J Mol Biol, 2001 Jul 27, 310(5), 965 - 71 Biogenesis of the dicarboxylate carrier (DIC): translocation across the mitochondrial outer membrane and subsequent release from the TOM channel are membrane potential-independent; Zara V et al.; The mitochondrial inner membrane of Saccharomyces cerevisiae contains a group of homologous carrier proteins that mediate the exchange of several metabolites . The members of this protein family are synthesized in the cytosol and reach their final topology after translocation across the mitochondrial outer membrane . Using the ADP/ATP carrier (AAC) as a model protein, previous studies have established four distinct steps of the import pathway (stages I-IV) . In the absence of the mitochondrial membrane potential (deltapsi), the AAC accumulates at the inner surface of the outer membrane (stage IIIa) and remains bound to the outer membrane import channel . Only in the presence of the membrane potential, can a complex of small Tim proteins mediate transfer of the AAC to the inner membrane . In this study, we characterized the import pathway of the dicarboxylate carrier (DIC) . Different from the AAC, the DIC showed complete deltapsi-independent translocation across the outer membrane, release from the import pore, and mainly accumulated in a soluble state in the intermembrane space, thus defining a new translocation intermediate (stage III*) . The DIC should be a suitable model protein for the characterization of deltapsi-independent functions of the intermembrane space Tim proteins. J Biol Regul Homeost Agents, 2001 Apr-Jun, 15(2), 123 - 9 Identification and characterization of androgen receptor associated coregulators in prostate cancer cells; Sampson ER et al.; The androgen receptor (AR) is a member of the nuclear receptor (NR) superfamily that mediates the effects of androgens on target tissues . Over the last decade, it has become apparent that NRs require accessory factors for optimal activation of target gene expression . Numerous NR coregulators have been identified, with diverse structures and potential mechanisms of coregulation, creating an increasingly complicated picture of NR action . Due to the expanding complexity of the coregulator field, this review will focus on the AR ligand-binding domain (LBD) and N-terminal interacting proteins identified by our lab . The LBD-interacting proteins ARA70, ARA55 and ARA54 were first characterized and ARA70 was found to have a relatively higher specificity for the AR in human prostate cancer DU145 cells . Characterization of the functional relationship between the AR and these coregulators indicated that ARA70 and ARA55 could enhance the androgenic effects of 17beta-estradiol (E2) and hydroxyflutamide (HF), an antiandrogen commonly used in the treatment of prostate cancer . ARA160, an AR N-terminal interacting protein also known as TATA element modulatory factor (TMF), was subsequently shown to cooperate with ARA70 in enhancing AR activity . Another AR N-terminal interacting protein, ARA24, interacted with the poly-Q tract, a region within the N-terminus of the AR linked to Kennedy's disease (X-linked spinal and bulbar muscular atrophy) . More recently, our lab has identified ARA267, a SET domain containing protein, and supervillin, an F-actin binding protein, as AR coregulators . Collectively, the data from these studies indicate that these coregulators are necessary for optimal AR transactivation . Interruption of the interaction between AR and these proteins may serve as a new therapeutic target in the treatment of prostate cancer. Biol Chem, 2001 Jun, 382(6), 891 - 902 Biological activity of mammalian transcriptional repressors; Thiel G et al.; Research on the regulation of transcription in mammals has focused in recent years mainly on the mechanism of transcriptional activation . However, transcriptional repression mediated by repressor proteins is a common regulatory mechanism in mammals and might play an important role in many biological processes . To understand the molecular mechanism of transcriptional repression, the activity of eight mammalian repressors or repressor domains was investigated using a set of model promoters in combination with two different transcriptional detection methods . The repressors studied were: REST, the thyroid hormone receptors alpha and beta, the zinc finger protein NK10 containing a 'kruppel-associated box' (KRAB), repressor domains derived from the proteins Egr-1, Oct2A and Dr1 and the repressor/activator protein YY1 . Here we show that the repressor domains of REST, Egr-1, the thyroid hormone receptors alpha< and beta and NK10 were transferable to a heterologous DNA-binding domain and repressed transcription from proximal and distal positions . Moreover, these repressor domains also blocked the activity of a strong viral enhancer in a 'remote position' . Thus, these domains are 'general' transcriptional repressor domains . The 'kruppel-associated box' was the most powerful repressor domain tested . In contrast, the repressor domains derived from Oct2A and Dr1 were inactive when fused to a heterologous DNA-binding domain . The repressor domain of YY1 exhibited transcriptional repression activity only in one of the transcriptional assay systems . The recruitment of histone deacetylases to the proximity of the basal transcriptional apparatus was recently discussed as a mechanism for some mammalian transcriptional repressor proteins . Here we show here that histone deacetylase 2, targeted to the reporter gene via DNA-protein interaction, functions as a transcriptional repressor protein regardless of the location of its binding site within the transcription unit. Plant Physiol, 2001 Aug, 126(4), 1519 - 26 Enhanced copper tolerance in Silene vulgaris (Moench) Garcke populations from copper mines is associated with increased transcript levels of a 2b-type metallothionein gene; van Hoof NA et al.; Silene vulgaris (Moench) Garcke has evolved populations with extremely high levels of copper tolerance . To evaluate the role of metallothioneins (MTs) in copper tolerance in S . vulgaris, we screened a cDNA library derived from a highly copper-tolerant population using Arabidopsis-based MT probes and identified an MT2b-like gene . When expressed in yeast, this gene, SvMT2b, restored cadmium and copper tolerance in different hypersensitive strains . Northern-blot analysis and quantitative reverse transcriptase-PCR showed that plants from the copper-tolerant S . vulgaris populations had significantly higher transcript levels of SvMT2b than plants from the copper-sensitive populations, both in roots and shoots and with and without copper exposure . Southern-blot analysis suggested that the higher expression of the latter allele was caused by gene amplification . Segregating families of crosses between copper-sensitive and copper-tolerant plants exhibited a 1 to 3 segregation for SvMT2b expression . Allele-specific PCR showed that low-expression F(3) plants were homozygous for the allele inherited from the copper-sensitive parent, whereas high-expression plants possessed at least one allele from the tolerant parent . SvMT2b expression did not cosegregate with copper tolerance in crosses between sensitive and tolerant plants . However, a significant cosegregation with copper tolerance did occur in families derived from crosses between moderately tolerant F(3) plants with different SvMT2b genotypes . Thus, overexpression of SvMT2b conferred copper tolerance although only within the genetic background of a copper tolerant plant. Plant Physiol, 2001 Aug, 126(4), 1480 - 92 Isolation and characterization of kinase interacting protein 1, a pollen protein that interacts with the kinase domain of PRK1, a receptor-like kinase of petunia; Skirpan AL et al.; Many receptor-like kinases have been identified in plants and have been shown by genetic or transgenic knockouts to play diverse physiological roles; however, to date, the cytosolic interacting proteins of relatively few of these kinases have been identified . We have previously identified a predominantly pollen-expressed receptor-like kinase of petunia (Petunia inflata), named PRK1, and we have shown by the antisense RNA approach that it is required for microspores to progress from the unicellular to bicellular stage . To investigate the PRK1-mediated signal transduction pathway, PRK1-K cDNA, encoding most of the cytoplasmic domain of PRK1, was used as bait in yeast (Saccharomyces cerevisiae) two-hybrid screens of pollen/pollen tube cDNA libraries of petunia . A protein named kinase interacting protein 1 (KIP1) was found to interact very strongly with PRK1-K . This interaction was greatly reduced when lysine-462 of PRK1-K, believed to be essential for kinase activity, was replaced with arginine (the resulting protein is named PRK1-K462R) . The amino acid sequence of KIP1 deduced from full-length cDNA contains an EF-hand Ca(2+)-binding motif and nine predicted coiled-coil regions . The yeast two-hybrid assay and affinity chromatography showed that KIP1 interacts with itself to form a dimer or higher multimer . KIP1 is present in a single copy in the genome, and is expressed predominantly in pollen with a similar temporal pattern to PRK1 . In situ hybridization showed that PRK1 and KIP1 transcripts were localized in the cytoplasm of pollen . PRK1-K phosphorylated KIP1-NT (amino acids 1--716), whereas PRK1-K462R only weakly phosphorylated KIP1-NT in vitro. J Biol Chem, 2001 Nov 2, 276(44), 41365 - 76 Epub 2001 Aug 10. Characterization of the binding interface between the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase; Arnesano F et al.; The interaction of the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase, Ccc2a, was investigated by NMR in solution . In particular, a solution of Cu(I)-15NAtx1 was titrated with apo-Ccc2a, and, vice versa, a solution of Cu(I)-15NCcc2a was titrated with apo-Atx1 . By following the 15N and 1H chemical shifts, a new species is detected in both experiments . This species is the same in both titrations and is in fast exchange with the parent species on the NMR time scale . Nuclear relaxation data are consistent with the formation of an adduct . Judging from the nuclear Overhauser effect spectroscopy patterns, the structure of Cu(I)-15NCcc2a in the presence of apo-Atx1 is not significantly altered, whereas Cu(I)-15NAtx1 in the presence of apo-Ccc2a experiences some changes with respect to both the apoproteins and the Cu(I)-loaded proteins . The structure of the Cu(I)-15NAtx1 moiety in the adduct was obtained from 1137 nuclear Overhauser effects to a final root mean square deviation to the mean structure of 0.76 +/- 0.13 A for the backbone and 1.11 +/- 0.11 A for the heavy atoms . 15N and 1H chemical shifts suggest the regions of interaction that, together with independent information, allow a structural model of the adduct to be proposed . The apo form of Atx1 displays significant mobility in loops 1 and 5, the N-terminal part of helix alpha1, and the C-terminal part of helix alpha2 on the ms-micros time scale . These regions correspond to the metal binding site . Such mobility is largely reduced in the free Cu(I)-Atx1 and in the adduct with apo-Ccc2a . The analogous mobility of Ccc2a in both Cu(I) and apo forms is reduced with respect to Atx1 . Such an adduct is relevant as a structural and kinetic model for copper transfer from Atx1 to Ccc2a in physiological conditions. EMBO J, 2001 Aug 15, 20(16), 4522 - 35 Sir2p exists in two nucleosome-binding complexes with distinct deacetylase activities; Ghidelli S et al.; The absolute requirement for the histone deacetylase activity of Sir2p in silencing coupled with the conservation of Sir2p-like proteins in larger eukaryotes suggests that this molecule plays an important role in gene regulation in all organisms . Here we report the purification and characterization of two Sir2p-containing protein complexes; one of which contains Sir4p and the other Net1p . The Sir4p-containing complex has an NAD-dependent histone deacetylase activity, while the Net1p-containing complex possesses deacetylase activity but only weak NAD-dependent histone deacetylase activity . Finally, we demonstrate that the Sir2p-containing complexes bind nucleosomes efficiently and partially restrict accessibility of the linker DNA to enzymatic probes. EMBO J, 2001 Aug 15, 20(16), 4423 - 31 Cic1, an adaptor protein specifically linking the 26S proteasome to its substrate, the SCF component Cdc4; Jager S et al.; In eukaryotes, the ubiquitin-proteasome system plays a major role in selective protein breakdown for cellular regulation . Here we report the discovery of a new essential component of this degradation machinery . We found the Saccharomyces cerevisiae protein Cic1 attached to 26S proteasomes playing a crucial role in substrate specificity for proteasomal destruction . Whereas degradation of short-lived test proteins is not affected, cic1 mutants stabilize the F-box proteins Cdc4 and Grr1, substrate recognition subunits of the SCF complex . Cic1 interacts in vitro and in vivo with Cdc4, suggesting a function as a new kind of substrate recruiting factor or adaptor associated with the proteasome. Biochem Biophys Res Commun, 2001 Aug 17, 286(2), 357 - 64 Occurrence of a putative SCF ubiquitin ligase complex in Drosophila; Bocca SN et al.; Many proteins are targeted to proteasome degradation by a family of E3 ubiquitin ligases, termed SCF complexes, that link substrate proteins to an E2 ubiquitin-conjugating enzyme . SCFs are composed of three core proteins-Skp1, Cdc53/Cull, Rbx1/Hrt1-and a substrate specific F-box protein . We have identified in Drosophila melanogaster the closest homologues to the human components of the SCF(betaTrCP) complex and the E2 ubiquitin-conjugating enzyme UbcH5 . We show that putative Drosophila SCF core subunits dSkpA and dRbx1 both interact directly with dCu11 and the F-box protein Slmb . We also describe the direct interaction of the UbcH5 related protein UbcD1 with dCul1 and Slmb . In addition, a functional complementation test performed on a Saccharomyces cerevisiae Hrt1p-deficient mutant showed that Drosophila Rbx1 is able to restore the yeast cells viability . Our results suggest that dRbx1, dSkpA, dCullin1, and