J Biol Chem, 2000 Jul 28, 275(30), 23247 - 52 Identification and cloning of two histone fold motif-containing subunits of HeLa DNA polymerase epsilon; Li Y et al.; HeLa DNA polymerase epsilon (pol epsilon), possibly involved in both DNA replication and DNA repair, was previously isolated as a complex of a 261-kDa catalytic subunit and a tightly bound 59-kDa accessory protein . Saccharomyces cerevisiae pol epsilon, however, consists of four subunits: a 256-kDa catalytic subunit with 39% identity to HeLa pol epsilon p261, a 80-kDa subunit (DPB2) with 26% identity to HeLa pol epsilon p59, a 23-kDa subunit (DPB3), and a 22-kDa subunit (DPB4) . We report here the identification and the cloning of two additional subunits of HeLa pol epsilon, p17, and p12 . Both proteins contain histone fold motifs which are present also in S . cerevisiae DPB4 and DPB3 . The histone fold motifs of p17 and DPB4 are related to that of subunit A of the CCAAT binding factor, whereas the histone fold motifs found in p12 and DPB3 are homologous to that in subunit C of CCAAT binding factor . p17 together with p12, but not p17 or p12 alone, interact with both p261 and p59 subunits of HeLa pol epsilon . The genes for p17 and p12 can be assigned to chromosome locations 9q33 and 2p12, respectively. Prog Lipid Res, 1999 Sep-Nov, 38(5-6), 361 - 99 Phospholipid biosynthesis in the yeast Saccharomyces cerevisiae and interrelationship with other metabolic processes; Carman GM et al.; In this review, we have discussed recent progress in the study of the regulation that controls phospholipid metabolism in S . cerevisiae . This regulation occurs on multiple levels and is tightly integrated with a large number of other cellular processes and related metabolic and signal transduction pathways . Progress in deciphering this complex regulation has been very rapid in the last few years, aided by the availability of the sequence of the entire Saccharomyces genome . The assignment of functions to the remaining unassigned open reading frames, as well as ascertainment of remaining gene-enzyme relationships in phospholipid biosynthesis in yeast, promises to provide detailed understanding of the genetic regulation of a crucial area of metabolism in a key eukaryotic model system . Since the processes of lipid metabolism, secretion, and signal transduction show fundamental similarities in all eukaryotes, the dissection of this regulation in yeast promises to have wide application to our understanding of metabolic control in all eukaryotes. J Biol Chem, 2000 Jul 7, 275(27), 20508 - 13 Cytochrome c methyltransferase, Ctm1p, of yeast; Polevoda B et al.; Cytochromes c from plants and fungi, but not higher animals, contain methylated lysine residues at specific positions, including for example, the trimethylated lysine at position 72 in iso-1-cytochrome c of the yeast Saccharomyces cerevisiae . Testing of 6,144 strains of S . cerevisiae, each overproducing a different open reading frame fused to glutathione S-transferase, previously revealed that YHR109w was associated with an activity that methylated horse cytochrome c . We show here that this open reading frame, denoted Ctm1p, is specifically responsible for trimethylating lysine 72 of iso-1-cytochrome c . Unmethylated forms of cytochrome c but not other proteins or nucleic acids are methylated in vitro by Ctm1p produced in S . cerevisiae or Escherichia coli . Iso-1-cytochrome c purified from a ctm1-Delta strain is not trimethylated in vivo, whereas the K72R mutant form, or the trimethylated Lys-72 form of iso-1-cytochrome c, are not significantly methylated by Ctm1p in vitro . Like apocytochrome c, but in contrast to holocytochrome c, Ctm lp is located in the cytosol, consistent with the view that the natural substrate is apocytochrome c . The ctm1-Delta strain lacking the methyltransferase did not exhibit any growth defect on a variety of media and growth conditions, and the unmethylated iso-1-cytochrome c was produced at the normal level and exhibited the normal activity in vivo . Ctm1p and cytochrome c were coordinately regulated during anaerobic to aerobic transition, a finding consistent with the view that this methyltransferase evolved to act on cytochrome c. Yeast, 2000 Apr, 16(6), 511 - 22 Two-dimensional gel analysis of the proteome of lager brewing yeasts; Joubert R et al.; Modern lager brewing yeasts used in beer production are hybrid strains consisting of at least two different genomes . To obtain information on the identity of the parental strains that gave rise to industrial lager yeasts, we used two-dimensional (2-D) gel electrophoresis and analysed the proteomes of different Saccharomyces species isolated from breweries . We found that the proteome of lager brewing yeasts and of the type strains of S . carlsbergensis, S . monacensis and S . pastorianus can be interpreted as the superimposition of two elementary patterns . One originates from proteins encoded by a S . cerevisiae-like genome . The other corresponds to a divergent Saccharomyces species whose best representative is a particular S . pastorianus strain, NRRL Y-1551 . A map of industrial lager brewing yeasts has been established, with the individual origin of proteins and with identification of protein spots by comparison to known S . cerevisiae proteins . This 2-D map can be accessed on the Lager Brewing Yeast Protein Map server through the World Wide Web . This study provides the first example of the use of proteome analysis for investigating taxonomic relationships between divergent yeast species . Lipids, 2000 Mar, 35(3), 243 - 7 Biochemical modifications and transcriptional alterations attendant to sterol feeding in Phytophthora parasitica; Dotson WD et al.; Phytophthora species are eukaryotic sterol auxotrophs that possess the ability to grow, albeit poorly, in the complete absence of sterols . Growth of Phytophthora is often improved substantially when an exogenous source of sterol is provided . Additionally, sterols may be required for sexual and asexual sporulation in Phytophthora . Our research has been focused on identifying and characterizing the immediate physiological effects following sterol addition to cultures of P . parasitica . Through gas chromatographic analysis of extracts from P . parasitica cultures that were fed various sterols, we have obtained evidence for sterol C5 desaturase and delta7 reductase activities in this organism . Zoo blots were probed with DNA sequences encoding these enzymes, from Saccharomyces cerevisiae and Arabidopsis thaliana . Hybridization of a S . cerevisiae ERG3 probe to P . parasitica DNA was observed, implicating sequence similarity between the sterol C5 desaturase encoding genes . Differential display experiments, using RNA from P . parasitica, have demonstrated a pattern of altered gene expression between cultures grown in the presence and absence of sitosterol . Characterization of sterol-related metabolic effects and sterol functions in Phytophthora should lead to improved measures for control of this important group of plant pathogens. Nucleic Acids Symp Ser, 1999, (42), 201 - 2 A role of the C-terminal region adjacent to the zinc-fingers in the DNA binding ability of Rme1p, a regulator of meiosis in S . cerevisiae; Shimizu M et al.; Rme1p is a zinc-finger protein and has a pivotal role in control of meiosis in Saccharomyces cerevisiae . The DNA binding domain of Rme1p consists of three zinc-finger segments and the C-terminal 16 amino acid residues (called C-TR) . To examine the role of C-TR, a series of mutant Rme1p fused with maltose binding protein (MBP) were constructed, purified, and characterized, in terms of the DNA binding ability . The basic amino acid residues R287 and K290, and the hydrophobic residues F288, L292, 1295, and L296 play an important role for DNA binding, suggesting that the C-TR forms an amphipathic alpha-helix . Also, it was shown that the mutations in the basic amino acid residues abolish the repression and inhibition of spore formation by Rme1p in vivo . Hence, the C-TR is important for in vivo function of Rme1p. EMBO J, 2000 Apr 17, 19(8), 1777 - 83 Supercomplexes in the respiratory chains of yeast and mammalian mitochondria; Schagger H et al.; Around 30-40 years after the first isolation of the five complexes of oxidative phosphorylation from mammalian mitochondria, we present data that fundamentally change the paradigm of how the yeast and mammalian system of oxidative phosphorylation is organized . The complexes are not randomly distributed within the inner mitochondrial membrane, but assemble into supramolecular structures . We show that all cytochrome c oxidase (complex IV) of Saccharomyces cerevisiae is bound to cytochrome c reductase (complex III), which exists in three forms: the free dimer, and two supercomplexes comprising an additional one or two complex IV monomers . The distribution between these forms varies with growth conditions . In mammalian mitochondria, almost all complex I is assembled into supercomplexes comprising complexes I and III and up to four copies of complex IV, which guided us to present a model for a network of respiratory chain complexes: a 'respirasome' . A fraction of total bovine ATP synthase (complex V) was isolated in dimeric form, suggesting that a dimeric state is not limited to S.cerevisiae, but also exists in mammalian mitochondria. Arch Biochem Biophys, 2000 Apr 15, 376(2), 288 - 98 Sds22p is a subunit of a stable isolatable form of protein phosphatase 1 (Glc7p) from Saccharomyces cerevisiae; Hong G et al.; Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells . PP1 activity is believed to be controlled by the interaction of PP1 catalytic subunit with various regulatory subunits . The essential gene GLC7 encodes the PP1 catalytic subunit in Saccharomyces cerevisiae . In this study, full-length GLC7(1-312), C-terminal deletion mutants, and C-terminally poly-his tagged mutants were constructed and expressed in a GLC7 knockout strain of S . cerevisiae . Viability studies of the GLC7 knockout strains carrying the plasmids expressing GLC7 C-terminal deletion mutants and their tagged forms showed that the mutants 1-295 and 1-304 were functional, whereas the mutant 1-245 was not . The C-terminally poly-his tagged Glc7p with and without an N-terminal hemagglutinin (HA) tag was partially purified by immobilized Ni(2+) affinity chromatography and further analyzed by gel filtration and ion exchange chromatography . Phosphatase activity assays, SDS-PAGE, and Western blot analyses of the chromatographic fractions suggested that the Glc7p associated with regulatory subunits in vivo . A 40-kDa protein was copurified with tagged Glc7p through several chromatographic procedures . Monoclonal antibody against the HA tag coimmunoprecipitated the tagged Glc7p and the 40-kDa protein . This protein was further purified by a reverse phase HPLC column . Analysis by CNBr digestion, peptide sequencing, and electrospray mass spectrometry showed that this 40-kDa protein is Sds22p, one of the proteins proposed to be a regulatory subunit of Glc7 . These results demonstrate that Sds22p forms a complex with Glc7p and that Sds22p:Glc7p is a stable isolatable form of yeast PP1 . Nucleic Acids Res, 2000 May 15, 28(10), 2099 - 107 Bi-directional replication and random termination; Santamaria D et al.; Two-dimensional (2D) agarose gel electrophoresis was used to study termination of DNA replication in a shuttle vector, YRp7', when it replicated in Escherichia coli, Saccharomyces cerevisiae and Xenopus egg extracts . In E . coli, the 2D gel patterns obtained were consistent with uni-directional replication initiated at a specific site, the ColE1 origin . In consequence, termination also occurred precisely at the ColE1 origin . In Xenopus egg extracts, the particular shape of the bubble arc as well as the triangular smear detected to the left of the simple-Y pattern indicated random initiation and termination . In S.cerevisiae, initiation occurred at the ARS1 origin and replication proceeded in a bi-directional manner . However, termination did not always occur at a specific site 180 degrees across from the origin, but almost all along the south hemisphere of the plasmid . Inversion, deletion or replacement of DNA sequences located throughout this hemisphere did not eliminate random termination . Analysis of the replication intermediates of another yeast plasmid bearing a different origin, ARS305, also exhibited random termination . We propose that the random termination events observed in S.cerevisiae could be due to an asynchronous departure of both forks from the bi-directional origin in addition to differences in the rate of fork progression . These observations could be extended to all bi-directional origins. J Biol Chem, 2000 Jul 7, 275(27), 20748 - 53 The interaction of TOGp with microtubules and tubulin; Spittle C et al.; TOGp is the human homolog of XMAP215, a Xenopus microtubule-associated protein that promotes rapid microtubule assembly at plus ends . These proteins are thought to be critical for microtubule assembly and/or mitotic spindle formation . To understand how TOGp interacts with the microtubule lattice, we cloned full-length TOGp and various truncations for expression in a reticulocyte lysate system . Based on microtubule co-pelleting assays, the microtubule binding domain is contained within a basic 600-amino acid region near the N terminus, with critical domains flanking a region homologous to the microtubule binding domain found in the related proteins Stu2p (S . cerevisiae) and Dis1 (S . pombe) . Both full-length TOGp and the N-terminal fragment show enhanced binding to microtubule ends . Full-length TOGp also binds altered polymer lattice structures including parallel protofilament sheets, antiparallel protofilament sheets induced with zinc ions, and protofilament rings, suggesting that TOGp binds along the length of individual protofilaments . The C-terminal region of TOGp has a low affinity for microtubule polymer but binds tubulin dimer . We propose a model to explain the microtubule-stabilizing and/or assembly-promoting functions of the XMAP215/TOGp family of microtubule-associated proteins based on the binding properties we have identified. J Cell Biol, 2000 Apr 17, 149(2), 397 - 410 The F-box protein Rcy1p is involved in endocytic membrane traffic and recycling out of an early endosome in Saccharomyces cerevisiae; Wiederkehr A et al.; In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole . In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1) . Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway . Mutation of RCY1 leads to the accumulation of an enlarged compartment that contains the t-SNARE Tlg1p and lies close to areas of cell expansion . In addition, endocytic markers such as Ste2p and the fluorescent dyes, Lucifer yellow and FM4-64, were found in a similar enlarged compartment after their internalization . To determine whether rcy1Delta is defective for recycling, we have developed an assay that measures the recycling of previously internalized FM4-64 . This method enables us to follow the recycling pathway in yeast in real time . Using this assay, it could be demonstrated that recycling of membranes is rapid in S . cerevisiae and that a major fraction of internalized FM4-64 is secreted back into the medium within a few minutes . The rcy1Delta mutant is strongly defective in recycling. Gene, 2000 Apr 4, 246(1-2), 37 - 48 New features of mitochondrial DNA replication system in yeast and man; Lecrenier N et al.; In this review, we sum up the research carried out over two decades on mitochondrial DNA (mtDNA) replication, primarily by comparing this system in Saccharomyces cerevisiae and Homo sapiens . Brief incursions into systems of other organisms have also been achieved when they provide new information.S . cerevisiae and H . sapiens mitochondrial DNA (mtDNA) have been thought for a long time to share closely related architecture and replication mechanisms . However, recent studies suggest that mitochondrial genome of S . cerevisiae may be formed, at least partially, from linear multimeric molecules, while human mtDNA is circular . Although several proteins involved in the replication of these two genomes are very similar, divergences are also now increasingly evident . As an example, the recently cloned human mitochondrial DNA polymerase beta-subunit has no counterpart in yeast . Yet, yeast Abf2p and human mtTFA are probably not as closely functionally related as thought previously . Some mtDNA metabolism factors, like DNA ligases, were until recently largely uncharacterized, and have been found to be derived from alternative nuclear products . Many factors involved in the metabolism of mitochondrial DNA are linked through genetic or biochemical interconnections . These links are presented on a map . Finally, we discuss recent studies suggesting that the yeast mtDNA replication system diverges from that observed in man, and may involve recombination, possibly coupled to alternative replication mechanisms like rolling circle replication. J Biol Chem, 2000 Apr 21, 275(16), 11626 - 30 D-tyrosyl-tRNA(Tyr) metabolism in Saccharomyces cerevisiae; Soutourina J et al.; The Saccharomyces cerevisiae YDL219w (DTD1) gene, which codes for an amino acid sequence sharing 34% identity with the Escherichia coli D-Tyr-tRNA(Tyr) deacylase, was cloned, and its product was functionally characterized . Overexpression in the yeast of the DTD1 gene from a multicopy plasmid increased D-Tyr-tRNA(Tyr) deacylase activity in crude extracts by two orders of magnitude . Upon disruption of the chromosomal gene, deacylase activity was decreased by more than 90%, and the sensitivity to D-tyrosine of the growth of S . cerevisiae was exacerbated . The toxicity of D-tyrosine was also enhanced under conditions of nitrogen starvation, which stimulate the uptake of D-amino acids . In relation with these behaviors, the capacity of purified S . cerevisiae tyrosyl-tRNA synthetase to produce D-Tyr-tRNA(Tyr) could be shown . Finally, the phylogenetic distribution of genes homologous to DTD1 was examined in connection with L-tyrosine prototrophy or auxotrophy . In the auxotrophs, DTD1-like genes are systematically absent . In the prototrophs, the putative occurrence of a deacylase is variable . It possibly depends on the L-tyrosine anabolic pathway adopted by the cell. FEBS Lett, 2000 Apr 7, 471(1), 113 - 8 The cytotoxic action of Bax on yeast cells does not require mitochondrial ADP/ATP carrier but may be related to its import to the mitochondria; Kissova I et al.; The effect of the expression of murine Bax protein on growth and vitality was examined in Saccharomyces cerevisiae and compared with the effect of Bax in mutant cells lacking functional mitochondria . The cytotoxic effect of Bax on yeast does not require functional oxidative phosphorylation, respiration, or mitochondrial proteins (ADP/ATP carriers) implicated in the formation of the permeability transition pore in mammalian mitochondria . In the wild type S . cerevisiae the expression of Bax does not result in a severe effect on mitochondrial membrane potential and respiration . On the basis of Bax induced differences in the fluorescence of green fluorescent protein fused to mitochondrial proteins, it is proposed that Bax may interfere with one essential cellular process in yeast: the mitochondrial protein import pathway that is specific for the proteins of the mitochondrial carrier family. Eur J Biochem, 2000 Apr, 267(8), 2409 - 18 Complementation of deletion mutants in the genes encoding the F1-ATPase by expression of the corresponding bovine subunits in yeast S . cerevisiae; Lai-Zhang J et al.; The F1F0 ATP synthase is composed of the F1-ATPase which is bound to F0, in the inner membrane of the mitochondrion . Assembly and function of the enzyme is a complicated task requiring the interactions of many proteins for the folding, import, assembly, and function of the enzyme . The F1-ATPase is a multimeric enzyme composed of five subunits in the stoichiometry of alpha3beta3gammadeltaepsilon . This study demonstrates that four of the five bovine subunits of the F1-ATPase can be imported and function in an otherwise yeast enzyme effectively complementing mutations in the genes encoding the corresponding yeast ATPase subunits . In order to demonstrate this, the coding regions of each of the five genes were separately deleted in yeast providing five null mutant strains . All of the strains displayed negative or a slow growth phenotype on medium containing glycerol as the carbon source and strains with a null mutation in the gene encoding the gamma-, delta- or epsilon-gene became completely, or at a high frequency, cytoplasmically petite . The subunits of bovine F1 were expressed individually in the yeast strains with the corresponding null mutations and targeted to the mitochondrion using a yeast mitochondrial leader peptide . Expression of the bovine alpha-, beta-, gamma-, and epsilon-, but not the delta-, subunit complemented the corresponding null mutations in yeast correcting the corresponding negative phenotypes . These results indicate that yeast is able to import, assemble subunits of bovine F1-ATPase in mitochondria and form a functional chimeric yeast/bovine enzyme complex. Biochemistry, 2000 Apr 18, 39(15), 4225 - 30 The whole is not the simple sum of its parts in calmodulin from S . cerevisiae; Lee SY et al.; Calmodulin is an essential Ca(2+)-binding protein involved in a multitude of cellular processes . The calmodulin sequence is highly conserved among all eukaryotic species; calmodulin from the yeast S . cerevisiae (yCaM) is the most divergent form, while still sharing 60% sequence identity with vertebrate calmodulin (vCaM) . Although yCaM can be functionally substituted by vCaM in vivo, the two calmodulin proteins possess significantly different Ca(2+)-binding properties as well as abilities to activate vertebrate target enzymes in vitro . In addition, it has been observed that certain properties of the N-terminal and C-terminal domains of Ca(2+)-yCaM differ depending on whether they are in the context of the whole protein or isolated as half-molecule fragments . To investigate the structural basis for these differing properties, we have undertaken nuclear magnetic resonance (NMR) studies on yCaM and the two half-molecule fragments representing its two individual domains, yTr1(residues 1-76) and yTr2 (residues 75-146) . We present direct evidence that the two domains of Ca(2+)-yCaM interact via their exposed hydrophobic surfaces . Thus, the Ca(2+)-bound form of yCaM exists in a novel compact structure in direct contrast to the well-established structure of Ca(2+)-vCaM comprised of two independent globular domains. J Chromatogr A, 2000 Mar 24, 873(2), 195 - 208 The influence of cell adsorbent interactions on protein adsorption in expanded beds; Fernandez-Lahore HM et al.; Expanded bed adsorption (EBA) is a primary recovery operation allowing the adsorption of proteins directly from unclarified feedstock, e.g . culture suspensions, homogenates or crude extracts . Thus solid-liquid separation is combined with adsorptive purification in a single step . The concept of integration requires that the solid components of the feed solution are regarded as a part of the process, which influences stability, reproducibility, and overall performance . This aspect is investigated here at the example of the influence of presence and concentration of intact yeast cells (S . cerevisiae) on the adsorption of model proteins (hen egg white lysozyme and bovine serum albumin) to various stationary phases (cation and anion-exchange, hydrophobic interaction, immobilised metal affinity) . The interaction of the cells with the adsorbents is determined qualitatively and quantitatively by a pulse response method as well as by a finite bath technique under different operating conditions . The consequence of these interactions for the stability of expanded beds in suspensions of varying cell concentration is measured by residence time distributions (RTDs) after tracer pulse injection (NaBr, LiCl) . Analysis of the measured RTD by the PDE model allows the calculation of the fraction of perfectly fluidised bed (phi), a parameter which may be regarded as a critical quantity for the estimation of the quality of fluidisation of adsorbents in cell containing suspensions . The correlation between bed stability and performance is made by analysing the breakthrough of model proteins during adsorption from unclarified yeast culture broth . A clear relationship is found between the degree of cell/adsorbent interaction, bed stability in terms of the phi parameter, and the sorption efficiency . Only beds characterised by a phi value larger than 0.8 in the presence of cells will show a conserved performance compared to adsorption from cell free solutions . A drop in phi, which is due to interactions of the fluidised adsorbent particles with cells from the feed, will directly result in a reduced breakthrough efficiency . The data presented highlight the importance of including the potential interaction of solid feedstock components and the expanded adsorbents into the design of EBA processes, as the interrelation found here is a key factor for the overall performance of EBA as a truly integrated operation. J Mol Biol, 2000 Apr 21, 298(1), 111 - 21 High-resolution crystal structure of S . cerevisiae Ypt51(DeltaC15)-GppNHp, a small GTP-binding protein involved in regulation of endocytosis; Esters H et al.; Ypt/Rab proteins are membrane-associated small GTP-binding proteins which play a central role in the coordination, activation and regulation of vesicle-mediated transport in eukaryotic cells . We present the 1.5 A high-resolution crystal structure of Ypt51 in its active, GppNHp-bound conformation . Ypt51 is an important regulator involved in the endocytic membrane traffic of Saccharomyces cerevisiae . The structure reveals small but significant structural differences compared with H-Ras p21 . The effector loop and the catalytic loop are well defined and stabilized by extensive hydrophobic interactions . The switch I and switch II regions form a well-defined epitope for hypothetical effector protein binding . Sequence comparisons between the different isoforms Ypt51, Ypt52 and Ypt53 provide the first insights into determinants for specific effector binding and for fine-tuning of the intrinsic GTP-hydrolysis rate . Nucleic Acids Res, 2000 May 1, 28(9), 1941 - 6 Identification of the Saccharomyces cerevisiae RNA:pseudouridine synthase responsible for formation of psi(2819) in 21S mitochondrial ribosomal RNA; Ansmant I et al.; So far, four RNA:pseudouridine (Psi)-synthases have been identified in yeast Saccharomyces cerevisiae . Together, they act on cytoplasmic and mitochondrial tRNAs, U2 snRNA and rRNAs from cytoplasmic ribosomes . However, RNA:Psi-synthases responsible for several U-->Psi conversions in tRNAs and UsnRNAs remained to be identified . Based on conserved amino-acid motifs in already characterised RNA:Psi-synthases, four additional open reading frames (ORFs) encoding putative RNA:Psi-synthases were identified in S.cerevisiae . Upon disruption of one of them, the YLR165c ORF, we found that the unique Psi residue normally present in the fully matured mitochondrial rRNAs (Psi(2819)in 21S rRNA) was missing, while Psi residues at all the tested pseudo-uridylation sites in cytoplasmic and mitochondrial tRNAs and in nuclear UsnRNAs were retained . The selective U-->Psi conversion at position 2819 in mitochondrial 21S rRNA was restored when the deleted yeast strain was transformed by a plasmid expressing the wild-type YLR165c ORF . Complementation