Anticancer Res, 1997 Mar-Apr, 17(2A), 843 - 63 Nuclear import of DNA repair proteins; Boulikas T; DNA repair enzymes play a pivotal role in the maintenance of chromosome integrity and in the elimination of premutagenic lesions from DNA by patrolling the genome; nuclear import mechanisms are implicated in molecular carcinogenesis . We have attempted to predict cell trafficking and the nuclear importation of proteins involved in DNA repair by sequence analysis aimed at identifying karyophilic clusters (arginines, lysines, histidines) flanked by the helix breakers proline or glycine that could function as nuclear localization signals (NLSs) . Most mammalian proteins that participate in DNA repair pathways seem to possess NLS peptides . Repair proteins with multiple nuclear signals are the ERCC6 helicase (eight signals), the XPC protein involved in the repair of the transcribed strand in active genes (eight strong and seven weak signals), and the Rep-3/Duc-1 mismatch repair protein (five strong one weak signal) . We propose that it is unlikely to identify mutations on the genes encoding these proteins resulting in cytoplalsmic retention . However, a number of mammalian DNA repair proteins lack NLS clusters; these proteins include ERCC1, ERCC2 (XPD), mouse RAD51, and the HHR23B/p58 and HHR23A subunits of XPC . NLS-less S . cerevisiae proteins include both RAD51 and RAD52 that function in the recombination and in the repair of double-strand breaks as well as the RAD23 and HRR25 molecules . We propose that these proteins depend on their complexation with other proteins in the cytoplasm for their nuclear localization . The hMSH2 human mismatch repair protein linked to the hereditary nonpolyposis colon cancer gene, has a weak nuclear signal containing two histidines. Genomics, 1997 Mar 1, 40(2), 362 - 6 SMT3A, a human homologue of the S . cerevisiae SMT3 gene, maps to chromosome 21qter and defines a novel gene family; Lapenta V et al.; cDNA selection was used to isolate coding sequences from cosmids mapping to the gene-rich telomeric region of human chromosome 21q . A novel cDNA, termed SMT3A, was isolated and mapped between the loci PFKL and D21S171, about 2.2 Mb proximal to the telomere . The predicted protein of 103 amino acids appears to be a homologue of the Saccharomyces cerevisiae SMT3 protein, whose gene was previously isolated as a suppressor of mutations in the MIF2 gene . The yeast MIF2 gene encodes an essential centromeric protein and shows homology to mammalian CENP-C, an integral component of active kinetochores . SMT3A was found to be highly homologous to two other recently isolated human genes, suggesting the presence of a new gene family . Homologous sequences were also found in protozoa, metazoa, and plants . Moreover, all predicted proteins show significant homology to ubiquitin . The proposed role of yeast SMT3 as centromeric protein and the strong evolutionary conservation of the SMT3A gene suggest an involvement of the encoded protein in the function and/or structure of the eukaryotic kinetochore. Mol Mar Biol Biotechnol, 1997 Mar, 6(1), 64 - 71 Adhesion of yeast isolated from fish gut to crude intestinal mucus of rainbow trout, Salmo gairdneri; Vazquez-Juarez R et al.; Two yeast strains, Saccharomyces cerevisiae CBS 7764 and Debaryomyces hansenii Hfl CBS 8339, with a high capacity to colonize fish intestine were used in this study . The ability to adhere to crude mucus obtained from fish intestine was demonstrated for both strains . Scatchard analysis of the binding indicated a positive cooperativity for D . hansenii Hfl and absence of cooperativity for S . cerevisiae CBS 7764 . In neither of the strains was adhesion extensively affected by reducing the hydrophobic interaction with p-nitrophenol, or by enhancing the hydrophobic interaction with ammonium sulfate . The adhesion was heat sensitive but resistant to trypsin treatment . We conclude that adhesion is mediated partly by specific mechanisms and partly by cell surface hydrophobicity. Curr Genet, 1997 Mar, 31(3), 235 - 40 Two types of alpha-factor receptor determinants for pheromone specificity in the mating-incompatible yeasts S . cerevisiae and S . kluyveri; Sen M et al.; The specificity of mating-pheromone systems helps prevent mating between different species of yeast . The mechanism of specificity of G protein-coupled receptors in other organisms may be similar . To determine the structural basis of species discrimination between alpha-factor variants, small extracellular segments of the Saccharomyces cerevisiae alpha-factor receptor were substituted with residues from the Saccharomyces kluyveri alpha-factor receptor . Three substitutions altered specificity towards S . cerevisiae and S . kluyveri alpha-factor pheromone peptides . A substitution at residues 47-49 affected specificity for pheromone binding but not for pheromone activation of response . Substitution of residues 267-269 affected pheromone specificity for activation of cellular responses, but not for pheromone binding (under the assay conditions used) . Substitution of residues 104-123 modestly affected both types of specificity . These results suggest two distinct types of receptor/ligand specificity (perhaps corresponding to ligand binding to G protein-coupled or uncoupled conformational states of the receptor) that evolved independently as S . cerevisiae and S . kluyveri diverged as species. Antimicrob Agents Chemother, 1997 Mar, 41(3), 672 - 6 Fungicidal action of aureobasidin A, a cyclic depsipeptide antifungal antibiotic, against Saccharomyces cerevisiae; Endo M et al.; Aureobasidin A, an antifungal antibiotic inhibiting a wide range of pathogenic fungi, is lethal for growing cells of susceptible fungi . We did cytological studies on the mechanism of its fungicidal action against Saccharomyces cerevisiae . When cultures were treated with 5.0 micrograms of aureobasidin A per ml, the numbers of viable cells started to decrease after 2 to 3 h of incubation, and most cells had lost viability after 5 to 6 h . When cell death in the treated cultures began, amino acids released by the cells could be detected, indicating disruption of the cell membrane . The proportion of cells with a single small bud or two or more buds increased as the population of viable cells decreased . Most such cells had the DNA content of cells in the G2 phase of the cell cycle, suggesting that the drug inhibited some cellular process involved in normal bud growth but did not affect DNA replication . Disruption of actin assembly was found in many cells treated for 2 to 3 h, as was chitin delocalization . The results suggest that aureobasidin A has a previously unknown mechanism of fungicidal action toward S . cerevisiae . It causes aberrant actin assembly, inhibiting the normal budding process and leading to cell death, probably through destruction of membrane integrity. Mol Cell Biol, 1997 Mar, 17(3), 1152 - 9 Regulation of gene expression during meiosis in Saccharomyces cerevisiae: SPR3 is controlled by both ABFI and a new sporulation control element; Ozsarac N et al.; The SPR3 gene encodes a sporulation-specific homolog of the yeast Cdc3/10/11/12 family of bud neck filament proteins . It is expressed specifically during meiosis and sporulation in Saccharomyces cerevisiae . Analysis of the sporulation-specific regulation of SPR3 has shown that it is strongly activated under sporulating conditions but shows low levels of expression under nonsporulating conditions . A palindromic sequence located near the TATA box is essential to the developmental regulation of this gene and is the only element directly activating SPR3 at the right time during sporulation . Within the palindrome is a 9-bp sequence, gNCRCAAA(A/T) (midsporulation element {MSE}), found in the known control regions of three other sporulation genes . A previously identified ABFI element is also needed for activation . The MSE has been shown to activate a heterologous promoter (CYC1) in a sporulation-specific manner . Related sequences, including an association of MSE and ABFI elements, have been found upstream of other genes activated during the middle stage of S . cerevisiae sporulation . One group of these may be involved in spore coat formation or maturation. J Cell Biol, 1997 Feb 24, 136(4), 761 - 73 A mouse cytoplasmic exoribonuclease (mXRN1p) with preference for G4 tetraplex substrates; Bashkirov VI et al.; Exoribonucleases are important enzymes for the turnover of cellular RNA species . We have isolated the first mammalian cDNA from mouse demonstrated to encode a 5'-3' exoribonuclease . The structural conservation of the predicted protein and complementation data in Saccharomyces cerevisiae suggest a role in cytoplasmic mRNA turnover and pre-rRNA processing similar to that of the major cytoplasmic exoribonuclease Xrn1p in yeast . Therefore, a key component of the mRNA decay system in S . cerevisiae has been conserved in evolution from yeasts to mammals . The purified mouse protein (mXRN1p) exhibited a novel substrate preference for G4 RNA tetraplex-containing substrates demonstrated in binding and hydrolysis experiments . mXRN1p is the first RNA turnover function that has been localized in the cytoplasm of mammalian cells . mXRN1p was distributed in small granules and was highly enriched in discrete, prominent foci . The specificity of mXRN1p suggests that RNAs containing G4 tetraplex structures may occur in vivo and may have a role in RNA turnover. Cell, 1997 Feb 21, 88(4), 493 - 502 Coordinate binding of ATP and origin DNA regulates the ATPase activity of the origin recognition complex; Klemm RD et al.; The Origin Recognition Complex (ORC) is a six-protein assembly that specifies the sites of DNA replication initiation in S . cerevisiae . Origin recognition by ORC requires ATP . Here, we demonstrate that two subunits, Orc1p and Orc5p, bind ATP and that Orc1p also hydrolyzes ATP . ATP binding and hydrolysis by Orc1p are both regulated by origin DNA in a sequence-specific manner . ATP binding to Orc1p, but not ATP hydrolysis, is responsible for the ATP dependence of the ORC-origin interaction, indicating that ATP is a cofactor that locks ORC on origin DNA . These data demonstrate that occupancy of the Orc1p ATP-binding site has a profound effect on ORC function and that ATP hydrolysis by Orc1p has the potential to drive transitions between different functional states of ORC. Biochemistry, 1997 Feb 18, 36(7), 1621 - 7 Crystal structure of a class I ubiquitin conjugating enzyme (Ubc7) from Saccharomyces cerevisiae at 2.9 angstroms resolution; Cook WJ et al.; Ubiquitin-conjugating enzymes are a family of related proteins that participate in the ubiquitination of proteins . Previous studies on the crystal structures of Saccharomyces cerevisiae Ubc4 and Arabidopsis thaliana Ubc1 indicated that the smallest enzymes (class I), which consist entirely of the conserved core domain, share a common tertiary fold . Here we report the three-dimensional structure of the S . cerevisiae class I enzyme encoded by the UBC7 gene . The crystal structure has been solved using molecular replacement techniques and refined by simulated annealing to an R-factor of 0.183 at 2.93 A resolution . Bond lengths and angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 2.3 degrees, respectively . Ubc7 is an alpha/beta protein with four alpha-helices and a four-stranded antiparallel beta-sheet . With the exception of two regions where extra residues are present, the tertiary folding of Ubc7 is similar to those of the other two enzymes . The ubiquitin-accepting cysteine is located in a cleft between two loops . One of these loops is nonconserved, as this region of the Ubc7 molecule differs from the other two enzymes by having 13 extra residues . There is also a second single amino acid insertion that alters the orientation of the turn between the first two beta-strands . Analysis of the 13 ubiquitin-conjugating enzyme sequences in S . cerevisiae indicates that there may be two other regions where extra residues could be inserted into the common tertiary fold . Both of these other regions exhibit significant deviations in the superposition of the three structures and, like the two insertion regions in Ubc7, may represent hypervariable regions within a common tertiary fold . As ubiquitin-conjugating enzymes interact with different substrates or other accessory proteins in the ubiquitination pathway, these variable surface regions may confer distinct specificity to individual enzymes. EMBO J, 1997 Feb 17, 16(4), 769 - 78 The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein; Martin F et al.; The hairpin structure at the 3' end of animal histone mRNAs controls histone RNA 3' processing, nucleocytoplasmic transport, translation and stability of histone mRNA . Functionally overlapping, if not identical, proteins binding to the histone RNA hairpin have been identified in nuclear and polysomal extracts . Our own results indicated that these hairpin binding proteins (HBPs) bind their target RNA as monomers and that the resulting ribonucleoprotein complexes are extremely stable . These features prompted us to select for HBP-encoding human cDNAs by RNA-mediated three-hybrid selection in Saccharomyces cerevesiae . Whole cell extract from one selected clone contained a Gal4 fusion protein that interacted with histone hairpin RNA in a sequence- and structure-specific manner similar to a fraction enriched for bovine HBP, indicating that the cDNA encoded HBP . DNA sequence analysis revealed that the coding sequence did not contain any known RNA binding motifs . The HBP gene is composed of eight exons covering 19.5 kb on the short arm of chromosome 4 . Translation of