Plant Mol Biol, 2003 Mar, 51(4), 577 - 87 Identification of a copper transporter family in Arabidopsis thaliana; Sancenon V et al.; Despite copper ions being crucial in proteins participating in plant processes such as electron transport, free-radical elimination and hormone perception and signaling, very little is known about copper inward transport across plant membranes . In this work, a five-member family (COPT1-5) of putative Arabidopsis copper transporters is described . We ascertain the ability of these proteins to functionally complement and transport copper in the corresponding Saccharomyces cerevisiae high-affinity copper transport mutant . The specific expression pattern of the Arabidopsis COPT1-5 mRNA in different tissues was analyzed by RT-PCR . Although all members are ubiquitously expressed, differences in their relative abundance in roots, leaves, stem and flowers have been observed . Moreover, steady-state COPT1 and COPT2 mRNA levels, the members that are most efficacious in complementing the S . cerevisiae high-affinity copper transport mutant, are down-regulated under copper excess, consistent with a role for these proteins in copper transport in Arabidopsis cells. J Biol Chem, 2003 May 23, 278(21), 18990 - 6 Epub 2003 Mar 20. Tpn1p, the plasma membrane vitamin B6 transporter of Saccharomyces cerevisiae; Stolz J et al.; Pyridoxine (PN) is a metabolic precursor of pyridoxal phosphate that functions as a cofactor of many enzymes in amino acid metabolism . PN, pyridoxal, and pyridoxamine are collectively referred to as vitamin B6, and mammalian organisms depend on its uptake from the diet . In addition to the ability to use extracellular vitamin B6, most unicellular organisms are also capable of synthesizing PN to generate pyridoxal phosphate . Here, we report the isolation of Saccharomyces cerevisiae mutants that have lost the ability to transport PN across the plasma membrane . We used these mutants to isolate TPN1, the first known gene encoding a transport protein for vitamin B6 . Tpn1p is a member of the purine-cytosine permease family within the major facilitator superfamily . The protein functions as a proton symporter, localizes to the plasma membrane, and has high affinity for PN . TPN1 mutants lost the ability to utilize extracellular PN, pyridoxal, and pyridoxamine, showing that there is no other transporter for vitamin B6 encoded in the genome . Amino acid substitutions that led to a loss of Tpn1p function localized to transmembrane domain 4 within the 12-transmembrane domain protein . Moreover, expression of TPN1 was regulated and increased with decreasing concentrations of vitamin B6 in the medium . We also provide evidence that of the highly conserved SNZ and SNO genes in S . cerevisiae, only the protein encoded by SNZ1 is required for vitamin B6 biosynthesis. J Soc Biol, 2002, 196(4), 349 - 54 {Nuclear pores: from yeast to higher eukaryotes}; Doye V; In eukaryotes, bidirectional transport of macro-molecules between the cytoplasm and the nucleus occurs through elaborate supramolecular structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs), whose overall architecture has been evolutionary conserved from yeast to vertebrates . In recent years, fast progress in characterizing the NPCs components (the nucleoporins or Nups) has been made in the yeast S . cerevisiae, and to a lesser extent in vertebrates . In addition, despite the low homology between most yeast and vertebrate nucleoporins, their organization and their topological mapping within the NPC substructures have been broadly conserved during evolution. Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku, 2002, (120), 53 - 74 {Functional analysis of yeast homologue gene associated with human DNA helicase causative syndromes}; Miyajima A; Proteins having DNA helicase activity play very important roles in many processes involving DNA workings such as replication, repair, and recombination . In this decade, many DNA helicase genes have been cloned as the causative genes of human recessive heredity diseases . These are the causative genes for Xeroderma pigmentosum (XPB and XPD), Cockayne syndrome (CSB), diffuse collagen disease (Ku80), alpha-thalassmia (ATR-X), Bloom syndrome (BLM), Werner syndrome (WRN) and Rothmund-Thomson syndrome (RTS) . The yeast homologue genes of these human DNA helicase genes exist . S . cerevisiae RAD25/SSL2, RAD3, RAD26, YKU80/HDF2 and RAD54 are the homologue for XPB/ERCC3, XPD/ERCC2, CSB/ERCC6, Ku80/XRCC5 and ATR-X/HX2, respectively . E coli . recQ gene and S . cerevisiae SGS1 are the homologue for all BLM, WRN and RTS . A search of whole genome of S . cerevisiae revealed that SGS1 is the sole homologue of recQ in S . cerevisiae . Thus it seems likely that SGS1 is a functional homologue of one or several human RecQ family genes . Many basic or essential functions are well conserved in the cells from lower eukaryotic to higher mammalian . The functional analysis in yeast could make an useful insight for the human homologue . To clarify the functions of S . cerevisiae Sgs1 and to get an insight into the functions of Blm, Wrn and Rts, in this study, we analyzed the phenotype of sgs1 disruptant and in detail the cause of the poor sporulation phenotype of sgs1 disruptants in relation to meiotic processes including meiotic recombination . The poor sporulation of sgs1 disruptants was complemented with a mutated SGS1 gene encoding a protein lacking DNA helicase activity; however, the mutated gene could suppress neither the sensitivity of sgs1 disruptants to methyl methanesulfonate (MMS) and hydroxyurea nor the mitotic hyperrecombination phenotype of sgs1 disruptants . The N-terminal 1-45 amino acid region and 698-1195 amino acid region of Sgs1, which including helicase domain and C-terminal RecQ conserved region with helicase activity, were required for complementation of MMS sensitivity and suppression of hyperrecombination of sgs1 disruptants in mitotic growth . The 126-400 and 596-1195 amino acid regions of Sgs1 were required for complementation of poor sporulation and of reduced meiotic functions . These regions required for the mitotic or meiotic functions of Sgs1 were well overlapped with the interaction regions of Top3 and Top2 . Some of these results might explain the mechanism of the symptom of RecQ-related syndromes. J Biol Chem, 2003 May 23, 278(21), 19079 - 86 Epub 2003 Mar 07. The Gly-952 residue of Saccharomyces cerevisiae DNA polymerase alpha is important in discriminating correct deoxyribonucleotides from incorrect ones; Limsirichaikul S et al.; Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha) that is strictly required for catalytic activity and for genetic complementation of a pol alpha-deficient yeast strain . This study analyzes the role of Gly-952 by characterizing the biochemical properties of Gly-952 mutants . Analysis of the nucleotide incorporation specificity of pol alpha G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity . In a steady-state kinetic assay to measure nucleotide misincorporation, pol alpha G952A incorporated incorrect nucleotides more efficiently than correct nucleotides opposite template C, G, and T . The fidelity of the G952A mutant polymerase was highest at template A, where the ratio of incorporation of dCMP to dTMP was as high as 0.37 . Correct nucleotide insertion was 500- to 3500-fold lower for G952A than for wild type pol alpha, with up to 22-fold increase in pyrimidine misincorporation . The Km for G952A pol alpha bound to mismatched termini T:T, T:C, C:A, and A:C was 71- to 460-fold lower than to a matched terminus . Furthermore, pol alpha G952A preferentially incorporated pyrimidine instead of dAMP opposite an abasic site, cis-syn cyclobutane di-thymine, or (6-4) di-thymine photoproduct . These data demonstrate that Gly-952 is a critical residue for catalytic efficiency and error prevention in S . cerevisiae pol alpha. J Biol Chem, 2003 May 23, 278(21), 19071 - 8 Epub 2003 Mar 07. Distinct function of conserved amino acids in the fingers of Saccharomyces cerevisiae DNA polymerase alpha; Ogawa M et al.; Structural differences between class A and B DNA polymerases suggest that the motif B region, a wall of the catalytic pocket, may have evolved differentially in the two polymerase families . This study examines the function of the motif B residues in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha) . Effects of the mutations were determined by biochemical analysis and genetic complementation of a yeast strain carrying a temperature-sensitive pol alpha mutant . Many conserved residues were viable with a variety of substitutions . Among them, mutations at Asn-948 or Tyr-951 conferred up to 8-fold higher colony formation frequency in a URA3 forward mutation assay, and 79-fold higher trp1 reversion frequency was observed for Y951P in yeast . Purified Y951P was as accurate as wild type in DNA synthesis but approximately 6-fold less processive and 22-fold less active in vitro . Therefore, Y951P may increase the frequency of mutant colony formation because of its low level of DNA polymerase activity in yeast . Mutations at Lys-944 or Gly-952 were not viable, which is consistent with the observation that mutants with substitutions at Gly-952 have strongly reduced catalytic activity in vitro . Gly-952 may provide a space for the nascent base pair and thus may play an essential function in S . cerevisiae DNA pol alpha . These results suggest that class B DNA polymerases have a unique structure in the catalytic pocket, which is distinct from the corresponding region in class A DNA polymerases. Folia Microbiol (Praha), 2002, 47(6), 663 - 6 The GCR1 gene function is essential for glycogen and trehalose metabolism in Saccharomyces cerevisiae; Turkel S; Trehalose (Tre) and glycogen (Glg) are synthesized in response to unfavorable growth conditions from glycolytic intermediates in Saccharomyces cerevisiae . Transcription of the glycolytic genes is activated by the Gcr1p complex, the DNA binding transcription factor that directly associates with the CT-box sequences on the promoter region of the glycolytic genes . gcr1 mutant yeast cells cannot utilize glucose effectively . Glg and Tre levels in stationary-phase gcr1 mutant yeast cells were 20-50% of those in the wild-type strain . Likewise, stress-induced accumulation of Tre and Glg in gcr1 mutant cells was significantly lower than in the wild type . In addition, both the synthesis and the degradation of Tre and Glg are very slow in the gcr1 mutant . It seems that Gcr1p function is essential for the coordinated regulation of glycolysis, Tre and Glg metabolism in S . cerevisiae. Cell, 2003 Mar 7, 112(5), 697 - 709 Exit from exit: resetting the cell cycle through Amn1 inhibition of G protein signaling; Wang Y et al.; In S . cerevisiae cells undergoing anaphase, a ras-related GTPase, Tem1, is located on the spindle pole body that enters the daughter cell and activates a signal transduction pathway, MEN, to allow mitotic exit . MEN activation must be reversed after mitotic exit to reset the cell cycle in G1 . We find that daughter cells activate an Antagonist of MEN pathway (AMEN) in part through induction of the Amn1 protein that binds directly to Tem1 and prevents its association with its target kinase Cdc15 . Failure of Amn1 function results in defects of both the spindle assembly and nuclear orientation checkpoints and delays turning off Cdc14 in G1 . Thus, Amn1 is part of a daughter-specific switch that helps cells exit from mitotic exit and reset the cell cycle. Yeast, 2003 Mar, 20(4), 343 - 50 RHO1 (YlRHO1) is a non-essential gene in Yarrowia lipolytica and complements rho1Delta lethality in Saccharomyces cerevisiae; Leon M et al.; The synthesis of beta-1,3-glucan, the structural component of the yeast cell wall that gives shape to the cell, occurs at the plasma membrane and is the result of the activity of at least a two-component complex . Fks1p is the catalytic subunit directly responsible for the synthesis of beta-1,3-glucan, whilst the second subunit, Rho1p, has a GTP-dependent regulatory role (Yamochi et al., 1994) . RHO1 has been characterized in Saccharomyces cerevisiae (Yamochi et al., 1994), and in several other fungal species . In this work, we have used degenerate oligonucleotides derived from the conserved regions of Rho1ps to isolate the RHO1 gene of Yarrowia lipolytica . The gene isolated in this way, which we have named YlRHO1, encodes a 204 amino acid protein that shows a high degree of homology with other Rho1ps . However, unlike S . cerevisiae, the ylrho1Delta disruptant strain in Y . lipolytica is viable, although it exhibits an increased sensitivity to Calcofluor white and Congo red . Also, YlRHO1 complements rho1 lethality in S . cerevisiae at both 28 degrees C and 37 degrees C . The complete sequence of YlRHO1 can be obtained from GenBank under Accession No . AF279915 . Proc Natl Acad Sci U S A, 2003 Mar 18, 100(6), 3107 - 12 Epub 2003 Mar 07. Protein pathway and complex clustering of correlated mRNA and protein expression analyses in Saccharomyces cerevisiae; Washburn MP et al.; The mRNA and protein expression in Saccharomyces cerevisiae cultured in rich or minimal media was analyzed by oligonucleotide arrays and quantitative multidimensional protein identification technology . The overall correlation between mRNA and protein expression was weakly positive with a Spearman rank correlation coefficient of 0.45 for 678 loci . To place the data sets in a proper biological context, a clustering approach based on protein pathways and protein complexes was implemented . Protein expression levels were transcriptionally controlled for not only single loci but for entire protein pathways (e.g., Met, Arg, and Leu biosynthetic pathways) . In contrast, the protein expression of loci in several protein complexes (e.g., SPT, COPI, and ribosome) was posttranscriptionally controlled . The coupling of the methods described provided insight into the biology of S . cerevisiae and a