S. cerevisiae

J Biol Chem, 1998 Mar 27, 273(13), 7235 - 43
Heat-induced elevation of ceramide in Saccharomyces cerevisiae via de novo synthesis; Wells GB et al.; Sphingolipid-related metabolites have been implicated as potential signaling molecules in many studies with mammalian cells as well as in some studies with yeast . Our previous work showed that sphingolipid-deficient strains of Saccharomyces cerevisiae are unable to resist a heat shock, indicating that sphingolipids are necessary for surviving heat stress . Recent evidence suggests that one role for the sphingolipid intermediate ceramide may be to act as a second messenger to signal accumulation of the thermoprotectant trehalose . We examine here the mechanism for generating the severalfold increase in ceramide observed during heat shock . As judged by compositional analysis and mass spectrometry, the major ceramides produced during heat shock are similar to those found in complex sphingolipids, a mixture of N-hydroxyhexacosanoyl C18 and C20 phytosphingosines . Since the most studied mechanism for ceramide generation in animal cells is via a phospholipase C-type sphingomyelin hydrolysis, we examined S . cerevisiae for an analogous enzyme . Using {3H}phytosphingosine and {3H}inositol-labeled yeast sphingolipids, a novel membrane-associated phospholipase C-type activity that generated ceramide from inositol-P-ceramide, mannosylinositol-P-ceramide, and mannose(inositol-P)2-ceramide was demonstrated . The sphingolipid head groups were concomitantly liberated with the expected stoichiometry . However, other data demonstrate that the ceramide generated during heat shock is not likely to be derived by breakdown of complex sphingolipids . For example, the water-soluble fraction of heat-shocked cells showed no increase in any of the sphingolipid head groups, which is inconsistent with complex sphingolipid hydrolysis . Rather, we find that de novo ceramide synthesis involving ceramide synthase appears to be responsible for heat-induced ceramide elevation . In support of this hypothesis, we find that the potent ceramide synthase inhibitor, australifungin, completely inhibits both the heat-induced increase in incorporation of {3H}sphinganine into ceramide as well as the heat-induced increase in ceramide as measured by mass . Thus, heat-induced ceramide most likely arises by temperature activation of the enzymes that generate ceramide precursors, activation of ceramide synthase itself, or both.

Int Endod J, 1997 Sep, 30(5), 313 - 7
Observation of Saccharomyces cerevisiae in blood of patient undergoing root canal treatment; Debelian GJ et al.; In this study, an unusual observation of Saccharomyces cerevisiae isolated from an infected root canal and from the blood of a patient undergoing endodontic therapy of a tooth with asymptomatic apical periodontitis is reported . Phenotypic (biochemical tests, antifungal susceptibility tests and SDS-PAGE of cellular proteins) and genetic (ribotyping) methods were used to characterize the strains . By using these methods it was found that the blood and root canal isolates were identical but differed from S . cerevisiae strains of other sources . It was therefore more than likely that the root canal was the source of the blood isolate and that it had been transferred unintentionally to the bloodstream during root canal treatment.

Eur J Biochem, 1998 Mar 15, 252(3), 391 - 9
Functional expression of recombinant N-methyl-D-aspartate receptors in the yeast Saccharomyces cerevisiae--localization and pharmacological characterization; Li Z et al.; The yeast Saccharomyces cerevisiae was used for expressing the genes encoding the ionotropic N-methyl-D-aspartate (NMDA) receptor subunits from rats (NR1a, NR2A, NR2C) and mice (NR2B) . Four plasmids were constructed by cloning the different NMDA receptor genes in the two multi-copy yeast-Escherichia coli shuttle vectors pMB01 (--> NR1a gene) and pMB02 (--> NR2A-2C genes) . The protease-deficient S . cerevisiae strain cI3-ABYS-86 (leu-, ura-) was transformed or co-transformed with the resulting plasmids pMBNR1a (leu+) or pMBNR1a/pMBNR2A-C (ura+) respectively . Western blotting analysis with antibodies raised against amino acid sequences at the C-termini of the respective subunits revealed that the recombinant receptor proteins were differently expressed and only partially glycosylated in the cell membranes of the recombinant yeast strains . The expression and localization of the recombinant NMDA receptor proteins were also proved by immunofluorescence microscopy which indicated a distinct expression of the different NMDA receptor subunits in the plasma membrane of the transformed yeast cells . Pharmacological characterization of crude membrane preparations of the recombinant yeast cells showed saturable binding of the glycine antagonist {3H}MDL105,519 with Kd values of 56.9 +/- 6.19 nM (NR1a/NR2A), 26.72 +/- 2.13 nM (NR1a/NR2B), and 21.22 +/- 1.64 nM (NR1a/NR2C), and bound capacities of 17.94 +/- 1.24 pmol/mg membrane protein (NR1a/NR2A), 11.45 +/- 0.67 pmol/mg (NR1a/NR2B), and 16.15 +/- 0.86 (NR1a/NR2C) pmol/mg . The {3H}MDL105,519 binding was inhibited by the glycine antagonist 5,7-dichlorokynurenate, ethyl-2-carboxy-4,6-dichloro-3-indoleacetate, and itself, but not by glycine, D-serine or 1-amino-cyclopropanecarboxylic acid . Specific binding of {3H}glycine or the NMDA channel blocker {3H}dizolcipine were not observed.

Appl Environ Microbiol, 1998 Apr, 64(4), 1303 - 7
Pyruvate decarboxylase catalyzes decarboxylation of branched-chain 2-oxo acids but is not essential for fusel alcohol production by Saccharomyces cerevisiae; ter Schure EG et al.; The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages . The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a decarboxylase, and an alcohol dehydrogenase . Partially purified preparations of pyruvate decarboxylase (EC 4.1.1.1) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine . Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type Saccharomyces cerevisiae strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids . Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all three PDC genes had been disrupted . Experiments with cell extracts from S . cerevisiae mutants expressing a single PDC gene demonstrated that both PDC1- and PDC5-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids . To investigate whether pyruvate decarboxylase is essential for fusel alcohol production by whole cells, wild-type S . cerevisiae and an isogenic pyruvate decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source . Surprisingly, the three corresponding fusel alcohols were produced in both strains . This result proves that decarboxylation of branched-chain 2-oxo acids via pyruvate decarboxylase is not an essential step in fusel alcohol production.

J Biol Chem, 1998 Mar 20, 273(12), 6844 - 52
Expression, purification, and characterization of choline kinase, product of the CKI gene from Saccharomyces cerevisiae; Kim KH et al.; In the yeast Saccharomyces cerevisiae, choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) is the product of the CKI gene . Choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine by the CDP-choline pathway . The yeast enzyme was overexpressed 106-fold in Sf-9 insect cells and purified 71.2-fold to homogeneity from the cytosolic fraction by chromatography with concanavalin A, Affi-Gel Blue, and Mono Q . The N-terminal amino acid sequence of purified choline kinase matched perfectly with the deduced sequence of the CKI gene . The minimum subunit molecular mass (73 kDa) of purified choline kinase was in good agreement with the predicted size (66.3 kDa) of the CKI gene product . Native choline kinase existed in oligomeric structures of dimers, tetramers, and octomers . The amounts of the tetrameric and octomeric forms increased in the presence of the substrate ATP . Antibodies were raised against the purified enzyme and were used to identify choline kinase in insect cells and in S . cerevisiae . Maximum choline kinase activity was dependent on Mg2+ ions (10 mM) at pH 9.5 and at 30 degrees C . The equilibrium constant (0.2) for the reaction indicated that the reverse reaction was favored in vitro . The activation energy for the reaction was 6.26 kcal/mol, and the enzyme was labile above 30 degrees C . Choline kinase exhibited saturation kinetics with respect to choline and positive cooperative kinetics with respect to ATP (n = 1.4-2.3) . Results of the kinetic experiments indicated that the enzyme catalyzes a sequential Bi Bi reaction . The Vmax for the reaction was 138.7 micromol/min/mg, and the Km values for choline and ATP were 0.27 mM and 90 microM, respectively . The turnover number per choline kinase subunit was 153 s-1 . Ethanolamine was a poor substrate for the purified choline kinase, and it was also poor inhibitor of choline kinase activity . ADP inhibited choline kinase activity (IC50 = 0.32 mM) in a positive cooperative manner (n = 1.5), and the mechanism of inhibition with respect to ATP and choline was complex . The regulation of choline kinase activity by ATP and ADP may be physiologically relevant.

Biochem J, 1998 Mar 1, 330 ( Pt 2), 811 - 7
Importance of glucose-6-phosphate dehydrogenase in the adaptive response to hydrogen peroxide in Saccharomyces cerevisiae; Izawa S et al.; Glucose-6-phosphate dehydrogenase (G6PDH)-deficient cells of Saccharomyces cerevisiae showed increased susceptibility and were unable to induce adaptation to oxidative stress . Historically, mainly in human erythrocytes, it has been suggested and accepted that decreased cellular GSH, due to loss of the NADPH-dependent activity of glutathione reductase (GR), is responsible for the increased sensitivity to oxidative stress in G6PDH-deficient cells . In the present study we investigated whether the increased susceptibility and the inability to induce adaptation to H2O2 stress of G6PDH-deficient yeast is caused by incompleteness of glutathione recycling . We constructed G6PDH- and GR-deficient mutants and analysed their adaptive response to H2O2 stress . Although G6PDH-deficient cells contained comparable amounts of GSH and GR activity to wild-type cells, GSSG was not reduced efficiently, and intracellular GSSG levels and the ratio of GSSG to total glutathione (GSSG/tGSH) were higher in G6PDH-deficient cells than in wild-type . On the other hand, GR-deficient cells showed a susceptibility identical with that of wild-type cells and induced adaptation to H2O2 stress, even though the GSSG/tGSH ratio in GR-deficient cells was higher than in G6PDH-deficient cells . These results indicate that incompleteness of glutathione recycling alone is not sufficient to account for the increased sensitivity and inability to induce adaptation to H2O2 stress of G6PDH-deficient yeast cells . In S . cerevisiae, G6PDH appears to play other important roles in the adaptive response to H2O2 stress besides supplying NADPH to the GR reaction.

Genes Cells, 1997 Dec, 2(12), 753 - 70
Gin4 of S . cerevisiae is a bud neck protein that interacts with the Cdc28 complex; Okuzaki D et al.; BACKGROUND: A number of proteins make up the Cdc28 complex in Saccharomyces cerevisiae, and regulate CDK activity . The cell cycle regulator Nik1 (Hsl1) is a protein kinase that interacts with the Cdc28 complex . The growth inhibitor Gin4 is structurally similar to Nik1 and may play a redundant role in the regulation of the cell cycle . We investigated the functions of Gin4 with respect to those of Nik1 . RESULTS: GIN4 was not essential for growth, and cells deficient in the GIN4 gene displayed no obvious defects in cell cycle regulation . The delta(gin)4 delta(nik)1 strain was temperature sensitive and showed an abnormal cell shape and FACS profile at permissive temperatures . GFP-fused Gin4 was localized at the bud-neck from late G1 to the M phase . Over-production of the C-terminal portion of Gin4 was toxic for cell growth, and this domain was required for the bud-neck localization of Gin4-GFP . High copy expression of Gin4-GFP disturbed the bud-neck localization of Gin4 in the abnormally elongated cells . Cytokinesis was defective in the delta(gin)4 cdc28 double mutants . The GST-Gin4 fusion protein physically associates with the Cdc28 complex . CONCLUSIONS: Gin4 is a bud-neck protein . GIN4 and NIK1 have distinct but partially overlapping functions . The major function of GIN4 is to ensure proper mitotic progression and cytokinesis.

Biochim Biophys Acta, 1998 Mar 5, 1401(3), 235 - 8
Correlation between stationary phase survival and acid trehalase activity in yeast; Roy A et al.; The levels of two trehalose hydrolysing enzymes, acid trehalase (AT) and neutral trehalase (NT), have been investigated in Candida utilis at different stages of growth; in complete contrast to Saccharomyces cerevisiae, significant AT activity appears to be absent at all stages of growth studied in C . utilis . In addition, presence of only very low amounts of iso-aspartyl methyl transferase (IMT) activity at the onset of stationary phase and lower survival ability in early stationary phase in contrast to that of S . cerevisiae lend support to the ideas that (a) lower degree of survival of C . utilis in the stationary phase may be a direct consequence of inability to mobilise stored trehalose due to absence of intracellular AT and reduced levels of IMT activities and (b) trehalose may have a dual role vis-a-vis stress resistance in yeasts.

Microbiology, 1998 Mar, 144 ( Pt 3), 671 - 80
Role of trehalose in survival of Saccharomyces cerevisiae under osmotic stress; Hounsa CG et al.; Trehalose is an enigmatic compound that accumulates in Saccharomyces cerevisiae and has been implicated in survival under various stress conditions by acting as membrane protectant, as a supplementary compatible solute or as a reserve carbohydrate that may be mobilized during stress . In this study, specific mutants in trehalose metabolism were used to evaluate whether trehalose contributes to survival under severe osmotic stress and generates the compatible solute glycerol under moderate osmotic stress . The survival under severe osmotic stress (0.866 aw' NaCl or sorbitol) of mutants was compared to that of the wild-type strain when cultivated to either the mid-exponential or the stationary growth phase on glucose, galactose or ethanol . Stationary-phase cells survived better than exponential-phase cells . The death rates of ethanol-grown cells were lower than those of galactose-grown cells, which in turn survived better than glucose-grown cells . There was a strong relationship between intracellular trehalose levels and resistance to osmotic stress . The mutant strains unable to produce trehalose (tps1 delta tps2 delta and tps1 delta hxk2 delta) were more sensitive to severe osmotic stress (0.866 aw) than the isogenic wild-type strain, confirming a role for trehalose in survival . Hyperaccumulation of trehalose found in the nth1 delta and the nth1 delta gpd1 delta mutant strains, however, did not improve survival rates compared to the wild-type strain . When wild-type, nth1 delta and nth1 delta gpd1 delta cells were exposed to moderate osmotic stress (0.98 and 0.97 aw' NaCl), which permits growth, glycerol production did not appear to be related to the intracellular trehalose levels although glycerol levels increased more rapidly in nth1 delta cells than in wild-type cells during the initial response to osmotic stress . These data indicate that trehalose does not act as a reserve compound for glycerol synthesis under these conditions . No evidence was found for solutes other than glycerol and trehalose being significant for the survival of or growth by S . cerevisiae under osmotic stress conditions.

Biochem J, 1998 Feb 15, 330 ( Pt 1), 421 - 7
The small GTPase Gsp1p binds to the repeat domain of the nucleoporin Nsp1p; Stochaj U et al.; The small GTPase Gsp1p of Saccharomyces cerevisiae and its homologue Ran play essential roles in several nuclear processes, such as cell-cycle progression, nuclear organization and nucleocytoplasmic traffic of RNA and proteins . Gsp1p/Ran is an abundant nuclear protein that interacts with different cytoplasmic and nuclear factors . Several of the previously identified Ran-binding proteins located at the nuclear-pore complex carry a specific Ran-binding domain . So far, direct interactions between the GTPase and other proteins have been mostly characterized in higher eukaryotes . Here we report that the yeast protein Gsp1p can directly bind to the nucleoporin Nsp1p in vitro . Nsp1p does not contain a Ran-binding domain and therefore represents a distinct type of nucleoporin that associates with Gsp1p . We demonstrate that the middle domain of Nsp1p is sufficient to mediate this interaction . Importantly, we show that a conserved cluster of positively charged amino acid residues of Gsp1p located at positions 142-144 is essential for the binding reaction . Thus we have identified Nsp1p as a new candidate protein located at the nuclear pore complex of the yeast S . cerevisiae that interacts directly with Gsp1p . We further demonstrate that both Gsp1p and Nsp1p are components of larger protein complexes in vivo, supporting the idea that the association between both proteins takes place in growing cells.

Microbiol Mol Biol Rev, 1998 Mar, 62(1), 230 - 47
Vacuole biogenesis in Saccharomyces cerevisiae: protein transport pathways to the yeast vacuole; Bryant NJ et al.; Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae provides an excellent model system in which to study vacuole and lysosome biogenesis and membrane traffic . This organelle receives proteins from a number of different routes, including proteins sorted away from the secretory pathway at the Golgi apparatus and endocytic traffic arising from the plasma membrane . Genetic analysis has revealed at least 60 genes involved in vacuolar protein sorting, numerous components of a novel cytoplasm-to-vacuole transport pathway, and a large number of proteins required for autophagy . Cell biological and biochemical studies have provided important molecular insights into the various protein delivery pathways to the yeast vacuole . This review describes the various pathways to the vacuole and illustrates how they are related to one another in the vacuolar network of S . cerevisiae.

Mol Cell Biol, 1998 Apr, 18(4), 2309 - 23
Ca2+ content and expression of an acidocalcisomal calcium pump are elevated in intracellular forms of Trypanosoma cruzi; Lu HG et al.; The survival of a eukaryotic protozoan as an obligate parasite in the interior of a eukaryotic host cell implies its adaptation to an environment with a very different ionic composition from that of its extracellular habitat . This is particularly important in the case of Ca2+, the intracellular concentration of which is 3 orders of magnitude lower than the extracellular value . Ca2+ entry across the plasma membrane is a widely recognized mechanism for Ca2+ signaling, needed for a number of intracellular processes, and obviously, it would be restricted in the case of intracellular parasites . Here we show that Trypanosoma cruzi amastigotes possess a higher Ca2+ content than the extracellular stages of the parasite . This correlates with the higher expression of a calcium pump, the gene for which was cloned and sequenced . The deduced protein product (Tca1) of this gene has a calculated molecular mass of 121,141 Da and exhibits 34 to 38% identity with vacuolar Ca2+-ATPases of Saccharomyces cerevisiae and Dictyostelium discoideum, respectively . The tca1 gene suppresses the Ca2+ hypersensitivity of a mutant of S . cerevisiae that has a defect in vacuolar Ca2+ accumulation . Indirect immunofluorescence and immunoelectron microscopy analysis indicate that Tca1 colocalizes with the vacuolar H+-ATPase to the plasma membrane and to intracellular vacuoles of T . cruzi . These vacuoles were shown to have the same size and distribution as the calcium-containing vacuoles identified by the potassium pyroantimoniate-osmium technique and as the electron-dense vacuoles observed in whole unfixed parasites by transmission electron microscopy and identified in a previous work (D . A . Scott, R . Docampo, J . A . Dvorak, S . Shi, and R . D . Leapman, J . Biol . Chem . 272:28020-28029, 1997) as being acidic and possessing a high calcium content (i.e., acidocalcisomes) . Together, these results suggest that acidocalcisomes are distinct from other previously recognized organelles present in these parasites and underscore the ability of intracellular parasites to adapt to the hostile environment of their hosts.

Nucleic Acids Symp Ser, 1997, (36), 42 - 4
Mitochondrial RNase P: the RNA family grows; Martin NC et al.; Recent results of biochemical approaches and genome sequencing approaches has extended the members of the family of mitochondrial RNase P RNA genes . So far all of them are AU rich, and most of their secondary structures are easier to predict than was the structure of the first mitochondrial RNase P RNA from S . cerevisiae . The recently sequenced protozoan R . americana mitochondrial gene displays many of the evolutionarily conserved primary sequence and secondary structure attributes of the well characterized bacterial RNase P RNAs . Continued addition of RNAs to this data base should allow increasingly informative alignments and an understanding of what structural elements are dispensable in the smallest mitochondrial RNAs . The only protein subunits identified to date are Rpm2p from S.cerevisiae and the homologous protein from S . douglasii.

Curr Genet, 1998 Jan, 33(1), 52 - 9
Evolution of mitochondrial DNA in yeast: gene order and structural organization of the mitochondrial genome of Saccharomyces uvarum; Cardazzo B et al.; We have determined the size, the restriction map and the gene order of the mitochondrial genome of the yeast Saccharomyces uvarum . Sequence analysis of the mitochondrial COXII gene confirmed the position of this yeast in the Saccharomyces cerevisiae-like group, near Saccharomyces cerevisiae and Saccharomyces douglasii . Most mitochondrial genes have been positioned on this approximately 57-kb long genome and three regions containing putative replication origins have been identified . The gene order of S . uvarum suggests that the mitochondrial genome of the S.cerevisiae-like yeasts could have evolved from an ancestral molecule, similar to that of S . uvarum, through specific genome rearrangements.

Curr Genet, 1998 Jan, 33(1), 1 - 3
A rapid method to monitor repair and mis-repair of DNA double-strand breaks by using cell extracts of the yeast Saccharomyces cerevisiae; Jha B et al.; We present a rapid in vitro method to scan the repair of DNA double-strand breaks (DSBs) . A DSB was introduced at the EcoRI site within the lacZ gene of the plasmid pUC18 and the plasmid was exposed to cellular extracts from a wild-type repair-competent (RAD) and a mutant (rad52Delta) strain of the yeast Saccharomyces cerevisiae . The fidelity of rejoining was determined by the expression of the lacZ gene after bacterial transformation with the treated plasmid . A cellular extract from the yeast S . cerevisiae was found to be capable of rejoining DNA DSBs . Breaks at the EcoRI site were rejoined by extracts from both wild-type and mutant strains to form circular plasmids with almost equal efficiency . However, the fidelity of rejoining was lower for the rad52Delta extract than for normal wild-type.

Gene, 1998 Jan 12, 206(2), 165 - 74
The DUG gene of Drosophila melanogaster encodes a structural and functional homolog of the S . cerevisiae SUG1 predicted ATPase associated with the 26S proteasome; Mounkes LC et al.; The DUG gene of Drosophila encodes a putative ATPase that is a structural and functional homolog of the yeast SUG1 product . When introduced into S . cerevisiae, the Drosophila DUG gene rescued the lethality associated with a SUG1 mutant . Anti-DUG antibodies recognized a protein that migrated in high molecular weight complexes, along with components of the 26S proteasome, and also immunoprecipitated components of the 26S proteasome from embryonic extracts . Proteins recognized by the affinity-purified antibody raised against DUG were localized in either a punctate cytoplasmic distribution or in the nucleus, depending on the cell type, consistent with the subcellular localization of the 26S proteasome in various cell types.

Oncogene, 1998 Feb 26, 16(8), 1085 - 9
The CXXC Zn binding motifs of the human papillomavirus type 16 E7 oncoprotein are not required for its in vitro transforming activity in rodent cells; Braspenning J et al.; The conserved region 3 (CR3) of the E7 protein of human papillomaviruses contains two CXXC motifs involved in zinc binding and in the homodimerization of the molecule . Studies have suggested that the intact CXXC motifs in the CR3 of HPV16 and HPV18 E7 are required for the in vitro transforming activity of these proteins . CR3 also contains a low affinity pRb binding site and is involved in the disruption of the E2F/Rb1 complex . E7 is structurally and functionally related to Adenovirus E1A protein, which also has two CXXC motifs in CR3 . However, the Ad E1A transforming activity appears to be independent of the presence of such domains . In fact, this viral protein exists in vivo as two different forms of 289 and 243 amino acids . The shorter Ad E1A form (Ad E1A243), where both CXXC motifs are deleted by internal splicing, retains its in vitro transforming activity . We have investigated if the HPV16 E7 CR3 can be functionally replaced by the Ad E1A243 CR3, which lacks both CXXC motifs . A chimeric protein (E7/E1A243) containing the CR1 and CR2 of HPV16 E7 fused to the CR3 of Ad E1A 243 was constructed . The E7/E1A243 while not able to homodimerize in the S . cerevisiae two-hybrid system retains several of the properties of the parental proteins, HPV16 E7 and Ad E1A . It associates with the 'pocket' proteins, induces growth in soft agar of NIH3T3 cells and immortalizes rat embryo fibroblasts . These data suggest that the CXXC motifs in CR3 of E7 do not play a direct role in the transforming properties of this viral protein but probably are important for maintaining the correct protein configuration.

Biochem Biophys Res Commun, 1998 Mar 17, 244(2), 505 - 13
A novel Dictyostelium discoideum gene required for cAMP-dependent cell aggregation; Nagasaki A et al.; Using a method of random insertional mutagenesis called REMI (restriction enzyme-mediated integration), we isolated two mutant strains of Dictyostelium discoideum with a defect in cAMP-dependent cell aggregation . On bacterial lawns, both of the cells formed large and smooth plaques . When starved in a non-nutrient medium, they became elongated and extended pseudopods very frequently like starved wild type cells . However, they never formed streams toward an aggregation center . Genomic DNA fragments flanking the sites of insertion of the REMI tag were rescued from the mutant cells . The fragments contained one common open reading frame encoding a protein of 1148 amino acid residues . The protein's sequence is homologous to those of two hypothetical proteins of S . cerevisiae and S . pombe.

Biochem Biophys Res Commun, 1998 Mar 6, 244(1), 102 - 9
Cloning and developmental expression of a nuclear ubiquitin-conjugating enzyme (DmUbc9) that interacts with small heat shock proteins in Drosophila melanogaster; Joanisse DR et al.; In a two hybrid screen designed to identify proteins that interact with small heat shock proteins (sHsps), a Drosophila melanogaster homologue of yeast and human ubc9 (Dmubc9) was found to interact with Drosophila Hsp23 . Further, two-hybrid system analysis reveals DmUbc9 interaction with Drosophila and mammalian Hsp27 . In situ hybridization localizes Dmubc9 as a doublet at locus 21D on chromosome 2L, and genomic cloning of the gene reveals a single open reading frame without introns . The predicted Dmubc9 protein sequence shares a very high level of homology with mouse (85.4%) and human (> or = 82.9%) Ubc9 . Genetic complementation analysis show that Dmubc9 functionally rescues a temperature-sensitive S . cerevisiae ubc9ts mutant . Co-immunoprecipitation with antibody raised against DmUbc9 confirms the interaction with Drosophila Hsp23 and Hsp26 and preferentially with Hsp27 . The DmUbc9 protein, which localizes primarily to the nucleus in Drosophila S2 cells, is found at high levels in embryos but is also present at lower levels throughout development . The significance of the sHsp-Ubc9 interaction is discussed.

J Mol Biol, 1998 Feb 20, 276(2), 449 - 59
The 2.3 A X-ray crystal structure of S . cerevisiae phosphoglycerate mutase; Rigden DJ et al.; The high resolution crystal structure of Saccharomyces cerevisiae phosphoglycerate mutase has been determined . This structure shows important differences from the lower resolution structure deposited in 1982 . The crystal used to determine the new structure was of a different form, having spacegroup P2(1) . The model was refined to a crystallographic R-factor of 18.9% and a free R-factor of 28.4% using all data between 25 and 2.3 A and employing a bulk solvent correction . The enzyme is a tetramer of identical, 246 amino acid subunits, whose structure is revealed to be a dimer of dimers, with four independent active sites located well away from the subunit contacts . Each subunit contains two domains, the larger with a typical nucleotide binding fold, although phosphoglycerate mutase has no physiological requirement to bind nucleotides . The catalytic-site histidine residues are no longer in a "clapping-hands" conformation, but more resemble the conformation seen in the distantly related enzymes prostatic acid phosphatase and fructose-2,6-bisphosphatase . However, the catalytic histidine residues in the mutase are found to be much closer to each other than in the phosphatase structures, perhaps due to the absence of bound ligands in the mutase crystal . An intricate web of H-bonds is found around the catalytic histidine residues, high-lighting residues probably important for maintaining their correct orientation and charge . The positions of certain other residues, including some found near the catalytic site and some lining the catalytic-site cleft, have been changed by the correction of registration errors between sequence and electron density in the original structure . Electron density was apparent for a portion of the functionally important C-terminal tail, which was absent from the earlier structure, showing it to adopt a mainly helical conformation.

Biochim Biophys Acta, 1998 Feb 11, 1395(3), 329 - 44
Correct targeting of a vacuolar tobacco chitinase in Saccharomyces cerevisiae--post-translational modifications are dependent on the host strain; Kunze I et al.; The chitinase gene FB7-1 of Nicotiana tabacum cv . samsun line 5 was expressed in the two Saccharomyces cerevisiae strains, INVSC2 and H4, under the control of the GAL1 promoter from S . cerevisiae and a multicopy plasmid vector . Both yeast strains express the plant gene as enzymatic active proteins . In transformants of the strain INVSC2, 94% of the total plant chitinase is contained inside the cells, probably within the vacuole which has been confirmed by subcellular fractionation as well as immunohistochemical experiments . This retention inside the cells is due to the C-terminally located 7 amino acids long vacuolar targeting peptide of the prochitinase . When this sequence was removed, chitinase was transported into the culture medium . Pulse-chase experiments revealed that during translation in transformants of both yeast strains one chitinase polypeptide can be immunoadsorbed with specific antibodies . In the case of INVSC2-transformants, newly formed chitinase is modified in a 60 min chase to slightly increase its molecular mass, whereas in H4-transformants the molecular mass constantly remained 32 kDa . By Western blot analysis two chitinase corresponding polypeptides of 32 and 37 kDa were accumulated in the culture medium of both transformants carrying the chitinase gene without the vacuolar targeting sequence . The larger one was very likely O-glycosylated . Whereas, both polypepitdes were also detected in cell extracts of the H4-transformant, only the smaller one was found in the INVSC2-transformant . The plant chitinase passed through the endoplasmic reticulum on its way to the vacuole . The N-terminal signal peptide responsible for the uptake into the endoplasmic reticulum is cleaved correctly . However, cleavage of the vacuolar targeting peptide located at the C-terminus, to give the mature chitinase is obviously influenced by the genetic background of the host strain . In INVSC2-transformants chitinase accumulates in its mature form whereas both the polypeptides of H4-transformants retain their vacuolar targeting peptide . Our results demonstrate that in the case of plant class I chitinase, the plant sorting signal is recognized in yeast cells but post-translational modifications are influenced by the host strain.

Bioessays, 1998 Jan, 20(1), 30 - 40
Silent chromatin in yeast: an orchestrated medley featuring Sir3p {corrected}; Stone EM et al.; Extensive regions of chromosomes can be transcriptionally repressed through silencing mechanisms mediated by complex chromatin structures . One of the most refined molecular portraits of silenced chromatin comes from studies of the silent mating-type loci and telomeres of S . cerevisiae . In this budding yeast, the Sir3p silent information regulator emerges as a critically important silencing component that interacts with nucleosomes and other silencing proteins . Not only is it essential for silencing, but Sir3p is also capable of spreading silenced chromatin when its dosage is increased . Sir3p is a target of mitogen-activated protein (MAP) kinase cascade regulation and has significant similarity to the Orc1p subunit of the DNA replication origin recognition complex . Thus, in concert with other silencing proteins, Sir3p appears poised to respond to cellular signals and reprogram silencing through replication-associated assembly of repressive chromatin structures.

Fungal Genet Biol, 1998 Feb, 23(1), 1 - 17
Cytochrome P450 enzyme systems in fungi; van den Brink HM et al.; The involvement of cytochrome P450 enzymes in many complex fungal bioconversion processes has been characterized in recent years . Accordingly, there is now considerable scientific interest in fungal cytochrome P450 enzyme systems . In contrast to S . cerevisiae, where surprisingly few P450 genes have been identified, biochemical data suggest that many fungi possess numerous P450 genes . This review summarizes the current information pertaining to these fungal cytochrome P450 systems, with emphasis on the molecular genetics . The use of molecular techniques to improve cytochrome P450 activities in fungi is also discussed.

Glycobiology, 1998 Feb, 8(2), 155 - 64
Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo; Jakob CA et al.; In higher eukaryotes a quality control system monitoring the folding state of glycoproteins is located in the ER and is composed of the proteins calnexin, calreticulin, glucosidase II, and UDP-glucose: glycoprotein glucosyltransferase . It is believed that the innermost glucose residue of the N- linked oligosaccharide of a glycoprotein serves as a tag in this control system and therefore performs an important function in the protein folding pathway . To address this function, we constructed Saccharomyces cerevisiae strains which contain nonglucosylated (G0), monoglucosylated (G1), or diglucosylated (G2) glycoproteins in the ER and used these strains to study the role of glucose residues in the ER processing of glycoproteins . These alterations of the oligosaccharide structure did not result in a growth phenotype, but the induction of the unfolded protein response upon treatment with DTT was much higher in G0 and G2 strains as compared to wild-type and G1 strains . Our results provide in vivo evidence that the G1 oligosaccharide is an active oligosaccharide structure in the ER glycoprotein processing pathway of S.cerevisiae . Furthermore, by analyzing N- linked oligosaccharides of the constructed strains we can directly show that no general glycoprotein glucosyltransferase exists in S . cerevisiae.

Essays Biochem, 1997, 32, 113 - 25
The co-ordination of nuclear and organellar genome expression in eukaryotic cells; Surpin M et al.; Mitochondria and chloroplasts contain their own genetic complement . Eukaryotic cells must establish mechanisms by which the organellar and nuclear genomes are co-ordinately expressed . In the yeast S . cerevisiae, mitochondrial mutants that have respiratory deficiencies have altered patterns of nuclear gene expression . In strains that are deficient in the mitochondrial isoform of citrate synthase there is an up-regulation of the peroxisomal isoform . There is evidence that the mitochondria send a signal to the nucleus which results in the increased expression of the peroxisomal form of citrate synthase . Oxygen induces the biosynthesis of haem . When yeast are grown in aerobic culture, mitochondrial haem is transduced to the nucleus where it induces the expression of nuclear genes which encode proteins important for cellular respiration . Plant cells in which the chloroplasts have suffered photo-oxidative damage do not express nuclear genes that encode photosynthetic protein . Genetic studies in Arabidopsis have resulted in the characterization of mutants that do express nuclear photosynthetic genes when the development of the chloroplast is inhibited . These mutants will be instrumental in identifying the components of the chloroplast-to-nucleus signal-transduction pathways in plant cells . The identity of the signal that informs the nucleus of the developmental site of the chloroplast is unknown . Chlorophyll precursors, haem and porphyrin are considered candidate signal molecules . Recent studies have implicated that the redox state of the plastoquinone pool in chloroplasts may be involved in regulating nuclear gene expression . These studies have also shown that kinases and phosphatases may participate in relaying signals to the nucleus about the functional state of the chloroplast.

Cell, 1998 Feb 20, 92(4), 489 - 99
Ump1p is required for proper maturation of the 20S proteasome and becomes its substrate upon completion of the assembly; Ramos PC et al.; We report the discovery of a short-lived chaperone that is required for the correct maturation of the eukaryotic 20S proteasome and is destroyed at a specific stage of the assembly process . The S . cerevisiae Ump1p protein is a component of proteasome precursor complexes containing unprocessed beta subunits but is not detected in the mature 20S proteasome . Upon the association of two precursor complexes, Ump1p is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated . Cells lacking Ump1p exhibit a lack of coordination between the processing of beta subunits and proteasome assembly, resulting in functionally impaired proteasomes . We also show that the propeptide of the Pre2p/Doa3p beta subunit is required for Ump1p's function in proteasome maturation.

Genes Cells, 1997 Nov, 2(11), 655 - 65
The efficiency and timing of initiation of replication of multiple replicons of Saccharomyces cerevisiae chromosome VI; Yamashita M et al.; BACKGROUND: A complete set of nine ARSs was identified (the tenth ARS in this paper), mapped on chromosome VI of Saccharomyces cerevisiae, and characterized for functional elements . RESULTS: The level of activity of all ARSs as chromosomal replication origins was determined by neutral/neutral 2D gel-electrophoresis . These origins were classified into three groups: (i) three high frequency origins used once nearly every cell cycle, (ii) four intermediate frequency origins used once in two to three cycles and (iii) two low frequency origins used in fewer than 5% of cell cycles . These variations in initiation frequency among origins of chromosome VI are present in three common laboratory wild-type strains . Each origin is initiated at a fixed time and passively replicated by incoming replication forks at a fixed time during a synchronous S phase . Replication of each arm of the chromosome starts from one major origin located one-fifth (left arm) and one-third (right arm) of the distance from the centromere, and expands sequentially in both directions . Two telomere vicinity origins are replicated last . Time of initiation and replication of the last replicating origin, Ori609, was remarkably variable from cell to cell . CONCLUSIONS: Chromosome VI of S . cerevisiae contains nine replication origins that comprise five active replicons under normal cell growth conditions . A clear correlation was found between the efficiency of initiation and the order of replication . The timing of initiation of most origins, except for the first and last, is coincident with the time of passive replication by incoming forks from neighbouring origins.

Mol Gen Genet, 1998 Jan, 257(2), 149 - 56
The RHC21 gene of budding yeast, a homologue of the fission yeast rad21+ gene, is essential for chromosome segregation; Heo SJ et al.; The Saccharomyces cerevisiae gene RHC21 is a homologue of the fission yeast rad21+ gene, which affects the sensitivity of cells to gamma-irradiation and is essential for cell growth in S . pombe . Disruption of the RHC21 gene showed that it is also essential in S . cerevisiae . To examine its function in cell growth further, we have isolated temperature-sensitive mutants for the RHC21 gene and characterized one of them, termed rhc21-sk16 . When this mutant was incubated at 36 degrees C, the percentage of large-budded cells was increased . Most of the large-budded cells had aberrant nuclear structures, such as unequally extended nuclear DNA with incompletely elongated spindles across the mother-daughter neck or only in a mother cell . Furthermore, a circular minichromosome is more unstable in the mutant than in the wild-type, even at 25 degrees C . Flow cytometry showed that the bulk of DNA replication takes place normally at the restrictive temperature in the mutant . These results indicated that the RHC21 gene is required for proper segregation of the chromosomes . In addition, we found that the mutant is sensitive not only to UV radiation and gamma-rays but also to the antimicrotubule agent nocodazole at 25 degrees C . This suggests that the RHC21 gene is involved in the microtubule function . We discuss how the RHC21 gene product may be involved in chromosome segregation and microtubule function.

J Biol Chem, 1998 Feb 6, 273(6), 3702 - 11
IDP3 encodes a peroxisomal NADP-dependent isocitrate dehydrogenase required for the beta-oxidation of unsaturated fatty acids; Henke B et al.; In Saccharomyces cerevisiae the metabolic degradation of saturated fatty acids is exclusively confined to peroxisomes . In addition to a functional beta-oxidation system, the degradation of unsaturated fatty acids requires auxiliary enzymes, including a Delta2, Delta3-enoyl-CoA isomerase and an NADPH-dependent 2,4-dienoyl-CoA reductase . We found both enzymes to be present in yeast peroxisomes . The impermeability of the peroxisomal membrane for pyrimidine nucleotides led to the question of how the NADPH needed by the reductase is regenerated in the peroxisomal lumen . We report the identification and functional analysis of the IDP3 gene product, which is a yeast peroxisomal NADP-dependent isocitrate dehydrogenase . The newly identified peroxisomal protein is homologous to the mitochondrial Idp1p and cytosolic Idp2p, which both are yeast NADP-dependent isocitrate dehydrogenases . Yeast cells lacking Idp3p grow normally on saturated fatty acids, but growth is impaired on unsaturated fatty acids, indicating that the peroxisomal Idp3p is involved in their metabolic utilization . The data presented are consistent with the assumption that peroxisomes of S . cerevisiae contain the enzyme equipment needed for the degradation of unsaturated fatty acids, including an NADP-dependent isocitrate dehydrogenase, a putative constituent of a peroxisomal NADPH-regenerating redox system.

J Biol Chem, 1998 Feb 6, 273(6), 3278 - 84
Isolation and characterization of the Saccharomyces cerevisiae DPP1 gene encoding diacylglycerol pyrophosphate phosphatase; Toke DA et al.; Diacylglycerol pyrophosphate (DGPP) is involved in a putative novel lipid signaling pathway . DGPP phosphatase (DGPP phosphohydrolase) is a membrane-associated 34-kDa enzyme from Saccharomyces cerevisiae which catalyzes the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyzes the dephosphorylation of PA to yield diacylglycerol . Amino acid sequence information derived from DGPP phosphatase was used to identify and isolate the DPP1 (diacylglycerol pyrophosphate phosphatase) gene encoding the enzyme . Multicopy plasmids containing the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity in S . cerevisiae . The heterologous expression of the S . cerevisiae DPP1 gene in Sf-9 insect cells resulted in a 500-fold overexpression of DGPP phosphatase activity over that expressed in wild-type S . cerevisiae . DGPP phosphatase possesses a Mg2+-independent PA phosphatase activity, and its expression correlated with the overexpression of DGPP phosphatase activity in S . cerevisiae and in insect cells . DGPP phosphatase was predicted to be an integral membrane protein with six transmembrane-spanning domains . The enzyme contains a novel phosphatase sequence motif found in a superfamily of phosphatases . A dpp1Delta mutant was constructed by deletion of the chromosomal copy of the DPP1 gene . The dpp1Delta mutant was viable and did not exhibit any obvious growth defects . The mutant was devoid of DGPP phosphatase activity and accumulated (4-fold) DGPP . Analysis of the mutant showed that the DPP1 gene was not responsible for all of the Mg2+-independent PA phosphatase activity in S . cerevisiae.

