S. cerevisiae

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 invasive growth by a cAMP-dependent mechanism .

FEBS Lett, 1999 Dec 3, 462(3), 472 - 6
Identification of the mitochondrial carnitine carrier in Saccharomyces cerevisiae; Palmieri L et al.; The mitochondrial carrier protein for carnitine has been identified in Saccharomyces cerevisiae . It is encoded by the gene CRC1 and is a member of the family of mitochondrial transport proteins . The protein has been over-expressed with a C-terminal His-tag in S . cerevisiae and isolated from mitochondria by nickel affinity chromatography . The purified protein has been reconstituted into proteoliposomes and its transport characteristics established . It transports carnitine, acetylcarnitine, propionylcarnitine and to a much lower extent medium- and long-chain acylcarnitines.

Yeast, 2000 Jan 15, 16(1), 11 - 22
Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast/mammalian G protein alpha-subunit chimeras; Brown AJ et al.; The expression of mammalian G protein coupled receptors (GPCRs) in S . cerevisiae provides a powerful assay system for functional analysis, ligand identification and pharmaceutical screening . However, relatively few receptors have been coupled to the pheromone response pathway via the yeast G(alpha), Gpa1p, or chimeric yeast/mammalian G(alpha) subunits containing long C-terminal regions of mammalian G(alpha) proteins . We tested an extended range of seven such chimeras for G(alpha) sub-types of three major classes (G(alphai/o), G(alphas) and G(alphaq)), against eight human GPCRs (SST(2), SST(5), 5-HT(1A), 5-HT(1Dalpha), ML(1B), P2Y(1) and P2Y(2)) . Although the G(alphai/o) chimeras increased the range of receptors that coupled efficiently, the G(alphas) and G(alphaq) chimeras were inactive when expressed using the GPA1 promoter . We describe 10 novel Gpa1p chimeras, designated 'transplants', in which the C-terminal five amino acids of Gpa1p were exchanged with mammalian residues . Coupling efficiency and ligand sensitivity improved significantly using the transplants . For the P2Y purinergic receptors, coupling could only be detected with the transplants; this is the first report of G(q) specificity coupling in yeast . Thus, the transplants offer major advantages over previously described approaches, in terms of both the range of receptors coupled and the efficiency of coupling .

Genes Cells, 1999 Nov, 4(11), 607 - 18
Mutation enhancement by DINB1, a mammalian homologue of the Escherichia coli mutagenesis protein dinB; Ogi T et al.; BACKGROUND: The Escherichia coli dinB gene is an SOS gene known to be required for lambda phage untargeted mutagenesis . When over-expressed, it exhibits a potent mutagenic activity without any exogenous treatment to damage DNA . Frameshift mutations at a run of identical bases are most enhanced . The product DinB is structurally related to the E . coli UmuC protein and the Saccharomyces cerevisiae Rev1 and Rad30 proteins, all of which are shown to be involved in bypass synthesis at a DNA lesion . RESULTS: We have cloned and sequenced human and mouse cDNAs encoding a DinB homologue . Their products are highly similar to DinB and less similar to UmuC, Rev1 or Rad30, and hence the genes were named DINB1 for human and Dinb1 for mouse . Both genes were expressed most abundantly in testis . Transient expression of the mouse cDNA in cultured mouse cells resulted in a nearly 10-fold increase in the incidence of point mutations, among which about 30% were frameshift mutations . CONCLUSIONS: The above results suggest that a mutagenic mechanism, a so-called untargeted type, also operates in mammalian cells . Taken together with recent findings that human cells have multiple DNA polymerases for translesion synthesis which are homologous to the S . cerevisiae Rev3 and Rad30 proteins, our results imply that multiple mutagenic pathways are conserved from bacteria to higher eukaryotes.

Cell, 1999 Dec 23, 99(7), 723 - 33
Telomerase-mediated telomere addition in vivo requires DNA primase and DNA polymerases alpha and delta; Diede SJ et al.; To better understand the requirements for telomerase-mediated telomere addition in vivo, we developed an assay in S . cerevisiae that creates a chromosome end immediately adjacent to a short telomeric DNA tract . The de novo end acts as a telomere: it is protected from degradation in a CDC13-dependent manner, telomeric sequences are added efficiently, and addition occurs at a faster rate in mutant strains that have long telomeres . Telomere addition was detected in M phase arrested cells, which permitted us to determine that the essential DNA polymerases alpha and delta and DNA primase were required . This indicates that telomeric DNA synthesis by telomerase is tightly coregulated with the production of the opposite strand . Such coordination prevents telomerase from generating excessively long single-stranded tails, which may be deleterious to chromosome stability in S . cerevisiae.

Mol Cell, 1999 Nov, 4(5), 873 - 81
Patching broken chromosomes with extranuclear cellular DNA; Yu X et al.; Chromosomal double-strand breaks (DSBs) can be repaired by either homology-dependent or homology-independent pathways . Using a novel intron-based genetic assay to identify rare homology-independent DNA rearrangements associated with repair of a chromosomal DSB in S . cerevisiae, we observed that approximately 20% of rearrangements involved endogenous DNA insertions at the break site . We have analyzed 37 inserts and find they fall into two distinct classes: Ty1 cDNA intermediates varying in length from 140 bp to 3.4 kb and short mitochondrial DNA fragments ranging in size from 33 bp to 219 bp . Several inserts consist of multiple noncontiguous mitochondrial DNA segments . These results demonstrate an ongoing mechanism for genome evolution through acquisition of organellar and mobile DNAs at DSB sites.

Mol Cell, 1999 Nov, 4(5), 805 - 14
The pachytene checkpoint in S . cerevisiae depends on Swe1-mediated phosphorylation of the cyclin-dependent kinase Cdc28; Leu JY et al.; Mutants defective in meiotic recombination and synaptonemal complex formation undergo checkpoint-mediated arrest in mid-meiotic prophase . In S . cerevisiae, this checkpoint requires Swe1, which phosphorylates and inactivates the cyclin-dependent kinase Cdc28 . A swe1 deletion allows mutants that normally arrest in meiotic prophase to sporulate at wild-type levels, though sporulation is delayed . This delay is eliminated by overproducing Clb1, the major cyclin required for meiosis I . The Swe1 protein accumulates and is hyperphosphorylated in checkpoint-arrested cells . Our results suggest that meiotic arrest is mediated both by increasing Swe1 activity and limiting cyclin production, with Swe1 being the primary downstream target of checkpoint control . The requirement for Swe1 distinguishes the pachytene checkpoint from the DNA damage checkpoints operating in vegetative cells.

Mol Cell, 1999 Nov, 4(5), 715 - 23
Two functionally distinct forms of the RSC nucleosome-remodeling complex, containing essential AT hook, BAH, and bromodomains; Cairns BR et al.; RSC is an essential 15 protein nucleosome-remodeling complex from S . cerevisiae . We have identified two closely related RSC members, Rsc1 and Rsc2 . Biochemical analysis revealed Rsc1 and Rsc2 in distinct complexes, defining two forms of RSC . Genetic analysis has shown that Rsc1 and Rsc2 possess shared and unique functions . Rsc1 and Rsc2 each contain two bromodomains, a bromo-adjacent homology (BAH) domain, and an AT hook . One of the bromodomains, the BAH domain, and the AT hook are each essential for Rsc1 and Rsc2 functions, although they are not required for assembly into RSC complexes . Therefore, these domains are required for RSC function . Additional genetic analysis provides further evidence that RSC function is related to transcriptional control.

J Bacteriol, 2000 Jan, 182(1), 76 - 80
Copper/zinc-Superoxide dismutase is required for oxytetracycline resistance of Saccharomyces cerevisiae; Avery SV et al.; Saccharomyces cerevisiae, along with other eukaryotes, is resistant to tetracyclines . We found that deletion of SOD1 (encoding Cu/Zn superoxide dismutase) rendered S . cerevisiae hypersensitive to oxytetracycline (OTC): a sod1Delta mutant exhibited a >95% reduction in colony-forming ability at an OTC concentration of 20 microg ml(-1), whereas concentrations of up to 1,000 microg ml(-1) had no effect on the growth of the wild type . OTC resistance was restored in the sod1Delta mutant by complementation with wild-type SOD1 . The effect of OTC appeared to be cytotoxic and was not evident in a ctt1Delta (cytosolic catalase) mutant or in the presence of tetracycline . SOD1 transcription was not induced by OTC, suggesting that constitutive SOD1 expression is sufficient for wild-type OTC resistance . OTC uptake levels in wild-type and sod1Delta strains were similar . However, lipid peroxidation and protein oxidation were both enhanced during exposure of the sod1Delta mutant, but not the wild type, to OTC . We propose that Sod1p protects S . cerevisiae against a mode of OTC action that is dependent on oxidative damage.

J Protein Chem, 1999 Aug, 18(6), 659 - 64
Characterization of the oxaloacetate decarboxylase and pyruvate kinase-like activities of Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinases; Jabalquinto AM et al.; Two members of the ATP-dependent class of phosphoenolpyruvate carboxykinases (PEPCKs) (Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens) have been comparatively studied with regard to their oxaloacetate (OAA) decarboxylase and pyruvate kinase-like activities . The pyruvate kinase-like activities were dependent on the presence of Mn2+; at the same concentrations Mg2+ was not effective . These activities were synergistically activated by a combination of both metal ions . Vmax for these activities in A . succiniciproducens and S . cerevisiae PEPCKs was 0.13% and 1.2% that of the principal reaction, respectively . The OAA decarboxylase activity was nucleotide independent and, with decreasing order of effectiveness, these activities were supported by Mn2+ and Mg2+ . AMP is an activator of these reactions . Vmax for the OAA decarboxylase activities in A . succiniciproducens and S . cerevisiae PEPCKs was 4% and 0.2% that of the PEP-forming reaction, respectively.

J Biol Chem, 1999 Dec 31, 274(53), 38027 - 31
Functional reconstitution of human telomerase expressed in Saccharomyces cerevisiae; Bachand F et al.; Telomerase is a ribonucleoprotein enzyme complex that adds DNA repeats at the ends of chromosomes . In an effort to establish an in vivo heterologous expression system for active human telomerase, we expressed human telomerase reverse transcriptase (hTERT) in Saccharomyces cerevisiae and affinity-purified the protein as a fusion with glutathione S-transferase (GST) . Addition of the GST moiety to the N terminus of hTERT did not interfere with telomerase activity when GST-hTERT was expressed in rabbit reticulocyte lysate (RRL) in the presence of the human telomerase RNA (hTR) . Active human telomerase was immunoprecipitated from yeast lysates that co-expressed GST-hTERT and hTR . In addition, telomerase activity could be reconstituted in vitro by the addition of hTR to GST-hTERT that was immunoprecipitated from either RRL or S . cerevisiae lysates . The expression and reconstitution of human telomerase activity in yeast will provide powerful biochemical and genetic tools to study the various components required for the assembly and function of this enzyme.

J Biol Chem, 1999 Dec 31, 274(53), 37565 - 74
A highly conserved mechanism of regulated ribosome stalling mediated by fungal arginine attenuator peptides that appears independent of the charging status of arginyl-tRNAs; Wang Z et al.; The Arg attenuator peptide (AAP) is an evolutionarily conserved peptide involved in Arg-specific negative translational control . It is encoded as an upstream open reading frame (uORF) in fungal mRNAs specifying the small subunit of Arg-specific carbamoyl phosphate synthetase . We examined the functions of the Saccharomyces cerevisiae CPA1 and Neurospora crassa arg-2 AAPs using translation extracts from S . cerevisiae, N . crassa, and wheat germ . Synthetic RNA containing AAP and firefly luciferase (LUC) sequences were used to program translation; analyses of LUC activity indicated that the AAPs conferred Arg-specific negative regulation in each system . The AAPs functioned either as uORFs or fused in-frame at the N terminus of LUC . Mutant AAPs lacking function in vivo did not function in vitro . Therefore, trans-acting factors conferring AAP-mediated regulation are in both fungal and plant systems . Analyses of ribosome stalling in the fungal extracts by primer extension inhibition (toeprint) assays showed that these AAPs acted similarly to stall ribosomes in the region immediately distal to the AAP coding region in response to Arg . The regulatory effect increased as the Arg concentration increased; all of the arginyl-tRNAs examined appeared maximally charged at low Arg concentrations . Therefore, AAP-mediated Arg-specific regulation appeared independent of the charging status of arginyl-tRNA.

Biochim Biophys Acta, 2000 Jan 3, 1476(1), 27 - 32
The FAD binding sites of human liver monoamine oxidases A and B: investigation of the role of flavin ribityl side chain hydroxyl groups in the covalent flavinylation reaction and catalytic activities; Miller JR et al.; The role of ribityl side chain hydroxyl groups of the flavin moiety in the covalent flavinylation reaction and catalytic activities of recombinant human liver monoamine oxidases (MAO) A and B have been investigated using the riboflavin analogue: N(10)-omega-hydroxypentyl-isoalloxazine . Using a rib5 disrupted strain of Saccharomyces cerevisiae which is auxotrophic for riboflavin, MAO A and MAO B were expressed separately under control of a galactose inducible GAL10/CYC1 promoter in the presence of N(10)-omega-hydroxypentyl-isoalloxazine as the only available riboflavin analogue . Analysis of mitochondrial membrane proteins shows both enzymes to be expressed at levels comparable to those cultures grown on riboflavin and to contain covalently bound flavin . Catalytic activities, as monitored by kynuramine oxidation, are equivalent to (MAO A) or 2-fold greater (MAO B) than control preparations expressed in the presence of riboflavin . Although N(10)-omega-hydroxypentyl-isoalloxazine is unable to support growth of riboflavin auxotrophic S . cerevisiae, it is converted to the FMN level by yeast cell free extracts . The FMN form of the analogue is converted to the FAD level by the yeast FAD synthetase, as shown by expression of the recombinant enzyme in Escherichia coli . These data show that the ribityl hydroxyl groups of the FAD moiety are not required for covalent flavinylation or catalytic activities of monoamine oxidases A and B . This is in contrast to the suggestion based on mutagenesis studies that an interaction between the 3'-hydroxyl group of the flavin and the beta-carbonyl of Asp(227) is required for the covalent flavinylation reaction of MAO B (Zhou et al., J . Biol . Chem . 273 (1998) 14862-14868).

J Exp Biol, 2000 Jan, 203 Pt 1, 89 - 95
The cellular biology of proton-motive force generation by V-ATPases; Nelson N et al.; The vacuolar H(+)-ATPase (V-ATPase) is one of the most fundamental enzymes in nature . It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes . In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force, V-ATPases function exclusively as ATP-dependent proton pumps . The proton-motive force generated by V-ATPases in organelles and across plasma membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes . The enzyme is also vital for the proper functioning of endosomes and the Golgi apparatus . In contrast to yeast vacuoles, which maintain an internal pH of approximately 5 . 5, it is believed that the vacuoles of lemon fruit may have a pH as low as 2 . Similarly, some brown and red algae maintain an internal pH as low as 1 in their vacuoles . It was yeast genetics that allowed the identification of the special properties of individual subunits and the discovery of the factors that are involved in V-ATPase biogenesis and assembly . Null mutations in genes encoding V-ATPase subunits of Saccharomyces cerevisiae result in a phenotype that is unable to grow at high pH and is sensitive to high and low metal-ion concentrations . Treatment of these null mutants with ethyl methanesulphonate causes mutations that suppress the V-ATPase null phenotype, and these cells are able to grow at pH 7.5 . The suppressor mutants were denoted as svf (Suppressor of V-ATPase Function) . The svf mutations are recessive: crossing the svf mutants with their corresponding V-ATPase null mutants resulted in diploid strains that were not able to grow at pH 7.5 . A novel gene family in which null mutations cause pleiotropic effects on metal-ion resistance or on the sensitivity and distribution of membrane proteins in different targets was discovered . We termed this gene family VTC (Vacuolar Transporter Chaperon) and discovered four genes in S . cerevisiae that belong to the family . Inactivation of one of them, VTC1, in the background of V-ATPase null mutations resulted in an svf phenotype that was able to grow at pH 7.5 . Apparently, Vtc1p is one of a few membrane organizers that determine the relative amounts of different membrane proteins in the various cellular membranes . We utilize the numerous yeast mutants generated in our laboratory to identify the specific organelle whose acidification is vital . The interaction between V-ATPase and the secretory pathway is investigated.

J Biol Chem, 1999 Dec 24, 274(52), 37443 - 9
His(73), often methylated, is an important structural determinant for actin . A mutagenic analysis of HIS(73) of yeast actin; Yao X et al.; His(73), has been proposed to regulate the release of P(i) from the interior of actin following polymerization-dependent hydrolysis of bound ATP . Although it is a 3-methylhistidine in the vast majority of actins, His(73) is unmethylated in S . cerevisiae actin . We mutated His(73) in yeast actin to Arg, Lys, Ala, Gln, and Glu and detected no altered phenotypes associated with the mutations in vivo . However, they significantly affect actin function in vitro . Substitution of the more basic residues resulted in enhanced thermal stability, decreased rate of nucleotide exchange, and decreased susceptibility to controlled proteolysis relative to wild-type actin . The opposite effects are observed with the neutral and anionic substitutions . All mutations reduced the rate of polymerization . Molecular dynamics simulations predict a new conformation for the His(73) imidazole in the absence of a methyl group . It also predicts that Arg(73) tightens and stabilizes the actin and that Glu(73) causes a rearrangement of the bottom of actin's interdomain cleft leading possibly to our observed destabilization of actin . Considering the exterior location of His(73), this work indicates a surprisingly important role for the residue as a major structural determinant of actin and provides a clue to the impact caused by methylation of His(73).

Gut, 2000 Jan, 46(1), 58 - 63
Familial expression of anti-Saccharomyces cerevisiae mannan antibodies in affected and unaffected relatives of patients with Crohn's disease; Sutton CL et al.; BACKGROUND: Crohn's disease is a familial disorder, and antiglycan antibodies to the cell wall mannan of Saccharomyces cerevisiae (ASCA) are highly correlated with Crohn's disease . AIMS: To determine whether there is a familial pattern for expression of serum levels of anti-mannan Ig, and whether this trait is expressed in clinically unaffected Crohn's disease family members . METHODS: 349 patients with Crohn's disease, 87 Crohn's disease affected relatives, 333 inflammatory bowel disease (IBD) free relatives, 58 spouses, and 190 healthy control patients were studied . Serum IgG and IgA binding activity to S cerevisiae cell wall mannan was quantitated by ELISA . RESULTS: A high percentage of patients with Crohn's disease (51.9%) and affected family members (56.3%) were seropositive for anti-mannan Ig, compared with the normal control population (3.7%) . Seropositive and seronegative phenotypes of Crohn's disease probands were correlated among all affected relatives, and this association was stronger in affected first degree relatives . Statistical intraclass correlations of quantitative anti-mannan Ig levels revealed significantly less variation within, rather than between families . A significant familial aggregation was observed for affected relatives; this was even stronger for unaffected relatives . While a significant familial aggregation was observed among unaffected siblings pairs, there was no significant correlation among marital pairs . CONCLUSION: Results show that anti-mannan Ig in family members affected and unaffected with Crohn's disease is a familial trait for both affected and unaffected relatives . The lack of concordance in marital pairs indicates that familiality is due in part to a genetic factor or childhood environmental exposure.

Oncogene, 1999 Nov 25, 18(50), 7046 - 54
An FH domain-containing Bnr1p is a multifunctional protein interacting with a variety of cytoskeletal proteins in Saccharomyces cerevisiae; Kikyo M et al.; Proteins containing formin homology domains, FH1 and FH2, are involved in cytokinesis or establishment of cell polarity in a variety of organisms . Bni1p and Bnr1p are FH proteins and potential targets of the Rho family small GTP-binding proteins in S . cerevisiae . We have shown that Bnr1p is localized at the bud neck to interact with Hof1p, involved in cytokinesis . We report here that the overexpression of BNR1 causes a cytokinesis deficiency which is similar to the phenotypes of the septin mutants, including cdc3, cdc10, cdc11, and cdc12 . The region required for the septin mutant phenotypes was mapped to Bnr1p (35-500), which coincided with the region required for the bud-neck localization . To further isolate a gene interacting with BNI1 or BNR1, a multicopy suppressor of the bni1 bnr1 mutant was isolated . This gene encoded Smy1p, a kinesin-related protein . Bnr1p, but not Bni1p, directly interacted with the C-terminal region of Smy1p . The Smy1p-interacting region of Bnr1p was mapped to a region containing the FH2 domain . Bnr1p also directly interacted with Bud6p, a novel actin-binding protein . Bnr1p is thus a multifunctional protein which interacts with the septin system, a microtubule-dependent motor protein, and the actin system, to regulate cytoskeletal functions in S . cerevisiae.

J Agric Food Chem, 1999 Jul, 47(7), 2860 - 4
Characterization by gas chromatography/mass spectrometry of sterols in saccharomyces cerevisiae during autolysis; Le Fur Y et al.; Yeast autolysis affects membrane stability and induces a release of vacuolar enzymes into the cell cytoplasm . Consecutively, it was important to study the evolution of sterol content in Saccharomycescerevisiae for a fourteen day period of accelerated autolysis . Unesterified and esterified sterols were analyzed both in the biomass and in the autolysis medium . Ten sterols were identified by gas chromatography/mass spectrometry . A second group of six sterols was separated and partially characterized . Among the first group of 10 sterols, a dehydroergosterol was identified as ergosta-5, 7,9(11),22-tetraen-3beta-ol, not yet charaterized in S . cerevisiae . Yeast autolysis induced a decrease of esterified sterol content, especially first intermediates in the sequence of the ergosterol biosynthesis, as zymosterol . In contrast, the yeast autolysis resulted in the release of a low quantity of sterols into the medium . At the end of the fourteenth day of autolysis, 0.015% of the total sterol content of the initial biomass was found in the medium.

J Biotechnol, 1999 Oct 8, 75(2-3), 195 - 208
The role of leaders in intracellular transport and secretion of the insulin precursor in the yeast Saccharomyces cerevisiae; Kjeldsen T et al.; Pulse-chase analysis of folded and misfolded insulin precursor (IP) expressed in Saccharomyces cerevisiae was performed to establish the requirements for intracellular transport and the influence of the secretory pathway quality control mechanisms on secretion . Metabolic labelling of the IP expressed in S . cerevisiae showed that the effect of a leader was to stabilise the IP in the endoplasmic reticulum (ER), and facilitate intracellular transport of the fusion protein and rapid secretion . The first metabolically labelled IP appeared in the culture supernatant within 2-4 min of chase, and most of the secreted IP appeared within the first 15 min of chase . After enzymatic removal of the leader in a late Golgi apparatus compartment, the IP followed one of two routes: (1) to the plasma membrane and hence to the culture supernatant, or (2) to a Golgi or post-Golgi compartment from which secretion was restricted . Combined secretion and intracellular retention of the IP reflected either saturation of a Golgi or post-Golgi compartment and secretion as a consequence of overexpression, or competition between secretion and intracellular retention . IP which was misfolded, either due to amino acid substitution or because disulphide bond formation had been prevented with dithiothreitol (DTT), was transported from the ER to the Golgi apparatus but then retained in a Golgi or post-Golgi compartment and not exported to the culture supernatant.

Curr Genet, 1999 Oct, 36(4), 201 - 7
Mutational analysis of yeast mitochondrial translational activator Cbs2p and of YHR063Cp, a protein with similarity to Cbs2p; Tzschoppe K et al.; Translation of mitochondrial cytochrome b in Saccharomyces cerevisiae requires the nuclearly encoded proteins Cbs1p, Cbs2p and Cbp6p . So far no homologs have been identified, except for the product of the S . cerevisiae orf YHR063C, which has some similarity to Cbs2p . Here we analyze the effect of a null mutation of YHR063C and show that it is not required for mitochondrial respiration . In addition, we report on the importance of the carboxyl-terminus of Cbs2p for its function . We show that truncations and some directed mutations in the carboxyl-terminal region of Cbs2p render the protein non-functional . The importance of the COOH-terminus is further underscored by the finding that mutational alteration of the cbs2-1 allele results in the substitution of Ile(372) by Lys.

Biochem Cell Biol, 1999, 77(5), 459 - 68
Differential transmission of G1 cell cycle arrest and mating signals by Saccharomyces cerevisiae Ste5 mutants in the pheromone pathway; Choi YJ et al.; Saccharomyces cerevisiae Ste5 is a scaffold protein that recruits many pheromone signaling molecules to sequester the pheromone pathway from other homologous mitogen-activated protein kinase pathways . G1 cell cycle arrest and mating are two different physiological consequences of pheromone signal transduction and Ste5 is required for both processes . However, the roles of Ste5 in G1 arrest and mating are not fully understood . To understand the roles of Ste5 better, we isolated 150 G1 cell cycle arrest defective STE5 mutants by chemical mutagenesis of the gene . Here, we found that two G1 cell cycle arrest defective STE5 mutants (ste5M(D248V) and ste5(delta-776)) retained mating capacity . When overproduced in a wild-type strain, several ste5 mutants also showed different dominant phenotypes for G1 arrest and mating . Isolation and characterization of the mutants suggested separable roles of Ste5 in G1 arrest and mating of S . cerevisiae . In addition, the roles of Asp-248 and Tyr-421, which are important for pheromone signal transduction were further characterized by site-directed mutagenesis studies.

Yeast, 1999 Dec, 15(16), 1747 - 59
Facilitating functional analysis of the Saccharomyces cerevisiae genome using an EGFP-based promoter library and flow cytometry; Bell PJ et al.; A promoter library was generated to facilitate identification of differentially regulated promoters in Saccharomyces cerevisiae . The library was constructed in a vector containing two reporter genes (EGFP and lacZ) divergently arranged about a unique cloning site . Approximately 2x10(5) clones were obtained and a flow cytometer was used to screen the library for copper-induced EGFP expression . A DNA fragment conferring copper-inducible expression of EGFP was rapidly identified . This DNA fragment, which contained several motifs associated with copper and oxidative stress homeostasis, lies upstream of two 'orphan' genes of unknown function . Further studies comparing expression from episomal vs . integrative vectors showed that construction of a similar library using an integrative vector would further enhance rapid identification of genes that are differentially regulated in S . cerevisiae . The ability to identify regulated promoters rapidly should facilitate the functional analysis of the yeast genome by identifying genes induced by specific physiological conditions .

Mol Gen Genet, 1999 Oct, 262(3), 473 - 80
Budding yeast Cdc6p induces re-replication in fission yeast by inhibition of SCF(Pop)-mediated proteolysis; Wolf DA et al.; In fission yeast, overexpression of the replication initiator protein Cdc18p induces re-replication, a phenotype characterized by continuous DNA synthesis in the absence of cell division . In contrast, overexpression of Cdc6p, the budding yeast homolog of Cdc18p, does not cause re-replication in S . cerevisiae . However, we have found that Cdc6p has the ability to induce rereplication in fission yeast . Cdc6p cannot functionally replace Cdc18p, but instead interferes with the proteolysis of both Cdc18p and Rum1p, the inhibitor of the protein kinase Cdc2p . This activity of Cdc6p is entirely contained within a short N-terminal peptide, which forms a tight complex with Cdc2p and the F-box/WD-repeat protein Sud1p/Pop2p, a component of the SCF(Pop) ubiquitin ligase in fission yeast . These interactions are mediated by two distinct regions within the N-terminal region of Cdc6p and depend on the integrity of its Cdc2p phosphorylation sites . The data suggest that disruption of re-replication control by overexpression of Cdc6p in fission yeast is a consequence of sequestration of Cdc2p and Pop2p, two factors involved in the negative regulation of Rum1p, Cdc18p and potentially other replication proteins.

