Anal Bioanal Chem, 2004 Feb, 378(3), 684 - 7 Epub 2003 Dec 05. Luminescent enzyme-linked receptor assay for estrogenic compounds; Seifert M; The analytics of endocrine-disrupting compounds has become a major issue during recent years . Several test systems have been developed for endocrine-disrupting chemicals . Yeast reporter gene assays and MCF-7 cell-based proliferation assays (E-screen) are particularly popular . A correlation of an enzyme-linked receptor assay (ELRA) with a yeast reporter gene assay is shown . In addition, the development of an ultra-sensitive luminescent ELRA with a detection limit of 20 ng/L for 17 beta-estradiol in the sample is reported . Data for real sample analysis are shown in this paper . ELRA characteristics are compared with cell-based assays, and the issue of detection limits is addressed . In this context, the detection limits of the cell-based assays have been claimed to be below the ELRA detection limits . However, it is clarified that the given detection limits for the yeast estrogen screen and the E-screen are usually based on concentrations of 17 beta-estradiol in the well, not in the sample, whereas ELRA detection limits are concentrations in the sample. Cell Cycle, 2004 Jan, 3(1), 61 - 3 Why do cells require heat shock proteins to survive heat stress? Riezman H. The cellular response to heat stress includes the induction of a group of proteins called the Heat Shock Proteins, whose functions include the synthesis of the thermoprotectant trehalose, refolding of denatured proteins, and ubiquitin- and proteasome-dependent degradation . Recent studies show that simply increasing the activity of ubiquitin- and proteasome-dependent degradation can replace the essential functions played by the induction of heat shock proteins during a heat stress . These results suggest that accumulation of denatured or aggregated proteins is the reason for the loss of cell viability due to heat stress. Science, 2003 Dec 5, 302(5651), 1765 - 8 Spatiotemporal rescue of memory dysfunction in Drosophila; McGuire SE et al.; We have developed a method for temporal and regional gene expression targeting (TARGET) in Drosophila and show the simultaneous spatial and temporal rescue of a memory defect . The transient expression of the rutabaga-encoded adenylyl cyclase in the mushroom bodies of the adult brain was necessary and sufficient to rescue the rutabaga memory deficit, which rules out a developmental brain defect in the etiology of this deficit and demonstrates an acute role for rutabaga in memory formation in these neurons . The TARGET system offers general utility in simultaneously addressing issues of when and where gene products are required. Mol Biol Cell, 2004 Feb, 15(2), 761 - 73 Epub 2003 Dec 02. Exchangeable chaperone modules contribute to specification of type I and type II Hsp40 cellular function; Fan CY et al.; Hsp40 family members regulate Hsp70s ability to bind nonnative polypeptides and thereby play an essential role in cell physiology . Type I and type II Hsp40s, such as yeast Ydj1 and Sis1, form chaperone pairs with cytosolic Hsp70 Ssa1 that fold proteins with different efficiencies and carry out specific cellular functions . The mechanism by which Ydj1 and Sis1 specify Hsp70 functions is not clear . Ydj1 and Sis1 share a high degree of sequence identity in their amino and carboxyl terminal ends, but each contains a structurally unique and centrally located protein module that is implicated in chaperone function . To test whether the chaperone modules of Ydj1 and Sis1 function in the specification of Hsp70 action, we constructed a set of chimeric Hsp40s in which the chaperone domains of Ydj1 and Sis1 were swapped to form YSY and SYS . Purified SYS and YSY exhibited protein-folding activity and substrate specificity that mimicked that of Ydj1 and Sis1, respectively . In in vivo studies, YSY exhibited a gain of function and, unlike Ydj1, could complement the lethal phenotype of sis1 Delta and facilitate maintenance of the prion {RNQ+} . Ydj1 and Sis1 contain exchangeable chaperone modules that assist in specification of Hsp70 function. Mol Biol Cell, 2004 Feb, 15(2), 468 - 80 Epub 2003 Dec 02. Multivesicular body sorting: ubiquitin ligase Rsp5 is required for the modification and sorting of carboxypeptidase S; Katzmann DJ et al.; The multivesicular body (MVB) sorting pathway provides a mechanism for delivering transmembrane proteins into the lumen of the lysosome/vacuole . Recent studies demonstrated that ubiquitin modification acts in cis as a signal for the sorting of cargoes into this pathway . Here, we present results from a genetic selection designed to identify mutants that missort MVB cargoes . This selection identified a point mutation in ubiquitin ligase Rsp5 (Rsp5-326) . At the permissive temperature, this mutant is specifically defective for ubiquitination and sorting of the ubiquitin-dependent MVB cargo precursor carboxypeptidase S (pCPS), but not ligand-induced ubiquitination of Ste2 . A previous study implicated Tul1 as the ubiquitin ligase responsible for MVB sorting of pCPS . However, we detected no defect in either the sorting or ubiquitination of pCPS in tul1 mutants . We had previously shown that Fab1 phosphatidylinositol 3-phosphate 5-kinase is also required for MVB sorting of pCPS, but not Ste2 . However, our analyses reveal that fab1 mutants do not exhibit a defect in ubiquitination of pCPS . Thus, both Rsp5 and Fab1 play distinct and essential roles in the targeting of biosynthetic MVB cargoes . However, whereas Rsp5 seems to be responsible for cargo ubiquitination, the precise role for Fab1 remains to be elucidated. Mol Biol Cell, 2004 Feb, 15(2), 896 - 907 Epub 2003 Dec 02. A conserved mechanism for Bni1- and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin; Moseley JB et al.; Formins have conserved roles in cell polarity and cytokinesis and directly nucleate actin filament assembly through their FH2 domain . Here, we define the active region of the yeast formin Bni1 FH2 domain and show that it dimerizes . Mutations that disrupt dimerization abolish actin assembly activity, suggesting that dimers are the active state of FH2 domains . The Bni1 FH2 domain protects growing barbed ends of actin filaments from vast excesses of capping protein, suggesting that the dimer maintains a persistent association during elongation . This is not a species-specific mechanism, as the activities of purified mammalian formin mDia1 are identical to those of Bni1 . Further, mDia1 partially complements BNI1 function in vivo, and expression of a dominant active mDia1 construct in yeast causes similar phenotypes to dominant active Bni1 constructs . In addition, we purified the Bni1-interacting half of the cell polarity factor Bud6 and found that it binds specifically to actin monomers and, like profilin, promotes rapid nucleotide exchange on actin . Bud6 and profilin show additive stimulatory effects on Bni1 activity and have a synthetic lethal genetic interaction in vivo . From these results, we propose a model in which Bni1 FH2 dimers nucleate and processively cap the elongating barbed end of the actin filament, and Bud6 and profilin generate a local flux of ATP-actin monomers to promote actin assembly. EMBO J, 2003 Dec 15, 22(24), 6621 - 30 Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis; Guo C et al.; Pol kappa and Rev1 are members of the Y family of DNA polymerases involved in tolerance to DNA damage by replicative bypass {translesion DNA synthesis (TLS)} . We demonstrate that mouse Rev1 protein physically associates with Pol kappa . We show too that Rev1 interacts independently with Rev7 (a subunit of a TLS polymerase, Pol zeta) and with two other Y-family polymerases, Pol iota and Pol eta . Mouse Pol kappa, Rev7, Pol iota and Pol eta each bind to the same approximately 100 amino acid C-terminal region of Rev1 . Furthermore, Rev7 competes directly with Pol kappa for binding to the Rev1 C-terminus . Notwithstanding the physical interaction between Rev1 and Pol kappa, the DNA polymerase activity of each measured by primer extension in vitro is unaffected by the complex, either when extending normal primer-termini, when bypassing a single thymine glycol lesion, or when extending certain mismatched primer termini . Our observations suggest that Rev1 plays a role(s) in mediating protein-protein interactions among DNA polymerases required for TLS . The precise function(s) of these interactions during TLS remains to be determined. EMBO J, 2003 Dec 15, 22(24), 6584 - 97 Nsl1p is essential for the establishment of bipolarity and the localization of the Dam-Duo complex; Scharfenberger M et al.; We identified a physical complex consisting of Mtw1p, an established kinetochore protein, with Nnf1p, Nsl1p and Dsn1p and have demonstrated that Nnf1p, Nsl1p and Dsn1p localize to the Saccharomyces cerevisiae kinetochore . When challenged prior to metaphase, the temperature-sensitive mutants nsl1-16 and nsl1-42 as well as Nsl1p-depleted cells failed to establish a bipolar spindle-kinetochore interaction and executed monopolar segregation of sister chromatids . In contrast, an nsl1-16 defect could not be evoked after the establishment of bipolarity . The observed phenotype is characteristic of that of mutants with defects in the protein kinase Ipl1p or components of the Dam-Duo kinetochore complex . However nsl1 mutants did not exhibit a defect in microtubule-kinetochore untethering as the ipl1-321 mutant does . Instead, they exhibited a severe defect in the kinetochore localization of the Dam-Duo complex suggesting this to be the cause for the failure of nsl1 cells to establish bipolarity . Moreover the analysis of Nsl1p-depleted cells indicated that Nsl1p is required for the spindle checkpoint and kinetochore integrity. EMBO J, 2003 Dec 15, 22(24), 6562 - 72 A La protein requirement for efficient pre-tRNA folding; Chakshusmathi G et al.; The La protein protects the 3' ends of many nascent small RNAs from exonucleases . Here we report that La is required for efficient folding of certain pre-tRNAs . A mutation in pre-tRNA(Arg)(CCG) causes yeast cells to be cold-sensitive and to require the La protein Lhp1p for efficient growth . When the mutant cells are grown at low temperature, or when Lhp1p is depleted, mature tRNA(Arg)(CCG) is not efficiently aminoacylated . The mutation causes the anticodon stem of pre-tRNA(Arg)(CCG) to misfold into an alternative helix in vitro . Intragenic suppressor mutations that disrupt the misfolded helix or strengthen the correct helix alleviate the requirement for Lhp1p, providing evidence that the anticodon stem misfolds in vivo . Chemical and enzymatic footprinting experiments suggest a model in which Lhp1p stabilizes the correctly folded stem . Lhp1p is also required for efficient aminoacylation of two wild-type tRNAs when yeast are grown at low temperature . These experiments reveal that pre-tRNAs can require protein assistance for efficient folding in vivo. EMBO J, 2003 Dec 15, 22(24), 6448 - 57 Ribosome binding to the Oxa1 complex facilitates co-translational protein insertion in mitochondria; Szyrach G et al.; The Oxa1 translocase of the mitochondrial inner membrane facilitates the insertion of both mitochondrially and nuclear-encoded proteins from the matrix into the inner membrane . Most mitochondrially encoded proteins are hydrophobic membrane proteins which are integrated into the lipid bilayer during their synthesis on mitochondrial ribosomes . The molecular mechanism of this co-translational insertion process is unknown . Here we show that the matrix-exposed C-terminus of Oxa1 forms an alpha-helical domain that has the ability to bind to mitochondrial ribosomes . Deletion of this Oxa1 domain strongly diminished the efficiency of membrane insertion of subunit 2 of cytochrome oxidase, a mitochondrially encoded substrate of the Oxa1 translocase . This suggests that co-translational membrane insertion of mitochondrial translation products is facilitated by a physical interaction of translation complexes with the membrane-bound translocase. Genome Res, 2003 Dec, 13(12), 2568 - 76 From gene networks to gene function; Schlitt T et al.; We propose a novel method to identify functionally related genes based on comparisons of neighborhoods in gene networks . This method does not rely on gene sequence or protein structure homologies, and it can be applied to any organism and a wide variety of experimental data sets . The character of the predicted gene relationships depends on the underlying networks;they concern biological processes rather than the molecular function . We used the method to analyze gene networks derived from genome-wide chromatin immunoprecipitation experiments, a large-scale gene deletion study, and from the genomic positions of consensus binding sites for transcription factors of the yeast Saccharomyces cerevisiae . We identified 816 functional relationships between 159 genes and show that these relationships correspond to protein-protein interactions, co-occurrence in the same protein complexes, and/or co-occurrence in abstracts of scientific articles . Our results suggest functions for seven previously uncharacterized yeast genes: KIN3 and YMR269W may be involved in biological processes related to cell growth and/or maintenance, whereas IES6, YEL008W, YEL033W, YHL029C, YMR010W, and YMR031W-A are likely to have metabolic functions. Int J Oncol, 2004 Jan, 24(1), 115 - 25 The role of p53 in the chemotherapeutic responses to cisplatin, doxorubicin and 5-fluorouracil treatment; Dart DA et al.; A panel of tumour models used extensively for in vivo evaluation of new drugs was characterised for their p53 status . Basal p53 protein levels were measured by immunodetection on both formalin-fixed tumour tissue and from protein extracts of fresh tumours . High levels of nuclear-specific staining, indicative of p53 mutation, was seen in 15/25 tumours, with the remainder showing intermittent or no staining . The functional status of p53 cDNA from these tumours was assayed within the functional analysis of separated alleles in yeast (F.A.S.A.Y.) reporter system . The cDNA from those tumours with high levels of p53 protein showed 