cerevisiae proteins with nuclear export activity: cell cycle-regulated transcription factor ace2p shows cell cycle-independent nucleocytoplasmic shuttling; Jensen TH et al.; Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p . We have carried out a yeast two-hybrid screen with Crm1p as a bait . The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB) . This approach identified three Saccharomyces cerevisiae proteins not previously known to have nuclear export activity . These proteins are the 5' RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W . Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs . Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues . However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity . Ace2p accumulates in the nucleus at the end of mitosis and activates early-G(1)-specific genes . We now provide evidence that Ace2p is nuclear not only in late M-early G(1) but also during other stages of the cell cycle . This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner.

Yeast, 2000 Sep 30, 17(3), 188 - 200
Predicting the function and subcellular location of Caenorhabditis elegans proteins similar to Saccharomyces cerevisiae beta-oxidation enzymes; Gurvitz A et al.; The role of peroxisomal processes in the maintenance of neurons has not been thoroughly investigated . We propose using Caenorhabditis elegans as a model organism for studying the molecular basis underlying neurodegeneration in certain human peroxisomal disorders, e.g . Zellweger syndrome, since the nematode neural network is well characterized and relatively simple in function . Here we have identified C . elegans PEX-5 (C34C6.6) representing the receptor for peroxisomal targeting signal type 1 (PTS1), defective in patients with such disorders . PEX-5 interacted strongly in a two-hybrid assay with Gal4p-SKL, and a screen using PEX-5 identified interaction partners that were predominantly terminated with PTS1 or its variants . A list of C . elegans proteins with similarities to well-characterized yeast beta-oxidation enzymes was compiled by homology probing . The possible subcellular localization of these orthologues was predicted using an algorithm based on trafficking signals . Examining the C termini of selected nematode proteins for PTS1 function substantiated predictions made regarding the proteins' peroxisomal location . It is concluded that the eukaryotic PEX5-dependent route for importing PTS1-containing proteins into peroxisomes is conserved in nematodes . C . elegans might emerge as an attractive model system for studying the importance of peroxisomes and affiliated processes in neurodegeneration, and also for studying a beta-oxidation process that is potentially compartmentalized in both mitochondria and peroxisomes .

Biochem J, 2000 Oct 15, 351 Pt 2, 477 - 84
The Fe(II) permease Fet4p functions as a low affinity copper transporter and supports normal copper trafficking in Saccharomyces cerevisiae; Hassett R et al.; The plasma-membrane of Saccharomyces cerevisiae contains high affinity permeases for Cu(I) and Fe(II) . A low affinity Fe(II) permease has also been identified, designated Fet4p . A corresponding low affinity copper permease has not been characterized, although yeast cells that lack high affinity copper uptake do accumulate this metal ion . We demonstrate in the present study that Fet4p can function as a low affinity copper permease . Copper is a non-competitive inhibitor of (55)Fe uptake through Fet4p (K(i)=22 microM) . Fet4p-dependent (67)Cu uptake was kinetically characterized, with K(m) and V(max) values of 35 microM and 8 pmol of copper/min per 10(6) cells respectively . A fet4-containing strain exhibited no saturable, low affinity copper uptake indicating that this uptake was attributable to Fet4p . Mutant forms of Fet4p that exhibited decreased efficiency in (55/59)Fe uptake were similarly compromised in (67)Cu uptake, indicating that similar amino acid residues in Fet4p contribute to both uptake processes . The copper taken into the cell by Fet4p was metabolized similarly to the copper taken into the cell by the high affinity permease, Ctr1p . This was shown by the Fet4p-dependence of copper activation of Fet3p, the copper oxidase that supports high affinity iron uptake in yeast . Also, copper-transported by Fet4p down-regulated the copper sensitive transcription factor, Mac1p . Whether supplied by Ctr1p or by Fet4p, an intracellular copper concentration of approx . 10 microM caused a 50% reduction in the transcriptional activity of Mac1p . The data suggest that the initial trafficking of newly arrived copper in the yeast cell is independent of the copper uptake pathway involved, and that this copper may be targeted first to a presumably small 'holding' pool prior to its partitioning within the cell.

Microbiology, 2000 Oct, 146 ( Pt 10), 2679 - 84
Dioctyl phthalate increases the percentage of unsaturated fatty acids with a concomitant decrease in cellular heat shock sensitivity in the yeast Saccharomyces cerevisiae; Curran BP et al.; In the past it has been reproducibly demonstrated that 37 degrees C-grown DBY747 yeast cells have 29% more unsaturated fatty acids and a 3 degrees C higher maximal heat shock response (HSR) than their 25 degrees C counterparts . Suddenly the HSR and lipid profiles of cells grown at 25 degrees C and 37 degrees C became indistinguishable from one another . This paper reports an aberrantly high level of unsaturated fatty acids and an abnormally insensitive HSR in cells grown at 25 degrees C in yeast nitrogen base (YNB) that has been reconstituted from dehydrated medium packaged in 'new' plastic containers . Effective even at a 1:600 dilution of reconstituted medium in laboratory-made YNB, the 'active ingredient' was identified using a combination of HPLC and mass spectroscopy as dioctyl phthalate (a plasticising agent) . Furthermore, the same levels of increase in the percentage of unsaturated fatty acids and decrease in the sensitivity of HSR were found in cells grown in laboratory-made YNB that contained as little as 36 microM pure dioctyl phthalate . This compound nevertheless failed to elicit an observable effect on cellular growth rate at levels up to and including 144 microM . These results suggest that dioctyl phthalate causes yeast cells to accumulate high levels of unsaturated fatty acids with a concomitant decrease in the sensitivity of the HSR, without compromising overall cellular function . They also support earlier work that suggested that the HSR is exquisitely sensitive to the level of unsaturated fatty acids present in yeast cells.

Nat Genet, 2000 Oct, 26(2), 187 - 90
Transposition of maize Ac/Ds transposable elements in the yeast Saccharomyces cerevisiae; Weil CF et al.; Excision by transposons is associated with chromosome breaks; generally, host-cell proteins repair this damage, often introducing mutations . Many transposons also use host proteins in the transposition mechanism or in regulation . Transposition in systems lacking host factors that influence the behaviour of these transpositions is useful in determining what those factors are and how they work . In addition, features of transposition and regulation intrinsic to the element itself can be determined . Maize Activator/Dissociation (Ac/Ds) elements transpose in a wide variety of heterologous plants, but their characteristics in these other systems differ from those in maize, including their response to increasing genetic dosage and the types of repair products recovered following excision . Two Arabidopsis thaliana mutants (iae1 and iae2) show increased Ac transposition frequencies . These mutants, and the differences mentioned above, suggest the involvement of host proteins in Ac/Ds activity and potential differences between these proteins among plant species . Here we report that Ac/Ds elements, members of the hAT (hobo, Ac, Tam3) superfamily, transpose in the yeast Saccharomyces cerevisiae, an organism lacking class II ('cut and paste') transposons . This demonstrates that plant-specific proteins are not essential for Ac/Ds transposition . The yeast system is valuable for dissecting the Ac/Ds transposition mechanism and identifying host factors that can influence transposition and the repair of DNA damage induced by Ac/Ds . Mutations caused by Ds excision in yeast suggest formation of a DNA-hairpin intermediate, and reinsertions occur throughout the genome with a frequency similar to that in plants . The high proportion of Ac/Ds reinsertions also makes this system an in vivo mutagenesis and reverse genetics tool in yeast and, presumably, other eukaryotic systems.

Mol Gen Genet, 2000 Sep, 264(1-2), 89 - 97
Genetic analysis of the Saccharomyces cerevisiae Sgs1 helicase defines an essential function for the Sgs1-Top3 complex in the absence of SRS2 or TOP1; Duno M et al.; The Saccharomyces cerevisiae gene SGS1 encodes a DNA helicase that shows homology to the Escherichia coli protein RecQ and the products of the BLM and WRN genes in humans, which are defective in Bloom's and Werner's syndrome, respectively . Recently, it has been proposed that this helicase is involved in maintaining the integrity of the rDNA and that loss of Sgs1 function leads to accelerated aging . Sgs1 has been isolated on the basis of its genetic interaction with both topoisomerase I and topoisomerase III, as well as in a two-hybrid screen for proteins that interact with the C-terminal portion of topoisomerase II . We have defined the minimal structural elements of Sgs1 required for its interactions with the three topoisomerases, and demonstrate that the complex phenotypes associated with sgs1 mutants are a consequence of a dysfunctional Sgs1-Top3 complex . We also report that the synthetic relationship between mutations in SGS1 and SRS2, which encodes another helicase implicated in recombinational repair, likewise result from a dysfunctional Sgs1-Top3 interaction . Our findings indicate that Sgs1 may act on different DNA structures depending on the activity of topoisomerase I, Srs2 and topoisomerase III.

Mol Gen Genet, 2000 Sep, 264(1-2), 64 - 74
Up-regulation of genes encoding glycosylphosphatidylinositol (GPI)-attached proteins in response to cell wall damage caused by disruption of FKS1 in Saccharomyces cerevisiae; Terashima H et al.; FKS1 and FKS2 encode alternative catalytic subunits of the glucan synthases that are responsible for synthesis of beta-1,3-glucan in the Saccharomyces cerevisiae cell wall . Disruption of FKS1 reduces the glucan content of the cell wall, increases chitin content and activates the expression of CWP1, which encodes a glycosylphosphatidylinositol (GPI)-dependent cell wall protein . These cellular responses have been regarded as compensating for cell wall damage in order to maintain cell wall integrity . Here, we report the identification, by genome-wide screening, of 22 genes that are transcriptionally up-regulated in fks1delta cells . Among them, five genes were found to encode GPI-attached proteins, three of which are covalently associated with the cell wall . Deletion and replacement analysis of the promoter regions identified Rlm1-binding sequences as being responsible for the up-regulation following disruption of FKS1 . Using the rlm1delta tetOp-FKS1 strain, in which the expression of FKS1 can be repressed by doxycycline, we examined the requirement for Rlm1 for the transcriptional up-regulation of these five genes . Three of the five genes were not up-regulated by doxycycline, indicating that Rlm1 mediates their up-regulation when FKS1 is inactivated . The remaining two genes were up-regulated by doxycycline, suggesting that a transcription factor other than Rlm1 is involved in their response to disruption of FKS1.

Mol Gen Genet, 2000 Sep, 264(1-2), 56 - 63
Effects of HDF1 (Ku70) and HDF2 (Ku80) on spontaneous and DNA damage-induced intrachromosomal recombination in Saccharomyces cerevisiae; Cervelli T et al.; The Ku heterodimer binds to the ends of double-stranded breaks (DSBs) in DNA, and is involved in nonhomologous end joining . HDF1 and HDF2, which have been identified in Saccharomyces cerevisiae as homologues of the Ku70 and Ku80 proteins of mammals, reduce radiosensitivity only when homologous recombination repair is impaired and, therefore, affect DSB repair via nonhomologous recombination . Although it has been reported that homologous recombination is defective in the hdf1 null mutant, the roles of HDF1 and HDF2 in this process are not completely clear . We investigated the effect of HDF1 and HDF2 on intrachromosomal recombination by measuring rates of deletion between direct repeats caused by spontaneous and DNA damage-induced events (DEL recombination) . We found a decrease in spontaneous DEL recombination in both TCY5 (hdf1delta) and TCY6 (hdf2delta) strains, suggesting that HDF1 and HDF2 play a role in homologous recombination . As DEL recombination events may occur by sister chromatid conversion and/or single-strand annealing, which is initiated by DSBs, HDF1 and HDF2 may be required to recruit proteins to the damaged ends so as to promote single-strand annealing . The strains TCY5 and TCY6 are also defective in methylmethane sulfonate (MMS)- and X-ray-induced, but not in UV-induced DEL recombination . This confirms that HDF1 and HDF2 are required for the completion of DEL recombination by single strand annealing.

Biochemistry, 2000 Oct 10, 39(40), 12149 - 61
Structure and calcium-binding properties of Frq1, a novel calcium sensor in the yeast Saccharomyces cerevisiae; Ames JB et al.; The FRQ1 gene is essential for growth of budding yeast and encodes a 190-residue, N-myristoylated (myr) calcium-binding protein . Frq1 belongs to the recoverin/frequenin branch of the EF-hand superfamily and regulates a yeast phosphatidylinositol 4-kinase isoform . Conformational changes in Frq1 due to N-myristoylation and Ca(2+) binding were assessed by nuclear magnetic resonance (NMR), fluorescence, and equilibrium Ca(2+)-binding measurements . For this purpose, Frq1 and myr-Frq1 were expressed in and purified from Escherichia coli . At saturation, Frq1 bound three Ca(2+) ions at independent sites, which correspond to the second, third, and fourth EF-hand motifs in the protein . Affinity of the second site (K(d) = 10 microM) was much weaker than that of the third and fourth sites (K(d) = 0.4 microM) . Myr-Frq1 bound Ca(2+) with a K(d)app of 3 microM and a positive Hill coefficient (n = 1.25), suggesting that the N-myristoyl group confers some degree of cooperativity in Ca(2+) binding, as seen previously in recoverin . Both the NMR and fluorescence spectra of Frq1 exhibited very large Ca(2+)-dependent differences, indicating major conformational changes induced upon Ca(2+) binding . Nearly complete sequence-specific NMR assignments were obtained for the entire carboxy-terminal domain (residues K100-I190) . Assignments were made for 20% of the residues in the amino-terminal domain; unassigned residues exhibited very broad NMR signals, most likely due to Frq1 dimerization . NMR chemical shifts and nuclear Overhauser effect (NOE) patterns of Ca(2+)-bound Frq1 were very similar to those of Ca(2+)-bound recoverin, suggesting that the overall structure of Frq1 resembles that of recoverin . A model of the three-dimensional structure of Ca(2+)-bound Frq1 is presented based on the NMR data and homology to recoverin . N-myristoylation of Frq1 had little or no effect on its NMR and fluorescence spectra, suggesting that the myristoyl moiety does not significantly alter Frq1 structure . Correspondingly, the NMR chemical shifts for the myristoyl group in both Ca(2+)-free and Ca(2+)-bound myr-Frq1 were nearly identical to those of free myristate in solution, indicating that the fatty acyl chain is solvent-exposed and not sequestered within the hydrophobic core of the protein, unlike the myristoyl group in Ca(2+)-free recoverin . Subcellular fractionation experiments showed that both the N-myristoyl group and Ca(2+)-binding contribute to the ability of Frq1 to associate with membranes.

Bioelectromagnetics, 2000 Oct, 21(7), 515 - 23
No mutagenic or recombinogenic effects of mobile phone fields at 900 MHz detected in the yeast Saccharomyces cerevisiae; Gos P et al.; Both actively growing and resting cells of the yeast Saccharomyces cerevisiae were exposed to 900-MHz fields that closely matched the Global System for Mobile Communication (GSM) pulsed modulation format signals for mobile phones at specific absorption rates (SAR) of 0.13 and 1.3 W/kg . Two identical anechoic test chambers were constructed to perform concurrent control and test experiments under well-controlled exposure conditions . Using specific test strains, we examined the genotoxic potential of mobile phone fields, alone and in combination, with a known genotoxic compound, the alkylating agent methyl methansulfonate . Mutation rates were monitored by two test systems, a widely used gene-specific forward mutation assay at CAN1 and a wide-range assay measuring the induction of respiration-deficient (petite) clones that have lost their mitochondrial function . In addition, two further assays measured the recombinogenic effect of mobile phone fields to detect possible effects on genomic stability: First, an intrachromosomal, deletion-formation assay previously developed for genotoxic screening; and second, an intragenic recombination assay in the ADE2 gene . Fluctuation tests failed to detect any significant effect of mobile phone fields on forward mutation rates at CAN1, on the frequency of petite formation, on rates of intrachromosomal deletion formation, or on rates of intragenic recombination in the absence or presence of the genotoxic agent methyl methansulfonate .

Genetics, 2000 Oct, 156(2), 579 - 92
Suppression of the profilin-deficient phenotype by the RHO2 signaling pathway in Saccharomyces cerevisiae; Marcoux N et al.; Profilin plays an important role in actin organization in all eukaryotic cells through mechanisms that are still poorly understood . We had previously shown that Mid2p, a transmembrane protein and a potential cell wall sensor, is an effective multicopy suppressor of the profilin-deficient phenotype in Saccharomyces cerevisiae . To better understand the role of Mid2p in the organization of the actin cytoskeleton, we isolated five additional multicopy suppressors of pfy1Delta cells that are Rom1p, Rom2p, Rho2p, Smy1p, and the previously uncharacterized protein Syp1p . The problems of caffeine and NaCl sensitivity, growth defects at 30 degrees and 37 degrees, the accumulation of intracellular vesicular structures, and a random budding pattern in pfy1Delta cells are corrected by all the suppressors tested . This is accompanied by a partial repolarization of the cortical actin patches without the formation of visible actin cables . The overexpression of Mid2p, Rom2p, and Syp1p, but not the overexpression of Rho2p and Smy1p, results in an abnormally thick cell wall in wild-type and pfy1Delta cells . Since none of the suppressors, except Rho2p, can correct the phenotype of the pfy1-111/rho2Delta strain, we propose a model in which the suppressors act through the Rho2p signaling pathway to repolarize cortical actin patches.

Genetics, 2000 Oct, 156(2), 571 - 8
Sequence composition and context effects on the generation and repair of frameshift intermediates in mononucleotide runs in Saccharomyces cerevisiae; Harfe BD et al.; DNA polymerase slippage occurs frequently in tracts of a tandemly repeated nucleotide, and such slippage events can be genetically detected as frameshift mutations . In long mononucleotide runs, most frameshift intermediates are repaired by the postreplicative mismatch repair (MMR) machinery, rather than by the exonucleolytic proofreading activity of DNA polymerase . Although mononucleotide runs are hotspots for polymerase slippage events, it is not known whether the composition of a run and the surrounding context affect the frequency of slippage or the efficiency of MMR . To address these issues, 10-nucleotide (10N) runs were inserted into the yeast LYS2 gene to create +1 frameshift alleles . Slippage events within these runs were detected as Lys(+) revertants . 10G or 10C runs were found to be more unstable than 10A or 10T runs, but neither the frequency of polymerase slippage nor the overall efficiency of MMR was greatly influenced by sequence context . Although complete elimination of MMR activity (msh2 mutants) affected all runs similarly, analyses of reversion rates in msh3 and msh6 mutants revealed distinct specificities of the yeast Msh2p-Msh3p and Msh2p-Msh6p mismatch binding complexes in the repair of frameshift intermediates in different sequence contexts.

Genetics, 2000 Oct, 156(2), 549 - 57
Involvement of very short DNA tandem repeats and the influence of the RAD52 gene on the occurrence of deletions in Saccharomyces cerevisiae; Welcker AJ et al.; Chromosomal rearrangements, such as deletions, duplications, or Ty transposition, are rare events . We devised a method to select for such events as Ura(+) revertants of a particular ura2 mutant . Among 133 Ura(+) revertants, 14 were identified as the result of a deletion in URA2 . Of seven classes of deletions, six had very short regions of identity at their junctions (from 7 to 13 bp long) . This strongly suggests a nonhomologous recombination mechanism for the formation of these deletions . The total Ura(+) reversion rate was increased 4.2-fold in a rad52Delta strain compared to the wild type, and the deletion rate was significantly increased . All the deletions selected in the rad52Delta context had microhomologies at their junctions . We propose two mechanisms to explain the occurrence of these deletions and discuss the role of microhomology stretches in the formation of fusion proteins.

Genetics, 2000 Oct, 156(2), 535 - 47
Synthetic lethal interactions suggest a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation; Costa PJ et al.; Strong evidence indicates that transcription elongation by RNA polymerase II (pol II) is a highly regulated process . Here we present genetic results that indicate a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation . A screen for synthetic lethal mutations was carried out with an rtf1 deletion mutation to identify factors that interact with Rtf1 or regulate the same process as Rtf1 . The screen uncovered mutations in SRB5, CTK1, FCP1, and POB3 . These genes encode an Srb/mediator component, a CTD kinase, a CTD phosphatase, and a protein involved in the regulation of transcription by chromatin structure, respectively . All of these gene products have been directly or indirectly implicated in transcription elongation, indicating that Rtf1 may also regulate this process . In support of this view, we show that RTF1 functionally interacts with genes that encode known elongation factors, including SPT4, SPT5, SPT16, and PPR2 . We also show that a deletion of RTF1 causes sensitivity to 6-azauracil and mycophenolic acid, phenotypes correlated with a transcription elongation defect . Collectively, our results suggest that Rtf1 may function as a novel transcription elongation factor in yeast.

Genetics, 2000 Oct, 156(2), 489 - 500
Saccharomyces cerevisiae BUB2 prevents mitotic exit in response to both spindle and kinetochore damage; Krishnan R et al.; The spindle assembly checkpoint-mediated mitotic arrest depends on proteins that signal the presence of one or more unattached kinetochores and prevents the onset of anaphase in the presence of kinetochore or spindle damage . In the presence of either damage, bub2 cells initiate a preanaphase delay but do not maintain it . Inappropriate sister chromatid separation in nocodazole-treated bub2 cells is prevented when mitotic exit is blocked using a conditional tem1(c) mutant, indicating that the preanaphase failure in bub2 cells is a consequence of events downstream of TEM1 in the mitotic exit pathway . Using a conditional bub2(tsd) mutant, we demonstrate that the continuous presence of Bub2 protein is required for maintaining spindle damage-induced arrest . BUB2 is not required to maintain a DNA damage checkpoint arrest, revealing a specificity for spindle assembly checkpoint function . In a yeast two-hybrid assay and in vitro, Bub2 protein interacts with the septin protein Cdc3, which is essential for cytokinesis . These data support the view that the spindle assembly checkpoint encompasses regulation of distinct mitotic steps, including a MAD2-directed block to anaphase initiation and a BUB2-directed block to TEM1-dependent exit.

Environ Mol Mutagen, 2000, 36(2), 113 - 20
Formation of circular amplifications in Saccharomyces cerevisiae by a breakage-fusion-bridge mechanism; Moore IK et al.; Primary gene amplification, the mutation from one gene copy per genome to two or more copies per genome, is a major mechanism of oncogene overexpression in human cancers . Analysis of the structures of amplifications can provide important evidence about the mechanism of amplification formation . We report here the analysis of the structures of four independent spontaneous circular amplifications of ADH4:CUP1 in the yeast Saccharomyces cerevisiae . The structures of all four amplifications are consistent with their formation by a breakage-fusion-bridge (BFB) mechanism . All four of these amplifications include a centromere as predicted by the BFB model . All four of the amplifications have a novel joint located between the amplified DNA and the telomere, which results in a dicentric chromosome, and is adjacent to all the copies of the amplified DNA as predicted by the BFB model . In addition we demonstrated that two of the amplifications contain most of chromosome VII in an unrearranged form in a 1:1 ratio with the normal copy of chromosome VII, again consistent with the predictions of the BFB model . Finally, all four amplifications are circular, one stable endpoint for molecules after breakage- fusion-bridge .

Environ Mol Mutagen, 2000, 36(2), 105 - 12
Telomere sequences at the novel joints of four independent amplifications in Saccharomyces cerevisiae; Moore IK et al.; Primary gene amplification, the mutation from one copy of a gene per genome to two or more genes per genome is a major mechanism of oncogene overexpression . We previously developed a system in the yeast Saccharomyces cerevisiae to phenotypically detect primary amplifications of a reporter cassette, ADH4:CUP1 . We present here the sequence analysis of novel joints from four independent, spontaneous circular amplifications identified by the ADH4:CUP1 system . All four novel joints consist of C(1-3) A telomeric repeats joined to short (14- to 16-bp) CA-rich tracts between ADH4 and the telomere of chromosome VII . In three of the four amplifications, the telomeric sequence and the CA-rich tract that are joined in the amplification are normally located in inverted orientation to each other on chromosome VII . In the fourth amplification, the CA-rich tract on chromosome VII is joined to telomere sequences from another chromosome . We suggest that formation of these amplifications was initiated by recombination between these CA-rich tracts and a telomere . The resulting dicentric chromosome could start a breakage-fusion-bridge cycle that could be resolved by the formation of a circular amplification structure .

Environ Mol Mutagen, 2000, 36(2), 97 - 104
Cloning and expression of rat CYP2E1 in Saccharomyces cerevisiae: detection of genotoxicity of N-alkylformamides; Del Carratore MR et al.; A cDNA coding for rat cytochrome P450 2E1 was cloned into the multicopy vector pYeDP60 and expressed in haploid RSY6 and diploid RS112 yeast strains of Saccharomyces cerevisiae under control of the GAL10-CYC1 promoter . Spectral and catalytic properties of the expressed 2E1 were examined in whole cells or microsomes of both strains . The level of CYP2E1 obtained in RS112 (200 pmol/mg microsomal protein) was the highest among CYP2E1 produced in the various expression systems . The monooxygenase activity in the microsomes of both strains, measured as aniline hydroxylase, was found comparable to that of control rat hepatic microsomes . In a reconstituted system in the presence of exogenous rat P450 reductase, their activity increased about 10-fold . When exposed to the carcinogen NDMA, a known 2E1 substrate, the recombination frequency determined in the 2E1-expressing RS112 cells was enhanced, in a dose-dependent manner, up to 20-fold . The exposure of the same cells to the hepatotoxic solvents, N-methyl- and N-ethylformamide, resulted in an induction of recombination frequency, which was not observed in the void plasmid containing RS112 cells in the presence of S9 hepatic fractions from pyrazole-induced rats, as a specific exogenous metabolic activation system . These results demonstrate that the 2E1-expressing cells metabolize the two N-alkylformamides to genotoxic intermediates and, therefore, they provide an useful tool to study the bioactivation mechanism of potential P450 2E1 substrates .

J Biol Chem, 2001 Jan 19, 276(3), 1916 - 22 Epub 2000 Sep 29.
Identification in Saccharomyces cerevisiae of two isoforms of a novel mitochondrial transporter for 2-oxoadipate and 2-oxoglutarate; Palmieri L et al.; The nuclear genome of Saccharomyces cerevisiae encodes 35 members of a family of membrane proteins . Known members transport substrates and products across the inner membranes of mitochondria . We have localized two hitherto unidentified family members, Odc1p and Odc2p, to the inner membranes of mitochondria . They are isoforms with 61% sequence identity, and we have shown in reconstituted liposomes that they transport the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a strict counter exchange mechanism . Intraliposomal adipate and glutarate and to a lesser extent malate and citrate supported {14C}oxoglutarate uptake . The expression of Odc1p, the more abundant isoform, made in the presence of nonfermentable carbon sources, is repressed by glucose . The main physiological roles of Odc1p and Odc2p are probably to supply 2-oxoadipate and 2-oxoglutarate from the mitochondrial matrix to the cytosol where they are used in the biosynthesis of lysine and glutamate, respectively, and in lysine catabolism.

J Biochem (Tokyo), 2000 Oct, 128(4), 665 - 71
Multiple actin-related proteins of Saccharomyces cerevisiae are present in the nucleus; Harata M et al.; An increasing number of actin-related proteins (Arps), which share the basal structure with skeletal actin but possess distinct functions, have been found in a wide variety of organisms . Individual Arps of Saccharomyces cerevisiae were classified into Arps 1-10 based on the relatedness of their sequences and functions, where Arp1 is the most similar to actin, and Arp10 is the least similar . While Arps 1-3 and their orthologs in other organisms are localized exclusively in the cytoplasm, Arp4 (also known as Act3) is localized in the nucleus and is involved in transcriptional regulation . Here we examined the more divergent Arps for possible nuclear functions . We show that Arps 5-9 are localized in the nucleus, but Arp10 is not . The nuclear export signals identified in actin are well conserved in the cytoplasmic Arps, Arps 1-3, but less conserved in the nuclear Arps . Gel filtration chromatography experiments show that the nuclear Arps are larger than monomer in size and thus are present in multi-protein complexes . Since nuclear protein complexes containing Arps are found to be responsible for histone acetylation and chromatin remodeling, it is suggested that most of the divergent Arps are involved in the !transcriptional regulation through chromatin modulation.

Biochemistry, 2000 Oct 3, 39(39), 11948 - 54
Characterization of the regiochemistry and cryptoregiochemistry of a Caenorhabditis elegans fatty acid desaturase (FAT-1) expressed in Saccharomyces cerevisiae; Meesapyodsuk D et al.; To characterize the fatty acid desaturase produced by the fat-1 gene from the nematode Caenorhabditis elegans, the functional expression of this enzyme was effected in the yeast Saccharomyces cerevisiae . The GC-MS analysis of desaturated products derived from various fatty acids, including deuterium-labeled thia fatty acids supplied to growing cultures of transformed yeast, has defined the substrate requirements, regiochemistry, and cryptoregiochemistry of the enzyme . The desaturase acts on substrates of 16-20 carbons with a preference for omega-6 fatty acids, and its regioselectivity was confirmed to be that of an omega-3 desaturase . (omega-x refers to a double bond or desaturation between carbons x and x+1, counting from the methyl end of a fatty acid.) The primary deuterium kinetic isotope effects (KIEs) at C-15 and C-16 of a C18 fatty acid analogue were measured via competitive incubation experiments: While k(H)/k(D) at the omega-3 position was shown to be large (7.8 +/- 0.4), essentially no KIE at the omega-2 position was observed (k(H)/k(D) = 0.99 +/- 0.04) . This result indicates that omega-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus . The results are discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring differing regioselectivities.

Mol Cell Biol, 2000 Oct, 20(20), 7559 - 71
Role of Cdc42p in pheromone-stimulated signal transduction in Saccharomyces cerevisiae; Moskow JJ et al.; CDC42 encodes a highly conserved GTPase of the Rho family that is best known for its role in regulating cell polarity and actin organization . In addition, various studies of both yeast and mammalian cells have suggested that Cdc42p, through its interaction with p21-activated kinases (PAKs), plays a role in signaling pathways that regulate target gene transcription . However, recent studies of the yeast pheromone response pathway suggested that prior results with temperature-sensitive cdc42 mutants were misleading and that Cdc42p and the Cdc42p-PAK interaction are not involved in signaling . To clarify this issue, we have identified and characterized novel viable pheromone-resistant cdc42 alleles that retain the ability to perform polarity-related functions . Mutation of the Cdc42p residue Val36 or Tyr40 caused defects in pheromone signaling and in the localization of the Ste20p PAK in vivo and affected binding to the Ste20p Cdc42p-Rac interactive binding (CRIB) domain in vitro . Epistasis analysis suggested that they affect the signaling step at which Ste20p acts, and overproduction of Ste20p rescued the defect . These results suggest that Cdc42p is in fact required for pheromone response and that interaction with the PAK Ste20p is critical for that role . Furthermore, the ste20DeltaCRIB allele, previously used to disrupt the Cdc42p-Ste20p interaction, behaved as an activated allele, largely bypassing the signaling defect of the cdc42 mutants . Additional observations lead us to suggest that Cdc42p collaborates with the SH3-domain protein Bem1p to facilitate signal transduction, possibly by providing a cell surface scaffold that aids in the local concentration of signaling kinases, thus promoting activation of a mitogen-activated protein kinase cascade by Ste20p.

Mol Cell Biol, 2000 Oct, 20(20), 7438 - 49
A motif shared by TFIIF and TFIIB mediates their interaction with the RNA polymerase II carboxy-terminal domain phosphatase Fcp1p in Saccharomyces cerevisiae; Kobor MS et al.; Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit . We have used deletion and point mutations in Fcp1p, a TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT domain, like that of its catalytic domain, is important for cell viability, mRNA synthesis, and CTD dephosphorylation in vivo . Although regions of Fcp1p carboxy terminal to its BRCT domain and at its amino terminus were not essential for viability, deletion of either of these regions affected the phosphorylation state of the CTD . Two portions of this carboxy-terminal region of Fcp1p bound directly to the first cyclin-like repeat in the core domain of the general transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF . These regulatory interactions with Fcp1p involved closely related amino acid sequence motifs in TFIIB and RAP74 . Mutating the Fcp1p-binding motif KEFGK in the RAP74 (Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate transcription when it is artificially tethered to a promoter . These results suggest strongly that this KEFGK motif in RAP74 mediates its interaction with Fcp1p in vivo.

Mol Cell Biol, 2000 Oct, 20(20), 7427 - 37
Saccharomyces cerevisiae transcription elongation mutants are defective in PUR5 induction in response to nucleotide depletion; Shaw RJ et al.; IMP dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides . It is a target of therapeutically useful drugs and is implicated in the regulation of cell growth rate . In the yeast Saccharomyces cerevisiae, mutations in components of the RNA polymerase II (Pol II) transcription elongation machinery confer increased sensitivity to a drug that inhibits IMPDH, 6-azauracil (6AU), by a mechanism that is poorly understood . This phenotype is thought to reflect the need for an optimally functioning transcription machinery under conditions of lowered intracellular GTP levels . Here we show that in response to the application of IMPDH inhibitors such as 6AU, wild-type yeast strains induce transcription of PUR5, one of four genes encoding IMPDH-related enzymes . Yeast elongation mutants sensitive to 6AU, such as those with a disrupted gene encoding elongation factor SII or those containing amino acid substitutions in Pol II subunits, are defective in PUR5 induction . The inability to fully induce PUR5 correlates with mutations that effect transcription elongation since 6AU-sensitive strains deleted for genes not related to transcription elongation are competent to induce PUR5 . DNA encompassing the PUR5 promoter and 5' untranslated region supports 6AU induction of a luciferase reporter gene in wild-type cells . Thus, yeast sense and respond to nucleotide depletion via a mechanism of transcriptional induction that restores nucleotides to levels required for normal growth . An optimally functioning elongation machinery is critical for this response.

Glycoconj J, 1999 Nov, 16(11), 673 - 9
Chromosomal promoter replacement in Saccharomyces cerevisiae: construction of conditional lethal strains for the cloning of glycosyltransferases from various organisms; Mazhari-Tabrizi R et al.; Heterologous complementation in yeast has been a successful tool for cloning and characterisation of genes from various organisms . Therefore we constructed conditionally lethal Saccharomyces cerevisiae strains by replacing the endogenous promoter from the genes of interest (glycosyltransferases) by the stringently regulated GAL1-promoter, by a technique called chromosomal promoter replacement . Such yeast strains were constructed for the genes Alg 1, Alg7, Sec59, Wbp1 involved in N-Glycosylation, the genes Gpi2, Gpi3/Spt14, Gaal, Pis1, involved in GPI-anchor biosynthesis and Dpm involved in both pathways . All strains show the expected conditionally lethal phenotype on glucose-containing medium when expression of the respective gene is turned off.

Nucleic Acids Res, 2000 Oct 1, 28(19), 3725 - 32
Phosphorylation of the replication protein A large subunit in the Saccharomyces cerevisiae checkpoint response; Brush GS et al.; The checkpoint mechanisms that delay cell cycle progression in response to DNA damage or inhibition of DNA replication are necessary for maintenance of genetic stability in eukaryotic cells . Potential targets of checkpoint-mediated regulation include proteins directly involved in DNA metabolism, such as the cellular single-stranded DNA (ssDNA) binding protein, replication protein A (RPA) . Studies in Saccharomyces cerevisiae have revealed that the RPA large subunit (Rfa1p) is involved in the G1 and S phase DNA damage checkpoints . We now demonstrate that Rfa1p is phosphorylated in response to various forms of genotoxic stress, including radiation and hydroxyurea exposure, and further show that phosphorylation of Rfa1p is dependent on the central checkpoint regulator Mec1p . Analysis of the requirement for other checkpoint genes indicates that different mechanisms mediate radiation- and hydroxyurea-induced Rfa1p phosphorylation despite the common requirement for functional Mec1p . In addition, experiments with mutants defective in the Cdc13p telomere-binding protein indicate that ssDNA formation is an important signal for Rfa1p phosphorylation . Because Rfa1p contains the major ssDNA binding activity of the RPA heterotrimer and is required for DNA replication, repair and recombination, it is possible that phosphorylation of this subunit is directly involved in modulating RPA activity during the checkpoint response.

Science, 2000 Sep 22, 289(5487), 2126 - 8
Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae; Lin SJ et al.; Calorie restriction extends life-span in a wide variety of organisms . Although it has been suggested that calorie restriction may work by reducing the levels of reactive oxygen species produced during respiration, the mechanism by which this regimen slows aging is uncertain . Here, we mimicked calorie restriction in yeast by physiological or genetic means and showed a substantial extension in life-span . This extension was not observed in strains mutant for SIR2 (which encodes the silencing protein Sir2p) or NPT1 (a gene in a pathway in the synthesis of NAD, the oxidized form of nicotinamide adenine dinucleotide) . These findings suggest that the increased longevity induced by calorie restriction requires the activation of Sir2p by NAD.

Biochem J, 2000 Oct 1, 351(Pt 1), 241 - 9
Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na+/H+ exchangers; Darley CP et al.; Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells . Sequestration of Na(+) ions into the vacuole is one mechanism to confer Na(+)-tolerance on these organisms . In the present study we provide direct evidence that the Arabidopsis thaliana At-NHX1 gene and the yeast NHX1 gene encode low-affinity electroneutral Na(+)/H(+) exchangers . We took advantage of the ability of heterologously expressed At-NHX1 to functionally complement the yeast nhx1-null mutant . Experiments on vacuolar vesicles isolated from yeast expressing At-NHX1 or NHX1 provided direct evidence for pH-gradient-energized Na(+) accumulation into the vacuole . A major difference between NHX1 and At-NHX1 is the presence of a cleavable N-terminal signal peptide (SP) in the former gene . Fusion of the SP to At-NHX1 resulted in an increase in the magnitude of Na(+)/H(+) exchange, indicating a role for the SP in protein targeting or regulation . Another distinguishing feature between the plant and yeast antiporters is their sensitivity to the diuretic compound amiloride . Whereas At-NHX1 was completely inhibited by amiloride, NHX1 activity was reduced by only 20-40% . These results show that yeast as a heterologous expression system provides a convenient model to analyse structural and regulatory features of plant Na(+)/H(+) antiporters.