Folia Microbiol (Praha), 1999, 44(2), 142 - 52
Protein kinases phosphorylating acidic ribosomal proteins from yeast cells; Szyszka R; Phosphorylation of ribosomal acidic proteins of Saccharomyces cerevisiae is an important mechanism regulating a number of active ribosomes . The key role in the regulatory mechanism is played by specific phosphoprotein kinases and phosphoprotein phosphatases . Three different cAMP-independent protein kinases phosphorylating acidic ribosomal proteins have been identified and characterized . The protein kinase 60S (PK60S), RAP kinase, and casein kinase type 2 (CK2) . All three protein kinases phosphorylate serine residues which are localized in the C-terminal end of phosphoproteins . Synthetic peptides were used to determinate the amino acid sequence of phosphoacceptor site for PK60S . Peptide AAEESDDD derived from phosphoproteins YP1 beta/beta' and YP2 alpha turned out to be the best substrate for PK60S . A number of halogenated benzimidazoles and 2-azabenzimidazoles were tested as inhibitors of the three protein kinases . 4,5,6,7-Tetrabromo-2-azabenzimidazole inhibits phosphorylation only of these polypeptides phosphorylated by protein kinase 60S, namely YP1 beta/beta' and YP2 alpha, but not the other, YP1 alpha and YP2 beta phosphorylated by protein kinases RAP and CK2 . RAP kinase has been found in an active form in the soluble fraction of S . cerevisiae . The enzyme uses ATP as a phosphate donor and is less sensitive to heparin than casein kinase 2 . RAP kinase monophosphorylates the four acidic proteins . The ribosome-bound proteins are a better substrate for the enzyme . Multifunctional CK2 kinase phosphorylate all four acidic proteins . The kinase phosphorylates preferentially serine or threonine residues surrounded by cluster of acidic residues . The enzyme activity is stimulated in vitro by the presence of polylysine and inhibited by heparin.

J Biol Chem, 1999 Dec 10, 274(50), 35975 - 84
Induction by adozelesin and hydroxyurea of origin recognition complex-dependent DNA damage and DNA replication checkpoints in Saccharomyces cerevisiae; Weinberger M et al.; DNA damaging agents induce a conserved intra-S-phase checkpoint that inhibits DNA replication in eukaryotic cells . To better understand this checkpoint and its role in determining the efficacy of antitumor drugs that damage DNA, we examined the effects of adozelesin, a DNA-alkylating antitumor agent that has a profound inhibitory effect on initiation of DNA replication in mammals, on the replication of Saccharomyces cerevisiae chromosomes . Adozelesin inhibited initiation of S . cerevisiae DNA replication by inducing an intra-S-phase DNA damage checkpoint . This inhibitory effect was abrogated in orc2-1 cells containing a temperature-sensitive mutation in a component of the origin recognition complex (ORC) that also causes a defect in initiation . The orc2-1 mutation also caused a defect in a checkpoint that regulates the activation of origins in late S phase in cells treated with hydroxyurea . Defects in both initiation and checkpoint regulation in the orc2-1 strain were suppressed by deletion of a gene encoding a putative acetyltransferase, SAS2 . Adozelesin also induced a cellular response that requires a function of ORC in G(1) . A similar G(1)-specific response in mammals may contribute to the cytotoxic and antitumor properties of this and other DNA-damaging drugs.

Appl Environ Microbiol, 1999 Dec, 65(12), 5451 - 8
Permeabilization of fungal membranes by plant defensins inhibits fungal growth; Thevissen K et al.; We used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in fungi . Membrane permeabilization induced by plant defensins in Neurospora crassa was biphasic, depending on the plant defensin dose . At high defensin levels (10 to 40 microM), strong permeabilization was detected that could be strongly suppressed by cations in the medium . This permeabilization appears to rely on direct peptide-phospholipid interactions . At lower defensin levels (0.1 to 1 microM), a weaker, but more cation-resistant, permeabilization occurred at concentrations that correlated with the inhibition of fungal growth . Rs-AFP2(Y38G), an inactive variant of the plant defensin Rs-AFP2 from Raphanus sativus, failed to induce cation-resistant permeabilization in N . crassa . Dm-AMP1, a plant defensin from Dahlia merckii, induced cation-resistant membrane permeabilization in yeast (Saccharomyces cerevisiae) which correlated with its antifungal activity . However, Dm-AMP1 could not induce cation-resistant permeabilization in the Dm-AMP1-resistant S . cerevisiae mutant DM1, which has a drastically reduced capacity for binding Dm-AMP1 . We think that cation-resistant permeabilization is binding site mediated and linked to the primary cause of fungal growth inhibition induced by plant defensins.

Bioessays, 1999 Dec, 21(12), 1004 - 10
Brave little yeast, please guide us to thebes: sphingolipid function in S . cerevisiae; Schneiter R; Sphingolipids typically cover the exoplasmic leaflet of the plasma membrane of eukaryotic cells . They differ from the more abundant glycerophospholipids in that they contain ceramide instead of diacylglycerol as a hydrophobic anchor . Why did nature choose to invent this complex class of lipids, and why do eukaryotic cells follow elaborate remodelling pathways in order to generate dozens to hundreds of different molecular species of sphingolipid, depending on cell type? Yeast may, once again, serve as a model to dissect sphingolipid function at various levels . Almost the complete pathway for sphingolipid synthesis in yeast has been uncovered during the past two decades . More recently, key enzymes in sphingolipid degradation and signalling have been identified . Together with a wealth of genetic data obtained from the characterization of various suppressor mutants, this information now allows for an unprecedented analysis of sphingolipid function in this organism . This overview summarizes recent data on sphingolipid function in cell signalling, their role in the heat-stress response and Ca(2+) homeostasis, and addresses their function in transport of glycosylphosphatidylinositol-anchored proteins .

Front Biosci, 1999 Dec 01, 4, D859 - 68
Replication origins of mammalian chromosomes: the happy few; Todorovic V et al.; Replication of eukaryotic cell genomes is a tightly controlled process occurring once and only once per cell cycle . Replication initiates at several thousand origins, whose cis-acting sequences and trans-acting proteins have been partially characterized in the yeast S . cerevisiae in the last few years . In contrast, identification of origins of DNA replication in mammalian cells have proven much more difficult . Currently, less then 20 bona fide mammalian origins have been identified, of which only few characterized in detail . Here we discuss the available methods for origin identification in mammalian DNA and the main results, sometimes controversial, so far generated by their application . In particular, we review the currently available information concerning the three best characterized origins, namely those in the lamin B2 and b-globin gene domains in human cells and the one located downstream of the dihydrofolate reductase gene in hamster cells.

J Biol Chem, 1999 Dec 3, 274(49), 34903 - 10
Patch clamp studies on V-type ATPase of vacuolar membrane of haploid Saccharomyces cerevisiae . Preparation and utilization of a giant cell containing a giant vacuole; Yabe I et al.; A method for obtaining giant protoplasts of Escherichia coli (the spheroplast incubation (SI) method: Kuroda et al . (Kuroda, T., Okuda, N., Saitoh, N., Hiyama, T., Terasaki, Y., Anazawa, H., Hirata, A., Mogi, T., Kusaka, I., Tsuchiya, T., and Yabe, I . (1998) J . Biol . Chem . 273, 16897-16904) was adapted to haploid cells of Saccharomyces cerevisiae . The yeast cell grew to become as large as 20 micrometer in diameter and to contain an oversized vacuole inside . A patch clamp technique in the whole cell/vacuole recording mode was applied for the vacuole isolated by osmotic shock . At zero membrane potential, ATP induced a strong current (as high as 100 pA; specific activity, 0.1 pA/micrometer(2)) toward the inside of the vacuole . Bafilomycin A(1,) a specific inhibitor of the V-type ATPase, strongly inhibited the activity (K(i) = 10 nM) . Complete inhibition at higher concentrations indicated that any other ATP-driven transport systems were not expressed under the present incubation conditions . This current was not observed in the vacuoles prepared from a mutant that disrupted a catalytic subunit of the V-type ATPase (RH105(Deltavma1::TRP)) . The K(m) value for the ATP dose response of the current was 159 microM and the H(+)/ATP ratio estimated from the reversible potential of the V-I curve was 3.5 +/- 0.3 . These values agreed well with those previously estimated by measuring the V-type ATPase activity biochemically . This method can potentially be applied to any type of ion channel, ion pump, and ion transporter in S . cerevisiae, and can also be used to investigate gene functions in various organisms by using yeast cells as hosts for homologous and heterogeneous expression systems.

J Biol Chem, 1999 Dec 3, 274(49), 34683 - 90
Cloning and characterization of a bifunctional leukotriene A(4) hydrolase from Saccharomyces cerevisiae; Kull F et al.; In mammals, leukotriene A(4) hydrolase is a bifunctional zinc metalloenzyme that catalyzes hydrolysis of leukotriene A(4) into the proinflammatory leukotriene B(4) and also possesses an arginyl aminopeptidase activity . We have cloned, expressed, and characterized a protein from Saccharomyces cerevisiae that is 42% identical to human leukotriene A(4) hydrolase . The purified protein is an anion-activated leucyl aminopeptidase, as assessed by p-nitroanilide substrates, and does not hydrolyze leukotriene A(4) into detectable amounts of leukotriene B(4) . However, the S . cerevisiae enzyme can utilize leukotriene A(4) as substrate to produce a compound identified as 5S,6S-dihydroxy-7,9-trans-11, 14-cis-eicosatetraenoic acid . Both catalytic activities are inhibited by 3-(4-benzyloxyphenyl)-2-(R)-amino-1-propanethiol (thioamine), a competitive inhibitor of human leukotriene A(4) hydrolase . Furthermore, the peptide cleaving activity of the S . cerevisiae enzyme was stimulated approximately 10-fold by leukotriene A(4) with kinetics indicating the presence of a lipid binding site . Nonenzymatic hydrolysis products of leukotriene A(4), leukotriene B(4), arachidonic acid, or phosphatidylcholine were without effect . Moreover, leukotriene A(4) could displace the inhibitor thioamine and restore maximal aminopeptidase activity, indicating that the leukotriene A(4) binding site is located at the active center of the enzyme . Hence, the S . cerevisiae leukotriene A(4) hydrolase is a bifunctional enzyme and appears to be an early ancestor to mammalian leukotriene A(4) hydrolases.

Yeast, 1999 Nov, 15(15), 1669 - 79
A series of protein phosphatase gene disruptants in Saccharomyces cerevisiae; Sakumoto N et al.; Thirty-two protein phosphatase (PPase) genes were identified in the genome nucleotide sequence of Saccharomyces cerevisiae . We constructed S . cerevisiae disruptants for each of the PPase genes and examined their growth under various conditions . The disruptants of six putative PPase genes, i.e . of YBR125c, YCR079w, YIL113w, YJR110w, YNR022c and YOR090c, were created for the first time in this study . The glc7, sit4 and cdc14 disruptants were lethal in our strain background . The remaining 29 PPase gene disruptants were viable at 30 degrees C and 37 degrees C, but only one disruptant, yvh1, showed intrinsic cold-sensitive growth at 13 degrees C . Transcription of the YVH1 gene was induced at 13 degrees C, consistent with an idea that Yvh1p has a specific role for growth at a low temperature . The viable disruptants grew normally on nutrient medium containing sucrose, galactose, maltose or glycerol as carbon sources . The ppz1 disruptant was tolerant to NaCl and LiCl, while the cmp2 disruptant was sensitive to these salts, as reported previously, and none of the other viable PPase disruptants exhibited the salt sensitivity . When the viable disruptants were tested for sensitivity to drugs, i.e . benomyl, caffeine and hydroxyurea, ppz1 and ycr079w disruptants exhibited sensitivity to caffeine .

Eur J Biochem, 1999 Dec, 266(2), 517 - 23
Alpha-complemented beta-galactosidase . An in vivo model substrate for the molecular chaperone heat-shock protein 90 in yeast; Abbas-Terki T et al.; Intracistronic complementation of N-terminally truncated beta-galactosidase mutants such as M15 by coexpressed alpha-peptide was originally discovered in Escherichia coli and exploited for plasmid cloning as the well-known blue-white screening method . We show here that alpha-complementation also works in the budding yeast Saccharomyces cerevisiae, and that it can be used as a simple nonselective enzymatic marker for a variety of in vivo studies, for example, on the role of molecular chaperones in protein folding and assembly . To be able to induce alpha-complementation post-translationally, we have constructed a hormone-inducible alpha-peptide by fusion of the DNA encoding the alpha-peptide to that of to the hormone binding domain of the estrogen receptor . The accumulation of both subunits, the alpha-peptide and M15, is severely compromised when they are expressed separately, presumably because their hydrophobic surfaces remain exposed . Moreover, alpha-complementation is defective in a strain of S . cerevisiae carrying a point mutant of the molecular chaperone heat-shock protein 90 . Heat-shock protein 90, which coprecipitates with M15, might be required in vivo to prevent the degradation of unassembled M15 and to hold it in an interaction-competent conformation.

Mol Cell Biol, 1999 Dec, 19(12), 8361 - 71
Saccharomyces cerevisiae RNase H(35) functions in RNA primer removal during lagging-strand DNA synthesis, most efficiently in cooperation with Rad27 nuclease; Qiu J et al.; Correct removal of RNA primers of Okazaki fragments during lagging-strand DNA synthesis is a critical process for the maintenance of genome integrity . Disturbance of this process has severe mutagenic consequences and could contribute to the development of cancer . The role of the mammalian nucleases RNase HI and FEN-1 in RNA primer removal has been substantiated by several studies . Recently, RNase H(35), the Saccharomyces cerevisiae homologue of mammalian RNase HI, was identified and its possible role in DNA replication was proposed (P . Frank, C . Braunshofer-Reiter, and U . Wintersberger, FEBS Lett . 421:23-26, 1998) . This led to the possibility of moving to the genetically powerful yeast system for studying the homologues of RNase HI and FEN-1, i.e., RNase H(35) and Rad27p, respectively . In this study, we have biochemically defined the substrate specificities and the cooperative as well as independent cleavage mechanisms of S . cerevisiae RNase H(35) and Rad27 nuclease by using Okazaki fragment model substrates . We have also determined the additive and compensatory pathological effects of gene deletion and overexpression of these two enzymes . Furthermore, the mutagenic consequences of the nuclease deficiencies have been analyzed . Based on our findings, we suggest that three alternative RNA primer removal pathways of different efficiencies involve RNase H(35) and Rad27 nucleases in yeast.

Mol Cell Biol, 1999 Dec, 19(12), 8016 - 27
Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton in Saccharomyces cerevisiae; Fujiwara T et al.; The RHO1 gene encodes a yeast homolog of the mammalian RhoA protein . Rho1p is localized to the growth sites and is required for bud formation . We have recently shown that Bni1p is one of the potential downstream target molecules of Rho1p . The BNI1 gene is implicated in cytokinesis and the establishment of cell polarity in Saccharomyces cerevisiae but is not essential for cell viability . In this study, we screened for mutations that were synthetically lethal in combination with a bni1 mutation and isolated two genes . They were the previously identified PAC1 and NIP100 genes, both of which are implicated in nuclear migration in S . cerevisiae . Pac1p is a homolog of human LIS1, which is required for brain development, whereas Nip100p is a homolog of rat p150(Glued), a component of the dynein-activated dynactin complex . Disruption of BNI1 in either the pac1 or nip100 mutant resulted in an enhanced defect in nuclear migration, leading to the formation of binucleate mother cells . The arp1 bni1 mutant showed a synthetic lethal phenotype while the cin8 bni1 mutant did not, suggesting that Bni1p functions in a kinesin pathway but not in the dynein pathway . Cells of the pac1 bni1 and nip100 bni1 mutants exhibited a random distribution of cortical actin patches . Cells of the pac1 act1-4 mutant showed temperature-sensitive growth and a nuclear migration defect . These results indicate that Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton . Bni1p lacking the Rho-binding region did not suppress the pac1 bni1 growth defect, suggesting a requirement for the Rho1p-Bni1p interaction in microtubule function.

Mol Biol Cell, 1999 Nov, 10(11), 3643 - 59
Adaptor complex-independent clathrin function in yeast; Yeung BG et al.; Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection . We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae . Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined . These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes . As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin . Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and alpha-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex . However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or alpha-factor maturation . Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1 . Similarly, combined deletion of genes encoding the beta subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles . We conclude that AP complexes are dispensable for clathrin function in S . cerevisiae under normal conditions . Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.

FEMS Microbiol Lett, 1999 Nov 15, 180(2), 229 - 33
Trifluoroleucine resistance as a dominant molecular marker in transformation of strains of Saccharomyces cerevisiae isolated from wine; Bendoni B et al.; The resistance to 5,5,5-trifluoro-DL-leucine, encoded by the dominant allele LEU4-1, was used as a selectable marker to transform laboratory and natural Saccharomyces cerevisiae strains by the lithium acetate procedure . Results of transformation of S . cerevisiae laboratory and wine natural strains showed that trifluoroleucine resistance is a very effective selection marker and can be widely used to transform prototrophic S . cerevisiae strains . The LEU4-1 gene could also be exploited to improve wine flavour, as indicated by the higher isoamyl alcohol content of the transformants compared to the parental strains.

Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1925 - 31
Karyotypes of Saccharomyces sensu lato species; Petersen RF et al.; An improved pulsed-field electrophoresis program was developed to study differently sized chromosomes within the genus Saccharomyces . The number of chromosomes in the type strains was shown to be nine in Saccharomyces castellii and Saccharomyces dairenensis, 12 in Saccharomyces servazzii and Saccharomyces unisporus, 16 in Saccharomyces exiguus and seven in Saccharomyces kluyveri . The sizes of individual chromosomes were resolved and the approximate genome sizes were determined by the addition of individual chromosomes of the karyotypes . Apparently, the genome of S . exiguus, which is the only Saccharomyces sensu lato yeast to contain small chromosomes, is larger than that of Saccharomyces cerevisiae . On the other hand, other species exhibited genome sizes that were 10-25% smaller than that of S . cerevisiae . Well-defined karyotypes represent the basis for future genome mapping and sequencing projects, as well as studies of the origin of the modern genomes.

Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1907 - 13
Distinctive electrophoretic isoenzyme profiles in Saccharomyces sensu stricto; Duarte FL et al.; Genetic variation among 35 strains representing the four currently recognized species of Saccharomyces sensu stricto (Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces pastorianus/carlsbergensis and Saccharomyces paradoxus) was estimated by analysing the electrophoretic mobilities of nonspecific esterases, acid phosphatase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase isoenzymes . Twenty-two electrophoretic types were identified, a result in agreement with the phenotypic and genetic polymorphisms reported for this group of yeasts . However, the four species were clearly distinguishable based on the patterns obtained using three of the enzymes assayed, the resolving power not being improved by the introduction of data correspondent to lactate dehydrogenase . The overall diversity was higher among S . cerevisiae isolates, in contrast with S . paradoxus which showed only two patterns, one of which was common to four of the five strains studied . Concordant results from the application of the method and DNA hybridization experiments demonstrate its value for identification purposes.

Cell, 1999 Oct 29, 99(3), 283 - 91
A molecular target for viral killer toxin: TOK1 potassium channels; Ahmed A et al.; Killer strains of S . cerevisiae harbor double-stranded RNA viruses and secrete protein toxins that kill virus-free cells . The K1 killer toxin acts on sensitive yeast cells to perturb potassium homeostasis and cause cell death . Here, the toxin is shown to activate the plasma membrane potassium channel of S . cerevisiae, TOK1 . Genetic deletion of TOK1 confers toxin resistance; overexpression increases susceptibility . Cells expressing TOK1 exhibit toxin-induced potassium flux; those without the gene do not . K1 toxin acts in the absence of other viral or yeast products: toxin synthesized from a cDNA increases open probability of single TOK1 channels (via reversible destabilization of closed states) whether channels are studied in yeast cells or X . laevis oocytes.

Biochem Cell Biol, 1999, 77(4), 355 - 65
Targeting of the mammalian nucleoporin p62 to the nuclear envelope in the yeast Saccharomyces cerevisiae and HeLa cells; Barth W et al.; We have analyzed the sorting of the mammalian nucleoporin p62 in human culture cells and in the yeast Saccharomyces cerevisiae . To this end, gene fusions were generated that carry Aequorea victoria green fluorescence protein and defined portions of p62 . Upon transient gene expression fluorescent fusion proteins were localized in HeLa cells . Likewise, fusion proteins were studied in S . cerevisiae using wild-type as well as mutant cells that cluster nuclear pore complexes . Our results demonstrate that evolutionarily distant organisms, such as humans and yeasts, recognize the same sequence elements of p62 for sorting to the nuclear envelope . Specifically, the entire sequence of p62 or its complete C-terminal domain targeted fusion proteins to the nuclear membranes . In contrast, truncations of the C-terminal domain or the N-terminal segment of p62 failed to associate with the nuclear envelope in either organism . In HeLa cells overexpression of several p62-containing fusion proteins resulted in nuclear fragmentation . The C-terminal domain of p62 caused this effect, and amino acid residues 477 to 525 were sufficient to induce aberrant nuclei . Thus, overexpression of 49 amino acid residues located at the C-terminal tail of p62 interferes with the nuclear integrity in human culture cells.

Biochemistry, 1999 Nov 2, 38(44), 14606 - 13
Mutagenesis of the phosphatase sequence motif in diacylglycerol pyrophosphate phosphatase from Saccharomyces cerevisiae; Toke DA et al.; Diacylglycerol pyrophosphate (DGPP) phosphatase, encoded by the DPP1 gene, is a membrane-associated enzyme in the yeast Saccharomyces cerevisiae . The enzyme removes the beta phosphate from DGPP to form phosphatidate . The substrate and product of the DGPP phosphatase reaction play roles in lipid signaling and in cell metabolism . The deduced primary structure of the DGPP phosphatase protein contains a three-domain phosphatase sequence motif . In this work, we examined the hypothesis that the phosphatase sequence motif in the enzyme is involved in the DGPP phosphatase reaction . The amino acid residues Arg(125), His(169), and His(223) in domains 1, 2, and 3, respectively, of the phosphatase sequence motif were changed to alanine residues by site-directed mutagenesis . The mutant DPP1(R125A), DPP1(H169A), and DPP1(H223A) alleles were cloned into a yeast shuttle vector and then expressed in a dpp1Delta lpp1Delta double mutant that lacks DGPP phosphatase activity . Northern blot and immunoblot analyses showed that the mutations in the phosphatase sequence motif did not affect the expression of the enzyme . The DGPP phosphatase activities of the R125A, the H169A, and the H223A mutant enzymes were 0.05, 9, and 0.03%, respectively, of the DGPP phosphatase activity of the wild-type enzyme . Enzymes with mutations in more than one domain of the phosphatase sequence motif had no measurable DGPP phosphatase activity . The R125A and H233A mutant DGPP phosphatase enzymes had reduced V(max) and elevated K(m) values for DGPP when compared with the wild-type enzyme . The H169A mutant enzyme had reduced V(max) and K(m) values when compared with the control . The specificity constants (V(max)/K(m)()) for DGPP of the R125A mutant and H233A mutant enzymes were 4610-fold and 15 367-fold lower, respectively, when compared to the wild-type enzyme . The studies reported here indicated that the phosphatase sequence motif played an important role in the reaction catalyzed by the S . cerevisiae DGPP phosphatase.

Genes Dev, 1999 Oct 15, 13(20), 2639 - 49
Drosophila ORC specifically binds to ACE3, an origin of DNA replication control element; Austin RJ et al.; In the yeast Saccharomyces cerevisiae, sequence-specific DNA binding by the origin recognition complex (ORC) is responsible for selecting origins of DNA replication . In metazoans, origin selection is poorly understood and it is unknown whether specific DNA binding by metazoan ORC controls replication . To address this problem, we used in vivo and in vitro approaches to demonstrate that Drosophila ORC (DmORC) binds to replication elements that direct repeated initiation of replication to amplify the Drosophila chorion gene loci in the follicle cells of egg chambers . Using immunolocalization, we observe that ACE3, a 440-bp chorion element that contains information sufficient to drive amplification, directs DmORC localization in follicle cells . Similarly, in vivo cross-linking and chromatin immunoprecipitation assays demonstrate association of DmORC with both ACE3 and two other amplification control elements, AER-d and ACE1 . To demonstrate that the in vivo localization of DmORC is related to its DNA-binding properties, we find that purified DmORC binds to ACE3 and AER-d in vitro, and like its S . cerevisiae counterpart, this binding is dependent on ATP . Our findings suggest that sequence-specific DNA binding by ORC regulates initiation of metazoan DNA replication . Furthermore, adaptation of this experimental approach will allow for the identification of additional metazoan ORC DNA-binding sites and potentially origins of replication.

Microbiology, 1999 Oct, 145 ( Pt 10), 2939 - 46
Two fatty acid delta9-desaturase genes, ole1 and ole2, from Mortierella alpina complement the yeast ole1 mutation; Wongwathanarat P et al.; Genes encoding two distinct fatty acid delta9-desaturases were isolated from strains of the oleaginous fungus Mortierella alpina . Two genomic sequences, delta9-1 and delta9-2, each containing a single intron, were cloned from strain CBS 528.72 while one cDNA clone, LM9, was isolated from strain CBS 210.32 . The delta9-1 gene encoded a protein of 445 aa which shared 99% identity with the LM9 gene product . These proteins also showed 40-60% identity to the delta9-desaturases (Ole1p) of other fungi and contained the three conserved histidine boxes, C-terminal cytochrome b5 fusion and transmembrane domains characteristic of endoplasmic reticulum membrane-bound delta9-desaturases . LM9 and delta9-1 are therefore considered to represent the same gene (ole1) . The ole1 gene was transcriptionally active in all M . alpina strains tested and its function was confirmed by complementation of the Saccharomyces cerevisiae ole1 mutation . Fatty acid analysis of yeast transformants expressing the CBS 210.32 ole1 gene showed an elevated level of oleic acid (18:1) compared to palmitoleic acid (16:1), the major fatty acid component of wild-type S . cerevisiae . This indicated that the M . alpina delta9-desaturase had a substrate preference for stearic acid (18:0) rather than palmitic acid (16:0) . Genomic clone delta9-2 (ole2) also encoded a protein of 445 aa which had 86% identity to the delta9-1 and LM9 proteins and whose ORF also complemented the yeast ole1 mutation . The transcript from this gene could only be detected in one of the six M . alpina strains tested, suggesting that its expression may be strain-specific or induced under certain physiological conditions.

Curr Biol, 1999 Oct 21, 9(20), 1180 - 2
A family of mammalian F-box proteins; Winston JT et al.; Ubiquitin-mediated destruction of regulatory proteins is a frequent means of controlling progression through signaling pathways {1} . F-box proteins {2} are components of modular E3 ubiquitin protein ligases called SCFs, which function in phosphorylation-dependent ubiquitination ({3} {4} {5}, reviewed in {6} {7}) . F-box proteins contain a carboxy-terminal domain that interacts with substrates and a 42-48 amino-acid F-box motif which binds to the protein Skp1 {2} {3} {4} . Skp1 binding links the F-box protein with a core ubiquitin ligase composed of the proteins Cdc53/Cul1, Rbx1 (also called Hrt1 and Roc1) and the E2 ubiquitin-conjugating enzyme Cdc34 {8} {9} {10} {11} . The genomes of the budding yeast Saccharomyces cerevisiae and the nematode worm Caenorhabditis elegans contain, respectively, 16 and more than 60 F-box proteins {2} {7}; in S . cerevisiae, the F-box proteins Cdc4, Grr1 and Met30 target cyclin-dependent kinase inhibitors, G1 cyclins and transcriptional regulators for ubiquitination ({3} {4} {5} {8} {10}, reviewed in {6} {7}) . Only four mammalian F-box proteins (Cyclin F, Skp1, beta-TRCP and NFB42) have been identified so far {2} {12} . Here, we report the identification of a family of 33 novel mammalian F-box proteins . The large number of these proteins in mammals suggests that the SCF system controls a correspondingly large number of regulatory pathways in vertebrates . Four of these proteins contain a novel conserved motif, the F-box-associated (FBA) domain, which may represent a new protein-protein interaction motif . The identification of these genes will help uncover pathways controlled by ubiquitin-mediated proteolysis in mammals.

