Microbiology, 1997 Jun, 143 ( Pt 6), 1877 - 83 Extrusion of benzoic acid in Saccharomyces cerevisiae by an energy-dependent mechanism; Henriques M et al.; When grown in the presence of benzoic acid, Saccharomyces cerevisiae was able to extrude {(14)C}benzoic acid when a pulse a glucose was given to preloaded cells . While octanoic, sorbic, hexanoic, salicylic, butyric and propionic acids were also inducers, ethanol and acetic acid were not . The mechanism of extrusion required energy and prior growth in the presence of the inducers . Diethylstilbestrol, an inhibitor of ATPases, prevented benzoic acid extrusion . Propionic acid was not actively extruded in cells adapted to either benzoic or propionic acid, behaving as an appropriate probe to measure intracellular pH . Even though the extrusion mechanism was active, benzoic acid entered the cells by a simple diffusion mechanism. Microbiology, 1997 Jun, 143 ( Pt 6), 1867 - 76 Filamentous growth of the budding yeast Saccharomyces cerevisiae induced by overexpression of the WHi2 gene; Radcliffe PA et al.; The WHI2 gene of the budding yeast Saccharomyces cerevisiae is required for the arrest of cell proliferation upon nutrient exhaustion: whi2 mutants carry on dividing and in the absence of growth become abnormally small . It is reported here that overexpression of Whi2 from the GAL1 promoter results in filamentous growth - cells fail to complete cytokinesis, the budding pattern changes from axial to polar, cells become elongated and cell size increases threefold . In many ways, these filaments resemble the pseudohyphae which result from nitrogen-limited growth and the filaments seen during the invasive growth of haploids . However, Whi2-induced filament formation is reduced, but not blocked, by mutations in STE7, STE12 or STE20 which do block pseudohypha formation . Furthermore, pseudohypha formation can still occur in a diploid in which both copies of the WHI2 gene have been deleted . Thus Whi2-induced filament formation and pseudohypha formation must come about through the action of different pathways . Despite this, a mutation in the STE11 gene, which is required for pseudohypha formation, does block Whi2-induced filament formation . Concanavalin A pulse-chase experiments show that new cell wall material is incorporated only into the tips of the apical cells . An extragenic suppressor of the Whi2 allele also results in filamentous growth . These results suggest that Whi2 negatively regulates a function required for the budding mode of cell proliferation including cytokinesis . This function is defined wholly or in part by the fsw1 allele. J Cell Sci, 1997 Jun, 110 ( Pt 11), 1299 - 306 Vam3p, a new member of syntaxin related protein, is required for vacuolar assembly in the yeast Saccharomyces cerevisiae; Wada Y et al.; Syntaxins are thought to participate in the specific interactions between vesicles and acceptor membranes in intracellular protein trafficking . VAM3 of Saccharomyces cerevisiae encodes a 33 kDa protein (Vam3p) with a hydrophobic transmembrane segment at its C terminus . Vam3p has structural similarities to syntaxins of yeast, animal and plant cells . delta vam3 cells accumulated spherical structures of 200-600 nm in diameter, but lacked normal large vacuolar compartments . Loss of function of Vam3p resulted in inefficient processing of vacuolar proteins proteinase A, proteinase B and carboxypeptidase Y, and defective maturation of alkaline phosphatase . Subcellular fractionation and immunofluorescence microscopy showed that Vam3p was localized to the vacuolar membranes . Vam3p was accumulated in certain regions of the vacuolar membranes . We conclude from these observations that Vam3p is a novel member of syntaxin in the vacuoles and it provides the t-SNARE function in a late step of the vacuolar assembly. Mol Biol Cell, 1997 Jun, 8(6), 1035 - 50 Saccharomyces cerevisiae genes required in the absence of the CIN8-encoded spindle motor act in functionally diverse mitotic pathways; Geiser JR et al.; Kinesin-related Cin8p is the most important spindle-pole-separating motor in Saccharomyces cerevisiae but is not essential for cell viability . We identified 20 genes whose products are specifically required by cell deficient for Cin8p . All are associated with mitotic roles and represent at least four different functional pathways . These include genes whose products act in two spindle motor pathways that overlap in function with Cin8p, the kinesin-related Kip1p pathway and the cytoplasmic dynein pathway . In addition, genes required for mitotic spindle checkpoint function and for normal microtubule stability were recovered . Mutant alleles of eight genes caused phenotypes similar to dyn1 (encodes the dynein heavy chain), including a spindle-positioning defect . We provide evidence that the products of these genes function in concept with dynein . Among the dynein pathway gene products, we found homologues of the cytoplasmic dynein intermediate chain, the p150Glued subunit of the dynactin complex, and human LIS-1, required for normal brain development . These findings illustrate the complex cellular interactions exhibited by Cin8p, a member of a conserved spindle motor family. Mol Biol Cell, 1997 Jun, 8(6), 1025 - 33 Mitotic spindle function in Saccharomyces cerevisiae requires a balance between different types of kinesin-related motors; Saunders W et al.; Two Saccharomyces cerevisiae kinesin-related motors, Cin8p and Kip1p, perform an essential role in the separation of spindle poles during spindle assembly and a major role in spindle elongation . Cin8p and Kip1p are also required to prevent an inward spindle collapse prior to anaphase . A third kinesin-related motor, Kar3p, may act antagonistically to Cin8p and Kip1p since loss of Kar3p partially suppresses the spindle collapse in cin8 kip1 mutants . We have tested the relationship between Cin8p and Kar3p by overexpressing both motors using the inducible GAL1 promoter . Overexpression of KAR3 results in a shrinkage of spindle size and a temperature-dependent inhibition of the growth of wild-type cells . Excess Kar3p has a stronger inhibitory effect on the growth of cin8 kip1 mutants and can completely block anaphase spindle elongation in these cells . In contrast, overexpression of CIN8 leads to premature spindle elongation in all cells tested . This is the first direct demonstration of antagonistic motors acting on the intact spindle and suggests that spindle length is determined by the relative activity of Kar3p-like and Cin8p/Kip1p-like motors. J Dairy Sci, 1997 Jun, 80(6), 1119 - 25 Effect of yeast culture (Saccharomyces cerevisiae) on adaptation of cows to diets postpartum; Robinson PH; For approximately 14 d prepartum and exactly 14 d postpartum, 20 multiparous Holstein cows were fed different basal diets that were supplemented, or not supplemented, with a yeast culture preparation . Cows supplemented with yeast culture lost less body condition prepartum, which was consistent with numerically higher body weight gain . No treatment differences were found in prepartum or postpartum dry matter intakes (DMI) or components of DMI . In addition, the extent of the depression in DMI prepartum and the rate of increase in DMI postpartum were not influenced by yeast culture supplementation . Milk and milk component yields were not influenced by yeast culture supplementation . Cows in both groups had higher calculated net energy for lactation for the diets postpartum than would have been expected based on values of the National Research Council for feedstuffs . The increased net energy for lactation seemed to be related to higher than expected metabolic efficiency during early lactation . Results of both the prepartum and postpartum periods were consistent with the hypothesis that supplementation of yeast culture in the diet increased net digestion in the forestomach, particularly of fiber, leading to increased energy output . However, there was no evidence to suggest that supplementation of yeast culture prepartum alleviated the reduction in DMI prepartum or improved the rate of increase in DMI postpartum. J Bacteriol, 1997 Jun, 179(12), 3875 - 83 AUT3, a serine/threonine kinase gene, is essential for autophagocytosis in Saccharomyces cerevisiae; Straub M et al.; Autophagocytosis is a starvation-induced process, carrying proteins destined for degradation to the lysosome . In the yeast Saccharomyces cerevisiae, the autophagic process is visualized by the appearance of autophagic vesicles in the vacuoles of proteinase yscB-deficient strains during starvation . aut3-1 mutant cells which exhibit a block in the autophagic process have been isolated previously . By using the drastically reduced sporulation frequency of homozygous aut3-1 diploid cells, the AUT3 gene was cloned by complementation . The Aut3 protein consists of 897 amino acids . The amino-terminal part of the protein shows significant homologies to serine/threonine kinases . aut3 null mutant cells are fully viable on rich media but show a reduced survival rate upon starvation . They are unable to accumulate autophagic vesicles in the vacuole during starvation . Starvation-induced vacuolar protein breakdown is almost completely impaired in aut3-deficient cells . Vacuolar morphology and acidification are not influenced in aut3-deficient cells . Also, secretion of invertase, endocytic uptake of Lucifer Yellow, and vacuolar protein sorting appear wild type like in aut3-deficient cells, suggesting autophagocytosis as a novel route for the transport of proteins from the cytosol to the vacuole . By using a fusion of Aut3p with green-fluorescent protein, Aut3p was localized to the cytosol. Arch Biochem Biophys, 1997 Jun 1, 342(1), 68 - 81 Substrate-based inhibitors of the (S)-adenosyl-L-methionine:delta24(25)- to delta24(28)-sterol methyl transferase from Saccharomyces cerevisiae; Nes WD et al.; A series of 31 side-chain-modified analogs of cholesterol, zymosterol, lanosterol, and cycloartenol and the steroidal alkaloids solasodine and solanidine were studied as inhibitors of (S)-adenosyl-L-methionine:delta24(25)-sterol methyl transferase (SMT) enzyme activity from Saccharomyces cerevisiae . Two classes of sterol methylation inhibitors were tested: substrate analogs, including mechanism-based inhibitors, and transition state analogs . Several novel sterol methylation inhibitors that contained an aza, aziridine, or ammonium group in the sterol side chain were prepared and tested for the first time . The degree and kinetic pattern of methylation inhibition were found to be influenced by the position and nature of the variant functional group introduced into the side chain . The most potent inhibitors of SMT enzyme activity were transition state analog inhibitors (Ki values of 5 to 10 nM) that mimicked the structure and conformation of the natural substrate presumed to form in the ternary complex generated in the transition state . Steroidal alkaloids were potent competitive inhibitors with Ki values ranging from 2 to 30 microM, which is about the Kmapp of zymosterol, ca . 27 microM . An isosteric analog of the natural substrate, zymosterol, in which the 26/27-gem-dimethyl groups were joined to form a cyclopropylidene function is shown to be a potent irreversible mechanism-based inactivator of SMT enzyme activity that exhibits competitive-type inhibition, Ki 48 microM with a K(inact) of 1.52 min(-1) . Mechanistic implications of these results provide new insights into the topology of the ternary complex involving sterol-AdoMet-enzyme. Glycobiology, 1997 Jun, 7(4), 487 - 97 Isolation of new nonconditional Saccharomyces cerevisiae mutants defective in asparagine-linked glycosylation; Manas P et al.; We describe the isolation and partial characterization of Saccharomyces cerevisiae nonconditional mutants that show defects in N-glycosylation of proteins . The selection method is based on the reduction of affinity for the ion exchanger QAE-Sephadex as a consequence of the decrease in the negative charge of the cell surface . This characteristic reflects a decrease in the incorporation of mannosylphosphate units into the N-linked oligosaccharides of the mannoproteins . The mutants exhibit low affinity for the basic dye alcian blue and for that reason we have called them Idb (low dye binding) mutants . Eight of the complementation groups seem to be new as shown by complementation studies with previously isolated mutants of similar phenotype . Four of the groups showed a significant reduction in the number and/or size of the N-linked oligosaccharides attached to secreted invertase . We have analyzed the N-linked oligosaccharides of Idb1 and Idb2, the mutants that show the most drastic reduction in the affinity for the alcian blue dye . In both cases, the purified endo H-released oligosaccharides from the mannoproteins lacked detectable amounts of phosphate groups as shown by ion exchange chromatography and the 1H NMR spectra . In addition, Ibd1 synthesizes a truncated and unbranched outer chain lacking any alpha (1,2) linked mannoses attached to the alpha (1,6) linear backbone. Genetics, 1997 Jun, 146(2), 491 - 8 Stabilization of microsatellite sequences by variant repeats in the yeast Saccharomyces cerevisiae; Petes TD et al.; We examined the effect of a single variant repeat on the stability of a 51-base pair (bp) microsatellite (poly GT) . We found that the insertion stabilizes the microsatellite about fivefold in wild-type strains . The stabilizing effect of the variant base was also observed in strains with mutations in the DNA mismatch repair genes pms1, msh2 and msh3, indicating that this effect does not require a functional DNA mismatch repair system . Most of the