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| 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 the activity of plasma membrane ATPase and on the cytosolic and vacuolar pH of Saccharomyces cerevisiae; Carmelo V et al.; The rapid in vivo activation of Saccharomyces cerevisiae plasma membrane H+-ATPase that has been attributed to medium acidification from pH 6.5 to pH 3.5 is not caused by the low pH itself but is induced by the weak organic acid (succinic) used as the acidulant . The activation induced by 50 mM succinic acid at pH 3.5 occurred in both the presence or absence of glucose . Activation at pH 3.5 was also induced by acetic acid and it was maximal at 50 mM concentration . To investigate the role of plasma membrane ATPase activation in pH homeostasis, the internal pH (cytosolic and vacuolar) of yeast cells incubated in media at pH 6.5 or at pH 3.5, acidified either with HCl or acetic acid, were compared by using in vivo (31)P-NMR . Despite plasma membrane ATPase activation by acetic acid, the decrease in cytosolic pH caused by external acidification was much more important when the permeant acetic acid was used instead of HCl as the acidulant . The supplementation of the incubation medium at pH 3.5 with glucose led to higher cytosolic pH values, consistent with the observed in vivo activation of plasma membrane ATPase by glucose . At the external pH value of 6.5 the vacuole was maintained at a mildly acidic pH (around 6) while the cytosol was at about neutral pH; however, when cytoplasmic pH decreased due to external acidification, vacuolar pH accompanied that decrease . Vacuolar pH reached 5.4-5.5 during incubation with HCI and dropped sharply to values below 4.4 in cells incubated with acetic acid . These results indicate that the vacuole also plays a role in homeostasis of the intracellular pH. Mol Biol Cell, 1997 Apr, 8(4), 647 - 62 Sec3p is involved in secretion and morphogenesis in Saccharomyces cerevisiae; Finger FP et al.; Two new temperature-sensitive alleles of SEC3, 1 of 10 late-acting SEC genes required for targeting or fusion of post-Golgi secretory vesicles to the plasma membrane in Saccharomyces cerevisiae, were isolated in a screen for temperature-sensitive secretory mutants that are synthetically lethal with sec4-8 . The new sec3 alleles affect early as well as late stages of secretion . Cloning and sequencing of the SEC3 gene revealed that it is identical to profilin synthetic lethal 1 (PSL1) . The SEC3 gene is not essential because cells depleted of Sec3p are viable although slow growing and temperature sensitive . All of the sec3 alleles genetically interact with a profilin mutation, pfy1-111 . The SEC3 gene in high copy suppresses pfy1-111 and sec5-24 and causes synthetic growth defects with ypt1, sec8-9, sec10-2, and sec15-1 . Actin structure is only perturbed in conditions of chronic loss of Sec3p function, implying that Sec3p does not directly regulate actin . All alleles of sec3 cause bud site selection defects in homozygous diploids, as do sec4-8 and sec9-4 . This suggests that SEC gene products are involved in determining the bud site and is consistent with a role for Sec3p in determining the correct site of exocytosis. J Photochem Photobiol B, 1997 Apr, 38(2-3), 123 - 8 Fluence rate as a determinant of synergistic interaction under simultaneous action of UV light and mild heat in Saccharomyces cerevisiae; Petin VG et al.; In experiments with wild-type diploid yeast cells of Saccharomyces cerevisiae, the synergistic lethal action of a simultaneous application of ultraviolet (UV) light (wavelength, 254 nm) and mild heat (45-57.5 degrees C) was studied . It was shown that, at any fixed UV light intensity, the synergistic effect occurred within the given temperature interval . The optimal temperature to achieve the greatest synergistic effect may be shown for every fluence rate examined . The correlation between the optimal temperature that maximized the synergy and UV light intensity was estimated: this temperature shifted towards higher temperature values with an increasing fluence rate . A possible interpretation of this effect is based on the supposition that the mechanism of the synergistic effect is related to additional lethal damage produced by the interaction of sublesions induced by each agent . These sublesions are supposed to be non-lethal when each agent is applied separately. Appl Biochem Biotechnol, 1997 Spring, 63-65, 203 - 12 Asparaginase II of Saccharomyces cerevisiae . GLN3/URE2 regulation of a periplasmic enzyme; Bon EP et al.; The production of some extracellular enzymes is known to be negatively affected by readily metabolized nitrogen sources such as NH4+ although there is no consensus regarding the involved mechanisms . Asparaginase II is a periplasmic enzyme of Saccharomyces cerevisiae encoded by the ASP3 gene . The enzyme activity is not found in cells grown in either ammonia, glutamine, or glutamate, but it is found in cells that have been subjected to nitrogen starvation or have been grown on a poor source of nitrogen such as proline . In this report it is shown that the formation of this enzyme is dependent upon the functional GLN3 gene and that the response to nitrogen availability is under the control of the URE2 gene product . In this respect the expression of ASP3 is similar to the system that regulates the GLN1, GDH2, GAP1, and PUT4 genes that codes for glutamine synthetase, NAD-linked glutamate dehydrogenase, general amino-acid permease, and high affinity proline permease, respectively. J Protein Chem, 1997 Apr, 16(3), 233 - 6 Site-directed mutagenesis in basic amino acid residues of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase; Chavez R et al.; Mutant Arg76Gln and Lys290Gln Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases have been prepared and analyzed . No alteration in the apparent kinetic constants were detected for the Arg76Gln mutant enzyme, while the Lys290Gln mutant showed a 12-fold decrease in V(max)/K(m)ADP . These results indicate that Arg76 is not involved in CO2 binding, but support the hypothesis that the binding of this substrate induces a conformational change that protects the region around Arg76 from trypsin action {Herrera et al . (1993) J . Protein Chem . 12, 413-418} . These findings also indicate that Lys290, a highly reactive residue against pyrydoxal phosphate {Bazaes et al . (1995), FEBS Lett . 360, 207-210}, does not perform an essential function for the enzyme activity. Yeast, 1997 Apr, 13(5), 441 - 7 Meiotic inheritance of functional Gal80S gene product in Saccharomyces cerevisiae; Keller AD et al.; Transcription factors inherited during meiosis play a crucial role directing subsequent gene activity . Factors of maternal origin have been shown to influence the pattern of early zygotic transcription during Drosophila and Xenopus embryogenesis . Nevertheless, little is known regarding the meiotic inheritance of the vast majority of transcription factors . In the case of yeast meiosis, for example, it is not yet known whether any of the multitude of transcription factors expressed in the diploid are transmitted to haploid spores in functional form . Here we use a GAL1-STE4 reporter whose activity is detectable in single living cells to identify a transcription factor inherited during sporulation in Saccharomyces cerevisiae . We show that functional Gal80S repressor is meiotically inherited at levels reflecting its expression in the diploid parent. Yeast, 1997 Apr, 13(5), 435 - 9 The branched-chain amino acid permease gene of Saccharomyces cerevisiae, BAP2, encodes the high-affinity leucine permease (S1); Schreve J et al.; The amino acid leucine has been shown previously to be transported into a yeast cell by at least three permeases: the general amino acid permease, a high-affinity permease (S1) and a low-affinity permease (S2) . We isolated the gene BAP2 as a multicopy suppressor of the YPD- phenotype of aat1leu2 yeast . BAP2 has been identified previously as encoding an amino acid permease which transports branched-chain amino acids . In order to align the genetic and biochemical studies of leucine uptake we completed a detailed kinetic analysis of yeast strains in which the BAP2 gene was disrupted and compared this to the kinetics of uptake of the parental strain . We demonstrate that BAP2 encodes the high-affinity leucine permease previously called S1. Biosci Biotechnol Biochem, 1997 Apr, 61(4), 704 - 9 Characterization of IKI1 and IKI3 genes conferring pGKL killer sensitivity on Saccharomyces cerevisiae; Yajima H et al.; The Saccharomyces cerevisiae iki mutants show an insensitive phenotype to the pGKL killer toxin, and we have cloned some IKI genes by complementation of this phenotype {Kishida et al., Biosci . Biotech . Biochem., 60, 798-801 (1996)} . Here, we identified and characterized the IKI1 and IKI3 genes . DNA sequencing of the genes showed that both have 100% identity with hypothetical genes identified by the yeast genome project, YHR187w (481,911-480,985 in chromosome VIII) for IKI1, and YLR384c (888,852-892,898 in chromosome XII) for IKI3 . Both are novel genes with no significant identity with other known genes and they do not belong to any homology domain group, gene family, or superfamily . The disruption of IKI1 is not lethal, but growth of the disruptant was slower than that of the wild type at all temperatures examined . The disruptant was the killer-insensitive phenotype . The sequence of the IKI1 gene predicted a hydrophilic protein with a molecular mass of 35 kDa (309 amino acids) . A 35-kDa protein band was also detected by immunoblotting the 25,000 x g pellet fraction of the wild type yeast cell lysate . Disruption of the IKI3 gene is also non-lethal and it has the killer-insensitive phenotype . Iki3p may contain a transmembrane domain near the NH2-terminal region (97-113 residues in a total of 1349 amino acids). Biosci Biotechnol Biochem, 1997 Apr, 61(4), 631 - 5 Changes in PAF (platelet-activating factor) production during cell cycle of yeast Saccharomyces cerevisiae; Nakayama R et al.; Yeast Saccharomyces cerevisiae cells were cultured synchronously and the change of platelet-activating factor (PAF) production during the cell cycle was investigated at each phase of the cycle . The basal PAF contents of diploid AKU4103 cells in G1 and M phases were higher than those of cells in S phase . Both diploid and haploid strains showed the same level of PAF production in response to the calcium ionophore A23187 . A23187-stimulated PAF productions of cells in G1 and M phases were about 20 times higher than that of cells in S phase . The contents of PAF precursor in G1 and M phases cells of AKU4103 were higher than those in S phase cells, and the ratio of A23187-stimulated PAF to the precursor was highest in G1 phase cells . We also examined the change in a PAF-synthesizing enzyme, acetyltransferase, activity during the cell cycle using a microsomal fraction . Specific activity was the highest at G1 phase, and total activity was higher at M phase . The enzyme activities of cells in S phase of strains AKU4103 and RAY-3Aa were one-third and one-tenth of those in G1 phase of corresponding cells, respectively . These results suggest that PAF production was higher at G1 and M phases and lower at S phase, and changes in PAF productivity during cell cycle were related to the precursor contents and the synthesizing enzyme activities in those cells . These data suggest that PAF may control the cell cycle phase in budding yeast. Microbiology, 1997 Apr, 143 ( Pt 4), 1263 - 70 The TBP gene from Aspergillus nidulans-structure and expression in Saccharomyces cerevisiae; Kucharski R et al.; The genomic and cDNA copy of the TATA-binding protein (TBP) gene from the filamentous fungus Aspergillus nidulans have been cloned . The gene is interrupted by four introns, one of which is in the long 5' untranslated region of 615 bp . The transcription initiation site was established and the levels of mRNA were analysed under diverse growth conditions and found to vary severalfold . The gene encodes a protein of 268 amino acids composed of an N-terminal domain of 88 amino acids with no significant homology to other TBPs and a C-terminal domain of 180 amino acids with about 95% homology to other fungal TBPs . A cDNA clone under the yeast ADH1 promoter was able to substitute for the yeast TBP gene in vivo; however, the transformants obtained grew poorly at 35 degrees C and on galactose and glycerol at 30 degrees C, though they could grow in the presence of copper ions or aminotriazole at this temperature . This phenotype may be the result of altered function of A . nidulans TBP in certain yeast transcription activation pathways. Mol Microbiol, 1997 Apr, 24(1), 53 - 9 The CIT3 gene of Saccharomyces cerevisiae encodes a second mitochondrial isoform of citrate synthase; Jia YK et al.; We have identified a third citrate synthase gene in Saccharomyces cerevisiae which we have called CIT3 . Complementation of a citrate synthase-deficient strain of Escherichia coli by lacZ::CIT3 gene fusions demonstrated that the CIT3 gene encodes an active citrate synthase . The CIT3 gene seems to be regulated in the same way as CIT1, which encodes the mitochondrial isoform of citrate synthase . Deletion of the CIT3 gene in a delta cit1 background severely reduced growth on the respiratory substrate glycerol, whilst multiple copies of the CIT3 gene in a delta cit1 background significantly improved growth on acetate . In vitro import experiments showed that cit3p is transported into the mitochondria . Taken together, these data show that the CIT3 gene encodes a second mitochondrial isoform of citrate synthase. EMBO J, 1997 Apr 1, 16(7), 1550 - 64 The spindle pole body component Spc97p interacts with the gamma-tubulin of Saccharomyces cerevisiae and functions in microtubule organization and spindle pole body duplication; Knop M et al.; Previously, we have shown that the gamma-tubulin Tub4p and the spindle pole body component Spc98p are involved in microtubule organization by the yeast microtubule organizing centre, the spindle pole body (SPB) . In this paper we report the identification of SPC97 encoding an essential SPB component that is in association with the SPB substructures that organize the cytoplasmic and nuclear microtubules . Evidence is provided for a physical and functional interaction between Tub4p, Spc98p and Spc97p: first, temperature-sensitive spc97(ts) mutants are suppressed by high gene dosage of SPC98 or TUB4 . Second, Spc97p interacts with Spc98p and Tub4p in the two-hybrid system . Finally, immunoprecipitation and fractionation studies revealed complexes containing Tub4p, Spc98p and Spc97p . Further support for a direct interaction of Tub4p, Spc98p and Spc97p comes from the toxicity of strong SPC97 overexpression which is suppressed by co-overexpression of TUB4 or SPC98 . Analysis of temperature-sensitive spc97(ts) alleles revealed multiple spindle defects . While spc97-14 cells are either impaired in SPB separation or mitotic spindle formation, spc97-20 cells show an additional defect in SPB duplication . We discuss a model in which the Tub4p-Spc98p-Spc97p complex is part of the microtubule attachment site at the SPB. Methods, 1997 Apr, 11(4), 353 - 60 Translation-competent extracts from Saccharomyces cerevisiae: effects of L-A RNA, 5' cap, and 3' poly(A) tail on translational efficiency of mRNAs; Iizuka N et al.; Yeast genetics has proven fruitful in the identification of key players that are involved in translational initiation . However, the exact roles of many translation initiation factors in translation initiation remain unknown . This has been due to lack of a suitable in vitro translation system in which the mode of action of certain translation factors can be studied . This report describes the preparation of cell-free Saccharomyces cerevisiae lysates that can mediate the translation of exogenously added mRNAs . Optimal translation required the absence of viral L-A RNA in the lysate and the presence of both a 5' cap and a 3' poly(A) tail on the mRNAs . A cooperative effect of cap and poly(A) tail on translation initiation was observed, a property that has been found to operate in intact yeast cells as well . In addition, the yeast lysates mediated translational initiation through several viral internal ribosome entry sites, demonstrating that the yeast translation apparatus can perform internal initiation . Thus, these lysates may be useful in the biochemical analysis of cap-dependent and cap-independent translation events. Arch Biochem Biophys, 1997 Apr 1, 340(1), 101 - 10 Characterization of a sphingomyelinase activity in Saccharomyces cerevisiae; Ella KM et al.; Sphingomyelinases (SMase), which hydrolyze sphingolipids to yield ceramide, participate in signal transduction pathways in mammalian cells . Although yeast express many homologs of mammalian signaling proteins, SMase activity had not been previously demonstrated in yeast . In this study, we used an in vitro assay to characterize yeast SMase activity . Activity was detected in yeast membranes at both acid and neutral pH . The enzyme exhibited a requirement for magnesium or manganese, and was sensitive to detergents . The pI of the enzyme was approximately 5.9 . SMase was separable from phospholipase D (PLD) activity, and was expressed at normal levels in yeast lacking expression of PLD1 . While sphingosine and phytosphingosine inhibited growth, other sphingolipid metabolites had no effect on yeast growth . Intact yeast generate ceramide from exogenous sphingomyelin . These studies demonstrate that yeast express a membrane-localized neutral SMase activity. Mol Cell Biol, 1997 Apr, 17(4), 2057 - 65 Saccharomyces cerevisiae BUR6 encodes a DRAP1/NC2alpha homolog that has both positive and negative roles in transcription in vivo; Prelich G; BUR3 and BUR6 were identified previously by selecting for mutations that increase transcription from an upstream activating sequence (UAS)-less promoter in Saccharomyces cerevisiae . The bur3-1 and bur6-1 mutations are recessive, increase transcription from a suc2 delta uas allele, and cause other mutant phenotypes, suggesting that Bur3p and Bur6p function as general repressors of the basal transcriptional machinery . The molecular cloning and characterization of BUR3 and BUR6 are presented here . BUR3 is identical to MOT1, a previously characterized essential gene that encodes an ATP-dependent inhibitor of the TATA box-binding protein . Cloning and nucleotide sequence analysis reveals that BUR6 encodes a homolog of DRAP1 (also called NC2alpha), a mammalian repressor of basal transcription . Strains that contain a bur6 null allele are viable but grow extremely poorly, demonstrating that BUR6 is critical for normal cell growth in yeast . The Bur6p histone fold domain is required for function; an extensive nonoverlapping set of deletion alleles throughout the histone fold domain impairs BUR6 function in vivo, whereas mutations in the amino- and carboxy-terminal tails have no detectable effect . BUR6 and BUR3/MOT1 have different functions depending on promoter context: although the bur3-1 and bur6-1 mutations increase transcription from delta uas promoters, they result in reduced transcription from the wild-type GAL1 and GAL10 promoters . This transcriptional defect is due to the inability of the GAL10 UAS to function in bur6-1 strains . The similar phenotypes of bur6 and bur3 (mot1) mutations suggest that Bur6p and Mot1p have related, but not identical, functions in modulating the activity of the general transcription machinery in vivo. Mol Cell Biol, 1997 Apr, 17(4), 1959 - 65 Ribosomal protein L32 of Saccharomyces cerevisiae influences both the splicing of its own transcript and the processing of rRNA; Vilardell J et al.; Ribosomal protein L32 of Saccharomyces cerevisiae binds to and regulates the splicing and the translation of the transcript of its own gene . Selecting for mutants deficient in the regulation of splicing, we have identified a mutant form of L32 that no longer binds to the transcript of RPL32 and therefore does not regulate its splicing . The mutation is the deletion of an isoleucine residue from a highly conserved hydrophobic domain near the middle of L32 . The mutant protein supports growth, at a reduced rate, and is found at normal levels in mature ribosomes . However, in cells homozygous for the mutant gene, the rate of processing of the ribosomal RNA component of the 60S ribosomal subunit is severely reduced, leading to an insufficiency of 60S subunits . L32 must be considered a remarkable protein . Composed of only 104 amino acids, it appears to interact with three distinct RNA molecules to influence three different elements of RNA processing and function in three different locations of the cell: the processing of pre-rRNA in the nucleolus, the splicing of the RPL32 transcript in the nucleus, and the translation of the spliced RPL32 mRNA in the cytoplasm. Curr Genet, 1997 Apr, 31(4), 292 - 301 Involvement of the RE V3 gene in the methylated base-excision repair system . Co-operation of two DNA polymerases, delta and Rev3p, in the repair of MMS-induced lesions in the DNA of Saccharomyces cerevisiae; Halas A et al.; The ability of four yeast DNA polymerase mutant strains to carry out the repair of DNA treated with MMS was studied . Mutation in DNA polymerase Rev3, as well as the already known mutation in the catalytic subunit of DNA polymerase delta, were both found to lead to the accumulation of single-strand breaks, which indicates defective repair . A double-mutant strain carrying mutations in DNA polymerase delta and a deletion in the REV3 gene had a complete repair defect, both at permissive (23 degrees C) and restrictive (38 degrees C) temperatures, which was not observed in other pairwise combinations of tested polymerase mutants . Other polymerases are not involved in the repair of exogenous DNA methylation damage, since neither mutation in the DNA polymerase epsilon, nor deletion in the DNA polymerase IV (beta70) gene, caused defective repair . The data obtained suggest that DNA polymerases delta and Rev3p are both necessary to perform repair synthesis in the base-excision repair of methylation damage . The results are discussed in the light of current concepts on the role of DNA polymerase Rev3 in mutagenesis. FEMS Microbiol Lett, 1997 Apr 1, 149(1), 85 - 8 HySP26 gene transcription is strongly induced during Saccharomyces cerevisiae growth at low pH; Carmelo V et al.; During exponential growth of Saccharomyces cerevisiae at the inhibitory pH 2.5, the transcription of the major small-heat-shock-protein-encoding gene HSP26 was strongly induced while at the optimal pH 5.0, the mRNA levels from the HSP26 gene were undetectable . When yeast cells entered the stationary phase of growth at pH 5.0, transcription was dramatically enhanced and the level of the HSP26 transcripts reached similar values in stationary cells grown at optimal or inhibitory low pH. Genetics, 1997 Apr, 145(4), 923 - 34 The role of Sas2, an acetyltransferase homologue of Saccharomyces cerevisiae, in silencing and ORC function; Ehrenhofer-Murray AE et al.; Silencing at the cryptic mating-type loci HML and HMR of Saccharomyces cerevisiae requires regulatory sites called silencers . Mutations in the Rap1 and Abf1 binding sites of the HMR-E silencer (HMRa-e**) cause the silencer to be nonfunctional, and hence, cause derepression of HMR . Here, we have isolated and characterized mutations in SAS2 as second-site suppressors of the silencing defect of HMRa-e** . Silencing conferred by the removal of SAS2 (sas2 delta) depended upon the integrity of the ARS consensus sequence of the HMR-E silencer, thus arguing for an involvement of the origin recognition complex (ORC) . Restoration of silencing by sas2 delta required ORC2 and ORC5, but not SIR1 or RAP1 . Furthermore, sas2 delta suppressed the temperature sensitivity, but not the silencing defect of orc2-1 and orc5-1 . Moreover, sas2 delta had opposing effects on silencing of HML and HMR . The putative Sas2 protein bears similarities to known protein acetyltransferases . Several models for the role of Sas2 in silencing are discussed. Genetics, 1997 Apr, 145(4), 891 - 902 Roles of replication protein-A subunits 2 and 3 in DNA replication fork movement in Saccharomyces cerevisiae; Maniar HS et al.; Replication Protein-A, the eukaryotic SSB, consists of a large subunit (RPA1) with strong ssDNA binding activity and two smaller subunits (RPA2 and 3) that may cooperate with RPA1 to bind ssDNA in a higher-order mode . To determine the in vivo function of the two smaller subunits and the potential role of higher-order ssDNA binding, we isolated an assortment of heat-lethal mutations in the genes encoding RPA2 and RPA3 . At the permissive temperature, the mutants show a range of effects on DNA replication fidelity and sensitivities to UV and MMS . At the nonpermissive temperature, four out of five RPA2 mutants show a fast-stop DNA synthesis phenotype typical of a replication fork block . In contrast, the fifth RPA2 mutant and all RPA3 mutants are able to complete at least one round of DNA replication at the nonpermissive temperature . The effect of these mutations on the stability of the RPA complex was tested using a coprecipitation assay . At the nonpermissive temperature, we find that RPA1 and RPA2 are dissociated in the fast-stop mutants, but not in the slow-stop mutants . Thus, replication fork movement in vivo requires the association of at least two subunits of RPA . This result is consistent with the hypothesis that RPA functions in vivo by binding ssDNA in a higher-order mode. J Bacteriol, 1997 Apr, 179(7), 2154 - 62 Identification of a mannoprotein present in the inner layer of the cell wall of Saccharomyces cerevisiae; Moukadiri I et al.; Cell wall extracts from the double-mutant mnn1 mnn9 strain were used as the immunogen to obtain a monoclonal antibody (MAb), SAC A6, that recognizes a specific mannoprotein--which we have named Icwp--in the walls of cells of Saccharomyces cerevisiae . Icwp runs as a polydisperse band of over 180 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Zymolyase extracts of cell walls, although an analysis of the secretory pattern of the mannoprotein shows that at the level of secretory vesicles, it behaves like a discrete band of 140 kDa . Immunofluorescence analysis with the MAb showed that Icwp lies at the inner layer of the cell wall, being accessible to the antibody only after the outer layer of mannoproteins is disturbed by treatment with tunicamycin . The screening of a lambda gt11 expression library enabled us to identify the open reading frame (ORF) coding for Icwp . ICWP (EMBL accession number YLR391w, frame +3) codes for 238 amino acids, of which over 40% are serine or threonine, and contains a putative N-glycosylation site and a putative glycosylphosphatidylinositol attachment signal . Both disruption and overexpression of the ORF caused increased sensitivities to calcofluor white and Congo red, while the disruption caused an increased sensitivity to Zymolyase digestion, suggesting for Icwp a structural role in association with glucan. Yeast, 1997 Mar 30, 13(4), 305 - 12 Saccharomyces cerevisiae cell lysis mutations cly5 and cly7 define temperature-sensitive alleles of PKC1, the gene encoding yeast protein kinase C; Baymiller J et al.; A set of temperature-sensitive Saccharomyces cerevisiae mutants designated cly (for cell lysis) 1-8 because the cells lyse at high temperature was isolated in a large screen for yeast temperature-sensitive (Hartwell, 1967) . Here we report the isolation of two plasmids, containing inserts that both the cly5 and cly7 mutations . DNA sequencing revealed that both of these inserts contain the gene encoding yeast protein kinase C (PKC1) (Levin et al., 1990) . Sequencing of the mutant alleles revealed that cly5 and cly7 contain distinct mutations separated by 194 base pairs . Consistent with this, the cly5 and cly7 ts alleles do not complement each other, and they are genetically linked to PKC1 and to each other . Like other temperature-sensitive pkc1 alleles, the temperature-sensitive phenotype is eliminated by growth in high osmotic strength media (Levin and Bartlett-Heubusch, 1992). J Mol Biol, 1997 Mar 28, 267(2), 324 - 37 Excision repair at the level of the nucleotide in the Saccharomyces cerevisiae MFA2 gene: mapping of where enhanced repair in the transcribed strand begins or ends and identification of only a partial rad16 requisite for repairing upstream control sequences; Teng Y et al.; We wished to determine where transcription enhanced nucleotide excision repair begins and ends for a Saccharomyces cerevisiae gene transcribed by RNA polymerase II, and to examine the role of the RAD16 gene in repairing upstream, non-transcribed control sequences of such a gene . To do so, we developed a method to study the repair of UV induced cyclobutane pyrimidine dimers (CPDs) at the level of the nucleotide in the control and coding sequences of the MFA2 gene . This gene is active in haploid a mating type cells but inactive in alpha cells: its regulation is mediated by changes in chromatin structure . DNA from UV irradiated cells was cut with a CPD-specific endonuclease, restricted and selected strands of the MFA2 gene separated from genomic DNA prior to end-labelling and resolution on a sequencing gel . We confirmed repair trends seen using Southern blotting to examine kilobase size fragments, but were additionally able to elucidate subtle differences in repairing portions of the transcribed strand (TS) of MFA2 . Enhanced repair of the TS when the gene is active, began well before the start of transcription . Clearly, enhanced repair in this region cannot be due to mRNA synthesis . The repair of CPDs is even further enhanced in the transcribed portion of the TS, and returns to a basal level after the termination of transcription . The approach also revealed that RAD16 has a role in the repair of the TS when MFA2 is active . Removal of CPDs from the TS control region was impaired but not totally defective in a rad16 a mutant . Repair from the TS coding sequence also has a Rad16 component, but a lesser one than for the upstream control sequences, and this was more marked for the sequences towards the end of the transcribed region . The system developed permits further dissection of the relationships between DNA repair, chromatin structure and transcription at the MFA2 locus. J Biol Chem, 1997 Mar 28, 272(13), 8256 - 62 Saccharomyces cerevisiae homologs of mammalian B and B' subunits of protein phosphatase 2A direct the enzyme to distinct cellular functions; Zhao Y et al.; Protein phosphatase 2A (PP2A) is a major cellular serine/threonine protein phosphatase, present in the cell in a variety of heterotrimeric forms that differ in their associated regulatory B-subunit . Cloning of the mammalian B' subunit has allowed the identification of a highly homologous Saccharomyces cerevisiae gene, RTS1 . Disruption of the gene results in a temperature-sensitive growth defect that can be suppressed by expression of rabbit B'alpha or B'gamma isoforms . The B'alpha subunit is much more effective in restoring normal growth at 37 degrees C than B'gamma . Immunoprecipitated Rts1p was found associated with type 2A-specific protein phosphatase activity that is sensitive to 2 nM okadaic acid, but not to 100 nM phosphatase inhibitor-2, and to be phosphorylated in vivo . However, overexpression of RTS1 was unable to suppress the cold sensitivity, defective cytokinesis, and abnormal cell morphology resulting from defects in the CDC55 gene, which encodes the yeast homolog of a different B subunit of another form of 2A phosphatase, PP2A1 . These results indicate that Rts1p is a yeast homolog of the mammalian B' subunit and that the various regulatory B-subunits of PP2A are not functionally redundant but direct the enzyme to distinct cellular functions. Mol Gen Genet, 1997 Mar 26, 254(2), 148 - 56 PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae; Carter AT et al.; In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis . Enzymes that can synthesize PRPP can be encoded by at least four genes . We have studied 5-phospho-ribosyl-1(alpha)-pyrophosphate synthetases (PRS) genetically and biochemically . Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has a significant effect on cell metabolism, whereas disruption of PRS2 or PRS4 has little measurable effect . Using Western blot analysis with antisera raised against peptides derived from the non-homology region (NHR) and the N-terminal half of the PRS1 gene product it has been shown that the NHR is not removed by protein splicing . However, the fact that disruption of this gene causes the most dramatic decrease in cell growth rate and enzyme activity suggests that Prs1p may have a key structural or regulatory role in the production of PRPP in the cell. Mutat Res, 1997 Mar 21, 374(2), 209 - 31 Structural and mechanistic bases for the induction of mitotic chromosomal loss and duplication ('malsegregation') in the yeast Saccharomyces cerevisiae: relevance to human carcinogenesis and developmental toxicology; Liu M et al.; MultiCASE has the ability to automatically determine the structural features responsible for the biological activity of chemicals . In the present study, 93 chemicals tested for their ability to induce chromosomal 'malsegregation' in the yeast Saccharomyces cerevisiae were analyzed . This 'malsegregation' mimics molecular events that occur during human development and carcinogenesis resulting in an effective loss of one chromosome of an autosomal pair and duplication of the homologue . Structural features associated with the ability to induce such chromosome loss and duplication were identified and compared with those obtained from examination of other toxicological data bases . The most significant structural similarities were identified between the induction of chromosomal malsegregation and several toxicological phenomena such as cellular toxicity, induction of sister chromatid exchanges in vitro and rodent developmental toxicity . Very significant structural similarities were also found with systemic toxicity, induction of micronuclei in vivo and human developmental toxicity . Less significant structural overlaps were found between yeast malsegregation and rodent carcinogenicity, DNA reactivity and mutagenicity, and the induction of chromosome aberrations in vitro and sister chromatid exchanges in vivo . These overlaps may indicate mechanistic similarities between the induction of chromosomal malsegregation and other toxicological phenomena . The predictivity of the SAR model derived from the present data base is relatively low, however . This may be merely a reflection of the small size and composition of the data base, however, further analyses suggest that it reflects primarily the multiple mechanisms responsible for the induction of chromosomal malsegregation in yeast and the complexity of the phenomenon. J Biol Chem, 1997 Mar 21, 272(12), 7940 - 5 A single-stranded DNA-binding protein is needed for efficient presynaptic complex formation by the Saccharomyces cerevisiae Rad51 protein; Sugiyama T et al.; Protein-promoted DNA strand exchange requires formation of an active presynaptic complex between the DNA-pairing protein and single-stranded DNA (ssDNA) . Formation of such a contiguous filament is stimulated by a ssDNA-binding protein . Here, the effects of replication protein A (RPA) on presynaptic complex formation and DNA strand exchange activities of Rad51 protein were examined . Presynaptic complex formation was assessed by measuring ATP hydrolysis . With phiX174 ssDNA, the ATPase activity of Rad51 protein is stimulated approximately 1.4-fold by RPA, provided that Rad51 protein is in excess of the ssDNA concentration; otherwise, RPA inhibits ATPase activity . In contrast, with ssDNA devoid of secondary structure (poly(dT), poly(dA), poly(dI), and etheno-M13 DNA), RPA does not stimulate the already elevated ATPase activity of Rad51 protein, but inhibits activity at low Rad51 protein concentrations . These results suggest that Rad51 protein and RPA exclude one another from ssDNA by competing for the same binding sites and that RPA exerts its effect on presynaptic complex formation by eliminating secondary structure to which Rad51 protein is bound nonproductively . DNA strand exchange catalyzed by Rad51 protein is also greatly stimulated by RPA . The optimal stoichiometry for stimulation is approximately 20-30 nucleotides of ssDNA/RPA heterotrimer . The ssDNA-binding protein of Escherichia coli can substitute for RPA, showing that the role of RPA is not specific . We conclude that RPA affects both presynaptic complex formation and DNA strand exchange via changes in DNA structure, employing the same mechanism used by the ssDNA-binding protein to effect change in E . coli RecA protein activity. J Biol Chem, 1997 Mar 21, 272(12), 7908 - 14 The Saccharomyces cerevisiae AP-1 protein discriminates between oxidative stress elicited by the oxidants H2O2 and diamide; Wemmie JA et al.; The Saccharomyces cerevisiae AP-1 protein (yAP-1) is a key mediator of oxidative stress tolerance . Transcriptional activation by yAP-1 has been shown to be inducible by exposure of cells to H2O2 and diamide, among other oxidative stress eliciting compounds . Here we define the segments of the yAP-1 protein that are required to respond to this environmental challenge . Western blotting analyses indicated that levels of yAP-1 do not change during oxidative stress . Deletion mutagenesis and gene fusion experiments indicate that two different segments of yAP-1 are required for oxidative stress inducibility . These two domains function differentially depending on the type of oxidant used to generate oxidative stress . Three repeated cysteine-serine-glutamate sequences located in the carboxyl terminus are required for normal regulation of yAP-1 function during oxidative stress . Replacement of these cysteine-serine-glutamate repeats by alanine residues does not similarly affect H2O2 and diamide regulation of yAP-1 function . While yAP-1 transactivation is enhanced by exposure to either H2O2 or diamide, the protein responds to the oxidative stress produced by these compounds in nonidentical ways. Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2266 - 71 UV-induced mutagenesis of human p53 in a vector replicated in Saccharomyces cerevisiae; Moshinsky DJ et al.; Mutation of the p53 tumor suppressor gene is the most common genetic alteration identified to date in human cancers . Similarities of p53 mutations found in human cancers with those induced in experimental systems have been interpreted as evidence supporting a causative role for environmental carcinogens in certain tumor types . We have developed and validated a method for generation of mutation spectra and measurement of mutation frequency directly on human p53 cDNA in a vector following treatment with mutagens and replication in yeast . Mutants that had lost the DNA binding/transcription activation function of p53 were detected by yeast colony color, isolated, and sequenced . UV light was used to characterize and validate the system, and a dose-dependent increase in mutation frequency was seen following exposure of the plasmid to increasing doses of UV, resulting in an 18-fold increase over the spontaneous frequency (3.2 x 10(-4)) at the highest level tested (300 J/m2) . Sequence analysis of p53 in the mutants revealed that the types of mutations induced were similar to those obtained in previous studies of UV mutagenesis in other model systems, and the types and positions of mutations were also similar to those found in human skin tumors . This experimental system will be useful in further evaluation of the importance of environmental agents as risk factors for cancer. Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2186 - 91 Silent information regulator protein complexes in Saccharomyces cerevisiae: a SIR2/SIR4 complex and evidence for a regulatory domain in SIR4 that inhibits its interaction with SIR3; Moazed D et al.; The SIR2, SIR3, and SIR4 silent information regulator proteins are involved in the assembly of silent chromatin domains in the budding yeast Saccharomyces cerevisiae . Using a series of biochemical experiments, we have studied protein-protein interactions involving these proteins . We found that yeast extracts contained a SIR2/SIR4 complex that was associated with little or no SIR3 . However, truncations of the N-terminal two-thirds of the SIR4 protein allowed it to efficiently associate with SIR3, suggesting that the N-terminal domain of SIR4 inhibited its interaction with SIR3 . We propose that the SIR3 and SIR4 proteins interact only during the assembly of the SIR protein complex at the silencer and that an early step in assembly unmasks the SIR4 protein to allow its association with SIR3 . To test whether the interactions observed in yeast extracts were direct, we tested these SIR-SIR interactions using bacterially expressed SIR proteins . We observed direct interactions between SIR4 and SIR2, SIR4 and SIR3, SIR2 and SIR3, SIR2 and SIR2, and SIR4 and SIR4, indicating that the associations observed in yeast extracts were direct. Mol Gen Genet, 1997 Mar 18, 254(1), 63 - 72 Aluminum-sensitive mutants of Saccharomyces cerevisiae; Schott EJ et al.; We are developing budding yeast, Saccharomyces cerevisiae, as a genetic system for the study of tolerance to the trivalent aluminum cation (Al3+) . We have isolated eight mutants that are more sensitive to Al3+ than the wild type . Each mutant represented a different complementation group . A number of the mutants were pleiotropic, and showed defects in other stress responses, changes in tolerance to other metal cations, or abnormal morphology . Two mutants also showed increased dependence on supplemental Mg2+ and Ca2+ . One mutant with a relatively specific sensitivity to Al3+ was chosen for molecular complementation . Normal Al3+ tolerance was restored by expression of the MAP kinase gene SLT2 . Strains carrying deletions of the SLT2 gene, or of the gene for the corresponding MAP kinase kinase SLK1, showed sensitivity to Al3+ . These results indicate that the SLT2 MAP kinase signal transduction pathway is required for yeast to sense and respond to Al3+ stress. Mol Gen Genet, 1997 Mar 18, 254(1), 43 - 53 Inducible nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers in the cell cycle of the budding yeast Saccharomyces cerevisiae: evidence that inducible NER is confined to the G1 phase of the mitotic cell cycle; Scott AD et al.; We previously reported on an inducible component of nucleotide excision repair in Saccharomyces cerevisiae that is controlled by the RAD16 gene . Here we describe a study of this event at the MAT alpha and HML alpha mating-type loci and on the transcribed (TS) and nontranscribed (NTS) strands of the RAD16 gene . Events were examined at various stages of the mitotic cycle in cells synchronised by centrifugal elutriation . Repair of cyclobutane pyrimidine dimers (CPDs) following a single UV dose does not vary significantly in different stages of the mitotic cell cycle . CPDs are removed more rapidly from the transcriptionally active MAT alpha locus than from the silent HML alpha locus, and the TS of RAD16 is repaired faster than the NTS in all stages of the cycle following a single UV irradiation . Enhanced excision of CPDs at MAT alpha and HML alpha can be induced only in the G1 and early S stages of the cell cycle . Here prior irradiation of cells with 25 J/m2 enhances the removal of CPDs following a second UV dose of 70 J/m2 . The level of enhancement of repair does not differ significantly between MAT alpha and HML alpha in G1 . Enhanced removal of CPDs is absent when cells receive the inducing dose in late S or G2/M . Repair of CPDs in both strands of RAD16 is similarly enhanced only if cells receive the initial irradiation in G1 and early S . The level of enhanced removal of CPDs is not significantly different in the TS and NTS of RAD16 either in asynchronous cells or in cells preirradiated in G1 and early S . It has been shown by others that UV-induced expression of RAD16 remains at high levels if cells are held in G1 by treatment with alpha factor . Therefore the increase in RAD16 transcript levels in G1 may be responsible for the ability to enhance NER solely in this stage of the cell cycle. Mol Gen Genet, 1997 Mar 18, 254(1), 21 - 8 A change in sister chromatid behavior precedes nuclear division in Saccharomyces cerevisiae; Burke DJ et al.; We used a genetic assay to monitor the behavior of sister chromatids during the cell cycle . We show that the ability to induce sister chromatid exchanges (SCE) with ionizing radiation is maximal in budded cells with undivided nuclei and then decreases prior to nuclear division . SCE can be induced in cells arrested in G2 using either nocodazole or cdc mutants . These data show that sister chromatids have two different states prior to nuclear division . We suggest that the sister chromatids of cir . III, a circular derivative of chromosome III, separate (anaphase A) prior to spindle elongation (anaphase B) . Other interpretations are also discussed . SCE can be induced in cdc mutants that arrest in G2 and in nocodazole-treated cells, suggesting that mitotic checkpoints arrest cells prior to sister chromatid separation. Gene, 1997 Mar 18, 187(2), 171 - 8 RIC1, a novel gene required for ribosome synthesis in Saccharomyces cerevisiae; Mizuta K et al.; We isolated a temperature-sensitive mutant of Saccharomyces cerevisiae in which transcription both of ribosomal protein genes and of ribosomal RNA is defective at the non-permissive temperature . Temperature-sensitivity for growth is recessive and segregates 2:2 . The wild type gene, termed RIC1 (for ribosome control) was cloned by complementation of the temperature-sensitive phenotype from a genomic DNA library based on the CEN plasmid . RIC1 encodes a protein of 1056 amino acid (aa) residues including a putative nuclear localization sequence . Data base searches revealed that RIC1 is a novel gene and predicted aa sequence share some sequence similarity with viral transcriptional regulatory proteins. EMBO J, 1997 Mar 17, 16(6), 1305 - 17 The CDK-activating kinase CAK1 can dosage suppress sporulation defects of smk1 MAP kinase mutants and is required for spore wall morphogenesis in Saccharomyces cerevisiae; Wagner M et al.; Mitogen-activated protein (MAP) kinase pathways are evolutionarily conserved kinase cascades that are required for the response of eukaryotic cells to a wide variety of environmental stimuli . MAP kinase pathways are also required for the execution of developmental and differentiative programs in a variety of cell and tissue types . SMK1 encodes a developmentally regulated MAP kinase in yeast that is required for spore wall morphogenesis . Cyclin-dependent kinase-activating kinases (CAKs) phosphorylate a conserved threonine residue in the activating loop of cyclin-dependent kinases . CAK1 encodes the major CAK activity in yeast and is required for cell cycle progression . The work presented here demonstrates that CAK1 functions positively in the spore wall morphogenesis pathway . First, CAK1 has been isolated as a dosage suppressor of a conditional smk1 mutant that is defective for spore wall morphogenesis . Second, CAK1 mRNA accumulates during spore development contemporaneously with SMK1 mRNA . Third, cak1 mutant strains have been isolated that are able to complete meiosis I and II but are specifically defective in assembly of the spore wall . These results show that cell cycle progression and morphogenetic pathways can be regulated by a single gene product and suggest mechanisms for coordinating these processes during development. Biochim Biophys Acta, 1997 Mar 15, 1334(2-3), 233 - 9 Modulation of trehalase activity in Saccharomyces cerevisiae by an intrinsic protein; de Mesquita JF et al.; The regulation of cytosolic trehalase activity in yeast has been described as cycles of activation by phosphorylation by cAMP protein kinase . In this paper, evidence is presented for another regulatory mechanism--the binding of an endogenous inhibitory protein . This negative modulator was isolated during the purification procedure of cytosolic cryptic trehalase from repressed wild-type cells of Saccharomyces cerevisiae . However, in derepressed cells the inhibitor was not found nor was it present in ras2 mutant cells submitted to a heat treatment . The trehalase inhibitory activity proved to be a calmodulin ligand protein and, therefore, involved in the modulation of trehalase activity by Ca2+ ions. Yeast, 1997 Mar 15, 13(3), 267 - 74 Completion of the Saccharomyces cerevisiae genome sequence allows identification of KTR5, KTR6 and KTR7 and definition of the nine-membered KRE2/MNT1 mannosyltransferase gene family in this organism; Lussier M et al.; The KRE2/MNT1 mannosyltransferase gene family of Saccharomyces cerevisiae currently consists of the KRE2, YUR1, KTR1, KTR2, KTR3 and KTR4 genes . All six encode putative type II membrane proteins with a short cytoplasmic N-terminus, a membrane-spanning region and a highly conserved catalytic lumenal domain . Here we report the identification of the three remaining members of this family in the yeast genome . KTR5 corresponds to an open reading frame (ORF) of the left arm of chromosome XIV, and KTR6 and KTR7 to ORFs on the left arms of chromosomes XVI and IX respectively . The KTR5, KTR6 and KTR7 gene products are highly similar to the Kre2p/Mnt1p family members . Initial functional characterization revealed that some mutant yeast strains containing null copies of these genes displayed cell wall phenotypes . None was K1 killer toxin resistant but ktr6 and ktr7 null mutants were found to be hypersensitive and resistant, respectively, to the drug Calcofluor White. Yeast, 1997 Mar 15, 13(3), 261 - 6 The DNA sequence of cosmid 14-13b from chromosome XIV of Saccharomyces cerevisiae reveals an unusually high number of overlapping open reading frames; De Antoni A et al.; This work is part of the effort for sequencing chromosome XIV of Saccharomyces cerevisiae . Cosmid 14-13b contains a 37.8 kb insert derived from a partial Sau3A digestion of the genome, cloned into the BamHI site of the vector Pou6 . The strategy used for sequencing is based on the fragmentation of the whole cosmid by sonication, followed by shotgun sequencing on an Applied Biosystem DNA sequencer . The clones with inserts corresponding to the vector were identified by dot-blot hybridization, without the need of sequencing . The analysis of the DNA sequence reveals 29 open reading frames (ORFs) longer than 300 bases . Nine ORFs are internal to some other ORFs . Similarity searches against DNA and protein data banks show that six ORFs correspond to already known yeast genes (OMP1, PSU1, MLS1, RPC19, DBP2, CYB5) and one ORF matches the sequence of a putative yeast gene (ESBP6). Yeast, 1997 Mar 15, 13(3), 251 - 9 Sequence analysis of a near-subtelomeric 35.4 kb DNA segment on the right arm of chromosome VII from Saccharomyces cerevisiae carrying the MAL1 locus reveals 15 complete open reading frames, including ZUO1, BGL2 and BIO2 genes and an ABC transporter gene; Volckaert G et al.; The nucleotide sequence of 35,400 bp at approximately 10 kb from the right telomere of chromosome VII was determined . The segment contains the MAL1 locus, one of the five unlinked loci sufficient for maltose utilization . Until now, each of these loci was considered to contain three genes (for regulator, permease and alpha-glucosidase), but a fourth gene, presumably an extra alpha-glucosidase gene, was found at MAL1 adjacent to the usual cluster of three genes . The two glucosidase genes are present in opposite orientation, forming an inverted repeat structure . In addition to the four genes at MAL1, there are 11 complete, non-overlapping open reading frames (ORFs) longer than 300 bp in the sequence presented here . A new ABC transporter gene (YGR281w), required for oligomycin resistance was found (YOR1; Katzman et al., 1995), and the previously sequenced BGL2 (YGR282c), ZUO1 (YGR285c) and BIO2 (YGR286c) genes were located . The sequence of BIO2, a biotin synthetase gene, required substantial correction and the size of Bio2p is 375, rather than 356, amino acids . Two ORFs show rather weak similarities to animal genes: YGR278w to an unknown ORF of Caenorhabditis elegans and YGR284c to the murine Surf-4, a member of a cluster of at least four housekeeping genes . The remaining five ORFs do not encode known functions, but three of these show weak to high similarities to other ORFs in the Saccharomyces cerevisiae genome and one (YGR280c) codes for a particularly lysine-rich protein. Yeast, 1997 Mar 15, 13(3), 241 - 50 Sequence analysis of a 37.6 kbp cosmid clone from the right arm of Saccharomyces cerevisiae chromosome XII, carrying YAP3, HOG1, SNR6, tRNA-Arg3 and 23 new open reading frames, among which several homologies to proteins involved in cell division control and to mammalian growth factors and other animal proteins are found; Verhasselt P et al.; The nucleotide sequence of 37,639 bp of the right arm of chromosome XII has been determined . Twenty-five open reading frames (ORFs) longer than 300 bp were detected, two of which extend into the flanking cosmids . Only two (L2931 and L2961) of the 25 ORFs correspond to previously sequenced genes (HOG1 and YAP3, respectively) . Another ORF is distinct from YAP3 but shows pronounced similarity to it . About half of the remaining ORFs show similarity to other genes or display characteristic protein signatures . In particular, ORF L2952 has striking homology with the probable cell cycle control protein crn of Drosophila melanogaster . L2949 has significant similarity to the human ZFM1 (related to a potential suppressor oncogene) and mouse CW17R genes, though it lacks the carboxy-terminal oligoproline and oligoglutamine stretches encoded by these mammalian genes . The small ORF L2922 is similar to part of the much larger yeast flocculation gene FLO1 . Other sequences found in the 37639 bp fragment are one delta and one solo-sigma element, the tRNA-Arg3 gene, the small nuclear RNA gene SNR6 and three ARS consensus sequences. Yeast, 1997 Mar 15, 13(3), 199 - 213 Multiple copies of PBS2, MHP1 or LRE1 produce glucanase resistance and other cell wall effects in Saccharomyces cerevisiae; Lai MH et al.; Five sequences were isolated by selection for multiple copy plasmids that conferred resistance to laminarinase, an enzyme that specifically degrades cell wall beta(1-3) glucan linkages . Strains carrying three of these plasmids showed alterations in cell wall glucan labelling . One of these plasmids carried PBS2, a previously identified, non-essential gene which produces a variety of phenotypes and encodes a mitogen-activated protein kinase kinase analogue (Boguslawski and Polazzi, 1987) . Cells carrying PBS2 at multiple copy show a small decrease in cell wall beta(1-6) glucans . Measurements of beta(1-3) glucan synthase activity in multi-copy PBS2 cells showed an approximate 30-45% increase in enzyme specific activity while a pbs2 delta disruption strain showed a decrease in glucan synthase activity of approximately 45% relative to control . A pbs2 delta disruption strain was laminarinase super-sensitive and supersensitive to K1 killer toxin while a strain carrying PBS2 at multiple copy was resistant to killer toxin . A second plasmid carried a portion of the MHP1 gene which has been reported to encode a microtubule-interacting protein (Irminger-Finger et al., 1996) . The MHP1 gene product is a predicted 1398 amino acid protein and only approximately 80% of the amino portion of this protein is required for laminarinase resistance . Cells carrying the amino portion of MHP1 at multiple copy show a decrease in high molecular weight cell wall beta(1-6) glucans and were killer toxin resistant while a disruption strain was viable and killer toxin super-sensitive . Cells carrying this plasmid showed decreased levels of high molecular weight beta(1-6) glucans and increased glucan synthase activity . The laminarinase resistance conferred by the third plasmid mapped to the previously uncharacterized YCL051W open reading frame and this gene was therefore named LRE1 (laminarinase resistance) . The LRE1 gene encodes a non-essential 604 amino acid hydrophilic protein . Unexpectedly, cells carrying LRE1 at multiple copy show no alteration in cell wall glucans or glucan synthase activity . Subcloning experiments demonstrated that the production of these cell wall effects requires the presence of both LRE1 and YCL052C (PBN1), a second open reading frame present on the original plasmid . Cells carrying multiple copies of PBN1 alone show no significant alterations in cell wall glucans or glucan synthase activity, indicating that these effects require the presence of multiple copies of both genes. Genes Dev, 1997 Mar 15, 11(6), 748 - 60 A novel Rap1p-interacting factor, Rif2p, cooperates with Rif1p to regulate telomere length in Saccharomyces cerevisiae; Wotton D et al.; The Saccharomyces cerevisiae Rap1 protein binds with high affinity to sites within the poly(C(1-3)A) tracts at telomeres, where it plays a role in both telomere length regulation and the initiation of telomeric silencing . Rap1p initiates silencing at telomeres by interacting through its carboxy-terminal domain with Sir3p and Sir4p, both of which are required for repression . This same domain of Rap1p also negatively regulates telomere elongation, through an unknown mechanism . We have identified a new Rap1-interacting factor (Rif2p) that plays a role in telomere length regulation . Rif2p has considerable functional similarities with a Rap1p-interacting factor (Rif1p) identified previously . Mutations in RIF1 or RIF2 (unlike mutations in the silencing genes SIR3 and SIR4) result in moderate telomere elongation and improved telomeric silencing . However, deletion of both RIF1 and RIF2 in the same cell results in a dramatic increase in telomere length, similar to that seen with a carboxy-terminal truncation of Rap1p . In addition, overexpression of either RIF1 or RIF2 decreases telomere length, and co-overexpression of these proteins can reverse the telomere elongation effect of overexpression of the Rap1p carboxyl terminus . Finally, we show that Rif1p and Rif2p can interact with each other in vivo . These results suggest that telomere length regulation is mediated by a protein complex consisting of Rif1p and Rif2p, each of which has distinct regulatory functions . One role of Rap1p in telomere length regulation is to recruit these proteins to the telomeres. Arch Biochem Biophys, 1997 Mar 15, 339(2), 344 - 52 Critical roles for arginine 1061/1060 and tyrosine 1057 in Saccharomyces cerevisiae arginine-specific carbamoyl-phosphate synthetase; Lim AL et al.; Carbamoyl-phosphate synthetases (CPSases) bind two molecules of ATP at two internally duplicated domains . Previous affinity labeling studies with the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA; Kim, H., Kelly, R . E., and Evans, D . R . (1991) Biochemistry 30, 10322-10329; Potter, M . D., and Powers-Lee, S . G . (1992) J . Biol . Chem . 