Yeast, 2003 Jan 15, 20(1), 79 - 88 Disruption of 12 ORFs located on chromosomes IV, VII and XIV of Saccharomyces cerevisiae reveals two essential genes; Volckaert G et al.; We describe the generation of null-mutants of 12 open reading frames (ORFs), discovered during the systematic sequencing of the Saccharomyces cerevisiae genome . These ORFs are located on chromosome IV (YDL183c), on chromosome VII (YGL139w, YGL140c, YGL141w, YGR280c and YGR284c) or on chromosome XIV (YNL006w, YNR004w, YNR007c, YNR008w, YNR009w and YNR013c) . Disruptants were generated using the PCR-based short flanking homology (SFH) strategy in yeast strain FY1679 . Tetrad analysis, following sporulation of the heterozygous disruptants, revealed that YGR280c and YNL006w are essential genes for vegetative yeast growth in rich medium . The lethality of the two genes was confirmed by gene complementation analysis . The protein encoded by YNL006w (LST8) is now known to be involved in transport of permeases from the Golgi to the plasma membrane . Basic phenotypic analyses were performed on haploid disruptants from both mating types of 10 non-essential genes . One disruptant (YNR004w) revealed a slow growth rate on glucose-minimal medium at 15 degrees C . For each of the individual ORFs, a disruption cassette and the corresponding cognate gene were cloned into appropriate plasmids . Yeast, 2003 Jan 15, 20(1), 53 - 67 Automated screening in environmental arrays allows analysis of quantitative phenotypic profiles in Saccharomyces cerevisiae; Warringer J et al.; A methodology for large-scale automated phenotypic profiling utilizing quantitative changes in yeast growth has been tested and applied to the analysis of some commonly used laboratory strains . This yeast-adjusted methodology is based on microcultivation in 350 microl liquid medium, where growth is frequently optically recorded, followed by automated extraction of relevant variables from obtained growth curves . We report that cultivation at this micro-scale displayed overall growth features and protein expression pattern highly similar to growth in well aerated medium-scale (10 ml) culture . However, differences were also encountered, mainly relating to the respiratory potential and the production of stress-induced proteins . Quantitative phenotypic profiles for the laboratory yeast strains W303, FY1679 and CEN-PK.2 were screened for in environmental arrays, including 98 different conditions composed of low, medium and high concentrations of 33 growth inhibitors . We introduce the concepts phenotypic index(rate) and phenotypic index(stationary), which relate to changes in rate of growth and the stationary phase optical density increment, respectively, in a particular environment relative a reference strain . The laboratory strains presented selective phenotypic profiles in both phenotypic indexes and the two features appeared in many cases to be independent characteristics . We propose the utilization of this methodology in large-scale screening of the complete collection of yeast deletion mutants . Yeast, 2003 Jan 15, 20(1), 31 - 7 The role of the GATA factors Gln3p, Nil1p, Dal80p and the Ure2p on ASP3 regulation in Saccharomyces cerevisiae; Oliveira EM et al.; The role of Gln3p, Nil1p, Dal80p and Ure2p in the nitrogen regulation of ASP3, which codes for the periplasmic Saccharomyces cerevisiae asparaginase II, was investigated . Analysis of enzyme levels and mRNA(ASP3) in two wild-type strains and gln3, nil1, gln3nil1, gln3ure2, nil1ure2, nil1dal80, ure2, dal80 and ure2dal80 mutant cells allowed the study of the qualitative and quantitative regulatory role of the GATA factors and Ure2p on ASP3 expression . The simultaneous presence of Gln3p and Nil1p is a required condition for full gene transcription . Enzyme activity doubled upon nitrogen starvation of either ammonium-grown (possibly due to Nil2p/Deh1p derepression) or proline-grown (due to Dal80p derepression) cells . The ure2 mutation increased enzyme levels five-fold in fresh ammonium-grown cells and ten-fold in fresh proline-grown cells . The combined effects of the ure2 mutation and nitrogen starvation on ammonium- or proline-grown cells resulted in an overall 10-20-fold enzyme activity increase, respectively, in comparison with the wild-type cells . J Clin Gastroenterol, 2003 Jan, 36(1), 39 - 40 Saccharomyces cerevisiae-associated diarrhea in an immunocompetent patient with ulcerative colitis; Candelli M et al.; Infectious diarrhea is common in cases of ulcerative colitis (UC), caused by resistance to treatment . In particular, it has been associated with cytomegalovirus or infection has rarely been observed in human beings, but has never been seen in immunocompetent patients . We report a case of intestinal infection presenting as acute diarrhea in a patient with a 10-year history of UC who was never treated with immunosuppressants. J Biol Chem, 2003 Feb 28, 278(9), 6687 - 95 Epub 2002 Dec 16. Rpn6p, a proteasome subunit from Saccharomyces cerevisiae, is essential for the assembly and activity of the 26 S proteasome; Santamaria PG et al.; We report the functional characterization of RPN6, an essential gene from Saccharomyces cerevisiae encoding the proteasomal subunit Rpn6p . For this purpose, conditional mutants that are able to grow on galactose but not on glucose were obtained . When these mutants are shifted to glucose, Rpn6p depletion induces several specific phenotypes . First, multiubiquitinated proteins accumulate, indicating a defect in proteasome-mediated proteolysis . Second, mutant yeasts are arrested as large budded cells with a single nucleus and a 2C DNA content; in addition, the spindle pole body is duplicated, indicating a general cell cycle defect related to the turnover of G(2)-cyclins after DNA synthesis . Clb2p and Pds1p, but not Sic1p, accumulate in the arrested cells . Depletion of Rpn6p affects both the structure and the peptidase activity of proteasomes in the cell . These results implicate Rpn6p function in the specific recognition of a subset of substrates and point to a role in maintaining the correct quaternary structure of the 26 S proteasome. EMBO J, 2002 Dec 16, 21(24), 6893 - 904 In Saccharomyces cerevisiae, ATP2 mRNA sorting to the vicinity of mitochondria is essential for respiratory function; Margeot A et al.; We recently demonstrated that polysome-associated mRNAs that co-isolate with mitochondria encode a subset of mitochondrial proteins, and that the 3' UTRs of these transcripts are essential for their localization to the vicinity of the organelle . To address the question of the involvement of the mRNA targeting process in mitochondrial biogenesis, we studied the role of ATP2 3' UTR . An altered ATP2 allele in which the 3' UTR was replaced by the ADH1 3' UTR exhibits properties supporting the importance of mRNA localization to the vicinity of mitochondria: (i) the mutated strain presents a respiratory dysfunction; (ii) mitochondrial import of the protein translated from the altered gene is strongly reduced, even though the precursor is addressed to the organelle surface; (iii) systematic deletions of ATP2 3' UTR revealed a 100 nucleotide element presenting RNA targeting properties . Additionally, when the ATM1 3' UTR was replaced by the ADH1 3' UTR, we obtained cells in which ATM1 mRNA is also delocalized, and presenting a respiratory dysfunction . This demonstrates that mRNA localization to the vicinity of mitochondria plays a critical role in organelle biogenesis. J Gen Appl Microbiol, 2001 Feb, 47(1), 21 - 26 Impact of mitochondrial activity on the cell wall composition and on the resistance to tannic acid in Saccharomyces cerevisiae; Wauters T et al.; In screening for resistance to tannic acid, mutants of Saccharomyces cerevisiae with an altered cell wall composition were recently isolated . Here we show that these mutants were all respiratory deficient . Cytoplasmic petite mutants isolated after ethidium bromide mutagenesis were resistant to tannic acid and had cell wall characteristics similar to the mutants isolated by screening for tannic acid resistance as shown by the lower sensitivity to zymolyase, a cell wall hydrolyzing enzyme, and by a changed sensitivity to calcofluor white, a molecule interfering with the cell wall assembly . Reintroducing active mitochondria to a tannic-acid-resistant mutant reduced the tannic acid resistance and zymolyase resistance to the wild-type level, showing that a mitochondrial mutation was responsible for the changes in cell wall composition and in tannic acid sensitivity. Microbiology, 2002 Dec, 148(Pt 12), 4035 - 48 The protein kinase Kic1 affects 1,6-beta-glucan levels in the cell wall of Saccharomyces cerevisiae; Vink E et al.; KIC1 encodes a PAK kinase that is involved in morphogenesis and cell integrity . Both over- and underexpressing conditions of KIC1 affected cell wall composition . Kic1-deficient cells were hypersensitive to the cell wall perturbing agent calcofluor white and had less 1,6-beta-glucan . When Kic1-deficient cells were crossed with various kre mutants, which also have less 1,6-beta-glucan in their wall, the double mutants displayed synthetic growth defects . However, when crossed with the 1,3-beta-glucan-deficient strain fks1delta, no synthetic growth defect was observed, supporting a specific role for KIC1 in regulating 1,6-beta-glucan levels . Kic1-deficient cells also became highly resistant to the cell wall-degrading enzyme mixture Zymolyase, and exhibited higher transcript levels of the cell wall protein-encoding genes CWP2 and SED1 . Conversely, overexpression of KIC1 resulted in increased sensitivity to Zymolyase and in a higher level of 1,6-beta-glucan . Multicopy suppressor analysis of a Kic1-deficient strain identified RHO3 . Consistent with this, expression levels of RHO3 correlated with 1,6-beta-glucan levels in the cell wall . Interestingly, expression levels of KIC1 and the MAP kinase kinase PBS2 had opposite effects on Zymolyase sensitivity of the cells and on cell wall 1,6-beta-glucan levels in the wall . It is proposed that Kic1 affects cell wall construction in multiple ways and in particular in regulating 1,6-beta-glucan levels in the wall. Dev Cell, 2002 Dec, 3(6), 815 - 24 Studies of cargo delivery to the vacuole mediated by autophagosomes in Saccharomyces cerevisiae; Suzuki K et al.; In the yeast Saccharomyces cerevisiae, aminopeptidase I (API), a vacuolar hydrolase, is selectively transported to the vacuole via the autophagosome . API forms a cytosol to vacuole targeting (Cvt) complex in the cytoplasm . The complex is engulfed by the autophagosome under starvation conditions . In this study, the Cvt complex is visualized as a dot in the cytoplasm by fluorescence microscopy with API-GFP . The Cvt complex associates with the preautophagosomal structure (PAS), which plays a central role in autophagosome formation . In a Deltacvt19 mutant, which is specifically defective in API transport, but not in autophagy, the Cvt complex forms normally but never associates with the PAS . This indicates that Cvt19p mediates association between the Cvt complex and the PAS. Yeast, 2002 Dec, 19(16), 1425 - 30 The identification of the Saccharomyces cerevisiae gene AYT1(ORF-YLL063c) encoding an acetyltransferase; Alexander NJ et al.; The recent isolation and characterization of Tri101 in Fusarium sporotrichioides has led to the functional identification of the yeast open reading frame (ORF) YLL063c, located on chromosome XII of Saccharomyces cerevisiae . The sequence of YLL063c predicts a protein of 474 residues that has 45% identity and 70% similarity to FsTri101 . FsTri101 encodes a trichothecene 3-O-acetyltransferase that functions in trichothecene biosynthesis . Feeding studies indicated low levels of C3-OH acetylation in cultures of the laboratory yeast strain, RW2802 . No similar activity was found in RW2802 transformed with an integrative plasmid carrying a disrupted YLL063c gene . Based on these results, which show structural and functional similarities between YLL063c and FsTri101, we propose that YLL063c encodes an acetyltransferase capable of trichothecene 3-O-acetylation and have named this gene AYT1 . Published in 2002 by John Wiley & Sons, Ltd. Curr Genet, 2002 Nov, 42(2), 85 - 93 Epub 2002 Nov 16. Mutation of TRS130, which encodes a component of the TRAPP II complex, activates transcription of OCH1 in Saccharomyces cerevisiae; Yamamoto K et al.; The Saccharomyces cerevisiae Och1p is required for the initiation of the mannose outer chain elongation of cell wall mannoproteins . A trs130(ts1) mutant that showed an elevated expression of the OCH1-reporters was isolated in our laboratory . In this study, we attempted to gain an understanding of the relationship between the defect in the Trs130p function and the elevated expression of OCH1 . The trs130(ts1) mutant showed calcofluor white-resistant and hygromycin B-sensitive phenotypes, indicating that the mutant is defective in cell wall integrity . YPT31 and YPT32, the multi-copy suppressors of the trs130(ts1) mutant, differed in the extent of suppression of the mutant phenotypes and in their genetic interaction with TRS130 . Our results suggest that the blocking of vesicle transport by a loss of the Trs130p function causes some defect in the cell wall mannoproteins, which leads to the elevated expression of OCH1 through the Skn7p function, to compensate for the defect. Eukaryot Cell, 2002 Dec, 1(6), 1041 - 4 ROX1 and ERG regulation in Saccharomyces cerevisiae: implications for antifungal susceptibility; Henry KW et al.; Yeasts respond to treatment with azoles and other sterol biosynthesis inhibitors by upregulating the expression of the ERG genes responsible for ergosterol production . Previous studies on Saccharomyces cerevisiae implicated the ROX1 repressor in ERG regulation . We report that