Biochemistry, 2000 Sep 12, 39(36), 11137 - 46
Biophysical characterization of elongin C from Saccharomyces cerevisiae; Buchberger A et al.; Elongin C (ELC) is an essential component of the mammalian CBC(VHL) E3 ubiquitin ligase complex . As a step toward understanding the role of ELC in assembly and function of CBC-type ubiquitin ligases, we analyzed the quaternary structure and backbone dynamics of the highly homologous Elc1 protein from Saccharomyces cerevisiae . Analytical ultracentrifugation experiments in conjunction with size exclusion chromatography showed that Elc1 is a nonglobular monomer over a wide range of concentrations . Pronounced line broadening in (1)H,(15)N-HSQC NMR spectra and failure to assign peaks corresponding to the carboxy-terminal helix 4 of Elc1 indicated that helix 4 is conformationally labile . Measurement of (15)N NMR relaxation parameters including T(1), T(2), and the (1)H-(15)N nuclear Overhauser effect revealed (i) surprisingly high flexibility of residues 69-77 in loop 5, and (ii) chemical exchange contributions for a large number of residues throughout the protein . Addition of 2,2,2-trifluoroethanol (TFE) stabilized helix 4 and reduced chemical exchange contributions, suggesting that stabilization of helix 4 suppresses the tendency of Elc1 to undergo conformational exchange on a micro- to millisecond time scale . Binding of a peptide representing the major ELC binding site of the von Hippel-Lindau (VHL) tumor suppressor protein almost completely eliminated chemical exchange processes, but induced substantial conformational changes in Elc1 leading to pronounced rotational anisotropy . These results suggest that elongin C interacts with various target proteins including the VHL protein by an induced fit mechanism involving the conformationally flexible carboxy-terminal helix 4.

Structure Fold Des, 2000 Aug 15, 8(8), 841 - 50
Crystal structure and mutational analysis of the Saccharomyces cerevisiae cell cycle regulatory protein Cks1: implications for domain swapping, anion binding and protein interactions; Bourne Y et al.; BACKGROUND: The Saccharomyces cerevisiae protein Cks1 (cyclin-dependent kinase subunit 1) is essential for cell-cycle progression . The biological function of Cks1 can be modulated by a switch between two distinct molecular assemblies: the single domain fold, which results from the closing of a beta-hinge motif, and the intersubunit beta-strand interchanged dimer, which arises from the opening of the beta-hinge motif . The crystal structure of a cyclin-dependent kinase (Cdk) in complex with the human Cks homolog CksHs1 single-domain fold revealed the importance of conserved hydrophobic residues and charged residues within the beta-hinge motif . RESULTS: The 3.0 A resolution Cks1 structure reveals the strict structural conservation of the Cks alpha/beta-core fold and the beta-hinge motif . The beta hinge identified in the Cks1 structure includes a novel pivot and exposes a cluster of conserved tyrosine residues that are involved in Cdk binding but are sequestered in the beta-interchanged Cks homolog suc1 dimer structure . This Cks1 structure confirms the conservation of the Cks anion-binding site, which interacts with sidechain residues from the C-terminal alpha helix of another subunit in the crystal . CONCLUSIONS: The Cks1 structure exemplifies the conservation of the beta-interchanged dimer and the anion-binding site in evolutionarily distant yeast and human Cks homologs . Mutational analyses including in vivo rescue of CKS1 disruption support the dual functional roles of the beta-hinge residue Glu94, which participates in Cdk binding, and of the anion-binding pocket that is located 22 A away and on an opposite face to Glu94 . The Cks1 structure suggests a biological role for the beta-interchanged dimer and the anion-binding site in targeting Cdks to specific phosphoproteins during cell-cycle progression.

Mol Genet Metab, 2000 Aug, 70(4), 272 - 80
Subcellular localization of galactose-1-phosphate uridylyltransferase in the yeast Saccharomyces cerevisiae; Christacos NC et al.; The enzyme galactose-1-phosphate uridylyltransferase (GALT) catalyzes the second step of the Leloir pathway of galactose metabolism, following galactokinase (GALK) and preceding UDP-galactose-4-epimerase (GALE) . Impairment of GALT in humans results in the potentially lethal disorder classic galactosemia . Standard lysis protocols of bacteria, yeast, or mammalian cells release all three Leloir enzymes in the soluble fraction, leading to the historical assumption that all three function as free cytosolic enzymes . We have tested this assumption with regard to GALT in vivo using the yeast Saccharomyces cerevisiae, by linking a GFP-tag onto the amino terminus of Gal7p, the endogenous yeast GALT . We find clear evidence of localization of the fusion protein to discrete spots in the cytoplasm of the majority of cells expressing all three Leloir enzymes, although GFP alone appears freely cytosolic . In contrast, yeast expressing GFP-Gal7p but lacking Gal1p (GALK), Gal10p (GALE), or both do not demonstrate spots in the majority of cells, implicating a role, either direct or indirect, for these other Leloir proteins in the Gal7p localization process . Preliminary truncation experiments reveal that amino acids 1-134 of Gal7p are sufficient to drive localization of the fusion protein, while amino acids 1-66 are not . Finally, GFP-tagged human GALT expressed in yeast also localizes to spots, demonstrating that at least some of the intrinsic determinants of localization have been conserved . These observations raise the intriguing possibility that GALT may function in a sequestered rather than a freely diffusible state, and that this subcellular organization may have been conserved through evolution .

Yeast, 2000 Sep 30, 16(13), 1185 - 90
Ultradian oscillation of Saccharomyces cerevisiae during aerobic continuous culture: hydrogen sulphide mediates population synchrony; Sohn HY et al.; Saccharomyces cerevisiae showed an ultradian respiratory oscillation during aerobic continuous culture . Analysis of the off-gas revealed that hydrogen sulphide production also oscillated . Production was first detected at the onset of low respiration and reached a maximum (1.5 microM) prior to minimum respiratory activity . Then H(2)S concentration fell rapidly to below 0.2 microM before the onset of high respiration . Injection of respiratory oscillation perturbation agents, such as glutathione (50 microM), NaNO(2) (50 microM) or acetaldehyde (4.5 mM),() transiently increased H(2)S production above 6 microM . The synchronization properties of H(2)S were analysed to reveal that changes of oscillation period and amplitude were dependent on H(2)S concentration in culture . It is concluded that H(2)S produced during oscillation produces population synchrony by respiratory chain inhibition .

J Biol Chem, 2000 Dec 8, 275(49), 38245 - 53
Cytosolic Ca2+ homeostasis is a constitutive function of the V-ATPase in Saccharomyces cerevisiae; Forster C et al.; The vacuole is the major site of intracellular Ca(2+) storage in yeast and functions to maintain cytosolic Ca(2+) levels within a narrow physiological range via a Ca(2+) pump (Pmc1p) and a H(+)/Ca(2+) antiporter (Vcx1p) driven by the vacuolar H(+)-ATPase (V-ATPase) . We examined the function of the V-ATPase in cytosolic Ca(2+) homeostasis by comparing responses to a brief Ca(2+) challenge of a V-ATPase mutant (vma2Delta) and wild-type cells treated with the V-ATPase inhibitor concanamycin A . The kinetics of the Ca(2+) response were determined using transgenic aequorin as an in vivo cytosolic Ca(2+) reporter system . In wild-type cells, the V-ATPase-driven Vcx1p was chiefly responsible for restoring cytosolic Ca(2+) concentrations after a brief pulse . In cells lacking V-ATPase activity, brief exposure to elevated Ca(2+) compromised viability, even when there was little change in the final cytosolic Ca(2+) concentration . vma2Delta cells were more efficient at restoring cytosolic {Ca(2+)} after a pulse than concanamycin-treated wild-type cells, suggesting long term loss of V-ATPase triggers compensatory mechanisms . This compensation was dependent on calcineurin, and was mediated primarily by Pmc1p.

Int J Radiat Biol, 2000 Sep, 76(9), 1273 - 9
Role of RAD9-dependent cell-cycle checkpoints in the adaptive response to ionizing radiation in yeast, Saccharomyces cerevisiae; Dolling JA et al.; PURPOSE: To determine whether yeast cells (Saccharomyces cerevisiae) defective in damage-inducible cell-cycle arrest can invoke an adaptive response and become resistant to normally lethal doses of ionizing radiation . MATERIALS AND METHODS: Wild-type yeast cells, cells defective for DNA-damage-responsive G1 and G2 cell-cycle arrest (rad9delta), and cells defective for recombinational repair of DNA damage (rad50, 51, 52) were subjected to adapting treatments of heat or radiation and subsequently exposed to normally lethal doses of radiation . Survival, as measured by colony-forming ability, was compared with non-adapted, control cells . RESULTS: Wild-type and rad9delta cells became more resistant to potentially lethal doses of radiation after exposure to conditions that are known to elicit the adaptive response . Further, the relative magnitude of resistance developed by the normal, wild-type and rad9delta yeast cells was similar, with a dose modifying factor (at D1) for radiation-induced radiation resistance of 1.3 for both strains . Dose modifying factors (at D1) for heat-induced radiation resistance were 1.7 and 1.6 for wild-type and rad9delta cells, respectively . In contrast, none of the recombinational repair-defective cells exhibited radiation resistance after an adapting treatment . CONCLUSIONS: The ability of yeast cells to arrest in cell-cycle gap phases did not appear to contribute significantly to radiation resistance induced by radiation or heat . Instead, it is suggested that the adaptive response was due mainly to the existence and enhancement of cellular recombinational repair capacity, which was sufficient to repair any DNA damage without the requirement of a detectable cell-cycle delay.

FEBS Lett, 2000 Sep 8, 481(1), 13 - 8
Caspase-3 and inhibitor of apoptosis protein(s) interactions in Saccharomyces cerevisiae and mammalian cells; Wright ME et al.; Using a heterologous yeast expression assay, we show that inhibitor of apoptosis proteins (IAPs) suppress caspase-3-mediated cytotoxicity in the order of XIAP>c-IAP2>c-IAP1>survivin . The same ordering of IAP activities was demonstrated in mammalian cells expressing an auto-activating caspase-3 . The relative anti-apoptotic activities of each IAP depended on the particular death stimulus . For IAP-expressing cells treated with camptothecin, survival correlated with their intrinsic anti-caspase-3 activity . However, c-IAP1-transfected cells were disproportionately resistant to tumor necrosis factor-alpha, suggesting that its anti-apoptotic activities extend beyond caspase-3 or -7 inhibition . Yeast-based caspase assays provide rapid, reliable information on specificity and activity of the IAPs and aid in identifying critical targets in mammalian apoptotic pathways.

J Biol Chem, 2000 Dec 1, 275(48), 37774 - 8
Use of the NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae as a possible cure for complex I defects in human cells; Seo BB et al.; The Ndi1 enzyme of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane . We have shown previously that the NDI1 gene can be functionally expressed in Chinese hamster cells (Seo, B . B., Kitajima-Ihara, T., Chan, E . K., Scheffler, I . E., Matsuno-Yagi, A., and Yagi, T . (1998) Proc . Natl . Acad . Sci . U . S . A . 95, 9167-9171) and human embryonal kidney 293 (HEK 293) cells (Seo, B . B., Matsuno-Yagi, A., and Yagi, T . (1999) Biochim . Biochem . Acta 1412, 56-65) and that the Ndi1 protein is capable of compensating respiratory deficiencies caused by defects in the host NADH-quinone oxidoreductase (complex I) . To extend the potential use of this enzyme to repair complex I deficiencies in vivo, we constructed a recombinant adeno-associated virus vector carrying the NDI1 gene (rAAV-NDI1) . With rAAV-NDI1 as the gene delivery method, we were able to achieve high transduction efficiencies (nearly 100%) even in 143B cells that are difficult to transfect by lipofection or calcium phosphate precipitation methods . The NDI1 gene was successfully introduced into non-proliferating human cells using rAAV-NDI1 . The expressed Ndi1 protein was shown to be functionally active just as seen for proliferating cells . Furthermore, when cells were cultured under the conditions where energy has to be provided by respiration, the NDI1-transduced cells were able to grow even in the presence of added complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion . In contrast, control cells that did not receive the NDI1 gene failed to survive as anticipated . The Ndi1 protein has a great potential as a molecular remedy for complex I defects, and it is highly likely that the same strategy can be extended to correction of other mitochondrial disorders.

Nucleic Acids Res, 2000 Sep 15, 28(18), 3649 - 56
Mutational spectrum analysis of RNase H(35) deficient Saccharomyces cerevisiae using fluorescence-based directed termination PCR; Chen JZ et al.; Mutational spectrum analysis has become an informative genetic tool to understand those protein functions involved in mutation avoidance pathways since specific types of mutations are often associated with particular protein defects involved in DNA replication and repair . In this study, we describe a novel, fluorescence-based procedure for direct determination of deletions and insertions with 100% accuracy . We performed two complementary directed termination PCR with near infrared dye-labeled primers, followed by visualization of termination fragments using an automated Li-cor DNA sequencer . This method is used for rapid analysis of mutational spectra generated in nuclease-defective strains of Saccharomyces cerevisiae to elucidate the role of RNase H(35) in RNA primer removal during DNA replication and in mutation avoidance . Strains deficient in RNH35 displayed a distinct spontaneous mutation spectrum of deletions characterized by a unique 4 bp deletion in a lys2-Bgl allele . This was in sharp contrast to strains deficient in rad27 that displayed duplication mutations . Further analysis of mutations in a rnh35/rad27 double mutant revealed a mixed spectrum . These results indicate that RNase H(35) may participate in a redundant pathway in Okazaki fragment processing and that mutational spectra caused by protein deficiencies may be more intermediate-specific than pathway-specific.

Mutat Res, 2000 Sep 15, 461(1), 1 - 13
Saccharomyces cerevisiae lacking Snm1, Rev3 or Rad51 have a normal S-phase but arrest permanently in G2 after cisplatin treatment; Grossmann KF et al.; The role of Snm1, Rev3 and Rad51 in S-phase after cisplatin (CDDP) DNA treatment has been examined . When isogenic deletion mutants snm1 delta, rev3 delta and rad51 delta were arrested in G1 and treated with doses of CDDP causing significant lethality (<20% survival in the mutant strains), they progressed through S-phase with normal kinetics . The mutants arrested in G2 like wild-type cells, however they did not exit the arrest and reenter the cell cycle . This finding demonstrates that these genes are not required to allow DNA replication in the presence of damage . Therefore, Snm1, Rev3 and Rad51 may act after S to allow repair . At high levels of damage (<40% survival in wild-type cells) S-phase was slowed in a MEC1-dependent fashion . The cross-link incision kinetics of snm1 delta and rev3 delta mutants were also examined; both showed no deficiencies in incision of cross-linked DNA.

Genetics, 2000 Sep, 156(1), 105 - 22
Pep3p/Pep5p complex: a putative docking factor at multiple steps of vesicular transport to the vacuole of Saccharomyces cerevisiae; Srivastava A et al.; Pep3p and Pep5p are known to be necessary for trafficking of hydrolase precursors to the vacuole and for vacuolar biogenesis . These proteins are present in a hetero-oligomeric complex that mediates transport at the vacuolar membrane . PEP5 interacts genetically with VPS8, implicating Pep5p in the earlier Golgi to endosome step and/or in recycling from the endosome to the Golgi . To understand further the cellular roles of Pep3p and Pep5p, we isolated and characterized a set of pep3 conditional mutants . Characterization of mutants revealed that pep3(ts) mutants are defective in the endosomal and nonendosomal Golgi to vacuole transport pathways, in the cytoplasm to vacuole targeting pathway, in recycling from the endosome back to the late Golgi, and in endocytosis . PEP3 interacts genetically with two members of the endosomal SNARE complex, PEP12 (t-SNARE) and PEP7 (homologue of mammalian EEA1); Pep3p and Pep5p associate physically with Pep7p as revealed by two-hybrid analysis . Our results suggest that a core Pep3p/Pep5p complex promotes vesicular docking/fusion reactions in conjunction with SNARE proteins at multiple steps in transport routes to the vacuole . We propose that this complex may be responsible for tethering transport vesicles on target membranes.

Genetics, 2000 Sep, 156(1), 45 - 58
Zinc-regulated genes in Saccharomyces cerevisiae revealed by transposon tagging; Yuan DS; The biochemistry of human nutritional zinc deficiency remains poorly defined . To characterize in genetic terms how cells respond to zinc deprivation, zinc-regulated genes (ZRG's) were identified in yeast . Gene expression was probed using random lacZ reporter gene fusions, integrated by transposon tagging into a diploid genome as previously described . About half of the genome was examined . Cells exhibiting differences in lacZ expression on low or moderate ( approximately 0 . 1 vs . 10 microm) zinc media were isolated and the gene fusions were sequenced . Ribonuclease protection assays demonstrated four- to eightfold increases for the RNAs of the ZAP1, ZRG17 (YNR039c), DPP1, ADH4, MCD4, and YEF3B genes in zinc-deficient cells . All but YEF3B were shown through reporter gene assays to be controlled by a master regulator of zinc homeostasis now known to be encoded by ZAP1 . ZAP1 mutants lacked the flocculence and distended vacuoles characteristic of zinc-deficient cells, suggesting that flocculation and vacuolation serve homeostatic functions in zinc-deficient cells . ZRG17 mutants required extra zinc supplementation to repress these phenotypes, suggesting that ZRG17 functions in zinc uptake . These findings illustrate the utility of transposon tagging as an approach for studying regulated gene expression in yeast.

Genetics, 2000 Sep, 156(1), 21 - 9
Important role for phylogenetically invariant PP2Acalpha active site and C-terminal residues revealed by mutational analysis in Saccharomyces cerevisiae; Evans DR et al.; PP2A is a central regulator of eukaryotic signal transduction . The human catalytic subunit PP2Acalpha functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo . Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues . The PP2Acalpha Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88 . The V159E substitution similarly impaired PP2Acalpha catalysis profoundly and may cause global disruption of the active site . Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro . Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity . Substitution of the PP2Acalpha C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo . Nevertheless, Y307D did not reduce PP2Acalpha catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions . Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

Curr Genet, 2000 Aug, 38(2), 78 - 86
Characterisation of a tripartite nuclear localisation sequence in the regulatory protein Lys14 of Saccharomyces cerevisiae; El Alami M et al.; The Lys14 regulatory protein of Saccharomyces cerevisiae activates the expression of the LYS genes involved in the lysine biosynthetic pathway . Studies with a fused Lys14-green fluorescent protein reveal that Lys14p is localised to the nucleus, even under growth conditions leading to the absence of LYS gene expression . Lys14p nuclear localisation is mediated by a tripartite sequence made up of three short basic motifs located on the C-terminal side of the Zn cluster domain of Lys14p . Substitution of basic residues by alanines in any of the three motifs partially prevents the nuclear import of the protein . Simultaneous mutations in the three basic domains are required to completely abolish the entry into the nucleus and to impair the Lys14 function.

Microbiology, 2000 Sep, 146 ( Pt 9), 2113 - 20
Hyperphosphorylation of Msn2p and Msn4p in response to heat shock and the diauxic shift is inhibited by cAMP in Saccharomyces cerevisiae; Garreau H et al.; In response to various stresses, as well as during the diauxic transition, the Msn2p and Msn4p transcription factors of Saccharomyces cerevisiae are activated and induce a large set of genes . This activation is inhibited by the Ras/cAMP/PKA (cAMP-dependent protein kinase) pathway . Here we show by immunoblotting experiments that Msn2p and Msn4p are phosphorylated in vivo during growth on glucose, and become hyperphosphorylated at the diauxic transition and upon heat shock . This hyperphosphorylation is correlated with activation of Msn2/4p-dependent transcription . An increased level of cAMP prevents and reverses these hyperphosphorylations, indicating that kinases other than PKA are involved . These results suggest that PKA and stress-activated kinases control Msn2/4p activity by antagonistic phosphorylation . It was also noted that Msn4p is transiently increased at the diauxic transition . Msn2p and Msn4p present different hyperphosphorylation patterns in response to different stresses.

J Biol Chem, 2000 Nov 24, 275(47), 37251 - 6
Functional connections between mediator components and general transcription factors of Saccharomyces cerevisiae; Sakurai H et al.; The yeast Gal11 protein is an important component of the Mediator complex in RNA polymerase II-directed transcription . Gal11 and the general transcription factor (TF) IIE are involved in regulation of the protein kinase activity of TFIIH that phosphorylates the carboxyl-terminal domain of RNA polymerase II . We have previously shown that Gal11 binds the small and large subunits of TFIIE at two Gal11 domains, A and B, respectively, which are important for normal function of Gal11 in vivo . Here we demonstrate that Gal11 binds directly to TFIIH through domain A in vitro . A null mutation in GAL11 caused lethality of cells when combined with temperature-sensitive mutations in the genes encoding TFIIE or the carboxyl-terminal domain kinase, indicating the presence of genetic interactions between Gal11 and these proteins . Mutational depletion of Gal11 or TFIIE caused inefficient opening of the transcription initiation region, but had no significant effect on TATA-binding protein occupancy of the TATA sequence in vivo . These results suggest that the functions of Gal11 and TFIIE are necessary after recruitment of TATA-binding protein to the TATA box presumably at the step of stable preinitiation complex formation and/or promoter melting . We illustrate genetic interactions between Gal11 and other Mediator components such as Med2 and Pgd1/Hrs1/Med3.

J Biol Chem, 2000 Oct 27, 275(43), 33197 - 200
Role of sterols in modulating the human mu-opioid receptor function in Saccharomyces cerevisiae; Lagane B et al.; This study provides evidence that the differences in membrane composition found from one cell type to another can represent a limiting factor to recovering the functionality of transmembrane proteins when expressed in heterologous systems . Restoring the properties of the human mu-opioid receptor in yeast (Saccharomyces cerevisiae), similar to those observed in native cells, was achieved by replacing ergosterol from yeast by cholesterol, which is normally found in mammalian plasma membranes . The results suggest that these two sterols have opposite effects with respect to the ligand binding function of the receptor . Ergosterol was found to constrain the mu-opioid receptor in an inactive state in yeast plasma membranes and cannot replace cholesterol in activating it . These data differ from previous works dealing with the function of related G-protein-coupled receptors (GPCR) in ergosterol-enriched membranes . This suggests that structural requirements of GPCR with respect to their modulation by lipid components differ from one protein to another . As a consequence, we assume that the presence of appropriate lipids around transmembrane proteins determines their function . This highlights the functional significance of lateral heterogeneities of membrane components within biological membranes.

Biotechnol Bioeng, 2000 Oct 20, 70(2), 187 - 96
Green fluorescent protein in Saccharomyces cerevisiae: real-time studies of the GAL1 promoter; Li J et al.; Green fluorescent protein (GFP) was used to study the regulation of the galactose-inducible GAL1 promoter in yeast Saccharomyces cerevisiae strains . GFP was cloned into the pGAL110 vector and transformed into the yeast strains . Time course studies comparing culture fluorescence intensity and GFP concentration were conducted along with on-line monitoring of GFP expression . Our results demonstrated that GFP fluorescence could be used as a quantifiable on-line reporter gene in yeast strains . The effect of an integrated GAL10p-GAL4 transcription cassette was investigated . Induction time studies showed that there was no significant difference in GFP expression level by adding galactose at different culture times . A wide range of galactose concentrations was used to study the initial galactose concentration effect on GFP expression kinetics . A minimum of 0.05 g/L galactose doubled the GFP fluorescence signal as compared to the control, whereas 0.1 g/L gave the highest specific GFP yield . A simple analytical model was proposed to describe GFP expression kinetics based on the experimental results . In addition, this GFP-based approach was shown to have potential use for high-throughput studies . The use of GFP as a generic tool provided important insights to the GAL expression system and has great potential for further process optimization applications .

EMBO J, 2000 Sep 1, 19(17), 4524 - 32
Cbk1p, a protein similar to the human myotonic dystrophy kinase, is essential for normal morphogenesis in Saccharomyces cerevisiae; Racki WJ et al.; We have studied the CBK1 gene of Saccharomyces cerevisiae, which encodes a conserved protein kinase similar to the human myotonic dystrophy kinase . We have shown that the subcellular localization of the protein, Cbk1p, varies in a cell cycle-dependent manner . Three phenotypes are associated with the inactivation of the CBK1 gene: large aggregates of cells, round rather than ellipsoidal cells and a change from a bipolar to a random budding pattern . Two-hybrid and extragenic suppressor studies have linked Cbk1p with the transcription factor Ace2p, which is responsible for the transcription of chitinase . Cbk1p is necessary for the activation of Ace2p and we have shown that the aggregation phenotype is due to a lack of chitinase expression . The random budding pattern and the round cell phenotype of the CBK1 deletion strain show that in addition to its role in regulating chitinase expression via Ace2p, Cbk1p is essential for a wild-type morphological development of the cell.

Annu Rev Biochem, 2000, 69, 571 - 95
Mechanisms and control of mRNA decapping in Saccharomyces cerevisiae; Tucker M et al.; The process of mRNA turnover is a critical component of the regulation of gene expression . In the past few years a discrete set of pathways for the degradation of polyadenylated mRNAs in eukaryotic cells have been described . A major pathway of mRNA degradation in yeast occurs by deadenylation of the mRNA, which leads to a decapping reaction, thereby exposing the mRNA to rapid 5' to 3' exonucleolytic degradation . A critical step in this pathway is decapping, since it effectively terminates the existence of the mRNA and is the site of numerous control inputs . In this review, we discuss the properties of the decapping enzyme and how its activity is regulated to give rise to differential mRNA turnover . A key point is that decapping appears to be controlled by access of the enzyme to the cap structure in a competition with the translation initiation complex . Strikingly, several proteins required for mRNA decapping show interactions with the translation machinery and suggest possible mechanisms for the triggering of mRNA decapping.

J Mol Biol, 2000 Sep 8, 302(1), 103 - 20
The Saccharomyces cerevisiae homologue YPA1 of the mammalian phosphotyrosyl phosphatase activator of protein phosphatase 2A controls progression through the G1 phase of the yeast cell cycle; Van Hoof C et al.; The Saccharomyces cerevisiae gene YPA1 encodes a protein homologous to the phosphotyrosyl phosphatase activator, PTPA, of the mammalian protein phosphatase type 2A (PP2A) . In order to examine the biological role of PTPA, we disrupted YPA1 and characterised the phenotype of the ypa1Delta mutant . Comparison of the growth rate of the wild-type strain and the ypa1Delta mutant on glucose-rich medium after nutrient depletion showed that the ypa1Delta mutant traversed the lag period more rapidly . This accelerated progression through "Start" was also observed after release from alpha-factor-induced G1 arrest as evidenced by a higher number of budding cells, a faster increase in CLN2 mRNA expression and a more rapid reactivation of Cdc28 kinase activity . This phenotype was specific for deletion of YPA1 since it was not observed when YPA2, the second PTPA gene in budding yeast was deleted . Reintroduction of YPA1 or the human PTPA cDNA in the ypa1Delta mutant suppressed this phenotype as opposed to overexpression of YPA2 . Disruption of both YPA genes is lethal, since sporulation of heterozygous diploids resulted in at most three viable spores, none of them with a ypa1Delta ypa2Delta genotype . This observation indicates that YPA1 and YPA2 share some essential functions . We compared the ypa1Delta mutant phenotype with a PP2A double deletion mutant and a PP2A temperature-sensitive mutant . The PP2A-deficient yeast strain also showed accelerated progression through the G1 phase . In addition, both PP2A and ypa1Delta mutants show similar aberrant bud morphology . This would support the notion that YPA1 may act as a positive regulator of PP2A in vivo .

J Bacteriol, 2000 Sep, 182(18), 5121 - 6
Cooperative regulation of DOG2, encoding 2-deoxyglucose-6-phosphate phosphatase, by Snf1 kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade in stress responses of Saccharomyces cerevisiae; Tsujimoto Y et al.; We screened the genome of Saccharomyces cerevisiae for the genes responsive to oxidative stress by using the lacZ transposon-insertion library . As a result, we found that expression of the DOG2 gene coding for 2-deoxyglucose-6-phosphate phosphatase was induced by oxidative stress . The expression of DOG2 was also induced by osmotic stress . We found a putative cis element (STRE, a stress response element) in the DOG2 promoter adjacent to a consensus sequence to which the Mig1p repressor is known to bind . The basal levels of DOG2 gene expression were increased in a mig1Delta mutant, while the derepression of DOG2 was not observed in a snf1Delta mutant under glucose-deprived conditions . Induction of the DOG2 gene expression by osmotic stress was observed in any of the three disruptants pbs2Delta, hog1Delta, and snf1Delta . However, the osmotic induction was completely abolished in both the snf1Delta pbs2Delta mutant and the snf1Delta hog1Delta mutant . Additionally, these single mutants as well as double mutants failed to induce DOG2 expression by oxidative stress . These results suggest that Snf1p kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade are likely to be involved in the signaling pathway of oxidative stress and osmotic stress in regulation of DOG2.

Yeast, 2000 Sep 15, 16(12), 1131 - 45
Relationship of codon bias to mRNA concentration and protein length in Saccharomyces cerevisiae; Coghlan A et al.; In 1982, Ikemura reported a strikingly unequal usage of different synonymous codons, in five Saccharomyces cerevisiae nuclear genes having high protein levels . To study this trend in detail, we examined data from three independent studies that used oligonucleotide arrays or SAGE to estimate mRNA concentrations for nearly all genes in the genome . Correlation coefficients were calculated for the relationship of mRNA concentration to four commonly used measures of synonymous codon usage bias: the codon adaptation index (CAI), the codon bias index (CBI), the frequency of optimal codons (F(op)), and the effective number of codons (N(c)) . mRNA concentration was best approximated as an exponential function of each of these four measures . Of the four, the CAI was the most strongly correlated with mRNA concentration (r(s)=0.62+/-0.01, n=2525, p<10(-17)) . When we controlled for CAI, mRNA concentration and protein length were negatively correlated (partial r(s)=-0.23+/-0.01, n=4765, p<10(-17)) . This may result from selection to reduce the size of abundant proteins to minimize transcriptional and translational costs . When we controlled for mRNA concentration, protein length and CAI were positively correlated (partial r(s)=0.16+/-0.01, n=4765, p<10(-17)) . This may reflect more effective selection in longer genes against missense errors during translation . The correlation coefficients between the mRNA levels of individual genes, as measured by different investigators and methods, were low, in the range r(s)=0.39-0.68 .

Yeast, 2000 Sep 15, 16(12), 1107 - 10
Cells of the yeast Saccharomyces cerevisiae are transformable by DNA under non-artificial conditions; Nevoigt E et al.; Transformants of bakers' yeast (Saccharomyces cerevisiae) can be generated when non-growing cells metabolize sugars (without additional nutrients) in the presence of plasmid DNA . These results suggest that there is a mechanism by which DNA can naturally be taken up by the yeast cell . Natural transformation does not take place in common complete or minimal yeast culture media such as YPD and YNB . The starvation conditions used in our experiments thus seem to be an important prerequisite for such transformation events .

J Cell Biol, 2000 Aug 21, 150(4), 707 - 18
Conserved SR protein kinase functions in nuclear import and its action is counteracted by arginine methylation in Saccharomyces cerevisiae; Yun CY et al.; Mammalian serine and arginine-rich (SR) proteins play important roles in both constitutive and regulated splicing, and SR protein-specific kinases (SRPKs) are conserved from humans to yeast . Here, we demonstrate a novel function of the single conserved SR protein kinase Sky1p in nuclear import in budding yeast . The yeast SR-like protein Npl3p is known to enter the nucleus through a composite nuclear localization signal (NLS) consisting of a repetitive arginine- glycine-glycine (RGG) motif and a nonrepetitive sequence . We found that the latter is the site for phosphorylation by Sky1p and that this phosphorylation regulates nuclear import of Npl3p by modulating the interaction of the RGG motif with its nuclear import receptor Mtr10p . The RGG motif is also methylated on arginine residues, but methylation does not affect the Npl3p-Mtr10p interaction in vitro . Remarkably, arginine methylation interferes with Sky1p-mediated phosphorylation, thereby indirectly influencing the Npl3p-Mtr10p interaction in vivo and negatively regulating nuclear import of Npl3p . These results suggest that nuclear import of Npl3p is coordinately influenced by methylation and phosphorylation in budding yeast, which may represent conserved components in the dynamic regulation of RNA processing in higher eukaryotic cells.

Electrophoresis, 2000 Jul, 21(13), 2610 - 6
Towards higher resolution: two-dimensional electrophoresis of Saccharomyces cerevisiae proteins using overlapping narrow immobilized pH gradients; Wildgruber R et al.; The rising number of proteome projects leads to new challenges for two-dimensional electrophoresis with immobilized pH gradients and different applications of this technique . Not only wide pH gradients such as 4-12 or 3-12 (Gorg et al., Electrophoresis 1999, 20, 712-717) which can give an overview of the total protein expressions of cells are in demand but also overlapping narrow immobilized pH gradients are to be used for more specialized and detailed research and micropreparative separations . The advantage of overlapping narrow pH gradients is the gain in higher resolution by stretching the protein pattern in the first dimension . This simplifies computer-aided image analysis and protein identification (e.g., by mass spectrometry) . In this study the protein patterns of yeast cells in pH gradients 4-5, 4.5-5.5, 5-6, 5.5-6.7 and 6-9 are presented and compared to the pH 4-7 and 3-10 gradients . This combination allowed us to reveal a total of 2286 yeast protein spots compared to 755 protein spots in the pH 3-10 gradient.

Eur J Clin Microbiol Infect Dis, 2000 Jun, 19(6), 468 - 70
Fungemia with Saccharomyces cerevisiae in two newborns, only one of whom had been treated with ultra-levura; Perapoch J et al.; Ultra-Levura (Upsamedica, Spain) is a yeast (Saccharomyces boulardii) widely used as a biotherapeutic agent . To date, few adverse effects have been reported, although fungemia with Saccharomyces cerevisiae can occur in weak and immunosuppressed patients . Reported here are two cases of fungemia with Saccharomyces cerevisiae . One patient had been treated with Ultra-Levura and the other contracted the infection from the first . This is the first report of infection with Saccharomyces boulardii (Saccharomyces cerevisiae) in a patient who was not being treated with the agent.

Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 1020 - 3
Crystallization and X-ray diffraction analysis of peroxisomal Delta3-Delta2-enoyl-CoA isomerase from Saccharomyces cerevisiae; Mursula AM et al.; The purification, crystallization and X-ray diffraction analysis of Saccharomyces cerevisiae Delta(3)-Delta(2)-enoyl-CoA isomerase is described . Delta(3)-Delta(2)-Enoyl-CoA isomerase is a member of the hydratase/isomerase protein family and is an auxiliary enzyme required for the beta-oxidation of unsaturated fatty acids . It is a hexameric enzyme consisting of six identical 32 kDa subunits of 280 residues each . In crystallization trials three crystal forms were obtained, with tetragonal and hexagonal lattices . A 2.5 A data set was collected from the unliganded hexagonal crystals with an R(merge) of 6.6% . The crystal, with unit-cell parameters a = 116.0, b = 116.0, c = 122.9 A, is likely to have P6(3)22 symmetry.

Proc Natl Acad Sci U S A, 2000 Aug 15, 97(17), 9537 - 42
Restriction of ectopic recombination by interhomolog interactions during Saccharomyces cerevisiae meiosis; Goldman AS et al.; In Saccharomyces cerevisiae meiosis, recombination occurs frequently between sequences at the same location on homologs (allelic recombination) and can take place between dispersed homologous sequences (ectopic recombination) . Ectopic recombination occurs less often than does allelic, especially when homologous sequences are on heterologous chromosomes . To account for this, it has been suggested that homolog pairing (homolog colocalization and alignment) either promotes allelic recombination or restricts ectopic recombination . The latter suggestion was tested by examining ectopic recombination in two cases where normal interhomolog relationships are disrupted . In the first case, one member of a homolog pair was replaced by a homologous (related but not identical) chromosome that has diverged sufficiently to prevent allelic recombination . In the second case, ndj1 mutants were used to delay homolog pairing and synapsis . Both circumstances resulted in a substantial increase in the frequency of ectopic recombination between arg4-containing plasmid inserts located on heterologous chromosomes . These findings suggest that, during normal yeast meiosis, progressive homolog colocalization, alignment, synapsis, and allelic recombination restrict the ability of ectopically located sequences to find each other and recombine . In the absence of such restrictions, the meiotic homology search may encompass the entire genome.

J Biol Chem, 2000 Nov 3, 275(44), 34140 - 6
Cpr6 and Cpr7, two closely related Hsp90-associated immunophilins from Saccharomyces cerevisiae, differ in their functional properties; Mayr C et al.; Hsp90 is an abundant cytosolic molecular chaperone . It controls the folding of target proteins including steroid hormone receptors and kinases in complex with several partner proteins . Prominent members of this protein family are large peptidyl prolyl cis/trans isomerases (PPIases), which catalyze the cis/trans isomerization of prolyl peptide bonds in proteins and possess chaperone activity . In Saccharomyces cerevisiae, two closely related large Hsp90-associated PPIases, Cpr6 and Cpr7, exist . We show here that these homologous proteins bind with comparable affinity to Hsp90 but exhibit significant structural and functional differences . Cpr6 is more stable than Cpr7 against thermal denaturation and displays an up to 100-fold higher PPIase activity . In contrast, the chaperone activity of Cpr6 is much lower than that of Cpr7 . Based on these results we suggest that the two immunophilins perform overlapping but not identical tasks in the Hsp90 chaperone cycle.

J Bacteriol, 2000 Sep, 182(17), 5017 - 9
Regulation of cation-coupled high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae; Pattison-Granberg J et al.; Studies of the high-affinity phosphate transporters in the yeast Saccharomyces cerevisiae using mutant strains lacking either the Pho84 or the Pho89 permease revealed that the transporters are differentially regulated . Although both genes are induced by phosphate starvation, activation of the Pho89 transporter precedes that of the Pho84 transporter early in the growth phase in a way which may possibly reflect a fine tuning of the phosphate uptake process relative to the availability of external phosphate.

J Bacteriol, 2000 Sep, 182(17), 4752 - 7
Chitin synthesis in a gas1 mutant of Saccharomyces cerevisiae; Valdivieso MH et al.; The existence of a compensatory mechanism in response to cell wall damage has been proposed in yeast cells . The increase of chitin accumulation is part of this response . In order to study the mechanism of the stress-related chitin synthesis, we tested chitin synthase I (CSI), CSII, and CSIII in vitro activities in the cell-wall-defective mutant gas1 delta . CSI activity increased twofold with respect to the control, a finding in agreement with an increase in the expression of the CHS1 gene . However, deletion of the CHS1 gene did not affect the phenotype of the gas1 delta mutant and only slightly reduced the chitin content . Interestingly, in chs1 gas1 double mutants the lysed-bud phenotype, typical of chs1 null mutant, was suppressed, although in gas1 cells there was no reduction in chitinase activity . CHS3 expression was not affected in the gas1 mutant . Deletion of the CHS3 gene severely compromised the phenotype of gas1 cells, despite the fact that CSIII activity, assayed in membrane fractions, did not change . Furthermore, in chs3 gas1 cells the chitin level was about 10% that of gas1 cells . Thus, CSIII is the enzyme responsible for the hyperaccumulation of chitin in response to cell wall stress . However, the level of enzyme or the in vitro CSIII activity does not change . This result suggests that an interaction with a regulatory molecule or a posttranslational modification, which is not preserved during membrane fractionation, could be essential in vivo for the stress-induced synthesis of chitin.

