FEBS Lett, 2002 Apr 24, 517(1-3), 144 - 50 A metacaspase of Trypanosoma brucei causes loss of respiration competence and clonal death in the yeast Saccharomyces cerevisiae; Szallies A et al.; Metacaspases constitute a new group of cysteine proteases homologous to caspases . Heterologous expression of Trypanosoma brucei metacaspase TbMCA4 in the budding yeast Saccharomyces cerevisiae resulted in growth inhibition, mitochondrial dysfunction and clonal death . The metacaspase orthologue of yeast, ScMCA1 (YOR197w), exhibited genetic interaction with WWM1 (YFL010c), which encodes a small WW domain protein . WWM1 overexpression resulted in growth arrest and clonal death, which was suppressed by concomitant overexpression of ScMCA1 . GFP-fusion reporters of WWM1, ScMCA1 and TbMCA4 localized to the nucleus . Taken together, we suggest that metacaspases may play a role in nuclear function controlling cellular proliferation coupled to mitochondrial biogenesis. Curr Biol, 2002 Jun 4, 12(11), 944 - 50 Mitotic exit network controls the localization of Cdc14 to the spindle pole body in Saccharomyces cerevisiae; Yoshida S et al.; Budding yeast Cdc14 phosphatase plays essential roles in mitotic exit . Cdc14 is sequestered in the nucleolus by its inhibitor Net1/Cfi1 and is only released from the nucleolus during anaphase to inactivate mitotic CDK . It is believed that the mitotic exit network (MEN) is required for the release of Cdc14 from the nucleolus because liberation of Cdc14 by net1/cfi1 mutations bypasses the essential role of the MEN . But how the MEN residing at the spindle pole body (SPB) controls the association of Cdc14 with Net1/Cfi1 in the nucleolus is not yet understood . We found that Cdc14-5GFP was released from the nucleolus in the MEN mutants (tem1, cdc15, dbf2, and nud1), but not in the cdc5 cells during early anaphase . The Cdc14 liberation from the nucleolus was inhibited by the Mad2 checkpoint and by the Bub2 checkpoint in a different manner when microtubule organization was disrupted . We observed Cdc14-5GFP at the SPB in addition to the nucleolus . The SPB localization of Cdc14 was significantly affected by the MEN mutations and the bub2 mutation . We conclude that Cdc14 is released from the nucleolus at the onset of anaphase in a CDC5-dependent manner and that MEN factors possibly regulate Cdc14 release from the SPB. Proc Natl Acad Sci U S A, 2002 Jun 11, 99(12), 7956 - 61 HIV type 1 Gag virus-like particle budding from spheroplasts of Saccharomyces cerevisiae; Sakuragi S et al.; Expression of retroviral Gag protein in yeast has previously shown Gag targeting to the plasma membrane but little or no production of Gag virus-like particles (VLPs) . Here we show that, after removal of the cell wall, the expression of HIV type 1 Gag protein in Saccharomyces cerevisiae spheroplasts allowed simultaneous budding of VLPs from the plasma membrane . Our data show that (i) the VLPs released from yeast spheroplasts were spherical and had morphological features, such as membrane apposed electron-dense layers, characteristic of the immature form of HIV particles; (ii) the VLPs were completely enclosed in the plasma membrane derived from yeast, which is denser than that of higher eukaryotic cells; (iii) the VLP Gag shells remained intact after treatment of nonionic detergent; and (iv) the VLPs were released soon after removal of the cell wall and accumulated up to 300 microg/liter of culture . Our results also show that VLP production was abolished by amino acid substitution of the Gag N-terminal myristoylglycine and impaired when Gag C-terminal deletions were extended beyond the nucleocapsid domain . These results were consistent with those obtained previously in higher eukaryotic expression systems, suggesting that similar Gag domains were used for VLP assembly . We suggest that the system described here offers significant advantages for studying host factors required for VLP budding . The system also may be available for production of vector virus-free VLPs for practical applications such as vaccine development. Biochemistry (Mosc), 2002 May, 67(5), 592 - 6 The content and chain length of polyphosphates from vacuoles of Saccharomyces cerevisiae VKM Y-1173; Trilisenko LV et al.; The content of inorganic polyphosphates (polyP) in vacuoles of the yeast Saccharomyces cerevisiae is approximately 15% of the total cellular polyP . Over 80% of the vacuole polyP are in an acid-soluble fraction . It was first established by (31)P-NMR spectroscopy that a polymeric degree (n) of two subfractions obtained by precipitation with Ba(2+) in succession at pH 4.5 and 8.2 was approximately 20 +/- 5 and 5 +/- 2 residues of ortho-phosphoric acid, respectively . Under a deficit of phosphate (P(i)) in the cultivation medium, the polyP content in vacuoles decreased approximately 7-fold with the same drastic reduction of their content in the cell . Unlike intact yeast cells, where polyP overcompensation is observed after their transfer from phosphate-free to phosphate-containing medium, the vacuoles do not show this effect . The data indicate the occurrence of special regulatory mechanisms of polyP synthesis in vacuoles differing from those in the whole cell. J Biol Chem, 2002 Aug 23, 277(34), 31079 - 88 Epub 2002 Jun 10. Genome-wide analysis of gene expression regulated by the calcineurin/Crz1p signaling pathway in Saccharomyces cerevisiae; Yoshimoto H et al.; In Saccharomyces cerevisiae, the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin, is activated by specific environmental conditions, including exposure to Ca(2+) and Na(+), and induces gene expression by regulating the Crz1p/Tcn1p transcription factor . We used DNA microarrays to perform a comprehensive analysis of calcineurin/Crz1p-dependent gene expression following addition of Ca(2+) (200 mm) or Na(+) (0.8 m) to yeast . 163 genes exhibited increased expression that was reduced 50% or more by calcineurin inhibition . These calcineurin-dependent genes function in signaling pathways, ion/small molecule transport, cell wall maintenance, and vesicular transport, and include many open reading frames of previously unknown function . Three distinct gene classes were defined as follows: 28 genes displayed calcineurin-dependent induction in response to Ca(2+) and Na(+), 125 showed calcineurin-dependent expression following Ca(2+) but not Na(+) addition, and 10 were regulated by calcineurin in response to Na(+) but not Ca(2+) . Analysis of crz1Delta cells established Crz1p as the major effector of calcineurin-regulated gene expression in yeast . We identified the Crz1p-binding site as 5'-GNGGC(G/T)CA-3' by in vitro site selection . A similar sequence, 5'-GAGGCTG-3', was identified as a common sequence motif in the upstream regions of calcineurin/ Crz1p-dependent genes . This finding is consistent with direct regulation of these genes by Crz1p. Free Radic Biol Med, 2002 Jun 15, 32(12), 1244 - 53 Repair of 8-oxoguanine in Saccharomyces cerevisiae: interplay of DNA repair and replication mechanisms; Boiteux S et al.; 8-Oxo-7,8-dihydroguanine (8-oxoG) is produced abundantly in DNA exposed to free radicals and reactive oxygen species . The biological relevance of 8-oxoG has been unveiled by the study of two mutator genes in Escherichia coli, fpg, and mutY . Both genes code for DNA N-glycosylases that cooperate to prevent the mutagenic effects of 8-oxoG in DNA . In Saccharomyces cerevisiae, the OGG1 gene encodes a DNA N-glycosylase/AP lyase, which is the functional homologue of the bacterial fpg gene product . The inactivation of OGG1 in yeast creates a mutator phenotype that is specific for the generation of GC to TA transversions . In yeast, nucleotide excision repair (NER) also contributes to the release of 8-oxoG in damaged DNA . Furthermore, mismatch repair (MMR) mediated by MSH2/MSH6/MLH1 plays a major role in the prevention of the mutagenic effect of 8-oxoG . Indeed, MMR acts as the functional homologue of the MutY protein of E . coli, excising the adenine incorporated opposite 8-oxoG . Finally, the efficient and accurate replication of 8-oxoG by the yeast DNA polymerase eta also prevents 8-oxoG-induced mutagenesis . The aim of this review is to summarize recent literature dealing with the replication and repair of 8-oxoG in Saccharomyces cerevisiae, which can be used as a paradigm for DNA repair in eukaryotes. Methods, 2002 Mar, 26(3), 281 - 90 Hybridization array technology coupled with chemostat culture: Tools to interrogate gene expression in Saccharomyces cerevisiae; Hayes A et al.; Hybridization array technology is increasingly being used for the analysis of gene expression in the yeast Saccharomyces cerevisiae . It is a powerful technique in which the relative abundance of all the mRNA molecules transcribed under a particular condition may be simultaneously measured . However, most studies performed using this technique are carried out in batch culture where the growth rate and environment are continuously changing . Often, the experimental condition being studied also impacts on the growth rate of the cells . Changes in growth rate affect the pattern of gene expression . Consequently, the analysis and interpretation of experimental results obtained in this way are inherently problematic due to the difficulty in discriminating between effects due to the experimental condition per se and concomitant growth rate-related effects . Here, we present a method that addresses this problem by exploiting chemostat culture, in which the cells can be grown at a fixed growth rate, in combination with hybridization array technology . We use two experimental examples to illustrate the advantages of using this approach and then describe a specific application of this approach to investigate the effect of carbon and nitrogen limitation at the transcriptome level. J Mol Biol, 2002 Jun 7, 319(3), 703 - 14 Mediator factor Med8p interacts with the hexokinase 2: implication in the glucose signalling pathway of Saccharomyces cerevisiae; de la Cera T et al.; In the presence of glucose the protein hexokinase 2 (Hxk2p), normally resident in the cytosol, is translocated to the nucleus where it impairs the activation of transcription of the glucose-repressed genes HXK1, GLK1 and SUC2, and promotes the activation of transcription of the glucose-induced genes HXK2 and HXT1 . Here, we demonstrate the involvement of an heptameric motif, named the MED8 site, in the direct binding of the mediator protein Med8p, either as a monomer or as a homodimer . Because this site was previously involved in the Hxk2p-dependent glucose-induced regulation of gene transcription, we tested whether Hxk2p interacts with Med8p . Our results show that Hxk2 and Med8 proteins are physically associated and that this Hxk2p-Med8p interaction is of physiological significance because both proteins have been found interacting together in a cluster with DNA fragments containing the MED8 site . We conclude that Hxk2p operates through the MED8 site, by interacting with Med8p, in the glucose signal transduction pathway of Saccharomyces cerevisiae . (c) 2002 Elsevier Science Ltd. Biochem Biophys Res Commun, 2002 Apr 26, 293(1), 139 - 44 Degradation of ornithine decarboxylase in Saccharomyces cerevisiae is ubiquitin independent; Gandre S et al.; Ornithine decarboxylase (ODC), the first rate-limiting enzyme in the polyamine biosynthesis is one of the most rapidly degraded proteins in eukaryotic cells . Mammalian ODC is a notable exception to the widely accepted dogma that ubiquitination is always required for targeting a protein to degradation by the 26S proteasome . However, while it is well established that in mammalian cells degradation of ODC is ubiquitin independent, the requirement of ubiquitination for degradation of ODC in yeast cells remained undetermined . We have investigated ODC degradation in three mutant strains of Saccharomyces cerevisiae in which ubiquitin-dependent protein degradation activity is severely compromised . While yeast ODC was rapidly degraded in all these mutant strains the degradation of N-end rule substrates was inhibited . A mutant mouse ODC that fails to interact with Az was rapidly degraded in yeast cells but was stable in mammalian cells suggesting that interaction with a mammalian Az like yeast protein is not necessary for the degradation of ODC in yeast cells . Deletion analysis revealed that sequences from its unique N-terminus are involved in targeting yeast ODC to rapid degradation in yeast cells. Mol Cell Biol, 2002 Jul, 22(13), 4739 - 49 A third osmosensing branch in Saccharomyces cerevisiae requires the Msb2 protein and functions in parallel with the Sho1 branch; O'Rourke SM et al.; Two Saccharomyces cerevisiae plasma membrane-spanning proteins, Sho1 and Sln1, function during increased osmolarity to activate a mitogen-activated protein (MAP) kinase cascade . One of these proteins, Sho1, utilizes the MAP kinase kinase kinase Ste11 to activate Pbs2 . We previously used the FUS1 gene of the pheromone response pathway as a reporter to monitor cross talk in hog1 mutants . Cross talk requires the Sho1-Ste11 branch of the HOG pathway, but some residual signaling, which is STE11 dependent, still occurs in the absence of Sho1 . These observations led us to propose the existence of another osmosensor upstream of Ste11 . To identify such an osmosensor, we screened for mutants in which the residual signaling in a hog1 sho1 mutant was further reduced . We identified the MSB2 gene, which encodes a protein with a single membrane-spanning domain and a large presumptive extracellular domain . Assay of the FUS1-lacZ reporter (in a hog1 mutant background) showed that sho1 and msb2 mutations both reduced the expression of the reporter partially and that the hog1 sho1 msb2 mutant was severely defective in the expression of the reporter . The use of DNA microarrays to monitor gene expression revealed that Sho1 and Msb2 regulate identical gene sets in hog1 mutants . A role for MSB2 in HOG1 strains was also seen in strains defective in the two known branches that activate Pbs2: an ssk1 sho1 msb2 strain was more osmosensitive than an ssk1 sho1 MSB2 strain . These observations indicate that Msb2 is partially redundant with the Sho1 osmosensing branch for the activation of Ste11. J Biol Chem, 2002 Aug 23, 277(34), 31062 - 71 Epub 2002 Jun 06. Fatty acid transport in Saccharomyces cerevisiae . Directed mutagenesis of FAT1 distinguishes the biochemical activities associated with Fat1p; Zou Z et al.; The fatty acid transport protein Fat1p functions as a component of the long-chain fatty acid transport apparatus in the yeast Saccharomyces cerevisiae . Fat1p has significant homologies to the mammalian fatty acid transport proteins (FATP) and the very long-chain acyl-CoA synthetases (VLACS) . In order to further understand the functional roles intrinsic to Fat1p (fatty acid transport and VLACS activities), a series of 16 alleles carrying site-directed mutations within FAT1 were constructed and analyzed . Sites chosen for the construction of amino acid substitutions were based on conservation between Fat1p and the mammalian FATP orthologues and included the ATP/AMP and FATP/VLACS signature motifs . Centromeric and 2 mu plasmids encoding mutant forms of Fat1p were transformed into a yeast strain containing a deletion in FAT1 (fat1Delta) . For selected subsets of FAT1 mutant alleles, we observed differences between the wild type and mutants in 1) growth rates when fatty acid synthase was inhibited with 45 microm cerulenin in the presence of 100 microm oleate (C(18:1)), 2) levels of fatty acid import monitored using the accumulation of the fluorescent fatty acid 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-S-indacene-3-dodecanoic acid and {(3)H}oleate, 3) levels of lignoceryl (C(24:0)) CoA synthetase activities, and 4) fatty acid profiles monitored using gas chromatography/mass spectrometry . In most cases, there was a correlation between growth on fatty acid/cerulenin plates, the levels of fatty acid accumulation, very long-chain fatty acyl-CoA synthetase activities, and the fatty acid profiles in the different FAT1 mutants . For several notable exceptions, the fatty acid transport and very long-chain fatty acyl-CoA synthetase activities were distinguishable . The characterization of these novel mutants provides a platform to more completely understand the role of Fat1p in the linkage between fatty acid import and activation to CoA thioesters. J Struct Biol, 2001 Dec, 136(3), 167 - 80 The first three-dimensional structure of phosphofructokinase from Saccharomyces cerevisiae determined by electron microscopy of single particles; Ruiz T et al.; Phosphofructokinaseis a key regulatory enzyme of the glycolytic pathway . We have determined the structure of this enzyme from Saccharomyces cerevisiae to a resolution of 2.0 nm . This is the first structure available for this family of enzymes in eukaryotic organisms . Phosphofructokinase is an octamer composed of 4alpha and 4beta subunits arranged in a dihedral point group symmetry D(2) . The enzyme has a very open and elongated structure, with dimensions of 24 nm in length and 17 nm in width . The final structure, calculated from 0 degrees tilt projections of the molecule at random orientations using as reference the volume obtained by the random conical reconstruction technique in ice, has allowed us to discern the shapes of the subunits and their mutual arrangement in the octamer . (c)2002 Elsevier Science (USA). Mol Cell, 2002 May, 9(5), 1055 - 65 Rad9 phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint; Schwartz MF et al.; Rad9 is required for the MEC1/TEL1-dependent activation of Saccharomyces cerevisiae DNA damage checkpoint pathways mediated by Rad53 and Chk1 . DNA damage induces Rad9 phosphorylation, and Rad53 specifically associates with phosphorylated Rad9 . We report here that multiple Mec1/Tel1 consensus {S/T}Q sites within Rad9 are phosphorylated in response to DNA damage . These Rad9 phosphorylation sites are selectively required for activation of the Rad53 branch of the checkpoint pathway . Consistent with the in vivo function in recruiting Rad53, Rad9 phosphopeptides are bound by Rad53 forkhead-associated (FHA) domains in vitro . These data suggest that functionally independent domains within Rad9 regulate Rad53 and Chk1, and support the model that FHA domain-mediated recognition of Rad9 phosphopeptides couples Rad53 to the DNA damage checkpoint pathway. J Biol Chem, 2002 Aug 9, 277(32), 28780 - 6 Epub 2002 Jun 04. Rpf2p, an evolutionarily conserved protein, interacts with ribosomal protein L11 and is essential for the processing of 27 SB Pre-rRNA to 25 S rRNA and the 60 S ribosomal subunit assembly in Saccharomyces cerevisiae; Morita D et al.; Saccharomyces cerevisiae Rrs1p is a nuclear protein that is essential for the maturation of 25 S rRNA and the 60 S ribosomal subunit assembly . In two-hybrid screening, using RRS1 as bait, we have cloned YKR081c/RPF2 . Rpf2p is essential for growth and is mainly localized in the nucleolus . The amino acid sequence of Rpf2p is highly conserved in eukaryotes from yeast to human . Similar to Rrs1p, Rpf2p shows physical interaction with ribosomal protein L11 and appears to associate with preribosomal subunits fairly tightly . Northern, methionine pulse-chase, and sucrose density gradient ultracentrifugation analyses reveal that the depletion of Rpf2p results in a delayed processing of pre-rRNA, a decrease of mature 25 S rRNA, and a shortage of 60 S subunits . An analysis of processing intermediates by primer extension shows that the Rpf2p depletion leads to an accumulation of 27 SB pre-rRNA, suggesting that Rpf2p is required for the processing of 27 SB into 25 S rRNA. J Cell Sci, 2002 Jun 15, 115(Pt 12), 2549 - 58 Mechanisms for targeting of the Saccharomyces cerevisiae GPI-anchored cell wall protein Crh2p to polarised growth sites; Rodriguez-Pena JM et al.; The cell wall is an essential structure that preserves the osmotic integrity of fungal cells and determines cellular morphology during developmental programs . The high number of different wall components demands a variety of processes to deliver precursors and synthetic proteins to the proper location at the right time for wall development and modification . Here, the specificity of the mechanisms that regulate the temporal and spatial localisation of cell wall proteins to sites of polarised growth in Saccharomyces cerevisiae is investigated . For this purpose, the localisation of Crh2p, a cell wall glycosylphosphatidylinositol (GPI)-anchored mannoprotein that we have recently described as involved in cell wall construction and localised to polarised growth sites, was followed using a Crh2p-GFP fusion protein . Crh2p distribution was studied in several genetic backgrounds affected in different steps of the cell polarity establishment machinery or/and bud morphogenesis . Crh2p is localised at the mother-bud neck in bud1 cells following the random budding pattern characteristic of this mutant . The Crh2p distribution was greatly altered in a cdc42-1 mutant, indicating complete dependence on an organised actin cytoskeleton for polarised Crh2p distribution . The usual deposition of Crh2p in a ring at the base of growing buds was lacking in cdc10-11 cells growing under restrictive temperature conditions, whereas Crh2p deposition at the septum region was absent in both cdc10-11 and cdc15-lyt1 cells . These results point to the dependence of Crh2p localisation at the bud-neck on both septins and septum integrity . Furthermore, in the absence of Bni4p, a scaffold protein involved in the targeting of the chitin synthase III complex to the bud neck, Crh2p was not longer found at the neck in large-budded cells undergoing cytokinesis . Finally, Crh2p was not properly localised in cells deleted in CHS5, which encodes a protein involved in the transport of Chs3p, and was completely mislocalised in sbe2/sbe22 mutants, suggesting that the transport systems for Chs3p and Crh2p are to a certain extent coincident . The transport of other GPI-cell wall proteins, such as Cwp1p, however, does not depend on these systems as the localisation of the latter protein was not affected in either of these mutants. J Biol Chem, 2002 Aug 16, 277(33), 29963 - 72 Epub 2002 May 31. Ntg2p, a Saccharomyces cerevisiae DNA N-glycosylase/apurinic or apyrimidinic lyase involved in base excision repair of oxidative DNA damage, interacts with the DNA mismatch repair protein Mlh1p . Identification of a Mlh1p binding motif; Gellon L et al.; Ntg2p is a DNA N-glycosylase/apurinic or apyrimidinic lyase involved in base excision repair of oxidatively damaged DNA in Saccharomyces cerevisiae . Using a yeast two-hybrid screen and a GST in vitro transcription and translation assay, the mismatch repair (MMR) protein Mlh1p was demonstrated to interact physically with Ntg2p . The Mlh1p binding site maps to amino acids residues 15-40 of Ntg2p . The Ntg2p binding site is localized in the C-terminal end (483-769) of Mlh1p . Overproduction of Ntg2p results in a mutator phenotype with enhanced frameshift reversion frequency, suggesting partial inhibition of the MMR pathway . In contrast, inactivation of NTG2 does not enhance mutagenesis, indicating that Ntg2p is not required for MMR . Site-directed mutagenesis of the Mlh1p binding domain of Ntg2p revealed three amino acids (Ser(24), Tyr(26), Phe(27)) that are absolutely required for Ntg2p-Mlh1p interaction . These residues are part of a motif found in Ntg2p (Arg(23)-Ser(24)-Lys(25)-Tyr(26)-Phe(27)), Exo1p (Arg(444)-Ser(445)-Lys(446)-Phe(447)-Phe(448)), and Sgs1p (Lys(1383)-Ser(1384)-Lys(1385)-Phe(1386)-Phe(1387)) . In these three proteins, the motif is part of the domain that interacts with the C-terminal end of Mlh1p . Furthermore, S445A, F447A, and F448A mutants of Exo1p do not bind Mlh1p, but the wild type Exo1p does . Therefore, we propose that the R/K-S-R/K-Y/F-Y/F sequence could define a Mhl1 binding motif . The results also suggest that base excision repair and MMR can cooperate to prevent deleterious effects of oxidative DNA damage. J Antimicrob Chemother, 2002 Jun, 49(6), 905 - 15 Response of gene expression in Saccharomyces cerevisiae to amphotericin B and nystatin measured by microarrays; Zhang L et al.; The response of the yeast Saccharomyces cerevisiae to two polyene antibiotics, amphotericin B and nystatin, was studied by genomic expression profiling . The two agents produced highly similar expression pattern changes, which was consistent with their known identical mechanisms of action on cell membranes . Detailed analysis was focused on the amphotericin B-treated sample in this study . Our data showed that genes involved in mitochondrial ribosomal protein synthesis were more severely repressed than those in the cytoplasm, which might contribute to the cytotoxicity of amphotericin B . To counteract the leakage of intracellular nutrients and ions from the pores in the cell membrane caused by amphotericin B, c . 17 genes involved in transport facilitation were induced, presumably to allow more efficient uptake of nutrients and ions . The expression level of five genes involved in ergosterol synthesis dropped and three genes related to cell wall biogenesis were induced, indicating that the cell membrane and cell wall were also affected by the presence of polyene antibiotics . It was observed that the pleiotropic drug resistance network in yeast cells was activated after exposure to amphotericin B, possibly contributing to the acquisition of amphotericin B resistance . Part of the gene expression alteration measured by microarray was confirmed by quantitative RT-PCR. J Biol Chem, 2002 Aug 30, 277(35), 31303 - 9 Epub 2002 May 29. Functional expression of phosphagen kinase systems confers resistance to transient stresses in Saccharomyces cerevisiae by buffering the ATP pool; Canonaco F et al.; Phosphagen kinase systems provide different advantages to tissues with high and fluctuating energy demands, in particular an efficient energy buffering system . In this study we show for the first time functional expression of two phosphagen kinase systems in Saccharomyces cerevisiae, which does not normally contain such systems . First, to establish the creatine kinase system, in addition to overexpressing creatine kinase isoenzymes, we had to install the biosynthesis pathway of creatine by co-overexpression of L-arginine:glycine amidinotransferase and guanidinoacetate methyltransferase . Although we could achieve considerable creatine kinase activity, together with more than 3 mM intracellular creatine, this was not sufficient to confer an obvious advantage to the yeast under the specific stress conditions examined here . Second, using arginine kinase, we successfully installed an intracellular phosphagen pool of about 5 mM phosphoarginine . Such arginine kinase-expressing yeast showed improved resistance under two stress challenges that drain cellular energy, which were transient pH reduction and starvation . Although transient starvation led to 50% reduced intracellular ATP concentrations in wild-type yeast, arginine kinase overexpression stabilized the ATP pool at the pre-stress level . Thus, our results demonstrate that temporal energy buffering is an intrinsic property of phosphagen kinases that can be transferred to phylogenetically very distant organisms. Biosci Biotechnol Biochem, 2002 Apr, 66(4), 906 - 9 5-Bromo-2'-deoxyuridine efficiently suppresses division potential of the yeast Saccharomyces cerevisiae; Fujii M et al.; We established a thymidine-auxotrophic strain of the yeast Saccharomyces cerevisiae to examine biological effects of thymidine analogues . 