Nucleic Acids Res, 1998 Jun 15, 26(12), 2865 - 72 Transcription of INO2 and INO4 is regulated by the state of protein N-myristoylation in Saccharomyces cerevisiae; Cok SJ et al.; Inositol regulates transcription of Saccharomyces cerevisiae genes required for de novo synthesis of acylCoAs and phospholipids . Removal of inositol results in transcriptional activation by heterodimeric complexes of two bHLH proteins, Ino2p and Ino4p . In the presence of inositol, transcription is repressed by Opi1p . MyristoylCoA:protein N-myristoyltransferase (Nmt1p) is an essential enzyme whose activity is influenced by cellular myristoylCoA pool size and availability . nmt451Dp contains a Gly451-->Asp substitution that produces temperature-dependent reductions in affinity for myristoylCoA and associated reductions in acylation of cellular N-myristoylproteins . The conditional lethality produced by nmt1-451D is rescued at temperatures up to 33 degreesC by withdrawal of inositol . We tested the hypothesis that N-myristoylproteins function to regulate INO2, INO4 and/or OPI1 transcription, thereby affecting the expression of inositol-sensitive genes that influence myristoylCoA metabolism . The effect of nmt1-451D on INO2 , INO4 and OPI1 promoter activities was examined by introducing episomes, containing their 5' non-transcribed domains linked to reporters, into isogenic NMT1 and nmt1-451D cells . The activity of INO2 is significantly higher, INO4 significantly lower and OPI1 unaffected in nmt1-451D cells, both in the presence and absence of inositol . These changes are associated with a net increase in expression of some inositol target genes, including FAS1 . FAS1 encodes one of the subunits of the fatty acid synthase complex that catalyzes de novo acylCoA (including myristoylCoA) biosynthesis . Augmented expression of FAS1 overcomes the kinetic defects in nmt451Dp . FAS1 expression is Ino2p-dependent in NMT1 cells at 24-33 degreesC . In contrast, FAS1 expression becomes Ino2p-independent in nmt1-451D cells at temperatures where efficient acylation of cellular N-myristoylproteins is jeopardized . The ability to maintain expression of FAS1 in nmt1-451Dino2 Delta cells suggests the existence of another transcription factor, or factors, whose expression/activity is inversely related to overall levels of cellular protein N-myristoy-lation . This factor is not functionally identical to Ino2p since other inositol-responsive genes (e.g . CHO1 ) maintain INO2 -dependent expression in nmt1-451D cells. Curr Opin Genet Dev, 1998 Apr, 8(2), 233 - 9 Mechanisms of silencing in Saccharomyces cerevisiae; Lustig AJ; In the yeast Saccharomyces cerevisiae, heterochromatin-like regions are formed at the silent mating type loci and at telomeres . The past year of investigations has led to a clearer understanding of the nature of nucleation and spreading of heterochromatin, as well as uncovering a fascinating link between silencing, the nucleolus and aging. Biochemistry, 1998 May 19, 37(20), 7572 - 7 Characterization of the copper chaperone Cox17 of Saccharomyces cerevisiae; Srinivasan C et al.; Assembly of functional cytochrome oxidase in yeast requires Cox17, which has been postulated to deliver copper ions to the mitochondrion for insertion into the enzyme . This role for Cox17 is supported by the observation that it binds copper as a binuclear cuprous-thiolate cluster . X-ray absorption spectroscopy, together with UV-visible absorption and emission spectroscopy, indicates the presence of bound cuprous ions, trigonally coordinated by thiolate ligands . Analysis of the EXAFS shows three Cu-S bonds at 2.26 A, plus a short Cu-Cu distance of 2.7 A, indicating a binuclear cluster in Cox17 . The cuprous-thiolate cluster in Cox17 is substantially more labile than structurally related clusters in metallothioneins. Biochemistry, 1998 May 19, 37(20), 7299 - 312 Pre-steady-state analysis of ATP hydrolysis by Saccharomyces cerevisiae DNA topoisomerase II . 2 . Kinetic mechanism for the sequential hydrolysis of two ATP; Harkins TT et al.; In the preceding paper, we showed that DNA topoisomerase II from Saccharomyces cerevisiae binds two ATP and rapidly hydrolyzes at least one of them before encountering a slow step in the reaction mechanism . These data are potentially consistent with two different types of reaction pathways: (1) sequential ATP hydrolysis or (2) simultaneous hydrolysis of both ATP . Here, we present results that are consistent only with topoisomerase II hydrolyzing its two bound ATP sequentially . Additionally, these results indicate that the products of the first hydrolysis are released from the enzyme before the second ATP is hydrolyzed . Release of products from both the first and second hydrolyses contributes to the rate-determining process . The proposed mechanism for ATP hydrolysis by topoisomerase II is complex, having nine rate constants . To calculate values for each of these rate constants, a technique of kinetic parameter estimation was developed . This technique involved using singular perturbation theory in order to estimate rate constants, and consequently identify kinetic steps following the rate-determining step. Biochemistry, 1998 May 19, 37(20), 7292 - 8 Pre-steady-state analysis of ATP hydrolysis by Saccharomyces cerevisiae DNA topoisomerase II . 1 . A DNA-dependent burst in ATP hydrolysis; Harkins TT et al.; When bound to DNA, topoisomerase II from Saccharomyces cerevisiae exhibits burst kinetics with respect to ATP hydrolysis . Pre-steady-state analysis shows that the enzyme binds and hydrolyzes two ATP per reaction cycle . Our data indicate that at least one of the two ATP is rapidly hydrolyzed prior to the rate-determining step in the reaction mechanism . When DNA is not bound to topoisomerase II, the rate-determining step shifts to become either ATP binding or hydrolysis . Two possible mechanisms are proposed that agree with our observations. Biochemistry, 1998 May 19, 37(20), 7268 - 76 Transfer RNA-pseudouridine synthetase Pus1 of Saccharomyces cerevisiae contains one atom of zinc essential for its native conformation and tRNA recognition; Arluison V et al.; RNA:pseudouridine synthetase (Pus1) from Saccharomyces cerevisiae is a multisite specific enzyme that catalyzes the formation of pseudouridine at positions 34 and 36 of intron-containing precursor tRNAIle and at positions 27 and/or 28 of several yeast tRNAs . In this paper we demonstrate that the purified recombinant Pus1, expressed in Escherichia coli, contains one atom of zinc per 63-kDa monomer, as determined by atomic absorption spectroscopy . This zinc ion could not be removed by treatment with EDTA or urea . However, a zinc-depleted enzyme was obtained after prolonged dialysis against the specific chelating agent 1,10-phenanthroline . Removal of the zinc ion resulted in inactivation of the enzyme with concomitant loss of its ability to bind tRNA . Dialysis of the zinc-depleted inactive enzyme against buffer containing zinc ions led to recovery of up to 25% of bound zinc in parallel with 25% of its initial activity . Removal of the tightly bound zinc atom resulted in a conformational change of the protein, as determined by analytical ultracentrifugation, with minor changes in the internal structure of the protein, as evidenced by circular dichroism and infrared and fluorescence spectroscopy . Our results are consistent with a structural role for the zinc in the tRNA-pseudouridine synthetase Pus1; zinc ion could maintain the association between domains structurally organized around the coordinated metal ion . Zinc chelation was never demonstrated for any of the tRNA-pseudouridine synthetases characterized so far. Yeast, 1998 Apr 30, 14(6), 587 - 91 A 9359 bp fragment from the right arm of Saccharomyces cerevisiae chromosome VII includes the FOL2 and YTA7 genes and three unknown open reading frames; Agostoni Carbone ML et al.; In the framework of the EU programme for systematic sequencing of the Saccharomyces cervisiae genome we determined the sequence of a 9359 bp fragment of the right arm of chromosome VII . Five open reading frames (ORFs) of at least 300 nucleotides were found in this region . YGR267c encodes a protein with significant similarity to the enzyme GTP-cyclohydrolase I, that controls the first step in the biosynthetic pathway leading to various pterins and shows a high degree of sequence conservation from bacteria to mammals . We have recently demonstrated (Nardese et al., 1996) that YGR267c corresponds to the FOL2 gene, previously localized in the same chromosomal region by genetic mapping . The protein deduced from YGR270w belongs to the superfamily of putative ATPases associated with diverse cellular activities . It corresponds to the YTA7 gene, a member of a set of yeast genes coding for putative ATPases with high similarity to constituents of the 26S protease . The three ORFs YGR266w, YGR268c and YGR269w encode putative products of unknown function, with neither significant similarity to proteins in databases nor recognizable domains . YGR268c and YGR269w are partially overlapping ORFs: YGR268c seems to correspond to a real gene . whereas YGR269w is probably a fortuitous ORF. Yeast, 1998 Apr 30, 14(6), 551 - 64 A family of laboratory strains of Saccharomyces cerevisiae carry rearrangements involving chromosomes I and III; Casaregola S et al.; In order to study meiotic segregation of chromosome length polymorphism in yeast, we analysed the progeny of a cross involving two laboratory strains FL100trp and YNN295 . Analysis of the parental strains led us to detect an important length polymorphism of chromosomes I and III in FL100trp . A reciprocal translocation involving 80 kb of the left arm of chromosome III and 45 kb of the right arm of chromosome I was shown to be the cause for the observed polymorphism in this strain . The characterization of the translocation breakpoints revealed the existence of a transposition hot-spot on chromosome I: the sequence of the translocation joints on chromosomes I and III suggests that the mechanism very likely involved homologous recombination between Ty2 transposable elements on each chromosome . Analysis of FL100, FL200 and FL100trp ura, which are related to FL100trp, shows that this reciprocal translocation is present in some of the strains of the FL series, whereas the parental strain FL100 does not carry the same rearrangement . We evidenced instead the duplication of 80 kb of chromosome III on chromosome I and a deletion of 45 kb of the right arm of chromosome I in this strain, indicating that secondary events might have taken place and that the strain currently named FL100 is not the common ancestor of the FL series. Mol Gen Genet, 1998 Apr, 257(6), 662 - 71 Switch-domain mutations in the Saccharomyces cerevisiae G protein alpha-subunit Gpa1p identify a receptor subtype-biased mating defect; DeSimone SM et al.; The response to pheromone in Saccharomyces cerevisiae involves a heterotrimeric G protein composed of Gpa1p (alpha subunit), Ste4p (beta) and Ste18p (gamma) . The switch II region of G alpha subunits is involved in several protein-protein interactions and an intrinsic GTPase activity . To investigate the role of this region of Gpa1p, we have analyzed the effect of switch II mutations . The Q323 analog in G alpha subunits and Ras is implicated in GTP hydrolysis . Mutation of the Q323 residue of Gpa1p resulted in constitutive activation of the pheromone response pathway and eliminated the ability to interact with Ste4p, consistent with a defect in GTPase activity . Mutation of residue A59 of Ras and the analogous G alphas residue has had quite different effects . The analogous Gpa1p G321T mutation resulted in phenotypes consistent with a less severe GTPase defect, but also led to an unexpected mating phenotype: mating was decreased in both mating types, but the defect was 1000-fold more severe in alpha cells than in a cells . In addition the G321T mutation resulted in an unusual pheromone response phenotype . We discuss the possibility that these phenotypes may reflect a differential role for the switch II region in activation by the a- and alpha-factor receptors. Mol Gen Genet, 1998 Apr, 257(6), 624 - 34 Nuclear protein import, but not mRNA export, is defective in all Saccharomyces cerevisiae mutants that produce temperature-sensitive forms of the Ran GTPase homologue Gsp1p; Oki M et al.; A series of ts mutations in the GSP1 gene of Saccharomyces cerevisiae was isolated by error-prone PCR . A total of 25 ts gsp1 strains was obtained . Each of these mutants showed between one and seven different amino acid alterations . In several of these ts gsp1 strains, the same amino acid residues in Gsp1p were repeatedly mutated, indicating that our screen for ts gsp1 mutations was saturating . All of the ts gsp1 strains isolated had a defect in nuclear protein import, but only 16 of the 25 ts gsp1 strains had a defect in mRNA export . Thus, Gsp1p is suggested to be directly involved in nuclear protein import, but not in mRNA export . Following release from alpha-factor arrest, 11 of the ts gsp1 mutants arrested in G1; the remainder did not show any specific cell-cycle arrest, at 37 degrees C, the nonpermissive temperature . While the mutants that are defective in both mRNA export and protein import have a tendency to arrest in G1, there was no clear correlation between the cell cycle phenotype and the defects in mRNA export and nuclear protein import . Based on this, we assume that Ran/Gsp1p GTPase regulates the cell cycle and the nucleus/cytosol exchange of macromolecules through interactions with effectors that were independent of each other, and are differentially affected by mutation. Mol Gen Genet, 1998 Apr, 257(6), 614 - 23 Mms4, a putative transcriptional (co)activator, protects Saccharomyces cerevisiae cells from endogenous and environmental DNA damage; Xiao W et al.; mms4-1 is one of several Saccharomyces cerevisiae mutants that exhibit an increased sensitivity to methyl methanesulfonate (MMS), but not to UV or X-rays . We have isolated the MMS4 gene by functional complementation of the MMS-sensitive phenotype in the mms4-1 strain . The MMS4 gene encodes a 691-amino acid, 78.7-kDa protein . The deduced Mms4 protein does not show significant homology to any of the known proteins in the database . However, several putative functional domains suggest that it may be a nuclear protein capable of interacting with other proteins . Examination of the mms4delta mutant phenotype indicates that the mutation not only sensitizes DNA to methylating and ethylating agents, but also to other DNA damage that blocks DNA replication . However, the mms4delta mutant appears to be more sensitive to chronic treatment than to acute treatment by DNA-damaging agents . Furthermore, the spontaneous mutation rate increases significantly in the mms4delta mutant . Mms4 alone, when fused to a Gal4 DNA-binding domain, is able to activate P(GAL1)-lacZ and P(GAL1)-HIS3 reporter genes in a two-hybrid system; the Mms4 transactivation domain maps to the highly acidic N-terminal region . These results collectively suggest that Mms4 may function as a transcriptional (co)activator and play an important role in DNA repair and/or synthesis. Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6245 - 50 Glucose-regulated interaction of a regulatory subunit of protein phosphatase 1 with the Snf1 protein kinase in Saccharomyces cerevisiae; Ludin K et al.; The Snf1 protein kinase family has been conserved in eukaryotes . In the yeast Saccharomyces cerevisiae, Snf1 is essential for transcription of glucose-repressed genes in response to glucose starvation . The direct interaction between Snf1 and its activating subunit, Snf4, within the kinase complex is regulated by the glucose signal . Glucose inhibition of the Snf1-Snf4 interaction depends on protein phosphatase 1 and its targeting subunit, Reg1 . Here we show that Reg1 interacts with the Snf1 catalytic domain in the two-hybrid system . This interaction increases in response to glucose limitation and requires the conserved threonine in the activation loop of the kinase, a putative phosphorylation site . The inhibitory effect of Reg1 appears to require the Snf1 regulatory domain because a reg1Delta mutation no longer relieves glucose repression of transcription when Snf1 function is provided by the isolated catalytic domain . Finally, we show that abolishing the Snf1 catalytic activity by mutation of the ATP-binding site causes elevated, constitutive interaction with Reg1, indicating that Snf1 negatively regulates its own interaction with Reg1 . We propose a model in which protein phosphatase 1, targeted by Reg1, facilitates the conformational change of the kinase complex from its active state to the autoinhibited state. J Biol Chem, 1998 Apr 24, 273(17), 10567 - 77 Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein; Chong S et al.; Protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein involves four highly coordinated reactions that result in precise cleavage and formation of peptide bonds . In this study, we investigated the roles of the last N-extein residue (-1 residue) and the intein penultimate residue in modulating splicing reactions . Most of the 20 amino acid substitutions at the -1 position had no effect on overall protein splicing but could lead to significant accumulation of thioester intermediates when splicing was blocked by mutation . A subset of -1 substitutions attenuated the initiation of protein splicing and enabled us to demonstrate in vitro splicing of a mesophilic intein containing all wild-type catalytic residues . Substitutions involving the intein penultimate residue allowed modulation of the branch resolution and C-terminal cleavage reaction . Our data suggest that the N-S acyl rearrangement, which initiates splicing, may also serve as the rate-limiting step . Through appropriate amino acid substitutions, we were able to modulate splicing reactions in vitro by change in pH or temperature or addition of thiol reagents . Both insertion and deletion were tolerated in the central region of the intein although splicing or structure of the intein may have been affected. J Biol Chem, 1998 May 22, 273(21), 12696 - 702 Monitoring the Gcn4 protein-mediated response in the yeast Saccharomyces cerevisiae; Albrecht G et al.; In Saccharomyces cerevisiae the GCN4 gene encodes the transcriptional activator of the "general control" system of amino acid bioynthesis, a network of at least 12 different biosynthetic pathways . We characterized the consequences of the general control response upon the signal "amino acid starvation" induced by the histidine analogue 3-aminotriazole with respect to Gcn4p levels in more detail . Therefore, we established test systems to monitor the time course of different parameters, including GCN4 mRNA, Gcn4 protein, Gcn4p DNA binding activity, as well as Gcn4p transactivation ability . We observed a biphasic response of Gcn4p activity in the cell . At first, translation of GCN4 mRNA is induced within 20 min after switch to starvation conditions . However, an additional increase in GCN4 transcript steady state level was observed, leading to an additional second phase of GCN4 expression after 3-4 h of starvation . The DNA binding activity of Gcn4p, as well as the ability to activate transcription of target genes, correlate with the amount of Gcn4 protein in the cell, suggesting that under the tested conditions there is no additional regulation of DNA binding or transactivation ability of Gcn4p, respectively. Curr Genet, 1998 Apr, 33(4), 239 - 47 The Tup1-Ssn6 general repressor is involved in repression of IME1 encoding a transcriptional activator of meiosis in Saccharomyces cerevisiae; Mizuno T et al.; Ime1 plays a pivotal role in the initiation of meiosis in a/alpha diploid cells of Saccharomyces cerevisiae . In the absence of glucose and nitrogen, IME1 expression is greater in a/alpha cells than in either a or alpha cells and therefore only a/alpha, but not a/a or alpha/alpha, cells are committed to sporulation . It is known that IME1 expression is positively regulated by Mck1, Rim1, Ime4 and the Swi-Snf complex but other factors may also be involved . In addition, Rme1 is assumed to repress IME1 expression . To provide more details of the repression of expression of IME1, we have isolated mutants in which the IME1p-PHO5 fusion gene integrated at the ura3 locus is expressed in alpha cells under nutritionally rich conditions . We found that mutations occurred in TUP1, SSN6, SIN4 and RGR1, among which TUP1 and SSN6 were identified for the first time as negative regulators of IME1 expression . Deletion of the Rme1-binding site from the IME1 promoter did not result in activation of the expression of IME1 under nutritionally rich conditions, suggesting that Rme1 does not function as a DNA-binding protein with the Tup1-Ssn6 repression complex . We also demonstrated that the 294-bp fragment from nucleotide position -914 to -621 and the 301-bp fragment from nucleotide position -1215 to -915 of the IME1 promoter region contain elements acting as URS and UAS in TUP1+ and tup1 mutant cells, respectively . These findings indicate that IME1 is negatively regulated by the Tup1-Ssn6 repressor complex through two distinct upstream regions in conjunction with unidentified DNA-binding proteins. Glycobiology, 1998 May, 8(5), 455 - 62 The ALG10 locus of Saccharomyces cerevisiae encodes the alpha-1,2 glucosyltransferase of the endoplasmic reticulum: the terminal glucose of the lipid-linked oligosaccharide is required for efficient N-linked glycosylation; Burda P et al.; The biosynthesis of the lipid-linked oligosaccharide substrate for N-linked protein glycosylation follows a highly conserved pathway at the membrane of the endoplasmic reticulum . Based on the synthetic growth defect in combination with a reduced oligosaccharyltransferase activity (wbp1), we have identified alg10 mutant strains which accumulate lipid-linked Glc2Man9GlcNAc2 . We cloned the corresponding wild-type gene and show in a novel in vitro assay that Alg10p is a dolichyl-phosphoglucose-dependent glucosyltransferase which adds the terminal alpha-1,2 glucose to the lipid-linked Glc2Man9GlcNAc2 oligosaccharide . Hypoglycosylation of secreted proteins in alg10 deletion strains demonstrates that the terminal alpha-1,2-linked glucose residue is a key element in substrate recognition by the oligosaccharyltransferase . This ensures that primarily completely assembled oligosaccharide is transferred to protein. Biochim Biophys Acta, 1998 Apr 10, 1380(2), 232 - 8 Adenosine 5'-tetraphosphate phosphohydrolase activity is an inherent property of soluble exopolyphosphatase from yeast Saccharomyces cerevisiae; Guranowski A et al.; Homogeneous soluble exopolyphosphatase (EC 3.6.1.11) from yeast Saccharomyces cerevisiae, (scPPX1) behaves as an adenosine 5'-tetraphosphate phosphohydrolase (EC 3.6.1.14) . The hydrolysis of adenosine 5'-tetraphosphate (p4A) to ATP and orthophosphate absolutely depends on one of the following cations: Co2+>Mn2+>Mg2+>Ni2+ . Optimum pH is around 4.75 and the Km for p4A estimated at that pH in 50 mM sodium acetate and at 5 mM CoCl2 is 80+/-10 microM . Adenosine 5'-pentaphosphate (p5A) is degraded under these conditions 18-fold more slowly than p4A . Assuming that the mass of scPPX1 is 45 kDa, the calculated kcat values for p4A and for p5A are 723 and 40 s-1, respectively . Two other nucleoside 5'-tetraphosphates (p4N), guanosine tetraphosphate (p4G) and inosine tetraphosphate (p4I), were hydrolyzed to Pi and either GTP or ITP, respectively, at the same rate as that observed for the hydrolysis of p4A . Ammonium molybdate, sodium o-vanadate and zinc chloride inhibit the hydrolysis of p4A (I50 values are 0.08, 0.3 and 0.4 mM, respectively) . This newly recognized 'acidic' adenosine tetraphosphatase activity from yeast is compared with two 'pH 8' adenosine tetraphosphatases described earlier in rabbit and yellow lupin . Biochemistry, 1998 Apr 28, 37(17), 6033 - 40 Saccharomyces cerevisiae possesses two functional homologues of Escherichia coli endonuclease III; You HJ et al.; We previously identified two distinct genes of Saccharomyces cerevisiae redoxyendonuclease (SCR1 and SCR2) which possess a high degree of sequence similarity to Escherichia coli endonuclease III {Augeri, L., Lee, Y . M., Barton, A . B., and Doetsch, P . W . (1997) Biochemistry 36, 721-729} . The proteins encoded by SCR1 and SCR2 were overexpressed in E . coli and purified to apparent homogeneity . Both proteins recognized and cleaved DNA substrates containing dihydrouracil, 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (FaPy-7-MeGua), and abasic sites but not DNA substrates containing uracil or 8-oxoguanine . Purified Scr2, but not Scr1, possesses spectral properties which indicate the presence of an iron-sulfur center . Kinetic parameters for Scr1 and Scr2 were determined by using an oligonucleotide containing a single dihydrouracil . Analysis of the deduced amino acid sequences of Scr1 and Scr2 suggests that Scr2 bears an iron-sulfur motif, while Scr1 does not have this motif . However, Scr1 has a long, positively charged N-terminus that could be a mitochondrial transit sequence . Targeted gene disruption of SCR1 and SCR2 produced a double mutant that had no detectable enzymatic activity against the dihydrouracil-containing substrate . Northern blot analysis showed that SCR1 was induced by menadione, but SCR2 was not . These results indicate that although Scr1 and Scr2 are both functional homologues of E . coli endonuclease III, they differ from each other with respect to their amino acid sequences and inducibility by DNA damaging agents, suggesting that their precise biological roles may be different. Biochim Biophys Acta, 1998 May 8, 1380(3), 320 - 8 Proteolytic processing of a secreted glycoprotein and O-glycosylation of mannoproteins are affected in the N-glycosylation mutant Saccharomyces cerevisiae ldb1; Manas P et al.; In a previous work {P.I . Manas, I . Olivero, M . Avalos, L.M . Hernandez, Glycobiology, 7 (1997) 487-497}, we described the isolation and characterization of the Saccharomyces cerevisiae ldb1 mutant which is affected in several steps of the N-glycosylation of mannoproteins probably due to a malfunction of the Golgi apparatus . Here, we found that two further functions assigned to the Golgi cisternae are also affected in the mutant: proteolytic processing of a secreted protein and O-glycosylation . We found that around 70% of the exoglucanase activity that is secreted into the culture medium by ldb1 bears an extra tetrapeptide in its NH2-terminus due to incomplete proteolytic processing . The O-linked oligosaccharides from ldb1 mnn1 were indistinguishable from those synthesized by the parental strain mnn1 . However, when the O-oligosaccharides from the wild type and ldb1 were compared, we found a significant decrease in the tetrasaccharide in the latter, as well as a concomitant increase in the disaccharide, suggesting a defect in the Kre2p/Mnt1p involved in the transfer of the third mannose of these residues . J Biol Chem, 1998 Apr 17, 273(16), 9912 - 20 Palindrome with spacer of one nucleotide is characteristic of the cis-acting unfolded protein response element in Saccharomyces cerevisiae; Mori K et