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| Nat Cell Biol, 2003 Mar, 5(3), 231 - 5 Spontaneous receptor-independent heterotrimeric G-protein signalling in an RGS mutant; Siekhaus DE et al.; Tripartite G-protein-coupled receptors (GPCRs) represent one of the largest groups of signal transducers, transmitting signals from hormones, neuropeptides, odorants, food and light . Ligand-bound receptors catalyse GDP/GTP exchange on the G-protein alpha-subunit (Galpha), leading to alpha-GTP separation from the betagamma subunits and pathway activation . Activating mutations in the receptors or G proteins underlie many human diseases, including some cancers, dwarfism and premature puberty . Regulators of G-protein signalling (RGS proteins) are known to modulate the level and duration of ligand-induced signalling by accelerating the intrinsic GTPase activity of the Galpha subunit, and thus reformation of the inactive GDP-bound Galpha . Here we find that even in the absence of receptor, mutation of the RGS family member Sst2 (refs 6-9) permits spontaneous activation of the G-protein-coupled mating pathway in Saccharomyces cerevisiae at levels normally seen only in the presence of ligand . Our work demonstrates the occurrence of spontaneous tripartite G-protein signalling in vivo and identifies a requirement for RGS proteins in preventing such receptor-independent activation. Nat Cell Biol, 2003 Mar, 5(3), 249 - 54 A non-proteolytic function of separase links the onset of anaphase to mitotic exit; Sullivan M et al.; Separase is a protease that triggers chromosome segregation at anaphase onset by cleaving cohesin, the chromosomal protein complex responsible for sister chromatid cohesion . After anaphase, cells exit from mitosis; that is, they complete downregulation of cyclin-dependent kinase activity, undergo cytokinesis and enter G1 of the next cell cycle . Here we show that separase activation at the onset of anaphase is sufficient to promote release from the nucleolus and activation of the budding yeast phosphatase, Cdc14, a key step in mitotic exit . The ability of separase to activate Cdc14 is independent of its protease function but may involve promoting phosphorylation of the Cdc14 inhibitor Net1 . This novel separase function is coregulated with its proteolytic activity by the separase inhibitor securin . This helps to explain the coupling of anaphase and mitotic exit--after securin degradation at anaphase onset, separase cleaves cohesin to trigger chromosome segregation and concurrently uses a non-proteolytic mechanism to initiate mitotic exit. J Biol Chem, 2003 Apr 25, 278(17), 15449 - 55 Epub 2003 Feb 21. Induction of nonmuscle myosin heavy chain II-C by butyrate in RAW 264.7 mouse macrophages; Buxton DB et al.; RAW 264.7 macrophages express nonmuscle myosin heavy chain II-A as the only significant nonmuscle myosin heavy chain isoform, with expression of nonmuscle myosin heavy chain II-B and II-C low or absent . Treatment of the cells with sodium butyrate, an inhibitor of histone deacetylase, led to the dose-dependent induction of nonmuscle myosin heavy chain II-C . Trichostatin A, another inhibitor of histone deacetylase, also induced nonmuscle myosin heavy chain II-C . Induction of nonmuscle myosin heavy chain II-C in response to these histone deacetylase inhibitors was attenuated by mithramycin, an inhibitor of Sp1 binding to GC-rich DNA sequences . Bacterial lipopolysaccharide alone had no effect on basal nonmuscle myosin heavy chain II-C expression, but attenuated butyrate-mediated induction of nonmuscle myosin heavy chain II-C . The effects of lipopolysaccharide were mimicked by the nitric oxide donors sodium nitroprusside and spermine NONOate, suggesting a role for nitric oxide in the lipopolysaccharide-mediated down-regulation of nonmuscle myosin heavy chain II-C induction . This was supported by experiments with the inducible nitric-oxide synthase inhibitor 1400W, which partially blocked the lipopolysaccharide-mediated attenuation of nonmuscle myosin heavy chain induction . 8-Bromo-cGMP had no effect on nonmuscle myosin heavy chain induction, consistent with a cGMP-independent mechanism for nitric oxide-mediated inhibition of nonmuscle myosin heavy chain II-C induction. Trends Microbiol, 2003 Feb, 11(2), 53 - 5 High-affinity ammonium transporters and nitrogen sensing in mycorrhizas; Javelle A et al.; Most terrestrial plants live in mutualistic symbiosis with root-infecting mycorrhizal fungi . This association requires a molecular dialogue between the two partners . However, the nature of the chemical signals that induce hyphal differentiation are not well characterized and the mechanisms for signal reception are still unknown . In addition to its role in ammonium scavenging, the Mep2 protein from Saccharomyces cerevisiae has been proposed to act as an ammonium sensor that is essential for pseudohyphal differentiation in response to ammonium limitation . We propose that the high-affinity ammonium transporters from mycorrhizal fungi act in a similar manner to sense the environment and induce, via as-yet-unidentified signal transduction cascades, the switch in the mode of fungal growth observed during the formation of mycorrhiza. Curr Top Microbiol Immunol, 2003, 274, 203 - 36 The MYST family of histone acetyltransferases; Utley RT et al.; Multiple chromatin modifying proteins and multisubunit complexes have been characterized in recent years . Histone acetyltransferase (HAT) activities have been the most thoroughly studied, both biochemically and functionally . This review sums up the current knowledge on a specific group of proteins that is extremely well conserved throughout evolution, the MYST family of histone acetyltransferases . These proteins play critical roles in various nuclear functions and the control of cell proliferation. Curr Top Microbiol Immunol, 2003, 274, 171 - 201 Changing the DNA landscape: putting a SPN on chromatin; Formosa T; In eukaryotic cells, transcription and replication each occur on DNA templates that are incorporated into nucleosomes . Formation of chromatin generally limits accessibility of specific DNA sequences and inhibits progression of polymerases as they copy information from the DNA . The processes that select sites for initiating either transcription or replication are therefore strongly influenced by factors that modulate the properties of chromatin proteins . Further, in order to elongate their products, both DNA and RNA polymerases must be able to overcome the inhibition presented by chromatin (Lipford and Bell 2001; Workman and Kingston 1998) . One way to adjust the properties of chromatin proteins is to covalently modify them by adding or removing chemical moieties . Both histone and non-histone chromatin proteins are altered by acetylation, methylation, and other changes, and the 'nucleosome modifying' complexes that perform these reactions are important components of pathways of transcriptional regulation (Cote 2002; Orphanides and Reinberg 2000; Roth et al . 2001; Strahl and Allis 2000; Workman and Kingston 1998) . Another way to alter the effects of nucleosomes is to change the position of the histone octamers relative to specific DNA sequences (Orphanides and Reinberg 2000; Verrijzer 2002; Wang 2002; Workman and Kingston 1998) . Since the ability of a sequence to be bound by specific proteins can vary significantly whether the sequence is in the linkers between nucleosomes or at various positions within a nucleosome, 'nucleosome remodeling' complexes that rearrange nucleosome positioning are also important regulators of transcription . Since the DNA replication machinery has to encounter many of the same challenges posed by chromatin, it seems likely that modifying and remodeling complexes also act during duplication of the genome, but most of the current information on these factors relates to regulation of transcription . This chapter describes the factor known variously as FACT in humans, where it promotes elongation of RNA polymerase II on nucleosomal templates in vitro (Orphanides et al . 1998, 1999), DUF in frogs, where it is needed for DNA replication in oocyte extracts (Okuhara et al . 1999), and CP or SPN in yeast, where it is linked in vivo to both transcription and replication (Brewster et al . 2001; Formosa et al . 2001) . Like the nucleosome modifying and remodeling complexes, it is broadly conserved among eukaryotes, affects a wide range of processes that utilize chromatin, and directly alters the properties of nucleosomes . However, it does not have nucleosome modifying or standard ATP-dependent remodeling activity, and therefore represents a third class of chromatin modulating factors . It is also presently unique in the extensive connections it displays with both transcription and replication: FACT/DUF/CP/SPN appears to modify nucleosomes in a way that is directly important for the efficient functioning of both RNA polymerases and DNA polymerases . While less is known about the mechanisms it uses to promote its functions than for other factors that affect chromatin, it is clearly an essential part of the complex mixture of activities that modulate access to DNA within chromatin . Physical and genetic interactions suggest that FACT/DUF/CP/SPN affects multiple pathways within replication and transcription as a member of several distinct complexes . Some of the interactions are easy to assimilate into models for replication or transcription, such as direct binding to DNA polymerase alpha (Wittmeyer and Formosa 1997; Wittmeyer et al . 1999), association with nucleosome modifying complexes (John et al . 2000), and interaction with factors that participate in elongation of RNA Polymerase II (Gavin et al . 2002; Squazzo et al . 2002) . Others are more surprising such as an association with the 19S complex that regulates the function of the 20S proteasome (Ferdous et al . 2001; Xu et al . 1995), and the indication that FACT/DUF/CP/SPN can act as a specificity factor for casein kinase II (Keller et al . 2001) . This chapter reviews the varied approaches that have each revealed different aspects of the function of FACT/DUF/CP/SPN, and presents a picture of a factor that can both alter nucleosomes and orchestrate the assembly or activity of a broad range of complexes that act upon chromatin. Curr Top Microbiol Immunol, 2003, 274, 23 - 52 Chromatin proteins are determinants of centromere function; Sharp JA et al.; Recent advances in the identification of molecular components of centromeres have demonstrated a crucial role for chromatin proteins in determining both centromere identity and the stability of kinetochore-microtubule attachments . Although we are far from a complete understanding of the establishment and propagation of centromeres, this review seeks to highlight the contribution of histones, histone deposition factors, histone modifying enzymes, and heterochromatin proteins to the assembly of this sophisticated, highly specialized chromatin structure . First, an overview of DNA sequence elements at centromeric regions will be presented . We will then discuss the contribution of chromatin to kinetochore function in budding yeast, and pericentric heterochromatin domains in other eukaryotic systems . We will conclude with discussion of specialized nucleosomes that direct kinetochore assembly and propagation of centromere-defining chromatin domains. J Mal Vasc, 2002 Jul, 27 Spec No, S19 - 23 {Fundamental aspects of extreme aging}; Treton J; Major developments in molecular biology in invertebrates have recently shown the determining effect of genetics on aging . The first finding was that artificial selection can highlight the genetic aspect of the aging process, demonstrating the polygenetic property of longevity . Another finding showed that certain gene transfers can modulate the lifespan of an organism . Recent progress has been made in three fields: genetic markers of aging, biological basis of cell maintenance, and hereditary factors contributing to late onset genetic disease . These new developments open new avenues of research in clinical biology . In regard to genetic markers of aging, it has been demonstrated that the ends of the chromosomes, telomeres, play a role in cell senescence . Telomeres can be viewed as markers of aging . Shortened telomeres are associated with replicative senescence and antitumor action . DNA anomalies are also more frequent: simple or double breaks, additions and base substitutions . Data on the biological basis of cell maintenance obtained in invertebrates show the polygenetic property of aging involving four significant mechanisms, control of metabolism, resistance to stress, chromatin-dependent gene regulation of genetic homeostasis . Finally, recent studies have shown that late onset hereditary diseases would be linked with particular genes, some of which have been identified . Two non-exclusive mechanisms could be involved: an adaptive mechanism involving gene selection during the evolutionary process, for example in obesity; and non-adaptive accumulation of gene expression during the post-reproductive phase, for example in Alzheimer's disease . These findings open a new era for the biology of aging. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 466 - 73 Epub 2003 Feb 21. Combinatorial crystallization of an RNA-protein complex; Hoggan DB et al.; One of the most difficult steps in X-ray crystallography of a ribonucleoprotein (RNP) complex is obtaining crystals that diffract to high resolution . This paper describes a procedure for identifying the optimal lengths of the nucleic acid components that provide high-quality crystals of the RNP . Both strands of an RNA duplex were varied in a systematic manner to generate a large number of unique RNPs that were screened for crystallization behavior . As observed in the crystallization of other nucleic acids and their complexes, the exact length of the RNA chains was found to be critical in obtaining diffraction-quality crystals, even though the relative molecular weights of the protein and RNA components were approximately 50 and approximately 10 kDa, respectively . In particular, the helix-loop-helix structure in the mRNA for the Saccharomyces cerevisiae ribosomal protein L30, which functions as an autoregulatory element for L30 expression, was synthesized as two separate RNA chains of variable length (12-14 and 15-17 nucletides) . Duplex formation of these RNAs formed the asymmetric, internal loop-binding site for L30 . 16 such RNA duplexes, varying by +/-1 residue at the 5' or 3' end of either chain, were used to prepare 16 unique complexes with a maltose-binding protein-L30 fusion protein . The complexes were screened against 48 standard crystallization conditions in 2304 experiments, yielding 30 conditions with single crystals in the initial screen . The most promising of these is being used for structure determination. Nucleic Acids Res, 2003 Mar 1, 31(5), 1565 - 70 RNA sequences that work as transcriptional activating regions; Saha S et al.; We describe a set of RNA molecules that work as transcriptional activators when tethered to DNA . These RNA activating regions were found amongst a randomized set of molecules bearing variants of a 10 nt loop attached to an RNA stem . The various RNA activating regions all bear an identical five- residue sequence with an interspersed sixth residue . The result shows that although all natural activating regions characterized thus far are peptidic, this function can be served by other kinds of moieties as well. Nucleic Acids Res, 2003 Mar 1, 31(5), 1470 - 80 The highly related DEAD box RNA helicases p68 and p72 exist as heterodimers in cells; Ogilvie VC et al.; The RNA helicases p68 and p72 are highly related members of the DEAD box family of proteins, sharing 90% identity across the conserved core, and have been shown to be involved in both transcription and mRNA processing . We previously showed that these proteins co-localise in the nucleus of interphase cells . In this study we show that p68 and p72 can interact with each other and self-associate in the yeast two-hybrid system . Co-immunoprecipitation experiments confirmed that p68 and p72 can interact in the cell and indicated that these proteins preferentially exist as hetero-dimers . In addition, we show that p68 can interact with NFAR-2, a protein that is also thought to function in mRNA processing . Moreover, gel filtration analysis suggests that p68 and p72 can exist in a variety of complexes in the cell (ranging from approximately 150 to approximately 400 kDa in size), with a subset of p68 molecules being in very large complexes (>2 MDa) . The potential to exist in different complexes that may contain p68 and/or p72, together with a range of other factors, would provide the potential for these proteins to interact with different RNA substrates and would be consistent with recent reports implying a wide range of functions for p68/p72. J Biol Chem, 2003 May 2, 278(18), 15744 - 8 Epub 2003 Feb 20. A native peptide ligation strategy for deciphering nucleosomal histone modifications; Shogren-Knaak MA et al.; Post-translational modifications of histones influence both chromatin structure and the binding and function of chromatin-associated proteins . A major limitation to understanding these effects has been the inability to construct nucleosomes in vitro that harbor homogeneous and site-specific histone modifications . Here, we describe a native peptide ligation strategy for generating nucleosomal arrays that can harbor a wide range of desired histone modifications . As a first test of this method, we engineered model nucleosomal arrays in which each histone H3 contains a phosphorylated serine at position 10 and performed kinetic analyses of Gcn5-dependent histone acetyltransferase activities . Recombinant Gcn5 shows increased histone acetyltransferase activity on nucleosomal arrays harboring phosphorylated H3 serine 10 and is consistent with peptide studies . However, in contrast to analyses using peptide substrates, we find that the histone acetyltransferase activity of the Gcn5-containing SAGA complex is not stimulated by H3 phosphorylation in the context of nucleosomal arrays . This difference between peptide and array substrates suggests that the ability to generate specifically modified nucleosomal arrays should provide a powerful tool for understanding the effects of post-translational histone modifications. Infect Immun, 2003 Mar, 71(3), 1155 - 60 Expression and immunogenicity of Mycoplasma hyopneumoniae heat shock protein antigen P42 by DNA vaccination; Chen YL et al.; Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia, a chronic nonfatal disease affecting pigs of all ages . The goal of this study was to design DNA vaccines by constructing plasmid pcDNA3/P42, carrying the heat shock protein gene P42 of M . hyopneumoniae, and to evaluate the immune responses elicited in BALB/c mice . The expression of P42 was first examined in transfected NIH 3T3 cells by reverse transcription-PCR to ensure that the construct was functional . The humoral and cell-mediated immune responses induced by the plasmid were further evaluated in BALB/c mice through intramuscular injection . Both immunoglobulin G1 (IgG1) and IgG2a levels were 64 times those of the control groups during the first 8 weeks . The levels of interleukin-2 (IL-2), IL-4, and gamma interferon mRNAs in the immunized animals were elevated, and the proliferation of spleen cells was also enhanced in the immunized animals . The results indicate that pcDNA3/P42 DNA immunization induces both Th1 and Th2 immune responses . In addition, antiserum from the immunized animals was found to inhibit the growth of M . hyopneumoniae . The present study reveals that DNA vaccination could be a new strategy against infection by M . hyopneumoniae and may have potential for developing vaccines for other infectious diseases as well. J Mol Biol, 2003 Mar 7, 326(5), 1427 - 35 The ScPex13p SH3 domain exposes two distinct binding sites for Pex5p and Pex14p; Pires JR et al.; Pex13p is an essential component of the peroxisomal protein import machinery and interacts via its C-terminal SH3 domain with the type II SH3-ligand Pex14p and the non-PXXP protein Pex5p . We report the solution structure of the SH3 domain of Pex13p from Saccharomyces cerevisiae and the identification of a novel-binding pocket, which binds a non-PXXP-peptide representing the binding site of Pex5p . Chemical shift assays revealed the binding sites for Pex5p and Pex14p ligand peptides to be distinct and spatially separated . Competition assays demonstrated that the two ligand peptides can bind simultaneously to the SH3 domain. Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2945 - 50 Epub 2003 Feb 19. Aquaglyceroporin AQP9: solute permeation and metabolic control of expression in liver; Carbrey JM et al.; Aquaglyceroporins form the subset of the aquaporin water channel family that is permeable to glycerol and certain small, uncharged solutes . AQP9 has unusually broad solute permeability and is expressed in hepatocyte plasma membranes . Proteoliposomes reconstituted with expressed, purified rat AQP9 protein were compared with simple liposomes for solute permeability . At pH 7.5, AQP9 proteoliposomes exhibited Hg(2+)-inhibitable glycerol and urea permeabilities that were increased 63-fold and 90-fold over background . beta-Hydroxybutyrate permeability was not increased above background, and osmotic water permeability was only minimally elevated . During starvation, the liver takes up glycerol for gluconeogenesis . Expression of AQP9 in liver was induced up to 20-fold in rats fasted for 24-96 h, and the AQP9 level gradually declined after refeeding . No changes in liver AQP9 levels were observed in rats fed ketogenic diets or high-protein diets, but AQP9 levels were elevated in livers of rats made diabetic by streptozotocin injection . When blood glucose levels of the diabetic rats were restored to normal by insulin treatments, the AQP9 levels returned to baseline . Confocal immunofluorescence revealed AQP9 immunostaining on the sinusoidal surfaces of hepatocyte plates throughout the livers of control rats . Denser immunostaining was observed in the same distribution in livers of fasted and streptozotocin-treated rats . We conclude that AQP9 serves as membrane channel in hepatocytes for glycerol and urea at physiological pH, but not for beta-hydroxybutyrate . In addition, levels of AQP9 expression fluctuate depending on the nutritional status of the subject and the circulating insulin levels. DNA Seq, 2002 Oct, 13(5), 271 - 5 Cloning and sequence analysis of a mitochondrial gene cluster encoding cytochrome C oxidase subunit III from Trichoderma pseudokoningii; Wang TH et al.; A mitochondrial gene cluster encoding cytochrome c oxidase subunit III (COX3), an ORF (called ORF250) similar to NADH dehydrogenase subunit VI (ND6), ten tRNA molecules, partial rRNA small subunit and rRNA large subunit from Trichoderma pseudokoningii S38 was cloned and sequenced . These genes are tandemly clustered on the mitochondrial genome of Trichoderma pseudokoningii S38 . Phylogenetic analysis showed that cytochrome C oxidase subunits III exhibited high degree of similarity to sequences from Hypocrea jecorina, Verticillium lecanii, Podospora anserine, Neurospora crassa and Magnaporthe grisea (99, 90, 84, 82 and 79% identity, respectively) . Prediction of transmembrane helices revealed that COX3 was a transmembrane protein . Northern dot blot analysis showed that the cytochrome c oxidase subunits III gene we had cloned is actively transcribed in the T . pseudokoningii mitochondria. Leukemia, 2003 Feb, 17(2), 359 - 65 Transcriptional activation is a key function encoded by MLL fusion partners; Zeisig BB et al.; Chromosomal translocations that fuse the mixed lineage leukemia gene (MLL) to a variety of unrelated partner genes are frequent in pediatric leukemias . The novel combination of genetic material leads to the production of active oncoproteins that depend on the contributions of both constituents . In a search for a common function amongst the diverse group of MLL fusion partners we constructed artificial fusions joining MLL with generic transactivator and repressor domains (acidic blob, GAL4 transactivator domain, Herpes simplex VP16 activation domain, KRAB repressor domain) . Of all constructs tested, only MLL-VP16 was able to transform primary bone marrow cells and to induce a block of early myeloid differentiation like an authentic MLL fusion . Interestingly, the transformation capability of the artificial MLL fusions was correlated with the transcriptional potential of the resulting chimeric protein but it was not related to the strength of the isolated transactivation domain that was joined to MLL . These results prove for the first time that a general biological function - transactivation - might be the common denominator of many MLL fusion partners. Protein Sci, 2003 Mar, 12(3), 491 - 500 WW domain sequence activity relationships identified using ligand recognition propensities of 42 WW domains; Otte L et al.; WW domains mediate protein-protein interactions in a number of different cellular functions by recognizing proline-containing peptide sequences . We determined peptide recognition propensities for 42 WW domains using NMR spectroscopy and peptide library screens . As potential ligands, we studied both model peptides and peptides based on naturally occurring sequences, including phosphorylated residues . Thirty-two WW domains were classified into six groups according to detected ligand recognition preferences for binding the motifs PPx(Y/poY), (p/phi)P(p,g)PPpR, (p/phi)PPRgpPp, PPLPp, (p/xi)PPPPP, and (poS/poT)P (motifs according to modified Seefeld Convention 2001) . In addition to these distinct binding motifs, group-specific WW domain consensus sequences were identified . For PPxY-recognizing domains, phospho-tyrosine binding was also observed . Based on the sequences of the PPx(Y/poY)-specific group, a profile hidden Markov model was calculated and used to predict PPx(Y/poY)-recognition activity for WW domains, which were not assayed . PPx(Y/poY)-binding was found to be a common property of NEDD4-like ubiquitin ligases. J Cell Biol, 2003 Feb 17, 160(4), 517 - 28 The molecular function of Ase1p: evidence for a MAP-dependent midzone-specific spindle matrix . Microtubule-associated proteins; Schuyler SC et al.; The midzone is the domain of the mitotic spindle that maintains spindle bipolarity during anaphase and generates forces required for spindle elongation (anaphase B) . Although there is a clear role for microtubule (MT) motor proteins at the spindle midzone, less is known about how microtubule-associated proteins (MAPs) contribute to midzone organization and function . Here, we report that budding yeast Ase1p is a member of a conserved family of midzone-specific MAPs . By size exclusion chromatography and velocity sedimentation, both Ase1p in extracts and purified Ase1p behaved as a homodimer . Ase1p bound and bundled MTs in vitro . By live cell microscopy, loss of Ase1p resulted in a specific defect: premature spindle disassembly in mid-anaphase . Furthermore, when overexpressed, Ase1p was sufficient to trigger spindle elongation in S phase-arrested cells . FRAP revealed that Ase1p has both a very slow rate of turnover within the midzone and limited lateral diffusion along spindle MTs . We propose that Ase1p functions as an MT cross-bridge that imparts matrix-like characteristics to the midzone . MT-dependent networks of spindle midzone MAPs may be one molecular basis for the postulated spindle matrix. J Bacteriol, 2003 Mar, 185(5), 1749 - 56 Molecular cloning of endo-beta-D-1,4-glucanase genes, rce1, rce2, and rce3, from Rhizopus oryzae; Moriya T et al.; Three endoglucanase genes, designated the rce1, rce2, and rce3 genes, were isolated from Rhizopus oryzae as the first cellulase genes from the subdivision ZYGOMYCOTA: All the amino acid sequences deduced from the rce1, rce2, and rce3 genes consisted of three distinct domains: cellulose binding domains, linker domains, and catalytic domains belonging to glycosyl hydrolase family 45 . The rce3 gene had two tandem repeated sequences of cellulose binding domains, while rce1 and rce2 had only one . rce1, rce2, and rce3 had various lengths of linker sequences. Biochemistry, 2003 Feb 25, 42(7), 2174 - 84 Effect of pH on the iso-1-cytochrome c denatured state: changing constraints due to heme ligation; Smith CR et al.; The effect of pH on the denatured state (3 M guanidine hydrochloride) was evaluated with fluorescence spectroscopy for four variants of iso-1-cytochrome c, AcTM (no surface histidines), AcH26 (surface histidine at position 26), AcH54 (surface histidine at position 54), and AcH54I52 (stabilizing I52 mutation added to AcH54) . Changes in the compactness and the heme ligation of the denatured state, as a function of pH, were monitored through changes in Trp 59-heme fluorescence quenching . With the AcTM and AcH26 variants, no change in the fluorescence intensity occurs from pH 4 to 10 . However, for the AcH54 and AcH54I52 variants the fluorescence intensity drops significantly between pH 4 and 6, consistent with His 54 binding to the heme of cytochrome c . Between pH 8 and 10 fluorescence intensity increases again, indicating that the His 54 is displaced from the heme . The data are consistent with lysines 4 and 5 being the primary heme ligands at alkaline pH, under denaturing conditions . This conclusion was confirmed by site-directed mutagenesis . Thermodynamic analysis indicates that heme-ligand affinity in the denatured state is controlled primarily by sequence position (loop size) and that when histidines are present they inhibit lysine ligation until approximately pH 8.5-9.0 as compared to pH 7.5 with the AcTM variant . Thus, at physiological pH, histidine ligands provide the primary constraint on the denatured state of cytochrome c . The heme-Trp 59 distance in the denatured state of iso-1-cytochrome c, derived from analysis by Forster energy transfer theory, is approximately 26 A at pH 4 and 10, much shorter than the random coil prediction of 56 A . Surprisingly, the heme-Trp 59 distance in the His 54 bound conformation only drops to approximately 21 A, consistent with an extended conformation for the short polypeptide segment separating heme and Trp 59. Nat Genet, 2003 Mar, 33(3), 349 - 55 Epub 2003 Feb 18. The study of macromolecular complexes by quantitative proteomics; Ranish JA et al.; We describe a generic strategy for determining the specific composition, changes in the composition, and changes in the abundance of protein complexes . It is based on the use of isotope-coded affinity tag (ICAT) reagents and mass spectrometry to compare the relative abundances of tryptic peptides derived from suitable pairs of purified or partially purified protein complexes . In a first application, the genuine protein components of a large RNA polymerase II (Pol II) preinitiation complex (PIC) were distinguished from a background of co-purifying proteins by comparing the relative abundances of peptides derived from a control sample and the specific complex that was purified from nuclear extracts by a single-step promoter DNA affinity procedure . In a second application, peptides derived from immunopurified STE12 protein complexes isolated from yeast cells in different states were used to detect quantitative changes in the abundance of the complexes, and to detect dynamic changes in the composition of the samples . The use of quantitative mass spectrometry to guide identification of specific complex components in partially purified samples, and to detect quantitative changes in the abundance and composition of protein complexes, provides the researcher with powerful new tools for the comprehensive analysis of macromolecular complexes. J Biol Chem, 2003 Apr 25, 278(17), 14949 - 55 Epub 2003 Feb 17. Identification of multiple RNA features that influence CCR4 deadenylation activity; Viswanathan P et al.; The CCR4 family proteins are 3'-5'-deadenylases that function in the first step of the degradation of poly(A) mRNA . Here we report the purification to homogeneity of the yeast CCR4 protein and the analysis of its substrate specificities . CCR4 deadenylated a 7N+23A substrate (seven nucleotides followed by 23 A residues) in a distributive manner . Only small differences in CCR4 activity for different A length substrates were observed until only 1 A residue remained . Correspondingly, the K(m) for a 25N+2A substrate was found to be at least 20-fold lower than that for a 26N+1A substrate, although their V(max) values differed by only 2-fold . In addition, the total length of the RNA was found to contribute to CCR4 activity: up to 17 nucleotides (not necessarily poly(A)) could be recognized by CCR4 . Poly(U), poly(C), and poly(G) were also found to be 12-30-fold better inhibitors of CCR4 compared with poly(A), supporting the observation that CCR4 contains a non-poly(A)-specific binding site . Surprisingly, even longer substrates (>/=45 nucleotides) stimulated CCR4 to become a processive enzyme, suggesting that CCR4 undergoes an additional transition in the presence of such substrates . CCR4 also displayed no difference in its activity with capped or uncapped RNA substrates . These results indicate that CCR4 recognition of its RNA substrates involves several features of the RNA that could be sites in vivo for controlling the rate of specific mRNA deadenylation. J Mol Biol, 2003 Feb 28, 326(4), 999 - 1004 Inherent protein structural flexibility at the RNA-binding interface of L30e; Chao JA et al.; The Saccharomyces cerevisiae ribosomal protein L30 autoregulates its own expression by binding to a purine-rich internal loop in its pre-mRNA and mRNA . NMR studies of L30 and its RNA complex showed that both the internal loop of the RNA as well as a region of the protein become substantially more ordered upon binding . A crystal structure of a maltose binding protein (MBP)-L30 fusion protein with two copies in the asymmetric unit has been determined . The flexible RNA-binding region in the L30 copies has two distinct conformations, one resembles the RNA bound form solved by NMR and the other is unique . Structure prediction algorithms also had difficulty accurately predicting this region, which is consistent with conformational flexibility seen in the NMR and X-ray crystallography studies . Inherent conformational flexibility may be a hallmark of regions involved in intermolecular interactions. Curr Genet, 2003 Jan, 42(4), 199 - 208 Epub 2002 Dec 13. Investigating the importance of proteasome-interaction for Rad23 function; Lambertson D et al.; Rad23 contributes to diverse cellular functions that include DNA repair, stress response and growth control . An amino-terminal ubiquitin-like (UbL) domain in Rad23 interacts with catalytically active proteasomes and internal sequences bind multi-ubiquitinated proteins . Rad23 regulates the assembly of substrate-linked multi-ubiquitin chains, promotes efficient degradation of model substrates, and plays an overlapping role with the proteasome subunit, Rpn10 . These and other results led to the hypothesis that Rad23 translocates proteolytic substrates to the proteasome to promote degradation . It was previously shown that the UbL domain in Rad23 could be functionally replaced by ubiquitin . However, monomeric ubiquitin does not bind the proteasome efficiently, and we therefore investigated whether proteasome interaction was required for all Rad23 functions . We report here that the ubiquitin moiety in Ub-rad23 is ubiquitinated in vivo and could provide an alternate mechanism for binding the proteasome . These results suggest that the localization of Rad23 to the proteasome, either by its UbL domain, or following ubiquitination of an amino-terminal ubiquitin moiety (Ub-rad23), is necessary for full activity. Mol Biol Cell, 2003 Feb, 14(2), 477 - 90 Chemical genetic analysis of Apg1 reveals a non-kinase role in the induction of autophagy; Abeliovich H et al.; Macroautophagy is a catabolic membrane trafficking phenomenon that is observed in all eukaryotic cells in response to various stimuli, such as nitrogen starvation and challenge with specific hormones . In the yeast Saccharomyces cerevisiae, the induction of autophagy involves a direct signal transduction mechanism that affects membrane dynamics . In this system, the induction process modifies a constitutive trafficking pathway called the cytoplasm-to-vacuole targeting (Cvt) pathway, which transports the vacuolar hydrolase aminopeptidase I, from the formation of small Cvt vesicles to the formation of autophagosomes . Apg1 is one of the proteins required for the direct signal transduction cascade that modifies membrane dynamics . Although Apg1 is required for both the Cvt pathway and autophagy, we find that Apg1 kinase activity is required only for Cvt trafficking of aminopeptidase I but not for import via autophagy . In addition, the data support a novel role for Apg1 in nucleation of autophagosomes that is distinct from its catalytic kinase activity and imply a qualitative difference in the mechanism of autophagosome and Cvt vesicle formation. Mol Cell Biol, 2003 Mar, 23(5), 1832 - 42 Multiple functional elements comprise a Mammalian chromosomal replicator; Liu G et al.; The structure of replication origins in metazoans is only nominally similar to that in model organisms, such as Saccharomyces cerevisiae . By contrast to the compact origins of budding yeast, in metazoans multiple elements act as replication start