Slimb proteins are components of a Drosophila SCF complex that functions in combination with the ubiquitin conjugating enzyme UbcD1 . Science, 2001 Aug 10, 293(5532), 1150 - 5 Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries; Noma K et al.; Eukaryotic genomes are organized into discrete structural and functional chromatin domains . Here, we show that distinct site-specific histone H3 methylation patterns define euchromatic and heterochromatic chromosomal domains within a 47-kilobase region of the mating-type locus in fission yeast . H3 methylated at lysine 9 (H3 Lys9), and its interacting Swi6 protein, are strictly localized to a 20-kilobase silent heterochromatic interval . In contrast, H3 methylated at lysine 4 (H3 Lys4) is specific to the surrounding euchromatic regions . Two inverted repeats flanking the silent interval serve as boundary elements to mark the borders between heterochromatin and euchromatin . Deletions of these boundary elements lead to spreading of H3 Lys9 methylation and Swi6 into neighboring sequences . Furthermore, the H3 Lys9 methylation and corresponding heterochromatin-associated complexes prevent H3 Lys4 methylation in the silent domain. Science, 2001 Aug 10, 293(5532), 1142 - 6 Snf1--a histone kinase that works in concert with the histone acetyltransferase Gcn5 to regulate transcription; Lo WS et al.; Modification of histones is an important element in the regulation of gene expression . Previous work suggested a link between acetylation and phosphorylation, but questioned its mechanistic basis . We have purified a histone H3 serine-10 kinase complex from Saccharomyces cerevisiae and have identified its catalytic subunit as Snf1 . The Snf1/AMPK family of kinases function in conserved signal transduction pathways . Our results show that Snf1 and the acetyltransferase Gcn5 function in an obligate sequence to enhance INO1 transcription by modifying histone H3 serine-10 and lysine-14 . Thus, phosphorylation and acetylation are targeted to the same histone by promoter-specific regulation by a kinase/acetyltransferase pair, supporting models of gene regulation wherein transcription is controlled by coordinated patterns of histone modification. Science, 2001 Aug 10, 293(5532), 1133 - 6 Coordination of a transcriptional switch by HMGI(Y) acetylation; Munshi N et al.; Dynamic control of interferon-beta (IFN-beta) gene expression requires the regulated assembly and disassembly of the enhanceosome, a higher-order nucleoprotein complex formed in response to virus infection . The enhanceosome activates transcription by recruiting the histone acetyltransferase proteins CREB binding protein (CBP) and p300/CBP-associated factors (PCAF)/GCN5, which, in addition to modifying histones, acetylate HMGI(Y), the architectural component required for enhanceosome assembly . We show that the accurate execution of the IFN-beta transcriptional switch depends on the ordered acetylation of the high-mobility group I protein HMGI(Y) by PCAF/GCN5 and CBP, which acetylate HMGI(Y) at distinct lysine residues on endogenous promoters . Whereas acetylation of HMGI(Y) by CBP at lysine-65 destabilizes the enhanceosome, acetylation of HMGI(Y) by PCAF/GCN5 at lysine-71 potentiates transcription by stabilizing the enhanceosome and preventing acetylation by CBP. Biochem Soc Trans, 2001 Aug, 29(Pt 4), 559 - 64 De novo design, synthesis and characterization of membrane-active peptides; Lear JD et al.; Our current level of understanding of membrane-protein folding is primitive, but it is beginning to advance . Previously {Choma, Gratkowski, Lear and DeGrado (2000) Nat . Struct . Biol . 7, 161-166}, we described studies of the association in detergent micelles of short, simple-sequence hydrophobic peptides modified from the sequence of the water-soluble, homodimeric coiled-coil GCN4-P1 peptide using the principle that the interiors of membrane proteins are similar to those of water-soluble proteins . Here, we discuss more quantitative aspects of the association equilibrium and compare the free energies of association of a number of mutant peptides designed to explore specific features responsible for the association. Biochem Soc Trans, 2001 Aug, 29(Pt 4), 446 - 51 Assembly of cytochrome c oxidase: what can we learn from patients with cytochrome c oxidase deficiency? Taanman JW, Williams SL. Cytochrome c oxidase is an intricate metalloprotein that transfers electrons from cytochrome c to oxygen in the last step of the mitochondrial respiratory chain . It uses the free energy of this reaction to sustain a transmembrane electrochemical gradient of protons . Site-directed mutagenesis studies of bacterial terminal oxidases and the recent availability of refined crystal structures of the enzyme are rapidly expanding the understanding of the coupling mechanism between electron transfer and proton translocation . In contrast, relatively little is known about the assembly pathway of cytochrome c oxidase . Studies in yeast have indicated that assembly is dependent on numerous proteins in addition to the structural subunits and prosthetic groups . Human homologues of a number of these assembly factors have been identified and some are now known to be involved in disease . To dissect the assembly pathway of cytochrome c oxidase, we are characterizing tissues and cell cultures derived from patients with genetically defined cytochrome c oxidase deficiency, using biochemical, biophysical and immunological techniques . These studies have allowed us to identify some of the steps of the assembly process. Biochem Soc Trans, 2001 Aug, 29(Pt 4), 431 - 6 AAA proteases of mitochondria: quality control of membrane proteins and regulatory functions during mitochondrial biogenesis; Langer T et al.; An ubiquitous and conserved proteolytic system regulates the stability of mitochondrial inner membrane proteins . Two AAA proteases with catalytic sites at opposite membrane surfaces form a membrane-integrated quality control system and exert crucial functions during the biogenesis of mitochondria . Their activity is modulated by another membrane-protein complex that is composed of prohibitins . Peptides generated upon proteolysis in the matrix space are transported across the inner membrane by an ATP-binding cassette transporter . The function of these conserved components is discussed in the present review. RNA, 2001 Aug, 7(8), 1153 - 64 Function and subnuclear distribution of Rpp21, a protein subunit of the human ribonucleoprotein ribonuclease P; Jarrous N et al.; Rpp21, a protein subunit of human nuclear ribonuclease P (RNase P) was cloned by virtue of its homology with Rpr2p, an essential subunit of Saccharomyces cerevisiae nuclear RNase P . Rpp21 is encoded by a gene that resides in the class I gene cluster of the major histocompatibility complex, is associated with highly purified RNase P, and binds precursor tRNA . Rpp21 is predominantly localized in the nucleoplasm but is also observed in nucleoli and Cajal bodies when expressed at high levels . Intron retention and splice-site selection in Rpp21 precursor mRNA regulate the intranuclear distribution of the protein products and their association with the RNase P holoenzyme . Our study reveals that dynamic nuclear structures that include nucleoli, the perinucleolar compartment and Cajal bodies are all involved in the production and assembly of human RNase P. Oncol Rep, 2001 Sep-Oct, 8(5), 1085 - 9 DNA methylation status of hMLH1, p16(INK4a), and CDH1 is not associated with mRNA expression levels of DNA methyltransferase and DNA demethylase in gastric carcinomas; Oue N et al.; DNA methyltransferase and DNA demethylase are enzymes potentially affecting promoter methylation status . We examined levels of DNA methyltransferase (DNMT1, DNMT3a, DNMT3b) and DNA demethylase (MBD2) mRNA expression by semi-quantitative RT-PCR . In addition, we examined promoter methylation status of hMLH1, p16(INK4a), and CDH1 by methylation-specific PCR since all three of these genes are reported to be hypermethylated in gastric carcinoma . MBD2 appeared to be down-regulated in neoplasms . The levels of DNMT1, DNMT3a, DNMT3b, and MBD2 mRNA expression were not associated with either tumor stage or histologic type . Promoter hypermethylation of hMLH1, p16(INK4a), and CDH1 was detected in 5/20 (25%), 8/20 (40%) and 8/20 (40%) of gastric carcinomas, respectively . There was no clear relation between DNA methylation status of hMLH1, p16(INK4a), and CDH1 and the mRNA expression levels of DNMT1, DNMT3a, DNMT3b or MBD2 . We divided the examined cases into two groups according to the number of hypermethylated genes . Cases with more than two hypermethylated genes comprised a hypermethylation group, and cases with no hypermethylation comprised a non-hypermethylation group . We found no group association for levels of DNMT1, DNMT3a, DNMT3b, and MBD2 mRNA expression . Our results suggest that the mRNA expression levels for pro-methylating (DNMT1, DNMT3a, DNMT3b) and anti-methylating (MBD2) enzymes is not a critical determinate of tumor-specific promoter hypermethylation of hMLH1, p(16INK4a), or CDH1 in gastric carcinoma. Trends Plant Sci, 2001 Aug, 6(8), 354 - 8 The U-box protein family in plants; Azevedo C et al.; The U-box is a highly conserved domain recently identified at the C terminus of yeast UFD2, an E4 ubiquitination factor . In yeast, UFD2 is the only U-box-containing protein, but there are two UFD2 homologs and several other proteins containing a U-box domain in humans . Intriguingly, a database search revealed 37 predicted proteins containing a U-box in Arabidopsis . The plant U-box (PUB) proteins form five distinct subclasses, suggesting that they play diverse roles . The ARC1 gene from Brassica, required for self-incompatibility, is currently the only PUB gene functionally characterized . Here, we discuss the characteristics and possible functions of the PUB gene family. Curr Opin Struct Biol, 2001 Aug, 11(4), 458 - 63 The design of self-replicating helical peptides; Issac R et al.; The self-assembly of helical peptides and information transfer through autocatalysis and cross-catalysis are the foundation of peptide-based molecular evolution models . Many fundamental properties of living systems, such as environmental sensitivity, chiroselectivity, cross-catalysis, dynamic error correction and conditional selection, are exhibited by various self-replicating peptide systems . Recently, advances have been made in the design of peptide systems with autocatalytic and cross-catalytic properties. Artif Cells Blood Substit Immobil Biotechnol, 2001 Jul, 29(4), 335 - 45 Study of interaction of mannan-BSA neoglycoconjugates with Concanavalin A; Mislovicova D et al.; Neoglycoconjugates prepared by synthesis of oxidized mannans from Saccharomyces cerevisiae with bovine serum albumin were studied for interaction with Concanavalin A . The mannan-bovine serum albumin neoglycoproteins, different in degree of mannan oxidation used for synthesis and its content in conjugates were prepared . The interaction of these glycoconjugates with Concanavalin A by using precipitation method was investigated . The conjugates prepared at high weight ratio mannan: protein (4:1) involved 47-65% of saccharides and formed by precipitation with Concanavalin A aggregates with low content of protein . The obtained results showed that conjugates with lower content of mannan (up to 30%) are more efficacious for their aggregation with Concanavalin A than the conjugates with high content of mannan. Oncogene, 2001 Jul 5, 20(30), 3995 - 4006 Multiple signaling interactions of Abl and Arg kinases with the EphB2 receptor; Yu HH et al.; The Eph family of receptor tyrosine kinases and the Abl family of non-receptor tyrosine kinases have both been implicated in tissue morphogenesis . They regulate the organization of the actin cytoskeleton in the developing nervous system and participate in signaling pathways involved in axon growth . Both Eph receptors and Abl are localized in the neuronal growth cone, suggesting that they play a role in axon pathfinding . Two-hybrid screens identified regions of Abl and Arg that bind to the EphB2 and EphA4 receptors, suggesting a novel signaling connection involving the two kinase families . The association of full-length Abl and Arg with EphB2 was confirmed by co-immunoprecipitation and found to involve several distinct protein interactions . The SH2 domains of Abl and Arg bind to tyrosine-phosphorylated motifs in the juxtamembrane region of EphB2 . A second, phosphorylation-independent interaction with EphB2 involves non-conserved sequences in the C-terminal tails of Abl and Arg . A third interaction between Abl and EphB2 is probably mediated by an intermediary protein because it requires tyrosine phosphorylation of EphB2, but not the binding sites for the Abl SH2 domain . The connection between EphB2 and Abl/Arg appears to be reciprocal . Activated EphB2 causes tyrosine phosphorylation of Abl and Arg, and vice versa . Interestingly, treatment of COS cells and B35 neuronal-like cells with ephrin-B1 to activate endogenous EphB2 decreased the kinase activity of endogenous Abl . These data are consistent with the opposite effects that Eph receptors and Abl have on neurite ougrowth and suggest that Eph receptors and Abl family kinases have shared signaling activities. Nature, 2001 Aug 9, 412(6847), 607 - 14 Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair; Walker JR et al.; The Ku heterodimer (Ku70 and Ku80 subunits) contributes to genomic integrity through its ability to bind DNA double-strand breaks and facilitate repair by the non-homologous end-joining pathway . The crystal structure of the human Ku heterodimer was determined both alone and bound to a 55-nucleotide DNA element at 2.7 and 2.5 A resolution, respectively . Ku70 and Ku80 share a common topology and form a dyad-symmetrical molecule with a preformed ring that encircles duplex DNA . The binding site can cradle two full turns of DNA while encircling only the central 3-4 base pairs (bp) . Ku makes no contacts with DNA bases and few with the sugar-phosphate backbone, but it fits sterically to major and minor groove contours so as to position the DNA helix in a defined path through the protein ring . These features seem well designed to structurally support broken DNA ends and to bring the DNA helix into phase across the junction during end processing and ligation. Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9648 - 53 Epub 2001 Aug 07. The Sec6/8 complex in mammalian cells: characterization of mammalian Sec3, subunit interactions, and expression of subunits in polarized cells; Matern HT et al.; The yeast exocyst complex (also called Sec6/8 complex in higher eukaryotes) is a multiprotein complex essential for targeting exocytic vesicles to specific docking sites on the plasma membrane . It is composed of eight proteins (Sec3, -5, -6, -8, -10, and -15, and Exo70 and -84), with molecular weights ranging from 70 to 144 kDa . Mammalian orthologues for seven of these proteins have been described and here we report the cloning and initial characterization of the remaining subunit, Sec3 . Human Sec3 (hSec3) shares 17% sequence identity with yeast Sec3p, interacts in the two-hybrid system with other subunits of the complex (Sec5 and Sec8), and is expressed in almost all tissues tested . In yeast, Sec3p has been proposed to be a spatial landmark for polarized secretion (1), and its localization depends on its interaction with Rho1p (2) . We demonstrate here that hSec3 lacks the potential Rho1-binding site and GFP-fusions of hSec3 are cytosolic . Green fluorescent protein (GFP)-fusions of nearly every subunit of the mammalian Sec6/8 complex were expressed in Madin-Darby canine kidney (MDCK) cells, but they failed to assemble into a complex with endogenous proteins and localized in the cytosol . Of the subunits tested, only GFP-Exo70 localized to lateral membrane sites of cell-cell contact when expressed in MDCK cells . Cells overexpressing GFP-Exo70 fail to form a tight monolayer, suggesting the Exo70 targeting interaction is critical for normal development of polarized epithelial cells. Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9694 - 9 Epub 2001 Aug 07. Functional analysis of the LACERATA gene of Arabidopsis provides evidence for different roles of fatty acid omega -hydroxylation in development; Wellesen K et al.; We describe lacerata (lcr) mutants of Arabidopsis, which display various developmental abnormalities, including postgenital organ fusions, and report cloning of the LCR gene by using the maize transposon Enhancer/Suppressor-mutator (En/Spm) . The pleiotropic mutant phenotype could be rescued by genetic complementation of lcr mutants with the wild-type LCR gene . The LCR gene encodes a cytochrome P450 monooxygenase, CYP86A8, which catalyzes omega-hydroxylation of fatty acids ranging from C12 to C18:1, as demonstrated by expression of the gene in yeast . Although palmitic and oleic acids were efficient substrates for LCR, 9,10-epoxystearate was not metabolized . Taken together with previous studies, our findings indicate that LCR-dependent omega-hydroxylation of fatty acids could be implicated in the biosynthesis of cutin in the epidermis and in preventing postgenital organ fusions . Strikingly, the same pathway seems to control trichome differentiation, the establishment of apical dominance, and senescence in plants. Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9587 - 92 Epub 2001 Aug 07. BRCA1 mediates ligand-independent transcriptional repression of the estrogen receptor; Zheng L et al.; Mutational inactivation of BRCA1 confers a cumulative lifetime risk of breast and ovarian cancers . However, the underlying basis for the tissue-restricted tumor-suppressive properties of BRCA1 remains poorly defined . Here we show that BRCA1 mediates ligand-independent transcriptional repression of the estrogen receptor alpha (ERalpha), a principal determinant of the growth, differentiation, and normal functional status of breasts and ovaries . In Brca1-null mouse embryo fibroblasts and BRCA1-deficient human ovarian cancer cells, ERalpha exhibited ligand-independent transcriptional activity that was not observed in Brca1-proficient cells . Ectopic expression in Brca1-deficient cells of wild-type BRCA1, but not clinically validated BRCA1 missense mutants, restored ligand-independent repression of ERalpha in a manner dependent upon apparent histone deacetylase activity . In estrogen-dependent human breast cancer cells, chromatin immunoprecipitation analysis revealed the association of BRCA1 with ERalpha at endogenous estrogen-response elements before, but not after estrogen stimulation . Collectively, these results reveal BRCA1 to be a ligand-reversible barrier to transcriptional activation by unliganded promoter-bound ERalpha and suggest a possible mechanism by which functional inactivation of BRCA1 could promote tumorigenesis through inappropriate hormonal regulation of mammary and ovarian epithelial cell proliferation. EMBO Rep, 2001 Aug, 2(8), 715 - 20 An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins; Lange H et al.; Biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins . Here, we identify the essential Erv1p of Saccharomyces cerevisia mitochondria as a novel component that is specifically required for the maturation of Fe/S proteins in the cytosol, but not in mitochondria . Furthermore, Erv1p was found to be important for cellular iron homeostasis . The homologous mammalian protein ALR ('augmenter of liver regeneration'), also termed hepatopoietin, can functionally replace defects in Erv1p and thus represents the mammalian orthologue of yeast Erv1p . Previously, a fragment of ALR was reported to exhibit an activity as an extracellular hepatotrophic growth factor . Both Erv1p and full-length ALR are located in the mitochondrial intermembrane space and represent the first components of this compartment with a role in the biogenesis of cytosolic Fe/S proteins . It is likely that Erv1p/ALR operates downstream of the mitochondrial ABC transporter Atm1p/ABC7/Sta1, which also executes a specific task in this essential biochemical process. Development, 2001 Jul, 128(13), 2593 - 602 A UAS site substitution approach to the in vivo dissection of promoters: interplay between the GATAb activator and the AEF-1 repressor at a Drosophila ecdysone response unit; Brodu V et al.; An ecdysone response unit (EcRU) directs the expression of the Fat body protein 1 (Fbp1) gene in the third instar larval Drosophila fat body . The tissue-specific activity of this regulatory element necessitates the binding of both the ligand-activated EcR/USP ecdysone receptor and GATAb . To analyze the role played by GATAb in the regulation of the Fbp1 EcRU activity, we have replaced the GATA-binding sites GBS1, GBS2 and GBS3 in the Fbp1 EcRU with UAS sites for the yeast GAL4 activator and tested the activity of the mutagenized Fbp1 EcRUs in transgenic lines, either in the presence or absence of ubiquitously expressed GAL4 . Our results reveal that GATAb plays two distinguishable roles at the Fbp1 EcRU that contribute to the tissue-specific activity of this regulatory element . On the one hand, GATAb mediates a fat body-specific transcriptional activation . On the other hand, it antagonizes specifically in the fat body a ubiquitous repressor that maintains the Fbp1 EcRU in an inactive state, refractory to activation by GAL4 . We identified this repressor as AEF-1, a factor previously shown to be involved in the regulation of the Drosophila Adh and yp1-yp2 genes . These results show that, for a functional dissection of complex promoter-dependent regulatory pathways, the replacement of specific regulatory target sites by UAS GAL4 binding sites is a powerful alternative to the widely used disruption approach. Development, 2001 Jul, 128(13), 2517 - 24 A new approach reveals syncytia within the visceral musculature of Drosophila melanogaster; Klapper R et al.; In order to reveal syncytia within the visceral musculature of Drosophila melanogaster, we have combined the GAL4/UAS system with the single-cell transplantation technique . After transplantation of single cells from UAS-GFP donor embryos into ubiquitously GAL4-expressing recipients, the expression of the reporter gene was exclusively activated in syncytia containing both donor- and recipient-derived nuclei . In the first trial, we tested the system in the larval somatic musculature, which is already known to consist of syncytia . By this means we could show that most of the larval somatic muscles are generated by clonally non-related cells . Moreover, using this approach we were able to detect syncytia within the visceral musculature - a tissue that has previously been described as consisting of mononuclear cells . Both the longitudinal visceral musculature of the midgut and the circular musculature of the hindgut consist of syncytia and persist through metamorphosis . This novel application of the transplantation technique might be a powerful tool to trace syncytia in any organism using the GAL4/UAS system. Development, 2001 Jun, 128(12), 2281 - 90 Two distinct domains of Bicoid mediate its transcriptional downregulation by the Torso pathway; Janody F et al.; The transcriptional activity of the Bicoid morphogen is directly downregulated by the Torso signal transduction cascade at the anterior pole of the Drosophila embryo . This regulation does not involve the homeodomain or direct phosphorylation of Bicoid . We analyse the transcriptional regulation of Bicoid in response to the Torso pathway, using Bicoid variants and fusion proteins between the Bicoid domains and the Gal4 DNA-binding domain . We show that Bicoid possesses three autonomous activation domains . Two of these domains, the serine/threonine-rich and the acidic domains, are downregulated by Torso, whereas the third activation domain, which is rich in glutamine, is not . The alanine-rich domain, previously described as an activation domain in vitro, has a repressive activity that is independent of Torso . Thus, Bicoid downregulation by Torso results from a competition between the glutamine-rich domain that is insensitive to Torso and the serine/threonine-rich and acidic activation domains downregulated by Torso . The alanine-rich domain contributes to this process indirectly by reducing the global activity of the protein and in particular the activity of the glutamine-rich domain that might otherwise prevent downregulation by Torso. J Mol Biol, 2001 Aug 17, 311(3), 543 - 8 Crystal structure of the 20 S proteasome:TMC-95A complex: a non-covalent proteasome inhibitor; Groll M et al.; The 20 S proteasome core particle (CP), a multicatalytic protease, is involved in a variety of biologically important processes, including immune response, cell-cycle control, metabolic adaptation, stress response and cell differentiation . Therefore, selective inhibition of the CP will be one possible way to influence these essential pathways . Recently, a new class of specific proteasome inhibitors, TMC-95s, was investigated and we now present a biochemical and crystallographic characterisation of the yeast proteasome core particle in complex with the natural product TMC-95A . This unusual heterocyclic compound specifically blocks the active sites of CPs non-covalently, without modifying the nucleophilic Thr1 residue . The inhibitor is bound to the CP by specific hydrogen bonds with the main-chain atoms of the protein . Analysis of the crystal structure of the complex has revealed which portions of TMC-95s are essential for binding to the proteasome . This will form the basis for the development of synthetic selective proteasome inhibitors as promising candidates for anti-tumoral or anti-inflammatory drugs . J Mol Biol, 2001 Aug 17, 311(3), 433 - 44 Structure of histone acetyltransferases; Marmorstein R; Histone acetyltranferase (HAT) enzymes are the catalytic subunits of multisubunit protein complexes that acetylate specific lysine residues on the N-terminal regions of the histone components of chromatin to promote gene activation . These enzymes, which now include more than 20 members, fall into distinct families that generally have high sequence similarity and related substrate specificity within families, but have divergent sequence and substrate specificity between families . Significant insights into the mode of catalysis and histone substrate binding have been provided by the structure determination of the divergent HAT enzymes Hat1, Gcn5/PCAF and Esa1 . A comparison of these structures reveals a structurally conserved central core domain that mediates extensive interactions with the acetyl-coenzyme A cofactor, and structurally divergent N and C-terminal domains . A correlation of these structures with other studies reveals that the core domain plays a particularly important role in histone substrate catalysis and that the N and C-terminal domains play important roles in histone substrate binding . These correlations imply a related mode of catalysis and histone substrate binding by a diverse group of HAT enzymes . Ann Med, 2001 Jul, 33(5), 313 - 8 The transcription factor Egr-1: a potential drug in wound healing and tissue repair; Braddock M; In the United States, between 40 and 90 million hospital days are lost per year as a result of trauma and surgical procedures which result in the loss of functional tissue . This is estimated to cost the economy and healthcare providers in excess of US$ 500 billion, a figure that is increasing because of extending population lifespan . Tissue engineering and gene therapies are radical new treatments that are aimed at tissue regeneration ranging from dermal, osteal and occular repair to the replacement of failing tissue with entire biosynthetic organs . Over the last decade, numerous proteins have been identified that are able to direct the synthesis of new tissue . Such proteins include growth factors, cytokines and, more recently, transcription factors. J Immunol, 2001 Aug 15, 167(4), 2343 - 8 Catalytic subunit of protein kinase A is an interacting partner of the inflammation-responsive transcription factor serum amyloid A-activating factor-1; Ray BK et al.; Serum amyloid A-activating factor-1 (SAF-1) is a zinc finger transcription factor that is activated by many mediators of inflammation including IL-1, IL-6, and bacterial LPS . However, the mechanism of activation is not fully understood . To