5-Bromo-2'-deoxyuridine efficiently suppressed the division potential of yeast showing morphology similar to senescent cells. J Biol Chem, 2002 Aug 16, 277(33), 30048 - 54 Epub 2002 May 28. Identification and characterization of a Saccharomyces cerevisiae gene, RSB1, involved in sphingoid long-chain base release; Kihara A et al.; Sphingoid long-chain bases (LCBs) and long-chain base phosphates (LCBPs) act as signaling molecules in eukaryotic cells . Accumulation of LCBPs results in cell growth inhibition in yeast, although the mechanism is unknown . Here, we identified a novel yeast gene, RSB1 (resistance to sphingoid long-chain base), by screening a multicopy suppressor of the LCB-sensitive phenotype of the LCBP lyase mutant . RSB1 encodes a polypeptide of 354 amino acids with a molecular mass of 40.4 kDa . Rsb1p is predicted to be an integral membrane protein with seven transmembrane-spanning domains . We demonstrated that cells overproducing Rsb1p showed a decrease in accumulation of exogenously added sphingosine and dihydrosphingosine because of their increased release . This release was ATP-dependent, and a mutant of the predicted ATP binding motif had no activity . Substrate specificity analysis of Rsb1p demonstrated that it is active on LCBs but not on LCBPs or other hydrophobic compounds . These results suggest that Rsb1p is a transporter or flippase that translocates LCBs from the cytoplasmic side toward the extracytoplasmic side of the membrane. Gene, 2002 Apr 17, 288(1-2), 19 - 27 DEAD-box RNA helicase Sub2 is required for expression of lacZ fusions in Saccharomyces cerevisiae and is a dosage-dependent suppressor of RLR1 (THO2); West RW Jr et al.; RLR1 (THO2) encodes a novel, phylogenetically-conserved KEKE motif protein involved in transcription and transcription-associated recombination in Saccharomyces cerevisiae . One characteristic aspect of RLR1 function is its requirement for expression of the Escherichia coli lacZ reporter gene regardless of the yeast promoter to which it is fused . rlr1-1 was originally isolated (employing lacZ as a transcriptional reporter) as a suppressor of a mutation in the gene encoding Sin4, a subunit of the Mediator subcomplex of the RNA polymerase II (PolII) transcriptional machinery . To clarify the function of Rlr1, we performed a genetic screen for dosage-dependent suppressors of the cold-sensitive phenotype of rlr1-1 . From this screen we isolated SUB2, encoding a conserved DEAD-box RNA helicase family member having roles in both pre-mRNA splicing and mRNA export in yeast, flies, and humans . We demonstrate that Sub2, like Rlr1, is required for lacZ to be expressed in yeast, and that sub2 mutants manifest rlr1-like growth defects . Our results are consistent with a hypothesis where expression of lacZ fusions in yeast preferentially requires a Sub2-mediated mRNP assembly/export pathway linked to transcription via Rlr1. Biochim Biophys Acta, 2002 Apr 22, 1554(1-2), 101 - 7 Mutations of cytochrome c oxidase subunits 1 and 3 in Saccharomyces cerevisiae: assembly defect and compensation; Meunier B et al.; Eukaryotic cytochrome oxidases are composed of up to 13 subunits, of which three, subunits 1, 2 and 3, are mitochondrially encoded . In this study, yeast mutants were used to investigate the role of subunits 1 and 3 domains on the enzyme assembly . Mutation S203L in subunit 3 which abolished the respiratory growth, decreased cytochrome oxidase content, as measured by optical spectroscopy and immunodetection . Secondary mutations in subunits 1 and 3 restored (partly) the enzyme level . Two reversions reintroduced residues with a hydroxyl group at the primary mutation site (S203T) or in a subunit 3 transmembrane helix close to subunit 1 (G104S) . These residues may be involved in hydrogen bonding which strengthen subunits 1-3 interaction . Two other reversions (A224V and Q137K) are located in P-side loops in subunit 1, which may be involved in the enzyme assembly . A mutation in residue A224 has been reported in a family presenting with encephalomyopathy . Surprisingly, the introduction of the 'human' mutation A224S and of a more drastic change A224F had no effect on the yeast enzyme . This might be explained by differences in local folding in the two enzymes. J Biol Chem, 2002 Aug 2, 277(31), 27991 - 5 Epub 2002 May 24. Kinetic regulation of the mitochondrial glycerol-3-phosphate dehydrogenase by the external NADH dehydrogenase in Saccharomyces cerevisiae; Pahlman IL et al.; In the yeast Saccharomyces cerevisiae, the two most important systems for conveying excess cytosolic NADH to the mitochondrial respiratory chain are external NADH dehydrogenase (Nde1p/Nde2p) and the glycerol-3-phosphate dehydrogenase shuttle . In the latter system, NADH is oxidized to NAD+ and dihydroxyacetone phosphate is reduced to glycerol 3-phosphate by the cytosolic Gpd1p; glycerol 3-phosphate gives two electrons to the respiratory chain via mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p)-regenerating dihydroxyacetone phosphate . Both Nde1p/Nde2p and Gut2p are located in the inner mitochondrial membrane with catalytic sites facing the intermembranal space . In this study, we showed kinetic interactions between these two enzymes . First, deletion of either one of the external dehydrogenases caused an increase in the efficiency of the remaining enzyme . Second, the activation of NADH dehydrogenase inhibited the Gut2p in such a manner that, at a saturating concentration of NADH, glycerol 3-phosphate is not used as respiratory substrate . This effect was not a consequence of a direct action of NADH on Gut2p activity because both NADH dehydrogenase and its substrate were needed for Gut2p inhibition . This kinetic regulation of the activity of an enzyme as a function of the rate of another having a similar physiological function may be allowed by their association into the same supramolecular complex in the inner membrane . The physiological consequences of this regulation are discussed. EMBO J, 2002 Jun 3, 21(11), 2833 - 41 Endogenous DNA abasic sites cause cell death in the absence of Apn1, Apn2 and Rad1/Rad10 in Saccharomyces cerevisiae; Guillet M et al.; In Saccharomyces cerevisiae, mutations in APN1, APN2 and either RAD1 or RAD10 genes are synthetic lethal . In fact, apn1 apn2 rad1 triple mutants can form microcolonies of approximately 300 cells . Expression of Nfo, the bacterial homologue of Apn1, suppresses the lethality . Turning off the expression of Nfo induces G(2)/M cell cycle arrest in an apn1 apn2 rad1 triple mutant . The activation of this checkpoint is RAD9 dependent and allows residual DNA repair . The Mus81/Mms4 complex was identified as one of these back-up repair activities . Furthermore, inactivation of Ntg1, Ntg2 and Ogg1 DNA N-glycosylase/AP lyases in the apn1 apn2 rad1 background delayed lethality, allowing the formation of minicolonies of approximately 10(5) cells . These results demonstrate that, under physiological conditions, endogenous DNA damage causes death in cells deficient in Apn1, Apn2 and Rad1/Rad10 proteins . We propose a model in which endogenous DNA abasic sites are converted into 3'-blocked single-strand breaks (SSBs) by DNA N-glycosylases/AP lyases . Therefore, we suggest that the essential and overlapping function of Apn1, Apn2, Rad1/Rad10 and Mus81/Mms4 is to repair 3'-blocked SSBs using their 3'-phosphodiesterase activity or their 3'-flap endonuclease activity, respectively. Biochim Biophys Acta, 2002 Jun 7, 1576(1-2), 23 - 9 Oxidant regulation of the Saccharomyces cerevisiae GSH1 gene; Dormer UH et al.; Glutathione (gamma-L-glutamyl-L-cysteinylglycine) is an important antioxidant molecule, helping to protect the cell against oxidative stress . Expression of the Saccharomyces cerevisiae GSH1 gene, coding for the first enzyme involved in glutathione biosynthesis, is regulated at the level of transcription by oxidants and heavy metals . We have characterised the sequences of the GSH1 promoter responsible for the amino acid-dependent H(2)O(2) regulation of transcription . We show that there are at least two H(2)O(2)-responsive elements in the promoter, neither of which map to the putative Yap1 binding site . Our results suggest that the Yap1 protein plays an important, but indirect role in the H(2)O(2)-dependent regulation of GSH1 transcription. Planta, 2002 Jun, 215(2), 293 - 303 Epub 2002 Feb 20. Targeting and membrane-insertion of a sunflower oleosin in vitro and in Saccharomyces cerevisiae: the central hydrophobic domain contains more than one signal sequence, and directs oleosin insertion into the endoplasmic reticulum membrane using a signal anchor sequence mechanism; Beaudoin F et al.; A range of N- and C-terminal deletions of an oleosin from Helianthus annuus L . were used to study the endoplasmic reticulum (ER) targeting and membrane insertion of this protein both in vitro and in vivo in yeast ( Saccharomyces cerevisiae) . Neither the N- nor the C-terminal hydrophilic domains are important for targeting and/or membrane insertion, with all the information required for these processes located within the central hydrophobic region of the protein . However, in vitro membrane-insertion experiments suggest that these domains are important for a correct topology of the oleosin within the ER membrane . The first half of the hydrophobic central domain, flanked by the positively charged N-terminal domain, is likely to function as a type-II signal-anchor (SAII) sequence . However, in the absence of the N-terminal 26 residues of this domain, the proline-knot region and the second half of this hydrophobic domain are sufficient to direct oleosin to the ER and to allow stable (but far less efficient) integration of the protein into the membrane . Taken together, these results indicate that oleosin contains more than one domain that is capable of interacting with the signal recognition particle to direct the protein to the ER membrane. Arch Microbiol, 2002 Jun, 177(6), 494 - 9 Epub 2002 Apr 04. Response to oxidative stress caused by H(2)O(2) in Saccharomyces cerevisiae mutants deficient in trehalase genes; Pedreno Y et al.; The role of trehalose as cell protector against oxidative stress induced by H(2)O(2) has been studied in Saccharomyces cerevisiae mutants in which the two trehalase genes ATH1 and NTH1 are deleted . The addition of low H(2)O(2) concentrations to proliferating cultures of either strain did not harm cell viability and induced a marked activity to Nth1p, but with no significant level of trehalose accumulation . This pattern was reversed after a more severe H(2)O(2) treatment that caused drastic cell killing . The most severe phenotype corresponded to the Delta nth1 mutant . Under these conditions, the increase in Nth1p was abolished and a three-fold rise in trehalose content was recorded concomitant with activation of the trehalose synthase complex . The behavior of the double-disruptant Delta ath1Delta nth1 mutant was identical to that of wild-type cells, although in exponential cultures Ath1p activity was virtually undetectable upon exposure to H(2)O(2) . Furthermore, these strains displayed an adaptive response to oxidative stress that was independent of intracellular trehalose synthesis . Our data strongly suggest that trehalose storage in budding yeasts is not an essential protectant in cell defense against oxidative challenge. BMC Mol Biol . 2002 May 20;3(1):7 . Epub 2002 May 20. Control of dinucleoside polyphosphates by the FHIT-homologous HNT2 gene, adenine biosynthesis and heat shock in Saccharomyces cerevisiae; Rubio-Texeira M et al.; BACKGROUND: The FHIT gene is lost early in the development of many tumors . Fhit possesses intrinsic ApppA hydrolase activity though ApppA cleavage is not required for tumor suppression . Because a mutant form of Fhit that is functional in tumor suppression and defective in catalysis binds ApppA well, it was hypothesized that Fhit-substrate complexes are the active, signaling form of Fhit . Which substrates are most important for Fhit signaling remain unknown . RESULTS: Here we demonstrate that dinucleoside polyphosphate levels increase 500-fold to hundreds of micromolar in strains devoid of the Saccharomyces cerevisiae homolog of Fhit, Hnt2 . Accumulation of dinucleoside polyphosphates is reversed by re-expression of Hnt2 and is active site-dependent . Dinucleoside polyphosphate levels depend on an intact adenine biosynthetic pathway and time in liquid culture, and are induced by heat shock to greater than 0.1 millimolar even in Hnt2+ cells . CONCLUSIONS: The data indicate that Hnt2 hydrolyzes both ApppN and AppppN in vivo and that, in heat-shocked, adenine prototrophic yeast strains, dinucleoside polyphosphates accumulate to levels in which they may saturate Hnt2. Mol Microbiol, 2002 Jun, 44(5), 1299 - 308 Hexose phosphorylation and the putative calcium channel component Mid1p are required for the hexose-induced transient elevation of cytosolic calcium response in Saccharomyces cerevisiae; Tokes-Fuzesi M et al.; Saccharomyces cerevisiae responds to environ-mental stimuli such as an exposure to pheromone or to hexoses after carbon source limitation with a transient elevation of cytosolic calcium (TECC) response . In this study, we examined whether hexose transport and phosphorylation are necessary for the TECC response . We found that a mutant strain lacking most of the known hexose transporters was unable to carry out the TECC response when exposed to glucose . A mutant strain that lacked the ability to phosphorylate glucose was unable to respond to glucose addition, but displayed a normal TECC response after the addition of galactose . These results indicate that hexose uptake and phosphorylation are required to trigger the hexose-induced TECC response . We also found