sites or control replication efficiency . We first reported that replication forks diverge from an origin 5' to the human c-myc gene and that a 2.4-kb core fragment of the origin displays autonomous replicating sequence activity in plasmids and replicator activity at an ectopic chromosomal site . Here we have used clonal HeLa cell lines containing mutated c-myc origin constructs integrated at the same chromosomal location to identify elements important for DNA replication . Replication activity was measured before or after integration of the wild-type or mutated origins using PCR-based nascent DNA abundance assays . We find that deletions of several segments of the c-myc origin, including the DNA unwinding element and transcription factor binding sites, substantially reduced replicator activity, whereas deletion of the c-myc promoter P1 had only a modest effect . Substitution mutagenesis indicated that the sequence of the DNA unwinding element, rather than the spacing of flanking sequences, is critical . These results identify multiple functional elements essential for c-myc replicator activity. Mol Cell Biol, 2003 Mar, 23(5), 1808 - 16 Cascade of distinct histone modifications during collagenase gene activation; Martens JH et al.; Gene activation in eukaryotes requires chromatin remodeling, in part via histone modifications . To study the events at the promoter of a mitogen-inducible gene, we examined the induction of expression of the collagenase gene . It has been established that the collagenase gene can be activated by c-Jun and c-Fos and that the transcriptional coactivator p300 is involved in the activation . As expected, we found histone acetyltransferase activity at the collagenase promoter during activation . Interestingly, we also found histone methyltransferase and kinase activity . Strikingly, the first modification observed is methylation of histone H3 lysine 4, which correlates with the binding of the SET9 methyltransferase and the assembly of a complex consisting of c-Jun, c-Fos, TATA binding protein, and RNA polymerase II . The assembly of the preinitiation complex also shows an ordered binding of the acetyltransferase p300, the RSK2 kinase, and the SWI/SNF component Brg-1 . Our results suggest that collagenase gene activation involves a dynamic recruitment of different factors and that in addition to acetylation, histone H3 lysine 4 di- and trimethylation and histone H3 serine 10 phosphorylation are important steps in the activation of this gene. Mol Cell Biol, 2003 Mar, 23(5), 1798 - 807 Nob1p is required for cleavage of the 3' end of 18S rRNA; Fatica A et al.; We report the characterization of a novel factor, Nob1p (Yor056c), which is essential for the synthesis of 40S ribosome subunits . Genetic depletion of Nob1p strongly inhibits the processing of the 20S pre-rRNA to the mature 18S rRNA, leading to the accumulation of high levels of the 20S pre-rRNA together with novel degradation intermediates . 20S processing occurs within a pre-40S particle after its export from the nucleus to the cytoplasm . Consistent with a direct role in this cleavage, Nob1p was shown to be associated with the pre-40S particle and to be present in both the nucleus and the cytoplasm . This suggests that Nob1p accompanies the pre-40S ribosomes during nuclear export . Pre-40S export is not, however, inhibited by depletion of Nob1p. Mol Cell Biol, 2003 Mar, 23(5), 1674 - 87 Beta-catenin binds to the activation function 2 region of the androgen receptor and modulates the effects of the N-terminal domain and TIF2 on ligand-dependent transcription; Song LN et al.; Beta-catenin is a multifunctional molecule that is activated by signaling through WNT receptors . beta-Catenin can also enhance the transcriptional activity of some steroid hormone receptors such as the androgen receptor and retinoic acid receptor alpha . Androgens can affect nuclear translocation of beta-catenin and influence its subcellular distribution . Using mammalian two-hybrid binding assays, analysis of reporter gene transcription, and coimmunoprecipitation, we now show that beta-catenin binds to the androgen receptor ligand-binding domain (LBD) and modulates the transcriptional effects of TIF2 and the androgen receptor N-terminal domain (NTD) . In functional assays, beta-catenin bound to androgen receptor only in the presence of ligand agonists, not antagonists . Beta-catenin binding to the androgen receptor LBD was independent of and cooperative with the androgen receptor NTD and the p160 coactivator TIF2, both of which bind to the activation function 2 (AF-2) region of the androgen receptor . Different mutations of androgen receptor helix 3 amino acids disrupted binding of androgen receptor NTD and beta-catenin . beta-Catenin, androgen receptor NTD, and TIF2 binding to the androgen receptor LBD were affected similarly by a subset of helix 12 mutations, but disruption of two sites on helix 12 affected only binding of beta-catenin and not of TIF2 or the androgen receptor NTD . Mutational disruption of each of five LXXLL peptide motifs in the beta-catenin armadillo repeats did not disrupt either binding to androgen receptor or transcriptional coactivation . ICAT, an inhibitor of T-cell factor 4 (TCF-4), and E-cadherin binding to beta-catenin also blocked binding of the androgen receptor LBD . We also demonstrated cross talk between the WNT and androgen receptor signaling pathways because excess androgen receptor could interfere with WNT signaling and excess TCF-4 inhibited the interaction of beta-catenin and androgen receptor . Taken together, the data show that beta-catenin can bind to the androgen receptor LBD and modulate the effects of the androgen receptor NTD and TIF2 on transcription. Mol Cell Biol, 2003 Mar, 23(5), 1656 - 65 MBDin, a novel MBD2-interacting protein, relieves MBD2 repression potential and reactivates transcription from methylated promoters; Lembo F et al.; We have identified a human gene encoding a novel MBD2-interacting protein (MBDin) that contains an N-terminal GTP-binding site, a putative nuclear export signal (NES), and a C-terminal acidic region . MBDin cDNA was isolated through a two-hybrid interaction screening using the methyl-CpG-binding protein MBD2 as bait . The presence of the C-terminal 46-amino-acid region of MBD2 and both the presence of the acidic C-terminal 128-amino-acid region and the integrity of the GTP-binding site of MBDin were required for the interaction . Interaction between MBD2 and MBDin in mammalian cells was confirmed by immunoprecipitation experiments . Fluorescence imaging experiments demonstrated that MBDin mainly localizes in the cytoplasm but accumulates in the nucleus upon disruption of the NES or treatment with leptomycin B, an inhibitor of NES-mediated transport . We also found that MBDin partially colocalizes with MBD2 at foci of heavily methylated satellite DNA . An MBD2 deletion mutant lacking the C-terminal region maintained its subnuclear localization but failed to recruit MBDin at hypermethylated foci . Functional analyses demonstrated that MBDin relieves MBD2-mediated transcriptional repression both when Gal4 chimeric constructs and when in vitro-methylated promoter-reporter plasmids were used in transcriptional assays . Southern blotting and bisulfite analysis showed that transcriptional reactivation occurred without changes of the promoter methylation pattern . Our findings suggest the existence of factors that could be targeted on methylated DNA by methyl-CpG-binding proteins reactivating transcription even prior to demethylation. Mol Cell Biol, 2003 Mar, 23(5), 1602 - 13 Delayed rRNA processing results in significant ribosome biogenesis and functional defects; Meskauskas A et al.; mof6-1 was originally isolated as a recessive mutation in Saccharomyces cerevisiae which promoted increased efficiencies of programmed -1 ribosomal frameshifting and rendered cells unable to maintain the killer virus . Here, we demonstrate that mof6-1 is a unique allele of the histone deacetylase RPD3, that the deacetylase function of Rpd3p is required for controlling wild-type levels of frameshifting and virus maintenance, and that the closest human homolog can fully complement these defects . Loss of the Rpd3p-associated histone deacetylase function, either by mutants of rpd3 or loss of the associated gene product Sin3p or Sap30p, results in a delay in rRNA processing rather than in an rRNA transcriptional defect . This results in production of ribosomes having lower affinities for aminoacyl-tRNA and diminished peptidyltransferase activities . We hypothesize that decreased rates of peptidyl transfer allow ribosomes with both A and P sites occupied by tRNAs to pause for longer periods of time at -1 frameshift signals, promoting increased programmed -1 ribosomal frameshifting efficiencies and subsequent loss of the killer virus . The frameshifting defect is accentuated when the demand for ribosomes is highest, suggesting that rRNA posttranscriptional modification is the bottleneck in ribosome biogenesis. Endocr Rev, 2003 Feb, 24(1), 78 - 90 Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha): transcriptional coactivator and metabolic regulator; Puigserver P et al.; Investigations of biological programs that are controlled by gene transcription have mainly studied the regulation of transcription factors . However, there are examples in which the primary focus of biological regulation is at the level of a transcriptional coactivator . We have reviewed here the molecular mechanisms and biological programs controlled by the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha) . Key cellular signals that control energy and nutrient homeostasis, such as cAMP and cytokine pathways, strongly activate PGC-1 alpha . Once PGC-1 alpha is activated, it powerfully induces and coordinates gene expression that stimulates mitochondrial oxidative metabolism in brown fat, fiber-type switching in skeletal muscle, and multiple aspects of the fasted response in liver . The regulation of these metabolic and cell fate decisions by PGC-1 alpha is achieved through specific interaction with a variety of transcription factors such as nuclear hormone receptors, nuclear respiratory factors, and muscle-specific transcription factors . PGC-1 alpha therefore constitutes one of the first and clearest examples in which biological programs are chiefly regulated by a transcriptional coactivator in response to environmental stimuli . Finally, PGC-1 alpha's control of energy homeostasis suggests that it could be a target for anti-obesity or diabetes drugs. Biochim Biophys Acta, 2003 Feb 17, 1610(1), 90 - 6 Semliki Forest virus vectors for rapid and high-level expression of integral membrane proteins; Lundstrom K; Semliki Forest virus (SFV) vectors have been applied for the expression of recombinant integral membrane proteins in a wide range of mammalian host cells . More than 50 G protein-coupled receptors (GPCRs), several ion channels and other types of transmembrane or membrane-associated proteins have been expressed at high levels . The establishment of large-scale SFV technology has facilitated the production of large quantities of recombinant receptors, which have then been subjected to drug screening programs and structure-function studies on purified receptors . The recent Membrane Protein Network (MePNet) structural genomics initiative, where 100 GPCRs are overexpressed from SFV vectors, will further provide new methods and technologies for expression, solubilization, purification and crystallization of GPCRs . Biochim Biophys Acta, 2003 Feb 17, 1610(1), 77 - 89 G protein-coupled receptor overexpression with the baculovirus-insect cell system: a tool for structural and functional studies; Massotte D; G protein-coupled receptors, whose topology shows seven transmembrane domains, form the largest known family of receptors involved in higher organism signal transduction . These receptors are generally of low natural abundance and overexpression is usually a prerequisite to their structural or functional characterisation . The baculovirus-insect cell system constitutes a versatile tool for the maximal production of receptors . This heterologous expression system also provides interesting alternatives for receptor functional studies in a well-controlled cellular context . J Hepatol, 2003 Mar, 38(3), 298 - 306 Positional mapping for amplified DNA sequences on 1q21-q22 in hepatocellular carcinoma indicates candidate genes over-expression; Wong N et al.; BACKGROUND/AIMS: Comparative genomic hybridization analysis on hepatocellular carcinoma (HCC) indicated frequent gains of 1q and an amplicon at 1q21-q22 . Current cytogenetic evidences confer much importance on 1q21-q22, where a role in drug resistance, tumor metastasis and shorter patient survival had been implicated . METHODS: Using positional mapping by interphase cytogenetics, we investigated the amplicon 1q21-q22 in five HCC cases . Three amplification maxima represented by yeast artificial chromosomes (YACs) 955E11, 876B11 and 945D5 that mapped to regions 1q21.1, 1q21.2 and 1q22, respectively, were indicated . We further investigated candidate genes expression in the mapped YACs by quantitative reverse-transcription-polymerase chain reaction . A panel of genes encoding protein transcripts involved in apoptosis, cell cycle progression, calcium binding and jumping translocation was studied . RESULTS: Among ten HCC cases with the amplicon 1q21-q22 examined, we found a significant gene expression level of JTB, SHC1, CCT3 and COPA in the tumors than the paired adjacent non-malignant liver tissues (P< or =0.04) . CONCLUSIONS: Our interphase findings on 1q21-q22 pinpointed three affected loci between D1S305 and D1S2369 . Up-regulation of candidate genes identified within these over-represented regions may represent targets in the progression of HCC and may carry prognostic significance. J Cell Sci, 2003 Mar 15, 116(Pt 6), 1107 - 15 Role of the Vtc proteins in V-ATPase stability and membrane trafficking; Muller O et al.; Vtc proteins have genetic and physical relations with the vacuolar H(+)-ATPase (V-ATPase), influence vacuolar H(+) uptake and, like the V-ATPase V(0) sectors, are important factors in vacuolar membrane fusion . Vacuoles from vtc1delta and vtc4delta mutants had slightly reduced H(+)-uptake activity . These defects could be separated from Vtc function in vacuole fusion, demonstrating that Vtc proteins have a direct role in membrane fusion . We analyzed their involvement in other membrane trafficking steps and in VATPase dynamics . Deletion of VTC genes did not impede endocytic trafficking to the vacuole . However, ER to Golgi trafficking and further transport to the vacuole was delayed in deltavtc3 cells . In accordance with that, deltavtc3 cells showed a reduced growth rate . Vtc mutations did not interfere with regulated assembly and disassembly of the V-ATPase, but they affected the number of peripheral V(1) subunits associated with the vacuoles . deltavtc3 vacuoles carried significantly more V(1) subunits, whereas deltavtc1, deltavtc2 and deltavtc4 had significantly less . The proteolytic sensitivity of the V(0) subunit Vph1p was different in deltavtc and wild-type cells in vivo, corroborating the physical interaction of Vtc proteins with the V-ATPase observed in vitro . We suggest that Vtc proteins affect the conformation of V(0) . They might thereby influence the stability of the VATPase holoenzyme and support the function of its V(0) sector in vacuolar membrane fusion. J Cell Sci, 2003 Mar 15, 116(Pt 6), 1023 - 33 Deletion of proteasomal subunit S5a/Rpn10/p54 causes lethality, multiple mitotic defects and overexpression of proteasomal genes in Drosophila melanogaster; Szlanka T et al.; The regulatory complex of the 26S proteasome is responsible for the selective recognition and binding of multiubiquitinated proteins . It was earlier shown that the subunit S5a/Rpn10/p54 of the regulatory complex is the only cellular protein capable of binding multiubiquitin chains in an in vitro overlay assay . The role of this subunit in substrate selection, however, is a subject of debate, following the observation that its deletion in Saccharomyces cerevisiae is not lethal and instead causes only a mild phenotype . To study the function of this subunit in higher eukaryotes, a mutant Drosophila strain was constructed by deleting the single copy gene encoding subunit S5a/Rpn10/p54 . This deletion caused larval-pupal polyphasic lethality, multiple mitotic defects, the accumulation of higher multimers of ubiquitinated proteins and a huge accumulation of defective 26S proteasome particles . Deletion of the subunit S5a/Rpn10/p54 does not destabilise the regulatory complex and does not disturb the assembly of the regulatory complex and the catalytic core . The pupal lethality is a consequence of the depletion of the maternally provided 26S proteasome during the larval stages and a sudden increase in the proteasomal activity demands during the first few hours of pupal development . The huge accumulation of the fully assembled 26S proteasome in the deletion mutant and the lack of free subunits or partially assembled particles indicate that there is a highly coordinated accumulation of all the subunits of the 26S proteasome . This suggests that in higher eukaryotes, as with yeast, a feedback circuit coordinately regulates the expression of the proteasomal genes, and this adjusts the actual proteasome concentration in the cells according to the temporal and/or spatial proteolytic demands. Bioinformatics, 2003 Feb 12, 19(3), 396 - 401 A literature-based method for assessing the functional coherence of a gene group; Raychaudhuri S et al.; MOTIVATION: Many experimental and algorithmic approaches in biology generate groups of genes that need to be examined for related functional properties . For example, gene expression profiles are frequently organized into clusters of genes that may share functional properties . We evaluate a method, neighbor divergence per gene (NDPG), that uses scientific literature to assess whether a group of genes are functionally related . The method requires only a corpus of documents and an index connecting the documents to genes . RESULTS: We evaluate NDPG on 2796 functional groups generated by the Gene Ontology consortium in four organisms: mouse, fly, worm and yeast . NDPG finds functional coherence in 96, 92, 82 and 45% of the groups (at 99.9% specificity) in yeast, mouse, fly and worm respectively. Bioinformatics, 2003 Feb 12, 19(3), 390 - 5 SimiTri--visualizing similarity relationships for groups of sequences; Parkinson J et al.; Global sequence comparisons between large datasets, such as those arising from genome projects, can be problematic to display and analyze . We have developed SimiTri, a Java/Perl-based application, which allows simultaneous display and analysis of relative similarity relationships of the dataset of interest to three different databases . We illustrate its utility in identifying Caenorhabditis elegans genes that have distinct patterns of phylogenetic affinity suggestive of horizontal gene transfer . SimiTri is freely downloadable from and the source code is freely available from the authors. Nucleic Acids Res . 