267, 2023-2031) have identified several peptides as being near the ATP sites, with most of the FSBA-labeled peptides localized to the internally duplicated domains . However, two of the FSBA-labeled peptides were localized to the third domain of CPSase, an autonomously folded but flexible domain at the extreme C-terminus of the protein . These findings suggested that the C-terminal domain is also involved in interaction with both molecules of ATP and that it might serve to complement the ATP binding sites on the duplicated domains by participating in catalytic processing of the ATP molecules . To further define the role of the C-terminal domain in ATP utilization, we have now carried out site-directed mutagenic analysis of peptide 1052-1061 of the Saccharomyces cerevisiae arginine-specific CPSase . Aspartate residues at positions 1053, 1054, and 1056 did not appear to play a significant role in CPSase structure or function . However, tyrosine 1057 was critical for CPSase structure and the presence of one of the tandem arginyl residues at positions 1061 and 1060 was critical for CPSase catalytic function. J Biol Chem, 1997 Mar 14, 272(11), 7106 - 13 Identification of cDNA clones for the large subunit of eukaryotic translation initiation factor 3 . Comparison of homologues from human, Nicotiana tabacum, Caenorhabditis elegans, and Saccharomyces cerevisiae; Johnson KR et al.; Initiation of translation in eukaryotes is mediated by a set of initiation factors . Mammalian initiation factor 3 is composed of at least 8 subunits, with the largest being about 180 kDa in size . Here we report the cloning of the p180 subunit of human eukaryotic translation initiation factor (eIF) 3 . The amino acid sequence deduced from the cDNA agrees with the sequences of CNBr fragments of eIF-3, confirming the identity of the clone . The 1382 amino acid open reading frame contains a high percentage of charged residues (48%) and an unusual repetitive domain near the carboxyl terminus composed of 25 repeats of 10 amino acids each . Data base searches identified related sequences found in members of the plant and fungal kingdoms as well as in other mammals and the nematode Caenorhabditis elegans . These sequences share significant identity with the human clone and probably represent the homologues of the p180 subunit in these organisms . This is the first report identifying the sequence of the large subunit of eIF-3. Mutat Res, 1997 Mar 12, 383(2), 125 - 35 Genomic characterization of the mouse homolog of the Saccharomyces cerevisiae recombination and double-strand break repair gene RAD52; van den Ouweland J et al.; The yeast Saccharomyces cerevisiae RAD52 gene is involved in recombination and DNA double-strand break repair . Recently, mouse and human homologs of the yeast RAD52 gene have been identified . Here we present the genomic organization of the mouse RAD52 gene . It consists of 12 exons ranging in size from 67 to 374 bp spread over a region of approximately 18 kb . The first ATG is located in exon 2 . Analysis of the promoter region revealed no classical promoter elements such as CCAAT or TATA boxes . Transcriptional mapping analysis revealed one major transcription start point . Analogous to the situation in yeast, transcription of the RAD52 gene in human skin fibroblasts and mouse Ltk- cells was not induced by methyl methanesulfonate treatment . Furthermore, no specific alteration in human RAD52 expression levels throughout the cell cycle was observed. Biochim Biophys Acta, 1997 Mar 10, 1345(1), 71 - 6 Requirement of heme to replace the sparking sterol function in the yeast Saccharomyces cerevisiae; Smith SJ et al.; At least four distinctive sterol functions have been defined in the yeast Saccharomyces cerevisiae . One of these functions, identified as sparking, has the lowest quantitative requirement for sterol, but has the greatest structural specificity . Based on studies utilizing a yeast strain auxotrophic for both heme and sterol biosynthesis, it had been reported that a delta 5-sterol was essential for the growth of the organism . We demonstrate here that heme, and not a heme precursor, can replace the delta 5-sparking sterol requirement of heme auxotrophic strains of yeast. Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1721 - 6 Galactose-dependent reversible interaction of Gal3p with Gal80p in the induction pathway of Gal4p-activated genes of Saccharomyces cerevisiae; Yano K et al.; When galactose is added to logarithmically growing culture of the yeast Saccharomyces cerevisiae, a set of genes encoding galactose-metabolizing enzymes (GAL genes) starts to be transcribed within a few minutes . This rapid induction involves a serial interplay of Gal3p, Gal80p, and Gal4p . Recent experiments have indicated that a direct interaction between Gal3p and Gal80p plays a pivotal role in an early step of GAL induction . Here we demonstrate that complex of Gal3p and Gal80p, otherwise unstable, is stabilized in the presence of 0.1 mM galactose and 0.5 mM ATP . The requirement for galactose and ATP for stable complex formation is also observed by using highly purified Gal3p and Gal80p from yeast . We further show that thus formed Gal3p/Gal80p complex can easily be dissociated when it is washed with buffer lacking galactose . Finally, we show that mutant proteins encoded by GAL80S or GAL80DE21, which confer galactose-uninducible phenotype, fail to interact with Gal3p . These results strongly suggest that Gal3p functions as the sensor and transducer of galactose signal in the induction pathway of Gal4p-activated genes. EMBO J, 1997 Mar 3, 16(5), 1035 - 44 The Skn7 response regulator controls gene expression in the oxidative stress response of the budding yeast Saccharomyces cerevisiae; Morgan BA et al.; Deletion of the bacterial two-component response regulator homologue Skn7 results in sensitivity of yeast to oxidizing agents indicating that Skn7 is involved in the response to this type of stress . Here we demonstrate that following oxidative stress, Skn7 regulates the induction of two genes: TRX2, encoding thioredoxin, and a gene encoding thioredoxin reductase . TRX2 is already known to be induced by oxidative stress dependent on the Yap1 protein, an AP1-like transcription factor responsible for the induction of gene expression in response to various stresses . The thioredoxin reductase gene has not previously been shown to be activated by oxidative stress and, significantly, we find that it too is regulated by Yap1 . The control of at least TRX2 by Skn7 is a direct mechanism as Skn7 binds to the TRX2 gene promoter in vitro . This shows Skn7 to be a transcription factor, at present the only such eukaryotic two-component signalling protein . Our data further suggest that Skn7 and Yap1 co-operate on the TRX2 promoter, to induce transcription in response to oxidative stress. Int J Biochem Cell Biol, 1997 Mar, 29(3), 505 - 12 RAS2/PKA pathway activity is involved in the nitrogen regulation of L-leucine uptake in Saccharomyces cerevisiae; Saenz DA et al.; The aim of the present work is to study the participation of RAS2/PKA signal pathway in the nitrogen regulation of L-leucine transport in yeast cells . The study was performed on Saccharomyces cerevisiae isogenic strains with the normal RAS2 gene, the RAS2val19 mutant and the disrupted ras2::LEU2 . These strains bring about different activities of the RAS2/PKA signal pathway, L-(14C)-Amino acid uptake measurements were determined in cells grown in a rich YPD medium with a mixed nitrogen source or in minimal media containing NH4+ or L-proline as the sole nitrogen source . We report herein that in all strains used, even in those grown in a minimal proline medium, the activity of the general amino acid permease (GAP1) was not detected . L-Leucine uptake in these strains is mediated by two kinetically characterized transport systems . Their KT values are of the same order as those of S1 and S2 L-leucine permeases . Mutation in the RAS2 gene alters initial velocities and Jmax values in both high and low affinity L-leucine transport systems . Activation of the RAS2/PKA signalling pathway by the RAS2val19 mutation, blocks the response to a poor nitrogen source whereas inactivation of RAS2 by gene disruption, results in an increase of the same response. J Biochem (Tokyo), 1997 Mar, 121(3), 578 - 84 Saccharomyces cerevisiae KAR2 (BiP) gene expression is induced by loss of cytosolic HSP70/Ssa1p through a heat shock element-mediated pathway; Oka M et al.; A family of highly-conserved 70 kDa stress proteins is localized in various intracellular compartments of Saccharomyces cerevisiae . Their gene expression is specifically and/or sometimes cooperatively regulated at the transcriptional level by cis-acting elements found in their respective promoters . Here, we find that depletion of cytosolic Ssa1p induced BiP(Kar2p) in the endoplasmic reticulum at the transcriptional level . By analyzing internal deletion mutants of the KAR2 promoter, we determined that the heat shock element (HSE) is necessary for KAR2 gene induction in response to the depletion of Ssa1p . Furthermore, either the KAR2HSE or SSA1HSE is sufficient for gene activation, as assayed using HSE-CYC1-lacZ fusion reporter plasmids . Finally, temperature-sensitive ssa1 mutants transformed with an HSE-CYC1-lacZ fusion vector exhibited strong induction of beta-galactosidase activity when shifted to a restrictive temperature . These results show that loss of functional Ssa1p from the cytosol up-regulates KAR2 gene expression through an HSE-mediated pathway and also support the idea that SSA1 gene expression is autoregulated. Mol Microbiol, 1997 Mar, 23(6), 1157 - 68 Gzf3p, a fourth GATA factor involved in nitrogen-regulated transcription in Saccharomyces cerevisiae; Soussi-Boudekou S et al.; In Saccharomyces cerevisiae, two positive transcription factors of the GATA family, Gln3p and Nil1p/Gat1p, upregulate the expression of multiple nitrogen pathway genes via upstream 5'-GATA-3' sequences . Another GATA factor, Uga43p/Dal80p, downregulates to varying degrees the expression of some nitrogen-regulated genes . Here, we report the functional analysis of a fourth GATA factor, Gzf3p/Nil2p, whose gene was discovered by systematic sequencing of chromosome X . The Gzf3 protein most closely resembles Uga43p . Similar to Uga43p, Gzf3p has the properties of a negative GATA factor . While Uga43p is active specifically under nitrogen-depression conditions, Gzf3p exerts its negative regulatory function specifically on preferred nitrogen sources: It is involved in nitrogen repression of Nil1p-dependent transcription . At least one positive GATA factor is required for the UGA43 and GZF3 genes to be expressed . The Uga43p factor negatively regulates GZF3 expression and vice versa . In addition, both Uga43p and Gzf3p moderately regulate expression of their own genes . These two proteins seem to be parts of a complex network of GATA factors which probably play a determining role in nitrogen-regulated transcription. Microbiologia, 1997 Mar, 13(1), 71 - 4 Transport of phosphate into vacuoles of Saccharomyces cerevisiae; Kulakovskaya TV et al.; Isolated vacuoles of the yeast Saccharomyces cerevisiae accumulated orthophosphate at pH 6-7 . This accumulation was inhibited by MgATP, and was insensitive to protonophores . Triton X-100 blocked this process . The accumulation increased linear by any phosphate concentrations employed (from 0.2 to 10 mM) . It is proposed that phosphate is transported into yeast vacuoles via a channel transport system independent of the electrochemical proton gradient on the vacuolar membrane. Methods, 1997 Mar, 11(3), 279 - 88 Approaches to the study of Rox1 repression of the hypoxic genes in the yeast Saccharomyces cerevisiae; Zitomer RS et al.; The yeast Saccharomyces cerevisiae is a facultative aerobe that responds to changes in oxygen tension by changing patterns of gene expression . One set of genes that responds to this environmental cue is the hypoxic genes . Oxygen levels are sensed by changes in heme biosynthesis, which controls the transcription of the ROX1 gene, encoding a protein that binds to the regulatory region of each hypoxic gene to repress transcription . Several experimental molecular and genetic approaches are described here to study Rox1 repression . Derepression of the hypoxic genes is rapid, and one model for such a response requires that Rox1 have a short half-life . This was demonstrated to be the case by immunoblotting using a c-myc epitope-tagged protein . Rox1 repression is mediated through the general repressors Ssn6 and Tup1 . To explore possible interactions among these proteins, all three were expressed and partially purified using a baculovirus expression system and histidine-tagged proteins . The effect of Ssn6 and Tup1 on the formation of Rox1-DNA complexes was explored using these purified proteins by both electrophoretic mobility shift and DNase I protection assays . We found that Rox1 DNA-binding activity decayed rapidly and that Ssn6 could stabilize and restore lost activity . Finally, genetic selections are described for the isolation of loss-of-function mutations in Rox1 . Also, schemes are proposed for the reversion of such mutations . These selections have been extended to genetic analyses of the TUP1 and SSN6 genes. FEMS Microbiol Lett, 1997 Mar 1, 148(1), 69 - 74 Delta sequence of Ty1 transposon can initiate transcription of the distal part of the URA2 gene complex in Saccharomyces cerevisiae; Roelants F et al.; Expression of a silent aspartate transcarbamylase (ATCase) domain can occur by insertion of a Tyl retrotransposon within the coding sequence of a mutated ura2 allele . This unusual type of Ty-mediated gene activation is possible as the URA2 gene product is a multifunctional protein containing the carbamoyl phosphate synthetase (CPSase), the ATCase and a cryptic dihydroorotase (DHOase) domain . The region in which transcription of the corresponding allele is initiated was determined by RT-PCR experiments . Expression is initiated by a sequence located in the delta element of the Tyl and not by a sequence of the URA2 gene itself . This situation differs with the Ty-mediated gene activation described thus far, in which the transposon substitutes only the 5' regulatory sequences and in which the normal transcription start point is used . The corresponding protein carries both the DHOase-like domain and the ATCase domain, suggesting that the DHOase-like domain is at least involved in the architecture of the protein and necessary to render the ATCase domain functional. Appl Environ Microbiol, 1997 Mar, 63(3), 910 - 5 Effect of aeration and unsaturated fatty acids on expression of the Saccharomyces cerevisiae alcohol acetyltransferase gene; Fujii T et al.; The reduction of acetate ester synthesis by aeration and the addition of unsaturated fatty acids to the medium has been reported to be the result of the reduction in alcohol acetyltransferase (AATase) activity induced by inhibition of this enzyme . However, regulation of the AATase gene ATF1 has not been reported . In this study, ATF1 gene expression was studied by Northern analysis, and the results showed that the ATF1 gene was repressed both by aeration and by unsaturated fatty acids . The results also showed that the reduction of AATase activity is closely related to the degree of repression of ATF1 mRNA, which suggested that the gene repression is the primary means of reducing AATase activity in vivo . Using the Escherichia coli lacZ gene as a reporter gene, it was shown that a 150-bp fragment of the 5' flanking sequence played a major role in the repression by aeration and unsaturated fatty acid addition. Genetics, 1997 Mar, 145(3), 671 - 84 Dissection of filamentous growth by transposon mutagenesis in Saccharomyces cerevisiae; Mosch HU et al.; Diploid Saccharomyces cerevisiae strains starved for nitrogen undergo a developmental transition from growth as single yeast form (YF) cells to a multicellular form consisting of filaments of pseudohyphal (PH) cells . Filamentous growth is regulated by an evolutionarily conserved signaling pathway that includes the small GTP-binding proteins Ras2p and Cdc42p, the protein kinases Ste20p, Ste11p and Ste7p, and the transcription factor Ste12p . Here, we designed a genetic screen for mutant strains defective for filamentous growth (dfg) to identify novel targets of the filamentation signaling pathway, and we thereby identified 16 different genes, CDC39, STE12, TEC1, WHI3, NAB1, DBR1, CDC55, SRV2, TPM1, SPA2, BNI1, DFG5, DFG9, DFG10, BUD8 and DFG16, mutations that block filamentous growth . Phenotypic analysis of dfg mutant strains genetically dissects filamentous growth into the cellular processes of signal transduction, bud site selection, cell morphogenesis and invasive growth . Epistasis tests between dfg mutant alleles and dominant activated alleles of the RAS2 and STE11 genes, RAS2Val19 and STE11-4, respectively, identify putative targets for the filamentation signaling pathway . Several of the genes described here have homologues in filamentous fungi, where they also regulate fungal development. Genetics, 1997 Mar, 145(3), 661 - 70 Competition between adjacent meiotic recombination hotspots in the yeast Saccharomyces cerevisiae; Fan QQ et al.; In a wild-type strain of Saccharomyces cerevisiae, a hotspot for meiotic recombination is located upstream of the HIS4 gene . An insertion of a 49-bp telomeric sequence into the coding region of HIS4 strongly stimulates meiotic recombination and the local formation of meiosis-specific double-strand DNA breaks (DSBs) . When strains are constructed in which both hotspots are heterozygous, hotspot activity is substantially less when the hotspots are on the same chromosome than when they are on opposite chromosomes. Genetics, 1997 Mar, 145(3), 647 - 59 Progression into the first meiotic division is sensitive to histone H2A-H2B dimer concentration in Saccharomyces cerevisiae; Tsui K et al.; The yeast Saccharomyces cerevisiae contains two genes for histone H2A and two for histone H2B located in two divergently transcribed gene pairs: HTA1-HTB1 and HTA2-HTB2 . Diploid strains lacking HTA1-HTB1 (hta1-htb1 delta/hta1-htb1 delta, HTA2-HTB2/HTA2-HTB2) grow vegetatively, but will not sporulate . This sporulation phenotype results from a partial depletion of H2A-H2B dimers . Since the expression patterns of HTA1-HTB1 and HTA2-HTB2 are similar in mitosis and meiosis, the sporulation pathway is therefore more sensitive than the mitotic cycle to depletion of H2A-H2B dimers . After completing premeiotic DNA replication, commitment to meiotic recombination, and chiasma resolution, the hta1-htb1 delta/hta1-htb1 delta, HTA2-HTB2/HTA2-HTB2 mutant arrests before the first meiotic division . The arrest is not due to any obvious disruptions in spindle pole bodies or microtubules . The meiotic block is not bypassed in backgrounds homozygous for spo13, rad50 delta, or rad9 delta mutations, but is bypassed in the presence of hydroxyurea, a drug known to inhibit DNA chain elongation . We hypothesize that the deposition of H2A-H2B dimers in the mutant is unable to keep pace with the replication fork, thereby leading to a disruption in chromosome structure that interferes with the meiotic divisions. Genetics, 1997 Mar, 145(3), 637 - 45 Saccharomyces cerevisiae HOC1, a suppressor of pkc1, encodes a putative glycosyltransferase; Neiman AM et al.; The Saccharomyces cerevisiae gene PKC1 encodes a protein kinase C isozyme that regulates cell wall synthesis . Here we describe the characterization of HOC1, a gene identified by its ability to suppress the cell lysis phenotype of pkc1-371 cells . The HOC1 gene (Homologous to OCH1) is predicted to encode a type II integral membrane protein that strongly resembles Och 1p, an alpha-1,6-mannosyltransferase . Immunofluorescence studies localized Hoc1p to the Golgi apparatus . While overexpression of HOC1 rescued the pkc1-371 temperature-sensitive cell lysis phenotype, disruption of HOC1 lowered the restrictive temperature of the pkc1-371 allele . Disruption of HOC1 also resulted in hypersensitivity to Calcofluor White and hygromycin B, phenotypes characteristic of defects in cell wall integrity and protein glycosylation, respectively . The function of HOC1 appears to be distinct from that of OCH1 . Taken together, these results suggest that HOC1 encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall. Genetics, 1997 Mar, 145(3), 627 - 35 Molecular characterization of two high affinity sulfate transporters in Saccharomyces cerevisiae; Cherest H et al.; Strains resistant to the toxic analogues of sulfate, selenate and chromate have been isolated . Their genetic analysis allowed us to identify four genes . One, called MET28, encodes a transcriptional factor . The three other genes, called SUL1, SUL2 and SUL3, encode proteins involved in sulfate transport . The sequence of Sul1p and Sul2p indicate that they are integral membrane proteins exhibiting, respectively, 11 and 10 transmembrane domains . Moreover, Sul1p and Sul2p share a high degree of similarity . Sulfate transport kinetic studies made with parental and mutant strains show that, as expected from genetic results, Saccharomyces cerevisiae has two high affinity sulfate transport systems . Sul3p has been shown to be involved in the transcriptional regulation of the SUL2 gene. Genetics, 1997 Mar, 145(3), 615 - 26 Alanine-scanning mutagenesis of protein phosphatase type 1 in the yeast Saccharomyces cerevisiae; Baker SH et al.; Protein phosphatase type 1, encoded by GLC7 in Saccharomyces cerevisiae, is an essential serine/threonine phosphatase implicated in the regulation of a diverse array of physiological functions . We constructed and examined 20 mutant alleles of GLC7 in which codons encoding clusters of charged residues were changed to alanine codons . Three of 20 mutant alleles alter residues in the active site of the phosphatase and are unable to rescue the lethality of a glc7::LEU2 disruption . The 17 alleles that support growth confer a range of mutant traits including cell cycle arrest, 2-deoxyglucose resistance, altered levels of glycogen, sensitivity to high salt, and sporulation defects . For some traits, such as 2-deoxyglucose resistance and cell cycle arrest, the mutated residues map to specific regions of the protein whereas the mutated residues in glycogen-deficient mutants and sporulation-defective mutants are more widely distributed over the protein surface . Many mutants have complex phenotypes, each displaying a diverse range of defects . The wide range of phenotypes identified from the collection of mutant alleles is consistent with the hypothesis that Glc7p-binding proteins, which are thought to regulate the specificity of Glc7p, have overlapping binding sites on the surface of Glc7p . This could account for the high level of sequence conservation found among type 1 protein phosphatases from different species. Genetics, 1997 Mar, 145(3), 595 - 603 MDP1, a Saccharomyces cerevisiae gene involved in mitochondrial/cytoplasmic protein distribution, is identical to the ubiquitin-protein ligase gene RSP5; Zoladek T et al.; Alteration of the subcellular distribution of Mod5p-I, a tRNA modification enzyme, member of the sorting isozyme family, affects tRNA-mediated nonsense suppression . Altered suppression efficiency was used to identify MDP genes, which, when mutant, change the mitochondrial/cytosolic distribution of Mod5p-I,KR6 . MDP2 is the previously identified VRP1, which encodes verprolin, required for proper organization of the actin cytoskeleton . MDP3 is identical to PAN1, which encodes a protein involved in initiation of translation and actin cytoskeleton organization . We report here the cloning and characterization of wild-type and mutant MDP1 alleles and the isolation and characterization of a multicopy suppressor of mdp1 mutations . MDP1 is identical to RSP5, which encodes ubiquitin-protein ligase, and mdp1 mutations are suppressed by high copy expression of ubiquitin . All four characterized mdp1 mutations cause missense changes located in the hect domain of Rsp5p that is highly conserved among ubiquitin-protein ligases . In addition to its well-known function in protein turnover, ubiquitination has been proposed to play roles in subcellular sorting of proteins via endocytosis and in delivery of proteins to peroxisomes, the endoplasmic reticulum and mitochondria . mdp1, as well as mdp2/vrp1 and mdp3/pan1 mutations, affect endocytosis . Further, mdp1 mutations show synthetic interactions with mdp2/vrp1 and mdp3/pan1 . Identification of MDP1 as RSP5, along with our previous identification of MDP2/VRP1 and MDP3/PAN1, implicate interactions of the ubiquitin system, the actin cytoskeleton and protein synthesis in the subcellular distribution of proteins. J Bacteriol, 1997 Mar, 179(5), 1541 - 9 Catabolite inactivation of the galactose transporter in the yeast Saccharomyces cerevisiae: ubiquitination, endocytosis, and degradation in the vacuole; Horak J et al.; When Saccharomyces cerevisiae cells growing on galactose are transferred onto glucose medium containing cycloheximide, an inhibitor of protein synthesis, a rapid reduction of Gal2p-mediated galactose uptake is observed . We show that glucose-induced inactivation of Gal2p is due to its degradation . Stabilization of Gal2p in pra1 mutant cells devoid of vacuolar proteinase activity is observed . Subcellular fractionation and indirect immunofluorescence showed that the Gal2 transporter accumulates in the vacuole of the mutant cells, directly demonstrating that its degradation requires vacuolar proteolysis . In contrast, Gal2p degradation is proteasome independent since its half-life is unaffected in pre1-1 pre2-2, cim3-1, and cim5-1 mutants defective in several subunits of the protease complex . In addition, vacuolar delivery of Gal2p was shown to be blocked in conditional end3 and end4 mutants at the nonpermissive temperature, indicating that delivery of Gal2p to the vacuole occurs via the endocytic pathway . Taken together, the results presented here demonstrate that glucose-induced proteolysis of Gal2p is dependent on endocytosis and vacuolar proteolysis and is independent of the functional proteasome . Moreover, we show that Gal2p is ubiquitinated under conditions of glucose-induced inactivation. J Bacteriol, 1997 Mar, 179(5), 1513 - 20 Suppressor gene analysis reveals an essential role for sphingolipids in transport of glycosylphosphatidylinositol-anchored proteins in Saccharomyces cerevisiae; Skrzypek M et al.; Sphingolipids are normally necessary for growth of Saccharomyces cerevisiae cells, but mutant strains that bypass the need for sphingolipids have been identified . Such bypass mutants fail to grow under stressful conditions, including low pH (pH 4.1), when they lack sphingolipids . To begin to understand why sphingolipids seem to be necessary for coping with low-pH stress, we screened a genomic library and selected a suppressor gene, CWP2 (cell wall protein 2), that when present in multiple copies partially compensates for the lack of sphingolipids and enhances survival at low pH . To explain these results, we present evidence that sphingolipids are required for a normal rate of transport of glycosylphosphatidylinositol (GPI)-anchored proteins, including Cwp2 and Gas1/Gpg1, from the endoplasmic reticulum (ER) to the Golgi apparatus . The effect of sphingolipids is specific for transport of GPI-anchored proteins because no effect on the rate of transport of carboxypeptidase Y, a non-GPI-anchored protein, was observed . Since the Gasl protein accumulated in the ER with a GPI anchor in cells lacking sphingolipids, we conclude that sphingolipids are not necessary for anchor attachment . Therefore, sphingolipids must be necessary for a step in formation of COPII vesicles or for their transport to the Golgi apparatus . Our data identify the Cwp2 protein as a vital component in protecting cells from the stress of low pH. J Bacteriol, 1997 Mar, 179(5), 1469 - 74 Regulation of the proteinase B structural gene PRB1 in Saccharomyces cerevisiae; Naik RR et al.; The expression of PRB1, the gene that encodes the precursor to the soluble vacuolar proteinase B (PrB) in Saccharomyces cerevisiae, is regulated by carbon and nitrogen sources and by growth phase . Little or no PRB1 mRNA is detectable during exponential growth on glucose as the carbon source; it begins to accumulate as cells exhaust the glucose . Previous work has shown that glucose repression of PRB1 transcription is not mediated by HXK2 or by the SNF1, SNF4, and SNF6 genes (C . M . Moehle and E . W . Jones, Genetics 124:39-55, 1990) . We analyzed the effects of mutations in the MIG1, TUP1, and GRR1 genes on glucose repression of PRB1 and found that mutations in each partially alleviate glucose repression . tup1 and mig1 mutants fail to translocate all of the Prb1p into the lumen of the endoplasmic reticulum . A screen for new mutants revealed mutations in MIG1 and REG1, genes already known to regulate glucose repression, as well as in three new genes that we have named PBD1 to PBD3; all cause derepressed expression . Mutations that result in failure to completely derepress PRB1 were also identified in two new genes, named PND1 and PND2 . Good nitrogen sources, like ammonia, repress PRB1 transcription; mutations in URE2 do not affect this response . Derepression upon transfer to a poor nitrogen source is dependent upon GLN3. Mol Cell Biol, 1997 Mar, 17(3), 1354 - 65 Dbp3p, a putative RNA helicase in Saccharomyces cerevisiae, is required for efficient pre-rRNA processing predominantly at site A3; Weaver PL et al.; In Saccharomyces cerevisiae, ribosomal biogenesis takes place primarily in the nucleolus, in which a single 35S precursor rRNA (pre-rRNA) is first transcribed and sequentially processed into 25S, 5.8S, and 18S mature rRNAs, leading to the formation of the 40S and 60S ribosomal subunits . Although many components involved in this process have been identified, our understanding of this important cellular process remains limited . Here we report that one of the evolutionarily conserved DEAD-box protein genes in yeast, DBP3, is required for optimal ribosomal biogenesis . DBP3 encodes a putative RNA helicase, Dbp3p, of 523 amino acids in length, which bears a highly charged amino terminus consisting of 10 tandem lysine-lysine-X repeats ({KKX} repeats) . Disruption of DBP3 is not lethal but yields a slow-growth phenotype . This genetic depletion of Dbp3p results in a deficiency of 60S ribosomal subunits and a delayed synthesis of the mature 25S rRNA, which is caused by a prominent kinetic delay in pre-rRNA processing at site A3 and to a lesser extent at sites A2 and A0 . These data suggest that Dbp3p may directly or indirectly facilitate RNase MRP cleavage at site A3 . The direct involvement of Dbp3p in ribosomal biogenesis is supported by the finding that Dbp3p is localized predominantly in the nucleolus . In addition, we show that the {KKX} repeats are dispensable for Dbp3p's function in ribosomal biogenesis but are required for its proper localization . The {KKX} repeats thus represent a novel signaling motif for nuclear localization and/or retention. Mol Cell Biol, 1997 Mar, 17(3), 1289 - 97 Regulation of the Saccharomyces cerevisiae HOG1 mitogen-activated protein kinase by the PTP2 and PTP3 protein tyrosine phosphatases; Wurgler-Murphy SM et al.; In response to increases in extracellular osmolarity, Saccharomyces cerevisiae activates the HOG1 mitogen-activated protein kinase (MAPK) cascade, which is composed of a pair of redundant MAPK kinase kinases, namely, Ssk2p and Ssk22p, the MAPK kinase Pbs2p, and the MAPK Hog1p . Hog1p is activated by Pbs2p through phosphorylation of specific threonine and tyrosine residues . Activated Hog1p is essential for survival of yeast cells at high osmolarity . However, expression of constitutively active mutant kinases, such as those encoded by SSK2deltaN and PBS2(DD), is toxic and results in a lethal level of Hog1p activation . Overexpression of the protein tyrosine phosphatase Ptp2p