was lost after point mutation (D71-->A) in the postulated active site of the YLR165c-encoded protein, indicating the direct role of the YLR165c protein in Psi(2819)synthesis in mitochondrial 21S rRNA . Hence, for nomenclature homogeneity the YLR165c ORF was renamed PUS5 and the corresponding RNA:Psi-synthase Pus5p . As already noticed for other mitochondrial RNA modification enzymes, no canonical mitochondrial targeting signal was identified in Pus5p . Our results also show that Psi(2819)in mitochondrial 21S rRNA is not essential for cell viability. J Biol Chem, 2000 May 26, 275(21), 16354 - 9 Siderophore-iron uptake in saccharomyces cerevisiae . Identification of ferrichrome and fusarinine transporters; Yun CW et al.; A family of four putative transporters (Arn1p-4p) in Saccharomyces cerevisiae is expressed under conditions of iron deprivation and is regulated by Aft1p, the major iron-dependent transcription factor in yeast . One of these, Arn3p/Sit1p, facilitates the uptake of ferrioxamine B, a siderophore of the hydroxamate class . Here we report that ARN family members facilitated the uptake of iron from the trihydroxamate siderophores ferrichrome, ferrichrome A, and triacetylfusarinine C . Uptake of siderophore-bound iron was dependent on either the high-affinity ferrous iron transport system or the ARN family of transporters . The specificity of each siderophore for individual transporters was determined . Uptake of ferrichrome and ferrichrome A was facilitated by both Arn1p and Arn3p . Uptake of triacetylfusarinine C was facilitated by Arn2p, although small amounts of uptake also occurred through Arn1p and Arn3p . In contrast to the trihydroxamates, uptake of iron from the dihydroxamate rhodotorulic acid occurred only via the high-affinity ferrous iron system . Epitope-tagged Arn1p was expressed in intracellular vesicles in a pattern that was indistinguishable from that of Arn3p, whereas Ftr1p, a component of the high-affinity ferrous system, was expressed on the plasma membrane . These data indicate that S . cerevisiae maintains two systems of siderophore uptake, only one of which is located on the plasma membrane. J Cell Biol, 2000 Apr 3, 149(1), 95 - 110 The role of the COOH terminus of Sec2p in the transport of post-Golgi vesicles; Elkind NB et al.; Sec2p is required for the polarized transport of secretory vesicles in S . cerevisiae . The Sec2p NH(2) terminus encodes an exchange factor for the Rab protein Sec4p . Sec2p associates with vesicles and in Sec2p COOH-terminal mutants Sec4p and vesicles no longer accumulate at bud tips . Thus, the Sec2p COOH terminus functions in targeting vesicles, however, the mechanism of function is unknown . We found comparable exchange activity for truncated and full-length Sec2 proteins, implying that the COOH terminus does not alter the exchange rate . Full-length Sec2-GFP, similar to Sec4p, concentrates at bud tips . A COOH-terminal 58-amino acid domain is necessary but not sufficient for localization . Sec2p localization depends on actin, Myo2p and Sec1p, Sec6p, and Sec9p function . Full-length, but not COOH-terminally truncated Sec2 proteins are enriched on membranes . Membrane association of full-length Sec2p is reduced in sec6-4 and sec9-4 backgrounds at 37 degrees C but unaffected at 25 degrees C . Taken together, these data correlate loss of localization of Sec2 proteins with reduced membrane association . In addition, Sec2p membrane attachment is substantially Sec4p independent, supporting the notion that Sec2p interacts with membranes via an unidentified Sec2p receptor, which would increase the accessibility of Sec2p exchange activity for Sec4p. Genetics, 2000 Apr, 154(4), 1533 - 48 Forkhead genes in transcriptional silencing, cell morphology and the cell cycle . Overlapping and distinct functions for FKH1 and FKH2 in Saccharomyces cerevisiae; Hollenhorst PC et al.; The SIR1 gene is one of four specialized genes in Saccharomyces cerevisiae required for repressing transcription at the silent mating-type cassettes, HMLalpha and HMRa, by a mechanism known as silencing . Silencing requires the assembly of a specialized chromatin structure analogous to heterochromatin . FKH1 was isolated as a gene that, when expressed in multiple copies, could substitute for the function of SIR1 in silencing HMRa . FKH1 (Forkhead Homologue One) was named for its homology to the forkhead family of eukaryotic transcription factors classified on the basis of a conserved DNA binding domain . Deletion of FKH1 caused a defect in silencing HMRa, indicating that FKH1 has a positive role in silencing . Significantly, deletion of both FKH1 and its closest homologue in yeast, FKH2, caused a form of yeast pseudohyphal growth, indicating that the two genes have redundant functions in controlling yeast cell morphology . By several criteria, fkh1Delta fkh2Delta-induced pseudohyphal growth was distinct from the nutritionally induced form of pseudohyphal growth observed in some strains of S . cerevisiae . Although FKH2 is redundant with FKH1 in controlling pseudohyphal growth, the two genes have different functions in silencing HMRa . High-copy expression of CLB2, a G2/M-phase cyclin, prevented fkh1Delta fkh2Delta-induced pseudohyphal growth and modulated some of the fkhDelta-induced silencing phenotypes . Interestingly, deletions in either FKH1 or FKH2 alone caused subtle but opposite effects on cell-cycle progression and CLB2 mRNA expression, consistent with a role for each of these genes in modulating the cell cycle and having opposing effects on silencing . The differences between Fkh1p and Fkh2p in vivo were not attributable to differences in their DNA binding domains. Genetics, 2000 Apr, 154(4), 1473 - 84 A role for the noncatalytic N terminus in the function of Cdc25, a Saccharomyces cerevisiae Ras-guanine nucleotide exchange factor; Chen RA et al.; The Saccharomyces cerevisiae CDC25 gene encodes a guanine nucleotide exchange factor (GEF) for Ras proteins . Its catalytic domain is highly homologous to Ras-GEFs from all eukaryotes . Even though Cdc25 is the first Ras-GEF identified in any organism, we still know very little about how its function is regulated in yeast . In this work we provide evidence for the involvement of the N terminus of Cdc25 in the regulation of its activity . A truncated CDC25 lacking the noncatalytic C-terminal coding sequence was identified in a screen of high-copy suppressors of the heat-shock-sensitive phenotype of strains in which the Ras pathway is hyper-activated . The truncated gene acts as a dominant-negative mutant because it only suppresses the heat-shock sensitivity of strains that require the function of CDC25 . Our two-hybrid assays and immunoprecipitation analyses show interactions between the N terminus of Cdc25 and itself, the C terminus, and the full-length protein . These results suggest that the dominant-negative effect may be a result of oligomerization with endogenous Cdc25 . Further evidence of the role of the N terminus of Cdc25 in the regulation of its activity is provided by the mapping of the activating mutation of CDC25HS20 to the serine residue at position 365 in the noncatalytic N-terminal domain . This mutation induces a phenotype similar to activating mutants of other genes in the Ras pathway in yeast . Hence, the N terminus may exert a negative control on the catalytic activity of the protein . Taken together these results suggest that the N terminus plays a crucial role in regulating Cdc25 and consequently Ras activity, which in S . cerevisiae is essential for cell cycle progression. Biol Chem, 2000 Feb, 381(2), 121 - 6 Responses to peroxynitrite in yeast: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a sensitive intracellular target for nitration and enhancement of chaperone expression and ubiquitination; Buchczyk DP et al.; Peroxynitrite (ONOO-), a potent oxidizing and nitrating species, has been linked to covalent modifications of biomolecules in a number of pathological conditions . In S . cerevisiae, a model eukaryotic cell system, ONOO- was found to be more potent than hydrogen peroxide in oxidizing thiols, inducing heat shock proteins (Hsp70) and enhancing the ubiquitination of proteins . As identified by microsequence analysis following immunoprecipitation with anti-nitrotyrosine antibodies, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was especially susceptible to nitration by ONOO- in yeast cells . The activity of this enzyme was strongly inhibited upon steady-state exposure of the cells to low doses of ONOO- in yeast and in cultured rat astrocytes . Thus, ONOO- is a potent stressor in yeast capable of inducing oxidative damage and protein nitration, with GAPDH being a preferential target protein that is efficiently inactivated. Biotechnol Appl Biochem, 2000 Apr, 31 ( Pt 2), 95 - 100 Yeast protease B-digested skimmed milk inhibits angiotensin-I-converting-enzyme activity; Roy MK et al.; Angiotensin-1-converting-enzyme (ACE) inhibitory activity was identified in skimmed milk digested with cell-free extract of yeast Saccharomyces cerevisiae . Simultaneously, a protease enzyme involved in the production of ACE-inhibition materials in digested skimmed milk was purified to homogeneity from the cell-free extracts of S . cerevisiae by ammonium sulphate fractionation and chromatography in DEAE-Sephacel, D-tryptophan methyl ester-Sepharose 4B, Hiload Superdex G-200 and HPLC Mono-Q chromatography . The purified enzyme was identified as protease B, based on the molecular mass on SDS/PAGE and the N-terminal amino acid sequence of the enzyme . The optimum pH for digestion of skimmed milk and production of ACE-inhibition materials was pH 4.8 . The IC(50) of the hydrolysate was 0.42 mg of protein/ml when skimmed milk was digested with yeast protease B. J Biol Chem, 2000 Apr 7, 275(14), 10709 - 15 Desferrioxamine-mediated iron uptake in Saccharomyces cerevisiae . Evidence for two pathways of iron uptake; Yun CW et al.; In the yeast Saccharomyces cerevisiae, uptake of iron is largely regulated by the transcription factor Aft1 . cDNA microarrays were used to identify new iron and AFT1-regulated genes . Four homologous genes regulated as part of the AFT1-regulon (ARN1-4) were predicted to encode members of a subfamily of the major facilitator superfamily of transporters . These genes were predicted to encode proteins with 14 membrane spanning domains and were from 26 to 53% identical at the amino acid level . ARN3 is identical to SIT1, which is reported to encode a ferrioxamine B permease . Deletion of ARN3 did not prevent yeast from using ferrioxamine B as an iron source; however, deletion of ARN3 and FET3, a component of the high affinity ferrous iron transport system, did prevent uptake of ferrioxamine-bound iron and growth on ferrioxamine as an iron source . The siderophore-mediated transport system and the high affinity ferrous iron transport system were localized to separate cellular compartments . Epitope-tagged Arn3p was expressed in intracellular vesicles that co-sediment with the endosomal protein Pep12 . In contrast, Fet3p was expressed on the plasma membrane and was digested by extracellular proteases . These data indicate that S . cerevisiae has two pathways for ferrrioxamine-mediated iron uptake, one occurring at the plasma membrane and the other occurring in an intracellular compartment. Mol Biochem Parasitol, 2000 Feb 25, 106(1), 21 - 35 Cell-cycle and developmental regulation of TbRAB31 localisation, a GTP-locked Rab protein from Trypanosoma brucei; Field H et al.; Rab proteins are small GTPases that control the direction and timing of vesicle fusion during intracellular trafficking between membraneous compartments . Genome sequencing and EST analysis of Trypanosoma brucei indicates that the trypanosome Rab (TbRAB) gene family, and hence complexity of intracellular transport pathways, is intermediate between Saccharomyces cerevisiae and mammals . TbRAB31 is a constitutively expressed T . brucei Rab protein (formerly Trab7p) and is the product of one of two closely linked TbRAB genes, the other being TbRAB2 (TbRab2p, in: Field H, Ali BRS, Sherwin T, Gull K, Croft SL, Field MC . TbRab2p, a marker for the endoplasmic reticulum of Trypanosoma brucei, localises to the ERGIC in mammalian cells . J Cell Sci 1999; 112:147-156), involved in ER to Golgi transport . TbRAB31 has high homology to members of the Sec4/Ypt1 subfamily of Rab proteins from S . cerevisiae and to Rab13 and Rab11 from higher eukaryotes . Recombinant TbRAB31 binds GTP but, unusually for a Rab protein, has undetectable GTPase activity resulting in a constitutively GTP-bound protein . Antibodies against TbRAB31 recognise a discrete structure located between the kinetoplast and nucleus in interphase procyclic cells; by contrast the structure is morphologically more complex in bloodstream form (BSF) parasites, consisting of at least two foci . TbRAB31 behaviour was also studied during the cell cycle; TbRAB31 always localised to a discrete structure that duplicated very early in mitosis and relocated to daughter cells in a coordinate manner with the basal body and kinetoplast, suggesting the involvement of microtubules . Additional evidence suggests that TbRAB31 localises to the trypanosome Golgi complex . Firstly, the interphase position of TbRAB31 is consistent with a Golgi location . Secondly, the TbRAB31 structure is also recognised by cross-reacting antibodies to mammalian beta-coatomer protein (beta-COP), which localises to the Golgi in mammalian cells . Thirdly, the fluorescent ceramide analogue, BODIPY-TR-ceramide, a reliable marker of the mammalian Golgi apparatus, exhibited overlapping distribution with TbRAB31 . The location of BODIPY-TR-ceramide was confirmed at the trypanosome Golgi by histochemistry with diaminobenzidine and electron microscopy. Curr Genet, 2000 Feb, 37(2), 79 - 86 The cadmium-resistant