clustering strategy by which future studies should be based. Glycobiology, 2003 Mar, 13(3), 169 - 77 Epub 2002 Oct 30. Structures of the glycosylphosphatidylinositol membrane anchors from Aspergillus fumigatus membrane proteins; Fontaine T et al.; Glycosylphosphatidylinositol (GPI)-anchored proteins have been identified in all eukaryotes . In fungi, structural and biosynthetic studies of GPIs have been restricted to the yeast Saccharomyces cerevisiae . In this article, four GPI-anchored proteins were purified from a membrane preparation of the human filamentous fungal pathogen Aspergillus fumigatus . Using new methodology applied to western blot protein bands, the GPI structures were characterized by ES-MS, fluorescence labeling, HPLC, and specific enzymatic digestions . The phosphatidylinositol moiety of the A . fumigatus GPI membrane anchors was shown to be an inositol-phosphoceramide containing mainly phytosphingosine and monohydroxylated C24:0 fatty acid . In constrast to yeast, only ceramide was found in the GPI anchor structures of A . fumigatus, even for Gel1p, a homolog of Gas1p in S . cerevisiae that contains diacylglycerol . The A . fumigatus GPI glycan moiety is mainly a linear pentomannose structure linked to a glucosamine residue: Manalpha1-3Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4GlcN. Mol Biol (Mosk), 2003 Jan-Feb, 37(1), 81 - 7 {A novel approach to isolation and functional characterization of genomic DNA from the methylotrophic yeast Hansenula polymorpha}; Agafonov MO et al.; A novel approach to isolation and functional characterization of the Hansenula polymorpha genes basing on the use of two strains of different origin is described . One of these strains is better suited for the isolation of genomic DNA fragments, while the other is preferable for their functional analysis . Thirty three genomic sequences governing expression of a reporter protein have been isolated . Analysis of the sequence encoding a homolog of the Saccharomyces cerevisiae cofilin revealed two introns . Another isolated DNA fragment encoded a homolog of the S . cerevisiae V ps10p . Disruption of the corresponding gene resulted in secretion of a vacuolar protein, carboxypeptidase Y, into the culture medium. Anal Chem, 2003 Feb 15, 75(4), 867 - 74 Abundance ratio-dependent proteomic analysis by mass spectrometry; Griffin TJ et al.; The goal of quantitative proteomics is to determine the identity and relative quantity of each protein present in two or more complex protein samples . Here we describe a novel approach to quantitative proteomics . It is based on a highly accurate algorithm for the automated quantification of chromatographically fractionated, isotope-coded affinity-tagged peptides and MALDI quadrupole time-of-flight tandem mass spectrometry for their identification . The method is capable of detecting and selectively identifying those proteins within a complex mixture that show a difference in relative abundance . We demonstrate the effectiveness and the versatility of this approach in the analysis of a standard protein mixture, protein expression profiling in a human prostate cancer cell line model, and identification of the specific components of the multiprotein transcriptional machinery in S . cerevisiae. Nature, 2003 Mar 6, 422(6927), 68 - 72 Engineering evolution to study speciation in yeasts; Delneri D et al.; The Saccharomyces 'sensu stricto' yeasts are a group of species that will mate with one another, but interspecific pairings produce sterile hybrids . A retrospective analysis of their genomes revealed that translocations between the chromosomes of these species do not correlate with the group's sequence-based phylogeny (that is, translocations do not drive the process of speciation) . However, that analysis was unable to infer what contribution such rearrangements make to reproductive isolation between these organisms . Here, we report experiments that take an interventionist, rather than a retrospective approach to studying speciation, by reconfiguring the Saccharomyces cerevisiae genome so that it is collinear with that of Saccharomyces mikatae . We demonstrate that this imposed genomic collinearity allows the generation of interspecific hybrids that produce a large proportion of spores that are viable, but extensively aneuploid . We obtained similar results in crosses between wild-type S . cerevisiae and the naturally collinear species Saccharomyces paradoxus, but not with non-collinear crosses . This controlled comparison of the effect of chromosomal translocation on species barriers suggests a mechanism for the generation of redundancy in the S . cerevisiae genome. Appl Environ Microbiol, 2003 Mar, 69(3), 1499 - 503 Ethanol tolerance in the yeast Saccharomyces cerevisiae is dependent on cellular oleic acid content; You KM et al.; In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol . The unsaturated fatty acid (UFA) composition of S . cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Delta(9)Z-C(16:1)) and oleic acid (Delta(9)Z-C(18:1)), with the former predominating . Both UFAs are formed in S . cerevisiae by the oxygen- and NADH-dependent desaturation of palmitic acid (C(16:0)) and stearic acid (C(18:0)), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene . We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Delta(9) and Delta(11)) and substrate chain-length preferences (i.e., C(16:0) and C(18:0)); and, (ii) by supplementation of the same strain with synthetic mono-UFAs . Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells . Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (Delta(11)Z-C(18:1)), whereas neither Delta(11)Z-C(16:1) nor palmitoleic acid (Delta(9)Z-C(16:1)) conferred any ethanol tolerance . We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol . These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol. Curr Biol, 2003 Mar 4, 13(5), 364 - 72 Determinants of S . cerevisiae dynein localization and activation: implications for the mechanism of spindle positioning; Sheeman B et al.; BACKGROUND: During anaphase in budding yeast, dynein inserts the mitotic spindle across the neck between mother and daughter cells . The mechanism of dynein-dependent spindle positioning is thought to involve recruitment of dynein to the cell cortex followed by capture of astral microtubules (aMTs) . RESULTS: We report the native-level localization of the dynein heavy chain and characterize the effects of mutations in dynein regulators on its intracellular distribution . Budding yeast dynein displays discontinuous localization along aMTs, with enrichment at the spindle pole body and aMT plus ends . Loss of Bik1p (CLIP-170), the cargo binding domain of Bik1p, or Pac1p (LIS1) resulted in diminished targeting of dynein to aMTs . By contrast, loss of dynactin or a mutation in the second P loop domain of dynein resulted in an accumulation of dynein on the plus ends of aMTs . Unexpectedly, loss of Num1p, a proposed dynein cortical anchor, also resulted in selective accumulation of dynein on the plus ends of anaphase aMTs . CONCLUSIONS: We propose that, rather than first being recruited to the cell cortex, dynein is delivered to the cortex on the plus ends of polymerizing aMTs . Dynein may then undergo Num1p-dependent activation and transfer to the region of cortical contact . Based on the similar effects of loss of Num1p and loss of dynactin on dynein localization, we suggest that Num1p might also enhance dynein motor activity or processivity, perhaps by clustering dynein motors. Lipids, 2002 Dec, 37(12), 1171 - 6 Subcellular localization of oxidosqualene cyclases from Arabidopsis thaliana, Trypanosoma cruzi, and Pneumocystis carinii expressed in yeast; Milla P et al.; Cycloartenol synthase from Arabidopsis thaliana and lanosterol synthase from Trypanosoma cruzi and Pneumocystis carinii were expressed in yeast, and their subcellular distribution in the expressing cells was compared . Determination of enzymatic (oxidosqualene cyclase, OSC) activity and SDS-PAGE analysis of subcellular fractions proved that enzymes from T . cruzi and A . thaliana have high affinity for lipid particles, a subcellular compartment rich in triacylglycerols, and steryl esters, harboring several enzymes of lipid metabolism . In lipid particles of strains expressing the P . carinii enzyme, neither OSC activity nor the electrophoretic band at the appropriate M.W . were detected . Microsomes from the three expressing strains retained some OSC activity . Affinity of enzymes from A . thaliana and T . cruzi for lipid particles is similar to that of OSC of Saccharomyces cerevisiae, which is mainly located in this compartment . A different distribution of OSC in yeast cells suggests that they differ in some structural features critical for the interaction with the surface of lipid particles . Computer analysis supports the hypothesis of the structural difference since OSC from S . cerevisiae, A . thaliana, and T . cruzi lack or contain only one transmembrane spanning domain (a structural feature that makes a protein poorly inclined to associate with lipid particles), whereas OSC from P . carinii possesses six transmembrane domains . In the strain expressing cycloartenol synthase from A . thaliana, the accumulation of lipid particles largely exceeded that of the other strains. Bioorg Med Chem, 2003 Mar 20, 11(6), 1117 - 22 Modes of antifungal action of alkanols against Saccharomyces cerevisiae; Kubo I et al.; Primary aliphatic alcohols from C(6) to C(13) were tested for their antifungal activity against Saccharomyces cerevisiae . Undecanol was found to be the most potent fungicide followed by decanol . The time-kill curve study showed that undecanol was fungicidal against S . cerevisiae at any growth stages . This fungicidal activity was not influenced by pH values . The alcohols tested inhibited glucose-induced acidification by inhibiting the plasma membrane H(+)-ATPase . The primary antifungal action of amphipathic medium-chain (C(9)-C(12)) alkanols comes mainly from their ability as nonionic surfactants to disrupt the native membrane-associated function of the integral proteins . Hence, the antifungal activity of alkanols is mediated by biophysical process, and the maximum activity can be obtained when balance between hydrophilic and hydrophobic portions becomes the most appropriate. Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2450 - 5 Epub 2003 Feb 25. Disorder in the nuclear pore complex: the FG repeat regions of nucleoporins are natively unfolded; Denning DP et al.; Nuclear transport proceeds through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope of eukaryotic cells . The Saccharomyces cerevisiae NPC is comprised of 30 nucleoporins (Nups), 13 of which contain phenylalanine-glycine repeats (FG Nups) that bind karyopherins and facilitate the transport of karyopherin-cargo complexes . Here, we characterize the structural properties of S . cerevisiae FG Nups by using biophysical methods and predictive amino acid sequence analyses . We find that FG Nups, particularly the large FG repeat regions, exhibit structural characteristics typical of "natively unfolded" proteins (highly flexible proteins that lack ordered secondary structure) . Furthermore, we use protease sensitivity assays to demonstrate that most FG Nups are disordered in situ within the NPCs of purified yeast nuclei . The conclusion that FG Nups constitute a family of natively unfolded proteins supports the hypothesis that the FG repeat regions of Nups form a meshwork of random coils at the NPC through which nuclear transport proceeds. Chem Biol Interact, 2003 Feb 1, 143-144, 271 - 8 Coenzyme-based functional assignments of short-chain dehydrogenases/reductases (SDRs); Persson B et al.; Short-chain dehydrogenases/reductases (SDRs) are enzymes of great functional diversity . In spite of a residue identity of only 15-30%, the folds are conserved to a large extent, with specific sequence motifs detectable . We have developed an assignment scheme based on these motifs and detect five families . Only two of these were known before, called 'Classical' and 'Extended', but are now distinguished at a further level based on patterns of charged residues in the coenzyme-binding region, giving seven subfamilies of classical SDRs and three subfamilies of extended SDRs . Three further families are novel entities, denoted 'Intermediate', 'Divergent' and 'Complex', encompassing short-chain alcohol dehydrogenases, enoyl reductases and multifunctional enzymes, respectively . The assignment scheme was applied to the genomes of human, mouse, D . melanogaster, C . elegans, A . thaliana and S . cerevisiae . In the animal genomes, genes corresponding to the extended SDRs amount to around one quarter or less of the total number of SDR genes, while in those of A . thaliana and S . cerevisiae, the extended members constitute about 40% of the SDR forms . The NAD(H)-dependent SDRs are about equally many as the NADP(H)-dependent ones in human, mouse and plant, while the proportions of NAD(H)-dependent enzymes are much lower in fruit fly, worm and yeast . We also find that NADP(H) is the preferred coenzyme among most classical SDRs, while NAD(H) is that preferred among most extended SDRs. Mol Biol Cell, 2003 Feb, 14(2), 810 - 21 Pex7p and Pex20p of Neurospora crassa function together in PTS2-dependent protein import into peroxisomes; Sichting M et al.; Recruiting matrix proteins with a peroxisomal targeting signal type 2 (PTS2) to the peroxisomal membrane requires species-specific factors . In Saccharomyces cerevisiae, the PTS2 receptor Pex7p acts in concert with the redundant Pex18p/Pex21p, whereas in Yarrowia lipolytica, Pex20p might unite the function of both S . cerevisiae peroxins . Herein, the genome of the filamentous fungus Neurospora crassa was analyzed for peroxin-encoding genes . We identified a set of 18 peroxins that resembles that of Y . lipolytica rather than that of S . cerevisiae . Interestingly, proteins homologous to both S . cerevisiae Pex7p and Y . lipolytica Pex20p exist in N . crassa . We report on the isolation of these PTS2-specific peroxins and demonstrate that NcPex20p can substitute for S . cerevisiae Pex18p/Pex21p, but not for ScPex7p . Like Pex18p, NcPex20p did not bind PTS2 protein or the docking proteins in the absence of ScPex7p . Rather, NcPex20p was required before docking to form an import-competent complex of cargo-loaded PTS2 receptors . NcPex7p did not functionally replace yeast Pex7p, probably because the N . crassa PTS2 receptor failed to associate with Pex18p/Pex21p . However, once NcPex7p and NcPex20p had been coexpressed, it proved possible to replace yeast Pex7p . Pex20p and Pex18p/Pex21p are therefore true orthologues, both of which are in need of Pex7p for PTS2 protein import. Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2766 - 70 Epub 2003 Feb 14. A Saccharomyces cerevisiae mutant with increased virulence; Wheeler RT et al.; Saccharomyces cerevisiae, bakers' yeast, is not a pathogen in healthy individuals, but is increasingly isolated from immunocompromised patients . The more frequent isolation of S . cerevisiae clinically raises a number of questions concerning the origin, survival, and virulence of this organism in human hosts . Here we compare the virulence of a human isolate, a strain isolated from decaying fruit, and a common laboratory strain in a mouse infection model . We find that the plant isolate is lethal in mice, whereas the laboratory strain is avirulent . A knockout of the SSD1 gene, which alters the composition and cell wall architecture of the yeast cell surface, causes both the clinical and plant isolates to be more virulent in the mouse model of infection . The hypervirulent ssd1 Delta/ssd1 Delta yeast strain is a more potent elicitor of proinflammatory cytokines from macrophages in vitro . Our data suggest that the increased virulence of the mutant strains is a consequence of unique surface characteristics that overstimulate the proinflammatory response. Mol Cell Biol, 2003 Mar, 23(5), 1726 - 36 Karyopherin-mediated nuclear import of the homing endonuclease VMA1-derived endonuclease is required for self-propagation of the coding region; Nagai Y et al.; VMA1-derived endonuclease (VDE), a site-specific endonuclease in Saccharomyces cerevisiae, enters the nucleus to generate a double-strand break in the VDE-negative allelic locus, mediating the self-propagating gene conversion called homing . Although VDE is excluded from the nucleus in mitotic cells, it relocalizes at premeiosis, becoming localized in both the nucleus and the cytoplasm in meiosis . The nuclear localization of VDE is induced by inactivation of TOR kinases, which constitute central regulators of cell differentiation in S . cerevisiae, and by nutrient depletion . A functional genomic approach revealed that at least two karyopherins, Srp1p and Kap142p, are required for the nuclear localization pattern . Genetic and physical interactions between Srp1p and VDE imply direct involvement of karyopherin-mediated nuclear transport in this process . Inactivation of TOR signaling or acquisition of an extra nuclear localization signal in the VDE coding region leads to artificial nuclear localization of VDE and thereby induces homing even during mitosis . These results serve as evidence that VDE utilizes the host systems of nutrient signal transduction and nucleocytoplasmic transport to ensure the propagation of its coding region. Genetics, 2003 Jan, 163(1), 35 - 46 SOD2 functions downstream of Sch9 to extend longevity in yeast; Fabrizio P et al.; Signal transduction pathways inactivated during periods of starvation are implicated in the regulation of longevity in organisms ranging from yeast to mammals, but the mechanisms responsible for life-span extension are poorly understood . Chronological life-span extension in S . cerevisiae cyr1 and sch9 mutants is mediated by the stress-resistance proteins Msn2/Msn4 and Rim15 . Here we show that mitochondrial superoxide dismutase (Sod2) is required for survival extension in yeast . Deletion of SOD2 abolishes life-span extension in sch9Delta mutants and decreases survival in cyr1:mTn mutants . The overexpression of Sods--mitochondrial Sod2 and cytosolic CuZnSod (Sod1)--delays the age-dependent reversible inactivation of mitochondrial aconitase, a superoxide-sensitive enzyme, and extends survival by 30% . Deletion of the RAS2 gene, which functions upstream of CYR1, also doubles the mean life span by a mechanism that requires Msn2/4 and Sod2 . These findings link mutations that extend chronological life span in S . cerevisiae to superoxide dismutases and suggest that the induction of other stress-resistance genes regulated by Msn2/4 and Rim15 is required for maximum longevity extension. FEMS Microbiol Lett, 2003 Jan 28, 218(2), 291 - 7 Role of Saccharomyces cerevisiae serine O-acetyltransferase in cysteine biosynthesis; Takagi H et al.; Some strains of Saccharomyces cerevisiae have detectable activities of L-serine O-acetyltransferase (SATase) and O-acetyl-L-serine/O-acetyl-L-homoserine sulfhydrylase (OAS/OAH-SHLase), but synthesize L-cysteine exclusively via cystathionine by cystathionine beta-synthase and cystathionine gamma-lyase . To untangle this peculiar feature in sulfur metabolism, we introduced Escherichia coli genes encoding SATase and OAS-SHLase into S . cerevisiae L-cysteine auxotrophs . While the cells expressing SATase grew on medium lacking L-cysteine, those expressing OAS-SHLase did not grow at all . The cells expressing both enzymes grew very well without L-cysteine . These results indicate that S . cerevisiae SATase cannot support L-cysteine biosynthesis and that S . cerevisiae OAS/OAH-SHLase produces L-cysteine if enough OAS is provided by E . coli SATase . It appears as if S . cerevisiae SATase does not possess a metabolic role in vivo either because of very low activity or localization . For example, S . cerevisiae SATase may be localized in the nucleus, thus controlling the level of OAS required for regulation of sulfate assimilation, but playing no role in the direct synthesis of L-cysteine. J Virol, 2003 Mar, 77(5), 2990 - 7 Mutation of host DnaJ homolog inhibits brome mosaic virus negative-strand RNA synthesis; Tomita Y et al.; The replication of positive-strand RNA viruses involves not only viral proteins but also multiple cellular proteins and intracellular membranes . In both plant cells and the yeast Saccharomyces cerevisiae, brome mosaic virus (BMV), a member of the alphavirus-like superfamily, replicates its RNA in endoplasmic reticulum (ER)-associated complexes containing viral 1a and 2a proteins . Prior to negative-strand RNA synthesis, 1a localizes to ER membranes and recruits both positive-strand BMV RNA templates and the polymerase-like 2a protein to ER membranes . Here, we show that BMV RNA replication in S . cerevisiae is markedly inhibited by a mutation in the host YDJ1 gene, which encodes a chaperone Ydj1p related to Escherichia coli DnaJ . In the ydj1 mutant, negative-strand RNA accumulation was inhibited even though 1a protein associated with membranes and the positive-strand RNA3 replication template and 2a protein were recruited to membranes as in wild-type cells . In addition, we found that in ydj1 mutant cells but not wild-type cells, a fraction of 2a protein accumulated in a membrane-free but insoluble, rapidly sedimenting form . These and other results show that Ydj1p is involved in forming BMV replication complexes active in negative-strand RNA synthesis and suggest that a chaperone system involving Ydj1p participates in 2a protein folding or assembly into the active replication complex. J Pathol, 2003 Mar, 199(3), 361 - 7 Cross-reactivity of yeast antigens in human colon and peripheral leukocytes; Oshitani N et al.; Elevation of the serum anti-Saccharomyces cerevisiae antibody (ASCA) level has been reported in patients with Crohn's disease . This study investigated the antigenic distribution of S . cerevisiae in human colon and peripheral leukocytes . ASCA was isolated from sera from patients with Crohn's disease using immuno-affinity chromatography and then biotinylated and assayed immunohistologically and immunocytologically to determine the distribution of antigens recognized by ASCA in human colon and peripheral leukocytes . Immunoblot analysis of yeast extract and human peripheral leukocytes was performed . Immunohistological study using biotinylated ASCA revealed the presence of yeast-like particles in the granulation tissue of inflamed colonic mucosa . Biotinylated ASCA also stained lymphocytes and polymorphonuclear cells infiltrating inflamed intestine . Monocytes in epithelioid granulomas of colon with Crohn's disease were also stained . Polymorphonuclear leukocytes in peripheral blood were also stained with biotinylated ASCA . The antigens reactive to ASCA among heat-extracted, non-heat-extracted yeast antigens, and human leukocyte extract differed . The findings of cross-reactivity of polymorphonuclear leukocytes with S . cerevisiae antigen and the presence of S . cerevisiae antigen in Crohn's disease granulomas suggest the possibility of involvement of S . cerevisiae in the pathogenesis of Crohn's disease . J Biol Chem, 2003 Apr 18, 278(16), 14082 - 6 Epub 2003 Feb 05. The quaternary structure of DNA polymerase epsilon from Saccharomyces cerevisiae; Chilkova O et al.; DNA polymerase epsilon (Pol epsilon) from Saccharomyces cerevisiae consists of four subunits (Pol2, Dpb2, Dpb3, and Dpb4) and is essential for chromosomal DNA replication . Biochemical characterizations of Pol epsilon have been cumbersome due to protease sensitivity and the limited amounts of Pol epsilon in cells . We have developed a protocol for overexpression and purification of Pol epsilon from S . cerevisiae . The native four-subunit complex was purified to homogeneity by conventional chromatography . Pol epsilon was characterized biochemically by sedimentation velocity experiments and gel filtration experiments . The stoichiometry of the four subunits was estimated to be 1:1:1:1 from colloidal Coomassie-stained gels . Based on the sedimentation coefficient (11.9 S) and the Stokes radius (74.5 A), a molecular mass for Pol epsilon of 371 kDa was calculated, in good agreement with the calculated molecular mass of 379 kDa for a heterotetramer . Furthermore, analytical equilibrium ultracentrifugation experiments support the proposed heterotetrameric structure of Pol epsilon . Thus, both DNA polymerase delta and Pol epsilon are purified as monomeric complexes, in agreement with accumulating evidence that Pol delta and Pol epsilon are located on opposite strands of the eukaryotic replication fork. Curr Drug Targets Infect Disord, 2002 Dec, 2(4), 309 - 29 Genomic pathways to antifungal discovery; Monk BC et al.; The limitations of the therapeutic antifungals are becoming increasingly apparent in the clinic due to their modest efficacy against life-threatening systemic fungal infections . These antifungals belong to only a few structural classes that affect a small range of targets, some are quite toxic in humans while the use of others, particularly the azole drugs, has encouraged the emergence of resistant clinical isolates and the selection of innately resistant fungal pathogens . Only a few new drugs based on novel targets are in clinical development, and these may be insufficient to overcome the changing tide of fungal disease . In parallel with the successful completion of the Saccharomyces cerevisiae and human genome sequencing projects, an increasing number of genome sequencing projects are being initiated and completed for significant fungal pathogens . The growing repository of genomic information, which is complemented by decades of genetic and biochemical study, is now available for genome-wide analysis of gene function and for incisive inter-genomic comparison, with the S . cerevisiae and human genomes providing key points of reference . Functional genomic and comparative genomic techniques, many of which were developed with S . cerevisiae, are being applied to fungal pathogens with the aim of obtaining an integrated view of fungal biology and to extract targets suitable for drug discovery . This review describes some of these techniques, their limitations and their increasing contribution to the antifungal discovery process through effective gene annotation, target identification and prioritization, and in the optimization of antifungal leads. J Mol Evol, 2003 Jan, 56(1), 28 - 37 Patterns of gene duplication in Saccharomyces cerevisiae and Caenorhabditis elegans; Cavalcanti AR et al.; In this paper we present a new method for detecting block duplications in a genome . It is more stringent than previous ones in that it requires a more rigorous definition of paralogous genes and that it requires the paralogous proteins on the two blocks to be contiguous . In addition, it provides three criterion choices: (1) the same composition (i.e., having the same paralogues in the two windows), (2) the same composition and gene order, and (3) the same composition, gene order, and gene orientation . The method is completely automated, requiring no visual inspection as in previous methods . We applied it to analyze the complete genomes of S . cerevisiae and C . elegans . In yeast we detected fewer duplicated blocks than previously reported . In C . elegans, however, we detected more block duplications than previously reported, indicating that although our method has a more stringent definition of block duplication than previous ones, it may be more sensitive in detection because it considers every possible window rather than only fixed nonoverlapping windows . Our results show that block duplication is a common phenomenon in both organisms . The patterns of block duplication in the two species are, however, markedly different . The yeast shows much more extensive block duplication than the nematode, with some chromosomes having more than 40% of the duplications