J Bacteriol, 2000 Sep, 182(17), 4730 - 7
The mitochondrial alcohol dehydrogenase Adh3p is involved in a redox shuttle in Saccharomyces cerevisiae; Bakker BM et al.; NDI1 is the unique gene encoding the internal mitochondrial NADH dehydrogenase of Saccharomyces cerevisiae . The enzyme catalyzes the transfer of electrons from intramitochondrial NADH to ubiquinone . Surprisingly, NDI1 is not essential for respiratory growth . Here we demonstrate that this is due to in vivo activity of an ethanol-acetaldehyde redox shuttle, which transfers the redox equivalents from the mitochondria to the cytosol . Cytosolic NADH can be oxidized by the external NADH dehydrogenases . Deletion of ADH3, encoding mitochondrial alcohol dehydrogenase, did not affect respiratory growth in aerobic, glucose-limited chemostat cultures . Also, an ndi1Delta mutant was capable of respiratory growth under these conditions . However, when both ADH3 and NDI1 were deleted, metabolism became respirofermentative, indicating that the ethanol-acetaldehyde shuttle is essential for respiratory growth of the ndi1 delta mutant . In anaerobic batch cultures, the maximum specific growth rate of the adh3 delta mutant (0.22 h(-1)) was substantially reduced compared to that of the wild-type strain (0.33 h(-1)) . This is consistent with the hypothesis that the ethanol-acetaldehyde shuttle is also involved in maintenance of the mitochondrial redox balance under anaerobic conditions . Finally, it is shown that another mitochondrial alcohol dehydrogenase is active in the adh3 delta ndi1 delta mutant, contributing to residual redox-shuttle activity in this strain.

Mol Cell Biol, 2000 Sep, 20(17), 6390 - 8
Functional studies on the candidate ATPase domains of Saccharomyces cerevisiae MutLalpha; Tran PT et al.; Saccharomyces cerevisiae MutL homologues Mlh1p and Pms1p form a heterodimer, termed MutLalpha, that is required for DNA mismatch repair after mismatch binding by MutS homologues . Recent sequence and structural studies have placed the NH(2) termini of MutL homologues in a new family of ATPases . To address the functional significance of this putative ATPase activity in MutLalpha, we mutated conserved motifs for ATP hydrolysis and ATP binding in both Mlh1p and Pms1p and found that these changes disrupted DNA mismatch repair in vivo . Limited proteolysis with purified recombinant MutLalpha demonstrated that the NH(2) terminus of MutLalpha undergoes conformational changes in the presence of ATP and nonhydrolyzable ATP analogs . Furthermore, two-hybrid analysis suggested that these ATP-binding-induced conformational changes promote an interaction between the NH(2) termini of Mlh1p and Pms1p . Surprisingly, analysis of specific mutants suggested differential requirements for the ATPase motifs of Mlh1p and Pms1p during DNA mismatch repair . Taken together, these results suggest that MutLalpha undergoes ATP-dependent conformational changes that may serve to coordinate downstream events during yeast DNA mismatch repair.

Metab Eng, 2000 Jan, 2(1), 49 - 68
Dynamic optimal control of homeostasis: an integrative system approach for modeling of the central nitrogen metabolism in Saccharomyces cerevisiae; van Riel NA et al.; The theory of dynamic optimal metabolic control (DOMC), as developed by Giuseppin and Van Riel (Metab . Eng., 2000), is applied to model the central nitrogen metabolism (CNM) in Saccharomyces cerevisiae . The CNM represents a typical system encountered in advanced metabolic engineering . The CNM is the source of the cellular amino acids and proteins, including flavors and potentially valuable biomolecules; therefore, it is also of industrial interest . In the DOMC approach the cell is regarded as an optimally controlled system . Given the metabolic genotype, the cell faces a control problem to maintain an optimal flux distribution in a changing environment . The regulation is based on strategies and balances feedback control of homeostasis and feedforward regulation for adaptation . The DOMC approach is an integrative, holistic approach, not based on mechanistic descriptions and (therefore) not biased by the variation present in biochemical and molecular biological data . It is an effective tool to structure the rapidly increasing amount of data on the function of genes and pathways . The DOMC model is used successfully to predict the responses of pulses of ammonia and glutamine to nitrogen-limited continuous cultures of a wild-type strain and a glutamine synthetase-negative mutant . The simulation results are validated with experimental data.

Metab Eng, 1999 Apr, 1(2), 128 - 40
In vivo dynamics of the pentose phosphate pathway in Saccharomyces cerevisiae; Vaseghi S et al.; The in vivo dynamics of the pentose phosphate pathway has been studied with transient experiments in continuous culture of Saccharomyces cerevisiae . Rapid sampling was performed with a special sampling device after disturbing the steady state with a pulse of glucose . The time span of observation was 120 s after the pulse . During this short time period the dynamic effect of protein biosynthesis can be neglected . The metabolites of interest (glucose 6-phosphate, NADP, NADPH, 6-phosphogluconate, and MgATP2-) we determined with enzymatic assays and HPLC . The experimental observations were then used for the identification of kinetic rate equations and parameters under in vivo conditions . In accordance with results from in vitro studies the in vivo diagnosis supports an ordered Bi-Bi mechanism with noncompetitive inhibition by MgATP2- for the enzyme glucose-6-phosphate dehydrogenase . In the case of 6-phosphogluconate dehydrogenase an ordered Bi-Ter mechanism with a competitive inhibition by MgATP2- has been found . Because the MgATP2- concentration decreases abruptly after the pulse of glucose the inhibitory effect vanishes and the flux through the pentose phosphate pathway increases . This regulation phenomenon guarantees the balance of fluxes through glycolysis and pentose phosphate pathway during the dynamic time period.

Protein Sci, 2000 Jul, 9(7), 1374 - 81
The X-ray structure of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae; Monzingo AF et al.; Eucaryotes possess one or more NADP-dependent methylene-THF dehydrogenases as part of multifunctional enzymes . In addition, yeast expresses an unusual monofunctional NAD-dependent enzyme, yMTD . We report X-ray structures for the apoenzyme and its complex with NAD+ at 2.8 and 3.0 A resolution, respectively . The protein fold resembles that seen for the human and Escherichia coli dehydrogenase/cyclohydrolase bifunctional enzymes . The enzyme has two prominent domains, with the active site cleft between them . yMTD has a noncanonical NAD-binding domain that has two inserted strands compared with the NADP-binding domains of the bifunctional enzymes . This insert precludes yMTD from dimerizing in the same way as the bifunctional enzymes . yMTD functions as a dimer, but the mode of dimerization is novel . It does not appear that the difference in dimerization accounts for the difference in cofactor specificity or for the loss of cyclohydrolase activity . These functional differences are probably accounted for by minor differences within the tertiary structure of the active site of the monomeric protein.

J Cell Biol, 2000 Aug 7, 150(3), 461 - 74
Mutants affecting the structure of the cortical endoplasmic reticulum in Saccharomyces cerevisiae; Prinz WA et al.; We find that the peripheral ER in Saccharomyces cerevisiae forms a dynamic network of interconnecting membrane tubules throughout the cell cycle, similar to the ER in higher eukaryotes . Maintenance of this network does not require microtubule or actin filaments, but its dynamic behavior is largely dependent on the actin cytoskeleton . We isolated three conditional mutants that disrupt peripheral ER structure . One has a mutation in a component of the COPI coat complex, which is required for vesicle budding . This mutant has a partial defect in ER segregation into daughter cells and disorganized ER in mother cells . A similar phenotype was found in other mutants with defects in vesicular trafficking between ER and Golgi complex, but not in mutants blocked at later steps in the secretory pathway . The other two mutants found in the screen have defects in the signal recognition particle (SRP) receptor . This receptor, along with SRP, targets ribosome-nascent chain complexes to the ER membrane for protein translocation . A conditional mutation in SRP also disrupts ER structure, but other mutants with translocation defects do not . We also demonstrate that, both in wild-type and mutant cells, the ER and mitochondria partially coalign, and that mutations that disrupt ER structure also affect mitochondrial structure . Our data suggest that both trafficking between the ER and Golgi complex and ribosome targeting are important for maintaining ER structure, and that proper ER structure may be required to maintain mitochondrial structure.

Mol Microbiol, 2000 Aug, 37(3), 549 - 54
Evidence for a second function for Saccharomyces cerevisiae Rev1p; Nelson JR et al.; The function of the Saccharomyces cerevisiae REV1 gene is required for translesion replication and mutagenesis induced by a wide variety of DNA-damaging agents . We showed previously that Rev1p possesses a deoxycytidyl transferase activity, which incorporates dCMP opposite abasic sites in the DNA template, and that dCMP insertion is the major event during bypass of an abasic site in vivo . However, we now find that Rev1p function is needed for the bypass of a T-T (6-4) UV photoproduct, a process in which dCMP incorporation occurs only very rarely, indicating that Rev1p possesses a second function . In addition, we find that Rev1p function is, as expected, required for bypass of an abasic site . However, replication past this lesion was also much reduced in the G-193R rev1-1 mutant, which we find retains substantial levels of deoxycytidyl transferase activity . This mutant is, therefore, presumably deficient principally in the second, at present poorly defined, function . The bypass of an abasic site and T-T (6-4) lesion also depended on REV3 function, but neither it nor REV1 was required for replication past the T-T dimer; bypass of this lesion presumably depends on another enzyme.

Mol Microbiol, 2000 Jul, 37(2), 382 - 97
Response of Saccharomyces cerevisiae to severe osmotic stress: evidence for a novel activation mechanism of the HOG MAP kinase pathway; Van Wuytswinkel O et al.; The HOG/p38 MAP kinase route is an important stress-activated signal transduction pathway that is well conserved among eukaryotes . Here, we describe a novel mechanism of activation of the HOG pathway in budding yeast . This mechanism operates upon severe osmostress conditions (1.4 M NaCl) and is independent of the Sln1p and Sho1p osmosensors . The alternative input feeds into the HOG pathway MAPKK Pbs2p and requires activation of Pbs2p by phosphorylation . We show that, upon severe osmotic shock, Hog1p nuclear accumulation and phosphorylation is delayed compared with mild stress . Moreover, both events lost their transient pattern, presumably because of the absence of negative feedback mediated by Ptp2p tyrosine phosphatase, which we found to be localized in the nucleus . Under severe osmotic stress conditions, the delayed nuclear accumulation correlates with a delay in stress-responsive gene expression . Severe osmoshock leads to a situation in which active and nuclear-localized Hog1p is transiently unable to induce transcription of osmotic stress-responsive genes . It also appeared from our studies that the Sho1p osmosensor is less active under severe osmotic stress conditions, whereas the Sln1p/Ypd1p/Ssk1p sensor and signal transducer functions normally under these circumstances.

Mol Microbiol, 2000 Jun, 36(6), 1403 - 14
Evidence for the genetic interaction between the actin-binding protein Vrp1 and the RhoGAP Rgd1 mediated through Rho3p and Rho4p in Saccharomyces cerevisiae; Roumanie O et al.; The non-essential RGD1 gene from Saccharomyces cerevisiae encodes a protein that has been characterized in vitro as a Rho GTPase activating protein (RhoGAP) for the Rho3 and Rho4 proteins . Rgd1p, which displays a conserved FCH-coiled coil-Rho-GAP domain organization, showed a patch-like distribution in the cell, including a localization in growing buds . Using a genetic screen, we found that rgd1delta and vrp1alpha mutations exhibited a synthetic lethality, thus revealing an interaction between these genes . The VRP1 product is an actin and myosin interacting protein involved in polarized growth . Using mutant forms of both Rho3 and Rho4 proteins, we provide evidence for the involvement of these two GTPases in RGD1-VRP1 co-lethality . In addition, these results strongly argue in favour of Rho3p and Rho4p being the targets of Rgd1p RhoGAP activity in vivo . Genetic relationships between either VRP1 or RGD1 and actin cytoskeleton-linked genes were also studied . These and other well-established data support the idea that Vrp1, Las17, Rvs167 proteins belong to the same complex . This protein structure might act with myosins in various actin cytoskeleton-based activities, in co-operation with a Rho3p/Rho4p signalling pathway that is negatively regulated by Rgd1p GAP activity.

Mol Microbiol, 2000 Jun, 36(6), 1381 - 90
The control of intracellular glycerol in Saccharomyces cerevisiae influences osmotic stress response and resistance to increased temperature; Siderius M et al.; Glycerol has been demonstrated to serve as the major osmolyte of Saccharomyces cerevisiae . Consistently, mutant strains gpd1gpd2 and gpp1gpp2, which are devoid of the main glycerol biosynthesis pathway, have been shown to be osmosensitive . In addition, the primary hyperosmotic stress response is affected in these strains . Hog1p phosphorylation turned out to be prolonged and osmostress-induced gene expression is delayed compared with the kinetics observed in wild-type cells . A hog1 deletion strain was previously found to contain lower internal glycerol and therefore displays an osmosensitive phenotype . Here, we show that the osmosensitivity of hog1 is suppressed by growth at 37 degrees C . We reasoned that this temperature-remedial osmoresistance might be caused by a higher intracellular glycerol level at the elevated temperature . This hypothesis was confirmed by measurement of the glycerol concentration, which was shown to be similar for wild type and hog1 cells only at elevated growth temperatures . In agreement with this finding, hog1 cells containing an fps1 allele, encoding a constitutively open glycerol channel, have lost their temperature-remedial osmoresistance . Furthermore, gpd1gpd2 and gpp1gpp2 strains were found to be temperature sensitive . The growth defect of these strains could be suppressed by adding external glycerol . In conclusion, the ability to control glycerol levels influences proper osmostress-induced signalling and the cellular potential to grow at elevated temperatures . These data point to an important, as yet unidentified, role of glycerol in cellular functioning.

Acta Crystallogr D Biol Crystallogr, 2000 Jul, 56 ( Pt 7), 902 - 3
Crystallization and preliminary X-ray diffraction analysis of the mitochondrial transcription factor sc-mtTFB from Saccharomyces cerevisiae; Schubot FD et al.; Eukaryotic mitochondria contain a distinct mini-chromosome . In yeast, transcription of the mitochondrial genome is mediated by a nuclear-encoded RNA polymerase consisting of a single polypeptide core enzyme and a specificity factor termed sc-mtTFB which bears some similarity to bacterial sigma-factors . sc-mtTFB from Saccharomyces cerevisiae has been cloned, expressed, purified and crystallized . The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 89.7, b = 44.6, c = 98.9 A, beta = 110 degrees . Based on one molecule per asymmetric unit, the solvent content is estimated to be 48% . Small crystals of dimensions 0.01 x 0.05 x 0.13 mm diffract to at least 2.7 A resolution on a rotating-anode X-ray source.

Exp Mol Med, 2000 Jun 30, 32(2), 61 - 6
Murine pro-tumor necrosis factor expressed in Saccharomyces cerevisiae HF7c localizes to membrane/particulate; Jeong JY et al.; Tumor necrosis factor (TNF) is a cytokine that is produced by immune cells in response to bacterial and viral stimuli and plays important roles in various inflammatory diseases . TNF is produced as a membrane-bound precursor, which is then cleaved to release soluble mature protein . We expressed murine pro-TNF in Saccharomyces cerevisiae and examined processing and cellular localization of the recombinant protein . Yeast cells were transformed with an expression construct carrying the pro-TNF gene under the control of alcohol dehydrogenase promoter . Immunoblotting analysis of cell homogenate revealed expression of 26 kD pro-TNF in transformed cells . Upon centrifugation, pro-TNF transformed cells fractionated into the membrane/particulate . In a clone that expresses a high level of pro-TNF, mature 17 kD TNF was detected in the culture medium, although the amount was far smaller than that of cell-associated pro-TNF . Flow cytometric analysis of yeast spheroplasts demonstrated the presence of TNF on the cell surface . Our results show that pro-TNF expressed in yeast mainly resides in the cellular membrane with an orientation similar to that of pro-TNF produced in mammalian cells . Our data suggest that the transformed yeast cells can be used for the genetic analysis of pro-TNF processing machinery in immune cells.

J Biol Chem, 2000 Oct 27, 275(43), 33244 - 51
Characterization of the Saccharomyces cerevisiae high affinity copper transporter Ctr3; Pena MM et al.; Copper is an essential nutrient required for the activity of a number of enzymes with diverse biological roles . In the bakers' yeast Saccharomyces cerevisiae, copper is transported into cells by two high affinity copper transport proteins, Ctr1 and Ctr3 . Although Ctr1 and Ctr3 are functionally redundant, they bear little homology at the amino acid sequence level . In this report, we characterize Ctr3 with respect to its localization, assembly, and post-transcriptional regulation . Ctr3 is an integral membrane protein that assembles as a trimer to form a competent copper uptake permease at the plasma membrane . Whereas the CTR1 and CTR3 genes are similarly regulated at the transcriptional level in response to copper, post-transcriptional regulation of these proteins is distinct . Unlike Ctr1, the Ctr3 transporter is neither regulated at the level of protein degradation nor endocytosis as a function of elevated copper levels . Our studies suggest that Ctr3 constitutes a fundamental module found in all eukaryotic high affinity copper transporters to date, which is sufficient for copper uptake but lacks elements for post-transcriptional regulation by copper.

Genetics, 2000 Aug, 155(4), 1643 - 55
Identification of domains required for developmentally regulated SNARE function in Saccharomyces cerevisiae; Neiman AM et al.; Saccharomyces cerevisiae cells contain two homologues of the mammalian t-SNARE protein SNAP-25, encoded by the SEC9 and SPO20 genes . Although both gene products participate in post-Golgi vesicle fusion events, they cannot substitute for one another; Sec9p is active primarily in vegetative cells while Spo20p functions only during sporulation . We have investigated the basis for the developmental stage-specific differences in the function of these two proteins . Localization of the other plasma membrane SNARE subunits, Ssop and Sncp, in sporulating cells suggests that these proteins act in conjunction with Spo20p in the formation of the prospore membrane . In vitro binding studies demonstrate that, like Sec9p, Spo20p binds specifically to the t-SNARE Sso1p and, once bound to Sso1p, can complex with the v-SNARE Snc2p . Therefore, Sec9p and Spo20p interact with the same binding partners, but developmental conditions appear to favor the assembly of complexes with Spo20p in sporulating cells . Analysis of chimeric Sec9p/Spo20p molecules indicates that regions in both the SNAP-25 domain and the unique N terminus of Spo20p are required for activity during sporulation . Additionally, the N terminus of Spo20p is inhibitory in vegetative cells . Deletion studies indicate that activation and inhibition are separable functions of the Spo20p N terminus . Our results reveal an additional layer of regulation of the SNARE complex, which is necessary only in sporulating cells.

Genetics, 2000 Aug, 155(4), 1633 - 41
The Saccharomyces cerevisiae RAD6 group is composed of an error-prone and two error-free postreplication repair pathways; Xiao W et al.; The RAD6 postreplication repair and mutagenesis pathway is the only major radiation repair pathway yet to be extensively characterized . It has been previously speculated that the RAD6 pathway consists of two parallel subpathways, one error free and another error prone (mutagenic) . Here we show that the RAD6 group genes can be exclusively divided into three rather than two independent subpathways represented by the RAD5, POL30, and REV3 genes; the REV3 pathway is largely mutagenic, whereas the RAD5 and the POL30 pathways are deemed error free . Mutants carrying characteristic mutations in each of the three subpathways are phenotypically indistinguishable from a single mutant such as rad18, which is defective in the entire RAD6 postreplication repair/tolerance pathway . Furthermore, the rad18 mutation is epistatic to all single or combined mutations in any of the above three subpathways . Our data also suggest that MMS2 and UBC13 play a key role in coordinating the response of the error-free subpathways; Mms2 and Ubc13 form a complex required for a novel polyubiquitin chain assembly, which probably serves as a signal transducer to promote both RAD5 and POL30 error-free postreplication repair pathways . The model established by this study will facilitate further research into the molecular mechanisms of postreplication repair and translesion DNA synthesis . In view of the high degree of sequence conservation of the RAD6 pathway genes among all eukaryotes, the model presented in this study may also apply to mammalian cells and predicts links to human diseases.

Genetics, 2000 Aug, 155(4), 1593 - 606
POB3 is required for both transcription and replication in the yeast Saccharomyces cerevisiae; Schlesinger MB et al.; Spt16 and Pob3 form stable heterodimers in Saccharomyces cerevisiae, and homologous proteins have also been purified as complexes from diverse eukaryotes . This conserved factor has been implicated in both transcription and replication and may affect both by altering the characteristics of chromatin . Here we describe the isolation and properties of a set of pob3 mutants and confirm that they have defects in both replication and transcription . Mutation of POB3 caused the Spt(-) phenotype, spt16 and pob3 alleles displayed severe synthetic defects, and elevated levels of Pob3 suppressed some spt16 phenotypes . These results are consistent with previous reports that Spt16 and Pob3 act in a complex that modulates transcription . Additional genetic interactions were observed between pob3 mutations and the genes encoding several DNA replication factors, including POL1, CTF4, DNA2, and CHL12 . pob3 alleles caused sensitivity to the ribonucleotide reductase inhibitor hydroxyurea, indicating a defect in a process requiring rapid dNTP synthesis . Mutation of the S phase checkpoint gene MEC1 caused pob3 mutants to lose viability rapidly under restrictive conditions, revealing defects in a process monitored by Mec1 . Direct examination of DNA contents by flow cytometry showed that S phase onset and progression were delayed when POB3 was mutated . We conclude that Pob3 is required for normal replication as well as for transcription.

RNA, 2000 Jul, 6(7), 937 - 51
Pentamidine inhibits mitochondrial intron splicing and translation in Saccharomyces cerevisiae; Zhang Y et al.; Pentamidine inhibits in vitro splicing of nuclear group I introns from rRNA genes of some pathogenic fungi and is known to inhibit mitochondrial function in yeast . Here we report that pentamidine inhibits the self-splicing of three group I and two group II introns of yeast mitochondria . Comparison of yeast strains with different configurations of mitochondrial introns (12, 5, 4, or 0 introns) revealed that strains with the most introns were the most sensitive to growth inhibition by pentamidine on glycerol medium . Analysis of blots of RNA from yeast strains grown in raffinose medium in the presence or absence of pentamidine revealed that the splicing of seven group I and two group II introns that have intron reading frames was inhibited by the drug to varying extents . Three introns without reading frames were unaffected by the drug in vivo, and two of these were inhibited in vitro, implying that the drug affects splicing by acting directly on RNA in vitro, but on another target in vivo . Because the most sensitive introns in vivo are the ones whose splicing depends on a maturase encoded by the intron reading frames, we tested pentamidine for effects on mitochondrial translation . We found that the drug inhibits mitochondrial but not cytoplasmic translation in cells at concentrations that inhibit mitochondrial intron splicing . Therefore, pentamidine is a potent and specific inhibitor of mitochondrial translation, and this effect explains most or all of its effects on respiratory growth and on in vivo splicing of mitochondrial introns.

J Pept Res, 2000 Jul, 56(1), 24 - 34
Position 13 analogs of the tridecapeptide mating pheromone from Saccharomyces cerevisiae: design of an iodinatable ligand for receptor binding; Liu S et al.; Analogs of the alpha-factor tridecapeptide mating pheromone (WHWLQLKPGQPMY) from Saccharomyces cerevisiae in which Tyr13 was replaced with Phe, p-F-Phe, m-F-Phe, p-NO2-Phe, p-NH2-Phe or Ser were synthesized and purified to >99% homogeneity . These analogs were bioassayed using a growth arrest assay and a gene induction assay and evaluated for their ability to compete with binding of tritiated alpha-factor to its receptor Ste2p . The results showed that the phenolic OH of Tyr13 is not required for either biological activity or receptor recognition . Analogs containing fluorine, amino, nitro or a hydrogen in place of OH had 80-120% of the biological activity of the parent pheromone in the gene induction assay and had receptor affinities from nearly equal to 6-fold lower than that of alpha-factor . In contrast, substitution of Ser or Ala at position 13 resulted in a >100-fold decrease in receptor affinity suggesting that the aromatic ring is involved in binding to the receptor . The lack of a strict requirement for Tyr13 allowed the design of several multiple replacement analogs in which Phe or p-F-Phe were substituted at position 13 and Tyr was placed in other positions of the peptide . These analogs could then be iodinated and used in the development of a highly sensitive receptor-binding assay . One potential receptor ligand {Tyr(125I)1,Nle12, Phe13} alpha-factor exhibited saturable binding with a KD of 81 nM and was competed by alpha-factor for binding in a whole-cell assay . Thus a new family of radioactive ligands for the alpha-factor receptor has been revealed . These ligands should be extremely useful in defining active site residues during mutagenesis and cross-linking studies.

Biosci Biotechnol Biochem, 2000 Jun, 64(6), 1238 - 46
Cloning and sequencing of an endoglucanase gene from Scopulariopsis brevicaulis TOF-1212, and its expression in Saccharomyces cerevisiae; Nakatani F et al.; The egI gene, encoding a major endoglucanase (EGI) of Scopulariopsis brevicaulis TOF-1212, was cloned and sequenced . The eglgene consisted of 868 bp with one intron and encoded a protein of 229 amino acids with a calculated molecular mass of 22,392 daltons . The EGI was assigned to a family 45 of glycosyl hydrolases and showed high similarity with other fungal endoglucanases, especially with those of Humicola grisea and Fusarium oxysporum, on the basis of hydrophobic cluster analysis . The egI gene was expressed under the promoter of the phosphoglycerate kinase gene (PGK) in Saccharomyces cerevisiae . The transformed cells were able to secrete the enzyme efficiently in an active form.

Genetika, 2000 Jun, 36(6), 767 - 73
{RAD29 and RAD31--new genes from Saccharomyces cerevisiae yeasts, participating in control of DNA repair . Isolation and genetic study of mutants}; Kozhina TN et al.; Base excision repair (BER) and nucleotide excision repair (NER) are two main cellular responses to DNA damage induced by various physical and chemical factors . After exposure of the strain that carries the NER-blocking rad2 mutation to UV light, several mutants hypersensitive to the UV light lethal action and simultaneously sensitive to methylmethanesulphonate (MMS) were isolated . Two of these mutants (Uvs64 and Uvs212) were examined in detail . The mutants were found to carry recessive, monogenically inherited lesions that had pleiotropic, though different, phenotypes: both mutants were also sensitive to nitrous acid (HNO2), whereas Uvs212 was sensitive to hydrogen peroxide as well . Moreover, the homozygote for the uvs212 mutation, but not for uvs64, blocks the sporulation . Since the mutations examined were not allelic to any of the known rad mutations that cause MMS sensitivity or to each other, it is concluded that two new genes involved in the control of yeast DNA repair were detected . Furthermore, these genes were mapped to different regions of the right arm of chromosome 2 where repair genes were not found . Thus, two new genes, designated RAD29(UVS64) and RAD31(UVS212) and probably involved in base excision repair, were identified.

Yeast, 2000 Aug, 16(11), 1025 - 33
Functional analysis of six genes from chromosomes XIV and XV of Saccharomyces cerevisiae reveals YOR145c as an essential gene and YNL059c/ARP5 as a strain-dependent essential gene encoding nuclear proteins; Grava S et al.; We report here basic functional analysis of strains deleted for six open reading frames (ORFs), YNL059c and YNL148c from chromosome XIV and YOR145c, YOR152c, YOR161c and YOR162c from chromosome XV of Saccharomyces cerevisiae . ORFs were replaced with the KanMX4 resistance marker using a long flanking homology PCR strategy in FY1679 and W303 diploid strains . Replacement cassettes were constructed in plasmid pUG7 and the cognate wild-type genes were recovered by gap repair . Sporulation and tetrad analysis revealed that deletion of YNL059c/ARP5 was lethal for vegetative growth in strain W303 and caused severe growth defects in strain FY1679 while YOR145c was essential for growth in both strains . Fusion of the green fluorescent protein (GFP) gene to the 3' ends of the YNL059c/ARP5 and YOR145c coding sequences created functional chimeric genes at the respective chromosomal loci . Both Arp5-GFP and Yor145-GFP localized to the nucleus, Yor145-GFP concentrating in the nucleolus . The vectors containing the deletion cassettes and the cognate wild-type genes, the oligonucleotides, and the deletant strains are available from the EUROFAN resource centre EUROSCARF (Frankfurt) .

FEBS Lett, 2000 Jul 28, 478(1-2), 84 - 8
Characterization of the chitin biosynthesis process as a compensatory mechanism in the fks1 mutant of Saccharomyces cerevisiae; Garcia-Rodriguez LJ et al.; Deletion of the 1,3-beta-D-glucan synthase gene FKS1 in Saccharomyces cerevisiae induces a compensatory mechanism that is reflected in a significant increase in chitin synthase III (CSIII) activity, leading to high rates of chitin synthesis . Deregulation of CSIII activity is mainly due to the intracellular delocalization of Chs3p and Chs4p, the two main components of the CSIII active complex.

J Biol Chem, 2000 Oct 20, 275(42), 32925 - 30
The Saccharomyces cerevisiae PCD1 gene encodes a peroxisomal nudix hydrolase active toward coenzyme A and its derivatives; Cartwright JL et al.; The PCD1 nudix hydrolase gene of Saccharomyces cerevisiae has been cloned and the Pcd1p protein characterized as a diphosphatase (pyrophosphatase) with specificity for coenzyme A and CoA derivatives . Oxidized CoA disulfide is preferred over CoA as a substrate with K(m) and k(cat) values of 24 micrometer and 5.0 s(-1), respectively, compared with values for CoA of 280 micrometer and 4.6 s(-1) respectively . The products of CoA hydrolysis were 3'-phosphoadenosine 5'-monophosphate and 4'-phosphopantetheine . F(-) ions inhibited the activity with an IC(50) of 22 micrometer . The sequence of Pcd1p contains a potential PTS2 peroxisomal targeting signal . When fused to the N terminus of yeast-enhanced green fluorescent protein, Pcd1p was shown to locate to peroxisomes by confocal microscopy . It was also shown to co-localize with peroxisomal thiolase by immunofluorescence microscopy . N-terminal sequence analysis of the expressed protein revealed the loss of 7 or 8 amino acids, suggesting processing of the proposed PTS2 signal after import . The function of Pcd1p may be to remove potentially toxic oxidized CoA disulfide from peroxisomes in order to maintain the capacity for beta-oxidation of fatty acids.

J Biol Chem, 2000 Nov 3, 275(44), 34068 - 72
Interactions between Spc2p and other components of the endoplasmic reticulum translocation sites of the yeast Saccharomyces cerevisiae; Antonin W et al.; In yeast, the endoplasmic reticulum membrane proteins Sec11p and Spc3p are essential for the cleavage of signal peptides of nascent polypeptide chains during their passage through translocation sites . Genetic and biochemical experiments demonstrate that Sec11p and Spc3p are tightly associated with two other proteins, Spc1p and Spc2p, whose functions are largely unknown . Using anti-Spc2p antibodies, we show here that this heterotetrameric complex associates with Sbh1p and Sbh2p, the beta-subunits of the Sec61p complex and the Ssh1p complex, respectively . Depletion of Spc2p decreased the enzymatic activity of the SPC in vitro, led to a loss of Spc1p, and led to a down-regulation of the amount of Sec11p and Spc3p in the endoplasmic reticulum . Moreover, the deletion of Spc2p also decreased the expression level of Sbh2p . These data implicate that Spc2p not only enhances the enzymatic activity of the SPC but also facilitates the interactions between different components of the translocation site.

J Biol Chem, 2000 Oct 6, 275(40), 30957 - 61
Regulation of monovalent ion homeostasis and pH by the Ser-Thr protein phosphatase SIT4 in Saccharomyces cerevisiae; Masuda CA et al.; A gene, SIT4, was identified as corresponding to a serine/threonine protein phosphatase and when overexpressed confers lithium tolerance in galactose medium to the budding yeast Saccharomyces cerevisiae . This gene has been previously identified as a regulator of the cell cycle and involved in nitrogen sensing . It is shown that the transcription levels of SIT4 are induced by low concentrations of Li(+) in a time-dependent manner . Na(+) and K(+) at high concentrations, but not sorbitol, also induce transcription . As a response to Na(+) or Li(+) stress, yeast cells lower the intracellular K(+) content . This effect is enhanced in cells overexpressing SIT4, which also increase (86)Rb efflux after the addition of Na(+) or Li(+) to the extracellular medium . Another feature of SIT4-overexpressing cells is that they maintain a more alkaline pH of 6.64 compared with 6.17 in the wild type cells . It has been proposed that the main pathway of salt tolerance in yeast is mediated by a P-type ATPase, encoded by PMR2A/ENA1 . However, our results show that in a sit4 strain, expression of ENA1 is still induced by monovalent cations, and overexpression of SIT4 does not alter the amount of ENA1 transcript . These results show that SIT4 acts in a parallel pathway not involving induction of transcription of ENA1 and suggest a novel function for SIT4 in response to salt stress.

Mutat Res, 2000 Jun 30, 451(1-2), 257 - 75
Recombination factors of Saccharomyces cerevisiae; Sung P et al.; The budding yeast Saccharomyces cerevisiae has been an excellent genetic and biochemical model for our understanding of homologous recombination . Central to the process of homologous recombination are the products of the RAD52 epistasis group of genes, whose functions we now know include the nucleolytic processing of DNA double-stand breaks, the ability to conduct a DNA homology search, and the capacity to promote the exchange of genetic information between homologous regions on recombining chromosomes . It is also clear that the basic functions of the RAD52 group of genes have been highly conserved among eukaryotes . Disruption of this important process causes genomic instability, which can result in a number of unsavory consequences, including tumorigenesis and cell death.

Mutat Res, 2000 Jun 30, 451(1-2), 187 - 96
DNA damage checkpoints and DNA replication controls in Saccharomyces cerevisiae; Foiani M et al.; In response to genotoxic agents and cell cycle blocks all eukaryotic cells activate a set of surveillance mechanims called checkpoints . A subset of these mechanisms is represented by the DNA damage checkpoint, which is triggered by DNA lesions . The activation of this signal transduction pathway leads to a delay of cell cycle progression to prevent replication and segregation of damaged DNA molecules, and to induce transcription of several DNA repair genes . The yeast Saccharomyces cerevisiae has been invaluable in genetically dissecting the DNA damage checkpoint pathway and recent findings have provided new insights into the architecture of checkpoint protein complexes, in their order of function and in the mechanisms controlling DNA replication in response to DNA damage.

Mutat Res, 2000 Jun 30, 451(1-2), 71 - 89
Tying up loose ends: nonhomologous end-joining in Saccharomyces cerevisiae; Lewis LK et al.; The ends of chromosomal DNA double-strand breaks (DSBs) can be accurately rejoined by at least two discrete pathways, homologous recombination and nonhomologous end-joining (NHEJ) . The NHEJ pathway is essential for repair of specific classes of DSB termini in cells of the budding yeast Saccharomyces cerevisiae . Endonuclease-induced DSBs retaining complementary single-stranded DNA overhangs are repaired efficiently by end-joining . In contrast, damaged DSB ends (e.g., termini produced by ionizing radiation) are poor substrates for this pathway . NHEJ repair involves the functions of at least 10 genes, including YKU70, YKU80, DNL4, LIF1, SIR2, SIR3, SIR4, RAD50, MRE11, and XRS2 . Most or all of these genes are required for efficient recombination-independent recircularization of linearized plasmids and for rejoining of EcoRI endonuclease-induced chromosomal DSBs in vivo . Several NHEJ mutants also display aberrant processing and rejoining of DSBs that are generated by HO endonuclease or formed spontaneously in dicentric plasmids . In addition, all NHEJ genes except DNL4 and LIF1 are required for stabilization of telomeric repeat sequences . Each of the proteins involved in NHEJ appears to bind, directly or through protein associations, with the ends of linear DNA . Enzymatic and/or structural roles in the rejoining of DSB termini have been postulated for several proteins within the group . Most yeast NHEJ genes have homologues in human cells and many biochemical activities and protein:protein interactions have been conserved in higher eucaryotes . Similarities and differences between NHEJ repair in yeast and mammalian cells are discussed.

Biochemistry, 2000 Aug 1, 39(30), 8935 - 43
Structural differences between Saccharomyces cerevisiae ribosomal stalk proteins P1 and P2 support their functional diversity; Zurdo J et al.; The eukaryotic acidic P1 and P2 proteins modulate the activity of the ribosomal stalk but playing distinct roles . The aim of this work was to analyze the structural features that are behind their different function . A structural characterization of Saccharomyces cerevisaie P1 alpha and P2 beta proteins was performed by circular dichroism, nuclear magnetic resonance, fluorescence spectroscopy, thermal denaturation, and protease sensitivity . The results confirm the low structure present in both proteins but reveal clear differences between them . P1 alpha shows a virtually unordered secondary structure with a residual helical content that disappears below 30 degrees C and a clear tendency to acquire secondary structure at low pH and in the presence of trifluoroethanol . In agreement with this higher disorder P1 alpha has a fully solvent-accessible tryptophan residue and, in contrast to P2 beta, is highly sensitive to protease degradation . An interaction between both proteins was observed, which induces an increase in the global secondary structure content of both proteins . Moreover, mixing of both proteins causes a shift of the P1 alpha tryptophan 40 signal, pointing to an involvement of this region in the interaction . This evidence directly proves an interaction between P1 alpha and P2 beta before ribosome binding and suggests a functional complementation between them . On a whole, the results provide structural support for the different functional roles played by the proteins of the two groups showing, at the same time, that relatively small structural differences between the two stalk acidic protein types can result in significant functional changes.