microsatellite alterations in the pms1, msh2 and msh3 strains were additions or deletions of single GT repeats, but about half of the alterations in the wild-type and msh6 strains were large (> 8 bp) deletions or additions. Genetics, 1997 Jun, 146(2), 481 - 9 Recombination and the progression of meiosis in Saccharomyces cerevisiae; Galbraith AM et al.; Recombination is an essential part of meiosis: in almost all organisms, including Saccharomyces cerevisiae, proper chromosome segregation and the viability of meiotic products is dependent upon normal levels of recombination . In this article we examine the kinetics of the meiotic divisions in four mutants defective in the initiation of recombination . We find that mutations in any of three Early Exchange genes (REC104, REC114 or REC102) confer a phenotype in which the reductional division occurs earlier than in an isogenic wild-type diploid . We also present data confirming previous reports that strains with a mutation in the Early Exchange gene . MEI4 undergo the first division at about the same time as wild-type cells . The rec104 mutation is epistatic to the mei4 mutation for the timing of the first division . These observations suggest a possible relationship between the initiation of recombination and the timing of the reductional division . These data also allow these four Early Exchange genes examined to be distinguished in terms of their role in coordinating recombination with the reductional division. J Bacteriol, 1997 Jun, 179(11), 3416 - 29 Cross regulation of four GATA factors that control nitrogen catabolic gene expression in Saccharomyces cerevisiae; Coffman JA et al.; Nitrogen catabolic gene expression in Saccharomyces cerevisiae has been reported to be regulated by three GATA family proteins, the positive regulators Gln3p and Gat1p/Nil1p and the negative regulator Dal80p/Uga43p . We show here that a fourth member of the yeast GATA family, the Dal80p homolog Deh1p, also negatively regulates expression of some, but not all, nitrogen catabolic genes, i.e., GAP1, DAL80, and UGA4 expression increases in a deh1 delta mutant . Consistent with Deh1p regulation of these genes is the observation that Deh1p forms specific DNA-protein complexes with GATAA-containing UGA4 and GAP1 promoter fragments in electrophoretic mobility shift assays . Deh1p function is demonstrable, however, only when a repressive nitrogen source such as glutamine is present; deh1 delta mutants exhibit no detectable phenotype with a poor nitrogen source such as proline . Our experiments also demonstrate that GATA factor gene expression is highly regulated by the GATA factors themselves in an interdependent manner . DAL80 expression is Gln3p and Gat1p dependent and Dal80p regulated . Moreover, Gln3p and Dal80p bind to DAL80 promoter fragments . In turn, GAT1 expression is Gln3p dependent and Dal80p regulated but is not autogenously regulated like DAL80 . DEH1 expression is largely Gln3p independent, modestly Gat1p dependent, and most highly regulated by Dal80p . Paradoxically, the high-level DEH1 expression observed in a dal80::hisG disruption mutant is highly sensitive to nitrogen catabolite repression. Mol Cell Biol, 1997 Jun, 17(6), 3429 - 39 Saccharomyces cerevisiae Mpt5p interacts with Sst2p and plays roles in pheromone sensitivity and recovery from pheromone arrest; Chen T et al.; SST2 plays an important role in the sensitivity of yeast cells to pheromone and in recovery from pheromone-induced G1 arrest . Recently, a family of Sst2p homologs that act as GTPase-activating proteins (GAPs) for G alpha subunits has been identified . We have identified an interaction between Sst2p and the previously identified Mpt5p by using the two-hybrid system . Loss of Mpt5p function resulted in a temperature-sensitive growth phenotype, an increase in pheromone sensitivity, and a defect in recovery from pheromone-induced G1 arrest, although the effects on pheromone response and recovery were mild in comparison to those of sst2 mutants . Overexpression of either Sst2p or Mpt5p promoted recovery from G1 arrest . Promotion of recovery by overexpression of Mpt5p required Sst2p, but the effect of overexpression of Sst2p was only partially dependent on Mpt5p . Mpt5p was also found to interact with the mitogen-activated protein kinase homologs Fus3p and Kss1p, and an mpt5 mutation was able to suppress the pheromone arrest and mating defects of a fus3 mutant . Because either mpt5 or cln3 mutations suppressed the fus3 phenotypes, interactions of Mpt5p with the G1 cyclins and Cdc28p were tested . An interaction between Mpt5p and Cdc28p was detected . We discuss these results with respect to a model in which Sst2p plays a role in pheromone sensitivity and recovery that acts through Mpt5p in addition to a role as a G alpha GAP suggested by the analysis of the Sst2p homologs. Mol Cell Biol, 1997 Jun, 17(6), 3382 - 7 Instability of CAG and CTG trinucleotide repeats in Saccharomyces cerevisiae; Miret JJ et al.; A quantitative genetic assay was developed to monitor alterations in tract lengths of trinucleotide repeat sequences in Saccharomyces cerevisiae . Insertion of (CAG)50 or (CTG)50 repeats into a promoter that drives expression of the reporter gene ADE8 results in loss of expression and white colony color . Contractions within the trinucleotide sequences to repeat lengths of 8 to 38 restore functional expression of the reporter, leading to red colony color . Reporter constructs including (CAG)50 or (CTG)50 repeat sequences were integrated into the yeast genome, and the rate of red colony formation was measured . Both orientations yielded high rates of instability (4 x 10(-4) to 18 x 10(-4) per cell generation) . Instability depended on repeat sequences, as a control harboring a randomized (C,A,G)50 sequence was at least 100-fold more stable . PCR analysis of the trinucleotide repeat region indicated an excellent correlation between change in color phenotype and reduction in length of the repeat tracts . No preferential product sizes were observed . Strains containing disruptions of the mismatch repair gene MSH2, MSH3, or PMS1 or the recombination gene RAD52 showed little or no difference in rates of instability or distributions of products, suggesting that neither mismatch repair nor recombination plays an important role in large contractions of trinucleotide repeats in yeast. Mol Cell Biol, 1997 Jun, 17(6), 3315 - 22 Differential requirements for DNA replication in the activation of mitotic checkpoints in Saccharomyces cerevisiae; Tavormina PA et al.; Checkpoints prevent inaccurate chromosome segregation by inhibiting cell division when errors in mitotic processes are encountered . We used a temperature-sensitive mutation, dbf4, to examine the requirement for DNA replication in establishing mitotic checkpoint arrest . We used gamma-irradiation to induce DNA damage and hydroxyurea to limit deoxyribonucleotides in cells deprived of DBF4 function to investigate the requirement for DNA replication in DNA-responsive checkpoints . In the absence of DNA replication, mitosis was not inhibited by these treatments, which normally activate the DNA damage and DNA replication checkpoints . Our results support a model that indicates that the assembly of replication structures is critical for cells to respond to defects in DNA metabolism . We show that activating the spindle checkpoint with nocodazole does not require prior progression through S phase but does require a stable kinetochore. Mol Cell Biol, 1997 Jun, 17(6), 3261 - 71 A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in Saccharomyces cerevisiae; Merchant AM et al.; We describe a new minichromosome maintenance factor, Mcm10, and show that this essential protein is involved in the initiation of DNA replication in Saccharomyces cerevisiae . The mcm10 mutant has an autonomously replicating sequence-specific minichromosome maintenance defect and arrests at the nonpermissive temperature with dumbbell morphology and 2C DNA content . Mcm10 is a nuclear protein that physically interacts with several members of the MCM2-7 family of DNA replication initiation factors . Cloning and sequencing of the MCM10 gene show that it is identical to DNA43, a gene identified independently for its putative role in replicating DNA . Two-dimensional DNA gel analysis reveals that the mcm10-1 lesion causes a dramatic reduction in DNA replication initiation at chromosomal origins, including ORI1 and ORI121 . Interestingly, the mcm10-1 lesion also causes replication forks to pause during elongation through these same loci . This novel phenotype suggests a unique role for the Mcm10 protein in the initiation of DNA synthesis at replication origins. Mol Cell Biol, 1997 Jun, 17(6), 3242 - 53 Molecular genetic analysis of Rts1p, a B' regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A; Shu Y et al.; The Saccharomyces cerevisiae gene RTS1 encodes a protein homologous to a variable B-type regulatory subunit of the mammalian heterotrimeric serine/threonine protein phosphatase 2A (PP2A) . We present evidence showing that Rts1p assembles into similar heterotrimeric complexes in yeast . Strains in which RTS1 has been disrupted are temperature sensitive (ts) for growth, are hypersensitive to ethanol, are unable to grow with glycerol as their only carbon source, and accumulate at nonpermissive temperatures predominantly as large-budded cells with a 2N DNA content and a nondivided nucleus . This cell cycle arrest can be overcome and partial suppression of the ts phenotype of rts1-null cells occurs if the gene CLB2, encoding a Cdc28 kinase-associated B-type cyclin, is expressed on a high-copy-number plasmid . However, CLB2 overexpression has no suppressive effects on other aspects of the rts1-null phenotype . Expression of truncated forms of Rts1p can also partially suppress the ts phenotype and can fully suppress the inability of cells to grow on glycerol and the hypersensitivity of cells to ethanol . By contrast, the truncated forms do not suppress the accumulation of large-budded cells at high temperatures . Coexpression of truncated Rts1p and high levels of Clb2p fully suppresses the ts phenotype, indicating that the inhibition of growth of rts1-null cells at high temperatures is due to both stress-related and cell cycle-related defects . Genetic analyses show that the role played by Rts1p in PP2A regulation is distinctly different from that played by the other known variable B regulatory subunit, Cdc55p, a protein recently implicated in checkpoint control regulation. Mol Cell Biol, 1997 Jun, 17(6), 2994 - 3004 The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae; Kaouass M et al.; Eukaryotic polyamine transport systems have not yet been characterized at the molecular level . We have used transposon mutagenesis to identify genes controlling polyamine transport in Saccharomyces cerevisiae . A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polyamine analog . A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2) . A 304-amino-acid stretch comprising 11 of the 12 catalytic subdomains of Stk2p is approximately 83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast . Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower Vmax than in the parental strain . Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild-type levels . Single mutants lacking the catalytic kinase subdomains of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs . Spermidine transport was repressed by prior incubation with exogenous spermidine . Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions . On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine . Interestingly, stk2::lacZ mutants showed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport . These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity. Curr Microbiol, 1997 Jun, 34(6), 382 - 4 Correlation of resistance to the alkaloid lycorine with the degree of suppressiveness in petite mutants of Saccharomyces cerevisiae; Del Giudice A et al.; In previous papers (Del Giudice et al . Curr Genet 8:493-497, 1984; Massardo et al . Curr Genet 17:455-457, 1985) we have shown that strains of Saccharomyces cerevisiae that are devoid of mitochondrial DNA (rhoo) are resistant to the alkaloid lycorine isolated from Amaryllis plants, whereas strains containing mitochondrial DNA (rho-, mit-, or rho+) are sensitive to this drug . In addition, we were able to show that the so-called hypersuppressive petites, whose mitochondrial genomes consist of short regions of DNA containing an ori sequence,show intermediate resistance . In this paper, we demonstrate that the degree of suppressiveness of a rho- mutant correlates with the degree of resistance to lycorine. Nature, 1997 May 29, 387(6632 Suppl), 103 - 5 The nucleotide sequence of Saccharomyces cerevisiae chromosome XVI; Bussey H et al.; The nucleotide sequence of the 948,061 base pairs of chromosome XVI has been determined, completing the sequence of the yeast genome . Chromosome XVI was the last yeast chromosome identified, and some of the genes mapped early to it, such as GAL4, PEP4 and RAD1 (ref . 