derived from block duplications . Moreover, in the yeast the majority of block duplications occurred between chromosomes, while in the nematode most block duplications occurred within chromosomes. Genome Res, 2003 Feb, 13(2), 244 - 53 Genome-scale reconstruction of the Saccharomyces cerevisiae metabolic network; Forster J et al.; The metabolic network in the yeast Saccharomyces cerevisiae was reconstructed using currently available genomic, biochemical, and physiological information . The metabolic reactions were compartmentalized between the cytosol and the mitochondria, and transport steps between the compartments and the environment were included . A total of 708 structural open reading frames (ORFs) were accounted for in the reconstructed network, corresponding to 1035 metabolic reactions . Further, 140 reactions were included on the basis of biochemical evidence resulting in a genome-scale reconstructed metabolic network containing 1175 metabolic reactions and 584 metabolites . The number of gene functions included in the reconstructed network corresponds to approximately 16% of all characterized ORFs in S . cerevisiae . Using the reconstructed network, the metabolic capabilities of S . cerevisiae were calculated and compared with Escherichia coli . The reconstructed metabolic network is the first comprehensive network for a eukaryotic organism, and it may be used as the basis for in silico analysis of phenotypic functions. Cancer Res, 2003 Feb 1, 63(3), 549 - 54 Disparity between DNA base excision repair in yeast and mammals: translational implications; Kelley MR et al.; One approach to the effective treatment of cancer requires the continued development of novel chemotherapeutic agents to kill tumor cells . Additionally, an element of cancer research has been devoted to understanding DNA repair pathways in hopes of defining the factors that confer resistance to anticancer drugs and developing strategies for modulating repair capacity as a means of overcoming resistance or enhancing sensitivity to cancer treatments . Historically, yeast, particularly Saccharomyces cerevisiae, has been used as a model system for DNA repair analyses . Additionally, it has been used to evaluate drug efficacy and selectivity, and to identify new targets for antitumor drugs . The usefulness of yeast for these types of analyses has been primarily because of it being considered to have well-conserved DNA repair processes among eukaryotes . However, as more information has accumulated in mammalian DNA repair, and particularly in DNA base excision repair (BER), a number of striking differences have emerged between yeast and mammalian (human) repair processes . The BER pathway is essential for the repair of damaged DNA induced by oxidizing and alkylating agents, which are the majority of chemotherapeutic drugs used currently in the clinic . The importance of this pathway in processing DNA damage makes its members potential targets for novel chemotherapeutic agents . However, because the BER process and its main players are remarkably divergent from S . cerevisiae to humans, it is worth keeping these differences in mind if yeast continues to be used as a model or primary system in the screening for potential new human therapeutics. Free Radic Biol Med, 2003 Feb 15, 34(4), 478 - 88 External alternative NADH dehydrogenase of Saccharomyces cerevisiae: a potential source of superoxide; Fang J et al.; Three rotenone-insensitive NADH dehydrogenases are present in the mitochondria of yeast Saccharomyces cerevisiae, which lack complex I . To elucidate the functions of these enzymes, superoxide production was determined in yeast mitochondria . The low levels of hydrogen peroxide (0.10 to 0.18 nmol/min/mg) produced in mitochondria incubated with succinate, malate, or NADH were stimulated 9-fold by antimycin A . Myxothiazol and stigmatellin blocked completely hydrogen peroxide formation with succinate or malate, indicating that the cytochrome bc(1) complex is the source of superoxide; however, these inhibitors only inhibited 46% hydrogen peroxide formation with NADH as substrate . Diphenyliodonium inhibited hydrogen peroxide formation (with NADH as substrate) by 64% . Superoxide formation, determined by EPR and acetylated cytochrome c reduction in mitochondria was stimulated by antimycin A, and partially inhibited by myxothiazol and stigmatellin . Proteinase K digestion of mitoplasts reduced 95% NADH dehydrogenase activity with a similar inhibition of superoxide production . Mild detergent treatment of the proteinase-treated mitoplasts resulted in an increase in NADH dehydrogenase activity due to the oxidation of exogenous NADH by the internal NADH dehydrogenase; however, little increase in superoxide production was observed . These results suggest that the external NADH dehydrogenase is a potential source of superoxide in S . cerevisiae mitochondria. J Biol Chem, 2003 Apr 4, 278(14), 12135 - 43 Epub 2003 Jan 31. Ubiquitin-independent mechanisms of mouse ornithine decarboxylase degradation are conserved between mammalian and fungal cells; Hoyt MA et al.; The polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is degraded by the 26 S proteasome via a ubiquitin-independent pathway in mammalian cells . Its degradation is greatly accelerated by association with the polyamine-induced regulatory protein antizyme 1 (AZ1) . Mouse ODC (mODC) that is expressed in the yeast Saccharomyces cerevisiae is also rapidly degraded by the proteasome of that organism . We have now carried out in vivo and in vitro studies to determine whether S . cerevisiae proteasomes recognize mODC degradation signals . Mutations of mODC that stabilized the protein in animal cells also did so in the fungus . Moreover, the mODC degradation signal was able to destabilize a GFP or Ura3 reporter in GFP-mODC and Ura3-mODC fusion proteins . Co-expression of AZ1 accelerated mODC degradation 2-3-fold in yeast cells . The degradation of both mODC and the endogenous yeast ODC (yODC) was unaffected in S . cerevisiae mutants with various defects in ubiquitin metabolism, and ubiquitinylated forms of mODC were not detected in yeast cells . In addition, recombinant mODC was degraded in an ATP-dependent manner by affinity-purified yeast 26 S proteasomes in the absence of ubiquitin . Degradation by purified yeast proteasomes was sensitive to mutations that stabilized mODC in vivo, but was not accelerated by recombinant AZ1 . These studies demonstrate that cell constituents required for mODC degradation are conserved between animals and fungi, and that both mammalian and fungal ODC are subject to proteasome-mediated proteolysis by ubiquitin-independent mechanisms. Indian J Exp Biol, 2002 Jan, 40(1), 95 - 100 Environmental radiation as the conditioning factor for the survival of yeast Saccharomyces cerevisiae; Gajendiran N et al.; Whether natural radiation can be a conditioning factor for the growth and survival of a living organism was investigated using diploid yeast S . cerevisiae D7 . Yeast cells were conditioned by growing them continuously for at least 100 generation in 3 different radiation background such as i) ambient radiation (1.1 mSv/y), ii) sub-ambient radiation (0.44 mSv/y, within a shielded chamber) and iii) an elevated background radiation (88 and 880 mSv/y in a gamma-field) . At the end, the cells were challenged with 60Co gamma, 100 Gy and the viable fractions were determined . Conditioning the cells in 880 mSv/y and in ambient radiation, enabled the cells to reduce the deleterious effect of the challenging dose significantly (P < 0.05) compared to that of sub-ambient radiation . The cellular viability of yeast cultures seems to be influenced by the prevailing radiation background, apart from starvation . Comparatively, a rapid decline in viability was noticed when the cultures were incubated for 60 days in the shielded chamber . The results indicate that some amount of radiation equivalent to background level or little above is needed to confer fitness in biological systems against stress factors, including radiation . The adaptive dose for the diploid yeast was also determined by single exposure . The priming dose ranged from 0.01 to 1.2 Gy . An adaptive dose of 0.25 or 0.4 Gy, almost nullified the deleterious effect of the challenging dose . The adaptive response may have a greater role in the field of cancer therapy and in radiation risk assessment . Understanding the response of an organism at different radiation-background will be helpful for successful space management. Gene, 2003 Jan 16, 303, 121 - 9 Using yeast to place human genes in functional categories; Zhang N et al.; The availability of the draft sequence of the human genome has created a pressing need to assign functions to each of the 35,000 or so genes that it defines . One useful approach for this purpose is to use model organisms for both bioinformatic and functional comparisons . We have developed a complementation system, based on the model eukaryote Saccharomyces cerevisiae, to clone human cDNAs that can functionally complement yeast essential genes . The system employs two regulatable promoters . One promoter, tetO (determining doxycycline-repressible expression), is used to control essential S . cerevisiae genes . The other, pMET3 (which is switched off in the presence of methionine), is employed to regulate the expression of mammalian cDNAs in yeast . We have demonstrated that this system is effective for both individual cDNA clones and for cDNA libraries, permitting the direct selection of functionally complementing clones . Three human cDNA libraries have been constructed and screened for clones that can complement specific essential yeast genes whose expression is switched off by the addition of doxycycline to the culture medium . The validity of each complementation was checked by showing that the yeast cells stop their growth in the presence of doxycycline and methionine, which represses the expression of the yeast and mammalian coding sequence, respectively . Using this system, we have screened 25 tetO replacement strains and succeeded in isolating human cDNAs complementing six essential yeast genes . In this way, we have uncovered a novel human ubiquitin-conjugating enzyme, have isolated a human cDNA clone that may function as a signal peptidase and have demonstrated that the functional segment of the human Psmd12 proteosome sub-unit contains a PINT domain. Wei Sheng Wu Xue Bao, 2002 Apr, 42(2), 242 - 5 {Sequencing and analysis of flocculation gene(FLO1G)}; He X et al.; The sequence of the flocculation gene (FLO1G) was determined . The result of sequcencing showed that: the cloned gene contains a large open reading frame (ORF) of 3936 bp and encodes for a protein of 1312 amino acid . According to the result of homologous analysis, the cloned gene is homologous to FLO1 but with 675 bp deletion in the ORF region . The missing part belongs to one of the four repeated sequence family of FLO1 . Since the cloned DNA fragment can trigger strong flocculence to non-flocculent strain S . cerevisiae YS58, we concluded that the missing part is not the crutical part for the flocculent ability of the gene. Yeast, 2003 Feb, 20(3), 249 - 61 Function of Candida glabrata ABC transporter gene, PDH1; Izumikawa K et al.; The rapid increase in azole resistance during treatment of patients infected with Candida glabrata may be due to increased azole efflux mediated by ABC transporters, as occurs with increased expression of PDR5 in Saccharomyces cerevisiae . Two known C . glabrata homologues of PDR5 influencing azole susceptibility are PDH1 (CgCDR2) and CgCDR1 . Disruption of PDH1 in a cgcdr1::ura3 strain increased susceptibility to rhodamine 6G, cycloheximide and chloramphenicol, and also increased rhodamine 6G accumulation, all properties of pdr5 null mutants . Overexpression of PDH1 in S . cerevisiae complemented the pdr5 mutation by reversing susceptibility to rhodamine 6G, chloramphenicol and cycloheximide, as well as by decreasing rhodamine 6G intracellular concentration . Expression of PDH1 in a C . glabrata cgcdr1::ura3 pdh1Delta::ura3 mutant using a multicopy plasmid almost completely restored the wild-type phenotype, showing that PDH1 at higher levels of expression can replace CgCDR1 . Because PDH1 and CgCDR1 have both been reported to have upstream sequences similar to the Pdr1p- and Pdr3p-binding elements of PDR5, we sought similarities in regulation between the three genes . Abundance of PDH1 and CgCDR1 mRNA in C . glabrata was increased by rhodamine 6G, cycloheximide and oligomycin, properties in common with PDR5 . PDH1, CgCDR1 and PDR5 have striking similarities in function and regulation. Wei Sheng Wu Xue Bao, 1999 Aug, 39(4), 321 - 6 {Cloning of a gene encoding cytoplasmic glycerol-3-phosphate dehydrogenase from Candida glycerolgenesis}; Wang Z et al.; The response of the yeast Saccharomyces cerevisiae to osmotic stress is to synthesis and accumulate the glycerol in order to increase the internal osmolarity and this response is controlled by the high-osmolarity glycerol (HOG) response pathway, whose important target gene is GPD1 . The increase of the activity of glycerol-3-phosphate dehydrogenase by over-expression of GPD1 gene can increase the glycerol yield greatly . In this study, a gene encoding cytoplasmic glycerol-3-phosphate dehydrogenase of Candida glycerolgenesis was cloned out by inserting Sau3AI-generated chromosomal DNA fragments into the BamHI site of a yeast-E . coli shuttle vector, YEp51 . Fifteen transformants were isolated on a supplemented minimal medium containing 50 g/L of sodium chloride from the constructed C . glycerol-genesis genomic library by using genetic complement approach . The recombinant plasmid, YEp0601, from transformant 0601, possessed the genetic markers of YEp51 and was able to restore the osmotolerance of S . cerevisiae 642(gpd1 delta, gpd2 delta) . These indicated that a gene coding for cytoplasmic glycerol-3-phosphate dehydrogenase of C . glycerolgenesis was successfully cloned out. Wei Sheng Wu Xue Bao, 2001 Aug, 41(4), 397 - 401 {Identification of Mortieralla isabellina M6-22 delta 6-fatty acid desaturase by heterologous expression in Saccharomyces cerevisiae}; Liu L et al.; Using plasmid pTMICL6 containing delta 6-fatty-acid desaturase gene from Mortieralla isabellina M6-22 as a template, 1.38 kb DNA fragment was amplified by PCR . The fragment was subcloned into the yeast-E . coli shuttle vector pYES2.0, then an expression recombinant plamid pYMID6 containing target gene was constructed . The pYMID6 was transformed into Saccharomyces cerevisiae for expression by LiAc method . It was found to exhibit delta 6-fatty acid desaturase activity in the recombinant S . cerevisiae YMID6 in the presence of exogenous fatty acid substrate linoleic acid under introduction of GAL . Expression of the delta 6-fatty acid desaturase gene under appropriate media and temperature conditions led to the production of gamma-linolenic acid reached 8.69% of the total yeast fatty acid by GC-MS detection . It is the first report about expression of M . isabellina D6D gene in S . cerevisiae. Wei Sheng Wu Xue Bao, 2001 Aug, 41(4), 391 - 6 {Cloning and expression of Trichoderma reesei endoglucanase III (EG III) gene in Saccharomyces cerevasiae}; Xiao Z et al.; Using Congo red-staining method, one positive clone with CMCase activity was isolated from the Trichoderma ressei cDNA gene bank constructed in Saccharomyces cerevasiae . Sequencing result showed that the 1.5 kb-length DNA fragment inserted in the recombinant plasmid encoded EG III gene from T . reesei . Enzymatic characterization of the EG III produced by recombinant S . cerevasiae was analyzed . The experimental results indicated that the optimum pH and temperature for EG III are 5.0 and 60 degrees C, respectively . The effects of secretory system components SSO 2 and SEB1 of S . cerevisiae on EG III secretion were examined . The results indicated that the amount of EG III secreted by the strain with SSO 2-overexpression was highest among the different recombinant S . cerevisiae strains, showed that SSO 2 is a rate-limiting component of the secretory machinery in the process of EG III secretion . Furthermore, the EG III expression level was increased 5.3 times by deletion . Furthermore, the EG III expression level was increased 5.3 times by deletion of the 98 bp in 5' untranslated region of eg3 mRNA sequence . This result suggested that the regulation region could exist in the 5' untranslated region of EG III mRNA, which is recognized by the gene expression related factors of S . cerevasiae. Wei Sheng Wu Xue Bao, 2001 Oct, 41(5), 587 - 91 {Effect of 3'-UTR of EG I from Trichoderma reesei on its gene expression in Saccharomyces cerevisiae}; Xiao Z et al.; Several industrial yeast are developed as ideal expression hosts for the production of the commercially useful proteins . The expression levels in yeast cells of the heterologous proteins are affected by the regulation factors of the genes themselves . The full-length cDNA coding for EG I from Trichoderma reesei, the cellulose-degrading filamentous fungus, was expressed in Saccharomyces cerevisiae H158 . EG I produced by the recombinant S . cerevisiae exhibits maximal activity at 50 degrees C-60 degrees C, pH 5.0 . It was observed that removal of the 3'-untranslated region (3'-UTR) from EG I cDNA resulted in no active EG I produced by recombinant yeast . RT-PCR analysis indicated that unlike the yeast cells harboring full-length EG I cDNA, there was no detectable EG I mRNA in the yeast cells harboring EG I cDNA without 3'-UTR . The data suggested that 3'-UTR is important for the expression of EG I in Saccharomyces cerevisiae. J Biol Chem, 2003 Apr 4, 278(14), 12554 - 62 Epub 2003 Jan 27. Members of the evolutionarily conserved PMT family of protein O-mannosyltransferases form distinct protein complexes among themselves; Girrbach V et al.; Protein O-mannosyltransferases (PMTs) initiate the assembly of O-mannosyl glycans, an essential protein modification . Since PMTs are evolutionarily conserved in fungi but are absent in green plants, the PMT family is a putative target for new antifungal drugs, particularly in fighting the threat of phytopathogenic fungi . The PMT family is phylogenetically classified into PMT1, PMT2, and PMT4 subfamilies, which differ in protein substrate specificity . In the model organism Saccharomyces cerevisiae as well as in many other fungi the PMT family is highly redundant, and only the simultaneous deletion of PMT1/PMT2 and PMT4 subfamily members is lethal . In this study we analyzed the molecular organization of PMT family members in S . cerevisiae . We show that members of the PMT1 subfamily (Pmt1p and Pmt5p) interact in pairs with members of the PMT2 subfamily (Pmt2p and Pmt3p) and that Pmt1p-Pmt2p and Pmt5p-Pmt3p complexes represent the predominant forms . Under certain physiological conditions, however, Pmt1p interacts also with Pmt3p, and Pmt5p with Pmt2p, suggesting a compensatory cooperation that guarantees the maintenance of O-mannosylation . Unlike the PMT1/PMT2 subfamily members, the single member of the PMT4 subfamily (Pmt4p) acts as a homomeric complex . Using mutational analyses we demonstrate that the same conserved protein domains underlie both heteromeric and homomeric interactions, and we identify an invariant arginine residue of transmembrane domain two as essential for the formation and/or stability of PMT complexes in general . Our data suggest that protein-protein interactions between the PMT family members offer a point of attack to shut down overall protein O-mannosylation in fungi. Wei Sheng Wu Xue Bao, 2000 Oct, 40(5), 518 - 22 {Effect of temperature on the activity of some enzymes representative of EMP pathway and TCA cycle in psychrophilic yeast}; Xin M et al.; Some key enzymes of EMP pathway and TCA cycle in a psychrophilic yeast Y18 were studied in this paper compared with those of Saccharomyces cerevisiae . The results indicated that fructose, 1,6-bisphosphate aldolase, succinate dehydrogenase, and hexokinase in Y18 were very thermolabile and have high activity at low temperature . These enzymes belong to cold-active enzymes . Alpha-ketoglutarate dehydrogenase existed possibly in isoenzyme which had different temperature characteristics . Citrayl synthetase was very similar in temperature characteristics to that of mesophiles . The Km value of succinate dehydrogenase both from Y18 and S . cerevisiae were studied and Some features of enzyme in psychrophiles were also discussed in this paper. Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 862 - 7 Epub 2003 Jan 22. Evolution of feedback-inhibited beta /alpha barrel isoenzymes by gene duplication and a single mutation; Hartmann M et al.; The betaalpha barrel is the common protein fold of numerous enzymes and was proposed recently to be the result of gene duplication and fusion of an ancient half-barrel . The initial enzyme of shikimate biosynthesis possesses the additional feature of feedback regulation . The crystal structure and kinetic studies on chimera and mutant proteins of yeast 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase from Saccharomyces cerevisiae inhibited by phenylalanine (Aro3p) and DAHP synthase S . cerevisiae inhibited by tyrosine (Aro4p) give insight into important regions for regulation in the enzyme: The loop, which is connecting the two half-barrels, and structural elements added to the barrel are prerequisites for regulation and form a cavity on the N-terminal side of the betaalpha barrel . In the cavity of Aro4p at position 226 is a glycine residue, which is highly conserved in all other tyrosine-regulated DAHP synthases as well . Sequence alignments with phenylalanine-regulated DAHP synthases including Aro3p show a highly conserved serine residue at this position . An exchange of glycine to serine and vice versa leads to a complete change in the regulation pattern . Therefore the evolution of these differently feedback-inhibited isoenzymes required gene duplication and a single mutation within the internal extra element . Numerous additional amino acid substitutions present in the contemporary isoenzymes are irrelevant for regulation and occurred independently. Bioinformatics, 2003 Jan 22, 19(2), 315 - 6 ARIA: automated NOE assignment and NMR structure calculation; Linge JP et al.; MOTIVATION: In the light of several ongoing structural genomics projects, faster and more reliable methods for structure calculation from NMR data are in great demand . The major bottleneck in the determination of solution NMR structures is the assignment of NOE peaks (nuclear Overhauser effect) . Due to the high complexity of the assignment problem, most NOEs cannot be directly converted into unambiguous inter-proton distance restraints . RESULTS: We present version 1.2 of our program ARIA (Ambiguous Restraints for Iterative Assignment) for automated assignment of NOE data and NMR structure calculation . We summarize recent progress in correcting for spin diffusion with a relaxation matrix approach, representing non-bonded interactions in the force field and refining final structures in explicit solvent . We also discuss book-keeping, data exchange with spectra assignment programs and deposition of the analysed experimental data to the databases . AVAILABILITY: ARIA 1.2 is available from: SUPPLEMENTARY INFORMATION: XML DTDs (for chemical shifts and NOE crosspeaks), Python scripts for the conversion of various NMR data formats and the results of example calculations using data from the S . cerevisiae HRDC domain are available from: http://www.pasteur.fr/recherche/unites/Binfs/aria/ Bioinformatics, 2003 Jan 22, 19(2), 173 - 7 Chemometric modelling based on 2D-fluorescence spectra without a calibration measurement; Solle D et al.; MOTIVATION: 2D fluorescence spectra provide information from intracellular compounds . Fluorophores like trytophan, tyrosine and phenylalanin as well as NADH and flavins make the corresponding measurement systems very important for bioprocess supervision and control . The evaluation is usually based on chemometric modelling using for their calibration procedure off-line measurements of the desired process variables . Due to the data driven approach lots of off-line measurements are required . Here a methodology is presented, which enables to perform a calibration procedure of chemometric models without any further measurement . RESULTS: The necessary information for the calibration procedure is provided by means of the a priori knowledge about the process, i.e . a mathematical model, whose model parameters are estimated during the calibration procedure, as well as the fact that the substrate should be consumed at the end of the process run . The new methodology for chemometric calibration is applied for a batch cultivation of aerobically grown S . cerevisiae on the glucose Schatzmann medium . As will be presented the chemometric models, which are determined by this method, can be used for prediction during new process runs . AVAILABILITY: The MATHLAB routine is free available on request from the authors. Biochem Biophys Res Commun, 2003 Jan 31, 301(1), 218 - 21 A yeast assay for high throughput screening of natural anti-viral agents; Srivastava R et al.; Over the last decade the yeast Saccharomyces cerevisiae has become a popular organism for studying heterologous gene expression and in vivo protein-protein interactions . Many variations of these basic systems have originated over the years . Besides these vast and varied applications of the yeast expression system, S . cerevisiae has also been used extensively in fundamental research as a model simple eukaryote . We have used the S . cerevisiae system to design a high throughput screen for anti-viral agents from natural sources . The design of the assay rests on the ability of the L-A helper virus and the M(1) satellite virus to detect small variations in -1 ribosomal frameshifting . A minor change in frameshifting efficiencies can be detected and clearly shown phenotypically in terms of zones of clearing on an agar plate . Using such a process, we have initiated a high throughput screening process for natural anti-viral agents. Mol Microbiol, 2003 Feb, 47(3), 729 - 43 The crk1 gene encodes an Ime2-related protein that is required for morphogenesis in the plant pathogen Ustilago maydis; Garrido E et al.; The fungal pathogen Ustilago maydis alternates between budding and filamentous growth during its life cycle . This dimorphic transition is regulated by environmental factors and mating . We cloned a new gene, crk1, which encodes a protein with sequence similarity to Ime2, a kinase involved in developmental choices in S . cerevisiae . Disruption of the crk1 gene in U . maydis resulted in cells that are unable to respond in an appropriate manner to environmental stimuli and show defects in morphogenesis and cell cycle adjustment to changing conditions . We have analysed the regulation of the crk1 gene and demonstrated that cAMP and MAPK pathways have opposite influences on the transcript levels for crk1 . Furthermore, we have shown that alterations in the components of these pathways impair the ability of the cellular machinery to adapt to changing conditions . These results demonstrate an important role for the crk1- encoded protein in the morphogenesis and environmental adaptation in Ustilago maydis. Anticancer Res, 2002 Sep-Oct, 22(5), 2797 - 804 Effect of S . cerevisiae APN1 protein on mammalian DNA base excision repair; Bogliolo M et al.; Mammalian cells transfected with the S . cerevisiae APN1 protein acquire resistance to oxidizing agents, the damage of which are mainly repaired via DNA base excision repair (BER) . We have recently hypothesized that this effect might be linked to the possible capacity of APN1 to accelerate mammalian BER by its 3' diesterase activity . We have investigated here the effect of pure APN1 protein on BER performed by mouse embryonic