S. cerevisiae

Mol Pharmacol, 2002 May, 61(5), 1146 - 53
Native CYP2C11: heterologous expression in Saccharomyces cerevisiae reveals a role for vacuolar proteases rather than the proteasome system in the degradation of this endoplasmic reticulum protein; Murray BP et al.; Cytochromes P450 (P450s) are hemoprotein enzymes committed to the metabolism of chemically diverse endo- and xenobiotics . They are anchored to the endoplasmic reticulum (ER) membrane with the bulk of their catalytic domain exposed to the cytosol, and thus they constitute excellent examples of integral monotopic ER proteins . Physiologically they are known to turn over asynchronously, but the determinants that trigger their proteolytic disposal and the pathways for such cellular disposal are not well defined . We recently showed that CYP3A4, the dominant human liver drug-metabolizing enzyme, and its rat liver orthologs undergo ubiquitin-dependent 26S proteasomal degradation not only after suicide inactivation, but also when CYP3A4 is expressed in Saccharomyces cerevisiae, presumably in its "native" form . The latter findings, obtained by the use of strains either with compromised proteasomal degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) or deficient in ubiquitin-conjugating enzymes (Ubc; UBC), revealed that this native monotopic P450 enzyme, in common with the polytopic HMGR, required the function of certain HRD (HMGR degradation) and UBC genes . In this study, we examined the degradation of CYP2C11, a male rat liver-specific P450, by heterologous expression in S . cerevisiae under comparable conditions . We report that unlike CYP3A4 and HMGR, the degradation of CYP2C11 in S . cerevisiae is independent of either HRD or UBC gene function, but it is largely dependent on vacuolar (lysosomal) proteolysis . These findings with two monotopic ER hemoproteins, CYP2C11 and CYP3A4, and the polytopic ER protein HMGR attest to the remarkable mechanistic diversity of cellular proteolytic disposal of ER proteins.

BMC Mol Biol . 2002 Apr 17;3(1):3 . Epub 2002 Apr 17.
Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex; Shou W et al.; BACKGROUND: In S . cerevisiae, the mitotic exit network (MEN) proteins, including the Polo-like protein kinase Cdc5 and the protein phosphatase Cdc14, are required for exit from mitosis . In pre-anaphase cells, Cdc14 is sequestered to the nucleolus by Net1 as a part of the RENT complex . When cells are primed to exit mitosis, the RENT complex is disassembled and Cdc14 is released from the nucleolus . RESULTS: Here, we show that Cdc5 is necessary to free nucleolar Cdc14 in late mitosis, that elevated Cdc5 activity provokes ectopic release of Cdc14 in pre-anaphase cells, and that the phosphorylation state of Net1 is regulated by Cdc5 during anaphase . Furthermore, recombinant Cdc5 and Xenopus Polo-like kinase can disassemble the RENT complex in vitro by phosphorylating Net1 and thereby reducing its affinity for Cdc14 . Surprisingly, although RENT complexes containing Net1 mutants (Net1(7m) and Net1(19m') lacking sites phosphorylated by Cdc5 in vitro are refractory to disassembly by Polo-like kinases in vitro, net1(7m) and net1(19m') cells grow normally and exhibit only minor defects in releasing Cdc14 during anaphase . However, net1(19m') cells exhibit a synergistic growth defect when combined with mutations in CDC5 or DBF2 (another MEN gene) . CONCLUSIONS: We propose that although Cdc5 potentially disassembles RENT by directly phosphorylating Net1, Cdc5 mediates exit from mitosis primarily by phosphorylating other targets . Our study suggests that Cdc5/Polo is unusually promiscuous and highlights the need to validate Cdc5/Polo in vitro phosphorylation sites by direct in vivo mapping experiments.

Biochem J, 2002 Jun 15, 364(Pt 3), 617 - 23
Two isoforms of Saccharomyces cerevisiae glutaredoxin 2 are expressed in vivo and localize to different subcellular compartments; Pedrajas JR et al.; Glutaredoxin (Grx)2 from Saccharomyces cerevisiae is a member of the two-cysteine (dithiol) subfamily of Grxs involved in the defence against oxidative stress in yeast . Recombinant yeast Grx2p, expressed in Escherichia coli, behaves as a 'classical' Grx that efficiently catalyses the reduction of hydroxyethyl disulphide by GSH . Grx2p also catalyses the reduction of GSSG by dihydrolipoamide with even higher efficiency . Western blot analysis of S . cerevisiae crude extracts identifies two isoforms of Grx2p of 15.9 and 11.9 kDa respectively . The levels of these two isoforms reach a peak during the exponential phase of growth in normal yeast extract/peptone/dextrose ('YPD') medium, with the long form predominating over the short one . From immunochemical analysis of subcellular fractions, it is shown that both isoforms are present in mitochondria, but only the short one is detected in the cytosolic fraction . On the other hand, only the long form is prominent in microsomes . Mitochondrial isoforms should represent the processed and unprocessed products of an open reading frame (YDR513W), with a putative start codon 99 bp upstream of the GRX2 start codon described thus far . These results indicate that GRX2 contains two in-frame start codons, and that translation from the first AUG results in a product that is targeted to mitochondria . The cytosolic form would result either by initiation from the second AUG, or by differential processing of one single translation product.

J Cell Sci, 2002 May 1, 115(Pt 9), 1895 - 905
Characterization of rat TOM70 as a receptor of the preprotein translocase of the mitochondrial outer membrane; Suzuki H et al.; We cloned a approximately 70 kDa rat mitochondrial outer membrane protein (OM70) with a sequence identity of 28.1% and 20.1% with N . crassa and S . cerevisiae Tom70, respectively . Even with this low sequence identity, however, the proteins share a remarkable structural similarity: they have 7-10 tetratricopeptide repeat motifs and are anchored to the outer membrane through the N-terminal transmembrane domain with the bulk portion located in the cytosol . Antibodies against OM70 inhibited import of preproteins, such as the ADP/ATP carrier and rTOM40, that use internal targeting signals but not the import of cleavable presequence-containing preproteins . Blue native gel electrophoresis and immunoprecipitation of digitoninsolubilized mitochondrial outer membranes revealed that OM70 was loosely associated with the approximately 400 kDa translocase complex of the mitochondrial outer membrane, which contains rTOM22 and rTOM40 . A yeast two-hybrid system demonstrated that OM70 interacted with rTOM20 and rTOM22 through the cytoplasmic domains . Thus, OM70 is a functional homologue of fungal Tom70 and functions as a receptor of the preprotein import machinery of the rat mitochondrial outer membrane . Furthermore, the N-terminal 66 residue region of OM70, which comprises a hydrophilic 41 residue N-terminal domain, a 22 residue transmembrane domain and three arginine residues, is sufficient to act as a mitochondria-targeting signal, and the arginine cluster is crucial for this function.

EMBO J, 2002 Apr 15, 21(8), 2009 - 18
Regulation of the ubiquitin-conjugating enzyme hHR6A by CDK-mediated phosphorylation; Sarcevic B et al.; Cell cycle progression in eukaryotes is mediated by phosphorylation of protein substrates by the cyclin-dependent kinases (CDKs) . We screened a cDNA library by solid-phase phosphorylation and isolated hHR6A as a CDK2 substrate . hHR6A is the human homologue of the product of the Saccharomyces cerevisiae RAD6/UBC2 gene, a member of the family of ubiquitin-conjugating enzymes . hHR6A is phosphorylated in vitro by CDK-1 and -2 on Ser120, a residue conserved in all hHR6A homologues, resulting in a 4-fold increase in its ubiquitin-conjugating activity . In vivo, hHR6A phosphorylation peaks during the G2/M phase of cell cycle transition, with a concomitant increase in histone H2B ubiquitylation . Mutation of Ser120 to threonine or alanine abolished hHR6A activity, while mutation to aspartate to mimic phosphorylated serine increased hHR6A activity 3-fold . Genetic complementation studies in S.cerevisiae demonstrated that hHR6A Ser120 is critical for cellular proliferation . This is the first study to demonstrate regulation of UBC function by phosphorylation on a conserved residue and suggests that CDK-mediated phosphorylation of hHR6A is an important regulatory event in the control of cell cycle progression.

J Cell Sci, 2002 Apr 15, 115(Pt 8), 1749 - 57
Robust G1 checkpoint arrest in budding yeast: dependence on DNA damage signaling and repair; Gerald JN et al.; Although most eukaryotes can arrest in G1 after ionizing radiation, the existence or significance of a G1 checkpoint in S . cerevisiae has been challenged . Previous studies of G1 response to chemical mutagens, X-ray or UV irradiation indicate that the delay before replication is transient and may reflect a strong intra-S-phase checkpoint . We examined the yeast response to double-stranded breaks in G1 using gamma irradiation . G1 irradiation induces repair foci on chromosome spreads and a Rad53 band shift characteristic of activation, which suggest an active DNA damage response . Consistent with a G1 arrest, bud emergence, spindle pole duplication and DNA replication are each delayed in a dose-dependent manner . Sensitivity to mating pheromone is prolonged to over 18 hours when G1 cells are lethally gamma or UV irradiated . Strikingly, G1 delay is the predominant response to continuous gamma irradiation at a dose that confers no loss of viability but delays cell division . Like the G2/M checkpoint, G1 delay is completely dependent on both RAD9 and RAD24 epistasis groups but independent of POL(epsilon) . Lethally irradiated rad9 mutants rapidly exit G1 but perform a slow S phase, whereas rad17 and rad24 mutants are completely arrest deficient . Distinct from gamma irradiation, G1 arrest after UV is RAD14 dependent, suggesting that DNA damage processing is required for checkpoint activation . Therefore, as in the yeast G2/M checkpoint response, free DNA ends and/or single-stranded DNA are necessary and sufficient to induce a bona fide G1 checkpoint arrest.

J Biol Chem, 2002 Jun 21, 277(25), 22822 - 8 Epub 2002 Apr 08.
Solution structure of the orphan PABC domain from Saccharomyces cerevisiae poly(A)-binding protein; Kozlov G et al.; We have determined the solution structure of the PABC domain from Saccharomyces cerevisiae Pab1p and mapped its peptide-binding site . PABC domains are peptide binding domains found in poly(A)-binding proteins (PABP) and are a subset of HECT-family E3 ubiquitin ligases (also known as hyperplastic discs proteins (HYDs)) . In mammals, the PABC domain of PABP functions to recruit several different translation factors to the mRNA poly(A) tail . PABC domains are highly conserved, with high specificity for peptide sequences of roughly 12 residues with conserved alanine, phenylalanine, and proline residues at positions 7, 10, and 12 . Compared with human PABP, the yeast PABC domain is missing the first alpha helix, contains two extra amino acids between helices 2 and 3, and has a strongly bent C-terminal helix . These give rise to unique peptide binding specificity wherein yeast PABC binds peptides from Paip2 and RF3 but not Paip1 . Mapping of the peptide-binding site reveals that the bend in the C-terminal helix disrupts binding interactions with the N terminus of peptide ligands and leads to greatly reduced binding affinity for the peptides tested . No high affinity or natural binding partners from S . cerevisiae could be identified by sequence analysis of known PABC ligands . Comparison of the three known PABC structures shows that the features responsible for peptide binding are highly conserved and responsible for the distinct but overlapping binding specificities.

Structure (Camb), 2002 Apr, 10(4), 569 - 79
Rab-subfamily-specific regions of Ypt7p are structurally different from other RabGTPases; Constantinescu AT et al.; The GTPase Ypt7p from S . cerevisiae is involved in late endosome-to-vacuole transport and homotypic vacuole fusion . We present crystal structures of the GDP- and GppNHp-bound conformation of Ypt7p solved at 1.35 and 1.6 A resolution, respectively . Despite the similarity of the overall structure to other Ypt/Rab proteins, Ypt7p displays small but significant differences . The Ypt7p-specific residues Tyr33 and Tyr37 cause a difference in the main chain trace of the RabSF2 region and form a characteristic surface epitope . Ypt7p*GppNHp does not display the helix alpha2, characteristic of the Ras-superfamily, but instead possess an extended loop L4/L5 . Due to insertions in loops L3 and L7, the neighboring RabSF1 and RabSF4 regions are different in their conformations to those of other Ypt/Rab proteins.

Appl Microbiol Biotechnol, 2002 Mar, 58(3), 370 - 7 Epub 2002 Jan 10.
Analysis of alkali-soluble glucan produced by Saccharomyces cerevisiae wild-type and mutants; Ha CH et al.; The alkali-soluble glucan of the yeast cell wall contains beta-(1,3)- and (1,6)-D-linkages and systemically enhances the immune system . To isolate Saccharomyces cerevisiae mutants producing glucan with a high degree of beta-(1,6)-D-glycosidic bonds, a wild-type strain was mutagenized with ultraviolet light . The mutants were then selected by treatment with 1.0 mg laminarinase, endo-beta-(1,3)-D-glucanase/ml . The alkali-soluble glucan was extracted by modified alkalysis followed by the Cetavlon method and concanavalin-A chromatography . The prepared alkali-soluble glucans from the wild-type and the mutants were compared with respect to yield and polymer structure using gas chromatography, 13C-NMR spectrometry, high performance liquid, and multi-angle laser light scattering and refractive index detectors . The results indicated that the S . cerevisiae mutants had ten-fold more alkali-soluble glucan than the wild-type . Structural analysis revealed that the alkali-soluble glucan from the mutants also had a higher degree of beta-(1,6)-D-linkage than that from the wild-type.

J Biol Chem, 2002 Jun 14, 277(24), 22103 - 6 Epub 2002 Apr 04.
SDT1/SSM1, a multicopy suppressor of S-II null mutant, encodes a novel pyrimidine 5'-nucleotidase; Nakanishi T et al.; SDT1 (suppressor of disruption of TFIIS 1, YGL224c, also known as SSM1, suppressor of S-II null mutant 1) is Saccharomyces cerevisiae gene identified as a multicopy suppressor of 6-azauracil sensitivity in a null mutant of the transcription elongation factor S-II . We found that overproduction of SDT1 caused hyposensitivity to not only 6-azauracil but also 5-fluorouracil and 5-fluorocytosine . This hyposensitivity was limited to pyrimidine derivatives, and no effect was observed for non-pyrimidine drugs including such clinically used anti-fungal drugs as amphotericin B and fluconazole . Purified recombinant SDT1 protein specifically dephosphorylated 5'-UMP and 5'-CMP . These results suggested that SDT1 conferred pyrimidine-specific hyposensitivity by dephosphorylating active metabolites of 6- or 5-modified pyrimidines, i.e . 6- or 5-modified UMP . This is the first description of a highly specific pyrimidine 5'-nucleotidase in S . cerevisiae.

Pac Symp Biocomput . 2002;:437-49.
Combining location and expression data for principled discovery of genetic regulatory network models; Hartemink AJ et al.; We develop principled methods for the automatic induction (discovery) of genetic regulatory network models from multiple data sources and data modalities . Models of regulatory networks are represented as Bayesian networks, allowing the models to compactly and robustly capture probabilistic multivariate statistical dependencies between the various cellular factors in these networks . We build on previous Bayesian network validation results by extending the validation framework to the context of model induction, leveraging heuristic simulated annealing search algorithms and posterior model averaging . Using expression data in isolation yields results inconsistent with location data so we incorporate genomic location data to guide the model induction process . We combine these two data modalities by allowing location data to influence the model prior and expression data to influence the model likelihood . We demonstrate the utility of this approach by discovering genetic regulatory models of thirty-three variables involved in S . cerevisiae pheromone response . The models we automatically generate are consistent with the current understanding regarding this regulatory network, but also suggest new directions for future experimental investigation.

Pac Symp Biocomput . 2002;:374-85.
Predicting the sub-cellular location of proteins from text using support vector machines; Stapley BJ et al.; We present an automatic method to classify the sub-cellular location of proteins based on the text of relevant medline abstracts . For each protein, a vector of terms is generated from medline abstracts in which the protein/gene's name or synonym occurs . A Support Vector Machine (SVM) is used to automatically partition the term space and to thus discriminate the textual features that define sub-cellular location . The method is benchmarked on a set of proteins of known sub-cellular location from S . cerevisiae . No prior knowledge of the problem domain nor any natural language processing is used at any stage . The method out-performs support vector machines trained on amino acid composition and has comparable performance to rule-based text classifiers . Combining text with protein amino-acid composition improves recall for some sub-cellular locations . We discuss the generality of the method and its potential application to a variety of biological classification problems.

Pac Symp Biocomput . 2002;:175-86.
Estimation of genetic networks and functional structures between genes by using Bayesian networks and nonparametric regression; Imoto S et al.; We propose a new method for constructing genetic network from gene expression data by using Bayesian networks . We use nonparametric regression for capturing nonlinear relationships between genes and derive a new criterion for choosing the network in general situations . In a theoretical sense, our proposed theory and methodology include previous methods based on Bayes approach . We applied the proposed method to the S . cerevisiae cell cycle data and showed the effectiveness of our method by comparing with previous methods.

Pac Symp Biocomput . 2002;:127-38.
Structure-based comparison of four eukaryotic genomes; Cline M et al.; The field of comparative genomics allows us to elucidate the molecular mechanisms necessary for the machinery of an organism by contrasting its genome against those of other organisms . In this paper, we contrast the genome of homo sapiens against C . Elegans, Drosophila melanogaster, and S . cerevisiae to gain insights on what structural domains are present in each organism . Previous work has assessed this using sequence-based homology recognition systems such as Pfam {1} and Interpro {2} . Here, we pursue a structure-based assessment, analyzing genomes according to domains in the SCOP structural domain dictionary . Compared to other eukaryotic genomes, we observe additional domains in the human genome relating to signal transduction, immune response, transport, and certain enzymes . Compared to the metazoan genomes, the yeast genome shows an absence of domains relating to immune response, cell-cell interactions, and cell signaling.

J Biol Chem, 2002 Jun 7, 277(23), 20205 - 13 Epub 2002 Mar 28.
Cloning and characterization of methenyltetrahydrofolate synthetase from Saccharomyces cerevisiae; Holmes WB et al.; The folate derivative 5-formyltetrahydrofolate (folinic acid; 5-CHO-THF) was discovered over 40 years ago, but its role in metabolism remains poorly understood . Only one enzyme is known that utilizes 5-CHO-THF as a substrate: 5,10-methenyltetrahydrofolate synthetase (MTHFS) . A BLAST search of the yeast genome using the human MTHFS sequence revealed a 211-amino acid open reading frame (YER183c) with significant homology . The yeast enzyme was expressed in Escherichia coli, and the purified recombinant enzyme exhibited kinetics similar to previously purified MTHFS . No new phenotype was observed in strains disrupted at MTHFS or in strains additionally disrupted at the genes encoding one or both serine hydroxymethyltransferases (SHMT) or at the genes encoding one or both methylenetetrahydrofolate reductases . However, when the MTHFS gene was disrupted in a strain lacking the de novo folate biosynthesis pathway, folinic acid (5-CHO-THF) could no longer support the folate requirement . We have thus named the yeast gene encoding methenyltetrahydrofolate synthetase FAU1 (folinic acid utilization) . Disruption of the FAU1 gene in a strain lacking both 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase isozymes (ADE16 and ADE17) resulted in a growth deficiency that was alleviated by methionine . Genetic analysis suggested that intracellular accumulation of the purine intermediate AICAR interferes with a step in methionine biosynthesis . Intracellular levels of 5-CHO-THF were determined in yeast disrupted at FAU1 and other genes encoding folate-dependent enzymes . In fau1 disruptants, 5-CHO-THF was elevated 4-fold over wild-type yeast . In yeast lacking MTHFS along with both AICAR transformylases, 5-CHO-THF was elevated 12-fold over wild type . 5-CHO-THF was undetectable in strains lacking SHMT activity, confirming SHMT as the in vivo source of 5-CHO-THF . Taken together, these results indicate that S . cerevisiae harbors a single, nonessential, MTHFS activity . Growth phenotypes of multiply disrupted strains are consistent with a regulatory role for 5-CHO-THF in one-carbon metabolism and additionally suggest a metabolic interaction between the purine and methionine pathways.

Yeast, 2002 Apr, 19(6), 509 - 20
Functional analysis of structural genes for NAD(+)-dependent formate dehydrogenase in Saccharomyces cerevisiae; Overkamp KM et al.; Co-consumption of formate by aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK 113-7D led to an increased biomass yield relative to cultures grown on glucose as the sole carbon and energy substrate . In this respect, this strain differed from two previously investigated S . cerevisiae strains, in which formate oxidation did not lead to an increased biomass yield on glucose . Enzyme assays confirmed the presence of a formate-inducible, cytosolic and NAD(+)-dependent formate dehydrogenase . To investigate whether this enzyme activity was entirely encoded by the previously reported FDH1 gene, an fdh1Delta null mutant was constructed . This mutant strain still contained formate dehydrogenase activity and remained capable of co-consumption of formate . The formate dehydrogenase activity in the mutant was demonstrated to be encoded by a second structural gene for formate dehydrogenase (FDH2) in S . cerevisiae CEN.PK 113-7D . FDH2 was highly homologous to FDH1 and consisted of a fusion of two open reading frames (ORFs) (YPL275w and YPL276w) reported in the S . cerevisiae genome databases . Sequence analysis confirmed that, in the database genetic background, the presence of two single-nucleotide differences led to two truncated ORFs rather than the full-length FDH2 gene present in strain CEN.PK 113-7D . In the latter strain background an fdh1Deltafdh2Delta double mutant lacked formate dehydrogenase activity and was unable to co-consume formate . Absence of formate dehydrogenase activity did not affect growth on glucose as sole carbon source, but led to a reduced biomass yield on glucose-formate mixtures . These findings are consistent with a role of formate dehydrogenase in the detoxification of exogenous formate .

Yeast, 2002 Apr, 19(6), 475 - 84
Increasing sulphite formation in Saccharomyces cerevisiae by overexpression of MET14 and SSU1; Donalies UE et al.; Saccharomyces cerevisiae produces sulphite as an intermediate product during the assimilatory reduction of sulphate to sulphide . Three genes, MET3, MET14 and MET16, are essential for this reduction . We investigated the level of transcription of these genes in strains of S . cerevisiae with high, medium and low sulphite formation . The level of MET14- and MET16-mRNA varied with sulphite production, whereas the level of MET3-mRNA was very weak in almost all strains . We also analysed the effect of overexpression of MET14 and MET16 on sulphite formation . Two strains with low sulphite production were transformed with high-copy plasmids containing either or both MET14 and MET16 . The overexpression of these two genes leads to a two- to three-fold sulphite formation . In addition, inactivation of MET10, encoding a subunit of the sulphite reductase, also leads to a distinct increase in sulphite formation; however, the cells became methionine auxotroph . The overexpression of SSU1, a gene encoding a putative sulphite pump, yields a slight increase in sulphite accumulation, whereas overexpression of SSU1, together with MET14, increases sulphite formation up to 10-fold . Furthermore, sulphite formation strongly depends on growth conditions, e.g . yeast transformants growing in wort produce much higher amounts of sulphite when compared to growth in minimal media . The addition of glucose can also increase the sulphite formation in strains overexpressing MET14 and/or SSU1 under oxygen-limiting conditions, while the addition of glucose has no significant effect under aerobic conditions .

Yeast, 2002 Mar 30, 19(5), 441 - 8
Tools for the study of genome rearrangements in laboratory and industrial yeast strains; Lockhart L et al.; In order to investigate the extent of genome rearrangements in laboratory and industrial yeast strains, a set of plasmids, containing ca . 300 bp fragments from highly conserved genes from S . cerevisiae, has been constructed . We chose three unique PCR products, each from a single gene, per chromosome: one from close to the centromere, and one from each chromosome end . Using these plasmids as probes to hybridize a Southern blot from a pulsed-field gel electrophoresis separation of the 16 yeast chromosomes, it is possible to identify large chromosomal rearrangements such as reciprocal translocations .

Mol Microbiol, 2002 Mar, 43(5), 1183 - 95
Control of chitin synthesis through Shc1p, a functional homologue of Chs4p specifically induced during sporulation; Sanz M et al.; The Saccharomyces cerevisiae SHC1 gene encodes a protein with a high homology to Chs4p, a positive regulator of chitin synthase III (CSIII) during vegetative growth . SHC1 is not expressed during vegetative growth but is strongly induced during sporulation as a mid-late gene . shc1/shc1 mutants do not show any defect in the total rate of sporulation and meiosis occurs normally . However, shc1/shc1 ascospores be-come highly permeable to DAPI, much more sensitive to glusulase treatment, and have very low levels of chitosan in their cell walls . All these observations indicate that Shc1p is required for proper maturation of the ascospore through its participation in the synthesis of the chitosan layer . Lack of SHC1 during sporulation can be partially compensated by over-expression of the CHS4 gene . During vegetative growth, SHC1 has no apparent function but, when ectopically overexpressed, it can substitute Chs4p as an activator of the CSIII activity; however, Shc1p fails to localize it properly, as Chs4p does . In conclusion, S . cerevisiae contains two functionally redundant genes in the control of CSIII activity: CHS4, whose function is restricted to vegetative growth because Chs4p is rapidly degraded during sporulation, and SHC1, whose function in cell wall ascospore assembly is transcriptionally restricted to the sporulation process.

BMC Genet . 2002 Mar 12;3(1):4.
Multiple telophase arrest bypassed (tab) mutants alleviate the essential requirement for Cdc15 in exit from mitosis in S . cerevisiae; Shou W et al.; BACKGROUND: The Mitotic Exit Network (MEN) proteins - including the protein kinase Cdc15 and the protein phosphatase Cdc14 - are essential for exit from mitosis in Saccharomyces cerevisiae . To identify downstream targets of the MEN, we sought telophase arrest bypassed (tab) mutations that bypassed the essential requirement for CDC15 . Previous studies identified net1(tab2-1) and CDC14(TAB6-1) as mutations in the RENT complex subunits Net1 and Cdc14, respectively, and revealed that the MEN acts by promoting release of Cdc14 from its nucleolar Net1 anchor during anaphase . However, the remaining tab mutants were not characterized . RESULTS: Fourteen out of fifteen tab mutants were mapped to three recessive (tab1-tab3) and three dominant (TAB5-TAB7) linkage groups . We show that net1(tab2-1) enables growth of tem1Delta, cdc15Delta, dbf2Delta dbf20Delta, and mob1Delta, but not cdc5Delta or cdc14Delta, arguing that whereas the essential task of the first four genes is to promote exit from mitosis, CDC5 possesses additional essential function(s) . net1(tab2-1) but not CDC14(TAB6-1) resulted in a high rate of chromosome loss, indicating that Net1 promotes accurate chromosome segregation in addition to its multiple known roles . Finally, TAB1 was shown to be MTR10, a gene encoding nuclear transport receptor/adaptor . In some of the tab mutants including mtr10(tab1-1), defective nuclear export of the ribosomal protein Rpl11b was observed . Furthermore, the transport-defective -31 allele of the karyopherin SRP1, but not the transport competent -49 allele, exhibited a tab phenotype . CONCLUSIONS: Transport-defective mutations in two karyopherins can bypass cdc15Delta, suggesting that the function of the MEN is to promote mitotic exit by regulating nuclear transport.

Mol Biochem Parasitol, 2002 Apr 9, 120(2), 187 - 94
Isolation of a Toxoplasma gondii cyclin by yeast two-hybrid interactive screen; Kvaal CA et al.; GAL4-based yeast two-hybrid cDNA libraries from Toxoplasma gondii RH strain were constructed and screened for interactors of a putative T . gondii cdc2-related kinase, TgCRK2 . A screen of 3.2 million transformants yielded a single yeast clone that harbored a protein fusion capable of specifically interacting with TgCRK2 . Sequencing revealed the cDNA insert (TgCYC1) had homology to the cyclin class of proteins . The TgCYC1 cDNA fragment was used to probe a conventional T . gondii cDNA library and a 2.65 kb cDNA coding for a predicted protein of 582 amino acids was obtained . Based on comparison with a 5'-RACE product from tachyzoite mRNA, the 2.65 kb cDNA for TgCYC1 appeared to be complete . TgCYC1 had the highest similarity to Plasmodium falciparum CYC1 and displayed sequence characteristics that place it in the cyclin H class of eukaryotic cyclins . In synchronous tachyzoite populations the level of TgCYC1 mRNA was unchanged indicating it is not cell cycle regulated at the mRNA level . TgCYC1 rescues the G(1)/S cyclin cell cycle defect in S . cerevisiae strain DL1 demonstrating that this apicomplexan cyclin can function in an established heterologous model system.

Genes Cells, 2002 Feb, 7(2), 99 - 113
Replication fork block protein, Fob1, acts as an rDNA region specific recombinator in S . cerevisiae; Johzuka K et al.; BACKGROUND: The analysis of homologous recombination in the tandemly repeating rDNA array of Saccharomyces cerevisiae should provide useful information about the stability of not only the rDNA repeat but also the abundant repeated sequences on higher eukaryotic genomes . However, the data obtained so far are not yet conclusive, due to the absence of a reliable assay for detecting products of recombination in the rDNA array . RESULTS: We developed an assay method to detect the products of unequal sister-chromatid recombination (marker-duplication products) in yeast rDNA . This assay, together with the circular rDNA detection assay, was used for the analysis . Marker-duplication occurred throughout the rDNA cluster, preferentially between nearby repeat units . The FOB1 and RAD52 genes were required for both types of recombinant formation . FOB1 showed a gene dosage effect on not only the amounts of both recombinants, but also on the copy number of the repeat . However, unlike the RAD52 gene, the FOB1 gene was not involved in homologous recombination in a non-rDNA locus . In addition, the marker-duplication products were drastically decreased in the mre11 mutant . CONCLUSION: Our data demonstrate that FOB1- and RAD52-dependent homologous recombination cause the gain and loss of a few copies of the rDNA unit, and this must be a basic mechanism responsible for amplification and reduction of the rDNA copy number . In addition, FOB1 may also play a role in the copy number regulation of rDNA tandem repeats.

EMBO J, 1983, 2(3), 353 - 9
Altered maturation of sequences at the 3' terminus of 5S gene transcripts in a Saccharomyces cerevisiae mutant that lacks a RNA processing endonuclease; Piper PW et al.; Sequences at the immediate 3' terminus of several eukaryotic primary transcripts, synthesised just before the termination of transcription, are often lost during RNA processing . The rna82.1 mutation in Saccharomyces cerevisiae appears to result in a deficiency of the endonuclease that removes such sequences from certain yeast transcripts . Some small RNAs of rna82.1 cells are a few nucleotides longer than their counterparts in wild-type S . cerevisiae . The 5S rRNAs made during very short pulse-labellings of the mutant have, relative to the mature 121 nucleotide 5S RNA of wild-type cells, an additional 7, 11 or 13 nucleotides at their 3' terminus . These 5S forms reveal sites upon 5S genes where transcription probably terminates in vivo . The extra nucleotides upon 5S RNAs in rna82.1 cells are lost very slowly by sequential removal from the 3' terminus . Through this 3'-5' exonuclease action the total 5S RNA of the mutant possesses several 3'-terminal sequences yet is mostly only 0-3 nucleotides longer than in wild-type S . cerevisiae . Just one or two of these 3'-terminal sequences serve as a substrate in vivo for a poly(A) polymerase since a small proportion of rna82.1 5S RNAs terminate in the sequence: CAAUCUUU(A)n.

Cell Stress Chaperones, 2002 Jan, 7(1), 47 - 54
The eukaryote chaperonin CCT is a cold shock protein in Saccharomyces cerevisiae; Somer L et al.; The eukaryotic Hsp60 cytoplasmic chaperonin CCT (chaperonin containing the T-complex polypeptide-1) is essential for growth in budding yeast, and mutations in individual CCT subunits have been shown to affect assembly of tubulin and actin . The present research focused mainly on the expression of the CCT subunits, CCTalpha and CCTbeta, in yeast (Saccharomyces cerevisiae) . Previous studies showed that, unlike most other chaperones, CCT in yeast does not undergo induction following heat shock . In this study, messenger ribonucleic acid (mRNA) and protein levels of CCT subunits following exposure to low temperatures, were examined . The Northern blot analysis indicated a 3- to 4-fold increase in mRNA levels of CCTalpha and CCTbeta genes after cold shock at 4 degrees C . Interestingly, Western blot analysis showed that cold shock induces an increase in the CCTalpha protein, which is expressed at 10 degrees C, but not at 4 degrees C . Transfer of 4 degrees C cold-shocked cells to 10 degrees C induced a 5-fold increase in the CCTalpha protein level . By means of fluorescent immunostaining and confocal microscopy, we found CCTalpha to be localized in the cortex and the cell cytoplasm of S . cerevisiae . Localization of CCTalpha was not affected at low temperatures . Co-localization of CCT and filaments of actin and tubulin was not observed by microscopy . The induction pattern of the CCTalpha protein suggests that expression of the chaperonin may be primarily important during the recovery from low temperatures and the transition to growth at higher temperatures, as found for other Hsps during the recovery phase from heat shock.

Proc Natl Acad Sci U S A, 2002 Mar 19, 99(6), 3690 - 4 Epub 2002 Mar 12.
Conflicting levels of selection in the accumulation of mitochondrial defects in Saccharomyces cerevisiae; Taylor DR et al.; The somatic accumulation of defective mitochondria causes human degenerative syndromes, senescence in fungi, and male sterility in plants . These diverse phenomena may result from conflicts between natural selection at different levels of organization . Such conflicts are fundamental to the evolution of cooperating groups, from cells to populations . We present a model in which defective mitochondrial genomes accumulate because of a within-cell replication advantage when among-cell selection for efficient respiration is relaxed . We tested the model by using experimental populations of the yeast Saccharomyces cerevisiae . We constructed yeast strains that were heteroplasmic for mitochondrial mutations that destroy the ability to respire (the petite phenotype) and followed the accumulation of mitochondrial defects in cultures with different effective population sizes . As predicted by the model, the inability to respire evolved only in small populations of S . cerevisiae, where among-cell selection favoring cells that can respire was reduced relative to within-cell selection favoring parasitic mitochondria . In a control experiment, mitochondrial point mutations that confer resistance to chloramphenicol showed no tendency to change in frequency under any culture conditions . The accumulation of some mitochondrial defects is therefore an evolutionary process, involving multiple levels of selection . The relative intensities of within- and among-cell selection may also explain the tissue specificity of human mitochondrial defects.

Proc Natl Acad Sci U S A, 2002 Mar 19, 99(6), 3740 - 5 Epub 2002 Mar 12.
Comprehensive analysis of CpG islands in human chromosomes 21 and 22; Takai D et al.; CpG islands are useful markers for genes in organisms containing 5-methylcytosine in their genomes . In addition, CpG islands located in the promoter regions of genes can play important roles in gene silencing during processes such as X-chromosome inactivation, imprinting, and silencing of intragenomic parasites . The generally accepted definition of what constitutes a CpG island was proposed in 1987 by Gardiner-Garden and Frommer {Gardiner-Garden, M . & Frommer, M . (1987) J . Mol . Biol . 196, 261-282} as being a 200-bp stretch of DNA with a C+G content of 50% and an observed CpG/expected CpG in excess of 0.6 . Any definition of a CpG island is somewhat arbitrary, and this one, which was derived before the sequencing of mammalian genomes, will include many sequences that are not necessarily associated with controlling regions of genes but rather are associated with intragenomic parasites . We have therefore used the complete genomic sequences of human chromosomes 21 and 22 to examine the properties of CpG islands in different sequence classes by using a search algorithm that we have developed . Regions of DNA of greater than 500 bp with a G+C equal to or greater than 55% and observed CpG/expected CpG of 0.65 were more likely to be associated with the 5' regions of genes and this definition excluded most Alu-repetitive elements . We also used genome sequences to show strong CpG suppression in the human genome and slight suppression in Drosophila melanogaster and Saccharomyces cerevisiae . This finding is compatible with the recent detection of 5-methylcytosine in Drosophila, and might suggest that S . cerevisiae has, or once had, CpG methylation.

Arch Microbiol, 2002 Apr, 177(4), 352 - 7 Epub 2002 Jan 31.
The SRD2 gene is involved in Saccharomyces cerevisiae morphogenesis; Canizares JV et al.; Saccharomyces cerevisiaepresents two alternative vegetative forms of growth, switching between yeast forms to pseudohyphal forms depending on the specific environmental conditions . To identify genes involved in cell wall morphogenesis, a haploid S . cerevisiae monomorphic mutant, W27, which exhibits pseudohyphal growth in the absence of the normal external signals that induce the formation of filamentous forms, was characterized . S . cerevisiaeW27 did not demonstrate agar-invasive growth, a characteristic of most filamentous strains . The mutant wall had no obvious alterations with respect to mannan and glucan content, but had three times more chitin than the parental strain . This produced an increase in the amount of proteins linked covalently to chitin . The same protein species, however, were released from the cell walls of the mutant and the parental strain . The W27 mutation was complemented with a genomic library and the SRD2/ECM23 gene was identified as the complementing ORF . Transformation of S . cerevisiaeW27 with the Ycplac33 vector carrying the SRD2 gene produced the original phenotype . These results suggest that the SRD2gene acts as a negative regulator of pseudohyphal growth.

J Virol, 2002 Apr, 76(7), 3359 - 64
Replication of bovine papillomavirus type 1 (BPV-1) DNA in Saccharomyces cerevisiae following infection with BPV-1 virions; Zhao KN et al.; Saccharomyces cerevisiae protoplasts exposed to bovine papillomavirus type 1 (BPV-1) virions demonstrated uptake of virions on electron microscopy . S . cerevisiae cells looked larger after exposure to BPV-1 virions, and cell wall regeneration was delayed . Southern blot hybridization of Hirt DNA from cells exposed to BPV-1 virions demonstrated BPV-1 DNA, which could be detected over 80 days of culture and at least 13 rounds of division . Two-dimensional gel analysis of Hirt DNA showed replicative intermediates, confirming that the BPV-1 genome was replicating within S . cerevisiae . Nicked circle, linear, and supercoiled BPV-1 DNA species were observed in Hirt DNA preparations from S . cerevisiae cells infected for over 50 days, and restriction digestion showed fragments hybridizing to BPV-1 in accord with the predicted restriction map for circular BPV-1 episomes . These data suggest that BPV-1 can infect S . cerevisiae and that BPV-1 episomes can replicate in the infected S . cerevisiae cells.

Curr Biol, 2002 Mar 5, 12(5), 389 - 95
Mks1 in concert with TOR signaling negatively regulates RTG target gene expression in S . cerevisiae; Dilova I et al.; The target of rapamycin (TOR) signaling pathway allows eukaryotic cells to regulate their growth in response to nutritional cues . In S . cerevisiae, TOR controls the expression of genes involved in several nutrient-responsive biosynthetic pathways . In particular, we have demonstrated that TOR negatively regulates a concise cluster of genes (termed RTG target genes) that encode mitochondrial and peroxisomal enzymes required for de novo amino acid biosynthesis . TOR acts in part by regulating the subcellular localization of the Rtg1/Rtg3 transcription factor complex . Nuclear entry of this complex requires the cytoplasmic protein Rtg2, whose precise function has remained ill defined . Here we establish that the likely role of Rtg2 is to antagonize the activity of another protein, Mks1, which we demonstrate is itself a negative regulator of RTG target gene activation . Results of epistasis analyses suggest that Rtg2 and Mks1 act downstream of TOR and upstream of Rtg1 and Rtg3 . Moreover, we find that Mks1 phosphorylation responds to TOR as well as to each of the Rtg1-Rtg3 proteins, indicative of complex regulation within this branch of TOR signaling . In addition to RTG target genes, microarray analysis reveals robust expression of lysine biosynthetic genes in mks1Delta cells, which depends on a functional RTG pathway . This latter result provides a molecular explanation for the previous identification of MKS1 as LYS80, a negative regulator of lysine biosynthesis {8}.

Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 2684 - 9
Interactions among the protein and RNA subunits of Saccharomyces cerevisiae nuclear RNase P; Houser-Scott F et al.; Ribonuclease P (RNase P) is a ubiquitous endoribonuclease that cleaves precursor tRNAs to generate mature 5' termini . Although RNase P from all kingdoms of life have been found to have essential RNA subunits, the number and size of the protein subunits ranges from one small protein in bacteria to at least nine proteins of up to 100 kDa . In Saccharomyces cerevisiae nuclear RNase P, the enzyme is composed of ten subunits: a single RNA and nine essential proteins . The spatial organization of these components within the enzyme is not yet understood . In this study we examine the likely binary protein-protein and protein-RNA subunit interactions by using directed two- and three-hybrid tests in yeast . Only two protein subunits, Pop1p and Pop4p, specifically bind the RNA subunit . Pop4p also interacted with seven of the other eight protein subunits . The remaining protein subunits all showed one or more specific protein-protein interactions with the other integral protein subunits . Of particular interest was the behavior of Rpr2p, the only protein subunit found in RNase P but not in the closely related enzyme, RNase MRP . Rpr2p interacts strongly with itself as well as with Pop4p . Similar interactions with self and Pop4p were also detected for Snm1p, the only unique protein subunit so far identified in RNase MRP . This observation is consistent with Snm1p and Rpr2p serving analogous functions in the two enzymes . This study provides a low-resolution map of the multisubunit architecture of the ribonucleoprotein enzyme, nuclear RNase P from S . cerevisiae.

J Biol Chem, 2002 May 24, 277(21), 18914 - 8 Epub 2002 Mar 04.
Subcellular localization of Aft1 transcription factor responds to iron status in Saccharomyces cerevisiae; Yamaguchi-Iwai Y et al.; The Aft1 transcription factor regulates the iron regulon in response to iron availability in Saccharomyces cerevisiae . Aft1 activates a battery of genes required for iron uptake under iron-starved conditions, whereas Aft1 function is inactivated under iron-replete conditions . Previously, we have shown that iron-regulated DNA binding by Aft1 is responsible for the controlled expression of target genes . Here we show that this iron-regulated DNA binding by Aft1 is not due to the change in the total expression level of Aft1 or alteration of DNA binding activity . Rather, nuclear localization of Aft1 responds to iron status, leading to iron-regulated expression of the target genes . We identified the nuclear export signal (NES)-like sequence in the AFT1 open reading frame . Mutation of the NES-like sequence causes nuclear retention of Aft1 and the constitutive activation of Aft1 function independent of the iron status of the cells . These results suggest that the nuclear export of Aft1 is critical for ensuring iron-responsive transcriptional activation of the Aft1 regulon and that the nuclear import/export systems are involved in iron sensing by Aft1 in S . cerevisiae.

Appl Environ Microbiol, 2002 Mar, 68(3), 1336 - 43
Saccharomyces cerevisiae URH1 (encoding uridine-cytidine N-ribohydrolase): functional complementation by a nucleoside hydrolase from a protozoan parasite and by a mammalian uridine phosphorylase; Mitterbauer R et al.; Nucleoside hydrolases catalyze the cleavage of N-glycosidic bonds in nucleosides, yielding ribose and the respective bases . While nucleoside hydrolase activity has not been detected in mammalian cells, many protozoan parasites rely on nucleoside hydrolase activity for salvage of purines and/or pyrimidines from their hosts . In contrast, uridine phosphorylase is the key enzyme of pyrimidine salvage in mammalian hosts and many other organisms . We show here that the open reading frame (ORF) YDR400w of Saccharomyces cerevisiae carries the gene encoding uridine hydrolase (URH1) . Disruption of this gene in a conditionally pyrimidine-auxotrophic S . cerevisiae strain, which is also deficient in uridine kinase (urk1), leads to the inability of the mutant to utilize uridine as the sole source of pyrimidines . Protein extracts of strains overexpressing YDR400w show increased hydrolase activity only with uridine and cytidine, but no activity with inosine, adenosine, guanosine, and thymidine as substrates, demonstrating that ORF YDR400w encodes a uridine-cytidine N-ribohydrolase . Expression of a homologous cDNA from a protozoan parasite (Crithidia fasciculata) in a ura3 urk1 urh1 mutant is sufficient to restore growth on uridine . Growth can also be restored by expression of a human uridine phosphorylase cDNA . Yeast strains expressing protozoan N-ribohydrolases or host phosphorylases could therefore become useful tools in drug screens for specific inhibitors.

Folia Biol (Praha), 2002, 48(1), 28 - 33
The regulatory region of Prague C v-Src inhibits the activity of the Schmidt-Ruppin A v-Src kinase domain; Brabek J et al.; Existing variants of the oncogene v-src differ in their transforming potential as well as in the range of their hosts . We compared the protein kinase activities of two Prague C v-Src variants (PRC and H19), reported to be of low oncogenic potential (Plachy et al., 1995), with the highly oncogenic Schmidt-Ruppin A v-Src (SRA) . We employed in vitro kinase assays of affinity-purified proteins expressed in rabbit reticulocyte lysate and in S . cerevisiae . In both systems used, the specific kinase activity of the Prague C v-Src kinases amounted to only ca 20% of the activity of SRA . This positions the PRC Src close to activated c-Src, despite the lack of the regulatory C-terminal tail in PRC . We constructed chimeras between PRC and SRA v-Src and tested them for specific kinase activity in S . cerevisiae . Remarkably, the regulatory N-terminal part of PRC, when fused to the SRA-derived kinase domain, lowered the chimeras' PK activity to ca 20%, suggesting that it is the regulatory part of PRC that is responsible for its low phosphotransferase activity.

J Cell Sci, 2002 Mar 1, 115(Pt 5), 881 - 6
Twinfilin, a molecular mailman for actin monomers; Palmgren S et al.; Twinfilin is a ubiquitous actin-monomer-binding protein that is composed of two ADF-homology domains . It forms a 1:1 complex with ADP-actin-monomers, inhibits nucleotide exchange on actin monomers and prevents assembly of the monomer into filaments . The two ADF-H domains in twinfilin probably have 3D structures similar to those of the ADF/cofilin proteins and overlapping actin-binding sites . Twinfilin also interacts with PtdIns(4,5)P(2), which inhibits its actin-monomer-sequestering activity in vitro . Mutations in the twinfilin gene result in defects in the bipolar budding pattern in S . cerevisiae and in a rough eye phenotype and aberrant bristle morphology in Drosophila melanogaster . These phenotypes are caused by the uncontrolled polymerization of actin filaments in the absence of twinfilin . Studies on budding yeast suggest that twinfilin contributes to actin filament turnover by localizing actin monomers, in their 'inactive' ADP-form, to the sites of rapid filament assembly . This is mediated through direct interactions between twinfilin and capping protein . Therefore, twinfilin might serve as a link between rapid actin filament depolymerization and assembly in cells.

J Biol Chem, 2002 May 17, 277(20), 18161 - 72 Epub 2002 Feb 26.
Accelerating the rate of disassembly of karyopherin.cargo complexes; Gilchrist D et al.; Transport of macromolecules across the nuclear pore complex (NPC) occurs in seconds and involves assembly of a karyopherin.cargo complex and docking to the NPC, translocation of the complex across the NPC via interaction with nucleoporins (Nups), and dissociation of the complex in the nucleoplasm . To identify rate-limiting steps in the Kap95p.Kap60p-mediated nuclear import pathway of Saccharomyces cerevisiae, we reconstituted key intermediate complexes and measured their rates of dissociation and affinities of interaction . We found that a nuclear localization signal-containing protein (NLS-cargo) dissociates slowly from Kap60p monomers and Kap60p.Kap95p heterodimers with half-lives (t(12)) of 7 and 73 min, respectively; that Kap60p and Kap60p.NLS-cargo complexes dissociate slowly from Kap95p (t(12) = 36 and 73 min, respectively); and that Kap95p.Kap60p.NLS-cargo complexes and Kap95p.Kap60p heterodimers dissociate rapidly from the nucleoporin Nup1p (t(12) < or = 21 s) and other Nups . A search for factors that accelerate disassembly of the long-lived intermediates revealed that Nup1p and Nup2p accelerate 16- and 19-fold the rate of dissociation of NLS-cargo from Kap60p.Kap95p heterodimers; that Gsp1p-GTP accelerates > or = 447-fold the rate of dissociation of Kap60p.NLS-cargo from Kap95p; and that Nup2p and the Cse1p.Gsp1p-GTP complex independently accelerate > or = 22- and > or = 39-fold the rate of dissociation of NLS-cargo from Kap60p . We suggest that Nup1p, Nup2p, Cse1p, and Gsp1p accelerate disassembly of Kap95p.Kap60p.NLS-cargo complexes by triggering allosteric mechanisms within Kaps that cause rapid release of binding partners . In that way, Nup1p, Nup2p, Cse1p, and Gsp1p may function as karyopherin release factors (or KaRFs) in the nuclear basket structure of the S . cerevisiae NPC.

J Mol Biol, 2002 Feb 22, 316(3), 489 - 99
The Saccharomyces cerevisiae histone acetyltransferase Gcn5 has a role in the photoreactivation and nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers in the MFA2 gene; Teng Y et al.; How DNA repair enzymes or complexes gain access to chromatin is still not understood . Here, we have studied the role of the S . cerevisiae histone acetyltransferase Gcn5 in photoreactivation (PR) and nucleotide excision repair (NER) at the level of the genome, the MFA2 and RPB2 genes, and at specific nucleotides within MFA2 . The deletion of GCN5 markedly reduced the PR and NER of UV-induced cyclobutane pyrimidine dimers in MFA2 but much less so in RPB2, whereas no detectable defect was seen for repair of the genome overall . In Delta(gcn5), the MFA2 mRNA level is reduced by fourfold, while transcription from RPB2 is reduced only to 80 % . These changes in transcription correlate with the changes in NER and PR found in the Delta(gcn5) mutant . However, changes in MFA2 transcription cannot account for the decrease in NER in the non-transcribed strand and the control region of MFA2 where global genome repair (GGR) operates . We conclude that the histone acetyltransferase Gcn5 influences PR and NER at MFA2 in both its transcribed and non-transcribed DNA, yet it has little effect on these processes for most of the yeast genome . As a result, we speculate that histone acetylation allows efficient access of the repair machinery to chromosomal DNA damages either indirectly via influencing transcription or directly via modifying chromatin structure irrespective of transcription .

Genetics, 2002 Feb, 160(2), 429 - 43
The cytoplasmic end of transmembrane domain 3 regulates the activity of the Saccharomyces cerevisiae G-protein-coupled alpha-factor receptor; Parrish W et al.; The binding of alpha-factor to its receptor (Ste2p) activates a G-protein-signaling pathway leading to conjugation of MATa cells of the budding yeast S . cerevisiae . We conducted a genetic screen to identify constitutively activating mutations in the N-terminal region of the alpha-factor receptor that includes transmembrane domains 1-5 . This approach identified 12 unique constitutively activating mutations, the strongest of which affected polar residues at the cytoplasmic ends of transmembrane domains 2 and 3 (Asn84 and Gln149, respectively) that are conserved in the alpha-factor receptors of divergent yeast species . Targeted mutagenesis, in combination with molecular modeling studies, suggested that Gln149 is oriented toward the core of the transmembrane helix bundle where it may be involved in mediating an interaction with Asn84 . These residues appear to play specific roles in maintaining the inactive conformation of the protein since a variety of mutations at either position cause constitutive receptor signaling . Interestingly, the activity of many mammalian G-protein-coupled receptors is also regulated by conserved polar residues (the E/DRY motif) at the cytoplasmic end of transmembrane domain 3 . Altogether, the results of this study suggest a conserved role for the cytoplasmic end of transmembrane domain 3 in regulating the activity of divergent G-protein-coupled receptors.

Bioresour Technol, 2002 Mar, 82(1), 79 - 85
Invertase and urease activities in the carotenogenic yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma); Persike DS et al.; Invertase and urease are enzyme entities highly associated with the cells of the astaxanthin-producer yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) during any stage of its cell growth cycle . In this study cellobiose was a more efficient carbon source than sucrose or its hexose counterparts for invertase expression . Extensive ultrasonication or abrasion with glass pearls were required in order to promote enzyme release . In contrast to the yeast whose growth declines above 27 degrees C, the released enzymes displayed a higher optimum temperature range when assayed in vitro . Isoforms from both enzymes could be resolved either by FPLC on DEAE-Sepharose or by an affinity approach on immobilized Concanavalin . The zymogram for invertase showed a pI somewhat less acidic than that of the similar enzyme from S . cerevisiae.

Genes Cells, 2002 Jan, 7(1), 11 - 7
Aurora-B phosphorylates Histone H3 at serine28 with regard to the mitotic chromosome condensation; Goto H et al.; BACKGROUND: Histone H3 (H3) phosphorylation plays important roles in mitotic chromosome condensation . We reported that H3 phosphorylation occurs at Ser28, as well as at Ser10 during mitosis, at least in mammals . Aurora B was recently demonstrated to be responsible for Ser10 phosphorylation in S . cerevisiae, C . elegans, Drosophila and Xenopus egg extract . RESULTS: We compared the distribution of Aurora-B with that of H3 phosphorylation . Aurora-B was primarily localized in the heterochromatin of late G2 phase cells, where only Ser10 phosphorylation was observed . The treatment of such cells with calyculin A induced Ser28 phosphorylation in the Aurora-B-localized area . During prophase to metaphase, Aurora-B was distributed in condensing chromosomes where Ser10 and Ser28 were phosphorylated . Aurora-B can phosphorylate H3-Ser10 and -Ser28 in nucleosomes in vitro . Transfection of a dominant-negative mutant of Aurora-B resulted in a reduction of H3 phosphorylation, not only at Ser10 but also Ser28, during mitosis . CONCLUSIONS: With regard to mitotic chromosome condensation, Aurora-B directly phosphorylated H3, not only at Ser10 but also at Ser28 . The level of Ser28 phosphorylation is diminished to undetectable levels by PP1 phosphatase prior to entry into mitosis.

Mol Pharmacol, 2002 Mar, 61(3), 659 - 66
Inactivation of the Saccharomyces cerevisiae SKY1 gene induces a specific modification of the yeast anticancer drug sensitivity profile accompanied by a mutator phenotype; Schenk PW et al.; The therapeutic potential of the highly active anticancer agent cisplatin is severely limited by the occurrence of cellular resistance . A better understanding of the molecular pathways involved in cisplatin-induced cell death could potentially indicate ways to overcome cellular unresponsiveness to the drug and thus lead to better treatment results . We used the budding yeast Saccharomyces cerevisiae as a model organism to identify and characterize novel genes involved in cisplatin-induced cell kill, and found that SKY1 (SR-protein-specific kinase from budding yeast) is a cisplatin sensitivity gene whose disruption conferred cisplatin resistance . In cross-resistance studies, we observed resistance of yeast sky1 Delta cells (i.e., cells from which the SKY1 gene had been disrupted) to cisplatin, carboplatin (but not oxaliplatin), doxorubicin and daunorubicin, and hypersensitivity to cadmium chloride and 5-fluorouracil . Furthermore, these cells did not display reduced platinum accumulation, DNA platination or doxorubicin accumulation, indicating that the resistance is unrelated to decreased drug import or increased drug export . Based on the modification of the anticancer drug sensitivity profile and our finding that sky1 Delta cells display a mutator phenotype, we propose that Sky1p might play a significant role in specific repair and/or tolerance pathways . Disruption of the S . cerevisiae SKY1 gene would thus result in deregulation of such mechanisms and, consequently, lead to altered drug sensitivity.

Mol Biol Cell, 2002 Feb, 13(2), 503 - 14
Pag1p, a novel protein associated with protein kinase Cbk1p, is required for cell morphogenesis and proliferation in Saccharomyces cerevisiae; Du LL et al.; Protein kinases in the Cot-1/Orb6/Ndr/Warts family are important regulators of cell morphogenesis and proliferation . Cbk1p, a member of this family in Saccharomyces cerevisiae, has previously been shown to be required for normal morphogenesis in vegetatively growing cells and in haploid cells responding to mating pheromone . A mutant of PAG1, a novel gene in S . cerevisiae, displayed defects similar to those of cbk1 mutants . pag1 and cbk1 mutants share a common set of suppressors, including the disruption of SSD1, a gene encoding an RNA binding protein, and the overexpression of Sim1p, an extracellular protein . These genetic results suggest that PAG1 and CBK1 act in the same pathway . Furthermore, we found that Pag1p and Cbk1p localize to the same polarized peripheral sites and that they coimmunoprecipitate with each other . Pag1p is a conserved protein . The homologs of Pag1p in other organisms are likely to form complexes with the Cbk1p-related kinases and function with those kinases in the same biological processes.

FEBS Lett, 2002 Feb 13, 512(1-3), 213 - 7
Different effects of calnexin deletion in Saccharomyces cerevisiae on the secretion of two glycosylated amyloidogenic lysozymes; Song Y et al.; Both glycosylated amyloidogenic lysozymes I55T/G49N and D66H/G49N were expressed in wild-type and calnexin-disrupted Saccharomyces cerevisiae . The secretion amounts of mutant I55T/G49N were almost similar in both wild-type and calnexin-disrupted S . cerevisiae . In contrast, the secretion of mutant D66H/G49N greatly increased in calnexin-disrupted S . cerevisiae, while the secretion was very low in the wild-type strain . In parallel, the induction level of the molecular chaperones BiP and PDI located in the endoplasmic reticulum (ER) was investigated when these glycosylated amyloidogenic lysozymes were expressed in wild-type and calnexin-disrupted S . cerevisiae . The mRNA concentrations of BiP and PDI were evidently increased when mutant lysozyme D66H/G49N was expressed in calnexin-disrupted S . cerevisiae, while they were not so increased when I55T/G49N mutant was expressed . This observation indicates that the conformation of mutant lysozyme D66H/G49N was less stable in the ER, thus leading to the higher-level expression of ER molecular chaperones via the unfolded protein response pathway . This suggests that glycosylated amyloidogenic lysozyme I55T/G49N may have a relatively stable conformation in the ER, thus releasing it from the quality control of calnexin compared with mutant lysozyme D66H/G49N.

Plant J, 2002 Jan, 29(2), 225 - 35
Trehalose-6-phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation; Eastmond PJ et al.; Despite the recent discovery that trehalose synthesis is widespread in higher plants very little is known about its physiological significance . Here we report on an Arabidopsis mutant (tps1), disrupted in a gene encoding the first enzyme of trehalose biosynthesis (trehalose-6-phosphate synthase) . The tps1 mutant is a recessive embryo lethal . Embryo morphogenesis is normal but development is retarded and stalls early in the phase of cell expansion and storage reserve accumulation . TPS1 is transiently up-regulated at this same developmental stage and is required for the full expression of seed maturation marker genes (2S2 and OLEOSN2) . Sucrose levels also increase rapidly in seeds during the onset of cell expansion . In Saccharomyces cerevisiae trehalose-6-phosphate (T-6-P) is required to regulate sugar influx into glycolysis via the inhibition of hexokinase and a deficiency in TPS1 prevents growth on sugars (Thevelein and Hohmann, 1995) . The growth of Arabidopsis tps1-1 embryos can be partially rescued in vitro by reducing the sucrose level . However, T-6-P is not an inhibitor of AtHXK1 or AtHXK2 . Nor does reducing hexokinase activity rescue tps1-1 embryo growth . Our data establish for the first time that an enzyme of trehalose metabolism is essential in plants and is implicated in the regulation of sugar metabolism/embryo development via a different mechanism to that reported in S . cerevisiae.

Oncogene, 2002 Jan 21, 21(4), 522 - 31
Telomere maintenance without telomerase; Lundblad V; Recombination-dependent maintenance of telomeres, first discovered in budding yeast, has revealed an alternative pathway for telomere maintenance that does not require the enzyme telomerase . Experiments conducted in two budding yeasts, S . cerevisiae and K . lactis, have shown recombination can replenish terminal G-rich telomeric tracts that would otherwise shorten in the absence of telomerase, as well as disperse and amplify sub-telomeric repeat elements . Investigation of the genetic requirements for this process have revealed that at least two different recombination pathways, defined by RAD50 and RAD51, can promote telomere maintenance . Although critically short telomeres are very recombinogenic, recombination among telomeres that have only partially shortened in the absence of telomerase can also contribute to telomerase-independent survival . These observations provide new insights into the mechanism(s) by which recombination can restore telomere function in yeast, and suggest future experiments for the investigation of potentially similar pathways in human cells.

Antimicrob Agents Chemother, 2002 Mar, 46(3), 787 - 96
Relationship between chloroquine toxicity and iron acquisition in Saccharomyces cerevisiae; Emerson LR et al.; Chloroquine is one of the most effective antimalarials, but resistance to it is becoming widespread . However, we do not fully understand either the drug's mode of action or the mechanism of resistance . In an effort to expand our understanding of the mechanism of action and resistance associated with chloroquine, we used Saccharomyces cerevisiae as a model eukaryotic system . To aid in the discovery of potential drug targets we applied the transcriptional profiling method to identify genes transcriptionally responsive to chloroquine treatment in S . cerevisiae . Among the genes that were differentially expressed with chloroquine treatment were a number of metal transporters involved in iron acquisition (SIT1, ARN2, ARN4, and SMF2) . These genes exhibit similar expression patterns, and several are known to be regulated by AFT1, a DNA binding protein, which responds to iron levels in the cell . We investigated the role of chloroquine in iron metabolism by using a variety of approaches, including pharmacological, genetic, and biochemical techniques . For these experiments, we utilized yeast lacking the major iron uptake pathways (FET3 and FET4) and yeast deficient in SIT1, encoding the major up-regulated iron siderophore transporter . Our experiments show that yeast genetically or environmentally limited in iron availability has increased sensitivity to chloroquine in pharmacological assays and that the addition of iron rescues these cells from chloroquine killing . 55FeCl3 accumulation was inhibited in the presence of chloroquine, and kinetic analysis demonstrated that inhibition was competitive . These results are consistent with deprivation of iron as a mechanism of chloroquine killing in yeast.

Mol Microbiol, 2002 Jan, 43(1), 227 - 37
Role of the ribosomal stalk components in the resistance of Aspergillus fumigatus to the sordarin antifungals; Santos C et al.; Aspergillus fumigatus, an important human nosocomial pathogen, is resistant to sordarin derivatives, a new family of antifungals that inhibit protein synthesis by interaction with the EF-2-ribosomal stalk complex . To explore the role of the A . fumigatus ribosome in the resistance mechanism, the fungal stalk proteins were biochemically and genetically characterized and expressed in the sensitive Saccharomyces cerevisiae . Two acidic phosphoproteins homologous to the 12 kDa P1 and P2 proteins described in other organisms were found together with the 34 kDa P0 protein, the third stalk component . The genes encoding each fungal stalk protein were expressed in mutant S . cerevisiae strains lacking the equivalent proteins . Both AfP1 and AfP2 proteins interact with their yeast counterparts of the opposite type and bind to the ribosomal particles in the presence of either the S . cerevisiae or the A . fumigatus P0 protein . The A . fumigatus acidic phosphoproteins did not alter the yeast ribosome sordarin sensitivity . On the contrary, the presence of the fungal P0 induces in vivo and in vitro resistance to sordarin derivatives when present in the yeast ribosome . The mutations A117-->E, P122-->R and G124-->V in A . fumigatus P0 reduce the resistance capacity of the protein . An S . cerevisiae strain with the complete ribosomal stalk of A . fumigatus was obtained, which could be useful for the screening of new antifungals against this pathogenic fungus.

Bioinformatics, 2002 Feb, 18(2), 287 - 97
Inference of a genetic network by a combined approach of cluster analysis and graphical Gaussian modeling; Toh H et al.; MOTIVATION: Recent advances in DNA microarray technologies have made it possible to measure the expression levels of thousands of genes simultaneously under different conditions . The data obtained by microarray analyses are called expression profile data . One type of important information underlying the expression profile data is the 'genetic network,' that is, the regulatory network among genes . Graphical Gaussian Modeling (GGM) is a widely utilized method to infer or test relationships among a plural of variables . RESULTS: In this study, we developed a method combining the cluster analysis with GGM for the inference of the genetic network from the expression profile data . The expression profile data of 2467 Saccharomyces cerevisiae genes measured under 79 different conditions (Eisen et al., PROC: Natl Acad . Sci . USA, 95, 14683-14868, 1998) were used for this study . At first, the 2467 genes were classified into 34 clusters by a cluster analysis, as a preprocessing for GGM . Then, the expression levels of the genes in each cluster were averaged for each condition . The averaged expression profile data of 34 clusters were subjected to GGM, and a partial correlation coefficient matrix was obtained as a model of the genetic network of S . cerevisiae . The accuracy of the inferred network was examined by the agreement of our results with the cumulative results of experimental studies.

J Cell Sci, 2002 Jan 15, 115(Pt 2), 421 - 31
Three proteins required for early steps in the protein secretory pathway also affect nuclear envelope structure and cell cycle progression in fission yeast; Matynia A et al.; The Ran GTPase is an essential protein that has multiple functions in eukaryotic cells . Fission yeast cells in which Ran is misregulated arrest after mitosis with condensed, unreplicated chromosomes and abnormal nuclear envelopes . The fission yeast sns mutants arrest with a similar cell cycle block and interact genetically with the Ran system . sns-A10, sns-B2 and sns-B9 have mutations in the fission yeast homologues of S . cerevisiae Sar1p, Sec31p and Sec53p, respectively, which are required for the early steps of the protein secretory pathway . The three sns mutants accumulate a normally secreted protein in the endoplasmic reticulum (ER), have an increased amount of ER membrane, and the ER/nuclear envelope lumen is dilated . Neither a post-ER block in the secretory pathway, nor ER proliferation caused by overexpression of an integral ER membrane protein, results in a cell cycle-specific defect . Therefore, the arrest seen in sns-A10, sns-B2 and sns-B9 is most likely due to nuclear envelope defects that render the cells unable to re-establish the interphase organization of the nucleus after mitosis . As a consequence, these mutants are unable to decondense their chromosomes or to initiate of the next round of DNA replication.

Biometals, 2001 Sep-Dec, 14(3-4), 251 - 70
Eukaryotic zinc transporters and their regulation; Gaither LA et al.; The last ten years have witnessed major advances in our understanding of zinc transporters and their regulation in eukaryotic organisms . Two families of transporters, the ZIP (Zrt-, Irt-like Protein) and CDF (Cation Diffusion Facilitator) families, have been found to play a number of important roles in zinc transport . These are ancient gene families that span all phylogenetic levels . The characterized members of each group have been implicated in the transport of metal ions, frequently zinc, across lipid bilayer membranes . This remarkable conservation of function suggests that other, as yet uncharacterized members of the family, will also be involved in metal ion transport . Many of the ZIP family transporters are involved in cellular zinc uptake and at least one member, the Zrt3 transporter of S . cerevisiae, transports stored zinc out of an intracellular compartment during adaptation to zinc deficiency . In contrast, CDF family members mediate zinc efflux out of cells or facilitate zinc transport into intracellular compartments for detoxification and/or storage . The activity of many of these transporters is regulated in response to zinc through transcriptional and post-transcriptional mechanisms to maintain zinc homeostasis at both the cellular and organismal levels.

J Mol Biol, 2002 Feb 8, 316(1), 35 - 49
Characterization of two highly similar Rad51 homologs of Physcomitrella patens; Ayora S et al.; The moss Physcomitrella patens, which is a land plant with efficient homologous recombination, encodes two Rad51 proteins (PpaRad51.1 and PpaRad51.2) . The PpaRad51.1 and PpaRad51.2 proteins, which share 94 % identity between them, interact with themselves and with each other . Both proteins bind ssDNA and dsDNA in a Mg(2+) and pH-dependent manner, with a stoichiometry of one PpaRad51.1 monomer per 3(+/-1) nt or bp and one PpaRad51.2 monomer per 1(+/-0.5) nt or bp, respectively . At neutral pH, a 1.6-fold excess of both proteins is required for ssDNA and dsDNA binding . PpaRad51.1 and PpaRad51.2 show ssDNA-dependent ATPase activity and efficiently promote strand annealing in a nucleotide-independent but in a Mg(2+)-dependent manner . Both proteins promote joint-molecule formation, DNA strand invasion and are able to catalyse strand exchange in the presence of Mg(2+) and ATP . No further increase in the activities is observed when both proteins are present in the same reaction . None of the PpaRad51 gene products complement the DNA repair and recombination phenotype of Saccharomyces cerevisiae rad51delta mutants . However, PpaRad51.1 confers a dominant-negative DNA repair phenotype, and both PpaRad51 proteins reduce the levels of double-strand break-induced recombination when overexpressed in S . cerevisiae wt cells . These results suggest that both PpaRad51 proteins are bona fide Rad51 proteins that may contribute, in a different manner, to homologous recombination, and that they might replace ScRad51 in a hypothetical yeast protein complex inactivating different functions required for recombinational repair .

Mol Cell Biochem, 2001 Nov, 227(1-2), 113 - 7
Mutation at the CK2 phosphorylation site on Cdc28 affects kinase activity and cell size in Saccharomyces cerevisiae; Russo GL et al.; We have recently reported that protein kinase CK2 phosphorylates both in vivo and in vitro residue serine-46 of the cell cycle regulating protein Cdc28 of budding yeast Saccharomyces cerevisiae, confirming a previous observation that the same site is phosphorylated in Cdc2/Cdk1, the human homolog of Cdc28 . In addition, S . cerevisiae in which serine-46 of Cdc28 has been mutated to alanine show a decrease of 33% in both cell volume and protein content, providing the genetic evidence that CK2 is involved in the regulation of budding yeast cell division cycle, and suggesting that this regulation may be brought about in G1 phase of the mammalian cell cycle . Here, we extended this observation reporting that the mutation of serine-46 of Cdc28 to glutamic acid doubles, at least in vitro, the H1-kinase activity of the Cdc28/cyclin A complex . Since this mutation has only little effects on the cell size of the cells, we hypothesize multiple roles of yeast CK2 in regulating the G1 transition in budding yeast.

Hum Mol Genet, 2002 Feb 1, 11(3), 217 - 27
Assembly and iron-binding properties of human frataxin, the protein deficient in Friedreich ataxia; Cavadini P et al.; Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused by a deficiency of frataxin, a conserved mitochondrial protein of unknown function . Mitochondrial iron accumulation, loss of iron-sulfur cluster-containing enzymes and increased oxidative damage occur in yeast and mouse frataxin-depleted mutants as well as tissues and cell lines from FRDA patients, suggesting that frataxin may be involved in export of iron from the mitochondria, synthesis of iron-sulfur clusters and/or protection from oxidative damage . We have previously shown that yeast frataxin has structural and functional features of an iron storage protein . In this study we have investigated the function of human frataxin in Escherichia coli and Saccharomyces cerevisiae . When expressed in E.coli, the mature form of human frataxin assembles into a stable homopolymer that can bind approximately 10 atoms of iron per molecule of frataxin . The iron-loaded homopolymer can be detected on non-denaturing gels by either protein or iron staining demonstrating a stable association between frataxin and iron . As analyzed by gel filtration and electron microscopy, the homopolymer consists of globular particles of approximately 1 MDa and ordered rod-shaped polymers of these particles that accumulate small electron-dense cores . When the human frataxin precursor is expressed in S.cerevisiae, the mitochondrially generated mature form is separated by gel filtration into monomer and a high molecular weight pool of >600 kDa . A high molecular weight pool of frataxin is also present in mouse heart indicating that frataxin can assemble under native conditions . In radiolabeled yeast cells, human frataxin is recovered by immunoprecipitation with approximately five atoms of (55)Fe bound per molecule . These findings suggest that FRDA results from decreased mitochondrial iron storage due to frataxin deficiency which may impair iron metabolism, promote oxidative damage and lead to progressive iron accumulation.

Curr Biol, 2002 Jan 22, 12(2), 165 - 70
Evidence that Set1, a factor required for methylation of histone H3, regulates rDNA silencing in S . cerevisiae by a Sir2-independent mechanism; Bryk M et al.; Several types of histone modifications have been shown to control transcription . Recent evidence suggests that specific combinations of these modifications determine particular transcription patterns . The histone modifications most recently shown to play critical roles in transcription are arginine-specific and lysine-specific methylation . Lysine-specific histone methyltransferases all contain a SET domain, a conserved 130 amino acid motif originally identified in polycomb- and trithorax-group proteins from Drosophila . Members of the SU(VAR)3-9 family of SET-domain proteins methylate K9 of histone H3 . Methylation of H3 has also been shown to occur at K4 . Several studies have suggested a correlation between K4-methylated H3 and active transcription . In this paper, we provide evidence that K4-methylated H3 is required in a negative role, rDNA silencing in Saccharomyces cerevisiae . In a screen for rDNA silencing mutants, we identified a mutation in SET1, previously shown to regulate silencing at telomeres and HML . Recent work has shown that Set1 is a member of a complex and is required for methylation of K4 of H3 at several genomic locations . In addition, we demonstrate that a K4R change in H3, which prevents K4 methylation, impairs rDNA silencing, indicating that Set1 regulates rDNA silencing, directly or indirectly, via H3 methylation . Furthermore, we present several lines of evidence that the role of Set1 in rDNA silencing is distinct from that of the histone deacetylase Sir2 . Together, these results suggest that Set1-dependent H3 methylation is required for rDNA silencing in a Sir2-independent fashion.

Yeast, 2002 Feb, 19(3), 241 - 56
Analysis of the RAP1 protein binding to homogeneous telomeric repeats in Saccharomyces castellii; Wahlin J et al.; The repressor activator protein 1 (RAP1) plays a role in telomere structure and function inS . cerevisiae . Here, the RAP1 homologue was identified and cloned from the budding yeast Saccharomyces castellii (scasRAP1) . The scasRAP1 gene encodes a protein of 826 amino acids and shares an overall high degree of similarity with the S . cerevisiae RAP1 (scerRAP1) . We demonstrate that the scasRAP1 is able to complement scerRAP1 in temperature-sensitive S . cerevisiae strains and is able to function as a regulator to maintain the original telomere lengths . Binding analyses of the E . coli-expressed scasRAP1 protein demonstrate that it needs two consecutive telomeric repeats in order to bind the S . castellii telomeric DNA sequences, and that it binds adjacent sites having a 16 bp centre-to-centre spacing . The binding affinity to telomeric DNA of several other yeasts is similar to that of scerRap1p . However, in contrast to scerRap1p, scasRap1p was found to bind the human telomeric sequence . Moreover, the scasRap1p was found to incorporate a variant repeat in its binding to the otherwise homogeneous telomeric DNA of S . castellii . This ability to bind various sites differing in DNA sequence indicates a high degree of adjustability in the binding of scasRap1p to DNA .

Yeast, 2002 Feb, 19(3), 225 - 31
Overproduction of pentose phosphate pathway enzymes using a new CRE-loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae; Johansson B et al.; Two new vectors are described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3 . The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator . The S . cerevisiae genes RKI1, RPE1, TAL1 and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in overexpression of the genes . S . cerevisiae TMB 3026, simultaneously overexpressing the RKI1, RPE1, TAL1 and TKL1 genes, was created by successive integrations and removal of the loxP-zeocin-loxP cassette using pCRE3 . The 2mu-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers . pCRE3 proved to be easily cured without active counter-selection . The zeocin marker is present on both the pB3 PGK and on pCRE3, so that screening for zeocin sensitivity indicates both chromosomal marker loss and loss of the pCRE3 vector . This feature saves time, since only one screening step is needed between successive chromosomal integrations . Marker recycling did not lead to increased zeocin resistance, indicating that the zeocin marker could be used for more than four rounds of transformation . The use of the CRE/loxP system proved to be a practical strategy to overexpress multiple genes without exhausting available markers .

Nucleic Acids Res, 2001 Dec 15, 29(24), 4935 - 40
Uracil-DNA glycosylase-deficient yeast exhibit a mitochondrial mutator phenotype; Chatterjee A et al.; Mutations in mitochondrial DNA (mtDNA) have been reported in cancer and are involved in the pathogenesis of many mitochondrial diseases . Uracil-DNA glycosylase, encoded by the UNG1 gene in Saccharomyces cerevisiae, repairs uracil in DNA formed due to deamination of cytosine . Our study demonstrates that inactivation of the UNG1 gene leads to at least a 3-fold increased frequency of mutations in mtDNA compared with the wild-type . Using a Ung1p-green fluorescent protein (GFP) fusion construct, we demonstrate that yeast yUng1-GFP protein localizes to both mitochondria and the nucleus, indicating that Ung1p must contain both a mitochondrial localization signal (MLS) and a nuclear localization signal . Our study reveals that the first 16 amino acids at the N-terminus contain the yUng1p MLS . Deletion of 16 amino acids resulted in the yUng1p-GFP fusion protein being transported to the nucleus . We also investigated the intracellular localization of human hUng1p-GFP in yeast . Our data indicate that hUng1p-GFP predominantly localizes to the mitochondria . Further analysis identified the N-terminal 16 amino acids as important for localization of hUng1 protein into the mitochondria . Expression of both yeast and human UNG1 cDNA suppressed the frequency of mitochondrial mutation in UNG1-deficient cells . However, expression of yUNG1 in wild-type cells increased the frequency of mutations in mtDNA, suggesting that elevated expression of Ung1p is mutagenic . An increase in the frequency of mitochondrial mutants was also observed when hUNG1 site-directed mutants (Y147C and Y147S) were expressed in mitochondria . Our study suggests that deamination of cytosine is a frequent event in S.cerevisiae mitochondria and both yeast and human Ung1p repairs deaminated cytosine in mitochondria.

Mol Genet Genomics, 2002 Jan, 266(5), 711 - 9 Epub 2001 Nov 07.
Subcellular localization of the homocitrate synthase in Penicillium chrysogenum; Banuelos O et al.; There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway . The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS(-)) of the Wis 54-1255 pyrG strain . The three mutants named HS1(-), HS2(-) and HS3(-) all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P . chrysogenum . The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria . Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase . Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety . Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P . chrysogenum; in S . cerevisiae the enzyme is targeted to the nucleus . The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P . chrysogenum . The difference in localization of homocitrate synthase between P . chrysogenum and S . cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S . cerevisiae but not in P . chrysogenum.

Genetics, 2002 Jan, 160(1), 75 - 82
Sls1p is a membrane-bound regulator of transcription-coupled processes involved in Saccharomyces cerevisiae mitochondrial gene expression; Bryan AC et al.; Mitochondrial translation is largely membrane-associated in S . cerevisiae . Recently, we discovered that the matrix protein Nam1p binds the amino-terminal domain of yeast mtRNA polymerase to couple translation and/or RNA-processing events to transcription . To gain additional insight into these transcription-coupled processes, we performed a genetic screen for genes that suppress the petite phenotype of a point mutation in mtRNA polymerase (rpo41-R129D) when overexpressed . One suppressor identified in this screen was SLS1, which encodes a mitochondrial membrane protein required for assembly of respiratory-chain enzyme complexes III and IV . The mtRNA-processing defects associated with the rpo41-R129D mutation were corrected in the suppressed strain, linking Sls1p to a pathway that includes mtRNA polymerase and Nam1p . This was supported by the observation that SLS1 overexpression rescued the petite phenotype of a NAM1 null mutation . In contrast, overexpression of Nam1p did not rescue the petite phenotype of a SLS1 null mutation, indicating that Nam1p and Sls1p are not functionally redundant but rather exist in an ordered pathway . On the basis of these data, a model in which Nam1p coordinates the delivery of newly synthesized transcripts to the membrane, where Sls1p directs or regulates their subsequent handling by membrane-bound factors involved in translation, is proposed.

Mutat Res, 2002 Jan 29, 499(1), 53 - 61
A role for Rad23 proteins in 26S proteasome-dependent protein degradation?
van Laar T, van der Eb AJ, Terleth C.
Treatment of cells with genotoxic agents affects protein degradation in both positive and negative ways . Exposure of S . cerevisiae to the alkylating agent MMS resulted in activation of genes that are involved in ubiquitin- and 26S proteasome-dependent protein degradation . This process partially overlaps with the activation of the ER-associated protein degradation pathway . The DNA repair protein Rad23p and its mammalian homologues have been shown to inhibit degradation of specific substrates in response to DNA damage . Particularly the recently identified inhibition of degradation by mouse Rad23 protein (mHR23) of the associated nucleotide excision repair protein XPC was shown to stimulate DNA repair.Recently, it was shown that Rad23p and the mouse homologue mHR23B also associate with Png1p, a deglycosylation enzyme . Png1p-mediated deglycosylation plays a role in ER-associated protein degradation after accumulation of malfolded proteins in the endoplasmic reticulum . Thus, if stabilization of proteins that are associated with the C-terminus of Rad23p is a general phenomenon, then Rad23 might be implicated in the stimulation of ER-associated protein degradation as well . Interestingly, the recently identified HHR23-like protein Mif1 is also thought to play a role in ER-associated protein degradation . The MIF1 gene is strongly activated in response to ER-stress . Mif1 contains a ubiquitin-like domain which is most probably involved in binding to S5a, a subunit of the 19S regulatory complex of the 26S proteasome . On the basis of its localization in the ER-membrane, it is hypothesized that Mif1 could play a role in the translocation of the 26S proteasome towards the ER-membrane, thereby enhancing ER-associated protein degradation.