suppresses the lethality of these mutations by dephosphorylating Hog1p . A catalytically inactive Cys-to-Ser Ptp2p mutant (Ptp2(C/S)p) is tightly bound to tyrosine-phosphorylated Hog1p in vivo . Disruption of PTP2 leads to elevated levels of tyrosine-phosphorylated Hog1p following exposure of cells to high osmolarity . Disruption of both PTP2 and another protein tyrosine phosphatase gene, PTP3, results in constitutive Hog1p tyrosine phosphorylation even in the absence of increased osmolarity . Thus, Ptp2p and Ptp3p are the major phosphatases responsible for the tyrosine dephosphorylation of Hog1p . When catalytically inactive Hog1(K/N)p is expressed in hog1delta cells, it is constitutively tyrosine phosphorylated . In contrast, Hog1(K/N)p, expressed together with wild-type Hog1p, is tyrosine phosphorylated only when cells are exposed to high osmolarity . Thus, the kinase activity of Hog1p is required for its own tyrosine dephosphorylation . Northern blot analyses suggest that Hog1p regulates Ptp2p and/or Ptp3p activity at the posttranscriptional level. J Biol Chem, 1997 Feb 28, 272(9), 6004 - 10 Ribosome concentration contributes to discrimination against poly(A)- mRNA during translation initiation in Saccharomyces cerevisiae; Proweller A et al.; Inactivation of Saccharomyces cerevisiae poly(A) polymerase in a strain bearing the temperature-sensitive lethal pap1-1 mutation results in the synthesis of poly(A)- mRNAs that initiate translation with surprising efficiency . Translation of poly(A)- mRNAs after polyadenylation shut-off might result from an increase in the ratio of ribosomes and associated translation factors to mRNA, caused by the inability of poly(A)- mRNAs to accumulate to normal levels . To test this hypothesis, we used ribosomal subunit protein gene mutations to decrease either 40 or 60 S ribosomal subunit concentrations in strains carrying the pap1-1 mutation . Polyadenylation shut-off in such cells results in a nearly normal ratio of ribosomes to mRNA as revealed by polyribosome sedimentation analysis . Ribonuclease protection and Northern blot analyses showed that a significant percentage of poly(A)-deficient and poly(A)- mRNA associate with smaller polyribosomes compared with cells with normal ribosome levels . Analysis of the ratio of poly(A)-deficient and poly(A)- forms of a specific mRNA showed relatively more poly(A)- mRNA sedimenting with 20-60 S complexes than do poly(A)+ forms, suggesting a block in an early step of the translation initiation of the poly(A)- transcripts . These findings support models featuring the poly(A) tail as an enhancer of translation and suggest that the full effect of a poly(A) tail on the initiation strength of a mRNA may require competition for a limited number of free ribosomes or translation factors. J Biol Chem, 1997 Feb 28, 272(9), 5757 - 64 On the unique structural organization of the Saccharomyces cerevisiae pyruvate dehydrogenase complex; Stoops JK et al.; Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components attach . We have determined the structures of the truncated 60-mer core dihydrolipoamide acetyltransferase (tE2) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex and complexes of the tE2 core associated with a truncated binding protein (tBP), intact binding protein (BP), and the BP associated with its dihydrolipoamide dehydrogenase (BP.E3) . The tE2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges . Previous studies have given rise to the generally accepted belief that the other components are bound on the outside of the E2 scaffold . However, this investigation shows that the 12 large openings in the tE2 core permit the entrance of tBP, BP, and BP.E3 into a large central cavity where the BP component apparently binds near the tip of the tE2 trimer . The bone-shaped E3 molecule is anchored inside the central cavity through its interaction with BP . One end of E3 has its catalytic site within the surface of the scaffold for interaction with other external catalytic domains . Though tE2 has 60 potential binding sites, it binds only about 30 copies of tBP, 15 of BP, and 12 of BP.E3 . Thus, E2 is unusual in that the stoichiometry and arrangement of the tBP, BP, and E3.BP components are determined by the geometric constraints of the underlying scaffold. J Biol Chem, 1997 Feb 28, 272(9), 5544 - 54 Metabolic and regulatory changes associated with growth of Saccharomyces cerevisiae in 1.4 M NaCl . Evidence for osmotic induction of glycerol dissimilation via the dihydroxyacetone pathway; Norbeck J et al.; The salt-instigated protein expression of Saccharomyces cerevisiae during growth in either 0.7 or 1.4 M NaCl was studied by two-dimensional polyacrylamide gel electrophoresis . The 73 protein spots that were identified as more than 3-fold responsive in 1.4 M NaCl were further grouped by response class (halometric, low-salt, and high-salt regulation) . Roughly 40% of these responsive proteins were found to decrease in expression, while at higher magnitudes of change (>8-fold) only induction was recorded . Enolase 1 (Eno1p) was the most increasing protein by absolute numbers per cell, but not by -fold change, and the enzymes involved in glycerol synthesis, Gpd1p and Gpp2p, were also induced to a similar degree as Eno1p . We furthermore present evidence for salt induction of glycerol dissimilation via dihydroxyacetone and also identify genes putatively encoding the two enzymes involved; dihydroxyacetone kinase (DAK1 and DAK2) and glycerol dehydrogenase (YPR1 and GCY1) . The GPD1, GPP2, GCY1, DAK1, and ENO1 genes all displayed a halometric increase in the amount of transcript . This increase was closely linked to the salt-induced rate of protein synthesis of the corresponding proteins, indicating mainly transcriptional regulation of expression for these genes . A consensus element with homology to the URS sequence of the ENO1 promoter was found in the promoters of the GPD1, GPP2, GCY1, and DAK1 genes. FEBS Lett, 1997 Feb 24, 403(3), 303 - 8 At-GDI1 from Arabidopsis thaliana encodes a rab-specific GDP dissociation inhibitor that complements the sec19 mutation of Saccharomyces cerevisiae; Zarsky V et al.; Rab GTPases play a central role in the control of vesicular membrane traffic . These proteins cycle between cytosolic and membrane-bound compartments in a guanine nucleotide-dependent manner, a process that is regulated by several accessory proteins . Of particular interest are the Rab guanosine nucleotide diphosphate dissociation inhibitor proteins (Rab-GDI) which bind to prenylated Rab GTPases, slow the rate of GDP dissociation and escort GDP bound Rab proteins to their target membranes and retrieve them after completion of their catalytic cycle . We have cloned from Arabidopsis thaliana a cDNA coding for the Rab guanosine diphosphate dissociation inhibitor (AtGDI1) by functional complementation of the Saccharomyces cerevisiae sec19-1 mutant . The Arabidopsis cDNA potentially encodes a 49850 Da protein which is homologous to yeast GDI (49%) and to other members of the Rab-GDI family (49-63%) . Northern blot analysis indicates that the mRNA is expressed in all tissues examined . The existence of a plant homologue of the Rab-GDI family indicates that the basic vesicle traffic control machinery may be highly conserved in plants as it is in yeast and mammals. Biochem Biophys Res Commun, 1997 Feb 24, 231(3), 535 - 9 A carboxy-terminal pro-sequence of aqualysin I prevents proper folding of the protease domain on its secretion by Saccharomyces cerevisiae; Kim DW et al.; The precursor of aqualysin I, a subtilisin-type protease secreted by Thermus aquaticus, consists of four domains, an N-terminal signal sequence, an N-terminal pro-sequence, a protease domain, and a C-terminal pro-sequence . A non-covalent N-terminal pro-sequence facilitates the production of active aqualsin I, when the C-terminal pro-sequence is deleted . The role of the C-terminal pro-sequence in protein secretion was analyzed using a Saccharomyces cerevisiae expression system . Deletion of the C-terminal pro-sequence resulted in increased secretion of aqualysin I, i.e., about three times as much as that in the case of the wild type . In the case of the wild-type precursor, non-secreted aqualysin I with the C-terminal pro-sequence was retained in the endoplasmic reticulum in an inactive form, suggesting that the C-terminal pro-sequence prevents the protease domain from taking on a properly folded structure, unlike the N-terminal pro-sequence. Biochim Biophys Acta, 1997 Feb 21, 1324(1), 111 - 9 Characterization of rat Glut4 glucose transporter expressed in the yeast Saccharomyces cerevisiae: comparison with Glut1 glucose transporter; Kasahara T et al.; Rat Glut4 glucose transporter was expressed in the yeast Saccharomyces cerevisiae, but was retained in an intracellular membranous compartment and did not contribute to glucose uptake by intact cells . A crude membrane fraction was prepared and reconstituted in liposome with the use of the freeze-thaw/sonication method . D-glucose-specific, cytochalasin B inhibitable glucose transport activity was observed . Kinetic analysis of D-glucose transport was performed by an integrated rate equation approach . The K(m) under zero-trans influx condition was 12 +/- 1 mM (mean +/- S.E., n = 3) and that under equilibrium exchange condition was 22 +/- 3 mM (n = 4) . D-glucose transport was inhibited by 2-deoxy-D-glucose or 3-O-methyl-D-glucose, but not by D-allose, D-fructose or L-glucose . Cytochalasin B, phloretin and phlorizin inhibited D-glucose transport, but neither p-chloromercuribenzoic acid (pCMB) (0-0.1 mM) nor p-chloromercuribenzene sulfonic acid (pCMBS) (0-1.0 mM) inhibited this activity . High concentrations of HgCl2 were required to inhibit D-glucose transport (IC50, 370 microM) . Comparing these properties to those of rat Glut1 we found two notable differences; (1) in Glut1, K(m) under zero-trans influx was significantly smaller than that under equilibrium exchange but in Glut4 less than two-fold difference was seen between these two K(m) values; and (2) Glut1 was inhibited with pCMB, pCMBS and low concentrations of HgCl2 (IC50, 3.5 microM), whereas Glut4 was almost insensitive to SH reagents . To examine the role of the exofacial cysteine, we replaced Met-455 of Glut4 (corresponding to Cys-429 of Glut1) with cysteine . The mutated Glut4 was inhibited by pCMB or pCMBS and the IC50 of HgCl2 decreased to 47 microM, whereas K(m), substrate specificity and the sensitivity to cytochalasin B were not significantly changed, indicating that the existence of exofacial cysteine contributed only to increase SH sensitivity in Glut4. J Biol Chem, 1997 Feb 21, 272(8), 4795 - 803 VMA11 and VMA16 encode second and third proteolipid subunits of the Saccharomyces cerevisiae vacuolar membrane H+-ATPase; Hirata R et al.; The vacuolar membrane H+-ATPase (V-ATPase) of the yeast Saccharomyces cerevisiae is composed of peripheral catalytic (V1) and integral membrane (V0) domains . The 17-kDa proteolipid subunit (VMA3 gene product; Vma3p) is predicted to constitute at least part of the proton translocating pore of V0 . Recently, two VMA3 homologues, VMA11 and VMA16 (PPA1), have been identified in yeast, and VMA11 has been shown to be required for the V-ATPase activity . Cells disrupted for the VMA16 gene displayed the same phenotypes as those lacking either Vma3p or Vma11p; the mutant cells lost V-ATPase activity and failed to assemble V-ATPase subunits onto the vacuolar membrane . Epitope-tagged Vma11p and Vma16p were detected on the vacuolar membrane by immunofluorescence microscopy . Density gradient fractionation of the solubilized vacuolar proteins demonstrated that the tagged proteins copurified with the V-ATPase complex . We conclude that Vma11p and Vma16p are essential subunits of the V-ATPase . Vma3p contains a conserved glutamic acid residue (Glu137) whose carboxyl side chain is predicted to be important for proton transport activity . Mutational analysis of Vma11p and Vma16p revealed that both proteins contain a glutamic acid residue (Vma11p Glu145 and Vma16p Glu108) functionally similar to Vma3p Glu137 . These residues could only be functionally substituted by an aspartic acid residue, because other mutations we examined inactivated the enzyme activity . Assembly and vacuolar targeting of the enzyme complex was not inhibited by these mutations . These results suggest that the three proteolipid subunits have similar but not redundant functions, each of which is most likely involved in proton transport activity of the enzyme complex . Yeast cells contain V0 and V1 subcomplexes in the vacuolar membrane and in the cytosol, respectively, that can be assembled into the active V0V1 complex in vivo . Surprisingly, loss-of-function mutations of either Vma11p Glu145 or Vma16p Glu108 resulted in a higher degree of assembly of the V1 subunits onto the V0 subcomplex in the vacuolar membrane. Mol Gen Genet, 1997 Feb 20, 253(5), 568 - 80 Integration of the multiple controls regulating the expression of the arginase gene CAR1 of Saccharomyces cerevisiae in response to differentnitrogen signals: role of Gln3p, ArgRp-Mcm1p, and Ume6p; Dubois E et al.; Expression of the catabolic gene encoding arginase in Saccharomyces cerevisiae, CAR1, is controlled by multiple nitrogen signals, such as the presence of the inducer, arginine, and the nature and amount of the nitrogen source . The present study has determined or confirmed the identity of the proteins involved in these different controls, as well as their targets in the CAR1 promoter . We show that Gln3p activates CAR1 expression through the GATAA sequences in the absence of an optimal nitrogen source, such as ammonia, glutamine or asparagine . Ume6p, which also controls the expression of early meiotic genes, represses CAR1 expression through a sequence called URS, as a function of nitrogen availability . Thus, the responses to the quality of the nitrogen source and to nitrogen starvation are achieved through different cis- and trans-regulatory elements . At least one of the multiple Rap1p and Abf1p binding sites is required for the basal transcription of the gene . The UAS(arg), containing the previously defined "arginine boxes" is the region that responds to the inducer through the action of the ArgRp-Mcm1p proteins, and its deletion alone significantly affects growth on arginine as sole nitrogen source . The functional UAS(arg) is about 60 nucleotides long, and contains two sequences homologous to the binding site for MADS-box proteins, to which ArgRIp and Mcm1p belong . No obvious palindromic sequence similar to the binding site of Gal4p, Ppr1p or Put3p is present in the UAS(arg), although ArgRIIp contains a Zn(II)2Cys6 motif . Interestingly, we have found that induction of CAR1 expression by arginine in the presence of an optimal nitrogen source is counteracted by Gln3p, independently of its action at the GATAA sequences. Gene, 1997 Feb 20, 186(1), 13 - 20 Cloning, and molecular characterization of the GCV1 gene encoding the glycine cleavage T-protein from Saccharomyces cerevisiae; McNeil JB et al.; We have isolated the gene encoding the glycine cleavage T-protein (GCV1) of the yeast Saccharomyces cerevisiae and shown through gene disruption and enzyme assays that inactivation of GCV1 destroys glycine cleavage function . A DNA fragment encoding the GCV1 gene was cloned by PCR amplification using degenerate oligodeoxyribonucleotides, and the cloned fragment was used as a probe to isolate the complete gene from a yeast genomic library . Growth with glycine stimulated expression of the GCV1 gene as determined by Northern analysis and increased the beta-galactosidase activity of a GCV1-lacZ fusion 30-fold . The URA3 gene was inserted into the coding sequence of GCV1 and the resulting construct was used to disrupt the chromosomal GCV1 gene in a diploid strain of yeast . gcv1::URA3 haploid derivatives grew normally or only slightly more slowly than the isogenic wild-type haploids . All gcv1 strains studied were unable to grow on glycine as a sole nitrogen source and lacked glycine cleavage enzyme activity . Growth of shm1 shm2 mutants was stimulated by glycine, whereas glycine could not supplement the growth of the isogenic gcv1 strain. EMBO J, 1997 Feb 17, 16(4), 779 - 92 The role of branchpoint-3' splice site spacing and interaction between intron terminal nucleotides in 3' splice site selection in Saccharomyces cerevisiae; Luukkonen BG et al.; A conserved 3' splice site YAG is essential for the second step of pre-mRNA splicing but no trans-acting factor recognizing this sequence has been found . A direct, non-Watson-Crick interaction between the intron terminal nucleotides was suggested to affect YAG selection . The mechanism of YAG recognition was proposed to involve 5' to 3' scanning originating from the branchpoint or the polypyrimidine tract . We have constructed a yeast intron harbouring two closely spaced 3' splice sites . Preferential selection of a wild-type site over mutant ones indicated that the two sites are competing . For two identical sequences, the proximal site is selected . As previously observed, an A at the first intron nucleotide spliced most efficiently with a 3' splice site UAC . In this context, UAA or UAU were also more efficient 3' splice sites than UAG and competed more efficiently than the wild-type sequence with a 3' splice site UAC . We observed that a U at the first intron nucleotide is used for splicing in combination with 3' splice sites UAG, UAA or UAU . Our data indicate that the 3' splice site is not primarily selected through an interaction with the first intron nucleotide . Selection of the 3' splice site depends critically on its distance from the branchpoint but does not occur by a simple leaky scanning mechanism. FEBS Lett, 1997 Feb 17, 403(2), 173 - 80 Osmoregulation and protein expression in a pbs2delta mutant of Saccharomyces cerevisiae during adaptation to hypersaline stress; Akhtar N et al.; We deleted the PBS2 gene encoding the MAP kinase activator of the osmosignaling HOG pathway in Saccharomyces cerevisiae and examined the effects on the kinetics of the osmoregulatory glycerol response and protein induction during adaptation to 0.7 M NaCl . Changes in protein expression as analyzed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) demonstrated that for the 29 proteins showing a 6-fold induction in wild-type cells during adaptation to NaCl stress, all displayed a decreased and delayed response in pbs2delta cells . Of the seven proteins that were identified, two were previously not known to be under HOG pathway control: Ald6p, an isoform of aldehyde dehydrogenase and Dak1p, a putative dihydroxyacetone kinase . The presence of a remaining significant induction in pbs2delta cells for about half of the examined proteins indicates existence of alternative osmosignaling pathway(s) . Northern analysis of the salt induced transcription of GPD1 and GPP2, encoding the cytosolic glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase involved in the osmostress induced glycerol production, demonstrated an about 20-fold PBS2-dependent transient activation, in agreement with the previously reported transient nature of the signal transduced by the HOG pathway. Genes Dev, 1997 Feb 15, 11(4), 512 - 27 Stn1, a new Saccharomyces cerevisiae protein, is implicated in telomere size regulation in association with Cdc13; Grandin N et al.; We have isolated STN1, an essential Saccharomyces cerevisiae gene, as a suppressor of the cdc13-1 mutation . A synthetic lethal interaction between a temperature-sensitive mutant allele of STN1, stn1-13, and cdc13-1 was observed . Stn1 and Cdc13 proteins displayed a physical interaction by two-hybrid analysis . As shown previously for cdc13-1, stn1-13 cells at the restrictive temperature accumulate single-stranded DNA in subtelomeric regions of the chromosomes, but to a lesser extent than cdc13-1 cells . In addition, both Cdc13 and Stn1 were found to be involved in the regulation of telomere length, mutations in STN1 or CDC13 conferring an increase in telomere size . Loss of Stn1 function activated the RAD9 and MEC3 G2/M checkpoints, therefore confirming that DNA damage is generated . We propose that Stn1 functions in telomere metabolism during late S phase in cooperation with Cdc13. Nucleic Acids Res, 1997 Feb 15, 25(4), 743 - 9 The RAD5 gene product is involved in the avoidance of non-homologous end-joining of DNA double strand breaks in the yeast Saccharomyces cerevisiae; Ahne F et al.; In wild-type yeast, the repair of a 169 bp double-strand gap induced by the restriction enzymes ApaI and NcoI in the URA3gene of the shuttle vector YpJA18 occurs with high fidelity according to the homologous chromosomal sequence . In contrast, only 25% of the cells of rad5-7 and rad5 Delta mutants perform correct gap repair . As has been proven by sequencing of the junction sites, the remaining cells recircularise the gapped plasmids by joining of the non-compatible, non-homologous ends . Thus, regarding the repair of DNA double-strand breaks, the rad5 mutants behave like mammalian cells rather than budding yeast . The majority of the end joined plasmids miss either one or both of the 3'and 5'protruding single-strands of the restriction ends completely and have undergone blunt-end ligation accompanied by fill-in DNA synthesis . These results imply an important role for the Rad5 protein (Rad5p) in the protection of protruding single-strand ends and for the avoidance of non-homologous end joining during repair of double-strand gaps in budding yeast . Alternatively, the Rad5p may be an accessory factor increasing the efficiency of homologous recombination in yeast, however, the molecular mechanism of Rad5p function requires further investigation. J Biol Chem, 1997 Feb 14, 272(7), 4444 - 50 Molecular characterization of GCV3, the Saccharomyces cerevisiae gene coding for the glycine cleavage system hydrogen carrier protein; Nagarajan L et al.; YAL044, a gene on the left arm of Saccharomyces cerevisiae chromosome one, is shown to code for the H-protein subunit of the multienzyme glycine cleavage system . The gene designation has therefore been changed to GCV3, reflecting its role in the glycine cleavage system . GCV3 encodes a 177-residue protein with a putative mitochondrial targeting signal at its amino terminus . Targeted gene replacement shows that GCV3 is not required for growth on minimal medium; however, it is essential when glycine serves as the sole nitrogen source . Studies of GCV3 expression revealed that it is highly regulated . Supplementation of minimal medium with glycine, the glycine cleavage system's substrate, induced expression at least 30-fold . In contrast, and consistent with the cleavage of glycine providing activated single-carbon units, the addition of the metabolic end products that require activated single-carbon units repressed expression about 10-fold . Finally, like many amino acid biosynthetic genes, GCV3 is subject to regulation by the general amino acid control system. Biochim Biophys Acta, 1997 Feb 8, 1337(2), 166 - 74 Identification of reactive conserved histidines in phosphoenolpyruvate carboxykinases from Escherichia coli and Saccharomyces cerevisiae; Bazaes S et al.; Escherichia coli and Saccharomyces cerevisiae phospho enol pyruvate (PEP) carboxykinases are inactivated by diethylpyrocarbonate (DEP) . Inactivation follows pseudo-first-order kinetics and exhibits a second order rate constant of 0.8 M-1 s-1 for the bacterial enzyme and of 3.3 M-1 s-1 for the yeast carboxykinase . A mixture of ADP + PEP + MnCl2 protects against inactivation by DEP, suggesting that residues within the active site are being modified . After digestion of the modified proteins with trypsin, the labeled peptides were isolated by reverse-phase high-performance liquid chromatography and sequenced by Edman degradation . His-271 of E . coli carboxykinase and His-273 of the yeast enzyme were identified as the reactive amino-acid residues . The modified histidine residues occupy equivalent positions in these enzymes, and they are located in a highly conserved region of all ATP-dependent phospho enol pyruvate carboxykinases described so far. Gene, 1997 Feb 7, 185(2), 291 - 6 Mutation in a new gene MAF1 affects tRNA suppressor efficiency in Saccharomyces cerevisiae; Boguta M et al.; Mutation in the MAF1 gene was identified in a screen for decreased efficiency of tRNA suppressor SUP11 in the yeast Saccharomyces cerevisiae (Sc) . maf1-1 mutation exerts a dual phenotypic effect: antisuppression and temperature sensitive (ts) respiratory growth . MAF1, cloned by complementation of the ts phenotype of maf1-1, also alleviates the antisuppressor effect . The coding sequence of MAF1 is interrupted by an intron of 80 bp . The putative gene product, Maf1p, is a hydrophilic protein of 395 amino acids (aa) not showing significant similarity to known proteins which indicates that MAF1 encodes a novel protein . Maf1p may play a role in the tRNA biosynthetic pathway since a fragment of the RPO31/RPC160 gene encoding the largest subunit of RNA polymerase III was cloned as a multicopy suppressor of mafl-1. J Mol Biol, 1997 Feb 7, 265(5), 541 - 52 Mge1 functions as a nucleotide release factor for Ssc1, a mitochondrial Hsp70 of Saccharomyces cerevisiae; Miao B et al.; Mge1, a GrpE-related protein in the mitochondrial matrix of the budding yeast Saccharomyces cerevisiae, is required for translocation of precursor proteins into mitochondria . The effect of Mge1 on nucleotide release from Ssc1, an Hsp70 of the mitochondrial matrix, was analyzed . The release of both ATP and ADP from Ssc1 was stimulated in the presence of Mge1, therefore we conclude that Mge1 functions as a nucleotide release factor for Ssc1 . Mge1 bound stably to Ssc1 in vitro; this interaction was resistant to high concentrations of salt but was disrupted by the addition of ATP . ADP was much less effective in releasing Mge1 from Ssc1 whereas ATP gamma S and AMPPNP could not disrupt the Ssc1/Mge1 complex . Ssc1-3, a temperature sensitive SSC1 mutant protein, did not form a detectable complex with Mge1 . Consistent with the lack of a detectable interaction, Mge1 did not stimulate nucleotide release from Ssc1-3 . A conserved loop structure on the surface of the ATPase domain of DnaK has been implicated in its interaction with GrpE . Since the single amino acid change in Ssc1-3 lies very close to the analogous loop in Ssc1, the role of this loop in the Ssc1:Mge1 interaction was investigated . Deletion of the loop abolished the physical and functional interaction of Ssc1 with Mge1, suggesting that the loop in Ssc1 is also important for the Ssc1:Mge1 interaction . Two mutants with single amino acid changes within the loop did not eliminate the stable binding of Mge1, yet the binding of Mge1 did not stimulate the release of nucleotides from the mutant SSC1 proteins . We propose that the loop region of Ssc1 is important for the physical interaction between Mge1 and Ssc1, and for generation of a conformational change necessary for Mge1-induced nucleotide release. Proc Natl Acad Sci U S A, 1997 Feb 4, 94(3), 867 - 72 DNA double-strand-break sensitivity, DNA replication, and cell cycle arrest phenotypes of Ku-deficient Saccharomyces cerevisiae; Barnes G et al.; In mammalian cells, the Ku heterodimer is involved in DNA double-strand-break recognition and repair . We have established in yeast a connection between Ku activity and DNA double-strand-break damage repair, and a connection between Ku activity and commitment to DNA replication . We generated double-stranded DNA breaks in yeast cells in vivo by expressing a restriction endonuclease and have shown that yeast mutants lacking Ku p70 activity died while isogenic wild-type cells did not . Moreover, we have discovered that DNA damage occurs spontaneously during normal yeast mitotic growth, and that Ku functions in repair of this damage . We also observed that mitotically growing Ku p70 mutants have an anomalously high DNA content, suggesting a role for Ku in regulation of DNA synthesis . Finally, we present evidence that Ku p70 function is conserved between yeast, Drosophila, and humans. FEBS Lett, 1997 Feb 3, 402(2-3), 251 - 5 Regulation of maltose utilization in Saccharomyces cerevisiae by genes of the RAS/protein kinase A pathway; Wanke V et al.; In Saccharomyces cerevisiae maltose utilization requires a functional MAL locus, each composed of three genes: MALR (gene 3) encoding a regulatory protein, MALT (gene 1) encoding maltose permease and MALS (gene 2) encoding maltase . We show that constitutive activation of the RAS/protein kinase A pathway severely reduces growth of MAL1 strains on maltose . This may be a consequence of reduction in MALT mRNA, reduced Vmax and increased catabolite inactivation of the MALT-encoded maltose transporter in the MAL1 strain . Mutations in the GGS1/TPS1 gene, which restricts glucose influx and possibly affects signalling, relieve carbon catabolite repression on both maltase and maltose permease and reduce maltose permease inactivation. Mol Biol Cell, 1997 Feb, 8(2), 367 - 85 Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton; Lila T et al.; In a variety of organisms, a number of proteins associated with the cortical actin cytoskeleton contain SH3 domains, suggesting that these domains may provide the physical basis for functional interactions among structural and regulatory proteins in the actin cytoskeleton . We present evidence that SH3 domains mediate at least two independent functions of the Saccharomyces cerevisiae actin-binding protein Abp1p in vivo . Abp1p contains a single SH3 domain that has recently been shown to bind in vitro to the adenylyl cyclase-associated protein Srv2p . Immunofluorescence analysis of Srv2p subcellular localization in strains carrying mutations in either ABP1 or SRV2 reveals that the Abp1p SH3 domain mediates the normal association of Srv2p with the cortical actin cytoskeleton . We also show that a site in Abp1p itself is specifically bound by the SH3 domain of the actin-associated protein Rvs167p . Genetic analysis provides evidence that Abp1p and Rvs167p have functions that are closely interrelated . Abp1 null mutations, like rvs167 mutations, result in defects in sporulation and reduced viability under certain suboptimal growth conditions . In addition, mutations in ABP1 and RVS167 yield similar profiles of genetic "synthetic lethal" interactions when combined