14/15 failing to activate the reporter gene . The cDNA from tumours with low or non-detectable p53 levels showed 8/10 with wild-type p53 . Tumours were grown subcutaneously in mice (n=10) . Each mouse was given maximum tolerated doses for either doxorubicin, 5-fluorouracil or cisplatin . Tumour volumes were measured daily, alongside untreated controls . The specific growth delay values for each tumour were separated into two groups, those with functional p53 (wild-type) and those without (mutant and null status) . The Mann-Whitney U test was performed on the groups of data, to evaluate differences in their response on the basis of p53 status . Cisplatin was moderately active against tumours with wild-type and mutant p53 genes with no significant difference seen between both groups . However, a significant difference in specific growth delay was seen between the two groups when treated with doxorubicin or 5-fluorouracil (P=0.05), indicating a role for p53 protein in modulating the in vivo efficacy of these agents. FEMS Yeast Res, 2003 Dec, 4(3), 323 - 7 Further development of the cassette-based pYC plasmid system by incorporation of the dominant hph, nat and AUR1-C gene markers and the lacZ reporter system; Hansen J et al.; Dominant selection markers encoding hygromycin B phosphotransferase (hph), nourseothricin N-acetyltransferase (nat) and a mutant inositol phosphoceramide synthase (AUR1-C) were all incorporated into the pYC yeast plasmid vector system, thus expanding this system with possible alternatives to the use of G418 resistance . We found the markers to be of use not only in standard laboratory strains of Saccharomyces cerevisiae but also in an industrial strain of S . carlsbergensis (syn . of S . pastorianus) brewing yeast as well as in Saccharomyces kluyveri . As the pYC system contains means of counter-selection for plasmid loss and loop-out of integrated plasmids, it now provides ample opportunities for genetic manipulation of industrial and non-conventional yeasts when the URA3 marker and FOA counter-selection is not an option . Furthermore, the lacZ system for analyzing gene expression was included in the system. FEMS Yeast Res, 2003 Dec, 4(3), 315 - 21 The delivery of ADP/ATP carrier protein to mitochondria probed by fusions with green fluorescent protein and beta-galactosidase; Polcicova K et al.; The import of proteins into mitochondria is an essential process, largely investigated in vitro with isolated mitochondria and radioactively labeled precursors . In this study, we used intact cells and fusions with genes encoding two reporter proteins, green fluorescent protein (GFP) and beta-galactosidase (lacZ), to probe the import of the ADP/ATP carrier (AAC) . Typical mitochondrial fluorescence was observed with AAC-GFP fusions containing at least one complete transmembrane loop . This confirms the results of in vitro analysis demonstrating that an internal targeting signal was present in each one of the three transmembrane loops of the carrier . The fusions of AAC fragments to beta-galactosidase demonstrated that the targeting signal was capable of delivering the reporter molecule to the mitochondrial surface, but not to internalize it to a protease-inaccessible location . The delivery to a protease-inaccessible location required the presence of more distal sequences present within the third (C-terminal) transmembrane loop of the carrier molecule . The results of our study provide an alternative for investigation in a natural context of mitochondrial protein import in cells when the isolation of intact, functional mitochondria is not achievable. Biochem J, 2004 Mar 15, 378(Pt 3), 889 - 98 Plant sterol biosynthesis: identification of two distinct families of sterol 4alpha-methyl oxidases; Darnet S et al.; In plants, the conversion of cycloartenol into functional phytosterols requires the removal of the two methyl groups at C-4 by an enzymic complex including a sterol 4alpha-methyl oxidase (SMO) . We report the cloning of candidate genes for SMOs in Arabidopsis thaliana, belonging to two distinct families termed SMO1 and SMO2 and containing three and two isoforms respectively . SMO1 and SMO2 shared low sequence identity with each other and were orthologous to the ERG25 gene from Saccharomyces cerevisiae which encodes the SMO . The plant SMO amino acid sequences possess all the three histidine-rich motifs (HX3H, HX2HH and HX2HH), characteristic of the small family of membrane-bound non-haem iron oxygenases that are involved in lipid oxidation . To elucidate the precise functions of SMO1 and SMO2 gene families, we have reduced their expression by using a VIGS (virus-induced gene silencing) approach in Nicotiana benthamiana . SMO1 and SMO2 cDNA fragments were inserted into a viral vector and N . benthamiana inoculated with the viral transcripts . After silencing with SMO1, a substantial accumulation of 4,4-dimethyl-9beta,19-cyclopropylsterols (i.e . 24-methylenecycloartanol) was obtained, whereas qualitative and quantitative levels of 4alpha-methylsterols were not affected . In the case of silencing with SMO2, a large accumulation of 4alpha-methyl-Delta7-sterols (i.e . 24-ethylidenelophenol and 24-ethyllophenol) was found, with no change in the levels of 4,4-dimethylsterols . These clear and distinct biochemical phenotypes demonstrate that, in contrast with animals and fungi, in photosynthetic eukaryotes, these two novel families of cDNAs are coding two distinct types of C-4-methylsterol oxidases controlling the level of 4,4-dimethylsterol and 4alpha-methylsterol precursors respectively. Biochem Biophys Res Commun, 2003 Dec 26, 312(4), 1266 - 72 WD dipeptide motifs and LXXLL motif of chicken HIRA are necessary for transcription repression and the latter motif is essential for interaction with histone deacetylase-2 in vivo; Ahmad A et al.; We previously reported not only that chicken HIRA, a homolog of Saccharomyces cerevisiae transcriptional corepressors Hir1p and Hir2p, possesses seven WD dipeptide motifs and a LXXLL motif in its N-terminal half and C-terminal half, respectively, but also that the N-terminal and C-terminal halves, respectively, bind to CAF-1p48 and HDAC-1 and -2 in vitro . Seven WD dipeptide motifs in the N-terminal half of HIRA are required for the in vitro interaction with CAF-1p48 . The LXXLL motif at positions 993-997 of HIRA is necessary for the in vitro interaction with HDAC-2 . Here we revealed not only that the N-terminal and C-terminal halves of HIRA mediate individually transcription repressions but also that even one of the seven WD dipeptide motifs and the LXXLL motif of HIRA are essential for the mediations in vivo . Moreover, the LXXLL motif is essential for the interaction with endogenous or recombinant HDAC-2 in vivo, probably resulting in formation of the active complex, harboring the HDAC activity . Taken together, these results indicate that HIRA should participate differentially in a number of DNA-utilizing processes, including transcription repressions, through interactions of its distinct regions with CAF-1p48 and HDAC-2, respectively. Cell, 2003 Nov 26, 115(5), 508 - 10 Rad53: a controller ensuring the fine-tuning of histone levels; Quivy JP et al.; Checkpoint proteins are activated in response to genotoxic insults or replication stress to maintain genome integrity . Their function is believed to depend largely on the detection of the DNA damage or defects occurring during replication fork progression. Biochem J, 2004 Mar 1, 378(Pt 2), 665 - 71 A novel omega3-fatty acid desaturase involved in the biosynthesis of eicosapentaenoic acid; Pereira SL et al.; Long-chain n-3 PUFAs (polyunsaturated fatty acids) such as EPA (eicosapentaenoic acid; 20:5 n-3) have important therapeutic and nutritional benefits in humans . In plants, cyanobacteria and nematodes, omega3-desaturases catalyse the formation of these n-3 fatty acids from n-6 fatty acid precursors . Here we describe the isolation and characterization of a gene ( sdd17 ) derived from an EPA-rich fungus, Saprolegnia diclina, that encodes a novel omega3-desaturase . This gene was isolated by PCR amplification of an S . diclina cDNA library using oligonucleotide primers corresponding to conserved regions of known omega3-desaturases . Expression of this gene in Saccharomyces cerevisiae, in the presence of various fatty acid substrates, revealed that the recombinant protein could exclusively desaturate 20-carbon n-6 fatty acid substrates with a distinct preference for ARA (arachidonic acid; 20:4 n-6), converting it into EPA . This activity differs from that of the known omega3-desaturases from any organism . Plant and cyanobacterial omega3-desaturases exclusively desaturate 18-carbon n-6 PUFAs, and a Caenorhabditis elegans omega3-desaturase preferentially desaturated 18-carbon PUFAs over 20-carbon substrates, and could not convert ARA into EPA when expressed in yeast . The sdd17 -encoded desaturase was also functional in transgenic somatic soya bean embryos, resulting in the production of EPA from exogenously supplied ARA, thus demonstrating its potential for use in the production of EPA in transgenic oilseed crops. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2140 - 9 Epub 2003 Nov 27. Refined structure of Pyrococcus furiosus ornithine carbamoyltransferase at 1.87 A; Massant J et al.; Using synchrotron radiation, X-ray data have been collected from Pyrococcus furiosus ornithine carbamoyltransferase (Pfu OTCase) to a maximal resolution of 1.87 A, allowing the refinement of a previous structure at 2.7 A {Villeret et al . (1998), Proc . Natl Acad . Sci . USA, 95, 2801-2806} . Thanks to the high resolution of this refined structure, two sulfate ions and 191 water molecules could be localized directly from the electron-density maps . The identification of these molecules allowed a more rigorous description of the active site and the identification of residues involved in binding carbamoyl phosphate . The improved quality of the model resulted in a better definition of several loops and the various interfaces . The dodecameric protein is composed of four catalytic trimers disposed in a tetrahedral manner . The extreme thermal stability of Pfu OTCase is mainly the result of the strengthening of the intersubunit interactions in a trimer and oligomerization of the trimers into a dodecamer . Interfaces between monomers in a catalytic trimer are characterized by an increase in ion-pair networks compared with mesophilic OTCases . However, the interfaces between catalytic trimers in the dodecameric oligomer are mainly hydrophobic and also involve aromatic-aromatic and cation-pi interactions. Mol Pharmacol, 2003 Dec, 64(6), 1549 - 56 Pharmacological and genetic analysis of 90-kDa heat shock isoprotein-aryl hydrocarbon receptor complexes; Cox MB et al.; The 90-kDa heat shock protein (Hsp90) is an abundant chaperone that regulates a diverse set of intracellular signaling proteins . Drugs that inhibit Hsp90 activity have been useful in the identification of novel Hsp90-dependent signaling pathways . One class of inhibitory compounds disrupts Hsp90-dependent processes by binding to the N-terminal ATPase/p23-binding domain of Hsp90, whereas a second inhibitor class binds within the C-terminal domain . We used signaling by aryl hydrocarbon receptor (AhR), an Hsp90-dependent transcription factor, as a functional probe to study the effects of Hsp90 inhibitors in yeast strains with deletion mutations of individual Hsp90 and p23 cochaperone genes . The more abundant and constitutively expressed Hsp90 isoform, Hsc82, functioned best in supporting AhR signaling . Deletion of the more inducible isoform, Hsp82, had no effect on signaling . AhR complexes containing Hsc82 were preferentially sensitive to the effects of low concentrations of the N-terminal inhibitors radicicol and herbimycin A . However, both Hsp90 isoforms were equally sensitive to the AhR-specific effects of novobiocin, which binds to the C terminus . Hsp90 inhibitors had no preferential effects on AhR signaling in strains that lacked p23, suggesting that the inhibitors exert their effects through a p23-independent mechanism . In contrast, overexpression of p23 buffered the effects of radicicol and herbimycin A, but not novobiocin, on AhR signaling . The data collectively suggest preferential use or function of the Hsc82 isoprotein in AhR signaling and provide new insight into the effects of three structurally unrelated Hsp90 inhibitors. Mol Cell Biol, 2003 Dec, 23(24), 9283 - 92 Novel methyltransferase for modified uridine residues at the wobble position of tRNA; Kalhor HR et al.; We have identified a novel tRNA methyltransferase in Saccharomyces cerevisiae that we designate Trm9 . This enzyme, the product of the YML014w gene, catalyzes the esterification of modified uridine nucleotides, resulting in the formation of 5-methylcarbonylmethyluridine in tRNA(Arg3) and 5-methylcarbonylmethyl-2-thiouridine in tRNA(Glu) . In intact yeast cells, disruption of the TRM9 gene results in the complete loss of these modified wobble bases and increased sensitivity at 37 degrees C to paromomycin, a translational inhibitor . These results suggest a role for this potentially reversible methyl esterification reaction when cells are under stress. Mol Cell Biol, 2003 Dec, 23(24), 9178 - 88 The replication fork barrier site forms a unique structure with Fob1p and inhibits the replication fork; Kobayashi T; The replication fork barrier site (RFB) is an approximately 100-bp DNA sequence located near the 3' end of the rRNA genes in the yeast Saccharomyces cerevisiae . The gene FOB1 is required for this RFB activity . FOB1 is also necessary for recombination in the ribosomal DNA (rDNA), including increase and decrease of rDNA repeat copy number, production of extrachromosomal rDNA circles, and possibly homogenization of the repeats . Despite the central role that Foblp plays in both replication fork blocking and rDNA recombination, the molecular mechanism by which Fob1p mediates these activities has not been determined . Here, I show by using chromatin immunoprecipitation, gel shift, footprinting, and atomic force microscopy assays that Fob1p directly binds to the RFB . Fob1p