Genetics, 1997 Dec, 147(4), 1545 - 56 Characterization of the roles of the Saccharomyces cerevisiae RAD54 gene and a homologue of RAD54, RDH54/TID1, in mitosis and meiosis; Shinohara M et al.; The RAD54 gene, which encodes a protein in the SWI2/SNF2 family, plays an important role in recombination and DNA repair in Saccharomyces cerevisiae . The yeast genome project revealed a homologue of RAD54, RDH54/TID1 . Properties of the rdh54/tid1 mutant and the rad54 rdh54/tid1 double mutant are shown for mitosis and meiosis . The rad54 mutant is sensitive to the alkylating agent, methyl methanesulfonate (MMS), and is defective in interchromosomal and intrachromosomal gene conversion . The rdh54/tid1 single mutant, on the other hand, does not show any significant deficiency in mitosis . However, the rad54 rdh54/tid1 mutant is more sensitive to MMS and more defective in interchromosomal gene conversion than is the rad54 mutant, but shows the same frequency of intrachromosomal gene conversion as the rad54 mutant . These results suggest that RDH54/TID1 is involved in a minor pathway of mitotic recombination in the absence of R4D54 . In meiosis, both single mutants produce viable spores at slightly reduced frequency . However, only the rdh54/tid1 mutant, but not the rad54 mutant, shows significant defects in recombination: retardation of the repair of meiosis-specific double-strand breaks (DSBs) and delayed formation of physical recombinants . Furthermore, the rad54 rdh54/tid1 double mutant is completely defective in meiosis, accumulating DSBs with more recessed ends than the wild type and producing fewer physical recombinants than the wild type . These results suggest that one of the differences between the late stages of mitotic recombination and meiotic recombination might be specified by differential dependency on the Rad54 and Rdh54/Tid1 proteins. Genetics, 1997 Dec, 147(4), 1533 - 43 RDH54, a RAD54 homologue in Saccharomyces cerevisiae, is required for mitotic diploid-specific recombination and repair and for meiosis; Klein HL; Most mitotic recombination and repair genes of Saccharomyces cerevisiae show no specificity of action for the genome ploidy . We describe here a novel repair and recombination gene that is specific for recombination and repair between homologous chromosomes . The RDH54 gene is homologous to the RAD54 gene, but rdh54 mutants do not show sensitivity to methyl methanesulfonate at concentrations that sensitize a rad54 mutant . However, the rdh54 null mutation enhances the methyl methanesulfonate sensitivity of a rad54 mutant and single rdh54 mutants are sensitive to prolonged exposure at high concentrations of methyl methanesulfonate . The RDH54 gene is required for recombination, but only in a diploid . We present evidence showing that the RDH54 gene is required for interhomologue gene conversion but not intrachromosomal gene conversion . The rdh54 mutation confers diploid-specific lethalities and reduced growth in various mutant backgrounds . These phenotypes are due to attempted recombination . The RDH54 gene is also required for meiosis as homozygous mutant diploids show very poor sporulation and reduced spore viability . The role of the RDH54 gene in mitotic repair and in meiosis and the pathway in which it acts are discussed. Gene, 1997 Nov 12, 201(1-2), 5 - 10 Suppression of the Saccharomyces cerevisiae hac1/ire15 mutation by yeast genes and human cDNAs; Nikawa J et al.; We previously reported that the Saccharomyces cerevisiae ire15 mutation results in an inositol-auxotrophic phenotype, and that human cDNAs can suppress the ire15 mutation (Nikawa, J., 1994 . A cDNA encoding the human transforming growth factor beta receptor suppresses the growth defect of a yeast mutant . Gene 149, 367 372; Nikawa, J., Nakano, H., Ohi, N., 1996b . Structural and functional conservation of human and yeast HAC1 genes which can suppress the growth defect of the Saccharomyces cerevisiae ire15 mutant . Gene 171, 107-111) . Herein, we present evidence that the gene responsible for the ire15 mutation is HAC1, which encodes a transcription factor for KAR2, obtained by isolating a yeast single-copy supressor gene and by performing complementation analysis . Sequencing analysis revealed that the mutant HAC1 gene obtained from the ire15 mutant contained an AAA codon at position 50 instead of the AGA codon observed in the wild-type gene, resulting in the alteration of the aa from Arg to Lys . All human cDNAs and yeast multicopy suppressors, which had been isolated as suppressors for the ire15 mutation, were able to suppress the inositol-auxotrophic phenotype but not the defect in KAR2 induction of the hac1-disrupted strain. J Biol Chem, 1997 Nov 28, 272(48), 30061 - 6 The Saccharomyces cerevisiae ACR3 gene encodes a putative membrane protein involved in arsenite transport; Wysocki R et al.; The cluster of three genes, ACR1, ACR2, and ACR3, previously was shown to confer arsenical resistance in Saccharomyces cerevisiae . The overexpression of ACR3 induced high level arsenite resistance . The presence of ACR3 together with ACR2 on a multicopy plasmid was conducive to increased arsenate resistance . The function of ACR3 gene has now been investigated . Amino acid sequence analysis of Acr3p showed that this hypothetical protein has hydrophobic character with 10 putative transmembrane spans and is probably located in yeast plasma membrane . We constructed the acr3 null mutation . The resulting disruptants were 5-fold more sensitive to arsenate and arsenite than wild-type cells . The acr3 disruptants showed wild-type sensitivity to antimony, tellurite, cadmium, and phenylarsine oxide . The mechanism of arsenical resistance was assayed by transport experiments using radioactive arsenite . We did not observe any significant differences in the accumulation of 76AsO33- in wild-type cells, acr1 and acr3 disruptants . However, the high dosage of ACR3 gene resulted in loss of arsenite uptake . These results suggest that arsenite resistance in yeast is mediated by an arsenite transporter (Acr3p). FEBS Lett, 1997 Oct 27, 416(3), 339 - 43 Mutational analysis of Yap1 protein, an AP-1-like transcriptional activator of Saccharomyces cerevisiae; Takeuchi T et al.; To define the essential amino acid residues of Yap1 in stress response, we generated yap1 mutations by in vitro mutagenesis, which cause defects in mediating resistance to the stress of H2O2, but not of CdCl2 . Sequence analysis of the mutant yap1 genes revealed three point mutations and two truncation mutations near the carboxy-terminus . The truncation mutations resulted in hyperresistance to cadmium . Northern blot analysis of stress-induced levels of TRX2 and GSH1 mRNAs indicated that the ability of the mutant Yap1 protein to induce transcriptional activation of target genes correlates well with its ability to confer stress resistance . The carboxy-terminal domain of Yap1 appears to act negatively in cadmium resistance. Mol Cell Biol, 1997 Dec, 17(12), 7283 - 94 Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae; Kressler D et al.; A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A) . Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family . Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization . An HA epitope-tagged Fal1p is localized predominantly in the nucleolus . Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S ribosomal subunits . Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin . Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2 . Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S ribosomal subunits . Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation. Mol Cell Biol, 1997 Dec, 17(12), 7029 - 39 SSP1, a gene necessary for proper completion of meiotic divisions and spore formation in Saccharomyces cerevisiae; Nag DK et al.; During meiosis, a diploid cell undergoes two rounds of nuclear division following one round of DNA replication to produce four haploid gametes . In yeast, haploid meiotic products are packaged into spores . To gain new insights into meiotic development and spore formation, we followed differential expression of genes in meiotic versus vegetatively growing cells in the yeast Saccharomyces cerevisiae . Our results indicate that there are at least five different classes of transcripts representing genes expressed at different stages of the sporulation program . Here we describe one of these differentially expressed genes, SSP1, which plays an essential role in meiosis and spore formation . SSP1 is expressed midway through meiosis, and homozygous ssp1 diploid cells fail to sporulate . In the ssp1 mutant, meiotic recombination is normal but viability declines rapidly . Both meiotic divisions occur at the normal time; however, the fraction of cells completing meiosis is significantly reduced, and nuclei become fragmented soon after meiosis II . The ssp1 defect does not appear to be related to a microtubule-cytoskeletal-dependent event and is independent of two rounds of chromosome segregation . The data suggest that Ssp1 is likely to function in a pathway that controls meiotic nuclear divisions and coordinates meiosis and spore formation. Mol Cell Biol, 1997 Dec, 17(12), 6982 - 93 Yap, a novel family of eight bZIP proteins in Saccharomyces cerevisiae with distinct biological functions; Fernandes L et al.; Saccharomyces cerevisiae contains eight members of a novel and fungus-specific family of bZIP proteins that is defined by four atypical residues on the DNA-binding surface . Two of these proteins, Yap1 and Yap2, are transcriptional activators involved in pleiotropic drug resistance . Although initially described as AP-1 factors, at least four Yap proteins bind most efficiently to TTACTAA, a sequence that differs at position +/-2 from the optimal AP-1 site (TGACTCA); further, a Yap-like derivative of the AP-1 factor Gcn4 (A239Q S242F) binds efficiently to the Yap recognition sequence . Molecular modeling suggests that the Yap-specific residues make novel contacts and cause physical constraints at the +/-2 position that may account for the distinct DNA-binding specificities of Yap and AP-1 proteins . To various extents, Yap1, Yap2, Yap3, and Yap5 activate transcription from a promoter containing a Yap recognition site . Yap-dependent transcription is abolished in strains containing high levels of protein kinase A; in contrast, Gcn4 transcriptional activity is stimulated by protein kinase A . Interestingly, Yap1 transcriptional activity is stimulated by hydrogen peroxide, whereas Yap2 activity is stimulated by aminotriazole and cadmium . In addition, unlike other yap mutations tested, yap4 (cin5) mutations affect chromosome stability, and they suppress the cold-sensitive phenotype of yap1 mutant strains . Thus, members of the Yap family carry out overlapping but distinct biological functions. J Biol Chem, 1997 Nov 21, 272(47), 29704 - 10 Hydroxylation of Saccharomyces cerevisiae ceramides requires Sur2p and Scs7p; Haak D et al.; The Saccharomyces cerevisiae SCS7 and SUR2 genes are members of a gene family that encodes enzymes that desaturate or hydroxylate lipids . Sur2p is required for the hydroxylation of C-4 of the sphingoid moiety of ceramide, and Scs7p is required for the hydroxylation of the very long chain fatty acid . Neither SCS7 nor SUR2 are essential for growth, and lack of the Scs7p- or Sur2p-dependent hydroxylation does not prevent the synthesis of mannosyldiinositolphosphorylceramide, the mature sphingolipid found in yeast . Deletion of either gene suppresses the Ca2+-sensitive phenotype of csg2Delta mutants, which arises from overaccumulation of inositolphosphorylceramide due to a defect in sphingolipid mannosylation . Characterization of scs7 and sur2 mutants is expected to provide insight into the function of ceramide hydroxylation. Genes Dev, 1997 Nov 15, 11(22), 3032 - 45 Rad53-dependent phosphorylation of Swi6 and down-regulation of CLN1 and CLN2 transcription occur in response to DNA damage in Saccharomyces cerevisiae; Sidorova JM et al.; Budding yeast possesses a checkpoint-dependent mechanism of delaying G1 progression in response to UV and ionizing radiation DNA damage . We have shown that after a pulse of DNA damage in G1 with the alkylating agent MMS, there is also a MEC1-, RAD53-, and RAD9-dependent delay in G1 . This delay occurs at or before Start, as the MMS-treated cells do not bud, remain sensitive to alpha-factor, and have low CLN1 and CLN2 transcript levels for a longer time than untreated cells . We further show that MMS directly and reversibly down-regulates CLN1 and CLN2 transcript levels . The initial drop in CLN transcript levels in MMS is not RAD53 dependent, but the kinetics of reaccumulation of CLN messages as cells recover from the damage is faster in rad53-11 cells than in wild type cells . This is not an indirect effect of faster progression through G1, because CLN transcripts reaccumulate faster in rad53-11 mutants arrested in G1 as well . In addition, the recovery of CLN mRNA levels can be also hastened by a SWI6 deletion or by overexpression of the truncated Swi4 (Swi4-t) that lacks the carboxy-terminal domain through which Swi4 associates with Swi6 . This indicates that both Rad53 and Swi6 are negative regulators of CLN expression after DNA damage . Finally, Swi6 undergoes an MMS-inducible, RAD53-dependent phosphorylation in G1 cells, and Rad53, immunoprecipitated from MMS-treated cells, phosphorylates Swi6 in vitro . On the basis of these observations, we suggest that the Rad53-dependent phosphorylation of Swi6 may delay the transition to S phase by inhibiting CLN transcription. Genes Dev, 1997 Nov 15, 11(22), 2958 - 71 The Cdc42 GTPase-associated proteins Gic1 and Gic2 are required for polarized cell growth in Saccharomyces cerevisiae; Chen GC et al.; BEM2 of Saccharomyces cerevisiae encodes a Rho-type GTPase-activating protein that is required for proper bud site selection at 26 degrees C and for bud emergence at elevated temperatures . We show here that the temperature-sensitive growth phenotype of bem2 mutant cells can be suppressed by increased dosage of the GIC1 gene . The Gic1 protein, together with its structural homolog Gic2, are required for cell size and shape control, bud site selection, bud emergence, actin cytoskeletal organization, mitotic spindle orientation/positioning, and mating projection formation in response to mating pheromone . Each protein contains a CRIB (Cdc42/Rac-interactive binding) motif and each interacts in the two-hybrid assay with the GTP-bound form of the Rho-type Cdc42 GTPase, a key regulator of polarized growth in yeast . The CRIB motif of Gic1 and the effector domain of Cdc42 are required for this association . Genetic experiments indicate that Gic1 and Gic2 play positive roles in the Cdc42 signal transduction pathway, probably as effectors of Cdc42 . Subcellular localization studies with a functional green fluorescent protein-Gic1 fusion protein indicate that this protein is concentrated at the incipient bud site of unbudded cells, at the bud tip and mother-bud neck of budded cells, and at cortical sites on large-budded cells that may delimit future bud sites in the two progeny cells . The ability of Gic1 to associate with Cdc42 is important for its function but is apparently not essential for its subcellular localization. FEMS Microbiol Rev, 1997 Sep, 21(2), 113 - 34 Classification of all putative permeases and other membrane plurispanners of the major facilitator superfamily encoded by the complete genome of Saccharomyces cerevisiae; Nelissen B et al.; On the basis of the complete genome sequence of the budding yeast Saccharomyces cerevisiae, a computer-aided analysis was carried out of all members of the major facilitator superfamily (MFS), which typically consists of permeases with 12 transmembrane spans . Analysis of all 5885 predicted open reading frames identified 186 potential MFS proteins . Binary sequence comparison made it possible to cluster 149 of them into 23 families . Putative permease functions could be assigned to 12 families, the largest including sugar, amino acid, and multidrug transport . Phylogenetic clustering of proteins allowed us to predict a possible permease function for a total of 119 proteins . Multiple sequence alignments were made for all families, and evolutionary trees were constructed for families with at least four members . The latter resulted in the identification of 21 subclusters with presumably tightly related permease function . No functional clues were predicted for a total of 41 clustered or unclustered proteins. Mol Biol Cell, 1997 Oct, 8(10), 1829 - 44 The Rho-GEF Rom2p localizes to sites of polarized cell growth and participates in cytoskeletal functions in Saccharomyces cerevisiae; Manning BD et al.; Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements . ROM2 and RHO2 were identified in a screen for high-copy number suppressors of cik1 delta, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae . A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections . Disruption of ROM2 results in temperature-sensitive growth defects at 11 degrees C and 37 degrees C . rom2 delta cells exhibit morphological defects . At permissive temperatures, rom2 delta cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate . High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1 delta and kar3 delta strains . KAR3 encodes a kinesin-related protein that interacts with Cik1p . Furthermore, rom2 delta strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl . These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton. Methods Cell Biol, 1998, 53, 545 - 57 In vivo nuclear transport kinetics in Saccharomyces cerevisiae; Roberts PM et al.; We have described a direct fluorescence assay to measure the relative rates of NLS-directed import and passive export of an NLS-GFP fusion protein in yeast . The design and construction of the reporter GFP fusion, its spectral qualities, size, use of inducible promoters, and the choice of NLS, are variables that could extend the method's utility . Future applications will almost certainly demand the quantification of transport rates in single cells using image analysis techniques . As is the case whenever cellular processes are studied in vivo, the in vivo nuclear trafficking properties of NLS-GFP are complicated and poorly understood . Some will be attracted to NLS-GFP kinetic assays simply because so little is known about the function and regulation of the transport apparatus in living cells . At the same time, the uncertainties that accompany in vivo work necessarily prevent the rigorous interpretation of data, which biochemists expect from experiments performed in vitro using highly purified enzymes. J Mol Biol, 1997 Oct 31, 273(3), 714 - 28 The 1.8 A crystal structure of the dimeric peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: implications for substrate binding and reaction mechanism; Mathieu M et al.; The dimeric, peroxisomal 3-ketoacyl-CoA thiolase catalyses the conversion of 3-ketoacyl-CoA into acyl-CoA, which is shorter by two carbon atoms . This reaction is the last step of the beta-oxidation pathway . The crystal structure of unliganded peroxisomal thiolase of the yeast Saccharomyces cerevisiae has been refined at 1.8 A resolution . An unusual feature of this structure is the presence of two helices, completely buried in the dimer and sandwiched between two beta-sheets . The analysis of the structure shows that the sequences of these helices are not hydrophobic, but generate two amphipathic helices . The helix in the N-terminal domain exposes the polar side-chains to a cavity at the dimer interface, filled with structured water molecules . The central helix in the C-terminal domain exposes its polar residues to an interior polar pocket . The refined structure has also been used to predict the mode of binding of the substrate molecule acetoacetyl-CoA, as well as the reaction mechanism . From previous studies it is known that Cys125, His375 and Cys403 are important catalytic residues . In the proposed model the acetoacetyl group fits near the two catalytic cysteine residues, such that the oxygen atoms point towards the protein interior . The distance between SG(Cys125) and C3(acetoacetyl-CoA) is 3.7 A . The O2 atom of the docked acetoacetyl group makes a hydrogen bond to N(Gly405), which would favour the formation of the covalent bond between SG(Cys125) and C3(acetoacetyl-CoA) of the intermediate complex of the two-step reaction . The CoA moiety is proposed to bind in a groove on the surface of the protein molecule . Most of the interactions of the CoA molecule are with atoms of the loop domain . The three phosphate groups of the CoA moiety are predicted to interact with side-chains of lysine and arginine residues, which are conserved in the dimeric thiolases . J Pept Res, 1997 Nov, 50(5), 319 - 28 Position one analogs of the Saccharomyces cerevisiae tridecapeptide pheromone; Zhang YL et al.; Analogs of the Saccharomyces cerevisiae alpha-mating factor {WHWLQLKPGQPMY}, in which a variety of residues replaced Trp1 were synthesized and assayed for biological activity and receptor affinity . Analogs containing Gly or Leu or many different aromatic residues in position 1 of the peptide exhibited bioactivity in a growth arrest assay slightly greater than, or equal to, that of the parent pheromone, whereas the Glu1 and Lys1 analogs exhibited significantly lower bioactivity . Analogs with an aromatic replacement at position 1 had 3- to 6-fold lower receptor affinity than the parent peptide, whereas analogs with a hydrophilic residue at the N-terminus exhibited large reductions in receptor affinity with the peptide with Glu in position 1 showing a 120-fold reduction . N alpha-Acetylation had little effect on bioactivity but lowered receptor affinity by 20- to 40-fold . Amidation of the carboxyl terminus resulted in a 10-fold decrease in activity and a 160-fold decrease in receptor affinity . These results indicate that the alpha-factor receptor has a large hydrophobic binding pocket, possibly containing a negatively charged side-chain, which interacts with the N-terminus of alpha-factor . The lack of correlation between activity and binding and several analogs suggests that small residues near the N-terminus of alpha-factor may be very efficient in triggering isomerization of the receptor to its activated state in the first step of the signal transduction pathway. J Bacteriol, 1997 Dec, 179(24), 7644 - 52 The minimal transactivation region of Saccharomyces cerevisiae Gln3p is localized to 13 amino acids; Svetlov V et al.; Regulated nitrogen catabolic gene transcription in Saccharomyces cerevisiae is mediated by four positive (Gln3p and Gat1p/Nil1p) and negative (Dal80p/Uga43p and Deh1p/Nil2p/GZF3p) regulators which function in opposition to one another . All four proteins contain GATA-type zinc finger domains, and three of them (Gln3p, Dal80p, and Deh1p) have been shown to bind to GATA sequences situated upstream of genes whose expression is sensitive to nitrogen catabolite repression (NCR) . The positive regulators, Gln3p and Gat1p, are able to support transcriptional activation when tethered by LexAp to the promoter of a reporter gene whose upstream activation sequences have been replaced with one or more lexA operator sites . Existing data suggest that these four proteins regulate transcription by competing with one another for binding to the GATA sequences which mediate NCR-sensitive gene expression . We show that the minimal Gln3p domain mediating transcriptional activation consists of 13 amino acids with a predicted propensity to form an alpha-helix . Genetic analysis of this region (Gln3p residues 126 to 138, QQNGEIAQLWDFN) demonstrated that alanine may be substituted for the aromatic and acidic amino acids without destroying transcriptional activation potential . Similar substitution of alanine for the two hydrophobic amino acids, isoleucine and leucine, however, destroys activation, as does introduction of basic amino acids in place of the acidic residues or introduction of proline into the center of the sequence . A point mutation in the Gln3p activation region destroys its in vivo ability to support NCR-sensitive DAL5 expression . We find no convincing evidence that NCR regulates Gln3p function by modulating the functioning of its activation region. J Bacteriol, 1997 Dec, 179(24), 7611 - 6 Biosynthesis of phosphatidic acid in lipid particles and endoplasmic reticulum of Saccharomyces cerevisiae; Athenstaedt K et al.; Lipid particles of the yeast Saccharomyces cerevisiae harbor two enzymes that stepwise acylate glycerol-3-phosphate to phosphatidic acid, a key intermediate in lipid biosynthesis . In lipid particles of the s1c1 disruptant YMN5 (M . M . Nagiec et al., J . Biol . Chem . 268:22156-22163, 1993) acylation stops after the first step, resulting in the accumulation of lysophosphatidic acid . Two-dimensional gel electrophoresis confirmed that S1c1p is a component of lipid particles . Lipid particles of a second mutant strain, TTA1 (T . S . Tillman and R . M . Bell, J . Biol . Chem . 