fibroblast extracts . No significant acceleration was observed in the repair of either a single AP site cleaved by the bifunctional glycosylase NTH of E . coli or the repair of a single 8-oxoguanine, initiated by the bifunctional glycosylase OGG1 . Similarly, no significant effect was observed on the repair of a single U (initiated by the monofunctional glycosylase U DNA glycosylase) or the repair of a single natural abasic site . The inability of APN1 to increase the efficiency of BER initiated by bifunctional glycosylases indicates that removal of 3' blocking fragments is not the rate limiting step of this repair pathway. J Biotechnol, 2003 Feb 27, 101(1), 81 - 7 Coexpression of BiP increased antithrombotic hirudin production in recombinant Saccharomyces cerevisiae; Kim MD et al.; In order to increase a production level of antithrombotic hirudin, BiP was simultaneously expressed in recombinant Saccharomyces cerevisiae strains carrying ten and 15 copies of the hirudin expression cassette integrated in the chromosome . Coexpression of BiP greatly enhanced both cell growth and hirudin production in recombinant S . cerevisiae . Maximum hirudin concentration of 36 mg l(-1) was obtained from batch culture of the ten copy-number transformant concomitantly harboring an episomal copy of the BiP gene under the control of the GAL1 promoter, which is corresponding to a 2.5-fold increase compared with the control strain carrying the genomic BiP gene only . The mean size of the recombinant yeast cells expressing the BiP gene remained at a relatively constant level compared with the control strains of which size increased after the onset of hirudin expression by the GAL10 promoter. Hum Genet, 2003 Feb, 112(2), 135 - 42 Epub 2002 Nov 28. Characterisation of mutations in 77 patients with X-linked myotubular myopathy, including a family with a very mild phenotype; Biancalana V et al.; X-linked myotubular myopathy is characterised by neonatal hypotonia, muscle weakness and respiratory distress in affected males, leading often to early death, although prolonged survival is observed in milder forms, or as a result of prolongation of ventilation support . It is caused by mutations in the MTM1 gene, which encodes a phosphatase called myotubularin, which has been highly conserved during evolution, down to yeasts ( S . cerevisiae and S . pombe) . To date, 251 mutations have been identified in unrelated families, corresponding to 158 different disease-associated mutations, which are widespread throughout the gene . We have found additional mutations in 77 patients, including 35 novel ones . We identified a missense mutation N180K in a 67-year-old grandfather (the oldest known patient with an MTM1 mutation), previously suspected to have autosomal centronuclear myopathy, and in his two grandsons also mildly affected . Mild and moderate phenotypes associated with novel missense mutations and with a translation initiation defect mutation are discussed, as well as severe phenotypes associated with particular novel mutations . With the present report, 192 different mutations in the MTM1 gene have been described in 328 families . The spectrum of mutations is now enlarged from the very severe classic neonatal phenotype to very mild phenotype allowing survival to the age of 67 years. Mol Microbiol, 2003 Jan, 47(2), 321 - 33 Autophagy is induced during cell death by incompatibility and is essential for differentiation in the filamentous fungus Podospora anserina; Pinan-Lucarre B et al.; In filamentous fungi, a cell death reaction occurs when cells of unlike genotype fuse . This cell death reaction, known as incompatibility reaction, is genetically controlled by a set of loci termed het loci (for heterokaryon incompatibility loci) . In Podospora anserina, genes induced during this cell death reaction (idi genes) have been identified . The idi-6/pspA gene encodes a serine protease that is the orthologue of the vacuolar protease B of Saccharomyces cerevisiae involved in autophagy . We report here that the PSPA protease participates in the degradative autophagic pathway in Podospora . We have identified the Podospora orthologue of the AUT7 gene of S . cerevisiae involved in the early steps of autophagy in yeast . This gene is induced during the development of the incompatibility reaction and was designated idi-7 . We have used a GFP-IDI7 fusion protein as a cytological marker of the induction of autophagy . Relocalization of this fusion protein and detection of autophagic bodies inside the vacuoles during the development of the incompatibility reaction provide cytological evidence of induction of autophagy during this cell death reaction . Therefore, cell death by incompatibility in fungi appears to be related to type II programmed cell death in metazoans . In addition, we found that pspA and idi-7 null mutations confer differentiation defects such as the absence of female reproductive structures, indicating that autophagy is required for differentiation in Podospora. Yeast, 2003 Jan 30, 20(2), 171 - 83 Structural characterization of chromosome I size variants from a natural yeast strain; Carro D et al.; Many yeast strains isolated from the wild show karyotype instability during vegetative growth, with rearrangement rates of up to 10(-2) chromosomal changes per generation . Physical isolation and analysis of several chromosome I size variants of one of these strains revealed that they differed only in their subtelomeric regions, leaving the central 150 Kb unaltered . Fine mapping of these subtelomeric variable regions revealed gross alterations of two very similar loci, FLO1 and FLO9 . These loci are located on the right and left arms, respectively, of chromosome I and encompass internal repetitive DNA sequences . Furthermore, some chromosome I variants lacking the FLO1 locus showed evidence of recombination at a DNA region on their right arm that is enriched in repeated sequences, including Ty LTRs . We propose that repetitive sequences in many subtelomeric regions in S . cerevisiae play a key role in karyotype hypervariability . As these regions encode several membrane-associated proteins, subtelomeric plasticity may allow rapid adaptive changes of the yeast strain to specific substrates . This pattern of semi-conservative chromosomal rearrangement may have profound implications, both in terms of evolution of wild strains and for biotechnological processes . Yeast, 2003 Jan 30, 20(2), 109 - 15 Molecular cloning and DNA analysis of a gene encoding alpha mating pheromone from the yeast Saccharomyces naganishii; Kodama T et al.; A DNA fragment encoding the precursor peptide for alpha mating pheromone was isolated from the S . naganishii genome based on the amino acid sequence of the mature pheromone . The precursor peptide contains three copies of the pheromone . Hydrophobicity analysis of the precursor peptide revealed an N-terminal signal sequence for translocation into the lumen of the endoplasmic reticulum and several signals for a series of secretion-related processes . However, upstream regulatory sequences necessary for expression of the S . cerevisiae alpha mating pheromone gene were not found, suggesting the divergence of systems that regulate alpha mating pheromone gene expression in S . naganishii and S . cerevisiae . Hybridization of a probe corresponding to the S . naganishii alpha mating pheromone nucleotide sequence to S . naganishii chromosomal DNA revealed a single gene located on either chromosome VI or VII . The S . naganishii alpha mating pheromone sequence has been deposited in the DDBJ/EMBL/GenBank data library under Accession No . AB086431 . Nature, 2003 Jan 2, 421(6918), 63 - 6 Role of duplicate genes in genetic robustness against null mutations; Gu Z et al.; Deleting a gene in an organism often has little phenotypic effect, owing to two mechanisms of compensation . The first is the existence of duplicate genes: that is, the loss of function in one copy can be compensated by the other copy or copies . The second mechanism of compensation stems from alternative metabolic pathways, regulatory networks, and so on . The relative importance of the two mechanisms has not been investigated except for a limited study, which suggested that the role of duplicate genes in compensation is negligible . The availability of fitness data for a nearly complete set of single-gene-deletion mutants of the Saccharomyces cerevisiae genome has enabled us to carry out a genome-wide evaluation of the role of duplicate genes in genetic robustness against null mutations . Here we show that there is a significantly higher probability of functional compensation for a duplicate gene than for a singleton, a high correlation between the frequency of compensation and the sequence similarity of two duplicates, and a higher probability of a severe fitness effect when the duplicate copy that is more highly expressed is deleted . We estimate that in S . cerevisiae at least a quarter of those gene deletions that have no phenotype are compensated by duplicate genes. DNA Repair (Amst), 2002 Aug 6, 1(8), 671 - 82 Characterization of the slow-growth phenotype of S . cerevisiae Whip/Mgs1 Sgs1 double deletion mutants; Branzei D et al.; RecQ DNA helicases from many organisms have been indicated to function in the maintenance of genomic stability . In human cells, mutation in the WRN helicase, a RecQ-like DNA helicase, results in the Werner syndrome (WS), a genetic disorder characterized by genomic instability and premature ageing . Similarly, mutation in SGS1, the RECQ homologue in budding yeast, results in genomic instability and accelerated ageing . We previously demonstrated that mouse WRN interacts physically with a novel, highly conserved protein that we named WHIP, and that in budding yeast cells, simultaneous deletion of WHIP/MGS1 and SGS1 results in slow growth and shortened life span . Here we show by using genetic analysis in Saccharomyces cerevisiae that mgs1Delta sgs1Delta cells have increased rates of terminal G2/M arrest, and show elevated rates of spontaneous sister chromatid recombination (SCR) and rDNA array recombination . Finally, we report that complementation of the synthetic relationship between SGS1 and WHIP/MGS1 requires both the helicase and Top3-binding activities of Sgs1, as well as the ATPase activity of Mgs1 . Our results suggest that Whip/Mgs1 is implicated in DNA metabolism, and is required for normal growth and cell cycle progression in the absence of Sgs1. DNA Repair (Amst), 2002 Aug 6, 1(8), 645 - 59 The S . cerevisiae Mag1 3-methyladenine DNA glycosylase modulates susceptibility to homologous recombination; Hendricks CA et al.; DNA glycosylases, such as the Mag1 3-methyladenine (3MeA) DNA glycosylase, initiate the base excision repair (BER) pathway by removing damaged bases to create abasic apurinic/apyrimidinic (AP) sites that are subsequently repaired by downstream BER enzymes . Although unrepaired base damage may be mutagenic or recombinogenic, BER intermediates (e.g . AP sites and strand breaks) may also be problematic . To investigate the molecular basis for methylation-induced homologous recombination events in Saccharomyces cerevisiae, spontaneous and methylation-induced recombination were studied in strains with varied MAG1 expression levels . We show that cells lacking Mag1 have increased susceptibility to methylation-induced recombination, and that disruption of nucleotide excision repair (NER; rad4) in mag1 cells increases cellular susceptibility to these events . Furthermore, expression of Escherichia coli Tag 3MeA DNA glycosylase suppresses recombination events, providing strong evidence that unrepaired 3MeA lesions induce recombination . Disruption of REV3 (required for polymerase zeta (Pol zeta)) in mag1 rad4 cells causes increased susceptibility to methylation-induced toxicity and recombination, suggesting that Pol zeta can replicate past 3MeAs . However, at subtoxic levels of methylation damage, disruption of REV3 suppresses methylation-induced recombination, indicating that the effects of Pol zeta on recombination are highly dose-dependent . We also show that overproduction of Mag1 can increase the levels of spontaneous recombination, presumably due to increased levels of BER intermediates . However, additional APN1 endonuclease expression or disruption of REV3 does not affect MAG1-induced recombination, suggesting that downstream BER intermediates (e.g . single strand breaks) are responsible for MAG1-induced recombination, rather than uncleaved AP sites . Thus, too little Mag1 sensitizes cells to methylation-induced recombination, while too much Mag1 can put cells at risk of recombination induced by single strand breaks formed during BER. DNA Repair (Amst), 2002 Aug 6, 1(8), 617 - 27 Effects of hexavalent chromium on the survival and cell cycle distribution of DNA repair-deficient S . cerevisiae; O'Brien TJ et al.; A broad spectrum of genetic damage results from exposure to hexavalent chromium . These lesions can result in DNA and RNA polymerase arrest, chromosomal aberrations, point mutations and deletions . Because of the complexity of Cr genotoxicity, the repair of Cr(VI)-induced DNA damage is poorly understood . Therefore, our aim was to investigate the sensitivities of DNA repair-deficient Saccharomyces cerevisiae strains to Cr(VI)-induced growth inhibition and lethality . Wild-type, translesion synthesis (rev3) and excision repair (apn1, ntg1, ntg2, rad1) mutants exhibited similar survival following Cr(VI) treatment (0-50mM) and underwent at least one population doubling within 2-4h post-treatment . The simultaneous loss of several excision repair genes (apn1 rad1 ntg1 ntg2) led to slower growth after Cr(VI) exposure (10mM) manifested as an initial delay in S phase progression . Higher concentrations of Cr(VI) (25mM) resulted in a prolonged transit through S phase in every strain tested . A G(2)/M arrest was evident within 1-2h after Cr(VI) treatment (10mM) in all strains and cells subsequently divided