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 Leeu