Biochemistry, 1999 Oct 5, 38(40), 13252 - 62
The N-terminal half of Cdc25 is essential for processing glucose signaling in Saccharomyces cerevisiae; Gross A et al.; Saccharomyces cerevisiae Cdc25 is the prototype Ras GDP/GTP exchange protein . Its C-terminal catalytic domain was found to be highly conserved in the homologues p140(ras-GRF) and Sos . The regulatory domains in each Ras exchanger mediate the signals arriving from upstream elements such as tyrosine kinases for Sos, or Ca2+ and G proteins for p140.(Ras-GRF) In this study, we show that the N-terminal half (NTH) of S . cerevisiae Cdc25, as well as the C-terminal 37 amino acids, is essential for processing the elevation of cAMP in response to glucose . The mammalian p140(ras-GRF) catalytic domain (CGRF) restores glucose signaling in S . cerevisiae only if tethered between the N-terminal half (NTH) of S . cerevisiae Cdc25 and the C-terminal 37 amino acids . The glucose-induced transient elevation in cAMP is nullified or severely hampered by the deletion of domains within the NTH of Cdc25 . These deletions, however, do not modify the intrinsic GDP/GTP exchange activity of mutant proteins as compared to native Cdc25 . We also show that 7 Ser to Ala mutations at the cAMP-dependent protein kinase putative phosphorylation sites within the NTH of Cdc25 eliminate the descending portion of the glucose response curve, responsible for signal termination . These findings support a dual role of the NTH of Cdc25 in both enabling the glucose signal and being responsible for its attenuation.

J Food Prot, 1999 Oct, 62(10), 1215 - 7
Saccharomyces cerevisiae thermal inactivation kinetics combined with ultrasound; Lopez-Malo A et al.; Inactivation kinetics of Saccharomyces cerevisiae during thermal treatments at moderate temperatures (45.0, 47.5, 50.0, 52.5, or 55.0 degrees C) combined with application of 20 kHz of ultrasound were evaluated . S . cerevisiae inactivation under the combined effects of heat and ultrasound followed first-order reaction kinetics, with decimal reduction times (D) that varied from 22.3 to 0.8 min . D values in treatments that combined heat and ultrasound were significantly smaller (P < 0.05) than D values obtained for thermal treatments and were more noticeable at temperatures below 50 degrees C . The dependence of the D value on temperature had a significantly (P < 0.05) greater z value for combined treatments . Yeast heat inactivation kinetics revealed decreased thermal resistance caused by ultrasound.

Mol Cell Biol, 1999 Nov, 19(11), 7681 - 7
The Mre11-Rad50-Xrs2 protein complex facilitates homologous recombination-based double-strand break repair in Saccharomyces cerevisiae; Bressan DA et al.; Saccharomyces cerevisiae mre11Delta mutants are profoundly deficient in double-strand break (DSB) repair, indicating that the Mre11-Rad50-Xrs2 protein complex plays a central role in the cellular response to DNA DSBs . In this study, we examined the role of the complex in homologous recombination, the primary mode of DSB repair in yeast . We measured survival in synchronous cultures following irradiation and scored sister chromatid and interhomologue recombination genetically . mre11Delta strains were profoundly sensitive to ionizing radiation (IR) throughout the cell cycle . Mutant strains exhibited decreased frequencies of IR-induced sister chromatid and interhomologue recombination, indicating a general deficiency in homologous recombination-based DSB repair . Since a nuclease-deficient mre11 mutant was not impaired in these assays, it appears that the role of the S . cerevisiae Mre11-Rad50-Xrs2 protein complex in facilitating homologous recombination is independent of its nuclease activities.

J Biol Chem, 1999 Oct 22, 274(43), 30826 - 31
Post-transcriptional adenylation of signal recognition particle RNA is carried out by an enzyme different from mRNA Poly(A) polymerase; Sinha K et al.; A fraction of the signal recognition particle (SRP) RNA from human, rat, Xenopus, and Saccharomyces cerevisiae cells contains a single post-transcriptionally added adenylic acid residue on its 3'-end; in the case of human SRP RNA, over 60% of the SRP RNA molecules contain a nontemplated adenylic acid residue on their 3'-ends (Sinha, K . M., Gu, J., Chen, Y., and Reddy, R . (1998) J . Biol . Chem . 273, 6853-6859) . In this study, we investigated the enzyme that is involved in this 3'-end adenylation of SRP RNA . A U1A protein peptide conjugated to albumin completely inhibited the polyadenylation of a SV40 mRNA by HeLa cell nuclear extract in vitro; however, the 3'-end adenylation of human SRP RNA or Alu RNA, which corresponds to 5' and 3'-ends of SRP RNA, was not affected by this U1A peptide conjugate . SRP RNA from mutant strains of S . cerevisiae with a temperature-sensitive mRNA poly(A) polymerase grown at a restrictive temperature of 37 degrees C also contained a post-transcriptionally added adenylic acid residue just like SRP RNA from wild-type cells and mutant cells grown at permissive temperature of 23 degrees C . In addition, binding of SRP 9/14-kDa protein heterodimer was required for adenylation of Alu RNA in vitro . These lines of evidence, along with other data, show that post-transcriptional adenylation of SRP and Alu RNAs is carried out by a novel enzyme that is distinct from the mRNA poly(A) polymerase, CCA-adding enzyme, and nonspecific terminal transferase.

Mol Cell, 1999 Sep, 4(3), 439 - 44
MSH2 and MSH6 are required for removal of adenine misincorporated opposite 8-oxo-guanine in S . cerevisiae; Ni TT et al.; Oxidation of G in DNA yields 8-oxo-G (GO), a mutagenic lesion that leads to misincorporation of A opposite GO . In E . coli, GO in GO:C base pairs is removed by MutM, and A in GO:A mispairs is removed by MutY . In S . cerevisiae, mutations in MSH2 or MSH6 caused a synergistic increase in mutation rate in combination with mutations in OGG1, which encodes a MutM homolog, resulting in a 140- to 218-fold increase in the G:C-to-T:A transversion rate . Consistent with this, MSH2-MSH6 complex bound to GO:A mispairs and GO:C base pairs with high affinity and specificity . These data indicate that in S . cerevisiae, MSH2-MSH6-dependent mismatch repair is the major mechanism by which misincorporation of A opposite GO is corrected.

J Bacteriol, 1999 Oct, 181(20), 6497 - 508
Divergent regulation of the evolutionarily closely related promoters of the Saccharomyces cerevisiae STA2 and MUC1 genes; Gagiano M et al.; The 5' upstream regions of the Saccharomyces cerevisiae glucoamylase-encoding genes STA1 to -3 and of the MUC1 (or FLO11) gene, which is critical for pseudohyphal development, invasive growth, and flocculation, are almost identical, and the genes are coregulated to a large extent . Besides representing the largest yeast promoters identified to date, these regions are of particular interest from both a functional and an evolutionary point of view . Transcription of the genes indeed seems to be dependent on numerous transcription factors which integrate the information of a complex network of signaling pathways, while the very limited sequence differences between them should allow the study of promoter evolution on a molecular level . To investigate the transcriptional regulation, we compared the transcription levels conferred by the STA2 and MUC1 promoters under various growth conditions . Our data show that transcription of both genes responded similarly to most environmental signals but also indicated significant divergence in some aspects . We identified distinct areas within the promoters that show specific responses to the activating effect of Flo8p, Msn1p (or Mss10p, Fup1p, or Phd2p), and Mss11p as well as to carbon catabolite repression . We also identified the STA10 repressive effect as the absence of Flo8p, a transcriptional activator of flocculation genes in S . cerevisiae.

J Bacteriol, 1999 Oct, 181(20), 6488 - 96
Horizontal transfer of genetic material among Saccharomyces yeasts; Marinoni G et al.; The genus Saccharomyces consists of several species divided into the sensu stricto and the sensu lato groups . The genomes of these species differ in the number and organization of nuclear chromosomes and in the size and organization of mitochondrial DNA (mtDNA) . In the present experiments we examined whether these yeasts can exchange DNA and thereby create novel combinations of genetic material . Several putative haploid, heterothallic yeast strains were isolated from different Saccharomyces species . All of these strains secreted an a- or alpha-like pheromone recognized by S . cerevisiae tester strains . When interspecific crosses were performed by mass mating between these strains, hybrid zygotes were often detected . In general, the less related the two parental species were, the fewer hybrids they gave . For some crosses, viable hybrids could be obtained by selection on minimal medium and their nuclear chromosomes and mtDNA were examined . Often the frequency of viable hybrids was very low . Sometimes putative hybrids could not be propagated at all . In the case of sensu stricto yeasts, stable viable hybrids were obtained . These contained both parental sets of chromosomes but mtDNA from only one parent . In the case of sensu lato hybrids, during genetic stabilization one set of the parental chromosomes was partially or completely lost and the stable mtDNA originated from the same parent as the majority of the nuclear chromosomes . Apparently, the interspecific hybrid genome was genetically more or less stable when the genetic material originated from phylogenetically relatively closely related parents; both sets of nuclear genetic material could be transmitted and preserved in the progeny . In the case of more distantly related parents, only one parental set, and perhaps some fragments of the other one, could be found in genetically stabilized hybrid lines . The results obtained indicate that Saccharomyces yeasts have a potential to exchange genetic material . If Saccharomyces isolates could mate freely in nature, horizontal transfer of genetic material could have occurred during the evolution of modern yeast species.

Yeast, 1999 Oct, 15(14), 1541 - 53
Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae; Goldstein AL et al.; Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae . Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers . We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat) . The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4 . Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids . These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus . These attributes make the cassettes ideally suited for creating S . cerevisiae strains with multiple mutations within a single strain .

Yeast, 1999 Sep 30, 15(13), 1323 - 9
One-step, PCR-mediated, gene disruption in the yeast Hansenula polymorpha; Gonzalez C et al.; Previous evidence based on the experience of our laboratory showed that one-step gene disruption in the yeast Hansenula polymorpha is not straightforward . A systematic study of several factors which could affect gene disruption frequency was carried out . We found that the more critical factor affecting one-step gene disruption in H . polymorpha is the length of the target gene region flanking the marker gene . Target gene regions of about 1 kb flanking the marker gene were necessary to obtain a disruption frequency of about 50% . However, the gene marker, either homologous or heterologous, the locus and the strain examined did not significantly affect the frequency of disruption; the highest disruption frequency obtained for the YNR1 gene was in the strain HMI39, using the Saccharomyces cerevisiae URA3 gene as a marker . Since long regions flanking the gene marker do not allow the easy PCR-mediated strategies, developed for S . cerevisiae, to obtain constructs to disrupt a given gene in H . polymorpha, an alternative PCR strategy was developed .

Curr Biol, 1999 Sep 9, 9(17), 975 - 8
Localization and dynamic relocalization of mammalian Rad52 during the cell cycle and in response to DNA damage; Liu Y et al.; The importance of RAD52 in establishment and maintenance of genomic structure has been established by genetic experiments in the yeast Saccharomyces cerevisiae, where mutation of RAD52 has been shown to diminish DNA repair and recombination of a variety of markers, including the rDNA {1} {2} {3} . Biochemical analysis has shown that yeast and mammalian Rad52 proteins have some identical functions in vitro {4} {5} {6}, but targeted deletion of Rad52 in vertebrates has little effect on repair and recombination {7} {8} . These results raise the question of whether mammalian Rad52 does indeed function in recombination and/or repair . Here we show that Rad52 is distributed throughout the nucleoplasm in actively cycling mammalian cells and is localized specifically to the nucleoli in S phase . In response to ionizing radiation, Rad52 relocalizes to form distinctive foci which are distributed throughout the nucleus and which colocalize with Rad50 foci in the DNA damage response . These data suggest that rDNA recombination and DNA repair are functions shared by mammalian Rad52 and its S . cerevisiae homolog, and provide evidence for the coordinated action of Rad50 and Rad52 in DNA repair.

J Clin Oncol, 1999 Oct, 17(10), 3025 - 32
Randomized, double-blind, placebo-controlled trial to evaluate the hematopoietic growth factor PIXY321 after moderate-dose fluorouracil, doxorubicin, and cyclophosphamide in stage II and III breast cancer; Jones SE et al.; PURPOSE: To measure the effect of PIXY321 (granulocyte-macrophage colony-stimulating factor/interleukin-3 S . cerevisiae fusion protein) on the incidence, duration, and complications of neutropenia and thrombocytopenia after moderate-dose fluorouracil 600 mg/m(2), doxorubicin 60 mg/m(2), and cyclophosphamide 750 mg/m(2) (FAC) chemotherapy in patients with stage II and III breast cancer . PATIENTS AND METHODS: In this multicenter, randomized, double-blind placebo-controlled trial, 71 women were to receive four 21-day cycles of treatment with moderate-dose FAC chemotherapy by short intravenous infusion on day 1, followed by either placebo or PIXY321 (375 microg/m(2) subcutaneously twice a day) on days 3 to 15 . All patients were to receive prophylactic oral ciprofloxacin when the absolute neutrophil count was less than 1,000/microL . RESULTS: PIXY321 significantly reduced the incidence and duration of grade 3 and grade 4 neutropenia in cycles 1 and 2 and the duration of grade 3 neutropenia in cycles 1 through 4 . In cycles 3 and 4, grade 3 thrombocytopenia was significantly more common with PIXY321 (P <.05) . Two patients, both in the PIXY321 group, required platelet transfusions . Fever and hospitalization for intravenous antibiotics were significantly more common in the PIXY321 group during cycle 1 only . More patients in the PIXY321 group achieved hematologic recovery by day 22 in cycles 1 through 3, and time to recovery was significantly shorter with PIXY321 in all cycles . FAC dose intensity was roughly 2% higher in the PIXY321 group (P = NS) . Nonhematologic events of any intensity occurring with significantly greater overall frequency in the PIXY321 group included injection-site reactions, fever, chills, abdominal pain, and arthralgia . No patient died on study or within 30 days of her last dose of study drug . CONCLUSION: PIXY321 decreased the incidence and duration of FAC-induced grade 3 and 4 neutropenia in cycles 1 and 2 and significantly shortened the time to hematologic recovery in all cycles . However, it produced more systemic toxicity as well as thrombocytopenia in cycles 3 and 4.

J Biol Chem, 1999 Oct 8, 274(41), 28865 - 74
Mutational analyses of yeast RNA triphosphatases highlight a common mechanism of metal-dependent NTP hydrolysis and a means of targeting enzymes to pre-mRNAs in vivo by fusion to the guanylyltransferase component of the capping apparatus; Pei Y et al.; Saccharomyces cerevisiae Cet1p is the prototype of a family of metal-dependent RNA 5'-triphosphatases/NTPases encoded by fungi and DNA viruses; the family is defined by conserved sequence motifs A, B, and C . We tested the effects of 12 alanine substitutions and 16 conservative modifications at 18 positions of the motifs . Eight residues were identified as important for triphosphatase activity . These were Glu-305, Glu-307, and Phe-310 in motif A (IELEMKF); Arg-454 and Lys-456 in motif B (RTK); Glu-492, Glu-494, and Glu-496 in motif C (EVELE) . Four acidic residues, Glu-305, Glu-307, Glu-494, and Glu-496, may comprise the metal-binding site(s), insofar as their replacement by glutamine inactivated Cet1p . E492Q retained triphosphatase activity . Basic residues Arg-454 and Lys-456 in motif B are implicated in binding to the 5'-triphosphate . Changing Arg-454 to alanine or glutamine resulted in a 30-fold increase in the K(m) for ATP, whereas substitution with lysine increased K(m) 6-fold . Changing Lys-456 to alanine or glutamine increased K(m) an order of magnitude; ATP binding was restored when arginine was introduced . Alanine in lieu of Phe-310 inactivated Cet1p, whereas Tyr or Leu restored function . Alanine mutations at aliphatic residues Leu-306, Val-493, and Leu-495 resulted in thermal instability in vivo and in vitro . A second S . cerevisiae RNA triphosphatase/NTPase (named Cth1p) containing motifs A, B, and C was identified and characterized . Cth1p activity was abolished by E87A and E89A mutations in motif A . Cth1p is nonessential for yeast growth and, by itself, cannot fulfill the essential role played by Cet1p in vivo . Yet, fusion of Cth1p in cis to the guanylyltransferase domain of mammalian capping enzyme allowed Cth1p to complement growth of cet1Delta yeast cells . This finding illustrates that mammalian guanylyltransferase can be used as a vehicle to deliver enzymes to nascent pre-mRNAs in vivo, most likely through its binding to the phosphorylated CTD of RNA polymerase II.

Mol Gen Genet, 1999 Aug, 262(1), 180 - 8
The MATA locus of the dimorphic yeast Yarrowia lipolytica consists of two divergently oriented genes; Kurischko C et al.; The MATA locus of Yarrowia lipolytica, which was on the basis of its ability to induce sporulation in a diploid B/B strain, represses the mating capacity of this strain . The gene functions required for induction of sporulation and repression of conjugation could be separated by subcloning . Sequence analysis revealed two ORFs in the MATA locus . One of them (MATA1) codes for a protein of 119 amino acids which is required to induce sporulation . The other (MATA2) codes for a protein of 291 amino acids that is able to repress conjugation . Both genes are oriented divergently from a central promoter region, which possesses putative TATA and CAAT boxes for both genes . The product of MATA1 shows no homology to any known protein and seems to represent a new class of mating-type genes . MATA2 contains a HMG box with homology to other mating-type genes . Both MATA1 and MATA2 are mating-type specific . In cells of both mating types, the regions flanking the MATA locus contain sequences with homology to either S . cerevisiae SLA2 and ORF YBB9, respectively . From hybridization and subcloning data we estimate that the MATA region is approximately 2 kb long and is present only once in the genome.

Curr Genet, 1999 Sep, 36(3), 113 - 23
The N-end rule pathway is required for import of histidine in yeast lacking the kinesin-like protein Cin8p; Xie Y et al.; The N-end rule pathway is a ubiquitin-dependent proteolytic system whose targets include proteins bearing destabilizing N-terminal residues . We carried out a synthetic lethal screen for Saccharomyces cerevisiae mutants that require the N-end rule pathway for cell viability . A mutant thus identified, termed sln2, could not grow in the absence of Ubr1p, the recognition component of the N-end rule pathway, which was not essential for viability of the parental strain under the same conditions . Further analysis showed that inviability of sln2ubr1Delta cells could be rescued either by the HIS3 gene (which was absent from the parental strain) or by a high concentration of histidine in the medium . This defect in histidine uptake, exhibited by the sln2 mutant in the absence but not in the presence of Ubr1p, was traced to the gene HIP1, which encodes the histidine transporter . HIP1 was underexpressed in sln2 ubr1Delta cells, in comparison to either sln2 UBR1 or SLN2 ubr1Delta cells . Yet another property of the sln2 mutant was its inviability at 37 degrees C, which could not be rescued by either UBR1 or HIS3 . This feature of sln2 allowed the cloning of SLN2, which was found to be a gene called CIN8, encoding a kinesin-like protein . Thus, either the N-end rule pathway or Cin8p must be present for the viability-sustaining rate of histidine import in S . cerevisiae auxotrophic for histidine . We consider possible mechanisms of this previously unsuspected link between kinesins, ubiquitin-dependent proteolysis, and the import of histidine.

Cell, 1999 Sep 17, 98(6), 847 - 58
Identification of cohesin association sites at centromeres and along chromosome arms; Tanaka T et al.; A multisubunit cohesin complex holds sister chromatids together after DNA replication . Using chromatin immunoprecipitation, we detected cohesin association with centromeres and with discrete sites along chromosome arms from S phase until metaphase in S . cerevisiae . Short DNA sequences (130-280 bp) are sufficient to confer cohesin association . Cohesin association with a centromere depends on Mif2p, the centromere binding factor CBF3, and a centromere-specific histone variant, Cse4p . Because only active centromeres confer cohesin association with centromeric DNA, we suggest that cohesin is recruited by the same chromatin structure that confers the attachment of microtubules . Propagation of this structure might be partly epigenetic . Finally, cohesion associated with "minimal" centromeres is insufficient to resist the splitting force exerted by microtubules and appears to be reinforced by cohesion provided by their flanking DNA sequences.

Mol Biochem Parasitol, 1999 Aug 20, 102(2), 283 - 95
Cloning and characterization of a gene encoding phosphatidyl inositol-specific phospholipase C from Trypanosoma cruzi; Nozaki T et al.; A gene encoding phosphatidyl inositol-4,5-bisphosphate phospholipase C (PLC) was cloned from the protozoan parasite Trypanosoma cruzi . A partial cDNA encoding putative PLC was obtained by a polymerase chain reaction (PCR) using degenerate oligonucleotide primers corresponding to conserved regions of PLCs . A 2178-bp protein coding region of the T . cruzi PLC gene, composed from cDNA and genomic clones, encodes a putative PLC with a calculated molecular mass of 82,032 Da and an isoelectric point of 5.93 . The deduced amino acid sequence of T . cruzi PLC exhibited 23-42% overall identities with the PLCs from other organisms . Among them, PLC from Ictalurus punctatus revealed the highest identity to T . cruzi PLC . The percentage identities of the entire proteins and the catalytic X/Y domains suggested that T . cruzi PLC is more evolutionarily related to the PLCs of higher eukaryotes than to those of lower unicellular eukaryotes . The tetrad analysis of the segregants of the Saccharomyces cerevisiae PLC1/plc1::HIS3 diploid strain transformed with the T . cruzi PLC-expressing plasmid showed that expression of T . cruzi PLC suppressed the growth defect caused by the plc1 disruption in yeasts . Temperature-sensitive phenotype of the S . cerevisiae plc1-mutant haploid strain was also suppressed by the expression of T . cruzi PLC . The phosphatidyl inositol-4,5-biphosphate (PtdIns(4,5)P2) hydrolyzing activity of T . cruzi PLC was demonstrated in the lysate from the plc1-temperature sensitive yeast mutant strain transformed with the T . cruzi PLC-expressing plasmid . The yeast-expressed T . cruzi PLC showed an absolute Ca2+ dependence which was similar to mammalian PLC isoforms: the half-maximal activity at 0.5-1 x 10(-5) M Ca2+ and the maximal activity at 1-2 x 10(-4) M Ca2+.

J Biol Chem, 1999 Oct 1, 274(40), 28779 - 86
The kinesin-related protein Kip1p of Saccharomyces cerevisiae is bipolar; Gordon DM et al.; Kip1p is a mitotic spindle-associated kinesin-related protein in Saccharomyces cerevisiae that participates in spindle pole separation . Here, we define the domain arrangement and polypeptide composition of the Kip1p holoenzyme . Electron microscopy of rotary shadowed Kip1p molecules revealed two globular domains 14 nm in diameter connected by a 73-nm long stalk . When the Kip1p domain homologous to the kinesin motor domain was decorated with an unrelated protein, the diameter of the globular domains at both ends of the stalk increased, indicating that Kip1p is bipolar . Soluble Kip1p isolated from S . cerevisiae cells was homomeric, based on the similarity of the sedimentation coefficients of native Kip1p from S . cerevisiae and Kip1p which was purified after expression in insect cells . The holoenzyme molecular weight was estimated using the sedimentation coefficient and Stokes radius, and was most consistent with a tetrameric composition . Kip1p exhibited an ionic strength-dependent transition in its sedimentation coefficient, revealing a potential regulatory mechanism . The position of kinesin motor-related domains at each end of the stalk may allow Kip1p to cross-link either parallel or antiparallel microtubules during mitotic spindle assembly and pole separation.

Mol Cell Biol, 1999 Oct, 19(10), 6621 - 31
The ADA complex is a distinct histone acetyltransferase complex in Saccharomyces cerevisiae; Eberharter A et al.; We have identified two Gcn5-dependent histone acetyltransferase (HAT) complexes from Saccharomyces cerevisiae, the 0.8-MDa ADA complex and the 1.8-MDa SAGA complex . The SAGA (Spt-Ada-Gcn5-acetyltransferase) complex contains several subunits which also function as part of other protein complexes, including a subset of TATA box binding protein-associated factors (TAFIIs) and Tra1 . These observations raise the question of whether the 0.8-MDa ADA complex is a subcomplex of SAGA or whether it is a distinct HAT complex that also shares subunits with SAGA . To address this issue, we sought to determine if the ADA complex contained subunits that are not present in the SAGA complex . In this study, we report the purification of the ADA complex over 10 chromatographic steps . By a combination of mass spectrometry analysis and immunoblotting, we demonstrate that the adapter proteins Ada2, Ada3, and Gcn5 are indeed integral components of ADA . Furthermore, we identify the product of the S . cerevisiae gene YOR023C as a novel subunit of the ADA complex and name it Ahc1 for ADA HAT complex component 1 . Biochemical functions of YOR023C have not been reported . However, AHC1 in high copy numbers suppresses the cold sensitivity caused by particular mutations in HTA1 (I . Pinto and F . Winston, personal communication), which encodes histone H2A (J . N . Hirschhorn et al., Mol . Cell . Biol . 15:1999-2009, 1995) . Deletion of AHC1 disrupted the integrity of the ADA complex but did not affect SAGA or give rise to classic Ada(-) phenotypes . These results indicate that Gcn5, Ada2, and Ada3 function as part of a unique HAT complex (ADA) and represent shared subunits between this complex and SAGA.

Eur J Biochem, 1999 Sep, 264(3), 825 - 32
Functional complementation analysis of yeast bc1 mutants . A study of the mitochondrial import of heterologous and hybrid proteins; van Wilpe S et al.; Previous complementation studies with yeast bc1 mutants, defective in subunit VII or VIII, using heterologous and hybrid subunits, suggested that the requirement for import into mitochondria might significantly restrict the scope of this test for compatible proteins . Prediction algorithms indicate that the N-terminal domain of subunit VII contains all known characteristics of a mitochondrial targeting signal, whereas in subunit VIII such a signal is absent from the N-terminal domain, but possibly present in an internal region of the protein . Despite the fact that the characteristics of a mitochondrial import signal are found in the N-terminus of all known subunit-VII orthologues, in vitro import experiments show that the protein of human origin is not imported into yeast mitochondria . In vitro import can be restored, however, by replacement of the N-terminal part of the human protein by the N-terminus of the Saccharomyces cerevisiae orthologue, indicating a requirement for species-specific elements . Similar experiments were performed with subunit VIII and orthologues thereof, including a hybrid protein in which the N-terminus of the bovine heart orthologue was replaced by that of S . cerevisiae . The ability of yeast mitochondria to import this hybrid protein, in contrast with the bovine subunit-VIII orthologue itself, indicates that for subunit VIII also the N-terminus, in contradiction of theoretical predictions, contributes to the targeting signal, most likely via species-specific elements . Our findings expose the limitations of the currently available criteria for prediction of the presence and location of a mitochondrial targeting sequence and highlight the necessity of performing separate import studies for interpreting complementation studies as long as the species-specific characteristics of the import signals have not been identified.