Yeast, 1998 Jan 15, 14(1), 67 - 76
Engineering yeast for efficient cellulose degradation; Van Rensburg P et al.; Saccharomyces cerevisiae produces several beta-1,3-glucanases, but lacks the multicomponent cellulase complexes that hydrolyse the beta-1,4-linked glucose polymers present in cellulose-rich biomass as well as in haze-forming glucans in certain wines and beers . We have introduced into S . cerevisiae a functional cellulase complex for efficient cellulose degradation by cloning the Endomyces fibuliger cellobiase (BGL1) gene and co-expressing it with the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase (END1), the Phanerochaete chrysosporium cellobiohydrolase (CBH1) and the Ruminococcus flavefacies cellodextrinase (CEL1) gene constructs in this yeast . The END1, CBH1 and CEL1 genes were inserted into yeast expression/secretion cassettes . Expression of END1, CBH1 and CEL1 was directed by the promoter sequences derived from the alcohol dehydrogenase II (ADH2), the phosphoglycerate kinase I (PKG1) and the alcohol dehydrogenase I (ADH1) genes, respectively . In contrast, BGL1 was expressed under the control of its native promoter . Secretion of End1p and Cel1p was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1), whereas Cbh1p and Bgl1p were secreted using their authentic leader peptides . The construction of a fur1 ura3 S . cerevisiae strain allowed for the autoselection of this multicopy URA3-based plasmid in rich medium . S . cerevisiae transformants secreting biologically active endo-beta-1,4-glucanase, cellobiohydrolase, cellodextrinase and cellobiase were able to degrade various substrates including carboxymethylcellulose, hydroxyethylcellulose, laminarin, barley glucan, cellobiose, polypectate, birchwood xylan and methyl-beta-D-glucopyranoside . This study could lead to the development of industrial strains of S . cerevisiae capable of converting cellulose in a one-step process into commercially important commodities.

J Biol Chem, 1998 Jan 30, 273(5), 2977 - 83
Expression of the glyoxalase I gene of Saccharomyces cerevisiae is regulated by high osmolarity glycerol mitogen-activated protein kinase pathway in osmotic stress response; Inoue Y et al.; Methylglyoxal is a cytotoxic metabolite derived from dihydroxyacetone phosphate, an intermediate of glycolysis . Detoxification of methylglyoxal is performed by glyoxalase I . Expression of the structural gene of glyoxalase I (GLO1) of Saccharomyces cerevisiae under several stress conditions was investigated using the GLO1-lacZ fusion gene, and expression of the GLO1 gene was found to be specifically induced by osmotic stress . The Hog1p is one of the mitogen-activated protein kinases (MAPKs) in S . cerevisiae, and both Msn2p and Msn4p are the transcriptional regulators that are thought to be under the control of Hog1p-MAPK . Expression of the GLO1 gene under osmotic stress was completely repressed in hog1Delta disruptant and was repressed approximately 80 and 50% in msn2Delta and msn4Delta disruptants, respectively . A double mutant of the MSN2 and MSN4 gene was unable to induce expression of the GLO1 gene under highly osmotic conditions . Glucose consumption increased approximately 30% during the adaptive period in osmotic stress in the wild type strain . On the contrary, it was reduced by 15% in the hog1Delta mutant . When the yeast cell is exposed to highly osmotic conditions, glycerol is synthesized as a compatible solute . Glycerol is synthesized from glucose, and a rate-limiting enzyme in glycerol biosynthesis is glycerol-3-phosphate dehydrogenase (GPD1 gene product), which catalyzes reduction of dihydroxyacetone phosphate to glycerol 3-phosphate . Expression of the GPD1 gene is also under the control of Hog1p-MAPK . Methylglyoxal is also synthesized from dihydroxyacetone phosphate; therefore, induction of the GLO1 gene expression by osmotic stress was thought to scavenge methylglyoxal, which increased during glycerol production for adaptation to osmotic stress.

EMBO J, 1998 Jan 15, 17(2), 396 - 405
Cell wall 1,6-beta-glucan synthesis in Saccharomyces cerevisiae depends on ER glucosidases I and II, and the molecular chaperone BiP/Kar2p; Simons JF et al.; The role of glucose trimming in the endoplasmic reticulum of Saccharomyces cerevisiae was investigated using glucosidase inhibitors and mutant strains devoid of glucosidases I and II . These glucosidases are responsible for removing glucose residues from the N-linked core oligosaccharides attached to newly synthesized polypeptide chains . In mammalian cells they participate together with calnexin, calreticulin and UDP-glucose:glycoprotein glucosyltransferase in the folding and quality control of newly synthesized glycoproteins . In S.cerevisiae, glucosidase II is encoded by the GLS2 gene, and glucosidase I, as suggested here, by the CWH41 gene . Using castanospermine (an alpha-glucosidase inhibitor) and yeast strains defective in glucosidase I, glucosidase II and BiP/Kar2p, it was demonstrated that cell wall synthesis depends on the two glucosidases and BiP/Kar2p . In double mutants with defects in both BiP/Kar2p and either of the glucosidases the phenotype was particularly clear: synthesis of 1,6-beta-glucan_a cell wall component_was reduced; the cell wall displayed abnormal morphology; the cells aggregated; and their growth was severely inhibited . No defects in protein folding or secretion could be detected . We concluded that glucose trimming in S.cerevisiae is necessary for proper cell wall synthesis, and that the glucosidases function synergistically with BiP/Kar2p in this process.

EMBO J, 1998 Jan 2, 17(1), 269 - 77
The N-end rule pathway controls the import of peptides through degradation of a transcriptional repressor; Byrd C et al.; Ubiquitin-dependent proteolytic systems underlie many processes, including the cell cycle, cell differentiation and responses to stress . One such system is the N-end rule pathway, which targets proteins bearing destabilizing N-terminal residues . Here we report that Ubr1p, the main recognition component of this pathway, regulates peptide import in the yeast Saccharomyces cerevisiae through degradation of Cup9p, a 35 kDa homeodomain protein . Cup9p was identified using a screen for mutants that bypass the previously observed requirement for Ubr1p in peptide import . We show that Cup9p is a short-lived protein (t1/2 approximately 5 min) whose degradation requires Ubr1p . Cup9p acts as a repressor of PTR2, a gene encoding the transmembrane peptide transporter . In contrast to engineered N-end rule substrates, which are recognized by Ubr1p through their destabilizing N-terminal residues, Cup9p is targeted by Ubr1p through an internal degradation signal . The Ubr1p-Cup9p-Ptr2p circuit is the first example of a physiological process controlled by the N-end rule pathway . An earlier study identified Cup9p as a protein required for an aspect of resistance to copper toxicity in S.cerevisiae . Thus, one physiological substrate of the N-end rule pathway functions as both a repressor of peptide import and a regulator of copper homeostasis.

Nucleic Acids Res, 1998 Jan 15, 26(2), 521 - 4
Human cytosolic asparaginyl-tRNA synthetase: cDNA sequence, functional expression in Escherichia coli and characterization as human autoantigen; Beaulande M et al.; The cDNA for human cytosolic asparaginyl-tRNA synthetase (hsAsnRSc) has been cloned and sequenced . The 1874 bp cDNA contains an open reading frame encoding 548 amino acids with a predicted M r of 62 938 . The protein sequence has 58 and 53% identity with the homologous enzymes from Brugia malayi and Saccharomyces cerevisiae respectively . The human enzyme was expressed in Escherichia coli as a fusion protein with an N-terminal 4 kDa calmodulin-binding peptide . A bacterial extract containing the fusion protein catalyzed the aminoacylation reaction of S.cerevisiae tRNA with {14C}asparagine at a 20-fold efficiency level above the control value confirming that this cDNA encodes a human AsnRS . The affinity chromatography purified fusion protein efficiently aminoacylated unfractionated calf liver and yeast tRNA but not E.coli tRNA, suggesting that the recombinant protein is the cytosolic AsnRS . Several human anti-synthetase sera were tested for their ability to neutralize hsAsnRSc activity . A human autoimmune serum (anti-KS) neutralized hsAsnRSc activity and this reaction was confirmed by western blot analysis . The human asparaginyl-tRNA synthetase appears to be like the alanyl- and histidyl-tRNA synthetases another example of a human Class II aminoacyl-tRNA synthetase involved in autoimmune reactions.

Nucleic Acids Res, 1998 Jan 15, 26(2), 477 - 85
The second subunit of DNA polymerase III (delta) is encoded by the HYS2 gene in Saccharomyces cerevisiae; Hashimoto K et al.; DNA polymerase III (delta) of Saccharomyces cerevisiae is purified as a complex of at least two polypeptides with molecular masses of 125 and 55 kDa as judged by SDS-PAGE . In this paper we determine partial amino acid sequences of the 125 and 55 kDa polypeptides and find that they match parts of the amino acid sequences predicted from the nucleotide sequence of the CDC2 and HYS2 genes respectively . We also show by Western blotting that Hys2 protein co-purifies with DNA polymerase III activity as well as Cdc2 polypeptide . The complex form of DNA polymerase III activity could not be detected in thermosensitive hys2 mutant cell extracts, although another form of DNA polymerase III was found . This form of DNA polymerase III, which could also be detected in wild-type extracts, was not associated with Hys2 protein and was not stimulated by addition of proliferating cell nuclear antigen (PCNA), replication factor A (RF-A) or replication factor C (RF-C) . The temperature-sensitive growth phenotype of hys2-1 and hys2-2 mutations could be suppressed by the CDC2 gene on a multicopy plasmid . These data suggest that the 55 kDa polypeptide encoded by the HYS2 gene is one of the subunits of DNA polymerase III complex in S.cerevisiae and is required for highly processive DNA synthesis catalyzed by DNA polymerase III in the presence of PCNA, RF-A and RF-C.

J Cell Sci, 1998 Jan, 111 ( Pt 1), 11 - 21
RagA is a functional homologue of S . cerevisiae Gtr1p involved in the Ran/Gsp1-GTPase pathway; Hirose E et al.; Human RagA and RagB is reported to be 52% identical to a putative GTPase of Saccharomyces cerevisiae, Gtr1p . According to the reported nucleotide sequence, we amplified human RagA and RagBs cDNAs from the human B cell cDNA library with PCR . Both cDNAs rescued a cold sensitivity of S . cerevisiae, gtr1-11 . Furthermore, we introduced into the cloned human RagA cDNA, the mutation 'T21L' corresponding to the gtr1-11 mutation which has been reported to suppress not only all of rcc1-, temperature-sensitive mutants of Ran/Gsp1p GTPase GDP/GTP-exchanging factor, but also rna1-1, a temperature-sensitive mutant of Ran/Gsp1p GTPase-activating protein . The resulting RagAgtr1-11 cDNA partially, but significantly, suppressed both rcc1- and rna1-1 mutations . These results indicated that RagA and RagBs are functional homologues of S . cervisiae Gtr1p . Interestingly, while wild-type human RagA and RagBs were localized within the cytoplasm, similar to S . cerevisiae Gtr1p, the mutated human RagAgtr1-11 corresponding to a dominant negative form of RagA was distributed in discrete speckles in the nucleus, being localized side by side with SC-35, a non-snRNP of the splicing complex . In contrast, a dominant positive form of RagA, Q66L was localized in the cytoplasm . Thus, RagA was suggested to shuttle between the cytoplasm and the nucleus, depending on the bound nucleotide state.

Biochem Biophys Res Commun, 1998 Feb 4, 243(1), 205 - 9
TAT1 encodes a low-affinity histidine transporter in Saccharomyces cerevisiae; Bajmoczi M et al.; Previous studies have revealed the presence of at least two histidine uptake systems in S . cerevisiae; one with high affinity and the other with low affinity for histidine . The HIP1 gene is known to encode the high affinity permease . The purpose of this study was to identify the gene that encodes the low affinity permease . A mutant strain of S . cerevisiae that is both a histidine auxotroph and a hip1 deletion mutant is unable to grow on low histidine media . This strain was transformed with a yeast cDNA library constructed in a yeast expression vector . Transformants with increased histidine transport were selected by their ability to grow on a low histidine media . Sequencing of the inserts revealed the presence of the HIP1 gene and also the presence of the TAT1 gene . Estimated Km and Vmax values for histidine transport by each system were determined . In a hip1 tat1 double mutant, the level of histidine required for growth increased eight-fold in comparison to the hip1 single mutant . Our results suggest that the TAT1-encoded protein, previously characterized as the high-affinity tyrosine permease, also acts as the low affinity histidine permease.

EMBO J, 1997 Dec 15, 16(24), 7326 - 41
Overexpression of Pex15p, a phosphorylated peroxisomal integral membrane protein required for peroxisome assembly in S.cerevisiae, causes proliferation of the endoplasmic reticulum membrane; Elgersma Y et al.; We have cloned PEX15 which is required for peroxisome biogenesis in Saccharomyces cerevisiae . pex15Delta cells are characterized by the cytosolic accumulation of peroxisomal matrix proteins containing a PTS1 or PTS2 import signal, whereas peroxisomal membrane proteins are present in peroxisomal remnants . PEX15 encodes a phosphorylated, integral peroxisomal membrane protein (Pex15p) . Using multiple in vivo methods to determine the topology, Pex15p was found to be a tail-anchored type II (Ncyt-Clumen) peroxisomal membrane protein with a single transmembrane domain near its carboxy-terminus . Overexpression of Pex15p resulted in impaired peroxisome assembly, and caused profound proliferation of the endoplasmic reticulum (ER) membrane . The lumenal carboxy-terminal tail of Pex15p protrudes into the lumen of these ER membranes, as demonstrated by its O-glycosylation . Accumulation in the ER was also observed at an endogenous expression level when Pex15p was fused to the N-terminus of mature invertase . This resulted in core N-glycosylation of the hybrid protein . The lumenal C-terminal tail of Pex15p is essential for targeting to the peroxisomal membrane . Furthermore, the peroxisomal membrane targeting signal of Pex15p overlaps with an ER targeting signal on this protein . These results indicate that Pex15p may be targeted to peroxisomes via the ER, or to both organelles.

Nucleic Acids Res, 1998 Jan 1, 26(1), 73 - 9
SGD: Saccharomyces Genome Database; Cherry JM et al.; The Saccharomyces Genome Database (SGD) provides Internet access to the complete Saccharomyces cerevisiae genomic sequence, its genes and their products, the phenotypes of its mutants, and the literature supporting these data . The amount of information and the number of features provided by SGD have increased greatly following the release of the S.cerevisiae genomic sequence, which is currently the only complete sequence of a eukaryotic genome . SGD aids researchers by providing not only basic information, but also tools such as sequence similarity searching that lead to detailed information about features of the genome and relationships between genes . SGD presents information using a variety of user-friendly, dynamically created graphical displays illustrating physical, genetic and sequence feature maps . SGD can be accessed via the World Wide Web at http://genome-www.stanford.edu/Saccharomyces/

Biochim Biophys Acta, 1998 Jan 8, 1379(1), 118 - 28
Expression of high-affinity trehalose-H+ symport in Saccharomyces cerevisiae; Stambuk BU et al.; The expression of the high-affinity trehalose-H+ symport was investigated in various Saccharomyces cerevisiae strains and culture conditions . Previous kinetic studies of trehalose transport in yeast have revealed the existence of at least two different uptake mechanisms: a high-affinity trehalose-H+ symport activity repressed by glucose, and a constitutive low-affinity transport activity, a putative facilitated diffusion process . Exogenously added trehalose was not an inducer of the high-affinity transport activity, and a correlation between trehalose and maltose uptake by yeast cells was found . Our results indicate that the maltose-H+ symporters encoded by MAL11, MAL21, and MAL41 are not responsible for the trehalose transport activity . The analysis of both trehalose and maltose transport activities in wild-type and in laboratory strains with defined MAL genes showed that the trehalose-H+ symporter was under control of MAL regulatory genes . Our results also suggest that the recently characterized AGT1 gene of S . cerevisiae may encode the high-affinity trehalose-H+ symporter . During diauxic growth on glucose the transport activity was low during the first exponential phase of growth, increased as glucose was exhausted from the medium, and decreased again as the cells reached the late stationary phase . This pattern was coincident with that of the intracellular levels of trehalose . The strong correlation between these two parameters may be of physiological significance during adaptation of yeast cells to stress conditions.

J Clin Microbiol, 1998 Feb, 36(2), 557 - 62
Epidemiological investigation of vaginal Saccharomyces cerevisiae isolates by a genotypic method; McCullough MJ et al.; Saccharomyces cerevisiae is a ubiquitous, ascomycetous yeast, and vaginitis caused by this organism has been reported only very rarely . The aim of the present investigation was to assess the epidemiological relatedness of a group of vaginal and commercial S . cerevisiae isolates by a previously reported genetic typing method, which divided the isolates into two broad groups with numerous subtypes . Nineteen S . cerevisiae isolates obtained from patients suffering from vaginitis and four isolates from commercial products in the same city were analyzed . The cellular DNA from each isolate was digested with the restriction endonuclease EcoRI, and restriction fragment length polymorphisms were generated by horizontal gel electrophoresis . The results showed that although vaginal isolates did not cluster in any particular genetic subtype, multiple patients were infected with indistinguishable strains (there were nine distinct strains among 23 isolates) . For two of three patients, all three with two episodes of S . cerevisiae vaginitis, different strains were isolated during the recurrence of this disease . Three other patients with indistinguishable isolates were epidemiologically related in that two were practitioners in the same clinic and the third was a patient at this clinic . We also found that one commercial strain was indistinguishable from the strain isolated from three different women at the time that they were suffering from vaginitis . The findings of the present study suggest that some S . cerevisiae strains may possess properties permitting persistence in the human host . Furthermore, person-to-person contact and the proliferation of the use of S . cerevisiae as a health-food product, in home baking, and in home brewing may be a contributing factor in human colonization and infection with this organism.

Appl Environ Microbiol, 1998 Feb, 64(2), 665 - 8
Effects of ethanol and other alkanols on transport of acetic acid in Saccharomyces cerevisiae; Casal M et al.; In glucose-grown cells of Saccharomyces cerevisiae IGC 4072, acetic acid enters only by simple diffusion of the undissociated acid . In these cells, ethanol and other alkanols enhanced the passive influx of labelled acetic acid . The influx of the acid followed first-order kinetics with a rate constant that increased exponentially with the alcohol concentration, and an exponential enhancement constant for each alkanol was estimated . The intracellular concentration of labelled acetic acid was also enhanced by alkanols, and the effect increased exponentially with alcohol concentration . Acetic acid is transported across the plasma membrane of acetic acid-, lactic acid-, and ethanol-grown cells by acetate-proton symports . We found that in these cells ethanol and butanol inhibited the transport of labelled acetic acid in a noncompetitive way; the maximum transport velocity decreased with alcohol concentration, while the affinity of the system for acetate was not significantly affected by the alcohol . Semilog plots of Vmax versus alcohol concentration yielded straight lines with negative slopes from which estimates of the inhibition constant for each alkanol could be obtained . The intracellular concentration of labelled acid was significantly reduced in the presence of ethanol or butanol, and the effect increased with the alcohol concentration . We postulate that the absence of an operational carrier for acetate in glucose-grown cells of S . cerevisiae, combined with the relatively high permeability of the plasma membrane for the undissociated acid and the inability of the organism to metabolize acetic acid, could be one of the reasons why this species exhibits low tolerance to acidic environments containing ethanol.

Appl Environ Microbiol, 1998 Feb, 64(2), 564 - 8
Intracellular signal triggered by cholera toxin in Saccharomyces boulardii and Saccharomyces cerevisiae; Brandao RL et al.; As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT) . The addition of glucose or dinitrophenol to cells of S . boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S.cerevisiae . The addition of CT to the same cells also resulted in trehalase activation . Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation . Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S . boulardii and S . cerevisiae . These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade . The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells . This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells . The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.

Appl Environ Microbiol, 1998 Feb, 64(2), 530 - 4
Measurement of the effects of acetic acid and extracellular pH on intracellular pH of nonfermenting, individual Saccharomyces cerevisiae cells by fluorescence microscopy; Guldfeldt LU et al.; The effects of acetic acid and extracellular pH (pHex) on the intracellular pH (pHi) of nonfermenting, individual Saccharomyces cerevisiae cells were studied by using a new experimental setup comprising a fluorescence microscope and a perfusion system . S . cerevisiae cells grown in brewer's wort to the stationary phase were stained with fluorescein diacetate and transferred to a perfusion chamber . The extracellular concentration of undissociated acetic acid at various pHex values was controlled by perfusion with 2 g of total acetic acid per liter at pHex 3.5, 4.5, 5.6, and 6.5 through the chamber by using a high-precision pump . The pHi of individual S . cerevisiae cells during perfusion was measured by fluorescence microscopy and ratio imaging . Potential artifacts, such as fading and efflux of fluorescein, could be neglected within the experimental time used . At pHex 6.5, the pHi of individual S . cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.035 g/liter, whereas at pHex 3.5, 4.5, and 5.6, the pHi of individual S . cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.10 g/liter . At concentrations of undissociated acetic acid of more than 0.10 g/liter, the pHi remained constant . The decreases in pHi were dependent on the pHex; i.e., the decreases in pHi at pHex 5.6 and 6.5 were significantly smaller than the decreases in pHi at pHex 3.5 and 4.5.

FEBS Lett, 1998 Jan 2, 421(1), 37 - 40
Rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria: the enzyme expressed in Escherichia coli acts as a member of the respiratory chain in the host cells; Kitajima-Ihara T et al.; The NDI1 gene encodes the internal rotenone-insensitive NADH-quinone oxidoreductase localized in the inner mitochondrial membranes of Saccharomyces cerevisiae . The T7 tag-fused mature NDI1 was overexpressed in Escherichia coli . The overexpressed NDI1 was exclusively found in the membrane fraction . The NDI1-overexpressed membranes showed significantly increased activities of NADH oxidase and NADH-ubiquinone-1 (UQ1) reductase when compared with the control membranes . Flavone, which is a specific inhibitor of the S . cerevisiae NDI1, inhibited almost completely NADH oxidase and NADH-UQ1 reductase activities of NDI1-overexpressed membranes but scarcely inhibited these activities of the control membranes . In addition, the NADH oxidase activity of the NDI1-overexpressed membranes was also inhibited by KCN as well as the control membranes . These results indicate that the overexpressed NDI1 worked as a member of the respiratory chain in the host cells, even though E . coli membranes are different from S . cerevisiae inner mitochondrial membranes in terms of quinones and lipid composition.

Biochemistry (Mosc), 1997 Oct, 62(10), 1146 - 51
Detection and some properties of membrane-bound and soluble polyphosphatases in mitochondria of the yeast Saccharomyces cerevisiae; Lichko LP et al.; Saccharomyces cerevisiae mitochondria possess polyphosphatases that are tightly bound to the membranes and differ from soluble polyphosphatase of these organelles in a number of properties . Molecular weights of the membrane-bound polyphosphatases are 120 and 76 kD, and the molecular weight of the soluble polyphosphatase is about 36 kD . All three enzymes are evidently monomers, since antibodies against purified cell-envelope polyphosphatase of S . cerevisiae reacted with 115, 78, and 37 kD polypeptides in immunoblotting . The activities of membrane-bound and soluble polyphosphatase are maximal at neutral pH . The soluble polyphosphatase activity is stimulated by divalent cations, unlike the membrane-bound enzymes which are inhibited by the same cations including Mg2+ . Monovalent cations do not affect the activity of the soluble enzyme but stimulate polyphosphatases in the membrane preparation . The specific activities for hydrolysis of polyphosphates with average chain lengths of 9 to 188 phosphate residues are enhanced by increasing the degree of substrate polymerization in the case of the membrane preparation and are unchanged in case of the soluble enzyme . Affinity of the soluble enzyme to polyphosphates is 5-10 times higher than that of the membrane-bound polyphosphatases . In the soluble fraction of mitochondria, high tripolyphosphatase activity is detected which is approximately 80% of that in isolated mitochondria.

Gene, 1998 Jan 5, 206(1), 107 - 16
Molecular cloning of YlPMR1, a S . cerevisiae PMR1 homologue encoding a novel P-type secretory pathway Ca2+ -ATPase, in the yeast Yarrowia lipolytica; Park CS et al.; A novel P-type ATPase gene, Saccharomyces cerevisiae PMR1 homologue (YlPMR1), has been cloned and sequenced in the yeast, Yarrowia lipolytica . The putative gene product has 928 amino acids with a calculated molecular mass of 100050 Da and a pI of 5.15 . The deduced amino-acid sequence analysis demonstrated that the cloned gene product contains all 10 of the conserved regions in P-type ATPases and exhibits 55% amino-acid identity to the S . cerevisiae PMR1 gene product; however, it shows a relatively lower homology to PMCA (24%) and SERCA (33%), confirming the presence of a third class of Ca2+-ATPase (secretory pathway Ca2+-ATPase, SPCA) . The YlPMR1-disrupted strain shows defective growth in low Ca2+ or EGTA-containing medium . In fact, a longer lag time (60 h) was observed in YlPMR1-defective mutant cells during cultivation in EGTA-containing YPD medium . These growth defects were overcome by adding Ca2+ and Mn2+ into the medium . Interestingly, whereas Mn2+ inhibits growth of the control strain, it significantly improves the growth of YlPMR1-disrupted cells . These results suggest an involvement of the YlPMR1 gene product in Ca2+ and Mn2+ ion homeostasis in Y . lipolytica.

J Biol Chem, 1997 Dec 12, 272(50), 31382 - 8
The carboxyl terminus of the Saccharomyces cerevisiae succinate dehydrogenase membrane subunit, SDH4p, is necessary for ubiquinone reduction and enzyme stability; Oyedotun KS et al.; The succinate dehydrogenase (SDH) of Saccharomyces cerevisiae is composed of four nonidentical subunits encoded by the nuclear genes SDH1, SDH2, SDH3, and SDH4 . The hydrophilic subunits, SDH1p and SDH2p, comprise the catalytic domain involved in succinate oxidation . They are anchored to the inner mitochondrial membrane by two small, hydrophobic subunits, SDH3p and SDH4p, which are required for electron transfer and ubiquinone reduction . Comparison of the deduced primary sequence of the yeast SDH4p subunit to SDH4p subunits from other species reveals the presence of an unusual 25-30 amino acid carboxyl-terminal extension following the last predicted transmembrane domain . The extension is predicted to be on the cytoplasmic side of the inner mitochondrial membrane . To investigate the extension's function, three truncations were created and characterized . The results reveal that the carboxyl-terminal extension is necessary for respiration and growth on nonfermentable carbon sources, for ubiquinone reduction, and for enzyme stability . Combined with inhibitor studies using a ubiquinone analog, our results suggest that the extension and more specifically, residues 128-135 are involved in the formation of a ubiquinone binding site . Our findings support a two-ubiquinone binding site model for the S . cerevisiae SDH.

Genes Dev, 1997 Dec 1, 11(23), 3218 - 31
A novel cytosolic regulator, Pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium; Chen MY et al.; Genetic analysis was applied to identify novel genes involved in G protein-linked pathways controlling development . Using restriction enzyme-mediated integration (REMI), we have identified a new gene, Pianissimo (PiaA), involved in cAMP signaling in Dictyostelium discoideum . PiaA encodes a 130-kD cytosolic protein required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase . In piaA- null mutants, neither chemoattractant stimulation of intact cells nor GTPgammaS treatment of lysates activates the enzyme; constitutive expression of PiaA reverses these defects . Cytosols of wild-type cells that contain Pia protein reconstitute the GTPgammaS stimulation of adenylyl cyclase activity in piaA- lysates, indicating that Pia is directly involved in the activation . Pia and CRAC, a previously identified cytosolic regulator, are both essential for activation of the enzyme as lysates of crac- piaA- double mutants require both proteins for reconstitution . Homologs of PiaA are found in Saccharomyces cerevisiae and Schizosaccaromyces pombe; disruption of the S . cerevisiae homolog results in lethality . We propose that homologs of Pia and similar modes of regulation of these ubiquitous G protein-linked pathways are likely to exist in higher eukaryotes.

Proc Natl Acad Sci U S A, 1997 Dec 23, 94(26), 14361 - 6
The anaphase inhibitor of Saccharomyces cerevisiae Pds1p is a target of the DNA damage checkpoint pathway; Cohen-Fix O et al.; Inhibition of DNA replication and physical DNA damage induce checkpoint responses that arrest cell cycle progression at two different stages . In Saccharomyces cerevisiae, the execution of both checkpoint responses requires the Mec1 and Rad53 proteins . This observation led to the suggestion that these checkpoint responses are mediated through a common signal transduction pathway . However, because the checkpoint-induced arrests occur at different cell cycle stages, the downstream effectors mediating these arrests are likely to be distinct . We have previously shown that the S . cerevisiae protein Pds1p is an anaphase inhibitor and is essential for cell cycle arrest in mitosis in the presence DNA damage . Herein we show that DNA damage, but not inhibition of DNA replication, induces the phosphorylation of Pds1p . Analyses of Pds1p phosphorylation in different checkpoint mutants reveal that in the presence of DNA damage, Pds1p is phosphorylated in a Mec1p- and Rad9p-dependent but Rad53p-independent manner . Our data place Pds1p and Rad53p on parallel branches of the DNA damage checkpoint pathway . We suggest that Pds1p is a downstream target of the DNA damage checkpoint pathway and that it is involved in implementing the DNA damage checkpoint arrest specifically in mitosis.

Curr Genet, 1997 Nov, 32(5), 309 - 14
Arabidopsis thaliana RAD6 homolog AtUBC2 complements UV sensitivity, but not N-end rule degradation deficiency, of Saccharomyces cerevisiae rad6 mutants; Zwirn P et al.; AtUBC2 of Arabidopsis thaliana encodes a structural homolog of the RAD6 gene of Saccharomyces cerevisiae with approximately 65% identical amino acids . Like structural homologs from other organisms, AtUBC2 lacks the carboxyl-terminal extension of mostly acidic amino acids which is present in Rad6p . AtUBC2 was expressed in S . cerevisiae rad6 mutants . It was found to partially complement the UV sensitivity and reduced growth rate of rad6 mutants at elevated temperatures . AtUBC2 however, has no apparent influence on the degradation of N-end rule substrates in the heterologous host.

FEMS Microbiol Rev, 1997 Nov, 21(3), 231 - 41
Osmoregulation and glycerol metabolism in the yeast Saccharomyces cerevisiae; Nevoigt E et al.; Glycerol is the main compatible solute in Saccharomyces cerevisiae . It is accumulated intracellularly when cells are exposed to decreased extracellular water activity . In general, increased intracellular accumulation of a solute may be caused by enhanced production, restricted dissimilation, increased retention by the plasma membrane and increased uptake from the medium . In this review, we evaluate current knowledge concerning mechanisms leading to the accumulation of glycerol in osmotically stressed cells of S . cerevisiae at the molecular and metabolic levels . An overview of glycerol metabolism in S . cerevisiae is provided.

Cell Mol Biol (Noisy-le-grand), 1997 Nov, 43(7), 1007 - 18
Cloning, characterization and identification of the gene encoding phosphatidylinositol 4-kinase; Pramanik A et al.; The vast majority of AIDS-related deaths are associated with opportunistic infections . For fungal infections, there are few effective antifungals, particularly for systemic use . The discovery that very low doses of the bleomycin family of anticancer chemical congeners compromise the integrity of fungal cell walls led to our approach to identify genes that complement-cell wall defects, and develop methods to facilitate the identification of new antifungals targeted to fungal cell walls . This report describes one of the genes cloned by complementation of the blm1-1 mutation of S . cerevisiae using a YCp50-based yeast genomic library . Characterization and identification of the gene were carried out using drug screening tests, Southern hybridization analyses, DNA sequencing and DNA sequence similarity searches in databases . The gene STT4, is essential for viability and encodes a phosphatidylinositol 4-kinase that plays an important role in the phosphatidylinositol-mediated signal transduction pathway required for cell wall integrity . Like blm1-1 mutant strains, stt4 cells arrest mostly in the G2/M phase of the cell cycle . Further studies using this approach should help us understand the role of PI4-K in maintaining fungal cell-wall integrity, identify additional genes affecting potential target structures in cell walls of opportunistic fungal pathogens in AIDS patients, and assist in drug discovery and antifungal drug design.

Antonie Van Leeuwenhoek, 1997 Nov, 72(4), 283 - 90
Modulation of sporulation and metabolic fluxes in Saccharomyces cerevisiae by 2 deoxy glucose; Aon JC et al.; Quantitative studies of metabolic fluxes during Saccharomyces cerevisiae sporulation on acetate in the presence of the glucose analog, 2-deoxy glucose (2dG) are reported . We have studied the inhibition of sporulation and associated catabolic or anabolic fluxes by 2dG . Sporulation frequencies decreased from 50% to 2% asci per cell at 2dG concentrations in the range of 0.03 to 0.30 g l-1, respectively . Under the same conditions, the acetate consumption flux was inhibited up to 60% and the glyoxylate cycle and gluconeogenic fluxes decreased from 0.7 and 0.3 mmol h-1 g-1 dw, respectively, to negligible values . We observed a linear correlation of the acetate consumption rate with the sporulation frequency by varying the 2dG concentration . The linear correlation was also verified between the frequency of sporulation and the fluxes through glyoxylate cycle and gluconeogenic pathways . In addition, the same association of inhibition of sporulation and metabolic fluxes was found in other S . cerevisiae strains displaying different potentials of sporulation . The results presented suggest that inhibition of sporulation in the presence of the glucose analog may be attributed, at least in part, to the inhibition of anabolic fluxes and might be associated with catabolite repression.

J Biol Chem, 1998 Jan 23, 273(4), 2402 - 8
Cardiolipin synthase is associated with a large complex in yeast mitochondria; Zhao M et al.; The phospholipid cardiolipin (CL) is ubiquitous in eucaryotes and is unique in structure, subcellular localization, and potential function . Previous studies have shown that CL is associated with major respiratory complexes in the mitochondrial membrane . To determine whether CL biosynthesis requires the presence of intact respiratory complexes, we measured activity of CL synthase, which catalyzes the synthesis of CL from cytidine diphosphate diacylglycerol and phosphatidylglycerol, in Saccharomyces cerevisiae strains with genetic defects in the oxidative phosphorylation system . Assembly mutants of cytochrome oxidase had significantly reduced CL synthase activity, while assembly mutants of respiratory complex III and the F0F1-ATPase were less inhibited . To obtain further information on the activity of CL synthase, we purified the enzyme and compared the size of the catalytic protein with the functional molecular mass . The enzyme was solubilized by Triton X-100 from KSCN-extracted mitochondrial membranes of S . cerevisiae . The functional molecular mass of Triton-solubilized CL synthase, determined by radiation inactivation, was 150-240 kDa, indicating that the functional enzyme was a large complex . After partial purification, the enzyme eluted from a Superose 12 gel filtration column with an apparent molecular mass of 70 kDa . CL synthase was further purified by hydroxylapatite and cytidine diphosphate diacylglycerol affinity chromatographies, Mono Q anion exchange FPLC, and preparative gel electrophoresis . These steps led to identification of a 28-kDa protein, which had catalytic activity when eluted from an SDS-polyacrylamide gel . This 28-kDa protein also reacted with an antiserum that inactivated the enzyme . We conclude that yeast CL synthase is a 28-kDa protein, which forms an oligomeric complex whose biogenesis and/or activity is influenced by the assembly of cyto-chrome oxidase.

J Biol Chem, 1998 Jan 16, 273(3), 1677 - 83
Deoxyhypusine synthase activity is essential for cell viability in the yeast Saccharomyces cerevisiae; Park MH et al.; Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein . The null mutation in the single copy gene, yDHS, encoding deoxyhypusine synthase results in the loss of viability in the yeast Saccharomyces cerevisiae . Upon depletion of deoxyhypusine synthase, and consequently of eIF-5A, cessation of growth was accompanied by a marked enlargement of cells, suggesting a defect in cell cycle progression or in cell division . Two residues of the yeast enzyme, Lys308 and Lys350, corresponding to Lys287 and Lys329, respectively, known to be critical for the activity of the human enzyme, were targeted for site-directed mutagenesis . The chromosomal ydhs null mutation was complemented by the plasmid-borne yDHS wild-type gene, but not by mutated genes encoding inactive proteins, including that with Lys350-->Arg substitution or with substitutions at both Lys308 and Lys350 . The mutated gene ydhs (K308R) encoding a protein with diminished activities (< 1% of wild type) could support growth but only to a very limited extent . These findings provide strong evidence that the hypusine modification is indeed essential for the survival of S . cerevisiae and imply a vital function for eIF-5A in all eukaryotes.

J Biol Chem, 1998 Jan 16, 273(3), 1506 - 10
Edg-2/Vzg-1 couples to the yeast pheromone response pathway selectively in response to lysophosphatidic acid; Erickson JR et al.; We have functionally expressed the human cDNA encoding the putative lysophosphatidic acid (LPA) receptor Edg-2 (Vzg-1) in Saccharomyces cerevisiae in an attempt to determine the agonist specificity of this G-protein-coupled receptor . LPA activated the pheromone response pathway in S . cerevisiae expressing Edg-2 in a time- and dose-dependent manner as determined by induction of a pheromone-responsive FUS1::lacZ reporter gene . LPA-mediated activation of the pheromone response pathway was dependent on mutational inactivation of the SST2 gene, the GTPase-activating protein for the yeast G alpha protein (the GPA1 gene product) . This indicates that, in sst2 delta yeast cells, Edg-2 can efficiently couple to the yeast heterotrimeric G-protein in response to LPA and activate the yeast mitogen-activated protein kinase pathway . The Edg-2 receptor showed a high degree of specificity for LPA; other lyso-glycerophospholipids, sphingosine 1-phosphate, and diacyl-glycerophospholipids did not activate FUS1::lacZ . LPA analogs including a cyclic phosphoester form and ether-linked forms of LPA activated FUS1::lacZ, although fatty acid chains of 6 and 10 carbons did not activate FUS1::lacZ, suggesting a role for the side chain in ligand binding or receptor activation . These results indicate that Edg-2 encodes a highly specific LPA receptor.

Chemosphere, 1997 Nov, 35(10), 2277 - 83
Comparative heavy metal biosorption study of brewery yeast and Myxococcus xanthus biomass; Ben Omar N et al.; The biosorption for La2+, Co2+, Mn2+, UO2(2+), Pb2+, Ag+, Zn2+, Cd2+ and Cr2+ by wet and dry biomass form Myxococcus xanthus obtained from laboratory cultures and Saccharomyces cerevisiae from the brewing industry has been studied . M . xanthus biomass was found to be the most efficient biosorbent for all of the metals assayed . However, due to the fact that S . cerevisiae is a low cost residual by-product from the brewing industry, and at the same time yields good levels of biosorption, it is considered in this work to be of great interest for use as a detoxifier of heavy metals contaminated waters . In addition, the use of sodium carbonate as a desorbent agent is discussed where it was possible to recover up to 94,53% of UO2(2+) by both M . xanthus and S . cerevisiae biomass.

Yeast, 1997 Dec, 13(15), 1409 - 21
Saccharomyces carlsbergensis contains two functional genes encoding the acyl-CoA binding protein, one similar to the ACB1 gene from S . cerevisiae and one identical to the ACB1 gene from S . monacensis; Borsting C et al.; Saccharomyces carlsbergensis is an amphiploid, and it has previously been suggested that the genomes of S . carlsbergensis originate from S . cerevisiae and S . monacensis . We have cloned the ACB1 genes encoding the acyl-CoA binding protein (ACBP) from S . carlsbergensis, S . cerevisiae and S . monacensis . Two genes were found in S . carlsbergensis and named ACB1 type 1 and type 2, respectively . The type 1 gene is identical to the S . cerevisiae ACB1 gene except for three substitutions, one single base pair deletion and one double base pair insertion, all located in the promoter region . The type 2 gene is completely identical to the S . monacensis ACB1 gene . These findings substantiate the notion that S . carlsbergensis is a hybrid between S . cerevisiae and S . monacensis . Both ACB1 type 1 and type 2 are actively transcribed in S . carlsbergensis and transcription is initiated at sites identical to those used for transcriptional initiation of the ACB1 genes in S . cerevisiae and S . monacensis, respectively . Two polyadenylation sites, spaced 225 bp apart, are present in the S . cerevisiae ACB1 gene . The upstream polyadenylation site is used exclusively during exponential growth, whereas both sites are utilized during later stages of growth.

