Mol Cell, 2001 Apr, 7(4), 767 - 77 Stress-induced map kinase Hog1 is part of transcription activation complexes; Alepuz PM et al.; In response to hyperosmotic environments, most eukaryotic cells activate a specialized mitogen-activated protein (MAP) kinase pathway . In S . cerevisiae, the key protein kinase, Hog1, coordinates the transcriptional induction of a variety of genes devoted to osmoadaptation and general stress protection . Depending on the promoter context, Hog1 can function through a variety of structurally unrelated transcription factors . Using chromatin precipitation assays, we discovered that the kinase itself becomes intimately linked with promoter regions during stress responses . This interaction is dependent on the presence of stress-mediating transcriptional activators . In turn, Hog1 modulates promoter association of at least one of these factors . Additional findings highlight the possibility that Hog1 constitutes an integral part of the upstream activation complex, perhaps targeting not only the activator but also components of the general transcription machinery. Trends Genet, 2001 May, 17(5), 239 - 43 Bakers yeast rises to the challenge: reconstitution of mammalian steroid receptor signalling in S . cerevisiae; McEwan IJ; Steroid hormones are an important class of signalling molecule, regulating a diverse range of processes in metazoan eukaryotes . The actions of these hormones are mediated by intracellular receptor proteins that act as ligand-activated transcription factors . The ability to reconstitute steroid receptor signalling in the budding yeast, Saccharomyces cerevisiae, provides a genetically tractable model system in which to investigate steroid receptor structure and function . Through targeted disruption and genetic screening, an increasing number of genes have been identified that are likely to have a role in steroid receptor action. RNA, 2001 Mar, 7(3), 445 - 56 Mammalian heat shock p70 and histone H4 transcripts, which derive from naturally intronless genes, are immune to nonsense-mediated decay; Maquat LE et al.; Nonsense-mediated decay (NMD), also called mRNA surveillance, is an evolutionarily conserved pathway that degrades mRNAs that prematurely terminate translation . To date, the pathway in mammalian cells has been shown to depend on the presence of a cis-acting destabilizing element that usually consists of an exon-exon junction generated by the process of pre-mRNA splicing . Whether or not mRNAs that derive from naturally intronless genes, that is, mRNAs not formed by the process of splicing, are also subject to NMD has yet to be investigated . The possibility of NMD is certainly reasonable considering that mRNAs of Saccharomyces cerevisiae are subject to NMD even though most derive from naturally intronless genes . In fact, mRNAs of S . cerevisiae generally harbor a loosely defined splicing-independent destabilizing element that has been proposed to function in NMD analogously to the spliced exon-exon junction of mammalian mRNAs . Here, we demonstrate that nonsense codons introduced into naturally intronless genes encoding mouse heat shock protein 70 or human histone H4 fail to elicit NMD . Failure is most likely because each mRNA lacks a cis-acting destabilizing element, because insertion of a spliceable intron a sufficient distance downstream of a nonsense codon within either gene is sufficient to elicit NMD. Yi Chuan Xue Bao, 2001, 28(4), 372 - 8 {Cloning and expression of TPS1 gene in Escherichia coli}; Yang B et al.; A S . cerevisiae TPS1 gene for trehalose-6-phosphate synthase was cloned by PCR amplification . The 1.5 kb DNA fragment was ligated to pUC19 and transformed into otsA deficient and deleted of E . coli strains FF4169 and FF4050 separately . otsA gene is encoding trehalose-6-phasphate synthase in E . coli . Restriction endonucleases digestion analysis of transformants' plasmid DNA showed that there was a cloned 1.5 kb fragment carried on the vector . The growth curve experiment result showed that the both transformants could grow well as wild type . Trehalose was synthesized and accumulated in these transformants during high osmotic stress by HPLC combined with ELSD (Evaporate light scatter detector) determination . From the result above, we could conclude that the TPS1 gene of S . cerevisiae was able to restore otsA gene function in E . coli for both osmotolerance and trehalose accumulation during salt stress. Yeast, 2001 May, 18(7), 611 - 20 Cytosolic redox metabolism in aerobic chemostat cultures of Saccharomyces cerevisiae; Pahlman IL et al.; Cytosolic redox balance has to be maintained in order to allow an enduring cellular metabolism . In other words, NADH generated in the cytosol has to be re-oxidized back to NAD(+) . Aerobically this can be done by respiratory oxidation of cytosolic NADH . However, NADH is unable to cross the mitochondrial inner membrane and mechanisms are required for conveying cytosolic NADH to the mitochondrial electron transport chain . At least two such systems have proved to be functional in S . cerevisiae, the external NADH dehydrogenase (Luttik et al., 1998; Small and McAlister-Henn, 1998) and the G3P shuttle (Larsson et al., 1998) . The aim of this investigation was to study the regulation and performance of these two systems in a wild-type strain of S . cerevisiae using aerobic glucose- and nitrogen-limited chemostat cultures . The rate of cytosolic NADH formation was calculated and as expected there was a continuous increase with increasing dilution rate . However, measurements of enzyme activities and respiratory activity on isolated mitochondria revealed a diminishing capacity at elevated dilution rates for both the external NADH dehydrogenase and the G3P shuttle . This suggests that adjustment of in vivo activities of these systems to proper levels is not achieved by changes in amount of protein but rather by, for example, activation/inhibition of existing enzymes . Adenine nucleotides are well-known allosteric regulators and both the external NADH and the G3P shuttle were sensitive to inhibition by ATP . The most severe inhibition was probably on the G3P shuttle, since one of its member proteins, Gpdp, turned out to be exceptionally sensitive to ATP . The external NADH dehydrogenase is suggested as the main system employed for oxidation of cytosolic NADH . The G3P shuttle is proposed to be of some importance at low growth rates and perhaps its real significance is only expressed during starvation conditions . Yeast, 2001 May, 18(7), 597 - 603 Isolation and sequence of the MIG1 homologue from the yeast Candida utilis; Delfin J et al.; The Mig1p repressor from the food yeast Candida utilis has been isolated using a homologous PCR hybridization probe . This probe was amplified with two sets of degenerate primers designed on the basis of highly conserved motifs in the DNA-binding region (zinc-finger domain) from yeast Mig1p and fungi CreA repressors . The cloned gene was sequenced and found to encode a polypeptide of 345 amino acids which shows significant identity with other yeast and fungus repressors in the DNA-binding domain and also with the yeast Mig1 proteins in the C-terminal region (effector domain) . The MIG1 repressor gene from C . utilis was able to complement functionally the mig1 mutation of S . cerevisiae . The sequence presented here has been deposited in the EMBL data library under Accession No . AJ277830 . J Bacteriol, 2001 May, 183(10), 3098 - 107 Tagging morphogenetic genes by insertional mutagenesis in the yeast Yarrowia lipolytica; Richard M et al.; The yeast Yarrowia lipolytica is distantly related to Saccharomyces cerevisiae, can be genetically modified, and can grow in both haploid and diploid states in either yeast, pseudomycelial, or mycelial forms, depending on environmental conditions . Previous results have indicated that the STE and RIM pathways, which mediate cellular switching in other dimorphic yeasts, are not required for Y . lipolytica morphogenesis . To identify the pathways involved in morphogenesis, we mutagenized a wild-type strain of Y . lipolytica with a Tn3 derivative . We isolated eight tagged mutants, entirely defective in hyphal formation, from a total of 40,000 mutants and identified seven genes homologous to S . cerevisiae CDC25, RAS2, BUD6, KEX2, GPI7, SNF5, and PPH21 . We analyzed their abilities to invade agar and to form pseudomycelium or hyphae under inducing conditions and their sensitivity to temperature and to Calcofluor white . Chitin staining was used to detect defects in their cell walls . Our results indicate that a functional Ras-cyclic AMP pathway is required for the formation of hyphae in Y . lipolytica and that perturbations in the processing of extracellular, possibly parietal, proteins result in morphogenetic defects. Biotechnol Bioeng, 2001 Jun 5, 73(5), 412 - 25 In vivo dynamics of galactose metabolism in Saccharomyces cerevisiae: metabolic fluxes and metabolite levels; Ostergaard S et al.; The dynamics of galactose metabolism in Saccharomyces cerevisiae was studied by analyzing the metabolic response of the CEN.PK 113-7D wild-type strain when exposed to a galactose pulse during aerobic growth in a galactose-limited steady-state cultivation at a dilution rate of 0.097 h(-1) . A fast sampling technique and subsequent methanol-chloroform/solid phase extractions were applied for in vivo measurements of the dynamic changes of the AMP, ADP, ATP levels and the sugar phosphates of the Leloir pathway . The ATP level was found to be significantly lower for yeast growing under galactose limitation (0.37 +/- 0.05 micromol/g CDW) than what has been reported for growth under glucose limitation . The galactose pulse of 5.58 mM was consumed within 40 min (t = 40) and 7 min after the pulse was added cell growth stopped . Subsequently, the cells started to grow and at t = 30 the specific growth rate had recovered to half the steady-state growth rate (0.047 h(-1)) . To evaluate the change in flux distribution at steady state and during the galactose transient, a stoichiometric model describing the aerobic metabolism of S . cerevisiae was set up for quantification of the metabolic fluxes . At t = 7 the flux entering the TCA cycle was low and acetate and ethanol started to be excreted to the extracellular medium . During recovery of cell growth the flux entering the TCA cycle increased again, and at t = 30 this flux exceeded the corresponding steady-state flux . During the pulse an enhanced level of Gal-1P was measured, which may be responsible for a toxic metabolic response in S . cerevisiae . The increase in the Gal-1P concentration is intensified by the low affinity of Gal7 towards Gal-1P and, hence, under the physiological conditions examined Gal7 seems to exert control over flux through the Leloir pathway . Biotechniques, 2001 Apr, 30(4), 816 - 20, 822-6, 828 passim Genetic transformation of yeast; Gietz RD et al.; Genetic transformation was first described by Griffith in 1928 and has since been demonstrated in a variety of organisms, including many species of fungi . This review focuses on the history and technology of the transformation of Saccharomyces cerevisiae . The application of protocols developed for S . cerevisiae to other important yeast species is discussed . The protocols for transformation by spheroplasting, LiAc/ssDNA/PEG, and electroporation are compared, and possible mechanisms for transformation are discussed. Oncogene, 2001 Mar 22, 20(12), 1519 - 24 An hsRPB4/7-dependent yeast assay for trans-activation by the EWS oncogene; Zhou H et al.; Chromosomal fusions of the N-terminal region of the Ewings Sarcoma Oncogene (EWS-Activation-Domain, EAD) to the DNA-binding domains of a variety of cellular transcription factors, produce oncogenic proteins (EWS-fusion proteins (EFPs)) that cause a variety of malignancies . The EAD can act as a potent transcriptional activation domain and is required for the oncogenic activity of EFPs . Previous studies demonstrating a physical interaction between the EAD and the human RNA Polymerase II subunit hsRPB7 suggest a crucial role for RPB7 and its partner, RPB4, in EAD function . Homologues of hsRPB4/7 exist in S . cerevisiae, and here we describe an RPB4/7-dependent yeast assay for EAD-mediated trans-activation . Conditional yeast strains lacking RPB4 are defective for trans-activation by a Gal4/EAD fusion protein at the permissive temperature . Introduction of hsRPB4 alone is unable to rescue trans-activation, while a combination of hsRPB4 and hsRPB7 significantly rescues activity . These findings provide the first functional evidence for a direct role of the RPB4/7 complex in EAD-mediated trans-activation . In addition, the yeast assay provides a tractable system for further molecular analysis of EAD and RPB4/7 action. J Cell Biol, 2001 Apr 16, 153(2), 435 - 42 Stu2 promotes mitotic spindle elongation in anaphase; Severin F et al.; During anaphase, mitotic spindles elongate up to five times their metaphase length . This process, known as anaphase B, is essential for correct segregation of chromosomes . Here, we examine the control of spindle length during anaphase in the budding yeast Saccharomyces cerevisiae . We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2 . We further show that the activity of Stu2 is opposed by the activity of the kinesin-related protein Kip3 . Reexamination of the kinesin homology tree suggests that KIP3 is the S . cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I) . We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B. Trends Cell Biol, 2001 Apr, 11(4), 160 - 6 Control of spindle polarity and orientation in Saccharomyces cerevisiae; Segal M et al.; Control of mitotic spindle orientation represents a major strategy for the generation of cell diversity during development of metazoans . Studies in the budding yeast Saccharomyces cerevisiae have contributed towards our present understanding of the general principles underlying the regulation of spindle positioning in an asymmetrically dividing cell . In S . cerevisiae, the mitotic spindle must orient along the cell polarity axis, defined by the site of bud emergence, to ensure correct nuclear division between the mother and daughter cells . Establishment of spindle polarity dictates this process and relies on the concerted control of spindle pole function and a precise program of cues originating from the cell cortex that directs cytoplasmic microtubule attachments during spindle morphogenesis . These cues cross talk with the machinery responsible for bud-site selection, indicating that orientation of the spindle in yeast cells is mechanistically coupled to the definition of a polarity axis and the division plane . Here, we propose a model integrating the inherently asymmetric properties of the spindle pathway with the program of positional information contributing towards orienting the spindle in budding yeast . Because the basic machinery orienting the spindle in higher-eukaryotic cells appears to be conserved, it might be expected that similar principles govern centrosome asymmetry in the course of metazoan development. Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 685 - 98 Three aldo-keto reductases of the yeast Saccharomyces cerevisiae; Ford G et al.; Saccharomyces cerevisiae is an industrially important yeast, which is also used extensively as a model eukaryote . The S . cerevisiae genome has been sequenced in its entirety and therefore represents an ideal organism in which to carry out functional analysis of genes . We have identified several open reading frames in the S . cerevisiae genome which show significant similarity to members of the aldo-keto reductase superfamily . The physiological roles of these gene products have not been previously determined, but their similarity to other enzymes suggests they may perform roles in carbohydrate metabolism and detoxification pathways . Cloning and expression of three of these enzymes has allowed their substrate specificities to be determined . Expression profiling and gene disruption analysis will allow potential roles for these enzymes within the cell to be examined. Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 527 - 36 The crystal structure of the GCY1 protein from S . cerevisiae suggests a divergent aldo-keto reductase catalytic mechanism; Hur E et al.; The crystal structure of the GCY1 gene product from Saccharomyces cerevisiae has been determined to 2.5 A and is being refined . The model includes two protein molecules, one apo and one holo, per asymmetric unit . Examination of the model reveals that the active site surface is somewhat flat when compared with the other aldo-keto reductase structures, possibly accommodating larger substrates . The K(m) for NADPH (28.5 microM) is higher than that seen for other family members . This can be explained structurally by the lack of the 'safety belt' of residues seen in other aldo-keto reductases with higher affinity for NADPH . Catalysis also differs from the other aldo-keto reductases . The tyrosine that acts as an acid in the reduction reaction is flipped out of the catalytic pocket . This implies that the protein must either undergo a conformational change before catalysis can take place or that there is an alternate acid moiety. Biochimie, 2001 Mar-Apr, 83(3-4), 319 - 23 Antizyme, a mediator of ubiquitin-independent proteasomal degradation; Coffino P; Ornithine decarboxylase (ODC) is among the small set of proteasome substrates that is not ubiquitinated . It is instead degraded in conjunction with the protein antizyme (AZ) . ODC and AZ are participants in a regulatory circuit that restricts pools of polyamines, the downstream products of ODC enzymatic activity . Functional studies using directed mutagenesis have identified regions of ODC and AZ required for the process of ODC degradation . Within ODC, there is a region that is required for AZ binding which lies on the surface of an alpha-beta barrel forming one domain of the ODC monomer . A carboxy-terminal ODC domain is needed for both AZ-dependent and AZ-independent degradation . Within AZ, the carboxy-terminal half molecule is sufficient for binding to ODC, but an additional domain found within the AZ amino terminus must be present for stimulation of ODC degradation by the proteasome . Recently, the AZs have been found to consist of an ancient gene family . Within vertebrate species, multiple isoforms are found, with distinct functions that remain to be sorted out . Although AZ homologs have been found in some yeast species, homology searches have failed to identify an AZ homolog in Saccharomyces cerevisiae . Nevertheless, the close parallel between polyamine-induced ODC degradation in S . cerevisiae and in animal cells suggests that this organism will also be found to harbor an AZ-like protein. Science, 2001 Apr 13, 292(5515), 288 - 90 Epub 2001 Apr 05. Regulation of longevity and stress resistance by Sch9 in yeast; Fabrizio P et al.; The protein kinase Akt/protein kinase B (PKB) is implicated in insulin signaling in mammals and functions in a pathway that regulates longevity and stress resistance in Caenorhabditis elegans . We screened for long-lived mutants in nondividing yeast Saccharomyces cerevisiae and identified mutations in adenylate cyclase and SCH9, which is homologous to Akt/PKB, that increase resistance to oxidants and extend life-span by up to threefold . Stress-resistance transcription factors Msn2/Msn4 and protein kinase Rim15 were required for this life-span extension . These results indicate that longevity is associated with increased investment in maintenance and show that highly conserved genes play similar roles in life-span regulation in S . cerevisiae and higher eukaryotes. Genetics, 2001 Apr, 157(4), 1543 - 53 Novel role for a Saccharomyces cerevisiae nucleoporin, Nup170p, in chromosome segregation; Kerscher O et al.; We determined that a mutation in the nucleoporin gene NUP170 leads to defects in chromosome transmission fidelity (ctf) and kinetochore integrity in Saccharomyces cerevisiae . A ctf mutant strain, termed s141, shows a transcription readthrough phenotype and stabilizes a dicentric chromosome fragment in two assays for kinetochore integrity . Previously, these assays led to the identification of two essential kinetochore components, Ctf13p and Ctf14p . Thus, s141 represents another ctf mutant involved in the maintenance of kinetochore integrity . We cloned and mapped the gene complementing the ctf mutation of s141 and showed that it is identical to the S . cerevisiae NUP170 gene . A deletion strain of NUP170 (nup170 Delta::HIS3) has a Ctf(-) phenotype similar to the s141 mutant (nup170-141) and also exhibits a kinetochore integrity defect . We identified a second nucleoporin, NUP157, a homologue of NUP170, as a suppressor of the Ctf(-) phenotype of nup170-141 and nup170 Delta::HIS3 strains . However, a deletion of NUP157 or several other nucleoporins did not affect chromosome segregation . Our data suggest that NUP170 encodes a specialized nucleoporin with a unique role in chromosome segregation and possibly kinetochore function. Genetics, 2001 Apr, 157(4), 1493 - 502 The spindle checkpoint of the yeast Saccharomyces cerevisiae requires kinetochore function and maps to the CBF3 domain; Gardner RD et al.; We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae . We constructed deletion mutants for nonessential genes by one-step gene replacements . We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele . In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles) . We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity . In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient . We conclude that the kinetochore plays a critical role in checkpoint signaling in S . cerevisiae . Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex. Gene, 2001 Mar 21, 266(1-2), 111 - 21 Conservation of the MORF4 related gene family: identification of a new chromo domain subfamily and novel protein motif; Bertram MJ et al.; The seven member, human MORF4 related gene (MRG) family was recently identified based on the ability of Mortality factor on chromosome 4 (MORF4) to induce replicative senescence in immortal cell lines assigned to complementation group B (Bertram et al., 1999 . Mol . Cell Biol . 19, 1479-1485) . Initial computer based similarity searches identified human retinoblastoma binding protein 1 (RBP-1), Drosophila melanogaster male specific lethal-3 (Msl-3), S . pombe altered polarity-13 (Alp13) and S . cerevisiae Eaf3p, a component of the yeast NuA4 HAT complex (Galarneau et al., 2000 . Mol . Cell 5, 927-937), as having similarity to the human MRG protein family . This suggested that the MRG family might be found in multiple species, and analysis of other homologs would provide functional and evolutionary insights into this gene family . Here, we report that MRG family members are present in twenty-three species based on molecular assays and sequence similarity searches . The new family members were divided into two groups based on similarity to the predominant human MRG family members, MRG15 and MRGX . The family members similar to MRG15 define a new, highly conserved subsection of the chromo domain superfamily . Additionally, conservation in the C-terminal two thirds of all the MRG family members and the Drosophila and human MSL-3 proteins defines a new protein domain, the MRG domain . These results indicate a highly conserved role for the MRG family in transcriptional regulation via chromatin remodeling by histone acetylation. Metab Eng, 2001 Apr, 3(2), 163 - 72 Signal transduction dynamics of the protein kinase-A/phosphofructokinase-2 system in Saccharomyces cerevisiae; Vaseghi S et al.; This work focuses on the phosphofructokinase-2-system dynamics in Saccharomyces cerevisiae, in vivo . The investigations were dedicated to the development and implementation of appropriate theoretical and experimental methods toward evaluation of a quantitative strategy for the characterization of systemic mechanisms involved in the cAMP/protein kinase-A/phosphofructokinase-2 signal transduction cascade in yeast . Upon glucose pulse experiments, applied to glucose-limited continuous cultures of S . cerevisiae, the system response was determined with respect to alterations of intracellular metabolite concentrations or in vivo enzyme activities . Phosphofructokinase-2, in vivo, was found to be saturated with respect to both its substrates, F6P and ATP . This restriction results in an uncoupling of the enzyme activity and the signal transduction cascade from glycolytic flux, concluding that activation of phosphofructokinase-2 is exclusively a result of phosphorylation by protein kinase-A, which in turn is activated by increasing intracellular cAMP concentration after an extracellular glucose pulse . Signal processing from cAMP versus phosphofructokinase-2 also displays peculiar features implicated in a hysteresis behavior: when increasing cAMP concentration achieves a certain critical value, protein kinase-A switches into an active state . Posterior to this activation, the signal transform maintains autonomy and functional independence of further alterations of the intracellular cAMP concentration . Our observations, finally, allow the establishment of a representative model for the description of the signal transduction process via protein kinase-A in yeast . Mol Cell Biol, 2001 May, 21(9), 3166 - 78 RNA polymerase III transcription complexes on chromosomal 5S rRNA genes in vivo: TFIIIB occupancy and promoter opening; Costanzo G et al.; Quantitative analysis of multiple-hit potassium permanganate (KMnO(4)) footprinting has been carried out in vivo on Saccharomyces cerevisiae 5S rRNA genes . The results fix the number of open complexes at steady state in exponentially growing cells at between 8 and 17% of the 150 to 200 chromosomal copies . UV and dimethyl sulfate footprinting set the transcription factor TFIIIB occupancy at 23 to 47% . The comparison between the two values suggests that RNA polymerase III binding or promoter opening is the rate-limiting step in 5S rRNA transcription in vivo . Inhibition of RNA elongation in vivo by cordycepin confirms this result . An experimental system that is capable of providing information on the mechanistic steps involved in regulatory events in S . cerevisiae cells has been established. J Virol, 2001 May, 75(9), 4444 - 7 Selection of apoptosis-deficient adenovirus E4orf4 mutants in Saccharomyces cerevisiae; Afifi R et al.; Adenovirus E4orf4 protein has been shown to induce p53-independent, protein phosphatase 2A (PP2A)-dependent apoptosis in transformed cells . Furthermore, E4orf4 also induces toxicity in Saccharomyces cerevisiae in a PP2A-dependent manner (D . Kornitzer and T . Kleinberger, submitted for publication) . In this work, we utilized yeast cells to select for nonapoptotic E4orf4 mutants which, in turn, were shown to possess a diminished ability to bind PP2A . The success of this selection system will provide additional apoptosis-relevant mutants for E4orf4 research and strongly supports the relevance of E4orf4-induced toxicity in S . cerevisiae to E4orf4-induced apoptosis in mammalian cells. Mol Genet Metab, 2001 Apr, 72(4), 336 - 42 Identification of the alpha-aminoadipic semialdehyde dehydrogenase-phosphopantetheinyl transferase gene, the human ortholog of the yeast LYS5 gene; Praphanphoj V et al.; In mammals, L-lysine is first catabolized to alpha-aminoadipate semialdehyde by the bifunctional enzyme alpha-aminoadipate semialdehyde synthase (AASS), followed by a conversion to alpha-aminoadipate by alpha-aminoadipate semialdehyde dehydrogenase . In Saccharomyces cerevisiae, which synthesize rather than degrade lysine, the latter activity requires two distinct genes . LYS2 encodes the alpha-aminoadipate reductase activity, while LYS5 encodes a phosphopantetheinyl transferase activity that is required to activate Lys2p . We have identified a full-length human cDNA homologous to the yeast LYS5 gene . The cDNA contains an open-reading frame of 930 bp predicted to encode 309 amino acids, and the human protein is 26% identical and 44% similar to its yeast counterpart . In Northern blot analysis the cDNA hybridizes to a single transcript of approximately 3 kb in all tissues except testis, where there is an additional transcript of 1.5 kb . Expression is highest in brain followed by heart and skeletal muscle, and to a lesser extent in liver . We further identified three human genomic BAC clones containing the human gene . Fluorescence in situ hybridization (FISH) analysis using the BAC clones mapped the gene to chromosome 11q22 while alignment of the cDNA and genomic sequences allowed partial identification of the intron-exon boundaries . Finally, using one-step homologous recombination in S . cerevisiae we generated a lys5 knockout strain . Complementation studies in the yeast knockout demonstrate that the human homolog encodes alpha-aminoadipate dehydrogenase phosphopantetheinyl transferase activity . We hypothesize that defects in this gene may result in pipecolic acidemia . J Cell Biol, 2001 Apr 2, 153(1), 47 - 62 A role for actin, Cdc1p, and Myo2p in the inheritance of late Golgi elements in Saccharomyces cerevisiae; Rossanese OW et al.; In Saccharomyces cerevisiae, Golgi elements are present in the bud very early in the cell cycle . We have analyzed this Golgi inheritance process using fluorescence microscopy and genetics . In rapidly growing cells, late Golgi elements show an actin-dependent concentration at sites of polarized growth . Late Golgi elements are apparently transported into the bud along actin cables and are also retained in the bud by a mechanism that may involve actin . A visual screen for mutants defective in the inheritance of late Golgi elements yielded multiple alleles of CDC1 . Mutations in CDC1 severely depolarize the actin cytoskeleton, and these mutations prevent late Golgi elements from being retained in the bud . The efficient localization of late Golgi elements to the bud requires the type V myosin Myo2p, further suggesting that actin plays a role in Golgi inheritance . Surprisingly, early and late Golgi elements are inherited by different pathways, with early Golgi elements localizing to the bud in a Cdc1p- and Myo2p-independent manner . We propose that early Golgi elements arise from ER membranes that are present in the bud . These two pathways of Golgi inheritance in S . cerevisiae resemble Golgi inheritance pathways in vertebrate cells. Mol Cell Biol, 2001 Apr, 21(8), 2790 - 801 Two compound replication origins in Saccharomyces cerevisiae contain redundant origin recognition complex binding sites; Theis JF et al.; While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different . As typified by ARS1, replication origins in Saccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences . DNA replication initiates from a discrete site . While the important sequences are currently less well defined, metazoan origins appear to be different . These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb . In this report, we characterize two S . cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm . These origins contain multiple, redundant binding sites for the origin recognition complex . Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1 . This redundant structure may be similar to that seen in metazoan origins. BMC Genet . 2001;2(1):5 . Epub 2001 Mar 19. NRG1 is required for glucose repression of the SUC2 and GAL genes of Saccharomyces cerevisiae; Zhou H et al.; BACKGROUND: Glucose repression of transcription in the yeast, Saccharomyces cerevisiae, has been shown to be controlled by several factors, including two repressors called Mig1 and Mig2 . Past results suggest that other repressors may be involved in glucose repression . RESULTS: By a screen for factors that control transcription of the glucose-repressible SUC2 gene of S . cerevisiae, the NRG1 gene was identified . Analysis of an nrg1Delta mutant has demonstrated that mRNA levels are elevated at both the SUC2 and of the GAL genes of S . cerevisiae when cells are grown under normally glucose-repressing conditions . In addition, genetic interactions have been detected between nrg1Delta and other factors that control SUC2 transcription . CONCLUSIONS: The analysis of nrg1Delta demonstrates that Nrg1 plays a role in glucose repression of the SUC2 and GAL genes of S . cerevisiae . Thus, three repressors, Nrg1, Mig1, and Mig2, are involved as the downstream targets of the glucose signaling in S . cerevisiae. Free Radic Biol Med, 2000 Jan 15, 28(2), 183 - 92 The biosynthesis of erythroascorbate in Saccharomyces cerevisiae and its role as an antioxidant; Spickett CM et al.; This study investigated the ability of the yeast Saccharomyces cerevisiae to synthesize ascorbate and its 5-carbon analogue erythroascorbate from a variety of precursors, and their importance as antioxidants in this organism . Studies of ascorbate and analogues in micro-organisms have been reported previously, but their function as antioxidants have been largely ignored . Ascorbate and erythroascorbate concentrations in yeast extracts were measured spectrophotometrically, and their levels and identity were checked using liquid chromatography-electrospray mass spectrometry . The yeast was readily able to synthesize ascorbate from L-galactono-1,4-lactone or erythroascorbate from D-arabinose and D-arabino-1,4-lactone, whereas L-gulono-1,4-lactone was a much poorer substrate for ascorbate biosynthesis . In untreated cells, the concentration of ascorbate-like compounds was below the level of detection of the methods of analysis used in this study (approximately 0.1 mM) . Intracellular ascorbate and erythroascorbate were oxidized at high concentrations of tert-butylhydroperoxide, but not hydrogen peroxide . Their synthesis was not increased in response to low levels of stress, however, and preloading with erythroascorbate did not protect glutathione levels during oxidative stress . This study provides new information on the metabolism of ascorbate and erythroascorbate in S . cerevisiae, and suggests that erythroascorbate is of limited importance as an antioxidant in S . cerevisiae. Genet Res, 2001 Feb, 77(1), 9 - 26 Masking and purging mutations following EMS treatment in haploid, diploid and tetraploid yeast (Saccharomyces cerevisiae); Mable BK et al.; The yeast, Saccharomyces cerevisiae, was used as a model to investigate theories of ploidy evolution . Mutagenesis experiments using the alkylating agent EMS (ethane methyl sulphonate) were conducted to assess the relative importance that masking of deleterious mutations has on response to and recovery from DNA damage . In particular, we tested whether cells with higher ploidy levels have relatively higher fitnesses after mutagenesis, whether the advantages of masking are more pronounced in tetraploids than in diploids, and whether purging of mutations allows more rapid recovery of haploid cells than cells with higher ploidy levels . Separate experiments were performed on asexually propagating stationary phase cells using (1) prototrophic haploid (MAT alpha) and diploid (MATa/alpha) strains and (2) isogenic haploid, diploid and tetraploid strains lacking a functional mating type locus . In both sets of experiments, haploids showed a more pronounced decrease in apparent growth rate than diploids, but both haploids and diploids appeared to recover very rapidly . Tetraploids did not show increased benefits of masking compared with diploids but volume measurements and FACScan analyses on the auxotrophic strains indicated that all treated tetraploid strains decreased in ploidy level and that some of the treated haploid lines increased in ploidy level . Results from these experiments confirm that while masking deleterious mutations provides an immediate advantage to higher ploidy levels in the presence of mutagens, selection is extremely efficient at removing induced mutations, leading growth rates to increase rapidly over time at all ploidy levels . Furthermore, ploidy level is itself a mutable trait in the presence of EMS, with both haploids and tetraploids often evolving towards diploidy (the ancestral state of S . cerevisiae) during the course of the experiment. J Biol Chem, 2001 May 11, 276(19), 16528 - 33 Epub 2001 Feb 05. Cloning and characterization of ELL-associated proteins EAP45 and EAP20 . a role for yeast EAP-like proteins in regulation of gene expression by glucose; Kamura T et al.; RNA polymerase II elongation factor ELL was recently purified from rat liver as a component of a multiprotein complex containing ELL and three ELL-associated proteins (EAPs) of approximately 45 (EAP45), approximately 30 (EAP30), and approximately 20 (EAP20) kDa (Shilatifard, A . (1998) J . Biol . Chem . 273, 11212-11217) . Cloning of cDNA encoding the EAP30 protein revealed that it shares significant sequence similarity with the product of the Saccharomyces cerevisiae SNF8 gene (Schmidt, A . E., Miller, T., Schmidt, S . L., Shiekhattar, R., and Shilatifard, A . (1999) J . Biol . Chem . 274, 21981-21985), which is required for efficient derepression of glucose-repressed genes . Here we report the cloning of cDNAs encoding the EAP45 and EAP20 proteins . In addition, we identify the S . cerevisiae VPS36 and YJR102c genes as potential orthologs of EAP45 and EAP20 and show that they are previously uncharacterized SNF genes with properties very similar to SNF8. Biochim Biophys Acta, 2001 Mar 26, 1535(3), 236 - 57 Molecular requirements for the internalisation step of endocytosis: insights from yeast; Munn AL; Molecular genetic studies of endocytosis using the unicellular eukaryote Saccharomyces cerevisiae (budding yeast) have led to the identification of many cellular components, both proteins and lipids, required for this process . While initially, many of these requirements (e.g . for actin, various actin-associated proteins, the ubiquitin conjugation system, and for ergosterol and sphingolipids) appeared to differ from known requirements for endocytosis in higher eukaryotes (e.g . clathrin, AP-2, dynamin), it now seems that endocytosis in higher and lower eukaryotes share many requirements . Often, what were initially identified as actin cytoskeleton-associated proteins in S . cerevisiae, are now revealing themselves as clathrin-coated pit- and vesicle-associated proteins in higher eukaryotes . So rather than delineating two endocytic pathways, one actin-based and one clathrin-based, the combined studies on higher and lower eukaryotes are revealing interesting interplay in both systems between the actin cytoskeleton, clathrin coats, and lipids in the formation of endocytic vesicles at the plasma membrane . Recent results from the yeast system show that the Arp2/3p complex, Wiskott-Aldrich syndrome protein (WASP), and WASP-interacting protein (WIP), proteins involved in the nucleation step of actin filament assembly, play a major role in the formation of endocytic vesicles . This discovery suggests models whereby endocytic vesicles may be actively pushed from the plasma membrane and into the cell by newly forming and rapidly extending actin filaments. FEBS Lett, 2001 Mar 23, 493(1), 1 - 5 Mutation Arg336 to Lys in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase originates an enzyme with increased oxaloacetate decarboxylase activity; Llanos L et al.; Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes one of the first reactions in the biosynthesis of carbohydrates . Apart from the physiologically important reaction, the enzyme also presents low oxaloacetate decarboxylase and pyruvate kinase-like activities . Data from the crystalline structure of homologous Escherichia coli PEP carboxykinase suggest that Arg(333) may be involved in stabilization of enolpyruvate, a postulated reaction intermediate . In this work, the equivalent Arg(336) from the S . cerevisiae enzyme was changed to Lys or Gln . Kinetic analyses of the varied enzymes showed that a positive charge at position 336 is critical for catalysis of the main reaction, and further suggested different rate limiting steps for the main reaction and the secondary activities . The Arg336Lys altered enzyme showed increased oxaloacetate decarboxylase activity and developed the ability to catalyze pyruvate enolization . These last results support the proposal that enolpyruvate is an intermediate in the PEP carboxykinase reaction and suggest that in the Arg336Lys PEP carboxykinase a proton donor group has appeared. Folia Microbiol (Praha), 2000, 45(2), 99 - 106 Mitochondria--tool for taxonomic identification of yeasts from Saccharomyces sensu stricto complex; Soltesova A et al.; Mitochondrial genomes of Saccharomyces and close relatives previously used for transplacement of mitochondria to S . cerevisiae were examined . The origins of replication in mitochondrial DNA, the presence of nuclear and mitochondrial polymorphic loci and the ability to produce mitochondrial respiration-deficient mutants were used to reclassify some collection yeasts and to assign others into four separate subgroups . The first included isolates identical to Saccharomyces cerevisiae (S . italicus, S . oviformis, S . chevalieri and S . capensis) which possess 5 or more replication origins . The second group consists of S paradoxus (var douglasii) mitochondrial genome with the equal number of ori sequences but incompatible mitochondria . The third group represents