that the TECC response was significantly smaller than normal when the level of environmental calcium was reduced, and was abolished in a mid1 mutant that lacked a subunit of the high-affinity calcium channel of the yeast plasma membrane . These results indicate that most or all of the TECC response is mediated by an influx of calcium from the extracellular space . Our results indicate that this transient increase in plasma membrane calcium permeability may be linked to the accumulation of Glc-1-P (or a related glucose metabolite) in yeast. Mol Cell Biol, 2002 Jun, 22(12), 4218 - 29 RSC2, encoding a component of the RSC nucleosome remodeling complex, is essential for 2 microm plasmid maintenance in Saccharomyces cerevisiae; Wong MC et al.; The stable maintenance of the 2 microm circle plasmid depends on its ability to overcome intrinsic maternal inheritance bias, which in yeast normally results in the failure to transmit DNA molecules efficiently to daughter cells . In addition to the plasmid proteins Rep1 and Rep2 acting on the plasmid DNA locus STB, it is likely that other chromosomally encoded yeast proteins are required . We have isolated mutants of yeast unable to maintain 2 microm and found that RSC2 is essential for 2 microm to overcome maternal inheritance bias . Rsc2 is part of a multisubunit RSC chromatin remodeling complex, and we show that in the absence of Rsc2 the chromatin structure of the STB region is significantly altered and the Rep1 protein loses its normal localization to subnuclear foci . Rsc1, a closely related homolog of Rsc2 present in an alternative form of the RSC complex, is not required for 2 microm maintenance and does not replace the requirement for Rsc2 when overexpressed . This represents the first specific role for Rsc2 that has been related to a change in chromatin structure, as well as the first direct evidence linking chromatin structure to 2 microm segregation. FEMS Microbiol Lett, 2002 Apr 23, 210(1), 81 - 5 Correct GPI-anchor synthesis is required for the incorporation of endoglucanase/glucanosyltransferase Bgl2p into the Saccharomyces cerevisiae cell wall; Kalebina TS et al.; The SSU21/MCD4 gene encodes an essential component of the glycosylphosphatidylinositol (GPI)-anchor synthesis pathway in Saccharomyces cerevisiae . Here we demonstrate that the ssu21 mutation affected the transport and the incorporation into the cell wall of the major non-GPI yeast cross-linker - endoglucanase/glucanosyltransferase Bgl2p . This mutation also led to a decrease in the levels of both known types of cell wall mannoproteins, those covalently linked with glucan and SDS-extractable proteins . Our results indicate that the precision of the GPI-anchor synthesis is essential for cell wall assembly and suggest the strong interdependence of different groups of cell wall proteins during their incorporation into the cell wall. FEMS Microbiol Lett, 2002 Apr 23, 210(1), 19 - 23 Carnitine biosynthesis in Neurospora crassa: identification of a cDNA coding for epsilon-N-trimethyllysine hydroxylase and its functional expression in Saccharomyces cerevisiae; Swiegers JH et al.; The biosynthesis of L-carnitine in eukaryotic organisms was first elucidated in the ascomycete Neurospora crassa . The first step of the pathway is catalysed by epsilon-N-trimethyllysine hydroxylase (TMLH), which converts epsilon-N-trimethyllysine into beta-hydroxy-N-epsilon-trimethyllysine in a reaction dependent on alpha-ketoglutarate, Fe2+ and oxygen . Here we report on the cloning of the N . crassa TMLH cDNA and its functional expression in Saccharomyces cerevisiae . The TMLH cDNA contains an open reading frame of 1413 base pairs encoding a predicted polypeptide of 471 amino acids . The Michaelis-Menten constants of the heterologously expressed enzyme were determined for epsilon-N-trimethyllysine, alpha-ketoglutarate, Fe2+ and correspond to 0.33 mM, 133 microM and 46 microM, respectively. Genetics, 2002 May, 161(1), 47 - 57 The mcm5-bob1 bypass of Cdc7p/Dbf4p in DNA replication depends on both Cdk1-independent and Cdk1-dependent steps in Saccharomyces cerevisiae; Sclafani RA et al.; The roles in DNA replication of two distinct protein kinases, Cdc7p/Dbf4p and Cdk1p/Clb (B-type cyclin), were studied . This was accomplished through a genetic and molecular analysis of the mechanism by which the mcm5-bob1 mutation bypasses the function of the Cdc7p/Dbf4p kinase . Genetic experiments revealed that loss of either Clb5p or Clb2p cyclins suppresses the mcm5-bob1 mutation and prevents bypass . These two cyclins have distinct roles in bypass and presumably in DNA replication as overexpression of one could not complement the loss of the other . Furthermore, the ectopic expression of CLB2 in G1 phase cannot substitute for CLB5 function in bypass of Cdc7p/Dbf4p by mcm5-bob1 . Molecular experiments revealed that the mcm5-bob1 mutation allows for constitutive loading of Cdc45p at early origins in arrested G1 phase cells when both kinases are inactive . A model is proposed in which the Mcm5-bob1 protein assumes a unique molecular conformation without prior action by either kinase . This conformation allows for stable binding of Cdc45p to the origin . However, DNA replication still cannot occur without the combined action of Cdk1p/Clb5p and Cdk1p/Clb2p . Thus Cdc7p and Cdk1p kinases catalyze the initiation of DNA replication at several distinct steps, of which only a subset is bypassed by the mcm5-bob1 mutation. Indian J Exp Biol, 2001 Dec, 39(12), 1254 - 7 Radioprotective action of caffeine: use of Saccharomyces cerevisiae as a test system; Vaidya PJ et al.; Mechanism of radioprotective action of caffeine by studying the gamma radiation -induced killing of yeast, S . cerevisae is reported . The results reveal that caffeine specifically protects aerobically (oxic) grown cells from gamma - radiation and sensitizes anaerobically (hypoxic) grown cells to some extent . Using radiation sensitive strains which lack recombinational pathway, it was found that protection by caffeine was solely brought about by reducing DNA damage, rather than by interfering with DNA repair process. Appl Microbiol Biotechnol, 2002 May, 58(6), 806 - 12 Epub 2002 Mar 23. Screening of a molecule endowing Saccharomyces cerevisiae with n-nonane-tolerance from a combinatorial random protein library; Zou W et al.; A combinatorial random protein library was constructed from random DNA fragments generated by "DNA random priming", an improved method of "random-priming recombination" using random-sequence primers and template cDNA from the yeast Saccharomyces cerevisiae . In order to express this library on the yeast cell surface, a yeast multicopy cassette vector was constructed, in which the random-protein-encoding DNA fragments were fused to a gene encoding the C-terminal 320 amino acids of alpha-agglutinin . Fluorescent labeling of the immuno-reaction of RGS(His)(6) epitope confirmed the surface display of random proteins . The surface display of heterologous random proteins on yeast cells will have a wide application . As an example, an n-nonane-tolerant yeast strain that could grow very well in nonane-overlaid culture medium was screened out from transformants displaying this combinatorial library . n-Nonane tolerance was dependent on the transformed plasmid, and the related protein was confirmed to localize on the cell surface by papain treatment and immunofluorescent labeling . Analysis of this displayed protein was also carried out . This strain is the first one to have been endowed artificially with organic solvent tolerance . This is a good example of creating cells exhibiting new phenotypes using a combinatorial protein library. Biochim Biophys Acta, 2002 Apr 15, 1570(3), 199 - 202 Effect of superoxide dismutase deficiency on the life span of the yeast Saccharomyces cerevisiae . An oxygen-independent role of Cu,Zn-superoxide dismutase; Wawryn J et al.; Effects of the absence of Cu,Zn-superoxide dismutase (CuZnSOD) on the replicative life span of the yeast Saccharomyces cerevisiae were studied under different oxygen conditions . In both strains, replicative life span and the rate of cell divisions were found to be similar under the atmosphere of air and under hypoxic (3% oxygen) and anoxic conditions . These results indicate that deleterious consequences of the lack of CuZnSOD are not limited to elevation of superoxide concentration and involve function(s) other than superoxide scavenging. J Virol Methods, 2002 Jun, 104(1), 93 - 8 Expression and antigenic characterization of the major capsid proteins of human polyomaviruses BK and JC in Saccharomyces cerevisiae; Hale AD et al.; BK and JC viruses are ubiquitous human polyomaviruses that are associated with post-transplant interstitial nephritis (BK virus) and progressive multifocal leucoencephalopathy (JC virus) . The use of a yeast system to express the major capsid protein (VP1) of two antigenic variants of BKV (strains SB and AS) and JCV is described . VP1s of AS and JCV expressed in Saccharomyces cerevisiae produced proteins of expected molecular weight as determined by gel electrophoresis whereas that of SB appeared to be lower than anticipated . However, all VP1s self-assembled into virus-like particles (VLP) retaining sialic acid-binding and antigenic properties of native virions . This method is highly efficient for producing recombinant proteins and therefore provides an alternative to the baculovirus system. Biochim Biophys Acta, 2002 Apr 15, 1581(3), 109 - 18 Mutations in erg4 affect the sensitivity of Saccharomyces cerevisiae to medium-chain fatty acids; McDonough V et al.; We have found that the medium-chain fatty acids (MCFAs) undecanoic acid (11:0), 10-undecenoic acid (11:1 Delta 10), and lauric acid (12:0) can affect the growth of Saccharomyces cerevisiae in a dose-dependent manner . The principal effect was a longer lag phase in MCFA-containing medium, although higher concentrations of 11:1 Delta 10 inhibited growth . Their relative order of inhibitory action was 11:1 Delta 10>11:0>12:0 . Cellular content with MCFA supplementation was dependent on the concentration and the particular species of fatty acid, with 12:0 showing the highest relative accumulation and 11:1 Delta 10 the lowest at all concentrations . We have isolated and characterized a mutant that is hypersensitive to MCFA supplementation and is unable to grow at the normally permissive condition of 1 mM 11:1 Delta 10 . However, it does not appear to accumulate higher relative levels of the fed MCFA compared to wild-type cells . Complementation of the mutant revealed that the ERG4 gene, encoding the enzyme that catalyzes the last step in ergosterol biosynthesis, had been mutated . The fatty acid composition of the erg4 Delta mutant differs only slightly from wild-type cells, mainly involving an increase in the relative amount of 12:0 . These results indicate that yeast require ergosterol for optimal growth on certain MCFAs . We discuss the role ergosterol may have in cells responding to exogenous MCFAs and in supporting optimal cell growth. Biotechniques . 2002 May;32(5):1036, 1038, 1040 passim. Higher-accuracy method for measuring minichromosome stability in Saccharomyces cerevisiae; Kramer DJ et al.; The minichromosome maintenance assay isfrequently used to characterize mutations genetically that affect the initiation of DNA replication or to decipherfunctional components in autonomously replicating sequences . The assay determines minichromosome loss by measuring the percentage of plasmid-containing cells in cultures after a period of growth in nonselective medium . Here we analyze data acquisition errors that contribute to the low accuracy of the routine versions of the assay . We propose modifications that eliminate errors in the acquisition of two variables and significantly improve the accuracy of the assay. J Biol Chem, 2002 Aug 2, 277(31), 28099 - 108 Epub 2002 May 15. The DNA polymerase domain of pol(epsilon) is required for rapid, efficient, and highly accurate chromosomal DNA replication, telomere length maintenance, and normal cell senescence in Saccharomyces cerevisiae; Ohya T et al.; Saccharomyces cerevisiae POL2 encodes the catalytic subunit of DNA polymerase epsilon . This study investigates the cellular functions performed by the polymerase domain of Pol2p and its role in DNA metabolism . The pol2-16 mutation has a deletion in the catalytic domain of DNA polymerase epsilon that eliminates its polymerase and exonuclease activities . It is a viable mutant, which displays temperature sensitivity for growth and a defect in elongation step of chromosomal DNA replication even at permissive temperatures . This mutation is synthetic lethal in combination with temperature-sensitive mutants or the 3'- to 5'-exonuclease-deficient mutant of DNA polymerase delta in a haploid cell . These results suggest that the catalytic activity of DNA polymerase epsilon participates in the same pathway as DNA polymerase delta, and this is consistent with the observation that DNA polymerases delta and epsilon colocalize in some punctate foci on yeast chromatids during S phase . The pol2-16 mutant senesces more rapidly than wild type strain and also has shorter telomeres . These results indicate that the DNA polymerase domain of Pol2p is required for rapid, efficient, and highly accurate chromosomal DNA replication in yeast. FEMS Microbiol Lett, 2002 Apr 9, 209(2), 223 - 8 Identification of lysine-78 as an essential residue in the Saccharomyces cerevisiae xylose reductase; Jeong EY et al.; Yeast xylose reductases are hypothesized as hybrid enzymes as their primary sequences contain elements of both the aldo-keto reductases (AKR) and short chain dehydrogenase/reductase (SDR) enzyme families . During catalysis by members of both enzyme families, an essential Lys residue