Prostate, 1999 Oct 1, 41(2), 134 - 42 Use of a yeast assay to detect functional alterations in p53 in prostate cancer: review and future directions; deVere White RW et al.; BACKGROUND: While many studies have suggested that p53 mutations are common in human cancers, the functional activity of these mutant alleles has not yet been fully addressed . We believe that information about the functional status of individual p53 mutants will prove to be important for a better understanding of the role of p53 in tumor development and progression . Ultimately, this information could also influence treatment decisions for individual cancer patients . METHODS: A recently developed yeast functional assay can be used to assess the transactivational activity of p53 mutants . Furthermore, this assay is more sensitive than single strand conformational polymorphism (SSCP) for detection of p53 mutations . In this review, we summarize the mechanism of this new technique and describe its applications in cancer research, with an emphasis on prostate cancer . RESULTS: The use of the yeast functional assay provides a simple, sensitive, and reproducible method for detecting p53 mutations and for determining the transactivational activity and dominant-negative role of individual p53 mutants . CONCLUSIONS: This method may be adapted to analyze other transcriptional factors, including the human androgen receptor . Mutat Res, 1999 Jul 21, 444(1), 117 - 22 Protective effect of the yeast glucomannan against cyclophosphamide-induced mutagenicity; Chorvatovicova D et al.; Glucomannan (GM) isolated from Candida utilis with molecular weight 30 kDa was administered either intraperitoneally or orally prior to cyclophosphamide (CP) injection and its effect on the frequency of micronuclei was evaluated in polychromatic erythrocytes of mouse bone marrow . GM administration by either route decreased significantly (p<0.002) the clastogenic effect of CP . The protective effect was concentration-dependent, with a higher decrease achieved by 200 mg/kg than by 100 mg/kg b . wt . (body weight) . The fact that GM was effective also at oral administration is indicative of the passage of GM molecules through the wall of the gastrointestinal tract . The important characteristics of GM isolated from C . utilis, such as good water solubility, relatively small molecular weight (30 kDa), and antimutagenic effect exerted also at oral administration, appear to be promising features for its prospective use as a natural protective agent. Nucleosides Nucleotides, 1999 Jun-Jul, 18(6-7), 1459 - 61 Interaction of complementary oligonucleotides with the 3'-end of yeast tRNA(Phe); Petyuk V et al.; Interaction of yeast tRNA(Phe) with oligodeoxyribonucleotides (ONs), complementary to the nucleotides 62-76 was investigated . Results of gel-mobility shift assay and RNase A probing evidence that the ONs containing the sequence complementary to the tRNA ACCA end can easily invade the hairpin structure under physiological conditions . The limiting step of association process is the tRNA unfolding. Nat Biotechnol, 1999 Sep, 17(9), 878 - 83 Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling; Cismowski MJ et al.; We describe genetic screens in Saccharomyces cerevisiae designed to identify mammalian nonreceptor modulators of G-protein signaling pathways . Strains lacking a pheromone-responsive G-protein coupled receptor and expressing a mammalian-yeast Galpha hybrid protein were made conditional for growth upon either pheromone pathway activation (activator screen) or pheromone pathway inactivation (inhibitor screen) . Mammalian cDNAs that conferred plasmid-dependent growth under restrictive conditions were identified . One of the cDNAs identified from the activator screen, a human Ras-related G protein that we term AGS1 (for activator of G-protein signaling), appears to function by facilitating guanosine triphosphate (GTP) exchange on the heterotrimeric Galpha . A cDNA product identified from the inhibitor screen encodes a previously identified regulator of G-protein signaling, human RGS5. Mol Biol Cell, 1999 Sep, 10(9), 2987 - 3001 Functional dissection and hierarchy of tubulin-folding cofactor homologues in fission yeast; Radcliffe PA et al.; We describe the isolation of fission yeast homologues of tubulin-folding cofactors B (Alp11) and E (Alp21), which are essential for cell viability and the maintenance of microtubules . Alp11(B) contains the glycine-rich motif (the CLIP-170 domain) involved in microtubular functions, whereas, unlike mammalian cofactor E, Alp21(E) does not . Both mammalian and yeast cofactor E, however, do contain leucine-rich repeats . Immunoprecipitation analysis shows that Alp11(B) interacts with both alpha-tubulin and Alp21(E), but not with the cofactor D homologue Alp1, whereas Alp21(E) also interacts with Alp1(D) . The cellular amount of alpha-tubulin is decreased in both alp1 and alp11 mutants . Overproduction of Alp11(B) results in cell lethality and the disappearance of microtubules, which is rescued by co-overproduction of alpha-tubulin . Both full-length Alp11(B) and the C-terminal third containing the CLIP-170 domain localize in the cytoplasm, and this domain is required for efficient binding to alpha-tubulin . Deletion of alp11 is suppressed by multicopy plasmids containing either alp21(+) or alp1(+), whereas alp21 deletion is rescued by overexpression of alp1(+) but not alp11(+) . Finally, the alp1 mutant is not complemented by either alp11(+) or alp21(+) . The results suggest that cofactors operate in a linear pathway (Alp11(B)-Alp21(E)-Alp1(D)), each with distinct roles. Mol Biol Cell, 1999 Sep, 10(9), 2879 - 89 Import into and degradation of cytosolic proteins by isolated yeast vacuoles; Horst M et al.; In eukaryotic cells, both lysosomal and nonlysosomal pathways are involved in degradation of cytosolic proteins . The physiological condition of the cell often determines the degradation pathway of a specific protein . In this article, we show that cytosolic proteins can be taken up and degraded by isolated Saccharomyces cerevisiae vacuoles . After starvation of the cells, protein uptake increases . Uptake and degradation are temperature dependent and show biphasic kinetics . Vacuolar protein import is dependent on cytosolic heat shock proteins of the hsp70 family and on protease-sensitive component(s) on the outer surface of vacuoles . Degradation of the imported cytosolic proteins depends on a functional vacuolar ATPase . We show that the cytosolic isoform of yeast glyceraldehyde-3-phosphate dehydrogenase is degraded via this pathway . This import and degradation pathway is reminiscent of the protein transport pathway from the cytosol to lysosomes of mammalian cells. J Biol Chem, 1999 Sep 10, 274(37), 26057 - 64 Substrate- and inhibitor-induced conformational changes in the yeast V-ATPase provide evidence for communication between the catalytic and proton-translocating sectors; Landolt-Marticorena C et al.; The vacuolar-type H(+)-ATPases (V-ATPases) are composed of two distinct sectors, a catalytic complex (V(1)) involved in ATP hydrolysis and a membrane-associated complex (V(0)) mediating proton translocation across a lipid bilayer . To date, little is known about the mechanism by which these two functions are coupled . We sought to examine the impact of nucleotide and cation binding on the structure of the core components of the catalytic complex and to determine whether conformational changes within the catalytic complex impact subunits of the membrane-associated complex . Nucleotide- and cation- induced changes in the catalytic core of the V-ATPase were investigated by monitoring changes in the rate and pattern of tryptic digests . ATP.Mg-induced changes were detected in both the catalytic (Vma1p or 69 kDa) and the regulatory subunits (Vma2p or 60 kDa) of the V(1) sector . ATP alone increased the rate of trypsinization of the regulatory subunit, but did not have any effect on Vma1p . Surprisingly, ATP also had an impact on the 95-kDa subunit, a component of the V(0) sector of the V-ATPase . Although the presence of divalent cations had no impact on the V(1) sector, the rate of trypsinization of the 95-kDa subunit was greatly enhanced . The effect of divalent cations on the structure of the 95-kDa subunit was abrogated when trypsinization was performed in the absence of the catalytic sector . Addition of bafilomycin A(1), a V-ATPase inhibitor that putatively binds to the 95-kDa subunit, increased the rate of trypsinization of the catalytic subunit . These data suggest that structural alterations within the V(1) sector result in alterations within the V(0) sector and vice versa . Clearly, a structural link must exist to couple the two sectors . The 95-kDa subunit is ideally suited to fulfill this role . Hydropathy analysis suggests a bipartite structure, with the NH(2)-terminal portion predicted to lie in an aqueous environment and the C-terminal portion predicted to contain 6 transmembrane segments . Tryptic digests of sealed vacuolar vesicles and immunofluorescence studies revealed that the large hydrophilic NH(2)-terminal domain of the 95-kDa subunit is localized toward the cytosol . This region therefore is ideally positioned to interact with components of the V(1) complex, potentially functioning as the elusive link between the two sectors of the V-ATPase. Plant Foods Hum Nutr, 1999, 53(2), 175 - 81 The relationship between soil copper content and copper resistance in yeast of an ultisol in midwestern Nigeria; Ekundayo EO et al.; Yeast isolates were obtained from the brewer's stock of the Guinness brewery in Benin City, midwestern Nigeria, and from local sources such as mango and sugar cane . The fruits were exposed to the immediate soil environment in eleven selected sites in the Ugbowo and Ikpoba Hill areas of Benin City, Edo State of midwestern Nigeria . A total of twenty-seven yeast isolates were obtained . Fourteen isolates were identified as Saccharomyces cerevisiae and 13 as S . carlsbergensis . Analysis of resistance to copper sulphate revealed that the two brewer's yeast isolates were resistant to 3.5 mM and 4.2 mM CuSO4, respectively, while the local yeasts were resistant to higher concentrations ranging from 6.5 to 16.5 mM . The percentage copper sulphate resistance of yeast isolates obtained from Mangifera inidica ranged from 52.4 to 73.8%, while the percentage copper sulphate resistance of yeast isolates obtained from Saccharum officinarum ranged from 51.8 to 62.6% . The two brewer's yeast isolates (BY2 and BY4) had a percentage CuSO4 resistance of 32.4 and 38.5%, respectively . Comparison of soil copper concentration and copper resistance levels in selected yeast isolates indicated that the soil copper concentrations generally influenced the ability of yeasts to resist copper. Neoplasma, 1999, 46(2), 80 - 8 FASAY: a simple functional assay in yeast for identification of p53 mutation in tumors; Smardova J; Alteration of the p53 tumor suppressor gene is the most common genetic defect known to occur in human tumors . Germ-line p53 mutations significantly increase the risk of developing diverse malignancies . FASAY is a simple functional assay for germ-line and somatic mutations in the p53 gene altering the transactivation capability of the p53 protein . The method was successfully used for mutation analysis of p53 in various cell lines, somatic tumor cells and blood cells . In addition, FASAY was also found effective as a tool for basic research of binding of mutant p53 proteins to promoters of different p53 target genes. Genes Dev, 1999 Aug 15, 13(16), 2148 - 58 The yeast exosome and human PM-Scl are related complexes of 3' --> 5' exonucleases; Allmang C et al.; We previously identified a complex of 3' --> 5' exoribonucleases, designated the exosome, that is expected to play a major role in diverse RNA processing and degradation pathways . Further biochemical and genetic analyses have revealed six novel components of the complex . Therefore, the complex contains 11 components, 10 of which are predicted to be 3' --> 5' exoribonucleases on the basis of sequence homology . Human homologs were identified for 9 of the 11 yeast exosome components, three of which complement mutations in the respective yeast genes . Two of the newly identified exosome components are homologous to known components of the PM-Scl particle, a multisubunit complex recognized by autoimmune sera of patients suffering from polymyositis-scleroderma overlap syndrome . We demonstrate that the homolog of the Rrp4p exosome subunit is also a component of the PM-Scl complex, thereby providing compelling evidence that the yeast exosome and human PM-Scl complexes are functionally equivalent . The two complexes are similar in size, and biochemical fractionation and indirect immunofluorescence experiments show that, in both yeast and humans, nuclear and cytoplasmic forms of the complex exist that differ only by the presence of the Rrp6p/PM-Scl100 subunit exclusively in the nuclear complex. J Biol Chem, 1999 Sep 3, 274(36), 25921 - 6 Regulated degradation of yeast ornithine decarboxylase; Toth C et al.; Ornithine decarboxylase (ODC) declines in cells that accumulate an excess of polyamines, the downstream products of the enzyme . Superfluous production of polyamines is