Mol Cell Biol, 1999 Oct, 19(10), 7123 - 37
Hsl7 localizes to a septin ring and serves as an adapter in a regulatory pathway that relieves tyrosine phosphorylation of Cdc28 protein kinase in Saccharomyces cerevisiae; Shulewitz MJ et al.; Successful mitosis requires faithful DNA replication, spindle assembly, chromosome segregation, and cell division . In the budding yeast Saccharomyces cerevisiae, the G(2)-to-M transition requires activation of Clb-bound forms of the protein kinase, Cdc28 . These complexes are held in an inactive state via phosphorylation of Tyr19 in the ATP-binding loop of Cdc28 by the Swe1 protein kinase . The HSL1 and HSL7 gene products act as negative regulators of Swe1 . Hsl1 is a large (1,518-residue) protein kinase with an N-terminal catalytic domain and a very long C-terminal extension . Hsl1 localizes to the incipient site of cytokinesis in the bud neck in a septin-dependent manner; however, the function of Hsl7 was not previously known . Using both indirect immunofluorescence with anti-Hsl7 antibodies and a fusion of Hsl7 to green fluorescent protein, we found that Hsl7 also localizes to the bud neck, congruent with the septin ring that faces the daughter cell . Both Swe1 and a segment of the C terminus of Hsl1 (which has no sequence counterpart in two Hsl1-related protein kinases, Gin4 and Kcc4) were identified as gene products that interact with Hsl7 in a two-hybrid screen of a random S . cerevisiae cDNA library . Hsl7 plus Swe1 and Hsl7 plus Hsl1 can be coimmunoprecipitated from extracts of cells overexpressing these proteins, confirming that Hsl7 physically associates with both partners . Also consistent with the two-hybrid results, Hsl7 coimmunoprecipitates with full-length Hsl1 less efficiently than with a C-terminal fragment of Hsl1 . Moreover, Hsl7 does not localize to the bud neck in an hsl1Delta mutant, whereas Hsl1 is localized normally in an hsl7Delta mutant . Phosphorylation and ubiquitinylation of Swe1, preludes to its destruction, are severely reduced in cells lacking either Hsl1 or Hsl7 (or both), as judged by an electrophoretic mobility shift assay . Collectively, these data suggest that formation of the septin rings provides sites for docking Hsl1, exposing its C terminus and thereby permitting recruitment of Hsl7 . Hsl7, in turn, presents its cargo of bound Swe1, allowing phosphorylation by Hsl1 . Thus, Hsl1 and Hsl7 promote proper timing of cell cycle progression by coupling septin ring assembly to alleviation of Swe1-dependent inhibition of Cdc28 . Furthermore, like septins and Hsl1, homologs of Hsl7 are found in fission yeast, flies, worms, and humans, suggesting that its function in this control mechanism may be conserved in all eukaryotes.

Mol Cell Biol, 1999 Oct, 19(10), 6796 - 802
Characterization of a protein complex containing spliceosomal proteins SAPs 49, 130, 145, and 155; Das BK et al.; SF3b is a U2 snRNP-associated protein complex essential for spliceosome assembly . Although evidence that SF3b contains the spliceosomal proteins SAPs 49, 130, 145, and 155 has accumulated, a protein-mediated association between all of these proteins has yet to be directly demonstrated . Here we report the isolation of a cDNA encoding SAP 130, which completes the cloning of the putative SF3b complex proteins . Using antibodies to SAP 130 and other putative SF3b components, we showed that SAPs 130, 145, and 155 are present in a protein complex in nuclear extracts and that these proteins associate with one another in purified U2 snRNP . Moreover, SAPs 155 and 130 interact with each other (directly or indirectly) within this complex, and SAPs 49 and 145 are known to interact directly with each other . Thus, together with prior work, our studies indicate that SAPs 49, 130, 145, and 155 are indeed components of SF3b . The Saccharomyces cerevisiae homologs of SAPs 49 and 145 are encoded by essential genes . We show here that the S . cerevisiae homologs of SAPs 130 and 155 (scSAP 130/RSE1 and scSAP 155, respectively) are also essential . Recently, the SF3b proteins were found in purified U12 snRNP, which functionally substitutes for U2 snRNP in the minor spliceosome . This high level of conservation, together with the prior observation that the SF3b proteins interact with pre-mRNA very close to the branch site, suggest that the SF3b complex plays a critical role near or at the spliceosome catalytic core.

Yeast, 1999 Sep 15, 15(12), 1287 - 96
Disruption of six novel ORFs on the left arm of chromosome XII reveals one gene essential for vegetative growth of Saccharomyces cerevisiae; Zhang N et al.; Deletion via PCR-mediated gene replacement, together with basic functional and bioinformatic analyses, have been performed on six novel open reading-frames (ORFs) on the left arm of chromosome XII of Saccharomyces cerevisiae(YLL033w, YLL032c, YLL031c, YLL030c, YLL029w and YLL028w) . ORF deletion was realized using either a short-flanking homology (SFH) or a long-flanking homology (LFH) replacement cassette in the diploid strain FY1679 . Sporulation and tetrad analysis showed that YLL031c is the only essential gene of the six . Microscopic examination of the non-growing spores carrying a disrupted copy of the essential gene showed that most of them were blocked after one or two cell divisions with heterogeneous bud size . The standard EUROFAN growth tests failed to reveal any obvious phenotype resulting from the deletion of each the five non-essential ORFs . Bioinformatic analysis revealed that YLL029w is probably an aminopeptidase for mitochondrial or nuclear protein processing and YLL028w may be involved in drug resistance in S . cerevisiae . Replacement cassettes, comprising the promoter and terminator regions of each of the six ORFs, were cloned into pUG7 and demonstrated to efficiently mediate gene replacement in an alternative diploid strain, W303 . All the cognate gene clones were constructed, using either PCR products amplified from genomic DNA, or gap-repair . All clones and strains generated have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt) .

Yeast, 1999 Sep 15, 15(12), 1237 - 42
Isolation and expression of the gene encoding mitochondrial ADP/ATP carrier (AAC) from the pathogenic yeast Candida parapsilosis; Nebohacova M et al.; A gene homologous to Saccharomyces cerevisiae AAC genes coding for mitochondrial ADP/ATP carriers has been cloned from the pathogenic yeast Candida parapsilosis . A probe obtained by PCR amplification from C . parapsilosis DNA, using primers derived from the conserved transmembrane region of yeast ADP/ATP carriers, was used for screening of the C . parapsilosis genomic library . The cloned gene was sequenced and found to encode a polypeptide of 303 amino acids that shows homology with other yeast and fungal mitochondrial ADP/ATP carriers . The gene was designated CpAAC1 and was able to complement the growth phenotypes of S . cerevisiae double deletion mutant (Deltaaac2; Deltaaac3) . The expression of the CpAAC1 gene was reduced under semi-anaerobic conditions and it was affected at normal aerobic conditions by the nature of carbon sources used for growth . Hybridization experiments indicate that C . parapsilosis possesses a single gene encoding a mitochondrial ADP/ATP carrier .

J Bacteriol, 1999 Sep, 181(18), 5876 - 9
Cloning and characterization of a sulfonate/alpha-ketoglutarate dioxygenase from Saccharomyces cerevisiae; Hogan DA et al.; The Saccharomyces cerevisiae open reading frame YLL057c is predicted to encode a gene product with 31.5% amino acid sequence identity to Escherichia coli taurine/alpha-ketoglutarate dioxygenase and 27% identity to Ralstonia eutropha TfdA, a herbicide-degrading enzyme . Purified recombinant yeast protein is shown to be an Fe(II)-dependent sulfonate/alpha-ketoglutarate dioxygenase . Although taurine is a poor substrate, a variety of other sulfonates are utilized, with the best natural substrates being isethionate and taurocholate . Disruption of the gene encoding this enzyme negatively affects the use of isethionate and taurine as sulfur sources by S . cerevisiae, providing strong evidence that YLL057c plays a role in sulfonate catabolism.

FEMS Microbiol Lett, 1999 Oct 1, 179(1), 107 - 13
By-product formation during exposure of respiring Saccharomyces cerevisiae cultures to excess glucose is not caused by a limited capacity of pyruvate carboxylase; Bauer J et al.; Upon exposure to excess glucose, respiring cultures of Saccharomyces cerevisiae produce substantial amounts of ethanol and acetate . A possible role of a limited anaplerotic capacity in this process was investigated by overexpressing pyruvate carboxylase and by replacing it with a heterologous enzyme (Escherichia coli phosphoenolpyruvate carboxylase) . Compared to the wild-type, neither the pyruvate carboxylase (Pyc)-overexpressing nor the transgenic strain exhibited reduced by-product formation after glucose pulses to aerobic glucose-limited chemostat cultures . An increased intracellular malate concentration was observed in the two engineered strains . It is concluded that by-product formation in S . cerevisiae is not caused by a limited anaplerotic capacity.

J Biol Chem, 1999 Sep 17, 274(38), 26885 - 93
A novel family of yeast chaperons involved in the distribution of V-ATPase and other membrane proteins; Cohen A et al.; Null mutations in genes encoding V-ATPase subunits in Saccharomyces cerevisiae result in a phenotype that is unable to grow at high pH and is sensitive to high and low metal-ion concentrations . Treatment of these null mutants with ethylmethanesulfonate causes mutations that suppress the V-ATPase null phenotype, and the mutant cells are able to grow at pH 7.5 . The suppressor mutants were denoted as svf (suppressor of V-ATPase function) . The frequency of svf is relatively high, suggesting a large target containing several genes for the ethylmethanesulfonate mutagenesis . The suppressors' frequency is dependent on the individual genes that were inactivated to manifest the V-ATPase null mutation . The svf mutations are recessive, because crossing the svf mutants with their corresponding V-ATPase null mutants resulted in diploid strains that are unable to grow at pH 7.5 . A novel gene family in which null mutations cause pleiotropic effects on metal-ion resistance or sensitivity and distribution of membrane proteins in different targets was discovered . The family was defined as VTC (Vacuolar Transporter Chaperon) and it contains four genes in the S . cerevisiae genome . Inactivation of one of them, VTC1, in the background of V-ATPase null mutations resulted in svf phenotype manifested by growth at pH 7.5 . Deletion of the VTC1 gene (DeltaVTC1) results in a reduced amount of V-ATPase in the vacuolar membrane . These mutant cells fail to accumulate quinacrine into their vacuoles, but they are able to grow at pH 7.5 . The VTC1 null mutant also results in a reduced amount of the plasma membrane H(+)-ATPase (Pma1p) in membrane preparations and possibly mis-targeting . This observation may provide an explanation for the svf phenotype in the double disruptant mutants of DeltaVTC1 and DeltaVMA subunits.

J Biol Chem, 1999 Sep 17, 274(38), 26697 - 704
The CLK family kinases, CLK1 and CLK2, phosphorylate and activate the tyrosine phosphatase, PTP-1B; Moeslein FM et al.; The protein-tyrosine phosphatase PTP-1B is an important regulator of intracellular protein tyrosine phosphorylation, and is itself regulated by phosphorylation . We report that PTP-1B and its yeast analog, YPTP, are phosphorylated and activated by members of the CLK family of dual specificity kinases . CLK1 and CLK2 phosphorylation of PTP-1B in vitro activated the phosphatase activity approximately 3-5-fold using either p-nitrophenol phosphate, or tyrosine-phosphorylated myelin basic protein as substrates . Co-expression of CLK1 or CLK2 with PTP-1B in HEK 293 cells led to a 2-fold stimulation of phosphatase activity in vivo . Phosphorylation of PTP-1B at Ser(50) by CLK1 or CLK2 is responsible for its enzymatic activation . These findings suggest that phosphorylation at Ser(50) by serine threonine kinases may regulate the activation of PTP-1B in vivo . We also show that CLK1 and CLK2 phosphorylate and activate the S . cerevisiae PTP-1B family member, YPTP1 . CLK1 phosphorylation of YPTP1 led to a 3-fold stimulation of phosphatase activity in vitro . We demonstrate that CLK phosphorylation of Ser(83) on YPTP1 is responsible for the activation of this enzyme . These findings demonstrate that the CLK kinases activate PTP-1B family members, and this phosphatase may be an important cellular target for CLK action.

Lipids, 1999 Jul, 34(7), 649 - 59
Cloning of delta12- and delta6-desaturases from Mortierella alpina and recombinant production of gamma-linolenic acid in Saccharomyces cerevisiae; Huang YS et al.; Two cDNA clones with homology to known desaturase genes were isolated from the fungus Mortierella alpina . The open reading frame in one clone encoded 399 amino acids and exhibited delta12-desaturase activity when expressed in Saccharomyces cerevisiae in the presence of endogenous fatty acid substrate oleic acid . The insert in another clone contained an open reading frame encoding 457 amino acids and exhibited delta6-desaturase activity in S . cerevisiae in the presence of exogenous fatty acid substrate linoleic acid . Expression of the delta12-desaturase gene under appropriate media and temperature conditions led to the production of linoleic acid at levels up to 25% of the total fatty acids in yeast . When linoleic acid was provided as an exogenous substrate to the yeast cultures expressing the delta6-desaturase activity, the level of gamma-linolenic acid reached 10% of the total yeast fatty acids . Co-expression of both the delta6- and delta12-desaturase cDNA resulted in the endogenous production of gamma-linolenic acid . The yields of gamma-linolenic acid reached as high as 8% of total fatty acids in yeast.

Development, 1999 Oct, 126(19), 4281 - 93
The Drosophila chiffon gene is required for chorion gene amplification, and is related to the yeast Dbf4 regulator of DNA replication and cell cycle; Landis G et al.; The Drosophila chorion genes encode the major protein components of the chorion (eggshell) and are arranged in two clusters in the genome . To meet the demand for rapid chorion synthesis, Drosophila ovary follicle cells amplify the chorion gene clusters approximately 80-fold . Amplification proceeds through repeated firing of one or more DNA replication origins located near the center of each gene cluster . Hypomorphic mutant alleles of the chiffon gene cause thin, fragile chorions and female sterility, and were found to eliminate chorion gene amplification . Null alleles of chiffon had the additional phenotypes of rough eyes and thin thoracic bristles: phenotypes often associated with disruption of normal cell cycle . The chiffon locus was cloned by chromosomal walking from the nearby cactus locus . A 6.5 kb transcript was identified and confirmed to be chiffon by sequencing of mutant alleles and by phenotypic rescue with genomic transformation constructs . The protein predicted by translation of the 5.1 kb chiffon ORF contains two domains related to the S . cerevisiae Dbf4 regulator of DNA replication origin firing and cell cycle progression: a 44 residue domain designated CDDN1 (43% identical) and a 41 residue domain designated CDDN2 (12% identical) . The CDDN domains were also found in the S . pombe homolog of Dbf4, Dfp1, as well as in the proteins predicted by translation of the Aspergillus nimO gene and specific human and mouse clones . The data suggest a family of eukaryotic proteins related to Dbf4 and involved in initiation of DNA replication.

Biochem J, 1999 Sep 15, 342 Pt 3, 667 - 75
The dihydrosphingosine-1-phosphate phosphatases of Saccharomyces cerevisiae are important regulators of cell proliferation and heat stress responses; Mao C et al.; We have identified YSR2 and YSR3 of Saccharomyces cerevisiae as genes encoding dihydrosphingosine-1-phosphate phophatases which are involved in regulation of sphingolipid metabolism {Mao, Wadleigh, Jenkins, Hannun and Obeid (1997) J . Biol . Chem . 272, 28690-28694} . In this study, we explored the physiological roles that these enzymes may have in S . cerevisiae . Deletion of either YSR2, YSR3 or both did not affect viability or growth rate of yeast cells . However, overexpression of YSR2 significantly prolonged the doubling time of cell growth, whereas overexpression of YSR3 affected cell growth only slightly . Cell cycle analysis suggested that overexpression of either YSR2 or, to a lesser extent, YSR3 caused cell cycle arrest at the G1 phase . Disruption of YSR2, but not YSR3, conferred increased thermotolerance . On the other hand, overexpression of either YSR2 or YSR3 diminished thermotolerance . Using labelled dihydrosphingosine and dihydrosphingosine-1-P (DHS-1-P), we found that overexpression of YSR2 significantly increased ceramide formation, whereas deletion of YSR2, YSR3, or both, accumulated DHS-1-P, and deletion of YSR2 decreased ceramide formation . Together, these results show that the phenotypes of YSR2 are associated with changes in endogenous levels of the different sphingolipids . Green fluorescent protein tagging showed that in the exponentially growing cells, YSR2 and YSR3 had the same cellular localization to endoplasmic reticulum . However, YSR2 and YSR3 differ in mRNA levels: YSR2 had significantly higher mRNA levels than YSR3 . This discrepancy might result in the functional differences that these proteins exhibited . In addition, this study implicates sphingolipids and their metabolism in the regulation of growth and heat stress responses of the yeast S . cerevisiae.

FEMS Microbiol Lett, 1999 Aug 15, 177(2), 211 - 6
Enolase is present in the cell wall of Saccharomyces cerevisiae; Edwards SR et al.; Non-covalently attached or soluble cell wall proteins of Saccharomyces cerevisiae were extracted using a high pH/2-mercaptoethanol procedure and were separated for peptide sequencing using 2D-PAGE . A partial N-terminal sequence of a major protein spot was obtained and showed high identity with enolase gene products . Western blotting with an anti-enolase antibody confirmed that enolase was present in the cell wall extract . Biotinylation of cells prior to protein extraction with a membrane impermeable biotinylating agent confirmed that the detection was not owing to cell lysis during extraction . Transmission immunoelectron microscopy showed enolase to be present in the cell wall . Enolase contains no known secretion signal that would localize it to the cell wall . Thus S . cerevisiae must have further mechanisms for targeting proteins to the cell wall.

Nucleic Acids Res, 1999 Sep 15, 27(18), 3736 - 44
Analysis of a YAC with human telomeres and oriP from epstein-barr virus in yeast and 293 cells; Tolmachova T et al.; One approach to the construction and propagation of a mammalian artificial chromosome is to build it up in Saccharomyces cerevisiae, using a yeast artificial chromosome (YAC) base . We have demonstrated that circular YACs carrying the Epstein-Barr virus origin of plasmid replication ( oriP ) are maintained as stable, episomal elements in human cells . We wished to determine whether this technology could be extended, to generate linear extrachromosomal elements . Here, we describe the generation of retrofitting constructs, which permit the addition of human telomeres and the oriP domain to YACs . The constructs contain 0.8 kb of human telomere sequence separated by a unique Not I site from 0.7 kb of Tetrahymena telomere sequence . These constructs seed telomere formation with approximately 40-60% efficiency in human 293-EBNA and HT1080 cells whether or not the Tetrahymena sequence is removed by Not I digestion . A detailed analysis demonstrates that YACs carrying the human telomere cassettes on both arms show instability of the telomere sequences in S.cerevisiae at a frequency of approximately 50% . Introduction of correctly retrofitted, linear oriP YACs into human 293-EBNA cells by lipofection resulted in the generation of circular extrachromosomal elements varying in size from 8 to 300 kb . However, no apparently linear YACs could be detected, suggesting that extrachromosomal maintenance of DNA with the oriP /EBNA-1 system is not compatible with linear molecules capped by telomeres.

Nat Genet, 1999 Sep, 23(1), 81 - 5
Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants; Chen C et al.; Cancer progression is often associated with the accumulation of gross chromosomal rearrangements (GCRs), such as translocations, deletion of a chromosome arm, interstitial deletions or inversions . In many instances, GCRs inactivate tumour-suppressor genes or generate novel fusion proteins that initiate carcinogenesis . The mechanism underlying GCR formation appears to involve interactions between DNA sequences of little or no homology . We previously demonstrated that mutations in the gene encoding the largest subunit of the Saccharomyces cerevisiae single-stranded DNA binding protein (RFA1) increase microhomology-mediated GCR formation . To further our understanding of GCR formation, we have developed a novel mutator assay in S . cerevisiae that allows specific detection of such events . In this assay, the rate of GCR formation was increased 600-5, 000-fold by mutations in RFA1, RAD27, MRE11, XRS2 and RAD50, but was minimally affected by mutations in RAD51, RAD54, RAD57, YKU70, YKU80, LIG4 and POL30 . Genetic analysis of these mutants suggested that at least three distinct pathways can suppress GCRs: two that suppress microhomology-mediated GCRs (RFA1 and RAD27) and one that suppresses non-homology-mediated GCRs (RAD50/MRE11/XRS2).

Biochemistry, 1999 Aug 31, 38(35), 11298 - 306
Saccharomyces cerevisiae Ntg1p and Ntg2p: broad specificity N-glycosylases for the repair of oxidative DNA damage in the nucleus and mitochondria; You HJ et al.; Saccharomyces cerevisiae possesses two functional homologues (Ntg1p and Ntg2p) of the Escherichia coli endonuclease III protein, a DNA base excision repair N-glycosylase with a broad substrate specificity directed primarily against oxidatively damaged pyrimidines . The substrate specificities of Ntg1p and Ntg2p are similar but not identical, and differences in their amino acid sequences as well as inducibility by DNA damaging agents suggest that the two proteins may have different biological roles and subcellular locations . Experiments performed on oligonucleotides containing a variety of oxidative base damages indicated that dihydrothymine, urea, and uracil glycol are substrates for Ntg1p and Ntg2p, although dihydrothymine was a poor substrate for Ntg2p . Vectors encoding Ntg1p-green fluorescent protein (GFP) and Ntg2p-GFP fusions under the control of their respective endogenous promoters were utilized to observe the subcellular targeting of Ntg1p and Ntg2p in S . cerevisiae . Fluorescence microscopy of pNTG1-GFP and pNTG2-GFP transformants revealed that Ntg1p localizes primarily to the mitochondria with some nuclear localization, whereas Ntg2p localizes exclusively to the nucleus . In addition, the subcellular location of Ntg1p and Ntg2p confers differential sensitivities to the alkylating agent MMS . These results expand the known substrate specificities of Ntg1p and Ntg2p, indicating that their base damage recognition ranges show distinct differences and that these proteins mediate different roles in the repair of DNA base damage in the nucleus and mitochondria of yeast.

Proc R Soc Lond B Biol Sci, 1999 Aug 7, 266(1428), 1555 - 60
Genomic demography: a life-history analysis of transposable element evolution; Promislow DE et al.; Retrotransposons are ubiquitous mobile genetic elements that have played a significant role in shaping eukaryotic genome evolution . The genome of the yeast Saccharomyces cerevisiae harbours five families of retrotransposons, Ty1-Ty5 . With the publication of the S . cerevisiae genome sequence, for the first time a full genomic complement of retrotransposon sequences is available . Analysis of these sequences promises to yield insight into the nature of host--transposon coevolution . Evolutionary change in Ty elements depends on their replication and excision rates, which have been determined in the laboratory . Rates measured in the laboratory may differ from those that have operated over evolutionary time . Based on an analysis of sequence data for the Ty1, Ty2 and hybrid Ty1/2 families, we develop a novel 'genomic demography' model to estimate long-term transposition and excision rates and to estimate how long ago these elements entered the yeast genome . We find that rates of excision and transposition have averaged 7.2-8.7 x 10(-8) per generation over evolutionary time . Two separate models provide upper- and lower-bound estimates for the age of the system, suggesting that the first elements entered the genome between approximately 50 million and 250 million generations ago.

Curr Genet, 1999 Jul, 35(6), 618 - 25
Compact organization of rRNA genes in the filamentous fungus Ashbya gossypii; Wendland J et al.; The rDNA cluster in the phytopathogenic fungus Ashbya gossypii consists of approximately 50 tandem repeat units of 8197 bp . Each unit carries a gene for the 35S pre-rRNA, processed into 18S, 5.8S and 25S rRNA, and a divergently transcribed gene for 5S rRNA . The well-characterized rDNA of the yeast Saccharomyces cerevisiae is the only other example of a completely sequenced rDNA unit (9137 bp) carrying both a 35S pre-rRNA and a 5S rRNA gene . The coding regions for the 5S, 5.8S, 18S and 25S rRNAs are 95-100% identical whereas transcribed and non-transcribed spacers show 43-66% sequence identity . Functionally characterized rDNA and rRNA elements of S . cerevisiae can be unambiguously recognized in the A . gossypii sequence, including the RNA polymerase-I transcription start site, two Reb1p enhancer binding sites and numerous recognition sequences for rRNA modification and processing . In addition to these functionally characterized sequences eight highly conserved elements from 10 to 71 bp were detected in the over 600-bp transcribed region upstream of the 18S rRNA gene which most likely play as yet uncharacterized functions at the DNA or RNA level . In addition to this work we started to identify A . gossypii homologs of S . cerevisiae nucleolar proteins involved in rDNA maturation.

Parasitology, 1999, 118 Suppl, S73 - 80
Whole genome genetic-typing in yeast using high-density oligonucleotide arrays; Winzeler EA et al.; Genome sequence information in combination with new technologies has allowed researchers to approach genetic problems in new ways . High-density oligonucleotide arrays were used to probe the genome content of the yeast Saccharomyces cerevisiae . We show that these arrays, containing oligonucleotides complementary to the sequenced strain of S . cerevisiae, can be used to identify open reading frames that are missing or present in higher or lower copy number in related isolates of S . cerevisiae . We apply this method to the characterization of the genome of a strain derived from a clinical isolate of S . cerevisiae . Our results show that the telomeres are the regions with the most variability between the two strains.

Yeast, 1999 Aug, 15(11), 1045 - 57
Transcription of the HXT4 gene is regulated by Gcr1p and Gcr2p in the yeast S . cerevisiae; Turkel S et al.; Glucose transport and glycolysis are two sequential events which are regulated by both physiological and environmental signals in the yeast Saccharomyces cerevisiae . Transcription of the HXT4 gene was found to be regulated by Gcr1p and Gcr2p, transcription factors that are required for the regulated high level transcriptions of glycolytic genes . Transcription of HXT4 decreased about 35-fold in gcr1 mutant and two-fold in gcr2 mutant yeast cells . However, transcription of other HXT genes was not affected at a significant level by gcr1 or gcr2 mutations . Overproduction of Gcr1p from an inducible promoter resulted in a 15-64% increase in transcription of HXT4, depending on the growth conditions . Gel mobility shift assays performed with the purified DNA binding domain of Gcr1p and the UAS region of the HXT4 gene showed that Gcr1p interacts directly with multiple sites on the HXT4 UAS region . These results indicate that Gcr1p and Gcr2p coordinate the transcription of HXT4 and glycolytic genes .

Yeast, 1999 Aug, 15(11), 1031 - 4
Total number of coding open reading frames in the yeast genome; Kowalczuk M et al.; At the end of 1996 we approximated the total number of protein coding ORFs in the Saccharomyces cerevisiae genome, based on their properties, as 4700-4800 . The number is much smaller than the 5800 which is widely accepted . According to our calculations, there remain about 200-300 orphans-ORFs without known function or homology to already discovered genes, which is only about 5% of the total number of genes . Our results would be questionable if the analysed set of known genes was not a statistically representative sample of the whole set of protein coding genes in the S . cerevisiae genome . Therefore, we repeated our estimation using recently updated databases . In the course of the last 18 months, previously unknown functions of about 500 genes have been found . We have used these to check our method, former results and conclusions . Our previous estimation of the total number of coding ORFs was confirmed .

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1997 Oct, 19(5), 389 - 94
{Erythropoietin gene cloning and expression in S . cerevisiae}; Zhang X et al.; Mature human erythropoietin gene was amplified from EPO cDNA by PCR methods . The PCR product was cloned into pUC18 plasmid at Sma I site, then precisely engineered into a intermidiate vector pSK43SB which were digested with Hind III, Mung bean nuclease, and Sal I . Then degest pSK43SB-EPO plasmid with EcoR I and Cla I, the EC fragment with an alpha-factor leading sequence, EPO gene and CYC1 terminater were produced . It was then cloned into a typical high efficiency episomal expression vector YEpHC8 . Human EPO protein with highly mannose glycosylated was identified by Western blot methods in both secreted and in cells proteins . N-Glycosidase F digested secreted EPO can produce 20,000 EPO without N-glycosylation similar with that produced in cells.