H-bonds to a Tyr residue that donates proton to the aldehyde substrate . In the Saccharomyces cerevisiae xylose reductase, Tyr49 has been identified as the proton donor . However, the primary sequence of the enzyme contains two Lys residues, Lys53 and Lys78, corresponding to the conserved motifs for SDR and AKR enzyme families, respectively, that may H-bond to Tyr49 . We used site-directed mutagenesis to substitute each of these Lys residues with Met . The activity of the K53M variant was slightly decreased as compared to the wild-type, while that of the K78M variant was negligible . The results suggest that Lys78 is the essential residue that H-bonds to Tyr49 during catalysis and indicate that the active site residues of yeast xylose reductases match those of the AKR, rather than SDR, enzymes . Intrinsic enzyme fluorescence spectroscopic analysis suggests that Lys78 may also contribute to the efficient binding of NADPH to the enzyme. FEMS Microbiol Lett, 2002 Mar 19, 209(1), 9 - 14 Osmolarity hypersensitivity of hog1 deleted mutants is suppressed by mutation in KSS1 in budding yeast Saccharomyces cerevisiae; Lee SJ et al.; An osmosensing mechanism of Saccharomyces cerevisiae involves a mitogen-activated protein kinase (MAPK) cascade (HOG pathway) . This study aimed to investigate the response of the yeast to osmotic stress . A mutant strain, in which the HOG1 gene was disrupted by TRP1, was constructed . A spontaneous mutant, named YJY45, which suppresses the osmosensitive growth phenotype of the hog1 deletion mutant, was selected and showed a secondary phenotype of temperature sensitivity on YPD containing 0.5 M NaCl at 37 degrees C . Our data indicate that the spontaneous mutation in YJY45 mutant was mapped in KSS1, which is one of the MAPK family . The mutation in KSS1 suppresses the osmolarity-hypersensitive phenotype of the hog1 deletion mutation and restores GPD1 induction. J Biol Chem, 2002 Jul 19, 277(29), 26177 - 84 Epub 2002 May 10. Sterol-dependent regulation of sphingolipid metabolism in Saccharomyces cerevisiae; Swain E et al.; We had previously isolated the temperature-sensitive erg26-1 mutant and characterized the sterol defects in erg26-1 cells (Baudry, K., Swain, E., Rahier, A., Germann, M., Batta, A., Rondet, S., Mandala, S., Henry, K., Tint, G . S., Edlind, T., Kurtz, M., and Nickels, J . T., Jr . (2001) J . Biol . Chem . 276, 12702-12711) . We have now determined the defects in sphingolipid metabolism in erg26-1 cells, examined their effects on cell growth, and initiated studies designed to elucidate how might changes in sterol levels coordinately regulate sphingolipid metabolism in Saccharomyces cerevisiae . Using {(3)H}inositol radiolabeling studies, we found that the biosynthetic rate and steady-state levels of specific hydroxylated forms of inositolphosphorylceramides were decreased in erg26-1 cells when compared with wild type cells . {(3)H}Dihydrosphingosine radiolabeling studies demonstrated that erg26-1 cells had decreased levels of the phytosphingosine-derived ceramides that are the direct precursors of the specific hydroxylated inositol phosphorylceramides found to be lower in these cells . Gene dosage experiments using the sphingolipid long chain sphingoid base (LCB) hydroxylase gene, SUR2, suggest that erg26-1 cells may accumulate LCB, thus placing one point of sterol regulation of sphingolipid synthesis possibly at the level of ceramide metabolism . The results from additional genetic studies using the sphingolipid hydroxylase and copper transporter genes, SCS7 and CCC2, respectively, suggest a second possible point of sterol regulation at the level of complex sphingolipid hydroxylation . In addition, {(3)H}inositol radiolabeling of sterol biosynthesis inhibitor-treated wild type cells and late sterol pathway mutants showed that additional blocks in sterol biosynthesis have profound effects on sphingolipid metabolism, particularly sphingolipid hydroxylation state . Finally, our genetic studies in erg26-1 cells using the LCB phosphate phosphatase gene, LBP1, suggest that increasing the levels of the LCB sphingoid base phosphate can remediate the temperature-sensitive phenotype of erg26-1 cells. J Biol Chem, 2002 Jul 26, 277(30), 26788 - 95 Epub 2002 May 10. Structural and enzymatic properties of the AAA protein Drg1p from Saccharomyces cerevisiae . Decoupling of intracellular function from ATPase activity and hexamerization; Zakalskiy A et al.; The AAA protein Drg1 from yeast was affinity-purified, and its ATPase activity and hexamerization properties were analyzed . The same parameters were also determined for several mutant proteins and compared in light of the growth characteristics of the corresponding cells . The protein from a thermosensitive mutant exhibited reduced ATPase activity and hexamerization . These defects were not reversed by an intragenic suppressor mutation, although this allele supported growth at the nonpermissive temperature . A different set of mutants was generated by site-specific mutagenesis intended to adjust the Walker A box of the D2 domain of Drg1p to that of the D1 domain . A S562G exchange in D2 produced a nonfunctional protein that did not hexamerize but showed above-normal ATPase activity . The C561T mutant protein, on the other hand, was functional but hexamerized less readily and had reduced ATPase activity . In contrast, the C561T/S562G protein hexamerized less than wild type but had much higher ATPase activity . We distinguished strong and weak ATP-binding sites in the wild type protein but two weak sites in the C561T/S562G protein, indicating that the stronger site resides in D2 . These observations are discussed in terms of the inter-relationship of ATPase activity per se, oligomeric status, and intracellular function for AAA proteins. Chemosphere, 2002 Feb, 46(7), 1045 - 51 Structure-activity relationships and response-surface analysis of nitroaromatics toxicity to the yeast (Saccharomyces cerevisiae); Wang X et al.; Inhibition of growth of the yeast Saccharomyces cerevisiae (Cmiz, the minimum concentration that produced a clear inhibition zone within 12 h) for 24 nitroaromatic compounds was investigated and a quantitative structure-activity relationship (QSAR) developed based on hydrophobicity expressed as the l-octanol/water partition coefficient in logarithm form, log K(ow), electrophilicity based on the energy of the lowest unoccupied orbital (E(lumo)) . All nitrobenzene derivatives exhibited enhanced reactive toxicity than baseline . The toxicities of mono-nitrobenzenes and di-nitrobenzenes were elicited by different mechanisms of toxic action . For mono-nitro-derivatives, both significant log K(ow) based and strong E(lumo)-dependent relationships were observed indicating that their toxicities were affected both by the penetration process and the interaction with target sites of interaction . The toxicities of di-nitrobenzenes were greater than mono-nitrobenzenes and no log K(ow)-dependent but highly significant E(lumo)-based relationship was obtained . This suggests that toxicity of di-nitrobenzenes was highly electrophilic and involved mainly their in vivo electrophilic interaction with biomacromolecules . In an effort to model the elevated toxicity of all nitrobenzenes, a response-surface analysis was performed and this resulted in a highly predictive two-variable QSAR without reference to their exact mechanisms (Cmiz = 0.41 log K(ow) - 0.89 E(lumo) - 0.46, r2 = 0.87, Q2 = 0.86, n = 24). Biosci Biotechnol Biochem, 2002 Feb, 66(2), 233 - 8 High-level TNF-alpha secretion and macrophage activity with soluble beta-glucans from Saccharomyces cerevisiae; Lee DY et al.; We have previously reported that water-soluble beta-glucan completely devoid of mannoprotein and purified from the yeast cell wall effectively stimulated the macrophage function (Biosci . Biotechnol., Biochem., 65, 4, 837-841 (2001)) . In this present study, to increase the yield of water-soluble beta-glucan, the wild type of Sacharomyces cerevisiae, JH, was treated with a combination of UV irradiation and laminarinase (endo-beta-(1,3)-glucanase) to yield the laminarinase-resistant mutants, JUL1 and JUL3 . Water-soluble beta-glucans that were free of mannoprotein from JH, JUL1 and JUL3 were purified and their effects on TNF-alpha secretion and phagocytosis by macrophages were evaluated . Crude beta-glucan was first solubilized from the yeast cell wall by alkaline extraction and then subjected to an acid treatment . The residual mannoprotein was completely removed by DEAE and ConA chromatography . The yield of water-soluble beta-glucan in both mutants (JUL1, 5.11%; JUL3, 5.76%) was about 5-fold higher than that of the wild type (1.16%) . The water-soluble beta-glucan from JH induced TNF-alpha secretion slightly more than that from JUL1 or JUL3: TNF-alpha secretion by JH at 50, 200, 500 microg/ml of beta-glucan was 11-17% more than that by JUL1 or JUL3 for the same treatment . Beta-glucan from the wild type stimulated phagocytosis slightly more than that from the mutants . These mutants could therefore effectively produce purified water-soluble beta-glucan with immune activity. Biochim Biophys Acta, 2002 Apr 12, 1574(3), 255 - 61 RPL29 codes for a non-essential protein of the 60S ribosomal subunit in Saccharomyces cerevisiae and exhibits synthetic lethality with mutations in genes for proteins required for subunit coupling; DeLabre ML et al.; RPL29 (YFR032c-a) is a non-essential gene that codes for a 60S ribosomal subunit protein in Saccharomyces cerevisiae . Deletion of RPL29 leads to a moderate accumulation of half-mer polysomes with little or no change in the amounts of free 60S subunits . In vitro translation and the growth rate are also delayed in the Deltarpl29 strain . Such a phenotype is characteristic of mutants defective in 60S to 40S subunit joining . The Deltarpl29 strain exhibits synthetic lethality with mutations in RPL10, the gene encoding an essential 60S ribosomal subunit protein that is required for 60S to 40S subunit joining . The Deltarpl29 strain also exhibits synthetic lethality with RSA1, a gene encoding a nucleoplasmic protein required for the loading of Rpl10p onto the 60S subunit . Over-expression of RPL10 suppresses the half-mer phenotype of the Deltarpl29 strain, but does not correct the growth defect of the deletion strain . We conclude that absence of Rpl29p impairs proper assembly of proteins onto the 60S subunit and that this retards subunit joining and additionally retards protein synthesis subsequent to subunit joining. Biochemistry (Mosc), 2002 Apr, 67(4), 485 - 90 Vesicular transport of extracellular acid phosphatases in yeast Saccharomyces cerevisiae; Blinnikova EI et al.; A method for isolation of secretory vesicles from the yeast Saccharomyces cerevisiae based on the disintegration of protoplasts by osmotic shock followed by separation of the vesicles by centrifugation in a density gradient of Urografin was developed in this study . Two populations of the secretory vesicles that differ in density and shape were separated . Acid phosphatases (EC 3.1.3.2) were used as markers of the secretory vesicles . It was shown that the constitutive acid phosphatase (PHO3 gene product) is mainly transported to the cell surface by a lower density population of vesicles, while the repressible acid phosphatase (a heteromer encoded by PHO5, PHO10, and PHO11 genes) by a vesicle population of higher density . These data provide evidence that at least two pathways of transport of yeast secretory proteins from the place of their synthesis and maturation to the cell surface may exist . To reveal the probable reasons for transport of Pho3p and Pho5p/Pho10p/Pho11p enzymes by two different kinds of vesicles, we isolated vesicles from strains that synthesize the homomeric forms of the repressible acid phosphatase . It was demonstrated that glycoproteins encoded by the PHO10 and/or PHO11 genes could be responsible for the choice of one of the alternative transport pathways of the repressible acid phosphatase . A high correlation coefficient between bud formation and secretion of Pho5p phosphatase and the absence of correlation between bud formation and secretion of minor phosphatases Pho10p and Pho11p suggests different functional roles of the polypeptides that constitute the native repressible acid phosphatase. Acta Biochim Pol, 2000, 47(4), 993 - 1005 The KRR1 gene encodes a protein required for 18S rRNA synthesis and 40S ribosomal subunit assembly in Saccharomyces cerevisiae; Gromadka R et al.; The newly discovered Saccharomyces cerevisiae gene KRR1 (YCL059c) encodes a protein essential for cell viability . Krr1p contains a motif of clustered basic amino acids highly conserved in the evolutionarly distant species from yeast to human . We demonstrate that Krr1p is localized in the nucleolus . The KRR1 gene is highly expressed in dividing cells and its expression ceases almost completely when cells enter the stationary phase . In vivo depletion of Krr1p leads to drastic reduction of 40S ribosomal subunits due to defective 18S rRNA synthesis . We propose that Krr1p is required for proper processing of pre-rRNA and the assembly of preribosomal 40S subunits. Acta Biochim Pol, 2001, 48(4), 1043 - 9 Anaerobic growth of Saccharomyces cerevisiae alleviates the lethal effect of phosphotyrosyl phosphatase activators depletion; Rempola B et al.; Saccharomyces cerevisiae homologues of phosphotyrosyl phosphatase activator (PTPA) are encoded byRRD1 and RRD2, genes whose combined deletion is synthetic lethal . Previously we have shown that the lethality of rrd1,2delta can be suppressed by increasing the osmolarity of the medium . Here we show that the lethality of rrd1,2delta is also suppressed under oxygen-limited conditions . The absence of respiration per se is not responsible for the