S. cerevisiae

DNA Repair (Amst), 2004 Feb 3, 3(2), 127 - 34
Nucleotide excision repair deficiency causes elevated levels of chromosome gain in Saccharomyces cerevisiae; Howlett NG et al.; Aneuploidy is the most frequent aberration observed in tumor cells, and underlies many debilitating and cancer-prone congenital disorders . Aneuploidy most often arises as a consequence of chromosomal non-disjunction, however, little is known about the genetic and epigenetic factors that affect the chromosomal segregation process . As many cancer-prone syndromes are associated with defects in DNA repair pathways we decided to investigate the relationship between DNA repair in mutation avoidance pathways, namely base and nucleotide excision, and mismatch repair (MMR), and aneuploidy in the yeast Saccharomyces cerevisiae . Isogenic haploid and diploid DNA repair deficient yeast strains were constructed, and spontaneous levels of intra- and inter-chromosomal recombination, forward mutation, chromosome gain, and loss were measured . We show that the nucleotide excision repair (NER) pathway is required for accurate chromosomal disjunction . In the absence of Rad1, Rad2, or Rad4, spontaneous levels of chromosome XV gain were significantly elevated in both haploid and diploid mutant strains . Thus, chromosome gain may be an additional cancer predisposing event in NER deficient patients.

Proc Natl Acad Sci U S A, 2004 Jan 13, 101(2), 434 - 9 Epub 2004 Jan 02.
Identification of Lhp1p-associated RNAs by microarray analysis in Saccharomyces cerevisiae reveals association with coding and noncoding RNAs; Inada M et al.; La is a conserved eukaryotic RNA-binding protein best known for its role in the biogenesis of noncoding RNAs transcribed by RNA polymerase III . To broaden our understanding of the function of the La homologous protein (Lhp1) in Saccharomyces cerevisiae, we have taken a genomics approach . Lhp1 ribonucleoprotein complexes were immunoprecipitated and bound RNAs were examined by hybridization to whole-genome microarrays that include >6,000 ORFs, documented noncoding RNAs, and the intervening intergenic regions . Demonstrating the validity of this approach, associations with previously known Lhp1p-associated RNAs were detected and associations with additional noncoding RNAs, including multiple tRNAs and small nucleolar RNAs, were revealed . Indicating that this approach provides a robust method for discovering RNAs, the data also identify associations between Lhp1p and several intergenic regions, three of which encode the recently annotated putative snoRNAs: RUF1, RUF2, and RUF3 . Unexpectedly, we find that Lhp1p is also associated with a subset of coding mRNAs . These mRNAs include many ribosomal protein transcripts as well as the mRNA encoding Hac1p, a transcription factor required during the unfolded protein stress response . In cells lacking LHP1, Hac1p levels are decreased 2- to 3-fold, whereas no changes are detected in the levels of spliced or unspliced HAC1 mRNA or in the stability of Hac1p . Finally, although LHP1 is dispensable for growth under standard conditions, we find that it is required when the unfolded protein response is induced at elevated temperatures . These results suggest that Lhp1p may play a novel role in the translation of one or more cellular mRNAs.

Proc Natl Acad Sci U S A, 2004 Jan 13, 101(2), 682 - 6 Epub 2004 Jan 02.
Functional expression in frog oocytes of human rho 1 receptors produced in Saccharomyces cerevisiae; Martinez-Martinez A et al.; The yeast Saccharomyces cerevisiae was engineered to express the rho 1 subunit of the human gamma-aminobutyric acid rho 1 (GABA rho 1) receptor . RNA that was isolated from several transformed yeast strains produced fully functional GABA receptors in Xenopus oocytes . The GABA currents elicited in the oocytes were fast, nondesensitizing chloride currents; and the order of agonist potency was GABA > beta-alanine > glycine . Moreover, the receptors were resistant to bicuculline, strongly antagonized by (1,2,5,6 tetrahydropyridine-4-yl)methylphosphinic acid, and modulated by zinc and lanthanum . Thus, the GABA receptors expressed by the yeast mRNA retained all of the principal characteristics of receptors expressed by cRNA or native retina mRNAs . Western blot assays showed immunoreactivity in yeast plasma membrane preparations, and a rho 1-GFP fusion gene showed mostly intracellular distribution with a faint fluorescence toward the plasma membrane . In situ immunodetection of rho 1 in yeast demonstrated that some receptors reach the plasma membrane . Furthermore, microtransplantation of yeast plasma membranes to frog oocytes resulted in the incorporation of a small number of functional yeast rho 1 receptors into the oocyte plasma membrane . These results show that yeast may be useful to produce complete functional ionotropic receptors suitable for structural analysis.

Genetics, 2003 Dec, 165(4), 1733 - 44
Role of mismatch repair in the fidelity of RAD51- and RAD59-dependent recombination in Saccharomyces cerevisiae; Spell RM et al.; To prevent genome instability, recombination between sequences that contain mismatches (homeologous recombination) is suppressed by the mismatch repair (MMR) pathway . To understand the interactions necessary for this regulation, the genetic requirements for the inhibition of homeologous recombination were examined using mutants in the RAD52 epistasis group of Saccharomyces cerevisiae . The use of a chromosomal inverted-repeat recombination assay to measure spontaneous recombination between 91 and 100% identical sequences demonstrated differences in the fidelity of recombination in pathways defined by their dependence on RAD51 and RAD59 . In addition, the regulation of homeologous recombination in rad51 and rad59 mutants displayed distinct patterns of inhibition by different members of the MMR pathway . Whereas the requirements for the MutS homolog, MSH2, and the MutL homolog, MLH1, in the suppression of homeologous recombination were similar in rad51 strains, the loss of MSH2 caused a greater loss in homeologous recombination suppression than did the loss of MLH1 in a rad59 strain . The nonequivalence of the regulatory patterns in the wild-type and mutant strains suggests an overlap between the roles of the RAD51 and RAD59 gene products in potential cooperative recombination mechanisms used in wild-type cells.

Genetics, 2003 Dec, 165(4), 1717 - 32
Functions of Saccharomyces cerevisiae 14-3-3 proteins in response to DNA damage and to DNA replication stress; Lottersberger F et al.; Two members of the 14-3-3 protein family, involved in key biological processes in different eukaryotes, are encoded by the functionally redundant Saccharomyces cerevisiae BMH1 and BMH2 genes . We produced and characterized 12 independent bmh1 mutant alleles, whose presence in the cell as the sole 14-3-3 source causes hypersensitivity to genotoxic agents, indicating that Bmh proteins are required for proper response to DNA damage . In particular, the bmh1-103 and bmh1-266 mutant alleles cause defects in G1/S and G2/M DNA damage checkpoints, whereas only the G2/M checkpoint is altered by the bmh1-169 and bmh1-221 alleles . Impaired checkpoint responses correlate with the inability to maintain phosphorylated forms of Rad53 and/or Chk1, suggesting that Bmh proteins might regulate phosphorylation/dephosphorylation of these checkpoint kinases . Moreover, several bmh1 bmh2Delta mutants are defective in resuming DNA replication after transient deoxynucleotide depletion, and all display synthetic effects when also carrying mutations affecting the polalpha-primase and RPA DNA replication complexes, suggesting a role for Bmh proteins in DNA replication stress response . Finally, the bmh1-169 bmh2Delta and bmh1-170 bmh2Delta mutants show increased rates of spontaneous gross chromosomal rearrangements, indicating that Bmh proteins are required to suppress genome instability.

Genetics, 2003 Dec, 165(4), 1703 - 15
The N-terminal DNA-binding domain of Rad52 promotes RAD51-independent recombination in Saccharomyces cerevisiae; Tsukamoto M et al.; In Saccharomyces cerevisiae, the Rad52 protein plays a role in both RAD51-dependent and RAD51-independent recombination pathways . We characterized a rad52 mutant, rad52-329, which lacks the C-terminal Rad51-interacting domain, and studied its role in RAD51-independent recombination . The rad52-329 mutant is completely defective in mating-type switching, but partially proficient in recombination between inverted repeats . We also analyzed the effect of the rad52-329 mutant on telomere recombination . Yeast cells lacking telomerase maintain telomere length by recombination . The rad52-329 mutant is deficient in RAD51-dependent telomere recombination, but is proficient in RAD51-independent telomere recombination . In addition, we examined the roles of other recombination genes in the telomere recombination . The RAD51-independent recombination in the rad52-329 mutant is promoted by a paralogue of Rad52, Rad59 . All components of the Rad50-Mre11-Xrs2 complex are also important, but not essential, for RAD51-independent telomere recombination . Interestingly, RAD51 inhibits the RAD51-independent, RAD52-dependent telomere recombination . These findings indicate that Rad52 itself, and more precisely its N-terminal DNA-binding domain, promote an essential reaction in recombination in the absence of RAD51.

Genetics, 2003 Dec, 165(4), 1687 - 702
The ESS1 prolyl isomerase and its suppressor BYE1 interact with RNA pol II to inhibit transcription elongation in Saccharomyces cerevisiae; Wu X et al.; Transcription by RNA polymerase II (pol II) requires the ordered binding of distinct protein complexes to catalyze initiation, elongation, termination, and coupled mRNA processing events . One or more proteins from each complex are known to bind pol II via the carboxy-terminal domain (CTD) of the largest subunit, Rpb1 . How binding is coordinated is not known, but it might involve conformational changes in the CTD induced by the Ess1 peptidyl-prolyl cis/trans isomerase . Here, we examined the role of ESS1 in transcription by studying one of its multicopy suppressors, BYE1 . We found that Bye1 is a negative regulator of transcription elongation . This led to the finding that Ess1 also inhibits elongation; Ess1 opposes elongation factors Dst1 and Spt4/5, and overexpression of ESS1 makes cells more sensitive to the elongation inhibitor 6-AU . In reporter gene assays, ess1 mutations reduce the ability of elongation-arrest sites to stall polymerase . We also show that Ess1 acts positively in transcription termination, independent of its role in elongation . We propose that Ess1-induced conformational changes attenuate pol II elongation and help coordinate the ordered assembly of protein complexes on the CTD . In this way, Ess1 might regulate the transition between multiple steps of transcription.

Genetics, 2003 Dec, 165(4), 1661 - 74
The Sla2p talin domain plays a role in endocytosis in Saccharomyces cerevisiae; Baggett JJ et al.; Clathrin-binding adaptors play critical roles for endocytosis in multicellular organisms, but their roles in budding yeast have remained unclear . To address this question, we created a quadruple mutant yeast strain lacking the genes encoding the candidate clathrin adaptors Yap1801p, Yap1802p, and Ent2p and containing a truncated version of Ent1p, Ent1DeltaCBMp, missing its clathrin-binding motif . This strain was viable and competent for endocytosis, suggesting the existence of other redundant adaptor-like factors . To identify these factors, we mutagenized the quadruple clathrin adaptor mutant strain and selected cells that were viable in the presence of full-length Ent1p, but inviable with only Ent1DeltaCBMp; these strains were named Rcb (requires clathrin binding) . One mutant strain, rcb432, contained a mutation in SLA2 that resulted in lower levels of a truncated protein lacking the F-actin binding talin homology domain . Analyses of this sla2 mutant showed that the talin homology domain is required for endocytosis at elevated temperature, that SLA2 exhibits genetic interactions with both ENT1 and ENT2, and that the clathrin adaptors and Sla2p together regulate the actin cytoskeleton and revealed conditions under which Yap1801p and Yap1802p contribute to viability . Together, our data support the view that Sla2p is an adaptor that links actin to clathrin and endocytosis.

Int J Radiat Biol, 2003 Sep, 79(9), 747 - 55
Effect of expression of the Escherichia coli nth gene in Saccharomyces cerevisiae on the toxicity of ionizing radiation and hydrogen peroxide; Skorvaga M et al.; PURPOSE: To examine the contribution of endonuclease III (Nth)-repairable lesions to the cytotoxicity of ionizing radiation (IR) and hydrogen peroxide (H2O2) in the yeast Saccharomyces cerevisiae . MATERIALS AND METHODS: A selectable expression vector containing the E . coli nth gene was transformed into two different wild-type strains (7799-4B and YNN-27) as well as one rad52 mutant strain (C5-6) . Nth expression was verified by Western analysis . Colony-forming assay was used to determine the sensitivity to IR and H2O2 in both stationary and exponentially growing cells . RESULTS: The pADHnth-transformed wild-type (77994B) strain was considerably more resistant than vector-only transformants to the toxic effects of IR, in both stationary and exponential growth phases, although this was not the case in another wild-type strain (YNN-27) . In contrast, there were no significant effects of nth expression on the sensitivity of the wild-type cells to H2O2 . Moreover, nth expression caused no effects on the H2O2 sensitivity in the rad52 mutant cells, but it led to a slight increase in sensitivity in these cells following IR, particularly at the highest dose levels used . CONCLUSIONS: Whilst other damage-processing systems may play a role, DNA lesions that are substrates for Nth can also make a contribution to the toxic effects of IR in certain wild-type yeast . Hence, DNA double-strand breaks should not be considered the sole lethal lesions following IR exposure.

J Biol Chem, 2004 Mar 19, 279(12), 10982 - 90 Epub 2003 Dec 29.
Characterization of Vta1p, a class E Vps protein in Saccharomyces cerevisiae; Shiflett SL et al.; We identified VTA1 in a screen for mutations that result in altered vacuole morphology . Deletion of VTA1 resulted in delayed trafficking of the lipophilic dye FM4-64 to the vacuole and altered vacuolar morphology when cells were exposed to the dye 5-(and 6)-carboxy-2',7'-dichlorofluorescein diacetate (CDCFDA) . Deletion of class E vacuolar protein sorting (VPS) genes, which encode proteins that affect multivesicular body formation, also showed altered vacuolar morphology upon exposure to high concentrations of CDCFDA . These results suggest a VPS defect for Deltavta1 cells . Deletion of VTA1 did not affect growth on raffinose and only mildly affected carboxypeptidase S sorting . Turnover of the surface protein Ste3p, the a-factor receptor, was affected in Deltavta1 cells with the protein accumulating on the vacuolar membrane . Likewise the alpha-factor receptor Ste2p accumulated on the vacuolar membrane in Deltavta1 cells . We demonstrated that many class E VPS deletion strains are hyper-resistant to the cell wall disruption agent calcofluor white . Deletion of VTA1 or VPS60, another putative class E gene, resulted in calcofluor white hypersensitivity . A Vta1p-green fluorescent protein fusion protein transiently associated with a Pep12p-positive compartment . This localization was altered by deletion of many of the class E VPS genes, indicating that Vta1p binds to endosomes in a manner dependent on the assembly of the endosomal sorting complexes required for transport . Membrane-associated Vta1p co-purified with Vps60p, suggesting that Vta1p is a class E Vps protein that interacts with Vps60p on a prevacuolar compartment.

Mol Cell Biol, 2004 Jan, 24(2), 757 - 64
Eaf3 regulates the global pattern of histone acetylation in Saccharomyces cerevisiae; Reid JL et al.; Saccharomyces cerevisiae has a global pattern of histone acetylation in which histone H3 and H4 acetylation levels are lower at protein-coding sequences than at promoter regions . The loss of Eaf3, a subunit of the NuA4 histone acetylase and Rpd3 histone deacetylase complexes, greatly alters the genomic profile of histone acetylation, with the effects on H4 appearing to be more pronounced than those on H3 . Specifically, the loss of Eaf3 causes increases in H3 and H4 acetylation at coding sequences and decreases at promoters, such that histone acetylation levels become evenly distributed across the genome . Eaf3 does not affect the overall level of H4 acetylation, the recruitment of the NuA4 catalytic subunit Esa1 to target promoters, or the level of transcription of the genes analyzed for histone acetylation . Whole-genome transcriptional profiling indicates that Eaf3 plays a positive, but quantitatively modest, role in the transcription of a small subset of genes, whereas it has a negative effect on very few genes . We suggest that Eaf3 regulates the genomic profile of histone H3 and H4 acetylation in a manner that does not involve targeted recruitment and is independent of transcriptional activity.

Mol Biol Cell, 2004 Mar, 15(3), 957 - 62 Epub 2003 Dec 29.
X-ray tomography generates 3-D reconstructions of the yeast, saccharomyces cerevisiae, at 60-nm resolution; Larabell CA et al.; We examined the yeast, Saccharomyces cerevisiae, using X-ray tomography and demonstrate unique views of the internal structural organization of these cells at 60-nm resolution . Cryo X-ray tomography is a new imaging technique that generates three-dimensional (3-D) information of whole cells . In the energy range of X-rays used to examine cells, organic material absorbs approximately an order of magnitude more strongly than water . This produces a quantifiable natural contrast in fully hydrated cells and eliminates the need for chemical fixatives or contrast enhancement reagents to visualize cellular structures . Because proteins can be localized in the X-ray microscope using immunogold labeling protocols (Meyer-Ilse et al., 2001 . J . Microsc . 201, 395-403), tomography enables 3-D molecular localization . The time required to collect the data for each cell shown here was <15 min and has recently been reduced to 3 min, making it possible to examine numerous yeast and to collect statistically significant high-resolution data . In this video essay, we show examples of 3-D tomographic reconstructions of whole yeast and demonstrate the power of this technology to obtain quantifiable information from whole, hydrated cells.

DNA Repair (Amst), 2004 Jan 5, 3(1), 51 - 9
Involvement of two endonuclease III homologs in the base excision repair pathway for the processing of DNA alkylation damage in Saccharomyces cerevisiae; Hanna M et al.; DNA base excision repair (BER) is initiated by DNA glycosylases that recognize and remove damaged bases . The phosphate backbone adjacent to the resulting apurinic/apyrimidinic (AP) site is then cleaved by an AP endonuclease or glycosylase-associated AP lyase to invoke subsequent BER steps . We have used a genetic approach in Saccharomyces cerevisiae to determine whether or not AP sites are blocks to DNA replication and the biological consequences if AP sites persist in the genome . We previously reported that yeast cells deficient in the two AP endonucleases (apn1 apn2 double mutant) are extremely sensitive to killing by a model DNA alkylating agent methyl methanesulfonate (MMS) and that this sensitivity can be reduced by deleting the MAG1 3-methyladenine DNA glycosylase gene . Here we report that in the absence of the AP endonucleases, deletion of two Escherichia coli endonuclease III homologs, NTG1 and NTG2, partially suppresses MMS-induced killing, which indicates that the AP lyase products are deleterious unless they are further processed by an AP endonuclease . The severe MMS sensitivity seen in AP endonuclease deficient strains can also be rescued by treatment of cells with the AP lyase inhibitor methoxyamine, which suggests that the product of AP lyase action on an AP site is indeed an extremely toxic lesion . In addition to the AP endonuclease interactions, deletion of NTG1 and NTG2 enhances the mag1 mutant sensitivity to MMS, whereas overexpression of MAG1 in either the ntg1 or ntg2 mutant severely affects cell growth . These results help to delineate alkylation base lesion flow within the BER pathway.

DNA Repair (Amst), 2004 Jan 5, 3(1), 1 - 12
Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae; Boiteux S et al.; Apurinic/apyrimidinic (AP) sites are one of the most frequent spontaneous lesions in DNA . They are potentially mutagenic and lethal lesions that can block DNA replication and transcription . In addition, cleavage of AP sites by AP endonucleases or AP lyases generates DNA single-strand breaks (SSBs) with 5'- or 3'-blocked ends, respectively . Therefore, we suggest that AP sites and 3'- or 5'-blocked SSBs, we name "honorary AP sites", constitute a single class of lesions . In this review, we describe the different mechanisms used by the budding yeast Saccharomyces cerevisiae to remove or tolerate AP sites and related SSBs . In wild-type cells, AP sites are primarily repaired by the base excision repair (BER) pathway, with the nucleotide excision repair (NER) pathway as a back up activity . BER is initiated by one of the two AP endonucleases, Apn1 or Apn2 . Three DNA N-glycosylases/AP lyases, Ntg1, Ntg2 and Ogg1, can also incise AP sites in DNA . Rad27, a structure specific endonuclease, is involved in the repair of 5'-blocked ends, whereas Apn1, Apn2 and Rad1-Rad10 are involved in the removal of 3'-blocked ends using their 3'-phosphodiesterase and 3'-flap endonuclease activities, respectively . AP sites can stall DNA replication forks, as well as they block in vitro DNA synthesis by DNA polymerase delta . Restart of stalled forks can occur through a recombination-associated pathway initiated by the Mus81-Mms4 endonuclease or mutagenic translesion DNA synthesis (TLS) . The mutagenic bypass of AP sites is a two-polymerases affair with an inserter DNA polymerase (Poldelta, Poleta or Rev1) and an extender DNA polymerase (Polzeta) . Under normal growth conditions, inactivation of Apn1, Apn2 and Rad1-Rad10 causes cell death . Therefore, the burden of spontaneous AP sites is not compatible with life, in the absence of excision repair pathways . These results in yeast demonstrate that AP sites are critical endogenous DNA damages that cause genetic instability and by analogy could be associated with degenerative pathologies in human.

Biophys J, 2004 Jan, 86(1 Pt 1), 371 - 83
Comparison of the effect of water release on the interaction of the Saccharomyces cerevisiae TATA binding protein (TBP) with "TATA Box" sequences composed of adenosine or inosine; Khrapunov S et al.; The formation of sequence-specific complexes of TATA binding protein (TBP) with the minor groove of DNA results in the burial of large nonpolar surfaces and the exclusion of water from these interfaces . The release of water is thus expected to provide a significant entropic driving force for formation of the transcription-preinitiated complexes mediated by the binding of TBP to specific sequences . In this article are described equilibrium-binding studies of Saccharomyces cerevisiae TBP to 14 bp oligonucleotides bearing either the tightly bound and efficiently transcribed adenovirus major late promoter (TATAAAAG) or its inosine-substituted derivative (TITIIIIG) as a function of neutral osmolyte concentration . These two DNA sequences present the same pattern of minor groove hydrogen-bond donors and acceptors to the protein . TBP-DNA complex formation was monitored by steady-state fluorescence resonance energy transfer measurements of the oligonucleotides end-labeled with fluorescein (donor) and TAMRA (acceptor) . Correct interpretation of the results obtained with the inosine-substituted sequence required careful consideration of the optical properties of the dyes as a function of osmolyte concentration to demonstrate that the relative change in the end-to-end distances for TATAAAAG- and TITIIIIG-bearing oligonucleotides is the same upon TBP binding . Although the affinity of TBP is slightly greater for the adenosine compared with the inosine-substituted TATA sequence in the absence of osmolyte, the end-to-end distances of the bound DNA in complex with TBP, the enthalpic and electrostatic components of binding, are identical within experimental precision . However, approximately 18 additional molecules of water are released upon TBP binding the TATAAAAG as compared with the TITIIIIG sequence resulting in an entropic advantage to the binding of the natural promoter sequence . These results are considered with regard to differences in the flexibility and hydration of the two DNA sequences.

J Cell Biol, 2003 Dec 22, 163(6), 1255 - 66
Myo4p and She3p are required for cortical ER inheritance in Saccharomyces cerevisiae; Estrada P et al.; Myo4p is a nonessential type V myosin required for the bud tip localization of ASH1 and IST2 mRNA . These mRNAs associate with Myo4p via the She2p and She3p proteins . She3p is an adaptor protein that links Myo4p to its cargo . She2p binds to ASH1 and IST2 mRNA, while She3p binds to both She2p and Myo4p . Here we show that Myo4p and She3p, but not She2p, are required for the inheritance of cortical ER in the budding yeast Saccharomyces cerevisiae . Consistent with this observation, we find that cortical ER inheritance is independent of mRNA transport . Cortical ER is a dynamic network that forms cytoplasmic tubular connections to the nuclear envelope . ER tubules failed to grow when actin polymerization was blocked with the drug latrunculin A (Lat-A) . Additionally, a reduction in the number of cytoplasmic ER tubules was observed in Lat-A-treated and myo4Delta cells . Our results suggest that Myo4p and She3p facilitate the growth and orientation of ER tubules.

Mol Cell, 2003 Dec, 12(6), 1525 - 36
The Saccharomyces cerevisiae helicase Rrm3p facilitates replication past nonhistone protein-DNA complexes; Ivessa AS et al.; The Saccharomyces cerevisiae RRM3 gene encodes a 5' to 3' DNA helicase . While replication of most of the yeast genome was not dependent upon Rrm3p, in its absence, replication forks paused and often broke at an estimated 1400 discrete sites, including tRNA genes, centromeres, inactive replication origins, and transcriptional silencers . These replication defects were associated with activation of the intra-S phase checkpoint . Activation of the checkpoint was critical for viability of rrm3Delta cells, especially at low temperatures . Each site whose replication was affected by Rrm3p is assembled into a nonnucleosomal protein-DNA complex . At tRNA genes and the silent mating type loci, disruption of these complexes eliminated dependence upon Rrm3p . These data indicate that the Rrm3p DNA helicase helps replication forks traverse protein-DNA complexes, naturally occurring impediments that are encountered in each S phase.

Curr Genet, 2004 Mar, 45(3), 121 - 8 Epub 2003 Dec 19.
Transcriptional activators Cat8 and Sip4 discriminate between sequence variants of the carbon source-responsive promoter element in the yeast Saccharomyces cerevisiae; Roth S et al.; The structural genes for gluconeogenesis in the yeast Saccharomyces cerevisiae are activated by the carbon source-responsive element (CSRE) found in the respective upstream regions . Regulatory genes CAT8 and SIP4 both encode zinc-cluster proteins which can bind to CSRE motifs and activate target genes under conditions of glucose deprivation . In this work, we describe a functional analysis of sequence variants containing single mutations within the strongly activating CSRE(ICL1) motif . While the sequence CCNNNNNNCCG was required as the minimal UAS for gene activation by both Cat8 and Sip4, the activators responded differently to sequence variations in the central part of the CSRE . Our results allowed us to derive a consensus sequence for efficient gene activation by Cat8 (YCCNYTNRKCCG), while a more specific motif is required for activation by Sip4 (TCCATTSRTCCGR) . Although their zinc cluster domains are clearly related, Cat8 and Sip4 are not isofunctional . This conclusion is further supported by the finding that biosynthetic derepression of Cat8 in the presence of a nonfermentable carbon source precedes that of Sip4 by about 90 min.

Cell Mol Biol (Noisy-le-grand), 2003 Nov, 49(7), 1049 - 56
Pleiotrophic cellular deficiencies conferred by the blm5-1 mutation of Saccharomyces cerevisiae; Martinez M et al.; Mutational alteration of the BLM5 gene of the model eukaryote, Saccharomyces cerevisiae, confers extreme hypersensitivities to lethal effects of ionizing radiation, anticancer bleomycins and structurally-related phleomycins . Additional properties conferred by the blm5-1 mutation in haploid and diploid strains were investigated for the current report . Only one copy of blm5-1 together with the normal BLM5 allele was sufficient to produce mitotic and meiotic defects in diploids, and greatly increase killing by bleomycin beyond wild type levels . Mitotic growth rates of blm5-1/blm5-1 homozygous mutant strains were slower than wild type or BLM5/blm5-1 heterozygous strains at 30 degrees C, and growth was nearly completely inhibited at 37 degrees C . Meiosis was inhibited at 30 degrees C and 37 degrees C in mutant homozygotes, and at 37 degrees C in BLM5/blm5-1 heterozygotes, while meiosis occurred at equivalent frequencies in wild type strains at both temperatures . Surprisingly, mutant strains were found to associate extremely low quantities of {S-methyl-3H}bleomycin A2, in contrast to normal strains that associated quite high amounts . However, the fractions of the total associated radioactivities that were released from normal and blm5-1 cells were equivalent . These results suggested that the extremely high killing suffered by blm5-1 mutant strains in response to bleomycin treatments results from something other than increased intracellular drug concentrations.

Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D319 - 22
SCMD: Saccharomyces cerevisiae Morphological Database; Saito TL et al.; To study the global regulation of cell morphology, a number of groups have recently reported genome-wide screening data for yeast mutants with abnormal morphology . Despite the relatively simple ellipsoidal shape of yeast cells, in the past, cell morphology researchers have processed information on cells manually . These time-consuming, entirely subjective tasks motivated us to develop image-processing software that automatically extracts yeast cells from micrographs and processes them to measure key morphological characteristics such as cell size, roundness, bud neck position angle, nuclear DNA localization and actin localization . To date, we have retrieved 960,609 cells from 52,988 micrographs of 2531 mutants using our software, and we have published the results in the Saccharomyces cerevisiae Morphological Database (SCMD), which facilitates the analysis of abnormal cells . Our system provides quantitative data for shapes of the daughter and mother cells, localization of the nuclear DNA and morphology of the actin patches . To search for mutants with similar morphological traits, the system outputs a list of mutants ranked by similarity of average morphological parameters . The SCMD is available at gi.k.u-tokyo.ac.jp/.

Protein Expr Purif, 2004 Jan, 33(1), 134 - 44
Purification and characterization of the DNA binding domain of Saccharomyces cerevisiae meiosis-specific transcription factor Ndt80; Sopko R et al.; Ndt80 is a Saccharomyces cerevisiae meiosis-specific transcription factor responsible for promoting the stage-specific expression of a family of genes referred to as middle sporulation genes . Many members of this gene family are essential for the completion of meiotic chromosome segregation . Thus, Ndt80 is essential for the completion of meiosis . Ndt80 is highly regulated both transcriptionally and post-translationally . To facilitate biochemical analysis of Ndt80, we have expressed the DNA binding domain in Escherichia coli and purified the recombinant protein with an affinity chromatography procedure . In addition we have dissected the amino-terminus of Ndt80 to delimit the functional DNA binding domain . This analysis shows that the amino-terminal 40 amino-acids of Ndt80, although not essential for its DNA binding activity, do have an effect on its ability to bind specifically to its target DNA sequence . In addition, we show that the Ndt80 DNA binding domain can be phosphorylated by the meiosis-specific protein kinase Ime2 in vitro, but contrary to our initial hypothesis this phosphorylation does not significantly affect the affinity of Ndt80 for its target DNA sequence.

FEMS Microbiol Lett, 2003 Dec 12, 229(2), 165 - 71
Glucose metabolism and cell size in continuous cultures of Saccharomyces cerevisiae; Porro D et al.; A detailed analysis of the cell size, monitored as protein content, has been performed in glucose-limited continuous cultures, so as to obtain the values of the average protein content for various subpopulations at different cell cycle stages, as a function of the growth rate . Glucose metabolism appears to affect cell size, since there is an increase of the average protein content of the population when cells produce ethanol above the critical dilution rate . If the production of ethanol is forced at low growth rates by the addition of formate, the average protein content increases . These results indicate a link between glucose metabolism and cell size in budding yeast, as observed for mammalian cells.

Biochem J, 2004 Apr 15, 379(Pt 2), 367 - 74
Expression of YAP4 in Saccharomyces cerevisiae under osmotic stress; Nevitt T et al.; YAP4, a member of the yeast activator protein ( YAP ) gene family, is induced in response to osmotic shock in the yeast Saccharomyces cerevisiae . The null mutant displays mild and moderate growth sensitivity at 0.4 M and 0.8 M NaCl respectively, a fact that led us to analyse YAP4 mRNA levels in the hog1 (high osmolarity glycerol) mutant . The data obtained show a complete abolition of YAP4 gene expression in this mutant, placing YAP4 under the HOG response pathway . YAP4 overexpression not only suppresses the osmosensitivity phenotype of the yap4 mutant but also relieves that of the hog1 mutant . Induction, under the conditions tested so far, requires the presence of the transcription factor Msn2p, but not of Msn4p, as YAP4 mRNA levels are depleted by at least 75% in the msn2 mutant . This result was further substantiated by the fact that full YAP4 induction requires the two more proximal stress response elements . Furthermore we find that GCY1, encoding a putative glycerol dehydrogenase, GPP2, encoding a NAD-dependent glycerol-3-phosphate phosphatase, and DCS2, a homologue to a decapping enzyme, have decreased mRNA levels in the yap4 -deleted strain . Our data point to a possible, as yet not entirely understood, role of the YAP4 in osmotic stress response.

J Biol Chem, 2004 Mar 12, 279(11), 10270 - 8 Epub 2003 Dec 16.
Gln3 phosphorylation and intracellular localization in nutrient limitation and starvation differ from those generated by rapamycin inhibition of Tor1/2 in Saccharomyces cerevisiae; Cox KH et al.; The ability of the cell to sense environmental conditions and alter gene expression in response to them is critical to its survival . In Saccharomyces cerevisiae, the Tor1/2 serine/threonine kinases are global regulators situated at the top of a signal cascade reported to receive and transmit nutritional signals associated with the nitrogen supply of the cell . At the other end of that cascade is Gln3, one of two transcriptional activators responsible for most nitrogen catabolic gene expression . When nitrogen is in excess, Tor1/2 are active, and Gln3 is phosphorylated and localizes to the cytoplasm . If Tor1/2 are inhibited by rapamycin or mutation, Gln3 becomes dephosphorylated, accumulates in the nucleus, and mediates nitrogen catabolite repression (NCR)-sensitive transcription . The observations that Gln3 also accumulates in the nuclei of cells provided with poor nitrogen sources or during nitrogen starvation has led to the conclusion that Tor1/2 control intracellular Gln3 localization and NCR-sensitive transcription by regulating Gln3 phosphorylation/dephosphorylation . To test this model, we compared Gln3 phosphorylation states and intracellular localizations under a variety of physiological conditions known to elicit different levels of NCR-sensitive transcription . Our data indicate that: (i) observable Gln3 phosphorylation levels do not correlate in a consistent way with the quality or quantity of the nitrogen source provided, the intracellular localization of Gln3, or the capacity to support NCR-sensitive transcription . (ii) Gln3-Myc(13) is hyperphosphorylated during nitrogen and carbon starvation, but this uniform response does not correlate with Gln3 intracellular localization . (iii) Gln3-Myc(13) dephosphorylation and nuclear localization correlate with one another at early but not late times after rapamycin treatment . These data suggest that rapamycin treatment and growth with poor nitrogen sources bring about nuclear accumulation of Gln3 but likely do so by different mechanisms or by a common mechanism involving molecules other than Gln3 and/or other than the levels of Gln3-Myc(13) phosphorylation thus far detected by others and ourselves.

Biotechnol Lett, 2003 Nov, 25(21), 1847 - 51
Construction of an amylolytic industrial strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis alpha-amylase gene; Kang NY et al.; The gene encoding Schwanniomyces occidentalis alpha-amylase (AMY) was introduced into the chromosomal delta sequences of an industrial strain of Saccharomyces cerevisiae . To obtain a strain suitable for commercial use, an delta-integrative cassette devoid of bacterial DNA sequences was constructed that contains the AMY gene and aureobasidin A resistance gene (AUR1-C) as the selection marker . The AMY gene was expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p) . The alpha-amylase activity of Sacc . cerevisiae transformed with this integrative cassette was 6 times higher than that of Sch . occidentalis . The transformants (integrants) were mitotically stable after 100 generations in nonselective medium.

Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15381 - 6 Epub 2003 Dec 15.
Genetic, biochemical, and morphological evidence for the involvement of N-glycosylation in biosynthesis of the cell wall beta1,6-glucan of Saccharomyces cerevisiae; Chavan M et al.; Recent evidence indicates that Stt3p plays a central role in the recognition and/or catalytic step in N-glycosylation (asparagine-linked glycosylation) in the lumen of the endoplasmic reticulum . It is known that stt3 mutants exhibit certain phenotypic features that are suggestive of a cell wall defect . To understand the basis of these phenotypes, we devised a genetic screen to isolate strains bearing mutations that lead to synthetic lethality in combination with the stt3-1 mutation . Using this screen, we were surprised to identify two KRE genes (KRE5 and KRE9) that are involved in the biosynthesis of the cell wall beta1,6-glucan . This finding led us to propose that the N-glycosylation process is essential in the biosynthesis of cell wall beta1,6-glucan . This proposal was supported by the observation that several stt3 mutants exhibited a 60-70% reduction in the content of cell wall beta1,6-glucan as compared with WT cells . Transmission electron microscopy revealed that the stt3 mutant strains exhibit a diffused cell wall with loss of the outer mannoprotein layer as compared with the WT cells . Thus, we provide genetic, morphological, and biochemical evidence for the critical involvement of N-glycosylation in some step in assembly of the cell wall beta1,6-glucan in Saccharomyces cerevisiae.

Biochemistry, 2003 Dec 23, 42(50), 14903 - 12
Characterization of Saccharomyces cerevisiae Ras1p and chimaeric constructs of Ras proteins reveals the hypervariable region and farnesylation as critical elements in the adenylyl cyclase signaling pathway; Crechet JB et al.; Ras1p and Ras2p, from Saccharomyces cerevisiae, are GTP-binding proteins that are essential elements in the signaling cascade leading to the activation of adenylyl cyclase . To overcome proteolytic activities that have hampered biochemical studies of Ras1p so far, its gene was genetically modified after which full-length Ras1p could be obtained . The interaction of farnesylated and unprenylated Ras1p with guanine nucleotides, guanine nucleotide exchange factors, GTPase activating proteins, and adenylyl cyclase was compared to Ras2p and human Ha-Ras interactions . Farnesylation of Ras proteins was demonstrated to be a prerequisite for membrane-bound guanine nucleotide exchange factor dependent formation of Ras-GTP complexes, and for efficient Ras-mediated adenylyl cyclase activation . To relate observed functional deviations with sequence differences between Ras1p and Ras2p, which reside almost exclusively within the hypervariable region, truncated versions and chimaeras of the Ras proteins were made . The characteristics of these constructs point to the presence of the hypervariable region of yeast Ras proteins for an efficient activation of adenylyl cyclase . The importance of the latter was confirmed as inhibition of the activation of adenylyl cyclase by an isolated farnesylated hypervariable region of Ras2p could be shown . This strongly suggests that the hypervariable region of Ras proteins can interact directly with adenylyl cyclase.

Mol Cell Biol, 2004 Jan, 24(1), 217 - 27
Noncompetitive counteractions of DNA polymerase epsilon and ISW2/yCHRAC for epigenetic inheritance of telomere position effect in Saccharomyces cerevisiae; Iida T et al.; Relocation of euchromatic genes near the heterochromatin region often results in mosaic gene silencing . In Saccharomyces cerevisiae, cells with the genes inserted at telomeric heterochromatin-like regions show a phenotypic variegation known as the telomere-position effect, and the epigenetic states are stably passed on to following generations . Here we show that the epigenetic states of the telomere gene are not stably inherited in cells either bearing a mutation in a catalytic subunit (Pol2) of replicative DNA polymerase epsilon (Pol epsilon) or lacking one of the nonessential and histone fold motif-containing subunits of Pol epsilon, Dpb3 and Dpb4 . We also report a novel and putative chromatin-remodeling complex, ISW2/yCHRAC, that contains Isw2, Itc1, Dpb3-like subunit (Dls1), and Dpb4 . Using the single-cell method developed in this study, we demonstrate that without Pol epsilon and ISW2/yCHRAC, the epigenetic states of the telomere are frequently switched . Furthermore, we reveal that Pol epsilon and ISW2/yCHRAC function independently: Pol epsilon operates for the stable inheritance of a silent state, while ISW2/yCHRAC works for that of an expressed state . We therefore propose that inheritance of specific epigenetic states of a telomere requires at least two counteracting regulators.

Mol Cell Biol, 2004 Jan, 24(1), 135 - 43
Spt10-dependent transcriptional activation in Saccharomyces cerevisiae requires both the Spt10 acetyltransferase domain and Spt21; Hess D et al.; Histone levels are a key factor in several nuclear processes, including transcription and chromosome segregation . Previous studies have demonstrated that Spt10 and Spt21 are required for the normal transcription of a subset of the histone genes in Saccharomyces cerevisiae, and sequence analysis has suggested that Spt10 is an acetyltransferase . We have now characterized several aspects of transcriptional activation of histone genes by Spt10 in vivo . Our results show that activation by Spt10 is dependent on its acetyltransferase domain . At HTA2-HTB2, the histone locus whose transcription is most strongly dependent on Spt10, Spt10 is physically recruited to the promoter in an Spt21-dependent and a cell cycle-dependent manner . Furthermore, Spt10 and Spt21 directly interact . These results, taken together with the identification of spt10 mutations that suppress an spt21Delta mutation, suggest a model for transcriptional activation by Spt10 and Spt21.