Saccharomyces sensu stricto petite-positive species (S . carlsbergensis, S . heterogenicus, S . uvarum, S . willianus) with 1-2 origins of replication significantly different from S . cerevisiae . In addition, the locus between tRNA(fMet) and tRNA(Pro) is about one-half of the 1400 bp members of S . cerevisiae complex . The last group includes isolates that do not belong to Saccharomyces sensu stricto group as they are petite-negative and devoid of any S . cerevisiae-like replication origins. Curr Biol, 2001 Mar 6, 11(5), R166 - 8 RNA localization: SHEdding light on the RNA-motor linkage; Kwon S et al.; Specific mRNAs achieve an asymmetric distribution in the cell by linking to molecular motors that walk along the cytoskeleton . Studies in S . cerevisiae have begun to define the nature of the RNA-motor linkage and identify She3p as an adaptor protein that links a type V myosin motor to specific ribonucleoproteins. Mutat Res, 2001 Apr 4, 485(3), 229 - 36 RAD9, RAD24, RAD16 and RAD26 are required for the inducible nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers from the transcribed and non-transcribed regions of the Saccharomyces cerevisiae MFA2 gene; Yu S et al.; In this study, the effect of a prior UV irradiation on the removal of cyclobutane pyrimidine dimers (CPDs) from the transcribed strand (TS) and non-transcribed strand (NTS) of the MFA2 gene in haploid Saccharomyces cerevisiae (S . cerevisiae) cells was investigated . In NER competent cells, the pre-irradiation with a dose of 20J/m2 enhances the removal of CPDs induced by a second UV dose of 100J/m2 in the TS and the NTS of MFA2 gene except for the CPDs in the region +258 to +298 in the NTS, where the enhanced repair was absent . No inducible repair was observed in rad9, rad24, rad16 and rad26 cells, indicating two checkpoint genes RAD9 and RAD24, the global repair gene RAD16 and the transcription coupled repair gene RAD26 are essential for inducible NER. Nucleic Acids Res, 2001 Apr 1, 29(7), 1582 - 9 Mitochondrial DNA ligase function in Saccharomyces cerevisiae; Donahue SL et al.; The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria . While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined . It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria . To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed . Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle . Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures . In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (Deltadnl4) . The Escherichia coli ECO:RI endonuclease was targeted to yeast mitochondria . Transient expression of this recombinant ECO:RI endonuclease led to the formation of mitochondrial DNA double-strand breaks . While wild-type and Deltadnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not . These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage. J Cell Biol, 2001 Feb 19, 152(4), 729 - 40 The karyopherin Kap142p/Msn5p mediates nuclear import and nuclear export of different cargo proteins; Yoshida K et al.; We have identified a novel pathway for protein import into the nucleus . Although the product of Saccharomyces cerevisiae gene MSN5 was previously shown to function as a karyopherin (Kap) for nuclear export of various proteins, we discovered a nuclear import pathway mediated by Msn5p (also referred to as Kap142p) . We have purified from yeast cytosol a complex containing Kap142p and the trimeric replication protein A (RPA), which is required for multiple aspects of DNA metabolism, including DNA replication, DNA repair, and recombination . In wild-type cells, RPA was localized primarily to the nucleus but, in a KAP142 deletion strain, RPA was mislocalized to the cytoplasm and the strain was highly sensitive to bleomycin (BLM) . BLM causes DNA double-strand breaks and, in S . cerevisiae, the DNA damage is repaired predominantly by RPA-dependent homologous recombination . Therefore, our results indicate that in wild-type cells a critical portion of RPA was imported into the nucleus by Kap142p . Like several other import-related Kap-substrate complexes, the endogenous RPA-Kap142p complex was dissociated by RanGTP, but not by RanGDP . All three RPA genes are essential for viability, whereas KAP142 is not . Perhaps explaining this disparity, we observed an interaction between RPA and Kap95p in a strain lacking Kap142p . This interaction could provide a mechanism for import of RPA into the nucleus and cell viability in the absence of Kap142p . Together with published results (Kaffman, A., N.M . Rank, E.M . O'Neill, L.S . Huang, and E.K . O'Shea . 1998 . Nature . 396:482-486; Blondel, M., P.M . Alepuz, L.S . Huang, S . Shaham, G . Ammerer, and M . Peter . 1999 . Genes Dev . 13:2284-2300; DeVit, M.J., and M . Johnston . 1999 . Curr . Biol . 9:1231-1241; Mahanty, S.K., Y . Wang, F.W . Farley, and E.A . Elion . 1999 . Cell . 98:501-512) our data indicate that the karyopherin Kap142p is able to mediate nuclear import of one set of proteins and nuclear export of a different set of proteins. Pac Symp Biocomput . 2001;:422-33. Using graphical models and genomic expression data to statistically validate models of genetic regulatory networks; Hartemink AJ et al.; We propose a model-driven approach for analyzing genomic expression data that permits genetic regulatory networks to be represented in a biologically interpretable computational form . Our models permit latent variables capturing unobserved factors, describe arbitrarily complex (more than pair-wise) relationships at varying levels of refinement, and can be scored rigorously against observational data . The models that we use are based on Bayesian networks and their extensions . As a demonstration of this approach, we utilize 52 genomes worth of Affymetrix GeneChip expression data to correctly differentiate between alternative hypotheses of the galactose regulatory network in S . cerevisiae . When we extend the graph semantics to permit annotated edges, we are able to score models describing relationships at a finer degree of specification. Pac Symp Biocomput . 2001;:151-63. Promoter region-based classification of genes; Pavlidis P et al.; In this paper we consider the problem of extracting information from the upstream untranslated regions of genes to make predictions about their transcriptional regulation . We present a method for classifying genes based on motif-based hidden Markov models (HMMs) of their promoter regions . Sequence motifs discovered in yeast promoters are used to construct HMMs that include parameters describing the number and relative locations of motifs within each sequence . Each model provides a Fisher kernel for a support vector machine, which can be used to predict the classifications of unannotated promoters . We demonstrate this method on two classes of genes from the budding yeast, S . cerevisiae . Our results suggest that the additional sequence features captured by the HMM assist in correctly classifying promoters. Biotechnol Bioeng, 2001 May 5, 73(3), 238 - 45 The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium; Gorgens JF et al.; Five recombinant S . cerevisiae strains were cultivated under identical conditions to quantify the molecular basis of the metabolic burden of heterologous gene expression, and to evaluate mechanisms for the metabolic burden . Two recombinant S . cerevisiae strains, producing Trichoderma reesei xylanase II under control of either the PGK1 or ADH2 promoters, were compared quantitatively with three references strains, where either the heterologous xylanase II (XYN2) gene, or the heterologous gene and the promoter and terminator were omitted from the recombinant plasmid . Neither the replication of multiple copies of the 2-microm-based YEp352 plasmid nor the replication the foreign XYN2 gene represented a metabolic burden to the cell, as the growth of the host organism was not affected . The inclusion of a glycolytic promoter on the recombinant plasmid, however, reduced the maximum specific growth rate (12% to 15%), biomass yield on glucose (8% to 11%), and specific glucose consumption rate (6% to 10%) of the recombinant strains . The presence of the heterologous XYN2 gene on the recombinant plasmid caused a further reduction in the maximum specific growth rate (11% to 14%), biomass yield (4%), and specific glucose consumption rate (12%) of the host strain during active gene expression, which was dictated by the regulatory characteristics of the promoter utilized . The metabolic effect of foreign gene expression was disproportionally large, with respect to on the amount of heterologous protein produced . This was most likely due to an increased energetic demand for the expression of a foreign gene and/or a competition for limiting amounts of transcription or translation factors, biosynthetic precursors or metabolic energy . Biochemistry (Mosc), 2001 Feb, 66(2), 147 - 53 Two exopolyphosphatases of the cytosol of the yeast S . cerevisiae: comparative characteristics; Andreeva NA et al.; In cell-free extracts of the yeast Saccharomyces cerevisiae that had been transferred from phosphate-deficient (-P) medium to complete (+P) medium ("hypercompensation" conditions), the specific and the total polyphosphatase activities increased (by 50 and 60%, respectively) compared to the control that was transferred from (+P) medium to (+P) medium . Specific and total polyphosphatase activities under "hypercompensation" conditions increased by 25 and 43% in cytosol, by 33 and 100% in vacuoles, and by 50 and 50% in the total membrane fraction, respectively . In contrast, the polyphosphatase activity in the cell envelope somewhat decreased compared to the control . Under the growth conditions indicated above, a novel high molecular weight polyphosphatase was revealed in the cytosol fraction along with the previously studied 40-kD polyphosphatase . Unlike the 40-kD polyphosphatase, which is most active with tripolyphosphate, this novel enzyme has a molecular mass of more than 440 kD and is most active with high molecular weight polyphosphates . This polyphosphatase is insensitive to antibodies that suppress the activity of the 40-kD polyphosphatase of the cytosol . In a number of properties, the high molecular weight polyphosphatase of the cytosol resembles the polyphosphatase of vacuoles, but it differs from the polyphosphatases of nuclei and mitochondria of S . cerevisiae . The ratio of the low and high molecular weight polyphosphatases depends on the culture growth conditions . Under "hypercompensation" conditions, the total activity of the high molecular weight polyphosphatase in the cytosol is five times higher than that of the 40-kD polyphosphatase . During growth without re-inoculation, the 40-kD polyphosphatase is predominant in the cytosol; its total activity in dependence on the growth stage is 3.5-12.5 times higher than the activity of the high molecular weight form. Mol Gen Genet, 2001 Feb, 264(6), 852 - 9 Isolation and characterization of the fission yeast gene Sprpa12+ reveals that the conserved C-terminal zinc-finger region is dispensable for the function of its product; Imazawa Y et al.; RNA polymerase I of Saccharomyces cerevisiae contains a small subunit, A12.2, encoded by RPA12, that was previously shown to be involved in the assembly and/or stabilization of the largest subunit, A190, of RNA polymerase I . To examine whether an equivalent subunit is present in another eukaryotic RNA polymerase I, we have cloned a Schizosaccahromyces pombe cDNA that is able to complement the rpa12 mutation in S . cerevisiae . The gene, named Sprpa12+, encodes a polypeptide of 119 amino acids that shows 55% identity to S . cerevisiae A12 . 2 over its entire length, including two zinc-finger motifs . Disruption of the chromosomal Sprpa12+ gene shows that it is required for growth at higher temperatures but not at lower temperatures . Expression of Sprpa190+/nuc1+, which encodes the largest subunit of the S . pombe RNA polymerase I, from a multicopy plasmid can partially suppress the growth defect of the Sprpa12 disruptant at higher temperatures . These findings suggest that A12.2 subunit is functionally and structurally conserved between S . cerevisiae and S . pombe . Finally, the analysis of mutants suggests that SpRPA12 requires the zinc-finger domain in the N-terminal region but not the one in the C-terminal region for its function. Mol Biol Rep, 2000 Sep, 27(3), 149 - 56 Identification of a approximately 30S size non-ribosomal Saccharomyces cerevisiae RNA that is rapidly labeled on its 3' end by ATP or UTP; Sinha K et al.; Cell-free extracts prepared from S . cerevisiae cells were incubated in the presence of {alpha-32P}-labeled ATP, CTP, GTP or UTP . An RNA larger than ribosomal 25S RNA with an apparent size of approximately 30S was prominently labeled on its 3' end in the presence of ATP or UTP but not with CTP or GTP . This labeled RNA was not hybrid-selected by cloned yeast ribosomal DNA; in addition, this approximately 30S RNA was not cleaved by RNase H in the presence of complementary deoxyribooligonucleotides to rRNA . These two lines of evidence show that this approximately 30S RNA is not structurally related to ribosomal RNA gene repeat . The cell-free extracts prepared from yeast cells containing temperature-sensitive poly(A) polymerase adenylated this novel yeast RNA at restrictive temperature with efficiency similar to extracts prepared from wild-type yeast cells . These data show that the enzyme responsible for adenylation of this approximately 30S RNA is distinct from mRNA poly(A) polymerase . While the human SRP RNA 3' adenylating enzyme in the HeLa cell extract adenylated human SRP or Alu RNAs, the yeast adenylating enzyme did not adenylate the human SRP or Alu RNAs in vitro; these data indicate species specificity for this adenylating enzyme. Curr Opin Genet Dev, 2001 