gene, CAD2, which is a mutated putative copper-transporter gene (PCA1), controls the intracellular cadmium-level in the yeast S . cerevisiae; Shiraishi E et al.; Yeast cells carrying the CAD2 gene exhibit a resistance to cadmium . We cloned this gene and demonstrated that it was a mutated form derived from the gene of a putative copper-transporting ATPase (PCA1) . By site-directed mutagenesis, it appeared that the mutation conferring cadmium resistance was a R970G-substitution in the C-terminal region of Pca1 protein . The intracellular cadmium level of cells carrying CAD2 was lower than that of cells carrying either PAC1 or delta cad2 . Furthermore, cells with overexpression of CAD2 showed a much lower intracellular cadmium level than that of cells with a single-copy CAD2 . From these results, we conclude that the Cad2 protein controls the intracellular cadmium level through an enhanced cadmium efflux system. Biochemistry (Mosc), 2000 Mar, 65(3), 355 - 60 Purification and characterization of a soluble polyphosphatase from mitochondria of Saccharomyces cerevisiae; Lichko LP et al.; A polyphosphatase with the specific activity 2.2 U/mg was purified to apparent homogeneity from a soluble preparation of mitochondria of Saccharomyces cerevisiae . The polyphosphatase is a monomeric protein of approximately 41 kD . The purified enzyme hydrolyzes polyphosphates with an average chain length of 9 to 208 phosphate residues to the same extent, but its activity is approximately 2-fold higher with tripolyphosphate . ATP, PPi, and p-nitrophenyl phosphate are not substrates of this enzyme . The apparent Km values are 300, 18, and 0.25 microM obtained at hydrolysis of polyphosphates with a chain length of 3, 15, and 188 phosphate residues, respectively . Several divalent cations stimulated the enzyme activity 1.2-27-fold (Mg2+ = Co2+ = Mn2+ > Zn2+) . Determination of the protein N-terminal sequence and its comparison with the EMBL data library indicates that the soluble polyphosphatase of mitochondria of S . cerevisiae is not encoded by the gene of the major yeast polyphosphatase PPX1. Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 4034 - 9 The p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae; Springer S et al.; To investigate the factors involved in the sorting of cargo proteins into COPII endoplasmic reticulum (ER) to Golgi apparatus transport vesicles, we have created a strain of S . cerevisiae (p24Delta8) that lacks all eight members of the p24 family of transmembrane proteins (Emp24p, Erv25p, and Erp1p to Erp6p) . The p24 proteins have been implicated in COPI and COPII vesicle formation, cargo protein sorting, and regulation of vesicular transport in eukaryotic cells . We find that p24Delta8 cells grow identically to wild type and show delays of invertase and Gas1p ER-to-Golgi transport identical to those seen in a single Deltaemp24 deletion strain . Thus, p24 proteins do not have an essential function in the secretory pathway . Instead, they may serve as quality control factors to restrict the entry of proteins into COPII vesicles. Bioseparation, 1999, 8(1-5), 145 - 51 Use of streamline chelating for capture and purification of poly-His-tagged recombinant proteins; Noronha S et al.; Expression of recombinant proteins with poly-histidine tags enables their convenient capture and purification using immobilized metal affinity chromatography (IMAC) . The 6 x His-tagged protein binds to a chelating resin charged with metal ions such as Ni2+, Cu2+ or Zn2+, and can therefore be separated from proteins which have lower, or no, affinity for the resin . Two recombinant proteins, a malaria transmission-blocking vaccine candidate secreted extracellularly by S . cerevisiae and a modified diphtheria toxin produced intracellularly by E . coli, were expressed with 6 x His tags and could therefore be purified using IMAC . In an effort to further simplify the initial capture of these proteins, an expanded bed adsorption technique using a chelating resin (Streamline Chelating) was introduced . It was possible to capture the intracellular diphtheria protein from E . coli directly after cell lysis, without prior centrifugation or filtration . The extracellular malaria vaccine candidate was also directly captured from a high cell density yeast culture . Detailed information on the experimental work performed, and the capture processes developed, is provided. J Biol Chem, 2000 Mar 31, 275(13), 9636 - 44 The Bloom's syndrome gene product interacts with topoisomerase III; Wu L et al.; Bloom's syndrome is a rare genetic disorder associated with loss of genomic integrity and a large increase in the incidence of many types of cancer at an early age . The Bloom's syndrome gene product, BLM, belongs to the RecQ family of DNA helicases, which also includes the human Werner's and Rothmund-Thomson syndrome gene products and the Sgs1 protein of Saccharomyces cerevisiae . This family shows strong evolutionary conservation of protein structure and function . Previous studies have shown that Sgs1p interacts both physically and genetically with topoisomerase III . Here, we have investigated whether this interaction has been conserved in human cells . We show that BLM and hTOPO IIIalpha, one of two human topoisomerase III homologues, co-localize in the nucleus of human cells and can be co-immunoprecipitated from human cell extracts . Moreover, the purified BLM and hTOPO IIIalpha proteins are able to bind specifically to each other in vitro, indicating that the interaction is direct . We have mapped two independent domains on BLM that are important for mediating the interaction with hTOPO IIIalpha . Furthermore, through characterizing a genetic interaction between BLM and TOP3 in S . cerevisiae, we have identified a functional role for the hTOPO IIIalpha interaction domains in BLM. Biochem Biophys Res Commun, 2000 Apr 2, 270(1), 112 - 8 The effects of RAD52 epistasis group genes on various types of spontaneous mitotic recombination in Saccharomyces cerevisiae; You JC; The role of RAD52 epistasis group genes on spontaneous mitotic recombination was examined using three different types of spontaneous mitotic recombination in Saccharomyces cerevisiae . The spontaneous recombination between homologous sequences in a plasmid and a chromosome was essentially unaffected by null mutations in any of the RAD52 epistasis group genes . Recombination between genes in separate autonomously replicating plasmids was reduced 833-fold in a rad52 null mutant, but only 2- to at most 20-fold in rad50, 51, 54, 55, 57 null mutants . Recombination between tandemly repeated heteroalleles in an autonomously replicating plasmid was reduced almost 100-fold in a rad52 null mutant, but is either unaffected or slightly increased in rad50, 51, 54, 55, 57 null mutants . The finding that RAD50, 51, 54, 55, 57 are dispensable or marginally involved in these spontaneous recombinations suggests further that spontaneous mitotic recombination in S . cerevisiae might be processed by other than RAD52 epistasis group . Protein Expr Purif, 2000 Apr, 18(3), 366 - 77 Expression and purification of imidazole glycerol phosphate synthase from Saccharomyces cerevisiae; Chittur SV et al.; Imidazole glycerol phosphate (IGP) synthase is a glutamine amidotransferase that catalyzes the formation of IGP and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) from N(1)-{(5'-phosphoribulosyl)formimino}-5-aminoimidazole-4-car boxamide ribonucleotide (PRFAR) . This enzyme represents a junction between histidine biosynthesis and de novo purine biosynthesis . The recent characterization of the HIS7 gene in the yeast Saccharomyces cerevisiae IGP synthase established that this protein is bifunctional, representing a fusion between the N-terminal HisH domain and a C-terminal HisF domain . Catalytically active yeast HIS7 was expressed in a bacterial system under the control of T7 polymerase promoter . The recombinant enzyme was purified to homogeneity and the native molecular weight and steady-state kinetic constants were determined . The yeast enzyme is distinguished from the Escherichia coli IGP synthase in its utilization of ammonia as a substrate . HIS7 displays a higher K(m) for glutamine and a lower turnover in the ammonia-dependent IGP synthase activity . As observed with the E . coli IGP synthase, HIS7 shows a low basal level glutaminase activity that can be enhanced 1000-fold in the presence of a nucleotide substrate or analog . The purification and characterization of the S . cerevisiae enzyme will enable a more detailed investigation of the biochemical mechanisms that mediate the ammonia-transfer process . The fused structural feature of the HIS7 protein and the development of a high-level production system for the active enzyme elevate the potential for determination of its three-dimensional structure through X-ray crystallography . Mol Cell Biol, 2000 Apr, 20(8), 2809 - 17 Dpb11 controls the association between DNA polymerases alpha and epsilon and the autonomously replicating sequence region of budding yeast; Masumoto H et al.; Dpb11 is required for chromosomal DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae . Here, we report detection of a physical complex containing Dpb11 and DNA polymerase epsilon (Dpb11-Polepsilon complex) . During the S phase of the cell cycle, Dpb11 associated preferentially with DNA fragments containing autonomously replicating sequences (ARSs), at the same time as Polepsilon associated with these fragments . Association of Dpb11 and Polepsilon with these fragments was mutually dependent, suggesting that the Dpb11-Polepsilon complex associates with the ARS . Moreover, Dpb11 was required for the association of Polalpha-primase with the fragments . Thus, it seems likely that association of the Dpb11-Polepsilon complex with the ARS fragments is required for the association of the Polalpha-primase complex . Hydroxyurea inhibits late-origin firing in S . cerevisiae, and the checkpoint genes, RAD53 and MEC1, are involved in this inhibition . In the presence of hydroxyurea at temperatures permissive for cell growth, Polepsilon in dpb11-1 cells associated with early- and late-origin fragments . In wild-type cells, however, it associated only with early-origin fragments . This indicates that Dpb11 may also be involved in the regulation of late-origin firing . Overall, these results suggest that Dpb11 controls the association between DNA polymerases alpha and epsilon and the ARS. FEMS Microbiol Lett, 2000 Apr 1, 185(1), 65 - 70 Protective effect of antioxidants against para-nonylphenol-induced inhibition of cell growth in Saccharomyces cerevisiae; Okai Y et al.; The cell growth-modulating activity of an endocrine disruptor, p-nonylphenol (NP), was estimated using the yeast Saccharomyces cerevisiae as a simple model of eukaryotic cells . NP caused a dose-dependent suppressive effect on cell growth of S . cerevisiae at 10, 25 and 50 microM . The NP-induced cell growth inhibition was restored when concomitantly lipophilic antioxidants such as alpha-tocopherol and beta-carotene were supplied, but not the hydrophilic antioxidants ascorbic acid or (-)epigallocatechin gallate (EGCG) . The cellular oxygen consumption of S . cerevisiae was also inhibited in a dose-dependent fashion by the extracellular addition of NP, and pretreatment with alpha-tocopherol and beta-carotene suppressed NP-induced inhibition of cellular oxygen consumption, but ascorbic acid and EGCG were not effective . Furthermore, NP caused a marked generation of radical oxygen species (ROS) in S . cerevisiae, which was suppressed by treatment with alpha-tocopherol and beta-carotene, but not with ascorbic acid and EGCG . However, NP did not show a significant inhibitory effect on cell growth and survival of mitochondria-deficient petite mutant cells and they showed a relatively weak ROS-generating activity compared with parent yeast cells . These results suggest that NP-induced inhibition of cell growth and oxygen consumption in S . cerevisiae might be possibly associated with ROS generation in yeast mitochondria . The significance of this finding is discussed from the viewpoint of NP-induced oxidative stress against eukaryotic cells. Biotechniques, 2000 Mar, 28(3), 552 - 4, 556, 558-60 GCN4-based expression system (pGES): translationally regulated yeast expression vectors; Mimran A et al.; The expression of foreign proteins in Saccharomyces cerevisiae is a powerful tool for basic research and the biotechnological industry . In spite of the potential of S . cerevisiae, only a few useful expression vectors have been developed for this yeast . These vectors are based on an increasing transcription rate in combination with an increase in gene dosage . Most vectors are maintained as plasmids, which forces growth of cultures on poor selective media . Expression of the yeast Gcn4 protein is regulated at the translational level and increases strongly under amino acid starvation . Because under these conditions protein synthesis in general ceases, it is conceivable that regulatory elements that control Gcn4 expression could support selective expression of foreign genes . We cloned DNA fragments residing upstream from the GCN4 coding sequence (including the 5' UTR) and ligated them to a cDNA that encodes the human serum albumin (HSA) gene . These GCN4 regulatory elements induced efficient HSA expression at the translational level under amino acid starvation . The GCN4/HSA cassette promoted efficient, inducible expression on either a multicopy or integrative plasmid . The integrated cassette induced a high level of HSA in dense cultures grown on rich media . Thus, the GCN4-based expression system (pGES) provides high protein quantities . pGES is the first expression vector to be induced at the translational level. J Biol Chem, 2000 Mar 24, 275(12), 8315 - 23 Isolation of Trypanosoma