identify possible activation partners for SAF-1, we used a yeast two-hybrid system that detected interaction between the catalytic subunit of cyclic AMP-dependent protein kinase (PKA-Calpha) and SAF-1 . Immunofluorescence and combined immunoprecipitation-Western blot analyses revealed colocalization and interaction between SAF-1 and PKA-Calpha . In vivo evidence of SAF-1 and PKA-Calpha interaction was further revealed by coimmunoprecipitation of these two proteins in cAMP-activated liver cells . We further show that SAF-1 is phosphorylated in vitro by PKA-Calpha and that addition of cAMP markedly induces in vivo phosphorylation of SAF-1 and transcription of SAF-regulated reporter genes . These results showed that SAF1-PKA-Calpha interaction is involved in functional activation of SAF-1. J Biol Chem, 2001 Oct 5, 276(40), 37327 - 34 Epub 2001 Aug 06. Role of the deafness dystonia peptide 1 (DDP1) in import of human Tim23 into the inner membrane of mitochondria; Rothbauer U et al.; Tim8 and Tim13 of yeast belong to a family of evolutionary conserved zinc finger proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space . Mutations in DDP1 (deafness dystonia peptide 1), the human homolog of Tim8, are associated with the Mohr-Tranebjaerg syndrome, a progressive neurodegenerative disorder . We show that DDP1 acts with human Tim13 in a complex in the intermembrane space . The DDP1.hTim13 complex is in direct contact with translocation intermediates of human Tim23 in mammalian mitochondria . The human DDP1.hTim13 complex complements the function of the TIM8.13 complex in yeast and facilitates import of yeast and human Tim23 . Thus, the pathomechanism underlying the Mohr-Tranebjaerg syndrome may involve an impaired biogenesis of the human TIM23 complex causing severe pleiotropic mitochondrial dysfunction. J Biol Chem, 2001 Oct 19, 276(42), 38820 - 9 Epub 2001 Aug 06. Functional analysis of the hydrophobic patch on nuclear transport factor 2 involved in interactions with the nuclear pore in vivo; Quimby BB et al.; Nuclear transport factor 2 (NTF2) is a small homodimeric protein that interacts simultaneously with both RanGDP and FxFG nucleoporins . The interaction between NTF2 and Ran is essential for the import of Ran into the nucleus . Here we use mutational analysis to dissect the in vivo role of the interaction between NTF2 and nucleoporins . We identify a series of surface residues that form a hydrophobic patch on NTF2, which when mutated disrupt the NTF2-nucleoporin interaction . Analysis of these mutants in vivo demonstrates that the strength of this interaction can be significantly reduced without affecting cell viability . However, cells cease to be viable if the interaction between NTF2 and nucleoporins is abolished completely, indicating that this interaction is essential for the function of NTF2 in vivo . In addition, we have isolated a dominant negative mutant of NTF2, N77Y, which has increased affinity for nucleoporins . Overexpression of the N77Y protein blocks nuclear protein import and concentrates Ran at the nuclear rim . These data support a mechanism in which NTF2 interacts transiently with FxFG nucleoporins to translocate through the pore and import RanGDP into the nucleus. Traffic, 2001 Aug, 2(8), 524 - 31 Approaching the molecular mechanism of autophagy; Stromhaug PE et al.; Autophagy is a complex cellular process that involves dynamic membrane rearrangements under a range of physiological conditions . It is a highly regulated process that plays a role in cellular maintenance and development, and has been implicated in a number of genetic diseases . Upon induction of autophagy, cytoplasm is sequestered into vesicles and delivered to a degradative organelle, the vacuole in yeast or the lysosome in mammalian cells . The process is unique in that it converts material that is topologically intracellular into topologically extracellular . Autophagy was first described more than 50 years ago, but it is since the discovery of the pathway in yeast cells that our knowledge about the molecular events taking place during the process has expanded . The generation of autophagy-specific mutants in a variety of yeast cell lines has provided insight into functional roles of more than 15 novel genes, double that number if we include genes whose products function also in other processes . Although we have learned much about autophagy, many questions remain to be answered . This review highlights the most recent advances in the autophagy field in both yeast and mammalian cells. Plant J, 2001 Jul, 27(2), 129 - 38 Two types of HKT transporters with different properties of Na+ and K+ transport in Oryza sativa; Horie T et al.; It is thought that Na+ and K+ homeostasis is crucial for salt-tolerance in plants . To better understand the Na+ and K+ homeostasis in important crop rice (Oryza sativa L.), a cDNA homologous to the wheat HKT1 encoding K+-Na+ symporter was isolated from japonica rice, cv Nipponbare (Ni-OsHKT1) . We also isolated two cDNAs homologous to Ni-OsHKT1 from salt-tolerant indica rice, cv Pokkali (Po-OsHKT1, Po-OsHKT2) . The predicted amino acid sequence of Ni-OsHKT1 shares 100% identity with Po-OsHKT1 and 91% identity with Po-OsHKT2, and they are 66-67% identical to wheat HKT1 . Low-K+ conditions (less than 3 mM) induced the expression of all three OsHKT genes in roots, but mRNA accumulation was inhibited by the presence of 30 mM Na+ . We further characterized the ion-transport properties of OsHKT1 and OsHKT2 using an expression system in the heterologous cells, yeast and Xenopus oocytes . OsHKT2 was capable of completely rescuing a K+-uptake deficiency mutation in yeast, whereas OsHKT1 was not under K+-limiting conditions . When OsHKTs were expressed in Na+-sensitive yeast, OsHKT1 rendered the cells more Na+-sensitive than did OsHKT2 in high NaCl conditions . The electrophysiological experiments for OsHKT1 expressed in Xenopus oocytes revealed that external Na+, but not K+, shifted the reversal potential toward depolarization . In contrast, for OsHKT2 either Na+ or K+ in the external solution shifted the reversal potential toward depolarization under the mixed Na+ and K+ containing solutions . These results suggest that two isoforms of HKT transporters, a Na+ transporter (OsHKT1) and a Na+- and K+-coupled transporter (OsHKT2), may act harmoniously in the salt tolerant indica rice. Arch Biochem Biophys, 2001 Aug 15, 392(2), 321 - 5 Rat liver mitochondrial contact sites and carnitine palmitoyltransferase-I; Hoppel C et al.; In hepatic mitochondria, the outer membrane enzyme, carnitine palmitoyltransferase-I (CPT-I), appears to colocalize with contact sites . We have prepared contact sites that are essentially devoid of noncontact site membranes . The contact site fraction has a high specific activity for CPT-I and contains a protein at 88 kDa that is recognized by antibodies directed at two different peptide epitopes on CPT-I . Similarly long-chain acyl-CoA synthetase (LCAS) specific activity is high in this fraction; a protein at 79 kDa is recognized by an antibody against LCAS . Although activity of carnitine palmitoyltransferase-II (CPT-II) is present, it is not enriched in the contact site fraction, and a protein of 68 kDa weakly reacted with anti-CPT-II antibody . Likewise, carnitine-acylcarnitine translocase (CACT) protein is present, but at a somewhat reduced level . Using an analytical continuous sucrose gradient, we demonstrate that the activities of CPT-I and LCAS and their associated immunoreactive proteins are present in a constant amount throughout the contact site subfractions . The enzymatic activity of CPT-II and its associated immunoreactive protein, as well as immunoreactive CACT, is absent in the lighter density gradient subfractions and is present in the higher density subfractions only in trace amounts . This heterogeneity of the contact site fraction is due to unvarying amounts of outer membrane and increasing amounts of attached inner membrane with increasing density of the subfractions . Arch Biochem Biophys, 2001 Aug 15, 392(2), 303 - 10 Molecular and catalytic properties of Arabidopsis thaliana adenylyl sulfate (APS)-kinase; Lillig CH et al.; A cDNA clone (Atakn1) from Arabidopsis thaliana encoding APS-kinase (EC 2.7.1.25) was investigated for structural and catalytic properties of the gene product . Recombinant his10-AtAkn1 formed PAPS at a Vmax of 7.35 U x mg(-1) . The Km for APS was 0.14 microM and for ATP 147 microM . APS caused a severe substrate inhibition (K(i) 4.5 microM) . The type of inhibition is uncompetitive with respect to MgATP . High ionic strength and reducing thiols stabilized the enzyme activity . Plant APS-kinase is regulated in vitro by the redox charge with thioredoxin as essential activator . Mutagenesis of a serine in S182C and S182F presumed to be involved in the transfer of the phosphoryl group had no effect upon catalytic activity . Using a yeast two-hybrid system with AtAkn1 as bait, an interacting clone was detected from a cDNA library of A . thaliana cv . Columbia that codes for an APS-kinase iso-form (Atakn2) . Complementation of APS-kinase-deficient Saccharomyces cerevisiae met14 showed that AtAkn2 is functionally active as APS-kinase . It was immunologically related to AtAkn1 and presumably represents a plastidal iso-form of the plant APS-kinase gene family . Plant Mol Biol, 2001 Jun, 46(3), 335 - 46 Two S-adenosylmethionine synthetase-encoding genes differentially expressed during adventitious root development in Pinus contorta; Lindroth AM et al.; Two S-adenosylmethionine synthetase (SAMS) cDNAs, PcSAMS1 and PcSAMS2, have been identified in Pinus contorta . We found that the two genes are differentially expressed during root development . Thus, PcSAMS1 is preferentially expressed in roots and exhibits a specific expression pattern in the meristem at the onset of adventitious root development, whereas PcSAMS2 is expressed in roots as well as in shoots and is down-regulated during adventitious root formation . The expression of the two SAMS genes is different from the SAMS activity levels during adventitious root formation . We conclude that other SAMS genes that remain to be characterized may contribute to the observed SAMS activity, or that the activities of PcSAMS1 and PcSAMS2 are affected by post-transcriptional regulation . The deduced amino acid sequences of PcSAMS1 and PcSAMS2 are highly divergent, suggesting different functional roles . However, both carry the two perfectly conserved motifs that are common to all plant SAMS . At the protein level, PcSAMS2 shares about 90% identity to other isolated eukaryotic SAMS, while PcSAMS1 shares less than 50% identity with other plant SAMS . In a phylogenetic comparison, PcSAMS1 seems to have diverged significantly from all other SAMS genes . Nevertheless, PcSAMS1 was able to complement a Saccharomyces cerevisiae sam1 sam2 double mutant, indicating that it encodes a functional SAMS enzyme. Plant Cell, 2001 Aug, 13(8), 1929 - 43 Dynamic recruitment of Cdc2 to specific microtubule structures during mitosis; Weingartner M et al.; A-type cyclin-dependent kinases (CDKs), also known as cdc2, are central to the orderly progression of the cell cycle . We made a functional Green Fluorescent Protein (GFP) fusion with CDK-A (Cdc2-GFP) and followed its subcellular localization during the cell cycle in tobacco cells . During interphase, the Cdc2-GFP fusion protein was found in both the cytoplasm and the nucleus, where it was highly resistant to extraction . In premitotic cells, a bright and narrow equatorial band appeared on the cell surface, resembling the late preprophase band, which disintegrated within 10 min as followed by time-lapse images . Cdc2-GFP was not found on prophase spindles but left the chromatin soon after this stage and associated progressively with the metaphase spindle in a microtubule-dependent manner . Arresting cells in mitosis through the stabilization of microtubules by taxol further enhanced the spindle-localized pool of Cdc2-GFP . Toward the end of mitosis, Cdc2-GFP was found at the midzone of the anaphase spindle and phragmoplast; eventually, it became focused at the midline of these microtubule structures . In detergent-extracted cells, the Cdc2-GFP remained associated with mitotic structures . Retention on spindles was prevented by pretreatment with the CDK-specific inhibitor roscovitine and was enhanced by the protein phosphatase inhibitor okadaic acid . Furthermore, we demonstrate that both the endogenous CDK-A and Cdc2-GFP were cosedimented with taxol-stabilized plant microtubules from cell extracts and that Cdc2 activity was detected together with a fraction of polymerized tubulin . These data provide evidence that the A-type CDKs associate physically with mitotic structures in a microtubule-dependent manner and may be involved in regulating the behavior of specific microtubule arrays throughout mitosis. Front Biosci, 2001 Aug 01, 6, D1019 - 23 The bromodomain: a regulator of ATP-dependent chromatin remodeling? Horn PJ, Peterson CL. In eukaryotes, processes requiring access to DNA are inhibited by the structural packaging of the genome . A number of specialized ATP-dependent chromatin remodeling enzymes have evolved to overcome this inhibition . One subset of these enzymes, SWI/SNF, plays a critical role in the regulation of transcription, often functioning in concert with nuclear histone acetyltransferases (HATs) . It remains unknown how these activities are coordinated . However, recent results revealing that the bromodomain, a motif common in these remodeling factors, constitutes an acetyl-lysine binding domain might provide insight into this process . Bromodomains may serve a role analogous to the signal transduction SH2 domain, by providing a means to recruit remodeling complexes to acetylated chromatin regions or to allosterically modify their function post-recruitment. Front Biosci, 2001 Aug 01, 6, D1008 - 18 You bet-cha: a novel family of transcriptional regulators; Florence B et al.; The BET proteins are a novel class of transcriptional regulators whose members can be found in animals, plants and fungi . Founding members are Human RING3, Drosophila Fsh and yeast Bdf1p . BET proteins are distinguished by