thus prevented . In animal cells, polyamines reduce ODC activity by accelerating its degradation . Similar down-regulation of ODC activity has been observed in the budding yeast Saccharomyces cerevisiae, but induced degradation has not been documented . Here we show using pulse-chase analysis that the loss of enzyme activity is the result of increased degradation of ODC . Polyamines reduce the half-life of the newly synthesized protein from 3 h to approximately 10 min . Degradation of bulk ODC pools is also accelerated by polyamines, but the absolute rate of turnover is slower, with a half-life of 5 h in untreated and 1 h in treated cells . Newly synthesized ODC polypeptide thus undergoes a process of maturation that renders it relatively resistant to both basal and polyamine-induced degradation . Proteasome mutants have a blunted or absent regulatory response, implicating both the core protease and the regulatory cap of the proteasome in induced degradation of yeast ODC. J Biol Chem, 1999 Sep 3, 274(36), 25682 - 90 Stable association of 70-kDa heat shock protein induces latent multisite specificity of a unisite-specific endonuclease in yeast mitochondria; Mizumura H et al.; The multisite-specific endonuclease Endo.SceI of yeast mitochondria is unique among endonucleases because its 50-kDa subunit forms a stable dimer with the mitochondrial 70-kDa heat shock protein (mtHSP70), which otherwise fulfills a chaperone function by binding transiently to unfolded proteins . Here we show that the mtHSP70 subunit confers broader sequence specificity, greater stability, and higher activity on the 50-kDa subunit . The 50-kDa subunit alone displayed weaker activity and highly sequence-specific endonuclease activity . The 50-kDa protein exists as a heterodimer with mtHSP70 in vivo, allowing Endo.SceI to cleave specifically at multiple sites on mitochondrial DNA . Endo.SceI may have evolved from a highly specific endonuclease that gained broader sequence specificity after becoming a stable partner of mtHSP70. J Pept Sci, 1999 Jun, 5(6), 245 - 50 Chemical synthesis of yeast mitochondrial ATP synthase membranous subunit 8; Goetz M et al.; Chemical synthesis of highly hydrophobic peptides and proteins remains a challenging problem . Strong interchain associations within the peptide-resin matrix have to be overcome . A synthetic strategy for solid phase peptide synthesis is proposed, mainly based on prolonged coupling time using aprotic polar solvent mixtures . A tailored chromatographic purification was required to obtain a sample sufficiently pure for structural analysis . In this work, the total chemical synthesis of the membrane-embedded yeast mitochondrial ATP synthase subunit 8 is described . The quality of the synthetic protein was checked by electrospray mass spectrometry, its tendency to adopt alpha-helical secondary structure is evidenced by circular dichroism spectroscopy. DNA Cell Biol, 1999 Aug, 18(8), 653 - 61 The product of the primary response gene BRF1 inhibits the interaction between 14-3-3 proteins and cRaf-1 in the yeast trihybrid system; Bustin SA et al.; The 14-3-3 proteins are small abundant cytosolic eukaryotic proteins that associate with and modulate the activity of numerous other proteins . The 14-3-3 beta isoform has been shown to bind to the product of the protooncogene cRaf-1 and to facilitate its activation by Ras . Using the yeast two-hybrid system, we have demonstrated that 14-3-3 beta and another isoform, 14-3-3 tau, bind to the product of the primary response gene BRF1 and that the interaction between each isoform and BRF1 is significantly stronger than that with cRaf-1 . We further demonstrated that the charge of residue 187 in 14-3-3 beta regulates its affinity for both BRF1 and cRaf-1 . The interaction of either isoform with BRF1 requires both proteins to be fully intact . When all three proteins are coexpressed in a yeast trihybrid system, BRF1 interferes significantly with the binding of 14-3-3 to full-length cRaf-1 as well as to its regulatory and kinase domains . Using quantitative reverse transcription-polymerase chain reaction, 14-3-3 beta and BRF1 were found to be coexpressed in four different human tissues, suggesting a biologic role for their interaction in the regulation of cRaf-1-mediated signal transduction processes. J Anim Sci, 1999 Aug, 77(8), 2172 - 9 Effects of dietary levels of selenium-enriched yeast and sodium selenite as selenium sources fed to growing-finishing pigs on performance, tissue selenium, serum glutathione peroxidase activity, carcass characteristics, and loin quality; Mahan DC et al.; This research evaluated the efficacy of inorganic and organic Se sources for growing-finishing pigs, as measured by performance and various tissue, serum, carcass, and loin quality traits . A total of 351 crossbred pigs were allotted at an average BW of 20.4 kg to six replicates of a 2x4 factorial experiment in a randomized complete block design . Pigs were fed diets containing Se-enriched yeast (organic) or sodium selenite (inorganic), each at .05, .10, .20, or .30 mg Se/kg diet . A non-Se-fortified basal diet was a ninth treatment group . Five pigs per pen were bled initially and at 30-d intervals with serum analyzed for Se and glutathione peroxidase (GSH-Px) activity . At 55 kg BW, one pig per pen from each of three replicates was killed, and tissues were collected for Se analysis . At 105 kg BW, the remaining pigs in the three replicates were killed, carcass measurements were collected, tissues were analyzed for Se, and loin quality was evaluated for pH, drip loss, and lightness . No performance or carcass measurement benefit resulted from either Se source or dietary Se levels . Pigs had a lower serum Se concentration and GSH-Px activity when the basal diet was fed, but both increased as dietary Se level increased (P<.01) . Serum GSH-Px activities were increased by pig age and reached a plateau when the diet contained approximately .10 mg Se/kg (P<.01) at d 30, and 60 of the trial, and at .05 mg Se/kg diet at d 90 of the trial . The organic Se group fed .05 and .10 mg Se/kg had serum GSH-Px activities that tended to be lower than those of pigs fed the inorganic Se source, but GSH-Px activities in both groups were similar at higher Se levels . Tissue Se contents increased linearly as the dietary Se level increased, but the increase was markedly higher when organic Se was fed, resulting in an interaction (P<.01) response . Loin drip loss, pH, and lightness were unaffected (P>.15) by organic Se source or level, but there was a trend for a higher drip loss (P = .11) and a linear (P<.01) increase in loin paleness when the inorganic Se level increased . These results indicate that neither Se source nor Se level had an effect on pig performance or carcass measurements, but organic Se source increased tissue Se concentrations . Inorganic Se may, however, have a detrimental effect on loin quality, as reflected by higher drip loss and a paler color . Using serum GSH-Px activity as the measurement criterion, the supplemental dietary Se requirement did not seem to exceed .10 and .05 mg Se/kg diet for the growing and finishing phases, respectively, when added to a basal diet containing .06 mg Se/kg. Trends Genet, 1999 Sep, 15(9), 337 - 40 Multi-organellar disorders of pigmentation: intracellular traffic jams in mammals, flies and yeast; Spritz RA; Several different mutant genes in humans, mice and Drosophila, most of which were identified initially on the basis of reduced pigmentation, have been associated with defects of multiple cytoplasmic organelles - melanosomes, lysosomes and granules . Recent discoveries show that several of these mutations directly affect components in the pathway of organelle-specific protein trafficking, and provide new insights into the relationships of these pathways in mammals, flies and yeast. J Cell Biol, 1999 Aug 23, 146(4), 741 - 54 Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane; Schneiter R et al.; Nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to determine qualitative differences in the lipid molecular species composition of a comprehensive set of organellar membranes, isolated from a single culture of Saccharomyces cerevisiae cells . Remarkable differences in the acyl chain composition of biosynthetically related phospholipid classes were observed . Acyl chain saturation was lowest in phosphatidylcholine (15.4%) and phosphatidylethanolamine (PE; 16.2%), followed by phosphatidylserine (PS; 29.4%), and highest in phosphatidylinositol (53.1%) . The lipid molecular species profiles of the various membranes were generally similar, with a deviation from a calculated average profile of approximately +/- 20% . Nevertheless, clear distinctions between the molecular species profiles of different membranes were observed, suggesting that lipid sorting mechanisms are operating at the level of individual molecular species to maintain the specific lipid composition of a given membrane . Most notably, the plasma membrane is enriched in saturated species of PS and PE . The nature of the sorting mechanism that determines the lipid composition of the plasma membrane was investigated further . The accumulation of monounsaturated species of PS at the expense of diunsaturated species in the plasma membrane of wild-type cells was reversed in elo3Delta mutant cells, which synthesize C24 fatty acid-substituted sphingolipids instead of the normal C26 fatty acid-substituted species . This observation suggests that acyl chain-based sorting and/or remodeling mechanisms are operating to maintain the specific lipid molecular species composition of the yeast plasma membrane. Curr Opin Microbiol, 1999 Aug, 2(4), 348 - 52 Cell wall dynamics in yeast; Smits GJ et al.; The yeast Saccharomyces cerevisiae is the first fungus for which the structure of the cell wall is known at the molecular level . It is a dynamic and highly regulated structure . This is vividly illustrated when the cell wall is damaged and a salvage pathway becomes active, resulting in compensatory changes in the wall. Cryobiology, 1999 Aug, 39(1), 80 - 7 A dual role for intracellular trehalose in the resistance of yeast cells to water stress; Sano F et al.; It is well known that yeast cells survive environmental stresses such as desiccation and freezing and there is evidence that these phenomena may be related to the presence of trehalose in the cells . However, the molecular mechanism by which trehalose might exert an influence on cell functions remains unknown . In this report, thermogravimetry and differential thermal analysis were used to estimate the amount of bound water in yeast cells . It is shown that when the trehalose content is greater than 2-3% of the cell dry weight, the amount of bound water is drastically decreased and the viability of the dried cells is increased . This implies that a major portion of the bound water is replaced by trehalose . In addition, measurements of the NMR spin-lattice relaxation time of the intracellular water protons show that trehalose acts as a water-structuring agent in hydrated yeast cells . This dual role is essential for high resistance to water stress in yeast cells . Biotechnol Bioeng, 1999 Oct 20, 65(2), 176 - 81 Slow heat rate increases yeast thermotolerance by maintaining plasma membrane integrity; Martinez de Maranon I et al.; Thermal resistance of Saccharomyces cerevisiae was found to be drastically dependent on the kinetics of heat perturbation . Yeasts were found to be more resistant to a plateau of 1 h at 50 degrees C after a slope of temperature increase (slow and linear temperature increments) than after a shock (sudden temperature change) . Thermotolerance was mainly acquired between 40-50 degrees C during a heat slope, i.e., above the maximal temperature of growth . The death of the yeasts subjected to a heat shock might be related to the loss of membrane integrity: intracellular contents extrusion, i.e., membrane permeabilization, was found to precede cell death . However, the permeabilization did not precede cell death during a heat slope and, therefore, membrane permeabilization was a consequence rather than a cause of cell death . During a slow temperature increase, yeasts which remain viable may have time to adapt their plasma membrane and thus maintain membrane integrity . Int J Biol Macromol, 1999 Aug, 25(4), 345 - 51 Identification of simian cyclophilin A as a calreticulin-binding protein in yeast two-hybrid screen and demonstration of cyclophilin A interaction with calreticulin; Reddy PA et al.; Cyclophilin A (CyPA) was identified as one of the calreticulin (CR)-binding proteins in a yeast two-hybrid screen utilizing simian cDNA expression-library . The simian CyPA protein had 96% identity with that of human, differing only at eight amino acid residues . We further established CyPA-CR interaction by incubation of glutathione transferase-fused CyPA (GST-CyPA) and CR proteins with CV-1 cyto-lysates, followed by CR and CyPA-specific immuno-blot analysis . The immunosuppressive drug cyclosporin A, a CyPA ligand, did not inhibit CyPA-CR interaction . Our results established a new property of CyPA binding activity to CR . Since CR is a Ca2+-binding protein, CR-CyPA interactions may be important in signaling pathways for induction of Ca2+-dependent