Gene, 1997 Dec 19, 204(1-2), 251 - 8
The Dictyostelium discoideum beta-1,4-mannosyltransferase gene, mntA, has two periods of developmental expression; Lee SK et al.; The precise roles of protein glycosylation in multicellular development are poorly understood . We have characterized the mntA gene from Dictyostelium discoideum which encodes the beta-1,4-mannosyltransferase enzyme that catalyzes the reaction: GDP-Man + dolichol-PP-GlcNAc2 --> dolichol-PP-GlcNAc2-Man + GDP . This gene has a central role in the synthesis of the lipid-linked oligosaccharide precursor which becomes the core of all asparagine-linked (N-linked) glycans . The mntA gene contains a single small intron and encodes a 493 aa protein with a predicted molecular size of 56 kDa . It is located 5' to the repE gene on chromosome IV and is transcribed in the opposite orientation to repE with which it shares a 585 bp of upstream intergenic region . The predicted mntA gene product shares 38% homology with the S . cerevisiae ALG1 gene product . The MntA protein has a region homologous to the putative dolichol-binding region in the yeast ALG1 protein, but it is located in a different part of the molecule . Northern analysis revealed that the expression of the mntA gene is regulated during multicellular development with two periods of mRNA accumulation . The mntA gene product has a classical endoplasmic reticulum retention motif, and is the first Dictyostelium gene encoding a protein that is active in this organelle . The identification of this gene will allow expanded studies of the role of N-linked glycans in multicellular development.

Prog Nucleic Acid Res Mol Biol, 1998, 59, 95 - 133
On the physiological role of casein kinase II in Saccharomyces cerevisiae; Glover CV 3rd; Casein kinase II (CKII) is a highly conserved serine/threonine protein kinase that is ubiquitous in eukaryotic organisms . This review summarizes available data on CKII of the budding yeast Saccharomyces cerevisiae, with a view toward defining the possible physiological role of the enzyme . Saccharomyces cerevisiae CKII is composed of two catalytic and two regulatory subunits encoded by the CKA1, CKA2, CKB1, and CKB2 genes, respectively . Analysis of null and conditional alleles of these genes identifies a requirement for CKII in at least four biological processes: flocculation (which may reflect an effect on gene expression), cell cycle progression, cell polarity, and ion homeostasis . Consistent with this, isolation of multicopy suppressors of conditional cka mutations has identified three genes that have a known or potential role in either the cell cycle or cell polarity: CDC37, which is required for cell cycle progression in both G1 and G2/M; ZDS1 and 2, which appear to have a function in cell polarity; and SUN2, which encodes a protein of the regulatory component of the 26S protease . The identity and properties of known CKII substrates in S . cerevisiae are also reviewed, and advantage is taken of the complete genomic sequence to predict globally the substrates of CKII in this organism . Although the combined data do not yield a definitive picture of the physiological role of CKII, it is proposed that CKII serves a signal transduction function in sensing and/or communicating information about the ionic status of the cell to the cell cycle machinery.

Gene, 1997 Dec 5, 203(1), 75 - 84
Isolation and cloning of the Yarrowia lipolytica SEC65 gene, a component of the yeast signal recognition particle displaying homology with the human SRP19 gene; Sanchez M et al.; The signal recognition particle (SRP) is a ribonucleoprotein composed of a 7SL RNA and six polypeptides . Here we report the results of a series of experiments carried out to define the function of the Yarrowia lipolytica homologue of the 19 kDa subunit of mammalian SRP . The YlSEC65 gene product is a 310 amino acid protein . Coimmuneprecipitation of Sec65p and 7SL RNA in Y . lipolytica revealed that these components are stable associated in a complex . Deletion of the YlSEC65 gene is lethal, in contrast with the results described for the Saccharomyces cerevisiae SEC65 gene, which is not essential for cell growth and whose deletion results in slowly growing strains . Using site-directed mutagenesis we demonstrate that the two arginine residues of the EGRR motif conserved in all SRP19 homologues are essential for SRP activity . By random mutagenesis of YlSEC65, we have isolated a temperature-sensitive mutant and shown that it was affected in protein secretion at the non-permissive temperature . We also show that the YlSEC65 gene is able to functionally complement the temperature-sensitive growth of S . cerevisiae sec65 mutants . Our results suggest that SRP-dependent targeting may be the main secretory pathway in Y lipolytica, as has been described for higher eukaryotes.

Cell Biochem Funct, 1997 Dec, 15(4), 265 - 9
The effects of selenium, vitamin E and their combination on the composition of fatty acids and proteins in Saccharomyces cerevisiae; Dilsiz N et al.; The aim of our studies was to test the effect and role of vitamin E and selenium supplements on yeast cell . In this study, the effects of selenium (Se), vitamin E (Vit . E), and their combination (Se plus Vit . E) on the composition of fatty acids and proteins were examined in Saccharomyces cerevisiae strains WET136 and 522 . S . cerevisiae cells were grown up in YEPD medium supplemented with Se, Vit . E or their combination . It was found that the level of stearic acid was increased in all supplemented groups (p < 0.05; p < 0.001) . The content of saturated and unsaturated fatty acids was decreased (p < 0.05; p < 0.01; p < 0.001) in Vit . E and Vit . E plus Se supplemented S . cerevisiae . On the other hand, Se alone caused an increase (p < 0.001) in the saturated fatty acids but a decrease (p < 0.05; p < 0.001) in the unsaturated fatty acids . Total proteins in S . cerevisiae were significantly increased (p < 0.001) by Vit . E supplement . There was no significant change observed in S . cerevisiae supplemented with Se . These findings indicate that membrane composition of S . cerevisiae is affected by both Vit . E and Se supplements.

Mol Cell Biol, 1998 Jan, 18(1), 353 - 60
Yeast pre-mRNA splicing requires a pair of U1 snRNP-associated tetratricopeptide repeat proteins; McLean MR et al.; The U1 snRNP functions to nucleate spliceosome assembly on newly transcribed pre-mRNA . Saccharomyces cerevisiae is unusual among eukaryotes in the greatly extended length of its U1 snRNA and the apparent increased polypeptide complexity of the corresponding U1 snRNP . In this paper, we report the identification of a novel U1 snRNP protein, Prp42p, with unexpected properties . Prp42p was identified by its surprising structural similarity to the essential U1 snRNP protein, Prp39p . Both Prp39p and Prp42p possess multiple copies of a variant tetratricopeptide repeat, an element implicated in a wide range of protein assembly events . Yeast strains depleted of Prp42p by transcriptional repression of a GAL1::PRP42 fusion gene arrest for splicing prior to pre-mRNA 5' splice site cleavage . Prp42p was not observed in a recent biochemical analysis of purified U1 snRNPs from S . cerevisiae (28) . Nevertheless, antibodies directed against an epitope-tagged version of Prp42p specifically precipitate U1 snRNA from yeast extracts . Furthermore, Prp42p is required for U1 snRNP biogenesis, because yeast strains depleted of Prp42p formed incomplete U1 snRNPs that failed to produce stable complexes with pre-mRNA in vitro . The evidence shows that Prp39p and Prp42p are both required to configure the atypical yeast U1 snRNP into a structure compatible with its evolutionarily conserved role in pre-mRNA splicing.

Genetics, 1997 Dec, 147(4), 1643 - 52
A MADS-box homologue in Ustilago maydis regulates the expression of pheromone-inducible genes but is nonessential; Kruger J et al.; Mating and pathogenic development in the smut fungus Ustilago maydis are controlled by a pheromone/receptor system and two homeodomain proteins, bEp and bWp, which form heterodimers in nonallelic combinations . We describe the isolation of a gene, umc1, encoding a MADS-box protein, which displays significant similarity to the Saccharomyces cerevisiae MCM1 gene . umc1 complemented the viability defect of yeast mcm1 mutants . In U . maydis, umc1 deletion mutants were viable and pathogenic development was unaffected . Nevertheless, the basal expression levels of several pheromone-inducible genes were significantly reduced leading to an attenuated mating reaction . In contrast to S . cerevisiae, where Mcm1p plays a crucial role in the cell-type specific expression of a- and alpha-specific genes, the U . maydis umc1 gene appears to have only a modulatory effect on the expression of mating type-specific genes.

Microbiol Mol Biol Rev, 1997 Dec, 61(4), 503 - 32
Metabolism of sulfur amino acids in Saccharomyces cerevisiae; Thomas D et al.; Sulfur amino acid biosynthesis in Saccharomyces cerevisiae involves a large number of enzymes required for the de novo biosynthesis of methionine and cysteine and the recycling of organic sulfur metabolites . This review summarizes the details of these processes and analyzes the molecular data which have been acquired in this metabolic area . Sulfur biochemistry appears not to be unique through terrestrial life, and S . cerevisiae is one of the species of sulfate-assimilatory organisms possessing a larger set of enzymes for sulfur metabolism . The review also deals with several enzyme deficiencies that lead to a nutritional requirement for organic sulfur, although they do not correspond to defects within the biosynthetic pathway . In S . cerevisiae, the sulfur amino acid biosynthetic pathway is tightly controlled: in response to an increase in the amount of intracellular S-adenosylmethionine (AdoMet), transcription of the coregulated genes is turned off . The second part of the review is devoted to the molecular mechanisms underlying this regulation . The coordinated response to AdoMet requires two cis-acting promoter elements . One centers on the sequence TCACGTG, which also constitutes a component of all S . cerevisiae centromeres . Situated upstream of the sulfur genes, this element is the binding site of a transcription activation complex consisting of a basic helix-loop-helix factor, Cbf1p, and two basic leucine zipper factors, Met4p and Met28p . Molecular studies have unraveled the specific functions for each subunit of the Cbf1p-Met4p-Met28p complex as well as the modalities of its assembly on the DNA . The Cbf1p-Met4p-Met28p complex contains only one transcription activation module, the Met4p subunit . Detailed mutational analysis of Met4p has elucidated its functional organization . In addition to its activation and bZIP domains, Met4p contains two regulatory domains, called the inhibitory region and the auxiliary domain . When the level of intracellular AdoMet increases, the transcription activation function of Met4 is prevented by Met30p, which binds to the Met4 inhibitory region . In addition to the Cbf1p-Met4p-Met28p complex, transcriptional regulation involves two zinc finger-containing proteins, Met31p and Met32p . The AdoMet-mediated control of the sulfur amino acid pathway illustrates the molecular strategies used by eucaryotic cells to couple gene expression to metabolic changes.

J Biol Chem, 1997 Nov 28, 272(48), 30196 - 200
Sphingolipids are potential heat stress signals in Saccharomyces; Dickson RC et al.; The ability of organisms to quickly respond to stresses requires the activation of many intracellular signal transduction pathways . The sphingolipid intermediate ceramide is thought to be particularly important for activating and coordinating signaling pathways during mammalian stress responses . Here we present the first evidence that ceramide and other sphingolipid intermediates are signaling molecules in the Saccharomyces cerevisiae heat stress response . Our data show a 2-3-fold transient increase in the concentration of C18-dihydrosphingosine and C18-phytosphingosine, more than a 100-fold transient increase in C20-dihydrosphingosine and C20-phytosphingosine, and a more stable 2-fold increase in ceramide containing C18-phytosphingosine and a 5-fold increase in ceramide containing C20-phytosphingosine following heat stress . Treatment of cells with dihydrosphingosine activates transcription of the TPS2 gene encoding a subunit of trehalose synthase and causes trehalose, a known thermoprotectant, to accumulate . Dihydrosphingosine induces expression of a STRE-LacZ reporter gene, showing that the global stress response element, STRE, found in many yeast promoter sequences can be activated by sphingolipid signals . The TPS2 promoter contains four STREs that may mediate dihydrosphingosine responsiveness . Using genetic and other approaches it should be possible to identify sphingolipid signaling pathways in S . cerevisiae and quantify the importance of each during heat stress.

J Biol Chem, 1997 Nov 21, 272(47), 29620 - 5
Synthesis of mannose-(inositol-P)2-ceramide, the major sphingolipid in Saccharomyces cerevisiae, requires the IPT1 (YDR072c) gene; Dickson RC et al.; Knowledge of the Saccharomyces cerevisiae genes and proteins necessary for sphingolipid biosynthesis is far from complete . Such information should expedite studies of pathway regulation and sphingolipid functions . Using the Aur1 protein sequence, recently identified as necessary for synthesis of the sphingolipid inositol-P-ceramide (IPC), we show that a homolog (open reading frame YDR072c), termed Ipt1 (inositolphosphotransferase 1) is necessary for synthesis of mannose-(inositol-P)2-ceramide (M(IP)2C), the most abundant and complex sphingolipid in S . cerevisiae . This conclusion is based upon analysis of an ipt1-deletion strain, which fails to accumulate M(IP)2C and instead accumulates increased amounts of the precursor mannose-inositol-P-ceramide . The mutant also fails to incorporate radioactive precursors into M(IP)2C, and membranes prepared from it do not incorporate {3H-inositol}phosphatidylinositol into M(IP)2C, indicating a lack of M(IP)2C synthase activity (putatively phosphatidylinositol:mannose-inositol-P-ceramide phosphoinositol transferase) . M(IP)2C synthase activity is inhibited in the micromolar range by aureobasidin A, but drug sensitivity is over 1000-fold lower than reported for IPC synthase activity . An ipt1-deletion mutant has no severe phenotypic effects but is slightly more resistant to growth inhibition by calcium ions . Identification of the IPT1 gene should be helpful in determining the function of the M(IP)2C sphingolipid and in determining the catalytic mechanism of IPC and M(IP)2C synthases.

Fungal Genet Biol, 1997 Oct, 22(2), 77 - 83
Expression of YWP1, a gene that encodes a specific Yarrowia lipolytica mycelial cell wall protein, in Saccharomyces cerevisiae; Ramon AM et al.; The YWP1 gene encoding a specific mycelial cell wall protein of Yarrowia lipolytica has been cloned and expressed in Saccharomyces cerevisiae using different episomal plasmids . Because the plasmids pYAE35BB and pYAE35ES carrying the YWP1 gene (including the 5' noncoding promoter sequences) failed to express it, the YWP1 gene was cloned under the control of GAL/CYC or ACT S . cerevisiae promoters . A main band with an apparent molecular mass of 70 kDa was detected by immunoblotting in the cell wall fraction of transformants . Ywp1 processing and incorporation to the cell wall were similar in both Y . lipolytica and S . cerevisiae but not in its final localization in the cell wall . In Y . lipolytica Ywp1 is covalently bound to the cell wall (it is released only by Zymolyase digestion), whereas in S . cerevisiae it was not (it was released by boiling SDS solutions) . These results suggest that the sequences involved in recognition, anchoring of a protein to the cell wall, or the catalytic activities implicated are different, at least for Ywp1, in Y . lipolytica and S . cerevisiae . Another possibility is that the target for attachment of Ywp1 is missing or cryptic in the cell wall of S . cerevisiae .

Arch Biochem Biophys, 1997 Nov 15, 347(2), 193 - 200
A dominant negative mutation in Saccharomyces cerevisiae methionine aminopeptidase-1 affects catalysis and interferes with the function of methionine aminopeptidase-2; Klinkenberg M et al.; Methionine aminopeptidase (MetAP) enzymes require the metal ion cobalt, but little is known about the role of cobalt in the structural stability or catalysis of these enzymes . In Escherichia coli MetAP, for which a crystal structure is available, the five amino acid residues liganding the two cobalt ions are Asp97, Asp108, His171, Glu204, and Glu235 . These five amino acids are conserved in all MetAPs sequenced to date . The C-terminal domain of the yeast Saccharomyces cerevisiae MetAP1 is 41% identical to E . coli MetAP and contains these cobalt coordinating residues . Using site-directed mutagenesis on the gene coding for yeast MetAP1, we replaced Asp219 (corresponding to Asp97 in E . coli MetAP) with Asn . The yeast D219N mutant enzyme has 10(3)-fold lower catalytic activity and a different substrate specificity when compared to wild-type yeast MetAP1 . These results indicate that the side-chain of Asp219 is important for catalysis . Expression of D219N-MetAP1 in yeast causes a slow-growth phenotype and interferes with wild-type MetAP1 in a dominant manner . Expression of D219N-MetAP1 also affects the function of S . cerevisiae MetAP2 .

RNA, 1997 Dec, 3(12), 1374 - 87
A new cyclophilin and the human homologues of yeast Prp3 and Prp4 form a complex associated with U4/U6 snRNPs; Horowitz DS et al.; We have purified three new human U4/U6-snRNP proteins from HeLa cells . The three proteins formed a tightly bound complex and behaved as a single species throughout the purification . All three proteins have been identified by peptide sequencing, and full-length cDNA sequences have been obtained for all of them . Two of the proteins are homologues of the Saccharomyces cerevisiae splicing factors Prp3 and Prp4, and the third protein is a cyclophilin . Both the human and S . cerevisiae Prp4 proteins have seven repeats of the WD motif and likely fold into structures very similar to those of the beta subunits of G proteins . The human Prp3 protein is highly basic and is closely related to S . cerevisiae Prp3 only in its carboxyl-terminal half . The human homologues of Prp3 and Prp4 are part of a stable complex in the absence of RNA . The third protein in the complex is a new cyclophilin . Cyclophilins have been proposed to act as chaperones in a variety of cellular processes, and we discuss some possible roles of this U4/U6 snRNP-associated cyclophilin.

Antonie Van Leeuwenhoek, 1997 Oct, 72(3), 229 - 37
The incorporation of mannoproteins in the cell wall of S . cerevisiae and filamentous Ascomycetes; Brul S et al.; In yeast, glucanase extractable cell wall proteins are anchored to the plasma membrane at an intermediate stage in their biogenesis via a glycosylphosphatidylinositol (GPI) moiety before they become anchored to the wall glucan via a beta 1,6-glucan linkage . The mechanism of the membrane processing step of cell wall proteins is not known . Here, we report that Ascomycete filamentous fungi involved in food spoilage such as Aspergillus, Paecilomyces and Penicillium, also contain GPI membrane-anchored proteins some of which are processed by an endogenous phospholipase C activity . Furthermore, similar to the situation in yeast, their cell walls contain mannoproteins which are linked to the glucan backbone through a beta 1,6-glucan linkage . Interestingly, one mould which contains a significant amount of non covalently linked beta 1,6-glucosylated cell wall proteins, is much more sensitive towards beta 1,3-glucanases and membrane perturbing peptides than the others.

J Bacteriol, 1997 Dec, 179(24), 7790 - 5
Characteristics of Fps1-dependent and -independent glycerol transport in Saccharomyces cerevisiae; Sutherland FC et al.; Eadie-Hofstee plots of glycerol uptake in wild-type Saccharomyces cerevisiae W303-1A grown on glucose showed the presence of both saturable transport and simple diffusion, whereas an fps1delta mutant displayed only simple diffusion . Transformation of the fps1delta mutant with the glpF gene, which encodes glycerol transport in Escherichia coli, restored biphasic transport kinetics . Yeast extract-peptone-dextrose-grown wild-type cells had a higher passive diffusion constant than the fps1delta mutant, and ethanol enhanced the rate of proton diffusion to a greater extent in the wild type than in the fps1delta mutant . In addition, the lipid fraction of the fps1delta mutant contained a lower percentage of phospholipids and a higher percentage of glycolipids than that of the wild type . Fps1p, therefore, may be involved in the regulation of lipid metabolism in S . cerevisiae, affecting membrane permeability in addition to fulfilling its specific role in glycerol transport . Simultaneous uptake of glycerol and protons occurred in both glycerol- and ethanol-grown wild-type and fps1delta cells and resulted in the accumulation of glycerol at an inside-to-outside ratio of 12:1 to 15:1 . Carbonyl cyanide m-chlorophenylhydrazone prevented glycerol accumulation in both strains and abolished transport in the fps1delta mutant grown on ethanol . Likewise, 2,4-dinitrophenol inhibited transport in glycerol-grown wild-type cells . These results indicate the presence of an Fps1p-dependent facilitated diffusion system in glucose-grown cells and an Fps1p-independent proton symport system in derepressed cells.

Biochem Biophys Res Commun, 1997 Nov 26, 240(3), 586 - 9
Expression and purification of His-tagged beta-1,4-galactosyltransferase in yeast and in COS cells; Borsig L et al.; His6-tag technology has been introduced for easy purification of recombinant proteins expressed in Escherichia coli . Aiming at extending this technology to purification of glycoproteins expressed in Saccharomyces cerevisiae or in animal cells, respectively, we adapted this protocol to recombinant soluble beta-1,4-galactosyltransferase (rgal-T) . A His6-tag was introduced to the N-terminus of the protein (hisGal-T) . The Histagged enzyme expressed in yeast S . cerevisiae was enzymically active but could not be purified from the cell extract by virtue of the His6-tag . Binding efficiency of hisGal-T was found to be impaired by a bulky N-glycan close to the His-tag . Removal of the unique site of N-glycosylation using site-directed mutagenesis restored binding of hisGal-T to the Ni-NTA resin . In comparison N-glycosylated hisGal-T transiently expressed in COS cells was secreted as a soluble active enzyme and could be purified in one single step by virtue of the His6-tag.

Proc Natl Acad Sci U S A, 1997 Nov 25, 94(24), 13122 - 7
Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1; Sargent RG et al.; Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in Saccharomyces cerevisiae and Drosophila melanogaster, but their role, if any, in mammalian cells is undefined . To investigate the role of the nucleotide excision repair gene ERCC1, the hamster homologue to the S . cerevisiae RADIO gene, we disabled the gene by targeted knockout . Partial tandem duplications of the adenine phosphoribosyltransferase (APRT) gene then were constructed at the endogenous APRT locus in ERCC1- and ERCC1+ cells . To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental APRT+ tandem duplication could give rise to APRT- cells by homologous recombination, gene rearrangement, or point mutation . Measurement of rates and analysis of individual APRT- products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little . The formation of deletions is not caused by a general effect of the ERCC1 deficiency on gene stability, because ERCC1- cell lines with a single wild-type copy of the APRT gene yielded no increase in deletions . Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination . Recombination-dependent deletion formation in ERCC1- cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination . We suggest that the ERCC1 gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in ERCC1- cells are repaired by illegitimate recombination.

Nat Med, 1997 Dec, 3(12), 1383 - 8
The human 37-kDa laminin receptor precursor interacts with the prion protein in eukaryotic cells; Rieger R et al.; Prions are thought to consist of infectious proteins that cause transmissible spongiform encephalopathies . According to overwhelming evidence, the pathogenic prion protein PrPSc converts its host encoded isoform PrPC into insoluble aggregates of PrPSc, concomitant with pathological modifications (for review, see refs . 1-3) . Although the physiological role of PrPC is poorly understood, studies with PrP knockout mice demonstrated that PrPC is required for the development of prion diseases . Using the yeast two-hybrid technology in Saccharomyces cerevisiae, we identified the 37-kDa laminin receptor precursor (LRP) as interacting with the cellular prion protein PrPC . Mapping analysis of the LRP-PrP interaction site in S . cerevisiae revealed that PrP and laminin share the same binding domain (amino acids 161 to 180) on LRP . The LRP-PrP interaction was confirmed in vivo in insect (Sf9) and mammalian cells (COS-7) . The LRP level was increased in scrapie-infected murine N2a cells and in brain and spleen of scrapie-infected mice . In contrast, the LRP concentration was not significantly altered in these organs from mice infected with the bovine spongiform encephalopathic agent (BSE), which have a lower PrPSc accumulation . LRP levels, however, were dramatically increased in brain and pancreas, slightly increased in the spleen and not altered in the liver of crapie-infected hamsters . These data show that enhanced LRP concentrations are correlated with PrPSc accumulation in organs from mice and hamsters . The laminin receptor precursor, which is highly conserved among mammals and is located on the cell surface, may act as a receptor or co-receptor for the prion protein on mammalian cells.

Can J Microbiol, 1997 Oct, 43(10), 963 - 70
Tyrosine is involved in protection from oxidative stress in Saccharomyces cerevisiae; Lupo S et al.; The phenotypic characterization of a Saccharomyces cerevisiae mutant unable to grow under agitated conditions is presented here . When this strain was incubated under aerobic conditions, it did not grow and the viability of the culture decreased . The loss in viability was prevented by the addition of antioxidants or chelating agents to the medium, indicating that this mutant was unable to withstand the oxidative stress generated by aerobic metabolism . This strain was complemented with plasmids from a yeast genomic library . The transformants that were obtained carried plasmids harbouring the TYR1 gene, which codes for one of the enzymes involved in tyrosine biosynthesis . A monogenic S . cerevisiae tyr1 mutant obtained from the Yeast Genetic Stock Center showed higher sensitivity to hydrogen peroxide than a TYR1 strain . This sensitivity was reverted when this strain was complemented with the TYR1 gene . Considering these results, we propose that tyrosine plays a role in the protection against oxidative stress.

Can J Microbiol, 1997 Oct, 43(10), 954 - 62
Influence of altered plasma membrane fatty acid composition on cesium transport characteristics and toxicity in Saccharomyces cerevisiae; Hoptroff MJ et al.; The influence of altered plasma membrane fatty acid composition on cesium uptake and toxicity was investigated in Saccharomyces cerevisiae . Detailed kinetic studies revealed that both the Vmax and Km values for Cs+ transport increased (by approximately twofold in the latter case) when S . cerevisiae was grown in medium supplemented with the polyunsaturated fatty acid linoleate . In addition, Cs+ uptake by linoleate-enriched cells was considerably less sensitive to the competitive effects of other monovalent cations (K+, Rb+, and NH4+) than that by unsupplemented cells . Stimulation of Cs+ uptake in the presence of certain K+ and Rb+ concentrations was only evident in linoleate-enriched S . cerevisiae . At 100 mM CsCl, the initial rate of Cs+ uptake was greater in linoleate-supplemented cells than in unsupplemented cells and this was reflected in a more rapid displacement of cellular K+ . However, little difference in net Cs+ accumulation between linoleate-supplemented and unsupplemented cells was evident during prolonged incubation in buffer or during growth . Thus, Cs+ toxicity was similar in linoleate-supplemented and unsupplemented cells . The results were consistent with the Cs+ (K+) transport mechanism adopting an altered conformational state in linoleate-enriched S . cerevisiae.

FEBS Lett, 1997 Nov 3, 417(1), 114 - 8
Identification of the yeast ACR1 gene product as a succinate-fumarate transporter essential for growth on ethanol or acetate; Palmieri L et al.; The protein encoded by the ACR1 gene in Saccharomyces cerevisiae belongs to a family of 35 related membrane proteins that are encoded in the fungal genome . Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 28 members of the family are unknown . The yeast ACR1 gene was introduced into Escherichia coli on an expression plasmid . The protein was over-produced as inclusion bodies, which were purified and solubilised in the presence of sarkosyl . The solubilised protein was reconstituted into liposomes and shown to transport fumarate and succinate . Its physiological role in S . cerevisiae is probably to transport cytoplasmic succinate, derived from isocitrate by the action of isocitrate lyase in the cytosol, into the mitochondrial matrix in exchange for fumarate . This exchange activity and the subsequent conversion of fumarate to oxaloacetate in the cytosol would be essential for the growth of S . cerevisiae on ethanol or acetate as the sole carbon source.

Cell, 1997 Nov 28, 91(5), 673 - 84
MAP kinases with distinct inhibitory functions impart signaling specificity during yeast differentiation; Madhani HD et al.; Filamentous invasive growth of S . cerevisiae requires multiple elements of the mitogen-activated protein kinase (MAPK) signaling cascade that are also components of the mating pheromone response pathway . Here we show that, despite sharing several constituents, the two pathways use different MAP kinases . The Fus3 MAPK regulates mating, whereas the Kss1 MAPK regulates filamentation and invasion . Remarkably, in addition to their kinase-dependent activation functions, Kss1 and Fus3 each have a distinct kinase-independent inhibitory function . Kss1 inhibits the filamentation pathway by interacting with its target transcription factor Ste12 . Fus3 has a different inhibitory activity that prevents the inappropriate activation of invasion by the pheromone response pathway . In the absence of Fus3, there is erroneous crosstalk in which mating pheromone now activates filamentation-specific gene expression using the Kss1 MAPK.

Mol Gen Genet, 1997 Oct, 256(4), 446 - 55
Isolation and analysis of functional homologues of the secretion-related SAR1 gene of Saccharomyces cerevisiae from Aspergillus niger and Trichoderma reesei; Veldhuisen G et al.; The Aspergillus niger and Trichoderma reesei genes encoding the functional homologues of the small GTP-binding protein SAR1p, which is involved in the secretion pathway in Saccharomyces cerevisiae, have been cloned and characterised . The A . niger gene (sarA) contains five introns, whereas the T . reesei gene (sar1) has only four . In both cases the first intron is at the same position as the single S . cerevisiae SAR1 intron . The encoded proteins show 70-80% identity to the SAR1 protein . Complementation of S . cerevisiae sar1 and sec12 mutants by expression vectors carrying the A . niger sarA and T . reesei sar1 cDNA clones confirmed that the cloned genes are functional homologues of the S . cerevisiae SAR1 gene . Three mutant alleles of the A . niger sarA gene (D29G, E109K, D29G/E109K), generated by site-directed mutagenesis, revealed a thermosensitive dominant-negative phenotype in the presence of the wild-type sarA allele . This result contrasts with the situation in S . cerevisiae, where similar mutations have a thermosensitive phenotype . Taken together, our results indicate that the sarA gene is involved in an essential function in A . niger.

Mol Gen Genet, 1997 Oct, 256(4), 436 - 45
Genetic and molecular characterization of Neurospora crassa mus-23: a gene involved in recombinational repair; Watanabe K et al.; A newly isolated mutant, mus-23, of Neurospora crassa was found to be highly sensitive to a wide variety of mutagens, including UV light, methyl methanesulfonate, 4-nitroquinoline 1-oxide, N-methyl-N'-nitro-N-nitrosoguanidine and tert-butyl hydroperoxide . This mutant was originally isolated as a mutant that could not grow on medium containing histidine . Meiosis and sporulation were defective in homozygous crosses between mus-23 haploids . The mus-23 gene is located on the right arm of LGII, between fl and trp-3 . Analyses of epistasis between mus-23 and other mutations that cause defects in DNA repair indicated that the mus-23 gene belongs to the same DNA repair group as mei-3, which is the Neurospora homolog of the Saccharomyces cerevisiae gene RAD51 . The double mutant carrying mus-23 and uvs-3 mutations was lethal . The mus-23 gene was cloned by complementation of the MMS-sensitive phenotype of the mus-23 mutant . The gene contained an open reading frame of 1578 bp and did not contain any introns . The molecular weight of the predicted mus-23 gene product was 60.4 kDa . Computer analyses revealed that the MUS23 protein has significant homology to Mre11p, which is known to be involved in recombinational repair in S . cerevisiae . The level of mus-23 transcripts increased significantly within 60 min of treatment with UV or MMS and then gradually decreased . The role of MUS23 protein in recombinational repair is discussed.

Mol Gen Genet, 1997 Oct, 256(4), 355 - 64
Cloning of the gene encoding the catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica; Benetti PH et al.; Casein kinase II from the yeast Yarrowia lipolytica is a heterotetramer of the form alpha alpha' beta 2 . We report on the cloning and sequencing of a partial cDNA and of the complete genomic DNA coding for the catalytic alpha subunit of the casein kinase II from this yeast species . The sequence of the gene coding for this enzyme has been analyzed . No intron was found in the gene, which is present in a single copy . The deduced amino acid sequence of the gene shows high similarity with those of alpha subunit described in other species, although, uniquely, Y . lipolytica CKII alpha lacks cysteines . We find that the alpha subunit sequence of Y . lipolytica CKII is shown greater homology with the corresponding protein from S . pombe than with that from S . cerevisiae . We have analyzed CKII alpha expression and CKII alpha activity . We show that expression of this enzyme is regulated . The catalytic subunit is translated from a single mRNA, and the enzyme is present at a very low level in Y . lipolytica, as in other yeasts.

Yeast, 1997 Nov, 13(14), 1357 - 62
Functional differences among the six Saccharomyces cerevisiae tRNATrp genes; Ong WC et al.; The Saccharomyces cerevisiae haploid genome includes six copies of the gene encoding tRNATrp which are scattered on five chromosomes . Other, non-functional tDNATrp fragments also occur in the genome . The segments of all six genes which encode the 72-nucleotide mature tRNATrp, as well as a 34-nucleotide intervening sequence, are identical . However, the 5' and 3' flanking sequences diverge virtually at the boundaries of the coding region . We have used an assay based on suppression of UGA mutations by multi-copy clones of tDNATrp to search for functional differences among these genes . Previous studies with one tDNATrp had demonstrated that moderate suppression of a UGA mutation, leu2-2, resulted from introduction of a multi-copy clone of the gene . Attempts to use this assay to select tDNATrp clones from a yeast genomic library yielded only four of the six different clones . The other two genes were amplified by PCR and cloned in pRS202, a 2 mu vector also used for the genomic library . Plasmids bearing the six tRNA genes were transformed into S . cerevisiae strain JG369.3B and scored for their ability to suppress the leu2-2 mutation as well as his4-260, another UGA marker . Two of the six tRNATrp clones were unable to suppress either marker, two evidenced weak suppression of the Leu auxotrophy, and two were able to suppress both markers . Growth rates in liquid media requiring suppression were measured for cell lines carrying each of the clones . Differences greater than 50-fold were observed in media lacking histidine . An evolutionary tree based on 5'-flanking sequence corresponds reasonably well with suppressor activity, while a similar analysis of 3'-flanking sequence does not . This suggests that the functional differences are based on divergence in the 5'-flanking sequences of the tRNATrp genes.

Yeast, 1997 Nov, 13(14), 1319 - 27
The ALD6 gene of Saccharomyces cerevisiae encodes a cytosolic, Mg(2+)-activated acetaldehyde dehydrogenase; Meaden PG et al.; The deduced translation product of an open reading frame on the left arm of chromosome XVI of Saccharomyces cerevisiae, with the systematic name of YPL061w, is 500 amino acids in length and shares significant homology with aldehyde dehydrogenases . Amino acids 2 to 16 of the protein encoded by YPL061w were found to be identical to the N-terminal 15 amino acids of the purified cytosolic, Mg(2+)-activated acetaldehyde dehydrogenase (ACDH) of S . cerevisiae . This enzyme is thought to be involved in the production of acetate from which cytosolic acetyl-CoA is then synthesized . Deletion of YPL061w was detrimental to the growth of haploid strains of yeast; an analysis of one deletion mutant revealed a maximum specific growth rate (in complex medium containing glucose) of one-third of that displayed by the wild-type strain . Mutants deleted in YPL061w were also unable to use ethanol as a carbon source . As expected, the cytosolic, Mg(2+)-activated ACDH activity had been lost from the mutants, although the mitochondrial, K(+)-activated ACDH was readily detected.

Am J Trop Med Hyg, 1997 Nov, 57(5), 626 - 33
Characterization of a recombinant Onchocerca volvulus antigen (Ov33) produced in yeast; Tume CB et al.; A yeast (Saccharomyces cerevisiae) expression system has been adapted to produce reagent quantities of a major Onchocerca antigen, Ov33 . Using a pool of monoclonal antibodies produced against third-stage larvae, a cDNA library constructed from adult O . volvulus worms was screened . Twenty-seven cDNAs were isolated, two of which had sequence homology to Ov33, a putative aspartyl protease inhibitor, which is the immunodominant antigen of O . volvulus . These cDNAs were expressed at high levels intracellularly or through the secretory pathway of S . cerevisiae . Localization studies using antisera produced against purified recombinant protein demonstrated that Ov33 is a very abundant parasite protein present in the hypodermis, muscle, and uterus of female worms, as well as in embryonic microfilariae . The soluble recombinant protein secreted by yeast (C71) demonstrated inhibitory activity against the aspartyl protease pepsin . Antibodies to the recombinant protein-mediated leukocyte adherence to and killing of skin microfilariae . The sensitivity of a diagnostic test using recombinant Ov33 was evaluated using sera from 441 patients . The mean sensitivities for the two recombinant constructs, C27 and C71, were 82.2% and 85.4%, respectively . The combined sensitivity using both recombinant proteins was 94%.

Cell, 1997 Nov 14, 91(4), 491 - 500
Regulating the yeast kinetochore by ubiquitin-dependent degradation and Skp1p-mediated phosphorylation; Kaplan KB et al.; In S . cerevisiae, the four-protein Cbf3 complex binds to the essential CDEIII region of centromeric DNA to initiate kinetochore assembly . We report the reconstitution of Cbf3p from recombinant proteins and an analysis of its p58Ctf13 and p23Skp1 subunits . p23Skp1 has both G1- and G2-specific functions in yeast and binds to p58Ctf13 and to the essential Cdc4p component of the ubiquitin conjugating complex Scul(Cdc4) . We show that the function of p23Skp1 in Cbf3p is to activate p58Ctf13 by phosphorylation . p58Ctf13 is an unstable protein that is targeted to the proteosome, probably by Scul(Cdc4)-mediated ubiquitination . Thus, p58 appears to be activated by phosphorylation in a p23Skp1-dependent step and degraded by the proteosome in a ubiquitin-dependent step . We propose that coupled activation and destruction link the assembly of Cbf3p to the duplication of centromeres in S phase.

Cell, 1997 Nov 14, 91(4), 457 - 66
The exosome: a conserved eukaryotic RNA processing complex containing multiple 3'-->5' exoribonucleases; Mitchell P et al.; We identified a complex in S . cerevisiae, the "exosome," consisting of the five essential proteins Rrp4p, Rrp41p, Rrp42p, Rrp43p, and Rrp44p (Dis3p) . Remarkably, four of these proteins are homologous to characterized bacterial 3'-->5' exoribonucleases; Rrp44p is homologous to RNase II, while Rrp41p, Rrp42p, and Rrp43p are related to RNase PH . Recombinant Rrp4p, Rrp44p, and Rrp41p are 3'-->5' exoribonucleases in vitro that have distributive, processive, and phosphorolytic activities, respectively . All components of the exosome are required for 3' processing of the 5.8S rRNA . Human Rrp4p is found in a comparably sized complex, and expression of the hRRP4 gene in yeast complements the rrp4-1 mutation . We conclude that the exosome constitutes a highly conserved eukaryotic RNA processing complex.

Appl Microbiol Biotechnol, 1997 Oct, 48(4), 539 - 45
Relationship between cadmium sensitivity and degree of plasma membrane fatty acid unsaturation in Saccharomyces cerevisiae; Howlett NG et al.; The sensitivity of Saccharomyces cerevisiae to the redox-active metal copper has recently been found to be influenced by cellular fatty acid composition . This study sought to investigate whether fatty acid composition affected plasma membrane permeabilisation and whole-cell toxicity induced by the redox-inactive metal cadmium . S . cerevisiae NCYC 1383 was enriched with the polyunsaturated fatty acids linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium . Incorporation of the exogenous fatty acids resulted in them comprising more than 65% of the total fatty acids in plasma membrane lipids . Inhibition of cell division in the presence of Cd(NO3)2 was accentuated by growth in the presence of a polyunsaturated fatty acid . Furthermore, susceptibility to Cd(2+)-induced plasma membrane permeabilisation increased with the degree of fatty acid unsaturation . Thus, during exposure to Cd2+, K+ efflux from 18:2- and 18:3-enriched cells was up to 2.5-fold or 3-fold greater, respectively than that from unsupplemented cells . In addition, reductions in cell viability during exposure to Cd2+ were most marked in polyunsaturated-fatty-acid-supplemented cells . At certain times, unsupplemented Cd(2+)-exposed cells displayed up to 7-fold greater viability than supplemented Cd(2+)-exposed cells . The study demonstrates that the toxicity of the redox-inactive metal Cd2+ towards S . cerevisiae becomes markedly amplified with increased cellular and plasma membrane fatty acid unsaturation.

Appl Microbiol Biotechnol, 1997 Oct, 48(4), 499 - 503
Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae; Murai T et al.; We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form . A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus, with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin a protein involved in mating and covalently anchored to the cell wall . The plasmid constructed containing this fusion gene was introduced into S . cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S . cerevisiae . The CMCase activity was detected in the cell pellet fraction . The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall . The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy . These results proved that the CMCase was anchored on the cell wall in its active form.