Cell Biol Int, 1998, 22(9-10), 709 - 14
Resumption of rapid proliferation from lag phase in cultures of Saccharomyces cerevisiae in poor nutrient conditions . Effect of surface and intracellular signalling mechanisms; Overgaard AK et al.; Saccharomyces cerevisiae was inoculated into a dilute synthetic minimal medium with glycerol as the carbon source . The number of live cells in the cultures was determined by colony counts on agar plates . Untreated control cells had doubled in number about once at the end of the first week and had gone through eight doublings by the end of the second week . Addition of either 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP) or human recombinant insulin, made the cells go through 12 and 10 doublings, respectively, by the end of the first week . In contrast, 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP) had only slight stimulating effects on cell multiplication, but if it was combined with phorbol-12-myristate-13-acetate (PMA) the cells went through about 12 doublings during the first week . Addition of LY 83583, an inhibitor of soluble guanylate cyclase, prevented cell proliferation . Further addition of 8-bromo-cGMP bypassed this inhibition . Singly, bradykinin or PMA did not affect cell multiplication . However, when these two compounds were combined, the cells went through about 10 doublings during the first week . Neither bradykinin, nor PMA had any releasing effect on the inhibition of LY 83583 . These results indicate the existence of several routes leading to cell proliferation in wildtype S . cerevisiae cells .

J Mol Biol, 1999 Aug 20, 291(3), 537 - 48
Human Rad51 amino acid residues required for Rad52 binding; Kurumizaka H et al.; The Rad51 protein, a homologue of the bacterial RecA protein, is an essential factor for both meiotic and mitotic recombination . The N-terminal domain of the human Rad51 protein (HsRad51) directly interacts with DNA . Based on a yeast two-hybrid analysis, it has been reported that the N-terminal region of the Saccharomyces cerevisiae Rad51 protein binds Rad52;S . cerevisiae Rad51 and Rad52 both activate the homologous pairing and strand exchange reactions . Here, we show that the HsRad51 N-terminal region, which corresponds to the Rad52-binding region of ScRad51, does not exhibit strong binding to the human Rad52 protein (HsRad52) . To investigate its function, the C-terminal region of HsRad51 was randomly mutagenized . Although this region includes the two segments corresponding to the putative DNA-binding sites of RecA, all seven of the mutants did not decrease, but instead slightly increased, the DNA binding . In contrast, we found that some of these HsRad51 mutations significantly decreased the HsRad52 binding . Therefore, we conclude that these amino acid residues are required for the HsRad51.HsRad52 binding . HsRad52, as well as S . cerevisiae Rad52, promoted homologous pairing between ssDNA and dsDNA, and higher homologous pairing activity was observed in the presence of both HsRad51 and HsRad52 than with either HsRad51 or HsRad52 alone . The HsRad51 F259V mutation, which strongly impaired the HsRad52 binding, decreased the homologous pairing in the presence of both HsRad51 and HsRad52, without affecting the homologous pairing by HsRad51 alone . This result suggests the importance of the HsRad51.HsRad52 interaction in homologous pairing .

J Biol Chem, 1999 Aug 20, 274(34), 23991 - 5
Identification of the human YVH1 protein-tyrosine phosphatase orthologue reveals a novel zinc binding domain essential for in vivo function; Muda M et al.; A human orthologue of the Saccharomyces cerevisiae YVH1 protein-tyrosine phosphatase is able to rescue the slow growth defect caused by the disruption of the S . cerevisiae YVH1 gene . The human YVH1 gene is located on chromosome 1q21-q22, which falls in a region amplified in human liposarcomas . The evolutionary conserved COOH-terminal noncatalytic domain of human YVH1 is essential for in vivo function . The cysteine-rich COOH-terminal domain is capable of coordinating 2 mol of zinc/mol of protein, defining it as a novel zinc finger domain . Human YVH1 is the first protein-tyrosine phosphatase that contains and is regulated by a zinc finger domain.

Mol Cell, 1999 Jul, 4(1), 55 - 62
An unusual eukaryotic protein phosphatase required for transcription by RNA polymerase II and CTD dephosphorylation in S . cerevisiae; Kobor MS et al.; The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II is phosphorylated soon after transcriptional initiation . We show here that the essential FCP1 gene of S . cerevisiae is linked genetically to RNA polymerase II and encodes a CTD phosphatase essential for dephosphorylation of RNA polymerase II in vivo . Fcp1p contains a phosphatase motif, psi psi psi DXDX(T/V)psi psi, which is novel for eukaryotic protein phosphatases and essential for Fcp1p to function in vivo . This motif is also required for recombinant Fcp1p to dephosphorylate the RNA polymerase II CTD or the artificial substrate p-nitrophenylphosphate in vitro . The effects of fcp1 mutations in global run-on and genome-wide expression studies show that transcription by RNA polymerase II in S . cerevisiae generally requires CTD phosphatase.

Mol Cell, 1999 Jul, 4(1), 11 - 9
Specialization and targeting of B-type cyclins; Cross FR et al.; The B-type cyclins of S . cerevisiae are diversified with respect to time of expression during the cell cycle as well as biological function . We replaced the early-expressed CLB5 coding sequence with the late-expressed CLB2 coding sequence, at the CLB5 locus . CLB5::CLB2 exhibited almost no rescue of clb5-specific replication defects, although it could rescue clb1 clb2 lethality, and in synchronized cells Clb2p-associated kinase activity from CLB5::CLB2 rose early in the cell cycle, similar to that of Clb5p . Mutagenesis of a potential substrate-targeting domain of CLB5 reduced biological activity without reducing Clb5p-associated kinase activity . Thus, Clb5p may have targeting domains required for CLB5-specific biological activity.

J Biol Chem, 1999 Aug 13, 274(33), 23558 - 64
Exo84p is an exocyst protein essential for secretion; Guo W et al.; The exocyst is a multiprotein complex that plays an important role in secretory vesicle targeting and docking at the plasma membrane . Here we report the identification and characterization of a new component of the exocyst, Exo84p, in the yeast Saccharomyces cerevisiae . Yeast cells depleted of Exo84p cannot survive . These cells are defective in invertase secretion and accumulate vesicles similar to those in the late sec mutants . Exo84p co-immunoprecipitates with the exocyst components, and a portion of the Exo84p co-sediments with the exocyst complex in velocity gradients . The assembly of Exo84p into the exocyst complex requires two other subunits, Sec5p and Sec10p . Exo84p interacts with both Sec5p and Sec10p in a two-hybrid assay . Overexpression of Exo84p selectively suppresses the temperature sensitivity of a sec5 mutant . Exo84p specifically localizes to the bud tip or mother/daughter connection, sites of polarized secretion in the yeast S . cerevisiae . Exo84p is mislocalized in a sec5 mutant . These studies suggest that Exo84p is an essential protein that plays an important role in polarized secretion.

J Biol Chem, 1999 Aug 13, 274(33), 23027 - 34
Histone acetyltransferase HBO1 interacts with the ORC1 subunit of the human initiator protein; Iizuka M et al.; The origin recognition complex (ORC) is an initiator protein for DNA replication, but also effects transcriptional silencing in Saccharomyces cerevisiae and heterochromatin function in Drosophila . It is not known, however, whether any of these functions of ORC is conserved in mammals . We report the identification of a novel protein, HBO1 (histone acetyltransferase binding to ORC), that interacts with human ORC1 protein, the largest subunit of ORC . HBO1 exists as part of a multisubunit complex that possesses histone H3 and H4 acetyltransferase activities . A fraction of the relatively abundant HBO1 protein associates with ORC1 in human cell extracts . HBO1 is a member of the MYST domain family that includes S . cerevisiae Sas2p, a protein involved in control of transcriptional silencing that also has been genetically linked to ORC function . Thus the interaction between ORC and a MYST domain acetyltransferase is widely conserved . We suggest roles for ORC-mediated acetylation of chromatin in control of both DNA replication and gene expression.

Science, 1999 Aug 6, 285(5429), 901 - 6
Functional characterization of the S . cerevisiae genome by gene deletion and parallel analysis; Winzeler EA et al.; The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown . New, whole-genome approaches are required to systematically determine their function . A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome) . Of the deleted ORFs, 17 percent were essential for viability in rich medium . The phenotypes of more than 500 deletion strains were assayed in parallel . Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

J Membr Biol, 1999 Jul 15, 170(2), 89 - 102
Mouse VDAC isoforms expressed in yeast: channel properties and their roles in mitochondrial outer membrane permeability; Xu X et al.; The channel-forming protein called VDAC forms the major pathway in the mitochondrial outer membrane and controls metabolite flux across that membrane . The different VDAC isoforms of a species may play different roles in the regulation of mitochondrial functions . The mouse has three VDAC isoforms (VDAC1, VDAC2 and VDAC3) . These proteins and different versions of VDAC3 were expressed in yeast cells (S . cerevisiae) missing the major yeast VDAC gene and studied using different approaches . When reconstituted into liposomes, each isoform induced a permeability in the liposomes with a similar molecular weight cutoff (between 3,400 and 6,800 daltons based on permeability to polyethylene glycol) . In contrast, electrophysiological studies on purified proteins showed very different channel properties . VDAC1 is the prototypic version whose properties are highly conserved among other species . VDAC2 also has normal gating activity but may exist in 2 forms, one with a lower conductance and selectivity . VDAC3 can also form channels in planar phospholipid membranes . It does not insert readily into membranes and generally does not gate well even at high membrane potentials (up to 80 mV) . Isolated mitochondria exhibit large differences in their outer membrane permeability to NADH depending on which of the mouse VDAC proteins was expressed . These differences in permeability could not simply be attributed to different amounts of each protein present in the isolated mitochondria . The roles of these different VDAC proteins are discussed.

Biol Chem, 1999 Jun, 380(6), 669 - 77
Cycloheximide, a new tool to dissect specific steps in ER-associated degradation of different substrates; Amshoff C et al.; To study the degradation requirements of unassembled immunoglobulin (Ig) chains, we heterologously expressed a cDNA encoding the secretory form of murine mu in the yeast S . cerevisiae . We found that mu chains were translocated into and retained in the endoplasmic reticulum (ER) as they were N-glycosylated and bound to the yeast homolog of BiP, Kar2p . Similar to mutant yeast carboxypeptidase Y (CPY*), known to undergo cytosolic degradation, mu protein is stabilized in yeast mutants lacking the ubiquitinating enzymes Ubc6p and Ubc7p or in cells overexpressing mutant ubiquitin . Unexpectedly, the translation inhibitor cycloheximide (CHX), but not puromycin, led to the accumulation of polyubiquitinated mu chains that were still glycosylated . By contrast, degradation of CPY* was not impaired by CHX, indicating that the drug affects a substrate-specific degradation step . In contrast to the situation for CPY*, the ER-transmembrane protein Der1p is not essential for mu degradation . Strikingly, however, the CHX-induced accumulation of polyubiquitinated Igmu chains was stronger in deltader1-mutants as compared to wild-type cells, indicating an additive effect of two inhibitory conditions . The results support a previously unknown activity of CHX, i.e . impairing the degradation of transport-incompetent secretory mu chains . Moreover, this activity will allow to dissect substrate-specific steps in ER associated protein degradation.

Eur J Biochem, 1999 Jul, 263(1), 254 - 63
Rapid transbilayer movement of fluorescent phospholipid analogues in the plasma membrane of endocytosis-deficient yeast cells does not require the Drs2 protein; Marx U et al.; Evidence is presented that endocytosis-deficient Saccharomyces cerevisiae end4 yeast cells rapidly internalize the fluorescent phospholipid analogues 1-palmitoyl-2- inverted question mark6-{7-nitro-2,1, 3-benzoxadiazol-4-yl(NBD)amino} caproyl inverted question markphosphatidylcholine (P-C6-NBD-PtdCho) and P-C6-NBD-phosphatidylserine (P-C6-NBD-PtdSer) . Both analogues redistributed between the exoplasmic and cytoplasmic leaflet with a half-time of < 15 min at 0 degrees C . The plateau of internalized analogues was about 70% . Transbilayer movement is probably protein-mediated, as the flip-flop of both analogues was very slow in liposomes composed of plasma-membrane lipids . Rapid analogue internalization was not abolished on depletion of intracellular ATP by about 90% . For P-C6-NBD-PtdCho only was a moderate decrease in the plateau of internalized analogues of about 20% observed, while that of P-C6-NBD-PtdSer was not affected . The Drs2 protein plays only a minor role, if any, in the rapid transbilayer movement of analogues in S . cerevisiae end4 cells . In S . cerevisiae end4 Deltadrs2 cells harbouring both an end4 allele and a drs2 null allele, about 60% and 50% of P-C6-NBD-PtdCho and P-C6-NBD-PtdSer, respectively, became internalized within 15 min at 0 degrees C . The preferential orientation of P-C6-NBD-PtdSer to the cytoplasmic leaflet is in qualitative agreement with the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet, as assessed by binding of annexin V . Virtually no binding of annexin V to spheroplasts of the parent wild-type strain or the mutant strains was observed . Likewise, no difference in the exposure of endogenous aminophospholipids to the exoplasmic leaflet between these strains was found by labelling with trinitrobenzenesulfonic acid . Thus, lipid asymmetry, at least of aminophospholipids, was preserved in S . cerevisiae end4 cells independently of the presence of the Drs2 protein.

J Biol Chem, 1999 Aug 6, 274(32), 22275 - 82
The aroC gene of Aspergillus nidulans codes for a monofunctional, allosterically regulated chorismate mutase; Krappmann S et al.; The cDNA and the chromosomal locus of the aroC gene of Aspergillus nidulans were cloned and is the first representative of a filamentous fungal gene encoding chorismate mutase (EC 5.4.99.5), the enzyme at the first branch point of aromatic amino acid biosynthesis . The aroC gene complements the Saccharomyces cerevisiae aro7Delta as well as the A . nidulans aroC mutation . The gene consists of three exons interrupted by two short intron sequences . The expressed mRNA is 0.96 kilobases in length and aroC expression is not regulated on the transcriptional level under amino acid starvation conditions . aroC encodes a monofunctional polypeptide of 268 amino acids . Purification of this 30-kDa enzyme allowed determination of its kinetic parameters (k(cat) = 82 s(-1), n(H) = 1 . 56, {S}(0.5) = 2.3 mM), varying pH dependence of catalytic activity in different regulatory states, and an acidic pI value of 4.7 . Tryptophan acts as heterotropic activator and tyrosine as negative acting, heterotropic feedback-inhibitor with a K(i) of 2.8 microM . Immunological data, homology modeling, as well as electron microscopy studies, indicate that this chorismate mutase has a dimeric structure like the S . cerevisiae enzyme . Site-directed mutagenesis of a crucial residue in loop220s (Asp(233)) revealed differences concerning the intramolecular signal transduction for allosteric regulation of enzymatic activity.

J Biol Chem, 1999 Aug 6, 274(32), 22184 - 90
Identification of the yeast mitochondrial transporter for oxaloacetate and sulfate; Palmieri L et al.; Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, including the OAC protein . The transport specificities of some family members are known, but most are not . The function of the OAC has been revealed by overproduction in Escherichia coli, reconstitution into liposomes, and demonstration that the proteoliposomes transport malonate, oxaloacetate, sulfate, and thiosulfate . Reconstituted OAC catalyzes both unidirectional transport and exchange of substrates . In S . cerevisiae, OAC is in inner mitochondrial membranes, and deletion of its gene greatly reduces transport of oxaloacetate sulfate, thiosulfate, and malonate . Mitochondria from wild-type cells swelled in isoosmotic solutions of ammonium salts of oxaloacetate, sulfate, thiosulfate, and malonate, indicating that these anions are cotransported with protons . Overexpression of OAC in the deletion strain increased greatly the {(35)S}sulfate/sulfate and {(35)S}sulfate/oxaloacetate exchanges in proteoliposomes reconstituted with digitonin extracts of mitochondria . The main physiological role of OAC appears to be to use the proton-motive force to take up into mitochondria oxaloacetate produced from pyruvate by cytoplasmic pyruvate carboxylase.

FEBS Lett, 1999 Jul 16, 455(1-2), 13 - 7
Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase . Expression and characterization of the recombinant enzyme; Emanuelli M et al.; The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD . A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation . Through data-base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S . cerevisiae YLR328W open reading frame (GenBank accession number U20618) . The YLR328W gene was isolated, cloned into a T7-based vector and successfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity . The purification of recombinant protein, by a two-step chromatographic procedure, resulted in a single polypeptide of 48 kDa under SDS-PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF . The N-terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence . Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources.

FEMS Microbiol Lett, 1999 Jul 15, 176(2), 379 - 86
Flow cytometric investigation of heterogeneous copper-sensitivity in asynchronously grown Saccharomyces cerevisiae; Howlett NG et al.; The variable stress-sensitivity of individual cells within pure cultures is widely noted but generally unexplained . Here, factors determining the heterogeneous susceptibility to copper toxicity in Saccharomyces cerevisiae were examined with a rapid non-perturbing approach based on flow cytometry . By determination of the DNA content (with propidium iodide) in cell fractions gated by forward angle light scatter (an indicator of the cell volume), it was shown that forward angle light scatter measurements gave an approximation of the cell cycle stage . Thus, our observation that cells in different forward angle light scatter fractions displayed differing Cu-sensitivities indicated that heterogeneous Cu-sensitivity is a function of the cell cycle stage . Furthermore, cells sorted by their Cu-sensitivity and-resistance and subsequently analyzed for DNA content were found predominantly to occupy G1/S and G2/M cell cycle stages, respectively . The oxidant-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate was used to show that the Cu-sensitivity of G2/M phase S . cerevisiae was correlated with greater levels of pre-existing reactive oxygen species in these cells . The results indicate that differential Cu-sensitivity in a S . cerevisiae culture is linked to the cell cycle stage and this link may be determined partly by cell cycle-dependent fluctuations in basal reactive oxygen species generation.

Biosci Biotechnol Biochem, 1999 Jun, 63(6), 1075 - 82
Nystatin effects on vacuolar function in Saccharomyces cerevisiae; Bhuiyan MS et al.; The effects of nystatin, a polyene antibiotic, was studied in Saccharomyces cerevisiae by isolating and characterizing nystatin-sensitive mutants . We isolated a number of nystatin-sensitive mutants by ethylmethane sulfonate mutagenesis . One of these mutants, the nss1 mutant, was characterized in detail . The mutant was sensitive to stresses such as high temperature or high concentrations of monovalent and divalent cations . The nss1 mutants showed severe vacuolar protein sorting and vacuolar morphology defects . The nss1 mutant was demonstrated to have a mutational lesion in the known VPS16 gene, which is essential for vacuolar protein sorting in S . cerevisiae . All of the vacuolar deficient mutants (vps11, vps16, vps18, and vps33) were sensitive to nystatin . Nystatin was found to cause extensive enlargement of the vacuole in wild-type S . cerevisiae cells . These results are discussed with special reference to the vacuolar function of S . cerevisiae.

Biosci Biotechnol Biochem, 1999 Jun, 63(6), 1025 - 31
Generation of free radicals during the death of Saccharomyces cerevisiae caused by lipid hydroperoxide; Aoshima H et al.; The exposure of Saccharomyces cerevisiae cells to 13-L-hydroperoxylinoleic acid (LOOH) caused their death, the degree of which was dependent on the growth phase of the cells . Pre-application of ethanol, hydrogen peroxide (H2O2) and LOOH to S . cerevisiae cells reduced the effect of LOOH on the cells, showing the transient cross adaptation to LOOH . Antioxidants such as N,N',-diphenyl-p-phenylenediamine (DPPD), melatonin and vitamin E, and inhibitors of permeability transition of mitochondria, cyclosporin A and trifluoperazine, inhibited the LOOH-triggered cell death, while an inhibitor of glutathione synthetase, buthionine sulfoximine (BSO), enhanced the cell death by LOOH . Reactive oxygen species (ROS) were detected by flow cytometry, using the ROS-specific fluorescent indicator . A ferric iron chelator, deferoxamine, inhibited the LOOH-triggered cell death, and peroxyl radicals (LOO.) were detected by a spin trapping method . These reactive radicals possibly induced the death of S . cerevisiae cells . However, the DNA fragmentation characteristic of apoptosis was not observed in S . cerevisiae cells after exposure to LOOH, staurosporine, dexamethasone or etoposide, which have been reported to cause apoptosis in mammalian cells.

Appl Environ Microbiol, 1999 Aug, 65(8), 3470 - 2
Purification and properties of an esterase from the yeast Saccharomyces cerevisiae and identification of the encoding gene; Degrassi G et al.; We purified an intracellular esterase that can function as an S-formylglutathione hydrolase from the yeast Saccharomyces cerevisiae . Its molecular mass was 40 kDa, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The isoelectric point was 5.0 by isoelectric focusing . The enzyme activity was optimal at 50 degrees C and pH 7.0 . The corresponding gene, YJLO68C, was identified by its N-terminal amino acid sequence and is not essential for cell viability . Null mutants have reduced esterase activities and grow slowly in the presence of formaldehyde . This enzyme may be involved in the detoxification of formaldehyde, which can be metabolized to S-formylglutathione by S . cerevisiae.

J Comput Biol, 1999 Summer, 6(2), 237 - 52
Construction of physical maps from oligonucleotide fingerprints data; Mayraz G et al.; A new algorithm for the construction of physical maps from hybridization fingerprints of short oligonucleotide probes has been developed . Extensive simulations in high-noise scenarios show that the algorithm produces an essentially completely correct map in over 95% of trials . Tests for the influence of specific experimental parameters demonstrate that the algorithm is robust to both false positive and false negative experimental errors . The algorithm was also tested in simulations using real DNA sequences of C . elegans, E . coli, S . cerevisiae, and H . sapiens . To overcome the non-randomness of probe frequencies in these sequences, probes were preselected based on sequence statistics and a screening process of the hybridization data was developed . With these modifications, the algorithm produced very encouraging results.

Plant J, 1999 Jun, 18(5), 541 - 50
Arabidopsis thaliana proteins related to the yeast SIP and SNF4 interact with AKINalpha1, an SNF1-like protein kinase; Bouly JP et al.; AKINalpha1, a Ser/Thr kinase from Arabidopsis thaliana belongs to the highly conserved SNF1 family of protein kinases in eukaryotes . Recent data suggest that the plant SNF1-related kinases (SnRK1 family) are key enzymes implicated in the regulation of carbohydrate and lipid metabolism . In Saccharomyces cerevisiae and mammals, the SNF1 and AMPKalpha protein kinases interact with two other families of proteins, namely SNF4/AMPKgamma and SIP1/SIP2/GAL83/AMPKbeta, to form active heterotrimeric complexes . In this paper, we describe the characterisation of three novel cDNAs . AKINbeta1 and AKINbeta2 encode proteins similar to SIP1, SIP2 and GAL83 and AKINgamma codes for a protein showing similarity with SNF4 . Using the two-hybrid system, specific interactions have been shown between A . thaliana AKINbeta1/beta2, AKINgamma and AKINgamma as well as between the A . thaliana and S . cerevisiae subunits . Interestingly, AKINbeta1, AKINbeta2 and AKINgamma mRNAs accumulate differentially in A . thaliana tissues and are modulated during development and under different growth conditions . These data suggest the presence in higher plants of a conserved heterotrimeric complex . Moreover, the differential transcription of different non-catalytic subunits can constitute a first level of regulation of the SNF1-like complex in plants.

Biochem J, 1999 Aug 1, 341 ( Pt 3), 537 - 43
Adenylosuccinate synthase from Saccharomyces cerevisiae: homologous overexpression, purification and characterization of the recombinant protein; Lipps G et al.; Adenylosuccinate synthase (EC 6.3.4.4) catalyses the first committed step in the synthesis of adenosine . We have overexpressed the cloned gene of Saccharomyces cerevisiae (ADE12) in S . cerevisiae . The recombinant enzyme exhibits similar kinetic behaviour to that of the native enzyme purified from S . cerevisiae . This ter-reactant dimeric enzyme shows Michaelis-Menten kinetics only with IMP . l-Aspartate and GTP display a weak negative co-operativity (Hill coefficient 0 . 8-0.9) . This negative co-operativity has not yet been reported for adenylosuccinate synthases from other organisms . Another unusual feature of the enzyme from S . cerevisiae is its negligible inhibition by adenine nucleotides and its pronounced inhibition by Cl(-) ions.

Fungal Genet Biol, 1999 Jun, 27(1), 49 - 54
Ploidy study in sporothrix schenkii
Torres-Guerrero H.
Sporothrix schenckii is a dimorphic fungus that is pathogenic for humans . No sexual cycle has been reported for this fungus and little is known of its genetic constitution . To inquire into the ploidy state of Sporothrix schenckii, different approaches were followed: DNA content during transition from conidia to yeast, survival to ultraviolet irradiation, chemical mutagenesis, and induction of mitotic recombination . No change in ploidy was detected between the conidia and yeast phases of the fungi . Resistance to cell inactivation by UV irradiation was higher in S . schenckii and in Sacharomyces cerevisiae in its diploid state than in isogenic haploids a and alpha from S . cerevisiae that were inactivated at lower doses . Two mutant phenotypes, auxotrophy and albinism, were screened after chemical mutagenesis . One-step mutagenesis with either nitrous acid or ultraviolet light was unsuccessful in inducing auxotrophy but was sufficient to induce albino colonies . Two-step mutagenesis with nitrous acid in combination with UV light was necessary to attain two auxotrophic requirements: adenine and methionine . Prototrophic and pigmented revertants behaved as heterozygotes; after exposure to UV light they gave rise to derivatives which resemble the original mutation . The experiments presented in this work suggest that S . schenckii is a diploid, although aneuploidy cannot be excluded .

Exp Cell Res, 1999 Aug 1, 250(2), 510 - 23
Chromosome separation and exit from mitosis in budding yeast: dependence on growth revealed by cAMP-mediated inhibition; Anghileri P et al.; Cell cycle progression of somatic cells depends on net mass accumulation . In Saccharomyces cerevisiae the cAMP-dependent kinases (PKAs) promote cytoplasmic growth and modulate the growth-regulated mechanism triggering the begin of DNA synthesis . By altering the cAMP signal in budding yeast cells we show here that mitotic events can also depend on growth . In fact, the hyperactivation of PKAs permanently inhibited both anaphase and exit from mitosis when cell growth was repressed . In S . cerevisiae the anaphase promoting complex (APC) triggers entry into anaphase by mediating the degradation of Pds1p . The cAMP pathway activation was lethal together with a partial impairment of the Cdc16p APC subunit, causing a preanaphase arrest, and conversely low PKA activity suppressed the lethality of cdc16-1 cells . Deregulated PKAs partially prevented the decrease of Pds1p intracellular levels concomitantly with the anaphase inhibition, and the PKA-dependent preanaphase arrest could be suppressed in pds1(-) cells . Thus, the cAMP pathway and APC functionally interact in S . cerevisiae and Pds1p is required for the cAMP-mediated inhibition of chromosome separation . Exit from mitosis requires APC, Cdc15p, and the polo-like Cdc5p kinase . PKA hyperactivation and a cdc15 mutation were synthetically lethal and brought to a telophase arrest . Finally, a low cAMP signal allowed cell division at a small cell size and suppressed the lethality of cdc15-2 or cdc5-1 cells . We propose that mitosis progression and the M/G1 phase transition specifically depend on cell growth through a mechanism modulated by PKAs and interacting with the APC/CDC15/CDC5 mitotic system . A possible functional antagonism between PKAs and the mitosis promoting factor is also discussed .