an N-terminal bromodomain or bromodomains and an ET domain . As predicted by the presence of the bromodomain(s), these proteins have been found to be associated with chromatin . The poorly characterized ET domain functions as a protein-protein interaction motif and may be part of a serine-kinase activity . Other regions ("modular domains"), which are conserved only in certain BET proteins, are likely to provide sub-family specific functions . Genetic, biochemical and molecular techniques have implicated BET proteins in functions as diverse as meiosis, cell cycle control and homeosis . The data suggest that BET proteins may modulate chromatin structure and affect transcription via a sequence-independent mechanism . This review will attempt to summarize current research on BET proteins and envision where future research is likely to lead. Front Biosci, 2001 Aug 01, 6, D866 - 76 The bromodomain: a chromatin browser? Filetici P, P O, Ballario P. Reversible modification of histone tails is a regulatory step in chromatin remodeling . The N-terminal tails of histones are signaling platforms that carry amino acid residues for post-translational modification and contribute to chromosomal higher order structure . These modifications are performed by a number of chromatin modulators such as histone (h) acetyltransferase, h-deacetylase, h-methyltransferase and h-kinase . Large numbers of these enzymes as well as other chromatin-associated proteins share the bromodomain, a signature protein motif . Structural studies reveal not only wide structural conservation of bromodomains but also envision a possible role of this domain in the recognition of specific modified residues in the histone tails . The widespread presence of bromodomains in leukemogenic and cancer genes has provided a fundamental tool for studies of the role of epigenetic and chromatin remodeling in malignant diseases. Front Biosci, 2001 Aug 01, 6, D853 - 65 Acetyllysine-binding and function of bromodomain-containing proteins in chromatin; Dyson MH et al.; Acetylated histones are generally associated with active chromatin . The bromodomain has recently been identified as a protein module capable of binding to acetylated lysine residues, and hence is able to mediate the recruitment of factors to acetylated chromatin . Functional studies of bromodomain-containing proteins indicate how this domain contributes to the activity of a number of nuclear factors including histone acetyltransferases and chromatin remodelling complexes . Here, we review the characteristics of acetyllysine-binding by bromodomains, discuss associated domains found in these proteins, and address the function of the bromodomain in the context of chromatin . Finally, the modulation of bromodomain binding by neighbouring post-translational modifications within histone tails might provide a mechanism through which combinations of covalent marks could exert control on chromatin function. Front Biosci, 2001 Aug 01, 6, D849 - 52 Duality in bromodomain-containing protein complexes; Denis GV; Proteins that contain a motif called a bromodomain are implicated in both transcriptional activation and repression . The bromodomain of p/CAF, the only solution structure of a bromodomain that has been solved to date, reveals that the motif binds N-acetyl-lysine groups, presumably to anchor enzymatic functions to histones and by extension to chromatin . The enzymatic activities can either be encoded within the same polypeptide as the bromodomain motif, or associated with a multiprotein complex . Thus, a wide variety of chromatin-directed functions, including but not limited to phosphorylation, acetylation, methylation, transcriptional co-activation or recruitment, characterize the complexes that contain bromodomain motifs . Their versatility and ubiquity ensures diverse, rapid and flexible transcriptional responses. Mol Cell Biol, 2001 Sep, 21(17), 5979 - 91 Regulation of transcription factor YY1 by acetylation and deacetylation; Yao YL et al.; YY1 is a sequence-specific DNA-binding transcription factor that has many important biological roles . It activates or represses many genes during cell growth and differentiation and is also required for the normal development of mammalian embryos . Previous studies have established that YY1 interacts with histone acetyltransferases p300 and CREB-binding protein (CBP) and histone deacetylase 1 (HDAC1), HDAC2, and HDAC3 . Here, we present evidence that the activity of YY1 is regulated through acetylation by p300 and PCAF and through deacetylation by HDACs . YY1 was acetylated in two regions: both p300 and PCAF acetylated the central glycine-lysine-rich domain of residues 170 to 200, and PCAF also acetylated YY1 at the C-terminal DNA-binding zinc finger domain . Acetylation of the central region was required for the full transcriptional repressor activity of YY1 and targeted YY1 for active deacetylation by HDACs . However, the C-terminal region of YY1 could not be deacetylated . Rather, the acetylated C-terminal region interacted with HDACs, which resulted in stable HDAC activity associated with the YY1 protein . Finally, acetylation of the C-terminal zinc finger domain decreased the DNA-binding activity of YY1 . Our findings suggest that in the natural context, YY1 activity is regulated through intricate mechanisms involving negative feedback loops, histone deacetylation, and recognition of the cognate DNA sequence affected by acetylation and deacetylation of the YY1 protein. Mol Cell Biol, 2001 Sep, 21(17), 5838 - 45 Chl12 (Ctf18) forms a novel replication factor C-related complex and functions redundantly with Rad24 in the DNA replication checkpoint pathway; Naiki T et al.; RAD24 has been identified as a gene essential for the DNA damage checkpoint in budding yeast . Rad24 is structurally related to subunits of the replication factor C (RFC) complex, and forms an RFC-related complex with Rfc2, Rfc3, Rfc4, and Rfc5 . The rad24Delta mutation enhances the defect of rfc5-1 in the DNA replication block checkpoint, implicating RAD24 in this checkpoint . CHL12 (also called CTF18) encodes a protein that is structurally related to the Rad24 and RFC proteins . We show here that although neither chl12Delta nor rad24Delta single mutants are defective, chl12Delta rad24Delta double mutants become defective in the replication block checkpoint . We also show that Chl12 interacts physically with Rfc2, Rfc3, Rfc4, and Rfc5 and forms an RFC-related complex which is distinct from the RFC and RAD24 complexes . Our results suggest that Chl12 forms a novel RFC-related complex and functions redundantly with Rad24 in the DNA replication block checkpoint. J Biol Chem, 2001 Sep 28, 276(39), 36460 - 6 Epub 2001 Aug 02. Regulation of Snf1 kinase . Activation requires phosphorylation of threonine 210 by an upstream kinase as well as a distinct step mediated by the Snf4 subunit; McCartney RR et al.; The yeast Snf1 kinase and its metazoan orthologues, the AMP-activated protein kinases, are activated in response to nutrient limitation . Activation requires the phosphorylation of a conserved threonine residue in the activation loop of the catalytic subunit . A phosphopeptide antibody was generated that specifically recognizes Snf1 protein that is phosphorylated in its activation loop on threonine 210 . Using this reagent, we show that phosphorylation of threonine 210 correlates with Snf1 activity, since it is detected in cells subjected to glucose limitation but not in cells grown in abundant glucose . A Snf1 mutant completely lacking kinase activity was phosphorylated normally on threonine 210 in glucose-starved cells, eliminating the possibility that the threonine 210 modification is due to an autophosphorylation event . Cells lacking the Reg1 protein, a regulatory subunit for the Glc7 phosphatase, showed constitutive phosphorylation of Snf1 threonine 210 . Exposure of cells to high concentrations of sodium chloride also induced phosphorylation of Snf1 . Interestingly, Mig1, a downstream target of Snf1 kinase, is phosphorylated in glucose-stressed but not sodium-stressed cells . Finally, cells lacking the gamma subunit of the Snf1 kinase complex encoded by the SNF4 gene exhibited normal regulation of threonine 210 phosphorylation in response to glucose limitation but are unable to phosphorylate Mig1 efficiently . Our data indicate that activation of the Snf1 kinase complex involves two steps, one that requires a distinct upstream kinase and one that is mediated by the gamma subunit of the kinase itself. J Biol Chem, 2001 Sep 21, 276(38), 35644 - 51 Epub 2001 Aug 02. Interactions in the error-prone postreplication repair proteins hREV1, hREV3, and hREV7; Murakumo Y et al.; Most mutations after DNA damage in yeast Saccharomyces cerevisiae are induced by error-prone translesion DNA synthesis employing scRev1 and DNA polymerase zeta that consists of scRev3 and scRev7 proteins . Recently, the human REV1 (hREV1) and REV3 (hREV3) genes were identified, and their products were revealed to be involved in UV-induced mutagenesis, as observed for their yeast counterparts . Human REV7 (hREV7) was also cloned, and its product was found to interact with hREV3, but the biological function of hREV7 remained unknown . We report here the analyses of precise interactions in the human REV proteins . The interaction between hREV1 and hREV7 was identified by the yeast two-hybrid library screening using a bait of hREV7, which was confirmed by in vitro and in vivo binding assays . The homodimerization of hREV7 was also detected in the two-hybrid analysis . In addition, the precise domains for interaction between hREV7 and hREV1 or hREV3 and for hREV7 homodimerization were determined . Although hREV7 interacts with both hREV1 and hREV3, a stable complex formation of the three proteins was undetectable in vitro . These findings suggest the possibility that hREV7 might play an important role in regulating the enzymatic activities of hREV1 and hREV3 for mutagenesis in response to DNA damage. Biochem J, 2001 Aug 15, 358(Pt 1), 7 - 16 A large family of endosome-localized proteins related to sorting nexin 1; Teasdale RD et al.; Sorting nexin 1 (SNX1), a peripheral membrane protein, has previously been shown to regulate the cell-surface expression of the human epidermal growth factor receptor {Kurten, Cadena and Gill (1996) Science 272, 1008-1010} . Searches of human expressed sequence tag databases with SNX1 revealed eleven related human cDNA sequences, termed SNX2 to SNX12, eight of them novel . Analysis of SNX1-related sequences in the Saccharomyces cerevisiae genome clearly shows a greatly expanded SNX family in humans in comparison with yeast . On the basis of the predicted protein sequences, all members of this family of hydrophilic molecules contain a conserved 70-110-residue Phox homology (PX) domain, referred to as the SNX-PX domain . Within the SNX family, subgroups were identified on the basis of the sequence similarities of the SNX-PX domain and the overall domain structure of each protein . The members of one subgroup, which includes human SNX1, SNX2, SNX4, SNX5 and SNX6 and the yeast Vps5p and YJL036W, all contain coiled-coil regions within their large C-terminal domains and are found distributed in both membrane and cytosolic fractions, typical of hydrophilic peripheral membrane proteins . Localization of the human SNX1 subgroup members in HeLa cells transfected with the full-length cDNA species revealed a similar intracellular distribution that in all cases overlapped substantially with the early endosome marker, early endosome autoantigen 1 . The intracellular localization of deletion mutants and fusions with green fluorescent protein showed that the C-terminal regions of SNX1 and SNX5 are responsible for their endosomal localization . On the basis of these results, the functions of these SNX molecules are likely to be unique to endosomes, mediated in part by interactions with SNX-specific C-terminal sequences and membrane-associated determinants. Nature, 2001 Aug 2, 412(6846), 557 - 61 The DNA replication checkpoint response stabilizes stalled replication forks; Lopes M et al.; In response to DNA damage and blocks to replication, eukaryotes activate the checkpoint pathways that prevent genomic instability and cancer by coordinating cell cycle progression with DNA repair . In budding yeast, the checkpoint response requires the Mec1-dependent activation of the Rad53 protein kinase . Active Rad53 slows DNA synthesis when DNA is damaged and prevents firing of late origins of replication . Further, rad53 mutants are unable to recover from a replication block . Mec1 and Rad53 also modulate the phosphorylation state of different DNA replication and repair enzymes . Little is known of the mechanisms by which checkpoint pathways interact with the replication apparatus when DNA is damaged or replication blocked . We used the two-dimensional gel technique to examine replication intermediates in response to hydroxyurea-induced replication blocks . Here we show that hydroxyurea-treated rad53 mutants accumulate unusual DNA structures at replication forks . The persistence of these abnormal molecules during recovery from the hydroxyurea block correlates with the inability to dephosphorylate Rad53 . Further, Rad53 is required to properly maintain stable replication forks during the block . We propose that Rad53 prevents collapse of the fork when replication pauses. Nature, 2001 Aug 2, 412(6846), 553 - 7 Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint; Tercero JA et al.; The checkpoint kinase proteins Mec1 and Rad53 are required in the budding yeast, Saccharomyces cerevisiae, to maintain cell viability in the presence of drugs causing damage to DNA or arrest of DNA replication forks . It is thought that they act by inhibiting cell cycle progression, allowing time for DNA repair to take place . Mec1 and Rad53 also slow S phase progression in response to DNA alkylation, although the mechanism for this and its relative importance in protecting cells from DNA damage have not been determined . Here we show that the DNA-alkylating agent methyl methanesulphonate (MMS) profoundly reduces the rate of DNA replication fork progression; however, this moderation does