2003 Feb 15;31(4):e18. Identification and removal of contaminating fluorescence from commercial and in-house printed DNA microarrays; Martinez MJ et al.; Microarray analysis is a critically important technology for genome-enabled biology, therefore it is essential that the data obtained be reliable . Current software and normalization techniques for microarray analysis rely on the assumption that fluorescent background within spots is essentially the same throughout the glass slide and can be measured by fluorescence surrounding the spots . This assumption is not valid if background fluorescence is spot-localized . Inaccurate estimates of background fluorescence under the spot create a source of error, especially for low expressed genes . We have identified spot-localized, contaminating fluorescence in the Cy3 channel on several commercial and in-house printed microarray slides . We determined through mock hybridizations (without labeled target) that pre-hybridization scans could not be used to predict the contribution of this contaminating fluorescence after hybridization because the change in spot-to-spot fluorescence after hybridization was too variable . Two solutions to this problem were identified . First, allowing 4 h of exposure to air prior to printing on to Corning UltraGAPS slides significantly reduced contaminating fluorescence intensities to approximately the value of the surrounding glass . Alternatively, application of a novel, hyperspectral imaging scanner and multivariate curve resolution algorithms, allowed the spectral contributions of Cy3 signal, glass, and contaminating fluorescence to be distinguished and quantified after hybridization. Nucleic Acids Res, 2003 Feb 15, 31(4), 1261 - 74 Mutations in the histone fold domain of the TAF12 gene show synthetic lethality with the TAF1 gene lacking the TAF N-terminal domain (TAND) by different mechanisms from those in the SPT15 gene encoding the TATA box-binding protein (TBP); Kobayashi A et al.; The general transcription factor TFIID, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), is important for both basal and regulated transcription by RNA polymerase II . Although it is well known that the TAF N-terminal domain (TAND) at the amino-terminus of the TAF1 protein binds to TBP and thereby inhibits TBP function in vitro, the physiological role of this domain remains obscure . In our previous study, we screened for mutations that cause lethality when co-expressed with the TAF1 gene lacking TAND (taf1-DeltaTAND) and identified two DeltaTAND synthetic lethal (nsl) mutations as those in the SPT15 gene encoding TBP . In this study we isolated another nsl mutation in the same screen and identified it to be a mutation in the histone fold domain (HFD) of the TAF12 gene . Several other HFD mutations of this gene also exhibit nsl phenotypes, and all of them are more or less impaired in transcriptional activation in vivo . Interestingly, a set of genes affected in the taf1-DeltaTAND mutant is similarly affected in the taf12 HFD mutants but not in the nsl mutants of TBP . Therefore, we discovered that the nsl mutations of these two genes cause lethality in the taf1-DeltaTAND mutant by different mechanisms. Nucleic Acids Res, 2003 Feb 15, 31(4), 1224 - 33 Rap1p and other transcriptional regulators can function in defining distinct domains of gene expression; Yu Q et al.; Barrier elements that are able to block the propagation of transcriptional silencing in yeast are functionally similar to chromatin boundary/insulator elements in metazoans that delimit functional chromosomal domains . We show that the upstream activating sequences of many highly expressed ribosome protein genes and glycolytic genes exhibit barrier activity . Analyses of these barriers indicate that binding sites for transcriptional regulators Rap1p, Abf1p, Reb1p, Adr1p and Gcn4p may participate in barrier function . We also present evidence suggesting that Rap1p is directly involved in barrier activity, and its barrier function correlates with local changes in chromatin structure . We further demonstrate that tethering the transcriptional activation domain of Rap1p to DNA is sufficient to recapitulate barrier activity . Moreover, targeting the activation domain of Adr1p or Gcn4p also establishes a barrier to silencing . These results support the notion that transcriptional regulators could also participate in delimiting functional domains in the genome. Eukaryot Cell, 2003 Feb, 2(1), 181 - 90 TOK homologue in Neurospora crassa: first cloning and functional characterization of an ion channel in a filamentous fungus; Roberts SK; In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology . The life cycle of most fungi depends on the "filamentous" polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made . In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K(+) channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa . The patch clamp technique was used to investigate the biophysical properties of the N . crassa K(+) channel (NcTOKA) after heterologous expression of NcTOKA in yeast . NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K(+) efflux . NcTOKA channel gating was sensitive to extracellular K(+) such that channel activation was dependent on the reversal potential for K(+) . However, expression of NcTOKA was able to overcome the K(+) auxotrophy of a yeast mutant missing the K(+) uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K(+) influx . Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K(+) currents at potentials negative of E(K) . NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS . NcTOKA was effectively blocked by extracellular Ca(2+), verapamil, quinine, and TEA(+) but was insensitive to Cs(+), 4-aminopyridine, and glibenclamide . The physiological significance of NcTOKA is discussed in the context of its biophysical properties. Eukaryot Cell, 2003 Feb, 2(1), 150 - 8 Transcriptional regulation of xyn2 in Hypocrea jecorina; Wurleitner E et al.; The xylanase system of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) consists of two specific xylanases, Xyn1 and Xyn2, which are simultaneously expressed during growth on xylan but respond differentially to low-molecular-weight inducers . Using in vivo footprinting analysis of xylan-induced and noninduced mycelia, we detected two adjacent nucleotide sequences (5'-AGAA-3' on the noncoding strand and 5'-GGGTAAATTGG-3', referred to as the xylanase-activating element {XAE}, on the coding strand, respectively) to bind proteins . Among these, binding to the AGAA-box is only observed under noninduced conditions, whereas binding to XAE is constitutive . Electrophoretic mobility shift assay with heterologously expressed components of the H . jecorina Hap2/3/5 protein complex and the cellulase regulator Ace2 suggests that these two transactivators form the protein complex binding to XAE . H . jecorina transformants, containing correspondingly mutated versions of the xyn2 promoter fused to the Aspergillus niger goxA gene as a reporter, revealed that the elimination of protein binding to the AGAA-box resulted in a threefold increase in both basal and induced transcription, whereas elimination of Ace2 binding to its target in XAE completely eliminated transcription under both conditions . Destruction of the CCAAT-box by insertion of a point mutation prevents binding of the Hap2/3/5 complex in vitro and results in a slight increase in both basal and induced transcription . These data support a model of xyn2 regulation based on the interplay of Hap2/3/5, Ace2 and the AGAA-box binding repressor. Eukaryot Cell, 2003 Feb, 2(1), 123 - 33 Overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p inhibits ubiquitin ligase activities of their SCF complexes; Dixon C et al.; Ubiquitin ligases direct the transfer of ubiquitin onto substrate proteins and thus target the substrate for proteasome-dependent degradation . SCF complexes are a family of ubiquitin ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity . Distinct SCF complexes, defined by a particular F-box protein, target different substrate proteins for degradation . Although a few have been identified to be involved in important biological pathways, such as the cell division cycle and coordinating cellular responses to changes in environmental conditions, the role of the overwhelming majority of F-box proteins is not clear . Creating inhibitors that will block the in vivo activities of specific SCF ubiquitin ligases may provide identification of substrates of these uncharacterized F-box proteins . Using Saccharomyces cerevisiae as a model system, we demonstrate that overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p results in specific inhibition of their SCF complexes . Analyses of mutant amino-terminal alleles demonstrate that the interaction of these polypeptides with their full-length counterparts is an important step in the inhibitory process . These results suggest a common means to inhibit specific SCF complexes in vivo. Eukaryot Cell, 2003 Feb, 2(1), 27 - 33 Ssk1p response regulator binding surface on histidine-containing phosphotransfer protein Ypd1p; Porter SW et al.; Ypd1p, a histidine-containing phosphotransfer protein, plays an important role in a branched His-Asp phosphorelay signal transduction pathway that regulates cellular responses to hyperosmotic stress in Saccharomyces cerevisiae . Ypd1p is required for phosphoryl group transfer from the membrane-bound Sln1p sensor histidine kinase to two downstream response regulator proteins, Ssk1p and Skn7p . To investigate the molecular basis for interaction of Ypd1p with these response regulator domains, we used an approach that coupled alanine-scanning mutagenesis of surface-exposed residues in Ypd1p with a yeast two-hybrid interaction screen . Mutated residues that adversely affected the interaction of Ypd1p with the C-terminal response regulator domain of Ssk1p were identified and found to cluster on or near the alphaA helix in Ypd1p . Our results, supported by analysis of a modeled complex, identify a binding site on Ypd1p for response regulators that is composed of a cluster of conserved hydrophobic residues surrounded by less conserved polar residues . We propose that molecular interactions involving Ypd1p are mediated primarily through hydrophobic contacts, whereas binding specificity and strength of interaction may be influenced by select polar side chain interactions. Di Yi Jun Yi Da Xue Xue Bao, 2003 Feb, 23(2), 124 - 6 {Two restriction fluorescence labeling methods for enhancing the signal-to-noise ratio of cDNA microarray hybridization}; Shi R et al.; OBJECTIVE: To study the signal-to-noise ratio (SNR) of two restricted fluorescence labeling methods for examining gene expression profile by microarray hybridization . METHOD: Samples of Saccharomyces cerevisiae mRNA was labeled by traditional reverse transcription method and 2 restriction fluorescent labeling methods using respectively Cy-universal primer and extension incorporated Cy-dNTP . The labeled samples were examined by the microarray, followed by washing and scanning under the same conditions . RESULTS: The two restriction labeling methods showed superior results with lowered background and enhanced SNR and sensitivity, and Cy-universal primer labeling presented the best results . CONCLUSION: SNR can be enhanced by the restriction labeling methods, which improve the applicability of microarray technology. Mol Microbiol, 2003 Feb, 47(4), 1149 - 61 Activation mechanisms of the HAC1-mediated unfolded protein response in filamentous fungi; Saloheimo M et al.; The unfolded protein response (UPR) is a regulatory pathway activating genes involved in multiple functions related to folding, quality control and transport of secreted proteins . Characterization of the hac1/hacA genes encoding the UPR transcription factors from the filamentous fungi Trichoderma reesei and Aspergillus nidulans is described in this article . The corresponding gene in Saccharomyces cerevisiae is activated through a non-spliceosomal intron-splicing reaction . The T . reesei hac1 and A . nidulans hacA mRNAs undergo an analogous splicing reaction of a 20-nt-long intron during UPR induction . This splicing changes the reading frame of the mRNA and thus could bring in an activation domain to the HACI/HACA proteins . In addition to the non-spliceosomal splicing, the hac1/A mRNAs of the filamentous fungi are truncated at the 5'-flanking region upon UPR induction . An upstream open reading frame is omitted from the mRNAs due to the truncation, and evidence is presented showing that the truncated T . reesei hac1 mRNA is translated more efficiently than a full-length mRNA . This paper reports a novel combination of two different regulatory mechanisms of a transcription factor gene, both operational at the mRNA level. Plant J, 2003 Feb, 33(3), 455 - 69 Alteration of GCN5 levels in maize reveals dynamic responses to manipulating histone acetylation; Bhat RA et al.; The role played by histone acetyltransferase (HAT), GCN5, in transcriptional co-activation has been analysed in detail in yeast and mammals . Here, we present the cloning and expression pattern of Zmgcn5, the maize homologue . The enzymatic activity of the recombinant ZmGCN5 was analysed with histone and nucleosome substrates . In situ hybridisation of developing maize kernels using Zmgcn5 as probe shows that the transcript is concentrated in rapidly dividing cells . To investigate the role of ZmGCN5 in the transcription of specific plant genes, direct protein-protein interactions were tested . A cDNA clone encoding a putative interacting partner in GCN5-adapter complexes, ZmADA2, was isolated and the interaction between ZmGCN5 and ZmADA2 was confirmed by a GST-spin down experiment . Co-immunoprecipitation of the plant transcriptional activator Opaque-2 and ZmADA2 in nuclear extracts suggests ADA2/GCN5-containing complexes to mediate transcriptional activation by binding of this bZIP factor . For a more general analysis of the effects of histone acetylation on plant gene expression, 2500 ESTs spotted on filters were hybridised with cDNA probes derived either from maize cell lines treated with Trichostatin A (TSA), or from a transgenic line expressing the ZmGCN5 antisense transcript . Several sequences showing marked changes in abundance were confirmed by RNA blot analysis . Inhibition of histone deacetylation with TSA is accompanied by a decrease in the abundance of ZmGCN5 acetylase protein, but by increases in mRNAs for histones H2A, H2B, H3 and H4 . The elevated histone mRNA levels were not reflected in increasing histone protein concentrations, suggesting hyperacetylated histones arising from TSA treatment may be preferentially degraded and substituted by de novo synthesised histones . The ZmGCN5 antisense material showed suppression of the endogenous ZmGCN5 transcript and the profiling analysis revealed increased mRNA levels for H2A, H2B and H4 . Furthermore, in the antisense line, a reduction in the amount of the RPD3-type HD1B-I histone deacetylase protein was observed . A model for linked regulation of histone acetylation and histone mRNA transcription is discussed. Genes Cells, 2003 Feb, 8(2), 145 - 61 CDK- and Cdc45-dependent priming of the MCM complex on chromatin during S-phase in Xenopus egg extracts: possible activation of MCM helicase by association with Cdc45; Masuda T et al.; BACKGROUND: MCM and Cdc45 are required for the initiation and elongation stages of eukaryotic DNA replication . Recent studies show that a purified Mcm4/6/7 complex has DNA helicase activity . However, the biochemical function of the MCM complex and Cdc45 bound to chromatin has not been elucidated . RESULTS: We have examined the biochemical properties of MCM proteins bound to chromatin fractions using Xenopus egg extracts . Immunoprecipitation of MCM proteins extracted under denaturing conditions reveals that all six subunits of MCM and Cdc45 form a tight complex following the initiation of DNA replication, and that both CDK activity and Cdc45 are essential for the complex formation . Chromatin immunoprecipitation of MCM proteins and Cdc45 shows that a complex containing MCM and Cdc45 has a DNA helicase activity which is dependent on CDK activity and Cdc45 in the extracts . Furthermore, both the complex and the helicase activity are resistant to treatment with phosphatase and high salt . CONCLUSIONS: Following the initiation of DNA replication, a tight MCM-Cdc45 complex is formed on chromatin and its formation is closely correlated with the DNA helicase activity of chromatin immunoprecipitates containing MCM and Cdc45 . We propose that the tight MCM-Cdc45 complex functions as a replicative DNA helicase in vivo. Biochemistry, 2003 Feb 18, 42(6), 1646 - 51 Mitochondrial form of a tRNA synthetase can be made bifunctional by manipulating its leader peptide; Wang CC et al.; Previous studies showed that yeast VAS1 encodes both the cytoplasmic and mitochondrial forms of valyl-tRNA synthetase (ValRS), using alternative transcription and translation . The ValRS isoforms have identical polypeptide sequences, except for a 46-amino acid leader peptide that functions as a mitochondrial targeting signal . Although the two forms of the enzyme exhibit indistinguishable tRNA specificities in vitro, they cannot substitute for each other in vivo because of their different localizations . Here we show that the 46-residue leader sequence can be divided into two nonoverlapping peptides, each of which retains the ability to target the enzyme into mitochondria . The engineered proteins (with truncated leader sequences) are dual-targeted, rescuing both the cytoplasmic and mitochondrial defects of a vas1 knockout strain . Thus, in addition to alternative splicing and alternative translation initiation as mechanisms by which a single gene can encode cytoplasmic and mitochondrial activities, the inherent characteristics of a single polypeptide may enable it to be distributed simultaneously between two cellular compartments . This mechanism may explain how certain other single genes in Saccharomyces cerevisiae provide dual functions. Biochemistry, 2003 Feb 18, 42(6), 1508 - 16 Altered Cu metabolism and differential transcription of Cu/ZnSod genes in a Cu/ZnSOD-deficient mutant of maize: evidence for a Cu-responsive transcription factor; Ruzsa SM et al.; Maize inbred line