suppression since elimination of the mitochondrial genome or a block in heme biosynthesis fail to rescue the rrd1,2delta double mutation. Biochemistry, 2002 May 14, 41(19), 6128 - 39 Identification of a contact region between the tridecapeptide alpha-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling; Henry LK et al.; Saccharomyces cerevisiae haploid cells communicate with their opposite mating type through peptide pheromones (alpha-factor and a-factor) that activate G protein-coupled receptors (GPCRs) . S . cerevisiaewas used as a model system for the study of peptide-responsive GPCRs . Here, we detail the synthesis and characterization of a number of alpha-factor (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) pheromone analogues containing the photo-cross-linkable group 4-benzoyl-L-phenylalanine (Bpa) . Following characterization, one analogue, {Bpa(1), Tyr(3), Arg(7), Phe(13)}alpha-factor, was radioiodinated and used as a probe for Ste2p, the GPCR for alpha-factor . Binding of the di-iodinated probe was saturable (K(d) = 200 nM) and competable by alpha-factor . Cross-linking into Ste2p was specific for this receptor and reversed by the wild-type pheromone . Chemical and enzymatic cleavage of the receptor/radioprobe complex indicated that cross-linking occurred on a portion of Ste2p spanning residues 251-294 which encompasses transmembrane domain 6, the extracellular loop between transmembrane domains 6 and 7, and transmembrane domain 7 . This fragment was verified using T7-epitope-tagged Ste2p and a biotinylated, photoactivatable alpha-factor . After cross-linking with the biotinylated photoprobe and trypsin cleavage, the cross-linked receptor fragment was revealed by both an anti T7-epitope antibody and a biotin probe . This is the first determination of a specific contact region between a Class IV GPCR and its ligand . The results demonstrate that Bpa alpha-factor probes are useful in determining contacts between alpha-factor and Ste2p and initiate mapping of the ligand binding site of this GPCR. Microbiology, 2002 May, 148(Pt 5), 1317 - 28 Mutational analysis of K28 preprotoxin processing in the yeast Saccharomyces cerevisiae; Riffer F et al.; K28 killer strains of Saccharomyces cerevisiae are permanently infected with a cytoplasmic persisting dsRNA virus encoding a secreted alpha/beta heterodimeric protein toxin that kills sensitive cells by cell-cycle arrest and inhibition of DNA synthesis . In vivo processing of the 345 aa toxin precursor (preprotoxin; pptox) involves multiple internal and carboxy-terminal cleavage events by the prohormone convertases Kex2p and Kex1p . By site-directed mutagenesis of the preprotoxin gene and phenotypic analysis of its in vivo effects it is now demonstrated that secretion of a biological active virus toxin requires signal peptidase cleavage after Gly(36) and Kex2p-mediated processing at the alpha subunit N terminus (after Glu-Arg(49)), the alpha subunit C terminus (after Ser-Arg(149)) and at the beta subunit N terminus (after Lys-Arg(245)) . The mature C terminus of the beta subunit is trimmed by Kex1p, which removes the terminal Arg(345) residue, thus uncovering the toxin's endoplasmic reticulum targeting signal (HDEL) which--in a sensitive target cell--is essential for retrograde toxin transport . Interestingly, both toxin subunits are covalently linked by a single disulfide bond between alpha-Cys(56) and beta-Cys(340), and expression of a mutant toxin in which beta-Cys(340) had been replaced by Ser(340) resulted in the secretion of a non-toxic alpha/beta heterodimer that is blocked in retrograde transport and incapable of entering the yeast cell cytosol, indicating that one important in vivo function of beta-Cys(340) might be to ensure accessibility of the toxin's beta subunit C terminus to the HDEL receptor of the target cell. J Biol Chem, 2002 Jul 12, 277(28), 25545 - 53 Epub 2002 May 01. Dominant Saccharomyces cerevisiae msh6 mutations cause increased mispair binding and decreased dissociation from mispairs by Msh2-Msh6 in the presence of ATP; Hess MT et al.; A previous study described four dominant msh6 mutations that interfere with both the Msh2-Msh6 and Msh2-Msh3 mismatch recognition complexes (Das Gupta, R., and Kolodner, R . D . (2000) Nat . Genet . 24, 53-56) . Modeling predicted that two of the amino acid substitutions (G1067D and G1142D) interfere with protein-protein interactions at the ATP-binding site-associated dimer interface, one (S1036P) similarly interferes with protein-protein interactions and affects the Msh2 ATP-binding site, and one (H1096A) affects the Msh6 ATP-binding site . The ATPase activity of the Msh2-Msh6-G1067D and Msh2-Msh6-G1142D complexes was inhibited by GT, +A, and +AT mispairs, and these complexes showed increased binding to GT and +A mispairs in the presence of ATP . The ATPase activity of the Msh2-Msh6-S1036P complex was inhibited by a GT mispair, and it bound the GT mispair in the presence of ATP, whereas its interaction with insertion mispairs was unchanged compared with the wild-type complex . The ATPase activity of the Msh2-Msh6-H1096A complex was generally attenuated, and its mispair-binding behavior was unaffected . These results are in contrast to those obtained with the wild-type Msh2-Msh6 complex, which showed mispair-stimulated ATPase activity and ATP inhibition of mispair binding . These results indicate that the dominant msh6 mutations cause more stable binding to mispairs and suggest that there may be differences in how base base and insertion mispairs are recognized. J Biochem (Tokyo), 2002 May, 131(5), 693 - 9 Phosphoinositide 3-OH kinase/protein kinase B inhibits apoptotic cell death induced by reactive oxygen species in Saccharomyces cerevisiae; Jeon BW et al.; Apoptosis is a common mode of programmed cell death in multicellular organisms . However, the recent observation of yeast cell death displaying the morphology of apoptosis has suggested the presence of an ancestral cell death machinery . Here we examined apoptotic features induced by reactive oxygen species (ROS) in yeast . Saccharomyces cerevisiae show typical apoptotic features upon exposure to ROS: membrane staining with annexin V and DNA fragmentation by the TUNEL assay . The detection of apoptotic features in yeast strongly support the existence of molecular machinery performing the basic pathways of apoptosis . The phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in a variety of cells . It is therefore of interest to determine whether the PI3K/PKB signaling pathway is capable of protecting yeast from apoptosis induced by ROS . We determined that PI3K/PKB is capable of significantly inhibiting ROS-evoked apoptosis in yeast . These results suggest that yeast may provide a suitable model system in which to study the apoptotic signaling pathway elicited by a variety of stimuli. Plasmid, 2002 Mar, 47(2), 94 - 107 Selective fitness of four episomal shuttle-vectors carrying HIS3, LEU2, TRP1, and URA3 selectable markers in Saccharomyces cerevisiae; Ugolini S et al.; A comparison of the selective fitness of four 2-microm-based shuttle-plasmids carrying the yeast genes HIS3, LEU2, TRP1, and URA3 was performed . The effect of each marker on long-term growth rate and plasmid maintenance was measured . In selective medium, the LEU2 and URA3 plasmids were maintained at the lowest and the highest levels, respectively, while the HIS3 and TRP1 plasmids were maintained at an intermediate level . In synthetic complete medium, plasmid loss rate was lower for the genes TRP1 and URA3 than for the other two markers, and a similar pattern was observed for cells growing in rich medium . These results were confirmed by competition experiments among transformants with different plasmids in complete and rich media, indicating a different degree of fitness for the markers used . A potential correlation of the energy cost of plasmid maintenance with the secondary DNA structure and the level of expression of the selective markers is also investigated . EMBO J, 2002 May 1, 21(9), 2068 - 75 The structure of Saccharomyces cerevisiae Met8p, a bifunctional dehydrogenase and ferrochelatase; Schubert HL et al.; Sirohaem is a tetrapyrrole-derived prosthetic group that is required for the essential assimilation of sulfur and nitrogen into all living systems as part of the sulfite and nitrite reductase systems . The final two steps in the biosynthesis of sirohaem involve a beta-NAD(+)-dependent dehydrogenation of precorrin-2 to generate sirohydrochlorin followed by ferrochelation to yield sirohaem . In Saccharomyces cerevisiae, Met8p is a bifunctional enzyme that carries out both of these reactions . Here, we report the 2.2 A resolution crystal structure of Met8p, which adopts a novel fold that bears no resemblance to the previously determined structures of cobalt- or ferro-chelatases . Analysis of mutant proteins suggests that both catalytic activities share a single active site, and that Asp141 plays an essential role in both dehydrogenase and chelatase processes. Genetics, 2002 Apr, 160(4), 1439 - 50 Ady3p links spindle pole body function to spore wall synthesis in Saccharomyces cerevisiae; Nickas ME et al.; Spore formation in Saccharomyces cerevisiae requires the de novo synthesis of prospore membranes and spore walls . Ady3p has been identified as an interaction partner for Mpc70p/Spo21p, a meiosis-specific component of the outer plaque of the spindle pole body (SPB) that is required for prospore membrane formation, and for Don1p, which forms a ring-like structure at the leading edge of the prospore membrane during meiosis II . ADY3 expression has been shown to be induced in midsporulation . We report here that Ady3p interacts with additional components of the outer and central plaques of the SPB in the two-hybrid assay . Cells that lack ADY3 display a decrease in sporulation efficiency, and most ady3Delta/ady3Delta asci that do form contain fewer than four spores . The sporulation defect in ady3Delta/ady3Delta cells is due to a failure to synthesize spore wall polymers . Ady3p forms ring-like structures around meiosis II spindles that colocalize with those formed by Don1p, and Don1p rings are absent during meiosis II in ady3Delta/ady3Delta cells . In mpc70Delta/mpc70Delta cells, Ady3p remains associated with SPBs during meiosis II . Our results suggest that Ady3p mediates assembly of the Don1p-containing structure at the leading edge of the prospore membrane via interaction with components of the SPB and that this structure is involved in spore wall formation. Genetics, 2002 Apr, 160(4), 1423 - 37 Protein phosphatase type 1 regulates ion homeostasis in Saccharomyces cerevisiae; Williams-Hart T et al.; Protein phosphatase type 1 (PP1) is encoded by the essential gene GLC7 in Saccharomyces cerevisiae . glc7-109 (K259A, R260A) has a dominant, hyperglycogen defect and a recessive, ion and drug sensitivity . Surprisingly, the hyperglycogen phenotype is partially retained in null mutants of GAC1, GIP2, and PIG1, which encode potential glycogen-targeting subunits of Glc7 . The R260A substitution in GLC7 is responsible for the dominant and recessive traits of glc7-109 . Another mutation at this residue, glc7-R260P, confers only salt sensitivity, indicating that the glycogen and salt traits of glc7-109 are due to defects in distinct physiological pathways . The glc7-109 mutant is sensitive to cations, aminoglycosides, and alkaline pH and exhibits increased rates of l-leucine and 3,3'-dihexyloxacarbocyanine iodide uptake, but it is resistant to molar concentrations of sorbitol or KCl, indicating that it has normal osmoregulation . KCl suppresses the ion and drug sensitivities of the glc7-109 mutant . The CsCl sensitivity of this mutant is suppressed by recessive mutations in PMA1, which encodes the essential plasma membrane H(+)ATPase . Together, these results indicate that Glc7 regulates ion homeostasis by controlling ion transport and/or plasma membrane potential, a new role for Glc7 in budding yeast. Genetics, 2002 Apr, 160(4), 1401 - 7 Domains of gene silencing near the left end of chromosome III in Saccharomyces cerevisiae; Bi X; In Saccharomyces cerevisiae the HM loci and regions adjacent to the telomeres are transcriptionally silent . HML is situated 11 kb from the left telomere of chromosome III . I have systematically examined gene silencing along this 11-kb chromosomal region . I found that silencing extends at least 1.1 kb beyond HML, indicating that the HML E silencer acts on both sides . Moreover, I obtained evidence indicating that a 0.71-kb sequence near the E silencer acts as a barrier to the spread of silencing and coincides with the left boundary of the silent HML domain . I also showed that silencing at the telomere is limited to an approximately 2-kb domain . On the other hand, an approximately 7-kb region between HML and the telomere is not silenced by HML or the telomere . These results provide a clear example of organization of the eukaryotic genome into interspersed domains with distinct potentials for gene expression. Genetics, 2002 Apr, 160(4), 1389 - 400 The mitochondrial nucleoid protein, Mgm101p, of Saccharomyces cerevisiae is involved in the maintenance of rho(+) and ori/rep-devoid petite genomes but is not required for hypersuppressive rho(-) mtDNA; Zuo XM et al.; The Saccharomyces cerevisiae MGM101 gene encodes a DNA-binding protein targeted to mitochondrial nucleoids . MGM101 is essential for maintenance of a functional rho(+) genome because meiotic segregants, with a disrupted mgm101 allele, cannot undergo more than 10 divisions on glycerol medium . Quantitative analysis of mtDNA copy number in a rho(+) strain carrying a temperature-sensitive allele, mgm101-1, revealed that the amount of mtDNA is halved each cell division upon a shift to the restrictive