the HBP open reading frame in vitro produced a 43 kDa protein with RNA binding specificity identical to murine or bovine HBP . In addition, recombinant HBP expressed in S . cerevisiae was functional in histone pre-mRNA processing, confirming that we have indeed identified the human HBP gene. Genes Dev, 1997 Feb 15, 11(4), 528 - 40 Telomerase RNA mutations in Saccharomyces cerevisiae alter telomerase action and reveal nonprocessivity in vivo and in vitro; Prescott J et al.; The ribonucleoprotein enzyme telomerase adds telomeric DNA to chromosomal ends . In most eukaryotes the telomeric repeat units are repeated precisely, consistent with the action of a telomerase that faithfully copies its RNA template . In contrast, Saccharomyces cerevisiae telomeric repeats are degenerate, suggesting that its telomerase has unusual mechanistic properties . We mutated the S . cerevisiae telomerase RNA (TLC1) with a series of 3-base (GUG) substitutions in and next to the 17-nucleotide templating domain . All mutant telomerases were active in TLC1/tlc1 diploids and synthesized patterns of mixed wild-type and mutant telomeric repeats into telomeric DNA, consistent with nonprocessive action . Telomerase isolated from cells containing each mutated tlc1 allele by itself had altered reaction properties in vitro . One mutant template enzyme, 476GUG, was active in vivo and in vitro in the presence of wild-type TLC1 RNA but lacked detectable activity in its absence . Haploid tlc1-476GUG cells containing only this mutant tlc1 allele underwent senescence . Other tlc1 template region mutations allowed maintenance of shortened telomeres in vivo but altered specific enzymatic properties of telomerase in vitro, including induction of primer-template slippage (472GUG) or alteration of the 5' boundary of the template (467GUG) . These data demonstrate that telomerase RNA bases influence enzyme activity profoundly, suggesting that their roles are not confined to serving simply as the template for this specialized reverse transcriptase. Cell, 1997 Feb 7, 88(3), 375 - 84 Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family; Keeney S et al.; Meiotic recombination in S . cerevisiae is initiated by double-strand breaks (DSBs) . In certain mutants, breaks accumulate with a covalently attached protein, suggesting that cleavage is catalyzed by the DSB-associated protein via a topoisomerase-like transesterase mechanism . We have purified these protein-DNA complexes and identified the protein as Spo11, one of several proteins required for DSB formation . These findings strongly implicate Spo11 as the catalytic subunit of the meiotic DNA cleavage activity . This is the first identification of a biochemical function for any of the gene products involved in DSB formation . Spo11 defines a protein family with other members in fission yeast, nematodes, and archaebacteria . The S . pombe homolog, rec12p, is also known to be required for meiotic recombination . Thus, these findings provide direct evidence that the mechanism of meiotic recombination initiation is evolutionarily conserved. Appl Microbiol Biotechnol, 1997 Feb, 47(2), 180 - 4 Manganese uptake and toxicity in magnesium-supplemented and unsupplemented Saccharomyces cerevisiae; Blackwell KJ et al.; The magnesium content of Saccharomyces cerevisiae was found to vary by up to fivefold at differing stages of batch growth and during growth in the presence of differing magnesium concentrations . Excess Mg was primarily sequestered in vacuoles . Mn(2+)-uptake experiments revealed that Mg-enriched cells had a markedly reduced capacity for Mn2+ accumulation . For example, after 6 h incubation in the presence of 50 microM Mn2+, Mn levels were approximately twofold higher in cells previously grown in unsupplemented medium than in those from Mg-supplemented medium . These differences were further accentuated at higher Mn2+ concentrations and were not attributable to altered cell-surface charge or altered cell-surface Mn2+ binding . Cellular Mg status also influenced Mn toxicity towards S . cerevisiae . During exposure to 5 mM Mn2+, 50% reductions in the viability of cells with initial Mg contents of approximately 1400 and 2700 nmol (10(9) cells)-1 occurred after approximately 1.6 h and 3.6 h respectively . In cells containing 3300 nmol Mg (10(9) cells)-1, more than 75% viability was still maintained after 7 h incubation with 5 mM Mn2+ . It is concluded that Mn2+ uptake and toxicity in S . cerevisiae are strongly influenced by intracellular Mg, possibly through Mg-dependent regulation of divalent-cation transport activity. Fungal Genet Biol, 1997 Feb, 21(1), 40 - 9 Cross-Pathway and Pathway-Specific Control of Amino Acid Biosynthesis in Magnaporthe grisea Shen WC, Ebbole DJ. The gene encoding the small subunit of the arginine-specific carbamoyl phosphate synthetase, ARG2, of Magnaporthe grisea was characterized to examine the basic regulation of biosynthetic genes in this plant pathogen . The transcript of the ARG2 gene contains an upstream open reading frame (uORF) that is similar to uORFs found in the homologous genes of Neurospora crassa (arg-2) and Saccharomyces cerevisiae (CPA1), suggesting that the M . grisea gene is translationally regulated by a mechanism that is conserved in these fungi . Amino acid imbalance leads to elevated levels of ARG2 mRNA, indicating that in addition to translational control, ARG2 is subject to cross-pathway transcriptional control . A DNA-binding activity that has properties similar to those of the global transcriptional regulator mediating cross-pathway control in N . crassa was detected in M . grisea cell extracts . Thus, it appears that both specific regulation of ARG2 by arginine and global regulation of amino acid biosynthesis are present in M . grisea and highly conserved among M . grisea, N . crassa, and S . cerevisiae. J Bioenerg Biomembr, 1997 Feb, 29(1), 97 - 102 Homologous and heterologous interactions between hexokinase and mitochondrial porin: evolutionary implications; Wilson JE; Binding of the Type I isozyme of mammalian hexokinase to mitochondria is mediated by the porin present in the outer mitochondrial membrane . Type I hexokinase from rat brain is avidly bound by rat liver mitochondria while, under the same conditions, there is no significant binding to mitochondria from S . cerevisiae . Previously published work demonstrates the lack of significant interaction of yeast hexokinase with mitochondria from either liver or yeast . Thus, structural features required for the interaction of porin and hexokinase must have emerged during evolution of the mammalian forms of these proteins . If these structural features serve no functional role other than facilitating this interaction of hexokinase with mitochondria, it seems likely that they evolved in synchrony since operation of selective pressures on the hexokinase-mitochondrial interaction would require the simultaneous presence of hexokinase and porin capable of at least minimal interaction, and be responsive to changes in either partner that affected this interaction . Recent studies have indicated that a second type of binding site, which may or may not involve porin, is present on mammalian mitochondria . There are also reports of hexokinase binding to mitochondria in plant tissues, but the nature of the binding site remains undefined. Mol Microbiol, 1997 Feb, 23(3), 431 - 44 Characterization of SKM1, a Saccharomyces cerevisiae gene encoding a novel Ste20/PAK-like protein kinase; Martin H et al.; Ste20/PAK serine/threonine protein kinases have been suggested as playing essential roles in cell signalling and morphogenesis as potential targets of Cdc42 and Rac GTPases . We have isolated and characterized the Saccharomyces cerevisiae SKM1 gene, which codes for a novel member of this family of protein kinases . The amino acid sequence analysis of Skm1p revealed the presence of a PH domain and a putative p21-binding domain near its amino terminus, suggesting its involvement in cellular signalling or cytoskeletal functions . However, deletion of SKM1 produced no detectable phenotype under standard laboratory conditions . Moreover, disruption of each of the two other S . cerevisiae Ste20/PAK-like kinase-encoding genes, STE20 and CLA4, in skm1 backgrounds, showed that Skm1p is not redundant with Ste20p or Cla4p . Interestingly, overexpression of SKM1 led to morphological alterations, indicating a possible role for this protein in morphogenetic control . Furthermore, overproduction of Skm1p lacking its N-terminus caused growth arrest . This effect was also seen when similarly truncated versions of Ste20p or Cla4p were overexpressed . We further observed that overproduction of this C-terminal fragment of Skm1p complements the mating defect of a ste20 mutant strain . These results suggest that the N-terminal domains of S . cerevisiae Ste20/ PAK-like protein kinases share a negative regulatory function and play a role in substrate specificity. Curr Genet, 1997 Feb, 31(2), 128 - 32 Cloning the Yarrowia lipolytica homologue of the Saccharomyces cerevisiae SEC62 gene; Swennen D et al.; Yarrowia lipolytica SEC62 cDNA was cloned by functional complementation of a thermo-sensitive sec62 Saccharomyces cerevisiae mutant strain . The Y . lipolytica SEC62 promoter region was amplified by the inverse polymerase chain reaction (PCR) . The cDNA codes for a 396 amino-acid protein with two potential trans-membrane domains . Y . lipolytica Sec62p behaves as an integral membrane protein as shown by Western blotting . Y . lipolytica SEC62 cDNA is able to complement a S . cerevisiae sec62 null mutant strain confirming functional conservation, although only 53.6% amino-acid similarity is observed between Y . lipolytica and S . cerevisiae Sec62. Curr Genet, 1997 Feb, 31(2), 122 - 7 Structural analysis of a Candida glabrata centromere and its functional homology to the Saccharomyces cerevisiae centromere; Kitada K et al.; A 451-bp fragment exhibiting centromere activity had been previously isolated from Candida glabrata genomic DNA . It contains three elements, CgCDEI, CgCDEII and CgCDEIII, highly homologous to those of Saccharomyces cerevisiae . In this study, the requirement of each element for centromere function was analyzed in detail . Deletion analysis identified a small fragment of 153 bp, which included all three elements, to be sufficient for centromere activity . Linker substitution analysis of CgCDEI and CgCDEIII revealed that both elements are required for centromere function . Some of the substitution mutations in CgCDEIII caused a complete loss of centromere activity . These results suggested a functional similarity of centromeres between C . glabrata and S . cerevisiae . However, the C . glabrata centromere did not function in S . cerevisiae cells, suggesting species specificity of the C . glabrata centromere . To examine whether species specificity of the centromeres between these two yeasts does exist, chimeric centromeres between the two species were constructed . Exchange of CgCDEII or CgCDEIII with CDEII or CDEIII of S . cerevisiae, respectively, increased C . glabrata centromere activity in S . cerevisiae, indicating participation of the two elements in determining the species specificity of centromere function. Biochemistry, 1997 Jan 28, 36(4), 721 - 9 Purification, characterization, gene cloning, and expression of Saccharomyces cerevisiae redoxyendonuclease, a homolog of Escherichia coli endonuclease III; Augeri L et al.; Saccharomyces cerevisiae redoxyendonuclease (Scr), a homolog of Escherichia coli endonuclease III, was purified from yeast deficient in the major apurinic/apyrimidinic endonuclease, Apnl . Studies of this highly purified preparation of Scr have revealed a number of similarities between this protein and endonuclease III as well as provided further evidence for a common mechanism of action for this class of DNA glycosylase/AP lyases . We have employed a sensitive and specific assay for Scr which utilizes oligonucleotide substrates containing a single 5,6-dihydrouracil base lesion or an abasic site . These substrates were utilized to investigate the mode of action of Scr on damaged DNA and to compare the kinetic properties of the yeast enzyme with its E . coli counterpart . Furthermore, we have identified two distinct genes, SCR1 and SCR2, which encode highly homologous proteins with similar activities in yeast . Analysis of the deduced amino acid sequences of SCR1 and SCR2 suggests that S . cerevisiae possesses two similar enzymes encoded on separate chromosomes: one which bears an Fe-S binding motif, while the other does not . The potential biological roles of these two forms of Scr are discussed. Cell, 1997 Jan 24, 88(2), 253 - 63 A novel mutation avoidance mechanism dependent on S . cerevisiae RAD27 is distinct from DNA mismatch repair; Tishkoff DX et al.; Mutations in the S . cerevisiae RAD27 (also called RTH1 or YKL510) gene result in a strong mutator phenotype . In this study we show that the majority of the resulting mutations have a structure in which sequences ranging from 5-108 bp flanked by direct repeats of 3-12 bp are duplicated . Such mutations have not been previously detected at high frequency in the mutation spectra of mutator strains . Epistasis analysis indicates that RAD27 does not play a major role in MSH2-dependent mismatch repair . Mutations in RAD27 cause increased rates of mitotic crossing over and are lethal in combination with mutations in RAD51 and RAD52 . These observations suggest that the majority of replication errors that accumulate in rad27 strains are processed by double-strand break repair, while a smaller percentage are processed by a mutagenic repair pathway . The duplication mutations seen in rad27 mutants occur both in human tumors and as germline mutations in inherited human diseases. J Biol Chem, 1997 Jan 24, 272(4), 2373 - 81 The influence of the proliferating cell nuclear antigen-interacting domain of p21(CIP1) on DNA synthesis catalyzed by the human and Saccharomyces cerevisiae polymerase delta holoenzymes; Gibbs E et al.; In eukaryotes, processive DNA synthesis catalyzed by DNA polymerases delta and epsilon (pol delta and epsilon) requires the proliferating cell nuclear antigen (PCNA) . It has recently been shown that in humans (h), the PCNA function, required for both DNA replication and nucleotide excision repair, can be inactivated by p21(CIP1) due to a specific interaction between hPCNA and the carboxyl terminus of p21(CIP1) . In this report, we show that Saccharomyces cerevisiae (S . cerevisiae) PCNA-dependent pol delta-catalyzed DNA synthesis was inhibited less efficiently than the human system by the intact p21(CIP1) protein and was unaffected by the p21(CIP1) carboxyl-terminal peptide (codons 139-160) . This species-specific response of PCNA to p21(CIP1)-mediated inhibition of DNA synthesis results from a marked difference in the ability of h and S . cerevisiae PCNA to interact with p21(CIP1) . As shown by binding studies using the surface plasmon resonance technique, hPCNA binds both full-length p21(CIP1) and the p21(CIP1) peptide-(139-160) stoichiometrically with a similar affinity (KD approximately 2.5 nM) while S . cerevisiae PCNA binds p21(CIP1) with approximately 10-fold less affinity and does not interact with the p21(CIP1) peptide-(139-160). J Biol Chem, 1997 Jan 24, 272(4), 2212 - 7 Two-step processing is not essential for the import and assembly of functionally active iron-sulfur protein into the cytochrome bc1 complex in Saccharomyces cerevisiae; Nett JH et al.; The iron-sulfur protein of the cytochrome bc1 complex is one of a small number of proteins that are processed in two sequential steps by matrix processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP) during import into Saccharomyces cerevisiae mitochondria . To test whether two-step processing is necessary for import and assembly of the iron-sulfur protein into the cytochrome bc1 complex, we mutagenized the presequence of the iron-sulfur protein to eliminate the original MPP site and replace the MIP site with a new MPP site . The mutated presequence is cleaved and forms mature-sized protein in a single step, and the mature-sized iron-sulfur protein is correctly targeted to the outer side of the inner mitochondrial membrane in vitro . Mutant iron-sulfur protein which is processed to mature size in one step complements the respiratory deficient phenotype of a yeast strain in which the endogenous gene for the iron-sulfur protein is deleted . These results establish that mature-sized iron-sulfur protein can be formed by single-step processing and assembled into a functionally active form in the cytochrome bc1 complex in S . cerevisiae. Genes Dev, 1997 Jan 15, 11(2), 241 - 54 An unusual form of transcriptional silencing in yeast ribosomal DNA; Smith JS et al.; Generalized transcriptional repression of large chromosomal regions in Saccharomyces cerevisiae occurs at the silent mating loci and at telomeres and is mediated by the silent information regulator (SIR) genes . We have identified a novel form of transcriptional silencing in S . cerevisiae in the ribosomal DNA (rDNA) tandem array . Ty1 retrotransposons marked with a weakened URA3 gene (Ty1-mURA3) efficiently integrated into rDNA . The mURA3 marker in rDNA was transcriptionally silenced in a SIR2-dependent manner . MET15 and LEU2 were also partially silenced, indicating that rDNA silencing may be quite general . Deletion of SIR4 enhanced mURA3 and MET15 silencing, but deletion of SIR1 or SIR3 did not affect silencing, indicating that the mechanism of silencing differs from that at telomeres and silent mating loci . Deletion of SIR2 resulted in increased psoralen cross-linking of the rDNA in vivo, suggesting that a specific chromatin structure in rDNA down-regulates polymerase II promoters. FEBS Lett, 1997 Jan 13, 401(1), 20 - 4 Siroheme biosynthesis in Saccharomyces cerevisiae requires the products of both the MET1 and MET8 genes; Hansen J et al.; Siroheme is a uroporphyrinogen III-derivative used by sulfite reductase as a prosthetic group . We investigated in Saccharomyces cerevisiae the possible involvement in siroheme biosynthesis of three genes, MET1, MET8 and MET20 . The MET1 gene from S . cerevisiae was cloned and shown to be the same gene as MET20 . Sequence similitudes as well as complementation studies indicate that Met1p and Met8p are both involved in siroheme biosynthesis . In addition, we show formally that S . cerevisiae does not need vitamin B12 for growth. Acta Microbiol Pol, 1997, 46(1), 105 - 14 High-glutathione containing yeast Saccharomyces cerevisiae: optimization of production; Udeh KO et al.; Experimental design procedure was used to search for optimal medium composition for maximization of glutathione yield using a high-glutathione containing yeast S . cerevisiae S-8H . The effects of the chosen factors (glucose, peptone, KN2PO4, biotin and cysteine) on the relationship between yeast biomass formation and the content of intracellular glutathione were examined . Results obtained showed that glucose and peptone had significant first- and second-order effects, as well as different interactive effects on these overall parameters . On the other hand, cysteine showed only second-order effect for glutathione content and interactive effects for both glutathione content and yield . Its presence had a marked positive effect on the content of glutathione, but reduced the biomass formation . The optimal levels of these factors found were in the range of 2.5%, 4%, 0.027%, 10 micrograms/cm3 and 0.08%, respectively . The average yield obtained at this optimium was 160.1 mg glutathione/dm3 and a glutathione content of 17 mg/g dry biomass. Biochimie, 1997, 79(1), 27 - 36 Expression of the release factor eRF1 (Sup45p) gene of higher eukaryotes in yeast and mammalian tissues; Urbero B et al.; Polypeptide chain termination in eukaryotic cells is mediated in part by the release factor eRF1 (Sup45p) . We have isolated and characterised cDNAs encoding this translation factor from Syrian hamster (Mesocricetus auratus) and human (Homo sapiens) Daudi cells . Comparison of the deduced amino acid sequence of these new eRF1 (Sup45p) sequences with those published for Saccharomyces cerevisiae, Arabidopsis thaliana, Xenopus laevis and human indicates a high degree of amino acid identity across a broad evolutionary range of species . Both the 5' and 3' UTRs of the mammalian eRF1 (Sup45p)-encoding cDNAs show an unusually high degree of conservation for non-coding regions . In addition, the presence of two different lengths of 3' UTR sequences in the mammalian eRF1 (Sup45p) cDNAs indicated that alternative polyadenylation sites might be used in vivo . Northern blot analysis demonstrated that eRF1 (Sup45p) transcripts of differing length, consistent with the use of alternative polyadenylation sites, were detectable in a wide range of mammalian tissues . The Xenopus, human and Syrian hamster eRF1 (Sup45p) cDNAs were shown to support the viability of a strain of S cerevisiae carrying an otherwise lethal sup45::HIS3 gene disruption indicating evolutionary conservation of function . However, the yeast strains expressing the heterogenous eRF1 (Sup45p) showed a defect in translation termination as defined by an enhancement of nonsense suppressor tRNA activity in vivo . Western blot analysis confirmed that Xenopus eRF1 (Sup45p) was primarily ribosome-associated when expressed in yeast indicating that the ribosome-binding domain of eRF1 (Sup45p) is also conserved. Adv Exp Med Biol, 1997, 419, 45 - 52 Selection of diphtheria toxin active-site mutants in yeast . Rediscovery of glutamic acid-148 as a key residue; Fu H et al.; Saccharomyces cerevisiae was transformed with expression plasmids carrying the DTA gene under control of the GAL1 promoter; colonies that formed under inducing conditions were selected; and plasmids from these colonies were screened for mutations in DTA that failed to block expression of the protein . Substitutions at three sites were identified, all of which are in the active-site cleft; and each of the substitutions reduced ADP-ribosyltransferase activity by > 10(5) . The substitutions include a charge reversal mutation of a catalytically important residue (Glu148Lys) and replacements of either of two glycines (Gly22 and Gly52) with bulky residues . The fact that multiple mutations were identified in these same residues implies that there are relatively few sites at which substitutions ablate ADP-ribosyltransferase activity without blocking expression of the full-length protein . Incorporation of a primary attenuating mutation into the DTA gene allowed S . cerevisiae also to be used to select complementary secondary mutations which altered activity less drastically . Besides elucidating structure-activity relationships, mutations identified by these approaches may be useful in designing new vaccines. Subcell Biochem, 1997, 28, 89 - 116 Antifungal sterol biosynthesis inhibitors; Guo DA et al.; During the course of the last decade, the development of SBIs, and particularly sterol biomethylation inhibitors, has been based on the rational design approach . Successful though this approach has been in elucidating sterol biomethylation enzymology, its limitations are becoming apparent from the findings that: (i) 24,25-double bond metabolism gives rise to cholesterol and ergosterol in a mechanistically similar manner, (ii) 25-azasterols are harmful to human physiology, and (iii) side-chain modified sterols designed to inhibit the SMT enzyme in S . cerevisiae may be ineffective or operate by another kinetic mechanism in a related organism, rendering it therapeutically nonuseful . Nevertheless, it may be possible to ultimately capitalize on the unique aspects of sterol biomethylation chemistry and enzymology to design taxa-specific inhibitors . With increased understanding of the structure and function of SMT enzymes in different fungi, it should be possible to prepare novel mechanism-based inactivators to control SMT activity uniquely and with high specific activity. Yeast, 1997 Jan, 13(1), 37 - 41 Identification and expression of the Saccharomyces cerevisiae cytoplasmic tryptophanyl-tRNA synthetase gene; John TR et al.; The enzymes that aminoacylate tRNAs have been studied extensively and can be organized into two distinct classes based on signature sequences and the position of aminoacylation . The class I enzymes have canonical HIGH and KMSKS sequences as part of a Rossman fold nucleotide-binding site . The tryptophan-specific enzymes have been placed in class I based on analysis of the cognate genes from Escherichia coli, B . stearothermophilus, B . taurus, and Homo sapiens . An unidentified open reading frame (ORF) on Saccharomyces cerevisiae chromosome XV, HRE342, has 46% identity with the bovine tryptophanyl-tRNA synthetase and possesses the appropriate signature sequences . The predicted molecular weight of the putative HRE342 protein also closely matched the expected monomer size of the S . cerevisiae enzyme . The HRE342 ORF plus about 250 bp of 5' and 3' flanking sequence was amplified by polymerase chain reaction, cloned into a 2 mu based vector, and transformed into a host strain, S . cerevisiae JG369.3B . Nucleotide sequence analysis of the clone confirmed the presence of HRE342 . Extracts from transformed yeast have a 30- to 100-fold increase in specific activity of the tryptophanyl-tRNA synthetase . An HRE342 locus in a diploid strain, PTY33XPTY44, was disrupted with a LEU2 insert . Sporulation and tetrad analysis of the HRE342::LEU2 strain demonstrated that HRE342 is an essential gene . We conclude that HRE342 is the S . cerevisiae gene encoding the cytoplasmic tryptophanyl-tRNA synthetase, WRS1 . A search of the Saccharomyces Genome Database using amino acid sequences from other eukaryotic aminoacyl-tRNA synthetase suggests there is sufficient similarity to identify both class I and class II genes. J Recept Signal Transduct Res, 1997 Jan-May, 17(1-3), 293 - 303 Investigation of growth hormone releasing hormone receptor structure and activity using yeast expression technologies; Kajkowski EM et al.; Growth hormone releasing hormone (GHRH) is the positive regulator of growth hormone synthesis and secretion in the anterior pituitary . The peptide confers activity by binding to a seven transmembrane domain G protein-coupled receptor . Signal transduction proceeds through subsequent G alpha s stimulation of adenylyl cyclase . To investigate ligand/receptor and receptor/G protein associations, the human GHRH receptor was expressed in a modified S . cerevisiae strain which allows for facile measurement of receptor activity by cell prototrophy mediated by a reporter gene coupled to the yeast pheromone response pathway . GHRH-dependent signal activation in this system required the substitution of yeast G alpha protein with proteins containing C-terminal regions of G alpha s . A D60G variant (analogous to the little mouse mutation) of the receptor failed to respond to agonist . In parallel studies, GHRH29 and the N-terminal extracellular region of the receptor were expressed as Gal4 fusion proteins in a 2-hybrid assay . A specific interaction between these proteins was readily observed . The D60G mutation was engineered into the receptor fusion protein . This protein failed to interact with the ligand fusion, confirming