ROX1 deletion resulted in 2.5- to 16-fold-lower susceptibilities to azoles and terbinafine . In untreated cultures, ERG11 was maximally expressed in mid-log phase and expression decreased in late log phase, while the inverse was observed for ROX1 . In azole-treated cultures, ERG11 upregulation was preceded by a decrease in ROX1 RNA . These inverse correlations suggest that transcriptional regulation of ROX1 is an important determinant of ERG expression and hence of azole and terbinafine susceptibilities. Eukaryot Cell, 2002 Dec, 1(6), 884 - 94 The Glc7p-interacting protein Bud14p attenuates polarized growth, pheromone response, and filamentous growth in Saccharomyces cerevisiae; Cullen PJ et al.; A genetic selection in Saccharomyces cerevisiae for mutants that stimulate the mating pathway uncovered a mutant that had a hyperactive pheromone response pathway and also had hyperpolarized growth . Cloning and segregation analysis demonstrated that BUD14 was the affected gene . Disruption of BUD14 in wild-type cells caused mild stimulation of pheromone response pathway reporters, an increase in sensitivity to mating factor, and a hyperelongated shmoo morphology . The bud14 mutant also had hyperfilamentous growth . Consistent with a role in the control of cell polarity, a Bud14p-green fluorescent protein fusion was localized to sites of polarized growth in the cell . Bud14p shared morphogenetic functions with the Ste20p and Bni1p proteins as well as with the type 1 phosphatase Glc7p . The genetic interactions between BUD14 and GLC7 suggested a role for Glc7p in filamentous growth, and Glc7p was found to have a positive function in filamentous growth in yeast. Mol Biol Cell, 2002 Dec, 13(12), 4141 - 55 Kar9p-independent microtubule capture at Bud6p cortical sites primes spindle polarity before bud emergence in Saccharomyces cerevisiae; Segal M et al.; Spindle orientation is critical for accurate chromosomal segregation in eukaryotic cells . In the yeast Saccharomyces cerevisiae, orientation of the mitotic spindle is achieved by a program of microtubule-cortex interactions coupled to spindle morphogenesis . We previously implicated Bud6p in directing microtubule capture throughout this program . Herein, we have analyzed cells coexpressing GFP:Bud6 and GFP:Tub1 fusions, providing a kinetic view of Bud6p-microtubule interactions in live cells . Surprisingly, even during the G1 phase, microtubule capture at the recent division site and the incipient bud is dictated by Bud6p . These contacts are eliminated in bud6 delta cells but are proficient in kar9 delta cells . Thus, Bud6p cues microtubule capture, as soon as a new cell polarity axis is established independent of Kar9p . Bud6p increases the duration of interactions and promotes distinct modes of cortical association within the bud and neck regions . In particular, microtubule shrinkage and growth at the cortex rarely occur away from Bud6p sites . These are the interactions selectively impaired at the bud cortex in bud6 delta cells . Finally, interactions away from Bud6p sites within the bud differ from those occurring at the mother cell cortex, pointing to the existence of an independent factor controlling cortical contacts in mother cells after bud emergence. J Biochem (Tokyo), 2002 Dec, 132(6), 967 - 73 Overexpression and functional characterization of a serine carboxypeptidase inhibitor (I(C)) from Saccharomyces cerevisiae; Mima J et al.; Carboxypeptidase Y (CPY) inhibitor, I(C), a cytoplasmic inhibitor of vacuolar proteinases in yeast, Saccharomyces cerevisiae, was purified by means of a high-level expression system using a proteinase-deficient strain, BJ2168, and an expression vector with the promoter GAL1 . The purified I(C) exists as a monomeric beta-protein in solution with a mole-cular weight of 24,398.4 as determined by gel filtration chromatography, MALDI-TOF mass spectrometry, and far-UV CD spectroscopy . The acetylated N-terminal methionine residue is the sole posttranslational modification . I(C) specifically inhibits both the peptidase and anilidase activities of CPY with inhibitor constants (K(i)) of approximately 1.0 x 10(-9) M . The chemical modification of I(C) with sulfhydryl reagents indicated that it lacks disulfide bonds and has two free SH groups, which are responsible, not for the inhibitory function, but, apparently, for the folding of the overall structure . The formation of a complex of I(C) with CPY was highly specific, as evidenced by no detectable interaction with pro-CPY . Chemical modification studies of the CPY-I(C) complex with specific reagents demonstrated that the catalytic Ser146 and S1 substrate-binding site of CPY are covered in the complex. Biochem J, 2003 Mar 15, 370(Pt 3), 805 - 15 Nucleoside diphosphate kinase of Saccharomyces cerevisiae, Ynk1p: localization to the mitochondrial intermembrane space; Amutha B et al.; Nucleoside diphosphate kinase (NDPK) is a highly conserved multifunctional enzyme . It catalyses the transfer of gamma phosphates from nucleoside triphosphates to nucleoside diphosphates by a mechanism that involves formation of an autophosphorylated enzyme intermediate . The phosphate is usually supplied by ATP . NDPK activity in different subcellular compartments may regulate the crucial balance between ATP and GTP or other nucleoside triphosphates . NDPKs are homo-oligomeric proteins and are predominantly localized in the cytosol . In this paper, we demonstrate that in Saccharomyces cerevisiae a small fraction of total NDPK activity encoded by YNK1 is present in the intermembrane space (IMS) of mitochondria, and the corresponding protein Ynk1p in the IMS represents approx . 0.005% of total mitochondrial proteins . Ynk1p, synthesized as a single gene product, must therefore be partitioned between cytoplasm and mitochondrial IMS fractions . A mechanism for this partitioning is suggested by our observations that interaction with a 40 kDa protein of the translocase of outer mitochondrial membrane (Tom40p), occurs preferentially with unfolded, unphosphorylated forms of Ynk1p . A population of newly translated, but not yet folded or autophosphorylated, Ynk1p intermediates may be imported into the IMS of mitochondria and trapped there by subsequent folding and oligomerization . Within the small volume of the IMS, Ynk1p may be more concentrated and may be required to supply GTP to several important proteins in this compartment. Proteomics, 2002 Dec, 2(12), 1706 - 14 Subproteomics: identification of plasma membrane proteins from the yeast Saccharomyces cerevisiae; Navarre C et al.; As a consequence of their poor solubility during isoelectric focusing, integral membrane proteins are generally absent from two-dimensional gel proteome maps . In order to analyze the yeast plasma membrane proteome, a plasma membrane purification protocol was optimized in order to reduce contaminating membranes and cytosolic proteins . Specifically, the new fractionation scheme largely depleted the plasma membrane fraction of cytosolic proteins by deoxycholate stripping and ribosomal proteins by sucrose gradient flotation . The plasma membrane complement was resolved by two-dimensional electrophoresis using the cationic detergent cetyl trimethyl ammonium bromide in the first, and sodium dodecyl sulfate in the second dimension, and fifty spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectometry . In spite of the presence of still contaminating ribosomal proteins, major proteins corresponded to known plasma membrane residents, the ABC transporters Pdr5p and Snq2p, the P-type H(+)-ATPase Pma1p, the glucose transporter Hxt7p, the seven transmembrane-span Mrh1p, the low affinity Fe(++) transporter Fet4p, the twelve-span Ptr2p, and the plasma membrane anchored casein kinase Yck2p . The four transmembrane-span proteins Sur7p and Nce102p were also present in the isolated plasma membranes, as well as the unknown protein Ygr266wp that probably contains a single transmembrane span . Thus, combining subcellular fractionation with adapted two-dimensional electrophoresis resulted in the identification of intrinsic plasma membrane proteins. J Gen Appl Microbiol, 2002 Aug, 48(4), 223 - 31 A promoter activity in Saccharomyces cerevisiae of the 3'-noncoding region of the basidiomycetous mushroom gene; Yamazaki T et al.; The 3'-noncoding region of the priB gene derived from the basidiomycete Lentinus edodes was found to contain one GC box-like sequence, two CAAT boxes, two TATA box-like sequences and two pyrimidine-rich stretches (CT-motifs) . It also contained a 16-bp sequence similar to the consensus sequence for PRIB protein binding and a short (61 bp) intron . Single-strand-specific S1 nuclease analysis of plasmid pBR322 DNA containing the 3'-noncoding region propagated in Escherichia coli revealed that this region forms an unusual, extended open structure within/around the downstream pyrimidine/purine (CT/AG)-biased sequence . To examine the promoter activity of the priB 3'-noncoding region in Saccharomyces cerevisiae, in which an autonomously replicating plasmid vector is available, we constructed two plasmids, YEp3'NCR-lacZ and YEp3'UTR-lacZ . The former contains the priB 3'-noncoding region and the reporter E . coli beta-galactosidase gene while the latter contains the priB 3'-noncoding region lacking the intron and the reporter gene . The yeast transformants obtained through introductions of these plasmids showed beta-galactosidase activity and the activity conferred by YEp3'NCR-lacZ was about 50% of that conferred by YEp3'UTR-lacZ . The primer extension analysis showed that transcription of the reporter gene on both plasmids starts at three alternative sites all of which are located within the downstream CT-motif, suggesting a role for the unusual structure of the CT/AG-biased sequence in the initiation of transcription. Gene, 2002 Oct 30, 300(1-2), 63 - 8 Structure, function and DNA composition of Saccharomyces cerevisiae chromatin loops; Filipski J et al.; Recent localization of cohesin association regions along the yeast chromatin fibre suggests that compositional variability of DNA in yeast is related to the function and organization of the chromosomal loops . The bases of the loops, where the chromatin fibre is attached to the chromosomal axis, are AT-rich, bind cohesin, and are flanked by genes transcribed convergently . The hotspots of meiotic recombination are mainly found in the GC-rich parts of the loops, 'external' with respect to the chromosomal axis, frequently in the vicinity of the promoters of divergently transcribed genes . There are two possible reasons why the regions of the hotspots of recombination were enriched in GC content during evolution . One is a biased repair of recombination intermediates, and the second is a selective advantage due to an increased chromatin accessibility, which may have the carriers of GC-enriched alleles over the carriers of AT-rich alleles. Nucleic Acids Res, 2002 Dec 1, 30(23), 5136 - 41 The vertebrate protein CTCF functions as an insulator in Saccharomyces cerevisiae; Defossez PA et al.; Insulators are elements that shelter genes from the effects of silencers or enhancers . CTCF is the only vertebrate protein that has a recognized role in transcriptional insulation, but how it exerts its effect is unknown . In an attempt to better understand how CTCF functions, we have used an insulation assay in Saccharomyces cerevisiae . We show that CTCF acts as an insulator in yeast, where it can efficiently block the spreading of repressive telomeric chromatin . We identify two domains of the protein that are responsible for this activity: a short and very potent N-terminal domain, as well as the C-terminus of the protein. Genes Dev, 2002 Dec 1, 16(23), 3017 - 33 Complex transcriptional circuitry at the G1/S transition in Saccharomyces cerevisiae; Horak CE et al.; In the yeast Saccharomyces cerevisiae, SBF (Swi4-Swi6 cell cycle box binding factor) and MBF (MluI binding factor) are the major transcription factors regulating the START of the cell cycle, a time just before DNA replication, bud growth initiation, and spindle pole body (SPB) duplication . These two factors bind to the promoters of 235 genes, but bind less than a quarter of the promoters upstream of genes with peak transcript levels at the G1 phase of the cell cycle . Several functional categories, which are known to be crucial for G1/S events, such as SPB duplication/migration and DNA synthesis, are under-represented in the list of SBF and MBF gene targets . SBF binds the promoters of several other transcription factors, including HCM1, PLM2, POG1, TOS4, TOS8, TYE7, YAP5, YHP1, and YOX1 . Here, we demonstrate that these factors are targets of SBF using an independent assay . To further elucidate the transcriptional circuitry that regulates the G1-to-S-phase progression, these factors were epitope-tagged and their binding targets were identified by chIp-chip analysis . These factors bind the promoters of genes with roles in G1/S events including DNA replication, bud growth, and spindle pole complex formation, as well as the general activities of mitochondrial function, transcription, and protein synthesis . Although functional overlap exists between these factors and MBF and SBF, each of these factors has distinct functional roles . Most of these factors bind the promoters of other transcription factors known to be cell cycle regulated or known to be important for cell cycle progression and differentiation processes indicating that a complex network of transcription factors coordinates the diverse activities that initiate a new cell cycle. Biol Res, 2002, 35(3-4), 347 - 57 Heterologous expression of syntaxin 6 in Saccharomyces cerevisiae; Gotte M et al.; The molecular mechanisms of vesicular protein transport in eukaryotic cells are highly conserved . Members of the syntaxin