261:9144-9149, 1986), which harbors a point mutation in the GAT gene, are essentially devoid of glycerol-3-phosphate acyltransferase activity in vitro . Synthesis of phosphatidic acid is reconstituted by combining lipid particles from YMN5 and TTA1 . These results indicate that two distinct enzymes are necessary for phosphatidic acid synthesis in lipid particles: the first step, acylation of glycerol-3-phosphate, is catalyzed by a putative Gat1p; the second step, acylation of lysophosphatidic acid, requires S1c1p . Surprisingly, YMN5 and TTA1 mutants grow like the corresponding wild types because the endoplasmic reticulum of both mutants has the capacity to form a reduced but significant amount of phosphatidic acid . As a consequence, an s1c1 gat1 double mutant is also viable . Lipid particles from this double mutant fail completely to acylate glycerol-3-phosphate, whereas endoplasmic reticulum membranes harbor residual enzyme activities to synthesize phosphatidic acid . Thus, yeast contains at least two independent systems of phosphatidic acid biosynthesis. Mol Biol Cell, 1997 Dec, 8(12), 2575 - 90 A mutational analysis identifies three functional regions of the spindle pole component Spc110p in Saccharomyces cerevisiae; Sundberg HA et al.; The central coiled coil of the essential spindle pole component Spc110p spans the distance between the central and inner plaques of the Saccharomyces cerevisiae spindle pole body (SPB) . The carboxy terminus of Spc110p, which binds calmodulin, resides at the central plaque, and the amino terminus resides at the inner plaque from which nuclear microtubules originate . To dissect the functions of Spc110p, we created temperature-sensitive mutations in the amino and carboxy termini . Analysis of the temperature-sensitive spc110 mutations and intragenic complementation analysis of the spc110 alleles defined three functional regions of Spc110p . Region I is located at the amino terminus . Region II is located at the carboxy-terminal end of the coiled coil, and region III is the previously defined calmodulin-binding site . Overexpression of SPC98 suppresses the temperature sensitivity conferred by mutations in region I but not the phenotypes conferred by mutations in the other two regions, suggesting that the amino terminus of Spc110p is involved in an interaction with the gamma-tubulin complex composed of Spc97p, Spc98p, and Tub4p . Mutations in region II lead to loss of SPB integrity during mitosis, suggesting that this region is required for the stable attachment of Spc110p to the central plaque . Our results strongly argue that Spc110p links the gamma-tubulin complex to the central plaque of the SPB. Mol Biol Cell, 1997 Dec, 8(12), 2519 - 37 Characterization of Saccharomyces cerevisiae dna2 mutants suggests a role for the helicase late in S phase; Fiorentino DF et al.; The TOR proteins, originally identified as targets of the immunosuppressant rapamycin, contain an ATM-like "lipid kinase" domain and are required for early G1 progression in eukaryotes . Using a screen to identify Saccharomyces cerevisiae mutants requiring overexpression of Tor1p for viability, we have isolated mutations in a gene we call ROT1 (requires overexpression of Tor1p) . This gene is identical to DNA2, encoding a helicase required for DNA replication . As with its role in cell cycle progression, both the N-terminal and C-terminal regions, as well as the kinase domain of Tor1p, are required for rescue of dna2 mutants . Dna2 mutants are also rescued by Tor2p and show synthetic lethality with tor1 deletion mutants under specific conditions . Temperature-sensitive (Ts) dna2 mutants arrest irreversibly at G2/M in a RAD9- and MEC1-dependent manner, suggesting that Dna2p has a role in S phase . Frequencies of mitotic recombination and chromosome loss are elevated in dna2 mutants, also supporting a role for the protein in DNA synthesis . Temperature-shift experiments indicate that Dna2p functions during late S phase, although dna2 mutants are not deficient in bulk DNA synthesis . These data suggest that Dna2p is not required for replication fork progression but may be needed for a later event such as Okazaki fragment maturation. Biochemistry, 1997 Dec 2, 36(48), 14956 - 64 Role of mitochondrial and cytoplasmic serine hydroxymethyltransferase isozymes in de novo purine synthesis in Saccharomyces cerevisiae; Kastanos EK et al.; One-carbon units are essential to a variety of anabolic processes which yield necessary cellular components including purines, pyrimidines, amino acids, and lipids . Serine hydroxymethyltransferase (SHMT) is the major provider of one-carbon units in the cell . The other product of this reaction is glycine . Both of these metabolites are required in de novo purine biosynthesis . In Saccharomyces cerevisiae, mitochondrial and cytoplasmic SHMT isozymes are encoded by distinct nuclear genes (SHM1 and SHM2) . Molecular genetic analyses have begun to define the roles of these two isozymes in folate-mediated one-carbon metabolism {McNeil, J . B., et al . (1996) Genetics 142, 371-381} . In our study, the SHM1 and SHM2 genes were disrupted singly and in combination to investigate the contributions of the two SHMT isozymes to the production of glycine and one-carbon units required in purine biosynthesis . Cell subfractionation experiments indicated that while only 5% of total activity was localized in the mitochondria, the specific activity in that compartment was much higher than in the cytoplasm . Growth and 13C NMR experiments indicate that the two isozymes function in different directions, depending on the nutritional conditions of the cell . When yeast was grown on serine as the primary one-carbon source, the cytoplasmic isozyme was the main provider of glycine and one-carbon groups for purine synthesis . When grown on glycine, the mitochondrial SHMT was the predominant isozyme catalyzing the synthesis of serine from glycine and one-carbon units . However, when both serine and glycine were present, the mitochondrial SHMT made a significant contribution of one-carbon units, but not glycine, for purine synthesis . Finally, NMR data are presented that suggest the existence of at least two sites of de novo purine biosynthesis in growing yeast cells, each being fed by distinct pools of precursors. Biochemistry, 1997 Nov 25, 36(47), 14439 - 46 Phosphorylation of the acidic ribosomal P proteins in Saccharomyces cerevisiae: a reappraisal; Zambrano R et al.; Previous reports had pointed to serines 62 and 71/79 as possible phosphorylation sites in the yeast acidic ribosomal proteins YP1 alpha and YP2 alpha, respectively . However, it has been found that mutation of these serine residues did not affect the phosphorylation level of the proteins . A detailed examination of the YP2 alpha tryptic digest from the in vivo labeled protein demonstrates the existence of a totally trypsin-insensitive site at lysine 88 that led to a misinterpretation of previous results . The unique YP2 alpha tryptic phosphopeptide obtained contains, in addition to serines 71 and 79, a serine at position 96 near the carboxyl end, which automatic Edman degradation confirmed as the phosphorylated residue . In addition, by using Staphyloccocus protease V8, it was possible to obtain phosphopeptides containing only serine 96, whose phosphorylation has likewise been confirmed by radioactive labeling as well as by chemical methods . A similar analysis of the other 12 kDa acidic proteins, YP1 alpha, YP1 beta, and YP2 beta, has shown the presence of equivalent phosphorylation sites in the four P proteins, which correspond to position 96 in proteins YP1 alpha, YP1 beta, and YP2 alpha and position 100 in YP2 beta . This conclusion has been confirmed by the fact that mutation of serine 96 in proteins YP1 alpha and YP2 alpha abolishes their capacity to be phosphorylated in vivo . The mutation of the phosphorylation site of the individual acidic proteins seems not to alter their interaction with the ribosome . However, it has been found that the level of phosphorylation of the stalk proteins has an effect on the response of the cells to some specific metabolic conditions, indicating that it may modulate the translation of specific proteins. Biochim Biophys Acta, 1997 Nov 10, 1322(1), 8 - 18 Contribution to the physiological characterization of glycerol active uptake in Saccharomyces cerevisiae; Lages F et al.; Evidence is presented here that in Saccharomyces cerevisiae IGC 3507, grown either on glycerol, ethanol or acetate, glycerol is transported by a high affinity uptake system of the electrogenic proton symport type, with Km of 1.7 +/- 0.7 mM, Vmax 441 +/- 19 micromolh(-1) g(-1) dry weight and a stoichiometry of 1:1 proton per molecule of glycerol, at 30 degrees C and pH 5.0 . No competitors were found among other polyols and sugars . Glycerol maximum accumulation ratios followed p.m.f . with extracellular pH . CCCP prevented glycerol accumulation, and inhibited uptake . NaCl did not interfere with H+/glycerol kinetics and energetics . This transport system was shown to be under glucose repression and inactivation . Glucose-grown cells presented, instead, a lower affinity permease for glycerol, probably a facilitated diffusion . Growth on glucose in the presence of NaCl did not induce the high affinity carrier . The stringent control of cell physiological condition over induction suggests for glycerol proton symport rather a physiological role connected with growth under gluconeogenic conditions. Proc Natl Acad Sci U S A, 1997 Nov 25, 94(24), 13093 - 8 All cyclophilins and FK506 binding proteins are, individually and collectively, dispensable for viability in Saccharomyces cerevisiae; Dolinski K et al.; The cyclophilins and FK506 binding proteins (FKBPs) bind to cyclosporin A, FK506, and rapamycin and mediate their immunosuppressive and toxic effects, but the physiological functions of these proteins are largely unknown . Cyclophilins and FKBPs are ubiquitous and highly conserved enzymes that catalyze peptidyl-prolyl isomerization, a rate-limiting step during in vitro protein folding . We have addressed their functions by a genetic approach in the yeast Saccharomyces cerevisiae . Five cyclophilins and three FKBPs previously were identified in yeast . We identified four additional enzymes: Cpr6 and Cpr7, which are homologs of mammalian cyclophilin 40 that have also recently been independently isolated by others, Cpr8, a homolog of the secretory pathway cyclophilin Cpr4, and Fpr4, a homolog of the nucleolar FKBP, Fpr3 . None of the eight cyclophilins or four FKBPs were essential . Surprisingly, yeast mutants lacking all 12 immunophilins were viable, and the phenotype of the dodecuplet mutant resulted from simple addition of the subtle phenotypes of each individual mutation . We conclude that cyclophilins and FKBPs do not play an essential general role in protein folding and find little evidence of functional overlap between the different enzymes . We propose that each cyclophilin and FKBP instead regulates a restricted number of unique partner proteins that remain to be identified. Proc Natl Acad Sci U S A, 1997 Nov 25, 94(24), 12949 - 56 In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone; Nathan DF et al.; In the highly concentrated environment of the cell, polypeptide chains are prone to aggregation during synthesis (as nascent chains await the emergence of the remainder of their folding domain), translocation, assembly, and exposure to stresses that cause previously folded proteins to unfold . A large and diverse group of proteins, known as chaperones, transiently associate with such folding intermediates to prevent aggregation, but in many cases the specific functions of individual chaperones are still not clear . In vivo, Hsp90 (heat shock protein 90) plays a role in the maturation of components of signal transduction pathways but also exhibits chaperone activity with diverse proteins in vitro, suggesting a more general function . We used a unique temperature-sensitive mutant of Hsp90 in Saccharomyces cerevisiae, which rapidly and completely loses activity on shift to high temperatures, to examine the breadth of Hsp90 functions in vivo . The data suggest that Hsp90 is not required for the de novo folding of most proteins, but it is required for a specific subset of proteins that have greater difficulty reaching their native conformations . Under conditions of stress, Hsp90 does not generally protect proteins from thermal inactivation but does enhance the rate at which a heat-damaged protein is reactivated . Thus, although Hsp90 is one of the most abundant chaperones in the cell, its in vivo functions are highly restricted. Proc Natl Acad Sci U S A, 1997 Nov 25, 94(24), 12898 - 903 Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II; Yue Z et al.; 5'-Capping is an early mRNA modification that has important consequences for downstream events in gene expression . We have isolated mammalian cDNAs encoding capping enzyme . They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily . The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical . Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5'-triphosphatase activity and catalyzed formation of RNA 5'-terminal GpppG . A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA . Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site . The K294A mutant retained RNA 5'-triphosphatase activity, which was eliminated by N-terminal truncation . Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase . The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation . These results also explain the selective capping of RNA polymerase II transcripts. J Bacteriol, 1997 Dec, 179(23), 7497 - 506 The 2microm-plasmid-encoded Rep1 and Rep2 proteins interact with each other and colocalize to the Saccharomyces cerevisiae nucleus; Ahn YT et al.; The efficient partitioning of the 2microm plasmid of Saccharomyces cerevisiae at cell division requires two plasmid-encoded proteins (Rep1p and Rep2p) and a cis-acting locus, REP3 (STB) . By using protein hybrids containing fusions of the Rep proteins to green fluorescent protein (GFP), we show here that fluorescence from GFP-Rep1p or GFP-Rep2p is almost exclusively localized in the nucleus in a cir+ strain . Nuclear localization of GFP-Rep1p and GFP-Rep2p, though discernible, is less efficient in a cir(0) host . GFP-Rep2p or GFP-Rep1p is able to promote the stability of a 2microm circle-derived plasmid harboring REP1 or REP2, respectively, in a cir(0) background . Under these conditions, fluorescence from GFP-Rep2p or GFP-Rep1p is concentrated within the nucleus, as is the case in cir+ cells . This characteristic nuclear accumulation is not dependent on the expression of the FLP or RAF1 gene of the 2microm circle . Nuclear colocalization of Rep1p and Rep2p is consistent with the hypothesis that the two proteins directly or indirectly interact to form a functional bipartite or high-order protein complex . Immunoprecipitation experiments as well as baiting assays using GST-Rep hybrid proteins suggest a direct interaction between Rep1p and Rep2p which, in principle, may be modulated by other yeast proteins . Furthermore, these assays provide evidence for Rep1p-Rep1p and Rep2p-Rep2p associations as well . The sum of these interactions may be important in controlling the effective cellular concentration of the Rep1p-Rep2p complex. Biochim Biophys Acta, 1997 Oct 20, 1321(3), 207 - 20 Patch clamp investigation into the phosphate carrier from Saccharomyces cerevisiae mitochondria; Herick K et al.; After heterologous expression in E . coli, functionally active phosphate carrier (PIC) from Saccharomyces cerevisiae mitochondria was purified and reconstituted into giant liposomes and used for patch clamp experiments . Single channel currents across excised patches revealed an anion channel function of the PIC protein . Besides the three transport modes known to date, namely phosphate/phosphate exchange, phosphate/OH exchange and mercurial-induced unidirectional transport, this channel activity represents the fourth transport mode of the PIC . The PIC channel activity was sensitive towards phosphate as its physiological substrate . Phosphate (10 mM) blocked in a specific but reversible manner the PIC channel, suggesting a phosphate-dependent conformational change of the protein into the carrier mode . Furthermore, the current through the channel and its gating activity were affected by divalent cations . In the presence of Ca2+ and Mg2+, the channel displayed a mean conductance of 25 +/- 5 pS whereas 40 +/- 10 pS was observed in the absence of divalent cations . Also, the dwell times in either the open or closed state of the PIC channel appeared to be prolonged in the presence of Ca2+ and Mg2+ . The observed PIC channel characteristics are discussed with respect to previously reported electrophysiological in situ measurements on anion channels of the inner mitochondrial membrane . Similarities of the PIC channel to the inner mitochondrial anion channel (IMAC) have been found. Mol Gen Genet, 1997 Oct, 256(4), 376 - 86 Identification of the Saccharomyces cerevisiae genes STB1-STB5 encoding Sin3p binding proteins; Kasten MM et al.; The yeast SIN3 gene functions as a transcriptional repressor, despite the fact that Sin3p does not bind DNA directly . We have conducted a two-hybrid screen to look for proteins that interact with Sin3p, using the PAH2 domain of Sin3p as bait . Five new genes, STB1-STB5 were identified, as well as the STB6 gene, which is similar to STB2 . STB1, STB2, STB3, and STB6 are novel genes, and STB4 and STB5 encode C6 zinc cluster DNA-binding proteins . None of these genes is essential for viability, and several of these genes may encode transcriptional activators . Several special problems were encountered in using a transcriptional repressor in a two-hybrid screen . For example, the STB genes will interact with a LexA-Sin3(PAH2) fusion protein containing a region of Sin3p, but a LexA-Sin3p fusion protein containing full-length Sin3p, along with a STB clone, does not produce two-hybrid activation of a transcriptional reporter . In addition, a sin3 mutation reduces the transcriptional activation by two-hybrid partners, suggesting that a sin3 mutation reduces the transcriptional efficiency of the Gal4p and VP16 activation domains . We have shown previously that Sin3p is part of a large multiprotein complex, and we show here that Stb1p and Stb2p are present in this complex. Yeast, 1997 Nov, 13(14), 1337 - 46 Lys80p of Saccharomyces cerevisiae, previously proposed as a specific repressor of LYS genes, is a pleiotropic regulatory factor identical to Mks1p; Feller A et al.; In Saccharomyces cerevisiae, an intermediate of the lysine pathway, alpha-aminoadipate semialdehyde (alpha AASA), acts as a coinducer for the transcriptional activation of LYS genes by Lys14p . The limitation of the production of this intermediate through feedback inhibition of the first step of the pathway results in apparent repression by lysine . Previously, the lys80 mutations, reducing the lysine repression and increasing the production of lysine, were interpreted as impairing a repressor of LYS genes expression . In order to understand the role of Lys80p in the control of the lysine pathway, we have analysed the effects of mutations epistatic to lys80 mutations . The effects of lys80 mutations on LYS genes expression were dependent on the integrity of the activation system (Lys14p and alpha AASA) . The increased production of lysine in lys80 mutants appeared to result from an improvement of the metabolic flux through the pathway and was correlated to an increase of the alpha-ketoglutarate pool and of the level of several enzymes of the tricarboxylic acid cycle . The LYS80 genes has been cloned and sequenced; it turned out to be identical to gene MKS1 cloned as a gene encoding a negative regulator of the RAS-cAMP pathway . We conclude that Lys80p is a pleiotropic regulatory factor rather than a specific repressor of LYS genes. Biochem Biophys Res Commun, 1997 Nov 17, 240(2), 287 - 92 G-protein coupled receptor from yeast Saccharomyces cerevisiae; Yun CW et al.; The Saccharomyces cerevisiae GPR1 (G-protein coupled receptor) gene was isolated using two-hybrid system with a heterotrimeric GTP binding protein alpha subunit Gpa2p as a bait . The GPR1 gene encodes 961 amino acids with predicted seven transmembrane segments and two large cytosolic regions as third cytosolic loop with 350 amino acids where asparagine-rich region was found and the C-terminal region with 283 amino acids . The Gpr1p interacted with Gpa2p at C-terminal region with 131 amino acid residues as well as third cytosolic loop . Disruption of the GPR1 gene was not lethal and did not affect to the cell growth . The Gpr1p-GFP fusion protein localized at the cell surface . These results suggest that Gpr1p is a G-protein coupled receptor which localized at plasma membrane . It is likely that a Gpr1p monitors the extracellular signal such as nutrition and transduce it via Gpa2p a possible positive regulator of cAMP level. Gene, 1997 Oct 1, 198(1-2), 165 - 9 Isocitrate lyase localisation in Saccharomyces cerevisiae cells; Chaves RS et al.; The isocitrate lyase from Saccharomyces cerevisiae was only located in the cell cytoplasm . This protein was found not to be associated with cell organelles, even under growth conditions that induce peroxisome proliferation . This conclusion is supported by experiments carried out by damaging the protoplast plasma membrane with DEAE-dextran, by differential centrifugation of osmotically lysed protoplast and by using the green fluorescent protein (GFP) of Aequorea victoria as a reporter fusion tag to localise the subcellular compartment to which isocitrate lyase is targeted. FEBS Lett, 1997 Oct 13, 416(1), 1 - 5 Effect of temperature on the role of Hsp104 and trehalose in barotolerance of Saccharomyces cerevisiae; Iwahashi H et al.; We have studied the effect of temperature on the contribution of Hsp104 and trehalose to barotolerance using mutants deficient in Hsp104 and trehalose synthesis . When compared with a corresponding wild type strain, mutants of Hsp104 did not show temperature dependent barotolerance when the incubation temperature during the hydrostatic pressure treatment was increased . However, a mutant deficient in trehalose synthesis showed features similar to a wild type strain . Furthermore, the Hsp104 level was low in the insoluble fraction of the wild type strain after pressure treatment at 35 degrees C but not at 4 degrees C, and the protein profiles in the insoluble fraction were different between 35 degrees C and 4 degrees C . In contrast to the Hsp104 deficient mutants, the protein profile of the wild type after pressure treatment at 35 degrees C favors the role of Hsp104 as a disaggregator of proteins during hydrostatic pressure stress . These results suggest that the role of Hsp104 in barotolerance is temperature dependent in contrast to trehalose. Cell Struct Funct, 1997 Aug, 22(4), 465 - 76 An altered nuclear migration into the daughter bud is induced by the cyclin A1-mediated Cdc28 kinase through an aberrant spindle movement in Saccharomyces cerevisiae; Sikder H et al.; A strain of Saccharomyces cerevisiae that contains an integrated copy of a Xenopus cyclin A1 gene under the control of the GAL1 promoter has been constructed . On inducing expression of cyclin A1, the nuclear migration that occurs prior to division becomes aberrant . Instead of migrating to the neck between the mother cell and daughter bud, the nucleus, the short mitotic spindle and its associated two spindle pole bodies entered the daughter bud . This phenotype was induced by expression of an indestructible cyclin mutant, but not by a mutated cyclin A1 unable to activate Cdc28 kinase . The nuclear abnormality induced by cyclin A1 was overcome by cdc28 mutations that abolish its ability to bind cyclin A1 . Both yeast cyclin Clb3 and Xenopus mitotic cyclin B produced the same phenotype, whereas G1 cyclin Cln2 did not . The results suggest that the proper movement of the nucleus through the spindle function during mitosis requires the appropriate activity of Cdc28 kinase mediated by specific cyclins. FEMS Microbiol Lett, 1997 Nov 1, 156(1), 147 - 52 Cytochrome P-450 reductase is responsible for the ferrireductase activity associated with isolated plasma membranes of Saccharomyces cerevisiae; Lesuisse E et al.; Cytochrome P-450 reductase (encoded by the NCP1 gene) was found to catalyse all the NADPH-dependent ferrireductase activities associated with isolated plasma membranes of the yeast Saccharomyces cerevisiae . We therefore examined the contribution of this enzyme to the ferrireductase activity of cells in vivo . Cytochrome P-450 reductase was shown to be not essential for the cell ferrireductase activity, but it influenced this activity, with different effects on the Fre1- and the Fre2-dependent reductase systems . Overexpression of FRE1 did not lead to an increased ferrireductase activity of the cells when NCP1 was repressed . In contrast, cells that overexpressed FRE2 had maximal ferrireductase activity when NCP1 was repressed . The degree of NCP1 expression also affected the amount of iron and copper accumulated by the cells during growth . The biochemical implications and the physiological significance of these observations are discussed. Genes Cells, 1997 Jul, 2(7), 443 - 55 Alteration of telomeric sequences and senescence caused by mutations in RAD50 of Saccharomyces cerevisiae; Kironmai KM et al.; BACKGROUND: Vegetatively dividing cells of Saccharomyces cerevisiae carrying a mutation in RAD50 grow significantly more slowly in rich medium and are sensitive to DNA damage inflicted by X-ray or chemical mutagens . RAD50 function is essential for the formation and repair of meiosis-specific double-strand breaks and chromosome stability . RESULTS: We present evidence for two new phenotypes associated with the rad50delta mutant; shortened telomeres and cell senescence . Comparison of TG1-3 telomeric sequences in an isogenic pair of RAD50 and rad50delta haploid strains showed that they were considerably shortened in the latter . Although rad50delta mutation conferred cell enlargement and slow growth, cell doubling was faster but caused an increase in the frequency of cell death . Telomeres were restored to the wild-type size in hemizygous RAD50/rad50delta and rad50S/rad50delta strains; however, they showed a significant increase in rad50S/rad50S diploid with a concomitant rise in cell viability . Telomeres were stabilized in hemizygous RAD50/rad50delta and rad50S/rad50delta diploids during prolonged growth, suggesting that even a half-dosage of RAD50 is sufficient to conserve the telomere size during successive cell divisions . Furthermore, cells bearing the rad50delta mutation revealed abnormalities in nuclear segregation and, in the presence of hydroxyurea, displayed phenotypes consistent with defects in S-phase checkpoint control . CONCLUSION: This report presents evidence of the involvement of a gene relevant to recombinational repair in the maintenance of telomeres . We conclude that the phenotypes displayed by yeast rad50delta cells have intriguing similarities among the human cell lines representing DNA repair-deficient chromosome instability syndromes. Biochim Biophys Acta, 1997 Sep 26, 1342(1), 62 - 72 Purification and characterization of a novel poly(U), poly(C) ribonuclease from Saccharomyces cerevisiae; Lalioti VS et al.; A new ribonuclease from Saccharomyces cerevisiae, specific for poly(U) and poly(C) substrate, was purified near to homogeneity by successive fractionation with DEAE-Sepharose, Heparin-Sepharose and CM-Sepharose chromatography . The purified molecule detected by SDS/polyacrylimide gel electrophoresis has a molecular mass of 29 kDa . The optimum pH for the enzyme activity is 5.5-7 and its isoelectric point is 7.5 . The purified enzyme was able to degrade 26S, 18S and 5S rRNAs as well as mRNA obtained from in vitro transcription . No catalytic activity was observed when the RNase was incubated with tRNA and double stranded substrate . Our findings suggest that this novel RNase may play an important role in the processing of RNA in Saccharomyces cerevisiae. Biochim Biophys Acta, 1997 Oct 11, 1358(3), 249 - 54 A novel dimeric oxovanadium (IV) species identified in Saccharomyces cerevisiae cells; Zoroddu MA et al.; Saccharomyces cerevisiae cells stored oxovanadium (IV) ions in a dimeric form . In the late stationary phase Saccharomyces cerevisiae cells grown in rich medium containing concentrations of oxovanadium (V), orthovanadate from 