cellular processes. FEBS Lett, 1999 Aug 6, 456(2), 281 - 4 Low iron concentration and aconitase deficiency in a yeast frataxin homologue deficient strain; Foury F; Deletion of the yeast frataxin homologue, YFH1, elicits accumulation of iron in mitochondria and mitochondrial defects . We report here that in the presence of an iron chelator in the culture medium, the concentration of iron in mitochondria is the same in wild-type and YFH1 deletant strains . Under these conditions, the activity of the respiratory complexes is restored . However, the activity of the mitochondrial aconitase, a 4Fe-4S cluster-containing protein, remains low . The frataxin family bears homology to a bacterial protein family which confers resistance to tellurium, a metal closely related to sulfur . Yfh1p might control the synthesis of iron-sulfur clusters in mitochondria. Methods, 1999 Jul, 18(3), 368 - 76, 323 In vitro DNA replication in yeast nuclear extracts; Pasero P et al.; Two assays have been developed for studying DNA replication in vitro based on nuclear extracts isolated from budding yeast cells synchronized in S phase . In the first, the template DNA for replication is provided in the form of intact yeast nuclei, usually from cells arrested in G(1) . In the second assay, bacterially produced supercoiled plasmid is replicated in an S-phase nuclear extract supplemented with nucleotides and an energy-regenerating system . Semiconservative DNA replication is monitored by substitution of newly synthesized DNA with bromodeoxyuridine 5'-triphosphate (BrdUTP) and density gradient analysis . In addition, neutral-neutral two-dimensional gel analyses and, in the case of nuclei, detection of newly synthesized DNA in replication foci by DIG-dUTP incorporation can be used to monitor replication . Methods, 1999 Jul, 18(3), 356 - 67 Genetic methods for characterizing the cis-acting components of yeast DNA replication origins; Huberman JA; Small circular plasmids containing replication origins and, in some cases, centromeres, can replicate autonomously in the nuclei of all tested yeast species . Because this autonomous replication is dependent on the replication origin within the plasmid, measurements of the efficiency of autonomous replication (by the methods summarized here) permit evaluation of the effects of mutations on origin function . Although alternative methods are available for genetic characterization of replication origins in other organisms, the simplicity of the autonomous replication assay in yeasts has permitted development of the deepest understanding to date of eukaryotic replication origin structure . This information has come primarily from studies with Saccharomyces cerevisiae . However, there are many other yeast species, each with its own variety of replication origins . Use of the methods summarized here to characterize origins in other yeast species is likely to provide additional insights into eukaryotic replication origin structure . Nucleic Acids Res, 1999 Aug 1, 27(15), 3001 - 8 New yeast genes important for chromosome integrity and segregation identified by dosage effects on genome stability; Ouspenski II et al.; Phenotypes produced by gene overexpression may provide important clues to gene function . Here, we have performed a search for genes that affect chromo-some stability when overexpressed in the budding yeast Saccharomyces cerevisiae . We have obtained clones encompassing 30 different genes . Twenty-four of these genes have been previously characterized . Most of them are involved in chromatin dynamics, cell cycle control, DNA replication or mitotic chromosome segregation . Six novel genes obtained in this screen were named CST (chromosome stability) . Based on the pattern of genomic instability, inter-action with checkpoint mutations and sensitivity to chromosome replication or segregation inhibitors, we conclude that overexpression of CST4 specifically interferes with mitotic chromosome segregation, and CST6 affects some aspect of DNA metabolism . The other CST genes had complex pleiotropic phenotypes . We have created deletions of five genes obtained in this screen, CST9, CST13, NAT1, SBA1 and FUN30 . None of these genes is essential for viability, and deletions of NAT1 and SBA1 cause chromosome instability, a phenotype not previously associated with these genes . This work shows that analysis of dosage effects is complementary to mutational analysis of chromosome transmission fidelity, as it allows the identification of chromosome stability genes that have not been detected in mutational screens. Mol Cell Biol, 1999 Sep, 19(9), 6110 - 9 The yeast trimeric guanine nucleotide-binding protein alpha subunit, Gpa2p, controls the meiosis-specific kinase Ime2p activity in response to nutrients; Donzeau M et al.; Saccharomyces cerevisiae Gpa2p, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein (G protein), is involved in the regulation of vegetative growth and pseudohyphal development . Here we report that Gpa2p also controls sporulation by interacting with the regulatory domain of Ime2p (Sme1p), a protein kinase essential for entrance of meiosis and sporulation . Protein-protein interactions between Gpa2p and Ime2p depend on the GTP-bound state of Gpa2p and correlate with down-regulation of Ime2p kinase activity in vitro . Overexpression of Ime2p inhibits pseudohyphal development and enables diploid cells to sporulate even in the presence of glucose or nitrogen . In contrast, overexpression of Gpa2p in cells simultaneously overproducing Ime2p results in a drastic reduction of sporulation efficiency, demonstrating an inhibitory effect of Gpa2p on Ime2p function . Furthermore, deletion of GPA2 accelerates sporulation on low-nitrogen medium . These observations are consistent with the following model . In glucose-containing medium, diploid cells do not sporulate because Ime2p is inactive or expressed at low levels . Upon starvation, expression of Gpa2p and Ime2p is induced but sporulation is prevented as long as nitrogen is present in the medium . The negative control of Ime2p kinase activity is exerted at least in part through the activated form of Gpa2p and is released as soon as nutrients are exhausted . This model attributes a switch function to Gpa2p in the meiosis-pseudohyphal growth decision. Mol Cell Biol, 1999 Sep, 19(9), 6098 - 109 Activation of silent replication origins at autonomously replicating sequence elements near the HML locus in budding yeast; Vujcic M et al.; In the budding yeast, Saccharomyces cerevisiae, replicators can function outside the chromosome as autonomously replicating sequence (ARS) elements; however, within chromosome III, certain ARSs near the transcriptionally silent HML locus show no replication origin activity . Two of these ARSs comprise the transcriptional silencers E (ARS301) and I (ARS302) . Another, ARS303, resides between HML and the CHA1 gene, and its function is not known . Here we further localized and characterized ARS303 and in the process discovered a new ARS, ARS320 . Both ARS303 and ARS320 are competent as chromosomal replication origins since origin activity was seen when they were inserted at a different position in chromosome III . However, at their native locations, where the two ARSs are in a cluster with ARS302, the I silencer, no replication origin activity was detected regardless of yeast mating type, special growth conditions that induce the transcriptionally repressed CHA1 gene, trans-acting mutations that abrogate transcriptional silencing at HML (sir3, orc5), or cis-acting mutations that delete the E and I silencers containing ARS elements . These results suggest that, for the HML ARS cluster (ARS303, ARS320, and ARS302), inactivity of origins is independent of local transcriptional silencing, even though origins and silencers share key cis- and trans-acting components . Surprisingly, deletion of active replication origins located 25 kb (ORI305) and 59 kb (ORI306) away led to detection of replication origin function at the HML ARS cluster, as well as at ARS301, the E silencer . Thus, replication origin silencing at HML ARSs is mediated by active replication origins residing at long distances from HML in the chromosome . The distal active origins are known to fire early in S phase, and we propose that their inactivation delays replication fork arrival at HML, providing additional time for HML ARSs to fire as origins. Mol Cell Biol, 1999 Sep, 19(9), 6029 - 40 Functional analysis of the yeast Glc7-binding protein Reg1 identifies a protein phosphatase type 1-binding motif as essential for repression of ADH2 expression; Dombek KM et al.; In Saccharomyces cerevisiae, the protein phosphatase type 1 (PP1)-binding protein Reg1 is required to maintain complete repression of ADH2 expression during growth on glucose . Surprisingly, however, mutant forms of the yeast PP1 homologue Glc7, which are unable to repress expression of another glucose-regulated gene, SUC2, fully repressed ADH2 . Constitutive ADH2 expression in reg1 mutant cells did require Snf1 protein kinase activity like constitutive SUC2 expression and was inhibited by unregulated cyclic AMP-dependent protein kinase activity like ADH2 expression in derepressed cells . To further elucidate the functional role of Reg1 in repressing ADH2 expression, deletions scanning the entire length of the protein were analyzed . Only the central region of the protein containing the putative PP1-binding sequence RHIHF was found to be indispensable for repression . Introduction of the I466M F468A substitutions into this sequence rendered Reg1 almost nonfunctional . Deletion of the central region or the double substitution prevented Reg1 from significantly interacting with Glc7 in two-hybrid analyses . Previous experimental evidence had indicated that Reg1 might target Glc7 to nuclear substrates such as the Snf1 kinase complex . Subcellular localization of a fully functional Reg1-green fluorescent protein fusion, however, indicated that Reg1 is cytoplasmic and excluded from the nucleus independently of the carbon source . When the level of Adr1 was modestly elevated, ADH2 expression was no longer fully repressed in glc7 mutant cells, providing the first direct evidence that Glc7 can repress ADH2 expression . These results suggest that the Reg1-Glc7 phosphatase is a cytoplasmic component of the machinery responsible for returning Snf1 kinase activity to its basal level and reestablishing glucose repression . This implies that the activated form of the Snf1 kinase complex must cycle between the nucleus and the cytoplasm. Mol Cell Biol, 1999 Sep, 19(9), 5943 - 51 SMG-2 is a phosphorylated protein required for mRNA surveillance in Caenorhabditis elegans and related to Upf1p of yeast; Page MF et al.; mRNAs that contain premature stop codons are selectively degraded in all eukaryotes tested, a phenomenon termed "nonsense-mediated mRNA decay" (NMD) or "mRNA surveillance." NMD may function to eliminate aberrant mRNAs so that they are not translated, because such mRNAs might encode deleterious polypeptide fragments . In both yeasts and nematodes, NMD is a nonessential system . Mutations affecting three yeast UPF genes or seven nematode smg genes eliminate NMD . We report here the molecular analysis of smg-2 of Caenorhabditis elegans . smg-2 is homologous to UPF1 of yeast and to RENT1 (also called HUPF1), a human gene likely involved in NMD . The striking conservation of SMG-2, Upf1p, and RENT1/HUPF1 in both sequence and function suggests that NMD is an ancient system, predating the divergence of most eukaryotes . Despite similarities in the sequences of SMG-2 and Upf1p, expression of Upf1p in C . elegans does not rescue smg-2 mutants . We have prepared anti-SMG-2 polyclonal antibodies and identified SMG-2 on Western blots . SMG-2 is phosphorylated, and mutations of the six other smg genes influence the state of SMG-2 phosphorylation . In smg-1, smg-3, and smg-4 mutants, phosphorylation of SMG-2 was not detected . In smg-5, smg-6, and smg-7 mutants, a phosphorylated isoform of SMG-2 accumulated to abnormally high levels . In smg-2(r866) and smg-2(r895) mutants, which harbor single amino acid substitutions of the SMG-2 nucleotide binding site, phosphorylated SMG-2 accumulated to abnormally high levels, similar to those observed in smg-5, smg-6, and smg-7 mutants . We discuss these results with regard to the in vivo functions of SMG-2 and NMD. Acta Biochim Pol, 1999, 46(1), 211 - 5 Phosphorylation of yeast ribosomal proteins by CKI and CKII in the presence of heparin; Wojda I et al.; We have found that heparin has a different effect on Trichosporon cutaneum ribosomal protein phosphorylation by CKI and by CKII . In the presence of heparin, modification of 13 kDa, 19 kDa and 38 kDa proteins catalyzed by CKII was inhibited, while in the case of CKI, in addition to protein of 15 kDa, phosphorylation of 20 kDa and 35 kDa proteins was detected . It was also found that, in the presence of heparin, phosphorylation of P proteins (13 kDa and 38 kDa) by ribosome-bound protein kinases was inhibited . Moreover at the same conditions modification of 40 kDa protein was observed in all four yeast species tested. Acta Biochim Pol, 1999, 46(1), 163 - 72 Thermodynamic contribution of nucleoside modifications to yeast tRNA(Phe) anticodon stem