Mol Cells, 2001 Dec 31, 12(3), 277 - 85
Yeast replicative DNA polymerases and their role at the replication fork; Kawasaki Y et al.; The budding yeast, Saccharomyces cerevisiae, is an excellent model system for the study of DNA polymerases and their roles in DNA replication, repair, and recombination . Presently ten DNA polymerases have been purified and characterized from S . cerevisiae . Rapid advances in genome sequencing projects for yeast and other organisms have greatly facilitated and accelerated the identification of yeast enzymes and their homologues in other eukaryotic species . This article reviews current available research on yeast DNA polymerases and their functional roles in DNA metabolism . Relevant information about eukaryotic homologues of these enzymes will also be discussed.

J Cell Sci, 2002 Jan 1, 115(Pt 1), 195 - 206
Essential functions of Sds22p in chromosome stability and nuclear localization of PP1; Peggie MW et al.; Sds22p is a conserved, leucine-rich repeat protein that interacts with the catalytic subunit of protein phosphatase 1 (PP1(C)) and which has been proposed to regulate one or more functions of PP1(C) during mitosis . Here we show that Saccharomyces cerevisiae Sds22p is a largely nuclear protein, most of which is present as a sTable 1:1 complex with yeast PP1(C) (Glc7p) . Temperature-sensitive (Ts(-)) S . cerevisiae sds22 mutants show profound chromosome instability at elevated growth temperatures but do not confer a cell cycle stage-specific arrest . In the sds22-6 Ts(-) mutant, nuclear Glc7p is both reduced in level and aberrantly localized at 37 degrees C and the interaction between Glc7p and Sds22p in vitro is reduced at higher temperatures, consistent with the in vivo Ts(-) growth defect . Like some glc7 mutations, sds22-6 can suppress the Ts(-) growth defect associated with ipl1-2, a loss of function mutation in a protein kinase that is known to work in opposition to PP1 on at least two nuclear substrates . This, together with reciprocal genetic interactions between GLC7 and SDS22, suggests that Sds22p functions positively with Glc7p to promote dephosphorylation of nuclear substrates required for faithful transmission of chromosomes during mitosis, and this role is at least partly mediated by effects of Sds22p on the nuclear distribution of Glc7p

Adv Space Res, 2001, 28(4), 555 - 61
The effects of microgravity on induced mutation in Escherichia coli and Saccharomyces cerevisiae; Takahashi A et al.; We examined whether microgravity influences the induced-mutation frequencies through in vivo experiments during space flight aboard the space shuttle Discovery (STS-91) . We prepared dried samples of repair-deficient strains and parental strains of Escherichia (E.) coli and Saccharomyces (S.) cerevisiae given DNA damage treatment . After culture in space, we measured the induced-mutation frequencies and SOS-responses under microgravity . The experimental findings indicate that almost the same induced-mutation frequencies and SOS-responses of space samples were observed in both strains compared with the ground control samples . It is suggested that microgravity might not influence induced-mutation frequencies and SOS-responses at the stages of DNA replication and/or DNA repair . In addition, we developed a new experimental apparatus for space experiments to culture and freeze stocks of E . coli and S . cerevisiae cells . c2001 COSPAR . Published by Elsevier Science Ltd . All rights reserved.

J Cell Sci, 2001 Dec, 114(Pt 24), 4599 - 611
Relationship between the function and the location of G1 cyclins in S . cerevisiae; Edgington NP et al.; The Saccharomyces cerevisiae cyclin-dependent kinase Cdc28 forms complexes with nine different cyclins to promote cell division . These nine cyclin-Cdc28 complexes have different roles, but share the same catalytic subunit; thus, it is not clear how substrate specificity is achieved . One possible mechanism is specific sub-cellular localization of specific complexes . We investigated the location of two G1 cyclins using fractionation and microscopy . In addition, we developed 'forced localization' cassettes, which direct proteins to particular locations, to test the importance of localization . Cln2 was found in both nucleus and cytoplasm . A substrate of Cln2, Sic1, was also in both compartments . Cytoplasmic Cln2 was concentrated at sites of polarized growth . Forced localization showed that some functions of Cln2 required a cytoplasmic location, while other functions required a nuclear location . In addition, one function apparently required shuttling between the two compartments . The G1 cyclin Cln3 required nuclear localization . An autonomous, nuclear localization sequence was found near the C-terminus of Cln3 . Our data supports the hypothesis that Cln2 and Cln3 have distinct functions and locations, and the specificity of cyclin-dependent kinases is mediated in part by subcellular location.

Genome Inform Ser Workshop Genome Inform, 2001, 12, 44 - 53
Extraction of correlated gene clusters by multiple graph comparison; Nakaya A et al.; This paper presents a new method to extract a set of correlated genes with respect to multiple biological features . Relationships among genes on a specific feature are encoded as a graph structure whose nodes correspond to genes . For example, the genome is a graph representing positional correlations of genes on the chromosome, the pathway is a graph representing functional correlations of gene products, and the expression profile is a graph representing gene expression similarities . When a set of genes are localized in a single graph, such as a gene cluster on the chromosome, an enzyme cluster in the metabolic pathway, or a set of coexpressed genes in the microarray gene expression profile, this may suggest a functional link among those genes . The functional link would become stronger when the clusters are correlated; namely, when a set of corresponding genes form clusters in multiple graphs . The newly introduced heuristic algorithm extracts such correlated gene clusters as isomorphic subgraphs in multiple graphs by using inter-graph links that are defined based on biological relevance . Using the method, we found E.coli correlated gene clusters in which genes are related with respect to the positions in the genome and the metabolic pathway, as well as the 3D structural similarity . We also analyzed protein-protein interaction data by two-hybrid experiments and gene coexpression data by microarrays in S.cerevisiae, and estimated the possibility of utilizing our method for screening the datasets that are likely to contain many false positive relations.

Int J Food Microbiol, 2001 Dec 30, 71(2-3), 219 - 34
Recovery of an oscillatory mode of batch yeast growth in water for a pure culture; Vadasz AS et al.; New experiments that we conducted show an oscillatory mode of batch yeast growth in water, for a pure culture of the T206 strain of Saccharomyces cerevisiae . The oscillations are damped over time, allowing the cell concentration to stabilize at the stationary equilibrium . A new proposed model that includes the complete cell growth dynamics is introduced and showed to recover the experimental oscillatory results . In addition the proposed model recovers effects that are frequently encountered in experiments such as a "Lag Phase" as well as an inflection point in the "ln curve" of the cell concentration . The proposed model recovers also the Logistic Growth Curve as a special case . For purposes of providing some interesting contrast we present additional experimental as well as computational results for the growth of the VIN7 strain of S . cerevisiae in a 5% grape juice medium . The latter indicates even stronger oscillations during the growth process . In order to capture experimentally the oscillatory growth behavior, very frequent readings are required (every 15-30 min) and the measurement process needs to be extended to longer than usual periods (over 250 h).

Cell Biol Toxicol, 2001, 17(6), 383 - 93
The myosin ATPase inhibitor 2,3-butanedione-2-monoxime disorganizes microtubules as well as F-actin in Saccharomyces cerevisiae; Chon K et al.; Interactions between microtubules and filamentous actin (F-actin) are essential to many cellular processes, but their mechanisms are poorly understood . We investigated possible roles of the myosin family of proteins in the interactions between filamentous actin (F-actin) and microtubules of budding yeast Saccharomyces cerevisiae with the general myosin ATPase inhibitor 2,3-butanedione-2-monoxime (BDM) . The growth of S . cerevisiae was completely inhibited by BDM at 20 mmol/L and the effect of BDM on cell growth was reversible . In more than 80% of BDM-treated budding yeast cells, the polarized distribution of F-actin was lost and fewer F-actin dots were observed . When cells were synchronized in G1 with alpha-factor and released in the presence of BDM, cell number did not increase and cells were mainly arrested in G1 DNA content without any bud, suggesting that myosin activity is required for new bud formation and the start of a new cell cycle . More than 10% of the BDM-treated cells also revealed defects in nuclear migration to the bud neck as well as in nuclear shape . Consistent with these defects, the orientation of mitotic spindles was random in the 57% of cells treated with 20 mmol/L BDM and immunostained with anti-tubulin antibody . Furthermore, microtubule structures were completely disorganized in most of the cells incubated in 50 mmol/L BDM, while similar amounts of tubulin proteins were present in both BDM-treated and untreated cells . These results show that the general myosin inhibitor BDM disorganizes microtubule structures as well as F-actin, and suggest that BDM-sensitive myosin activities are necessary for the interaction of F-actin and microtubules to coordinate polarized bud growth and the shape and migration of the nucleus in S . cerevisiae.

Bioseparation, 2001, 10(1-3), 7 - 19
Minimising biomass/adsorbent interactions in expanded bed adsorption processes: a methodological design approach; Lin DQ et al.; Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from crude feedstock . Interactions between solid matter in the feed suspension and fluidised adsorbent particles influence bed stability and therefore have a significant impact on protein adsorption in expanded beds . In order to design efficient and reliable EBA processes a strategy is needed, which allows to find operating conditions, where these adverse events do not take place . In this paper a methodological approach is presented, which allows systematic characterisation and minimisation of cell/adsorbent interactions with as little experimental effort as possible . Adsorption of BSA to the anion exchanger Streamline Q XL from a suspension containing S . cerevisiae cells was chosen as a model system with a strong affinity of the biomass towards the stationary phase . Finite bath biomass adsorption experiments were developed as an initial screening method to estimate a potential interference . The adhesiveness of S . cerevisiae to the anion exchanger could be reduced significantly by increasing the conductivity of the feedstock . A biomass pulse response method was used to find optimal operation conditions showing no cell/adsorbent interactions . A good correlation was found between the finite bath test and the pulse experiment for a variety of suspensions (intact yeast cells, E . coli homogenate and hybridoma cells) and adsorbents (Streamline Q XL, DEAE and SP), which allows to predict cell/adsorbent interactions in expanded beds just from finite bath adsorption tests . Under the optimised operating conditions obtained using the prior methods, the stability of the expanded bed was investigated during fluidisation in biomass containing feedstock (up to 15% yeast on wet weight basis) employing residence time distribution analysis and evaluation by an advanced model . Based on these studies threshold values were defined for the individual experiments, which have to be achieved in order to obtain an efficient EBA process . Breakthrough experiments were conducted to characterise the efficiency of BSA adsorption from S . cerevisiae suspensions in EBA mode under varying operating conditions . This allowed to correlate the stability of the expanded bed with its sorption efficiency and therefore could be used to verify the threshold values defined . The approach presented in this work provides a fast and simple way to minimise cell/adsorbent interactions and to define a window of operation for protein purification using EBA.

Genes Dev, 2002 Jan 1, 16(1), 85 - 100
Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S . cerevisiae; Sharp JA et al.; Budding yeast centromeres are comprised of approximately 125-bp DNA sequences that direct formation of the kinetochore, a specialized chromatin structure that mediates spindle attachment to chromosomes . We report here a novel role for the histone deposition complex chromatin assembly factor I (CAF-I) in building centromeric chromatin . The contribution of CAF-I to kinetochore function overlaps that of the Hir proteins, which have also been implicated in nucleosome formation and heterochromatic gene silencing . cacDelta hirDelta double mutant cells lacking both CAF-I and Hir proteins are delayed in anaphase entry in a spindle assembly checkpoint-dependent manner . Further, cacDelta and hirDelta deletions together cause increased rates of chromosome missegregation, genetic synergies with mutations in kinetochore protein genes, and alterations in centromeric chromatin structure . Finally, CAF-I subunits and Hir1 are enriched at centromeres, indicating that these proteins make a direct contribution to centromeric chromatin structures.

RNA, 2001 Dec, 7(12), 1693 - 701
A genome-wide survey of RS domain proteins; Boucher L et al.; Domains rich in alternating arginine and serine residues (RS domains) are frequently found in metazoan proteins involved in pre-mRNA splicing . The RS domains of splicing factors associate with each other and are important for the formation of protein-protein interactions required for both constitutive and regulated splicing . The prevalence of the RS domain in splicing factors suggests that it might serve as a useful signature for the identification of new proteins that function in pre-mRNA processing, although it remains to be determined whether RS domains also participate in other cellular functions . Using database search and sequence clustering methods, we have identified and categorized RS domain proteins encoded within the entire genomes of Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae . This genome-wide survey revealed a surprising complexity of RS domain proteins in metazoans with functions associated with chromatin structure, transcription by RNA polymerase II, cell cycle, and cell structure, as well as pre-mRNA processing . Also identified were RS domain proteins in S . cerevisiae with functions associated with cell structure, osmotic regulation, and cell cycle progression . The results thus demonstrate an effective strategy for the genomic mining of RS domain proteins . The identification of many new proteins using this strategy has provided a database of factors that are candidates for forming RS domain-mediated interactions associated with different steps in pre-mRNA processing, in addition to other cellular functions.

Genome Res, 2002 Jan, 12(1), 177 - 89
ARACHNE: a whole-genome shotgun assembler; Batzoglou S et al.; We describe a new computer system, called, for assembling genome sequence using paired-end whole-genome shotgun reads . has several key features, including an efficient and sensitive procedure for finding read overlaps, a procedure for scoring overlaps that achieves high accuracy by correcting errors before assembly, read merger based on forward-reverse links, and detection of repeat contigs by forward-reverse link inconsistency . To test, we created simulated reads providing approximately 10-fold coverage of the genomes of H . influenzae, S . cerevisiae, and D . melanogaster, as well as human chromosomes 21 and 22 . The assemblies of these simulated reads yielded nearly complete coverage of the respective genomes, with a small number of contigs joined into a smaller number of supercontigs (or scaffolds) . For example, analysis of the D . melanogaster genome yielded approximately 98% coverage with an N50 contig length of 324 kb and an N50 supercontig length of 5143 kb . The assembly accuracy was high, although not perfect: small errors occurred at a frequency of roughly 1 per 1 Mb (typically, deletion of approximately 1 kb in size), with a very small number of other misassemblies . The assembly was rapid: the Drosophila assembly required only 21 hours on a single 667 MHz processor and used 8.4 Gb of memory.

Genetics, 2001 Dec, 159(4), 1595 - 604
The Aspergillus nidulans musN gene encodes a RecQ helicase that interacts with the PI-3K-related kinase UVSB; Hofmann AF et al.; In Aspergillus nidulans, the uvsB gene encodes a member of the PI-3K-related kinase family of proteins . We have recently shown that UVSB is required for multiple aspects of the DNA damage response . Since the musN227 mutation is capable of partially suppressing defects caused by uvsB mutations, we sought to understand the mechanism underlying the suppression by cloning the musN gene . Here, we report that musN encodes a RecQ helicase with homology to S . pombe rqh1, S . cerevisiae sgs1, and human BLM and WRN . Phenotypic characterization of musN mutant alleles reveals that MUSN participates in the response to a variety of genotoxic agents . The slow growth and genotoxin sensitivity of a musN null mutant can be partially suppressed by a defect in homologous recombination caused by the uvsC114 mutation . In addition, we present evidence suggesting that MUSN may promote recovery from the DNA damage response . We suggest that a block to recovery caused by the musN227 mutation, coupled with the modest accumulation of recombination intermediates, can suppress defects caused by uvsB mutations . Finally, we report that another RecQ helicase, ORQA, performs a function that partially overlaps that of MUSN.

Appl Microbiol Biotechnol, 2001 Dec, 57(5-6), 702 - 7
Intelligent yeast strains with the ability to self-monitor the concentrations of intra- and extracellular phosphate or ammonium ion by emission of fluorescence from the cell surface; Shibasaki S et al.; Saccharomyces cerevisiae strains that respond to environmental changes and transmit the information by emission of fluorescence from the cell surface were constructed . The technique of cell surface engineering enabled the yeast cells to display enhanced cyan blue fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) on the surface under the control of promoters that sense environmental changes . Two model promoters were examined in this study . For monitoring the intra- and extracellular concentrations of phosphate ion, the PHO5 promoter was chosen to display ECFP . The MEP2 promoter was used to display EYFP to sense the concentrations of ammonium ion . Fluorescence was observed by fluorescence microscopy and immunofluorescence microscopy, and the intensity was measured by a flow cytometer . The relationship between ion concentration inside and outside the cells was evaluated by the change in the rate of fluorescence . This S . cerevisiae system enables environmental changes to be transmitted as intra- and extracellular information using a suitable promoter functioning at real time and in a non-invasive manner.

J Bioenerg Biomembr, 2000 Feb, 32(1), 111 - 21
Cloning and characterization of a 35-kDa mouse mitochondrial outer membrane protein MOM35 with high homology to Tom40; Rivera IL et al.; We have cloned a 35-kDa protein from a mouse cDNA library with a 25% overall amino acid identity to yTom40 and 27% identity to nTom40 . This homolog toTom40 was named MOM35 . It contains two possible start codons 36 amino acids apart from each other . Both the long and the short version of MOM35 can be imported in vitro into mouse mitochondria . The identified protein is imported into the outer mitochondrial membrane and comprises a trypsin-resistance pattern similar to that of nTom40 . Tom40 of N . crassa, S . cerevisiae, and the protein identified herein contains a highly conserved region with possible physiological importance . Subsequent investigation has revealed that this region interacts specifically in vitro with preproteins proposed to be imported by a Tom40-dependent pathway.

Can J Microbiol, 2001 Nov, 47(11), 969 - 78
Phosphite disrupts the acclimation of Saccharomyces cerevisiae to phosphate starvation; McDonald AE et al.; The influence of phosphite (H2PO3-) on the response of Saccharomyces cerevisiae to orthophosphate (HPO4(2-); Pi) starvation was assessed . Phosphate-repressible acid phosphatase (rAPase) derepression and cell development were abolished when phosphate-sufficient (+Pi) yeast were subcultured into phosphate-deficient (-Pi) media containing 0.1 mM phosphite . By contrast, treatment with 0.1 mM phosphite exerted no influence on rAPase activity or growth of +Pi cells . 31P NMR spectroscopy revealed that phosphite is assimilated and concentrated by yeast cultured with 0.1 mM phosphite, and that the levels of sugar phosphates, pyrophosphate, and particularly polyphosphate were significantly reduced in the phosphite-treated -Pi cells . Examination of phosphite's effects on two PHO regulon mutants that constitutively express rAPase indicated that (i) a potential target for phosphite's action in -Pi yeast is Pho84 (plasmalemma high-affinity Pi transporter and component of a putative phosphate sensor-complex), and that (ii) an additional mechanism exists to control rAPase expression that is independent of Pho85 (cyclin-dependent protein kinase) . Marked accumulation of polyphosphate in the delta pho85 mutant suggested that Pho85 contributes to the control of polyphosphate metabolism . Results are consistent with the hypothesis that phosphite obstructs the signaling pathway by which S . cerevisiae perceives and responds to phosphate deprivation at the molecular level.

Antonie Van Leeuwenhoek, 2001 Oct, 80(1), 11 - 7
Human acylphosphatase cannot replace phosphoglycerate kinase in Saccharomyces cerevisiae; Van Hoek P et al.; Human acylphosphatase (h-AP, EC 3.6.1.7) has been reported to catalyse the hydrolysis of the 1-phosphate group of 1,3-diphosphoglycerate . In vivo operation of this reaction in the yeast Saccharomyces cerevisiae would bypass phosphoglycerate kinase and thus reduce the ATP yield from glycolysis . To investigate whether h-AP can indeed replace the S . cerevisiae phosphoglycerate kinase, a multi-copy plasmid carrying the h-AP gene under control of the yeast TDH3 promoter was introduced into a pgk1 delta mutant of S . cerevisiae . A strain carrying the expression vector without the h-AP cassette was used as a reference . For both strains, steady-state carbon- and energy-limited chemostat cultures were obtained at a dilution rate of 0.10 h(-1) on a medium containing a mixture of glucose and ethanol (15% and 85% on a carbon basis, respectively) . Although the h-AP strain exhibited a high acylphosphatase activity in cell extracts, switching to glucose as sole carbon and energy source resulted in a complete arrest of glucose consumption and growth . The lack of a functional glycolytic pathway was further evident from the absence of ethanol formation in the presence of excess glucose in the culture . As h-AP cannot replace yeast phosphoglycerate kinase in vivo, the enzyme is not a useful tool to modify the ATP yield of glycolysis in S . cerevisiae.

Biotechnol Bioeng, 2002 Feb 5, 77(3), 290 - 5
Self-assembly and two-dimensional patterning of cell arrays by electrophoretic deposition; Brisson V et al.; Using Saccharomyces cerevisiae as a demonstration system, we present a method to form two-dimensional, patternable cellular arrays . The method does not require surface chemical templating of the substratum to produce arrays or patterns . By virtue of their colloidal characteristics, S . cerevisiae cells may be induced to form dense, quasi-ordered two-dimensional clusters adjacent to an electrode surface by electrophoretic deposition (EPD) . Using ac EPD, dense two-dimensional cell clusters may be formed in minutes from extremely dilute cell suspensions . The arrays may be induced to form geometric patterns by focusing the electric field during deposition . These monolayer arrays are reversible, dissipating by diffusion on removal of the electric field, and are not in adhesive contact with the electrode surface . Brief application of a modest dc current density adheres the arrays tightly to the surface .

Nucleic Acids Res, 2002 Jan 1, 30(1), 69 - 72
Saccharomyces Genome Database (SGD) provides secondary gene annotation using the Gene Ontology (GO); Dwight SS et al.; The Saccharomyces Genome Database (SGD) resources, ranging from genetic and physical maps to genome-wide analysis tools, reflect the scientific progress in identifying genes and their functions over the last decade . As emphasis shifts from identification of the genes to identification of the role of their gene products in the cell, SGD seeks to provide its users with annotations that will allow relationships to be made between gene products, both within Saccharomyces cerevisiae and across species . To this end, SGD is annotating genes to the Gene Ontology (GO), a structured representation of biological knowledge that can be shared across species . The GO consists of three separate ontologies describing molecular function, biological process and cellular component . The goal is to use published information to associate each characterized S.cerevisiae gene product with one or more GO terms from each of the three ontologies . To be useful, this must be done in a manner that allows accurate associations based on experimental evidence, modifications to GO when necessary, and careful documentation of the annotations through evidence codes for given citations . Reaching this goal is an ongoing process at SGD . For information on the current progress of GO annotations at SGD and other participating databases, as well as a description of each of the three ontologies, please visit the GO Consortium page at SGD gene associations to GO can be found by visiting our site at http://genome-www.stanford.edu/Saccharomyces/.

Biochim Biophys Acta, 2001 Dec 3, 1522(2), 67 - 73
Molecular cloning, expression, and site-directed mutations of oxidosqualene cyclase from Cephalosporium caerulens; Abe I et al.; A cDNA for oxidosqualene:lanosterol cyclase (OSLC) was cloned and sequenced from the fungus Cephalosporium caerulens, that produces a steroidal antibiotic, helvolic acid . A 2280 bp open reading frame encoded an M(r) 87078 protein with 760 amino acids . The cDNA was functionally expressed in the OSLC-deficient mutant GIL77 strain of Saccharomyces cerevisiae . A truncated recombinant enzyme (Delta49N) starting from the second methionine (M50) residue was completely inactive, suggesting that ca . 30 additional hydrophilic amino acid residues at the N-terminal are essential for the folding of the enzyme . Furthermore, the active site residues, H234 and D456 (numbering in S . cerevisiae OSLC), were chosen for site-directed mutagenesis experiments; H234E, H234Y, H234F, D456E, D456N, and D456H mutants were inactive, while H234W and H234K mutants retained lanosterol-forming activity.

Acta Pharmacol Sin, 2001 Aug, 22(8), 741 - 6
DNA topoisomerase II as the primary cellular target for salvicine in Saccharomyces cerevisiae; Meng LH et al.; AIM: To identify whether DNA topoisomerase II (Topo II) is the primary cellular target of salvicine in Saccharomyces cerevisiae (S cerevisiae) and the action mode of salvicine . METHODS: The catalytic activity of Topo II was determined by Topo II mediated supercoiled pBR322 relaxation . The effects of salvicine on the growth of four strains of S cerevisiae were assessed by clone forming assay . RESULTS: Salvicine inhibited Topo II mediated supercoiled pBR322 relaxation in cell-free system . Cytotoxicities of salvicine to parent (JN394) and TOP1 deleted (JN394top1-) yeast cells were at the same level, suggesting Topo I might not be the cellular target of salvicine . Salvicine displayed high activity against JN394t2-1 cells at 25 degrees C, while no growth inhibition was observed at 30 degrees C in the concentration range of interest . Furthermore, JN394t2-5 cells which expressed top2-5 mutant allele were highly resistant to salvicine and etoposide (VP16) . CONCLUSION: Topo II was the primary cellular target of salvicine in vivo and salvicine killed yeast cells mainly by trapping the DNA-Topo II cleavage complex . Salvicine and VP16 might share some similar action locus on Topo II.

Eur J Biochem, 2001 Dec, 268(23), 6097 - 104
Expression of V-ATPase proteolipid subunit of Acetabularia acetabulum in a VMA3-deficient strain of Saccharomyces cerevisiae and its complementation study; Ikeda M et al.; The function of the translation products of six different cDNAs for Acetabularia V-ATPase proteolipid subunit (AACEVAPD1 to AACEVAPD6) was examined using a Saccharomyces cerevisiae VMA3-deficient strain that lacked its own gene for one of the proteolipid subunits of V-ATPase . Expression of the cDNAs in the strain revealed that four cDNAs from the six complemented the proton transport activity into the vacuole, visualized by fluorescence microscopy . The vacuolar-membrane-enriched fractions from the four transformants showed cross-reactivity with antibodies against the subunits a and A of S . cerevisiae V-ATPase . Two translation products from the other two cDNAs were demonstrated not to be localized in vacuolar membranes, and thus could not complement the function of the VMA3-deficient strain . As the primary structures deduced from the former four cDNAs are similar but clearly different from those of the latter two, the latter two translation products may not be able to substitute for theVMA3 gene product.

Yeast, 2001 Dec, 18(16), 1493 - 503
Cooperative functions of the mannoprotein-encoding genes in the biogenesis and maintenance of the cell wall in Saccharomyces cerevisiae; Horie T et al.; To elucidate the roles of genes involved in the cell wall biogenesis and function in Saccharomyces cerevisiae, we isolated and characterized mutants that were lethal in a strain in which the SED1 gene encoding a cell wall mannoprotein was disrupted . Thus, double mutants of SED1 and either MNN9 or MNN10 were unable to grow and YOL155c on a multicopy plasmid could suppress their synthetic lethality . A Yol155cp-GFP fusion protein was found to localize to the cell wall, suggesting that it might also be a cell wall mannoprotein . Subsequently, we analysed the effects of the shut-off of SED1 in a sed1 and mnn9 double mutant: cells after the shut-off showed anomalous cellular morphology and died in the mitotic M phase . From these and other results, we postulate that these genes function cooperatively with each other and in a cell cycle-dependent manner in the biogenesis and maintenance of cell wall in S . cerevisiae .

Curr Biol, 2001 Dec 11, 11(24), 1934 - 44
C . elegans RAD-5/CLK-2 defines a new DNA damage checkpoint protein; Ahmed S et al.; BACKGROUND: In response to genotoxic stress, cells activate checkpoint pathways that lead to a transient cell cycle arrest that allows for DNA repair or to apoptosis, which triggers the demise of genetically damaged cells . RESULTS: During positional cloning of the C . elegans rad-5 DNA damage checkpoint gene, we found, surprisingly, that rad-5(mn159) is allelic with clk-2(qm37), a mutant previously implicated in regulation of biological rhythms and life span . However, clk-2(qm37) is the only C . elegans clock mutant that is defective for the DNA damage checkpoint . We show that rad-5/clk-2 acts in a pathway that partially overlaps with the conserved C . elegans mrt-2/S . cerevisiae RAD17/S . pombe rad1(+) checkpoint pathway . In addition, rad-5/clk-2 also regulates the S phase replication checkpoint in C . elegans . Positional cloning reveals that the RAD-5/CLK-2 DNA damage checkpoint protein is homologous to S . cerevisiae Tel2p, an essential DNA binding protein that regulates telomere length in yeast . However, the partial loss-of-function C . elegans rad-5(mn159) and clk-2(qm37) checkpoint mutations have little effect on telomere length, and analysis of the partial loss-of-function of S . cerevisiae tel2-1 mutant failed to reveal typical DNA damage checkpoint defects . CONCLUSIONS: Using C . elegans genetics we define the novel DNA damage checkpoint protein RAD-5/CLK-2, which may play a role in oncogenesis . Given that Tel2p has been shown to bind to a variety of nucleic acid structures in vitro, we speculate that the RAD-5/CLK-2 checkpoint protein may act at sites of DNA damage, either as a sensor of DNA damage or to aid in the repair of damaged DNA.

Yeast, 2001 Nov, 18(15), 1371 - 81
The catabolic capacity of Saccharomyces cerevisiae is preserved to a higher extent during carbon compared to nitrogen starvation; Nilsson A et al.; A comparison of catabolic capacity was made between S . cerevisiae cells subjected to 24 h carbon or nitrogen starvation . The cells were shifted to starvation conditions at the onset of respiratory growth on ethanol in aerobic batch cultures, using glucose as the carbon and energy source . The results showed that the catabolic capacity was preserved to a much larger extent during carbon compared to nitrogen starvation . Nitrogen starvation experiments were made in the presence of ethanol (not glucose) to exclude the effect of glucose transport inactivation (Busturia and Lagunas, 1986) . Hence, the difference in catabolic capacity could not be attributed to differences in glucose transport capacity during these conditions . In order to understand the reason for this difference in starvation response, measurement of protein composition, adenine nucleotides, inorganic phosphate, polyphosphate and storage carbohydrates were performed . No clear correlation between any of these variables and catabolic capacity after starvation could be obtained . However, there was a positive correlation between total catabolic activity and intracellular ATP concentration when glucose was added to starved cells . The possible mechanism for this correlation, as well as what determines the ATP level, is discussed .

Biotechnol Bioeng, 2002 Jan 5, 77(1), 61 - 72
Characterization of null mutants of the glyoxylate cycle and gluconeogenic enzymes in S . cerevisiae through metabolic network modeling verified by chemostat cultivation; Stuckrath I et al.; Biomass yields for several null mutants in Saccharomyces cerevisiae were successfully predicted with a metabolic network model . Energetic parameters of the model were obtained from growth data in C-limited aerobic chemostat cultures of the corresponding wild-type strain, which exhibited a P/O ratio of 1.46, a non-growth-related maintenance of 56 mmol ATP/C-mol biomass/h, and a growth-related requirement of 655 mmol ATP/C-mol biomass . Biomass yields and carbon uptake rates were modeled for different mutants incapacitated in their glyoxylate cycle and their gluconeogenesis . Biomass yields were calculated for different feed ratios of glucose to ethanol, and decreases for higher ethanol fractions were correctly predicted for mutants with deletions of the malate synthase, the isocitrate lyase, or the phosphoenolpyruvate carboxykinase . The growth of the fructose- 1,6-bisphosphatase deletion mutant was anticipated less accurate, but the tendency was modeled correctly .

Planta Med, 2001 Dec, 67(9), 820 - 4
Photosensitization and mutation induced in Escherichia coli and Saccharomyces cerevisiae strains by dorstenin, a psoralen analog isolated from Dorstenia bahiensis; Lopes D et al.; Dorstenin, 5-{3-(4,5-dihydro-5,5-dimethyl-4-oxo-2-furanyl)-butoxy}-7H-furo{3, 2-g} benzopyran-7-one, is a psoralen analog recently isolated from Dorstenia species (Moraceae) . In order to characterize its biological activity, its photosensitizing and mutational properties were measured in wild-type E . coli and S . cerevisiae and also in strains carrying mutations which affect DNA repair . Compared to the high activities of psoralen and bergapten, dorstenin showed lower genotoxic effect.

Science, 2001 Dec 14, 294(5550), 2357 - 60
Genome-wide distribution of ORC and MCM proteins in S . cerevisiae: high-resolution mapping of replication origins; Wyrick JJ et al.; DNA replication origins are fundamental to chromosome organization and duplication, but understanding of these elements is limited because only a small fraction of these sites have been identified in eukaryotic genomes . Origin Recognition Complex (ORC) and minichromosome maintenance (MCM) proteins form prereplicative complexes at origins of replication . Using these proteins as molecular landmarks for origins, we identified ORC- and MCM-bound sites throughout the yeast genome . Four hundred twenty-nine sites in the yeast genome were predicted to contain replication origins, and approximately 80% of the loci identified on chromosome X demonstrated origin function . A substantial fraction of the predicted origins are associated with repetitive DNA sequences, including subtelomeric elements (X and Y') and transposable element-associated sequences (long terminal repeats) . These findings identify the global set of yeast replication origins and open avenues of investigation into the role(s) ORC and MCM proteins play in chromosomal architecture and dynamics.

J Bacteriol, 2002 Jan, 184(1), 250 - 65
Genomic analyses of anaerobically induced genes in Saccharomyces cerevisiae: functional roles of Rox1 and other factors in mediating the anoxic response; Kwast KE et al.; DNA arrays were used to investigate the functional role of Rox1 in mediating acclimatization to anaerobic conditions in Saccharomyces cerevisiae . Multiple growth conditions for wild-type and rox1 null strains were used to identify open reading frames with a statistically robust response to this repressor . These results were compared to those obtained for a wild-type strain in response to oxygen availability . Transcripts of nearly one-sixth of the genome were differentially expressed (P < 0.05) with respect to oxygen availability, the majority (>65%) being down-regulated under anoxia . Of the anaerobically induced genes, about one-third (106) contain putative Rox1-binding sites in their promoters and were significantly (P < 0.05) up-regulated in the rox1 null strains under aerobiosis . Additional promoter searches revealed that nearly one-third of the anaerobically induced genes contain an AR1 site(s) for the Upc2 transcription factor, suggesting that Upc2 and Rox1 regulate the majority of anaerobically induced genes in S . cerevisiae . Functional analyses indicate that a large fraction of the anaerobically induced genes are involved in cell stress (approximately 1/3), cell wall maintenance (approximately 1/8), carbohydrate metabolism (approximately 1/10), and lipid metabolism (approximately 1/12), with both Rox1 and Upc2 predominating in the regulation of this latter group and Upc2 predominating in cell wall maintenance . Mapping the changes in expression of functional regulons onto metabolic pathways has provided novel insight into the role of Rox1 and other trans-acting factors in mediating the physiological response of S . cerevisiae to anaerobic conditions.

Mol Cell, 2001 Nov, 8(5), 1105 - 15
Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes; Chen L et al.; S . cerevisiae RAD50, MRE11, and XRS2 genes are required for telomere maintenance, cell cycle checkpoint signaling, meiotic recombination, and the efficient repair of DNA double-strand breaks (DSB)s by homologous recombination and nonhomologous end-joining (NHEJ) . Here, we demonstrate that the complex formed by Rad50, Mre11, and Xrs2 proteins promotes intermolecular DNA joining by DNA ligase IV (Dnl4) and its associated protein Lif1 . Our results show that the Rad50/Mre11/Xrs2 complex juxtaposes linear DNA molecules via their ends to form oligomers and interacts directly with Dnl4/Lif1 . We also demonstrate that Rad50/Mre11/Xrs2-mediated intermolecular DNA joining is further stimulated by Hdf1/Hdf2, the yeast homolog of the mammalian Ku70/Ku80 heterodimer . These studies reveal specific functional interplay among the Hdf1/Hdf2, Rad50/Mre11/Xrs2, and Dnl4/Lif1 complexes in NHEJ.

Mol Biol Cell, 2001 Dec, 12(12), 4103 - 13
The TOR signal transduction cascade controls cellular differentiation in response to nutrients; Cutler NS et al.; Rapamycin binds and inhibits the Tor protein kinases, which function in a nutrient-sensing signal transduction pathway that has been conserved from the yeast Saccharomyces cerevisiae to humans . In yeast cells, the Tor pathway has been implicated in regulating cellular responses to nutrients, including proliferation, translation, transcription, autophagy, and ribosome biogenesis . We report here that rapamycin inhibits pseudohyphal filamentous differentiation of S . cerevisiae in response to nitrogen limitation . Overexpression of Tap42, a protein phosphatase regulatory subunit, restored pseudohyphal growth in cells exposed to rapamycin . The tap42-11 mutation compromised pseudohyphal differentiation and rendered it resistant to rapamycin . Cells lacking the Tap42-regulated protein phosphatase Sit4 exhibited a pseudohyphal growth defect and were markedly hypersensitive to rapamycin . Mutations in other Tap42-regulated phosphatases had no effect on pseudohyphal differentiation . Our findings support a model in which pseudohyphal differentiation is controlled by a nutrient-sensing pathway involving the Tor protein kinases and the Tap42-Sit4 protein phosphatase . Activation of the MAP kinase or cAMP pathways, or mutation of the Sok2 repressor, restored filamentation in rapamycin treated cells, supporting models in which the Tor pathway acts in parallel with these known pathways . Filamentous differentiation of diverse fungi was also blocked by rapamycin, demonstrating that the Tor signaling cascade plays a conserved role in regulating filamentous differentiation in response to nutrients.

Mutat Res, 2001 Dec 19, 487(3-4), 73 - 83
S . cerevisiae has three pathways for DNA interstrand crosslink repair; Grossmann KF et al.; Yeast mutants, snm1 (pso2-1), rev3 (pso1-1), and rad51, which display significant sensitivity to interstrand crosslinks (ICLs) have low relative sensitivity to other DNA damaging agents . SNM1, REV3, and RAD51 were disrupted in the same haploid strain, singly and in combination . The double mutants, snm1 Delta rev3 Delta, snm1 Delta rad51 Delta and rev3 Delta rad51 Delta were all more sensitive to ICLs than any of the single mutants, indicating that they are in separate epistasis groups for survival . A triple mutant displayed greater sensitivity to ICLs than any of the double mutants, with one ICL per genome being lethal . Therefore, Saccharomyces cerevisiae appears to have three separate ICL repair pathways, but no more . S-phase delay was not observed after ICL damage introduced by cisplatin (CDDP) or 8-methoxypsoralen (8-MOP) during the G1-phase, in any of the above mutants, or in an isogenic rad14 Delta mutant deficient in nucleotide excision repair . However, the psoralen analog angelicin (monoadduct damage) induced a significant S-phase delay in the rad14 Delta mutant . Thus, normal S-phase in the presence of ICLs does not seem to be due to rapid excision repair . The results also indicate that monoadduct formation by CDDP or 8-MOP at the doses used is not sufficient to delay S-phase in the rad14 Delta mutant . While the sensitivity of a rev3 Delta mutant indicates Pol zeta is needed for optimal ICL repair, isogenic cells deficient in Pol eta (rad30 Delta cells) were not significantly more sensitive to ICL agents than wild-type cells, and have no S-phase delay.

Gene, 2001 Dec 12, 280(1-2), 9 - 17
Identification of transcriptionally expressed pheromone receptor genes in filamentous ascomycetes; Poggeler S et al.; Detection of pheromone genes in filamentous ascomycetes implicated the presence of pheromone receptor genes . Similar to yeasts and basidiomycetes, these might be involved in a G-protein triggered signal transduction pathway during mating . We have identified two pheromone receptor genes, named pre1 and pre2, in the genome of the heterothallic filamentous ascomycete Neurospora crassa and the closely related homothallic Sordaria macrospora . The deduced pre1 gene product is a putative seven-transmembrane protein, which displays a high-level amino acid identity with the a-factor receptor Ste3p of Saccharomyces cerevisiae, and is also homologous to lipopeptide pheromone receptors of basidiomycetes . The deduced pre2 product displays significant sequence similarity with the S . cerevisiae STE2 gene product, the alpha-factor receptor . Pair-wise comparisons between pheromone receptor genes of N . crassa and S . macrospora revealed an extremely low degree of nucleotide conservation in these genes, suggesting that they evolved very rapidly . The two genes are transcriptionally expressed in both N . crassa and S . macrospora . Northern and reverse transcription-polymerase chain reaction analyses indicate that in N . crassa, expression of the receptor genes does not occur in a mating type specific manner . Thus, filamentous ascomycetes appear to posses and express pheromone receptor genes.