A351 exhibits extremely low levels of Cu/Zn superoxide dismutase (SOD) isozymes, three cytosolic and one chloroplastic, which are increased by supplying copper to near-toxic concentrations . Activities of the copper enzymes cytochrome c oxidase and ascorbate oxidase are also reduced . The level of expression of the maize copper chaperone for SOD is normal to elevated . The gene transcript encoding chloroplastic SOD-1 is present at normal levels, whereas RNA levels of the cytosolic SODs are low and increase with added copper, suggesting a promoter element and copper-dependent transcription factor common to the three genes . Although a reduced level of high-affinity copper transport in A351 cannot be ruled out, high transcript levels of a constitutively expressed metallothionein, suggesting increased copper chelation capacity and creating a general copper-deprivation effect, seem to be a likely cause of the reduced levels of copper enzyme activity and Cu/ZnSod gene transcripts . While exogenous copper does not affect the wild-type SOD activity or protein, it increases wild-type Cu/ZnSod transcript levels in a response similar to that of several yeast genes involved in copper sequestration and antioxidant defense . A sequence that is highly homologous to those of the copper-responsive transcription factors ACE1 (Saccharomyces cerevisiae) and AMT1 (Candida glabrata) is present in the promoters of three maize Cu/ZnSod genes. Nat Genet, 2003 Mar, 33(3), 392 - 5 Epub 2003 Feb 10. Evidence for co-evolution of gene order and recombination rate; Pal C et al.; There is increasing evidence in eukaryotic genomes that gene order is not random, even allowing for tandem duplication . Notably, in numerous genomes, genes of similar expression tend to be clustered . Are there other reasons for clustering of functionally similar genes? If genes are linked to enable genetic, rather than physical clustering, then we also expect that clusters of certain genes might be associated with blocks of reduced recombination rates . Here we show that, in yeast, essential genes are highly clustered and this clustering is independent of clustering of co-expressed genes and of tandem duplications . Adjacent pairs of essential genes are preferentially conserved through evolution . Notably, we also find that clusters of essential genes are in regions of low recombination and that larger clusters have lower recombination rates . These results suggest that selection acts to modify both the fine-scale intragenomic variation in the recombination rate and the distribution of genes and provide evidence for co-evolution of gene order and recombination rate. Nat Struct Biol, 2003 Mar, 10(3), 182 - 6 Rad54 protein possesses chromatin-remodeling activity stimulated by the Rad51-ssDNA nucleoprotein filament; Alexeev A et al.; In Saccharomyces cerevisiae, the Rad54 protein participates in the recombinational repair of double-strand DNA breaks together with the Rad51, Rad52, Rad55 and Rad57 proteins . In vitro, Rad54 interacts with Rad51 and stimulates DNA strand exchange promoted by Rad51 protein . Rad54 is a SWI2/SNF2-related protein that possesses double-stranded DNA-dependent ATPase activity and changes DNA topology in an ATP hydrolysis-dependent manner . Here we show that Rad54 catalyzes bidirectional nucleosome redistribution by sliding nucleosomes along DNA . Nucleosome redistribution is greatly stimulated by the Rad51 nucleoprotein filament but does not require the presence of homologous single-stranded DNA within the filament . On the basis of these data, we propose that Rad54 facilitates chromatin remodeling and, perhaps more generally, protein clearing at the homology search step of genetic recombination. Cell Struct Funct, 2002 Dec, 27(6), 421 - 9 Autophagosome formation in mammalian cells; Mizushima N et al.; Macroautophagy is an intracellular degradation system for the majority of proteins and some organelles . The molecular mechanism of autophagy has been extensively studied using the yeast, Saccharomyces cerevisiae, during these past 10 years . These studies suggested that the molecular machinery of autophagosome formation is well conserved from yeast to higher eukaryotes . Identification and characterization of the mammalian counterparts of the yeast autophagy proteins has facilitated our understanding of mammalian autophagy, particularly of autophagosome formation . These findings are now being applied to studies on the physiological roles of autophagy in mammals. J Biol Chem, 2003 Apr 18, 278(16), 14174 - 84 Epub 2003 Feb 07. The Caenorhabditis elegans mRNA 5'-capping enzyme . In vitro and in vivo characterization; Takagi T et al.; Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities . The Caenorhabditis elegans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity . Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not originally predicted from the genomic sequence . Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae . The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases; therefore, mutagenesis of residues predicted to be important for RTPase activity was carried out . CEL-1 uses a mechanism similar to protein-tyrosine phosphatases, except that there was not an absolute requirement for a conserved acidic residue that acts as a proton donor . CEL-1 shows a strong preference for RNA substrates of at least three nucleotides in length . RNA-mediated interference in C . elegans embryos shows that lack of CEL-1 causes development to arrest with a phenotype similar to that seen when RNA polymerase II elongation activity is disrupted . Therefore, capping is essential for gene expression in metazoans. J Biol Chem, 2003 Apr 18, 278(16), 14168 - 73 Epub 2003 Feb 07. mRNA capping enzyme requirement for Caenorhabditis elegans viability; Srinivasan P et al.; Capping of the initiated 5' ends of RNA polymerase II products is evolutionarily and functionally conserved from yeasts to humans . The m(7)GpppN cap promotes RNA stability, processing, transport, and translation . Deletion of capping enzymes in yeasts was shown to be lethal due to rapid exonucleolytic degradation of uncapped transcripts or failure of capped but unmethylated RNA to initiate protein synthesis . Using RNA interference and Caenorhabditis elegans we have found that RNA capping is also essential for metazoan viability . C . elegans bifunctional capping enzyme was cloned, and capping activity by the expressed protein as well as growth complementation of yeast deletion strains missing either RNA triphosphatase or guanylyltransferase required terminal sequences not present in the previously isolated cel-1 clone . By RNA interference analysis we show that cel-1 is required for embryogenesis . cel-1(RNAi) embryos formed cytoplasmic granules characteristic of a phenocluster of RNA processing genes and died early in development. Neuron, 2003 Feb 6, 37(3), 433 - 47 Mutations in the exocyst component Sec5 disrupt neuronal membrane traffic, but neurotransmitter release persists; Murthy M et al.; The exocyst (Sec6/8) complex is necessary for secretion in yeast and has been postulated to establish polarity by directing vesicle fusion to specific sites along the plasma membrane . The complex may also function in the nervous system, but its precise role is unknown . We have investigated exocyst function in Drosophila with mutations in one member of the complex, sec5 . Null alleles die as growth-arrested larvae, whose neuromuscular junctions fail to expand . In culture, neurite outgrowth fails in sec5 mutants once maternal Sec5 is exhausted . Using a trafficking assay, we found impairments in the membrane addition of newly synthesized proteins . In contrast, synaptic vesicle fusion was not impaired . Thus, Sec5 differentiates between two forms of vesicle trafficking: trafficking for cell growth and membrane protein insertion depend on sec5, whereas transmitter secretion does not . In this regard, sec5 differs from the homologs of other yeast exocytosis genes that are required for both neuronal trafficking pathways. J Biol Chem, 2003 Apr 11, 278(15), 13033 - 8 Epub 2003 Feb 05. A hybrid IRF9-STAT2 protein recapitulates interferon-stimulated gene expression and antiviral response; Kraus TA et al.; Type I interferon (IFN) signaling induces the heterotrimeric transcription complex, IFN-stimulated gene factor (ISGF) 3, which contains STAT1, STAT2, and the DNA binding subunit, interferon regulatory factor (IRF) 9 . Because IRF9 is targeted to the nucleus in the absence of IFN stimulation, the potential of IRF9 protein for gene regulation was examined using a GAL4 DNA binding domain fusion system . GAL4-IRF9 was transcriptionally active in reporter gene assays but not in the absence of cellular STAT1 and STAT2 . However, the inert IRF9 protein was readily converted to a constitutively active ISGF3-like activator by fusion with the C-terminal transcriptional activation domain of STAT2 or the acidic activation domain of herpesvirus VP16 . The IRF9 hybrids are targeted to endogenous ISGF3 target loci and can activate their transcription . Moreover, expression of the IRF9-STAT2 fusion can recapitulate the type I IFN biological response, producing a cellular antiviral state that protects cells from virus-induced cytopathic effects and inhibits virus replication . The antiviral state generated by regulated IRF9-STAT2 hybrid protein expression is independent of autocrine IFN signaling and inhibits both RNA and DNA viruses. EMBO J, 2003 Feb 17, 22(4), 816 - 25 Tim50, a novel component of the TIM23 preprotein translocase of mitochondria; Mokranjac D et al.; The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane . We isolated the TIM23 complex of Neurospora crassa . Besides Tim23 and Tim17, it contained a novel component, referred to as Tim50 . Tim50 spans the inner membrane with a single transmembrane segment and exposes a large hydrophilic domain in the intermembrane space . Tim50 is essential for viability of yeast . Mitochondria from cells depleted of Tim50 displayed strongly reduced import kinetics of preproteins using the TIM23 complex . Tim50 could be cross-linked to preproteins that were halted at the level of the translocase of the outer membrane (TOM complex) or spanning both TOM and TIM23 complexes . We suggest that Tim50 plays a crucial role in the transfer of preproteins from the TOM complex to the TIM23 complex through the intermembrane space. EMBO J, 2003 Feb 17, 22(4), 786 - 96 Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition; Passmore LA et al.; The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets specific cell cycle-related proteins for degradation, regulating progression from metaphase to anaphase and exit from mitosis . The APC is regulated by binding of the coactivator proteins Cdc20p and Cdh1p, and by phosphorylation . We have developed a purification strategy that allowed us to purify the budding yeast APC to near homogeneity and identify two novel APC-associated proteins, Swm1p and Mnd2p . Using an in vitro ubiquitylation system and a native gel binding assay, we have characterized the properties of wild-type and mutant APC . We show that both the D and KEN boxes contribute to substrate recognition and that coactivator is required for substrate binding . APC lacking Apc9p or Doc1p/Apc10 have impaired E3 ligase activities . However, whereas Apc9p is required for structural stability and the incorporation of Cdc27p into the APC complex, Doc1p/Apc10 plays a specific role in substrate recognition by APC-coactivator complexes . These results imply that Doc1p/Apc10 may play a role to regulate the binding of specific substrates, similar to that of the coactivators. Biochim Biophys Acta, 2003 Feb 20, 1631(1), 1 - 11 The OSBP-related proteins: a novel protein family involved in vesicle transport, cellular lipid metabolism, and cell signalling; Lehto M et al.; Proteins/genes showing high sequence homology to the mammalian oxysterol binding protein (OSBP) have been identified in a variety of eukaryotic organisms from yeast to man . The unifying feature of the gene products denoted as OSBP-related proteins (ORPs) is the presence of an OSBP-type ligand binding (LB) domain . The LB domains of OSBP and its closest homologue bind oxysterols, while data on certain other family members suggest interaction with phospholipids . Many ORPs also have a pleckstrin homology (PH) domain in the amino-terminal region . The PH domains of the family members studied in detail are known to interact with membrane phosphoinositides and play an important role in the intracellular targeting of the proteins . It is plausible that the ORPs constitute a regulatory apparatus that senses the status of specific lipid ligands in membranes, using the PH and/or LB domains, and mediates information to yet poorly known downstream machineries . Functional studies carried out on the ORP proteins in different organisms indicate roles of the gene family in diverse cellular processes including control of lipid metabolism, regulation of vesicle transport, and cell signalling events. Curr Biol, 2003 Feb 4, 13(3), 258 - 62 Ubiquitin signals protein trafficking via interaction with a novel ubiquitin binding domain in the membrane fusion regulator, Vps9p; Donaldson KM et al.; The conserved vacuolar protein-sorting (Vps) pathway controls the trafficking of proteins to the vacuole/lysosome . Both the internalization of ubiquitylated cargo from the plasma membrane and its sorting at the late endosome via the Vps pathway depend on ubiquitin (Ub) binding motifs present in trafficking regulators . Here we report that Ub controls yet a third step in the Vps pathway . Vps9p, which promotes endosomal and Golgi-derived vesicle fusion, binds directly to Ub via a Cue1p-homologous (CUE) domain . The CUE domain is structurally related to the Ub-associated (UBA) domain . In an assay for vacuolar delivery of a transmembrane receptor fused to Ub, a Ub mutation impairing interaction with Vps9p led to a cytoplasmic block in receptor trafficking . This block resembled that of a receptor fused to wild-type Ub but expressed in a vps9-null background . Strikingly, this trafficking defect caused by a mutant Ub was rescued by deletion of the Vps9p CUE domain, indicating that lack of the CUE domain renders Vps9p independent of Ub for activation in vivo . We thus provide evidence for biochemical and genetic interactions between Ub and a novel Ub binding domain in Vps9p . Ub plays a positive role, whereas the CUE domain plays both positive and negative roles in Vps9p function in trafficking. Biometals, 2003 Mar, 16(1), 175 - 84 Copper-induced trafficking of the cU-ATPases: a key mechanism for copper homeostasis; Mercer JF et al.; The Menkes protein (MNK) and Wilson protein (WND) are transmembrane, CPX-type Cu-ATPases with six metal binding sites (MBSs) in the N-terminal region containing the motif GMXCXXC . In cells cultured in low copper concentration MNK and WND localize to the transGolgi network but in high copper relocalize either to the plasma membrane (MNK) or a vesicular compartment (WND) . In this paper we investigate the role of the MBSs in Cu-transport and trafficking . The copper transport activity of MBS mutants of MNK was determined by their ability to complement a strain of Saccharomyces cerevisiae deficient in CCC2 (delta ccc2), the yeast MNK/WND homologue . Mutants (CXXC to SXXS) of MBS1, MBS6, and MBSs1-3 were able to complement delta ccc2 while mutants of MBS4-6, MBS5-6 and all six MBS inactivated the protein . Each of the inactive mutants also failed to display Cu-induced trafficking suggesting a correlation between trafficking and transport activity . A similar correlation was found with mutants of MNK in which various MBSs were deleted, but two constructs with deletion of MBS5-6 were unable to traffic despite retaining 25% of copper transport activity . Chimeras in which the N-terminal MBSs of MNK were replaced with the corresponding MBSs of WND were used to investigate the region of the molecules that is responsible for the difference in Cu-trafficking of MNK and WND . The chimera which included the complete WND N-terminus localized to a vesicular compartment, similar to WND in elevated copper . Deletions of various MBSs of the WND N-terminus in the chimera indicate that a targeting signal in the region of MBS6 directs either WND/MNK or WND to a vesicular compartment of the cell. J Biol Chem, 2003 Apr 11, 278(15), 12710 - 5 Epub 2003 Feb 04. Protein kinase CK2 phosphorylates the high mobility group domain protein SSRP1, inducing the recognition of UV-damaged DNA; Krohn NM et al.; The structure-specific recognition protein SSRP1 plays a role in transcription and replication in the chromatin context . Mediated by its C-terminal high mobility group (HMG) box domain, SSRP1 binds DNA non-sequence specifically but recognizes certain DNA structures . Using acetic acid urea polyacrylamide gel electrophoresis and mass spectrometry, we have examined the phosphorylation of maize SSRP1 by protein kinase CK2 alpha . The kinase phosphorylated several amino acid residues in the C-terminal part of the SSRP1 protein . Two phosphorylation sites were mapped in the very C-terminal region next to the HMG box domain, and about seven sites are localized within the acidic domain . Circular dichroism showed that the phosphorylation of the two C-terminal sites by CK2 alpha resulted in a structural change in the region of HMG box domain, because the negative peak of the CD spectrum at 222 nm was decreased by approximately 10% . In parallel, the phosphorylation induced the recognition of UV-damaged DNA, whereas the non-phosphorylated protein does not discriminate between UV-damaged DNA and control DNA . The affinity of CK2 alpha-phosphorylated SSRP1 for the DNA correlates with the degree of UV-induced DNA damage . Moreover, maize SSRP1 can restore the increased UV-sensitivity of a yeast strain lacking the NHP6A/B HMG domain proteins to levels of the control strain . Collectively, these findings indicate a role for SSRP1 in the UV response of eukaryotic cells. J Biol Chem, 2003 Apr 11, 278(15), 13216 - 26 Epub 2003 Feb 04. Mot1 regulates the DNA binding activity of free TATA-binding protein in an ATP-dependent manner; Darst RP et al.; Mot1 is an essential Snf2/Swi2-related Saccharomyces cerevisiae protein that binds the TATA-binding protein (TBP) and removes TBP from DNA using ATP hydrolysis . Mot1 functions in vivo both as a repressor and as an activator of transcription . Mot1 catalysis of TBP.DNA disruption is consistent with its function as a repressor, but the Mot1 mechanism of activation is unknown . To better understand the physiologic role of Mot1 and its enzymatic mechanism, MOT1 mutants were generated and tested for activity in vitro and in vivo . The results demonstrate a close