temperature . These data suggest that mtDNA replication is rapidly blocked in cells lacking MGM101 . However, a small proportion of meiotic segregants, disrupted in MGM101, have rho(-) genomes that are stably maintained . Interestingly, all surviving rho(-) mtDNAs contain an ori/rep sequence . Disruption of MGM101 in hypersuppressive (HS) strains does not have a significant effect on the propagation of HS rho(-) mtDNA . However, in petites lacking an ori/rep, disruption of MGM101 leads to either a complete loss or a dramatically decreased stability of mtDNA . This discriminatory effect of MGM101 suggests that replication of rho(+) and ori/rep-devoid rho(-) mtDNAs is carried out by the same process . By contrast, the persistence of ori/rep-containing mtDNA in HS petites lacking MGM101 identifies a distinct replication pathway . The alternative mtDNA replication mechanism provided by ori/rep is independent of mitochondrial RNA polymerase encoded by RPO41 as a HS rho(-) genome is stably maintained in a mgm101, rpo41 double mutant. Genetics, 2002 Apr, 160(4), 1375 - 87 A role for histone H2B during repair of UV-induced DNA damage in Saccharomyces cerevisiae; Martini EM et al.; To investigate the role of the nucleosome during repair of DNA damage in yeast, we screened for histone H2B mutants that were sensitive to UV irradiation . We have isolated a new mutant, htb1-3, that shows preferential sensitivity to UV-C . There is no detectable difference in bulk chromatin structure or in the number of UV-induced cis-syn cyclobutane pyrimidine dimers (CPD) between HTB1 and htb1-3 strains . These results suggest a specific effect of this histone H2B mutation in UV-induced DNA repair processes rather than a global effect on chromatin structure . We analyzed the UV sensitivity of double mutants that contained the htb1-3 mutation and mutations in genes from each of the three epistasis groups of RAD genes . The htb1-3 mutation enhanced UV-induced cell killing in rad1Delta and rad52Delta mutants but not in rad6Delta or rad18Delta mutants, which are defective in postreplicational DNA repair (PRR) . When combined with other mutations that affect PRR, the histone mutation increased the UV sensitivity of strains with defects in either the error-prone (rev1Delta) or error-free (rad30Delta) branches of PRR, but did not enhance the UV sensitivity of a strain with a rad5Delta mutation . When combined with a ubc13Delta mutation, which is also epistatic with rad5Delta, the htb1-3 mutation enhanced UV-induced cell killing . These results suggest that histone H2B acts in a novel RAD5-dependent branch of PRR. Genetics, 2002 Apr, 160(4), 1363 - 73 A novel selection system for chromosome translocations in Saccharomyces cerevisiae; Tennyson RB et al.; Chromosomal translocations are common genetic abnormalities found in both leukemias and solid tumors . While much has been learned about the effects of specific translocations on cell proliferation, much less is known about what causes these chromosome rearrangements . This article describes the development and use of a system that genetically selects for rare translocation events using the yeast Saccharomyces cerevisiae . A translocation YAC was created that contains the breakpoint cluster region from the human MLL gene, a gene frequently involved in translocations in leukemia patients, flanked by positive and negative selection markers . A translocation between the YAC and a yeast chromosome, whose breakpoint falls within the MLL DNA, physically separates the markers and forms the basis for the selection . When RAD52 is deleted, essentially all of the selected and screened cells contain simple translocations . The detectable translocation rates are the same in haploids and diploids, although the mechanisms involved and true translocation rates may be distinct . A unique double-strand break induced within the MLL sequences increases the number of detectable translocation events 100- to 1000-fold . This novel system provides a tractable assay for answering basic mechanistic questions about the development of chromosomal translocations. Genetics, 2002 Apr, 160(4), 1353 - 61 Differential regulation of Saccharomyces cerevisiae phospholipase D in sporulation and Sec14-independent secretion; Rudge SA et al.; Saccharomyces cerevisiae Spo14, a phosphatidylcholine-specific, phosphatidylinositol (4,5) bisphosphate-activated phospholipase D (PLD), is essential for meiosis and spore formation . Spo14 is also required for secretion in the absence of the phosphatidylinositol/phosphatidylcholine transfer protein Sec14 (i.e., Sec14-independent secretion) . In sporulating cells Spo14 is phosphorylated and relocalized within the cell . In contrast, Spo14 does not relocalize and is not phosphorylated in Sec14-independent secretion . Analysis of a partially phosphatidylinositol (4,5) bisphosphate-activated Spo14 mutant, spo14(R894G), revealed that Spo14 function in Sec14-independent secretion, unlike the situation in meiosis, requires fully stimulated PLD activity . Consistent with the differential regulation of Spo14 function during sporulation and secretion, we isolated a mutant allele, spo14-S251P, the product of which is improperly phosphorylated and fails to relocalize and rescue the sporulation phenotype of homozygous spo14 diploids, but supports Sec14-independent secretion . Furthermore, we show that the N-terminal domain of Spo14 is both phosphorylated and sufficient for prospore membrane localization during sporulation . These data indicate that Spo14 phosphorylation and relocalization are essential for the process of sporulation, but dispensable for Sec14-independent secretion . Finally, we demonstrate that Spo14 phosphorylation and relocalization are initiated by nitrogen and glucose limitation and occur independently of the process of meiosis. Genetics, 2002 Apr, 160(4), 1335 - 52 Mutations that affect vacuole biogenesis inhibit proliferation of the endoplasmic reticulum in Saccharomyces cerevisiae; Koning AJ et al.; In yeast, increased levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase isozyme, Hmg1p, induce assembly of nuclear-associated ER membranes called karmellae . To identify additional genes involved in karmellae assembly, we screened temperature-sensitive mutants for karmellae assembly defects . Two independently isolated, temperature-sensitive strains that were also defective for karmellae biogenesis carried mutations in VPS16, a gene involved in vacuolar protein sorting . Karmellae biogenesis was defective in all 13 other vacuole biogenesis mutants tested, although the severity of the karmellae assembly defect varied depending on the particular mutation . The hypersensitivity of 14 vacuole biogenesis mutants to tunicamycin was well correlated with pronounced defects in karmellae assembly, suggesting that the karmellae assembly defect reflected alteration of ER structure or function . Consistent with this hypothesis, seven of eight mutations causing defects in secretion also affected karmellae assembly . However, the vacuole biogenesis mutants were able to proliferate their ER in response to Hmg2p, indicating that the mutants did not have a global defect in the process of ER biogenesis. Mol Cell Biol, 2002 May, 22(10), 3281 - 91 Saccharomyces cerevisiae Mer3 is a DNA helicase involved in meiotic crossing over; Nakagawa T et al.; Crossing over is regulated to occur at least once per each pair of homologous chromosomes during meiotic prophase to ensure proper segregation of chromosomes at the first meiotic division . In a mer3 deletion mutant of Saccharomyces cerevisiae, crossing over is decreased, and the distribution of the crossovers that occur is random . The predicted Mer3 protein contains seven motifs characteristic of the DExH box type of DNA/RNA helicases . The mer3G166D and the mer3K167A mutation, amino acid substitutions of conserved residues in a putative nucleotide-binding domain of the helicase motifs caused a defect in the transition of meiosis-specific double-strand breaks to later intermediates, decreased crossing over, and reduced crossover interference . The purified Mer3 protein was found to have DNA helicase activity . This helicase activity was reduced by the mer3GD mutation to <1% of the wild-type activity, even though binding of the mutant protein to single- and double-strand DNA was unaffected . The mer3KA mutation eliminated the ATPase activity of the wild-type protein . These results demonstrate that Mer3 is a DNA helicase that functions in meiotic crossing over. Glycobiology, 2002 Mar, 12(3), 229 - 34 Overexpression, purification, and partial characterization of Saccharomyces cerevisiae processing alpha glucosidase I; Dhanawansa R et al.; The gene encoding yeast processing alpha glucosidase I, CWH41, was overexpressed in Saccharomyces cerevisiae AH22, resulting in a 28-fold increase in expression of the soluble form of the enzyme . The soluble enzyme results from proteolytic cleavage between residues Ala 24 and Thr 25 of the transmembrane sequence of the membrane-bound form of the enzyme . This cleavage could be partially inhibited by addition of leupeptin and pepstatin during the enzyme isolation . The enzyme was purified to a final specific activity of 8550 U/mg protein using a combination of ammonium sulfate precipitation, anion exchange, concanavalin A, and gel filtration chromatography . The soluble form of the enzyme is a monomer with a molecular weight of 98 kDa by SDS-PAGE, and 89 kDa by gel filtration . The molecular weight decreased by approximately 5 kDa after treatment with N-glycosidase F, indicating that it is a glycoprotein . Soluble glucosidase I was sensitive to diethyl pyrocarbonate and not affected by N-ethylmaleimide, suggesting that mechanistically it is more similar to the plant than the mammalian form of the enzyme. Biochemistry, 2002 Apr 30, 41(17), 5404 - 14 Binding to cisplatin-modified DNA by the Saccharomyces cerevisiae HMGB protein Nhp6A; Wong B et al.; Nhp6A is an abundant non-histone chromatin-associated protein in Saccharomyces cerevisiae that contains a minor groove DNA binding motif called the HMG box . In this report, we show that Nhp6Ap binds to cisplatin intrastrand cross-links on duplex DNA with a 40-fold greater affinity than to unmodified DNA with the same sequence . Nevertheless, Nhp6Ap bound to cisplatinated DNA readily exchanges onto unmodified DNA . Phenanthroline-copper footprinting and two-dimensional NMR on complexes of wild-type and mutant Nhp6Ap with DNA were employed to probe the mode of binding to the cisplatin lesion . Recognition of the cisplatin adduct requires a surface-exposed phenylalanine on Nhp6Ap that promotes bending of DNA by inserting into the helix from the minor groove . We propose that Nhp6Ap targets the cisplatin adduct by means of intercalation by the phenylalanine and that it can bind in either orientation with respect to the DNA lesion . A methionine, which also inserts between base pairs and functions in target selection on unmodified DNA, plays no apparent role in recognition of the cisplatin lesion . Basic amino acids within the N-terminal arm of Nhp6Ap are required for high-affinity binding to the cisplatin adduct as well as to unmodified DNA . Cisplatin mediates its cytotoxicity by forming covalent adducts on DNA, and we find that Deltanhp6a/b mutants are hypersensitive to cisplatin in comparison with the wild-type strain . In contrast, Deltanhp6a/b mutants are slightly more resistant to hydrogen peroxide and ultraviolet irradiation . Therefore, Nhp6A/Bp appears to directly or indirectly function in yeast to enhance cellular resistance to cisplatin. Yeast, 2002 May, 19(7), 619 - 29 How many protein-coding genes are there in the Saccharomyces cerevisiae genome? Mackiewicz P, Kowalczuk M, Mackiewicz D, Nowicka A, Dudkiewicz M, Laszkiewicz A, Dudek MR, Cebrat S. We have compared the results of estimations of the total number of protein-coding genes in the Saccharomyces cerevisiae genome, which have been obtained by many laboratories since the yeast genome sequence was published in 1996 . We propose that there are 5300-5400 genes in the genome . This makes the first estimation of the number of intronless ORFs longer than 100 codons, based on the features of the set of genes with phenotypes known in 1997 to be correct . This estimation assumed that the set of the first 2300 genes with known phenotypes was representative for the whole set of protein-coding genes in the genome . The same method used in this paper for the approximation of the total number of protein-coding sequences among more than 40 000 ORFs longer than 20 codons gives a result that is only slightly higher . This suggests that there are still some non-coding ORFs in the databases and a few dozen small ORFs, not yet annotated, which probably code for proteins . Yeast, 2002 May, 19(7), 587 - 99 A series of double disruptants for protein phosphatase genes in Saccharomyces cerevisiae and their phenotypic analysis; Sakumoto N et al.; Thirty-two protein phosphatase (PPase) genes were identified in Saccharomyces cerevisiae based on the nucleotide sequences of the entire genome . In an effort to understand the role of PPases and their functional redundancy in the cellular physiology of one of the reference eukaryotic organisms, a series of single and double PPase gene disruptants were constructed in the W303 strain background . Two single disruptants for the CDC14 and GLC7 genes were lethal . Double disruptants for 30 non-essential PPase genes were constructed in all possible 435 combinations . No double disruptant showed synthetic lethality . Several phenotypes of the viable 30 single and 435 double disruptants were examined; temperature-sensitive growth, utilization of carbon sources and sensitivity to cations and drugs . Four double disruptants