family play a pivotal role in the membrane fusion process . We have expressed rat syntaxin 6 and its cytoplasmic domain in wild-type and pep12 mutant strains of Saccharomyces cerevisiae to elucidate the role of the syntaxin 6-dependent vesicular trafficking step in yeast . Immunofluorescence microscopy revealed a punctate, Golgi-like staining pattern for syntaxin 6, which only partially overlapped with Pep12p in wild-type yeast cells . In contrast to Pep12p, syntaxin 6 was not mislocalized to the vacuole upon expression from 2 micron vectors, which might be attributed to conserved sorting and retention signals . Syntaxin 6 was not capable of complementing the sorting and maturation defects of the vacuolar hydrolase CPY in pep12 null mutants . No dominant negative effects of either syntaxin 6 or syntaxin 6 delta C overexpression on CPY sorting and maturation were observed in wild-type yeast cells . We conclude that syntaxin 6 and Pep12p do not act at the same vesicular trafficking step(s) in yeast and higher eukaryotes. Glycobiology, 2002 Nov, 12(11), 749 - 62 The Saccharomyces cerevisiae alg12delta mutant reveals a role for the middle-arm alpha1,2Man- and upper-arm alpha1,2Manalpha1,6Man- residues of Glc3Man9GlcNAc2-PP-Dol in regulating glycoprotein glycan processing in the endoplasmic reticulum and Golgi apparatus; Cipollo JF et al.; N-glycosylation in nearly all eukaryotes proceeds in the endoplasmic reticulum (ER) by transfer of the precursor Glc(3)Man(9)GlcNAc(2) from dolichyl pyrophosphate (PP-Dol) to consensus Asn residues in nascent proteins . The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide lipid properly, and the alg12 mutant accumulates a Man(7)GlcNAc(2)-PP-Dol intermediate . We show that the Man(7)GlcNAc(2) released from alg12Delta-secreted invertase is Manalpha1,2Manalpha1,2Manalpha1,3(Manalpha1,2Manalpha1,3Manalpha1,6)-Manbeta1,4-GlcNAcbeta1-4GlcNAcalpha/beta, confirming that the Man(7)GlcNAc(2) is the product of the middle-arm terminal alpha1,2-mannoslytransferase encoded by the ALG9 gene . Although the ER glucose addition and trimming events are similar in alg12Delta and wild-type cells, the central-arm alpha1,2-linked Man residue normally removed in the ER by Mns1p persists in the alg12Delta background . This confirms in vivo earlier in vitro experiments showing that the upper-arm Manalpha1,2Manalpha1,6-disaccharide moiety, missing in alg12Delta Man(7)GlcNAc(2), is recognized and required by Mns1p for optimum mannosidase activity . The presence of this Man influences downstream glycan processing by reducing the efficiency of Ochlp, the cis-Golgi alpha1,6-mannosyltransferase responsible for initiating outer-chain mannan synthesis, leading to hypoglycosylation of external invertase and vacuolar protease A. Protein Expr Purif, 2002 Dec, 26(3), 416 - 24 Purification and some properties of Saccharomyces cerevisiae meiosis-specific protein kinase Ime2; Hui CM et al.; Ime2 is the founding member of a family of protein kinases that are required for effective progression through meiotic development . Ime2 is essential for the induction of meiosis-specific genes and for the activation of meiotic DNA replication in the budding yeast Saccharomyces cerevisiae . Aside from the fact that Ime2 is a protein kinase and shares several amino acid motifs with cyclin dependent kinases, virtually nothing is known about its enzymatic properties or substrates . Biochemical characterization of Ime2 has been hindered by its low abundance and short half-life . We have created baculovirus expression vectors to produce recombinant Ime2 in insect cells . In this report, we describe the overproduction of Ime2 and its purification using affinity chromatography . Using this procedure, we have been able to purify up to 2mg Ime2 from 1L of infected insect cells . The Ime2 isolated by this method displays properties similar to those of the native enzyme that has been immunoprecipitated from yeast . The high level expression of Ime2 in this system and its ease of purification will be beneficial for more extensive biochemical analysis of Ime2 and related meiosis-specific kinases. Lipids, 2002 Sep, 37(9), 863 - 8 Isolation and characterization of a delta5 FA desaturase from Pythium irregulare by heterologous expression in Saccharomyces cerevisiae and oilseed crops; Hong H et al.; By using the polymerase chain reaction approach with two degenerate primers targeting the heme-binding and the third histidine-rich motifs in microsomal carboxyl-directed desaturases, we identified a cDNA PiD5 from Pythium irregulare encoding a delta5 desaturase . The substrate specificity of the enzyme was studied in detail by expressing PiD5 in a yeast (Saccharomyces cerevisiae) mutant strain, AMY-2alpha, where ole1, a delta9 desaturase gene, is disrupted . The result revealed that the encoded enzyme could desaturate unsaturated FA from 16 to 20 carbons beginning with delta9 and delta11 as well as delta8 ethylenic double bonds . Introduction of PiD5 into Brassica juncea under the control of a CaMV 35S constitutive promoter resulted in accumulation of several delta5-unsaturated polymethylene-interrupted FA (delta5-UPIFA) including 18:2-5,9, 18:2-5,11, 18:3-5,9,12, and 18:4-5,9,12,15 in vegetative tissues . The transgenic enzyme could also desaturate the exogenously supplied homo-gamma-linolenic acid (20:3-8,11,14) to arachidonic acid (20:4-5,8,11,14) . Introduction of PiD5 into B . juncea and flax under the control of seed-specific promoters resulted in production of delta5-UPIFA, representing more than 10% of the total FA in the seeds. Int J Antimicrob Agents, 2002 Dec, 20(6), 444 - 50 Expression profiling of the response of Saccharomyces cerevisiae to 5-fluorocytosine using a DNA microarray; Zhang L et al.; 5-Fluorocytosine is a commonly used antifungal agent . It acts by inhibiting the synthesis of fungal DNA and protein . In order to understand the global response of Saccharomyces cerevisiae to changes in DNA and protein synthesis caused by 5-fluorocytosine, genome-wide transcript profiling following 5-fluorocytosine exposure was obtained . A total of 96 genes were identified as responsive to 25 microg/ml fluorocytosine treatment for 90 min, which caused approximately 17% specific growth inhibition . The transcript levels of 57 genes were increased more than 2-fold, while it was found that the transcript levels of the other 39 genes decreased to a similar extent . Genes involved in DNA repair, synthesis and replication represented the highest proportion of induced genes identified, which may account for the easily acquired resistance to 5-fluorocytosine . Two enzyme encoding genes CTS1 and EGT2, which function in the separation of daughter cells from their mother cells, were down-regulated by a factor of 3.7 and 10.2, respectively, indicating that 5-fluorocytosine may also inhibit the separation of fungal cells . Eukaryot Cell, 2002 Aug, 1(4), 606 - 12 Interaction with Btn2p is required for localization of Rsglp: Btn2p-mediated changes in arginine uptake in Saccharomyces cerevisiae; Chattopadhyay S et al.; Btn2p, a novel coiled-coil protein, is up-regulated in btn1delta yeast strains, and this up-regulation is thought to contribute to maintaining a stable vacuolar pH in btn1delta strains (D . A . Pearce, T . Ferea, S . A . Nosel, B . Das, and F . Sherman, Nat . Genet . 22:55-58, 1999) . We now report that Btn2p interacts biochemically and functionally with Rsglp, a down-regulator of the Can1p arginine and lysine permease . Rsglp localizes to a distinct structure toward the cell periphery, and strains lacking Btn2p (btn2delta strains) fail to correctly localize Rsg1p . btn2delta strains, like rsg1delta strains, are sensitive for growth in the presence of the arginine analog canavanine . Furthermore, btn2delta strains, like rsg1delta strains, demonstrate an elevated rate of uptake of {14C}arginine, which leads to increased intracellular levels of arginine . Overexpression of BTN2 results in a decreased rate of arginine uptake . Collectively, these results indicate that altered levels of Btn2p can modulate arginine uptake through localization of the Can1p-arginine permease regulatory protein, Rsglp . Our original identification of Btn2p was that it is up-regulated in the btn1delta strain which serves as a model for the lysosomal storage disorder Batten disease . Btnlp is a vacuolar/lysosomal membrane protein, and btn1delta suppresses both the canavanine sensitivity and the elevated rate of uptake of arginine displayed by btn2delta rsg1delta strains . We conclude that Btn2p interacts with Rsglp and modulates arginine uptake . Up-regulation of BTN2 expression in btn1delta strains may facilitate either a direct or indirect effect on intracellular arginine levels. Eukaryot Cell, 2002 Jun, 1(3), 481 - 90 Mga2p processing by hypoxia and unsaturated fatty acids in Saccharomyces cerevisiae: impact on LORE-dependent gene expression; Jiang Y et al.; In Saccharomyces cerevisiae, OLE1 encodes a delta9 fatty acid desaturase, an enzyme that plays a critical role in maintaining the correct ratio of saturated to monounsaturated fatty acids in the cell membrane . Previous studies have demonstrated that (i) OLE1 expression is repressed by unsaturated fatty acids (UFAs) and induced by low oxygen tension, (ii) a component of this regulation is mediated through the same low oxygen response element (LORE) in the OLE1 promoter, and (iii) Mga2p is involved in LORE-dependent hypoxic induction of OLE1 . We now report that LORE-CYC1 basal promoter-lacZ fusion reporter assays demonstrate that UFAs repress the reporter expression under hypoxic conditions in a dose-dependent manner via LORE . Electrophoretic mobility shift assays show that UFAs repress the hypoxia-induced complex formation with LORE . Studies with a construct encoding a truncated form of Mga2p support the hypothesis that both hypoxia and UFA signals affect the processing of Mga2p and the UFA repression of OLE1 hypoxic induction is mediated through Mga2p . Data from Western blot assays provide evidence that under normoxic conditions, Mga2p processing produces approximately equimolar levels of the membrane-bound and processed forms and is unaffected by UFAs . Hypoxic induction of OLE1, however, is associated with increased processing of the protein, resulting in an approximately fivefold increase in the soluble active form that is counteracted by exposure of the cells to unsaturated fatty acids . Data from this study suggest that the Mga2p-LORE interaction plays an important role in OLE1 expression under both normoxic and hypoxic conditions. Eukaryot Cell, 2002 Jun, 1(3), 458 - 68 Saccharomyces cerevisiae Cdc42p localizes to cellular membranes and clusters at sites of polarized growth; Richman TJ et al.; The Cdc42p GTPase controls polarized growth and cell cycle progression in eukaryotes from yeasts to mammals, and its precise subcellular localization is essential for its function . To examine the cell cycle-specific targeting of Cdc42p in living yeast cells, a green fluorescent protein (GFP)-Cdc42 fusion protein was used . In contrast to previous immunolocalization data, GFP-Cdc42p was found at the plasma membrane around the entire cell periphery and at internal vacuolar and nuclear membranes throughout the cell cycle, and it accumulated or clustered at polarized growth sites, including incipient bud sites and mother-bud neck regions . These studies also showed that C-terminal CAAX and polylysine domains were sufficient for membrane localization but not for clustering . Time-lapse fluorescence microscopy showed that GFP-Cdc42p clustered at the incipient bud site prior to bud emergence and at the mother-bud neck region postanaphase as a diffuse, single band and persisted as two distinct bands on mother and daughter cells following cytokinesis and cell separation . Initial clustering occurred immediately prior to actomyosin ring contraction and persisted postcontraction . These results suggest that Cdc42p targeting occurs through a novel mechanism of membrane localization followed by cell cycle-specific clustering at polarized growth sites. Eukaryot Cell, 2002 Jun, 1(3), 391 - 400 Localization, regulation, and substrate transport properties of Bpt1p, a Saccharomyces cerevisiae MRP-type ABC transporter; Sharma KG et al.; Saccharomyces cerevisiae Bpt1p is an ATP-binding cassette (ABC) protein that belongs to the MRP subfamily and is a close homologue of the glutathione conjugate (GS conjugate) transporter Ycf1p . The function of Bpt1p has previously been evaluated only in vitro, by using nonphysiological substrates . In the present study we examined the localization, regulation, and transport properties of Bpt1p in vivo, as well as its capacity to transport a set of prototypical MRP substrates in vitro . Our results show that Bpt1p, like Ycf1p, localizes to the yeast vacuolar membrane, plays a role in cadmium detoxification and ade2 pigmentation in vivo, and can participate in the transport of GS conjugates and glucuronate conjugates, as well as free glutathione, in vitro . However, in all of these cases the contribution of Bpt1p is substantially less than that of Ycf1p . In addition, the expression patterns of YCF1 and BPT1 differ significantly . Whereas YCF1 expression is markedly increased by cadmium, adenine limitation in an ade2 strain, or overexpression of the stress-responsive transcription factor Yap1p, BPT1 expression is only modestly affected under these conditions . Thus, although the functional capabilities of Bpt1p and Ycf1p overlap, their differences in regulation and substrate preference imply that they contribute to cellular detoxification processes in different ways. Eukaryot Cell, 2002 Feb, 1(1), 66 - 74 Saccharomyces cerevisiae C-type cyclin Ume3p/Srb11p is required for efficient induction and execution of meiotic development; Cooper