Mol Cell Biol, 2002 Jun, 22(12), 4402 - 18 The G(1) cyclin Cln3 promotes cell cycle entry via the transcription factor Swi6; Wijnen H et al.; In Saccharomyces cerevisiae (budding yeast), commitment to cell division in late G(1) is promoted by the G(1) cyclin Cln3 and its associated cyclin-dependent kinase, Cdc28 . We show here that all known aspects of the function of Cln3 in G(1) phase, including control of cell size, pheromone sensitivity, cell cycle progress, and transcription, require the protein Swi6 . Swi6 is a component of two related transcription factors, SBF and MBF, which are known to regulate many genes at the G(1)-S transition . The Cln3-Cdc28 complex somehow activates SBF and MBF, but there was no evidence for direct phosphorylation of SBF/MBF by Cln3-Cdc28 or for a stable complex between SBF/MBF and Cln3-Cdc28 . The activation also does not depend on the ability of Cln3 to activate transcription when artificially recruited directly to a promoter . The amino terminus and the leucine zipper of Swi6 are important for the ability of Swi6 to respond to Cln3 but are not essential for the basal transcriptional activity of Swi6 . Cln3-Cdc28 may activate SBF and MBF indirectly, perhaps by phosphorylating some intermediary protein. Mol Cell Biol, 2002 Jun, 22(12), 4202 - 17 Dynamic localization of an Okazaki fragment processing protein suggests a novel role in telomere replication; Choe W et al.; We have found that the Dna2 helicase-nuclease, thought to be involved in maturation of Okazaki fragments, is a component of telomeric chromatin . We demonstrate a dynamic localization of Dna2p to telomeres that suggests a dual role for Dna2p, one in telomere replication and another, unknown function, perhaps in telomere capping . Both chromatin immunoprecipitation (ChIP) and immunofluorescence show that Dna2p associates with telomeres but not bulk chromosomal DNA in G(1) phase, when there is no telomere replication and the telomere is transcriptionally silenced . In S phase, there is a dramatic redistribution of Dna2p from telomeres to sites throughout the replicating chromosomes . Dna2p is again localized to telomeres in late S, where it remains through G(2) and until the next S phase . Telomeric localization of Dna2p required Sir3p, since the amount of Dna2p found at telomeres by two different assays, one-hybrid and ChIP, is severely reduced in strains lacking Sir3p . The Dna2p is also distributed throughout the nucleus in cells growing in the presence of double-strand-break-inducing agents such as bleomycin . Finally, we show that Dna2p is functionally required for telomerase-dependent de novo telomere synthesis and also participates in telomere lengthening in mutants lacking telomerase. Mol Cell Biol, 2002 Jun, 22(12), 4086 - 93 Mitochondrial dysfunction due to oxidative mitochondrial DNA damage is reduced through cooperative actions of diverse proteins; O'Rourke TW et al.; The mitochondrial genome is a significant target of exogenous and endogenous genotoxic agents; however, the determinants that govern this susceptibility and the pathways available to resist mitochondrial DNA (mtDNA) damage are not well characterized . Here we report that oxidative mtDNA damage is elevated in strains lacking Ntg1p, providing the first direct functional evidence that this mitochondrion-localized, base excision repair enzyme functions to protect mtDNA . However, ntg1 null strains did not exhibit a mitochondrial respiration-deficient (petite) phenotype, suggesting that mtDNA damage is negotiated by the cooperative actions of multiple damage resistance pathways . Null mutations in ABF2 or PIF1, two genes implicated in mtDNA maintenance and recombination, exhibit a synthetic-petite phenotype in combination with ntg1 null mutations that is accompanied by enhanced mtDNA point mutagenesis in the corresponding double-mutant strains . This phenotype was partially rescued by malonic acid, indicating that reactive oxygen species generated by the electron transport chain contribute to mitochondrial dysfunction in abf2 Delta strains . In contrast, when two other genes involved in mtDNA recombination, CCE1 and NUC1, were inactivated a strong synthetic-petite phenotype was not observed, suggesting that the effects mediated by Abf2p and Pif1p are due to novel activities of these proteins other than recombination . These results document the existence of recombination-independent mechanisms in addition to base excision repair to cope with oxidative mtDNA damage in Saccharomyces cerevisiae . Such systems are likely relevant to those operating in human cells where mtDNA recombination is less prevalent, validating yeast as a model system in which to study these important issues. Mol Cell Biol, 2002 Jun, 22(12), 4011 - 9 The E2 ubiquitin conjugase Rad6 is required for the ArgR/Mcm1 repression of ARG1 transcription; Turner SD et al.; Transcription of the Saccharomyces cerevisiae ARG1 gene is under the control of both positive and negative elements . Activation of the gene in minimal medium is induced by Gcn4 . Repression occurs in the presence of arginine and requires the ArgR/Mcm1 complex that binds to two upstream arginine control (ARC) elements . With the recent finding that the E2 ubiquitin conjugase Rad6 modifies histone H2B, we examined the role of Rad6 in the regulation of ARG1 transcription . We find that Rad6 is required for repression of ARG1 in rich medium, with expression increased approximately 10-fold in a rad6 null background . Chromatin immunoprecipitation analysis indicates increased binding of TATA-binding protein in the absence of Rad6 . The active-site cysteine of Rad6 is required for repression, implicating ubiquitination in the process . The effects of Rad6 at ARG1 involve two components . In one of these, histone H2B is the likely target for ubiquitination by Rad6, since a strain expressing histone H2B with the principal ubiquitination site converted from lysine to arginine shows a fivefold relief of repression . The second component requires Ubr1 and thus likely the pathway of N-end rule degradation . Through the analysis of promoter constructs with ARC deleted and an arg80 rad6 double mutant, we show that Rad6 repression is mediated through the ArgR/Mcm1 complex . In addition, analysis of an ada2 rad6 deletion strain indicated that the SAGA acetyltransferase complex and Rad6 act in the same pathway to repress ARG1 in rich medium. J Biol Chem, 2002 Aug 2, 277(31), 28051 - 7 Epub 2002 May 22. Involvement of TRAF4 in oxidative activation of c-Jun N-terminal kinase; Xu YC et al.; We previously found that the angiogenic factors TNFalpha and HIV-1 Tat activate an NAD(P)H oxidase in endothelial cells, which operates upstream of c-Jun N-terminal kinase (JNK), a MAPK involved in the determination of cell fate . To further understand oxidant-related signaling pathways, we screened lung and endothelial cell libraries for interaction partners of p47(phox) and recovered the orphan adapter TNF receptor-associated factor 4 (TRAF4) . Domain analysis suggested a tail-to-tail interaction between the C terminus of p47(phox) and the conserved TRAF domain of TRAF4 . In addition, TRAF4, like p47(phox), was recovered largely in the cytoskeleton/membrane fraction . Coexpression of p47(phox) and TRAF4 increased oxidant production and JNK activation, whereas each alone had minimal effect . In addition, a fusion between p47(phox) and the TRAF4 C terminus constitutively activated JNK, and this activation was decreased by the antioxidant N-acetyl cysteine . In contrast, overexpression of the p47(phox) binding domain of TRAF4 blocked endothelial cell JNK activation by TNFalpha and HIV-1 Tat, suggesting an uncoupling of p47(phox) from upstream signaling events . A secondary screen of endothelial cell proteins for TRAF4-interacting partners yielded a number of proteins known to control cell fate . We conclude that endothelial cell agonists such as TNFalpha and HIV-1 Tat initiate signals that enter basic signaling cassettes at the level of TRAF4 and an NAD(P)H oxidase . We speculate that endothelial cells may target endogenous oxidant production to specific sites critical to cytokine signaling as a mechanism for increasing signal specificity and decreasing toxicity of these reactive species. Biochem J, 2002 Aug 15, 366(Pt 1), 23 - 34 Phosphatidylinositol transfer protein beta displays minimal sphingomyelin transfer activity and is not required for biosynthesis and trafficking of sphingomyelin; Segui B et al.; Mammalian phosphatidylinositol transfer proteins (PITPs) alpha and beta, which share 77% identity, have been shown to exhibit distinct lipid-transfer activities . In addition to transferring phosphatidylinositol (PI) and phosphatidylcholine (PC), PITPbeta has been shown to transfer sphingomyelin (SM), and this has led to the suggestion that PITPbeta is important for the regulation of SM metabolism . In the present study, we have analysed the ability of human PITPbeta to transfer and regulate the metabolism of cellular SM . We report that, in vitro, the two PITP isoforms were comparable in mediating PI, PC or SM transfer . Using permeabilized HL-60 cells as the donor compartment, both PITP isoforms efficiently transferred PI and PC, and were slightly active towards SM, with the activity of PITPbeta being slightly greater . To identify which cellular lipids were selected by PITPs, PITPalpha and PITPbeta were exposed to permeabilized HL-60 cells, and subsequently repurified and analysed for their bound lipids . Both PITPs were able to select only PI and PC, but not SM . SM synthesis takes place at the Golgi, and PITPbeta was shown to localize in that compartment . To examine the role of PITPbeta in SM biosynthesis, Golgi membranes were used . Purified Golgi membranes had lost their endogenous PITPbeta, but were able to recruit PITPbeta when added exogenously . However, PITPbeta did not enhance the activities of either SM synthase or glucosylceramide synthase . Further analysis in COS-7 cells overexpressing PITPbeta showed no effects on (a) SM and glucosylceramide biosynthesis, (b) diacylglycerol or ceramide levels, (c) SM transport from the Golgi to the plasma membrane, or (d) resynthesis of SM after exogenous sphingomyelinase treatment . Altogether, these observations do not support the suggestion that PITPbeta participates in the transfer of SM, the regulation of SM biosynthesis or its intracellular trafficking. Biochem Soc Trans, 2002 Apr, 30(2), 307 - 11 Role of AMP-activated protein kinase in the regulation of gene transcription; Leclerc I et al.; AMP-activated protein kinase (AMPK) is a regulator of cellular metabolism in response to changes in the energy status of the cells . AMPK was known to shut down energy-consuming pathways in response to a fall in the ATP/AMP ratio by phosphorylating key enzymes of intermediate metabolism . Here we will discuss the recent evidence implicating AMPK in the regulation of gene expression in mammals, mainly in the liver and in the pancreatic beta-cells. Biochem Soc Trans, 2002 Apr, 30(2), 258 - 64 Lactate shuttles in nature; Brooks GA; Once thought to be the consequence of oxygen lack in contracting skeletal muscle, the glycolytic product lactate is formed and utilized continuously under fully aerobic conditions . "Cell-cell" and "intracellular lactate shuttle" concepts describe the roles of lactate in the delivery of oxidative and gluconeogenic substrates, as well as in cell signalling . Examples of cell-cell shuttles include lactate exchanges between white-glycolytic and red-oxidative fibres within a working muscle bed, between working skeletal muscle and heart, and between tissues of net lactate release and gluconeogenesis . Lactate exchange between astrocytes and neurons that is linked to glutamatergic signalling in the brain is an example of a lactate shuttle supporting cell-cell signalling . Lactate uptake by mitochondria and pyruvate-lactate exchange in peroxisomes are examples of intracellular lactate shuttles . Lactate exchange between sites of production and removal is facilitated by monocarboxylate transport proteins, of which there are several isoforms, and, probably, also by scaffolding proteins . The mitochondrial lactate-pyruvate transporter appears to work in conjunction with mitochondrial lactate dehydrogenase, which permits lactate to be oxidized within actively respiring cells . Hence mitochondria function to establish the concentration and proton gradients necessary for cells with high mitochondrial densities (e.g . cardiocytes) to take up and oxidize lactate . Arteriovenous difference measurements on working cardiac and skeletal muscle beds as well as NMR spectral analyses of these tissues show that lactate is formed and oxidized within the cells of formation in vivo . Glycolysis and lactate oxidation within cells permits high flux rates and the maintenance of redox balance in the cytosol and mitochondria . Other examples of intracellular lactate shuttles include lactate uptake and oxidation in sperm mitochondria and the facilitation of beta-oxidation in peroxisomes by pyruvate-lactate exchange . An ancient origin to the utility of lactate shuttling is implied by the observation that mitochondria of Saccharomyces cerevisiae contain flavocytochrome b(2), a lactate-cytochrome c oxidoreductase that couples lactate dehydrogenation to the reduction of cytochrome c . The presence of cell-cell and intracellular lactate shuttles gives rise to the notion that glycolytic and oxidative pathways can be viewed as linked, as opposed to alternative, processes, because lactate, the product of one pathway, is the substrate for the other. Biochem Soc Trans, 2002 Apr, 30(2), 155 - 62 Translation initiation and surface plasmon resonance: new technology applied to old questions; von der Haar T et al.; Translation initiation in eukaryotes is a complicated process involving some of the largest cellular structures, the ribosomes, together with approx . 