exhibited synthetic phenotypes in addition to eight single ones: the pph21 pph22 double disruptant showed slow growth on complete medium, as did the sit4 and yvh1 single ones . In addition to the ptc1, ynr022c and ycr079w single disruptants, the ppz1 ppz2 double disruptant showed temperature-sensitive slow growth . The msg5 ptp2 double disruptant, like the ynr022c single one, did not grow on complete medium containing 0.3 M CaCl(2) . The double msg5 ptc2 disruptant failed to grow on medium containing 1.0 M NaCl and, like the ynr022c single deletion, also could not grow on medium containing 0.3 M CaCl(2) . The synthetic phenotypes in the two latter cases where each of the PPases is categorized in a different phosphatase family led us to discuss the novel mechanism involved in the functional redundancy of the PPases . J Biol Chem, 2002 Jul 12, 277(28), 25011 - 9 Epub 2002 Apr 19. Saccharomyces cerevisiae acyl-CoA oxidase follows a novel, non-PTS1, import pathway into peroxisomes that is dependent on Pex5p; Klein AT et al.; The peroxisomal protein acyl-CoA oxidase (Pox1p) of Saccharomyces cerevisiae lacks either of the two well characterized peroxisomal targeting sequences known as PTS1 and PTS2 . Here we demonstrate that peroxisomal import of Pox1p is nevertheless dependent on binding to Pex5p, the PTS1 import receptor . The interaction between Pex5p and Pox1p, however, involves novel contact sites in both proteins . The interaction region in Pex5p is located in a defined area of the amino-terminal part of the protein outside of the tetratricopeptide repeat domain involved in PTS1 recognition; the interaction site in Pox1p is located internally and not at the carboxyl terminus where a PTS1 is normally found . By making use of pex5 mutants that are either specifically disturbed in binding of PTS1 proteins or in binding of Pox1p, we demonstrate the existence of two independent, Pex5p-mediated import pathways into peroxisomes in yeast as follows: a classical PTS1 pathway and a novel, non-PTS1 pathway for Pox1p. Biochem J, 2002 May 1, 363(Pt 3), 737 - 44 Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae; Paiva S et al.; Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae . The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123 s(-1) . It is expressed and tagged to the plasma membrane under induction conditions . The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p-GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways . The chimaeric protein Jen1p-GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole . Experiments performed in a Deltadoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease. Genetika, 2002 Mar, 38(3), 293 - 9 {Factors affecting the frequency of concealed heterokaryosis in Saccharomyces cerevisiae}; Nevzgliadova OV et al.; In this work, studies on the phenomenon of concealed heterokaryosis that we previously detected in the saccharomycetes yeast strains were continued . New approaches to high effectiveness of isolation of cytoductants carrying the concealed nucleus were implemented, and the composition of individual concealed heterokaryons, zygotic clones, and the first zygotic buds was analyzed by a micromanipulator . The relationship between a delay in the growth of the parental strain (a potential donor of the concealed nucleus) and a decline in the frequency of the appearance of concealed heterokaryons (HKC) was observed . It is assumed that different replication rates of two nuclei of the heterokaryon probably underlie the appearance of HKC . A drastically decreased level of replication of one of the parental nuclei may be connected with the fact that binuclear buds appear extremely rarely and give rise to the rapidly "purified" progeny consisting of cells carrying the second nucleus with normal replication . A lack of the phenotype allows rare binuclear cells to persist as concealed heterokaryons . HKC may be detected only when cells of either parental type are isolated on the corresponding selective media. Proc Natl Acad Sci U S A, 2002 Apr 16, 99(8), 5436 - 41 Bfa1 can regulate Tem1 function independently of Bub2 in the mitotic exit network of Saccharomyces cerevisiae; Ro HS et al.; In budding yeast, exit from mitosis is achieved by inactivation of Cdc28/Clb2 activity . Although it is not clear at present how mitotic exit is triggered, a growing body of evidence suggests that the Tem1 GTPase plays a critical role in mediating this pathway and that Bfa1 and Bub2 constitute a two-component GTPase-activating protein to negatively regulate Tem1 . Here, we have demonstrated that introduction of bfa1 Delta suppresses the growth defects associated with the cdc5-1 mutation significantly better than that of bub2 Delta, suggesting that Bfa1 may have a previously uncharacterized role in this pathway . Overexpression of BFA1 efficiently arrested the cell cycle at postanaphase even in the absence of BUB2, whereas overexpression of BUB2 weakly induced mitotic arrest only in the presence of BFA1 . Coimmunoprecipitation and in vitro binding studies indicate that Bfa1 binds strongly to Tem1 independently of Bub2 . Provision of GDP+AlF(4)(-), which mimics the GTPase transition state, enhanced the Bub2-Tem1 interaction both in vitro and in vivo . Interestingly, introduction of bfa1 Delta, but not bub2 Delta, greatly increased the interaction between Tem1 and Cdc15, a step that is thought to be critical for activating the mitotic exit network . Our data suggest that, in addition to its role as a putative, two-component GTPase-activating protein with Bub2, Bfa1 also can play a role in the regulation of mitotic exit by directly inhibiting the interaction between Tem1 and Cdc15 even in the absence of Bub2. Proc Natl Acad Sci U S A, 2002 Apr 16, 99(8), 5253 - 60 Mechanism of inactivation on prion conversion of the Saccharomyces cerevisiae Ure2 protein; Baxa U et al.; The {URE3} infectious protein (prion) of Saccharomyces cerevisiae is a self-propagating amyloid form of Ure2p . The C-terminal domain of Ure2p controls nitrogen catabolism by complexing with the transcription factor, Gln3p, whereas the asparagine-rich N-terminal "prion" domain is responsible for amyloid filament formation (prion conversion) . On filament formation, Ure2p is inactivated, reflecting either a structural change in the C-terminal domain or steric blocking of its interaction with Gln3p . We fused the prion domain with four proteins whose activities should not be sterically impeded by aggregation because their substrates are very small: barnase, carbonic anhydrase, glutathione S-transferase, and green fluorescent protein . All formed amyloid filaments in vitro, whose diameters increased with the mass of the appended enzyme . The helical repeat lengths were consistent within a single filament but varied with the construct and between filaments from a single construct . CD data suggest that, in the soluble fusion proteins, the prion domain has no regular secondary structure, whereas earlier data showed that in filaments, it is virtually all beta-sheet . In filaments, the activity of the appended proteins was at most mildly reduced, when substrate diffusion effects were taken into account, indicating that they retained their native structures . These observations suggest that the amyloid content of these filaments is confined to their prion domain-containing backbones and imply that Ure2p is inactivated in {URE3} cells by a steric blocking mechanism. FEMS Microbiol Lett, 2002 Mar 5, 208(2), 227 - 32 Vacuolar H(+)-ATPase, but not mitochondrial F(1)F(0)-ATPase, is required for NaCl tolerance in Saccharomyces cerevisiae; Hamilton CA et al.; Salt tolerance in Saccharomyces cerevisiae is a complex trait, involving regulation of membrane polarization, Na(+) efflux and sequestration of Na(+) in the vacuole . Since transmembrane transport energized by H(+)-adenosine triphosphatases (ATPases) is common to all of these tolerance mechanisms, the objective of this study was to characterize the responses of the plasma membrane H(+)-ATPase, vacuolar H(+)-ATPase and mitochondrial F(1)F(0)-ATPase to NaCl stress . We hypothesized that since the vacuolar ATPase is responsible for generating the proton motive force required for import of cations (such as Na(+)) into the vacuole, strains lacking this activity should be hypersensitive to NaCl . We found that strains lacking vacuolar ATPase activity were in fact hypersensitive to NaCl, while strains lacking ATP synthase were not . This effect was specific to the ionic component of NaCl stress, since the mutant strains were indistinguishable from wild-type and complemented strains in the presence of sorbitol. FEBS Lett, 2002 Apr 10, 516(1-3), 119 - 23 Regulation of the heme A biosynthetic pathway in Saccharomyces cerevisiae; Barros MH et al.; Biosynthesis of heme A, a prosthetic group of cytochrome oxidase (COX), involves an initial farnesylation of heme B . The heme O product formed in this reaction is modified by hydroxylation of the methyl group at carbon C-8 of the porphyrin ring . This reaction was proposed to be catalyzed by Cox15p, ferredoxin, and ferredoxin reductase . Oxidation of the alcohol to the corresponding aldehyde yields heme A . In the present study we have assayed heme A and heme O in yeast COX mutants . The steady state concentrations of the two hemes in the different strains studied indicate that hydroxylation of heme O, catalyzed by Cox15p, is regulated either by a subunit or assembly intermediate of COX . The heme profiles of the mutants also suggest positive regulation of heme B farnesylation by the hydroxylated intermediate formed at the subsequent step or by Cox15p itself. Funct Integr Genomics, 2002 Apr, 1(6), 345 - 56 Epub 2001 Dec 21. Large-scale identification of genes important for apical growth in Saccharomyces cerevisiae by directed allele replacement technology (DART) screening; Bidlingmaier S et al.; In Saccharomyces cerevisiae, apical bud growth occurs for a brief period in G1 when the deposition of membrane and cell wall is restricted to the tip of the growing bud . To identify genes important for apical bud growth, we have utilized a novel transposon-based mutagenesis system termed DART (Directed Allele Replacement Technology) that allows the rapid transfer of defined insertion alleles into any strain background . A total of 4,810 insertion alleles affecting 1,392 different yeast genes were transferred into a cdc34-2 mutant strain that arrests in the apical growth phase when grown at the restrictive temperature of 37 degrees C . We identified 29 insertion alleles, containing mutations in 17 different genes ( SMY1, SPA2, PAN1, SLA1, SLA2, CBK1, SEC22, FAB1, VPS36, VID22, RAS2, ECM33, OPI3, API1/YDR372c, API2/YDR525w, API3/YKR020w, and API4/YNL051w), which alter the elongated bud morphology of cdc34-2 cells arrested in the apical growth phase . Upon treatment with mating pheromone at 25 degrees C, cells containing insertion alleles affecting ten of these genes ( SMY1, SPA2, PAN1, SLA1, SLA2, CBK1, FAB1, VPS36, VID22, and API2/YDR525w) form abnormal mating projections . Additionally, cells containing insertion alleles affecting SEC22, RAS2, API1/YDR372c, API3/YKR020w,and API4/YNL051display severe mating projection formation defects at the elevated temperature of 37 degrees C . DART mutagenesis has many advantages over traditional mutagenesis methods and will be a useful tool for dissecting gene networks important for biological processes. Appl Microbiol Biotechnol, 2002 Apr, 58(5), 637 - 44 Epub 2002 Feb 12. Spacer-elongated cell wall fusion proteins improve cell surface expression in the yeast Saccharomyces cerevisiae; Breinig F et al.; Fusion proteins for cell surface expression in the yeast Saccharomyces cerevisiae were constructed that consisted of the N-terminal leader sequence of Kre1p, followed by the nine amino acid viral epitope hemagglutinin (HA), and the carboxyterminal anchoring domain of either Cwp2p or Flo1p . All fusions were constitutively expressed under transcriptional control of the phosphoglycerate kinase promoter and immunofluorescence analysis indicated that in each construct the HA peptide was correctly anchored to the outer yeast cell surface . Successful solubilization of the cell wall fusions by laminarinase treatment indicated that the fusions are covalently linked to cell wall beta-1,3- D-glucans in vivo . FACS analyses further demonstrated that 70% of the yeast cell population expressed the corresponding cell wall fusion . Neither the number of positive cells within the population nor the distribution of the fusion at the single-cell level were negatively affected by replacing the "heterologous" Kre1p leader by the "native" Cwp2p leader . Insertion of a 350 amino acid Ser/Thr-rich spacer sequence into the fusions led to a dramatic increase in HA peptide accessibility on the yeast cell surface . Our data show that FACS analyses represent a valuable means for investigating cell surface expression, and indicate that artificial-spacer-elongated cell wall fusions might raise novel possibilities for cell surface expression of heterologous proteins in yeast. J Biol Chem, 2002 Jun 28, 277(26), 23755 - 63 Epub 2002 Apr 15. In Saccharomyces cerevisiae, the inositol polyphosphate kinase activity of Kcs1p is required for resistance to salt stress, cell wall integrity, and vacuolar morphogenesis; Dubois E et al.; A problem for inositol signaling is to understand the significance of the kinases that convert inositol hexakisphosphate to diphosphoinositol polyphosphates . This kinase activity is catalyzed by Kcs1p in the yeast Saccharomyces cerevisiae . A kcs1Delta yeast strain that was transformed with a specifically "kinase-dead" kcs1p mutant did not synthesize diphosphoinositol polyphosphates, and the cells contained a fragmented vacuolar compartment . Biogenesi