2) have played important roles in the development of yeast biology . The architecture of this final chromosome seems to be typical of the large yeast chromosomes, and shows large duplications with other yeast chromosomes . Chromosome XVI contains 487 potential protein-encoding genes, 17 tRNA genes and two small nuclear RNA genes; 27% of the genes have significant similarities to human gene products, and 48% are new and of unknown biological function . Systematic efforts to explore gene function have begun. Nature, 1997 May 29, 387(6632 Suppl), 98 - 102 The nucleotide sequence of Saccharomyces cerevisiae chromosome XV; Dujon B et al.; Chromosome XV was one of the last two chromosomes of Saccharomyces cerevisiae to be discovered . It is the third-largest yeast chromosome after chromosomes XII and IV, and is very similar in size to chromosome VII . It alone represents 9% of the yeast genome (8% if ribosomal DNA is included) . When systematic sequencing of chromosome XV was started, 93 genes or markers were identified, and most of them were mapped . However, very little else was known about chromosome XV which, in contrast to shorter chromosomes, had not been the object of comprehensive genetic or molecular analysis . It was therefore decided to start sequencing chromosome XV only in the third phase of the European Yeast Genome Sequencing Programme, after experience was gained on chromosomes III, XI and II . The sequence of chromosome XV has been determined from a set of partly overlapping cosmid clones derived from a unique yeast strain, and physically mapped at 3.3-kilobase resolution before sequencing . As well as numerous new open reading frames (ORFs) and genes encoding tRNA or small RNA molecules, the sequence of 1,091,283 base pairs confirms the high proportion of orphan genes and reveals a number of ancestral and successive duplications with other yeast chromosomes. Nature, 1997 May 29, 387(6632 Suppl), 93 - 8 The nucleotide sequence of Saccharomyces cerevisiae chromosome XIV and its evolutionary implications; Philippsen P et al.; In 1992 we started assembling an ordered library of cosmid clones from chromosome XIV of the yeast Saccharomyces cerevisiae . At that time, only 49 genes were known to be located on this chromosome and we estimated that 80% to 90% of its genes were yet to be discovered . In 1993, a team of 20 European laboratories began the systematic sequence analysis of chromosome XIV . The completed and intensively checked final sequence of 784,328 base pairs was released in April, 1996 . Substantial parts had been published before or had previously been made available on request . The sequence contained 419 known or presumptive protein-coding genes, including two pseudogenes and three retrotransposons, 14 tRNA genes, and three small nuclear RNA genes . For 116 (30%) protein-coding sequences, one or more structural homologues were identified elsewhere in the yeast genome . Half of them belong to duplicated groups of 6-14 loosely linked genes, in most cases with conserved gene order and orientation (relaxed interchromosomal synteny) . We have considered the possible evolutionary origins of this unexpected feature of yeast genome organization. Nature, 1997 May 29, 387(6632 Suppl), 87 - 90 The nucleotide sequence of Saccharomyces cerevisiae chromosome XII; Johnston M et al.; The yeast Saccharomyces cerevisiae is the pre-eminent organism for the study of basic functions of eukaryotic cells . All of the genes of this simple eukaryotic cell have recently been revealed by an international collaborative effort to determine the complete DNA sequence of its nuclear genome . Here we describe some of the features of chromosome XII. Nature, 1997 May 29, 387(6632 Suppl), 78 - 81 The nucleotide sequence of Saccharomyces cerevisiae chromosome V; Dietrich FS et al.; Here we report the sequence of 569,202 base pairs of Saccharomyces cerevisiae chromosome V . Analysis of the sequence revealed a centromere, two telomeres and 271 open reading frames (ORFs) plus 13 tRNAs and four small nuclear RNAs . There are two Tyl transposable elements, each of which contains an ORF (included in the count of 271) . Of the ORFs, 78 (29%) are new, 81 (30%) have potential homologues in the public databases, and 112 (41%) are previously characterized yeast genes. Nature, 1997 May 29, 387(6632 Suppl), 67 - 73 Genetic and physical maps of Saccharomyces cerevisiae; Cherry JM et al.; Genetic and physical maps for the 16 chromosomes of Saccharomyces cerevisiae are presented . The genetic map is the result of 40 years of genetic analysis . The physical map was produced from the results of an international systematic sequencing effort . The data for the maps are accessible electronically from the Saccharomyces Genome Database (SGD: edu/Saccharomyces/). J Biol Chem, 1997 May 23, 272(21), 13647 - 52 Potassium transport by amino acid permeases in Saccharomyces cerevisiae; Wright MB et al.; Deletion of the potassium transporter genes TRK1 and TRK2 impairs potassium uptake in Saccharomyces cerevisiae, resulting in a greatly increased requirement for the ion and the inability to grow on low pH medium . Selection for mutations that restored growth of trk1Delta trk2Delta cells on low pH (3.0) medium led to the isolation of a dominant suppressor that also partially suppressed the increased K+ requirement of these cells . Molecular analysis revealed the suppressor to be an allele of BAP2 that encodes a permease for branched chain amino acids . The suppressor mutation (BAP2-1) converts a phenylalanine codon, highly conserved among the amino acid permease genes, to a serine codon in a region predicted to lie within the sixth membrane-spanning domain . Generation of the analogous mutation in the histidine permease produced an allele, HIP1-293, that similarly suppressed the low pH sensitivity of trk1Delta trk2Delta cells . Suppression of trk1Delta trk2Delta phenotypes by BAP2-1 or HIP1-293 was correlated with increased Rb+ uptake . The presence of the substrate amino acids enhanced but was not essential for suppression of trk1Delta trk2Delta phenotypes and increased Rb+ uptake . The conserved site altered by the suppressor mutations appears to be important; his4 HIP1-293 cells show an increased requirement for histidine compared with his4 HIP1 cells. Biochemistry, 1997 May 20, 36(20), 6107 - 14 Type II DNA topoisomerase from Saccharomyces cerevisiae is a stable dimer; Tennyson RB et al.; Type II DNA topoisomerases function as homodimeric enzymes in transiently cleaving double-stranded DNA to catalyze unlinking and unknotting reactions . The dimeric enzyme creates a DNA double-strand break by forming a covalent attachment between an active site tyrosine from each monomer and a 5'-phosphate from each strand of DNA . The dimer must be very stable to dissociation or subunit exchange when covalently attached to DNA to prevent directly or indirectly catalyzed rearrangements of the genome . Past studies have indicated conflicting results for the monomer-dimer stability of topoisomerase II in solution . Here, we report results from sedimentation equilibrium studies and two different subunit exchange assays indicating that purified Saccharomyces cerevisiae DNA topoisomerase II exists as a stable dimer in solution, with a Kd estimated to be < or = 10(-11) M . This high dimer stability is not detectably altered by a change of ionic strength or by the presence of ATP, ADP, or DNA. Biochemistry, 1997 May 20, 36(20), 6100 - 6 Partial purification of Pde1 from Saccharomyces cerevisiae: enzymatic redundancy for the repair of 3'-terminal DNA lesions and abasic sites in yeast; Sander M et al.; Earlier work indicates that the major DNA repair phosphodiesterase (PDE) in yeast cells is the well-characterized Apn1 protein . Apn1 demonstrates both Mg2+-independent PDE activity and Mg2+-independent class II apurinic/apyrimidinic (AP) endonuclease activity and represents greater than 90% of the activity detected in crude extracts from wild-type yeast cells . Apn1 is related to Echerichia coli endonuclease IV, both in its enzymatic properties and its amino acid sequence . In this work, we report the partial purification of a novel yeast protein, Pde1, present in Apn1-deficient cells . Pde1 is purified by sequential BioRex-70, PBE118, and MonoS chromatography steps using a sensitive and highly specific 3'-phosphoglycolate-terminated oligonucleotide-based assay as a measure of PDE activity . Mg2+-stimulated PDE and Mg2+-stimulated class II AP endonuclease copurify during this procedure . These results indicate that yeast, like many other organisms studied to date, has enzymatic redundancy for the repair of 3'-blocking groups and abasic sites. Biochemistry, 1997 May 20, 36(20), 5955 - 62 Stimulation of RTH1 nuclease of the yeast Saccharomyces cerevisiae by replication protein A; Biswas EE et al.; The RTH1 nuclease is involved in the replication of chromosomal DNA as well as in the repair of DNA damage . Replication protein A (RPA) is also an integral part of the DNA replication and repair processes . We have investigated the roles(s) of RPA in the function of RTH1 nuclease, including its structure specific endonuclease activity . Initial in vitro studies, which employed a "flap" or a "pseudo Y" substrate containing a short 14 bp duplex region, showed the effect of RPA to be minimal or inhibitory . As RPA inhibition is unwarranted for a protein participating in the DNA replication process, we have further investigated the mechanism of such inhibition . Alternate flap and pseudo Y substrates with a long duplex region (50 bp) were prepared using M13mp19 ssDNA and synthetic oligonucleotides . Yeast RPA stimulated the endonuclease activity of RTH1 endonuclease with these substrates in a dose-dependent manner . Kinetic analysis suggested that yRPA exerted a bipartite effect on the nuclease reaction: (i) the "load time" of RTH1 nuclease onto the DNA substrate decreased from approximately 5 to 2 min in the presence of RPA, and (ii) following initiation of the nuclease reaction, the initial rate of the reaction increased 10-fold in the presence of yRPA . Further analysis of the interaction of RPA with various endonuclease substrates indicated that RPA has a weak helix destabilizing effect and could melt small, 14 bp, regions of duplex DNA . RTH1 endonuclease cleaves the DNA strand at the junction of single- and double-stranded DNA; consequently, the observed inhibition with small duplex substrates was likely due to duplex melting . Our studies also demonstrated that RPA stimulated the RNase H activity of RTH1 nuclease significantly . In both instances (RTH1 endonuclease and RNase H), the stimulation may involve a specific interaction of RPA with the RTH1 nuclease rather than a structural positioning of the DNA substrate by RPA. FEBS Lett, 1997 May 19, 408(2), 217 - 20 Mitochondrial acyl carrier protein is involved in lipoic acid synthesis in Saccharomyces cerevisiae; Brody S et al.; The yeast gene, ACP1, encoding the mitochondrial acyl carrier protein, was deleted by gene replacement . The resulting acp1-deficient mutants had only 5-10% of the wild-type lipoic acid content remaining, and exhibited a respiratory-deficient phenotype . Upon meiosis, the lipoate deficiency co-segregated with the acp1 deletion . The role of ACP1 in long-chain fatty acid synthesis was studied in fast and fas2 null mutants completely lacking cytoplasmic fatty acid synthase . When grown on odd-chain (13:0 and 15:0) fatty acids, these cells showed less than 1% of C-16 and C-18 acids in their total lipids . Mitochondrial ACP is therefore suggested to be involved with the biosynthesis of octanoate, a precursor to lipoic acid. J Exp Med, 1997 May 19, 185(10), 1827 - 36 A new mouse gene, SRG3, related to the SWI3 of Saccharomyces cerevisiae, is required for apoptosis induced by glucocorticoids in a thymoma cell line; Jeon SH et al.; We isolated a new mouse gene that is highly expressed in thymocytes, testis, and brain . This gene, SRG3, showed a significant sequence homology to SWI3, a yeast transcriptional activator, and its human homolog BAF155 . SRG3 encodes 1,100 amino acids and has 33-47% identity with SWI3 protein over three regions . The SRG3 protein contains an acidic NH2 terminus, a myb-like DNA binding domain, a leucine-zipper motif, and a proline- and glutamine-rich region at its COOH terminus . Rabbit antiserum raised against a COOH-terminal polypeptide of the SRG3 recognized a protein with an apparent molecular mass of 155 kD . The serum also detected a 170-kD protein that seems to be a mouse homologue of human BAF170 . Immunoprecipitation of cell extract with the antiserum against the mouse SRG3 also brought down a 195-kD protein that could be recognized by an antiserum raised against human SWI2 protein . The results suggest that the SRG3 protein associates with a mouse SWI2 . The SRG3 protein is expressed about three times higher in thymocytes than in peripheral lymphocytes . The expression of anti-sense RNA to SRG3 mRNA in a thymoma cell line, S49.1, reduced the expression level of the SRG3 protein, and decreased the apoptotic cell death induced by glucocorticoids . These results suggest that the SRG3 protein is involved in the glucocorticoid-induced apoptosis in the thymoma cell line . This implicates that the SRG3 may play an important regulatory role during T cell development in thymus. J Biol Chem, 1997 May 16, 272(20), 13180 - 8 Overexpression of the G1-cyclin gene CLN2 represses the mating pathway in Saccharomyces cerevisiae at the level of the MEKK Ste11; Wassmann K et al.; Basal and induced transcription of pheromone-dependent genes is regulated in a cell cycle-dependent way . FUS1, a gene strongly induced after pheromone treatment, shows high mRNA levels in mitosis and early G1 phase of the cell cycle, a decrease in G1 after START and again an increase in S phase . Overexpression of CLN2 was shown to repress the transcript number of pheromone-dependent genes (1) . We asked whether the activities of components of the mating pathway fluctuate during the cell cycle . We were also interested in determining at what level Cln2 represses the signal transduction machinery . Here we show that the activity of the mitogen-activated protein kinase Fus3 indeed fluctuates during the cell cycle, reflecting the