Mol Cell Biol, 2004 Jan, 24(1), 46 - 57
Aberrant processing of the WSC family and Mid2p cell surface sensors results in cell death of Saccharomyces cerevisiae O-mannosylation mutants; Lommel M et al.; Protein O mannosylation is a crucial protein modification in uni- and multicellular eukaryotes . In humans, a lack of O-mannosyl glycans causes congenital muscular dystrophies that are associated with brain abnormalities . In yeast, protein O mannosylation is vital; however, it is not known why impaired O mannosylation results in cell death . To address this question, we analyzed the conditionally lethal Saccharomyces cerevisiae protein O-mannosyltransferase pmt2 pmt4Delta mutant . We found that pmt2 pmt4Delta cells lyse as small-budded cells in the absence of osmotic stabilization and that treatment with mating pheromone causes pheromone-induced cell death . These phenotypes are partially suppressed by overexpression of upstream elements of the protein kinase C (PKC1) cell integrity pathway, suggesting that the PKC1 pathway is defective in pmt2 pmt4Delta mutants . Congruently, induction of Mpk1p/Slt2p tyrosine phosphorylation does not occur in pmt2 pmt4Delta mutants during exposure to mating pheromone or elevated temperature . Detailed analyses of the plasma membrane sensors of the PKC1 pathway revealed that Wsc1p, Wsc2p, and Mid2p are aberrantly processed in pmt mutants . Our data suggest that in yeast, O mannosylation increases the activity of Wsc1p, Wsc2p, and Mid2p by enhancing their stability . Reduced O mannosylation leads to incorrect proteolytic processing of these proteins, which in turn results in impaired activation of the PKC1 pathway and finally causes cell death in the absence of osmotic stabilization.

J Biol Chem, 2004 Feb 27, 279(9), 7785 - 91 Epub 2003 Dec 12.
Alternative start sites in the Saccharomyces cerevisiae GLR1 gene are responsible for mitochondrial and cytosolic isoforms of glutathione reductase; Outten CE et al.; To combat oxidative damage, eukaryotic cells have evolved with numerous anti-oxidant factors that are often distributed between cytosolic and mitochondrial pools . Glutathione reductase, which regenerates the reduced form of glutathione, represents one such anti-oxidant factor, yet nothing is known regarding the partitioning of this enzyme within the cell . Using the bakers' yeast Saccharomyces cerevisiae as a model, we provide evidence that a single gene, namely GLR1, encodes both the mitochondrial and cytosolic forms of glutathione reductase . A deletion in GLR1 drastically increases levels of oxidized glutathione in these two subcellular compartments . The GLR1 gene has two inframe start codons that are both used as translation initiation sites . Translation from the first codon generates the mitochondrial form that includes a mitochondrial targeting signal, whereas translation from the second codon produces the cytosolic form that lacks this sequence . Our results indicate that the sequence context of the two AUG codons influences the efficiency of translation initiation at each site, which in turn affects the relative levels of cytosolic and mitochondrial Glr1p . This method of subcellular distribution of glutathione reductase may be conserved in mammalian cells as well.

J Biol Chem, 2004 Mar 5, 279(10), 9424 - 31 Epub 2003 Dec 12.
The quaternary structure of the Saccharomyces cerevisiae succinate dehydrogenase . Homology modeling, cofactor docking, and molecular dynamics simulation studies; Oyedotun KS et al.; Succinate dehydrogenases and fumarate reductases are complex mitochondrial or bacterial respiratory chain proteins with remarkably similar structures and functions . Succinate dehydrogenase oxidizes succinate and reduces ubiquinone using a flavin adenine dinucleotide cofactor and iron-sulfur clusters to transport electrons . A model of the quaternary structure of the tetrameric Saccharomyces cerevisiae succinate dehydrogenase was constructed based on the crystal structures of the Escherichia coli succinate dehydrogenase, the E . coli fumarate reductase, and the Wolinella succinogenes fumarate reductase . One FAD and three iron-sulfur clusters were docked into the Sdh1p and Sdh2p catalytic dimer . One b-type heme and two ubiquinone or inhibitor analog molecules were docked into the Sdh3p and Sdh4p membrane dimer . The model is consistent with numerous experimental observations . The calculated free energies of inhibitor binding are in excellent agreement with the experimentally determined inhibitory constants . Functionally important residues identified by mutagenesis of the SDH3 and SDH4 genes are located near the two proposed quinone-binding sites, which are separated by the heme . The proximal quinone-binding site, located nearest the catalytic dimer, has a considerably more polar environment than the distal site . Alternative low energy conformations of the membrane subunits were explored in a molecular dynamics simulation of the dimer embedded in a phospholipid bilayer . The simulation offers insight into why Sdh4p Cys-78 may be serving as the second axial ligand for the heme instead of a histidine residue . We discuss the possible roles of heme and of the two quinone-binding sites in electron transport.

J Biol Chem, 2004 Feb 27, 279(9), 7678 - 84 Epub 2003 Dec 11.
A short-range gradient of histone H3 acetylation and Tup1p redistribution at the promoter of the Saccharomyces cerevisiae SUC2 gene; Boukaba A et al.; Chromatin immunoprecipitation assays are used to map H3 and H4 acetylation over the promoter nucleosomes and the coding region of the Saccharomyces cerevisiae SUC2 gene, under repressed and derepressed conditions, using wild type and mutant strains . In wild type cells, a high level of H3 acetylation at the distal end of the promoter drops sharply toward the proximal nucleosome that covers the TATA box, a gradient that become even steeper on derepression . In contrast, substantial H4 acetylation shows no such gradient and extends into the coding region . Overall levels of both H3 and H4 acetylation rise on derepression . Mutation of GCN5 or SNF2 lead to substantially reduced SUC2 expression; in gnc5 there is no reduction in basal H3 acetylation, but large reductions occur on derepression . SNF2 mutation has little effect on H3 acetylation, so SAGA and SWI/SNF recruitment seem to be independent events . H4 acetylation is little affected by either GCN5 or SNF2 mutation . In a double snf2/gcn5 mutant (very low SUC2 expression), H3 acetylation is at the minimal level, but H4 acetylation remains largely unaffected . Transcription is thus linked to H3 but not H4 acetylation . Chromatin immunoprecipitation assays show that Tup1p is evenly distributed over the four promoter nucleosomes in repressed wild type cells but redistributes upstream on derepression, a movement probably linked to its conversion from a repressor to an activator.

Int Rev Cytol, 2003, 229, 1 - 42
Polarized distribution of intracellular components by class V myosins in Saccharomyces cerevisiae; Matsui Y; The budding yeast Saccharomyces cerevisiae has three classes of myosins corresponding to three actin structures: class I myosin for endocytic actin structure, actin patches; class II myosin for contraction of the actomyosin contractile ring around the bud neck; and class V myosin for transport along a cable-like actin structure (actin cables), extending toward the growing cortex . Myo2p and Myo4p constitute respective class V myosins as the heavy chain and, like class V myosins in other organisms, function as actin-based motors for polarized distribution of organelles and intracellular molecules . Proper distribution of organelles is essential for autonomously replicating organelles that cannot be reproduced de novo, and is also quite important for other organelles to ensure their efficient segregation and proper positioning, even though they can be newly synthesized, such as those derived from endoplasmic reticulum . In the budding yeast, microtubule-based motors play limited roles in the distribution . Instead, the actin-based motor myosins, especially Myo2p, play a major role . Studies on Myo2p have revealed a wide variety of Myo2p cargo and Myo2p-interacting proteins and have established that Myo2p interacts with cargo and transfers it along actin cables . Moreover, recent findings suggest that Myo2p has another way to distribute cargo in that Myo2p conveys the attaching cargo along the actin track . Thus, the myosin have "dual paths" for distribution of a cargo . This dual path mechanism is proposed in the last section of this review.

Cell Mol Biol Lett, 2003, 8(4), 1005 - 11
Dicarboxylate platinum(II) complexes as inhibitors of plasma membrane H(+)-ATPase in the yeast Saccharomyces cerevisiae; Oblak E et al.; A series of cytotoxic neutral dicarboxylatoplatinum(II) complexes containing D(+), L(-) or DL-malate dianion and ethylenediamine or 1-ethylimidazole as ligands were examined using ATPase activity assays and the proton extrusion test . ATPase activity assays in vitro on plasma membrane H+-ATPase and on mitochondrial ATPase were carried out . The concentrations of compounds inhibiting enzyme activity to 50 per cent (J50) was determined . The new platinum complexes showed a stronger level of inhibition of both ATPases than the reference carboplatin; this inhibitory activity is related to a stereoisomeric form of anionic platinum ligands . ATPase inhibition in vivo was tested by glucose-stimulated proton extrusion and the influence of platinum compounds on this process in yeast cells was determined . Significant differences in activity levels were observed between those complexes with 1-ethylimidazole and those with ethylenediamine.

Mol Biol Cell, 2004 Feb, 15(2), 908 - 21 Epub 2003 Dec 10.
A striking quality control subcompartment in Saccharomyces cerevisiae: the endoplasmic reticulum-associated compartment; Huyer G et al.; The folding of nascent secretory and membrane proteins is monitored by the endoplasmic reticulum (ER) quality control system . Misfolded proteins are retained in the ER and can be removed by ER-associated degradation . As a model for the ER quality control of multispanning membrane proteins in yeast, we have been studying mutant forms of Ste6p . Here, we identify mislocalized mutant forms of Ste6p that induce the formation of, and localize to, prominent structures that are absent in normal cells . We have named these structures ER-associated compartments (ERACs), based on their juxtaposition to and connection with the ER, as observed by fluorescence and electron microscopy . ERACs comprise a network of tubulo-vesicular structures that seem to represent proliferated ER membranes . Resident ER lumenal and membrane proteins are present in ERACs in addition to their normal ER localization, suggesting there is no barrier for their entry into ERACs . However, the forms of Ste6p in ERACs are excluded from the ER and do not enter the secretory pathway; instead, they are ultimately targeted for ER-associated degradation . The presence of ERACs does not adversely affect secretory protein traffic through the ER and does not lead to induction of the unfolded protein response . We propose that ERACs may be holding sites to which misfolded membrane proteins are specifically diverted so as not to interfere with normal cellular functions . We discuss the likelihood that related ER membrane proliferations that form in response to certain other mutant or unassembled membrane proteins may be substantially similar to ERACs.

Mol Biol Cell, 2004 Mar, 15(3), 1233 - 43 Epub 2003 Dec 10.
Transcriptional remodeling in response to iron deprivation in Saccharomyces cerevisiae; Shakoury-Elizeh M et al.; The budding yeast Saccharomyces cerevisiae responds to depletion of iron in the environment by activating Aft1p, the major iron-dependent transcription factor, and by transcribing systems involved in the uptake of iron . Here, we have studied the transcriptional response to iron deprivation and have identified new Aft1p target genes . We find that other metabolic pathways are regulated by iron: biotin uptake and biosynthesis, nitrogen assimilation, and purine biosynthesis . Two enzymes active in these pathways, biotin synthase and glutamate synthase, require an iron-sulfur cluster for activity . Iron deprivation activates transcription of the biotin importer and simultaneously represses transcription of the entire biotin biosynthetic pathway . Multiple genes involved in nitrogen assimilation and amino acid metabolism are induced by iron deprivation, whereas glutamate synthase, a key enzyme in nitrogen assimilation, is repressed . A CGG palindrome within the promoter of glutamate synthase confers iron-regulated expression, suggesting control by a transcription factor of the binuclear zinc cluster family . We provide evidence that yeast subjected to iron deprivation undergo a transcriptional remodeling, resulting in a shift from iron-dependent to parallel, but iron-independent, metabolic pathways.

Genetics, 2003 Nov, 165(3), 1059 - 70
The Ras/PKA signaling pathway may control RNA polymerase II elongation via the Spt4p/Spt5p complex in Saccharomyces cerevisiae; Howard SC et al.; The Ras signaling pathway in Saccharomyces cerevisiae controls cell growth via the cAMP-dependent protein kinase, PKA . Recent work has indicated that these effects on growth are due, in part, to the regulation of activities associated with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II . However, the precise target of these Ras effects has remained unknown . This study suggests that Ras/PKA activity regulates the elongation step of the RNA polymerase II transcription process . Several lines of evidence indicate that Spt5p in the Spt4p/Spt5p elongation factor is the likely target of this control . First, the growth of spt4 and spt5 mutants was found to be very sensitive to changes in Ras/PKA signaling activity . Second, mutants with elevated levels of Ras activity shared a number of specific phenotypes with spt5 mutants and vice versa . Finally, Spt5p was efficiently phosphorylated by PKA in vitro . Altogether, the data suggest that the Ras/PKA pathway might be directly targeting a component of the elongating polymerase complex and that this regulation is important for the normal control of yeast cell growth . These data point out the interesting possibility that signal transduction pathways might directly influence the elongation step of RNA polymerase II transcription.

Genetics, 2003 Nov, 165(3), 1017 - 29
The global transcriptional activator of Saccharomyces cerevisiae, Gcr1p, mediates the response to glucose by stimulating protein synthesis and CLN-dependent cell cycle progression; Willis KA et al.; Growth of Saccharomyces cerevisiae requires coordination of cell cycle events (e.g., new cell wall deposition) with constitutive functions like energy generation and duplication of protein mass . The latter processes are stimulated by the phosphoprotein Gcr1p, a transcriptional activator that operates through two different Rap1p-mediated mechanisms to boost expression of glycolytic and ribosomal protein genes, respectively . Simultaneous disruption of both mechanisms results in a loss of glucose responsiveness and a dramatic drop in translation rate . Since a critical rate of protein synthesis (CRPS) is known to mediate passage through Start and determine cell size by modulating levels of Cln3p, we hypothesized that GCR1 regulates cell cycle progression by coordinating it with growth . We therefore constructed and analyzed gcr1delta cln3delta and gcr1delta cln1delta cln2delta strains . Both strains are temperature and cold sensitive; interestingly, they exhibit different arrest phenotypes . The gcr1delta cln3delta strain becomes predominantly unbudded with 1N DNA content (G1 arrest), whereas gcr1delta cln1delta cln2delta cells exhibit severe elongation and apparent M phase arrest . Further analysis demonstrated that the Rap1p/Gcr1p complex mediates rapid growth in glucose by stimulating both cellular metabolism and CLN transcription.

Genetics, 2003 Nov, 165(3), 985 - 95
A role for GEA1 and GEA2 in the organization of the actin cytoskeleton in Saccharomyces cerevisiae; Zakrzewska E et al.; Profilin is an actin monomer-binding protein implicated in the polymerization of actin filaments . In the budding yeast Saccharomyces cerevisiae, the pfy1-111 rho2delta double mutant has severe growth and actin cytoskeletal defects . The GEA1 and GEA2 genes, which code for paralog guanosine exchange factors for Arf proteins, were identified as multicopy suppressors of the mutant phenotype . These two genes restored the polarized distribution of actin cortical patches and produced visible actin cables in both the pfy1-111 rho2delta and pfy1delta cells . Thus, overexpression of GEA1 or GEA2 bypassed the requirement for profilin in actin cable formation . In addition, gea1 gea2 double mutants showed defects in budding and in actin cytoskeleton organization, while overexpression of GEA1 or GEA2 led to the formation of supernumerary actin cable-like structures in a Bni1p/Bnr1p-dependent manner . The ADP-ribosylation factor Arf3p may be a target of Gea1p/Gea2p, since overexpression of ARF3 partially suppressed the profilin-deficient phenotype and a deletion of ARF3 exacerbated the phenotype of a pfy1-111 mutant . Gea1p, Gea2p, Arf1p, and Arf2p but not Arf3p are known to function in vesicular transport between the endoplasmic reticulum and the Golgi . In this work, we demonstrate a role for Gea1p, Gea2p, and Arf3p in the organization of the actin cytoskeleton.

Genetics, 2003 Nov, 165(3), 975 - 83
Evolution in Saccharomyces cerevisiae: identification of mutations increasing fitness in laboratory populations; Blanc VM et al.; Since the publication of the complete sequence of the genome of Saccharomyces cerevisiae, a number of comprehensive investigations have been initiated to gain insight into cellular function . The focus of these studies has been to identify genes essential for survival in specific environments or those that when mutated cause gross phenotypic defects in growth . Here we describe Ty1-based mutational approaches designed to identify genes, which when mutated generate evolutionarily significant phenotypes causing small but positive increments on fitness . As expected, Ty1 mutations with a positive fitness effect were in the minority . However, mutations in two loci, one inactivating FAR3 and one upstream of CYR1, identified in evolving populations, were shown to have small but significantly positive fitness effects.

Genetics, 2003 Nov, 165(3), 961 - 74
Activity of mitochondrially synthesized reporter proteins is lower than that of imported proteins and is increased by lowering cAMP in glucose-grown Saccharomyces cerevisiae cells; Demlow CM et al.; We selected for increased phenotypic expression of a synthetic cox2::arg8m-G66S reporter gene inserted into Saccharomyces cerevisiae mtDNA in place of COX2 . Recessive mutations in ras2 and cyr1, as well as elevated dosage of PDE2, allowed cox2::arg8m-G66S to support Arg prototrophy . Each of these genetic alterations should decrease cellular cAMP levels . The resulting signal was transduced through redundant action of the three cAMP-dependent protein kinases, TPK1, TPK2, and TPK3 . ras2 had little or no effect on the level of wild-type Arg8p encoded by cox2::ARG8m, but did increase Arg8p activity, as judged by growth phenotype . ras2 also caused increased fluorescence in cells carrying the synthetic cox3::GFPm reporter in mtDNA, but had little effect on the steady-state level of GFP polypeptide detected immunologically . Thus, decreased cAMP levels did not affect the synthesis of mitochondrially coded protein reporters in glucose-grown cells, but rather elevated activities in the matrix that promote efficient folding . Furthermore, we show that when Arg8p is synthesized in the cytoplasm and imported into mitochondria, it has greater activity than when it is synthesized in the matrix . Thus, mitochondrially synthesized proteins may not have the same access to matrix chaperones as cytoplasmically synthesized proteins emerging from the import apparatus.

Nucleic Acids Res, 2003 Dec 15, 31(24), 7199 - 207
The linker histone homolog Hho1p from Saccharomyces cerevisiae represents a winged helix-turn-helix fold as determined by NMR spectroscopy; Ono K et al.; Hho1p is assumed to serve as a linker histone in Saccharomyces cerevisiae and, notably, it possesses two putative globular domains, designated HD1 (residues 41-118) and HD2 (residues 171-252), that are homologous to histone H5 from chicken erythrocytes . We have determined the three-dimensional structure of globular domain HD1 with high precision by heteronuclear magnetic resonance spectroscopy . The structure had a winged helix-turn-helix motif composed of an alphabetaalphaalphabetabeta fold and closely resembled the structure of the globular domain of histone H5 . Interestingly, the second globular domain, HD2, in Hho1p was unstructured under physiological conditions . Gel mobility assay demonstrated that Hho1p preferentially binds to supercoiled DNA over linearized DNA . Furthermore, NMR analysis of the complex of a deletion mutant protein (residues 1-118) of Hho1p with a linear DNA duplex revealed that four regions within the globular domain HD1 are involved in the DNA binding . The above results suggested that Hho1p possesses properties similar to those of linker histones in higher eukaryotes in terms of the structure and binding preference towards supercoiled DNA.

Curr Genet, 2004 Feb, 45(2), 90 - 5 Epub 2003 Dec 02.
NADH-reductive stress in Saccharomyces cerevisiae induces the expression of the minor isoform of glyceraldehyde-3-phosphate dehydrogenase (TDH1); Valadi H et al.; A strain of Saccharomyces cerevisiae lacking the GPD2 gene, encoding one of the glycerol-3-phosphate dehydrogenases, grows slowly under anaerobic conditions, due to reductive stress caused by the accumulation of cytoplasmic NADH . We used 2D-PAGE to study the effect on global protein expression of reductive stress in the anaerobically grown gpd2Delta strain . The most striking response was a strongly elevated expression of Tdh1p, the minor isoform of glyceraldehyde-3-phosphate dehydrogenase . This increased expression could be reversed by the addition of acetoin, a NADH-specific redox sink, which furthermore largely restored anaerobic growth of the gpd2Delta strain . Additional deletion of the TDH1 gene (but not of TDH2 or TDH3) improved anaerobic growth of the gpd2Delta strain . We therefore propose that TDH1 has properties not displayed by the other TDH isogenes and that its expression is regulated by reductive stress caused by an excess of cytoplasmic NADH.

Biochem Biophys Res Commun, 2003 Dec 26, 312(4), 1317 - 24
Activation and significance of vacuolar H+-ATPase in Saccharomyces cerevisiae adaptation and resistance to the herbicide 2,4-dichlorophenoxyacetic acid; Fernandes AR et al.; The stimulation of the activity of the H(+)-ATPase present in the vacuolar membrane (V-ATPase) of Saccharomyces cerevisiae is here described in response to a moderate stress induced by 2,4-dichlorophenoxyacetic acid (2,4-D) . This in vivo activation (up to 5-fold) took place essentially during the adaptation period, preceding cell division under herbicide stress, in coordination with a marked activation of plasma membrane H(+)-ATPase (PM-ATPase) (up to 30-fold) and the decrease of intracellular and vacuolar pH values, suggesting that activation may be triggered by acidification . Single deletion of VMA1 and genes encoding other V-ATPase subunits led to a more extended period of adaptation and to slower growth under 2,4-D stress . Results suggest that a functional V-ATPase is required to counteract, more rapidly and efficiently, the dissipation of the physiological H(+)-gradient across vacuolar membrane registered during 2,4-D adaptation.

Mol Pharmacol, 2003 Dec, 64(6), 1512 - 20
Uridine binding motifs of human concentrative nucleoside transporters 1 and 3 produced in Saccharomyces cerevisiae; Zhang J et al.; An extensive series of structural analogs of uridine that differed in substituents in the sugar and/or base moieties were subjected to inhibitor-sensitivity assays in a yeast expression system to define uridine structural determinants for inhibitors of human concentrative nucleoside transporters 1 and 3 (hCNT1 and hCNT3) . The production of recombinant hCNT1 and hCNT3 in a nucleoside-transporter deficient strain of yeast was confirmed by immunoblotting, and uridine transport parameters (Km, Vmax) were determined by defining the concentration dependence of initial rates of uptake of {3H}uridine by intact yeast . The Ki values of uridine analogs were obtained from inhibitory-effect curves and converted to binding energies . hCNT1 and hCNT3 recognized uridine through distinguishable binding motifs . hCNT1 was sensitive to modifications at C(3), less sensitive at C(5') or N(3), and much less sensitive at C(2') . hCNT3 was sensitive to modifications at C(3'), but much less sensitive at N(3), C(5') or C(2') . The changes of binding energy between transporter proteins and different uridine analogs suggested that hCNT1 formed hydrogen bonds (H-bonds) with C(3')-OH, C(5')-OH, or N(3)-H of uridine, but not with C(2')-OH, whereas hCNT3 formed H-bonds to C(3')-OH, but not to N(3)-H, C(5')-OH, and C(2')-OH . Both transporters barely tolerated modifications at C(3') or inversion of configurations at C(2')orC(3') . The binding profiles identified in this study can be used to predict the potential transportability of nucleoside analogs, including anticancer or antiviral nucleoside drugs, by hCNT1 and hCNT3.

FEBS Lett, 2003 Dec 4, 555(2), 397 - 404
Inhibition of ubiquitin/proteasome-dependent proteolysis in Saccharomyces cerevisiae by a Gly-Ala repeat; Heessen S et al.; The glycine-alanine (GA) repeat of the Epstein-Barr virus nuclear antigen-1 inhibits in cis ubiquitin-dependent proteolysis in mammalian cells through a yet unknown mechanism . In the present study we demonstrate that the GA repeat targets an evolutionarily conserved step in proteolysis since it can prevent the degradation of proteasomal substrates in the yeast Saccharomyces cerevisiae . Insertion of yeast codon-optimised recombinant GA (rGA) repeats of different length in green fluorescent protein reporters harbouring N-end rule or ubiquitin fusion degradation signals resulted in efficient stabilisation of these substrates . Protection was also achieved in rpn10delta yeast suggesting that this polyubiquitin binding protein is not required for the rGA effect . The conserved effect of the GA repeat in yeast opens the possibility for the use of genetic screens to unravel its mode of action.

FEBS Lett, 2003 Dec 4, 555(2), 268 - 73
Mouse polyomavirus large T antigen inhibits cell growth and alters cell and colony morphology in Saccharomyces cerevisiae; Adamec T et al.; The gene for mouse polyomavirus large tumor (LT) antigen, a potent oncoprotein, was expressed in Saccharomyces cerevisiae from the inducible GAL1 promoter . Substantial cell growth inhibition as well as colony and cell morphology changes dependent on cyclic adenosine monophosphate (cAMP) were observed . In contrast to cell and colony morphology alterations, the growth inhibition appeared to be transient, thus indicating the existence of an active adaptation of yeast cells to the LT antigen presence.

Mutat Res, 2003 Nov 27, 532(1-2), 41 - 58
The S-phase checkpoint and its regulation in Saccharomyces cerevisiae; Longhese MP et al.; Cells are never more vulnerable than during DNA replication, which represents a major moment of potential genetic instability . Genotoxic insults induce many different forms of DNA damage that may interfere with the ability of cells to properly duplicate their genome . Primary damage may in turn undergo structural transformations during DNA replication, thus generating secondary lesions that may be even more dangerous . Cells experiencing replication of damaged DNA or replication blocks activate an S-phase checkpoint response that assures the fidelity and completion of DNA replication before cells enter M-phase . The S-phase checkpoint pathway regulates not only progress through the cell cycle but also DNA repair and DNA replication itself.

Biochim Biophys Acta, 2003 Nov 30, 1635(1), 1 - 9
Diacylglycerol pyrophosphate phosphatase in Saccharomyces cerevisiae; Oshiro J et al.; Diacylglycerol pyrophosphate (DGPP) phosphatase in the yeast Saccharomyces cerevisiae is a Mg(2+)-independent and N-ethylmaleimide-insensitive 34-kDa vacuolar membrane-associated enzyme . It catalyzes the dephosphorylation of DGPP to form phosphatidate (PA) and then removes the phosphate from PA to form diacylglycerol (DAG) . The enzyme is a member of the lipid phosphate phosphatase superfamily that contains a three-domain lipid phosphatase motif required for catalytic activity . Expression of the DPP1 gene, which encodes DGPP phosphatase, is induced by zinc depletion, by inositol supplementation, and when cells enter the stationary phase . Induction by zinc depletion is mediated by the transcription factor Zap1p, which binds to a zinc-responsive element in the DPP1 promoter . Repression of DPP1 expression is mediated by the transcription factor Gis1p, which binds to three post-diauxic shift elements in the promoter . Regulation of DPP1 correlates with the expression of DGPP phosphatase activity and the cellular levels of DGPP and PA.

DNA Repair (Amst), 2003 Dec 9, 2(12), 1435 - 47
Functional domains required for the Saccharomyces cerevisiae Mus81-Mms4 endonuclease complex formation and nuclear localization; Fu Y et al.; The Saccharomyces cerevisiae Mms4 and Mus81 proteins form a specific complex, which functions as an endonuclease specific for branched DNA molecules and protects cells from killing by DNA alkylation damage, but not damage induced by ionizing radiations . In an effort to further understand the structure and functions of the Mus81-Mms4 complex, we attempted to define domains required for complex formation and nuclear localization through deletion and mutagenesis analyses . Combined yeast two-hybrid and co-immunoprecipitation experiments indicate that the C-terminal 100 amino acids of both Mus81 and Mms4 are required and sufficient for heterodimer formation . However, a single amino acid substitution in Mms4 in the N-terminal region is able to abolish the interaction, which suggests that the three-dimensional structure is also important for Mms4 to interact with Mus81 . By fusion to green fluorescent protein and in vivo subcellular localization studies, we demonstrate that Mms4 and Mus81 are nuclear proteins and can be localized to the nucleus independently . Deletion analyses indicate that one of two putative nuclear localization signals (residues 244-263) in Mms4 is required for localization, whereas the N-terminal half of Mus81 is necessary and sufficient for its localization to the nucleus.

Mol Genet Genomics, 2004 Feb, 271(1), 72 - 81 Epub 2003 Nov 27.
Evidence for control of nitrogen metabolism by a START-dependent mechanism in Saccharomyces cerevisiae; Bryan BA et al.; It is generally thought that cell growth and metabolism regulate cell division and not vice versa . Here, we examined Saccharomyces cerevisiae cells growing under conditions of continuous culture in a chemostat . We found that loss of G1 cyclins, or inactivation of the cyclin-dependent kinase Cdc28p, reduced the activity of glutamate synthase (Glt1p), a key enzyme in nitrogen assimilation . We also present evidence indicating that the G1 cyclin-dependent control of Glt1p may involve Jem1p, a DnaJ-type chaperone . Our results suggest that completion of START may be linked to nitrogen metabolism.

Nat Biotechnol, 2004 Jan, 22(1), 86 - 92 Epub 2003 Nov 30.
Principles of transcriptional control in the metabolic network of Saccharomyces cerevisiae; Ihmels J et al.; Cellular networks are subject to extensive regulation, which modifies the availability and efficiency of connections between components in response to external conditions . Thus far, studies of large-scale networks have focused on their connectivity, but have not considered how the modulation of this connectivity might also determine network properties . To address this issue, we analyzed how the coordinated expression of enzymes shapes the metabolic network of Saccharomyces cerevisiae . By integrating large-scale expression data with the structural description of the metabolic network, we systematically characterized the transcriptional regulation of metabolic pathways . The analysis revealed recurrent patterns, which may represent design principles of metabolic gene regulation . First, we find that transcription regulation biases metabolic flow toward linearity by coexpressing only distinct branches at metabolic branchpoints . Second, individual isozymes were often separately coregulated with distinct processes, providing a means of reducing crosstalk between pathways using a common reaction . Finally, transcriptional regulation defined a hierarchical organization of metabolic pathways into groups of varying expression coherence . These results emphasize the utility of incorporating regulatory information when analyzing properties of large-scale cellular networks.

J Biol Chem, 2004 Feb 20, 279(8), 7072 - 81 Epub 2003 Nov 26.
Identification of a novel one-carbon metabolism regulon in Saccharomyces cerevisiae; Gelling CL et al.; Glycine specifically induces genes encoding subunits of the glycine decarboxylase complex (GCV1, GCV2, and GCV3), and this is mediated by a fall in cytoplasmic levels of 5,10-methylenetetrahydrofolate caused by inhibition of cytoplasmic serine hydroxymethyltransferase . Here it is shown that this control system extends to genes for other enzymes of one-carbon metabolism and de novo purine biosynthesis . Northern analysis of the response to glycine demonstrated that the induction of the GCV genes and the induction of other amino acid metabolism genes are temporally distinct . The genome-wide response to glycine revealed that several other genes are rapidly co-induced with the GCV genes, including SHM2, which encodes cytoplasmic serine hydroxymethyltransferase . These results were refined by examining transcript levels in an shm2Delta strain (in which cytoplasmic 5,10-methylenetetrahydrofolate levels are reduced) and a met13Delta strain, which lacks the main methylenetetrahydrofolate reductase activity of yeast and is effectively blocked at consumption of 5,10-methylene tetrahydrofolate for methionine synthesis . Glycine addition also caused a substantial transient disturbance to metabolism, including a sequence of changes in induction of amino acid biosynthesis and respiratory chain genes . Analysis of the glycine response in the shm2Delta strain demonstrated that apart from the one-carbon regulon, most of these transient responses were not contingent on a disturbance to one-carbon metabolism . The one-carbon response is distinct from the Bas1p purine biosynthesis regulon and thus represents the first example of transcriptional regulation in response to activated one-carbon status.

J Biol Chem, 2004 Feb 20, 279(8), 6501 - 6 Epub 2003 Nov 26.
Decrease of H2O2 plasma membrane permeability during adaptation to H2O2 in Saccharomyces cerevisiae; Branco MR et al.; Contrary to what is widely believed, recent published results show that H2O2 does not freely diffuse across biomembranes . The fast removal of H2O2 by antioxidant enzymes is able to generate a gradient if H2O2 is produced in a different compartment from that containing the enzymes (Antunes, F., and Cadenas, E . (2000) FEBS Lett . 475, 121-126) . In this work, we extended these studies and tested whether an active regulation of biomembranes permeability characteristics is part of the cell response to oxidative stress . Using Saccharomyces cerevisiae as a model, we showed that: (a) H2O2 gradients across the plasma membrane are formed upon exposure to external H2O2; (b) there is a correlation between the magnitude of the gradients and the resistance to H2O2; (c) there is not a correlation between the intracellular capacity to remove H2O2 and the resistance to H2O2; (d) the plasma membrane permeability to H2O2 decreases by a factor of two upon acquisition of resistance to this agent by pre-exposing cells either to nonlethal doses of H2O2 or to cycloheximide, an inhibitor of protein synthesis; and (e) erg3Delta and erg6Delta mutants, which have impaired ergosterol biosynthesis pathways, show higher plasma membrane permeability to H2O2 and are more sensitive to H2O2 . Altogether, the regulation of the plasma membrane permeability to H2O2 emerged as a new mechanism by which cells respond and adapt to H2O2 . The consequences of the results to cellular redox compartmentalization and to the origin and evolution of the eukaryotic cell are discussed.

Biochemistry, 2003 Dec 9, 42(48), 14207 - 13
Fidelity of DNA polymerase delta holoenzyme from Saccharomyces cerevisiae: the sliding clamp proliferating cell nuclear antigen decreases its fidelity; Hashimoto K et al.; DNA polymerases delta and epsilon (pol delta and epsilon) are the two major replicative polymerases in the budding yeast Saccharomyces cerevisiae . The fidelity of pol delta is influenced by its 3'-5' proofreading exonuclease activity, which corrects misinsertion errors, and by enzyme cofactors . PCNA is a pol delta cofactor, called the sliding clamp, which increases the processivity of pol delta holoenzyme . This study measures the fidelity of 3'-5' exonuclease-proficient and -deficient pol delta holoenzyme using a synthetic 30mer primer/100mer template in the presence and absence of PCNA . Although PCNA increases pol delta processivity, the presence of PCNA decreased pol delta fidelity 2-7-fold . In particular, wild-type pol delta demonstrated the following nucleotide substitution efficiencies for mismatches in the absence of PCNA: G.G, 0.728 x 10(-4); T.G, 1.82 x 10(-4); A.G, <0.01 x 10(-4) . In the presence of PCNA these values increased as follows: G.G, 1.30 x 10(-4); T.G, 2.62 x 10(-4); A.G, 0.074 x 10(-4) . A similar but smaller effect was observed for exonuclease-deficient pol delta (i.e., 2-4-fold increase in nucleotide substitution efficiencies in the presence of PCNA) . Thus, the fidelity of wild-type pol delta in the presence of PCNA is more than 2 orders of magnitude lower than the fidelity of wild-type pol epsilon holoenzyme and is comparable to the fidelity of exonuclease-deficient pol epsilon holoenzyme.

J Biol Chem, 2004 Feb 13, 279(7), 5846 - 51 Epub 2003 Nov 24.
A mammalian mediator subunit that shares properties with Saccharomyces cerevisiae mediator subunit Cse2; Tomomori-Sato C et al.; The multiprotein Mediator complex is a coactivator required for activation of RNA polymerase II transcription by DNA bound transcription factors . We previously identified and partially purified a mammalian Mediator complex from rat liver nuclei (Brower, C.S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R.D., Malik, S., Lane, W.S., Sorokina, I., Roeder, R.G., Conaway, J.W., and Conaway, R.C . (2002) Proc . Natl . Acad . Sci . U . S . A . 99, 10353-10358) . Analysis by tandem mass spectrometry of proteins present in the most highly purified rat Mediator fractions led to the identification of a collection of new mammalian Mediator subunits, as well as several potential Mediator subunits including a previously uncharacterized protein encoded by the FLJ10193 open reading frame . In this study, we present direct biochemical evidence that the FLJ10193 protein, which we designate Med25, is a bona fide subunit of the mammalian Mediator complex . In addition, we present evidence that Med25 shares structural and functional properties with Saccharomyces cerevisiae Mediator subunit Cse2 and may be a mammalian Cse2 ortholog . Taken together, our findings identify a novel mammalian Mediator subunit and shed new light on the architecture of the mammalian Mediator complex.

Antimicrob Agents Chemother, 2003 Dec, 47(12), 3890 - 900
Molecular mechanism of terbinafine resistance in Saccharomyces cerevisiae; Leber R et al.; Ten mutants of the yeast Saccharomyces cerevisiae resistant to the antimycotic terbinafine were isolated after chemical or UV mutagenesis . Molecular analysis of these mutants revealed single base pair exchanges in the ERG1 gene coding for squalene epoxidase, the target of terbinafine . The mutants did not show cross-resistance to any of the substrates of various pleiotropic drug resistance efflux pumps tested . The ERG1 mRNA levels in the mutants did not differ from those in the wild-type parent strains . Terbinafine resistance was transmitted with the mutated alleles in gene replacement experiments, proving that single amino acid substitutions in the Erg1 protein were sufficient to confer the resistance phenotype . The amino acid changes caused by the point mutations were clustered in two regions of the Erg1 protein . Seven mutants carried the amino acid substitutions F402L (one mutant), F420L (one mutant), and P430S (five mutants) in the C-terminal part of the protein; and three mutants carried an L251F exchange in the central part of the protein . Interestingly, all exchanges identified involved amino acids which are conserved in the squalene epoxidases of yeasts and mammals . Two mutations that were generated by PCR mutagenesis of the ERG1 gene and that conferred terbinafine resistance mapped in the same regions of the Erg1 protein, with one resulting in an L251F exchange and the other resulting in an F433S exchange . The results strongly indicate that these regions are responsible for the interaction of yeast squalene epoxidase with terbinafine.

FEMS Microbiol Rev, 2003 Dec, 27(5), 629 - 49
Exploiting the yeast Saccharomyces cerevisiae for the study of the organization and evolution of complex genomes; Kouprina N et al.; Yeast artificial chromosome (YAC) cloning systems have advanced the analysis of complex genomes considerably . They permit the cloning of larger fragments than do bacterial artificial chromosome systems, and the cloned material is more easily modified . We recently developed a novel YAC cloning system called transformation-associated recombination (TAR) cloning . Using in vivo recombination in yeast, TAR cloning selectively isolates, as circular YACs, desired chromosome segments or entire genes from complex genomes . The ability to do that without constructing a representative genomic library of random clones greatly facilitates analysis of gene function and its role in disease . In this review, we summarize how recombinational cloning techniques have advanced the study of complex genome organization, gene expression, and comparative genomics.

Gene, 2003 Dec 4, 321, 123 - 9
Functional diversity of potato SNF1-related kinases tested in Saccharomyces cerevisiae; Lovas A et al.; Sucrose nonfermenting 1 catalytic subunit (SNF1)-type protein kinases are members of a metabolite-sensing protein kinase family distributed ubiquitously from yeast to plants and animals . In yeast cells, SNF1 acts in complex with the activator subunit SNF4 and a member of the SIP1/SIP2/GAL83 family responsible for substrate definition . The potato (Solanum tuberosum) genome possesses at least two SnRK1s, designated PKIN1 and StubSNF1 . In this study, potato kinase 1 (PKIN1) and StubSNF1 were analysed in the yeast two-hybrid system and characterised by suppression of yeast mutations . It was shown that StubSNF1 interacted with the GAL83 ortholog of potato, StubGAL83, and complemented the Delta snf1 mutation . Moreover, it suppressed Delta snf4 and Delta sip1,Delta sip2,Delta gal83 deficiencies . In contrast, PKIN1 was unable to interact with StubGAL83 and did not rescue the yeast mutants . These data suggest different functions for PKIN1 and StubSNF1 in potato.

J Mol Biol, 2003 Dec 5, 334(4), 769 - 80
The Sir4 C-terminal coiled coil is required for telomeric and mating type silencing in Saccharomyces cerevisiae; Murphy GA et al.; Saccharomyces cerevisiae Sir4p plays important roles in silent chromatin at telomeric and silent mating type loci . The C terminus of Sir4p (Sir4CT) is critical for its functions in vivo because over-expression or deletion of Sir4CT fragments disrupts normal telomeric structure and abolishes the telomere position effect . The 2.5A resolution X-ray crystal structure of an Sir4CT fragment (Sir4p 1217-1358) reveals a 72 residue homodimeric, parallel coiled coil, burying an extensive 3600A(2) of surface area . The crystal structure is consistent with results of protein cross-linking and analytical ultracentrifugation results demonstrating that Sir4CT exists as a dimer in solution . Disruption of the coiled coil in vivo by point mutagenesis results in total derepression of telomeric and HML silent mating marker genes, suggesting that coiled coil dimerization is essential for Sir4p-mediated silencing . In addition to the coiled coil dimerization interface (Sir4CC interface), a crystallographic interface between pairs of coiled coils is significantly hydrophobic and buries 1228A(2) of surface area (interface II) . Remarkably, interface II mutants are deficient in telomeric silencing but not in mating type silencing in vivo . However, point mutants of interface II do not affect the oligomerization state of Sir4CT in solution . These results are consistent with the hypothesis that interface II mimics a protein interface between Sir4p and one of its protein partners that is essential for telomeric silencing but not mating type silencing.