KF et al.; The yeast C-type cyclin Ume3p/Srb11p and its cyclin-dependent kinase partner Ume5p/Srb10p repress the transcription of several genes required for meiotic recombination or meiosis I nuclear division . To relieve this repression, Srbllp is destroyed early in meiosis, prior to the first meiotic division . This report identifies two roles for Srb11p in regulating meiotic development . First, SRB11 is required for the normal exit from the mitotic cell cycle prior to meiotic induction . Specifically, mutants lacking SRB11 (srb11delta) uncouple bud growth from chromosome segregation, producing small buds with nuclei . The bud growth defect is most likely due to the failure of srb11delta mutants to reestablish polarized actin fibers at the bud tip following exposure to sporulation medium . Second, Srb11p is required for the efficient execution of meiosis I . srb11delta mutants either exhibited a delay in performing meiosis I and meiosis II or skipped meiosis I entirely . This meiotic defect is not due to the activation of the recombination or spindle assembly checkpoint pathways . However, the expression of several meiotic genes is delayed and reduced in the mutant strains . These results suggest a positive role for Srb10-Srb11p in regulating the transcription program . This model is supported by the finding that overexpression of the meiotic inducer IME2 partially restored the ability of srb11 mutants to perform meiosis I . In conclusion, these findings indicate that Srb11p is required for both entry into and execution of the meiotic program, thus describing multiple roles for a C-type cyclin in the regulation of a developmental pathway. Eukaryot Cell, 2002 Apr, 1(2), 229 - 40 Nnf1p, Dsn1p, Mtw1p, and Nsl1p: a new group of proteins important for chromosome segregation in Saccharomyces cerevisiae; Euskirchen GM; Previously, antibodies were raised against a nuclear envelope-enriched fraction of yeast, and the essential gene NNF1 was cloned by reverse genetics . Here it is shown that the conditional nnf1-17 mutant has decreased stability of a minichromosome in addition to mitotic spindle defects . I have identified the novel essential genes DSN1, DSN3, and NSL1 through genetic interactions with nnf1-17 . Dsn3p was found to be equivalent to the kinetochore protein Mtw1p . By indirect immunofluorescence, all four proteins, Nnf1p, Mtw1p, Dsn1p, and Nsl1p, colocalize and are found in the region of the spindle poles . Based on the colocalization of these four proteins, the minichromosome instability and the spindle defects seen in nnf1 mutants, I propose that Nnf1p is part of a new group of proteins necessary for chromosome segregation. Eukaryot Cell, 2002 Apr, 1(2), 200 - 12 Involvement of RAD9-dependent damage checkpoint control in arrest of cell cycle, induction of cell death, and chromosome instability caused by defects in origin recognition complex in Saccharomyces cerevisiae; Watanabe K et al.; Perturbation of origin firing in chromosome replication is a possible cause of spontaneous chromosome instability in multireplicon organisms . Here, we show that chromosomal abnormalities, including aneuploidy and chromosome rearrangement, were significantly increased in yeast diploid cells with defects in the origin recognition complex . The cell cycle of orc1-4/orc1-4 temperature-sensitive mutant was arrested at the G2/M boundary, after several rounds of cell division at the restrictive temperature . However, prolonged incubation of the mutant cells at 37 degrees C led to abrogation of G2 arrest, and simultaneously the cells started to lose viability . A sharp increase in chromosome instability followed the abrogation of G2 arrest . In orc1-4/orc1-4 rad9delta/rad9delta diploid cells grown at 37 degrees C, G2 arrest and induction of cell death were suppressed, while chromosome instability was synergistically augmented . These findings indicated that DNA lesions caused by a defect in Orc1p function trigger the RAD9-dependent checkpoint control, which ensures genomic integrity either by stopping the cell cycle progress until lesion repair, or by inducing cell death when the lesion is not properly repaired . At semirestrictive temperatures, orc2-1/orc2-1 diploid cells demonstrated G2 arrest and loss of cell viability, both of which require RAD9-dependent checkpoint control . However, chromosome instability was not induced in orc2-1/orc2-1 cells, even in the absence of the checkpoint control . These data suggest that once cells lose the damage checkpoint control, perturbation of origin firing can be tolerated by the cells . Furthermore, although a reduction in origin-firing capacity does not necessarily initiate chromosome instability, the Orc1p possesses a unique function, the loss of which induces instability in the chromosome. Eukaryot Cell, 2002 Apr, 1(2), 174 - 80 Altered phosphotransfer in an activated mutant of the Saccharomyces cerevisiae two-component osmosensor Sln1p; Ault AD et al.; The SLN1 two-component signaling pathway of Saccharomyces cerevisiae utilizes a multistep phosphorelay mechanism to control osmotic stress responses via the HOG1 mitogen-activated protein kinase pathway and the transcription factor Skn7p . Sln1p consists of a sensor kinase module that undergoes histidine autophosphorylation and a receiver module that autocatalytically transfers the phosphoryl group from histidine to aspartate . The Sln1p aspartyl phosphate is then transferred to Ypd1p, which in turn transfers the phosphoryl group to a conserved aspartate on one of two response regulators, Ssk1p and Skn7p . Activated alleles of SLN1 (sln1*) were previously identified that appear to increase the level of phosphorylation of downstream targets Ssk1p and Skn7p . In principle, the phenotype of sln1* alleles could arise from an increase in autophosphorylation or phosphotransfer activities or a decrease in an intrinsic or extrinsic dephosphorylation activity . Genetic analysis of the activated mutants has been unable to distinguish between these possibilities . In this report, we address this issue by analyzing phosphorelay and phosphohydrolysis reactions involving the Sln1p-associated receiver . The results are consistent with a model in which the activated phenotype of the sln1* allele, sln-22, arises from a shift in the phosphotransfer equilibrium from Sln1p to Ypd1p, rather than from impaired dephosphorylation of the system in response to osmotic stress. Eukaryot Cell, 2002 Oct, 1(5), 843 - 5 Role of Fig2p in agglutination in Saccharomyces cerevisiae; Jue CK et al.; In W303-derived strains, disruption of FIG2 increased agglutinability of alpha cells, but not a cells, and did not alter expression of alpha-agglutinin, binding of 125I-labeled alpha-agglutinin, or mating efficiency . Fig2p overexpression led to alpha-cell-specfic suppression of agglutinability . These results imply that Fig2p is an indirect masker of the active sites in alpha-agglutinin. Eukaryot Cell, 2002 Oct, 1(5), 774 - 86 Eng1p, an endo-1,3-beta-glucanase localized at the daughter side of the septum, is involved in cell separation in Saccharomyces cerevisiae; Baladron V et al.; ENG1 (YNR067c), a gene encoding a new endo-1,3-beta-glucanase, was cloned by screening a genomic library with a DNA probe obtained by PCR with synthetic oligonucleotides designed according to conserved regions found between yeast exo-1,3-beta-glucanases (Exglp, Exg2p, and Ssglp) . Eng1p shows strong sequence similarity to the product of the Saccharomyces cerevisiae ACF2 gene, involved in actin assembly "in vitro," and to proteins present in other yeast and fungal species . It is also related to plant glucan-binding elicitor proteins, which trigger the onset of a defense response upon fungal infection . Eng1p and Acf2p/Eng2p are glucan-hydrolyzing proteins that specifically act on 1,3-beta linkages, with an endolytic mode of action . Eng1p is an extracellular, heavily glycosylated protein, while Acf2p/Eng2p is an intracellular protein with no carbohydrate linked by N-glycosidic bonds . ENG1 transcription fluctuates periodically during the cell cycle; maximal accumulation occurs during the M/G1 transition and is dependent on the transcription factor Ace2p . Interestingly, eng1 deletion mutants show defects in cell separation, and Eng1p localizes asymmetrically to the daughter side of the septum, suggesting that this protein is involved, together with chitinase, in the dissolution of the mother-daughter septum. Eukaryot Cell, 2002 Oct, 1(5), 673 - 86 Dual role of the Saccharomyces cerevisiae TEA/ATTS family transcription factor Tec1p in regulation of gene expression and cellular development; Kohler T et al.; In Saccharomyces cerevisiae, the transcription factors Tec1p and Ste12p are required for haploid invasive and diploid pseudohyphal growth . Tec1p and Ste12p have been postulated to regulate these developmental processes primarily by cooperative binding to filamentous and invasion-responsive elements (FREs), which are combined enhancer elements that consist of a Tec1p-binding site (TCS) and an Stel2p-binding site (PRE) . They are present in the promoter regions of target genes, e.g., FLO11 . Here, we show that Tec1p efficiently activates target gene expression and cellular development in the absence of Stel2p . We further demonstrate that TCS elements alone are sufficient to mediate Tec1p-driven gene expression by a mechanism termed TCS control that is operative even when Stel2p is absent . Mutational analysis of TEC1 revealed that TCS control, FLO11 expression, and haploid invasive growth require the C terminus of Tec1p . In contrast, the Ste12p-dependent FRE control mechanism is sufficiently executed by the N-terminal portion of Tec1p, which contains the TEA/ATTS DNA-binding domain . Our study suggests that regulation of haploid invasive and diploid pseudohyphal growth by Stel2p and Tec1p is not only executed by combinatorial control but involves additional control mechanisms in which Stel2p activates TEC1 expression via clustered PREs and where Tec1p regulates expression of target genes, e.g., FLO11, by TCS control. Genetics, 2002 Nov, 162(3), 1147 - 56 Maintenance of mitochondrial morphology is linked to maintenance of the mitochondrial genome in Saccharomyces cerevisiae; Hanekamp T et al.; In the yeast Saccharomyces cerevisiae, certain mutant alleles of YME4, YME6, and MDM10 cause an increased rate of mitochondrial DNA migration to the nucleus, carbon-source-dependent alterations in mitochondrial morphology, and increased rates of mitochondrial DNA loss . While single mutants grow on media requiring mitochondrial respiration, any pairwise combination of these mutations causes a respiratory-deficient phenotype . This double-mutant phenotype allowed cloning of YME6, which is identical to MMM1 and encodes an outer mitochondrial membrane protein essential for maintaining normal mitochondrial morphology . Yeast strains bearing null mutations of MMM1 have altered mitochondrial morphology and a slow growth rate on all carbon sources and quantitatively lack mitochondrial DNA . Extragenic suppressors of MMM1 deletion mutants partially restore mitochondrial morphology to the wild-type state and have a corresponding increase in growth rate and mitochondrial DNA stability . A dominant suppressor also suppresses the phenotypes caused by a point mutation in MMM1, as well as by specific mutations in YME4 and MDM10. Genetics, 2002 Nov, 162(3), 1101 - 15 The Est1 subunit of Saccharomyces cerevisiae telomerase makes multiple contributions to telomere length maintenance; Evans SK et al.; The telomerase-associated Est1 protein of Saccharomyces cerevisiae mediates enzyme access by bridging the interaction between the catalytic core of telomerase and the telomere-binding protein Cdc13 . In addition to recruiting telomerase, Est1 may act as a positive regulator of telomerase once the enzyme has been brought to the telomere, as previously suggested by the inability of a Cdc13-Est2 fusion protein to promote extensive telomere elongation in an est1-Delta strain . We report here three classes of mutant Est1 proteins that retain association with the telomerase enzyme but confer different in vivo consequences . Class 1 mutants display a telomere replication defect but are capable of promoting extensive telomere elongation in the presence of a Cdc13-Est2 fusion protein, consistent with a defect in telomerase recruitment . Class 2 mutants fail to elongate telomeres even in the presence of the Cdc13-Est2 fusion, which is the phenotype predicted for a defect in the proposed second regulatory function of EST1 . A third class of mutants impairs an activity of Est1 that is potentially required for the Ku-mediated pathway of telomere length maintenance . The isolation of mutations that perturb separate functions of Est1 demonstrates that a telomerase holoenzyme subunit can contribute multiple regulatory roles to telomere length maintenance. Genetics, 2002 Nov, 162(3), 1063 - 77 The roles of REV3 and RAD57 in double-strand-break-repair-induced mutagenesis of Saccharomyces cerevisiae; Rattray AJ et al.; The DNA synthesis associated with recombinational repair of chromosomal double-strand breaks (DSBs) has a lower fidelity than normal replicative DNA synthesis . Here, we use an inverted-repeat substrate to monitor the fidelity of repair of a site-specific DSB . DSB induction made by the HO endonuclease stimulates recombination >5000-fold and is associated with a >1000-fold increase in mutagenesis of an adjacent gene . We demonstrate that most break-repair-induced mutations (BRIMs) are point mutations and have a higher proportion of frameshifts than do spontaneous mutations of the same substrate . Although the REV3 translesion DNA polymerase is not required for recombination, it introduces approximately 75% of the BRIMs and approximately 90% of the base