Biochim Biophys Acta, 1997 Sep 4, 1348(1-2), 117 - 23
CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase; McMaster CR et al.; Ethanolaminephosphotransferase catalyzes the final step of the CDP-ethanolamine pathway for the de novo synthesis of phosphatidylethanolamine (PtdEtn) via transfer of a phosphoethanolamine moiety from CDP-ethanolamine to diacylglycerol for the formation of PtdEtn and CMP . Ethanolaminephosphotransferase is an integral membrane-bound enzyme whose intracellular location defines the site of PtdEtn synthesis by the CDP-ethanolamine pathway . Subcellular fractionation experiments have yet to resolve the precise subcellular location of ethanolaminephosphotransferase, although it is routinely associated with the microsomal fraction . Ethanolaminephosphotransferase has yet to be purified from any source and its cDNA has not been isolated from any mammalian source, thus preventing the generation of antibodies necessary to directly examine its intracellular location through immunofluorescence or electron microscopy approaches . An ethanolaminephosphotransferase gene has recently been isolated from the yeast Saccharomyces cerevisiae and structure/function analyses of the encoded enzyme identified several important characteristics including the catalytic site . The predicted amino acid sequence of the S . cerevisiae ethanolaminephosphotransferase gene should allow for the generation of antibodies required to directly define the site of PtdEtn synthesis in this organism, and it has provided the necessary information to pursue the isolation of a mammalian cDNA.

Biochim Biophys Acta, 1997 Sep 4, 1348(1-2), 100 - 10
CDP-choline:1,2-diacylglycerol cholinephosphotransferase; McMaster CR et al.; Cholinephosphotransferase transfers a phosphocholine moiety from CDP-choline to diacylglycerol thus forming phosphatidylcholine (PtdCho) and CMP . This reaction defines the ultimate step in the Kennedy pathway for the genesis of de novo synthesized PtdCho . Hence, the intracellular location of cholinephosphotransferase identifies both the site from which de novo synthesized PtdCho is transported to other organelles and the site from which it is assembled with proteins and other lipids for secretion from the cell during the generation of lung surfactant, lipoproteins, and bile . Most subcellular fractionation studies observed the majority of cholinephosphotransferase activity in the endoplasmic reticulum, although the method of subcellular fractionation was found to grossly affect these results with activity alternately dispersed within Golgi, nuclear, and mitochondrial fractions . Coupling subcellular fractionation results with immunofluorescence or electron microscopy studies would resolve the issue of the site of PtdCho synthesis . However, antibodies have yet to be generated to cholinephosphotransferase since its integral membrane-bound nature has prevented its purification from any source and a mammalian cholinephosphotransferase cDNA has also yet to be isolated . However, cholinephosphotransferase genes have recently been isolated from the yeast Saccharomyces cerevisiae . Structure/function analysis of the S . cerevisiae cholinephosphotransferase has allowed for an in depth molecular examination resulting in the identification of the catalytic site . In addition, this analysis has generated the predicted amino acid data necessary to produce antibodies to pursue the site of PtdCho synthesis in this organism, as well as to provide information that should allow for the isolation of mammalian cholinephosphotransferase cDNA(s).

Biochim Biophys Acta, 1997 Sep 4, 1348(1-2), 45 - 55
Phosphatidate phosphatases and diacylglycerol pyrophosphate phosphatases in Saccharomyces cerevisiae and Escherichia coli; Carman GM; Phosphatidate phosphatase plays a major role in the synthesis of phospholipids and triacylglycerols in the yeast Saccharomyces cerevisiae . Membrane- and cytosolic-associated forms of the enzyme have been isolated and characterized . These enzymes are Mg2+-dependent and N-ethylmaleimide-sensitive . The expression of a membrane-associated form of phosphatidate phosphatase is regulated by growth phase and inositol supplementation, whereas enzyme activity is regulated by lipids, nucleotides, and by phosphorylation . Phosphatidate phosphatase is coordinately regulated with other phospholipid biosynthetic enzymes including phosphatidylserine synthase . Diacylglycerol pyrophosphate phosphatase is a novel enzyme of phospholipid metabolism which is present in S . cerevisiae, Escherichia coli, and mammalian cells . This enzyme possesses a phosphatidate phosphatase activity which is Mg2+-independent and N-ethylmaleimide-insensitive and is distinct from the Mg2+-dependent and N-ethylmaleimide-sensitive form of phosphatidate phosphatase . Genes encoding for diacylglycerol pyrophosphate phosphatase have been isolated from S . cerevisiae and E . coli . The deduced protein sequences of these genes show homology to the sequence of the mouse PAP2 (Mg2+-independent and N-ethylmaleimide-insensitive phosphatidate phosphatase) protein, especially in a novel phosphatase sequence motif . Rat liver PAP2 displays diacylglycerol pyrophosphate phosphatase activity.

FASEB J, 1997 Nov, 11(13), 1067 - 75
Cell cycle regulation by the ubiquitin pathway; Pagano M; In the past 2 years, two ubiquitin-dependent proteolytic pathways have been established as important players in the regulation of the cell division cycle . In S . cerevisiae, the entry into S phase requires ubiquitin-mediated degradation of a cdk inhibitor, p40Sic1, in a pathway that involves the E2 enzyme Cdc34 . Recent studies reviewed herein show that the Cdc34 pathway targets phosphorylated substrates . A second pathway that regulates chromosome segregation and mitotic exit by degrading anaphase inhibitors and mitotic cyclins involves a different E2 and a large molecular weight E3 complex, called the anaphase-promoting complex or cyclosome . This pathway targets substrates containing one or more destruction box motif.

J Biol Chem, 1997 Nov 14, 272(46), 28918 - 24
Characterization of the Saccharomyces cerevisiae cytosine transporter using energizable plasma membrane vesicles; Pinson B et al.; The purine-cytosine permease is a carrier localized in the plasma membrane of the yeast Saccharomyces cerevisiae . The energetics of cytosine transport catalyzed by this permease has been studied in an artificial system obtained by fusion between proteoliposomes containing beef heart cytochrome c oxidase and plasma membrane-enriched fractions of a S . cerevisiae strain overexpressing the permease . Upon addition of an energy donor, a proton-motive force (inside alkaline and negative) is created in this system and promotes cytosine accumulation . By using different phospholipids, it is shown that cytosine uptake is dependent on the phospholipids surrounding the carrier . It was demonstrated that the purine-cytosine permease is able to catalyze a secondary active transport of cytosine . By using nigericin and valinomycin, the DeltapH component of the proton-motive force is shown to be the only force driving nucleobase accumulation . Moreover, transport measurements done at two pH values have shown that alkalinization of intravesicular pH leads to a significant increase in cytosine uptake rate . Finally, no specific role of K+ ions on cytosine transport could be demonstrated in this system.

Yeast, 1997 Oct, 13(13), 1251 - 63
Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: characterization of the 54 kb right terminal CDC15-FLO1-PHO11 region; Barton AB et al.; Gene density near the ends of Saccharomyces cerevisiae chromosomes is much lower than on the rest of the chromosome . Non-functional gene-fragments are common and a high proportion of the sequences are repeated elsewhere in the genome . This sequence arrangement suggests that the ends of chromosomes play a structural rather than a coding role and may be analogous to the highly repeated heterochromatic DNA of higher organisms . In order to evaluate the function of the ends of S . cerevisiae chromosomes, the rightmost 54-kb of DNA from chromosome I was investigated . The region contains 16 open reading frames (ORFs) and two tRNA genes . Gene-disruption studies indicated that none of these genes are essential for growth on rich or minimal medium, mating or sporulation . In contrast to the central region where 80% of the genes are transcribed when cells are grown on rich medium, only seven ORFs and the two tRNA genes appeared to produce transcripts . Six of the transcribed ORFs were from the centromere-proximal part of the region, leaving the rightmost 35-kb with only a single sequence that is transcribed during vegetative growth . Two genes located 3 and 10-kb from the chromosome I telomere are almost identical to two genes located somewhat further from the chromosome VIII telomere . Surprisingly, the chromosome VIII copies were transcribed while the chromosome I genes were not . These results suggest that the chromosome I genes may be repressed by a natural telomere position effect . The low level of transcription, absence of essential genes as well as the repetitive nature of these sequences are consistent with their having a structural role in chromosome function.

Yeast, 1997 Oct, 13(13), 1243 - 50
A phylogenetic analysis of Saccharomyces species by the sequence of 18S-28S rRNA spacer regions; Oda Y et al.; Sequences of two internally transcribed spacer regions between 18S and 28S rRNA genes were determined to assess the phylogenetic relationship in the strains belonging to the genus Saccharomyces . The sequences of S . bayanus and S . pastorianus were quite similar, but not identical . Two phylogenetic trees constructed by the neighbor-joining method showed that all the species examined were distinguished from one another . The Saccharomyces sensu stricto species: S . cerevisiae, S . bayanus, S . paradoxus and S . pastorianus, were closely related and far from the Saccharomyces sensu lato species including S . barnetti, S . castellii, S . dairensis, S . exiguus, S . servazzii, S . spencerorum and S . unisporus, and an outlying species, S . kluyveri.

Nature, 1997 Nov 6, 390(6655), 85 - 8
Inhibitory and activating functions for MAPK Kss1 in the S . cerevisiae filamentous-growth signalling pathway; Cook JG et al.; Mitogen-activated protein kinase (MAPK) cascades are conserved signalling modules that regulate responses to diverse extracellular stimuli, developmental cues and environmental stresses . A MAPK is phosphorylated and activated by a MAPK kinase (MAPKK), which is activated by an upstream protein kinase, such as Raf, Mos or a MAPKK kinase . Ste7, a MAPKK in the yeast Saccharomyces cerevisiae, is required for two developmental pathways: mating and invasive (filamentous) growth . Kss1 and Fus3, the MAPK targets of Ste7, are required for mating, but their role in invasive growth has been unclear . Because no other S . cerevisiae MAPK has been shown to function in invasive growth, it was proposed that Ste7 may have non-MAPK targets . We show instead that Kss1 is the principal target of Ste7 in the invasive-growth response in both haploids and diploids . We demonstrate further that Kss1 in its inactive form is a potent negative regulator of invasive growth . Ste7 acts to relieve this negative regulation by switching Kss1 from an inhibitor to an activator . These results indicate that this MAPK has a physiologically important function in its unactivated state . Comparison of normal and MAPK-deficient cells indicates that nitrogen starvation and activated Ras stimulate filamentous growth through both MAPK-independent and MAPK-dependent means.

Cell, 1997 Oct 31, 91(3), 311 - 23
Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes; Pak DT et al.; The origin recognition complex (ORC) is required to initiate eukaryotic DNA replication and also engages in transcriptional silencing in S . cerevisiae . We observed a striking preferential but not exclusive association of Drosophila ORC2 with heterochromatin on interphase and mitotic chromosomes . HP1, a heterochromatin-localized protein required for position effect variegation (PEV), colocalized with DmORC2 at these sites . Consistent with this localization, intact DmORC and HP1 were found in physical complex . The association was shown biochemically to require the chromodomain and shadow domains of HP1 . The amino terminus of DmORC1 contained a strong HP1-binding site, mirroring an interaction found independently in Xenopus by a yeast two-hybrid screen . Finally, heterozygous DmORC2 recessive lethal mutations resulted in a suppression of PEV . These results indicate that ORC may play a widespread role in packaging chromosomal domains through interactions with heterochromatin-organizing factors.

Biochim Biophys Acta, 1997 Sep 5, 1341(2), 173 - 82
Purification and characterization of adenine phosphoribosyltransferase from Saccharomyces cerevisiae; Alfonzo JD et al.; Adenine phosphoribosyltransferase (APRT) from Saccharomyces cerevisiae was purified approximately 1500-fold . The enzyme catalyzes the Mg-dependent condensation of adenine and 5-phosphoribosylpyrophosphate (PRPP) to yield AMP . The purification procedure included anion exchange chromatography, chromatofocusing and gel filtration . Elution of the enzyme from the chromatofocusing column indicated a pI value of 4.7 . The molecular mass for the native enzyme was 50 kDa; however, upon electrophoresis under denaturing conditions two bands of apparent molecular mass of 29 and 20 kDa were observed . We have previously reported the presence of two separate coding sequences for APRT, APT1 and APT2 in S . cerevisiae . The appearance of two bands under denaturing conditions suggests that, unlike other APRTs, this enzyme could form heterodimers . This may be the basis for substrate specificity differences between this enzyme and other APRTs . Substrate kinetics and product inhibition patterns are consistent with a ping-pong mechanism . The Km for adenine and PRPP were 6 microM and 15 microM, respectively and the Vmax was 15 micromol/min . These kinetic constants are comparable to the constants of APRT from other organisms.

J Mol Biol, 1997 Oct 31, 273(3), 552 - 71
An in vivo and in vitro structure-function analysis of the Saccharomyces cerevisiae U3A snoRNP: protein-RNA contacts and base-pair interaction with the pre-ribosomal RNA; Mereau A et al.; The structure and accessibility of the S . cerevisiae U3A snoRNA was studied in semi-purified U3A snoRNPs using both chemical and enzymatic probes and in vivo using DMS as the probe . The results obtained show that S . cerevisiae U3A snoRNA is composed of a short 5' domain with two stem-loop structures containing the phylogenetically conserved boxes A' and A and a large cruciform 3' domain containing boxes B, C, C' and D . A precise identification of RNA-protein contacts is provided . Protection by proteins in the snoRNP and in vivo are nearly identical and were exclusively found in the 3' domain . There are two distinct protein anchoring sites: (i), box C' and its surrounding region, this site probably includes box D, (ii) the boxes B and C pair and the bases of stem-loop 2 and 4 . Box C' is wrapped by the proteins . RNA-protein interactions are more loose at the level of boxes C and D and a box C and D interaction is preserved in the snoRNP . In accord with this location of the protein binding sites, an in vivo mutational analysis showed that box C' is important for U3A snoRNA accumulation, whereas mutations in the 5' domain have little effect on RNA stability . Our in vivo probing experiments strongly suggest that, in exponentially growing cells, most of the U3A snoRNA molecules are involved in the 10-bp interaction with the 5'-ETS region and in two of the interactions recently proposed with 18S rRNA sequences . Our experimental study leads to a slightly revised version of the model of interaction proposed by J . Hughes . Single-stranded segments linking the heterologous helices are highly sensitive to DMS in vivo and their functional importance was tested by a mutational analysis .

J Biol Chem, 1997 Nov 7, 272(45), 28690 - 4
Identification and characterization of Saccharomyces cerevisiae dihydrosphingosine-1-phosphate phosphatase; Mao C et al.; We have identified the yeast sphingosine resistance gene (YSR2) of Saccharomyces cerevisiae as encoding a protein that specifically dephosphorylates dihydrosphingosine 1-phosphate (DHS-1-P), and we refer to this protein as dihydrosphingosine-1-phosphate phosphatase . Overexpression of YSR2 conferred sphingosine resistance to the dihydrosphingosine-1-P lyase-defective mutant (JS16) of S . cerevisiae, which is hypersensitive to sphingosine . The ysr2Delta deletion mutant of S . cerevisiae accumulated DHS-1-P compared with its wild type strain upon labeling with D-erythro-{4, 5-3H}dihydrosphingosine, whereas overexpression of YSR2 increased dephosphorylation of DHS-1-P . An epitope-tagged fusion protein (YSR2-Flag) was partially purified and found to specifically dephosphorylate DHS-1-P to yield dihydrosphingosine . YSR2 failed to dephosphorylate ceramide 1-phosphate or phosphatidic acid . Functionally, the mutant bearing the ysr2Delta deletion decreased labeling of sphingolipids and increased labeling of glycerolipids dramatically following in vivo labeling with D-erythro-{3H}dihydrosphingosine, but it slightly affected labeling of sphingolipids with inositol . Taken together, these results identify YSR2 as dihydrosphingosine-1-phosphate phosphatase . They also raise the intriguing possibility that phosphorylation followed by dephosphorylation is required for incorporation of exogenous long chain sphingoid bases into sphingolipids.

J Biol Chem, 1997 Nov 7, 272(45), 28646 - 51
Human Prk is a conserved protein serine/threonine kinase involved in regulating M phase functions; Ouyang B et al.; Human prk encodes a novel protein serine/threonine kinase capable of strongly phosphorylating casein but not histone H1 in vitro . prk expression is tightly regulated at various levels during different stages of the cell cycle in lung fibroblasts . The Prk kinase activity is relatively low during mitosis, G1, and G1/S, and peaks during late S and G2 stages of the cell cycle . Recombinant human Prk expressed through the baculoviral vector system is capable of phosphorylating Cdc25C, a positive regulator for the G2/M transition . Human prk shares significant sequence homology with Saccharomyces cerevisiae CDC5 and Drosophila melanogaster polo, both of which are essential for mitosis and meiosis . Full-length prk transcripts greatly potentiate progesterone-induced meiotic maturation of Xenopus laevis oocytes . On the other hand, antisense prk transcripts significantly delay and reduce the rate of oocyte maturation . When expressed in a CDC5 mutant strain of S . cerevisiae, human Prk, but not a deletional mutant protein, fully rescues the temperature-sensitive phenotype of the budding yeast . Taken together, prk may represent a new protein kinase, playing an important role in regulating the onset and/or progression of mitosis in mammalian cells.

J Biol Chem, 1997 Nov 7, 272(45), 28281 - 8
Fah1p, a Saccharomyces cerevisiae cytochrome b5 fusion protein, and its Arabidopsis thaliana homolog that lacks the cytochrome b5 domain both function in the alpha-hydroxylation of sphingolipid-associated very long chain fatty acids; Mitchell AG et al.; A search of the Saccharomyces cerevisiae genome data base for cytochrome b5-like sequences identified a 1.152-kilobase pair open reading frame, located on chromosome XIII at locus YMR272C (FAH1) . That gene encodes a putative 384-amino acid protein with an amino-terminal cytochrome b5 domain . The b5 core domain shows a 52% identity and 70% similarity to that of the yeast microsomal cytochrome b5 and a 35% identity and 54% similarity to the b5 core domain of OLE1, the S . cerevisiae Delta-9 fatty acid desaturase . Expression of the S . cerevisiae FAH1 cytochrome b5 domain in Escherichia coli produces a soluble protein that exhibits the typical oxidized versus reduced differential absorbance spectra of cytochrome b5 . Sequence analysis of Fah1p reveals other similarities to Ole1p . Both proteins are predicted to have two hydrophobic domains, each capable of spanning the membrane twice, and both have the HX(2-3)(XH)H motifs that are characteristic of membrane-bound fatty acid desaturases . These similarities to Ole1p suggested that Fah1p played a role in the biosynthesis or modification of fatty acids . Disruption of the FAH1 gene in S . cerevisiae did not give any visible phenotype, and there was no observable difference in content or distribution of the most abundant long chain saturated and unsaturated 14-18-carbon fatty acid species . Northern blot analysis, however, showed that this gene is expressed at much lower levels ( approximately 150-fold) than the OLE1 gene, suggesting that it might act on a smaller subset of fatty acids . Analysis of sphingolipid-derived very long chain fatty acids revealed an approximately 40-fold reduction of alpha-HO 26:0 and a complementary increase in 26:0 in the gene-disrupted fah1Delta strain . GAL1 expression of the S . cerevisiae FAH1 genes in the fah1Delta strain restores alpha-HO 26:0 fatty acids to wild type levels . Also identified are a number of homologs to this gene in other species . Expression of an Arabidopsis thaliana FAH1 gene, which does not contain the cytochrome b5 domain, in the fah1Delta strain produced an approximately 25-fold increase in alpha-HO 26:0 and reduced the levels of its 26-carbon precursor, suggesting that it functions in very long chain fatty acid hydroxylation using an alternate electron transfer mechanism.

Genes Dev, 1997 Nov 1, 11(21), 2790 - 800
Functionally interacting telomerase RNAs in the yeast telomerase complex; Prescott J et al.; The ribonucleoprotein (RNP) enzyme telomerase from Saccharomyces cerevisiae adds telomeric DNA to chromosomal ends in short increments both in vivo and in vitro . Whether or not telomerase functions as a multimer has not been addressed previously . Here we show, first, that following polymerization, the telomerase RNP remains stably bound to its telomeric oligonucleotide reaction product . We then exploit this finding and a previously reported mutant telomerase RNA to demonstrate that, unexpectedly, the S . cerevisiae telomerase complex contains at least two functionally interacting RNA molecules that both act as templates for DNA polymerization . Here, functional telomerase contains at least two active sites.

J Membr Biol, 1997 Nov 1, 160(1), 77 - 83
Activation of divalent cation influx into S . cerevisiae cells by hypotonic downshift; Beeler T et al.; Subjecting Saccharomyces cerevisiae cells to a hypotonic downshift by transferring cells form YPD medium containing 0.8 M sorbitol to YPD medium without sorbitol induces a transient rapid influx of Ca2+ and other divalent cations into the cell . For cells grown in YPD at 37 degrees C, this hypotonic downshift increases Ca2+ accumulation 6.7-fold . Hypotonic downshift-induced Ca2+ accumulation and steady-state Ca2+ accumulation in isotonic YPD medium are differentially affected by dodecylamine and Mg2+ . The Ca(2+)-influx pathway responsible for hypotonic-induced Ca2+ influx may account for about 10-35% of Ca2+ accumulation by cells growing in YPD . Ca2+ influx is not required for cells to survive a hypotonic downshift . Hypotonic downshift greatly reduces the ability of S . cerevisiae cells to survive a 5-min exposure to 10 mM Cd2+ suggesting that mutants resistant to acute Cd2+ exposure may help identify genes required for hypotonic downshift-induced divalent cation influx.

Cell, 1997 Oct 17, 91(2), 221 - 30
A complex of Cdc4p, Skp1p, and Cdc53p/cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1p; Feldman RM et al.; In S . cerevisiae, the G1/S transition requires Cdc4p, Cdc34p, Cdc53p, Skp1p, and the Cln/Cdc28p cyclin-dependent kinase (Cdk) . These proteins are thought to promote the proteolytic inactivation of the S-phase Cdk inhibitor Sic1p . We show here that Cdc4p, Cdc53p, and Skp1p assemble into a ubiquitin ligase complex named SCFCdc4p . When mixed together, SCFCdc4p subunits, E1 enzyme, the E2 enzyme Cdc34p, and ubiquitin are sufficient to reconstitute ubiquitination of Cdk-phosphorylated Sic1p . Phosphorylated Sic1p substrate is specifically targeted for ubiquitination by binding to a Cdc4p/Skp1p subcomplex . Taken together, these data illuminate the molecular basis for the G1/S transition in budding yeast and suggest a general mechanism for phosphorylation-targeted ubiquitination in eukaryotes.

Biochem Biophys Res Commun, 1997 Oct 9, 239(1), 116 - 22
Isolation and characterization of the yeast mRNA capping enzyme beta subunit gene encoding RNA 5'-triphosphatase, which is essential for cell viability; Tsukamoto T et al.; The yeast Saccharomyces cerevisiae mRNA capping enzyme is composed of two subunits of alpha (52 kDa, mRNA guanylyltransferase) and beta (80 kDa, RNA 5'-triphosphatase) . We have isolated the alpha subunit gene (CEG1) by immunological screening . In this report, with the aid of partial amino acid sequences of purified yeast capping enzyme, we isolated the gene, designated CET1, encoding the S . cerevisiae capping enzyme beta subunit . Amino acid sequence analysis revealed that the gene encodes for 549 amino acids with a calculated M(r) of 61,800 which is unexpectedly smaller than the size estimated by SDS-PAGE . Gene disruption experiment showed that CET1 is essential for yeast cell growth . The purified recombinant CET1 gene product, Cet1, exhibited an RNA 5'-triphosphatase activity which specifically removed the gamma-phosphate from the triphosphate-terminated RNA substrate, but not from nucleoside triphosphates, confirming the identity of the gene . Interaction between the Cet1 and the Ceg1 was also studied by the West-Western procedure using recombinant Ceg1-{32P}GMP as probe.

J Biol Chem, 1997 Oct 10, 272(41), 25445 - 8
Requirement of yeast DNA polymerase delta in post-replicational repair of UV-damaged DNA; Torres-Ramos CA et al.; DNA lesions in the template strand pose a block to the replication machinery . Replication across such lesions may occur by a mutagenic bypass process in which a wrong base is inserted opposite the lesion or may involve processes that are relatively error-free . Genetic studies in the yeast Saccharomyces cerevisiae have indicated the requirement of REV3-encoded DNA polymerase in mutagenic bypass . The DNA polymerase responsible for error-free bypass, however, has not been identified, but genetic studies implicating proliferating cell nuclear antigen in this process have suggested that either DNA polymerase delta or DNA polymerase epsilon may be involved . Here, we use temperature-sensitive (ts) conditional lethal mutations of the S . cerevisiae POL2 and POL3 genes, which encode DNA polymerase epsilon and delta, respectively, and show that post-replicational bypass of UV-damaged DNA is severely inhibited in the pol3-3 mutant at the restrictive temperature . By contrast, the pol-2-18 mutation has no adverse effect on this process at the restrictive temperature . From these observations, we infer a requirement of DNA polymerase delta in post-replicative bypass of UV-damaged DNA.

Virology, 1997 Sep 29, 236(2), 374 - 81
Assembly of hepatitis delta virus-like empty particles in yeast; Wu HL et al.; Large delta antigen (L-HDAg) of hepatitis delta virus (HDV) and small-form hepatitis B surface antigen (HBsAg) of helper hepatitis B virus have previously been shown to be the minimum components for the assembly of HDV-like particles in mammalian cells . Extending from this finding, we coexpressed L-HDAg and small HBsAg in Saccharomyces cerevisiae to study their assembly in yeast cells . The assembly of virus particles from L-HDAg and HBsAg in yeast was demonstrated by their coexistence in the same isopycnic fractions and by the coimmunoprecipitation of L-HDAg with HBsAg using an antibody against HBsAg (anti-HBs) . Furthermore, after purification by affinity chromatography with anti-HBs, HDV-like particles with size and morphology similar to those derived from mammalian cells could be visualized by electron microscopy . Mice immunized with yeast-derived HDV-like particles simultaneously acquired antibodies against HBsAg and HDAg, indicating that both viral proteins are antigenic . The results indicated that S . cerevisiae could serve as a host for the assembly of HDV-like empty particles . This system may be useful in investigating cellular processes involved in HDV assembly and in producing ample amount of HDV-like particles for structural and immunological studies.

J Basic Microbiol, 1997, 37(4), 295 - 303
Trehalases from spores and vegetative cells of yeast Saccharomyces cerevisiae; Wolska-Mitaszko B; Trehalase (THA) activity from S . cerevisiae spores and vegetative cells could be differentiated in cell-free extracts . THA from the vegetative cells has an optimal activity at neutral pH whereas biphase pH optimum in the spores was observed . The enzyme from the spores exhibited higher thermostability than that from the vegetative cells . The presence of magnesium ions was necessary mainly for THA activity from the vegetative cells . The effect of the other metal ions studied: Hg2+, Ag2+, Cu2+, Fe3+, Ni2+, Cd2+ etc . (Table II), on THA from both sources was almost the same, however, the spores THA was resistant to Pb2+ and especially to Zn2+ . Moreover, the influence of inorganic polyphosphates and polyamines was also quite dissimilar . Polyphosphates inhibited THA from the vegetative cells and to a smaller extent from the spores . On the other hand, polyamines stimulated highly THA activity from vegetative yeast cells in contrast to spores one . The effect of these ions modulators would facilitate differentiating of THA activity in the cell-free extracts from both sources . These data could be interpreted as phenotypic reflections of trehalase genes expression in the S . cerevisiae cells.

Mol Cell Biol, 1997 Nov, 17(11), 6359 - 66
Mutations in genes encoding the mitochondrial outer membrane proteins Tom70 and Mdm10 of Podospora anserina modify the spectrum of mitochondrial DNA rearrangements associated with cellular death; Jamet-Vierny C et al.; Tom70 and Mdm10 are mitochondrial outer membrane proteins . Tom70 is implicated in the import of proteins from the cytosol into the mitochondria in Saccharomyces cerevisiae and Neurospora crassa . Mdm10 is involved in the morphology and distribution of mitochondria in S . cerevisiae . Here we report on the characterization of the genes encoding these proteins in the filamentous fungus Podospora anserina . The two genes were previously genetically identified through a systematic search for nuclear suppressors of a degenerative process displayed by the AS1-4 mutant . The PaTom70 protein shows 80% identity with its N . crassa homolog . The PaMdm10 protein displays 35.9% identity with its S . cerevisiae homolog, and cytological analyses show that the PaMDM10-1 mutant exhibits giant mitochondria, as does the S . cerevisiae mdm10-1 mutant . Mutations in PaTOM70 and PaMDM10 result in the accumulation of specific deleted mitochondrial genomes during the senescence process of the fungus . The phenotypic properties of the single- and double-mutant strains suggest a functional relationship between the Tom70 and Mdm10 proteins . These data emphasize the role of the mitochondrial outer membrane in the stability of the mitochondrial genome in an obligate aerobe, probably through the import process.

Mol Cell Biol, 1997 Nov, 17(11), 6223 - 35
Role of UME6 in transcriptional regulation of a DNA repair gene in Saccharomyces cerevisiae; Sweet DH et al.; In Saccharomyces cerevisiae UV radiation and a variety of chemical DNA-damaging agents induce the transcription of specific genes, including several involved in DNA repair . One of the best characterized of these genes is PHR1, which encodes the apoenzyme for DNA photolyase . Basal-level and damage-induced expression of PHR1 require an upstream activation sequence, UAS(PHR1), which has homology with DRC elements found upstream of at least 19 other DNA repair and DNA metabolism genes in yeast . Here we report the identification of the UME6 gene of S . cerevisiae as a regulator of UAS(PHR1) activity . Multiple copies of UME6 stimulate expression from UAS(PHR1) and the intact PHR1 gene . Surprisingly, the effect of deletion of UME6 is growth phase dependent . In wild-type cells PHR1 is induced in late exponential phase, concomitant with the initiation of glycogen accumulation that precedes the diauxic shift . Deletion of UME6 abolishes this induction, decreases the steady-state concentration of photolyase molecules and PHR1 mRNA, and increases the UV sensitivity of a rad2 mutant . Despite the fact that UAS(PHR1) does not contain the URS1 sequence, which has been previously implicated in UME6-mediated transcriptional regulation, we find that Ume6p binds to UAS(PHR1) with an affinity and a specificity similar to those seen for a URS1 site . Similar binding is also seen for DRC elements from RAD2, RAD7, and RAD53, suggesting that UME6 contributes to the regulated expression of a subset of damage-responsive genes in yeast.

J Virol, 1997 Nov, 71(11), 8852 - 5
Unique double-stranded RNAs responsible for the anti-Candida activity of the yeast Hanseniaspora uvarum; Schmitt MJ et al.; Killer strains of the yeast Hanseniaspora uvarum contain cytoplasmic double-stranded RNAs (dsRNAs) of 4.7-kbp L and 1.0-kbp M species, which were shown to be separately packaged into icosahedral virus-like particles exhibiting RNA-dependent RNA polymerase activity . The L genome of the H . uvarum L-dsRNA virion HuV-L was shown to encode a 77-kDa major capsid protein . Peptide maps of the purified HuV coat protein and the 81-kDa major capsid protein from K1 killer viruses of Saccharomyces cerevisiae revealed distinctly different peptide patterns, suggesting significant sequence divergence at the level of the capsid-coding L-dsRNAs . In vitro transcripts from purified HuV-L particles showed no cross-hybridization to denatured L(A), L(B), or L(C), indicating that L from H . uvarum represents a unique L-dsRNA species . Weak, but clearly detectable cross-hybridization of the 1.0-kb dsRNA of HuV-M, encoding the secreted 18-kDa anti-Candida toxin, to the toxin-coding M genomes of S . cerevisiae K1, K2, and K28 killers indicated partial sequence homology among all of the M-dsRNAs tested.

Curr Genet, 1997 Oct, 32(4), 260 - 6
MSS11, a novel yeast gene involved in the regulation of starch metabolism; Webber AL et al.; Expression of the STA1-3 glucoamylase genes, responsible for starch degradation in Saccharomyces cerevisiae, is down regulated by the presence of STA10 . In order to elucidate the role of STA10 in the regulation of the glucoamylase system, a multicopy genomic library was constructed and screened for genes that enhanced growth of a STA2-STA10 S . cerevisiae strain on starch media . This screen allowed us to clone and characterize a novel activator gene of STA2 (and by extrapolation, STA1 and STA3), designated MSS11 . A strain transformed with multiple copies of MSS11 exhibits increased levels of STA2 mRNA and, consequently, increased glucoamylase activity . Deletion of MSS11, located on chromosome XIII, results in media-dependent absence of glucoamylase synthesis . MSS11 has not been cloned previously and the encoded protein, Mss11p, is not homologous to any other known protein . An outstanding feature of Mss11p is that the protein contains regions of 33 asparagine residues interrupted by only three serine residues, and 35 glutamine residues interrupted by a single histidine residue . Epistasis studies showed that deletion of MSS11 abolishes the activation of STA2 caused by the over-expression of MSS10, a previously identified gene . In turn, it was found that deletion of MSS10 still allows activation of STA2 by over-expression of MSS11 . Mss11p therefore appears to be positioned below Mss10p in a signal transduction pathway.

Curr Genet, 1997 Oct, 32(4), 247 - 59
Chromosomal reorganization during meiosis of Saccharomyces cerevisiae baker's yeasts; Codon AC et al.; The genomic constitution of two S . cerevisiae baker's yeasts and their meiotic products have been analyzed by pulsed-field gel-electrophoresis, hybridization with specific gene probes, marker segregation, and flow cytometry . The parental strains have chromosomal patterns substantially different from those of laboratory strains used as controls . This pattern is partly the result of there being more than one copy of homologous chromosomes of different size, as judged by Southern-blot hybridization carried out with specific gene probes . Flow cytometry indicated that the strains have a 2.7 C DNA content . Tetrad analysis showed disomy for some chromosomes and tetrasomy for others . When two complete tetrads were subjected to molecular analysis the results confirmed instances of segregation of homologous chromosomes of different size . However, the presence of chromosomal bands absent in the parentals and the disappearance of chromosomal bands present in the parental strains were frequently seen . This result was attributed to two different phenomena: (1) the presence of multiple Ty1 and Ty2 transposable elements which seem to undergo interchromosomal translocation together with amplification, giving rise to differences in chromosomal size; (2) the presence of multiple Y' subtelomeric regions, giving rise to asymmetrical homologous recombination and, as a consequence, differences between the size of the recombinant chromosomes and the non-recombinant parental chromosomes . Chromosomal reorganization occurs with a very high frequency during meiosis . By contrast, mitosis is very stable, as judged by the reproducible electrophoretic karyotype shown by the parental strains in successive generations.

Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 11803 - 7
Cloning of mDEAH9, a putative RNA helicase and mammalian homologue of Saccharomyces cerevisiae splicing factor Prp43; Gee S et al.; Yeast splicing factor Prp43, a DEAH box protein of the putative RNA helicase/RNA-dependent NTPase family, is a splicing factor that functions late in the pre-mRNA splicing pathway to facilitate spliceosome disassembly . In this paper we report cDNA cloning and characterization of mDEAH9, an apparent mammalian homologue of Prp43 . Amino acid sequence comparison revealed that the two proteins are approximately 65% identical over a 500-aa region spanning the central helicase domain and the C-terminal region . Expression of mDEAH9 in S . cerevisiae bearing a temperature-sensitive mutation in prp43 was sufficient to restore growth at the nonpermissive temperature . This functional complementation was specific, as mouse mDEAH9 failed to complement mutations in related splicing factor genes prp16 or prp22 . Finally, double label immunofluorescence experiments performed with mammalian cells revealed colocalization of mDEAH9 and splicing factor SC35 in punctate nuclear speckles . Thus, the hypothesis that mDEAH9 represents the mammalian homologue of yeast Prp43 is supported by its high sequence homology, functional complementation, and colocalization with a known splicing factor in the nucleus . Our results provide additional support for the hypothesis that the spliceosomal machinery that mediates regulated, dynamic changes in conformation of pre-mRNA and snRNP RNAs has been highly conserved through evolution.

J Biol Chem, 1997 Oct 24, 272(43), 27042 - 52
Structure of cDNAs encoding human eukaryotic initiation factor 3 subunits . Possible roles in RNA binding and macromolecular assembly; Asano K et al.; The mammalian translation initiation factor 3 (eIF3), is a multiprotein complex of approximately 600 kDa that binds to the 40 S ribosome and promotes the binding of methionyl-tRNAi and mRNA . cDNAs encoding 5 of the 10 subunits, namely eIF3-p170, -p116, -p110, -p48, and -p36, have been isolated previously . Here we report the cloning and characterization of human cDNAs encoding the major RNA binding subunit, eIF3-p66, and two additional subunits, eIF3-p47 and eIF3-p40 . Each of these proteins is present in immunoprecipitates formed with affinity-purified anti-eIF3-p170 antibodies . Human eIF3-p66 shares 64% sequence identity with a hypothetical Caenorhabditis elegans protein, presumably the p66 homolog . Deletion analyses of recombinant derivatives of eIF3-p66 show that the RNA-binding domain lies within an N-terminal 71-amino acid region rich in lysine and arginine . The N-terminal regions of human eIF3-p40 and eIF3-p47 are related to each other and to 17 other eukaryotic proteins, including murine Mov-34, a subunit of the 26 S proteasome . Phylogenetic analyses of the 19 related protein sequences, called the Mov-34 family, distinguish five major subgroups, where eIF3-p40, eIF3-p47, and Mov-34 are each found in a different subgroup . The subunit composition of eIF3 appears to be highly conserved in Drosophila melanogaster, C . elegans, and Arabidopsis thaliana, whereas only 5 homologs of the 10 subunits of mammalian eIF3 are encoded in S . cerevisiae.

J Biol Chem, 1997 Oct 24, 272(43), 26799 - 802
Ubc9p is the conjugating enzyme for the ubiquitin-like protein Smt3p; Johnson ES et al.; At least one essential function of Smt3p, a Saccharomyces cerevisiae ubiquitin-like protein similar to the mammalian protein SUMO-1, involves its posttranslational covalent attachment to other proteins . Using Smt3p affinity chromatography, we have isolated the second enzyme of the Smt3p conjugation pathway and have found that it is identical to Ubc9p, a previously identified protein that has extensive sequence similarity to the ubiquitin-conjugating enzymes (E2s) and that is required for yeast to progress through mitosis . A hallmark of E2s is the ability to form a thioester bond-containing covalent intermediate with ubiquitin (Ub) . While we were unable to detect formation of a Ub approximately Ubc9p thioester, Ubc9p was found to form a thioester with Smt3p, indicating that Ubc9p is the functional analog of E2s in the Smt3p pathway and that this step is distinct from the ubiquitin pathway . Ubc9p is required for attachment of Smt3p to other proteins in vitro, suggesting that it is the only such enzyme in S . cerevisiae . These results suggest that, like ubiquitination, Smt3p conjugation may be a critical modification in cell cycle regulation.

Curr Genet, 1997 Sep, 32(3), 197 - 202
Development of pseudohyphae by embedded haploid and diploid yeast; Lo WS et al.; Diploid strains of S . cerevisiae are known to develop pseudohyphae in response to starvation for nitrogen . We report that both haploid and diploid yeast grow in a filamentous form when embedded in solid media . This is not a response to starvation, since yeast grown on rich media and overlaid with rich agar grow within the agar as pseudohyphae . While we find that the only element of diploidy required for formation of pseudohyphae in response to nitrogen starvation is the a1/alpha 2 repressor, pseudohyphal development by embedded cells does not require a1/alpha 2 . Deletion of BUD 5 prevented the formation of pseudohyphae by embedded cells, suggesting that these structures are the result of ordered filament formation rather than agar penetration . Deletion of STE 12 prevented the formation of pseudohyphae by all cell types, showing that the same signal transduction pathway is used by embedded cells as by those responding to nitrogen starvation . Different cell types of yeast thus form filaments in response to several kinds of environmental stimuli.

Cell, 1997 Oct 3, 91(1), 59 - 69
Components and dynamics of DNA replication complexes in S . cerevisiae: redistribution of MCM proteins and Cdc45p during S phase; Aparicio OM et al.; In S . cerevisiae, the chromatin structure of DNA replication origins changes as cells become competent for DNA replication, suggesting that G1 phase-specific association of replication factors with origin DNA regulates entry into S phase . We demonstrate that ORC, Cdc45p, and MCM proteins are components of prereplication complexes (pre-RC) . The MCM-origin association is dependent upon ORC and Cdc6p . During S phase, MCM proteins and Cdc45p dissociate from origin DNA and associate with nonorigin DNA with similar kinetics as DNA Polymerase epsilon, which is present at DNA replication forks . Our results identify protein components of the pre-RC and a novel replication complex appearing at the G1/S transition (the RC), and suggest that after initiation MCM proteins and Cdc45p move with eukaryotic replication forks.