loop analogs; Agris PF et al.; The determination of the structural and functional contributions of natural modified nucleosides to tRNA has been limited by lack of an approach that can systematically incorporate the modified units . We have produced a number of oligonucleotide analogs, of the anticodon of yeast tRNA(Phe) by, combining standard automated synthesis for the major nucleosides with specialty chemistries for the modified nucleosides . In this study, both naturally occurring and unnatural modified nucleotides were placed in native contexts . Each oligonucleotide was purified and the nucleoside composition determined to validate the chemistry . The RNAs were denatured and analyzed to determine the van't Hoff thermodynamic parameters . Here, we report the individual thermodynamic contributions for Cm, Gm, m1G, m5C, psi . In addition m5m6U, m1psi, and m3psi, were introduced to gain additional understanding of the physicochemical contribution of psi and m5C at an atomic level . These oligonucleotides demonstrate that modifications have measurable thermodynamic contributions and that loop modifications have global contributions. Hum Genet, 1999 Jun, 104(6), 443 - 8 Hereditary spastic paraplegia: mitochondrial metalloproteases of yeast; Pearce DA; Hereditary spastic paraplegia (HSP) is a genetically heterogenous group of inherited neurodegenerative disorders . Recently, an autosomal recessive form of HSP was mapped to 16q24.3, and subsequently the defective gene associated to HSP was identified and designated SPG7 . The SPG7 gene product was predicted to encode a protein of 795 amino acids, and is called paraplegin . Paraplegin is highly homologous to a class of well studied yeast ATP-dependent zinc metalloproteases, which show 55%, 55% and 52% identity, respectively, to Afg3p, Rca1p and Yme1p . Mutation of either Afg3p, Rca1p or Yme1p in yeast results in pleiotropic effects with regard to growth, respiration and, particularly, in the assembly and/or degradation of more than one mitochondrial protein complex . Taking into account the homology of paraplegin to these yeast ATP-dependent zinc metalloproteases and what is known about their function, allows us to speculate as to what function paraplegin plays in normal individuals. Can J Microbiol, 1999 Jun, 45(6), 472 - 9 Stress tolerance in a yeast lipid mutant: membrane lipids influence tolerance to heat and ethanol independently of heat shock proteins and trehalose; Swan TM et al.; The response of a yeast unsaturated fatty acid auxotroph, defective in delta 9-desaturase activity, to heat and ethanol stresses was examined . The most heat- and ethanol-tolerant cells had membranes enriched with oleic acid (C18:1), followed in order by cells enriched with linoleic (C18:2) and linolenic (C18:3) acids . Cells subjected to a heat shock (25-37 degrees C for 30 min) accumulated trehalose and synthesized typical heat shock proteins . Although there were no obvious differences in protein profiles attributable to lipid supplementation of the mutant, relative protein synthesis as determined by densitometric analysis of autoradiograms suggested that hsp expression was different . However, there was no consistent relationship between the synthesis of heat shock proteins and the acquisition of thermotolerance in the lipid supplemented auxotroph or related wild type . Furthermore, trehalose accumulation was also not closely related to stress tolerance . On the other hand, the data presented indicated a more consistent role for membrane lipid composition in stress tolerance than trehalose, heat shock proteins, or ergosterol . We suggest that the sensitivity of C18:3-enriched cells to heat and ethanol may be attributable to membrane damage associated with increases in membrane fluidity and oxygen-derived free radical attack of membrane lipids. J Mol Biol, 1999 Aug 27, 291(4), 761 - 73 Yeast aspartyl-tRNA synthetase residues interacting with tRNA(Asp) identity bases connectively contribute to tRNA(Asp) binding in the ground and transition-state complex and discriminate against non-cognate tRNAs; Eriani G et al.; Crystallographic studies of the aspartyl-tRNA synthetase-tRNA(Asp)complex from yeast identified on the enzyme a number of residues potentially able to interact with tRNA(Asp) . Alanine replacement of these residues (thought to disrupt the interactions) was used in the present study to evaluate their importance in tRNA(Asp)recognition and acylation . The results showed that contacts with the acceptor A of tRNA(Asp)by amino acid residues interacting through their side-chain occur only in the acylation transition state, whereas those located near the G73 discriminator base occur also during initial binding of tRNA(Asp) . Interactions with the anticodon bases provide the largest free energy contribution to stability of the enzyme-tRNA complex in its ground state . These contacts also favour catalysis, by acting connectively with each other and with those of G73, as shown by multiple mutant analysis . This implies structural communication transmitting the anticodon recognition signal to the distally located acylation site . This signal might be conveyed via tRNA(Asp)as suggested by the observed conformational change of this molecule upon interaction with AspRS . From binding free energy values corresponding to the different AspRS-tRNA(Asp)interaction domains, it might be concluded that upon complex formation, the anticodon interacts first . Finally, acylation efficiencies of AspRS mutants in the presence of pure tRNA(Asp)and non-fractionated tRNAs indicate that residues involved in the binding of identity bases also discriminate against non-cognate tRNAs . Cytogenet Cell Genet, 1999, 85(3-4), 217 - 20 A novel human gene, encoding a potential membrane protein conserved from yeast to man, is strongly expressed in testis and cancer cell lines; Veitia RA et al.; We have characterized a novel human gene (C14orf1) which codes for a polypeptide homologous to the yeast protein Yer044c . Both the human and yeast proteins are predicted to be highly basic and to present several potential, evolutionarily conserved, transmembrane domains . C14orf1 mRNA was found to be particularly abundant in the adult testis and in several cancer cell lines . The gene maps to chromosome band 14q24 . Further investigations should be performed to understand the role of C14orf1 in the testis and the significance of its strong expression in the cell lines studied here. Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9533 - 8 Studying interactions of four proteins in the yeast two-hybrid system: structural resemblance of the pVHL/elongin BC/hCUL-2 complex with the ubiquitin ligase complex SKP1/cullin/F-box protein; Pause A et al.; The yeast two-hybrid system is a powerful technique that detects interactions between two proteins and has been useful in identifying new binding partners . However, the system fails to detect protein-protein interactions that require the presence of additional components of a multisubunit complex . Here we demonstrate that the vector YIpDCE1 can be used to express elongins B and C in yeast, and that these proteins form a stable complex that interacts with the von Hippel-Lindau tumor-suppressor gene product (pVHL) . Only when pVHL and elongins B and C (VBC) are present does an interaction with the cullin family member, hCUL-2, occur, forming the heterotetrameric pVHL/elongin BC/hCUL-2 complex . This system was then used to map the binding region of hCUL-2 for the VBC complex . The first amino-terminal 108 aa of hCUL-2 are necessary for interaction with the VBC complex . The elongin BC dimer acts as a bridge between pVHL and hCUL-2 because pVHL and hCUL-2 can form distinct complexes with elongins B and C . These results reveal a striking structural resemblance of pVHL/elongin BC/hCUL-2 complex with the E3-like ubiquitin ligase complex SKP1/Cullin/F-box protein with respect to protein composition and sites of interactions . Thus, it seems possible that pVHL/elongin BC/hCUL-2 complex will possess ubiquitin ligase activity targeting specific proteins for degradation by the proteasome. Nature, 1999 Aug 5, 400(6744), 573 - 6 Oligopeptide-repeat expansions modulate 'protein-only' inheritance in yeast; Liu JJ et al.; The yeast {PSI+} element represents a new type of genetic inheritance, in which changes in phenotype are transmitted by a 'protein only' mechanism reminiscent of the 'protein-only' transmission of mammalian prion diseases . The underlying molecular mechanisms for both are poorly understood and it is not clear how similar they might be . Sup35, the {PSI+} protein determinant, and PrP, the mammalian prion determinant, have different functions, different cellular locations and no sequence similarity; however, each contains five imperfect oligopeptide repeats-PQGGYQQYN in Sup35 and PHGGGWGQ in PrP . Repeat expansions in PrP produce spontaneous prion diseases . Here we show that replacing the wild-type SUP35 gene with a repeat-expansion mutation induces new {PSI+} elements, the first mutation of its type among these newly described elements of inheritance . In vitro, fully denatured repeat-expansion peptides can adopt conformations rich in beta-sheets and form higher-order structures much more rapidly than wild-type peptides . Our results provide insight into the nature of the conformational changes underlying protein-based mechanisms of inheritance and suggest a link between this process and those producing neurodegenerative prion diseases in mammals. EMBO J, 1999 Aug 16, 18(16), 4485 - 97 Cell cycle progression in the presence of irreparable DNA damage is controlled by a Mec1- and Rad53-dependent checkpoint in budding yeast; Neecke H et al.; We studied the response of nucleotide excision repair (NER)-defective rad14Delta cells to UV irradiation in G(1) followed by release into the cell cycle . Only a subset of checkpoint proteins appears to mediate cell cycle arrest and regulate the timely activation of replication origins in the presence of unrepaired UV-induced lesions . In fact, Mec1 and Rad53, but not Rad9 and the Rad24 group of checkpoint proteins, are required to delay cell cycle progression in rad14Delta cells after UV damage in G(1) . Consistently, Mec1-dependent Rad53 phosphorylation after UV irradiation takes place in rad14Delta cells also in the absence of Rad9, Rad17, Rad24, Mec3 and Ddc1, and correlates with entry into S phase . Two-dimensional gel analysis indicates that late replication origins are not fired in rad14Delta cells UV-irradiated in G(1) and released into the cell cycle, which instead initiate DNA replication from early origins and accumulate replication and recombination intermediates . Progression through S phase of UV-treated NER-deficient mec1 and rad53 mutants correlates with late origin firing, suggesting that unregulated DNA replication in the presence of irreparable UV-induced lesions might result from a failure to prevent initiation at late origins. EMBO J, 1999 Aug 16, 18(16), 4383 - 93 Yeast epsins contain an essential N-terminal ENTH domain, bind clathrin and are required for endocytosis; Wendland B et al.; The mammalian protein epsin is required for endocytosis . In this study, we have characterized two homologous yeast proteins, Ent1p and Ent2p, which are similar to mammalian epsin . An essential function for the highly conserved N-terminal epsin N-terminal homology (ENTH) domain was revealed using deletions and randomly generated temperature-sensitive ent1 alleles . Changes in conserved ENTH domain residues in ent1(ts) cells revealed defects in endocytosis and actin cytoskeleton structure . The Ent1 protein was localized to peripheral and internal punctate structures, and biochemical fractionation studies found the protein associated with a large, Triton X-100-insoluble pellet . Finally, an Ent1p clathrin-binding domain was mapped to the final eight amino acids (RGYTLIDL*) in the Ent1 protein sequence . Based on these and other data, we propose that the yeast epsin-like proteins are essential components of an endocytic complex that may act at multiple stages in the endocytic pathway. J Bioenerg Biomembr, 1999 Apr, 31(2), 105 - 17 Activation and deactivation of F0F1-ATPase in yeast mitochondria; Schouppe C et al.; The regulation of membrane-bound proton F0F1 ATPase by the protonmotive force and nucleotides was studied in yeast mitochondria . Activation occurred in whole mitochondria and the ATPase activity was measured just after disrupting the membranes with Triton X-100 . Deactivation occurred either in whole mitochondria uncoupled with FCCP, or in disrupted membranes . No effect of Triton X-100 on the ATPase was observed, except a slow reactivation observed only in the absence of MgADP . Both AMPPNP and ATP increased the ATPase deactivation rate, thus indicating that occupancy of nucleotidic sites by ATP is more decisive than catalytic turnover for this process . ADP was found to stimulate the energy-dependent ATPase activation . ATPase deactivated at the same rate in uncoupled and disrupted mitochondria This suggests that deactivation is not controlled by rebinding of some soluble factor, like IF1, but rather by the conversion of the F1.IF1 complex into an inactive form. J Bioenerg Biomembr, 1999 Apr, 31(2), 85 - 94 Subunit f of the