substitution mutations . Recombinational repair of the DSB is strongly dependent upon genes of the RAD52 epistasis group; however, the residual recombinants present in rad57 mutants are associated with a 5- to 20-fold increase in BRIMs . The spectrum of mutations in rad57 mutants is similar to that seen in the wild-type strain and is similarly affected by REV3 . We also find that REV3 is required for the repair of MMS-induced lesions when recombinational repair is compromised . Our data suggest that Rad55p/Rad57p help limit the generation of substrates that require pol zeta during recombination. Mol Microbiol, 2002 Dec, 46(5), 1319 - 33 The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediated signalling; Serrano R et al.; The short-time transcriptional response of yeast cells to a mild increase in external pH (7.6) has been investigated using DNA microarrays . A total of 150 genes increased their mRNA level at least twofold within 45 min . Alkalinization resulted in the repression of 232 genes . The response of four upregulated genes, ENA1 (encoding a Na+-ATPase also induced by saline stress) and PHO84, PHO89 and PHO12 (encoding genes upregulated by phosphate starvation), was characterized further . The alkaline response of ENA1 was not affected by mutation of relevant genes involved in osmotic or oxidative signalling, but was decreased in calcineurin and rim101 mutants . Mapping of the ENA1 promoter revealed two pH-responsive regions . The response of the upstream region was fully abolished by the drug FK506 or mutation of CRZ1 (a transcription factor activated by calcium/calcineurin), whereas the response of the downstream region was essentially calcium independent . PHO84 and PHO12 responses were unaffected in crz1 cells, but required the presence of Pho2 and Pho4 . In contrast, part of the alkali-induced expression of PHO89 was maintained in pho4 or pho2 cells, but was fully abolished in a crz1 strain or in the presence of FK506 . Heterologous promoters carrying the minimal calcineurin-dependent response elements found in ENA1 or FKS2 were able to drive alkaline pH-induced expression . These results demonstrate that the transcriptional response to alkaline pH involves different signalling mechanisms, and that calcium signalling is a relevant component of this response. Biol Chem, 2002 Oct, 383(10), 1475 - 80 Lipid rafts in protein sorting and cell polarity in budding yeast Saccharomyces cerevisiae; Bagnat M et al.; Cellular membranes contain many types and species of lipids . One of the most important functional consequences of this heterogeneity is the existence of microdomains within the plane of the membrane . Sphingolipid acyl chains have the ability of forming tightly packed platforms together with sterols . These platforms or lipid rafts constitute segregation and sorting devices into which proteins specifically associate . In budding yeast, Saccharomyces cerevisiae, lipid rafts serve as sorting platforms for proteins destined to the cell surface . The segregation capacity of rafts also provides the basis for the polarization of proteins at the cell surface during mating . Here we discuss some recent findings that stress the role of lipid rafts as key players in yeast protein sorting and cell polarity. Genetika, 2002 Oct, 38(10), 1330 - 6 {Yeast comparative genetics . A new MEL15 alpha-galactosidase gene of Saccharomyces cerevisiae}; Naumov GI et al.; To supplement the earlier identified European family of the highly homologous alpha-galactosidase MEL1-MEL11 genes and the African family of the divergent MEL12-MEL14 genes, a new MEL gene (MEL15) was found in several Saccharomyces cerevisiae strains isolated from maize dough in Ghana . Southern blotting and restriction enzyme analysis assigned the MEL15 gene to the African family and mapped it to chromosomes IV/XII, which migrate together in electrophoresis . Tetrad analysis ruled out the MEL15 location in the left arm of chromosome IV or the right arm of chromosome XII, which respectively contain the known MEL5 and MEL10 genes. Genetika, 2002 Oct, 38(10), 1324 - 9 {Coregulation of alternative transcripts of the STA2 gene in Saccharomyces cerevisiae}; Kozlov DG et al.; It is known that upon STA2 gene expression in Saccharomyces cerevisiae cells, two transcripts of different lengths are formed . These fragments contain different start codons of translation (AUG1 and AUG2) located in the same reading frame . The ratio between expression levels of proteins translated from AUG1 and AUG2 was invariably about 2:7 and did not depend on the choice of the reporter product (secreted glucoamylase (GA) or beta-galactosidase accumulated in cells) . Neither this ratio depended on glucose repression/derepression . Based on the assumed proportional relationship between levels of transcription and translation of the STA2 gene in yeast cells, all these results cumulatively indicate that the two STA2 transcripts are coregulated . The production of secreted GA was also shown to be markedly stimulated at the posttranscriptional level under conditions of glucose repression. Mol Cell Biol, 2002 Dec, 22(24), 8763 - 73 Role of transcription in plasmid maintenance in the hpr1Delta mutant of Saccharomyces cerevisiae; Merker RJ et al.; The Saccharomyces cerevisiae hyperrecombination mutation hpr1Delta results in instability of sequences between direct repeats that is dependent on transcription of the repeat . Here it is shown that the HPR1 gene also functions in plasmid stability in the presence of destabilizing transcription elongation . In the hpr1Delta mutant, plasmid instability results from unchecked transcription elongation, which can be suppressed by a strong transcription terminator . The plasmid system has been used to examine in vivo aspects of transcription in the absence of Hpr1p . Nuclear run-on studies suggest that there is an increased RNA polymerase II density in the hpr1Delta mutant strain, but this is not accompanied by an increase in accumulation of cytoplasmic mRNA . Suppression of plasmid instability in hpr1Delta can also be achieved by high-copy expression of the RNA splicing factor SUB2, which has recently been proposed to function in mRNA export, in addition to its role in pre-mRNA splicing . High-copy-number SUB2 expression is accompanied by an increase in message accumulation from the plasmid, suggesting that the mechanism of suppression by Sub2p involves the formation of mature mRNA . Models for the role of Hpr1p in mature mRNA formation and the cause of plasmid instability in the absence of the Hpr1 protein are discussed. Mol Cell Biol, 2002 Dec, 22(24), 8756 - 62 Base composition of mononucleotide runs affects DNA polymerase slippage and removal of frameshift intermediates by mismatch repair in Saccharomyces cerevisiae; Gragg H et al.; The postreplicative mismatch repair (MMR) system is important for removing mutational intermediates that are generated during DNA replication, especially those that arise as a result of DNA polymerase slippage in simple repeats . Here, we use a forward mutation assay to systematically examine the accumulation of frameshift mutations within mononucleotide runs of variable composition in wild-type and MMR-defective yeast strains . These studies demonstrate that (i) DNA polymerase slippage occurs more often in 10-cytosine/10-guanine (10C/10G) runs than in 10-adenine/10-thymine (10A/10T) runs, (ii) the MMR system removes frameshift intermediates in 10A/10T runs more efficiently than in 10C/10G runs, (iii) the MMR system removes -1 frameshift intermediates more efficiently than +1 intermediates in all 10-nucleotide runs, and (iv) the repair specificities of the Msh2p-Msh3p and Msh2p-Msh6p mismatch recognition complexes with respect to 1-nucleotide insertion/deletion loops vary dramatically as a function of run composition . These observations are relevant to issues of genome stability, with both the rates and types of mutations that accumulate in mononucleotide runs being influenced by the primary sequence of the run as well as by the status of the MMR system. J Biol Chem, 2003 Feb 7, 278(6), 3770 - 5 Epub 2002 Nov 22. COX16 encodes a novel protein required for the assembly of cytochrome oxidase in Saccharomyces cerevisiae; Carlson CG et al.; We have characterized Cox16p, a new cytochrome oxidase (COX) assembly factor . This protein is encoded by COX16, corresponding to the previously uncharacterized open reading frame YJL003w of the yeast genome . COX16 was identified in studies of COX-deficient mutants previously assigned to complementation group G22 of a collection of yeast pet mutants . To determine its location, Cox16p was tagged with a Myc epitope at the C terminus . The fusion protein, when expressed from a low-copy plasmid, complements the mutant and is detected solely in mitochondria . Cox16p-myc is an integral component of the mitochondrial inner membrane, with its C terminus exposed to the intermembrane space . Cox16 homologues are found in both the human and murine genomes, although human COX16 does not complement the yeast mutant . Cox16p does not appear to be involved in maturation of subunit 2, copper recruitment, or heme A biosynthesis . Cox16p is thus a new protein in the growing family of eukaryotic COX assembly factors for which there are as yet no specific functions known . Like other recently described nuclear gene products involved in expression of cytochrome oxidase, COX16 is a candidate for screening in inherited human COX deficiencies. Free Radic Biol Med, 2002 Dec 1, 33(11), 1507 - 15 Hydrogen peroxide-induced carbonylation of key metabolic enzymes in Saccharomyces cerevisiae: the involvement of the oxidative stress response regulators Yap1 and Skn7; Costa VM et al.; H(2)O(2) induces a specific protein oxidation in yeast cells, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Tdh) is a major target . Using a 2D-gel system to study protein carbonylation, it is shown in this work that both Tdh2p and Tdh3p isozymes were oxidized during exposure to H(2)O(2) . In addition, we identified two other proteins carbonylated and inactivated: Cu,Zn-superoxide dismutase and phosphoglycerate mutase . The oxidative inactivation of Cu,Zn-superoxide dismutase decreases the antioxidant capacity of yeast cells and probably contributes to H(2)O(2)-induced cell death . Cyclophilin 1 was also carbonylated, but CPH1 gene disruption did not affect peroxide stress sensitivity . The correlation between H(2)O(2) sensitivity and the accumulation of oxidized proteins was evaluated by assaying protein carbonyls in mutants deficient in the stress response regulators Yap1p and Skn7p . The results show that the high sensitivity of yap1delta and skn7delta mutants to H(2)O(2) was correlated with an increased induction of protein carbonylation . In wild-type cells, the acquisition of stress resistance by pre-exposure to a sublethal H(2)O(2) stress was associated with a lower accumulation of oxidized proteins . However, pre-exposure of yap1delta and skn7delta cells to 0.4 mM H(2)O(2) decreased protein carbonylation induced by 1.5 mM H(2)O(2), indicating that the adaptive mechanism involved in the protection of proteins from carbonylation is Yap1p- and Skn7p-independent. Med Sci Monit, 2002 Nov, 8(11), BR460 - 70 Functional consequences of the in-frame insertion of a transposon into the mutated gamma amino butyric acid transporter of Saccharomyces cerevisiae; Naghibalhossaini F et al.; BACKGROUND: The Saccharomyces cerevisiae strain, 22574d, lacks gamma amino butyric acid (GABA) transport activity and cannot grow on this amino acid as sole nitrogen source . Both transport of and growth on this amino acid are restored when the yeast is transformed with a form of mouse gamma actin containing an extended C-terminal sequence (M-g-A) . The nature of the mutation in the transporter as well as the complementation mode are addressed . MATERIAL/METHODS: The cDNA sequence of the mutated transporter was achieved using reverse transcription, 3'-Race and cloning . For detection, Northern blotting and labeling with 32-P were used . The putative ability of the transporter to interact with gamma actin in vivo was examined by following the interaction in vitro of synthetic peptides corresponding to the C-termini of the gamma actin and the transporter . RESULTS: Up to codon 394 the mutated and native transporters are identical . At the 3' end, the mutant is by extended by 32 codons from the delta region of a Ty1 transposon, giving an open reading frame of 426 codons, and a predicted structure of 9 of the 11 transmembrane domains . Peptides corresponding to the carboxy terminal regions of the truncated transporter and the elongated species of gamma actin show the potential to interact in vitro . CONCLUSIONS: The mutated GABA transporter mRNA contains an insert from the delta region of a Ty1 transposon . This insertion allows expression of a transporter capable of interacting with elongated gamma actin to rehabilitate the transporter. Genes Genet Syst, 2002 Oct, 77(5), 309 - 22 Genetic characterization of genes encoding enzymes catalyzing addition of phospho-ethanolamine to the glycosylphosphatidylinositol anchor in Saccharomyces cerevisiae; Toh-e A et al.; MPC1/GPI13/YLL031C, one of the genes involved in the addition of phospho-ethanolamine to the glycosylphosphatidylinositol (GPI) anchor core, is an essential gene . Three available temperature-sensitive mutant alleles, mpc1-3, mpc1-4, and mpc1-5, displayed different phenotypes to each other and, correspondingly, these mutants were found to have different mutations in the MPC1 ORF . Temperature-sensitivity of mpc1-5 mutants was suppressed by 5 mM ZnSO(4) and by 5 mM MnCl(2) . Multicopy suppressors were isolated from mpc1-5 mutant . Suppressors commonly effective to mpc1-4 and mpc1-5 mutations are PSD1, encoding phosphatidylserine