the specificity of the association between unmodified proteins . These two yeast expression technologies should prove invaluable in additional structure/activity analyses of this ligand/receptor pair as well as other peptide ligands and receptors. Curr Genet, 1997 Jan, 31(1), 7 - 14 Isolation of early meiotic recombination genes analogous to S . cerevisiae REC104 from the yeasts S . paradoxus and S . pastorianus; Nau JJ et al.; The REC104 gene of Saccharomyces cerevisiae is required to initiate recombination in meiosis . Mutations in REC104 eliminate meiotic recombination and lead to the production of inviable spores . To determine if analogous genes exist in other yeasts, clones that hybridized to a REC104 probe were isolated from the yeasts S . paradoxus and S . pastorianus . When transformed into a rec104 strain, the REC104 analogs from these two yeasts restored spore viability and meiotic recombination to the same level as a REC104 gene cloned from S . cerevisiae . Compared to S . cerevisiae, the S . paradoxus gene codes for 79% identical amino acids and has 86% nucleic-acid identity in the promoter region and 84% in the coding region . The S . pastorianus gene codes for 63% identical amino acids and has 59% and 71% identity in the promoter and the coding regions, respectively. FEMS Microbiol Lett, 1997 Jan 1, 146(1), 109 - 15 Cloning of a new FLO gene from the flocculating Saccharomyces cerevisiae IM1-8b strain; Sieiro C et al.; A flocculation conferring gene was cloned from a genomic library of the flocculating strain Saccharomyces cerevisiae IM1-8b as a 5 kb DNA fragment . The shortest DNA fragment (XbaI-XbaI) able to confer the flocculating phenotype was 3.1 kb . Southern analysis revealed that this gene was not homologous to the already reported FLO1 gene since strong hybridization signals were obtained when chromosomes IV and XII were probed with a digoxygenin-labelled fragment and no signal at all was detected for chromosome I . Partial sequencing data unequivocally ascribed the cloned fragment to chromosome XII . The gene was detected in a variety of S . cerevisiae strains regardless of their being phenotypically flocculating . This gene which, we propose as FLO2, is able to complement the flo1 mutation and is suppressed by suppressors (fsu3) that do not affect other FLO genes. J Bacteriol, 1997 Jan, 179(1), 157 - 62 Expression of bacterial mtlD in Saccharomyces cerevisiae results in mannitol synthesis and protects a glycerol-defective mutant from high-salt and oxidative stress; Chaturvedi V et al.; Polyols, or polyhydroxy alcohols, are produced by many fungi . Saccharomyces cerevisiae produces large amounts of glycerol, and several fungi that cause serious human infections produce D-arabinitol and mannitol . Glycerol functions as an intracellular osmolyte in S . cerevisiae, but the functions of D-arabinitol and mannitol in pathogenic fungi are not yet known . To investigate the functions of mannitol, we constructed a new mannitol biosynthetic pathway in S . cerevisiae . S . cerevisiae transformed with multicopy plasmids encoding the mannitol-1-phosphate dehydrogenase of Escherichia coli produced mannitol, whereas S . cerevisiae transformed with control plasmids did not . Although mannitol production had no obvious phenotypic effects in wild-type S . cerevisiae, it restored the ability of a glycerol-defective, osmosensitive osg1-1 mutant to grow in the presence of high NaCl concentrations . Moreover, osg1-1 mutants producing mannitol were more resistant to killing by oxidants produced by a cell-free H2O2-FeSO4-NaI system than were controls . These results indicate that mannitol can (i) function as an intracellular osmolyte in S . cerevisiae, (ii) substitute for glycerol as the principal intracellular osmolyte in S . cerevisiae, and (iii) protect S . cerevisiae from oxidative damage by scavenging toxic oxygen intermediates. Mol Cell Biol, 1997 Jan, 17(1), 69 - 80 A complex containing two transcription factors regulates peroxisome proliferation and the coordinate induction of beta-oxidation enzymes in Saccharomyces cerevisiae; Karpichev IV et al.; Expression of the POX1 gene, which encodes peroxisomal acyl coenzyme A oxidase in the yeast Saccharomyces cerevisiae, is tightly regulated and can be induced by fatty acids such as oleate . Previously we have shown that this regulation is brought about by interactions between trans-acting factor(s) and an upstream activating sequence (UAS1) in the POX1 promoter . We recently identified and isolated a transcription factor, Oaf1p, that binds to the UAS1 of POX1 and mediates its induction . A screening strategy has been developed and used to identify eight S . cerevisiae mutants, from three complementation groups, that are defective in the oleate induction of POX1 . Characterization of one such mutant led to the identification of Oaf2p, a protein that is 39% identical to Oaf1p . Oaf1p and Oaf2p form a protein complex that is required for the activation of POX1 and FOX3 and for proliferation of peroxisomes . We propose a model in which these two transcription factors heterodimerize and mediate this activation process . The mutants that we have isolated, and further identification of the corresponding defective genes, provide us with an opportunity to characterize the mechanisms involved in the coordinate regulation of peroxisomal beta-oxidation enzymes. Cell, 1996 Dec 27, 87(7), 1249 - 60 RSC, an essential, abundant chromatin-remodeling complex; Cairns BR et al.; A novel 15-subunit complex with the capacity to remodel the structure of chromatin, termed RSC, has been isolated from S . cerevisiae on the basis of homology to the SWI/SNF complex . At least three RSC subunits are related to SWI/SNF polypeptides: Sth1p, Rsc6p, and Rsc8p are significantly similar to Swi2/Snf2p, Swp73p, and Swi3p, respectively, and were identified by mass spectrometric and sequence analysis of peptide fragments . Like SWI/SNF, RSC exhibits a DNA-dependent ATPase activity stimulated by both free and nucleosomal DNA and a capacity to perturb nucleosome structure . RSC is, however, at least 10-fold more abundant than SWI/SNF complex and is essential for mitotic growth . Contrary to a report for SWII/SNF complex, no association of RSC (nor of SWI/SNF complex) with RNA polymerase II holoenzyme was detected. Eur J Biochem, 1996 Dec 15, 242(3), 770 - 8 Mathematical models for determining metabolic fluxes through the citric acid and the glyoxylate cycles in Saccharomyces cerevisiae by 13C-NMR spectroscopy; Tran-Dinh S et al.; We propose, first, a practical method for studying the isotopic transformation of glutamate or any other metabolite isotopomers in the citric acid and the glyoxylate cycles; second, two mathematical models, one for evaluating the flux through the citric acid cycle and the other for evaluating the flux through the latter coupled to the glyoxylate cycle in yeast . These models are based on the analysis of 13C-NMR spectra of glutamate obtained from Saccharomyces cerevisiae, NCYC strain, fed with 100% enriched {2-13C}acetate . The population of each glutamate isotopomer, the change in intensity of each multiplet component or the enrichment of any glutamate carbon is expressed by a specific analytical equation from which the flux in the citric acid and the glyoxylate cycles can be deduced . The aerobic metabolism of 100% {2-13C}acetate in acetate-grown S . cerevisiae cells was studied as a function of time using 13C-NMR . 1H-NMR and biochemical techniques . The C1 and C6 doublet and singlet of labeled trehalose increase continuously with time indicating that there is no isotopic transformation between trehalose isotopomers even though the corresponding formation rates are different . By contrast, the glutamate C4 singlet increases then decreases with time . The C4 doublet, which is lower than the singlet for t < 60 min, increases continuously and becomes higher than the singlet for t > 90 min . A similar observation was made for the C2 resonance singlet and doublet . In addition, the glutamate C2 multiplet consists of only seven instead of nine peaks as in random labeling . These results agree well with our models and demonstrate that, in the presence of acetate, anaplerotic carbon sources involved in the synthesis of acetyl-CoA are negligible in yeast . The flux in the citric acid cycle was deduced from a plot of the C4 area versus incubation time, while the flux within the glyoxylate cycle was determined from the relative intensity of the glutamate C4 doublet and singlet . The fluxes in the citric acid and the glyoxylate cycles were found to be comparable . The proportion of glutamate in isotopic exchange with the citric acid cycle is about 2.5% min1 in yeast. Experientia, 1996 Dec 15, 52(12), 1042 - 9 Polypeptide translocation machinery of the yeast endoplasmic reticulum; Lyman SK et al.; Proteins enter the secretory pathway by two general routes . In one, the complete polypeptide is made in the cytoplasm and held in an incompletely folded state by chaperoning adenosine triphosphatases (ATPases) such as hsp70 . In Saccharomyces cerevisiae, fully synthesized secretory precursors engage the endoplasmic reticulum (ER) membrane by interaction with a set of Sec proteins comprising the polypeptide translocation apparatus (Sec61p, Sec62p, Sec63p, Sec71p, Sec72p) . Productive interaction requires displacement of hsp70 from the precursor, a reaction that is facilitated by Ydj1p, a homologue of the Escherichia coli DnaJ protein . Both DnaJ and Ydj1p regulate chaperone activity by stimulating the ATPase activity of their respective hsp70 partners (E . coli DnaK and S . cerevisiae Ssa1p, respectively) . In the ER lumen, another hsp70 chaperone, BiP, binds ATP and interacts with the ER membrane via its contact with a peptide loop of Sec63p . This loop represents yet another DnaJ homologue in that it contains a region of approximately 70 residue similarity to the 'J box', the most conserved region of the DnaJ family of proteins . In the presence of ATP, under conditions in which BiP can bind to Sec63p, the secretory precursor passes from the cytosol into the lumen through a membrane channel formed by Sec61p . A second route to the membrane pore that is used by many other secretory precursors, particularly in mammalian cells, requires that the polypeptide engage the ER membrane as the nascent chain emerges from the ribosome . Such cotranslational translocation bypasses the need for certain Sec proteins, instead utilizing an alternate set of cytosolic and membrane factors that allows the nascent chain to be inserted directly into the Sec61p channel. Gene, 1996 Dec 12, 183(1-2), 183 - 90 Cloning of a cDNA encoding DNA topoisomerase I in Daucus carota and expression analysis in relation to cell proliferation; Balestrazzi A et al.; DNA topoisomerase I is an enzyme involved in several processes related to DNA metabolism . Despite the physiological importance, the regulation of top1 gene expression has not yet been investigated in plants . In order to monitor the possible correlation between levels of top1 transcripts and the proliferative state of the cell, two partially overlapping cDNAs encoding DNA topoisomerase I from Daucus carota have been isolated from a poly(A)(+)-primed library, using an Arabidopsis thaliana probe, and from a cDNA library spanning the 5' region of the top1 transcript, which was constructed using an antisense specific oligonucleotide . The top1 nucleotide sequence encoded an open reading frame of 2370 bp, predicting a protein of 90 kDa . The deduced amino acid sequence showed a similarity of 51% with A . thaliana, 41% with S . cerevisiae, 40% with S . pombe and 31% with H . sapiens, respectively . Southern blot analysis, performed under moderate stringency conditions, showed the presence of a single-copy gene . Evaluation of the top1 mRNA steady-state level revealed, besides a constitutive expression in vegetative carrot tissues, an induced expression related to cell proliferation. Gene, 1996 Dec 5, 182(1-2), 77 - 80 Reexamining interaction of the SH2 domains of SYP and GAP with insulin and IGF-1 receptors in the two-hybrid system; Lamothe B et al.; Direct interaction of effector proteins such as the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), SYP (SH2-domain-containing tyrosine phosphatase) and GAP (Ras-GTPase activating protein) with the insulin receptor (IR) and insulin-like growth factor-1 (IGF-1) type 1 receptor (IGF-1R) has been reported in some studies . Interaction of SYP and GAP with IR and IGF-1R was re-investigated here in the two-hybrid system by assessing his3/lacZ activation in S . cerevisiae . The experiments were performed with the cytoplasmic beta domain of IR and IGF-1R and various SH2-subdomains of SYP and GAP . None of the subdomains of SYP and GAP tested were able to activate his3/lacZ, whereas these reporter genes were strongly activated when p85 was used as we have recently shown . Thus, interaction of SYP and GAP with IR and IGF-1R, if any, would be weak and/or transient as compared to that of p85. EMBO J, 1996 Dec 2, 15(23), 6483 - 94 The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae; TerBush DR et al.; In the yeast Saccharomyces cerevisiae, the products of at least 15 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane . Previously, we have shown that three of these genes, SEC6, SEC8 and SEC15, encode components of a multisubunit complex which localizes to the tip of the bud, the predominant site of exocytosis in S . cerevisiae . Mutations in three more of these genes, SEC3, SEC5 and SEC10, were found to disrupt the subunit integrity of the Sec6-Sec8-Sec15 complex, indicating that these genes may encode some of the remaining components of this complex . To examine this possibility, we cloned and sequenced the SEC5 and SEC10 genes, disrupted them, and either epitope tagged them (Sec5p) or prepared polyclonal antisera (Sec10p) to them for co-immunoprecipitation studies . Concurrently, we biochemically purified the remaining unidentified polypeptides of the Sec6-Sec8-Sec15 complex for peptide microsequencing . The genes encoding these components were identified by comparison of predicted amino acid sequences with those obtained from peptide microsequencing of the purified complex components . In addition to Sec6p, Sec8p and Sec15p, the complex contains the proteins encoded by SEC3, SEC5, SEC10 and a novel gene, EXO70 . Since these seven proteins function together in a complex required for exocytosis, and not other intracellular trafficking steps, we have named it the Exocyst. Genetics, 1996 Dec, 144(4), 1425 - 36 Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast; James P et al.; The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae . However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system . These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives . We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S . cerevisiae . In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter . This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays . Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system . Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections. Genetics, 1996 Dec, 144(4), 1343 - 53 Evidence that the transcriptional regulators SIN3 and RPD3, and a novel gene (SDS3) with similar functions, are involved in transcriptional silencing in S . cerevisiae; Vannier D et al.; In a screen for extragenic suppressors of a silencing defective rap 1s hmr delta A strain, recessive mutations in 21 different genes were found that restored repression to HMR . We describe the characterization of three of these SDS (suppressors of defective silencing) genes . SDS16 and SDS6 are known transcriptional modifiers, SIN3(RPD1/UME4/SDI1/GAM2) and RPD3(SDI2), respectively, while the third is a novel gene, SDS3 . SDS3 shares the meiotic functions of SIN3 and RPD3 in that it represses IME2 in haploid cells and is necessary for sporulation in diploid cells . However, sds3 mutations differ from sin3 and rpd3 mutations in that they do not derepress TRK2 . These sds mutations suppress a variety of cis- and trans-defects, which impair the establishment of silencing at HMR . Any one of the sds mutations slightly increases telomere position effect while a striking synergistic increase in repression is observed in a rap 1s background . Epistasis studies suggest that SDS3 works in a different pathway from RPD3 and SIN3 to affect silencing at HMR . Together these results show that defects in certain general transcriptional modifiers can have a pronounced influence on position-effect gene silencing in yeast . Mechanisms for this increase in position effect are discussed. Nucleic Acids Res, 1996 Dec 1, 24(23), 4825 - 31 The Saccharomyces retrotransposon Ty5 influences the organization of chromosome ends; Zou S et al.; Retrotransposons are ubiquitous components of eukaryotic genomes suggesting that they have played a significant role in genome organization . In Saccharomyces cerevisiae, eight of 10 endogenous insertions of the Ty5 retrotransposon family are located within 15 kb of chromosome ends, and two are located near the subtelomeric HMR locus . This genomic organization is the consequence of targeted transposition, as 14 of 15 newly transposed Ty5 elements map to telomeric regions on 10 different chromosomes . Nine of these insertions are within 0.8 kb and three are within 1.5 kb of the autonomously replicating consensus sequence in the subtelomeric X repeat . This suggests that the X repeat plays an important role in directing Ty5 integration . Analysis of endogenous insertions from S.cerevisiae and its close relative S.paradoxus revealed that only one of 12 insertions has target site duplications, indicating that recombination occurs between elements . This is further supported by the observation that Ty5 insertions mark boundaries of sequence duplications and rearrangements in these species . These data suggest that transposable elements like Ty5 can shape the organization of chromosome ends through both transposition and recombination. Nucleic Acids Res, 1996 Dec 1, 24(23), 4791 - 7 Pat1: a topoisomerase II-associated protein required for faithful chromosome transmission in Saccharomyces cerevisiae; Wang X et al.; Saccharomyces cerevisiae top2 mutants deficient in topoisomerase II activity are defective in chromosome segregation during both mitotic and meiotic cell divisions . To identify proteins that act in concert with topoisomerase II during chromosome segregation in S.cerevisiae, we have used a two-hybrid cloning approach . We report the isolation of the PAT1 gene (for protein associated with topoisomerase II), which encodes a novel 90 kDa proline- and glutamine-rich protein that interacts with a highly conserved, leucine-rich region of topoisomerase II in vivo . Strains lacking Pat1p exhibit a slow growth rate and a phenotype reminiscent of conditional top2 mutants grown at the semi-permissive temperature; most notably, a reduced fidelity of chromosome segregation during both mitosis and meiosis . These findings indicate that the PAT1 gene is necessary for accurate chromosome transmission during cell division in eukaryotic cells and suggest that the interaction of Pat1p and topoisomerase II is an important component of this function. Mol Microbiol, 1996 Dec, 22(5), 859 - 66 Saccharomyces cerevisiae chorismate synthase has a flavin reductase activity; Henstrand JM et al.; Chorismate synthase (CS) catalyses the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to form chorismate, which is the last common intermediate in the synthesis of the three aromatic amino acids phenylalanine, tyrosine and tryptophan . Despite the overall redox-neutral reaction, catalysis has an absolute requirement for reduced flavin . In the fungus Neurospora crassa, a flavin reductase (FR) activity able to generate reduced flavin mononucleotide in the presence of NADPH is an intrinsic feature of a bifunctional CS . In all bacterial and plant species investigated to date, purified CSs lack an FR activity and are correspondingly 8-10 kDa smaller than the N . crassa CS (on the basis of SDS-PAGE) . The cloning of N . crassa CS and subsequent characterization of the purified heterologously expressed enzyme indicates that, surprisingly, the FR probably resides within a region conserved amongst both mono- and bifunctional CSs and is not related to non-homologous sequences which contribute to the larger molecular mass of the N . crassa CS . This information directed this work towards the smaller Saccharomyces cerevisiae CS, the sequence of which was known, although the protein has not been extensively characterized biochemically . Here the characterization of the S . cerevisiae CS is reported in more detail and it is shown that the protein is also bifunctional . With this knowledge, S . cerevisiae could be used as a genetic system for studying the physiological consequences of bifunctionality . The phylogenetic relationship amongst known CSs is discussed. Cancer Res, 1996 Dec 1, 56(23), 5457 - 65 Genotoxicity of aflatoxin B1: evidence for a recombination-mediated mechanism in Saccharomyces cerevisiae; Sengstag C et al.; The potent liver carcinogen aflatoxin B1 (AFB1) is metabolized by cytochrome P450 to the mutagenic epoxide . We have observed that activated AFB1 also strongly induced mitotic recombination in the yeast Saccharomyces cerevisiae . To compare the recombinogenicity of AFB1 to its mutagenicity, three metabolically competent S . cerevisiae strains have been constructed . The frequencies of induced recombinants resulting from gene conversion or chromosomal translocations were determined by different prototrophic selections using two strains, whereas the inducibility of forward mutations was determined by the frequency of drug resistance in the third strain . Human cytochrome P4501A1- (CYP1A) and NADPH-cytochrome P450-oxidoreductase cDNAs were expressed in the strains to ensure intracellular metabolism to the epoxide . Exposure of the strains to AFB1 resulted in a 139- and 24-fold increase in the translocation and gene conversion frequencies, respectively, whereas the mutation frequency was increased only 3-fold . In contrast, benzo{a}pyrene-7,8-dihydrodiol and ethyl methanesulfonate induced mutation and mitotic recombination to similar degrees . We conclude that AFB1 exerted a strong recombinogenic, but only a weak mutagenic, effect . The recombinogenicity of AFB1 in yeast may indicate a mechanism for the high proportion of loss of heterozygosity that has been detected in AFB1-related human liver cancers. Appl Environ Microbiol, 1996 Dec, 62(12), 4309 - 13 Polyunsaturated fatty acid biosynthesis in Saccharomyces cerevisiae: expression of ethanol tolerance and the FAD2 gene from Arabidopsis thaliana; Kajiwara S et al.; The Arabidopsis thaliana delta-12 fatty acid desaturase gene (FAD2) was overexpressed in Saccharomyces cerevisiae by using the GAL1 promoter . S . cerevisiae harboring the FAD2 gene was capable of forming hexadecadienoyl (16:2) and linoleoyl (18:2) residues in the membrane lipid when cultured in medium containing galactose . Gas-liquid chromatography analysis of total lipids indicated that the transformed S . cerevisiae accumulated these dienoic fatty acyl residues and that they accounted for approximately 50% of the total fatty acyl residues . Phospholipid analysis of this strain indicated that the oleoyl (18:1) residue binding phosphatidylcholine (PC) was mostly converted to the 18:2 residue binding PC, whereas 50% of the palmitoleoyl (16:1) residue binding PC was converted to the 16:2 residue binding PC . A marked effect on the unsaturation of 16:1 and 18:1 was observed when S . cerevisiae harboring the FAD2 gene was cultured at 8 degrees C . To assess the ethanol tolerance of S . cerevisiae producing polyunsaturated fatty acids, the cell viability of this strain in the presence of ethanol was examined . The results indicated that S . cerevisiae cells overexpressing the FAD2 gene had greater resistance to 15% (vol/vol) ethanol than did the control cells. Infect Immun, 1996 Dec, 64(12), 5269 - 73 Disruption of the SNF1 gene abolishes trehalose utilization in the pathogenic yeast Candida glabrata; Petter R et al.; The SNF1 gene product, a serine/threonine protein kinase, is a global regulatory protein which has been isolated from several organisms . In Saccharomyces cerevisiae the SNF1 gene product is essential for the derepression of glucose repression since snf1 strains are unable to utilize sucrose, galactose, maltose, melibiose, or nonfermentable carbohydrates . Moreover, the SNF1 gene product was suggested to interact with additional regulatory pathways and to affect the expression of multiple target genes as reflected by the pleiotropic nature of the snf1 mutation . Here we report the characterization of the SNF1 homolog of Candida glabrata, a pathogenic yeast phylogenetically related to S . cerevisiae . The carbon utilization spectrum of C . glabrata is considerably narrower than that of other pathogenic yeasts, and the majority of the strains utilize solely glucose and trehalose from among 20 of the most commonly tested carbohydrates . Disruption of the C . glabrata SNF1 homolog resulted in the loss of the ability to utilize trehalose, indicating that even in an organism with such a limited carbon utilization spectrum, the regulatory mechanism governing catabolic repression is preserved. Mol Cell Biol, 1996 Dec, 16(12), 7144 - 50 A role for nucleoporin FG repeat domains in export of human immunodeficiency virus type 1 Rev protein and RNA from the nucleus; Stutz F et al.; The human immunodeficiency virus type 1 Rev protein contains a nuclear export signal (NES) that is required for Rev-mediated RNA export in mammals as well as in the yeast Saccharomyces cerevisiae . The Rev NES has been shown to specifically interact with a human (hRIP/RAB1) and a yeast (yRip1p) protein in the two-hybrid assay . Both of these interacting proteins are related to FG nucleoporins on the basis of the presence of typical repeat motifs . This paper shows that Rev is able to interact with multiple FG repeat-containing nucleoporins from both S . cerevisiae and mammals; moreover, the ability of Rev NES mutants to interact with these FG nucleoporins parallels the ability of the mutants to promote RNA export in yeast and mammalian cells . The data also show that, after Xenopus oocyte nuclear injection, several FG nucleoporin repeat domains inhibit the export of both Rev protein and U small nuclear RNAs, suggesting that these nucleoporins participate in Rev-mediated and cellular RNA export . Interestingly, not all FG nucleoporin repeat domains produced the same pattern of RNA export inhibition . The results suggest that Rev and cellular mediators of RNA export can interact with multiple components of the nuclear pore complex during transport, analogous to the proposed mode of action of the nuclear protein import receptor. Mol Cell Biol, 1996 Dec, 16(12), 6810 - 9 Spliceosome activation by PRP2 ATPase prior to the first transesterification reaction of pre-mRNA splicing; Kim SH et al.; In addition to small nuclear RNAs and spliceosomal proteins, ATP hydrolysis is needed for nuclear pre-mRNA splicing . A number of RNA-dependent ATPases which are involved in several distinct ATP-dependent steps in splicing have been identified in