J Biol Chem, 2004 Feb 13, 279(7), 5338 - 45 Epub 2003 Nov 20.
Vacuole membrane topography of the DPP1-encoded diacylglycerol pyrophosphate phosphatase catalytic site from Saccharomyces cerevisiae; Han GS et al.; The Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate phosphatase is a vacuole membrane-associated enzyme that catalyzes the removal of the beta-phosphate from diacylglycerol pyrophosphate to form phosphatidate, and it then removes the phosphate from phosphatidate to form diacylglycerol . The enzyme has six putative transmembrane domains and a hydrophilic region that contains a phosphatase motif required for its catalytic activity . In this work, we examined the topography of diacylglycerol-pyrophosphate phosphatase catalytic site within the transverse plane of the vacuole membrane . Results of protease protection analysis using endoproteinase Lys-C and labeling of cysteine residues using sulfhydryl reagents were consistent with a model where the catalytic site of diacylglycerol-pyrophosphate phosphatase was oriented to the cytosolic face of the vacuole membrane . In addition, diacylglycerol-pyrophosphate phosphatase activity was found with intact vacuoles . The phospholipids diacylglycerol pyrophosphate (0.6 mol %) and phosphatidate (1.4 mol %) were found in the vacuole membrane, and their levels decreased to an undetectable level and by 79%, respectively, when cells were depleted for zinc . The reduced levels of diacylglycerol pyrophosphate and phosphatidate correlated with the induced expression of diacylglycerol-pyrophosphate phosphatase . This work suggested that diacylglycerol pyrophosphate phosphatase functions to regulate the levels of diacylglycerol pyrophosphate and phosphatidate on the cytosolic face of the vacuole membrane.

Water Res, 2004 Jan, 38(1), 61 - 70
Toxicity of anionic detergents determined by Saccharomyces cerevisiae microarray analysis; Sirisattha S et al.; Sodium n-dodecyl benzene sulfonate (LAS) and sodium dodecyl sulfate (SDS) are popular anionic detergents (surfactants) that are used worldwide and the toxicities of these chemicals have been characterized . We applied these chemicals in a DNA microarray bioassay and determined that the microarray data reflects previous findings and also provides some new information about anionic detergent toxicity . The mRNA expression profiles suggest that LAS and SDS cause damage to membranes and alterations in carbon metabolism, and induce the oxidative stress response . We also found that LAS and SDS induce the pleiotropic drug-resistance network, and that LAS and SDS may be pumped out of yeast cells by this network . Hierarchical clustering of the expression profiles showed that LAS and SDS cause similar features of toxicity and that the toxicity is similar to that of capsaicin but different from that of cadmium and mercury.

Nucleic Acids Res, 2003 Dec 1, 31(23), 6798 - 805
Ribosomal proteins Rps0 and Rps21 of Saccharomyces cerevisiae have overlapping functions in the maturation of the 3' end of 18S rRNA; Tabb-Massey A et al.; The Rps0 proteins of Saccharomyces cerevisiae are components of the 40S ribosomal subunit required for maturation of the 3' end of 18S rRNA . Drosophila and human homologs of the Rps0 proteins physically interact with Rps21 proteins, and decreased expression of both proteins in Drosophila impairs control of cellular proliferation in hematopoietic organs during larval development . Here, we characterize the yeast RPS21A/B genes and show that strains where both genes are disrupted are not viable . Relative to the wild type, cells with disrupted RPS21A or RPS21B genes exhibit a reduction in growth rate, a decrease in free 40S subunits, an increase in the amount of free 60S subunits, and a decrease in polysome size . Ribosomal RNA processing studies reveal RPS21 and RPS0 mutants have virtually identical processing defects . The pattern of processing defects observed in RPS0 and RPS21 mutants is not a general characteristic of strains with suboptimal levels of small subunit ribosomal proteins, since disruption of the RPS18A or RPS18B genes results in related but distinct processing defects . Together, these data link the Rps0 and Rps21 proteins together functionally in promoting maturation of the 3' end of 18S rRNA and formation of active 40S ribosomal subunits.

J Basic Microbiol, 2003, 43(6), 522 - 9
Effect of oxythiamin on growth rate, survival ability and pyruvate decarboxylase activity in Saccharomyces cerevisiae; Tylicki A et al.; Oxythiamin is one of the antivitamin derivatives of thiamin which, after phosphorylation, can be bound to the catalytic centre of thiamin-dependent enzymes and inhibit these enzymes . In this work the influence of oxythiamin on the growth rate, survival and the activity of pyruvate decarboxylase of Saccharomyces cerevisiae (s288c) was investigated . Oxythiamin decreased both the growth rate and survival ability of yeast cells . Moreover, in three-day-old cultures on a medium with oxythiamin, an increase of pyruvate decarboxylase activity was observed . This unusual effect may be in response to the earlier inhibition of pyruvate decarboxylase . A high concentration of pyruvate in the cell extracts taken from the medium with oxythiamin was found . This accumulation of pyruvate could provide for enhanced biosynthesis of the pyruvate decarboxylase apoform and an increase of enzyme activity.

Biochem Biophys Res Commun, 2003 Nov 28, 311(4), 1143 - 50
Calcineurin signaling in Saccharomyces cerevisiae: how yeast go crazy in response to stress; Cyert MS; In the yeast Saccharomyces cerevisiae, Ca(2+) signaling mediated by the Ca(2+)/calmodulin dependent phosphatase, calcineurin, is required for survival during environmental stress . One role of the phosphatase under these conditions is to activate gene expression through its regulation of the Crz1p ("crazy") transcription factor . Calcineurin dephosphorylates Crz1p and causes its rapid translocation from the cytosol to the nucleus . Crz1p then activates the transcription of genes whose products promote cell survival . Recent studies concerning the regulation of Crz1p by calcineurin are discussed in this review and the mechanisms by which calcineurin controls gene expression in yeast and mammalian cells are compared.

FEBS Lett, 2003 Nov 20, 554(3), 295 - 300
Genetic interactions among ZDS1,2, CDC37, and protein kinase CK2 in Saccharomyces cerevisiae; Bandhakavi S et al.; We report here the identification of the homologous gene pair ZDS1,2 as multicopy suppressors of a temperature-sensitive allele (cka2-13(ts)) of the CKA2 gene encoding the alpha' catalytic subunit of protein kinase CK2 . Overexpression of ZDS1,2 suppressed the temperature sensitivity, geldanamycin (GA) sensitivity, slow growth, and flocculation of multiple cka2 alleles and enhanced CK2 activity in vivo toward a known physiological substrate, Fpr3 . Consistent with the existence of a recently described positive feedback loop between CK2 and Cdc37, overexpression of ZDS1,2 also suppressed the temperature sensitivity, abnormal morphology, and GA sensitivity of a CK2 phosphorylation-deficient mutant of CDC37, cdc37-S14A, as well as the GA sensitivity of a cdc37-1 allele . A likely basis for all of these effects is our observation that ZDS1,2 overexpression enhances Cdc37 protein levels . Activation of the positive feedback loop between CK2 and Cdc37 likely contributes to the pleiotropic nature of ZDS1,2, as both CK2 and Cdc37 regulate diverse cellular functions.

Mol Microbiol, 2003 Nov, 50(4), 1257 - 69
Identification and metabolic role of the mitochondrial aspartate-glutamate transporter in Saccharomyces cerevisiae; Cavero S et al.; The malate-aspartate NADH shuttle in mammalian cells requires the activity of the mitochondrial aspartate-glutamate carrier (AGC) . Recently, we identified in man two AGC isoforms, aralar1 and citrin, which are regulated by calcium on the external face of the inner mitochondrial membrane . We have now identified Agc1p as the yeast counterpart of the human AGC . The corresponding gene was overexpressed in bacteria and yeast mitochondria, and the protein was reconstituted in liposomes where it was identified as an aspartate-glutamate transporter from its transport properties . Furthermore, yeast cells lacking Agc1p were unable to grow on acetate and oleic acid, and had reduced levels of valine, ornithine and citrulline; in contrast they grew on ethanol . Expression of the human AGC isoforms can replace the function of Agc1p . However, unlike its human orthologues, yeast Agc1p catalyses both aspartate-glutamate exchange and substrate uniport activities . We conclude that Agc1p performs two metabolic roles in Saccharomyces cerevisiae . On the one hand, it functions as a uniporter to supply the mitochondria with glutamate for nitrogen metabolism and ornithine synthesis . On the other, the Agc1p, as an aspartate-glutamate exchanger, plays a role within the malate-aspartate NADH shuttle which is critical for the growth of yeast on acetate and fatty acids as carbon sources . These results provide strong evidence of the existence of a malate-aspartate NADH shuttle in yeast.

Mol Microbiol, 2003 Nov, 50(4), 1155 - 71
The nuclear actin-related protein Act3p/Arp4p of Saccharomyces cerevisiae is involved in transcription regulation of stress genes; Gorzer I et al.; A mutational analysis of the essential nuclear actin-related protein of Saccharomyces cerevisiae, Act3p/Arp4p, was performed . The five residues chosen for substitution were amino acids conserved between actin and Act3p/Arp4p, the tertiary structure of which most probably resembles that of actin . Two thermosensitive (ts) mutants, a single and a double point mutant, and one lethal double point mutant were obtained . Both ts mutants were formamide-sensitive which supports a structural relatedness of Act3p/Arp4p to actin; they were also hypersensitive against hydroxyurea and ultraviolet irradiation pointing to a possible role of Act3p/Arp4p in DNA replication and repair . Their 'suppressor of Ty' (SPT) phenotype, observed with another ts mutant of Act3p/Arp4p before, suggested involvement of Act3p/Arp4p in transcription regulation . Accordingly, genome-wide expression profiling revealed misregulated transcription in a ts mutant of a number of genes, among which increased expression of various stress-responsive genes (many of them requiring Msn2p/Msn4p for induction) was the most salient result . This provides an explanation for the mutant's enhanced resistance to severe thermal and oxidative stress . Thus, Act3p/Arp4p takes an important part in the repression of stress-induced genes under non-stress conditions.

Genes Cells, 2003 Nov, 8(11), 873 - 88
Double-stranded DNA binding properties of Saccharomyces cerevisiae DNA polymerase epsilon and of the Dpb3p-Dpb4p subassembly; Tsubota T et al.; BACKGROUND: DNA polymerase epsilon (Pol epsilon) of Saccharomyces cerevisiae participates in many aspects of DNA replication, as well as in DNA repair . In order to clarify molecular mechanisms employed in the multiple tasks of Pol epsilon, we have been characterizing the interaction between Pol epsilon and DNA . RESULTS: Analysis of the four-subunit Pol epsilon complex by gel mobility shift assay revealed that the complex binds not only to single-stranded (ss) DNA but also equally well to double-stranded (ds) DNA . A truncated polypeptide consisting of the N-terminal domain of Pol2p catalytic subunit binds to ssDNA but not to dsDNA, indicating that the Pol2p C-terminal domain and/or the auxiliary subunits are involved in the dsDNA-binding . The dsDNA-binding by Pol epsilon does not require DNA ends or specific DNA sequences . Further analysis by competition experiments indicated that Pol epsilon contains at least two distinct DNA-binding sites, one of which binds exclusively to ssDNA and the other to dsDNA . The dsDNA-binding site, however, is suggested to also bind ssDNA . The DNA polymerase activity of Pol epsilon is inhibited by ssDNA but not by dsDNA . Furthermore, purification of the Pol epsilon auxiliary subunits Dpb3p and Dpb4p revealed that these proteins form a heterodimer and associate with dsDNA . CONCLUSIONS: Pol epsilon has multiple sites at which it interacts with DNA . One of these sites has a strong affinity for dsDNA, a feature that is not generally associated with DNA polymerases . Involvement of the Dpb3p-Dpb4p complex in the dsDNA-binding of Pol epsilon is inferred.

Genet Res, 2003 Aug, 82(1), 19 - 31
Small fitness effects and weak genetic interactions between deleterious mutations in heterozygous loci of the yeast Saccharomyces cerevisiae; Szafraniec K et al.; Rare, random mutations were induced in budding yeast by ethyl methanesulfonate (EMS) . Clones known to bear a single non-neutral mutation were used to obtain mutant heterozygotes and mutant homozygotes that were later compared with wild-type homozygotes . The average homozygous effect of mutation was an approximately 2% decrease in the growth rate . In heterozygotes, the harmful effect of these relatively mild mutations was reduced approximately fivefold . In a test of epistasis, two heterozygous mutant loci were paired at random . Fitness of the double mutants was best explained by multiplicative action of effects at single loci, with little evidence for epistasis and essentially excluding synergism . In other experiments, the same mutations in haploid and heterozygous diploid clones were compared . Regardless of the haploid phenotypes, mildly deleterious or lethal, fitness of the heterozygotes was decreased by less than half a per cent on average . In general, the results presented here suggest that most mutations tend to exhibit small and weakly interacting effects in heterozygous loci regardless of how harmful they are in haploids or homozygotes.

Mol Biol Cell, 2004 Feb, 15(2), 706 - 20 Epub 2003 Nov 14.
Global phenotypic analysis and transcriptional profiling defines the weak acid stress response regulon in Saccharomyces cerevisiae; Schuller C et al.; Weak organic acids such as sorbate are potent fungistatic agents used in food preservation, but their intracellular targets are poorly understood . We thus searched for potential target genes and signaling components in the yeast genome using contemporary genome-wide functional assays as well as DNA microarray profiling . Phenotypic screening of the EUROSCARF collection revealed the existence of numerous sorbate-sensitive strains . Sorbate hypersensitivity was detected in mutants of the shikimate biosynthesis pathway, strains lacking the PDR12 efflux pump or WAR1, a transcription factor mediating stress induction of PDR12 . Using DNA microarrays, we also analyzed the genome-wide response to acute sorbate stress, allowing for the identification of more than 100 genes rapidly induced by weak acid stress . Moreover, a novel War1p- and Msn2p/4p-independent regulon that includes HSP30 was identified . Although induction of the majority of sorbate-induced genes required Msn2p/4p, weak acid tolerance was unaffected by a lack of Msn2p/4p . Ectopic expression of PDR12 from the GAL1-10 promoter fully restored sorbate resistance in a strain lacking War1p, demonstrating that PDR12 is the major target of War1p under sorbic acid stress . Interestingly, comparison of microarray data with results from the phenotypic screening revealed that PDR12 remained as the only gene, which is both stress inducible and required for weak acid resistance . Our results suggest that combining functional assays with transcriptome profiling allows for the identification of key components in large datasets such as those generated by global microarray analysis.

Mol Biol Cell, 2004 Feb, 15(2), 665 - 77 Epub 2003 Nov 14.
Pex30p, Pex31p, and Pex32p form a family of peroxisomal integral membrane proteins regulating peroxisome size and number in Saccharomyces cerevisiae; Vizeacoumar FJ et al.; The peroxin Pex23p of the yeast Yarrowia lipolytica exhibits high sequence similarity to the hypothetical proteins Ylr324p, Ygr004p, and Ybr168p encoded by the Saccharomyces cerevisiae genome . Ylr324p, Ygr004p, and Ybr168p are integral to the peroxisomal membrane and act to control peroxisome number and size . Synthesis of Ylr324p and Ybr168p, but not of Ygr004p, is induced during incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes . Cells deleted for YLR324w exhibit increased numbers of peroxisomes, whereas cells deleted for YGR004w or YBR168w exhibit enlarged peroxisomes . Ylr324p and Ybr168p cannot functionally substitute for one another or for Ygr004p, whereas Ygr004p shows partial functional redundancy with Ylr324p and Ybr168p . Ylr324p, Ygr004p, and Ybr168p interact within themselves and with Pex28p and Pex29p, which have been shown also to regulate peroxisome size and number . Systematic deletion of genes demonstrated that PEX28 and PEX29 function upstream of YLR324w, YGR004w, and YBR168w in the regulation of peroxisome proliferation . Our data suggest a role for Ylr324p, Ygr004p, and Ybr168p--now designated Pex30p, Pex31p, and Pex32p, respectively--together with Pex28p and Pex29p in controlling peroxisome size and proliferation in Saccharomyces cerevisiae.

Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 13869 - 74 Epub 2003 Nov 14.
Spermidine but not spermine is essential for hypusine biosynthesis and growth in Saccharomyces cerevisiae: spermine is converted to spermidine in vivo by the FMS1-amine oxidase; Chattopadhyay MK et al.; In our earlier work we showed that either spermidine or spermine could support the growth of spe2Delta or spe3Delta polyamine-requiring mutants, but it was unclear whether the cells had a specific requirement for either of these amines . In the current work, we demonstrate that spermidine is specifically required for the growth of Saccharomyces cerevisiae . We were able to show this specificity by using a spe3Delta fms1Delta mutant that lacked both spermidine synthase and the FMS1-encoded amine oxidase that oxidizes spermine to spermidine . The polyamine requirement for the growth of this double mutant could only be satisfied by spermidine; i.e., spermine was not effective because it cannot be oxidized to spermidine in the absence of the FMS1 gene . We also showed that at least one of the reasons for the absolute requirement for spermidine for growth is the specificity of its function as a necessary substrate for the hypusine modification of eIF5A . Spermine itself cannot be used for the hypusine modification, unless it is oxidized to spermidine by the Fms1 amine oxidase . We have quantified the conversion of spermine in vivo and have shown that this conversion is markedly increased in a strain overexpressing the Fms1 protein . We have also shown this conversion in enzymatic studies by using the purified amine oxidase from yeast.

Mol Microbiol, 2003 Nov, 50(3), 883 - 96
The omega-site sequence of glycosylphosphatidylinositol-anchored proteins in Saccharomyces cerevisiae can determine distribution between the membrane and the cell wall; Frieman MB et al.; Glycosylphosphatidylinositol (GPI)-anchored cell wall proteins play an important role in the structure and function of the cell wall in yeast and other fungi . Although the majority of characterized fungal GPI-anchored proteins do in fact localize to the cell wall, some are believed to reside at the plasma membrane and not to traffic significantly to the cell wall . There is evidence suggesting that the amino acids immediately upstream of the site of GPI anchor addition (the omega site) serve as the signal determining whether a GPI protein localizes to the cell wall or to the plasma membrane, although this remains controversial . Here, we examine in detail the functional and biochemical differences between the GPI anchor addition signals of putative cell wall (CW) and plasma membrane (PM) GPI proteins . We find strong evidence for the existence of PM-class and CW-class GPI proteins . We show that the biological function of a GPI-CWP is strongly compromised by changing the GPI anchor signal from a CW-class signal to a PM-class signal . Biochemically, this abrogation of function corresponds to a change in the protein from a cell wall form to a membrane form . To understand better the basis for the difference between the two classes of proteins, we mutated the amino acids upstream of the omega site in a GPI-PM protein and selected mutant proteins that were now localized to the cell wall . We were also able to design simple amino acid mutations in a GPI-CW protein that efficiently redirected the protein to the plasma membrane . These studies make clear that different GPI anchor sequences can have dramatic effects on localization of the proteins and help to define the GPI anchor addition signal sequences that distinguish the PM-class and CW-class GPI proteins.

Biol Proced Online, 2003, 5, 162 - 169 Epub 2003 Jul 3.
Methods designed for the identification and characterization of in vitro and in vivo chromatin assembly mutants in Saccharomyces cerevisiae; Harkness TA et al.; Assembly of DNA into chromatin allows for the formation of a barrier that protects naked DNA from protein and chemical agents geared to degrade or metabolize DNA . Chromatin assembly occurs whenever a length of DNA becomes exposed to the cellular elements, whether during DNA synthesis or repair . This report describes tools to study chromatin assembly in the model system Saccharomyces cerevisiae . Modifications to an in vitro chromatin assembly assay are described that allowed a brute force screen of temperature sensitive (ts) yeast strains in order to identify chromatin assembly defective extracts . This screen yielded mutations in genes encoding two ubiquitin protein ligases (E3s): RSP5, and a subunit of the Anaphase Promoting Complex (APC), APC5 . Additional modifications are described that allow for a rapid analysis and an in vivo characterization of yeast chromatin assembly mutants, as well as any other mutant of interest . Our analysis suggests that the in vitro and invivo chromatin assembly assays are responsive to different cellular signals, including cell cycle cues that involve different molecular networks.

BMC Genomics . 2003 Nov 13;4(1):45.
In silico and in vivo analysis reveal a novel gene in Saccharomyces cerevisiae trehalose metabolism; De Mesquita JF et al.; BACKGROUND: The ability to respond rapidly to fluctuations in environmental changes is decisive for cell survival . Under these conditions trehalose has an essential protective function and its concentration increases in response to enhanced expression of trehalose synthase genes, TPS1, TPS2, TPS3 and TSL1 . Intriguingly, the NTH1 gene, which encodes neutral trehalase, is highly expressed at the same time . We have previously shown that trehalase remains in its inactive non-phosphorylated form by the action of an endogenous inhibitor . Recently, a comprehensive two-hybrid analysis revealed a 41-kDa protein encoded by the YLR270w ORF, which interacts with NTH1p . RESULTS: In this work we investigate the correlation of this Trehalase Associated Protein, in trehalase activity regulation . The neutral trehalase activity in the ylr270w mutant strain was about 4-fold higher than in the control strain . After in vitro activation by PKA the ylr270w mutant total trehalase activity increased 3-fold when compared to a control strain . The expression of the NTH1 gene promoter fused to the heterologous reporter lacZ gene was evaluated . The mutant strain lacking YLR270w exhibited a 2-fold increase in the NTH1-lacZ basal expression when compared to the wild type strain . CONCLUSIONS: These results strongly indicate a central role for Ylr270p in inhibiting trehalase activity, as well as in the regulation of its expression preventing a wasteful futile cycle of synthesis-degradation of trehalose.

FEMS Microbiol Lett, 2003 Nov 7, 228(1), 105 - 10
The GTS1 gene product facilitates the self-organization of the energy metabolism oscillation in the continuous culture of the yeast Saccharomyces cerevisiae; Akiyama S et al.; To study the role of the GTS1 gene in the energy metabolism oscillation in continuous cultures of yeast from the physical aspect, time-series data of dissolved oxygen oscillations were analyzed by transforming them into power spectra and by creating two-dimensional trajectories using time delay embedding technique . We found that the wild-type cells organized themselves into a stable limit cycle oscillation and that the GTS1-deleted mutant, gts1Delta, usually showed transient oscillations whose power spectra resembled those of 1/f noise . Thus, we suggested that GTS1 plays an important role in the self-organization of the energy metabolism oscillation.

Genomics, 2003 Dec, 82(6), 606 - 18
Quantitative comparison of cDNA-AFLP, microarrays, and GeneChip expression data in Saccharomyces cerevisiae; Reijans M et al.; cDNA-AFLP is a genome-wide expression analysis technology that does not require any prior knowledge of gene sequences . This PCR-based technique combines a high sensitivity with a high specificity, allowing detection of rarely expressed genes and distinguishing between homologous genes . In this report, we validated quantitative expression data of 110 cDNA-AFLP fragments in yeast with DNA microarrays and GeneChip data . The best correlation was found between cDNA-AFLP and GeneChip data . The cDNA-AFLP data revealed a low number of inconsistent profiles that could be explained by gel artifact, overexposure, or mismatch amplification . In addition, 18 cDNA-AFLP fragments displayed homology to genomic yeast DNA, but could not be linked unambiguously to any known ORF . These fragments were most probably derived from 5' or 3' noncoding sequences or might represent previously unidentified ORFs . Genes liable to cross hybridization showed identical results in cDNA-AFLP and GeneChip analysis . Three genes, which were readily detected with cDNA-AFLP, showed no significant expression in GeneChip experiments . We show that cDNA-AFLP is a very good alternative to microarrays and since no preexisting biological or sequence information is required, it is applicable to any species.

J Biochem (Tokyo), 2003 Oct, 134(4), 607 - 13
Conversion of the aminocrotonate intermediate limits the rate of gamma-elimination reaction catalyzed by L-cystathionine gamma-lyase of the yeast Saccharomyces cerevisiae; Yamagata S et al.; L-Cystathionine gamma-lyase {EC 4.4.1.1} of Saccharomyces cerevisiae was shown to bind cofactor pyridoxal 5'-phosphate, up to 2 molecules/subunit . The association constants of the enzyme for the cofactor were estimated to be 3.67 x 10(5) M(-1) and 9.05 x 10(3) M(-1) . However, the latter value was too small for the binding to play a catalytic role . Changes in the absorption spectra of the enzyme in gamma-elimination reaction mixtures with various amino acids as substrates were observed at 10 degrees C to elucidate the reaction mechanism of the enzyme . The enzyme formed a chromophore exhibiting absorption at approximately 480 nm, which is characteristic of an aminocrotonate intermediate with O-succinyl-L-homoserine, L-cystathionine, L-homoserine, or O-acetyl-L-homoserine, at rates in this order . The intermediate was consumed at much lower rates than those of formation . The order of the rates of consumption was the same as the order of the formation rates and the order of the gamma-elimination activity of the enzyme with the above-mentioned substrates . These results strongly suggested that the intermediate was essential for gamma-elimination and that the reaction was rate-limited by its conversion into the product alpha-ketobutyrate . L-Cysteine sensitively inhibited the alpha, gamma-elimination activity of the enzyme, and also retarded the formation of the chromophore when it was provided to the enzyme together with a substrate . The reason for these phenomena is discussed.

J Biol Chem, 2004 Jan 23, 279(4), 2600 - 7 Epub 2003 Nov 07.
Reconstitution of the entry point of plant phenylpropanoid metabolism in yeast (Saccharomyces cerevisiae): implications for control of metabolic flux into the phenylpropanoid pathway; Ro DK et al.; Phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and the C4H redox partner cytochrome p450 reductase (CPR) are important in allocating significant amounts of carbon from phenylalanine into phenylpropanoid biosynthesis in plants . It has been proposed that multienzyme complexes (MECs) containing PAL and C4H are functionally important at this entry point into phenylpropanoid metabolism . To evaluate the MEC model, two poplar PAL isoforms presumed to be involved in either flavonoid (PAL2) or in lignin biosynthesis (PAL4) were independently expressed together with C4H and CPR in Saccharomyces cerevisiae, creating two yeast strains expressing either PAL2, C4H and CPR or PAL4, C4H and CPR . When {(3)H}Phe was fed, the majority of metabolized {(3)H}Phe was incorporated into p-{(3)H}coumarate, and Phe metabolism was highly reduced by inhibiting C4H activity . PAL alone expressers metabolized very little phenylalanine into cinnamic acid . To test for intermediate channeling between PAL and C4H, we fed {(3)H}Phe and {(14)C}cinnamate simultaneously to the triple expressers, but found no evidence for channeling of the endogenously synthesized {(3)H}cinnamate into p-coumarate . Therefore, efficient carbon flux from Phe to p-coumarate via reactions catalyzed by PAL and C4H does not appear to require channeling through a MEC in yeast, and instead biochemical coupling of PAL and C4H is sufficient to drive carbon flux into the phenylpropanoid pathway . This may be the primary mechanism by which carbon allocation into phenylpropanoid metabolism is controlled in plants.

Mol Genet Genomics, 2003 Dec, 270(4), 324 - 36 Epub 2003 Nov 07.
Activity of phosphoforms and truncated versions of Ndt80, a checkpoint-regulated sporulation-specific transcription factor of Saccharomyces cerevisiae; Shubassi G et al.; Ndt80 contributes to the highly regulated cascade of sequential gene expression that directs spore formation in Saccharomyces cerevisiae . This DNA-binding transcriptional activator, which is responsible for the expression of a set of middle sporulation-specific genes, is a target of the meiotic recombination checkpoint . Triggering of this checkpoint prevents phosphorylation and accumulation of active Ndt80 . In this study we have investigated the requirements for the activation function of Ndt80 by exploring the role of phosphorylation in the regulation of its activity and by examining the effect of C-terminal truncations . Of three phosphoforms of Ndt80 that we resolved, which we refer to as P approximately Ndt80", P approximately Ndt80', and P approximately Ndt80 in order of increasing electrophoretic mobility, the P approximately Ndt80" and P approximately Ndt80' isoforms correlated with active Ndt80 . In particular, P approximately Ndt80" was present in lysates from wild-type sporulating cells and in cells that bypassed checkpoint-mediated arrest as a result of mutations in RAD17, SUM1, or SWE1, or overexpression of NDT80 . P approximately Ndt80' was the slowest-migrating isoform that accumulated in Delta ime2/Delta ime2 Delta sum1/Delta sum1 cells in sporulation medium and in mitotic cells that ectopically expressed NDT80 . Nonphosphorylated Ndt80 and P approximately Ndt80, which had a slightly lower mobility than nonphosphorylated Ndt80 and was the predominant phosphoform present in checkpoint-arrested cells, correlated with inactive Ndt80 . These data are consistent with the notion that extensive phosphorylation, but not Ime2-dependent phosphorylation, of Ndt80 is required for its activity . Examination of the effect of increasingly extensive truncation of the C terminal region of Ndt80 revealed that some functions of Ndt80 were more sensitive to a reduction in its activity than others . In particular, we found that a truncated version of Ndt80 that lacked the last 110 residues was able to promote expression of some middle sporulation-specific genes, but could not direct spore formation . Full activity, however, could be restored to this version of Ndt80 by increasing its level of expression.

J Clin Microbiol, 2003 Nov, 41(11), 5340 - 3
Outbreak of Saccharomyces cerevisiae subtype boulardii fungemia in patients neighboring those treated with a probiotic preparation of the organism; Cassone M et al.; We report an outbreak of Saccharomyces cerevisiae subtype boulardii fungemia among three intensive care unit roommates of patients receiving lyophilized preparations of this fungus . The fungemia was probably due to central venous catheter contamination and resolved after fluconazole treatment . The need for stringent application of proper hygiene when using a probiotic preparation of this organism is emphasized.

Appl Environ Microbiol, 2003 Nov, 69(11), 6527 - 32
Gene dosage effect of L-proline biosynthetic enzymes on L-proline accumulation and freeze tolerance in Saccharomyces cerevisiae; Terao Y et al.; We have previously reported that L-proline has cryoprotective activity in Saccharomyces cerevisiae . A freeze-tolerant mutant with L-proline accumulation was recently shown to carry an allele of the PRO1 gene encoding gamma-glutamyl kinase, which resulted in a single amino acid substitution (Asp154Asn) . Interestingly, this mutation enhanced the activities of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase, both of which catalyze the first two steps of L-proline synthesis and which together may form a complex in vivo . Here, we found that the Asp154Asn mutant gamma-glutamyl kinase was more thermostable than the wild-type enzyme, which suggests that this mutation elevated the apparent activities of two enzymes through a stabilization of the complex . We next examined the gene dosage effect of three L-proline biosynthetic enzymes, including Delta(1)-pyrroline-5-carboxylate reductase, which converts Delta(1)-pyrroline-5-carboxylate into L-proline, on L-proline accumulation and freeze tolerance in a non-L-proline-utilizing strain . Overexpression of the wild-type enzymes has no influence on L-proline accumulation, which suggests that the complex is very unstable in nature . However, co-overexpression of the mutant gamma-glutamyl kinase and the wild-type gamma-glutamyl phosphate reductase was effective for L-proline accumulation, probably due to a stabilization of the complex . These results indicate that both enzymes, not Delta(1)-pyrroline-5-carboxylate reductase, are rate-limiting enzymes in yeast cells . A high tolerance for freezing clearly correlated with higher levels of L-proline in yeast cells . Our findings also suggest that, in addition to its cryoprotective activity, intracellular L-proline could protect yeast cells from damage by oxidative stress . The approach described here provides a valuable method for breeding novel yeast strains that are tolerant of both freezing and oxidative stresses.

Mol Genet Genomics, 2003 Dec, 270(4), 287 - 95 Epub 2003 Nov 05.
Twofold symmetries in nucleotide distribution in large domains of Saccharomyces cerevisiae Chromosome I; Conde J; Single stranded chains of biological DNA show a widespread occurrence of parity for complementary nucleotides, i.e., A=T, G=C . This has been referred to as A-T, G-C symmetry . A distinction must be made between this, which this paper calls mirror symmetry, and twofold symmetry, where complementary nucleotide parity occurs between two segments, of the same length and equidistant from a symmetry center, along a single-stranded DNA chain . I have analysed the sequence of Chromosome I of Saccharomyces cerevisiae for the occurrence of complementary nucleotide symmetry . Open reading frame (ORF) sequences made up 63% of the total chromosome length and most of them were asymmetric for both A-T and G-C . The sign of A-T asymmetry was correlated with transcriptional orientation (A>T for sense and A<T for antisense ORFs), whereas G-C asymmetry was not . However, long single-stranded segments of Chromosome I were A-T mirror symmetric because they contained similar frequencies of ORFs in both transcriptional orientations . The same results were obtained with the AA-TT pair of complementary dinucleotides . Profiling of AA-TT symmetry along Chromosome I showed this chromosome to be organized as a succession of five domains that were twofold symmetric for AA-TT, placed between two subtelomeric regions without clear symmetry properties . This pattern was destroyed when ORF sequences were randomly repositioned along the chromosome . Based on the above findings, an architectural model is proposed for Chromosome I, in which the twofold symmetric domains, from 30 to 50 kb long, correspond to chromosome loops.

Microbiology, 2003 Nov, 149(Pt 11), 3129 - 37
Chitin scar breaks in aged Saccharomyces cerevisiae; Powell CD et al.; Ageing in budding yeast is not determined by chronological lifespan, but by the number of times an individual cell is capable of dividing, termed its replicative capacity . As cells age they are subject to characteristic cell surface changes . Saccharomyces cerevisiae reproduces asexually by budding and as a consequence of this process both mother and daughter cell retain chitinous scar tissue at the point of cytokinesis . Daughter cells exhibit a frail structure known as the birth scar, while mother cells display a more persistent bud scar . The number of bud scars present on the cell surface is directly related to the number of times a cell has divided and thus constitutes a biomarker for replicative cell age . It has been proposed that the birth scar may be subject to stretching caused by expansion of the daughter cell; however, no previous analysis of the effect of cell age on birth or bud scar size has been reported . This paper provides evidence that scar tissue expands with the cell during growth . It is postulated that symmetrically arranged breaks in the bud scar allow these rigid chitinous structures to expand without compromising cellular integrity.

J Cell Biol, 2003 Nov 10, 163(3), 457 - 61 Epub 2003 Nov 03.
A function for the mitochondrial chaperonin Hsp60 in the structure and transmission of mitochondrial DNA nucleoids in Saccharomyces cerevisiae; Kaufman BA et al.; The yeast mitochondrial chaperonin Hsp60 has previously been implicated in mitochondrial DNA (mtDNA) transactions: it is found in mtDNA nucleoids associated with single-stranded DNA; it binds preferentially to the template strand of active mtDNA ori sequences in vitro; and wild-type (rho+) mtDNA is unstable in hsp60 temperature-sensitive (ts) mutants grown at the permissive temperature . Here we show that the mtDNA instability is caused by a defect in mtDNA transmission to daughter cells . Using high resolution, fluorescence deconvolution microscopy, we observe a striking alteration in the morphology of mtDNA nucleoids in rho+ cells of an hsp60-ts mutant that suggests a defect in nucleoid division . We show that rho- petite mtDNA consisting of active ori repeats is uniquely unstable in the hsp60-ts mutant . This instability of ori rho- mtDNA requires transcription from the canonical promoter within the ori element . Our data suggest that the nucleoid dynamics underlying mtDNA transmission are regulated by the interaction between Hsp60 and mtDNA ori sequences.

Exp Cell Res, 2003 Nov 15, 291(1), 46 - 55
Reduction of Htt inclusion formation in strains of Saccharomyces cerevisiae deficient in certain DNA repair functions: a statistical analysis of phenotype; Hu Y et al.; Saccharomyces cerevisiae has been used as a model system to examine the aggregation of the huntingtin protein (Htt), a well-established marker in the pathology of the triplet expansion disorder Huntington's disease (HD) . Several genetic backgrounds, such as Deltahsp104, have proven to be refractory to inclusion formation through a process yet to be fully elucidated . These results have prompted a wide-ranging search for other mutant strains that exhibit a lower level of Htt aggregation . A novel assay system in which Htt is expressed as a fusion protein containing eGFP enables an analysis of aggregation and the factors that suppress it . We have examined several strains that are devoid of certain mismatch repair genes and find that some of these support a reduced level of inclusion body formation . We apply a detailed and stringent statistical analysis to the results obtained for all yeast strains that exhibit a definable phenotype . Such analyses should be useful and applicable to other in vivo analyses of related phenomena.

Vet Immunol Immunopathol, 2003 Dec 15, 96(3-4), 183 - 92
Immunostimulant properties of a cell wall-modified whole Saccharomyces cerevisiae strain administered by diet to seabream (Sparus aurata L.); Rodriguez A et al.; The possible differences in the immunostimulant effects between whole wild Saccharomyces cerevisiae and a mutant strain (fks-1) administered in the diet to gilthead seabream (Sparus aurata L.) were studied . Fish were fed diets containing 0 (control) or 10 g whole wild yeast or fks-1 strain per kilogram feed for 2, 4 or 6 weeks . After each sampling, the innate humoral (complement, lysozyme and peroxidases) and cellular (intracellular peroxidases, phagocytosis, respiratory burst activity and cytotoxicity) responses were determined . Among the humoral responses, serum peroxidases and complement activity were significantly decreased after 6 weeks of feeding with the fks-1 strain-supplemented diet, while lysozyme activity increased after 2 and 4 weeks of feeding the fks-1 strain . Of the cellular responses studied, phagocytosis was increased to a significant degree at all the assayed times but only by the fks-1 strain-supplemented diet, while respiratory burst activity (after 4 weeks) and natural cytotoxicity (after 4 and 6 weeks) increased with either yeast strain . The intracellular peroxidase content was not affected by the dietary supplementation of either yeast strain . These results are discussed with a view to assessing the possible use of wild and/or mutant yeast strains for immunostimulant purposes in aquaculture.

Mol Cell Biol, 2003 Nov, 23(22), 8386 - 94
Origin of endogenous DNA abasic sites in Saccharomyces cerevisiae; Guillet M et al.; Abasic (AP) sites are among the most frequent endogenous lesions in DNA and present a strong block to replication . In Saccharomyces cerevisiae, an apn1 apn2 rad1 triple mutant is inviable because of its incapacity to repair AP sites and related 3'-blocked single-strand breaks (M . Guillet and S . Boiteux, EMBO J . 21:2833, 2002) . Here, we investigated the origin of endogenous AP sites in yeast . Our results show that the deletion of the UNG1 gene encoding the uracil DNA glycosylase suppresses the lethality of the apn1 apn2 rad1 mutant . In contrast, inactivation of the MAG1, OGG1, or NTG1 and NTG2 genes encoding DNA glycosylases involved in the repair of alkylation or oxidation damages does not suppress lethality . Although viable, the apn1 apn2 rad1 ung1 mutant presents growth delay due to a G(2)/M checkpoint . These results point to uracil as a critical source of the formation of endogenous AP sites in DNA . Uracil can arise in DNA by cytosine deamination or by the incorporation of dUMP during replication . Here, we show that the overexpression of the DUT1 gene encoding the dUTP pyrophosphatase (Dut1) suppresses the lethality of the apn1 apn2 rad1 mutant . Therefore, this result points to the dUTP pool as an important source of the formation of endogenous AP sites in eukaryotes.

J Biol Chem, 2004 Jan 9, 279(2), 1020 - 9 Epub 2003 Oct 28.
Protein sorting in the late Golgi of Saccharomyces cerevisiae does not require mannosylated sphingolipids; Lisman Q et al.; Glycosphingolipids are widely viewed as integral components of the Golgi-based machinery by which membrane proteins are targeted to compartments of the endosomal/lysosomal system and to the surface domains of polarized cells . The yeast Saccharomyces cerevisiae creates glycosphingolipids by transferring mannose to the head group of inositol phosphorylceramide (IPC), yielding mannosyl-IPC (MIPC) . Addition of an extra phosphoinositol group onto MIPC generates mannosyldi-IPC (M(IP)2C), the final and most abundant sphingolipid in yeast . Mannosylation of IPC is partially dependent on CSG1, a gene encoding a putative sphingolipidmannosyltransferase . Here we show that open reading frame YBR161w, renamed CSH1, is functionally homologous to CSG1 and that deletion of both genes abolishes MIPC and M(IP)2C synthesis without affecting protein mannosylation . Csg1p and Csh1p are closely related polytopic membrane proteins that co-localize with IPC synthase in the medial-Golgi . Loss of Csg1p and Csh1p has no effect on clathrin- or AP-3 adaptor-mediated protein transport from the Golgi to the vacuole . Moreover, segregation of the periplasmic enzyme invertase, the plasma membrane ATPase Pma1p and the glycosylphosphatidylinositol-anchored protein Gas1p into distinct classes of secretory vesicles occurs independently of Csg1p and Csh1p . Our results indicate that protein sorting in the late Golgi of yeast does not require production of mannosylated sphingolipids.