12 to 18 mM, causing growth stasis, a dimeric oxovanadium (IV) species was identified by EPR spectroscopy . The EPR spectrum exhibited at 110 K the low-field forbidden deltaMs = +/-2 transition at g around 4 and the half-field deltaMs = +/-1 15-lines feature at g around 2 out of the presence of a triplet state by the coupling of the oxovanadium (IV) ions in a dimeric form . Hyperfine splitting of 75.2 x 10(-4) cm(-1) and an interionic distance of about 4.4 angstroms was calculated . The dimeric species was localized in the cellular cytoplasmic space. Yeast, 1997 Oct, 13(13), 1275 - 90 Expression profiles of transcripts from 126 open reading frames in the entire chromosome VI of Saccharomyces cerevisiae by systematic northern analyses; Naitou M et al.; Chromosome VI of Saccharomyces cerevisiae contains 126 open reading frames (ORFs), and the functions of proteins encoded by 80 ORFs are still unknown . In this report, we have systematically examined the expression profiles of all 126 ORFs on chromosome VI under five kinds of growth conditions by quantitative Northern hybridization . A series of Northern analyses and reverse transcription polymerase chain reactions have revealed that more than 64 novel ORFs are transcribed . Two ORFs (YFL059w and YFR011c) are specifically expressed in the presence of galactose . Two ORFs (YFL012w and YFR032c) are specifically transcribed in sporulation . Six ORFs (YFL049w, YFL035c, YFL010c, YFR006w, YFR010w and YFR017c) are abundantly expressed in many growth conditions. Yeast, 1997 Oct, 13(13), 1265 - 74 Transcriptional regulation of SUP35 and SUP45 in Saccharomyces cerevisiae; Dagkessamanskaya A et al.; SUP35 and SUP45 encode translational release factors in the yeast Saccharomyces cerevisiae . In addition, Sup35p is related to the cytoplasmically inherited prion-like phenotype {PSI+} . The vital cellular role of Sup35p and Sup45p prompted us to study the regulation of transcription of the corresponding genes . Since the {PSI} state of the yeast strain affects the abundance of Sup35p and Sup45p, both {PSI+} and {psi-} variants were included in these analyses . It turned out that SUP35 and SUP45 transcript levels are regulated by nutritional changes and stress in a way strikingly similar to those of ribosomal protein genes . The {PSI} state did not influence the respective transcript levels nor their regulation, although HSP12 (as a monitor of general stress-responsive) gene expression appeared to differ in the two variant strains . The transcription activation sites of SUP35 and SUP45 were mapped using deletion analysis of the respective promoter-reporter fusion genes . The UAS in both cases was found to consist of an Abf1p-site and a T-rich element . Also in this respect SUP35 and SUP45 show a notable resemblance with ribosomal protein genes . Evidence was found that SUP35 in addition harbors a potential internal promoter element which became active after progressive 5'-deletion removing the first of the three in-frame ATGs. Eur J Biochem, 1997 Oct 1, 249(1), 343 - 9 Identification and analysis of a static culture-specific cell wall protein, Tir1p/Srp1p in Saccharomyces cerevisiae; Kitagaki H et al.; A 100-kDa protein was found to be a major cell wall protein in Saccharomyces cerevisiae cells cultured without shaking, but was not present in cells cultured with shaking . The amino acid sequence of this protein was identical to the sequence of Tir1p/Srp1p . TIR1/SRP1 has previously been identified as a gene induced by glucose, cold shock or anaerobiosis and was believed to be a cell membrane protein but not a cell wall protein . However, we found that beta-1,3-glucanase solubilized Tir1p/Srp1p from the cell wall and the purified Tir1p/Srp1p reacted with antiserum to beta-1,6-glucan and contained glucose . These results suggest that Tir1p/Srp1p is a major structural cell wall protein in the static-cultured yeast cells and is bound to the cell wall through beta-1,6-glucan . TIR1/SRP1 mRNA was transcribed only in the static culture and its transcription was regulated by the ROX1 repressor. Eur J Biochem, 1997 Oct 1, 249(1), 309 - 17 Purification and nucleic-acid-binding properties of a Saccharomyces cerevisiae protein involved in the control of ploidy; Weber V et al.; Scp160p (Saccharomyces cerevisiae protein involved in the control of ploidy), a polypeptide with a molecular mass of around 160 kDa, is associated with the nuclear envelope and the endoplasmic reticulum . The most noteworthy phenotype of SCP160 deletion mutants is a decrease in viability and an increased number of chromosomes in the surviving cells {Wintersberger, U., Kuhne, C . & Karwan, A . (1995) Yeast 11, 929-944} . Scp160p contains 14 KH domains, conserved motifs that have lately been identified in a variety of RNA-binding proteins . In this report, we demonstrate that the Scp160p sequence shows nearly perfect colinearity with the putative gene product of C08H9.2 from the nematode Caenorhabditis elegans as well as with the vigilins, vertebrate RNA-binding proteins with a cellular location similar to that of Scp160p . Moreover, we found that Scp160p contains a potential nuclear-export signal (NES) near its N-terminus and a potential nuclear-localization signal (NLS) between KH domains 3 and 4 . To determine whether the protein is able to bind to RNA, we purified Scp160p from yeast cell extract by DNA-cellulose and anti-Scp160p affinity chromatography . In northwestern blotting experiments, the electrophoretically homogeneous protein bound to ribohomopolymers and ribosomal RNA as well as to single-stranded and double-stranded DNA . Subcellular fractionation studies revealed that the major part of Scp160p is membrane associated via ionic interactions and can be released from the membrane fraction under conditions that lead to a dissociation of ribosomes . Together, our findings suggest that Scp160p is the yeast homologue of the vigilins, and point to a role for Scp160p in nuclear RNA export or in RNA transport within the cytoplasm. Int J Biochem Cell Biol, 1997 Apr, 29(4), 635 - 48 The role of the Src homology-2 domain in the lethal effect of Src expression in the yeast Saccharomyces cerevisiae; Trager JB et al.; Expression of the retroviral transforming gene v-src arrests the proliferation of the yeast Saccharomyces cerevisiae . A functional Src SH2 (Src homology 2) domain is required for this arrest . To examine the mechanism by which Src blocks yeast cell proliferation, and to determine the role of the Src SH2 domain in the growth arrest, src variants were expressed in yeast under the control of the galactose-inducible GAL1 promoter . Following galactose induction of Src expression, phosphotyrosyl-proteins were isolated by immunoprecipitation with beads coupled to either anti-phosphotyrosine antibody or to a recombinant fusion protein containing the Src SH2 domain . A group of SH2-binding phosphotyrosyl proteins was detected in cells expressing toxic forms of Src, but were not detected in cells expressing non-toxic variants . This group of phosphotyrosyl-proteins represents a minor subset of the proteins phosphorylated by v-Src . The lethality of v-Src and the phosphorylation of SH2-binding proteins were co-ordinately affected by alterations in phosphotyrosine-phosphatase activity . These observations indicate that the lethality of Src is correlated with the phosphorylation of proteins that bind to the Src SH2 domain . The role of the SH2 domain in determining the lethal effects of Src in yeast may be similar to its role in targeting Src to substrates necessary for its biological effects in vertebrate cells. Mol Biol Cell, 1997 Nov, 8(11), 2119 - 32 Nuclear pore complex number and distribution throughout the Saccharomyces cerevisiae cell cycle by three-dimensional reconstruction from electron micrographs of nuclear envelopes; Winey M et al.; The number of nuclear pore complexes (NPCs) in individual nuclei of the yeast Saccharomyces cerevisiae was determined by computer-aided reconstruction of entire nuclei from electron micrographs of serially sectioned cells . Nuclei of 32 haploid cells at various points in the cell cycle were modeled and found to contain between 65 and 182 NPCs . Morphological markers, such as cell shape and nuclear shape, were used to determine the cell cycle stage of the cell being examined . NPC number was correlated with cell cycle stage to reveal that the number of NPCs increases steadily, beginning in G1-phase, suggesting that NPC assembly occurs continuously throughout the cell cycle . However, accumulation of nuclear envelope observed during the cell cycle, indicated by nuclear surface area, is not continuous at the same rate, such that the density of NPCs per unit area of nuclear envelope peaks in apparent S-phase cells . Analysis of the nuclear envelope reconstructions also revealed no preferred NPC-to-NPC distance . However, NPCs were found in large clusters over regions of the nuclear envelope . Interestingly, clusters of NPCs were most pronounced in early mitotic nuclei and were found to be associated with the spindle pole bodies, but the functional significance of this association is unknown. Mol Biol Cell, 1997 Nov, 8(11), 2291 - 306 End4p/Sla2p interacts with actin-associated proteins for endocytosis in Saccharomyces cerevisiae; Wesp A et al.; end4-1 was isolated as a temperature-sensitive endocytosis mutant . We cloned and sequenced END4 and found that it is identical to SLA2/MOP2 . This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis . We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable . The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization . A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex . Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2 . The endocytic function of Abp1p depends on its SH3 domain . In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis . Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis. Mol Biol Cell, 1997 Nov, 8(11), 2267 - 80 Functions of FKBP12 and mitochondrial cyclophilin active site residues in vitro and in vivo in Saccharomyces cerevisiae; Dolinski K et al.; Cyclophilin and FK506 binding protein (FKBP) accelerate cis-trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506 . The normal cellular functions of these proteins, however, are unknown . We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes . Surprisingly, most of these mutant enzymes were biologically active in vivo . In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay . However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5-20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading . In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases . Thus, a number of cyclophilin and FKBP12 "active-site" mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo . One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast . Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions. Acta Biochim Pol, 1997, 44(2), 191 - 200 The phosphorylation sites of ribosomal P proteins from Saccharomyces cerevisiae cells by endogenous CK-2, PK60S and RAP protein kinases; Boguszewska A et al.; The phosphorylation sites of ribosomal acidic proteins (P proteins) from Saccharomyces cerevisiae were studied in vivo and in vitro by using CK-2, PK60S and RAP protein kinases . The three enzymes phosphorylate the last serine residues located in a highly conserved carboxyl end of the polypeptide chains . This was established by two-dimensional analysis of tryptic phosphopeptides from 32P-labelled proteins YP1 alpha, YP1 beta, YP2 alpha and YP2 beta, and by kinetic studies of the protein kinases with synthetic peptides corresponding to the fragments of endogenous ribosomal acidic polypeptides . In experiments with both endogenous P proteins and synthetic peptides as substrates protein kinase PK60S demonstrated unusual substrate specificity . In contrast to CK-2 and RAP protein kinases, PK60S phosphorylates predominantly two of the four P proteins, YP1 alpha and YP2 beta, with kinetic constants dependent on the primary structure of the N-terminal region of the polypeptide containing the target residue . The neutral amino acid, alanine, at position 3 in the peptide AAEESDDD (polypeptide fragments of YP1 beta and YP2 alpha) decreases the K(m) value more than 10-fold by comparison with the basic lysine residue at the same position in the peptide AKEESDDD (polypeptide fragments of YP1 alpha and YP2 beta). J Biochem (Tokyo), 1997 Jun, 121(6), 1182 - 9 The transcriptional activators of the PHO regulon, Pho4p and Pho2p, interact directly with each other and with components of the basal transcription machinery in Saccharomyces cerevisiae; Magbanua JP et al.; The transcriptional regulators Pho4p and Pho2p are involved in transcription of several genes in the PHO regulon of Saccharomyces cerevisiae . Genetic evidence with temperature-sensitive pho4 and pho2 mutants suggested that Pho4p and Pho2p interact with each other . Immunoprecipitation experiments showed that Pho4p and Pho2p form a complex on a 36-bp sequence bearing an upstream activation site (UAS) and protein binding assays indicated that these proteins interact directly . DNA-binding experiments with crude extracts prepared from yeast strains expressing T7-PHO4, encoding Pho4p tagged with the T7 epitope, indicated that Pho2p interacts with T7-Pho4p and enhances the binding affinity of T7-Pho4p to the UAS . Protein binding experiments also showed that both