decarboxylase, and ECM33, which were found to suppress the temperature-sensitive phenotype shown by the fsr2-1 and las21delta mutants, those of which have defects in the GPI anchor synthesis . PSD2, encoding another phosphatidylserine decarboxylase that is localized in Golgi/vacuole, was found to be able to serve as a multicopy suppressor of mpc1 and fsr2-1 mutants but not of the las21 delta mutant . In contrast to psd1delta, psd2delta showed a synthetic growth defect with mpc1 mutants but not with fsr2-1 or las21delta . Furthermore, psd1delta psd2delta mpc1 triple mutants did not form colonies on nutrient medium unless ethanolamine was supplied to the medium, whereas psd1delta psd2 delta fsr2-1 or psd1delta psd2 delta las21delta triple mutants grew on nutrient medium without supplementation of ethanolamine . These observations suggest that Mpc1 preferentially utilizes phosphatidylethanolamine produced by Psd2 that is localized in Golgi/vacuole . fsr2-1 dpl1 Delta psd1delta strains showed slower growth than fsr2-1 dpl1delta psd2 delta, suggesting that Fsr2 enzyme depends more on Dpl1 and Psd1 for production of phosphatidylethanolamine . Las21 did not show preference for the metabolic pathway to produce phosphatidylethanolamine. Biochem Cell Biol, 2002, 80(5), 639 - 44 Comparison of the interaction of uridine, cytidine, and other pyrimidine nucleoside analogues with recombinant human equilibrative nucleoside transporter 2 (hENT2) produced in Saccharomyces cerevisiae; Vickers MF et al.; The human equilibrative nucleoside transporters I and 2 (hENT1, hENT2) share 50% amino acid identity and exhibit broad selectivities, accepting purine and pyrimidine nucleosides as permeants . The permeant selectivity of hENT2 is less well understood because of the low abundance of the native transporter in cells amenable to functional analysis . Recent studies of hENT2 produced in recombinant form in functional expression systems have shown that it differs from hENT1 in that it transports nucleobases . To further understand the structural requirements for permeant interaction with hENT2, we compared the relative abilities of uridine, cytidine, and their analogues to inhibit transport of {3H}uridine by recombinant hENT1 and hENT2 produced in yeast . hENT1 and hENT2 tolerated halogen modification at the 5 position of the base and the 2' and 5' positions of the ribose moieties of uridine whereas removal of the hydroxyl group at the 3' position of the ribose moiety of uridine eliminated interaction with both transporters . hENT2 displayed a lower ability, compared with hENT1, to interact with cytidine and cytidine analogues, suggesting a low tolerance for the presence of the amino group at the 4 position of the base. Mol Genet Genomics, 2002 Nov, 268(3), 371 - 86 Epub 2002 Oct 08. The product of Saccharomyces cerevisiae WHIP/MGS1, a gene related to replication factor C genes, interacts functionally with DNA polymerase delta; Branzei D et al.; The Saccharomyces cerevisiae gene WHIP/ MGS1 encodes a protein related to the subunits of Replication Factor C (RFC) . We found that the RFC-like motifs in Whip/Mgs1 are essential for its function . Furthermore, by screening for synthetic dosage lethality, we have shown that overexpression of MGS1 causes lethality in combination with mutations in genes that encode replication proteins such as DNA polymerase delta, RFC, PCNA and RPA . Moreover, loss of MGS1 function interferes with the ability of multicopy PCNA to suppress the replication defect of the rfc5-1 mutant . At permissive temperatures, deletion of MGS1 suppresses the hydroxyurea (HU) sensitivity of pol31 and pol32 mutants, which bear mutations in the smaller subunits of DNA polymerase delta, and at semipermissive and non-permissive temperatures mgs1delta partially alleviates the growth defects of the pol31 mutant . We also report that the growth defect and HU sensitivity of the pol31 mutant are suppressed by mms2delta and rad18delta mutations . We suggest that Mgs1 interacts with the DNA replication machinery to modulate the function of DNA polymerase delta during replication or replication-associated repair, and influences the choice of the pathway employed for replication fork reactivation . Possible roles of Mgs1, DNA polymerase delta, Rad18 and Mms2 in replication and replication fork restart are discussed. FEMS Microbiol Lett, 2002 Nov 5, 216(2), 165 - 70 SUT1 suppresses sec14-1 through upregulation of CSR1 in Saccharomyces cerevisiae; Regnacq M et al.; SUT1 constitutive expression in aerobiosis suppressed the ts phenotype of the sec14-1 mutation, restored growth of the sec14-null mutant and corrected the translocation defect of the vacuolar carboxypeptidase Y . Therefore SUT1 was shown to be a novel potent sec14-1 suppressor . Further, the hypoxic gene CSR1 (YLR380W), a Sec14 homolog, was upregulated upon SUT1 constitutive expression . In addition, SUT1 effects on both sec14-1 suppression and on free sterol composition were abolished in a csr1-null background, showing that this gene acts downstream of SUT1. Biochem J, 2003 Mar 1, 370(Pt 2), 641 - 9 In vitro fusion between Saccharomyces cerevisiae secretory vesicles and cytoplasmic-side-out plasma membrane vesicles; Arrastua L et al.; The final step in the secretory pathway, which is the fusion event between secretory vesicles and the plasma membrane, was reconstructed using highly purified secretory vesicles and cytoplasmic-side-out plasma membrane vesicles from the yeast Saccharomyces cerevisiae . Both organelle preparations were obtained from a sec 6-4 temperature-sensitive mutant . Fusion was monitored by means of a fluorescence assay based on the dequenching of the lipophilic fluorescent probe octadecylrhodamine B-chloride (R18) . The probe was incorporated into the membrane of secretory vesicles, and it diluted in unlabelled cytoplasmic-side-out plasma membrane vesicles as the fusion process took place . The obtained experimental dequenching curves were found by mathematical analysis to consist of two independent but simultaneous processes . Whereas one of them reflected the fusion process between both vesicle populations as confirmed by its dependence on the assay conditions, the other represented a non-specific transfer of the probe . The fusion process may now be examined in detail using the preparation, validation and analytical methods developed in this study. Nucleic Acids Res, 2002 Nov 15, 30(22), 4993 - 5003 Thermoconditional modulation of the pleiotropic sensitivity phenotype by the Saccharomyces cerevisiae PRP19 mutant allele pso4-1; Revers LF et al.; The conditionally-lethal pso4-1 mutant allele of the spliceosomal-associated PRP19 gene allowed us to study this gene's influence on pre-mRNA processing, DNA repair and sporulation . Phenotypes related to intron-containing genes were correlated to temperature . Splicing reporter systems and RT-PCR showed splicing efficiency in pso4-1 to be inversely correlated to growth temperature . A single amino acid substitution, replacing leucine with serine, was identified within the N-terminal region of the pso4-1 allele and was shown to affect the interacting properties of Pso4-1p . Amongst 24 interacting clones isolated in a two-hybrid screening, seven could be identified as parts of the RAD2, RLF2 and DBR1 genes . RAD2 encodes an endonuclease indispensable for nucleotide excision repair (NER), RLF2 encodes the major subunit of the chromatin assembly factor I, whose deletion results in sensitivity to UVC radiation, while DBR1 encodes the lariat RNA splicing debranching enzyme, which degrades intron lariat structures during splicing . Characterization of mutagen-sensitive phenotypes of rad2Delta, rlf2Delta and pso4-1 single and double mutant strains showed enhanced sensitivity for the rad2Delta pso4-1 and rlf2Delta pso4-1 double mutants, suggesting a functional interference of these proteins in DNA repair processes in Saccharomyces cerevisiae. J Cell Sci, 2002 Dec 15, 115(Pt 24), 4977 - 91 Spatial regulation of the guanine nucleotide exchange factor Lte1 in Saccharomyces cerevisiae; Jensen S et al.; In budding yeast, activation of the small Ras-like GTPase Tem1 triggers exit from mitosis and cytokinesis . Tem1 is regulated by Bub2/Bfa1, a two-component GTPase-activating protein (GAP), and by Lte1, a putative guanine nucleotide exchange factor . Lte1 is confined to the bud cortex, and its spatial separation from Tem1 at the spindle pole body (SPB) is important to prevent untimely exit from mitosis . The pathways contributing to Lte1 asymmetry have not been elucidated . Here we show that establishment of Lte1 at the cortex occurs by an actin-independent mechanism, which requires activation of Cdc28/Cln kinase at START and Cdc42, a key regulator of cell polarity and cytoskeletal organisation . This defines a novel role for Cdc42 in late mitotic events . In turn, dissociation of Lte1 from the cortex in telophase depends on activation of the Cdc14 phosphatase . Ectopic expression of Cdc14 at metaphase results in premature dephosphorylation of Lte1 coincident with its release from the cortex . In vitro phosphatase assays confirm that Lte1 is a direct substrate for Cdc14 . Our results suggest that the asymmetry in Lte1 localisation is imposed by Cdc28-dependent phosphorylation . Finally, we report a mutational analysis undertaken to investigate intrinsic Lte1 determinants for localisation . Our data suggest that an intrameric interaction between the N-and C-terminal regions of Lte1 is important for cortex association. FEMS Microbiol Lett, 2002 Oct 29, 216(1), 33 - 8 KEM1 is involved in filamentous growth of Saccharomyces cerevisiae; Kim J et al.; The KEM1/XRN1 gene was originally identified because of its functions in microtubule-mediated processes, and is also known to be a major cytoplasmic 5'-3' exoribonuclease gene, which is involved in RNA turnover . Here we present evidence that KEM1 plays a role in filamentous growth . In Saccharomyces cerevisiae, the filamentation signalling shares multiple components of the MAP kinase cascade (STE7, STE11, and KSS1) and the transcription factor STE12 with mating process . Both haploid invasive growth and diploid pseudohyphal growth were found to be greatly impaired in kem1 mutant strains . KEM1 affected the level of FLO11 transcripts and the expression of the filamentation-associated reporter genes, Ty1-lacZ and FLO11-lacZ . Suppression analysis implies that KEM1 does not affect the RAS/PKA pathway, but that it possibly functions downstream of the MAP kinase pathway during filamentation. Eur J Biochem, 2002 Nov, 269(22), 5738 - 45 Characterization of a Saccharomyces cerevisiae NADP(H)-dependent alcohol dehydrogenase (ADHVII), a member of the cinnamyl alcohol dehydrogenase family; Larroy C et al.; A new NADP(H)-dependent alcohol dehydrogenase (the YCR105W gene product, ADHVII) has been identified in Saccharomyces cerevisiae . The enzyme has been purified to homogeneity and found to be a homodimer of 40 kDa subunits and a pI of 6.2-6.4 . ADHVII shows a broad substrate specificity similar to the recently characterized ADHVI (64% identity), although they show some differences in kinetic properties . ADHVI and ADHVII are the only members of the cinnamyl alcohol dehydrogenase family in yeast . Simultaneous deletion of ADH6 and ADH7 was not lethal for the yeast . Both enzymes could participate in the synthesis of fusel alcohols, ligninolysis and NADP(H) homeostasis. Biochem J, 2003 Feb 15, 370(Pt 1), 141 - 7 The TATA-binding protein is not an essential target of the transcriptional activators Gal4p and Gcn4p in Saccharomyces cerevisiae; Bongards C et al.; According to the recruitment model, transcriptional activators work by increasing the local concentration of one or several limiting factors for the transcription process at the target promoter . The TATA-binding protein Tbp1 has been considered as a likely candidate for such a limiting factor . We have used a series of Gal4p and Tbp1 mutants to correlate the in vivo interaction between the two proteins with the strength of activation . We find a clear correlation between activation strength and in vivo interaction for the series of Gal4p mutants . Consistently, the weaker activator Gcn4p does not interact with Tbp1 . However, a corresponding analysis of the series of Tbp1 mutants revealed that Tbp1 is not an essential target of the acidic activators Gal4p and Gcn4p . Furthermore, detailed analysis of a Tbp1 mutant deficient for transcriptional activation by Gal4p revealed that the mutant is defective in interactions with five other proteins involved in the process of transcription. Acta Biochim Pol, 2002, 49(3), 781 - 7 Induction of the synthesis of an additional family of long-chain dolichols in the yeast Saccharomyces cerevisiae . Effect of starvation and ageing; Szkopinska A et al.; The yeast Saccharomyces cerevisiae strain W303 synthesizes in the early logarithmic phase of growth dolichols of 14-18 isoprene residues . The analysis of the polyisoprenoids present in the stationary phase revealed an additional family which proved to be also dolichols but of 19-24 isoprene residues, constituting 39% of the total dolichols . The transfer of early logarithmic phase cells to a starvation medium lacking glucose or nitrogen resulted in the synthesis of the longer chain dolichols . The additional family of dolichols represented 13.8% and 10.3% of total dolichols in the glucose and nitrogen deficient media, respectively . The level of dolichols in yeast cells increased with the age of the cultures . Since both families of dolichols are present in stationary phase cells we postulate that the longer chain dolichols may be responsible for the physico-chemical changes in cellular membranes allowing yeast cells to adapt to nutrient deficient conditions to maintain long-term viability. Mol