brucei CYC2 and CYC3 cyclin genes by rescue of a yeast G(1) cyclin mutant . Functional characterization of CYC2; Van Hellemond JJ et al.; Two Trypanosoma brucei cyclin genes, CYC2 and CYC3, have been isolated by rescue of the Saccharomyces cerevisiae mutant DL1, which is deficient in CLN G(1) cyclin function . CYC2 encodes a 24-kDa protein that has sequence identity to the Neurospora crassa PREG1 and the S . cerevisiae PHO80 cyclin . CYC3 has the most sequence identity to mitotic B-type cyclins from a variety of organisms . Both CYC2 and CYC3 are single-copy genes and expressed in all life cycle stages of the parasite . To determine if CYC2 is found in a complex with previously identified trypanosome cdc2-related kinases (CRKs), the CYC2 gene was fused to the TY epitope tag, integrated into the trypanosome genome, and expressed under inducible control . CYC2ty was found to associate with an active trypanosome CRK complex since CYC2ty bound to leishmanial p12(cks1), and histone H1 kinase activity was detected in CYC2ty immune-precipitated fractions . Gene knockout experiments provide evidence that CYC2 is an essential gene, and co-immune precipitations together with a two-hybrid interaction assay demonstrated that CYC2 interacts with CRK3 . The CRK3 x CYC2ty complex, the first cyclin-dependent kinase complex identified in trypanosomes, was localized by immune fluorescence to the cytoplasm throughout the cell cycle. Biochem Biophys Res Commun, 2000 Mar 24, 269(3), 692 - 6 Studies on the relationships between the synonymous codon usage and protein secondary structural units; Gupta SK et al.; The relationship between the synonymous codon usage and protein secondary structural elements (alpha helices and beta sheets) were reinvestigated by taking structural information of proteins from Protein Data Bank (PDB) and their corresponding mRNA sequences from GenBank for four different organisms E . coli, B . subtilis, S . cerevisiae, and Homo sapiens . It was observed that synonymous codon families have non-random codon usage, but there does not exist any species invariant universal correlation between the synonymous codon usage and protein secondary structural elements . The secondary structural units of proteins can be distinguished from the occurrences of bases at the second codon position . Xenobiotica, 2000 Feb, 30(2), 155 - 77 DNA arrays: technology, options and toxicological applications; Rockett JC et al.; The human genome contains an estimated 3 billion bases of DNA making up some 100000 genes, and the variation within this genome accounts for human diversity and, in many cases, disease . Defining and understanding the expression profile of given genotypes is essential to understanding adverse effects from acute or chronic exposure to environmental toxicants or other stimuli . DNA array technology could help researchers understand how organisms function in response to exposure by elucidating the molecular mechanisms that underlie them . DNA arrays have been developed and refined over the past 5 years and matured into a relatively accessible and affordable technology . They vary in design from membrane-based filters with a few hundred cDNAs, to glass-based 'chips' with tens of thousands of genetic elements . Mammalian DNA arrays will soon allow expression analysis on a genome-wide scale, similar to that already accomplished in some lower organisms (e.g . S . cerevisiae, E . coli) . These whole-genome arrays will be powerful tools for identifying and characterizing toxicants in environmental and pharmaceutical science . This review discusses the technology behind the production of DNA arrays, the options available to those interested in applying them to their own research, and the possible toxicological applications of this exciting new technology. Gene, 2000 Mar 21, 245(2), 299 - 309 Evolution of a glucose-regulated ADH gene in the genus Saccharomyces; Young ET et al.; To determine when a glucose-repressed alcohol dehydrogenase isozyme and its regulatory gene, ADR1, arose during evolution, we surveyed species of the genus Saccharomyces for glucose-repressed ADH isozymes and for ADR1 homologues . Glucose-repressed ADH isozymes were present in all species of Saccharomyces sensu strictu and also in Saccharomyces kluyveri, the most distant member of the Saccharomyces clade . We cloned and characterized ADH promoters from S . bayanus, S . douglasii, and S . kluyveri . The ADH promoters from S . bayanus and S . douglasii had conserved sequences, including upstream regulatory elements, and an extended polydA tract . The expression of a reporter gene driven by the S . bayanus promoter was glucose-repressed and dependent on the major activator of transcription, ADR1, when it was introduced into S . cerevisiae . One S . kluyveri promoter was also glucose-repressed and ADR1-dependent in S . cerevisiae . The other S . kluyveri ADH promoter was expressed constitutively and was ADR1-independent . Although showing little sequence conservation with the S . cerevisiae ADH2 promoter, the glucose-repressed S . kluyveri promoter contains numerous potential binding sites for Adr1 . The glucose-repressed ADH from S . kluyveri is a mitochondrial isozyme most closely related to S . cerevisiae ADHIII . ADR1 homologues from S . douglasii and S . paradoxus contain a trinucleotide repeat encoding polyAsn that is lacking in S . cerevisiae and S . bayanus . No ADR1 homologue could be detected in S . kluyveri, suggesting that the potential for Adr1 regulation may have arisen first, before ADR1 evolved. Biochimie, 2000 Jan, 82(1), 71 - 8 Characterization of genetic interactions with RFA1: the role of RPA in DNA replication and telomere maintenance; Smith J et al.; Replication protein A (RPA) is a heterotrimeric single-stranded DNA binding protein whose role in DNA replication, recombination and repair has been mainly elucidated through in vitro biochemical studies utilizing the mammalian complex . However, the identification of homologs of all three subunits in Saccharomyces cerevisiae offers the opportunity of examining the in vivo role of RPA . In our laboratory, we have previously isolated a missense allele of the RFA1 gene, encoding the p70 subunit of the RPA complex . Strains containing this mutant allele, rfa1-D228Y, display increased levels of direct-repeat recombination, decreased levels of heteroallelic recombination, UV sensitivity and a S-phase delay . In this study, we have characterized further the role of RPA by screening other replication and repair mutants for a synthetic lethal phenotype in combination with the rfa1-D228Y allele . Among the replication mutants examined, only one displayed a synthetic lethal phenotype, pol12-100, a conditional allele of the B subunit of pol alpha-primase . In addition, a delayed senescence phenotype was observed in raf1-D228Y strains containing a null mutation of HDF1, the S . cerevisiae homolog of the 70 kDa subunit of Ku . Interestingly, a synergistic reduction in telomere length observed in the double mutants suggests that the shortening of telomeres may be the cause of the decreased viability in these strains . Furthermore, this result represents the first evidence of a role for RPA in telomere maintenance. J Biol Chem, 2000 Mar 17, 275(11), 7612 - 8 Two distinct nucleosome alterations characterize chromatin remodeling at the Saccharomyces cerevisiae ADH2 promoter; Di Mauro E et al.; Glucose depletion derepresses the Saccharomyces cerevisiae ADH2 gene; this metabolic change is accompanied by chromatin structural modifications in the promoter region . We show that the ADR6/SWI1 gene is not necessary for derepression of the wild type chromosomal ADH2, whereas the transcription factor Adr1p, which regulates several S . cerevisiae functions, plays a major role in driving nucleosome reconfiguration and ADH2 expression . When we tested the effect of individual domains of the regulatory protein Adr1p on the chromatin structure of ADH2, a remodeling consisting of at least two steps was observed . Adr1p derivatives were analyzed in derepressing conditions, showing that the Adr1p DNA binding domain alone causes an alteration in chromatin organization in the absence of transcription . This alteration differs from the remodeling observed in the presence of the Adr1p activation domain when the promoter is transcriptionally active. Mol Microbiol, 2000 Mar, 35(5), 1079 - 88 Molecular cloning of the calcium and sodium ATPases in Neurospora crassa; Benito B et al.; Using PCR, reverse transcription-PCR (RT-PCR) and colony hybridization in a genomic library, we isolated six genes which encode type II P-type ATPases in Neurospora crassa . The six full-length cDNAs were cloned in a yeast expression vector and transformed into Saccharomyces cerevisiae null Ca2+- or Na+-ATPase mutants . Three cDNAs suppressed the defect of the Ca2+ mutant and two of these protected from Mn2+ toxicity . One cDNA suppressed the defect of the Na+ mutant and two cDNAs were not functional in S . cerevisiae . The expression of the transcripts of the six genes in the presence of Ca2+, Na+, high pH or supporting an osmotic shock indicated that, with the exception of one of the Ca2+-ATPases, the main function of the cloned ATPases is the adaptation to stress conditions . The relationship between the cloned fungal Ca2+- and Na+-ATPases and plant type II P-ATPases is discussed. Eur J Biochem, 2000 Mar, 267(6), 1607 - 18 Effects of singlet oxygen on membrane sterols in the yeast Saccharomyces cerevisiae; Bocking T et al.; Photodynamic treatment of the yeast Saccharomyces cerevisiae with the singlet oxygen sensitizer toluidine blue and visible light leads to rapid oxidation of ergosterol and accumulation of oxidized ergosterol derivatives in the plasma membrane . The predominant oxidation product accumulated was identified as 5alpha, 6alpha-epoxy-(22E)-ergosta-8,22-dien-3beta,7a lpha-diol (8-DED) . 9(11)-dehydroergosterol (DHE) was identified as a minor oxidation product . In heat inactivated cells ergosterol is photooxidized to ergosterol epidioxide (EEP) and DHE . Disrupted cell preparations of S . cerevisiae convert EEP to 8-DED, and this activity is abolished in a boiled control indicating the presence of a membrane associated enzyme with an EEP isomerase activity . Yeast selectively mobilizes ergosterol from the intracellular sterol ester pool to replenish the level of free ergosterol in the plasma membrane during singlet oxygen oxidation . The following reaction pathway is proposed: singlet oxygen-mediated oxidation of ergosterol leads to mainly the formation of EEP, which is enzymatically rearranged to 8-DED . Ergosterol 7-hydroperoxide, a known minor product of the reaction of singlet oxygen with ergosterol, is formed at a much lower rate and decomposes to give DHE . Changes of physical properties of the plasma membrane are induced by depletion of ergosterol and accumulation of polar derivatives . Subsequent permeation of photosensitizer through the plasma membrane into the cell leads to events including impairment of mitochondrial function and cell inactivation. J Recept Signal Transduct Res, 2000 Jan, 20(1), 61 - 73 Golf complements a GPA1 null mutation in Saccharomyces cerevisiae and functionally couples to the STE2 pheromone receptor; Crowe ML et al.; We have produced a plasmid designed for the expression of heterologous G protein alpha subunits in the yeast Saccharomyces cerevisiae . Introduction of these genes is by simple cassette replacement using unique restriction sites, and their expression is controlled by the regulatory sequences of the S . cerevisiae GPA1 gene . Levels of expression are therefore suitable for interaction of these heterologous proteins with elements of the yeast pheromone response pathway . We believe that this plasmid will facilitate the coupling of more members of the seven transmembrane domain superfamily of receptors, through their native G protein alpha subunit, to the yeast pheromone response pathway . The plasmid pRGP, is a stable centromeric shuttle vector with a HIS3-selectable marker . We have demonstrated that production of GPA1 from this plasmid functionally complements a gpal1- null mutation . A similar response is obtained when an alternative G protein alpha subunit, G(olf), is introduced using pRGP . We believe that this is the first example of a heterologous G protein shown to couple to a yeast pheromone receptor. Biosci Biotechnol Biochem, 2000 Jan, 64(1), 167 - 72 Phosphatidylserine synthesis required for the maximal tryptophan transport activity in Saccharomyces cerevisiae; Nakamura H et al.; Saccharomyces cerevisiae cho1/pss mutants, which are severely impaired in phosphatidylserine (PS) synthesis, do not have detectable amounts of PS in their lipid fractions . Their derivatives with mutations that cause defects in tryptophan synthesis grew poorly in a medium containing 5 micrograms/ml of L-tryptophan, a concentration that met the requirements of tryptophanauxotrophic CHO1/PSS strains . The rates of tryptophan uptake of trp1 cho1/pss mutants were low at low tryptophan concentrations . This defect in the use of tryptophan was restored either by expression of CHO1/PSS or by introduction of a gene encoding tryptophan transporter, TAT1 or TAT2 . These results indicate that PS synthesis is required for the maximal tryptophan-transporting activity of S . cerevisiae at low tryptophan concentrations. Glycobiology, 2000 Mar, 10(3), 321 - 7 Cloning of the human cDNA which can complement the defect of the yeast mannosyltransferase I-deficient mutant alg 1; Takahashi T et al.; The assembly of the lipid-linked oligosaccharide, Glc(3)Man(9)GlcNAc(2)-P-P-Dol, occurs on the rough ER membrane in an ordered stepwise manner . The process is highly conserved among eukaryotes . In order to isolate the human mannosyltransferase I (MT-I) gene involved in the process, we