Mol Cell Biol, 2000 Aug, 20(16), 5888 - 96
Rfc5, in cooperation with rad24, controls DNA damage checkpoints throughout the cell cycle in Saccharomyces cerevisiae; Naiki T et al.; RAD24 and RFC5 are required for DNA damage checkpoint control in the budding yeast Saccharomyces cerevisiae . Rad24 is structurally related to replication factor C (RFC) subunits and associates with RFC subunits Rfc2, Rfc3, Rfc4, and Rfc5 . rad24Delta mutants are defective in all the G(1)-, S-, and G(2)/M-phase DNA damage checkpoints, whereas the rfc5-1 mutant is impaired only in the S-phase DNA damage checkpoint . Both the RFC subunits and Rad24 contain a consensus sequence for nucleoside triphosphate (NTP) binding . To determine whether the NTP-binding motif is important for Rad24 function, we mutated the conserved lysine(115) residue in this motif . The rad24-K115E mutation, which changes lysine to glutamate, confers a complete loss-of-function phenotype, while the rad24-K115R mutation, which changes lysine to arginine, shows no apparent phenotype . Although neither rfc5-1 nor rad24-K115R single mutants are defective in the G(1)- and G(2)/M-phase DNA damage checkpoints, rfc5-1 rad24-K115R double mutants become defective in these checkpoints . Coimmunoprecipitation experiments revealed that Rad24(K115R) fails to interact with the RFC proteins in rfc5-1 mutants . Together, these results indicate that RFC5, like RAD24, functions in all the G(1)-, S- and G(2)/M-phase DNA damage checkpoints and suggest that the interaction of Rad24 with the RFC proteins is essential for DNA damage checkpoint control.

Mol Biol Evol, 2000 Aug, 17(8), 1268 - 75
Analysis of intrachromosomal duplications in yeast Saccharomyces cerevisiae: a possible model for their origin; Achaz G et al.; The complete genome of the yeast Saccharomyces cerevisiae was investigated for intrachromosomal duplications at the level of nucleotide sequences . The analysis was performed by looking for long approximate repeats (from 30 to 3,885 bp) present on each of the chromosomes . We show that direct and inverted repeats exhibit very different characteristics: the two copies of direct repeats are more similar and longer than those of inverted repeats . Furthermore, contrary to the inverted repeats, a large majority of direct repeats appear to be closely spaced . The distance (delta) between the two copies is generally smaller than 1 kb . Further analysis of these "close direct repeats" shows a negative correlation between delta and the percentage of identity between the two copies, and a positive correlation between delta and repeat length . Moreover, contrary to the other categories of repeats, close direct repeats are mostly located within coding sequences (CDSs) . We propose two hypotheses in order to interpret these observations: first, the deletion/conversion rate is negatively correlated with delta; second, there exists an active duplication mechanism which continuously creates close direct repeats, the other intrachromosomal repeats being the result, by chromosomal rearrangements of these "primary repeats."

Adv Microb Physiol, 2000, 43, 75 - 115
Dynamics of metabolism and its interactions with gene expression during sporulation in Saccharomyces cerevisiae; Cortassa S et al.; The dynamics of metabolism has been shown to be involved in the triggering of events that are concurrent with sporulation of the budding yeast Saccharomyces cerevisiae . Indeed, quantitative correlations have been demonstrated between sporulation and the rate of carbon substrate or oxygen consumption, and the fluxes through gluconeogenic and glyoxylate cycle pathways . The results suggest that an imbalance between catabolic and anabolic fluxes influences the occurrence of the differentiation process . The hypothesis that the initiation of sporulation is triggered by the accumulation of an intracellular metabolite is confronted with the notion that intermediary metabolism and the expression of genes involved in sporulation interact to trigger the differentiation process . Several pieces of evidence indicate that derepression of the gluconeogenic pathway is crucial for the initiation of sporulation . One of the possible pathways through which glucose repression hampers sporulation might be the repression of gluconeogenesis as well as that of respiratory activity, in turn modulating the expression of IMEL++ . The stages defined in the dynamics of sporulating cultures, namely readiness and commitment, are related to metabolic events associated with sporulation . An interpretation in terms of metabolic flux dynamics is given to the reversal of commitment occurring when the normal progression to sporulation is somehow blocked . The quantitative data are here integrated in a model attempting to simulate the dynamics of metabolic as well as cellular events during sporulation . The model is envisaged as a test of the hypothesis that an imbalance between anabolism and catabolism is involved in initiation of the sporulation process . It is proposed that such an imbalance may be a signal for differential gene expression associated with the differentiation pathway.

J Biol Chem, 2000 Oct 6, 275(40), 30886 - 93
Roles of the Dal82p domains in allophanate/oxalurate-dependent gene expression in Saccharomyces cerevisiae; Scott S et al.; Allophanate/oxalurate-induced gene expression in Saccharomyces cerevisiae requires at least five transcription factors, four of which act positively (Gln3p, Gat1p, Dal81p, and Dal82p) and one negatively (Dal80p) . Gln3p binds to and Gat1p is proposed to bind to single GATA sequences; Dal80p binds to pairs of specifically oriented and spaced GATA sequences, and Dal82p binds to a pathway-specific element, UIS(ALL) . Dal82p consists of at least three domains as follows: (i) UIS(ALL) DNA-binding, (ii) transcriptional activation, and (iii) coiled-coil(DAL82) . Here we show that the coiled-coil(DAL82) domain possesses two demonstrable functions . (i) It prevents Dal82p-mediated transcription when inducer is absent . (ii) It is a major, although not exclusive, domain through which the inducer signal is received . Supporting the latter conclusion, a 38-amino acid fragment, containing little more than the coiled-coil(DAL82) domain, supports oxalurate-inducible, Dal81p-dependent, reporter gene transcription . Dal81p is required for inducer responsiveness of LexAp-Dal82p and LexAp coiled-coil(DAL82)-mediated transcription but isn't needed for inducer-dependent activation mediated by a Dal82p containing deletions in both the coiled-coil(DAL82), UIS(ALL)-binding domains . There may be an interaction between Dal81p and the coiled-coil(DAL82) domain since (i) Dal81p is required for transcription mediated by LexA-coiled-coil(DAL82)p and (ii) a Dal81p-Dal82p complex is detected by two-hybrid assay.

Mol Gen Genet, 2000 Jun, 263(5), 877 - 88
Characterization of staurosporine-sensitive mutants of Saccharomyces cerevisiae: vacuolar functions affect staurosporine sensitivity; Yoshida S et al.; Mutations at several loci affect the sensitivity of the yeast Saccharomyces cerevisiae to staurosporine . We report here the characterization of novel staurosporine- and temperature-sensitive mutants (stt) . Cloning and integration mapping showed that the genes STT2/ STT6, STT5, STT7, STT8 and STT9 are allelic to VPS18, ERG10, GPI1, VPS34 and VPS11, respectively . The products of ERG10 and GPI1, respectively, catalyze mevalonate and glycosyl phosphatidylinositol anchor synthesis, while VPS18 and VPS11 genes belong to the class C VPS (Vacuolar Protein Sorting) genes, and the VPS34 gene is classified as a class D VPS . Therefore, staurosporine sensitivity is affected by ergosterol and glycolipid biosynthesis and by vacuolar functions . We found that other vps mutants belonging to classes C and D exhibit staurosporine sensitivity, and that they show calcium sensitivity and fail to grow on glycerol as the sole carbon source; both of the last two characteristics are shared by vacuolar H+-ATPase mutants (vma) . As vma mutants were also found to show staurosporine-sensitive growth, staurosporine sensitivity is likely to be affected by acidification of the vacuole . Moreover, wild type yeast cells are more sensitive to staurosporine in alkaline media than in acidic media, suggesting that staurosporine is exported from the cytosol by H+/drug antiporters . Pleiotropic drug resistance (PDR) genes also provide some resistance to staurosporine, because deltapdr5, deltasnq2 and deltayor1 strains are more sensitive to staurosporine than the wild-type strain . This suggests that staurosporine is also exported by the ATP-binding cassette (ABC) transporters on the plasma membrane . vma mutants and vps mutants of classes C and D vps are sensitive to hygromycin B and vanadate, while ABC transporter-depleted mutants do not show such sensitivity, indicating that two systems differ in their ability to protect the cell against different types of drug.

Mol Gen Genet, 2000 Jun, 263(5), 854 - 66
Structure-function relationships in replication origins of the yeast Saccharomyces cerevisiae: higher-order structural organization of DNA in regions flanking the ARS consensus sequence; Marilley M; In order to better understand the involvement of the DNA molecule in the replication initiation process we have characterized the structure of the DNA at Autonomously Replicating Sequences (ARSs) in Saccharomyces cerevisiae . Using a new method for anti-bent DNA analysis, which allowed us to take into account the bending contribution of each successive base plate, we have investigated the higher-order structural organization of the DNA in the region which immediately surrounds the ARS consensus sequence (ACS) . We have identified left- and right-handed anti-bent DNAs which flank this consensus sequence . The data show that this organization correlates with an active ACS . Analysis of the minimum nucleotide sequence providing ARS function to plasmids reveals an example where the critical nucleotides are restricted to the ACS and the right-handed anti-bent DNA domain, although most of the origins considered contained both left- and right-handed anti-bent DNAs . Moreover, mutational analysis shows that the right-handed form is necessary in order to sustain a specific DNA conformation which is correlated with the level of plasmid maintenance . A model for the role of these individual structural components of the yeast replication origin is presented . We discuss the possible role of the right-handed anti-bent DNA domain, in conjunction with the ACS, in the process of replication initiation, and potentialities offered by the combination of left- and right-handed structural components in origin function.

Anal Chem, 2000 Jul 1, 72(13), 2856 - 60
Detection of atrazine based on the SPR determination of P450 mRNA levels in Saccharomyces cerevisiae; Lim T et al.; We describe a novel method for quantification of atrazine based on detection of P450 mRNA levels in Saccharomyces cerevisiae . The selected oligonucleotide probe exhibited specificity against P450 mRNA and was successfully immobilized on the sensor chip . The mRNA was subsequently quantified by RU change using a SPR system . When the cells were disrupted by boiling, mRNA could be measured without further purification at reduced sensitivity . This simple technique permits the detection of atrazine within 15 min . This rapid and highly sensitive method can be used for the detection of atrazine.

Eur J Biochem, 2000 Aug, 267(15), 4825 - 30
Generation of catalytically active 6-phosphofructokinase from Saccharomyces cerevisiae in a cell-free system; Edelmann A et al.; PFK1 and PFK2 coding for the subunits of 6-phosphofructokinase from Saccharomyces cerevisiae were cloned into plasmids suitable for runoff transcription . In vitro translation products of both kinds of subunit were obtained using rabbit reticulocyte lysate as the synthesis and folding system . They were monitored by chemiluminescent Western-blot analysis . Folding and assembly of the alpha-subunit and beta-subunit of 6-phosphofructokinase were found to occur in the cell-free system resulting in an enzymatically active protein . The in vitro generated enzyme exhibits a folding state that is similar to that of the heterooctameric form of 6-phosphofructokinase in the presence of fructose 6-phosphate, ATP and ammonium sulfate, as demonstrated by size-exclusion HPLC followed by ELISA.

Mol Cells, 2000 Jun 30, 10(3), 301 - 8
Saccharomyces cerevisiae Ste5 is important for induction and substrate specificity of Fus3 MAP kinase in the pheromone signaling pathway; Choi YJ et al.; The pheromone pathway is one of the mitogen activated protein kinase (MAPK) signaling pathways identified in Saccharomyces cerevisiae and is involved in both G1 cell cycle arrest and mating of cells . Fus3 functions at a branching point for G1 cell cycle arrest and mating responses in the signaling cascade, and the Fus3 MAPK uses components of both G1 arrest and mating routes as substrates . The Ste5 is a scaffold protein of the MAPK module and is essential for the activation of Fus3 . However, it is not known how Ste5 is involved in the specific activation of Fus3 in G1 arrest and mating . In this study, we characterized several G1 arrest defective Ste5 mutants to better understand the roles of Ste5 in the regulation of Fus3 . The level of Fus3 increased by treatment with alpha-factor . However, the alpha-factor effects were not readily apparent in the observation of yeast cells containing G1 arrest defective ste5 mutant . This suggests that Ste5 plays an essential role in Fus3 induction . Fus3 immune kinase assay of G1 arrest defective ste5 transformants revealed that Ste5 is important for substrate specificity of Fus3 for G1 arrest and/or mating.

FEBS Lett, 2000 Jul 14, 477(1-2), 62 - 6
Erv1p from Saccharomyces cerevisiae is a FAD-linked sulfhydryl oxidase; Lee J et al.; The yeast ERV1 gene encodes a small polypeptide of 189 amino acids that is essential for mitochondrial function and for the viability of the cell . In this study we report the enzymatic activity of this protein as a flavin-linked sulfhydryl oxidase catalyzing the formation of disulfide bridges . Deletion of the amino-terminal part of Erv1p shows that the enzyme activity is located in the 15 kDa carboxy-terminal domain of the protein . This fragment of Erv1p still binds FAD and catalyzes the formation of disulfide bonds but is no longer able to form dimers like the complete protein . The carboxy-terminal fragment contains a conserved CXXC motif that is present in all homologous proteins from yeast to human . Thus Erv1p represents the first FAD-linked sulfhydryl oxidase from yeast and the first of these enzymes that is involved in mitochondrial biogenesis.

Int J Food Microbiol, 2000 Jun 30, 58(1-2), 117 - 21
Mitochondrial DNA restriction enzyme analysis and evaluation of the enological characteristics of Saccharomyces cerevisiae strains isolated from grapes of the wine-producing area of Collio (Italy); Comi G et al.; A total of 70 strains of Saccharomyces cerevisiae were isolated from different grapes from the Collio Region . Chemical parameters and mitochondrial DNA (mtDNA) restriction patterns were determined . Higher alcohols were the main parameter useful for differentiating between strains, whereas the mtDNA analysis demonstrated a high genetic variability between strains . A weak correlation was observed when the dendrograms obtained from the chemical and genetic results were compared.

Mol Cell Biol, 2000 Aug, 20(15), 5766 - 76
Activation of the Kss1 invasive-filamentous growth pathway induces Ty1 transcription and retrotransposition in Saccharomyces cerevisiae; Morillon A et al.; Using a set of genomic TY1A-lacZ fusions, we show that Ste12 and Tec1, two transcription factors of the Kss1 mitogen-activated protein kinase (MAPK) cascade activate Ty1 transcription in Saccharomyces cerevisiae . This result strongly suggests that the invasive-filamentous pathway regulates Ty1 transcription . Since this pathway is active in diploid cells, we suspected that Ty1 transposition might occur in this cell type, despite the fact that this event has been never reported before (unless activated by heterologous promoters such as that of GAL1) . We demonstrate here that constitutive activation of the invasive-filamentous pathway by the STE11-4 allele or by growth in low-nitrogen medium induces Ty1 transcription and retrotransposition in diploid cells . We show that Ty1 retrotransposition can be activated by STE11-4 in haploid cells as well . Our findings provide the first evidence that Ty1 retrotransposition can be activated by environmental signals that affect differentiation . Activation of the Kss1 MAPK cascade by stress is known to cause filament formation that permits the search for nutrients away from the colonization site . We propose that activation of Ty1 retrotransposition by this cascade could play a role in adaptive mutagenesis in response to stress.

Biochem Biophys Res Commun, 2000 Jul 14, 273(3), 824 - 8
Disruption of galactokinase signature sequence in gal3p of Saccharomyces cerevisiae does not lead to loss of signal transduction function; Murthy TV et al.; Gal3p of Saccharomyces cerevisiae is a 520-amino-acid residue protein, which activates the GAL genes in the presence of galactose by relieving the repression of Gal80p . It shows significant amino acid sequence homology to galactokinases but does not possess galactokinase activity . Deletion mutants of Gal3p were generated to identify the role of N-terminal amino acid residues required for function . The mutant versions of Gal3p could be detected on a Western blot . The Gal3p mutant lacking N-terminal 50-amino-acid residues which is disrupted for galactokinase signature sequence was found to be functional . These results suggest that the evolutionarily conserved galactokinase signature sequence present in known galactokinases may not have a role in Gal3p function .

Proc Natl Acad Sci U S A, 2000 Jul 18, 97(15), 8415 - 20
Balancing transcriptional interference and initiation on the GAL7 promoter of Saccharomyces cerevisiae; Greger IH et al.; Transcriptional termination of the GAL10 gene in Saccharomyces cerevisiae depends on the efficiency of polyadenylation . Either cis mutations in the poly(A) signal or trans mutations of mRNA 3' end cleavage factors result in GAL10 read-through transcripts into the adjacent GAL7 gene and inactivation (occlusion) of the GAL7 promoter . Herein, we present a molecular explanation of this transcriptional interference phenomenon . In vivo footprinting data reveal that GAL7 promoter occlusion is associated with the displacement of Gal4p transcription factors from the promoter . Interestingly, overexpression of Gal4p restores promoter occupancy, activates GAL7 expression, and rescues growth on the otherwise toxic galactose substrate . Our data therefore demonstrate a precise balance between transcriptional interference and initiation.

Mol Biol Cell, 2000 Jul, 11(7), 2445 - 57
Nucleus-vacuole junctions in Saccharomyces cerevisiae are formed through the direct interaction of Vac8p with Nvj1p; Pan X et al.; Vac8p is a vacuolar membrane protein that is required for efficient vacuole inheritance and fusion, cytosol-to-vacuole targeting, and sporulation . By analogy to other armadillo domain proteins, including beta-catenin and importin alpha, we hypothesize that Vac8p docks various factors at the vacuole membrane . Two-hybrid and copurfication assays demonstrated that Vac8p does form complexes with multiple binding partners, including Apg13p, Vab2p, and Nvj1p . Here we describe the surprising role of Vac8p-Nvj1p complexes in the formation of nucleus-vacuole (NV) junctions . Nvj1p is an integral membrane protein of the nuclear envelope and interacts with Vac8p in the cytosol through its C-terminal 40-60 amino acids (aa) . Nvj1p green fluorescent protein (GFP) concentrated in small patches or rafts at sites of close contact between the nucleus and one or more vacuoles . Previously, we showed that Vac8p-GFP concentrated in intervacuole rafts, where is it likely to facilitate vacuole-vacuole fusion, and in "orphan" rafts at the edges of vacuole clusters . Orphan rafts of Vac8p red-sifted GFP (YFP) colocalize at sites of NV junctions with Nvj1p blue-sifted GFP (CFP) . GFP-tagged nuclear pore complexes (NPCs) were excluded from NV junctions . In vac8-Delta cells, Nvj1p-GFP generally failed to concentrate into rafts and, instead, encircled the nucleus . NV junctions were absent in both nvj1-Delta and vac8-Delta cells . Overexpression of Nvj1p caused the profound proliferation of NV junctions . We conclude that Vac8p and Nvj1p are necessary components of a novel interorganelle junction apparatus.

Mol Biol Cell, 2000 Jul, 11(7), 2335 - 47
The Skn7 response regulator of Saccharomyces cerevisiae interacts with Hsf1 in vivo and is required for the induction of heat shock genes by oxidative stress; Raitt DC et al.; The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide . The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes . These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein-protein interactions . Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements . Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress . Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress . We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.

Mol Biol Cell, 2000 Jul, 11(7), 2267 - 81
Inositol phosphorylceramide synthase is located in the Golgi apparatus of Saccharomyces cerevisiae; Levine TP et al.; The plasma membrane of eukaryotic cells differs in lipid composition from most of the internal organelles, presumably reflecting differences in many of its functions . In particular, the plasma membrane is rich in sphingolipids and sterols, one property of which is to decrease the permeability and increase the thickness of lipid bilayers . In this paper, we examine the length of transmembrane domains throughout the yeast secretory pathway . Although the transmembrane domains of cis and medial Golgi residents are similar to those of endoplasmic reticulum proteins, these domains lengthen substantially beyond the medial Golgi, suggesting a thickening of the bilayer . Yeast sphingolipids have particularly long acyl chains, and Aur1p, the inositol phosphorylceramide synthase that initiates yeast sphingolipid synthesis, was found to be located in the Golgi apparatus by both immunofluorescence and membrane fractionation, with its active site apparently in the Golgi lumen . Thus, it appears that sphingolipid synthesis in yeast takes place in the Golgi, separated from glycerophospholipid synthesis in the endoplasmic reticulum . A similar separation has been found in mammalian cells, and this conservation suggests that such an arrangement of enzymes within the secretory pathway could be important for the creation of bilayers of different thickness within the cell.

Mol Biol Cell, 2000 Jul, 11(7), 2221 - 33
Exo1 roles for repair of DNA double-strand breaks and meiotic crossing over in Saccharomyces cerevisiae; Tsubouchi H et al.; The MRE11, RAD50, and XRS2 genes of Saccharomyces cerevisiae are involved in the repair of DNA double-strand breaks (DSBs) produced by ionizing radiation and by radiomimetic chemicals such as methyl methanesulfonate (MMS) . In these mutants, single-strand DNA degradation in a 5' to 3' direction from DSB ends is reduced . Multiple copies of the EXO1 gene, encoding a 5' to 3' double-strand DNA exonuclease, were found to suppress the high MMS sensitivity of these mutants . The exo1 single mutant shows weak MMS sensitivity . When an exo1 mutation is combined with an mre11 mutation, both repair of MMS-induced damage and processing of DSBs are more severely reduced than in either single mutant, suggesting that Exo1 and Mre11 function independently in DSB processing . During meiosis, transcription of the EXO1 gene is highly induced . In meiotic cells, the exo1 mutation reduces the processing of DSBs and the frequency of crossing over, but not the frequency of gene conversion . These results suggest that Exo1 functions in the processing of DSB ends and in meiotic crossing over.

J Biol Chem, 2000 Jul 14, 275(28), 21158 - 68
Purification and enzymic properties of Mot1 ATPase, a regulator of basal transcription in the yeast Saccharomyces cerevisiae; Adamkewicz JI et al.; The 1867-residue Mot1 protein is a member of a superfamily of ATPases, some of which are helicases, that interact with protein-nucleic acid assemblies . Mot1 is an essential regulator of RNA polymerase II-dependent transcription in vivo and dissociates TATA box-binding protein (TBP)-DNA complexes in vitro . Mot1-(His)(6) was purified to apparent homogeneity from yeast extracts . The preparation efficiently dissociated TBP.TATA complexes, suggesting that no other protein or cofactor is required . Mot1 behaved as a non-globular monomer in hydrodynamic studies, and no association was detected between differentially tagged co-expressed Mot1 constructs . ATPase activity was stimulated about 10-fold by high ionic strength or alkaline pH, or by deletion of the N-terminal TBP-binding segment, suggesting that the N-terminal domain negatively regulates the C-terminal ATPase domain (Mot1C) . Correspondingly, at moderate salt concentration, Mot1 ATPase (but not Mot1C) was stimulated >/=10-fold by yeast TBP, suggesting that interaction with TBP relieves a conformational constraint in Mot1 . Double- or single-stranded TATA-containing DNA did not affect ATPase activity of Mot1 or Mot1C, with or without TBP . Mot1 did not exhibit detectable helicase activity in strand displacement assays using substrates with flush ends or 5'- or 3'-overhangs . Mot1-catalyzed dissociation of TBP from DNA was not prevented by a psoralen cross-link positioned immediately preceding the TATA sequence . Thus, Mot1 most likely promotes release of TBP from TATA-containing DNA by causing a structural change in TBP itself, rather than by strand unwinding.

Cell Struct Funct, 2000 Apr, 25(2), 125 - 31
Ca2+ signal is generated only once in the mating pheromone response pathway in Saccharomyces cerevisiae; Nakajima-Shimada J et al.; The mating pheromone, alpha-factor, of the yeast Saccharomyces cerevisiae binds to the heterotrimeric G protein-coupled cell surface receptor of MATa cells and induces cellular responses necessary for mating . In higher eukaryotic cells, many hormones and growth factors rapidly mobilize a second messenger, Ca2+, by means of receptor-G protein signaling . Although striking similarities between the mechanisms of the receptor-G protein signaling in yeast and higher eukaryotes have long been known, it is still uncertain whether the pheromone rapidly mobilizes Ca2+ necessary for early events of the pheromone response . Here we reexamine this problem using sensitive methods for detecting Ca2+ fluxes and mobilization, and find no evidence that there is rapid Ca2+ influx leading to a rapid increase in the cytosolic free Ca2+ concentration . In addition, the yeast PLC1 deletion mutant lacking phosphoinositide-specific phospholipase C, a key enzyme for generating Ca2+ signals in higher eukaryotic cells, responds normally to the pheromone . These findings suggest that the receptor-G protein signaling does not utilize Ca2+ as a second messenger in the early stage of the pheromone response pathway . Since the receptor-G protein signaling does stimulate Ca2+ influx after early events have finished and this stimulation is essential for late events in the pheromone response pathway {Iida et al., (1990) J . Biol . Chem., 265: 13391-13399} Ca2+ may be used only once in the signal transduction pathway in unicellular eukaryotes such as yeast.

Mol Cell, 2000 May, 5(5), 789 - 99
The Saccharomyces cerevisiae Msh2 mismatch repair protein localizes to recombination intermediates in vivo; Evans E et al.; Mismatch repair proteins act during double-strand break repair (DSBR) to correct mismatches in heteroduplex DNA, to suppress recombination between divergent sequences, and to promote removal of nonhomologous DNA at DSB ends . We investigated yeast Msh2p association with recombination intermediates in vivo using chromatin immunoprecipitation . During DSBR involving nonhomologous ends, Msh2p localized strongly to recipient and donor sequences . Localization required Msh3p and was greatly reduced in rad50delta strains . Minimal localization of Msh2p was observed during fully homologous repair, but this was increased in rad52delta strains . These findings argue that Msh2p-Msh3p associates with intermediates early in DSBR to participate in the rejection of homeologous pairing and to stabilize nonhomologous tails for cleavage by Rad1p-Rad10p endonuclease.

Genetics, 2000 Jul, 155(3), 1033 - 44
Isolation and characterization of HRT1 using a genetic screen for mutants unable to degrade Gic2p in saccharomyces cerevisiae; Blondel M et al.; Skp1p-cullin-F-box (SCF) protein complexes are ubiquitin ligases required for degradation of many regulatory proteins involved in cell cycle progression, morphogenesis, and signal transduction . Using a genetic screen, we have isolated a novel allele of the HRT1/RBX1 gene in budding yeast (hrt1-C81Y) . hrt1-C81Y mutant cells exhibited an aberrant morphology but were viable at all temperatures . The cells displayed multiple genetic interactions with mutations in known SCF components and were defective for the degradation of several SCF targets including Gic2p, Far1p, Sic1p, and Cln2p . In addition, they also failed to degrade the F-box proteins Grr1p, Cdc4p, and Met30p . Wild-type Hrt1p but not Hrt1p-C81Y was able to bind multiple F-box proteins in an F-box-dependent manner . Hrt1p-C81Y harbors a single mutation in its ring-finger domain, which is conserved in subunits of distinct E3 ligases . Finally, Hrt1p was localized in both nucleus and cytoplasm and despite a short half-life was expressed constitutively throughout the cell cycle . Taken together, these results suggest that Hrt1p is a core subunit of multiple SCF complexes.

Genetics, 2000 Jul, 155(3), 1019 - 32
The Saccharomyces cerevisiae RDN1 locus is sequestered from interchromosomal meiotic ectopic recombination in a SIR2-dependent manner; Davis ES et al.; Meiotic ectopic recombination occurs at similar frequencies among many sites in the yeast genome, suggesting that all loci are similarly accessible to homology searching . In contrast, we found that his3 sequences integrated in the RDN1 (rDNA) locus were unusually poor participants in meiotic recombination with his3 sequences at other sites . We show that the low rate of meiotic ectopic recombination resulted from the poor ability of RDN1::his3 to act as a donor sequence . SIR2 partially repressed interchromosomal meiotic ectopic recombination at RDN1, consistent with its role in regulating recombination, gene expression, and retrotransposition within RDN1 . We propose that RDN1 is physically sequestered from meiotic homology searching mechanisms.

J Biol Chem, 2000 Sep 15, 275(37), 28816 - 25
Subunit interactions within the Saccharomyces cerevisiae DNA polymerase epsilon (pol epsilon ) complex . Demonstration of a dimeric pol epsilon; Dua R et al.; Saccharomyces cerevisiae DNA polymerase epsilon (pol epsilon) is essential for chromosomal replication . A major form of pol epsilon purified from yeast consists of at least four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p . We have investigated the protein/protein interactions between these polypeptides by using expression of individual subunits in baculovirus-infected Sf9 insect cells and by using the yeast two-hybrid assay . The essential subunits, Pol2p and Dpb2p, interact directly in the absence of the other two subunits, and the C-terminal half of POL2, the only essential portion of Pol2p, is sufficient for interaction with Dpb2p . Dpb3p and Dpb4p, non-essential subunits, also interact directly with each other in the absence of the other two subunits . We propose that Pol2p.Dpb2p and Dpb3p.Dpb4p complexes interact with each other and document several interactions between individual members of the two respective complexes . We present biochemical evidence to support the proposal that pol epsilon may be dimeric in vivo . Gel filtration of the Pol2p.Dpb2p complexes reveals a novel heterotetrameric form, consisting of two heterodimers of Pol2p.Dpb2p . Dpb2p, but not Pol2p, exists as a homodimer, and thus the Pol2p dimerization may be mediated by Dpb2p . The pol2-E and pol2-F mutations that cause replication defects in vivo weaken the interaction between Pol2p and Dpb2p and also reduce dimerization of Pol2p . This suggests, but does not prove, that dimerization may also occur in vivo and be essential for DNA replication.

J Biol Chem, 2000 Jul 7, 275(27), 20920 - 7
Characterization of two 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase isozymes from Saccharomyces cerevisiae; Tibbetts AS et al.; The Saccharomyces cerevisiae ADE16 and ADE17 genes encode 5-aminoimidazole-4-carboxamide ribonucleotide transformylase isozymes that catalyze the penultimate step of the de novo purine biosynthesis pathway . Disruption of these two chromosomal genes results in adenine auxotrophy, whereas expression of either gene alone is sufficient to support growth without adenine . In this work, we show that an ade16 ade17 double disruption also leads to histidine auxotrophy, similar to the adenine/histidine auxotrophy of ade3 mutant yeast strains . We also report the purification and characterization of the ADE16 and ADE17 gene products (Ade16p and Ade17p) . Like their counterparts in other organisms, the yeast isozymes are bifunctional, containing both 5-aminoimidazole-4-carboxamide ribonucleotide transformylase and inosine monophosphate cyclohydrolase activities, and exist as homodimers based on cross-linking studies . Both isozymes are localized to the cytosol, as shown by subcellular fractionation experiments and immunofluorescent staining . Epitope-tagged constructs were used to study expression of the two isozymes . The expression of Ade17p is repressed by the addition of adenine to the media, whereas Ade16p expression is not affected by adenine . Ade16p was observed to be more abundant in cells grown on nonfermentable carbon sources than in glucose-grown cells, suggesting a role for this isozyme in respiration or sporulation.

Biochem, Educ. . 2000 May 1, 28(3), 169 - 170
A simple protocol to evaluate nitrogen utilisation in Saccharomyces cerevisiae; Stella CA et al.; The present article describes a simple protocol that allows the study of nitrogen source utilisation by cells and its relation with cellular growth . The assay can be performed using the minimum of laboratory equipment.

Structure Fold Des, 2000 Jun 15, 8(6), 669 - 84
Structure at 2.3 A resolution of the cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae co-crystallized with an antibody Fv fragment; Hunte C et al.; BACKGROUND: The cytochrome bc(1) complex is part of the energy conversion machinery of the respiratory and photosynthetic electron transfer chains . This integral membrane protein complex catalyzes electron transfer from ubiquinol to cytochrome c . It couples the electron transfer to the electrogenic translocation of protons across the membrane via a so-called Q cycle mechanism . RESULTS: The cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae was crystallized together with a bound antibody Fv fragment . The structure was determined at 2.3 A resolution using multiple isomorphous replacement, and refined to a crystallographic R factor of 22.2% (R(free) = 25.4%) . The complex is present as a homodimer . Each 'monomer' of the refined model includes 2178 amino acid residues of subunits COR1, QCR2, COB, CYT1, RIP1, QCR6, QCR7, QCR8 and QCR9 of the cytochrome bc(1) complex and of the polypeptides V(H) and V(L) of the Fv fragment, the cofactors heme b(H), heme b(L), heme c(1), the {2Fe-2S} cluster and 346 water molecules . The Fv fragment binds to the extrinsic domain of the {2Fe-2S} Rieske protein and is essential for formation of the crystal lattice . CONCLUSIONS: The approach to crystallize membrane proteins as complexes with specific antibody fragments appears to be of general importance . The structure of the yeast cytochrome bc(1) complex reveals in detail the binding sites of the natural substrate coenzyme Q6 and the inhibitor stigmatellin . Buried water molecules close to the binding sites suggest possible pathways for proton uptake and release . A comparison with other cytochrome bc(1) complexes shows features that are specific to yeast.

Biochem Biophys Res Commun, 2000 Jul 5, 273(2), 753 - 8
Cloning and characterization of the NADH pyrophosphatases from Caenorhabditis elegans and Saccharomyces cerevisiae, members of a Nudix hydrolase subfamily; Xu W et al.; Two genes from Caenorhabditis elegans and Saccharomyces cerevisiae, coding for enzymes homologous to the Nudix hydrolase family of nucleotide pyrophosphatases, have been cloned and expressed in Escherichia coli . The purified enzymes are homodimers of 39.1 and 43 . 5 kDa, respectively, are activated by Mg(2+) and Mn(2+), and are 30 to 50 times more active on NADH than on NAD(+) . They both have a conserved array of amino acids downstream of the Nudix box first seen in the orthologous enzyme from E . coli which designates them as members of an NADH pyrophosphatase subfamily of the Nudix hydrolases .

Cell Immunol, 2000 May 25, 202(1), 1 - 5
Mouse homolog of Saccharomyces cerevisiae spo11 is induced in normal mu(+)B-cells by stimuli that cause germline C(H) transcription and subsequent class switch recombination; Tokuyama H et al.; The first step of Ig heavy chain class switch recombination (CSR) is considered to be DNA double strand break (DSB) formation in the two switch (S) regions (S(mu) and downstream S(H)), although the underlying mechanism is unknown . Recently, it has been demonstrated that at least Spo11, a homolog of the novel type II topoisomerase (topo VI) that catalyzes DSB formation, is involved in the initiation of meiotic recombination of Saccaromyces cerevisiae . In the present study, we examined whether the mouse homolog of Spo11 is induced in normal mouse mu(+)B-cells by stimuli that cause an early step of CSR, germline C(H) transcription, and subsequent CSR . Two CSR systems were used: IgA CSR induced by all-trans retinoic acid, IL-5, and LPS, and IgG1 CSR induced by IL-4 and LPS . Germline transcript and mouse Spo11 expression were analyzed by RT-PCR . In both systems, first germline transcripts were clearly detected on day 2 and then Spo11 was detected on day 3, increasing thereafter with time . The time course of changes in Spo11 expression coincided with that of CSR . Spo11 seems to be induced by CSR-inducing stimuli, regardless of the direction of CSR . These results suggested that mouse Spo11 might participate in the initiation step of CSR .

J Biol Chem, 2000 Oct 6, 275(40), 30987 - 95
Regulation of the balance of one-carbon metabolism in Saccharomyces cerevisiae; Piper MD et al.; One-carbon metabolism in yeast is an essential process that relies on at least one of three one-carbon donor molecules: serine, glycine, or formate . By a combination of genetics and biochemistry we have shown how cells regulate the balance of one-carbon flow between the donors by regulating cytoplasmic serine hydroxymethyltransferase activity in a side reaction occurring in the presence of excess glycine . This control governs the level of 5,10-methylene tetrahydrofolate (5,10-CH(2)-H(4)folate) in the cytoplasm, which has a direct role in signaling transcriptional control of the expression of key genes, particularly those encoding the unique components of the glycine decarboxylase complex (GCV1, GCV2, and GCV3) . Based on these and other observations, we propose a model for how cells balance the need to supplement their one-carbon pools when charged folates are limiting or when glycine is in excess . We also propose that under normal conditions, cytoplasmic 5,10-CH(2)-H(4)folate is mainly directed to generating methyl groups via methionine, whereas one-carbon units generated from glycine in mitochondria are more directed to purine biosynthesis . When glycine is in excess, 5, 10-CH(2)-H(4)folate is decreased, and the regulation loop shifts the balance of generation of one-carbon units into the mitochondrion.

Yeast, 2000 Jul, 16(10), 921 - 32
Possible regulatory function of the Saccharomyces cerevisiae Ty1 retrotransposon core protein; Roth JF et al.; The yeast Ty1 retrotransposon encodes proteins and RNA that assemble into virus-like particles (VLPs) as part of the life cycle of the retro-element . The Tya protein, which is equivalent to the retroviral Gag, is the major structural component of these particles . In this work, we demonstrate that Tya proteins fulfil other functions apart from their structural role . We show that Tya interacts in vitro with the Ty1 RNA domain required for RNA packaging, suggesting that this RNA-protein interaction may direct the packaging process . Furthermore, the overexpression of both Tya proteins, i.e . p1, the primary translation product, and p2, the mature form, increases endogenous Ty1 RNA levels in trans without increasing translation significantly . These observations suggest that Tya may exert a regulatory function during transposition . Interestingly, however, only p2, the mature form of Tya, trans-activates transposition of a marked genomic Ty element . This confirms that processing is required for transposition .

Yeast, 2000 Jul, 16(10), 897 - 903
Polymorphism of Saccharomyces cerevisiae aquaporins; Laize V et al.; Aquaporin water channels facilitate the transmembrane diffusion of water and higher organisms possess a large number of isoforms . The genome of the yeast Saccharomyces cerevisiae contains two highly similar aquaporin genes, AQY1 and AQY2 . AQY1 has been shown to encode a functional water channel but only in certain laboratory strains . Here we show that the AQY2 gene is interrupted by an 11 bp deletion in 23 of the 27 laboratory strains tested, with the exception of strains from the sigma 1278b background, which also exhibit a functional Aqy1p . However, although the AQY2 gene from sigma 1278b is highly homologous to functional aquaporins, we did not observe Aqy2p-mediated water transport in Xenopus oocytes . A survey of 52 yeast strains revealed that all industrial and wild yeasts carry the allele encoding a functional Aqy1p, while none of these strains appear to have a functional Aqy2p . We conclude that natural and industrial conditions provide selective pressure to maintain AQY1 but apparently not AQY2 .

Yeast, 2000 Jul, 16(10), 881 - 8
SSU1 mediates sulphite efflux in Saccharomyces cerevisiae; Park H et al.; Ssu1p, a plasma membrane protein involved in sulphite metabolism in Saccharomyces cerevisiae, was found to be required for efficient sulphite efflux . An SSU1 null mutant accumulated significantly more sulphite than wild-type, whereas cells expressing multicopy SSU1 accumulated significantly less . Cells expressing FZF1-4, a dominant allele of a transcriptional activator of SSU1 that confers sulphite resistance, also accumulated less sulphite . beta-galactosidase activity in the FZF1-4 strain carrying an SSU1::lacZ fusion was found to be 8.5-fold higher than in a strain carrying wild-type FZF1, confirming that the heightened resistance was correlated with hyperactivation of SSU1 . Multicopy SSU1 was also found to increase the sulphite resistance of a number of unrelated sulphite-sensitive strains by a factor of 3- to 8-fold . Rates of efflux of free sulphite from cells expressing multicopy SSU1 or FZF1-4 were significantly greater than that from wild-type or from a SSU1 null mutant . Rates of efflux of bound sulphite from wild-type, a SSU1 null mutant, a FZF1-4 mutant, or cells expressing multicopy SSU1 were not significantly different, suggesting that Ssu1p specifically mediates efflux of the free form of sulphite .