binds to two separated sequences in the RFB . A predicted zinc finger motif in Fob1p was shown to be essential for the RFB binding, replication fork blocking, and rDNA recombination activities . The RFB seems to wrap around Fob1p, and this wrapping structure may be important for function in the rDNA repeats. Mol Cell Biol, 2003 Dec, 23(24), 9251 - 61 Ubiquitin depletion as a key mediator of toxicity by translational inhibitors; Hanna J et al.; Cycloheximide acts at the large subunit of the ribosome to inhibit translation . Here we report that ubiquitin levels are critical for the survival of Saccharomyces cerevisiae cells in the presence of cycloheximide: ubiquitin overexpression confers resistance to cycloheximide, while a reduced ubiquitin level confers sensitivity . Consistent with these findings, ubiquitin is unstable in yeast (t(1/2) = 2 h) and is rapidly depleted upon cycloheximide treatment . Cycloheximide does not noticeably enhance ubiquitin turnover, but serves principally to block ubiquitin synthesis . Cycloheximide also induces UBI4, the polyubiquitin gene . The cycloheximide-resistant phenotype of ubiquitin overexpressors is also characteristic of partial-loss-of-function proteasome mutants . Ubiquitin is stabilized in these mutants, which may account for their cycloheximide resistance . Previous studies have reported that ubiquitin is destabilized in the absence of Ubp6, a proteasome-associated deubiquitinating enzyme, and that ubp6 mutants are hypersensitive to cycloheximide . Consistent with the model that cycloheximide-treated cells are ubiquitin deficient, the cycloheximide sensitivity of ubp6 mutants can be rescued either by ubiquitin overexpression or by mutations in proteasome subunit genes . These results also show that ubiquitin wasting in ubp6 mutants is proteasome mediated . Ubiquitin overexpression rescued cells from additional translational inhibitors such as anisomycin and hygromycin B, suggesting that ubiquitin depletion may constitute a widespread mechanism for the toxicity of translational inhibitors. Mol Cell Biol, 2003 Dec, 23(24), 9136 - 49 Global control of histone modification by the anaphase-promoting complex; Ramaswamy V et al.; Acetylation and phosphorylation of the amino-terminal tails of the core histones fluctuate on a global scale in concert with other major events in chromosome metabolism . A ubiquitin ligase, the anaphase-promoting complex (APC), controls events in chromosome metabolism such as sister chromatid cohesion and may regulate H3 phosphorylation by targeting Aurora A, one of several S10-directed H3 kinases in vertebrate cells, for destruction by the proteasome . Our analysis of apc10Delta and apc11(ts) loss-of-function mutants reveals that the APC controls the global level of H3 S10 phosphorylation in cycling yeast cells . Surprisingly, it also regulates dephosphorylation of H3 and global deacetylation of H2B, H3, and H4 during exit from the cell cycle into G(0) . Genetic, biochemical, and microarray analyses suggest that APC-dependent cell cycle control of H3 phosphorylation is exerted at the level of an Aurora H3 kinase, Ipl1p, while APC-dependent transcriptional induction of GLC7, an essential H3 phosphatase, contributes to sustained H3 dephosphorylation upon cell cycle withdrawal . Collectively, our results establish that core histone acetylation state and H3 phosphorylation are physiologically regulated by the APC and suggest a model in which global reconfiguration of H3 phosphorylation state involves APC-dependent control of both an H3 kinase and a conserved phosphatase. Biosystems, 2003 Dec, 72(3), 229 - 39 Genetic network inference: the effects of preprocessing; Lindlof A et al.; Clustering of gene expression data and gene network inference from such data has been a major research topic in recent years . In clustering, pairwise measurements are performed when calculating the distance matrix upon which the clustering is based . Pairwise measurements can also be used for gene network inference, by deriving potential interactions above a certain correlation or distance threshold . Our experiments show how interaction networks derived by this simple approach exhibit low-but significant-sensitivity and specificity . We also explore the effects that normalization and prefiltering have on the results of methods for identifying interactions from expression data . Before derivation of interactions or clustering, preprocessing is often performed by applying normalization to rescale the expression profiles and prefiltering where genes that do not appear to contribute to regulation are removed . In this paper, different ways of normalizing in combination with different distance measurements are tested on both unfiltered and prefiltered data, different prefiltering criteria are considered. Mutat Res, 2003 Nov 27, 532(1-2), 245 - 53 G2 and spindle assembly checkpoint adaptation, and tetraploidy arrest: implications for intrinsic and chemically induced genomic instability; Andreassen PR et al.; While checkpoints that act in S-phase are essential to the maintenance of genomic stability, these checkpoints do not act alone . Additionally, G2 DNA damage checkpoints, the spindle assembly checkpoint, and a post-mitotic G1 tetraploidy checkpoint act subsequent to DNA replication to ensure genetic fidelity in cell division . In this review, we will examine how these checkpoints cooperate in the maintenance of genomic stability in response to either DNA damage or cytoskeletal disruption . Since the G2 and spindle assembly checkpoints are subject to adaptation, we will discuss how the G1 tetraploidy checkpoint acts in concert with these checkpoints to mediate stable arrest . We will also probe the relationship of these checkpoints by exploring common features of their regulation . Finally, the consequences of malfunction of these checkpoints for both intrinsic and chemically induced genomic instability will be examined . Among these consequences are aneuploidization, extranumerary centrosomes, and micronucleation. Mutat Res, 2003 Nov 27, 532(1-2), 117 - 35 Role of the error-free damage bypass postreplication repair pathway in the maintenance of genomic stability; Smirnova M et al.; The postreplication repair pathway (PRR) is composed of error-free and error-prone sub-pathways that allow bypass of DNA damage-induced replication-blocking lesions . The error-free sub-pathway is also used for bypass of spontaneous DNA damage and functions in cooperation with recombination pathways . In diploid yeast cells, error-free PRR is needed to prevent genomic instability, which is manifest as loss of heterozygosity (LOH) events of increased chromosome loss and recombination . Homologous recombination acts synergistically with the error-free damage avoidance branch of PRR to prevent chromosome loss . The DNA damage checkpoint gene MEC1 acts synergistically with the PRR pathway in maintaining genomic stability . Integration of the PRR pathway with other cellular pathways for preventing genomic instability is discussed . In diploid strains, the most dramatic increase is in the abnormality of chromosome loss when a repair or damage detection pathway is defective. Mutat Res, 2003 Nov 27, 532(1-2), 29 - 40 Functions of mammalian Cdc7 kinase in initiation/monitoring of DNA replication and development; Kim JM et al.; Cdc7 kinase plays an essential role in firing of replication origins by phosphorylating components of the replication complexes . Cdc7 kinase has also been implicated in S phase checkpoint signaling downstream of the ATR and Chk1 kinases . Inactivation of Cdc7 in yeast results in arrest of cell growth with 1C DNA content after completion of the ongoing DNA replication . In contrast, conditional inactivation of Cdc7 in undifferentiated mouse embryonic stem (ES) cells leads to growth arrest with rapid cessation of DNA synthesis, suggesting requirement of Cdc7 functions for continuation of ongoing DNA synthesis . Furthermore, loss of Cdc7 function induces recombinational repair (nuclear Rad51 foci) and G2/M checkpoint responses (inhibition of Cdc2 kinase) . Eventually, p53 becomes highly activated and the cells undergo massive p53-dependent apoptosis . Thus, defective origin activation in mammalian cells can generate DNA replication checkpoint signals . Efficient removal of those cells in which replication has been perturbed, through cell death, may be beneficial to maintain the highest level of genetic integrity in totipotent stem cells . Partial, rather than total, loss of Cdc7 kinase expression results in retarded growth at both cellular and whole body levels, with especially profound impairment of germ cell development. Fungal Genet Biol, 2004 Jan, 41(1), 75 - 88 Aspergillus nidulans hypA regulates morphogenesis through the secretion pathway; Shi X et al.; Aspergillus nidulans hypA encodes a predicted 1474 amino acid, 161.9 kDa cytoplasmic peptide . Strains with hypA1 and hypA6 alleles are wild type at 28 degrees C but have wide, slow-growing hyphae and thick walls at 42 degrees C . hypA1 and hypA6 have identical genetic lesions . hypA1 and hypA6 restrictive phenotypes have statistically similar morphometry, and strains with either allele can conidiate at 42 degrees C . hypA deletion strains require osmotic support and have aberrant morphology, but produce viable spores at 28 degrees C . hypA has full-length orthologs in filamentous fungi and yeasts and a 200 amino acid region with similarity to sequences in plants and animals . The Saccharomyces cerevisiae hypA ortholog is TRS120, a regulatory subunit in the TRAPP II complex that mediates traffic through the Golgi equivalent . Enzyme secretion is reduced in hypA1 cells at 42 degrees C . Endomembranes and cytoplasmic actin arrays in hypA1 have weak polarity at 42 degrees C and cytoplasmic microtubules have reduced number and normal distribution. Fungal Genet Biol, 2004 Jan, 41(1), 13 - 22 Aspergillus nidulans RhoA is involved in polar growth, branching, and cell wall synthesis; Guest GM et al.; Growth of the filamentous fungus Aspergillus nidulans begins when the conidium breaks dormancy and grows isotropically . Eventually a germ tube emerges and the axis of growth remains fixed in the primary hypha while new growth axes are established basally to form secondary germ tubes and lateral branches . Rho1 is a Rho family GTPase that has been shown to be involved in polarity establishment and cell wall deposition in Saccharomyces cerevisiae . A gene predicted to encode a Rho1 homolog was cloned from A . nidulans and named rhoA . Strains carrying ectopic copies of the constitutively active rhoA(G14V) allele or the dominant rhoA(E40I) allele were created and characterized . The constitutively active rhoA(G14V) strain grew slowly relative to wild type and showed an abnormal clustered pattern of branch emergence . The rhoA(G14V) strain also labeled intensely with calcofluor, showed elevated levels of cell wall N-acetylglucosamine and had unusually thick cell walls . The dominant rhoA(E40I)strain was accelerated in the emergence of secondary and tertiary germ tubes, and lateral branches relative to wild type and showed lysis with prolonged incubation . The rhoA(E40I) strain also was hypersensitive to the cell wall disrupting agents calcofluor and caspofungin acetate and showed an increase in cell wall N-acetylglucosamine levels . Our results suggest that rhoA plays a role in polarity, proper branching pattern, and cell wall deposition. Biochem Soc Trans, 2003 Dec, 31(Pt 6), 1140 - 2 Mechanism of glucose sensing in the small intestine; Dyer J et al.; Sensing nutrients is a fundamental task for all living cells . For most eukaryotic cells glucose is a major source of energy, having significant and varied effects on cell function . Interest in identifying mechanisms by which cells sense and respond to variations in glucose concentration has increased recently . The epithelial cells lining the intestinal tract are exposed, from the luminal domain, to an environment with continuous and massive fluctuations in the levels of dietary monosaccharides . Enterocytes therefore have to sense and respond to the significant changes in the levels of luminal sugars, and regulate the expression of the intestinal glucose transporter (Na+/glucose co-transporter, SGLT1) accordingly . Our data, using a combination of in vivo and in vitro model systems, suggest that glucose in the lumen of the intestine is sensed by a glucose sensor residing on the external face of the enterocyte luminal membrane . Glucose binds to the sensor and generates an intracellular signal leading to enhancement in the expression of SGLT1 . The generated signal is independent of glucose metabolism and is likely to operate via a G-protein-coupled receptor and cAMP/protein kinase A signalling cascade. Arzneimittelforschung, 2003, 53(10), 738 - 43 Synthesis, in vitro/in vivo antifungal evaluation and structure-activity relationship study of 3(2H)-pyridazinones; Karolyhazy L et al.; The synthesis, in vitro/in vivo antifungal evaluation and a structure-activity relationship (SAR) study of 3(2H)-pyridazinones was carried out . The results reported here may be helpful in the structural identification and understanding of the minimum structural requirements for these molecules acting as antifungal agents . In addition, the most active structure in this series was tested for its capacity of inhibiting Saccharomyces cerevisiae beta 1,3-glucan synthase and chitin synthase, enzymes that catalyze the synthesis of the major polymers of the fungal cell wall. Planta, 2004 Mar, 218(5), 784 - 92 Epub 2003 Nov 26. Kinetic properties of a micronutrient transporter from Pisum sativum indicate a primary function in Fe uptake from the soil; Cohen CK et al.; Fe uptake in dicotyledonous plants is mediated by a root plasma membrane-bound ferric reductase that reduces extracellular Fe(III)-chelates, releasing Fe(2+) ions, which are then absorbed via a metal ion transporter . We previously showed that Fe deficiency induces an increased capacity to absorb Fe and other micronutrient and heavy metals such as Zn(2+) and Cd(2+) into pea ( Pisum sativum L.) roots {Cohen et al . (1998) Plant Physiol 116:1063-1072) . To