with mutations in genes encoding other cytoskeletal components . Mutations which specifically disrupt the SH3 domain-mediated interaction between Abp1p and Srv2p, however, show none of the shared phenotypes of abp1 and rvs167 mutations . We conclude that the Abp1p SH3 domain mediates the association of Srv2p with the cortical actin cytoskeleton, and that Abp1p performs a distinct function that is likely to involve binding by the Rvs167p SH3 domain . Overall, work presented here illustrates how SH3 domains can integrate the activities of multiple actin cytoskeleton proteins in response to varying environmental conditions. Mol Microbiol, 1997 Feb, 23(4), 777 - 89 Bidirectional regulation of two DNA-damage-inducible genes, MAG1 and DDI1, from Saccharomyces cerevisiae; Liu Y et al.; MAG1 encodes a Saccharomyces cerevisiae 3-methyladenine DNA glycosylase that initiates a base-excision-repair pathway and protects yeast cells from killing by methylating agents such as methyl methanesulphonate (MMS) . In the promoter region of the MAG1 gene, there is an 8 bp GC-rich direct repeat (DR) . Here we report that the DR sequence functions as an upstream activating site (UAS) that upregulates the expression of MAG1 as well as another DNA-damage-inducible gene, DDI1, which is transcribed divergently from MAG1 . Deletions, or point mutations, within this repeat completely abolished DNA-damage induction and resulted in a reduced basal-level expression for both MAG1 and DDI1 genes . Furthermore, yeast cells carrying the MAG1 gene with the DR deletion displayed an increased sensitivity to MMS compared with wildtype cells . The DR sequence alone can activate transcription of a CYC1 minimal promoter and confer a partial DNA-damage responsiveness . Electrophoretic mobility-shift assays indicate that the DR function is not due to interaction with the yeast RPA . Like MAG1, the DDI1 gene is also controlled by an upstream repressing site (URS) located 5' to the direct repeat . Based on this and previous studies, a model is proposed whereby the constitutive expression of MAG1 and DDI1 is controlled by two functionally opposite regulatory elements, UAS and URS, probably through an antagonistic mechanism, whereas the damage-induced expression appears to be regulated by mechanisms of derepression at the URS as well as activation at the UAS. J Biochem (Tokyo), 1997 Feb, 121(2), 338 - 44 Acidification of vacuoles is required for autophagic degradation in the yeast, Saccharomyces cerevisiae; Nakamura N et al.; Acidification inside vacuoles has been shown to play a key role in a number of physiologically important cellular events . We studied the role of vacuolar membrane H(+)-ATPase in the autophagic process of Saccharomyces cerevisiae . Mutants lacking VMA genes which encode their subunits of the vacuolar H(+)-ATPase accumulated autophagic bodies in vacuoles on starvation . vma mutants also had a defect in protein degradation induced by starvation . In vma mutants, the activities of vacuolar proteases were remarkably lower than those of the wild-type . Overexpression of vacuolar proteases did not overcome the defect in the disintegration of autophagic bodies in vma mutant, even the overexpressed proteinase A and proteinase B being substantially localized to the vacuolar compartment and undergoing proper proteolytic maturation . Our results showed that the acidification of vacuoles is not required for the formation and delivery of autophagosomes to vacuoles, but is essential for the disintegration of autophagic bodies. Fungal Genet Biol, 1997 Feb, 21(1), 141 - 52 Aspergillus fumigatus chsE: A Gene Related to CHS3 of Saccharomyces cerevisiae and Important for Hyphal Growth and Conidiophore Development but Not Pathogenicity Aufauvre-Brown A, Mellado E, Gow NAR, Holden DW. The Aspergillus fumigatus chsE (AfchsE) gene was isolated from an A . fumigatus DNA library on the basis of hybridization to a segment of Saccharomyces cerevisiae CHS3 (ScCHS3) . The amino acid sequence derived from AfchsE is 28% identical with ScCHS3 and 80% identical with the product of Aspergillus nidulans chsD (AnchsD) . A mutant strain constructed by disruption of AfchsE has reduced levels of mycelial chitin, periodic swellings along the length of hyphae, and a block in conidiation that can be partially restored by growth in osmotic stabilizer . This phenotype is different from that reported for an AnchsD mutant, in which germinating conidia and hyphal tips undergo lysis and the colonial growth rate is significantly reduced . Despite the defects associated with the AfchsE- strain, its virulence was not significantly reduced when compared with the wild-type parental strain in a mouse model of pulmonary aspergillosis. Gan To Kagaku Ryoho, 1997 Feb, 24(4), 466 - 70 {Functional screening of p53 status in tumor cells using Saccharomyces cerevisiae}; Jia LQ et al.; We have detected both germ-line and somatic p53 mutations in lymphocytes, cell lines and tumor tissues using a functional analysis of p53 tumor suppressor gene based on yeast transcription assay . Through our screening projects of the p53 gene, a number of missense p53 mutations were identified as loss-of-function mutations . This method, previously termed FASAY, is rapid, sensitive, less-expensive and can be automated for screening both somatic and germ-line p53 mutations. Genome, 1997 Feb, 40(1), 151 - 64 Analysis of a 103 kbp cluster homology region from the left end of Saccharomyces cerevisiae chromosome I; Storms RK et al.; The DNA sequence and preliminary functional analysis of a 103-kbp section of the left arm of yeast chromosome I is presented . This region, from the left telomere to the LTE1 gene, can be divided into two distinct portions . One portion, the telomeric 29 kbp, has a very low gene density (only five potential genes and 21 kbp of noncoding sequence), does not encode any "functionally important" genes, and is rich in sequences repeated several times within the yeast genome . The other portion, with 37 genes and only 14.5 kbp of noncoding sequence, is gene rich and codes for at least 16 "functionally important" genes . The entire gene-rich portion is apparently duplicated on chromosome XV as an extensive region of partial gene synteney called a cluster homology region . A function can be assigned with varying degrees of precision to 23 of the 42 potential genes in this region; however, the precise function is know for only eight genes . Nineteen genes encode products presently novel to yeast, although five of these have homologs elsewhere in the yeast genome. J Exp Zool, 1997 Feb 1, 277(2), 120 - 38 Mini review: mitosis and the spindle pole body in Saccharomyces cerevisiae; Sobel SG; Cell duplication is characteristic of life . The coordination of cell growth with cell duplication and, specifically, the ordered steps necessary for this process are termed the cell cycle . Central to this process is the faithful replication and segregation of the chromosomes . The cycle consists of four phases: G1, where the decision to enter the cell cycle, which is known as Start, is made; S phase, during which the DNA is replicated; G2, during which controls assuring the completion of S phase operate; and M, or the mitotic phase, which is characterized by chromosome segregation, nuclear division, and cytokinesis . The budding yeast Saccharomyces cerevisiae has been developed into a model genetic system for the study of the cell division cycle (Hartwell et al . {"73} Genetics, 74:267-286) . Here I review the basic processes by which chromosomes are segregated, with an emphasis on the physical structures fundamental to this process. Yeast, 1997 Feb, 13(2), 177 - 82 The sequence of a nearly unclonable 22.8 kb segment on the left arm chromosome VII from Saccharomyces cerevisiae reveals ARO2, RPL9A, TIP1, MRF1 genes and six new open reading frames; Voet M et al.; The nucleotide sequence of 22,803 bp on the left arm of chromosome VII was determined by polymerase chain reaction-based approaches to compensate for the unstable character of cosmid clones from this region of the chromosome . The coding density of the sequence is particularly high (more than 83%) . Twelve open reading frames (ORFs) longer than 300 bp were found, two of which (at the left side) have been described previously (James et al., 1995) after sequencing of an overlapping cosmid . Four other ORFs correspond to published sequences of the known genes ARO2, RPL9A, TIP1 and MRF1 . ARO2 codes for chorismate synthetase . RPL9A for protein L9 of the large ribosomal subunit and MRF1 for a mitochondrial translation release factor . The TIP1 product interacts with Sec20p and is thus involved in transport from endoplasmic reticulum to Golgi . Five of the remaining ORFs have not been identified previously, while the sixth (YGL142c) has been partially sequenced as it lies 5' upstream of MRF1 . These six ORFs are relatively large (between 933 and 3657 nucleotides) . YGL146c, YGL142c, YGL140c and YGL139w have no significant homology to any protein sequence presently available in the public databases, but show two, nine, nine and eight putative transmembrane spans, respectively . YGL144c has a serine active site signature of lipases . YGL141w has limited homology to several human proteins, one of which mediates complex formation between papillomavirus E6 oncoprotein and tumor suppressor protein p53. Yeast, 1997 Feb, 13(2), 151 - 61 Histone H1 in Saccharomyces cerevisiae; Ushinsky SC et al.; The existence of histone H1 in the yeast, Saccharomyces cerevisiae, has long been debated . In this report we describe the presence of histone H1 in yeast . YPL127c, a gene encoding a protein with a high degree of similarity to histone H1 from other species was sequenced as part of the contribution of the Montreal Yeast Genome Sequencing Group to chromosome XVI . To reflect this similarity, the gene designation has been changed HHO1 (Histone H One) . The HHO1 gene is highly expressed as poly A+ RNA in yeast . Although deletion of this gene had no detectable effect on cell growth, viability or mating, it significantly altered the expression of beta-galactosidase from a CYC1-lacZ reporter . Fluorescence observed in cells expressing a histone H1-GFP protein fusion indicated that histone H1 is localized to the nucleus. Yeast, 1997 Feb, 13(2), 127 - 37 Expression of the SUC2 gene of Saccharomyces cerevisiae is induced by low levels of glucose; Ozcan S et al.; High levels of glucose repress expression of the SUC2 gene in the yeast Saccharomyces cerevisiae . We have discovered that low levels of glucose are required for maximal transcription of SUC2: SUC2 expression is induced about five- to ten-fold in cells growing on low levels of glucose (0.1%) compared to cells growing on galactose or glycerol . Two pieces of evidence suggest that this low-glucose-induced expression is mediated by a repression mechanism that involves an upstream repression site in the SUC2 promoter (URS(SUC2)) . First, deletion of the URS(SUC2) results in expression of the SUC2 gene in the absence of glucose, and second the URS(SUC2) mediates a six-fold repression of a reporter gene when inserted into a heterologous promoter . However, this URS(SUC2) mediated repression occurs on all tested carbon sources, suggesting that this URS element acts in concert with all other promoter elements to respond to low concentrations of glucose . This repression requires the general repressor SSn6p . SNF3, which encodes a glucose transporter that appears to be a sensor of low levels of glucose, is also required for low-glucose-induced expression of SUC2. Yeast, 1997 Feb, 13(2), 119 - 25 The fate of glucose in strains S288C and S173-6B of the yeast Saccharomyces cerevisiae; Pedler SM et al.; Intracellular metabolic flux has been investigated in two strains of Saccharomyces cerevisiae grown into stationary phase under both glucose-repressed and glucose-derepressed conditions . By employing a variety of simple methodologies (manometry, enzymatic analysis and colorimetric analysis) we have been able to identify and quantitate carbon flow from glucose without the need for isotopically labelled substrate . We can account for 88-98% (depending on strain and growth conditions) of the carbon products of glucose metabolism under both glycolytic and oxidative conditions as ethanol (27-40%), carbon dioxide (15-26%), acetate (2-3%), glycerol (5-11%), glycogen (5-13%) and trehalose (9-39%). Protein Sci, 1997 Feb, 6(2), 481 - 3 Crystallization and preliminary structural analysis of catalase A from Saccharomyces cerevisiae; Berthet S et al.; Yeast peroxisomal catalase A, obtained at high yields by over expression of the C-terminally modified gene from a 2 mu-plasmid, has been crystallized in a form suitable for high resolution X-ray diffraction studies . Brownish crystals with bipyrimidal morphology and reaching ca . 0.8 mm in size were produced by the hanging drop method using ammonium sulphate as precipitant . These crystals diffract better than 2.0 A resolution and belong to the hexagonal space group P6(1)22 with unit cell parameters a = b = 184.3 A and c = 305.5 A . An X-ray data set with 76% completeness at 3.2 A resolution was collected in a rotating anode generator using mirrors to improve the collimation of the beam . An initial solution was obtained by molecular replacement only when using a beef liver catalase tetramer model in which fragments with no sequence homology had been omitted, about 150 residues per subunit . In the structure found a single molecule of catalase A (a tetramer with accurate 222 molecular symmetry) is located in the asymmetric unit of the crystal with an estimated solvent content of about 61% . The preliminary analysis of the structure confirms the absence of a carboxy terminal domain as the one found in the catalase from Penicillium vitalae, the only other fungal catalase structure available . The NADPH binding site appears to be involved in crystal contacts, suggesting that heterogeneity in the occupancy of the nucleotide can be a major difficulty during crystallization. FEMS Microbiol Lett, 1997 Feb 1, 147(1), 17 - 22 Inhibitory effects of nucleoside 5'-alkylphosphates on sexual agglutination in Saccharomyces cerevisiae; Machida K et al.; Among various nucleoside 5'-alkylphosphates, uridine 5'-hexadecylphosphate (UMPC16) and adenosine 5'-hexadecylphosphate (AMPC16) inhibited the sexual agglutination between a and alpha haploid cells of Saccharomyces cerevisiae . The effect of AMPC16 accompanied severe growth inhibition of the yeast cells but it was not observed with UMPC16 . Sexual agglutination was not inhibited by the presence of UMPC16 or AMPC16 when the yeast cells had been pretreated with the mating pheromone . UMPC16 was characterized as a specific inhibitor of sexual agglutination without direct influence on the agglutinin function, being distinguishable from any of those ever known. Genes Dev, 1997 Feb 1, 11(3), 345 - 57 Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I; Kaufman PD et al.; In vivo, nucleosomes are formed rapidly on newly synthesized DNA after polymerase passage . Previously, a protein complex from human cells, termed chromatin assembly factor-I (CAF-I), was isolated that assembles nucleosomes preferentially onto SV40 DNA templates that undergo replication in vitro . Using a similar assay, we now report the purification of CAF-I from the budding yeast Saccharomyces cerevisiae . Amino acid sequence data from purified yeast CAF-I led to identification of the genes encoding each subunit in the yeast genome data base . The CAC1 and CAC2 (chromatin assembly complex) genes encode proteins similar to the p150 and p60 subunits of human CAF-I, respectively . The gene encoding the p50 subunit of yeast CAF-I (CAC3) is similar to the human p48 CAF-I subunit and was identified previously as MSI1, a member of a highly conserved subfamily of WD repeat proteins implicated in histone function in several organisms . Thus, CAF-I has been conserved functionally and structurally from yeast to human cells . Genes encoding the CAF-I subunits (collectively referred to as CAC genes) are not essential for cell viability . However, deletion of any CAC gene causes an increase in sensitivity to ultraviolet radiation, without significantly increasing sensitivity to gamma rays . This is consistent with previous biochemical data demonstrating the ability of CAF-I to assemble nucleosomes on templates undergoing nucleotide excision repair . Deletion of CAC genes also strongly reduces silencing of genes adjacent to telomeric DNA; the CAC1 gene is identical to RLF2 (Rap1p localization factor-2), a gene required for the normal distribution of the telomere-binding Rap1p protein within the nucleus . Together, these data suggest that CAF-I plays a role in generating chromatin structures in vivo. Appl Environ Microbiol, 1997 Feb, 63(2), 615 - 20 Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins; Van der Vaart JM et al.; The carboxyl-terminal regions of five cell wall proteins (Cwp1p, Cwp2p, Ag alpha 1p, Tip1p, and Flo1p) and three potential cell wall proteins (Sed1p, YCR89w, and Tir1p) all proved capable of immobilizing alpha-galactosidase in the cell wall of Saccharomyces cerevisiae . The fraction of the total amount of fusion protein that was localized to the cell wall varied depending on the anchor domain used . The highest proportion of cell wall incorporation was achieved with Cwp2p, Ag alpha 1p, or Sed1p as an anchor . Although 80% of these fusion proteins were incorporated in the cell wall, the total production of alpha-galactosidase-Ag alpha 1p was sixfold lower than that of alpha-galactosidase-Cwp2p and eightfold lower than that of alpha-galactosidase-Sed1p . Differences in mRNA levels were not responsible for this discrepancy, nor was an intracellular accumulation of alpha-galactosidase-Ag alpha 1p detectable . A lower translation efficiency of the alpha-galactosidase-AG alpha 1 fusion construct is most likely to be responsible for the low level of protein production . alpha-Galactosidase immobilized by the carboxyl-terminal 67 amino acids of Cwp2p was most effective in the hydrolysis of the high-molecular-weight substrate guar gum from Cyamopsis tetragonoloba . This indicates that the use of a large anchoring domain does not necessarily result in a better exposure of the immobilized enzyme to the exterior of the yeast cell. J Bacteriol, 1997 Feb, 179(4), 1096 - 101 Saccharomyces cerevisiae exhibits a yAP-1-mediated adaptive response to malondialdehyde; Turton HE et al.; Malondialdehyde (MDA) is a highly reactive aldehyde generally formed as a consequence of lipid peroxidation . MDA has been inferred to have mutagenic and cytotoxic roles and possibly to be a participant in the onset of atherosclerosis . Wild-type Saccharomyces cerevisiae acquires resistance to a lethal dose (5 mM) of MDA following prior exposure to a nonlethal concentration (1 mM) . This response was completely inhibited by cycloheximide (50 microg ml(-1)), indicating a requirement for protein synthesis for adaptation . Furthermore, we have examined the roles of glutathione (GSH), mitochondrial function, and yAP-1-mediated transcription in conferring resistance and adaptation to MDA . A yap1 disruption mutant exhibited the greatest sensitivity and was unable to adapt to MDA, implicating yAP-1 in both the adaptive response and constitutive survival . The effect of MDA on GSH mutants indicated a role for GSH in initial resistance, whereas resistance acquired through adaptation was independent of GSH . Likewise, respiratory mutants (petite mutants) were sensitive to MDA but were still able to mount an adaptive response similar to that of the wild type, excluding mitochondria from any role in adaptation . MDA was detected in yeast cells by the thiobarbituric acid test and subsequent high-pressure liquid chromatography separation . Elevated levels were detected following treatment with hydrogen peroxide . However, the MDA-adaptive response was independent of that to H2O2. J Bacteriol, 1997 Feb, 179(4), 1068 - 76 AUT1, a gene essential for autophagocytosis in the yeast Saccharomyces cerevisiae; Schlumpberger M et al.; Autophagocytosis is a starvation-induced process responsible for transport of cytoplasmic proteins to the vacuole . In Saccharomyces cerevisiae, autophagy is characterized by the phenotypic appearance of autophagic vesicles inside the vacuole of strains deficient in proteinase yscB . The AUT1 gene, essential for autophagy, was isolated by complementation of the sporulation deficiency of a diploid aut1-1 mutant strain by a yeast genomic library and characterized . AUT1 is located on the right arm of chromosome XIV, 10 kb from the centromere, and encodes a protein of 310 amino acids, with an estimated molecular weight of 36 kDa . Cells carrying a chromosomal deletion of AUT1 are defective in the starvation-induced bulk flow transport of cytoplasmic proteins to the vacuole . aut1 null mutant strains are completely viable but show decreased survival rates during starvation . Homozygous delta aut1 diploid cells fail to sporulate . The selective cytoplasm-to-vacuole transport of aminopeptidase I is blocked in logarithmically growing and in starved delta autl cells . Deletion of the AUT1 gene had no obvious influence on secretion, fluid phase endocytosis, or vacuolar protein sorting . This supports the idea of autophagocytosis as being a novel route transporting proteins from the cytoplasm to the vacuole. Mol Cell Biol, 1997 Feb, 17(2), 819 - 32 Multiple phosphorylated forms of the Saccharomyces cerevisiae Mcm1 protein include an isoform induced in response to high salt concentrations; Kuo MH et al.; The Saccharomyces cerevisiae Mcm1 protein is an essential multifunctional transcription factor which is highly homologous to human serum response factor . Mcm1 protein acts on a large number of distinctly regulated genes: haploid cell-type-specific genes, G2-cell-cycle-regulated genes, pheromone-induced genes, arginine metabolic genes, and genes important for cell wall and cell membrane function . We show here that Mcm1 protein is phosphorylated in vivo . Several (more than eight) isoforms of Mcm1 protein, resolved by isoelectric focusing, are present in vivo; two major phosphorylation sites lie in the N-terminal 17 amino acids immediately adjacent to the conserved MADS box DNA-binding domain . The implications of multiple species of Mcm1, particularly the notion that a unique Mcm1 isoform could be required for regulation of a specific set of Mcm1's target genes, are discussed . We also show here that Mcm1 plays an important role in the response to stress caused by NaCl . G . Yu, R . J . Deschenes, and J . S . Fassler (J . Biol . Chem . 270:8739-8743, 1995) showed that Mcm1 function is affected by mutations in the SLN1 gene, a signal transduction component implicated in the response to osmotic stress . We find that mcm1 mutations can confer either reduced or enhanced survival on high-salt medium; deletion of the N terminus or mutation in the primary phosphorylation site results in impaired growth on high-salt medium . Furthermore, Mcm1 protein is a target of a signal transduction system responsive to osmotic stress: a new isoform of Mcm1 is induced by NaCl or KCl; this result establishes that Mcm1 itself is regulated. Mol Cell Biol, 1997 Feb, 17(2), 635 - 43 The RAD7, RAD16, and RAD23 genes of Saccharomyces cerevisiae: requirement for transcription-independent nucleotide excision repair in vitro and interactions between the gene products; Wang Z et al.; Nucleotide excision repair (NER) is a biochemical process required for the repair of many different types of DNA lesions . In the yeast Saccharomyces cerevisiae, the RAD7, RAD16, and RAD23 genes have been specifically implicated in NER of certain transcriptionally repressed loci and in the nontranscribed strand of transcriptionally active genes . We have used a cell-free system to study the roles of the Rad7, Rad16, and Rad23 proteins in NER . Transcription-independent NER of a plasmid substrate was defective in rad7, rad16, and rad23 mutant extracts . Complementation studies with a previously purified NER protein complex (nucleotide excision repairosome) indicate that Rad23 is a component of the repairosome, whereas Rad7 and Rad16 proteins were not found in this complex . Complementation studies with rad4, rad7, rad16, and rad23 mutant extracts suggest physical interactions among these proteins . This conclusion was confirmed by experiments using the yeast two-hybrid assay, which demonstrated the following pairwise interactions: Rad4 with Rad23, Rad4 with Rad7, and Rad7 with Rad16 . Additionally, interaction between the Rad7 and Rad16 proteins was demonstrated in vitro . Our results show that Rad7, Rad16, and Rad23 are required for transcription-independent NER in vitro . This process may involve a unique protein complex which is distinct from the repairosome and which contains at least the Rad4, Rad7, and Rad16 proteins. Mol Cell Biol, 1997 Feb, 17(2), 620 - 6 Cdc55p, the B-type regulatory subunit of protein phosphatase 2A, has multiple functions in mitosis and is required for the kinetochore/spindle checkpoint in Saccharomyces cerevisiae; Wang Y et al.; Saccharomyces cerevisiae, like most eucaryotic cells, can prevent the onset of anaphase until chromosomes are properly aligned on the mitotic spindle . We determined that Cdc55p (regulatory B subunit of protein phosphatase 2A {PP2A}) is required for the kinetochore/spindle checkpoint regulatory pathway in yeast . ctf13 cdc55 double mutants could not maintain a ctf13-induced mitotic delay, as determined by antitubulin staining and levels of histone H1 kinase activity . In addition, cdc55::LEU2 mutants and tpd3::LEU2 mutants (regulatory A subunit of PP2A) were nocodazole sensitive and exhibited the phenotypes of previously identified kinetochore/spindle checkpoint mutants . Inactivating CDC55 did not simply bypass the arrest that results from inhibiting ubiquitin-dependent proteolysis because cdc16-1 cdc55::LEU2 and cdc23-1 cdc55::LEU2 double mutants arrested normally at elevated temperatures . CDC55 is specific for the kinetochore/spindle checkpoint because cdc55 mutants showed normal sensitivity to gamma radiation and hydroxyurea . The conditional lethality and the abnormal cellular morphogenesis of cdc55::LEU2 were suppressed by cdc28F19, suggesting that the cdc55 phenotypes are dependent on the phosphorylation state of Cdc28p . In contrast, the nocodazole sensitivity of cdc55::LEU2 was not suppressed by cdc28F19 . Therefore, the mitotic checkpoint activity of CDC55 (and TPD3) is independent of regulated phosphorylation of Cdc28p . Finally, cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1, suggesting additional roles for CDC55 in mitosis. Mol Cell Biol, 1997 Feb, 17(2), 545 - 52 Hir1p and Hir2p function as transcriptional corepressors to regulate histone gene transcription in the Saccharomyces cerevisiae cell cycle; Spector MS et al.; The HIR/HPC (histone regulation/histone periodic control) negative regulators play important roles in the transcription of six of the eight core histone genes during the Saccharomyces cerevisiae cell cycle . The phenotypes of hir1 and hir2 mutants suggested that the wild-type HIR1 and HIR2 genes encode transcriptional repressors that function in the absence of direct DNA binding . When Hir1p and Hir2p were artificially tethered to yeast promoters, each protein repressed transcription, suggesting that they represent a new class of transcriptional corepressors . The two proteins might function as a complex in vivo: Hir2p required both Hir1p and another Hir protein, Hir3p, to repress transcription when it was tethered to an HTA1-lacZ reporter gene, and Hir1p and Hir2p could be coimmunoprecipitated from yeast cell extracts . Tethered Hir1p also directed the periodic transcription of the HTA1 gene and repressed HTA1 transcription in response to two cell cycle regulatory signals . Thus, it represents the first example of a transcriptional corepressor with a direct role in cell cycle-regulated transcription. Mutat Res, 1997 Jan 31, 383(1), 39 - 48 The Saccharomyces cerevisiae RAD7 and RAD16 genes are required for inducible excision of endonuclease III sensitive-sites, yet are not needed for the repair of these lesions following a single UV dose; Scott AD et al.; The RAD7 and RAD16 genes of Saccharomyces cerevisiae have roles in the repair of UV induced CPDs in nontranscribed genes {1}, and in the repair of CPDs in the nontranscribed strand of transcribed genes {2} . Previously, we identified an inducible component to nucleotide excision repair (NER), which is absent in a rad16 delta strain {3} . We have examined the repair of UV induced endonuclease III sensitive-sites (EIIISS), and have shown repair of these lesions to proceed by NER but their removal from nontranscribed regions is independent of RAD7 and RAD16 . Furthermore, EIIISS are repaired with equal efficiency from both transcribed and nontranscribed genes {4} . In order to dissect the roles of RAD7 and RAD16 in the above processes we examined the repair of EIIISS in the MAT alpha and HML alpha loci, which are, respectively, transcriptionally active and inactive in alpha haploid cells . These loci have elevated levels of these lesions after UV (in genomic DNA EIIISS constitute about 10% of total lesions, whereas CPDs are about 70% of total lesions) . We have shown that excision of UV induced EIIISS is enhanced following a prior UV irradiation . No enhancement of repair was detected in either the rad7 delta or the rad16 delta mutant . The fact that RAD7 and RAD16 are not required for the repair of EIIISS per se yet are required for the enhanced excision of these lesions from MAT alpha and HML alpha suggests two possibilities . These genes have two roles in NER, namely in the repair of CPDs from nontranscribed sequences, and in enhancing NER itself regardless of whether these genes' products are required for the excision of the specific lesion being repaired . In the latter case, the induction of RAD7 and RAD16 may increase the turnover of complexes stalled in nontranscribed DNA so as to increase the availability of NER proteins for the repair of CPDs and EIIISS in all regions of the genome. J Cell Biol, 1997 Jan 27, 136(2), 251 - 69 Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor; Chen P et al.; The Saccharomyces cerevisiae mating pheromone a-factor is a prenylated and carboxyl methylated extracellular peptide