Lett Appl Microbiol, 2001 Dec, 33(6), 461 - 6
Detection, distribution and selection of microsatellites (SSRs) in the genome of the yeast Saccharomyces cerevisiae as molecular markers; Perez MA et al.; AIMS: The aim of this work was the selection of six polymorphic microsatellite loci for their use as molecular markers in the identification, typification and genetic differentiation of S . cerevisiae strains . METHODS AND RESULTS: The selection was undertaken following a search of the genomic DNA database of Saccharomyces cerevisiae for simple tandem repeat sequences (microsatellites) of di- and trinucleotides . The genetic variability generated by these markers was evaluated in 51 isolates . The discriminatory power produced by combining the information obtained by the six microsatellites was very high . A total of 57 alleles, which generated 44 genotypes, were found . CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The multiple analysis of microsatellites proved to be a powerful and agile tool for analysing the genome of S . cerevisiae populations.

Cell, 2001 Nov 30, 107(5), 563 - 5
New glimpses of an old machine; Paull TT; Two recent studies illustrate the ability of the Mre11/Rad50 DNA repair complex to bind and connect DNA ends . Specific stimulation of DNA ligase IV-mediated end-joining by Mre11 complexes from S . cerevisiae suggests the possibility of a direct role in nonhomologous end-joining in eukaryotic cells.

Curr Biol, 2001 Nov 27, 11(23), 1825 - 35
Separase is required for chromosome segregation during meiosis I in Caenorhabditis elegans; Siomos MF et al.; BACKGROUND: Chromosome segregation during mitosis and meiosis is triggered by dissolution of sister chromatid cohesion, which is mediated by the cohesin complex . Mitotic sister chromatid disjunction requires that cohesion be lost along the entire length of chromosomes, whereas homolog segregation at meiosis I only requires loss of cohesion along chromosome arms . During animal cell mitosis, cohesin is lost in two steps . A nonproteolytic mechanism removes cohesin along chromosome arms during prophase, while the proteolytic cleavage of cohesin's Scc1 subunit by separase removes centromeric cohesin at anaphase . In Saccharomyces cerevisiae and Caenorhabditis elegans, meiotic sister chromatid cohesion is mediated by Rec8, a meiosis-specific variant of cohesin's Scc1 subunit . Homolog segregation in S . cerevisiae is triggered by separase-mediated cleavage of Rec8 along chromosome arms . In principle, chiasmata could be resolved proteolytically by separase or nonproteolytically using a mechanism similar to the mitotic "prophase pathway." RESULTS: Inactivation of separase in C . elegans has little or no effect on homolog alignment on the meiosis I spindle but prevents their timely disjunction . It also interferes with chromatid separation during subsequent embryonic mitotic divisions but does not directly affect cytokinesis . Surprisingly, separase inactivation also causes osmosensitive embryos, possibly due to a defect in the extraembryonic structures, referred to as the "eggshell." CONCLUSIONS: Separase is essential for homologous chromosome disjunction during meiosis I . Proteolytic cleavage, presumably of Rec8, might be a common trigger for the first meiotic division in eukaryotic cells . Cleavage of proteins other than REC-8 might be necessary to render the eggshell impermeable to solutes.

J Biochem (Tokyo), 2001 Dec, 130(6), 757 - 64
Effects of calnexin deletion in Saccharomyces cerevisiae on the secretion of glycosylated lysozymes; Song Y et al.; Disruption of the calnexin gene in Saccharomyces cerevisiae did not lead to gross effects on the levels of cell growth and secretion of wild-type hen egg white lysozymes (HEWL) . To investigate the function of calnexin in relation to the secretion of glycoproteins, we expressed both stable and unstable mutant glycosylated lysozymes in calnexin-disrupted S . cerevisiae . The secreted amounts of stable mutant glycosylated lysozymes (G49N and S91T/G49N) were almost the same in both wild-type and calnexin-disrupted S . cerevisiae . In contrast, the secretion of unstable mutant glycosylated lysozymes (K13D/G49N, C76A/G49N, and D66H/G49N) greatly increased in calnexin-disrupted S . cerevisiae, although their secretion was very low in the wild-type strain . This indicates that calnexin may act in the quality control of glycoproteins . We further investigated the expression level of the mRNA of the molecular chaperones BiP and PDI, which play a major role in the protein folding process in the ER, when glycosylated lysozymes were expressed in wild-type and calnexin-disrupted S . cerevisiae . The mRNA concentrations of BiP and PDI were evidently increased when the glycosylated lysozymes were expressed in calnexin-disrupted S . cerevisiae . This observation indicates that BiP and PDI may be induced by the accumulation of unfolded glycosylated lysozymes due to the deletion of calnexin.

Appl Environ Microbiol, 2001 Dec, 67(12), 5512 - 9
Degradation of xylan to D-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger beta-xylosidase (xlnD) and the Trichoderma reesei xylanase II (xyn2) genes; La Grange DC et al.; The beta-xylosidase-encoding xlnD gene of Aspergillus niger 90196 was amplified by the PCR technique from first-strand cDNA synthesized on mRNA isolated from the fungus . The nucleotide sequence of the cDNA fragment was verified to contain a 2,412-bp open reading frame that encodes a 804-amino-acid propeptide . The 778-amino-acid mature protein, with a putative molecular mass of 85.1 kDa, was fused in frame with the Saccharomyces cerevisiae mating factor alpha1 signal peptide (MFalpha1(s)) to ensure correct posttranslational processing in yeast . The fusion protein was designated Xlo2 . The recombinant beta-xylosidase showed optimum activity at 60 degrees C and pH 3.2 and optimum stability at 50 degrees C . The K(i(app)) value for D-xylose and xylobiose for the recombinant beta-xylosidase was determined to be 8.33 and 6.41 mM, respectively . The XLO2 fusion gene and the XYN2 beta-xylanase gene from Trichoderma reesei, located on URA3-based multicopy shuttle vectors, were successfully expressed and coexpressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II gene (ADH2) promoter and terminator . These recombinant S . cerevisiae strains produced 1,577 nkat/ml of beta-xylanase activity when expressing only the beta-xylanase and 860 nkat/ml when coexpressing the beta-xylanase with the beta-xylosidase . The maximum beta-xylosidase activity was 5.3 nkat/ml when expressed on its own and 3.5 nkat/ml when coexpressed with the beta-xylanase . Coproduction of the beta-xylanase and beta-xylosidase enabled S . cerevisiae to degrade birchwood xylan to D-xylose.

Anal Chem, 2001 Nov 1, 73(21), 5228 - 31
Characterization of protein biomarkers desorbed by MALDI from whole fungal cells; Amiri-Eliasi B et al.; In this publication, the use of ultraviolet (UV) matrix-assisted laser desorption/ionization (MALDI) time-of-fight (TOF) mass spectrometry (MS) for rapid identification and characterization of Saccharomyces cerevisiae, a fungus, is reported . S . cerevisiae is a unicellular eukaroyte that can serve as a model to study more complex organisms . We have determined that the best technique for cell wall lyses for MALDI involves the use of high concentrations of formic acid solutions . We also have shown that different fungal species exhibit different mass spectra, which can be used to distinguish them readily . Protein peaks from S . cerevisiae spectra have been tentatively identified using bioinformatics and are mainly assigned to ribosomal and mitochondrion-related proteins.

Mutat Res, 2002 Jan 15, 513(1-2), 183 - 91
Cell cycle and morphological alterations as indicative of apoptosis promoted by UV irradiation in S . cerevisiae; Del Carratore R et al.; An apoptotic phenotype induced by oxygen radicals or Bax expression has been observed in Saccharomyces cerevisiae yeast cells by electron and fluorescence microscopy . In this work, we analyzed DNA content and cellular morphology of S . cerevisiae after H(2)O(2) or UV treatment by TdT-mediated dUTP nick end labeling (TUNEL)-test and flow cytofluorimetry . A TUNEL-positive phenotype was observed in both cases, on the same samples a dose-dependent increase in the sub-G(1) population was pointed out by flow cytometry . Sub-G(1) cells were isolated by flow sorting and analyzed by electron microscopy . This population showed condensed chromatin in the nucleus and cell shrinking . This paper reports the first evidence of apoptosis in yeast cells induced by DNA damage after UV irradiation.

Mutat Res, 2002 Jan 15, 513(1-2), 69 - 74
Application of the single cell gel electrophoresis on yeast cells; Miloshev G et al.; In the present paper, we have applied the single cell gel electrophoresis (SCGE) assay on yeast cells treating Saccharomyces cerevisiae cells with hydrogen peroxide and methyl methanesulfonate (MMS), two DNA damaging agents . In order to overcome the problem with the yeast cell wall that prevented DNA to be extended by the electric field, we disintegrated the cell wall after embedding the cells in agarose . A characteristic picture of comets with residual nuclei and tails was observed and the length of the comet tails was dependent on the concentration of the damaging agents . Yeast cells developed comets at concentrations at least 10 times lower than the concentrations at which comets begin to appear in mammalian cells after treatment with the two genotoxic agents . The higher sensitivity of the yeast comet assay and the fact that S . cerevisiae is one of the most thoroughly studied and easy to work with eukaryotic model system suggest that the proposed method could be an useful tool for investigation of the DNA damaging activity of potential genotoxins.

Trends Genet, 2001 Dec, 17(12), 682 - 4
Fungi and humans: closer than you think; Zeng Q et al.; The budding yeast, Saccharomyces cerevisiae, has long been used as a model system to study the functions of human genes . Now that the genome sequences from several other fungal species are nearly complete, we can characterize the genetic diversity in the fungal kingdom at the genomic level . This diversity means that the number of human genes with homologues in the fungal kingdom is double that with homologues in S . cerevisiae only . Therefore, functional studies of human genes in the fungal model systems should look beyond S . cerevisiae.

Exp Cell Res, 2001 Dec 10, 271(2), 296 - 304
Differentiated gene expression in cells within yeast colonies; Minarikova L et al.; Yeast cells growing on solid media organize themselves into multicellular structures, colonies, exhibiting patterns specific for particular yeast strains . With the aim of identifying genes involved in regulations of the colony formation, we applied a new approach enabling the extensive screening of Saccharomyces cerevisiae genes, the expression of which is changed during colony development . We used the library of S . cerevisiae DNA fragments inserted in front of the lacZ gene lacking its own promoter . Colonies of transformants with a blue/white patterned morphotype, implying that the expression of the lacZ gene from the inserted yeast promoter is switched on and off during the colony formation, were isolated . We identified several genes with variable expression during colony morphogenesis, including CCR4, PAM1, MEP3, ADE5,7 and CAT2 . S . cerevisiae strain deleted in the CCR4 gene forms colonies with less organized morphology when compared with the isogenic parental strain . The synchronization of the expression patterns of some of the isolated genes in neighboring colonies was observed.

Zhonghua Yi Xue Za Zhi, 1999 Dec, 79(12), 941 - 3
{Human hR24L gene is involved in DNA excision repair and recombination repair}; Han Y et al.; OBJECTIVE: To study the correction phenotype of temperature sensitive mutant rad24LR of S . cerevisiae and to clone wild human functional homologue . METHODS: The temperature sensitive mutant rad24LR was studied by complementation test . Transformation of rad24LR was performed with human cDNA expression library to screen for reversion phenotype . RESULTS: RAD24L can complement the radiation sensitive phenotype of the mutation . Its homologue of human cDNA was cloned by screening transformed rad24LR with human expression library for reversion phenotype . CONCLUSION: Human DNA repair gene hR24L was successfully cloned in this study . The product hR24L is involved in both DNA excision repair and recombination repair.

Genes Dev, 2001 Nov 15, 15(22), 3005 - 12
NHEJ regulation by mating type is exercised through a novel protein, Lif2p, essential to the ligase IV pathway; Frank-Vaillant M et al.; In the yeast Saccharomyces cerevisiae, DNA double strand break (DSB) repair by nonhomologous end-joining (NHEJ) requires the DNA end-binding heterodimer Yku70p-Yku80p and the ligase Dnl4p associated with its cofactor Lif1p . NHEJ efficiency is down-regulated in MATa/MATalpha cells relative to MATa or MATalpha cells, but the mechanism of this mating type regulation is unknown . Here we report the identification of Lif2p, a S . cerevisiae protein that interacts with Lif1p in a two-hybrid system . Disruption of LIF2 abolishes the capacity of cells to repair DSBs by end-joining to the same extent than lif1 and dnl4 mutants . In MATa/MATalpha cells, Lif2p steady-state level is strongly repressed when other factors involved in NHEJ are unaffected . Increasing the dosage of the Lif2p protein can suppress the NHEJ defect in a/alpha cells . Together, these results indicate that NHEJ regulation by mating type is achieved, at least in part, by a regulation of Lif2p activity.

Genes Dev, 2001 Nov 15, 15(22), 2991 - 3004
The S . cerevisiae SET3 complex includes two histone deacetylases, Hos2 and Hst1, and is a meiotic-specific repressor of the sporulation gene program; Pijnappel WW et al.; Set3 is one of two proteins in the yeast Saccharomyces cerevisiae that, like Drosophila Trithorax, contains both SET and PHD domains . We found that Set3 forms a single complex, Set3C, with Snt1, YIL112w, Sif2, Cpr1, and two putative histone deacetylases, Hos2 and NAD-dependent Hst1 . Set3C includes NAD-dependent and independent deacetylase activities when assayed in vitro . Homology searches suggest that Set3C is the yeast analog of the mammalian HDAC3/SMRT complex . Set3C represses genes in early/middle of the yeast sporulation program, including the key meiotic regulators ime2 and ndt80 . Whereas Hos2 is only found in Set3C, Hst1 is also present in a complex with Sum1, supporting previous characterizations of Hst1 and Sum1 as repressors of middle sporulation genes during vegetative growth . However, Hst1 is not required for meiotic repression by Set3C, thus implying that Set3C (-Hst1) and not Hst1-Sum1, is the meiotic-specific repressor of early/middle sporulation genes.

FEBS Lett, 2001 Nov 9, 508(1), 99 - 102
The role of PDR13 in tolerance to high copper stress in budding yeast; Kim DY et al.; PDR13 in Saccharomyces cerevisiae contributes to drug resistance via sequential activation of PDR1 and PDR5 . In this study, we found that a PDR13 deletion mutant was hypersensitive to Cu(2+) compared to the wild-type counterpart . The Cu(2+) tolerance mechanism mediated by Pdr13 does not seem to involve Pdr1 or Pdr5, since mutants harboring a deletion of either the PDR1 or PDR5 gene did not show elevated Cu(2+) sensitivity . Instead, we found that the PDR13 null mutant could not express CUP1 or CRS5 metallothionein at wild-type levels when subjected to high Cu(2+) stress . These results suggest that Pdr13 contributes to high Cu(2+) tolerance of S . cerevisiae, at least in part, via a mechanism involving metallothionein expression.

Annu Rev Genet, 2001, 35, 647 - 72
Genetic analysis of calmodulin and its targets in Saccharomyces cerevisiae; Cyert MS; Calmodulin, a small, ubiquitous Ca2+-binding protein, regulates a wide variety of proteins and processes in all eukaryotes . CMD1, the single gene encoding calmodulin in S . cerevisiae, is essential, and this review discusses studies that identified many of calmodulin's physiological targets and their functions in yeast cells . Calmodulin performs essential roles in mitosis, through its regulation of Nuf1p/Spc110p, a component of the spindle pole body, and in bud growth, by binding Myo2p, an unconventional class V myosin required for polarized secretion . Surprisingly, mutant calmodulins that fail to bind Ca2+ can perform these essential functions . Calmodulin is also required for endocytosis in yeast and participates in Ca2+-dependent, stress-activated signaling pathways through its regulation of a protein phosphatase, calcineurin, and the protein kinases, Cmk1p and Cmk2p . Thus, calmodulin performs important physiological functions in yeast cells in both its Ca2+-bound and Ca2+-free form.

J Mol Biol, 2001 Nov 9, 313(5), 955 - 63
UBA domains mediate protein-protein interactions between two DNA damage-inducible proteins; Bertolaet BL et al.; The Saccharomyces cerevisiae genes RAD23 and DDI1 were identified in a screen for multicopy suppressors of the temperature-sensitivity of a mutant allele of S . cerevisiae PDS1 . Pds1 is a regulator of anaphase that needs to accumulate and then be degraded by the ubiquitin-proteasome pathway at the metaphase-anaphase transition for cells to progress normally through mitosis . Both the Rad23 and Ddi1 pds1 suppression phenotypes depend on a shared motif known as a UBA domain found in a variety of proteins associated with ubiquitin metabolism . UBA domains were found to be essential for homodimerization of Rad23 and heterodimerization between Rad23 and Ddi1, but not for homodimerization of Ddi1 . This observation, coupled with the findings that Rad23 and Ddi1 UBA domains bind ubiquitin and that dimerization of Rad23 blocks ubiquitin binding, suggests a possible mechanism for regulating Rad23 and Ddi1 function .

J Gen Microbiol, 1989 Feb, 135(Pt 2), 257 - 63
Reductive and non-reductive mechanisms of iron assimilation by the yeast Saccharomyces cerevisiae; Lesuisse E et al.; Iron reduction and uptake was studied in wild-type and haem-deficient strains of Saccharomyces cerevisiae . Haem-deficient strains lacked inducible ferri-reductase activity and were unable to take up iron from different ferric chelates such as Fe(III)-citrate or rhodoturulic acid . In contrast, ferrioxamine B was taken up actively by the mutants as well as by the wild-type strains . At a low extracellular concentration, uptake was insensitive to ferrozine and competitively inhibited by Ga(III)-desferrioxamine B . Extracellular reductive dissociation of the siderophore occurred at higher extracellular concentrations . Two mechanisms appear to contribute to the uptake of ferrioxamine B by S . cerevisiae: one with high affinity, by which the siderophore is internalized as such and another with lower affinity by which iron is dissociated from the ligand prior to uptake.

DNA Seq, 2001, 11(6), 463 - 73
Isolation and sequence analysis of the arbuscular mycorrhizal fungus Glomus mosseae (Nicol & Gerd.) Gerdemann & Trappe 3-phosphoglycerate kinase (PGK) gene promoter region; Harrier LA; The Glomus mosseae 3-phosphoglycerate kinase (GmPGK) gene promoter has been isolated from a phage genomic library and represents one of the few promoter elements to be isolated and analysed from these symbiotic fungi . The analysis revealed the presence of several motifs which are found in the promoter region of other fungal PGK genes . In particular, DNA sequences homologous to segments of the S . cerevisiae and Rhizopus niveus upstream activating elements (UAS) . The importance of these UAS sequences in regulating carbon source in PGK genes is known and the presence of two carbon source regulated UAS sequences in the GmPGK gene promoter and its role in the biology of AM fungi is discussed briefly.

Mol Biol Cell, 2001 Nov, 12(11), 3317 - 27
Completion of replication map of Saccharomyces cerevisiae chromosome III; Poloumienko A et al.; In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of approximately 40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements . The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis . To complete the systematic identification of ARS elements on S . cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids . Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in < or = 10% of cells in the population and two ARS elements active in > or = 90% of the population . Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome.

Mol Cell Biol, 2001 Dec, 21(23), 8203 - 12
Characterization of a six-subunit holo-elongator complex required for the regulated expression of a group of genes in Saccharomyces cerevisiae; Krogan NJ et al.; The Elongator complex associated with elongating RNA polymerase II in Saccharomyces cerevisiae was originally reported to have three subunits, Elp1, Elp2, and Elp3 . Using the tandem affinity purification (TAP) procedure, we have purified a six-subunit yeast Holo-Elongator complex containing three additional polypeptides, which we have named Elp4, Elp5, and Elp6 . TAP tapping and subsequent purification of any one of the six subunits result in the isolation of all six components . Purification of Elongator in higher salt concentrations served to demonstrate that the complex could be separated into two subcomplexes: one consisted of Elp1, -2, and -3, and the other consisted of Elp4, -5, and -6 . Deletions of the individual genes encoding the new Elongator subunits showed that only the ELP5 gene is essential for growth . Disruption of the two nonessential new Elongator-encoding genes, ELP4 and ELP6, caused the same phenotypes observed with knockouts of the original Elongator-encoding genes . Results of microarray analyses demonstrated that the gene expression profiles of strains containing deletions of genes encoding subunits of either Elongator subcomplex, in which we detected significantly altered mRNA expression levels for 96 genes, are very similar, implying that all the Elongator subunits likely function together to regulate a group of S . cerevisiae genes in vivo.

Mol Cell Biol, 2001 Dec, 21(23), 8007 - 21
Construction and analysis of mouse strains lacking the ubiquitin ligase UBR1 (E3alpha) 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 . In the yeast Saccharomyces cerevisiae, the UBR1-encoded ubiquitin ligase (E3) of the N-end rule pathway mediates the targeting of substrate proteins in part through binding to their destabilizing N-terminal residues . The functions of the yeast N-end rule pathway include fidelity of chromosome segregation and the regulation of peptide import . Our previous work described the cloning of cDNA and a gene encoding the 200-kDa mouse UBR1 (E3alpha) . Here we show that mouse UBR1, in the presence of a cognate mouse ubiquitin-conjugating (E2) enzyme, can rescue the N-end rule pathway in ubr1Delta S . cerevisiae . We also constructed UBR1(-/-) mouse strains that lacked the UBR1 protein . UBR1(-/-) mice were viable and fertile but weighed significantly less than congenic +/+ mice . The decreased mass of UBR1(-/-) mice stemmed at least in part from smaller amounts of the skeletal muscle and adipose tissues . The skeletal muscle of UBR1(-/-) mice apparently lacked the N-end rule pathway and exhibited abnormal regulation of fatty acid synthase upon starvation . By contrast, and despite the absence of the UBR1 protein, UBR1(-/-) fibroblasts contained the N-end rule pathway . Thus, UBR1(-/-) mice are mosaics in regard to the activity of this pathway, owing to differential expression of proteins that can substitute for the ubiquitin ligase UBR1 (E3alpha) . We consider these UBR1-like proteins and discuss the functions of the mammalian N-end rule pathway.

J Biol Chem, 2002 Jan 4, 277(1), 395 - 401 Epub 2001 Oct 31.
Mutation of residue 33 of human equilibrative nucleoside transporters 1 and 2 alters sensitivity to inhibition of transport by dilazep and dipyridamole; Visser F et al.; Human equilibrative nucleoside transporters (hENT) 1 and 2 differ in that hENT1 is inhibited by nanomolar concentrations of dipyridamole and dilazep, whereas hENT2 is 2 and 3 orders of magnitude less sensitive, respectively . When a yeast expression plasmid containing the hENT1 cDNA was randomly mutated and screened by phenotypic complementation in Saccharomyces cerevisiae to identify mutants with reduced sensitivity to dilazep, clones with a point mutation that converted Met33 to Ile (hENT1-M33I) were obtained . Characterization of the mutant protein in S . cerevisiae and Xenopus laevis oocytes revealed that the mutant had less than one-tenth the sensitivity to dilazep and dipyridamole than wild type hENT1, with no change in nitrobenzylmercaptopurine ribonucleoside (NBMPR) sensitivity or apparent uridine affinity . To determine whether the reciprocal mutation in hENT2 (Ile33 to Met) also altered sensitivity to dilazep and dipyridamole, hENT2-I33M was created by site-directed mutagenesis . Although the resulting mutant (hENT2-I33M) displayed >10-fold higher dilazep and dipyridamole sensitivity and >8-fold higher uridine affinity compared with wild type hENT2, it retained insensitivity to NBMPR . These data established that mutation of residue 33 (Met versus Ile) of hENT1 and hENT2 altered the dilazep and dipyridamole sensitivities in both proteins, suggesting that a common region of inhibitor interaction has been identified.

Proc Natl Acad Sci U S A, 2001 Nov 6, 98(23), 12972 - 7 Epub 2001 Oct 30.
Internal initiation in Saccharomyces cerevisiae mediated by an initiator tRNA/eIF2-independent internal ribosome entry site element; Thompson SR et al.; Internal initiation of translation can be mediated by specific internal ribosome entry site (IRES) elements that are located in certain mammalian and viral mRNA molecules . Thus far, these mammalian cellular and viral IRES elements have not been shown to function in the yeast Saccharomyces cerevisiae . We report here that a recently discovered IRES located in the genome of cricket paralysis virus can direct the efficient translation of a second URA3 cistron in dicistronic mRNAs in S . cerevisiae, thereby conferring uracil-independent growth . Curiously, the IRES functions poorly in wild-type yeast but functions efficiently either in the presence of constitutive expression of the eIF2 kinase GCN2 or in cells that have two initiator tRNA(met) genes disrupted . Both of these conditions have been shown to lower the amounts of ternary eIF2-GTP/initiator tRNA(met) complexes . Furthermore, tRNA(met)-independent initiation was also observed in translation-competent extracts prepared from S . cerevisiae in the presence of edeine, a compound that has been shown to interfere with start codon recognition by ribosomal subunits carrying ternary complexes . Therefore, the cricket paralysis virus IRES is likely to recruit ribosomes by internal initiation in S . cerevisiae in the absence of eIF2 and initiator tRNA(met), by the same mechanism of factor-independent ribosome recruitment used in mammalian cells . These findings will allow the use of yeast genetics to determine the mechanism of internal ribosome entry.

Anal Chem, 2001 Oct 15, 73(20), 4891 - 902
Peptide mass mapping constrained with stable isotope-tagged peptides for identification of protein mixtures; Hunter TC et al.; Through proteolysis and peptide mass determination using mass spectrometry, a peptide mass map (PMM) can be generated for protein identification . However, insufficient peptide mass accuracy and protein sequence coverage limit the potential of the PMM approach for high-throughput, large-scale analysis of proteins . In our novel approach, nonlabile protons in particular amino acid residues were replaced with deuteriums to mass-tag proteins of the S . cerevisiae proteome in a sequence-specific manner . The resulting mass-tagged proteolytic peptides with characteristic mass-split patterns can be identified in the data search using constraints of both amino acid composition and mass-to-charge ratio . More importantly, the mass-tagged peptides can further act as internal calibrants with high confidence in a PMM to identify the parent proteins at modest mass accuracy and low sequence coverage . As a result, the specificity and accuracy of a PMM was greatly enhanced without the need for peptide sequencing or instrumental improvements to obtain increased mass accuracy . The power of PMM has been extended to the unambiguous identification of multiple proteins in a 1D SDS gel band including the identification of a membrane protein.

Curr Genet, 2001 Sep, 40(2), 119 - 27
Trichoderma reesei rho3 a homologue of yeast RH03 suppresses the growth defect of yeast sec15-1 mutation; Vasara T et al.; The Trichoderma reesei gene, rho3, encoding the functional homologue of the Saccharomyces cerevisiae small GTP-binding protein Rho3p was cloned as a suppressor of the secretion-deficient mutation sec15-1 in yeast . The encoded protein showed 61% amino acid identity to the Rho3 protein . Rescue of the growth defect of a RHO3 disruption strain by an expression vector carrying rho3 cDNA confirmed the functional homology with the S . cerevisiae RHO3 gene . In addition, overproduction of T . reesei RHOIII in this yeast strain appeared to improve the actin organization and chitin localization of the cells . Three putative mutant (rho3Gly20Val alleles of the T . reesei rho3 gene rho3 Thr25Asn, rho3Asp12Ala) were introduced into the wild-type yeast, in yeast with sec15 mutation and in yeast with Rho3p depletion . Cells expressing rho3Gly20Val displayed wild-type growth and those expressing rho3 Thr25Asn and rho3Asp126Ala had a loss-of-function phenotype.

Curr Biol, 2001 Oct 16, 11(20), 1624 - 30
spRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function in fission yeast; Kanoh J et al.; Telomeres are essential for genome integrity . scRap1 (S . cerevisiae Rap1) directly binds to telomeric DNA and regulates telomere length and telomere position effect (TPE) by recruiting two different groups of proteins to its RCT (Rap1 C-terminal) domain . The first group, Rif1 and Rif2, regulates telomere length . The second group, Sir3 and Sir4, is involved in heterochromatin formation . On the other hand, human TRF1 and TRF2, as well as their fission yeast homolog, Taz1, directly bind to telomeric DNA and negatively regulate telomere length . Taz1 also plays important roles in TPE and meiosis . Human Rap1, the ortholog of scRap1, negatively regulates telomere length and appears to be recruited to telomeres by interacting with TRF2 . Here, we describe two novel fission yeast proteins, spRap1 (S . pombe Rap1) and spRif1 (S . pombe Rif1), which are orthologous to scRap1 and scRif1, respectively . spRap1 and spRif1 are independently recruited to telomeres by interacting with Taz1 . The rap1 mutant is severely defective in telomere length control, TPE, and telomere clustering toward the spindle pole body (SPB) at the premeiotic horsetail stage, indicating that spRap1 has critical roles in these telomere functions . The rif1 mutant also shows some defects in telomere length control and meiosis . Our results indicate that Taz1 provides binding sites for telomere regulators, spRap1 and spRif1, which perform the essential telomere functions . This study establishes the similarity of telomere organization in fission yeast and humans.

Curr Biol, 2001 Oct 16, 11(20), 1600 - 5
Characterization of MTMR3 . an inositol lipid 3-phosphatase with novel substrate specificity; Walker DM et al.; Inositol lipids play key roles in many fundamental cellular processes that include growth, cell survival, motility, and membrane trafficking . Recent studies on the PTEN and Myotubularin proteins have underscored the importance of inositol lipid 3-phosphatases in cell function . Inactivating mutations in the genes encoding PTEN and Myotubularin are key steps in the progression of some cancers and in the onset of X-linked myotubular myopathy, respectively . Myotubularin-related protein 3 (MTMR3) shows extensive homology to Myotubularin, including the catalytic domain, but additionally possesses a C-terminal extension that includes a FYVE domain . We show that MTMR3 is an inositol lipid 3-phosphatase, with a so-far-unique substrate specificity . It is able to hydrolyze PtdIns3P and PtdIns3,5P2, both in vitro and when heterologously expressed in S . cerevisiae, and to thereby provide the first clearly defined route for the cellular production of PtdIns5P . Overexpression of a catalytically dead MTMR3 (C413S) in mammalian cells induces a striking formation of vacuolar compartments that enclose membranous structures that are highly concentrated in mutant proteins.

J Org Chem, 1999 Mar 5, 64(5), 1535 - 1542
Site-Directed Mutagenesis of the Sterol Methyl Transferase Active Site from Saccharomyces cerevisiae Results in Formation of Novel 24-Ethyl Sterols; Nes WD et al.; Delta(24(28))-Sterols are end products of a mono C-methylation pathway catalyzed by the native Delta(24(25))- to Delta(24(28))-sterol methyl transferase (SMT) enzyme from Saccharomyces cerevisiae . Using a Tyr(81) to Phe mutant SMT enzyme of S . cerevisiae, generated by site-directed mutagenesis of a highly conserved residue in the sterol binding site, we found that several Delta(24(25))- and Delta(24(28))-sterols, which are not substrates for the native protein, were catalyzed to mono- and bis-C24-alkylated side chains . The mutant protein behaved similarly to the native protein in chromatography and in binding zymosterol, the preferred substrate . Zymosterol was converted to fecosterol by the Y81F mutant protein with similar turnover efficiency as the native protein (K(m) = 12 &mgr;M and k(cat) = 0.01 s(-)(1)); trace 24-ethyl sterols were detected from these incubations . 4alpha-Methyl zymosterol, which is not a normal substrate for the wild-type SMT enzyme, was converted to 4alpha-methyl fecosterol in high yield . When fecosterol and 4alpha-methyl fecosterol were assayed individually at saturating concentrations only fecosterol served as an effective substrate for the second C-transfer step (K(m) = 38 &mgr;M and k(cat) = 0.002 s(-)(1)), suggesting that successive C-methylation of Delta(24(28))-substrates is limited by product release and that molecular recognition of sterol features involves hydrogen bond formation . Isomeric 24-ethyl sterol olefins generated from 24(28)-methylene cholesterol were characterized by chromatographic (GC and HPLC) and spectral methods (MS and (1)H NMR), viz., fucosterol, isofucosterol, and clerosterol . Changes in rate of C-methylation and product distributions resulting from deuterium substitution at C28 were used to establish the kinetic isotope effects (KIEs) for the various deprotonations leading to C24-methylene, C24-ethylidene, and C24-ethyl sterols . An isotope effect on C28 methyl deprotonation generated during the first C(1)-transfer was detected with zymosterol and desmosterol paired with AdoMet and {(2)H(3)-methyl}AdoMet . A similar experiment to test for a KIE generated during the second C(1)-transfer reaction with AdoMet paired with 24(28)-methylenecholesterol and {28-(2)H(2)}24(28)-methylene cholesterol indicated an inverse isotope effect associated with C27 deprotonation . Alteration in the proportion of the C24 alkylated olefinic products generated by the pure Y81F mutant resulted from the suppression of the formation of Delta(24(28))-ethylidene sterols (C28 deprotonation) by a primary deuterium isotope effect with a compensating stimulation of the formation of 24-ethyl sterols (C27 deprotonation) . Kinetic study on the rate of product formation indicated a normal KIE of k(H)/k(D) = 2.62 for the first C(1)-transfer . Alternatively, an inverse KIE was established with k(H)/k(D) = 0.9 for the second C(1)-transfer resulting from conversion of the 24(28)-double bond (sp(2) hybridization) to a 24beta-ethyl group (sp(3) hybridization) . From the structures and stereochemical assignments of the C-ethyl olefin products, the stereochemistry of the attack of AdoMet in the second C(1)-transfer was found to operate a Si-face (backside) attack at C24, analogous to the first C(1)-transfer reaction.

Mutat Res, 2001 Oct, 489(1), 79 - 96
The pso mutants of Saccharomyces cerevisiae comprise two groups: one deficient in DNA repair and another with altered mutagen metabolism; Brendel M et al.; Yeast mutants that are sensitive to photoactivated psoralens, named pso mutants, were isolated and described more than 20 years ago . Nine genes responsible for the pso phenotypes were identified and seven of them cloned and molecularly characterized . Of the nine PSO genes of yeast seven apparently encode proteins involved in the repair of DNA lesions generated by photoinduced psoralens and by other mutagens, while two, PSO6 and PSO7, are responsible for structural elements of the membrane and for a functional respiratory chain, respectively . Of the seven proven or putative DNA repair genes six directly or indirectly control induced mutagenesis . Four of these PSO loci were found allelic to already known repair genes, whereas two, PSO2 and PSO4, represent new genes involved in DNA repair and in repair/pre-mRNA processing in S . cerevisiae . Gene PSO2 encodes a protein indispensable for repair of DNA interstrand cross-links that are produced by a variety of bi- and poly-functional mutagens and that appears to be important for a likewise repair function in humans as well.

Cell, 2001 Oct 19, 107(2), 149 - 59
hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase; Vaziri H et al.; DNA damage-induced acetylation of p53 protein leads to its activation and either growth arrest or apoptosis . We show here that the protein product of the gene hSIR2(SIRT1), the human homolog of the S . cerevisiae Sir2 protein known to be involved in cell aging and in the response to DNA damage, binds and deacetylates the p53 protein with a specificity for its C-terminal Lys382 residue, modification of which has been implicated in the activation of p53 as a transcription factor . Expression of wild-type hSir2 in human cells reduces the transcriptional activity of p53 . In contrast, expression of a catalytically inactive hSir2 protein potentiates p53-dependent apoptosis and radiosensitivity . We propose that hSir2 is involved in the regulation of p53 function via deacetylation.

Biotechnol Bioeng, 2001 Nov, 76(3), 247 - 58
Quantitative analysis of the regulation scheme of invertase expression in Saccharomyces cerevisiae; Herwig C et al.; In Saccharomyces cerevisiae, the expression of invertase, which is the hydrolyzing enzyme of sucrose, is controlled by the presence of monosaccharides, such as glucose and fructose, and referred to as carbon catabolite repression . To date, efforts have been made to identify the mechanism by which cells sense extracellular monosaccharide concentrations and trigger the genes involved in the repression pathway . The aim of the present work was to quantitatively investigate the cellular regulation of invertase expression in the wild-type strain S . cerevisiae CEN.PK113-7D during batch growth containing mixed sugar substrates under different initial conditions . Because of the high frequency and accurate online analysis of multiple components, a tight control of invertase expression could be observed, and threshold concentrations of the monosaccharides for derepression could be determined to 0.5 gl(-1) for glucose and 2 gl(-1) for fructose . Also, the existence of a hitherto undescribed regulatory state, in which cells regulate invertase expression very precisely and operate over long periods at monosaccharide concentrations lower than the above thresholds, could be demonstrated . All experimental observations could be summarized in a formulation of the cellular regulation scheme of invertase expression . A simple kinetic model could show that the regulation scheme explains the observed behavior very well . Additionally, the model was able to explain consequences of the regulation on the global metabolism .

Development, 2001 Oct, 128(20), 4045 - 55
The C . elegans maternal-effect gene clk-2 is essential for embryonic development, encodes a protein homologous to yeast Tel2p and affects telomere length; Benard C et al.; The Caenorhabditis elegans maternal-effect clk genes are involved in the temporal control of development and behavior . We report the genetic and molecular characterization of clk-2 . A temperature-sensitive mutation in the gene clk-2 affects embryonic and post-embryonic development, reproduction, and rhythmic behaviors . Yet, virtually all phenotypes are fully maternally rescued . Embryonic development strictly requires the activity of maternal clk-2 during a narrow time window between oocyte maturation and the two- to four-cell embryonic stage . Positional cloning of clk-2 reveals that it encodes a protein homologous to S . cerevisiae Tel2p . In yeast, the gene TEL2 regulates telomere length and participates in gene silencing at subtelomeric regions . In C . elegans, clk-2 mutants have elongated telomeres, and clk-2 overexpression can lead to telomere shortening . Tel2p has been reported to bind to telomeric DNA repeats in vitro . However, we find that a functional CLK-2::GFP fusion protein is cytoplasmic in worms . We discuss how the phenotype of clk-2 mutants could be the result of altered patterns of gene expression.

Genetics, 2001 Oct, 159(2), 527 - 35
Molecular population genetics and evolution of a prion-like protein in Saccharomyces cerevisiae; Jensen MA et al.; The prion-like behavior of Sup35p, the eRF3 homolog in the yeast Saccharomyces cerevisiae, mediates the activity of the cytoplasmic nonsense suppressor known as {PSI(+)} . Sup35p is divided into three regions of distinct function . The N-terminal and middle (M) regions are required for the induction and propagation of {PSI(+)} but are not necessary for translation termination or cell viability . The C-terminal region encompasses the termination function . The existence of the N-terminal region in SUP35 homologs of other fungi has led some to suggest that this region has an adaptive function separate from translation termination . To examine this hypothesis, we sequenced portions of SUP35 in 21 strains of S . cerevisiae, including 13 clinical isolates . We analyzed nucleotide polymorphism within this species and compared it to sequence divergence from a sister species, S . paradoxus . The N domain of Sup35p is highly conserved in amino acid sequence and is highly biased in codon usage toward preferred codons . Amino acid changes are under weak purifying selection based on a quantitative analysis of polymorphism and divergence . We also conclude that the clinical strains of S . cerevisiae are not recently derived and that outcrossing between strains in S . cerevisiae may be relatively rare in nature.

Eur J Biochem, 2001 Oct, 268(20), 5375 - 85
Phosphorylation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required for protein synthesis in vitro and in vivo; McKendrick L et al.; Eukaryotic translation initiation factor 4E (eIF4E) is essential for efficient translation of the vast majority of capped cellular mRNAs; it binds the 5'-methylated guanosine cap of mRNA and serves as a nucleation point for the assembly of the 48S preinitiation complex . eIF4E is phosphorylated in vivo at residue 209 of the human sequence . The phosphorylated form is often regarded as the active state of the protein, with ribosome-associated eIF4E enriched for the phosphorylated form and increased phosphorylation often correlated with upregulation of rates of protein synthesis . However, the only reported measured effect attributable to phosphorylation at the physiological site has been a relatively small increase in the affinity of eIF4E for the mRNA m7GTP cap structure . Here, we provide data to suggest that phosphorylation of eIF4E at Ser209 is not required for translation . eIF4E that is modified such that it cannot be phosphorylated (Ser209-->Ala), is unimpaired in its ability to restore translation to an eIF4E-dependent in vitro translation system . In addition, both the wild-type and mutant forms of eIF4E interact equally well with eIF4G, with the phosphorylation of eIF4E not required to effect the change in conformation of eIF4G that is required for efficient cleavage of eIF4G by L-protease . Furthermore, we show that wild-type and phosphorylation-site variants of eIF4E protein are equally able to rescue the lethal phenotype of eIF4E deletion in S . cerevisiae.