investigate the molecular basis for this phenomenon, an Fe-regulated transporter that is a homologue of the Arabidopsis IRT1 micronutrient transporter was isolated from pea seedlings . This cDNA clone, designated RIT1 for root iron transporter, encodes a 348 amino acid polypeptide with eight putative membrane-spanning domains that is induced under Fe deficiency and can functionally complement yeast mutants defective in high- and low-affinity Fe transport . Chelate buffer techniques were used to control Fe(2+) in the uptake solution at nanomolar activities representative of those found in the rhizosphere, and radiotracer methodologies were employed to show that RIT1 is a very high-affinity (59)Fe(2+) uptake system ( K(m) =54-93 nM) . Additionally, radiotracer ((65)Zn, (109)Cd) flux techniques were used to show that RIT can also mediate a lower affinity Zn and Cd influx ( K(m) of 4 and 100 microM, for Zn(2+) and Cd(2+), respectively) . These findings suggest that, in typical agricultural soils, RIT1 functions primarily as a high-affinity Fe(2+) transporter that mediates root Fe acquisition . This is consistent with recent findings with Arabidopsis IRT1 knockout mutants that strongly suggest that this transporter plays a key role in root Fe uptake and nutrition . However, the ability of RIT1 to facilitate Zn and Cd uptake when these metals are present at elevated concentrations suggests that RIT1 may be one pathway for the entry of toxic metals into the food chain . Furthermore, the finding that plant Fe deficiency status may promote heavy metal uptake via increased expression of this transporter could have implications both for human nutrition and also for phytoremediation, the use of terrestrial plants to sequester toxic metals from contaminated soil. Curr Genet, 2004 Feb, 45(2), 96 - 103 Epub 2003 Nov 26. A novel beta-glucosidase in Uromyces fabae: feast or fight? Haerter AC, Voegele RT. Efficient nutrient mobilization is a key element for biotrophic plant parasites such as the rust fungi . In the course of a cDNA library screen for elements involved in sugar utilization in Uromyces fabae, we identified a sequence with homology to beta-glucosidases . Full-length genomic and cDNA clones of the gene, termed BGL1, were isolated and sequenced . The BGL1 gene comprises 3,372 nucleotides, including nine introns . The open reading frame encompasses 2,532 bases and codes for a polypeptide of 843 amino acids with an apparent molecular mass of 92.4 kDa . Analysis of the polypeptide revealed a potential secretion signal, indicating an extracellular localization of mature BGL1p (89.8 kDa) . BGL1 seems to be expressed in all stages of growth, including haustoria, the feeding structures of rust fungi . In the course of immunolocalization studies, the gene product BGL1p was localized in the periphery of intercellular hyphae and haustoria . On the basis of sequence homology, the BGL1 gene was identified as a fungal beta-glucosidase. Oncogene, 2004 Feb 12, 23(6), 1206 - 13 Checkpoint-mediated control of replisome-fork association and signalling in response to replication pausing; Lucca C et al.; The replication checkpoint controls the integrity of replicating chromosomes by stabilizing stalled forks, thus preventing the accumulation of abnormal replication and recombination intermediates that contribute to genome instability . Checkpoint-defective cells are susceptible to rearrangements at chromosome fragile sites when replication pauses, and certain human cancer prone diseases suffer checkpoint abnormalities . It is unclear as to how the checkpoint stabilizes stalled forks and how cells sense replication blocks . We have analysed the checkpoint contribution in controlling replisome-fork association when replication pauses . We show that in yeast wild-type cells, stalled forks exhibit stable replisome complexes and the checkpoint sensors Ddc1 and Ddc2, thus activating Rad53 checkpoint kinase . Ddc1/Ddc2 recruitment on stalled forks and Rad53 activation are influenced by the single-strand-binding protein replication factor A (RFA) . rad53 forks exhibit a defective association with DNA polymerases alpha, epsilon and delta . Further, in rad53 mutants, stalled forks progressively generate abnormal structures that turn into checkpoint signals by accumulating RFA, Ddc1 and Ddc2 . We suggest that, following replication blocks, checkpoint activation mediated by RFA-ssDNA filaments stabilizes stalled forks by controlling replisome-fork association, thus preventing unscheduled recruitment of recombination enzymes that could otherwise cause the pathological processing of the forks. Nat Genet, 2004 Jan, 36(1), 69 - 76 Epub 2003 Nov 30. Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex; Sheen VL et al.; Disruption of human neural precursor proliferation can give rise to a small brain (microcephaly), and failure of neurons to migrate properly can lead to an abnormal arrest of cerebral cortical neurons in proliferative zones near the lateral ventricles (periventricular heterotopia) . Here we show that an autosomal recessive condition characterized by microcephaly and periventricular heterotopia maps to chromosome 20 and is caused by mutations in the gene ADP-ribosylation factor guanine nucleotide-exchange factor-2 (ARFGEF2) . By northern-blot analysis, we found that mouse Arfgef2 mRNA levels are highest during embryonic periods of ongoing neuronal proliferation and migration, and by in situ hybridization, we found that the mRNA is widely distributed throughout the embryonic central nervous system (CNS) . ARFGEF2 encodes the large (>200 kDa) brefeldin A (BFA)-inhibited GEF2 protein (BIG2), which is required for vesicle and membrane trafficking from the trans-Golgi network (TGN) . Inhibition of BIG2 by BFA, or by a dominant negative ARFGEF2 cDNA, decreases cell proliferation in vitro, suggesting a cell-autonomous regulation of neural expansion . Inhibition of BIG2 also disturbed the intracellular localization of such molecules as E-cadherin and beta-catenin by preventing their transport from the Golgi apparatus to the cell surface . Our findings show that vesicle trafficking is an important regulator of proliferation and migration during human cerebral cortical development. Science, 2004 Jan 16, 303(5656), 343 - 8 Epub 2003 Nov 26. ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex; Mizuguchi G et al.; The conserved histone variant H2AZ has an important role in the regulation of gene expression and the establishment of a buffer to the spread of silent heterochromatin . How histone variants such as H2AZ are incorporated into nucleosomes has been obscure . We have found that Swr1, a Swi2/Snf2-related adenosine triphosphatase, is the catalytic core of a multisubunit, histone-variant exchanger that efficiently replaces conventional histone H2A with histone H2AZ in nucleosome arrays . Swr1 is required for the deposition of histone H2AZ at specific chromosome locations in vivo, and Swr1 and H2AZ commonly regulate a subset of yeast genes . These findings define a previously unknown role for the adenosine triphosphate-dependent chromatin remodeling machinery. Plant Physiol, 2003 Dec, 133(4), 1630 - 42 Epub 2003 Nov 26. Overexpression of a mutant basic helix-loop-helix protein HFR1, HFR1-deltaN105, activates a branch pathway of light signaling in Arabidopsis; Yang KY et al.; The HFR1, a basic helix-loop-helix protein, is required for a subset of phytochrome A-mediated photoresponses in Arabidopsis . Here, we show that overexpression of the HFR1-deltaN105 mutant, which lacks the N-terminal 105 amino acids, confers exaggerated photoresponses even in darkness . Physiological analysis implied that overexpression of HFR1-deltaN105 activated constitutively a branch pathway of light signaling that mediates a subset of photomorphogenic responses, including germination, de-etiolation, gravitropic hypocotyl growth, blocking of greening, and expression of some light-regulated genes such as CAB, DRT112, PSAE, PSBL, PORA, and XTR7, without affecting the light-responsiveness of anthocyanin accumulation and expression of other light-regulated genes such as CHS and PSBS . Although the end-of-day far-red light response and petiole elongation were suppressed in the HFR1-deltaN105-overexpressing plants, flowering time was not affected by HFR1-deltaN105 . In addition, the HFR1-deltaN105-overexpressing plants showed hypersensitive photoresponses in the inhibition of hypocotyl elongation, dependently on phytochrome A, FHY1, and FHY3 under FR light or phyB under R light, respectively . Moreover, our double mutant analysis suggested that the hypersensitive photoresponse is due to functional cooperation between HFR1-deltaN105 and other light-signaling components including HY5, a basic leucine zipper protein . Taken together, our results of gain-of-function approach with HFR1-deltaN105 suggest the existence of a complex and important basic helix-loop-helix protein-mediated transcriptional network controlling a branch pathway of light signaling and provide a useful framework for further genetic dissection of light-signaling network in Arabidopsis. J Neurosci, 2003 Nov 26, 23(34), 10756 - 64 Mechanism of toxicity in rotenone models of Parkinson's disease; Sherer TB et al.; Exposure of rats to the pesticide and complex I inhibitor rotenone reproduces features of Parkinson's disease, including selective nigrostriatal dopaminergic degeneration and alpha-synuclein-positive cytoplasmic inclusions (Betarbet et al., 2000; Sherer et al., 2003) . Here, we examined mechanisms of rotenone toxicity using three model systems . In SK-N-MC human neuroblastoma cells, rotenone (10 nm to 1 microm) caused dose-dependent ATP depletion, oxidative damage, and death . To determine the molecular site of action of rotenone, cells were transfected with the rotenone-insensitive single-subunit NADH dehydrogenase of Saccharomyces cerevisiae (NDI1), which incorporates into the mammalian ETC and acts as a "replacement" for endogenous complex I . In response to rotenone, NDI1-transfected cells did not show mitochondrial impairment, oxidative damage, or death, demonstrating that these effects of rotenone were caused by specific interactions at complex I . Although rotenone caused modest ATP depletion, equivalent ATP loss induced by 2-deoxyglucose was without toxicity, arguing that bioenergetic defects were not responsible for cell death . In contrast, reducing oxidative damage with antioxidants, or by NDI1 transfection, blocked cell death . To determine the relevance of rotenone-induced oxidative damage to dopaminergic neuronal death, we used a chronic midbrain slice culture model . In this system, rotenone caused oxidative damage and dopaminergic neuronal loss, effects blocked by alpha-tocopherol . Finally, brains from rotenone-treated animals demonstrated oxidative damage, most notably in midbrain and olfactory bulb, dopaminergic regions affected by Parkinson's disease . These results, using three models of increasing complexity, demonstrate the involvement of oxidative damage in rotenone toxicity and support the evaluation of antioxidant therapies for Parkinson's disease. J Biol Chem, 2004 Feb 20, 279(8), 7055 - 63 Epub 2003 Nov 25. Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling; Varadan R et al.; Diverse cellular events are regulated by post-translational modification of substrate proteins via covalent attachment of one or a chain of ubiquitin molecules . The outcome of (poly)ubiquitination depends upon the specific lysine residues involved in the formation of polyubiquitin chains . Lys48-linked chains act as a universal signal for proteasomal degradation, whereas Lys63-linked chains act as a specific signal in several non-degradative processes . Although it has been anticipated that functional diversity between alternatively linked polyubiquitin chains relies on linkage-dependent differences in chain conformation/topology, direct structural evidence in support of this model has been lacking . Here we use NMR methods to determine the structure of a Lys63-linked di-ubiquitin chain . The structure is characterized by an extended conformation, with no direct contact between the hydrophobic residues Leu8, Ile44, and Val70 on the ubiquitin units . This structure contrasts with the closed conformation observed for Lys48-linked di-ubiquitin wherein these residues form the interdomain interface (Cook, W . J., Jeffrey, L . C., Carson, M., Zhijian, C., and Pickart, C . M . (1992) J . Biol . Chem . 267, 16467-16471; Varadan, R., Walker, O., Pickart, C., and Fushman, D . (2002) J . Mol . Biol . 