al.; When unfolded proteins are accumulated in the endoplasmic reticulum (ER), an intracellular signaling pathway termed the unfolded protein response (UPR) is activated to induce transcription of ER-localized molecular chaperones and folding enzymes in the nucleus . In Saccharomyces cerevisiae, at least six lumenal proteins including essential Kar2p and Pdi1p are known to be regulated by the UPR . We and others recently demonstrated that the basic-leucine zipper protein Hac1p/Ern4p functions as a trans-acting factor responsible for the UPR . Hac1p binds directly to the cis-acting unfolded protein response element (UPRE) responsible for Kar2p induction . Moreover, we showed that the KAR2 UPRE contains an E box-like palindrome separated by one nucleotide (CAGCGTG) that is essential for its function . We report here that the promoter regions of each of five target proteins (Kar2p, Pdi1p, Eug1p, Fkb2p, and Lhs1p) contain a single UPRE sequence that is necessary and sufficient for induction and that binds specifically to Hac1p in vitro . All of the five functional UPRE sequences identified contain a palindromic sequence that has, in four cases, a spacer of one C nucleotide . This unique characteristic of UPRE explains why only a specific set of proteins are induced in the UPR to cope with ER stress. J Biol Chem, 1998 Apr 17, 273(16), 9644 - 50 Purification and characterization of the Sgs1 DNA helicase activity of Saccharomyces cerevisiae; Bennett RJ et al.; The yeast Saccharomyces cerevisiae Sgs1 protein is a member of a family of DNA helicases that include the Escherichia coli RecQ protein and the products of human Bloom's syndrome and Werner's syndrome genes . To study the enzymatic characteristics of the protein, a recombinant Sgs1 fragment (amino acids 400-1268 of the 1447-amino acid full-length protein) was overexpressed in yeast and purified to near homogeneity . The purified protein exhibits an ATPase activity in the presence of single- or double-stranded DNA . In the presence of ATP or dATP, unwinding of duplex DNA or a DNA-RNA heteroduplex by the recombinant Sgs1 fragment was readily observed . Similar to the E . coli RecQ helicase, displacement of the DNA strand occurs in the 3' to 5' direction with respect to the single-stranded DNA flanking the duplex . The efficiency of unwinding was found to correlate inversely with the length of the duplex region and was enhanced by the presence of E . coli single-stranded DNA-binding protein . In addition, the recombinant Sgs1 fragment was found to bind more tightly to a forked DNA substrate than to either single- or double-stranded DNA. J Biol Chem, 1998 Apr 17, 273(16), 9534 - 8 Functional correlation among Gal11, transcription factor (TF) IIE, and TFIIH in Saccharomyces cerevisiae . Gal11 and TFIIE cooperatively enhance TFIIH-mediated phosphorylation of RNA polymerase II carboxyl-terminal domain sequences; Sakurai H et al.; Saccharomyces cerevisiae Gal11, a component of the holoenzyme of RNA polymerase II, interacts through its functional domains A and B with the small (Tfa2) and large (Tfa1) subunits of the general transcription factor (TF) IIE, respectively . We have recently suggested that Gal11 functions through a common pathway with TFIIE in transcriptional regulation (Sakurai, H., and Fukasawa, T . (1997) J . Biol . Chem . 272, 32663-32669) . Here, we report that the activity of the TFIIH-associated kinase, responsible for phosphorylation of the largest subunit of RNA polymerase II at the carboxyl-terminal domain (CTD), is enhanced cooperatively by Gal11 and TFIIE . The enhancement of CTD phosphorylation was observed in the holoenzyme of RNA polymerase II, but not in its core enzyme . The stimulatory effect was completely abolished in the absence of either domain B of Gal11 or the Tfa1 subunit of TFIIE, suggesting that the domain B-Tfa1 interaction is necessary, if not sufficient, for an extensive phosphorylation of the CTD by TFIIH . Stimulation of basal transcription by Gal11 was coupled with enhancement of TFIIH-catalyzed CTD phosphorylation in a cell-free transcription system, suggesting that Gal11 activates transcription by stimulating the CTD phosphorylation in the cell. J Biol Chem, 1998 Apr 17, 273(16), 9527 - 33 Flavohemoglobin expression and function in Saccharomyces cerevisiae . No relationship with respiration and complex response to oxidative stress; Buisson N et al.; The yeast Saccharomyces cerevisiae contains a flavohemoglobin, encoded by the gene YHB1, whose function is unclear . Previous reports presented evidence that its maximal expression requires disruption of mitochondrial respiration and that it plays a role in the response to oxidative stress . We have studied the expression of YHB1 in respiratory deficient cells and in cells exposed to various compounds causing oxidative stress . Several different strains and approaches (spectroscopic detection of the oxygenated form of Yhb1p, beta-galactosidase activity of a YHB1-lacZ fusion, and Northern blot analysis) were used to demonstrate that YHB1 expression and Yhb1p production are not increased by respiration deficiency . YHB1 expression was unchanged in cells challenged with antimycin A or menadione, while it decreased in cells exposed to H2O2, diamide, dithiothreitol, and Cu2+ . Transcription of YHB1 is not under the control of the transcriptional factor Yap1p . These results do not support a participation of YHB1 in the genetic response to oxidative stress . We also analyzed the growth phenotypes associated with altered Yhb1p production using YHB1-deleted strains and strains that greatly overproduced Yhb1p . Yhb1p appears to protect cells against the damage caused by Cu2+ and dithiothreitol, while sensitizing them to H2O2 . Yhb1p overproduction in a glucose-6-phosphate dehydrogenase-deficient mutant decreased its growth rate . These data indicate that there is a complex relationship(s) between Yhb1p function(s) and cell defense reactions against various stresses. J Biol Chem, 1998 Apr 17, 273(16), 9472 - 9 Amino acid residues that define both the isoprenoid and CAAX preferences of the Saccharomyces cerevisiae protein farnesyltransferase . Creating the perfect farnesyltransferase; Caplin BE et al.; Studies of the yeast protein farnesyltransferase (FTase) have shown that the enzyme preferentially farnesylates proteins ending in CAAX (C = cysteine, A = aliphatic residue, X = cysteine, serine, methionine, alanine) and to a lesser degree CAAL . Furthermore, like the type I protein geranylgeranyltransferase (GGTase-I), FTase can also geranylgeranylate methionine- and leucine-ending substrates both in vitro and in vivo . Substrate overlap of FTase and GGTase I has not been determined to be biologically significant . In this study, specific residues that influence the substrate preferences of FTase have been identified using site-directed mutagenesis . Three of the mutations altered the substrate preferences of the wild type enzyme significantly . The ram1p-74D FTase farnesylated only Ras-CIIS and not Ras-CII(M,L), and it geranylgeranylated all three substrates as well or better than wild type . The ram1p-206DDLF FTase farnesylated Ras-CII(S,M,L) at wild type levels but could no longer geranylgeranylate the Ras-CII(M,L) substrates . The ram1p-351FSKN FTase farnesylated Ras-CIIS and Ras-CIIM but not Ras-CIIL . The ram1p-351FSKN FTase was not capable of geranylgeranylating the Ras-CII(M,L) substrates, giving this mutant the attributes of the dogmatic FTase that only farnesylates non-leucine-ending CAAX substrates and does not geranylgeranylate any substrate . These results suggest that the isoprenoid and protein substrate specificities of FTase are interrelated . The availability of a mutant FTase that lacked substrate overlap with the protein GGTase-I made possible an analysis of the role of substrate overlap in normal cellular processes of yeast, such as mating and growth at elevated temperatures . Our findings suggest that neither farnesylation of leucine-ending CAAX substrates nor geranylgeranylation by the FTase is necessary for these cellular processes. Curr Genet, 1998 Mar, 33(3), 171 - 7 Dip5p mediates high-affinity and high-capacity transport of L-glutamate and L-aspartate in Saccharomyces cerevisiae; Regenberg B et al.; Genes encoding homologues of known amino-acid permeases were deleted in a strain also deficient in the general amino-acid permease . The uptake capacity of the mutants was investigated for several L-alpha-amino acids . Deletion of a gene denoted DIP5 results in the loss of L-aspartate and L-glutamate uptake . The dip5 mutation caused a several hundred-fold reduction of uptake of the two amino acids, both in cells grown on proline as a nitrogen source and in cells grown on ammonium . DIP5-dependent uptake of L-aspartate and L-glutamate was somewhat lower in ammonium-grown cells than in proline-grown cells . Transcriptional regulation is at least partially responsible for this difference, as shown by assaying the DIP5 promoter fused to lacZ . This suggests that the promoter is subject to nitrogen catabolite repression . Transport of a few other amino acids was moderately affected by dip5 but was not competed by L-aspartate in the DIP5 parental strain; transport of these amino acids is therefore unlikely to be mediated by Dip5p . Our results suggest that DIP5 encodes an amino-acid permease with a high transport capacity and a high affinity for L-glutamate and L-aspartate, with a Kt of about 50 microM for both. Curr Genet, 1998 Feb, 33(2), 110 - 6 Ts mutations in mitochondrial tRNA genes: characterization and effects of two point mutations in the mitochondrial gene for tRNAphe in Saccharomyces cerevisiae; Francisci S et al.; Two new mitochondrial mutations conferring heat sensitivity on glycerol medium to the cells that carry them and affecting mitochondrial protein synthesis were investigated . Both map in the mitochondrial tRNAphe gene and have C-to-U transitions, one at position 2 (ts22b16) and the other at 62 (ts1345) . The latter mutation clearly affects the 3' end-maturation of tRNAphe, while the former presents normal patterns of both tRNA processing and amino-acylation . The defective phenotype resulting from the ts22b16 mutation can be corrected by over-expressing either the mitochondrial elongation factor EF-Tu or the mutated form of the tRNA . These results suggest that this mutation's primary effect might involve modified interactions during the ternary complex formation. Curr Genet, 1998 Feb, 33(2), 100 - 9 RPG1: an essential gene of saccharomyces cerevisiae encoding a 110-kDa protein required for passage through the G1 phase; Kovarik P et al.; In Saccharomyces cerevisiae cells a number of genes are required for progression through, or else to pass beyond, the G1 phase . We characterized a novel gene, RPG1, which is also involved in this phase . RPG1 is an essential gene encoding a 110-kDa evolutionarily conserved protein . Elutriated or alpha-factor-synchronized cells of the rpg1-1 temperature-sensitive mutant were arrested in the first cell cycle when shifted to a non-permissive temperature . The cells remained unbudded and neither grew nor duplicated DNA . rpg1-1 cells synchronized in S phase completed mitosis and arrested as unseparated G1 cells after a shift to a non-permissive temperature . Similarly, the asynchronous rpg1-1 cells accumulated in G1 at the non-permissive temperature, but mother and daughter cells did not separate . A bulk of Calcofluor-stained material was localized in the region adjacent to the cell septum . Our data show that Rpg1p is required for passage through the G1 phase and may be involved in growth control . Data published recently indicate that Rpg1p exhibits significant sequence similarity to a subunit of the mammalian translation initiation factor 3. Biochem J, 1998 Mar 15, 330 ( Pt 3), 1333 - 40 Expression, processing and secretion of a proteolytically-sensitive insect diuretic hormone by Saccharomyces cerevisiae requires the use of a yeast strain lacking genes encoding the Yap3 and Mkc7 endoproteases found in the secretory pathway; Copley KS et al.; A system is described for the heterologous expression of peptides in Saccharomyces cerevisiae . A synthetic gene encoding a precursor of the 41 amino acid Manduca sexta diuretic hormone (Mas-DH) was expressed at 0.8 mg/l purified peptide . A precursor of a mutant peptide of Mas-DH, Mas-DH{K22Q} was also expressed . The peptides were purified, then treated with peptidylglycine alpha-amidating enzyme to generate the alpha-amidated, mature, form of Mas-DH or Mas-DH{K22Q}, which were biologically active . Successful expression of full-length Mas-DH+Gly depended upon the use of a protease-deficient yeast strain . In wild-type strains, Mas-DH+Gly was recovered only as proteolytic fragments, even in the presence of various protease inhibitors . Expression of Mas-DH+Gly in strains deficient in either the Mkc7 or the Yap3 protease reduced proteolysis, while no proteolysis of Mas-DH+Gly was detectable in a strain lacking both proteases . This protease-deficient strain may prove of general utility for expression of peptides . Analysis of recovered proteolytic fragments revealed a complex pattern of cleavage sites . Both the Yap3 and Mkc7 proteases preferred to cleave at a single Glu-Lys downward arrow-Glu-Arg site . Analysis of secondary cleavage sites showed that Yap3 preferred to cleave after either Lys or Arg and Mkc7 after Lys . This paper is the first report on the in vivo activity and specificity of Yap3 and Mkc7 expressed at physiological levels. Mol Biol Cell, 1998 May, 9(5), 1221 - 33 Rho1p-Bni1p-Spa2p interactions: implication in localization of Bni1p at the bud site and regulation of the actin cytoskeleton in Saccharomyces cerevisiae; Fujiwara T et al.; Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein . Rho1p is localized at the growth sites and required for bud formation . We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein . Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p . This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity . The C-terminal 254 amino acid region of Spa2p, Spa2p(1213-1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826-987) . Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p . Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells . However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm . A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip . These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p. Mol Biol Cell, 1998 May, 9(5), 1081 - 91 The yeast Saccharomyces cerevisiae contains two glutaredoxin genes that are required for protection against reactive oxygen species; Luikenhuis S et al.; Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases . Two genes, designated GRX1 and GRX2, which share 40-52% identity and 61-76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae . Strains deleted for both GRX1 and GRX2 were viable but lacked heat-stable oxidoreductase activity using beta-hydroxyethylene disulfide as a substrate . Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1 mutant was unaffected in oxidoreductase activity, whereas the grx2 mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo . Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1 and GRX2 . In addition, a grx1 mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide . Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains . The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth . Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins. Mol Biol Cell, 1998 May, 9(5), 977 - 91 Saccharomyces cerevisiae cells with defective spindle pole body outer plaques accomplish nuclear migration via half-bridge-organized microtubules; Brachat A et al.; Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB) . Deletion of CNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%) . Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules . Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Delta1 cells that lack a detectable outer plaque . These microtubules are attached to the SPB half- bridge throughout the cell cycle . This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque . Although CNM67 is not strictly required for mitosis, it is essential for sporulation . Time-lapse microscopy of cnm67Delta1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation . This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating. Mol Microbiol, 1998 Apr, 28(1), 69 - 79 A role for the Pcl9-Pho85 cyclin-cdk complex at the M/G1 boundary in Saccharomyces cerevisiae; Tennyson CN et al.; PHO85 is a cyclin-dependent kinase (CDK) with roles in phosphate and glycogen metabolism and cell cycle progression . As a CDK, Pho85 is activated by association with Pho85 cyclins (Pcls), of which 10 are known . PCL1, PCL2 and PCL9 are the only members of the Pho85 cyclin family that are expressed in a cell cycle-regulated pattern . We found that PCL9 is expressed in late M/early G1 phase of the cell cycle and is activated by the transcription factor, Swi5 . This pattern of regulation is different from PCL1 and PCL2, which are expressed later in G1 phase and are regulated primarily by the transcription factor SBF . Co-immunoprecipitation experiments using in vitro translated proteins showed that Pcl9 and Pho85 form a complex . Furthermore, immunoprecipitated Pcl9 complexes from yeast lysates were capable of phosphorylating the exogenous substrate Pho4 . The Pcl9-associated kinase activity was dependent on PHO85, showing that Pcl9 and Pho85 form a functionally active kinase complex in vivo . Deletion of PCL9 in diploid cells caused random, rather than bipolar, budding in 18% of cells . In contrast, deletion of PCL2, the closest relative of PCL9, had no effect on the budding pattern . Deleting more members of the PCL1,2 subfamily (which includes PCL9) increased the percentage of random budding in the cell population . When all members of the PCL1,2 subfamily were deleted, 73% of cells budded randomly, a value similar to that obtained when the CDK partner PHO85 was deleted . Our results show that PCL9 and PHO85 form a functional kinase complex and suggest a role for Pho85 CDKs at the M/G1 boundary. Oncogene, 1998 Apr 16, 16(15), 2011 - 6 Interaction of Rho1p target Bni1p with F-actin-binding elongation factor 1alpha: implication in Rho1p-regulated reorganization of the actin cytoskeleton in Saccharomyces cerevisiae; Umikawa M et al.; The RHO1 gene encodes a homolog of mammalian RhoA small G protein in the yeast Saccharomyces cerevisiae . We have shown that Bni1p is one of the downstream targets of Rho1p and regulates reorganization of the actin cytoskeleton through the interaction with profilin, an actin monomer-binding protein . A Bni1p-binding protein was affinity purified from the yeast cytosol fraction and was identified to be Tef1p/Tef2p, translation elongation factor 1alpha (EF1alpha) . EF1alpha is an essential component of the protein synthetic machinery and also possesses the actin filament (F-actin)-binding and -bundling activities . EF1alpha bound to the 186 amino acids region of Bni1p, located between the FH1 domain, the proline-rich profilin-binding domain, and the FH2 domain, of which function is not known . The binding of Bni1p to EF1alpha inhibited its F-actin-binding and -bundling activities . The BNI1 gene deleted in the EF1alpha-binding region did not suppress the bni1 bnr1 mutation in which the actin organization was impaired . These results suggest that the Rho1p-Bni1p system regulates reorganization of the actin cytoskeleton through the interaction with both EF1alpha and profilin. Genetika, 1998 Feb, 34(2), 190 - 7 {New phenotypic manifestation of the ad2 mutation in Saccharomyces cerevisiae yeast--the inability to grow on a synthetic medium with glycerol and hypoxanthine}; Zekhnov AM et al.; The ADE2 gene of Saccharomyces cerevisiae yeast encodes aminoimidazole ribonucleotide-carboxylase (AIR-carboxylase), an enzyme catalyzing the sixth stage of purine nucleotide biosynthesis . Strains bearing the ade2 mutation are able to grow on a glucose-containing synthetic medium with the addition of adenine or hypoxanthine, which under the action of the cellular phosphoribosyltransferases are converted into adenosine monophosphate and inosine monophosphate, respectively . Our studies showed that ade2 mutants were unable to grow on a synthetic medium with glycerol and hypoxanthine . This newly described feature is not constitutively manifested, because some strains can contain suppressor mutations which restore the ability to grow on a synthetic medium with glycerol and hypoxanthine . The ade4, ade5, ade8, ade6, and ade7 mutations were found to suppress the phenotypic manifestation of the ade2 mutations via inactivation of enzymes catalyzing the first, second, third, fourth and fifth stages of purine biosynthesis, while the ade1 mutation, which inactivates enzyme of the seventh stage, lacks suppressive activity . Strains with single adenine mutations, ade4, ade5, ade8, ade6, ade7, or ade1 grow on glycerol- and hypoxanthine-containing media . Our data suggest that the new property of the ade2 mutations could be associated with the accumulation of the AIR-carbole-ribonucleotide . A mutation resulting in the requirement for serine on the medium with glycerol, but not glucose, is described. Arch Biochem Biophys, 1998 Apr 15, 352(2), 175 - 81 One of the fumarate reductase isoenzymes from Saccharomyces cerevisiae is encoded by the OSM1 gene; Muratsubaki H et al.; Soluble fumarate reductase from yeast irreversibly catalyzes the reduction of fumarate to succinate and has noncovalently bound flavin adenine dinucleotide . In yeast, there are two isoenzymes of fumarate reductase, which can be distinguished on the basis of their absorption or nonabsorption to DE-52 columns . Previously, we have purified FRDS1 and isolated its gene (FRDS) from Saccharomyces cerevisiae . In the present study, FRDS2 was purified to homogeneity by four chromatography steps . The N-terminal and C-terminal amino acid sequences of FRDS2 were identical to the deduced amino acid sequence of the OSM1 gene (EMBL Database Accession No . L-26347), whose isolation and biochemical properties have not been studied up until now . From these results, we conclude that FRDS2 is encoded by the OSM1 gene . The deduced amino acid sequence of the OSM1 gene revealed that FRDS2 is synthesized as a precursor protein containing a presequence composed of 32 amino acid residues . The mature enzyme consists of a protein of 469 amino acid residues with a molecular weight of 51,370 . The N-terminal extension had the characteristics of a typical signal sequence required for targeting and sorting to a noncytosolic destination . In fact, FRDS2 was found to be located in promitochondria. Proc Natl Acad Sci U S A, 1998 May 12, 95(10), 5584 - 9 Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae; Srinivasa SP et al.; RGS4, a mammalian GTPase activating protein for G protein alpha subunits, was identified by its ability to inhibit the pheromone response pathway in Saccharomyces cerevisiae . To define regions of RGS4 necessary for its function in vivo, we assayed mutants for activity in this system . Deletion of the N-terminal 33 aa of RGS4 (Delta1-33) yielded a nonfunctional protein and loss of plasma membrane localization . These functions were restored by addition of a C-terminal membrane-targeting sequence to RGS4 (Delta1-33) . Thus, plasma membrane localization is tightly coupled with the ability of RGS4 to inhibit signaling . Fusion of the N-terminal 33 aa of RGS4 to green fluorescent protein was sufficient to localize an otherwise soluble protein to the plasma membrane, defining this N-terminal region as a plasma membrane anchorage domain . RGS4 is palmitoylated, with Cys-2 and Cys-12 the likely sites of palmitoylation . Surprisingly, mutation of the cysteine residues within the N-terminal domain of RGS4 did not affect plasma membrane localization in yeast or the ability to inhibit signaling . Features of the N-terminal domain other than palmitoylation are responsible for the plasma membrane association of RGS4 and its ability to inhibit pheromone response in yeast. Cell Stress Chaperones, 1998 Mar, 3(1), 37 - 43 Identification of an integral membrane 80 kDa protein of Saccharomyces cerevisiae induced in response to dehydration; Eleutherio EC et al.; Using SDS-PAGE gels we observed the induced synthesis of a protein with a molecular mass of 80 kDa when cells of strains of Saccharomyces cerevisiae were subjected to dehydration . Physiological analysis showed that this protein is not present during growth on glucose but was found in derepressed cells from stationary phase . Furthermore, its synthesis was induced when cells were grown on medium containing alpha-methyl-glucoside as carbon source . However, the 80 kDa protein was not found in cells of mutants unable to transport trehalose . This protein was localized in the cytoplasmic membrane and showed trehalose-binding activity, determined by its partial purification on a trehalose-Sepharose 6B affinity column . The possible involvement of the 80 kDa protein with the trehalose transport system is discussed. Biochem Mol Biol Int, 1998 Apr, 44(4), 747 - 59 Menadione toxicity in Saccharomyces cerevisiae cells: activation by conjugation with glutathione; Zadzinski R et al.; Menadione (2-methyl-1,4-naphthoquinone) has been used extensively as an oxidant stressor at the cellular level . However, the mechanism of cytotoxicity of this compound still remains controversial . This study deals with the role of intracellular glutathione in the resistance of the yeast Saccharomyces cerevisiae to menadione . Incubation with 0.5 mM menadione resulted in a decrease of total glutathione concentration in yeast cells, intracellular formation of menadione S-glutathione conjugate and export of the conjugate from cells . GSH-deficient mutants showed lower stimulation of superoxide and hydrogen peroxide production upon exposure to menadione and were more resistant to menadione than wild-type isogenic strains . These results indicate that in yeast cells the formation of S-glutathione conjugate is a major pathway of menadione metabolism and that this reaction leads to redox activation of menadione but permits its removal from the cells. Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4264 - 9 The TOR (target of rapamycin) signal transduction pathway regulates the stability of translation initiation factor eIF4G in the yeast Saccharomyces cerevisiae; Berset C et al.; Initiation factor eIF4G is an essential protein required for initiation of mRNA translation via the 5' cap-dependent pathway . It interacts with eIF4E (the mRNA 5' cap-binding protein) and serves as an anchor for the assembly of further initiation factors . With treatment of Saccharomyces cerevisiae with rapamycin or with entry of cells into the diauxic phase, eIF4G is rapidly degraded, whereas initiation factors eIF4E and eIF4A remain stable . We propose that nutritional deprivation or interruption of the TOR signal transduction pathway induces eIF4G degradation. J Biol Chem, 1998 Apr 10, 273(15), 9306 - 11 The Gag domain of the Gag-Pol fusion protein directs incorporation into the L-A double-stranded RNA viral particles in Saccharomyces cerevisiae; Ribas JC et al.; The L-A double-stranded RNA virus of yeast encodes its major coat protein, Gag, and a Gag-Pol fusion protein made by a -1 ribosomal frameshift, a coding strategy used by many retroviruses . We find that cells expressing only Gag from one plasmid and only Gag-Pol (in frame) from a separate plasmid can support the propagation of M1 double-stranded RNA, encoding the killer toxin . We use this system to separately investigate the functions of Gag and the Gag part of Gag-Pol . L-A contains two fusion protein molecules per particle, and although N-terminal acetylation of Gag is essential for viral assembly, it is completely dispensable for function of Gag-Pol . In general, the requirements on Gag for viral assembly and propagation are more stringent than on the Gag part of Gag-Pol . Finally, we directly show that it is Gag that instructs the incorporation of Gag-Pol into the viral particles. J Biol Chem, 1998 Apr 10, 273(15), 9249 - 54 A homologue of Saccharomyces cerevisiae Dpm1p is not sufficient for synthesis of dolichol-phosphate-mannose in mammalian cells; Tomita S et al.; Dolichol-phosphate-mannose (Dol-P-Man) serves as a donor of mannosyl residues in major eukaryotic glycoconjugates . It donates four mannosyl residues in the N-linked oligosaccharide precursor and all three mannosyl residues in the core of the glycosylphosphatidylinositol anchor . In yeasts it also donates one mannose to the O-linked oligosaccharide . The yeast DPM1 gene encodes a Dol-P-Man synthase that is a transmembrane protein expressed in the endoplasmic reticulum . We cloned human and mouse homologues of DPM1, termed hDPM1 and mDPM1, respectively, both of which encode proteins of 260 amino acids, having 30% amino acid identity with yeast Dpm1 protein but lacking a hydrophobic transmembrane domain, which exists in the yeast synthase . Human and mouse DPM1 cDNA restored Dol-P-Man synthesis in mouse Thy-1-deficient mutant class E cells . Mouse class E mutant cells had an inactivating mutation in the mDPM1 gene, indicating that mDPM1 is the gene for class E mutant . In contrast, hDPM1 and mDPM1 cDNA did not complement another Dol-P-Man synthesis mutant, hamster Lec15 cells, whereas yeast DPM1 restored both mutants . Therefore, in contrast to yeast, mammalian cells require hDPM1/mDPM1 protein and a product of another gene that is defective in Lec15 mutant cells for synthesis of Dol-P-Man. J Biol Chem, 1998 Apr 10, 273(15), 8564 - 71 Hydrogen peroxide causes RAD9-dependent cell cycle arrest in G2 in Saccharomyces cerevisiae whereas menadione causes G1 arrest independent of RAD9 function; Flattery-O'Brien JA et al.; This study shows differences at the level of cell cycle arrest between the response of yeast cells to hydrogen peroxide and superoxide stress . These include both cell cycle phases at which arrest occurs and the involvement of the RAD9 checkpoint gene . Wild-type and rad9 cells were treated with hydrogen peroxide or the superoxide-generating agent menadione . rad9 mutants were up to 100-fold more sensitive to hydrogen peroxide but not affected in their resistance to menadione . Hydrogen peroxide caused G2-phase arrest, whereas menadione-treated cells arrested in G1 . G2 arrest, induced by methyl 2-benzimidazil carbamate, increased cellular resistance to hydrogen peroxide but not to menadione . G1 arrest mediated by alpha-factor caused an increase in survival of wild-type cells treated with menadione but not with hydrogen peroxide . A cdc28 mutant arrested in G1 was significantly more sensitive to hydrogen peroxide than other cdc mutants arrested in later phases, including G2 . rad9 cells have normal stationary phase resistance to hydrogen peroxide, the ability to adapt to it, glutathione content and induction of genes via the stress responsive element . Although rad9-dependent G2 arrest is important, other rad9-dependent factors may be involved in the resistance of cells to hydrogen peroxide since arrest in G2 did not make rad9 cells fully resistant. J Biol Chem, 1998 Apr 10, 273(15), 8556 - 9 The role of Cdc42 in signal transduction and mating of the budding yeast Saccharomyces cerevisiae; Oehlen LJ et al.; The small G-protein Cdc42 functions in many eukaryotic signal transduction pathways . In the budding yeast Saccharomyces cerevisiae, cells with defective Cdc42 fail to induce mating-specific genes in response to mating factor and to adopt the proper morphology for conjugation . Here we show that the failure of mating factor-induced transcription is largely the indirect result of arrest at a specific cell cycle position and/or the accumulation of high levels of the Cln1/2-Cdc28 kinase, a known repressor of mating factor signal transduction . Cdc42-defective cells with restored transcriptional induction have a partially restored mating ability but are still defective in the morphological response to mating factor . These results show that Cdc42 is not required for transduction of the mating factor signal per se but that it is essential for proper mating factor-induced morphogenesis. Mol Biol Cell, 1998 Apr, 9(4), 917 - 30 Mitochondrial inheritance is delayed in Saccharomyces cerevisiae cells lacking the serine/threonine phosphatase PTC1; Roeder AD et al.; In wild-type yeast mitochondrial inheritance occurs early in the cell cycle concomitant with bud emergence . Cells lacking the PTC1 gene initially produce buds without a mitochondrial compartment; however, these buds later receive part of the mitochondrial network from the mother cell . Thus, the loss of PTC1 causes a delay, but not a complete block, in mitochondrial transport . PTC1 encodes a serine/threonine phosphatase in the high-osmolarity glycerol response (HOG) pathway . The mitochondrial inheritance delay in the ptc1 mutant is not attributable to changes in intracellular glycerol concentrations or defects in the organization of the actin cytoskeleton . Moreover, epistasis experiments with ptc1delta and mutations in HOG pathway kinases reveal that PTC1 is not acting through the HOG pathway to control the timing of mitochondrial inheritance . Instead, PTC1 may be acting either directly or through a different signaling pathway to affect the mitochondrial transport machinery in the cell . These studies indicate that the timing of mitochondrial transport in wild-type cells is genetically controlled and provide new evidence that mitochondrial inheritance does not depend on a physical link between the mitochondrial network and the incipient bud site. Gene, 1998 Mar 16, 209(1-2), 247 - 54 Activation of rat androgen receptor by androgenic ligands is unaffected by antiandrogens in Saccharomyces cerevisiae; Rana S et al.; The E . coli lacZ has been utilized as a reporter to evaluate ligand-mediated activation of the rat androgen receptor (AR) in Saccharomyces cerevisiae strain YCR1 . beta-galactosidase activity was androgen-specific and was found to be inducible approximately 260-fold by dihydrotestosterone (DHT), testosterone and R1881 . None of the antiandrogens tested was able to antagonize the DHT-dependent induction of beta-galactosidase activity . In the gel retardation assay, exposure of the receptor to DHT in vitro led to the formation of a protein-DNA complex that was not detected in yeast extracts unexposed to hormone . However, activation of AR by a steroidal (cyproterone acetate) and a non-steroidal antiandrogen (flutamide) either alone or in combination with DHT also results in a similar migration pattern . Additionally, LEM1, the ABC transporter that selectively modulates the biological potency of steroids in yeast, although operative in YCR1, was not responsible for antiandrogen resistance . These results thus indicate the involvement of other non-receptor factor(s) in mediating the effect of antiandrogens in yeast. RNA, 1998 May, 4(5), 566 - 81 Dbp7p, a putative ATP-dependent RNA helicase from Saccharomyces cerevisiae, is required for 60S ribosomal subunit assembly; Daugeron MC et al.; Putative ATP-dependent RNA helicases are ubiquitous, highly conserved proteins that are found in most organisms and they are implicated in all aspects of cellular RNA metabolism . Here we present the functional characterization of the Dbp7 protein, a putative ATP-dependent RNA helicase of the DEAD-box protein family from Saccharomyces cerevisiae . The complete deletion of the DBP7 ORF causes a severe slow-growth phenotype . In addition, the absence of Dbp7p results in a reduced amount of 60S ribosomal subunits and an accumulation of halfmer polysomes . Subsequent analysis of pre-rRNA processing indicates that this 60S ribosomal subunit deficit is due to a strong decrease in the production of 27S and 7S precursor rRNAs, which leads to reduced levels of the mature 25S and 5.8S rRNAs . Noticeably, the overall decrease of the 27S pre-rRNA species is neither associated with the accumulation of preceding precursors nor with the emergence of abnormal processing intermediates, suggesting that these 27S pre-rRNA species are degraded rapidly in the absence of Dbp7p . Finally, an HA epitope-tagged Dbp7 protein is localized in the nucleolus . We propose that Dbp7p is involved in the assembly of the pre-ribosomal particle during the biogenesis of the 60S ribosomal subunit. RNA, 1998 May, 4(5), 551 - 65 Conservation of functional domains involved in RNA binding and protein-protein interactions in human and Saccharomyces cerevisiae pre-mRNA splicing factor SF1; Rain JC et al.; The modular structure of splicing factor SF1 is conserved from yeast to man and SF1 acts at early stages of spliceosome assembly in both organisms . The hnRNP K homology (KH) domain of human (h) SF1 is the major determinant for RNA binding and is essential for the activity of hSF1 in spliceosome assembly, supporting the view that binding of SF1 to RNA is essential for its function . Sequences N-terminal to the KH domain mediate the interaction between hSF1 and U2AF65, which binds to the polypyrimidine tract upstream of the 3' splice site . Moreover, yeast (y) SF1 interacts with Mud2p, the presumptive U2AF65 homologue in yeast, and the interaction domain is conserved in ySF1 . The C-terminal degenerate RRMs in U2AF65 and Mud2p mediate the association with hSF1 and ySF1, respectively . Analysis of chimeric constructs of hSF1 and ySF indicates that the KH domain may serve a similar function in both systems, whereas sequences C-terminal to the KH domain are not exchangeable . Thus, these results argue for hSF1 and ySF1, as well as U2AF65 and Mud2p, being functional homologues. Genetics, 1998 Apr, 148(4), 1763 - 76 Functions of the high mobility group protein, Abf2p, in mitochondrial DNA segregation, recombination and copy number in Saccharomyces cerevisiae; Zelenaya-Troitskaya O et al.; Previous studies have established that the mitochondrial high mobility group (HMG) protein, Abf2p, of Saccharomyces cerevisiae influences the stability of wild-type (rho+) mitochondrial DNA (mtDNA) and plays an important role in mtDNA organization . Here we report new functions for Abf2p in mtDNA transactions . We find that in homozygous deltaabf2 crosses, the pattern of sorting of mtDNA and mitochondrial matrix protein is altered, and mtDNA recombination is suppressed relative to homozygous ABF2 crosses . Although Abf2p is known to be required for the maintenance of mtDNA in rho+ cells growing on rich dextrose medium, we find that it is not required for the maintenance of mtDNA in p cells grown on the same medium . The content of both rho+ and rho- mtDNAs is increased in cells by 50-150% by moderate (two- to threefold) increases in the ABF2 copy number, suggesting that Abf2p plays a role in mtDNA copy control . Overproduction of Abf2p by > or = 10-fold from an ABF2 gene placed under control of the GAL1 promoter, however, leads to a rapid loss of rho+ mtDNA and a quantitative conversion of rho+ cells to petites within two to four generations after a shift of the culture from glucose to galactose medium . Overexpression of Abf2p in rho- cells also leads to a loss of mtDNA, but at a slower rate than was observed for rho+ cells . The mtDNA instability phenotype is related to the DNA-binding properties of Abf2p because a mutant Abf2p that contains mutations in residues of both HMG box domains known to affect DNA binding in vitro, and that binds poorly to mtDNA in vivo, complements deltaabf2 cells only weakly and greatly lessens the effect of overproduction on mtDNA instability . In vivo binding was assessed by colocalization to mtDNA of fusions between mutant or wild-type Abf2p and green fluorescent protein.These findings are discussed in the context of a model relating mtDNA copy number control and stability to mtDNA recombination. Mol Cell Biochem, 1998 Jan, 178(1-2), 27 - 31 The use of molecular modelling in the understanding of configurational specificity (R or S) in asymmetric reactions catalyzed by Saccharomyces cerevisiae or isolated dehydrogenases; de Souza Pereira R et al.; This method gives a general ideal how to use crystallographic information of enzymes to understand reactions catalyzed by these biocatalysts, commonly used by biochemists to produce chiral products . The interactions of three acetoacetic esters with the enzymes L-lactate dehydrogenase and alcohol dehydrogenase were studied through molecular modelling computer program . These artificial substrates have been widely used to produce chiral synthons . Through this methodology it was possible to understand the conformational specificity of these enzymes with respect to the products and how these enzymes can be inhibited by modifying the structures of the artificial substrates . Also, it was possible to predict whether