not require Rad53 or Mec1 . The accelerated S phase in checkpoint mutants, therefore, is primarily a consequence of inappropriate initiation events . Wild-type cells ultimately complete DNA replication in the presence of MMS . In contrast, replication forks in checkpoint mutants collapse irreversibly at high rates . Moreover, the cytotoxicity of MMS in checkpoint mutants occurs specifically when cells are allowed to enter S phase with DNA damage . Thus, preventing damage-induced DNA replication fork catastrophe seems to be a primary mechanism by which checkpoints preserve viability in the face of DNA alkylation. Nat Rev Mol Cell Biol, 2001 Aug, 2(8), 621 - 7 A bouquet makes ends meet; Scherthan H; The 'chromosomal bouquet' is a polarized chromosomal arrangement that is highly conserved among eukaryotes . There have been many hypotheses about its role in the pairing of meiotic chromosomes, but until recently these have been difficult to test. Nat Rev Mol Cell Biol, 2001 Aug, 2(8), 589 - 98 Four deaths and a funeral: from caspases to alternative mechanisms; Leist M et al.; A single family of proteases, the caspases, has long been considered the pivotal executioner of all programmed cell death . However, recent findings of evolutionarily conserved, caspase-independent controlled death mechanisms have opened new perspectives on the biology of cell demise, with particular implications for neurobiology, cancer research and immunological processes. EMBO J, 2001 Aug 1, 20(15), 4183 - 93 An essential nuclear envelope integral membrane protein, Brr6p, required for nuclear transport; de Bruyn Kops A et al.; Despite rapid advances in our understanding of the function of the nuclear pore complex in nuclear transport, little is known about the role the nuclear envelope itself may play in this critical process . A small number of integral membrane proteins specific to the envelope have been identified in budding yeast, however, none has been reported to affect transport . We have identified an essential gene, BRR6, whose product, Brr6p, behaves like a nuclear envelope integral membrane protein . Notably, the brr6-1 mutant specifically affects transport of mRNA and a protein reporter containing a nuclear export signal . In addition, Brr6p depletion alters nucleoporin distribution and nuclear envelope morphology, suggesting that the protein is required for the spatial organization of nuclear pores . BRR6 interacts genetically with a subset of nucleoporins, and Brr6-green fluorescent protein (GFP) localizes in a punctate nuclear rim pattern, suggesting location at or near the nuclear pore . However, Brr6-GFP fails to redistribute in a (Delta)nup133 mutant, distinguishing Brr6p from known proteins of the pore membrane domain . We hypothesize that Brr6p is located adjacent to the nuclear pore and interacts functionally with the pore and transport machinery. EMBO J, 2001 Aug 1, 20(15), 4088 - 98 PIG-S and PIG-T, essential for GPI anchor attachment to proteins, form a complex with GAA1 and GPI8; Ohishi K et al.; Many eukaryotic cell surface proteins are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI) . The GPI transamidase mediates GPI anchoring in the endoplasmic reticulum, by replacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GPI . During this transamidation reaction, the GPI transamidase forms a carbonyl intermediate with a substrate protein . It was known that the GPI transamidase is a complex containing GAA1 and GPI8 . Here, we report two new components of this enzyme: PIG-S and PIG-T . To determine roles for PIG-S and PIG-T, we disrupted these genes in mouse F9 cells by homologous recombination . PIG-S and PIG-T knockout cells were defective in transfer of GPI to proteins, particularly in formation of the carbonyl intermediates . We also demonstrate that PIG-S and PIG-T form a protein complex with GAA1 and GPI8, and that PIG-T maintains the complex by stabilizing the expression of GAA1 and GPI8 . Saccharomyces cerevisiae Gpi16p (YHR188C) and Gpi17p (YDR434W) are orthologues of PIG-T and PIG-S, respectively. EMBO J, 2001 Aug 1, 20(15), 4076 - 87 The Ras-like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin-like protein KIF9; Piddini E et al.; Gem belongs to the Rad/Gem/Kir (RGK) subfamily of Ras-related GTPases, which also comprises Rem, Rem2 and Ges . The RGK family members Ges and Rem have been shown to produce endothelial cell sprouting and reorganization of the actin cytoskeleton upon overexpression . Here we show that high intracellular Gem levels promote profound changes in cell morphology and we investigate how this phenotype arises dynamically . We also show that this effect requires intact microtubules and microfilaments, and that Gem is associated with both cytoskeletal components . In order to investigate the mechanisms of Gem recruitment to the cytoskeleton, we performed a yeast two-hybrid screen and identified a novel kinesin-like protein, termed KIF9, as a new Gem interacting partner . We further show that Gem and KIF9 interact by co-immunoprecipitation . Furthermore, Gem and KIF9 display identical patterns of gene expression in different tissues and developmental stages . The Gem- KIF9 interaction reported here is the first molecular link between RGK family members and the microtubule cytoskeleton. EMBO J, 2001 Aug 1, 20(15), 4041 - 54 Mammalian Golgi-associated Bicaudal-D2 functions in the dynein-dynactin pathway by interacting with these complexes; Hoogenraad CC et al.; Genetic analysis in Drosophila suggests that Bicaudal-D functions in an essential microtubule-based transport pathway, together with cytoplasmic dynein and dynactin . However, the molecular mechanism underlying interactions of these proteins has remained elusive . We show here that a mammalian homologue of Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin . This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays . In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co-localizes with dynactin at microtubule plus ends . Overexpression studies using green fluorescent protein-tagged forms of BICD2 verify its intracellular distribution and co-localization with dynactin, and indicate that the C-terminus of BICD2 is responsible for Golgi targeting . Overexpression of the N-terminal domain of BICD2 disrupts minus-end-directed organelle distribution and this portion of BICD2 co-precipitates with cytoplasmic dynein . Nocodazole treatment of cells results in an extensive BICD2-dynactin-dynein co-localization . Taken together, these data suggest that mammalian BICD2 plays a role in the dynein- dynactin interaction on the surface of membranous organelles, by associating with these complexes. EMBO J, 2001 Aug 1, 20(15), 4035 - 40 Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion; Kato M et al.; In vitro homotypic fusion of yeast vacuoles occurs in three stages: priming, the Sec18 (NSF)-mediated changes that precede vacuole association; docking, the Ypt7 and SNARE-mediated pairing of vacuoles; and fusion, mediated by calmodulin/V0/t-SNARE interactions . Defects in catalysts of each stage result in fragmented (unfused) vacuoles . Strains with deletions in any of ERG genes 3-6, lacking normal ergosterol biosynthesis, have fragmented vacuoles . The ergosterol ligands filipin, nystatin and amphotericin B block the in vitro fusion of vacuoles from wild-type cells . Each of these inhibitors acts at the priming stage to inhibit Sec17p release from vacuoles . A reversible delay in Sec18p action prevents vacuoles from acquiring resistance to any of these three drugs, confirming that their action is on the normal fusion pathway . Ergosterol or cholesterol delivery to wild-type vacuoles stimulates their in vitro fusion, and the in vitro fusion of ergDelta vacuoles requires added sterol . The need for ergosterol for vacuole priming underscores the role of lipids in organizing the membrane elements of this complex reaction. EMBO J, 2001 Aug 1, 20(15), 3947 - 56 Structure and ligand recognition of the PB1 domain: a novel protein module binding to the PC motif; Terasawa H et al.; PB1 domains are novel protein modules capable of binding to target proteins that contain PC motifs . We report here the NMR structure and ligand-binding site of the PB1 domain of the cell polarity establishment protein, Bem1p . In addition, we identify the topology of the PC motif-containing region of Cdc24p by NMR, another cell polarity establishment protein that interacts with Bem1p . The PC motif-containing region is a structural domain offering a scaffold to the PC motif . The chemical shift perturbation experiment and the mutagenesis study show that the PC motif is a major structural element that binds to the PB1 domain . A structural database search reveals close similarity between the Bem1p PB1 domain and the c-Raf1 Ras-binding domain . However, these domains are functionally distinct from each other. EMBO J, 2001 Aug 1, 20(15), 3938 - 46 Novel modular domain PB1 recognizes PC motif to mediate functional protein-protein interactions; Ito T et al.; Modular domains mediating specific protein-protein interactions play central roles in the formation of complex regulatory networks to execute various cellular activities . Here we identify a novel domain PB1 in the budding yeast protein Bem1p, which functions in polarity establishment, and mammalian p67(phox), which activates the microbicidal phagocyte NADPH oxidase . Each of these specifically recognizes an evolutionarily conserved PC motif to interact directly with Cdc24p (an essential protein for cell polarization) and p40(phox) (a component of the signaling complex for the oxidase), respectively . Swapping the PB1 domain of Bem1p with that of p67(phox), which abolishes its interaction with Cdc24p, confers on cells temperature- sensitive growth and a bilateral mating defect . These phenotypes are suppressed by a mutant Cdc24p harboring the PC motif-containing region of p40(phox), which restores the interaction with the altered Bem1p . This domain-swapping experiment demonstrates that Bem1p function requires interaction with Cdc24p, in which the PB1 domain and the PC motif participate as responsible modules. Insect Biochem Mol Biol, 2001 Sep, 31(10), 949 - 64 Structural and functional conservation and divergence among acyl-CoA desaturases of two noctuid species, the corn earworm, Helicoverpa zea, and the cabbage looper, Trichoplusia ni; Rosenfield CL et al.; In this report, we describe the structural and functional analyses of four acyl-CoA desaturase-encoding cDNAs that we isolated from RNA expressed in the pheromone gland of the corn earworm, Helicoverpa zea . We deduced the homology relationships of the encoded proteins, designated HzPGDs1, HzPGDs2, HzPGDs3 and HzFBDs, to each other and to previously described desaturases of the cabbage looper moth, Trichoplusia ni, the fly, Drosophila melanogaster, and other more distantly related organisms . We also isolated genomic DNA fragments of the four H . zea desaturase-encoding genes, determined the locations of introns present in them, and compared them to conserved intron positions in reported desaturase genes of other species . We measured the levels of the four desaturase mRNAs in H . zea pheromone glands and larval fat bodies by RT-PCR . We established the functional identities of the deduced proteins HzPGDs1 and HzPGDs2, encoded by the two desaturase mRNAs that are differentially and abundantly expressed in pheromone glands of sexually mature adult H . zea females, by functional expression of their encoding cDNAs in a desaturase-deficient mutant, ole1, of the yeast Saccharomyces cerevisiae . We compared the unique unsaturated fatty acid profiles of HzPGDs1- and HzPGDs2-expressing transformants to those of strains expressing previously described Delta11 and Delta9 desaturases of T . ni. Brain Res Mol Brain Res, 2001 Aug 15, 92(1-2), 98 - 106 The cyclic AMP response element in the Bcl-2 promoter confers inducibility by hypoxia in neuronal cells; Freeland K et al.; In neuronal cells, expression of the anti-apoptotic Bcl-2 gene is induced by hypoxia and produces a protective effect . We show here that this effect is dependent upon the cyclic AMP response element (CRE) in the Bcl-2 promoter since mutation of this element abolishes the response and the isolated CRE can confer the response on a heterologous promoter . Interestingly however, the CRE in the Bcl-2 promoter does not render the promoter responsive to cyclic AMP and is not essential for its response to nerve growth factor . Despite the lack of cyclic AMP responsiveness, activation of the Bcl-2 promoter via the CRE in response to hypoxia requires the CREB transcription factor and is associated with the enhanced phosphorylation of CREB on serine 133 and enhanced transcriptional activation by the CREB-binding protein, CBP, in response to hypoxia . This finding establishes the importance of the CRE in the induction of Bcl-2 gene expression by hypoxia, allowing the Bcl-2 protein to protect neuronal cells against this damaging stimulus. Proc Natl Acad Sci U S A, 2001 Jul 31, 98(16), 9032 - 7 Substrate conformational transitions in the active site of chorismate mutase: their role in the catalytic mechanism; Guo H et al.; Chorismate mutase acts at the first branch-point of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate . The results of molecular dynamics simulations of the substrate in solution and in the active site of chorismate mutase are reported . Two nonreactive conformers of chorismate are found to be more stable than the reactive pseudodiaxial chair conformer in solution . It is shown by QM/MM molecular dynamics simulations, which take into account the motions of the enzyme, that when these inactive conformers are bound to the active site, they are rapidly converted to the reactive chair conformer . This result suggests that one contribution of the enzyme is to bind the more prevalent nonreactive conformers and transform them into the active form in a step before the chemical reaction . The motion of the reactive chair conformer in the active site calculated by using the QM/MM potential generates transient structures that are closer to the transition state than is the stable CHAIR conformer. Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9713 - 8 Epub 2001 Jul 31. Regulation of the transcriptional coactivator PGC-1 via MAPK-sensitive interaction with a repressor; Knutti D et al.; Mechanisms and signals that regulate transcriptional coactivators are still largely unknown . Here we provide genetic evidence for a repressor that interacts with and regulates the nuclear receptor coactivator PGC-1 . Association with the repressor requires a PGC-1 protein interface that is similar to the one used by nuclear receptors . Removal of the repressor enhances PGC-1 coactivation of steroid hormone responses . We also provide evidence that interaction of the repressor with PGC-1 is regulated by mitogen-activated protein kinase (MAPK) signaling . Activation of the MAPK p38 enhances the activity of wild-type PGC-1 but not of a PGC-1 variant that no longer interacts with the repressor . Finally, p38 activation enhances steroid hormone response in a PGC-1-dependent manner . Our data suggest a model where the repressor and nuclear receptors compete for recruiting PGC-1 to an inactive and active state, respectively . Extracellular signals such as nuclear receptor ligands or activators of the MAPK p38 can shift the equilibrium between the two states. Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9995 - 10000 Epub 2001 Jul 31. Functional activity and role of cation-efflux family members in Ni hyperaccumulation in Thlaspi goesingense; Persans MW et al.; The ability of Thlaspi goesingense to hyperaccumulate Ni seems to be governed in part by enhanced accumulation of Ni within leaf vacuoles . We have characterized genes from T . goesingense encoding putative vacuolar metal ion transport proteins, termed metal tolerance proteins (TgMTPs) . These proteins contain all of the features of cation-efflux family members, and evidence indicates they are derived from a single genomic sequence (TgMTP1) that gives rise to an unspliced (TgMTP1t1) and a spliced (TgMTP1t2) transcript . Heterologous expression of these transcripts in yeast lacking the TgMTP1 orthologues COT1 and ZRC1 complements the metal sensitivity of these yeast strains, suggesting that TgMTP1s are able to transport metal ions into the yeast vacuole in a manner similar to COT1 and ZRC1 . The unspliced and spliced TgMTP1 variants differ within a histidine-rich putative metal-binding domain, and these sequence differences are reflected as alterations in the metal specificities of these metal ion transporters . When expressed in yeast, TgMTP1t1 confers the highest level of tolerance to Cd, Co, and Zn, whereas TgMTP1t2 confers the highest tolerance to Ni . TgMTP1 transcripts are highly expressed in T . goesingense compared with orthologues in the nonaccumulators Arabidopsis thaliana, Thlaspi arvense, and Brassica juncea . We propose that the high-level expression of TgMTP1 in T . goesingense accounts for the enhanced ability of this hyperaccumulator to accumulate metal ions within shoot vacuoles. Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9581 - 6 Epub 2001 Jul 31. Kinetic trapping of DNA by transcription factor IIIB; Cloutier TE et al.; High levels of RNA polymerase III gene transcription are achieved by facilitated recycling of the polymerase on transcription factor IIIB (TFIIIB)-DNA complexes that are stable through multiple rounds of initiation . TFIIIB-DNA complexes in yeast comprise the TATA-binding protein (TBP), the TFIIB-related factor TFIIIB70, and TFIIIB90 . The high stability of the TFIIIB-DNA complex is conferred by TFIIIB90 binding to TFIIIB70-TBP-DNA complexes . This stability is thought to result from compound bends introduced in the DNA by TBP and TFIIIB90 and by protein-protein interactions that obstruct DNA dissociation . Here we present biochemical evidence that the high stability of TFIIIB-DNA complexes results from kinetic trapping of the DNA . Thermodynamic analysis shows that the free energies of formation of TFIIIB70-TBP-DNA (DeltaG degrees = -12.10 +/- 0.12 kcal/mol) and TFIIIB-DNA (DeltaG degrees = -11.90 +/- 0.14 kcal/mol) complexes are equivalent whereas a kinetic analysis shows that the half-lives of these complexes (46 +/- 3 min and 95 +/- 6 min, respectively) differ significantly . The differential stability of these isoenergetic complexes demonstrates that TFIIIB90 binding energy is used to drive conformational changes and increase the barrier to complex dissociation. Histochem Cell Biol, 2001 Jul, 116(1), 31 - 9 Protein phosphatase 2Calpha expression in normal human tissues: an immunohistochemical study; Lifschitz-Mercer B et al.; Protein phosphatase (PP2Calpha) is a member of the mammalian serine threonine-specific protein phosphatases family . We produced monoclonal antibodies against the recombinant PP2Calpha and evaluated the immunoreactivity of normal human tissues . The reactivity was strong in normal skin, the digestive tract, lung, kidney, breast, prostate, endocrine glands, and brain, while it was moderate in the ovary, testis, and liver . Epithelial cells revealed high levels of PP2Calpha expression, but stromal cells, including fibroblasts and endothelial cells, showed no or little PP2Calpha expression . Given the broad reactivity in endocrine and secreting epithelial cells, we propose that PP2Calpha expression might contribute to secretory cell function. Nat Genet, 2001 Aug, 28(4), 345 - 9 A novel pantothenate kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome; Zhou B et al.; Hallervorden-Spatz syndrome (HSS) is an autosomal recessive neurodegenerative disorder associated with iron accumulation in the brain . Clinical features include extrapyramidal dysfunction, onset in childhood, and a relentlessly progressive course . Histologic study reveals iron deposits in the basal ganglia . In this respect, HSS may serve as a model for complex neurodegenerative diseases, such as Parkinson disease, Alzheimer disease, Huntington disease and human immunodeficiency virus (HIV) encephalopathy, in which pathologic accumulation of iron in the brain is also observed . Thus, understanding the biochemical defect in HSS may provide key insights into the regulation of iron metabolism and its perturbation in this and other neurodegenerative diseases . Here we show that HSS is caused by a defect in a novel pantothenate kinase gene and propose a mechanism for oxidative stress in the pathophysiology of the disease. Nat Genet, 2001 Aug, 28(4), 303 - 4 To bind or not to bind; Biggin MD; Gene expression is regulated by transcription factors binding selectively to particular portions of the genome . To what extent are these protein-DNA interactions influenced by the intrinsic sequence-specific recognition properties at each protein, and to what extent are they affected by other factors, such as chromatin structure or cooperative interactions with other proteins . Genome-wide surveys of DNA binding by transcription factors in vivo are beginning to provide some answers. J Exp Bot, 2001 Aug, 52(361), 1739 - 40 ZMPP2, a novel type-2C protein phosphatase from maize; Broz AK et al.; A cDNA clone was selected as a candidate for the catalytic subunit of phospho-pyruvate dehydrogenase phosphatase (PDP) by screening a Zea mays expressed sequence tag database with the bovine PDP deduced amino acid sequence . Both strands of the cDNA were completely sequenced . The maize clone contains an open reading frame of 1098 base pairs that encodes a polypeptide of 40 127 Da, ZMPP2 . The deduced amino acid sequence of ZMPP2 contains the five PP2C signature domains, as does PDP . However, the expression pattern of ZMPP2, determined by reverse transcriptase-polymerase chain reaction, was different from those of the maize pyruvate dehydrogenase E1 alpha subunit and pyruvate dehydrogenase kinase . Additionally, the predicted subcellular location of ZMPP2 is cytoplasmic, while the pyruvate dehydrogenase complex, regulated by reversible phosphorylation, is mitochondrial . Thus, ZMPP2 is a PP2C-type protein phosphatase related to but distinct from PDP. J Biol Chem, 2001 Oct 12, 276(41), 38307 - 19 Epub 2001 Jul 30. Regulation of global acetylation in mitosis through loss of histone acetyltransferases and deacetylases from chromatin; Kruhlak MJ et al.; Histone acetylation, a reversible modification of the core histones, is widely accepted to be involved in remodeling chromatin organization for genetic reprogramming . Histone acetylation is a dynamic process that is regulated by two classes of enzymes, the histone acetyltransferases (HATs) and histone deacetylases (HDACs) . Although promoter-specific acetylation and deacetylation has received most of the recent attention, it is superimposed upon a broader acting and dynamic acetylation that profoundly affects many nuclear processes . In this study, we monitored this broader histone acetylation as cells enter and exit mitosis . In contrast to the hypothesis that HATs and HDACs remain bound to mitotic chromosomes to provide an epigenetic imprint for postmitotic reactivation of the genome, we observed that HATs and HDACs are spatially reorganized and displaced from condensing chromosomes as cells progress through mitosis . During mitosis, HATs and HDACs are unable to acetylate or deacetylate chromatin in situ despite remaining fully catalytically active when isolated from mitotic cells and assayed in vitro . Our results demonstrate that HATs and HDACs do not stably bind to the genome to function as an epigenetic mechanism of selective postmitotic gene activation . Our results, however, do support a role for spatial organization of these enzymes within the cell nucleus and their relationship to euchromatin and heterochromatin postmitotically in the reactivation of the genome. FEBS Lett, 2001 Jul 27, 502(1-2), 57 - 62 The cAMP response element binding protein-2 (CREB-2) can interact with the C/EBP-homologous protein (CHOP); Gachon F et al.; cAMP response element binding protein-2 (CREB-2) is a basic leucine zipper (bZIP) factor that was originally described as a repressor of CRE-dependent transcription but that can also act as a transcriptional activator . Moreover, CREB-2 is able to function in association with the viral Tax protein as an activator of the human T-cell leukemia virus type I (HTLV-I) promoter . Here we show that CREB-2 is able to interact with C/EBP-homologous protein (CHOP), a bZIP transcription factor known to inhibit CAAT/enhancer-dependent transcription . Cotransfection of CHOP with CREB-2 results in decreased activation driven by the cellular CRE motif or the HTLV-I proximal Tax-responsive element, confirming that CREB-2 and CHOP can interact with each other in vivo. Biochemistry, 2001 Aug 7, 40(31), 9421 - 7 Evidence that Gal11 protein is a target of the Gal4 activation domain in the mediator; Jeong CJ et al.; The mediator is an approximately 20 protein complex that is essential for the transcription of most genes in yeast . It is contacted by a number of gene-specific activators, but the details of these interactions are not well understood in most cases . Here, evidence is presented that the mediator component Gal11 represents at least one target of the Gal4 activation domain (AD) . Deletion of Gal11 is shown to decrease the affinity of the Gal4 AD for the mediator, and direct binding of an N-terminal domain of Gal11 with the Gal4 AD is demonstrated . Quantitative studies, however, indicate that the K(D) of the 1:1 Gal4 AD--Gal11 complex is modest . Combined with in vivo data showing that Delta gal11 cells exhibit reduced, but still significant, Gal4-mediated gene expression, these results suggest that the dimeric activator might also contact another protein in the mediator in addition to Gal11. Biochemistry, 2001 Aug 7, 40(31), 9151 - 8 Modifications of cysteine residues in the solution and membrane-associated conformations of phosphatidylinositol transfer protein have differential effects on lipid transfer activity; Tremblay JM et al.; The alpha isoforms of mammalian phosphatidylinositol transfer protein (PITP) contain four conserved Cys residues . In this investigation, a series of thiol-modifying reagents, both alkylating and mixed disulfide-forming, was employed to define the accessibility of these residues and to evaluate their role in protein-mediated intermembrane phospholipid transport . Isolation and analysis of chemically modified peptides and site-directed mutagenesis of each Cys residue to Ala were also performed . Soluble, membrane-associated, and denatured preparations of wild-type and mutant rat PITPs were studied . Under denaturing conditions, all four Cys residues could be detected spectrophotometrically by chemical reaction with 4,4'-dipyridyl disulfide or 5,5'-dithiobis(2-nitrobenzoate) . In the native protein, two of the four Cys residues were sensitive to some but not all thiol-modifying reagents, with discrimination based on the charge and hydrophobicity of the reagent and the conformation of the protein . With the soluble conformation of PITP, achieved in the absence of phospholipid vesicles, the surface-exposed Cys(188) was chemically modified without consequence to lipid transfer activity . Cys(188) exhibited an apparent pK(a) of 7.6 . The buried Cys(95), which constitutes part of the phospholipid substrate binding site, was covalently modified upon transient association of PITP with a membrane surface . The Cys-to-Ala mutations showed that neither Cys(95) nor Cys(188) was essential for lipid transfer activity . However, chemical modification of Cys(95) resulted in the loss of lipid transfer activity . These results demonstrate that the Cys residues of PITP can be assigned to several different classes of chemical reactivity . Of particular interest is Cys(95), whose sulfhydryl group becomes exposed to modification in the membrane-associated conformation of PITP . Furthermore, the inhibition of PITP activity by thiol-modifying reagents is a result of steric hindrance of phospholipid substrate binding. J Biol Chem, 2001 Sep 28, 276(39), 36295 - 302 Epub 2001 Jul 26. Stimulation of eukaryotic flap endonuclease-1 activities by proliferating cell nuclear antigen (PCNA) is independent of its in vitro interaction via a consensus PCNA binding region; Frank G et al.; Interaction between human flap endonuclease-1 (hFEN-1) and proliferating