signaling molecule . Biogenesis of the a-factor precursor proceeds via a distinctive multistep pathway that involves COOH-terminal modification . NH2-terminal proteolysis, and a nonclassical export mechanism . In this study, we examine the formation and fate of a-factor biosynthetic intermediates to more precisely define the events that occur during a-factor biogenesis . We have identified four distinct a-factor biosynthetic intermediates (P0, P1, P2, and M) by metabolic labeling, immunoprecipitation, and SDS-PAGE . We determined the biochemical composition of each by defining their NH2-terminal amino acid and COOH-terminal modification status . Unexpectedly, we discovered that not one, but two NH2-terminal cleavage steps occur during the biogenesis of a-factor . In addition, we have shown that COOH-terminal prenylation is required for the NH2-terminal processing of a-factor and that all the prenylated a-factor intermediates (P1, P2, and M) are membrane bound, suggesting that many steps of a-factor biogenesis occur in association with membranes . We also observed that although the biogenesis of a-factor is a rapid process, it is inherently inefficient, perhaps reflecting the potential for regulation . Previous studies have identified gene products that participate in the COOH-terminal modification (Ram1p, Ram2p, Ste14p), NH2-terminal processing (Ste24p, Axl1p), and export (Ste6p) of a-factor . The intermediates defined in the present study are discussed in the context of these biogenesis components to formulate an overall model for the pathway of a-factor biogenesis. J Mol Biol, 1997 Jan 24, 265(3), 289 - 301 Saccharomyces cerevisiae MSH2, a mispaired base recognition protein, also recognizes Holliday junctions in DNA; Alani E et al.; Genetic and biochemical studies have suggested that mismatch repair proteins interact with recombination intermediates to prevent recombination, or to limit the extent of formation of heteroduplex DNA during recombination between divergent DNA sequences . To test the idea that mismatch repair proteins regulate recombination by interacting with recombination intermediates, we investigated whether the Saccharomyces cerevisiae MutS homolog MSH2 could interact with Holliday junctions . Both filter-binding and electron-microscopic analysis showed that MSH2 bound to duplex DNA molecules containing Holliday junctions with a higher affinity than to control duplex DNA, single-stranded DNA or a control duplex DNA containing a mispaired base . The MSH2-Holliday junction complexes were also more stable than MSH2-duplex DNA complexes . This observation suggests that MSH2 protein could directly coordinate the interaction between mismatch repair and genetic recombination observed in genetic studies. Proc Natl Acad Sci U S A, 1997 Jan 21, 94(2), 581 - 6 Role of the casein kinase I isoform, Hrr25, and the cell cycle-regulatory transcription factor, SBF, in the transcriptional response to DNA damage in Saccharomyces cerevisiae; Ho Y et al.; In the budding yeast, Saccharomyces cerevisiae, DNA damage or ribonucleotide depletion causes the transcriptional induction of an array of genes with known or putative roles in DNA repair . The ATM-like kinase, Mec1, and the serine/threonine protein kinases, Rad53 and Dun1, are required for this transcriptional response . In this paper, we provide evidence suggesting that another kinase, Hrr25, is also involved in the transcriptional response to DNA damage through its interaction with the transcription factor, Swi6 . The Swi6 protein interacts with Swi4 to form the SBF complex and with Mbp1 to form the MBF complex . SBF and MBF are required for the G1-specific expression of G1 cyclins and genes required for S-phase . We show that Swi6 associates with and is phosphorylated by Hrr25 in vitro . We find that swi4, swi6, and hrr25 mutants, but not mbp1 mutants, are sensitive to hydroxyurea and the DNA-damaging agent methyl methane-sulfonate and are defective in the transcriptional induction of a subset of DNA damage-inducible genes . Both the sensitivity of swi6 mutants to methyl methanesulfonate and hydroxyurea and the transcriptional defect of hrr25 mutants are rescued by overexpression of SWI4, implicating the SBF complex in the Hrr25/Swi6-dependent response to DNA damage. J Cell Biol, 1997 Jan 13, 136(1), 111 - 23 A role for the actin cytoskeleton of Saccharomyces cerevisiae in bipolar bud-site selection; Yang S et al.; Saccharomyces cerevisiae cells select bud sites according to one of two predetermined patterns . MATa and MAT alpha cells bud in an axial pattern, and MATa/alpha cells bud in a bipolar pattern . These budding patterns are thought to depend on the placement of spatial cues at specific sites in the cell cortex . Because cytoskeletal elements play a role in organizing the cytoplasm and establishing distinct plasma membrane domains, they are well suited for positioning bud-site selection cues . Indeed, the septin-containing neck filaments are crucial for establishing the axial budding pattern characteristic of MATa and MAT alpha cells . In this study, we determined the budding patterns of cells carrying mutations in the actin gene or in genes encoding actin-associated proteins: MATa/alpha cells were defective in the bipolar budding pattern, but MATa and MAT alpha cells still exhibit a normal axial budding pattern . We also observed that MATa/alpha actin cytoskeleton mutant daughter cells correctly position their first bud at the distal pole of the cell, but mother cells position their buds randomly . The actin cytoskeleton therefore functions in generation of the bipolar budding pattern and is required specifically for proper selection of bud sites in mother MATa/alpha cells . These observations and the results of double mutant studies support the conclusion that different rules govern bud-site selection in mother and daughter MATa/alpha cells . A defective bipolar budding pattern did not preclude an sla2-6 mutant from undergoing pseudohyphal growth, highlighting the central role of daughter cell bud-site selection cues in the formation of pseudohyphae . Finally, by examining the budding patterns of mad2-1 mitotic checkpoint mutants treated with benomyl to depolymerize their microtubules, we confirmed and extended previous evidence indicating that microtubules do not function in axial or bipolar bud-site selection. Biochim Biophys Acta, 1996 Jan 10, 1310(1), 110 - 8 In vivo processing of the precursor of the major exoglucanase by KEX2 endoprotease in the Saccharomyces cerevisiae secretory pathway; Basco RD et al.; We have established the main post-translational modification of the major exoglucanase of Saccharomyces cerevisiae as the enzyme progresses through the secretory pathway . The protein portion of the enzyme accumulated by sec18 cells was about 2 kDa larger than that of the secreted enzyme . This precursor (form A) was stable when maintained in the endoplasmic reticulum but was processed to the mature form (form B) before the block imposed by the sec7 mutation . Sec7 cells, when incubated at 37 degrees C, accumulated form B first, but upon prolonged incubation, form A was preferentially accumulated . When the supply of newly synthesized exoglucanase was prevented by the addition of cycloheximide, the accumulated A was transformed into B in the presence of altered Sec7p that still prevented secretion . Conversion of A into B was prevented in the double mutant sec7 kex2-1, indicating that Kex2p is central to the in vivo processing . Consistent with this, a KEX2 deletion mutant secreted form A exclusively . Conversion of A into B was also prevented in sec7 cells by the presence of dinitrophenol, a poison that depletes ATP levels, indicating that processing is dependent upon intracellular transport which involves ER --> Golgi and/or, at least, one intra-Golgi step(s) . It follows that this transport step(s) is independent of functional Sec7p. J Biol Chem, 1997 Jan 10, 272(2), 1256 - 62 Overproduction and affinity purification of Saccharomyces cerevisiae replication factor C; Gerik KJ et al.; Yeast replication factor C (RF-C) is a heteropentamer encoded by the RFC1-5 genes . RF-C activity in yeast extracts was overproduced about 80-fold after induction of a strain containing all five genes on a single plasmid, with expression of each gene placed under control of the galactose-inducible GAL1-10 promoter . This strongly indicates that overexpression of the five known RFC genes is sufficient for overproduction of RF-C . Overexpression of all five genes was also necessary to achieve overproduction of RF-C as omission of any single gene from the plasmid gave uninduced, i.e . normal cellular levels of RF-C . The interaction between RF-C and proliferating cell nuclear antigen (PCNA) was studied with PCNA-agarose beads . Binding of RF-C to PCNA-agarose beads is negligible in buffers containing 0.3 M NaCl . However, addition of Mg-ATP to the binding buffer caused strong binding of RF-C to the beads even at 0.8 M NaCl . Binding of ATP, but not its hydrolysis, was required for the strong binding mode as nonhydrolyzable analogs were also effective . The existence of two distinct binding modes between PCNA and RF-C was used as the key step in a greatly improved procedure for the purification of RF-C . RF-C from the overproduction strain purified by this procedure was essentially homogeneous and had a severalfold higher specific activity than RF-C preparations that had previously been purified through multicolumn procedures. Proc Natl Acad Sci U S A, 1997 Jan 7, 94(1), 190 - 5 A multipurpose transposon system for analyzing protein production, localization, and function in Saccharomyces cerevisiae; Ross-Macdonald P et al.; Analysis of the function of a particular gene product typically involves determining the expression profile of the gene, the subcellular location of the protein, and the phenotype of a null strain lacking the protein . Conditional alleles of the gene are often created as an additional tool . We have developed a multifunctional, transposon-based system that simultaneously generates constructs for all the above analyses and is suitable for mutagenesis of any given Saccharomyces cerevisiae gene . Depending on the transposon used, the yeast gene is fused to a coding region for beta-galactosidase or green fluorescent protein . Gene expression can therefore be monitored by chemical or fluorescence assays . The transposons create insertion mutations in the target gene, allowing phenotypic analysis . The transposon can be reduced by cre-lox site-specific recombination to a smaller element that leaves an epitope tag inserted in the encoded protein . In addition to its utility for a variety of immunodetection purposes, the epitope tag element also has the potential to create conditional alleles of the target gene . We demonstrate these features of the transposons by mutagenesis of the SPA2, ARP100, SER1, and BDF1 genes. Proc Natl Acad Sci U S A, 1997 Jan 7, 94(1), 42 - 7 A new pathway for vacuolar cadmium sequestration in Saccharomyces cerevisiae: YCF1-catalyzed transport of bis(glutathionato)cadmium; Li ZS et al.; The yeast cadmium factor (YCF1) gene encodes an MgATP-energized glutathione S-conjugate transporter responsible for the vacuolar sequestration of organic compounds after their S-conjugation with glutathione . However, while YCF1 was originally isolated according to its ability to confer resistance to cadmium salts, neither its mode of interaction with Cd2+ nor the relationship between this process and organic glutathione-conjugate transport are known . Here we show through direct comparisons between vacuolar membrane vesicles purified from Saccharomyces cerevisiae strain DTY167, harboring a deletion of the YCF1 gene, and the isogenic wild-type strain DTY165 that YCF1 mediates the MgATP-energized vacuolar accumulation of Cd-glutathione complexes . The substrate requirements, kinetics and Cd2+/glutathione stoichiometry of cadmium uptake and the molecular weight of the transport-active complex demonstrate that YCF1 selectively catalyzes the transport of bis(glutathionato)cadmium (Cd x +GS2) . On the basis of these results--the Cd2+ hypersensitivity of DTY167, versus DTY165, cells, the inducibility of YCF1-mediated transport, and the rapidity and spontaneity of Cd-GS2 formation--this new pathway is concluded to contribute substantially to Cd2+ detoxification. C R Seances Soc Biol Fil, 1997, 191(2), 221 - 35 {Ras proteins in Saccharomyces cerevisiae, their partners and their activation}; Jacquet M; Ras proteins play the role of molecular switches by conformational change between a GTP and a GDP-bound state . In the yeast Saccharomyces cerevisiae, they are encoded by two partially redundant genes RAS1 and RAS2 with a different pattern of gene expression . They are essential for growth because they are required for the activation of the adenylate cyclase and thus the protein kinase A pathway . Other possible biological functions remains to be established . To achieve their biological function, they need to be processed after their synthesis, they are modified farnesylated and palmitoylated at their C-terminal end at their CaaX box . Palmitoylation, involved in membrane localization, is not essential for growth but required for glucose signaling whereas farnesylation appears to participate in adenylate cyclase activation . In the GTP-bound state ras proteins interact through their conserved effector domain with the adenylate cyclase, the product of the CYR1/CDC35 gene . They also interact with GTPase activating proteins encoded by IRA1 and IRA2 . These proteins are specific for yeast ras . It has been shown that Ira2p recognizes specific residues of yeast ras not shared by mammalian ras . The interaction with the guanine nucleotide exchange factor (GEF) of the CDC25 family is enhanced by dominant negative mutations such as RAS2ala22 . Using the two hybrid approach, we have showed the key role of position 80 in Ras2p and confirmed the involvement of the a2 helix, the other switching part of ras, in this interaction and the induced effect . As a counterpart we have identified positions in HGRF55 conserved in other GEF involved in ras interaction . The triggering elements of ras activation: the GEF Cdc25p and Sdc25p are limiting components of the ras system . Cdc25p is part of a multimolecular complex associated with the membrane . We have shown that it can form homodimers and heterodimers with Sdc25p . It is an unstable protein containing a cyclin destruction box . Therefore its activity on ras could be regulated by controlling its cellular content. Cell Motil Cytoskeleton, 1997, 37(3), 199 - 210 Mitochondrial inheritance: cell cycle and actin cable dependence of polarized mitochondrial movements in Saccharomyces cerevisiae; Simon VR et al.; Asymmetric growth and division of budding yeast requires the vectorial transport of growth components and organelles from mother to daughter cells . Time lapse video microscopy and vital staining were used to study motility events which result in partitioning of mitochondria in dividing yeast . We identified four different stages in the mitochondrial inheritance cycle: (1) mitochondria align along the mother-bud axis prior to bud emergence in G1 phase, following polarization of the actin cytoskeleton; (2) during S phase, mitochondria undergo linear, continuous and polarized transfer from mother to bud; (3) during S and G2 phases, inherited mitochondria accumulate in the bud tip . This event occurs concomitant with accumulation of actin patches in this region; and (4) finally, during M phase prior to cytokinesis, mitochondria are released from the bud tip and redistribute throughout the bud . Previous studies showed that yeast mitochondria colocalize with actin cables and that isolated mitochondria contain actin binding and motor activities on their surface . We find that selective destabilization of actin cables in a strain lacking the tropomyosin 1 gene (TPM1) has no significant effect on the velocity of mitochondrial motor activity in vivo or in vitro . However, tpm1 delta mutants display abnormal mitochondrial distribution and morphology; loss of long distance, directional mitochondrial movement; and delayed transfer of mitochondria from the mother cell to the bud . Thus, cell cycle-linked mitochondrial motility patterns which lead to inheritance are strictly dependent on organized and properly oriented actin cables. Folia Microbiol (Praha), 1997, 42(1), 47 - 51 Accumulation of Ade+ reversions in isoauxotrophic stains of Saccharomyces cerevisiae allelic in RAD6 during adenine starvation; Storchova Z et al.; A comparative method based on an analysis of accumulation of starvation-induced Ade+ reversions and cell death during adenine starvation was developed and exploited for estimating the role of RAD6 in the starvation-induced reversions . It was shown that inactivation of RAD6 function in Saccharomyces cerevisiae markedly enhances the accumulation of Ade+ reversions, and therefore it is likely that this gene is taking part in maintaining the low level of starvation-induced mutations in yeast cells. J Ind Microbiol Biotechnol, 1997 Jan, 18(1), 30 - 6 Stress co-tolerance and trehalose content in baking strains of Saccharomyces cerevisiae; Lewis JG et al.; Fourteen wild-type baking strains of Saccharomyces cerevisiae were grown in batch culture to true stationary phase (exogenous carbon source exhausted) and tested for their trehalose content and their tolerance to heat (52 degrees C for 4.5 min), ethanol (20% v/v for 30 min), H2O2 (0.3 M for 60 min), rapid freezing (-196 degrees C for 20 min, cooling rate 200 degrees C min-1), slow freezing (-20 degrees C for 24 h, cooling rate 3 degrees C min(-1)), salt (growth in 1.5 M NaCl agar) or acetic acid (growth in 0.4% w/v acetic acid agar) stresses . Stress tolerance among the strains was highly variable and up to 1000-fold differences existed between strains for some types of stress . Compared with previously published reports, all strains were tolerant to H2O2 stress . Correlation analysis of stress tolerance results demonstrated relationships between tolerance to H2O2 and tolerance to all stresses except ethanol . This may imply that oxidative processes are associated with a wide variety of cellular stresses and also indicate that the general robustness associated with industrial yeast may be a result of their oxidative stress tolerance . In addition, H2O2 tolerance might be a suitable marker for the general assessment of stress tolerance in yeast strains . Trehalose content failed to correlate with tolerance to any stress except acetic acid . This may indicate that the contribution of trehalose to tolerance to other stresses is either small or inconsistent and that trehalose may not be used as a general predictor of stress tolerance in true stationary phase yeast. Yeast, 1997 Jan, 13(1), 85 - 91 The genes encoding the transcription factor yTAFII60, the G4p1 protein and a putative glucose transporter are contained in a 12.3 kb DNA fragment on the left arm of Saccharomyces cerevisiae chromosome VII; Paoluzi S et al.; We report the nucleotide sequence of a DNA fragment of 12,325 base pairs from the left arm of the Saccharomyces cerevisiae chromosome VII . Inspection of the coding capacity revealed 11 open reading frames (ORFs) longer than 100 amino acids . Five ORFs are significantly homologous to known proteins . The region encoding ORF G2985 corresponds (100%) to the gene encoding the yeast TATA binding protein-associated factor TAFII60 . The G3075 ORF is 47.8% identical to the hypothetical yeast protein YB88 . G3089 shows 36.7% identity to the eel calmodulin . G3085 shows 94.9% identity with the published sequence of the quadruplex DNA binding protein G4p1 . G3090 reveals 46.7% identity with the probable glucose transport protein yBR1625. Yeast, 1997 Jan, 13(1), 73 - 83 Analysis of a 35.6 kb region on the right arm of Saccharomyces cerevisiae chromosome XV; Bordonne R et al.; We report the sequence of a 35,600 bp fragment covering the PET123 region on the right arm of chromosome XV from Saccharomyces cerevisiae . This region contains 19 possible open reading frames (ORFs) of which 16 are non-overlapping ORFs . Eight ORFs correspond to the SPP2, SMP3, PDR5, NFI1, PUP1, PET123 and MTR10 loci, described previously . Two ORFs correspond to yeast homologues of genes from other organisms: O3530 is a member of the large ribosomal subunit protein L13 family and O3560 (SME1 gene) is a 94-codon ORF and is a homologue of the mammalian SmE spliceosomal core protein . Three ORFs (O3513, O3521, O3548) present significant similarities to proteins of unknown function and three ORFs (O3510, O3536, O3545) lack homology to sequences within the databases screened. Yeast, 1997 Jan, 13(1), 65 - 71 The sequence of a 36.7 kb segment on the left arm of chromosome IV from Saccharomyces cerevisiae reveals 20 non-overlapping open reading frames (ORFs) including SIT4, FAD1, NAM1, RNA11, SIR2, NAT1, PRP9, ACT2 and MPS1 and 11 new ORFs; Saren AM et al.; A 36,688 bp fragment from the left arm of chromosome IV of saccharomyces cerevisiae was sequenced . Sequence analysis identified 20 complete non-overlapping open reading frames (ORFs) of at least 100 amino acids . Nine of these correspond to previously identified and sequenced genes: SIT4/PH1, FAD1, NAM1/MTF2, RNA11, SIR2/MAR1, NAT1/AAA1, PRP9, ACT2 and MPS1/RPK1 . Three ORFs show homology to previously sequenced genes . One ORF exhibits a hypothetical yabO/yceC/YfiI family signature and one has the ATP-dependent helicase signature of the DEAD and DEAH box families . Six ORFs show no appreciable homology to any proteins in the database . One of these is identical to yeast expressed sequence tags and therefore corresponds to and expressed gene . In addition, two partial ORFs and 11 ORFs that are totally internal and are not likely to be functional were detected. Yeast, 1997 Jan, 13(1), 55 - 64 Sequencing of a 40.5 kb fragment located on the left arm of chromosome VII from Saccharomyces cerevisiae; Coglievina M et al.; The nucleotide sequence of a 40.5 kb DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae was determined and analysed . Twenty-eight open reading frames (ORFs) longer than 300 nucleotides were identified . Eight of the them correspond to the following known yeast genes: EMP24, GCN1, SPO8, COX13, CDC55, RPS26, COX4 and LSR1, also called GTS1 . Twelve ORFs are new, among them eight show homology with other genes while four have no homology with any sequence in the databases . Eight additional ORFs are internal to or partially overlapping with other ORFs. Yeast, 1997 Jan, 13(1), 31 - 6 Characterization of the rad14-2 mutant of Saccharomyces cerevisiae: implications for the recognition of UV photoproducts by the Rad14 protein; Jones GW et al.; The RAD14 gene of Saccharomyces cerevisiae is required for the incision step of the nucleotide excision repair process . The Rad14 protein can bind zinc, possesses a potential zinc finger DNA binding domain and has been shown to bind specifically to damaged DNA . Differences in UV sensitivity exist between a rad14 deletion strain and a putative rad14 point mutant, the point mutant being more resistant to UV than the deletion strain . Here, we confirm that the rad14 deletion strain repairs neither UV-induced cyclobutane pyrimidine dimers (CPDs) nor endonuclease III-sensitive damage sites, whereas the point mutant cannot repair the former but can repair the latter . From this it can be inferred that the point mutant produces an altered protein product allowing recognition of endonuclease III sensitive sites but not CPDs . To investigate this, the rad14 mutant allele was sequenced . It contained two GC-AT transition mutations when compared to the wild-type RAD14 gene sequence . When the rad14 point mutant sequence is translated, alterations within the putative zinc finger binding domain are observed, with one of the cysteine residues of the zinc binding motif being replaced by tyrosine . This suggests that alterations within the zinc finger binding domain of the Rad14 protein cause changes to the damage recognition properties of the protein . The use of the Rad14 protein from the point mutant should assist in experiments investigating the in vitro binding properties of the Rad14 protein to different types of DNA damage. Yeast, 1997 Jan, 13(1), 9 - 20 The C-terminal domain of Snf3p is sufficient to complement the growth defect of snf3 null mutations in Saccharomyces cerevisiae: SNF3 functions in glucose recognition; Coons DM et al.; The SNF3 protein, Snf3p, of Saccharomyces cerevisiae was initially thought to be a high affinity glucose transporter required for efficient catabolism of low glucose concentrations . We now report evidence suggesting that Snf3p is a regulatory protein and not a catabolic transporter . The C-terminal domain of Snf3p is able to complement the growth defect on solid media of snf3 null mutants independent of attachment to the membrane-spanning domains . However, the C-terminal domain is unable to fully restore high affinity glucose transport to a snf3 null strain . Examination of deletions of the C-terminal domain of intact SNF3 demonstrates that this region is required for both the growth and transport functions of Snf3p . Loss of the SNF3 gene leads to a long-term adaptation phenotype for cells grown in liquid medium at low substrate concentrations in the presence of the respiratory inhibitor, antimycin A . The presence of the C-terminal domain shortens the time required for adaptation in a snf3 null strain . Thus, Snf3p appears to affect ability to adapt to low substrate conditions, but does not confer an absolute defect in uptake of substrate . Taken together, these data suggest that Snf3p is a regulatory protein likely functioning in the detection of glucose. Mol Microbiol, 1997 Jan, 23(2), 203 - 10 Amino acid-dependent regulation of the Saccharomyces cerevisiae GSH1 gene by hydrogen peroxide; Stephen DW et al.; The tripeptide gamma-L-glutamyl-L-cysteinylglycine (glutathione) is one of the major antioxidant molecules of cells and plays a vital role in buffering the cell against reactive oxygen species and toxic electrophiles . In the yeast Saccharomyces cerevisiae, the first enzyme involved in glutathione biosynthesis, gamma-glutamylcysteine synthetase, is encoded by the GSH1 gene . This study shows that the regulation of the yeast GSH1 gene by oxidants and the heavy metal cadmium is at the level of transcription . We also demonstrate that the regulation of the GSH1 gene by H2O2 depends upon the presence of the amino acids glutamate, glutamine and lysine in the media . Moreover, regulation of the GSH1 gene by H2O2, although requiring the Yap1 protein, appears to be mediated by a mechanism distinct from that which regulates the Yap1-dependent induction of genes encoding thioredoxin (TRX2) and a stress-inducible HSP70 (SSA1) by H2O2. Cancer Chemother Pharmacol, 1997, 39(4), 367 - 75 Complementation of temperature-sensitive topoisomerase II mutations in Saccharomyces cerevisiae by a human TOP2 beta construct allows the study of topoisomerase II beta inhibitors in yeast; Meczes EL et al.; We show herein that human DNA topoisomerase II beta is functional in yeast . It can complement a yeast temperature-sensitive mutation in topoisomerase II . The effect on human topoisomerase II beta of a number of topoisomerase II inhibitors was analysed in a yeast in vivo system and compared with that of human topoisomerase II alpha and wild-type yeast topoisomerase II . A drug permeable yeast strain (JN394 top2-4) was used to analyse the in vivo effects of known anti-topoisomerase II agents on human topoisomerase II beta transformants . A parallel analysis on human topoisomerase II alpha transformants provides the first in vivo analysis of the responses of yeast bearing the individual isoforms to these drugs . The strain was analysed at 35 degrees C, a non-permissive temperature at which only plasmid-borne topoisomerase II is active . A shuttle vector with either human topoisomerase II beta, human topoisomerase II alpha or yeast topoisomerase II under the control of a GAL1 promoter was used . The key findings were that amsacrine produced comparable levels of cell killing with both alpha and beta, whilst etoposide, doxorubicin and mitoxantrone produced higher degrees of cell killing with alpha than with beta or yeast topoisomerase II . Merbarone had the greatest effect on the yeast strain bearing plasmid-borne yeast topoisomerase II . Suramin, quercetin and genistein showed little cell killing in this system . This yeast in vivo system provides a powerful way to analyse the effects of anti-topoisomerase II agents on transformants bearing the individual human isoforms . This system also provides a means of analysing putative drug-resistance mutations in human topoisomerase II beta or to select for drug-resistance mutations in human topoisomerase II beta. Microbiology, 1997 Jan, 143 ( Pt 1), 219 - 29 Expulsion of uracil and thymine from the yeast Saccharomyces cerevisiae: contrasting responses to changes in the proton electrochemical gradient; Eddy AA; The outflow of uracil from the yeast Saccharomyces cerevisiae is known to be relatively fast in certain circumstances, to be retarded by proton conductors and to occur in strains lacking a uracil proton symport . In the present work, it was shown that uracil exit from washed yeast cells is an active process, creating a uracil gradient of the order of -80 mV relative to the surrounding medium . Glucose accelerated uracil exit, while retarding its entry . DNP or sodium azide each lowered the gradient to about -30 mV, simultaneously increasing the rate of uracil entry . They also lowered cellular ATP content . Manipulation of the external ionic conditions governing delta mu H+ at the plasma membrane had no detectable effect on uracil transport in yeast preparations thoroughly depleted of ATP . It was concluded that uracil exit is probably not driven by the proton gradient but may utilize ATP directly . It is known that thymine is not normally absorbed by yeast . However, thymine expulsion was here observed during deamination of the substrate 5-methylcytosine in the presence of glucose . In the absence of glucose, or following ATP depletion, thymine uptake from the medium only occurred when delta mu H+ was dissipated, either by DNP or azide, or by manipulation of the external ionic environment . The yeast expelled