yeast mitochondrial ATP synthase: topological and functional studies; Roudeau S et al.; Modified versions of subunit f were produced by mutagenesis of the ATP17 gene of Saccharomyces cerevisiae . A version of subunit f devoid of the last 28 amino acid residues including the unique transmembranous domain complemented the oxidative phosphorylation of the null mutant . However, a two-fold decrease in the specific ATP synthase activity was measured and attributed to a decrease in the stability of the mutant ATP synthase complex as shown by the low oligomycin-sensitive ATPase activity at alkaline pH . The modification or not by nonpermeant maleimide reagents of cysteine residues introduced at the N and C termini of subunit f indicated a Nin-Cout orientation . From the C terminus of subunit f it was possible to cross-link subunit 4 (also called subunit b), which is another component of the F0 sector and which also displays a short hydrophilic segment exposed to the intermembrane space. Can J Microbiol, 1999 May, 45(5), 413 - 7 Clues to the origin of high external invertase activity in immobilized growing yeast: prolonged SUC2 transcription and less susceptibility of the enzyme to endogenous proteolysis; de Alteriis E et al.; Expression of the SUC2 gene encoding invertase was studied using free and gelatin-immobilized yeast cells to try to explain the high activity of this enzyme exhibited by immobilized cells when allowed to grow in a nutrient medium . The results indicated that at least two factors are probably responsible for the accumulation of invertase in immobilized cells . First, the expression of the SUC2 gene was maintained throughout growth in immobilized cells, whereas its expression was only transient in free cells . Second, invertase of immobilized cells was shown to be less susceptible to endogenous proteolytic attack than that of the corresponding free cells . These results have been interpreted, respectively, in terms of diffusional limitations and changes in the pattern of invertase glycosylation due to growth of yeast in an immobilized state. Nucleic Acids Res, 1999 Sep 1, 27(17), 3610 - 20 Nucleotide excision repair in a constitutive and inducible gene of a yeast minichromosome in intact cells; Li S et al.; Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in a yeast minichromosome, having a galactose-inducible GAL1:URA3 fusion gene, a constitutively expressed HIS3 gene and varied regions of chromatin structure . Transcription of GAL1:URA3 increased >150-fold, while HIS3 expression decreased <2-fold when cells were switched from glucose to galactose medium . Following galactose induction, four nucleosomes were displaced or rearranged in the GAL3-GAL10 region . However, no change in nucleosome arrangement was observed in other regions of the minichromosome following induction, indicating that only a few plasmid molecules actively transcribe at any one time . Repair at 269 cis-syn CPD sites revealed moderate preferential repair of the transcribed strand of GAL1:URA3 in galactose, consistent with transcription-coupled repair in a fraction of these genes . Many sites upstream of the transcription start site in the transcribed strand were also repaired faster upon induction . There is remarkable repair heterogeneity in the HIS3 gene and preferential repair is seen only in a short sequence immediately downstream of the transcription start site . Finally, a mild correlation of repair heterogeneity with nucleosome positions was observed in the transcribed strand of the inactive GAL1:URA3 gene and this correlation was abolished upon galactose induction. Nucleic Acids Res, 1999 Sep 1, 27(17), 3503 - 9 Origin and properties of non-coding ORFs in the yeast genome; Mackiewicz P et al.; In a recent paper we have estimated the total number of protein coding open reading frames (ORFs) in the Saccharomyces cerevisiae genome, based on their properties, at about 4800 . This number is much smaller than the 5800-6000 which is widely accepted . In this paper we analyse differences between the set of ORFs with known phenotypes annotated in the Munich Information Centre for Protein Sequences (MIPS) database and ORFs for which the probability of coding, counted by us, is very low . We have found that many of the latter ORFs have properties of antisense sequences of coding ORFs, which suggests that they could have been generated by duplication of coding sequences . Since coding sequences generate ORFs inside themselves, with especially high frequency in the antisense sequences, we have looked for homology between known proteins and hypothetical polypeptides generated by ORFs under consideration in all the six phases . For many ORFs we have found paralogues and orthologues in phases different than the phase which had been assumed in the MIPS database as coding. J Biol Chem, 1999 Aug 20, 274(34), 24431 - 7 Purification and characterization of the yeast glycosylphosphatidylinositol-anchored, monobasic-specific aspartyl protease yapsin 2 (Mkc7p); Komano H et al.; The Saccharomyces cerevisiae YPS2 (formerly MKC7) gene product is a glycosylphosphatidylinositol-linked aspartyl protease that functions as a yeast secretase . Here, the glycosylphosphatidylinositol-linked form of yapsin 2 (Mkc7p) was purified to homogeneity from the membrane fraction of an overexpressing yeast strain . Purified yapsin 2 migrated diffusely in SDS-polyacrylamide gel electrophoresis (molecular mass approximately 200 kDa), suggesting extensive, heterogeneous glycosylation . Studies using internally quenched fluorogenic peptide substrates revealed cleavage by the enzyme carboxyl to Lys or Arg . No cleavage was seen when both Lys and Arg were absent . No significant enhancement was seen with multiple basic residues . However, cleavage always occurred carboxyl to the most COOH-terminal basic residue . V(max)/K(m) was insensitive to P(2) and P(3) residues except that Pro at P(2) blocked cleavage entirely . These results suggest that yapsin 2 is a monobasic amino acid-specific protease that requires a basic residue at P(1) and excludes basic residues from P(1)' . The pH dependence of V(max)/K(m) for a substrate containing a pro-alpha factor cleavage site was bell-shaped, with a maximum near pH 4.0 . However, V(max)/K(m) for a substrate mimicking the alpha-secretase site in human beta amyloid precursor protein was optimal near pH 6.0, consistent with cleavage of beta amyloid precursor protein by yapsin 2 when expressed in yeast. J Biol Chem, 1999 Aug 20, 274(34), 24257 - 62 Synergistic interaction between yeast nucleotide excision repair factors NEF2 and NEF4 in the binding of ultraviolet-damaged DNA; Guzder SN et al.; Saccharomyces cerevisiae RAD4, RAD7, RAD16, and RAD23 genes function in the nucleotide excision repair (NER) of ultraviolet light (UV)-damaged DNA . Previous biochemical studies have shown that the Rad4 and Rad23 proteins are associated in a stoichiometric complex named NEF2, and the Rad7 and Rad16 proteins form another stoichiometric complex named NEF4 . While NEF2 is indispensable for the incision of UV-damaged DNA in the in vitro reconstituted system, NEF4 stimulates the incision reaction . Both NEF2 and NEF4 bind UV-damaged DNA, which raises the intriguing possibility that these two complexes cooperate to achieve the high degree of specificity for DNA damage demarcation required for nucleotide excision repair in vivo . Consistent with this hypothesis, we find that NEF2 and NEF4 bind in a synergistic fashion to UV-damaged DNA in a reaction that is dependent on ATP . We also purify the Rad7 protein and show that it binds DNA but has no preference for UV-damaged DNA . Rad7 physically interacts with NEF2, suggesting a role for Rad7 in linking NEF2 with NEF4. J Biol Chem, 1999 Aug 20, 274(34), 24220 - 31 Recovery of the yeast cell cycle from heat shock-induced G(1) arrest involves a positive regulation of G(1) cyclin expression by the S phase cyclin Clb5; Li X et al.; In the yeast Saccharomyces cerevisiae, heat shock stress induces a variety of cellular responses including a transient cell cycle arrest before G(1)/S transition . Previous studies have suggested that this G(1) delay is probably attributable to a reduced level of the G(1) cyclin gene (CLN1 and CLN2) transcripts . Here we report our finding that the G(1) cyclin Cln3 and the S cyclin Clb5 are the key factors required for recovery from heat shock-induced G(1) arrest . Heat shock treatment of G(1) cells lacking either CLN3 or CLB5/CLB6 functions leads to prolonged cell cycle arrest before the initiation of DNA synthesis, concomitant with a severe deficiency in bud formation . The inability of the clb5 clb6 mutant to resume normal budding after heat shock treatment is unanticipated, since the S phase cyclins are generally thought to be required mainly for initiation of DNA synthesis and have no significant roles in bud formation in the presence of functional G(1) cyclins . Further studies reveal that the accumulation of G(1) cyclin transcripts is markedly delayed in the clb5 clb6 mutant following heat shock treatment, indicating that the CLN gene expression may require Clb5/Clb6 to attain a threshold level for driving the cell cycle through G(1)/S transition . Consistent with this assumption, overproduction of Clb5 greatly enhances the transcription of at least two G(1) cyclin genes (CLN1 and CLN2) in heat-shocked G(1) cells . These results suggest that Clb5 may positively regulate the expression of G(1) cyclins during cellular recovery from heat shock-induced G(1) arrest . Additional evidence is presented to support a role for Clb5 in maintaining the synchrony between budding and DNA synthesis during normal cell division as well. J Biol Chem, 1999 Aug 20, 274(34), 24038 - 46 Functional expression of human PP2Ac in yeast permits the identification of novel C-terminal and dominant-negative mutant forms; Evans DR et al.; The protein phosphatase 2A (PP2A) holoenzyme is structurally conserved among eukaryotes . This reflects a conservation of function in vivo because the human catalytic subunit (PP2Ac) functionally replaced the endogenous PP2Ac of Saccharomyces cerevisiae and bound the yeast regulatory PR65/A subunit (Tpd3p) forming a dimer . Yeast was employed as a novel system for mutagenesis and functional analysis of human PP2Ac, revealing that the invariant C-terminal leucine residue, a site of regulatory methylation, is apparently dispensable for protein function . However, truncated forms of human PP2Ac lacking larger portions of the C terminus exerted a dominant interfering effect, as did several mutant forms containing a substitution mutation . Computer modeling of PP2Ac structure revealed that interfering amino acid substitutions clustered to the active site, and consistently, the PP2Ac-L199P mutant protein was catalytically impaired despite binding Tpd3p . Thus, interfering forms of PP2Ac titrate regulatory subunits and/or substrates into non-productive complexes and will serve as useful tools for studying PP2A function in mammalian cells . The transgenic approach employed here, involving a simple screen for interfering mutants, may be applicable generally to the analysis of structure-function relationships within protein phosphatases and other conserved proteins and demonstrates further the utility of yeast for analyzing gene function. J Biol Chem, 1999 Aug 20, 274(34), 23752 - 60 Functional analysis of a homopolymeric (dA-dT) element that provides nucleosomal access to yeast and mammalian transcription factors; Koch KA et al.; Eukaryotic organisms ranging from yeast to humans maintain a large amount of genetic information in the highly compact folds of chromatin, which poses a large DNA accessibility barrier to rapid changes in gene expression . The ability of the yeast Candida glabrata to survive copper insult requires rapid transcriptional autoactivation of the AMT1 copper-metalloregulatory transcription factor gene . The kinetics of AMT1 autoactivation is greatly enhanced by homopolymeric (dA-dT) element (A16)-mediated nucleosomal accessibility for Amt1p to a metal response element in this promoter . Analysis of the nucleosomal positional requirements for the A16 element reveal an impaired ability of the A16 element to stimulate AMT1 autoregulation when positioned downstream of the metal response element within the nucleosome, implicating an inherent asymmetry to the nucleosome positioned within the AMT1 promoter . Importantly, we demonstrate that the A16 element functions to enhance nucleosomal access and hormone-stimulated transcriptional activation for the mammalian glucocorticoid receptor, in a rotational phase-dependent manner . These data provide compelling evidence that nucleosomal homopolymeric (dA-dT) elements provide enhanced DNA access to diverse classes of transcription factors and suggest that these elements may function in this manner to elicit rapid transcriptional responses in higher eukaryotic organisms. RNA, 1999 Aug, 5(8), 1055 - 70 Overexpression of truncated Nmd3p inhibits protein synthesis in yeast; Belk JP et al.; The yeast NMD3 gene was identified in a two-hybrid screen using the nonsense-mediated mRNA decay factor, Upf1p, as bait . NMD3 was shown to encode an essential, highly conserved protein that associated principally with free 60S ribosomal subunits . Overexpression of a truncated form of Nmd3p, lacking 100 C-terminal amino acids and most of its Upf1p-interacting domain, had dominant-negative effects on both cell growth and protein synthesis and promoted the formation of polyribosome half-mers . These effects were eliminated by truncation of an additional 100 amino acids from Nmd3p . Overexpression of the nmd3delta100 allele also led to increased synthesis and destabilization of some ribosomal protein mRNAs, and increased synthesis and altered processing of 35S pre-rRNA . Our data suggest that Nmd3p has a role in the formation, function, or maintenance of the 60S ribosomal subunit and may provide a link for Upf1p to 80S monosomes. RNA, 1999 Aug, 5(8), 1042 - 54 Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition; Chung S et al.; Mutants in the Drosophila crooked neck (crn) gene show an embryonic lethal phenotype with severe developmental defects . The unusual crn protein consists of sixteen tandem repeats of the 34 amino acid tetratricopeptide (TPR) protein recognition domain . Crn-like TPR elements are found in several RNA processing proteins, although it is unknown how the TPR repeats or the crn protein contribute to Drosophila development . We have isolated a Saccharomyces cerevisiae gene, CLF1, that encodes a crooked neck-like factor . CLF1 is an essential gene but the lethal phenotype of a clf1::HIS3 chromosomal null mutant can be rescued by plasmid-based expression of CLF1 or the Drosophila crn open reading frame . Clf1p is required in vivo and in vitro for pre-mRNA 5' splice site cleavage . Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 association . Yeast two-hybrid analyses and in vitro binding studies show that Clf1p interacts specifically and differentially with the U1 snRNP-Prp40p protein and the yeast U2AF65 homolog, Mud2p . Intriguingly, Prp40p and Mud2p also bind the phylogenetically conserved branchpoint binding protein (BBP/SF1) . Our results indicate that Clf1p acts as a scaffolding protein in spliceosome assembly and suggest that Clf1p may support the cross-intron bridge during the prespliceosome-to-spliceosome transition. Mol Cell, 1999 Jul, 4(1), 75 - 83 A role for transcriptional repressors in targeting the yeast Swi/Snf complex; Dimova D et al.; Genetic and biochemical studies indicate that the evolutionarily conserved Swi/Snf complex acts at a subset of genes to help transcriptional activators function on chromatin templates . The mechanism by which this complex is targeted to specific chromosomal loci remains unknown . We show that Swi/Snf is required for expression of the yeast histone HTA1-HTB1 locus because of the role of Hir1p and Hir2p corepressors in negatively regulating transcription . Snf5p, Snf2p/Swi2p, and Swi3p, three components of the yeast Swi/Snf complex, coimmunoprecipitate with each Hir protein, and Snf5p is maximally associated with the HTA1-HTB1 promoter when the Hir-based repression system is intact and the Swi/Snf complex is functional . The data support a role for the Hir repressors in the gene-specific targeting of Swi/Snf. Mol Cell, 1999 Jul, 4(1), 21 - 33 SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex; Kitagawa K et al.; We have identified SGT1 as a dosage suppressor of skp1-4, a mutation causing defects in yeast kinetochore function . Sgt1p physically associates with Skp1p in vivo and in vitro . SGT1 is an essential gene, and different sgt1 conditional mutants arrest with either a G1 or G2 DNA content . Genetic and phenotypic analyses of sgt1-3 (G2 allele) mutants support an essential role in kinetochore function . Sgt1p is required for assembling the yeast kinetochore complex, CBF3, via activation of Ctf13p . Sgt1p also associates with SCF (Skp1p/Cdc53p/F box protein) ubiquitin ligase . sgt1-5 (G1 allele) mutants are defective in Sic1p turnover in vivo and Cln1p ubiquitination in vitro . Human SGT1 rescues an sgt1 null mutation, suggesting that the function of SGT1 is conserved in evolution. Genes Dev, 1999 Aug 1, 13(15), 1950 - 9 Pds1p of budding yeast has dual roles: inhibition of anaphase initiation and regulation of mitotic exit; Cohen-Fix O et al.; Progression through mitosis is controlled by protein degradation that is mediated by the anaphase-promoting complex/cyclosome (APC/C) and its associated specificity factors . In budding yeast, APC/C(Cdc20) promotes the degradation of the Pds1p anaphase inhibitor at the metaphase-to-anaphase transition, whereas APC/C(Cdh1) promotes the degradation of the mitotic cyclins at the exit from mitosis . Here we show that Pds1p has a novel activity as an inhibitor of mitotic cyclin destruction, apparently by preventing the activation of APC/C(Cdh1) . This activity of Pds1p is independent of its activity as an anaphase inhibitor . We propose that the dual role of Pds1p as an inhibitor of anaphase and of cyclin degradation allows the cell to couple the exit from mitosis to the prior completion of anaphase . Finally, these observations provide a novel regulatory paradigm in which the sequential degradation of two substrates is determined by the substrates themselves, such that an early substrate inhibits the degradation of a later one. Genes Dev, 1999 Aug 1, 13(15), 1912 - 7 Localization of bud2p, a GTPase-activating protein necessary for programming cell polarity in yeast to the presumptive bud site; Park HO et al.; Yeast cells of different cell type exhibit distinct budding patterns that reflect the organization of the actin cytoskeleton . Bud1p (Rsr1p), a Ras-like GTPase, and Bud2p, a GTPase-activating protein for Bud1p, are essential for proper budding pattern . We show that Bud2p is localized at the presumptive bud site in G(1) cells in all cell types and that this localization is independent of Bud1p . Bud2p subsequently localizes to the mother-bud neck after bud emergence; this localization depends on the integrity of the septins . These observations indicate that Bud2p becomes positioned in G(1) cells by recognizing cell type-specific landmarks at the presumptive bud site. Plant Physiol, 1999 Aug, 120(4), 1193 - 204 Determination of the motif responsible for interaction between the rice APETALA1/AGAMOUS-LIKE9 family proteins using a yeast two-hybrid system; Moon YH et al.; A MADS family gene, OsMADS6, was isolated from a rice (Oryza sativa L.) young flower cDNA library using OsAMDS1 as a probe . With this clone, various MADS box genes that encode for protein-to-protein interaction partners of the OsMADS6 protein were isolated by the yeast two-hybrid screening method . On the basis of sequence homology, OsMADS6 and the selected partners can be classified in the APETALA1/AGAMOUS-LIKE9 (AP1/AGL9) family . One of the interaction partners, OsMADS14, was selected for further study . Both genes began expression at early stages of flower development, and their expression was extended into the later stages . In mature flowers the OsMADS6 transcript was detectable in lodicules and also weakly in sterile lemmas and carpels, whereas the OsMADS14 transcript was detectable in sterile lemmas, paleas/lemmas, stamens, and carpels . Using the yeast two-hybrid system, we demonstrated that the region containing of the 109th to 137th amino acid residues of OsMADS6 is indispensable in the interaction with OsMADS14 . Site-directed mutation analysis revealed that the four periodical leucine residues within the region are essential for this interaction . Furthermore, it was shown that the 14 amino acid residues located immediately downstream of the K domain enhance the interaction, and that the two leucine residues within this region play an important role in that enhancement. Curr Microbiol, 1999 Sep, 39(3), 168 - 72 Stochastic nucleosome positioning in a yeast chromatin region is not dependent on histone H1; Puig S et al.; To gain a better understanding of the function of the yeast histone H1, its role in nucleosome positioning was studied . With this objective in mind, we analyzed a chromatin region of the yeast Chromosome (Chr) IX, in which there are two closely packed open reading frames (ORFs), POT1 and YIL161w . This locus shows a regular ladder of 13 stochastically positioned nucleosomes, which is unaffected by the absence of the HHO1 gene . This suggests that histone H1 has no effect on nucleosome positioning in yeast. Biochem Biophys Res Commun, 1999 Aug 11, 261(3), 734 - 9 Activator-specific requirement for the general transcription factor IIE in yeast; Sakurai H et al.; The general transcription factor (TF) IIE is required for mRNA synthesis of many, but not all, genes in yeast . In the transcription process, TFIIE regulates TFIIH kinase activity that phosphorylates the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II . The CTD and the CTD kinase Kin28, a subunit of TFIIH, have been shown to be dispensable for activation of several heat shock genes and the copper metallothionein gene CUP1 . Here we analyzed requirement of TFIIE for transcription of these genes and found that TFIIE is necessary for activation of the heat shock genes by heat shock transcription factor Hsf1 . By contrast, transcription of CUP1 mediated by both Hsf1 and copper-activated transcription factor Ace1 was inducible after inactivating TFIIE . These results show that both TFIIE and the CTD/the CTD kinase exhibit "gene specificities" which are overlapping, but not identical to each other, and thereby suggest that TFIIE functions with or without involvement of the CTD/the CTD kinase depending on the gene to be transcribed . Arch Biochem Biophys, 1999 Aug 15, 368(2), 338 - 46 Mechanism of inhibition of yeast squalene synthase by substrate analog inhibitors; Kalinowski SS et al.; Squalene synthase catalyzes the reductive condensation of two identical substrate molecules, farnesyl diphosphate, to the hydrocarbon squalene via an obligatory intermediate, presqualene pyrophosphate . Since the kinetic mechanism of the transformation is sequential, two substrate binding pockets that recognize the same molecule must exist in the enzyme active site . This raises the possibility of a choice of binding pockets for inhibitors that are designed as substrate or reaction intermediate analogs and thus may provide some information on the mechanism of differentiation of the two identical molecules . In this report, we have investigated the mechanism of inhibition of a series of farnesyl diphosphate analog inhibitors . The inhibitors fall into two categories . One class of compounds binds to free enzyme as well as the enzyme substrate complex, and the binding is refractory to the concentration of the substrate . The second class binds only to the free enzyme, and its binding is significantly modulated by the substrate concentration . Very modest structural changes in the compounds appear to dictate which class of inhibitor any compound may fall into . The significance of these observations with respect to the mechanism of the enzyme are discussed . J Med Virol, 1999 Sep, 59(1), 78 - 83 Ability of yeast Ty-VLPs (virus-like particles) containing varicella-zoster virus (VZV)gE and assembly protein fragments to induce in vitro proliferation of human lymphocytes from VZV immune patients; Welsh MD et al.; Yeast Ty virus-like particles (VLPs) containing viral protein inserts have previously been shown to be potent immunogens, inducing both humoral and cell mediated immunity (CMI) . The antigenicity of hybrid VLPs containing fragments of the varicella-zoster virus (VZV) gE protein or the assembly protein (AP) was assessed by lymphocyte proliferation . Peripheral blood mononuclear cells (PBMCs) from patients with a recent natural VZV infection were stimulated in vitro with VZV-VLPs together with control antigens . PBMC samples from both varicella (85%) and zoster (75%) patients proliferated in responses to at least one of the gE VZV-VLPs . As reported for the first time, VZV specific lymphocyte responses were also identified towards the VZV AP in two varicella and two zoster patient samples . The results demonstrate specific CMI recognition of the VZV gE fragments tested and the VZV AP delivered in the form of recombinant Ty-VLPs, and highlights their potential use as a recombinant antigen delivery system for vaccination. J Biol Chem, 1999 Aug 13, 274(33), 23599 - 609 Efficient processing of DNA ends during yeast nonhomologous end joining . Evidence for a DNA polymerase beta (Pol4)-dependent pathway; Wilson TE et al.; Repair of DNA double strand breaks by nonhomologous end joining (NHEJ) requires enzymatic processing beyond simple ligation when the terminal bases are damaged or not fully compatible . We transformed yeast with a series of linearized plasmids to examine the role of Pol4 (Pol IV, DNA polymerase beta) in repair at a variety of end configurations . Mutation of POL4 did not impair DNA polymerase-independent religation of fully compatible ends and led to at most a 2-fold reduction in the frequency of joins that require only DNA polymerization . In contrast, the frequency of joins that also required removal of a 5'- or 3'-terminal mismatch was markedly reduced in pol4 (but not rev3, exo1, apn1, or rad1) yeast . In a chromosomal double strand break assay, pol4 mutation conferred a marked increase in sensitivity to HO endonuclease in a rad52 background, due primarily to loss of an NHEJ event that anneals