11 initiation factors, and a poorly characterized set of other proteins . The concerted action of all these components ultimately results in the formation of an 80 S ribosomal complex on the AUG codon of an mRNA, which is competent to start polypeptide production . In this brief overview, we describe the strategies developed by our laboratory to apply surface plasmon resonance (SPR)-based technology to the problem of elucidating kinetic aspects of substeps within the translation-initiation reaction . We then review how other groups have used similar SPR-based techniques to study related interactions. J Biol Chem, 2002 Jul 26, 277(30), 27162 - 8 Epub 2002 May 21. Conversion of phosphoglycolate to phosphate termini on 3' overhangs of DNA double strand breaks by the human tyrosyl-DNA phosphodiesterase hTdp1; Inamdar KV et al.; Mammalian cells contain potent activity for removal of 3'-phosphoglycolates from single-stranded oligomers and from 3' overhangs of DNA double strand breaks, but no specific enzyme has been implicated in such removal . Fractionated human whole-cell extracts contained an activity, which in the presence of EDTA, catalyzed removal of glycolate from phosphoglycolate at a single-stranded 3' terminus to leave a 3'-phosphate, reminiscent of the human tyrosyl-DNA phosphodiesterase hTdp1 . Recombinant hTdp1, as well as Saccharomyces cerevisiae Tdp1, catalyzed similar removal of glycolate, although less efficiently than removal of tyrosine . Moreover, glycolate-removing activity could be immunodepleted from the fractionated extracts by antiserum to hTdp1 . When a plasmid containing a double strand break with a 3'-phosphoglycolate on a 3-base 3' overhang was incubated in human cell extracts, phosphoglycolate processing proceeded rapidly for the first few minutes but then slowed dramatically, suggesting that the single-stranded overhangs gradually became sequestered and inaccessible to hTdp1 . The results suggest a role for hTdp1 in repair of free radical-mediated DNA double strand breaks bearing terminally blocked 3' overhangs. J Biol Chem, 2002 Aug 2, 277(31), 27801 - 8 Epub 2002 May 21. A bichaperone (Hsp70-Hsp78) system restores mitochondrial DNA synthesis following thermal inactivation of Mip1p polymerase; Germaniuk A et al.; Mitochondrial DNA synthesis is a thermosensitive process in the yeast Saccharomyces cerevisiae . We found that restoration of mtDNA synthesis following heat treatment of cells is dependent on reactivation of the mtDNA polymerase Mip1p through the action of a mitochondrial bichaperone system consisting of the Hsp70 system and the Hsp78 oligomeric protein . mtDNA synthesis was inefficiently restored after heat shock in yeast lacking either functional component of the bichaperone system . Furthermore, the activity of purified Mip1p was also thermosensitive; however, the purified components of the mitochondrial bichaperone system (Ssc1p, Mdj1p, Mge1p, and Hsp78p) were able to protect its activity under moderate heat shock conditions as well as to reactivate thermally inactivated Mip1p . Interestingly, the reactivation of endogenous Mip1p contributed more significantly to the restoration of mtDNA synthesis than did import of newly synthesized Mip1p from the cytosol . These observations suggest an important link between function of mitochondrial chaperones and the propagation of mitochondrial genomes under ever-changing environmental conditions. Biophys J, 2002 Jun, 82(6), 2928 - 33 The role of proofreading in signal transduction specificity; Swain PS et al.; Many intracellular signaling proteins such as MAP kinases and transcription factors require multiple covalent modifications before activating downstream targets . This property suggests that signaling pathways are organized to facilitate proofreading, which expends energy to enhance the specificity of the pathway for the appropriate effector . Focusing on MAP kinases, we show that each phosphorylation of the kinase can act as an independent specificity test for that kinase . This is independent of whether MAP kinase activation is distributive, processive, or confined to a protein scaffold . We also highlight the importance of phosphatases in developing and maintaining specificity . Support for our proposals can be drawn from the existing literature. FEBS Lett, 2002 May 22, 519(1-3), 227 - 30 Involvement of decreased myo-inositol transport in lipopolysaccharide-induced depression of phosphoinositide hydrolysis in vascular smooth muscle; Sotoda Y et al.; The mechanism underlying lipopolysaccharide (LPS)-induced depression of phosphoinositide (PI) hydrolysis was investigated using rat aortas . In LPS-pretreated aortas, the 5-hydroxytryptamine-stimulated accumulation of inositol monophosphate and incorporation of exogenous myo-inositol into PIs were significantly less than those in control aortas . Both sodium-myo-inositol cotransporter (SMIT) and phosphatidylinositol transfer protein (PITP) genes were constituently expressed in rat aortas . The mRNA level of SMIT was remarkably lower in LPS-pretreated aortas, while that of PITP mRNA was not affected by LPS . These results suggest that LPS-induced depression of SMIT expression is involved in inhibition of agonist-stimulated PI hydrolysis by LPS. FEBS Lett, 2002 May 22, 519(1-3), 178 - 80 An EVH1/WH1 domain as a key actor in TGFbeta signalling; Callebaut I; EVH1 (enabled VASP (vasodilator-stimulated protein) homology 1)/WH1 (WASP (Wiskott-Aldrich syndrome protein) homology 1) domains, present in Ena VASP and WASP, are protein interaction modules specialised in binding proline-rich ligands . An EVH1/WH1 domain is here identified in the recently cloned SMIF protein, a key protein in transforming growth factor-beta (TGFbeta) signalling which was not yet related to defined domains . The SMIF EVH1/WH1 domain interacts with the proline-rich Smad4 activation domain, leading to translocation of so-formed complex to the nucleus where SMIF possesses strong intrinsic TGFbeta-inducible transcriptional activity . This finding highlights the pivotal role that the EVH1/WH1 family of domains play in multiple eukaryotic signal transduction pathways. FEBS Lett, 2002 May 22, 519(1-3), 159 - 63 Diphosphonucleotide phosphatase/phosphodiesterase from yellow lupin (Lupinus luteus L.) belongs to a novel group of specific metallophosphatases; Olczak M et al.; A cDNA encoding previously purified and characterized diphosphonucleotide phosphatase/phosphodiesterase (PPD1) from yellow lupin (Lupinus luteus L.) was identified . The ppd1 gene encodes a protein containing a cleavable signal sequence . A functional expression of PPD1 in Saccharomyces cerevisiae confirmed the proper gene identification . A gene homologous to ppd1, encoding a putative membrane protein (PPD2), as well as fragments of two other genes encoding PPD3 and PPD4 proteins were also isolated . Amino acids composing the putative active center of PPD1 and PPD2 are similar to those present in known purple acid phosphatases, which suggests that the reported genes might encode a novel group of specific metallophosphatases . RT-PCR revealed that the corresponding PPD1 mRNA accumulates in stems and leaves, and PPD2 mRNA in stems, leaves and seedlings. FEBS Lett, 2002 May 22, 519(1-3), 59 - 65 Degradation of human Aurora-A protein kinase is mediated by hCdh1; Taguchi S et al.; Human Aurora-A is related to a protein kinase originally identified by its close homology to Ipl1p from Saccharomyces cerevisiae and aurora from Drosophila melanogaster, which are key regulators of the structure and function of the mitotic spindle . We previously showed that human Aurora-A is turned over through the anaphase promoting complex/cyclosome (APC/C)-ubiquitin-proteasome pathway . The association of two distinct WD40 repeat proteins known as Cdc20 and Cdh1, respectively, sequentially activates the APC/C . The present study shows that Aurora-A degradation is dependent on hCdh1 in vivo, not on hCdc20, and that Aurora-A is targeted for proteolysis through distinct structural features of the destruction box, the KEN box motifs and its kinase activity. RNA, 2002 May, 8(5), 579 - 86 Branchpoint selection in the splicing of U12-dependent introns in vitro; McConnell TS et al.; In metazoans, splicing of introns from pre-mRNAs can occur by two pathways: the major U2-dependent or the minor U12-dependent pathways . Whereas the U2-dependent pathway has been well characterized, much about the U12-dependent pathway remains to be discovered . Most of the information regarding U12-type introns has come from in vitro studies of a very few known introns of this class . To expand our understanding of U12-type splicing, especially to test the hypothesis that the simple base-pairing mechanism between the intron and U12 snRNA defines the branchpoint of U12-dependent introns, additional in vitro splicing substrates were created from three putative U12-type introns: the third intron of the Xenopus RPL1 a gene (XRP), the sixth intron of the Xenopus TFIIS.oA gene (XTF), and the first intron of the human Sm E gene (SME) . In vitro splicing in HeLa nuclear extract confirmed U12-dependent splicing of each of these introns . Surprisingly, branchpoint mapping of the XRP splicing intermediate shows use of the upstream rather than the downstream of two consecutive adenosines within the branchpoint sequence (BPS), contrary to the prediction based on alignment with the sixth intron of human P120, a U12-dependent intron whose branch site was previously determined . Also, in the SME intron, the position of the branchpoint A residue within the region base paired with U12 differs from that in P120 and XTF . Analysis of these three additional introns therefore rules out simple models for branchpoint selection by the U12-type spliceosome. Hum Mol Genet, 2002 May 15, 11(11), 1351 - 62 The mitochondrial protein frataxin prevents nuclear damage; Karthikeyan G et al.; The mitochondrial protein frataxin helps maintain appropriate iron levels in the mitochondria of yeast and humans . A deficiency of this protein in humans causes Friedreich's ataxia, while its complete absence in yeast (Delta yfh1 mutant) results in loss of mitochondrial DNA, apparently due to radicals generated by excess iron . We found that the absence of frataxin in yeast also leads to nuclear damage, as evidenced by inducibility of a nuclear DNA damage reporter, increased chromosomal instability including recombination and mutation, and greater sensitivity to DNA-damaging agents, as well as slow growth . Addition of a human frataxin mutant did not prevent nuclear damage, although it partially complemented the Delta yfh1 mutant in preventing mitochondrial DNA loss . The effects in Delta yfh1 mutants result from reactive oxygen species (ROS), since (i) Delta yfh1 cells produce more hydrogen peroxide, (ii) the effects are alleviated by a radical scavenger and (iii) the glutathione peroxidase gene prevents an increase in mutation rates . Thus, the frataxin protein is concluded to have a protective role for the nucleus as well as the mitochondria. Cancer Res, 2002 May 15, 62(10), 2791 - 7 Myeloid leukemias have increased activity of the nonhomologous end-joining pathway and concomitant DNA misrepair that is dependent on the Ku70/86 heterodimer; Gaymes TJ et al.; Human myeloid leukemias are characterized by chromosomal abnormalities, including translocations, deletions, and allelic loss . These alterations are known to disrupt the function of genes that contribute to tumor initiation and progression . The mechanism underlying the appearance of these chromosomal alterations is poorly understood . Recent evidence suggests that altered nonhomologous end joining (NHEJ) is associated with the incidence of chromosome abnormalities in mutant rodent cells . This pathway is thought to provide a major mechanism for the repair of double-strand breaks (DSB) in higher eukaryotes . Here, we show that in an in vitro assay for DSB end ligation, nuclear extracts prepared from cultured and primary myeloid leukemia cells show a 2-7-fold increase in end-ligation efficiency as compared with mobilized peripheral CD34+ blood progenitor cells (CD34+) and interleukin-2-stimulated peripheral blood lymphocytes from normal healthy donors (P < 0.001) . Furthermore, using an in vitro plasmid LacZ gene reactivation assay to determine DSB repair fidelity, nuclear extracts prepared from myeloid leukemia cells showed an increased frequency of misrepair compared with normal control cells (P < 0.001) . Most importantly, this misrepair in myeloid leukemia cells is associated with large deletions (30-400 bp) within the test plasmids used in our assay . These deletions were not observed using normal hematopoietic cells (<28 bp) . Strikingly, we show that the NHEJ proteins, Ku70 and 86, are required for the deletions in myeloid leukemias because preincubating nuclear extracts from leukemic cells with antisera against Ku86 and Ku70 inhibits plasmid reactivation and restores the frequency and size of deletions to control levels . Our findings suggest that an overactive NHEJ system and, specifically, aberrant Ku70/86 activity is a candidate mechanism for chromosomal instability in myeloid leukemias. Antimicrob Agents Chemother, 2002 Jun, 46(6), 1785 - 92 Antifungal activity of eupolauridine and its action on DNA topoisomerases; Khan SI et al.; The azafluoranthene alkaloid eupolauridine has previously been shown to have in vitro antifungal activity and selective inhibition of fungal topoisomerase I . The present study was undertaken to examine further its selectivity and mode of action . Eupolauridine completely inhibits the DNA relaxation activity of purified fungal topoisomerase I at 50 microg/ml, but it does not stabilize the cleavage complex of either human or fungal topoisomerase I . Cleavage complex stabilization is the mode of action of topoisomerase I targeting drugs of the camptothecin family . Also, unlike camptothecin, eupolauridine does not cause significant cytotoxicity in mammalian cells . To determine if the inhibition of topoisomerase I is the principal mode of antifungal action of eupolauridine, Saccharomyces cerevisiae strains with alterations in topoisomerase genes were used in clonogenic assays . The antifungal activity of eupolauridine was not diminished in the absence of topoisomerase I; rather, the cells lacking the enzyme were more sensitive to the drug . Cell-killing activity of eupolauridine was also more pronounced in cells that overexpressed topoisomerase II . In vitro assays with the purified yeast enzyme