Nucleic Acids Res, 2000 Jul 1, 28(13), 2551 - 6
Identification of proteins of Escherichia coli and Saccharomyces cerevisiae that specifically bind to C/C mismatches in DNA; Nakahara T et al.; The pathways leading to G:C-->C:G transversions and their repair mechanisms remain uncertain . C/C and G/G mismatches arising during DNA replication are a potential source of G:C-->C:G transversions . The Escherichia coli mutHLS mismatch repair pathway efficiently corrects G/G mismatches, whereas C/C mismatches are a poor substrate . Escherichia coli must have a more specific repair pathway to correct C/C mismatches . In this study, we performed gel-shift assays to identify C/C mismatch-binding proteins in cell extracts of E . COLI: By testing heteroduplex DNA (34mers) containing C/C mismatches, two specific band shifts were generated in the gels . The band shifts were due to mismatch-specific binding of proteins present in the extracts . Cell extracts of a mutant strain defective in MutM protein did not produce a low-mobility complex . Purified MutM protein bound efficiently to the C/C mismatch-containing heteroduplex to produce the low-mobility complex . The second protein, which produced a high-mobility complex with the C/C mismatches, was purified to homogeneity, and the amino acid sequence revealed that this protein was the FabA protein of E.COLI: The high-mobility complex was not formed in cell extracts of a fabA mutant . From these results it is possible that MutM and FabA proteins are components of repair pathways for C/C mismatches in E.COLI: Furthermore, we found that Saccharomyces cerevisiae OGG1 protein, a functional homolog of E.COLI: MutM protein, could specifically bind to the C/C mismatches in DNA.

Nucleic Acids Res, 2000 Jun 15, 28(12), 2315 - 23
Dbp10p, a putative RNA helicase from Saccharomyces cerevisiae, is required for ribosome biogenesis; Burger F et al.; Ribosome biogenesis requires, in addition to rRNA molecules and ribosomal proteins, a multitude of trans-acting factors . Recently it has become clear that in the yeast Saccharomyces cerevisiae many RNA helicases of the DEAD-box and related families are involved in ribosome biogenesis . Here we show that the previously uncharacterised open reading frame YDL031w (renamed DBP10 for DEAD-box protein 10) encodes an essential putative RNA helicase that is required for accurate ribosome biogenesis . Genetic depletion of Dbp10p results in a deficit in 60S ribosomal subunits and an accumulation of half-mer polysomes . Furthermore, pulse-chase analyses of pre-rRNA processing reveal a strong delay in the maturation of 27SB pre-rRNA intermediates into 25S rRNA and 7S pre-rRNA . Northern blot analyses indicate that this delay leads to higher steady-state levels of 27SB species and reduced steady-state levels of 7S pre-rRNA and 25S/5.8S mature rRNAs, thus explaining the final deficit in 60S subunit and the formation of half-mer polysomes . Consistent with a direct role in ribosome biogenesis, Dbp10p was found to be located predominantly in the nucleolus.

Nucleic Acids Res, 2000 Jun 15, 28(12), 2293 - 301
Saccharomyces cerevisiae RAP1 binds to telomeric sequences with spatial flexibility
Wahlin J, Cohn M.
A wide divergence has been detected in the telomeric sequences among budding yeast species . Despite their length and homogeneity differences, all these yeast telomeric sequences show a conserved core which closely matches the consensus RAP1-binding sequence . We demonstrate that the RAP1 protein binds this sequence core, without involving the diverged sequences outside the core . In Saccharomyces castellii and Saccharomyces dairensis specific classes of interspersed variant repeats are present . We show here that a RAP1-binding site is formed in these species by connecting two consecutive 8 bp telomeric repeats . DNase I footprint analyses specify the binding site as the 13 bp sequence CTGGGTGTCTGGG . The RAP1 protein also binds the variant repeats, although with a lowered affinity . However, a split footprint is produced when RAP1 binds a variant repeat where the two half-sites of the binding site are separated by an additional 6 nt . This is probably caused by the intervening sequence looping out of the RAP1-DNA complex . We suggest that the bipartite subdomain structure of the RAP1 protein allows it to remodel telomeric chromatin, a feature which may be of great relevance for telomeric chromatin assembly and structure in vivo.

Nucleic Acids Res, 2000 Jun 15, 28(12), 2292 - 301
Saccharomyces cerevisiae RAP1 binds to telomeric sequences with spatial flexibility; Wahlin J et al.; A wide divergence has been detected in the telomeric sequences among budding yeast species . Despite their length and homogeneity differences, all these yeast telomeric sequences show a conserved core which closely matches the consensus RAP1-binding sequence . We demonstrate that the RAP1 protein binds this sequence core, without involving the diverged sequences outside the core . In Saccharomyces castellii and Saccharomyces dairensis specific classes of interspersed variant repeats are present . We show here that a RAP1-binding site is formed in these species by connecting two consecutive 8 bp telomeric repeats . DNase I footprint analyses specify the binding site as the 13 bp sequence CTGGGTGTCTGGG . The RAP1 protein also binds the variant repeats, although with a lowered affinity . However, a split footprint is produced when RAP1 binds a variant repeat where the two half-sites of the binding site are separated by an additional 6 nt . This is probably caused by the intervening sequence looping out of the RAP1-DNA complex . We suggest that the bipartite subdomain structure of the RAP1 protein allows it to remodel telomeric chromatin, a feature which may be of great relevance for telomeric chromatin assembly and structure in vivo.

FEBS Lett, 2000 Jun 23, 475(3), 237 - 41
Transcriptional regulation of the Saccharomyces cerevisiae DAL5 gene family and identification of the high affinity nicotinic acid permease TNA1 (YGR260w); Llorente B et al.; We have studied the transcript levels of YGR260w and YLR004c, two genes encoding members of the yeast Dal5p subfamily of the major facilitator family, and we show that they increase when extracellular nicotinic acid and thiamine, respectively, are absent . The deletion of YGR260w in a bna1 auxotrophic mutant for nicotinic acid prevents growth at low nicotinic acid concentration . This suggests that YGR260w is necessary for nicotinic acid import into the cell . The direct measurement of nicotinic acid uptake on whole cells demonstrates that YGR260w encodes the yeast high affinity nicotinic acid permease . Its apparent K(m) of 1.7 microM is low enough to allow the uptake of the low concentrations of nicotinic acid normally secreted by wild type cells.

Genetika, 2000 May, 36(5), 644 - 50
{Interaction of ATP17 gene with SUP45 and SUP35 genes in Saccharomyces cerevisiae yeast}; Shumov NN et al.; Genes SUP35 and SUP45 have been identified in the saccharomycete yeast as genes controlling termination of translation in cytoplasmic ribosomes . However, many facts indicate that the control of translation termination is not the only function of these genes . This work is devoted to studying one of the pleiotropic effects of sup35 and sup45 mutations, a respiratory deficiency . The compensation for this deficiency in mutants for either gene can occur due to a mutation in the ATP17 gene encoding the f-subunit of mitochondrial F1F0 ATP synthase . It is assumed that the observed interaction can be related to the system of co-translational protein import into mitochondria.

Mol Cell Biol, 2000 Jul, 20(14), 5321 - 9
The C terminus of the Saccharomyces cerevisiae alpha-factor receptor contributes to the formation of preactivation complexes with its cognate G protein; Dosil M et al.; Binding of the alpha-factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway in MATa yeast cells . To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant Galpha subunits in an alpha-factor-independent manner . Although the amino acid substitution L236H in the third intracellular loop of the receptor impairs G-protein activation, this substitution had no influence on the ability of the dominant-negative receptors to sequester G proteins or on the ability of receptors to suppress the GPA1-A345T mutant Galpha subunit . In contrast, removal of the cytoplasmic C-terminal domain of the receptor eliminated both of these activities even though the C-terminal domain is unnecessary for G-protein activation . Moreover, the alpha-factor-independent signaling activity of ste2-P258L mutant receptors was inhibited by the coexpression of wild-type receptors but not by coexpression of truncated receptors lacking the C-terminal domain . Deletion analysis suggested that the distal half of the C-terminal domain is critical for sequestration of G proteins . The C-terminal domain was also found to influence the affinity of the receptor for alpha-factor in cells lacking G proteins . These results suggest that the C-terminal cytoplasmic domain of the alpha-factor receptor, in addition to its role in receptor downregulation, promotes the formation of receptor-G-protein preactivation complexes.

Mol Cell Biol, 2000 Jul, 20(14), 5300 - 9
DNA length dependence of the single-strand annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-strand break repair; Sugawara N et al.; A DNA double-strand break (DSB) created by the HO endonuclease in Saccharomyces cerevisiae will stimulate recombination between flanking repeats by the single-strand annealing (SSA) pathway, producing a deletion . Previously the efficiency of SSA, using homologous sequences of different lengths, was measured in competition with that of a larger repeat further from the DSB, which ensured that nearly all cells would survive the DSB if the smaller region was not used (N . Sugawara and J . E . Haber, Mol . Cell . Biol . 12:563-575, 1992) . Without competition, the efficiency with which homologous segments of 63 to 205 bp engaged in SSA was significantly increased . A sequence as small as 29 bp was used 0.2% of the time, and homology dependence was approximately linear up to 415 bp, at which size almost all cells survived . A mutant with a deletion of RAD59, a homologue of RAD52, was defective for SSA, especially when the homologous-sequence length was short; however, even with 1.17-kb substrates, SSA was reduced fourfold . DSB-induced gene conversion also showed a partial dependence on Rad59p, again being greatest when the homologous-sequence length was short . We found that Rad59p plays a role in removing nonhomologous sequences from the ends of single-stranded DNA when it invades a homologous DNA template, in a manner similar to that previously seen with srs2 mutants . Deltarad59 affected DSB-induced gene conversion differently from msh3 and msh2, which are also defective in removing nonhomologous ends in both DSB-induced gene conversion and SSA . A msh3 rad59 double mutant was more severely defective in SSA than either single mutant.

Mol Cell Biol, 2000 Jul, 20(14), 5235 - 47
Polarized growth controls cell shape and bipolar bud site selection in Saccharomyces cerevisiae; Sheu YJ et al.; We examined the relationship between polarized growth and division site selection, two fundamental processes important for proper development of eukaryotes . Diploid Saccharomyces cerevisiae cells exhibit an ellipsoidal shape and a specific division pattern (a bipolar budding pattern) . We found that the polarity genes SPA2, PEA2, BUD6, and BNI1 participate in a crucial step of bud morphogenesis, apical growth . Deleting these genes results in round cells and diminishes bud elongation in mutants that exhibit pronounced apical growth . Examination of distribution of the polarized secretion marker Sec4 demonstrates that spa2Delta, pea2Delta, bud6Delta, and bni1Delta mutants fail to concentrate Sec4 at the bud tip during apical growth and at the division site during repolarization just prior to cytokinesis . Moreover, cell surface expansion is not confined to the distal tip of the bud in these mutants . In addition, we found that the p21-activated kinase homologue Ste20 is also important for both apical growth and bipolar bud site selection . We further examined how the duration of polarized growth affects bipolar bud site selection by using mutations in cell cycle regulators that control the timing of growth phases . The grr1Delta mutation enhances apical growth by stabilizing G(1) cyclins and increases the distal-pole budding in diploids . Prolonging polarized growth phases by disrupting the G(2)/M cyclin gene CLB2 enhances the accuracy of bud site selection in wild-type, spa2Delta, and ste20Delta cells, whereas shortening the polarized growth phases by deleting SWE1 decreases the fidelity of bipolar budding . This study reports the identification of components required for apical growth and demonstrates the critical role of polarized growth in bipolar bud site selection . We propose that apical growth and repolarization at the site of cytokinesis are crucial for establishing spatial cues used by diploid yeast cells to position division planes.

Mol Cell Biol, 2000 Jul, 20(14), 5140 - 8
Evidence for Gal3p's cytoplasmic location and Gal80p's dual cytoplasmic-nuclear location implicates new mechanisms for controlling Gal4p activity in Saccharomyces cerevisiae; Peng G et al.; Genetics and in vitro studies have shown that the direct interaction between Gal3p and Gal80p plays a central role in galactose-dependent Gal4p-mediated GAL gene expression in the yeast Saccharomyces cerevisiae . Precisely how Gal3p-Gal80p interaction effects induction is not clear . It has been assumed that Gal3p interacts with Gal80p in the nucleus upon galactose addition to release Gal80p inhibition of Gal4p . Although Gal80p has been shown to possess nuclear localization signal (NLS) peptides, the subcellular distribution of neither Gal80p nor Gal3p was previously determined . Here we report that Gal3p is located in the cytoplasm and apparently excluded from the nucleus . We show that Gal80p is located in both the cytoplasm and the nucleus . Converting Gal80p into a nucleus-localized protein (NLS-Gal80p) by exogenous NLS addition impairs GAL gene induction . The impaired induction can be partially suppressed by targeting Gal3p to the nucleus (NLS-Gal3p) . We document a very rapid association between NLS-Gal3p and Gal80p in vivo in response to galactose, illustrating that the nuclear import of Gal80p is very rapid and efficient . We also demonstrate that nucleus-localized NLS-Gal80p can move out of the nucleus and shuttle between nuclei in yeast heterokaryons . These results are the first indication that the subcellular distribution dynamics of the Gal3 and Gal80 proteins play a role in regulating Gal4p-mediated GAL gene expression in vivo.

Arch Biochem Biophys, 2000 Jul 1, 379(1), 127 - 36
Functional expression of regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha spp.) in Escherichia coli and saccharomyces cerevisiae; Haudenschild C et al.; The oxygenation pattern of the essential oil monoterpenes of commercial mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (-)-4S-limonene . In spearmint (M . spicata), C6-allylic hydroxylation leads to (-)-trans-carveol and thence (-)-carvone, whereas in peppermint (M . x piperita), C3-allylic hydroxylation leads to (-)-trans-isopiperitenol and ultimately (-)-menthol . cDNAs encoding the C6-hydroxylase and C3-hydroxylase from spearmint and peppermint, respectively, were isolated by a combination of reverse genetic and homology-based cloning methods (S . Lupien, F . Karp, M . Wildung, and R . Croteau, Arch . Biochem . Biophys . 368, 181-192, 1999) . Although both hydroxylase genes were confirmed by functional expression using the baculovirus-Spodoptera system, too little protein was available by this approach to permit detailed study of the structure-function relationships of these catalysts, especially the substrate binding determinants that underlie the regiochemistry and stereochemistry of the reactions . Therefore, heterologous overexpression systems based on Escherichia coli and Saccharomyces cerevisiae were developed to produce several N-terminally modified versions of the recombinant hydroxylases . Ancillary methods for the solubilization, purification, and reconstitution (with recombinant spearmint cytochrome P450 reductase) of the limonene hydroxylases were also devised, with which substrate binding behavior and precise regiochemistry and stereochemistry of product formation were determined .

RNA, 2000 Jun, 6(6), 844 - 60
Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae; Nordlund ME et al.; The presence of 5-methyluridine (m5U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs . In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m5U54)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae . Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m5U54 nucleoside . Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain . In contrast to what is found for the tRNA(m5U54)methyltransferase encoding gene trmA+ in E . coli, the TRM2 gene is not essential for cell viability and a deletion strain shows no obvious phenotype . Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease . The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene . In contrast, we find that the expression of a trm2-LacZ fusion and the activity of the tRNA(m5U54)methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene . Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein . The purified yNucR endo-exonuclease has been reported to have an NH2-D-E-K-N-L motif, which is not found in the Trm2p . Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2.

J Pept Res, 2000 May, 55(5), 372 - 83
Structure, biological activity and membrane partitioning of analogs of the isoprenylated a-factor mating peptide of Saccharomyces cerevisiae; Xie H et al.; Previous biochemical investigations on the Saccharomyces cerevisiae a-factor indicated that this lipopeptide pheromone {YIIKGVFWDPAC(farnesyl)OMe} might adopt a type II beta-turn at positions 4 and 5 of the peptide sequence . To test this hypothesis, we synthesized five analogs of a-factor, in which residues at positions 4 and 5 were replaced with: L-Pro4(I); D-Pro4(II); L-Pro4-D-Ala5(III); D-Pro4-L-Ala5(IV); or Nle4(V) . Analogs were purified to > 99% homogeneity as evidenced by HPLC and TLC and were characterized by mass spectrometry and amino acid analysis . Using a growth arrest assay the conformationally restricted a-factor analogs I and III were found to be almost 50-fold more active than the diastereometric homologs II and IV and were equally active to wild-type a-factor . Replacement of Lys4 with the isosteric Nle4 almost abolished the activity of the pheromone . Thus, the incorporation of residues that promote a type II beta-turn compensated for the loss of the favorable contribution of the Lys4 side chain to pheromone activity . CD spectra on these peptides suggested that they were essentially disordered in both TFE/H2O and in the presence of DMPC vesicles . There was no correlation between CD peak shape and biological activity . Using fluorescence spectroscopy we measured the interaction of lipid vesicles with these position 4 and 5 analogs as well as with three a-factor analogs with a modified farnesyl group . The results indicated that modifications of both the peptide sequence and the lipid moiety affect partitioning into lipid, and that no correlation existed between the propensity of a pheromone to partition into the lipid and its biological activity.

Enzyme Microb Technol, 2000 Jun 1, 26(9-10), 785 - 792
The role of metabolic engineering in the improvement of Saccharomyces cerevisiae: utilization of industrial media; Olsson L et al.; Metabolic engineering has become a very important approach to strain improvement in parallel with classical strain development . Although Saccharomyces cerevisiae has been domesticated for ethanol and bread production, there are still some fundamental problems associated with its industrial use . The industrially used carbon sources often consist of a sugar mixture, and due to glucose repression these sugars are utilized sequentially, resulting in prolonged production time . In this article we discuss the application of metabolic engineering for construction of glucose-derepressed strains and specify advantages as well as difficulties associated with this approach.

Enzyme Microb Technol, 2000 Jun 1, 26(9-10), 763 - 770
Enrichment of threonine content in Saccharomyces cerevisiae by pathway engineering; Farfan M et al.; In a previous work, we have investigated the effect of amplifying individually the genes of the threonine biosynthetic pathway on threonine accumulation by yeast . Here, we present the results of the simultaneous amplification of these genes in strains with different genetic backgrounds . These strains carry a mutant HOM3-R2 allele (coding for a feedback-insensitive aspartate kinase), and/or a mutant cha1 allele that makes it defective in threonine degradation by the catabolic L-serine (L-threonine) deaminase . The results show that the amplification of the clustered genes affects threonine and homoserine accumulation only when it includes the HOM3 gene, or when combined with a HOM3-R2 mutation . Similarly, the cha1 mutation is only effective when a certain amount of threonine is reached . Threonine overproduction affects other cellular functions such as the accumulation of other amino acids, the cell growth and metabolite excretion, probably reflecting a redirection of the carbon flux in the central metabolism.

Enzyme Microb Technol, 2000 Jun 1, 26(9-10), 748 - 755
Mutations in GAL2 or GAL4 alleviate catabolite repression produced by galactose in Saccharomyces cerevisiae; Rodriguez C et al.; Galactose does not allow growth of pyruvate carboxylase mutants in media with ammonium as a nitrogen source, and inhibits growth of strains defective in phosphoglyceromutase in ethanol-glycerol mixtures . Starting with pyc1, pyc2, and gpm1 strains, we isolated mutants that eliminated those galactose effects . The mutations were recessive and were named dgr1-1 and dgr2-1 . Strains bearing those mutations in an otherwise wild-type background grew slower than the wild type in rich galactose media, and their growth was dependent on respiration . Galactose repression of several enzymes was relieved in the mutants . Biochemical and genetic evidence showed that dgr1-1 was allelic with GAL2 and dgr2-1 with GAL4 . The results indicate that the rate of galactose consumption is critical to cause catabolite repression.

Enzyme Microb Technol, 2000 Jun 1, 26(9-10), 688 - 698
Simultaneous genomic overexpression of seven glycolytic enzymes in the yeast Saccharomyces cerevisiae; Hauf J et al.; Fusions of the glycolytic genes TPI1, PGK1, ENO1, PYK1, PDC1, and ADH1 with the lacZ reporter gene of Escherichia coli and a lacZ fusion construct of a 390-bp fragment from the promoter of the HXT7 gene were assayed for beta-galactosidase activity . The glycolytic promoters were induced after addition of glucose to ethanol-grown cells, whereas the HXT7 promoter fragment showed a constitutive beta-galactosidase expression on both carbon sources . The genes coding for the seven enzymes of lower glycolysis Tdh, Pgk, Gpm, Eno, Pyk, Pdc, and Adh were simultaneously put under the control of the same strong promoter, a truncated HXT7 promoter that is constitutively active on ethanol as well as on glucose medium . Genomic expression of the glycolytic genes under the control of this promoter, resulted in an at least 2-fold overexpression . The gene MSG5 was isolated, coding for a protein phosphatase normally involved in cell cycle regulation, as a factor that possibly influences the expression of the HXT7 gene . However, overexpression of MSG5 had no effect on the expression of the HXT7/lacZ fusion, whereas a deletion of this gene resulted in a decreased expression of beta-galactosidase.

Yeast, 2000 Jun 30, 16(9), 797 - 809
The importance of ATP as a regulator of glycolytic flux in Saccharomyces cerevisiae; Larsson C et al.; The control of glycolytic flux in the yeast Saccharomyces cerevisiae was studied by using permeabilized cells . Cells were harvested from chemostat cultures and, after removal of the cell wall, nystatin was used to permeabilize the spheroplasts . By this method it is possible to study the performance and regulation of a complete and functional metabolic pathway and not only a single enzymatic step . The results showed that ATP has a strong negative effect on glycolytic activity affecting several of the glycolytic enzymes . However, the main targets for ATP inhibition was phosphofructokinase and pyruvate kinase . Phospofructokinase was inhibited by ATP concentrations starting at about 1-2 mM, while pyruvate kinase required ATP levels above 2.5 mM before any inhibition was visible . These ATP concentrations were in the same range as measured for nitrogen- and glucose-limited cells cultivated in chemostat cultures . Other potential candidates as enzymes susceptible to ATP inhibition included hexokinase and enolase . The ATP:ADP ratio, as well as trehalose-6-phosphate levels, did not seem to influence the glycolytic activity .

Anal Biochem, 2000 Jun 15, 282(1), 54 - 64
Histidine-tagged ubiquitin substitutes for wild-type ubiquitin in Saccharomyces cerevisiae and facilitates isolation and identification of in vivo substrates of the ubiquitin pathway; Ling R et al.; A general method for purification of any substrate of the ubiquitin pathway, the major eukaryotic proteolytic pathway, should utilize the common characteristic of covalent linkage of ubiquitin to substrate lysyl residues . The utility of a N-terminal histidine-tagged ubiquitin (HisUb) for in vivo conjugation and isolation of ubiquitinated proteins by metal chelation chromatography is conditioned by the requirement that HisUb conjugate to the same set of proteins as wild-type ubiquitin . Stringent in vivo tests with Saccharomyces cerevisiae strains expressing ubiquitins only from plasmids were performed to show that HisUb could substitute for wild-type ubiquitin . The utility of HisUb as a method for purification of proteins ubiquitinated in vivo was demonstrated by metal chelation chromatography of yeast extracts expressing HisUb and immunoblotting for Rpb1, the largest subunit of RNA polymerase II . A fraction of Rpb1 was present in the ubiquitinated form in vivo . The ability to use HisUb expression in transgenic organisms that retain expression of their endogenous ubiquitin genes was demonstrated through transgenic Arabidopsis thaliana expressing HisUb or its variant HisUbK48R . UbK48R is a version of ubiquitin capable of conjugation to proteins, but cannot serve as an attachment site for ubiquitin via the major in vivo interubiquitin linkage . Whereas transgenic plants expressing HisUb showed insignificant enrichment of ubiquitinated proteins, transgenic Arabidopsis lines expressing HisUbK48R gave a much better yield .

Biochem Biophys Res Commun, 2000 Jun 16, 272(3), 946 - 50
Porphyrin biosynthesis intermediates are not regulating delta-aminolevulinic acid transport in Saccharomyces cerevisiae; Moretti MB et al.; In Saccharomyces cerevisiae, as in all eukaryotic organisms, delta-aminolevulinic acid (ALA) is a precursor of porphyrin biosynthesis, a very finely regulated pathway . ALA enters yeast cells through the gamma-aminobutyric acid (GABA) permease Uga4 . The incorporation of a metabolite into the cells may be a limiting step for its intracellular metabolization . To determine the relationship between ALA transport and ALA metabolization, ALA incorporation was measured in yeast mutant strains deficient in the delta-aminolevulinic acid-synthase, uroporphyrinogen III decarboxylase, and ferrochelatase, three enzymes involved in porphyrin biosynthesis . Results presented here showed that neither intracellular ALA nor uroporphyrin or protoporphyrin regulates ALA incorporation, indicating that ALA uptake and its subsequent metabolization are not related to each other . Thus a key metabolite as it is, ALA does not have a transport system regulated according to its role .

Antimicrob Agents Chemother, 2000 Jul, 44(7), 1943 - 53
Effect of polygodial on the mitochondrial ATPase of Saccharomyces cerevisiae; Lunde CS et al.; The fungicidal mechanism of a naturally occurring sesquiterpene dialdehyde, polygodial, was investigated in Saccharomyces cerevisiae . In an acidification assay, polygodial completely suppressed the glucose-induced decrease in external pH at 3.13 microgram/ml, the same as the fungicidal concentration . Acidification occurs primarily through the proton-pumping action of the plasma membrane ATPase, Pma1p . Surprisingly, this ATPase was not directly inhibited by polygodial . In contrast, the two other membrane-bound ATPases in yeast were found to be susceptible to the compound . The mitochondrial ATPase was inhibited by polygodial in a dose-dependent manner at concentrations similar to the fungicidal concentration, whereas the vacuolar ATPase was only slightly inhibited . Cytoplasmic petite mutants, which lack mitochondrial DNA and are respiration deficient, were significantly less susceptible to polygodial than the wild type, as was shown in time-kill curves . A pet9 mutant which lacks a functional ADP-ATP translocator and is therefore respiration dependent was rapidly inhibited by polygodial . The results of these susceptibility assays link enzyme inhibition to physiological effect . Previous studies have reported that plasma membrane disruption is the mechanism of polygodial-induced cell death; however, these results support a more complex picture of its effect . A major target of polygodial in yeast is mitochondrial ATP synthase . Reduction of the ATP supply leads to a suppression of Pma1 ATPase activity and impairs adaptive responses to other facets of polygodial's cellular inhibition.

FEBS Lett, 2000 Jun 16, 475(2), 111 - 6
The mnn2 mutant of Saccharomyces cerevisiae is affected in phosphorylation of N-linked oligosaccharides; Olivero I et al.; We studied the phosphorylation of the inner core region of N-linked oligosaccharides in the mannan defective mutant Saccharomyces cerevisiae mnn2 which was described as unable to synthesize branches on the outer chain . We performed structural studies of the N-oligosaccharides synthesized by the strains mnn2, mnn1mnn2mnn9 and mnn1mnn9ldb8, and the results are compared with previously published structural data of mnn1mnn2mnn10 and mnn1mnn9 {Hernandez, L.M., Ballou, L., Alvarado, E., Tsai, P.-K . and Ballou, C.E . (1989) J . Biol . Chem . 264, 13648-13659} . We conclude that the mnn2/ldb8 mutation is responsible for the inhibition of incorporation of phosphate to mannose A(3) (see below), a particular phosphorylation site of the inner core, while phosphorylation at the other possible site (mannose C(1)) is allowed, although it is also reduced . *Phosphorylation sites in mnn1mnn9 . (see structure below)

FEMS Microbiol Lett, 2000 Jun 15, 187(2), 179 - 84
Phosphorylation of the GTS1 gene product of the yeast Saccharomyces cerevisiae and its effect on heat tolerance and flocculation; Yaguchi S et al.; The GTS1 gene from the yeast Saccharomyces cerevisiae showed pleiotropic effects on yeast phenotypes, including an increase of heat tolerance in stationary-phase cells and an induction of flocculation . Here, we found that the GTS1 product, Gts1p, was partially phosphorylated at some serine residue(s) in cells grown on glucose . Studies using mutants of protein kinase A (PKA) and CDC25, the Ras-GTP exchange activator, showed that PKA positively regulated the phosphorylation level of Gts1p . Overexpression of Gts1p in a mutant with attenuated PKA activity did not show any increase of heat tolerance and partially decreased flocculation inducibility, suggesting that phosphorylation of Gts1p is required for induction of these phenomena.

EMBO J, 2000 Jun 15, 19(12), 2845 - 55
Zinc transporters that regulate vacuolar zinc storage in Saccharomyces cerevisiae; MacDiarmid CW et al.; All cells regulate their intracellular zinc levels . In yeast, zinc uptake is mediated by Zrt1p and Zrt2p, which belong to the ZIP family of metal transporters . Under zinc limitation, ZRT1 and ZRT2 transcription is induced by the Zap1p transcriptional activator . We describe here a new component of zinc homeostasis, vacuolar zinc storage, that is also regulated by Zap1p . Zinc-replete cells accumulate zinc in the vacuole via the Zrc1p and Cot1p transporters . Our results indicate that another zinc transporter, Zrt3p, mobilizes this stored zinc in zinc-limited cells . ZRT3 is a Zap1p-regulated gene whose transcription increases in low zinc . Zrt3p is also a member of the ZIP family and it localizes to the vacuolar membrane . The effects of ZRT3 mutation and overexpression on cell growth, cellular zinc accumulation and intracellular labile zinc pools are all consistent with its proposed role . Furthermore, we demonstrate that zrt3 mutants inefficiently mobilize stored zinc to offset deficiency . Thus, our studies define a system of zinc influx and efflux transporters in the vacuole that play important roles in zinc homeostasis.

EMBO J, 2000 Jun 15, 19(12), 2824 - 33
Sphingoid base synthesis requirement for endocytosis in Saccharomyces cerevisiae; Zanolari B et al.; The internalization step of endocytosis in yeast requires actin and sterols for maximum efficiency . In addition, many receptors and plasma membrane proteins must be phosphorylated and ubiquitylated prior to internalization . The Saccharomyces cerevisiae end8-1 mutant is allelic to lcb1, a mutant defective in the first step of sphingoid base synthesis . Upon arrest of sphingoid base synthesis a rapid block in endocytosis is seen . This block can be overcome by exogenous sphingoid base . Under conditions where endogenous sphingosine base synthesis was blocked and exogenous sphingoid bases could not be converted to phosphorylated sphingoid bases or to ceramide, sphingoid bases could still suppress the endocytic defect . Therefore, the required lipid is most likely a sphingoid base . Interestingly, sphingoid base synthesis is required for proper actin organization, but is not required for receptor phosphorylation . This is the first case of a physiological role for sphingoid base synthesis, other than as a precursor for ceramide or phosphorylated sphingoid base synthesis.

Chromosoma, 2000, 109(1-2), 86 - 93
Meiotic recombination in RAD54 mutants of Saccharomyces cerevisiae; Schmuckli-Maurer J et al.; The Rad54 protein is an important component of the recombinational DNA repair pathway in vegetative Saccharomyces cerevisiae cells . Unlike those in other members of the RAD52 group, the meiotic defect in rad54 is rather mild, reducing spore viability only to 26%-65% . A consistently greater requirement for Rad54p during meiosis was observed in hybrid strains, suggesting that Rad54p has a certain role in interhomolog interactions . Such a role is probably minor as no recombination defects were found in the surviving gametes in three genetic intervals on chromosome V . Also, the spore viability pattern in tetrads did not reflect an increase in nondisjunction at meiosis I indicative of a meiotic recombination defect . We suggest that the meiotic defect of rad54 cells lies in the failure to repair meiosis-specific double-strand breaks outside the context of the highly differentiated pathway leading to interhomolog joint molecules and meiotic crossovers that ensure accurate segregation at meiosis I.

Chromosoma, 2000, 109(1-2), 72 - 85
Spo1, a phospholipase B homolog, is required for spindle pole body duplication during meiosis in Saccharomyces cerevisiae; Tevzadze GG et al.; The SPO1 gene was cloned and shown to encode an early meiotic transcript specifying a nuclear protein with extensive similarity to fungal and vertebrate phospholipase enzymes . Alteration of a conserved serine residue in the putative phospholipase active site, and presence of the spo1-1 temperature-sensitive mutation, which resides near this site, each result in loss of SPO1 function . The phenotype of a complete deletion indicates that SPO1 is dispensable for vegetative growth, premeiotic DNA synthesis and meiotic recombination . In contrast, it is required for Meiosis I (MI) and Meiosis II (MII) chromosome segregation and spore formation . In a null mutant approximately 75% of cells arrest early at MI spindle pole body (SPB) duplication, approximately 20% arrest at MII, and approximately 5% arrest at spore formation . Progression beyond the first arrest point suggests the existence of functions partially redundant to Spo1 and that Spo1 is required at multiple stages . At present SPO1 is the only known gene required for SPB duplication in meiosis but not in mitosis . Its product may thus play a regulatory (rather than a structural) role in SPB function . The transcriptional program in the spo1 null is similar to the wild type early in meiosis but is significantly delayed at later stages of sporulation . A single gene, CWP1, was recovered as a multicopy suppressor of the spo1 null . CWP1 encodes a cell wall protein with a glycolipid moiety . We propose that, when modified by other lipases, this moiety may substitute for the product(s) of Spo1p lipase activity in meiosis . Based on the similarity of Spo1p to phospholipase B enzymes, its unique role in SPB duplication, and pleiotropic effects on MII, late gene expression and spore formation, we propose that the Spo1 protein participates in a novel meiotic pathway that functions through the SPB to coordinate nuclear division with spore development.

Curr Genet, 2000 May, 37(5), 285 - 91
Recessive mutations in SUP35 and SUP45 genes coding for translation release factors affect chromosome stability in Saccharomyces cerevisiae; Borchsenius AS et al.; Chromosome stability in suppressor mutants for SUP35 and SUP45 genes coding for translation release factors was studied . We obtained spontaneous and UV-induced sup35 or sup45 mutants in a haploid strain disomic for chromosome III and tested the stability of an extra copy of this chromosome . The majority of the mutants showed increased chromosome instability . This phenotype was correlated with an increased sensitivity to the microtubule-poisoning drug benomyl which affects chromosome segregation at anaphase . Our data suggest that termination-translation factors eRF3 and eRF1 control chromosome transmission at mitotic anaphase in Saccharomyces cerevisiae.

J Biol Chem, 2000 Aug 25, 275(34), 26011 - 7
Purification and kinetic analysis of eIF2B from Saccharomyces cerevisiae; Nika J et al.; Eukaryotic translation initiation factor 2B (eIF2B) is the heteropentameric guanine nucleotide exchange factor for translation initiation factor 2 (eIF2) . Recent studies in the yeast Saccharomyces cerevisiae have served to characterize genetically the exchange factor . However, enzyme kinetic studies of the yeast enzyme have been hindered by the lack of sufficient quantities of protein suitable for biochemical analysis . We have purified yeast eIF2B and characterized its catalytic properties in vitro . Values for K(m) and V(max) were determined to be 12.2 nm and 250.7 fmol/min, respectively, at 0 degrees C . The calculated turnover number (K(cat)) of 43.2 pmol of GDP released per min/pmol of eIF2B at 30 degrees C is approximately 1 order of magnitude lower than values previously reported for the mammalian factor . Reciprocal plots at varying fixed concentrations of the second substrate were linear and intersected to the left of the y axis . This is consistent with a sequential catalytic mechanism and argues against a ping-pong mechanism similar to that proposed for EF-Tu/EF-Ts . In support of this model, our yeast eIF2B preparations bind guanine nucleotides, with an apparent dissociation constant for GTP in the low micromolar range.

J Bacteriol, 2000 Jun, 182(12), 3517 - 28
The dual-specificity protein phosphatase Yvh1p regulates sporulation, growth, and glycogen accumulation independently of catalytic activity in Saccharomyces cerevisiae via the cyclic AMP-dependent protein kinase cascade; Beeser AE et al.; Yvh1p, a dual-specific protein phosphatase induced specifically by nitrogen starvation, regulates cell growth as well as initiation and completion of sporulation . We demonstrate that yvh1 disruption mutants are also unable to accumulate glycogen in stationary phase . A catalytically inactive variant of yvh1 (C117S) and a DNA fragment encoding only the Yvh1p C-terminal 159 amino acids (which completely lacks the phosphatase domain) complement all three phenotypes as well as the wild-type allele; no complementation occurs with a fragment encoding only the C-terminal 74 amino acids . These observations argue that phosphatase activity is not required for the Yvh1p functions we measured . Mutations which decrease endogenous cyclic AMP (cAMP) levels partially suppress the sporulation and glycogen accumulation defects . In addition, reporter gene expression supported by a DRR2 promoter fragment, containing two stress response elements known to respond to cAMP-protein kinase A, decreases in a yvh1 disruption mutant . Therefore, our results identify three cellular processes that both require Yvh1p and respond to alterations in cAMP, and they lead us to suggest that Yvh1p may be a participant in and/or a contributor to regulation of the cAMP-dependent protein kinase cascade . The fact that decreasing the levels of cAMP alleviates the need for Yvh1p function supports this suggestion.

Mol Gen Genet, 2000 May, 263(4), 722 - 32
Homologous recombination and transposition generate chromosome I neopolymorphism during meiosis in Saccharomyces cerevisiae; Neuveglise C et al.; We have studied the meiotic segregation of a chromosome length polymorphism (CLP) in the yeast Saccharomyces cerevisiae . The neopolymorphism frequently observed within the smallest chromosomes (I, VI, III and IX) is not completely understood . We focused on the analysis of the structure of chromosome I in 88 segregants from a cross between YNN295 and FL100trp . Strain FL100trp is known to carry a reciprocal translocation between the left arm of chromosome III and the right arm of chromosome I . PCR and Southern hybridization analyses were performed and a method for the rapid detection of chromosome I rearrangements was developed . Seven chromosome I types were identified among the 88 segregants . We detected 22 recombination events between homologous chromosomes I and seven ectopic recombination events between FL100trp chromosome III and YNN295 chromosome I . These recombination events occurred in 20 of the 22 tetrads studied (91%) . Nine tetrads (41%) showed two recombination events . This showed that homologous recombination involving polymorphic homologues or heterologous chromosomes is the main source of neopolymorphism . Only one of the seven chromosome I variants resulted from a transposition event rather than a recombination event . We demonstrated that a Tyl element had transposed within the translocated region of chromosome I, generating mutations in the 3' LTR, at the border between U5 and PBS.