oscillations of the gene transcripts . CLN2 overexpression represses Fus3 kinase activity, independently of the phosphatase Msg5 . Additionally, we show that the activity of the MEK Ste7 also fluctuates during the cell cycle . Increased Cln2 levels repress the ability of hyperactive STE11 alleles to induce the pathway . G protein-independent activation of Ste11 caused by an rga1 pbs2 mutation is resistant to high levels of Cln2 kinase . Therefore our results suggest that Cln2-dependent repression of the mating pathway occurs at the level of Ste11. Science, 1997 May 16, 276(5315), 1116 - 9 Inhibition of pathogenicity of the rice blast fungus by Saccharomyces cerevisiae alpha-factor; Beckerman JL et al.; Magnaporthe grisea is a fungal pathogen with two mating types, MAT1-1 and MAT1-2, that forms a specialized cell necessary for pathogenesis, the appressorium . Saccharomyces cerevisiae alpha-factor pheromone blocked appressorium formation in a mating type-specific manner and protected plants from infection by MAT1-2 strains . Experiments with alpha-factor analogs suggest that the observed activity is due to a specific interaction of alpha-factor with an M . grisea receptor . Culture filtrates of a MAT1-1 strain contained an activity that inhibited appressorium formation of mating type MAT1-2 strains . These findings provide evidence that a pheromone response pathway exists in M . grisea that can be exploited for plant protection. FEBS Lett, 1997 May 12, 408(1), 109 - 14 Serine-threonine protein kinase activity of Elm1p, a regulator of morphologic differentiation in Saccharomyces cerevisiae; Koehler CM et al.; The Saccharomyces cerevisiae gene ELM1 regulates morphologic differentiation and its nucleotide sequence predicts a novel protein kinase . Elm1p was expressed in yeast and insect cells and purified . Elm1p displayed protein kinase activity in autophosphorylation assays and towards exogenous substrates . Serine and threonine residues were identified as the acceptors in these reactions . These data together with previous genetic analysis of ELM1 function indicate that phosphorylation on serine and/or threonine residues of a particular substrate or set of substrates by Elm1p is required for repression of the filamentous growth differentiation state. J Mol Biol, 1997 May 2, 268(2), 322 - 30 Transfer RNA gene redundancy and translational selection in Saccharomyces cerevisiae; Percudani R et al.; A total of 274 transfer RNA genes, representing the entire tRNA gene set of the yeast Saccharomyces cerevisiae, has been extracted from the whole genome sequence of this organism using a dedicated search algorithm (Pol3scan) . All tRNA genes were assigned to 42 classes of distinct codon specificity . Accordingly, four deviations from previously proposed rules for third position wobble pairing in yeast, three G:U and one A:I codon-anticodon pairings, were found to be required to account for the reading of 61 coding triplets . The gene copy number for individual tRNA species, which ranges from one to 16, correlates well with both the frequency of codon occurrence in a sample of 1756 distinct protein coding sequences (r = 0.82) and the previously measured intracellular content of 21 tRNA species . A close link between tRNA gene redundancy and the overall amino acid composition of yeast proteins was also observed . Regression analysis values for individual protein coding sequences proved to be effective descriptions of the translational selective pressure operating on a particular gene . A significantly stronger co-adaptation between codon choice and tRNA gene copy number was observed in highly expressed genes . These observations strongly support the notion that intracellular tRNA levels in normally growing yeast cells are mainly determined by gene copy number, which, along with codon choice, is the key parameter acted upon by translational selection. Genetika, 1997 May, 33(5), 591 - 8 {Genetic control of metabolism of a mutagenic analog of 6-N-hydroxylaminopurine bases in Saccharomyces cerevisiae yeasts}; Koz'min SG et al.; Yeasts were shown to utilize 6-substituted adenine analogues as a purine source via the reutilization pathway leading to the formation of inosine monophosphate (IMP) . This occurs because the ade12 strains with blocked conversion of IMP into adenosine monophosphate (AMP) cannot grow on media containing the above analogues as a sole purine source . Haploid strains with the double mutation ham1 ade2 or ham1 ade5 were also incapable of growing on a medium with 6-N-hydroxylaminopurine (HAP) as a sole purine source . However, in this case, this inability was caused by the occurrence of recessive lethal mutations rather than by a defect in purine reutilization . Yeast adenine aminohydrolase (AAH) can deaminate HAP to hypoxanthine . Adenine aminohydrolase (AAH) was uniformly active both in strains with a mutation in the HAMI gene and in strains wild-type with respect to this trait. Z Naturforsch {C}, 1997 May-Jun, 52(5-6), 373 - 9 A particulate guanylate cyclase (EC 4.6.1.2) from growing yeast cells (Saccharomyces cerevisiae); Eckstein H et al.; The detection of cGMP in yeast (Eckstein 1988), but lacking hints at guanylate cyclase from sequencing of the yeast genome, raised questions about existence, isoform, and regulation of guanylate cyclase from this organism . We found a particulate guanylate cyclase activity in yeast extracts, exhibiting properties of an integral membrane protein . Characteristics are: pH-optimum at pH 6.8, temperature-optimum around 60 degrees C, only slight stimulation by Mn2+ . Sigmoidal enzyme kinetics indicate allosteric regulation, ATP and Ca2+ act as negative allosteric effectors . The enzyme activity is increased by yeast alpha-1 mating factor, and by sodium nitrite, thus showing properties of particulate as well as of soluble isoforms from other eukaryotes . The activation by alpha-1 mating factor suggests receptor functions, and a role in ascospore conjugation. Mol Gen Genet, 1997 May, 254(6), 681 - 8 A simple signal element mediates transcription termination and mRNA 3' end formation in the DEG1 gene of Saccharomyces cerevisiae; Brambilla A et al.; DEG1 is a weakly transcribed gene of Saccharomyces cerevisiae, closely associated with CEN6 . We mapped its major poly(A) site only 24 nucleotides (nt) downstream of the stop codon, and only 26 nt upstream of the CDEI centromere element . The deletion of this 50 nt stretch completely abolishes formation of the mRNA 3' end . A shorter deletion of a 16 nt sequence in the 3'-untranslated region has the same effect on transcription termination and 3'-maturation function . A TATATA sequence within this 16 nt region is essential for both functions, while a TGTATA sequence has a weak compensating activity in 3' end maturation if the TATATA stretch is deleted . We assume that the 3' end formation signals of the DEG1 gene have this simple structure: a single essential element (TATATA, whether alone or with the few surrounding nucleotides), probably, but not necessarily, cooperating with the sequence at the poly(A) site . This simple structure differs from the emerging model for 3' end-processing signals in that (i) it is shorter: 24 nt long at the most, while the model suggests 39 nt; (ii) there is no element located downstream of the TATATA signal to position the poly(A) site; and (iii) unlike the other naturally occurring signals studied, no cooperation among multiple TATATA-like elements is observed . We found that the same TATATA sequence also directs transcription termination, irrespective of promoter strength, and presumably without the cooperation of a downstream polymerase II pausing site . Taken together, these findings support the hypothesis that the DEG1 3' end-forming signals are more condensed than in other yeast genes, probably because of their proximity to CEN6. Mol Microbiol, 1997 May, 24(4), 711 - 21 Normal processing of AP sites in Apn1-deficient Saccharomyces cerevisiae is restored by Escherichia coli genes expressing either exonuclease III or endonuclease III; Masson JY et al.; Escherichia coli exonuclease III and endonuclease III are two distinct DNA-repair enzymes that can cleave apurinic/apyrimidinic (AP) sites by different mechanisms . While the AP endonuclease activity of exonuclease III generates a 3'-hydroxyl group at AP sites, the AP lyase activity of endonuclease III produces a 3'-alpha,beta unsaturated aldehyde that prevents DNA-repair synthesis . Saccharomyces cerevisiae Apn1 is the major AP endonuclease/3'-diesterase that also produces a 3'-hydroxyl group at the AP site, but it is unrelated to either exonuclease III or endonuclease III . apn1 deletion mutants are unable to repair AP sites generated by the alkylating agent methyl methane sulphonate and display a spontaneous mutator phenotype . This work shows that either exonuclease III or endonuclease III can functionally replace yeast Apn1 in the repair of AP sites . Two conclusions can be derived from these findings . The first of these conclusions is that yeast cells can complete the repair of AP sites even though they are cleaved by AP lyase . This implies that AP lyase can contribute significantly to the repair of AP sites and that yeast cells have the ability to process the alpha,beta unsaturated aldehyde produced by endonuclease III . The second of these conclusions is that unrepaired AP sites are strictly the cause of the high spontaneous mutation rate in the apn1 deletion mutant. Mol Microbiol, 1997 May, 24(4), 687 - 95 Structural analysis of the subunits of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock; Reinders A et al.; Synthesis of trehalose in the yeast Saccharomyces cerevisiae is catalysed by the trehalose-6-phosphate (Tre6P) synthase/phosphatase complex, which is composed of at least three different subunits encoded by the genes TPS1, TPS2, and TSL1 . Previous studies indicated that Tps1 and Tps2 carry the catalytic activities of trehalose synthesis, namely Tre6P synthase (Tps1) and Tre6P phosphatase (Tps2), while TsI1 was suggested to have regulatory functions . In this study two different approaches have been used to clarify the molecular composition of the trehalose synthase complex as well as the functional role of its potential subunits . Two-hybrid analyses of the in vivo interactions of Tps1, Tps2, TsI1, and Tps3, a protein with high homology to TsI1, revealed that both TsI1 and Tps3 can interact with Tps1 and Tps2; the latter two proteins also interact with each other . In addition, trehalose metabolism upon heat shock was analysed in a set of 16 isogenic yeast strains carrying deletions of TPS1, TPS2, TSL1, and TPS3 in all possible combinations . These results not only confirm the previously suggested roles for Tps1 and Tps2, but also provide, for the first time, evidence that TsI1 and Tps3 may share a common function with respect to regulation and/or structural stabilization of the Tre6P synthase/phosphatase complex in exponentially growing, heat-shocked cells. J Lipid Res, 1997 May, 38(5), 962 - 8 Polyprenol formation in the yeast Saccharomyces cerevisiae: effect of farnesyl diphosphate synthase overexpression; Szkopinska A et al.; Biosynthesis of polyprenols was followed in the erg mutants of Saccharomyces cerevisiae impaired in various steps of the mevalonate pathway . The end products of the enzymatic reaction carried out in vitro, in the wild type yeast and all mutants tested, were identified as dehydrodolichols (alpha-unsaturated polyprenols) whereas in vivo, yeast synthesize dolichols (alpha-saturated polyprenols) (Biochimie, 1996.78:111-112.) The strain defective in the farnesyl diphosphate (FPP) synthase, (coded by the erg20-2 gene) required the presence of exogenous FPP for synthesis of dehydrodolichols to occur in vitro . Overexpression of the ERG20 gene restored synthesis of polyprenols in vitro indicating that FPP is the allylic "starter" for cis-prenyltransferase in yeast . Overexpression of the ERG20 gene in the erg 9 mutant, defective in squalene synthase activity, not only restored synthesis of dehydrodolichols in vitro, but also increased the synthesis of dolichols in vivo, almost 10-fold in comparison with wild type yeast . On the other hand overexpression of the mutated FPP synthase, coded by the gene erg20-2 in the same genetic background, resulted in a 100-fold increase of the amount of dehydrodolichols . Interestingly, in addition to the family of typical for yeast C60-C80 compounds, dehydrodolichols of chain length up to C135 were synthesized both in vitro and in vivo. Eur J Biochem, 1997 May 1, 245(3), 589 - 99 Addition of acetaldehyde to the N-terminus of a recombinant Schistosoma mansoni glutathione S-transferase upon high-level expression in Saccharomyces cerevisiae; Roecklin D et al.; Intracellular expression of recombinant Schistosoma mansoni protein p28 (Smp28) in soluble form to a concentration of more than 6 g/l culture in Saccharomyces cerevisiae was accompanied by a post-translational modification, which occurred during the late stage of the culture . The modified protein, which had a reduced isoelectric point, was isolated by anion-exchange HPLC and characterized by tryptic mapping by means of on-line reversed-phase HPLC/electrospray mass spectrometry . Comparison with non-modified recombinant Smp28 allowed us to localize the modification to the N-terminal hexapeptide AGEHIK, which had an increased mass of 26 Da . Reversed-phase HPLC of the modified peptide with a shallow acetonitrile gradient revealed the presence of two components of identical mass and amino acid composition . Both peptides were inaccessible to N-terminal Edman sequencing, indicating that a rearrangement of the N-terminal region of