Pho4p and Pho2p could bind with the general transcription factors, TBP, TFIIB, and TFIIEbeta, suggesting that the Pho4p-Pho2p complex bound to the UAS activates transcription of the PHO genes by direct interaction with the general transcription factors. Biometals, 1997 Oct, 10(4), 239 - 46 Reduction of vanadate to vanadyl by a strain of Saccharomyces cerevisiae; Bisconti L et al.; Three strains of Saccharomyces cerevisiae, SC-1, DBVPG 6173 and DBVPG 6037, were studied for vanadate resistance in complex Sabouraud medium since they did not thrive in different minimal media (yeast nitrogen base with and without amino acids) . The strain SC-1 was resistant up to 16 mM of vanadate, whereas the strains DBVPG 6173 and DBVPG 6037 were inhibited by 8 mM and 4 mM vanadate, respectively . The vanadate resistance in strain SC-1 was constitutive and due to the reduction of this oxyanion to vanadyl, which was detected by EPR spectroscopy and visible spectroscopy . The transformation of vanadate to vanadyl took place during the exponential growth phase; 10 mM of vanadate was reduced to vanadyl outside the cells since the oxyanion was not detected in the cell biomass and only a negligible concentration of vanadyl (25 nmoles mg-1 cells dry weight) was found in the biomass . The other two vanadate-sensitive yeast strains only accumulated vanadate and did not reduce the oxyanion to vanadyl. J Biol Chem, 1997 Nov 7, 272(45), 28539 - 44 Purification and biochemical properties of Saccharomyces cerevisiae Mdj1p, the mitochondrial DnaJ homologue; Deloche O et al.; The DnaK/DnaJ/GrpE heat shock proteins of Escherichia coli constitute the prototype DnaK chaperone machine . Various studies have shown that these three proteins work synergistically in a diverse array of biological functions, including protein folding and disaggregation, proteolysis, and transport across biological membranes . We have overexpressed and purified the mitochondrial Saccharomyces cerevisiae DnaJ homologue, Mdj1pDelta55, which lacks the mitochondrial presequence, and studied its biochemical properties in well defined in vitro systems . We find that Mdj1pDelta55 interacts with DnaK as judged both by an enzyme-linked immunosorbent assay, as well as stimulation of DnaK's weak ATPase activity in the presence of GrpE . In addition, Mdj1pDelta55 not only interacts with denatured firefly luciferase on its own, but also enables DnaK to bind to it in an ATP-dependent mode . Using co-immunoprecipitation assays we can demonstrate the presence of a stable Mdj1pDelta55-luciferase-DnaK complex . However, in contrast to DnaJ, Mdj1pDelta55 does not appear to interact well with certain seemingly folded proteins, such as the sigma32 heat shock transcription factor or the lambdaP DNA replication protein . Finally, Mdj1pDelta55 can substitute perfectly well for DnaJ in the refolding of denatured firefly luciferase by the DnaK chaperone machine . These studies demonstrate that Mdj1pDelta55 has conserved most of DnaJ's known biological properties, thus supporting an analogous functional role in yeast mitochondria. Genes Dev, 1997 Nov 1, 11(21), 2926 - 37 Rpp1, an essential protein subunit of nuclear RNase P required for processing of precursor tRNA and 35S precursor rRNA in Saccharomyces cerevisiae; Stolc V et al.; The gene for an essential protein subunit of nuclear RNase P from Saccharomyces cerevisiae has been cloned . The gene for this protein, RPP1, was identified by virtue of its homology with a human scleroderma autoimmune antigen, Rpp30, which copurifies with human RNase P . Epitope-tagged Rpp1 can be found in association with both RNase P RNA and a related endoribonuclease, RNase MRP RNA, in immunoprecipitates from crude extracts of cells . Depletion of Rpp1 in vivo leads to the accumulation of precursor tRNAs with unprocessed 5' and 3' termini and reveals rRNA processing defects that have not been described previously for proteins associated with RNase P or RNase MRP . Immunoprecipitated complexes cleave both yeast precursor tRNAs and precursor rRNAs. J Bacteriol, 1997 Nov, 179(21), 6560 - 5 Effects of different carbon fluxes on G1 phase duration, cyclin expression, and reserve carbohydrate metabolism in Saccharomyces cerevisiae; Sillje HH et al.; By controlled addition of galactose to synchronized galactose-limited Saccharomyces cerevisiae cultures, the growth rate could be regulated while external conditions were kept constant . By using this method, the G1 phase duration was modulated and expression of cell cycle-regulated genes was investigated . The expression of the cyclin genes CLN1 and CLN2 was always induced just before bud emergence, indicating that this event marks the decision to pass Start . Thus, G1 phase elongation was not due to a slower accumulation of the CLN1 and CLN2 mRNA levels . Only small differences in CLN3 expression levels were observed . The maximal SWI4 expression preceded maximal CLN1 and CLN2 expression under all conditions, as expected for a transcriptional activator . But whereas SWI4 was expressed at about 10 to 20 min, before CLN1 and CLN2 expression at high growth rates, this time increased to about 300 min below a particular consumption rate at which the G1 phase strongly elongated . In the slower-growing cultures, also an increase in SWI6 expression was observed in the G1 phase . The increase in G1 phase duration below a particular consumption rate was accompanied by a strong increase in the reserve carbohydrate levels . These carbohydrates were metabolized again before bud emergence, indicating that below this consumption rate, a transient increase in ATP flux is required for progression through the cell cycle . Since Start occurred at different cell sizes under different growth conditions, it is not just a certain cell size that triggers passage through Start. Appl Microbiol Biotechnol, 1997 Sep, 48(3), 339 - 45 Improved efficiency and stability of multiple cloned gene insertions at the delta sequences of Saccharomyces cerevisiae; Lee FW et al.; Two delta-integration vectors were evaluated for the insertion of an inducible expression cassette (the yeast CUP1 promoter fused to the Escherichia coli lacZ structural gene, CUP1p-lacZ) and a bacterial neomycin-resistance gene (neo) into the genome of Saccharomyces cerevisiae via homologous recombination . Cells containing integrations were selected by resistance to the aminoglycoside G418 . The first vector was a traditional construct containing only one delta sequence; with this vector, the transformation efficiency and the number of integrations per cell were quite low . The second carried two delta sequences flanking the desired insert, and the unneeded bacterial sequences were removed by restriction-enzyme digestion immediately before transformation . When this double delta vector was employed, the integrated copy number was more than doubled relative to the single delta system and final beta-galactosidase levels exceeded those obtained with the 2 mu-based plasmid . Furthermore, the integrations appeared more stable in long-term sequential culture (both with and without induction of the lacZ gene) than those obtained via the single delta vector. J Biol Chem, 1997 Oct 31, 272(44), 27671 - 7 STT4 is an essential phosphatidylinositol 4-kinase that is a target of wortmannin in Saccharomyces cerevisiae; Cutler NS et al.; Wortmannin is a natural product that inhibits signal transduction . One target of wortmannin in mammalian cells is the 110-kDa catalytic subunit of phosphatidylinositol 3-kinase (PI 3-kinase) . We show that wortmannin is toxic to the yeast Saccharomyces cerevisiae and present genetic and biochemical evidence that a phosphatidylinositol 4-kinase (PI 4-kinase), STT4, is a target of wortmannin in yeast . In a strain background in which stt4 mutants are rescued by osmotic support with sorbitol, the toxic effects of wortmannin are similarly prevented by sorbitol . In contrast, in a different strain background, STT4 is essential under all conditions and wortmannin toxicity is not mitigated by sorbitol . Overexpression of STT4 confers wortmannin resistance, but overexpression of PIK1, a related PI 4-kinase, does not . In vitro, the PI 4-kinase activity of STT4, but not of PIK1, was potently inhibited by wortmannin . Overexpression of the phosphatidylinositol 4-phosphate 5-kinase homolog MSS4 conferred wortmannin resistance, as did deletion of phospholipase C-1 . These observations support a model for a phosphatidylinositol metabolic cascade involving STT4, MSS4, and phospholipase C-1 and provide evidence that an essential product of this pathway is the lipid phosphatidylinositol 4,5-bisphosphate. Arch Biochem Biophys, 1997 Nov 1, 347(1), 78 - 84 Purification and characterization of UBP6, a new ubiquitin-specific protease in Saccharomyces cerevisiae; Park KC et al.; Ubiquitin-specific protease-6 (UBP6) in Saccharomyces cerevisiae was expressed in Escherichia coli and purified from the cells using 125I-labeled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a model substrate . The purified UBP6 behaved as a 58-kDa under both nondenaturing and denaturing conditions, indicating that the enzyme comprises a single polypeptide . It was maximally active at pH levels between 8.5 and 9, but showed little or no activity at pH below 7 and above 9.5 . As with other UBPs, its activity was strongly inhibited by sulfhydryl-blocking reagents, such as N-ethylmaleimide, and by ubiquitin-aldehyde . In addition to the model substrate, UBP6 hydrolyzed ubiquitin-alphaNH-protein extensions, such as the ubiquitin-alphaNH-carboxyl extension protein of 80 amino acids and ubiquitin-alphaNH-dihydrofolate reductase, but not poly-His-tagged diubiquitin . It was also capable of releasing free ubiquitin from branched polyubiquitin chains that are ligated to proteins through epsilonNH-isopeptide bonds, although to a limited extent . These results suggest that UBP6 may play an important role in the generation of free ubiquitins and certain ribosomal proteins from ubiquitin-ribosomal fusion proteins as well as in deubiquitination of certain polyubiquitinated proteins targeted for degradation by the 26S proteasomes . FEBS Lett, 1997 Sep 22, 415(1), 16 - 20 The acetyl-CoA synthetase gene ACS2 of the yeast Saccharomyces cerevisiae is coregulated with structural genes of fatty acid biosynthesis by the transcriptional activators Ino2p and Ino4p; Hiesinger M et al.; The yeast Saccharomyces cerevisiae contains two acetyl-CoA synthetase genes, ACS1 and ACS2 . While ACS1 transcription is glucose repressible, ACS2 shows coregulation with structural genes of fatty acid biosynthesis . The ACS2 upstream region contains an ICRE (inositol/choline-responsive element) as an activating sequence and requires the regulatory genes INO2 and INO4 for maximal expression . We demonstrate in vitro binding of the heterodimeric activator protein Ino2p/Ino4p to the ACS2 promoter . In addition, the pleiotropic transcription factor Abf1p also binds to the ACS2 control region . The identification of ACS2 activating elements also found upstream of ACC1, FAS1 and FAS2 suggests a role of this acetyl-CoA synthetase isoenzyme for the generation of the acetyl-CoA pool required for fatty acid biosynthesis. J Biol Chem, 1997 Oct 10, 272(41), 25787 - 93 Restriction of copper export in Saccharomyces cerevisiae to a late Golgi or post-Golgi compartment in the secretory pathway; Yuan DS et al.; The CCC2 gene in the yeast Saccharomyces cerevisiae encodes a P-type ATPase (Ccc2p) required for the export of cytosolic copper to the extracytosolic domain of a copper-dependent oxidase, Fet3p . Ccc2p appears to be both a structural and functional homolog of ATPases impaired in two human disorders of intracellular copper transport, Menkes disease and Wilson disease . In the present work, three approaches were used to determine the locus of Ccc2p-dependent copper export within the secretory pathway . First, like ccc2 mutants, sec mutants blocked in the secretory pathway at steps prior to and including the Golgi complex failed to deliver radioactive copper to Fet3p . Second, also like ccc2 mutants, vps33 and certain other mutants with defects in post-Golgi sorting exhibited phenotypes traceable to deficient copper delivery to Fet3p . These findings were sufficient to explain the respiratory deficiency of these mutants . Third, immunofluorescence microscopy revealed that Ccc2p was distributed among several punctate foci within wild-type cells, consistent with late Golgi or post-Golgi localization . Thus, copper export by Ccc2p appears to be restricted to a late or post-Golgi compartment in the secretory pathway. Mol Gen Genet, 1997 Aug, 255(6), 561 - 9 Locus-specific suppression of ilv1 in Saccharomyces cerevisiae by deregulation of CHA1 transcription; Pedersen JO et al.; The ILV1 gene of Saccharomyces cerevisiae encodes the anabolic threonine deaminase, which catalyzes the first committed step in isoleucine biosynthesis . Strains devoid of a functional Ilv1p have a requirement for isoleucine . Threonine can also be deaminated by a second serine/threonine deaminase encoded by the CHA1 gene . CHA1 is regulated by transcriptional induction by serine and threonine, and enables yeast to utilize the hydroxyamino acids as sole nitrogen source . Phenotypic suppression of ilv1 can occur by inducer-mediated transcriptional activation of the CHA1 gene . To identify mutations in putative trnas-acting factors regulating CHA1 expression, we