J Soc Biol, 1999, 193(1), 23 - 7
{Mechanisms and control of meiotic recombination in the yeast Saccharomyces cerevisiae}; Mezard C et al.; Recent studies in Saccharomyces cerevisiae have provided new insights in our understanding of the molecular mechanisms of meiotic recombination . Meiosis-specific DNA double-strand breaks have been detected and have been shown to be the lesions that initiate recombination events . These are located mostly in promoter regions where the chromatin is in an open configuration, and cluster in domains along the chromosome . They are likely to be made by a topoisomerase II-like protein encoded by the SPO11 gene . Several DNA intermediates in the meiotic double strand-break repair pathway have been characterised and several multi-protein complexes have been identified and shown to be involved at different steps in the repair pathway . The conservation of these protein complexes in higher eukaryotes suggests that the meiotic recombination mechanism could be conserved . With the application of the well characterised genetical, molecular, cytological and biochemical techniques and the recently developed technology for genomic studies (biochips), we can expect a rapid increase in our comprehension of the meiotic recombination process.

J Bacteriol, 2000 Jul, 182(13), 3877 - 80
Organization and cell-cell interaction in starved Saccharomyces cerevisiae colonies; Varon M et al.; Cell growth in yeast colonies is a complex process, the control of which is largely unknown . Here we present scanning electron micrographs of Saccharomyces cerevisiae colonies, showing changes in the pattern of cell organization and cell-cell interactions during colony development . In young colonies (</=36 h), cell density is relatively low, and the cells seem to divide in a random orientation . However, as the colonies age, cell density increases and the cells seem to be oriented in a more orderly fashion . Unexpectedly, cells in starved colonies form connecting fibrils . A single connecting fibril 180 +/- 50 nm wide is observed between any two neighboring cells, and the fibrils appear to form a global network . The results suggest a novel type of communication between cells within a colony that may contribute to the ability of the community to cope with starvation.

J Bacteriol, 2000 Jul, 182(13), 3748 - 53
Cross-pathway regulation in Saccharomyces cerevisiae: activation of the proline utilization pathway by Ga14p in vivo; D'Alessio M et al.; The Put3p and Gal4p transcriptional activators are members of a distinct class of fungal regulators called the Cys(6) Zn(II)(2) binuclear cluster family . This family includes over 50 different Saccharomyces cerevisiae proteins that share a similar domain organization . Gal4p activates the genes of the galactose utilization pathway permitting the use of galactose as the sole source of carbon and energy . Put3p controls the expression of the proline utilization pathway that allows yeast cells to grow on proline as the sole nitrogen source . We report that Gal4p can activate the PUT structural genes in a strain lacking Put3p . We also show that the activation of PUT2 by Gal4p depends on the presence of the inducer galactose and the Put3p binding site and that activation increases with increased dosage of Gal4p . Put3p cannot activate the GAL genes in the absence of Gal4p . Our in vivo results confirm previously published in vitro data showing that Gal4p is more promiscuous than Put3p in its DNA binding ability . The results also suggest that under appropriate circumstances, Gal4p may be able to function in place of a related family member to activate expression.

J Bacteriol, 2000 Jul, 182(13), 3655 - 60
Elo1p-dependent carboxy-terminal elongation of C14:1Delta(9) to C16:1Delta(11) fatty acids in Saccharomyces cerevisiae; Schneiter R et al.; Saccharomyces cerevisiae medium-chain acyl elongase (ELO1) mutants have previously been isolated in screens for fatty acid synthetase (FAS) mutants that fail to grow on myristic acid (C14:0)-supplemented media . Here we report that wild-type cells cultivated in myristoleic acid (C14:1Delta(9))-supplemented media synthesized a novel unsaturated fatty acid that was identified as C16:1Delta(11) fatty acid by gas chromatography-mass spectroscopy . Synthesis of C16:1Delta(11) was dependent on a functional ELO1 gene, indicating that Elo1p catalyzes carboxy-terminal elongation of unsaturated fatty acids (alpha-elongation) . In wild-type cells, the C16:1Delta(11) elongation product accounted for approximately 12% of the total fatty acids . This increased to 18% in cells that lacked a functional acyl chain desaturase (ole1Delta mutants) and hence were fully dependent on uptake and elongation of C14:1 . The observation that ole1Delta mutant cells grew almost like wild type on medium supplemented with C14:1 indicated that uptake and elongation of unsaturated fatty acids were efficient . Interestingly, wild-type cells supplemented with either C14:1 or C16:1 fatty acids displayed dramatic alterations in their phospholipid composition, suggesting that the availability of acyl chains is a dominant determinant of the phospholipid class composition of cellular membranes . In particular, the relative content of the two major phospholipid classes, phosphatidylethanolamine and phosphatidylcholine, was strongly dependent on the chain length of the supplemented fatty acid . Moreover, analysis of the acyl chain composition of individual phospholipid classes in cells supplemented with C14:1 revealed that the relative degree of acyl chain saturation characteristic for each phospholipid class appeared to be conserved, despite the gross alteration in the cellular acyl chain pool . Comparison of the distribution of fatty acids that were taken up and elongated (C16:1Delta(11)) to those that were endogenously synthesized by fatty acid synthetase and then desaturated by Ole1p (C16:1Delta(9)) in individual phospholipid classes finally suggested the presence of two different pools of diacylglycerol species . These results will be discussed in terms of biosynthesis of different phospholipid classes via either the de novo or the Kennedy pathway.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 247 - 54
Nitrogen regulation of Saccharomyces cerevisiae invertase . Role of the URE2 gene; Silveira MC et al.; The regulation of extracellular enzymes is of great biotechnological interest . We studied the regulatory role of the URE2 gene on the periplasmic invertase of Saccharomyces cerevisiae, because its periplasmic asparaginase is regulated by the URE2/GLN3 system . Enzymatic activity was measured in the isogenic strains P40-1B, the ure2 mutant P40-3C, and the P40-3C strain transformed with the pIC-CS plasmid carrying the URE2 gene . The assays were performed using midlog and stationary phase cells and nitrogen-starved cells from these growth phases . During exponential growth, the level of invertase in both wild-type and ure2 mutant cells was comparable . However, the invertase activity in ure2 mutant cells from stationary phase was sixfold lower than in the wild-type cells . When P40-3C cells were transformed with the pIC-CS plasmid, the wild-type phenotype was restored . On nitrogen starvation in the presence of sucrose, the invertase activity in wild-type cells from midlog phase decreased three times, whereas in stationary cells, the activity decreased eight times . However, invertase activity doubled in ure2 mutant cells from both phases . When these cells were transformed with the aforementioned plasmid, the wild-type phenotype was restored, although a significant invertase decrease in stationary cell was not observed . These results suggested that the URE2 protein plays a role in invertase activity.

Mol Biol Cell, 2000 Jun, 11(6), 2175 - 89
Assembly and functional organization of the nucleolus: ultrastructural analysis of Saccharomyces cerevisiae mutants; Trumtel S et al.; Using Saccharomyces cerevisiae strains with genetically modified nucleoli, we show here that changing parameters as critical as the tandem organization of the ribosomal genes and the polymerase transcribing rDNA, although profoundly modifying the position and the shape of the nucleolus, only partially alter its functional subcompartmentation . High-resolution morphology achieved by cryofixation, together with ultrastructural localization of nucleolar proteins and rRNA, reveals that the nucleolar structure, arising upon transcription of rDNA from plasmids by RNA polymerase I, is still divided in functional subcompartments like the wild-type nucleolus . rRNA maturation is restricted to a fibrillar component, reminiscent of the dense fibrillar component in wild-type cells; a granular component is also present, whereas no fibrillar center can be distinguished, which directly links this latter substructure to rDNA chromosomal organization . Although morphologically different, the mininucleoli observed in cells transcribing rDNA with RNA polymerase II also contain a fibrillar subregion of analogous function, in addition to a dense core of unknown nature . Upon repression of rDNA transcription in this strain or in an RNA polymerase I thermosensitive mutant, the nucleolar structure falls apart (in a reversible manner), and nucleolar constituents partially relocate to the nucleoplasm, indicating that rRNA is a primary determinant for the assembly of the nucleolus.

Mol Microbiol, 2000 Jun, 36(5), 1167 - 74
A single glutaredoxin or thioredoxin gene is essential for viability in the yeast Saccharomyces cerevisiae; Draculic T et al.; Glutaredoxins and thioredoxins are small heat-stable oxidoreductases that have been conserved throughout evolution . The yeast Saccharomyces cerevisiae contains two gene pairs encoding cytoplasmic glutaredoxins (GRX1, GRX2) and thioredoxins (TRX1, TRX2) . We report here that the quadruple trx1 trx2 grx1 grx2 mutant is inviable and that either a single glutaredoxin or a single thioredoxin (i.e . grx1 grx2 trx1, grx1 grx2 trx2, grx1 trx1 trx2, grx2 trx1 trx2) is essential for viability . Loss of both thioredoxins has been reported previously to lead to methionine auxotrophy consistent with thioredoxins being the sole reductants for 3'-phosphoadenosine 5'-phosphosulphate reductase (PAPS) in yeast . However, we present evidence for the existence of a novel yeast hydrogen donor for PAPS reductase, as strains lacking both thioredoxins assimilated sulphate under conditions that minimized the generation of reactive oxygen species (low aeration and absence of functional mitochondria) . In addition, the assimilation of {35S}-sulphate was approximately 60-fold higher in the trx1 trx2 grx1 and trx1 trx2 grx2 mutants compared with the trx1 trx2 mutant . Furthermore, in contrast to the trx1 trx2 mutant, the trx1 trx2 grx2 mutant grew on minimal agar plates, and the trx1 trx2 grx1 mutant grew on minimal agar plates under anaerobic conditions . We propose a model in which the novel reductase activity normally functions in the repair of oxidant-mediated protein damage but, under conditions that minimize the generation of reactive oxygen species, it can serve as a hydrogen donor for PAPS reductase.

Mol Microbiol, 2000 May, 36(3), 679 - 87
Identification of genes affecting selenite toxicity and resistance in Saccharomyces cerevisiae; Pinson B et al.; Recent studies associating dietary selenium with reduced cancer susceptibility have aroused interest in this substance . In the millimolar range, selenite is toxic and slightly mutagenic for yeast . We show that selenite-treated yeast cells tend to arrest as large budded cells and that this arrest is abolished in a rad9 mutant that is significantly sensitive to selenite . Interestingly, a rev3 mutant affected in the error-prone repair pathway is also sensitive to selenite, whereas mutations in the other DNA repair pathways do not strongly affect resistance to selenite . We propose that selenite treatment leads to DNA damage inducing the RAD9-dependent cell cycle arrest . Selenite-induced DNA damage could be converted to mutations by the Rev3p-dependent lesion bypass system, thus allowing the cell cycle to progress . We have also investigated the selenite detoxification mechanisms and identified three genes involved in this process . In the present study, we show that lack of the cadmium glutathione-conjugate vacuolar pump Ycf1p or overexpression of the sulphite resistance membrane protein Ssu1p enhance the capacity of yeast cells to resist selenite treatment . Finally, we show that overexpression of the glutathione reductase Glr1p increases resistance to selenite, suggesting that selenite toxicity in yeast is closely linked to its oxidative capacity.

Biochemistry, 2000 Jun 13, 39(23), 6910 - 7
Prenyl-flavonoids as potent inhibitors of the Pdr5p multidrug ABC transporter from Saccharomyces cerevisiae; Conseil G et al.; The Pdr5p multidrug ABC ("ATP-binding cassette) transporter was highly overexpressed in plasma membranes from a yeast strain exhibiting both pdr1-3 gain-of-function mutation in the transcription factor-encoding gene PDR1 and disruption of genes encoding other plasma membrane ABC transporters . Solubilized and purified Pdr5p displayed a tryptophan-characteristic intrinsic fluorescence, whose quenching was used to monitor interactions with substrates and effectors . The transporter exhibited a magnesium-dependent binding affinity for ATP and its fluorescent analogue 2'(3')-N-methylanthraniloyl-ATP, producing a marked fluorescence resonance-energy transfer . It also bound a series of known drug substrates and modulators . Interestingly, yeast Pdr5p interacted with flavonoids recently found to bind to cancer cell P-glycoprotein and to the protozoan parasite multidrug transporter . The extent of high-affinity binding of prenyl-flavonoids to purified Pdr5p was correlated to their efficiency to inhibit energy-dependent quenching of rhodamine 6G fluorescence catalyzed by Pdr5p-enriched plasma membranes . The hydrophobic flavonoid derivative 6-(3, 3-dimethylallyl)galangin was the most efficient, with a K(i) of 0.18 microM for competitive inhibition of the MgATP-dependent quenching of rhodamine 6G fluorescence . In contrast, inhibition of either ATP or UTP hydrolysis occurred at much higher concentrations and appeared to be noncompetitive . Prenyl-flavonoids therefore behave as potent inhibitors of drug binding to the yeast Pdr5p ABC transporter.

Biochemistry, 2000 Jun 13, 39(23), 6960 - 8
Denaturation of phosphofructokinase-1 from Saccharomyces cerevisiae by guanidinium chloride and reconstitution of the unfolded subunits to their catalytically active form; Bar J et al.; Unfolding and refolding of heterooctameric phosphofructokinase-1 from Saccharomyces cerevisiae were investigated by application of kinetic, hydrodynamic, and spectroscopic methods and by use of guanidinium chloride (GdmCl) as denaturant . Inactivation of the enzyme starts at about 0.3 M GdmCl and undergoes a sharp unfolding transition in a narrow range of the denaturant concentration . The inactivation is accompanied by a dissociation of the enzyme into dimers (at 0.6 M GdmCl), which could be detected by changes of the circular dichroism and intrinsic fluorescence . Protein aggregates were observed from 0.7 to 1.5 M GdmCl that unfold at higher denaturant concentrations . Refolding of chemically denatured phosphofructokinase proceeds as a stepwise process via the generation of elements of secondary structure, the formation of assembly-competent monomers that associate to heterodimers and the assembly of dimers to heterotetramers and heterooctamers . The assembly reactions seem to be rate-limiting . Recovery of the enzyme activity (maximum 65%) competes with an nonproductive aggregation of the subunits . alpha-Cyclodextrin functions as an artificial chaperone by preventing aggregation of the subunits, whereas ATP is suggested to support the generation of heterodimers that are competent to a further assembly.

Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6457 - 62
Identification of the single-strand telomeric DNA binding domain of the Saccharomyces cerevisiae Cdc13 protein; Hughes TR et al.; The CDC13 gene of Saccharomyces cerevisiae is required both to protect telomeric DNA and to ensure proper function of yeast telomerase in vivo . We have previously demonstrated that Cdc13p has a high affinity single-strand telomeric DNA binding activity, although the primary amino acid sequence of Cdc13p has no previously characterized DNA binding motifs . We report here mapping of the Cdc13 DNA binding domain by a combination of proteolysis mapping and deletion cloning . The DNA binding domain maps to residues 557-694 of the 924-amino acid Cdc13 polypeptide, within the most basic region of Cdc13p . A slightly larger version of this domain can be efficiently expressed in Escherichia coli as a soluble small protein, with DNA binding properties comparable to those of the full-length protein . A single amino acid missense mutation within this domain results in thermolabile DNA binding and conditional lethality in yeast, consistent with the prediction that DNA binding should be essential for CDC13 function . These results show that Cdc13p contains a discrete substructure responsible for DNA binding and should facilitate structural characterization of this telomere binding protein.

Genetics, 2000 Jun, 155(2), 989 - 92
A glucose transporter chimera confers a dominant negative glucose starvation phenotype in Saccharomyces cerevisiae; Sherwood PW et al.; A family of glucose transporters mediates glucose uptake in Saccharomyces cerevisiae . We show that the dominant mutation GSF4-1, which impairs glucose repression of SUC2, results in a nonfunctional chimera of the transporters Hxt1p and Hxt4p . Hxt1/4p inhibits the function of wild-type glucose transporters . Similar mutations may facilitate analysis of the major facilitator superfamily.

Genetics, 2000 Jun, 155(2), 589 - 99
EXO1 and MSH6 are high-copy suppressors of conditional mutations in the MSH2 mismatch repair gene of Saccharomyces cerevisiae; Sokolsky T et al.; In Saccharomyces cerevisiae, Msh2p, a central component in mismatch repair, forms a heterodimer with Msh3p to repair small insertion/deletion mismatches and with Msh6p to repair base pair mismatches and single-nucleotide insertion/deletion mismatches . In haploids, a msh2Delta mutation is synthetically lethal with pol3-01, a mutation in the Poldelta proofreading exonuclease . Six conditional alleles of msh2 were identified as those that conferred viability in pol3-01 strains at 26 degrees but not at 35 degrees . DNA sequencing revealed that mutations in several of the msh2(ts) alleles are located in regions with previously unidentified functions . The conditional inviability of two mutants, msh2-L560S pol3-01 and msh2-L910P pol3-01, was suppressed by overexpression of EXO1 and MSH6, respectively . Partial suppression was also observed for the temperature-sensitive mutator phenotype exhibited by msh2-L560S and msh2-L910P strains in the lys2-Bgl reversion assay . High-copy plasmids bearing mutations in the conserved EXO1 nuclease domain were unable to suppress msh2-L560S pol3-01 conditional lethality . These results, in combination with a genetic analysis of msh6Delta pol3-01 and msh3Delta pol3-01 strains, suggest that the activity of the Msh2p-Msh6p heterodimer is important for viability in the presence of the pol3-01 mutation and that Exo1p plays a catalytic role in Msh2p-mediated mismatch repair.

Genetics, 2000 Jun, 155(2), 577 - 87
The Saccharomyces cerevisiae centromere protein Slk19p is required for two successive divisions during meiosis; Zeng X et al.; Meiotic cell division includes two separate and distinct types of chromosome segregation . In the first segregational event the sister chromatids remain attached at the centromere; in the second the chromatids are separated . The factors that control the order of chromosome segregation during meiosis have not yet been identified but are thought to be confined to the centromere region . We showed that the centromere protein Slk19p is required for the proper execution of meiosis in Saccharomyces cerevisiae . In its absence diploid cells skip meiosis I and execute meiosis II division . Inhibiting recombination does not correct this phenotype . Surprisingly, the initiation of recombination is apparently required for meiosis II division . Thus Slk19p appears to be part of the mechanism by which the centromere controls the order of meiotic divisions.

Genetics, 2000 Jun, 155(2), 569 - 76
The Saccharomyces cerevisiae mre11(ts) allele confers a separation of DNA repair and telomere maintenance functions; Chamankhah M et al.; The yeast Mre11 protein participates in important cellular functions such as DNA repair and telomere maintenance . Analysis of structure-function relationships of Mre11 has led to identification of several separation-of-function mutations as well as N- and C-terminal domains essential for Mre11 meiotic and mitotic activities . Previous studies have established that there is a strong correlation between Mre11 DNA repair and telomere maintenance functions and that Mre11-Rad50-Xrs2 complex formation appears to be essential for both of these activities . Here we report that the mre11(ts) allele, previously shown to cause temperature-dependent defects in DNA repair and meiosis, confers a temperature-independent telomere shortening, indicating that mre11(ts) is a separation-of-function mutation with respect to DNA repair and telomere maintenance . In a yeast two-hybrid system, Mre11(ts) fails to form a homodimer or interact with Rad50 and Xrs2 irrespective of experimental temperatures . These observations collectively suggest that the Pro(162)Ser substitution in Mre11(ts) confers a novel separation of Mre11 mitotic functions . Moreover, we observed that while overexpression of the 5'-3' exonuclease gene EXO1 partially complements the MMS sensitivity of mre11, rad50, and xrs2 null mutants, it has no effect on telomere shortening in these strains . This result provides additional evidence on possible involvement of distinctive mechanisms in DNA repair and telomere maintenance by the Mre11-Rad50-Xrs2 complex.

Genetics, 2000 Jun, 155(2), 523 - 38
Identification of a novel allele of SIR3 defective in the maintenance, but not the establishment, of silencing in Saccharomyces cerevisiae; Enomoto S et al.; Using a screen for genes that affect telomere function, we isolated sir3-P898R, an allele of SIR3 that reduces telomeric silencing yet does not affect mating . While sir3-P898R mutations cause no detectable mating defect in quantitative assays, they result in synergistic mating defects in combination with mutations such as sir1 that affect the establishment of silencing . In contrast, sir3-P898R in combination with a cac1 mutation, which affects the maintenance of silencing, does not result in synergistic mating defects . MATa sir3-P898R mutants form shmoo clusters in response to alpha-factor, and sir3-P898R strains are capable of establishing silencing at a previously derepressed HML locus with kinetics like that of wild-type SIR3 strains . These results imply that Sir3-P898Rp is defective in the maintenance, but not the establishment of silencing . In addition, overexpression of a C-terminal fragment of Sir3-P898R results in a dominant nonmating phenotype: HM silencing is completely lost at both HML and HMR . Furthermore, HM silencing is most vulnerable to disruption by the Sir3-P898R C terminus immediately after S-phase, the time when new silent chromatin is assembled onto newly replicated DNA.

Genetics, 2000 Jun, 155(2), 509 - 22
Two classes of sir3 mutants enhance the sir1 mutant mating defect and abolish telomeric silencing in Saccharomyces cerevisiae; Stone EM et al.; Silent information regulators, or Sir proteins, play distinct roles in chromatin-mediated transcriptional control at the silent mating-type loci, telomeres, and within the rDNA repeats of Saccharomyces cerevisiae . An unusual collection of sir3 mutant alleles was identified in a genetic screen for enhancers of the sir1 mutant mating-defective phenotype . These sir3-eso mutants, like the sir1 mutant, exhibit little or no mating defects alone, but the sir1 sir3-eso double mutants are essentially nonmating . All of the sir3-eso mutants are defective in telomeric silencing . In some mutants, this phenotype is suppressed by tethering Sir1p to telomeres; other mutants are dominant for mating and telomeric silencing defects . Additionally, several sir3-eso mutants are nonmating in combination with the nat1 N-terminal acetyltransferase mutant . The temperature-sensitive allele sir3-8 has an eso phenotype at permissive temperature, yet acts as a null allele at restrictive temperature due to loss of sir3-8 protein . Sequence analysis showed that eight of the nine sir3-eso alleles have mutations within the N-terminal region that is highly similar to the DNA replication initiation protein Orc1p . Together, these data reveal modular domains for Sir3p and further define its function in silencing chromatin.

EMBO J, 2000 Jun 1, 19(11), 2710 - 8
Three-dimensional cryo-electron microscopy localization of EF2 in the Saccharomyces cerevisiae 80S ribosome at 17.5 A resolution; Gomez-Lorenzo MG et al.; Using a sordarin derivative, an antifungal drug, it was possible to determine the structure of a eukaryotic ribosome small middle dotEF2 complex at 17.5 A resolution by three-dimensional (3D) cryo-electron microscopy . EF2 is directly visible in the 3D map and the overall arrangement of the complex from Saccharomyces cerevisiae corresponds to that previously seen in Escherichia coli . However, pronounced differences were found in two prominent regions . First, in the yeast system the interaction between the elongation factor and the stalk region of the large subunit is much more extensive . Secondly, domain IV of EF2 contains additional mass that appears to interact with the head of the 40S subunit and the region of the main bridge of the 60S subunit . The shape and position of domain IV of EF2 suggest that it might interact directly with P-site-bound tRNA.

EMBO J, 2000 Jun 1, 19(11), 2569 - 79
Saccharomyces cerevisiae Ras/cAMP pathway controls post-diauxic shift element-dependent transcription through the zinc finger protein Gis1; Pedruzzi I et al.; The Saccharomyces cerevisiae protein kinase Rim15 was identified previously as a component of the Ras/cAMP pathway acting immediately downstream of cAMP-dependent protein kinase (cAPK) to control a broad range of adaptations in response to nutrient limitation . Here, we show that the zinc finger protein Gis1 acts as a dosage-dependent suppressor of the rim15Delta defect in nutrient limitation-induced transcriptional derepression of SSA3 . Loss of Gis1 results in a defect in transcriptional derepression upon nutrient limitation of various genes that are negatively regulated by the Ras/cAMP pathway (e.g . SSA3, HSP12 and HSP26) . Tests of epistasis as well as transcriptional analyses of Gis1-dependent expression indicate that Gis1 acts in this pathway downstream of Rim15 to mediate transcription from the previously identified post-diauxic shift (PDS) element . Accordingly, deletion of GIS1 partially suppresses, and overexpression of GIS1 exacerbates the growth defect of mutant cells that are compromised for cAPK activity . Moreover, PDS element-driven expression, which is negatively regulated by the Ras/cAMP pathway and which is induced upon nutrient limitation, is almost entirely dependent on the presence of Gis1.

Hum Gene Ther, 2000 May 20, 11(8), 1165 - 76
Assembly of human papillomavirus type 16 pseudovirions in Saccharomyces cerevisiae; Rossi JL et al.; Studies of the encapsidation of papillomavirus (PV) DNA, and production of preparative amounts of PVs in vitro, have met with only limited success . To circumvent this problem we established a system in yeast to generate infectious HPV-16 pseudovirions . Saccharomyces cerevisiae strain 1699 was transformed with a construct to allow production of HPV-16 virus-like particles (VLPs) . This strain was then transformed with a second construct (target plasmid), the same size as the HPV-16 genome and containing the HPV-16 upstream regulatory region (URR) and the HPV-16 E2 open reading frame . In addition, the target plasmid contained the green fluorescent protein gene to monitor delivery of the target plasmid into mammalian cells after infection . We conclude that this system allows HPV DNA encapsidation because (1) HPV-16 VLPs of two different types (heavy and light) were detected by CsCl gradient centrifugation, (2) DNase I-resistant DNA was detected by PCR/Southern blot analysis in fractions of CsCl gradients at a density corresponding to heavy VLPs, (3) in vitro infection of mammalian cells, including primary mouse splenocytes, with pseudovirions resulted in delivery of the reporter gene as demonstrated by FACS analysis for GFP expression, and (4) after injection of pseudovirions into mice, in vivo reporter gene expression was detected by confocal microscopy in sections of muscle tissue . We conclude that HPV-16 pseudovirions produced in yeast may be useful both for in vitro transduction and for gene delivery in vivo.

Biochem Biophys Res Commun, 2000 Jun 7, 272(2), 403 - 9
Functional expression of the pore forming subunit of the ATP-sensitive potassium channel in Saccharomyces cerevisiae; Graves FM et al.; We have expressed the pore-forming subunits (Kir 6.1 and Kir 6.2) of the mammalian ATP-sensitive potassium channel in a potassium-transport deficient yeast strain (trk1 trk2) . Functional expression of Kir 6.2 and Kir 6.1 can complement growth deficiency weakly and strongly respectively of the yeast strain on low-potassium medium . Mutations of Kir 6.2 that abolish ATP sensitivity (K185Q, I182Q) and enhance trafficking to the plasma membrane surface (Kir 6.2DeltaC36) lead to significantly better growth rescue . Growth rescue of Kir 6.1, Kir 6.2 and the above mutants can be inhibited by pharmacological agents (cesium ions, phentolamine and quinine) known to decrease channel activity by direct interaction with the pore forming subunit . Thus we have developed a system in yeast that can report both loss and gain of function mutations in these subunits and pharmacological interventions .

J Biol Chem, 2000 Aug 25, 275(34), 25931 - 8
Nucleoside transporter proteins of Saccharomyces cerevisiae . Demonstration of a transporter (FUI1) with high uridine selectivity in plasma membranes and a transporter (FUN26) with broad nucleoside selectivity in intracellular membranes; Vickers MF et al.; FUI1 and function unknown now 26 (FUN26) are proteins of uncertain function with sequence similarities to members of the uracil/allantoin permease and equilibrative nucleoside transporter families of transporter proteins, respectively . {(3)H}Uridine influx was eliminated by disruption of the gene encoding FUI1 (fui1) and restored by expression of FUI1 cDNA, whereas influx in transport-competent and fui1-negative yeast were unaffected, respectively, by disruption of the FUN26 gene or overexpression of FUN26 cDNA . FUI1 transported uridine with high affinity (K(m), 22 +/- 3 micrometer) and was unaffected or inhibited only partially by high concentrations (1 mm) of a variety of ribo- and deoxyribonucleosides or nucleobases . When FUN26 cDNA was expressed in oocytes of Xenopus laevis, inward fluxes of {(3)H}uridine, {(3)H}adenosine, and {(3)H}cytidine were stimulated, and uridine influx was independent of pH and not inhibited by dilazep, dipyridamole, or nitrobenzylmercaptopurine ribonucleoside . Fractionation of yeast membranes containing immunotagged recombinant FUN26 (shown to be functional in oocytes) demonstrated that the protein was primarily in intracellular membranes . These results indicated that FUI1 has high selectivity for uracil-containing ribonucleosides and imports uridine across cell-surface membranes, whereas FUN26 has broad nucleoside selectivity and most likely functions to transport nucleosides across intracellular membranes.

Acta Biochim Pol, 1999, 46(4), 901 - 10
Extraribosomal function of the acidic ribosomal P1-protein YP1alpha from Saccharomyces cerevisiae; Tchorzewski M et al.; The yeast acidic ribosomal P-proteins YP1alpha, YP1beta, YP2alpha and YP2beta were studied for a possible transactivation potential beside their ribosomal function . The fusions of P-proteins with the GAL4 DNA-binding domain were assayed toward their transcriptional activity with the aid of reporter genes in yeast . Two of the P-proteins, YP1alpha and YP1beta, exhibited transactivation potential, however, only YP1alpha can be regarded as a potent transactivator . This protein was able to transactivate a reporter gene associated with two distinct promoter systems, GAL1 or CYC1 . Additionally, truncated proteins of YP1alpha and YP1beta were analyzed . The N-terminal part of YP1alpha fused to GAL4-BD showed transactivation potential but the C-terminal part did not . Our results suggest a putative extraribosomal function for these ribosomal proteins which consequently may be classified as "moonlighting" proteins.

J Biol Chem, 2000 May 26, 275(21), 16296 - 301
Mitochondria of Saccharomyces cerevisiae contain one-conserved cysteine type peroxiredoxin with thioredoxin peroxidase activity; Pedrajas JR et al.; Peroxiredoxins are ubiquitously expressed proteins that reduce hydroperoxides using disulfur-reducing compounds as electron donors . Peroxiredoxins (Prxs) have been classified in two groups dependent on the presence of either one (1-Cys Prx) or two (2-Cys Prx) conserved cysteine residues . Moreover, 2-Cys Prxs, also named thioredoxin peroxidases, have peroxide reductase activity with the use of thioredoxin as biological electron donor . However, the biological reducing agent for the 1-Cys Prx has not yet been identified . We report here the characterization of a 1-Cys Prx from yeast Saccharomyces cerevisiae that we have named Prx1p . Prx1p is located in mitochondria, and it is overexpressed when cells use the respiratory pathway, as well as in response to oxidative stress conditions . We show also that Prx1p has peroxide reductase activity in vitro using the yeast mitochondrial thioredoxin system as electron donor . In addition, a mutated form of Prx1p containing the absolutely conserved cysteine as the only cysteine residue also shows thioredoxin-dependent peroxide reductase activity . This is the first example of 1-Cys Prx that has thioredoxin peroxidase activity . Finally, exposure of null Prx1p mutant cells to oxidant conditions reveals an important role of the mitochondrial 1-Cys Prx in protection against oxidative stress.

Genetika, 2000 Apr, 36(4), 470 - 81
{The phenomenon of predetermination of the cytoplasm upon interaction between alleles of the ADE2 and ADE13 in the yeast Saccharomyces cerevisiae}; Zekhnov AM et al.; Our previous data showed that mutation ade13-1, blocking steps 8 and 12 of purine biosynthesis in the yeast Saccharomyces cerevisiae, caused the inability of strains manifesting this activity to grow on the complete nutrient medium with glucose in addition to the loss of adenylosuccinate lyase activity . It was also determined that the ade2-D mutation, inactivating aminoimidasole ribonucleotide carboxylase (the enzyme of step 6), suppressed this phenotypic manifestation of ade13-1; i.e., the ade2-D mutation restores the ability to grow on this medium . When spores of a hybrid that contained both mutations in the heterozygote were germinated on the YEPD medium, almost complete viability of segregants with genotypes ADE2 ADE13 and ade2-D ADE13 and the absence of ADE2 ade13-1 growth were observed . The number of growing segregants ade2-D ade13-1 amounted to approximately half of the possible number . In this work, a decrease in the proportion of segregants with this genotype was shown to occur only when they were obtained as a result of the segregation of hybrids with the normal allele (ADE2) in the heterozygote . The proportion of segregants with genotype ade2-D ade13-1 did not decrease upon segregation of hybrids similar in the genetic background and containing the ade2-D mutation in the homozygote and ade13-1 in the heterozygote . Spores with this genotype formed in the diheterozygous diploid were able to germinate on a medium containing glycerol and to further grow on a medium with glucose . The data suggest that, when a product of the normal ADE2 allele or of another gene, the synthesis of which is stimulated in the presence of this allele, enters spores with genotype ade2-D ade13-1 during meiosis, some of these spores lose their ability to grow on the medium with glucose; i.e., the ADE2 allele can be phenotypically expressed in the spores that did not contain this allele . This phenomenon is similar to the maternal effect known in some species of animals from various systematic groups.

Mol Gen Genet, 2000 Apr, 263(3), 535 - 42
Cysteine is essential for transcriptional regulation of the sulfur assimilation genes in Saccharomyces cerevisiae; Hansen J et al.; Transcription of the genes for sulfur assimilation and methionine biosynthesis in Saccharomyces cerevisiae is regulated by the size of the intracellular pool of an organic sulfur compound . The identity of this compound is not clear, but suggestions include S-adenosylmethionine (SAM) and cysteine . By studying the repression of selected sulfur assimilation (MET) genes, we found that the ability to form cysteine from homocysteine is crucial for methionine-mediated repression to take place . The transcription of MET14 and MET25 could not be repressed by methionine in strains in which either STR4 (which encodes cystathionine beta-synthase) or STR1 (cystathionine gamma-lyase) was disrupted, whereas the repression was independent of GSH1 (which encodes the enzyme responsible for the first step in glutathione biosynthesis from cysteine) . In contrast, cysteine could repress the MET genes in all of these strains . Two genes that presumably encode cystathionine gamma-synthase and cystathionine beta-lyase were identified by genetic disruption (ORFs YJR130c and YGL184c), yielding yeast strains that cannot convert cysteine into homocysteine . Repression by cysteine was possible in either disruptant, suggesting a role in repression for cysteine alone . While some repression of MET genes could be accomplished by homocysteine in a strain that cannot form SAM from methionine, a low intracellular level of SAM seems to be necessary for full cysteine-mediated repression to take place.

Mol Gen Genet, 2000 Apr, 263(3), 527 - 34
Functional specificity of the mitochondrial DnaJ protein, Mdj1p, in Saccharomyces cerevisiae; Lisse T et al.; Inactivation of the gene for the mitochondrial DnaJ homolog, Mdj1p, in Saccharomyces cerevisiae results in temperature sensitivity and the loss of respiratory activity; the latter phenotype has been attributed to the loss of mitochondrial DNA . To investigate the functional specificity of Mdj1p, non-mitochondrial DnaJ proteins were targeted to mitochondria and tested for their ability to substitute for Mdj1p . The tested DnaJ proteins were able to complement the two Mdj1p-linked phenotypes, i.e., respiratory activity and growth at 37 degrees C, to different extents, ranging from full to very poor complementation . All DnaJ homologs ensured faithful propagation of the mitochondrial genome . N-terminal fragments of Mdjlp and Escherichia coli DnaJ comprising the well-characterized J domain partially substituted for Mdj1p . As the only hitherto known function of the N-terminal fragment is modulation of the substrate binding activity of the cognate Hsp70, we conclude that both Mdj1p-linked phenotypes - maintenance of respiratory activity and the ability to grow at elevated temperature - involve a mitochondrial Hsp70 partner protein.

Mol Gen Genet, 2000 Apr, 263(3), 455 - 62
The isolation and characterization of missense mutants in the general repressor protein Ssn6 of Saccharomyces cerevisiae; Limbach MP et al.; Ssn6, a TPR repeat-containing protein, associates with the Tup1 protein to form a general transcriptional repression complex in Saccharomyces cerevisiae . As part of a genetic analysis of this complex, we targeted mutations to the TPR repeat-coding region of the SSN6 gene, and applied selection for constitutive expression of the hypoxic gene ANB1 . All but one of the resulting mutants failed to express full-length Ssn6 protein, indicating that they harbored deletion, frameshift, or nonsense mutations . The one missense mutation encoded a protein with three amino acid substitutions, and the combination was required for the mutant phenotype . One mutation, a proline substitution for a serine at codon 51, was used in a second round of mutagenesis in which six further multiple-substitution alleles were obtained . These were separated into their component mutations, and again, all but one of the single substitutions displayed the wild-type phenotype . The single and multiple mutants were characterized in terms of their effects on the repression of the glucose-repressible SUC2 gene and the a mating-type gene STE2 . The mutant Ssn6 proteins were also tested for their ability to associate with Tup1 . The S5 P mutation, despite its lack of a mutant phenotype, had lost the ability to fully associate with Tupl in vitro . In general, those single substitutions that fell within the first two TPR repeats impaired Tup1-associating activity, while the two that fell in TPR repeats five and eight retained this activity . Overexpression of TUP1 partially suppressed the mutant phenotype in only some of the multiple mutants . The results are discussed in terms of the current models of Ssn6 function and the structure of TPR repeats.