cell nuclear antigen (PCNA) represents a good model for interactions between multiple functional proteins involved in DNA metabolic pathways . A region of 9 conserved amino acid residues (residues Gln-337 through Lys-345) in the C terminus of human FEN-1 (hFEN-1) was shown to be responsible for the interaction with PCNA . Our current study indicates that 4 amino acid residues in hFEN-1 (Leu-340, Asp-341, Phe-343, and Phe-344) are critical for human PCNA (hPCNA) interaction . A conserved PCNA interaction motif in various proteins from assorted species has been defined as Q(1)X(2)X(3)(L/I)(4)X(5)X(6)F(7)(F/Y)(8), although our results fail to implicate Q(1) (Gln-337 in hFEN-1) as a crucial residue . Surprisingly, all hFEN-1 mutants, including L340A, D341A, F343A, and F344A, retained hPCNA-mediated stimulation of both exo- and flap endonuclease activities . Furthermore, our in vitro assay showed that hPCNA failed to bind to the scRad27 (yeast homolog of FEN-1) nuclease . However, its nuclease activities were significantly enhanced in the presence of hPCNA . Four additional Saccharomyces cerevisiae scRad27 mutants, including multiple alanine mutants and a deletion mutant of the entire PCNA binding region, were constructed to confirm this result . All of these mutants retained PCNA-driven nuclease activity stimulation . We therefore conclude that stimulation of eukaryotic hFEN-1 nuclease activities by PCNA is independent of its in vitro interaction via the PCNA binding region. Int J Cancer, 2001 Aug 20, 96(4), 238 - 42 Two metachronous tumors in the radiotherapy fields of a patient with Li-Fraumeni syndrome; Limacher JM et al.; A woman with a family history of brain tumors in her daughter and sister presented with a breast cancer . She subsequently developed two metachronous primary tumors: a small-cell lung cancer and a colon carcinoma . These tumors arose within the internal mammary radiotherapy field and within the field irradiated for ovariolysis . The p53 gene was analyzed in whole blood lymphocytes using a functional assay developed in yeast Saccharomyces cerevisiae, which tests the transcriptional competence of p53 . DNA from the colon cancer cells was analyzed by polymerase chain reaction and sequencing . The patient had a germline-inactivating p53 mutation, confirming the diagnosis of Li-Fraumeni syndrome (LFS) . The colon tumor and the lung tumor both conserved the mutant p53 allele but had lost the wild-type allele . This observation and the experimental data suggest an abnormal sensitivity of LFS patients to radiogenic carcinogenesis . The indications and extent of radiotherapy in patients with a clinical or molecular diagnosis of LFS should be discussed individually and should take into account the risk of secondary neoplasms arising in the radiation fields . Cytogenet Cell Genet, 2001, 93(1-2), 109 - 13 Sequence polymorphisms, allelic expression status and chromosome locations of the chicken IGF2 and MPR1 genes; Yokomine T et al.; By screening 26 chicken breeds and lines, DNA polymorphisms were identified in the IGF2 and MPR1 genes, of which mammalian homologues are parentally imprinted, and the GAPD gene, a housekeeping control . Using the polymorphisms as genetic markers, we found that all three genes are expressed biallelically in embryonic tissues . IGF2 and MPR1 were mapped on chicken chromosomes 5 and 3, respectively, by fluorescence in situ hybridization, demonstrating conserved linkage homology between mammals and birds . Cytogenet Cell Genet, 2001, 93(1-2), 52 - 6 Expression and chromosome location of hamster Ku70 and Ku80; Koike M et al.; Ku proteins play an important role in DNA double-strand break (DSB) repair, chromosome maintenance, and growth regulation . To understand the fundamental characteristics of Ku proteins, we examined the electrophoretic mobility and expression of hamster Ku70 and Ku80 and determined the chromosome locations of their genes . The electrophoretic mobility of hamster Ku proteins are different from that of human Ku proteins . No significant changes in the quantity of Ku proteins were observed in CHO-K1 cells treated with 10 Gy of ionizing radiation, suggesting that both proteins are expressed constitutively in amounts adequate to repair DNA DSBs . The chromosome locations of the Ku genes were determined by direct R-banding fluorescence in situ hybridization . The Ku70 gene was localized to Syrian hamster chromosome 4qa4.1--> qa4.2 and Chinese hamster chromosome 2p3.1, and the Ku80 gene was localized to Syrian hamster chromosome 4qb5--> qb6.1 and Chinese hamster chromosome 2p3.5-->p3.6 . These results provide clues to the biological functions of Ku, as well as useful information for constructing comparative chromosome maps between hamsters and other mammalian species, including human, mouse, and rat . Science, 2001 Jul 27, 293(5530), 698 - 702 An autoinhibitory mechanism for nonsyntaxin SNARE proteins revealed by the structure of Ykt6p; Tochio H et al.; Ykt6p is a nonsyntaxin SNARE implicated in multiple intracellular membrane trafficking steps . Here we present the structure of the NH2-terminal domain of Ykt6p (Ykt6pN, residues 1 to 140) . The structure of Ykt6pN differed entirely from that of syntaxin and resembled the overall fold of the actin regulatory protein, profilin . Like some syntaxins, Ykt6p adopted a folded back conformation in which Ykt6pN bound to its COOH-terminal core domain . The NH2-terminal domain plays an important biological role in the function of Ykt6p, which in vitro studies revealed to include influencing the kinetics and proper assembly of SNARE complexes. Science, 2001 Sep 14, 293(5537), 2101 - 5 Epub 2001 Jul 26. Global analysis of protein activities using proteome chips; Zhu H et al.; To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins . The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids . We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins . Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities . The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications. Nat Struct Biol, 2001 Aug, 8(8), 669 - 73 Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13; Moraes TF et al.; The ubiquitin conjugating enzyme complex Mms2-Ubc13 plays a key role in post-replicative DNA repair in yeast and the NF-kappaB signal transduction pathway in humans . This complex assembles novel polyubiquitin chains onto yet uncharacterized protein targets . Here we report the crystal structure of a complex between hMms2 (Uev1) and hUbc13 at 1.85 A resolution and a structure of free hMms2 at 1.9 A resolution . These structures reveal that the hMms2 monomer undergoes a localized conformational change upon interaction with hUbc13 . The nature of the interface provides a physical basis for the preference of Mms2 for Ubc13 as a partner over a variety of other structurally similar ubiquitin-conjugating enzymes . The structure of the hMms2-hUbc13 complex provides the conceptual foundation for understanding the mechanism of Lys 63 multiubiquitin chain assembly and for its interactions with the RING finger proteins Rad5 and Traf6. J Biol Chem, 2001 Nov 9, 276(45), 41629 - 37 Epub 2001 Jul 25. Recruitment of multiple interferon regulatory factors and histone acetyltransferase to the transcriptionally active interferon a promoters; Au WC et al.; Type I interferon (IFN) plays a critical role in the innate immunity against viral infection . Expression of IFNA genes in infected cells is cell type-dependent and is regulated at the transcriptional level . The present study is focused on the molecular mechanism underlying the differential expression of human IFNA1 and A2 genes . Two nucleotides, at positions -98 and -81 of IFNA1 and A2 promoter, were pivotal to the differential expression . The DNA pull-down and chromatin precipitation assays have shown that nuclear interferon regulatory factor (IRF)-3 and IRF-7 as well as IRF-1 bind to IFNA1 virus-responsive element (VRE) . Interestingly, overexpression of IRF-7 increased the otherwise weak binding of both IRF-3 and IRF-7 to IFNA2 VRE . These data together with the results of two-step chromatin immunoprecipitation strongly suggest that the IRF-3 and IRF-7 bind to IFNA1 promoter as a dimer . Furthermore, binding of IRF-3 and IRF-7 to IFNA VRE is associated with the presence of acetylated histone H3, suggesting that histone acetyltransferase(s) is tethered together with virus-activated IRF-3 and IRF-7 to the IFNA1 promoter . In addition, the constitutively active IRF-3 (5D) and IRF-7 (2D) mutants activate the endogenous IFNA genes in uninfected cells; however, the expression profile of IFNA is not identical to that induced by viral infection. J Biol Chem, 2001 Oct 19, 276(42), 38410 - 6 Epub 2001 Jul 25. Copper stabilizes a heterodimer of the yCCS metallochaperone and its target superoxide dismutase; Torres AS et al.; The copper chaperone for superoxide dismutase (CCS) activates the antioxidant enzyme Cu,Zn-SOD (SOD1) by directly inserting the copper cofactor into the apo form of SOD1 . Neither the mechanism of protein-protein recognition nor of metal transfer is clear . The metal transfer step has been proposed to occur within a transient copper donor/acceptor complex that is either a heterodimer or heterotetramer (i.e . a dimer of dimers) . To determine the nature of this intermediate, we generated a mutant form of SOD1 by replacing a copper binding residue His-48 with phenylalanine . This protein cannot accept copper from CCS but does form a stable complex with apo- and Cu-CCS, as observed by immunoprecipitation and native gel electrophoresis . Fluorescence anisotropy measurements corroborate the formation of this species and further indicate that copper enhances the stability of the dimer by an order of magnitude . The copper form of the heterodimer was isolated by gel filtration chromatography and contains one copper and one zinc atom per heterodimer . These results support a mechanism for copper transfer in which CCS and SOD1 dock via their highly conserved dimer interfaces in a manner that precisely orients the Cys-rich copper donor sites of CCS and the His-rich acceptor sites of SOD1 to form a copper-bridged intermediate. Bioinformatics, 2001, 17 Suppl 1, S270 - 8 Computational expansion of genetic networks; Tanay A et al.; We present a new methodology for computational analysis of gene and protein networks . The aim is to generate new educated hypotheses on gene functions and on the logic of the biological network circuitry, based on gene expression profiles . The framework supports the incorporation of biologically motivated network constraints and rules to improve specificity . Since current data is insufficient for de-novo reconstruction, the method receives as input a known pathway core and suggests likely expansions to it . Network modeling is combinatorial, yet data can be probabilistic . At the heart of the approach are a fitness function which estimates the quality of suggested network expansions given the core and the data, and a specificity measure of the expansions . The approach has been implemented in an interactive software tool called GENESYS . We report encouraging results in preliminary analysis of yeast ergosterol pathway based on transcription profiles . In particular, the analysis suggests a novel ergosterol transcription factor. Bioinformatics, 2001, 17 Suppl 1, S243 - 52 Rich probabilistic models for gene expression; Segal E et al.; Clustering is commonly used for analyzing gene expression data . Despite their successes, clustering methods suffer from a number of limitations . First, these methods reveal similarities that exist over all of the measurements, while obscuring relationships that exist over only a subset of the data . Second, clustering methods cannot readily incorporate additional types of information, such as clinical data or known attributes of genes . To circumvent these shortcomings, we propose the use of a single coherent probabilistic model, that encompasses much of the rich structure in the genomic expression data, while incorporating additional information such as experiment type, putative binding sites, or functional information . We show how this model can be learned from the data, allowing us to discover patterns in the data and dependencies between the gene expression patterns and additional attributes . The learned model reveals context-specific relationships, that exist only over a subset of the experiments in the dataset . We demonstrate the power of our approach on synthetic data and on two real-world gene expression data sets for yeast . For example, we demonstrate a novel functionality that falls naturally out of our framework: predicting the "cluster" of the array resulting from a gene mutation based only on the gene's expression pattern in the context of other mutations. Bioinformatics, 2001, 17 Suppl 1, S115 - 22 GEST: a gene expression search tool based on a novel Bayesian similarity metric; Hunter L et al.; Gene expression array technology has made possible the assay of expression levels of tens of thousands of genes at a time; large databases of such measurements are currently under construction . One important use of such databases is the ability to search for experiments that have similar gene expression levels as a query, potentially identifying previously unsuspected relationships among cellular states . Such searches depend crucially on the metric used to assess the similarity between pairs of experiments . The complex joint distribution of gene expression levels, particularly their correlational structure and non-normality, make simple similarity metrics such as Euclidean distance or correlational similarity scores suboptimal for use in this application . We present a similarity metric for gene expression array experiments that takes into account the complex joint distribution of expression values . We provide a computationally tractable approximation to this measure, and have implemented a database search tool based on it . We discuss implementation issues and efficiency, and we compare our new metric to other standard metrics. Bioinformatics, 2001, 17 Suppl 1, S49 - 55 Visualizing associations between genome sequences and gene expression data using genome-mea