Apr, 11(2), 199 - 204 Chromosomal boundaries in S . cerevisiae; Bi X et al.; Chromatin boundary elements or insulators in metazoans delimit distinct chromosomal domains of gene expression . Recently, DNA sequences with properties similar to boundary elements were also discovered in Saccharomyces cerevisiae . These sequences block the spread of transcriptionally silent chromatin, the yeast equivalent of metazoan heterochromatin, and are referred to as 'heterochromatin barriers' . These barriers share no sequence homology but all consist of multiple binding sites for various regulatory proteins . Current data suggest that barriers may function in yeast by recruiting a protein complex that precludes nucleosome assembly and thereby disrupts a contiguous array of nucleosomes required for the spread of silent chromatin. Int J Biochem Cell Biol, 2001 Feb, 33(2), 175 - 80 Supercoiling unwinds two-micrometer plasmid yeast DNA at the origin of replication; Stoynov SS et al.; All studied origins of replication of DNA in Saccharomyces cerevisiae contain DNA unwinding elements . The introduction of unrestrained negative supercoiling leads to melting of the two DNA strands in DNA unwinding elements . To understand the mechanism of DNA replication it is important to know whether the most unstable region of DNA coincides with the origin of replication . Two-micrometer plasmid DNA from S . cerevisiae inserted in pBR322 was investigated by cleaving with snake venom phosphodiesterase . Its single-strand endonucleolytic activity allows cutting of negatively supercoiled DNA in the DNA unwinding elements . The sites of the venom phosphodiesterase hydrolysis were mapped by restriction enzymes . This study shows that the unwinding of the two-micrometers plasmid DNA of S . cerevisiae takes place only in the origin of replication as a result of unrestrained negative supercoiling. Mol Cell, 2001 Feb, 7(2), 309 - 18 Nhp6, an HMG1 protein, functions in SNR6 transcription by RNA polymerase III in S . cerevisiae; Kruppa M et al.; Nhp6A and Nhp6B are HMG1-like proteins required for the growth of S . cerevisiae at elevated temperatures . We show that the conditional lethality of an nhp6 strain results from defective transcription of SNR6 (U6 snRNA) by RNA polymerase III . Overexpression of U6 snRNA or Brf1, a limiting component of TFIIIB, and an activating mutation (PCF1-1) in TFIIIC were each found to suppress the nhp6 growth defect . Additionally, U6 snRNA levels, which are reduced over 10-fold in nhp6 cells at 37 degrees C, were restored by Brf1 overexpression and by PCF1-1 . Nhp6A protein specifically enhanced TFIIIC-dependent, but not TATA box-dependent, SNR6 transcription in vitro by facilitating TFIIIC binding to the SNR6 promoter . Thus, Nhp6 has a direct role in transcription complex assembly at SNR6. Mol Cell Biol, 2001 Mar, 21(6), 1953 - 61 A truncated form of the human CAF-1 p150 subunit impairs the maintenance of transcriptional gene silencing in mammalian cells; Tchenio T et al.; Chromatin assembly factor 1 (CAF-1) is a protein complex formed of three subunits, p150, p60, and p48, conserved from the yeast Saccharomyces cerevisiae to humans, which can promote nucleosome assembly onto newly replicated DNA . In S . cerevisiae, deletion of the genes encoding any of the three CAF-1 subunits (cacDelta mutants), although nonlethal, results in a silencing defect of genes packaged into heterochromatin . Here we report on a mammalian cell model that we devised to monitor gene silencing and its reversal in a quantitative manner . This model relies on the use of a cell line stably transfected with a reporter gene in a silenced state . Reversal of reporter gene silencing was achieved upon treatment of the cells with 5-azacytidine, which resulted in the demethylation of the reporter gene copies . We show that expression of a cDNA for the human p150 CAF-1 subunit harboring 5' truncations, but not that of a cDNA encoding the full-length p150 CAF-1 subunit, increases by more than 500-fold the frequency at which transcriptional silencing of the reporter gene copies is reversed in these cells . Reversal of gene silencing is dependent upon expression of a truncated protein, possibly acting as a dominant negative mutant of the wild-type CAF-1, is associated with alterations in chromatin structure as measured by an endonuclease sensitivity assay and is not associated with detectable changes in the methylation status of the silenced genes . These results suggest that the role of CAF-1 in the epigenetic control of gene expression has been conserved between yeast and mammals, despite the lack of DNA methylation in yeast chromatin. Genetics, 2001 Mar, 157(3), 1067 - 75 Analysis of the pdx-1 (snz-1/sno-1) region of the Neurospora crassa genome: correlation of pyridoxine-requiring phenotypes with mutations in two structural genes; Bean LE et al.; We report the analysis of a 36-kbp region of the Neurospora crassa genome, which contains homologs of two closely linked stationary phase genes, SNZ1 and SNO1, from Saccharomyces cerevisiae . Homologs of SNZ1 encode extremely highly conserved proteins that have been implicated in pyridoxine (vitamin B6) metabolism in the filamentous fungi Cercospora nicotianae and in Aspergillus nidulans . In N . crassa, SNZ and SNO homologs map to the region occupied by pdx-1 (pyridoxine requiring), a gene that has been known for several decades, but which was not sequenced previously . In this study, pyridoxine-requiring mutants of N . crassa were found to possess mutations that disrupt conserved regions in either the SNZ or SNO homolog . Previously, nearly all of these mutants were classified as pdx-1 . However, one mutant with a disrupted SNO homolog was at one time designated pdx-2 . It now appears appropriate to reserve the pdx-1 designation for the N . crassa SNZ homolog and pdx-2 for the SNO homolog . We further report annotation of the entire 36,030-bp region, which contains at least 12 protein coding genes, supporting a previous conclusion of high gene densities (12,000-13,000 total genes) for N . crassa . Among genes in this region other than SNZ and SNO homologs, there was no evidence of shared function . Four of the genes in this region appear to have been lost from the S . cerevisiae lineage. Genetics, 2001 Mar, 157(3), 969 - 77 Characterization of the Aspergillus nidulans septin (asp) gene family; Momany M et al.; Members of the septin gene family are involved in cytokinesis and the organization of new growth in organisms as diverse as yeast, fruit fly, worm, mouse, and human . Five septin genes have been cloned and sequenced from the model filamentous fungus A . nidulans . As expected, the A . nidulans septins contain the highly conserved GTP binding and coiled-coil domains seen in other septins . On the basis of hybridization of clones to a chromosome-specific library and correlation with an A . nidulans physical map, the septins are not clustered but are scattered throughout the genome . In phylogenetic analysis most fungal septins could be grouped with one of the prototypical S . cerevisiae septins, Cdc3, Cdc10, Cdc11, and Cdc12 . Intron-exon structure was conserved within septin classes . The results of this study suggest that most fungal septins belong to one of four orthologous classes. Appl Microbiol Biotechnol, 2001 Jan, 55(1), 108 - 11 Genetic reidentification of the pectinolytic yeast strain SCPP as Saccharomyces bayanus var . uvarum; Naumov GI et al.; Using genetic hybridization analysis, pulsed-field gel electrophoresis of chromosomal DNA and PCR/RFLP analysis of the MET2 gene, we reidentified 11 Champagne yeast strains . Two of them, SCPP and SC4, were found to belong to Saccharomyces bayanus var . uvarum and the remaining strains to S . cerevisiae . Strain Prikl Biokhim Mikrobiol, 2001 Jan-Feb, 37(1), 90 - 5 {Comparative study of some parameters of energy metabolism in two strains of Saccharomyces cerevisiae}; Aliverdieva DA; A comparative study of energy metabolism in two strains Saccharomyces cerevisiae (the initial strain N 73 and laser-irradiated mutant strain Y-503) was performed . In all growth phases, the rates of oxygen consumption by cells of Y-503 were higher than in the initial strain . The maximum (threefold) increase in the rate of oxygen consumption was observed in the linear phase . The effects of respiratory chain inhibitors rotenone, antimycin A, and cyanide on cellular and mitochondrial respiration were identical . There are two sites of energy coupling in the respiratory chain of mitochondria in S . cerevisiae N 73 and Y-503, and electron flow mainly is mainly mediated by cytochrome oxidase . The data suggest that a higher respiratory activity of S . cerevisiae Y-503 cells in comparison with N 73 is associated with greater amounts of mitochondria and total surface area of coupling mitochondrial membranes, which appears to be a factor contributing to a high physiological and biochemical activity of this strain. Curr Biol, 2001 Jan 23, 11(2), 125 - 9 SGS1 is required for telomere elongation in the absence of telomerase; Huang P et al.; In S . cerevisiae, mutations in genes that encode telomerase components, such as the genes EST1, EST2, EST3, and TLC1, result in the loss of telomerase activity in vivo . Two telomerase-independent mechanisms can overcome the resulting senescence . Type I survival is characterized by amplification of the subtelomeric Y' elements with a short telomere repeat tract at the terminus . Type II survivors arise through the abrupt addition of long tracts of telomere repeats . Both mechanisms are dependent on RAD52 and on either RAD50 or RAD51 . We show here that the telomere elongation pathway in yeast (type II) is dependent on SGS1, the yeast homolog of the gene products of Werner's (WRN) and Bloom's (BLM) syndromes . Survival in the absence of SGS1 and EST2 is dependent upon RAD52 and RAD51 but not RAD50 . We propose that the RecQ family helicases are required for processing a DNA structure specific to eroding telomeres. FEBS Lett, 2001 Feb 23, 491(1-2), 63 - 6 In vivo glycosylation suppresses the aggregation of amyloidogenic hen egg white lysozymes expressed in yeast; Song Y et al.; The mutant hen egg white lysozymes Ile55Thr and Asp66His, corresponding to human amyloidogenic mutant lysozymes Ile56Thr and Asp67His, respectively, were secreted in Saccharomyces cerevisiae . The amyloidogenic mutants (I55T and D66H) of hen egg white lysozymes were remarkably less soluble than that of the wild-type protein . To enhance the secretion of these mutants, we constructed the glycosylated amyloidogenic lysozymes (I55T/G49N and D66H/G49N) having the N-glycosylation signal sequence (Asn-X-Ser) by the substitution of glycine with asparagine at position 49 . The secretion of these glycosylated mutant proteins is greatly increased in S . cerevisiae, compared with that of non-glycosylated type . Both the glycosylated mutants retained about 40% enzymatic activity when incubated at pH 7.4 for 1 h at the physiological temperature of 37 degrees C whereas the non-glycosylated proteins eventually lost all activity under these conditions . These results suggest that the glycosylated chains could mask the beta-strand of amyloidogenic lysozymes from the intermolecular cross-beta-sheet association, thus improving the solubility of amyloidogenic lysozymes. EMBO J, 2001 Jan 15, 20(1-2), 55 - 64 A plasma membrane-type Ca(2+)-ATPase co-localizes with a vacuolar H(+)-pyrophosphatase to acidocalcisomes of Toxoplasma gondii; Luo S et al.; Ca(2+)-ATPases are likely to play critical roles in the biochemistry of Toxoplasma gondii, since these protozoa are obligate intracellular parasites and the Ca(2+) concentration in their intracellular location is three orders of magnitude lower than in the extracellular medium . Here, we report the cloning and sequencing of a gene encoding a plasma membrane-type Ca(2+)-ATPase (PMCA) of T.gondii (TgA1) . The predicted protein (TgA1) exhibits 32-36% identity to vacuolar Ca(2+)-ATPases of Trypanosoma cruzi, Saccharomyces cerevisiae, Entamoeba histolytica and Dictyostelium discoideum . Sequencing of both cDNA and genomic DNA from T.gondii indicated that TgA1 contains two introns near the C-terminus . A hydropathy profile of the protein suggests 10 transmembrane domains . TgA1 suppresses the Ca(2+) hypersensitivity of a mutant of S.cerevisiae that has a defect in vacuolar Ca(2+) accumulation . Indirect immunofluorescence and immunoelectron microscopy analysis indicate that TgA1 localizes to the plasma membrane and co-localizes with the vacuolar H(+)-pyrophosphatase to intracellular vacuoles identified morphologically and by X-ray microanalysis as the acidocalcisomes . This vacuolar-type Ca(2+)-ATPase could play an important role in Ca(2+) homeostasis in T.gondii. Yeast, 2001 Mar 15, 18(4), 335 - 42 Role of adenosine kinase in Saccharomyces cerevisiae: identification of the ADO1 gene and study of the mutant phenotypes; Lecoq K et al.; Sequencing of the Saccharomyces cerevisiae genome revealed an open reading frame (YJR105w) encoding a putative protein highly similar to adenosine kinases from other species . Disruption of this gene (renamed ADO1) affected utilization of S-adenosyl methionine (AdoMet) as a purine source and resulted in a severe reduction of adenosine kinase activity in crude extracts . Furthermore, knock-out of ADO1 led to adenosine excretion in the medium and resistance to the toxic adenosine analogue cordycepin . From these data we conclude that ADO1 encodes yeast adenosine kinase . We also show that ADO1 does not play a major role in adenine utilization in yeast and we propose that the physiological role of adenosine kinase in S . cerevisiae could primarily be to recycle adenosine produced by the methyl cycle . Nucleic Acids Res, 2001 Mar 1, 29(5), 1054 - 60 Determination of