correlation between the TBP.DNA disruption activity of Mot1 and its essential in vivo function . Previous results demonstrated a large overlap in the gene sets controlled by Mot1 and NC2 . Mot1 and NC2 can co-occupy TBP.DNA in vitro, and NC2 binding does not impair Mot1-catalyzed disruption of the complex . Residues on the DNA-binding surface of TBP are important for Mot1 binding and the Mot1.TBP binary complex binds very poorly to DNA and does not dissociate in the presence of ATP . However, the binary complex binds DNA well in the presence of the transition state analog ADP-AlF(4) . A model for Mot1 action is proposed in which ATP hydrolysis causes the Mot1 N terminus to displace the TATA box, leading to ejection of Mot1 and TBP from DNA. Appl Environ Microbiol, 2003 Feb, 69(2), 754 - 9 Identification of a high-affinity phosphate transporter gene in a prasinophyte alga, Tetraselmis chui, and its expression under nutrient limitation; Chung CC et al.; A high-affinity phosphate transporter gene, TcPHO, was isolated from a growth-dependent subtracted cDNA library of the marine unicellular alga Tetraselmis chui . The full-length cDNA of TcPHO obtained by 5' and 3' rapid amplification of cDNA ends was 1,993 bp long and encoded an open reading frame consisting of 610 amino acids . The deduced amino acid sequence of TcPHO exhibited 51.6 and 49.8% similarity to the amino acid sequences of PHO89 from Saccharomyces cerevisiae and PHO4 from Neurospora crassa, respectively . In addition, hydrophobicity and secondary structure analyses revealed 12 conserved transmembrane domains that were the same as those found in PHO89 and PHO4 . The expression of TcPHO mRNA was dependent on phosphate availability . With a low-phosphate treatment, the TcPHO mRNA concentration increased sharply to 2.72 fmol micro g of total RNA(-1) from day 1 to day 2 and remained at this high level from days 2 to 4 . Furthermore, rescue treatment with either phosphate or p-nitrophenyl phosphate effectively inhibited TcPHO mRNA expression . In contrast, TcPHO mRNA expression stayed at a low level (range, 0.25 to 0.28 fmol micro g of total RNA(-1)) under low-nitrate conditions . The expression pattern suggests that TcPHO can be used as a molecular probe for monitoring phosphorus stress in T . chui. Comb Chem High Throughput Screen, 2003 Feb, 6(1), 67 - 78 Design and peptide-based validation of phage display antibodies for proteomic biochips; Stich N et al.; To validate potential application of phage display-antibody arrays for high-throughput screening on a novel proteomics biochip, we examined the epitopes versus the full protein of glucose-6-phosphate-dehydrogenase (G6PD) from yeast . In a predictive approach, we used the Hopp-Woods method and compared the results with antibodies directed against the entire enzyme . In total, 16 peptides of a length of 11 amino acids each fulfilling the desired criteria were identified and synthesized . Subsequently, antibodies against G6PD were raised using a phage display library . Selective interaction of the antibodies with certain peptides facilitated the identification of epitopes predicted by the hydropathic profile . The setup was adapted to a novel biochip system based on surface-enhanced absorption for direct CCD-camera based screening. Comb Chem High Throughput Screen, 2003 Feb, 6(1), 37 - 50 DBU-assisted cyclorelease elimination: combinatorial synthesis and gamma-glutamyl cysteine synthetase and glutathione-S-transeferase modulatory effect of C-nucleoside analogs; Mishra RC et al.; A combinatorial library of 60C- nucleoside analogs was synthesized by sequential coupling of building blocks followed by cyclative cleavage with DBU in an efficient manner . Only DMSO soluble compounds were tested for their modulatory effect against filarial gamma-glutamyl cysteine synthetase (gamma-GCase) and glutathione-S-transeferases (GSTs) . Several compounds were found to be weak inhibitors of filarial gamma-GCase, whereas, most of them stimulated filarial GSTs. Curr Drug Targets Infect Disord, 2003 Mar, 3(1), 77 - 91 Chitin synthesis as target for antifungal drugs; Ruiz-Herrera J et al.; Human mycoses have become a threat to health world-wide . Unfortunately there are only a limited number of antimycotic drugs in use . Promising targets for drugs specific against fungi are those affecting chitin synthesis . Chitin is absent in vertebrates, and is essential for fungal wall integrity . A thorough knowledge of the mechanism of chitin synthesis is required to design specific inhibitors . We review here our current understanding of the process, and the most promising drugs that inhibit it . Chitin is made by chitin synthases requiring specific microvesicles, the chitosomes, for intracellular transport . Fungi contain several chitin synthases, some of which may be essential at a certain stage . This phenomenon is important to take into account for drug design . The most widely studied chitin synthase inhibitors are polyoxins and nikkomycins that probably bind to the catalytic site of chitin synthases . These are not equally susceptible to the drugs . In Saccharomyces cerevisiae the order of sensitivity is: Chs3p>Chs1p>Chs2p . Main problems for their succesful use in vivo are: low permeability, and different susceptibility of fungal species, and variable responses in animal models . Chemical modifications have been proposed to make more potent derivatives . Other synthetic or natural compounds are also promising as possible inhibitors, but their properties are less well known . Rational drug design has proceeded only on the basis of existing inhibitors, because the structure of the active site of chitin synthase is unknown . Undoubtedly, determination of this, and the biosynthetic mechanism will reveal unexpected drug targets in the future. Biotechnol Bioeng, 2003 Apr 5, 82(1), 38 - 46 Inhibition of an activated Ras protein with genetically selected peptide aptamers; Kurtz SE et al.; Mutant alleles of Ras maintain an activated, GTP-bound conformation and relay mitogenic signals that cannot be turned off . A genetic selection in Saccharomyces cerevisiae was used to identify peptide aptamers that suppress the growth arrest phenotype of an activated Ras allele . Peptide aptamers were expressed as C-terminal fusions to glutathione-S-transferase . Modifications that alter the coding capacity of the peptide aptamer indicate it is necessary for Ras2-Val19 suppression . Aptamer expression also reduces the elevated levels of cAMP and suppresses the heat shock sensitivity characteristic of Ras-activated yeast cells . The peptide aptamer retains suppressor activity when fused to thioredoxin . The peptide aptamer expression strategy described here indicates that aptamers presented as unconstrained peptides have functional capacity in vivo . Planta, 2003 Feb, 216(4), 604 - 13 Epub 2002 Nov 30. Effects of co-expressing the plant CDK inhibitor ICK1 and D-type cyclin genes on plant growth, cell size and ploidy in Arabidopsis thaliana; Zhou Y et al.; The cyclin-dependent kinase (CDK) plays a crucial role in regulating the cell cycle of eukaryotic organisms including plants . From previous studies, it is known that ICK1, the first plant CDK inhibitor identified in Arabidopsis plants, interacts with Arath;CycD3;1 (CycD3) and Arath;CDKA;1 (Cdc2a) . Overexpression of ICK1 has major effects on cell division, plant growth, and morphology . In this study, approaches were taken to determine the effects on transgenic 35S::ICK1 Arabidopsis plants of introducing another gene that could potentially modulate the activity of ICK1 . F1 plants were obtained by crossing 35S::ICK1 plants with wild type (Wt) and transgenic plants expressing 35::GUS, 35S::CycD3, 35S::CycD2, or 35S::antiICK1 ( antiICK1 refers to antisense- ICK1) . The major effects on plant growth and morphology observed in the 35S::ICK1 plants were partially reversed in the F1 plants from the crosses {35S::ICK1 x 35S::CycD2} and {35S::ICK1 x 35S::CycD3}, and completely restored in the F1 plants from the cross {35S::ICK1 x 35S::antiICK1} . This observation was further supported by the results of ploidy analysis and structural characterization . Overexpression of CycD2 and CycD3 had the opposite effect on leaf cell size to the overexpression of ICK1 . In addition, in ICK1-overexpressing plants, the CycD2 and CycD3 transcript levels increased, indicating a possible feedback regulation . The present results demonstrate that the interactions between ICK1 and D-type cyclins previously observed by the yeast two-hybrid and in vitro techniques are biologically relevant . These results illustrate the possibility of modifying plant growth and architecture dynamically by adjusting the levels of positive and negative cell-cycle regulators. J Biol Chem, 2003 Apr 11, 278(15), 12696 - 702 Epub 2003 Feb 04. Phosphorylation of serine 1106 in the catalytic domain of topoisomerase II alpha regulates enzymatic activity and drug sensitivity; Chikamori K et al.; Topoisomerases alter DNA topology and are vital for the maintenance of genomic integrity . Topoisomerases I and II are also targets for widely used antitumor agents . We demonstrated previously that in the human leukemia cell line, HL-60, resistance to topoisomerase (topo) II-targeting drugs such as etoposide is associated with site-specific hypophosphorylation of topo II alpha . This effect can be mimicked in sensitive cells treated with the intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) . Here we identify Ser-1106 as a major phosphorylation site in the catalytic domain of topo II alpha . This site lies within the consensus sequence for the acidotrophic kinases, casein kinase I and casein kinase II . Mutation of serine 1106 to alanine (S1106A) abrogates phosphorylation of phosphopeptides that were found to be hypophosphorylated in resistant HL-60 cells or sensitive cells treated with BAPTA-AM . Purified topo II alpha containing a S1106A substitution is 4-fold less active than wild type topo II alpha in decatenating kinetoplast DNA and also exhibits a 2-4-fold decrease in the level of etoposide-stabilized DNA cleavable complex formation . Saccharomyces cerevisiae (JN394t2-4) cells expressing S1106A mutant topo II alpha protein are more resistant to the cytotoxic effects of etoposide or amsacrine . These results demonstrate that Ca(2+)-regulated phosphorylation of Ser-1106 in the catalytic domain of topo II alpha modulates the enzymatic activity of this protein and sensitivity to topo II-targeting drugs. J Agric Food Chem, 2003 Feb 12, 51(4), 1077 - 80 Sequestering ability of butylated hydroxytoluene, propyl gallate, resveratrol, and vitamins C and E against ABTS, DPPH, and hydroxyl free radicals in chemical and biological systems; Soares DG et al.; The antioxidant capacity of butylated hydroxytoluene (BHT; 2,6-di-tert-butyl-p-cresol), propyl gallate (3,4,5-trihydroxybenzoic acid n-propyl ester), resveratrol (trans-3,4',5-trihydroxystilbene), and vitamins C (l-ascorbic acid) and E {(+)-alpha-tocopherol} was studied in chemical and biological systems . The chemical assays evaluated the capacity of these antioxidants to sequester 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS.) and 1,1 diphenyl-2-picrylhydrazyl (DPPH.) . A new colorimetric method to determine hydroxyl radical scavenging is also described . The biological tests use the eucaryotic cells of Saccharomyces cerevisiae treated with the antioxidants in the presence of the stressing agents apomorphine, hydrogen peroxide, and paraquat dichloride (methylviologen; 1,1'-dimethyl-4,4'-bipyridinium dichloride) . The results in chemical systems showed that all of the antioxidants were able to significantly inhibit the oxidation of beta-carotene by hydroxyl free radicals . The assays in yeast showed that the antioxidant activity of the tested compounds depended on the stressing agent used and the mechanism of action of the antioxidant. Cell Mol Life Sci, 2002 Dec, 59(12), 2144 - 54 Structural features of prions explored by sequence analysis . II . A PrP(Sc) model; Mornon JP et al.; Prion diseases are neurodegenerative disorders associated with a conformational conversion of the prion PrP protein, in which the beta strand content increases and that of the a helix decreases . However, the structure of the pathogenous form PrP(Sc), occurring after conformational conversion of the normal cellular form PrP(C), is not yet known . From sequence analysis, we have previously proposed that helix H2 of the prion PrP(C) structure might be a key region for this structural conversion . More recently, we identified the TATA box-binding protein fold as a putative scaffold that may locally satisfy the predicted secondary-structure organisation of PrP(Sc) . In the present analysis, we detail the schematic construction of PrP(Sc) monomeric and dimeric models, based on this hypothesis . These models are globally compatible with available data and therefore may provide further insights into the structurally and functionally elusive PrP protein . Some comments are also devoted to a comparison of the yeast Ure2p prion and animal prions. Planta Med, 2003 Jan, 69(1), 94 - 6 A new diepoxy-ent-kauranoid, rugosinin, from Isodon rugosus; Abbaskhan A et al.; A new diterpenoid, rugosinin (1), isolated from Isodon rugosus, with absolute configuration was proved by single-crystal X-ray diffraction analysis, to be the member of a rare class of C-20/C-7 and C-20/C-14 diepoxy- ent-kauranoids . Effusanin A (2), effusanin B (3), effusanin E (4), lasiokaurin (5) and oridonin (6) were found as known constituents of the genus Isodon with C-20/C-7 epoxy function . These compounds have exhibited DNA-damaging activity in assay which employed DNA-repair deficient (RAD 52Y) and repair proficient (RAD +) yeast strains. J Biol Chem, 2003 Apr 18, 278(16), 14029 - 36 Epub 2003 Feb 03. A novel function of Rad54 protein . Stabilization of the Rad51 nucleoprotein filament; Mazin AV et al.; Homologous recombination is important for the repair of double-stranded DNA breaks in all organisms . Rad51 and Rad54 proteins are two key components of the homologous recombination machinery in eukaryotes . In vitro, Rad51 protein assembles with single-stranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of homologous recombination . Rad54 protein interacts with this Rad51 nucleoprotein filament and stimulates its DNA pairing activity, suggesting that Rad54 protein is a component of the nucleoprotein complex involved in the DNA homology search . Here, using physical criteria, we demonstrate directly the formation of Rad54-Rad51-DNA nucleoprotein co-complexes that contain equimolar amounts of each protein . The binding of Rad54 protein significantly stabilizes the Rad51 nucleoprotein filament formed on either single-stranded DNA or double-stranded DNA . The Rad54-stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions . Thus, the co-assembly of an interacting partner with the Rad51 nucleoprotein filament represents a novel means of stabilizing the biochemical entity central to homologous recombination, and reveals a new function of Rad54 protein. J Biol Chem, 2003 Apr 18, 278(16), 13775 - 83 Epub 2003 Feb 03. Biochemical properties of mammalian neutral sphingomyelinase 2 and its role in sphingolipid metabolism; Marchesini N et al.; Neutral sphingomyelinase (N-SMase) is one of the key enzymes involved in the generation of ceramide; however, the gene(s) encoding for the mammalian N-SMase is still not well defined . Previous studies on the cloned nSMase1 had shown that the protein acts primarily as lyso-platelet-activating factor-phospholipase C . Recently the cloning of another putative N-SMase, nSMase2, was reported . In this study, biochemical characterization of the mouse nSMase2 was carried out using the overexpressed protein in yeast cells in which the inositol phosphosphingolipid phospholipase C (Isc1p) was deleted . N-SMase activity was dependent on Mg(2+) and was activated by phosphatidylserine and inhibited by GW4869 . The ability of nSMase2 to recognize endogenous sphingomyelin (SM) as substrate was investigated by overexpressing nSMase2 in MCF7 cells . Mass measurements showed a 40% decrease in the SM levels in the overexpressor cells, and labeling studies demonstrated that nSMase2 accelerated SM catabolism . Accordingly, ceramide measurement showed a 60 +/- 15% increase in nSMase2-overexpressing cells compared with the vector-transfected MCF7 . The role of nSMase2 in cell growth was next investigated . Stable overexpression of nSMase2 resulted in a 30-40% decrease in the rate of growth at the late exponential phase . Moreover, tumor necrosis factor induced approximately 50% activation of nSMase2 in MCF7 cells overexpressing the enzyme, demonstrating that nSMase2 is a tumor necrosis factor-responsive enzyme . In conclusion, these results 1) show that nSMase2 is a structural gene for nSMase, 2) suggest that nSMase2 acts as a bona fide N-SMase in cells, and 3) implicate nSMase2 in the regulation of cell growth and cell signaling. J Cell Biol, 2003 Feb 3, 160(3), 303 - 11 The intramitochondrial dynamin-related GTPase, Mgm1p, is a component of a protein complex that mediates mitochondrial fusion; Wong ED et al.; A balance between fission and fusion events determines the morphology of mitochondria . In yeast, mitochondrial fission is regulated by the outer membrane-associated dynamin-related GTPase, Dnm1p . Mitochondrial fusion requires two integral outer membrane components, Fzo1p and Ugo1p . Interestingly, mutations in a second mitochondrial-associated dynamin-related GTPase, Mgm1p, produce similar phenotypes to fzo1 and ugo cells . Specifically, mutations in MGM1 cause mitochondrial fragmentation and a loss of mitochondrial DNA that are suppressed by abolishing DNM1-dependent fission . In contrast to fzo1ts mutants, blocking DNM1-dependent fission restores mitochondrial fusion in mgm1ts cells during mating . Here we show that blocking DNM1-dependent fission in Deltamgm1 cells fails to restore mitochondrial fusion during mating . To examine the role of Mgm1p in mitochondrial fusion, we looked for molecular interactions with known fusion components . Immunoprecipitation experiments revealed that Mgm1p is associated with both Ugo1p and Fzo1p in mitochondria, and that Ugo1p and Fzo1p also are associated with each other . In addition, genetic analysis of specific mgm1 alleles indicates that Mgm1p's GTPase and GTPase effector domains are required for its ability to promote mitochondrial fusion and that Mgm1p self-interacts, suggesting that it functions in fusion as a self-assembling GTPase . Mgm1p's localization within mitochondria has been controversial . Using protease protection and immuno-EM, we have shown previously that Mgm1p localizes to the intermembrane space, associated with the inner membrane . To