used the Saccharomyces cerevisiae MT-I gene ( ALG1 ), which has already been cloned . On searching the EST database with the amino acid sequence of the ALG1 gene product, we detected seven related human EST clones . A human fetal brain cDNA library was screened by PCR using gene-specific primers based on the EST nucleotide sequences and a 430 bp cDNA fragment was amplified . The cDNA library was rescreened with this 430 bp cDNA, and two cDNA clones (HR1-3 and HR1-4) were isolated and sequenced . On a homology search of the EST database with the nucleotide sequence of HR1-3, we detected a novel human EST clone, AA675921 (GenBank accession number) . Based on the nucleotide sequences of AA675921 and HR1-4, we designed gene-specific PCR primers, which allowed to amplify a 1.8 kb cDNA from human fetal brain cDNA . This cDNA was cloned and shown to contain an ORF encoding a protein of 464 amino acids . We designated this ORF as Hmat-1 . The amino acid sequence deduced from the Hmat-1 gene showed several highly conserved regions shared with the yeast and nematode MT-I sequences . Furthermore, this 1.8 kb cDNA successfully complemented the S . cerevisiae alg1-1 mutation, indicating that the Hmat-1 gene encodes the human MT-I and that the function of this enzyme was conserved between yeast and human. J Biotechnol, 2000 Feb 28, 78(1), 11 - 21 Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae; Frenken LG et al.; Recently the existence of 'heavy chain' immunoglobulins in Camelidae has been described . However, as yet there is no data on the binding of this type of antibody to haptens . In addition, it was not a priori predictable whether the binding domains (VHH) of these antibodies could be produced and secreted by the lower eukaryotic micro-organism Saccharomyces cerevisiae . In the present study these questions are addressed . Heavy chain immunoglobulins directed against two hapten molecules, the azo-dyes RR6 and RR120 as well as the (proteinaceous) human pregnancy hormone, have been raised in Lama glama . We were able to select specific VHH fragments for all three antigens by direct screening of Escherichia coli or yeast libraries, even without prior enrichment via bio-panning . This is the first example of the isolation of llama anti-hapten VHH domains . Surprisingly, the affinities of the llama VHHs for the RR6 hapten obtained in this way are in the low nM range . Furthermore, some of the antigen specific VHHs were secreted by S . cerevisiae at levels over 100 mg l-1 in shake flask cultures . These two findings extend the possible application areas for the llama VHH fragments significantly. J Biol Chem, 2000 Mar 3, 275(9), 6530 - 6 The 8-nucleotide-long RNA:DNA hybrid is a primary stability determinant of the RNA polymerase II elongation complex; Kireeva ML et al.; The sliding clamp model of transcription processivity, based on extensive studies of Escherichia coli RNA polymerase, suggests that formation of a stable elongation complex requires two distinct nucleic acid components: an 8-9-nt transcript-template hybrid, and a DNA duplex immediately downstream from the hybrid . Here, we address the minimal composition of the processive elongation complex in the eukaryotes by developing a method for promoter-independent assembly of functional elongation complex of S . cerevisiae RNA polymerase II from synthetic DNA and RNA oligonucleotides . We show that only one of the nucleic acid components, the 8-nt RNA:DNA hybrid, is necessary for the formation of a stable elongation complex with RNA polymerase II . The double-strand DNA upstream and downstream of the hybrid does not affect stability of the elongation complex . This finding reveals a significant difference in processivity determinants of RNA polymerase II and E . coli RNA polymerase . In addition, using the imperfect RNA:DNA hybrid disturbed by the mismatches in the RNA, we show that nontemplate DNA strand may reduce the elongation complex stability via the reduction of the RNA:DNA hybrid length . The structure of a "minimal stable" elongation complex suggests a key role of the RNA:DNA hybrid in RNA polymerase II processivity. J Med Chem, 2000 Feb 24, 43(4), 683 - 9 Benzyl derivatives of 2,1,3-benzo- and benzothieno{3,2-a}thiadiazine 2,2-dioxides: first phosphodiesterase 7 inhibitors; Martinez A et al.; The synthesis of a new family of benzyl derivatives of 2,1,3-benzo- and benzothieno{3,2-a}thiadiazine 2,2-dioxides was achieved . The biological data revealed the first heterocyclic family of compounds with PDE 7 inhibitory properties appearing to be a new objective for the treatment of T-cell-dependent disorders . The IC(50) values or percent inhibition values of the compounds against PDE 7 were calculated by testing them against human recombinant PDE 7 expressed in S . cerevisiae . In this expression system the only cyclic nucleotide hydrolyzing activity present in cell extracts corresponded to human PDE 7 . Isoenzyme selectivity PDE 7 versus PDE 4 and PDE 3 was also measured . Considering simultaneously inhibition of the three different isoenzymes, monobenzyl derivatives 15 and 23 showed interesting PDE 7 potency (around 10 microM); although not statistically significant, a trend toward selectivity with respect to PDE 3 and PDE 4 was obtained . Benzothiadiazine 16, although less potent at PDE 7 (IC(50) = 25 microM), also showed a trend of selectivity toward PDE 3 and PDE 4 . These compounds are considered the best leads for further optimization. Gene, 2000 Feb 22, 244(1-2), 109 - 18 Isolation and functional characterization of a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae in translation initiation factor eIF5: an eIF5-dependent cell-free translation system; Maiti T et al.; Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S ribosomal initiation complex (40S.eIF3.AUG.Met-tRNA(f).eIF2.GTP) to promote the hydrolysis of bound GTP . In Saccharomyces cerevisiae, eIF5, a protein of 45346Da, is encoded by a single-copy essential gene, TIF5 . In this paper, we have isolated a temperature-sensitive S . cerevisiae strain, TMY5-1, by replacing the wild-type chromosomal copy of TIF5 with one mutagenized in vitro . The mutant yeast cells rapidly cease protein synthesis when grown under non-permissive conditions, lose polyribosomes and accumulate free 80S ribosomes . Further characterization of mutant eIF5 showed that the mutant protein, expressed in Escherichia coli, is defective both in its interaction with eIF2 as well as in mediating the hydrolysis of GTP bound to the 40S initiation complex and consequently in the formation of the 80S initiation complex . Additionally, the availability of a yeast strain containing temperature-sensitive mutation in the eIF5 gene allowed us to construct a cell-free translation system that was dependent on exogenously added eIF5 for translation of mRNAs in vitro. Nature, 2000 Feb 10, 403(6770), 623 - 7 A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae; Uetz P et al.; Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence . In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain . This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array . In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library . We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis . These approaches resulted in the detection of 957 putative interactions involving 1,004 S . cerevisiae proteins . These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes . The results of these screens are shown here. Int J Biochem Cell Biol, 2000 Feb, 32(2), 215 - 24 The overexpression of the CDC25 gene of Saccharomyces cerevisiae causes a derepression of GAL system and an increase of GAL4 transcription; Rudoni S et al.; The CDC25 gene product is an exchange factor for Ras proteins and it activates the Ras/cAMP pathway in the yeast Saccharomyces cerevisiae . The overexpression of the CDC25 gene in S . cerevisiae cells causes a partial glucose-derepressed phenotype which is particularly evident for expression of invertase . To define domains of Cdc25 protein relevant for this derepression and to test another glucose repressed system, different to invertase, we have overexpressed different regions of the CDC25 gene under the control of a GAL-promoter . We found that a derepression of both GAL regulated promoters and invertase was related to the overexpression of CDC25 regions that contain a functional guanine nucleotide exchange (GEF) domain . The effect on GAL-promoters was particular evident when the CDC25 gene was under the control of a UASgal element and operates at transcriptional level, although a moderate derepression was found also for UASgal/lacZ reporter gene . Finally, the overexpression of the GEF domain of CDC25 also caused an increase in the expression of the GAL4 regulatory gene, while a constitutive activation of the Ras/cAMP pathway did not produce any increase in GAL4 expression . These findings indicate that the overexpression of the catalytic domain of CDC25 gene is necessary and sufficient to give a glucose-derepression of GAL promoters and of invertase . They also suggest that the derepression of GAL promoters occurs through an increase of GAL4 expression in a Ras cAMP independent way. Biochim Biophys Acta, 2000 Feb 24, 1484(1), 71 - 82 Enzymological properties of the LPP1-encoded lipid phosphatase from Saccharomyces cerevisiae; Furneisen JM et al.; The product of the LPP1 gene in Saccharomyces cerevisiae is a membrane-associated enzyme that catalyzes the Mg(2+)-independent dephosphorylation of phosphatidate (PA), diacylglycerol pyrophosphate (DGPP), and lysophosphatidate (LPA) . The LPP1-encoded lipid phosphatase was overexpressed 681-fold in Sf-9 insect cells and used to examine the enzymological properties of the enzyme using PA, DGPP, and LPA as substrates . The optimum pH values for PA phosphatase, DGPP phosphatase, and LPA phosphatase activities were 7 . 5, 7.0, and 7.0, respectively . Divalent cations (Mn(2+), Co(2+), and Ca(2+)), NaF, heavy metals, propranolol, phenylglyoxal, and N-ethylmaleimide inhibited the PA phosphatase, DGPP phosphatase, and LPA phosphatase activities of the enzyme . The inhibitory effects of N-ethylmaleimide and phenylglyoxal on the LPP1-encoded enzyme were novel properties when compared with other Mg(2+)-independent lipid phosphate phosphatases from S . cerevisiae and mammalian cells . The LPP1-encoded enzyme exhibited saturation kinetics with respect to the surface concentrations of PA (K(m)=0.05 mol%), DGPP (K(m)=0.07 mol%), and LPA (K(m)=0.08 mol%) . Based on specificity constants (V(max)/K(m)LPA (1.3 units/mg/mol%) . DGPP (K(i)=0.12 mol%) was a competitive inhibitor with respect to PA, and PA (K(i)=0.12 mol%) was a competitive inhibitor with respect to DGPP . This suggested that the binding sites for these substrates were the same . The enzymological properties of the LPP1-encoded enzyme differed significantly from those of the S . cerevisiae DPP1-encoded lipid phosphatase, a related enzyme that also utilizes PA, DGPP, and LPA as substrates. J Biol Chem, 2000 Feb 25, 275(8), 5817 - 25 Isolation of the ace1 gene encoding a Cys(2)-His(2) transcription factor involved in regulation of activity of the cellulase promoter cbh1 of Trichoderma reesei; Saloheimo A et al.; A genetic selection method was developed for the cloning of positive-acting transcriptional regulatory genes in Saccharomyces cerevisiae . The method was applied for the isolation of activators of Trichoderma reesei (Hypocrea jecorina) cellulase genes . Activator genes were isolated from a T . reesei expression cDNA library on the basis of the ability of their translation products to activate transcription from the full-length T . reesei cbh1 promoter coupled to the S . cerevisiae HIS3 gene and to support the growth of the yeast colonies in the absence of histidine . Among the clones obtained was the ace1 gene encoding a novel polypeptide, ACEI, that contains three zinc finger motifs of Cys(2)-His(2) type . Possible ACEI homologues were found among expressed sequence tags of Aspergillus and Neurospora . The ability of ACEI to bind to the cbh1 promoter was further confirmed in the yeast one-hybrid system . In vitro binding and gel mobility shift assays revealed several binding sites for the ACEI protein in the cbh1 promoter . Disruption of the ace1 gene in T . reesei resulted in retarded growth of the fungus on a cellulose-containing medium, on which cellulases are normally highly expressed. Trends Cell Biol, 2000 Mar, 10(3), 98 - 105 Protein-only inheritance in yeast: something to get {PSI+}-ched about; Serio TR et al.; Recent work suggests that two unrelated phenotypes, {PSI+} and {URE3}, in the yeast Saccharomyces cerevisiae are transmitted by non-covalent changes in the physical states of their protein determinants, Sup35p and Ure2p, rather than by changes in the genes that encode these proteins . The mechanism by which alternative protein states are self-propagating is the key to understanding how proteins function as elements of epigenetic inheritance . Here, we focus on recent molecular-genetic analysis of the inheritance of the {PSI+} factor of S . cerevisiae . Insights into this process might be extendable to a group of mammalian diseases (the amyloidoses), which are also believed to be a manifestation of self-perpetuating changes in protein conformation. Biochim Biophys Acta, 2000 Feb 15, 1463(2), 407 - 18 Properties of a reconstituted eukaryotic hexose/proton symporter solubilized by structurally related non-ionic detergents: specific requirement of phosphatidylcholine for permease stability; Robl I et al.; Overexpression of the hexose/proton symporter HUP1 from Chlorella kessleri in S . cerevisiae permits a one-step purification via a biotinylation domain . Milligram amounts of the protein are obtained starting from 2 l of yeast culture . The HUP1 protein is used as a model