recombinant Smp28 had taken place during tryptic digestion leading to two isomeric, N-terminally blocked peptides . Deuterium-exchange mass spectrometry showed that the modified peptides lacked two exchangeable protons, suggesting cyclic modifications implying the N-terminal amino group . Tandem mass spectrometry by means of the nano-electrospray technique and collision-induced dissociation allowed us to identify the modified sites as Ala1, His4 and Lys6 based on a characteristic modified a1 ion of Ala1 (70.0 Da), a modified immonium ion of His4 (136.0 Da) and a modified y1" ion (173.2 Da) of Lys6 . Combination of all the above results led to the conclusion that recombinant Smp28 was initially modified at its N-terminus by addition of acetaldehyde to form an aldimine which rearranged during tryptic digestion to two different cyclic peptides. Eur J Biochem, 1997 May 1, 245(3), 557 - 63 A conditional-lethal translation termination defect in a sup45 mutant of the yeast Saccharomyces cerevisiae; Stansfield I et al.; Genetic studies have indicated that the product of the yeast SUP45 gene encodes a component of the translational-termination machinery . In higher eukaryotes, genes similar to SUP45 encode eukaryote release factor 1 (eRF1), which has a stop-codon-dependent peptidyl-release activity . Using a conditional-lethal mutant allele of SUP45 (sup45-2) and a combination of in vivo and in vitro approaches, we demonstrate that the product of the SUP45 gene (Sup45p or eRF1) is a factor required for translation termination in yeast . A homologous in vitro assay based on suppressor-tRNA-mediated readthrough of stop codons is used to show that a translating lysate from a sup45-2 mutant strain exhibits a termination defect when heated for short periods to greater than the non-permissive temperature (37 degrees C) . This defect can be complemented with a purified preparation of Sup45p (eRF1) expressed in Eschericha coli . The termination defect in this strain appears to be due to an inability of the Sup45p protein to bind the ribosome, resulting in vivo in a reduced ability of Sup45p to release nascent polypeptides from the ribosome at the non-permissive temperature . Cell-free translation lysates from the sup45-2 strain do not show a defect in sense-codon translation at the non-permissive temperature . These data demonstrate that yeast eRF1 plays a role in translation termination and is functionally equivalent to its higher eukaryotic homologues. Mol Microbiol, 1997 May, 24(3), 607 - 16 A C-terminal di-leucine motif and nearby sequences are required for NH4(+)-induced inactivation and degradation of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae; Hein C et al.; The general amino acid permease, Gap1, of Saccharomyces cerevisiae is very active in cells grown on proline as the sole nitrogen source . Adding NH4+ to the medium triggers inactivation and degradation of the permease via a regulatory process involving Npi1p/Rsp5p, a ubiquitin-protein ligase . In this study, we describe several mutations affecting the C-terminal region of Gap1p that render the permease resistant to NH4(+)-induced inactivation . An in vivo isolated mutation (gap1pgr) causes a single Glu-->Lys substitution in an amino acid context similar to the DXKSS sequence involved in ubiquitination and endocytosis of the yeast alpha-factor receptor, Ste2p . Another replacement, substitution of two alanines for a di-leucine motif, likewise protects the Gap1 permease against NH4(+)-induced inactivation . In mammalian cells, such a motif is involved in the internalization of several cell-surface proteins . These data provide the first indication that a di-leucine motif influences the function of a plasma membrane protein in yeast . Mutagenesis of a putative phosphorylation site upstream from the di-leucine motif altered neither the activity nor the regulation of the permease . In contrast, deletion of the last eleven amino acids of Gap1p, a region conserved in other amino acid permeases, conferred resistance to NH4+ inactivation . Although the C-terminal region of Gap1p plays an important role in nitrogen control of activity, it was not sufficient to confer this regulation to two NH4(+)-insensitive permeases, namely the arginine (Can1p) and uracil (Fur4p) permeases. Yeast, 1997 May, 13(6), 573 - 81 Phylogenetic classification of the mitochondrial carrier family of Saccharomyces cerevisiae; el Moualij B et al.; The screening of the open reading frames identified in the whole yeast genome has allowed us to discover 34 proteins belonging to the mitochondrial carrier family . By phylogenetic study, they can be divided into 27 subfamilies including ADP/ATP, phosphate and citrate carriers, putative oxoglutarate and GDC carriers and 22 new subfamilies . Topology predictions using the 'positive inside rule' approach have shown that the yeast carriers are similarly oriented with both extremities exposed to the cytosol . In each subfamily, a strict conservation of the charged residues in the six transmembrane alpha-helices is observed, suggesting a functional role for these residues and the existence of 27 functionally distinct carriers. Yeast, 1997 May, 13(6), 515 - 28 Mechanisms of salt tolerance conferred by overexpression of the HAL1 gene in Saccharomyces cerevisiae; Rios G et al.; Overexpression of the HAL1 gene improves the tolerance of Saccharomyces cerevisiae to NaCl by increasing intracellular K+ and decreasing intracellular Na+ . The effect of HAL1 on intracellular Na+ was mediated by the PMR2/ENA1 gene, corresponding to a major Na+ efflux system . The expression level of ENA1 was dependent on the gene dosage of HAL1 and overexpression of HAL1 suppressed the salt sensitivity of null mutants in calcineurin and Hal3p, other known regulators of ENA1 expression . The effect of HAL1 on intracellular K+ was independent of the TRK1 and TOK1 genes, corresponding to a major K+ uptake system and to a K+ efflux system activated by depolarization, respectively . Overexpression of HAL1 reduces K+ loss from the cells upon salt stress, a phenomenon mediated by an unidentified K+ efflux system . Overexpression of HAL1 did not increase NaCl tolerance in galactose medium . NaCl poses two types of stress, osmotic and ionic, counteracted by glycerol synthesis and sodium extrusion, respectively . As compared to glucose, with galactose as carbon source glycerol synthesis is reduced and the expression of ENA1 is increased . As a consequence, osmotic adjustment through glycerolsynthesis, a process not affected by HAL1, is the limiting factor for growth on galactose under NaCl stressed. Microbiology, 1997 May, 143 ( Pt 5), 1665 - 71 Near-UV-induced absorbance change and photochemical decomposition of ergosterol in the plasma membrane of the yeast Saccharomyces cerevisiae; Arami S et al.; When cells of the yeast Saccharomyces cerevisiae were exposed to near-UV (300-400 nm), their absorption spectra changed slightly within the range 220-300 nm with increasing dosage . Difference spectra, calculated by substracting the curve recorded in cells exposed to near-UV from the curve of unexposed cells, decreased with increasing dosage over a broad band with peaks at 272, 282 and 295 nm and a shoulder at 265 nm . These peaks were in agreement with the absorption maxima of ergosterol, which is one of the major components of the plasma membrane of yeast . Near-UV radiation induced a simultaneous decrease in absorption spectra and reduction of ergosterol content in the plasma membrane . Photochemical decomposition of ergosterol by near-UV radiation was revealed in vivo, although ergosterol is generally known to be photoconverted to previtamin D2 industrially by UV radiation in vitro . In order to remove photosensitizers, liposomes were prepared from phospholipids and glycolipids, with or without ergosterol from purified yeast plasma membranes . Liposomal ergosterol in the orientated state was photochemically decomposed by near-UV radiation but ergosterol in the disorientated state in a homogeneous solution was not . Near-UV radiation also induced a decrease in activity of membrane-bound ATPase . Dose-response curves for the reduction of ATPase activity were similar to that for decomposition of ergosterol, suggesting that near-UV caused membrane function damage. Microbiology, 1997 May, 143 ( Pt 5), 1657 - 64 Cloning of a protopectinase gene of Trichosporon penicillatum and its expression in Saccharomyces cerevisiae; Iguchi K et al.; A protopectinase (PPase)-encoding gene, PSE3, from Trichosporon penicillatum was cloned by colony hybridization using two oligonucleotide probes synthesized from the N-terminal amino acid sequences of native PPase SE1 and one peptide from a lysyl endopeptidase digest . Nucleotide sequencing revealed that PSE3 contains an ORF encoding a 367 amino acid protein . Mature PPase SE3 is composed of 340 amino acids and the N-terminus of the ORF appeared to correspond to a signal peptide and a propeptide processed by a KEX2-like proteinase . The deduced amino acid sequence of PSE3 was 65.4, 56.7, 58.1, 61.8 and 48.9% homologous to the polygalacturonases of Aspergillus oryzae, Aspergillus niger, Aspergillus tubigensis, Cochliobolus carbonum and Fusarium moniliforme, respectively . One domain, which might interact with polygalacturonic acid, is highly conserved not only in fungal polygalacturonases but also in bacterial and plant polygalacturonases . PSE3 was expressed in Saccharomyces cerevisiae, but three forms (the mature form, a glycosylated form and an uncharacterized processed form) of PPase SE3 were present among the PSE3 products. Microbiology, 1997 May, 143 ( Pt 5), 1649 - 56 Mitochondrial superoxide dismutase is essential for ethanol tolerance of Saccharomyces cerevisiae in the post-diauxic phase; Costa V et al.; This work reports the role of both superoxide dismutases-CuZnSOD (encoded by SOD1) and MnSOD (encoded by SOD2)-in the build-up of tolerance to ethanol during growth of Saccharomyces cerevisiae from exponential to post-diauxic phase . Both enzyme activities increase from the exponential phase to the diauxic shift and from the diauxic shift to the post-diauxic phase . The levels of mRNA-SOD1 and mRNA-SOD2 increase from the exponential phase to the diauxic shift; however, during the post-diauxic phase mRNA-SOD1 levels decrease while mRNA-SOD2 levels remain unchanged . These data indicate the existence of two regulatory mechanisms involved in the induction of SOD activity during growth: synthesis de novo of the proteins (until the diauxic shift), and post-transcriptional or post-translational regulation (during the post-diauxic phase) . Ethanol does not alter the activities of either enzyme in cells from the diauxic shift or post-diauxic-phases, although the respective mRNA levels decrease in post-diauxic-phase cells treated with ethanol (14% or 20%) . Results of experiments with sod1 and sod2 mutants show that MnSOD, but not CuZnSOD, is essential for ethanol tolerance of diauxic-shift and post-diauxic-phase cells . Evidence that ethanol toxicity is correlated with the production of reactive oxygen species in the mitochondria is obtained from results with respiration-deficient mutants . In these cells, the induction of superoxide dismutase activity by ethanol is low; also, the respiratory deficiency restores the capacity of sod2 cells to acquire ethanol tolerance. FEMS Microbiol Lett, 1997 May 1, 150(1), 55 - 60 Identification of Saccharomyces cerevisiae GLY1 as a threonine aldolase: a key enzyme in glycine biosynthesis; Monschau N et al.; Determination of enzyme-specific activities revealed that GLY1 encodes a threonine aldolase (TA) in Saccharomyces cerevisiae . A knock-out mutant auxotrophic for glycine lacked detectable activity . After transformation with YEp24GLY1 glycine prototrophy was restored and TA-specific activity was 16-fold higher than in the wild type . Growth experiments using glucose as the sole carbon source showed that GLY1 is more important for glycine biosynthesis than SHM1 and SHM2 encoding alternative serine hydroxymethyltransferases . On ethanol as carbon source simultaneous disruption of GLY1, SHM1 and SHM2 did not lead to glycine auxotrophy because glycine biosynthesis proceeds via alanine glyoxylate aminotransferase. FEMS Microbiol Lett, 1997 May 1, 150(1), 43 - 7 Inhibition of ergosterol biosynthesis is not accompanied by a change in fatty acid composition in Saccharomyces cerevisiae treated with the antifungal agent 6-amino-2-n-pentylthiobenzothiazole; Kuchta T et al.; The antifungal agent 6-amino-2-n-pentylthiobenzothiazole at a concentration of 40 microM lowered the specific growth rate of exponentially growing cultures of Saccharomyces cerevisiae by 36% . Treatment with 6-amino-2-n-pentylthiobenzothiazole inhibited the biosynthesis of ergosterol and caused an accumulation of the methylated sterol precursors ergosta-5,7-dienol and squalene, but had no significant effect on the composition and the rate of biosynthesis of fatty acids . The results indicate that neither the inhibition of ergosterol biosynthesis, nor the slowing-down of culture growth by this antifungal agent, led to a compensatory alteration in the pattern of fatty-acyl chains in membrane lipids . This finding contradicts the accepted wisdom for the action of a number of antifungal agents. Curr Genet, 1997 May, 31(5), 408 - 13 Starvation for a specific amino acid induces high frequencies of rho- mutants in Saccharomyces cerevisiae; Heidenreich E et al.; Auxotrophic yeast cells were starved on solid media for their respective essential amino acid in the course of "adaptive mutation" experiments . Thereby, high proportions of mitochondrially respiratory deficient (rho-) mutants