Cell, 1997 Oct 3, 91(1), 47 - 57
A direct link between sister chromatid cohesion and chromosome condensation revealed through the analysis of MCD1 in S . cerevisiae; Guacci V et al.; The S . cerevisiae MCD1 (mitotic chromosome determinant) gene was identified in genetic screens for genes important for chromosome structure . MCD1 is essential for viability and homologs are found from yeast to humans . Analysis of the mcd1 mutant and cell cycle-dependent expression pattern of Mcd1p suggest that this protein functions in chromosome morphogenesis from S phase through mitosis . The mcd1 mutant is defective in sister chromatid cohesion and chromosome condensation . The physical association between Mcd1p and Smc1p, one of the SMC family of chromosomal proteins, further suggests that Mcd1p functions directly on chromosomes . These data implicate Mcd1p as a nexus between cohesion and condensation . We present a model for mitotic chromosome structure that incorporates this previously unsuspected link.

Micron, 1997 Jun, 28(3), 221 - 30
Cerium-based ultracytochemical localization of aspartate transcarbamylase activity in the cell membrane complex of Saccharomyces cerevisiae; Vorisek J et al.; Aspartate transcarbamylase (ATCase) activity was localized ultracytochemically in the yeast Saccharomyces cerevisiae by precipitation of its reaction product orthophosphate as cerium phosphate . We prefixed yeast cells with ice-cold 1% glutaraldehyde for 30 min which preserved 80% of ATCase activity . Cells were washed and incubated with ATCase substrates (aspartate, carbamyl phosphate) plus cerium chloride, and postfixed by osmium tetroxide . In cells from exponential batch cultures, deposits of cerium phosphate delineated simultaneously or alternatively membranes of the secretory pathway: nuclear envelope, endoplasmic reticulum, Golgi complex and the plasmalemma; mitochondrial membranes and intramitochondrial fibrous component were labelled as well . Deposits of cerium phosphate were never observed in the nucleoplasm . Cells incubated in the absence of cerium or ATCase substrates and mutant S . cerevisiae cells lacking ATCase activity served as controls . Small round electron-dense condensates were found to be randomly distributed within some cells, both in control and experimental runs, in the nucleoplasm, cytoplasm and mitochondrial matrix and represented undefined osmicated endogenous compounds . Our results suggest that the synthesis of pyrimidine precursors occurs in membranes, where compounds such as UDP-glucose and CDP-diglycerides are needed for membrane and/or yeast cell wall synthesis . The possible contribution of ATCase activity found in the nuclear envelope to nucleic acid synthesis remains to be clarified.

DNA Res, 1997 Jun 30, 4(3), 205 - 13
Identification of likely genes on chromosome VI of Saccharomyces cerevisiae by correlating transcripts and nucleotide sequence data; Baba T et al.; Most of the 97 transcripts of the genes on chromosome VI of Saccharomyces cerevisiae that were identified by a series of Northern hybridization experiments (Yoshikawa and Isono, Nucl . Acids Res., 19, 1189-1195, 1991) have been correlated with the open reading frames (ORFs) deduced from the nucleotide sequence data of this chromosome (Murakami et al., Nature Genet., 10, 261-268, 1995) . This was performed by comparing the experimentally constructed physical map and the one produced from the nucleotide sequence data, as well as the sizes and positions of observed transcripts and those of sequenced ORFs . Thus, 75 ORFs of chromosome VI were correlated uniquely with the corresponding transcripts and 3 ORFs with two transcripts of different sizes . Comparing the relative abundance levels of individual transcripts with that of the RPO41 transcript, highly expressed genes of chromosome VI were found to be located almost exclusively on the Crick strand . Based on the correlation between the abundance level of the experimentally identified transcripts and the codon adaptation indices of the corresponding gene, the genes on chromosome VI of S . cerevisiae were classified into three groups . The data thus provides information concerning their chromosomal locations as well as their likely levels of expression in vegetatively growing cells.

Biochem Biophys Res Commun, 1997 Sep 29, 238(3), 811 - 6
Functional complementation of a membrane transport deficiency in Saccharomyces cerevisiae by recombinant ND4 fusion protein; Hogue DL et al.; ND4 is a mitochondrially encoded component of NADH dehydrogenase (Complex I) of the respiratory chain . A cDNA encoding a fusion protein comprised of the N-terminus of GAL10 of Saccharomyces cerevisiae and an N-terminally truncated form of human ND4 was isolated by its ability to functionally complement the thymidine transport deficiency of S . cerevisiae grown under conditions of thymidylate starvation . Epitope-tagged GAL10-ND4 was shown by immunological methods to be present in the plasma membranes of yeast expressing the GAL10-ND4 encoding cDNA . The ability of the GAL10-ND4 fusion protein to induce uptake of thymidine raises the possibility that native ND4, which is predicted to have 12 transmembrane domains, may function as a transporter or channel in the mitochondrial inner membrane.

Cell, 1997 Sep 19, 90(6), 1041 - 50
Exportin 1 (Crm1p) is an essential nuclear export factor; Stade K et al.; Nuclear protein export is mediated by nuclear export signals (NESs), but the mechanisms governing this transport process are not well understood . Using a novel protein export assay in S . cerevisiae, we identify CRM1 as an essential mediator of nuclear protein export in yeast . Crm1p shows homology to importin beta-like transport factors and is able to specifically interact with both the NES motif and the Ran GTPase . A mutation in the shuttling protein Crm1p affects not only protein export, but also mRNA export, indicating that these pathways are tightly coupled in S . cerevisiae . The presented data are consistent with the conclusion that Crm1p is a carrier for the NES-mediated protein export pathway . We propose CRM1 be renamed exportin 1 (XPO1).

FEBS Lett, 1997 Sep 8, 414(2), 439 - 43
Isolation and expression of the gene which encodes a novel enzyme with polymethoxygalacturonate-degrading activity in Trichosporon penicillatum; Sakai T et al.; The novel gene named PSX1, encoding a new protopectinase with the polymethoxygalacturonase activity, was isolated from Trichosporon penicillatum . Nucleotide sequencing revealed that the PSX1 gene is composed of 1080 bases (360 amino acids, 38,747 Da) . The N-terminal amino acid sequences of the open reading frame correspond to a signal peptide and propeptide processed by a Kex2-like proteinase . Mature PPase SX1 was composed of 334 amino acids (36,121 Da) . PPase SX1 produced by a S . cerevisiae transformant harboring the PSX1 gene degraded methoxylated polygalacturonic acid as a substrate, but not degraded unmethoxylated polygalacturonic acid.

FEBS Lett, 1997 Sep 8, 414(2), 353 - 8
Involvement of CIF1 (GGS1/TPS1) in osmotic stress response in Saccharomyces cerevisiae; Hazell BW et al.; The transcriptional responses of the osmotically induced genes ALD2, CTT1, ENA1, GPD1, HSP12 and HSP104, were studied in Saccharomyces cerevisiae strains differing in CIF1 gene function following application of osmotic stress . The CIF1 gene (allelic to GGS1 and TPS1) encodes a subunit of the trehalose synthase complex that affects trehalose synthesis . Recent work has implicated this gene in various signalling events in the cell, including transcriptional response to heat-shock treatment . Because many genetic factors can influence S . cerevisiae osmoresponse, we have compared the expression of osmotically induced genes and glycerol production in isogenic strains differing only in functionality of CIF1, growing logarithmically on galactose medium . When cultures were exposed to 0.8 M NaCl or 1.5 M sorbitol the cif1 strain showed greatly reduced transcription of osmotically induced genes compared to the wild type . These treatments did not affect viability of the yeast strains . Treatment with 0.3 M NaCl produced no significant differences in transcription of these genes in CIF1 or cif1 strains . Treatment with 0.6 M sorbitol induced small but reproducible differences, with gene expression higher in the CIF1 strain compared to the cif1 mutant . When cultures were treated with 0.3 M NaCl or 0.6 M sorbitol for 1 h, glycerol production was similar for both strains, but after 3 h of the same treatment, total glycerol production was higher in the CIF1 strain . When cultures were treated with 0.8 M NaCl for 3 h, the wild type strain produced more glycerol than the mutant strain . Both strains produced similar amounts of glycerol following exposure to 1.5 M sorbitol for 3 h, although the wild type strain showed enhanced ability to retain glycerol inside the cell . The results are discussed in the context of the possible role that the CIF1 gene product has in response to osmotic stress.

Mol Cell Biol, 1997 Oct, 17(10), 5803 - 12
Mpp10p, a U3 small nucleolar ribonucleoprotein component required for pre-18S rRNA processing in yeast; Dunbar DA et al.; We have isolated and characterized Mpp10p, a novel protein component of the U3 small nucleolar ribonucleoprotein (snoRNP) from the yeast Saccharomyces cerevisiae . The MPP10 protein was first identified in human cells by its reactivity with an antibody that recognizes specific sites of mitotic phosphorylation . To study the functional role of MPP10 in pre-rRNA processing, we identified the yeast protein by performing a GenBank search . The yeast Mpp10p homolog is 30% identical to the human protein over its length . Antibodies to the purified yeast protein recognize a 110-kDa polypeptide in yeast extracts and immunoprecipitate the U3 snoRNA, indicating that Mpp10p is a specific protein component of the U3 snoRNP in yeast . As a first step in the genetic analysis of Mpp10p function, diploid S . cerevisiae cells were transformed with a null allele . Sporulation and tetrad analysis indicate that MPP10 is an essential gene . A strain was constructed where Mpp10p is expressed from a galactose-inducible, glucose- repressible promoter . After depletion of Mpp10p by growth in glucose, cell growth is arrested and levels of 18S and its 20S precursor are reduced or absent while the 23S and 35S precursors accumulate . This pattern of accumulation of rRNA precursors suggests that Mpp10p is required for cleavage at sites A0, A1, and A2 . Pulse-chase analysis of newly synthesized pre-rRNAs in Mpp10p-depleted yeast confirms that little mature 18S rRNA formed . These results reveal a novel protein essential for ribosome biogenesis and further elucidate the composition of the U3 snoRNP.

J Cell Biol, 1997 Oct 6, 139(1), 75 - 93
A septin-based hierarchy of proteins required for localized deposition of chitin in the Saccharomyces cerevisiae cell wall; DeMarini DJ et al.; Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell . The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S . cerevisiae . The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the "neck filaments" that lie immediately subjacent to the plasma membrane in the mother-bud neck . In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III . Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III . Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs . Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology . Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants . Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition) . This localization depends on the septins but not on Chs4p or Chs3p . A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side . This localization is dependent on the septins, Bni4p, and Chs3p . Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p . In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p . Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.

Eur J Cell Biol, 1997 Sep, 74(1), 31 - 40
Human Rer1 is localized to the Golgi apparatus and complements the deletion of the homologous Rer1 protein of Saccharomyces cerevisiae; Fullekrug J et al.; Sec12p is a type II membrane glycoprotein in the endoplasmic reticulum (ER) of Saccharomyces cerevisiae which is essential for transport vesicle budding . It is the guanine nucleotide exchange factor for the small GTP-binding protein Sar1p which is a constituent of COP II ER to Golgi vesicles . We report the sequence and localization of the human homologue to yeast Rer1p, which has recently been identified genetically as an essential component for retention of Sec12p in the ER . Reverse polymerase chain reaction was used to obtain cDNAs from HeLa cells . They code for a protein of 196 amino acids, corresponding to a molecular mass of 23 kDa . The translated sequence is 44% identical and 65% similar to yeast Rer1 protein . The four putative transmembrane domains are predicted to form a W-topology with both N- and C-terminus facing the cytosol . The functional activity of myc-tagged human Rer1 was demonstrated by the complementation of the RER1 deletion in S . cerevisiae . Mislocalization of the Sec12-reporter protein was reduced similar to the results obtained with yeast Rer1p . Human Rer1 protein was expressed in HeLa cells and the subcellular distribution analyzed by double immunofluorescence and immunoelectron microscopy of thawed cryosections . The tagged protein was localized to the Golgi apparatus and peripheral elements of the ER-Golgi interface . High overexpression leads to relocation of human Rer1 to ER-like structures together with KDEL-receptor and affects the structural organization of the Golgi apparatus . Under conditions of brefeldin A treatment, human Rer1 distributes together with recycling Golgi proteins.

Prog Nucleic Acid Res Mol Biol, 1998, 58, 197 - 237
Molecular biology of trehalose and the trehalases in the yeast Saccharomyces cerevisiae; Nwaka S et al.; The present state of knowledge of the role of trehalose and trehalose hydrolysis catalyzed by trehalase (EC 3.2.1.28) in the yeast Saccharomyces cerevisiae is reviewed . Trehalose is believed to function as a storage carbohydrate because its concentration is high during nutrient limitations and in resting cells . It is also believed to function as a stress metabolite because its concentration increases during certain adverse environmental conditions, such as heat and toxic chemicals . The exact way trehalose may perform the stress function is not understood, and conditions exist under which trehalose accumulation and tolerance to certain stress situations cannot be correlated . Three trehalases have been described in S . cerevisiae: 1) the cytosolic neutral trehalase encoded by the NTH1 gene, and regulated by cAMP-dependent phosphorylation process, nutrients, and temperature; 2) the vacuolar acid trehalase encoded by the ATH1 gene, and regulated by nutrients; and 3) a putative trehalase Nth1p encoded by the NTH2 gene (homolog of the NTH1 gene) and regulated by nutrients and temperature . The neutral trehalase is responsible for intracellular hydrolysis of trehalose, in contrast to the acid trehalase, which is responsible for utilization of extracellular trehalose . The role of the putative trehalase Nth2p in trehalose metabolism is not known . The NTH1 and NTH2 genes are required for recovery of cells after heat shock at 50 degrees C, consistent with their heat inducibility and sequence similarity . Other stressors, such as toxic chemicals, also induce the expression of these genes . We therefore propose that the NTH1 and NTH2 genes have stress-related function and the gene products may be called stress proteins . Whether the stress function of the trehalase genes is linked to trehalose is not clear, and possible mechanisms of stress protective function of the trehalases are discussed.

Prog Nucleic Acid Res Mol Biol, 1998, 58, 165 - 95
Copper-regulatory domain involved in gene expression; Winge DR; Copper ion homeostasis in yeast is maintained through regulated expression of genes involved in copper ion uptake, Cu(I) sequestration, and defense against reactive oxygen intermediates . Positive and negative copper ion regulation is observed, and both effects are mediated by Cu(I)-sensing transcription factors . The mechanism of Cu(I) regulation is distinct for transcriptional activation versus transcriptional repression . Cu(I) activation of gene expression in S . cerevisiae and C . glabrata occurs through Cu-regulated DNA binding . The activation process involves Cu(I) cluster formation within the regulatory domain in Ace1 and Amt1 . Cu(I) binding stabilizes a specific conformation capable of high-affinity interaction with specific DNA promoter sequences . Cu(I)-activated transcription factors are modular proteins in which the DNA-binding domain is distinct from the domain that mediates transcriptional activation . The all-or-nothing formation of the polycopper cluster permits a graded response of the cell to environmental copper . Cu(I) triggering may involve a metal exchange reaction converting Ace1 from a Zn(II)-specific conformer to a clustered Cu(I) conformer . The Cu(I) regulatory domain occurs in transcription factors from S . cerevisiae and C . glabrata . Sequence homologs are also known in Y . lipolytica and S . pombe, although no functional information is available for these candidate regulatory molecules . The presence of the Cu(I) regulatory domain in four distinct yeast strains suggests that this Cu-responsive domain may occur in other eukaryotes . Cu-mediated repression of gene expression in S . cerevisiae occurs through Cu(I) regulation of Mac1 . Cu(I) binding to Mac1 appears to inhibit the transactivation domain . The Cu(I) specificity of this repression is likely to arise from formation of a polycopper thiolate cluster.

J Biol Chem, 1997 Sep 26, 272(39), 24530 - 5
The AtVAM3 encodes a syntaxin-related molecule implicated in the vacuolar assembly in Arabidopsis thaliana; Sato MH et al.; The vacuole constitutes a large compartment in plant and fungal cells . The VAM3 gene of Saccharomyces cerevisiae encodes a syntaxin-related protein required for vacuolar assembly . An Arabidopsis thaliana cDNA library, designed for expression in S . cerevisiae, was screened for cDNAs able to complement defective vacuolar assembly of the Deltavam3 mutation . One cDNA, encoding a 33-kDa protein with structural similarities to the other syntaxins, was identified . The product of AtVAM3 (AtVam3p) was expressed in various tissues including roots, leaves, inflorescence stems, flower buds, and young siliques . The AtVAM3 transcripts were abundant in undifferentiated cells in the meristematic region . AtVam3p fractionated predominantly to an 8,000 x g pellet fraction where a vacuolar membrane protein H+-translocating inorganic pyrophosphatase (H+-PPase) also fractionated . Immunoelectron microscopy showed that AtVam3p was localized to restricted regions on the vacuolar membranes . We propose that AtVam3p provides the t-SNARE function in the vacuolar assembly in A . thaliana.

Mol Microbiol, 1997 Aug, 25(3), 541 - 6
Regulation of inositol monophosphatase in Saccharomyces cerevisiae; Murray M et al.; Inositol monophosphatase is a key enzyme in the de novo biosynthesis of inositol and in the phosphoinositide second-messenger signalling pathway . Inhibition of this enzyme is a proposed mechanism for lithium's pharmacological action in bipolar illness (manic depression) . Very little is known about how expression of this enzyme is regulated . Because the yeast Saccharomyces cerevisiae has been shown to be an excellent model system in which to understand the regulation of inositol metabolism, we characterized inositol monophosphatase in this yeast . Lithium inhibited monophosphatase activity in vitro . Growth in the presence of inositol resulted in increased expression of the enzyme in vivo, although inositol had no effect on enzyme activity in vitro . The inositol effect was apparent when cells were grown in glucose but not in glycerol/ethanol . Monophosphatase activity was derepressed as cells entered stationary phase . This effect was apparent only during growth in glucose plus inositol . The results demonstrate that S . cerevisiae monophosphatase is inhibited by lithium and regulated by factors affecting phospholipid biosynthesis.

Yeast, 1997 Sep 30, 13(12), 1155 - 66
Ribosomal protein L9 is the product of GRC5, a homolog of the putative tumor suppressor QM in S . cerevisiae; Nika J et al.; Genes encoding members of the highly conserved QM family have been identified in eukaryotic organisms from yeast to man . Results of previous studies have suggested roles for QM in control of cell growth and proliferation, perhaps as a tumor suppressor, and in energy metabolism . We identified recessive lethal alleles of the Saccharomyces cerevisiae QM homolog GRC5 that increased GCN4 expression when present in multiple copies . These alleles encode truncated forms of the yeast QM protein Grc5p . Using a functional epitope-tagged GRC5 allele, we localized Grc5p to a 60S fraction that contained the large ribosomal subunit . Two-dimensional gel analysis of highly purified yeast ribosomes indicated that Grc5p corresponds to 60S ribosomal protein L9 . This identification is consistent with the predicted physical characteristics of eukaryotic QM proteins, the highly biased codon usage of GRC5, and the presence of putative Rap1p-binding sites in the 5' sequences of the yeast GRC5 gene.

Biol Chem, 1997 Aug, 378(8), 893 - 8
Synthesis, kinetic characterization and X-ray analysis of peptide aldehydes as inhibitors of the 20S proteasomes from Thermoplasma acidophilum and Saccharomyces cerevisiae; Escherich A et al.; A comparative kinetic characterization of the peptide aldehydes Ac-Leu-Leu-X-H {X = Trp, Tyr and Tyr(tBu)} and Z-Gly-Pro-Gly-Gly-Leu-Leu-Nle-H as inhibitors of the chymotryptic activity of 20S proteasomes from the archaebacterium T . acidophilum and yeast S . cerevisiae revealed significantly differentiated inhibitory potencies that can be rationalized on the basis of X-ray crystallographic data.

Appl Microbiol Biotechnol, 1997 Aug, 48(2), 204 - 7
A counter-selectable marker for genetic transformation of the yeast Schwanniomyces alluvius; Dave MN et al.; We report here a counter-selectable marker system for genetic transformation of the yeast Schwanniomyces alluvius, based on the complementation of uracil auxotrophs defective in either orotidine-5'-phosphate decarboxylase (URA3) or orotidine-5'-pyrophosphate (URA5) . Uracil auxotrophs of S . alluvius were obtained by ethyl methanesulphonate mutagenesis and complemented using the ura3 gene from S . cerevisiae . A transformation frequency of approximately 10(4)/micrograms DNA was obtained, which is tenfold higher than results described in either reports . Transformants were analysed by Southern blot hybridisation and were found to be mitotically stable . The extrachromosomal nature of the transforming DNA was confirmed by Southern hybridisation and plasmid rescue . The rescued plasmid DNA had a restriction pattern identical to that of the parent plasmid.

Genomics, 1997 Sep 1, 44(2), 188 - 94
Cloning and expression analysis of a meiosis-specific MutS homolog: the human MSH4 gene; Paquis-Flucklinger V et al.; The Escherichia coli MutHLS system has been highly conserved throughout evolution . The eukaryotic pathway results in a specialization of MutS homologs that have evolved to play crucial roles in both DNA mismatch repair and meiotic recombination . So far, meiosis-specific genes belonging to this family have only been identified in yeast . In Saccharomyces cerevisiae, MSH4 (MutS homolog 4) is a meiosis-specific protein that is not involved in mismatch correction . This protein is required for reciprocal recombination and proper segregation of homologous chromosomes at meiosis I . We have identified the human MSH4 homolog gene . The predicted amino acid sequence shows 28.7% identity with the S . cerevisiae MSH4 protein . By Northern blot analysis, human MSH4 transcripts are only detectable in testis and in ovary with a lower level of expression . We have mapped MSH4 to human chromosome 1 at band p31 by fluorescence in situ hybridization . The identification of such a gene provides a powerful tool for clarifying the respective roles of MSH and MLH genes in mammalian meiosis .

Mol Biochem Parasitol, 1997 Oct, 89(1), 73 - 83
Unusual genome organisation in Entamoeba histolytica leads to two overlapping transcripts; Gangopadhyay SS et al.; We have isolated homologs of the mini chromosome maintenance (MCM) gene family from the parasitic protozoan Entamoeba histolytica . The full length genomic and cDNA clones for the Eh MCM3 gene have been characterised . The Eh MCM3 gene is much smaller than the Saccharomyces cerevisiae MCM3 gene and other eukaryotic homologs of the MCM3/P1 family . The predicted Eh Mcm3 protein was 597 amino acids long and showed 37 and 46% positional identity with the Sc Mcm3 and the mouse P1 homologs respectively . While proceeding along the chromosome from the Eh MCM3 gene, we have identified a homolog (Eh PAK) of the murine p21 activated kinase (Rn KPAK), or S . cerevisiae STE20 . Eh PAK lies 126 bp upstream of the Eh MCM3 gene . The predicted Eh p21 activated kinase protein was 459 amino-acids long and showed 33% positional identity with the murine p21 activated kinase and its yeast homolog Ste20 . Analysis of cDNA and genomic sequences shows that the 3' untranslated region (UTR) of the Eh PAK mRNA and the 5' UTR of the Eh MCM3 mRNA are transcribed from a common 40 bp genomic segment . This is the first report of an amoeba gene being physically linked to a second gene such that their transcripts are overlapping and there is no non-transcribed intergenic region between the two genes . Primer extension studies have confirmed that unlike most E . histolytica genes, which have short 5' UTRs, the Eh MCM3 mRNA has a 126 bp long 5' UTR and the Eh PAK mRNA has a 265 bp long 5' UTR.

Prikl Biokhim Mikrobiol, 1997 May-Jun, 33(3), 325 - 8
{Morphological and biochemical characteristics of new isolates Saccharomyces cerevisiae U-503}; Abramov ShA et al.; Compared with S . cerevisiae N73, its laser irradiation-induced mutant S . cerevisiae U-503 exhibited a significantly higher respiration rate . Electron microscopic changes consistent with this finding were found in the mitochondrial system of mutant cells . The mutant strain retained its physiological and biochemical properties over a nine-year storage period.

Mol Gen Genet, 1997 Aug, 255(5), 533 - 42
UAS(MAG1), a yeast cis-acting element that regulates the expression of MAG1, is located within the protein coding region of DDI1; Liu Y et al.; MAG1 and DDI1 are two divergently transcribed DNA damage-inducible genes from Saccharomyces cerevisiae . Previous studies have shown that MAG1 induction requires an upstream activating site (UAS) located between nucleotides -376 and -330 . Here we show that a 24-bp oligonucleotide from within the UAS(MAG1) region forms a sequence-specific DNA-protein complex with partially purified proteins from S . cerevisiae . Point mutations introduced into the 24-bp oligonucleotide inhibited the formation of the DNA-protein complex and decreased the level of induction of MAG1-lacZ . By determining the transcription and translation start points of both MAG1 and DDI1, an interesting, indeed unprecedented feature of genome organization in eukaryotes was revealed: UAS(MAG1) actually lies within the protein-coding region of DDI1 . Although tightly linked to each other, and co-induced upon treatment with DNA-damaging agents, DDI1 does not share the UAS(MAG1) required for DNA damage induction of MAG1 . Furthermore, MAG1 and DDI1 respond differently in the presence of the protein synthesis inhibitor cycloheximide, suggesting that these two genes are regulated by different mechanisms in the absence of de novo protein synthesis.

Yeast, 1997 Sep 15, 13(11), 1077 - 90
Sequence analysis of 203 kilobases from Saccharomyces cerevisiae chromosome VII; Rieger M et al.; The nucleotide sequences of five major regions from chromosome VII of Saccharomyces cerevisiae have been determined and analysed . These regions represent 203 kilobases corresponding to approximately one-fifth of the complete yeast chromosome VII . Two fragments originate from the left arm of this chromosome . The first one of about 15.8 kb starts approximately 75 kb from the left telomere and is bordered by the SK18 chromosomal marker . The second fragment covers the 72.6 kb region between the chromosomal markers CYH2 and ALG2 . On the right chromosomal arm three regions, a 70.6 kb region between the MSB2 and the KSS1 chromosomal markers and two smaller regions dominated by the KRE11 marker and another one in the vicinity of the SER2 marker were sequenced . We found a total of 114 open reading frames (ORFs), 13 of which were completely overlapping with larger ORFs running in the opposite direction . A total of 44 yeast genes, the physiological functions of which are known, could be precisely mapped on this chromosome . Of the remaining 57 ORFs, 26 shared sequence homologies with known genes, among which were 13 other S . cerevisiae genes and five genes from other organisms . No homology with any sequence in the databases could be found for 31 ORFs . Furthermore, five Ty elements were found, one of which may not be functional due to a frame shift in its Ty1B amino acid sequence . The five chromosomal regions harboured five potential ARS elements and one sigma element together with eight tRNA genes and two snRNAs, one of which is encoded by an intron of a protein-coding gene.

Yeast, 1997 Sep 15, 13(11), 1065 - 75
Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae; Wach A et al.; We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosac-charomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii . Both chimeric modules and the cognate S . kluyveri HIS3 gene were tested in transformations of his3 S . cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences . The 1.4 kb chimeric Sz . pombe module (HIS3MX6) performed best . With less than 5% incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX . The rare false-positive His+ transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele . We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA-plasmids with HIS3MX6 and kanMX markers . The 0.9 kb GFP reporters consist of wild-type GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S . cerevisiae ADH1 terminator . PCR-synthesized 2.4 kb-long double modules flanked by 40-45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S . cerevisiae genes . We could estimate that only about 10% of the transformants carried inactivating mutations in the GFP reporter.

J Cell Biol, 1997 Sep 8, 138(5), 1041 - 53
Mitotic spindle positioning in Saccharomyces cerevisiae is accomplished by antagonistically acting microtubule motor proteins; Cottingham FR et al.; Proper positioning of the mitotic spindle is often essential for cell division and differentiation processes . The asymmetric cell division characteristic of budding yeast, Saccharomyces cerevisiae, requires that the spindle be positioned at the mother-bud neck and oriented along the mother-bud axis . The single dynein motor encoded by the S . cerevisiae genome performs an important but nonessential spindle-positioning role . We demonstrate that kinesin-related Kip3p makes a major contribution to spindle positioning in the absence of dynein . The elimination of Kip3p function in dyn1Delta cells severely compromised spindle movement to the mother-bud neck . In dyn1Delta cells that had completed positioning, elimination of Kip3p function caused spindles to mislocalize to distal positions in mother cell bodies . We also demonstrate that the spindle-positioning defects exhibited by dyn1 kip3 cells are caused, to a large extent, by the actions of kinesin- related Kip2p . Microtubules in kip2Delta cells were shorter and more sensitive to benomyl than wild-type, in contrast to the longer and benomyl-resistant microtubules found in dyn1Delta and kip3Delta cells . Most significantly, the deletion of KIP2 greatly suppressed the spindle localization defect and slow growth exhibited by dyn1 kip3 cells . Likewise, induced expression of KIP2 caused spindles to mislocalize in cells deficient for dynein and Kip3p . Our findings indicate that Kip2p participates in normal spindle positioning but antagonizes a positioning mechanism acting in dyn1 kip3 cells . The observation that deletion of KIP2 could also suppress the inviability of dyn1Delta kar3Delta cells suggests that kinesin-related Kar3p also contributes to spindle positioning.

Genetics, 1997 Sep, 147(1), 87 - 100
In vivo analysis of Saccharomyces cerevisiae COX2 mRNA 5'-untranslated leader functions in mitochondrial translation initiation and translational activation; Dunstan HM et al.; We have used mutational and revertant analysis to study the elements of the 54-nucleotide COX2 5'-untranslated leader involved in translation initiation in yeast mitochondria and in activation by the COX2 translational activator . Pet111p . We generated a collection of mutants with substitutions spanning the entire COX2 5'-UTL by in vitro mutagenesis followed by mitochondrial transformation and gene replacement . The phenotypes of these mutants delimit a 31-nucleotide segment, from -16 to -46, that contains several short sequence elements necessary for COX2 5'-UTL function in translation . The sequences from -16 to -47 were shown to be partially sufficient to promote translation in a foreign context . Analysis of revertants of both the series of linker-scanning alleles and two short deletion/ insertion alleles has refined the positions of several possible functional elements of the COX2 5'-untranslated leader, including a putative RNA stem-loop structure that functionally interacts with Pet111p and an octanucleotide sequence present in all S . cerevisiae mitochondrial mRNA 5'-UTLs that is a potential rRNA binding site.

FASEB J, 1997 Sep, 11(11), 917 - 21
S . cerevisiae and sulfur: a unique way to deal with the environment; Scheibel T et al.; Saccharomyces cerevisiae is by far the best-studied unicellular eukaryote . Although yeast cells are very similar to higher eukaryotes in many respects, there is striking evidence that S . cerevisiae is not a perfect model for a eukaryotic cell (cf . 1) . Here we report that yeast proteins contain a significantly lower amount of cysteine residues compared to other eukaryotes . Explanations for this phenomenon could not be found in the sulfur metabolism of yeast, which showed no major differences from other organisms (2-4) . However, previous examinations could link a defect in sulfate uptake of S . cerevisiae to an increased resistance against toxic substances like selenate and chromate in the environment, which share the same permeases (5-7) . This environmental problem might have caused S . cerevisiae to down-regulate its sulfate uptake and therefore lead to a lower amount of available sulfur in the cell, making it necessary to replace all dispensable sulfur amino acids in proteins . We show in two examples that S . cerevisiae proteins contain only such cysteine residues that are structurally or functionally needed . Therefore, we conclude that S . cerevisiae has solved a widespread environmental problem in a specific way which might be unique among eukaryotes.

Cell, 1997 Aug 22, 90(4), 649 - 60
Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs; Tanaka T et al.; In eukaryotic cells, firing of DNA replication origins normally does not recur until after M phase . This characteristic is thought to be due to the properties of "initiation" proteins like Orc, Cdc6, and Mcms . Using formaldehyde cross-linking, we show that Cdc6p and Mcm7p associate specifically with replication origins during G1 but not during G2 in S . cerevisiae . Mcm7p's association with origins depends on Cdc6p . Ectopic expression of Cdc6p enables it to associate with origins during G2, but this fails to recruit Mcm7p . Our data suggest that the loading of Mcm proteins onto origins is regulated by two mechanisms: first, by Cdc6p occupancy, and second, by S- and M-CDKs, whose activity during S, G2, and M phases prevents Mcm loading.

J Biol Chem, 1997 Aug 22, 272(34), 21534 - 9
High levels of the GTPase Ran/TC4 relieve the requirement for nuclear protein transport factor 2; Paschal BM et al.; The GTPase Ran/TC4 and the 14-kDa protein nuclear transport factor 2 (NTF2) are two of the cytosolic factors that mediate nuclear protein import in vertebrates . Previous biochemical studies have shown that NTF2 binds directly to the GDP-bound form of Ran/TC4 and to proteins of the nuclear pore complex that contain phenylalanine-glycine repeats . In the present study we have used molecular genetic approaches to study the Saccharomyces cerevisiae homologue of NTF2 . The scNTF2 gene encodes a protein that is 44% identical to the human protein . We found that deletion of the scNTF2 gene is lethal and that repression of NTF2p expression by a regulatable promoter results in gross structural distortions of the nuclear envelope . In a screen for high copy number suppressors of a scNTF2 deletion, the only gene we isolated other than scNTF2 itself was GSP1, the S . cerevisiae homologue of Ran/TC4 . Furthermore, we found that high levels of Ran/TC4 can relieve the requirement for NTF2 in a mammalian-permeabilized cell assay for nuclear protein import . These data suggest that certain of the nuclear protein import functions of NTF2 and Ran/TC4 are closely linked and that NTF2 may serve to modulate a transport step involving Ran/TC4.

J Biol Chem, 1997 Aug 22, 272(34), 21381 - 7
Crystal structure of murine/human Ubc9 provides insight into the variability of the ubiquitin-conjugating system; Tong H et al.; Murine/human ubiquitin-conjugating enzyme Ubc9 is a functional homolog of Saccharomyces cerevisiae Ubc9 that is essential for the viability of yeast cells with a specific role in the G2-M transition of the cell cycle . The structure of recombinant mammalian Ubc9 has been determined from two crystal forms at 2.0 A resolution . Like Arabidopsis thaliana Ubc1 and S . cerevisiae Ubc4, murine/human Ubc9 was crystallized as a monomer, suggesting that previously reported hetero- and homo-interactions among Ubcs may be relatively weak or indirect . Compared with the known crystal structures of Ubc1 and Ubc4, which regulate different cellular processes, Ubc9 has a 5-residue insertion that forms a very exposed tight beta-hairpin and a 2-residue insertion that forms a bulge in a loop close to the active site . Mammalian Ubc9 also possesses a distinct electrostatic potential distribution that may provide possible clues to its remarkable ability to interact with other proteins . The 2-residue insertion and other sequence and structural heterogeneity observed at the catalytic site suggest that different Ubcs may utilize catalytic mechanisms of varying efficiency and substrate specificity.

Biochem Biophys Res Commun, 1997 Aug 18, 237(2), 362 - 6
Modulation of lipid unsaturation and membrane fluid state in mammalian cells by stable transformation with the delta9-desaturase gene of Saccharomyces cerevisiae; Gyorfy Z et al.; The composition and physical state of membrane lipids determine the dynamic nature of membranes, which in turn, could directly be linked to the activity of various membrane-associated cellular functions . To better understand the molecular basis of different membrane-related phenomena we established a novel strategy to alter unsaturation of mammalian cell membranes with an identical genetic background . We transfected L929 mouse fibroblastoid cells with DNA constructs containing the Delta9-fatty acid desaturase gene (Ole1) of S . cerevisiae under the control of desaturase promoters derived either from wild type or mutant strains of the dimorphic fungus H . capsulatum.

Biochemistry, 1997 Aug 12, 36(32), 9867 - 77
Solution structure of oxidized horse heart cytochrome c; Banci L et al.; The solution structure of oxidized horse heart cytochrome c was obtained at pH 7.0 in 100 mM phosphate buffer from 2278 NOEs and 241 pseudocontact shift constraints . The final structure was refined through restrained energy minimization . A 35-member family, with RMSD values with respect to the average structure of 0.70 +/- 0.11 A and 1.21 +/- 0.14 A for the backbone and all heavy atoms, respectively, and with an average penalty function of 130 +/- 4.0 kJ/mol and 84 +/- 3.7 kJ/mol for NOE and pseudocontact shift constraints, respectively (corresponding to a target function of 0.9 A2 and 0.2 A2), was obtained . The solution structure is somewhat different from that recently reported (Qi et al., 1996) and appears to be similar to the X-ray structure of the same oxidation state (Bushnell et al., 1990) . A noticeable difference is a rotation of 17 +/- 8 degrees of the imidazole plane between solid and solution structure . Detailed and accurate structural determinations are important within the frame of the current debate of the structural rearrangements occurring upon oxidation or reduction . From the obtained magnetic susceptibility tensor a separation of the hyperfine shifts into their contact and pseudocontact contributions is derived and compared to that of the analogous isoenzyme from S . cerevisiae and to previous results.

Mol Biol Cell, 1997 Aug, 8(8), 1587 - 601
Mcm2 and Mcm3 are constitutive nuclear proteins that exhibit distinct isoforms and bind chromatin during specific cell cycle stages of Saccharomyces cerevisiae; Young MR et al.; The Mcm2-7 proteins are a family of conserved proteins whose functions are essential for the initiation of DNA synthesis in all eukaryotes . These patients are constitutively present in high abundance in actively proliferating cells . In Saccharomyces cerevisiae, the intracellular concentrations of Mcms are between 100 and 500 times the number of replication origins . However, these proteins are limiting for the initiation of DNA synthesis at replication origins . Our studies indicate that only a small fraction of Mcm2 and Mcm3 tightly associates with chromatin, from late M phase to the beginning of the S phase . The rest of the Mcm2 and Mcm3 proteins are disturbed to both the cytoplasm and nucleoplasm in relatively constant levels throughout the cell cycle . We also show that S . cerevisiae Mcm3 is a phosphoprotein that exists in multiple isoforms and that distinct isoforms of Mcm2 and Mcm3 can be detected at specific stages of the cell cycle . These results suggest that the localization and function of the Mcm proteins are regulated by posttranslational phosphorylation in a manner that is consistent with a role for the Mcm proteins in restricting DNA replication to once per cell cycle.

Yeast, 1997 Aug, 13(10), 973 - 84
Identification of genes with nutrient-controlled expression by PCR-mapping in the yeast Saccharomyces cerevisiae; Crauwels M et al.; We have used RNA fingerprinting by the mRNA Differential Display technique to identify new genes in the yeast Saccharomyces cerevisiae, expression of which is controlled by specific nutrient conditions . mRNA was isolated from cells grown on glucose medium into exponential and stationary phase, and from cells starved for nitrogen on glucose-containing medium . To avoid interference with the large number of glucose-repressible genes, a glucose-repression-deficient strain was used . Twenty different sets of arbitrary primers chosen at random were used for PCR-amplification of reverse transcriptase generated cDNAs, which resulted in six highly reproducible gene expression patterns . The validity of the approach was confirmed by sequencing PCR products of genes with known expression patterns, SUP44/RPS4, CTT1, SSA3, HSP30 and HSP104, and genes with related functions, TEF1 and TEF3, encoding translation elongation factors . In all cases the specificity of the responses was confirmed by Northern blot analysis . The results show that the PCR-mapping method is highly useful for the identification of new genes expressed under specific conditions in the yeast S . cerevisiae.

J Cell Sci, 1997 Aug, 110 ( Pt 15), 1813 - 20
DNA damage inhibits proteolysis of the B-type cyclin Clb5 in S . cerevisiae; Germain D et al.; Cell cycle progression is mediated by waves of specific cyclin dependent kinases (CDKs) in all eukaryotes . Cyclins are degraded by the ubiquitin pathway of proteolysis . The recent identification of several components of the cyclin proteolysis machinery has highlighted both the importance of proteolysis at multiple transition points in the cell cycle and the involvement of other substrates degraded by the same machinery . In this study, we have investigated the effects of DNA damage on the cyclin proteolytic machinery in Saccharomyces cerevisiae . We find that the half-life of the B-type cyclin Clb5 is markedly increased following DNA damage while that of G1 cyclins is not . This effect is independent of cell cycle phase . Clb5 turnover requires p34CDC28 activity . Stabilisation of Clb5 correlates with an increase in tyrosine phosphorylation of p34CDC28, but stabilisation does not require this tyrosine phosphorylation . The stabilisation is independent of the checkpoint genes Mec1 and Rad53 . These observations establish a new link between the regulation of proteolysis and DNA damage.