J Biol Chem, 2001 Dec 21, 276(51), 48526 - 31 Epub 2001 Oct 15.
Cell cycle-dependent proteolysis and phosphorylation of human Mcm10; Izumi M et al.; Mcm10 (Dna43) is an essential protein for chromosomal DNA replication in Saccharomyces cerevisiae . Recently, we identified a human Mcm10 homolog that interacts with the mammalian Orc2 and Mcm2-7 complex . We additionally demonstrated that human Mcm10 binds nuclease-resistant nuclear structures during S phase and dissociates from them in G(2) phase . In this study, we have further characterized the subcellular localization, modification, and expression levels of human Mcm10 protein throughout the cell cycle . Human Mcm10 protein decreased in late M phase, remained low during G(1) phase, started to accumulate, and bound chromatin at the onset of S phase . Proteasome inhibitors stabilized Mcm10 levels, suggesting that proteolysis is involved in the down-regulation of the protein in late M/G(1) phase . Dissociation of Mcm10 from chromatin in G(2)/M phase was concomitant with alterations in the electrophoretic mobility of the protein . Treatment with lambda phosphatase revealed that mobility shifts were due to hyperphosphorylation . These results indicate that human Mcm10 is regulated by proteolysis and phosphorylation in a cell cycle-dependent manner . It is further suggested that mammalian Mcm10 is involved in S phase progression, and not the formation of a prereplicative complex, as previously proposed from data on the S . cerevisiae protein.

Biochemistry, 2001 Oct 23, 40(42), 12695 - 703
Saccharomyces cerevisiae leukotriene A4 hydrolase: formation of leukotriene B4 and identification of catalytic residues; Kull F et al.; Leukotriene A(4) hydrolase in mammals is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of leukotriene A(4) into the proinflammatory mediator leukotriene B(4), and also possesses an aminopeptidase activity . Recently we cloned and characterized an leukotriene A(4) hydrolase from Saccharomyces cerevisiae as a leucyl aminopeptidase with an epoxide hydrolase activity . Here we show that S . cerevisiae leukotriene A(4) hydrolase is a metalloenzyme containing one zinc atom complexed to His-340, His-344, and Glu-363 . Mutagenetic analysis indicates that the aminopeptidase activity follows a general base mechanism with Glu-341 and Tyr-429 as the base and proton donor, respectively . Furthermore, the yeast enzyme hydrolyzes leukotriene A(4) into three compounds, viz., 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid, leukotriene B(4), and Delta(6)-trans-Delta(8)-cis-leukotriene B(4), with a relative formation of 1:0.2:0.1 . In addition, exposure of S . cerevisiae leukotriene A(4) hydrolase to leukotriene A(4) selectively inactivates the epoxide hydrolase activity with a simultaneous stimulation of the aminopeptidase activity . Moreover, kinetic analyses of wild-type and mutated S . cerevisiae leukotriene A(4) hydrolase suggest that leukotriene A(4) binds in one catalytic mode and one tight-binding, regulatory mode . Exchange of a Phe-424 in S . cerevisiae leukotriene A(4) hydrolase for a Tyr, the corresponding residue in human leukotriene A(4) hydrolase, results in a protein that converts leukotriene A(4) into leukotriene B(4) with an improved efficiency and specificity . Hence, by a single point mutation, we could make the active site better suited to bind and turn over the substrate leukotriene A(4), thus mimicking a distinct step in the molecular evolution of S . cerevisiae leukotriene A(4) hydrolase toward its mammalian counterparts.

Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 776 - 9
Quantification of intracellular amino acids in batch cultures of Saccharomyces cerevisiae; Hans MA et al.; The dynamics of intracellular amino acid pools were determined in batch cultures of Saccharomyces cerevisiae . Immediate termination of metabolic activity was found to be necessary for accurate quantification of in vivo concentrations of intracellular amino acids, due to significant changes in most intracellular amino acid pools observed during extraction without an instantaneous stop of the metabolism . The method applied to batch-cultures of S . cerevisiae on glucose revealed complex dynamics in intracellular amino acid pools . The most drastic changes were observed during the diauxic shift and at the entry into the stationary phase . Even during phases of exponential growth on glucose and ethanol, cells showed significant variations in intracellular amino acid concentrations . The method presented can be used to investigate the physiology of yeast cultures, including industrially relevant batch and fed-batch processes.

Nucleic Acids Res, 2001 Oct 15, 29(20), 4215 - 23
Degeneration of a homing endonuclease and its target sequence in a wild yeast strain; Gimble FS; Mobile introns and inteins self-propagate by 'homing', a gene conversion process initiated by site-specific homing endonucleases . The VMA intein, which encodes the PI-SceI endonuclease in Saccharomyces cerevisiae, is present in several different yeast strains . Surprisingly, a wild wine yeast (DH1-1A) contains not only the intein(+) allele, but also an inteinless allele that has not undergone gene conversion . To elucidate how these two alleles co-exist, we characterized the endonuclease encoded by the DH1-1A intein(+) allele and the target site in the intein(-) allele . Sequence analysis reveals seven mutations in the 31 bp recognition sequence, none of which occurs at positions that are individually critical for activity . However, binding and cleavage of the sequence by PI-SceI is reduced 10-fold compared to the S.cerevisiae target . The PI-SceI analog encoded by the DH1-1A intein(+) allele contains 11 mutations at residues in the endonuclease and protein splicing domains . None affects protein splicing, but one, a R417Q substitution, accounts for most of the decrease in DNA cleavage and DNA binding activity of the DH1-1A protein . Loss of activity in the DH1-1A endonuclease and target site provides one explanation for co-existence of the intein(+) and intein(-) alleles.

Transgenic Res, 2001 Aug, 10(4), 317 - 28
FLP/FRT-mediated restoration of normal phenotypes and clonal sectors formation in rolC transgenic tobacco; Gidoni D et al.; Site-specific recombination systems have been shown to excise transgene DNA sequences positioned between their cognate target sites, and thus be used to generate clonal sectors in transgenic plants . Here we characterized clonal sectors derived from genetic reversion of rolC (A . rhizogenes)--induced vegetative and reproductive phenotypes, mediated by FLP recombinase from S . cerevisiae, in tobacco . The constitutive expression of rolC induces pleiotropic effects including reduced apical dominance and plant height, lanceolate and pale green leaves and small, male-sterile flowers . Two transgenic male-sterile tobacco lines (N . tabacum, Samsun NN) expressing a 35sP-rolC gene construct flanked by two FRT (FLP recombinase target) sites, were cross-pollinated with pollen from a constitutive 35sP-FLP expressing line . Three main phenotypes were generated in result of recombinase-mediated excision of the 35sP-rolC locus in the F1 (FLP x FRT-35sP-rolC-FRT) hybrid progenies: (a) restoration of male fertility, associated with reversion to normal leaf phenotypes prior to flower bud formation, (b) development of normal and fertile lateral shoot sectors on the background of rolC-type plants, (c) restoration of partially fertile flowers, associated with display of peripheral normal leaf sectors surrounding rolC-type inner-leaf tissues, consistent with periclinal chimeras . These results, supported by DNA molecular analysis, indicate that site-specific recombination might be used as a relatively efficient tool for generation of transgenic periclinal chimeric plants.

FEBS Lett, 2001 Oct 5, 506(2), 140 - 2
Toxicity of the bacterial luciferase substrate, n-decyl aldehyde, to Saccharomyces cerevisiae and Caenorhabditis elegans; Hollis RP et al.; This study determined that the bacterial luciferase fusion gene (luxAB) was not a suitable in vivo gene reporter in the model eukaryotic organisms Saccharomyces cerevisiae and Caenorhabditis elegans . LuxAB expressing S . cerevisiae strains displayed distinctive rapid decays in luminescence upon addition of the bacterial luciferase substrate, n-decyl aldehyde, suggesting a toxic response . Growth studies and toxicity bioassays have subsequently confirmed, that the aldehyde substrate was toxic to both organisms at concentrations well tolerated by Escherichia coli . As the addition of aldehyde is an integral part of the bacterial luciferase activity assay, our results do not support the use of lux reporter genes for in vivo analyses in these model eukaryotic organisms.

Structure (Camb), 2001 Oct, 9(10), 897 - 904
Structure of a conjugating enzyme-ubiquitin thiolester intermediate reveals a novel role for the ubiquitin tail; Hamilton KS et al.; BACKGROUND: Ubiquitin-conjugating enzymes (E2s) are central enzymes involved in ubiquitin-mediated protein degradation . During this process, ubiquitin (Ub) and the E2 protein form an unstable E2-Ub thiolester intermediate prior to the transfer of ubiquitin to an E3-ligase protein and the labeling of a substrate for degradation . A series of complex interactions occur among the target substrate, ubiquitin, E2, and E3 in order to efficiently facilitate the transfer of the ubiquitin molecule . However, due to the inherent instability of the E2-Ub thiolester, the structural details of this complex intermediate are not known . RESULTS: A three-dimensional model of the E2-Ub thiolester intermediate has been determined for the catalytic domain of the E2 protein Ubc1 (Ubc1(Delta450)) and ubiquitin from S . cerevisiae . The interface of the E2-Ub intermediate was determined by kinetically monitoring thiolester formation by 1H-(15)N HSQC spectra by using combinations of 15N-labeled and unlabeled Ubc1(Delta450) and Ub proteins . By using the surface interface as a guide and the X-ray structures of Ub and the 1.9 A structure of Ubc1(Delta450) determined here, docking simulations followed by energy minimization were used to produce the first model of a E2-Ub thiolester intermediate . CONCLUSIONS: Complementary surfaces were found on the E2 and Ub proteins whereby the C terminus of Ub wraps around the E2 protein terminating in the thiolester between C88 (Ubc1(Delta450)) and G76 (Ub) . The model supports in vivo and in vitro experiments of E2 derivatives carrying surface residue substitutions . Furthermore, the model provides insights into the arrangement of Ub, E2, and E3 within a ternary targeting complex.

J Cell Sci, 2001 Sep, 114(Pt 17), 3137 - 45
The Ccz1 protein interacts with Ypt7 GTPase during fusion of multiple transport intermediates with the vacuole in S . cerevisiae; Kucharczyk R et al.; Previously we have shown that the Saccharomyces cerevisiae CCZ1 (YBR131w) gene encodes a protein involved in protein trafficking . Deletion of this gene leads to fragmentation of the vacuole typical of the class B vps mutants . Genetic and biochemical data indicated that Ccz1p is required for fusion of various transport intermediates with the vacuole . Here we present data indicating that CCZ1 is a close partner of the YPT7 gene, which encodes Rab GTPase and is required for fusion of transport vesicles to vacuole and homotypic vacuole fusion . We isolated extragenic suppressors of CCZ1 deletion . All these suppressors belong to one complementation group and correspond to mutated alleles of the YPT7 gene . The mutated residues are located in two Ypt7p domains responsible for guanine binding . These data suggest that Ccz1p and Ypt7p interact physically . Coimmunoprecipitation experiments provide direct evidence that this indeed is the case . A possible mechanism of Ccz1p action is discussed.

Eur J Paediatr Neurol, 2001, 5 Suppl A, 127 - 9
A yeast model for classical juvenile Batten disease (CLN3); Barwell KJ et al.; The gene involved in the classical juvenile form of Batten disease, CLN3 has been identified as being highly homologous to the Saccharomyces cerevisiae YHC3 gene . To provide a simple model for the biochemical events underlying this disease, several disruptions have been made in YHC3 in three different S . cerevisiae strains . No obvious growth differences were observed, and neither was the previously reported phenotypic difference between wild-type and yeast disrupted in YHC3.

Mutat Res, 2001 May 10, 485(4), 345 - 55
Increased DNA double strand breakage is responsible for sensitivity of the pso3-1 mutant of Saccharomyces cerevisiae to hydrogen peroxide; Brozmanova J et al.; Escherichia coli endonuclease III (endo III) is the key repair enzyme essential for removal of oxidized pyrimidines and abasic sites . Although two homologues of endo III, Ntgl and Ntg2, were found in Saccharomyces cerevisiae, they do not significantly contribute to repair of oxidative DNA damage in vivo . This suggests that an additional activity(ies) or a regulatory pathway(s) involved in cellular response to oxidative DNA damage may exist in yeast . The pso3-1 mutant of S . cerevisiae was previously shown to be specifically sensitive to toxic effects of hydrogen peroxide (H2O2) and paraquat . Here, we show that increased DNA double strand breakage is very likely the basis of sensitivity of the pso3-1 mutant cells to H2O2 . Our results, thus, indicate an involvement of the Pso3 protein in protection of yeast cells from oxidative stress presumably through its ability to prevent DNA double strand breakage . Furthermore, complementation of the repair defects of the pso3-1 mutant cells by E . coli endo III has been examined . It has been found that expression of the nth gene in the pso3-1 mutant cells recovers survival, decreases mutability and protects yeast genomic DNA from breakage following H2O2 treatment . This might suggest some degree of functional similarity between Pso3 and Nth.

Plant Mol Biol, 2001 Aug, 46(6), 673 - 82
Establishment of Arabidopsis thaliana ribosomal protein RPL23A-1 as a functional homologue of Saccharomyces cerevisiae ribosomal protein L25; McIntosh KB et al.; Arabidopsis thaliana ribosomal protein (r-protein) RPL23A-1 shows 54% amino acid sequence identity to the Saccharomyces cerevisiae equivalent r-protein, L25 . AtRPL23A-1 also shows high amino acid sequence identity to members of the L23/L25 r-protein family in other species . R-protein L25 in S . cerevisiae has been identified as a primary rRNA-binding protein that directly binds to a specific site on yeast 26S rRNA . It is translocated to the nucleolus where it binds to 26S rRNA during early large ribosome subunit assembly; this binding is thought to play an important role in ribosome assembly . The S . cerevisiae mutant strain YCR61 expresses L25 when grown on galactose, but not glucose, medium . Transformation of YCR61 with a shuttle vector containing the AtRPL23A-1 cDNA allowed transformed colonies to grow in and on glucose selection medium . R-protein AtRPL23A-1 can complement the L25 mutation, demonstrating the functional equivalence of the two r-proteins and introducing AtRPL23A-1 as the first plant member of the L23/L25 r-protein family.

Gene, 2001 Sep 5, 275(1), 39 - 46
Cloning and characterization of the gene encoding human NPL4, a protein interacting with the ubiquitin fusion-degradation protein (UFD1L); Botta A et al.; The ubiquitin fusion-degradation gene (UFD1L) encodes the human homologue of the yeast ubiquitin fusion-degradation 1 protein, an essential component of the ubiquitin-dependent proteolytic turnover and mRNA processing . Although the UFD1L gene has been mapped in the region commonly deleted in patients with DiGeorge syndrome (DGS)/velocardiofacial syndrome (VCFS), correlation between its haploinsufficiency and the phenotype has not yet been established . The only functional data available about mammalian Ufd1p is the ability to form a complex with the rat Npl4 protein, a component of the nuclear pore complex . In this paper we report the cloning and molecular characterization of the human NPL4 gene . This gene encodes for a protein 96% homologous to the rat Npl4, and 44 and 34% homologous to the C . elegans and S . cerevisiae Npl4 gene products, respectively . Fluorescence in situ hybridization experiments on human metaphases localized the NPL4 gene on the most telomeric region of chromosome 17q . Northern blots analysis on foetal and adult human tissues revealed a major approximately 4.5 kb transcript most abundant in heart, brain, kidney and skeletal muscle . In order to test a potential relationship between nuclear transport defects and some aspect of the DGS/VCFS phenotype, we also exclude the presence of mutations in the NPL4 coding sequence in a subset of patients with DGS/VCFS and no detectable 22q11 deletion or mutations at the UFD1L locus.

Cell, 2001 Sep 21, 106(6), 735 - 44
An E3-like factor that promotes SUMO conjugation to the yeast septins; Johnson ES et al.; Covalent attachment of the ubiquitin-related protein SUMO to other proteins participates in many processes including signal transduction, transcriptional regulation, and growth control . We report the characterization of Siz1 as an E3-like factor in the SUMO pathway . Siz1 is required for SUMO attachment to the S . cerevisiae septins in vivo and strongly stimulates septin sumoylation in vitro . Siz1 and the related protein Siz2 promote SUMO conjugation to different substrates at different stages of the cell cycle and, together, are required for most SUMO conjugation in yeast . Siz1, Siz2, and the PIAS (protein inhibitor of activated STAT) proteins form a conserved family defined by an unusual RING-related motif . Our results suggest that this family functions by promoting SUMO conjugation to specific substrates.

Yeast, 2001 Oct, 18(14), 1277 - 84
Isolation of a Candida glabrata homologue of RAP1, a regulator of transcription and telomere function in Saccharomyces cerevisiae; Haw R et al.; To study the function of RAP1, an essential gene involved in the regulation of transcriptional activation, silencing and the telomere function in Saccharomyces cerevisiae, we isolated a Candida glabrata gene that complements the growth defect of a S . cerevisiae rap1 conditional mutant . The DNA sequence of the cloned gene, which we designated CgRAP1, predicted a 2064 bp open reading frame encoding a 687 amino acid protein with an overall identity of 65% and a similarity of 78% to Rap1p from S . cerevisiae .

EMBO J, 2001 Sep 17, 20(18), 5049 - 59
Identification and functional reconstitution of the yeast peroxisomal adenine nucleotide transporter; Palmieri L et al.; The requirement for small molecule transport systems across the peroxisomal membrane has previously been postulated, but not directly proven . Here we report the identification and functional reconstitution of Ant1p (Ypr128cp), a peroxisomal transporter in the yeast Saccharomyces cerevisiae, which has the characteristic sequence features of the mitochondrial carrier family . Ant1p was found to be an integral protein of the peroxisomal membrane and expression of ANT1 was oleic acid inducible . Targeting of Ant1p to peroxisomes was dependent on Pex3p and Pex19p, two peroxins specifically required for peroxisomal membrane protein insertion . Ant1p was essential for growth on medium-chain fatty acids as the sole carbon source . Upon reconstitution of the overexpressed and purified protein into liposomes, specific transport of adenine nucleotides could be demonstrated . Remarkably, both the substrate and inhibitor specificity differed from those of the mitochondrial ADP/ATP transporter . The physiological role of Ant1p in S.cerevisiae is probably to transport cytoplasmic ATP into the peroxisomal lumen in exchange for AMP generated in the activation of fatty acids.

Biophys J, 2001 Oct, 81(4), 2331 - 8
Resolving the individual components of a pH-induced conformational change; Blouin C et al.; This communication introduces a simple method to determine the pKs of microscopic ionizations from complex titration curves . We used this approach to study the alkaline transition (pH-dependent ligand exchange) of mitochondrial cytochrome c . The linearization of titration curves permitted resolution of two to three limiting microscopic ionizations . By combining these data with studies of the temperature dependence of ligand-exchange equilibria, we found evidence that the alkaline transition comprises two chemically distinct processes: the deprotonation of the alternative ligands and the break of the iron-methionine ligation bond . We also noted that, in the horse and untrimethylated S . cerevisiae iso-1 cytochromes c, the permissible deprotonation of the epsilon-amino group of Lys(72) allows formation of an alkaline isomer at lower pH, with lesser stability, which leads to hysteresis in the titration curves . The linearization of the titration curves for different cytochromes c thus brings insight on the microscopic contributions to conformational stability.

Electrophoresis, 2001 Aug, 22(14), 2969 - 82
Identification by mass spectrometry of two-dimensional gel electrophoresis-separated proteins extracted from lager brewing yeast; Joubert R et al.; As two-dimensional (2-D) electrophoresis allows the separation of several hundred proteins in a single gel, this technique has become an important tool for proteome studies and for investigating the cellular physiology . In order to take advantage of information provided by the comparison of proteome pictures, the mass spectrometry technique is the way chosen for a rapid and an accurate identification of proteins of interest . Unfortunately, in the case of industrial yeasts, due to the high level of complexity of their genome, the whole DNA sequence is not yet available and all encoded protein sequences are still unknown . Nevertheless, this study presents here 30 lager brewing yeast proteins newly identified with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), tandem mass spectrometry (MS/MS) and database searching against the protein sequences of Saccharomyces cerevisiae . The identified proteins of the industrial strain correspond to proteins which do not comigrate with known proteins of S . cerevisiae separated on 2-D gels . This study presents an application of the MS technique for the identification of industrial yeast proteins which are only homologous to the corresponding S . cerevisiae proteins.

Mol Cell Biol, 2001 Oct, 21(20), 6972 - 83
Saccharomyces cerevisiae Mob1p is required for cytokinesis and mitotic exit; Luca FC et al.; The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G(1) by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK) . Here, we demonstrate that, in addition to mitotic exit, S . cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation . The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect . Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble . The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings . The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly . We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis . Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1 . The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants . These results suggest that the MEN functions during the mitosis-to-G(1) transition to control cyclin-CDK inactivation and cytokinesis.

Nucleosides Nucleotides Nucleic Acids, 2001 Apr-Jul, 20(4-7), 873 - 7
Inhibition of in vitro pre-mRNA splicing in S . cerevisiae by branched oligonucleotides; Carriero S et al.; A series of V- and Y-shaped nucleic acids, related to the splicing intermediates derived from S . cerevisiae actin pre-mRNA, were prepared . The effects of such branched nucleic acids (bNAs) on the efficiency of in vitro pre-mRNA splicing in yeast were studied . The exogenous bNAs each effect the efficiency of splicing, yet to different degrees, depending on the sugar composition and topology of the molecules . Y-shaped RNAs inhibited the formation of mRNA (i.e . RNA splicing) to the greatest extent.

J Gen Virol, 2001 Oct, 82(Pt 10), 2353 - 62
E5 transforming proteins of papillomaviruses do not disturb the activity of the vacuolar H(+)-ATPase; Ashby AD et al.; Papillomaviruses contain a gene, E5, that encodes a short hydrophobic polypeptide that has transforming activity . E5 proteins bind to the 16 kDa subunit c (proteolipid) of the eukaryotic vacuolar H(+)-ATPase (V-ATPase) and this binding is thought to disturb the V-ATPase and to be part of transformation . This link has been examined in the yeast Saccharomyces cerevisiae . The E5 proteins from human papillomavirus (HPV) type 16, bovine papillomavirus (BPV) type 1, BPV-4 E5 and various mutants of E5 and the p12' polypeptide from human T-lymphotropic virus (HTLV) type I all bound to the S . cerevisiae subunit c (Vma3p) and could be found in vacuolar membranes . However, none affected the activity of the V-ATPase . In contrast, a dominant-negative mutant of Vma3p (E137G) inactivated the enzyme and gave the characteristic VMA phenotype . A hybrid V-ATPase containing a subunit c from Norway lobster also showed no disruption . Sedimentation showed that HPV-16 E5 was not part of the active V-ATPase . It is concluded that the binding of E5 and E5-related proteins to subunit c does not affect V-ATPase activity or function and it is proposed that the binding may be due to a chaperone function of subunit c.

Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11075 - 80 Epub 2001 Sep 18.
Gene transcription analysis of Saccharomyces cerevisiae exposed to neocarzinostatin protein-chromophore complex reveals evidence of DNA damage, a potential mechanism of resistance, and consequences of prolonged exposure; Schaus SE et al.; The natural product neocarzinostatin (NCS), a protein-small molecule complex, exhibits potent antiproliferative activity in mammalian cells but has little apparent effect on the growth of the unicellular eukaryotic organism, Saccharomyces cerevisiae . Here, we show by whole-genome transcription profiling experiments that incubation of S . cerevisiae with NCS leads to dramatic and wide-ranging modifications in the expression profile of yeast genes . Approximately 18% of yeast transcripts are altered by 2-fold or more within 4 h of treatment with NCS . Analysis of the observed transcription profile provides evidence that yeast rapidly and continuously overexpress multiple DNA-damage repair genes during NCS exposure . Perhaps to meet the energetic requirements of continuous DNA-damage repair, yeast cells enter respiration upon prolonged exposure to NCS, although grown in nutrient-rich medium . The NCS protein component is readily transported into S . cerevisiae, as demonstrated by fluorescence microscopy of yeast treated with fluorescently labeled NCS . Transcription profiling experiments with neocarzinostatin protein alone implicate a specific resistance mechanism in yeast that targets the NCS protein component, one involving the nonclassical export pathway . These experiments provide a detailed picture of the effects of exposure to NCS upon yeast and the mechanisms they engage as a response to this protein-small molecule DNA-damaging agent.

Mol Cells, 2001 Aug 31, 12(1), 84 - 90
Yeast ribosomal protein S3 has an endonuclease activity on AP DNA; Jung SO et al.; The mammalian ribosomal protein S3 (rpS3) is a component of the 40S ribosomal subunit . It is known to function as a DNA repair enzyme, UV endonuclease III, which cleaves DNA that is irradiated by UV . It also has an endonuclease activity on AP DNA . In this report, the yeast ribosomal protein S3 (Rps3p) in S . cerevisiae was cloned, expressed in E . coli, and affinity-purified by 285 fold . Rps3p is composed of 240 amino acids and has a 78% amino acid similarity with the human counterpart that has 243 amino acids . The major difference in the amino acid sequence between the two proteins lies in most of the C-terminal 50 residues . Surprisingly, Rps3p only showed an endonuclease activity on AP DNA, but not on DNA that was irradiated with UV . The AP endonuclease activity of Rps3p was affected by pH, KCl, and beta-mercaptoethanol, but Triton X-100 and EDTA did not affect the enzyme activity . From these results, both the mammalian rpS3 and Rps3p appear to be involved in DNA damage processing, but in different modes.

J Biol Chem, 2001 Dec 14, 276(50), 47239 - 47 Epub 2001 Sep 14.
Characterization of the Saccharomyces cerevisiae homolog of the melatonin rhythm enzyme arylalkylamine N-acetyltransferase (EC 2.3.1.87); Ganguly S et al.; Arylalkylamine N-acetyltransferase (AANAT, serotonin N-acetyltransferase, EC ) plays a unique transduction role in vertebrate physiology by converting information about day and night into a hormonal signal: melatonin . Only vertebrate members of the AANAT family have been functionally characterized . Here a putative AANAT from Saccharomyces cerevisiae (scAANAT) was studied to determine whether it possessed the catalytic activity of the vertebrate enzyme . scAANAT is 47% similar to ovine AANAT, but lacks the regulatory N- and C-terminal flanking regions conserved in all vertebrate AANATs . It was found to have enzyme activity generally typical for AANAT family members, although the substrate preference pattern was somewhat broader, the specific activity was lower, and the pH optimum was higher . Deletion of scAANAT reduced arylalkylamine acetylation by S . cerevisiae extracts, indicating that scAANAT contributes significantly to this process . The scAANAT sequence conformed to the three-dimensional structure of ovine AANAT catalytic core; however, an important structural element (loop 1) was found to be shorter and to lack a proline involved in substrate binding . These differences could explain the lower specific activity of scAANAT, because of the importance of loop 1 in catalysis . Data base analysis revealed the presence of putative AANATs in other fungi but not in the nearly complete genomes of Drosophila melanogaster or Caenorhabditis elegans . These studies indicate that the catalytic and kinetic characteristics of fungal and vertebrate enzymes can be considered to be generally similar, although some differences exist that appear to be linked to changes in one structural element . Perhaps the most striking difference is that fungal AANATs lack the regulatory domains of the vertebrate enzyme, which appear to be essential for the regulatory role the enzyme plays in photochemical transduction.

J Biol Chem, 2001 Nov 23, 276(47), 44179 - 84 Epub 2001 Sep 13.
All phox homology (PX) domains from Saccharomyces cerevisiae specifically recognize phosphatidylinositol 3-phosphate; Yu JW et al.; Phox homology (PX) domains are named for a 130-amino acid region of homology shared with part of two components of the phagocyte NADPH oxidase (phox) complex . They are found in proteins involved in vesicular trafficking, protein sorting, and lipid modification . It was recently reported that certain PX domains specifically recognize phosphatidylinositol 3-phosphate (PtdIns-3-P) and drive recruitment of their host proteins to the cytoplasmic leaflet of endosomal and/or vacuolar membranes where this phosphoinositide is enriched . We have analyzed phosphoinositide binding by all 15 PX domains encoded by the Saccharomyces cerevisiae genome . All yeast PX domains specifically recognize PtdIns-3-P in protein-lipid overlay experiments, with just one exception (a significant sequence outlier) . In surface plasmon resonance studies, four of the yeast PX domains bind PtdIns-3-P with high (micromolar range) affinity . Although the remaining PX domains specifically recognize PtdIns-3-P, they bind this lipid with only low affinity . Interestingly, many proteins with "low affinity" PX domains are known to form large multimeric complexes, which may increase the overall avidity for membranes . Our results establish that PtdIns-3-P, and not other phosphoinositides, is the target of all PX domains in S . cerevisiae and suggest a role for PX domains in assembly of multiprotein complexes at specific membrane surfaces.

Arch Biochem Biophys, 2001 Sep 15, 393(2), 215 - 21
The N-terminus of the Qcr7 protein of the cytochrome bc(1) complex in S . cerevisiae may be involved in facilitating stability of the subcomplex with the Qcr8 protein and cytochrome b; Lee SY et al.; Subunit 7 or the Qcr7 protein of the cytochrome bc(1) complex in yeast is a nuclear-encoded 14-kDa protein and is essential for formation of a functional bc(1) complex and respiration . It was shown previously that the N-terminal region of the Qcr7 protein might play little role in import but may be essential for correct assembly of the bc(1) complex . To examine the role of the N-terminus in assembly of the bc(1) complex, various N-terminus deletion and point mutants of the QCR7 gene were expressed in yeast strains where the endogenous QCR7 gene has been inactivated . Deletion of the first 8-15 residues after methionine at the N-terminus of the Qcr7 protein was studied and it was shown that except for the Qcr7p-Delta7 mutant, the strains overexpressing deletion mutants (Qcr7p-Delta8 to Qcr7p-Delta14) displayed decreased steady-state levels of iron-sulfur protein (ISP) and 14-kDa (Qcr7) and 11-kDa (Qcr8) subunits, as well as a respiratory defect . It was shown that introducing mutations at the N-terminus of the QCR7 gene in the Delta7 context had more drastic effects on respiration and assembly than in the full-length context, suggesting that the first seven residues at the N-terminus have a role in increasing the stability of the holocomplex . This led to a respiratory-deficient phenotype and drastic decrease in the steady-state levels of Qcr7 (14-kDa) and Qcr8 (11-kDa) proteins . The mutants Qcr7p-Delta7 (R10I), Qcr7p-Delta7 (G12V), Qcr7p-Delta7 (D13G), Qcr7p-Delta7 (D13V), and Qcr7p-Delta7 (D13Y) showed decreases in the steady-state levels of ISP and 14- and 11-kDa subunits, as well as defects in respiration . These results are interpreted in the light of the X-ray crystal structure of the yeast bc(1) complex .

J Biol Chem, 2001 Nov 9, 276(45), 41998 - 2002 Epub 2001 Sep 12.
A novel acetyltransferase found in Saccharomyces cerevisiae Sigma1278b that detoxifies a proline analogue, azetidine-2-carboxylic acid; Shichiri M et al.; L-Azetidine-2-carboxylic acid (AZC), a toxic four-membered ring analogue of L-proline, is transported into the cells via proline transporters . It causes misfolding of the proteins into which it is incorporated competitively with L-proline and thereby inhibits the growth of the cells . We recently have discovered, on the chromosome of Saccharomyces cerevisiae Sigma1278b, a novel gene MPR1 required for the resistance of Sigma1278 background strains to toxic AZC . This gene was missing in the particular yeast strain used for the genomic sequence determination . Although the protein sequence was homologous to that of the S . cerevisiae transcriptional regulator, Mpr1p did not affect the expression of genes involved in proline uptake . However, gene expression in Escherichia coli and enzymatic analysis showed that the MPR1 gene encodes a novel AZC acetyltransferase, by which L-proline itself and other L-proline analogues are not acetylated . Mpr1p was considered to be a member of the N-acetyltransferase superfamily based on the results of an Ala-scan mutagenesis through the highly conserved region involved in binding acetyl-CoA in members of the superfamily . Our findings suggest that Mpr1p detoxifies AZC by acetylating it in the cytoplasm . This enzyme might be utilized as a selective marker in a wide variety of organisms, because the cells expressing the MPR1 gene acquire the AZC-resistant phenotype.

Lett Appl Microbiol, 2001 Sep, 33(3), 206 - 10
Heteroduplex mobility assay of the D1/D2 region of the 26S rDNA for differentiation of Saccharomyces species; Ramos JP et al.; AIMS: We present the HMA method for Saccharomyces differentiation using the PCR amplified D1/D2 26S rDNA . METHODS AND RESULTS: This methodology is based on heteroduplex formation when two different DNAs are hybridized . We tested 11 type cultures of Saccharomyces, 27 different cultures of S . cerevisiae and four other ascomycetic genera . CONCLUSION: The method was capable of differentiating Saccharomyces species and was mainly very efficient for S . cerevisiae identification . HMA can probably be applied in other genera, where identification is sometimes difficult only by conventional traits, which are based on physiology and morphology . SIGNIFICANCE AND IMPACT OF THE STUDY: HMA provides a rapid and relatively simple molecular tool, contributing for yeast taxonomy.

Prog Nucleic Acid Res Mol Biol, 2001, 68, 29 - 39
Yeast base excision repair: interconnections and networks; Doetsch PW et al.; The removal of oxidative base damage from the genome of Saccharomyces cerevisiae is thought to occur primarily via the base excision repair (BER) pathway in a process initiated by several DNA N-glycosylase/AP lyases . We have found that yeast strains containing simultaneous multiple disruptions of BER genes are not hypersensitive to killing by oxidizing agents, but exhibit a spontaneous hyperrecombinogenic (hyper-rec) and mutator phenotype . The hyper-rec and mutator phenotypes are further enhanced by elimination of the nucleotide excision repair (NER) pathway . Furthermore, elimination of either the lesion bypass (REV3-dependent) or recombination (RAD52-dependent) pathway results in a further, specific enhancement of the hyper-rec or mutator phenotypes, respectively . Sensitivity (cell killing) to oxidizing agents is not observed unless multiple pathways are eliminated simultaneously . These data suggest that the BER, NER, recombination, and lesion bypass pathways have overlapping specificities in the removal of, or tolerance to, exogenous or spontaneous oxidative DNA damage in S . cerevisiae . Our results also suggest a physiological role for the AP lyase activity of certain BER N-glycosylases in vivo.

Mol Biol Cell, 2001 Sep, 12(9), 2672 - 87
Single site alpha-tubulin mutation affects astral microtubules and nuclear positioning during anaphase in Saccharomyces cerevisiae: possible role for palmitoylation of alpha-tubulin; Caron JM et al.; We generated a strain of Saccharomyces cerevisiae in which the sole source of alpha-tubulin protein has a cys-to-ser mutation at cys-377, and then we examined microtubule morphology and nuclear positioning through the cell cycle . During G1 of the cell cycle, microtubules in the C377S alpha-tubulin (C377S tub1) mutant were indistinguishable from those in the control (TUB1) strain . However, mitotic C377S tub1 cells displayed astral microtubules that often appeared excessive in number, abnormally long, and/or misoriented compared with TUB1 cells . Although mitotic spindles were always correctly aligned along the mother-bud axis, translocation of spindles through the bud neck was affected . In late anaphase, spindles were often not laterally centered but instead appeared to rest along the sides of cells . When the doubling time was increased by growing cells at a lower temperature (15 degrees C), we often found abnormally long mitotic spindles . No increase in the number of anucleate or multinucleate C377S mutant cells was found at any temperature, suggesting that, despite the microtubule abnormalities, mitosis proceeded normally . Because cys-377 is a presumptive site of palmitoylation in alpha-tubulin in S . cerevisiae, we next compared in vivo palmitoylation of wild-type and C377S mutant forms of the protein . We detected palmitoylated alpha-tubulin in TUB1 cells, but the cys-377 mutation resulted in approximately a 60% decrease in the level of palmitoylated alpha-tubulin in C377S tub1 cells . Our results suggest that cys-377 of alpha-tubulin, and possibly palmitoylation of this amino acid, plays a role in a subset of astral microtubule functions during nuclear migration in M phase of the cell cycle.

Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 420 - 4
Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast; Geerlings A et al.; A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for strictosidine synthase (STR) and strictosidine beta-glucosidase (SGD) from the medicinal plant Catharanthus roseus . Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids . The yeast culture was found to express high levels of both enzymes . STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight) . Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of strictosidine in the medium; levels up to 2 g/l were measured . Inside the yeast cells strictosidine was also detected, although in much lower amounts (0.2 mg/g cells) . This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine . However, the strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells . Furthermore, transgenic S . cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates . Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids . Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.

Mol Cell, 2001 Aug, 8(2), 417 - 26
Structure of the catalytic core of S . cerevisiae DNA polymerase eta: implications for translesion DNA synthesis; Trincao J et al.; DNA polymerase eta is unique among eukaryotic polymerases in its proficient ability to replicate through a variety of distorting DNA lesions . We report here the crystal structure of the catalytic core of S . cerevisiae DNA polymerase eta, determined at 2.25A resolution . The structure reveals a novel polydactyl right hand-shaped molecule with a unique polymerase-associated domain . We identify the catalytic residues and show that the fingers and thumb domains are unusually small and stubby . In particular, the unexpected absence of helices "O" and "O1" in the fingers domain suggests that openness of the active site is the critical feature which enables DNA polymerase eta to replicate through DNA lesions such as a UV-induced cis-syn thymine-thymine dimer.

Int J Food Microbiol, 2001 Aug 15, 68(1-2), 75 - 81
A simplified procedure to analyse mitochondrial DNA from industrial yeasts; Lopez V et al.; A rapid method based on mtDNA restriction analysis is described for yeast strain identification . The method is an adaptation of that devised by Querol et a} . {Syst . Appl . Microbiol . 15 (1992) 439} for Saccharomyces cerevisiae wine strains, and consists of the standard miniprep isolation of yeast total DNA, and the use of restriction endonucleases that recognise a large number of sites in yeast nuclear DNA, but few sites in the mitochondrial DNA . In the adapted method, the propagation of yeast cells and restriction analysis were the steps mainly affected: cell growth was reduced to 36 h by using microfuge tubes, and the restriction analysis was carried out in just 33 min using a microwave oven for DNA digestion, and minigels for restriction fragment separation . The DNA extraction procedure was performed in the same way as in the original protocol . but slightly reducing the duration of each step and scaling down the volumes of the different solutions . enzymes and reagents used . As result, a large time reduction (52.5 h) was obtained compared to the original method . The DNA obtained can be directly digested with endonucleases displaying clear restriction patterns useful for S . cerevisiae yeast strain differentiation . In addition, strains belonging to other foodborne yeast species, including spoilage yeast species, can also be identified.

Can J Gastroenterol, 2001 Aug, 15(8), 499 - 504
Development of an assay for antibodies to Saccharomyces cerevisiae: Easy, cheap and specific for Crohn's disease; Bernstein CN et al.; OBJECTIVE: To develop a serological test to measure antibodies to Saccharomyces cerevisiae in patients with inflammatory bowel disease . METHODS: An ELISA to the mannan of S cerevisiae that is commercially available was developed . Sera were tested from randomly chosen sera specimens kept frozen at the University of Manitoba Inflammatory Bowel Disease Clinical and Research Centre, Winnipeg, Manitoba . Clinical diagnoses were kept blinded until the assay results were finalized . One hundred thirty-six sera were tested, including 51 with Crohn's disease, 32 with ulcerative colitis, one with indeterminate colitis and 16 other control subjects . Thirty-six samples were duplicates from patients already studied but were either run on separate days or drawn on different days . RESULTS: Using a cutoff of 15 binding units as a positive result, Crohn's disease was found to have a sensitivity of 53% but a specificity of 100% compared with ulcerative colitis . Compared with all other diagnoses (including ulcerative colitis), Crohn's disease had a sensitivity of 53% and a specificity of 96% . For patients with Crohn's disease only, those who were anti-S cerevisiae antibody (ASCA) positive (n=27) were significantly more likely to have proximal gastrointestinal disease and significantly less likely to have colonic or inflammatory type disease than those who were ASCA negative (n=24) . The direct cost of this assay was $6.00 per positive test, and the total charge was set at $38.15 . CONCLUSIONS: A reasonably inexpensive, easy and reproducible assay to assess for antibodies to S cerevisiae has been developed . Using a cutoff for positivity of 15 binding units, this test had a specificity of 100% for ruling out Crohn's disease and a lower (60%) sensitivity compared with ulcerative colitis . This test could identify a specific phenotype of patients with Crohn's disease as being more likely to have small bowel Crohn's disease and less likely to have colonic (isolated) or inflammatory disease, as opposed to fibrostenotic disease or penetrating disease . The test proved reliable when assaying samples drawn or assayed on different days.