324, 637-647) . Consistent with the open conformation of the Lys(63)-linked di-ubiquitin, our binding studies show that both ubiquitin domains in this chain can bind a ubiquitin-associated domain from HHR23A independently and in a mode similar to that for mono-ubiquitin . In contrast, Lys48-linked di-ubiquitin binds in a different, higher affinity mode that has yet to be determined . This is the first experimental evidence that alternatively linked polyubiquitin chains adopt distinct conformations. Adv Food Nutr Res, 2003, 47, 73 - 112 The nutritional significance, metabolism and toxicology of selenomethionine; Schrauzer GN; SeMet is a naturally occurring toxic amino acid but at the same time represents the major nutritional source of selenium for higher animals and humans . The ability of SeMet to be incorporated into the body proteins in place of Met furthermore provides a means of reversible Se storage in organs and tissues . This property is not shared by any other naturally occurring selenoamino acid and thus could be associated with a specific physiological function of SeMet . Since higher animals cannot synthesize SeMet, yet from it all needed forms of Se are produced, SeMet meets the criteria of an essential amino acid . Accordingly, SeMet, or enriched food sources thereof, are appropriate forms of Se for human nutritional Se supplementation . However, while SeMet or Se yeast are already widely used in over-the-counter nutritional supplements, infant formulas and parenteral feeding mixtures still contain Se in the form of sodium selenate or sodium selenite, even though these are not the normal nutritional forms of Se . In animal nutrition, these inorganic selenium salts are increasingly replaced by food sources of SeMet such as Se yeast . Synthetic SeMet could also be employed as a feed additive, but its regulatory status is as yet undetermined . The optimal nutritional levels of SeMet for different animal species still need to be determined . The expectation is that lower additions to feedstock of equivalent levels of SeMet will suffice to achieve adequacy than currently approved maximum levels of Se in the form of inorganic Se salts. J Cell Biol, 2003 Nov 24, 163(4), 707 - 13 A J-protein is an essential subunit of the presequence translocase-associated protein import motor of mitochondria; Truscott KN et al.; Transport of preproteins into the mitochondrial matrix is mediated by the presequence translocase-associated motor (PAM) . Three essential subunits of the motor are known: mitochondrial Hsp70 (mtHsp70); the peripheral membrane protein Tim44; and the nucleotide exchange factor Mge1 . We have identified the fourth essential subunit of the PAM, an essential inner membrane protein of 18 kD with a J-domain that stimulates the ATPase activity of mtHsp70 . The novel J-protein (encoded by PAM18/YLR008c/TIM14) is required for the interaction of mtHsp70 with Tim44 and protein translocation into the matrix . We conclude that the reaction cycle of the PAM of mitochondria involves an essential J-protein. Infect Immun, 2003 Dec, 71(12), 7109 - 18 Phenotypic switching and mating type switching of Candida glabrata at sites of colonization; Brockert PJ et al.; Candida glabrata switches spontaneously at high frequency among the following four graded phenotypes discriminated on agar containing 1 mM CuSO(4): white, light brown, dark brown (DB), and very dark brown . C . glabrata also contains three mating type loci with a configuration similar to that of the Saccharomyces cerevisiae mating type cassette system, suggesting it may also undergo cassette switching at the expression locus MTL1 . To analyze both reversible, high-frequency phenotypic switching and mating type switching at sites of colonization, primary samples from the oral cavities and vaginal canals of three patients suffering from C . glabrata vaginitis were clonally plated on agar containing CuSO(4) . It was demonstrated that (i) in each vaginitis patient, there was only one colonizing strain; (ii) an individual could have vaginal colonization without oral colonization; (iii) phenotypic switching occurred at sites of colonization; (iv) the DB phenotype predominated at the site of infection in all three patients; (v) genetically unrelated strains switched in similar, but not identical, fashions and caused vaginal infection; (vi) different switch phenotypes of the same strain could simultaneously dominate different body locations in the same host; (vii) pathogenesis could be caused by cells in different mating type classes; and (viii) mating type switching demonstrated at both the genetic and transcription levels occurred in one host. Curr Opin Genet Dev, 2003 Dec, 13(6), 636 - 43 Turning the clock back on ancient genome duplication; Seoighe C; Complete genome sequence data led rapidly to the conclusion that ancient genome duplications had shaped the genomes of the model organisms Saccharomyces cerevisiae and Arabidopsis thaliana . Recent contributions have gone on to refine date estimates for these duplications and, in the case of Arabidopsis, to infer additional, more ancient, rounds of duplication by reconstructing gene order before the most recent duplication event . It is becoming widely accepted that an ancient duplication occurred before the radiation of the ray-finned fish . However, despite methodological advances and the availability of complete genome sequence data the debate over whether very ancient genome duplications have occurred early in the vertebrate lineage has not yet been fully resolved. Gene, 2003 Dec 4, 321, 173 - 83 MdAP, a novel protein in apple, is associated with the major allergen Mal d 1; Puehringer HM et al.; Mal d 1, an 18-kDa intracellular pathogenesis-related protein (PR-10), has been known since long as the major apple allergen in Middle and Northern Europe . However, its biological function, as that of many other PR-10 proteins, is still unknown . In order to identify proteins putatively interacting with Mal d 1, an expression library of Malus domestica was screened using the yeast-two-hybrid (Y2H) system . A novel protein binding to two isoforms of Mal d 1 being used as 'bait' was isolated . The deduced amino acid sequence from the corresponding full-length cDNA of the predicted Mal d 1-Associated-Protein (MdAP) do not display any homology to known proteins, but shares 45% identity with a 'hypothetical protein' in Arabidopsis thaliana . Southern analysis of the apple genome indicated that MdAP, comprising 190 amino acids, is encoded by a single gene . The expression pattern of the 1-kb MdAP transcript resembled the expression profile of the different Mal d 1 isoforms in various apple organs, however at a much lower level . Furthermore, a huge variation in transcription levels of Mal d 1 isoforms was observed in apple tissue . For both, Mal d 1 and MdAP highest amounts of mRNAs were measured in ripe fruits and significantly lower amounts in vegetative tissue by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Mol Cell, 2003 Nov, 12(5), 1333 - 40 Chromatin remodeling in vivo: evidence for a nucleosome sliding mechanism; Fazzio TG et al.; Members of the ISWI family of chromatin remodeling factors exhibit ATP-dependent nucleosome sliding, loading, and spacing activities in vitro . However, it is unclear which of these activities are utilized by ISWI complexes to remodel chromatin in vivo . We therefore sought to identify the mechanisms of chromatin remodeling by Saccharomyces cerevisiae Isw2 complex at its known sites of action in vivo . To address this question, we developed a method of identifying intermediates of the Isw2-dependent chromatin remodeling reaction as it proceeded . We show that Isw2 complex catalyzes nucleosome sliding at two different classes of target genes in vivo, in each case sliding nucleosomes closer to the promoter regions . In contrast to its biochemical activities in vitro, nucleosome sliding by Isw2 complex in vivo is unidirectional and localized to a few nucleosomes at each site, suggesting that Isw2 activity is constrained by cellular factors. Mol Cell, 2003 Nov, 12(5), 1325 - 32 Methylation of histone H3 K4 mediates association of the Isw1p ATPase with chromatin; Santos-Rosa H et al.; Set1p methylates lysine 4 (K4) of histone H3 and regulates the expression of many genes in yeast . Here we use a biochemical approach to identify a protein, Isw1p, which recognizes chromatin preferentially when it is di- and trimethylated at K4 H3 . We show that on certain actively transcribed genes, the Isw1p chromatin remodeling ATPase requires K4 H3 methylation to associate with chromatin in vivo . Analysis of one such gene, MET16, shows that the enzymatic activities of Set1p and Isw1p are functionally connected: Set1p methylation and Isw1p ATPase generate specific chromatin changes at the 5' end of the gene, are necessary for the correct distribution of RNA polymerase II over the coding region, and are required for the recruitment of the cleavage and polyadenylation factor Rna15p . These results indicate that K4 H3 methylation and Isw1p ATPase activity are intimately linked in regulating transcription of certain genes in yeast. Mol Cell, 2003 Nov, 12(5), 1239 - 50 Active and inactive orientations of the transmembrane and cytosolic domains of the erythropoietin receptor dimer; Seubert N et al.; Binding of erythropoietin to the erythropoietin receptor (EpoR) extracellular domain orients the transmembrane (TM) and cytosolic regions of the receptor dimer into an unknown activated conformation . By replacing the EpoR extracellular domain with a dimeric coiled coil, we engineered TM EpoR fusion proteins where the helical TM domains were constrained into seven possible relative orientations . We identify one dimeric TM conformation that imparts full activity to the cytosolic domain of the receptor and signals via JAK2, STAT proteins, and MAP kinase, one partially active orientation that preferentially activates MAP kinase, and one conformation corresponding to the inactive receptor . The active and inactive conformations were independently identified by computational searches for low-energy TM dimeric structures . We propose a specific EpoR-activated interface and suggest its use for structural and signaling studies. Cell, 2003 Oct 31, 115(3), 355 - 67 The AAA-ATPase Cdc48/p97 regulates spindle disassembly at the end of mitosis; Cao K et al.; Spindle disassembly at the end of mitosis is a complex and poorly understood process . Here, we report that the AAA-ATPase Cdc48/p97 and its adapters Ufd1-Npl4, which have a well-established role in membrane functions, also regulate spindle disassembly by modulating microtubule dynamics and bundling at the end of mitosis . In the absence of p97-Ufd1-Npl4 function, microtubules in Xenopus egg extracts remain as monopolar spindles attached to condensed chromosomes after Cdc2 kinase activity has returned to the interphase level . Consequently, interphase microtubule arrays and nuclei are not established . Genetic analyses of Cdc48, the yeast homolog of p97, reveal that Cdc48 is also required for disassembly of mitotic spindles after execution of the mitotic exit pathway . Furthermore, Cdc48/p97-Ufd1-Npl4 directly binds to spindle assembly factors and regulates their interaction with microtubules at the end of mitosis . Therefore, Cdc48/p97-Ufd1-Npl4 is an essential chaperone that regulates transformation of the microtubule structure as cells reenter interphase. Biochemistry, 2003 Dec 2, 42(47), 13869 - 78 Near native structure in an RNA collapsed state; Buchmueller KL et al.; Many large RNAs form conformationally collapsed, but non-native, states prior to folding to the native state or assembling with protein cofactors . Although RNA collapsed states play fundamental roles in RNA folding and ribonucleoprotein assembly processes, their structures have been poorly understood . We obtained 12 high-quality structural constraints for the collapsed state formed by the catalytic core of the bI5 intron RNA using site-specific cross-linking mediated by a short-lived reactant . RNA tertiary structures in the collapsed and native states are indistinguishable, even though only the native state forms a solvent-inaccessible core . Thus, structural neighbors in the collapsed state, including several long-range tertiary interactions, are approximately as close in space as in the native state, but RNA packing is sufficiently loose or dynamic to allow access by solvent . Binding by the obligate CBP2 protein cofactor has almost no effect on structural neighbors reported by cross-linking, even though protein binding chases the RNA from the collapsed state to the native state . Protein binding thus appears to promote only the final few angstroms of RNA folding rather than mediate global conformational rearrangements in the catalytic core . The bI5 RNA collapsed state functions to self-chaperone ribonucleoprotein assembly because this conformationally restrained structure lies very near that of the native state and excludes structures that otherwise misassemble efficiently. Nat Cell Biol, 2003 Dec, 5(12), 1090 - 4 Epub 2003 Nov 23. Securin and B-cyclin/CDK are the only essential targets of the APC; Thornton BR et al.; The anaphase-promoting complex/cyclosome (APC) is a highly conserved ubiquitin ligase that controls passage through the cell cycle by targeting many proteins for proteolysis . The complex is composed of at least thirteen core subunits, eight of which are essential, and two activating subunits, Cdc20 (essential) and Cdh1/Hct1 (non-essential) . Previously, it was not known which APC targets are sufficient to explain the essential nature of the complex . Here, we show that each of the eight normally essential APC subunits is rendered non-essential ('bypass-suppressed') by the simultaneous removal/inhibition of the APC substrates securin (Pds1) and B-type cyclin/CDK (Clb/CDK) . In strains lacking the APC, levels of Clb2 and Clb3 remain constant, but Clb/CDK activity oscillates as cells cycle . This suggests that in the absence of B-type cyclin destruction, oscillation of the Clb/CDK-inhibitor Sic1 is sufficient to trigger the feedback loops necessary for the bi-stable nature of Clb/CDK activity . These results strongly suggest that securin and B-type cyclin/CDK activity are the only obligatory targets of the APC in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A, 2003 Dec 9, 100(25), 14695 - 700 Epub 2003 Nov 21. The RNA polymerase III transcriptome revealed by genome-wide localization and activity-occupancy relationships; Roberts DN et al.; RNA polymerase III (Pol III) transcribes small untranslated RNAs, such as tRNAs . To define the Pol III transcriptome in Saccharomyces cerevisiae, we performed genome-wide chromatin immunoprecipitation using subunits of Pol III, TFIIIB and TFIIIC . Virtually all of the predicted targets of Pol III, as well as several novel candidates, were occupied by Pol III machinery . Interestingly, TATA box-binding protein occupancy was greater at Pol III targets than virtually all Pol II targets, and the highly occupied Pol II targets are generally strongly transcribed . The temporal relationships between factor occupancy and gene activity were then investigated at selected targets . Nutrient deprivation rapidly reduced both Pol III transcription and Pol III occupancy of both a tRNA gene and RPR1 . In contrast, TFIIIB remained bound, suggesting that TFIIIB release is not a critical aspect of the onset of repression . Remarkably, TFIIIC occupancy increased dramatically during repression . Nutrient addition generally reestablished transcription and initial occupancy levels . Our results are consistent with active Pol III displacing TFIIIC, and with inactivation/release of Pol III enabling TFIIIC to bind, marking targets