after this transient delay . In contrast to all other strains, only recombination-deficient (rad52, rad52 rev3) yeast were markedly hypersensitive towards Cr(VI) lethality . RAD52 mutant strains (rad52, rad52 rev3) also exhibited a significant delay (>6h) in the resumption of replication after Cr(VI) exposure which was related to the immediate and apparently terminal arrest of these yeast in G(2)/M after Cr(VI) treatment . These results, taken together with the recombinogenic effects of Cr(VI) in yeast containing a functional RAD52 gene, suggest that RAD52-mediated recombination is critical for the normal processing of lethal Cr-induced genetic lesions and exit from G(2) arrest . Furthermore, only the combined inactivation of multiple excision repair genes affects cell growth after Cr(VI) treatment. DNA Repair (Amst), 2003 Jan 2, 2(1), 121 - 9 The beta-lactamase motif in Snm1 is required for repair of DNA double-strand breaks caused by interstrand crosslinks in S . cerevisiae; Li X et al.; The SNM1 gene of Saccharomyces cerevisiae is specific for repair of DNA interstrand crosslinks (ICLs) . We report that the SNM1 gene functions in steps needed for the reformation of chromosomal DNA after double-strand breaks (DSBs) made in the process of ICL repair . However, SNM1 function is not needed for repair of HO endonuclease-generated DSBs . Therefore, the function of the SNM1 gene appears to act in the processing of the intermediates of the DSB repair, since the rate and extent of DSB appearance after ICL formation is normal in mutants lacking SNM1 function . The action of the SNM1 gene does not appear to depend on homologous recombination, but it does depend on an intact beta-lactamase domain conserved with Artemis, a protein required for processing of V(D)J recombination intermediates. DNA Repair (Amst), 2002 Apr 29, 1(4), 311 - 5 Epistatic analysis of the roles of the RAD27 and POL4 gene products in DNA base excision repair in S . cerevisiae; McInnis M et al.; The cellular role of the DNA polymerase encoded by the Saccharomyces cerevisiae POL4 gene is unclear . We have used an epistasis analysis to investigate whether the proteins encoded by the POL4 and RAD27 genes participate in alternative, non-redundant subpathways of DNA base excision repair (BER) . We constructed strains in which the genes were deleted singly or in combination and have examined their sensitivity to DNA damaging agents as well as spontaneous mutation frequency . The double deletion strain is no more sensitive to damaging agents and has no higher spontaneous mutation frequency than the most sensitive single mutant . These data indicate that the protein encoded by the POL4 gene does not participate in a non-redundant subpathway of base excision repair under these conditions . We discuss the implications of these results in light of the recent classification of the POL4 gene product as a member of the DNA polymerase lambda family. Mol Cell, 2002 Dec, 10(6), 1441 - 52 A role for chromatin remodeling in transcriptional termination by RNA polymerase II; Alen C et al.; Chromatin remodeling can facilitate the recruitment of RNA polymerase II (Pol II) to targeted promoters, as well as enhancing the level of transcription . Here, we describe a further key role for chromatin remodeling in transcriptional termination . Using a genetic screen in S . pombe, we identified the CHD-Mi2 class chromatin remodeling ATPase, Hrp1, as a termination factor . In S . cerevisiae, we show that transcriptional termination and chromatin structure at the 3' ends of three genes all depend on the activity of the Hrp1 homolog, Chd1p, either alone or redundantly with the ISWI ATPases, Isw1p, and Isw2p . We suggest that chromatin remodeling of termination regions is a necessary prelude to efficient Pol II termination. Glycobiology, 2002 Dec, 12(12), 821 - 8 Production in yeast of alpha-galactosidase A, a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease; Chiba Y et al.; A mammalian-like sugar moiety was created in glycoprotein by Saccharomyces cerevisiae in combination with bacterial alpha-mannosidase to produce a more economic enzyme replacement therapy for patients with Fabry disease . We introduced the human alpha-galactosidase A (alpha-GalA) gene into an S . cerevisiae mutant that was deficient in the outer chains of N-linked mannan . The recombinant alpha-GalA contained both neutral (Man(8)GlcNAc(2)) and acidic ({Man-P}(1-2)Man(8)GlcNAc(2)) sugar chains . Because an efficient incorporation of alpha-GalA into lysosomes of human cells requires mannose-6-phosphate (Man-6-P) residues that should be recognized by the specific receptor, we trimmed down the sugar chains of the alpha-GalA by a newly isolated bacterial alpha-mannosidase . Treatment of the alpha-GalA with the alpha-mannosidase resulted in the exposure of a Man-6-P residue on a nonreduced end of oligosaccharide chains after the removal of phosphodiester-linked nonreduced-end mannose . The treated alpha-GalA was efficiently incorporated into fibroblasts derived from patients with Fabry disease . The uptake was three to four times higher than that of the nontreated alpha-GalA and was inhibited by the addition of 5 mM Man-6-P . Incorporated alpha-GalA was targeted to the lysosome, and hydrolyzed ceramide trihexoside accumulated in the Fabry fibroblasts after 5 days . This method provides an effective and economic therapy for many lysosomal disorders, including Fabry disease. J Protein Chem, 2002 Aug, 21(6), 419 - 25 Increased expression and secretion of recombinant alpha-amylase in Saccharomyces cerevisiae by using glycerol as the carbon source; Wong DW et al.; Saccharomyces cerevisiae transformed with plasmids containing the barley alpha-amylase gene was cultured, and enzyme activity and cell density were monitored at various time intervals . Proteins in yeast extract and culture medium were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . Western blots of intra- and extracellular proteins were sequentially probed with anti-amylase antibody and anti-rabbit horseradish peroxidase conjugate, followed by chemiluminescent detection . The enzyme activity of recombinant barley alpha-amylase secreted by the yeast clone DY150{pYEX-Amyl} showed a significant increase when the culture medium included glycerol as the carbon source . The enhancement reached a 4.5-fold increase at 120 hr, and the effect was strain-nonspecific . Intra- and extracellular proteins increased significantly with time in both the yeast clone and the control grown in YEPG (2% yeast extract, 1% bacto-peptone, 2% glycerol) . Proteins in YEPD (2% yeast extract,1% bacto-peptone, 2% glucose) and YEPG cultures showed very different band patterns, indicating that the metabolic pathway was altered . Western blot analysis indicated that the recombinant amylase accumulated inside yeast cells, at a relatively low level, compared with that in the culture medium . The transcript level of the alpha-amylase gene was significantly increased in the clone cultured in YEPG . This investigation demonstrates that the use of glycerol as a carbon source for S . cerevisiae enhances the synthesis and secretion of the recombinant enzyme while suppressing cell growth. Curr Genet, 2002 Dec, 42(3), 169 - 78 Epub 2002 Nov 26. Inducibility studies with the arbuscular mycorrhizal fungus Glomus mosseae 3-phosphoglycerate kinase (PGK) gene promoter; Harrier LA et al.; Arbuscular mycorrhizal (AM) fungi are a multifaceted group of mutualistic symbionts that are common to terrestrial ecosystems . The role of AM fungi within processes related to carbon (C) dynamics of the plant-root-soil system are of global significance . An understanding of the C metabolism of AM fungi within the symbiotic and asymbiotic stages of their lifecycle is a necessary pre-requisite to understanding the mechanisms of C movement . This investigation studies the regulation of the Glomus mosseae (Gm) 3-phosphoglycerate kinase ( PGK) gene promoter by different C sources . The suitability of studying the GmPGK promoter (P(GmPGK)) within Saccharomyces cerevisiae was confirmed by complementation of a S . cerevisiae pgk (-) mutant with an expression vector carrying the P(GmPGK) driving the expression of the S . cerevisiae PGK gene . The inducibility of the P(GmPGK) to different C sources was studied by creating a P(GmPGK)-luciferase fusion, which was introduced into a S . cerevisiae expression vector and used to transform S . cerevisiae . The P(GmPGK)-luciferase fusion was tested for expression within S . cerevisiae by RT-PCR analysis and was shown to be expressing the luciferase gene . C upshift studies were completed to evaluate the inducibility of this promoter by different C sources . The sources raffinose, glycerol and glucose significantly induced the P(GmPGK), whereas the sources galactose, succinate and sucrose had the lowest transcriptional responses . Semi-quantitative RT-PCR was used to establish gene expression patterns of GmPGK during spore germination; and up-regulation of the GmPGK gene was observed. Curr Genet, 2002 Dec, 42(3), 153 - 60 Epub 2002 Nov 29. Genomic evidence for mating abilities in the asexual pathogen Aspergillus fumigatus; Poggeler S; The filamentous fungus Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals . It is classified as asexual because no direct observation of mating or meiosis has been reported . Sequencing of the complete genome by an international collaboration, including the Wellcome Trust Sanger Institute (UK) and The Institute for Genomic Research (TIGR, USA), has made most of the genomic sequence information from A . fumigatus publicly available . By searching the incomplete genome sequence of A . fumigatus, I have identified the coding capacity for a set of proteins that could be involved in mating and the pheromone response pathway . These include one putative mating-type gene, one gene encoding a pheromone and two pheromone-receptor genes . The mating-type gene encodes a high-mobility group domain protein exhibiting significant similarity with mating-type proteins from sexually reproducing filamentous ascomycetes . The pheromone gene is predicted to encode a precursor pheromone that is processed by a KEX2-like protease to yield a pheromone that is structurally similar to the alpha-factor of the yeast Saccharomyces cerevisiae . In addition, the deduced gene products of the receptor genes are putative seven-transmembrane proteins, which display a high-level amino acid identity with the a-factor receptor Ste3p and the alpha-receptor Ste2p of S . cerevisiae, respectively . The identification of these homologues suggests the existence of a sexual cycle in A . fumigatus. Nucleic Acids Res, 2002 Dec 15, 30(24), 5549 - 60 Discovery of novel transcription factor binding sites by statistical overrepresentation; Sinha S et al.; Understanding the complex and varied mechanisms that regulate gene expression is an important and challenging problem . A fundamental sub-problem is to identify DNA binding sites for unknown regulatory factors, given a collection of genes believed to be co-regulated . We discuss a computational method that identifies good candidates for such binding sites . Unlike local search techniques such as expectation maximization and Gibbs samplers that may not reach a global optimum, the method discussed enumerates all motifs in the search space, and is guaranteed to produce the motifs with greatest z-scores . We discuss the results of validation experiments in which this algorithm was used to identify candidate binding sites in several well studied regulons of Saccharomyces cerevisiae, where the most prominent transcription factor binding sites are largely known . We then discuss the results on gene families in the functional and mutant phenotype catalogs of S.cerevisiae, where the algorithm suggests many promising novel transcription factor binding sites . The program is available at http://bio.cs.washington.edu/software.html. J Clin Gastroenterol, 2003 Jan, 36(1), 41 - 3 Saccharomyces cerevisiae fungemia after Saccharomyces boulardii treatment in immunocompromised patients; Riquelme AJ et al.; Saccharomyces cerevisiae is widely used as a probiotic compound . Clinical data suggest that this agent is safe and effective . We report two cases of fungemia caused by S . cerevisiae occurring in immunosuppressed patients treated orally with S . boulardii Molecular typing confirmed clonality in isolate strains from patients and the capsule . Physicians caring for immunosuppressed patients must be aware of this potential serious complication of probiotic use. Xenobiotica, 2002 Nov, 32(11), 937 - 47 Comparative study of the metabolism of drug substrates by human cytochrome P450 3A4 expressed in bacterial, yeast and human lymphoblastoid cells; Andrews J et al.; 1 . The aim was to compare the metabolic activity of human CYP3A4 expressed in bacteria (E . coli), yeast (S . cerevisiae) and human lymphoblastoid cells (hBl), with the native CYP3A4 activity observed in a panel of human livers . 2 . Three CYP3A4 substrates were selected for study: dextromethorphan (DEM), midazolam (MDZ) and diazepam (DZ) . The substrate metabolism in each of the four systems was characterized by deriving the kinetic parameters K(m) or S(50), V(max) and intrinsic clearance (CL(int)) or maximum clearance (CL(max)) from the kinetic profiles; the latter differing by 100-fold across the three substrates . 3 . The K(m) or S(50) for the formation of metabolites 3-methoxymorphinan (MEM), 1'-hydroxymidazolam (1'-OH MDZ) and 3-hydroxydiazepam (3HDZ) compared well in all systems . For CYP3A4-mediated metabolism of DEM, MDZ and DZ, the V(max) for hBl microsomes were generally 2-9-fold higher than the respective yeast and human liver microsomes and E . coli membrane preparations, resulting in greater CL(int) or CL(max) . In the case of 3HDZ formation, non-linear kinetics were observed for E . coli, hBl microsomes and human liver microsomes, whereas the kinetics observed for S . cerevisiae were linear . 