Yeast, 2001 Sep 15, 18(12), 1145 - 54
AFLP analysis of type strains and laboratory and industrial strains of Saccharomyces sensu stricto and its application to phenetic clustering; Azumi M et al.; Using nine primer pairs, amplified fragment length polymorphism (AFLP) analysis was conducted to characterize industrial, laboratory and type strains of Saccharomyces sensu stricto . S . cerevisiae, S . bayanus, S . carlsbergensis and S . paradoxus had species-specific AFLP profiles, with some variations among the strains . Nineteen wine, ale, bakery, whisky and laboratory strains of S . cerevisiae were differentiated by two primer pairs, while out of 19 strains of sake yeast, two groups consisting of two and eight strains were not differentiated using nine primer pairs . A phenogram of 41 strains of S . cerevisiae, two strains of S . bayanus, the type strain of S . pastorianus, three strains of S . carlsbergensis, one hybrid strain of S . cerevisiae and S . bayanus and the type strain of S . paradoxus was obtained by the unweighted pair group method, using arithmetic averages (UPGMA) based on the percentage of shared AFLP fragments of each sample pair . This phenogram demonstrated clear separations of S . cerevisiae, S . bayanus, S . carlsbergensis and S . paradoxus . However, S . pastorianus ATCC 12752(T) showed the highest percentages of shared fragments with the strains of S . bayanus, and formed a cluster with them . Except for the type strain of S . pastorianus, the percentages of shared fragments showed a similar tendency with reported data of DNA relatedness . The cluster of S . cerevisiae separated into three subclusters: one consisting of sake and shochu strains and a whisky strain; another consisting of bakery, wine, ale and whisky strains; and a third consisting of laboratory strains .

Yeast, 2001 Sep 15, 18(12), 1131 - 43
The putative monocarboxylate permeases of the yeast Saccharomyces cerevisiae do not transport monocarboxylic acids across the plasma membrane; Makuc J et al.; We have characterized the monocarboxylate permease family of Saccharomyces cerevisiae comprising five proteins . We could not find any evidence that the monocarboxylate transporter-homologous (Mch) proteins of S . cerevisiae are involved in the uptake or secretion of monocarboxylates such as lactate, pyruvate or acetate across the plasma membrane . A yeast mutant strain deleted for all five MCH genes exhibited no growth defects on monocarboxylic acids as the sole carbon and energy sources . Moreover, the uptake and secretion rates of monocarboxylic acids were indistinguishable from the wild-type strain . Additional deletion of the JEN1 lactate transporter gene completely blocked uptake of lactate and pyruvate . However, uptake of acetate was not even affected after the additional deletion of the gene YHL008c, which had been proposed to code for an acetate transporter . The mch1-5 mutant strain showed strongly reduced biomass yields in aerobic glucose-limited chemostat cultures, pointing to the involvement of Mch transporters in mitochondrial metabolism . Indeed, intracellular localization studies indicated that at least some of the Mch proteins reside in intracellular membranes . However, pyruvate uptake into isolated mitochondria was not affected in the mch1-5 mutant strain . It is concluded that the yeast monocarboxylate transporter-homologous proteins perform other functions than do their mammalian counterparts .

Proc Natl Acad Sci U S A, 2001 Sep 11, 98(19), 10620 - 3 Epub 2001 Sep 04.
Effect of spermidine on the in vivo degradation of ornithine decarboxylase in Saccharomyces cerevisiae; Gupta R et al.; As part of our studies on the regulation of polyamine biosynthesis in Saccharomyces cerevisiae, we have investigated the effect of spermidine on the degradation of ornithine decarboxylase in this organism . We have found that in S . cerevisiae, as in other eukaryotic cells, the rate of degradation of ornithine decarboxylase, measured either enzymatically or immunologically, is increased by the addition of spermidine to a yeast culture . It is noteworthy that this effect of added spermidine is found even when the experiments are conducted with strains in which the ornithine decarboxylase is overexpressed several hundred-fold more than the wild-type level . The effect of added spermidine in the overexpressed SPE1 strains is best seen in spe2 mutants in which the initial intracellular spermidine is very low or absent . Experiments with cycloheximide show that new protein synthesis is required to effect the breakdown of the ornithine decarboxylase . These results indicate that S . cerevisiae contains an antizyme-like mechanism for the control of the level of ornithine decarboxylase by spermidine, even though, as contrasted with other eukaryotic cells, no specific antizyme homologue has been detected either in in vitro experiments or in the S . cerevisiae genome.

Microbiology, 2001 Sep, 147(Pt 9), 2409 - 15
Saccharomyces cerevisiae commits to a programmed cell death process in response to acetic acid; Ludovico P et al.; Recent evidence has revealed the occurrence of an apoptotic phenotype in Saccharomyces cerevisiae that is inducible with oxidative stress . Here, exposure of S . cerevisiae to 20-200 mM acetic acid for 200 min at pH 3.0 resulted in cell death . Yeast mortality induced by 120-200 mM acid was not inhibited by cycloheximide and was accompanied by ultrastructural alterations typical of necrosis . In contrast, alterations associated with cell death induced by 20-80 mM acetic acid included: (i) cycloheximide-inhibitable chromatin condensation along the nuclear envelope; (ii) exposure of phosphatidylserine on the surface of the cytoplasmic membrane, revealed by the FITC-annexin V reaction; and (iii) the occurrence of DNA strand breaks, demonstrated by the TUNEL assay . These results show that a programmed cell death process sharing common features with an apoptotic phenotype can be induced by acetic acid in S . cerevisiae . This observation raises the possibility of this mode of cell death being more generalized in yeasts than previously considered and extended to cell death induced by other stress agents.

Vaccine, 2001 Sep 14, 19(32), 4661 - 8
Comparison of the protective efficacy of yeast-derived and Escherichia coli-derived recombinant merozoite surface protein 4/5 against lethal challenge by Plasmodium yoelii; Kedzierski L et al.; The gene encoding the Plasmodium yoelii homologue of P . falciparum merozoite surface proteins 4 (MSP4) and 5 (MSP5) has been expressed in Escherichia coli and Saccharomyces cerevisiae . The protein contains a single epidermal growth factor (EGF)-like domain and is expressed in a form lacking the predicted N-terminal signal and glycosyl phosphatidylinositol (GPI) attachment sequences . The recombinant protein derived from E . coli (EcMSP4/5) was highly effective at protecting mice against lethal challenge with 10(5) parasites of the P . yoelii YM strain . In contrast, the protective efficacy of yeast-derived MSP4/5 (yMSP4/5) was considerably less . The antibody titres in both groups were significantly different with mice immunised with yeast-derived protein showing significantly lower pre-challenge antibody responses . There was a significant inverse correlation between antibody levels as measured by ELISA and peak parasitaemia . Mice immunised with EcMSP4/5 produced anti-PyMSP4/5 antibodies predominantly of the IgG2a and IgG2b isotypes, whereas, mice immunised with yMSP4/5 mainly produced antibodies of the IgG1 isotype . The differences in antibody titres and subtype distribution may account for the observed differences in protective efficacy of these protein preparations . Levels of protective efficacy of MSP4/5 were compared with that obtained using P . yoelii MSP1 produced in S . cerevisiae . Levels of protection induced by E . coli derived MSP4/5 were superior to those induced by MSP1 which in turn were better than those induced by yeast-derived MSP4/5.

J Comput Biol, 2001, 8(3), 235 - 48
Fast approximate motif statistics; Nicodeme P; We present in this article a fast approximate method for computing the statistics of a number of non-self-overlapping matches of motifs in a random text in the nonuniform Bernoulli model . This method is well suited for protein motifs where the probability of self-overlap of motifs is small . For 96% of the PROSITE motifs, the expectations of occurrences of the motifs in a 7-million-amino-acids random database are computed by the approximate method with less than 1% error when compared with the exact method . Processing of the whole PROSITE takes about 30 seconds with the approximate method . We apply this new method to a comparison of the C . elegans and S . cerevisiae proteomes.

Appl Microbiol Biotechnol, 2001 Jun, 55(6), 734 - 41
Molecular characterization of the actin-encoding gene and the use of its promoter for a dominant selection system in the methylotrophic yeast Hansenula polymorpha; Kang HA et al.; The actin gene (ACT) from the methylotrophic yeast Hansenula polymorpha was cloned and its structural feature was characterized . In contrast to the actin genes of other ascomycetous yeasts, which have only one large intron, the H . polymorpha ACT gene was found to be split by two introns . The H . polymorpha ACT introns were correctly processed in the heterologous host Saccharomyces cerevisiae despite appreciable differences in the splice site sequences . The promoter region of H . polymorpha ACT displayed two CCAAT motifs and two TATA-like sequences in a configuration similar to that observed in the S . cerevisiae actin promoter . A set of deleted H . polymorpha ACT promoters was exploited to direct expression of the bacterial hygromycin B resistance (hph) gene as a dominant selectable marker in the transformation of H . polymorpha . The resistance level of H . polymorpha transformants to the antibiotic was shown to be dependent on the integration copy number of the hph cassette . The selectivity of the hygromycin B resistance marker for transformants of higher copy number was remarkably increased with the deletion of the upstream TATA-like sequence, but not with the removal of either CCAAT motif, from the H . polymorpha promoter . The dosage-dependent selection system developed in this study should be useful for genetic manipulation of H . polymorpha as an industrial strain to produce recombinant proteins.

Appl Microbiol Biotechnol, 2001 Jun, 55(6), 712 - 20
Characterization of a Saccharomyces cerevisiae mutant with oversecretion phenotype; Wang BD et al.; An oversecreting mutant of Saccharomyces cerevisiae was obtained from about 400 meiotic segregants derived from thediploid cells made by crossing the HBsAg-induced mutant NI-C with the wild-type strain Sey6211 . When transformed with a plasmid containing mouse alpha-amylase cDNA, the mutant (NI-C-D4) exhibited an increased capacity (up to 13-fold) for the secretion of mouse alpha-amylase, higher than the parental strains and other standard wild-type strains . It was also shown that alpha-amylase secreted by the oversecreting mutant had a higher activity and contained more of the non-glycosylated form than the glycosylated form . This isolated oversecreting, low-glycosylation mutant may prove to be a potential S . cerevisiae host for the production of foreign proteins . Further genetic analysis suggested that the mutation responsible for the mutant's oversecretion was partially dominant and that both the oversecretion and low-glycosylation phenotypes were governed by a single chromosome mutation . These pleiotrophic phenotypes may be attributed to a defect in the synthesis of an ER-resident chaperone.

J Exp Bot, 2001 Sep, 52(362), 1817 - 26
Trehalose metabolism in Arabidopsis: occurrence of trehalose and molecular cloning and characterization of trehalose-6-phosphate synthase homologues; Vogel G et al.; Axenically grown Arabidopsis thaliana plants were analysed for the occurrence of trehalose . Using gas chromatography-mass spectrometry (GC-MS) analysis, trehalose was unambiguously identified in extracts from Arabidopsis inflorescences . In a variety of organisms, the synthesis of trehalose is catalysed by trehalose-6-phosphate synthase (TPS; EC 2.4.1.15) and trehalose-6-phosphate phosphatase (TPP; EC 3.1.3.12) . Based on EST (expressed sequence tag) sequences, three full-length Arabidopsis cDNAs whose predicted protein sequences show extensive homologies to known TPS and TPP proteins were amplified by RACE-PCR . The expression of the corresponding genes, AtTPSA, AtTPSB and AtTPSC, and of the previously described TPS gene, AtTPS1, was analysed by quantitative RT-PCR . All of the genes were expressed in the rosette leaves, stems and flowers of Arabidopsis plants and, to a lower extent, in the roots . To study the role of the Arabidopsis genes, the AtTPSA and AtTPSC cDNAs were expressed in Saccharomyces cerevisiae mutants deficient in trehalose synthesis . In contrast to AtTPS1, expression of AtTPSA and AtTPSC in the tps1 mutant lacking TPS activity did not complement trehalose formation after heat shock or growth on glucose . In addition, no TPP function could be identified for AtTPSA and AtTPSC in complementation studies with the S . cerevisiae tps2 mutant lacking TPP activity . The results indicate that while AtTPS1 is involved in the formation of trehalose in Arabidopsis, some of the Arabidopsis genes with homologies to known TPS/TPP genes encode proteins lacking catalytic activity in trehalose synthesis.

EMBO Rep, 2001 Sep, 2(9), 787 - 93 Epub 2001 Aug 23.
Impact of the six nucleotides downstream of the stop codon on translation termination; Namy O et al.; The efficiency of translation termination is influenced by local contexts surrounding stop codons . In Saccharomyces cerevisiae, upstream and downstream sequences act synergistically to influence the translation termination efficiency . By analysing derivatives of a leaky stop codon context, we initially demonstrated that at least six nucleotides after the stop codon are a key determinant of readthrough efficiency in S . cerevisiae . We then developed a combinatorial-based strategy to identify poor 3' termination contexts . By screening a degenerate oligonucleotide library, we identified a consensus sequence -CA(A/G)N(U/C/G)A-, which promotes >5% readthrough efficiency when located downstream of a UAG stop codon . Potential base pairing between this stimulatory motif and regions close to helix 18 and 44 of the 18S rRNA provides a model for the effect of the 3' stop codon context on translation termination.

Gen Physiol Biophys, 2001 Jun, 20(2), 131 - 44
The effect of azalomycin F on Ca2+ homeostasis in Trichoderma viride and Saccharomyces cerevisiae; Simkovic M et al.; Azalomycin F (AMF), a macrocyclic lactone antibiotic, in concentrations of 10(-5) g/ml (10(-6) - 10(-5) mol/l) was found to stimulate both the 45Ca2+ influx and efflux in intact Trichoderma viride submerged mycelium and in cells of Saccharomyces cerevisiae without having Ca2+ ionophoric properties . AMF also inhibited ATP-dependent Ca2+ uptake in membrane fractions prepared from T . viride submerged mycelium . 45Ca2+ which had been accumulated in membrane fractions in an ATP-dependent manner was released upon addition of AMF . This release was observed in light organellar fractions (LOF) of S . cerevisiae and of T . viride submerged mycelium and, to a small extent, in heavy organellar fraction (HOF) of S . cerevisiae . No Ca2+ releasing effect of AMF was observed in HOF from T . viride submerged mycelium . In S . cerevisiae expressing Ca2+-dependent photoprotein aequorin, AMF induced transients of luminescence which reflect changes in the cytoplasmic Ca2+ concentration . The results suggest that the stimulation by AMF of the Ca2+ efflux from the mycelium (cells) could be explained by an increase of the cytoplasmic Ca2+ concentration due to the release of Ca2+ from microsomal membranes or to the stimulation of Ca2+ influx.

Arch Biochem Biophys, 2001 Sep 1, 393(1), 106 - 16
Ubiquitin-dependent 26S proteasomal pathway: a role in the degradation of native human liver CYP3A4 expressed in Saccharomyces cerevisiae?
Murray BP, Correia MA.
Cytochrome P450, CYP3A4, is the dominant human liver endoplasmic reticulum (ER) hemoprotein enzyme, responsible for the metabolism of over 60% of clinically relevant drugs . We have previously shown that mechanism-based suicide inactivation of CYP3A4 and its rat liver ER orthologs, CYPs 3A, via heme-modification of their protein moieties, results in their ubiquitin (Ub)-dependent 26S proteasomal degradation (Korsmeyer et al . (1999) Arch . Biochem . Biophys . 365, 31; Wang et al . (1999) Arch . Biochem . Biophys . 365, 45) . This is not surprising given that the heme-modified CYP3A proteins are structurally damaged . To determine whether the turnover of the native enzyme similarly recruited this pathway, we heterologously expressed this protein in wild-type Saccharomyces cerevisiae and mutant strains (hrd1Delta, hrd2-1, and hrd3Delta) previously shown to be deficient in the Ub-dependent 26S proteasomal degradation of the polytopic ER protein 3-hydroxy-3-methylglutaryl-CoA reductase (isoform Hmg2p), the rate-limiting enzyme in sterol biosynthesis, as well as in strains deficient in ER-associated Ub-conjugating enzymes, Ubc6p and/or Ubc7p (Hampton et al . (1996) Mol . Biol . Cell 7, 2029; Hampton and Bhakta (1997) Proc . Natl . Acad . Sci . USA 94, 12,944) . Our findings reveal that in common with the degradation of Hmg2p, that of native CYP3A4 also requires Hrd2p (a subunit of the 19S cap complex of the 26S proteasome) and Ubc7p, and to a much lesser extent Hrd3p, a component of the ER-associated Ub-ligase complex . In contrast to Hmg2p-degradation, that of native CYP3A4 does not appear to absolutely require Hrd1p, another component of the ER-associated Ub-ligase complex . Furthermore, studies in a S . cerevisiae pep4Delta strain proven to be deficient in the vacuolar degradation of carboxypeptidase Y indicated that CYP3A4 degradation is also largely independent of vacuolar (lysosomal) proteolytic function . The degradation of two other native ER proteins, Sec61p and Sec63p, normal components of the ER translocon, were also examined in parallel and found to be stabilized to some extent in HRD2- and UBC7-deficient strains . Together these findings attest to the remarkable mechanistic diversity in the normal degradation of ER proteins .

Cell, 2001 Jul 27, 106(2), 195 - 206
Molecular analysis of kinetochore-microtubule attachment in budding yeast; He X et al.; The complex series of movements that mediates chromosome segregation during mitosis is dependent on the attachment of microtubules to kinetochores, DNA-protein complexes that assemble on centromeric DNA . We describe the use of live-cell imaging and chromatin immunoprecipitation in S . cerevisiae to identify ten kinetochore subunits, among which are yeast homologs of microtubule binding proteins in animal cells . By analyzing conditional mutations in several of these proteins, we show that they are required for the imposition of tension on paired sister kinetochores and for correct chromosome movement . The proteins include both molecular motors and microtubule associated proteins (MAPs), implying that motors and MAPs function together in binding chromosomes to spindle microtubules.

Mol Cell Biol, 2001 Sep, 21(18), 6161 - 9
MGA2 is involved in the low-oxygen response element-dependent hypoxic induction of genes in Saccharomyces cerevisiae; Jiang Y et al.; Eukaryotes have the ability to respond to changes in oxygen tension by alterations in gene expression . For example, OLE1 expression in Saccharomyces cerevisiae is upregulated under hypoxic conditions . Previous studies have suggested that the pathway regulating OLE1 expression by unsaturated fatty acids may involve Mga2p and Spt23p, two structurally and functionally related proteins . To define the possible roles of each of these genes on hypoxia-induced OLE1 expression, we examined OLE1 expression under normoxia, hypoxia, and cobalt treatment conditions in Deltamga2 or Deltaspt23 deletion strains . The results of OLE1 promoter-lacZ reporter gene and Northern blot analyses showed that hypoxia- and cobalt-induced OLE1 expression was dramatically decreased in a Deltamga2 strain but not in a Deltaspt23 strain . Further analyses using low-oxygen response element (LORE)-CYC1-lacZ fusion reporter assays and electrophoretic mobility shift assays (EMSAs) demonstrated that MGA2 significantly affects the LORE-dependent hypoxic induction pathway of gene expression . When MGA2 was supplied by a plasmid, the LORE-dependent hypoxia-inducible reporter expression was recovered, as was the hypoxia-inducible complex in EMSAs in the S . cerevisiae Deltamga2 strain . Supershift analysis of EMSAs using crude extracts containing mycMga2p indicated that Mga2p is a component of the LORE-binding complex . Another LORE-dependent, hypoxia-inducible gene, ATF1, was similarly affected in the Deltamga2 strain . These results indicate that MGA2 is required for the LORE-dependent hypoxic gene induction in S . cerevisiae.

Cell, 2001 Aug 10, 106(3), 275 - 86
Telomere dysfunction increases mutation rate and genomic instability; Hackett JA et al.; The increased tumor incidence in telomerase null mice suggests that telomere dysfunction induces genetic instability . To test this directly, we examined mutation rate in the absence of telomerase in S . cerevisiae . The mutation rate in the CAN1 gene increased 10- to 100-fold in est1Delta strains as telomeres became dysfunctional . This increased mutation rate resulted from an increased frequency of terminal deletions . Chromosome fusions were recovered from est1Delta strains, suggesting that the terminal deletions may occur by a breakage-fusion-bridge type mechanism . At one locus, chromosomes with terminal deletions gained a new telomere through a Rad52p-dependent, Rad51p-independent process consistent with break-induced replication . At a second locus, more complicated rearrangements involving multiple chromosomes were seen . These data suggest that telomerase can inhibit chromosomal instability.

Rapid Commun Mass Spectrom, 2001, 15(16), 1416 - 21
Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Katayama H et al.; High-sensitivity, high-throughput analysis of proteins for proteomics studies is usually performed by polyacrylamide gel electrophoresis in combination with mass spectrometry . However, the quality of the data obtained depends on the in-gel digestion procedure employed . This work describes an improvement in the in-gel digestion efficiency for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis . A dramatic improvement in the coverage of tryptic peptides was observed when n-octyl glucoside was added to the buffer . Whole cell extracted proteins from S . cerevisiae were separated by two-dimensional gel electrophoresis and stained with silver . Protein spots were identified using our improved in-gel digestion method and MALDI-TOFMS . In addition, the mass spectra obtained by using the matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) were compared with those obtained using 2,5-dihydroxybenzoic acid (DHB) . The DHB matrix usually gave more peaks, which led to higher sequence coverage and, consequently, to higher confidence in protein identification . This improved in-gel digestion protocol is simple and useful for protein identification by MALDI-TOFMS .

Fed Regist, 2000 Nov 22, 65(226), 70305 - 7
Immunology and Microbiology Devices; Classification of Anti-Saccharomyces cerevisiae (S . cerevisiae) Antibody (ASCA) Test Systems . Food and Drug Administration, HHS . Final rule; Effects of the Fenton reagent on transport in yeast; Department of Biochemistry, Faculty of Medicine, University of Chiang Mai, Chiang Mai 50200, ThailandIn the facultatively anaerobic yeast Saccharomyces cerevisiae the uptake rate and the accumulation ratio of 2-aminoisobutyric acid was decreased by some 30% by Fenton's reagent (FR), a powerful source of OH . radicals . Likewise, the uptake of glutamic acid, leucine and arginine was diminished . The mediated diffusion of 6-deoxy-D-glucose was not affected . The H+ symport of maltose and trehalose was inhibited by some 40% both in the initial rate and in the accumulation ratio . FR had a dramatic inhibitory effect when present during preincubation with 50 mmol/L glucose . In the obligately aerobic Lodderomyces elongisporus the uptake of all amino acids tested was decreased by 15-30%, that of 6-deoxy-D-glucose by about 10% . The initial rates of uptake of maltose and trehalose were depressed by FR by 40% and the acceleration of uptake observed after 8 min of incubation, was abolished by FR completely . Acidification rate of the external medium by S . cerevisiae in the presence of glucose or galactose was enhanced three-fold, that after subsequently added K+ was substantially decreased . FR appears to have a dual effect on sugar and amino acid transport processes in yeast: (1) it blocks carrier protein synthesis; (2) it inhibits the source of energy for transport . It does not appreciably affect the carrier proteins themselves.

J Biol Chem, 2001 Oct 12, 276(41), 37950 - 61 Epub 2001 Aug 08.
Identification of residues of the Saccharomyces cerevisiae G protein-coupled receptor contributing to alpha-factor pheromone binding; Lee BK et al.; The Saccharomyces cerevisiae pheromone, alpha-factor (WHWLQLKPGQPMY), and Ste2p, its G protein-coupled receptor, were studied as a model for peptide ligand-receptor interaction . The affinities and activities of various synthetic position-10 alpha-factor analogs with Ste2p expressing mutations at residues Ser47 and Thr48 were investigated . All mutant receptors were expressed at a similar level in the cytoplasmic membrane, and their efficacies of signal transduction were similar to that of the wild-type receptor . Mutant receptors differed in binding affinity (Kd) and potency (EC50) for gene induction by alpha-factor . One mutant receptor (S47K,T48K) had dramatically reduced affinity and activity for {Lys10}- and {Orn10}alpha-factor, whereas the affinity for Saccharomyces kluyveri alpha-factor (WHWLSFSKGEPMY) was increased over 20-fold compared with that of wild-type receptor . In contrast, the affinity of {Lys10}- and {Orn10}alpha-factor was increased greatly in a S47E,T48E mutant receptor, whereas the binding of the S . kluyveri alpha-factor was abolished . The affinity of {Lys10}- and {Orn10}alpha-factor for the S47E,T48E receptor dropped 4-6-fold in the presence of 1 m NaCl, whereas the affinity of alpha-factor was not affected by this treatment . These results demonstrate that when bound to its receptor the 10th residue (Gln) of the S . cerevisiae alpha-factor is adjacent to Ser47 and Thr48 residues in the receptor and that the 10th residue of alpha-factors from two Saccharomyces species is responsible for the ligand selectivity to their cognate receptors . Based on these data, we have developed a two-dimensional model of alpha-factor binding to its receptor.

Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1607 - 18
Analysis of the constitution of the beer yeast genome by PCR, sequencing and subtelomeric sequence hybridization; Casaregola S et al.; The lager brewing yeasts, Saccharomyces pastorianus (synonym Saccharomyces carlsbergensis), are allopolyploid, containing parts of two divergent genomes . Saccharomyces cerevisiae contributed to the formation of these hybrids, although the identity of the other species is still unclear . The presence of alleles specific to S . cerevisiae and S . pastorianus was tested for by PCR/RFLP in brewing yeasts of various origins and in members of the Saccharomyces sensu stricto complex . S . cerevisiae-type alleles of two genes, HIS4 and YCL008c, were identified in another brewing yeast, S . pastorianus CBS 1503 (Saccharomyces monacensis), thought to be the source of the other contributor to the lager hybrid . This is consistent with the hybridization of S . cerevisiae subtelomeric sequences X and Y' to the electrophoretic karyotype of this strain . S . pastorianus CBS 1503 (S . monacensis) is therefore probably not an ancestor of S . pastorianus, but a related hybrid . Saccharomyces bayanus, also thought to be one of the contributors to the lager yeast hybrid, is a heterogeneous taxon containing at least two subgroups, one close to the type strain, CBS 380T, the other close to CBS 395 (Saccharomyces uvarum) . The partial sequences of several genes (HIS4, MET10, URA3) were shown to be identical or very similar (over 99%) in S . pastorianus CBS 1513 (S . carlsbergensis), S . bayanus CBS 380T and its close derivatives, showing that S . pastorianus and S . bayanus have a common ancestor . A distinction between two subgroups within S . bayanus was made on the basis of sequence analysis: the subgroup represented by S . bayanus CBS 395 (S . uvarum) has 6-8% sequence divergence within the genes HIS4, MET10 and MET2 from S . bayanus CBS 380T, indicating that the two S . bayanus subgroups diverged recently . The detection of specific alleles by PCR/RFLP and hybridization with S . cerevisiae subtelomeric sequences X and Y' to electrophoretic karyotypes of brewing yeasts and related species confirmed our findings and revealed substantial heterogeneity in the genome constitution of Czech brewing yeasts used in production.

J Biol Chem, 2001 Oct 26, 276(43), 40008 - 17 Epub 2001 Aug 06.
A membrane protein enriched in endoplasmic reticulum exit sites interacts with COPII; Tang BL et al.; Although all mammalian COPII components have now been cloned, little is known of their interactions with other regulatory proteins involved in exit from the endoplasmic reticulum (ER) . We report here that a mammalian protein (Yip1A) that is about 31% identical to S . cerevisiae and which interacts with and modulates COPII-mediated ER-Golgi transport . Yip1A transcripts are ubiquitously expressed . Transcripts of a related mammalian homologue, Yip1B, are found specifically in the heart . Indirect immunofluorescence microscopy revealed that Yip1A is localized to vesicular structures that are concentrated at the perinuclear region . The structures marked by Yip1A co-localized with Sec31A and Sec13, components of the COPII coat protein complex . Immunoelectron microscopy also showed that Yip1A co-localizes with Sec13 at ER exit sites . Overexpression of the hydrophilic N terminus of Yip1A arrests ER-Golgi transport of the vesicular stomatitis G protein and causes fragmentation and dispersion of the Golgi apparatus . A glutathione S-transferase fusion protein with the hydrophilic N terminus of Yip1A (GST-Yip1A) is able to bind to and deplete vital components from rat liver cytosol that is essential for in vitro vesicular stomatitis G transport . Peptide sequence analysis of cytosolic proteins that are specifically bound to GST-Yip1A revealed, among other proteins, mammalian COPII components Sec23 and Sec24 . A highly conserved domain at the N terminus of Yip1A is required for Sec23/Sec24 interaction . Our results suggest that Yip1A is involved in the regulation of ER-Golgi traffic at the level of ER exit sites.

Mol Microbiol, 2001 Jul, 41(2), 513 - 25
Identification and characterization of a family of secretion-related small GTPase-encoding genes from the filamentous fungus Aspergillus niger: a putative SEC4 homologue is not essential for growth; Punt PJ et al.; DNA fragments containing genetic information for five secretion-related small GTPases of Aspergillus niger (srgA-E) were isolated and identified as members of different Rab/Ypt subfamilies . This isolation and the search for similar sequences in fungal genomic and EST databases showed that, in contrast to Saccharomyces cerevisiae, filamentous fungi also possess homologues of mammalian Rab2 GTPases . Multiple transcripts with unusually long 5' and 3' untranslated regions were found for all srg genes . Their level of expression was independent of the type of carbon source used for growth . Although the transcripts of srgA and srgB were abundant to the same extent throughout the cultivation, that of the other genes peaked during the early growth phase and then declined . Two genes, srgA and srgB, were characterized further . The protein encoded by srgA exhibited relatively low identity (58%) to its closest S . cerevisiae homologue SEC4, whereas the protein encoded by srgB showed 73% identity with S . cerevisiae YPT1 . In contrast to other SEC4 homologues, srgA was unable to complement an S . cerevisiae sec4 mutant, and its disruption was not lethal in A . niger . SrgA mutants displayed a twofold increase in their hyphal diameter, unusual apical branching and strongly reduced protein secretion during growth on glucose.

Eur J Biochem, 2001 Aug, 268(15), 4352 - 8
Improving solubility of catalytic domain of human beta-1,4-galactosyltransferase 1 through rationally designed amino acid replacements; Malissard M et al.; beta-1,4-galactosyltransferase 1 (beta4gal-T1, EC 2.4.1.38) transfers galactose from UDP-galactose to free N-acetyl-D-glucosamine or bound N-acetyl-D-glucosamine-R . Soluble beta4gal-T1, purified from human milk has been refractory to structural studies by X-ray or NMR . In a previous study (Malissard et al . 1996, Eur . J . Biochem . 239, 340-348) we produced in the yeast Saccaromyces cerevisiae an N-deglycosylated form of soluble beta4gal-T1 that was much more homogeneous than the human enzyme, as it displayed only two isoforms when analysed by IEF as compared to 13 isoforms for the native beta4gal-T1 . The propensity of recombinant beta4gal-T1 to aggregate at concentrations > 1 mg.mL(-1) prevented structural and biophysical studies . In an attempt to produce a beta4gal-T1 form suitable for structural studies, we combined site-directed mutagenesis and heterologous expression in Escherichia coli . We produced a mutated form of the catalytic domain of beta4gal-T1 (sfbeta4gal-T1mut) in which seven mutations were introduced at nonconserved sites (A155E, N160K, M163T, A168T, T242N, N255D and A259T) . Sfbeta4gal-T1mut was shown to be much more soluble than beta4gal-T1 expressed in S . cerevisiae (8.5 mg.mL(-1) vs . 1 mg.mL(-1)) . Catalytic activity and kinetic parameters of sfbeta4gal-T1mut produced in E . coli were shown not to differ to any significant extent from those of the native enzyme.

Mol Cell Biol, 2001 Sep, 21(17), 5733 - 41
Evidence that fungal MEP proteins mediate diffusion of the uncharged species NH(3) across the cytoplasmic membrane; Soupene E et al.; Methylammonium and ammonium (MEP) permeases of Saccharomyces cerevisiae belong to a ubiquitous family of cytoplasmic membrane proteins that transport only ammonium (NH(4)(+) + NH(3)) . Transport and accumulation of the ammonium analog {(14)C}methylammonium, a weak base, led to the proposal that members of this family were capable of energy-dependent concentration of the ammonium ion, NH(4)(+) . In bacteria, however, ATP-dependent conversion of methylammonium to gamma-N-methylglutamine by glutamine synthetase precludes its use in assessing concentrative transport across the cytoplasmic membrane . We have confirmed that methylammonium is not metabolized in the yeast S . cerevisiae and have shown that it is little metabolized in the filamentous fungus Neurospora crassa . However, its accumulation depends on the energy-dependent acidification of vacuoles . A Deltavph1 mutant of S . cerevisiae and a Deltavma1 mutant, which lack vacuolar H(+)-ATPase activity, had large (fivefold or greater) defects in the accumulation of methylammonium, with little accompanying defect in the initial rate of transport . A vma-1 mutant of N . crassa largely metabolized methylammonium to methylglutamine . Thus, in fungi as in bacteria, subsequent energy-dependent utilization of methylammonium precludes its use in assessing active transport across the cytoplasmic membrane . The requirement for a proton gradient to sequester the charged species CH(3)NH(3)(+) in acidic vacuoles provides evidence that the substrate for MEP proteins is the uncharged species CH(3)NH(2) . By inference, their natural substrate is NH(3), a gas . We postulate that MEP proteins facilitate diffusion of NH(3) across the cytoplasmic membrane and speculate that human Rhesus proteins, which lie in the same domain family as MEP proteins, facilitate diffusion of CO(2).

Genes Dev, 2001 Aug 1, 15(15), 1957 - 70
Characterization of U4 and U6 interactions with the 5' splice site using a S . cerevisiae in vitro trans-splicing system; Johnson TL et al.; Spliceosome assembly has been characterized as the ordered association of the snRNP particles U1, U2, and U4/U6.U5 onto pre-mRNA . We have used an in vitro trans-splicing/cross-linking system in Saccharomyces cerevisiae nuclear extracts to examine the first step of this process, 5' splice site recognition . This trans-splicing reaction has ATP, Mg(2+), and splice-site sequence requirements similar to those of cis-splicing reactions . Using this system, we identified and characterized a novel U4-5' splice site interaction that is ATP-dependent, but does not require the branch point, the 3' splice site, or the 5' end of the U1 snRNA . Additionally, we identified several ATP-dependent U6 cross-links at the 5' splice site, indicating that different regions of U6 sample it before a U6-5' splice site interaction is stabilized that persists through the first step of splicing . This work provides evidence for ATP-dependent U4/U6 association with the 5' splice site independent of ATP-mediated U2 association with the branch point . Furthermore, it defines specific nucleotides in U4 and U6 that interact with the 5' splice site at this early stage, even in the absence of base-pairing with the U1 snRNA.

Genes Dev, 2001 Aug 1, 15(15), 1946 - 56
The S . cerevisiae SAGA complex functions in vivo as a coactivator for transcriptional activation by Gal4; Larschan E et al.; Previous studies demonstrated that the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex facilitates the binding of TATA-binding protein (TBP) during transcriptional activation of the GAL1 gene of Saccharomyces cerevisiae . TBP binding was shown to require the SAGA components Spt3 and Spt20/Ada5, but not the SAGA component Gcn5 . We have now examined whether SAGA is directly required as a coactivator in vivo by using chromatin immunoprecipitation analysis . Our results demonstrate that SAGA is physically recruited in vivo to the upstream activation sequence (UAS) regions of the galactose-inducible GAL genes . This recruitment is dependent on both induction by galactose and the Gal4 activation domain . Furthermore, we demonstrate that another well-characterized activator, Gal4-VP16, also recruits SAGA in vivo . Finally, we provide evidence that a specific interaction between Spt3 and TBP in vivo is important for Gal4 transcriptional activation at a step after SAGA recruitment . These results, taken together with previous studies, demonstrate a dependent pathway for the recruitment of TBP to GAL gene promoters consisting of the recruitment of SAGA by Gal4 and the subsequent recruitment of TBP by SAGA.

Lipids, 2001 Jun, 36(6), 629 - 36
Vinyl sulfide derivatives of truncated oxidosqualene as selective inhibitors of oxidosqualene and squalene-hopene cyclases; Ceruti M et al.; Various vinyl sulfide and ketene dithioacetal derivatives of truncated 2,3-oxidosqualene were developed . These compounds, having the reactive functions at positions C-2, C-15 and C-19 of the squalene skeleton, were studied as inhibitors of pig liver and Saccharomyces cerevisiae oxidosqualene cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalene hopene cyclase (SHC) (EC 5.4.99.-) . They contain one or two sulfur atoms in alpha-skeletal position to carbons considered to be cationic during enzymatic cyclization of the substrate and should strongly interact with enzyme nucleophiles of the active site . Most of the new compounds are inhibitors of the OSC and of SHC, with various degrees of selectivity . The methylthiovinyl derivative, having the reactive group at position 19, was the most potent and selective inhibitor of the series toward S . cerevisiae OSC, with a concentration inhibiting 500% of the activity of 50 nM, while toward the animal enzyme it was 20 times less potent . These results could offer new insight for the design of antifungal drugs.

J Lipid Res, 2001 Aug, 42(8), 1203 - 13
The OSBP-related protein family in humans; Lehto M et al.; Oxysterols are oxygenated derivatives of cholesterol that have a number of biological effects and play a key role in the maintenance of the body cholesterol balance . In this study, we describe the cDNA sequences and genomic structures of the recently identified human oxysterol-binding protein (OSBP)-related protein (ORP) family (Laitinen, S . et al . 1999 . J . Lipid Res . 40: 2204-2211) . The family now includes 12 genes/proteins, which can be divided into six distinct subfamilies . The ORP have two major structural features: a highly conserved OSBP-type sterol-binding domain in the C-terminal half and a pleckstrin homology domain present in the N-terminal region of most family members . Several ORP genes are present in S . cerevisiae, D . melanogaster, and C . elegans, suggesting that the protein family has functions of fundamental importance in the eukaryotic kingdom . Analysis of ORP mRNA levels in unloaded or acetylated LDL-loaded human macrophages revealed that the expression of ORP genes was not significantly affected by the loading, with the exception of ORP6, which was up-regulated 2-fold . The present study summarizes the basic characteristics of the OSBP-related gene/protein family in humans, and provides tools for functional analysis of the encoded proteins.

J Biol Chem, 2001 Oct 5, 276(40), 37722 - 9 Epub 2001 Aug 01.
A single gene produces mitochondrial, cytoplasmic, and peroxisomal NADP-dependent isocitrate dehydrogenase in Aspergillus nidulans; Szewczyk E et al.; NADP-dependent isocitrate dehydrogenase enzymes catalyze the decarboxylation of isocitrate to 2-oxoglutarate accompanied by the production of NADPH . In mammals two different genes encode mitochondrial and cytoplasmic/peroxisomal located enzymes, whereas in Saccharomyces cerevisiae three separate genes specify compartment specific enzymes . We have identified a single gene, idpA, in the filamentous fungus Aspergillus nidulans that specifies a protein with a high degree of identity to mammalian and S . cerevisiae enzymes . Northern blot analysis and reverse transcription-polymerase chain reaction revealed the presence of two idpA transcripts and two transcription start points were identified by sequencing cDNA clones and by 5'-rapid amplification of cDNA ends . The shorter transcript was found to be inducible by acetate and by fatty acids while the longer transcript was present in higher amounts during growth in glucose containing media . The longer transcript is predicted to encode a polypeptide containing an N-terminal mitochondrial targeting sequence as well as a C-terminal tripeptide (ARL) as a potential peroxisomal targeting signal . The shorter transcript is predicted to encode a polypeptide lacking the mitochondrial targeting signal but retaining the C-terminal sequence . Immunoblotting using antibody raised against S . cerevisiae Idp1p detected two polypeptides consistent with these predictions . The functions of the predicted targeting sequences were confirmed by microscopic analysis of transformants containing fluorescent protein fusion constructs . Using anti-Idp1p antibodies, protein localization to mitochondria and peroxisomes was observed during growth on glucose whereas cytoplasmic and peroxisomal localization was found upon acetate or fatty acid induction . Therefore, we have established that by the use of two transcription start points a single gene is sufficient to specify localization of NADP-dependent isocitrate dehydrogenase to three different cellular compartments in A . nidulans.