absorbed thymine when delta mu H+ was restored to the physiological range . The properties of the system corresponded to those of an H+/thymine antiport that is distinct from the mechanism expelling uracil. Mol Biol Cell, 1997 Jan, 8(1), 1 - 11 Three-dimensional analysis and ultrastructural design of mitotic spindles from the cdc20 mutant of Saccharomyces cerevisiae; O'Toole ET et al.; The three-dimensional organization of mitotic microtubules in a mutant strain of Saccharomyces cerevisiae has been studied by computer-assisted serial reconstruction . At the nonpermissive temperature, cdc20 cells arrested with a spindle length of approximately 2.5 microns . These spindles contained a mean of 81 microtubules (range, 56-100) compared with 23 in wild-type spindles of comparable length . This increase in spindle microtubule number resulted in a total polymer length up to four times that of wild-type spindles . The spindle pole bodies in the cdc20 cells were approximately 2.3 times the size of wild-type, thereby accommodating the abnormally large number of spindle microtubules . The cdc20 spindles contained a large number of interpolar microtubules organized in a "core bundle." A neighbor density analysis of this bundle at the spindle midzone showed a preferred spacing of approximately 35 nm center-to-center between microtubules of opposite polarity . Although this is evidence of specific interaction between antiparallel microtubules, mutant spindles were less ordered than the spindle of wild-type cells . The number of noncore microtubules was significantly higher than that reported for wild-type, and these microtubules did not display a characteristic metaphase configuration . cdc20 spindles showed significantly more cross-bridges between spindle microtubules than were seen in the wild type . The cross-bridge density was highest between antiparallel microtubules . These data suggest that spindle microtubules are stabilized in cdc20 cells and that the CDC20 gene product may be involved in cell cycle processes that promote spindle microtubule disassembly. Genetics, 1997 Jan, 145(1), 85 - 96 The Swi5 transcription factor of Saccharomyces cerevisiae has a role in exit from mitosis through induction of the cdk-inhibitor Sic1 in telophase; Toyn JH et al.; Deactivation of the B cyclin kinase (Cdc28/Clb) drives the telophase to G1 cell cycle transition . Here we investigate one of the control pathways than contributes to kinase deactivation, involving the cell cycle-regulated production of the cdk inhibition Sic1 . We show that the cell cycle timing of SIC1 expression depends on the transcription factor Swi5, and that Swi5-dependent SIC1 expression begins during telophase . In contrast to Swi5, the related transcription factor Ace2, which can also induce SIC1 expression, is not active during telophase . The functional consequence of Swi5-regulated SIC1 expression in vivo is that both sic1 delta and swi5 delta strains have identical mitotic exit-related phenotypes . First, both are synthetically lethal with dbj2 delta, resulting in cell cycle arrest in telophase . Second, both are hypersensitive to overexpression of the B cyclin CLB2 . Thus Swi5-dependent activation of the SIC1 gene contributes to the deactivation of the B cyclin kinase, and hence exit from mitosis. Genetics, 1997 Jan, 145(1), 45 - 62 RAD9, RAD17, and RAD24 are required for S phase regulation in Saccharomyces cerevisiae in response to DNA damage; Paulovich AG et al.; We have previously shown that a checkpoint dependent on MEC1 and RAD53 slows the rate of S phase progression in Saccharomyces cerevisiae in response to alkylation damage . Whereas wild-type cells exhibit a slow S phase in response to damage, mec1-1 and rad53 mutants replicate rapidly in the presence or absence of DNA damage . In this report, we show that other genes (RAD9, RAD17, RAD24) involved in the DNA damage checkpoint pathway also play a role in regulating S phase in response to DNA damage . Furthermore, RAD9, RAD17, and RAD24 fall into two groups with respect to both sensitivity to alkylation and regulation of S phase . We also demonstrate that the more dramatic defect in S phase regulation in the mec1-1 and rad53 mutants is epistatic to a less severe defect seen in rad9 delta, rad 17 delta, and rad24 delta . Furthermore, the triple rad9 delta rad17 delta rad24 delta mutant also has a less severe defect than mec1-1 or rad53 mutants . Finally, we demonstrate the specificity of this phenotype by showing that the DNA repair and/or checkpoint mutants mgt1 delta, mag1 delta, apn1 delta, rev3 delta, rad18 delta, rad16 delta, dun1-delta 100, sad4-1, tel1 delta, rad26 delta, rad51 delta, rad52-1, rad54 delta, rad14 delta, rad1 delta, pol30-46, pol30-52, mad3 delta, pds1 delta/esp2 delta, pms1 delta, mlh1 delta, and msh2 delta are all proficient at S phase regulation, even though some of these mutations confer sensitivity to alkylation. Nucleic Acids Res, 1997 Jan 1, 25(1), 57 - 62 Yeast Protein database (YPD): a database for the complete proteome of Saccharomyces cerevisiae; Payne WE et al.; The Yeast Protein Database (YPD) is a database for the proteins of the budding yeast,Saccharomyces cerevisiae . YPD is the first annotated database for the complete proteome of any organism . Now that the complete genome sequence of yeast is available, YPD contains entries for each of the characterized proteins and for each of the uncharacterized proteins predicted from the sequence . Contained in YPD are the calculated properties of each protein such as molecular weight and isoelectric point, experimentally determined properties such as subcellular localization and post-translational modifications, and extensive annotations from the yeast literature . YPD contains 25 000 lines of textual annotation that describe the known functions, mutant phenotypes, interactions, and other properties for the approximately 6000 proteins in the yeast proteome . The information in YPD is updated daily, and it is available on the World Wide Web at . J Gen Virol, 1997 Jan, 78 ( Pt 1), 39 - 43 Purification and characterization of the NS3 serine protease domain of hepatitis C virus expressed in Saccharomyces cerevisiae; Markland W et al.; cDNA encoding the putative core of the hepatitis C virus NS3 serine protease domain (residues 1-181 of NS3; NS3 (181)) was expressed as an N-terminally (His)6-tagged fusion protein in Saccharomyces cerevisiae . NS3 (181) protease activity was found in soluble cell lysates, and the N-terminal metal-chelating domain facilitated the efficient purification of active enzyme, using immobilized metal affinity chromatography . The purified NS3(181), protease activity was characterized by assaying the trans-cleavage of in vitro transcription-translation generated substrates, and subsequently a previously unobserved cleavage site within the NS5A region was identified . The inhibitory effect of known protease inhibitors was also examined . It is hoped that availability of this method for the expression and purification of the NS3(181) protease will facilitate the development of anti-hepatitis C therapies. FEMS Microbiol Lett, 1997 Jan 1, 146(1), 39 - 46 Ca(2+)-transporting ATPase(s) of the reticulum type in intracellular membranes of Saccharomyces cerevisiae: biochemical identification; Okorokov LA et al.; Several lines of evidence are presented to show that the Ca(2+)-ATPase activity of total yeast membranes is due to the reticulum (R) type of Ca(2+)-ATPase: (1) Neither calmodulin nor low concentrations of calmodulin antagonists change Ca2+ uptake; (2) removal of plasma membranes (PM) following Con A treatment of spheroplasts (SP) does not significantly alter Ca2+ uptake by the remaining membranes, but increases its specific activity 3.5-fold; (3) after incubation of membranes with {gamma-32P}ATP, SDS-PAGE shows the formation of acyl phosphate intermediates with molecular masses of around 100, 180-190 and 205 kDa; formation of these acyl phosphates requires Ca2+ and is blocked by cyclopiazonic acid, La3+ ions and in the absence of Ca2+ . The data on fractionation of yeast membranes are consistent with the suggestion that both the ER and the Golgi are equipped with Ca(2+)-ATPase(s). J Bacteriol, 1997 Jan, 179(2), 463 - 9 Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae; Popolo L et al.; The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae . The ggp1delta mutant shows morphogenetic defects which suggest changes in the cell wall matrix . In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1delta mutant cells . In these cells, the level of alkali-insoluble 1,6-beta-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan . Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility . The increase of chitin in ggp1delta cells was found to be essential since the chs3delta ggp1delta mutations determined a severe reduction in the growth rate and in cell viability . Electron microscopy analysis showed the loss of the typical structure of yeast cell walls . Furthermore, in the chs3delta ggp1delta cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled . We tested the effect of inhibition of chitin synthesis also by a different approach . The ggp1delta cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug . In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1delta kre6delta and the ggp1delta pkc1delta combined mutations . Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-beta-D-glucans with 1,6-beta-D-glucans and with chitin) . Chitin is essential to compensate for the defects due to the lack of Ggp1p . Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells. Arch Biochem Biophys, 1997 Jan 1, 337(1), 27 - 33 Mutagenesis of beta-V198 in the F1-ATPase of yeast Saccharomyces cerevisiae and its role in binding nucleotide; Sosa-Peinado A et al.; Residue beta-V198 of the yeast mitchondrial F1-ATPase abuts the P-loop motif and the side chain is within 3.8 A of the nucleotide as shown in the crystal structure of the bovine ATPase {J . P . Abrahams, A . G . W . Leslie, R . Lutter, and J . E . Walker (1984) Nature 370,621-628} . This study has made and analyzed 17 replacements of V198 to understand the importance of the side chain in the nucleotide binding site . In addition, a suppressor of V198S, beta-L390F, was studied in the presence of various replacements at position 198 . In vivo and in vitro analyses indicate that the Val side chain is critical for forming a stable and active enzyme . Biochemical analysis of mitochondria isolated from the mutant strains indicates that amino acids with hydrophobic side chains are the most effective replacements . In addition, size is important, but a large side chain can be largely compensated for until the size reaches that of the Phe and Trp . A methyl group is the minimal side chain necessary for function, as the beta-subunit is not stable in vivo with Gly at position 198 . These results indicate that V198 forms critical hydrophobic interactions with the adenine ring of the nucleotide. Mol Cell Biol, 1997 Jan, 17(1), 287 - 95 Evidence that Spt3 functionally interacts with Mot1, TFIIA, and TATA-binding protein to confer promoter-specific transcriptional control in Saccharomyces cerevisiae; Madison JM et al.; Spt3 of Saccharomyces cerevisiae is a factor required for normal transcription from particular RNA polymerase II-dependent promoters . Previous genetic and biochemical analyses have shown that Spt3 interacts with the yeast TATA-binding protein (TBP) . To identify other factors that might interact with Spt3, we have screened for mutations that, in combination with an spt3 null mutation, lead to inviability . In this way, we have identified a mutation in MOT1, which encodes an ATP-dependent inhibitor of TBP binding to TATA boxes: Previous analyses suggested that Mot1 causes repression in vivo . However, our analysis of mot1 mutants shows that, similar to spt3 mutants, they have decreased levels of transcription from certain genes, suggesting that Mot1 may function as an activator in vivo . In addition, mot1 mutants have other phenotypes in common with spt3 delta mutants, including suppression of the insertion mutation his4-912 delta . Motivated by these Spt3-Mot1 genetic interactions, we tested for genetic interactions between Spt3 and the general transcription factor TFIIA . TFIIA has been shown previously to be functionally related to Mot1 . We found that overexpression of TFIIA partially suppresses an spt3 delta mutation, that toa1 mutants have Spt-phenotypes, and that spt3 delta toa1 double mutants are inviable . We believe that, taken together, these data suggest that Spt3, Mot1, and TFIIA cooperate to regulate TBP-DNA interactions, perhaps at the level of TATA box selection in vivo. Mol Cell Biol, 1997 Jan, 17(1), 145 - 53 The 39-kilodalton subunit of eukaryotic translation initiation factor 3 is essential for the complex's integrity and for cell viability in Saccharomyces cerevisiae; Naranda T et al.; Eukaryotic translation initiation factor 3 (eIF3) in the yeast Saccharomyces cerevisiae comprises about eight polypeptides and plays a central role in the binding of methionyl-tRNAi and mRNA to the 40S ribosomal subunit . The fourth largest subunit, eIF3-p39, was gel purified, and a 12-amino-acid tryptic peptide was sequenced, enabling the cloning of the TIF34 gene . TIF34 encodes a 38,753-Da protein that corresponds to eIF3-p39 in size and antigenicity . Disruption of TIF34 is lethal, and depletion of eIF3-p39 by glucose repression of TIF34 expressed from a GAL promoter results in cessation of cell growth . As eIF3-p39 levels fall, polysomes become smaller, indicating a role for eIF3-p39 in the initiation phase of protein synthesis . Unexpectedly, depletion results in degradation of all of the subunit proteins of eIF3 at a rate much faster than the normal turnover rates of these proteins . eIF3-p39 has 46% sequence identity with the p36 subunit of human eIF3 . Both proteins are members of the WD-repeat family of proteins, possessing five to seven repeat elements . Taken together, the results indicate that eIF3-p39 plays an important, although not necessarily direct, role in the initiation phase of protein synthesis and suggest that it may be required for the assembly and maintenance of the eIF3 complex in eukaryotic cells. Mol Cell Biol, 1997 Jan, 17(1), 123 - 34 An Ssn6-Tup1-dependent negative regulatory element controls sporulation-specific expression of DIT1 and DIT2 in Saccharomyces cerevisiae; Friesen H et al.; Sporulation of the yeast Saccharomyces cerevisiae is a process of cellular differentiation that occurs in MATa/MAT alpha diploid cells in response to starvation . The sporulation-specific genes DIT1 and DIT2, which are required for spore wall formation, are activated midway through the sporulation program, with maximal transcript accumulation occurring at the time of prospore enclosure . In this study, we have identified a negative regulatory element, termed NREDIT, that is located between the start sites of transcription of these divergently transcribed genes . This element, which prevents expression of the DIT1 and DIT2 genes during vegetative growth, reduces expression of a CYC1-lacZ reporter gene more than 1,000-fold and acts in an orientation- and position-independent manner . We found that the ability of NREDIT to turn of expression of the reporter gene and the chromosomal DIT1 and DIT2 genes in vegetative cells requires the Ssn6-Tup1 repression complex . Interestingly, NREDIT-mediated repression of the reporter gene is maintained during sporulation . Derepression during sporulation requires complex interactions among several cis-acting elements . These are present on an approximately 350-bp DNA fragment extending from NREDIT to the TATA box and an approximately 125-bp fragment spanning the TATA box of DIT1 . Additionally, a region of NREDIT which is very similar in sequence to UASSPS4, an element that activates gene expression midway through sporulation, contributes both to vegetative repression and to sporulation-specific induction of DIT1 . We propose a model to explain the requirement for multiple elements in overcoming NREDIT-mediated repression during sporulation. J Biol Chem, 1996 Dec 27, 271(52), 33261 - 7 The Saccharomyces cerevisiae Prp5 protein has RNA-dependent ATPase activity with specificity for U2 small nuclear RNA; O'Day CL et al.; The Saccharomyces cerevisiae protein Prp5 is a member of the "DEAD box" family of putative RNA-dependent ATPases and helicases . The protein was purified from Escherichia coli and determined to be an RNA-dependent ATPase . The ATPase activity is 7-fold more specific for full-length U2 than for any of the other small nuclear RNAs or nonspecific RNAs tested . An RNaseH assay in extracts was used to demonstrate that Prp5 mediates an ATP-dependent conformational change in the intact U2 small nuclear ribonucleoprotein . We propose that this conformational change makes the branch point pairing sequence of U2 RNA accessible for pairing with the intron allowing formation of the pre-spliceosome. J Biol Chem, 1996 Dec 20, 271(51), 33131 - 40 Biochemical studies of Saccharomyces cerevisiae myristoyl-coenzyme A:protein N-myristoyltransferase mutants; Zhang L et al.; Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential 455-residue, monomeric enzyme that catalyzes the transfer of myristate from myristoyl-CoA to the NH2-terminal Gly residue of cellular proteins . Nmt1p has an ordered Bi Bi reaction mechanism with binding of myristoyl-CoA occurring before binding of peptide substrates . To define residues important for function, the polymerase chain reaction was used to generate random mutations in the NMT1 gene . A colony color sectoring assay was used to screen a library of 52,000 transformants for nmt1 alleles encoding enzymes with reduced activity . nmt1 alleles were identified that produced temperature-sensitive (ts) growth arrest due to substitutions affecting eight residues conserved in orthologous Nmts: Asn102, Ala202, Cys217, Ser328, Val395, Asn404, Leu420, and Asn426 . Ala202 --> Thr, Cys217 --> Arg, Ser328 --> Pro, Asn404 --> Tyr, and Asn426 --> Ile produced the most severe ts phenotype . Their effects on the functional properties of the enzyme's myristoyl-CoA and peptide binding sites were defined by purifying each mutant from Escherichia coli and conducting in vitro kinetic analyses with acyl-CoA and peptide substrates and with two competitive inhibitors: S-(2-oxo)pentadecyl-CoA, a nonhydrolyzable myristoyl-CoA analog, and SC-58272, a peptidomimetic derived from the NH2-terminal sequence of an Nmt1p substrate (ADP-ribosylation factor-2, Arf2p) . None of the substitutions affect the enzyme's acyl chain length selectivity . When compared with wild type Nmt1p, Cys217 --> Arg produces 3- and 6-fold increases in Ki for SC-58272 at 24 and 37 degrees C but no change in Ki for S-(2-oxo)pentadecyl-CoA, indicating that the substitution selectively affects Nmt1p's peptide binding site . Asn426 --> Ile selectively perturbs the myristoyl-CoA binding site, resulting in the most pronounced reduction in affinity for S-(2-oxo)pentadecyl-CoA (12- and 20-fold) . Ala202 --> Thr, which confers the most severe ts phenotype, provides an example of a substitution that affects both sites, producing 3- and 6-fold increases in the Ki for S-(2-oxo)pentadecyl-CoA and 6- and 9-fold increases in the Ki for SC-58272 at 24 and 37 degrees C . An N-myristoylation-dependent change in the electrophoretic mobility of Arf1p was used to assay the effects of the mutants on cellular levels of protein N-myristoylation under a variety of growth conditions . The ts growth arrest produced by nmt1 alleles correlates with a reduction in myristoyl-Arf1p to </=50% of total cellular Arf1p. J Biol Chem, 1996 Dec 20, 271(51), 33123 - 30 The light chain subunit is required for clathrin function in Saccharomyces cerevisiae; Chu DS et al.; Clathrin, a multimeric protein involved in intracellular protein trafficking, is composed of three heavy chains (Chc) and three light chains (Clc) . Upon disruption (clc1Delta) of the single Clc-encoding gene (CLC1) in yeast, the steady state protein levels of Chc decreased 5-10-fold compared with wild type cells; consequently, phenotypes exhibited by clc1Delta cells may result indirectly from the loss of Chc as opposed to the absence of Clc . As an approach to directly examine Clc function, clc1Delta strains were generated that carry a multicopy plasmid containing the clathrin heavy chain gene (CHC1), resulting in levels of Chc 5-10-fold elevated over wild-type levels . As with deletion of CHC1, deletion of CLC1 results in defects in growth, receptor-mediated endocytosis, and maturation of the mating pheromone alpha-factor . However, elevated Chc expression in clc1Delta cells partially suppresses the growth and alpha-factor maturation defects displayed by clc1Delta cells alone . Biochemical analyses indicate that trimerization and assembly of Chc are perturbed in the absence of Clc, resulting in vesiculation defects . Our results demonstrate that the light chain subunit of clathrin is required for efficient Chc trimerization, proper formation of clathrin coats, and the generation of clathrin-coated vesicles. J Biol Chem, 1996 Dec 20, 271(51), 32975 - 80 Identification of Triton X-100 insoluble membrane domains in the yeast Saccharomyces cerevisiae . Lipid requirements for targeting of heterotrimeric G-protein subunits; Kubler E et al.; Low density Triton X-100 insoluble (LDTI) membrane domains are found in most mammalian cell types . Previous biochemical and immunolocalization studies have revealed the presence of G-protein coupled receptors and heterotrimeric G-protein subunits (Galpha and Gbetagamma subunits) within these structures, implicating mammalian LDTI membrane domains in G-protein coupled signaling . Here, we present biochemical evidence that similar LDTI structures exist in a genetically tractable organism, the yeast Saccharomyces cerevisiae . Yeast LDTI membranes were purified based on the known biochemical properties of mammalian LDTI membranes: (i) their Triton X-100 insolubility; and (ii) their discrete buoyant density in sucrose gradients . As with purified mammalian LDTI membranes, these yeast LDTI membranes harbor the subunits of the heterotrimeric G-proteins (Galpha and Gbetagamma subunits) . Other plasma membrane marker proteins (the plasma membrane H+-ATPase and a GPI-linked protein Gas1p) are preferentially excluded from these purified fractions . Mutational and genetic analyses were performed to define the requirements for the targeting of G-protein subunits to these yeast membrane domains . We find that the targeting of Galpha is independent of myristoylation, whereas targeting of Ggamma requires prenylation . Perhaps surprisingly, the targeting of Gbeta to this membrane domain did not require coexpression of Ggamma . It should now be possible to dissect the function of LDTI membrane domains using yeast as a model genetic system. J Biol Chem, 1996 Dec 20, 271(51), 32695 - 701 Expression and purification of the RNA polymerase III transcription specificity factor IIIB70 from Saccharomyces cerevisiae and its cooperative binding with TATA-binding protein; Librizzi MD et al.; Transcription by RNA polymerase III (pol III) in yeast requires the assembly of an initiation complex comprising the TATA-binding protein (TBP), a 90-kDa polypeptide (TFIIIB90), and a 70-kDa polypeptide (TFIIIB70) . TFIIIB70 interacts with TBP, a unique pol III subunit, C34, and the 131-kDa subunit of the pol III-specific complex, TFIIIC . TFIIIB70 was expressed in Escherichia coli and purified to homogeneity . The specific transcription activity of rTFIIIB70 is 22-58% that of the native yeast and in vitro synthesized factor . However, only a small fraction (0.07-0.32%) of the TFIIIB70 from these sources results in the synthesis of full-length RNA . The data suggest that TFIIIB70 function may be limited by an unfavorable recruitment equilibrium into the preinitiation complex . Quantitative DNase I "footprint" titrations of yeast TBP to the adenovirus major late promoter were conducted at a series of constant TFIIIB70 concentrations . A value of -0.7 +/- 0.2 kcal/mol was determined for the cooperative free energy of formation of the TBP.TFIIIB70.DNA complex at concentrations of TFIIIB70 sufficient to partition all of the binding cooperativity to the TBP binding isotherm . A Kd of 44 +/- 23 nM characterizes the TFIIIB70 concentration dependence of the TBP.TFIIIB70 cooperativity . The relationship deltalog K/deltalog (TFIIIB70) is consistent with the linkage of a single molecule of TFIIIB70 with the TBP-promoter binding reaction. Biochim Biophys Acta, 1996 Dec 18, 1277(3), 209 - 16 Kinetic analysis of hexose uptake in Saccharomyces cerevisiae cultivated in continuous culture; Meijer MM et al.; As the expression of the hexose transporters in Saccharomyces cerevisiae is regulated by the external fructose concentration, in vivo fructose transport was investigated at a number of different but constant fructose concentrations in a continuous culture . The in vitro uptake rate and kinetic parameters were studied by uptake of 14C-labelled fructose . From Eadie-Hofstee plots, based on the results of the in vitro uptake experiment, K(m) and Vmax for the different steady-state conditions were determined . These results demonstrate that as fructose concentrations increase, fructose consumption rates and K(m) increase and Vmax slightly decreases . However, remarkably a large discrepancy was observed between in vivo determined consumption rates in the continuous culture and the in vitro uptake rates from the 14C-labelled fructose uptake experiment on cells from an identical steady-state condition . In every condition the in vivo consumption rate was approx . 2-3 times higher than the in vitro uptake rate . A comparable discrepancy was observed for glucose uptake rates; however, in vivo and in vitro galactose uptake rates were identical . These observations imply that kinetic analyses of sugar transport mechanisms can only be performed correctly when accompanied by a comparison with in vivo determined consumption rates. FEBS Lett, 1996 Dec 16, 399(3), 259 - 63 The heat shock factor and mitochondrial Hsp70 are necessary for survival of heat shock in Saccharomyces cerevisiae; Nwaka S et al.; A heat shock recovery assay on solid medium (Nwaka et al . (1995) J . Biol . Chem . 