Acta Crystallogr D Biol Crystallogr, 2000 Jun, 56 ( Pt 6), 778 - 80
Crystallization and preliminary X-ray diffraction analysis of Saccharomyces cerevisiae Ygr203p, a homologue of Acr2 arsenate reductase; Moon J et al.; Ygr203p, a 148-residue protein encoded by the ygr203w gene of Saccharomyces cerevisiae, is a homologue of the yeast Acr2 arsenate reductase encoded by the acr2 (or ypr200c) gene . It also shows significant sequence similarity to the human cell-cycle control Cdc25 phosphatase family . It has been overexpressed in soluble form in Escherichia coli with a His(6) tag at its C-terminus . The recombinant protein has been crystallized at 296 K using sodium chloride as precipitant . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 40.48, b = 50.95, c = 91.95 A . The asymmetric unit contains a monomer, giving a crystal volume per protein mass (V(m)) of 2.61 A(3) Da(-1) and a solvent content of 53.8% . The crystals diffract to better than 1.9 A resolution with Cu Kalpha X-rays . They are therefore suitable for high-resolution structure determination.

Acta Crystallogr D Biol Crystallogr, 2000 Jun, 56 ( Pt 6), 763 - 5
Crystallization and aldo-keto reductase activity of Gcy1p from Saccharomyces cerevisiae; Hur E et al.; Crystallization and preliminary X-ray diffraction studies of Gcy1p, an aldo-keto reductase from Saccharomyces cerevisiae, have been performed . Both the wild type and a double-mutant form of Gcy1p were crystallized using the hanging-drop method at 298 K; however, only the double-mutant form has so far yielded crystals suitable for X-ray diffraction analysis . These crystals belonged to the primitive monoclinic space group P2(1), with unit-cell parameters a = 50.94, b = 65.64, c = 86.23 A, beta = 92.64 degrees . Diffraction data were collected to 2.5 A . Assuming two 35 kDa subunits in the asymmetric unit yielded a V(m) of 2.06 A(3) Da(-1) . Additionally, a kinetic study performed by measuring the rate of oxidation of NADPH in the presence of several substrates indicates that both wild-type and double-mutant proteins are enzymes possessing NADPH-dependent reductase activity.

J Biol Chem, 2000 Aug 4, 275(31), 23471 - 5
Absence of the mitochondrial AAA protease Yme1p restores F0-ATPase subunit accumulation in an oxa1 deletion mutant of Saccharomyces cerevisiae; Lemaire C et al.; The nuclear gene OXA1 encodes a protein located within the mitochondrial inner membrane that is required for the biogenesis of both cytochrome c oxidase (Cox) and ATPase . In the absence of Oxa1p, the translocation of the mitochondrially encoded subunit Cox2p to the intermembrane space (also referred to as export) is prevented, and it has been proposed that Oxa1p could be a component of a general mitochondrial export machinery . We have examined the role of Oxa1p in light of its relationships with two mitochondrial proteases, the matrix protease Afg3p-Rca1p and the intermembrane space protease Yme1p, by analyzing the assembly and activity of the Cox and ATPase complexes in Deltaoxa1, Deltaoxa1Deltaafg3, and Deltaoxa1Deltayme1 mutants . We show that membrane subunits of both complexes are specifically degraded in the absence of Oxa1p . Neither Afg3p nor Yme1p is responsible for the degradation of Cox subunits . However, the F(0) subunits Atp4p, Atp6p, and Atp17p are stabilized in the Deltaoxa1Deltayme1 double mutant, and oligomycin-sensitive ATPase activity is restored, showing that the increased stability of the ATPase subunits allows significant translocation and assembly to occur even in the absence of Oxa1p . These results suggest that Oxa1p is not essential for the export of ATPase subunits . In addition, although respiratory function is dispensable in Saccharomyces cerevisiae, we show that the simultaneous inactivation of AFG3 and YME1 is lethal and that the essential function does not reside in their protease activity.

Biochem J, 2000 Jun 1, 348 Pt 2, 263 - 72
Saccharomyces cerevisiae Yak1p protein kinase autophosphorylates on tyrosine residues and phosphorylates myelin basic protein on a C-terminal serine residue; Kassis S et al.; The serine/threonine protein kinase, Yak1p, functions as a negative regulator of the cell cycle in Saccharomyces cerevisiae, acting downstream of the cAMP-dependent protein kinase . In the present work we report that overexpression of haemagglutinin-tagged full-lengthYak1p and an N-terminally truncated form (residues 148-807) lead to growth arrest in PKA compromised yak1 null yeast cells . Both forms of recombinant Yak1p kinase were catalytically active and preferred myelin basic protein (MBP) as a substrate over several other proteins . Phosphopeptide analysis of bovine MBP by tandem MS revealed two major Yak1p phosphorylation sites, Thr-97 and Ser-164 . Peptides containing each site were obtained and tested as Yak1p substrates . Both forms of Yak1p phosphorylated a peptide containing the Ser-164 residue with far more efficient kinetics than MBP . The maximal velocity (V(max)) values of the full-length Yak1p reaction were 110+/-21 (Ser-164) and 8.7+/-1.7 (MBP), and those of N-terminally truncated Yak1p were 560.7+/-74.8 (Ser-164) and 34 . 4+/-2.2 (MBP) pmol/min per mg of protein . Although neither form of Yak1p was able to phosphorylate two generic protein tyrosine kinase substrates, both were phosphorylated on tyrosine residues in vivo and underwent tyrosine autophosphorylation when reacted with ATP in vitro . Tandem MS showed that Tyr-530 was phosphorylated both in vivo and in vitro after reaction with ATP . Pre-treatment with protein tyrosine phosphatase 1B removed all of Yak1p phosphotyrosine content and drastically reduced Yak1p activity against exogenous substrates, suggesting that the phosphotyrosine content of the enzyme is essential for its catalytic activity . Although the N-terminally truncated Yak1p was expressed at a lower level than the full-length protein, its catalytic activity and phosphotyrosine content were significantly higher than those of the full-length enzyme . Taken together, our results suggest that Yak1p is a dual specificity protein kinase which autophosphorylates on Tyr-530 and phosphorylates exogenous substrates on Ser/Thr residues.

Arch Microbiol, 2000 Apr, 173(4), 262 - 8
Modification of plasma membrane lipid order and H+-ATPase activity as part of the response of Saccharomyces cerevisiae to cultivation under mild and high copper stress; Fernandes AR et al.; Plasma membrane lipid disorganization takes place in cells of Saccharomyces cerevisiae grown under copper stress, as shown by fluorescence anisotropy measurements with the lipid reporter probe 1,6-diphenyl-1,3,5-hexatriene . The extent of plasma membrane disorganization, presumably due to copper-induced lipid peroxidation, was discontinuous when measured in cells grown in media supplemented with different concentrations of CuSO4 . Results suggested the existence of adaptive mechanisms that cells employ to protect themselves against the deleterious effects of copper . The adaptive mechanisms examined in this study included the coordinate increase in the activities of Cu,Zn-superoxide dismutase (up to five-fold), glutathione reductase (up to 1.7-fold), and plasma membrane H+-ATPase (up to three-fold) . These enzyme activities showed maximal levels in cells grown with copper supplied at intermediate concentrations, within the range that allowed growth . Significantly, at these concentrations, plasma membrane disorganization did not increase when increasing amounts of CuSO4 were supplied . However, at copper concentrations close to the maximal that allowed growth, the capacity of the yeast cell response to cope with the deleterious effects of copper was exceeded; plasma membrane lipid organization and plasma-membrane-bound H+-ATPase activity drastically declined in response to the increased levels of copper stress and the consequences on growth kinetics were even more severe . Our results clearly suggest that modification of plasma membrane H+-ATPase activity is either part of or the result of the global response of yeast to mild or high copper stress.

Org Lett, 2000 Feb 10, 2(3), 339 - 41
Steric bulk at position 454 in Saccharomyces cerevisiae lanosterol synthase influences B-ring formation but not deprotonation; Joubert BM et al.; {reaction: see text} Lanosterol synthase converts oxidosqualene to the tetracyclic sterol precursor lanosterol . The mutation experiments described here show that an active-site valine residue in lanosterol synthase contributes to cyclization control through steric effects . Mutating to smaller alanine or glycine residues allows formation of the monocyclic achilleol A, whereas the leucine and isoleucine mutants make exclusively lanosterol . The phenylalanine mutant is inactive.

Mutat Res, 2000 Apr 28, 459(3), 203 - 9
Elevation of sister chromatid exchange in Saccharomyces cerevisiae sgs1 disruptants and the relevance of the disruptants as a system to evaluate mutations in Bloom's syndrome gene; Onoda F et al.; The SGS1 of Saccharomyces cerevisiae is a homologue of the Bloom's syndrome and Werner's syndrome genes . The sgs1 disruptants show hyperrecombination, higher sensitivity to methyl methanesulfonate and hydroxyurea, and poor sporulation . In this study, we found that sister chromatid exchange was increased in sgs1 disruptants . We made mutated SGS1 genes coding a protein proved to lack DNA helicase activity (sgs1-hd), having equivalent missense mutations found in Bloom's syndrome patients (sgs1-BS1, sgs1-BS2) . None of the mutated genes could suppress the higher sensitivity to methyl methanesulfonate and hydroxyurea and the increased frequency of interchromosomal recombination and sister chromatid exchange of sgs1 disruptants . On the other hand, all of the mutant genes were able to complement the poor sporulation phenotype of sgs1 disruptants, although the values were not as high as that of wild-type SGS1.

J Cell Biol, 2000 May 15, 149(4), 863 - 74
Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae; Adames NR et al.; During mitosis in budding yeast the nucleus first moves to the mother-bud neck and then into the neck . Both movements depend on interactions of cytoplasmic microtubules with the cortex . We investigated the mechanism of these movements in living cells using video analysis of GFP-labeled microtubules in wild-type cells and in EB1 and Arp1 mutants, which are defective in the first and second steps, respectively . We found that nuclear movement to the neck is largely mediated by the capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization . Efficient microtubule interactions with the capture site require that microtubules be sufficiently long and dynamic to probe the cortex . In contrast, spindle movement into the neck is mediated by microtubule sliding along the bud cortex, which requires dynein and dynactin . Free microtubules can also slide along the cortex of both bud and mother . Capture/shrinkage of microtubule ends also contributes to nuclear movement into the neck and can serve as a backup mechanism to move the nucleus into the neck when microtubule sliding is impaired . Conversely, microtubule sliding can move the nucleus into the neck even when capture/shrinkage is impaired.

J Biol Chem, 2000 May 19, 275(20), 15535 - 40
The Yap1p-dependent induction of glutathione synthesis in heat shock response of Saccharomyces cerevisiae; Sugiyama K et al.; Glutathione is synthesized in two sequential reactions catalyzed by gamma-glutamylcysteine synthetase (GSH1 gene product) and glutathione synthetase (GSH2 gene product) . The expression of GSH1 in Saccharomyces cerevisiae has been known to be up-regulated by Yap1p, a critical transcription factor for the oxidative stress response in yeast . The present study demonstrates that GSH2 expression is also regulated by Yap1p under oxidative stress-induced conditions . In addition to oxidative stress, expression of GSH1 and GSH2 was induced by heat shock stress in a Yap1p-dependent manner with subsequent increases in intracellular glutathione content . Oxygen respiration rate increased when cells were exposed to higher temperatures, and as a result, intracellular oxidation levels were increased . The heat shock-induced expression of GSH1 and GSH2 did not occur under anaerobic conditions . Furthermore, even under aerobic conditions, the heat shock response of these genes was not observed when cells were pretreated with KCN to block oxygen respiration . We speculate that heat shock stress enhances oxygen respiration, which in turn results in an increase in the generation of reactive oxygen species in mitochondria . This signal may be mediated by Yap1p, resulting in the elevation of intracellular glutathione levels.

J Biol Chem, 2000 May 19, 275(20), 14898 - 902
Cloning and characterization of COX18, a Saccharomyces cerevisiae PET gene required for the assembly of cytochrome oxidase; Souza RL et al.; Nuclear mutants of Saccharomyces cerevisiae assigned to complementation group G34 are respiratory-deficient and lack cytochrome oxidase activity and the characteristic spectral peaks of cytochromes a and a(3) . The corresponding gene was cloned by complementation, sequenced, and identified as reading frame YGR062C on chromosome VII . This gene was named COX18 . The COX18 gene product is a polypeptide of 316 amino acids with a putative amino-terminal mitochondrial targeting sequence and predicted transmembrane domains . Respiratory chain carriers other than cytochromes a and a(3) and the ATPase complex are present at near wild-type levels in cox18 mutants, indicating that the mutations specifically affect cytochrome oxidase . The synthesis of Cox1p and Cox3p in mutant mitochondria is normal whereas Cox2p is barely detected among labeled mitochondrial polypeptides . Transcription of COX2 does not require COX18 function, and a chimeric COX3-COX2 mRNA did not suppress the respiratory defect in the null mutant, indicating that the mutation does not impair transcription or translation of the mRNA . Western analysis of cytochrome oxidase subunits shows that inactivation of the COX18 gene greatly reduces the steady state amounts of subunit 2 and results in variable decreases in other subunits of cytochrome oxidase . A gene fusion expressing a biotinylated form of Cox18p complements cox18 mutants . Biotinylated Cox18p is a mitochondrial integral membrane protein . These results indicate Cox18p to be a new member of a group of mitochondrial proteins that function at a late stage of the cytochrome oxidase assembly pathway.

J Bacteriol, 2000 Jun, 182(11), 3158 - 64
In Saccharomyces cerevisiae, expression of arginine catabolic genes CAR1 and CAR2 in response to exogenous nitrogen availability is mediated by the Ume6 (CargRI)-Sin3 (CargRII)-Rpd3 (CargRIII) complex; Messenguy F et al.; The products of three genes named CARGRI, CARGRII, and CARGRIII were shown to repress the expression of CAR1 and CAR2 genes, involved in arginine catabolism . CARGRI is identical to UME6 and encodes a regulator of early meiotic genes . In this work we identify CARGRII as SIN3 and CARGRIII as RPD3 . The associated gene products are components of a high-molecular-weight complex with histone deacetylase activity and are recruited by Ume6 to promoters containing a URS1 sequence . Sap30, another component of this complex, is also required to repress CAR1 expression . This histone deacetylase complex prevents the synthesis of the two arginine catabolic enzymes, arginase (CAR1) and ornithine transaminase (CAR2), as long as exogenous nitrogen is available . Upon nitrogen depletion, repression at URS1 is released and Ume6 interacts with ArgRI and ArgRII, two proteins involved in arginine-dependent activation of CAR1 and CAR2, leading to high levels of the two catabolic enzymes despite a low cytosolic arginine pool . Our data also show that the deletion of the UME6 gene impairs cell growth more strongly than the deletion of the SIN3 or RPD3 gene, especially in the Sigma1278b background.

Mikrobiologiia, 2000 Jan-Feb, 69(1), 34 - 7
{Mutual effect of invertase and acid phosphatase from the yeast Saccharomyces cerevisiae on their secretion into culture media}; Egorov SN et al.; The hypothesis that various extracellular enzymes produced by the yeast Saccharomyces cerevisiae exert a mutual influence on their secretion into the culture medium was tested experimentally . The statistically processed results indicate that extracellular invertase affects the secretion of acid phosphatase, and acid phosphatase affects the secretion of invertase . In addition, the secretion of each of these enzymes was shown to be subject to autoregulation.

Yeast, 2000 May, 16(7), 621 - 30
Disruption of six novel ORFs from Saccharomyces cerevisiae chromosome VII and phenotypic analysis of the deletants; Victoria Escribano M et al.; We describe the disruption and basic phenotypic analysis of six open reading frames (ORFs) of unknown function located in the left arm of Saccharomyces cerevisiae chromosome VII, namely YGL133w, YGL134w, YGL136c, YGL138c, YGL142c and YGL144c . Disruptions were made using the short flanking homology PCR replacement strategy in the FY1679 and CEN.PK2 diploid strains . Sporulation and tetrad analysis of the heterozygous deletants was performed, as well as phenotypic analysis of the corresponding deleted haploid strains . No obvious phenotypes could be attributed to the strains deleted in any of the genes YGL134w, YGL138c and YGL144c under the conditions tested . YGL142c was shown to be an essential gene . Segregants bearing a deletion in YGL136c grew slowly in complete glycerol medium at 37 degrees C . Cells deleted in YGL133w showed abnormal morphology and reduced mating efficiency, but these phenotypes were observed only when the YGL133w disruption was in a MATalpha background . Ygl133 protein was found to localize to the nucleus .

Mol Cell Biol, 2000 Jun, 20(11), 4006 - 15
Saccharomyces cerevisiae RAI1 (YGL246c) is homologous to human DOM3Z and encodes a protein that binds the nuclear exoribonuclease Rat1p; Xue Y et al.; The RAT1 gene of Saccharomyces cerevisiae encodes a 5'-->3' exoribonuclease which plays an essential role in yeast RNA degradation and/or processing in the nucleus . We have cloned a previously uncharacterized gene (YGL246c) that we refer to as RAI1 (Rat1p interacting protein 1) . RAI1 is homologous to Caenorhabditis elegans DOM-3 and human DOM3Z . Deletion of RAI1 confers a growth defect which can be complemented by an additional copy of RAT1 on a centromeric vector or by directing Xrn1p, the cytoplasmic homolog of Rat1p, to the nucleus through the addition of a nuclear targeting sequence . Deletion of RAI1 is synthetically lethal with the rat1-1(ts) mutation and shows genetic interaction with a deletion of SKI2 but not XRN1 . Polysome analysis of an rai1 deletion mutant indicated a defect in 60S biogenesis which was nearly fully reversed by high-copy RAT1 . Northern blot analysis of rRNAs revealed that rai1 is required for normal 5.8S processing . In the absence of RAI1, 5.8S(L) was the predominant form of 5.8S and there was an accumulation of 3'-extended forms but not 5'-extended species of 5 . 8S . In addition, a 27S pre-rRNA species accumulated in the rai1 mutant . Thus, deletion of RAI1 affects both 5' and 3' processing reactions of 5.8S rRNA . Consistent with the in vivo data suggesting that RAI1 enhances RAT1 function, purified Rai1p stabilized the in vitro exoribonuclease activity of Rat1p.

Mol Cell Biol, 2000 Jun, 20(11), 3918 - 27
Role of Saccharomyces cerevisiae ISA1 and ISA2 in iron homeostasis; Jensen LT et al.; The budding yeast Saccharomyces cerevisiae contains two homologues of bacterial IscA proteins, designated Isa1p and Isa2p . Bacterial IscA is a product of the isc (iron-sulfur cluster) operon and has been suggested to participate in Fe-S cluster formation or repair . To test the function of yeast Isa1p and Isa2p, single or combinatorial disruptions were introduced in ISA1 and ISA2 . The resultant isaDelta mutants were viable but exhibited a dependency on lysine and glutamate for growth and a respiratory deficiency due to an accumulation of mutations in mitochondrial DNA . As with other yeast genes proposed to function in Fe-S cluster assembly, mitochondrial iron concentration was significantly elevated in the isa mutants, and the activities of the Fe-S cluster-containing enzymes aconitase and succinate dehydrogenase were dramatically reduced . An inspection of Isa-like proteins from bacteria to mammals revealed three invariant cysteine residues, which in the case of Isa1p and Isa2p are essential for function and may be involved in iron binding . As predicted, Isa1p is targeted to the mitochondrial matrix . However, Isa2p is present within the intermembrane space of the mitochondria . Our deletion analyses revealed that Isa2p harbors a bipartite N-terminal leader sequence containing a mitochondrial import signal linked to a second sequence that targets Isa2p to the intermembrane space . Both signals are needed for Isa2p function . A model for the nonredundant roles of Isa1p and Isa2p in delivering iron to sites of the Fe-S cluster assembly is discussed.

Curr Genet, 2000 Apr, 37(4), 213 - 20
Mss51p, a putative translational activator of cytochrome c oxidase subunit-1 (COX1) mRNA, is required for synthesis of Cox1p in Saccharomyces cerevisiae; Siep M et al.; Mutants of Saccharomyces cerevisiae that lack a functional MSS51 gene are respiratory deficient due to the absence of cytochrome c oxidase subunit 1 (Cox1p) . It has been previously suggested, but not formally proven, that Mss51p is required for translational activation of COX1 mRNA, rather than being involved in a subsequent step in the synthesis of Cox1p or its assembly into cytochrome c oxidase . Pulse-chase labelling experiments now show that the absence of detectable levels of Cox1p in mss51-null strains is indeed due to the lack of synthesis of Cox1p, and is not caused by reduced stability of the protein . To gain more insight into the exact function of Mss51p, we determined the subcellular localization of the protein . We were able to show that an epitope-tagged version of Mss51p (Mss51HA) complements the mutation and can be localized in mitochondria, where it is firmly associated with the mitochondrial inner membrane . In addition, we characterized the previously identified mutant allele mss51-3 . Sequence analysis revealed the presence of a short open reading frame upstream of MSS51 resulting from the creation of an extra ATG start-codon.

FEMS Microbiol Lett, 2000 May 15, 186(2), 221 - 7
Identification and substrate specificity of a ferrichrome-type siderophore transporter (Arn1p) in Saccharomyces cerevisiae; Heymann P et al.; Genes encoding transporters for heterologous siderophores have been identified in Saccharomyces cerevisiae, of which SIT1, TAF1, and ENB1 encode the transporters for ferrioxamines, ferric triacetylfusarinine C and ferric enterobactin, respectively . In the present communication we have shown that a further gene encoding a member of the major facilitator superfamily, ARN1 (YHL040c), is involved in the transport of a specific class of ferrichromes, possessing anhydromevalonyl residues linked to N(delta)-ornithine (ARN) . Ferrirubin and ferrirhodin, which both are produced by filamentous fungi, are the most common representatives of this class of ferrichromes . A strain possessing a disruption in the ARN1 gene was unable to transport ferrirubin, ferrirhodin and also ferrichrome A, indicating that the encoded transporter recognizes anhydromevalonyl and the structurally-related methylglutaconyl side-chains surrounding the iron center . Ferrichromes possessing short-chain ornithine-N(delta)-acetyl residues such as ferrichrome, ferricrocin and ferrichrysin, were excluded by the Arn1 transporter . Substitution of the iron-surrounding N-acyl chains of ferrichromes by propionyl residues had no effect, whereas substitution by butyryl residues led to recognition by the Arn1 transporter . This would indicate that a chain length of four C-atoms is sufficient to allow binding . Using different asperchromes (B1, D1) we also found that a minimal number of two anhydromevalonyl residues is sufficient for recognition by Arn1p . Contrary to the iron-surrounding N-acyl residues, the peptide backbone of ferrichromes was not an important determinant for the Arn1 transporter.

J Biol Chem, 2000 Jul 28, 275(30), 23303 - 9
Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae; Toman PD et al.; The expression of stable recombinant human collagen requires an expression system capable of post-translational modifications and assembly of the procollagen polypeptides . Two genes were expressed in the yeast Saccharomyces cerevisiae to produce both propeptide chains that constitute human type I procollagen . Two additional genes were expressed coding for the subunits of prolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformation of the collagen molecule . Type I procollagen was produced as a stable heterotrimeric helix similar to type I procollagen produced in tissue culture . A key requirement for glutamate was identified as a medium supplement to obtain high expression levels of type I procollagen as heat-stable heterotrimers in Saccharomyces . Expression of these four genes was sufficient for correct assembly and processing of type I procollagen in a eucaryotic system that does not produce collagen.

J Biol Chem, 2000 May 12, 275(19), 14408 - 14
Nitrogen catabolite repression of DAL80 expression depends on the relative levels of Gat1p and Ure2p production in Saccharomyces cerevisiae; Cunningham TS et al.; GATA family activators (Gln3p and Gat1p) and repressors (Dal80p and Deh1p) regulate nitrogen catabolite repression (NCR)-sensitive transcription in Saccharomyces cerevisiae presumably via their competitive binding to the GATA sequences upstream of NCR-sensitive genes . Ure2p, which is not a GATA family member, inhibits Gln3p/Gat1p from functioning in the presence of good nitrogen sources . We show that NCR-sensitive DAL80 transcription can be influenced by the relative levels of GAT1 and URE2 expression . NCR, normally observed with ammonia or glutamine, is severely diminished when Gat1p is overproduced, and this inhibition is overcome by simultaneously increasing URE2 expression . Further, overproduction of Ure2p nearly eliminates NCR-sensitive transcription under derepressive growth conditions, i.e . with proline as the sole nitrogen source . Enhanced green fluorescent protein-Gat1p is nuclear when Gat1p-dependent transcription is high and cytoplasmic when it is inhibited by overproduction of Ure2p.

J Biol Chem, 2000 May 12, 275(19), 14056 - 63
Polyglutamylation of folate coenzymes is necessary for methionine biosynthesis and maintenance of intact mitochondrial genome in Saccharomyces cerevisiae; Cherest H et al.; One-carbon metabolism is essential to provide activated one-carbon units in the biosynthesis of methionine, purines, and thymidylate . The major forms of folates in vivo are polyglutamylated derivatives . In organisms that synthesize folate coenzymes de novo, the addition of the glutamyl side chains is achieved by the action of two enzymes, dihydrofolate synthetase and folylpolyglutamate synthetase . We report here the characterization and molecular analysis of the two glutamate-adding enzymes of Saccharomyces cerevisiae . We show that dihydrofolate synthetase catalyzing the binding of the first glutamyl side chain to dihydropteroate yielding dihydrofolate is encoded by the YMR113w gene that we propose to rename FOL3 . Mutant cells bearing a fol3 mutation require folinic acid for growth and have no dihydrofolate synthetase activity . We show also that folylpolyglutamate synthetase, which catalyzes the extension of the glutamate chains of the folate coenzymes, is encoded by the MET7 gene . Folylpolyglutamate synthetase activity is required for methionine synthesis and for maintenance of mitochondrial DNA . We have tested whether two folylpolyglutamate synthetases could be encoded by the MET7 gene, by the use of alternative initiation codons . Our results show that the loss of mitochondrial functions in met7 mutant cells is not because of the absence of a mitochondrial folylpolyglutamate synthetase.

Curr Genet, 2000 Mar, 37(3), 168 - 74
UBC13, a DNA-damage-inducible gene, is a member of the error-free postreplication repair pathway in Saccharomyces cerevisiae; Brusky J et al.; The Ubc13 protein was recently identified for its unique role in ubiquitin (Ub) chain assembly at the Ub Lys-63 residue instead of the conventional Lys-48 residue . This activity requires Ubc13 to form a complex with Mms2 and indeed ubc13 and mms2 mutations have been shown elsewhere to be epistatic with respect to UV sensitivity . The MMS2 gene is known to be a member of the error-free DNA postreplication repair (PRR) pathway . By contrast, the Ub Lys-63 residue has been previously implicated in the error-prone PRR pathway, since yeast cells carrying the ubiK63R mutation are defective in UV-induced mutagenesis . In the present study, we attempted to define the role of UBC13 within the PRR pathway . We found that the ubc13 mutation is epistatic to mms2 and rad6, confirming that UBC13 belongs to the PRR-pathway . We also found that ubc13 is synergistic to the error-prone PRR pathway mutation rev3, indicating that UBC13 is in a pathway alternative to REV3 mutagenesis . The ubc13 mutant displays up to a 30-fold increase in the spontaneous mutation rate, and this increase is largely REV3 dependent . In addition, UV-induced mutagenesis is fully functional in the ubc13 mutant . These results together demonstrate that UBC13 is a member of the error-free PRR pathway . The involvement of UBC13 in cellular tolerance to DNA-damage is further implicated by our finding that the UBC13 transcript level is increased up to 6-fold in response to DNA-damage.

Mol Biol Cell, 2000 May, 11(5), 1727 - 37
Role of actin and Myo2p in polarized secretion and growth of Saccharomyces cerevisiae; Karpova TS et al.; We examined the role of the actin cytoskeleton in secretion in Saccharomyces cerevisiae with the use of several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells . In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level . Thus, filamentous actin is necessary for polarized secretion but not for secretion per se . Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell . Cortical actin patches cluster at locations that correlate with sites of polarized secretion . However, we found that actin patch polarization is not necessary for polarized secretion because a mutant, bee1Delta(las17Delta), which completely lacks actin patch polarization, displayed polarized growth . In contrast, a mutant lacking actin cables, tpm1-2 tpm2Delta, had a severe defect in polarized growth . The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion . A mutation in the motor domain of Myo2p, myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth . This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin . However, inhibition of Myo2p function by expression of its tail domain completely abolished growth.

Mol Biol Cell, 2000 May, 11(5), 1597 - 609
Activating phosphorylation of the Saccharomyces cerevisiae cyclin-dependent kinase, cdc28p, precedes cyclin binding; Ross KE et al.; Eukaryotic cell cycle progression is controlled by a family of protein kinases known as cyclin-dependent kinases (Cdks) . Two steps are essential for Cdk activation: binding of a cyclin and phosphorylation on a conserved threonine residue by the Cdk-activating kinase (CAK) . We have studied the interplay between these regulatory mechanisms during the activation of the major Saccharomyces cerevisiae Cdk, Cdc28p . We found that the majority of Cdc28p was phosphorylated on its activating threonine (Thr-169) throughout the cell cycle . The extent of Thr-169 phosphorylation was similar for monomeric Cdc28p and Cdc28p bound to cyclin . By varying the order of the addition of cyclin and Cak1p, we determined that Cdc28p was activated most efficiently when it was phosphorylated before cyclin binding . Furthermore, we found that a Cdc28p(T169A) mutant, which cannot be phosphorylated, bound cyclin less well than wild-type Cdc28p in vivo . These results suggest that unphosphorylated Cdc28p may be unable to bind tightly to cyclin . We propose that Cdc28p is normally phosphorylated by Cak1p before it binds cyclin . This activation pathway contrasts with that in higher eukaryotes, in which cyclin binding appears to precede activating phosphorylation.

J Cell Biol, 2000 May 1, 149(3), 553 - 66
Partitioning of the 2-microm circle plasmid of Saccharomyces cerevisiae . Functional coordination with chromosome segregation and plasmid-encoded rep protein distribution; Velmurugan S et al.; The efficient partitioning of the 2-microm plasmid of Saccharomyces cerevisiae at cell division is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB) . In addition, host encoded factors are likely to contribute to plasmid segregation . Direct observation of a 2-microm-derived plasmid in live yeast cells indicates that the multiple plasmid copies are located in the nucleus, predominantly in clusters with characteristic shapes . Comparison to a single-tagged chromosome or to a yeast centromeric plasmid shows that the segregation kinetics of the 2-microm plasmid and the chromosome are quite similar during the yeast cell cycle . Immunofluorescence analysis reveals that the plasmid is colocalized with the Rep1 and Rep2 proteins within the yeast nucleus . Furthermore, the Rep proteins (and therefore the plasmid) tend to concentrate near the poles of the yeast mitotic spindle . Depolymerization of the spindle results in partial dispersion of the Rep proteins in the nucleus concomitant with a loosening in the association between plasmid molecules . In an ipl1-2 yeast strain, shifted to the nonpermissive temperature, the chromosomes and plasmid almost always missegregate in tandem . Our results suggest that, after DNA replication, plasmid distribution to the daughter cells occurs in the form of specific DNA-protein aggregates . They further indicate that the plasmid partitioning mechanism may exploit at least some of the components of the cellular machinery required for chromosomal segregation.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 275 - 9
Influence of thermal and osmotic stresses on the viability of the yeast Saccharomyces cerevisiae; Beney L et al.; This work studies the effect of thermal and dehydration kinetics on the viability of Saccharomyces cerevisiae . The influence of the rate of temperature (T) and osmotic pressure (pi) increases are first investigated . Results showed that yeast viability is preserved by slow variations of temperature or osmotic pressure in a precise range of T or pi . The influence of a previous thermal stress on the resistance to a hyperosmotic stress is also studied . Temperatures equal to or lower than 10 degrees C allowed the preservation of viability after an osmotic stress whereas temperatures above 10 degrees C did not preserve yeast survival.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 33 - 40
Trehalose reserve in Saccharomyces cerevisiae: phenomenon of transport, accumulation and role in cell viability; Plourde-Owobi L et al.; Strains of Saccharomyces cerevisiae deleted for TPS1 encoding trehalose-6-phosphate synthase still accumulate trehalose when harbouring a functional MAL locus . We demonstrate that this accumulation results from an active uptake of trehalose present in the 'yeast extract' used to make the enriched culture media and that no accumulation is observed in mineral media . The uptake of trehalose was shown to be mediated by the alpha-glucoside transporter encoded by AGT1, the expression of which is linked to the presence of a functional MAL locus . Deletion of this gene in a MAL+ tps1 mutant abolished trehalose accumulation on a maltose or galactose mineral medium . However, small amounts of disaccharide were still detected in a agt1 tps1 double mutant when the medium was supplemented with 10 g trehalose l(-1), indicating the existence of a non-concentrative low-affinity sugar transporter . The presence of the high-affinity trehalose permease allowed us to investigate the effect of increasing exogenous trehalose from 0 to 10 g(-1) on intracellular accumulation . A maximum of ca . 10% (wt/wt dry cells) trehalose was attained in the presence of only 1 g l(-1) of disaccharide in the medium . The capability to monitor the intracellular content of trehalose by varying its extracellular concentration, independent of genetic alterations of the trehalose metabolic machinery, allowed the remarkable contribution of this molecule in stress tolerance to be demonstrated, as the higher the trehalose content, the longer the cell survival to a severe heat shock and to glucose starvation.

Yeast, 2000 Apr, 16(6), 547 - 52
Systematic disruption of 456 ORFs in the yeast Saccharomyces cerevisiae; Lucau-Danila A et al.; In the framework of the European Network for Functional Analysis (EUROFAN), five packages of 96 ORFs from chromosomes III, IV, VII, XIII, XIV and XV were subjected to systematic deletions in an isogenic derivative of strain S288c . Deletions were constructed in diploid and haploid strains . Two questionable ORFs overlapping with larger ORFs and seven TY ORFs were discarded . A total of 456 heterozygous and 385 homozygous deletant diploids were obtained . Sixty-nine deletions, 25 of which had never been published before, were lethal in haploid strains and 30 caused slow cellular growth .

Genetics, 2000 May, 155(1), 69 - 83
Genetic interactions between GLC7, PPZ1 and PPZ2 in saccharomyces cerevisiae; Venturi GM et al.; GLC7 encodes an essential serine/threonine protein type I phosphatase in Saccharomyces cerevisiae . Three other phosphatases (Ppz1p, Ppz2p, and Sal6p) share >59% identity in their catalytic region with Glc7p . ppz1 ppz2 null mutants have no apparent growth defect on rich media . However, null alleles of PPZ1 and PPZ2, in combination with mutant alleles of GLC7, confer a range of growth defects varying from slow growth to lethality . These results indicate that Glc7p, Ppz1p, and Ppz2p may have overlapping functions . To determine if this overlap extends to interaction with targeting subunits, Glc7p-binding proteins were tested for interaction in the two-hybrid system with the functional catalytic domain of Ppz1p . Ppz1p interacts strongly with a number of Glc7p regulatory subunits, including Glc8p, a protein that shares homology with mammalian PP1 inhibitor I2 . Genetic data suggest that Glc8p positively affects both Glc7p and Ppz1p functions . Together our data suggest that Ppz1p and Ppz2p may have overlapping functions with Glc7p and that all three phosphatases may act through common regulatory proteins.

Genetics, 2000 May, 155(1), 43 - 55
Point mutations identify a conserved region of the saccharomyces cerevisiae AFR1 gene that is essential for both the pheromone signaling and morphogenesis functions; DeMattei CR et al.; Mating pheromone receptors activate a G protein signal pathway that leads to the conjugation of the yeast Saccharomyces cerevisiae . This pathway also induces the production of Afr1p, a protein that negatively regulates pheromone receptor signaling and is required to form pointed projections of new growth that become the site of cell fusion during mating . Afr1p lacks strong similarity to any well-characterized proteins to help predict how it acts . Therefore, we investigated the relationship between the different functions of Afr1p by isolating and characterizing seven mutants that were defective in regulating pheromone signaling . The AFR1 mutants were also defective when expressed as fusions to STE2, the alpha-factor receptor, indicating that the mutant Afr1 proteins are defective in function and not in co-localizing with receptors . The mutant genes contained four distinct point mutations that all occurred between codons 254 and 263, identifying a region that is critical for AFR1 function . Consistent with this, we found that the corresponding region is very highly conserved in the Afr1p homologs from the yeasts S . uvarum and S . douglasii . In contrast, there were no detectable effects on pheromone signaling caused by deletion or overexpression of YER158c, an open reading frame with overall sequence similarity to Afr1p that lacks this essential region . Interestingly, all of the AFR1 mutants showed a defect in their ability to form mating projections that was proportional to their defect in regulating pheromone signaling . This suggests that both functions may be due to the same action of Afr1p . Thus, these studies identify a specific region of Afr1p that is critical for its function in both signaling and morphogenesis.

FEBS Lett, 2000 Apr 28, 472(2-3), 283 - 6
The essential role of Glu-185 and Tyr-354 residues in the ferroxidase activity of Saccharomyces cerevisiae Fet3; Bonaccorsi di Patti MC et al.; The structural determinants required for ferroxidase activity by the yeast multicopper oxidase Fet3 have been partially clarified by site-directed mutagenesis based on homology modeling . Glu-185 and Tyr-354 were substituted with Ala and Phe, respectively . Fet3 E185A retained ca . 5% residual ferroxidase catalytic efficiency, and almost 40% oxidase efficiency . On the other hand, Fet3 Y354F exhibited 50% residual efficiency as a ferroxidase and more than 70% as an oxidase . These results provide new insights in the mechanism of iron binding and oxidation by Fet3, establishing the essential role of Glu-185 and Tyr-354, and allowing to dissect ferroxidase from non-iron oxidase activity.

J Biol Chem, 2000 May 5, 275(18), 13895 - 900
Identification of two novel TAF subunits of the yeast Saccharomyces cerevisiae TFIID complex; Sanders SL et al.; Using a combination of ion exchange and immunoaffinity chromatography we have purified the general transcription initiation factor TFIID to near homogeneity from Saccharomyces cerevisiae . Yeast TFIID is composed of TBP, the TATA box binding protein, and 14 distinct TBP-associated factors (TAFs), which range in size from 17 to 150 kDa . Twelve of the TAF subunits have been previously identified, but two, TAF48p and TAF65p, are novel . TAF48p exhibits significant sequence similarity to the conserved C-terminal region of Drosophila TAF110p, human TAF130p, and human TAF105p and is encoded by a previously identified gene MPT1 . TAF65p shows no significant sequence homology to any previously identified TAFp . The genes encoding TAF48p and TAF65p are single copy and essential for normal yeast cell growth . Furthermore, neither TAF48p nor TAF65p are associated with the histone acetylase Spt-Ada-Gcn5 complex or other non-TFIID TBF.TAF complexes . The significance of these results in terms of TFIID structure, function, and organization is discussed.