have isolated and characterized three extragenic suppressors of ilv1 . A dominant mutation, SIL4 (suppressor of ilv1), is allelic to HOM3 . It increases the size of the threonine pool, by 15- to 20-fold, which is sufficient to induce CHA1 transcription, thereby creating a metabolic bypass of ilv1 . A second dominant mutation, SIL3, and a recessive mutation, sil2, both suppress ilv1 by causing inducer-independent, constitutive transcription of CHA1 . Importantly, sil2 and SIL3 increase the expression of a CHA1p-lacZ translational gene fusion, demonstrating that they exert their action through the CHA1 promoter . Genetic analysis showed that both SIL3 and sil2 are alleles of CHA4, a positive regulator of CHA1, i.e., they convert Cha4p to a constitutive activator. J Mol Biol, 1997 Oct 3, 272(4), 477 - 83 Mdj2p, a novel DnaJ homolog in the mitochondrial inner membrane of the yeast Saccharomyces cerevisiae; Westermann B et al.; Members of the heat shock protein 70 (Hsp70) family mediate import, folding, assembly and degradation of proteins in mitochondria . The function of Hsp70 proteins is dependent on their interaction with cofactors, including members of the DnaJ protein family . The mitochondrial DnaJ homolog, Mdj1p, has been shown to cooperate with the major mitochondrial Hsp70, mt-Hsp70 . We describe the identification of a second mitochondrial DnaJ homolog, Mdj2p, in the yeast Saccharomyces cerevisiae . The protein possesses an N-terminal transmembrane domain that anchors it in the mitochondrial inner membrane . The C-terminal J-domain shares 30% amino acid identity with the J-domain of Escherichia coli DnaJ and is exposed to the mitochondrial matrix . Mdj2p carries a putative internal mitochondrial targeting signal and is imported into mitochondria in a membrane potential-dependent manner . Deletion of the MDJ2 gene did not result in a detectable growth defect . Double mutants of mdj1 and mdj2 showed severe growth defects at elevated temperature, indicating a distinct overlap of the functions of Mdj1p and Mdj2p . Mol Cell Biol, 1997 Nov, 17(11), 6765 - 71 Two pathways for removal of nonhomologous DNA ends during double-strand break repair in Saccharomyces cerevisiae; Paques F et al.; During repair of a double-strand break (DSB) by gene conversion, one or both 3' ends of the DSB invade a homologous donor sequence and initiate new DNA synthesis . The use of the invading DNA strand as a primer for new DNA synthesis requires that any nonhomologous bases at the 3' end be removed . We have previously shown that removal of a 3' nonhomologous tail in Saccharomyces cerevisiae depends on the nucleotide excision repair endonuclease Rad1/Rad10, and also on the mismatch repair proteins Msh2 and Msh3 . We now report that these four proteins are needed only when the nonhomologous ends of recombining DNA are 30 nucleotides (nt) long or longer . An additional protein, the helicase Srs2, is required for the RAD1-dependent removal of long 3' tails . We suggest that Srs2 acts to extend and stabilize the initial nascent joint between the invading single strand and its homolog . 3' tails shorter than 30 nt are removed by another mechanism that depends at least in part on the 3'-to-5' proofreading activity of DNA polymerase delta. Mol Cell Biol, 1997 Nov, 17(11), 6693 - 9 The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae; Huang H et al.; It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres . This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci . Here, we show that a null mutation of the DNA repair gene RAD6 reduces silencing of the HM loci and lowers the mating efficiency of MATa strains . Likewise, rad6-delta reduces silencing of the telomere-located RNAP II-transcribed genes URA3 and ADE2 . We also show that the RNAP III-transcribed tyrosyl tRNA gene, SUP4-o, is subject to position effect variegation when located near a telomere and that this silencing requires the RAD6 and SIR genes . Neither of the two known Rad6 binding factors, Rad18 and Ubr1, is required for telomeric silencing . Since Ubrl is the recognition component of the N-end rule-dependent protein degradation pathway, this suggests that N-end rule-dependent protein degradation is not involved in telomeric silencing . Telomeric silencing requires the amino terminus of Rad6 . Two rad6 point mutations, rad6(C88A) and rad6(C88S), which are defective in ubiquitin-conjugating activity fail to complement the silencing defect, indicating that the ubiquitin-conjugating activity of RAD6 is essential for full telomeric silencing. Mol Cell Biol, 1997 Nov, 17(11), 6517 - 25 Coordination of the mating and cell integrity mitogen-activated protein kinase pathways in Saccharomyces cerevisiae; Buehrer BM et al.; Mating pheromone stimulates a mitogen-activated protein (MAP) kinase activation pathway in Saccharomyces cerevisiae that induces cells to differentiate and form projections oriented toward the gradient of pheromone secreted by a mating partner . The polarized growth of mating projections involves new cell wall synthesis, a process that relies on activation of the cell integrity MAP kinase, Mpk1 . In this report, we show that Mpk1 activation during pheromone induction requires the transcriptional output of the mating pathway and protein synthesis . Consequently, Mpk1 activation occurs subsequent to the activation of the mating pathway MAP kinase cascade . Additionally, Spa2 and Bni1, a formin family member, are two coil-coil-related proteins that are involved in the timing and other aspects of mating projection formation . Both proteins also affect the timing and extent of Mpk1 activation . This correlation suggests that projection formation comprises part of the pheromone-induced signal that coordinates Mpk1 activation with mating differentiation . Stimulation of Mpk1 activity occurs through the cell integrity phosphorylation cascade and depends on Pkc1 and the redundant MAP/Erk kinases (MEKs), Mkk1 and Mkk2 . Surprisingly, Mpk1 activation by pheromone was only partially impaired in cells lacking the MEK kinase Bck1 . This Bck1-independent mechanism reveals the existence of an alternative activator of Mkk1/Mkk2 in some strain backgrounds that at least functions under pheromone-induced conditions. Mol Cell Biol, 1997 Nov, 17(11), 6491 - 501 Xbp1, a stress-induced transcriptional repressor of the Saccharomyces cerevisiae Swi4/Mbp1 family; Mai B et al.; We have identified Xbp1 (XhoI site-binding protein 1) as a new DNA-binding protein with homology to the DNA-binding domain of the Saccharomyces cerevisiae cell cycle regulating transcription factors Swi4 and Mbp1 . The DNA recognition sequence was determined by random oligonucleotide selection and confirmed by gel retardation and footprint analyses . The consensus binding site of Xbp1, GcCTCGA(G/A)G(C/A)g(a/g), is a palindromic sequence, with an XhoI restriction enzyme recognition site at its center . This Xbpl binding site is similar to Swi4/Swi6 and Mbp1/Swi6 binding sites but shows a clear difference from these elements in one of the central core bases . There are binding sites for Xbp1 in the G1 cyclin promoter (CLN1), but they are distinct from the Swi4/Swi6 binding sites in CLN1, and Xbp1 will not bind to Swi4/Swi6 or Mbp1/Swi6 binding sites . The XBP1 promoter contains several stress-regulated elements, and its expression is induced by heat shock, high osmolarity, oxidative stress, DNA damage, and glucose starvation . When fused to the LexA DNA-binding domain, Xbp1 acts as transcriptional repressor, defining it as the first repressor in the Swi4/Mbp1 family and the first potential negative regulator of transcription induced by stress . Overexpression of XBP1 results in a slow-growth phenotype, lengthening of G1, an increase in cell volume, and a repression of G1 cyclin expression . These observations suggest that Xbp1 may contribute to the repression of specific transcripts and cause a transient cell cycle delay under stress conditions. Mol Cell Biol, 1997 Nov, 17(11), 6410 - 8 Transcriptional activation upon pheromone stimulation mediated by a small domain of Saccharomyces cerevisiae Ste12p; Pi H et al.; In the yeast Saccharomyces cerevisiae, Ste12p induces transcription of pheromone-responsive genes by binding to a DNA sequence designated the pheromone response element . We generated a series of hybrid proteins of Ste12p with the DNA-binding and activation domains of the transcriptional activator Gal4p to define a pheromone induction domain of Ste12p sufficient to mediate pheromone-induced transcription by these hybrid proteins . A minimal pheromone induction domain, delineated as residues 301 to 335 of Ste12p, is dependent on the pheromone mitogen-activated protein (MAP) kinase pathway for induction activity . Mutation of the three serine and threonine residues within the minimal pheromone induction domain did not affect transcriptional induction, indicating that the activity of this domain is not directly regulated by MAP kinase phosphorylation . By contrast, mutation of the two tyrosines or their preceding acidic residues led to a high level of transcriptional activity in the absence of pheromone and consequently to the loss of pheromone induction . This constitutively high activity was not affected by mutations in the MAP kinase cascade, suggesting that the function of the pheromone induction domain is normally repressed in the absence of pheromone . By two-hybrid analysis, this minimal domain interacts with two negative regulators, Dig1p and Dig2p (also designated Rst1p and Rst2p), and the interaction is abolished by mutation of the tyrosines . The pheromone induction domain itself has weak and inducible transcriptional activity, and its ability to potentiate transcription depends on the activity of an adjacent activation domain . These results suggest that the pheromone induction domain of Ste12p mediates transcriptional induction via a two-step process: the relief of repression and synergistic transcriptional activation with another activation domain. Mol Cell Biol, 1997 Nov, 17(11), 6203 - 11 Genetic evidence for interaction between Cbp1 and specific nucleotides in the 5' untranslated region of mitochondrial cytochrome b mRNA in Saccharomyces cerevisiae; Chen W et al.; The cytochrome b (COB) gene is encoded by the mitochondrial genome; however, its expression requires the participation of several nuclearly encoded protein factors . The yeast Cbp1 protein, which is encoded by the nuclear CBP1 gene, is required for the stabilization of COB mRNA . A previous deletion analysis identified an 11-nucleotide-long sequence within the 5' untranslated region of COB mRNA that is important for Cbp1-dependent COB mRNA stability . In the present study, site-directed mutagenesis experiments were carried out to define further the features of this cis element . The CCG sequence within this region was shown to be necessary for stability . A change in residue 533 of Cbp1 from aspartate to tyrosine suppresses the effects of a single-base change in the CCG element . This is strong genetic evidence that the nuclearly encoded Cbp1 protein recognizes and binds directly to the sequence containing CCG and thus protects COB mRNA from degradation. Curr Genet, 1997 Oct, 32(4), 273 - 80 The Neurospora crassa cya-5 nuclear gene encodes a protein with a region of homology to the Saccharomyces cerevisiae PET309 protein and is required in a post-transcriptional step for the expression of the mitochondrially encoded COXI protein; Coffin JW et al.; The cya-5 nuclear mutant of Neurosopora crassa was previously shown to be deficient in cytochrome aa3, cytochrome c oxidase activity, and the immunologically detectable COXI protein . We have now demonstrated that the mitochondria of this mutant contain mRNA for the COXI protein and that COXI cannot be detected during pulse-chase labeling experiments of mitochondrial translation products . Cloning and analysis of the cya-5 gene reveal a long open reading frame capable of encoding a 1136 amino-acid protein . Sequence analysis suggests that the potential CYA-5 protein contains a mitochondrial targeting sequence at its amino-terminus . The long open reading frame also contains a 200 amino-acid region with homology to the PET309 protein, which is required for the production or stability of intron-containing coxI mRNAs, as well as the translation of mature coxI mRNAs, in the yeast Saccharomyces cerevisiae . These data suggest that the CYA-5 protein of N . crassa is required in a post-transcriptional step for COXI expression, most probably for the efficient translation of coxI mRNA. Mol Gen Genet, 1997 Sep, 256(1), 88 - 91 Mitochondrial protein synthesis is not required for efficient excision of intron aI5 beta from COX1 pre-mRNA in Saccharomyces cerevisiae; Johnson CH et al.; Splicing of the group I intron aI5 beta from the yeast mitochondrial COX1 transcript requires at least four proteins, encoded by the nuclear genes PET54, MRS1/PET157, SUV3 and MSS18 . These proteins either act directly to facilitate intron aI5 beta excision, or indirectly in some manner . One possible indirect mode of action of these nuclear gene products is in stimulation of expression of a mitochondrial protein, such as a maturase, that is necessary for intron aI5 beta excision . To test this possibility, splicing of intron aI5 beta was examined in a rho-strain, which is incapable of mitochondrial protein synthesis . A quantitative RT-PCR assay was set up to