with a 3'-terminal mismatch . The NHEJ activity of Pol4 was dependent on its nucleotidyl transferase function, as well as its unique amino terminus . Paradoxically, in vitro analyses with oligonucleotide substrates demonstrated that although Pol4 fills gaps with displacement of mismatched but not matched 5' termini, it lacks both 5'- and 3'-terminal nuclease activities . Pol4 is thus specifically recruited to perform gap-filling in an NHEJ pathway that must also involve as yet unidentified nucleases. J Biol Chem, 1999 Aug 13, 274(33), 23203 - 9 An interaction between the N-terminal region and the core domain of yeast TFIIB promotes the formation of TATA-binding protein-TFIIB-DNA complexes; Bangur CS et al.; The general transcription factor IIB (TFIIB) plays an essential role in transcription of protein-coding genes by eukaryotic RNA polymerase II . We previously identified a yeast TFIIB mutant (R64E) that exhibited increased activity in the formation of stable TATA-binding protein-TFIIB-DNA (DB) complexes in vitro . We report here that the homologous human TFIIB mutant (R53E) also displayed increased activity in DB complex formation in vitro . Biochemical analyses revealed that the increased activity of the R64E mutant in DB complex formation was associated with an altered protease sensitivity of the protein and an enhanced interaction between the N-terminal region and the C-terminal core domain . These results suggest that the intramolecular interaction in yeast TFIIB stabilizes a productive conformation of the protein for the association with promoter-bound TATA-binding protein. Mol Biol Cell, 1999 Aug, 10(8), 2745 - 57 Fission yeast Pob1p, which is homologous to budding yeast Boi proteins and exhibits subcellular localization close to actin patches, is essential for cell elongation and separation; Toya M et al.; The fission yeast pob1 gene encodes a protein of 871 amino acids carrying an SH3 domain, a SAM domain, and a PH domain . Gene disruption and construction of a temperature-sensitive pob1 mutant indicated that pob1 is essential for cell growth . Loss of its function leads to quick cessation of cellular elongation . Pob1p is homologous to two functionally redundant Saccharomyces cerevisiae proteins, Boi1p and Boi2p, which are necessary for cell growth and relevant to bud formation . Overexpression of pob1 inhibits cell growth, causing the host cells to become round and swollen . In growing cells, Pob1p locates at cell tips during interphase and translocates near the division plane at cytokinesis . Thus, this protein exhibits intracellular dynamics similar to F-actin patches . However, Pob1p constitutes a layer, rather than patches, at growing cell tips . It generates two split discs flanking the septum at cytokinesis . The pob1-defective cells no longer elongate but swell gradually at the middle, eventually assuming a lemon-like morphology . Analysis using the pob1-ts allele revealed that Pob1p is also essential for cell separation . We speculate that Pob1p is located on growing plasma membrane, possibly through the function of actin patches, and may recruit proteins required for the synthesis of cell wall. Mol Biol Cell, 1999 Aug, 10(8), 2607 - 18 The spindle checkpoint of budding yeast depends on a tight complex between the Mad1 and Mad2 proteins; Chen RH et al.; The spindle checkpoint arrests the cell cycle at metaphase in the presence of defects in the mitotic spindle or in the attachment of chromosomes to the spindle . When spindle assembly is disrupted, the budding yeast mad and bub mutants fail to arrest and rapidly lose viability . We have cloned the MAD2 gene, which encodes a protein of 196 amino acids that remains at a constant level during the cell cycle . Gel filtration and co-immunoprecipitation analyses reveal that Mad2p tightly associates with another spindle checkpoint component, Mad1p . This association is independent of cell cycle stage and the presence or absence of other known checkpoint proteins . In addition, Mad2p binds to all of the different phosphorylated isoforms of Mad1p that can be resolved on SDS-PAGE . Deletion and mutational analysis of both proteins indicate that association of Mad2p with Mad1p is critical for checkpoint function and for hyperphosphorylation of Mad1p. Mol Biol Cell, 1999 Aug, 10(8), 2583 - 94 The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast; Swaminathan S et al.; Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation . In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome . In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked . We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome . Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome . Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well . In the doa4Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase . Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Delta cells . This loss of viability and several other defects of doa4Delta cells are rescued by provision of additional ubiquitin . Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells . Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis . We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole. Mol Biol Cell, 1999 Aug, 10(8), 2559 - 72 Multiple sex pheromones and receptors of a mushroom-producing fungus elicit mating in yeast; Fowler TJ et al.; The mushroom-producing fungus Schizophyllum commune has thousands of mating types defined, in part, by numerous lipopeptide pheromones and their G protein-linked receptors . Compatible combinations of pheromones and receptors encoded by different mating types regulate a pathway of sexual development leading to mushroom formation and meiosis . A complex set of pheromone-receptor interactions maximizes the likelihood of outbreeding; for example, a single pheromone can activate more than one receptor and a single receptor can be activated by more than one pheromone . The current study demonstrates that the sex pheromones and receptors of Schizophyllum, when expressed in Saccharomyces cerevisiae, can substitute for endogenous pheromone and receptor and induce the yeast pheromone response pathway through the yeast G protein . Secretion of active Schizophyllum pheromone requires some, but not all, of the biosynthetic machinery used by the yeast lipopeptide pheromone a-factor . The specificity of interaction among pheromone-receptor pairs in Schizophyllum was reproduced in yeast, thus providing a powerful system for exploring molecular aspects of pheromone-receptor interactions for a class of seven-transmembrane-domain receptors common to a wide range of organisms. Mol Biol Cell, 1999 Aug, 10(8), 2531 - 46 Protein kinase activity and identification of a toxic effector domain of the target of rapamycin TOR proteins in yeast; Alarcon CM et al.; In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1 . The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton . A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified . We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin . We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast . Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect . Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation . Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains . In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM. Analyst, 1998 Dec, 123(12), 2585 - 8 Determination of oestrogen concentrations in bovine plasma by a recombinant oestrogen receptor-reporter gene yeast bioassay; Burdge GC et al.; A recombinant cell yeast bioassay (RCBA) was applied to the generic measurement of bovine plasma oestrogen concentration . Samples were prepared by diethyl ether extraction of plasma following addition of {3H}17 beta-oestradiol as internal standard; organic and aqueous phases were separated by freezing (recovery 97.1 +/- 0.7%) and dried extract reconstituted in culture medium (recovery 31.4 +/- 4.5%) . Plasma oestrogen concentrations were measured by incubation of extracts with yeast containing a stable human oestrogen receptor (hER) and a reporter construct comprising an hER response element regulating beta-galactosidase expression . The linearity of response for the analysis of spiked plasma samples using the RCBA, following corrections, is described by y = 0.8994x - 0.111 (r2 = 0.9776, P < 0.0001) . Inter-assay variation for endogenous oestrogen was 11.5% for > 1 pg ml-1 . Plasma oestrogen concentrations for intact (n = 5) and castrated (n = 3) males were < 0.5 pg ml-1, and 3.7 +/- 2.6 pg ml-1 for luteal phase females (n = 10) . Analysis by RCBA of sequential samples from heifers during the reproductive cycle failed to detect the pre-ovulatory increase in plasma 17 beta-oestradiol as determined by radioimmunoassay (RIA) (maximal concentrations 2.09 +/- 2.1 pg ml-1 and 32.6 +/- 14.6 pg ml-1, respectively) . Interestingly, when samples were hydrolysed using Helix pomatia glucuronidase the RCBA gave concentrations (29.5 +/- 8.9 pg ml-1) not significantly different to those obtained by RIA . These preliminary findings suggest that a substantial proportion of plasma oestrogen during the pre-ovulatory period may be conjugated . These data indicate the potential of the RCBA to measure biologically active and physiological levels of plasma oestrogens in cattle . One potentially valuable application of this generic oestrogen assay could be in surveillance programmes to detect illegal use of anabolic oestrogens in live-stock where the identity of the analyte may be unknown. Mutat Res, 1999 Aug 11, 429(1), 13 - 26 Cell division transforms mutagenic lesions into deletion-recombinagenic lesions in yeast cells; Galli A et al.; Cell proliferation has been recognized as an important factor in human and experimental carcinogenesis . Point mutations as well as larger chromosomal rearrangements are involved in the initiation of cancer . In this paper we compared the relative potencies of radiation and chemical carcinogens for inducing point mutations vs . deletions in cell cycle arrested with dividing cells of Saccharomyces cerevisiae . Point mutation substrates and deletion (DEL) recombination substrates were constructed with the genes CDC28 and TUB2 that are required for cell cycle progression through G1 and G2, respectively . The carcinogens ionizing radiation, UV, MMS, EMS and 4-NQO induced point mutations in G1 and in G2 arrested as well as in dividing cells . UV, MMS, EMS and 4-NQO caused very weak if any increases in DEL recombination in G1 or G2 arrested cells, but large increases in dividing cells . When cells treated with carcinogen either in G1 or G2 were allowed to progress through the cell cycle, a time-dependent increase in DEL recombination was seen . Ionizing radiation and the site-specific endonuclease I-SceI, which both directly create double-strand breaks, induced DEL recombination in G1 as well as in G2 arrested cells . In conclusion, UV-, MMS-, EMS- and 4-NQO-induced DNA damage was converted during DNA replication to a lesion capable of inducing DEL recombination which is probably a DNA strand break . Thus, cell proliferation is not necessary to turn DNA alkylation or UV damage into a mutagenic lesion but to convert the damage into a lesion that induces DNA deletions . These results are discussed with respect to mechanisms of carcinogenesis . Gene, 1999 Aug 5, 236(1), 53 - 61 Insertion of expanded CAG trinucleotide repeat motifs into a yeast artificial chromosome containing the human Machado-Joseph disease gene; Cemal CK et al.; Machado-Joseph disease or spinocerebellar ataxia 3 (SCA3) is a progressive neurodegenerative disorder caused by pathological expansion of a trinucleotide repeat motif present within exon 4 of the MJD1 gene . Previous attempts to create a transgenic animal model have failed to produce a neurological deficit truly representative of the disease phenotype . This appears to be the result of inappropriate expression of the mutant protein in neuronal populations generally spared in the disease state . Introduction of a human disease gene in the context of a yeast artificial chromosome clone containing endogenous regulatory elements would enhance the potential for correct tissue/cell-specific expression at physiological levels . We report the introduction of expanded CAG repeat motifs into a 250kb yeast artificial chromosome clone spanning the MJD1 locus using two rounds of homologous recombination . Transformants exhibited both expansions and contractions of the motif with alleles ranging in size from 48 to 84 repeat units . The availability of these clones for modelling of the disease in transgenic animals should allow elucidation of the role of repeat length in the phenotypic spectrum of the disease. Gene, 1999 Aug 5, 236(1), 33 - 42 Monitoring dynamics of gene expression in yeast during stationary phase; Paz I et al.; The commonly used genetic approaches in yeast are designed to identify defects in cell/colony growth . In order to identify genes which control molecular mechanisms during quiescence ('stationary phase'), different tactics are required . We describe the development of a new genetic approach based on the previous observations that gene expression in quiescent Saccharomyces cerevisiae cells is largely repressed . For studying the mechanism controlling the repression of gene expression in