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 m