Biochemistry, 1999 Jul 13, 38(28), 8961 - 71
Spt16 and Pob3 of Saccharomyces cerevisiae form an essential, abundant heterodimer that is nuclear, chromatin-associated, and copurifies with DNA polymerase alpha; Wittmeyer J et al.; Previously we showed that the yeast proteins Spt16 (Cdc68) and Pob3 are physically associated, and interact physically and genetically with the catalytic subunit of DNA polymerase alpha, Pol1 {Wittmeyer and Formosa (1997) Mol . Cell . Biol . 17, 4178-4190} . Here we show that purified Spt16 and Pob3 form a stable, abundant, elongated heterodimer and provide evidence that this is the functional form of these proteins . Genetic interactions between mutations in SPT16 and POB3 support the importance of the Spt16-Pob3 interaction in vivo . Spt16, Pob3, and Pol1 proteins were all found to localize to the nucleus in S . cerevisiae . A portion of the total cellular Spt16-Pob3 was found to be chromatin-associated, consistent with the proposed roles in modulating chromatin function . Some of the Spt16-Pob3 complex was found to copurify with the yeast DNA polymerase alpha/primase complex, further supporting a connection between Spt16-Pob3 and DNA replication.

Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8522 - 7
Hsl7p, a negative regulator of Ste20p protein kinase in the Saccharomyces cerevisiae filamentous growth-signaling pathway; Fujita A et al.; In the budding yeast, Saccharomyces cerevisiae, protein kinases Ste20p (p21(Cdc42p/Rac)-activated kinase), Ste11p {mitogen-activated protein kinase (MAPK) kinase kinase}, Ste7p (MAPK kinase), Fus3p, and Kss1p (MAPKs) are utilized for haploid mating, invasive growth, and diploid filamentous growth . Members of the highly conserved Ste20p/p65(PAK) protein kinase family regulate MAPK signal transduction pathways from yeast to man . We describe here a potent negative regulator of Ste20p in the yeast filamentous growth-signaling pathway . We identified a mutant, hsl7, that exhibits filamentous growth on rich medium . Hsl7p belongs to a highly conserved protein family in eukaryotes . Hsl7p associates with the noncatalytic region within the amino-terminal half of Ste20p as well as Cdc42p . Deletions of HSL7 in haploid and diploid strains led to cell elongation and enhancement of both haploid invasive growth and diploid pseudohyphal growth . However, deletions of STE20 in haploid and diploid greatly diminished these hsl7-associated phenotypes . In addition, overexpression of HSL7 inhibited pseudohyphal growth . Thus, Hsl7p may inhibit the activity of Ste20p in the S . cerevisiae filamentous growth-signaling pathway . Our genetic analyses suggest the possibility that Cdc42p and Hsl7p compete for binding to Ste20p for pseudohyphal development when starved for nitrogen.

Indian J Biochem Biophys, 1998 Oct, 35(5), 260 - 5
Channeling of TCA cycle intermediates in Saccharomyces cerevisiae; Ira et al.; Metabolism of 13C labeled substrates viz . glucose and pyruvate in S . cerevisiae has been studied by 13C Nuclear Magnetic Resonance Spectroscopy . C3-Pyruvate, alanine and lactate, and C2-acetate are produced from {1-13C}glucose . The pyruvate, entering TCA cycle, leads to preferential labeling of C2-glutamate . {2-13C}Glucose results in labeling of C2-pyruvate, alanine and lactate . Some C3-pyruvate is also produced, indicating the routing of the label from glucose through pentose phosphate pathway (PPP) . In TCA cycle the C2-pyruvate preferentially labels the C3-glutamate . The NMR spectra, obtained with {2-13C}pyruvate as substrate, confirm the above observations . These results suggest that the intermediates of TCA cycle are transferred from one enzyme active site to another in a manner that allows only restricted rotation of the intermediates . That is, the intermediates are partially channeled.

Biochem Mol Biol Int, 1999 Jun, 47(6), 971 - 7
Expression of the divergent transcription unit containing the yeast PET122 and OXA1 genes; Marathe SV et al.; The nuclear PET122 gene of S . cerevisiae encodes a mitochondrial-localized protein that activates initiation of translation of the mitochondrial mRNA from the COX3 gene, which encodes subunit III of cytochrome c oxidase . The PET122 locus contains two divergent transcription units: one is involved in expression of PET122 mRNA and the mRNA for an adjacent gene OXA1, which is also required for cytochrome c oxidase biogenesis, and the other is involved in expression of an antisense RNA that is complementary to about two thirds of the PET122 mRNA and an adjacent gene YER152C of unknown function . Steady state levels of OXA1, PET122 sense and PET122 antisense RNAs were measured after growth of yeast cells under catabolite repressing or derepressing conditions, or after deletion of portions of the 5' flanking DNA of the genes . The results reported here indicate that the PET122 and OXA1 genes are unconventional in terms of the control of their transcription . Neither possesses a canonical TATA element and they exhibit no apparent need for native upstream DNA . These results raise the interesting possibility that PET122 and OXA1 transcription is controlled by downstream DNA, perhaps located within the coding regions of the respective genes.

J Biol Chem, 1999 Jul 23, 274(30), 21297 - 304
The Cap-binding protein eIF4E promotes folding of a functional domain of yeast translation initiation factor eIF4G1; Hershey PE et al.; The association of eucaryotic translation initiation factor eIF4G with the cap-binding protein eIF4E establishes a critical link between the mRNA and the ribosome during translation initiation . This association requires a conserved seven amino acid peptide within eIF4G that binds to eIF4E . Here we report that a 98-amino acid fragment of S . cerevisiae eIF4G1 that contains this eIF4E binding peptide undergoes an unfolded to folded transition upon binding to eIF4E . The folding of the eIF4G1 domain was evidenced by the eIF4E-dependent changes in its protease sensitivity and (1)H-(15)N HSQC NMR spectrum . Analysis of a series of charge-to-alanine mutations throughout the essential 55.4-kDa core of yeast eIF4G1 also revealed substitutions within this 98-amino acid region that led to reduced eIF4E binding in vivo and in vitro . These data suggest that the association of yeast eIF4E with eIF4G1 leads to the formation of a structured domain within eIF4G1 that could serve as a specific site for interactions with other components of the translational apparatus . They also suggest that the stability of the native eIF4E-eIF4G complex is determined by amino acid residues outside of the conserved seven-residue consensus sequence.

Yeast, 1999 Jul, 15(10B), 1009 - 19
A fast method to diagnose chromosome and plasmid loss in Saccharomyces cerevisiae strains; Hegemann JH et al.; We have developed a simple, fast and reliable method for the analysis of genetic stability in budding yeast strains . The assay relies on our previous finding that cells expressing the green fluorescent protein (GFP) can be detected and counted by flow cytometric analysis (FACS) (Niedenthal et al., 1996) . Expression of a gfp-carrying CEN-plasmid in a wild-type strain resulted in the emission of strong fluorescence from 80% of the cell population . Strong fluorescence and presence of the plasmid, determined by the presence of the URA3 genetic marker, was strictly correlated . Expression of this plasmid in 266 yeast strains, each carrying a complete deletion of a novel, non-essential gene identified in the S . cerevisiae sequencing project, pinpointed 12 strains with an increased level of mitotic plasmid loss . Finally we have shown that measurement of mitotic loss of artificial chromosome fragments equipped with the gfp expression cassette can be performed quantitatively using FACS .

Yeast, 1999 Jul, 15(10B), 955 - 61
Construction and genetic analysis of S . cerevisiae deletants of six novel ORFs from chromosome II; Malagon F et al.; We have constructed S . cerevisiae strains carrying genomic deletions of six ORFs from the left arm of chromosome II (YBL018c, YBL019w, YBL024w, YBL042c, YBL043w and YBL046w) in both FY1679 and W303 backgrounds . We have found that YBL018c is an essential gene in yeast, whereas the other five genes are non-essential . We have developed plasmids carrying deletion cassettes that can be used to delete any of the six genes in S . cerevisiae by transforming to G418-resistance, as well as centromeric plasmids containing the cognate genes .

Yeast, 1999 Jul, 15(10B), 921 - 33
Disruption of six open reading frames on chromosome X of Saccharomyces cerevisiae reveals a cluster of four essential genes; Esser K et al.; In this study we report the construction and basic phenotypic analysis of six Saccharomyces cerevisiae deletion mutants . The open reading frames (ORFs) YJL008C (gene symbol CCT8), YJL010C, YJL011C, YJL012C, YJL017W, and YJL020C from chromosome X have been disrupted by integration of deletion cassettes, comprising the bacterial KanMX4 marker gene and terminal long (LFH) or short (SFH) flanking sequences that are homologous to the 5' and 3' untranslated regions of the respective ORFs . For correct disruption of ORF YJL008C, it was necessary to construct a deletion cassette flanked by 300-350 bp long target guide sequences by LFH-PCR . Transformations using ORF YJL008C gene disruption cassettes synthesized by standard SFH-PCR exclusively resulted in false-positive or multiple integration events, probably because seven additional genes homologous to CCT8 exist in the yeast genome . The other five ORFs have been disrupted using cassettes generated by SFH-PCR, comprising terminal homologous regions of approximately 50 bp to each target site . Correct genomic integration of the reporter modules was verified by analytical PCR and Southern hybridization . Deletion of YJL008C, YJL010C, YJL011C, and YJL012C was found to be lethal, as shown by sporulation and tetrad analysis . This result is in contrast to the finding that only 16-20% of the genes in S . cerevisiae are estimated to be essential . The four essential genes described in this work are clustered, while the two other non-essential ORFs are separated by further ORFs . Although the two viable deletion mutants were tested against 60 different inhibitors, heavy metal ions and salts, no phenotype could be detected that co-segregated with the deletion during meiosis .

Yeast, 1999 Jul, 15(10A), 873 - 8
The three copies of the ATP1 gene are arranged in tandem on chromosome II of Saccharomyces cerevisiae S288C; Takeda M et al.; In the yeast Saccharomyces cerevisiae there are three copies of the F(1)F(0)-ATPase alpha-subunit gene ATP1 on chromosome II (Takeda et al., 1995) . However, after genome analysis using S . cerevisiae strain S288C, only one ATP1 gene sequence was observed (Feldman et al., 1994; Obermaier et al., 1995) . To check whether the number of copies of ATP1 is strain-dependent or not, we carried out three different experiments: (a) long-PCR analyses of total DNAs isolated from several reference strains, carried out by preparing 29-mer oligonucleotides based on the 5'- and 3'- up- and downstream regions of the ATP1 nucleotide sequence using the data from the genome project to synthesize primers; (b) restriction analyses of chromosome II from the reference strains with SplI; and (c) long-PCR analyses of prime clones 70113 and 70804, both of which contained two ATP1 gene copies, ATP1a and ATP1b, and ATP1b and ATP1c, respectively, using 30 nucleotides just inside the 3'-end (sense) and 5'-end (antisense) of the ATP1-coding region as primers . In the case of the long-PCR experiments, the reference strains DC5, SEY2102, W303-1A, W303-1B, LL20 and DBY746, as well as strain S288C, generated a DNA fragment of approximately 32 kb, which hybridized with ATP1 . During SplI digestion, a DNA fragment of more than 50 kb which hybridized with ATP1, was obtained from all reference strains . In the case of prime clone analyses using the long-PCR experiments, the distance between ATP1a and ATP1b or ATP1b and ATP1c was approximately 10 kb or 7 kb, respectively . The S288C strain generated these two DNA fragments, as do the other strains . These results showed that all these strains contained three copies of ATP1 on chromosome II .

Crit Rev Biotechnol, 1999, 19(2), 113 - 43
The molecular biology of Schwanniomyces occidentalis klocker; Wang TT et al.; This review describes the molecular studies of Schwanniomyces occidentalis (Debaryomyces occidentalis) concerning transformation, genome, gene cloning, gene structure, gene expression and its characteristics to application . Schw . occidentalis appears to have at least five or seven chromosomes and no native plasmid from the yeast has been reported . Four transformation systems based on complement of Schw . occidentalis auxotrophic mutants were established . Vectors with the replicon of 2-micron plasmid and autonomous replication sequences (ARS) of Saccharomyces cerevisiae and Schw . occidentalis ARS replicated extrachromosomally in Schw . occidentalis transformants, without modification of the transformed vector DNA . So far, at least 21 Schw . occidentalis genes encoding 14 different proteins have been cloned . Most of the Schw . occidentalis genes have shown homologies (45 to 91%) with the corresponding genes of other organisms, especially of S . cerevisiae . However, some Schw . occidentalis genes possess other unique structures for their operators, promoters, transcription initiation sites, and terminators . Some foreign genes were expressed in Schw . occidentalis, while Schw . occidentalis genes functioned in other yeasts and bacteria, Escherichia coli, and Streptomyces lividans . Due to a strong ability of secretion and low level of glycosylation, Schw . occidentalis might be a promising host to produce heterologous proteins.

Comput Chem, 1999 Jun 15, 23(3-4), 303 - 15
Applications of the pyramidal clustering method to biological objects; Aude JC et al.; In conventional hierarchical clustering methods, any object can belong to only one class or cluster . We present here an application of the pyramidal classification method to biological objects, which illustrates the intuitively appealing idea that some objects may belong simultaneously to two classes . In a first step, we performed an all-by-all comparison of all the open reading frames in the genomes from S . cerevisiae, M . jannaschii, E . coli, H . influenzae and Synechocystis . In a second step, a series of connex classes was built, each connex class containing all those sequences that were linked by a Z-value (obtained after 100 sequence shufflings) greater than a given threshold . Finally, each connex class was submitted to a pyramidal classification . Three examples of such classifications are given, concerning two sets of multi-domains protein sequences and a family of aminoacyl-tRNA synthetases . They make it clear that the linear order among the classified objects that results from the pyramidal classification is useful in deciphering the multiple relationships that can exist between the objects under study . A program for calculating and displaying a pyramidal classification from a dissimilarity matrix is available from The pyramidal classifications of the connex classes from the five organisms (intra- and inter-genomic comparisons) are available from under the family item.

FEBS Lett, 1999 Jun 18, 453(1-2), 54 - 8
The glucose repressor CRE1 from Sclerotinia sclerotiorum is functionally related to CREA from Aspergillus nidulans but not to the Mig proteins from Saccharomyces cerevisiae; Vautard G et al.; We isolated the putative glucose repressor gene cre1 from the phytopathogenic fungus Sclerotinia sclerotiorum . cre1 encodes a 429 amino acid protein 59% similar to the carbon catabolite repressor CREA from Aspergillus nidulans . In addition to the overall amino acid sequence relatedness between CRE1 and CREA proteins, cre1 can functionally complement the A . nidulans creAd30 mutation as assessed by repression of the alcohol dehydrogenase I gene expression . The CREI region carrying the two zinc fingers is also very similar to the DNA binding domains of the Saccharomyces cerevisiae glucose repressors Mig1p and Mig2p . Despite the presence in the CRE1 protein of several motifs involved in the regulation of Miglp activity, cre1 cannot complement mig deficiencies in S . cerevisiae . These data suggest that glucose repression pathways may have evolved differently in yeasts and filamentous fungi.

Cell Biochem Biophys, 1999, 30(3), 389 - 411
How many is enough? Exploring the myosin repertoire in the model eukaryote Dictyostelium discoideum; Soldati T et al.; The cytoplasm of eukaryotic cells is a very complex milieu and unraveling how its unique cytoarchitecture is achieved and maintained is a central theme in modern cell biology . It is crucial to understand how organelles and macro-complexes of RNA and/or proteins are transported to and/or maintained at their specific cellular locations . The importance of filamentous-actin-directed myosin-powered cargo transport was only recently realized, and after an initial explosion in the identification of new molecules, the field is now concentrating on their functional dissection . Direct connections of myosins to a variety of cellular tasks are now slowly emerging, such as in cytokinesis, phagocytosis, endocytosis, polarized secretion and exocytosis, axonal transport, etc . Unconventional myosins have been identified in a wide variety of organisms, making the presence of actin and myosins a hallmark of eukaryotism . The genome of S . cerevisiae encodes only five myosins, whereas a mammalian cell has the capacity to express between two and three dozen myosins . Why is it so crucial to arrive at this final census? The main questions that we would like to discuss are the following . How many distinct myosin-powered functions are carried out in a typical higher eukaryote? Or, in other words, what is the minimal set of myosins essential to accomplish the multitude of tasks related to motility and intracellular dynamics in a multicellular organism? And also, as a corollary, what is the degree of functional redundancy inside a given myosin class? In that respect, the choice of a model organism suitable for such an investigation is more crucial than ever . Here we argue that Dictyostelium discoideum is affirming its position as an ideal system of intermediate complexity to study myosin-powered trafficking and is or will soon become the second eukaryote for which complete knowledge of the whole repertoire of myosins is available.

J Bacteriol, 1999 Jul, 181(14), 4437 - 40
Water transport across yeast vacuolar and plasma membrane-targeted secretory vesicles occurs by passive diffusion; Coury LA et al.; To determine whether solute transport across yeast membranes was facilitated, we measured the water and solute permeations of vacuole-derived and late secretory vesicles in Saccharomyces cerevisiae; all permeations were consistent with passive diffusive flow . We also overexpressed Fps1p, the putative glycerol facilitator in S . cerevisiae, in secretory vesicles but observed no effect on water, glycerol, formamide, or urea permeations . However, spheroplasts prepared from the strain overexpressing Fps1p showed enhanced glycerol uptake, suggesting that Fps1p becomes active only upon insertion in the plasma membrane.

Int J Biochem Cell Biol, 1999 May, 31(5), 575 - 84
Factors and processes involved in membrane potential build-up in yeast: diS-C3(3) assay; Gaskova D et al.; No methods are currently available for fully reliable monitoring of membrane potential changes in suspensions of walled cells such as yeast . Our method using the Nernstian cyanine probe diS-C3(3) monitors even relatively fast changes in membrane potential delta psi by recording the shifts of probe fluorescence maximum lambda max consequent on delta psi-dependent probe uptake into, or exit from, the cells . Both increased {K+}out and decreased pHout, but not external NaCl or choline chloride depolarise the membrane . The major ion species contributing to the diS-C3(3)-reported membrane potential in S . cerevisiae are thus K+ and H+, whereas Na+ and Cl- do not perceptibly contribute to measured delta psi . The strongly pHout-dependent depolarisation caused by the protonophores CCCP and FCCP, lack of effect of the respiratory chain inhibitors rotenone and HQNO on the delta psi, as well as results obtained with a respiration-deficient rho- mutant show that the major component of the diS-C3(3)-reported membrane potential is the delta psi formed on the plasma membrane while mitochondrial potential forms a minor part of the delta psi . Its role may be reflected in the slight depolarisation caused by the F1F0-ATPase inhibitor azide in both rho- mutant and wildtype cells . Blocking the plasma membrane H(+)-ATPase with the DMM-11 inhibitor showed that the enzyme participates in delta psi build-up both in the absence and in the presence of added glucose . Pore-forming agents such as nystatin cause a fast probe entry into the cells signifying membrane damage and extensive binding of the probe to cell constituents reflecting obviously disruption of ionic balance in permeabilised cells . In damaged cells the probe therefore no longer reports on membrane potential but on loss of membrane integrity . The delta psi-independent probe entry signalling membrane damage can be distinguished from the potential-dependent diS-C3(3) uptake into intact cells by being insensitive to the depolarising action of CCCP.

Biotechnol Bioeng, 1999 Jul 5, 64(1), 54 - 60
Production of S-lactoylglutathione by high activity whole cell biocatalysts prepared by permeabilization of recombinant saccharomyces cerevisiae with alcohols
Liu Y, Hama H, Fujita Y, Kondo A, Inoue Y, Kimura A, Fukuda H.
The permeabilization of yeast cells with methanol, ethanol, and isopropyl alcohol under various conditions was studied to develop the preparation method of high activity whole cell biocatalysts . Recombinant Saccharomyces cerevisiae, which intracellularly overexpresses glyoxalase I and catalyzes the conversion of methylglyoxal to S-lactoylglutathione in the presence of glutathione, was used as the model system . The permeabilization treatments with alcohols significantly enhanced the activities of yeast cells . Especially, the initial S-lactoylglutathione production rates of cells permeabilized with 40% ethanol and isopropyl alcohol solutions for 10 min at 4 degrees C were high and were 364 and 582 times larger than those of untreated cells, respectively . These permeabilized yeast cells retained high activities during repeated batch reactions . Even in third batch reaction, they showed approximately 70-80% of the activity in the first batch . The plasma membrane of S . cerevisiae cells was damaged by the treatment with alcohol solutions in such a way that leakage of glyoxalase I from the cells is rather small and that both substrate and product show very high permeability . The initial S-lactoylglutathione production rates of these permeabilized cells were 1.5-2.5 times larger than those of glyoxalase I in cell extracts prepared by ethyl acetate method from the same amount of cells . These results demonstrate that the recombinant S . cerevisiae cells permeabilized with alcohol solutions under the optimum condition are very effective whole cell biocatalysts .

Yeast, 1999 Jun 30, 15(9), 799 - 804
Isolation of a Histoplasma capsulatum cDNA that complements a mitochondrial NAD(+)-isocitrate dehydrogenase subunit I-deficient mutant of Saccharomyces cerevisiae; Johnson CH et al.; A cDNA library was prepared from Histoplasma capsulatum strain G-217B yeast cells and an apparently full-length cDNA for a subunit of the citric acid cycle enzyme NAD(+)-isocitrate dehydrogenase was identified by sequence analysis . Its predicted amino acid sequence is more similar to the IDH1 regulatory subunit of S . cerevisiae NAD(+)-isocitrate dehydrogenase than to the IDH2 catalytic subunit . After expression in S . cerevisiae from an S . cerevisiae promoter, it was shown to functionally complement an S . cerevisiae idh1 mutant, but not an idh2 mutant, for growth on acetate as a carbon source and for production of NAD(+)-isocitrate dehydrogenase enzyme activity . These results confirm that the H . capsulatum cDNA encodes a homologue of subunit I of the S . cerevisiae mitochondrial isocitrate dehydrogenase isozyme that functions in the citric acid cycle .

Yeast, 1999 Jun 30, 15(9), 793 - 8
Cloning of a centromere binding factor 3d (CBF3D) gene from Candida glabrata; Stoyan T et al.; The gene encoding centromere binding factor 3d (CBF3D) of the human pathogenic yeast Candida glabrata has been isolated by hybridization of Saccharomyces cerevisiae CBF3D (ScCBF3D) DNA to a C . glabrata partial genomic library . Sequence analysis revealed a 540 bp open reading frame encoding a protein of 179 amino acids with a calculated molecular mass of 20.9 kDa . The amino acid sequence is highly homologous (78.6% identity) to ScCbf3d and 48.3% identical to the human homologue p19 (SKP1) . Southern blot analysis indicates that CgCbf3d is encoded by an unique nuclear gene . The cloned CgCBF3D gene can functionally substitute the S . cerevisiae homologue in a S . cerevisiae CBF3D-deletion mutant . The GenBank Accession No . for this gene is AF 072472 .

Yeast, 1999 Jun 30, 15(9), 741 - 54
The Hansenula polymorpha PDD1 gene product, essential for the selective degradation of peroxisomes, is a homologue of Saccharomyces cerevisiae Vps34p; Kiel JA et al.; Via functional complementation we have isolated the Hansenula polymorpha PDD1 gene essential for selective, macroautophagic peroxisome degradation . HpPDD1 encodes a 116 kDa protein with high similarity (42% identity) to Saccharomyces cerevisiae Vps34p, which has been implicated in vacuolar protein sorting and endocytosis . Western blotting experiments revealed that HpPDD1 is expressed constitutively . In a H . polymorpha pdd1 disruption strain peroxisome degradation is fully impaired . Sequestered peroxisomes, typical for the first stage of peroxisome degradation in H . polymorpha, were never observed, suggesting that HpPdd1p plays a role in the tagging of redundant peroxisomes and/or sequestration of these organelles from the cytosol . Possibly, HpPdd1p is the functional homologue of ScVps34p, because-like S . cerevisiae vps34 mutants-H . polymorpha pdd1 mutants are temperature-sensitive for growth and are impaired in the sorting of vacuolar carboxypeptidase Y . Moreover, HpPdd1p is associated to membranes, as was also observed for ScVps34p .

Mol Cell Biochem, 1999 May, 195(1-2), 219 - 26
The DHHC domain: a new highly conserved cysteine-rich motif; Putilina T et al.; A unique clone from a human pancreatic cDNA library was isolated and sequenced . Examination of the deduced polypeptide sequence of the clone showed a new form of cysteine-rich domain that included a region with the form of a Cys4 zinc-finger-like metal binding site followed by a complex Cys-His region . Searches of the Swiss-Protein data bank found a similar 48-residue domain in fifteen open reading frames deduced from A . thaliana, C . elegans, S . cerevisiae and S . pombe genomic sequences . The high degree of conservation of this domain (13 absolutely conserved and 17 highly conserved positions) suggests that it has an important function in the cell, possibly related to protein-protein or protein-DNA interactions . The gene recognized by the clone is is localized to human chromosome 16, and is conserved in vertebrates . The 2 Kb message is expressed in various human fetal and adult tissues . An antibody made to a peptide sequence of the deduced protein showed reactivity in immunoblots of monkey lung and retinal subcellular fractions and immunohistochemically in late fetal mouse tissues and a limited number of adult mouse tissues, including pancreatic islets, Leydig cells of the testis, and the plexiform layers of the retina.

Mol Gen Genet, 1999 Jun, 261(4-5), 601 - 9
A mutation in the secretion pathway of the yeast Yarrowia lipolytica that displays synthetic lethality in combination with a mutation affecting the signal recognition particle; Boisrame A et al.; In an attempt to identify proteins involved in the translocation step of protein secretion, a genetic screen was carried out in the yeast Yarrowia lipolytica . A conditional lethal mutant which has a defect in the 7S RNA of the signal recognition particle was mutagenized and screened for second-site mutations that specifically exacerbate its temperature sensitivity . This approach had previously allowed the characterization of an endoplasmic reticulum component, Sls1p, involved in protein translocation . A second mutation, sls2-1, was isolated that causes synthetic lethality when combined with the 7S RNA mutation . On its own, the sls2-1 mutation confers a temperature-sensitive growth phenotype . The secretory phenotype of the sls2 mutant consists in abnormal secretion of several polypeptides, and thus differs from the defect in secretory protein synthesis associated with the 7S RNA and sls1-1 mutations . Two new Y . lipolytica genes were identified which can relieve the growth defect of sls2-1 cells: SLS2 itself and SSL2, a multicopy suppressor of the temperature sensitivity of the sls2 mutant . The SLS2 gene encodes a polypeptide that can potentially be farnesylated and phosphorylated, and shares some homology with an S . cerevisiae protein of unknown function . Ssl2p resembles calmodulin-dependent serine/threonine protein kinases . These two proteins may interact to regulate protein sorting.

Yeast, 1999 Jun 15, 15(8), 703 - 13
Genome-wide transcriptional analysis in S . cerevisiae by mini-array membrane hybridization; Cox KH et al.; Access to the powerful micro-array analytical methods used for genome-wide transcriptional analysis has so far been restricted by the high cost and/or lack of availability of the sophisticated instrumentation and materials needed to perform it . Mini-array membrane hybridization provides a less expensive alternative . The reliability of this technique, however, is not well documented and its reported use has, up to this point, been very limited . Our objective was to test whether or not mini-array membrane hybridization would reliably identify genes whose expression was controlled by a specific set of genetic and/or physiological signals . Our results demonstrate that mini-array hybridization can correctly identify genes whose expression is known to be controlled by the GATA-factor regulatory network in S . cerevisiae and in addition can reliably identify genes not previously reported to be associated with this nitrogen control system.