confirmed that eupolauridine stabilized topoisomerase II covalent complexes . These results indicate that a major target for fungal cell killing by eupolauridine is DNA topoisomerase II rather than topoisomerase I, but does not exclude the possibility that the drug also acts against other targets. J Bioenerg Biomembr, 2002 Apr, 34(2), 81 - 8 Molecular modeling studies of the DCCD-treated cytochrome bc1 complex: predicted conformational changes and inhibition of proton translocation; Wang Y et al.; Dicyclohexylcarbodiimide (DCCD) binds covalently to an acidic amino acid located in the cd loop connecting membrane-spanning helices C and D of cytochrome b resulting in an inhibition of proton translocation in the cytochrome bc1 complex with minimal effects on the steady state rate of electron transfer . Single turnover studies performed with the yeast cytochrome bc1 complex indicated that the initial phase of cytochrome b reduction was inhibited 25-45% in the DCCD-treated cytochrome bc1 complex, while the rate of cytochrome c1 reduction was unaffected . Simulations by molecular modeling predict that binding of DCCD to glutamate 163 located in the cd2 loop of cytochrome b of chicken liver mitochondria results in major conformational changes in the protein . The conformation of the cd loop and the end of helix C appeared twisted with a concomitant rearrangement of the amino acid residues of both cd1 and cd2 loops . The predicted rearrangement of the amino acid residues of the cd loop results in disruptions of the hydrogen bonds predicted to form between amino acid residues of the cd and ef loops . Simultaneously, two new hydrogen bonds are predicted to form between glutamate 272 and two residues, aspartate 253 and tyrosine 272 . Formation of these new hydrogen bonds would restrict the rotation and protonation of glutamate 272, which may be necessary for the release of the second electrogenic proton obtained during ubiquinol oxidation in the bc1 complex. IUBMB Life, 2002 Jan, 53(1), 25 - 36 Inositol polyphosphate 5-phosphatases: lipid phosphatases with flair; Mitchell CA et al.; Recent studies have identified the inositol polyphosphate 5-phosphatases as a large family of signal modifying enzymes comprising 10 mammalian and 4 yeast family members . A number of investigations including gene-targeted deletion of 5-phosphatases in mice have demonstrated that these enzymes regulate many important cellular events including hematopoietic cell proliferation and activation, insulin signaling, endocytosis, and actin polymerization. Anticancer Res, 2002 Jan-Feb, 22(1A), 193 - 6 Decreased expression of Ku70/Ku80 proteins in malignant melanomas of the oral cavity; Korabiowska M et al.; Ku70/80 are genes responsible for the repairing of DNA double-strand breaks and they function as a regulatory subunit of the DNA-dependent protein kinase . Their expression has not yet been investigated in malignant melanomas of the oral cavity . These tumours are characterized by very poor prognosis and etiology independent of UV-radiation . We investigated 29 malignant melanomas of the oral cavity for the expression of Ku70/80 proteins . Ku70 expression was preserved in 21 out of 29 tumours and the percentage of Ku70-positive cells did not exceed 76% . Ku80 was found in 19 out of 29 tumours and the percentage of Ku80-positive cells peaked at 62% . Correlations between Ku70 and Ku80 expression were lost (p>0.05) . We conclude that decreased Ku70/80 expression in malignant melanomas of the oral cavity and loss of correlation between these markers may influence progression of oral melanomas. Shi Yan Sheng Wu Xue Bao, 1998 Sep, 31(3), 217 - 21 {Mismatch repair in MNNG-induced genetically unstable monkey kidney vero cell}; Feng ZH et al.; Gel retardation analysis and in vitro DNA mismatch repair system were used to examine whether there were mismatch repair deficiency in MNNG-induced genetically unstable vero cell, which was manifested by a delayed and highly increased rate of non-targeted mutation . A mismatch binding protein which could selectively bind to G.T mispair in DNA was identified in its whole-cell extracts . It was also identified that G.T mispair could be specifically and effectively corrected into G.C pair in its nuclear extracts . Compared with normal vero cell, there were no functional deficiency of the above mismatch repair mechanisms . So it could be excluded the possibility that the functional deficiency of mismatch binding protein or G.T mismatch repair pathway participated in the induction of genetic instability in vero cell by MNNG. Bioinformatics, 2002 Apr, 18(4), 566 - 75 Application of Bayesian decomposition for analysing microarray data; Moloshok TD et al.; MOTIVATION: Microarray and gene chip technology provide high throughput tools for measuring gene expression levels in a variety of circumstances, including cellular response to drug treatment, cellular growth and development, tumorigenesis, among many other processes . In order to interpret the large data sets generated in experiments, data analysis techniques that consider biological knowledge during analysis will be extremely useful . We present here results showing the application of such a tool to expression data from yeast cell cycle experiments . RESULTS: Originally developed for spectroscopic analysis, Bayesian Decomposition (BD) includes two features which make it useful for microarray data analysis: the ability to assign genes to multiple coexpression groups and the ability to encode biological knowledge into the system . Here we demonstrate the ability of the algorithm to provide insight into the yeast cell cycle, including identification of five temporal patterns tied to cell cycle phases as well as the identification of a pattern tied to an approximately 40 min cell cycle oscillator . The genes are simultaneously assigned to the patterns, including partial assignment to multiple patterns when this is required to explain the expression profile . AVAILABILITY: The application is available free to academic users under a material transfer agreement . Go to for more details. Bioinformatics, 2002 Apr, 18(4), 536 - 45 Clustering gene expression data using a graph-theoretic approach: an application of minimum spanning trees; Xu Y et al.; MOTIVATION: Gene expression data clustering provides a powerful tool for studying functional relationships of genes in a biological process . Identifying correlated expression patterns of genes represents the basic challenge in this clustering problem . RESULTS: This paper describes a new framework for representing a set of multi-dimensional gene expression data as a Minimum Spanning Tree (MST), a concept from the graph theory . A key property of this representation is that each cluster of the expression data corresponds to one subtree of the MST, which rigorously converts a multi-dimensional clustering problem to a tree partitioning problem . We have demonstrated that though the inter-data relationship is greatly simplified in the MST representation, no essential information is lost for the purpose of clustering . Two key advantages in representing a set of multi-dimensional data as an MST are: (1) the simple structure of a tree facilitates efficient implementations of rigorous clustering algorithms, which otherwise are highly computationally challenging; and (2) as an MST-based clustering does not depend on detailed geometric shape of a cluster, it can overcome many of the problems faced by classical clustering algorithms . Based on the MST representation, we have developed a number of rigorous and efficient clustering algorithms, including two with guaranteed global optimality . We have implemented these algorithms as a computer software EXpression data Clustering Analysis and VisualizATiOn Resource (EXCAVATOR) . To demonstrate its effectiveness, we have tested it on three data sets, i.e . expression data from yeast Saccharomyces cerevisiae, expression data in response of human fibroblasts to serum, and Arabidopsis expression data in response to chitin elicitation . The test results are highly encouraging . AVAILABILITY: EXCAVATOR is available on request from the authors. Dev Cell, 2002 May, 2(5), 593 - 605 Stt4 PI 4-kinase localizes to the plasma membrane and functions in the Pkc1-mediated MAP kinase cascade; Audhya A et al.; Production of the essential phospholipid PI4P at the Golgi by the Pik1 kinase is required for protein secretion, while a distinct pool of PI4P generated by the Stt4 kinase is critical for normal actin cytoskeleton organization . We identify a transmembrane protein that stabilizes Stt4 at the plasma membrane where it directs synthesis of PI4P, which is required for activation of the Rho1/Pkc1-mediated MAP kinase cascade . Inactivation of Stt4 or the PI4P 5-kinase Mss4 results in mislocalization of the Rho-GTPase GEF Rom2 . Rom2 binds PI4,5P(2) through its PH domain and represents the first identified effector in the Stt4-Mss4 pathway . Based on these results, we propose that Stt4-Mss4 generates PI4,5P(2) at the plasma membrane, required to recruit/activate effector proteins such as Rom2. J Comput Biol, 2002, 9(2), 401 - 11 Learning gene functional classifications from multiple data types; Pavlidis P et al.; In our attempts to understand cellular function at the molecular level, we must be able to synthesize information from disparate types of genomic data . We consider the problem of inferring gene functional classifications from a heterogeneous data set consisting of DNA microarray expression measurements and phylogenetic profiles from whole-genome sequence comparisons . We demonstrate the application of the support vector machine (SVM) learning algorithm to this functional inference task . Our results suggest the importance of exploiting prior information about the heterogeneity of the data . In particular, we propose an SVM kernel function that is explicitly heterogeneous . In addition, we describe feature scaling methods for further exploiting prior knowledge of heterogeneity by giving each data type different weights. J Comput Biol, 2002, 9(2), 317 - 30 Analysis techniques for microarray time-series data; Filkov V et al.; We address possible limitations of publicly available data sets of yeast gene expression . We study the predictability of known regulators via time-series analysis, and show that less than 20% of known regulatory pairs exhibit strong correlations in the Cho/Spellman data sets . By analyzing known regulatory relationships, we designed an edge detection function which identified candidate regulations with greater fidelity than standard correlation methods . We develop general methods for integrated analysis of coarse time-series data sets . These include 1) methods for automated period detection in a predominately cycling data set and 2) phase detection between phase-shifted cyclic data sets . We show how to properly correct for the problem of comparing correlation coefficients between pairs of sequences of different lengths and small alphabets . Finally, we note that the correlation coefficient of sequences over alphabets of size two can exhibit very counterintuitive behavior when compared with the Hamming distance. J Biol Chem, 2002 Aug 9, 277(32), 28870 - 6 Epub 2002 May 20. Aph2, a protein with a zf-DHHC motif, interacts with c-Abl and has pro-apoptotic activity; Li B et al.; c-Abl is a non-receptor tyrosine kinase implicated in DNA damage-induced cell death and in growth factor receptor signaling . To further understand the function and regulation of c-Abl, a yeast two-hybrid screen was performed to identify c-Abl-interacting proteins . Here we report the identification of Abl-philin 2 (Aph2), encoding a novel protein with a unique cysteine-rich motif (zf-DHHC) and a 53-amino acid stretch sharing homology with the creatine kinase family . The zf-DHHC domain is highly conserved from yeast to human . Two proteins containing this motif, Akr1p and Erf2p, have been characterized in Saccharomyces cerevisiae, both implicated in signaling pathways . Deletion analysis by two-hybrid assays revealed that the N-terminal portion of Aph2 interacts with the C terminus of c-Abl . Aph2 was demonstrated to interact with c-Abl by co-immunoprecipitation assays . Aph2 is expressed in most tissues tested and is localized in the cytoplasm, mainly in the endoplasmic reticulum (ER) . The sequences required for ER location reside in the N terminus and the zf-DHHC motif of Aph2 . It has been reported that a portion of c-Abl is localized in the ER . We demonstrate here that Aph2 and c-Abl are co-localized in the ER region . Overexpression of Aph2 leads to apoptosis as justified by TUNEL assays, and the induction of apoptosis requires the N terminus . Co-expression of c-Abl and Aph2 had a synergistic effect on apoptosis induction and led to a decreased expression of both proteins, suggesting either that these two proteins are mutually down-regulated or that cells expressing both c-Abl and Aph2 rapidly disappeared from the culture . These results suggest that Aph2 may be involved in ER stress-induced apoptosis in which c-Abl plays an important role. J Biol Chem, 2002 Aug 2, 277(31), 28212 - 21 Epub 2002 May 20. Huntingtin-associated protein 1 interacts with hepatocyte growth factor-regulated tyrosine kinase substrate and functions in endosomal trafficking; Li Y et al.; Huntingtin-associated protein 1 (HAP1) is a novel protein of unknown function with a higher binding affinity for the mutant form of Huntington's disease protein huntingtin . Here we report that HAP1 interacts with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a mammalian homologue of yeast vacuolar protein sorting protein Vps27p involved in the endosome-to-lysosome trafficking . This novel interaction was identified in a yeast two-hybrid screen using full-length Hrs as bait, and confirmed by in vitro binding assays and co-immunoprecipitation experiments . Deletion analysis reveals that the association of HAP1 with Hrs is mediated via a coiled-coil interaction between the central coiled-coil domains of both proteins . Immunofluorescence and subcellular fractionation studies show that HAP1 co-localizes with Hrs on early endosomes . Like Hrs, overexpression of HAP1 causes the formation