for later activation . These studies reveal new aspects of the kinetics, dynamics, and targets of the Pol III system. J Biol Chem, 2004 Feb 13, 279(7), 6163 - 70 Epub 2003 Nov 21. The meiosis-specific protein kinase Ime2 directs phosphorylation of replication protein A; Clifford DM et al.; In Saccharomyces cerevisiae, the cellular single-stranded DNA-binding protein replication protein A (RPA) becomes phosphorylated during meiosis in two discrete reactions . The primary reaction is first observed shortly after cells enter the meiotic program and leads to phosphorylation of nearly all the detectable RPA . The secondary reaction, which requires the ATM/ATR homologue Mec1, is induced upon initiation of recombination and only modifies a fraction of the total RPA . We now report that correct timing of both RPA phosphorylation reactions requires Ime2, a meiosis-specific protein kinase that is critical for proper initiation of meiotic progression . Expression of Ime2 in vegetative cells leads to an unscheduled RPA phosphorylation reaction that does not require other tested meiosis-specific kinases and is distinct from the RPA phosphorylation reaction that normally occurs during mitotic growth . In addition, immunoprecipitated Ime2 catalyzes phosphorylation of purified RPA . Our data strongly suggest that Ime2 is an RPA kinase in vivo . We propose that Ime2 directly catalyzes RPA phosphorylation in the primary reaction and indirectly promotes the Mec1-dependent secondary reaction by advancing cells through meiotic progression . Our studies have identified a novel meiosis-specific reaction that targets a key protein required for DNA replication, repair, and recombination . This pathway could be important in differentiating mitotic and meiotic DNA metabolism. J Biol Chem, 2004 Feb 20, 279(8), 6794 - 804 Epub 2003 Nov 21. DNA recognition by the homing endonuclease PI-SceI involves a divalent metal ion cofactor-induced conformational change; Noel AJ et al.; PI-SceI, a homing endonuclease of the LAGLIDADG family, consists of two domains involved in DNA cleavage and protein splicing, respectively . Both domains cooperate in binding the recognition sequence . Comparison of the structures of PI-SceI in the absence and presence of substrate reveals major conformational changes in both the protein and DNA . Notably, in the protein-splicing domain the loop comprising residues 53-70 and adopts a "closed" conformation, thus enabling it to interact with the DNA . We have studied the dynamics of DNA binding and subsequent loop movement by fluorescence techniques . Six amino acids in loop53-70 were individually replaced by cysteine and modified by fluorescein . The interaction of the modified PI-SceI variants with the substrate, unlabeled or labeled with tetramethylrhodamine, was analyzed in equilibrium and stopped-flow experiments . A kinetic scheme was established describing the interaction between PI-SceI and DNA . It is noteworthy that the apparent hinge-flap motion of loop53-70 is only observed in the presence of a divalent metal ion cofactor . Substitution of the major Mg2+-binding ligands in PI-SceI, Asp-218 and Asp-326, by Asn or "nicking" PI-SceI with trypsin at Arg-277, which interferes with formation of an active enzyme.substrate complex, both prevent the conformational change of loop53-70 . Deletion of the loop inactivates the enzyme . We conclude that loop53-70 is an important structural element that couples DNA recognition by the splicing domain with DNA cleavage by the catalytic domain and as such "communicates" with the Mg2+ binding sites at the catalytic centers. J Comput Biol, 2003, 10(5), 791 - 802 PaTre: a method for paralogy trees construction; Pisanti N et al.; Genomes can be described as a collection of clusters, the gene families, whose members are called paralogs . Paralogs are genes that most probably share duplication history and show a significant similarity in their sequences, even if they perform slightly different biological function . Among the different mechanisms that have led to an increase of the genomic information during biological evolution, gene duplication is probably the most important . To better understand duplication events, the first step is to investigate the history of the gene families in order to detect which duplication events have taken place, and in which relative (partial) order . Here we present a method, called PaTre, that, given a gene family, attempts to construct the paralogy tree of the family . We will work under the hypothesis that every family member derives from a duplication process of another member . By the term paralogy tree, we mean a directed tree in which the root represents the most ancient paralog of the family and each oriented arc (a, b) represents the existence of a duplication event from the template gene a to its copy b . Notice that gene a survives the event and can serve as a template of more than one duplication event; in fact, there can be more than one arc leaving a . PaTre uses new algorithmic techniques motivated by the specific application at hand . The reliability of the inferential process has been tested by means of a simulator that implements different hypotheses on the duplication-with-modification paradigm and on three examples of different biological gene families, belonging either to lower and higher organisms. Anal Chem, 2003 Aug 15, 75(16), 4081 - 6 Web and database software for identification of intact proteins using "top down" mass spectrometry; Taylor GK et al.; For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available . We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions . Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker . Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results . The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search . Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer . A +142-Da delta(m) on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp90 and Asp192, consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation . ProSight PTM is the first search engine and Web environment for identification of intact proteins . Science, 2003 Nov 21, 302(5649), 1399 - 401 Nucleolar clustering of dispersed tRNA genes; Thompson M et al.; Early transfer RNA (tRNA) processing events in Saccharomyces cerevisiae are coordinated in the nucleolus, the site normally associated with ribosome biosynthesis . To test whether spatial organization of the tRNA pathway begins with nucleolar clustering of the genes, we have probed the subnuclear location of five different tRNA gene families . The results show that tRNA genes, though dispersed in the linear genome, colocalize with 5S ribosomal DNA and U14 small nucleolar RNA at the nucleolus . Nucleolar localization requires tRNA gene transcription-complex formation, because inactivation of the promoter at a single locus removes its nucleolar association . This organization of tRNA genes must profoundly affect the spatial packaging of the genome and raises the question of whether gene types might be coordinated in three dimensions to regulate transcription. Plant Physiol, 2004 Jan, 134(1), 147 - 60 Epub 2003 Nov 20. Differential expression of sucrose transporter and polyol transporter genes during maturation of common plantain companion cells; Ramsperger-Gleixner M et al.; The cDNAs of two sorbitol transporters, common plantain (Plantago major) polyol transporter (PLT) 1 and 2 (PmPLT1 and PmPLT2), were isolated from a vascular bundle-specific cDNA library from common plantain, a dicot plant transporting Suc plus sorbitol in its phloem . Here, we describe the kinetic characterization of these sorbitol transporters by functional expression in Brewer's yeast (Saccharomyces cerevisiae) and in Xenopus sp . oocytes and for the first time the localization of plant PLTs in specific cell types of the vascular tissue . In the yeast system, both proteins were shown to be uncoupler sensitive and could be characterized as low-affinity and low-specificity polyol symporters . The Km value for the physiological substrate sorbitol is 12 mm for PmPLT1 and even higher for PmPLT2, which showed an almost linear increase in sorbitol transport rates up to 20 mm . These data were confirmed in the Xenopus sp . system, where PmPLT1 was analyzed in detail and characterized as a H+ symporter . Using peptide-specific polyclonal antisera against PmPLT1 or PmPLT2 and simultaneous labeling with the monoclonal antiserum 1A2 raised against the companion cell-specific PmSUC2 Suc transporter, both PLTs were localized to companion cells of the phloem in common plantain source leaves . These analyses revealed two different types of companion cells in the common plantain phloem: younger cells expressing PmSUC2 at higher levels and older cells expressing lower levels of PmSUC2 plus both PLT genes . The putative role of these low-affinity transporters in phloem loading is discussed. Genes Dev, 2003 Nov 15, 17(22), 2798 - 811 Lipoprotein receptors and a disabled family cytoplasmic adaptor protein regulate EGL-17/FGF export in C . elegans; Kamikura DM et al.; Growth factors and morphogens need to be secreted to act on distant cells during development and in response to injury . Here, we report evidence that efficient export of a fibroblast growth factor (FGF), EGL-17, from the Caenorhabditis elegans developing vulva requires the lipoprotein receptor-related proteins Ce-LRP-1 and Ce-LRP-2 and a cytoplasmic adaptor protein, Ce-DAB-1 (Disabled) . Lipoprotein receptors are transmembrane proteins best known for their roles in endocytosis . Ce-LRP-1 and Ce-LRP-2 possess a conserved intraluminal domain that can bind to EGL-17, as well as a cytosolic FXNPXY motif that can bind to Ce-DAB-1 . Ce-DAB-1 contains signals that confer subcellular localization to Golgi-proximal vesicles . These results suggest a model in which Ce-DAB-1 coordinates selection of receptors and cargo, including EGL-17, for transport through the secretory pathway. Genes Dev, 2003 Nov 15, 17(22), 2747 - 52 Template boundary definition in mammalian telomerase; Chen JL et al.; Telomerase uses a short template sequence in its intrinsic RNA component to synthesize telomere repeats . Disruption of the helix P1b in human telomerase RNA or alteration of its distance from the template resulted in telomerase copying residues past the normal template boundary both in vivo and in vitro . Therefore, helix P1b is important for template boundary definition in human telomerase . Mouse telomerase RNA lacks helix P1b, and the boundary is established at 2 nt downstream of the 5'-end . The divergent structure of boundary definition elements in mammals, yeast, and ciliates suggests diverse mechanisms for template boundary definition in telomerase. Biochem Biophys Res Commun, 2003 Dec 5, 312(1), 121 - 30 Conformational heterogeneity of cytochrome P450 3A4 revealed by high pressure spectroscopy; Davydov DR et al.; We applied hydrostatic pressure perturbation to study substrate-induced transitions in human cytochrome P450 3A4 (CYP3A4) with bromocriptine (BCT) as a substrate . The barotropic behavior of the purified enzyme in solution was compared with that observed in recombinant microsomes of Saccharomyces cerevisiae coexpressing CYP3A4, cytochrome b(5), (b(5)) and NADPH-cytochrome P450 reductase (CPR) . Important barotropic heterogeneity of CYP3A4 was detected in both cases . Only about 70% of CYP3A4 in solution and about 50% of the microsomal enzyme were susceptible to a pressure-induced P450-->P420 transition . The results suggest that both in solution and in the membrane CYP3A4 is represented by two conformers with different positions of spin equilibrium and different barotropic properties . No interconversion between these conformers was observed within the time frame of the experiment . Importantly, a pressure-induced spin shift, which is characteristic of all cytochromes P450 studied to date, was detected in CYP3A4 in solution only; the P450-->P420 transition was the sole pressure-induced process detected in microsomes . This fact suggests unusual stabilization of the high-spin state of CYP3A4, which is assumed to reflect decreased water accessibility of the heme moiety due to specific interactions of the hemoprotein with the protein partners (b(5) and CPR) and/or membrane lipids. J Microsc, 2003 Dec, 212(Pt 3), 254 - 63 Monitoring enzymatic reactions in nanolitre wells; Young IT et al.; We have developed a laboratory-on-a-chip microarray system based on nanolitre-capacity wells etched in silicon . We have devised methods for dispensing reagents as well as samples, for preventing evaporation, for embedding electronics in each well to measure fluid volume per well in real-time, and for monitoring the fluorescence associated with the production or consumption of NADH in enzyme-catalysed reactions . Such reactions can be found in the glycolytic pathway of yeast . We describe the design, construction and testing of our laboratory-on-a-chip . We also describe the use of these chips to measure both fluorescence (such as that evidenced in NADH) as well as bioluminescence (such as evidenced in ATP assays) . We show that our detection limit for NADH fluorescence is 5 micro m with a microscope-based system and 100 micro m for an embedded photodiode system . The photodiode system also provides a detection limit of 2.4 micro m for ATP/luciferase bioluminescence. Neoplasma, 2003, 50(5), 311 - 8 Artemis, a novel guardian of the genome; Dudasova Z et al.; B and T lymphocytes recognize foreign antigen through specialized receptors: the immunoglobulins and the T cell receptors, respectively . The highly polymorphic antigen-recognition regions of these receptors are composed of variable (V), diversity (D), and joining (J) gene segments that undergo somatic rearrangement prior to their expression by the V(D)J recombination process . Proper joining of the V, D, and J segments requires the participation of the Rag proteins as well as the non-homologous end-joining (NHEJ) factors . Recently, a novel V(D)J recombination/NHEJ factor, Artemis, has been identified . Mutations in the ARTEMIS gene cause human severe combined immunodeficiency with increased radiosensitivity (RS-SCID), an autosomal recessive disease characterized by the absence of the T and B lymphocytes and by a defect in the V(D)J recombination . This minireview compiles all mutations in the ARTEMIS gene identified so far . Furthermore, phenotypes of RS-SCID patients and links to the particular mutations are described . Biochemical and structural properties of the Artemis proteins are reviewed and integrated into the processes of V(D)J recombination and NHEJ . A genomic caretaker function is assigned to Artemis. Nucleic Acids Res . 