270, 10193-10198) as well as the classical cell counting method were used to investigate the function of some heat shock proteins in thermotolerance . We show that expression of intact heat shock factor protein (HSF), which regulates the stress induced expression of heat shock proteins (HSPs), is necessary for recovery from heat shock . A HSF1 mutant (hsf1-m3) which does not induce the expression of some heat shock proteins at heat stress (37-40 degrees C) is defective in recovery after heat shock at 50-52 degrees C compared to a corresponding wild-type strain in both stationary and exponentially growing cells . Using two temperature sensitive mutants of the mitochondrial Hsp70 (ssc1-2 and ssc1-3) encoded by the SSC1 gene, we show that the ssc1-3 mutant, which has a mutation in the ATPase domain, is defective in recovery after heat shock in contrast to the ssc1-2 mutant, which has a mutation in the peptide binding domain . Different binding capacities for unfolded proteins are shown to be the molecular reason for the observed phenotypes . The thermotolerance defect of the hsf1-m3 and ssc1-3 mutants is demonstrated for both glucose and glycerol media. Eur J Biochem, 1996 Dec 15, 242(3), 747 - 59 Pre-tRNA 3'-processing in Saccharomyces cerevisiae . Purification and characterization of exo- and endoribonucleases; Papadimitriou A et al.; We investigated ribonucleases from Saccharomyces cerevisiae which are active in pre-tRNA 3'-processing in vitro . Two pre-tRNA 3'-exonucleases with molecular masses of 33 and 60 kDa, two pre-tRNA 3'-endonucleases with molecular masses of 45 kDa/60 kDa and 55 kDa and 70-kDa 3'-pre-tRNase were purified from yeast whole cell extracts by several successive chromatographic purification steps . The purified exonucleases are non-processive 3'-exonucleases that catalyze the exonucleolytic processing of 3'-trailer sequences of pre-tRNAs to produce mature tRNAs . The 45-kDa/60-kDa 3'-endonuclease is tRNA-specific and catalyzes the processing of pre-tRNAs in a single endonucleolytic step . Two isoenzymes of this activity (p45 and p60) were identified by chromatography . The second endonuclease, p55, is dependent on monovalent ions and cleaves about three nucleotides downstream the mature 3'-end . All of the purified 3'-pre-tRNases accept homologous as well as heterologous pre-tRNA substrates . Pre-tRNAs carrying a 5'-leader are processed with almost the same efficiency as those lacking this 5'-leader . Mature tRNAs carrying the CCA 3'-sequence and tRNA pseudogene products carrying mutations in the mature domain are processed by the 3'-exonucleases, not by the 3'-endonucleases . The specific endonuclease p45/p60 discriminates between UUUOH as a 3'-flank, which is cleaved, and the CCA 3'-end of mature tRNAs, which is not cleaved . This study suggests that several 3'-pre-tRNases are active on tRNA precursors in vitro and might therefore in pre-tRNA 3'-processing in yeast, partly in a cooperative manner. Genes Dev, 1996 Dec 15, 10(24), 3081 - 93 Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p; Cohen-Fix O et al.; Anaphase initiation has been postulated to be controlled through the ubiquitin-dependent proteolysis of an unknown inhibitor . This process involves the anaphase promoting complex (APC), a specific ubiquitin ligase that has been shown to be involved in mitotic cyclin degradation . Previous studies demonstrated that in Saccharomyces cerevisiae, Pds1 protein is an anaphase inhibitor and suggested that it may be an APC target . Here we show that in yeast cells and in mitotic Xenopus extracts Pds1p is degraded in an APC-dependent manner . In addition, Pds1p is directly ubiquitinated by the Xenopus APC . In budding yeast Pds1p is degraded at the time of anaphase initiation and nondegradable derivatives of Pds1p inhibit the onset of anaphase . We conclude that Pds1p is an anaphase inhibitor whose APC-dependent degradation is required for the initiation of anaphase. FEMS Microbiol Lett, 1996 Dec 15, 145(3), 401 - 7 The beta-1, 6-glucan containing side-chain of cell wall proteins of Saccharomyces cerevisiae is bound to the glycan core of the GPI moiety; Van Der Vaart JM et al.; Cell wall proteins of Saccharomyces cerevisiae are anchored by means of a beta-1, 6-glucan-containing side-chain . It is not known whether this chain is linked to the protein part (e.g . through carbohydrate side-chains) or to the glycosylphosphatidylinositol (GPI) moiety of cell wall proteins . An IgA protease recognition site was introduced in Cwp2p, a beta-1, 6-glucosylated cell wall protein, immediately N-terminal from the omega amino acid (the attachment site of the GPI moiety) . Proteolytic cleavage of this site revealed that the beta-1, 6-glucan epitope was not linked to the protein part . We conclude that neither N-or O-glycosylation is involved in beta-glucosylation of cell wall proteins . This confirms that the glycan core of the GPI moiety is the probable beta-1, 6-glucan attachment site. Arch Biochem Biophys, 1996 Dec 15, 336(2), 268 - 74 Molecular cloning and kinetic characterization of a flavin-containing monooxygenase from Saccharomyces cerevisiae; Suh JK et al.; An open reading frame from yeast coding for a homologue of flavin containing monooxygenase (FMO) has been cloned into several Escherichia coli expression vectors . A His10 peptide attached to the amino terminus produced a high yield of soluble protein when coexpressed with GroEL and GroES . The protein was purified on an affinity column and characterized . The protein binds one mole per mole of flavin but the binding is relatively weak and 50 microM exogenous FAD is used to maintain full occupancy . The yeast enzyme, like mammalian enzymes, exhibits NADPH oxidase activity . The enzyme does not catalyze the oxidation of amines, but thiols, including glutathione, cysteine, and cysteamine, show substrate activity . The Km values for these are 7.0, 9.9, and 1.3 mM, respectively; kcat values are 94, 246, and 94 per min, respectively . The enzyme apparently does not accept xenobiotic compounds but may be involved in maintaining cellular reducing potential, probably through its action on cysteamine . This activity may represent the initial role of the FMO family of enzymes, giving rise to the multigene family of drug metabolizing enzymes seen in modern mammals. Biochem Biophys Res Commun, 1996 Dec 13, 229(2), 540 - 7 Structure and functional analysis of the multistress response gene DDR2 from Saccharomyces cerevisiae; Kobayashi N et al.; The DDR2 gene is a multistress response gene in Saccharomyces cerevisiae that is transcriptionally activated by more than thirteen xenobiotic agents and environmental or physiological stresses . The DDR2 gene encodes a small hydrophobic 61 amino acid polypeptide located on chromosome XV adjacent to the SPE2 locus . Disruption alleles of the DDR2 gene have been constructed and these ddr2 delta mutants show no defect in heat shock recovery or thermotolerance and appear normal for DNA damage sensitivity and mutagenesis. Biochemistry, 1996 Dec 10, 35(49), 15704 - 14 Expression and biochemical characterization of iron regulatory proteins 1 and 2 in Saccharomyces cerevisiae; Phillips JD et al.; Iron-regulatory proteins (IRPs) 1 and 2 are cytosolic RNA-binding proteins that bind to specific stem-loop structures, termed iron-responsive elements (IREs) that are located in the untranslated regions of specific mRNAs encoding proteins involved in iron metabolism . The binding of IRPs to IREs regulates either translation or stabilization of mRNA . Although IRP1 and IRP2 are similar proteins in that they are ubiquitously expressed and are negatively regulated by iron, they are regulated by iron by different mechanisms . IRP1, the well-characterized IRP in cells, is a dual-function protein exhibiting either aconitase activity when cellular iron is abundant or RNA-binding activity when cellular iron is scarce . In contrast, IRP2 lacks detectable aconitase activity and functions exclusively as an RNA-binding protein . To study and compare the biochemical characteristics of IRP1 and IRP2, we expressed wild-type and mutant rat IRP1 and IRP2 in the yeast Saccharomyces cerevisiae . IRP1 and IRP2 expressed in yeast bind the IRE RNA with high affinity, resulting in the inhibition of translation of an IRE-reporter mRNA . Mutant IRP2s lacking a 73 amino acid domain unique to IRP2 and a mutant IRP1 containing an insertion of this domain bound RNA, but lacked detectable aconitase activity, suggesting that the presence of this domain prevents aconitase activity . Like IRP1, the RNA-binding activity of IRP2 was sensitive to inactivation by N-ethylmaleimide (NEM) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), indicating IRP2 contains a cysteine(s) that is (are) necessary for RNA binding . However, unlike IRP1, where reconstitution of the 4Fe-4S cluster resulted in a loss in RNA-binding activity, the RNA-binding activity of IRP2 was unaffected using the same iron treatment . These data suggested that IRP2 does not contain a 4Fe-4S cluster similar to the cluster in IRP1, indicating that they sense iron by different mechanisms. Biochim Biophys Acta, 1996 Dec 6, 1291(3), 206 - 14 The retention mechanism of cell wall proteins in Saccharomyces cerevisiae . Wall-bound Cwp2p is beta-1,6-glucosylated; van der Vaart JM et al.; It has been proposed that the cell wall proteins of Saccharomyces cerevisiae are anchored by means of a beta-1,6-glucose-containing side chain . Recently, we have identified three cell wall mannoproteins . Two of these mannoproteins are recognized in their cell wall bound form by an antiserum raised against beta-1,6-glucan but the third, Cwp2p, is not . This could indicate the existence of alternative retention mechanisms for cell wall proteins . Western analysis of a fusion protein consisting of Cwp2p and the reporter enzyme alpha-galactosidase revealed that this protein is glycosyl phosphatidylinositol-anchored in the intracellular precursor form and is recognized by an anti beta-1,6-glucan antiserum in the cell wall bound form . The cell wall bound forms of fusion proteins consisting of the anchor regions of Sed1p or Flo1p and alpha-galactosidase were also recognized by an anti beta-1,6-glucan antiserum . This is consistent with the existence of a general anchoring mechanism of proteins to the cell wall by means of a beta-1,6-glucose-containing carbohydrate chain . Western analysis of a yeast strain producing c-myc epitope tagged Cwp2p revealed that this protein is only detectable if fatty acid chains are present on the protein, indicating that the lack of recognition of Cwp2p by an anti beta-1,6-glucan antiserum is caused by a blotting artefact of the mature protein. J Biol Chem, 1996 Dec 6, 271(49), 31420 - 5 Two sporulation-specific chitin deacetylase-encoding genes are required for the ascospore wall rigidity of Saccharomyces cerevisiae; Christodoulidou A et al.; The formation of the ascospore wall of Saccharomyces cerevisiae requires the coordinate activity of enzymes involved in the biosynthesis of its components such as chitosan, the deacetylated form of chitin . We have cloned the CDA1 and CDA2 genes which together account for the total chitin deacetylase activity of the organism . We have shown that expression of these genes is restricted to a distinct time period during sporulation . The two genes are functionally redundant, each contributing equally to the total chitin deacetylase activity . Diploids disrupted for both genes sporulate as efficiently as wild type cells, and the resulting mutant spores are viable under standard laboratory conditions . However, they fail to emit the natural fluorescence of yeast spores imparted by the dityrosine residues of the outermost ascospore wall layer . Moreover, mutant spores are relatively sensitive to hydrolytic enzymes, ether, and heat shock, a fact that underscores the importance of the CDA genes for the proper formation of the ascospore wall. EMBO J, 1996 Dec 2, 15(23), 6671 - 9 ORC- and Cdc6-dependent complexes at active and inactive chromosomal replication origins in Saccharomyces cerevisiae; Santocanale C et al.; We have developed a genomic footprinting protocol which allows us to examine protein-DNA interactions at single copy chromosomal origins of DNA replication in the budding yeast Saccharomyces cerevisiae . We show that active replication origins oscillate between two chromatin states during the cell cycle: an origin recognition complex (ORC)-dependent post-replicative state and a Cdc6p-dependent pre-replicative state . Furthermore, we show that both post- and pre-replicative complexes can form efficiently on closely apposed replicators . Surprisingly, ARS301 which is active as an origin on plasmids but not in its normal chromosomal location, forms ORC- and Cdc6p-dependent complexes in both its active and inactive contexts . Thus, although ORC and Cdc6p are essential for initiation, their binding is not sufficient to dictate origin use. Mutat Res, 1996 Dec 2, 364(3), 209 - 15 Positional cloning of the Drosophila melanogaster mei-9 gene, the putative homolog of the Saccharomyces cerevisiae RAD1 gene; Araj H et al.; Mutation of the Drosophila melanogaster mei-9 gene causes enhanced mutagen sensitivity, DNA excision repair incision deficiency, germ line hypermutability, mitotic chromosome breakage and instability, and, in females, increased levels of meiotic non-disjunction and decreased levels of meiotic recombination . This paper reports on the utilization of a positional mapping strategy combined with a differential expression assay to clone the mei-9 gene . We report that mei-9 is homologous to the yeast repair gene, RAD1, and demonstrate that the mei-9 gene transcript is absent in the mei-9AT3 mutant. Plant Cell Physiol, 1996 Dec, 37(8), 1090 - 3 Vacuolar function in the phosphate homeostasis of the yeast Saccharomyces cerevisiae; Shirahama K et al.; We studied physiological roles of the yeast vacuole in the phosphate metabolism using 31P-in vivo nuclear magnetic resonance (NMR) spectroscopy . Under phosphate starvation wild-type yeast cells continued to grow for two to three generations, implying that wild-type cells contain large phosphate pool to sustain the growth . During the first four hours under the phosphate starved condition, the cytosolic phosphate level was maintained almost constant, while the vacuolar pool of phosphate decreased significantly . 31P-NMR spectroscopy on the intact cells and perchloric acid (PCA) extracts showed that drastic decrease of polyphosphate took place during this phase . In contrast, delta slp1 cells, which were defective in the vacuolar compartment, thus lacked polyphosphate, ceased their growth immediately when they faced to phosphate starvation . Taken together, we conclude that vacuolar polyphosphate provides an active pool for phosphate and is mobilized to cytosol during phosphate starvation and sustained cell growth for a couple rounds of cell cycle. Glycobiology, 1996 Dec, 6(8), 805 - 10 Cloning and analysis of the MNN4 gene required for phosphorylation of N-linked oligosaccharides in Saccharomyces cerevisiae; Odani T et al.; The Saccharomyces cerevisiae MNN4 gene, which is involved in mannosylphosphate transfer from GDP-mannose to N-linked oligosaccharide, has been cloned from a lambda phage containing a yeast chromosome XI DNA fragment . The MNN4 ORF encodes a protein of 1178 amino acids . The deduced amino acid sequence shows a topology of type II membrane proteins and has a unique repeated sequence of lysine and glutamic acid at the C-terminus . Disruption and overexpression of MNN4 led to a decrease and increase, respectively, of the mannosylphosphate content in cell wall mannans prepared from both mnn4 and wild type strains . A dramatic decrease of mannosylphosphate occurs in delta mnn4 mutans . The results from genetic and biochemical experiments combine to suggest that Mnn4p is required to mediate mannosylphosphate transfer in both the core and outer chain portions of N-linked oligosaccharides. J Cell Biol, 1996 Dec, 135(6 Pt 2), 1789 - 800 The sequence NPFXD defines a new class of endocytosis signal in Saccharomyces cerevisiae; Tan PK et al.; The yeast membrane protein Kex2p uses a tyrosine-containing motif within the cytoplasmic domain for localization to a late Golgi compartment . Because Golgi membrane proteins mislocalized to the plasma membrane in yeast can undergo endocytosis, we examined whether the Golgi localization sequence or other sequences in the Kex2p cytoplasmic domain mediate endocytosis . To assess endocytic function, the Kex2p cytoplasmic domain was fused to an endocytosis-defective form of the alpha-factor receptor . Ste2p . Like intact Ste2p, the chimeric protein, Stex22p, undergoes rapid endocytosis that is dependent on clathrin and End3p . Uptake of Stex22p does not require the Kex2p Golgi localization motif . Instead, the sequence NPFSD, located 37 amino acids from the COOH terminus, is essential for Stex22p endocytosis . Internalization was abolished when the N, P, or F residues were converted to alanine and severely impaired upon conversion of D to A . NPFSD restored uptake when added to the COOH terminus of an endocytosis-defective Ste2p chimera lacking lysine-based endocytosis signals present in wild-type Ste2p . An NPF sequence is present in the cytoplasmic domain of the a-factor receptor, Ste3p . Mutation of this sequence prevented pheromone-stimulated endocytosis of a truncated form of Ste3p . Our results identify NPFSD as a clathrin-dependent endocytosis signal that is distinct from the aromatic amino acid-containing Golgi localization motif and lysine-based, ubiquitin-dependent endocytosis signals in yeast. Proteins, 1996 Dec, 26(4), 481 - 2 Crystallization of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae; Monzingo AF et al.; Saccharomyces cerevisiae possesses three isozymes of 5,10-methylenetetrahydrofolate dehydrogenase (MTD) . The NAD-dependent enzyme is the first monofunctional form found in eukaryotes . Here we report its crystallization in a form suitable for high-resolution structure . The space group is P4(2)2(1)2 with cell constants a = b = 75.9, c = 160.0 A, and there is one 36 kDa molecule in the asymmetric unit . Crystals diffract to 2.9 A resolution. FEMS Microbiol Rev, 1996 Dec, 19(2), 117 - 36 The transcriptional apparatus required for mRNA encoding genes in the yeast Saccharomyces cerevisiae emerges from a jigsaw puzzle of transcription factors; Kunzler M et al.; The number of identified yeast factors involved in transcription has dramatically increased in recent years and the understanding of the interplay between the different factors has become more and more puzzling . Transcription initiation at the core promoter of mRNA encoding genes consisting of upstream, TATA and initiator elements requires an approximately ribosome-sized complex of more than 50 polypeptides . The recent identification and isolation of an RNA polymerase holoenzyme which seems to be preassembled before interacting with a promoter allowed a better understanding of the roles, assignments and interplays of the various constituents of the basal transcription machinery . Recruitment of this complex to the promoter is achieved by numerous interactions with a variety of DNA-bound proteins . These interactions can be direct or mediated by additional adaptor proteins . Other proteins negatively affect transcription by interrupting the recruitment process through protein-protein or protein-DNA interactions . Some basic features of cis-acting elements, the transcriptional apparatus and various trans-acting factors involved in the initiation of mRNA synthesis in yeast are summarized. Lett Appl Microbiol, 1996 Dec, 23(6), 448 - 52 Characteristics of maltose transporter activity in an ale and large strain of the yeast Saccharomyces cerevisiae; Crumplen RM et al.; Kinetic studies on maltose uptake by ale and larger yeasts showed lower Km and higher Vmax values than for laboratory yeasts . Eadie-Hofstee plots were biphasic and inhibition by sulphydryl reagents was consistent with low affinity uptake by a specific transporter rather than by non-specific adsorption . High affinity maltose transport by both strains was inhibited by galactose, CCCP, 2, 4,-DNP and NEM . In contrast, glucose and pCMBS inhibited high affinity transport by the ale yeast but not by the larger yeast . In the case of pCMBS the difference could result from a single amino acid change in the maltose transporter. J Cell Biol, 1996 Dec, 135(6 Pt 1), 1535 - 49 Actin and myosin function in directed vacuole movement during cell division in Saccharomyces cerevisiae; Hill KL et al.; During cell division, cytoplasmic organelles are not synthesized de novo, rather they are replicated and partitioned between daughter cells . Partitioning of the vacuole in the budding yeast Saccharomyces cerevisiae is coordinated with the cell cycle and involves a dramatic translocation of a portion of the parental organelle from the mother cell into the bud . While the molecular mechanisms that mediate this event are unknown, the vacuole's rapid and directed movements suggest cytoskeleton involvement . To identify cytoskeletal components that function in this process, vacuole inheritance was examined in a collection of actin mutants . Six strains were identified as being defective in vacuole inheritance . Tetrad analysis verified that the defect cosegregates with the mutant actin gene . One strain with a deletion in a myosin-binding region was analyzed further . The vacuole inheritance defect in this strain appears to result from the loss of a specific actin function; the actin cytoskeleton is intact and protein targeting to the vacuole is normal . Consistent with these findings, a mutation in the actin-binding domain of Myo2p, a class V unconventional myosin, abolishes vacuole inheritance . This suggests that Myo2p serves as a molecular motor for vacuole transport along actin filaments . The location of actin and Myo2p relative to the vacuole membrane is consistent with this model . Additional studies suggest that the actin filaments used for vacuole transport are dynamic, and that profilin plays a critical role in regulating their assembly . These results present the first demonstration that specific cytoskeletal proteins function in vacuole inheritance. Genetics, 1996 Dec, 144(4), 1399 - 412 Senescence mutants of Saccharomyces cerevisiae with a defect in telomere replication identify three additional EST genes; Lendvay TS et al.; The primary determinant for telomere replication is the enzyme telomerase, responsible for elongating the G-rich strand of the telomere . The only component of this enzyme that has been identified in Saccharomyces cerevisiae is the TLC1 gene, encoding the telomerase RNA subunit . However, a yeast strain defective for the EST1 gene exhibits the same phenotypes (progressively shorter telomeres and a senescence phenotype) as a strain deleted for TLC1, suggesting that EST1 encodes either a component of telomerase or some other factor essential for telomerase function . We designed a multitiered screen that led to the isolation of 22 mutants that display the same phenotypes as est1 and tlc1 mutant strains . These mutations mapped to four complementation groups: the previously identified EST1 gene and three additional genes, called EST2, EST3 and EST4 . Cloning of the EST2 gene demonstrated that it encodes a large, extremely basic novel protein with no motifs that provide clues as to function . Epistasis analysis indicated that the four EST genes function in the same pathway for telomere replication as defined by the TLC1 gene, suggesting that the EST genes encode either components of telomerase or factors that positively regulate telomerase activity. Genetics, 1996 Dec, 144(4), 1387 - 97 CDP1, a novel Saccharomyces cerevisiae gene required for proper nuclear division and chromosome segregation; Foreman PK et al.; To identify new gene products involved in chromosome segregation, we isolated Saccharomyces cerevisiae mutants that require centromere binding factor I (Cbf1p) for viability . One Cbf1p-dependent mutant (denoted cdp1-1) was selected for further analysis . The CDP1 gene encodes a novel 125-kD protein that is notably similar to previously identified mouse, human and Caenorhabditis elegans proteins . CDP1 delta and cdp1-1 mutant cells were temperature sensitive for growth . At the permissive temperature, cdp1-1 and cdp1 delta cells lost chromosomes at a frequencies approximately 20-fold and approximately 110-fold higher than wild-type cells, respectively . These mutants also displayed unusually long and numerous bundles of cytoplasmic microtubules as revealed by immunofluorescent staining . In addition, we occasionally observed improperly oriented mitotic spindles, residing entirely within one of the cells . Presumably as a result of undergoing nuclear division with improperly oriented spindles, a large percentage of cdp1 cells had accumulated multiple nuclei . While cdp1 mutant cells were hypersensitive to the microtubule-disrupting compound thiabendazole, they showed increased resistance to the closely related compound benomyl relative to wild-type cells . Taken together, these results suggest that Cdp1p plays a role in governing tubulin dynamics within the cell and may interact directly with microtubules or tubulin. Genetics, 1996 Dec, 144(4), 1375 - 86 Genesis and variability of {PSI} prion factors in Saccharomyces cerevisiae; Derkatch IL et al.; We have previously shown that multicopy plasmids containing the complete SUP35 gene are able to induce the appearance of the non-Mendelian factor {PSI} . This result was later interpreted by others as a crucial piece of evidence for a model postulating that {PSI} is a self-modified, prion-like conformational derivative of the Sup35 protein . Here we support this interpretation by proving that it is the overproduction of Sup35 protein, and not the excess of SUP35 DNA or mRNA that causes the appearance of {PSI} . We also show that the "prion-inducing domain" of Sup35p is in the N-terminal region, which, like the "prion-inducing domain" of another yeast prion, Ure2p, was previously shown to be distinct from the functional domain of the protein . This suggests that such a chimeric organization may be a common pattern of some prion elements . Finally, we find that {PSI} factors of different efficiencies and different mitotic stabilities are induced in the same yeast strain by overproduction of the identical Sup35 protein . We suggest that the different {PSI}-containing derivatives are analogous to the mysterious mammalian prion strains and result from different conformational variants of Sup35p. Protein Sci, 1996 Dec, 5(12), 2651 - 2 Saccharomyces cerevisiae mitochondria lack a bacterial-type sec machinery; Glick BS et al.; The bacterial Sec genes encode a generalized protein export machinery . Although the mitochondria present in eukaryotic cells are derived from bacterial ancestors, a comprehensive search of the complete genomic sequence for the eukaryotic yeast Saccharomyces cerevisiae did not reveal any close homologs of the bacterial Sec genes, strongly suggesting that yeast mitochondria lack a generalized bacterial-type export system . This finding has implications for the sorting of imported mitochondrial proteins to the intermembrane space compartment, and also for the insertion of mitochondrially encoded proteins into the inner membrane. Biochem J, 1996 Dec 1, 320 ( Pt 2), 401 - 12 Comparative studies on the 5-aminolaevulinic acid dehydratases from Pisum sativum, Escherichia coli and Saccharomyces cerevisiae; Senior NM et al.; 5-Aminolaevulinic acid dehydratase (ALAD) is an essential enzyme in most organisms, catalysing an inaugural step in the tetrapyrrole biosynthetic pathway, the Knorr-type condensation reaction of two molecules of 5-aminolaevulinic acid (ALA) to form the monopyrrole porphobilinogen . ALADs can be conveniently separated into two main groups: those requiring Zn2+ for activity (typified here by the enzymes from Escherichia coli and Saccharomyces cerevisiae, yeast) and those requiring Mg2+ (represented here by the enzyme from Pisum sativum, pea) . Here we describe a detailed comparison of these two metal-dependent systems . Kinetically influential ionizations were identified by using pH-dependent kinetics . Groups with pKa values of approx . 7 and 10 (assigned to cysteine and lysine residues) were detected in the free enzyme and enzyme-substrate states of all three enzymes, and a further ionizable group with a pKa of approx . 8.5 (assigned to histidine) was found to be additionally important to the yeast enzyme . The importance of these residues was confirmed by using protein modifying reagents . Shifts in the pKa values of the pea and E . coli enzymes consequent on E-S complex formation suggest a change to a less hydrophobic micro-environment when substrate binds . Studies with inhibitors revealed that the three enzymes exhibit differential susceptibilities and, in the case of succinylacetone, this is reflected in Ki values that vary by three orders of magnitude . In addition, the crystallization of the yeast ALAD is described, raising the possibility of an X-ray-derived three-dimensional structure of this enzyme. Nucleic Acids Res, 1996 Dec 1, 24(23), 4639 - 48 Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double strand break rejoining and in telomeric maintenance; Boulton SJ et al.; Ku is a heterodimer of polypeptides of approximately 70 and 80 kDa (Ku70 and Ku80, respectively) that binds to DNA ends . Mammalian cells lacking Ku are defective in DNA double-strand break (DSB) repair and in site-specific V(D)J recombination . Here, we describe the identification and characterisation of YKU80, the gene for the Saccharomyces cerevisiae Ku80 homologue . Significantly, we find that YKU80 disruption enhances the radiosensitivity of rad52 mutant strains, suggesting that YKU80 functions in a DNA DSB repair pathway that does not rely on homologous recombination . Indeed, through using an in vivo plasmid rejoining assay, we find that YKU80 plays an essential role in illegitimate recombination events that result in the accurate repair of restriction enzyme generated DSBs . Interestingly, in the absence of YKU80function, residual repair operates through an error-prone pathway that results in recombination between short direct repeat elements . This resembles closely a predominant DSB repair pathway in vertebrates . Together, our data suggest that multiple, evolutionarily conserved mechanisms for DSB repair exist in eukaryotes . Furthermore, they imply that Ku binds to DSBs in vivo and promotes repair both by enhancing accurate DNA end joining and by suppressing alternative error-prone repair pathways . Finally, we report that yku80 mutant yeasts display dramatic telomeric shortening, suggesting that, in addition to recognising DNA damage, Ku also binds to naturally occurring chromosomal ends . These findings raise the possibility that Ku protects chromosomal termini from nucleolytic attack and functions as part of a telomeric length sensing system. Photochem Photobiol, 1996 Dec, 64(6), 957 - 62 Saccharomyces cerevisiae mutants defective in heme biosynthesis as a tool for studying the mechanism of phototoxicity of porphyrins; Zoladek T et al.; Mutants of Saccharomyces cerevisiae accumulating uroporphyrin (UP) or protoporphyrin (PP) were used as a model for the in vivo phototoxic effect of porphyrins observed in the human skin photosensitivity associated with porphyrias (porphyria cutanea tarda and erythropoietic protoporphyria) . We have found that UP is localized in vacuoles and PP is present in all compartments except vacuoles in yeast cells . Endogenous PP is much more effective as a photosensitizer of yeast cells than UP . Protoporphyrin action is strictly dependent on the presence of oxygen . In contrast, UP displays a phototoxic effect even if oxygen is not present in the suspension, implicating a free radical mechanism that operates in anaerobiosis upon photosensitization by UP . Catalase or superoxide dismutase deficiency affects photosensitization by UP . A possible mechanism of UP photosensitizing activity is discussed. Yeast, 1996 Dec, 12(15), 1575 - 86 Sequence and analysis of a 26.9 kb fragment from chromosome XV of the yeast Saccharomyces cerevisiae; Boyer J et al.; We have determined the nucleotide sequence of a fragment of chromosome XV of Saccharomyces cerevisiae cloned into cosmid pEOA048 . The analysis of the 26,857 bp sequence reveals the presence of 19 open reading frames (ORFs), and of one RNA-coding gene (SNR17A) . Six ORFs correspond to previously known genes (MKK1/SSP32, YGE1/GRPE/MGE1, KIN4/KIN31/KIN3, RPL37B, DFR1 and HES1, respectively), all others were discovered in this work . Only five of the new ORFs have significant homologs in public databases, the remaining eight correspond to orphans (two of them are questionable) . O5248 is a probable folypolyglutamate synthetase, having two structural homologs already sequenced in the yeast genome . O5273 shows homology with a yeast protein required for vanadate resistance . O5268 shows homology with putative oxidoreductases of different organisms . O5257 shows homology with the SAS2 protein and another hypothetical protein from yeast . The last one, O5245, shows homology with a putative protein of Caenorhabditis elegans of unknown function . The present sequence corresponds to coordinates 772,331 to 799,187 of the entire chromosome XV sequence which can be retrieved by anonymous ftp (ftp . mips . embnet . org). Yeast, 1996 Dec, 12(15), 1563 - 73 Analysis of a 22,956 bp region on the right arm of Saccharomyces cerevisiae chromosome XV; Madania A et al.; We present here the sequence analysis of a DNA fragment (cosmid pUOA1258) located on the right arm of chromosome XV . The 22,956 bp sequence reveals 14 open reading frames (ORFs) longer than 300 bp and the 201 bp RPS33 gene . Among the 14 large ORFs, two overlapping frames are likely to be non-expressed and one corresponds to the known GLN4 gene encoding glutaminyl-tRNA synthetase . Two ORFs, O3571 and O3620, encode putative transcriptional regulators with a Zn(2)-Cys(6) DNA binding domain characteristic of members of the GAL4 family . Among the nine remaining ORFs, five (O3568, O3575, O3590, O3615 and O3625) present significant similarity to proteins of unknown function and four (O3580, O3595, O3630 and O3635) lack homology to sequences present in the databases screened.
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