of enlarged early endosomes, and inhibits the degradation of internalized epidermal growth factor receptors . Whereas overexpression of HAP1 does not affect either constitutive or ligand-induced receptor-mediated endocytosis, it potently blocks the trafficking of endocytosed epidermal growth factor receptors from early endosomes to late endosomes . These findings implicate, for the first time, the involvement of HAP1 in the regulation of vesicular trafficking from early endosomes to the late endocytic compartments. Endocrinology, 2002 Jun, 143(6), 2208 - 15 Endocrine disrupter bisphenol a induces orphan nuclear receptor Nur77 gene expression and steroidogenesis in mouse testicular Leydig cells; Song KH et al.; The orphan nuclear receptor Nur77 (NR4A1) is a member of the nuclear receptor superfamily, which plays an important role in the regulation of LH-mediated steroidogenesis in testicular Leydig cells . The aim of the current study was to investigate the potential role of bisphenol A (BPA) on orphan nuclear receptor Nur77 gene expression and steroidogenesis . Northern blot analysis demonstrated that BPA transiently induced Nur77 mRNA expression, and protein kinase inhibitor H-89 and PD98059 strongly inhibited the induction of BPA-mediated Nur77 gene expression in mouse Leydig tumor cell line, K28 . Moreover, BPA increased the activation of mitogen-activated protein kinase . Transient transfection assay demonstrated that BPA increased Nur77 gene promoter activity and Nur77 transactivation, whereas BPA did not significantly affect the interaction of Nur77 with its corepressor . Furthermore, BPA increased progesterone biosynthesis in K28 cells, which was suppressed by overexpression of dominant negative Nur77 . Finally, BPA injection to prepubertal mice revealed that the expression of Nur77 mRNA was elevated, and this induction was correlated with increased concentration of testicular T in vivo . Taken together, these results demonstrated that BPA induces Nur77 gene expression and subsequently alters the steroidogenesis in testicular Leydig cells . These observations provide a novel mechanism by which BPA acts as an endocrine disrupting chemical. J Ethnopharmacol, 2002 Jun, 81(1), 17 - 22 Antitumour and anticarcinogenic activity of Phyllanthus amarus extract; Rajeshkumar NV et al.; Aqueous extract of Phyllanthus amarus (P . amarus) treatment exhibited potent anticarcinogenic activity against 20-methylcholanthrene (20-MC) induced sarcoma development and increased the survival of tumour harboring mice . The extract administration (p.o) was also found to prolong the life span of Dalton's Lymphoma Ascites (DLA) and Ehrlich Ascites Carcinoma (EAC) bearing mice and reduced the volume of transplanted solid tumours . The extract inhibited aniline hydroxylase, a P-450 enzyme . The concentration required for 50% inhibition (IC(50)) was found to be 540 microg/ml . The extract was found to inhibit DNA topoisomerase II of Saccharomyces cerevisiae mutant cell cultures and inhibited cell cycle regulatory enzyme cdc25 tyrosine phosphatase (IC(50-25) microg/ml) . Antitumour and anticancer activity of P . amarus may be related with the inhibition of metabolic activation of carcinogen as well as the inhibition of cell cycle regulators and DNA repair. Biochim Biophys Acta, 2002 May 3, 1575(1-3), 26 - 30 Characterization of the sequences encoding for Xenopus laevis box C/D snoRNP Nop56 protein; Renzi F et al.; Nop56p was initially identified in yeast as the third common component of the ribonucleoprotein particles (snoRNPs) assembled on box C/D small nucleolar RNAs (snoRNAs) . Thereafter, the characterization of Nop56p homologs in Archaea and in several eukaryotes pointed to the highly conserved structure of this factor . Studies in yeast indicate that Nop56 is not required for the stability of box C/D snoRNAs . Through the isolation of a Xenopus laevis Nop56 cDNA clone, we have been able to characterize the X . laevis Nop56 protein (XNop56p) . We showed that it is a common component of X . laevis box C/D snoRNPs and that it displays the same electrophoretic mobility of p62 protein that we previously characterized as a box C/D snoRNP component, not essential for snoRNA stability in X . laevis . Mapping the 5' end of X . laevis Nop56 transcript indicates that it starts with a pyrimidine tract and the analysis of genomic clones revealed a snoRNA encoded in one of NOP56 introns . Although these two characteristics could suggest that XNOP56 is a TOP gene, it is not translationally controlled in a growth-dependent manner. Int J Radiat Biol, 2002 May, 78(5), 417 - 24 Examining the non-homologous repair process following cisplatin and radiation treatments; Myint WK et al.; PURPOSE: To investigate the extent of non-homologous end-joining (NHEJ) in the mechanism of cisplatin radiosensitization . MATERIALS AND METHODS: Ku80-deficient cells are deficient in the non-homologous DNA double-strand break repair process, while the wild-type MEF cells maintain full mammalian cell repair capabilities . Both cell lines were exposed to clinically applicable doses of cisplatin (1, 3 and 6 microg x ml(-1)) for 1 h immediately before exposure to 250 kV X-rays . Radiation responses were plotted for each cell line and for all doses of cisplatin to observe relative levels of radiosensitization . Split-dose experiments were also performed on each cell line to measure levels of sublethal damage repair . RESULTS: Radiosensitization was observed in the wild-type cells but not in the Ku80 cells when treated with a combination of 1 microg x ml(-1) cisplatin followed by X-rays, implying that the NHEJ pathway may play a large role in cisplatin radiosensitization . Concurrent administration of this cisplatin dose with radiation produced similar levels of radiosensitization . Conversely, 3 and 6 microg x ml(-1) cisplatin applied immediately before radiation revealed an increasing resistance to radiation in both cell lines--possibly due to resistant subpopulations of cells remaining after subsequent lethal doses of cisplatin . Further experiments revealed that the high concentrations of cisplatin did not alter cell cycle distribution . Finally, split-dose experiments revealed that the NHEJ pathway also plays a significant role in sublethal damage repair . CONCLUSIONS: The study reveals that clinically applicable doses of cisplatin treatment results in the radiosensitization of mammalian cells due to the inhibition of the operation of NHEJ. Biochem J, 2002 Sep 1, 366(Pt 2), 501 - 10 A novel role for farnesyl pyrophosphate synthase in fibroblast growth factor-mediated signal transduction; Reilly JF et al.; Farnesyl pyrophosphate synthase (FPPS) catalyses the formation of a key cellular intermediate in isoprenoid metabolic pathways . Here we describe a novel role for this enzyme in fibroblast growth factor (FGF)-mediated signalling . We demonstrate the binding of FPPS to FGF receptors (FGFRs) using the yeast two-hybrid assay, pull-down assays and co-immunoprecipitation . The interaction between FPPS and FGFR is regulated by the cellular metabolic state and by treatment with FGF-2 . Overexpression of FPPS inhibits FGF-2-induced cell proliferation, accompanied by a failure of the FGF-2-mediated induction of cyclins D1 and E . Overexpression of FPPS in fibroblasts also promotes increased farnesylation of Ras, and temporally extends FGF-2-stimulated activation of the Ras/ERK (extracellular-signal-regulated kinase) cascade . These data suggest that, in addition to its role in isoprenoid biosynthesis, FPPS may function as a modulator of the cellular response to FGF treatment. J Biol Chem, 2002 Aug 16, 277(33), 29832 - 9 Epub 2002 May 15. Human ING1 proteins differentially regulate histone acetylation; Vieyra D et al.; ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems . Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300 . Human ING1 immunocomplexes contain HAT activity, and overexpression of p33(ING1b), but not of p47(ING1a), induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level . p47(ING1a) inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1 . Finally, we present evidence indicating that p33(ING1b) affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling . Together with the finding that human ING1 proteins bind PCNA in a DNA damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT.ING1.PCNA protein complexes. Hum Mol Genet, 2002 May 15, 11(10), 1241 - 9 Understanding anesthesia: making genetic sense of the absence of senses; Humphrey JA et al.; The discovery of the phenomenon of anesthesia over 150 years ago was a watershed event that revolutionized the practice of medicine . Despite their annual use in millions of patients, the mechanism by which volatile anesthetics produce reversible loss of consciousness remains a mystery . The inherent problems in studying loss of consciousness in humans are legion . However, multiple model organisms are currently being exploited to apply the powerful tools of modern molecular genetics to this question . Mutants in yeast, nematodes, fruit flies and mice have been produced that display abnormalities in their response to volatile anesthetics . Each organism possesses unique advantages and difficulties as a model system, and each reveals different molecules that control its response to anesthetics . Nonetheless, the accumulating body of genetic evidence points to multiple targets for volatile anesthetics . Not only will understanding how volatile anesthetics work yield better and safer anesthetics, but, in addition, these remarkable compounds may ultimately serve as probes to understand the nature of consciousness itself. Curr Biol, 2002 May 14, 12(10), R366 - 71 Cdc48-Ufd1-Npl4: stuck in the middle with Ub; Bays NW et al.; The ubiquitin-proteasome pathway has a well-defined beginning and end . Target proteins are initially recognized by upstream components and tagged with polyubiquitin chains . The 26S proteasome then degrades these polyubiquitinated proteins . Until recently, it was not known what, if any, steps occurred between the initial polyubiquitination of target proteins and their final degradation . Several new papers investigating the function of the Cdc48-Ufd1-Npl4 complex indicate that there is indeed a middle to the ubiquitin-proteasome pathway . The Cdc48-Ufd1-Npl4 complex functions in the recognition of several polyubiquitin-tagged proteins and facilitates their presentation to the 26S proteasome for processive degradation or even more specific processing . The elucidation of Cdc48, Ufd1 and Npl4 action not only provides long-sought functions for these specific proteins, but illuminates a poorly understood part of the ubiquitin-proteasome pathway. Biopolymers, 2002 Jul 15, 64(3), 161 - 76 High resolution NMR analysis of the seven transmembrane domains of a heptahelical receptor in organic-aqueous medium; Arshava B et al.; The NMR properties of seven peptides representing the transmembrane domains of the alpha-factor receptor from Saccharomyces cerevisiae were examined in trifluoroethanol/water (4:1) at 10 to 55 degrees C . The parameters extracted indicated all peptides were helical in this membrane mimetic solvent . Using chemical shift indices as the criterion, helicity varied from 64 to 83% . The helical residues in the peptides corresponded to the region predicted to cross the hydrocarbon interior of the bilayer . A study of a truncated 25-residue peptide corresponding to domain 2 gave evidence that the helix extended all the way to the N-terminus of this peptide, indicating that sequence and not chain end effects are very important in helix termination for our model peptides . Both nuclear Overhauser effect spectroscopy (NOESY) connectivities and chemical shift indices revealed significant perturbations around prolyl residues in the helices formed by transmembrane domains 6 and 7 . Molecular models of the transmembrane domains indicate that helices for domains 6 and 7 are severely kinked at these prolyl residues . The helix perturbation around proline 258 in transmembrane domain 6 correlates with mutations that cause phenotypic changes in this receptor . Int J Oncol, 2002 Jun, 20(6), 1219 - 25 The rgl-1 is a legitimate homologue of lethal giant larvae recessive oncogene in rat; Kim YS et al.; We have cloned a rat homologue of the Drosophila recessive oncogene lethal (2) giant larvae from rat brain by RT-PCR using primers prepared from sequences conserved amongst lgl family genes . Sequence analysis predicts that the rat rgl-1 gene encodes a 1,036 amino acid polypeptide with a molecular weight of approximately 112 kDa, which contains a domain characteristic of WD-40 proteins . Northern blot analysis revealed that the highest expression of rgl-1 is detected in the testis, with moderate expression in ovary, brain, spleen, and kidney . Since there is a high degree of amino acid similarity among lgl proteins in various species, it is likely that there is an evolutionary relationship among these proteins . The amino acid identity of rgl-1 to Drosophila l(2)gl and mouse mgl-1 proteins showed 30.6 and 96.8%, respectively . The rat tomosyn, previously known as a homologue of Drosophila l(2)gl, showed much lower amino acid identity to Drosophila l(2)gl and mouse mgl-1 proteins (17.8 and 20%, respectively) . Functional analysis showed that the expression of a rat rgl-1 cDNA in Saccharomyces cerevisiae missing sop genes, the yeast homologues of the Drosophila l(2)gl, restored partially the Na+ tolerance of the cell . Taken together, these results indicate that rgl-1, not tomosyn, is the legitimate homologue of lgl gene. Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 6544 - 9 Gaussian fluctuations and linear response in an electron transfer protein; Simonson T; In response to charge separation or transfer, polar liquids respond in a simple linear fashion . A similar linear response for proteins might be expected from the central limit theorem and is postulated in widely used theories of protein electrostatics, including the Marcus electron transfer theory and dielectric continuum theories . Although these theories are supported by a variety of experimental data, the exact validity of a linear protein dielectric response has been difficult to determine . Molecular dynamics simulations are presented that establish a linear dielectric response of both protein and surrounding solvent over the course of a biologically relevant electron transfer reaction: oxido-reduction of yeast cytochrome c in solution . Using an umbrella-sampling free energy approach with long simulations, an accurate treatment of long-range electrostatics and both classical and quantum models of the heme, good agreement is obtained with experiment for the redox potential relative to a heme-octapeptide complex . We obtain a reorganization free energy that is only half that for heme-octapeptide and is reproduced with a dielectric continuum model where the heme vicinity has a dielectric constant of only 1.1 . This value implies that the contribution of protein reorganization to the electron transfer free energy barrier is reduced almost to the theoretical limit (a dielectric of one), and that the fluctuations of the electrostatic potential on the heme have a simple harmonic form, in accord with Marcus theory, even though the fluctuations of many individual protein groups (especially at the protein surface) are anharmonic. Plant Physiol, 2002 May, 129(1), 354 - 62 High-level production of gamma-linolenic acid in Brassica juncea using a delta6 desaturase from Pythium irregulare; Hong H et al.; gamma-Linolenic acid (GLA), a nutritionally important fatty acid in mammals, is synthesized by a delta6 desaturase . Here, we report identification of PiD6, a new cDNA from the oleaginous fungus, Pythium irregulare, encoding a 459-amino acid protein that shares sequence similarity to carboxyl-directed desaturases from various species . Expression of PiD6 in yeast (Saccharomyces cerevisiae) revealed that it converts exogenously supplied linoleic acid into GLA, indicating that it encodes a delta6 fatty acid desaturase . Expression of the desaturase in Brassica juncea under the control of the Brassica napus napin promoter resulted in production of three delta6 unsaturated fatty acids (18:2-6, 9; 18:3-6, 9, 12; and 18:4-6, 9, 12, 15) in seeds . Among them, GLA (18:3-6, 9, 12) is the most abundant and accounts for up to 40% of the total seed fatty acids . Lipid class and positional analysis indicated that GLA is almost exclusively incorporated into triacylglycerol (98.5%) with only trace amounts found in the other lipids . Within triacylglycerols, GLA is more abundant at the sn-2 position. Plant Physiol, 2002 May, 129(1), 300 - 9 Molecular and genetic characterization of a non-climacteric phenotype in melon reveals two loci conferring altered ethylene response in fruit; Perin C et al.; Fruit ripening and abscission are associated with an ethylene burst in several melon (Cucumis melo) genotypes . In cantaloupe as in other climacteric fruit, exogenous ethylene can prematurely induce abscission, ethylene production, and ripening . Melon genotypes without fruit abscission or without ethylene burst also exist and are, therefore, non-climacteric . In the nonabscising melon fruit PI 161375, exogenous ethylene failed to stimulate abscission, loss of firmness, ethylene production, and expression of all target genes tested . However, the PI 161375 etiolated seedlings displayed the usual ethylene-induced triple response . Genetic analysis on a population of recombinant cantaloupe Charentais x PI 161375 inbred lines in segregation for fruit abscission and ethylene production indicated that both characters are controlled by two independent loci, abscission layer (Al)-3 and Al-4 . The non-climacteric phenotype in fruit tissues is attributable to ethylene insensitivity conferred by the recessive allelic forms from PI 161375 . Five candidate genes (two ACO, two ACS, and ERS) that were localized on the melon genetic map did not exhibit colocalization with Al-3 or Al-4. Plant Physiol, 2002 May, 129(1), 85 - 94 Characterization of FRO1, a pea ferric-chelate reductase involved in root iron acquisition; Waters BM et al.; To acquire iron, many plant species reduce soil Fe(III) to Fe(II) by Fe(III)-chelate reductases embedded in the plasma membrane of root epidermal cells . The reduced product is then taken up by Fe(II) transporter proteins . These activities are induced under Fe deficiency . We describe here the FRO1 gene from pea (Pisum sativum), which encodes an Fe(III)-chelate reductase . Consistent with this proposed role, FRO1 shows similarity to other oxidoreductase proteins, and expression of FRO1 in yeast conferred increased Fe(III)-chelate reductase activity . Furthermore, FRO1 mRNA levels in plants correlated with Fe(III)-chelate reductase activity . Sites of FRO1 expression in roots, leaves, and nodules were determined . FRO1 mRNA was detected throughout the root, but was most abundant in the outer epidermal cells . Expression was detected in mesophyll cells in leaves . In root nodules, mRNA was detected in the infection zone and nitrogen-fixing region . These results indicate that FRO1 acts in root Fe uptake and they suggest a role in Fe distribution throughout the plant . Characterization of FRO1 has also provided new insights into the regulation of Fe uptake . FRO1 expression and reductase activity was detected only in Fe-deficient roots of Sparkle, whereas both were constitutive in brz and dgl, two mutants with incorrectly regulated Fe accumulation . In contrast, FRO1 expression was responsive to Fe status in shoots of all three plant lines . These results indicate differential regulation of FRO1 in roots and shoots, and improper FRO1 regulation in response to a shoot-derived signal of iron status in the roots of the brz and dgl mutants. J Cell Biol, 2002 May 13, 157(4), 631 - 43 Epub 2002 May 13. The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins; Suvorova ES et al.; The Sec34/35 complex was identified as one of the evolutionarily conserved protein complexes that regulates a cis-Golgi step in intracellular vesicular transport . We have identified three new proteins that associate with Sec35p and Sec34p in yeast cytosol . Mutations in these Sec34/35 complex subunits result in defects in basic Golgi functions, including glycosylation of secretory proteins, protein sorting, and retention of Golgi resident proteins . Furthermore, the Sec34/35 complex interacts genetically and physically with the Rab protein Ypt1p, intra-Golgi SNARE molecules, as well as with Golgi vesicle coat complex COPI . We propose that the Sec34/35 protein complex acts as a tether that connects cis-Golgi membranes and COPI-coated, retrogradely targeted intra-Golgi vesicles. J Biol Chem, 2002 Jul 19, 277(29), 26185 - 93 Epub 2002 May 13. Inactivation of Mre11 does not affect VSG gene duplication mediated by homologous recombination in Trypanosoma brucei; Robinson NP et al.; We demonstrate, by gene deletion analysis, that Mre11 has a critical role in maintaining genomic integrity in Trypanosoma brucei . mre11(-/-) null mutant strains exhibited retarded growth but no delay or disruption of cell cycle progression . They showed also a weak hyporecombination phenotype and the accumulation of gross chromosomal rearrangements, which did not involve sequence translocation, telomere loss, or formation of new telomeres . The trypanosome mre11(-/-) strains were hypersensitive to phleomycin, a mutagen causing DNA double strand breaks (DSBs) but, in contrast to mre11(-/-) null mutants in other organisms and T . brucei rad51(-/-) null mutants, displayed no hypersensitivity to methyl methanesulfonate, which causes point mutations and DSBs . Mre11 therefore is important for the repair of chromosomal damage and DSBs in trypanosomes, although in this organism the intersection of repair pathways appears to differ from that in other organisms . Mre11 inactivation appears not to affect VSG gene switching during antigenic variation of a laboratory strain, which is perhaps surprising given the importance of homologous recombination during this process. J Biol Chem, 2002 Jul 26, 277(30), 27256 - 64 Epub 2002 May 14. Isolation and characterization of the putative nuclear modifier gene MTO1 involved in the pathogenesis of deafness-associated mitochondrial 12 S rRNA A1555G mutation; Li X et al.; The human mitochondrial 12 S rRNA A1555G mutation has been found to be associated with aminoglycoside-induced and non-syndromic deafness . However, putative nuclear modifier gene(s) have been proposed to regulate the phenotypic expression of this mutation . In yeast, the mutant alleles of MTO1, encoding a mitochondrial protein, manifest respiratory-deficient phenotype only when coupled with the mitochondrial 15 S rRNA P(R)454 mutation corresponding to human A1555G mutation . This suggests that the MTO1-like modifier gene may influence the phenotypic expression of human A1555G mutation . Here we report the identification of full-length cDNA and elucidation of genomic organization of the human MTO1 homolog . Human Mto1 is an evolutionarily conserved protein that implicates a role in the mitochondrial tRNA modification . Functional conservation of this protein is supported by the observation that isolated human MTO1 cDNA can complement the respiratory deficient phenotype of yeast mto1 cells carrying P(R)454 mutation . MTO1 is ubiquitously expressed in various tissues, but with a markedly elevated expression in tissues of high metabolic rates including cochlea . These observations suggest that human MTO1 is a structural and functional homolog of yeast MTO1 . Thus, it may play an important role in the pathogenesis of deafness-associated A1555G mutation in 12 S rRNA gene or mutations in tRNA genes. J Biol Chem, 2002 Jul 26, 277(30), 26944 - 9 Epub 2002 May 14. Maturation of cytosolic iron-sulfur proteins requires glutathione; Sipos K et al.; Glutathione is the major protective agent against oxidative stress in Saccharomyces cerevisiae . Deletion of the GSH1 gene (strain Deltagsh1) encoding the enzyme that catalyzes the first step of glutathione biosynthesis leads to growth arrest, which can be relieved by either glutathione or reducing agents such as dithiothreitol . Because defects in the biosynthesis of cellular iron-sulfur (Fe/S) proteins are associated with increases in glutathione levels, we examined the consequences of glutathione depletion on this essential process . No significant defects were detected in the amounts, activities, and maturation of mitochondrial Fe/S proteins in glutathione-depleted Deltagsh1 cells . On the contrary, the maturation of extra-mitochondrial Fe/S proteins was decreased substantially . The defect was rectified neither by addition of dithiothreitol nor under anaerobic conditions excluding oxidative damage of Fe/S clusters . A double mutant in GSH1 and ATM1 encoding a mitochondrial ATP binding cassette (ABC) transporter involved in cytosolic Fe/S protein maturation is nonviable even in the presence of dithiothreitol . Similar to atm1 and other mutants defective in cytosolic Fe/S protein maturation, mitochondria from glutathione-depleted Deltagsh1 cells accumulated high amounts of iron . Together, our data demonstrate that glutathione, in addition to its protective role against oxidative damage, performs a novel and specific function in the maturation of cytosolic Fe/S proteins. J Biol Chem, 2002 Jul 19, 277(29), 26609 - 17 Epub 2002 May 14. Hansenula polymorpha Pex3p is a peripheral component of the peroxisomal membrane; Haan GJ et al.; Hansenula polymorpha Pex3p plays an essential role in the biogenesis and maintenance of the peroxisomal membrane . In the initial report, bakers' yeast Pex3p was suggested to represent an integral component of the peroxisomal membrane, containing one membrane-spanning region that exposes the N terminus of the protein into the organellar matrix . Biochemically, HpPex3p behaved like an integral membrane protein as it was resistant toward high salt and carbonate treatment . However, urea fully removed Pex3p from the membrane under conditions in which the integral membrane protein Pex10p was resistant to this treatment . Additional experiments, including protease protection assays and pre-embedding labeling experiments on purified organellar fractions from cells that produced Pex3ps carrying Myc epitopes at various selected locations in the protein, revealed that invariably all Myc tags were accessible for externally added proteases and antibodies, independent of the presence of detergents . Also, overproduction of Pex3p failed to demonstrate the typical integral membrane protein structures in fracture faces of freeze-fractured peroxisomes . Taken together, our data suggest that HpPex3p does not span the peroxisomal membrane but instead is tightly associated to the cytosolic face of the organelle where it may be present in focal protein clusters. J Biol Chem, 2002 Jul 5, 277(27), 23981 - 4 Epub 2002 May 14. Copper ion-sensing transcription factor Mac1p post-translationally controls the degradation of its target gene product Ctr1p; Yonkovich J et al.; Copper ion uptake must be regulated to avoid both deficiency and excess because its essential yet toxic biological nature depends on the concentration . Yeast copper uptake is controlled at both the transcriptional and post-translational levels . The transcription of CTR1 and CTR3, encoding high affinity copper ion transporters, is regulated by the copper ion-sensing transcription factor Mac1p through the cis-acting copper ion-responsive elements in CTR1 and CTR3 promoters . Ctr1p is known to undergo degradation in cells exposed to high copper levels . We report that Mac1p is also required for copper-dependent Ctr1p degradation . Both mutations within a conserved copper ion binding motif, the "Cu-fist" in the Mac1p DNA-binding domain, and within a metal ion binding motif, REP-III located in the cytosolic domain of Ctr1p, cause defects in Ctr1p turnover . Furthermore, we show that the Mac1p limits intracellular copper accumulation likely by controlling Ctr1p degradation . The findings have uncovered an unprecedented mechanism by which a transcription factor not only regulates its target gene transcription but also controls the degradation of its target gene product. Biochemistry, 2002 May 21, 41(20), 6469 - 76 Structures of the cuprous-thiolate clusters of the Mac1 and Ace1 transcriptional activators; Brown