2003 Dec 1;31(23):e151. Validation of a novel, fully integrated and flexible microarray benchtop facility for gene expression profiling; Baum M et al.; Here we describe a novel microarray platform that integrates all functions needed to perform any array-based experiment in a compact instrument on the researcher's laboratory benchtop . Oligonucle otide probes are synthesized in situ via a light- activated process within the channels of a three-dimensional microfluidic reaction carrier . Arrays can be designed and produced within hours according to the user's requirements . They are processed in a fully automatic workflow . We have characterized this new platform with regard to dynamic range, discrimination power, reproducibility and accuracy of biological results . The instrument detects sample RNAs present at a frequency of 1:100 000 . Detection is quantitative over more than two orders of magnitude . Experiments on four identical arrays with 6398 features each revealed a mean coefficient of variation (CV) value of 0.09 for the 6398 unprocessed raw intensities indicating high reproducibility . In a more elaborate experiment targeting 1125 yeast genes from an unbiased selection, a mean CV of 0.11 on the fold change level was found . Analyzing the transcriptional response of yeast to osmotic shock, we found that biological data acquired on our platform are in good agreement with data from Affymetrix GeneChips, quantitative real-time PCR and--albeit somewhat less clearly--to data from spotted cDNA arrays obtained from the literature. Nucleic Acids Res, 2003 Dec 1, 31(23), 6953 - 62 Exhaustive identification of interaction domains using a high-throughput method based on two-hybrid screening and PCR-convergence: molecular dissection of a kinetochore subunit Spc34p; Ikeuchi A et al.; The Dam1 complex, also known as DASH complex, is the outer kinetochore protein complex of yeast that plays a crucial role in attachment of kinetochore to microtubule . The Dam1 complex is formed by at least nine proteins including Dam1p, Duo1p, Dad1p, Spc19p and Spc34p . In this study, domains of Spc34p that physically interact with other subunits of the complex were mapped using a high-throughput methodology . The method is a combination of two-hybrid screening of a random truncation library of the Spc34 gene and a unique PCR-based amplification that converge the selected DNA fragments to a few short fragments . Duo1p, Dam1p, Dad1p and Spc19p binding domains of Spc34p were mapped on M1-E59, M1-D47, M1-D47 or T207-E295 and S154-Q294, respectively . Most of the boundaries were located at less conserved regions among fungal Spc34p homologs, which is consistent with the boundaries of the putative secondary structures . The accuracy of the mapped domain boundaries was verified using truncated Spc34p polypeptides . The results and methodology we demonstrated herein not only shed light on the molecular architecture of the protein complex but also pave the road to the high-throughput identification of specific interaction domains of proteins whose possible interaction partners have been identified in genome-scale analyses. Nucleic Acids Res, 2003 Dec 1, 31(23), 6741 - 7 Dynamic methylation of histone H3 at lysine 4 in transcriptional regulation by the androgen receptor; Kim J et al.; The methylation of histone H3 correlates with either gene expression or silencing depending on the residues modified . Methylated lysine 4 (H3-K4) is associated with transcription at active gene loci . Furthermore, it was reported that trimethylated but not dimethylated H3-K4 is exclusively associated with active chromatin in Saccharomyces cerevisiae . In the present study, we investigated the H3-K4 methylation at the human prostate specific antigen (PSA) locus following gene activation and repression via androgen receptor (AR) . We show that ligand-induced, AR-mediated transcription was accompanied by rapid decreases in di- and trimethylated H3-K4 at the PSA enhancer and promoter . Moreover, the observed decreases in H3-K4 methylation were reversed when AR was inhibited by a specific AR antagonist, bicalutamide . In contrast to the decreases in methylation at the 5' transcriptional control regions of the PSA gene, H3-K4 methylation in the coding region steadily increased after a lag period of approximately 4 h . The results suggest a novel role of methylated H3-K4 in transcriptional regulation. Cell Stress Chaperones, 2003 Summer, 8(2), 108 - 13 p23, a simple protein with complex activities; Felts SJ et al.; p23 is a small but important cochaperone for the Hsp90 chaperoning pathway . It appears to facilitate the adenosine triphosphate-driven cycle of Hsp90 binding to client proteins . It enters at a late stage of the cycle and enhances the maturation of client proteins . Although this role of p23 is fairly well established, recent studies suggest that it may have additional functions in the cell that merit further exploration. Cell Mol Life Sci, 2003 Nov, 60(11), 2303 - 18 Long-range silencing and position effects at telomeres and centromeres: parallels and differences; Perrod S et al.; Most of the human genome is compacted into heterochromatin, a form that encompasses multiple forms of inactive chromatin structure . Transcriptional silencing mechanisms in budding and fission yeasts have provided genetically tractable models for understanding heritably repressed chromatin . These silent domains are typically found in regions of repetitive DNA, that is, either adjacent to centromeres or telomeres or within the tandemly repeated ribosomal DNA array . Here we address the mechanisms of centromeric, telomeric and locus-specific gene silencing, comparing simple and complex animals with yeast . Some aspects are universally shared, such as histone-tail modifications, while others are unique to either centromeres or telomeres . These may reflect roles for heterochromatin in other chromosomal functions, like kinetochore attachment and DNA ends protection. Cell Mol Life Sci, 2003 Nov, 60(11), 2295 - 302 Composition and conservation of the telomeric complex; Kanoh J et al.; The telomere is composed of telomeric DNA and telomere-associated proteins . Recently, many telomere-associated proteins have been identified, and various telomere functions have been uncovered . In budding yeast, scRap1 binds directly to telomeric DNA, and other telomere regulators (Sir proteins and Rif proteins) are recruited to the telomeres by interacting with scRap1 . Cdc13 binds to the most distal end of the chromosome and recruits telomerase to the telomeres . In fission yeast and humans, TTAGGG repeat binding factor (TRF) family proteins bind directly to telomeric DNA, and Rap1 proteins and other telomere regulators are recruited to the telomeres by interacting with the TRF family proteins . Both organisms have Pot1 proteins at the most distal end of the telomere instead of a budding-yeast Cdc13-like protein . Therefore, fission yeast and humans have in part common telomeric compositions that differ from that of budding yeast, a result that suggests budding yeast has lost some telomere components during the course of evolution. Nat Cell Biol, 2003 Dec, 5(12), 1062 - 70 Epub 2003 Nov 16. Scaffold-mediated symmetry breaking by Cdc42p; Irazoqui JE et al.; Cell polarization generally occurs along a single well-defined axis that is frequently determined by environmental cues such as chemoattractant gradients or cell-cell contacts, but polarization can also occur spontaneously in the apparent absence of such cues, through a process called symmetry breaking . In Saccharomyces cerevisiae, cells are born with positional landmarks that mark the poles of the cell and guide subsequent polarization and bud emergence to those sites, but cells lacking such landmarks polarize towards a random cortical site and proliferate normally . The landmarks employ a Ras-family GTPase, Rsr1p, to communicate with the conserved Rho-family GTPase Cdc42p, which is itself polarized and essential for cytoskeletal polarization . We found that yeast Cdc42p was effectively polarized to a single random cortical site even in the combined absence of landmarks, microtubules and microfilaments . Among a panel of Cdc42p effectors and interacting proteins, we found that the scaffold protein Bem1p was uniquely required for this symmetry-breaking behaviour . Moreover, polarization was dependent on GTP hydrolysis by Cdc42p, suggesting that assembly of a polarization site involves cycling of Cdc42p between GTP- and GDP-bound forms, rather than functioning as a simple on/off switch. J Biol Chem, 2004 Feb 27, 279(9), 8469 - 77 Epub 2003 Nov 18. Analysis on origin recognition complex containing Orc5p with defective Walker A motif; Takahashi N et al.; Orc5p is one of six proteins that make up the origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes . To investigate the role of ATP binding to Orc5p in cells, we constructed orc5-A, a strain of Saccharomyces cerevisiae having a mutation in the Walker A motif of Orc5p (K43E) . The strain showed temperature-sensitive growth . Incubation at a nonpermissive temperature (37 degrees C) caused accumulation of cells with nearly 2C DNA content . Overproduction of Orc4p, another subunit of ORC, suppresses this temperature sensitivity, but overproduction of other subunits did not . Overproduction of Orc4p did not suppress the temperature sensitivity of another orc5 mutant, orc5-1, whose mutation, L331P, is outside the ATP-binding motif . These results suggest that Orc4p is specifically involved in ATP binding to Orc5p itself or its function in DNA replication . Immunoblotting experiments revealed that in the orc5-A strain at a nonpermissive temperature, all ORC subunits gradually disappeared, suggesting that ORC5-A becomes degraded at nonpermissive temperatures . We therefore consider that ATP binding to Orc5p is involved in efficient ORC formation and that Orc4p is involved in this process. J Cell Biochem, 2003 Dec 1, 90(5), 884 - 91 Identification of a novel protein from glial cells based on its ability to interact with NF-kappaB subunits; Sweet T et al.; Nuclear factor kappaB (NF-kappaB) represents a family of inducible DNA-binding transcription factors whose activity is critical for expression of the HIV-1 genome in a broad range of cells . In addition to its interaction with the kappaB DNA sequence, the association of NF-kappaB subunits with other cellular proteins plays an important role in stimulation of HIV-1 gene transcription in astrocytic cells . Here, we utilized a yeast two-hybrid system to screen a cDNA library from a human astrocytic cell line and were able to isolate a partial cDNA belonging to a gene with an open reading frame of 1,871 amino acid residues which binds to both the p50 and p65 subunits of NF-kappaB . This gene, named NF-kappaB-binding protein (NFBP) is located on chromosome 10q24.2-25.1 and hybridized to a single transcript of nearly 6 kb in size . It is localized to the nucleus, specifically the nucleolus of cells . Extensive computer analysis was performed with the sequence of the full length NFBP and significant homology was found between NFBP, and yeast and mouse proteins . A discussion of the potential roles of NFBP in normal and viral infected cells is included . RNA, 2003 Dec, 9(12), 1476 - 90 Evidence that poly(A) binding protein has an evolutionarily conserved function in facilitating mRNA biogenesis and export; Chekanova JA et al.; Eukaryotic poly(A) binding protein (PABP) is a ubiquitous, essential cellular factor with well-characterized roles in translational initiation and mRNA turnover . In addition, there exists genetic and biochemical evidence that PABP has an important nuclear function . Expression of PABP from Arabidopsis thaliana, PAB3, rescues an otherwise lethal phenotype of the yeast pab1Delta mutant, but it neither restores the poly(A) dependent stimulation of translation, nor protects the mRNA 5' cap from premature removal . In contrast, the plant PABP partially corrects the temporal lag that occurs prior to the entry of mRNA into the decay pathway in the yeast strains lacking Pab1p . Here, we examine the nature of this lag-correction function . We show that PABP (both PAB3 and the endogenous yeast Pab1p) act on the target mRNA via physically binding to it, to effect the lag correction . Furthermore, substituting PAB3 for the yeast Pab1p caused synthetic lethality with rna15-2 and gle2-1, alleles of the genes that encode a component of the nuclear pre-mRNA cleavage factor I, and a factor associated with the nuclear pore complex, respectively . PAB3 was present physically in the nucleus in the complemented yeast strain and was able to partially restore the poly(A) tail length control during polyadenylation in vitro, in a poly(A) nuclease (PAN)-dependent manner . Importantly, PAB3 in yeast also promoted the rate of entry of mRNA into the translated pool, rescued the conditional lethality, and alleviated the mRNA export defect of the nab2-1 mutant when overexpressed . We propose that eukaryotic PABPs have an evolutionarily conserved function in facilitating mRNA biogenesis and export. RNA, 2003 Dec, 9(12), 1431 - 6 Cic1p/Nsa3p is required for synthesis and nuclear export of 60S ribosomal subunits; Fatica A et al.; Cic1p/Nsa3p was previously reported to be associated with the 26S proteasome and required for the degradation of specific substrates, but was also shown to be associated with early pre-60S particles and to be localized to the nucleolus . Here we report that Cic1p/Nsa3p is required for the synthesis of 60S ribosome subunits . A temperature-sensitive lethal cic1-2 point mutation inhibits synthesis of the mature 5.8S and 25S rRNAs . Release of the pre-60S particles from the nucleolus to the nucleoplasm was also inhibited as judged by the nuclear accumulation of an Rpl11b-GFP reporter construct . We suggest that Cic1p/Nsa3p associates early with nascent preribosomal particles and is required for correct processing and nuclear release of large ribosomal subunit precursors. Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 13887 - 91 Epub 2003 Nov 17. Association of the Mediator complex with enhancers of active genes; Kuras L et al.; The multiprotein Mediator complex has been shown to interact with gene-specific regulatory proteins and RNA polymerase II in vitro . Here, we use chromatin immunoprecipitation to analyze the recruitment of Mediator to GAL genes of yeast in vivo . We find that Mediator associates exclusively with transcriptionally active and not inactive GAL genes . This association maps to the upstream activating sequence, rather than the core promoter, and is independent of RNA polymerase II, general transcription factors, and core promoter sequences . These findings support the idea of Mediator as a primary conduit of regulatory information from enhancers to promoters in eukaryotic cells. J Biol Chem, 2004 Feb 20, 279(8), 6414 - 25 Epub 2003 Nov 17. Transcriptional profiling of ubp10 null mutant reveals altered subtelomeric gene expression and insurgence of oxidative stress response; Orlandi I et al.; UBP10 codes for a deubiquitinating enzyme of Saccharomyces cerevisiae whose loss of function determines slow growth rate and partial impairment of silencing at telomeres and HM loci . A genome-wide analysis performed on a ubp10 disruptant revealed alterations in expression of subtelomeric genes together with a broad change in the whole transcriptional profile, closely parallel to that induced by oxidative stress . This response was accompanied by intracellular accumulation of reactive oxygen species as well as by DNA fragmentation and phosphatidylserine externalization, two markers of apoptosis . SIR4 inactivation mitigated the wide transcriptome remodeling of the ubp10 null mutant affecting particularly the stress transcriptional profile . Moreover, the ubp10sir4 disruptant did not display apoptotic markers . These results argue in favor of an involvement of deubiquitination in transcriptional control and suggest a linkage between oxidative stress and apoptotic pathway in budding yeast. J Cell Biol, 2003 Nov 24, 163(4), 729 - 41 Epub 2003 Nov 17. Pds5p regulates the maintenance of sister chromatid cohesion and is sumoylated to promote the dissolution of cohesion; Stead K et al.; Pds5p and the cohesin complex are required for sister chromatid cohesion and localize to the same chromosomal loci over the same cell cycle window . However, Pds5p and the cohesin complex likely have distinct roles in cohesion . We report that pds5 mutants establish cohesion, but during mitosis exhibit precocious sister dissociation . Thus, unlike the cohesin complex, which is required for cohesion establishment and maintenance, Pds5p is required only for maintenance . We identified SMT4, which encodes a SUMO isopeptidase, as a high copy suppressor of both the temperature sensitivity and precocious sister dissociation of pds5 mutants . In contrast, SMT4 does not suppress temperature sensitivity of cohesin complex mutants . Pds5p is SUMO conjugated, with sumoylation peaking during mitosis . SMT4 overexpression reduces Pds5p sumoylation, whereas smt4 mutants have increased Pds5p sumoylation . smt4 mutants were previously shown to be defective in cohesion maintenance during mitosis . These data provide the first link between a protein required for cohesion, Pds5p, and sumoylation, and suggest that Pds5p sumoylation promotes the dissolution of cohesion. J Cell Biol, 2003 Nov 24, 163(4), 777 - 87 Epub 2003 Nov 17. Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia; Atorino L et al.; Mmutations in paraplegin, a putative mitochondrial metallopeptidase of the AAA family, cause an autosomal recessive form of hereditary spastic paraplegia (HSP) . Here, we analyze the function of paraplegin at the cellular level and characterize the phenotypic defects of HSP patients' cells lacking this protein . We demonstrate that paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane . These two proteins form a high molecular mass complex, which we show to be aberrant in HSP fibroblasts . The loss of this complex causes a reduced complex I activity in mitochondria and an increased sensitivity to oxidant stress, which can both be rescued by exogenous expression of wild-type paraplegin . Furthermore, complementation studies in yeast demonstrate functional conservation of the human paraplegin-AFG3L2 complex with the yeast m-AAA protease and assign proteolytic activity to this structure . These results shed new light on the molecular pathogenesis of HSP and functionally link AFG3L2 to this neurodegenerative disease. Biochem Biophys Res Commun, 2003 Nov 21, 311(3), 577 - 82 Identification of mouse Vps16 and biochemical characterization of mammalian class C Vps complex; Kim BY et al.; Many multiprotein complexes mediate the fusion of the intracellular membranes . The question how the specificity of the membrane fusion is controlled has not been fully elucidated . Here we report the identification of a mouse homologue Vps16p (mVps16), which exhibits a high homology to the yeast Vps16p, a component of Class C vacuolar protein sorting (Vps) complex implicated in the yeast vacuole membrane fusion . Northern and Western blot analyses reveal that mVps16 is ubiquitously expressed in the mouse peripheral tissues . Biochemical analyses show that mammalian Class C Vps proteins interact with multiple syntaxins and Vps45p, which localizes in the endosomal compartments . The internalization of transferrin (Tf) is not affected by the overexpression of mammalian class C Vps proteins, but the recycling was inhibited . Taken together, this study provides biochemical characteristics of mVps16p in mammalian cells and the potential roles of mammalian Class C Vps proteins in membrane trafficking. Dev Biol, 2003 Dec 1, 264(1), 50 - 63 Golgi dynamics during meiosis are distinct from mitosis and are coupled to endoplasmic reticulum dynamics until fertilization; Payne C et al.; One current theory of the Golgi apparatus views its organization as containing both a matrix fraction of structural proteins and a reservoir of cycling enzymes . During mitosis, the putative matrix protein GM130 is phosphorylated and relocalized to spindle poles . When the secretory pathway is inhibited during interphase, GM130 redistributes to regions adjacent to vesicle export sites on the endoplasmic reticulum (ER) . Strikingly, meiotic maturation and fertilization in nonrodent mammalian eggs presents a unique experimental environment for the Golgi apparatus, because secretion is inhibited until after fertilization, and because the centrosome is absent until introduced by the sperm . Here, we test the hypothesis that phosphorylated GM130 associates not with meiotic spindle poles, but with ER clusters in the mature bovine oocyte . At the germinal vesicle stage, phosphorylated GM130 is observed as fragments dispersed throughout the cytoplasm . During meiotic maturation, GM130 reorganizes into punctate foci that associate near the ER-resident protein calreticulin and is notably absent from the meiotic spindle . GM130 colocalizes with Sec23, a marker for ER vesicle export sites, but not with Lens culinaris agglutinin, a marker for cortical granules . Because disruption of vesicle transport has been shown to block meiotic maturation and embryonic cleavage in some species, we also test the hypothesis that fertilization and cytokinesis are inhibited with membrane trafficking disruptor brefeldin A (BFA) . Despite Golgi fragmentation after BFA treatment, pronuclei form and unite, and embryos cleave and develop through the eight-cell stage . We conclude that, while the meiotic phosphorylation cycle of GM130 mirrors that of mitosis, absence of a maternal centrosome precludes Golgi association with the meiotic spindle . Fertilization introduces the sperm centrosome that can reorganize Golgi proteins, but neither fertilization nor cytokinesis prior to compaction requires a functional Golgi apparatus. Cell, 2003 Nov 14, 115(4), 475 - 87 A pathway for association of receptors, adaptors, and actin during endocytic internalization; Kaksonen M et al.; In budding yeast, many proteins involved in endocytic internalization, including adaptors and actin cytoskeletal proteins, are localized to cortical patches of differing protein composition . Using multicolor real-time fluorescence microscopy and particle tracking algorithms, we define an early endocytic pathway wherein an invariant sequence of changes in cortical patch protein composition correlates with changes in patch motility . Three Arp2/3 activators each showed a distinct behavior, suggesting distinct patch-related endocytic functions . Actin polymerization occurs late in the endocytic pathway and is required both for endocytic internalization and for patch disassembly . In cells lacking the highly conserved endocytic protein Sla2p, patch motility was arrested and actin comet tails associated with endocytic patch complexes . Fluorescence recovery after photobleaching of the actin comet tails revealed that endocytic complexes are nucleation sites for rapid actin polymerization . Attention is now focused on the mechanisms by which the order and timing of events in this endocytic pathway are achieved. Cell, 2003 Nov 14, 115(4), 425 - 35 Isw1 chromatin remodeling ATPase coordinates transcription elongation and termination by RNA polymerase II; Morillon A et al.; We demonstrate that distinct forms of the yeast chromatin-remodeling enzyme Isw1p sequentially regulate each stage of the transcription cycle . The Isw1a complex (Iswlp/Ioc3p) represses gene expression at initiation through specific positioning of a promoter proximal dinucleosome, whereas the Isw1b complex (Iswlp/Ioc2p/Ioc4p) acts within coding regions to control the amount of RNA polymerase (RNAPII) released into productive elongation and to coordinate elongation with termination and pre-mRNA processing . These effects of Isw1b are controlled via phosphorylation of the heptad repeat carboxy-terminal domain (CTD) of RNAPII and methylation of the chromatin template . The transcription elongation factor Spt4p antagonizes Isw1p and overcomes the Isw1p dependent pausing of RNAPII at the onset of the elongation cycle . Overall these studies establish the central role played by Isw1p in the coordination of transcription. Mol Microbiol, 2003 Nov, 50(4), 1309 - 18 Candida glabrata STE12 is required for wild-type levels of virulence and nitrogen starvation induced filamentation; Calcagno AM et al.; The highly conserved fungal Ste12 transcription factor family of proteins play critical roles in the regulation of many cellular processes including mating, cell wall biosynthesis, filamentation and invasive growth . They are also important mediators of fungal virulence . The Candida glabrata STE12 homologue was cloned . The encoded protein has a single DNA binding homeodomain but lacks both a C2H2 zinc finger DNA binding domain and an apparent Dig1/Dig2 regulatory motif . Candida glabrata STE12 can functionally complement the nitrogen starvation induced filamentation and mating defects of Saccharomyces cerevisiae ste12 mutants . We also show that C . glabrata STE12 is required for nitrogen starvation-induced filamentation as ste12 mutants rarely produce pseudohyphae on nitrogen depleted media . Finally we describe a novel murine model of C . glabrata systemic disease and use this to demonstrate that C . glabrata ste12 mutants, although still able to cause disease, are attenuated for virulence compared with STE12 reconstituted strains . Candida glabrata STE12 is therefore the first virulence factor encoding gene to be described in this increasingly important fungal pathogen. Eur J Biochem, 2003 Nov, 270(21), 4254 - 63 Identification and characterization of eukaryotic initiation factor 5A-2; Clement PM et al.; The phylogenetically conserved eukaryotic translation initiation factor 5A (eIF5A) is the only known cellular protein to contain the post-translationally derived amino acid hypusine {Nepsilon-(4-amino-2-hydroxybutyl)lysine} . Both eIF5A and its hypusine modification are essential for sustained cell proliferation . Normally only one eIF5A protein is expressed in human cells . Recently, we identified a second human EIF5A gene that would encode an isoform (eIF5A-2) of 84% sequence identity . Overexpression of eIF5A-2 mRNA in certain human cancer cells, in contrast to weak normal expression limited to human testis and brain, suggests EIF5A2 as a potential oncogene . However, eIF5A-2 protein has not been described in human or mammalian cells heretofore . Here, we describe the identification of eIF5A-2 protein in human colorectal and ovarian cancer lines, SW-480 and UACC-1598, that overexpress eIF5A-2 mRNAs . Functional characterization of the human isoforms revealed that either human EIF5A gene can complement growth of a yeast strain in which the yeast EIF5A genes were disrupted . This indicates functional similarity of the human isoforms in yeast and suggests that eIF5A-2 has an important role in eukaryotic cell survival similar to that of the ubiquitous eIF5A-1 . Detectable structural differences were also noted, including lack of immunological cross-reactivity, formation of different complexes with deoxyhypusine synthase, and Km values (1.5 +/- 0.2 vs . 8.3 +/- 1.4 microm for eIF5A-1 and -2, respectively) as substrates for deoxyhypusine synthase in vitro . These physical characteristics and distinct amino acid sequences in the C-terminal domain together with differences in gene expression patterns imply differentiated, tissue-specific functions of the eIF5A-2 isoform in the mammalian organism and in cancer. Eur J Biochem, 2003 Nov, 270(22), 4587 - 93 The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure; Kato M et al.; The heat- and pressure-induced unfolding of the glycosylated and