some type of artificial substrate will suffer reduction by cells that contain these dehydrogenases and what kind of configuration (R or S) the final product will have. Microbiology, 1998 Apr, 144 ( Pt 4), 1103 - 11 Lack of correlation between trehalose accumulation, cell viability and intracellular acidification as induced by various stresses in Saccharomyces cerevisiae; Alexandre H et al.; A pma1-1 mutant of Saccharomyces cerevisiae with reduced H(+)-ATPase activity and the isogenic wild-type strain accumulated high levels of trehalose in response to a temperature upshift to 40 degrees C and after addition of 10% ethanol, but only modest levels in response to a rapid drop in external pH and after addition of decanoic acid . There was, however, no correlation between the absolute levels of trehalose in the stressed cells and their viability . All these treatments induced a significant decrease in intracellular pH, and surprisingly, this decrease was very similar in both strains, indicating that intracellular acidification could not be the triggering mechanism for trehalose accumulation in response to stress . A careful investigation of metabolic parameters was carried out to explain how trehalose accumulated under the four different stress conditions tested . No single and common mechanism for trehalose accumulation could be put forward and the transcriptional activation of TPS1 was not unequivocally related to trehalose accumulation . Another finding was that a pma1-1 mutant exhibited a two- to threefold greater capacity to accumulate trehalose than the isogenic wild-type . This enhanced disaccharide synthesis could be attributed to a twofold higher trehalose-6-phosphate synthase activity, together with a fourfold higher content of intracellular UDP-Glc . In addition, this mutant showed 1.5-fold higher levels of ATP compared to the wild-type . The various stress treatments studied showed that a drop in intracellular pH does not correlate with trehalose accumulation . It is suggested that plasma membrane alteration could be the physiological trigger inducing trehalose accumulation in yeast. J Exp Biol, 1998 Jun, 201 ( Pt 11), 1729 - 37 Neither respiration nor cytochrome c oxidase affects mitochondrial morphology in Saccharomyces cerevisiae; Church C et al.; Previous studies have reported that mitochondrial morphology and volume in yeast cells are linked to cellular respiratory capacity . These studies revealed that mitochondrial morphology in glucose-repressed or anaerobically grown cells, which lack or have reduced levels of respiration, is different from that in fully respiring cells . Although both oxygen deprivation and glucose repression decrease the levels of respiratory chain proteins, they decrease the expression of many non-mitochondrial proteins as well, making it difficult to determine whether it is a defect in respiration or something else that effects mitochondrial morphology . To determine whether mitochondrial morphology is dependent on respiration per se, we used a strain with a null mutation in PET100, a nuclear gene that is specifically required for the assembly of cytochrome c oxidase . Although this strain lacks respiration, the mitochondrial morphology and volumes are both comparable to those found in its respiration-proficient parent . These findings indicate that respiration is not involved in the establishment or maintenance of yeast mitochondrial morphology, and that the previously observed effects of oxygen availability and glucose repression on mitochondrial morphology are not exerted through the respiratory chain . By applying the principle of symmorphosis to these findings, we conclude that the shape and size of the mitochondrial reticulum found in respiring yeast cells is maintained for reasons other than respiration. Biochem J, 1998 May 15, 332 ( Pt 1), 153 - 9 Saccharomyces cerevisiae GPI10, the functional homologue of human PIG-B, is required for glycosylphosphatidylinositol-anchor synthesis; Sutterlin C et al.; An increasing number of plasma membrane proteins have been shown to be attached to the membrane via a glycosylphosphatidylinositol (GPI) moiety . All eukaryotes share a highly conserved GPI-core structure EthN-P-Man3-GlcN-PI, where EthN is ethanolamine . We have identified a protein encoded by the yeast open reading frame YGL142C that shares 33% identity with the human Pig-B protein . Deletion of this essential gene leads to a block in GPI anchor biosynthesis . We therefore named the gene GPI10 . Gpi10p and Pig-B are functional homologues and the lethal deletion of GPI10 can be rescued by expression of the PIG-B cDNA . As found for PIG-B mutant cells, gpi10 deletant cells cannot attach the third mannose in an alpha-1,2 linkage to the GPI core-structure intermediate . Overexpression of GPI10 gives partial resistance to the GPI-synthesis inhibitor YW3548, suggesting that this gene product may affect the target of the inhibitor. J Basic Microbiol, 1998, 38(1), 27 - 31 Depression by miconazole of heat-induced respiratory-deficiency in Saccharomyces cerevisiae; Madeira-Lopes A et al.; Miconazole, at 0.2 microM, decreased, by two orders of magnitude, the specific mutation rate of Saccharomyces cerevisiae to respiratory-deficient mutants (petites), which had been induced at either 37 degrees C or 39 degrees C . Identical concentrations of ketoconazole did not change the mutation rate . The results fit in the mechanisms of action which have been proposed for imidazole antimycotics, and constitute further support for the hypothesis that the targets of thermal death, in petite-positive associately-profiled yeasts, lie in the mitochondria. J Bacteriol, 1998 May, 180(9), 2556 - 9 The Saccharomyces cerevisiae YCC5 (YCL025c) gene encodes an amino acid permease, Agp1, which transports asparagine and glutamine; Schreve JL et al.; The yeast YCC5 gene encodes a putative amino acid permease and is homologous to GNP1 (encoding a high-affinity glutamine permease) . Using strains with disruptions in the genes for multiple permeases, we demonstrated that Ycc5 (which we have renamed Agp1) is involved in the transport of asparagine and glutamine, performed a kinetic analysis of this activity, and showed that AGP1 expression is subject to nitrogen repression. J Biol Chem, 1998 Apr 3, 273(14), 8425 - 33 Increased camptothecin toxicity induced in mammalian cells expressing Saccharomyces cerevisiae DNA topoisomerase I; Hann C et al.; The yeast Saccharomyces cerevisiae has been useful in establishing the phenotypic effects of specific mutations on the enzymatic activity and camptothecin sensitivity of yeast and human DNA topoisomerase I . To determine whether these phenotypes were faithfully reiterated in higher eukaryotic cells, wild-type and mutant yeast Top1 proteins were epitope-tagged at the amino terminus and transiently overexpressed in mammalian COS cells . Camptothecin preferentially induced apoptosis in cells expressing wild-type eScTop1p yet did not appreciably increase the cytotoxic response of cells expressing a catalytically inactive (eSctop1Y727F) or a catalytically active, camptothecin-resistant eSctop1vac mutant . Using an epitope-specific antibody, immobilized precipitates of eScTop1p were active in DNA relaxation assays, whereas immunoprecipitates of eScTop1Y727Fp were not . Thus, the enzyme retained catalytic activity while tethered to a support . Interestingly, the mutant eSctop1T722A, which mimics camptothecin-induced cytotoxicity in yeast through stabilization of the covalent enzyme-DNA intermediate, induced apoptosis in COS cells in the absence of camptothecin . This correlated with increased DNA cleavage in immunoprecipitates of eScTop1T722Ap, in the absence of the drug . The observation that the phenotypic consequences of expressing wild-type and mutant yeast enzymes were reiterated in mammalian cells suggests that the mechanisms underlying cellular responses to DNA topoisomerase I-mediated DNA damage are conserved between yeast and mammalian cells. RNA, 1998 Feb, 4(2), 167 - 80 Genetic interaction between U6 snRNA and the first intron nucleotide in Saccharomyces cerevisiae; Luukkonen BG et al.; Nuclear pre-mRNA splicing necessitates specific recognition of the pre-mRNA splice sites . It is known that 5' splice site selection requires base pairing of U6 snRNA with intron positions 4-6 . However, no factor recognizing the highly conserved 5' splice site GU has yet been identified . We have tested if the known U6 snRNA-pre-mRNA interaction could be extended to include the first intron nucleotides and the conserved 50GAG52 sequence of U6 snRNA . We observe that some combinations of 5' splice site and U6 snRNA mutations produce a specific synthetic block to the first splicing step . In addition, the U6-G52U allele can switch between two competing 5' splice sites harboring different nucleotides following the cleavage site . These results indicate that U6 snRNA position 52 interacts with the first nucleotide of the intron before 5' splice site cleavage . Some combinations of U6 snRNA and pre-mRNA mutations also blocked the second splicing step, suggesting a role for the corresponding nucleotides in a proofreading step before exon ligation . From studies in diverse organisms, various functions have been ascribed to the conserved U6 snRNA 47ACAGAG52 sequence . Our results suggest that these discrepancies might reflect variations between different experimental systems and point to an important conserved role of this sequence in the splicing reaction. FEMS Microbiol Lett, 1998 Apr 15, 161(2), 359 - 64 Identification of the mgc1 mutation which affects mating-pheromone-induced morphogenesis in the yeast Saccharomyces cerevisiae; Fujimura HA; Cells of the yeast Saccharomyces cerevisiae undergo morphogenesis in response to the mating pheromones a- and alpha-factors . The 'shmoo' morphology involves localized cell surface projection formation and cytoskeleton protein synthesis . This polarization is presumed to be a prelude to mating between mating partners with opposite mating types, a and alpha . To identify genes involved in pheromone-induced morphogenesis, a system in which a gpa1 fus3 double mutant was used as a parent strain was developed, and mutants which showed altered morphogenesis in response to mating pheromone were identified . The mutation was designated mgc1 for morphogenesis control by mating pheromones . The mgc1 mutant arrested cell division in response to alpha-factor and mated with cells of the opposite mating type, but did not form a typical projection in response to pheromones. FEMS Microbiol Lett, 1998 Apr 15, 161(2), 345 - 9 Transcription of multiple cell wall protein-encoding genes in Saccharomyces cerevisiae is differentially regulated during the cell cycle; Caro LH et al.; The yeast cell wall consists of an internal skeletal layer and an outside protein layer . The synthesis of both beta-1,3-glucan and chitin, which together from the cell wall skeleton, is cell cycle-regulated . We show here that the expression of five cell wall protein-encoding genes (CWP1, CWP2, SED1, TIP1 and TIR1) is also cell cycle-regulated . TIP1 is expressed in G1 phase, CWP1, CWP2 and TIR1 are expressed in S/G2 phase, and SED1 in M phase . The data suggest that these proteins fulfil distinct functions in the cell wall. Insect Biochem Mol Biol, 1997 Dec, 27(12), 999 - 1006 Functional expression of house fly (Musca domestica) cytochrome P450 CYP6D1 in yeast (Saccharomyces cerevisiae); Smith FF et al.; Cytochrome P450 CYP6D1 from the house fly is important in the detoxication of xenobiotics and in resistance to pyrethroid insecticides . In house fly microsomes CYP6D1 requires cytochrome b5 for the metabolism of some substrates, such as benzo{a}pyrene, but does not require cytochrome b5 for the metabolism of other substrates such as methoxyresorufin . To examine the molecular mechanisms involved in its metabolism of pyrethroids and other substrates, a system for the heterologous expression of CYP6D1 in the yeast Saccharomyces cerevisiae was developed . Heterologous CYP6D1 can be inducibly expressed by culture in media with galactose as the sole carbon source, and is successfully inserted into the yeast microsomes . CYP6D1 is enzymatically active, as measured by methoxyresorufin-O-demethylation, indicating that CYP6D1 is able to interact with yeast P450 reductase . However, CYP6D1 expression did not result in measurable benzo{a}pyrene hydroxylation, suggesting that CYP6D1 cannot interact with yeast cytochrome b5, or that there is insufficient cytochrome b5 in the yeast microsomes to support this CYP6D1-mediated activity . Some suggestions are made for improving the yeast microsomal oxidoreductase environment in order to optimize CYP6D1 function. Biotechnol Appl Biochem, 1998 Apr, 27 ( Pt 2), 109 - 15 alpha-Factor pro-peptide N-linked oligosaccharides facilitate secretion of the insulin precursor in Saccharomyces cerevisiae; Kjeldsen T et al.; To evaluate the possible relationship between N-linked glycosylation of the Saccharomyces cerevisiae alpha-factor pro-peptide and transport of the alpha-factor pro-peptide/insulin precursor fusion protein through the Saccharomyces cerevisiae secretory pathway, we analysed secretion of insulin precursor facilitated by alpha-factor pro-peptides with one or more of the three N-linked glycosylation sites removed . Mutation of the three alpha-factor pro-peptide N-linked glycosylation sites drastically decreased insulin precursor secretion . The three alpha-factor pro-peptide N-linked glycosylation sites differ in their ability to facilitate secretion of the insulin precursor . The two alpha-factor pro-peptide