J Biol Chem, 2004 Jan 30, 279(5), 3651 - 61 Epub 2003 Oct 28.
Evidence for antagonistic regulation of cell growth by the calcineurin and high osmolarity glycerol pathways in Saccharomyces cerevisiae; Shitamukai A et al.; Because Ca(2+) signaling of budding yeast, through the activation of calcineurin and the Mpk1/Slt2 mitogen-activated protein kinase cascade, performs redundant function(s) in the events essential for growth, the simultaneous deletion of both these pathways (Delta cnb1 Delta mpk1) leads to lethality . A PTC4 cDNA that encodes a protein phosphatase belonging to the PP2C family was obtained as a high dosage suppressor of the lethality of Delta cnb1 Delta mpk1 strain . Overexpression of PTC4 led to a decrease in the high osmolarity-induced Hog1 phosphorylation, and HOG1 deletion remarkably suppressed the synthetic lethality, indicating an antagonistic role of the high osmolarity glycerol (HOG) pathway and the Ca(2+) signaling pathway in growth regulation . The calcineurin-Crz1 pathway was required for the down-regulation of the HOG pathway . Analysis of the time course of actin polarization, bud formation, and the onset of mitosis in synchronous cell cultures demonstrated that calcineurin negatively regulates actin polarization at the bud site, whereas the HOG pathway positively regulates bud formation at a later step after actin has polarized.

Mol Cell Biol, 1982 Dec, 2(12), 1532 - 49
Identification of proteins whose synthesis is modulated during the cell cycle of Saccharomyces cerevisiae; Lorincz AT et al.; We examined the synthesis and turnover of individual proteins in the Saccharomyces cerevisiae cell cycle . Proteins were pulse-labeled with radioactive isotope (35S or 14C) in cells at discrete cycle stages and then resolved on two-dimensional gels and analyzed by a semiautomatic procedure for quantitating gel electropherogram-autoradiographs . The cells were obtained by one of three methods: (i) isolation of synchronous subpopulations of growing cells by zonal centrifugation.; (ii) fractionation of pulse-labeled steady-state cultures according to cell age; and (iii) synchronization of cells with the mating pheromone, alpha-factor . In confirmation of previous studies, we found that the histones H4, H2A, and H2B were synthesized almost exclusively in the late G1 and early S phases . In addition, we identified eight proteins whose rates of synthesis were modulated in the cell cycle, and nine proteins (of which five, which may well be related, were unstable, with half-lives of 10 to 15 min) that might be regulated in the cell cycle by periodic synthesis, modification, or degradation . Based on the time of maximal labeling in the cell cycle and on experiments with alpha-factor and hydroxyurea, we assigned the cell cycle proteins to two classes: proteins in class I were labeled principally in early G1 phase and at a late stage of the cycle, whereas those in class II were primarily synthesized at times ranging from late G1 to mid S phase . At least one major control point for the cell cycle proteins occurred between "start" and early S phase . A set of stress-responsive proteins was also identified and analyzed . The rates of synthesis of these proteins were affected by certain perturbations that resulted during selection of synchronous cell populations and by heat shock.

Mol Cell Biol, 1982 Dec, 2(12), 1514 - 23
Isolation of the CAR1 gene from Saccharomyces cerevisiae and analysis of its expression; Sumrada RA et al.; We isolated the CAR1 gene from Saccharomyces cerevisiae on a recombinant plasmid and localized it to a 1.58-kilobase DNA fragment . The cloned gene was used as a probe to analyze polyadenylated RNA derived from wild-type and mutant cells grown in the presence and absence of an inducer . Wild-type cells grown without the inducer contained very little polyadenylated RNA capable of hybridizing to the isolated CAR1 gene . A 1.25-kilobase CAR1-specific RNA species was markedly increased, however, in wild-type cells grown in the presence of inducer and in constitutive, regulatory mutants grown without it . No CAR1-specific RNA was observed when one class of constitutive mutant was grown in medium containing a good nitrogen source, such as asparagine . Two other mutants previously shown to be resistant to nitrogen repression contained large quantities of CAR1 RNA regardless of the nitrogen source in the medium . These data point to a qualitative correlation between the steady-state levels of CAR1-specific, polyadenylated RNA and the degree of arginase induction and repression observed in the wild type and in strains believed to carry regulatory mutations . Therefore, they remain consistent with our earlier suggestion that arginase production is probably controlled at the level of gene expression.

Mol Cell Biol, 1982 Dec, 2(12), 1501 - 13
Mutations at the Saccharomyces cerevisiae SUP4 tRNA(Tyr) locus: isolation, genetic fine-structure mapping, and correlation with physical structure; Kurjan J et al.; The SUP4 tRNA(Tyr) locus in Saccharomyces cerevisiae has been studied by the isolation and characterization of mutations at the SUP4 gene which result in the loss of suppressor function . Most of the mutations act as single-site mutations, whereas about a third of the mutations are deletions of the entire gene . Two meiotic fine-structure maps of the gene were made . The first mapping technique placed 10 mutations plus the sup4+ anticodon on a map by a measurement of levels of recombination between pairs of mutations . The second map utilized a more qualitative estimate of recombination frequency, allowing 69 mutations and the sup4+ anticodon to be mapped . The maps were compared with the physical structure of the gene for the 34 mutations whose nucleotide alteration has been determined by DNA sequencing (Koski et al., Cell 22:415-425, 1980; Kurjan et al., Cell 20:701-709, 1980) . Both maps show a good correlation with the physical structure of the gene, even though certain properties of genetic fine-structure maps, such as marker effects and "map expansion," were seen.

Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13207 - 12 Epub 2003 Oct 23.
The proteome of Saccharomyces cerevisiae mitochondria; Sickmann A et al.; We performed a comprehensive approach to determine the proteome of Saccharomyces cerevisiae mitochondria . The proteins of highly pure yeast mitochondria were separated by several independent methods and analyzed by tandem MS . From >20 million MS spectra, 750 different proteins were identified, indicating an involvement of mitochondria in numerous cellular processes . All known components of the oxidative phosphorylation machinery, the tricarboxylic acid cycle, and the stable mitochondria-encoded proteins were found . Based on the mitochondrial proteins described in the literature so far, we calculate that the identified proteins represent approximately 90% of all mitochondrial proteins . The function of a quarter of the identified proteins is unknown . The mitochondrial proteome will provide an important database for the analysis of new mitochondrial and mitochondria-associated functions and the characterization of mitochondrial diseases.

J Biol Chem, 2004 Jan 23, 279(4), 2817 - 24 Epub 2003 Oct 23.
Molecular basis for atovaquone resistance in Pneumocystis jirovecii modeled in the cytochrome bc(1) complex of Saccharomyces cerevisiae; Kessl JJ et al.; Atovaquone is a substituted hydroxynaphthoquinone that is widely used to prevent and clear Plasmodium falciparum malaria and Pneumocystis jirovecii pneumonia . Atovaquone inhibits respiration in target organisms by specifically binding to the ubiquinol oxidation site at center P of the cytochrome bc(1) complex . The failure of atovaquone treatment and mortality of patients with malaria and P . jirovecii pneumonia has been linked to the appearance of mutations in the cytochrome b gene . To better understand the molecular basis of atovaquone resistance, we have introduced seven of the mutations from atovaquone-resistant P . jirovecii into the cytochrome b gene of Saccharomyces cerevisiae and thus obtained cytochrome bc(1) complexes resistant to inhibition by atovaquone . In these enzymes, the IC(50) for atovaquone increases from 25 nm for the enzyme from wild-type yeast to >500 nm for some of the mutated enzymes . Modeling of the changes in cytochrome b structure and atovaquone binding with the mutated bc(1) complexes provides the first quantitative explanation for the molecular basis of atovaquone resistance.

Biotechnol Bioeng, 2003 Dec 5, 84(5), 573 - 82
Efficient anaerobic whole cell stereoselective bioreduction with recombinant Saccharomyces cerevisiae; Katz M et al.; In this study we investigate the NADPH-dependent stereoselective reduction of the bicyclic diketone bicyclo{2.2.2}octane-2,6-dione (BCO2,6D) to the chiral ketoalcohol (1R,4S,6S)-6-hydroxybicyclo{2.2.2}octane-2-one (BCO2one6ol) . Our aim was to develop a whole cell batch process for reduction of carbonyl substrates with (i) a high cosubstrate yield (formed product/consumed cosubstrate) and (ii) a high conversion rate under anaerobic conditions with Saccharomyces cerevisiae as biocatalyst and glucose as cosubstrate . Five open reading frames (ORFs), YMR226c, YDR368w, YOR120w, YGL157w, and YGL039w, encoding reductases involved in the conversion of BCO2,6D were identified using cell-free extract from strains belonging to the ExClone collection (yeast ORF expression clones; ResGen, Invitrogen Corp., UK) . We report the one-step purification and characterization of three major BCO2,6D reductases, YMR226cp, YDR368wp (YPR1p), and YOR120wp (GCY1p) . The reductases were overexpressed under a strong constitutive promoter and the impact on cosubstrate yield, conversion time, glucose consumption rate, and reduction rate was investigated when reductases were overexpressed either alone or in combination with low phosphoglucose isomerase activity (encoded by YBR196c) . Combining overexpression of BCO2,6D reductase with reduced glycolytic rate (low phosphoglucose isomerase activity) offers a fast whole cell stereoselective bioreduction system useful for facilitated anaerobic batch conversions .

J Biol Chem, 2004 Jan 2, 279(1), 619 - 25 Epub 2003 Oct 21.
Crystal structure of the bifunctional chorismate synthase from Saccharomyces cerevisiae; Quevillon-Cheruel S et al.; Chorismate synthase (EC 4.2.3.5), the seventh enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate, which is the last common precursor in the biosynthesis of numerous aromatic compounds in bacteria, fungi, and plants . The chorismate synthase reaction involves a 1,4-trans-elimination of phosphoric acid from EPSP and has an absolute requirement for reduced FMN as a cofactor . We have determined the three-dimensional x-ray structure of the yeast chorismate synthase from selenomethionine-labeled crystals at 2.2-A resolution . The structure shows a novel betaalphabetaalpha fold consisting of an alternate tight packing of two alpha-helical and two beta-sheet layers, showing no resemblance to any documented protein structure . The molecule is arranged as a tight tetramer with D2 symmetry, in accordance with its quaternary structure in solution . Electron density is missing for 23% of the amino acids, spread over sequence regions that in the three-dimensional structure converge on the surface of the protein . Many totally conserved residues are contained within these regions, and they probably form a structured but mobile domain that closes over a cleft upon substrate binding and catalysis . This hypothesis is supported by previously published spectroscopic measurements implying that the enzyme undergoes considerable structural changes upon binding of both FMN and EPSP.

Genetics, 2003 Oct, 165(2), 929 - 34
A function for subtelomeric DNA in Saccharomyces cerevisiae; Barton AB et al.; The subtelomeric DNA sequences from chromosome I of Saccharomyces cerevisiae are shown to be inherently poor substrates for meiotic recombination . On the basis of these results and prior observations that crossovers near telomeres do not promote efficient meiosis I segregation, we suggest that subtelomeric sequences evolved to prevent recombination from occurring where it cannot promote efficient segregation.

Genetics, 2003 Oct, 165(2), 467 - 76
The G1 cyclin Cln3p controls vacuolar biogenesis in Saccharomyces cerevisiae; Han BK et al.; How organelle biogenesis and inheritance is linked to cell division is poorly understood . In the budding yeast Saccharomyces cerevisiae the G(1) cyclins Cln1,2,3p control initiation of cell division . Here we show that Cln3p controls vacuolar (lysosomal) biogenesis and segregation . First, loss of Cln3p, but not Cln1p or Cln2p, resulted in vacuolar fragmentation . Although the vacuoles of cln3delta cells were fragmented, together they occupied a large space, which accounted for a significant fraction of the overall cell size increase in cln3delta cells . Second, cytosol prepared from cells lacking Cln3p had reduced vacuolar homotypic fusion activity in cell-free assays . Third, vacuolar segregation was perturbed in cln3delta cells . Our findings reveal a novel role for a eukaryotic G(1) cyclin in cytoplasmic organelle biogenesis and segregation.

Genetics, 2003 Oct, 165(2), 457 - 66
Recombinogenic effects of DNA-damaging agents are synergistically increased by transcription in Saccharomyces cerevisiae . New insights into transcription-associated recombination; Garcia-Rubio M et al.; Homologous recombination of a particular DNA sequence is strongly stimulated by transcription, a phenomenon observed from bacteria to mammals, which we refer to as transcription-associated recombination (TAR) . TAR might be an accidental feature of DNA chemistry with important consequences for genetic stability . However, it is also essential for developmentally regulated processes such as class switching of immunoglobulin genes . Consequently, it is likely that TAR embraces more than one mechanism . In this study we tested the possibility that transcription induces recombination by making DNA more susceptible to recombinogenic DNA damage . Using different plasmid-chromosome and direct-repeat recombination constructs in which transcription is driven from either the P(GAL1)- or the P(tet)-regulated promoters, we have shown that either 4-nitroquinoline-N-oxide (4-NQO) or methyl methanesulfonate (MMS) produces a synergistic increase of recombination when combined with transcription . 4-NQO and MMS stimulated recombination of a transcriptionally active DNA sequence up to 12,800- and 130-fold above the spontaneous levels observed in the absence of transcription, whereas 4-NQO and MMS alone increased recombination 193- and 4.5-fold, respectively . Our results provide evidence that TAR is due, at least in part, to the ability of transcription to enhance the accessibility of DNA to exogenous chemicals and internal metabolites responsible for recombinogenic lesions . We discuss possible parallelisms between the mechanisms of induction of recombination and mutation by transcription.

Physiol Genomics, 2003 Dec 16, 16(1), 107 - 18
Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/cAMP pathway; Jones DL et al.; Often changes in gene expression levels have been considered significant only when above/below some arbitrarily chosen threshold . We investigated the effect of applying a purely statistical approach to microarray analysis and demonstrated that small changes in gene expression have biological significance . Whole genome microarray analysis of a pde2Delta mutant, constructed in the Saccharomyces cerevisiae reference strain FY23, revealed altered expression of approximately 11% of protein encoding genes . The mutant, characterized by constitutive activation of the Ras/cAMP pathway, has increased sensitivity to stress, reduced ability to assimilate nonfermentable carbon sources, and some cell wall integrity defects . Applying the Munich Information Centre for Protein Sequences (MIPS) functional categories revealed increased expression of genes related to ribosome biogenesis and downregulation of genes in the cell rescue, defense, cell death and aging category, suggesting a decreased response to stress conditions . A reduced level of gene expression in the unfolded protein response pathway (UPR) was observed . Cell wall genes whose expression was affected by this mutation were also identified . Several of the cAMP-responsive orphan genes, upon further investigation, revealed cell wall functions; others had previously unidentified phenotypes assigned to them . This investigation provides a statistical global transcriptome analysis of the cellular response to constitutive activation of the Ras/cAMP pathway.

J Biol Chem, 2003 Dec 26, 278(52), 52537 - 45 Epub 2003 Oct 21.
Synthesis and biophysical characterization of a multidomain peptide from a Saccharomyces cerevisiae G protein-coupled receptor; Naider F et al.; We attached peptides corresponding to the seventh transmembrane domain (TMD7) of the alpha-mating factor receptor (Ste2p) of Saccharomyces cerevisiae to a hydrophilic, 40-residue fragment of the carboxyl terminus of this G protein-coupled receptor . Peptides corresponding to (a) the 40-residue portion of the carboxyl tail (T-40), (b) the tail plus a part of TMD7 (M7-12-T40), and (c) to the tail plus the full TMD7 (M7-24-T40) were chemically synthesized and purified . The molecular mass and primary sequence of these peptides were confirmed by mass spectrometry and tandem mass spectrometry procedures . Circular dichroism (CD) revealed that T-40 was disordered in phosphate buffer and in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-{phospho-racemic-(1-glycerol)} bilayers . In contrast, M7-12-T40 and M7-24-T40 peptides were partially helical in the presence of vesicles, and difference CD spectroscopy showed that the transmembrane regions of these peptides were 42 and 94% helical, respectively . CD analysis also demonstrated that M7-24-T40 retained its secondary structure in the presence of 1-palmitoyl-2-hydroxy-sn-glycero-3-{phospho-racemic-(1-glycerol)} micelles at 0.5 mm concentration . Thus, the tail and the transmembrane domain of the multidomain 64-amino acid residue peptide manifest individual conformational preferences . Measurement of tryptophan fluorescence indicated that the transmembrane domain integrated into bilayers in a manner similar to that expected for this region in the native state of the receptor . This study demonstrated that the tail of Ste2p can be used as a hydrophilic template to study transmembrane domain structure using techniques such as CD and NMR spectroscopy.

Curr Microbiol, 2003 Sep, 47(3), 192 - 7
Overexpression of peroxisomal malate dehydrogenase MDH3 gene enhances cell death on H2O2 stress in the ald5 mutant of Saccharomyces cerevisiae; Kurita O; Mitochondrial aldehyde dehydrogenase ALD5 of Saccharomyces cerevisiae is involved in the biosynthesis of mitochondrial electron transport chain, and the ald5 mutant is incompetent for respiration . With use of the mutant, we examined the detoxication of H2O2 generation by fatty acid beta-oxidation in peroxisome . The ald5 mutant (AKD321), as well as the 746 rho0 mutant, was more resistant to H2O2 stress than the wild type . However, overexpression of the MDH3 gene that was involved in the reoxidation of NADH during fatty acid beta-oxidation caused a decrease in cell viability of AKD321 to H2O2 stress, while the 746 rho0 mutant had no such effect . Intracellular H2O2 concentration increased approximately fourfold in MDH3 overexpressing ald5 strain (MD3-AKD321), compared with AKD321 . The peroxisomal catalase activity of MD3-AKD321 decreased by 83% to that of AKD321 . And also, the overexpression of MDH3 had only a weak effect in MDH3 overexpressing 746 rho0 strain, decreasing by 14% to that of 746 rho0 mutant . The increased palmitoyl CoA oxidation by overexpression of MDH3 gene was the same in both strains . Under conditions of MDH3 overexpression, peroxisomal catalase (CTA1) appears to be a limiting factor to oxidative stress . These observations point to an important, as yet unidentified, role of mitochondrial aldehyde dehydrogenase (ALD5) to endogeneous oxidative stress in peroxisome.

World J Gastroenterol, 2003 Oct, 9(10), 2308 - 12
Seroreactivity against Saccharomyces cerevisiae in patients with Crohn's disease and celiac disease; Barta Z et al.; AIM: To explore whether there was anti-Saccharomyces cerevisiae antibodies (ASCA) positivity in our patients with biopsy-confirmed celiac disease . METHODS: A cohort of patients with inflammatory bowel diseases (42 patients with Crohn's disease and 10 patients with ulcerative colitis) and gluten sensitive enteropathy (16 patients) from Debrecen, Hungary were enrolled in the study . The diagnosis was made using the formally accepted criteria . Perinuclear antineutrophil cytoplasmic antibodies (pANCA) and anti-Saccharomyces cerevisiae antibodies (ASCA), antiendomysium antibodies (EMA), antigliadin antibodies (AGA) and anti human tissue transglutaminase antibodies (tTGA) were investigated . RESULTS: The results showed that ASCA positivity occurred not only in Crohn's disease but also in Celiac disease and in these cases both the IgG and IgA type antibodies were proved . CONCLUSION: It is conceivable that ASCA positivity correlates with the (auto-) immune inflammation of small intestines and it is a specific marker of Crohn's disease.

RNA, 2003 Nov, 9(11), 1371 - 82
The Saccharomyces cerevisiae U2 snRNA:pseudouridine-synthase Pus7p is a novel multisite-multisubstrate RNA:Psi-synthase also acting on tRNAs; Behm-Ansmant I et al.; The Saccharomyces cerevisiae Pus7 protein was recently characterized as a novel RNA:pseudouridine (Psi)-synthase acting at position 35 in U2 snRNA . However, U2 snRNA was the only potential substrate tested for this enzyme . In this work, we demonstrated that although Pus7p is responsible for the formation of only one of the six Psi residues present in yeast UsnRNAs, it catalyzes U to Psi conversion at position 13 in cytoplasmic tRNAs and at position 35 in pre-tRNA(Tyr) . Sites of RNA modification by Pus7p were identified by analysis of the in vivo RNA modification defects resulting from the absence of active Pus7p production and by in vitro tests using extracts from WT and genetically modified yeast cells . For demonstration of the direct implication of Pus7p in RNA modification, the activity of the WT and mutated Pus7p recombinant proteins was tested on in vitro produced tRNA and pre-tRNA transcripts . Mutation of an aspartic acid residue (D256) that is conserved in all Pus7 homologs abolishes the enzymatic activity both in vivo and in vitro . This suggests the direct involvement of D256 in catalysis . Target sites of Pus7p in RNAs share a common sequence Pu(G/C)UNPsiAPu (Pu = purine, N = any nucleotide), which is expected to be important for substrate recognition . Modification of tRNAs by Pus7p explains the presence of Pus7p homologs in archaea and some bacteria species, which do not have U2 snRNA, and in vertebrates, where Psi34 (equivalent to Psi35 in yeast) formation in U2 snRNA is an H/ACA snoRNA guided process . Our results increase the number of known RNA modification enzymes acting on different types of cellular RNAs.

Genome Res, 2003 Nov, 13(11), 2450 - 4 Epub 2003 Oct 14.
Bioinformatics analysis of experimentally determined protein complexes in the yeast Saccharomyces cerevisiae; Dezso Z et al.; Many important cellular functions are implemented by protein complexes that act as sophisticated molecular machines of varying size and temporal stability . Here we demonstrate quantitatively that protein complexes in the yeast Saccharomyces cerevisiae are comprised of a core in which subunits are highly coexpressed, display the same deletion phenotype (essential or nonessential), and share identical functional classification and cellular localization . This core is surrounded by a functionally mixed group of proteins, which likely represent short-lived or spurious attachments . The results allow us to define the deletion phenotype and cellular task of most known complexes, and to identify with high confidence the biochemical role of hundreds of proteins with yet unassigned functionality.

Yeast, 2003 Oct 15, 20(13), 1145 - 50
Expression in Escherichia coli of a recombinant adenosine kinase from Saccharomyces cerevisiae: purification, kinetics and substrate analyses; Barrado P et al.; The Saccharomyces cerevisiae ADO1 gene is known to encode a homologue of eukaryotic adenosine kinases . This gene was expressed in Escherichia coli as a recombinant protein fused to a polyhistidine tag by using the rhamnose-inducible bacterial promoter rhaB . The recombinant protein was purified to apparent homogeneity and its ability to phosphorylate different substrates was evaluated . Adenosine (Km 3 microM) is its primary substrate . In addition, it also phosphorylates, albeit less efficiently, 3'-deoxyadenosine (cordycepin; Km 1.84 mM) and 3'-amino-3'-deoxyadenosine (Km 0.26 mM) . Other kinetic properties of the recombinant enzyme have also been determined .

Yeast, 2003 Oct 15, 20(13), 1085 - 96
MIG1-dependent and MIG1-independent regulation of GAL gene expression in Saccharomyces cerevisiae: role of Imp2p; Alberti A et al.; Imp2p (Yil154c) is a transcriptional activator involved in glucose derepression of the maltose, galactose and raffinose utilization pathways and in resistance to thermal, oxidative or osmotic stress . We analysed the role of Imp2 in the regulation of GAL genes . Imp2 was shown to have a positive effect on glucose derepression of Leloir pathway genes and their activator gene GAL4 . The effect of Imp2 on galactose metabolism was shown to be partially dependent on Mig1p . The Mig1-independent role depends on Nrg1p . However, disruption of both MIG1 and NRG1 only partially relieves the glucose repression of GAL genes in the Deltaimp2 mutant, indicating that Imp2 must also have other function(s) . Moreover, the interaction between IMP2 and GAL6/BLH1, a recently isolated gene involved in the regulation of GAL genes that shares with Imp2 the ability to protect cells from the glycopeptide bleomycin, was also analysed . The results suggest a major role of Imp2 in a GAL6-independent pathway .

Biochemistry, 2003 Oct 21, 42(41), 12038 - 49
Topological and functional study of subunit h of the F1Fo ATP synthase complex in yeast Saccharomyces cerevisiae; Fronzes R et al.; Subunit h, a 92-residue-long, hydrophilic, acidic protein, is a component of the yeast mitochondrial F1Fo ATP synthase . This subunit, homologous to the mammalian factor F6, is essential for the correct assembly and/or functioning of this enzyme since yeast cells lacking it are not able to grow on nonfermentable carbon sources . Chemical cross-links between subunit h and subunit 4 have previously been shown, suggesting that subunit h is a component of the peripheral stalk of the F1Fo ATP synthase . The construction of cysteine-containing subunit h mutants and the use of bismaleimide reagents provided insights into its environment . Cross-links were obtained between subunit h and subunits alpha, f, d, and 4 . These results and secondary structure predictions allowed us to build a structural model and to propose that this subunit occupies a central place in the peripheral stalk between the F1 sector and the membrane . In addition, subunit h was found to have a stoichiometry of one in the F1Fo ATP synthase complex and to be in close proximity to another subunit h belonging to another F1Fo ATP synthase in the inner mitochondrial membrane . Finally, functional characterization of mitochondria from mutants expressing different C-terminal shortened subunit h suggested that its C-terminal part is not essential for the assembly of a functional F1Fo ATP synthase.

J Biol Chem, 2004 Jan 2, 279(1), 95 - 102 Epub 2003 Oct 10.
Riboflavin uptake and FAD synthesis in Saccharomyces cerevisiae mitochondria: involvement of the Flx1p carrier in FAD export; Bafunno V et al.; We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf) . Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of (at least two) carrier systems . FAD was synthesized inside mitochondria by a mitochondrial FAD synthetase (EC 2.7.7.2), and it was exported into the cytosol via an export system that was inhibited by lumiflavin, and which was different from the riboflavin uptake system . To understand the role of the putative mitochondrial FAD carrier, Flx1p, in this pathway, an flx1Delta mutant strain was constructed . Coupled mitochondria isolated from flx1Delta mutant cells were compared with wild-type mitochondria with respect to the capability to take up Rf, to synthesize FAD from it, and to export FAD into the extramitochondrial phase . Mitochondria isolated from flx1Delta mutant cells specifically lost the ability to export FAD, but did not lose the ability to take up Rf, FAD, or FMN and to synthesize FAD from Rf . Hence, Flx1p is proposed to be the mitochondrial FAD export carrier . Moreover, deletion of the FLX1 gene resulted in a specific reduction of the activities of mitochondrial lipoamide dehydrogenase and succinate dehydrogenase, which are FAD-binding enzymes . For the flavoprotein subunit of succinate dehydrogenase we could demonstrate that this was not due to a changed level of mitochondrial FAD or to a change in the degree of flavinylation of the protein . Instead, the amount of the flavoprotein subunit of succinate dehydrogenase was strongly reduced, indicating an additional regulatory role for Flx1p in protein synthesis or degradation.

Eukaryot Cell, 2003 Oct, 2(5), 962 - 70
Ask10p mediates the oxidative stress-induced destruction of the Saccharomyces cerevisiae C-type cyclin Ume3p/Srb11p; Cohen TJ et al.; Srb11p-Srb10p is the budding yeast C-type cyclin-cyclin-dependent kinase that is required for the repression of several stress response genes . To relieve this repression, Srb11p is destroyed in cells exposed to stressors, including heat shock and oxidative stress . In the present study, we identified Ask10p (for activator of Skn7) by two-hybrid analysis as an interactor with Srb11p . Coimmunoprecipitation studies confirmed this association, and we found that, similar to Srb11p-Srb10p, Ask10p is a component of the RNA polymerase II holoenzyme . Ask10p is required for Srb11p destruction in response to oxidative stress but not heat shock . Moreover, this destruction is important since the hypersensitivity of an ask10 mutant strain to oxidative stress is rescued by deleting SRB11 . We further show that Ask10p is phosphorylated in response to oxidative stress but not heat shock . This modification requires the redundant mitogen-activated protein (MAP) kinase kinase Mkk1/2 but not their normal MAP kinase target Slt2p . Moreover, the other vegetative MAP kinases--Hog1p, Fus3p, or Kss1p--are not required for Ask10p phosphorylation, suggesting the existence of an alternative pathway for transducing the Pkc1p-->Bck1-->Mkk1/2 oxidative stress signal . In conclusion, Ask10p is a new component of the RNA polymerase II holoenzyme and an important regulator of the oxidative stress response . In addition, these results define a new role for the Pkc1p MAP kinase cascade (except the MAP kinase itself) in transducing the oxidative damage signal directly to the RNA polymerase II holoenzyme, thereby bypassing the stress-activated transcription factors.

Eukaryot Cell, 2003 Oct, 2(5), 949 - 61
Phosphorylation of the MAPKKK regulator Ste50p in Saccharomyces cerevisiae: a casein kinase I phosphorylation site is required for proper mating function; Wu C et al.; The Ste50 protein of Saccharomyces cerevisiae is a regulator of the Ste11p protein kinase . Ste11p is a member of the MAP3K (or MEKK) family, which is conserved from yeast to mammals . Ste50p is involved in all the signaling pathways that require Ste11p function, yet little is known about the regulation of Ste50p itself . Here, we show that Ste50p is phosphorylated on multiple serine/threonine residues in vivo . Threonine 42 (T42) is phosphorylated both in vivo and in vitro, and the protein kinase responsible has been identified as casein kinase I . Replacement of T42 with alanine (T42A) compromises Ste50p function . This mutation abolishes the ability of overexpressed Ste50p to suppress either the mating defect of a ste20 ste50 deletion mutant or the mating defect of a strain with a Ste11p deleted from its sterile-alpha motif domain . Replacement of T42 with a phosphorylation-mimetic aspartic acid residue (T42D) permits wild-type function in all assays of Ste50p function . These results suggest that phosphorylation of T42 of Ste50p is required for proper signaling in the mating response . However, this phosphorylation does not seem to have a detectable role in modulating the high-osmolarity glycerol synthesis pathway.

Eukaryot Cell, 2003 Oct, 2(5), 922 - 9
Constitutive and hyperresponsive signaling by mutant forms of Saccharomyces cerevisiae amino acid sensor Ssy1; Gaber RF et al.; Sensing of extracellular amino acids results in transcriptional induction of amino acid permease genes in yeast . Ssy1, a membrane protein resembling amino acid permeases, is required for signaling but is apparently unable to transport amino acids and is thus believed to be a sensor . By using a novel genetic screen in which potassium uptake was made dependent on amino acid signaling, we obtained gain-of-function mutations in SSY1 . Some alleles confer inducer-independent signaling; others increase the apparent affinity for inducers . The results reveal that amino acid transport is not required for signaling and support the notion that sensing by Ssy1 occurs via its direct interaction with extracellular amino acids.

FEMS Yeast Res, 2003 Oct, 4(1), 119 - 22
Comparative analyses of Saccharomyces cerevisiae RNAs using Agilent RNA 6000 Nano Assay and agarose gel electrophoresis; Ricicova M et al.; Precise quantification and quality characterisation of isolated RNAs are prerequisites for their further exploitation in genome-wide microarrays, Northern blots, cDNA library preparation and others . Our data indicate that RNA analyses using Agilent RNA Nano Assay exhibit several advantages when compared with those performed on ethidium bromide-stained agarose gel electrophoresis or on a spectrophotometer . The RNA Nano Assay makes it possible to estimate RNA concentrations in the range from 1000 ng microl(-1) to 17 ng microl(-1) . The presence of impurities including traces of DNA within RNA samples does not influence the concentration measurements . Like agarose gel electrophoresis, RNA Nano Assay allows to analyse RNAs dissolved in formamide and therefore protected against RNase action . Moreover, it allows a clearer distinction of partially degraded samples . The limitation of RNA Nano Assay is the impossibility to detect and to analyse double-stranded RNAs.

FEMS Yeast Res, 2003 Oct, 4(1), 87 - 95
Combined overexpression of genes of the ergosterol biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae; Veen M et al.; Genes of the post-squalene ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been overexpressed in a systematic approach with the aim to construct yeast strains that produce high amounts of sterols from a squalene-accumulating strain . This strain had previously been deregulated by overexpressing a truncated HMG-CoA reductase (tHMG1) in the main bottleneck of the early ergosterol pathway . The overexpression of the gene ERG1 (squalene epoxidase) induced a significant decrease of the direct substrate squalene, a high increase of lanosterol, and a small increase of later sterols . The overexpression of the ERG11 gene encoding the sterol-14alpha-demethylase resulted in a decrease of lanosterol and an increase of downstream sterols . When these two genes were simultaneously overexpressed, later sterols from zymosterol to ergosterol accumulated and the content of squalene was decreased about three-fold, indicating that these steps had limited the transformation of squalene into sterols . The total sterol content in this strain was three-fold higher than in a wild-type strain.

Biotechnol Adv, 1989, 7(2), 155 - 85
Expression of proteins encoded by foreign genes in Saccharomyces cerevisiae; Martin CE et al.; The yeast, Saccharomyces cerevisiae is currently used for the production of recombinant DNA-generated proteins derived from a variety of eukaryotic organisms . The applications of a yeast-based technology in the production of proteins for pharmaceutical and industrial purposes is discussed including current methods for introducing recombinant genes into yeast and strategies for maximizing their expression.

Appl Environ Microbiol, 2003 Oct, 69(10), 6216 - 24
Nuclease activity of Saccharomyces cerevisiae Mre11 functions in targeted nucleotide alteration; Liu L et al.; Oligonucleotides can be used to direct site-specific changes in genomic DNA through a process in which mismatched base pairs in the oligonucleotide and the target DNA are created . The mechanism by which these complexes are developed and resolved is being studied by using Saccharomyces cerevisiae as a model system . Genetic analyses have revealed that in all likelihood the reaction occurs in two phases: DNA pairing and DNA repair . While the former phase involves strand assimilation, the latter phase likely involves an endonucleolytic processing step that leads to joint resolution . In this study, we established the importance of a functioning MRE11 gene in the overall reaction, as yeast strains deficient in MRE11 exhibited severely reduced activity . The activity could be rescued by complementation with wild-type MRE11 genes but not with MRE11 alleles lacking the nuclease function . Taken together, the data suggest that Mre11 provides nuclease activity for targeted nucleotide exchange, a process that could be used to reengineer yeast genes.

Nucleic Acids Res, 2003 Oct 15, 31(20), 5831 - 7
Synergistic repression of anaerobic genes by Mot3 and Rox1 in Saccharomyces cerevisiae; Sertil O et al.; Two groups of anaerobic genes (genes induced in anaerobic cells and repressed in aerobic cells) are negatively regulated by heme, a metabolite present only in aerobic cells . Members of both groups, the hypoxic genes and the DAN/TIR/ERG genes, are jointly repressed under aerobic conditions by two factors . One is Rox1, an HMG protein, and the second, originally designated Rox7, is shown here to be Mot3, a global C2H2 zinc finger regulator . Repression of anaerobic genes results from co-induction of Mot3 and Rox1 in aerobic cells . Repressor synthesis is triggered by heme, which de-represses a mechanism controlling expression of both MOT3 and ROX1 in anaerobic cells; it includes Hap1, Tup1, Ssn6 and a fourth unidentified factor . The constitutive expression of various anaerobic genes in aerobic rox1Delta or mot3Delta cells directly implies that neither factor can repress by itself at endogenous levels and that stringent aerobic repression results from the concerted action of both . Mot3 and Rox1 are not essential components of a single complex, since each can repress independently in the absence of the other, when artificially induced at high levels . Moreover, the two repression mechanisms appear to be distinct: as shown here repression of ANB1 by Rox1 alone requires Tup1-Ssn6, whereas repression by Mot3 does not . Though artificially high levels of either factor can repress well, the absolute efficiency observed in normal cells when both are present-at much lower levels-demonstrates a novel inhibitory synergy . Evidently, expression levels for the two mutually dependent repressors are calibrated to permit a range of variation in basal aerobic expression at different promoters with differing operator site combinations.

J Biol Chem, 2003 Dec 19, 278(51), 51566 - 76 Epub 2003 Oct 06.
The conserved and non-conserved regions of Rpb4 are involved in multiple phenotypes in Saccharomyces cerevisiae; Sampath V et al.; Rpb4, the fourth largest subunit of RNA polymerase II in Saccharomyces cerevisiae, is required for many phenotypes, including growth at high and low temperatures, sporulation, pseudohyphal growth, activated transcription of a subset of genes, and efficient carbon and energy metabolism . We have used deletion analysis to delineate the domains of the protein involved in these multiple phenotypes . The scRpb4 protein is conserved at the N and C termini but possesses certain non-conserved regions in the central portion . Our deletion analysis and molecular modeling results show that the N- and C-terminal conserved regions of Rpb4 are involved in interaction with Rpb7, the Rpb4 interacting partner in the RNA polymerase II . We further show that the conserved N terminus is required for efficient activated transcription from the INO1 promoter but not the GAL10- or the HSE-containing promoters . The N terminus is not required for any of the stress responses tested: growth at high temperatures, sporulation, and pseudohyphal growth . The conserved C-terminal 23 amino acids are not required for the role of Rpb4 in the pseudohyphal growth phenotype but might play a role in other stress responses and activated transcription . From the deletion analysis of the non-conserved regions, we report that they influence phenotypes involving both the N and C termini (interaction with Rpb7 and transcription from the INO1 promoter) but not any of the stress-responsive phenotypes tested suggesting that they might be involved in maintaining the two conserved domains in an appropriate conformation for interaction with Rpb7 and other proteins . Taken together, our results allow us to assign phenotype-specific roles for the different conserved and non-conserved regions of Rpb4.

Gene, 2003 Sep 18, 314, 173 - 9
Protein BmP0 from the silkworm Bombyx mori can be assembled and is functional in the Saccharomyces cerevisiae ribosomal stalk in the absence of the acidic P1 and P2 proteins; Kouyanou S et al.; The DNA complementary to RNA (cDNA) of the ribosomal stalk protein BmP0 of the silkworm Bombyx mori was isolated from a cDNA library and was subsequently expressed in the conditional P0-null mutant Saccharomyces cerevisiae D67dGP0, whose ribosomes also lack the other stalk components, proteins P1/P2 . The transformed strain was able to grow under restrictive conditions, indicating that in the absence of the P1/P2 proteins BmP0 can bind to the yeast ribosomes and complement the lack of the endogenous YP0 protein . In addition, the binding capacity of the B . mori ribosomal stalk components to the ribosomal particle was studied by means of high salt treatment of purified ribosomes . The BmP0 protein retained its binding to the ribosome, suggesting a stable association with the rRNA, in contrast to the acidic proteins BmP1 and BmP2, which were easily released . The results clearly indicate that, as opposed to recent in vitro results, BmP0 does not require the presence of P1/P2 proteins in order to bind to the ribosome.

Curr Genet, 2003 Dec, 44(5), 261 - 7 Epub 2003 Oct 02.
STA10 repression of STA gene expression is caused by a defective activator, flo8, in Saccharomyces cerevisiae; Kim TS et al.; The expression of STA genes that encode extracellular glucoamylase isozymes is repressed in most laboratory Saccharomyces cerevisiae strains, which are believed to contain an undefined repressor, designated STA10 . To identify the regulator involved in STA10 repression, we investigate the FLO8, MSN1, MSS11, STE12, and TEC1 genes . The Deltaflo8 or Deltamss11 deletion mutants in the sta10 genetic background exhibit both a loss of flocculation ability and a reduction in extracellular glucoamylase activity, as in the STA10 strain . Moreover, the STA10 repression is suppressed completely or partially by the introduction of a single copy of the FLO8 or MSS11 genes . Sequence analysis and complementation testing of the STA10 strain reveal that it has an inactive, mutated flo8-1 allele . A random spore analysis and transplacement (allele replacement) experiment confirms that the repressive phenotype of STA10 is due to the amber mutation of the transcriptional activator, FLO8.