KR et al.; X-ray absorption spectroscopy on the minimal copper-regulatory domains of the two copper-regulated transcription factors (Ace1 and Mac1) in Saccharomyces cerevisiae revealed the presence of a remarkably similar polycopper cluster in both proteins . The Cu-regulatory switch motif of Mac1 consisting of the C-terminal first Cys-rich motif, designated the C1 domain, binds four Cu(I) ions as does the Cu-regulatory domain of Ace1 . The four Cu(I) ions are bound to each molecule in trigonal geometry . An extended X-ray absorption fine structure (EXAFS) arising from outer-shell Cu...Cu interactions at 2.7 and 2.9 A was apparent in each Cu(I) complex indicative of a polycopper cluster . The intensity of the 2.9 A Cu...Cu backscatter peak, apparently diminished by partial cancellation, dominates the EXAFS . The results suggest that CuAce1 and CuMac1(C1) contain somewhat distorted forms of a known {Cu(4)-S(6)} cage in which a core of Cu atoms forming an approximate tetrahedron is bound by bridging thiolates above each of the six edges . The tetracopper clusters bound by Ace1 and Mac1 differ in that the Ace1 cluster is coordinated entirely by cysteinyl thiolate, whereas the cysteine-deficient Mac1 cluster appears to consist of a Cu(4)(S-Cys)(5)(N-His) cluster with a bridging histidyl-derived nitrogen. Biochemistry, 2002 May 21, 41(20), 6438 - 48 Spectroscopic characterization and O2 reactivity of the trinuclear Cu cluster of mutants of the multicopper oxidase Fet3p; Palmer AE et al.; Fet3p is a multicopper oxidase that uses four copper ions (one type 1, one type 2, and one type 3 binuclear site) to couple substrate oxidation to the reduction of O(2) to H(2)O . The type 1 Cu site shuttles electrons between the substrate and the type 2/type 3 Cu sites which form a trinuclear Cu cluster that is the active site for O(2) reduction . This study extends the spectroscopic and reactivity studies that have been conducted with type 1-substituted Hg (T1Hg) laccase to Fet3p and a mutant of Fet3p in which the trinuclear Cu cluster is perturbed . To examine the reaction between the trinuclear Cu cluster and O(2), the type 1 Cu Cys(484) was mutated to Ser, resulting in a type 1-depleted (T1D) form of the enzyme . Additional His to Gln mutations were made at the trinuclear cluster to further probe specific contributions to reactivity . One of these mutants (His(126)Gln) produces the first stable but perturbed trinuclear Cu cluster (T1DT3' Fet3p) . Spectroscopic characterization (absorption, circular dichroism, magnetic circular dichroism, and electron paramagnetic resonance) of the resting trinuclear sites in T1D and T1DT3' Fet3p reveal that the His(126)Gln mutation changes the electronic structure of both the type 3 and type 2 Cu sites . The trinuclear clusters in T1D and T1DT3' Fet3p react with O(2) to produce peroxide intermediates analogous to that observed in T1Hg laccase . Spectroscopic data on the peroxide intermediates in the three forms provide further insight into the structure of this intermediate . In T1D Fet3p, the decay of this peroxide intermediate is pH-dependent, and the rate of decay is 10-fold higher at low pH . In T1DT3' Fet3p, the decay of the peroxide intermediate is pH-independent and is slow at all pH's . This change in the pH dependence provides new insight into the mechanism of intermediate decay involving reductive cleavage of the O-O bond. Plant Mol Biol, 2002 May, 49(1), 31 - 44 Identification of SnIP1, a novel protein that interacts with SNF1-related protein kinase (SnRK1); Slocombe SP et al.; Plant SNF1-related protein kinase (SnRK1) phosphorylates 3-hydroxy-3-methylglutaryl-Coenzyme A, nitrate reductase and sucrose phosphate synthase in vitro, and is required for expression of sucrose synthase in potato tubers and excised leaves . In this study, a barley (Hordeum vulgare) endosperm cDNA, SnIP1, was isolated by two-hybrid screening with barley SnRK1b, a seed-specific form of SnRK1 . The protein encoded by the SnIP1 cDNA was found to interact with barley SnRK1b protein in vitro . Southern analysis suggested that barley contains a single SnIP1 gene or small gene family . SnIP1 transcripts were detected in RNA isolated from leaf, root and mid-maturation seed . Sequence similarity searches against protein, nucleotide and expressed sequence tag databases identified hitherto uncharacterized sequences related to SnIP1 from maize (Zea mays, accession number AI691404), arabidopsis (Arabidopsis thaliana . AC079673 and AB016886) and poplar (Populus balsamifera, AI166543) . No homologous sequences were identified from outside the plant kingdom, but weak sequence similarity was found between the SnIP1 peptide and yeast (Saccharomyces cerevisiae) SNF4 and its mammalian homologue AMPKy . Nevertheless, SnIP1 failed to complement a yeast snf4 mutant . SnIP1 was found to have little overall sequence similarity with the PV42 family of SNF4-like plant proteins, but proteins of both the SnIP1 and PV42 families contain a conserved hydrophobic sequence we named the SnIP motif. Curr Biol, 2002 Apr 30, 12(9), R316 - 8 Mitosis: aurora gives chromosomes a healthy stretch; Stern BM; Attachment of sister chromatids to microtubules from opposite spindle poles--bi-orientation--generates tension at the kinetochores . The Ipl1/Aurora B kinase responds to the absence of tension at mono-oriented chromosomes and promotes microtubule turnover and spindle checkpoint activation until a stable bi-oriented attachment is achieved. Curr Biol, 2002 Apr 30, 12(9), 762 - 6 The MYST domain acetyltransferase Chameau functions in epigenetic mechanisms of transcriptional repression; Grienenberger A et al.; Reversible acetylation of histone tails plays an important role in chromatin remodelling and regulation of gene activity . While modification by histone acetyltransferase (HAT) is usually linked to transcriptional activation, we provide here evidence for HAT function in several types of epigenetic repression . Chameau (Chm), a new Drosophila member of the MYST HAT family, dominantly suppresses position effect variegation (PEV), is required for the maintenance of Hox gene silencing by Polycomb group (PcG) proteins, and can partially substitute for the MYST Sas2 HAT in yeast telomeric position effect (TPE) . Finally, we provide in vivo evidence that the acetyltransferase activity of Chm is required in these processes, since a variant protein mutated in the catalytic domain no longer rescues PEV modification, telomeric silencing of SAS2-deficient yeast cells, nor lethality of chm mutant flies . These findings emphasize the role of an acetyltransferase in gene silencing, which supports, according to the histone code hypothesis, that transcription at a particular locus is determined by a precise combination of histone tail modifications rather than by overall acetylation levels. Pathobiology, 2001, 69(4), 219 - 24 Expression of MRE11 complex (MRE11, RAD50, NBS1) and hRap1 and its relation with telomere regulation, telomerase activity in human gastric carcinomas; Matsutani N et al.; The MRE11 complex (MRE11, RAD50, NBS1) are required for the repair of DNA double-strand breaks and have another important function in regulating telomere length . The silent information regulator (Sir) proteins required for telomere position effect also bind telomeres . hRap1 protein is a human ortholog of yeast Rap1 which regulates telomere length by interacting with TRF2 and is recruited to telomeres by TRF2 . We examined the expression of the MRE11 complex (MRE11, RAD50, NBS1), Sir2 and hRAP1 in 20 gastric carcinomas by reverse transcription polymerase chain reaction and then analyzed the relation between telomerase activity and other telomerase components such as human telomerase reverse transcriptase (TERT), human telomerase RNA component (hTR), human telomerase-associated protein (TEP1), telomeric repeat binding factor 1 (TRF1), TRF2- and TRF1-interacting, ankyrin-related ADP-ribose polymerase (tankyrase) as well as TRF1-interacting nuclear protein 2 (TIN2) . Of twenty gastric carcinomas examined, 13 (65%), 14 (70%), 16 (80%), 12 (60%) and 13 (65%) expressed MRE11, RAD50, NBS1, Sir2 and hRap1 at higher levels than corresponding nonneoplastic gastric mucosa, respectively . No obvious correlation was observed between MRE11 complex expression and telomerase activity or expression of TERT, hTR, TEP1, tankyrase and TIN2 . Carcinomas with high TRF1 expression expressed significantly higher levels of MRE11 and RAD50 than those with low TRF1 expression (p < 0.05) . On the other hand, carcinomas with high TRF2 expression expressed significantly higher levels of MRE11, NBS1 and hRap1 than those with low TRF2 expression (p < 0.05) . These results suggest that gastric carcinomas with high TRF1 and TRF2 expression may need a large quantity of the MRE11 complex . Moreover, gastric carcinomas with high TRF1 expression may require a large quantity of hRap1 . Genes Chromosomes Cancer, 2002 Jul, 34(3), 285 - 98 The cancer-related protein SSX2 interacts with the human homologue of a Ras-like GTPase interactor, RAB3IP, and a novel nuclear protein, SSX2IP; de Bruijn DR et al.; The SSX gene family is composed of at least five functional and highly homologous members, SSX1 to SSX5, that are normally expressed in only the testis and thyroid . SSX1, SSX2, or SSX4 may be fused to the SYT gene as a result of the t(X;18) translocation in synovial sarcoma . In addition, the SSX1, SSX2, SSX4, and SSX5 genes were found to be aberrantly expressed in several other malignancies, including melanoma . The SSX proteins are localized in the nucleus and are diffusely distributed . In addition, they may be included in polycomb-group nuclear bodies . Other studies have indicated that the SSX proteins may act as transcriptional repressors . As a first step toward the elucidation of the cellular signaling networks in which the SSX proteins may act, we used the yeast two-hybrid system to identify SSX2-interacting proteins . By doing so, two novel human proteins were detected: RAB3IP, the human homolog of an interactor of the Ras-like GTPase Rab3A; and a novel protein, SSX2IP . RAB3IP did not interact with either SSX1, SSX3, or SSX4 in the yeast two-hybrid system, whereas SSX2IP interacted with SSX3 but not with either SSX1 or SSX4 . Further analysis of deletion mutants showed that both RAB3IP and SSX2IP interact with the N-terminal moiety of the SSX2 protein . Immunofluorescence analyses of transfected cells revealed that the RAB3IP protein is normally localized in the cytoplasm . However, coexpression of both RAB3IP and SSX2 led to colocalization of both proteins in the nucleus . Likewise, the SSX2IP protein was found to be colocalizing with SSX2 in the nucleus . By performing glutathione-S-transferase pull-down assays, we found that both RAB3IP and SSX2IP interact directly with SSX2 in vitro . These newly observed protein/protein interactions may have important implications for the mechanisms underlying normal and malignant cellular growth . Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Mar, 34(2), 231 - 5 {Interaction between HTLV-1 transcription activator tax and Taxreb107}; Yang X et al.; Tax is a transcription activator encoded by human T-cell leukemia virus (HTLV)-1 . Ribosomal protein L6 was also defined as Taxreb107 (Tax responsible element binding protein 107) for its activity of binding to the long terminal repeats of HTLV-1 . To investigate the relationship between Tax and Taxreb107/RpL6, yeast two hybrid and GST pull-down assays were used . Results suggest that Tax can interact with Taxreb107/RpL6 directly and Taxreb107/RpL6 may regulate the function of Tax in HTLV-1 proliferation. Mol Biol Cell, 2002 May, 13(5), 1709 - 21 Repression and activation domains of RME1p structurally overlap, but differ in genetic requirements; Blumental-Perry A et al.; Rme1p, a repressor of meiosis in the yeast Saccharomyces cerevisiae, acts as both a transcriptional repressor and activator . Rme1p is a zinc-finger protein with no other homology to any protein of known function . The C-terminal DNA binding domain of Rme1p is essential for function . We find that mutations and progressive deletions in all three zinc fingers can be rescued by fusion of RME1 to the DNA binding domain of another protein . Thus, structural integrity of the zinc fingers is not required for the Rme1p-mediated effects on transcription . Using a series of mutant Rme1 proteins, we have characterized domains responsible for repression and activation . We find that the minimal transcriptional repression and activation domains completely overlap and lie in an 88-amino-acid N-terminal segment (aa 61-148) . An additional transcriptional effector determinant lies in the first 31 amino acids of the protein . Notwithstanding the complete overlap between repression and activation domains of Rme1p, we demonstrated a functional difference between repression and activation: Rgr1p and Sin4p are absolutely required for repression but dispensable for activation. Mol Biol Cell, 2002 May, 13(5), 1615 - 25 Characterization of signal that directs C-tail-anchored proteins to mammalian mitochondrial outer membrane; Horie C et al.; We analyzed the signal that directs the outer membrane protein with the C-terminal transmembrane segment (TMS) to mammalian mitochondria by using yeast Tom5 as a model and green fluorescent protein as a reporter . Deletions or mutations were systematically introduced into the TMS or the flanking regions and their intracellular localization in COS-7 cells was examined using confocal microscopy and cell fractionation . 1) Three basic amino acid residues within the C-terminal five-residue segment (C-segment) contained the information required for mitochondrial-targeting . Reduction of the net positive charge in this segment decreased mitochondrial specificity, and the mutants were distributed throughout the intracellular membranes . 2) Elongation of the TMS interfered with the function of the C-segment and the mutants were delivered to the intracellular membranes . 3) Separation of the TMS and C-segment by linker insertion severely impaired mitochondrial targeting function, leading to mislocalization to the cytoplasm . 