Microbiol, 2002 Nov, 46(4), 1011 - 22 Two homologous genes, DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae; Kitagaki H et al.; The cell wall of Saccharomyces cerevisiae consists of glucan, chitin and various kinds of mannoproteins . Major parts of mannoproteins are synthesized as glycosylphosphatidylinositol (GPI)-anchored proteins and are then transferred to cell wall beta-1,6-glucan . A glycosyltransferase has been hypothesized to catalyse this transfer reaction . A database search revealed that the products of YKL046c and DFG5 are homologous to bacterial mannosidase . These genes are homologous to each other and have primary structures characteristic of GPI-anchored proteins . Although single disruptants of ykl046c and dfg5 were viable, ykl046cDelta was hypersensitive to a cell wall-digesting enzyme (zymolyase), suggesting that this gene is involved in cell wall biosynthesis . We therefore designated this gene as DCW1 (defective cell wall) . A double disruptant of dcw1 and dfg5 was synthetically lethal, indicating that the functions of these gene products are redundant, and at least one of them is required for cell growth . Cells deficient in both Dcw1p and Dfg5p were round and large, had cell walls that contained an increased amount of chitin and secreted a major cell wall protein, Cwp1p, into the medium . Biochemical analyses showed that epitope-tagged Dcw1p is an N-glycosylated, GPI-anchored membrane protein and is localized in the membrane fraction including the cell surface . These results suggest that both Dcw1p and Dfg5p are GPI-anchored membrane proteins and are required for normal biosynthesis of the cell wall. Mol Microbiol, 2002 Nov, 46(4), 947 - 57 Saccharomyces cerevisiae C1D is implicated in both non-homologous DNA end joining and homologous recombination; Erdemir T et al.; C1D is a gamma-irradiation inducible nuclear matrix protein that interacts with and activates the DNA-dependent protein kinase (DNA-PK) that is essential for the repair of the DNA double-strand breaks and V(D)J recombination . Recently, it was demonstrated that C1D can also interact with TRAX and prevent the association of TRAX with Translin, a factor known to bind DNA break-point junctions, and that over expression of C1D can induce p53-dependent apoptosis . Taken together, these findings suggest that mammalian C1D could be involved in maintenance of genome integrity by regulating the activity of proteins involved in DNA repair and recombination . To obtain direct evidence for the biological function of C1D that we show is highly conserved between diverse species, we have analysed the Saccharomyces cerevisiae C1D homologue . We report that the disruption of the YC1D gene results in a temperature sensitivity and that yc1d mutant strains exhibit defects in non-homologous DNA end joining (NHEJ) and accurate DNA repair . In addition, using a novel plasmid-based in vivo recombination assay, we show that yc1d mutant strains are also defective in homologous recombination . These results indicate that YC1D is implicated in both homologous recombination and NHEJ pathways for the repair of DNA double-strand breaks. Mol Cell Biol, 2002 Dec, 22(23), 8165 - 74 HTL1 encodes a novel factor that interacts with the RSC chromatin remodeling complex in Saccharomyces cerevisiae; Romeo MJ et al.; RSC is an essential chromatin remodeling complex in Saccharomyces cerevisiae that performs central roles in transcriptional regulation and cell cycle progression . Here we identify Htl1 as a novel factor that associates with the RSC complex both physically and functionally . We isolated HTL1 through a genetic screen for mutants that displayed additive growth defects with a conditional mutation in the protein kinase C gene (PKC1), which has been suggested through genetic connections to interact functionally with RSC . Several lines of evidence connect HTL1 to RSC function . First, an htl1Delta mutant displayed temperature-sensitive growth and a G(2)/M cell cycle arrest at restrictive temperatures, a phenotype similar to that of strains with conditional mutations in essential RSC components . Second, we isolated RSC3, which encodes a component of the RSC complex, as a dosage suppressor of the htl1Delta growth arrest . Third, an htl1Delta mutant displayed additive growth defects with conditional rsc3 alleles . Fourth, overexpression of HTL1 suppressed the growth defect of a strain with a conditional mutation in another RSC component, RSC8 . Finally, we demonstrate that Htl1 is a nuclear protein that can associate in vivo with a fraction of the RSC complex . We propose that an RSC-Htl1 complex acts coordinately with protein kinase C to regulate the G(2)/M transition. Mol Cell Biol, 2002 Dec, 22(23), 8122 - 34 Mot1 associates with transcriptionally active promoters and inhibits association of NC2 in Saccharomyces cerevisiae; Geisberg JV et al.; Mot1 stably associates with the TATA-binding protein (TBP), and it can dissociate TBP from DNA in an ATP-dependent manner . Mot1 acts as a negative regulator of TBP function in vitro, but genome-wide transcriptional profiling suggests that Mot1 positively affects about 10% of yeast genes and negatively affects about 5% . Unexpectedly, Mot1 associates with active RNA polymerase (Pol) II and III promoters, and it is rapidly recruited in response to activator proteins . At Pol II promoters, Mot1 association requires TBP and is strongly correlated with the level of TBP occupancy . However, the Mot1/TBP occupancy ratio at both Mot1-stimulated and Mot1-inhibited promoters is high relative to that at typical promoters, strongly suggesting that Mot1 directly affects transcriptional activity in a positive or negative manner, depending on the gene . The effect of Mot1 at the HIS3 promoter region depends on the functional quality and DNA sequence of the TATA element . Unlike TBP, Mot1 association is largely independent of the Srb4 component of Pol II holoenzyme, and it also can occur downstream of the promoter region . Mot1 removes TBP, but not TBP complexes or preinitiation complexes, from inappropriate genomic locations . Mot1 inhibits the association of NC2 with promoters, suggesting that the TBP-Mot1 and TBP-NC2 complexes compete for promoter occupancy in vivo . We speculate that Mot1 does not form transcriptionally active TBP complexes but rather regulates transcription in vivo by modulating the activity of free TBP and/or by affecting promoter DNA structure. FEBS Lett, 2002 Nov 6, 531(2), 215 - 21 Abundance of the RSC nucleosome-remodeling complex is important for the cells to tolerate DNA damage in Saccharomyces cerevisiae; Koyama H et al.; The essential Nps1p/Sth1p is a catalytic subunit of the nucleosome-remodeling complex, RSC, of Saccharomyces cerevisiae that can alter nucleosome structure by using the energy of ATP hydrolysis . Besides the ATPase domain, Nps1p harbors the bromodomain, of which the function(s) have not yet been defined . We have isolated a temperature-sensitive mutant allele of NPS1, nps1-13, which has amino acid substitutions within the bromodomain . This mutation perturbed the interaction between the RSC components and enhanced the sensitivity of the cells to several DNA-damaging treatments at the permissive temperature . Reduced expression of NPS1 also caused DNA damage sensitivity . These results suggest the importance of the Nps1p bromodomain in RSC integrity and a model in which high amounts of RSC would be required for the cells to overcome DNA damage. J Biol Chem, 2003 Jan 31, 278(5), 3265 - 74 Epub 2002 Oct 31. The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur; Boer VM et al.; Profiles of genome-wide transcriptional events for a given environmental condition can be of importance in the diagnosis of poorly defined environments . To identify clusters of genes constituting such diagnostic profiles, we characterized the specific transcriptional responses of Saccharomyces cerevisiae to growth limitation by carbon, nitrogen, phosphorus, or sulfur . Microarray experiments were performed using cells growing in steady-state conditions in chemostat cultures at the same dilution rate . This enabled us to study the effects of one particular limitation while other growth parameters (pH, temperature, dissolved oxygen tension) remained constant . Furthermore, the composition of the media fed to the cultures was altered so that the concentrations of excess nutrients were comparable between experimental conditions . In total, 1881 transcripts (31% of the annotated genome) were significantly changed between at least two growth conditions . Of those, 484 were significantly higher or lower in one limitation only . The functional annotations of these genes indicated cellular metabolism was altered to meet the growth requirements for nutrient-limited growth . Furthermore, we identified responses for several active transcription factors with a role in nutrient assimilation . Finally, 51 genes were identified that showed 10-fold higher or lower expression in a single condition only . The transcription of these genes can be used as indicators for the characterization of nutrient-limited growth conditions and provide information for metabolic engineering strategies. EMBO J, 2002 Nov 1, 21(21), 5921 - 9 Rpb4 and Rpb9 mediate subpathways of transcription-coupled DNA repair in Saccharomyces cerevisiae; Li S et al.; Rpb9, a non-essential subunit of RNA polymerase II, mediates a transcription-coupled repair (TCR) subpathway in Saccharomyces cerevisiae . This subpathway initiates at the same upstream site as the previously identified Rad26 subpathway . However, the Rpb9 subpathway operates more effectively in the coding region than in the region upstream of the transcription start site, whereas the Rad26 subpathway operates equally in the two regions . Rpb4, another non-essential subunit of RNA polymerase II, plays a dual role in regulating the two subpathways, suppressing the Rpb9 subpathway and facilitating the Rad26 subpathway . Simultaneous deletion of RPB9 and RAD26 genes completely abolishes TCR in both the coding and upstream regions, indicating that no other TCR subpathway exists in RNA polymerase II-transcribed genes. Mol Microbiol, 2002 Nov, 46(3), 869 - 78 Thioredoxins are required for protection against a reductive stress in the yeast Saccharomyces cerevisiae; Trotter EW et al.; Thioredoxins are small, highly conserved oxidoreductases that are required to maintain the redox homeostasis of the cell . They have been best characterized for their role as antioxidants in protection against reactive oxygen species . We show here that thioredoxins (TRX1, TRX2) and thioredoxin reductase (TRR1) are also required for protection against a reductive stress induced by exposure to dithiothreitol (DTT) . This sensitivity to reducing conditions is not a general property of mutants affected in redox control, as mutants lacking components of the glutathione/glutaredoxin system are unaffected . Furthermore, TRX2 gene expression is induced in response to DTT treatment, indicating that thioredoxins form part of the cellular response to a reductive challenge . Our data indicate that the sensitivity of thioredoxin mutants to reducing stress appears to be a consequence of elevated glutathione levels, which is present predominantly in the reduced form (GSH) . The elevated GSH levels also result in a constitutively high unfolded protein response (UPR), indicative of an accumulation of unfolded proteins in the endoplasmic reticulum (ER) . However, there does not appear to be a general defect in ER function in thioredoxin mutants, as oxidative protein folding of the model protein carboxypeptidase Y occurs with similar kinetics to the wild-type strain, and trx1 trx2 mutants are unaffected in sensitivity to the glycosylation inhibitor tunicamycin . Furthermore, trr1 mutants are resistant to tunicamycin, consistent with their high UPR . The high UPR seen in trr1 mutants can be abrogated by the GSH-specific reagent 1-chloro-2,4-dinitrobenzene . In summary, thioredoxins are required to maintain redox homeostasis in response to both oxidative and reductive stress conditions. J Biol Chem, 2003 Jan 3, 278(1), 679 - 85 Epub 2002 Oct 29. 20 S proteasome from Saccharomyces cerevisiae is responsive to redox modifications and is S-glutathionylated; Demasi M et al.; The 20 S proteasome core purified from Saccharomyces cerevisiae is inhibited by reduced glutathione (GSH), cysteine (Cys), or the GSH precursor gamma-glutamylcysteine . Chymotrypsin-like activity was more affected by GSH than trypsin-like activity, whereas the peptidylglutamyl-hydrolyzing activity (caspase-like) was not inhibited by GSH . Cys-sulfenic acid formation in the 20 S core was demonstrated by spectral characterization of the Cys-S(O)-4-nitrobenzo-2-oxa-1,3-diazole adduct, indicating that 20 S proteasome Cys residues might react with reduced sulfhydryls (GSH, Cys, and gamma-glutamylcysteine) through the oxidized Cys-sulfenic acid form . S-Glutahionylation of the 20 S core was demonstrated in vitro by GSH-biotin incorporation and by decreased alkylation with monobromobimane . Compounds such as N-ethylmaleimide (-S-sulfhydril H alkylating), dimedone (-SO sulfenic acid H reactant), or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (either -SH or -SOH reactant) highly inhibited proteasomal chymotrypsin-like activity . In vivo experiments revealed that 20 S proteasome extracted from H(2)O(2)-treated cells showed decreased chymotrypsin-like activity accompanied by S-glutathionylation as demonstrated by GSH release from the 20 S core after reduction with NaBH(4) . Moreover, cells pretreated with H(2)O(2) showed decreased reductive capacity assessed by determination of the GSH/oxidized glutathione ratio and increased protein carbonyl levels . The present results indicate that at the physiological level the yeast 20 S proteasome is regulated by its sulfhydryl content, thereby coupling intracellular redox signaling to proteasome-mediated proteolysis. Appl Environ Microbiol, 2002 Nov, 68(11), 5693 - 7 Engineering of polyploid Saccharomyces cerevisiae for secretion of large amounts of fungal glucoamylase; Ekino K et al.; We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var . kawachi . To do this, we used the delta-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method . delta-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable . In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other . The cells were then allowed to form spores, which was followed by dissection . Haploid cells containing GAI genes on multiple chromosomes were obtained in this way . One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome . Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes . The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted. Mol Microbiol, 2002 Oct, 46(2), 531 - 44 Intracellular translation initiation factor levels in Saccharomyces cerevisiae and their role in cap-complex function; von der Haar T et al.; Knowledge of the balance of activities of eukaryotic initiation factors (eIFs) is critical to our understanding of the mechanisms underlying translational control . We have therefore estimated the intracellular levels of 11 eIFs in logarithmically growing cells of Saccharomyces cerevisiae using polyclonal antibodies raised in rabbits against recombinant proteins . Those factors involved in 43S complex formation occur at levels comparable (i.e . within a 0.5- to 2.0-fold range) to those published for ribosomes . In contrast, the subunits of the cap-binding complex eIF4F showed considerable variation in their abundance . The helicase eIF4A was the most abundant eIF of the yeast cell, followed by eIF4E at multiple copies per ribosome, and eIF4B at approximately one copy per ribosome . The adaptor protein eIF4G was the least abundant of the eIF4 factors, with a copy number per cell that is substoichiometric to the ribosome and similar to the abundance of mRNA . The observed excess of eIF4E over its functional partner eIF4G is not strictly required during exponential growth: at eIF4E levels artificially reduced to 30% of those in wild-type yeast, growth rates and the capacity for general protein synthesis are only minimally affected . This demonstrates that eIF4E does not exercise a higher level of rate control over translation than other eIFs . However, other features of the yeast life cycle, such as the control of cell size, are more sensitive to changes in eIF4E abundance . Overall, these data constitute an important basis for developing a quantitative model of the workings of the eukaryotic translation apparatus. RNA, 2002 Oct, 8(10), 1280 - 93 Mutational analysis identifies two separable roles of the Saccharomyces cerevisiae splicing factor Prp18; Bacikova D et al.; Prp18 functions in the second step of pre-mRNA splicing, joining the spliceosome just prior to the transesterification reaction that creates the mature mRNA . Prp18 interacts with Slu7, and the functions of the two proteins are intertwined . Using the X-ray structure of Prp18, we have designed mutants in Prp18 that imply that Prp18 has two distinct roles in splicing . Deletion mutations were used to delineate the surface of Prp18 that interacts with Slu7, and point mutations in Prp18 were used to define amino acids that contact Slu7 . Experiments in which Slu7 and mutant Prp18 proteins were expressed at different levels support a model in which interaction between the proteins is needed for stable binding of both proteins to the spliceosome . Mutations in an evolutionarily conserved region show that it is critical for Prp18 function but is not involved in binding Slu7 . Alleles with mutations in the conserved region are dominant negative, suggesting that the resulting mutant prp18 proteins make proper contacts with the spliceosome, but fail to carry out a Prp18-specific function . Prp18 thus appears to have two separable roles in splicing, one in stabilizing interaction of Slu7 with the spliceosome, and a second that requires the conserved loop. Yeast, 2002 Nov, 19(15), 1323 - 33 Proteins interacting with Lin 1p, a putative link between chromosome segregation, mRNA splicing and DNA replication in Saccharomyces cerevisiae; Bialkowska A et al.; Proteins involved in chromosome segregation during mitosis are likely to participate in other cell cycle-coordinated processes . Using a two-hybrid screen we identified a novel nuclear protein, Lin1, interacting with Irr1p/Scc3p, a component of the cohesin complex . The second round of two-hybrid assay with Lin1p as the bait resulted in the identification of six proteins: Prp8, Slx5, Siz2, Wss1, Rfc1 and YIL149w . These proteins have previously been shown to participate in mRNA splicing, DNA replication, chromosome condensation, chromatid separation and alternative cohesion . We propose that Lin1p may constitute a link among these processes . Yeast, 2002 Nov, 19(15), 1299 - 321 Non-respiratory oxygen consumption pathways in anaerobically-grown Saccharomyces cerevisiae: evidence and partial characterization; Rosenfeld E et al.; Despite the absence of an alternative mitochondrial ubiquinol oxidase, Saccharomyces cerevisiae consumes oxygen in an antimycin A- and cyanide-resistant manner . Cyanide-resistant respiration is typically used when the classical respiratory chain is impaired or absent (i.e in anaerobically-grown cells shifted to normoxia or in respiratory-deficient cells) . We characterized the non-respiratory oxygen consumption pathways operating during anoxic-normoxic transitions in glucose-repressed resting cells . High-resolution oxygraphy confirmed that the cellular non-respiratory oxygen consumption pathway is sensitive to high concentrations of cyanide, azide, SHAM and TTFA, and revealed several new characteristics . First, the use of sterol biosynthesis inhibitors showed that this pathway makes a considerable contribution (about 25%) to both endogenous and glucose-dependent oxygen consumption . Anaerobically-grown glucose-repressed cells exhibited high apparent oxygen affinities (K(m) for oxygen = 0.5-1 micro M), even in mutants deficient in respiration or sterol synthesis . Exogeneously added glucose and endogenous stored carbohydrates were the only substrates that were efficient for cellular oxygen consumption (apparent K(m) for exogenous glucose = 2-3 mM) . On the other hand, fluorimetric measurements of the cellular NAD(P)H pool showed that the cellular oxygen consumption (sterol biosynthesis and unknown pathways) was dependent more on the intracellular level of NADPH than of NADH . High oxygen affinity NADPH-dependent oxygen consumption systems were thought to be mainly localized in microsomal membranes, and several data indicated a significant contribution made by uncoupled p450 systems, together with still uncharacterized systems . Such activities are associated in vitro with a massive production of O(2) (.-) and, to a lower extent, H(2)O(2) and a likely concomitant production of H(2)O . J Biol Chem, 2003 Jan 17, 278(3), 1415 - 23 Epub 2002 Oct 24. Dihydroxyacetone kinases in Saccharomyces cerevisiae are involved in detoxification of dihydroxyacetone; Molin M et al.; The genes YML070W/DAK1 and YFL053W/DAK2 in the yeast Saccharomyces cerevisiae were characterized by a combined genetic and biochemical approach that firmly functionally classified their encoded proteins as dihydroxyacetone kinases (DAKs), an enzyme present in most organisms . The kinetic properties of the two isoforms were similar, exhibiting K(m)((DHA)) of 22 and 5 microm and K(m)((ATP)) of 0.5 and 0.1 mm for Dak1p and Dak2p, respectively . We furthermore show that their substrate, dihydroxyacetone (DHA), is toxic to yeast cells and that the detoxification is dependent on functional DAK . The importance of DAK was clearly apparent for cells where both isogenes were deleted (dak1 Delta dak2 Delta), since this strain was highly sensitive to DHA . In the opposite case, overexpression of either DAK1 or DAK2 made the dak1 Delta dak2 Delta highly resistant to DHA . In fact, overexpression of either DAK provided cells with the capacity to grow efficiently on DHA as the only carbon and energy source, with a generation time of about 5 h . The DHA toxicity was shown to be strongly dependent on the carbon and energy source utilized, since glucose efficiently suppresses the lethality, whereas galactose or ethanol did so to a much lesser extent . However, this suppression was found not to be explained by differences in DHA uptake, since uptake kinetics revealed a simple diffusion mechanism with similar capacity independent of carbon source . Salt addition strongly aggravated the DHA toxicity, independent of carbon source . Furthermore, the DHA toxicity was not linked to the presence of oxygen or to the known harmful agents methylglyoxal and formaldehyde . It is proposed that detoxification of DHA may be a vital part of the physiological response during diverse stress conditions in many species. Chem Biol, 2002 Oct, 9(10), 1073 - 84 Targeted nucleotide exchange in Saccharomyces cerevisiae directed by short oligonucleotides containing locked nucleic acids; Parekh-Olmedo H et al.; Locked nucleic acids (LNAs) are novel base modifications containing a methylene bridge uniting the 2'-oxygen and the 4'-carbon . In this study, LNA-modified single-stranded molecules directed the repair of single base mutations in a yeast chromosomal gene . Using a genetic assay involving a mutant hygromycin-resistance gene, correction of point and frameshift mutations was facilitated by vectors containing an LNA residue on each terminus . Increasing the number of LNA bases on each terminus reduced the correction frequency progressively . When the LNA vector is used in combination with a phosphorothioate-modified vector (74-mer), however, a high level of gene-repair activity occurs; hence, short LNA-based vectors can augment the activity of other types of targeting vectors . These data suggest that oligonucleotides containing locked nucleic acid residues can be used to direct single nucleotide exchange reactions in vivo. J Microbiol Methods, 2003 Jan, 52(1), 141 - 5 Generation of disruption cassettes in vivo using a PCR product and Saccharomyces cerevisiae; Zaragoza O; A method to obtain disruption cassettes based on the homologous recombination in Saccharomyces cerevisiae is described . The disruption marker is amplified by PCR using oligonucleotides containing 50 bp homologous to the disruptable gene and 20 bp from the marker . The PCR product is cotransformed into yeast with a plasmid containing the gene . After recombination, a plasmid that carries the disruption cassette for the gene is produced. Science, 2002 Oct 25, 298(5594), 799 - 804 Transcriptional regulatory networks in Saccharomyces cerevisiae; Lee TI et al.; We have determined how most of the transcriptional regulators encoded in the eukaryote Saccharomyces cerevisiae associate with genes across the genome in living cells . Just as maps of metabolic networks describe the potential pathways that may be used by a cell to accomplish metabolic processes, this network of regulator-gene interactions describes potential pathways yeast cells can use to regulate global gene expression programs . We use this information to identify network motifs, the simplest units of network architecture, and demonstrate that an automated process can use motifs to assemble a transcriptional regulatory network structure . Our results reveal that eukaryotic cellular functions are highly connected through networks of transcriptional regulators that regulate other transcriptional regulators. Genetics, 2002 Oct, 162(2), 721 - 36 A unique class of conditional sir2 mutants displays distinct silencing defects in Saccharomyces cerevisiae; Garcia SN et al.; Silencing provides a critical means of repressing transcription through the assembly and modification of chromatin proteins . The NAD(+)-dependent deacetylation of histones by the Sir2p family of proteins lends mechanistic insight into how SIR2 contributes to silencing . Here we describe three locus-specific sir2 mutants that have a spectrum of silencing phenotypes in yeast . These mutants are dependent on SIR1 for silencing function at the HM silent mating-type loci, display distinct phenotypes at the rDNA, and have dominant silencing defects at the telomeres . Telomeric silencing is restored if the mutant proteins are directly tethered to subtelomeric regions, via a Gal4p DNA-binding domain (GBD), or are recruited by tethered GBD-Sir1p . These sir2 mutations are found within conserved residues of the SIR2 family and lead to defects in catalytic activity . Since one of the mutations lies outside the previously defined minimal catalytic core, our results show that additional regions of Sir2p can be important for enzymatic activity and that differences in levels of activity may have distinct effects at the silenced loci. Genetics, 2002 Oct, 162(2), 705 - 20 Mutational analysis reveals a role for the C terminus of the proteasome subunit Rpt4p in spindle pole body duplication in Saccharomyces cerevisiae; McDonald HB et al.; The ubiquitin/proteasome pathway plays a key role in regulating cell cycle progression . Previously, we reported that a conditional mutation in the Saccharomyces cerevisiae gene RPT4/PCS1, which encodes one of six ATPases in the proteasome 19S cap complex/regulatory particle (RP), causes failure of spindle pole body (SPB) duplication . To improve our understanding of Rpt4p, we created 58 new mutations, 53 of which convert clustered, charged residues to alanine . Virtually all mutations that affect the N-terminal region, which contains a putative nuclear localization signal and