J Biol Chem, 2003 Dec 12, 278(50), 50120 - 7 Epub 2003 Oct 01.
Pse1p mediates the nuclear import of the iron-responsive transcription factor Aft1p in Saccharomyces cerevisiae; Ueta R et al.; In Saccharomyces cerevisiae, the iron-responsive transcription factor Aft1p plays a critical role in maintaining iron homeostasis . The activity of Aft1p is induced in response to iron starvation and as a consequence the expression of the iron-regulon is increased . We have shown previously that Aft1p is localized to the cytoplasm under iron-replete conditions but that it is localized to the nucleus under iron-depleted conditions . In this study, we identified the transport receptor that mediates the import of Aft1p into the nucleus, located the nuclear localization signal (NLS) sequences of Aft1p, and examined whether the nuclear import of Aft1p is affected by iron status . In pse1-1 cells, which bear a temperature-sensitive mutation of PSE1, Aft1p was misdirected to the cytoplasm during iron starvation at the restrictive temperature . Aft1p could also directly bind to Pse1p and was dissociated from the complex by Ran-GTP in vitro . These results indicate that Aft1p is imported into the nucleus by Pse1p . Supporting this is that the induction of an Aft1p target gene, FTR1, in response to iron starvation was greatly reduced in pse1-1 cells . Furthermore, we demonstrated that the nuclear localization of a mutant Aft1 protein that contains an NLS derived from SV40 was regulated by iron status regardless of whether Pse1p could interact with Aft1p . This suggests that the interaction between Aft1p and Pse1p is not a critical step that controls the iron-regulated nucleo-cytoplasmic transport of Aft1p.

Biochim Biophys Acta, 2003 Oct 1, 1629(1-3), 34 - 43
Comparative expression of the human beta(2) and beta(3) adrenergic receptors in Saccharomyces cerevisiae; Duport C et al.; The beta(3) adrenergic receptor (beta(3)AR) is the predominant beta subtype in human brown adipocytes and is essential for regulating thermogenic lipolysis . To establish a novel experimental system for the biochemical analysis of this protein, we engineered several yeast strains . We show that the sterol background of the host strain greatly modulates the beta(3)AR expression but not in the same way as it modulates the beta(2) adrenergic receptor (beta(2)AR), the other main studied adipocyte subtype . The human beta(3)AR expressed in yeast is N-glycosylated but not phosphorylated . This latter characteristic distinguishes it from the beta(2)AR . We showed that both beta(2)AR and beta(3)AR follow the secretory pathway to the yeast plasma membrane (PM) and are degraded in the vacuole . In the yeast strains used in this work, the two receptors also share a common mechanism of direct signal transduction through the yeast G(alpha) protein, Gpa1p . These strains thus appear to be useful for biochemical and structural studies of the human beta(3)AR in an in vivo reconstitution system.

Genome Biol . 2003;4(10):230 . Epub 2003 Sep 24.
Tackling an essential problem in functional proteomics of Saccharomyces cerevisiae; Aparicio OM; Gene inactivation is the cornerstone of functional genetic analysis, but the analysis of essential genes requires conditional inactivation of the gene product . A new study has adapted a simple method for creating conditional alleles to allow large-scale analysis of essential genes in Saccharomyces cerevisiae and has identified a role in DNA replication for a newly identified protein complex.

Biochem J, 2004 Jan 15, 377(Pt 2), 395 - 405
Analysis of the interaction between piD261/Bud32, an evolutionarily conserved protein kinase of Saccharomyces cerevisiae, and the Grx4 glutaredoxin; Lopreiato R et al.; The Saccharomyces cerevisiae piD261/Bud32 protein and its structural homologues, which are present along the Archaea-Eukarya lineage, constitute a novel protein kinase family (the piD261 family) distantly related in sequence to the eukaryotic protein kinase superfamily . It has been demonstrated that the yeast protein displays Ser/Thr phosphotransferase activity in vitro and contains all the invariant residues of the family . This novel protein kinase appears to play an important cellular role as deletion in yeast of the gene encoding piD261/Bud32 results in the alteration of fundamental processes such as cell growth and sporulation . In this work we show that the phosphotransferase activity of Bud32 is relevant to its functionality in vivo, but is not the unique role of the protein, since mutants which have lost catalytic activity but not native conformation can partially complement the disruption of the gene encoding piD261/Bud32 . A two-hybrid approach has led to the identification of several proteins interacting with Bud32; in particular a glutaredoxin (Grx4), a putative glycoprotease (Ykr038/Kae1) and proteins of the Imd (inosine monophosphate dehydrogenase) family seem most plausible interactors . We further demonstrate that Grx4 directly interacts with Bud32 and that it is phosphorylated in vitro by Bud32 at Ser-134 . The functional significance of the interaction between Bud32 and the putative protease Ykr038/Kae1 is supported by its evolutionary conservation.

Genetika, 2003 Aug, 39(8), 1039 - 45
{Genetic analysis of pleiotropic effects of pho85 mutations in yeast Saccharomyces cerevisiae}; Sambuk EV et al.; The cyclin-dependent phosphoprotein kinase Pho85p is involved in the regulation of metabolism and cell cycle in the yeast Saccharomyces cerevisiae . It is known that mutations in the PHO85 gene lead to constitutive synthesis of Pho5p acidic phosphatase, a delay in cell growth on media containing nonfermentable carbon sources, sensitivity to high temperature, and other phenotypic effects . A lack of growth at 37 degrees C and on a medium with alcohol as the carbon source was shown to be associated with the rapid accumulation of nuclear ts and mitochondrial {rho-} mutations occurring in the background of gene PHO85 inactivation . Thus, Pho85p seems to play an important role in the maintenance of yeast genome stability.

Appl Biochem Biotechnol, 2003 Sep, 110(3), 151 - 64
Ethanol production by Saccharomyces cerevisiae grown in sugarcane blackstrap molasses through a fed-batch process: optimization by response surface methodology; Carvalho JC et al.; We studied the effect of reactor filling time (T) (3-5 h), initial mass of inoculum (M) (1000-2100 g), and exponential time decay constant for the substrate feed rate (K) (0.6-1.6 h-1) on ethanol production by Saccharomyces cerevisiae grown in sugarcane blackstrap molasses through a fed-batch culture . The highest ethanol productivity (16.9 g/{L x h}) occurred at T = 3 h, K = 1.6 h-1, and M = 1300 g . In addition, productivity was affected by both M (for T = 3 and 4 h) and K (for T = 3 h) and varied inversely with T under any value fixed for M and K . By the quadratic regression multivariable analysis method, equations were determined to estimate ethanol yield and productivity as function of the variables studied (T, K, and M).

J Biol Chem, 2003 Dec 5, 278(49), 48764 - 9 Epub 2003 Sep 25.
Quantitative analysis of GAL genetic switch of Saccharomyces cerevisiae reveals that nucleocytoplasmic shuttling of Gal80p results in a highly sensitive response to galactose; Verma M et al.; The nucleocytoplasmic shuttling of the repressor Gal80p is known to play a pivotal role in the signal transduction process of GAL genetic switch of Saccharomyces cerevisiae (Peng, G., and Hopper, J . E . (2002) Proc . Natl . Acad . Sci . U . S . A . 99, 8548-8553) . We have developed a comprehensive model of this GAL switch to quantify the expression from the GAL promoter containing one or two Gal4p-binding sites and to understand the biological significance of the shuttling process . Our experiments show that the expression of proteins from the GAL promoter containing one and two binding sites for Gal4p is ultrasensitive (a steep response to a given input) . Furthermore, the model revealed that the shuttling of Gal80p is the key step in imparting ultrasensitive response to the inducer . During induction, free Gal80p concentration is altered by sequestration, without any change in the distribution coefficient across the nuclear membrane . Furthermore, the estimated concentrations of Gal80p and Gal3p allow basal expression of alpha-galactosidase, but not beta-galactosidase, from the GAL promoter containing one and two binding sites for Gal4p, respectively . Conversely, the expression from genes with two binding sites is more sensitive to inducer concentration as compared with one binding site . We show that autoregulation of Gal80p is coincidental to the autoregulation of Gal3p, and it does not impart ultrasensitivity . We conclude from our analysis that the ultrasensitivity of the GAL genetic switch is solely because of the shuttling phenomena of the repressor Gal80p across the nuclear membrane.

Curr Genet, 2003 Oct, 44(1), 26 - 37 Epub 2003 Jul 09.
Effects of ploidy, growth conditions and the mitochondrial nucleoid-associated protein Ilv5p on the rate of mutation of mitochondrial DNA in Saccharomyces cerevisiae; Sia RA et al.; Microsatellites, or simple repetitive sequences, are abundant in eukaryotic genomes and in the mitochondrial genome of Saccharomyces cerevisiae . These sequences alter at rates significantly higher than non-repetitive sequences of comparable size . The stability of a mitochondrial microsatellite is nearly 100-fold greater in diploid yeast cells than in isogenic haploid cells . We were able to demonstrate that this effect is likely due to ploidy alone, rather than mating-type-specific gene expression . In addition, we demonstrated that amino acid starvation affects the organization of the mitochondrial DNA and its segregation into the bud . We also tested the effect of amino acid starvation on the copy number and the mutation rate of mitochondrial DNA in both haploid and diploid yeast cells . Yeast cells grown in rich medium have a lower mitochondrial DNA content than cells starved for amino acids and have a correspondingly higher mutation rate for both frameshift mutations and point mutations in mitochondrial DNA . These effects appear to be dependent on the mitochondrial nucleoid-associated protein Ilv5p.

OMICS, 2003 Summer, 7(2), 193 - 202
Large-scale evaluation of in silico gene deletions in Saccharomyces cerevisiae; Forster J et al.; A large-scale in silico evaluation of gene deletions in Saccharomyces cerevisiae was conducted using a genome-scale reconstructed metabolic model . The effect of 599 single gene deletions on cell viability was simulated in silico and compared to published experimental results . In 526 cases (87.8%), the in silico results were in agreement with experimental observations when growth on synthetic complete medium was simulated . Viable phenotypes were correctly predicted in 89.4% (496 out of 555) and lethal phenotypes were correctly predicted in 68.2% (30 out of 44) of the cases considered . The in silico evaluation was solely based on the topological properties of the metabolic network which is based on well-established reaction stoichiometry . No interaction or regulatory information was accounted for in the in silico model . False predictions were analyzed on a case-by-case basis for four possible inadequacies of the in silico model: (1) incomplete media composition, (2) substitutable biomass components, (3) incomplete biochemical information, and (4) missing regulation . This analysis eliminated a number of false predictions and suggested a number of experimentally testable hypotheses . A genome-scale in silico model can thus be used to systematically reconcile existing data and fill in our knowledge gaps about an organism.

J Biol Chem, 2003 Nov 28, 278(48), 47744 - 52 Epub 2003 Sep 23.
Molecular characterization of Ypi1, a novel Saccharomyces cerevisiae type 1 protein phosphatase inhibitor; Garcia-Gimeno MA et al.; The Saccharomyces cerevisiae open reading frame YFR003c encodes a small (155-amino acid) hydrophilic protein that we identified as a novel, heat-stable inhibitor of type 1 protein phosphatase (Ypi1) . Ypi1 interacts physically in vitro with both Glc7 and Ppz1 phosphatase catalytic subunits, as shown by pull-down assays . Ypi1 inhibits Glc7 but appears to be less effective toward Ppz1 phosphatase activity under the conditions tested . Ypi1 contains a 48RHNVRW53 sequence, which resembles the characteristic consensus PP1 phosphatase binding motif . A W53A mutation within this motif abolishes both binding to and inhibition of Glc7 and Ppz1 phosphatases . Deletion of YPI1 is lethal, suggesting a relevant role of the inhibitor in yeast physiology . Cells overexpressing Ypi1 display a number of phenotypes consistent with an inhibitory role of this protein on Glc7, such as decreased glycogen content and an increased growth defect in a slt2/mpk1 mitogen-activated protein kinase-deficient background . Taking together, these results define Ypi1 as the first inhibitory subunit of Glc7 identified in budding yeast.

Genetics, 2003 Sep, 165(1), 47 - 63
Patterns of heteroduplex formation associated with the initiation of meiotic recombination in the yeast Saccharomyces cerevisiae; Merker JD et al.; The double-strand break repair (DSBR) model of recombination predicts that heteroduplexes will be formed in regions that flank the double-strand break (DSB) site and that the resulting intermediate is resolved to generate either crossovers or noncrossovers for flanking markers . Previous studies in Saccharomyces cerevisiae, however, failed to detect heteroduplexes on both sides of the DSB site . Recent physical studies suggest that some recombination events involve heterodupex formation by a mechanism, synthesis-dependent strand annealing (SDSA), that is inherently asymmetric with respect to the DSB site and that leads exclusively to noncrossovers of flanking markers . Below, we demonstrate that many of the recombination events initiated at the HIS4 recombination hotspot are consistent with a variant of the DSBR model in which the extent of heteroduplex on one side of the DSB site is much greater than that on the other . Events that include only one flanking marker in the heteroduplex (unidirectional events) are usually resolved as noncrossovers, whereas events that include both flanking markers (bidirectional events) are usually resolved as crossovers . The unidirectional events may represent SDSA, consistent with the conclusions of others, although other possibilities are not excluded . We also show that the level of recombination reflects the integration of events initiated at several different DSB sites, and we identify a subset of gene conversion events that may involve break-induced replication (BIR) or repair of a double-stranded DNA gap.

Nucleic Acids Res . 2003 Oct 1;31(19):e120.
Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae; Vernis L et al.; The budding yeast Saccharomyces cerevisiae is unable to incorporate exogenous nucleosides into DNA . We have made a number of improvements to existing strategies to reconstitute an efficient thymidine salvage pathway in yeast . We have constructed strains that express both a nucleoside kinase as well as an equilibrative nucleoside transporter . By also deleting the gene encoding thymidylate synthase (CDC21) we have constructed strains that are entirely dependent upon exogenous thymidine for viability and that can grow with normal kinetics at low thymidine concentrations . Using this novel approach, we show that depletion of a single deoxyribonucleoside causes reversible arrest of cells in S phase with concomitant phosphorylation and activation of the S phase checkpoint kinase, Rad53 . We show that this strain also efficiently incorporates the thymidine analogue, BrdU, into DNA and can be used for pulse-chase labelling.

Nucleic Acids Res, 2003 Oct 1, 31(19), 5560 - 7
Characterization of AP lyase activities of Saccharomyces cerevisiae Ntg1p and Ntg2p: implications for biological function; Meadows KL et al.; Saccharomyces cerevisiae possesses two Escherichia coli endonuclease III homologs, NTG1 and NTG2, whose gene products function in the base excision repair pathway and initiate removal of a variety of oxidized pyrimidines from DNA . Although the glycosylase activity of these proteins has been well studied, the in vivo importance of the AP lyase activity has not been determined . Previous genetic studies have suggested that the AP lyase activities of Ntg1p and Ntg2p may be major contributors in the initial processing of abasic sites . We conducted a biochemical characterization of the AP lyase activities of Ntg1p and Ntg2p via a series of kinetic experiments . Such studies were designed to determine if Ntg1p and Ntg2p prefer specific bases located opposite abasic sites and whether these lesions are processed with a catalytic efficiency similar to Apn1p, the major hydrolytic AP endonuclease of yeast . Our results indicate that Ntg1p and Ntg2p are equally effective in processing four types of abasic site-containing substrates . Certain abasic site substrates were processed with greater catalytic efficiency than others, a situation similar to Apn1p processing of such substrates . These biochemical studies strongly support an important biological role for Ntg1p and Ntg2p in the initial processing of abasic sites and maintenance of genomic stability.

Biotechnol Adv, 2003 May, 21(3), 183 - 92
Oscillatory metabolism of Saccharomyces cerevisiae: an overview of mechanisms and models; Patnaik PR; The budding yeast Saccharomyces cerevisiae displays steady oscillations in continuous cultures under certain conditions . Oscillatory responses are important both metabolically and in process applications . Although much information has become available, a definitive theory to explain and model these oscillations is yet to be formulated . Models of oscillatory cultivation have focussed primarily either on intracellular reactions or on transport processes coupled to substantially lumped intracellular kinetics . This review discusses the development of the models and the directions they provide for a comprehensive model of oscillatory metabolism.

BMC Genomics . 2003 Sep 22;4(1):38.
A novel design of whole-genome microarray probes for Saccharomyces cerevisiae which minimizes cross-hybridization; Talla E et al.; BACKGROUND: Numerous DNA microarray hybridization experiments have been performed in yeast over the last years using either synthetic oligonucleotides or PCR-amplified coding sequences as probes . The design and quality of the microarray probes are of critical importance for hybridization experiments as well as subsequent analysis of the data . RESULTS: We present here a novel design of Saccharomyces cerevisiae microarrays based on a refined annotation of the genome and with the aim of reducing cross-hybridization between related sequences . An effort was made to design probes of similar lengths, preferably located in the 3'-end of reading frames . The sequence of each gene was compared against the entire yeast genome and optimal sub-segments giving no predicted cross-hybridization were selected . A total of 5660 novel probes (more than 97% of the yeast genes) were designed . For the remaining 143 genes, cross-hybridization was unavoidable . Using a set of 18 deletant strains, we have experimentally validated our cross-hybridization procedure . Sensitivity, reproducibility and dynamic range of these new microarrays have been measured . Based on this experience, we have written a novel program to design long oligonucleotides for microarray hybridizations of complete genome sequences . CONCLUSIONS: A validated procedure to predict cross-hybridization in microarray probe design was defined in this work . Subsequently, a novel Saccharomyces cerevisiae microarray (which minimizes cross-hybridization) was designed and constructed . Arrays are available at Eurogentec S . A . Finally, we propose a novel design program, OliD, which allows automatic oligonucleotide design for microarrays . The OliD program is available from authors.

Curr Genet, 2003 Dec, 44(4), 184 - 94 Epub 2003 Sep 13.
Saccharomyces cerevisiae Ats1p interacts with Nap1p, a cytoplasmic protein that controls bud morphogenesis; Shields CM et al.; Saccharomyces cerevisiae ATS1 (alpha-tubulin suppressor 1) was originally identified as a high-copy suppressor of class two alpha-tubulin mutations and was proposed to have a regulatory role in coordinating the microtubule state with the cell cycle . Here, we show that Ats1p interacts with Nap1p, a cytoplasmic protein that regulates the activity of the Cdc28p/Clb2p complex . Loss of Nap1p results in a delayed switch from polar to isotropic bud growth . The delayed switch results in elongated buds . Nap1p and Ats1p interact in two-hybrid and co-immunoprecipitation assays . Both nap1Delta and ats1Delta cells have a Clb2p-dependent elongated bud morphology . Deletion of ATS1 partially suppresses the elongated bud morphology and benomyl resistance of nap1Delta mutants . Our results suggest Ats1p might regulate coordination of the microtubule state with the cell cycle through an interaction with Nap1p.

Biochem Biophys Res Commun, 2003 Oct 3, 309(4), 999 - 1004
Mutations in Saccharomyces cerevisiae sterol C5-desaturase conferring resistance to the CYP51 inhibitor fluconazole; Jackson CJ et al.; Understanding fluconazole resistance is important as it emerged as a serious clinical problem for this CYP51, sterol 14alpha-demethylase, inhibitor . One mechanism, observed first in Saccharomyces cerevisiae, was through defective sterol C5-desaturase (Erg3p) required to form the fungistatic sterol end-product resulting from CYP51 inhibition, 14alpha-methylergosta-8,24(28)-dien-3beta,6alpha-diol . Here, we report molecular changes resulting in both blocked mutants and also leaky mutants in which reduced ergosterol levels were detected . Blocked mutants exhibited nonsense and frameshift mutations, while leaky mutants contained missense mutations that were generally in conserved positions based on the alignment of sterol C5-desaturases and located mainly between residues 250 and 282.

J Protein Chem, 2003 May, 22(4), 311 - 5
Thermal stability of phosphoenolpyruvate carboxykinases from Escherichia coli, Trypanosoma brucei, and Saccharomyces cerevisiae; Ravanal MC et al.; The quaternary structure of ATP-dependent phosphoenolpyruvate (PEP) carboxykinases is variable . Thus, the carboxykinases from Escherichia coli, Trypanosoma brucei, and Saccharomyces cerevisiae are monomer, homodimer, and homotetramer, respectively . In this work, we studied the effect of temperature on the stability of the enzyme activity of these three carboxykinases, and have found that it follows the order monomer > dimer > tetramer . The inactivation processes are first order with respect to active enzyme . The presence of substrates leads to an increase in the thermal stability of all three PEP carboxykinases . The protection effect of the substrates on the thermal inactivation of these enzymes suggests similarities in the substrate-bound form of these proteins . We propose that the higher structural complexity of some PEP carboxykinases could be related to the acquisition of properties of relevance in vivo.

J Bioenerg Biomembr, 2003 Jun, 35(3), 231 - 41
In Saccharomyces cerevisiae, cations control the fate of the energy derived from oxidative metabolism through the opening and closing of the yeast mitochondrial unselective channel; Perez-Vazquez V et al.; The yeast mitochondrial unspecific channel (YMUC) sensitivity to inorganic (Ca2+ or Mg2+) or organic (hexyl or octyl-guanidine) cations was measured . The rate of oxygen consumption in State 3 and State 4, the transmembrane potential (deltapsi), mitochondrial swelling, and the polyethylene-glycol mediated recontraction were used to follow opening of the YMUC . Addition of 0.4 mM PO4 did not close the YMUC, although it did enhance the sensitivity to Ca2+ (I50 decreased from 50 to 0.3 mM) and Mg2+ (I50 decreased from 5 to 0.83 mM Mg2+) . The Ca2+ concentration needed to close the YMUC was higher than the concentrations usually observed in the cell . Nonetheless, Mg2+, Ca2+, and PO4 exhibited additive effects . These cations did not inhibit contraction of preswollen mitochondria, suggesting that the YMUC/cation interaction was labile . Octyl-guanidine (OG-I50 7.5 microM) was the only cation which inhibited mitochondrial recontraction, probably as a result of membrane binding stabilization through its hydrophobic tail . The PO4-dependent, Ca(2+)/Mg(2+)-mediated closure of the YMUC may be a means to control the proportion of oxidative energy producing ATP or being lost as heat.

J Biol Chem, 2003 Nov 28, 278(48), 47629 - 35 Epub 2003 Sep 16.
The ubiquinone-binding site of the Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase is a source of superoxide; Guo J et al.; The mitochondrial succinate dehydrogenase (SDH) is a tetrameric iron-sulfur flavoprotein of the Krebs cycle and of the respiratory chain . A number of mutations in human SDH genes are responsible for the development of paragangliomas, cancers of the head and neck region . The mev-1 mutation in the Caenorhabditis elegans gene encoding the homolog of the SDHC subunit results in premature aging and hypersensitivity to oxidative stress . It also increases the production of superoxide radicals by the enzyme . In this work, we used the yeast succinate dehydrogenase to investigate the molecular and catalytic effects of paraganglioma- and mev-1-like mutations . We mutated Pro-190 of the yeast Sdh2p subunit to Gln (P190Q) and recreated the C . elegans mev-1 mutation by converting Ser-94 in the Sdh3p subunit into a glutamate residue (S94E) . The P190Q and S94E mutants have reduced succinate-ubiquinone oxidoreductase activities and are hypersensitive to oxygen and paraquat . Although the mutant enzymes have lower turnover numbers for ubiquinol reduction, larger fractions of the remaining activities are diverted toward superoxide production . The P190Q and S94E mutations are located near the proximal ubiquinone-binding site, suggesting that the superoxide radicals may originate from a ubisemiquinone intermediate formed at this site during the catalytic cycle . We suggest that certain mutations in SDH can make it a significant source of superoxide production in mitochondria, which may contribute directly to disease progression . Our data also challenge the dogma that superoxide production by SDH is a flavin-mediated event rather than a quinone-mediated one.

Proc Natl Acad Sci U S A, 2003 Sep 30, 100(20), 11529 - 34 Epub 2003 Sep 12.
A genomewide screen in Saccharomyces cerevisiae for genes that suppress the accumulation of mutations; Huang ME et al.; A genomewide screen of a collection of 4,847 yeast gene deletion mutants was carried out to identify the genes required for suppressing mutations in the CAN1 forward-mutation assay . The primary screens and subsequent analysis allowed (i) identification of 18 known mutator mutants, providing a solid means for checking the efficiency of the screen, and (ii) identification of a number of genes not known previously to be involved in suppressing mutations . Among the previously uncharacterized mutation-suppressing genes were six genes of unknown function including four (CSM2, SHU2, SHU1, and YLR376c) encoding proteins that interact with each other and promote resistance to killing by methyl methanesulfonate, one gene (EGL1) previously identified as suppressing Ty1 mobility and recombination between repeated sequences, and one gene (YLR154c) that was not associated with any known processes . In addition, five genes (TSA1, SOD1, LYS7, SKN7, and YAP1) implicated in the oxidative-stress responses were found to play a significant role in mutation suppression . Furthermore, TSA1, which encodes thioredoxin peroxidase, was found to strongly suppress gross chromosomal rearrangements . These results provide a global view of the nonessential genes involved in preventing mutagenesis . Study of such genes should provide useful clues in identification of human genes potentially involved in cancer predisposition and in understanding their mechanisms of action.

Mol Cell Biol, 2003 Oct, 23(19), 7044 - 54
NAD+-dependent deacetylase Hst1p controls biosynthesis and cellular NAD+ levels in Saccharomyces cerevisiae; Bedalov A et al.; Nicotine adenine dinucleotide (NAD(+)) performs key roles in electron transport reactions, as a substrate for poly(ADP-ribose) polymerase and NAD(+)-dependent protein deacetylases . In the latter two processes, NAD(+) is consumed and converted to ADP-ribose and nicotinamide . NAD(+) levels can be maintained by regeneration of NAD(+) from nicotinamide via a salvage pathway or by de novo synthesis of NAD(+) from tryptophan . Both pathways are conserved from yeast to humans . We describe a critical role of the NAD(+)-dependent deacetylase Hst1p as a sensor of NAD(+) levels and regulator of NAD(+) biosynthesis . Using transcript arrays, we show that low NAD(+) states specifically induce the de novo NAD(+) biosynthesis genes while the genes in the salvage pathway remain unaffected . The NAD(+)-dependent deacetylase activity of Hst1p represses de novo NAD(+) biosynthesis genes in the absence of new protein synthesis, suggesting a direct effect . The known Hst1p binding partner, Sum1p, is present at promoters of highly inducible NAD(+) biosynthesis genes . The removal of HST1-mediated repression of the NAD(+) de novo biosynthesis pathway leads to increased cellular NAD(+) levels . Transcript array analysis shows that reduction in cellular NAD(+) levels preferentially affects Hst1p-regulated genes in comparison to genes regulated with other NAD(+)-dependent deacetylases (Sir2p, Hst2p, Hst3p, and Hst4p) . In vitro experiments demonstrate that Hst1p has relatively low affinity toward NAD(+) in comparison to other NAD(+)-dependent enzymes . These findings suggest that Hst1p serves as a cellular NAD(+) sensor that monitors and regulates cellular NAD(+) levels.

Mol Biol Cell, 2003 Sep, 14(9), 3834 - 47 Epub 2003 Jun 13.
Role for Arf3p in development of polarity, but not endocytosis, in Saccharomyces cerevisiae; Huang CF et al.; ADP-ribosylation factors (ARFs) are ubiquitous regulators of virtually every step of vesicular membrane traffic . Yeast Arf3p, which is most similar to mammalian ARF6, is not essential for cell viability and not required for endoplasmic reticulum-to-Golgi protein transport . Although mammalian ARF6 has been implicated in the regulation of early endocytic transport, we found that Arf3p was not required for fluid-phase, membrane internalization, or mating-type receptor-mediated endocytosis . Arf3p was partially localized to the cell periphery, but was not detected on endocytic structures . The nucleotide-binding, N-terminal region, and N-terminal myristate of Arf3p are important for its proper localization . C-Terminally green fluorescent protein-tagged Arf3, expressed from the endogenous promoter, exhibited a polarized localization to the cell periphery and buds, in a cell cycle-dependent manner . Arf3-GFP achieved its proper localization during polarity growth through an actin-independent pathway . Both haploid and homologous diploid arf3 mutants exhibit a random budding defect, and the overexpression of the GTP-bound form Arf3p(Q71L) or GDP-binding defective Arf3p(T31N) mutant interfered with budding-site selection . We conclude that the GTPase cycle of Arf3p is likely to be important for the function of Arf3p in polarizing growth of the emerging bud and/or an unidentified vesicular trafficking pathway.

J Cell Sci, 2003 Oct 15, 116(Pt 20), 4119 - 30
Differential cellular localization among mitotic cyclins from Saccharomyces cerevisiae: a new role for the axial budding protein Bud3 in targeting Clb2 to the mother-bud neck; Bailly E et al.; The mitotic cyclin Clb2 plays a major role in promoting M-phase in budding yeast, despite its functional redundancy with three closely related cyclins Clb1, Clb3 and Clb4 . Here, we further investigate the mechanisms controlling the cellular distribution of Clb2 in living cells . In agreement with observations recently made by Hood et al., we find that GFP-tagged Clb2 expressed from its natural promoter localizes to various cellular compartments, including the nucleus, the mitotic spindle, the spindle pole bodies as well as the mother-bud neck . The neck localization is specific to Clb2 as Clb1, Clb3 and Clb4 are never observed there, even when over-expressed . Mutational analysis identifies a central region of Clb2, comprising residues 213-255 and a phylogenetically conserved hydrophobic patch, as an essential cis-acting determinant . Clb2 co-localizes with the bud site selection protein Bud3 . Consistent with a role of Bud3 in targeting Clb2 to the bud neck, we report a two-hybrid interaction between these proteins . Furthermore, Clb2 is shown to be specifically delocalized in Deltabud3 cells and in a bud3 mutant deleted for its C-terminal Clb2-interacting domain (bud3(Delta1221)), but not in a Deltabud10 mutant . Correlating with this phenotype, bud3(Delta1221) cells exhibit a pronounced (15-30 minutes) delay in cytokinesis and/or cell separation, suggesting an unanticipated function of Clb2 in these late mitotic events . Taken together, our data uncover a new role for Bud3 in cytokinesis that correlates with its capacity to target Clb2 at the neck, independently of its well established cell-type-specific function in bud site selection.

J Biol Chem, 2003 Nov 21, 278(47), 47190 - 8 Epub 2003 Sep 12.
The crystal structure of the glutathione S-transferase-like domain of elongation factor 1Bgamma from Saccharomyces cerevisiae; Jeppesen MG et al.; The crystal structure of the N-terminal 219 residues (domain 1) of the conserved eukaryotic translation elongation factor 1Bgamma (eEF1Bgamma), encoded by the TEF3 gene in Saccharomyces cerevisiae, has been determined at 3.0 A resolution by the single wavelength anomalous dispersion technique . The structure is overall very similar to the glutathione S-transferase proteins and contains a pocket with architecture highly homologous to what is observed in glutathione S-transferase enzymes . The TEF3-encoded form of eEF1Bgamma has no obvious catalytic residue . However, the second form of eEF1Bgamma encoded by the TEF4 gene contains serine 11, which may act catalytically . Based on the x-ray structure and gel filtration studies, we suggest that the yeast eEF1 complex is organized as an {eEF1A.eEF1Balpha.eEF1Bgamma}2 complex . A 23-residue sequence in the middle of eEF1Bgamma is essential for the stable dimerization of eEF1Bgamma and the quaternary structure of the eEF1 complex.

J Struct Biol, 2003 Aug, 143(2), 124 - 34
The 10.8-A structure of Saccharomyces cerevisiae phosphofructokinase determined by cryoelectron microscopy: localization of the putative fructose 6-phosphate binding sites; Ruiz T et al.; Phosphofructokinase plays a key role in the regulation of the glycolytic pathway and is responsible for the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate . Although the structure of the bacterial enzyme is well understood, the knowledge is still quite limited for higher organisms given the larger size and complexity of the eukaryotic enzymes . We have studied phosphofructokinase from Saccharomyces cerevisiae in the presence of fructose 6-phosphate by cryoelectron microscopy and image analysis of single particles and obtained the structure at 10.8A resolution . This was achieved by optimizing the illumination conditions to obtain routinely 8-A data from hydrated samples in an electron microscope equipped with an LaB(6) and by improving the image alignment techniques . The analysis of the structure has evidenced that the homology of the subunits at the sequence level has transcended to the structural level . By fitting the X-ray structure of the bacterial tetramer into each dimer of the yeast octamer the putative binding sites for fructose 6-phosphate were revealed . The data presented here in combination with molecular replacement techniques have served to provide the initial phases to solve the X-ray structure of the yeast phosphofructokinase.

DNA Repair (Amst), 2003 Sep 18, 2(9), 1041 - 64
Amino acid changes in Xrs2p, Dun1p, and Rfa2p that remove the preferred targets of the ATM family of protein kinases do not affect DNA repair or telomere length in Saccharomyces cerevisiae; Mallory JC et al.; In eukaryotes, mutations in a number of genes that affect DNA damage checkpoints or DNA replication also affect telomere length {Curr . Opin . Cell Biol . 13 (2001) 281} . Saccharomyces cerevisae strains with mutations in the TEL1 gene (encoding an ATM-like protein kinase) have very short telomeres, as do strains with mutations in XRS2, RAD50, or MRE11 (encoding members of a trimeric complex) . Xrs2p and Mre11p are phosphorylated in a Tel1p-dependent manner in response to DNA damage {Genes Dev . 15 (2001) 2238; Mol . Cell 7 (2001) 1255} . We found that Xrs2p, but not Mre11p or Rad50p, is efficiently phosphorylated in vitro by immunopreciptated Tel1p . Strains with mutations eliminating all SQ and TQ motifs in Xrs2p (preferred targets of the ATM kinase family) had wild-type length telomeres and wild-type sensitivity to DNA damaging agents . We also showed that Rfa2p (a subunit of RPA) and the Dun1p checkpoint kinase, which are required for DNA damage repair and which are phosphorylated in response to DNA damage in vivo, are in vitro substrates of the Tel1p and Mec1p kinases . In addition, Dun1p substrates with no SQ or TQ motifs are phosphorylated by Mec1p in vitro very inefficiently, but retain most of their ability to be phosphorylated by Tel1p . We demonstrated that null alleles of DUN1 and certain mutant alleles of RFA2 result in short telomeres . As observed with Xrs2p, however, strains with mutations of DUN1 or RFA2 that eliminate SQ motifs have no effect on telomere length or DNA damage sensitivity.

J Biol Chem, 2003 Nov 7, 278(45), 44331 - 7 Epub 2003 Sep 08.
ATPase and DNA helicase activities of the Saccharomyces cerevisiae anti-recombinase Srs2; Van Komen S et al.; Saccharomyces cerevisiae SRS2 encodes an ATP-dependent DNA helicase that is needed for DNA damage checkpoint responses and that modulates the efficiency of homologous recombination . Interestingly, strains simultaneously mutated for SRS2 and a variety of DNA repair genes show low viability that can be overcome by inactivating homologous recombination, thus implicating inappropriate recombination as the cause of growth impairment in these mutants . Here, we report on our biochemical characterization of the ATPase and DNA helicase activities of Srs2 . ATP hydrolysis by Srs2 occurs efficiently only in the presence of DNA, with ssDNA being considerably more effective than dsDNA in this regard . Using homopolymeric substrates, the minimal DNA length for activating ATP hydrolysis is found to be 5 nucleotides, but a length of 10 nucleotides is needed for maximal activation . In its helicase action, Srs2 prefers substrates with a 3' ss overhang, and approximately 10 bases of 3' overhanging DNA is needed for efficient targeting of Srs2 to the substrate . Even though a 3' overhang serves to target Srs2, under optimized conditions blunt-end DNA substrates are also dissociated by this protein . The ability of Srs2 to unwind helicase substrates with a long duplex region is enhanced by the inclusion of the single-strand DNA-binding factor replication protein A.

J Biol Chem, 2003 Nov 21, 278(47), 46440 - 5 Epub 2003 Sep 08.
Kinetics of ATP binding to the origin recognition complex of Saccharomyces cerevisiae; Makise M et al.; Origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes, binds specifically to ATP through two of its subunits (Orc1p and Orc5p) . In this study, we investigated the kinetics of ATP binding to ORC by a filter binding assay . The Kd values for the ATP of wild-type ORC and ORC-1A (mutant ORC containing Orc1p with a defective Walker A motif) were less than 10 nm, suggesting that the affinity of Orc5p for ATP is very high . On the other hand, the Kd values for the ATP of ORC-5A (mutant ORC containing Orc5p with a defective Walker A motif) was much higher (about 1.5 microm), suggesting that the affinity of Orc1p for ATP is relatively low in the absence of origin DNA . ATP dissociated more rapidly from its complex with ORC-5A than from its complex with ORC-1A, suggesting that the ATP-Orc5p complex is more stable than ATP-Orc1p complex . Origin DNA fragments decreased the Kd value of ORC-5A for ATP and stabilized the complex of ATP with ORC-5A . Wild-type ORC, ORC-1A, and ORC-5A required different concentrations of ATP for specific binding to origin DNA . All of these results imply that ATP binding to Orc5p, ATP binding to Orc1p, and origin DNA binding to ORC are co-operatively regulated, which may be important for the initiation of DNA replication.

Biochim Biophys Acta, 2003 Sep 8, 1623(1), 1 - 5
Oxidative stresses elevate the expression of cytochrome c peroxidase in Saccharomyces cerevisiae; Kwon M et al.; Cytochrome c peroxidase (CcP) uses hydrogen peroxide as an electron acceptor to oxidize cytochrome c (Cc) in the mitochondrial intermembrane space . A null allele of yeast CCP1 gene encoding CcP was created by one-step gene disruption method in a diploid yeast strain . Haploid yeast cells with the disrupted CCP1 gene were viable and able to grow in a medium containing lactic acid or glycerol as an energy source, indicating that CcP is not essential for both cell viability and respiration . However, CCP1-disrupted cells were more sensitive to H2O2 than wild-type cells . We also constructed a CCP1-lacZ fused gene and integrated this gene into yeast chromosomal DNA to monitor the expression of CCP1 gene . We found that expression of CCP1 gene increases under respiratory culture conditions and by treatments with H2O2 . These results hint that the biological function of CcP is to reduce H2O2 generated during aerobic respiratory process . Moreover, expression of CCP1 gene increased by treatments with peroxynitrite, indicating that CcP may act as a peroxynitrite scavenger.

J Clin Virol, 2003 Oct, 28(2), 121 - 9
Development of a measles specific IgM ELISA for use with serum and oral fluid samples using recombinant measles nucleoprotein produced in Saccharomyces cerevisiae; Samuel D et al.; In order to develop sensitive assays for detecting measles antibodies in oral fluid specimens, we have produced recombinant measles virus nucleoprotein (rMVN) in a yeast expression system and prepared monoclonal antibodies to the protein . Measles nucleoprotein gene from the Schwarz vaccine strain was cloned into a yeast expression vector, pFX7 under the control of the hybrid GAL10-PYK1 promoter . High levels of rMVN (20 mg/litre of yeast culture) were generated . Electron microscopy showed that the purified rMVN assembled into typical herring-bone structures . Monoclonal antibodies produced to the rMVN also reacted with native measles virus N in immunofluorescence tests . The purified rMVN and a monoclonal antibody to the rMVN conjugated to horseradish peroxidase were used to develop a measles specific IgM capture EIA (MACEIA) in both serum and oral fluid specimens . Evaluations of the MACEIA were performed by testing a) serum samples (n=80) and b) paired oral fluid/serum samples from measles cases (n=50, representing 16 cases) and oral fluids from controls with non-measles rash (n=59, representing 48 cases) . The samples were also tested for measles IgM, using a reference radioimmunoassay (MACRIA) . The sensitivity and specificity of the MACEIA compared with MACRIA for a) the serum samples were 100 and 96.6% respectively and b) for paired serum/oral fluids samples 100 and 100%, respectively.