stationary phase, we use UBI4-lacZ as a reporter gene . The product of this fusion gene was shown previously to encode an unstable protein in dividing cells . We show here that it is also unstable in stationary cells . We demonstrate that the relatively short half-life of this reporter protein can be utilized to monitor the dynamics of the repression of gene expression during stationary phase in liquid culture, using ACT1 or SSA3 promoters as the model promoters . By adapting a colony color test, we show that the reporter gene can also be used to monitor gene expression in quiescent colonies, thus serving as a tool to screen for defects in the regulation of this process during growth arrest . The utility of the approach was demonstrated by confirming the defects of top1Delta and bcy1Delta cells to appropriately express the ACT1p-UBI4-lacZ in stationary phase . The mutant colonies were easily discernible from wild-type colonies by our color test . Finally, using SSA3p-UBI4-lacZ as the reporter gene, we found that the 5'-untranslated region of SSA3 mRNA is sufficient to repress translation of the reporter mRNA after entry of the cells into stationary phase . The possibility that the short length of the SSA3 5'-untranslated region is a major determinant of the inefficient translation of SSA3p-UBI4-lacZ in stationary phase is discussed. Biochemistry, 1999 Aug 3, 38(31), 10004 - 12 Effects of substitution of tryptophan 412 in the substrate activation pathway of yeast pyruvate decarboxylase; Li H et al.; Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at W412, located on the putative substrate activation pathway and linking E91 on the alpha domain with W412 on the gamma domain of the enzyme . While C221 on the beta domain is the residue at which substrate activation is triggered {Baburina, I., et al . (1994) Biochemistry 33, 5630-5635; Baburina, I., et al . (1996) Biochemistry 35, 10249-10255}, that information, via the substrate bound at C221, is transmitted to H92 on the alpha domain, across the domain divide from C221 {Baburina, I., et al . (1998) Biochemistry 37, 1235-1244; Baburina, I., et al . (1998) Biochemistry 37, 1245-1255}, thence to E91 on the alpha domain {Li, H., and Jordan, F . (1999) Biochemistry 38, 9992-10003}, and then on to W412 on the gamma domain and to the active site thiamin diphosphate located at the interface of the alpha and gamma domains {Arjunan, D., et al . (1996) J . Mol . Biol . 256, 590-600} . Substitution at W412 with F and A was carried out, resulting in active enzymes with specific activities about 4- and 10-fold lower than that of the wild-type enzyme . Even though W412 interacts with E91 and H115 via a main chain hydrogen bond donor and acceptor, respectively, there is clear evidence for the importance of the indole side chain of W412 from a variety of experiments: thermostability, fluorescence quenching, and the binding constants of the thiamin diphosphate, and circular dichroism spectroscopy, in addition to conventional steady-state kinetic measurements . While the substrate activation is still prominent in the W412F variant, its level is very much reduced in the W412A variant, signaling that the size of the side chain is also important in positioning the amino acids surrounding the active center to achieve substrate activation . The fluorescence studies demonstrate that W412 is a relatively minor contributor to the well-documented fluorescence of apopyruvate decarboxylase in its native state . The information about the W412 variants provides strong additional support for the putative substrate activation pathway from C221 --> H92 --> E91 --> W412 --> G413 --> thiamin diphosphate . The accumulating evidence for the central role of the beta domain in stabilizing the overall structure is summarized. Biochemistry, 1999 Aug 3, 38(31), 9992 - 10003 Role of glutamate 91 in information transfer during substrate activation of yeast pyruvate decarboxylase; Li H et al.; Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at E91, located on the putative substrate activation pathway and linking the alpha and gamma domains of the enzyme . While C221 on the beta domain is the residue at which substrate activation is triggered {Baburina, I., et al . (1994) Biochemistry 33, 5630-5635; Baburina, I., et al . (1996) Biochemistry 35, 10249-10255}, that information, via the substrate bound at C221, is transmitted to H92 on the alpha domain, across the domain divide from C221 {Baburina, I . , et al . (1998) Biochemistry 37, 1235-1244}, thence to E91 on the alpha domain, and then on to W412 on the gamma domain {Li, H., and Jordan, F . (1999) Biochemistry 38, 10004-10012} and to the active site thiamin diphosphate located at the interface of the alpha and gamma domains {Arjunan, D., et al . (1996) J . Mol . Biol . 256, 590-600} . Substitution at E91 with Q, D, or A led to modest reductions in the specific activity (4-, 5-, and 30-fold), as well as in both the turnover number and the catalytic efficiency, in that order . Interestingly, the Hill coefficient was only slightly reduced for the E91D variant, but cooperativity was virtually abolished for the E91Q and E91A variants . The thermal stability of the variants was reduced in the following order: wild type > E91Q > E91D > E91A; circular dichroism and fluorescence experiments also demonstrated that the tertiary structure of the enzyme was affected by these substitutions . The variants could be purified as apoenzymes, demonstrating their impaired ability to bind thiamin diphosphate . Apparently, the charge at residue 91 is quite important for maintaining optimal cooperativity . To maintain strong domain-domain interactions, the length of the side chain at position 91 with hydrogen bonding potential to W412 is sufficient. J Appl Microbiol, 1999 Jul, 87(1), 1 - 7 Yeast cell mortality related to a high-pressure shift: occurrence of cell membrane permeabilization; Perrier-Cornet JM et al.; The shrinkage of yeast cells caused by high-pressure treatment (250 MPa, 15 min) was investigated using direct microscopic observation . A viable staining method after treatment allowed the volume variation of two populations to be distinguished: an irreversible volume decrease (about 35% of the initial volume) of pressure-inactivated cells during pressure holding time, and viable cells, which were less affected . A mass transfer was then induced during high-pressure treatment . Causes of this transfer seem to be related to a pressure-induced membrane permeabilization, allowing a subsequent leakage of internal solutes, where three ions (Na+, K+ and Ca2+), plus endogenous glycerol, were verified . This glycerol leakage was found to occur after yeast pressurization in a medium having low water activity, although the yeast was not inactivated . All these observations lead to the hypothesis that pressure-induced cell permeabilization could be the cause of yeast inactivation under pressure. FEBS Lett, 1999 Jul 9, 454(3), 201 - 6 Assessment of uncoupling activity of the human uncoupling protein 3 short form and three mutants of the uncoupling protein gene using a yeast heterologous expression system; Hagen T et al.; The human uncoupling protein 3 gene generates two mRNA transcripts, uncoupling protein 3L and uncoupling protein 3S, which are predicted to encode long and short forms of the uncoupling protein 3 protein, respectively . While uncoupling protein 3L is similar in length to the other known uncoupling proteins, uncoupling protein 3S lacks the last 37 C-terminal residues . A splice site mutation in the human uncoupling protein 3 gene, resulting in the exclusive expression of uncoupling protein 3S, and a number of point mutations in the uncoupling protein 3 gene have been described . This study compares the biochemical activity of uncoupling protein 3S as well as three mutants of the uncoupling protein 3 gene (V9M, V102I, R282C) with that of uncoupling protein 3L utilizing a yeast expression system . All proteins were expressed at similar levels and had qualitatively similar effects on parameters related to the uncoupling function . Both uncoupling protein 3S and uncoupling protein 3L decreased the yeast growth rate by 35 and 52%, increased the whole yeast basal O2 consumption by 26 and 48%, respectively, and decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3'3-dihexyloxacarbocyanine iodide . In isolated mitochondria, uncoupling protein 3S and uncoupling protein 3L caused a similar (33 and 35%, respectively) increase in state 4 respiration, which was relatively small compared to uncoupling protein 1 (102% increase) . A truncated version of uncoupling protein 3S, lacking the last three C-terminal residues, Tyr, Lys and Gly, that are part of a carrier motif that is highly conserved among all mitochondrial carriers, had a greatly reduced uncoupling activity . The two naturally occurring uncoupling protein 3 mutants, V9M and V102I, were similar to uncoupling protein 3L with respect to effects on the yeast growth and whole yeast O2 consumption . The R282C mutant had a reduced effect compared to uncoupling protein 3L . In summary, uncoupling protein 3S and the three mutants of uncoupling protein 3 appear to be functional proteins with biochemical activities similar to uncoupling protein 3L, although uncoupling protein 3S and the R282C mutant have a modestly reduced function. FEBS Lett, 1999 Jul 9, 454(3), 181 - 6 Induction of vacuolar Ca2+-ATPase and H+/Ca2+ exchange activity in yeast mutants lacking Pmr1, the Golgi Ca2+-ATPase; Marchi V et al.; We have analyzed Ca2+ transport activity in defined subcellular fractions of an isogenic set of wild-type and mutant yeast . The results, together with measurements of polypeptide expression levels and promoter::reporter gene activity, show that the Golgi Ca2+-ATPase, Pmr1, is the major Ca2+ pump under normal growth conditions . In the absence of Pmr1, we show a massive, calcineurin-dependent compensatory induction of the vacuolar Ca2+-ATPase, Pmc1 . In addition, H+/Ca2+ exchange activity, that may be distinct from the vacuolar exchanger Vcx1, is also increased. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 9033 - 8 Binding of elongin A or a von Hippel-Lindau peptide stabilizes the structure of yeast elongin C; Botuyan MV et al.; Elongin is a heterotrimeric transcription elongation factor composed of subunits A, B, and C in mammals . Elongin A and C are F-box-containing and SKP1 homologue proteins, respectively, and are therefore of interest for their potential roles in cell cycle-dependent proteolysis . Mammalian elongin C interacts with both elongin A and elongin B, as well as with the von Hippel-Lindau tumor suppressor protein VHL . To investigate the corresponding interactions in yeast, we have utilized NMR spectroscopy combined with ultracentrifugal sedimentation experiments to examine complexes of yeast elongin C (Elc1) with yeast elongin A (Ela1) and two peptides from homologous regions of Ela1 and human VHL . Elc1 alone is a homotetramer composed of subunits with a structured N-terminal region and a dynamically unstable C-terminal region . Binding of a peptide fragment of the Elc1-interaction domain of Ela1 or with a homologous peptide from VHL promotes folding of the C-terminal region of Elc1 into two regular helical structures and dissociates Elc1 into homodimers . Moreover, analysis of the complex of Elc1 with the full Elc1-interaction domain of Ela1 reveals that the Elc1 homodimer is dissociated to preferentially form an Ela1/Elc1 heterodimer . Thus, elongin C is found to oligomerize in solution and to undergo significant structural rearrangements upon binding of two different partner proteins . These results suggest a structural basis for the interaction of an F-box-containing protein with a SKP1 homologue and the modulation of this interaction by the tumor suppressor VHL. Genetics, 1999 Aug, 152(4), 1543 - 56 Genetic interactions in yeast between Ypt GTPases and Arf guanine nucleotide exchangers; Jones S et al.; Two families of GTPases, Arfs and Ypt/rabs, are key regulators of vesicular transport . While Arf proteins are implicated in vesicle budding from the donor compartment, Ypt/rab proteins are involved in the targeting of vesicles to the acceptor compartment . Recently, we have shown a role for Ypt31/32p in exit from the yeast trans-Golgi, suggesting a possible function for Ypt/rab proteins in vesicle budding as well . Here we report the identification of a new member of the Sec7-domain family, SYT1, as a high-copy suppressor of a ypt31/32 mutation . Several proteins that belong to the Sec7-domain family, including the yeast Gea1p, have recently been shown to stimulate nucleotide exchange by Arf GTPases . Nucleotide exchange by Arf GTPases, the switch from the GDP- to the GTP-bound form, is thought to be crucial for their function . Sec7p itself has an important role in the yeast secretory pathway . However, its mechanism of action is not yet understood . We show that all members of the Sec7-domain family exhibit distinct genetic interactions with the YPT genes . Biochemical assays demonstrate that, although the homology between the members of the Sec7-domain family is relatively low (20-35%) and limited to a small domain, they all can act as guanine nucleotide exchange factors (GEFs) for Arf proteins,