Genetics, 1997 Aug, 146(4), 1239 - 51
Involvement of the yeast DNA polymerase delta in DNA repair in vivo; Giot L et al.; The POL3 encoded catalytic subunit of DNA polymerase delta possesses a highly conserved C-terminal cysteine-rich domain in Saccharomyces cerevisiae . Mutations in some of its cysteine codons display a lethal phenotype, which demonstrates an essential function of this domain . The thermosensitive mutant pol3-13, in which a serine replaces a cysteine of this domain, exhibits a range of defects in DNA repair, such as hypersensitivity to different DNA-damaging agents and deficiency for induced mutagenesis and for recombination . These phenotypes are observed at 24 degrees, a temperature at which DNA replication is almost normal; this differentiates the functions of POL3 in DNA repair and DNA replication . Since spontaneous mutagenesis and spontaneous recombination are efficient in pol3-13, we propose that POL3 plays an important role in DNA repair after irradiation, particularly in the error-prone and recombinational pathways . Extragenic suppressors of pol3-13 are allelic to sdp5-1, previously identified as an extragenic suppressor of pol3-11 . SDP5, which is identical to HYS2, encodes a protein homologous to the p50 subunit of bovine and human DNA polymerase delta . SDP5 is most probably the p55 subunit of Pol delta of S . cerevisiae and seems to be associated with the catalytic subunit for both DNA replication and DNA repair.

RNA, 1997 Aug, 3(8), 926 - 41
The human U4/U6 snRNP contains 60 and 90kD proteins that are structurally homologous to the yeast splicing factors Prp4p and Prp3p; Lauber J et al.; Immunoaffinity-purified human 25S {U4/U6.U5} tri-snRNPs harbor a set of polypeptides, termed the tri-snRNP proteins, that are not present in Mono Q-purified 20S U5 snRNPs or 10S U4/U6 snRNPs and that are important for tri-snRNP complex formation (Behrens SE, Luhrmann R, 1991, Genes & Dev 5:1439-1452) . Biochemical and immunological characterization of HeLa {U4/U6.U5} tri-snRNPs led to the identification of two novel proteins with molecular weights of 61 and 63kD that are distinct from the previously described 15.5, 20, 27, 60, and 90kD tri-snRNP proteins . For the initial characterization of tri-snRNP proteins that interact directly with U4/U6 snRNPs, immunoaffinity chromatography with an antibody directed against the 60kD protein was performed . We demonstrate that the 60 and 90kD tri-snRNP proteins specifically associate with the U4/U6 snRNP at salt concentrations where the tri-snRNP complex has dissociated . The primary structures of the 60kD and 90kD proteins were determined by cloning and sequencing their respective cDNAs . The U4/U6-60kD protein possesses a C-terminal WD domain that contains seven WD repeats and thus belongs to the WD-protein family, whose best-characterized members include the Gbeta subunits of heterotrimeric G proteins . A database homology search revealed a significant degree of overall homology (57.8% similarity, 33.9% identity) between the human 60kD protein and the Saccharomyces cerevisiae U4/U6 snRNP protein Prp4p . Two additional, previously undetected WD repeats (with seven in total) were also identified in Prp4p, consistent with the possibility that 60kD/Prp4p, like beta-transducin, may adopt a propeller-like structure . The U4/U6-90kD protein was shown to exhibit significant homology, particularly in its C-terminal half, with the S . cerevisiae splicing factor Prp3p, which also associates with the yeast U4/U6 snRNP . Interestingly, U4/U6-90kD shares short regions of homology with E . coli RNase III, including a region encompassing its double-stranded RNA binding domain . Based on their structural similarity with essential splicing factors in yeast, the human U4/U6-60kD and 90kD proteins are likely also to play important roles in the mammalian splicing process.

FEMS Microbiol Lett, 1997 Aug 1, 153(1), 75 - 81
The Saccharomyces cerevisiae acetyl-coenzyme A synthetase encoded by the ACS1 gene, but not the ACS2-encoded enzyme, is subject to glucose catabolite inactivation; de Jong-Gubbels P et al.; In Saccharomyces cerevisiae, the structural genes ACS1 and ACS2 each encode an isoenzyme of acetyl-CoA synthetase (ACS; EC 6.2.1.1) . Involvement of glucose catabolite repression in regulation of the two isoenzymes was investigated by following ACS activity after glucose pulses (100 mM) to ethanol-limited chemostat cultures . In wild-type S . cerevisiae and in an isogenic strain in which ACS2 had been disrupted, ACS activity decreased after a glucose pulse . No such inactivation was observed in a strain in which ACS1 was disrupted . Western blots demonstrated that the ACS1 product, but not the ACS2 product, was degraded after a glucose pulse . Inactivation kinetics of the ACS1 product resembled those of isocitrate lyase.

Mol Cell Biol, 1997 Aug, 17(8), 4718 - 29
Genetic redundancy between SPT23 and MGA2: regulators of Ty-induced mutations and Ty1 transcription in Saccharomyces cerevisiae; Zhang S et al.; SPT23 was isolated as a dosage-dependent suppressor of Ty-induced mutations in Saccharomyces cerevisiae . SPT23 shows considerable sequence homology with MGA2, a gene identified as a dosage-dependent suppressor of a snf2-imposed block on STA1 transcription in S . cerevisiae var . diastaticus . Although single mutations in either of these genes have only modest effects on cell growth, spt23 mga2 double mutants are inviable . Unlike SPT23, multicopy expression of a truncated form of MGA2 suppresses a narrow subset of Ty-induced mutations . SPT23/MGA2 and the SNF/SWI genes affect transcription of certain target genes in similar ways . Spt23p appears to be a rate-limiting component required for functional HIS4 expression of his4-912delta, a promoter insertion mutation induced by the Ty1-912 long terminal repeat . Furthermore, both Spt23p and Mga2p can activate transcription when fused to the Gal4p DNA-binding domain, as previously observed with Snf2p and Snf5p . A 50-amino-acid region in the N terminus of the predicted Spt23p protein is necessary and sufficient for the transactivation and necessary for suppression of Ty1-induced mutations and the essential function of Spt23p . Cell fractionation and cytological experiments suggest that Spt23p is associated with the nucleus . Our results suggest that SPT23/MGA2 affects transcription of a subset of genes in yeast, perhaps by changing chromatin accessibility.

Nature, 1997 Jul 31, 388(6641), 495 - 8
Yeast DNA ligase IV mediates non-homologous DNA end joining; Wilson TE et al.; The discovery of homologues from the yeast Saccharomyces cerevisiae of the human Ku DNA-end-binding proteins (HDF1 and KU80) has established that this organism is capable of non-homologous double-strand end joining (NHEJ), a form of DNA double-strand break repair (DSBR) active in mammalian V(D)J recombination . Identification of the DNA ligase that mediates NHEJ in yeast will help elucidate the function of the four mammalian DNA ligases in DSBR, V(D)J recombination and other reactions . Here we show that S . cerevisiae has two typical DNA ligases, the known DNA ligase I homologue CDC9 and the previously unknown DNA ligase IV homologue DNL4 . dnl4 mutants are deficient in precise and end-processed NHEJ . DNL4 and HDF1 are epistatic in this regard, with the mutation of each having equivalent effects . dnl4 mutants are complemented by overexpression of Dnl4 but not of Cdc9, and deficiency of Dnl4 alone does not impair either cell growth or the Cdc9-mediated responses to ionizing and ultraviolet radiation . Thus, S . cerevisiae has two distinct and separate ligation pathways.

Arch Biochem Biophys, 1997 Jul 15, 343(2), 215 - 24
Establishment of a novel host, high-red yeast that stably expresses hamster NADPH-cytochrome P450 oxidoreductase: usefulness for examination of the function of mammalian cytochrome P450; Ohgiya S et al.; A novel strain of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal cytochrome P450s was established and named High-red yeast . Hamster NADPH-cytochrome P450 oxidoreductase (P450 reductase) cDNA to be introduced into yeast was isolated from a hamster liver cDNA library . The cDNA was 2421 bp long and contained an entire coding region for 667 amino acids . The NH2-terminal amino acid sequence deduced from the hamster P450 reductase cDNA was identical with that of the enzyme purified from hamster livers except for deletion of the initial methionine . A delta-sequence derived from yeast retrotransposon Ty was cloned and used as a sequence for homologous recombination in a yeast genome . S . cerevisiae YPH500 was transformed with a multi-integration cassette containing the expression unit of the hamster P450 reductase and the delta-sequence . The transformant showing the highest activity of the P450 reductase was named High-red yeast . High-red yeast carried more than six copies of the multi-integration cassettes in a single chromosome and retained the multi-integration cassettes over a period of 100 generations under nonselective culture conditions, indicating that this yeast was a mitotically stable transformant . The microsomes prepared from High-red yeast had 20 times the P450 reductase activity of the microsomes prepared from the parental yeast . Due to the high activity of the hamster P450 reductase, the 7-ethoxycoumarin deethylase activity of mouse CYP1A1 expressed in High-red yeast was 250 times higher than the activity of mouse CYP1A1 expressed in the parental yeast.

Biochem Biophys Res Commun, 1997 Jul 9, 236(1), 50 - 3
Sac1p of Saccharomyces cerevisiae is not involved in ATP release to the extracellular fluid; Boyum R et al.; One activity ascribed to Sac1p is the transport of ATP into the lumen of the endoplasmic reticulum of Saccharomyces cerevisiae; therefore, the question of whether this protein plays a role in ATP efflux from yeast was addressed . Preliminary results suggested that deletion of the SAC1 gene eliminated nigericin-stimulated ATP efflux . However, further experimentation revealed that this result was caused by a pronounced extracellular ATPase activity for sac1delta cells at alkaline pH, conditions required to measure extracellular ATP in wild type cells . At acid pH, sac1delta cells exhibit glucose-dependent, nigericin-stimulated ATP efflux . sac1delta cells express less acid phosphatase activity in the periplasm than do wild type cells, thus increasing the stability of extracellular ATP . At alkaline pH, however, sac1delta cells tend to lose structural integrity and release lactate dehydrogenase as well as an unidentified ATPase activity to the extracellular fluid . Therefore, Sac1p is not involved in ATP efflux from S . cerevisiae.

Mol Gen Genet, 1997 Jul, 255(3), 237 - 47
The Aspergillus nidulans lysF gene encodes homoaconitase, an enzyme involved in the fungus-specific lysine biosynthesis pathway; Weidner G et al.; In filamentous fungi, lysine is synthesized via the alpha-aminoadipate pathway . In order to gain insight into this fungus-specific pathway (to date, no genes for enzymes of this pathway in filamentous fungi have been cloned) the lysine auxotrophic mutant LysF88 of Aspergillus nidulans was studied . HPLC and 1H-NMR analyses revealed that LysF88 accumulated homocitric acid in the culture supernatant . In addition, both the LysF88 mutant strain and LysF deletion strain (LysFKO) described here showed hardly any homoaconitase activity, indicating that lysF encodes homoaconitase . The lysF gene was cloned by complementation of the LysF88 mutant and sequenced . It has a size of 2397 bp, including a single intron of 72 bp . The two exons encode an open reading frame (ORF) of 2325 bp . The calculated M(r) of the homoaconitase protein (775 amino acids) is 83,943 . A major and a minor transcript begin at positions -28 and -32, respectively . The 3' end of the lysF cDNA showed a poly(A) tail commencing at position +2647 following a 250 bp untranslated region after the lysF stop codon . A putative polyadenylation signal sequence (TATAAA) is located 49 bp upstream of the polyadenylation site . Computer analysis revealed 55% amino acid sequence identity between the products of the putative homoaconitase ORF of A . nidulans and that of the recently sequenced homologous Saccharomyces cerevisiae . The similarity was particularly obvious in a region of cysteine residues, which are characteristic of an iron-sulfur cluster, implying that homoaconitase contains such a cluster . The homoaconitases of A . nidulans and S . cerevisiae share only 20% sequence identity with S . cerevisiae aconitase . The pH optimum for the activity of A . nidulans homoaconitase in 0.1 M potassium phosphate buffer is between pH 8.1 and pH 8.6 . Homoaconitase exhibited an apparent K(m) of 1.1 mM toward homoisocitric acid . The specific activity of homoaconitase was reduced by up to six-fold in mycelia grown in the presence of L-lysine, suggesting that it is regulated by lysine.

Mol Gen Genet, 1997 Jul, 255(4), 372 - 5
Regulation by tetracycline of gene expression in Saccharomyces cerevisiae; Nagahashi S et al.; A convenient system for the control of gene expression in Saccharomyces cerevisiae was developed . Tetracycline-responsive promoters were constructed by fusing the tetracycline operator (tetO) to the S . cerevisiae HOP1 promoter . When fused to the tetracycline repressor (tetR), trans-activation domains of both GAL4 and HAP4 were capable of promoting transcription from the tetO-HOP1 chimeric promoter, but the tetR-HAP4 fusion activator was the more efficient transcriptional activator . Addition of tetracycline nearly completely repressed activator-dependent transcription from the tetO-HOP1 promoter . Moreover, tetracycline-dependent repression of YEF3, CDC28 and RAM2 expression impaired cell growth . Thus, this system is useful for the elucidation of gene function in S . cerevisiae.

Biosci Biotechnol Biochem, 1997 Jul, 61(7), 1221 - 4
Thiamine increases expression of yeast gene; Ichikawa K et al.; We found that CPY production in Saccharomyces cerevisiae KS58-2D/pCY303 was increased by the addition of thiamine into the medium, while the addition of thiamine had no effect on cell growth . It became clear that the positive effect of thiamine was due to transcriptional increase, because the levels of CPYmRNA were increased according to the amount of thiamine added . Furthermore, it was suggested that thiamine generally increases the expression of yeast genes, since the expression of the luciferase gene that was artificially constructed was also increased to some extent by thiamine in S . cerevisiae.

Genes Dev, 1997 Jul 1, 11(13), 1703 - 16
The 30-kD subunit of mammalian cleavage and polyadenylation specificity factor and its yeast homolog are RNA-binding zinc finger proteins; Barabino SM et al.; Cleavage and polyadenylation specificity factor (CPSF), a key component of the mammalian RNA 3'-end processing machinery, consists of four subunits of 160, 100, 73, and 30 kD . Here we report the isolation and characterization of a cDNA encoding the 30-kD polypeptide . Antibodies raised against this protein inhibit cleavage and polyadenylation and coimmunoprecipitate the other CPSF subunits . The protein sequence contains five C3H-zinc-finger repeats and a putative RNA-binding zinc knuckle motif at the carboxyl terminus . Consistent with this observation, the in vitro translated 30-kD protein binds RNA polymers with a distinct preference for poly(U) . In addition, an essential S . cerevisiae gene, YTH1, was cloned which is 40% identical to CPSF 30K at the protein level . Extracts prepared from a conditional yth1 mutant have normal cleavage activity, but fail to polyadenylate the upstream cleavage product . Efficient polyadenylation activity can be restored by the addition of purified polyadenylation factor I (PF I) . We demonstrate that Yth1p is a component of PF I that interacts in vivo and in vitro with Fip1p, a known PF I subunit.

Genetics, 1997 Jul, 146(3), 835 - 47
A 140-bp-long palindromic sequence induces double-strand breaks during meiosis in the yeast Saccharomyces cerevisiae; Nag DK et al.; Palindromic sequences have the potential to form hairpin or cruciform structures, which are putative substrates for several nucleases and mismatch repair enzymes . A genetic method was developed to detect such structures in vivo in the yeast Saccharomyces cerevisiae . Using this method we previously showed that short hairpin structures are poorly repaired by the mismatch repair system in S . cerevisiae . We show here that mismatches, when present in the stem of the hairpin structure, are not processed by the repair machinery, suggesting that they are treated differently than those in the interstrand base-paired duplex DNA . A 140-bp-long palindromic sequence, on the contrary, acts as a meiotic recombination hotspot by generating a site for a double-strand break, an initiator of meiotic recombination . We suggest that long palindromic sequences undergo cruciform extrusion more readily than short ones . This cruciform structure then acts as a substrate for structure-specific nucleases resulting in the formation of a double-strand break during meiosis in yeast . In addition, we show that residual repair of the short hairpin structure occurs in an MSH2-independent pathway.

Appl Environ Microbiol, 1997 Jul, 63(7), 2897 - 905
Development of the FUN-1 family of fluorescent probes for vacuole labeling and viability testing of yeasts; Millard PJ et al.; A new family of fluorescent probes has been developed for assessing the viability and metabolic activity of yeasts . This class of halogenated unsymmetric cyanine dyes is exemplified by the FUN-1 {2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)- methylidene)-1-phenylquinolinium iodide} stain, a membrane-permeant nucleic acid-binding dye that has been found to give rise to cylindrical intravacuolar structures (CIVS) in Saccharomyces cerevisiae . Biochemical processing of the dye by active yeasts yielded CIVS that were markedly red shifted in fluorescence emission and therefore spectrally distinct from the nucleic acid-bound form of the dye . The formation of CIVS occurred under both aerobic and anaerobic conditions and was highly temperature dependent . Treatment of yeasts with the nonmetabolizable glucose analog 2-deoxy-D-glucose reduced cellular ATP levels approximately 6-fold and completely inhibited CIVS formation . Under aerobic conditions, the formation of CIVS was abrogated by the cytochrome oxidase inhibitors azide and cyanide; however, the H+ transport uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited CIVS formation under both aerobic and anaerobic conditions . Depletion of cellular thiols, including glutathione, with millimolar concentrations of N-ethylmaleimide, iodoacetamide, or allyl alcohol completely inhibited CIVS production . Marked reduction in the formation of CIVS by ethacrynic acid and sulfobromophthalein, inhibitors of glutathione S-transferase, suggested that dye processing can involve enzyme-mediated formation of glutathione conjugates . The conversion of FUN-1 by S . cerevisiae was studied quantitatively by using several techniques, including fluorometry, flow cytometry, and wide-field and confocal laser scanning fluorescence microscopy.

J Clin Microbiol, 1997 Jul, 35(7), 1822 - 8
Application of DNA typing methods and genetic analysis to epidemiology and taxonomy of Saccharomyces isolates; Clemons KV et al.; We have previously described differences in phenotype and virulence among clinical and nonclinical isolates of Saccharomyces . To further characterize these isolates, a comparison of restriction fragment length polymorphism (RFLP) patterns and genetic analysis were done . The cellular DNA of each of 49 clinical and 11 nonclinical isolates of Saccharomyces was digested with the endonuclease EcoRI, and the resultant fragments were separated by electrophoresis . Sixty isolates were grouped on the basis of the presence (group B) or absence (group A) of a 3-kb band . Group A contained 43 isolates (35 clinical and 8 nonclinical isolates) in 31 discernible subgroups, and group B had 17 isolates (14 clinical and 3 nonclinical isolates) in 10 subgroups . Interestingly, six of eight known vaginal isolates were group B, with four of those six being identical . Virulence of isolates was associated with membership in group A (P = 0.03) . Comparison of known members of sibling species within the genus Saccharomyces, which cannot be distinguished by standard biochemical tests, showed that S . paradoxus, S . bayanus, and S . cerevisiae could be differentiated by RFLP analysis . Genetic analysis of the isolates forming viable spores showed that most group A isolates were diploid and members of the species S . cerevisiae . Those group A and B isolates unable to form viable spores may be diploid hybrids between Saccharomyces species . The group B isolates that formed viable spores were tetraploid and may also be interspecific hybrids . Overall, clinical isolates of Saccharomyces were very heterogeneous and exhibited little clonality . RFLP pattern analysis could be a useful method of demonstrating transmission in patients with infection or between environmental sources and patients.

FEBS Lett, 1997 Jun 30, 410(2-3), 461 - 6
RNA polymerase I from S . cerevisiae depends on an additional factor to release terminated transcripts from the template; Tschochne H et al.; Terminated transcripts were generated at the ends of linearized DNA templates and at DNA-bound lac repressor by in vitro transcription with highly enriched or purified yeast RNA polymerase I (pol I) . The release of the synthesized transcripts from the DNA was analyzed using immobilized DNA as template for the transcription reaction . An additional activity distinguishable from pol I was necessary to remove the terminated RNA from the template . Efficiency of transcript release could be improved if a thymidine-rich DNA fragment was located upstream of the transcriptional arrest caused by the DNA-bound lac repressor . The release activity interacted with different forms of polymerases, pol I able to initiate on the ribosomal gene promoter and pol I only active in non-specific transcription.

Cell, 1997 Jun 27, 89(7), 1055 - 65
14-3-3 proteins are essential for RAS/MAPK cascade signaling during pseudohyphal development in S . cerevisiae; Roberts RL et al.; 14-3-3 proteins are highly conserved ubiquitous proteins whose explicit functions have remained elusive . Here, we show that the S . cerevisiae 14-3-3 homologs BMH1 and BMH2 are not essential for viability or mating MAPK cascade signaling, but they are essential for pseudohyphal-development MAPK cascade signaling and other processes . Activated alleles of RAS2 and CDC42 induce pseudohyphal development and FG(TyA)-lacZ signaling in Bmh+ strains but not in ste20 (p65PAK) or bmh1 bmh2 mutant strains . Moreover, Bmh1p and Bmh2p associate with Ste20p in vivo . Three alleles of BMH1 encode proteins defective for FG(TyA)-lacZ signaling and association with Ste20p, yet these alleles complement other 14-3-3 functions . Therefore, the 14-3-3 proteins are specifically required for RAS/MAPK cascade signaling during pseudohyphal development in S . cerevisiae.

J Biol Chem, 1997 Jun 27, 272(26), 16308 - 14
Saccharomyces cerevisiae VIG9 encodes GDP-mannose pyrophosphorylase, which is essential for protein glycosylation; Hashimoto H et al.; A genomic DNA fragment that complements a newly identified protein glycosylation-defective mutation, vig9, of Saccharomyces cerevisiae was cloned . Chromosomal integration of this fragment by homologous recombination indicated that it contains the wild type VIG9 gene . The nucleotide sequence was determined . A predicted gene product showed significant amino acid sequence homology with several bacterial enzymes that catalyze the synthesis of (deoxy)ribonucleotide diphosphate sugars from sugar phosphates and (deoxy)ribonucleotide triphosphate . We examined the enzyme activity to synthesize GDP-mannose in the cell extracts of the wild type, vig9-1 mutant, and VIG9 transformant yeasts . Reduction of the activity in the mutant cell and its restoration by VIG9 suggested that the VIG9 gene is the structural gene for GDP-mannose pyrophosphorylase of S . cerevisiae which catalyzes the production of GDP-mannose . We demonstrated the enzyme activity of Vig9 protein using a recombinant fusion protein produced in Escherichia coli.

J Biol Chem, 1997 Jun 27, 272(26), 16110 - 7
Identification of a Saccharomyces gene, LCB3, necessary for incorporation of exogenous long chain bases into sphingolipids; Qie L et al.; To identify genes necessary for sphingolipid synthesis in Saccharomyces cerevisiae we developed a procedure to enrich for mutants unable to incorporate exogenous long chain base into sphingolipids . We show here that a mutant strain, AG84-3, isolated by using the enrichment procedure, makes sphingolipids from endogenously synthesized but not from exogenously supplied long chain base . A gene termed LCB3 (YJL134W, GenBank designation X87371x21), which complements the long chain base utilization defect of strain AG84-3, was isolated from a genomic DNA library . The gene is predicted to encode a protein with multiple membrane-spanning domains and a COOH-terminal glycosylphosphatidylinositiol cleavage/attachment site . Deletion of the lcb3 gene in a wild type genetic background reduces the rate of exogenous long chain base incorporation into sphingolipids and makes the host strain more resistant to growth inhibition by long chain bases . Only one protein in current data bases, the S . cerevisiae open-reading frame YKR053C, whose function is unknown, shows homology to the Lcb3 protein . The two proteins are not, however, functional homologs because deletion of the YKR053C open reading frame does not impair long chain base utilization or enhance resistance of cells to growth inhibition by long chain bases . Based upon these data we hypothesize that the Lcb3 protein is a plasma membrane transporter capable of transporting sphingoid long chain bases into cells . It is the first candidate for such a transporter and the first member of what appears to be a new class of membrane-bound proteins.

Gene, 1997 Jun 19, 192(2), 199 - 205
The DAN1 gene of S . cerevisiae is regulated in parallel with the hypoxic genes, but by a different mechanism; Sertil O et al.; The DAN1 gene is expressed under anaerobic conditions in yeast and completely repressed during aerobic growth . The function of the gene is unknown, and genetic disruption had no effect on fitness which could be detected, even upon prolonged anaerobic growth . Expression of DAN1 was constitutive in a heme-deficient strain, indicating that heme participates in repression . Expression was blocked by heme in anaerobic medium, suggesting that heme acts as a negative co-effector rather than through its metabolic functions, i.e., in the production of a co-effector . Expression of DAN1 was regulated in parallel with the hypoxic gene ANB1, showing identical kinetics of induction and dose response to heme . However, unlike ANB1, DAN1 is not regulated by the repressor of the hypoxic regulon, ROX1, as shown by observation of normal aerobic repression of DAN1 in a strain carrying a deletion of ROX1 . These results indicate the existence of a parallel regulatory system which produces an identical response to oxygen by a different mechanism than that controlling the hypoxic regulon.

Nucleic Acids Res, 1997 Jun 15, 25(12), 2319 - 25
Cloning and characterization of a cDNA encoding a bacteriophage-type RNA polymerase from the higher plant Chenopodium album; Weihe A et al.; We have cloned a full-length cDNA from the higher plant Chenopodium album coding for a single subunit bacteriophage-type RNA polymerase . The cDNA isolated from an actively growing cell suspension culture recognized a 3.8 kb transcript on Northern blots . The open reading frame comprises 987 amino acids with a predicted molecular mass of 112 kDa . A comparison of the protein sequence with those of the two known fungal mitochondrial RNA polymerases, from Saccharomyces cerevisiae and Neurospora crassa , reveals extensive homology between the three enzymes . with complete conservation of all catalytically essential amino acids . The putative mitochondrial RNA polymerase from C.album , as well as homologous sequences from rice and barley, which have been partially cloned, lack two catalytically non-essential regions of up to 176 amino acids near the C-terminus present in the two fungal mitochondrial RNA polymerases . The extreme N-terminus of the cloned C.album RNA polymerase displays features of a potential mitochondrial transit sequence . In phylogenetic trees constructed to compare the evolutionary relationships between the different single subunit RNA polymerases the C.album sequence forms a subgroup together with the S.cerevisiae and the N.crassa mitochondrial RNA polymerases, well separating from both bacteriophage enzymes and plasmid-encoded RNA polymerases found in mitochondria of many fungi and some higher plants.

Proc Natl Acad Sci U S A, 1997 Jun 10, 94(12), 6099 - 103
The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, MetAP-2; Sin N et al.; The inhibition of new blood vessel formation (angiogenesis) is an effective means of limiting both the size and metastasis of solid tumors . The leading anti-angiogenic compound, TNP-470, has proven to be effective in in vitro and in animal model studies, and is currently being tested in phase III antitumor clinical trials . Despite many detailed pharmacological studies, little is known of the molecular mode of action of TNP-470 . Using a derivative of the TNP-470 parent compound, the fungal metabolite, fumagillin, we have purified a mammalian protein that is selectively and covalently bound by this natural product . This fumagillin binding protein was found to be a metalloprotease, methionine aminopeptidase (MetAP-2), that is highly conserved between human and Saccharomyces cerevisiae . In the absence of MetAP-1, a distantly related methionine aminopeptidase, MetAP-2 function is essential for vegetative growth in yeast . We demonstrate that fumagillin selectively inhibits the S . cerevisiae MetAP-2 protein in vivo . The binding is highly specific as judged by the failure of fumagillin to inhibit MetAP-1 in vivo . Hence, these results identify MetAP-2 as an important target of study in the analysis of the potent biological activities of fumagillin.

Biochim Biophys Acta, 1997 Jun 6, 1335(3), 273 - 82
Possible role of isoaspartyl methyltransferase towards regulation of acid trehalase activity in Saccharomyces cerevisiae; Biswas N et al.; Logarithmically growing cells of S . cerevisiae contained high neutral trehalase (NT) activity while stationary-phase cells had high acid trehalase (AT) activity . Change in activity profile of AT and NT were different during growth under different conditions, particularly during growth in acetate medium and up to 1 h of germination period, but that for AT and isoaspartyl methyltransferase (IMT) were found to be almost identical . Concomitant increase in NT activity as well as increase in cAMP level was noticed at the onset of spore germination . Increase in AT and IMT activities as well as decrease in S-adenosyl-L-methionine (AdoMet) level were noticed during stationary phase of growth . Acidic polyacrylamide gel electrophoresis and subsequent autoradiography revealed that substrate of IMT was a protein of molar mass around 82 kDa which could be an AT . Methylated AT was found to be more active while non-methylated AT was relatively less active in comparison to the untreated sample . Since AT existed as an equilibrium mixture of protomer and oligomer, it was suggested that IMT catalysed carboxyl methylation might have some contribution towards the regulation of AT activity.

EMBO J, 1997 Jun 2, 16(11), 3219 - 32
Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S . cerevisiae telomeres; Laible G et al.; Gene silencing is required to stably maintain distinct patterns of gene expression during eukaryotic development and has been correlated with the induction of chromatin domains that restrict gene activity . We describe the isolation of human (EZH2) and mouse (Ezh1) homologues of the Drosophila Polycomb-group (Pc-G) gene Enhancer of zeste {E(z)}, a crucial regulator of homeotic gene expression implicated in the assembly of repressive protein complexes in chromatin . Mammalian homologues of E(z) are encoded by two distinct loci in mouse and man, and the two murine Ezh genes display complementary expression profiles during mouse development . The E(z) gene family reveals a striking functional conservation in mediating gene repression in eukaryotic chromatin: extra gene copies of human EZH2 or Drosophila E(z) in transgenic flies enhance position effect variegation of the heterochromatin-associated white gene, and expression of either human EZH2 or murine Ezh1 restores gene repression in Saccharomyces cerevisiae mutants that are impaired in telomeric silencing . Together, these data provide a functional link between Pc-G-dependent gene repression and inactive chromatin domains, and indicate that silencing mechanism(s) may be broadly conserved in eukaryotes.

Biochemistry (Mosc), 1997 Jun, 62(6), 636 - 40
Oxidation of exogenous formaldehyde in methylotrophic and nonmethylotrophic yeast cells; Maidan NN et al.; Nonmethylotrophic (Candida maltosa and Saccharomyces cerevisiae) and methylotrophic (Hansenula polymorpha) yeast cells acidified their incubation media in the presence of formaldehyde . This was associated with the release of formate . We studied the formaldehyde-dependent production of formic acid and the enzymatic properties of these strains grown on media containing various carbon sources . The acidifying potential was considerably lower in formaldehyde dehydrogenase-deficient cells of mutant strains of H . polymorpha . The rates of acidification by C . maltosa and S . cerevisiae depended on the activity of their nonspecific aldehyde dehydrogenases . We suggest that accumulation of formate by yeast cells incubated in the presence of formaldehyde is caused by the total activity of formaldehyde dehydrogenase and nonspecific aldehyde dehydrogenase in methylotrophic yeasts or aldehyde dehydrogenase only in nonmethylotrophic yeasts . This is probably an additional mechanism for detoxification of formaldehyde.

Environ Health Perspect, 1997 Jun, 105 Suppl 4, 931 - 4
Regulation of eukaryotic abasic endonucleases and their role in genetic stability; Demple B et al.; Abasic (AP) sites in DNA arise from spontaneous reactions or the action of DNA glycosylases and represent a loss of genetic information . The AP sites can be mutagenic or cytotoxic, and their repair is initiated by class II AP endonucleases, which incise immediately 5' to AP sites . The main enzyme of S . cerevisiae . Apn1, provides cellular resistance to oxidants (e.g., H2O2) or alkylating agents, and limits the spontaneous mutation rate . AP endonucleases from other species can replace Apn1 function in yeast to different extents . We studied the main human enzyme, Ape, with respect to its incision specificity in vitro and the expression of the APE gene in vivo . The results suggest that Ape evolved to act preferentially on AP sites compared to deoxyribose fragments located at oxidative strand breaks and that the incision modes of Ape and Apn1 may be fundamentally different . We also defined the functional APE promoter, and showed that APE expression is transiently downregulated during the regeneration of epidermis after wounding . This latter effect may lead to a window of vulnerability for DNA damage and perhaps mutagenesis during the healing of epidermal and other wounds . Such unexpected effects on the expression of DNA repair enzymes need to be taken into account in analyzing the susceptibility of different tissues to carcinogens.

Plant Mol Biol, 1997 Jun, 34(3), 497 - 506
Isolation of a cDNA encoding an Arabidopsis galactokinase by functional expression in yeast; Kaplan CP et al.; A cDNA clone encoding Arabidopsis thaliana galactokinase was fortuitously isolated during the course of a screen for plant homologues of a Saccharomyces cerevisiae peroxisome assembly gene, PAS9 . Clones were sought which restored the ability of pas9 cells to grow on oleate as a sole carbon source, as oleate metabolism requires peroxisomal beta-oxidation and therefore functional peroxisomes . Subsequent experiments showed that high level expression of the galactokinase cDNA did not complement the peroxisomal assembly defect, but instead permitted the cells to grow on agar plates in the absence of an external carbon source . Agar plates were shown to contain a small amount of galactose released from the agar as a result of autoclaving . The galactokinase clone was shown to be functional, as it could complement a S . cerevisiae galactokinase mutant . Galactokinase is a single copy gene in Arabidopsis, which has been designated AGK1, and is expressed in all the major organs of the plant.

Chromosoma, 1997 Jun, 105(7-8), 391 - 400
Eukaryotic ribosomal RNA: the recent excitement in the nucleotide modification problem; Maden BE et al.; Eukaryotic ribosomal RNA (rRNA) contains numerous modified nucleotides: about 115 methyl groups and some 95 pseudouridines in vertebrates; about 65 methyl groups and some 45 pseudouridines in Saccharomyces cerevisiae . All but about ten of the methyl groups are ribose methylations . The remaining ten are on heterocyclic bases . The ribose methylations occur very rapidly upon the primary rRNA transcript in the nucleolus, probably on nascent chains, and they appear to play an important role in ribosome maturation, at least in vertebrates . All of the methyl groups occur in the conserved core of rRNA . However, there is no consensus feature of sequence or secondary structure for the methylation sites; thus the nature of the signal(s) for site-specific methylations had until recently remained a mystery . The situation changed dramatically with the discovery that many of the ribose methylation sites are in regions that are precisely complementary to small nucleolar RNA (snoRNA) species . Experimental evidence indicates that structural motifs within the snoRNA species do indeed pinpoint the precise nucleotides to be methylated by the putative 2'-O-methyl transferase(s) . Regarding base methylations, the gene DIM1, responsible for modification of the conserved dimethyladenosines near the 3' end of 18S rRNA, has been shown to be essential for viability in S . cerevisiae and is suggested to play a role in the nucleocytoplasmic transport of the small ribosomal subunit . Recently nearly all of the pseudouridines have also been mapped in the rRNA of several eukaryotic species . As is the case for ribose methylations, most pseudouridine modifications occur rapidly upon precursor rRNA, within core sequences, and in a variety of local primary and secondary structure environments . In contrast to ribose methylation, no potentially unifying process has yet been identified for the enzymic recognition of the many pseudouridine modification sites . However, the new data afford the basis for a search for any potential involvement of snoRNAs in the recognition process.

Curr Genet, 1997 Jun, 31(6), 457 - 61
Incidence of SUC-RTM telomeric repeated genes in brewing and wild wine strains of Saccharomyces; Denayrolles M et al.; When over-expressed, RTM yeast genes confer resistance to the toxicity of molasses . They are found in distiller's and baker's industrial yeasts in multiple copies, scattered on the telomeres and physically linked to the telomeric SUC genes . Because these genes are absent from some laboratory strains, we explored the genomes of other industrial yeasts (brewing strains) and wine wild strains . A collection of 47 wine yeast strains (S . cerevisiae and S . bayanus) and 15 brewing strains, lager, ale and possible ancestors (S . monacensis, S . paradoxus and S . carlsbergensis) were screened for the presence of RTM genes . Only three wine strains and all brewing strains proved to contain RTM sequences in different copy numbers . PCR and chromosome blotting confirm the presence of SUC sequences in tandem with RTM . Moreover, analysis of the entire S . cerevisiae genome sequence shows that three other, non-telomeric, genes related to RTM are scattered on different chromosomes.

Semin Immunol, 1997 Jun, 9(3), 181 - 8
The RAD52 epistasis group in mammalian double strand break repair; Petrini JH et al.; The S . cerevisiae RAD52 epistasis group gene products mediate DNA double strand break repair and recombination . These proteins and their modes of action have been extensively characterized . The existence of highly conserved mammalian RAD52 epistasis group homologues suggests that information regarding the functions and mechanisms of double strand break repair proteins in yeast may be applicable to mammalian recombinational DNA repair . Herein, we provide an overview of the S . cerevisiae RAD52 epistasis group and describe the characterization of the five mammalian RAD52 epistasis group homologues identified to date . In the context of their expression patterns and other functional analyses, we discuss potential roles for these proteins in mammalian recombinational DNA repair and specialized recombination events such as V(D)J recombination.

J Biomol Struct Dyn, 1997 Jun, 14(6), 667 - 75
Homology modeling of adenylosuccinate synthetase from Saccharomyces cerevisiae reveals a possible binding region for single-stranded ARS sequences; Sticht H et al.; Adenylosuccinate synthetase from Saccharomyces cerevisiae was investigated in order to find a structural explanation for its ability to bind specifically to single-stranded ARS elements (autonomously replicating sequences) . Using the E . coli enzyme as template, a model for the structure of adenylosuccinate synthetase from S . cerevisiae was generated and subsequently refined by molecular dynamics techniques . The resulting three-dimensional structure offers an explanation for the DNA binding activity of the yeast enzyme by revealing a distinct basic region that is not present in the homologous enzymes from other organisms . The model is also in good agreement with biochemical data available for a mutant protein in which Glycine 252 is replaced by Aspartate . On the basis of the model a significant structural distortion near the catalytic center was predicted for this mutant, corresponding well to the enzymatic inactivity observed . The mutant enzyme shows larger structural fluctuations than the wild-type protein according to the results of two independent molecular dynamics simulations.

Glycobiology, 1997 Jun, 7(4), 481 - 6
Protein-O-glycosylation in yeast: protein-specific mannosyltransferases; Gentzsch M et al.; S . cerevisiae contains at least six genes (PMT1-6) for dolicholphosphate-D-mannose: protein-O-D-mannosyltransferases . The in vivo mannosylation of seven O-mannosylated yeast proteins has been analyzed in a number of pmt mutants . The results clearly indicate that the various protein O-mannosyltransferases have different specificities for protein substrates . Five of the proteins tested (chitinase, a-agglutinin, Kre9p, Bar1p, Pir2p/hsp 150) are mainly underglycosylated in pmt1 and pmt2 mutants, whereby qualitative differences exist among the various proteins . Two of the O-mannosylated proteins (Ggp1p and Kex2p) are not at all affected in pmt1 and pmt2 mutants but are clearly underglycosylated when PMT4 is mutated . Although the PMT4 gene product is shown to be responsible for O-mannosylating a Ser-rich region of Ggp1p in vivo, a penta-seryl-peptide is not an in vitro substrate for this transferase . A PMT3 mutation does affect O-mannosylation of chitinase only in the genetic background of a pmt1pmt2 double mutation, indicating that PMT1 and PMT2 can compensate for a deleted PMT3 gene.

Mol Cell Biol, 1997 Jun, 17(6), 3037 - 46
Reconstitution of a MEC1-independent checkpoint in yeast by expression of a novel human fork head cDNA; Pati D et al.; A novel human cDNA, CHES1 (checkpoint suppressor 1), has been isolated by suppression of the mec1-1 checkpoint mutation in Saccharomyces cerevisiae . CHES1 suppresses a number of DNA damage-activated checkpoint mutations in S . cerevisiae, including mec1, rad9, rad24, dun1, and rad53 . CHES1 suppression of sensitivity to DNA damage is specific for checkpoint-defective strains, in contrast to DNA repair-defective strains . Presence of CHES1 but not a control vector resulted in G2 delay after UV irradiation in checkpoint-defective strains, with kinetics, nuclear morphology, and cycloheximide resistance similar to those of a wild-type strain . CHES1 can also suppress the lethality, UV sensitivity, and G2 checkpoint defect of a mec1 null mutation . In contrast to this activity, CHES1 had no measurable effect on the replication checkpoint as assayed by hydroxyurea sensitivity of a mec1 strain . Sequence analysis demonstrates that CHES1 is a novel member of the fork head/Winged Helix family of transcription factors . Suppression of the checkpoint-defective phenotype requires a 200-amino-acid domain in the carboxy terminus of the protein which is distinct from the DNA binding site . Analysis of CHES1 activity is most consistent with activation of an alternative MEC1-independent checkpoint pathway in budding yeast.