J Biol Chem, 2003 Dec 12, 278(50), 50309 - 15 Epub 2003 Sep 03.
Fre1p Cu2+ reduction and Fet3p Cu1+ oxidation modulate copper toxicity in Saccharomyces cerevisiae; Shi X et al.; Fre1p is a metalloreductase in the yeast plasma membrane that is essential to uptake of environmental Cu2+ and Fe3+ . Fet3p is a multicopper oxidase in this membrane essential for high affinity iron uptake . In the uptake of Fe3+, Fre1p produces Fe2+ that is a substrate for Fet3p; the Fe3+ produced by Fet3p is a ligand for the iron permease, Ftr1p . Deletion of FET3 leads to iron deficiency; this deletion also causes a copper sensitivity not seen in wild type . Deletion of FTR1 leads to copper sensitivity also . Production in the ftr1delta strain of an iron-uptake negative Ftr1p mutant, Ftr1p(RAGLA), suppressed this copper sensitivity . This Ftr1p mutant supported the plasma membrane targeting of active Fet3p that is blocked in the parental ftr1delta strain . A ferroxidase-negative Fet3p did not suppress the copper sensitivity in a fet3delta strain, although it supported the plasma membrane localization of the Fet3p.Ftr1p complex . Thus, loss of membrane-associated Fet3p oxidase activity correlated with copper sensitivity . Furthermore, in vitro Cu1+ was shown to be an excellent substrate for Fet3p . Last, the copper sensitivity of the fet3delta strain was suppressed by co-deletion of FRE1, suggesting that the cytotoxic species was Cu1+ . In contrast, deletion of CTR1 or of FET4 did not suppress the copper sensitivity in the fet3delta strain; these genes encode the two major copper transporters in laboratory yeast strains . This result indicated that the apparent cuprous ion toxicity was not due to excess intracellular copper . These biochemical and physiologic results indicate that at least with respect to cuprous and ferrous ions, Fet3p can be considered a metallo-oxidase and appears to play an essential role in both iron and copper homeostasis in yeast . Its functional homologs, e.g . ceruloplasmin and hephaestin, could play a similar role in mammals.

J Biol Chem, 2003 Nov 7, 278(45), 44727 - 34 Epub 2003 Sep 02.
Ume1p represses meiotic gene transcription in Saccharomyces cerevisiae through interaction with the histone deacetylase Rpd3p; Mallory MJ et al.; Ume1p is a member of a conserved protein family including RbAp48 that associates with histone deacetylases . Consistent with this finding, Ume1p is required for the full repression of a subset of meiotic genes during vegetative growth in budding yeast . In addition to mitotic cell division, this report describes a new role for Ume1p in meiotic gene repression in precommitment sporulating cultures returning to vegetative growth . However, Ume1p is not required to re-establish repression as part of the meiotic transient transcription program . Mutational analysis revealed that two conserved domains (NEE box and a WD repeat motif) are required for Ume1p-dependent repression . Co-immunoprecipitation studies revealed that both the NEE box and the WD repeat motif are essential for normal Rpd3p binding . Finally, Ume1p-Rpd3p association is dependent on the global co-repressor Sin3p . Moreover, this activity was localized to one of the four paired amphipathic-helix domains of Sin3p shown previously to be required for transcriptional repression . These findings support a model that Ume1p binding to Rpd3p is required for its repression activity . In addition, these results suggest that Rpd3-Ume1p-Sin3p comprises an interdependent complex required for mediating transcriptional repression.

J Cell Sci, 2003 Oct 1, 116(Pt 19), 3957 - 70
Vps20p and Vta1p interact with Vps4p and function in multivesicular body sorting and endosomal transport in Saccharomyces cerevisiae; Yeo SC et al.; Vps4p (End13p) is an AAA-family ATPase that functions in membrane transport through endosomes, sorting of soluble vacuolar proteins to the vacuole, and multivesicular body (MVB) sorting of membrane proteins to the vacuole lumen . In a yeast two-hybrid screen with Vps4p as bait we isolated VPS20 (YMR077c) and the novel open reading frame YLR181c, for which the name VTA1 has recently been assigned (Saccharomyces Genome Database) . Vps4p directly binds Vps20p and Vta1p in vitro and binding is not dependent on ATP - conversely, Vps4p binding to Vps20p is partially sensitive to ATP hydrolysis . Both ATP binding {Vps4p-(K179A)} and ATP hydrolysis {Vps4p-(E233Q)} mutant proteins exhibit enhanced binding to Vps20p and Vta1p in vitro . The Vps4p-Vps20p interaction involves the coiled-coil domain of each protein, whereas the Vps4p-Vta1p interaction involves the (non-coiled-coil) C-terminus of each protein . Deletion of either VPS20 (vps20Delta) or VTA1 (vta1Delta) leads to similar class E Vps- phenotypes resembling those of vps4Delta, including carboxypeptidase Y (CPY) secretion, a block in ubiquitin-dependent MVB sorting, and a delay in both post-internalisation endocytic transport and biosynthetic transport to the vacuole . The vacuole resident membrane protein Sna3p (whose MVB sorting is ubiquitin-independent) does not appear to exit the class E compartment or reach the vacuole in cells lacking Vps20p, Vta1p or Vps4p, in contrast to other proteins whose delivery to the vacuole is only delayed . We propose that Vps20p and Vta1p regulate Vps4p function in vivo.

Microbiology, 2003 Sep, 149(Pt 9), 2487 - 99
A synthetic analysis of the Saccharomyces cerevisiae stress sensor Mid2p, and identification of a Mid2p-interacting protein, Zeo1p, that modulates the PKC1-MPK1 cell integrity pathway; Green R et al.; Mid2p is a plasma membrane protein that functions in Saccharomyces cerevisiae as a sensor of cell wall stress, activating the PKC1-MPK1 cell integrity pathway via the small GTPase Rho1p during exposure to mating pheromone, calcofluor white, and heat . To examine Mid2p signalling, a global synthetic interaction analysis of a mid2 mutant was performed; this identified 11 interacting genes . These include WSC1 and ROM2, upstream elements in cell integrity pathway signalling, and FKS1 and SMI1, required for 1,3-beta-glucan synthesis . These synthetic interactions indicate that the Wsc1p sensor acts through Rom2p to activate the Fks1p glucan synthase in a Mid2p-independent way . To further explore Mid2p signalling a two-hybrid screen was done using the cytoplasmic tail of Mid2p; this identified ZEO1 (YOL109w), encoding a 12 kDa peripheral membrane protein that localizes to the plasma membrane . Disruption of ZEO1 leads to resistance to calcofluor white and to a Mid2p-dependent constitutive phosphorylation of Mpk1p, supporting a role for Zeo1p in the cell integrity pathway . Consistent with this, zeo1-deficient cells suppress the growth defect of mutants in the Rho1p GDP-GTP exchange factor Rom2p, while exacerbating the growth defect of sac7delta mutants at 37 degrees C . In contrast, mid2delta mutants have opposing effects to zeo1delta mutants, being synthetically lethal with rom2delta, and suppressing an 18 degrees C growth defect of sac7delta, while overexpression of MID2 rescues a rom2delta 37 degrees C growth defect . Thus, MID2 and ZEO1 appear to play reciprocal roles in the modulation of the yeast PKC1-MPK1 cell integrity pathway.

J Biol Chem, 2003 Nov 14, 278(46), 45269 - 79 Epub 2003 Aug 28.
Maintenance and regulation of mRNA stability of the Saccharomyces cerevisiae OLE1 gene requires multiple elements within the transcript that act through translation-independent mechanisms; Vemula M et al.; The Saccharomyces cerevisiae OLE1 gene encodes a membrane-bound Delta-9 fatty acid desaturase, whose expression is regulated by unsaturated fatty acids through both transcriptional and mRNA stability controls . In fatty acid-free medium, the mRNA has a half-life of 10 +/- 1.5 min (basal stability) that drops to 2 +/- 1.5 min when cells are exposed to unsaturated fatty acids (regulated stability) . A deletion analysis of elements within the transcript revealed that the sequences within the protein-coding region that encode transmembrane sequences and a part of the cytochrome b5 domain are essential for the basal stability of the transcript . Deletion of any of the three essential elements produced unstable transcripts and loss of regulated instability . By contrast, substitution of the 3'-untranslated region with that of the stable PGK1 gene did not affect the basal stability of the transcript and did not block regulated decay . Given that Ole1p is a membrane-bound protein whose activities are a major determinant of membrane fluidity, we asked whether membrane-associated translation of the protein was essential for basal and regulated stability . Insertion of stop codons within the transcript that blocked either translation of the entire protein or parts of the protein required for co-translation insertion of Ole1p had no effect . We conclude that the basal and regulated stability of the OLE1 transcript is resistant to the nonsense-mediated decay pathway and that the essential protein-encoding elements for basal stability act cooperatively as stabilizing sequences through RNA-protein interactions via a translation-independent mechanism.

Mol Cell Biol, 2003 Sep, 23(18), 6585 - 96
The checkpoint protein Rad24 of Saccharomyces cerevisiae is involved in processing double-strand break ends and in recombination partner choice; Aylon Y et al.; Upon chromosomal damage, cells activate a checkpoint response that includes cell cycle arrest and a stimulation of DNA repair . The checkpoint protein Rad24 is key to the survival of a single, repairable double-strand break (DSB) . However, the low survival of rad24 cells is not due to their inability to arrest cell cycle progression . In rad24 mutants, processing of the broken ends is delayed and protracted, resulting in extended kinetics of DSB repair and in cell death . The limited resection of rad24 mutants also affects recombination partner choice by a mechanism dependent on the length of the interacting homologous donor sequences . Unexpectedly, rad24 cells with a DSB eventually accumulate and die at the G(2)/M phase of the cell cycle . This arrest depends on the spindle checkpoint protein Mad2.

Mol Cell Biol, 2003 Sep, 23(18), 6574 - 84
Palmitoylation and plasma membrane localization of Ras2p by a nonclassical trafficking pathway in Saccharomyces cerevisiae; Dong X et al.; Subcellular localization of Ras proteins to the plasma membrane is accomplished in part by covalent attachment of a farnesyl moiety to the conserved CaaX box cysteine . Farnesylation targets Ras to the endoplasmic reticulum (ER), where additional processing steps occur, resulting in translocation of Ras to the plasma membrane . The mechanism(s) by which this occurs is not well understood . In this report, we show that plasma membrane localization of Ras2p in Saccharomyces cerevisiae does not require the classical secretory pathway or a functional Golgi apparatus . However, when the classical secretory pathway is disrupted, plasma membrane localization requires Erf2p, a protein that resides in the ER membrane and is required for efficient palmitoylation of Ras2p . Deletion of ERF2 results in a Ras2p steady-state localization defect that is more severe when combined with sec-ts mutants or brefeldin A treatment . The Erf2p-dependent localization of Ras2p correlates with the palmitoylation of Cys-318 . An Erf2p-Erf4p complex has recently been shown to be an ER-associated palmitoyltransferase that can palmitoylate Cys-318 of Ras2p (S . Lobo, W . K . Greentree, M . E . Linder, and R . J . Deschenes, J . Biol . Chem . 277:41268-41273, 2002) . Erf2-dependent palmitoylation as well as localization of Ras2p requires a region of the hypervariable domain adjacent to the CaaX box . These results provide evidence for the existence of a palmitoylation-dependent, nonclassical endomembrane trafficking system for the plasma membrane localization of Ras proteins.

Mol Cell Biol, 2003 Sep, 23(18), 6363 - 72
Rad52-independent accumulation of joint circular minichromosomes during S phase in Saccharomyces cerevisiae; Wellinger RE et al.; We investigated the formation of X-shaped molecules consisting of joint circular minichromosomes (joint molecules) in Saccharomyces cerevisiae by two-dimensional neutral/neutral gel electrophoresis of psoralen-cross-linked DNA . The appearance of joint molecules was found to be replication dependent . The joint molecules had physical properties reminiscent of Holliday junctions or hemicatenanes, as monitored by strand displacement, branch migration, and nuclease digestion . Physical linkage of the joint molecules was detected along the entire length of the minichromosome and most likely involved newly replicated sister chromatids . Surprisingly, the formation of joint molecules was found to be independent of Rad52p as well as of other factors associated with a function in homologous recombination or in the resolution of stalled replication intermediates . These findings thus imply the existence of a nonrecombinational pathway(s) for the formation of joint molecules during the process of DNA replication or minichromosome segregation.

J Biol Chem, 2003 Oct 31, 278(44), 43110 - 3 Epub 2003 Aug 27.
Iron requirement for GAL gene induction in the yeast Saccharomyces cerevisiae; Shi X et al.; Iron is an essential nutrient . Its deficiency hinders the synthesis of ATP and DNA . We report that galactose metabolism is defective when iron availability is restricted . Our data support this connection because 1) galactose-mediated induction of GAL promoter-dependent gene expression was diminished by iron limitation, and 2) iron-deficient mutants grew slowly on galactose-containing medium . These two defects were immediately corrected by iron replacement . Inherited defects in human galactose metabolism are characteristic of the disease called galactosemia . Our findings suggest that iron-deficient galactosemic individuals might be more severely compromised than iron-replete individuals . This work shows that iron homeostasis and galactose metabolism are linked with one another.

J Steroid Biochem Mol Biol, 2003 Jul, 86(1), 15 - 26
Potentiation of human estrogen receptor alpha-mediated gene expression by steroid receptor coactivator-1 (SRC-1) in Saccharomyces cerevisiae; Ellison AR et al.; The yeast Saccharomyces cerevisiae was used to reconstruct a human estrogen receptor alpha (ERalpha)-mediated transcription activation system . The level of reporter gene activation was dependent on both the position of the estrogen response element (ERE) relative to the translation start site and the number of EREs in the hybrid promoter . A G400V amino acid alteration in the ERalpha polypeptide decreased sensitivity to 17beta-estradiol (E(2)), demonstrating the hormone responsiveness of ERalpha to be qualitatively and quantitatively similar in yeast and mammalian cells . Coexpression of SRC-1a, a potent stimulator of ERalpha function in mammalian cells, potentiated ERalpha-mediated gene expression over fivefold in a E(2)-dependent manner . Deletion of 56 amino acids at the C-terminal end of SRC-1a resulted in a protein with enhanced ability to potentiate ERalpha-mediated gene expression, which mimics the activity of the same truncation in human SRC-1a as well as the SRC-1e isoform that has the 56 C-terminal residues replaced with a different 14 amino acid peptide . The selective estrogen receptor modulator tamoxifen acted as a weak agonist of ERalpha-mediated gene expression and this weak activity was potentiated by SRC-1 . Tamoxifen had no effect on E(2)-induced gene activation in either the presence or absence of SRC-1 . In contrast to previously reported yeast-based ERalpha-transactivation systems, the system reported here in which SRC-1 functions as a bona fide coactivator should permit a more thorough dissection of the factors involved in ERalpha-mediated transcriptional activation.

Mol Genet Genomics, 2003 Nov, 270(2), 165 - 72 Epub 2003 Aug 26.
Mapping candidate hotspots of meiotic recombination in segments of human DNA cloned in the yeast Saccharomyces cerevisiae; Mucha M et al.; The hotspots of meiotic recombination in the human genome can be localized by genetic techniques . The resolution of these techniques is in the range of kilobases and depends on the density of the physical markers identifying allelic variants of the chromosomal loci . We thought it would be interesting to localize these sites with higher resolution . Assuming that some human chromosomal sites conserve their propensity for recombination when cloned in yeast, we localized the hotspots of recombination in several yeast artificial chromosomes (YACs) carrying human DNA . A number of potential recombination hotspots could be identified in the clones studied . Among them there are two classes of sites that are particularly recombination prone also in human meiotic cells: sites associated with CpG islands and sites located in the vicinity of long minisatellite sequences.

Mol Microbiol, 2003 Sep, 49(5), 1321 - 32
The link between 20S proteasome activity and post-replication DNA repair in Saccharomyces cerevisiae; Podlaska A et al.; We have shown previously that deletion of the Saccharomyces cerevisiae UMP1 gene encoding the 20S proteasome maturase causes sensitivity to UV radiation . In the current report, we have extended this finding to show that mutations specifically compromising chymotrypsin-like or trypsin-like activity of 20S proteasome peptidases also result in increased UV sensitivity . We have also established that mutations affecting proteasome activity, namely ump1Delta, pre2-K108R and pup1-T20A, result in spontaneous and UV-induced mutator phenotypes . To elucidate the origin of these DNA repair phenotypes of the proteasomal mutants, we performed epistasis analysis, with respect to UV sensitivity, using yeast strains with the UMP1 deletion in different DNA repair backgrounds . We show that UMP1 is not epistatic to RAD23 and RAD2, which are involved in the nucleotide excision repair (NER) pathway . Instead, our results indicate that UMP1 as well as PUP1 and PRE2 (encoding catalytic subunits of 20S proteasome) belong to an epistatic group of genes functioning in post-replication DNA repair (PRR) and are hypostatic to RAD18, which, in complex with RAD6, plays a central role in PRR . We also show that UMP1 is epistatic to REV3 and RAD30, although the relationship of UMP1 with these genes is different.

Mol Microbiol, 2003 Sep, 49(5), 1267 - 85
Mos10 (Vps60) is required for normal filament maturation in Saccharomyces cerevisiae; Kohler JR; Early pseudohyphal growth of Saccharomyces cerevisiae is well described, and is known to be subject to a complex web of developmental regulation . In maturing filaments, young cells differ significantly from their pseudohyphal progenitors, in their shape, and in their timing and direction of cell division . The changes that occur during filament maturation result in round and oval cells surrounding and covering the pseudohyphal filament . In a screen for mutants that affect this process, a vacuolar protein sorting gene, MOS10 (VPS60), and a gene encoding an alpha subunit of the proteasome core, PRE9, were isolated . Characterization of the mos10/mos10 phenotype showed that the process of filament maturation is regulated differently from early filamentous growth, and that the requirement for Mos10 is limited to the maturation stage of pseudohyphal development . The mos10/mos10 phenotype is unlikely to be an unspecific effect of disruption of endocytosis or vacuolar protein sorting, because it is not recapitulated by mutants in other genes required for these processes . Disruption of homologues of MOS10, which act as components of the ESCRT-III complex in targeting proteins for vacuolar degradation, results in abnormal early pseudohyphal growth, not in the filament maturation defect seen in mos10/mos10 . Thus, Mos10 may function in targeting of specific cargo proteins for degradation, under conditions particular to maturing filaments.

Nature, 2003 Sep 11, 425(6954), 191 - 6 Epub 2003 Aug 24.
Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan; Howitz KT et al.; In diverse organisms, calorie restriction slows the pace of ageing and increases maximum lifespan . In the budding yeast Saccharomyces cerevisiae, calorie restriction extends lifespan by increasing the activity of Sir2 (ref . 1), a member of the conserved sirtuin family of NAD(+)-dependent protein deacetylases . Included in this family are SIR-2.1, a Caenorhabditis elegans enzyme that regulates lifespan, and SIRT1, a human deacetylase that promotes cell survival by negatively regulating the p53 tumour suppressor . Here we report the discovery of three classes of small molecules that activate sirtuins . We show that the potent activator resveratrol, a polyphenol found in red wine, lowers the Michaelis constant of SIRT1 for both the acetylated substrate and NAD(+), and increases cell survival by stimulating SIRT1-dependent deacetylation of p53 . In yeast, resveratrol mimics calorie restriction by stimulating Sir2, increasing DNA stability and extending lifespan by 70% . We discuss possible evolutionary origins of this phenomenon and suggest new lines of research into the therapeutic use of sirtuin activators.

J Biol Chem, 2003 Nov 14, 278(46), 45888 - 902 Epub 2003 Aug 25.
Identification of a novel TATA element-binding protein binding region at the N terminus of the Saccharomyces cerevisiae TAF1 protein; Takahata S et al.; TFIID, a multiprotein complex composed of TATA element-binding protein (TBP) and 14 TBP-associated factors (TAFs), can directly recognize core promoter elements and mediate transcriptional activation . The TAF N-terminal domain (TAND) of TAF1 may play a significant role in these two principal TFIID functions by regulating the access of TBP to the TATA element . In yeast, TAND consists of two subdomains, TAND1 (10-37 amino acids (aa)) and TAND2 (46-71 aa), which interact with the concave and convex surfaces of TBP, respectively . Here we demonstrate that another region located on the C-terminal side of TAND2 (82-139 aa) can also bind to TBP and induce transcriptional activation when tethered to DNA as a GAL4 fusion protein . As these properties are the same as those of TAND1, we denoted this sequence as TAND3 . Detailed mutational analyses revealed that three blocks of hydrophobic amino acid residues located within TAND3 are required not only for TBP binding and transcriptional activation but also for supporting cell growth and the efficient transcription of a subset of genes . We also show that the surface of TBP recognized by TAND3 is broader than that recognized by TAND1, although these regions overlap partially . Supporting these observations is that TAND1 can be at least partly functionally substituted by TAND3.

Bioessays, 2003 Sep, 25(9), 833 - 6
Mum, this bud's for you: where do you want it? Roles for Cdc42 in controlling bud site selection in Saccharomyces cerevisiae; Nelson WJ; The generation of asymmetric cell shapes is a recurring theme in biology . In budding yeast, one form of cell asymmetry occurs for division and is generated by anisotropic growth of the mother cell to form a daughter cell bud . Previous genetic studies uncovered key roles for the small GTPase Cdc42 in organizing the actin cytoskeleton and vesicle delivery to the site of bud growth, but a recent paper has also raised questions about how control of Cdc42 activity is integrated into a proposed hierarchical regulatory pathway that specifies a unique site of bud formation .

Proc Natl Acad Sci U S A, 2003 Sep 2, 100(18), 10275 - 80 Epub 2003 Aug 22.
ACE2 is required for daughter cell-specific G1 delay in Saccharomyces cerevisiae; Laabs TL et al.; Saccharomyces cerevisiae cells reproduce by budding to yield a mother cell and a smaller daughter cell . Although both mother and daughter begin G1 simultaneously, the mother cell progresses through G1 more rapidly . Daughter cell G1 delay has long been thought to be due to a requirement for attaining a certain critical cell size before passing the commitment point in the cell cycle known as START . We present an alternative model in which the daughter cell-specific Ace2 transcription factor delays G1 in daughter cells . Deletion of ACE2 produces daughter cells that proceed through G1 at the same rate as mother cells, whereas a mutant Ace2 protein that is not restricted to daughter cells delays G1 equally in both mothers and daughters . The differential in G1 length between mothers and daughters requires the Cln3 G1 cyclin, and CLN3-GFP reporter expression is reduced in daughters in an ACE2-dependent manner . Specific daughter delay elements in the CLN3 promoter are required for normal daughter G1 delay, and these elements bind to an unidentified 127-kDa protein . This DNA-binding activity is enhanced by deletion of ACE2 . These results support a model in which daughter cell G1 delay is determined not by cell size but by an intrinsic property of the daughter cell generated by asymmetric cell division.

J Biol Chem, 2003 Oct 31, 278(44), 43051 - 9 Epub 2003 Aug 22.
Regulation of S-adenosylmethionine levels in Saccharomyces cerevisiae; Chan SY et al.; Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, used to methylate homocysteine in methionine biosynthesis . Methionine can be activated by ATP to give rise to the universal methyl donor, S-adenosylmethionine (AdoMet) . Previously, a chimeric MTHFR (Chimera-1) comprised of the yeast Met13p N-terminal catalytic domain and the Arabidopsis thaliana MTHFR (AtMTHFR-1) C-terminal regulatory domain was constructed (Roje, S., Chan, S . Y., Kaplan, F., Raymond, R . K., Horne, D . W., Appling, D . R., and Hanson, A . D . (2002) J . Biol . Chem . 277, 4056-4061) . Engineered yeast (SCY4) expressing Chimera-1 accumulated more than 100-fold more AdoMet and 7-fold more methionine than the wild type . Surprisingly, SCY4 showed no appreciable growth defect . The ability of yeast to hyperaccumulate AdoMet was investigated by studying the intracellular compartmentation of AdoMet as well as the mode of hyperaccumulation . Previous studies have established that AdoMet is distributed between the cytosol and the vacuole . A strain expressing Chimera-1 and lacking either vacuoles (vps33 mutant) or vacuolar polyphosphate (vtc1 mutant) was not viable when grown under conditions that favored AdoMet hyperaccumulation . The hyperaccumulation of AdoMet was a robust phenomenon when these cells were grown in medium containing glycine and formate but did not occur when these supplements were replaced by serine . The basis of the nutrient-dependent AdoMet hyperaccumulation effect is discussed in relation to homocysteine biosynthesis and sulfur metabolism.

FEBS Lett, 2003 Aug 28, 550(1-3), 41 - 5
Polyphosphates strongly inhibit the tRNA dependent synthesis of poly(A) catalyzed by poly(A) polymerase from Saccharomyces cerevisiae; Sillero MA et al.; Polyphosphates of different chain lengths (P(3), P(4), P(15), P(35)), (1 microM) inhibited 10, 60, 90 and 100%, respectively, the primer (tRNA) dependent synthesis of poly(A) catalyzed poly(A) polymerase from Saccharomyces cerevisiae . The relative inhibition evoked by p(4)A and P(4) (1 microM) was 40 and 60%, respectively, whereas 1 microM Ap(4)A was not inhibitory . P(4) and P(15) were assayed as inhibitors of the enzyme in the presence of (a) saturating tRNA and variable concentrations of ATP and (b) saturating ATP and variable concentrations of tRNA . In (a), P(4) and P(15) behaved as competitive inhibitors, with K(i) values of 0.5 microM and 0.2 microM, respectively . In addition, P(4) (at 1 microM) and P(15) (at 0.3 microM) changed the Hill coefficient (n(H)) from 1 (control) to about 1.3 and 1.6, respectively . In (b), the inhibition by P(4) and P(15) decreased V and modified only slightly the K(m) values of the enzyme towards tRNA.

Genetics, 2003 Aug, 164(4), 1333 - 44
Depletion of H2A-H2B dimers in Saccharomyces cerevisiae triggers meiotic arrest by reducing IME1 expression and activating the BUB2-dependent branch of the spindle checkpoint; Hanlon SE et al.; In the yeast Saccharomyces cerevisiae, diploid strains carrying homozygous hta1-htb1Delta mutations express histone H2A-H2B dimers at a lower level than do wild-type cells . Although this mutation has only minor effects on mitotic growth, it causes an arrest in sporulation prior to the first meiotic division . In this report, we show that the hta1-htb1Delta mutant exhibits reduced expression of early and middle-sporulation-specific genes and that the meiotic arrest of the hta1-htb1Delta mutant can be partially bypassed by overexpression of IME1 . Additionally, deletions of BUB2 or BFA1, components of one branch of the spindle checkpoint pathway, bypass the meiotic arrest . Mutations in the other branch of the pathway or in the pachytene checkpoint are unable to suppress the meiotic block . These observations indicate that depletion of the H2A-H2B dimer blocks sporulation by at least two mechanisms: disruption of the expression of meiotic regulatory genes and activation of the spindle checkpoint . Our results show that the failure to progress through the meiotic pathway is not the result of global chromosomal alterations but that specific aspects of meiosis are sensitive to depletion of the H2A-H2B dimer.

Appl Microbiol Biotechnol, 2004 Mar, 64(1), 120 - 4 Epub 2003 Aug 19.
Nuclear thioredoxin peroxidase Dot5 in Saccharomyces cerevisiae: roles in oxidative stress response and disruption of telomeric silencing; Izawa S et al.; The DOT5 gene was originally cloned as one of the DOT (disrupter of telomeric silencing) genes; and later it was re-discovered as a nuclear thioredoxin peroxidase in Saccharomyces cerevisiae . Here, we demonstrate that the telomeric-silencing disruption activity of Dot5 is independent of thioredoxin peroxidase activity . In addition, Dot5 cannot suppress the increased susceptibility to peroxides of mutants defected in cytosolic thioredoxin peroxidase, even when Dot5 is expressed in the cytoplasm . Furthermore, Dot5 does not affect redox regulation of the Yap1 transcription factor . These results suggest that Dot5 is less important as an antioxidant in yeast cells.

Mol Biol Cell, 2003 Aug, 14(8), 3494 - 505 Epub 2003 May 03.
The Saccharomyces cerevisiae spindle pole body is a dynamic structure; Yoder TJ et al.; During spindle pole body (SPB) duplication, the new SPB is assembled at a distinct site adjacent to the old SPB . Using quantitative fluorescence methods, we studied the assembly and dynamics of the core structural SPB component Spc110p . The SPB core exhibits both exchange and growth in a cell cycle-dependent manner . During G1/S phase, the old SPB exchanges approximately 50% of old Spc110p for new Spc110p . In G2 little Spc110p is exchangeable . Thus, Spc110p is dynamic during G1/S and becomes stable during G2 . The SPB incorporates additional Spc110p in late G2 and M phases; this growth is followed by reduction in the next G1 . Spc110p addition to the SPBs (growth) also occurs in response to G2 and mitotic arrests but not during a G1 arrest . Our results reveal several dynamic features of the SPB core: cell cycle-dependent growth and reduction, growth in response to cell cycle arrests, and exchange of Spc110p during SPB duplication . Moreover, rather than being considered a conservative or dispersive process, the assembly of Spc110p into the SPB is more readily considered in terms of growth and exchange.

Mol Biol Cell, 2003 Aug, 14(8), 3126 - 43 Epub 2003 May 03.
Sudden telomere lengthening triggers a Rad53-dependent checkpoint in Saccharomyces cerevisiae; Viscardi V et al.; Telomeres are specialized functional complexes that ensure chromosome stability by protecting chromosome ends from fusions and degradation and avoiding chromosomal termini from being sensed as DNA breaks . Budding yeast Tel1 is required both for telomere metabolism and for a Rad53-dependent checkpoint responding to unprocessed double-strand breaks . We show that overexpression of a GAL1-TEL1 fusion causes transient telomere lengthening and activation of a Rad53-dependent G2/M checkpoint in cells whose telomeres are short due to the lack of either Tel1 or Yku70 . Sudden telomere elongation and checkpoint-mediated cell cycle arrest are also triggered in wild-type cells by overproducing a protein fusion between the telomeric binding protein Cdc13 and the telomerase-associated protein Est1 . Checkpoint activation by GAL1-TEL1 requires ongoing telomere elongation . In fact, it is turned off concomitantly with telomeres reaching a new stable length and is partially suppressed by deletion of the telomerase EST2 gene . Moreover, both telomere length rebalancing and checkpoint inactivation under galactose-induced conditions are accelerated by high levels of either the Sae2 protein, involved in double-strand breaks processing, or the negative telomere length regulator Rif2 . These data suggest that sudden telomere lengthening elicits a checkpoint response that inhibits the G2/M transition.

Mol Biol Cell, 2003 Aug, 14(8), 3097 - 113 Epub 2003 May 03.
Suppression of coatomer mutants by a new protein family with COPI and COPII binding motifs in Saccharomyces cerevisiae; Sandmann T et al.; Protein trafficking is achieved by a bidirectional vesicle flow between the various compartments of the eukaryotic cell . COPII coated vesicles mediate anterograde protein transport from the endoplasmic reticulum to the Golgi apparatus, whereas retrograde Golgi-to-endoplasmic reticulum vesicles use the COPI coat . Inactivation of COPI vesicle formation in conditional sec21 (gamma-COP) mutants rapidly blocks transport of certain proteins along the early secretory pathway . We have identified the integral membrane protein Mst27p as a strong suppressor of sec21-3 and ret1-1 mutants . A C-terminal KKXX motif of Mst27p that allows direct binding to the COPI complex is crucial for its suppression ability . Mst27p and its homolog Yar033w (Mst28p) are part of the same complex . Both proteins contain cytoplasmic exposed C termini that have the ability to interact directly with COPI and COPII coat complexes . Site-specific mutations of the COPI binding domain abolished suppression of the sec21 mutants . Our results indicate that overexpression of MST27 provides an increased number of coat binding sites on membranes of the early secretory pathway and thereby promotes vesicle formation . As a consequence, the amount of cargo that can bind COPI might be important for the regulation of the vesicle flow in the early secretory pathway.

Mol Cell Biol, 2003 Sep, 23(17), 6187 - 99
Phosphorylation of mammalian eukaryotic translation initiation factor 6 and its Saccharomyces cerevisiae homologue Tif6p: evidence that phosphorylation of Tif6p regulates its nucleocytoplasmic distribution and is required for yeast cell growth; Basu U et al.; The synthesis of 60S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eukaryotic translation initiation factor 6 (eIF6) . In the present work, we have isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate recombinant human eIF6 . Mass spectrometric analysis as well as antigenic properties of the purified kinase identified it as casein kinase I . The site of in vitro phosphorylation, which is highly conserved from yeast to mammals, was identified as the serine residues at positions 174 (major site) and 175 (minor site) . The homologous yeast protein Tif6p was also phosphorylated in vivo in yeast cells . Mutation of Tif6p at serine-174 to alanine reduced phosphorylation drastically and caused loss of cell growth and viability . When both Ser-174 and Ser-175 were mutated to alanine, phosphorylation of Tif6p was completely abolished . Furthermore, while wild-type Tif6p was distributed both in nuclei and the cytoplasm of yeast cells, the mutant Tif6p (with Ser174Ala and Ser175Ala) became a constitutively nuclear protein . These results suggest that phosphorylatable Ser-174 and Ser-175 play a critical role in the nuclear export of Tif6p.

Mol Cell Biol, 2003 Sep, 23(17), 5972 - 8
Set2-catalyzed methylation of histone H3 represses basal expression of GAL4 in Saccharomyces cerevisiae; Landry J et al.; Recent work has shown that histone methylation is an important regulator of transcription . While much is known about the roles of histone methyltransferases (HMTs) in the establishment of heterochromatin, little is known of their roles in the regulation of actively transcribed genes . We describe an in vivo role of the Saccharomyces cerevisiae HMT, Set2 . We identified SET2 as a gene necessary for repression of GAL4 basal expression and show that the evolutionarily conserved SACI, SACII, and SET domains of Set2 are necessary for this repression . We confirm that Set2 catalyzes methylation of lysine 36 on the N-terminal tail of histone H3 . Conversion of lysine 36 to an unmethylatable arginine causes a decrease in the repression of GAL4 transcription, as does a Delta set2 mutation . We further show that lysine 36 of histone H3 at GAL4 is methylated and that this methylation is dependent upon the presence of SET2.

Plant Physiol, 2003 Aug, 132(4), 2152 - 65
Systematic trans-genomic comparison of protein kinases between Arabidopsis and Saccharomyces cerevisiae; Wang D et al.; The genome of the budding yeast (Saccharomyces cerevisiae) provides an important paradigm for transgenomic comparisons with other eukaryotic species . Here, we report a systematic comparison of the protein kinases of yeast (119 kinases) and a reference plant Arabidopsis (1,019 kinases) . Using a whole-protein-based, hierarchical clustering approach, the complete set of protein kinases from both species were clustered . We validated our clustering by three observations: (a) clustering pattern of functional orthologs proven in genetic complementation experiments, (b) consistency with reported classifications of yeast kinases, and (c) consistency with the biochemical properties of those Arabidopsis kinases already experimentally characterized . The clustering pattern identified no overlap between yeast kinases and the receptor-like kinases (RLKs) of Arabidopsis . Ten more kinase families were found to be specific for one of the two species . Among them, the calcium-dependent protein kinase and phosphoenolpyruvate carboxylase kinase families are specific for plants, whereas the Ca(2+)/calmodulin-dependent protein kinase and provirus insertion in mouse-like kinase families were found only in yeast and animals . Three yeast kinase families, nitrogen permease reactivator/halotolerance-5), polyamine transport kinase, and negative regulator of sexual conjugation and meiosis, are absent in both plants and animals . The majority of yeast kinase families (21 of 26) display Arabidopsis counterparts, and all are mapped into Arabidopsis families of intracellular kinases that are not related to RLKs . Representatives from 11 of the common families (54 kinases from Arabidopsis and 17 from yeast) share an extremely high degree of similarity (blast E value < 10(-80)), suggesting the likelihood of orthologous functions . Selective expansion of yeast kinase families was observed in Arabidopsis . This is most evident for yeast genes CBK1, HRR25, and SNF1 and the kinase family S6K . Reduction of kinase families was also observed, as in the case of the NEK-like family . The distinguishing features between the two sets of kinases are the selective expansion of yeast families and the generation of a limited number of new kinase families for new functionality in Arabidopsis, most notably, the Arabidopsis RLKs that constitute important components of plant intercellular communication apparatus.

J Biol Chem, 2003 Oct 24, 278(43), 41849 - 55 Epub 2003 Aug 11.
Lipid hydroperoxides activate the mitogen-activated protein kinase Mpk1p in Saccharomyces cerevisiae; Alic N et al.; Saccharomyces cerevisiae is capable of responding to oxidants, including lipid peroxidation products . We investigate here the role of the mitogen-activated protein kinase Mpk1p in protection against linoleic acid hydroperoxide (LoaOOH), a product of radical attack on an unsaturated lipid . MPK1 was found to be required for resistance to LoaOOH . Furthermore, Mpk1p was rapidly and transiently phosphorylated in response to LoaOOH . This phosphorylation was dose-dependent and stimulated by sublethal concentrations as low as 1 mum in the external medium . Such low doses have been shown to result in resistance to subsequent challenge with a higher dose through the process of adaptation . However MPK1 was not essential for this adaptive response . MPK1 was also not involved in cell cycle modulation and acted independently of the cell cycle-regulating Oca1p . Transcriptional profiling of the mpk1Delta cells during LoaOOH stress indicated that Mpk1p may be important in effecting changes to the cell surface and metabolism during LoaOOH exposure . Furthermore, it revealed that Mpk1p is required for the regulation of 97 LoaOOH-responsive transcripts . Evidence is presented that the activation of Mpk1p may be caused by the activation of protein kinase C by LoaOOH.

Eukaryot Cell, 2003 Aug, 2(4), 821 - 5
Specific protein targeting during cell differentiation: polarized localization of Fus1p during mating depends on Chs5p in Saccharomyces cerevisiae; Santos B et al.; In budding yeast, chs5 mutants are defective in chitin synthesis and cell fusion during mating . Chs5p is a late-Golgi protein required for the polarized transport of the chitin synthase Chs3p to the membrane . Here we show that Chs5p is also essential for the polarized targeting of Fus1p, but not of other cell fusion proteins, to the membrane during mating.

Eukaryot Cell, 2003 Aug, 2(4), 809 - 20
POS5 gene of Saccharomyces cerevisiae encodes a mitochondrial NADH kinase required for stability of mitochondrial DNA; Strand MK et al.; In a search for nuclear genes that affect mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae, an ATP-NAD (NADH) kinase, encoded by POS5, that functions exclusively in mitochondria was identified . The POS5 gene product was overproduced in Escherichia coli and purified without a mitochondrial targeting sequence . A direct biochemical assay demonstrated that the POS5 gene product utilizes ATP to phosphorylate both NADH and NAD(+), with a twofold preference for NADH . Disruption of POS5 increased minus-one frameshift mutations in mitochondrial DNA 50-fold, as measured by the arg8(m) reversion assay, with no increase in nuclear mutations . Also, a dramatic increase in petite colony formation and slow growth on glycerol or limited glucose were observed . POS5 was previously described as a gene required for resistance to hydrogen peroxide . Consistent with a role in the mitochondrial response to oxidative stress, a pos5 deletion exhibited a 28-fold increase in oxidative damage to mitochondrial proteins and hypersensitivity to exogenous copper . Furthermore, disruption of POS5 induced mitochondrial biogenesis as a response to mitochondrial dysfunction . Thus, the POS5 NADH kinase is required for mitochondrial DNA stability with a critical role in detoxification of reactive oxygen species . These results predict a role for NADH kinase in human mitochondrial diseases.

Biochemistry, 2003 Aug 19, 42(32), 9575 - 85
Nucleotide-induced conformational changes in the Saccharomyces cerevisiae SR protein kinase, Sky1p, revealed by X-ray crystallography; Nolen B et al.; Conformational changes are thought to play a key role in the function of active protein kinases, although little is known about how these changes relate to the mechanism of phosphorylation . Here we present four high-resolution structures of a single crystal form of Sky1p, a constitutively active serine kinase implicated in yeast RNA processing, each in a different state of nucleotide binding . By comparing the apoenzyme structure to the ADP- and ATP-bound Sky1p structures, we have revealed conformational changes caused by ATP binding or conversion from nucleotide reactant to product . Rotation of the small lobe of the kinase closes the cleft upon binding, allowing the nucleotide to interact with residues from both lobes of the kinase, although some interactions thought to be important for phosphotransfer are missing in the ATP-containing structure . In the apoenzyme, a kinase-conserved phosphate-anchoring loop is in a twisted conformation that is incompatible with ADP and ATP binding, providing a potential mechanism for facilitating ADP release in Sky1p . The nonhydrolyzable ATP analogue AMP-PNP binds in a unique mode that fails to induce lobe closure . This observation, along with comparisons between the two independent molecules in the asymmetric unit of each structure, has provided new molecular details about how the nucleotide binds and induces closure . Finally, we have used mutational analysis to establish the importance of a glycine within the linker that connects the two lobes of Sky1p.