accumulated among the cells stressed on selective plates . Using a strain with a plus-four frameshift mutation in a chromosomal gene involved in lysine biosynthesis, we observed that many of the revertant colonies which arose late under the selective pressure were composed of mixtures of rho+ and rho- cells, indicating that they originated from founder cells containing intact as well as defective mitochondrial genomes . We show that in spite of the slower growth of rho- cells the late-appearing colonies cannot be interpreted as descending from rho- revertants present before selective plating. Curr Genet, 1997 May, 31(5), 375 - 9 Translational activator proteins required for cytochrome b synthesis in Saccharomyces cerevisiae; Rodel G; In the yeast Saccharomyces cerevisiae translation of apocytochrome b, the only mitochondrially encoded subunit of ubiquinol-cytochrome c oxidoreductase, requires the products of at least three nuclear genes, CBP6, CBS1 and CBS2 . In this article I review available data on CBS1p and CBS2p from the initial detection of the genes up to the current investigations on interacting components and the proteins' topology. RNA, 1997 May, 3(5), 476 - 88 Variable regions V13 and V3 of Saccharomyces cerevisiae contain structural features essential for normal biogenesis and stability of 5.8S and 25S rRNA; Jeeninga RE et al.; The homologous ribosomal RNA species of all organisms can be folded into a common "core" secondary structure . In addition, eukaryotic rRNAs contain a large number of segments, located at fixed positions, that are highly variable in size and sequence from one organism to another . We have investigated the role of the two largest of these variable regions in Saccharomyces cerevisiae 25S rRNA, V13, and V3, by mutational analysis in a yeast strain that can be rendered completely dependent on the synthesis of mutant (pre-)rRNA . We found that approximately half of variable region V13 can be deleted without any phenotypic effect . The remaining portion, however, contains multiple structural features whose disturbance causes serious growth defects or lethality . Accumulation of 25S rRNA is strongly reduced by these mutations, at least in part because they inhibit processing of ITS2 . Removal of even a relatively small portion of V3 also strongly reduces the cellular growth rate and larger deletions are lethal . Interestingly, some of the deletions in V3 cause accumulation of 27S(A) pre-rRNA and, moreover, appear to interfere with the close coupling between the processing cleavages at sites A3 and B1(S) . These results demonstrate that both variable regions play an important role in 60S subunit formation. Appl Environ Microbiol, 1997 May, 63(5), 1959 - 64 Influence of cosubstrate concentration on xylose conversion by recombinant, XYL1-expressing Saccharomyces cerevisiae: a comparison of different sugars and ethanol as cosubstrates; Meinander NQ et al.; Conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae expressing the XYL1 gene, encoding xylose reductase, was investigated by using different cosubstrates as generators of reduced cofactors . The effect of a pulse addition of the cosubstrate on xylose conversion in cosubstrate-limited fed-batch cultivation was studied . Glucose, mannose, and fructose, which are transported with high affinity by the same transport system as is xylose, inhibited xylose conversion by 99, 77, and 78%, respectively, reflecting competitive inhibition of xylose transport . Pulse addition of maltose, which is transported by a specific transport system, did not inhibit xylose conversion . Pulse addition of galactose, which is also transported by a specific transporter, inhibited xylose conversion by 51%, in accordance with noncompetitive inhibition between the galactose and glucose/ xylose transport systems . Pulse addition of ethanol inhibited xylose conversion by 15%, explained by inhibition of xylose transport through interference with the hydrophobic regions of the cell membrane . The xylitol yields on the different cosubstrates varied widely . Galactose gave the highest xylitol yield, 5.6 times higher than that for glucose . The difference in redox metabolism of glucose and galactose was suggested to enhance the availability of reduced cofactors for xylose reduction with galactose . The differences in xylitol yield observed between some of the other sugars may also reflect differences in redox metabolism . With all cosubstrates, the xylitol yield was higher under cosubstrate limitation than with cosubstrate excess. J Bacteriol, 1997 May, 179(9), 2987 - 93 Characterization of a glucose-repressed pyruvate kinase (Pyk2p) in Saccharomyces cerevisiae that is catalytically insensitive to fructose-1,6-bisphosphate; Boles E et al.; We have characterized the gene YOR347c of Saccharomyces cerevisiae and shown that it encodes a second functional pyruvate kinase isoenzyme, Pyk2p . Overexpression of the YOR347c/PYK2 gene on a multicopy vector restored growth on glucose of a yeast pyruvate kinase 1 (pyk1) mutant strain and could completely substitute for the PYK1-encoded enzymatic activity . PYK2 gene expression is subject to glucose repression . A pyk2 deletion mutant had no obvious growth phenotypes under various conditions, but the growth defects of a pyk1 pyk2 double-deletion strain were even more pronounced than those of a pyk1 single-mutation strain . Pyk2p is active without fructose-1,6-bisphosphate . However, overexpression of PYK2 during growth on ethanol did not cause any of the deleterious effects expected from a futile cycling between pyruvate and phosphoenolpyruvate . The results indicate that the PYK2-encoded pyruvate kinase may be used under conditions of very low glycolytic flux. Genetics, 1997 May, 146(1), 39 - 55 Genetic analysis of default mating behavior in Saccharomyces cerevisiae; Dorer R et al.; Haploid Saccharomyces cerevisiae cells find each other during conjugation by orienting their growth toward each other along pheromone gradients (chemotropism) . However, when their receptors are saturated for pheromone binding, yeast cells must select a mate by executing a default pathway in which they choose a mating partner at random . We previously demonstrated that this default pathway requires the SPA2 gene . In this report we show that the default mating pathway also requires the AXL1, FUS1, FUS2, FUS3, PEA2, RVS161, and BNI1 genes . These genes, including SPA2, are also important for efficient cell fusion during chemotropic mating . Cells containing null mutations in these genes display defects in cell fusion that subtly affect mating efficiency . In addition, we found that the defect in default mating caused by mutations in SPA2 is partially suppressed by multiple copies of two genes, FUS2 and MFA2 . These findings uncover a molecular relationship between default mating and cell fusion . Moreover, because axl1 mutants secrete reduced levels of a-factor and are defective at both cell fusion and default mating, these results reveal an important role for a-factor in cell fusion and default mating . We suggest that default mating places a more stringent requirement on some aspects of cell fusion than does chemotropic mating. Mol Cell Biol, 1997 May, 17(5), 2816 - 24 Pet127p, a membrane-associated protein involved in stability and processing of Saccharomyces cerevisiae mitochondrial RNAs; Wiesenberger G et al.; Nuclear mutations that inactivate the Saccharomyces cerevisiae gene PET127 dramatically increased the levels of mutant COX3 and COX2 mitochondrial mRNAs that were destabilized by mutations in their 5' untranslated leaders . The stabilizing effect of pet127 delta mutations occurred both in the presence and in the absence of translation . In addition, pet127 delta mutations had pleiotropic effects on the stability and 5' end processing of some wild-type mRNAs and the 15S rRNA but produced only a leaky nonrespiratory phenotype at 37 degrees C . Overexpression of PET127 completely blocked respiratory growth and caused cells to lose wild-type mitochondrial DNA, suggesting that too much Pet127p prevents mitochondrial gene expression . Epitope-tagged Pet127p was specifically located in mitochondria and associated with membranes . These findings suggest that Pet127p plays a role in RNA surveillance and/or RNA processing and that these functions may be membrane bound in yeast mitochondria. Mol Cell Biol, 1997 May, 17(5), 2615 - 23 Characterization of a serum response factor-like protein in Saccharomyces cerevisiae, Rlm1, which has transcriptional activity regulated by the Mpk1 (Slt2) mitogen-activated protein kinase pathway; Watanabe Y et al.; The Mpk1 (Slt2) mitogen-activated protein (MAP) kinase has been implicated in several biological processes in Saccharomyces cerevisiae . The Rlm1 protein, a member of the MADS box family of transcription factors, functions downstream of Mpk1 in the pathway . To characterize the role of Rlm1 in mediating the transcriptional activation by the Mpk1 pathway, we constructed a LexA-Rlm1 deltaN chimera in which sequences, including the MADS box domain of the Rlm1 protein, were replaced by the LexA DNA binding domain and tested the ability of this chimera to activate a LexA operator-controlled reporter gene . In this assay, the Rlm1 protein was found to activate transcription in a manner regulated by the Mpk1 pathway . The Mpk1 protein kinase phosphorylated Rlm1 deltaN in vitro and the LexA-Rlm1 deltaN chimera protein was phosphorylated in vivo in a Mpk1-dependent manner . These results suggest that Mpk1 regulates the transcriptional activity of Rlm1 by directly phosphorylating it . We identified a Mpk1-like protein kinase, Mlp1, as an Rlm1-associated protein by using the yeast two-hybrid system . Overexpression of MLP1 suppresses the caffeine-sensitive phenotype of the bck1 delta mutation . The additivity of the mlp1 delta defect with the Mpk1 delta defect with regard to the caffeine sensitivity, combined with the results of genetic epistasis experiments, suggested that the activity of Rlm1 is regulated independently by Mpk1 MAP kinase and the Mlp1 MAP kinase-like kinase. Mol Cell Biol, 1997 May, 17(5), 2566 - 75 Novel Gal3 proteins showing altered Gal80p binding cause constitutive transcription of Gal4p-activated genes in Saccharomyces cerevisiae; Blank TE et al.; Gal4p-mediated activation of galactose gene expression in Saccharomyces cerevisiae normally requires both galactose and the activity of Gal3p . Recent evidence suggests that in cells exposed to galactose, Gal3p binds to and inhibits Ga180p, an inhibitor of the transcriptional activator Gal4p . Here, we report on the isolation and characterization of novel mutant forms of Gal3p that can induce Gal4p activity independently of galactose . Five mutant GAL3(c) alleles were isolated by using a selection demanding constitutive expression of a GAL1 promoter-driven HIS3 gene . This constitutive effect is not due to overproduction of Gal3p . The level of constitutive GAL gene expression in cells bearing different GAL3(c) alleles varies over more than a fourfold range and increases in response to galactose . Utilizing glutathione S-transferase-Gal3p fusions, we determined that the mutant Gal3p proteins show altered Gal80p-binding characteristics . The Gal3p mutant proteins differ in their requirements for galactose and ATP for their Gal80p-binding ability . The behavior of the novel Gal3p proteins provides strong support for a model wherein galactose causes an alteration in Gal3p that increases either its ability to bind to Gal80p or its access to Gal80p . With the Gal3p-Gal80p interaction being a critical step in the induction process, the Gal3p proteins constitute an important new reagent for studying the induction mechanism through both in vivo and in vitro methods. Mol Cell Biol, 1997 May, 17(5), 2502 - 10 Glucose derepression of gluconeogenic enzymes in Saccharomyces cerevisiae correlates with phosphorylation of the gene activator Cat8p; Randez-Gil F et al.; The Cat8p zinc cluster protein is essential for growth of Saccharomyces cerevisiae with nonfermentable carbon sources . Expression of the CAT8 gene is subject to glucose repression mainly caused by Mig1p . Unexpectedly, the deletion of the Mig1p-binding motif within the CAT8 promoter did not increase CAT8 transcription; moreover, it resulted in a loss of CAT8 promoter activation . Insertion experiments with a promoter test plasmid confirmed that this regulatory 20-bp element influences glucose repression and derepression as well . This finding suggests an upstream activating function of this promoter region, which is Mig1p independent, as delta mig1 mutants are still able to derepress the CAT8 promoter . No other putative binding sites such as a Hap2/3/4/5p site and an Abf1p consensus site were functional with respect to glucose-regulated CAT8 expression . Fusions of Cat8p with the Gal4p DNA-binding domain mediated transcriptional activation . This activation capacity was still carbon source regulated and depended on the Cat1p (Snf1p) protein kinase, which indicated that Cat8p needs posttranslational modification to reveal its gene-activating function . Indeed, Western blot analysis on sodium dodecyl sulfate-gels revealed a single band (Cat8pI) with crude extracts from glucose-grown cells, whereas three bands (Cat8pI, -II, and -III) were identified in derepressed cells . Derepression-specific Cat8pII and -III resulted from differential phosphorylation, as shown by phosphatase treatment . Only the most extensively phosphorylated modification (Cat8pIII) depended on the Cat1p (Snf1p) kinase, indicating that another protein kinase is responsible for modification form Cat8pII . The occurrence of Cat8pIII was