Saccharomyces cerevisiae and mammals . These so-called DEAD/H ATPases contain conserved RNA helicase motifs, although RNA unwinding activity has not been demonstrated in purified proteins . Here we report the role of one such DEAH protein, PRP2 of S . cerevisiae, in spliceosome activation . PRP2 bound to a precatalytic spliceosome prior to the first step of splicing . By blocking the activity of a novel splicing factor(s), HP, which was involved in a post-PRP2 step, we found that PRP2 hydrolyzed ATP to cause a change in the spliceosome without the occurrence of splicing . The change was quite dramatic and could account for the previously reported differences between the precatalytic, pre-mRNA-containing spliceosome and the "active," intermediate-containing spliceosome . The post-PRP2-ATP spliceosome was further isolated and could carry out the subsequent reaction apparently in the absence of PRP2 and ATP . We hypothesize that PRP2 functions as a molecular motor, similar to some DExH ATPases in transcription, in the activation of the precatalytic spliceosome for the transesterification reaction. Mol Cell Biol, 1996 Dec, 16(12), 6668 - 76 The NOT, SPT3, and MOT1 genes functionally interact to regulate transcription at core promoters; Collart MA; Previous studies demonstrated that mutations in the Saccharomyces cerevisiae NOT genes increase transcription from TATA-less promoters . In this report, I show that in contrast, mutations in the yeast MOT1 gene decrease transcription from TATA-less promoters . I also demonstrate specific genetic interactions between the Not complex, Mot1p, and another global regulator of transcription in S . cerevisiae, Spt3p . Five distinct genetic interactions have been established . First, a null allele of SPT3, or a mutation in SPT15 that disrupts the interaction between Spt3p and TATA-binding protein (TBP), allele specifically suppressed the not1-2 mutation . Second, in contrast to not mutations, mutations in MOT1 decreased HIS3 and HIS4 TATA-less transcription . Third, not mutations suppressed toxicity due to overexpression of TBP in mot1-1 mutants . Finally, overexpression of SPT3 caused a weak Not- mutant phenotype in mot1-1 mutants . Collectively, these results suggest a novel type of transcriptional regulation whereby the distribution of limiting TBP (TFIID) on weak and strong TBP-binding core promoters is regulated: Mot1p releases stably bound TBP to allow its redistribution to low-affinity sites, and the Not proteins negatively regulate the activity of factors such as Spt3p that favor distribution of TBP to these low-affinity sites. J Clin Microbiol, 1996 Dec, 34(12), 3031 - 4 In vitro susceptibility testing and DNA typing of Saccharomyces cerevisiae clinical isolates; Zerva L et al.; Saccharomyces spp . are widely distributed in nature and may colonize the normal human gastrointestinal tract . Although Saccharomyces cerevisiae isolates have been previously considered nonpathogenic, they appear to be increasingly associated with infections in immunocompromised or otherwise debilitated patients . The antifungal susceptibility and epidemiology of S . cerevisiae are poorly defined at present . A series of 76 isolates (mostly stool surveillance and throat swab isolates) from 70 bone marrow transplant patients hospitalized at two different medical centers were characterized by antifungal susceptibility testing and restriction endonuclease analysis of chromosomal DNA . For DNA typing, digestion with NotI followed by pulsed-field gel electrophoresis was applied . Typing results revealed 62 distinct DNA types among the 76 clinical isolates . Despite this genomic diversity, clusters of identical isolates were identified among different patients hospitalized concurrently in the same unit, indicating possible nosocomial transmission . The MICs of amphotericin B, 5-fluorocytosine, fluconazole, and itraconazole were determined by a broth microdilution method, as recommended by the National Committee for Clinical Laboratory Standards . The MICs at which 90% of the strains were inhibited were as follows: amphotericin B, 1.0 micrograms/ml; 5-fluorocytosine, 0.25 micrograms/ml; fluconazole, 8.0 micrograms/ml; and itraconazole, 1.0 micrograms/ml . The relative resistance of S . cerevisiae to fluconazole and itraconazole may promote the emergence of this species as a pathogen among immunosuppressed patients. Mol Gen Genet, 1996 Nov 27, 253(1-2), 149 - 56 Genetic interaction of DED1 encoding a putative ATP-dependent RNA helicase with SRM1 encoding a mammalian RCC1 homolog in Saccharomyces cerevisiae; Hayashi N et al.; The Saccharomyces cerevisiae temperature-sensitive mutants srm1-1, mtr1-2 and prp20-1 carry alleles of a gene encoding a homolog of mammalian RCC1 . In order to identify a protein interacting with RCC1, a series of suppressors of the srm1-1 mutation were isolated as cold-sensitive mutants and one of the mutants, designated ded1-21, was found to be defective in the DED1 gene . The double mutant, srm1-1 ded1-21, could grow at 35 degrees C, but not at 37 degrees C . A revertant of srm1-1 ded1-21 that became able to grow at 37 degrees C acquired another mutation in the SRM1 gene, indicating the tight relationship between SRM1 and DED1 . In all the rcc1- strains examined, the amount of mutated SRM1 proteins was reduced or not detectable at the nonpermissive temperature . While mutated SRM1 protein was stabilized in all of the rcc1- strains by the ded1-21 mutation, the ded1-21 mutation suppressed both srm1-1 and mtr1-2, but not the prp20-1 mutation, contrary to the previous finding that overproduction of the S . cerevisiae Ran homolog GSP1 suppresses prp20-1, but not srm1-1 or mtr1-2. Mol Gen Genet, 1996 Nov 27, 253(1-2), 111 - 7 Superfamily of alpha-galactosidase MEL genes of the Saccharomyces sensu stricto species complex; Naumova ES et al.; In order to study the molecular evolution of the yeasts grouped in the Saccharomyces sensu stricto species complex by analysis of the MEL gene family, we have cloned and sequenced two new species-specific MEL genes from Saccharomyces yeasts: S . paradoxus (MELp) and a Japanese Saccharomyces sp . (MELj) . The clones were identified by sequence homology to the S . cerevisiae MEL1 gene . Both clones revealed an ORF of 1413 bp coding for a protein of 471 amino acids . The deduced molecular weights of the alpha-galactosidase enzymes were 52,767 for MELp and 52,378 for MELj . The nucleotide sequences of the MELp (EMBL accession no . X95505) and the MELj (EMBL accession no . X95506) genes showed 74.7% identity . The degree of identity of MELp to the MEL1 gene was 76.8% and to the S . pastorianus MELx gene, 75.7% . The MELj coding sequence was 75.1% identical to the MEL1 gene and 80.7% to the MELx gene . The data suggest that MEL1, MELj, MELp, and MELx genes are species-specific MEL genes . The strains studied each have only one MEL locus . The MELp gene is located on the S . paradoxus equivalent of S . cerevisiae chromosome X; the MELj gene was on the chromosome that comigrates with the S . cerevisiae chromosome VII/XV doublet and hybridizes to the S . cerevisiae chromosome XV marker HIS3. J Biol Chem, 1996 Nov 22, 271(47), 29958 - 68 Identification and characterization of the CLK1 gene product, a novel CaM kinase-like protein kinase from the yeast Saccharomyces cerevisiae; Melcher ML et al.; The CLK1 gene of Saccharomyces cerevisiae encodes a 610-residue protein kinase that resembles known type II Ca2+/calmodulin-dependent protein kinases (CaM kinases), including the CMK1 and CMK2 gene products from the same yeast . The Clk1 kinase domain is preceded by a 162-residue N-terminal extension, followed by a 132-residue C-terminal extension (which contains a basic segment resembling known calmodulin-binding sites) and is as similar to mammalian CaM kinase (38% identity to rat CaM kinase alpha) as it is to yeast CaM kinase (37% identity to Cmk2) . However, Clk1 shares 52% identity with Rck1, another putative protein kinase encoded in the S . cerevisiae genome . Clk1 tagged with a c-myc epitope (expressed in yeast) and a GST-Clk1 fusion (expressed in bacteria) underwent autophosphorylation and phosphorylated an exogenous substrate (yeast protein synthesis elongation factor 2), primarily on Ser . Neither Clk1 activity was stimulated by purified yeast calmodulin (CMD1 gene product), with or without Ca2+; no association of Clk1 with Cmd1 was detectable by other methods . C-terminally truncated Clk1(Delta487-610) was growth-inhibitory when overexpressed, whereas catalytically inactive Clk1(K201R Delta487-610) was not, suggesting that the C terminus is a negative regulatory domain . Using immunofluorescence, Clk1 was localized to the cytosol and excluded from the nucleus . A clk1Delta mutant, a clk1Delta rck1Delta double mutant, a clk1Delta cmk1Delta cmk2Delta triple mutant, and a clk1Delta rck1Delta cmk1Delta cmk2Delta quadruple mutant were all viable and manifested no other overt growth phenotype. Biochem J, 1996 Nov 15, 320 ( Pt 1), 61 - 7 Importance of catalase in the adaptive response to hydrogen peroxide: analysis of acatalasaemic Saccharomyces cerevisiae; Izawa S et al.; Controversy about the importance of catalase in the detoxification of H2O2 in human erythrocytes continues . It has been suggested that catalase has no role in the clearance of H2O2 in erythrocytes . In the present study we investigated the role of catalase in the defence mechanism against oxidative stress using Saccharomyces cerevisiae . S . cerevisiae has two catalases, catalase A and catalase T . We constructed a double mutant (acatalasaemic mutant) unable to produce either catalase A or catalase T, and compared it with wild-type and single-mutant cells . The acatalasaemic mutant cells showed a similar growth rate to wild-type cells under non-oxidative stress conditions, and showed a similar susceptibility to H2O2 stress in the exponential growth phase . The acatalasaemic mutant cells at stationary phase were, however, much more sensitive to H2O2 stress than wild-type and single-mutant cells . Moreover, the ability of acatalasaemic and single-mutant cells to show adaptation to 2 mM H2O2 was distinctly inferior to that of wild-type cells . These results suggest that catalase is not essential for yeast cells under normal conditions, but plays an important role in the acquisition of tolerance to oxidative stress in the adaptive response of these cells. EMBO J, 1996 Nov 15, 15(22), 6060 - 8 Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae; Kohno H et al.; The RHO1 gene encodes a homolog of mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae . Rho1p is localized at the growth sites, including the bud tip and the cytokinesis site, and is required for bud formation . We have recently shown that Pkc1p, a yeast homolog of mammalian protein kinase C, and glucan synthase are targets of Rho1p . Using the two-hybrid screening system, we cloned a gene encoding a protein which interacted with the GTP-bound form of Rho1p . This gene was identified as BNI1, known to be implicated in cytokinesis or establishment of cell polarity in S.cerevisiae . Bni1p shares homologous domains (FH1 and FH2 domains) with proteins involved in cytokinesis or establishment of cell polarity, including formin of mouse, capu and dia of Drosophila and FigA of Aspergillus . A temperature-sensitive mutation in which the RHO1 gene was replaced by the mammalian RhoA gene showed a synthetically lethal interaction with the bni1 mutation and the RhoA bni1 mutant accumulated cells with a deficiency in cytokinesis . Furthermore, this synthetic lethality was caused by the incapability of RhoA to activate Pkc1p, but not glucan synthase . These results suggest that Rho1p regulates cytoskeletal reorganization at least through Bni1p and Pkc1p. J Biol Chem, 1996 Nov 15, 271(46), 28777 - 83 Phosphorylation and regulation of CTP synthetase from Saccharomyces cerevisiae by protein kinase A; Yang WL et al.; The phosphorylation and regulation of the URA7-encoded CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) from Saccharomyces cerevisiae by cAMP-dependent protein kinase (protein kinase A) were examined . Protein kinase A is the principal mediator of signals transmitted through the RAS/cAMP pathway in S . cerevisiae . The results of labeling experiments indicated that the phosphorylation of CTP synthetase was mediated by the RAS/cAMP pathway in vivo . In vitro, protein kinase A phosphorylated CTP synthetase at a serine residue with a stoichiometry consistent with one phosphorylation site per CTP synthetase subunit . Protein kinase A activity was dose- and time-dependent using CTP synthetase as a substrate . The dependence of protein kinase A activity on CTP synthetase was cooperative (n = 1.8) and the Km value for CTP synthetase was 73 nM . Phosphorylation of CTP synthetase with protein kinase A resulted in the stimulation (190%) of activity . The mechanism of this stimulation included an increase in the Vmax of the reaction with respect to UTP and ATP, a decrease in the Km for ATP, and a decrease in the cooperative kinetic behavior of the enzyme . Phosphorylated CTP synthetase was less sensitive to product inhibition by CTP . Protein kinase C also phosphorylates and activates CTP synthetase . Phosphorylation of CTP synthetase with protein kinases A and C together resulted in an increase in CTP synthetase activity that was slightly greater than that obtained when the enzyme was phosphorylated with either protein kinase alone. Gene, 1996 Nov 14, 179(2), 291 - 3 The Caenorhabditis elegans gene CeAPN1 encodes a homolog of Escherichia coli and yeast apurinic/apyrimidinic endonuclease; Masson JY et al.; The Saccharomyces cerevisiae APN1 gene, encoding the bifunctional DNA repair enzyme apurinic/apyrimidinic (AP) endonuclease/3'-repair diesterase, was used as a probe to isolate a gene homolog, CeAPN1, from a Caenorhabditis elegans cDNA library . The CeAPN1 gene is predicted to encode a protein 30 kDa in size, which shares 40.4% and 44.9% identity at the amino acid level with, respectively, S . cerevisiae Apn1 and Escherichia coli endonuclease IV . We suggest that CeApn1 protein is a member of the endonuclease IV family of DNA repair enzymes. Biochemistry, 1996 Nov 12, 35(45), 14054 - 61 Kinetics and thermodynamics of phalloidin binding to actin filaments from three divergent species; De La Cruz EM et al.; We compared the kinetics and thermodynamics of rhodamine phalloidin binding to actin purified from rabbit skeletal muscle, Acanthamoeba castellanii, and Saccharomyces cerevisiae in 50 mM KCl, 1 mM MgCl2, and pH 7.0 buffer at 22 degrees C . Filaments of S . cerevisiae actin bind rhodamine phalloidin more weakly than Acanthamoeba and rabbit skeletal muscle actin filaments due to a more rapid dissociation rate in spite of a significantly faster association rate constant . The higher dissociation rate constant and lower binding affinity of rhodamine phalloidin for S . cerevisiae actin filaments provide a quantitative explanation for the inefficient staining of yeast actin filaments, compared with that of rabbit skeletal muscle actin filaments {Kron et al . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 4466-4470} . The temperature dependence of the rate constants was interpreted according to transition state theory . There is a small enthalpic difference (delta H++) between the ground states and the transition state . Consequently, the free energy of activation (delta G++) for association and dissociation of rhodamine phalloidin is dominated by entropic changes (delta S++) . At equilibrium, rhodamine phalloidin binding generates a positive entropy change (delta S0) . The rates of rhodamine phalloidin binding are independent of the pH, ionic strength, and filament length . Rhodamine covalently bound decreases the association rate and affinity of phalloidin for actin . The association rate constant is low for both phalloidin and rhodamine phalloidin because the filaments must undergo conformational changes (i.e . "breathe") to expose the phalloidin binding site {De La Cruz, E . M., & Pollard, T . D . (1994) Biochemistry 33, 14387-14392} . Raising the solvent microviscosity, but not the macroviscosity, dampens these conformational fluctuations, and phalloidin binding kinetics are inhibited . Yeast actin filaments bind rhodamine phalloidin more rapidly, suggesting that perhaps they are more flexible and can breathe more easily than rabbit or Acanthamoeba actin filaments. Mutat Res, 1996 Nov 11, 372(1), 133 - 9 Anti-recombinogenic and convertible co-mutagenic effects of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and other 5-substituted pyrimidine nucleoside analogs in S . cerevisiae MP1; Fahrig R; In experiments using yeast, without addition of an external metabolic activation system, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was co-mutagenic and showed an insignificant anti-recombinogenic effect in combination with triethylene melamine (TEM) . In the presence of activating S9-mix, the anti-recombinogenicity and co-mutagenicity could clearly be seen . At higher concentrations the co-mutagenic effect was converted into anti-mutagenicity . The other three 5-substituted pyrimidine nucleoside analogs were tested only in the presence of activating S9-mix and showed similar effects . As TEM is a direct alkylating agent that is inactivated by liver microsomes, the higher activity in presence of S9-mix can be interpreted as resulting from metabolic activation of the 5-substituted pyrimidine nucleoside analogs . In previous experiments using yeast bacteria, Drosophila or mice, tumor promoters were co-recombinogenic/anti-mutagenic, and co-carcinogens were co-mutagenic/anti-recombinogenic . Thus, there is not only an operational difference between tumor promoters and co-carcinogens but a real difference in respect to their genetic effectiveness . As up to now only co-carcinogens have shown co-mutagenic and anti-recombinogenic effects, it is perhaps possible that, within a certain concentration range, 5-substituted pyrimidine nucleoside analogs may have co-carcinogenic activity in carcinogenicity tests . At higher concentrations the co-carcinogenic effect may be converted into an anti-carcinogenic one. J Biol Chem, 1996 Nov 8, 271(45), 28521 - 32 A mouse amidase specific for N-terminal asparagine . The gene, the enzyme, and their function in the N-end rule pathway; Grigoryev S et al.; The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue . In both fungi and mammals, the tertiary destabilizing N-terminal residues asparagine and glutamine function through their conversion, by enzymatic deamidation, into the secondary destabilizing residues aspartate and glutamate, whose destabilizing activity requires their enzymatic conjugation to arginine, one of the primary destabilizing residues . We report the isolation and analysis of a mouse cDNA and the corresponding gene (termed Ntan1) that encode a 310-residue amidohydrolase (termed NtN-amidase) specific for N-terminal asparagine . The approximately 17-kilobase pair Ntan1 gene is located in the proximal region of mouse chromosome 16 and contains 10 exons ranging from 54 to 177 base pairs in length . The approximately 1.4-kilobase pair Ntan1 mRNA is expressed in all of the tested mouse tissues and cell lines and is down-regulated upon the conversion of myoblasts into myotubes . The Ntan1 promoter is located approximately 500 base pairs upstream of the Ntan1 start codon . The deduced amino acid sequence of mouse NtN-amidase is 88% identical to the sequence of its porcine counterpart, but bears no significant similarity to the sequence of the NTA1-encoded N-terminal amidohydrolase of the yeast Saccharomyces cerevisiae, which can deamidate either N-terminal asparagine or glutamine . The expression of mouse NtN-amidase in S . cerevisiae nta1Delta was used to verify that NtN-amidase retains its asparagine selectivity in vivo and can implement the asparagine-specific subset of the N-end rule . Further dissection of mouse Ntan1, including its null phenotype analysis, should illuminate the functions of the N-end rule, most of which are still unknown. J Biol Chem, 1996 Nov 8, 271(45), 28243 - 9 Assessing the requirements for nucleotide excision repair proteins of Saccharomyces cerevisiae in an in vitro system; He Z et al.; Nucleotide excision repair (NER) is the primary mechanism by which both Saccharomyces cerevisiae and human cells remove the DNA lesions caused by ultraviolet light and other mutagens . This complex process involves the coordinated actions of more than 20 polypeptides . To facilitate biochemical studies of NER in yeast, we have established a simple protocol for preparing whole cell extracts which perform NER in vitro . As expected, this assay of in vitro repair was dependent on the products of RAD genes such as RAD14, RAD4, and RAD2 . Interestingly, it was also dependent upon proteins encoded by the RAD7, RAD16, and RAD23 genes whose precise roles in NER are uncertain, but not the RAD26 gene whose product is believed to participate in coupling NER to transcription . Replication protein A (RPA/Rpa), known to be required for NER in human cell extracts, was also shown by antibody inhibition and immunodepletion experiments to be required for NER in our yeast cell extracts . Moreover, yeast cells with temperature-sensitive mutations in the RFA2 gene, which encodes the 34-kDa subunit of Rpa, had increased sensitivity to UV and yielded extracts defective in NER in vitro . These data indicate that Rpa is an essential component of the NER machinery in S . cerevisiae as it is in mammalian cells. J Biochem (Tokyo), 1996 Nov, 120(5), 1040 - 7 Isolation and characterization of ECT1 gene encoding CTP: phosphoethanolamine cytidylyltransferase of Saccharomyces cerevisiae; Min-Seok R et al.; Saccharomyces cerevisiae mutants that were unable to utilize extracellular ethanolamine for phosphatidylethanolamine synthesis were isolated . Two of them carried recessive chromosomal mutations in a same gene and were defective in CTP:phosphoethanolamine cytidylyltransferase (ECT) activity in vitro (Ect-) . In an Ect- mutant that also carried the cho1 mutation, phosphatidylethanolamine accounted for less than 2% of total phospholipids, suggesting the importance of ECT in phosphatidylethanolamine synthesis . By screening a genomic library on a low copy number vector, three complementary clones of different size were isolated . A 2.8-kb common DNA region carried an open reading frame (ORF) of 969 bp in length, of which a truncated from failed to complement the Ect- mutation . This ORF was identical to the previously isolated MUQ1 gene of unknown function . Its deduced amino acid sequence had significant similarity to CTP: phosphocholine cytidylyl-transferases of yeast and rat . The entire ORF, when combined with the glutathione S-transferase gene and expressed in Escherichia coli, exhibited ECT activity . These results indicate that the cloned gene encodes a catalytic subunit of ECT of S . cerevisiae. Curr Biol, 1996 Nov 1, 6(11), 1416 - 25 The Xenopus origin recognition complex is essential for DNA replication and MCM binding to chromatin; Romanowski P et al.; BACKGROUND: The origin recognition complex (ORC) and the minichromosome maintenance (MCM) protein complex were initially discovered in yeast and shown to be essential for DNA replication . Homologues of ORC and MCM proteins exist in higher eukaryotes, including Xenopus . The Xenopus MCM proteins and the Xenopus homologues of Saccharomyces cerevisiae Orc 1p and Orc2p (XOrc1 and XOrc2) have recently been shown to be essential for DNA replication . Here, we describe the different but interdependent functions of the ORC and MCM complexes in DNA replication in Xenopus egg extracts . RESULTS: The XOrc1 and XOrc2 proteins are present in the same multiprotein complex in Xenopus egg extracts . Immunodepletion of ORC inhibits DNA replication of Xenopus sperm nuclei . Mixing MCM-depleted and ORC-depleted extracts restores replication capacity . ORC does not co-localize with sites of DNA replication during elongation . However, at initiation the two staining patterns overlap . In contrast to MCMs, which are displaced from chromatin during S phase, XOrc1 and XOrc2 are nuclear chromatin-bound proteins throughout interphase and move to the cytoplasm in mitosis . Permeable HeLa G1- and G2-phase nuclei can replicate in ORC-depleted extract, consistent with the presence of chromatin-bound ORC in both pre-replicative and post-replicative nuclei . Interestingly, the binding of ORC to chromatin does not require the presence of MCMs; however, the binding of MCM proteins to chromatin is dependent on the presence of ORC . CONCLUSIONS: The Xenopus ORC and the MCM protein complex perform essential, non-redundant functions in DNA replication . Xenopus ORC is bound to chromatin throughout interphase but, in contrast to S . cerevisiae ORC, it appears to be, at least partly, displaced from chromatin during mitosis . The binding of MCM proteins requires the presence of ORC . Thus, the assembly of replication-competent chromatin involves the sequential binding of ORC and MCMs to DNA. Protein Sci, 1996 Nov, 5(11), 2353 - 7 Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3-kinases: binding partners of SH3 domains? Ponting CP. Two SH3 domain-containing cytosolic components of the NADPH oxidase, p47phox and p40phox, are shown by analyses of their sequences to contain single copies of a novel class of domain, the PX (phox) domain . Homologous domains are demonstrated to be present in the Cpk class of phosphatidylinositol 3-kinase, S . cerevisiae Bem1p, and S . pombe Scd2, and a large family of human sorting nexin 1 (SNX1) homologues . The majority of these domains contains a polyproline motif, typical of SH3 domain-binding proteins . Two further findings are reported . A third NADPH oxidase subunit, p67phox, is shown to contain four tetratricopeptide repeats (TPRs) within its N-terminal RaclGTP-binding region, and a 28 residue motif in p40phox is demonstrated to be present in protein kinase C isoforms iota/lambda and zeta, and in three ZZ domain-containing proteins. Arch Microbiol, 1996 Nov, 166(5), 283 - 92 The hexose transporter family of Saccharomyces cerevisiae; Kruckeberg AL; Saccharomyces cerevisiae accomplishes high rates of hexose transport . The kinetics of hexose transport are complex . The capacity and kinetic complexity of hexose transport in yeast are reflected in the large number of sugar transporter genes in the genome . Twenty hexose transporter genes exist in S . cerevisiae . Some of these have been found by genetic means; many have been discovered by the comprehensive sequencing of the yeast genome . This review codifies the nomenclature of the hexose transporter genes and describes the sequence homology and structural similarity of the proteins they encode . Information about the expression and function of the transporters is presented . Access to the sequences of the genes and proteins at three sequence databases is provided via the World Wide Web. Genetics, 1996 Nov, 144(3), 935 - 45 SGS1, a homologue of the Bloom's and Werner's syndrome genes, is required for maintenance of genome stability in Saccharomyces cerevisiae; Watt PM et al.; The Saccharomyces cerevisiae SGS1 gene is homologous to Escherichia coli RecQ and the human BLM and WRN proteins that are defective in the cancer-prone disorder Bloom's syndrome and the premature aging disorder Werner's syndrome, respectively . While recQ mutants are deficient in conjugational recombination and DNA repair, Bloom's syndrome cell lines show hyperrecombination . Bloom's and Werner's syndrome cell lines both exhibit chromosomal instability, sgs1 delta strains show mitotic hyperrecombination, as do Bloom's cells . This was manifested as an increase in the frequency of interchromosomal homologous recombination, intrachromosomal excision recombination, and ectopic recombination . Hyperrecombination was partially independent of both RAD52 and RAD1 . Meiotic recombination was not increased in sgs1 delta mutants, although meiosis I chromosome missegregation has been shown to be elevated sgs1 delta suppresses the slow growth of a top3 delta strain lacking topoisomerase III . Although there was an increase in subtelomeric Y' instability in sgs1 delta strains due to hyperrecombination, no evidence was found for an increase in the instability of terminal telomeric sequences in a top3 delta or a sgs1 delta strain . This contrasts with the telomere maintenance defects of Werner's patients . We conclude that the SGS1 gene product is involved in the maintenance of genome stability in S . cerevisiae. J Biol Chem, 1996 Nov 1, 271(44), 27829 - 37 Gpi1, a Saccharomyces cerevisiae protein that participates in the first step in glycosylphosphatidylinositol anchor synthesis; Leidich SD et al.; The temperature-sensitive Saccharomyces cerevisiae gpi1 mutant is blocked in {3H}inositol incorporation into protein and defective in the synthesis of N-acetylglucosaminylphosphatidylinositol, the first step in glycosylphosphatidylinositol (GPI) anchor assembly (Leidich, S . D., Drapp, D . A., and Orlean, P . (1994) J . Biol . Chem . 