Yeast, 1999 Jun 15, 15(8), 657 - 68
Copper-zinc superoxide dismutase from the marine yeast Debaryomyces hansenii; Hernandez-Saavedra NY et al.; We have isolated the cytosolic form of Cu-Zn superoxide dismutase (SOD) from the marine yeast Debaryomyces hansenii . This enzyme has a subunit mass of 18 kDa . The preparation was found to be heterogeneous by IF electrophoresis with two pI ranges: 5.14-4.0 and 1.6-1.8 . The enzyme preparation had a remarkably strong stability at pH 6.0-7.0, surviving boiling for 10 min without losing more than 60% of activity . On Western blots, this enzyme was recognized by antibodies raised in rabbits against D . hansenii extracts, while only a weak cross-reaction could be detected using antibodies generated against either Saccharomyces cerevisiae or bovine erythrocyte Cu-Zn SODs . In sequencing analysis, a peptide obtained by trypsin digestion was found to have 85% identity to the S . cerevisiae Cu-Zn SOD.

Gene, 1999 Jun 24, 234(1), 35 - 44
Functional expression of human and Arabidopsis protein phosphatase 2A in Saccharomyces cerevisiae and isolation of dominant-defective mutants; Lizotte DL et al.; Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic subunit and two distinct regulatory subunits, A and B . The primary sequence of the catalytic (C) subunit is highly conserved in evolution, and its function has been shown to be essential in yeast, Drosophila and mice . In many eukaryotes, the C subunit is encoded by at least two nearly identical genes, impeding conventional loss-of-function genetic analysis . We report here the development of a functional complementation assay in S . cerevisiae that has allowed us to isolate dominant-defective alleles of human and Arabidopsis C subunit genes . Wild-type human and Arabidopsis C subunit genes can complement the lethal phenotype of S . cerevisiae PP2A-C mutations . Site-directed mutagenesis was used to create two distinct, catalytically impaired C subunit mutants of the human and Arabidopsis genes . In both cases, expression of the mutant subunit in yeast prevented growth, even in the presence of functional C subunit proteins . This dominant growth defect is consistent with a dominant-interfering mode of action . Thus, we have shown that S . cerevisiae provides a rapid system for the functional analysis of heterologous PP2A genes, and that two mutations that abrogate phosphatase activity exhibit dominant-defective phenotypes in S . cerevisiae.

J Appl Microbiol, 1999 Jun, 86(6), 1047 - 52
Relationship between lipid composition, frequency of ethanol-induced respiratory deficient mutants, and ethanol tolerance in Saccharomyces cerevisiae; Chi Z et al.; The frequency of ethanol-induced respiratory deficient mutants and lipid composition in two Saccharomyces cerevisiae strains showing different degrees of ethanol tolerance were investigated . The more ethanol-tolerant strain exhibited a lower frequency of ethanol-induced respiratory deficient mutants than the less ethanol-tolerant strain . In addition, the more ethanol-tolerant strain contained a higher ergosterol/phospholipid ratio, a higher proportion of phosphatidylcholine, a lower proportion of phosphatidylethanolamine, a higher incorporation of long-chain fatty acids in total phospholipids, and a slightly higher proportion of unsaturated fatty acids in total phospholipids than the less ethanol-tolerant strain . These results show a clear relationship between the lipid composition, the frequency of ethanol-induced respiratory deficient mutants, and the ethanol tolerance of S . cerevisiae . A possible explanation of this relationship is discussed.

Genetics, 1999 Jul, 152(3), 943 - 52
DNA sequence and functional analysis of homologous ARS elements of Saccharomyces cerevisiae and S . carlsbergensis; Theis JF et al.; ARS elements of Saccharomyces cerevisiae are the cis-acting sequences required for the initiation of chromosomal DNA replication . Comparisons of the DNA sequences of unrelated ARS elements from different regions of the genome have revealed no significant DNA sequence conservation . We have compared the sequences of seven pairs of homologous ARS elements from two Saccharomyces species, S . cerevisiae and S . carlsbergensis . In all but one case, the ARS308-ARS308(carl) pair, significant blocks of homology were detected . In the cases of ARS305, ARS307, and ARS309, previously identified functional elements were found to be conserved in their S . carlsbergensis homologs . Mutation of the conserved sequences in the S . carlsbergensis ARS elements revealed that the homologous sequences are required for function . These observations suggested that the sequences important for ARS function would be conserved in other ARS elements . Sequence comparisons aided in the identification of the essential matches to the ARS consensus sequence (ACS) of ARS304, ARS306, and ARS310(carl), though not of ARS310.

Genetics, 1999 Jul, 152(3), 933 - 41
Conservation of ARS elements and chromosomal DNA replication origins on chromosomes III of Saccharomyces cerevisiae and S . carlsbergensis; Yang C et al.; DNA replication origins, specified by ARS elements in Saccharomyces cerevisiae, play an essential role in the stable transmission of chromosomes . Little is known about the evolution of ARS elements . We have isolated and characterized ARS elements from a chromosome III recovered from an alloploid Carlsberg brewing yeast that has diverged from its S . cerevisiae homeologue . The positions of seven ARS elements identified in this S . carlsbergensis chromosome are conserved: they are located in intergenic regions flanked by open reading frames homologous to those that flank seven ARS elements of the S . cerevisiae chromosome . The S . carlsbergensis ARS elements were active both in S . cerevisiae and S . monacensis, which has been proposed to be the source of the diverged genome present in brewing yeast . Moreover, their function as chromosomal replication origins correlated strongly with the activity of S . cerevisiae ARS elements, demonstrating the conservation of ARS activity and replication origin function in these two species.

Genetics, 1999 Jul, 152(3), 853 - 67
Saccharomyces cerevisiae putative G protein, Gtr1p, which forms complexes with itself and a novel protein designated as Gtr2p, negatively regulates the Ran/Gsp1p G protein cycle through Gtr2p; Nakashima N et al.; Prp20p and Rna1p are GDP/GTP exchanging and GTPase-activating factors of Gsp1p, respectively, and their mutations, prp20-1 and rna1-1, can both be suppressed by Saccharomyces cerevisiae gtr1-11 . We found that gtr1-11 caused a single amino acid substitution in Gtr1p, forming S20L, which is a putative GDP-bound mutant protein, while Gtr1p has been reported to bind to GTP alone . Consistently, gtr1-S20N, another putative GDP-bound mutant, suppressed both prp20-1 and rna1-1 . On the other hand, gtr1-Q65L, a putative GTP-bound mutant, was inhibitory to prp20-1 and rna1-1 . Thus, the role that Gtr1p plays in vivo appears to depend upon the nucleotide bound to it . Our data suggested that the GTP-bound Gtr1p, but not the GDP-bound Gtr1p, interacts with itself through its C-terminal tail . S . cerevisiae possesses a novel gene, GTR2, which is homologous to GTR1 . Gtr2p interacts with itself in the presence of Gtr1p . The disruption of GTR2 suppressed prp20-1 and abolished the inhibitory effect of gtr1-Q65L on prp20-1 . This finding, taken together with the fact that Gtr1p-S20L is a putative, inactive GDP-bound mutant, implies that Gtr1p negatively regulates the Ran/Gsp1p GTPase cycle through Gtr2p.

Protein Sci, 1999 Jun, 8(6), 1268 - 75
The Ubp6 family of deubiquitinating enzymes contains a ubiquitin-like domain: SUb; Wyndham AM et al.; A sequence motif that is Similar to Ubiquitin (SUb) has been identified in the Saccharomyces cerevisiae ubiquitin-specific protease Ubp6 . SUb is conserved in all known Ubp6 homologues from a spectrum of eukaryotic species and is also present in a group of hypothetical proteins of unknown function (Unk1-3) present in sequence databases . An N-terminal deletion mutant of Ubp6 that lacks SUb is still capable of cleaving alpha-linked ubiquitin fusions, suggesting that SUb forms a separate domain to the catalytic core of Ubp6 and demonstrating that it is not required for in vitro cleavage activity . A homology model of the 78 N-terminal amino acids of human Ubp6, based on the known fold of ubiquitin, is presented . In human Ubp6, SUb shares only 20% sequence identity with ubiquitin . Even weaker similarity occurs between S . cerevisiae SUb and ubiquitin . The homology model supports a ubiquitin-like fold for SUb and suggests that two conserved Lys residues, corresponding to Lys48 and Lys63 of ubiquitin, are functionally important.

Biosci Biotechnol Biochem, 1999 May, 63(5), 847 - 50
Construction of an effective host-vector system for the yeast Saccharomyces exiguus Yp74L-3; Hisatomi T et al.; An effective host-vector system specific to the yeast Saccharomyces exiguus Yp74L-3 was constructed to promote the molecular genetic analyses for the yeast . To obtain a stable reversionless host strain, we constructed an S . exiguus strain carrying leu2::ScURA3 by disrupting the S . exiguus LEU2 gene with the S . cerevisiae URA3 gene . A vector plasmid unique to S . exiguus was subsequently developed by inserting both the LEU2 gene and an ARS cloned from S . exiguus into an Escherichia coli phagemid, pUC119 . The vector constructed, pTH119 was able to transform the S . exiguus leu2::ScURA3 strain to Leu+ efficiently . The stability of the vector in the S . exiguus host cells resembled that of a YRp-type vector in S . cerevisiae.

Mutat Res, 1999 May 14, 434(1), 29 - 39
Cisplatin DNA cross-links do not inhibit S-phase and cause only a G2/M arrest in Saccharomyces cerevisiae; Grossmann KF et al.; Cisplatin (CDDP) has been used as a DNA cross-linking agent to evaluate whether there is a specific cell cycle checkpoint response to such damage in Saccharomyces cerevisiae (S . cerevisiae) . Fluorescent-activated cell sorting (FACS) analysis showed only a G2/M checkpoint, normal exit from G1 and progression through S-phase following alpha-factor arrest and CDDP treatment . Of the checkpoint mutants tested, rad9, rad17 and rad24, did not show increased sensitivity to CDDP compared to isogenic wild-type cells . However, other checkpoint mutants tested (mec1, mec3 and rad53) showed increased sensitivity to CDDP, as did controls with a defect in excision repair (rad1 and rad14) or a defect in recombination (rad51 and rad52) . Thus, by survival and cell cycle kinetics, it appears that DNA cross-links do not inhibit entry into S-phase or slow DNA replication and that replication continues after cisplatin treatment in yeast.

Ecotoxicol Environ Saf, 1999 Jun, 43(2), 149 - 55
Toxicity of organic and inorganic mercury to Saccharomyces cerevisiae; Kungolos A et al.; In this study the effect if six different forms of mercury on the growth of the yeast Saccharomyces cerevisiae is presented . Five kinds of strains of S . cerevisiae were used . They were a wild type, a mercury-resistant type, and three mutants: mutation repair-deficient mutant, excision repair-deficient mutant, and recombination repair-deficient mutant . In terms of EC50 toward the wild-type strain, the toxicity order for the inorganic forms was Hg(NO3)2>HgSO4>HgCl2 . Monovalent nitrate mercury Hg(NO3)2 was more toxic than bivalent Hg(NO3)2 . The toxicity of organic mercury CH3HgCl on cell growth was two orders of magnitude higher than that of inorganic HgCl2 . Between the two organic forms, CH3HgCl was more toxic than CH3HgOH . The survival rate in the presence of a certain concentration of CH3HgCl was about one-hundredth of the survival in presence of the same concentration of HgCl2 . On the other hand, the concentration of CH3HgCl in the cell was about 170 times that of HgCl2 . The addition of chelating agents, EDTA and methyl-penicillamine, to the medium did not reduce the toxicity of mercury . Among the three mutants tested, the one deficient in recombination repair systems was the most sensitive to mercury .

Genomics, 1999 Jun 15, 58(3), 293 - 301
Orthologues of the Caenorhabditis elegans longevity gene clk-1 in mouse and human; Asaumi S et al.; The clk-1 gene was isolated from the long-lived mutant of Caenorhabditis elegans and was suggested to play a biological role in longevity (Ewbank et al., 1997, Science 275: 980-983) . The primary structure of CLK-1 showed a significant homology to Saccharomyces cerevisiae Coq7p/Cat5p, which is required for the biosynthesis of ubiquinone and the derepression of gluconeogenic genes . In the present study, we isolated and characterized human and mouse orthologues of the COQ7/CLK-1 gene . Sequence analysis of both the human and the mouse COQ7 cDNAs showed an open reading frame composed of 217 amino acids with calculated molecular mass of 24,309 and 24,044 Da, respectively . Homology search revealed that human COQ7 showed 85% identity to mouse COQ7, 89% identity to rat COQ7, 53% identity to C . elegans CLK-1, and 37% identity to S . cerevisiae Coq7p/Cat5p . Zoo blot analysis implied that the COQ7 gene was well conserved among mammal, bird, and reptile genomes . Tissue blot analysis showed that human COQ7 is dominantly transcribed in heart and skeletal muscle . Genomic analyses revealed that the human COQ7 gene is composed of six exons spanning 11 kb of human genome as a single-copy gene . Radiation hybrid mapping assigned the COQ7 gene to human chromosome 16p12.3-p13.11 .

FEBS Lett, 1999 May 21, 451(2), 132 - 6
The vacuolar Ca2+/H+ exchanger Vcx1p/Hum1p tightly controls cytosolic Ca2+ levels in S . cerevisiae; Miseta A et al.; It is well established that the vacuole plays an important role in the cellular adaptation to growth in the presence of elevated extracellular Ca2+ concentrations in Saccharomyces cerevisiae . The Ca2+ ATPase Pmc1p and the Ca2+/H+ exchanger Vcx1p/Hum1p have been shown to facilitate Ca2+ sequestration into the vacuole . However, the distinct physiological roles of these two vacuolar Ca2+ transporters remain uncertain . Here we show that Vcx1p can rapidly sequester a sudden pulse of cytosolic Ca2+ into the vacuole, while Pmc1p carries out this function much less efficiently . This finding is consistent with the postulated role of Vcx1p as a high capacity, low affinity Ca2+ transporter and suggests that Vcx1p may act to attenuate the propagation of Ca2+ signals in this organism.

J Cell Biol, 1999 Jun 14, 145(6), 1153 - 63
Chs7p, a new protein involved in the control of protein export from the endoplasmic reticulum that is specifically engaged in the regulation of chitin synthesis in Saccharomyces cerevisiae; Trilla JA et al.; The Saccharomyces cerevisiae CHS7 gene encodes an integral membrane protein located in the ER which is directly involved in chitin synthesis through the regulation of chitin synthase III (CSIII) activity . In the absence of CHS7 product, Chs3p, but not other secreted proteins, is retained in the ER, leading to a severe defect in CSIII activity and consequently, to a reduced rate of chitin synthesis . In addition, chs7 null mutants show the yeast phenotypes associated with a lack of chitin: reduced mating efficiency and lack of the chitosan ascospore layer, clear indications of Chs7p function throughout the S . cerevisiae biological cycle . CHS3 overexpression does not lead to increased levels of CSIII because the Chs3p excess is retained in the ER . However, joint overexpression of CHS3 and CHS7 increases the export of Chs3p from the ER and this is accompanied by a concomitant increase in CSIII activity, indicating that the amount of Chs7p is a limiting factor for CSIII activity . Accordingly, CHS7 transcription is increased when elevated amounts of chitin synthesis are detected . These results show that Chs7p forms part of a new mechanism specifically involved in Chs3p export from the ER and consequently, in the regulation of CSIII activity.

Virology, 1999 Jun 5, 258(2), 271 - 81
Activation of Ste20 by Nef from human immunodeficiency virus induces cytoskeletal rearrangements and downstream effector functions in Saccharomyces cerevisiae; Plemenitas A et al.; The negative factor (Nef) from human and simian immunodeficiency viruses is important for the pathogenesis of acquired immune deficiency syndrome . Among other targets, it activates the Nef-associated kinase, which is related to the p21-activated kinase . In this study, we demonstrate that Nef activates Ste20, the homolog of p21-activated kinase in Saccharomyces cerevisiae . Nef binds to the adaptor proteins Bem1 and Ste20 via its proline-rich (PXXP) and diarginine (RR) motifs, respectively . These interactions induce the mitogen-activated protein kinase and increase the rates of budding, sizes of cells, and patterns of mating projections . These effects of Nef depend on the small GTPase Cdc42 and guanine nucleotide exchange factor Cdc24 . Thus, studies in S . cerevisiae identified specific interactions between Nef and cellular proteins and their associated signaling cascade .

J Mol Biol, 1999 Jun 4, 289(2), 235 - 48
Binding specificity determines polarity of DNA unwinding by the Sgs1 protein of S . cerevisiae; Bennett RJ et al.; Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ DNA helicase family which also includes the products of the human Bloom's syndrome and Werner's syndrome genes . We have studied the substrate specificity of a recombinant Sgs1 helicase (amino acid residues 400-1268 of the Sgs1 protein) . Sgs1 shows a strong preference for binding branched DNA substrates, including duplex structures with a 3' single-stranded overhang and DNA junctions with multiple branches . Duplex DNA with a 5' rather than a 3' single-stranded tail is not recognized or unwound by Sgs1 . DNase I and hydroxyl radical footprinting of the Sgs1-DNA complex shows that the protein binds specifically to the junction of a double-stranded DNA and its 3' overhang . Binding and unwinding of duplex DNA with a 3' overhang are much reduced if the backbone polarity of the 3' overhang is reversed in the junction region, but are unaffected if polarity reversal occurs four nucleotides away from the junction . These results indicate that the 3' to 5' polarity of unwinding by the recombinant Sgs1 protein is a direct consequence of the binding of the helicase to the single-stranded/double-stranded DNA junction and its recognition of the polarity of the single-stranded DNA at the junction . The recombinant Sgs1 also unwinds four-way junctions (synthetic Holliday junctions), a result that may be significant in terms of its role in suppressing DNA recombination in vivo .

J Clin Microbiol, 1999 Jul, 37(7), 2230 - 5
Molecular and epidemiological characterization of vaginal Saccharomyces cerevisiae isolates; Posteraro B et al.; Although vaginitis caused by Saccharomyces cerevisiae is extremely rare, in recent years we have experienced an increasing frequency of S . cerevisiae isolation from the vaginas of fertile-age women . In order to investigate the epidemiology of these vaginal infections, a total of 40 isolates of S . cerevisiae derived from symptomatic and asymptomatic women were characterized by two DNA typing approaches, named ribosomal DNA (rDNA) hybridization and Ty917 hybridization, based on the Southern blotting technique . After transfer, the polymorphic DNA restriction fragments were hybridized with the entire repeat of S . cerevisiae rDNA for one method and with the entire sequence of the Ty917 retrotransposon for the other . After elaboration with computer-assisted analysis, the results of each method showed that Ty917 hybridization is endowed with a discriminatory power higher than that of rDNA hybridization . With the Ty917 hybridization method, all of the S . cerevisiae isolates tested appeared very heterogeneous, with the exception of those collected from individual patients with recurrent vaginitis . This allowed us to exclude a possible common source of infection while the high relatedness among S . cerevisiae sequential isolates from recurrent-vaginitis patients could suggest a pattern of relapse rather than frequent reinfection.

J Biol Chem, 1999 Jun 18, 274(25), 18135 - 9
Bivalent inhibitor of the N-end rule pathway; Kwon YT et al.; The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue . Ubr1p, the recognition (E3) component of the Saccharomyces cerevisiae N-end rule pathway, contains at least two substrate-binding sites . The type 1 site is specific for N-terminal basic residues Arg, Lys, and His . The type 2 site is specific for N-terminal bulky hydrophobic residues Phe, Leu, Trp, Tyr, and Ile . Previous work has shown that dipeptides bearing either type 1 or type 2 N-terminal residues act as weak but specific inhibitors of the N-end rule pathway . We took advantage of the two-site architecture of Ubr1p to explore the feasibility of bivalent N-end rule inhibitors, whose expected higher efficacy would result from higher affinity of the cooperative (bivalent) binding to Ubr1p . The inhibitor comprised mixed tetramers of beta-galactosidase that bore both N-terminal Arg (type 1 residue) and N-terminal Leu (type 2 residue) but that were resistant to proteolysis in vivo . Expression of these constructs in S . cerevisiae inhibited the N-end rule pathway much more strongly than the expression of otherwise identical beta-galactosidase tetramers whose N-terminal residues were exclusively Arg or exclusively Leu . In addition to demonstrating spatial proximity between the type 1 and type 2 substrate-binding sites of Ubr1p, these results provide a route to high affinity inhibitors of the N-end rule pathway.

Mol Biol Rep, 1999 Apr, 26(1-2), 21 - 8
Functional analysis of the proteasome regulatory particle; Glickman MH et al.; We have developed S . cerevisiae as a model system for mechanistic studies of the 26S proteasome . The subunits of the yeast 19S complex, or regulatory particle (RP), have been defined, and are closely related to those of mammalian proteasomes . The multiubiquitin chain binding subunit (S5a/Mcb1/Rpn10) was found, surprisingly, to be nonessential for the degradation of a variety of ubiquitin-protein conjugates in vivo . Biochemical studies of proteasomes from deltarpn10 mutants revealed the existence of two structural subassemblies within the RP, the lid and the base . The lid and the base are both composed of 8 subunits . By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively . The base is sufficient to activate the 20S core particle for degradation of peptides, but the lid is required for ubiquitin-dependent degradation . The lid subunits share sequence motifs with components of the COP9/signalosome complex, suggesting that these functionally diverse particles have a common evolutionary ancestry . Analysis of equivalent point mutations in the six ATPases of the base indicate that they have well-differentiated functions . In particular, mutations in one ATPase gene, RPT2, result in an unexpected defect in peptide hydrolysis by the core particle . One interpretation of this result is that Rpt2 participates in gating of the channel through which substrates enter the core particle.

Fungal Genet Biol, 1999 Apr, 26(3), 190 - 7
Ligand recognition in multiallelic pheromone receptors from the basidiomycete Schizophyllum commune studied in yeast; Hegner J et al.; The homobasidiomycete Schizophyllum commune encodes a multiallelic pheromone receptor system that distinguishes more than 20 nonself from at least 2 self pheromones . The well-investigated pheromone response system of the yeast Saccharomyces cerevisiae was used to link the FUS1::lacZ reporter system to the heterologous pheromone receptors from S . commune . To investigate yeast G-protein binding, the unchanged heterologous receptor was compared to constructs carrying an exchange of the 3rd cytoplasmatic loop for the Ste2 sequence . A better coupling could be achieved with the altered constructs . In order to examine activation by single pheromones, an artificial peptide based on the sequence of a new putative pheromone gene, bap2(1), in the Balpha2 mating-type locus encoding the shortest pheromone found so far in fungal mating types was used . Thus, we have reassembled the pheromone recognition of the basidiomycete S . commune and constructed a system ideal for specificity analysis in the yeast S . cerevisiae .

Mol Cell, 1999 May, 3(5), 611 - 20
RecQ helicase and topoisomerase III comprise a novel DNA strand passage function: a conserved mechanism for control of DNA recombination; Harmon FG et al.; E . coli RecQ protein is a multifunctional helicase with homologs that include the S . cerevisiae Sgs1 helicase and the H . sapiens Wrn and Blm helicases . Here we show that RecQ helicase unwinds a covalently closed double-stranded DNA (dsDNA) substrate and that this activity specifically stimulates E . coli topoisomerase III (Topo III) to fully catenate dsDNA molecules . We propose that these proteins functionally interact and that their shared activity is responsible for control of DNA recombination . RecQ helicase has a comparable effect on the Topo III homolog of S . cerevisiae, consistent with other RecQ and Topo III homologs acting together in a similar capacity . These findings highlight a novel, conserved activity that offers insight into the function of the other RecQ-like helicases.

Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 6982 - 7
A mutation in the human ortholog of the Saccharomyces cerevisiae ALG6 gene causes carbohydrate-deficient glycoprotein syndrome type-Ic; Imbach T et al.; Carbohydrate-deficient glycoprotein syndrome (CDGS) represents a class of genetic diseases characterized by abnormal N-linked glycosylation . CDGS patients show a large number of glycoprotein abnormalities resulting in dysmorphy, encephalopathy, and other organ disorders . The majority of CDGSs described to date are related to an impaired biosynthesis of dolichyl pyrophosphate-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum . Recently, we identified in four related patients a novel type of CDGS characterized by an accumulation of dolichyl pyrophosphate-linked Man9GlcNAc2 . Elaborating on the analogy of this finding with the phenotype of alg5 and alg6 Saccharomyces cerevisiae strains, we have cloned and analyzed the human orthologs to the ALG5 dolichyl phosphate glucosyltransferase and ALG6 dolichyl pyrophosphate Man9GlcNAc2 alpha1,3-glucosyltransferase in four novel CDGS patients . Although ALG5 was not altered in the patients, a C-->T transition was detected in ALG6 cDNA of all four CDGS patients . The mutation cosegregated with the disease in a Mendelian recessive manner . Expression of the human ALG5 and ALG6 cDNA could partially complement the respective S . cerevisiae alg5 and alg6 deficiency . By contrast, the mutant ALG6 cDNA of CDGS patients failed to revert the hypoglycosylation observed in alg6 yeasts, thereby proving a functional relationship between the alanine to valine substitution introduced by the C-->T transition and the CDGS phenotype . The mutation in the ALG6 alpha1,3-glucosyltransferase gene defines an additional type of CDGS, which we propose to refer to as CDGS type-Ic.

FEBS Lett, 1999 May 7, 450(3), 251 - 6
Purification of Saccharomyces cerevisiae RNase H(70) and identification of the corresponding gene; Frank P et al.; We purified Saccharomyces cerevisiae RNase H(70) to homogeneity, using an optimized chromatographic purification procedure . Renaturation gel assay assigned RNase H activity to a 70 kDa polypeptide . Sequencing of tryptic peptides identified the open reading frame YGR276c on chromosome VII of the S . cerevisiae genome as the corresponding gene, which encodes a putative polypeptide of molecular mass of 62849 . We therefore renamed this gene RNH70 . Immunofluorescence microscopy using a RNH70-EGFP fusion construct indicates nuclear localization of RNase H(70) . Deletion of RNH70 from the yeast genome did not result in any serious phenotype under the conditions tested . Homology searches revealed striking similarity with a number of eukaryotic proteins and open reading frames, among them the chimpanzee GOR protein, a homolog of a human autoimmune antigen, found to elicit autoimmune response in patients infected with hepatitis C virus.

J Biol Chem, 1999 Jun 11, 274(24), 16861 - 70
The Cdc42p GTPase is involved in a G2/M morphogenetic checkpoint regulating the apical-isotropic switch and nuclear division in yeast; Richman TJ et al.; The Cdc42p GTPase is involved in the signal transduction cascades controlling bud emergence and polarized cell growth in S . cerevisiae . Cells expressing the cdc42(V44A) effector domain mutant allele displayed morphological defects of highly elongated and multielongated budded cells indicative of a defect in the apical-isotropic switch in bud growth . In addition, these cells contained one, two, or multiple nuclei indicative of a G2/M delay in nuclear division and also a defect in cytokinesis and/or cell separation . Actin and chitin were delocalized, and septin ring structure was aberrant and partially delocalized to the tips of elongated cdc42(V44A) cells; however, Cdc42(V44A)p localization was normal . Two-hybrid protein analyses showed that the V44A mutation interfered with Cdc42p's interactions with Cla4p, a p21(Cdc42/Rac)-activated kinase (PAK)-like kinase, and the novel effectors Gic1p and Gic2p, but not with the Ste20p or Skm1p PAK-like kinases, the Bni1p formin, or the Iqg1p IQGAP homolog . Furthermore, the cdc42(V44A) morphological defects were suppressed by deletion of the Swe1p cyclin-dependent kinase inhibitory kinase and by overexpression of Cla4p, Ste20p, the Cdc12 septin protein, or the guanine nucleotide exchange factor Cdc24p . In sum, these results suggest that proper Cdc42p function is essential for timely progression through the apical-isotropic switch and G2/M transition and that Cdc42(V44A)p differentially interacts with a number of effectors and regulators.