Cell, 1997 May 30, 89(5), 811 - 9
Self-seeded fibers formed by Sup35, the protein determinant of {PSI+}, a heritable prion-like factor of S . cerevisiae; Glover JR et al.; The {PSI+} factor of S . cerevisiae represents a new form of inheritance: cytosolic transmission of an altered phenotype is apparently based upon inheritance of an altered protein structure rather than an altered nucleic acid . The molecular basis of its propagation is unknown . We report that purified Sup35 and subdomains that induce {PSI+} elements in vivo form highly ordered fibers in vitro . Fibers bind Congo red and are rich in beta sheet, characteristics of amyloids found in certain human diseases, including the prion diseases . Some fibers have distinct structures and these, once initiated, are self-perpetuating . Preformed fibers greatly accelerate fiber formation by unpolymerized protein . These data support a "protein-only" seeded polymerization model for the inheritance of {PSI+}.

Nature, 1997 May 29, 387(6632 Suppl), 90 - 3
The nucleotide sequence of Saccharomyces cerevisiae chromosome XIII; Bowman S et al.; Systematic sequencing of the genome of Saccharomyces cerevisiae has revealed thousands of new predicted genes and allowed analysis of long-range features of chromosomal organization . Generally, genes and predicted genes seem to be distributed evenly throughout the genome, having no overall preference for DNA strand . Apart from the smaller chromosomes, which can have substantially lower gene density in their telomeric regions, there is a consistent average of one open reading frame (ORF) approximately every two kilobases . However, one of the most surprising findings for a eukaryote with approximately 6,000 genes was the amount of apparent redundancy in its genome . This redundancy occurs both between individual ORFs and over more extensive chromosome regions, which have been duplicated preserving gene order and orientation . Here we report the entire nucleotide sequence of chromosome XIII, the sixth-largest S . cerevisiae chromosome, and demonstrate that its features and organization are consistent with those observed for other S . cerevisiae chromosomes . Analysis revealed 459 ORFs, 284 have not been identified previously . Both intra- and interchromosomal duplications of regions of this chromosome have occurred.

Nature, 1997 May 29, 387(6632 Suppl), 81 - 4
The nucleotide sequence of Saccharomyces cerevisiae chromosome VII; Tettelin H et al.; The complete nucleotide sequence of Saccharomyces cerevisiae chromosome VII has 572 predicted open reading frames (ORFs), of which 341 are new . No correlation was found between G+C content and gene density along the chromosome, and their variations are random . Of the ORFs, 17% show high similarity to human proteins . Almost half of the ORFs could be classified in functional categories, and there is a slight increase in the number of transcription (7.0%) and translation (5.2%) factors when compared with the complete S . cerevisiae genome . Accurate verification procedures demonstrate that there are less than two errors per 10,000 base pairs in the published sequence.

Nature, 1997 May 29, 387(6632 Suppl), 75 - 8
The nucleotide sequence of Saccharomyces cerevisiae chromosome IV; Jacq C et al.; The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome IV has been determined . Apart from chromosome XII, which contains the 1-2 Mb rDNA cluster, chromosome IV is the longest S . cerevisiae chromosome . It was split into three parts, which were sequenced by a consortium from the European Community, the Sanger Centre, and groups from St Louis and Stanford in the United States . The sequence of 1,531,974 base pairs contains 796 predicted or known genes, 318 (39.9%) of which have been previously identified . Of the 478 new genes, 225 (28.3%) are homologous to previously identified genes and 253 (32%) have unknown functions or correspond to spurious open reading frames (ORFs) . On average there is one gene approximately every two kilobases . Superimposed on alternating regional variations in G+C composition, there is a large central domain with a lower G+C content that contains all the yeast transposon (Ty) elements and most of the tRNA genes . Chromosome IV shares with chromosomes II, V, XII, XIII and XV some long clustered duplications which partly explain its origin.

J Biotechnol, 1997 May 23, 55(1), 9 - 20
Enhancement of recombinant glucoamylase expression by introducing yeast GAL7 mRNA termination sequence; Cho KM et al.; Glucoamylase gene (STA1) of Saccharomyces diastaticus was expressed in recombinant Saccharomyces cerevisiae systems . The yeast, GAL7 mRNA termination sequence, was introduced in the 3' noncoding region of the STA1 structural gene which was under the control of the SUC2 promoter and STA1 secretion signal sequence . This plasmid was named YEpSSG7 and was introduced into yeast S . cerevisiae MMY2 to construct recombinant S . cerevisiae MMY2SSG7 . The GAL7 mRNA termination sequence enhanced the glucoamylase expression level by 3-5 times depending on the culture conditions compared to the result from the strain S . cerevisiae MMY2SUCSTA which did not contain the GAL7 mRNA termination sequence . Such an enhancement was not due to plasmid stability or plasmid copy number effects . Such an enhancement was primarily due to the fact that GAL7 mRNA termination sequence stabilized the STA1 mRNA 3' end.

Mol Gen Genet, 1997 May 20, 254(5), 562 - 70
Analyses of Saccharomyces cerevisiae Cdc7 kinase point mutants: dominant-negative inhibition of DNA replication on overexpression of kinase-negative Cdc7 proteins; Ohtoshi A et al.; Saccharomyces cerevisiae Cdc7 kinase is required for initiation of S phase, and its kinase activity, which is positively regulated by Dbf4 protein, reaches maximum at the G1/S boundary . In this study, we constructed Cdc7 point mutants (T281E, T281A, D182N, D163N, and T167E) and examined the effect of each mutant on growth . All the mutants lost the ability to complement temperature-sensitive growth of cdc7(ts) mutants at a low protein level, whereas T281A (putative target of phosphorylation) and T167E (residue involved in substrate recognition) restored the growth of cdc7(ts) when overproduced to a high level . Three putative kinase-negative mutants (T281E, D182N, and D163N) inhibited growth when overexpressed in a wild-type strain . Analyses of DNA content and morphology revealed that most cells were arrested as dumbbells with 1C DNA, indicative of a block in the G1 to S transition . This growth inhibition was suppressed by co-overexpression of the wild-type Cdc7 or Dbf4 protein . Furthermore, deletion of the Dbf4 protein-binding region in each Cdc7 mutant resulted in loss of growth inhibitory effect . Thus, dominant-negative effects of T281E, D182N, and D163N on growth can be best explained by inactivation of the wild-type Cdc7 function through titration of Dbf4 by these inactive kinases . Our results are consistent with the notion that association of Dbf4 with Cdc7 is essential for the G1 to S transition in S . cerevisiae.

Mol Gen Genet, 1997 May 20, 254(5), 486 - 94
Molecular cloning and characterization of Drosophila genes encoding small GTPases of the rab and rho families; Sasamura T et al.; We have isolated eight genes from Drosophila, small GTPases . They can be classified into three rab family genes (Drab2, Drab5, Drab11) and five rho family genes (Drac1a, Drac1b, Drac3, Dcdc42, DrhoA) . While Drac3 is a novel type of rac gene, others are homologues of known mammalian genes for small GTPases . Northern blot analyses showed that all the genes are expressed throughout all developmental stages from embryo to adult . In situ hybridization to embryos revealed that Drab2, Drac1b, and Drac3 are highly expressed in the nervous system, in the trunk mesoderm, and in the cephalic mesoderm, respectively . Since hemocytes are derived from the cephalic mesoderm, we carried out double stainings using a hemocyte marker anti-peroxidasin antibody and Drac3 in situ hybridization . We found that Drac3 is expressed in hemocyte precursor cells . In the Drac3 deficiency embryos, the hemocyte precursor cells start to differentiate normally, but never develop into mature hemocytes, indicating that Drac3 is essential for their maturation . The DrhoA and Dcdc42 genes complemented S . cerevisiae rho1 and cdc42 mutations in the same manner as human rhoA and CDC42, respectively . These results suggest functional similarity between Drosophila and mammalian small GTPase genes.

Biochemistry, 1997 May 20, 36(20), 5947 - 54
Purification and characterization of the DNA polymerase alpha associated exonuclease: the RTH1 gene product; Zhu FX et al.; We report here the purification and mechanistic characterization of a 5'-3' exonuclease associated with DNA polymerase alpha from the yeast Saccharomyces cerevisiae . Earlier, we identified a 5' --> 3' exonuclease activity that copurified with yeast DNA polymerase alpha-primase in a multiprotein complex {Biswas, E . E., et al . (1993) Biochemistry, 32, 3020-3027} . Peptide sequence analysis of the purified 47 kDa exonuclease was carried out, and the peptide sequence was found to be identical to the S . cerevisiae gene YKL510 encoded polypeptide, which is also known as yeast RAD2 homolog 1 or RTH1 nuclease . The native exonuclease also had strong flap endonuclease activity similar to that observed with RTH1 nuclease and homologous yeast (RAD2) and mammalian enzymes . During our studies, we have discovered certain unique features of the mechanism of action of the native RTH1 nuclease . Studies presented here indicated that the exonuclease had specific pause sites during its 5'-3' exonuclease nucleotide excision . These pause sites were easily detected with long (approximately 50 bp) oligonucleotide substrates during exonucleolytic excision by the formation of a discontinuous ladder of excision product . We have further analyzed the mechanism of generation of the pause sites, as they could occur through a number of different pathways . Alignment of the pause sites with the nucleotide sequence of the oligonucleotide substrate indicated that the pause sites were dependent on the nucleotide sequence . Our analysis revealed that RTH1 nuclease pauses predominantly at G:C rich sequences . With poly(dA):oligo(dT)50 as substrate, the exonucleolytic products formed a continuous ladder with no evidence of pausing . The G:C rich DNA sequences are thermodynamically more stable than the A:T rich sequences, which may be in part responsible for pausing of the RTH1 5' --> 3' exonuclease at these sites.

J Cell Biol, 1997 May 19, 137(4), 813 - 24
The Hsp70 homologue Lhs1p is involved in a novel function of the yeast endoplasmic reticulum, refolding and stabilization of heat-denatured protein aggregates; Saris N et al.; Heat stress is an obvious hazard, and mechanisms to recover from thermal damage, largely unknown as of yet, have evolved in all organisms . We have recently shown that a marker protein in the ER of Saccharomyces cerevisiae, denatured by exposure of cells to 50 degrees C after preconditioning at 37 degrees C, was reactivated by an ATP-dependent machinery, when the cells were returned to physiological temperature 24 degrees C . Here we show that refolding of the marker enzyme Hsp150Delta-beta-lactamase, inactivated and aggregated by the 50 degrees C treatment, required a novel ER-located homologue of the Hsp70 family, Lhs1p . In the absence of Lhs1p, Hsp150Delta-beta-lactamase failed to be solubilized and reactivated and was slowly degraded . Coimmunoprecipitation experiments suggested that Lhs1p was somehow associated with heat-denatured Hsp150Delta- beta-lactamase, whereas no association with native marker protein molecules could be detected . Similar findings were obtained for a natural glycoprotein of S . cerevisiae, pro-carboxypeptidase Y (pro-CPY) . Lhs1p had no significant role in folding or secretion of newly synthesized Hsp150Delta-beta-lactamase or pro-CPY, suggesting that the machinery repairing heat-damaged proteins may have specific features as compared to chaperones assisting de novo folding . After preconditioning and 50 degrees C treatment, cells lacking Lhs1p remained capable of protein synthesis and secretion for several hours at 24 degrees C, but only 10% were able to form colonies, as compared to wild-type cells . We suggest that Lhs1p is involved in a novel function operating in the yeast ER, refolding and stabilization against proteolysis of heatdenatured protein . Lhs1p may be part of a fundamental heat-resistant survival machinery needed for recovery of yeast cells from severe heat stress.

EMBO J, 1997 May 15, 16(10), 2745 - 55
Bni1p and Bnr1p: downstream targets of the Rho family small G-proteins which interact with profilin and regulate actin cytoskeleton in Saccharomyces cerevisiae; Imamura H et al.; The RHO1 gene encodes a homologue of mammalian RhoA small G-protein in the yeast Saccharomyces cerevisiae . Rho1p is required for bud formation and is localized at a bud tip or a cytokinesis site . We have recently shown that Bni1p is a potential target of Rho1p . Bni1p shares the FH1 and FH2 domains with proteins involved in cytokinesis or establishment of cell polarity . In S . cerevisiae, there is an open reading frame (YIL159W) which encodes another protein having the FH1 and FH2 domains and we have named this gene BNR1 (BNI1 Related) . Bnr1p interacts with another Rho family member, Rho4p, but not with Rho1p . Disruption of BNI1 or BNR1 does not show any deleterious effect on cell growth, but the bni1 bnr1 mutant shows a severe temperature-sensitive growth phenotype . Cells of the bni1 bnr1 mutant arrested at the restrictive temperature are deficient in bud emergence, exhibit a random distribution of cortical actin patches and often become multinucleate . These phenotypes are similar to those of the mutant of PFY1, which encodes profilin, an actin-binding protein . Moreover, yeast two-hybrid and biochemical studies demonstrate that Bni1p and Bnr1p interact directly with profilin at the FH1 domains . These results indicate that Bni1p and Bnr1p are potential targets of the Rho family members, interact with profilin and regulate the reorganization of actin cytoskeleton.

Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5201 - 6
The p20 and Ded1 proteins have antagonistic roles in eIF4E-dependent translation in Saccharomyces cerevisiae; de la Cruz J et al.; The translation initiation factor eIF4E mediates the binding of the small ribosomal subunit to the cap structure at the 5' end of the mRNA . In Saccharomyces cerevisiae, the cap-binding protein eIF4E is mainly associated with eIF4G, forming the cap-binding complex eIF4F . Other proteins are detected upon purification of the complex on cap-affinity columns . Among them is p20, a protein of unknown function encoded by the CAF20 gene . Here, we show a negative regulatory role for the p20 protein in translation initiation . Deletion of CAF20 partially suppresses mutations in translation initiation factors . Overexpression of the p20 protein results in a synthetic enhancement of translation mutation phenotypes . Similar effects are observed for mutations in the DED1 gene, which we have isolated as a multicopy suppressor of a temperature-sensitive eIF4E mutation . The DED1 gene encodes a putative RNA helicase of the DEAD-box family . The analyses of its suppressor activity, of polysome profiles of ded1 mutant strains, and of synthetic lethal interactions with different translation mutants indicate that the Ded1 protein has a role in translation initiation in S . cerevisiae.

J Biotechnol, 1997 May 9, 54(3), 161 - 74
Metabolic flux distributions in recombinant Saccharomyces cerevisiae during foreign protein production; Jin S et al.; A stoichiometric flux balancing analysis was applied to the recombinant yeast cultivation to examine the cellular physiology and relationship between the production of heterologous protein and metabolic fluxes . The fluxes in the metabolic pathway within a recombinant S . cerevisiae grown on galactose alone or mixtures of galactose and ethanol medium were calculated . It is found that an amplification of the PP (Pentose Phosphate) pathway activity resulted in an improvement of the foreign protein expression and cell yield on ATP . The carbon source used for biosynthesis from TCA cycle in the exponential growth phase was 2 and 5-fold higher, respectively, as compared with that in the late exponential growth phase and stationary phase in batch culture with galactose minimum medium . The metabolism of ethanol together with galactose in the recombinant cell looks like increasing the flux from Acetyl-CoA to TCA cycle, and amplifying the flux directing the synthesis of various kinds of precursors such as amino acids and nucleic acid which are necessary for production of a foreign protein . Metabolic flux distribution analysis also shows that the ATP synthesis rate under substrate-level phosphorylation in the mixed carbon source cultivation was lower than that in the sole carbon source (galactose) during the expression of foreign protein . However, the total ATP production rate was higher in the mixed carbon source cultivation.

J Biol Chem, 1997 May 9, 272(19), 12683 - 91
The yeast Saccharomyces cerevisiae as a genetic system for obtaining variants of poliovirus protease 2A; Barco A et al.; The inducible expression of poliovirus protease 2A (2Apro) blocks the growth of Saccharomyces cerevisiae . A number of yeast colonies that grow after 2Apro induction have been isolated . The majority of these clones express 2Apro to control levels, suggesting that their ability to divide is not due to the loss of 2Apro gene inducibility . The sequences of the 2Apro genes isolated from 22 clones were determined . Most of the 2Apro sequences from these colonies contain point mutations in the poliovirus protease . The different variant protease sequences were transferred to an infectious poliovirus cDNA clone . Translation of genomic RNA obtained from these poliovirus mutants in cell-free systems revealed that some of them had defects in their ability to cleave P1-2A in cis . In addition, several of these variants cleaved the translation initiation factor eIF-4G inefficiently . Transfection of the RNA generated from the full-length poliovirus genomes mutated in 2Apro yielded five viable polioviruses with a small plaque phenotype . These five polioviruses efficiently cleaved p220 but showed defects in viral protein synthesis, transactivation of a leader-luciferase mRNA, and 3CD cleavage to 3C' and 3D' . All 2Apro mutant sequences, including those that did not yield viable viruses, were cloned in pTM1 vector under a T7 promoter . Only the 2Apro variants that have activity to cleave 3CD produced viable poliovirus . Our findings indicate that S . cerevisiae represents a useful system for obtaining poliovirus 2Apro variants that may provide further insight into the role of this protease during the poliovirus replication cycle.

J Biol Chem, 1997 May 9, 272(19), 12616 - 9
Functional expression of the murine Golgi CMP-sialic acid transporter in saccharomyces cerevisiae; Berninsone P et al.; We have functionally expressed the murine Golgi putative CMP-sialic acid transporter in Saccharomyces cerevisiae . Using a galactose-inducible expression system, S . cerevisiae vesicles were able to transport CMP-sialic acid . Transport was dependent on galactose induction and was temperature-dependent and saturable with an apparent Km of 2.9 microM . Transport was inhibited by CMP, and upon vesicle disruption with Triton X-100 parameters were very similar to the previously described CMP-sialic acid transport characteristics observed with mammalian Golgi vesicles . CMP-sialic acid transport induction was specific as no transport of UDP-galactose was observed even though the latter putative transporter has a high degree of amino acid sequence identity with the CMP-sialic acid transporter . Together, the above results demonstrate that the previously described cDNA encoding the putative CMP-sialic acid transporter encodes the transporter protein per se and suggests that this heterologous expression system may be used for further structural and functional studies of other Golgi membrane transporter proteins.

J Cell Biol, 1997 May 5, 137(3), 539 - 53
A proteasome cap subunit required for spindle pole body duplication in yeast; McDonald HB et al.; Proteasome-mediated protein degradation is a key regulatory mechanism in a diversity of complex processes, including the control of cell cycle progression . The selection of substrates for degradation clearly depends on the specificity of ubiquitination mechanisms, but further regulation may occur within the proteasomal 19S cap complexes, which attach to the ends of the 20S proteolytic core and are thought to control entry of substrates into the core . We have characterized a gene from Saccharomyces cerevisiae that displays extensive sequence similarity to members of a family of ATPases that are components of the 19S complex, including human subunit p42 and S . cerevisiae SUG1/CIM3 and CIM5 products . This gene, termed PCS1 (for proteasomal cap subunit), is identical to the recently described SUG2 gene (Russell, S.J., U.G . Sathyanarayana, and S.A . Johnston . 1996 . J . Biol . Chem . 271:32810-32817) . We have shown that PCS1 function is essential for viability . A temperature-sensitive pcs1 strain arrests principally in the second cycle after transfer to the restrictive temperature, blocking as large-budded cells with a G2 content of unsegregated DNA . EM reveals that each arrested pcs1 cell has failed to duplicate its spindle pole body (SPB), which becomes enlarged as in other monopolar mutants . Additionally, we have shown localization of a functional Pcs1-green fluorescent protein fusion to the nucleus throughout the cell cycle . We hypothesize that Pcs1p plays a role in the degradation of certain potentially nuclear component(s) in a manner that specifically is required for SPB duplication.

Cell, 1997 May 2, 89(3), 403 - 12
Cross-intron bridging interactions in the yeast commitment complex are conserved in mammals; Abovich N et al.; The commitment complex is the first defined step in the yeast (S . cerevisiae) splicing pathway . It contains U1 snRNP as well as Mud2p, which resembles human U2AF65 . In a genetic screen, we identified the yeast gene MSL-5, which is a novel commitment complex component . Genetic and biochemical criteria indicate a direct interaction between Msl5p and both Mud2p and the U1 snRNP protein Prp40p . This defines a bridge between the two ends of the intron . Msl5p (renamed BBP for branchpoint bridging protein) has a mammalian ortholog, the splicing factor SF1 . Our results show that SF1 interacts strongly with human U2AF65, and that SF1 is a bona fide E complex component . This implies that aspects of these novel cross-intron protein-protein interactions are conserved between yeast and mammals.

Cell, 1997 May 2, 89(3), 393 - 402
The yeast La protein is required for the 3' endonucleolytic cleavage that matures tRNA precursors; Yoo CJ et al.; Although the La autoantigen binds to the 3' ends of all nascent polymerase III transcripts, its function in vivo has long been unclear . Although S . cerevisiae cells lacking the La protein homolog Lhp1p are viable, cells containing a mutation that disrupts the anticodon stem of tRNA(Ser)CGA require Lhp1p for growth . We demonstrate that for the wild-type pre-tRNA(Ser)CGA and other pre-tRNAs, Lhp1p is required for the normal endonucleolytic removal of the 3' trailer sequence . In cells lacking Lhp1p, the 3' trailer is removed by exonuclease(s) . Although maturation of the mutant pre-tRNA(Ser)CGA requires Lhp1p, introduction of a second mutation that restores base pairing eliminates the requirement . We propose that binding by Lhp1p stabilizes pre-tRNAs in conformations that allow the 3' endonucleolytic cleavage to occur.

Cell, 1997 May 2, 89(3), 381 - 91
Redistribution of silencing proteins from telomeres to the nucleolus is associated with extension of life span in S . cerevisiae; Kennedy BK et al.; A prior genetic study indicated that activity of Sir silencing proteins at a hypothetical AGE locus is essential for long life span . In this model, the SIR4-42 mutation would direct the Sir protein complex to the AGE locus, giving rise to a long life span . We show by indirect immunofluorescence that Sir3p and Sir4p are redirected to the nucleolus in the SIR4-42 mutant . Furthermore, this relocalization is dependent on both UTH4 a novel yeast gene that extends life span, and its homologue YGL023 . Strikingly, the Sir complex is relocalized from telomeres to the nucleolus in old wild-type cells . We propose that the rDNA is the AGE locus and that nucleolar function is compromised in old yeast cells in a way that may be mitigated by targeting of Sir proteins to the nucleolus.

Cell, 1997 May 2, 89(3), 349 - 56
Histone deacetylases associated with the mSin3 corepressor mediate mad transcriptional repression; Laherty CD et al.; Transcriptional repression by Mad-Max heterodimers requires interaction of Mad with the corepressors mSin3A/B . Sin3p, the S . cerevisiae homolog of mSin3, functions in the same pathway as Rpd3p, a protein related to two recently identified mammalian histone deacetylases, HDAC1 and HDAC2 . Here, we demonstrate that mSin3A and HDAC1/2 are associated in vivo . HDAC2 binding requires a conserved region of mSin3A capable of mediating transcriptional repression . In addition, Mad1 forms a complex with mSin3 and HDAC2 that contains histone deacetylase activity . Trichostatin A, an inhibitor of histone deacetylases, abolishes Mad repression . We propose that Mad-Max functions by recruiting the mSin3-HDAC corepressor complex that deacetylates nucleosomal histones, producing alterations in chromatin structure that block transcription.

J Ind Microbiol Biotechnol, 1997 May, 18(5), 319 - 25
Effects of medium composition and nutrient limitation on loss of the recombinant plasmid pLG669-z and beta-galactosidase expression by Saccharomyces cerevisiae; O'Kennedy RD et al.; The effects of medium composition, nutrient limitation and dilution rate on the loss of the recombinant plasmid pLG669-z and plasmid-borne beta-galactosidase expression were studied in batch and chemostat cultures of Saccharomyces cerevisiae strain CGpLG . The difference in growth rates between plasmid-free and plasmid-containing cells (delta mu) and the rate of segregation (R) were determined and some common factors resulting from the effect of medium composition on plasmid loss were identified . Glucose-limited chemostat cultures of CGpLG grown on defined medium were more stable at higher dilution rates and exhibited delta mu-dominated plasmid loss kinetics . Similar cultures grown on complex medium were more stable at lower dilution rates and exhibited R-dominated plasmid loss kinetics . Overall plasmid stability was greatest in phosphate-limited chemostat cultures grown on defined medium and was least stable in magnesium-limited cultures grown on defined medium . delta mu decreased and R increased with increased dilution rate, irrespective of medium composition . Increased plasmid loss rates at high or low dilution rates would appear to be characteristic of loss kinetics dominated by R or delta mu, respectively . Growth of glucose-limited chemostat cultures on complex medium decreased delta mu values but increased R values, in comparison to those cultures grown on defined medium . Any increased stability that a complex medium-induced reduction of delta mu may have conferred was counteracted by an increased R value . Increased beta-galactosidase productivity was correlated with increased plasmid stability only in glucose-limited chemostat cultures grown on defined medium and not in those grown on complex medium . Previous studies have yielded contrasting responses with regard to the effect of dilution rate on recombinant plasmid loss from S . cerevisiae . Our findings can account for these differences and may be generally valid for the stability of similar yeast plasmid constructs . This information would facilitate the design of bioprocesses, where recombinant plasmid instability results in reduced culture productivity.

Yeast, 1997 May, 13(6), 551 - 9
Expression cassettes for formaldehyde and fluoroacetate resistance, two dominant markers in Saccharomyces cerevisiae; van den Berg MA et al.; We employed two genes in constructing yeast expression cassettes for dominant, selectable markers . The Saccharomyces cerevisiae gene SFA1, encoding formaldehyde dehydrogenase, was placed under the control of the GPD1 promoter and CYC1 terminator . The Moraxella sp . strain B gene dehH1, encoding fluoroacetate dehalogenase, was placed under the control of both the GPD1 and CYC1 promoters . With these constructs it was possible to select directly for yeast strains resistant to either formaldehyde or fluoroacetate . Both selective agents are completely metabolized and inexpensive, making them very useful in the pursuit of yeast gene functions and for industrial applications . An additional advantage of the formaldehyde dehydrogenase marker is that it is an S . cerevisiae gene, thus allowing 'all yeast' constructs.

RNA, 1997 May, 3(5), 527 - 37
YRA1, an essential Saccharomyces cerevisiae gene, encodes a novel nuclear protein with RNA annealing activity; Portman DS et al.; The complexity of eukaryotic mRNA processing suggests a need for certain factors, called RNA chaperones, that can modulate RNA secondary structure as well as the interactions between pre-mRNA and trans-acting components . To identify factors that may fulfill this role in the yeast Saccharomyces cerevisiae, we fractionated whole-cell extracts and assayed for activity that could facilitate a specific RNA-RNA annealing reaction . We detected one strong RNA annealing activity and purified it to homogeneity . This previously undescribed factor, Yra1p, is localized to the nucleus; its sequence contains one RNP-motif RNA-binding domain . The YRA1 gene contains a 766-nt intron, the second-largest identified in this organism, and Yra1p serves an essential, nonredundant function . Taken together, our findings indicate that Yra1p is likely to have an important role in S . cerevisiae nuclear pre-mRNA metabolism.

Br J Pharmacol, 1997 May, 121(1), 1 - 6
High affinity of sigma 1-binding sites for sterol isomerization inhibitors: evidence for a pharmacological relationship with the yeast sterol C8-C7 isomerase; Moebius FF et al.; 1 . The sigma-drug binding site of guinea-pig liver is carried by a protein which shares significant amino acid sequence similarities with the yeast sterol C8-C7 isomerase (ERG2 protein) . Pharmacologically-but not structurally-the sigma 1-site is also related to the emopamil binding protein, the mammalian sterol C8-C7 isomerase . We therefore investigated if sterol C8-C7 isomerase inhibitors are high affinity ligands for the (+)-{3H}-pentazocine labelled sigma 1-binding site . 2 . Among the compounds which bound with high affinity to native hepatic and cerebral as well as to yeast expressed sigma 1-binding sites were the agricultural fungicide fenpropimorph (Ki 0.005 nM), the antihypocholesterinaemic drugs triparanol (Ki 7.0 nM), AY-9944 (Ki, 0.46 nM) and MDL28,815 (Ki 0.16 nM), the enantiomers of the ovulation inducer clomiphene (Ki 5.5 and 12 nM, respectively) and the antioestrogene tamoxifen (Ki 26 nM) . 3 . Except for tamoxifen these affinities are essentially identical with those for the {3H}-ifenprodil labelled sterol C8-C7 isomerase of S . cerevisiae . This demonstrates that sigma 1-binding protein and yeast isomerase are not only structurally but also pharmacologically related . Because of its affiliations with yeast and mammalian sterol isomerases we propose that the sigma 1-binding site is localized on a sterol isomerase related protein, involved in postsqualene sterol biosynthesis.

Nat Genet, 1997 May, 16(1), 44 - 53
HIP1, a human homologue of S . cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain; Kalchman MA et al.; Huntington disease (HD) is associated with the expansion of a polyglutamine tract, greater than 35 repeats, in the HD gene product, huntingtin . Here we describe a novel huntingtin interacting protein, HIP1, which co-localizes with huntingtin and shares sequence homology and biochemical characteristics with Sla2p, a protein essential for function of the cytoskeleton in Saccharomyces cerevisiae . The huntingtin-HIP1 interaction is restricted to the brain and is inversely correlated to the polyglutamine length in huntingtin . This provides the first molecular link between huntingtin and the neuronal cytoskeleton and suggests that, in HD, loss of normal huntingtin-HIP1 interaction may contribute to a defect in membrane-cytoskeletal integrity in the brain.

J Biol Chem, 1997 Apr 18, 272(16), 10839 - 46
Homocitrate synthase is located in the nucleus in the yeast Saccharomyces cerevisiae; Chen S et al.; We have generated monoclonal antibodies against nuclear proteins from the yeast Saccharomyces cerevisiae . The monoclonal antibodies react with proteins of 47 and 49 kDa on immunoblots and with partially overlapping sets of proteins on two-dimensional nonequilibrium pH gradient electrophoresis-SDS blots . Immunofluorescence localization shows a nuclear staining pattern . Immunoscreening a yeast expression library yielded five independent full-length clones of two open reading frames from chromosome IV, corresponding to YDL182w (LYS20) and YDL131w in the Saccharomyces genome data base . These two open reading frames are predicted to encode homocitrate synthase isozymes of 47 and 49 kDa, respectively . A clone carrying YDL182w was sequenced in its entirety and directs the expression of a 47-kDa protein in Escherichia coli . A clone carrying YDL131w expresses a 49-kDa protein in E . coli . Yeast grown in minimal medium plus lysine show significant reductions in nuclear immunofluorescence staining . Cell fractionation studies localize the 47- and 49-kDa proteins to the nucleus . Nuclear fractionation studies reveal that a portion of the 47- and 49-kDa proteins can only be extracted with DNase digestion and high salt . The localization of homocitrate synthase to the nucleus is unexpected given previous reports that homocitrate synthase is present in mitochondria and the cytoplasm in S . cerevisiae.

J Biol Chem, 1997 Apr 18, 272(16), 10361 - 6
Mammalian Mg2+-independent phosphatidate phosphatase (PAP2) displays diacylglycerol pyrophosphate phosphatase activity; Dillon DA et al.; Recent studies indicate that the metabolism of diacylglycerol pyrophosphate (DGPP) is involved in a novel lipid signaling pathway . DGPP phosphatases (DGPP phosphohydrolase) from Saccharomyces cerevisiae and Escherichia coli catalyze the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyze the dephosphorylation of PA to yield diacylglycerol . We demonstrated that the Mg2+-independent form of PA phosphatase (PA phosphohydrolase, PAP2) purified from rat liver catalyzed the dephosphorylation of DGPP . This reaction was Mg2+-independent, insensitive to inhibition by N-ethylmaleimide and bromoenol lactone, and inhibited by Mn2+ ions . PAP2 exhibited a high affinity for DGPP (Km = 0.04 mol %) . The specificity constant (Vmax/Km) for DGPP was 1 . 3-fold higher than that of PA . DGPP inhibited the ability of PAP2 to dephosphorylate PA, and PA inhibited the dephosphorylation of DGPP . Like rat liver PAP2, the Mg2+-independent PA phosphatase activity of DGPP phosphatase purified from S . cerevisiae was inhibited by lyso-PA, sphingosine 1-phosphate, and ceramide 1-phosphate . Mouse PAP2 showed homology to DGPP phosphatases from S . cerevisiae and E . coli, especially in localized regions that constitute a novel phosphatase sequence motif . Collectively, our work indicated that rat liver PAP2 is a member of a phosphatase family that includes DGPP phosphatases from S . cerevisiae and E . coli . We propose a model in which the phosphatase activities of rat liver PAP2 and the DGPP phosphatase of S . cerevisiae regulate the cellular levels of DGPP, PA, and diacylglycerol.

Biochem Biophys Res Commun, 1997 Apr 17, 233(2), 480 - 6
Phase-specific protein expression in the dimorphic yeast Saccharomyces cerevisiae; Viard B et al.; In oxygen-limited continuous culture, Saccharomyces cerevisiae formed pseudohyphae by unipolar budding . We developed a continuous cultivation sequence to discriminate phase-specific from metabolically regulated proteins during dimorphism . Computer-aided substractive analysis of 2D-PAGE protein patterns allowed the detection of proteins specifically expressed during yeast and pseudohyphal phases . Image analysis resolved 3 spots that were specific to the pseudohyphal phase and 2 spots that were specific to yeast phase . In addition to phase-specific proteins, important regulation of protein expression took place . A group of 9 proteins was highly over-expressed during the yeast phase when another group of 12 was underexpressed . This phenomenon was reversed during the pseudohyphal phase . These experiments showed that dimorphism in S . cerevisiae is associated with the expression of specific proteins and suggest that yeast phase-specific proteins maintain the yeast form or repress pseudohyphae formation.

Eur J Biochem, 1997 Apr 15, 245(2), 449 - 56
Identification and characterization of the genes for mitochondrial ribosomal proteins of Saccharomyces cerevisiae; Kitakawa M et al.; We have purified 13 large subunit proteins of the mitochondrial ribosome of the yeast Saccharomyces cerevisiae and determined their partial amino acid sequences . To elucidate the structure and function of these proteins, we searched for their genes by comparing our sequence data with those deduced from the genomic nucleotide sequence data of S . cerevisiae and analyzed them . In addition, we searched for the genes encoding proteins whose N-terminal amino acid sequences we have reported previously {Grohmann, L., Graack, H.-R., Kruft, V., Choli, T., Goldschmidt-Reisin, S . & Kitakawa, M . (1991) FEBS Lett . 284, 51-56} . Thus, we were able to identify and characterize 12 new genes for large subunit proteins of the yeast mitochondrial ribosome . Furthermore, we determined the N-terminal amino acid sequences of seven small subunit proteins and subsequently identified the genes for five of them, three of which were found to be new.

Eur J Biochem, 1997 Apr 15, 245(2), 373 - 80
Functional expression of Fasciola hepatica cathepsin L1 in Saccharomyces cerevisiae; Roche L et al.; A cDNA encoding the complete precursor of a Fasciola hepatica cathepsin L protease was isolated and sequenced . Functionally active enzyme was expressed and secreted by Saccharomyces cerevisiae transformed with a plasmid carrying the complete gene . Experiments with temperature-sensitive yeast mutants showed that the enzyme is trafficked through the yeast secretory pathway . Yeast transformed with a truncated gene, which lacked the pre-peptide-encoding and most of the pro-peptide-encoding sequences, did not express funtionally active enzyme . The yeast-expressed enzyme exhibited physicochemical properties in common with the native enzyme including, pH optimum for activity, stability at 37 degrees C and ability to cleave gelatin and immunoglobulin . Enzyme kinetic data showed that the native and yeast-expressed cathepsin L1 have similar specificities for substrates with hydrophobic residues in the P2 position . This is the first report of the functional expression of a cathepsin L proteinase in S . cerevisiae that did not require the use of yeast secretory signal sequences.

Arch Biochem Biophys, 1997 Apr 15, 340(2), 195 - 200
Saccharomyces cerevisiae expresses two genes encoding isozymes of 5-aminoimidazole-4-carboxamide ribonucleotide transformylase; Tibbetts AS et al.; We have isolated and cloned two Saccharomyces cerevisiae genes which encode isozymes of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase, the ninth step of the de novo purine biosynthesis pathway . This reaction involves the formylation of AICAR using 10-formyltetrahydrofolate as the formyl donor . ADE16 is located on chromosome XII and encodes an open reading frame of 591 amino acids . ADE17 is located on chromosome XIII and encodes an open reading frame of 592 amino acids . The deduced amino acid sequences of the two genes are 84% identical to each other and are 60-63% identical to the chicken and human bifunctional AICAR transformylase/IMP cyclohydrolase amino acid sequences . Disruption of the two chromosomal yeast genes resulted in adenine auxotrophy, while the expression of either gene alone was sufficient to support growth without adenine . In vitro assays of AICAR transformylase activity demonstrated the lack of IMP production in the double disruptant strain . S . cerevisiae is the only organism known thus far to possess isozymes of this protein . Because it is likely that the proteins encoded by ADE16 and ADE17 also contain IMP cyclohydrolase activity, these two genes complete the set of clones and mutants for the entire de novo purine biosynthesis pathway in yeast.

FEMS Microbiol Lett, 1997 Apr 15, 149(2), 279 - 84
Properties of yeast expressed Aspergillus nidulans chitin synthase B which is essential for hyphal growth; Tatsuno K et al.; A complementary DNA of the Aspergillus nidulans chsB gene encoding chitin synthase, an essential gene for hyphal growth, was obtained by RT-PCR and expressed in Saccharomyces cerevisiae by using the GAL1 promoter in a multicopy plasmid . The biochemical characteristics of chitin synthase B (ChsB) expressed in S . cerevisiae were examined . The chitin synthase B produced in galactose medium showed zymogenicity due to activation by trypsin treatment and required Mg2+ ion to exert maximal activity . It was competitively inhibited by polyoxin D . The Ki value of the inhibitor was 10 microM, and the K(m) for the substrate was 1.6 mM . The activity was enhanced by the addition of N-acetylglucosamine . The optimal pH is 7.5 when Mg2+ is used . These characteristics are the same as those of other chitin synthases.

Nucleic Acids Res, 1997 Apr 15, 25(8), 1485 - 92
Two distinct DNA ligase activities in mitotic extracts of the yeast Saccharomyces cerevisiae; Ramos W et al.; Four biochemically distinct DNA ligases have been identified in mammalian cells . One of these enzymes, DNA ligase I, is functionally homologous to the DNA ligase encoded by the Saccharomyces cerevisiae CDC9 gene . Cdc9 DNA ligase has been assumed to be the only species of DNA ligase in this organism . In the present study we have identified a second DNA ligase activity in mitotic extracts of S . cerevisiae with chromatographic properties different from Cdc9 DNA ligase, which is the major DNA joining activity . This minor DNA joining activity, which contributes 5-10% of the total cellular DNA joining activity, forms a 90 kDa enzyme-adenylate intermediate which, unlike the Cdc9 enzyme-adenylate intermediate, reacts with an oligo (pdT)/poly (rA) substrate . The levels of the minor DNA joining activity are not altered by mutation or by overexpression of the CDC9 gene . Furthermore, the 90 kDa polypeptide is not recognized by a Cdc9 antiserum . Since this minor species does not appear to be a modified form of Cdc9 DNA ligase, it has been designated as S . cerevisiae DNA ligase II . Based on the similarities in polynucleotide substrate specificity, this enzyme may be the functional homolog of mammalian DNA ligase III or IV.