Yeast, 2001 Aug, 18(11), 1081 - 9
Mutational study of the role of tyrosine-49 in the Saccharomyces cerevisiae xylose reductase; Jeong EY et al.; The xyl1 gene encoding xylose reductase was cloned from Saccharomyces cerevisiae and expressed in Escherichia coli . The purified enzyme readily carried out xylose reduction in vitro . It prefers NADPH as the co-enzyme by about 80-fold over NADH . Compared to the native enzyme purified from S . cerevisiae (Kuhn et al., 1995), the recombinant xylose reductase displayed slightly higher (about two-fold) affinities (K(m)) for the substrate (xylose) and co-factor (NADPH), as well as a 3.9-fold faster turnover number (K(cat)) and 7.4-fold greater catalytic efficiency (K(cat)/K(m)) . The reason for the apparent discrepancies in kinetic constants between the recombinant and native S . cerevisiae xylose reductases is not known . Replacement of Tyr49 by Phe in the recombinant enzyme led to greater than 98% loss of activity, suggesting that this residue plays a critical role in catalysis . Intrinsic enzyme fluorescence spectroscopic analysis showed that the wild-type and the Y49F variant both bound the co-enzyme NADPH with similar affinity . This supports the view that Tyr49 is involved in interaction with the substrate and not the co-factor during catalysis .

Yeast, 2001 Aug, 18(11), 1023 - 33
Co-consumption of sugars or ethanol and glucose in a Saccharomyces cerevisiae strain deleted in the HXK2 gene; Raamsdonk LM et al.; In previous studies it was shown that deletion of the HXK2 gene in Saccharomyces cerevisiae yields a strain that hardly produces ethanol and grows almost exclusively oxidatively in the presence of abundant glucose . This paper reports on physiological studies on the hxk2 deletion strain on mixtures of glucose/sucrose, glucose/galactose, glucose/maltose and glucose/ethanol in aerobic batch cultures . The hxk2 deletion strain co-consumed galactose and sucrose, together with glucose . In addition, co-consumption of glucose and ethanol was observed during the early exponential growth phase . In S.cerevisiae, co-consumption of ethanol and glucose (in the presence of abundant glucose) has never been reported before . The specific respiration rate of the hxk2 deletion strain growing on the glucose/ethanol mixture was 900 micromol.min(-1).(g protein)(-1), which is four to five times higher than that of the hxk2 deletion strain growing oxidatively on glucose, three times higher than its parent growing on ethanol (when respiration is fully derepressed) and is almost 10 times higher than its parent growing on glucose (when respiration is repressed) . This indicates that the hxk2 deletion strain has a strongly enhanced oxidative capacity when grown on a mixture of glucose and ethanol .

Arch Microbiol, 2001 Jul, 176(1-2), 96 - 105
Regulation of maltose transport in Saccharomyces cerevisiae; Brondijk TH et al.; Solute transport in Saccharomyces cerevisiae can be regulated through mechanisms such as trans-inhibition and/or catabolite inactivation by nitrogen or carbon sources . Studies in hybrid membranes of S . cerevisiae suggested that the maltose transport system Mal61p is fully reversible and capable of catalyzing both influx and efflux transport . This conclusion has now been confirmed by studies in a S . cerevisiae strain lacking the maltase enzyme . Whole cells of this strain, wherein the orientation of the maltose transporter is fully preserved, catalyze fully reversible maltose transport . Catabolite inactivation of the maltose transporter Mal61p was studied in the presence and absence of maltose metabolism and by the use of different glucose analogues . Catabolite inactivation of Mal61p could be triggered by maltose, provided the sugar was metabolized, and the rate of inactivation correlated with the rate of maltose influx . We also show that 2-deoxyglucose, unlike 6-deoxyglucose, can trigger catabolite inactivation of the maltose transporter . This suggests a role for early glycolytic intermediates in catabolite inactivation of the Mal61 protein . However, there was no correlation between intracellular glucose-6-phosphate or ATP levels and the rate of catabolite inactivation of Mal61p . On the basis of their identification in cell extracts, we speculate that (dideoxy)-trehalose and/or (deoxy)-trehalose-6-phosphate trigger catabolite inactivation of the maltose transporter.

Bioinformatics, 2001, 17 Suppl 1, S215 - 24
Inferring subnetworks from perturbed expression profiles; Pe'er D et al.; Genome-wide expression profiles of genetic mutants provide a wide variety of measurements of cellular responses to perturbations . Typical analysis of such data identifies genes affected by perturbation and uses clustering to group genes of similar function . In this paper we discover a finer structure of interactions between genes, such as causality, mediation, activation, and inhibition by using a Bayesian network framework . We extend this framework to correctly handle perturbations, and to identify significant subnetworks of interacting genes . We apply this method to expression data of S . cerevisiae mutants and uncover a variety of structured metabolic, signaling and regulatory pathways.

Bioinformatics, 2001, 17 Suppl 1, S30 - 8
Separating real motifs from their artifacts; Blanchette M et al.; The typical output of many computational methods to identify binding sites is a long list of motifs containing some real motifs (those most likely to correspond to the actual binding sites) along with a large number of random variations of these . We present a statistical method to separate real motifs from their artifacts . This produces a short list of high quality motifs that is sufficient to explain the over-representation of all motifs in the given sequences . Using synthetic data sets, we show that the output of our method is very accurate . On various sets of upstream sequences in S . cerevisiae, our program identifies several known binding sites, as well as a number of significant novel motifs.

Appl Environ Microbiol, 2001 Aug, 67(8), 3693 - 701
Induction of a mitosis delay and cell lysis by high-level secretion of mouse alpha-amylase from Saccharomyces cerevisiae; Wang BD et al.; Some foreign proteins are produced in yeast in a cell cycle-dependent manner, but the cause of the cell cycle dependency is unknown . In this study, we found that Saccharomyces cerevisiae cells secreting high levels of mouse alpha-amylase have elongated buds and are delayed in cell cycle completion in mitosis . The delayed cell mitosis suggests that critical events during exit from mitosis might be disturbed . We found that the activities of PP2A (protein phosphatase 2A) and MPF (maturation-promoting factor) were reduced in alpha-amylase-oversecreting cells and that these cells showed a reduced level of assembly checkpoint protein Cdc55, compared to the accumulation in wild-type cells . MPF inactivation is due to inhibitory phosphorylation on Cdc28, as a cdc28 mutant which lacks an inhibitory phosphorylation site on Cdc28 prevents MPF inactivation and prevents the defective bud morphology induced by overproduction of alpha-amylase . Our data also suggest that high levels of alpha-amylase may downregulate PPH22, leading to cell lysis . In conclusion, overproduction of heterologous alpha-amylase in S . cerevisiae results in a negative regulation of PP2A, which causes mitotic delay and leads to cell lysis.

J Biol Chem, 2001 Oct 5, 276(40), 37027 - 33 Epub 2001 Jul 24.
Structure of the Rad50 x Mre11 DNA repair complex from Saccharomyces cerevisiae by electron microscopy; Anderson DE et al.; The RAD50 gene of Saccharomyces cerevisiae is one of several genes required for recombinational repair of double-strand DNA breaks during vegetative growth and for initiation of meiotic recombination . Rad50 forms a complex with two other proteins, Mre11 and Xrs2, and this complex is involved in double-strand break formation and processing . Rad50 has limited sequence homology to the structural maintenance of chromosomes (SMC) family of proteins and shares the same domain structure as SMCs: N- and C-terminal globular domains separated by two long coiled-coils . However, a notable difference is the much smaller non-coil hinge region between the two coiled-coils . We report here a structural analysis of full-length S . cerevisiae Rad50, alone and in a complex with yeast Mre11 by electron microscopy . Our results confirm that yeast Rad50 does have the same antiparallel coiled-coil structure as SMC proteins, but with no detectable globular hinge domain . However, the molecule is still able to bend sharply in the middle to bring the two catalytic domains together, indicating that the small hinge domain is flexible . We also demonstrate that Mre11 binds as a dimer between the catalytic domains of Rad50, bringing the nuclease activities of Mre11 in close proximity to the ATPase and DNA binding activities of Rad50.

Acta Crystallogr D Biol Crystallogr, 2001 Aug, 57(Pt 8), 1147 - 9 Epub 2001 Jul 23.
Crystallization and preliminary X-ray crystallographic analysis of deoxyuridine triphosphate nucleotidohydrolase from Saccharomyces cerevisiae; Han BW et al.; Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) from Saccharomyces cerevisiae is essential for cell viability . It has been overexpressed in Escherichia coli and has been crystallized at 296 K using polyethylene glycol (PEG) 1500 as a precipitant . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 59.48, b = 138.54, c = 157.91 A, alpha = beta = gamma = 90 degrees . Two molecules of trimeric dUTPase from S . cerevisiae are present in the asymmetric unit, giving a crystal volume per protein mass (V(M)) of 3.36 A(3) Da(-1) and a solvent content of 63% . The diffraction limit of the crystals could be significantly extended by the crystal-annealing procedure . A set of native data extending to 2.7 A resolution has been collected at 100 K using synchrotron X-rays.

Biochemistry, 2001 Jul 31, 40(30), 8945 - 54
ATR-FTIR study of the structure and orientation of transmembrane domains of the Saccharomyces cerevisiae alpha-mating factor receptor in phospholipids; Ding FX et al.; The structures of seven synthetic transmembrane domains (TMDs) of the alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae were studied in phospholipid multilayers by transmission Fourier transform infrared (FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopies . Peptide conformation assumed in multilayers depended on the method of sample preparation . Amide proton H/D exchange experiments showed that 60-80% of the NH bonds in these TMDs did not exchange with bulk water in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilayers . FTIR results showed that peptides corresponding to TMDs one, two, and seven were mostly alpha-helical in DMPC multilayers . Peptides corresponding to TMDs three and six assumed predominantly beta-sheet structures, whereas those corresponding to TMDs four and five were a mixture of alpha-helices and beta-sheets . ATR-FTIR showed that in DMPC the alpha-helices of TMDs two and five oriented with tilt angles of 34 degrees and 32 degrees, respectively, with respect to the multilayer normal . Similar results were obtained for six of the transmembrane domains in DMPC/DMPG (4:1) multilayers . In a mixture {POPC/POPE/POPS/PI/ergosterol (30:20:5:20:25)} which mimicked the lipid composition of the S . cerevisiae cell membrane, the percentage of alpha-helical structures found for TMDs one and five increased compared to those in DMPC and DMPC/DMPG (4:1) multilayers, and TMD six exhibited a mixture of beta-sheet ( approximately 60%) and alpha-helical ( approximately 40%) structure . These experiments provide biophysical evidence that peptides representing the seven transmembrane domains in Ste2p assume different structures and tilt angles within a membrane multilayer.

J Biol Chem, 2001 Sep 21, 276(38), 36000 - 7 Epub 2001 Jul 18.
Enhanced gluconeogenesis and increased energy storage as hallmarks of aging in Saccharomyces cerevisiae; Lin SS et al.; A relationship between life span and cellular glucose metabolism has been inferred from genetic manipulations and caloric restriction of model organisms . In this report, we have used the Snf1p glucose-sensing pathway of Saccharomyces cerevisiae to explore the genetic and biochemical linkages between glucose metabolism and aging . Snf1p is a serine/threonine kinase that regulates cellular responses to glucose deprivation . Loss of Snf4p, an activator of Snf1p, extends generational life span whereas loss of Sip2p, a presumed repressor of the kinase, causes an accelerated aging phenotype . An annotated data base of global age-associated changes in gene expression in isogenic wild-type, sip2Delta, and snf4Delta strains was generated from DNA microarray studies . The transcriptional responses suggested that gluconeogenesis and glucose storage increase as wild-type cells age, that this metabolic evolution is exaggerated in rapidly aging sip2Delta cells, and that it is attenuated in longer-lived snf4Delta cells . To test this hypothesis directly, we applied microanalytic biochemical methods to generation-matched cells from each strain and measured the activities of enzymes and concentrations of metabolites in the gluconeogenic, glycolytic, and glyoxylate pathways, as well as glycogen, ATP, and NAD(+) . The sensitivity of the assays allowed comprehensive biochemical profiling to be performed using aliquots of the same cell populations employed for the transcriptional profiling . The results provided additional evidence that aging in S . cerevisiae is associated with a shift away from glycolysis and toward gluconeogenesis and energy storage . They also disclosed that this shift is forestalled by two manipulations that extend life span, caloric restriction and genetic attenuation of the normal age-associated increase in Snf1p activity . Together, these findings indicate that Snf1p activation is not only a marker of aging but also a candidate mediator, because a shift toward energy storage over expenditure could impact myriad aspects of cellular maintenance and repair.

Nature, 2001 Jul 19, 412(6844), 355 - 8
Cdc6 cooperates with Sic1 and Hct1 to inactivate mitotic cyclin-dependent kinases; Calzada A et al.; Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinases (CDKs) . In the budding yeast, Saccharomyces cerevisiae, inactivation of CDKs during late mitosis involves degradation of B-type cyclins as well as direct inhibition of cyclin-CDK complexes by the CDK-inhibitor protein Sic1 (refs 1,2,3) . Several striking similarities exist between Sic1 and Cdc6, a DNA replication factor essential for the formation of pre-replicative complexes at origins of DNA replication . Transcription of both genes is activated during late mitosis by a process dependent on Swi5 (ref . 10) . Like Sic1, Cdc6 binds CDK complexes in vivo and downregulates them in vitro . Here we show that Cdc6, like Sic1, also contributes to inactivation of CDKs during late mitosis in S . cerevisiae . Deletion of the CDK-interacting domain of Cdc6 does not inhibit the function of origins of DNA replication during S phase, but instead causes a delay in mitotic exit; this delay is accentuated in the absence of Sic1 or of cyclin degradation . By contributing to mitotic exit and inactivation of CDKs, Cdc6 helps to create the conditions that are required for its subsequent role in the formation of pre-replicative complexes at origins of DNA replication.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8276 - 82
Rad52 forms DNA repair and recombination centers during S phase; Lisby M et al.; Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes . Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases . In the eukaryote Saccharomyces cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks . The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands . In this study, we find that S . cerevisiae Rad52 fused to green fluorescent protein (GFP) is fully functional in DNA repair and recombination . After induction of DNA double-strand breaks by gamma-irradiation, meiosis, or the HO endonuclease, Rad52-GFP relocalizes from a diffuse nuclear distribution to distinct foci . Interestingly, Rad52 foci are formed almost exclusively during the S phase of mitotic cells, consistent with coordination between recombinational repair and DNA replication . This notion is further strengthened by the dramatic increase in the frequency of Rad52 focus formation observed in a pol12-100 replication mutant and a mec1 DNA damage checkpoint mutant . Furthermore, our data indicate that each Rad52 focus represents a center of recombinational repair capable of processing multiple DNA lesions.

Mol Genet Genomics, 2001 Jun, 265(4), 705 - 10
Genetic interactions within TFIIIC, the promoter-binding factor of yeast RNA polymerase III; Rozenfeld S et al.; TFIIIC is a heteromultimeric protein, made of six distinct subunits in Saccharomyces cerevisiae, that binds to RNA polymerase III promoters and triggers the assembly of the transcription complex . The largest yeast subunit tau138, encoded by TFC3, binds to the B-box promoter element . This binding is defective in the temperature-sensitive mutant tfc3-G349E; the mutation responsible is located in one of two conserved motifs shared with the B-binding component of human TFIIIC . Rare dominant gain-of-function mutations that restore growth at high temperature were obtained following ultraviolet mutagenesis of tfc3-G349E . All of them resulted from single amino acid substitutions that alter the structure of TFIIIC . Three were due to reversion or intragenic suppression (TFC3-K754E and TFC3-L804H) events . Three were identical isolates of TFC6-E330K, a previously described mutation of the tau91 subunit . The remaining suppressors mapped in TFC4, and resulted in amino acid replacements in the second largest subunit of TFIIIC (tau131) . With the exception TFC4-E711K, these affect positions that are invariant between the S . cerevisiae and Homo sapiens proteins, and are localised in conserved tetratricopeptide motifs . These findings demonstrate a close functional interaction between the two largest subunits of TFIIIC and underscore the importance of the tetratricopeptide motif of tau131.

Mol Genet Genomics, 2001 Jun, 265(4), 636 - 46
Functional analysis of TamA, a coactivator of nitrogen-regulated gene expression in Aspergillus nidulans; Small AJ et al.; The tam A gene of Aspergillus nidulans encodes a 739-amino acid protein with similarity to Uga35p/Dal81p/DurLp of Saccharomyces cerevisiae . It has been proposed that TamA functions as a co-activator of AreA, the major nitrogen regulatory protein in A . nidulans . Because AreA functions as a transcriptional activator under nitrogen-limiting conditions, we investigated whether TamA was also present in the nucleus . We found that a GFP-TamA fusion protein was predominantly localised to the nucleus in the presence and absence of ammonium, and that AreA was not required for this distribution . As the predicted DNA-binding domain of TamA is not essential for function, we have used a number of approaches to further define functionally important regions . We have cloned the tamA gene of A . oryzae and compared its functional and sequence characteristics with those of A . nidulans tamA and S . cerevisiae UGA35/DAL81/DURL . The Aspergillus homologues are highly conserved and functionally interchangeable, whereas the S . cerevisiae gene does not complement a tamA mutant when expressed in A . nidulans . Uga35p/Dal81p/DurLp was also found to be unable to recruit AreA . The sequence changes in a number of tamA mutant alleles were determined, and altered versions of TamA were tested for tamA complementation and interaction with AreA . Changes in most regions of TamA appeared to destroy its function, suggesting that the overall conformation of the protein may be critical for its activity.

J Bioenerg Biomembr, 2001 Apr, 33(2), 119 - 26
Under conditions of insufficient permeability of VDAC1, external NADH may use the TOM complex channel to cross the outer membrane of Saccharomyces cerevisiae mitochondria; Antos N et al.; Thus far, only three channel-forming activities have been identified in the outer membrane of the yeast Saccharomyces cerevisiae mitochondria . Two of them, namely the TOM complex channel (translocase of the outer membrane) and the PSC (peptide-sensitive channel) participate in protein translocation and are probably identical, whereas a channel-forming protein called VDAC (voltage-dependent anion channel) serves as the major pathway for metabolites . The VDAC is present in two isoforms (VDAC1 and VDAC2) of which only VDAC1 has been shown to display channel-forming activity . Moreover, the permeability of VDAC1 has been reported to be limited in uncoupled mitochondria of S . cerevisiae . The presented data indicate that in S . cerevisiae-uncoupled mitochondria, external NADH, applied at higher concentrations (above 50 nmoles per 0.1 mg of mitochondrial protein), may use the TOM complex channel, besides VDAC1, to cross the outer membrane . Thus, the permeability of VDAC1 could be a limiting step in transport of external NADH across the outer membrane and might be supplemented by the TOM complex channel.

Genetics, 2001 Jul, 158(3), 999 - 1011
Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity; Gojkovic Z et al.; beta-Alanine synthase (EC 3.5.1.6), which catalyzes the final step of pyrimidine catabolism, has only been characterized in mammals . A Saccharomyces kluyveri pyd3 mutant that is unable to grow on N-carbamyl-beta-alanine as the sole nitrogen source and exhibits diminished beta-alanine synthase activity was used to clone analogous genes from different eukaryotes . Putative PYD3 sequences from the yeast S . kluyveri, the slime mold Dictyostelium discoideum, and the fruit fly Drosophila melanogaster complemented the pyd3 defect . When the S . kluyveri PYD3 gene was expressed in S . cerevisiae, which has no pyrimidine catabolic pathway, it enabled growth on N-carbamyl-beta-alanine as the sole nitrogen source . The D . discoideum and D . melanogaster PYD3 gene products are similar to mammalian beta-alanine synthases . In contrast, the S . kluyveri protein is quite different from these and more similar to bacterial N-carbamyl amidohydrolases . All three beta-alanine synthases are to some degree related to various aspartate transcarbamylases, which catalyze the second step of the de novo pyrimidine biosynthetic pathway . PYD3 expression in yeast seems to be inducible by dihydrouracil and N-carbamyl-beta-alanine, but not by uracil . This work establishes S . kluyveri as a model organism for studying pyrimidine degradation and beta-alanine production in eukaryotes.

Genetics, 2001 Jul, 158(3), 959 - 72
Saccharomyces cerevisiae rad51 mutants are defective in DNA damage-associated sister chromatid exchanges but exhibit increased rates of homology-directed translocations; Fasullo M et al.; Saccharomyces cerevisiae Rad51 is structurally similar to Escherichia coli RecA . We investigated the role of S . cerevisiae RAD51 in DNA damage-associated unequal sister chromatid exchanges (SCEs), translocations, and inversions . The frequency of these rearrangements was measured by monitoring mitotic recombination between two his3 fragments, his3-Delta5' and his3-Delta3'::HOcs, when positioned on different chromosomes or in tandem and oriented in direct or inverted orientation . Recombination was measured after cells were exposed to chemical agents and radiation and after HO endonuclease digestion at his3-Delta3'::HOcs . Wild-type and rad51 mutant strains showed no difference in the rate of spontaneous SCEs; however, the rate of spontaneous inversions was decreased threefold in the rad51 mutant . The rad51 null mutant was defective in DNA damage-associated SCE when cells were exposed to either radiation or chemical DNA-damaging agents or when HO endonuclease-induced double-strand breaks (DSBs) were directly targeted at his3-Delta3'::HOcs . The defect in DNA damage-associated SCEs in rad51 mutants correlated with an eightfold higher spontaneous level of directed translocations in diploid strains and with a higher level of radiation-associated translocations . We suggest that S . cerevisiae RAD51 facilitates genomic stability by reducing nonreciprocal translocations generated by RAD51-independent break-induced replication (BIR) mechanisms.

Mol Microbiol, 2001 Jul, 41(1), 1 - 8
Mediator--a universal complex in transcriptional regulation; Gustafsson CM et al.; The Mediator complex is essential for basal and regulated expression of nearly all RNA polymerase II-dependent genes in the Saccharomyces cerevisiae genome . Mediator acts as a bridge, conveying regulatory information from enhancers and other control elements to the promoter . It is now clear that Mediator-like complexes also exist in higher eukaryotic cells and that they have an important role in metazoan transcriptional regulation . However, the exact mechanism of Mediator-dependent transcriptional regulation remains unclear . We review here some recent advances in our understanding of Mediator structure and function . We also discuss a model to account for the functional and evolutionary relationship between yeast and metazoan Mediators . As an appendix to this review, we have created a database, MEDB, in which we have compiled information about all the S . cerevisiae Mediator subunits and their homologues in other eukaryotic cells .

J Biol Chem, 2001 Sep 7, 276(36), 33821 - 5 Epub 2001 Jul 12.
Expression of a micro-protein; Yu X et al.; The smallest known open reading frame encodes the ribosomal protein L41, which in yeast is composed of only 24 amino acids, 17 of which are arginine or lysine . Because of the unique problems that might attend the translation of such a short open reading frame, we have investigated the properties and the translation of the mRNAs encoding L41 . In Saccharomyces cerevisiae L41 is encoded by two linked genes, RPL41A and RPL41B . These genes give rise to mRNAs that have short 5' leaders of 18 and 22 nucleotides and rather long 3' leaders of 203 and 210 nucleotides not including their poly(A) tails . The mRNAs are translated exclusively on monosomes, suggesting that ribosomes do not remain attached to the mRNA after termination of translation . Calculations based on the abundance of ribosomes and of L41 mRNA indicate that the entire translation event, from initiation through termination, must occur in approximately 2 s . Termination of translation after only 25 codons does not subject the mRNAs encoding L41 to nonsense-mediated decay . Surprisingly, despite the L41 ribosomal protein being conserved from the archaea through the mammalia, S . cerevisiae can grow relatively normally after deletion of both RPL41A and RPL41B.

Mikrobiologiia, 2001 May-Jun, 70(3), 300 - 4
{Effect of sodium azide on heat-shock resistance in Saccharomyces cerevisiae and Debaryomyces vanriji yeasts}; Rikhvanov EG et al.; The pretreatment of Saccharomyces cerevisiae and Debaryomyces vanriji with sodium azide was found to induce thermotolerance in both yeasts, whereas sodium azide used in combination with heat shock enhanced the thermotolerance of S . cerevisiae and substantially decreased the thermotolerance of D . vanriji . It is suggested that the different responses of the yeasts to sodium azide during heat shock are due to the different functional organizations of their mitochondrial apparatus.

Yeast, 2001 Jul, 18(10), 931 - 41
Functional analysis of six ORFs from Saccharomyces cerevisiae chromosome IV: two-spored asci produced by disruptant of YDR027c and strain-dependent DNA heterogeneity around YDR036c; Aittamaa M et al.; Six S . cerevisiae FY1679 heterozygous deletion mutants were made by replacing six open reading frames (ORFs) of the chromosome IV right arm with kanMX4 selection marker . Haploid and homozygous diploid deletion mutants were obtained from sporulation, dissection and mating experiments . No essential genes were found . The basic phenotypic analysis showed that the haploid and homozygous deletants for the ORF YDR027c (LUV1, VSP54 or RKI1) grew slowly . The diploid homozygous deletants for this ORF had a low frequency of sporulation . They produced asci with no more than one or two haploid spores and the majority of these spores formed were not viable . The deletion of the other ORFs, YDR022c (CIS1), YDR030c (RAD28), YDR032c (PST2), YDR033w (MRH1) and YDR036c, did not change the phenotypes tested in strain FY1679 or the first four ORFs in strain CEN.PK2 . This work showed some differences in the DNA sequences between FY1679 and CEN.PK2: the regions immediately 1 kb upstream from YDR036c in these two strains are too different to hybridize properly, preventing deletion of YDR036c in the CEN.PK2 background by recombination with a disruption cassette designed for FY1679 . In addition, there are different sets of transposable elements on the other side of the ORF, the differences starting at about 3.5 kb downstream from YDR036c .

J Biol Chem, 2001 Sep 7, 276(36), 33730 - 5 Epub 2001 Jul 09.
Saccharomyces cerevisiae expresses three phospholipid hydroperoxide glutathione peroxidases; Avery AM et al.; The GPX1, GPX2, and GPX3 genes of Saccharomyces cerevisiae have been reported previously to encode glutathione peroxidases (GPxs) . We re-examined the sequence alignments of these proteins with GPxs from higher eukaryotes . Sequence identities, particularly with phospholipid hydroperoxide glutathione peroxidases (PHGPxs), were enhanced markedly by introduction to the yeast sequences of gaps that are characteristic of PHGPxs . PHGPx-like activity was detectable in extracts from wild-type S . cerevisiae and was diminished in extracts from gpx1 Delta, gpx2 Delta, and gpx3 Delta deletion mutants; PHGPx activity was almost absent in a gpx1 Delta/gpx2 Delta/gpx3 Delta triple mutant . Studies with cloned GPX1, GPX2, and GPX3 expressed heterologously in Escherichia coli confirmed that these genes encode proteins with PHGPx activity . An S . cerevisiae gpx1 Delta/gpx2 Delta/gpx3 Delta mutant was defective for growth in medium supplemented with the oxidation-sensitive polyunsaturated fatty acid linolenate (18:3) . This sensitivity to 18:3 was more marked than sensitivity to H(2)O(2) . Unlike H(2)O(2) toxicity, delayed toxicity of 18:3 toward gpx1 Delta/gpx2 Delta/gpx3 Delta cells was correlated with the gradual incorporation of 18:3 into S . cerevisiae membrane lipids and was suppressible with alpha-tocopherol, an inhibitor of lipid peroxidation . The results show that the GPX genes of S . cerevisiae, previously reported to encode GPxs, encode PHGPxs (PHGPx1, PHGPx2, and PHGPx3) and that these enzymes protect yeast against phospholipid hydroperoxides as well as nonphospholipid peroxides during oxidative stress . This is the first report of an organism that expresses PHGPx from more than one gene and produces PHGPx in the absence of a GPx.

J Bacteriol, 2001 Aug, 183(15), 4636 - 42
Green fluorescent protein-Dal80p illuminates up to 16 distinct foci that colocalize with and exhibit the same behavior as chromosomal DNA proceeding through the cell cycle of Saccharomyces cerevisiae; Distler M et al.; Four GATA family DNA binding proteins mediate nitrogen catabolite repression-sensitive transcription in Saccharomyces cerevisiae . Gln3p and Gat1p are transcriptional activators, while Dal80p and Deh1p repress Gln3p- and Gat1p-mediated transcription by competing with these activators for binding to DNA . Strong Dal80p binding to DNA is thought to result from C-terminal leucine zipper-mediated dimerization . Many Dal80p binding site-homologous sequences are relatively evenly distributed across the S . cerevisiae genome, raising the possibility that Dal80p might be able to "stain" DNA . We demonstrate that cells containing enhanced green fluorescent protein-Dal80p (EGFP-Dal80p) exhibit up to 16 fluorescent foci that colocalize with DAPI (4',6'-diamidino-2-phenylindole)-positive material and follow DNA movement through the cell cycle, suggesting that EGFP-Dal80p may indeed be useful for monitoring yeast chromosomes in live cells and in real time.

Lett Appl Microbiol, 2001 Jul, 33(1), 71 - 5
Differentiation of industrial wine yeast strains using microsatellite markers; Gonzalez Techera A et al.; AIMS: To differentiate nine industrial wine strains of Saccharomyces cerevisiae using microsatellite (simple sequence repeats, SSR) markers . METHODS AND RESULTS: Six of the strains were indigenous yeasts currently used as high-density starter monocultures by the Uruguayan wine industry . Unequivocal differentiation of these six native strains and three commercial S . cerevisiae wine strains was achieved by PCR amplification and polymorphism analysis of loci containing microsatellite markers . CONCLUSION: We recommend the use of this reproducible and simple molecular method to routinely discriminate wine yeast strains . SIGNIFICANCE AND IMPACT OF THE STUDY: Microsatellites are superior to other methods for typing yeasts because the results can be exchanged as quantitative data . Knowledge of the frequencies of the alleles for different SSR markers will eventually lead to an accurate typing method to identify industrial wine yeast strains.

Mol Cell Biol, 2001 Aug, 21(15), 4875 - 88
Multifunctional centromere binding factor 1 is essential for chromosome segregation in the human pathogenic yeast Candida glabrata; Stoyan T et al.; The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant . The C . glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins . CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M(-1)) . Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S . cerevisiae . We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay . Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function . Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates . Deletion of the CBF1 gene proved to be lethal in C . glabrata . C . glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline . DAPI (4',6'-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle . Thus, we conclude that Cbf1p is required for chromosome segregation in C . glabrata.

Cytogenet Cell Genet, 2001, 92(3-4), 279 - 82
Cloning and molecular characterization of three ubiquitin fusion degradation 1 (Ufd1) ortholog genes from Xenopus laevis, Gallus gallus and Drosophila melanogaster; Ratti A et al.; The yeast ubiquitin fusion degradation 1 (Ufd1) protein is involved in a degradation pathway for ubiquitin fused products . The human ortholog gene (UFD1-like, UFD1L) is deleted in patients affected by the DiGeorge/velocardiofacial syndromes . We report the cloning of UFD1L orthologs from Drosophila melanogaster (dufd1l), Xenopus laevis and Gallus gallus . The 1,125-bp Drosophila cDNA encodes a protein of 316 amino acids, showing 60% identity with the human and murine proteins . The identity to the G . gallus, X . laevis, C . elegans and S . cerevisiae proteins is 95%, 83%, 32%, and 36%, respectively . Northern expression data in Drosophila indicate that dufd1l is expressed through embryonic, larval and pupal development, as well as in the adult fly .

Genome Res, 2001 Jul, 11(7), 1175 - 86
Surveying Saccharomyces genomes to identify functional elements by comparative DNA sequence analysis; Cliften PF et al.; Comparative sequence analysis has facilitated the discovery of protein coding genes and important functional sequences within proteins, but has been less useful for identifying functional sequence elements in nonprotein-coding DNA because the relatively rapid rate of change of nonprotein-coding sequences and the relative simplicity of non-coding regulatory sequence elements necessitates the comparison of sequences of relatively closely related species . We tested the use of comparative DNA sequence analysis to aid identification of promoter regulatory elements, nonprotein-coding RNA genes, and small protein-coding genes by surveying random DNA sequences of several Saccharomyces yeast species, with the goal of learning which species are best suited for comparisons with S . cerevisiae . We also determined the DNA sequence of a few specific promoters and RNA genes of several Saccharomyces species to determine the degree of conservation of known functional elements within the genome . Our results lead us to conclude that comparative DNA sequence analysis will enable identification of functionally conserved elements within the yeast genome, and suggest a path for obtaining this information.

J Biol Chem, 2001 Aug 31, 276(35), 32474 - 9 Epub 2001 Jun 28.
Asymmetric interactions between the acidic P1 and P2 proteins in the Saccharomyces cerevisiae ribosomal stalk; Guarinos E et al.; The Saccharomyces cerevisiae ribosomal stalk is made of five components, the 32-kDa P0 and four 12-kDa acidic proteins, P1alpha, P1beta, P2alpha, and P2beta . The P0 carboxyl-terminal domain is involved in the interaction with the acidic proteins and resembles their structure . Protein chimeras were constructed in which the last 112 amino acids of P0 were replaced by the sequence of each acidic protein, yielding four fusion proteins, P0-1alpha, P0-1beta, P0-2alpha, and P0-2beta . The chimeras were expressed in P0 conditional null mutant strains in which wild-type P0 is not present . In S . cerevisiae D4567, which is totally deprived of acidic proteins, the four fusion proteins can replace the wild-type P0 with little effect on cell growth . In other genetic backgrounds, the chimeras either reduce or increase cell growth because of their effect on the ribosomal stalk composition . An analysis of the stalk proteins showed that each P0 chimera is able to strongly interact with only one acidic protein . The following associations were found: P0-1alpha.P2beta, P0-1beta.P2alpha, P0-2alpha.P1beta, and P0-2beta.P1alpha . These results indicate that the four acidic proteins do not form dimers in the yeast ribosomal stalk but interact with each other forming two specific associations, P1alpha.P2beta and P1beta.P2alpha, which have different structural and functional roles.

Mol Cell, 2001 Jun, 7(6), 1255 - 66
A DNA damage response pathway controlled by Tel1 and the Mre11 complex; Usui T et al.; We define a DNA damage checkpoint pathway in S . cerevisiae governed by the ATM homolog Tel1 and the Mre11 complex . In mitotic cells, the Tel1-Mre11 complex pathway triggers Rad53 activation and its interaction with Rad9, whereas in meiosis it acts via Rad9 and the Rad53 paralog Mre4/Mek1 . Activation of the Tel1-Mre11 complex pathway checkpoint functions appears to depend upon the Mre11 complex as a damage sensor and, at least in meiotic cells, to depend on unprocessed DNA double-strand breaks (DSBs) . The DSB repair functions of the Mre11 complex are enhanced by the pathway, suggesting that the complex both initiates and is regulated by the Tel1-dependent DSB signal . These findings demonstrate that the diverse functions of the Mre11 complex in the cellular DNA damage response are conserved in mammals and yeast.

Yeast, 2001 Jun 30, 18(9), 789 - 95
A Saccharomyces servazzii clone homologous to Saccharomyces cerevisiae chromosome III spanning KAR4, ARS 304 and SPB1 lacks the recombination enhancer but contains an unknown ORF; Zhou Z et al.; In order to learn about the evolutionary conservation of the recombination enhancer (RE) that controls donor preference during mating type switching in Saccharomyces cerevisiae, we have cloned a 13 kb region from S . servazzii . We find that the order of four genes surrounding the RE in S . cerevisiae (PRD1, KAR4, SPB1 and PBN1) is preserved in S.servazzii . However, there is an additional ORF in S . servazzii between PRD1 and KAR4 that is not homologous to any gene in S . cerevisiae or to genes in other organisms . Despite a 75-79% amino acid identity for KAR4 and SPB1, respectively, the S . servazzii sequence did not carry a well-conserved RE sequence and these sequences lacked RE function when introduced into S . cerevisiae . The S . servazzii region contains a sequence that supports autonomous DNA replication in S . cerevisiae and may represent a homologue of ARS304 . The S . servazziii sequence has Genbank Accession No . BankIt359091 AF307954 .

Biochim Biophys Acta, 2001 Jul 2, 1527(1-2), 31 - 8
Mitochondrial NADH-cytochrome b(5) reductase plays a crucial role in the reduction of D-erythroascorbyl free radical in Saccharomyces cerevisiae; Lee JS et al.; The relevance of NADH-cytochrome b(5) reductase to the NADH-dependent reduction of D-erythroascorbyl free radical was investigated in Saccharomyces cerevisiae . MCR1, which is known to encode NADH-cytochrome b(5) reductase in S . cerevisiae, was disrupted by the insertion of URA3 gene into the gene of MCR1 . In the mcr1 disruptant cells, the activity of NADH-D-erythroascorbyl free radical reductase almost disappeared and the intracellular level of D-erythroascorbic acid was about 11% of that of the congenic wild-type strain . In the transformant cells carrying MCR1 in multicopy plasmid, the intracellular level of D-erythroascorbic acid and the activity of NADH-D-erythroascorbyl free radical reductase increased up to 1.7-fold and 2.1-fold, respectively . Therefore, it indicated that the MCR1 product, mitochondrial NADH-cytochrome b(5) reductase, plays a key role in the NADH-dependent reduction of D-erythroascorbyl free radical in S . cerevisiae . On the other hand, the mcr1 disruptant cells were hypersensitive to hydrogen peroxide and menadione, and overexpression of MCR1 made the cells more resistant against oxidative stress . These results suggested that the mitochondrial NADH-cytochrome b(5) reductase functions as NADH-D-erythroascorbyl free radical reductase and plays an important role in the response to oxidative damage in S . cerevisiae.

FEBS Lett, 2001 Jun 15, 499(1-2), 41 - 4
In situ analysis of methylglyoxal metabolism in Saccharomyces cerevisiae; Martins AM et al.; Methylglyoxal metabolism was studied during Saccharomyces cerevisiae grown with D-glucose as the sole carbon and energy source . Using for the first time a specific assay for methylglyoxal in yeast, metabolic fluxes of its formation and D-lactate production were determined . D-Glucose consumption and ethanol production were determined during growth . Metabolic fluxes were also determined in situ, at the glycolytic triose phosphate levels and glyoxalase pathway . Maximum fluxes of ethanol production and glucose consumption correspond to maxima of methylglyoxal and D-lactate formation fluxes during growth . Methylglyoxal formation is quantitatively related to glycolysis, representing 0.3% of the total glycolytic flux in S . cerevisiae.

Mol Cell Biol, 2001 Jul, 21(14), 4427 - 40
Promoter-specific shifts in transcription initiation conferred by yeast TFIIB mutations are determined by the sequence in the immediate vicinity of the start sites; Faitar SL et al.; The general transcription factor IIB (TFIIB) is required for transcription of class II genes by RNA polymerase II . Previous studies demonstrated that mutations in the Saccharomyces cerevisiae SUA7 gene, which encodes TFIIB, can alter transcription initiation patterns in vivo . To further delineate the functional domain and residues of TFIIB involved in transcription start site utilization, a genetic selection was used to isolate S . cerevisiae TFIIB mutants exhibiting downstream shifts in transcription initiation in vivo . Both dominant and recessive mutations conferring downstream shifts were identified at multiple positions within a highly conserved homology block in the N-terminal region of the protein . The TFIIB mutations conferred downstream shifts in transcription initiation at the ADH1 and CYC1 promoters, whereas no significant shifts were observed at the HIS3 promoter . Analysis of a series of ADH1-HIS3 hybrid promoters and variant ADH1 and HIS3 promoters containing insertions, deletions, or site-directed base substitutions revealed that the feature that renders a promoter sensitive to TFIIB mutations is the sequence in the immediate vicinity of the normal start sites . We discuss these results in light of possible models for the mechanism of start site utilization by S . cerevisiae RNA polymerase II and the role played by TFIIB.