4) Mutations or small deletions in the region of the TMS flanking the C-segment also impaired the mitochondrial targeting . Therefore, the moderate length of the TMS, the positive charges in the C-segment, and the distance between or context of the TMS and C-segment are critical for the targeting signal . The structural characteristics of the signal thus defined were also confirmed with mammalian C-tail-anchored protein OMP25. Mol Biol Cell, 2002 May, 13(5), 1608 - 14 Discrimination between paralogs using microarray analysis: application to the Yap1p and Yap2p transcriptional networks; Cohen BA et al.; Ohno {Ohno, S . (1970) in Evolution by Gene Duplication, Springer, New York} proposed that gene duplication with subsequent divergence of paralogs could be a major force in the evolution of new gene functions . In practice the functional differences between closely related homologues produced by duplications can be subtle and difficult to separate experimentally . Here we show that DNA microarrays can distinguish the functions of two closely related homologues from the yeast Saccharomyces cerevisiae, Yap1p and Yap2p . Although Yap1p and Yap2p are both bZIP transcription factors involved in multiple stress responses and are 88% identical in their DNA binding domains, our work shows that these proteins activate nonoverlapping sets of genes . Yap1p controls a set of genes involved in detoxifying the effects of reactive oxygen species, whereas Yap2p controls a set of genes over represented for the function of stabilizing proteins . In addition we show that the binding sites in the promoters of the Yap1p-dependent genes differ from the sites in the promoters of Yap2p-dependent genes and we validate experimentally that these differences are important for regulation by Yap1p . We conclude that while Yap1p and Yap2p may have some overlapping functions they are clearly not redundant and, more generally, that DNA microarray analysis will be an important tool for distinguishing the functions of the large numbers of highly conserved genes found in all eukaryotic genomes. J Biol Chem, 2002 Jul 12, 277(28), 24938 - 48 Epub 2002 May 02. Functional and molecular characterization of nucleobase transport by recombinant human and rat equilibrative nucleoside transporters 1 and 2 . Chimeric constructs reveal a role for the ENT2 helix 5-6 region in nucleobase translocation; Yao SY et al.; The human (h) and rat (r) equilibrative (Na(+)-independent) nucleoside transporters (ENTs) hENT1, rENT1, hENT2, and rENT2 belong to a family of integral membrane proteins with 11 transmembrane domains (TMs) and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine and coronary vasoactive drugs . Structurally, the proteins have a large glycosylated loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7 . In the present study, hENT1, rENT1, hENT2, and rENT2 were produced in Xenopus laevis oocytes and investigated for their ability to transport pyrimidine and purine nucleobases . hENT2 and rENT2 efficiently transported radiolabeled hypoxanthine, adenine, guanine, uracil, and thymine (apparent K(m) values 0.7-2.6 mm), and hENT2, but not rENT2, also transported cytosine . These findings were independently confirmed by hypoxanthine transport experiments with recombinant hENT2 produced in purine-cytosine permease (FCY2)-deficient Saccharomyces cerevisiae and provide the first direct demonstration that the ENT2 isoform is a dual mechanism for the cellular uptake of nucleosides and nucleobases, both of which are physiologically important salvage metabolites . In contrast, recombinant hENT1 and rENT1 mediated negligible oocyte fluxes of hypoxanthine relative to hENT2 and rENT2 . Chimeric experiments between rENT1 and rENT2 using splice sites at rENT1 residues 99 (end of TM 2), 171 (between TMs 4 and 5), and 231 (end of TM 6) identified TMs 5-6 of rENT2 (amino acid residues 172-231) as a determinant of nucleobase transport activity, suggesting that this domain forms part(s) of the ENT2 substrate translocation channel. J Biol Chem, 2002 Jul 19, 277(29), 26379 - 88 Epub 2002 May 10. Phosphatidylinositol 3-phosphate induces the membrane penetration of the FYVE domains of Vps27p and Hrs; Stahelin RV et al.; The FYVE domain mediates the recruitment of proteins involved in membrane trafficking and cell signaling to phosphatidylinositol 3-phosphate (PtdIns(3)P)-containing membranes . To elucidate the mechanism by which the FYVE domain interacts with PtdIns(3)P-containing membranes, we measured the membrane binding of the FYVE domains of yeast Vps27p and Drosophila hepatocyte growth factor-regulated tyrosine kinase substrate and their mutants by surface plasmon resonance and monolayer penetration analyses . These measurements as well as electrostatic potential calculation show that PtdIns(3)P specifically induces the membrane penetration of the FYVE domains and increases their membrane residence time by decreasing the positive charge surrounding the hydrophobic tip of the domain and causing local conformational changes . Mutations of hydrophobic residues located close to the PtdIns(3)P-binding pocket or an Arg residue directly involved in PtdIns(3)P binding abrogated the penetration of the FYVE domains into the monolayer, the packing density of which is comparable with that of biological membranes and large unilamellar vesicles . Based on these results, we propose a mechanism of the membrane binding of the FYVE domain in which the domain first binds to the PtdIns(3)P-containing membrane by specific PtdIns(3)P binding and nonspecific electrostatic interactions, which is then followed by the PtdIns(3)P-induced partial membrane penetration of the domain. EMBO J, 2002 May 15, 21(10), 2397 - 406 Structure and functional interactions of the Tsg101 UEV domain; Pornillos O et al.; Human Tsg101 plays key roles in HIV budding and in cellular vacuolar protein sorting (VPS) . In performing these functions, Tsg101 binds both ubiquitin (Ub) and the PTAP tetrapeptide 'late domain' motif located within the viral Gag protein . These interactions are mediated by the N-terminal domain of Tsg101, which belongs to the catalytically inactive ubiquitin E2 variant (UEV) family . We now report the structure of Tsg101 UEV and chemical shift mapping of the Ub and PTAP binding sites . Tsg101 UEV resembles canonical E2 ubiquitin conjugating enzymes, but has an additional N-terminal helix, an extended beta-hairpin that links strands 1 and 2, and lacks the two C-terminal helices normally found in E2 enzymes . PTAP-containing peptides bind in a hydrophobic cleft exposed by the absence of the C-terminal helices, whereas ubiquitin binds in a novel site surrounding the beta-hairpin . These studies provide a structural framework for understanding how Tsg101 mediates the protein-protein interactions required for HIV budding and VPS. EMBO J, 2002 May 15, 21(10), 2383 - 96 Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence; Langley E et al.; The yeast Sir2 protein mediates chromatin silencing through an intrinsic NAD-dependent histone deacetylase activity . Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in budding yeast and worms . Here, we show that SIRT1, the human Sir2 homolog, is recruited to the promyelocytic leukemia protein (PML) nuclear bodies of mammalian cells upon overexpression of either PML or oncogenic Ras (Ha-rasV12) . SIRT1 binds and deacetylates p53, a component of PML nuclear bodies, and it can repress p53-mediated transactivation . Moreover, we show that SIRT1 and p53 co-localize in nuclear bodies upon PML upregulation . When overexpressed in primary mouse embryo fibroblasts (MEFs), SIRT1 antagonizes PML-induced acetylation of p53 and rescues PML-mediated premature cellular senescence . Taken together, our data establish the SIRT1 deacetylase as a novel negative regulator of p53 function capable of modulating cellular senescence. EMBO J, 2002 May 15, 21(10), 2343 - 53 Essential role of calcineurin in response to endoplasmic reticulum stress; Bonilla M et al.; Depletion of calcium ions (Ca2+) from the endoplasmic reticulum (ER) of yeast cells resulted in the activation of the unfolded protein response (UPR) signaling pathway involving Ire1p and Hac1p . The depleted ER also stimulated Ca2+ influx at the plasma membrane through the Cch1p-Mid1p Ca2+ channel and another system . Surprisingly, both Ca2+ influx systems were stimulated upon accumulation of misfolded proteins in the ER even in the presence of Ca2+ . The ability of misfolded ER proteins to stimulate Ca2+ influx at the plasma membrane did not require Ire1p or Hac1p, and Ca2+ influx and signaling factors were not required for initial UPR signaling . However, activation of the Ca2+ channel, calmodulin, calcineurin and other factors was necessary for long-term survival of cells undergoing ER stress . A similar calcium cell survival (CCS) pathway operates in the pathogenic fungi and promotes resistance to azole antifungal drugs . These findings reveal an unanticipated new regulatory mechanism that couples ER stress to Ca2+ influx and signaling pathways, which help to prevent cell death and promote resistance to an important class of fungistatic drugs. Mol Cell Probes, 2002 Feb, 16(1), 31 - 9 Efficacy of a novel reverse transcriptase-polymerase chain reaction (RT-PCR) for detecting Toxoplasma gondii bradyzoite gene expression in human clinical specimens; Cultrera R et al.; A reverse transcriptase-polymerase chain reaction (RT-PCR) assay, was performed to evaluate the transcription degree of bradyzoite- or tachyzoite-specific genes of Toxoplasma gondii on cerebrospinal fluid (CSF) specimens from AIDS patients with toxoplasmic encephalitis (TE), and to distinguish an asymptomatic latent infection from a reactivated disease . This method was compared with nested DNA amplification (n)-PCR . The mRNA expression of the representative T . gondii cystic matrix (MAG1) or bradyzoite-specific (SAG4) genes was investigated on CSF obtained from AIDS patients with first episode (no . 11) or relapse (no . 8) of TE . The mRNA expression of tachyzoite-specific (SAG1) gene was also studied . New designed oligonucleotide primers and probes, which identify a 212 bp fragment inside to the open reading MAG1 sequence, were employed in both RT-PCR and n-PCR assays . Oligo-dT primed cDNA synthesis appeared a suitable method for subsequent analysis by n-PCR . RT-PCR has been shown to be more sensitive and specific than n-PCR . MAG1 and SAG4 gene expression was detected in 8 (100%) and 6 (75%) patients with TE relapses, respectively, while SAG1 detected 7 (63%) patients with TE first episode . These findings suggest that RT-PCR method is able to identify the bradyzoite stage of T . gondii especially in patients who are at risk for TE relapse. Biosci Biotechnol Biochem, 2002 Mar, 66(3), 628 - 31 Construction of protease-deficient Candida boidinii strains useful for recombinant protein production: cloning and disruption of proteinase A gene (PEP4) and proteinase B gene (PRBI); Komeda T et al.; The yeast Candida boidinii PEP4 and PRB1 genes, encoding proteinase A (PrA) and proteinase B (PrB), respectively, have been cloned and their primary structures were analyzed . The open reading frames of the PEP4 gene (1263 bp encoding a protein of 420 amino acids) and the PRBI gene (1683 bp encoding a protein of 560 amino acids) were found . The deduced amino acid sequences of PrA and PrB are very similar to Saccharomyces cerevisiae PrA and PrB (64% and 61% identities, respectively) . Both PEP4 and PRBI genes were disrupted in the C . boidinii genome by one-step gene disruption . The resultant pep4delta and the pep4delta prb1delta strains lost protease activity when compared with the wild-type original strain . The constructed C . boidinii strains are expected to be useful hosts for heterologous protein production. Biosci Biotechnol Biochem, 2002 Mar, 66(3), 613 - 21 Identification and expression of a rat fatty acid elongase involved in the biosynthesis of C18 fatty acids; Inagaki K et al.; A major part of the palmitic acid (C16:0) generated by fatty acid synthase is converted into stearic acid (C18:0) via carbon chain elongation . Here, we describe the cloning and expression of a rat hepatic enzyme, rELO2, responsible for the elongation of C16:0, presumably at the condensing reaction . Heterologous expression experiments in a yeast, Saccharomyces cerevisiae, demonstrated the elongation activity of rELO2 on C16:0 and to a lesser extent, C18:0 and fatty acids with low desaturation degree . This was distinct from that rELO1, a rat homolog of HELO1, which preferably catalyzed the elongation of mono- and polyunsaturated fatty acids of C16-C20 . The Northern analysis showed that the expression of rELO2, but not rELO1, in hepatocytes was activated by the cycles of fasting and refeeding rats on a fat-free diet . Under these conditions, the rELO1 was expressed constitutively in various tissues but the rELO2 transcripts were detected predominantly in liver. Breast Cancer Res Treat, 2002 Mar, 72(1), 61 - 8 Functional mutations of estrogen receptor protein: assay for detection; Nichols M et al.; Antiestrogens block the function of estrogen receptor (ER) by binding and misfolding the AF-2 transcriptional activation region in the ligand-binding domain, inhibiting or altering its association with coactivator proteins . We describe a novel assay uniquely configured to identify aberrations in this function that may lead to antiestrogen resistance . The identification of mutations of ER that affect its function is important to current breast cancer therapies . Standard methods to detect these mutations are cumbersome and the number of described mutations is limited, reflecting this difficulty . Conventional ER analysis in the clinic demonstrates the presence of antigenic determinants of the receptor protein or estrogenic ligand binding without reflection on the critical ability of the liganded receptor to interact with transcription cofactors . Here, we describe the use of estrogenic regulation of a site-specific recombinase activity, measuring deletion of a color marker gene via FLP-ER fusion proteins, to detect functional changes in ER protein folding that affects the site where cofactors interact . The assay provides a method to readily detect single amino acid