J Biol Chem, 2000 May 5, 275(18), 13879 - 87
Interaction of p55 reverse transcriptase from the Saccharomyces cerevisiae retrotransposon Ty3 with conformationally distinct nucleic acid duplexes; Rausch JW et al.; The 55-kDa reverse transcriptase (RT) domain of the Ty3 POL3 open reading frame was purified and evaluated on conformationally distinct nucleic acid duplexes . Purified enzyme migrated as a monomer by size exclusion chromatography . Enzymatic footprinting indicate Ty3 RT protects template nucleotides +7 through -21 and primer nucleotides -1 through -24 . Contrary to previous data with retroviral enzymes, a 4-base pair region of the template-primer duplex remained nuclease accessible . The C-terminal portion of Ty3 RT encodes a functional RNase H domain, although the hydrolysis profile suggests an increased spatial separation between the catalytic centers . Despite conservation of catalytically important residues in the RNase H domain, Fe(2+) fails to replace Mg(2+) in the RNase H catalytic center for localized generation of hydroxyl radicals, again suggesting this domain may be structurally distinct from its retroviral counterparts . RNase H specificity was investigated using a model system challenging the enzyme to select the polypurine tract primer from within an RNA/DNA hybrid, extend this into (+) DNA, and excise the primer from nascent DNA . Purified RT catalyzed each of these three steps but was almost inactive on a non-polypurine tract RNA primer . Our studies provide the first detailed characterization of the enzymatic activities of a retrotransposon reverse transcriptase.

J Biol Chem, 2000 May 5, 275(18), 13259 - 65
Hgt1p, a high affinity glutathione transporter from the yeast Saccharomyces cerevisiae; Bourbouloux A et al.; A high affinity glutathione transporter has been identified, cloned, and characterized from the yeast Saccharomyces cerevisiae . This transporter, Hgt1p, represents the first high affinity glutathione transporter to be described from any system so far . The strategy for the identification involved investigating candidate glutathione transporters from the yeast genome sequence project followed by genetic and physiological investigations . This approach revealed HGT1 (open reading frame YJL212c) as encoding a high affinity glutathione transporter . Yeast strains deleted in HGT1 did not show any detectable plasma membrane glutathione transport, and hgt1Delta disruptants were non-viable in a glutathione biosynthetic mutant (gsh1Delta) background . The glutathione repressible transport activity observed in wild type cells was also absent in the hgt1Delta strains . The transporter was cloned and kinetic studies indicated that Hgt1p had a high affinity for glutathione (K(m) = 54 micrometer)) and was not sensitive to competition by amino acids, dipeptides, or other tripeptides . Significant inhibition was observed, however, with oxidized glutathione and glutathione conjugates . The transporter reveals a novel class of transporters that has homologues in other yeasts and plants but with no apparent homologues in either Escherichia coli or in higher eukaryotes other than plants.

J Biol Chem, 2000 Jul 7, 275(27), 20638 - 46
Human ERK1 induces filamentous growth and cell wall remodeling pathways in Saccharomyces cerevisiae; Atienza JM et al.; Expression of an activated extracellular signal-regulated kinase 1 (ERK1) construct in yeast cells was used to examine the conservation of function among mitogen-activated protein (MAP) kinases . Sequence alignment of the human MAP kinase ERK1 with all Saccharomyces cerevisiae kinases reveals a particularly strong kinship with Kss1p (invasive growth promoting MAP kinase), Fus3p (pheromone response MAP/ERK kinase), and Mpk1p (cell wall remodeling MAP kinase) . A fusion protein of constitutively active human MAP/ERK kinase 1 (MEK) and human ERK1 was introduced under regulated expression into yeast cells . The fusion protein (MEK/ERK) induced a filamentation response element promoter and led to a growth retardation effect concomitant with a morphological change resulting in elongated cells, bipolar budding, and multicell chains . Induction of filamentous growth was also observed for diploid cells following MEK/ERK expression in liquid culture . Neither haploids nor diploids, however, showed marked penetration of agar medium . These effects could be triggered by either moderate MEK/ERK expression at 37 degrees C or by high level MEK/ERK expression at 30 degrees C . The combination of high level MEK/ERK expression and 37 degrees C resulted in cell death . The deleterious effects of MEK/ERK expression and high temperature were significantly mitigated by 1 m sorbitol, which also enhanced the filamentous phenotype . MEK/ERK was able to constitutively activate a cell wall maintenance reporter gene, suggesting misregulation of this pathway . In contrast, MEK/ERK effectively blocked expression from a pheromone-responsive element promoter and inhibited mating . These results are consistent with MEK/ERK promoting filamentous growth and altering the cell wall through its ability to partially mimic Kss1p and stimulate a pathway normally controlled by Mpk1p, while appearing to inhibit the normal functioning of the structurally related yeast MAP kinase Fus3p.

J Biol Chem, 2000 Jul 21, 275(29), 22409 - 17
The Saccharomyces cerevisiae RuvB-like protein, Tih2p, is required for cell cycle progression and RNA polymerase II-directed transcription; Lim CR et al.; Two highly conserved RuvB-like putative DNA helicases, p47/TIP49b and p50/TIP49a, have been identified in the eukaryotes . Here, we study the function of Saccharomyces cerevisiae TIH2, which corresponds to mammalian p47/TIP49b . Tih2p is required for vegetative cell growth and localizes in the nucleus . Immunoprecipitation analysis revealed that Tih2p tightly interacts with Tih1p, the counterpart of mammalian p50/TIP49a, which has been shown to interact with the TATA-binding protein and the RNA polymerase II holoenzyme complex . Furthermore, the mutational study of the Walker A motif, which is required for nucleotide binding and hydrolysis, showed that this motif plays indispensable roles in the function of Tih2p . When a temperature-sensitive tih2 mutant, tih2-160, was incubated at the nonpermissive temperature, cells were rapidly arrested in the G(1) phase . Northern blot analysis revealed that Tih2p is required for transcription of G(1) cyclin and of several ribosomal protein genes . The similarities between the mutant phenotypes of tih2-160 and those of taf145 mutants suggest a role for TIH2 in the regulation of RNA polymerase II-directed transcription.

FASEB J, 2000 May, 14(7), 968 - 72
GFAT as a target molecule of methylmercury toxicity in Saccharomyces cerevisiae; Naganuma A et al.; Using a genomic library constructed from Saccharomyces cerevisiae, we have identified a gene GFA1 that confers resistance to methylmercury toxicity . GFA1 encodes L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) and catalyzes synthesis of glucosamine-6-phosphate . Transformed yeast cells expressing GFA1 demonstrated resistance to methylmercury but no resistance to p-chloromercuribenzoate, a GFAT inhibitor . The cytotoxicity of methylmercury was inhibited by loading excess glucosamine 6-phosphate into yeast . Considering that GFAT is an essential cellular enzyme, our findings suggest that GFAT is the major target molecule of methylmercury in yeasts . This report is the first to identify the target molecule of methylmercury toxicity in eukaryotic cells.

J Bacteriol, 2000 May, 182(10), 2886 - 92
Initiation of protein synthesis in Saccharomyces cerevisiae mitochondria without formylation of the initiator tRNA; Li Y et al.; Protein synthesis in eukaryotic organelles such as mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl tRNA (fMet-tRNA(fMet)) for initiation . Here we show that initiation of protein synthesis in yeast mitochondria can occur without formylation of the initiator methionyl-tRNA (Met-tRNA(fMet)) . The formylation reaction is catalyzed by methionyl-tRNA formyltransferase (MTF) located in mitochondria and uses N(10)-formyltetrahydrofolate (10-formyl-THF) as the formyl donor . We have studied yeast mutants carrying chromosomal disruptions of the genes encoding the mitochondrial C(1)-tetrahydrofolate (C(1)-THF) synthase (MIS1), necessary for synthesis of 10-formyl-THF, and the methionyl-tRNA formyltransferase (open reading frame YBL013W; designated FMT1) . A direct analysis of mitochondrial tRNAs using gel electrophoresis systems that can separate fMet-tRNA(fMet), Met-tRNA(fMet), and tRNA(fMet) shows that there is no formylation in vivo of the mitochondrial initiator Met-tRNA in these strains . In contrast, the initiator Met-tRNA is formylated in the respective "wild-type" parental strains . In spite of the absence of fMet-tRNA(fMet), the mutant strains exhibited normal mitochondrial protein synthesis and function, as evidenced by normal growth on nonfermentable carbon sources in rich media and normal frequencies of generation of petite colonies . The only growth phenotype observed was a longer lag time during growth on nonfermentable carbon sources in minimal media for the mis1 deletion strain but not for the fmt1 deletion strain.

Can J Microbiol, 2000 Apr, 46(4), 383 - 6
Inactivation of maltose permease and maltase in sporulating Saccharomyces cerevisiae; Ferreira JC et al.; Maltose transport and maltase activities were inactivated during sporulation of a MAL constitutive yeast strain harboring different MAL loci . Both activities were reduced to almost zero after 5 h of incubation in sporulation medium . The inactivation of maltase and maltose permease seems to be related to optimal sporulation conditions such as a suitable supply of oxygen and cell concentration in the sporulating cultures, and occurs in the fully derepressed conditions of incubation in the sporulation acetate medium . The inactivation of maltase and maltose permease under sporulation conditions in MAL constitutive strains suggests an alternative mechanism for the regulation of the MAL gene expression during the sporulation process.

Proc Natl Acad Sci U S A, 2000 May 9, 97(10), 5173 - 8
The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae; Anderson J et al.; The modified nucleoside 1-methyladenosine (m(1)A) is found at position 58 in the TPsiC loop of many eukaryotic tRNAs . The absence of m(1)A from all tRNAs in Saccharomyces cerevisiae mutants lacking Gcd10p elicits severe defects in processing and stability of initiator methionine tRNA (tRNA(i)(Met)) . Gcd10p is found in a complex with Gcd14p, which contains conserved motifs for binding S-adenosylmethionine (AdoMet) . These facts, plus our demonstration that gcd14Delta cells lacked m(1)A, strongly suggested that Gcd10p/Gcd14p complex is the yeast tRNA(m(1)A)methyltransferase {(m(1)A)MTase} . Supporting this prediction, affinity-purified Gcd10p/Gcd14p complexes used AdoMet as a methyl donor to synthesize m(1)A in either total tRNA or purified tRNA(i)(Met) lacking only this modification . Kinetic analysis of the purified complex revealed K(M) values for AdoMet or tRNA(i)(Met) of 5.0 microM and 2.5 nM, respectively . Mutations in the predicted AdoMet-binding domain destroyed GCD14 function in vivo and (m(1)A)MTase activity in vitro . Purified Flag-tagged Gcd14p alone had no enzymatic activity and was severely impaired for tRNA-binding compared with the wild-type complex, suggesting that Gcd10p is required for tight binding of the tRNA substrate . Our results provide a demonstration of a two-component tRNA MTase and suggest that binding of AdoMet and tRNA substrates depends on different subunits of the complex.

Biochim Biophys Acta, 2000 May 1, 1474(3), 269 - 72
Dynamics of ethanol translocation in Saccharomyces cerevisiae as detected by (13)C-NMR; Yuan YJ et al.; (13)C-NMR has yielded to the dynamics study of ethanol as carbon and energy source in the metabolic oscillation of Saccharomyces cerevisiae . Three ethanol fractions such as media, cytoplasm and mitochondria were observed and characterised by different longitudinal relaxation times and chemical shifts.

Mol Cell Biol, 2000 May, 20(10), 3597 - 607
Phospholipase C is involved in kinetochore function in Saccharomyces cerevisiae; Lin H et al.; The budding yeast PLC1 gene encodes a homolog of the delta isoform of mammalian phosphoinositide-specific phospholipase C . Here, we present evidence that Plc1p associates with the kinetochore complex CBF3 . This association is mediated through interactions with two established kinetochore proteins, Ndc10p and Cep3p . We show by chromatin immunoprecipitation experiments that Plc1p resides at centromeric loci in vivo . Deletion of PLC1, as well as plc1 mutations which abrogate the interaction of Plc1p with the CBF3 complex, results in a higher frequency of minichromosome loss, nocodazole sensitivity, and mitotic delay . Overexpression of Ndc10p suppresses the nocodazole sensitivity of plc1 mutants, implying that the association of Plc1p with CBF3 is important for optimal kinetochore function . Chromatin extracts from plc1Delta cells exhibit reduced microtubule binding to minichromosomes . These results suggest that Plc1p associates with kinetochores and regulates some aspect of kinetochore function and demonstrate an intranuclear function of phospholipase C in eukaryotic cells.

Mol Cell Biol, 2000 May, 20(10), 3425 - 33
Repair of intermediate structures produced at DNA interstrand cross-links in Saccharomyces cerevisiae; McHugh PJ et al.; Bifunctional alkylating agents and other drugs which produce DNA interstrand cross-links (ICLs) are among the most effective antitumor agents in clinical use . In contrast to agents which produce bulky adducts on only one strand of the DNA, the cellular mechanisms which act to eliminate DNA ICLs are still poorly understood, although nucleotide excision repair is known to play a crucial role in an early repair step . Using haploid Saccharomyces cerevisiae strains disrupted for genes central to the recombination, nonhomologous end-joining (NHEJ), and mutagenesis pathways, all these activities were found to be involved in the repair of nitrogen mustard (mechlorethamine)- and cisplatin-induced DNA ICLs, but the particular pathway employed is cell cycle dependent . Examination of whole chromosomes from treated cells using contour-clamped homogenous electric field electrophoresis revealed the intermediate in the repair of ICLs in dividing cells, which are mostly in S phase, to be double-strand breaks (DSBs) . The origin of these breaks is not clear since they were still efficiently induced in nucleotide excision and base excision repair-deficient, mismatch repair-defective, rad27 and mre11 disruptant strains . In replicating cells, RAD52-dependent recombination and NHEJ both act to repair the DSBs . In contrast, few DSBs were observed in quiescent cells, and recombination therefore seems dispensable for repair . The activity of the Rev3 protein (DNA polymerase zeta) is apparently more important for the processing of intermediates in stationary-phase cells, since rev3 disruptants were more sensitive in this phase than in the exponential growth phase.

J Biol Chem, 2000 Apr 28, 275(17), 13007 - 11
Steady-state levels of histone acetylation in Saccharomyces cerevisiae; Waterborg JH; The importance of control of the levels of histone acetylation for the control of gene expression in eukaryotic chromatin is being elucidated, and the yeast Saccharomyces cerevisiae has proven to be an important model system . The level of histone acetylation in yeast is the highest known . However, only acetylation of H4 has been quantified, and reports reveal loss of acetylation in histone preparations . A chaotropic guanidine-based method for histone isolation from intact wild-type cells or from a single-step nuclear preparation with butyrate preserves acetylation of all core histones . Histone H4 has an average of more than 2 acetylated lysines per molecule, distributed over 4 sites . Histones H2A, H3, and H2B have 0 . 2, approximately 2, and >2 acetylated lysines per molecule, respectively, distributed across 2, 5, and 6 sites . Thus, yeast nucleosomes carry, on average, 13 acetylated lysines per octamer, i . e . just above the threshold of 10-12 deduced for transcriptionally activated chromatin of animals, plants, and algae . Following M(r) 100,000 ultrafiltration in 2.5% acetic acid, yeast histone H3 was purified to homogeneity by reversed-phase high pressure liquid chromatography . Other core histones were obtained at 80-95% purity.

J Agric Food Chem, 2000 Apr, 48(4), 1086 - 95
Saccharomyces cerevisiae mannoproteins that protect wine from protein haze: evaluation of extraction methods and immunolocalization; Dupin IV et al.; Yeast-derived haze-protective mannoprotein material (HPM) offers protection to white wines from commercially unacceptable turbidities . HPM extraction methods have been evaluated using three winemaking strains of Saccharomyces cerevisiae . Digestion with Zymolyase of cells pretreated with DTE and EDTA gave the greatest yields of active material . Heat treatment of cells with SDS also released active material but the quantities were low . Treatment of the cells in an autoclave or with a French pressure device was less effective . A detailed study was conducted on the strain Maurivin PDM . SDS was not necessary to extract HPM from PDM; boiling the cells for 5 min in Tris buffer was sufficient . HPM could also be extracted with EDTA during the pretreatment of the cells prior to Zymolyase digestion . The data suggest that HPM was noncovalently linked to other cell wall components and loosely associated with the cell wall . An immunological investigation showed that a specific mannoprotein with haze-protective activity, HPF1, was located primarily on the outermost and innermost layers of the cell wall.

Gene, 2000 Apr 18, 247(1-2), 45 - 52
Mass-murder deletion of 19 ORFs from Saccharomyces cerevisiae chromosome XI; Vandenbol M et al.; Nineteen open reading frames (ORFs) in the left arm of chromosome XI of the yeast Saccharomyces cerevisiae were inactivated . This was done by producing single-gene or contiguous-gene deletions in haploid and diploid strains . Four deletions are lethal to the corresponding haploid strains, and two result in a failure to grow on a rich glycerol medium . Complementation experiments showed that five of the six identified phenotypes were due to deletion of a single gene (ORFs YKL173w, YKL172w, YKL165c, YKL154w are essential, and YKL160w is required for growth on glycerol medium) . One of the phenotypes observed on glycerol medium was not suppressed by the corresponding deleted genes . None of the other deletions, covering 13 ORFs in all, gave rise to any obvious phenotype when the cells were grown at three different temperatures on rich glycerol or glucose medium or on minimal synthetic medium.

Arch Biochem Biophys, 2000 Apr 15, 376(2), 399 - 408
Identification and characterization of an animal delta(12) fatty acid desaturase gene by heterologous expression in Saccharomyces cerevisiae; Peyou-Ndi MM et al.; We have cloned a Caenorhabditis elegans cDNA encoding a Delta12 fatty acid desaturase and demonstrated its activity by heterologous expression in Saccharomyces cerevisiae . The predicted protein is highly homologous both to the cloned plant genes with similar function and to the published sequence of the C . elegans omega-3 fatty acid desaturase . In addition, it conforms to the structural constraints expected of a membrane-bound fatty acid desaturase including the canonical histidine-rich regions . This is the first report of a cloned animal Delta(12) desaturase gene . Expression of this cDNA in yeast resulted in the accumulation of 16:2 and 18:2 (linoleic) acids . The increase of membrane fluidity brought about by this change in unsaturation was measured . The production of polyunsaturated fatty acids in yeast cells and the concomitant increase in membrane fluidity was correlated with a modest increase in growth rate at low temperature and with increased resistance to ethanol and oxidative stress .

Antimicrob Agents Chemother, 2000 May, 44(5), 1255 - 65
Genome-wide expression patterns in Saccharomyces cerevisiae: comparison of drug treatments and genetic alterations affecting biosynthesis of ergosterol; Bammert GF et al.; Enzymes in the ergosterol-biosynthetic pathway are the targets of a number of antifungal agents including azoles, allylamines, and morpholines . In order to understand the response of Saccharomyces cerevisiae to perturbations in the ergosterol pathway, genome-wide transcript profiles following exposure to a number of antifungal agents targeting ergosterol biosynthesis (clotrimazole, fluconazole, itraconazole, ketoconazole, voriconazole, terbinafine, and amorolfine) were obtained . These profiles were compared to the transcript profiles of strains containing deletions of one of the late-stage ergosterol genes: ERG2, ERG5, or ERG6 . A total of 234 genes were identified as responsive, including the majority of genes from the ergosterol pathway . Expression of several responsive genes, including ERG25, YER067W, and YNL300W, was also monitored by PCR over time following exposure to ketoconazole . The kinetics of transcriptional response support the conditions selected for the microarray experiment . In addition to ergosterol-biosynthetic genes, 36 mitochondrial genes and a number of other genes with roles related to ergosterol function were responsive, as were a number of genes responsive to oxidative stress . Transcriptional changes related to heme biosynthesis were observed in cells treated with chemical agents, suggesting an additional effect of exposure to these compounds . The expression profile in response to a novel imidazole, PNU-144248E, was also determined . The concordance of responsive genes suggests that this compound has the same mode of action as other azoles . Thus, genome-wide transcript profiles can be used to predict the mode of action of a chemical agent as well as to characterize expression changes in response to perturbation of a metabolic pathway.

Antimicrob Agents Chemother, 2000 May, 44(5), 1174 - 80
Syringomycin E inhibition of Saccharomyces cerevisiae: requirement for biosynthesis of sphingolipids with very-long-chain fatty acids and mannose- and phosphoinositol-containing head groups; Stock SD et al.; Syringomycin E is an antifungal cyclic lipodepsinonapeptide that inhibits the growth of Saccharomyces cerevisiae by interaction with the plasma membrane . A screen conducted to find the yeast genes necessary for its fungicidal action identified two novel syringomycin E response genes, SYR3 and SYR4 . A syr3 mutant allele was complemented by ELO2 and ELO3 . These genes encode enzymes that catalyze the elongation of sphingolipid very long chain fatty acids . Tetrad analysis showed that SYR3 was ELO2 . Strains with deletions of SYR3/ELO2 and ELO3 were resistant to syringomycin E, and lipid analyses of both mutants revealed shortened fatty acid chains and lower levels of sphingolipids . SYR4 was identified by Tn5 inactivation of genomic library plasmids that complemented a syr4 mutant allele . SYR4 was found to be identical to IPT1, which encodes the terminal sphingolipid biosynthetic enzyme, mannosyl-diinositolphosphoryl-ceramide synthase . Deletion Deltasyr4/ipt1 strains were viable, were resistant to syringomycin E, did not produce mannosyl-diinositolphosphoryl-ceramide, and accumulated mannosyl-inositolphosphoryl-ceramide . Accumulation of mannosyl-inositolphosphoryl-ceramide was not responsible for resistance since a temperature-sensitive secretory pathway mutant (sec14-3(ts)) accumulated this sphingolipid and was sensitive to syringomycin E . Finally, Deltacsg1/sur1 and Deltacsg2 strains defective in the transfer of mannose to inositolphosphoryl-ceramide were resistant to syringomycin E . These findings show that syringomycin E growth inhibition of yeast is promoted by the production of sphingolipids with fully elongated fatty acid chains and the mannosyl and terminal phosphorylinositol moieties of the polar head group.

Biochem J, 2000 May 1, 347 Pt 3, 837 - 43
Purification and cloning of phosphatidylinositol transfer proteins from Dictyostelium discoideum: homologues of both mammalian PITPs and Saccharomyces cerevisiae sec14p are found in the same cell; Swigart P et al.; Soluble phosphatidylinositol transfer proteins (PITPs) have important roles in lipid-mediated signalling as well as in membrane traffic . Two PITPs (alpha and beta) have been cloned from mammalian cells, which are unrelated in sequence to yeast PITP (the product of the SEC14 gene) . However, all three PITPs can perform interchangeably to reconstitute function in mammalian cells . We have now purified the major PITP from the cytoplasm of Dictyostelium discoideum and cloned the gene . This protein, DdPITP1, is homologous with mammalian PITPalpha and PITPbeta . We have also cloned a second gene (DdPITP2) related in sequence to DdPITP1 . In addition, an independently cloned cDNA encodes a relative of the SEC14 family of yeast PITPs . DdPITP1, DdPITP2 and DdSec14 proteins were all able to mediate the transfer of PtdIns from one membrane compartment to another; they thus exhibited the hallmark of PITPs . Secondly, all three PITPs were able to rescue phospholipase C-mediated phosphoinositide hydrolysis in PITP-depleted HL60 cells, indicating that all three PITPs were capable of stimulating phosphoinositide synthesis . The identification of PITPs related to both mammalian PITPs and yeast Sec14p in a single organism will provide a unique opportunity to examine the functions of this class of protein with genetic approaches.

Gene, 2000 Apr 4, 246(1-2), 395 - 404
NBP1 (Nap1 binding protein 1), an essential gene for G2/M transition of Saccharomyces cerevisiae, encodes a protein of distinct sub-nuclear localization; Shimizu Y et al.; Nap1p is identified in mammalian cell extract by its intrinsic activity to facilitate nucleosome assembly in vitro in the physiological ionic condition . The homologous proteins are present in most eukaryotes, and their functional analyses in vitro have suggested that they are necessary to keep proper nucleosome structures in transcription and replication . This protein is also identified for its interaction with Clb2p in vitro . To address the function of Nap1p in vivo, we have surveyed for proteins to interact with Nap1p by two-hybrid system and obtained two genes, NBP1 and NBP2 (Nap1 Binding Protein 1 and 2) . NBP1 is an essential gene and encodes a novel protein consisting of 319 amino acids, with a coiled-coil structure in the center of the predicted amino acid sequence . Several A-kinase dependent phosphorylation sites and Cdc28p kinase-dependent sites are also observed . By isolating the temperature-sensitive mutant, we demonstrate that the nuclear division at a non-permissive temperature is delayed and that the population of cells with a large bud carrying a single nucleus with a short spindle are increased . This mutant also confers resistance against benomyl, a microtubule-destabilizing agent . Judging from the green fluorescent protein (GFP) signal fused with Nbp1p, this protein localizes in the nucleus as one or two tiny dots.

Gene, 2000 Apr 4, 246(1-2), 179 - 85
A novel multi-purpose cassette for repeated integrative epitope tagging of genes in Saccharomyces cerevisiae; De Antoni A et al.; Gene tagging can be achieved by homologous recombination in yeast . The kan(r) marker gene plays an important role in PCR-mediated gene disruption and PCR-mediated epitope tagging experiments . In this paper, new modules containing a tag-loxP-kanMX-loxP cassette are described that allow tagging of different genes by using the kan(r) marker repeatedly.

J Biol Chem, 2000 Jul 21, 275(29), 21817 - 26
The Saccharomyces cerevisiae RNA-binding protein Rbp29 functions in cytoplasmic mRNA metabolism; Winstall E et al.; Here we report that the Saccharomyces cerevisiae RBP29 (SGN1, YIR001C) gene encodes a 29-kDa cytoplasmic protein that binds to mRNA in vivo . Rbp29p can be co-immunoprecipitated with the poly(A) tail-binding protein Pab1p from crude yeast extracts in a dosage- and RNA-dependent manner . In addition, recombinant Rbp29p binds preferentially to poly(A) with nanomolar binding affinity in vitro . Although RBP29 is not essential for cell viability, its deletion exacerbates the slow growth phenotype of yeast strains harboring mutations in the eIF4G genes TIF4631 and TIF4632 . Furthermore, overexpression of RBP29 suppresses the temperature-sensitive growth phenotype of specific tif4631, tif4632, and pab1 alleles . These data suggest that Rbp29p is an mRNA-binding protein that plays a role in modulating the expression of cytoplasmic mRNA.

J Bacteriol, 2000 May, 182(9), 2428 - 37
Calcofluor antifungal action depends on chitin and a functional high-osmolarity glycerol response (HOG) pathway: evidence for a physiological role of the Saccharomyces cerevisiae HOG pathway under noninducing conditions; Garcia-Rodriguez LJ et al.; We have isolated several Saccharomyces cerevisiae mutants resistant to calcofluor that contain mutations in the PBS2 or HOG1 genes, which encode the mitogen-activated protein kinase (MAPK) and MAP kinases, respectively, of the high-osmolarity glycerol response (HOG) pathway . We report that blockage of either of the two activation branches of the pathway, namely, SHO1 and SLN1, leads to partial resistance to calcofluor, while simultaneous disruption significantly increases resistance . However, chitin biosynthesis is independent of the HOG pathway . Calcofluor treatment also induces an increase in salt tolerance and glycerol accumulation, although no activation of the HOG pathway is detected . Our results indicate that the antifungal effect of calcofluor depends on its binding to cell wall chitin but also on the presence of a functional HOG pathway . Characterization of one of the mutants isolated, pbs2-14, revealed that resistance to calcofluor and HOG-dependent osmoadaptation are two different physiological processes . Sensitivity to calcofluor depends on the constitutive functionality of the HOG pathway; when this is altered, the cells become calcofluor resistant but also show very low levels of basal salt tolerance . Characterization of some multicopy suppressors of the calcofluor resistance phenotype indicated that constitutive HOG functionality participates in the maintenance of cell wall architecture, a conclusion supported by the antagonism observed between the protein kinase and HOG signal transduction pathways.

Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 4186 - 91
The function of the human homolog of Saccharomyces cerevisiae REV1 is required for mutagenesis induced by UV light; Gibbs PE et al.; In Saccharomyces cerevisiae, most mutations induced by a wide range of mutagens arise during translesion replication employing the REV1 gene product and DNA polymerase zeta . As part of an effort to investigate mammalian mutagenic mechanisms, we have identified cDNA clones of the human homologs of the yeast REV genes and examined their function in UV mutagenesis . Previously, we described the isolation of a human homolog of yeast REV3, the catalytic subunit of pol zeta, and here report the identification and sequence of a human homolog of yeast REV1 . This gene was isolated by identifying an expressed sequence tag encoding a peptide with similarity to the C terminus of yeast Rev1p, followed by sequencing of the clone and retrieval of the remaining cDNA by 5' rapid amplification of cDNA ends . The human gene encodes an expected protein of 1,251 residues, compared with 985 residues in the yeast protein . The proteins share two amino-terminal regions of approximately 100 residues with 41% and 20% identity, a region of approximately 320 residues with 31% identity, and a central motif in which 11 of 13 residues are identical . Human cells expressing high levels of an hREV1 antisense RNA grew normally, and were not more sensitive to the cytotoxic effect of 254 nm UV radiation than cells lacking antisense RNA . However, the frequencies of 6-thioguanine resistance mutants induced by UV in the cells expressing antisense hREV1 RNA were significantly lower than in the control (P = 0.01), suggesting that the human gene has a function similar to that of the yeast homolog.

Plant Physiol, 2000 Apr, 122(4), 1249 - 59
The Arabidopsis HKT1 gene homolog mediates inward Na(+) currents in xenopus laevis oocytes and Na(+) uptake in Saccharomyces cerevisiae; Uozumi N et al.; The Na(+)-K(+) co-transporter HKT1, first isolated from wheat, mediates high-affinity K(+) uptake . The function of HKT1 in plants, however, remains to be elucidated, and the isolation of HKT1 homologs from Arabidopsis would further studies of the roles of HKT1 genes in plants . We report here the isolation of a cDNA homologous to HKT1 from Arabidopsis (AtHKT1) and the characterization of its mode of ion transport in heterologous systems . The deduced amino acid sequence of AtHKT1 is 41% identical to that of HKT1, and the hydropathy profiles are very similar . AtHKT1 is expressed in roots and, to a lesser extent, in other tissues . Interestingly, we found that the ion transport properties of AtHKT1 are significantly different from the wheat counterpart . As detected by electrophysiological measurements, AtHKT1 functioned as a selective Na(+) uptake transporter in Xenopus laevis oocytes, and the presence of external K(+) did not affect the AtHKT1-mediated ion conductance (unlike that of HKT1) . When expressed in Saccharomyces cerevisiae, AtHKT1 inhibited growth of the yeast in a medium containing high levels of Na(+), which correlates to the large inward Na(+) currents found in the oocytes . Furthermore, in contrast to HKT1, AtHKT1 did not complement the growth of yeast cells deficient in K(+) uptake when cultured in K(+)-limiting medium . However, expression of AtHKT1 did rescue Escherichia coli mutants carrying deletions in K(+) transporters . The rescue was associated with a less than 2-fold stimulation of K(+) uptake into K(+)-depleted cells . These data demonstrate that AtHKT1 differs in its transport properties from the wheat HKT1, and that AtHKT1 can mediate Na(+) and, to a small degree, K(+) transport in heterologous expression systems.

Genetics, 2000 Mar, 154(3), 1101 - 14
Bipartite structure of the SGS1 DNA helicase in Saccharomyces cerevisiae; Mullen JR et al.; SGS1 in yeast encodes a DNA helicase with homology to the human BLM and WRN proteins . This group of proteins is characterized by a highly conserved DNA helicase domain homologous to Escherichia coli RecQ and a large N-terminal domain of unknown function . To determine the role of these domains in SGS1 function, we constructed a series of truncation and helicase-defective (-hd) alleles and examined their ability to complement several sgs1 phenotypes . Certain SGS1 alleles showed distinct phenotypes: sgs1-hd failed to complement the MMS hypersensitivity and hyper-recombination phenotypes, but partially complemented the slow-growth suppression of top3 sgs1 strains and the top1 sgs1 growth defect . Unexpectedly, an allele that encodes the amino terminus alone showed essentially complete complementation of the hyper-recombination and top1 sgs1 defects . In contrast, an allele encoding the helicase domain alone was unable to complement any sgs1 phenotype . Small truncations of the N terminus resulted in hyper-recombination and slow-growth phenotypes in excess of the null allele . These hypermorphic phenotypes could be relieved by deleting more of the N terminus, or in some cases, by a point mutation in the helicase domain . Intragenic complementation experiments demonstrate that both the amino terminus and the DNA helicase are required for full SGS1 function . We conclude that the amino terminus of Sgs1 has an essential role in SGS1 function, distinct from that of the DNA helicase, with which it genetically interacts.

Genetics, 2000 Mar, 154(3), 1069 - 83
Analysis of Sir2p domains required for rDNA and telomeric silencing in Saccharomyces cerevisiae; Cockell MM et al.; Silent information regulator (Sir) 2 is a limiting component of the Sir2/3/4 complex, which represses transcription at subtelomeric and HM loci . Sir2p also acts independently of Sir3p and Sir4p to influence chromatin organization in the rDNA locus . Deleted and mutated forms of Sir2p have been tested for their ability to complement and/or to disrupt silencing . The highly conserved C-terminal domain of Sir2p (aa 199-562) is insufficient to restore repression at either telomeric or rDNA reporters in a sir2Delta background and fails to nucleate silencing when targeted to an appropriate reporter gene . However, its expression in an otherwise wild-type strain disrupts telomeric repression . Similarly, a point mutation (P394L) within this conserved core inactivates the full-length protein but renders it dominant negative for all types of silencing . Deletion of aa 1-198 from Sir2(394L) eliminates its dominant negative effect . Thus we define two distinct functional domains in Sir2p, both essential for telomeric and rDNA repression: the conserved core domain found within aa 199-562 and a second domain that encompasses aa 94-198 . Immunolocalization and two-hybrid studies show that aa 94-198 are required for the binding of Sir2p to Sir4p and for the targeting of Sir2p to the nucleolus through another ligand . The globular core domain provides an essential silencing function distinct from that of targeting or Sir complex formation that may reflect its reported mono-ADP-ribosyl transferase activity.

Genetics, 2000 Mar, 154(3), 1053 - 68
Trinucleotide repeats are clustered in regulatory genes in Saccharomyces cerevisiae; Young ET et al.; The genome of Saccharomyces cerevisiae contains numerous unstable microsatellite sequences . Mononucleotide and dinucleotide repeats are rarely found in ORFs, and when present in an ORF are frequently located in an intron or at the C terminus of the protein, suggesting that their instability is deleterious to gene function . DNA trinucleotide repeats (TNRs) are found at a higher-than-expected frequency within ORFs, and the amino acids encoded by the TNRs represent a biased set . TNRs are rarely conserved between genes with related sequences, suggesting high instability or a recent origin . The genes in which TNRs are most frequently found are related to cellular regulation . The protein structural database is notably lacking in proteins containing amino acid tracts, suggesting that they are not located in structured regions of a protein but are rather located between domains . This conclusion is consistent with the location of amino acid tracts in two protein families . The preferred location of TNRs within the ORFs of genes related to cellular regulation together with their instability suggest that TNRs could have an important role in speciation . Specifically, TNRs could serve as hot spots for recombination leading to domain swapping, or mutation of TNRs could allow rapid evolution of new domains of protein structure.

Genetics, 2000 Mar, 154(3), 1039 - 51
Subtelomeric repeat amplification is associated with growth at elevated temperature in yku70 mutants of Saccharomyces cerevisiae; Fellerhoff B et al.; Inactivation of the Saccharomyces cerevisiae gene YKU70 (HDF1), which encodes one subunit of the Ku heterodimer, confers a DNA double-strand break repair defect, shortening of and structural alterations in the telomeres, and a severe growth defect at 37 degrees . To elucidate the basis of the temperature sensitivity, we analyzed subclones derived from rare yku70 mutant cells that formed a colony when plated at elevated temperature . In all these temperature-resistant subclones, but not in cell populations shifted to 37 degrees, we observed substantial amplification and redistribution of subtelomeric Y' element DNA . Amplification of Y' elements and adjacent telomeric sequences has been described as an alternative pathway for chromosome end stabilization that is used by postsenescence survivors of mutants deficient for the telomerase pathway . Our data suggest that the combination of Ku deficiency and elevated temperature induces a potentially lethal alteration of telomere structure or function . Both in yku70 mutants and in wild type, incubation at 37 degrees results in a slight reduction of the mean length of terminal restriction fragments, but not in a significant loss of telomeric (C(1-3)A/TG(1-3))(n) sequences . We propose that the absence of Ku, which is known to bind to telomeres, affects the telomeric chromatin so that its chromosome end-defining function is lost at 37 degrees.

Free Radic Biol Med, 2000 Mar 1, 28(5), 659 - 64
Oxidative stress during aging of stationary cultures of the yeast Saccharomyces cerevisiae; Jakubowski W et al.; Comparison of 5 d old stationary cultures of Saccharomyces cerevisiae and of cultures aged for 3 months revealed increased generation of reactive oxygen species assessed by 2', 7'-dichlorofluorescin oxidation, decreased activity of superoxide dismutase, decreased content of glutathione and increased protein carbonyl content during prolonged incubation of stationary yeast cultures . These results point to the occurrence of oxidative stress during aging of stationary cultures of the yeast . The magnitude of this stress was augmented in antioxidant-deficient strains, devoid of superoxide dismutases and catalases, and of decreased glutathione content.

Genetika, 2000 Feb, 36(2), 155 - 8
{Polymorphism of the SUP35 gene and its product in the Saccharomyces cerevisiae yeasts}; Volkov KV et al.; The product of the SUP35 gene of the saccharomycete yeast, the translation termination eRF3 factor, can be converted in prion, the heritable determinant of protein nature . The nucleotide sequence of this gene from the strain belonging to Peterhof genetic lines of the yeast Saccharomyces cerevisiae was determined . A comparison of the identified sequence with SUP35 sequences in the database of GenBank allowed the detection of polymorphic sites both in the SUP35 gene and its product . The location of polymorphic sites in the evolutionarily nonconserved N-terminal protein region confirmed that this eRF3 fragment lacks functions vital to life activity . Nevertheless, these sites are located in the vicinity of sites, whose role in the prion conversion of eRF3 has been established . Based on this, natural polymorphism of the primary eRF3 structure is assumed to be connected with the existence of different variants (strains) of its prion analog.

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