Phys Rev Lett, 2001 Jun 18, 86(25), 5815 - 8
New stopping criteria for segmenting DNA sequences; Li W; We propose a solution on the stopping criterion in segmenting inhomogeneous DNA sequences with complex statistical patterns . This new stopping criterion is based on Bayesian information criterion in the model selection framework . When this criterion is applied to telomere of S . cerevisiae and the complete sequence of E . coli, borders of biologically meaningful units were identified, and a more reasonable number of domains was obtained . We also introduce a measure called segmentation strength which can be used to control the delineation of large domains . The relationship between the average domain size and the threshold of segmentation strength is determined for several genome sequences.

Curr Genet, 2001 May, 39(3), 156 - 65
Cis-acting sites contributing to expression of divergently transcribed DAL1 and DAL4 genes in S . cerevisiae: a word of caution when correlating cis-acting sequences with genome-wide expression analyses; van der Merwe GK et al.; Correlating genome-wide expression profiles with sequence searches of promoter regions is being used as a technique to identify putative binding sites for transacting factors or to refine consensus sequences of those already known . To evaluate the limitations of such an approach in our studies of GATA-mediated transcription in Saccharomyces cerevisiae, we identified the relative contributions made to DAL1 and DAL4 expression by each of five Gln3p-, and/or Gat1p-, and three Dal82p-binding site homologous sequences situated in the 829-bp intergenic region separating these highly related, divergently transcribed genes . Our data suggest that although the correlation of repeated sequences or sequence homologies appearing within promoter regions with expression profiles obtained from genome-wide transcription analyses can provide useful starting points for analyses of cis-acting sites, significant limitations and possibilities for misinterpretation also abound.

Sheng Wu Gong Cheng Xue Bao, 2001 Mar, 17(2), 161 - 4
{Expression of delta 6-fatty acid desaturase gene from Mortierella alpina in Saccharomyces cerevisiae}; Liu L et al.; delta 6-fatty acid desaturase is the rate-limiting enzyme of the desaturation of linoleic acid in the production of an essential fatty acid, gamma-linolenic acid . The 1.4 kb fragment in plasmid pTMACL6 encoding delta 6-fatty acid desaturase from Mortieralla alpina ATCC16266 was subcloned into the yeast-E . coli shuttle vector pYES2.0, thus an expression recombinant plasmid pYMAD6 containing target gene was constructed and obtained in the SC-Ura media . The pYMAD6 was introduced into defective mutant INCSc1 of Saccharomyces cerevisiae by LiAc method . When linoleic acid was provided as an exogenous substrate to the yeast cultures expressing delta 6-fatty acid desaturase activity under appropriate media and temperature condition, the level of gamma-linolenic acid reached 31.6% of the total yeast fatty acids by GC-MS detecting, which is the highest report of delta 6-fatty-acid desaturase gene in S . cerevisiae.

EMBO J, 2001 Jun 15, 20(12), 3177 - 86
Transcript analysis of 1003 novel yeast genes using high-throughput northern hybridizations; Brown AJ et al.; The expression of 1008 open reading frames (ORFs) from the yeast Saccharomyces cerevisiae has been examined under eight different physiological conditions, using classical northern analysis . These northern data have been compared with publicly available data from a microarray analysis of the diauxic transition in S.cerevisiae . The results demonstrate the importance of comparing biologically equivalent situations and of the standardization of data normalization procedures . We have also used our northern data to identify co-regulated gene clusters and define the putative target sites of transcriptional activators responsible for their control . Clusters containing genes of known function identify target sites of known activators . In contrast, clusters comprised solely of genes of unknown function usually define novel putative target sites . Finally, we have examined possible global controls on gene expression . It was discovered that ORFs that are highly expressed following a nutritional upshift tend to employ favoured codons, whereas those overexpressed in starvation conditions do not . These results are interpreted in terms of a model in which competition between mRNA molecules for translational capacity selects for codons translated by abundant tRNAs.

J Inherit Metab Dis, 2001 Apr, 24(2), 166 - 80
Import of proteins into mitochondria: a novel pathomechanism for progressive neurodegeneration; Bauer MF et al.; The vast majority of mitochondrial proteins are encoded as precursors by the nuclear genome . A major aspect of mitochondrial biogenesis is therefore the transfer of nuclear-encoded, cytosplasmically synthesized precursor proteins across and into the mitochondrial membranes . During the past years the use of simple model organisms such as the yeasts S . cerevisiae and N . crassa has helped considerably to identify and unravel the structure and function of a substantial number of components involved in targeting of nuclear-encoded preproteins to mitochondria . Several pathways and a number of components were characterized that are involved in guiding mitochondrial preproteins to their specific sites of function . In particular, import of nuclear-encoded precursor proteins into and across the mitochondrial inner membrane is mediated by two distinct translocases, the TIM23 complex and the TIM22 complex . Both TIM complexes cooperate with the general preprotein translocase of the outer membrane, TOM complex . The TIM complexes differ in the their substrate specificity . While the TIM23 complex mediates import of preproteins with a positively charged matrix targeting signal, the TIM22 complex facilitates the insertion of a class of hydrophobic proteins with internal targeting signals into the inner membrane . Most recently the rapid progress of research has allowed elucidation of a new mitochondrial disease on the molecular level . This rare X-linked progressive neurodegenerative disorder, named Mohr-Tranebjaerg (MT syndrome), is caused by mutations in the DDP1 gene and includes sensorineural deafness, blindness, mental retardation and a complex movement disorder . The analysis of the novel pathomechanism is based on the homology of the affected DDP1 protein to a family of conserved yeast components acting along the TIM22 pathway . This contribution briefly summarizes the current knowledge of the pathways of protein import and proposes a mechanism to explain how defective import leads to neurodegeneration.

Genetics, 2001 Jun, 158(2), 613 - 25
The Saccharomyces cerevisiae small GTPase, Gsp1p/Ran, is involved in 3' processing of 7S-to-5.8S rRNA and in degradation of the excised 5'-A0 fragment of 35S pre-rRNA, both of which are carried out by the exosome; Suzuki N et al.; Dis3p, a subunit of the exosome, interacts directly with Ran . To clarify the relationship between the exosome and the RanGTPase cycle, a series of temperature-sensitive Saccharomyces cerevisiae dis3 mutants were isolated and their 5.8S rRNA processing was compared with processing in strains with mutations in a S . cerevisiae Ran homologue, Gsp1p . In both dis3 and gsp1 mutants, 3' processing of 7S-to-5.8S rRNA was blocked at three identical sites in an allele-specific manner . In contrast, the 5' end of 5.8S rRNA was terminated normally in gsp1 and in dis3 . Inhibition of 5.8S rRNA maturation in gsp1 was rescued by overexpression of nuclear exosome components Dis3p, Rrp4p, and Mtr4p, but not by a cytoplasmic exosome component, Ski2p . Furthermore, gsp1 and dis3 accumulated the 5'-A0 fragment of 35S pre-rRNA, which is also degraded by the exosome, and the level of 27S rRNA was reduced . Neither 5.8S rRNA intermediates nor 5'-A0 fragments were observed in mutants defective in the nucleocytoplasmic transport, indicating that Gsp1p regulates rRNA processing through Dis3p, independent of nucleocytoplasmic transport.

J Biol Chem, 2001 Sep 14, 276(37), 34934 - 40 Epub 2001 Jun 13.
Identification and characterization of the tRNA:Psi 31-synthase (Pus6p) of Saccharomyces cerevisiae; Ansmant I et al.; To characterize the substrate specificity of the putative RNA:pseudouridine (Psi)-synthase encoded by the Saccharomyces cerevisiae open reading frame (ORF) YGR169c, the corresponding gene was deleted in yeast, and the consequences of the deletion on tRNA and small nuclear RNA modification were tested . The resulting DeltaYGR169c strain showed no detectable growth phenotype, and the only difference in Psi formation in stable cellular RNAs was the absence of Psi at position 31 in cytoplasmic and mitochondrial tRNAs . Complementation of the DeltaYGR169c strain by a plasmid bearing the wild-type YGR169c ORF restored Psi(31) formation in tRNA, whereas a point mutation of the enzyme active site (Asp(168)-->Ala) abolished tRNA:Psi(31)-synthase activity . Moreover, recombinant His(6)-tagged Ygr169 protein produced in Escherichia coli was capable of forming Psi(31) in vitro using tRNAs extracted from the DeltaYGR169c yeast cells as substrates . These results demonstrate that the protein encoded by the S . cerevisiae ORF YGR169c is the Psi-synthase responsible for modification of cytoplasmic and mitochondrial tRNAs at position 31 . Because this is the sixth RNA:Psi-synthase characterized thus far in yeast, we propose to rename the corresponding gene PUS6 and the expressed protein Pus6p . Finally, the cellular localization of the green fluorescent protein-tagged Pus6p was studied by functional tests and direct fluorescence microscopy.

J Chromatogr A, 2001 May 11, 917(1-2), 29 - 42
New family of glutathionyl-biomimetic ligands for affinity chromatography of glutathione-recognising enzymes; Melissis SC et al.; Three anthraquinone glutathionyl-biomimetic dye ligands, comprising as terminal biomimetic moiety glutathione analogues (glutathionesulfonic acid, S-methyl-glutathione and glutathione) were synthesised and characterised . The biomimetic ligands were immobilised on agarose gel and the affinity adsorbents, together with a nonbiomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their purifying ability for the glutathione-recognising enzymes, NAD+-dependent formaldehyde dehydrogenase (FaDH) from Candida boidinii, NAD(P)+-dependent glutathione reductase from S . cerevisiae (GSHR) and recombinant maize glutathione S-transferase I (GSTI) . All biomimetic adsorbents showed higher purifying ability for the target enzymes compared to the nonbiomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials . In particular, the affinity adsorbent comprising as terminal biomimetic moiety glutathionesulfonic acid (BM1), exhibited the highest purifying ability for FaDH and GSTI, whereas, the affinity adsorbent comprising as terminal biomimetic moiety methyl-glutathione (BM2) exhibited the highest purifying ability for GSHR . The BM1 adsorbent was integrated in a facile two-step purification procedure for FaDH . The purified enzyme showed a specific activity equal to 79 U/mg and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis . Molecular modelling was employed to visualise the binding of BM1 with FaDH, indicating favourable positioning of the key structural features of the biomimetic dye . The anthraquinone moiety provides the driving force for the correct positioning of the glutathionyl-biomimetic moiety in the binding site . It is located deep in the active site cleft forming many favourable hydrophobic contacts with hydrophobic residues of the enzyme . The positioning of the glutathione-like biomimetic moiety is primarily achieved by the strong ionic interactions with the Zn2+ ion of FaDH and Arg 114, and by the hydrophobic contacts made with Tyr 92 and Met 140 . Molecular models were also produced for the binding of BM1 and BM3 (glutathione-substituted) to GSTI . In both cases the biomimetic dye forms multiple hydrophobic interactions with the enzyme through binding to a surface pocket . While the glutathioine moiety of BM3 is predicted to bind in the crystallographically observed way, an alternative, more favourable mode seems to be responsible for the better purification results achieved with BM1.

J Mol Biol, 2001 Jun 22, 309(5), 1087 - 100
Protein-dependent transition states for ribonucleoprotein assembly; Webb AE et al.; Native folding and splicing by the Saccharomyces cerevisiae mitochondrial bI5 group I intron RNA is facilitated by both the S . cerevisiae CBP2 and Neurospora crassa CYT-18 protein cofactors . Both protein-bI5 RNA complexes splice at similar rates, suggesting that the RNA active site structure is similar in both ribonucleoproteins . In contrast, the two proteins assemble with the bI5 RNA by distinct mechanisms and bind opposing, but partially overlapping, sides of the group I intron catalytic core . Assembly with CBP2 is limited by a slow, unimolecular RNA folding step characterized by a negligible activation enthalpy . We show that assembly with CYT-18 shows four distinctive features . (1) CYT-18 binds stably to the bI5 RNA at the diffusion controlled limit, but assembly to a catalytically active RNA structure is still limited by RNA folding, as visualized directly using time-resolved footprinting . (2) This mechanism of rapid stable protein binding followed by subsequent assembly steps has a distinctive kinetic signature: the apparent ratio of k(off) to k(on), determined in a partitioning experiment, differs from the equilibrium K(d) by a large factor . (3) Assembly with CYT-18 is characterized by a large activation enthalpy, consistent with a rate limiting conformational rearrangement . (4) Because assembly from the kinetically trapped state is faster at elevated temperature, we can identify conditions where CYT-18 accelerates (catalyzes) bI5 RNA folding relative to assembly with CBP2 .

Bioresour Technol, 2001 Aug, 79(1), 99 - 102
Inhibition effect of initial Pb2+ concentration on Pb2+ accumulation by Saccharomyces cerevisiae and Aureobasidium pullulans; Suh JH et al.; Pb2+ accumulation by Saccharomyces cerevisiae and Aureobasidium pullulans was inhibited by the initial Pb2+ concentration . In the case of S . cerevisiae, as initial Pb2+ concentrations increased, the accumulated Pb2+ per unit cell dry weight at equilibrium and the time required to reach an equilibrium state increased at low initial Pb2+ concentration . On the contrary, the accumulated Pb2+ decreased at high initial Pb2+ concentration at all pH values . The inhibition effect of initial Pb2+ concentration was delayed by the decrease of pH . However, the maximal Pb2+ accumulation capacity of S . cerevisiae was almost constant regardless of pH values . In the case of A . pullulans, the time required to reach an equilibrium state was independent of the initial Pb2+ concentration . The maximal Pb2+ accumulation capacity of A . pullulans decreased according to the decrease of pH values . However, the initial Pb2+ concentration needed to reach maximal Pb2+ accumulation amount was almost constant.

Mol Membr Biol, 2001 Jan-Mar, 18(1), 73 - 9
Functional production of mammalian concentrative nucleoside transporters in Saccharomyces cerevisiae; Vickers MF et al.; The transport of nucleosides and nucleobases in the yeast Saccharomyces cerevisiae is reviewed and the use of this organism to study recombinant mammalian concentrative nucleoside transport (CNT) proteins is described . A selection strategy based on the ability of an expressed nucleoside transporter cDNA to mediate thymidine uptake by yeast under a selective condition that depletes endogenous thymidylate was used to assess the transport capacity of heterologous transporter proteins . The pyrimidine-nucleoside selective concentrative transporters from human (hCNT1) and rat (rCNT1) complemented the imposed thymidylate depletion in S . cerevisiae, as did N-terminally truncated versions of hCNT1 and rCNT1 lacking up to 31 amino acids . Transporter-mediated rescue of S . cerevisiae by both nucleoside transporters was inhibited by cytidine, uridine and adenosine, but not by guanosine or inosine . This work represents the development of a new model system for the functional production of recombinant nucleoside transporters of the CNT family of membrane proteins.

Mol Membr Biol, 2001 Jan-Mar, 18(1), 105 - 12
Multiplicity and regulation of genes encoding peptide transporters in Saccharomyces cerevisiae; Hauser M et al.; The model eukaryote Saccharomyces cerevisiae has two distinct peptide transport mechanisms, one for di-/tripeptides (the PTR system) and another for tetra-/pentapeptides (the OPT system) . The PTR system consists of three genes, PTR1, PTR2 and PTR3 . The transporter (Ptr2p), encoded by the gene PTR2, is a 12 transmembrane domain (TMD) integral membrane protein that translocates di-/tripeptides . Homologues to Ptr2p have been identified in virtually all organisms examined to date and comprise the PTR family of transport proteins . In S . cerevisiae, the expression of PTR2 is highly regulated at the cellular level by complex interactions of many genes, including PTR1, PTR3, CUP9 and SSY1 . Oligopeptides, consisting of four to five amino acids, are transported by the 12-14 TMD integral membrane protein Opt1p . Unlike Ptr2p, distribution of this protein appears limited to fungi and plants, and there appears to be three paralogues in S . cerevisiae . This transporter has an affinity for enkephalin, an endogenous mammalian pentapeptide, as well as for glutathione . Although it is known that OPT1 is normally expressed only during sporulation, to date little is known about the genes and proteins involved in the regulation of OPT1 expression.

Mol Cell Biol, 2001 Jul, 21(13), 4089 - 96
Phosphorylation of the RNA polymerase II carboxy-terminal domain by the Bur1 cyclin-dependent kinase; Murray S et al.; BUR1, which was previously identified by a selection for mutations that have general effects on transcription in Saccharomyces cerevisiae, encodes a cyclin-dependent kinase that is essential for viability, but none of its substrates have been identified to date . Using an unbiased biochemical approach, we have identified the carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II, as a Bur1 substrate . Phosphorylation of Rpb1 by Bur1 is likely to be physiologically relevant, since bur1 mutations interact genetically with rpb1 CTD truncations and with mutations in other genes involved in CTD function . Several genetic interactions are presented, implying a role for Bur1 during transcriptional elongation . These results identify Bur1 as a fourth S . cerevisiae CTD kinase and provide striking functional similarities between Bur1 and metazoan P-TEFb.

Mol Cell, 2001 May, 7(5), 1003 - 11
Transcriptional termination factors for RNA polymerase II in yeast; Aranda A et al.; The molecular connections between mRNA 3' end processing and transcriptional termination have been investigated in S . pombe using a genetic screen . By this approach, we have identified a RNAP II termination domain in the well-defined cleavage polyadenylation factor called CstF-64 in metazoans and Rna15p in S . cerevisiae . Furthermore, this C-terminal domain interacts with Res2, previously identified as a component of the G1/S transition-specific transcription factor MBF . Deletion of res2 in both fission and budding yeast results in a defect in 3' end formation . This raises the possibility that RNAP II transcriptional termination may in some situations be integrated with cell cycle events.

Mol Cell, 2001 May, 7(5), 959 - 70
Identification of RFC(Ctf18p, Ctf8p, Dcc1p): an alternative RFC complex required for sister chromatid cohesion in S . cerevisiae; Mayer ML et al.; We have identified and characterized an alternative RFC complex RFC(Ctf18p, Ctf8p, Dcc1p) that is required for sister chromatid cohesion and faithful chromosome transmission . Ctf18p, Ctf8p, and Dcc1p interact physically in a complex with Rfc2p, Rfc3p, Rfc4p, and Rfc5p but not with Rfc1p or Rad24p . Deletion of CTF18, CTF8, or DCC1 singly or in combination (ctf18Deltactf8Deltadcc1Delta) leads to sensitivity to microtubule depolymerizing drugs and a severe sister chromatid cohesion defect . Furthermore, temperature-sensitive mutations in RFC4 result in precocious sister chromatid separation . Our results highlight a novel function of the RFC proteins and support a model in which sister chromatid cohesion is established at the replication fork via a polymerase switching mechanism and a replication-coupled remodeling of chromatin.

Protein Expr Purif, 2001 Jun, 22(1), 101 - 7
Refolding and purification of yeast carboxypeptidase Y expressed as inclusion bodies in Escherichia coli; Hahm MS et al.; The genes encoding carboxypeptidase Y (CPY) and CPY propeptide (CPYPR) from Saccharomyces cerevisiae were cloned and expressed in Escherichia coli . Six consecutive histidine residues were fused to the C-terminus of the CPYPR for facilitated purification . High-level expression of CPY and CPYPR-His(6) was achieved but most of the expressed proteins were present in the form of inclusion bodies in the bacterial cytoplasm . The CPY and CPYPR-His(6) produced as inclusion bodies were separated from the cells and solubilized in 6 and 3 M guanidinium chloride, respectively . The denatured CPYPR-His(6) was refolded by dilution 1:30 into the renaturation buffer (50 mM Tris-HCl containing 0.5 M NaCl and 3 mM EDTA, pH 8.0), and the refolded CPYPR-His(6) was further purified to 90% purity by single-step immobilized metal ion affinity chromatography . The denatured CPY was refolded by dilution 1:60 into the renaturation buffer containing CPYPR-His(6) at various concentrations . Increasing the molar ratio of CPYPR-His(6) to CPY resulted in an increase in the CPY refolding yield, indicating that the CPYPR-His(6) plays a chaperone-like role in in vitro folding of CPY . The refolded CPY was purified to 92% purity by single-step p-aminobenzylsuccinic acid affinity chromatography . When refolding was carried out in the presence of 10 molar eq CPYPR-His(6), the specific activity, N-(2-furanacryloyl)-l-phenylalanyl-l-phenylalanine hydrolysis activity per milligram of protein, of purified recombinant CPY was found to be about 63% of that of native S . cerevisiae CPY .

Biosci Biotechnol Biochem, 2001 Apr, 65(4), 766 - 73
Molecular cloning and characterization of the fructooligosaccharide-producing beta-fructofuranosidase gene from Aspergillus niger ATCC 20611; Yanai K et al.; The fopA gene encoding a fructooligosaccharide-producing beta-fructofuranosidase was isolated from Aspergillus niger ATCC 20611 . The primary structure deduced from the nucleotide sequence showed considerable similarity to those of two other beta-fructofuranosidases from A . niger, but the fopA gene product had several amino acid insertions and an extra C-terminal polypeptide consisting of 38 amino acids that could not be found in the two others . We could successfully express the fopA gene in S . cerevisiae and the fopA gene product obtained from the culture supernatant of the S . cerevisiae transformant had similar characteristics to the beta-fructofuranosidase purified from A . niger ATCC 20611 . However, we could not detect any beta-fructofuranosidase activity in either the culture supernatant or cell lysate when the C-terminal truncated fopA gene product by 38 amino acids was used to transform S . cerevisiae . In western analysis of those samples, there was no protein product that is cross-reacted with anti-beta-fructofuranosidase antibody . These results suggested that the C-terminal region of the fopA gene product consisting of 38 amino acids was essential for the enzyme production.

Virus Res, 2001 Jul, 76(1), 17 - 29
Nucleolin stimulates viral internal ribosome entry site-mediated translation; Izumi RE et al.; Previous results from our laboratory have identified a small (60 nt) RNA from the yeast S . cerevisiae that specifically inhibits internal ribosome entry site (IRES)-mediated translation programmed by poliovirus (PV) and hepatitis C virus (HCV) 5'-untranslated region (5'UTR) . The yeast inhibitor RNA (called IRNA) was found to efficiently compete with viral 5'UTR for binding of several cellular polypeptides that presumably play important roles in IRES-mediated translation . One such IRNA (and 5'UTR)-binding protein has previously been identified as the La autoantigen . In this report, we have identified a 110-kDa IRNA-binding protein (which also interacts with viral 5'UTR) as nucleolin, a nucleolar RNA binding protein that was previously shown to translocate into the cytoplasm following infection of cells with poliovirus . We demonstrate that nucleolin (called C23) stimulates viral IRES-mediated translation both in vitro and in vivo . We also show that nucleolin mutants containing the carboxy-terminal RNA binding domains but lacking the amino terminal domain inhibit IRES-mediated translation in vitro . The translation inhibitory activity of these mutants correlates with their ability to bind the 5'UTR sequence . These results suggest a role of nucleolin/C23 in viral IRES-mediated translation.

J Biol Chem, 2001 Aug 24, 276(34), 31487 - 93 Epub 2001 May 25.
The MER3 helicase involved in meiotic crossing over is stimulated by single-stranded DNA-binding proteins and unwinds DNA in the 3' to 5' direction; Nakagawa T et al.; The meiosis-specific MER3 protein of Saccharomyces cerevisiae is required for crossing over, which ensures faithful segregation of homologous chromosomes at the first meiotic division . The predicted sequence of the MER3 protein contains the seven motifs characteristic of the DExH-box type of DNA/RNA helicases . The purified MER3 protein is a DNA helicase, which can displace a 50-nucleotide fragment annealed to a single-stranded circular DNA . MER3 was found to have ATPase activity, which was stimulated either by single- or double-stranded DNA . The turnover rate, k(cat), of ATP hydrolysis was approximately 500/min in the presence of either DNA . MER3 was able to efficiently displace relatively long 631-nucleotide fragments from single-stranded circular DNA only in the presence of the S . cerevisiae single-stranded DNA-binding protein, RPA (replication protein A) . It appears that RPA inhibits re-annealing of the single-stranded products of the MER3 helicase . The MER3 helicase was found to unwind DNA in the 3' to 5' direction relative to single-stranded regions in the DNA substrates . Possible roles for the MER3 helicase in meiotic crossing over are discussed.

Microbiol Res, 2001, 156(1), 9 - 12
Protein overexport in a Saccharomyces cerevisiae mutant depends on mitochondrial genome integrity and function; Alexandar I et al.; The wild-type yeast Saccharomyces cerevisiae (S . cerevisiae) is able to export less than 1 percent of the protein to be secreted . The reasons for retention of most of the secretory proteins on the cell surface of S . cerevisiae are unknown . Recently, temperature-sensitive (ts) mutants of S . cerevisiae showing an oversecretion phenotype were isolated . In order to study the influence of the mitochondrial genome status on protein export in yeast cells, we have isolated several types of respiratory impaired mitochondrial mutants of either the parental S . cerevisiae strain or their derivative ts protein-overexporting mutants . In this paper we demonstrate by quantitative analyses of exported proteins and by SDS-PAGE analysis that protein overexport in ts mutants requires mitochondrial genome integrity and function.

Biochemistry, 2001 May 29, 40(21), 6257 - 66
Kinetic preference for oriented DNA binding by the yeast TATA-binding protein TBP; Liu Y et al.; In solution, the TATA box binding protein from S . cerevisiae (yTBP) is only minimally oriented when bound to the adenovirus major late promoter (AdMLP) and the yeast CYC1 promoter . At equilibrium, approximately 60% of the complexes are assembled in the orientation observed within crystal structures; 40% are assembled in the opposite orientation . Here we use stopped-flow fluorescence resonance energy transfer (FRET) to study the association kinetics of the two TBP.TATA box orientational isomers . Kinetics were determined by monitoring FRET between a unique tryptophan residue engineered into either the C- or the N-terminal stirrup of the conserved C-terminal subunit of yeast TBP (yTBPc) and an aminocoumarin moiety appended either upstream or downstream of the TATA box . Together, these constructs permitted a simultaneous yet independent monitor of the kinetics of TBP binding in both orientations . Not only did our results provide an independent confirmation of the free energy difference between the two orientational isomers, but they also showed that the orientational binding preference at equilibrium is a result of a faster association rate when TBP binds DNA in the orientation observed in the crystal structure.

Eur J Cell Biol, 2001 Apr, 80(4), 257 - 70
Pex12p of Saccharomyces cerevisiae is a component of a multi-protein complex essential for peroxisomal matrix protein import; Albertini M et al.; We have isolated the Saccharomyces cerevisiae pex12-1 mutant from a screen to identify mutants defective in peroxisome biogenesis . The pex12delta deletion strain fails to import peroxisomal matrix proteins through both the PTS1 and PTS2 pathway . The PEX12 gene was cloned by functional complementation of the pex12-1 mutant strain and encodes a polypeptide of 399 amino acids . ScPex12p is orthologous to Pex12 proteins from other species and like its orthologues, S . cerevisiae Pex12p contains a degenerate RING finger domain of the C3HC4 type in its essential carboxy-terminus . Localization studies demonstrate that Pex12p is an integral peroxisomal membrane protein, with its NH2-terminus facing the peroxisomal lumen and with its COOH-terminus facing the cytosol . Pex12p-deficient cells retain particular structures that contain peroxisomal membrane proteins consistent with the existence of peroxisomal membrane remnants ("ghosts") in pex12A null mutant cells . This finding indicates that pex12delta cells are not impaired in peroxisomal membrane biogenesis . In immunoisolation experiments Pex12p was co-purified with the RING finger protein Pex10p, the PTS1 receptor Pex5p and the docking proteins for the PTS1 and the PTS2 receptor at the peroxisomal membrane, Pex13p and Pex14p . Furthermore, two-hybrid experiments suggest that the two RING finger domains are sufficient for the Pex10p-Pex12p interaction . Our results suggest that Pex12p is a component of the peroxisomal translocation machinery for matrix proteins.

Arch Biochem Biophys, 2001 May 1, 389(1), 7 - 14
Kinetic characterization of the rotenone-insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae; Velazquez I et al.; Saccharomyces cerevisiae mitochondria contain an NADH:Q6 oxidoreductase (internal NADH dehydrogenase) encoded by NDI1 gene in chromosome XIII . This enzyme catalyzes the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane . From a structural point of view, the mature enzyme has a single subunit of 53 kDa with FAD as the only prosthetic group . Due to the fact that S . cerevisiae cells lack complex I, the expression of this protein is essential for cell growth under respiratory conditions . The results reported in this work show that the internal NADH dehydrogenase follows a ping-pong mechanism, with a Km for NADH of 9.4 microM and a Km for oxidized 2,6-dichorophenolindophenol (DCPIP) of 6.2 microM . NAD+, one of the products of the reaction, did not inhibit the enzyme while the other product, reduced DCPIP, inhibited the enzyme with a Ki of 11.5 microM . Two dead-end inhibitors, AMP and flavone, were used to further characterize the kinetic mechanism of the enzyme . AMP was a linear competitive inhibitor of NADH (Ki = 5.5 mM) and a linear uncompetitive inhibitor of oxidized DCPIP (Ki = 11.5 mM), in agreement with the ping-pong mechanism . On the other hand, flavone was a partial inhibitor displaying a hyperbolic uncompetitive inhibition regarding NADH, and a hyperbolic noncompetitive inhibition with respect to oxidized DCPIP . The apparent intercept inhibition constant (Kii = 5.4 microM) and the slope inhibition constant (Kis = 7.1 microM) were obtained by non linear regression analysis . The results indicate that the ternary complex F-DCPIPox-flavone catalyzes the reduction of DCPIP, although with lower efficiency . The effect of pH on Vmax was studied . The Vmax profile shows two groups with pKa values of 5.3 and 7.2 involved in the catalytic process.

Gene, 2001 May 2, 268(1-2), 23 - 30
Cloning and characterization of Aspergillus nidulans vpsA gene which is involved in vacuolar biogenesis; Tarutani Y et al.; In Saccharomyces cerevisiae, vacuoles play very important roles in pH and osmotic regulation, protein degradation and storage of amino acids, small ions as well as polyphosphates . In filamentous fungi, however, little is known about vacuolar functions at a molecular level . In this paper, we report the isolation of the vpsA gene from the filamentous fungus Aspergillus nidulans as a homologue of the VPS1 gene of S . cerevisiae which encodes a dynamin-related protein . The vpsA gene encodes a polypeptide consisting of 696 amino acids that is nearly 60% homologous to the S . cerevisiae Vps1 . Similar to Vps1, VpsA contains a highly conserved tripartite GTPase domain but lacks the pleckstrin homology domain and proline-rich region . The vpsA disruptant shows poor growth and contains highly fragmented vacuoles . These results suggest that A . nidulans VpsA functions in the vacuolar biogenesis.

Mol Microbiol, 2001 May, 40(3), 656 - 68
Functional study of the Saccharomyces cerevisiae Nha1p C-terminus; Kinclova O et al.; Saccharomyces cerevisiae cells possess an alkali metal cation antiporter encoded by the NHA1 gene . Nha1p is unique in the family of yeast Na+/H+ antiporters on account of its broad substrate specificity (Na+, Li+, K+) and its long C-terminus (56% of the whole protein) . In order to study the role of the C-terminus in Nha1p function, we constructed a series of 13 truncated NHA1 versions ranging from the complete one (2958 nucleotides, 985 amino acids) down to the shortest version (1416 nucleotides, 472 amino acids), with only 41 amino acid residues after the last putative transmembrane domain . Truncated NHA1 versions were expressed in an S . cerevisiae alkali metal cation-sensitive strain (B31; ena1-4Delta nha1Delta) . We found that the entire Nha1p C-terminus domain is not necessary for either the proper localization of the antiporter in the plasma membrane or the transport of all four substrates (we identified rubidium as the fourth Nha1p substrate) . Partial truncation of the C-terminus of about 70 terminal amino acids improves the tolerance of cells to Na+, Li+ and Rb+ compared with cells expressing the complete Nha1p . The presence of the neighbouring part of the C-terminus (amino acids 883-928), rich in aspartate and glutamate residues, is necessary for the maintenance of maximum Nha1p activity towards sodium and lithium . In the case of potassium, the participation of the long C-terminus in the regulation of intracellular potassium content is demonstrated . We also present evidence that the Nha1p C-terminus is involved in the cell response to sudden changes in environmental osmolarity.

J Biol Chem, 2001 Jul 20, 276(29), 27026 - 33 Epub 2001 May 17.
RACK1 interacts with E1A and rescues E1A-induced yeast growth inhibition and mammalian cell apoptosis; Sang N et al.; The adenoviral E1A proteins are able to promote proliferation and transformation, inhibit differentiation, induce apoptosis, and suppress tumor growth . The extreme N terminus and conserved region one of E1A, which are indispensable for transcriptional regulation and for binding to p300/CBP, TBP, and pCAF, play essential roles in these abilities . These observations strongly suggest an intrinsic link between E1A-mediated transcriptional regulation and other effects . In this report, we show that E1A inhibits the normal growth of Saccharomyces cerevisiae HF7c, and this inhibition also depends on the domains required for transcriptional regulation . We demonstrate that E1A associates with histone acetyltransferase activity and represses the transactivation activity of transcription factor in S . cerevisiae, suggesting that E1A may suppress the expression of genes required for normal growth . Based on yeast growth rescue, we present a genetic screening strategy that identified RACK1 as an E1A antagonizing factor . Expression of human RACK1 efficiently relieves E1A-mediated growth inhibition in HF7c and protects human tumor cells from E1A-induced apoptosis . Finally, we show that RACK1 decreases E1A-associated histone acetyltransferase activity in yeast and mammalian cells, and physically interacts with E1A . Our data demonstrate that RACK1 is a repressor of E1A, possibly by antagonizing the effects of E1A on host gene transcription.

Eur J Biochem, 2001 May, 268(10), 3083 - 90
CYP94A5, a new cytochrome P450 from Nicotiana tabacum is able to catalyze the oxidation of fatty acids to the omega-alcohol and to the corresponding diacid; Le Bouquin R et al.; A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A5) was isolated from a tobacco cDNA library . CYP94A5 was expressed in S . cerevisiae strain WAT11 containing a P450 reductase from Arabidopsis thaliana necessary for catalytic activity of cytochrome P450 enzymes . When incubated for 10 min in presence of NADPH with microsomes of recombinant yeast, 9,10-epoxystearic acid was converted into one major metabolite identified by GC/MS as 18-hydroxy-9,10-epoxystearic acid . The kinetic parameters of the reaction were Km,app = 0.9 +/- 0.2 microM and Vmax,app = 27 +/- 1 nmol x min(-1) x nmol(-1) P450 . Increasing the incubation time to 1 h led to the formation of a compound identified by GC/MS as 9,10-epoxy-octadecan-1,18-dioic acid . The diacid was also produced in microsomal incubations of 18-hydroxy-9,10-epoxystearic acid . Metabolites were not produced in incubations with microsomes of yeast transformed with a control plasmid lacking CYP94A5 and their production was inhibited by antibodies raised against the P450 reductase, demonstrating the involvement of CYP94A5 in the reactions . The present study describes a cytochrome P450 able to catalyze the complete set of reactions oxidizing a terminal methyl group to the corresponding carboxyl . This new fatty acid hydroxylase is enantioselective: after incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was 40/60 in favor of 9R,10S enantiomer . CYP94A5 also catalyzed the omega-hydroxylation of saturated and unsaturated fatty acids with aliphatic chain ranging from C12 to C18.

Biochim Biophys Acta, 2001 Feb 9, 1510(1-2), 426 - 41
Vesicular monoamine transporters heterologously expressed in the yeast Saccharomyces cerevisiae display high-affinity tetrabenazine binding; Yelin R et al.; A mammalian vesicular neurotransmitter transporter has been expressed in the yeast Saccharomyces cerevisiae . The gene encoding the rat vesicular monoamine transporter (rVMAT(1)) was cloned in several expression plasmids . The transporter was expressed at detectable levels only when short sequences using codons favored by S . cerevisiae were fused preceding the start of translation of rVMAT(1) . The scarce expression of the wild-type protein was, most likely, due to the fact that part of the N-terminus of the protein is encoded by codons not preferred in S . cerevisiae . Furthermore, low growth temperatures increased rVMAT(1) expression and altered its processing . Whereas at 30 degrees C the protein is not glycosylated, at lower temperatures ( approximately 16 degrees C) half of the expressed transporters undergo core glycosylation . In addition, under these conditions the levels of protein expression significantly increase . Using a functional chimeric protein composed by VMAT and the green fluorescent protein (GFP), it is shown that the punctate pattern of intracellular distribution remains invariable at the different temperatures . Using a similar fusion sequence, the bovine VMAT isoform 2 (bVMAT(2)) was also expressed in yeast . The yeast-expressed bVMAT(2) binds {(3)H}dihydrotetrabenazine ({(3)H}TBZOH) with the same characteristics found in the native protein from bovine chromaffin granules . Dodecyl maltoside-solubilized bVMAT(2) retains the conformation required for {(3)H}TBZOH binding . We exploited the robust binding to follow the transporter during purification assays on a Ni(2+)-chelating column . In this report we describe for the first time the heterologous expression of a neurotransmitter transporter in the yeast S . cerevisiae.

Structure (Camb), 2001 Jan 10, 9(1), 39 - 46
Structure of the globular region of the prion protein Ure2 from the yeast Saccharomyces cerevisiae; Bousset L et al.; BACKGROUND: The {URE3} non-Mendelian element of the yeast S . cerevisiae is due to the propagation of a transmissible form of the protein Ure2 . The infectivity of Ure2p is thought to originate from a conformational change of the normal form of the prion protein . This conformational change generates a form of Ure2p that assembles into amyloid fibrils . Hence, knowledge of the three-dimensional structure of prion proteins such as Ure2p should help in understanding the mechanism of amyloid formation associated with a number of neurodegenerative diseases . RESULTS: Here we report the three-dimensional crystal structure of the globular region of Ure2p (residues 95--354), also called the functional region, solved at 2.5 A resolution by the MAD method . The structure of Ure2p 95--354 shows a two-domain protein forming a globular dimer . The N-terminal domain is composed of a central 4 strand beta sheet flanked by four alpha helices, two on each side . In contrast, the C-terminal domain is entirely alpha-helical . The fold of Ure2p 95--354 resembles that of the beta class glutathione S-transferases (GST), in line with a weak similarity in the amino acid sequence that exists between these proteins . Ure2p dimerizes as GST does and possesses a potential ligand binding site, although it lacks GST activity . CONCLUSIONS: The structure of the functional region of Ure2p is the first crystal structure of a prion protein . Structure comparisons between Ure2p 95--354 and GST identified a 32 amino acid residues cap region in Ure2p exposed to the solvent . The cap region is highly flexible and may interact with the N-terminal region of the partner subunit in the dimer . The implication of this interaction in the assembly of Ure2p into amyloid fibrils is discussed.

Mol Cell, 2001 Apr, 7(4), 887 - 98
A block to mRNA nuclear export in S . cerevisiae leads to hyperadenylation of transcripts that accumulate at the site of transcription; Jensen TH et al.; Several factors contribute to nuclear mRNA export in Saccharomyces cerevisiae, including Mex67p, Mtr2p, Gle1p, Nup159p, Dbp5p, and Rip1p . Strains carrying mutations in these factors show rapid and dramatic nuclear accumulation of poly(A)(+) RNA . We have characterized two heat shock mRNAs, SSA4 and HSP104, in these mutant backgrounds; each transcript concentrates in a single intranuclear focus . Evidence suggests that it coincides with the site of transcription . Interestingly, all detectable SSA4 transcripts have undergone 3'-end formation, indicating that RNAs in the foci are no longer nascent . Poly(A) tails of the transcripts are also dramatically longer in all of these export mutants . Based on all of the data, we suggest that very early mRNA maturation events determine transcript export competence.

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