strongly correlated with the derepression of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) and gluconeogenic PCK1 mRNA . Furthermore, glucose triggered the dephosphorylation of Cat8pIII, but this did not depend on the Glc7p (Cid1p) phosphatase previously described as being involved in invertase repression . These results confirm our current model that glucose derepression of gluconeogenic genes needs Cat8p phosphorylation and additionally show that a still unknown transcriptional activator is also involved. Mol Cell Biol, 1997 May, 17(5), 2485 - 96 CHS5, a gene involved in chitin synthesis and mating in Saccharomyces cerevisiae; Santos B et al.; The CHS5 locus of Saccharomyces cerevisiae is important for wild-type levels of chitin synthase III activity . chs5 cells have reduced levels of this activity . To further understand the role of CHS5 in yeast, the CHS5 gene was cloned by complementation of the Calcofluor resistance phenotype of a chs5 mutant . Transformation of the mutant with a plasmid carrying CHS5 restored Calcofluor sensitivity, wild-type cell wall chitin levels, and chitin synthase III activity levels . DNA sequence analysis reveals that CHS5 encodes a unique polypeptide of 671 amino acids with a molecular mass of 73,642 Da . The predicted sequence shows a heptapeptide repeated 10 times, a carboxy-terminal lysine-rich tail, and some similarity to neurofilament proteins . The effects of deletion of CHS5 indicate that it is not essential for yeast cell growth; however, it is important for mating . Deletion of CHS3, the presumptive structural gene for chitin synthase III activity, results in a modest decrease in mating efficiency, whereas chs5delta cells exhibit a much stronger mating defect . However, chs5 cells produce more chitin than chs3 mutants, indicating that CHS5 plays a role in other processes besides chitin synthesis . Analysis of mating mixtures of chs5 cells reveals that cells agglutinate and make contact but fail to undergo cell fusion . The chs5 mating defect can be partially rescued by FUS1 and/or FUS2, two genes which have been implicated previously in cell fusion, but not by FUS3 . In addition, mating efficiency is much lower in fus1 fus2 x chs5 than in fus1 fus2 x wild type crosses . Our results indicate that Chs5p plays an important role in the cell fusion step of mating. Arch Microbiol, 1997 May, 167(5), 284 - 8 Genetic determination of polygalacturonase production in wild-type and laboratory strains of Saccharomyces cerevisiae; Blanco P et al.; The genetic determination of polygalacturonase (PG) production in Saccharomyces cerevisiae was studied by biochemical and classical genetic techniques . Crosses of PG+ strains with PG- strains showed that in the haploid wild-type-derived strain, two structural genes were involved in the production of a hydrolysis halo on plates with polygalacturonic acid . However, in the case of PG+ laboratory strain IM1-8b, the phenotype was controlled by only one structural gene although the analysis of PG- IM1-8b mutants demonstrated the existence of at least two complementation groups . All these genetic results were assessed biochemically by means of cation-exchange chromatography . Two enzymes were separated in the wild-type strain, and only one in the laboratory strain . The three enzymes had different Km values, molecular masses, and optimal pHs for activity. J Biol Chem, 1997 Apr 25, 272(17), 11344 - 9 Vam2/Vps41p and Vam6/Vps39p are components of a protein complex on the vacuolar membranes and involved in the vacuolar assembly in the yeast Saccharomyces cerevisiae; Nakamura N et al.; The VAM2/VPS41 and VAM6/VPS39 were shown to encode hydrophilic proteins of 113 and 123 kDa, respectively . Deletion of the VAM2 and VAM6 functions resulted in accumulation of numerous vacuole-related structures of 200-400 nm in diameter that were much smaller than the normal vacuoles . Loss of functions of Vam2p and Vam6p resulted in inefficient processings of a set of vacuolar proteins, including proteinase A, proteinase B, and carboxypeptidase Y (CPY), and in severely defective maturation of another vacuolar protein, alkaline phosphatase . A part of newly synthesized CPY was missorted to the cell surface in the mutants . Epitope-tagged versions of Vam2p and Vam6p retained their functions, and they were found mostly in sedimentable fractions . The epitope-tagged Vam2p and Vam6p remained in the sedimentable fractions in the presence of Triton X-100, but they were extracted by urea or NaCl . Vam2p and Vam6p were cross-linked by the treatment of a chemical cross-linker . These observations indicated that Vam2p and Vam6p physically interact with each other and exist as components of a large protein complex . Vam6p fused with a green fluorescent protein were highly accumulated in a few specific regions of the vacuolar membranes . Large portions of Vam2p and Vam6p were fractionated into a vacuolar enriched fraction, indicating that they were localized mainly in the vacuolar membranes . These results showed that Vam2p and Vam6p execute their function in the vacuolar assembly as the components of a protein complex reside on the vacuolar membranes. Mutat Res, 1997 Apr 24, 390(1-2), 113 - 20 Modulation of mitomycin C mutagenicity on Saccharomyces cerevisiae by glutathione, cytochrome P-450, and mitochondria interactions; Rossi C et al.; It is well established that most anticancer drugs also have mutagenic effects and require metabolic activation before exerting their mutagenic/antiblastic activity . Antitumoral compound effects strongly depend on the biochemical/physiological conditions of the tumoral cells, and especially on the activation of specific drugs metabolizing enzymes and on respiration . We examined the mitomycin C-induced mutagenic effects on the D7 strain of Saccharomyces cerevisiae and on its derivative mitochondrial mutant p degrees at different contents of glutathione and cytochrome P-450, molecules able to activate/detoxicate xenobiotics . The mutagenic activity of the drug was evaluated as frequency of mitotic gene conversion and reversion in different physiological conditions . The highest frequencies of reversion and especially of gene conversion were observed at the highest cytochrome P-450 contents in the D7 strain with a further increase at high glutathione level . In the respiratory-deficient strain, the highest frequency of convertants was shown at low glutathione level and lack of cytochrome P-450 . These results suggest the relevance of mitochondrial functionality for the expression of genotoxic activity of this anticancer drug. Carbohydr Res, 1997 Apr 21, 299(3), 203 - 8 The application of various protic acids in the extraction of (1-->3)-beta-D-glucan from Saccharomyces cerevisiae; Muller A et al.; Glucans are (1-->3)-beta-linked glucose polymers which have immune-stimulating capability . The extraction of water-insoluble (1-->3)-beta-D-glucan form Saccharomyces cerevisiae employs hydrochloric acid . Hydrochloric acid is difficult to employ in the large-scale pharmaceutical extraction of glucans due to its corrosive nature and toxicity . To address these concerns, we determined whether acetic, formic or phosphoric acid can be substituted for hydrochloric acid in the process for the isolation of (1-->3)-beta-D-glucan . The resulting microparticulate glucans were employed as the starting material for the production of (1-->3)-beta-D-glucan phosphate . 13C NMR analysis of the glucan phosphates derived from the acetic, formic or phosphoric acid-extracted microparticulate glucan show excellent correspondence to hydrochloric acid extracted glucan and laminarin, a (1-->3)-beta-D-glucan standard, indicating that the primary structure is not altered by the acid used for extraction . Glucan phosphate prepared from hydrochloric acid had a Mw of 7.2 x 10(4) g/mol, rmsz of 17.7 nm, of 1.50 and (eta) of 49.0 mL/g . Glucan phosphate prepared from acetic acid had a primary polymer peak with a Mw of 1.4 x 10(6) g/mol, rmsz of 23.6 nm, I of 1.93 and (eta) of 62.4 mL/g . Glucan phosphate prepared from formic acid had a main polymer peak with a Mw of 1.2 x 10(6) g/mol, rmsz 27.1 nm, I of 1.56 and (eta) of 89.0 mL/g . Glucan phosphate prepared from phosphoric acid had a primary polymer peak with a Mw of 6.6 x 10(5) g/mol, rmsz of 32.3 nm, I of 2.70 and (eta) of 91.3 mL/g . These data indicate that the molecular mass, size, polydispersity and intrinsic viscosity of the glucan phosphate obtained is influenced by the pKa of protic acid employed to extract the microparticulate glucan . However, the primary structure and side-chain branching are not substantially altered regardless of the acid employed. J Cell Biol, 1997 Apr 21, 137(2), 417 - 31 The Saccharomyces cerevisiae kinesin-related motor Kar3p acts at preanaphase spindle poles to limit the number and length of cytoplasmic microtubules; Saunders W et al.; The Saccharomyces cerevisiae kinesin-related motor Kar3p, though known to be required for karyogamy, plays a poorly defined, nonessential role during vegetative growth . We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle-specific manner . Deletion of KAR3 leads to a dramatic increase in cytoplasmic microtubules, a phenotype which is most pronounced from START through the onset of anaphase but less so during late anaphase in synchronized cultures . We have immunolocalized HA-tagged Kar3p to the spindle pole body region, and fittingly, Kar3p was not detected by late anaphase . A microtubule depolymerizing activity may be the major vegetative role for Kar3p . Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential alpha-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization . Microtubule depolymerization may also be the mechanism by which Kar3p acts in opposition to the anaphase B motors Cin8p and Kip1p . A preanaphase spindle collapse phenotype of cin8 kip1 mutants, previously shown to involve Kar3p, is markedly delayed when microtubule depolymerization is inhibited by the tub2-150 mutation . These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle. J Biol Chem, 1997 Apr 18, 272(16), 10831 - 8 Cleavage factor II of Saccharomyces cerevisiae contains homologues to subunits of the mammalian Cleavage/ polyadenylation specificity factor and exhibits sequence-specific, ATP-dependent interaction with precursor RNA; Zhao J et al.; Cleavage of pre-mRNA during 3'-end formation in yeast requires two protein factors, cleavage factor I (CF I) and cleavage factor (CF II) . A 5300-fold purification of CF II indicates that four polypeptides of 150, 105, 100, and 90 kDa copurify with CF II activity . The 150-kDa protein is recognized by antibodies against Cft1, the yeast homologue of the 160-kDa subunit of the mammalian cleavage/polyadenylation specificity factor (CPSF) . The 100-kDa subunit is identical to Brr5/Ysh1, a yeast protein with striking similarity to the 73-kDa subunit of CPSF . The 105-kDa protein, designated Cft2 (cleavage factor two) exhibits significant homology to the CPSF 100-kDa subunit . Cft2 is cross-linked to pre-mRNA substrate containing the poly(A) site and wild type upstream and downstream flanking sequences, but not to precleaved RNA lacking downstream sequences or to substrate in which the (UA)6 processing signal has been deleted . The specific binding of Cft2 to the RNA substrate is ATP-dependent, in agreement with the requirement of ATP for cleavage . The sequence-specific binding of Cft2 and the similarities of CF II subunits to those of CPSF supports the hypothesis that CF II functions in the cleavage of yeast mRNA 3'-ends in a manner analagous to that of CPSF in the mammalian system . These results provide additional evidence that certain features of the molecular mechanism of mRNA 3'-end formation are conserved between yeast and mammals, but also highlight unexpected differences. Eur J Biochem, 1997 Apr 15, 245(2), 324 - 33 Kinetic characterization of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression; Reifenberger E et al.; In Saccharomyces cerevisiae, there are a large number of genes (HXT1-HXT17/SNF3/RGT2) encoding putative hexose transporters which, together with a galactose permease gene (GAL2), belong to a superfamily of monosaccharide facilitator genes . We have performed a systematic analysis of the HXT1-7 and GAL2 genes and their function in hexose transport . Glucose uptake was below the detection level in the hxt1-7 null strain growing on maltose . Determination of the kinetic parameters of individual hexose transporter-related proteins (Hxtp) expressed in the hxt null background revealed Hxt1p and Hxt3p as low-affinity transporters (Km(glucose) = 50-100 mM), Hxt2p and Hxt4p as moderately low in affinity (Km(glucose) about 10 mM), and Hxt6p, Hxt7p as well as Gal2p as high-affinity transporters (Km(glucosse) = 1-2 mM) . However, Hxt2p kinetics in cells grown on low glucose concentrations showed a high-affinity (Km = 1.5 mM) and a low-affinity component (Km = 60 mM) . Furthermore, we investigated the involvement of glucose transport in glucose signalling . Glucose repression of MAL2, SUC2 and GAL1 was not dependent on a specific transporter but, instead, the strength of the repression signal was dependent on the level of expression, the properties of the individual transporters and the kind of sugar transported . The strength of the glucose repression signal correlated with the glucose consumption rates in the different strains, indicating that glucose transport limits the provision of a triggering signal rather then being directly involved in the triggering mechanism. J Mol Biol, 1997 Apr 11, 267(4), 899 - 915 The three-dimensional structure at 2.4 A resolution of glycosylated proteinase A from the lysosome-like