the binding constants of the centromere protein Cbf1 to all 16 centromere DNAs of Saccharomyces cerevisiae; Wieland G et al.; Cbf1p is a Saccharomyces cerevisiae chromatin protein belonging to the basic region helix-loop-helix leucine zipper (bHLHzip) family of DNA binding proteins . Cbf1p binds to a conserved element in the 5'-flanking region of methionine biosynthetic genes and to centromere DNA element I (CDEI) of S.cerevisiae centromeric DNA . We have determined the apparent equilibrium dissociation constants of Cbf1p binding to all 16 CDEI DNAs in gel retardation assays . Binding constants of full-length Cbf1p vary between 1.7 and 3.8 nM . However, the dissociation constants of a Cbf1p deletion variant that has been shown to be fully sufficient for Cbf1p function in vivo vary in a range between 3.2 and 12 nM . In addition, native polyacrylamide gel electrophoresis revealed distinct changes in the 3D structure of the Cbf1p/CEN complexes . We also show that the previously reported DNA binding stimulation activity of the centromere protein p64 functions on both the Cbf1 full-length protein and a deletion variant containing only the bHLHzip domain of Cbf1p . Our results suggest that centromeric DNA outside the consensus CDEI sequence and interaction of Cbf1p with adjacent centromere proteins contribute to the complex formation between Cbf1p and CEN DNA. Yao Xue Xue Bao, 2000 Nov, 35(11), 860 - 3 {Antifungal effect of three natural products on the genetic substance of Saccharomyces cerevisiae GL7 and Prototheca wickerhamii}; Wang LD et al.; AIM: To investigate the antifungal effect of three natural products on the genetic substance of Saccharomyces cerevisiae GL7 and Prototheca wickerhamii . METHODS: The normal and treated cells were observed by confocal laser scanning microscope (CLSM) and image analysis to quantitatively described the cell morphology, area, DNA and RNA content . RESULTS: The morphology, area, DNA and RNA contents were changed greatly in the treated cells . CONCLUSION: Solasodine, 4'-hydroxy-3, 5-dimethoxystilbene and dictamnine directly or indirectly interfered the synthesis and function of genetic substance in S . cerevisiae and P . wickerhamii. J Chromatogr A, 2001 Jan 26, 908(1-2), 243 - 50 Continuous separation of green fluorescent protein by annular chromatography; Uretschlager A et al.; The concept of annular chromatography was tested by separation of a real protein solution used in biotechnology . Green fluorescent protein was expressed in S . cerevisiae and the extract was continuously separated by a pressurized annular chromatograph packed with a Superdex 200 prep grade size-exclusion chromatography medium . Purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and fluorescent intensity . The continuous mode was compared to batchwise operation . Under the assumption that equivalent fractions are collected, both modes are comparable. Antonie Van Leeuwenhoek, 2000 Aug, 78(2), 187 - 94 Pseudohyphal growth is induced in Saccharomyces cerevisiae by a combination of stress and cAMP signalling; Zaragoza O et al.; In Saccharomyces cerevisiae pseudohyphae formation may be triggered by nitrogen deprivation and is stimulated by cAMP . It was observed that even in a medium with an adequate nitrogen supply, cAMP can induce pseudohyphal growth when S . cerevisiae uses ethanol as carbon source . This led us to investigate the effects of the carbon source and of a variety of stresses on yeast morphology . Pseudohyphae formation and invasive growth were observed in a rich medium (YP) with poor carbon sources such as lactate or ethanol . External cAMP was required for the morphogenetic transition in one genetic background, but was dispensable in strain sigma 1278b which has been shown to have an overactive Ras2/cAMP pathway . Pseudohyphal growth and invasiveness also took place in YPD plates when the yeast was subjected to different stresses: a mild heat-stress (37 degrees C), an osmotic stress (1 m NACl), or addition of compounds which affect the lipid bilayer organization of the cell membrane (aliphatic alcohols at 2%) or alter the glucan structure of the cell wall (Congo red) . We conclude that pseudohyphal growth is a physiological response not only to starvation but also to a stressful environment; it appears to require the coordinate action of a MAP kinase cascade and a cAMP-dependent pathway. Mol Pharmacol, 2001 Mar, 59(3), 470 - 4 Two nuclear proteins, Cin5 and Ydr259c, confer resistance to cisplatin in Saccharomyces cerevisiae; Furuchi T et al.; In an attempt to identify genes that can confer resistance to cisplatin, we introduced a yeast genomic library into Saccharomyces cerevisiae and selected for transformants that grew in the presence of a normally toxic concentration of cisplatin . Plasmids were rescued from the transformants and were analyzed for the presence of individual open reading frames that conferred resistance to cisplatin . We isolated two genes, CIN5 and YDR259c, that increased resistance to cisplatin when overexpressed in Saccharomyces cerevisiae . These genes encoded two proteins, Cin5 and Ydr259c, that were homologous to yAP-1, a basic leucine zipper transcriptional factor that is known to mediate cellular resistance to various toxic agents . The two proteins exhibited stronger homology to each other (33.2% identity, 49.2% similarity) than to all other gene products in S . cerevisiae . Overexpression of each of these proteins also conferred resistance to two DNA-alkylating agents, methylmethanesulfonate and mitomycin C . An experiment with fusion proteins with green fluorescent protein revealed that Cin5 and Ydr259c were localized constitutively in the nuclei of yeast cells . Our results suggest that Cin5 and Ydr259c might be involved in pleiotropic drug-resistance and might protect yeast against the toxicity of cisplatin and other alkylating agents via a single mechanism . These two nuclear proteins might act as transcriptional factors, regulating the expression of certain genes that confer resistance to DNA-alkylating agents. ORL J Otorhinolaryngol Relat Spec, 2001 Jan-Feb, 63(1), 19 - 24 No increased serum levels of antifood antibodies in patients with Ménière's disease; Boulassel MR et al.; The etiology of Meniere's disease (MD) is still unknown, but it is likely to be multifactorial, one of the factors being an immunological causation . Antifood allergens as well as anti-baker's yeast antibodies are humoral factors that may be linked with allergenic disorders and other autoimmune conditions . To determine their possible role in MD activity, we investigated 29 MD sera for the presence of antibodies against gliadin, beta-lactoglobulin, albumin, ovalbumin, soya, and Dermatophagoides pteronyssinus and Saccharomyces cerevisiae strains using an ELISA technique . The patients were compared with 29 healthy individuals matched for sex and age . A serum was regarded as positive if the absorbance was two standard deviations higher than values obtained with sera from healthy subjects . Historical data, including factors which the patients believed to provoke their Meniere's symptoms, were obtained from patients' questionnaires . MD patients showed no significant symptoms of allergenic disorders suggesting allergies when compared to controls (p > 0.05) . IgG and IgA antibody levels were not significantly raised in MD patients as compared with healthy controls (p > 0.05) for gliadin, beta-lactoglobulin, soya, albumin, ovalbumin, and D . pteronyssinus and S . cerevisiae strains . These data do not convincingly support a hypothesis of increased serum levels of antifood antibodies in patients with MD, as very few patients were antibody positive . Mol Cell, 2001 Jan, 7(1), 21 - 30 Nucleosomes positioned by ORC facilitate the initiation of DNA replication; Lipford JR et al.; The packaging of eukaryotic DNA into nucleosomes is a critical regulator of nuclear events . To address the interplay between chromatin and replication initiation, we have assessed the determinants and function of the nucleosomal configuration of S . cerevisiae replication origins . Using in vitro and in vivo assays, we demonstrate that the yeast initiator, the origin recognition complex (ORC), is required to maintain the nucleosomal configuration adjacent to origins . Disruption of the ORC-directed nucleosomal arrangement at an origin interferes with initiation of replication, but does not alter the association of ORC with the origin . Instead, the nucleosomes positioned by ORC are important for prereplicative complex formation . These findings suggest that origin-proximal nucleosomes facilitate replication initiation, and that local chromatin structure affects origin function. Microsc Res Tech, 2000 Dec 15, 51(6), 547 - 62 The yeast endocytic membrane transport system; Munn AL; Progress has been made recently in visualizing the structures and organelles responsible for endocytic membrane traffic from the cell surface to the lysosome-like vacuole in Saccharomyces cerevisiae . This, together with the recent discovery of several new membrane trafficking pathways connecting these organelles, has led to a quantum leap in our understanding of the S . cerevisiae endocytic pathway . We now know that although the cortical actin cytoskeleton is required for the internalization step of endocytosis, the internalization event occurs at furrow-like invaginations of the plasma membrane, which are distinct from cortical actin patches . Internalized material is taken into the cell in the form of small (30-50 nm diameter) vesicles and delivered to tubulo-vesicular early endosomes at the cell periphery . Subsequently, the internalized material arrives in multivesicular late endosomes adjacent to the vacuole . Recent microscopy evidence suggests that transfer from late endosomes to the vacuole may involve direct fusion of late endosomes with the vacuole . The visualization of the S . cerevisiae endocytic pathway has revealed similarities to endocytic pathways visualized in higher eukaryotes . Microsc Res Tech, 2000 Dec 15, 51(6), 530 - 46 Functional morphology of the secretory pathway organelles in yeast; Vorisek J; The glycoprotein secretory pathway of yeast serves mainly for cell surface growth and cell division . It involves a centrifugal transport of transit macromolecules among organelles, whose membranes contain resident proteins needed for driving the transport . These resident membrane proteins return by retrograde vesicular transport . Apart from this, the pathway involves endocytosis . The model yeast Saccharomyces cerevisiae and vertebrate cells were found to contain very similar gene products regulating the molecular mechanism of glycoprotein transport, and the cellular mechanism of their secretion pathways was therefore also presumed to be identical . Biochemists have postulated that, in S . cerevisiae, the translocation of peptides through the endoplasmic reticulum membranes into the lumen of ER cisternae and the core glycosylation is followed by a vector-mediated transport into the functional cascade of the Golgi system cisternae and between them . This is the site of maturation and sorting of glycoproteins, before the ultimate transport by other vectors involving either secretion from the cells (exocytosis across the plasmalemma into the cell wall) or transport into the lysosome-like vacuole via a prevacuolar compartment, which serves at the same time as a primary endosome . The established cellular model of secretion deals with budding yeast; interphase yeast cells, in which the secretion is limited and which predominate in exponential cultures, have not been taken into consideration . The quality of organelle imaging in S . cerevisiae ultra-thin sections depends on the fixation technique used and on specimen contrasting by metals . The results achieved by combinations of different techniques differ mostly in the imaging of bilayers of membrane interfaces and the transparence of the matrix phase . Fixation procedures are decisive for the results of topochemical localisations of cellular antigenic components or enzyme activities, which form the basis of the following survey of functional morphology of organelles involved in the yeast secretory pathway . The existing results of these studies do not confirm all aspects of the vertebrate model of the Golgi apparatus proposed by molecular geneticists to hold for S . cerevisiae, and alternative models of the cellular mechanism of secretion in this yeast are, therefore, also discussed . Yeast, 2001 Jan 30, 18(2), 137 - 50 Structural conformers produced during malaria vaccine production in yeast; Stowers AW et al.; A recombinant protein expression system based on Saccharomyces cerevisiae has been used to express malarial vaccine candidate antigens . The antigens so produced have been used in three Phase 1 clinical trials and numerous preclinical non-human primate trials . Further Phase I trials are planned using these candidate vaccine antigens . These molecules were identified as attractive candidates for antimalarial vaccines, as they are all surface-exposed at some stage in the parasite's life cycle . They all share an unusual structural feature: epidermal growth factor (EGF)-like motifs . When these proteins are expressed in our S . cerevisiae expression system, they are produced as a series of stable structural conformers, each with a different disulphide bonding pattern . This leads to both biochemical and, more importantly, antigenic differences between the conformers (e.g . presence or absence of an antibody B cell epitope) . These findings have important ramifications for other EGF-domain-containing proteins expressed in S . cerevisiae, or for proteins which contain other cysteine-folding motifs not normally expressed by this organism, both for vaccine production or for research/reagent purposes . Mol Biochem Parasitol, 2001 Jan 15, 112(1), 19 - 28 Spontaneous dimerization and leucine-zipper induced activation of the recombinant catalytic domain of a new adenylyl cyclase of Trypanosoma brucei, GRESAG4.4B; Naula C et al.; In this study, we describe the isolation and characterization of a new adenylyl cyclase from Trypanosoma brucei and its activation by dimerization of the catalytic domain . In agreement with the current nomenclature of trypanosomal adenylyl cyclases, this new gene is termed GRESAG4.4B . The complete ORF of the GRESAG4.4B gene encodes a protein of 1291 amino acids . Its predicted protein structure is consistent with the structure of other trypanosomal cyclases, and with the cyclases of L . donovani . GRESAG 4.4B is constitutively expressed during the life cycle of trypanosomes . GRESAG4.4B is a member of a gene family, which contains at least six members, which are all clustered on chromosome IV . The catalytic domain of GRESAG4.4B is able to dimerize spontaneously . However, these spontaneously formed, stable dimers only show minimal enzymatic activity . The addition of a leucine zipper (LZ) derived from the S . cerevisiae GCN 4 gene to the N-terminus of the catalytic domain of GRESAG4.4B strongly activated its enzymatic activity . The LZ appears to enforce a distinct conformation of the dimer, which leads to an increased enzymatic activity, and thus may mimic the effect of ligand-induced dimerization of adenylyl cyclase in vivo. J Biotechnol, 2001 Jan 23, 85(1), 41 - 8 Enhanced production of anticoagulant hirudin in recombinant Saccharomyces cerevisiae by chromosomal delta-integration; Kim MD et al.; Recombinant Saccharomyces cerevisiae strains were developed to overproduce an anticoagulant hirudin . The delta-sequences of the yeast retrotransposon Ty1 and URA3 were used as target sites for a hirudin expression cassette . High copy-number transformants were successfully selected using a dominant selection antibiotic, G418 . The copy numbers of the hirudin expression cassette integrated into delta-sequences of the yeast chromosome ranged from five to ten copies per cell . Production of hirudin in the delta-integrated recombinant S . cerevisiae system increased over two-fold compared with the YEp-based episomal hirudin expression system . A linear relationship between the copy number of the hirudin expression cassette and hirudin expression level was observed up to 10 copies . The hirudin expression cassettes integrated into the yeast chromosome were stably maintained in non-selective culture conditions. Mol Biochem Parasitol, 2000 Dec, 111(2), 309 - 17 Genetic manipulation indicates that ARD1 is an essential N(infinity)-acetyltransferase in Trypanosoma brucei; Ingram AK et al.; N(infinity)-acetylation, the most common protein modification, involves the transfer of an acetyl group from acetyl-coenzyme A to the N-terminus of a protein or peptide . The major N(infinity)-acetyltransferase in Saccharomyces cerevisiae is the ARDI-NATI complex . To investigate N(infinity) -acetylation in Trypanosoma brucei we have cloned and characterised genes encoding putative homologues of ARD1 and NAT1 . Both genes are single copy and ARD1, the putative catalytic component, is expressed in both bloodstream-form and insect-stage cells . In either of these life-cycle stages, disruption of both ARD1 alleles was only possible when another copy was generated via gene duplication or when ARD1 was expressed from elsewhere in the genome . These genetic manipulations demonstrate that, unlike the situation in S . cerevisiae, ARD1 is an essential gene in T . brucei . We propose that protein modification by ARD1 is essential for viability in mammalian and insect-stage T . brucei cells. Biochem Biophys Res Commun, 2001 Feb 9, 280(5), 1292 - 9 Mutations in the yeast Nhp6 protein can differentially affect its in vivo functions; Kruppa M et al.; Nhp6A and Nhp6B from S . cerevisiae are required for viability at 38 degrees C because they are involved in transcription of SNR6 by RNA polymerase III . Nhp6A also represses transcription of NHP6B by RNA polymerase II . Nhp6 belongs to the HMG1 family, defined by an 80 amino acid DNA binding domain, which includes six highly conserved residues . These amino acids were mutated in Nhp6A and their affects on Nhp6 function were assessed in vivo . Surprisingly, most of the changes allowed Nhp6A to function normally in supporting growth at 38 degrees C . However, six mutants had differential effects on in vivo function . Finally, two of the mutant proteins that did not restore Nhp6A function in vivo were shown to bind and bend DNA in vitro as well as wild type . Together, these results suggest that Nhp6 interacts with another protein(s) to carry out some of its biological functions and that this interaction might differ at promoters transcribed by RNA polymerase II versus RNA polymerase III. Protein Expr Purif, 2001 Feb, 21(1), 105 - 14 Expression of the Aspergillus aculeatus endo-beta-1,4-mannanase encoding gene (man1) in Saccharomyces cerevisiae and characterization of the recombinant enzyme; Setati ME et al.; The endo-beta-1,4-mannanase encoding gene man1 of Aspergillus aculeatus MRC11624 was amplified from mRNA by polymerase chain reaction using sequence-specific primers designed from the published sequence of man1 from A . aculeatus KSM510 . The amplified fragment was cloned and expressed in Saccharomyces cerevisiae under the gene regulation of the alcohol dehydrogenase (ADH2(PT)) and phosphoglycerate kinase (PGK1(PT)) promoters and terminators, respectively . The man1 gene product was designated Man5A . Subsequently, the FUR1 gene of the recombinant yeast strains was disrupted to create autoselective strains: S . cerevisiae Man5ADH2 and S . cerevisiae Man5PGK1 . The strains secreted 521 nkat/ml and 379 nkat/ml of active Man5A after 96 h of growth in a complex medium . These levels were equivalent to 118 and 86 mg/l of Man5A protein produced, respectively . The properties of the native and recombinant Man5A were investigated and found to be similar . The apparent molecular mass of the recombinant enzyme was 50 kDa compared to 45 kDa of the native enzyme due to glycosylation . The determined K(m) and V(max) values were 0.3 mg/ml and 82 micromol/min/mg for the recombinant and 0.15 mg/ml and 180 micromol/min/mg for the native Man5A, respectively . The maximum pH and thermal stability were observed within the range of pH 4-6 and 50 degrees C and below . The pH and temperature optima and stability were relatively similar for recombinant and native Man5A . Hydrolysis of an unbranched beta-1,4-linked mannan polymer released mannose, mannobiose, and mannotriose as the main products . Protein Expr Purif, 2001 Feb, 21(1), 1 - 7 Transmembrane-sequence-dependent overexpression and secretion of glycoproteins in Saccharomyces cerevisiae; Schuster M et al.; Protein expression using the secretory pathway in Saccharomyces cerevisiae can lead to high amounts of overexpressed and secreted proteins in culture supernatants in a short period of time . These post-translational modified expression products can be purified up to >90% in a single step . The overexpression and secretion of the transmembrane glycoprotein signaling lymphocytic activation molecule (SLAM) was studied . SLAM belongs to the immunoglobulin superfamily and its engagement results in T-cell expansion and INF-gamma production . The molecule is composed of an extracellular, a single-span transmembrane and a cytoplasmatic domain . The extracellular part may be relevant for stimulation studies in vitro since SLAM is a high-affinity self-ligand . Therefore several fragments of this region have been expressed as Flag-fusions in S . cerevisiae: a full-length fragment containing the transmembrane region and the autologous signal sequence, another without the transmembrane region, and two fragments without the autologous signal sequence with and without the transmembrane region . By molecular cloning, the different deletion mutants of the cDNA encoding the full-length construct have been inserted in a yeast episomal plasmid . Upstream of the cDNA, the alpha-leader sequence of a yeast mating pheromone has been cloned to direct the fusion proteins into the secretory protein maturation pathway . All four fragments were expressed but yield, location, and maturation were highly influenced by the transmembrane domain and the autologous signal sequence . Only the fragment without autologous signal sequence and transmembrane domain could be efficiently secreted . High-mannose glycosylation was analyzed by lectin mapping and digestion with specific glycosidases . After enzyme treatment, a single band product with the theoretical size could be detected and identified as SLAM by a specific monoclonal antibody . The fusion protein concentration in the supernatant was 30 microg/ml . The affinity-purified and deglycosylated protein is a tool for further biochemical and biophysical characterization of SLAM . Exp Parasitol, 2000 Dec, 96(4), 195 - 201 Cryptosporidium parvum: functional complementation of a parasite transcriptional coactivator CpMBF1 in yeast; Zhu G et al.; We report here the identification of a novel multiprotein bridging factor type 1 from the apicomplexan Cryptosporidium parvum (CpMBF1), one of the opportunistic pathogens in AIDS patients . In slime molds, insects, and humans, MBF1-regulated systems have been associated with cell differentiation, which indicates that CpMBF1 could be responsible for the activation of similar systems in C . parvum during its complex life cycle . Because of the difficulties and high cost in obtaining sufficient and purified C . parvum material for molecular and biochemical analyses, well-characterized yeast genetic systems may be useful for investigating the functions of C . parvum genes . In this study, the function of CpMBF1 as an interconnecting element between a DNA-binding regulator and TATA-box-binding protein (TBP) was confirmed using a yeast complementation assay . Under conditions of histidine starvation, an MBF1-deficient strain of Saccharomyces cerevisiae was unable to activate the HIS3 gene, which encodes imidazoleglycerol-phosphate dehydratase (IGPDH), and thus became sensitive to 3-amino triazole, an inhibitor of this enzyme . Upon introduction of parasite CpMBF1 into S . cerevisiae, 3-amino triazole resistance of the MBF1-deficient strain was restored to wild-type levels, and Northern blot analysis revealed that CpMBF1 was able to activate HIS3 transcription in response to histidine starvation . Exp Cell Res, 2001 Feb 1, 263(1), 43 - 54 Catalytic activity of protein kinase CK1 delta (casein kinase 1delta) is essential for its normal subcellular localization; Milne DM et al.; Mammalian casein kinase 1delta (CK1delta) is a homologue of the S . cerevisiae Hrr25p protein kinase . Hrr25p is involved in regulating diverse events including vesicular trafficking, gene expression, DNA repair, and chromosome segregation . In contrast to Hrr25p, little is known about the function, regulation, or subcellular localization of CK1delta . In the present study, we show that CK1delta in mammalian cells is mainly cytoplasmic and enriched within the Golgi and/or ER-Golgi transport vesicles, consistent with a role in vesicular trafficking . Transient expression of green fluorescent protein (GFP)- or FLAG peptide-tagged CK1delta showed localization similar to that of the endogenous CK1delta . GFP-CK1delta was also enriched at the centrosomes in interphase cells . Strikingly, two inactive mutant CK1delta proteins (K38M and T176I) showed almost exclusive nuclear staining, suggesting that protein kinase activity is required for normal localization of CK1delta and prevention of nuclear accumulation . The nuclear export inhibitor leptomycin B promoted nuclear enrichment of CK1delta indicating that nuclear localization of CK1delta occurs physiologically . Both endogenous CK1delta and GFP-CK1delta are enriched on the spindle poles in mitotic cells, consistent with a role in regulating spindle formation . Localization is a property of the protein kinase domain and is independent of the C-terminal noncatalytic domain . These data are consistent with roles for CK1delta in mammalian cells analogous to those of its yeast counterparts. Science, 2001 Jan 26, 291(5504), 646 - 50 DNA replication-independent silencing in S . cerevisiae; Kirchmaier AL et al.; In Saccharomyces cerevisiae, the silent mating loci are repressed by their assembly into heterochromatin . The formation of this heterochromatin requires a cell cycle event that occurs between early S phase and G(2)/M phase, which has been widely assumed to be DNA replication . To determine whether DNA replication through a silent mating-type locus, HMRa, is required for silencing to be established, we monitored heterochromatin formation at HMRa on a chromosome and on a nonreplicating extrachromosomal cassette as cells passed through S phase . Cells that passed through S phase established silencing at both the chromosomal HMRa locus and the extrachromosomal HMRa locus with equal efficiency . Thus, in contrast to the prevailing view, the establishment of silencing occurred in the absence of passage of the DNA replication fork through or near the HMR locus, but retained a cell cycle dependence. Microbiology, 2001 Feb, 147(Pt 2), 411 - 7 The glyceraldehyde-3-phosphate dehydrogenase polypeptides encoded by the Saccharomyces cerevisiae TDH1, TDH2 and TDH3 genes are also cell wall proteins; Delgado ML et al.; The authors show that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Saccharomyces cerevisiae, previously thought to be restricted to the cell interior, is also present in the cell wall . GAPDH activity, proportional to cell number and time of incubation, was detected in intact wild-type yeast cells . Intact cells of yeast strains containing insertion mutations in each of the three structural TDH genes (tdh1, tdh2 and tdh3) and double mutants (tdh1 tdh2 and tdh1 tdh3) also displayed a cell-wall-associated GAPDH activity, in the range of parental wild-type cells, although with significant differences among strains . A cell wall location of GAPDH was further confirmed in wild-type and tdh mutants by indirect immunofluorescence and flow cytometry analysis with a polyclonal antibody against S . cerevisiae GAPDH . By immunoelectron microscopy, the GAPDH protein was detected at the outer surface of the cell wall of wild-type cells, as well as in the cytoplasm . Western immunoblot analysis of cell wall extracts and cytosol showed that Tdh2 and Tdh3 polypeptides are present in the cell wall, as well as in the cytosol, of exponentially growing cells . Tdh1 is only detected in stationary-phase cells, again in both cytosol and cell wall extracts . The results incorporate the GAPDH of S . cerevisiae, encoded by TDH1-3, into the newly emerging family of multifunctional cell-wall-associated GAPDHs which retain their catalytic activity. Microbiology, 2001 Feb, 147(Pt 2), 383 - 90 Species-specific inhibition of fungal protein synthesis by sordarin: identification of a sordarin-specificity region in eukaryotic elongation factor 2; Shastry M et al.; The sordarin class of natural products selectively inhibits fungal protein synthesis by impairing the function of eukaryotic elongation factor 2 (eEF2) . Mutations in Saccharomyces cerevisiae eEF2 or the ribosomal stalk protein rpP0 can confer resistance to sordarin, although eEF2 is the major determinant of sordarin specificity . It has been shown previously that sordarin specifically binds S . cerevisiae eEF2 while there is no detectable binding to eEF2 from plants or mammals, despite the high level of amino acid sequence conservation among these proteins . In both whole-cell assays and in vitro translation assays, the efficacy of sordarin varies among different species of pathogenic fungi . To investigate the basis of sordarin's fungal selectivity, eEF2 has been cloned and characterized from several sordarin-sensitive and -insensitive fungal species . Results from in vivo expression of Candida species eEF2s in S . cerevisiae and in vitro translation and growth inhibition assays using hybrid S . cerevisiae eEF2 proteins demonstrate that three amino acid residues within eEF2 account for the selectivity of this class of compounds . It is also shown that the corresponding residues at these positions in human eEF2 are sufficient to confer sordarin insensitivity to S . cerevisiae identical to that observed with mammalian eEF2. J Bacteriol, 2001 Feb, 183(4), 1441 - 51 Network identification and flux quantification in the central metabolism of Saccharomyces cerevisiae under different conditions of glucose repression; Gombert AK et al.; The network structure and the metabolic fluxes in central carbon metabolism were characterized in aerobically grown cells of Saccharomyces cerevisiae . The cells were grown under both high and low glucose concentrations, i.e., either in a chemostat at steady state with a specific growth rate of 0.1 h(-1) or in a batch culture with a specific growth rate of 0.37 h(-1) . Experiments were carried out using {1-(13)C}glucose as the limiting substrate, and the resulting summed fractional labelings of intracellular metabolites were measured by gas chromatography coupled to mass spectrometry . The data were used as inputs to a flux estimation routine that involved appropriate mathematical modelling of the central carbon metabolism of S . cerevisiae . The results showed that the analysis is very robust, and it was possible to quantify the fluxes in the central carbon metabolism under both growth conditions . In the batch culture, 16.2 of every 100 molecules of glucose consumed by the cells entered the pentose-phosphate pathway, whereas the same relative flux was 44.2 per 100 molecules in the chemostat . The tricarboxylic acid cycle does not operate as a cycle in batch-growing cells, in contrast to the chemostat condition . Quantitative evidence was also found for threonine aldolase and malic enzyme activities, in accordance with published data . Disruption of the MIG1 gene did not cause changes in the metabolic network structure or in the flux pattern. Genetics, 2001 Feb, 157(2), 601 - 10 Cell polarity and hyphal morphogenesis are controlled by multiple rho-protein modules in the filamentous ascomycete Ashbya gossypii; Wendland J et al.; Polarized cell growth requires a polarized organization of the actin cytoskeleton . Small GTP-binding proteins of the Rho-family have been shown to be involved in the regulation of actin polarization as well as other processes . Hyphal growth in filamentous fungi represents an ideal model to investigate mechanisms involved in generating cell polarity and establishing polarized cell growth . Since a potential role of Rho-proteins has not been studied so far in filamentous fungi we isolated and characterized the Ashbya gossypii homologs of the Saccharomyces cerevisiae CDC42, CDC24, RHO1, and RHO3 genes . The AgCDC42 and AgCDC24 genes can both complement conditional mutations in the S . cerevisiae CDC42 and CDC24 genes and both proteins are required for the establishment of actin polarization in A . gossypii germ cells . Agrho1 mutants show a cell lysis phenotype . Null mutant strains of Agrho3 show periodic swelling of hyphal tips that is overcome by repolarization and polar hyphal growth in a manner resembling the germination pattern of spores . Thus different Rho-protein modules are required for distinct steps during polarized hyphal growth of A . gossypii. J Biol Chem, 2001 Apr 6, 276(14), 10794 - 800 Epub 2001 Jan 11. Saccharomyces cerevisiae is capable of de Novo pantothenic acid biosynthesis involving a novel pathway of beta-alanine production from spermine; White WH et al.; Pantothenic acid and beta-alanine are metabolic intermediates in coenzyme A biosynthesis . Using a functional screen in the yeast Saccharomyces cerevisiae, a putative amine oxidase, encoded by FMS1, was found to be rate-limiting for beta-alanine and pantothenic acid biosynthesis . Overexpression of FMS1 caused excess pantothenic acid to be excreted into the medium, whereas deletion mutants required beta-alanine or pantothenic acid for growth . Furthermore, yeast genes ECM31 and YIL145c, which both have structural homology to genes of the bacterial pantothenic acid pathway, were also required for pantothenic acid biosynthesis . The homology of FMS1 to FAD-containing amine oxidases and its role in beta-alanine biosynthesis suggested that its substrates are polyamines . Indeed, we found that all the enzymes of the polyamine pathway in yeast are necessary for beta-alanine biosynthesis; spe1Delta, spe2Delta, spe3Delta, and spe4Delta are all beta-alanine auxotrophs . Thus, contrary to previous reports, yeast is naturally capable of pantothenic acid biosynthesis, and the beta-alanine is derived from methionine via a pathway involving spermine . These findings should facilitate the identification of further enzymes and biochemical pathways involved in polyamine degradation and pantothenic acid biosynthesis in S . cerevisiae and raise questions about these pathways in other organisms. J Biol Chem, 2001 Apr 13, 276(15), 11559 - 66 Epub 2000 Dec 27. Characterization of TbPDE2A, a novel cyclic nucleotide-specific phosphodiesterase from the protozoan parasite Trypanosoma brucei; Zoraghi R et al.; This study reports the identification and characterization of a cAMP-specific phosphodiesterase from the parasitic hemoflagellate Trypanosoma brucei . TbPDE2A is a class I phosphodiesterase . Its catalytic domain exhibits 30-40% sequence identity with those of all 11 mammalian phosphodiesterase (PDE) families, as well as with PDE2 from Saccharomyces cerevisiae, dunce from Drosophila melanogaster, and regA from Dictyostelium discoideum . The overall structure of TbPDE2A resembles that of human PDE11A in that its N-terminal region contains a single GAF domain . This domain is very similar to those of the mammalian PDE2, -5, -6, -10, and -11, where it constitutes a potential cGMP binding site . TbPDE2A can be expressed in S . cerevisiae, and it complements an S . cerevisiae PDE deletion strain . Recombinant TbPDE2A is specific for cAMP, with a K(m) of approximately 2 micrometer . It is entirely resistant to the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine, but it is sensitive to trequinsin, dipyridamole, sildenafil, and ethaverine with IC(50) values of 5.4, 5.9, 9.4, and 14.2 micrometer, respectively . All four compounds inhibit proliferation of bloodstream form trypanosomes in culture, indicating that TbPDE2A is an essential enzyme. FEBS Lett, 2000 Dec 22, 487(1), 134 - 49 Genomic exploration of the hemiascomycetous yeasts: 21 . Comparative functional classification of genes; Gaillardin C et al.; We explored the biological diversity of hemiascomycetous yeasts using a set of 22000 newly identified genes in 13 species through BLASTX searches . Genes without clear homologue in Saccharomyces cerevisiae appeared to be conserved in several species, suggesting that they were recently lost by S . cerevisiae . They often identified well-known species-specific traits . Cases of gene acquisition through horizontal transfer appeared to occur very rarely if at all . All identified genes were ascribed to functional classes . Functional classes were differently represented among species . Species classification by functional clustering roughly paralleled rDNA phylogeny . Unequal distribution of rapidly evolving, ascomycete-specific, genes among species and functions was shown to contribute strongly to this clustering . A few cases of gene family amplification were documented, but no general correlation could be observed between functional differentiation of yeast species and variations of gene family sizes . Yeast biological diversity seems thus to result from limited species-specific gene losses or duplications, and for a large part from rapid evolution of genes and regulatory factors dedicated to specific functions. FEBS Lett, 2000 Dec 22, 487(1), 122 - 33 Genomic exploration of the hemiascomycetous yeasts: 20 . Evolution of gene redundancy compared to Saccharomyces cerevisiae; Llorente B et al.; We have evaluated the degree of gene redundancy in the nuclear genomes of 13 hemiascomycetous yeast species . Saccharomyces cerevisiae singletons and gene families appear generally conserved in these species as singletons and families of similar size, respectively . Variations of the number of homologues with respect to that expected affect from 7 to less than 24% of each genome . Since S . cerevisiae homologues represent the majority of the genes identified in the genomes studied, the overall degree of gene redundancy seems conserved across all species . This is best explained by a dynamic equilibrium resulting from numerous events of gene duplication and deletion rather than by a massive duplication event occurring in some lineages and not in others. FEBS Lett, 2000 Dec 22, 487(1), 113 - 21 Genomic exploration of the hemiascomycetous yeasts: 19 . Ascomycetes-specific genes; Malpertuy A et al.; Comparisons of the 6213 predicted Saccharomyces cerevisiae open reading frame (ORF) products with sequences from organisms of other biological phyla differentiate genes commonly conserved in evolution from 'maverick' genes which have no homologue in phyla other than the Ascomycetes . We show that a majority of the 'maverick' genes have homologues among other yeast species and thus define a set of 1892 genes that, from sequence comparisons, appear 'Ascomycetes-specific' . We estimate, retrospectively, that the S . cerevisiae genome contains 5651 actual protein-coding genes, 50 of which were identified for the first time in this work, and that the present public databases contain 612 predicted ORFs that are not real genes . Interestingly, the sequences of the 'Ascomycetes-specific' genes tend to diverge more rapidly in evolution than that of other genes . Half of the 'Ascomycetes-specific' genes are functionally characterized in S . cerevisiae, and a few functional categories are over-represented in them. FEBS Lett, 2000 Dec 22, 487(1), 101 - 12 Genomic exploration of the hemiascomycetous yeasts: 18 . Comparative analysis of chromosome maps and synteny with Saccharomyces cerevisiae; Llorente B et al.; We have analyzed the evolution of chromosome maps of Hemiascomycetes by comparing gene order and orientation of the 13 yeast species partially sequenced in this program with the genome map of Saccharomyces cerevisiae . From the analysis of nearly 8000 situations in which two distinct genes having homologs in S . cerevisiae could be identified on the sequenced inserts of another yeast species, we have quantified the loss of synteny, the frequency of single gene deletion and the occurrence of gene inversion . Traces of ancestral duplications in the genome of S . cerevisiae could be identified from the comparison with the other species that do not entirely coincide with those identified from the comparison of S . cerevisiae with itself . From such duplications and from the correlation observed between gene inversion and loss of synteny, a model is proposed for the molecular evolution of Hemiascomycetes . This model, which can possibly be extended to other eukaryotes, is based on the reiteration of events of duplication of chromosome segments, creating transient merodiploids that are subsequently resolved by single gene deletion events. FEBS Lett, 2000 Dec 22, 487(1), 95 - 100 Genomic exploration of the hemiascomycetous yeasts: 17 . Yarrowia lipolytica; Casaregola S et al.; A total of 4940 random sequence tags of the dimorphic yeast Ya