Can J Microbiol, 1999 Jan, 45(1), 31 - 7
PKA from Saccharomyces cerevisiae can be activated by cyclic AMP and cyclic GMP; Cytrynska M et al.; Analysis of Saccharomyces cerevisiae genome revealed no sequence homologous to cyclic GMP (cGMP) dependent protein kinase from other organisms . Here we demonstrate that cyclic AMP (cAMP) dependent protein kinase purified from S . cerevisiae was almost equally activated by cAMP and cGMP in 3 x 10(-6) M concentrations of either nucleotide in the presence of Mg2+ ions . Interestingly, if Mn2+ ions were used instead of Mg2+, cGMP was only 30% as effective as cAMP in the activation of cAMP-dependent protein kinase . Analogs of cAMP such as 8-chloro-cAMP and 3':5'-cyclic monophosphate of ribofuranosylbenzimidazole were as potent as cAMP in the enzyme activation, while N6,2'-O-dibutyryl-cAMP activated the enzyme to a lower extent . It was also found that yeast cAMP-dependent protein kinase can be activated by limited proteolytic digestion . The results presented were obtained with protamine and ribosomal protein S10 used as phosphorylation substrates.

Mech Dev, 1998 Dec, 79(1-2), 51 - 5
Identification and developmental expression of a 5'-3' exoribonuclease from Drosophila melanogaster; Till DD et al.; In multicellular organisms, very little is known about the role of mRNA stability in development, and few proteins involved in degradation pathways have been characterized . We have identified the Drosophila homologue of XRN1, which is the major cytoplasmic 5'-3' exoribonuclease in Saccharomyces cerevisiae . The protein sequence of this homologue (pacman) has 59% identity to S . cerevisiae XRN1 and 67% identity to the mouse homologue (mXRN1p) in certain regions . Sequencing of this cDNA revealed that it includes a trinucleotide repeat (CAG)9 which encodes polyglutamine . By directly measuring pacman exoribonuclease activity in yeast, we demonstrate that pacman can complement the yeast XRN1 mutation . Northern blots show a single transcript of approximately 5.2 kb which is abundant only in 0-8-h embryos and in adult males and females . In situ hybridization analysis revealed that the pcm transcripts are maternally derived, and are expressed at high levels in nurse cells . During early embryonic syncytial nuclear divisions, pcm transcripts are homogenously distributed . pcm mRNA is expressed abundantly and ubiquitously throughout the embryo during gastrulation, with high levels in the germ band and head structures . After germ band retraction, pcm transcripts are present at much lower levels, in agreement with the Northern results . Our experiments provide the first example of an exoribonuclease which is differentially expressed throughout development.

Appl Environ Microbiol, 1999 Jun, 65(6), 2508 - 12
The carboxy-terminal portion of the aflatoxin pathway regulatory protein AFLR of Aspergillus parasiticus activates GAL1::lacZ gene expression in Saccharomyces cerevisiae; Chang PK et al.; AFLR, a DNA-binding protein of 444 amino acids, transactivates the expression of aflatoxin biosynthesis genes in Aspergillus parasiticus and Aspergillus flavus, as well as the sterigmatocystin synthesis genes in Aspergillus nidulans . We show here by fusion of various aflR coding regions to the GAL4 DNA-binding coding region that the AFLR carboxyl terminus contained a region that activated GAL1::lacZ gene expression in Saccharomyces cerevisiae and that the AFLR internal region was required for the activation activity . Compared to the AFLR carboxy-terminal fusion protein (AFLRC), a mutant AFLRC retained approximately 75% of the activation activity after deletion of three acidic amino acids, Asp365, Glu366, and Glu367, in a previously identified acidic stretch . Removal of the carboxy-terminal amino acid, Glu444, did not affect the activation activity . Substitutions of acidic Glu423, Asp439, or Asp436/Asp439 with basic amino acids, Lys and His, resulted in 10- to 15-fold-lower activation activities . Strikingly, the Asp436His mutation abolished the activation activity . Substitutions of basic His428 and His442 with acidic Asp resulted in 20 and 40% decreases in the activation activities, respectively . Simultaneous substitutions of Arg427, Arg429, and Arg431 with Leu also significantly decreased the activation activity; the decrease was approximately 50-fold . Results suggest that the AFLR carboxy-terminal region is involved in transcription activation and that total acidity in this region is not a major determinant of AFLR's activation ability in S . cerevisiae.

Electrophoresis, 1999 Apr-May, 20(4-5), 826 - 9
Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis; Harder A et al.; Three different procedures for the solubilization of yeast (S . cerevisiae) cell proteins were compared on the basis of the obtained two-dimensional (2-D) polypeptide patterns . Major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high molecular mass proteins . The procedures employed were sonication, followed by (i) protein solubilization with "standard" lysis buffer (9 M urea, 2% 3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), 2% v/v carrier ampholytes, (ii) presolubilization of proteins with sodium dodecyl sulfate (SDS) buffer, consisting of 1% SDS and 100 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.0, followed by dilution with "standard" lysis buffer, and (iii) boiling the sample with SDS during cell lysis, followed by dilution with thiourea/urea lysis buffer (2 M thiourea/ 7 M urea, 4% w/v CHAPS, 1% w/v DTT, 2% v/v carrier ampholytes) . All procedures tested were rapid and simple . However, with the first procedure (i), considerable degradation of high Mr proteins occurred . In contrast, protein degradation was minimized by boiling the sample in SDS buffer immediately after sonication (method ii) . Protein disaggregation and solubilization of high Mr proteins were further improved by pre-boiling with SDS and using thiourea/urea lysis buffer instead of "standard" lysis buffer (procedure iii).

Plant Mol Biol, 1999 Mar, 39(5), 953 - 67
Heterologous expression in Saccharomyces cerevisiae of an Arabidopsis thaliana cDNA encoding mevalonate diphosphate decarboxylase; Cordier H et al.; Sequence comparison with the mevalonate diphosphate decarboxylase (MVD) amino acid sequence of Saccharomyces cerevisiae identified an EST clone corresponding to a cDNA that may encode Arabidopsis thaliana MVD (AtMVD1) . This enzyme catalyses the synthesis of isopentenyl diphosphate, the building block of sterol and isoprenoid biosynthesis, and uses mevalonate diphosphate as a substrate . Sequencing of the full-length cDNA was performed . The predicted amino acid sequence presents about 55% identity with the yeast, human and rat MVDs . The sequence of the genomic region of A . thaliana MVD was also obtained and Southern blot analysis on genomic DNA showed that A . thaliana could have at least one homologous MVD gene . In order to allow heterologous expression in S . cerevisiae, the MVD open reading frame (ORF) was then cloned under the control of the yeast PMA1 strong promoter . When expressed in yeast, the A . thaliana cDNA complemented both the thermosensitive MN19-34 strain deficient in MVD, and the lethal phenotype of an ERG19 deleted strain . However, the wild-type sterol content was not fully restored suggesting that the A . thaliana MVD activity may not be optimal in yeast . A two-hybrid assay was also performed to evaluate homodimer formation of the A . thaliana MVD and heterodimer formation between the plant and yeast heterologous enzymes.

Curr Biol, 1999 May 20, 9(10), 551 - 4
The BRCT domain of the S . cerevisiae checkpoint protein Rad9 mediates a Rad9-Rad9 interaction after DNA damage; Soulier J et al.; The Saccharomyces cerevisiae checkpoint protein Rad9 is required for transient cell-cycle arrest and transcriptional induction of DNA-repair genes in response to DNA damage {1} . It contains a carboxyterminal tandem repeat of the BRCT (BRCA1 carboxyl terminus) motif, a motif that is also found in many proteins involved in various aspects of DNA repair, recombination and checkpoint control {2}{3} . We produced yeast strains expressing Rad9 in which the BRCT domain had been deleted or which harboured point mutations in the highly conserved aromatic residue of each BRCT motif . Rates of survival and checkpoint delay of the mutants after ultraviolet (UV) irradiation were essentially equivalent to those of rad9Delta (null) cells, demonstrating that the BRCT domain is required for Rad9 function . Rad9 hyperphosphorylation, which occurs after DNA damage {4}{5}{6}, was absent in the BRCT mutants, as was Rad9-dependent phosphorylation of the Rad53 protein . A two-hybrid approach identified a specific interaction between the Rad9 BRCT domain and itself . Biochemical analysis in vitro and in vivo confirmed this interaction and, furthermore, demonstrated that the Rad9 BRCT domain preferentially interacted with the hyperphosphorylated forms of Rad9 . This interaction was suppressed by mutations of the BRCT motifs that caused null phenotypes in vivo, suggesting that Rad9 oligomerization is required for Rad9 function after DNA damage.

Genes Cells, 1999 Apr, 4(4), 197 - 203
Incorporation of Drosophila TAF110 into the yeast TFIID complex does not permit the Sp1 glutamine-rich activation domain to function in vivo; Keaveney M et al.; BACKGROUND: Acidic activation domains function across eukaryotic species, and hence stimulate transcription by a conserved molecular mechanism . In contrast, glutamine-rich activation domains function in flies, mammals, and fission yeasts but not in the budding yeast Saccharomyces cerevisiae . The glutamine-rich activation domain of Sp1 interacts with TAF110, and it has been suggested that this interaction is important for transcriptional activation . S . cerevisiae does not contain a homologue of TAF110, suggesting a potential mechanism to account for the failure of glutamine-rich activation domains to stimulate transcription . RESULTS: Here, we have artificially recruited Drosophila TAF110 into the yeast TFIID complex by fusing it to yeast TBP . The resulting TFIID complex supports normal cell growth, but it is unable to mediate Sp1-dependent activation . CONCLUSIONS: Thus, the interaction of glutamine-rich activation domains with TAF110 is insufficient for transcriptional activation in vivo, indicating that other targets within the PolII machinery are necessary.

Am J Med Genet, 1999 May 28, 84(3), 272 - 6
KH domain-containing proteins of yeast: absence of a fragile X gene homologue; Currie JR et al.; The KH domain is a region defined by its homology to the RNA-binding domains of the heterogeneous nuclear ribonucleoprotein K (hnRNPK) . There are two such domains in the FMR1 protein which is underexpressed in the fragile X syndrome . We developed a computer method to search the S . cerevisiae protein sequences as they became available for the KH domain of the FMR1 protein . Using our motif and FINDPATTERNS of the Wisconsin Package of GCG, nine proteins were identified in the completed yeast ORF database that contain KH domains . Five proteins have known or predicted functions; four await functional analysis . Using GeneWorks and GeneJockeyII alignments, we found that the yeast protein KH domain showing the most similarity to either FMR1P KH domain was a KH domain in HX/SCP160 . Its sequence is 50% identical to the second KH domain of FMR1P . However, SCP160 contains eight conserved and six degenerate KH domains . Further analysis showed that SCP160 is a better match overall to the vertebrate and C . elegans protein Vigilin, which also contains 14 KH domains . The next most similar yeast KH domain was found in YB83, a protein shorter than FMR1P and containing three KH domains, one of which shares 45% identity with the second KH domain in FMR1P . There is no significant overall sequence similarity between this yeast protein and FMR1P . Thus, while several proteins in yeast contain KH domains, no apparent yeast homologue exists for the FMR1 protein of the fragile X gene family.

Mol Cell Biochem, 1999 Feb, 192(1-2), 41 - 52
Long-chain fatty acid transport in bacteria and yeast . Paradigms for defining the mechanism underlying this protein-mediated process; DiRusso CC et al.; Protein-mediated transport of exogenous long-chain fatty acids across the membrane has been defined in a number of different systems . Central to understanding the mechanism underlying this process is the development of the appropriate experimental systems which can be manipulated using the tools of molecular genetics . Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both {1} exhibit saturable long-chain fatty acid transport at low ligand concentration; {2} have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus; and {3} can be easily manipulated using the tools of molecular genetics . In E . coli, this process requires the outer membrane-bound fatty acid transport protein FadL and the inner membrane associated fatty acyl CoA synthetase (FACS) . FadL appears to represent a substrate specific channel for long-chain fatty acids while FACS activates these compounds to CoA thioesters thereby rendering this process unidirectional . This process requires both ATP generated from either substrate-level or oxidative phosphorylation and the proton electrochemical gradient across the inner membrane . In S . cerevisiae, the process of long-chain fatty acid transport requires at least the membrane-bound protein Fat1p . Exogenously supplied fatty acids are activated by the fatty acyl CoA synthetases Faa1p and Faa4p but unlike the case in E . coli, there is not a tight linkage between transport and activation . Studies evaluating the growth parameters in the presence of long-chain fatty acids and long-chain fatty acid transport profiles of a fat1delta strain support the hypothesis that Fatlp is required for optimal levels of long-chain fatty acid transport.

Acta Crystallogr D Biol Crystallogr, 1999 Jun, 55 ( Pt 6), 1234 - 6
Purification, crystallization and preliminary X-ray crystallographic studies of S . cerevisiae Hsp40 Sis1; Sha B et al.; Heat-shock protein 70 (Hsp70), one of the major molecular chaperones, has been shown to play a central role in many cellular processes . Heat-shock protein 40 (Hsp40) works as a co-chaperone for Hsp70 . Hsp40, bound by unfolded polypeptide, can interact directly with Hsp70 to stimulate the ATPase activity of Hsp70 . Hsp40 can also bind to unfolded polypeptides and prevent them from aggregating in vitro, thus acting as an independent molecular chaperone . The S . cerevisiae Hsp40 Sis1 C-terminal peptide-binding domain has been crystallized . The crystals diffract to 2.7 A and belong to space group P41212 or P43212 with a = 73.63, c = 80.16 A . The structure determination by the MAD method is under way.

J Biol Chem, 1999 May 21, 274(21), 14857 - 66
Isolation and characterization of the Saccharomyces cerevisiae EKI1 gene encoding ethanolamine kinase; Kim K et al.; Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1 . 82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway . The gene encoding ethanolamine kinase (EKI1) was identified from the Saccharomyces Genome Data Base (locus YDR147W) based on its homology to the Saccharomyces cerevisiae CKI1-encoded choline kinase, which also exhibits ethanolamine kinase activity . The EKI1 gene was isolated and used to construct eki1Delta and eki1Delta cki1Delta mutants . A multicopy plasmid containing the EKI1 gene directed the overexpression of ethanolamine kinase activity in wild-type, eki1Delta mutant, cki1Delta mutant, and eki1Delta cki1Delta double mutant cells . The heterologous expression of the S . cerevisiae EKI1 gene in Sf-9 insect cells resulted in a 165,500-fold overexpression of ethanolamine kinase activity relative to control insect cells . The EKI1 gene product also exhibited choline kinase activity . Biochemical analyses of the enzyme expressed in insect cells, in eki1Delta mutants, and in cki1Delta mutants indicated that ethanolamine was the preferred substrate . The eki1Delta mutant did not exhibit a growth phenotype . Biochemical analyses of eki1Delta, cki1Delta, and eki1Delta cki1Delta mutants showed that the EKI1 and CKI1 gene products encoded all of the ethanolamine kinase and choline kinase activities in S . cerevisiae . In vivo labeling experiments showed that the EKI1 and CKI1 gene products had overlapping functions with respect to phospholipid synthesis . Whereas the EKI1 gene product was primarily responsible for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, the CKI1 gene product was primarily responsible for phosphatidylcholine synthesis via the CDP-choline pathway . Unlike cki1Delta mutants, eki1Delta mutants did not suppress the essential function of Sec14p.

Genes Dev, 1999 May 1, 13(9), 1089 - 101
UASrpg can function as a heterochromatin boundary element in yeast; Bi X et al.; The HM loci in Saccharomyces cerevisiae constitute region-specific but gene-nonspecific repression domains, as a number of heterologous genes transcribed by RNA polymerase II or III are silenced when placed at these loci . The promoters of the Ashbya gossypii TEF gene and the S . cerevisiae TEF1 and TEF2 genes, however, are resistant to transcriptional silencing by the HM silencers in yeast . Moreover, when interposed between the HML alpha genes and the E silencer, certain segments of these promoters block the repression effect of the silencer on the alpha genes . All of these fragments contain UASrpg (upstream activation sequence of ribosome protein genes) composed of multiple binding sites for Rap1 . In fact, a 149-bp segment consisting essentially of only three tandem Rap1-binding sites from the UASrpg of yeast TEF2 exhibits silencer-blocking activity . This element also exhibits insulating activity and orientation dependence characteristic of known chromatin boundary elements . Finally, the element blocks the physical spread of heterochromatin initiated at a silencer . This segment provides the first example of chromatin domain boundary or insulator elements in yeast.

Biochemistry, 1999 May 11, 38(19), 6171 - 7
Lysine biosynthesis in Saccharomyces cerevisiae: mechanism of alpha-aminoadipate reductase (Lys2) involves posttranslational phosphopantetheinylation by Lys5; Ehmann DE et al.; A key step in fungal biosynthesis of lysine, enzymatic reduction of alpha-aminoadipate at C6 to the semialdehyde, requires two gene products in Saccharomyces cerevisiae, Lys2 and Lys5 . Here, we show that the 31-kDa Lys5 is a specific posttranslational modification catalyst, using coenzyme A (CoASH) as a cosubstrate to phosphopantetheinylate Ser880 of the 155-kDa Lys2 and activate it for catalysis . Lys2 was subcloned from S . cerevisiae and expressed in and purified from Escherichia coli as a full-length 155-kDa enzyme, as a 105-kDa adenylation/peptidyl carrier protein (A/PCP) fragment (residues 1-924), and as a 14-kDa PCP fragment (residues 809-924) . The apo-PCP fragment was covalently modified to phosphopantetheinylated holo-PCP by pure Lys5 and CoASH with a Km of 1 microM and kcat of 3 min-1 for both the PCP and CoASH substrates . The adenylation domain of the A/PCP fragment activated S-carboxymethyl-L-cysteine (kcat/Km = 840 mM-1 min-1) at 16% the efficiency of L-alpha-aminoadipate in {32P}PPi/ATP exchange assays . The holo form of the A/PCP 105-kDa fragment of Lys2 covalently aminoacylated itself with {35S}S-carboxymethyl-L-cysteine . Addition of NADPH discharged the covalent acyl-S-PCP Lys2, consistent with a reductive cleavage of the acyl-S-enzyme intermediate . These results identify the Lys5/Lys2 pair as a two-component system in which Lys5 covalently primes Lys2, allowing alpha-aminoadipate reductase activity by holo-Lys2 with catalytic cycles of autoaminoacylation and reductive cleavage . This is a novel mechanism for a fungal enzyme essential for amino acid metabolism.

Proc Natl Acad Sci U S A, 1999 May 11, 96(10), 5440 - 5
Conservation in budding yeast of a kinase specific for SR splicing factors; Siebel CW et al.; SR protein kinases (SRPKs) and their substrates, the SR family of serine/arginine-rich pre-mRNA splicing factors, appear to be key regulators of alternative splicing . Although SR proteins have been well characterized through biochemical experiments in metazoans, their functions in vivo are unclear . Because of the strict splice site consensus and near absence of alternative splicing in Saccharomyces cerevisiae, it had been thought that budding yeast would lack an SRPK and its substrates . Here, we present structural, biochemical, and cell-biological evidence that directly demonstrates an SR protein kinase, Sky1p, as well as a number of SRPK substrates in S . cerevisiae . One of these substrates is Npl3p, an SR-like protein involved in mRNA export . This finding raises the provocative possibility that Sky1p, and by extension metazoan SRPKs, regulates mRNA export or the nucleocytoplasmic shuttling of RS domain proteins . The unexpected discovery of an SR protein kinase in budding yeast provides a foundation for genetic dissection of the biological functions of SR proteins and their kinases.

J Biol Chem, 1999 May 14, 274(20), 14337 - 43
The role of human TFIIB in transcription start site selection in vitro and in vivo; Hawkes NA et al.; The general transcription factor TFIIB plays a crucial role in selecting the transcription initiation site in yeast . We have analyzed the human homologs of TFIIB mutants that have previously been shown to affect transcription start site selection in the yeast Saccharomyces cerevisiae . Despite the distinct mechanisms of transcription start site selection observed in S . cerevisiae and humans, the role of TFIIB in this process is similar . However, unlike their yeast counterparts, the human mutants do not show a severe defect in supporting either basal transcription or transcription stimulated by an acidic activator in vitro . Transient transfection analysis revealed that, in addition to a role in transcription start site selection, human TFIIB residue Arg-66 performs a critical function in vivo that is bypassed in vitro . Furthermore, although correct transcription start site selection is dependent upon an arginine residue at position 66 in human TFIIB, innate function in vivo is determined by the charge of the residue alone . Our observations raise questions as to the evolutionary conservation of TFIIB and uncover an additional function for TFIIB that is required in vivo but can be bypassed in vitro.

Yeast, 1999 Apr, 15(6), 481 - 96
Analysis of genetic interactions between DHH1, SSD1 and ELM1 indicates their involvement in cellular morphology determination in Saccharomyces cerevisiae; Moriya H et al.; The DHH1 gene of Saccharomyces cerevisiae belongs to a family of genes that encode highly conserved DEAD-box proteins commonly present in various eukaryotic organisms . Its precise function in yeast has not yet been well documented . To investigate its role in vivo, we constructed a DHH1 disruptant, characterized it genetically and searched for genes the mutations in which would cause synthetic lethality in combination with the DHH1 disruption . CDC28, ELM1 and SSD1 were thus found to be such candidates and we subsequently analysed their interactions . Mutations in ELM1 were previously reported to result in the elongation of cells . We confirmed this phenotype and observed in addition elongated bud formation in an Elm1p overproducing strain . Also, Elm1p fused with the green fluorescent protein (GFP) was found to be localized at the bud neck . These and other observations seem to suggest that Elm1p plays a role during cytokinesis in S . cerevisiae . The phenotypes of strains harbouring either delta dhh1 delta elm1 or ssd1-d delta elm1 were very similar to each other, showing abnormal cellular morphology and defects in cytokinesis and mitosis . Furthermore, DHH1 and SSD1 could functionally complement each other in the ade2 red colour pigment formation, hypersensitivity to SDS, growth on synthetic media and at high temperature . A triple mutant, delta dhh1 ssd1-d delta elm1, apparently had very fragile cell walls and could grow only in a medium supplemented with 1 M sorbitol.

FEMS Microbiol Lett, 1999 May 1, 174(1), 73 - 9
Growth requirements of pyruvate-decarboxylase-negative Saccharomyces cerevisiae; Flikweert MT et al.; Pyruvate-decarboxylase (Pdc)-negative Saccharomyces cerevisiae has been reported to grow in batch cultures on glucose-containing complex media, but not on defined glucose-containing media . By a combination of batch and chemostat experiments it is demonstrated that even in complex media, Pdc- S . cerevisiae does not exhibit prolonged growth on glucose . Pdc- strains do grow in carbon-limited cultures on defined media containing glucose-acetate mixtures . The acetate requirement for glucose-limited growth, estimated experimentally by continuously decreasing the acetate feed to chemostat cultures, matched the theoretical acetyl-CoA requirement for lipid and lysine synthesis, consistent with the proposed role of pyruvate decarboxylase in the synthesis of cytosolic acetyl-CoA.

Planta Med, 1999 Apr, 65(3), 261 - 3
Chitin synthase II inhibitory activity of ursolic acid, isolated from Crataegus pinnatifida; Jeong TS et al.; Two triterpenoid compounds, ursolic acid and uvaol, were isolated from Crataegus pinnatifida Bunge leaves . Ursolic acid inhibits chitin synthase II from S . cerevisiae with an IC50 value of 0.84 microgram/ml and the inhibition appears to be selective for chitin synthase II, whereas uvaol has no inhibitory activity up to 280 micrograms/ml . Oleanolic acid, alpha-hederin hydrate, and betulic acid inhibited the chitin synthase II activity under the same conditions with an IC50 of 5.6, 64.3, and 98.7 micrograms/ml, respectively.

Biochemistry, 1999 May 4, 38(18), 5864 - 71
The PLB2 gene of Saccharomyces cerevisiae confers resistance to lysophosphatidylcholine and encodes a phospholipase B/lysophospholipase; Fyrst H et al.; The PLB1 gene of Saccharomyces cerevisiae encodes a protein that demonstrates phospholipase B, lysophospholipase, and transacylase activities . Several genes with significant homology to PLB1 exist in the S . cerevisiae genome, raising the possibility that other proteins may contribute to the total phospholipase B/lysophospholipase/transacylase activities of the cell . We report the isolation of a previously uncharacterized gene that is highly homologous to PLB1 and that, when overexpressed, confers resistance to 1-palmitoyllysophosphatidylcholine . This gene, which is located adjacent to the PLB1 gene on the left arm of chromosome XIII and which we refer to as PLB2, encodes a phospholipase B/lysophospholipase . Unlike PLB1, this gene product does not contain significant transacylase activity . The PLB2 gene product shows lysophospholipase activity toward lysophosphatidylcholine, lysophosphatidylserine, and lysophosphatidylethanolamine . Whereas deletion of either PLB1 or PLB2 resulted in the loss of 80% of cellular lysophospholipase activity, a plb1/plb2 double deletion mutant is completely devoid of lysophospholipase activity toward the preferred substrate lysophosphatidylcholine . Overexpression of PLB2 was associated with an increase in total cellular phospholipase B/lysophospholipase activity, as well as the appearance of significant lysophospholipase activity in the medium . Moreover, overexpression of PLB2 was associated with saturation at a higher cell density, and an increase in total cellular phospholipid content, but no change in phospholipid composition or fatty acid incorporation into cellular lipids . Deletion of PLB2 was not lethal and did not result in alteration of membrane phospholipid composition or content . PLB2 gene expression was found to be maximal during exponential growth conditions and was decreased in late phase, in a manner similar to other genes involved in phospholipid metabolism.

Mol Cell, 1999 Apr, 3(4), 477 - 86
Chromosomal ARS1 has a single leading strand start site; Bielinsky AK et al.; Initiation sites for DNA synthesis in the chromosomal autonomously replicating sequence (ARS)1 of Saccharomyces cerevisiae were detected at the nucleotide level . The transition from discontinuous to continuous synthesis defines the origin of bidirectional replication (OBR), which mapped adjacent to the origin recognition complex binding site . To ascertain which sites represented starts for leading or lagging strands, we characterized DNA replication from ARS1 in a cdc9 (DNA ligase I) mutant, defective for joining Okazaki fragments . Leading strand synthesis in ARS1 initiated at only a single site, the OBR . Thus, replication in S . cerevisiae is not initiated stochastically by choosing one out of multiple possible sites but, rather, is a highly regulated process with one precise start point.

Mutagenesis, 1999 Mar, 14(2), 233 - 8
Bleomycin genotoxicity alteration by glutathione and cytochrome P-450 cellular content in respiratory proficient and deficient strains of Saccharomyces cerevisiae; Poli P et al.; The genotoxic effects of the antiblastic drug bleomycin were studied in the D7 strain of Saccharomyces cerevisiae and on its derivative mitochondrial mutant rho degree at different cellular concentrations of two drug metabolizing systems, glutathione (GSH) and cytochrome P-450 . Bleomycin mutagenic activity was evaluated as frequencies of mitotic gene conversion, reversion and total aberrations under different physiological conditions . In the D7 strain, petite mutant induction was also detected . This is important due to the role of the mitochondrial genome in cancer induction, ageing and degenerative diseases . Both strains showed higher convertant than revertant induction . At high cytochrome P-450 levels, bleomycin-induced gene conversion was enhanced in both strains although mitochondrial functionality showed a detoxicant role while cellular GSH content decreased the induction of convertants only in the respiratory proficient strain . Cell metabolic conditions, such as cell cycle, aerobic/hypoxic conditions of the cell and content of drug metabolizing enzymes, appeared to interact with the genotoxic effectiveness of bleomycin . Moreover, the usefulness of S.cerevisiae as a model organism for drug assessment for mutagenicity was emphasized.

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