Extremophiles, 2003 Aug, 7(4), 291 - 8 Epub 2003 Apr 09.
Piezophysiology of genome wide gene expression levels in the yeast Saccharomyces cerevisiae; Iwahashi H et al.; Hydrostatic pressure is one of the physical factors affecting cellular physiology . Hydrostatic pressure of a few hundred MPa decreases the viability of yeast cells, and pressure of a few tens MPa decreases the growth rate . To understand the effect of hydrostatic pressure, we employed yeast DNA microarrays and analyzed genome-wide gene-expression levels after the pressure treatment with 180 MPa (immediate) at 4 degrees C and recovery incubation for 1 h and 40 MPa (16 h) at 4 degrees C and recovery incubation for 1 h . The transcription of genes involved in energy metabolism, cell defense, and protein metabolism was significantly induced by the pressure treatment . Genome-wide expression profiles suggested that high pressure caused damage to cellular organelles, since the induced gene products were localized in the membrane structure and/or cellular organelles . Hierarchical clustering analysis suggested that the damage caused by the pressure was similar to that caused by detergents, oils, and freezing/thawing . We also estimated the contribution of induced genes to barotolerance using some strains that have the deletion in the corresponding genes.

Appl Microbiol Biotechnol, 2004 Feb, 63(6), 734 - 41 Epub 2003 Aug 09.
Response to different environmental stress conditions of industrial and laboratory Saccharomyces cerevisiae strains; Garay-Arroyo A et al.; Two sets of Saccharomyces cerevisiae strains were compared for their physiological responses to different stress conditions . One group is composed of three strains adapted to controlled laboratory conditions (CEN.PK, LR88 and RS58), whereas the other consisted of five industrial strains (IND1101, SuperStart, LO24, LO41 and Azteca) . Most industrial strains showed higher tolerance to heat shock and to an oxidative environment than laboratory strains . Excluding CEN.PK, a similar behavior was observed regarding ethanol production in high sugar concentrations (180 g/l glucose) . Addition of acetate (10 g/l) or furfural (2 g/l), in concentrations similar to those found in sugar cane bagasse hydrolysates, decreased cell mass formation and growth rate in almost all strains . CEN.PK and SuperStart showed the highest sensitivity when grown in furfural-containing medium . Acetic acid treatment severely affected cell mass formation and reduced growth rate in all strains; CEN.PK and LO24 were the most resistant . The specific ethanol production rate was not affected by furfural addition . However, specific ethanol production rates decreased in response to acetic acid in four industrial strains, and increased in all laboratory strains and in LO24 . No significant correlation was found between the stress tolerance of the strains tested and the transcript accumulation of genes selected by their involvement in the response to each of the stressful environments applied.

J Biol Chem, 2003 Oct 24, 278(43), 41607 - 17 Epub 2003 Aug 06.
Plasmid accumulation reduces life span in Saccharomyces cerevisiae; Falcon AA et al.; Aging in the yeast Saccharomyces cerevisiae is under the control of multiple pathways . The production and accumulation of extrachromosomal rDNA circles (ERCs) is one pathway that has been proposed to bring about aging in yeast . To test this proposal, we have developed a plasmid-based model system to study the role of DNA episomes in reduction of yeast life span . Recombinant plasmids containing different replication origins, cis-acting partitioning elements, and selectable marker genes were constructed and analyzed for their effects on yeast replicative life span . Plasmids containing the ARS1 replication origin reduce life span to the greatest extent of the plasmids analyzed . This reduction in life span is partially suppressed by a CEN4 centromeric element on ARS1 plasmids . Plasmids containing a replication origin from the endogenous yeast 2 mu circle also reduce life span, but to a lesser extent than ARS1 plasmids . Consistent with this, ARS1 and 2 mu origin plasmids accumulate in approximately 7-generation-old cells, but ARS1/CEN4 plasmids do not . Importantly, ARS1 plasmids accumulate to higher levels in old cells than 2 mu origin plasmids, suggesting a correlation between plasmid accumulation and life span reduction . Reduction in life span is neither an indirect effect of increased ERC levels nor the result of stochastic cessation of growth . The presence of a fully functional 9.1-kb rDNA repeat on plasmids is not required for, and does not augment, reduction in life span . These findings support the view that accumulation of DNA episomes, including episomes such as ERCs, cause cell senescence in yeast.

Mikrobiologiia, 2003 May-Jun, 72(3), 308 - 11
{Arsenite-induced lipid peroxidation in Saccharomyces cerevisiae}; Samokhvalov VA et al.; The ability of sodium arsenite at concentrations of 10(-2), 10(-4), and 10(-6) M to induce lipid peroxidation in Saccharomyces cerevisiae cells was studied . Arsenite at the concentrations 10(-2) and 10(-4) M enhanced lipid peroxidation and inhibited the growth of yeast cells . Enhanced lipid peroxidation likely induced oxidative damage to various cellular structures, which led to suppression of the metabolic activity of cells . Arsenite at the concentration 10(-6) M did not activate lipid peroxidation in cells . All of the tested arsenite concentrations inhibited the activity of alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase in cells . The inference is made that the toxicity of arsenite may be related to its stimulating effect on intracellular lipid peroxidation.

J Biol Chem, 2003 Oct 10, 278(41), 39632 - 6 Epub 2003 Aug 04.
Involvement of the VDE homing endonuclease and rapamycin in regulation of the Saccharomyces cerevisiae GSH11 gene encoding the high affinity glutathione transporter; Miyake T et al.; The Saccharomyces cerevisiae gene HGT1/GSH11 encodes the high affinity glutathione transporter and is repressed by cysteine added to the culture medium . It has been found previously that a 5'-upstream cis-element, CCGCCACAC, is responsible for regulating GSH11 expression and that several proteins bind to this element (Miyake, T., Kanayama, M., Sammoto, H., and Ono, B . (2002) Mol . Genet . Genomics 266, 1004-1011) . In this report we present evidence that the most prominent of these proteins is VDE, known previously as the homing endonuclease encoded by VMA1 . We show also that GSH11 is not expressed in a VDE-deleted strain and that inability to express the GSH11 of this strain is overcome by introduction of the coding region of VDE or the entire VMA1 gene . It is also found that VDE does not cut DNA in the vicinity of the GSH11 cis-element . Rapamycin, an inhibitor of the target of rapamycin (TOR) signal-transduction system, is found to enhance expression of GSH11 in a VDE-dependent manner under conditions of sulfur starvation . These results indicate that GSH11 is regulated by a system sensitive to sulfur starvation (presumably via cysteine depletion) and a more general system involving the nutritional starvation signal mediated by the TOR system . Both systems need to be operational (inhibition of TOR and sulfur starvation) for full expression of GSH11.

Yeast, 2003 Aug, 20(11), 943 - 54
Studies on the ATP3 gene of Saccharomyces cerevisiae: presence of two closely linked copies, ATP3a and ATP3b, on the right arm of chromosome II; Ohnishi K et al.; In this paper, we present evidence that there are two closely linked copies of the ATP3 gene coding for the gamma subunit of the F(1)F(0)-ATPase complex (EC3.6.1.34) in four laboratory strains of Saccharomyces cerevisiae, even though the yeast genome project has reported that ATP3 is a single-copy gene on chromosome II . We previously reported that the gene dosage (three copies) of ATP1 and ATP2 is coincident with the subunit number of F(1)-alpha and F(1)-beta, but that the gene dosage of ATP3 was not consistent with the subunit stoichiometry of F(1)F(0)-ATPase . By applying long PCR and gene walking analyses, we estimated that the two copies of ATP3 were approximately 20 kb apart, and we designated that which is proximal to the centromere ATP3a, while we named that which is distal ATP3b . The nucleotide sequences of the two copies of ATP3 were identical to the reported sequence in the W303-1A, W303-1B and LL20 strains, while only the DC5 strain had a single base substitution in its ATP3a . With the exception of this substitution, the other nucleotide sequences were identical to the upstream 860 bp and the downstream 150 bp . The differences between ATP3 with the single base substitution (Ser(308) to Phe) and ATP3 without the substitution on the complementation of the ATP3 disruptant and on the maintenance of the mitochondrial DNA were observed, suggesting that Atp3ap and Atp3bp in the DC5 strain might have different functions . However, it should not always be necessary for yeast cells to carry different types of ATP3 because the other three strains carry the same type of ATP3 . It was also demonstrated that the disruption of the ATP3 genes basically leads to a loss of wild-type mtDNA, but the stability of the mtDNA is not dependent on the ATP3 alone .

Can J Microbiol, 2003 May, 49(5), 336 - 43
Toxic effects caused by heavy metals in the yeast Saccharomyces cerevisiae: a comparative study; Soares EV et al.; The decreasing order of toxicity of select heavy metals on the yeast Saccharomyces cerevisiae, in 10 mM MES (2-(N-morpholino)ethanesulfonic acid) pH buffer at pH 6.0, was found to be copper, lead, and nickel . Heavy metal (200 microM) induced a decrease in the number of viable cells by about 50% in the first 5 min for copper and in 4 h for lead, while nickel was not toxic up to a 200 microM concentration over a period of 48 h . Glucose (25 mM) strongly enhanced the toxic effect of 50 microM copper but had little or no effect on the toxicity of 200 microM lead or nickel . Copper, lead, and nickel induced the leakage of UV260-absorbing compounds from cells with different kinetics . The addition of 0.5 mM calcium, before addition of 200 microM copper, showed a protective action against cell death and decreased the release of UV-absorbing compounds, while no effect was observed against lead or nickel toxic effects . Copper complexation capacities of the filtrates of cells exposed for 2 h in 200 microM copper and 24 h in 200 microM lead were 51 and 14 microM, respectively . The implication of the complexation shown by these soluble compounds in the bioavailability of heavy metals is discussed.

Nat Genet, 2003 Sep, 35(1), 57 - 64 Epub 2003 Aug 03.
Trans-acting regulatory variation in Saccharomyces cerevisiae and the role of transcription factors; Yvert G et al.; Natural genetic variation can cause significant differences in gene expression, but little is known about the polymorphisms that affect gene regulation . We analyzed regulatory variation in a cross between laboratory and wild strains of Saccharomyces cerevisiae . Clustering and linkage analysis defined groups of coregulated genes and the loci involved in their regulation . Most expression differences mapped to trans-acting loci . Positional cloning and functional assays showed that polymorphisms in GPA1 and AMN1 affect expression of genes involved in pheromone response and daughter cell separation, respectively . We also asked whether particular classes of genes were more likely to contain trans-regulatory polymorphisms . Notably, transcription factors showed no enrichment, and trans-regulatory variation seems to be broadly dispersed across classes of genes with different molecular functions.

Mol Cell Biol, 2003 Aug, 23(16), 5502 - 15
Degradation of normal mRNA in the nucleus of Saccharomyces cerevisiae; Das B et al.; A nuclear mRNA degradation (DRN) system was identified from analysis of mRNA turnover rates in nup116-Delta strains of Saccharomyces cerevisiae lacking the ability to export all RNAs, including poly(A) mRNAs, at the restrictive temperature . Northern blotting, in situ hybridization, and blocking transcription with thiolutin in nup116-delta strains revealed a rapid degradation of mRNAs in the nucleus that was suppressed by the rrp6-delta, rai1-delta, and cbc1-delta deletions, but not by the upf1-delta deletion, suggesting that DRN requires Rrp6p, a 3'-to-5' nuclear exonuclease, the Rat1p, a 5'-to-3' nuclear exonuclease, and Cbc1p, a component of CBC, the nuclear cap binding complex, which may direct the mRNAs to the site of degradation . We propose that certain normal mRNAs retained in the nucleus are degraded by the DRN system, similar to degradation of transcripts with 3' end formation defects in certain mutants.

Biochim Biophys Acta, 2003 Aug 7, 1614(2), 131 - 4
Potent competitive inhibition of drug binding to the Saccharomyces cerevisiae ABC exporter Pdr5p by the hydrophobic estradiol-derivative RU49953; Conseil G et al.; The hydrophobic estradiol-derivative RU49953 inhibits the energy-dependent interaction of yeast multidrug-transporter Pdr5p with its fluorescent drug-substrate rhodamine 6G . The potent inhibition is competitive towards drug binding (Ki=23+/-6 nM), whereas nucleoside-triphosphate hydrolysis is two-orders-of-magnitude less sensitive . RU49953 constitutes the most efficient inhibitor of drug binding to a yeast multidrug ABC exporter reported so far.

J Biol Chem, 2003 Sep 26, 278(39), 36985 - 8 Epub 2003 Jul 31.
Projection structure of the atractyloside-inhibited mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae; Kunji ER et al.; ADP/ATP carriers in the inner mitochondrial membrane catalyze the exchange of cytosolic ADP for ATP synthesized in the mitochondrial matrix by ATP synthase and thereby replenish the eukaryotic cell with metabolic energy . The yeast ADP/ATP carrier (AAC3) was overexpressed, inhibited by atractyloside, purified, and reconstituted into two-dimensional crystals . Images of frozen hydrated crystals were recorded by electron microscopy, and a projection structure was calculated to 8-A resolution . The AAC3 molecule has pseudo 3-fold symmetry in agreement with the 3-fold sequence repeats that are typical of members of the mitochondrial carrier family . The density distribution is consistent with a bundle of six transmembrane alpha-helices with two or three short alpha-helical extensions closing the central pore on the matrix side . The AAC3 molecules in the crystal are arranged in symmetrical homo-dimers, but the translocation pore for adenine nucleotides lies in the center of the molecule and not along the dyad axis of the dimer.

DNA Repair (Amst), 2003 Aug 12, 2(8), 925 - 40
Complementary functions of the Saccharomyces cerevisiae Rad2 family nucleases in Okazaki fragment maturation, mutation avoidance, and chromosome stability; Sun X et al.; Rad2 family nucleases, identified by sequence similarity within their catalytic domains, function in multiple pathways of DNA metabolism . Three members of the Saccharomyces cerevisiae Rad2 family, Rad2, Rad27, and exonuclease 1 (Exo1), exhibit both 5' exonuclease and flap endonuclease activities . Deletion of RAD27 results in defective Okazaki fragment maturation, DNA repair, and subsequent defects in mutation avoidance and chromosomal stability . However, strains lacking Rad27 are viable . The expression profile of EXO1 during the cell cycle is similar to that of RAD27 and other genes encoding proteins that function in DNA replication and repair, suggesting Exo1 may function as a back up nuclease for Rad27 in DNA replication . We show that overexpression of EXO1 suppresses multiple rad27 null mutation-associated phenotypes derived from DNA replication defects, including temperature sensitivity, Okazaki fragment accumulation, the rate of minichromosome loss, and an elevated mutation frequency . While generally similar findings were observed with RAD2, overexpression of RAD2, but not EXO1, suppressed the MMS sensitivity of the rad27 null mutant cells . This suggests that Rad2 can uniquely complement Rad27 in base excision repair (BER) . Furthermore, Rad2 and Exo1 complemented the mutator phenotypes and cell cycle defects of rad27 mutant strains to differing extents, suggesting distinct in vivo nucleic acid substrates.

Proc Natl Acad Sci U S A, 2003 Sep 2, 100(18), 10353 - 7 Epub 2003 Jul 30.
Manganese activation of superoxide dismutase 2 in Saccharomyces cerevisiae requires MTM1, a member of the mitochondrial carrier family; Luk E et al.; Manganese-containing superoxide dismutase (SOD2) plays a critical role in guarding against mitochondrial oxidative stress and is essential for survival of many organisms . Despite the recognized importance of SOD2, nothing is known regarding the mechanisms by which this nuclear-encoded protein is converted to an active enzyme in the mitochondrial matrix . To search for factors that participate in the posttranslational activation of SOD2, we screened for yeast genes that when mutated lead to SOD2 inactivation and identified a single ORF, YGR257c . The encoded protein localizes to the mitochondria and represents a member of the yeast mitochondrial carrier family . YGR257c was previously recognized as the homologue to human CGI-69, a widely expressed mitochondrial carrier family of unknown function . Our studies suggest a connection with SOD2, and we have named the yeast gene MTM1 for manganese trafficking factor for mitochondrial SOD2 . Inactivation of yeast MTM1 leads to loss of SOD2 activity that is restored only when cells are treated with high supplements of manganese, but not other heavy metals, indicative of manganese deficiency in the SOD2 polypeptide . Surprisingly, the mitochondrial organelle of mtm1 Delta mutants shows no deficiency in manganese levels . Moreover, mtm1 Delta mutations do not impair activity of a cytosolic version of manganese SOD . We propose that Mtm1p functions in the mitochondrial activation of SOD2 by specifically facilitating insertion of the essential manganese cofactor.

Nucleic Acids Res . 2003 Aug 1;31(15):e84.
Parallel competition analysis of Saccharomyces cerevisiae strains differing by a single base using polymerase colonies; Merritt J et al.; We describe a strategy to analyze the impact of single nucleotide mutations on protein function . Our method utilizes a combination of yeast functional complementation, growth competition of mutant pools and polyacrylamide gel immobilized PCR . A system was constructed in which the yeast PGK1 gene was expressed from a plasmid-borne copy of the gene in a PGK1 deletion strain of Saccharomyces cerevisiae . Using this system, we demonstrated that the enrichment or depletion of PGK1 point mutants from a mixed culture was consistent with the expected results based on the isolated growth rates of the mutants . Enrichment or depletion of individual point mutants was shown to result from increases or decreases, respectively, in the specific activities of the encoded proteins . Further, we demonstrate the ability to analyze the functional effect of many individual point mutations in parallel . By functional complementation of yeast deletions with human homologs, our technique could be readily applied to the functional analysis of single nucleotide polymorphisms in human genes of medical interest.

Nucleic Acids Res, 2003 Aug 1, 31(15), 4541 - 52
Roles of Saccharomyces cerevisiae DNA polymerases Poleta and Polzeta in response to irradiation by simulated sunlight; Kozmin SG et al.; Sunlight causes lesions in DNA that if unrepaired and inaccurately replicated by DNA polymerases yield mutations that result in skin cancer in humans . Two enzymes involved in translesion synthesis (TLS) of UV-induced photolesions are DNA polymerase eta (Poleta) and polymerase zeta (Polzeta), encoded by the RAD30A and REV3 genes, respectively . Previous studies have investigated the TLS roles of these polymerases in human and yeast cells irradiated with monochromatic, short wavelength UVC radiation (254 nm) . However, less is known about cellular responses to solar radiation, which is of higher and mixed wavelengths (310-1100 nm) and produces a different spectrum of DNA lesions, including Dewar photoproducts and oxidative lesions . Here we report on the comparative cytotoxic and mutagenic effects of simulated sunlight (SSL) and UVC radiation on yeast wild-type, rad30Delta, rev3Delta and rev3Delta rad30Delta strains . The results with SSL support several previous interpretations on the roles of these two polymerases in TLS of photodimers and (6-4) photoproducts derived from studies with UVC . They further suggest that Poleta participates in the non-mutagenic bypass of SSL-dependent cytosine-containing Dewar photoproducts and 8-oxoguanine, while Polzeta is mainly responsible for the mutagenic bypass of all types of Dewar photoproducts . They also suggest that in the absence of Polzeta, Poleta contributes to UVC- and SSL-induced mutagenesis, possibly by the bypass of photodimers containing deaminated cytosine.

Biochem J, 2003 Sep 15, 374(Pt 3), 607 - 11
Identification of the mitochondrial pyruvate carrier in Saccharomyces cerevisiae; Hildyard JC et al.; Mitochondrial pyruvate transport is fundamental for metabolism and mediated by a specific inhibitable carrier . We have identified the yeast mitochondrial pyruvate carrier by measuring inhibitor-sensitive pyruvate uptake into mitochondria from 18 different Saccharomyces cerevisiae mutants, each lacking an unattributed member of the mitochondrial carrier family (MCF) . Only mitochondria from the YIL006w deletion mutant exhibited no inhibitor-sensitive pyruvate transport, but otherwise behaved normally . YIL006w encodes a 41.9 kDa MCF member with homologous proteins present in both the human and mouse genomes.

Genetika, 2003 Jun, 39(6), 739 - 47
{The genetic control of cell growth and development in yeast Saccharomyces cerevisiae . Disturbed sporulation in diploids with decreased activity of the Ras/cAMP signal transduction pathway}; Rakauskaite R et al.; Seven haploid strains (four with the MAT alpha mating type and three with the MATa mating type) were selected from the Peterhof genetic collection of yeast . Previous phenotypic analysis assigned six of these strains to a physiological group of strains with a lower activity of the Ras/cAMP signal transduction pathway . The haploids were crossed, and the resulting 12 diploids showed higher glycogen accumulation, tolerance to heat shock and nitrogen starvation, and sporulation in complete media . Ten of the diploids expressed the hypersporulation phenotype (higher sporulation efficiency) . The phenotypic characters of these ten diploids suggested a reduced activity of the Ras/cAMP pathway . All 12 diploids were tested for sporulation and production of two groups of asci (those with one or two spores and those with three or four spores) as dependent on culture conditions (21, 30, or 34 degrees C; standard sporulation medium or a complete medium containing potassium acetate or glycerol in place of glucose) . Sporulation proved to depend on temperature and medium composition . The results are collated with the data on yeast phenotypes associated with a lower activity of the Ras/cAMP signal transduction pathway.

Genetika, 2003 Jun, 39(6), 732 - 8
{Effect of the pho85 mutation on the catabolite repression of the CIT1 gene in yeasts Saccharomyces cerevisiae}; Padkina MV et al.; The Krebs cycle is one of the major metabolic pathways in a cell, which includes both catabolic and anabolic reactions . The first enzyme of the Krebs cycle, citrate synthase, catalyzes one of a few irreversible reactions of the cycle, citrate formation from acetyl-CoA and oxaloacetate . Expression of the CIT1 gene encoding the mitochondrial form of this enzyme in Saccharomyces cerevisiae is repressed on glucose- and glutamate-containing medium and activated on the raffinose-containing medium . In this work, the dependence of glucose repression of the CIT1 gene on the content of phosphate in the medium was studied . On the phosphate-deficient medium, the level of the CIT1 gene expression was increased twice . A low-molecular-weight (about 34 kDa) protein was identified and shown to interact with a region of the CIT1 gene promoter (from -367 to -346 bp), which controls the glucose repression . The results obtained suggest that the Pho4 protein is involved in regulation of the CIT1 gene expression on the glucose-containing and phosphate-deficient medium . Disruption of the PHO85 gene encoding phosphoprotein kinase (Pho4p is the substrate of this enzyme) leads to alleviation of glucose repression of the CIT1 gene . Thus, in yeast cells grown in the presence of glucose, the PHO85 gene mediates downregulation of the CIT1 expression.

Biochemistry (Mosc), 2003 May, 68(5), 577 - 81
Effect of inhibitors on polyphosphate metabolism in the yeast Saccharomyces cerevisiae under hypercompensation conditions; Trilisenko LV et al.; After re-inoculation of the yeast Saccharomyces cerevisiae from phosphate-deficient to complete medium, the total content of polyphosphates increased tenfold during 2 h (hypercompensation), but the content of certain fractions increased differently . The content of acid-soluble polyphosphate increased to the maximal extent . The ratio of the activities of two exopolyphosphatases also changed in the cytosol . Activity of a low molecular weight exopolyphosphatase (40 kD) decreased almost twice, whereas activity of a high molecular weight exopolyphosphatase (830 kD) increased tenfold . Cycloheximide blocks the increase in activity of high molecular weight exopolyphosphatase and hence, under these conditions the latter is synthesized de novo . Inhibitors of energy metabolism and cycloheximide, an inhibitor of protein synthesis, differently influence accumulation of certain polyphosphate fractions under hypercompensation conditions . The effect of iodoacetamide, an inhibitor of glycolysis, on any fraction is negligible, while cycloheximide suppresses accumulation of only polyP4 fraction associated with the cell envelope and bafilomycin A1, an inhibitor of vacuolar H+-ATPase, suppresses accumulation of polyP3 fraction . The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) to variable extent inhibits accumulation of all the fractions . Analysis of the effect of inhibitors on accumulation of polyphosphates under hypercompensation conditions confirms various localization, heterogeneity, and multiplicity of the routes of biosynthesis of certain fractions of these macroergic phosphorus compounds and also suggests interrelation between their biosynthesis and the gradient of H+ electrochemical potential.

Biotechnol Lett, 2003 Feb, 25(4), 331 - 4
Saccharomyces cerevisiae expression vectors with thrombin-cleavable N- and C-terminal 6x(His) tags; Patel O et al.; General-purpose yeast expression vectors for convenient cloning and production of proteins with N- or C-terminal His6 tags that can be efficiently removed with thrombin have been developed . To the parental yeast-E . coli shuttle vectors that have convenient copper-inducible expression, two selectable markers and LEU2d vector amplification, this development adds substantial versatility to product recovery.

Biotechnol Lett, 2003 May, 25(9), 671 - 4
Sol-gel immobilisation of Saccharomyces cerevisiae enhances viability in organic media; Desimone MF et al.; The polyhydroxylated silane network of a sol-gel protected immobilised Saccharomyces cerevisiae against the effects of five organic solvents . The viability of immobilised yeast directly correlated with the logarithm of the partition coefficient of the solvent in an octanol/water two phase system increasing the decimal reduction time (D) and reaching the maximum with octanol, the most hydrophobic solvent assayed . The D value increased from 0.16 min for free yeast to 1.9 and to 22 min for immobilised yeast exposed to ethanol and 1-octanol respectively.

Biotechnol Lett, 2003 May, 25(10), 783 - 9
A novel technique to evaluate interactions between Saccharomyces cerevisiae cell wall and mycotoxins: application to zearalenone; Yiannikouris A et al.; Three models based on sigmoidal plotting were tested for their ability to describe zearalenone adsorption on Saccharomyces cerevisiae cell walls in vitro . All three models closely fitted the experimental data, but Hill's equation gave the most accurate parameters, and provided information on the physical and chemical mechanisms involved in the adsorption of mycotoxin on yeast cell walls.

Biotechnol Lett, 2003 May, 25(10), 773 - 8
Overexpression of a sterol C-24(28) reductase increases ergosterol production in Saccharomyces cerevisiae; He X et al.; Three plasmids, pHX4, pHXA4 and pHXC4, containing sterol C-24(28) reductase gene (ERG4) under the control of ERG4, ADH1 or CUP1 promoters, respectively, and the copper resistance gene as the selection marker were constructed, and they were then introduced into Saccharomyces cerevisiae . Ergosterol production in recombinant strains was enhanced . Under the optimal culture condition, ergosterol content in recombinant strains YEH56(pHX4), YEH56(pHXA4) and YEH56(pHXC4) was 1.2, 1.4 and 1.5-fold (47 mg g-1) of that in the original strain.

J Biol Chem, 2003 Oct 3, 278(40), 38461 - 9 Epub 2003 Jul 23.
Fig1p facilitates Ca2+ influx and cell fusion during mating of Saccharomyces cerevisiae; Muller EM et al.; During the mating process of yeast cells, two Ca2+ influx pathways become activated . The resulting elevation of cytosolic free Ca2+ activates downstream signaling factors that promote long term survival of unmated cells, but the roles of Ca2+ in conjugation have not been described . The high affinity Ca2+ influx system is composed of Cch1p and Mid1p and sensitive to feedback inhibition by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase . To identify components and regulators of the low affinity Ca2+ influx system (LACS), we screened a collection of pheromone-responsive genes that when deleted lead to defects in LACS activity but not high affinity Ca2+ influx system activity . Numerous factors implicated in polarized morphogenesis and cell fusion (Fus1p, Fus2p, Rvs161p, Bni1p, Spa2p, and Pea2p) were found to be necessary for LACS activity . Each of these factors was also required for activation of the cell integrity mitogen-activated protein kinase cascade during the response to alpha-factor . Interestingly a polytopic plasma membrane protein, Fig1p, was required for LACS activity but not required for activation of Mpk1p mitogen-activated protein kinase . Mpk1p was not required for LACS activity, suggesting Mpk1p and Fig1p define two independent branches in the pheromone response pathways . Fig1p-deficient mutants exhibit defects in the cell-cell fusion step of mating, but unlike other fus1 and fus2 mutants the fusion defect of fig1 mutants can be largely suppressed by high Ca2+ conditions, which bypass the requirement for LACS . These findings suggest Fig1p is an important component or regulator of LACS and provide the first evidence for a role of Ca2+ signals in the cell fusion step of mating.

Mech Ageing Dev, 2003 Jul, 124(7), 839 - 46
DNA damage and stress transcripts in Saccharomyces cerevisiae mutant sgs1; Fry RC et al.; The human aging diseases Werner and Bloom syndromes are a result of mutation of the WRN and BLM genes, respectively . The SGS1 gene of Saccharomyces cerevisiae is homologous to the human WRN and BLM genes of the RecQ DNA helicase family . Deletion of SGS1 results in accelerated yeast aging and a reduction in life span as well as cell cycle arrest . We demonstrate that SGS1 deletion, DNA damage, and stress show similar transcriptional responses in yeast . Our comparative analysis of the genome-wide expression response of SGS1 deletion, stress and DNA damage indicates parallel transcriptional responses to cellular insult and aging in yeast.

J Cell Biochem, 2003 Aug 1, 89(5), 964 - 74
N-terminal methionine removal and methionine metabolism in Saccharomyces cerevisiae; Dummitt B et al.; Methionine aminopeptidase (MetAP) catalyzes removal of the initiator methionine from nascent polypeptides . In eukaryotes, there are two forms of MetAP, type 1 and type 2, whose combined activities are essential, but whose relative intracellular roles are unclear . Methionine metabolism is an important aspect of cellular physiology, involved in oxidative stress, methylation, and cell cycle . Due to the potential of MetAP activity to provide a methionine salvage pathway, we evaluated the relationship between methionine metabolism and MetAP activity in Saccharomyces cerevisiae . We provide the first demonstration that yeast MetAP1 plays a significant role in methionine metabolism, namely, preventing premature activation of MET genes through MetAP function in methionine salvage . Interestingly, in cells lacking MetAP1, excess methionine dramatically inhibits cell growth . Growth inhibition is independent of the ability of methionine to repress MET genes and does not result from inhibition of synthesis of another metabolite, rather it results from product inhibition of MetAP2 . Inhibition by methionine is selective for MetAP2 over MetAP1 . These results provide an explanation for the previously observed dominance of MetAP1 in terms of N-terminal processing and cell growth in yeast . Additionally, differential regulation of the two isoforms may be indicative of different intracellular roles for the two enzymes .

Genetics, 2003 Jul, 164(3), 909 - 21
Telomerase-independent proliferation is influenced by cell type in Saccharomyces cerevisiae; Lowell JE et al.; Yeast strains harboring mutations in genes required for telomerase function (TLC1 and the EST genes) exhibit progressive shortening of telomeric DNA and replicative senescence . A minority of cells withstands loss of telomerase through RAD52-dependent amplification of telomeric and subtelomeric sequences; such survivors are now capable of long-term propagation with telomeres maintained by recombination rather than by telomerase . Here we report that simultaneous expression in haploid cells of both MATa and MATalpha information suppresses the senescence of telomerase-deficient mutants, with suppression occurring via the RAD52-dependent survivor pathway(s) . Such suppression can be mimicked by deletion of SIR1-SIR4, genes that function in transcriptional silencing of several loci including the silent mating-type loci . Furthermore, telomerase-defective diploid strains that express only MATa or MATalpha information senesce at a faster rate than telomerase-defective diploids that are heterozygous at the MAT locus . This suggests that the RAD52-dependent pathway(s) for telomere maintenance respond to changes in the levels of recombination, a process regulated in part by the hierarchy of gene control that includes MAT regulation . We propose that cell-type-specific regulation of recombination at human telomeres may similarly contribute to the tissue-specific patterns of disease found in telomerase-deficient tumors.

RNA, 2003 Aug, 9(8), 1019 - 24
An in vivo dual-luciferase assay system for studying translational recoding in the yeast Saccharomyces cerevisiae; Harger JW et al.; A new in vivo assay system has been developed to study programmed frameshifting in the yeast Saccharomyces cerevisiae . Frameshift signals are inserted between the Renilla and firefly luciferase reporter genes contained in a yeast expression vector and the two activities are directly measured from cell lysates in one tube . Similar to other bicistronic reporter systems, this one allows the efficient estimation of recoding efficiency by comparison of the normalized activity ratios from each luciferase protein . The assay system has been applied to HIV-1 and L-A directed programmed -1 frameshifting and Ty1 and Ty3 directed +1 frameshifting . The assay system is amenable to high-throughput screening.

RNA, 2003 Aug, 9(8), 993 - 1006
Perturbation of transcription elongation influences the fidelity of internal exon inclusion in Saccharomyces cerevisiae; Howe KJ et al.; Unknown mechanisms exist to ensure that exons are not skipped during biogenesis of mRNA . Studies have connected transcription elongation with regulated alternative exon inclusion . To determine whether the relative rates of transcription elongation and spliceosome assembly might play a general role in enforcing constitutive exon inclusion, we measured exon skipping for a natural two-intron gene in which the internal exon is constitutively included in the mRNA . Mutations in this gene that subtly reduce recognition of the intron 1 branchpoint cause exon skipping, indicating that rapid recognition of the first intron is important for enforcing exon inclusion . To test the role of transcription elongation, we treated cells to increase or decrease the rate of transcription elongation . Consistent with the "first come, first served" model, we found that exon skipping in vivo is inhibited when transcription is slowed by RNAP II mutants or when cells are treated with inhibitors of elongation . Expression of the elongation factor TFIIS stimulates exon skipping, and this effect is eliminated when lac repressor is targeted to DNA encoding the second intron . A mutation in U2 snRNA promotes exon skipping, presumably because a delay in recognition of the first intron allows elongating RNA polymerase to transcribe the downstream intron . This indicates that the relative rates of elongation and splicing are tuned so that the fidelity of exon inclusion is enhanced . These findings support a general role for kinetic coordination of transcription elongation and splicing during the transcription-dependent control of splicing.

RNA, 2003 Aug, 9(8), 982 - 92
Decreased peptidyltransferase activity correlates with increased programmed -1 ribosomal frameshifting and viral maintenance defects in the yeast Saccharomyces cerevisiae; Meskauskas A et al.; Increased efficiencies of programmed -1 ribosomal frameshifting in yeast cells expressing mutant forms of ribosomal protein L3 are unable to maintain the dsRNA "Killer" virus . Here we demonstrate that changes in frameshifting and virus maintenance in these mutants correlates with decreased peptidyltransferase activities . The mutants did not affect Ty1-directed programmed +1 ribosomal frameshifting or nonsense-mediated mRNA decay . Independent experiments demonstrate similar programmed -1 ribosomal frameshifting specific defects in cells lacking ribosomal protein L41, which has previously been shown to result in peptidyltransferase defects in yeast . These findings are consistent with the hypothesis that decreased peptidyltransferase activity should result in longer ribosome pause times after the accommodation step of the elongation cycle, allowing more time for ribosomal slippage at programmed -1 ribosomal frameshift signals.

Mol Pharmacol, 2003 Aug, 64(2), 259 - 68
Anticancer drug resistance induced by disruption of the Saccharomyces cerevisiae NPR2 gene: a novel component involved in cisplatin- and doxorubicin-provoked cell kill; Schenk PW et al.; The therapeutic potential of antitumor drugs is seriously limited by the manifestation of cellular drug resistance . We used the budding yeast Saccharomyces cerevisiae as a model system to identify novel mechanisms of resistance to one of the most active anticancer agents, cisplatin . We pinpointed NPR2 (nitrogen permease regulator 2) as a gene whose disruption conferred resistance to cisplatin . In addition, we observed a 4-fold cross-resistance of yeast npr2Delta cells (i.e., cells from which the NPR2 gene had been disrupted) to the anticancer drug doxorubicin, in combination with hypersensitivity to cadmium chloride . Furthermore, npr2Delta cells displayed unaltered cellular cisplatin and doxorubicin accumulation and showed an enhanced rate of spontaneous mutation compared with the isogenic parent . These data indicate that the npr2Delta phenotype overlaps that of the sky1Delta cells that we characterized previously (Mol Pharmacol 61:659-666, 2002) . Therefore, we generated yeast npr2Delta sky1Delta double-knockout cells and performed clonogenic survival assays for cisplatin and doxorubicin, which revealed that NPR2 and SKY1 (SR-protein-specific kinase from budding yeast) are epistatic . The double-knockout strain was just as resistant to cisplatin and doxorubicin as the single-knockout strain that was most resistant to either drug . In conclusion, we identified NPR2 as a novel component involved in cell kill provoked by cisplatin and doxorubicin, and our data support the hypothesis that NPR2 and SKY1 may use mutual regulatory routes to mediate the cytotoxicity of these anticancer drugs.

Yeast, 2003 Jul 30, 20(10), 857 - 63
Investigations into the polymorphisms at the ECM38 locus of two widely used Saccharomyces cerevisiae S288C strains, YPH499 and BY4742; Kumar C et al.; The ECM38 gene encodes the gamma-glutamyl transpeptidase enzyme, an enzyme involved in glutathione turnover . The enzyme was found to be present in the S288C strain, BY4742, but absent in another widely used strain congenic to S288C, YPH499 . Cloning and sequencing the genes from these yeasts indicated the presence of 11 single nucleotide polymorphisms in the coding region and eight single nucleotide polymorphisms in the promoter region of the ECM38 gene of YPH499 (but none in that of BY4742) . One of the SNPs in the ECM38 ORF led to a G --> D conversion in a region conserved in all gamma-GT enzymes and was found to be responsible for the loss of activity in this strain . The presence of gamma-GT activity in other YPH strains led us to trace the origins of the polymorphisms in YPH499 . Our results indicated that among the progenitor strains, YPH1 and YPH2, YPH1 carried the polymorphisms seen in YPH499 and also lacked the gamma-GT activity . The implications of these results for the use of these widely used S288C strains and the origin of these single nucleotide polymorphisms are presented .

J Mol Microbiol Biotechnol, 2003, 5(4), 199 - 205
The cyclin in the RNA polymerase holoenzyme is a target for the transcriptional repressor Tup1p in Saccharomyces cerevisiae; Schuller J et al.; The general transcriptional repressor Tup1p requires the cyclin/cyclin-dependent kinase pair Srb11p/Srb10p in the holoenzyme of transcription . We used the split-ubiquitin system to demonstrate that Tup1p interacts with Srb11p in vivo . We confirmed our observation in vitro with the help of purified proteins, and we compared the de-repression effect of deleting TUP1, SRB10, and SRB11 on different promoters . We propose that Tup1p targets the cyclin Srb11p to affect the cyclin-dependent kinase Srb10p .

Genes Dev, 2003 Jul 15, 17(14), 1741 - 54
Chromosome integrity in Saccharomyces cerevisiae: the interplay of DNA replication initiation factors, elongation factors, and origins; Huang D et al.; The integrity of chromosomes during cell division is ensured by both trans-acting factors and cis-acting chromosomal sites . Failure of either these chromosome integrity determinants (CIDs) can cause chromosomes to be broken and subsequently misrepaired to form gross chromosomal rearrangements (GCRs) . We developed a simple and rapid assay for GCRs, exploiting yeast artificial chromosomes (YACs) in Saccharomyces cerevisiae . We used this assay to screen a genome-wide pool of mutants for elevated rates of GCR . The analyses of these mutants define new CIDs (Orc3p, Orc5p, and Ycs4p) and new pathways required for chromosome integrity in DNA replication elongation (Dpb11p), DNA replication initiation (Orc3p and Orc5p), and mitotic condensation (Ycs4p) . We show that the chromosome integrity function of Orc5p is associated with its ATP-binding motif and is distinct from its function in controlling the efficiency of initiation of DNA replication . Finally, we used our YAC assay to assess the interplay of trans and cis factors in chromosome integrity . Increasing the number of origins on a YAC suppresses GCR formation in our dpb11 mutant but enhances it in our orc mutants . This result provides potential insights into the counterbalancing selective pressures necessary for the evolution of origin density on chromosomes.

Biochem Biophys Res Commun, 2003 Jul 25, 307(2), 308 - 14
Growth temperature downshift induces antioxidant response in Saccharomyces cerevisiae; Zhang L et al.; A rapid downshift in the growth temperature of Saccharomyces cerevisiae from 30 to 10 degrees C resulted in an increase in transcript levels of the antioxidation genes SOD1 {encoding Cu-Zn superoxide dismutase (SOD)}, CTT1 (encoding catalase T), and GSH1 (encoding gamma-glutamylcysteine synthetase) . The cellular activities of SOD and catalase were also increased, indicating that the temperature downshift caused an antioxidant response . In support of this, a simultaneous increase in the intracellular level of H(2)O(2) was observed . The level of YAP1 mRNA, encoding a transcription factor critical for the oxidative stress response in this yeast, was also increased by the temperature downshift . However, deletion of YAP1 did not reduce the elevated mRNA levels of the antioxidant genes . This suggests that the temperature downshift-induced increase in the mRNA level of anti-oxidant genes is YAP1-independent.