N-linked glycosylation sites localized closest to the insulin precursor contributed significantly to secretion, whereas the most N-terminally linked glycosylation site did not appear to facilitate secretion . Only correctly folded insulin precursor was found in the culture supernatant, regardless of the pro-peptide used for secretion, indicating that alpha-factor pro-peptide N-linked oligosaccharide chains are not necessary for correct folding of the insulin precursor . Thus, N-linked glycosylation facilitates intracellular transport of the alpha-factor propeptide/insulin precursor fusion protein through the Saccharomyces cerevisiae secretory pathway and secretion of the insulin precursor . N-linked glycosylation per se is not sufficient to facilitate secretion of the insulin precursor; the position of the N-linked oligosaccharide chain on the alpha-factor pro-peptide is important for facilitating efficient secretion. Biotechnol Appl Biochem, 1998 Apr, 27 ( Pt 2), 81 - 8 Expression and secretion of beta-galactosidase in Saccharomyces cerevisiae using the signal sequences of GgpI, the major yeast glycosylphosphatidylinositol-containing protein; Pignatelli R et al.; New secretory signals and strategies can be attempted to improve the secretion of heterologous proteins of biotechnological interest which encounter difficulties being exported in yeast . The GGPI gene of Saccharomyces cerevisiae codes for a 125 kDa glycoprotein transported through the secretory pathway and anchored to the plasma membrane by means of a glycosylphosphatidylinositol . The regions coding for the secretory signal or also for the first 46 amino acids were tested for efficiency in secretion by fusion to the lacZ gene of Escherichia coli resulting in the synthesis of the endoplasmic reticulum-targeted 1-22- and 1-68-GgpIp/beta-gal hybrids . A cytoplasmic form was also examined . The 1-22 beta gal is partially transported to the cell surface and in the medium in an unglycosylated form . The 1-68 beta gal is completely retained in the intracellular membranes and is N-glycosylated in the GgpIp moiety . The amount of hybrid protein produced is similar and independent from its targeted site, suggesting that translocation through endoplasmic reticulum is not a limiting step, whereas the amount of active enzyme is from 50 to 80% lower for the endoplasmic reticulum forms compared with the cytoplasmic form . BiP/Kar2p putative precursor is accumulated in cells expressing the endoplasmic reticulum-targeted forms but not in those producing the cytosolic beta-galactosidase or over-expressing an endogenous secretory protein . Thus, glycosylation and abnormal folding rather than over-expression are among the factors responsible for the decreased activity and exit of beta-galactosidase from the endoplasmic reticulum and for induction of BiP . The results obtained indicate that the sole secretory signal of GgpIp is suitable to drive secretion of foreign products with complex folding and point to the importance of the endoplasmic reticulum quality control in the secretion of heterologous proteins in yeast. Mol Cell Biol, 1998 May, 18(5), 2884 - 91 Ash1, a daughter cell-specific protein, is required for pseudohyphal growth of Saccharomyces cerevisiae; Chandarlapaty S et al.; Ash1 (for asymmetric synthesis of HO) was first uncovered in genetic screens that revealed its role in mating-type switching . Ash1 prevents HO expression in daughter cells . Because Ash1 has a zinc finger-like domain related to that of the GATA family of transcription factors, it presumably acts by repressing HO transcription . Nonswitching diploid cells also express Ash1, suggesting it could have functions in addition to regulation of HO expression . We show here that Ash1 has an essential function for pseudohyphal growth . Our epistasis analyses are consistent with the deduction that Ash1 acts separately from the mitogen-activated protein kinase cascade and Ste12 . Similarly to the case in yeast form cells, Ash1 is asymmetrically localized to the nuclei of daughter cells during pseudohyphal growth . This asymmetric localization reveals that there is a previously unsuspected daughter cell-specific function necessary for pseudohyphal growth. Mol Cell Biol, 1998 May, 18(5), 2738 - 47 Bub1p kinase activates the Saccharomyces cerevisiae spindle assembly checkpoint; Farr KA et al.; Saccharomyces cerevisiae BUB1 encodes a protein kinase required for spindle assembly checkpoint function . In the presence of spindle damage, BUB1 is required to prevent cell cycle progression into anaphase . We have identified a dominantly acting BUB1 allele that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles . High-level expression of BUB1-5 did not cause detectable spindle damage, yet it delayed yeast cells in mitosis at a stage following bipolar spindle assembly but prior to anaphase spindle elongation . Delayed cells possessed a G2 DNA content and elevated Clb2p mitotic cyclin levels . Unlike cells delayed in mitosis by spindle damage or MPS1 kinase overexpression, hyperphosphorylated forms of the Mad1p checkpoint protein did not accumulate . Similar to cells overexpressing MPS1, the BUB1-5 delay was dependent upon the functions of the other checkpoint genes, including BUB2 and BUB3 and MAD1, MAD2, and MAD3 . We found that the mitotic delay caused by BUB1-5 or MPS1 overexpression was interdependent upon the function of the other . This suggests that the Bub1p and Mps1p kinases act together at an early step in generating the spindle damage signal. FEBS Lett, 1998 Apr 3, 425(3), 475 - 8 Hexokinase PII has a double cytosolic-nuclear localisation in Saccharomyces cerevisiae; Randez-Gil F et al.; We describe here that the HXK2 gene product, isoenzyme PII of hexokinase, is located in both the nucleus and the cytoplasm of Saccharomyces cerevisiae cells . This conclusion is supported by assays of hexokinase-specific activity in isolated nuclei from wild-type and hxk1lhxk2 double mutant strains, by immunoblot experiments using anti-Hxk2 antibodies and by observation of the fluorescence distribution of a Hxk2-GFP fusion protein in cells transformed with the HXK2::gfp gene. Genetics, 1998 Apr, 148(4), 1787 - 98 Cdc1 and the vacuole coordinately regulate Mn2+ homeostasis in the yeast Saccharomyces cerevisiae; Paidhungat M et al.; The yeast CDC1 gene encodes an essential protein that has been implicated in the regulation of cytosolic {Mn2+} . To identify factors that impinge upon Cdc1 or the Cdc1-dependent process, we isolated second-site suppressors of the conditional cdc1-1(Ts) growth defect . Recessive suppressors define 15 COS (CdcOne Suppressor) genes . Seven of the fifteen COS genes are required for biogenesis of the vacuole, an organelle known to sequester intracellular Mn2+ . An eighth gene, COS16, encodes a vacuolar membrane protein that seems to be involved in Mn2+ homeostasis . These results suggest mutations that block vacuolar Mn2+ sequestration compensate for defects in Cdc1 function . Interestingly, Cdc1 is dispensable in a cos16delta deletion strain, and a cdc1delta cos16delta double mutant exhibits robust growth on medium supplemented with Mn2+ . Thus, the single, essential function of Cdc1 is to regulate intracellular, probably cytosolic, Mn2+. Genetics, 1998 Apr, 148(4), 1777 - 86 Cdc1 is required for growth and Mn2+ regulation in Saccharomyces cerevisiae; Paidhungat M et al.; Cdc1 function was initially implicated in bud formation and nuclear division because cdc1(Ts) cells arrested with a small bud, duplicated DNA, and undivided nucleus . Our studies show that Cdc1 is necessary for cell growth at several stages of the cell cycle, as well as in pheromone-treated cells . Thus, Cdc1 depletion might affect bud formation and nuclear division, as well as other cellular processes, by blocking a process involved in general cell growth . Cells depleted of intracellular Mn2+ also exhibit a cdc1-like phenotype and recent results suggested Cdc1 might be a Mn2+-dependent protein . We show that all of the conditional Cdc1(Ts) alleles tested cause cells to become sensitive to Mn2+ depletion . In addition, Cdc1 overproduction alleviates the chelator sensitivity of several Mn2+ homeostasis mutants . These findings are compatible with a model in which Cdc1 regulates intracellular, and in particular cytosolic, Mn2+ levels which, in turn, are necessary for cell growth. Genetics, 1998 Apr, 148(4), 1701 - 13 Cell cycle arrest in cdc20 mutants of Saccharomyces cerevisiae is independent of Ndc10p and kinetochore function but requires a subset of spindle checkpoint genes; Tavormina PA et al.; The spindle checkpoint ensures accurate chromosome segregation by inhibiting anaphase onset in response to altered microtubule function and impaired kinetochore function . In this study, we report that the ability of the anti-microtubule drug nocodazole to inhibit cell cycle progression in Saccharomyces cerevisiae depends on the function of the kinetochore protein encoded by NDC10 . We examined the role of the spindle checkpoint in the arrest in cdc20 mutants that arrest prior to anaphase with an aberrant spindle . The arrest in cdc20 defective cells is dependent on the BUB2 checkpoint and independent of the BUB1, BUB3, and MAD spindle checkpoint genes . We show that the lesion recognized by Bub2p is not excess microtubules, and the cdc20 arrest is independent of kinetochore function . We show that Cdc20p is not required for cyclin proteolysis at two points in the cell cycle, suggesting that CDC20 is distinct from genes encoding integral proteins of the anaphase promoting complex. Genetics, 1998 Apr, 148(4), 1647 - 54 Topical reversion at the HIS1 locus of Saccharomyces cerevisiae . A tale of three mutants; von Borstel RC et al.; Mutants of the HIS1 locus of the yeast Saccharomyces cerevisiae are suitable reporters for spontaneous reversion events because most reversions are topical, that is, within the locus itself . Thirteen mutations of his1-1 now have been identified with respect to base sequence . Revertants of three mutants and their spontaneous reversion rates are presented: (1) a chain termination mutation (his1-208, nee his1-1) that does not revert by mutations of tRNA loci and reverts only by intracodonic suppression; (2) a missense mutation (his1-798, nee his1-7) that can revert by intragenic suppression by base substitutions of any sort, including a back mutation as well as one three-base deletion; and (3) a -1 frameshift mutation (his1-434, nee his1-19) that only reverts topically by +1 back mutation, +1 intragenic suppression, or a -2 deletion . Often the +1 insertion is accompanied by base substitution events at one or both ends of a run of A's . Missense suppressors of his1-798 are either feeders or nonfeeders, and at four different locations within the locus, a single base substitution encoding an amino acid alteration will suffice to turn the nonfeeder phenotype into a feeder phenotype . Late-appearing revertants of his1-798 were found to be slowly growing leaky mutants rather than a manifestation of adaptive mutagenesis . Spontaneous revertants of his1-208 and his1-434 produced no late-arising colonies. Genetics, 1998 Apr, 148(4), 1525 - 33 The chromosome bias of misincorporations during double-strand break repair is not altered in mismatch repair-defective strains of Saccharomyces cerevisiae; McGill CB et al.; Recombinational repair of a site-specific, double-strand DNA break (DSB) results in increased reversion frequency for nearby mutations . Although some models for DSB repair predict that newly synthesized DNA will be inherited equally by both the originally broken chromosome and the chromosome that served as a template, the DNA synthesis errors are almost exclusively found on the chromosome that had the original DSB (introduced by the HO endonuclease) . To determine whether mismatch repair acts on the template chromosome in a directed fashion to restore mismatches to the initial sequence, these experiments were repeated in mismatch repair-defective (pms1, mlh1, and msh2) backgrounds . The results suggest that mismatch repair is not responsible for the observed bias. Genetics, 1998 Apr, 148(4), 1507 - 24 Factors affecting inverted repeat stimulation of recombination and deletion in Saccharomyces cerevisiae; Lobachev KS et al.; Inverted DNA repeats are an at-risk motif for genetic instability that can induce both deletions and recombination in yeast . We investigated the role of the length of inverted repeats and size of the DNA separating the repeats for deletion and recombination . Stimulation of both deletion and recombination was directly related to the size of inverted repeats and inversely related to the size of intervening spacers . A perfect palindrome, formed by two 1.0-kb URA3-inverted repeats, increased intra- and interchromosomal recombination in the adjacent region 2,400-fold and 17,000-fold, respectively . The presence of a strong origin of replication in the spacer reduced both rates of deletion and recombination . These results support a model in which the stimulation of deletion and recombination by inverted repeats is initiated by a secondary structure formed between single-stranded DNA of inverted repeats during replication. Genetics, 1998 Apr, 148(4), 1491 - 505 DNA sequence analysis of spontaneous mutagenesis in Saccharomyces cerevisiae; Kunz BA et al.; To help elucidate the mechanisms involved in spontaneous mutagenesis, DNA sequencing has been applied to characterize the types of mutation whose rates are increased or decreased in mutator or antimutator strains, respectively . Increased spontaneous mutation rates point to malfunctions in genes