eukaryotic membrane protein of the 'major facilitator superfamily' (MFS) to study specific lipid requirements for activity and stability . Testing two series of detergents revealed that n-nonyl-beta-D-glucoside (NG) and n-octyl-beta-D-glucoside (OG) solubilize the HUP1 protein efficiently . Only the use of NG resulted in long-term stabilization of the HUP1 protein in the absence of external lipids . When affinity purified protein was extracted with organic solvents, a stoichiometric amount of phosphatidyl choline, phosphatidyl ethanolamine and ergosterol in the ratio of close to 2:1 was detected . These lipids were only observed, however, when the protein purification was carried out in the presence of NG; no lipids were copurified with the HUP1 protein in the presence of OG . Of the three lipids copurified, phosphatidyl choline showed a crucial role in ensuring maximal HUP1 permease activity and stability when added back to the OG-protein . The requirement of phosphatidylcholine documents a specific effect of lipids on vectorial transport mediated by a eukaryotic membrane protein of the MFS family. Eur J Cell Biol, 1999 Dec, 78(12), 892 - 902 Comparative analysis of Ca2+ and H+ flux magnitude and location along growing hyphae of Saprolegnia ferax and Neurospora crassa; Lew RR; Calcium and proton ion fluxes were mapped at the growing apices of two hyphal organisms, the oomycete Saprolegnia ferax and the ascomycete Neurospora crassa and pseudohyphal Saccharomyces cerevisiae using self-referencing ion-selective probes . S . ferax exhibited well-defined transport zones absent in N . crassa . Ca2+ fluxes were located within 8 microm of the growing hyphal tip; the net Ca2+ flux was either inward (75% of all experiments) or outward . The inward component of the net flux was inhibited by Gd3+, known to inhibit Ca2+ permeable stretch-activated channels . Because the Ca2+ flux is located at the region of maximal hyphal expansion, exocytosis may contribute to Ca2+ efflux, in addition to the stretch-activated channel mediated influx . Maximal inward H+ flux was observed 10-30 microm behind the hyphal tip where peak mitochondria densities taper off at the onset of a vacuolation zone, presumably due to highly localized H+ cotransporter activity . By contrast, N . crassa exhibited no net Ca2+ flux and a consistently inward H+ flux (93% of all experiments) that was homogeneously distributed up to 60 microm behind the hyphal apex . Both hyphal organisms have similar tip morphology and growth rates, and are reported to have tip-high cytosolic Ca2+ gradients associated with growth . Only S . ferax exhibited tip-localized Ca2+ fluxes and a well defined H+ influx zone just behind the tip . Differences in ecological habitats and cytology--S . ferax is an aquatic organism that grows as a migrating plug of cytoplasm while N . crassa is normally terrestrial with a cytoplasm-rich mycelium and highly active cytoplasmic streaming behind the growing margin--may account for the differences in the 'architecture' of ion transport occurring during the process of tip growth . Net Ca2+ efflux and H+ influx of growing S . cerevisiae pseudohyphae were also measured but localization was not possible due to small cell size. J Cell Biol, 2000 Feb 7, 148(3), 405 - 16 A role for centrin 3 in centrosome reproduction; Middendorp S et al.; Centrosome reproduction by duplication is essential for the bipolarity of cell division, but the molecular basis of this process is still unknown . Mutations in Saccharomyces cerevisiae CDC31 gene prevent the duplication of the spindle pole body (SPB) . The product of this gene belongs to the calmodulin super-family and is concentrated at the half bridge of the SPB . We present a functional analysis of HsCEN3, a human centrin gene closely related to the CDC31 gene . Transient overexpression of wild-type or mutant forms of HsCen3p in human cells demonstrates that centriole localization depends on a functional fourth EF-hand, but does not produce mitotic phenotype . However, injection of recombinant HsCen3p or of RNA encoding HsCen3p in one blastomere of two-cell stage Xenopus laevis embryos resulted in undercleavage and inhibition of centrosome duplication . Furthermore, HsCEN3 does not complement mutations or deletion of CDC31 in S . cerevisiae, but specifically blocks SPB duplication, indicating that the human protein acts as a dominant negative mutant of CDC31 . Several lines of evidence indicate that HsCen3p acts by titrating Cdc31p-binding protein(s) . Our results demonstrate that, in spite of the large differences in centrosome structure among widely divergent species, the centrosome pathway of reproduction is conserved. J Biol Chem, 2000 Feb 11, 275(6), 4391 - 7 A human REV7 homolog that interacts with the polymerase zeta catalytic subunit hREV3 and the spindle assembly checkpoint protein hMAD2; Murakumo Y et al.; Widespread alteration of the genomic DNA is a hallmark of tumors, and alteration of genes involved in DNA maintenance have been shown to contribute to the tumorigenic process . The DNA polymerase zeta of Saccharomyces cerevisiae is required for error-prone repair following DNA damage and consists of a complex between three proteins, scRev1, scRev3, and scRev7 . Here we describe a candidate human homolog of S . cerevisiae Rev7 (hREV7), which was identified in a yeast two-hybrid screen using the human homolog of S . cerevisiae Rev3 (hREV3) . The hREV7 gene product displays 23% identity and 53% similarity with scREV7, as well as 23% identity and 54% similarity with the human mitotic checkpoint protein hMAD2 . hREV7 is located on human chromosome 1p36 in a region of high loss of heterozygosity in human tumors, although no alterations of hREV3 or hREV7 were found in primary human tumors or human tumor cell lines . The interaction domain between hREV3 and hREV7 was determined and suggests that hREV7 probably functions with hREV3 in the human DNA polymerase zeta complex . In addition, we have identified an interaction between hREV7 and hMAD2 but not hMAD1 . While overexpression of hREV7 does not lead to cell cycle arrest, we entertain the possibility that it may act as an adapter between DNA repair and the spindle assembly checkpoint. J Mol Biol, 2000 Jan 28, 295(4), 1073 - 87 PYD2 encodes 5,6-dihydropyrimidine amidohydrolase, which participates in a novel fungal catabolic pathway; Gojkovic Z et al.; Most fungi cannot use pyrimidines or their degradation products as the sole nitrogen source . Previously, we screened several yeasts for their ability to catabolise pyrimidines . One of them, Saccharomyces kluyveri, was able to degrade the majority of pyrimidines . Here, a series of molecular techniques have been modified to clone pyrimidine catabolic genes, study their expression and purify the corresponding enzymes from this yeast . The pyd2-1 mutant, which lacked the 5,6-dihydropyrimidine amidohydrolase (DHPase) activity, was transformed with wild-type S . kluyveri genomic library . The complementing plasmid contained the full sequence of the PYD2 gene, which exhibited a high level of homology with mammalian DHPases and bacterial hydantoinases . The organisation of PYD2 showed a couple of specific features . The 542-codons open reading frame was interrupted by a 63 bp intron, which does not contain the Saccharomyces cerevisiae branch-point sequence, and the transcripts contained a long 5' untranslated leader with five or six AUG codons . The derived amino acid sequence showed similarities with dihydroorotases, allantoinases and uricases from various organisms . Surprisingly, the URA4 gene from S . cerevisiae, which encodes dihydroorotase, shows greater similarity to PYD2 and other catabolic enzymes than to dihydroorotases from several other non-fungal organisms . The S . kluyveri DHPase was purified to homogeneity and sequencing of the N-terminal region revealed that the purified enzyme corresponds to the PYD2 gene product . The enzyme is a tetramer, likely consisting of similar if not identical subunits each with a molecular mass of 59 kDa . The S . kluyveri DHPase was capable of catalysing both dihydrouracil and dihydrothymine degradation, presumably by the same reaction mechanism as that described for mammalian DHPase . On the other hand, the regulation of the yeast PYD2 gene and DHPase seem to be different from that in other organisms . DHPase activity and Northern analysis demonstrated that PYD2 expression is inducible by dihydrouracil, though not by uracil . Apparently, dihydrouracil and DHPase represent an important regulatory checkpoint of the pyrimidine catabolic pathway in S . kluyveri . Curr Genet, 1999 Dec, 36(6), 339 - 44 Use of sulfite resistance in Saccharomyces cerevisiae as a dominant selectable marker; Park H et al.; Two S . cerevisiae genes were found to exhibit dominant phenotypes useful for selecting transformants of industrial and laboratory strains of S . cerevisiae . FZF1-4, which confers sulfite resistance, was originally isolated and identified as RSU1-4, but the two genes are shown here to be allelic . Cysteine 57 in wild-type Fzf1p was found to be replaced by tyrosine in Fzf1-4p . Multicopy SSU1, which also confers sulfite resistance, was found to be somewhat less efficient . In both cases, a period of outgrowth in non-selective medium following transformation was found to be necessary . The number of transformants obtained was found to be strain-dependent, and also to depend on the sulfite concentration used during selection . Undesirable background growth of non-transformants was not observed at cell densities as high as 2.5 x 10(7)/plate . In two ura3 laboratory strains where selection for URA3 was applied independently of that for sulfite, the transformation efficiency for sulfite resistance was about 50% that for uracil prototrophy. Curr Genet, 1999 Dec, 36(6), 317 - 28 Substrate specificity and gene expression of the amino-acid permeases in Saccharomyces cerevisiae; Regenberg B et al.; All known amino-acid permeases (AAPs) in Saccharomyces cerevisiae belong to a single family of homologous proteins . Genes of 15 AAPs were overexpressed in different strains, and the ability to take up one or more of the 20 common L-alpha-amino acids was studied in order to obtain a complete picture of the substrate specificity for these permeases . Radiolabelled amino-acid uptake measurements showed that Agp1p is a general permease for most uncharged amino acids (Ala, Gly, Ser, Thr, Cys, Met, Phe, Tyr, Ile, Leu, Val, Gln and Asn) . Gnp1p, which is closely related to Agp1p, has a somewhat less-broad specificity, transporting Leu, Ser, Thr, Cys, Met, Gln and Asn, while Bap2p and Bap3p, which are also closely related to Agp1p, are able to transport Ile, Leu, Val, Cys, Met, Phe, Tyr and Trp . All four permeases are transcriptionally induced by an extracellular amino acid, but differ in expression with respect to the nitrogen source . On a non-repressive nitrogen source, AGP1 is induced, while GLN1, BAP2 and BAP3 are not . Except for Dip5p, which is a transporter for Glu, Asp, Gln, Asn, Ser, Ala and Gly, the rest of the permeases exhibit narrow specificity . Tat2p can take up Phe, Trp and Tyr; Put4p can transport Ala, Gly and Pro; while Can1p, Lyp1p and the previously uncharacterized Alp1p are specific for the cationic amino acids . These findings modify the prevalent view that S . cerevisiae only contains one general amino-acid permease, Gap1p, and a number of permeases that are specific for a single or a few amino acids. Appl Environ Microbiol, 2000 Feb, 66(2), 775 - 82 Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger; Ngiam C et al.; Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes . A gene, pdiA, encoding PDIA was previously isolated from Aspergillus niger, and we report its functional characterization here . Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A . pdiA also complemented PDI function in a Saccharomyces cerevisiae Deltapdi1 mutant in a yeast-based killer toxin assay . Levels of pdiA mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum . This response of pdiA mRNA levels was slower and lower in magnitude than that of A . niger bipA, suggesting that the induction of pdiA is not part of the primary stress response . An increased level of pdiA transcripts was also observed in two A . niger strains overproducing a heterologous protein, hen egg white lysozyme (HEWL) . Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S . cerevisiae, overexpression of PDIA did not increase secreted yields of HEWL in A . niger, suggesting that PDIA itself is not limiting for secretion of this protein . Downregulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%. Appl Biochem Biotechnol, 1999 Sep, 81(3), 153 - 60 Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems; Wang H et al.; Functional expression of a beta-D-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413 . Optimal activity of the E . coli-expressed enzyme was found at incubation temperatures of 60 degrees C, whereas the enzyme activity was optimal at 40 degrees C when expressed by S . cerevisiae . Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0 . Yeast expression resulted in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa . The hyperglycosylated protein had reduced enzyme activity, indicating that E . coli is a preferred vehicle for production scale-up. Crit Rev Eukaryot Gene Expr, 1999, 9(3-4), 221 - 30 Interplay between chromatin modifying and remodeling complexes in transcriptional regulation; Belotserkovskaya R et al.; The question of a possible functional relationship between different chromatin-altering enzymatic activities is of great interest . Several remarkable parallels have been revealed regarding the action of the remodeling complex SWI/SNF and the histone acetylation complex SAGA during transcriptional activation in S . cerevisiae . Many promoters, but not all, that require one complex require the other as well . Mutations that disrupt both complexes cause much more severe phenotypes than single mutations . Both types of complexes are recruited to specific promoters by interaction with DNA-bound acidic activators, resulting in targeted acetylation and transcriptional activation . Taken together the data argue for independent mechanisms, but similar recruitment and functional interplay between these two types of chromatin-altering activities. Genes Genet Syst, 1999 Aug, 74(4), 159 - 67 Meiotic contraction of CAG repeats in Saccharomyces cerevisiae; Arai N et al.; Several human neurodegenerative disorders are caused by expansion of CAG repeats that occurs during meiosis or gametogenesis . We anticipated that the CAG repeats cloned in a plasmid of Saccharomyces cerevisiae might undergo a change in the number of repeats during meiosis and sporulation . To test this possibility, we devised a new method to change in vitro the number of CAG repeats and constructed plasmids carrying (CAG)39, (CAG)65 or (CAG)123 from a plasmid carrying (CAG)18 . We monitored the number of colonies showing an altered length of the repeat tracts during mitosis and meiotic growth . Contraction of long CAG repeat was found to occur frequently, whereas a few cases of expansion were observed . The contraction was equally enhanced in both orientations when the host cells grew through meiosis . Thus, our results suggest that long CAG repeats are destabilized during meiosis or gametogenesis in S . cerevisiae. FEMS Microbiol Lett, 2000 Feb 1, 183(1), 131 - 5 Purification and characterization of acidic endo-polygalacturonase encoded by the PGL1-1 gene from Saccharomyces cerevisiae; Gainvors A et al.; The PGL1 gene of the yeast Saccharomyces cerevisiae has been shown to encode polygalacturonase . Cloning of the PGL1 open reading frame behind the ADH1 promoter allowed overexpression of polygalacturonase activity in S . cerevisiae . This enzyme was purified to apparent homogeneity from cultures of recombinant S . cerevisiae on synthetic medium using one-step purification by anionic exchange chromatography . The enzyme, named Pgl1P, had an apparent M(r) of 42 kDa as shown by SDS-PAGE . Pgl1P was active from pH 3 to 5.5, with an optimum temperature at 25 degrees C . This enzyme hydrolyzed polygalacturonic acid as an endo-polygalacturonase as demonstrated by independent methods . The purified protein was N-glycosylated . However, the activity remained in the N-deglycosylated form . The N-terminal amino acid sequence was also determined as D-S-C-T-L-T-G-S-S-L. Yeast, 2000 Feb, 16(3), 255 - 65 Inactivation of six genes from chromosomes VII and XIV of Saccharomyces cerevisiae and basic phenotypic analysis of the mutant strains; Sartori G et al.; Within the frame of the EUROFAN project, aimed at the functional analysis of the novel ORFs revealed by the systematic sequencing of the Saccharomyces cerevisiae genome, we have inactivated six ORFs encoding putative proteins with unknown function in the two S . cerevisiae strains FY1679 and W303-1B . Five ORFs are located on chromosome VII (YGR250c, YGR251w, YGR260w, YGR262c, YGR263c) and one on chromosome XIV (YNL234w) . The genes have been inactivated in the FY1679 strain by a strategy that makes use of deletion cassettes containing the kanMX4 module, which confers resistance to geneticin to yeast cells, and short flanking regions homologous to the target locus (SFH) . Tetrad dissection of heterozygous mutants and basic phenotypic analysis of the spores revealed that ORF YGR251w is an essential gene, while the disruption of YGR262c causes a severe slow-growth phenotype . Deletion of the remaining ORFs did not give rise to a detectable phenotype in the mutant strains . For each ORF we have cloned, in the pUG7 plasmid, a replacement cassette that possesses long flanking regions homologous to the target locus (LFH) and, in the pRS416 plasmid, the cognate wild-type gene . The LFH replacement cassettes were used to inactivate the respective genes in the W303-1B strain . This work has been performed in the framework of the B0 Consortium of the EUROFAN I project . J Biol Chem, 2000 Jan 21, 275(3), 2130 - 6 The RNA interacting domain but not the protein interacting domain is highly conserved in ribosomal protein P0; Rodriguez-Gabriel MA et al.; Protein P0 interacts with proteins P1alpha, P1beta, P2alpha, and P2beta, and forms the Saccharomyces cerevisiae ribosomal stalk . The capacity of RPP0 genes from Aspergillus fumigatus, Dictyostelium discoideum, Rattus norvegicus, Homo sapiens, and Leishmania infantum to complement the absence of the homologous gene has been tested . In S . cerevisiae W303dGP0, a strain containing standard amounts of the four P1/P2 protein types, all heterologous genes were functional except the one from L . infantum, some of them inducing an osmosensitive phenotype at 37 degrees C . The polymerizing activity and the elongation factor-dependent functions but not the peptide bond formation capacity is affected in the heterologous P0 containing ribosomes . The heterologous P0 proteins bind to the yeast ribosomes but the composition of the ribosomal stalk is altered . Only proteins P1alpha and P2beta are found in ribosomes carrying the A . fumigatus, R . norvegicus, and H . sapiens proteins . When the heterologous genes are expressed in a conditional null-P0 mutant whose ribosomes are totally deprived of P1/P2 proteins, none of the heterologous P0 proteins complemented the conditional phenotype . In contrast, chimeric P0 proteins made of different amino-terminal fragments from mammalian origin and the complementary carboxyl-terminal fragments from yeast allow W303dGP0 and D67dGP0 growth at restrictive conditions . These results indicate that while the P0 protein RNA-binding domain is functionally conserved in eukaryotes, the regions involved in protein-protein interactions with either the other stalk proteins or the elongation factors have notably evolved. J Biol Chem, 2000 Jan 21, 275(3), 1615 - 24 Identification of the Xenopus laevis homolog of Saccharomyces cerevisiae DNA2 and its role in DNA replication; Liu Q et al.; The DNA2 gene of Saccharomyces cerevisiae is essential for growth and appears to be required for a late stage of chromosomal DNA replication . S . cerevisiae Dna2p (ScDna2p) is a DNA helicase and also a nuclease . We have cloned and sequenced the homologous gene from Xenopus (Xenopus Dna2) . Xenopus Dna2p (XDna2p) is 32% identical to ScDna2p, and the similarity extends over the entire length, including but not limited to the five conserved helicase motifs . XDna2p is even more closely related (60% identical) to a partial human cDNA . The Xenopus Dna2 (XDna2) gene was able to complement an S . cerevisiae dna2-1 mutant strain for growth at the nonpermissive temperature, suggesting that XDna2p is a functional as well as a structural homolog of the yeast protein . Recombinant XDna2p was expressed in insect cells and purified . Like the ScDna2p purified from yeast, it is a single-stranded DNA endonuclease and a DNA-dependent ATPase, suggesting that both of these activities are part of the essential function of Dna2p . However, unlike ScDna2p from yeast, recombinant XDna2p showed no DNA helicase activity . When XDna2 was immunodepleted from interphase egg extracts, chromosomal DNA replication was almost completely inhibited . From the size of the residually synthesized DNA from the XDna2-depleted egg extracts, it seems that initiation of DNA replication may be impaired . This interpretation is also supported by the normal DNA replication of M13 single-stranded DNA in the XDna2-depleted egg extracts. Mol Cell, 1999 Dec, 4(6), 1005 - 15 Near-cognate peptidyl-tRNAs promote +1 programmed translational frameshifting in yeast; Sundararajan A et al.; Translational frameshifting is a ubiquitous, if rare, form of alternative decoding in which ribosomes spontaneously shift reading frames during translation elongation . In studying +1 frameshifting in Ty retrotransposons of the yeast S . cerevisiae, we previously showed that unusual P site tRNAs induce frameshifting . The frameshift-inducing tRNAs we show here are near-cognates for the P site codon . Their abnormal decoding induces frameshifting in either of two ways: weak codon-anticodon pairing allows the tRNA to disengage from the mRNA and slip +1, or an unusual codon-anticodon structure interferes with cognate in-frame decoding allowing out-of-frame decoding in the A site . We draw parallels between this mechanism and a proposed mechanism of frameshift suppression by mutant tRNAs. Mol Cell, 1999 Dec, 4(6), 925 - 34 The engagement of Sec61p in the ER dislocation process; Zhou M et al.; Sec61p comprises the endoplasmic reticulum (ER) channel through which nascent polypeptides are imported and from which malfolded proteins have been suggested to be exported, or dislocated, back to the cytoplasm . We have devised a genetic screen for dislocation-specific mutant alleles of SEC61 from S . cerevisiae by employing the unfolded protein response to report on the accumulation of misfolded proteins in the ER . Three of the isolated sec61 alleles are fully proficient in protein translocation into the ER, but defective in the elimination of a misfolded ER luminal substrate and a short-lived ER membrane-spanning model protein, which are otherwise rapidly degraded by cytoplasmic proteolysis in wild-type cells . Our results point to the fourth luminal loop and third transmembrane domain of Sec61p that markedly influence dislocation . We suggest that distinct features of the Sec61-translocon direct the two-way translocation processes. Arch Microbiol, 2000 Nov, 174(5), 340 - 5 Glucose does not activate the plasma-membrane-bound H+-ATPase but affects pmaA transcript abundance in Aspergillus nidulans; Abdallah BM et al.; The addition of glucose to starved cells of Aspergillus nidulans increased the abundance of the pmaA transcript only transiently (15 min) and to a very low degree (1.3-fold), but strongly decreased its abundance during further incubation . This down-regulation was CreA (carbon catabolite repressor protein)-dependent . Glucose failed to stimulate the plasma membrane (PM)-ATPase activity of A . nidulans, whereas under the same experimental conditions the activity of the enzyme from Saccharomyces cerevisiae was enhanced four-fold within 5-10 min following glucose addition . Glucose stimulated the PM-ATPase of Neurospora crassa only 1.3-fold . Sequence comparison of the C-terminal end of the PM-ATPase from S . cerevisiae, N . crassa, A . nidulans, Fusarium sporotrichoides and Penicillium simplicissimum showed that the two regulatory sites necessary for glucose stimulation in S . cerevisiae are conserved in N . crassa and F . sporotrichoides but not in A . nidulans and P . simplicissimum, and their presence therefore does not correlate with glucose stimulation . We conclude that, in contrast to S . cerevisiae, which has become a paradigm of fungal glucose metabolism, glucose does not up-regulate the activity of the plasma membrane ATPase in the filamentous fungi examined. Genetics, 2000 Jan, 154(1), 73 - 81 The product of the DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, specifically functions in mitochondria; Fikus MU et al.; We reported previously that the product of the DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, belongs to a family of proteins that are involved in DNA repair and replication . The family includes S . cerevisiae proteins Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I) . Here, we report that Din7p specifically affects metabolism of mitochondrial DNA (mtDNA) . We have found that dun1 strains, defective in the transcriptional activation of the DNA damage-inducible genes RNR1, RNR2, and RNR3, exhibit an increased frequency in the formation of the mitochondrial petite (rho(-)) mutants . This high frequency of petites arising in the dun1 strains is significantly reduced by the din7::URA3 allele . On the other hand, overproduction of Din7p from the DIN7 gene placed under control of the GAL1 promoter dramatically increases the frequency of petite formation and the frequency of mitochondrial mutations conferring resistance to erythromycin (E(r)) . The frequencies of chromosomal mutations conferring resistance to canavanine (Can(r)) or adenine prototrophy (Ade(+)) are not affected by enhanced synthesis of Din7p . Experiments using Din7p fused to the green fluorescent protein (GFP) and cell fractionation experiments indicate that the protein is located in mitochondria . A possible mechanism that may be responsible for the decreased stability of the mitochondrial genome in S . cerevisiae cells with elevated levels of Din7p is discussed. Biochem Biophys Res Commun, 2000 Jan 7, 267(1), 164 - 8 GPR1 regulates filamentous growth through FLO11 in yeast Saccharomyces cerevisiae; Tamaki H et al.; Cell growth and differentiation are regulated by nutrient availability in the yeast Saccharomyces cerevisiae . Under conditions of nitrogen limitation, diploid cells of S . cerevisiae differentiate to a filamentous growth known as a pseudohyphal growth, while haploid cells produce invasive filaments which penetrate the agar in nutrient-rich medium . We have found that GPR1, which encodes a putative G-protein-coupled receptor, is required for both pseudohyphal and invasive growth . Pseudohyphal growth was defective in Deltagpr1/Deltagpr1 mutant strain and this defect was reversed by addition of cAMP . Also, haploid Deltagpr1 mutant strain was defective in invasive growth . Northern blot analysis revealed that the transcriptional level of FLO11, which encodes a recently identified cell surface flocculin required for pseudohyphal growth, was reduced in Deltagpr1 mutant strain . These results indicate that GPR1 regulates both pseudohyphal and invas