4 . The use of native human liver microsomes for drug metabolic studies will always be preferable . However, owing to the limited availability of human tissues, we find it is reasonable to use any of the recombinant systems described herein, since all three recombinant systems gave good predictions of the native human liver enzyme activities. Biochem J, 2003 Apr 15, 371(Pt 2), 505 - 13 Characterization of the Aspergillus nidulans transporters for the siderophores enterobactin and triacetylfusarinine C; Haas H et al.; The filamentous ascomycete Aspergillus nidulans produces three major siderophores: fusigen, triacetylfusarinine C, and ferricrocin . Biosynthesis and uptake of iron from these siderophores, as well as from various heterologous siderophores, is repressed by iron and this regulation is mediated in part by the transcriptional repressor SREA . Recently we have characterized a putative siderophore-transporter-encoding gene ( mirA ) . Here we present the characterization of two further SREA- and iron-regulated paralogues (mirB and mirC ), including the chromosomal localization and the complete exon/intron structure . Expression of mirA and mirB in a Saccharomyces cerevisiae strain, which lacks high affinity iron transport systems, showed that MIRA transports specifically the heterologous siderophore enterobactin and that MIRB transports exclusively the native siderophore triacetylfusarinine C . Construction and analysis of an A . nidulans mirA deletion mutant confirmed the substrate specificity of MIRA . Phylogenetic analysis of the available sequences suggests that the split of the species A . nidulans and S . cerevisiae predates the divergence of the paralogous Aspergillus siderophore transporters. Biochim Biophys Acta, 2002 Sep 23, 1599(1-2), 102 - 5 Effect of PPX1 inactivation on exopolyphosphatases of different cell compartments of the yeast Saccharomyces cerevisiae; Lichko L et al.; Inactivation of PPX1 encoding a major exopolyphosphatase (PPX1) in Saccharomyces cerevisiae results in a change of exopolyphosphatase spectra in the yeast cells . In the PPX1-deficient strain, an elimination of approximately 45 kDa enzyme is observed in cytosol and cell envelopes, and the activity of an exopolyphosphatase with a molecular mass of approximately 830 kDa increases 5-fold in the cytosol . These two enzyme activities differ greatly from each other not only in molecular masses but also in biochemical properties . Inactivation of PPX1 does not result in any changes in the content and properties of vacuolar exopolyphosphatase as compared with the wild strain of S . cerevisiae . In response to PPX1 mutation, exopolyphosphatase properties in the cell as a whole undergo modifications including the ability to hydrolyze polyphosphates with different lengths of the chain. Biochim Biophys Acta, 2002 Sep 23, 1599(1-2), 65 - 71 Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: theoretical and experimental study of the effect of glutamic acid 284 on the protonation state of lysine 213; Gonzalez-Nilo FD et al.; The crystal structure of Escherichia coli phosphoenolpyruvate (PEP) carboxykinase shows Lys213 is one of the ligands of enzyme-bound Mn2+ {Nat . Struct . Biol . 4 (1997) 990} . The direct coordination of Mn2+ by N(epsilon) of Lys213 is only consistent with a neutral (uncharged) Lys213, suggesting a low pKa for this residue . This work shows, through theoretical calculations and experimental analyses on homologous Saccharomyces cerevisiae PEP carboxykinase, how the microenvironment affects Mn2+ binding and the protonation state of Lys213 . We show that Glu284, a residue close to Lys212, is required for correct protonation states of Lys212 and Lys213, and for Mn2+ binding . deltaG and deltaH values for the proton reorganization processes were calculated to analyze the energetic stability of the two different protonation states of Lys212 and Lys213 in wild-type and Glu284Gln S . cerevisiae PEP carboxykinase . Calculations were done using two modeling approaches, ab-initio density functional calculations and free energy perturbation (FEP) calculations . Both methods suggest that Lys212 must be protonated and Lys213 neutral in the wild-type enzyme . On the other hand, the calculations on the Glu284Gln mutant suggest a more stable neutral Lys212 and protonated Lys213 . Experimental measurements showed 3 orders of magnitude lower activity and a threefold increase in Km for Mn2+ for Glu284Gln S . cerevisiae PEP carboxykinase when compared to wild type . The data here presented suggest that Glu284 is required for Mn2+ binding by S . cerevisiae PEP carboxykinase . We propose that Glu284 modulates the pKa value of Lys213 through electrostatic effects mediated by Yeast, 2002 Dec, 19(16), 1431 - 5 Isolation and characterization of the gene URA3 encoding the orotidine-5'-phosphate decarboxylase from Torulaspora delbrueckii; Hernandez-Lopez MJ et al.; A DNA fragment containing the URA3 gene from Torulaspora delbrueckii was isolated by complementation cloning in Saccharomyces cerevisiae . DNA sequence analysis revealed the presence of an ORF of 795 bp, encoding a 264 amino acid protein, which shares a high similarity to the Saccharomycetaceae Ura3 proteins . Furthermore, the cloned ORF fully complemented the ura3 mutation of S . cerevisiae, confirming that it encodes for the TdUra3 protein . The GeneBank Accession No . for TdURA3 is AF518402 . Yeast, 2002 Dec, 19(16), 1413 - 24 Progression of the stacked Golgi compartments in the yeast Saccharomyces cerevisiae by overproduction of GDP-mannose transporter; Hashimoto H et al.; The Golgi compartments of the yeast Saccharomyces cerevisiaeare dispersed within the cytoplasm, in contrast to the stacked cisternae in the mammalian cell, and consequently are observed as a punctate pattern by immunofluorescent staining of Golgi-marker proteins . The VIG4/VRG4 gene encodes the essential yeast GDP-mannose transporter, which is a polytopic membrane protein in the early and medial Golgi compartments . Upon overexpression of this gene by the aid of a strong promoter and multicopy vector, we found that stacked multivesicular structures, which resembled the cisternae of mammalian Golgi apparatus, had developed in S . cerevisiae . Immuno-electron microscopy showed that the GDP-mannose transporter was located on the stacked cisternae . Immuno-isolation and immunoblotting analyses of the vesicles showed that the overproduced GDP-mannose transporter also co-localized with the Golgi glycosyltransferases, but not with the ER- or late Golgi-marker proteins as in the control cell . We propose that the localization mechanism of the GDP-mannose transporter in the Golgi compartment would be efficient and hardly saturable, and therefore the overproduced protein induced a progression of Golgi-like compartments rather than being mislocalized in other compartments, such as the ER or a vacuole . Curr Biol, 2002 Dec 10, 12(23), 1992 - 2001 Genomic scale mutant hunt identifies cell size homeostasis genes in S . cerevisiae; Zhang J et al.; BACKGROUND: In most eukaryotic cells, there is a relationship between cell size and proliferative capacity . For example, in order to commit to cell division, the yeast Saccharomyces cerevisiae must attain a "critical cell size." This mechanism coordinates growth with cell division to maintain cell size homeostasis . Because very few cell size control genes are known, the genetic pathways responsible for cell size homeostasis remain obscure . Furthermore, elucidation of the mechanism of cell size homeostasis has been recalcitrant to genetic analysis primarily due to the difficulty in cloning cell size control genes . RESULTS: To identify new size control genes, the effect of 5958 single gene deletions (4792 homozygous and 1166 heterozygous gene deletions) on cell size in yeast grown to saturation was systematically determined . From these data, 49 genes were identified that dramatically altered cell size . Of these, 34 are involved in transcription, signal transduction, or cell cycle control; 88% of these genes have putative human homologs . Sixteen genes regulate cell size in a dosage-dependent manner, and the majority of mutants identified fail to correctly exit the cell cycle . Many of these genes are components of Ccr4-Not transcriptional complexes or function in the PKC-MAP kinase pathway . These genes may modulate cell size by altering the expression or activity of G1-phase cyclins . CONCLUSIONS: These results illustrate how systematic genetic screens can be used to dissect intricate biological processes that are refractory to classic genetic approaches . This genomic-wide genetic screen yielded 46 new cell size mutants and systematically assessed the effect of 5958 single gene deletions on cell size as cells exited the cell cycle. Mutagenesis, 2003 Jan, 18(1), 25 - 36 Saccharomyces cerevisiae as an eukaryotic cell model to assess cytotoxicity and genotoxicity of three anticancer anthraquinones; Buschini A et al.; The toxicity of most drugs is associated with their enzymatic conversion to toxic metabolites . Bioactivation reactions occur in a range of cellular organs and organelles, including mitochondria . We have investigated different effects (i.e . growth inhibition, mortality and genotoxicity) of doxorubicin, epirubicin and mitoxantrone on the D7 strain of Saccharomyces cerevisiae and on its petite (rho degrees ) respiratory-deficient mutant at various cellular concentrations of cytochrome P450 and glutathione (GSH) . The data confirmed the importance of oxygen production for doxorubicin toxicity . The complete absence, or a very low level, of cytochrome oxidase subunit IV conferred some resistance to doxorubicin . Low GSH levels decreased resistance to doxorubicin in both strains, suggesting that thiol depletion could potentiate membrane lipid peroxidation . Doxorubicin induction of petite colonies suggests that the drug is able to select rather than induce respiratory-deficient mutants . Epirubicin induced levels of cytotoxicity similar to those of doxorubicin . The effects did not appear to be significantly dependent on mitochondrial function or GSH levels, whereas cells were strongly protected by cytochrome P450 . GSH did not induce an evident alteration . Neither were genotoxic effects induced . Mitoxantrone had reduced levels of both growth inhibition and cytotoxicity in comparison to anthracyclines and induced convertants, revertants and aberrants . All the effects considered were amplified at high cytochrome P450 cellular concentrations, although the drug was also shown to act without previous metabolism via cytochrome P450 . Anthracenedione effectiveness was increased by metabolism via cytochrome P450 and partially reduced by GSH . However, further mechanisms were suggested, which might implicate mitochondrial function and/or production of electrophilic cytotoxic and/or genotoxic intermediates by means of GSH conjugation . The biological effectiveness of doxorubicin, epirubicin and mitoxantrone on S.cerevisiae was shown to be strictly dependent on cell-specific physiological/biochemical conditions, such as a functional respiratory chain and levels of cytochrome P450 and GSH. J Biol Chem, 2003 May 2, 278(18), 15983 - 90 Epub 2002 Dec 08. Helicase and nuclease activities of hyperthermophile Pyrococcus horikoshii Dna2 inhibited by substrates with RNA segments at 5'-end; Higashibata H et al.; Dna2 protein plays an important role in Okazaki fragment maturation on the lagging strand and also participates in DNA repair in Eukarya . Herein, we report the first biochemical characterization of a Dna2 homologue from Archaea, the hyperthermophile Pyrococcus horikoshii (Dna2Pho) . Dna2Pho has both a RecB-like nuclease motif and seven conserved helicase motifs similar to Dna2 from Saccharomyces cerevisiae . Dna2Pho has single-stranded (ss) DNA-stimulated ATPase activity, DNA helicase activity (5' to 3' direction) requiring ATP, and nuclease activity, which prefers free 5'-ends of ssDNA as substrate . These activities depend on MgCl(2) concentrations . Dna2Pho requires a higher concentration of MgCl(2) for the nuclease than helicase activity . Both the helicase and nuclease activities of Dna2Pho were inhibited by substrates with RNA segments at the 5'-end of flap DNA, whereas the nuclease activity of Dna2 from S . cerevisiae was reported to be stimulated by RNA segments in the 5'-tail (Bae, S.-H., and Seo, Y . S . (2000) J . Biol . Chem . 38022-38031). J Hosp Infect, 2002 Dec, 52(4), 268 - 72 Nosocomial transmission of Saccharomyces cerevisiae in bone marrow transplant patients; Olver WJ et al.; Saccharomyces cerevisiae is an unusual cause of clinical infection . We describe three bone marrow transplant patients on a haematology unit who developed possible invasive disease with the organism . Two patients died and both these patients appeared to have a related strain of S . cerevisiae . Screening for S . cerevisiae from throat and stool samples revealed four further patients who were carriers . Genotyping of the invasive and carriage strains demonstrated an indistinguishable strain from patients who had been on the unit at the same time, suggesting cross-infection . Int J Mol Med, 2003 Jan, 11(1), 99 - 104 Analysis of intestinal HLA-DR bound peptides and dysregulated immune responses to enteric flora in the pathogenesis of inflammatory bowel disease; Oshitani N et al.; Isolation of antigenic peptides from the MHC-groove has contributed to the understanding of T cell responses . However, these MHC-associated peptides have been isolated from various murine and human cell lines . The specific antigen responsible for the pathogenesis of inflammatory bowel disease is unknown . We examined antigenic peptides bound to the class II major histocompatibility complex (MHC) groove in human intestine by ion-trap tandem mass spectrometry equipped with online reverse-phase high performance liquid chromatography . We detected 55 parent proteins from 4 controls,