changes in ER, some with biologically important consequences . Without such a functional assay as described, phenotypic changes are likely to remain undetected and under-evaluated . It is probable that some human tumors have antihormone resistance resulting from ER mutations that either block antihormone binding or transmit antihormone binding as a positive transcriptional signal via cofactor interaction . An assay to evaluate functional ER will lead to better predictive tests of treatment modalities. RNA, 2002 Mar, 8(3), 324 - 35 Dual function of the tRNA(m(5)U54)methyltransferase in tRNA maturation; Johansson MJ et al.; A 5-methyluridine (m(5)U) residue at position 54 is a conserved feature of bacterial and eukaryotic tRNAs . The methylation of U54 is catalyzed by the tRNA(m5U54)methyltransferase, which in Saccharomyces cerevisiae is encoded by the nonessential TRM2 gene . In this study, we identified four different strains with mutant forms of tRNA(Ser)CGA . The absence of the TRM2 gene in these strains decreased the stability of tRNA(Ser)CGA and induced lethality . Two alleles of TRM2 encoding catalytically inactive tRNA(m5U54)methyltransferases were able to stabilize tRNA(Ser)CGA in one of the mutants, revealing a role for the Trm2 protein per se in tRNA maturation . Other tRNA modification enzymes interacting with tRNA(Ser)CGA in the maturation process, such as Pus4p, Trm1 p, and Trm3p were essential or important for growth of the tRNA(Ser)CGA mutants . Moreover, Lhp1p, a protein binding RNA polymerase III transcripts, was required to stabilize the mutant tRNAs . Based on our results, we suggest that tRNA modification enzymes might have a role in tRNA maturation not necessarily linked to their known catalytic activity. J Microsc, 2002 Apr, 206(Pt 1), 24 - 32 Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment; Egner A et al.; We compare the axial sectioning capability of multifocal confocal and multifocal multiphoton microscopy in theory and in experiment, with particular emphasis on the background arising from the cross-talk between adjacent imaging channels . We demonstrate that a time-multiplexed non-linear excitation microscope exhibits significantly less background and therefore a superior axial resolution as compared to a multifocal single-photon confocal system . The background becomes irrelevant for thin (< 15 microm) and sparse fluorescent samples, in which case the confocal parallelized system exhibits similar or slightly better sectioning behaviour due to its shorter excitation wavelength . Theoretical and experimental axial responses of practically implemented microscopes are given. Plant Mol Biol, 2002 May, 49(2), 137 - 47 Do 14-3-3 proteins and plasma membrane H+-AtPases interact in the barley epidermis in response to the barley powdery mildew fungus? Finni C, Andersen CH, Borch J, Gjetting S, Christensen AB, de Boer AH, Thordal-Christensen H, Collinge DB. 14-3-3 proteins form a family of highly conserved proteins with central roles in many eukaryotic signalling networks . In plants, they bind to and activate the plasma membrane H+-ATPase, creating a binding site for the phytotoxin fusicoccin . Barley 14-3-3 transcripts accumulate in the epidermis upon inoculation with the powdery mildew fungus . We have isolated a cDNA encoding a plasma membrane H+-ATPase (HvHAI), which is also induced by powdery mildew attack . The C-terminal domain of this H+-ATPase interacts with 14-3-3 proteins in the yeast two-hybrid system . Inoculation with the powdery mildew fungus, or treatment with fusicoccin, results in an increase in fusicoccin binding ability of barley leaf membranes . Overlay assays show a fungus-induced increase in binding of digoxygenin-labelled 14-3-3 protein to several proteins including a 100 kDa membrane protein, probably the plasma membrane H+-ATPase . These effects are seen specifically in the inoculated epidermis and not in the whole leaf . We propose that 14-3-3 proteins are involved in an epidermis-specific response to the powdery mildew fungus, possibly via an activation of the plasma membrane H+-ATPase. Life Sci Space Res, 1973, 11, 105 - 10 Effects of space flight factors on the heredity of higher and lower plants; Dubinin NP et al.; The effects were studied of a long-term space exposure (72 days) aboard the Salyut orbital station on the following: dry seeds of Crepis capillaris (L.) Wallr . and Arabidopsis thaliana (L.) Heynh, haploid and diploid strains of Saccharomyces cerevisiae mutant for adenine locus (ad) and strain LARG-I of Chlorella vulgaris Beijer . A modifying effect of space factors on radiobiological action of gamma-irradiation from 137Cs was determined with the higher plant seeds and Chlorella culture . For this, the material was partly irradiated prior to, and partly after the flight at doses of 3, 30 and 10 kr for C . capillaris, A . thaliana and Chl . vulgaris, respectively . It was shown that: (1) Space factors caused reduced survival of Arabidopsis seedlings and Chlorella and yeast cultures, reduced productivity of Chlorella cells, increased mutability of Chlorella and yeast cells and increased frequency of chromosome aberrations in cells of Crepis root meristem . There are, however, a few exceptions: cell germination of C . capillaris was enhanced while mutability of A . thaliana seeds declined . (2) Space factors increased the adverse effects of the pre-flight irradiation on all the parameters of the cultures tested . (3) Space factors had no genetic effect on the objects tested . They have, however, caused a stronger inhibition of seed germination and augmenting survival of A . thaliana seedlings and Chlorella cells. Mol Cell Biol, 2002 Jun, 22(11), 3852 - 63 Strand bias in targeted gene repair is influenced by transcriptional activity; Liu L et al.; Modified single-stranded DNA oligonucleotides can direct nucleotide exchange in Saccharomyces cerevisiae . Point and frameshift mutations are corrected in a reaction catalyzed by cellular enzymes involved in various DNA repair processes . The present model centers on the annealing of the vector to one strand of the helix, followed by the correction of the designated base . The choice of which strand to target is a reaction parameter that can be controlled, so here we investigate the properties of strand bias in targeted gene repair . An in vivo system has been established in which a plasmid containing an actively transcribed, but mutated, hygromycin-enhanced green fluorescent protein fusion gene is targeted for repair and upon conversion will confer hygromycin resistance on the cell . Overall transcriptional activity has a positive influence on the reaction, elevating the frequency . If the targeting vector is synthesized so that it directs nucleotide repair on the nontranscribed strand, the level of gene repair is higher than if the template strand is targeted . We provide data showing that the targeting vector can be displaced from the template strand by an active T7 phage RNA polymerase . The strand bias is not influenced by which strand serves as the leading or lagging strand during DNA synthesis . These results may provide an explanation for the enhancement of gene repair observed when the non-template strand is targeted. Mol Cell Biol, 2002 Jun, 22(11), 3794 - 802 Growth and early postimplantation defects in mice deficient for the bromodomain-containing protein Brd4; Houzelstein D et al.; In a gene trap screen we recovered a mouse mutant line in which an insertion generated a null allele of the Brd4 gene . Brd4 belongs to the Fsh/Brd family, a group of structurally related proteins characterized by the association of two bromodomains and one extraterminal domain . Members of this family include Brd2/Ring3/Fsrg1 in mammals, fs(1)h in Drosophila, and Bdf1 in Saccharomyces cerevisiae . Brd4 heterozygotes display pre- and postnatal growth defects associated with a reduced proliferation rate . These mice also exhibit a variety of anatomical abnormalities: head malformations, absence of subcutaneous fat, cataracts, and abnormal liver cells . In primary cell cultures, heterozygous cells also display reduced proliferation rates and moderate sensitivity to methyl methanesulfonate . Embryos nullizygous for Brd4 die shortly after implantation and are compromised in their ability to maintain an inner cell mass in vitro, suggesting a role in fundamental cellular processes . Finally, sequence comparisons suggest that Brd4 is likely to correspond to the Brd-like element of the mediator of transcriptional regulation isolated by Y . W . Jiang, P . Veschambre, H . Erdjument-Bromage, P . Tempst, J . W . Conaway, R . C . Conaway, and R . D . Kornberg (Proc . Natl . Acad . Sci . USA 95:8538-8543, 1998) and the Brd4 mutant phenotype is discussed in light of this result . Together, our results provide the first genetic evidence for an in vivo role in mammals for a member of the Fsh/Brd family. Mol Cell Biol, 2002 Jun, 22(11), 3757 - 68 CK2 forms a stable complex with TFIIIB and activates RNA polymerase III transcription in human cells; Johnston IM et al.; CK2 is a highly conserved protein kinase with growth-promoting and oncogenic properties . It is known to activate RNA polymerase III (PolIII) transcription in Saccharomyces cerevisiae and is shown here to also exert a potent effect on PolIII in mammalian cells . Peptide and chemical inhibitors of CK2 block PolIII transcription in human cell extracts . Furthermore, PolIII transcription in mammalian fibroblasts is decreased significantly when CK2 activity is compromised by chemical inhibitors, antisense oligonucleotides, or kinase-inactive mutants . Coimmunoprecipitation and cofractionation show that endogenous human CK2 associates stably and specifically with the TATA-binding protein-containing factor TFIIIB, which brings PolIII to the initiation site of all class III genes . Serum stimulates TFIIIB phosphorylation in vivo, an effect that is diminished by inhibitors of CK2 . Binding to TFIIIC2 recruits TFIIIB to most PolIII promoters; this interaction is compromised specifically by CK2 inhibitors . The data suggest that CK2 stimulates PolIII transcription by binding and phosphorylating TFIIIB and facilitating its recruitment by TFIIIC2 . CK2 also activates PolI transcription in mammals and may therefore provide a mechanism to coregulate the output of PolI and PolIII . CK2 provides a rare example of an endogenous activity that operates on the PolIII system in both mammals and yeasts . Such evolutionary conservation suggests that this control may be of fundamental importance. Mol Cell Biol, 2002 Jun, 22(11), 3698 - 706 Ligand-selective potentiation of rat mineralocorticoid receptor activation function 1 by a CBP-containing histone acetyltransferase complex; Kitagawa H et al.; The rat mineralocorticoid receptor (MR) has two activation functions in distinct regions of the A/B domain, designated activation function 1a (AF-1a; amino acids 1 to 169) and AF-1b (amino acids 451 to 600) . Since the p160 family protein TIF2, a known component of the AF-2 coactivator complex, potentiates the transactivation function of AF-1b but not that of AF-1a, it is likely that some other, novel protein complex interacts with the AF-1a region . Therefore, we attempted to identify such coactivator complexes from HeLa nuclear extracts by biochemical purification using a glutathione S-transferase-MR AF-1a fusion protein . Purified AF-1a region-interacting proteins were found to contain RNA helicase A (RHA) and CBP . Further analysis showed that RHA interacted with the AF-1a region directly and then recruited a complex with histone acetyltransferase (HAT) activity that contained CBP . For full-length MR, aldosterone, but not hydrocortisone, was found to induce the binding of RHA/CBP complexes to the AF-1a region, as well as to allow the cooperative potentiation of MR transcriptional activity by RHA and CBP . In addition, a chromatin immunoprecipitation assay showed that aldosterone-bound MR, but not hydrocortisone-bound MR, recruited RHA/CBP complexes to native MR target gene promoters . Our results suggested that an altered conformation of the A/B region induced by aldosterone, but not hydrocortisone, might determine the accessibility of MR AF-1a to RHA/CBP complexes. Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 6784 - 9 Epub 2002 May 07. The TOR-controlled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine; Crespo JL et al.; The essential, rapamycin-sensitive TOR kinases regulate a diverse set of cell growth-related readouts in response to nutrients . Thus, the yeast TOR proteins function as nutrient sensors, in particular as sensors of nitrogen and possibly carbon . However, the nutrient metabolite(s) that acts upstream of TOR is unknown . We investigated the role of glutamine, a preferred nitrogen source and a key intermediate in yeast nitrogen metabolism, as a possible regulator of TOR . We show that the glutamine synthetase inhibitor L-methionine sulfoximine (MSX) specifically provokes glutamine depletion in yeast cells . MSX-induced glutamine starvation caused nuclear localization and activation of the TOR-inhibited transcription factors GLN3, RTG1, and RTG3, all of which mediate glutamine synthesis . The MSX-induced nuclear localization of GLN3 required the TOR-controlled, type 2A-related phosphatase SIT4 . Other TOR-controlled transcription factors, GAT1/NIL1, MSN2, MSN4, and an unknown factor involved in the expression of ribosomal protein genes, were not affected by glutamine starvation . These findings suggest that the TOR pathway senses glutamine . Furthermore, as glutamine starvation affects only a subset of TOR-controlled transcription factors, TOR appears to discriminate between different nutrient conditions to elicit a response appropriate to a given condition. Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 6631 - 6 Epub 2002 May 07. Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase; Helmstaedt K et al.; The yeast chorismate mutase is regulated by tyrosine as feedback inhibitor and tryptophan as crosspathway activator . The monomer consists of a catalytic and a regulatory domain covalently linked by the loop L220s (212-226), which functions as a molecular hinge . Two monomers form the active dimeri