Gene, 1997 Apr 11, 189(1), 13 - 8
Satellite DNA from the brine shrimp Artemia affects the expression of a flanking gene in yeast; Maiorano D et al.; We have previously revealed that in the brine shrimp Artemia franciscana an AluI DNA family of repeats, 113 bp in length, is the major component of the constitutive heterochromatin and that this repetitive DNA shows a stable curvature that confers a solenoidal geometry on the double helix in vitro . It was suggested that this particular structure may play a relevant role in determining the condensation of the heterochromatin . In this report we have cloned hexamers of highly-repetitive sequence (AluI-satellite DNA) in proximity to a yeast lacZ reporter gene on a plasmid . We find that the expression of the reporter gene is affected by the presence of this DNA in a dose- and orientation-dependent manner in the yeast, S . cerevisiae . We show that this effect is not dependent on under-replication or re-arrangements of the repetitive DNA in the cell but is due to decreased expression of the reporter gene . Our results indicate that the AluI-satellite DNA of Artemia per se is able to influence gene expression.

J Biol Chem, 1997 Apr 11, 272(15), 9720 - 7
Heterologous expression of human cholecystokinin in Saccharomyces cerevisiae . Evidence for a lysine-specific endopeptidase in the yeast secretory pathway; Rourke IJ et al.; Precursors of the human regulatory peptide cholecystokinin (CCK) have been expressed in Saccharomyces cerevisiae, and the post-translational processing of secreted CCK-related products analyzed . Recombinant plasmids expressing native human prepro-CCK and a hybrid molecule encompassing the prepro leader of the yeast alpha-mating pheromone fused to pro-CCK were examined . The latter construct resulted in considerably higher levels of pro-CCK secretion and was therefore analyzed in more detail . Two of the protein modifications essential for CCK bioactivity, C-terminal alpha-amidation and tyrosyl sulfation, were not detected in S . cerevisiae . Proteolytic cleavage of pro-CCK occurred C-terminally of three basic sites; (i) Arg105-Arg106 which, upon exposure to carboxypeptidase activity, leads to the production of glycine-extended CCK; (ii) Arg95 to produce CCK-8 related processing intermediates; and (iii) Lys81 resulting in CCK-22 related products . To elucidate which protease(s) are involved in these endoproteolytic cleavage events, pro-CCK was expressed in yeast mutants lacking various combinations of the Mkc7, Yap3, and Kex2 proteases . Only in S . cerevisiae strains deficient in Kex2 function was any of the above mentioned pro-CCK cleavages abolished, namely processing at the Arg105-Arg106 and Arg95 sites . This suggests that mammalian Kex2-like serine proteases may process pro-CCK at single arginine residues . Our data suggests that an as yet uncharacterized endopeptidase(s) in the S . cerevisiae secretory pathway is responsible for the lysine-specific cleavage of pro-CCK.

J Cell Biol, 1997 Apr 7, 137(1), 79 - 92
Endosome to Golgi retrieval of the vacuolar protein sorting receptor, Vps10p, requires the function of the VPS29, VPS30, and VPS35 gene products; Seaman MN et al.; Mutations in the S . cerevisiae VPS29 and VPS30 genes lead to a selective protein sorting defect in which the vacuolar protein carboxypeptidase Y (CPY) is missorted and secreted from the cell, while other soluble vacuolar hydrolases like proteinase A (PrA) are delivered to the vacuole . This phenotype is similar to that seen in cells with mutations in the previously characterized VPS10 and VPS35 genes . Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for soluble vacuolar hydrolases like CPY and PrA, while Vps35p is a peripheral membrane protein which cofractionates with membranes enriched in Vps10p . The sequences of the VPS29, VPS30, and VPS35 genes do not yet give any clues to the functions of their products . Each is predicted to encode a hydrophilic protein with homologues in the human and C . elegans genomes . Interestingly, mutations in the VPS29, VPS30, or VPS35 genes change the subcellular distribution of the Vps10 protein, resulting in a shift of Vps10p from the Golgi to the vacuolar membrane . The route that Vps10p takes to reach the vacuole in a vps35 mutant does not depend upon Sec1p mediated arrival at the plasma membrane but does require the activity of the pre-vacuolar endosomal t-SNARE, Pep12p . A temperature conditional allele of the VPS35 gene was generated and has been found to cause missorting/secretion of CPY and also Vps10p to mislocalize to a vacuolar membrane fraction at the nonpermissive temperature . Vps35p continues to cofractionate with Vps10p in vps29 mutants, suggesting that Vps10p and Vps35p may directly interact . Together, the data indicate that the VPS29, VPS30, and VPS35 gene products are required for the normal recycling of Vps10p from the prevacuolar endosome back to the Golgi where it can initiate additional rounds of vacuolar hydrolase sorting.

Cell, 1997 Apr 4, 89(1), 83 - 92
Pex14p, a peroxisomal membrane protein binding both receptors of the two PTS-dependent import pathways; Albertini M et al.; Pex14p, an S . cerevisiae peroxin, is attached to the outer face of the peroxisomal membrane and is a component of the protein import machinery . Pex14p interacts with both the PTS1 and PTS2 receptors . It is the only known peroxisomal membrane protein that binds the PTS2 receptor and might thus mediate the membrane docking event of PTS2-dependent protein import . These results suggest that the two import pathways overlap and, furthermore, that Pex14p represents the point of convergence . Pex14p also interacts with two other membrane-bound peroxins including Pex13p, another binding protein for the PTS1 receptor . The data presented here are consistent with the idea of a common translocation machinery for both PTS-dependent protein import pathways in the peroxisomal membrane.

C R Acad Sci III, 1997 Apr, 320(4), 299 - 305
A second DNA polymerase activity in yeast mitochondria; Lucas P et al.; In eukaryotic cells, there is much evidence to indicate that the replication of the mitochondrial genome is carried out by a specific DNA polymerase named DNA polymerase gamma . In the yeast S . cerevisiae, a DNA polymerase gamma has been partially purified and the gene encoding the catalytic subunit identified . The characteristics of this enzyme are the same as those found in higher eukaryotes, except for the requirement for a higher magnesium concentration . During a purification procedure of yeast mitochondrial DNA polymerase, we have isolated a second DNA polymerase activity . Using different approaches ive have ruled out the possibility of nuclear contamination or a product of proteolysis . From its properties, this new DNA polymerase activity seems to be different from any yeast DNA polymerase . This new mitochondrial DNA polymerase activity provides evidence that the animal model of mitochondrial DNA replication cannot be generalized . The presence of two DNA polymerases in yeast mitochondria could reflect a different replication or repair mechanism.

Appl Microbiol Biotechnol, 1997 Apr, 47(4), 405 - 11
Isolation of freeze-tolerant laboratory strains of Saccharomyces cerevisiae from proline-analogue-resistant mutants; Takagi H et al.; Since some amino acids, polyols and sugars in cells are thought to be osmoprotectants, we expected that several amino acids might also contribute to enhancing freeze tolerance in yeast cells . In fact, proline and charged amino acids such as glutamate, arginine and lysine showed a marked cryoprotective activity nearly equivalent to that of glycerol or trehalose, both known as major cryoprotectants for Saccharomyces cerevisiae . To investigate the cryoprotective effect of proline on the freezing stress of yeast, we isolated proline-analogue-resistant mutants derived from a proline-non-utilizing strain of S . cerevisiae . When cultured in liquid minimal medium, many mutants showed a prominent increase, two- to approximately tenfold, in cell viability compared to the parent after freezing in the medium at -20 degrees C for 1 week . Some of the freeze-tolerant mutants were found to accumulate a higher amount of proline, as well as of glutamate and arginine which are involved in proline metabolism . It was also observed that proline-non-utilizer and the freeze-tolerant mutants were able to grow against osmotic stress . These results suggest that the increased flux in the metabolic pathway of specific amino acids such as proline is effective for breeding novel freeze-tolerant yeasts.

Plant Mol Biol, 1997 Apr, 33(6), 1025 - 35
The presence of a Sar1 gene family in Brassica campestris that suppresses a yeast vesicular transport mutation Sec12-1; Kim WY et al.; Two new members (Bsar1a and Bsar1b) of the Sar1 gene family have been identified from a flower bud cDNA library of Brassica campestris and their functional characteristics were analyzed . The two clones differ from each other at 14 positions of the 193 amino acid residues deduced from their coding region . The amino acid sequences of Bsar1a and Bsar1b are most closely related to the Sar1 family, genes that function early in the process of vesicle budding from the endoplasmic reticulum (ER) . The sequences contain all the conserved motifs of the Ras superfamily (G1-G4 motifs) as well as the distinctive structural feature near the C-terminus that is Sar1 specific . Our phylogenetic analysis confirmed that these two clones can indeed be considered members of the Sar1 family and that they have a close relationship to the ARF family . The Bsar1 proteins, expressed in Escherichia coli, cross-reacted with a polyclonal antibody prepared against Saccharomyces cerevisiae Sar1 protein . It also exhibited GTP-binding activity . Genomic Southern blot analysis, using the 3'-gene-specific regions of the Bsar1 cDNAs as probes, revealed that the two cDNA clones are members of a B . campestris Sar1 family that consists of 2 to 3 genes . RNA blot analysis, using the same gene-specific probes, showed that both genes are expressed with similar patterns in most tissues of the plant, including leaf, stem, root, and flower buds . Furthermore, when we placed the two Bsar1 genes under the control of the yeast pGK1 promoter into the temperature-sensitive mutant yeast strain S . cerevisiae Sec12-1, they suppressed the mutation which consists of a defect in vesicle transport . The amino acid sequence similarity, the GTP-binding activity, and the functional suppression of the yeast mutation suggest that the Bsar1 proteins are functional homologues of the Sar1 protein in S . cerevisiae and that they may perform similar biological functions.

DNA Cell Biol, 1997 Apr, 16(4), 501 - 14
Protein quality--a determinant of the intracellular fate of membrane-bound cytochromes P450 in yeast; Zimmer T et al.; To elucidate mechanisms determining the intracellular localization of cytochromes P450, authentic and mutant cytochromes P450 52A4 (P450Cm2) and P450 52A5 (P450Alk2A) were heterologously expressed in Saccharomyces cerevisiae and the ultrastructure of the respective transformants was investigated by means of immunoelectron microscopy . As a result, overproduction of both wild-type P450 forms resulted in a massive proliferation of tubular membrane structures distributed over the whole cytoplasm . In contrast, all mutant P450Cm2 and Alk2A forms tested were mainly localized within stacks of paired membranes which often occurred in close vicinity to the nucleus . As found by serial sectioning of a single cell, these stacked membranes bearing the mutant P450 actually represented plates of consecutive membranes arranged one upon the other . A tubular network of endoplasmic reticulum membranes as observed after expression of the wild-type proteins could not be detected . Generally, the kind of mutation introduced into the P450 forms did not influence the morphology of the induced membranes . Even single amino acid exchanges in the cytosolic domain caused the formation of membrane stacks . The common feature of all mutant P450 forms causing the formation of stacked membranes was, however, their lower protein stability after heterologous expression in the S . cerevisiae host cells, compared to the stability of the authentic cytochromes P450 . Furthermore, the proliferated membranes containing the different P450 forms were characterized by means of subcellular fractionation experiments . Using this approach, clear differences in the distribution of spectrally active and inactive P450 molecules were found . The results obtained suggest the presence of an intracellular sorting mechanism based on the protein quality, which finally leads to the differences in the intracellular distribution of wild-type and mutant cytochromes P450.

Methods, 1997 Apr, 11(4), 343 - 52
Translation initiation factor-dependent extracts from yeast Saccharomyces cerevisiae; Altmann M et al.; Translation initiation factor-dependent extracts are prepared from Saccharomyces cerevisiae strains that have no or reduced activity of a translation initiation factor . Elimination of factor activity can be achieved by deletion of the gene encoding the factor if it is not essential for the survival of the strain . If the gene is essential it is placed under the control of the regulatable GAL1 promoter and its expression is shut off in vivo . Alternatively, a temperature-sensitive mutation can be introduced into the gene and the activity of the gene product eliminated in vitro by preincubation of the extract at the nonpermissive temperature . Factor-dependent extracts can be complemented in vitro with purified initiation factor preparations isolated from S . cerevisiae or from Escherichia coli cells expressing them from plasmid-encoded constructs . To simplify the purification of the factors they may be expressed as fusion proteins with N- or C-terminal tags . Initiation factor-dependent extracts can be used to study initiation factor structure-function relationships and initiation factor requirements for specific mRNA translation.

Mol Cell Biol, 1997 Apr, 17(4), 1848 - 59
The Saccharomyces cerevisiae MADS-box transcription factor Rlm1 is a target for the Mpk1 mitogen-activated protein kinase pathway; Dodou E et al.; Mutation of Saccharomyces cerevisiae RLM1, which encodes a MADS-box transcription factor, confers resistance to the toxic effects of constitutive activity of the Mpk1 mitogen-activated kinase (MAPK) pathway . The Rlm1 DNA-binding domain, which is similar to that of the metazoan MEF2 transcription factors, is also closely related to that of a second S . cerevisiae protein, Smp1 (second MEF2-like protein), encoded by the YBR182C open reading frame (N . Demolis et al., Yeast 10:1511-1525, 1994; H . Feldmann et al., EMBO J . 13:5795-5809, 1994) . We show that Rlm1 and Smp1 have MEF2-related DNA-binding specificities: Rlm1 binds with the same specificity as MEF2, CTA(T/A)4TAG, while SMP1 binds a more extended consensus sequence, ACTACTA(T/A)4TAG . The two DNA-binding domains can heterodimerize with each other and with MEF2A . Deletion of RLM1 enhances resistance to cell wall disruptants, increases saturation density, reduces flocculation, and inactivates reporter genes controlled by the Rlm1 consensus binding site . Deletion of SMP1 neither causes these phenotypes nor enhances the Rlm1 deletion phenotype . However, overexpression of the DNA-binding domain of either protein causes an osmoremedial phenotype . Synthetic and naturally occurring MEF2 consensus sequences exhibit strong RLM1- and MPK1-dependent upstream activation sequence activity . Transcriptional activation by Rlm1 requires its C-terminal sequences, and Gal4 fusion proteins containing Rlm1 C-terminal sequences also act as MPK1-dependent transcriptional activators . These results establish the Rlm1 C-terminal sequences as a target for the Mpk1 MAPK pathway.

Mol Cell Biol, 1997 Apr, 17(4), 1768 - 75
Interaction of a Swi3 homolog with Sth1 provides evidence for a Swi/Snf-related complex with an essential function in Saccharomyces cerevisiae; Treich I et al.; The Saccharomyces cerevisiae Swi/Snf complex has a role in remodeling chromatin structure to facilitate transcriptional activation . The complex has 11 components, including Swi1/Adr6, Swi2/Snf2, Swi3, Snf5, Snf6, Snf11, Swp73/Snf12, and Tfg3 . Mammalian homologs of these proteins have been shown to form multiple Swi/Snf-related complexes . Here we characterize an S . cerevisiae Swi3 homolog (Swh3) and present evidence that it associates in a complex with a Snf2 homolog, Sthl . We identified Swh3 as a protein that interacts with the N terminus of Snf2 in the two-hybrid system . Swh3 and Swi3 are functionally distinct, and overexpression of one does not compensate for loss of the other . Swh3 is essential for viability and does not activate transcription of reporters . The Snf2 sequence that interacts with Swh3 was mapped to a region conserved in Sth1 . We show that Swh3 and Sth1 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts . We also map interactions between Swh3 and Sth1 and examine the role of a leucine zipper motif in self-association of Swh3 . These findings, together with previous analysis of Sth1, indicate that Swh3 and Sth1 are associated in a complex that is functionally distinct from the Swi/Snf complex and essential for viability.

Appl Environ Microbiol, 1997 Apr, 63(4), 1362 - 6
Construction of a starch-utilizing yeast by cell surface engineering; Murai T et al.; We have engineered the cell surface of the yeast Saccharomyces cerevisiae by anchoring active glucoamylase protein on the cell wall, and we have endowed the yeast cells with the ability to utilize starch directly as the sole carbon source . The gene encoding Rhizopus oryzae glucoamylase with its secretion signal peptide was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin, a protein involved in mating and covalently anchored to the cell wall . The constructed plasmid containing this fusion gene was introduced into S . cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S . cerevisiae . The glucoamylase activity as not detected in the culture medium, but it was detected in the cell pellet fraction . The glucoamylase protein transferred to the soluble fraction from the cell wall fraction after glucanase treatment but not after sodium dodecyl sulfate treatment, indicating the covalent binding of the fusion protein to the cell wall . Display of the fused protein was further confirmed by immunofluorescence microscopy and immunoelectron microscopy . The transformant cells could surely grow on starch as the sole carbon source . These results showed that the glucoamylase was anchored on the cell wall and displayed as its active form . This is the first example of an application of cell surface engineering to utilize and improve the metabolic ability of cells.

Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 3151 - 5
mcm5/cdc46-bob1 bypasses the requirement for the S phase activator Cdc7p; Hardy CF et al.; Cdc7p is a protein kinase that is required for G1/S transition and initiation of DNA replication in Saccharomyces cerevisiae . The mechanisms whereby Cdc7p and its substrates exerts their effects are unknown . We report here the characterization in S . cerevisiae of a recessive mutation in a member of the MCM family, MCM5/CDC46, which bypasses the requirement for Cdc7p and its interacting factor Dbf4p . Because the MCM family of evolutionarily conserved proteins have been implicated in restricting DNA replication to once per cell cycle, our studies suggest that Cdc7p is required late in G1 because in its absence the Mcm5p/Cdc46p blocks the initiation of DNA replication . Moreover, Mcm5p/Cdc46p may have both positive and negative effects on the ability of cell to initiate replication.

Mamm Genome, 1997 Apr, 8(4), 274 - 6
A yeast artificial chromosome (YAC) library containing 10 haploid chicken genome equivalents; Toye AA et al.; We report the construction of a YAC library that provides 10-fold redundant coverage of the chicken genome . The library was made by transforming S . cerevisiae AB1380 with YAC constructs consisting of partially digested and size fractionated (>465 kb) EcoRI genomic fragments ligated to pCGS966 YAC vector arms . The primary library provides 8.5-fold redundant coverage and consists of 16,000 clones arrayed in duplicate 96-well microtiter plates and gridded on nylon membranes at high density (18,000 clones/484cm2) . The average insert size, 634 kb, was derived from size fractionation of a random sample of 218 YACs . Hybridization of five unlinked chicken genes to colony blots revealed six or more positive clones . This is consistent with the theoretical expectation from average insert sizes and number of clones . A second collection of clones consists of a further 20,000 colonies, of which 20% contain inserts larger than 450 kb and 80% contain only coligated vector arms . We estimate that these clones provide a further 1.5-fold redundant coverage of the chicken genome; thus, the total collection of 36,000 clones provides 10-fold redundant coverage of the chicken genome . The library is intended as a resource for fine-scale analysis of the organization of the chicken genome and is presently being used to construct a contig map of chicken Chromosome (Chr) 16, which contains the MHC and nucleolar organizer.

Curr Biol, 1997 Apr 1, 7(4), 228 - 38
Regulation of the mating pheromone and invasive growth responses in yeast by two MAP kinase substrates; Tedford K et al.; BACKGROUND: In the budding yeast Saccharomyces cerevisiae, components of a single mitogen-activated protein (MAP) kinase pathway transduce two distinct signals, each of which activates an independent developmental programme: peptide mating pheromones initiate the mating response, whereas nutrient limitation initiates filamentous growth . One of the MAP kinases in this pathway, Fus3, triggers mating but antagonizes filamentous growth, while the other, Kss 1, preferentially triggers filamentous growth . Both kinases activate the same transcription factor, Ste 12, which can stimulate gene expression specific to each of the developmental programmes . The precise mechanism by which these MAP kinases activate Ste 12, however, is not clear . RESULTS: Two newly identified proteins, Rst 1 and Rst 2 (also known as Dig1 and Dig2), were found to associate physically with Fus3 and Ste12 . Rst1 and Rst2 were prominent substrates in kinase reactions of Fus3 immune complexes from pheromone-treated cells . Association of Fus3 with Ste12 required Rst1 and Rst2, and activation of Fus3 by pheromone caused release of Ste12 from the Fus3 complex . Although rst1 and rst2 single mutants had no obvious phenotype, both filamentous growth and mating-specific gene expression were constitutive in rst1 rst2 double mutants . The phenotype of rst1 rst2 cells required Ste12 function, but did not require the function of upstream kinases . Consistent with Rst1 and Rst2 having a role in Ste12 regulation, both proteins were localized to the nucleus . CONCLUSIONS: Rst1 and Rst2 repress the mating and filamentous growth responses of S . cerevisiae by directly inhibiting Ste12 . Activation of Fus3 or Kss1 may cause phosphorylation-dependent release of Ste12 from Rst1/Rst2 and thereby activate Ste12-dependent transcription.

Genetics, 1997 Apr, 145(4), 903 - 10
Deletion of the leader peptide of the mitochondrially encoded precursor of Saccharomyces cerevisiae cytochrome c oxidase subunit II; Torello AT et al.; Cytochrome c oxidase subunit II (Cox2p) of Saccharomyces cerevisiae is synthesized within mitochondria as a precursor, pre-Cox2p . The 15-amino acid leader peptide is processed after export to the intermembrane space . Leader peptides are relatively unusual in mitochondrially coded proteins: indeed mammalian Cox2p lacks a leader peptide . We generated two deletions in the S . cerevisiae COX2 gene, removing either the leader peptide (cox2-20) or the leader peptide and processing site (cox2-21) without altering either the promoter or the mRNA-specific translational activation site . When inserted into mtDNA, both deletions substantially reduced the steady-state levels of Cox2p and caused a tight nonrespiratory phenotype . A respiring pseudorevertant of the cox2-20 mutant was heteroplasmic for the original mutant mtDNA and a p- mtDNA whose deletion fused the first 251 codons of the mitochondrial gene encoding cytochrome b to the cox2-20 sequence . The resulting fusion protein was processed to yield functional Cox2p . Thus, the presence of amino-terminal cytochrome b sequence bypassed the need for the pre-Cox2p leader peptide . We propose that the pre-Cox2p leader peptide contains a targeting signal necessary for membrane insertion, without which it remains in the matrix and is rapidly degraded.

Nat Genet, 1997 Apr, 15(4), 377 - 80
Rhizomelic chondrodysplasia punctata is a peroxisomal protein targeting disease caused by a non-functional PTS2 receptor; Motley AM et al.; Rhizomelic chondrodysplasia punctata (RCDP) is an autosomal recessive disease characterized clinically by a disproportionately short stature primarily affecting the proximal parts of the extremities, typical dysmorphic facial appearance, congenital contractures and severe growth and mental retardation . Although some patients have single enzyme deficiencies, the majority of RCDP patients (86%) belong to a single complementation group (CG11, also known as complementation group I, Amsterdam nomenclature) . Cells from CG11 show a tetrad of biochemical abnormalities: a deficiency of i) dihydroxyacetonephosphate acyltransferase, ii) alkyldihydroxyacetonephosphate synthase, iii) phytanic acid alpha-oxidation and iv) inability to import peroxisomal thiolase . These deficiencies indicate involvement of a component required for correct targeting of these peroxisomal proteins . Deficiencies in peroxisomal targeting are also found in Saccharomyces cerevisiae pex5 and pex7 mutants, which show differential protein import deficiencies corresponding to two peroxisomal targeting sequences (PTS1 and PTS2) . These mutants lack their PTS1 and PTS2 receptors, respectively . Like S . cerevisiae pex cells, RCDP cells from CG11 cannot import a PTS2 reporter protein . Here we report the cloning of PEX7 encoding the human PTS2 receptor, based on its similarity to two yeast orthologues . All RCDP patients from CG11 with detectable PEX7 mRNA were found to contain mutations in PEX7 . A mutation resulting in C-terminal truncation of PEX7 cosegregates with the disease and expression of PEX7 in RCDP fibroblasts from CG11 rescues the PTS2 protein import deficiency . These findings prove that mutations in PEX7 cause RCDP, CG11.

Antimicrob Agents Chemother, 1997 Apr, 41(4), 776 - 80
Characterization of Saccharomyces cerevisiae CYP51 and a CYP51 fusion protein with NADPH cytochrome P-450 oxidoreductase expressed in Escherichia coli; Venkateswarlu K et al.; Saccharomyces cerevisiae CYP51, target of azole antifungal agents, and CYP51 fused with S . cerevisiae cytochrome P-450 oxidoreductase (FUS protein) were expressed in active forms in Escherichia coli by cloning into pET15b . The expression was monitored immunologically, catalytically, and by using reduced carbon monoxide difference and type II binding spectra . CYP51 and FUS enzymes were located in membranes and produced a Soret peak at 448 nm in the reduced CO difference spectrum . The cytochrome P-450 contents in the membrane fractions containing CYP51 and FUS proteins were 12.8 +/- 2.6 and 17.4 +/- 3.7 pmol/mg of protein, respectively . The NADPH cytochrome P-450 oxidoreductase (CPR) content was estimated to be 15.7 +/- 1.1 pmol/mg of protein in FUS membrane fractions . FUS protein catalyzed the demethylation of substrate at the 14alpha position, with a turnover number of 1.96 +/- 0.37 min(-1) in the presence of NADPH . No reductase activity was observed in membrane fractions containing CYP51, and therefore, CYP51 did not function catalytically in the presence of NADPH, but in the presence of an artificial electron donor, cumene hydroperoxide, activity was comparable to that of the FUS enzyme . Further support for a normal structure for the hemoproteins was obtained from type II binding spectra, in which the spectral response was saturated with an equimolar concentration of ketoconazole.

Yeast, 1997 Mar 30, 13(4), 391 - 7
Identification of gene encoding a putative RNA-helicase, homologous to SKI2, in chromosome VII of Saccharomyces cerevisiae; Martegani E et al.; We have determined the nucleotide sequence of a segment of VII of the yeast Saccharomyces cerevisiae contained in the cosmid clone pEGH101 for a total of 7 kbp . This sequence contains a large open reading frame (ORF) called G9365, coding for a protein of 1967 amino acids that shows a significant homology with the product of the SKI2 gene of S . cerevisiae and contains domains characteristics of RNA-helicases . The ORF is transcribed in vegetative cells but it is not essential for viability as demonstrated by gene disruption.

Yeast, 1997 Mar 30, 13(4), 369 - 72
Sequence analysis of a 10.5 kb DNA fragment from the yeast chromosome VII reveals the presence of three new open reading frames and of a tRNAThr gene; Mazzoni C et al.; We report the sequence analysis of a 10,531 bp DNA of Saccharomyces cerevisiae chromosome VII . This sequence contains five complete open reading frames (ORFs) potentially encoding proteins longer than 100 amino acids and incomplete ORF encoding for the 3' part of the GCN5 gene (Georgakopoulos and Thireos, 1992) . ORFs G9160 and G9155 correspond to the genes ENO1 (Holland et al . 1981) and PUP2 (Gergatsou et al., 1992) respectively . ORF G9165 codes for a protein which shares significant homology with known proteins present in databases (see below) . The translated sequence of ORF G9170 shows 88% identity to the 6-phosphogluconate dehydrogenase encoded by the gene 6PGD from S . cerevisiae present in the SwissProt data library (P38720) . This indicates that G9170 might code for a second 6-phosphogluconate dehydrogenase . ORF G9175 codes for a putative new member of the mitochondrial carrier family . A hypothetical tRNAThr (TGT) is also present in position 6842-6913.

Yeast, 1997 Mar 30, 13(4), 357 - 63
DNA sequence analysis of a 23,002 bp DNA fragment of the right arm of Saccharomyces cerevisiae chromosome VII; Arroyo J et al.; We report the sequence of a 23,002 bp fragment located on the right arm of Saccharomyces cerevisiae chromosome VII . Analysis of this region revealed 14 complete open reading frames (ORFs) wit more than 300 base pairs . Six of them correspond to previously known genes . G7164 is the QCR9 gene coding for subunit 9 of the cytochrome c reductase; G7168 is UBR1, encoding an ubiquitin protein ligase; G7522 is the TYS1 gene, which encodes for the tyrosyl tRNA synthetase; G7526 is TFG1, the gene coding for the RNA polymerase transcription initiation factor TFIIF (factor G); G7538 is the gene HGH1 which encodes a protein related to the mammalian HMG1 and HMG2 proteins . G7542 is the BUB1 gene which encodes a ser/thr protein kinase involved in spindle assembly during the cell cycle . One of the ORFs, G7553, shares significant homologies with the gene UTR2 from S . cerevisiae . None of the seven remaining ORFs shows similarity to any of the sequences within the public databases . Three ORFs are internal ORFs of the above-described known genes, and two small ORFs are completely contained in larger ORFs on the complementary strand, and therefore probably do not correspond to real genes . This region also contains three genes specifying tRNAs for Leu, Lys and Trp, and several LTR elements.

Yeast, 1997 Mar 30, 13(4), 327 - 36
Cloning and expression of two chitin deacetylase genes of Saccharomyces cerevisiae; Mishra C et al.; Chitin deacetylase (EC 3.5.1.41), which hydrolyses the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, has been demonstrated in crude extracts from sporulating Saccharomyces cerevisiae . Two S . cerevisiae open reading frames (ORFs), identified by the Yeast Genome Project, have protein sequence homology to a chitin deacetylase from Mucor rouxii . Northern blot hybridizations show each ORF was transcribed in diploid cells after transfer to sporulation medium and prior to formation of asci . Each ORF was cloned in a vector under transcriptional control of the GAL 1, 10 promoter and introduced back into haploid strains of S . cerevisiae . Chitin deacetylase activity was detected by in vitro assays from vegetative cells grown in galactose . Chemical analysis of these cells also demonstrated the synthesis of chitosam in vivo . Both recombinant chitin deacetylases showed similar qualitative and quantitative activities toward chitooligosaccharides in vitro . A diploid strain deleted to both ORFs, when sporulated, did not show deacetylase activity . The mutant spores were hypersensitive to lytic enzymes (Glusulase or Zymolyase).

J Biol Chem, 1997 Mar 28, 272(13), 8531 - 8
Disruption of the Saccharomyces cerevisiae homologue to the murine fatty acid transport protein impairs uptake and growth on long-chain fatty acids; Faergeman NJ et al.; The yeast Saccharomyces cerevisiae is able to utilize exogenous fatty acids for a variety of cellular processes including beta-oxidation, phospholipid biosynthesis, and protein modification . The molecular mechanisms that govern the uptake of these compounds in S . cerevisiae have not been described . We report the characterization of FAT1, a gene that encodes a putative membrane-bound long-chain fatty acid transport protein (Fat1p) . Fat1p contains 623 amino acid residues that are 33% identical and 54% with similar chemical properties as compared with the fatty acid transport protein FATP described in 3T3-L1 adipocytes (Schaffer and Lodish (1994) Cell 79, 427-436), suggesting a similar function . Disruption of FAT1 results in 1) an impaired growth in YPD medium containing 25 microM cerulenin and 500 microM fatty acid (myristate (C14:0), palmitate (C16:0), or oleate (C18:1)); 2) a marked decrease in the uptake of the fluorescent long-chain fatty acid analogue boron dipyrromethene difluoride dodecanoic acid (BODIPY-3823); 3) a reduced rate of exogenous oleate incorporation into phospholipids; and 4) a 2-3-fold decrease in the rates of oleate uptake . These data support the hypothesis that Fat1p is involved in long-chain fatty acid uptake and may represent a long-chain fatty acid transport protein.

Nature, 1997 Mar 27, 386(6623), 414 - 7
An atypical topoisomerase II from Archaea with implications for meiotic recombination; Bergerat A et al.; Type II topoisomerases help regulate DNA topology during transcription, replication and recombination by catalysing DNA strand transfer through transient double-stranded breaks . All type II topoisomerases described so far are members of a single protein family . We have cloned and sequenced the genes encoding the A and B subunits of topoisomerase II from the archaeon Sulfolobus shibatae . This enzyme is the first of a new family . It has no similarity with other type II topoisomerases, except for three motifs in the B subunit probably involved in ATP binding and hydrolysis . We also found these motifs in proteins of the Hsp90 and MutL families . The A subunit has similarities with four proteins of unknown function . One of them, the Saccharomyces cerevisiae Spo11 protein, is required for the initiation of meiotic recombination . Mutagenesis, performed on SPO11, of the single tyrosine conserved between the five homologues shows that this amino acid is essential for Spo11 activity . By analogy with the mechanism of action of known type II topoisomerases, we suggest that Spo11 catalyses the formation of double-strand breaks that initiate meiotic recombination in S . cerevisiae.

Biochim Biophys Acta, 1997 Mar 27, 1356(1), 23 - 34
An amino terminal prosequence is required for efficient synthesis of S . cerevisiae a-factor; Quinby GE et al.; The Saccharomyces cerevisiae a-mating pheromones are 12 amino acid lipopeptides whose secretion is dependent on the ABC transporter, Ste6p . The pheromones are synthesized as 36 and 38 amino acid precursors that terminate in a CaaX box (C is Cys, a is an aliphatic residue, and X is the C-terminal amino acid) . Posttranslational processing of the a-factor precursors includes at least 5 events . C-terminal processing of the CaaX box includes farnesylation of Cys, removal of the -aaX residues, and methylation of the cysteine alpha-carboxyl group . The N-terminal steps involve proteolytic cleavages that remove the prosequences . In this report, we have investigated the role of posttranslational modification in the generation of functional a-factor . Wild type, mutant and chimeric forms of a-factor have been expressed in yeast and assessed for their abilities to serve as sources of functional a-factor . We have found that although modification of the CaaX box is necessary, it is not sufficient to generate bioactive a-factor . The amino terminal prosequences are also required . Deletion of these sequences reduces intracellular levels of a-factor resulting in sterility . Glutathione-S-transferase (GST)-a-factor fusions undergo CaaX box processing and membrane localization, but are not substrates for the N-terminal proteases and fail to interact with Ste6p . These results suggest that the amino terminal precursor sequences play a direct role in the generation of functional a-factor.

Mol Gen Genet, 1997 Mar 26, 254(2), 171 - 8
Inactivation of OGG1 increases the incidence of G . C-->T . A transversions in Saccharomyces cerevisiae: evidence for endogenous oxidative damage to DNA in eukaryotic cells; Thomas D et al.; The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine . To investigate the biological role of the OGG1 gene, mutants were constructed by partial deletion of the coding sequence and insertion of marker genes, yielding ogg1::TRP1 and ogg1::URA3 mutant strains . The disruption of the OGG1 gene does not compromise the viability of haploid cells, therefore it is not an essential gene . The capacity to repair 8-OxoG has been measured in cell-free extracts of wild-type and ogg1 strains using a 34mer DNA fragment containing a single 8-OxoG residue paired with a cytosine (8-OxoG/C) as a substrate . Cell-free extracts of the wild-type strain efficiently cleave the 8-OxoG-containing strand of the 8-OxoG/C duplex . In contrast, cell-free extracts of the Ogg1-deficient strain have no detectable activity that can cleave the 8-OxoG/C duplex . The biological properties of the ogg1 mutant have also been investigated . The results show that the ogg1 disruptant is not hypersensitive to DNA-damaging agents such as ultraviolet light at 254 nm, hydrogen peroxide or methyl methanesulfonate . However, the ogg1 mutant exhibits a mutator phenotype . When compared to those of a wild-type strain, the frequencies of mutation to canavanine resistance (CanR) and reversion to Lys+ are sevenfold and tenfold higher for the ogg1 mutant strain, respectively . Moreover, using a specific tester system, we show that the Ogg1-deficient strain displays a 50-fold increase in spontaneously occurring G x C-->T x A transversions compared to the wild-type strain . The five other base substitution events are not affected by the disruption of the OGG1 gene . These results strongly suggest that endogeneous reactive oxygen species cause DNA damage and that the excision of 8-OxoG catalyzed by the Ogg1 protein contributes to the maintenance of genetic stability in S . cerevisiae.

Mol Gen Genet, 1997 Mar 26, 254(2), 139 - 47
A novel yeast gene, RHK1, is involved in the synthesis of the cell wall receptor for the HM-1 killer toxin that inhibits beta-1,3-glucan synthesis; Kimura T et al.; The HM-1 killer toxin from Hansenula mrakii is known to inhibit cell wall beta-1,3-glucan synthase of Saccharomyces cerevisiae and other sensitive strains of yeast . A number of mutants of Saccharomyces cerevisiae that show resistance to this toxin were isolated in order to clarify the killing mechanism of the toxin . These mutants, designated rhk (resistant to Hansenula killer), were classified into three complementation groups . A novel gene RHK1, which complements the killer-resistant phenotype of the largest complementation group rhk1, was isolated . DNA sequence analysis revealed an open reading frame that encodes a hydrophobic protein composed of 458 amino acids . Gene disruption followed by tetrad analysis showed that RHK1 is not essential and loss of RHK1 function endowed S . cerevisiae cells with complete killer resistance . A biochemical analysis suggested that RHK1 does not participate directly in the synthesis of beta-1,3-glucan but is involved in the synthesis of the receptor for the HM-1 killer toxin.

Gene, 1997 Mar 18, 187(2), 201 - 9
Identification of a profilin homologue in Trypanosoma brucei by complementation screening; Wilson W et al.; Using genetic complementation in Saccharomyces cerevisiae, we have isolated a Trypanosoma brucei gene encoding profilin . Overexpression of trypanosome profilin suppresses defects that are associated with the loss of the C-terminal domain of the adenylyl cyclase-associated protein in S . cerevisiae . Similarly, the T . brucei gene complements a profilin-deletion mutant of S . cerevisiae . The full-length cDNA clone isolated contains an open reading frame of 150 amino acids, with a predicted molecular mass of 16.1 kDa . The gene appears to be present at single copy and is expressed at approximately equal levels in both mammalian and insect forms of the parasite.

FEBS Lett, 1997 Mar 17, 405(1), 11 - 5
Increased stress parameter synthesis in the yeast Saccharomyces cerevisiae after treatment with 4-hydroxy-2-nonenal; Wonisch W et al.; The metabolism of glutathione (GSH), a marker of oxidative stress and trehalose, a rather general physiological stress marker, was examined in exponentially growing Saccharomyces cerevisiae cells after treatment with 4-hydroxynonenal (HNE) . GSH was entirely depleted within a 2 h incubation with 250 microM HNE . After removal of the aldehyde it was replenished by de novo synthesis leading to an overshooting GSH level, which later decreased to the basal level . In addition, trehalose was elevated 4-fold in HNE-treated yeast cells compared to control cells . We conclude that increased GSH levels upon HNE treatment are a general phenomenon of eukaryotic cells to ensure protection and survival during further harsh conditions . Furthermore, we have discovered a new indication for the stress marker trehalose in S . cerevisiae.

Yeast, 1997 Mar 15, 13(3), 287 - 90
Analysis of an 11.6 kb region from the right arm of chromosome VII of Saccharomyces cerevisiae between the RAD2 and the MES1 genes reveals the presence of three new genes; Clemente ML et al.; Sequence analysis of an 11,628 bp DNA segment from the right arm of Saccharomyces cerevisiae chromosome VII revealed the presence of the 5' end of the RAD2 gene, the MES1 gene and six open reading frames (ORFs) each longer than 300 bp . Four of these ORFs are expressed genes, as indicated by transcript analysis . One of them, YGR261c, which specifies a putative beta-adaptine, corresponds to gene YKS5, which has recently been identified as a suppressor of loss of casein kinase 1 function . The remaining three ORFs are new genes; of these, YGR260w encodes a protein showing similarity to the S . cerevisiae allantoate permease and YGR262c specifies a putative protein kinase.

J Cell Biol, 1997 Mar 10, 136(5), 957 - 67
The yeast Red1 protein localizes to the cores of meiotic chromosomes; Smith AV et al.; Mutants in the meiosis-specific RED1 gene of S . cerevisiae fail to make any synaptonemal complex (SC) or any obvious precursors to the SC . Using antibodies that specifically recognize the Red1 protein, Red1 has been localized along meiotic pachytene chromosomes . Red1 also localizes to the unsynapsed axial elements present in a zip1 mutant, suggesting that Red1 is a component of the lateral elements of mature SCs . Anti-Red1 staining is confined to the cores of meiotic chromosomes and is not associated with the loops of chromatin that lie outside the SC . Analysis of the spo11 mutant demonstrates that Red1 localization does not depend upon meiotic recombination . The localization of Red1 has been compared with two other meiosis-specific components of chromosomes, Hop1 and Zip1; Zip1 serves as a marker for synapsed chromosomes . Double labeling of wild-type meiotic chromosomes with anti-Zip1 and anti-Red1 antibodies demonstrates that Red1 localizes to chromosomes both before and during pachytene . Double labeling with anti-Hop1 and anti-Red1 antibodies reveals that Hop1 protein localizes only in areas that also contain Red1, and studies of Hop1 localization in a red1 null mutant demonstrate that Hop1 localization depends on Red1 function . These observations are consistent with previous genetic studies suggesting that Red1 and Hop1 directly interact . There is little or no Hop1 protein on pachytene chromosomes or in synapsed chromosomal regions.

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