further test our conclusions, we have used a novel method using the tobacco etch virus protease and confirm that Mgm1p is present in the intermembrane space compartment in vivo . Taken together, these data suggest a model where Mgm1p functions in fusion to remodel the inner membrane and to connect the inner membrane to the outer membrane via its interactions with Ugo1p and Fzo1p, thereby helping to coordinate the behavior of the four mitochondrial membranes during fusion. J Cell Biol, 2003 Feb 3, 160(3), 289 - 90 LIS1 at the microtubule plus end and its role in dynein-mediated nuclear migration; Xiang X; The cytoplasmic dynein complex and its accessory dynactin complex are involved in many cellular activities including nuclear migration in fungi (for review see Karki and Holzbaur, 1999) . LIS1, the product of a causal gene for human lissencephaly (smooth brain), has also been implicated in dynein function based on studies in fungi and more recent studies in higher eukaryotic systems (for review see Gupta et al., 2002) . Exactly how LIS1 may regulate the behavior of cytoplasmic dynein in various organisms is a fascinating question . In this issue, Lee et al . (2003) describe important new findings in Saccharomyces cerevisiae regarding the role of LIS1 (Pac1) in dynein-mediated nuclear migration. Genome Res, 2003 Feb, 13(2), 294 - 307 Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH; Lage JM et al.; Structural genetic alterations in cancer often involve gene loss or gene amplification . With the advent of microarray approaches for the analysis of the genome, as exemplified by array-CGH (Comparative Genomic Hybridization), scanning for gene-dosage alterations is limited only by issues of DNA microarray density . However, samples of interest to the pathologist often comprise small clusters of just a few hundred cells, which do not provide sufficient DNA for array-CGH analysis . We sought to develop a simple method that would permit amplification of the whole genome without the use of thermocycling or ligation of DNA adaptors, because such a method would lend itself to the automated processing of a large number of tissue samples . We describe a method that permits the isothermal amplification of genomic DNA with high fidelity and limited sequence representation bias . The method is based on strand displacement reactions that propagate by a hyperbranching mechanism, and generate hundreds, or even thousands, of copies of the genome in a few hours . Using whole genome isothermal amplification, in combination with comparative genomic hybridization on cDNA microarrays, we demonstrate the ability to detect gene losses in yeast and gene dosage imbalances in human breast tumor cell lines . Although sequence representation bias in the amplified DNA presents potential problems for CGH analysis, these problems have been overcome by using amplified DNA in both control and tester samples . Gene-dosage alterations of threefold or more can be observed with high reproducibility with as few as 1000 cells of starting material. Biochem Biophys Res Commun, 2003 Feb 7, 301(2), 572 - 7 The effect of oxidative stress on histone acetylation and IL-8 release; Tomita K et al.; Acetylation of histone residues regulates the expression of inflammatory genes and is controlled by the activities of histone acetyltransferases (HAT) and histone deacetylases (HDAC) . Analysis of histone acetylation in human cells is limited by the large numbers needed to perform activity assays or Western blotting . We have used flow cytometry to investigate changes in HAT and HDAC activities at the single cell level and to investigate the effect of hydrogen peroxide (H(2)O(2)) on histone H4 acetylation and cell-cycle progression . Using an anti-acetylated histone H4 antibody we show that H(2)O(2) induced a time-dependent increase in histone acetylation that was maintained for 12h . This was associated with increased IL-8 production . H(2)O(2) also affected cell-cycle progression . HAT activity was found to be highest in G2/M and equivalent in G0/G1 and S phases of the cell cycle . These data show that detection of acetylated histone residues at the single cell level using FACs may be a powerful new tool for the analysis of modulation of cell proliferation and gene transcription. Biochem Biophys Res Commun, 2003 Feb 14, 301(3), 633 - 40 Global insights into protein complexes through integrated analysis of the reliable interactome and knockout lethality; Saito R et al.; We performed an integrated computational analysis of data derived from a comprehensive set of protein-protein interactions (interactome) and a phenotype dataset on lethality in Saccharomyces cerevisiae . For the analysis, we selected reliable interactome data using our previous 'interaction generality,' a computational approach to assess reliability of interactions . Those efforts gave clear evidence that proteins with lethal phenotypes in knockout studies (lethal proteins) may interact with each other to form functional protein complexes to perform their cellular roles . However, our analysis indicates that interactions between lethal proteins are rather restricted to the same cellular pathway or function, and it is quite unlikely that they interact with other lethal proteins functioning in different cellular roles . Furthermore, our results allowed us predictions on the functions of thus far uncharacterized lethal proteins with an estimated 93% accuracy . Thus, the analysis described in here can provide global insights into the biological features of the protein complexes. Can J Physiol Pharmacol, 2002 Dec, 80(12), 1167 - 72 Models of depressed hepatic mrp2 activity reveal bromosulphophthalein-sensitive passive K+ flux; Li Q et al.; Bile acid independent flow composes up to 40% of hepatic bile secretory capacity . Apical (canalicular) efflux of non-bile-acid organic anions provides the major osmotic driving force for bile acid independent flow . Organic anion accumulation in the hepatocyte is accompanied by increases in both K+ conductance in isolated hepatocytes and passive K+ flux in the perfused rat liver, which are indicative of K+ channel activation . We used two models of disrupted canalicular anion transport to test whether organic anion stimulated K+ efflux occurs independently of anion excretion . In both wild type (wt) and mrp2 mutant (transport minus, tr-) rat liver, bromosulfophthalein (BSP; 0.5 mM) caused a reversible increase in K+ flux that (i) was outwardly directed with low external K+ and (ii) depended upon the electrochemical potential for K+ . K+ efflux from wt livers of both sexes was about 1.5 times larger than that from tr- livers . Further, K+ release from female rat livers was about three times higher than that from male livers, independent of phenotype . Two transcripts of the rat hepatocyte K+ channel (Kir4.2) were expressed in hepatocytes of all rats . The results demonstrate that BSP stimulates basolateral (sinusoidal) K+ channels independently of its canalicular excretion, revealing an early event in BAIF and suggesting that Kir4.2 may mediate BSP-sensitive K+ flux. World Health Organ Tech Rep Ser, 2002, 909, i - x, 1-171, back cover Evaluation of certain food additives and contaminants; Tor signalling in bugs et al.; Division of Biochemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, SwitzerlandTOR--a highly conserved atypical protein kinase and the 'target of rapamycin', an immunosuppressant and anti-cancer drug--controls cell growth . TOR controls the growth of proliferating yeast, fly and mammalian cells in response to nutrients . Recent findings, however, indicate that TOR also controls the growth of non-proliferating cells, such as neurons and muscle cells . Furthermore, TOR, by associating with regulatory proteins and inhibiting phosphatases, controls the activity of multiphosphorylated effectors. J Lipid Res, 2003 Mar, 44(3), 640 - 4 Epub 2002 Dec 01. A novel HPLC-based method to diagnose peroxisomal D-bifunctional protein enoyl-CoA hydratase deficiency; Gloerich J et al.; D-bifunctional protein (D-BP) plays an indispensable role in peroxisomal beta-oxidation, and its inherited deficiency in humans is associated with severe clinical abnormalities . Three different subtypes of D-BP deficiency can be distinguished: 1) a complete deficiency of D-BP (type I), 2) an isolated D-BP enoyl-CoA hydratase deficiency (type II), and 3) an isolated D-BP 3-hydroxyacyl-CoA dehydrogenase deficiency (type III) . In this study, we developed a method to measure D-BP dehydrogenase activity independent of D-BP hydratase (D-BP HY) activity to distinguish between D-BP deficiency type I and type II, which until now was only possible by mutation analysis . For this assay, the hydratase domain of D-BP was expressed in the yeast Saccharomyces cerevisiae . After a coincubation of yeast homogenate expressing D-BP HY with fibroblast homogenate of patients using the enoyl-CoA ester of the bile acid intermediate trihydroxycholestanoic acid as substrate, D-BP dehydrogenase activity was measured . Fibroblasts of patients with a D-BP deficiency type II displayed D-BP dehydrogenase activity, whereas type I and type III patients did not . This newly developed assay to measure D-BP dehydrogenase activity in fibroblast homogenates provides a quick and reliable method to assign patients with deficient D-BP HY activity to the D-BP deficiency subgroups type I or type II. J Biol Chem, 2003 Apr 11, 278(15), 12805 - 12 Epub 2003 Jan 31. Molecular evolution by change of function . Alkaloid-specific homospermidine synthase retained all properties of deoxyhypusine synthase except binding the eIF5A precursor protein; Ober D et al.; Deoxyhypusine synthase participates in the post-translational activation of the eukaryotic initiation factor 5A (eIF5A) . The enzyme transfers the aminobutyl moiety of spermidine to a specific lysine residue in the eIF5A precursor protein, i.e . eIF5A(lys) . Homospermidine synthase catalyzes an analogous reaction but uses putrescine instead of eIF5A(lys) as substrate yielding the rare polyamine homospermidine as product . Homospermidine is an essential precursor in the biosynthesis of pyrrolizidine alkaloids, an important class of plant defense compounds against herbivores . Sequence comparisons of the two enzymes indicate an evolutionary origin of homospermidine synthase from ubiquitous deoxyhypusine synthase . The two recombinant enzymes from Senecio vernalis were purified, and their properties were compared . Protein-protein binding and kinetic substrate competition studies confirmed that homospermidine synthase, in comparison to deoxyhypusine synthase, lost the ability to bind the eIF5A(lys) to its surface . The two enzymes show the same unique substrate specificities, catalyze the aminobutylation of putrescine with the same specific activities, and exhibit almost identical Michaelis kinetics . In conclusion, homospermidine synthase behaves like a deoxyhypusine synthase that lost its major function (aminobutylation of eIF5A precursor protein) but retained unaltered its side activity (aminobutylation of putrescine) . It is suggested as having evolved from deoxyhypusine synthase by gene duplication and being recruited for a new function. J Neurochem, 2003 Feb, 84(4), 829 - 39 Cloning and characterization of a novel variant (mM-rdgBbeta1) of mouse M-rdgBs, mammalian homologs of Drosophila retinal degeneration B gene proteins, and its mRNA localization in mouse brain in comparison with other M-rdgBs; Takano N et al.; We report the cloning, characterization and localization in the brain of a novel isoform termed mM-rdgBbeta1 (mouse type of mammalian retinal degeneration Bbeta1 protein) in comparison with the localization of three known mammalian homologs (M-rdgBbeta, M-rdgB1, M-rdgB2) . mM-rdgBbeta1 cDNA contains a sequence of 119 bp as a form of insertion in the open reading frame of the known mM-rdgBbeta, and encodes a protein of 269 amino acids with a calculated molecular mass of 31.7 kDa, different from the molecular mass of 38.3 kDa of mM-rdgBbeta . It also contains a phosphatidylinositol transfer protein (PITP)-like domain similar to the known three homologs, as well as D-rdgB . The recombinant mM-rdgBbeta1 protein shows the specific binding activity to phosphatidylinositol but not to other phospholipids . This novel molecule is localized not only in the cytoplasm but also in the nucleus, different from the cytoplasmic localization of mM-rdgBbeta . In in situ hybridization analysis, the gene expression for mM-rdgBbeta1 in the brain, though weak, is rather confined to the embryonic stage, different from wider expression of mM-rdgBbeta in the gray matters of pre- and post-natal brains . Taken together, mM-rdgBbeta1 is suggested to play a role in the phosphoinositide-mediated signaling in the neural development. Plast Reconstr Surg, 2003 Feb, 111(2), 763 - 72; discussion 773-4 Fate of donor cells in vascularized bone grafts: identification of systemic chimerism by the polymerase chain reaction; Muramatsu K et al.; Systemic chimerism, or the movement of cells from a transplanted tissue into host organs, is a phenomenon known to occur in association with development of immunological tolerance in allotransplantation . However, little is known about the fate and movement of cells into or out of autogenous free tissue transfers, including vascularized bone grafts . The purpose of this study was to identify systemic chimerism in vascularized bone grafts by transplantation of a vascularized tibiofibular graft from isogenous (inbred) male Lewis rats to female recipients . Donor (male) cells could be identified in the recipient (female) tissues by semiquantitative polymerase chain reaction analysis for a Y chromosome-specific DNA sequence . Chimerism was assessed at 1, 12, 18, and 24 weeks after transplantation . Competitive polymerase chain reaction study using the specific primers for a Y-chromosome marker ( gene) and an autosomal gene (GAPDH) allowed detection of small amounts of male cells in a large pool of female cells and measurement of their relative proportions as a function of time . Of 19 nonimmunosuppressed recipients, nine animals (47 percent) showed low-level chimerism (<0.1 percent) in the peripheral blood . Nine (47 percent), three (16 percent), and two (11 percent) recipients showed high-level chimerism (>1 percent) in the spleen, liver, and thymus, respectively, at final assessment . Donor cells were detected in all bone grafts and in six contralateral tibial bones (i.e., 67 percent of sampled contralateral tibial bones) at 18 and 24 weeks after transplantation . Twenty-four recipients were immunosuppressed with FK506 (tacrolimus) to suppress reaction to a minor histocompatibility barrier present on the Y chromosome . In this group, 14 animals (58 percent) showed low-level chimerism in peripheral blood and 12 (50 percent), eight (33 percent), and one (4 percent) recipients showed high-level chimerism in the spleen, thymus, and liver, respectively . Transplanted cells were detected in nine contralateral tibial bones (i.e., 60 percent of sampled contralateral tibial bones) at 12 and 18 weeks after surgery . The results indicate that polymerase chain reaction for the Y chromosome is a useful tool for differentiating between donor and recipient cell populations experimentally using sex-mismatched tissues in a rat model . This study demonstrated that systemic chimerism occurs after successful vascularized bone transplantation . Transplanted cells not only survive in the graft but also gradually migrate into the recipient's body. Science, 2003 Feb 21, 299(5610), 1231 - 5 Epub 2003 Jan 30. Spontaneous cell polarization through actomyosin-based delivery of the Cdc42 GTPase; Wedlich-Soldner R et al.; Cell polarization can occur in the absence of any spatial cues . To investigate the mechanism of spontaneous cell polarization, we used an assay in yeast where expression of an activated form of Cdc42, a Rho-type guanosine triphosphatase (GTPase) required for cell polarization, could generate cell polarity without any recourse to a preestablished physical cue . The polar distribution of Cdc42 in this assay required targeted secretion directed by the actin cytoskeleton . A mathematical simulation showed that a stable polarity axis could be generated through a positive feedback loop in which a stochastic increase in the local concentration of activated Cdc42 on the plasma membrane enhanced the probability of actin polymerization and increased the probability of further Cdc42 accumulation to that site. J Biol Chem, 2003 Apr 4, 278(14), 12530 - 6 Epub 2003 Jan 29. Nuclear localization of the cell cycle regulator CDH1 and its regulation by phosphorylation; Zhou Y et al.; The anaphase-promoting complex activated by CDC20 and CDH1 is a major ubiquitination system that controls the destruction of cell cycle regulators . Exactly how ubiquitination is regulated in time and space is incompletely understood . Here we report on the cell cycle-dependent localization of CDH1 and its regulation by phosphorylation . CDH1 localizes dynamically to the nucleus during interphase and to the centrosome during metaphase and anaphase . The nuclear accumulation of CDH1 correlates with a reduction in the steady-state amount of cyclin A, but not of cyclin E . A nuclear localization signal conserved in various species was identified in CDH1, and it sufficiently targets green fluorescent protein to the nucleus . Interestingly, a CDH1-4D mutant mimicking the hyperphosphorylated form was constitutively found in the cytoplasm . In further support of the notion that phosphorylation inhibits nuclear import, the nuclear localization signal of CDH1 with two phospho-accepting serine/threonine residues changed into aspartates was unable to drive heterologous protein into the nucleus . On the other hand, abolition of the cyclin-binding ability of CDH1 has no influence on its nuclear localization . Taken together, our findings document the phosphorylation-dependent localization of CDH1 in vertebrate cells. FEBS Lett, 2003 Jan 30, 535(1-3), 77 - 81 Ubiquitin binding proteins protect ubiquitin conjugates from disassembly; Hartmann-Petersen R et al.; As a step in their turnover proteins in eukaryotic cells are coupled to a small protein, ubiquitin, before they are recognised by 26S proteasomes and degraded . However, cells also contain many deubiquitinating enzymes, which can rescue proteins by cleaving off the ubiquitin chains . Here we report that three ubiquitin binding proteins, Rhp23, Dph1 and Pus1, from fission yeast can protect multiubiquitin conjugates against deubiquitination . This protection depends on the ubiquitin binding domains and may promote degradation of ubiquitinated proteins. Lancet, 2003 Jan 25, 361(9354), 301 - 7 A new subtype of autosomal