vacuole of Saccharomyces cerevisiae; Aguilar CF et al.; The crystal structures of glycosylated native proteinase A, an aspartic proteinase found in the vacuole of Saccharomyces cerevisiae, and its complex with a difluorostatone-containing tripeptide have been determined by molecular replacement to 3.5 A and 2.4 A resolutions, respectively . Superposition of the bound and native forms gave an r.m.s . difference of 0.6 A largely reflecting the poor resolution of the native crystal structure . The secondary and tertiary structures are highly similar to those found in porcine pepsin and lysosomal cathepsin D; superposition of the structure of proteinase A bound to the difluorostatone inhibitor on those of pepsin and cathepsin D gave pairwise r.m.s . differences for C(alpha) atoms of 1.36 A and 0.88 A . Most differences occur in loop regions . Comparison of the structure of the proteinase A-difluorostatone complex with that of endothiapepsin bound with the same inhibitor shows that the conformation and hydrogen bond interactions of the inhibitor in the active site are very similar, even though the enzymes have only 27% sequence identity . Electron density for the crystal structure of the proteinase A complex reveals five residues of the oligosaccharide structure attached to Asn67: Man-(1 --> 2)-alpha-Man-(1 --> 3)-beta-Man-(1 --> 4)-beta-GlcNAc-(1 --> 4)-beta-GlcNAc-Asn-67 . The first three residues of the oligosaccharide cover the same region of the protein surface as those of the oligosaccharide attached to the equivalent position in cathepsin D . The second carbohydrate attachment site is disordered beyond the first carbohydrate residue in both enzymes. J Biol Chem, 1997 Apr 11, 272(15), 9986 - 8 Characterization of Saccharomyces cerevisiae CYP61, sterol delta22-desaturase, and inhibition by azole antifungal agents; Kelly SL et al.; Cytochrome P-45061 (CYP61) was a cytochrome P-450 revealed during the yeast genome project when chromosome XIII was sequenced . Here we report on the properties of this second microsomal P-450 of vegetatively growing yeast . The enzyme kinetics associated with its endogenous role in sterol Delta22-desaturation revealed a Km of 20.4 microM and a Vmax of 2.9nmol/min/nmol CYP61 . The affinity of the enzyme for antifungal drugs was characterized to investigate its potential role in determining tolerance to these sterol 14alpha-demethylase (CYP51) inhibitors . Drug binding induced a type II spectral change, which became saturated at equimolar concentrations of azole drug and P-450 . Fluconazole exhibited slightly reduced affinity in comparison to ketoconazole as indicated by carbon monoxide displacement . These and Ki determination for fluconazole (0.14 nM) revealed CYP61 to have a similar affinity to azole drugs when compared with data available for CYP51, and the implications for antifungal treatment were considered. J Biol Chem, 1997 Apr 11, 272(15), 9809 - 17 Sphingolipid synthesis as a target for antifungal drugs . Complementation of the inositol phosphorylceramide synthase defect in a mutant strain of Saccharomyces cerevisiae by the AUR1 gene; Nagiec MM et al.; We have identified a Saccharomyces cerevisiae gene necessary for the step in sphingolipid synthesis in which inositol phosphate is added to ceramide to form inositol-P-ceramide, a reaction catalyzed by phosphatidylinositol:ceramide phosphoinositol transferase (IPC synthase) . This step should be an effective target for antifungal drugs . A key element in our experiments was the development of a procedure for isolating mutants defective in steps in sphingolipid synthesis downstream from the first step including a mutant defective in IPC synthase . An IPC synthase defect is supported by data showing a failure of the mutant strain to incorporate radioactive inositol or N-acetylsphinganine into sphingolipids and, by using an improved assay, a demonstration that the mutant strain lacks enzyme activity . Furthermore, the mutant accumulates ceramide when fed exogenous phytosphingosine as expected for a strain lacking IPC synthase activity . Ceramide accumulation is accompanied by cell death, suggesting the presence of a ceramide-activated death response in yeast . A gene, AUR1 (YKL004w), that complements the IPC synthase defect and restores enzyme activity and sphingolipid synthesis was isolated . Mutations in AUR1 had been shown previously to give resistance to the antifungal drug aureobasidin A, leading us to predict that the drug should inhibit IPC synthase activity . Our data show that the drug is a potent inhibitor of IPC synthase with an IC50 of about 0.2 nM . Fungal pathogens are an increasing threat to human health . Now that IPC synthase has been shown to be the target for aureobasidin A, it should be possible to develop high throughput screens to identify new inhibitors of IPC synthase to combat fungal diseases. J Biol Chem, 1997 Apr 11, 272(15), 9697 - 702 Functional analysis of mutated purine-cytosine permease from Saccharomyces cerevisiae . A possible role of the hydrophilic segment 371-377 in the active carrier conformation; Ferreira T et al.; The purine-cytosine permease (PCP) is an active transporter located in the plasma membrane of the yeast Saccharomyces cerevisiae . This protein mediates purine (adenine, guanine, and hypoxanthine) and cytosine accumulation in the cell by using an electrochemical potential difference in proton as the energy source . Various mutant strains, with altered Kt(app) (apparent Michaelis constant of transport) of uptake for one or several bases, have already been selected . Their cloning and sequencing revealed that three of them presented substitutions in the same region of the putative sequence of the PCP: this region might correspond to the hydrophilic segment 371-377 (I-A-N-N-I-P-N) . Two mutants displayed single mutations, resulting in only one amino acid residue change (N377I and N374I, respectively), and the other displayed three amino acid substitutions (I371V, I375V, and N377G) . Therefore, to analyze the contribution of individual amino acid changes to the phenotype of the complex mutant, single (N377G) and double (I371V,I375V) mutants were constructed by site-directed mutagenesis . The influence of single mutations in this region was studied by measuring, for adenine, hypoxanthine, and cytosine, the uptake constants on cells and equilibrium binding parameters on plasma membrane-enriched fractions . Uptake and binding constant determinations showed that all the variations observed for the Kt(app) of uptake were correlated with variations of the binding Kd(app) for the corresponding solutes . Thus, our results emphasize the role of the two asparagine residues, located at positions 374 and 377, respectively, in the binding of the bases . In addition, the sole substitution of the 377 asparagine residue by glycine is responsible for the phenotype of the triple mutant . The effect of pH on the apparent hypoxanthine binding dissociation constant showed that the effects of N377G and N377I changes were, at least partially, due to a shift of the pKa of an ionizable amino acid residue of the unliganded permease . These two amino acid residue changes induced a shift of the pKa of this group in the unliganded, deprotonated permease about two units toward acidic pH . This result suggests that the 371-377 segment might play a key role in the proper three-dimensional structure of the active purine-cytosine permease. J Biol Chem, 1997 Apr 4, 272(14), 9436 - 42 Cloning and characterization of an essential Saccharomyces cerevisiae gene, TAF40, which encodes yTAFII40, an RNA polymerase II-specific TATA-binding protein-associated factor; Klebanow ER et al.; In this report we describe the cloning and initial characterization of TAF40, a gene that encodes a yeast TATA-binding protein-associated factor (yTAF) of Mr = approximately 40,000 . This gene has many similarities to other yTAFs described thus far in that it is present at a single copy per haploid genome, it is essential for viability, and the deduced protein sequence of yTAF40 exhibits similarity to previously described human and Drosophila TAFIIs . Immunological studies confirm that yTAF40 protein is a subunit of a large multiprotein TATA-binding protein-TAF complex that contains a subset of the total number of the yTAFs present in yeast cell extracts . Transcription reactions performed using yeast whole cell extracts reveal that of the three nuclear RNA polymerases only RNA polymerase II function is abrogated when yTAF40 and associated proteins are immunodepleted from solution, indicating that the functionality of the multiprotein complex containing yTAF40 is RNA polymerase II-specific . By these criteria yTAF40 appears to encode a bona fide RNA polymerase II-specific TAF, and thus the protein that it encodes has been termed yTAFII40. J Biol Chem, 1997 Apr 4, 272(14), 9215 - 20 A role for the Saccharomyces cerevisiae ATX1 gene in copper trafficking and iron transport; Lin SJ et al.; The ATX1 gene of Saccharomyces cerevisiae was originally identified as a multi-copy suppressor of oxidative damage in yeast lacking superoxide dismutase . We now provide evidence that Atx1p helps deliver copper to the copper requiring oxidase Fet3p involved in iron uptake . atx1Delta null mutants are iron-deficient and are defective in the high affinity uptake of iron . These defects due to ATX1 inactivation are rescued by copper treatment, and the same has been reported for strains lacking either the cell surface copper transporter, Ctr1p, or the putative copper transporter in the secretory pathway, Ccc2p . Atx1p localizes to the cytosol, and our studies indicate that it functions as a carrier for copper that delivers the metal from the cell surface Ctr1p to Ccc2p and then to Fet3p within the secretory pathway . The iron deficiency of atx1 mutants is augmented by mutations in END3 blocking endocytosis, suggesting that a parallel pathway for intracellular copper trafficking is mediated by endocytosis . As additional evidence for the role of Atx1p in iron metabolism, we find that the gene is induced by the same iron-sensing trans-activator, Aft1p, that regulates CCC2 and FET3. J Biol Chem, 1997 Apr 4, 272(14), 9131 - 40 Disruption of ribosomal scanning on the 5'-untranslated region, and not restriction of translational initiation per se, modulates the stability of nonaberrant mRNAs in the yeast Saccharomyces cerevisiae; Linz B et al.; Translation and mRNA decay constitute key players in the post-transcriptional control of gene expression . We examine the mechanisms by which the 5'-untranslated region (UTR) of nonaberrant mRNAs acts to modulate both these processes in Saccharomyces cerevisiae . Two classes of functional relationship between ribosome-5'-UTR interactions and mRNA decay are identifiable . In the first of these, elements in the main open reading frame (ORF) dictate how the decay process reacts to inhibitory structures in the 5'-UTR . The same types of stability modulation can be elicited by trans-regulation of translation via inducible binding of the iron-regulatory protein to an iron-responsive element located 9 nucleotides from the 5' cap . A eukaryotic translational repressor can therefore modulate mRNA decay via the 5'-UTR . In contrast, translational regulation mediated via changes in the activity of the cap-binding eukaryotic translation initiation factor eIF-4E bypasses translation-dependent pathways of mRNA degradation . Thus modulation of mRNA stability via the 5'-UTR depends on disruption of the scanning process, rather than changes in translational initiation efficiency per se . In the second class of pathway, an upstream ORF (uORF) functions as a powerful destabilizing element, inducing termination-dependent degradation that is apparently independent of any main ORF determinants but influenced by the efficiencies of ribosomal recognition of the uORF start and stop codons . This latter mechanism provides a regulatable means to modulate the stability of nonaberrant mRNAs via a UPF-dependent pathway. Biochim Biophys Acta, 1997 Apr 3, 1325(1), 126 - 34 Different activation energies in glucose uptake in Saccharomyces cerevisiae DFY1 suggest two transport systems; Reinhardt C et al.; The analysis of initial glucose uptake in Saccharomyces cerevisiae at 25 degrees, 20 degrees, 15 degrees and 10 degrees C by computer-assisted nonlinear regression analysis predicts two transport systems . The first demonstrates Michaelis-Menten kinetics and the second shows first order behaviour . The activation energies of these two systems were calculated by the Arrhenius equation at four different growth phases, namely early exponential (EE), middle exponential (ME2), late exponential (LE) and early stationary (ES) with 2% glucose in the batch medium . The activation energies calculated from the V(m) values in EE, ME, LE and ES growth phases were 15.8 +/- 1.7, 13.5 +/- 1.0, 15.1 +/- 0.8 and 13.5 +/- 0.7 kcal/mol . These values are in agreement with activation energies calculated for the first mechanism, facilitated diffusion, which is the mechanism deduced from countertransport experiments . The activation energies derived for the second transport system from the first order rate constants in cells grown to EE, ME2, LE and ES were 8.0 +/- 2.1, 8.1 +/- 1.3, 9.6 +/- 3.0 and 7.5 +/- 2.6 kcal/mol . These values are still significantly higher than for free diffusion of glucose in water and lower as predicted for passage of glucose through the lipid phase . Therefore, we assume in addition to carrier-mediated facilitated diffusion the entrance of glucose into the cell through a pore. Biochim Biophys Acta, 1997 Apr 3, 1325(1), 63 - 70 Effect of extracellular acidification on