Mol Biol Cell, 2003 Jul, 14(7), 2617 - 29 Epub 2003 Apr 04.
The Saccharomyces cerevisiae calponin/transgelin homolog Scp1 functions with fimbrin to regulate stability and organization of the actin cytoskeleton; Goodman A et al.; Calponins and transgelins are members of a conserved family of actin-associated proteins widely expressed from yeast to humans . Although a role for calponin in muscle cells has been described, the biochemical activities and in vivo functions of nonmuscle calponins and transgelins are largely unknown . Herein, we have used genetic and biochemical analyses to characterize the budding yeast member of this family, Scp1, which most closely resembles transgelin and contains one calponin homology (CH) domain . We show that Scp1 is a novel component of yeast cortical actin patches and shares in vivo functions and biochemical activities with Sac6/fimbrin, the one other actin patch component that contains CH domains . Purified Scp1 binds directly to filamentous actin, cross-links actin filaments, and stabilizes filaments against disassembly . Sequences in Scp1 sufficient for actin binding and cross-linking reside in its carboxy terminus, outside the CH domain . Overexpression of SCP1 suppresses sac6Delta defects, and deletion of SCP1 enhances sac6Delta defects . Together, these data show that Scp1 and Sac6/fimbrin cooperate to stabilize and organize the yeast actin cytoskeleton.

Environ Sci Technol, 2003 Jun 15, 37(12), 2788 - 93
Correlation of the structures of agricultural fungicides to gene expression in Saccharomyces cerevisiae upon exposure to toxic doses; Kitagawa E et al.; Correlations between the chemical structures of agricultural fungicides and mRNA expression levels following exposure of Saccharomyces cerevisiae to toxic doses of thiuram, zineb, maneb, TPN, and PCP were examined . Structurally, thiuram, zineb, and maneb are dithiocarbamate fungicides, whereas TPN and PCP are not . To characterize chemical toxicity, genes expression was classified according to the functional groups used by the MIPS database . However, no correlations between the classification scheme and chemical structures were found . Hierarchical clustering of gene expression profiles was performed to characterize the effects of the five chemicals . According to this analysis the similarity of gene expression profiles depended on the similarity of chemical structures . These results suggest that DNA microarray technology has potential for predicting the major chemicals which will cause environmental toxicity and will provide information on new biomonitoring methods.

Nucleic Acids Res, 2003 Jul 15, 31(14), 4119 - 28
Computational identification of non-coding RNAs in Saccharomyces cerevisiae by comparative genomics; McCutcheon JP et al.; We screened for new structural non-coding RNAs (ncRNAs) in the genome sequence of the yeast Saccharomyces cerevisiae using computational comparative analysis of genome sequences from five related species of Saccharomyces . The screen identified 92 candidate ncRNA genes . Thirteen showed discrete transcripts when assayed by northern blot . Of these, eight appear to be novel ncRNAs ranging in size from 268 to 775 nt, including three new H/ACA box small nucleolar RNAs.

Nucleic Acids Res, 2003 Jul 15, 31(14), 3909 - 17
Mitochondria-mediated nuclear mutator phenotype in Saccharomyces cerevisiae; Rasmussen AK et al.; Using Saccharomyces cerevisiae as a model organism, we analyzed the consequences of disrupting mitochondrial function on mutagenesis of the nuclear genome . We measured the frequency of canavanine-resistant colonies as a measure of nuclear mutator phenotype . Our data suggest that mitochondrial dysfunction leads to a nuclear mutator phenotype (i) when oxidative phosphorylation is blocked in wild-type yeast at mitochondrial complex III by antimycin A and (ii) in mutant strains lacking the entire mitochondrial genome (rho(0)) or those with deleted mitochondrial DNA (rho(-)) . The nuclear mutation frequencies obtained for antimycin A-treated cells as well as for rho(-) and rho(0) cells were approximately 2- to 3-fold higher compared to untreated control and wild-type cells, respectively . Blockage of oxidative phosphorylation by antimycin A treatment led to increased intracellular levels of reactive oxygen species (ROS) . In contrast, inactivation of mitochondrial activity (rho(-) and rho(0)) led to decreased intracellular levels of ROS . We also demonstrate that in rho(0) cells the REV1, REV3 and REV7 gene products, all implicated in error-prone translesion DNA synthesis (TLS), mediate mutagenesis in the nuclear genome . However, TLS was not involved in nuclear DNA mutagenesis caused by inhibition of mitochondrial function by antimycin A . Together, our data suggest that mitochondrial dysfunction is mutagenic and multiple pathways are involved in this nuclear mutator phenotype.

Free Radic Biol Med, 2003 Jul 15, 35(2), 179 - 88
H(2)O(2) generation in Saccharomyces cerevisiae respiratory pet mutants: effect of cytochrome c; Barros MH et al.; Impaired electron transport chain function has been related to increases in reactive oxygen species (ROS) generation . Here we analyzed different pet mutants of Saccharomyces cerevisiae in order to determine the relative contribution of respiratory chain components in ROS generation and removal . We found that the maintenance of respiration strongly prevented mitochondrial H(2)O(2) release and increased cellular H(2)O(2) removal . Among all respiratory-deficient strains analyzed, cells lacking cytochrome c (cyc3 point mutants) presented the highest level of H(2)O(2) synthesis, indicating that the absence of functional cytochrome c in mitochondria leads to oxidative stress . This finding was supported by the presence of high levels of catalase and peroxidase activity despite the lack of respiration . Furthermore, the addition of exogenous cytochrome c to isolated yeast mitoplasts significantly reduced H(2)O(2) detection in a manner enhanced by cytochrome c reduction and the presence of a functional respiratory chain . Together, our results indicate that the maintenance of electron transport by cytochrome c prevents ROS generation by the respiratory chain.

J Cell Biol, 2003 Jul 7, 162(1), 85 - 97
Lack of GTP-bound Rho1p in secretory vesicles of Saccharomyces cerevisiae; Abe M et al.; Rho1p, an essential Rho-type GTPase in Saccharomyces cerevisiae, activates its effectors in the GTP-bound form . Here, we show that Rho1p in secretory vesicles cannot activate 1,3-beta-glucan synthase, a cell wall synthesizing enzyme, during vesicular transport to the plasma membrane . Analyses with an antibody preferentially reacting with the GTP-bound form of Rho1p revealed that Rho1p remains in the inactive form in secretory vesicles . Rom2p, the GDP/GTP exchange factor of Rho1p, is preferentially localized on the plasma membrane even when vesicular transport is blocked . Overexpression of Rom2p results in delocalization of Rom2p and accumulation of 1,3-beta-glucan in secretory vesicles . Based on these results, we propose that Rho1p is kept inactive in intracellular secretory organelles, resulting in repression of the activity of the cell wall-synthesizing enzyme within cells.

Yeast, 2003 Jul 15, 20(9), 797 - 801
SIR-dependent repression of non-telomeric genes in Saccharomyces cerevisiae?
Marchfelder U, Rateitschak K, Ehrenhofer-Murray AE.
The proteins Sir2, Sir3 and Sir4 repress transcription of the silent mating-type loci HML and HMR and of reporter genes inserted at telomeres . Previous microarray analyses suggested that additional non-telomeric genes exist which are repressed by the Sir proteins . In this study, we tested the expression of 12 such genes by Northern analysis and RT-PCR . However, we were unable to verify their SIR-dependent regulation, which suggests that SIR-mediated repression may be restricted to the known repressed regions .

J Biol Chem, 2003 Sep 12, 278(37), 35204 - 10 Epub 2003 Jul 02.
Cardiolipin is not required to maintain mitochondrial DNA stability or cell viability for Saccharomyces cerevisiae grown at elevated temperatures; Zhang M et al.; In eukaryotic cells, the phospholipid cardiolipin (CL) is primarily found in the inner mitochondrial membrane . Saccharomyces cerevisiae mutants, unable to synthesize CL because of a null allele of the CRD1 gene (encodes CL synthase), have been reported with different phenotypes . Some mutants, when grown on a nonfermentable carbon source at elevated temperatures, exhibit mitochondrial DNA instability, loss of viability, and significant defects in several functions that rely on the mitochondrial energy transducing system (ETS) . These mutants also lack the immediate precursor to CL, phosphatidylglycerol (PG), when grown on glucose as a carbon source . Other mutants show reduced growth efficiency on a nonfermentable carbon source but much milder phenotypes associated with growth at elevated temperatures and increased levels of PG when grown on glucose . We present evidence that mitochondrial DNA instability, loss of viability, and defects in the ETS exhibited at elevated temperatures by some mutants are caused by the reduced expression of the PET56 gene in the presence of the his3 Delta 200 allele and not the lack of CL alone . We also found that PG is present and elevated in all crd1 Delta strains when grown on glucose . A supermolecular complex between complex III and complex IV of the mitochondrial ETS detected in wild type cells was missing in all of the above crd1 Delta cells . The level of components of the ETS was also reduced in crd1 Delta cells grown at elevated temperatures because of reduced gene expression and not reduced stability . These results suggest that all phenotypes reported for cells carrying the his3 Delta 200 allele and lacking CL should be re-evaluated.

Appl Environ Microbiol, 2003 Jul, 69(7), 4019 - 28
Adaptation of Saccharomyces cerevisiae to the herbicide 2,4-dichlorophenoxyacetic acid, mediated by Msn2p- and Msn4p-regulated genes: important role of SPI1; Simoes T et al.; The possible roles of 13 Msn2p- and Msn4p-regulated genes in the adaptation of Saccharomyces cerevisiae to the herbicide 2,4-D-dichlorophenoxyacetic acid (2,4-D) were examined . Single deletion of genes involved in defense against oxidizing agents (CTT1, GRX1, and GRX2/TTR1) or encoding chaperones of the HSP70 family (SSA1, SSA4, and SSE2) showed a slight effect . A more significant role was observed for the heat shock genes HSP78, HSP26, HSP104, HSP12, and HSP42, most of which encode molecular chaperones . However, the SPI1 gene, encoding a member of the glycosylphosphatidylinositol-anchored cell wall protein family, emerged as the major determinant of 2,4-D resistance . SPI1 expression reduced the loss of viability of an unadapted yeast population suddenly exposed to the herbicide, allowing earlier growth resumption . Significantly, yeast adaptation to 2,4-D involves the rapid and transient Msn2p- and Msn4p-mediated activation (fivefold) of SPI1 transcription . SPI1 mRNA levels were reduced to values slightly above those in unstressed cells when the adapted population started duplication in the presence of 2,4-D . Since SPI1 deletion leads to the higher beta-1,3-glucanase sensitivity of 2,4-D-stressed cells, it was hypothesized that adaptation may involve an Spi1p-mediated increase in the diffusional restriction of the liposoluble acid form of the herbicide across the cell envelope . Such a cell response would avoid a futile cycle due to acid reentry into the cell counteracting the active export of the anionic form, presumably through an inducible plasma membrane transporter(s) . Consistent with this concept, the concentration of (14)C-labeled 2,4-D in 2,4-D-energized adapted Deltaspi1 mutant cells and the consequent intracellular acidification are higher than in wild-type cells.

Traffic, 2003 Aug, 4(8), 566 - 75
The HECT ubiquitin ligase Rsp5p is required for proper nuclear export of mRNA in Saccharomyces cerevisiae; Rodriguez MS et al.; The nuclear transport of both proteins and RNAs has attracted considerable interest in recent years . However, regulation pathways of the nuclear transport machineries are still not well characterized . Previous studies indicated that ubiquitination is involved in poly(A)+ RNA nuclear export . For this reason, we systematically investigated ubiquitin-protein ligasess from the homologous to E6-AP carboxy terminus (HECT) family for potential individual roles in nuclear transport in Saccharomyces cerevisiae . Here we report that Rsp5, an essential yeast ubiquitin ligase involved in many cellular functions, when deleted or mutated in ligase activity, blocks the nuclear export of mRNAs . Affected messenger RNAs include both total poly(A)+ mRNA and heat-shock mRNAs . Mutation of Rsp5 does not affect nuclear protein import or export . Deletion of RSP5 blocks mRNA export, even under conditions where its essential role in unsaturated fatty acids biosynthesis is bypassed . Using domain mapping, we find that the ligase activity is required for proper mRNA export, indicating that ubiquitination by Rsp5 acts directly or indirectly to affect RNA export . The finding that Rsp5p ligase mutations cause a more pronounced defect at high temperatures suggests that ubiquitination of transport factors by Rsp5p may also be essential during stress conditions.

Chem Biol, 2003 Jun, 10(6), 521 - 31
A small molecule suppressor of FK506 that targets the mitochondria and modulates ionic balance in Saccharomyces cerevisiae; Butcher RA et al.; FK506 inhibits the evolutionarily conserved, Ca(2+)-dependent phosphatase calcineurin, which in yeast is essential for growth during sodium stress . We undertook a chemical genetic modifier screen to identify small molecules that suppress the ability of FK506 to inhibit yeast growth in high NaCl . One of these small molecule suppressors, SFK1 (suppressor of FK506 1), causes a mitochondrially induced death in low salt, concomitant with the release of reactive oxygen species . Biochemically, SFK1 interacts with Por1p, a channel protein in the outer mitochondrial membrane, suggesting that SFK1 interacts with the mitochondria directly . A genome-wide screen of yeast deletion strains for hypersensitivity to SFK1 yielded several strains with impaired mitochondrial function, as well as several with reduced sodium tolerance . Our data link ionic balance to mitochondrial function and suggest a role for calcineurin in mediating this signaling network.

J Struct Funct Genomics, 2002, 2(3), 129 - 34
Structural analysis of Saccharomyces cerevisiae myo-inositol phosphate synthase; Kniewel R et al.; The New York Structural Genomics Research Consortium has targeted highly conserved but uncharacterized enzyme families for structure determination . As part of this effort, the 2.65-A crystal structure has been determined for Saccharomyces cerevisiae myo-inositol 1-phosphate synthase (MIP), an essential enzyme that catalyzes critical steps in inositol biosynthesis . The structure determination of four independent monomers in the asymmetric unit (240 kDa) reveals atomic details and residue composition for the partially closed NAD-containing active sites in apo-configuration . The structure further reveals extensive interactions involved in tetrameric assembly of the enzyme complex.

J Struct Funct Genomics, 2003, 3(1-4), 213 - 24
Approach of the functional evolution of duplicated genes in Saccharomyces cerevisiae using a new classification method based on protein-protein interaction data; Brun C et al.; The concept of protein function is widely used and manipulated by biologists . However, the means of the concept and its understanding may vary depending on the level of functionality one considers (molecular, cellular, physiological, etc.) . Genomic studies and new high-throughput methods of the post-genomic era provide the opportunity to shed a new light on the concept of protein function: protein-protein interactions can now be considered as pieces of incomplete but still gigantic networks and the analysis of these networks will permit the emergence of a more integrated view of protein function . In this context, we propose a new functional classification method, which, unlike usual methods based on sequence homology, allows the definition of functional classes of protein based on the identity of their interacting partners . An example of such classification will be shown and discussed for a subset of Saccharomyces cerevisiae proteins, accounting for 7% of the yeast proteome . The genome of the budding yeast contains 50% of protein-coding genes that are paralogs, including 457 pairs of duplicated genes coming probably from an ancient whole genome duplication . We will comment on the functional classification of the duplicated genes when using our method and discuss the contribution of these results to the understanding of function evolution for the duplicated genes.

Mol Genet Genomics, 2003 Aug, 269(5), 649 - 57 Epub 2003 Jun 27.
Binding of calmodulin to Nuf1p is required for karyogamy in Saccharomyces cerevisiae; Okano H et al.; The role of calmodulin (CaM) during mating in Saccharomyces cerevisiae was examined by using a set of Phe-to-Ala substitutions . We identified ten CaM mutants that exhibited significantly reduced mating efficiencies when crossed to a strain of the opposite mating type harboring the same CaM mutation . Most of the mating-defective CaM mutants were bilateral, i.e., they also exhibited mating defects, albeit minor ones, when crossed to the wild type . When strains carrying different bilateral CaM mutations were mated, the mating efficiencies recovered dramatically . We termed this phenomenon "intragenic mating complementation", and classified the mating-defective CaM mutations into two intragenic mating complementation groups . Two mutant alleles belonging to different groups showed minor defects in cell adhesion and cell fusion, but exhibited severe defects in karyogamy . CaM is known to bind to the essential spindle pole body component Nuf1p . This binding appears to be important for karyogamy because the nuf1(C911R) mutation, which impairs CaM-Nuf1p binding, resulted in a severe defect in karyogamy . Indeed, the two mating-defective CaM mutations were found to compromise formation of the CaM/Nuf1p complex, and the mating defects of these two CaM mutants were suppressible by a dominant, CaM-independent, mutation in NUF1 . Taken together, these results suggest that loss of CaM binding to Nuf1p causes a defect in karyogamy, thereby inhibiting productive mating.

Proteomics, 2003 Jun, 3(6), 906 - 16
Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis; Zischka H et al.; The analysis of complex cellular proteomes by means of two-dimensional gel electrophoresis (2-DE) is significantly limited by the power of resolution of this technique . Although subcellular fractionation can be a fundamental first step to increase resolution, it frequently leads to preparations contaminated with other cellular structures . Here, we chose mitochondria of Saccharomyces cerevisiae to demonstrate that an integrated zone-electrophoretic purification step (ZE), with a free-flow electrophoresis device (FFE), can assist in overcoming this problem, while significantly improving their degree of purity . Whereas mitochondrial preparations isolated by means of differential centrifugation include a considerable degree of non-mitochondrial proteins (16%), this contamination could be effectually removed by the inclusion of a ZE-FFE purification step (2%) . This higher degree of purity led to the identification of many more proteins from ZE-FFE purified mitochondrial protein extracts (n = 129), compared to mitochondrial protein extracts isolated by differential centrifugation (n = 80) . Moreover, a marked decrease of degraded proteins was found in the ZE-FFE purified mitochondrial protein extracts . It is noteworthy that even at a low 2-DE resolution level, a four-fold higher number (17 versus 4) of presumably low abundance proteins could be identified in the ZE-FFE purified mitochondrial protein extracts . Therefore these results represent a feasible approach for an in-depth proteome analysis of mitochondria and possibly other organelles.

Mol Cell Biol, 2003 Jul, 23(14), 5064 - 77
G1 transcription factors are differentially regulated in Saccharomyces cerevisiae by the Swi6-binding protein Stb1; Costanzo M et al.; Stage-specific transcriptional programs are an integral feature of cell cycle regulation . In the budding yeast Saccharomyces cerevisiae, over 120 genes are coordinately induced in late G(1) phase by two heterodimeric transcription factors called SBF and MBF . Activation of SBF and MBF is an upstream initiator of key cell cycle events, including budding and DNA replication . SBF and MBF regulation is complex and genetically redundant, and the precise mechanism of G(1) transcriptional activation is unclear . Assays using SBF- and MBF-specific reporter genes revealed that the STB1 gene specifically affected MBF-dependent transcription . STB1 encodes a known Swi6-binding protein, but an MBF-specific function had not been previously suspected . Consistent with a specific role in regulating MBF, a STB1 deletion strain requires SBF for viability and microarray studies show a decrease in MBF-regulated transcripts in a swi4Delta mutant following depletion of Stb1 . Chromatin immunoprecipitation experiments confirm that Stb1 localizes to promoters of MBF-regulated genes . Our data indicate that, contrary to previous models, MBF and SBF have unique components and might be distinctly regulated.

Biochim Biophys Acta, 2003 Jun 27, 1613(1-2), 1 - 6
Functional analysis of the M2(D) helix of the TRK1 potassium transporter of Saccharomyces cerevisiae; Haro R et al.; Eukaryotic KcsA-related K+ transporters mediate physiologically relevant K+ and Na+ fluxes in fungi and plants . ScTRK1 is a characteristic member of the group, and here we report a mutational analysis of the unique M2(D) helix of this transporter . Our results support the theoretical models placing this helix in a relevant position in the pore and interacting with P segments . Most single mutations eliminating positively charged or introducing negatively charged residues reduced the V(max) of Rb+ influx to a half, several together showed an additive effect, and four practically suppressed transport . In contrast, the introduction of only one positively charged residue practically abolished the function of the transporter . Almost all mutations in the M2(D) helix affected the two Rb+ binding sites of the transporter, mimicking mutations in the selectivity filter.

FEMS Microbiol Lett, 2003 Jun 27, 223(2), 253 - 8
The budding index of Saccharomyces cerevisiae deletion strains identifies genes important for cell cycle progression; Zettel MF et al.; Budding marks initiation of cell division in Saccharomyces cerevisiae . Consequently, cell cycle progression can be monitored by the fraction of budded cells (budding index) in a proliferating cell population . We determined the budding index of a large collection of deletion strains, to systematically identify genes involved in cell cycle progression.

Hepatogastroenterology, 2003 May-Jun, 50(51), 718 - 20
Antibodies to Saccharomyces cerevisiae: are they useful in clinical practice?
Candelli M, Papa A, Nista EC, Danese S, Armuzzi A, Bartolozzi F, Tondi P, Ojetti V, Gasbarrini G, Gasbarrini A.
BACKGROUND/AIMS: Antibodies to Saccharomyces cerevisiae are associated with Crohn's disease . The aim of this study was to assess the sensitivity and specificity of antibodies to Saccharomyces cerevisiae for Crohn's disease in an Italian population and to evaluate their clinical role . METHODOLOGY: Antibodies to Saccharomyces cerevisiae serum levels were assessed in 72 inflammatory bowel disease patients (30 Crohn's disease; 42 ulcerative colitis) and 35 age-matched controls . Patients were divided into subgroups on the basis of disease behavior and clinical feature were evaluated . RESULTS: Antibodies to Saccharomyces cerevisiae sensitivity and specificity for Crohn's disease patients were 50% (31-68) and 91% (77-98), for IgG and 56% (37-64) and 94% (80-99), respectively, for IgA . No correlation was observed between the presence of antibodies to Saccharomyces cerevisiae and clinical features . Antibodies to Saccharomyces cerevisiae were more prevalent in Crohn's disease than in ulcerative colitis patients (IgA: 57% vs . 26%; IgG: 50% vs . 17%; p < 0.05) . Interestingly, in Crohn's disease patients, the prevalence of antibodies to Saccharomyces cerevisiae was higher in patients with small bowel involvement than in patients with pure colonic disease (IgA: 66% vs . 17%; IgG: 58% vs . 17%) . No difference in antibodies to Saccharomyces cerevisiae prevalence was observed between ulcerative colitis and pure colonic Crohn's disease patients . CONCLUSIONS: Our data show an association between Crohn's disease and antibodies to Saccharomyces cerevisiae . However, it does not seem useful in discriminating between ulcerative colitis and colic Crohn's disease.

Mol Genet Genomics, 2003 Jul, 269(4), 562 - 73 Epub 2003 Jun 25.
Mutations that are synthetically lethal with a gas1Delta allele cause defects in the cell wall of Saccharomyces cerevisiae; Tomishige N et al.; The GAS1-related genes of fungi encode GPI-anchored proteins with beta-1,3-glucanosyltransferase activity . Loss of this activity results in defects in the assembly of the cell wall . We isolated mutants that show a synthetic defect when combined with a gas1Delta allele in Saccharomyces cerevisiae, and identified nine wild-type genes that rescue this defect . The indispensability of BIG1 and KRE6 for the viability of gas1Delta cells confirmed the important role of beta-1,6-glucan in cells that are defective in the processing of beta-1,3-glucan . The identification of the Wsc1p hypo-osmotic stress sensor and components of the PKC signal transduction pathway in our screen also confirmed that the cell wall integrity response attenuates the otherwise lethal gas1Delta defect . Unexpectedly, we found that the KEX2 gene is also required for the viability of the gas1Delta mutant . Kex2p is a Golgi/endosome-membrane-anchored protease that processes secretory preproteins . A cell wall defect was also found in the kex2Delta mutant, which was suppressible by multiple copies of the MKC7 or YAP3 gene, both of which encode other GPI-anchored proteases . Therefore, normal cell wall assembly requires proteolytic processing of secretory preproteins . Furthermore, the genes CSG2 and IPT1 were found to be required for normal growth of gas1Delta cells in the presence of 1 M sorbitol . This finding suggests that complex sphingolipids play a role in the hyper-osmotic response.

Proc Natl Acad Sci U S A, 2003 Jul 8, 100(14), 8199 - 204 Epub 2003 Jun 24.
A homologue of elongation factor 1 gamma regulates methionine sulfoxide reductase A gene expression in Saccharomyces cerevisiae; Hanbauer I et al.; Methionine sulfoxide reductase A (MsrA) maintains the function of many proteins by reversing oxidation of methionine residues . Lack of this repair mechanism very likely increases aging-related disease susceptibility . In Saccharomyces cerevisiae, disruption of the msrA gene increases free and protein-bound methionine sulfoxide and decreases cell viability . Although the underlying mechanisms in the induction of the msrA gene are still unknown, a transcriptional regulation may be involved . Hence, a search of nuclear proteins regulating the msrA gene is a major target of the experiments reported in this article . Using protein purification combined with MS, we discovered that calcium phospholipid-binding protein (CPBP), a homologue of elongation factor-1 gamma, is a component of a complex that binds to the msrA promoter . By measuring CPBP cooperative binding to the msrA promoter, we have mapped the CPBP binding site to a 39-bp sequence at the 3' end of the promoter . In a mutant yeast strain lacking the CPBP-encoding gene, the ability to overexpress msrA mRNA and MsrA protein was impaired and MsrA catalytic activity was greatly reduced, suggesting that CPBP may enhance msrA gene expression.

J Biol Chem, 2003 Sep 12, 278(37), 34952 - 8 Epub 2003 Jun 24.
The protein interaction of Saccharomyces cerevisiae cytoplasmic thiol peroxidase II with SFH2p and its in vivo function; Cha MK et al.; Previously, we reported that the yeast cytoplasmic thiol peroxidase type II isoform (cTPx II), a member of the TSA/AhpC family, showed a very low peroxidase activity when compared with other cytoplasmic yeast isoforms, and that cTPx II mutant (cTPx II Delta) showed a severe growth retardation compared with that of the wild-type cells . To reveal the physiological function of cTPx II in yeast cell growth, we searched for proteins which react with cTPx II . In this study, we identified a novel interaction between cTPx II and CSR1p using the yeast two-hybrid system . CSR1p (SFH2p) has been known to be one member of Sec14 homologous (SFH2) proteins . SFH2p exhibits phosphatidylinositol transfer protein activity . Interestingly, we found that cTPx II selectively bound to SFH2p among the five types of SFH proteins and Sec14p . The interaction required the dimerization of cTPx II . In addition, SFH2p also specifically bound to cTPx II among the yeast thiol peroxidase isoforms . The selective interaction of the dimer form of cTPx II (the oxidized form) with SFH2p was also confirmed by glutathione S-transferase pull-down and immunoprecipitation assays . The growth retardation, clearly reflected by the length of the lag phase, of cTPx II Delta was rescued by deleting SFH2p in the cTPx II Delta strain . The SFH2 Delta strain did not show any growth retardation . In addition, the double mutant showed a higher susceptibility to oxidative stress . This finding provides the first in vivo demonstration of the specific interaction of cTPx II with SFH2p in an oxidative stress-sensitive manner and a novel physiological function of the complex of cTPx II and SFH2p.

J Biol Chem, 2003 Sep 12, 278(37), 34998 - 5015 Epub 2003 Jun 24.
Genome-wide expression profiling of the response to polyene, pyrimidine, azole, and echinocandin antifungal agents in Saccharomyces cerevisiae; Agarwal AK et al.; Antifungal compounds exert their activity through a variety of mechanisms, some of which are poorly understood . Novel approaches to characterize the mechanism of action of antifungal agents will be of great use in the antifungal drug development process . The aim of the present study was to investigate the changes in the gene expression profile of Saccharomyces cerevisiae following exposure to representatives of the four currently available classes of antifungal agents used in the management of systemic fungal infections . Microarray analysis indicated differential expression of 0.8, 4.1, 3.0, and 2.6% of the genes represented on the Affymetrix S98 yeast gene array in response to ketoconazole, amphotericin B, caspofungin, and 5-fluorocytosine (5-FC), respectively . Quantitative real time reverse transcriptase-PCR was used to confirm the microarray analyses . Genes responsive to ketoconazole, caspofungin, and 5-FC were indicative of the drug-specific effects . Ketoconazole exposure primarily affected genes involved in ergosterol biosynthesis and sterol uptake; caspofungin exposure affected genes involved in cell wall integrity; and 5-FC affected genes involved in DNA and protein synthesis, DNA damage repair, and cell cycle control . In contrast, amphotericin B elicited changes in gene expression reflecting cell stress, membrane reconstruction, transport, phosphate uptake, and cell wall integrity . Genes with the greatest specificity for a particular drug were grouped together as drug-specific genes, whereas genes with a lack of drug specificity were also identified . Taken together, these data shed new light on the mechanisms of action of these classes of antifungal agents and demonstrate the potential utility of gene expression profiling in antifungal drug development.

Mol Microbiol, 2003 Jul, 49(1), 23 - 35
The interaction of Slt2 MAP kinase with Knr4 is necessary for signalling through the cell wall integrity pathway in Saccharomyces cerevisiae; Martin-Yken H et al.; In budding yeast, PKC1 plays an essential role in cell integrity and proliferation through a linear MAP (Mitogen Activated Protein) kinase phosphorylation cascade, which ends up with the activation of the Slt2-MAP kinase by dual phosphorylation on two conserved threonine and tyrosine residues . In this phosphorylated form, Slt2p kinase activates by phosphorylation at least two known downstream targets: Rlm1p, which is implicated in the expression of cell wall-related genes, and SBF, required for transcription activation of cell cycle-regulated genes at the G1 to S transition . In this paper, we demonstrate by two-hybrid, in vitro immunoprecipitation and tandem affinity purification (TAP) methods that Knr4p physically interacts with Slt2p . Moreover, we show that the absence of Knr4p alters proper signalling of Slt2p to its two known downstream targets . In a knr4 null mutant, the SLT2-dependent activation of Rlm1p is strongly reduced and the transcriptional activity of Rlm1p is decreased, although the phosphorylated form of Slt2p is more abundant than in wild-type cells . On the contrary, SBF is abnormally activated in this mutant, as shown by a more abundant phosphorylated form of Swi6p, by higher beta-galactosidase levels from a SCB-lacZ gene fusion, and by deregulation of the cyclic behaviour of several cell cycle-regulated genes . These results, taken together with our recent finding that Bck2p requires Knr4p to activate additively with Cln3-Cdc28p SBF target genes, lead to a model in which Knr4p is involved in co-ordinating the Slt2p-mediated cell wall integrity pathway with progression of the cell cycle.

J Biol Chem, 2003 Sep 5, 278(36), 33978 - 84 Epub 2003 Jun 23.
Regulation of phosphatidylglycerophosphate synthase by inositol in Saccharomyces cerevisiae is not at the level of PGS1 mRNA abundance; Zhong Q et al.; Phosphatidylglycerophosphate synthase catalyzes the committed step in the synthesis of the mitochondrial phospholipid cardiolipin . We showed previously that phosphatidylglycerophosphate synthase activity in Saccharomyces cerevisiae is increased in conditions favoring mitochondrial development and during growth in the absence of inositol . Interestingly, the regulatory effects of inositol were not altered in ino2, ino4, or opi1 mutants suggesting that regulation in response to inositol is not at the level of gene transcription . We report here that steady state mRNA levels of the PGS1 gene, which encodes phosphatidylglycerophosphate synthase, were not altered by inositol or choline . Growth in the presence of the inositol-depleting drug valproate led to an increase in phosphatidylglycerophosphate synthase activity unaccompanied by increased PGS1 mRNA . PGS1 mRNA abundance was not decreased in ino2 or ino4 mutants and was unaffected in an opi1 mutant . Therefore, regulation of phosphatidylglycerophosphate synthase by inositol is not mediated at the level of mRNA abundance and does not require the INO2-INO4-OPI1 regulatory circuit . PGS1 was increased in glycerol/ethanol compared with glucose media and was maximally expressed as cells entered the stationary phase . Deletion of the mitochondrial genome did not affect PGS1 expression . Thus, whereas inositol controls phosphatidylglycerophosphate synthase activity, regulation of PGS1 expression occurs primarily in response to mitochondrial development cues.

Biochem Biophys Res Commun, 2003 Jul 11, 306(4), 843 - 50
Transcriptional regulation of phosphate-responsive genes in low-affinity phosphate-transporter-defective mutants in Saccharomyces cerevisiae; Auesukaree C et al.; In Saccharomyces cerevisiae, two systems have been shown to be involved in the active transport of inorganic phosphate (P(i)) across the plasma membrane, the high-affinity system and the low-affinity system . The high-affinity system consists of Pho84p and Pho89p . The low-affinity system has recently been shown to be composed of Pho87p, Pho90p, and Pho91p . In this study, we found that the Deltapho87Deltapho90Deltapho91 strain which shows repressed PHO5 expression under high-P(i) condition has, unlike the wild-type strain, increased levels of PHO5 expression at an intermediate P(i) concentration of 0.5mM, whereas it is not defective in terms of P(i) uptake under the same conditions . Moreover, we observed that the transcription levels of PHO84 and PHO89 are also increased in low-affinity P(i)-transporter-defective mutants, indicating that the inactivation of low-affinity P(i) transporters leads to the activation of the PHO pathway . In contrast to that of PHO5, PHO84, and PHO89, the transcription of PHO87, PHO90, and PHO91 genes is independent of P(i) concentration and Pho4p activity, and the increased expression level of these transporters does not occur when other transporters including PHO84 are inactivated . The fact that low-affinity P(i)-transporter-defective mutants exhibit a derepression of P(i)-responsive genes suggests that low-affinity transporters play a role not only in P(i) transport but also in the regulation of the P(i) signal transduction pathway.

Mol Cell, 2003 Jun, 11(6), 1685 - 92
Suppression of homologous recombination by the Saccharomyces cerevisiae linker histone; Downs JA et al.; The basic unit of chromatin in eukaryotes is the nucleosome, comprising 146 bp of DNA wound around two copies of each of four core histones . Chromatin is further condensed by association with linker histones . Saccharomyces cerevisiae Hho1p has sequence homology to other known linker histones and interacts with nucleosomes in vitro . However, disruption of HHO1 results in no significant changes in the phenotypes examined thus far . Here, we show that Hho1p is inhibitory to DNA repair by homologous recombination (HR) . We find Hho1p is abundant and associated with the genome, consistent with a global role in DNA repair . Furthermore, we establish that Hho1p is required for a full life span and propose that this is mechanistically linked to its role in HR . Finally, we show that Hho1p is inhibitory to the recombination-dependent mechanism of telomere maintenance . The role of linker histones in genome stability, aging, and tumorigenesis is discussed.

J Biol Chem, 2003 Aug 29, 278(35), 32596 - 601 Epub 2003 Jun 20.
Futile cycling of intermediates of fatty acid biosynthesis toward peroxisomal beta-oxidation in Saccharomyces cerevisiae; Marchesini S et al.; The flux of fatty acids toward beta-oxidation was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate synthesis in the peroxisome from the polymerization, by a bacterial polyhydroxyalkanoate synthase, of the beta-oxidation intermediates 3-hydroxyacyl-CoAs . Synthesis of polyhydroxyalkanoate was dependent on the beta-oxidation enzymes acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional protein, which are involved in generating 3-hydroxyacyl-CoAs, and on the peroxin PEX5, which is involved in the import of proteins into the peroxisome . In wild type cells grown in media containing fatty acids, the polyhydroxyalkanoate monomer composition was largely influenced by the nature of the external fatty acid, such that even-chain monomers are generated from oleic acid and odd-chain monomers are generated from heptadecenoic acid . In contrast, polyhydroxyalkanoate containing predominantly 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate was synthesized in a mutant deficient in the peroxisomal 3-ketothiolase (fox3 Delta 0) growing either on oleic acid or heptadecenoic acid as well as in wild type and fox3 Delta 0 mutants grown on glucose or raffinose, indicating that 3-hydroxyacyl-CoAs used for polyhydroxyalkanoate synthesis were generated from the degradation of intracellular short- and medium-chain fatty acids by the beta-oxidation cycle . Inhibition of fatty acid biosynthesis with cerulenin blocked the synthesis of polyhydroxyalkanoate from intracellular fatty acids but still enabled the use of extracellular fatty acids for polymer production . Mutants affected in the synthesis of lipoic acid showed normal polyhydroxyalkanoate synthesis capacity . Together, these results uncovered the existence of a substantial futile cycle whereby short- and medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed toward the peroxisomal beta-oxidation pathway.

J Altern Complement Med, 2003 Jun, 9(3), 379 - 87
Antioxidant activity of a botanical extract preparation of Ilex paraguariensis: prevention of DNA double-strand breaks in Saccharomyces cerevisiae and human low-density lipoprotein oxidation; Bracesco N et al.; We analyzed the antioxidant properties of Ilex paraguariensis infusion (Ip) popularly known as mate (ma'ta), by using two experimental models: the induction of DNA double-strand breaks (DSB) by hydrogen peroxide (H(2)O(2)) and lethality in Saccharomyces cerevisiae, as well as peroxide and lipoxygenase-induced human low-density lipoprotein (LDL) oxidation . Diploid yeast cells were exposed to different concentrations of H(2)O(2) (5-10 mmol/L) in the absence or presence of Ip infusion (10(-1) v/v) or alpha-tocopherol (10(-2) mol/L) . Both mate infusion and alpha-tocopherol significantly decreased the dose dependent DSB number, and the lethality induced by H(2)O(2) . Peroxynitrite and lipoxygenase-induced human LDL oxidation are inhibited by Ip extracts in a potent, dose-dependent fashion . Dilutions of 5 x 10(-3) v/v provide 50% +/- 10% inhibition . Finally, Ip extracts are potent direct quenchers of the free radical 1,1-diphenyl-2-picrylhydrazyl . Dilutions of 2 x 10(-2) v/v produced quenching of more than 30%, which was comparable to that obtained with 0.5-1 mmol/L alpha-tocopherol or the quercetin aglycone, respectively . For comparison, total polyphenol content of Ip, green, and black tea (Camelia sinensis) were 6.5 +/- 0.8; 1.8 +/- 0.5; and 1.13 +/- 0.3 mmol of quercetin equivalents per liter, respectively . Their respective free radical quenching activities at dilutions of 1 x 10(-1) v/v were 75% +/- 5%; 35% +/- 5%; and 2% +/- 5% . Ip is thus a rich source of polyphenols and has antioxidant properties comparable to those of green tea which merit further in vivo intervention and cross-sectional studies.

Radiat Res, 2003 Jul, 160(1), 25 - 37
Effect of power-frequency magnetic fields on genome-scale gene expression in Saccharomyces cerevisiae; Nakasono S et al.; To estimate the effect of 50 Hz magnetic-field exposure on genome-wide gene expression, the yeast Saccharomyces cerevisiae was used as a model for eukaryotes . 2D PAGE (about 1,000 spots) for protein and cDNA microarray (about 5,900 genes) analysis for mRNA were performed . The cells were exposed to 50 Hz vertical magnetic fields at 10, 150 or 300 mT r.m.s . for 24 h . As positive controls, the cells were exposed to aerobic conditions, heat (40 degrees C) or minimal medium . The 2D PAGE and microarray analyses for the positive controls showed high-confidence differential expression of many genes including those for known or unknown proteins and mRNAs . For magnetic-field exposure, no high-confidence changes in expression were observed for proteins or genes that were related to heat-shock response, DNA repair, respiration, protein synthesis and the cell cycle . Principal component analysis showed no statistically significant difference in principal components, with only insignificant differences between the magnetic-field intensities studied . In contrast, the principal components for the positive controls were significantly different . The results indicate that a 50 Hz magnetic field below 300 mT did not act as a general stress factor like heat shock or DNA damage, as had been reported previously by others . This study failed to find a plausible differential gene expression that would point to a possible mechanism of an effect of magnetic fields . The findings provide no evidence that the magnetic-field exposure alters the fundamental mechanism of translation and transcription in eukaryotic cells.

What is Food Microbiology?, What Is Environmental Microbiology?, What Is Bioassay?, What Is Listeria Monocytogenes?, What Is Water Purification?, e, Bacteria, i, Bacterium, a, Microbiology, e, Bacteriology, c, Microbe, a, Streptococcal, e, Escherichia coli, c, Antibiotic prophylaxis, s, Antibiotics, e, Antibiotics, o, Bacillus subtilis, a, Staphylococcus, s, Fermentations, e, Meningococcus, s, Cryptococci, i, Campylobacter, i, Yeasts, o, Multidrug resistant, e, Antibiotics, n, Antibiotics, e, Vibriosis, n, Cryptococci, s, Botulin, i, Escherichia coli, i, Streptococcal, n, Escherichia coli

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005