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