compare levels of spliced COX1 mRNA present in three strains: a wild-type rho + strain; the rho-strain 7-49b-11, which retains the entire COX1 transcription unit; and a strain bearing a null mutation in the nuclear PET54 gene . The results showed that excision of aI5 beta occurs relatively efficiently in the rho-strain, and therefore does not require any mitochondrial-encoded proteins. J Biol Chem, 1997 Oct 24, 272(43), 27091 - 8 Diazaborine resistance in the yeast Saccharomyces cerevisiae reveals a link between YAP1 and the pleiotropic drug resistance genes PDR1 and PDR3; Wendler F et al.; We have investigated the mechanisms underlying resistance to the drug diazaborine in Saccharomyces cerevisiae . We used UV mutagenesis to generate resistant mutants, which were divided into three different complementation groups . The resistant phenotype in these groups was found to be caused by allelic forms of the genes AFG2, PDR1, and PDR3 . The AFG2 gene encodes an AAA (ATPases associated to a variety of cellular activities) protein of unknown function, while PDR1 and PDR3 encode two transcriptional regulatory proteins involved in pleiotropic drug resistance development . The isolated PDR1-12 and PDR3-33 alleles carry mutations that lead to a L1044Q and a Y276H exchange, respectively . In addition, we report that overexpression of Yap1p, the yeast homologue of the transcription factor AP1, results in a diazaborine-resistant phenotype . The YAP1-mediated diazaborine resistance is dependent on the presence of functional PDR1 and PDR3 genes, although PDR3 had a more pronounced effect . These results provide the first evidence for a functional link between the Yap1p-dependent stress response pathway and Pdr1p/Pdr3p-dependent development of pleiotropic drug resistance. J Biol Chem, 1997 Oct 24, 272(43), 26871 - 8 A 13C nuclear magnetic resonance investigation of the metabolism of leucine to isoamyl alcohol in Saccharomyces cerevisiae; Dickinson JR et al.; The metabolism of leucine to isoamyl alcohol in yeast was examined by 13C nuclear magnetic resonance spectroscopy . The product of leucine transamination, alpha-ketoisocaproate had four potential routes to isoamyl alcohol . The first, via branched-chain alpha-keto acid dehydrogenase to isovaleryl-CoA with subsequent conversion to isovalerate by acyl-CoA hydrolase operates in wild-type cells where isovalerate appears to be an end product . This pathway is not required for the synthesis of isoamyl alcohol because abolition of branched-chain alpha-keto acid dehydrogenase activity in an lpd1 disruption mutant did not prevent the formation of isoamyl alcohol . A second possible route was via pyruvate decarboxylase; however, elimination of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant did not decrease the levels of isoamyl alcohol produced . A third route utilizes alpha-ketoisocaproate reductase (a novel activity in Saccharomyces cerevisiae) but with no role in the formation of isoamyl alcohol from alpha-hydroxyisocaproate because cell homogenates could not convert alpha-hydroxyisocaproate to isoamyl alcohol . The final possibility was that a pyruvate decarboxylase-like enzyme encoded by YDL080c appears to be the major route of decarboxylation of alpha-ketoisocaproate to isoamyl alcohol although disruption of this gene reveals that at least one other unidentified decarboxylase can substitute to a minor extent. Mol Cells, 1997 Aug 31, 7(4), 532 - 6 Molecular cloning and characterization of the Saccharomyces cerevisiae SAB1 gene that suppresses a temperature-sensitive phenotype of the ARS-binding factor 1 mutant; So IS et al.; A high-copy number suppressor gene of the yeast temperature-sensitive lethal abf1 mutant was isolated and named SAB1 (suppressor of ABF1) . Chromoblot hybridization and grid-filter hybridization analyses showed that the SAB1 gene was located on chromosome IV . Deletion analyses of the SAB1 plasmid revealed that the suppressor activity was contained in a 1.1 kb DNA region . The nucleotide sequence of the 1.1 kb DNA fragment was determined and turned out to be identical to that of the yeast phosphoribosylanthranilate isomerase gene (TRP1) . A binding site for ARS-Binding Factor 1 was located in the coding sequence of the TRP1 gene, which has been known to be a part of the B domain of yeast autonomously replicating sequence 1 (ARS1) . Our results suggest that ABF1 might be important for the transcription of the yeast TRP1 gene in addition to having important roles in the stimulation of replication at the ARS1 locus. J Bacteriol, 1997 Oct, 179(19), 6066 - 75 Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli; Deloche O et al.; The DnaK, DnaJ, and GrpE proteins of Escherichia coli have been universally conserved across the biological kingdoms and work together to constitute a highly efficient molecular chaperone machine . We have examined the extent of functional conservation of Saccharomyces cerevisiae Ssc1p, Mdj1p, and Mge1p by analyzing their ability to substitute for their corresponding E . coli homologs in vivo . We found that the expression of yeast Mge1p, the GrpE homolog, allowed for the deletion of the otherwise essential grpE gene of E . coli, albeit only up to 40 degrees C . The inability of Mge1p to substitute for GrpE at very high temperatures is consistent with our previous finding that it specifically failed to stimulate DnaK's ATPase at such extreme conditions . In contrast to Mge1p, overexpression of Mdj1p, the DnaJ homolog, was lethal in E . coli . This toxicity was specifically relieved by mutations which affected the putative zinc binding region of Mdj1p . Overexpression of a truncated version of Mdj1p, containing the J- and Gly/Phe-rich domains, partially substituted for DnaJ function at high temperature . A chimeric protein, consisting of the J domain of Mdj1p coupled to the rest of DnaJ, acted as a super-DnaJ protein, functioning even more efficiently than wild-type DnaJ . In contrast to the results with Mge1p and Mdj1p, both the expression and function of Ssc1p, the DnaK homolog, were severely compromised in E . coli . We were unable to demonstrate any functional complementation by Ssc1p, even when coexpressed with its Mdj1p cochaperone in E . coli. Nucleic Acids Res, 1997 Nov 1, 25(21), 4257 - 63 Recovery of RNA polymerase II synthesis following DNA damage in mutants of Saccharomyces cerevisiae defective in nucleotide excision repair; Reagan MS et al.; We have measured the kinetics of the recovery of mRNA synthesis in the inducible GAL10 and RNR3 genes after exposure of yeast cells to ultraviolet (UV) radiation . Such recovery is abolished in mutant strains defective in nucleotide excision repair (NER) of DNA, including a rad23 mutant . Mutants defective in the RAD7 or RAD16 genes, which are required for the repair of the non-transcribed strand but not the transcribed strand of transcriptionally active genes, show slightly faster recovery of RNA synthesis than wild-type strains . A strain deleted of the RAD26 gene, which is known to be required for strand-specific NER in yeast, manifested delayed recovery of mRNA synthesis, whereas a rad28 mutant, which does not show defective strand-specific repair, showed normal kinetics of recovery . Measurement of the recovery of expression of selected individual yeast genes by Northern analysis following exposure of cells to UV radiation apparently correlates directly with the capacity of cells for strand-specific NER. Genetics, 1997 Oct, 147(2), 557 - 66 Mutations in GSF1 and GSF2 alter glucose signaling in Saccharomyces cerevisiae; Sherwood PW et al.; One function of the Saccharomyces cerevisiae Snf1 protein kinase is to relieve glucose repression of SUC, GAL, and other genes in response to glucose depletion . To identify genes that regulate Snf1 kinase activity, we have selected mutants that inappropriately express a SUC2promoter::HIS3 gene fusion when grown in glucose and that require Snf1 function for this phenotype . Mutations representing two new complementation groups (gsf1 and gsf2) were isolated . gsf1 mutations affect two distinct responses to glucose: the Snf1-regulated glucose repression of SUC2 and GAL10 transcription and the Snf1-independent induction by glucose of HXT1 transcription . gsf2 mutations relieve glucose repression of SUC2 and GAL10 transcription and, in combination with snf1 delta, cause an extreme slow growth phenotype . The GSF2 gene was cloned by complementation of the gsf2-1 snf1 delta slow growth phenotype and encodes a previously uncharacterized 46kD protein. Genetics, 1997 Oct, 147(2), 533 - 44 DMC1 functions in a Saccharomyces cerevisiae meiotic pathway that is largely independent of the RAD51 pathway; Dresser ME et al.; Meiotic recombination in the yeast Saccharomyces cerevisiae requires two similar recA-like proteins, Dmc1p and Rad51p . A screen for dominant meiotic mutants provided DMC1-G126D, a dominant allele mutated in the conserved ATP-binding site (specifically, the A-loop motif) that confers a null phenotype . A recessive null allele, dmc1-K69E, was isolated as an intragenic suppressor of DMC1-G126D . Dmc1-K69Ep, unlike Dmc1p, does not interact homotypically in a two-hybrid assay, although it does interact with other fusion proteins identified by two-hybrid screen with Dmc1p . Dmc1p, unlike Rad51p, does not interact in the two-hybrid assay with Rad52p or Rad54p . However, Dmc1p does interact with Tid1p, a Rad54p homologue, with Tid4p, a Rad16p homologue, and with other fusion proteins that do not interact with Rad51p, suggesting that Dmc1p and Rad51p function in separate, though possibly overlapping, recombinational repair complexes . Epistasis analysis suggests that DMC1 and RAD51 function in separate pathways responsible for meiotic recombination . Taken together, our results are consistent with a requirement for DMC1 for meiosis-specific entry of DNA double-strand break ends into chromatin . Interestingly, the pattern on CHEF gels of chromosome fragments that result from meiotic DNA double-strand break formation is different in DMC1 mutant strains from that seen in rad50S strains. Genetics, 1997 Oct, 147(2), 521 - 32 The role of Gcr1p in the transcriptional activation of glycolytic genes in yeast Saccharomyces cerevisiae; Uemura H et al.; To study the interdependence of Gcr1p and Rap1p, we prepared a series of synthetic regulatory sequences that contained various numbers and combinations of CT-boxes (Gcr1p-binding sites) and RPG-boxes (Rap1p-binding sites) . The ability of the synthetic oligonucleotides to function as regulatory sequences was tested using an ENO1-lacZ reporter gene . As observed previously, synthetic oligonucleotides containing both CT- and RPG-boxes conferred strong UAS activity . Likewise, a lone CT-box did not show any UAS activity . By contrast, oligonucleotides containing tandem Ct-boxes but no RPG-box conferred strong promoter activity . This UAS activity was not dependent on position or orientation of the oligonucleotides in the 5' noncoding region . However, it was dependent on both GCR1 and GCR2 . These results suggest that the ability of Gcr1p to bind Gcr1p-binding sites in vivo is not absolutely dependent on Rap1p . Eleven independent mutants of GCR1 were isolated that conferred weak UAS activity to a single CT-box . Five mutants has single mutations in Gcr1p's DNA-binding domain and displayed slightly higher affinity for the CT-box . These results support the hypothesis that Gcr1p and Gcr2p play the central role in glycolytic gene expression and that the function of Rap1p is to facilitate the binding of Gcr1p to its target. Genetics, 1997 Oct, 147(2), 507 - 19 Genetic and environmental factors affecting the de novo appearance of the {PSI+} prion in Saccharomyces cerevisiae; Derkatch IL et al.; It has previously been shown that yeast prion {PSI+} is cured by GuHCl, although reports on reversibility of curing were contradictory . Here we show that GuHCl treatment of both {PSI+} and {psi-} yeast strains results in two classes of {psi-} derivatives: Pin+, in which {PSI+} can be reinduced by Sup35p overproduction, and Pin-, in which overexpression of the complete SUP35 gene does not lead to the {PSI+} appearance . However, in both Pin+ and Pin- derivatives {PSI+} is reinduced by overproduction of a short Sup35p N-terminal fragment, thus, in principle, {PSI+} curing remains reversible in both cases . Neither suppression nor growth inhibition caused by SUP35 overexpression in Pin+ {psi-} derivatives are observed in Pin- {psi-} derivatives . Genetic analyses show that the Pin+ phenotype is determined by a non-Mendelian factor, which, unlike the {PSI+} prion, is independent of the Sup35p N-terminal domain . A Pin- {psi-} derivative was also generated by transient inactivation of the heat shock protein, Hsp104, while {PSI+} curing by Hsp104 overproduction resulted exclusively in Pin+ {psi-} derivatives . We hypothesize that in addition to the {PSI+} prion-determining domain in the Sup35p N-terminus, there is another self-propagating conformational determinant in the C-proximal part of Sup35p and that this second prion is responsible for the Pin+ phenotype. Genetics, 1997 Oct, 147(2), 479 - 92 Mutational analysis of STE5 in the yeast Saccharomyces cerevisiae: application of a differential interaction trap assay for examining protein-protein interactions; Inouye C et al.; Ste5 is essential for the yeast mating pheromone response pathway and is thought to function as a scaffold