269, 10193-10196) . The GPI1 gene, which encodes a 609-amino acid membrane protein, was cloned by complementation of the temperature sensitivity of gpi1 and corrects the mutant's {3H}inositol labeling and enzymatic defects . Disruption of GPI1 yields viable haploid cells that are temperature-sensitive for growth, for {3H}inositol incorporation into protein, and for GPI anchor-dependent processing of the Gas1/Ggp1 protein and that lack in vitro N-acetylglucosaminylphosphatidylinositol synthetic activity . The Gpi1 protein thus participates in GPI synthesis and is required for growth at 37 degrees C . When grown at a semipermissive temperature of 30 degrees C, gpi1 cells and gpi1::URA3 disruptants form large, round, multiply budded cells with a separation defect . Homozygous gpi1/gpi1, gpi1::URA3/gpi1::URA3, gpi2/gpi2, and gpi3/gpi3 diploids undergo meiosis, but are defective in ascospore wall maturation for they fail to give the fluorescence due to the dityrosine-containing layer in the ascospore wall . These findings indicate that GPIs have key roles in the morphogenesis and development of S . cerevisiae. J Cell Biol, 1996 Nov, 135(3), 585 - 95 Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro; Kuehn MJ et al.; In S . cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane . We examined the requirements of transport of the permeases from the ER to the Golgi in vitro . Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease . Gap1p, and the histidine permease, Hip1p . Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles . In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p . In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion . Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles. J Virol, 1996 Nov, 70(11), 7478 - 84 The oncoprotein Tax of the human T-cell leukemia virus type 1 activates transcription via interaction with cellular ATF-1/CREB factors in Saccharomyces cerevisiae; Shnyreva M et al.; The transcription factor Tax of the oncogenic human T-cell leukemia virus type 1 is likely to be responsible for viral replication in the host organism and for the induction of proliferation in infected cells . To investigate Tax-mediated transcription in vivo, we expressed Tax as well as CREB in Saccharomyces cerevisiae . The activity of these proteins was monitored by expression of a beta-galactosidase reporter gene, which was fused to two viral 21-bp repeats located upstream of the yeast cytochrome c1 oxidase minimal promoter . Coexpression of Tax and CREB in S . cerevisiae led to a 20-fold increase in beta-galactosidase activity in comparison with that in strains expressing either Tax or CREB alone . By screening a human cDNA library, we were able to demonstrate that the Tax transactivation assay using S . cerevisiae can be successfully applied to identify other cellular proteins forming ternary complexes with Tax and 21-bp repeats in vivo . Upon transformation in S . cerevisiae, 1 of 13,500 clones tested positive . Sequencing of the cDNA insert of the rescued plasmid revealed that this DNA encoded the ATF-1 protein . beta-Galactosidase induction was comparable to that of the Tax/CREB coexpression system . This indicates that Tax-mediated transcription is critically dependent on the presence of cellular CREB or ATF-1 in vivo . Stimulation of transcription initiation required an unmasked NH2 terminus of Tax . Fusion of Tax to the yeast Gal4 protein abolished the transactivation potential of Tax . Reconstitution of the transcriptional properties of viral Tax together with the cellular proteins of the ATF-1/CREB family in S . cerevisiae allows the functional characterization of these proteins in vivo. Mol Cell Biol, 1996 Nov, 16(11), 6444 - 56 SSI1 encodes a novel Hsp70 of the Saccharomyces cerevisiae endoplasmic reticulum; Baxter BK et al.; The endoplasmic reticulum (ER) of the budding yeast Saccharomyces cerevisiae contains a well-characterized, essential member of the Hsp70 family of molecular chaperones, Kar2p . Kar2p has been shown to be involved in the translocation of proteins into the ER as well as the proper folding of proteins in that compartment . We report the characterization of a novel Hsp70 of the ER, Ssi1p . Ssi1p, which shares 24% of the amino acids of Kar2p, is not essential for growth under normal conditions . However, deletion of SSI1 results in cold sensitivity as well as enhanced resistance to manganese . The localization of Ssi1p to the ER, suggested by the presence of a conserved S . cerevisiae ER retention signal at its C terminus, was confirmed by subcellular fractionation, protease protection assays, and immunofluorescence . The SSI1 promoter contains an element with similarity to the unfolded protein response element of KAR2 . Like KAR2, SSI1 is induced both in the presence of tunicamycin and in a kar2-159 mutant strain, conditions which lead to an accumulation of unfolded proteins in the ER . Unlike KAR2, however, SSI1 is not induced by heat shock . Deletion of SSI1 shows a complex pattern of genetic interactions with various conditional alleles of KAR2, ranging from synthetic lethality to synthetic rescue . Interestingly, SSI1 deletion strains show a partial block in translocation of multiple proteins into the ER, suggesting that Ssi1p plays a direct role in the translocation process. Mol Cell Biol, 1996 Nov, 16(11), 6325 - 37 The mating-specific G(alpha) protein of Saccharomyces cerevisiae downregulates the mating signal by a mechanism that is dependent on pheromone and independent of G(beta)(gamma) sequestration; Stratton HF et al.; It has been inferred from compelling genetic evidence that the pheromone-responsive G(alpha) protein of Saccharomyces cerevisiae, Gpa1, directly inhibits the mating signal by binding to its own beta(gamma) subunit . Gpa1 has also been implicated in a distinct but as yet uncharacterized negative regulatory mechanism . We have used three mutant alleles of GPA1, each of which confers resistance to otherwise lethal doses of pheromone, to explore this possibility . Our results indicate that although the G322E allele of GPA1 completely blocks the pheromone response, the E364K allele promotes recovery from pheromone treatment rather than insensitivity to it . This observation suggests that Gpa1, like other G(alpha) proteins, interacts with an effector molecule and stimulates a positive signal--in this case, an adaptive signal . Moreover, the Gpa1-mediated adaptive signal is itself induced by pheromone, is delayed relative to the mating signal, and does not involve sequestration of G(beta)(gamma) . The behavior of N388D, a mutant form of Gpa1 predicted to be activated, strongly supports these conclusions . Although N388D cannot sequester beta(gamma), as evidenced by two-hybrid analysis and its inability to complement a Gpa1 null allele under normal growth conditions, it can stimulate adaptation and rescue a gpa1(delta) strain when cells are exposed to pheromone . Considered as a whole, our data suggest that the pheromone-responsive heterotrimeric G protein of S . cerevisiae has a self-regulatory signaling function . Upon activation, the heterotrimer dissociates into its two subunits, one of which stimulates the pheromone response, while the other slowly induces a negative regulatory mechanism that ultimately shuts off the mating signal downstream of the receptor. Mol Cell Biol, 1996 Nov, 16(11), 6303 - 12 Suppression of oxidative damage by Saccharomyces cerevisiae ATX2, which encodes a manganese-trafficking protein that localizes to Golgi-like vesicles; Lin SJ et al.; Oxygen toxicity in Saccharomyces cerevisiae lacking the copper/zinc superoxide dismutase (SOD1) can be suppressed by overexpression of the S . cerevisiae ATX2 gene . Multiple copies of ATX2 were found to reverse the aerobic auxotrophies of sod1(delta) mutants for lysine and methionine and also to enhance the resistance of these yeast strains to paraquat and atmospheric levels of oxygen . ATX2 encodes a novel 34.4-kDa polypeptide with a number of potential membrane-spanning domains . Our studies indicate that Atx2p localizes to the membrane of a vesicular compartment in yeast cells reminiscent of the Golgi apparatus . With indirect immunofluorescence microscopy, Atx2p exhibited a punctate pattern of staining typical of the Golgi apparatus, and upon subcellular fractionation, Atx2p colocalized with a biochemical marker for the yeast Golgi apparatus . We demonstrate here that this vesicle protein normally functions in the homeostasis of manganese ions and that this role in metal metabolism is necessary for the ATX1 suppression of SOD1 deficiency . First, overexpression of ATX2 caused cells to accumulate increased levels of manganese . Second, a deletion in ATX2 caused a decrease in the apparent available level of intracellular manganese and caused sod1(delta) mutants to become dependent upon exogenous manganese for aerobic growth . Third, ATX2 was incapable of suppressing oxidative damage in cells depleted of manganese ions or lacking the plasma membrane transporter for manganese . The effect of ATX2 overexpression on manganese accumulation and oxygen resistance is similar to what we have previously reported for mutations in PMR1, which encodes a manganese-trafficking protein that also resides in a vesicular compartment . Our studies are consistent with a model in which Atx2p and Pmr1p work in opposite directions to control manganese homeostasis. Mol Cell Biol, 1996 Nov, 16(11), 6020 - 8 The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover; van Nocker S et al.; The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin . It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals . In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1 . Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography . In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex . Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains . Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress . These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae . However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates . Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized . Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates. J Biol Chem, 1996 Oct 18, 271(42), 25958 - 65 Identification of the structural gene for glyoxalase I from Saccharomyces cerevisiae; Inoue Y et al.; The structural gene for glyoxalase I (GLO1) of Saccharomyces cerevisiae was identified . The GLO1 gene contained an open reading frame with 326 amino acids, and the molecular weight of the gene product (Glo1p) deduced from the DNA sequence was calculated to be 37,207.06 . Glyoxalase I activity increased approximately 95-fold when the GLO1 gene was introduced into the yeast cell with a multicopy plasmid, and the resultant transformant showed the increased resistance against methylglyoxal . Since the knockout mutant of the GLO1 gene of haploid strain of S . cerevisiae was still viable, the GLO1 gene was thought to be unnecessary for growth of the yeast . The GLO1 gene was overexpressed in two kinds of glutathione-deficient mutants, gamma-glutamylcysteine synthetase-deficient (gsh1(-)) and glutathione synthetase-deficient (gsh2(-)), respectively, and the sensitivites to methylglyoxal were compared . The gsh1-deficient mutant, which could not produce glutathione at all, was hypersensitive to methylglyoxal, and overproduction of the Glo1p did not restore the growth arrest caused by exogenously added methylglyoxal . The gsh2-deficient mutant, which accumulates gamma-glutamylcysteine (an intermediate of glutathione biosynthesis), was also sensitive to methylglyoxal compared with the isogenic wild type strain, although the growth arrest caused by methylglyoxal was partially restored by overexpression of the GLO1 gene . Purified glyoxalase I from yeast could use gamma-glutamylcysteine as a substrate (kcat/Km = 1.89 x 10(7) M-1 s-1,