that normally act to reduce spontaneous mutation, whereas decreased rates are associated with defects in genes whose products are necessary for spontaneous mutagenesis . In this article, we survey and discuss the mutational specificities conferred by mutator and antimutator genes in the budding yeast Saccharomyces cerevisiae . The implications of selected aspects of the data are considered with respect to the mechanisms of spontaneous mutagenesis. Yeast, 1998 Mar 30, 14(5), 471 - 7 The list of cytoplasmic ribosomal proteins of Saccharomyces cerevisiae; Planta RJ et al.; Screening of the complete genome sequence from the yeast Saccharomyces cerevisiae has enabled us to compile a complete list of the genes encoding cytoplasmic ribosomal proteins in this organism . Putative ribosomal protein genes were selected primarily on the basis of the sequence similarity of their products with ribosomal proteins from other eukaryotic organisms, in particular the rat . These genes were subsequently screened for typical yeast rp-gene characteristics, viz . (1) a high codon adaptation index; (2) their promoter structure and (3) their responses to changes in growth conditions . The yeast genome appears to carry 78 different genes, of which 59 are duplicated, encoding 32 different small-subunit and 46 large-subunit proteins . A new nomenclature for these ribosomal proteins is proposed. Yeast, 1998 Mar 15, 14(4), 383 - 90 Purification and properties of polyphosphatase from Saccharomyces cerevisiae cytosol; Andreeva N et al.; A homogenous polyphosphatase preparation was obtained from Saccharomyces cerevisiae cytosol with a 3.8% yield and 3540-fold purification . The enzyme hydrolysed polyphosphate (polyP) with various chain lengths, including polyP3, and split Pi off the end of the chain . It was inactive with respect to ATP, PPi, and p-nitrophenylphosphate . Its specific activity with polyP15 was 283 U/mg protein . The polyphosphatase was a monomeric protein with a molecular mass of 40 kDa . This enzyme was inactive without divalent cations and with Cu2+ and Ca2+ . The ability of other divalent cations to activate the enzyme decreased in the following order; Co2+ > Mn2+ > Mg2+ > Zn2+ . A kinetic model of the hydrolysis of polyP3 and action of Mg2+ is proposed. Yeast, 1998 Mar 15, 14(4), 359 - 69 The SKS1 gene of Saccharomyces cerevisiae is required for long-term adaptation of snf3 null strains to low glucose; Vagnoli P et al.; The SKS1 gene was originally identified as a multicopy suppressor of the growth defect of snf3 null mutations on low glucose concentrations . Snf3p is required for the rapid induction of HXT2 during growth on low substrate concentrations . Loss of Snf3p leads to a dramatic delay in expression of HXT2 . Adaptation to low substrate concentrations does not occur in snf3 sks1 double null mutant strains, suggesting that SKS1 is required for the glucose-dependent expression of HXT2 in the absence of Snf3p activity . Over-expression of SKS1 leads to over-expression of Hxt2p, thus explaining the mechanism of suppression of the snf3 defect . SKS1 defines a novel, Snf3p-independent pathway for the expression of Hxt2p . Under certain growth conditions, over-expression of SKS1 itself leads to a growth defect which is diminished in snf3 hxt2 double mutants . This suggests that over-expression of Hxt2p at physiologically inappropriate times is detrimental to the cells. Yeast, 1998 Mar 15, 14(4), 347 - 57 The importance of the glycerol 3-phosphate shuttle during aerobic growth of Saccharomyces cerevisiae; Larsson C et al.; Maintenance of a cytoplasmic redox balance is a necessity for sustained cellular metabolism . Glycerol formation is the only way by which Saccharomyces cerevisiae can maintain this balance under anaerobic conditions . Aerobically, on the other hand, several different redox adjustment mechanisms exist, one of these being the glycerol 3-phosphate (G3P) shuttle . We have studied the importance of this shuttle under aerobic conditions by comparing growth properties and glycerol formation of a wild-type strain with that of gut2 delta mutants, lacking the FAD-dependent glycerol 3-phosphate dehydrogenase, assuming that the consequent blocking of G3P oxidation is forcing the cells to produce glycerol from G3P . To impose different demands on the redox adjustment capability we used various carbon sources having different degrees of reduction . The results showed that the shuttle was used extensively with reduced substrate such as ethanol, whereas the more oxidized substrates lactate and pyruvate, did not provoke any activity of the shuttle . However, the absence of a functional G3P shuttle did not affect the growth rate or growth yield of the cells, not even during growth on ethanol . Presumably, there must be alternative systems for maintaining a cytoplasmic redox balance, e.g . the so-called external NADH dehydrogenase, located on the outer side of the inner mitochondrial membrane . By comparing the performance of the external NADH dehydrogenase and the G3P shuttle in isolated mitochondria, it was found that the former resulted in high respiratory rates but a comparably low P/O ratio of 1.2, whereas the shuttle gave low rates but a high P/O ratio of 1.7 . Our results also demonstrated that of the two isoforms of NAD-dependent glycerol 3-phosphate dehydrogenase, only the enzyme encoded by GPD1 appeared important for the shuttle, since the enhanced glycerol production that occurs in a gut2 delta strain proved dependent on GPD1 but not on GPD2. Yeast, 1998 Mar 15, 14(4), 311 - 21 Synthesis of monohydroxylated inositolphosphorylceramide (IPC-C) in Saccharomyces cerevisiae requires Scs7p, a protein with both a cytochrome b5-like domain and a hydroxylase/desaturase domain; Dunn TM et al.; Saccharomyces cerevisiae mutants lacking Scs7p fail to accumulate the inositolphosphorylceramide (IPC) species . IPC-C, which is the predominant form found in wild-type cells . Instead scs7 mutants accumulate an IPC-B species believed to be unhydroxylated on the amide-linked C26-fatty acid . Elimination of the SCS7 gene suppresses the Ca(2+)-sensitive phenotype of csg1 and csg2 mutants . The CSG1 and CSG2 genes are required for mannosylation of IPC-C and accumulation of IPC-C by the csg mutants renders them Ca(2+)-sensitive . The SCS7 gene encodes a protein that contains both a cytochrome b5-like domain and a domain that resembles the family of cytochrome b5-dependent enzymes that use iron and oxygen to catalyse desaturation or hydroxylation of fatty acids and sterols . Scs7p is therefore likely to be the enzyme that hydroxylates the C26-fatty acid of IPC-C. Yeast, 1998 Mar 15, 14(4), 303 - 10 Mam33p, an oligomeric, acidic protein in the mitochondrial matrix of Saccharomyces cerevisiae is related to the human complement receptor gC1q-R; Seytter T et al.; Mam33p (mitochondrial acidic matrix protein) is a soluble protein, located in mitochondria of Saccharomyces cerevisiae . It is synthesized as a precursor with an N-terminal mitochondrial targeting sequence that is processed on import . Mam33p assembles to a homo-oligomeric complex in the mitochondrial matrix . It can bind to the sorting signal of cytochrome b2 that directs this protein into the intermembrane space . Mam33p is encoded by an 801 bp open reading frame . Gene disruption did not result in a significant growth defect . Mam33p exhibits sequence similarity to gC1q-R, a human protein that has been implicated in the binding of complement factor C1q and kininogen. J Bacteriol, 1998 Apr, 180(8), 2253 - 6 Physiological regulation of the derepressible phosphate transporter in Saccharomyces cerevisiae; Martinez P et al.; The extracellular phosphate concentration permissive for the expression of different amounts of the active high-affinity Pho84 phosphate transporter in the plasma membrane as well as the PHO84 messenger RNA levels in low-phosphate-grown Saccharomyces cerevisiae cells is very narrow and essential for a tight regulation of the transporter . The Pho84 transporter undergoes a rapid degradation once the supply of phosphate and/or carbon source is exhausted. Prog Cell Cycle Res, 1997, 3, 143 - 56 Cell cycle regulation of S phase entry in Saccharomyces cerevisiae; Piatti S; Eukaryotic DNA replication is restricted to a narrow window of the cell cycle called S phase, and occurs once and only once during each cell cycle . The combination of genetic and biochemical approaches in the budding yeast Saccharomyces cerevisiae has proven extremely helpful for studying the cell cycle regulation of S phase entry . This review will try to summarise the most recent discoveries which led to a new model to explain how entry into S phase is regulated in eukaryotic cells. Mutat Res, 1998 Feb 2, 397(2), 271 - 8 Chloroform and carbon tetrachloride induce intrachromosomal recombination and oxidative free radicals in Saccharomyces cerevisiae; Brennan RJ et al.; Chlorination of drinking water results in the generation of low levels of numerous chlorinated hydrocarbons due to the reaction of chlorine with naturally occurring organic compounds in the water . Concern has been raised about the safety of these chlorinated contaminants as several of them, most notably chloroform (trichloromethane), have been shown to be carcinogenic in long-term rodent bioassays and weak correlations between trihalomethane levels in drinking water and an increased risk of bladder and colorectal cancer in humans have been found . Chloroform and carbon tetrachloride induce liver cancer in rats and mice only at doses where significant hepatotoxicity is observed and have been classed as non-genotoxic carcinogens . We have investigated the ability of chloroform, carbon tetrachloride and 1,1,1-trichloroethane to induce deletions via intrachromosomal recombination in the yeast Saccharomyces cerevisiae . Chloroform and carbon tetrachloride induced this genotoxic recombination event at similar doses, 1,1,1-Trichloroethane gave only a weak response in the DEL recombination assay and only at the highest dose . We further show that chloroform and carbon tetrachloride, but not trichloroethane, induced oxidative free radical species in our yeast strain . The free radical scavenger N-acetylcysteine reduced chloroform-induced toxicity and recombination, and both chloroform and carbon tetrachloride were able to oxidize the free radical-sensitive reporter compound dichlorofluorescein diacetate in vivo . The implications of these findings to the carcinogenic activities of the three compounds are discussed. Biochemistry, 1998 Mar 31, 37(13), 4437 - 50 Mutation and modeling analysis of the Saccharomyces cerevisiae Swi6 ankyrin repeats; Ewaskow SP et al.; The Swi4-Swi6 family of transcription factors confers G1/S specific transcription in budding and fission yeast . These proteins contain four ankyrin repeats, which are present in a large number of functionally diverse proteins and have been shown to be important for protein-protein interaction . However, no specific sequence has been identified that is diagnostic of an ankyrin repeat-interacting protein . To determine the function of the ankyrin repeats of Swi6, we generated both random and site-directed mutations within the ankyrin repeat domain of Swi6 and assayed the transcriptional function of these mutant swi6 alleles . We found six single mutations, scattered within the first and the fourth repeats, that generate a temperature-sensitive Swi6 protein . In addition, we found that alanine substitutions for the most conserved residues in each repeat were highly deleterious and also confer temperature sensitivity . Most of these swi6 alleles are able to form ternary complexes with Swi4 and DNA, but these complexes display reduced mobility in band-shift gels, suggesting a dramatic conformational change . We have modeled the ankyrin repeats of Swi6 using the coordinates derived for 53BP2 and find that, despite its low level of sequence conservation, these modeling studies and our mutation data are consistent with Swi6 having a structure very similar to that of 53BP2 . Moreover, all but one of our single mutants and all of the site-directed mutants disrupt critical structural features of the predicted folding pattern of these repeats . We conclude that the ankyrin repeats play a major structural role in Swi6 . Ankyrin repeats are unlikely to have inherent protein or DNA binding properties . However, they form a characteristic and stable structure with surfaces that may be tailored for many different macromolecular interactions. J Biol Chem, 1998 Mar 27, 273(13), 7628 - 36 Regulation of high affinity iron uptake in the yeast Saccharomyces cerevisiae . Role of dioxygen and Fe; Hassett RF et al.; High affinity iron uptake in Saccharomyces cerevisiae requires a metal reductase, a multicopper ferroxidase, and an iron permease . Fet3, the apparent ferroxidase, is proposed to facilitate iron uptake by catalyzing the oxidation of reductase-generated Fe(II) to Fe(III) by O2; in this model, Fe(III) is the substrate for the iron permease, encoded by FTR1 (Kaplan, J., and O'Halloran, T . V . (1996) Science 271, 1510-1512) . We show here that dioxygen also plays an essential role in the expression of these iron uptake activities . Cells grown anaerobically exhibited no Fe(III) reductase or high affinity iron uptake activity, even if assayed for these activities under air . Northern blot analysis showed that the amount of those mRNAs encoding proteins associated with this uptake was repressed in anaerobic cultures but was rapidly induced by exposure of the culture to dioxygen . The anaerobic repression was reduced in cells expressing an iron-independent