dominant diabetes attributable to a mutation in the gene for sulfonylurea receptor 1; Huopio H et al.; BACKGROUND: ATP-sensitive potassium (KATP) channels are major regulators of glucose-induced insulin secretion in pancreatic beta cells . We have described a dominant heterozygous mutation--E1506K--in the sulfonylurea receptor 1 (SUR1) gene (ABCC8) in a Finnish family, which leads to congenital hyperinsulinaemia due to reduction of K(ATP)-channel activity . We aimed to characterise glucose metabolism in adults heterozygous for the E1506K mutation . METHODS: Glucose tolerance was assessed by an oral glucose tolerance test, insulin secretion by the intravenous glucose tolerance test and hyperglycaemic clamp, and insulin sensitivity by hyperinsulinaemic euglycaemic clamp in 11 people heterozygous for the E1506K mutation and 19 controls . FINDINGS: Four people who were heterozygous for the SUR1 E1506K mutation had diabetes, five had impaired glucose tolerance, one had impaired fasting glucose, and one had normal glucose tolerance . Although glucose-induced, first-phase insulin secretion was normal in children younger than 10 years of age who were heterozygous for the SUR1 E1506K mutation (n=2; 66 and 334 pmol/L), it fell rapidly after puberty (n=3; 12-32 pmol/L), and was almost completely lost in adulthood (n=11; 12-32 pmol/L) . Furthermore, these heterozygous people had a substantial reduction in maximum glucose-stimulated insulin secretion during hyperglycemic clamp (carriers without diabetes 422 pmol/L; carriers with diabetes 97 pmol/L) . By contrast, insulin sensitivity (M/I value) was normal in carriers of the E1506K mutation who did not have diabetes and was reduced by 15% in those who were heterozygous with diabetes (0.07 in those without diabetes and 0.05 in those with the disorder; not significantly different from controls) . INTERPRETATION: Heterozygous E1506K substitution in the SUR1 gene causes congenital hyperinsulinism in infancy, loss of insulin secretory capacity in early adulthood, and diabetes in middle-age . This variant represents a new subtype of autosomal dominant diabetes. Trends Cell Biol, 2003 Feb, 13(2), 59 - 60 Vesicle budding: a coat for the COPs; Haucke V; Although vesicular transport in eukaryotic cells involves a number of different carriers, one common feature is that most of them use small GTPases to direct coat assembly at the donor membrane . COPII coated vesicles bud from the endoplasmic reticulum to selectively export secretory cargo en route to the Golgi complex . Vesicle formation involves the stepwise recruitment of the small GTPase Sar1 and two large heterodimeric complexes Sec23-Sec24 and Sec13-Sec31 to the membrane . A new structural study now provides breathtaking molecular insights into the formation of the Sec23-Sec24-Sar1 pre-budding complex and into COPII coat assembly. Gene, 2003 Jan 16, 303, 47 - 54 A novel nuclear protein, Twa1, and Muskelin comprise a complex with RanBPM; Umeda M et al.; A truncated human RanBPM has been isolated as a protein binding to Ran, Ras-like nuclear small GTPase . Full-sized human RanBPM cDNA which was recently isolated, was found to encode a protein of 90 kDa which comprises a large protein complex . Consistent with this finding, several proteins were found to be co-precipitated with RanBPM by immunoprecipitation analysis . Accordingly, in the present study, we screened the human cDNA library by the two-hybrid method using RanBPM cDNA as bait . One novel protein designated as Twa1 (Two hybrid associated protein No . 1 with RanBPM), and two known proteins, a human homologue (hMuskelin) of mouse Muskelin and HSMpp8 were isolated repeatedly . Twa1 was well conserved through evolution and was localized within the nucleus . Interestingly, in addition to Muskelin and RanBPM, Twa1 was found to possess the LisH-CTLH motif which is detected in proteins involved in microtubule dynamics, cell migration, nucleokinesis and chromosome segregation . These functions overlap with functions suggested for the RanGTPase cycle . Immunoprecipitation and gel-filtration analyses indicated that both Twa1 and hMuskelin did indeed comprise a protein complex with RanBPM . Taken together with the fact that RanBPM interacts with Ran, our present findings suggested that there is an as yet uncovered function of the RanGTPase cycle. Gene, 2003 Jan 16, 303, 35 - 46 Identification and characterization of the novel centrosome-associated protein CCCAP; Kenedy AA et al.; We have cloned and characterized the cDNA, expression pattern, and subcellular localization of the human and murine orthologs of the centrosomal colon cancer autoantigen protein (CCCAP) . We identified both the transcriptional start site of murine CCCAP (mCCCAP) and its TATA-less promoter within BAC genomic clones of the mCCCAP 5' region . The mCCCAP transcript is ubiquitously present in mouse tissues, but at very low copy number . The 2151 bp open reading frame of mCCCAP encodes an 83 kDa protein that possesses a large C-terminal coiled-coil domain, which is able to homo-oligomerize in the yeast 2-hybrid system . Endogenous mCCCAP localizes to the centrosomes of murine BALB/c 3T3 fibroblasts during both interphase and mitosis . This centrosomal localization was not disrupted by nocodazole-induced depolymerization of the microtubule cytoskeleton, suggesting that mCCCAP is an integral component of the centrosome rather than simply a microtubule-associated protein . We also cloned human CCCAP (hCCCAP) . The 2139 bp open reading frame of hCCCAP encodes an 82.5 kDa protein that is 71% identical to mCCCAP at the amino acid level and has the same predicted secondary structure . Ectopically expressed full-length hCCCAP in human U2-osteosarcoma cells also displayed centrosomal localization during interphase and mitosis . This pattern of localization was abolished by truncations of the N- and C-terminus of the protein . We further discovered that the C-terminal portion of hCCCAP is identical to the human colon cancer autoantigen NY-CO-8 (Human Gene Nomenclature symbol SDCCAG8). Genes Cells, 2003 Jan, 8(1), 41 - 50 Artificial recruitment of certain Mediator components affects requirement of basal transcription factor IIE; Sakurai H et al.; BACKGROUND: Basal transcription factors are essential for RNA polymerase II (RNAPII)-catalysed transcription of many but not all the mRNA-encoding genes in vivo as well as in vitro . For example, copper-inducible transcription of the copper metallothionein gene CUP1 occurs independently of basal factor TFIIE in budding yeast . To gain insight into the mechanism by which the requirement for TFIIE is bypassed, we artificially recruited certain constituents of Mediator, a large protein complex transmitting signals from various activators to the RNAPII machinery, to the CUP1 promoter by protein fusions with Ace1, the copper-inducible activator . RESULTS: Fusions with Med2 or Pgd1 activated CUP1 independently of TFIIE . Surprisingly, fusions with neither Srb5 nor Med9 circumvented TFIIE requirement for the CUP1 activation . Components of TFIID were similarly recruited to the CUP1 promoter without activation . By using a chromatin immunoprecipitation technique, we found that TFIIE is necessary for stable binding of TFIIH and RNAPII to the ADH1 promoter, whose activation requires TFIIE . However, binding of TFIIH and RNAPII to CUP1 upon its activation did not require TFIIE . CONCLUSIONS: Our results strongly suggest that the TFIIE requirement of a gene is determined by a target(s) in Mediator through which the signal of the cognate activator is transmitted. Yeast, 2003 Feb, 20(3), 233 - 48 Identification of a triacylglycerol lipase gene family in Candida deformans: molecular cloning and functional expression; Bigey F et al.; The yeast Candida deformans CBS 2071 produces an extracellular lipase which was shown to catalyse the production of various esters by the esterification of free fatty acids, even in the presence of a large molar excess of water . To clone the gene encoding this extracellular lipase, Saccharomyces cerevisiae was transformed with C . deformans genomic libraries and screened for lipolytic activity on a medium containing rapeseed oil emulsion and rhodamine B . Three members of a lipase gene family (CdLIP1, CdLIP2 and CdLIP3) were cloned and characterized . Each deduced lipase sequence has a Gly-His-Ser-Leu-Gly-(Gly/Ala)-Ala conserved motif, eight cysteine residues and encodes an N-terminal signal sequence . MALDI-TOF mass spectrometry analysis of a proteolytic digest of the lipase produced was used to obtain experimental evidence that the CdLIP1 gene encoded the extracellular lipase . Recombinant expression studies confirmed that the cloned genes encoded functional lipases . The three lipases are very similar to lipases from the related species Yarrowia lipolytica . Significant homologies were also found with several yeast and fungal lipases . As C . deformans CBS 2071 was previously considered to be synonymous with Y . lipolytica, the strains were compared for the extent of nucleotide divergence in the variable regions (D1/D2) at the 5'-end of the large-subunit (26S) ribosomal DNA (rDNA) gene . This rDNA region has diverged sufficiently to suggest that C . deformans is a separate species . The nucleotide sequences of the CdLIP1, CdLIP2 and CdLIP3 genes will appear in the EMBL nucleotide sequence database under Accession Nos AJ428393, AJ428394 and AJ428395, respectively . Mol Cancer Res, 2003 Jan, 1(3), 207 - 18 DNA replication-dependent nuclear dynamics of the Mre11 complex; Mirzoeva OK et al.; The Mre11 complex undergoes dramatic relocalization in the nuclei of gamma-irradiated and replicating human cells . In this study, we examined Mre11 complex localization and chromatin association in synchronous cultures to examine the molecular determinants of relocalization . The data indicate that the complex is deposited on chromatin in an S phase-specific manner . Mre11 complex chromatin association in S phase was resistant to detergent extraction, in contrast to that in gamma-irradiated cells . The complex exhibits extensive colocalization with proliferating cell nuclear antigen throughout S phase, and chromatin loading is enhanced by replication fork stalling, suggesting that the replication fork is a site of Mre11 complex chromatin loading . This is supported by the observation that the complex localized to single-stranded DNA arising in hydroxyurea-treated cells . Although the Mre11 complex appears to function as a DNA damage sensor, limited colocalization with Brca1 or gamma-H2AX was observed, arguing that neither DNA damage nor gamma-H2AX is required for Mre11 complex chromatin loading . These data provide a potential molecular basis for promotion of sister chromatid association and recombination by the Mre11 complex as well as for ATM-Mre11 complex-dependent activation of cell cycle checkpoints. Mol Cell Biol, 2003 Feb, 23(4), 1470 - 6 Disruption of the pelota gene causes early embryonic lethality and defects in cell cycle progression; Adham IM et al.; Mutations in either the Drosophila melanogaster pelota or pelo gene or the Saccharomyces cerevisiae homologous gene, DOM34, cause defects of spermatogenesis and oogenesis in Drosophila, and delay of growth and failure of sporulation in yeast . These phenotypes suggest that pelota is required for normal progression of the mitotic and meiotic cell cycle . To determine the role of the pelota in mouse development and progression of cell cycle, we have established a targeted disruption of the mouse PELO: Heterozygous animals are variable and fertile . Genotyping of the progeny of heterozygous intercrosses shows the absence of Pelo(-/-) pups and suggests an embryo-lethal phenotype . Histological analyses reveal that the homozygous Pelo deficient embryos fail to develop past day 7.5 of embryogenesis (E7.5) . The failure of mitotic active inner cell mass of the Pelo(-/-) blastocysts to expand in growth after 4 days in culture and the survival of mitotic inactive trophoplast indicate that the lethality of Pelo-null embryos is due to defects in cell proliferation . Analysis of the cellular DNA content reveals the significant increase of aneuploid cells in Pelo(-/-) embryos at E7.5 . Therefore, the percent increase of aneuploid cells at E7.5 may be directly responsible for the arrested development and suggests that Pelo is required for the maintenance of genomic stability. Mol Cell Biol, 2003 Feb, 23(4), 1368 - 78 Dual roles for Spt5 in pre-mRNA processing and transcription elongation revealed by identification of Spt5-associated proteins; Lindstrom DL et al.; During transcription elongation, eukaryotic RNA polymerase II (Pol II) must contend with the barrier presented by nucleosomes . The conserved Spt4-Spt5 complex has been proposed to regulate elongation through nucleosomes by Pol II . To help define the mechanism of Spt5 function, we have characterized proteins that coimmunopurify with Spt5 . Among these are the general elongation factors TFIIF and TFIIS as well as Spt6 and FACT, factors thought to regulate elongation through nucleosomes . Spt5 also coimmunopurified with the mRNA capping enzyme and cap methyltransferase, and spt4 and spt5 mutations displayed genetic interactions with mutations in capping enzyme genes . Additionally, we found that spt4 and spt5 mutations lead to accumulation of unspliced pre-mRNA . Spt5 also copurified with several previously unstudied proteins; we demonstrate that one of these is encoded by a new member of the SPT gene family . Finally, by immunoprecipitating these factors we found evidence that Spt5 participates in at least three Pol II complexes . These observations provide new evidence of roles for Spt4-Spt5 in pre-mRNA processing and transcription elongation. Mol Cell Biol, 2003 Feb, 23(4), 1341 - 8 Snf1 kinases with different beta-subunit isoforms play distinct roles in regulating haploid invasive growth; Vyas VK et al.; The Snf1 protein kinase of Saccharomyces cerevisiae has been shown to have a role in regulating haploid invasive growth in response to glucose depletion . Cells contain three forms of the Snf1 kinase, each with a different beta-subunit isoform, either Gal83, Sip1, or Sip2 . We present evidence that different Snf1 kinases play distinct roles in two aspects of invasive growth, namely, adherence to the agar substrate and filamentation . The Snf1-Gal83 form of the kinase is required for adherence, whereas either Snf1-Gal83 or Snf1-Sip2 is sufficient for filamentation . Genetic evidence indicates that Snf1-Gal83 affects adherence by antagonizing Nrg1- and Nrg2-mediated repression of the FLO11 flocculin and adhesin gene . In contrast, the mechanism(s) by which Snf1-Gal83 and Snf1-Sip2 affect filamentation is independent of FLO11 . Thus, the Snf1 kinase regulates invasive growth by at least two distinct mechanisms. Mol Cell Biol, 2003 Feb, 23(4), 1151 - 62 Loss of HR6B ubiquitin-conjugating activity results in damaged synaptonemal complex structure and increased crossing-over frequency during the male meiotic prophase; Baarends WM et al.; The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6 . In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation . HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility . In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase . Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes . These complexes were found to be longer . In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei . Finally, we detected an increased number of foci containing the mismatch DNA repair protein MLH1 in these nuclei, reflecting a remarkable and consistent increase (20 to 25%) in crossing-over frequency . The present findings reveal a specific requirement for the ubiquitin-conjugating activity of HR6B in relation to dynamic aspects of the synaptonemal complex and meiotic recombination in spermatocytes. J Biol Chem, 2003 Apr 11, 278(15), 12786 - 95 Epub 2003 Jan 29. The LIM protein FHL3 binds basic Krüppel-like factor/Krüppel-like factor 3 and its co-repressor C-terminal-binding protein 2; Turner J et al.; The ability of DNA-binding transcription factors to recruit specific cofactors is central to the mechanism by which they regulate gene expression . BKLF/KLF3, a member of the Kruppel-like factor family of zinc finger proteins, is a potent transcriptional repressor that recruits a CtBP co-repressor . We show here that BKLF also recruits the four and a half LIM domain protein FHL3 . Different but closely linked regions of BKLF mediate contact with CtBP2 and FHL3 . We present evidence that CtBP2 also interacts with FHL3 and demonstrate that the three proteins co-elute in gel filtration experiments . CtBP and FHL proteins have been implicated in both nuclear and cytoplasmic functions, but expression of BKLF promotes the nuclear accumulation of both FHL3 and CtBP2 . FHL proteins have been shown to act predominantly as co-activators of transcription . However, we find FHL3 can repress transcription . We suggest that LIM proteins like FHL3 are important in assembling specific repression or activation complexes, depending on conditions such as cofactor availability and promoter context. J Neurobiol, 2003 Mar, 54(4), 618 - 27 Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase; Friggi-Grelin F et al.; Dopamine (DA) is the only catecholaminergic neurotransmitter in the fruit fly Drosophila melanogaster . Dopaminergic neurons have been identified in the larval and adult central nervous system (CNS) in Drosophila and other insects, but no specific genetic tool was available to study their development, function, and degeneration in vivo . In Drosophila as in vertebrates, the rate-limiting step in DA biosynthesis is catalyzed by the enzyme tyrosine hydroxylase (TH) . The Drosophila TH gene (DTH) is specifically expressed in all dopaminergic cells and the corresponding mutant, pale (ple), is embryonic lethal . We have performed ple rescue experiments with modified DTH transgenes . Our results indicate that partially redundant regulatory elements located in DTH introns are required for proper expression of this gene in the CNS . Based on this study, we generated a GAL4 driver transgene, TH-GAL4, containing regulatory sequences from the DTH 5' flanking and downstream coding regions . TH-GAL4 specifically expresses in dopaminergic cells in embryos, larval CNS, and adult brain when introduced into the Drosophila genome . As a first application of this driver, we observed that in vivo inhibition of DA release induces a striking hyperexcitability behavior in adult flies . We propose that TH-GAL4 will be useful for studies of the role of DA in behavior and disease models in Drosophila . RNA, 2003 Jan, 9(1), 138 - 50 Identification and characterization of Prp45p and Prp46p, essential pre-mRNA splicing factors; Albers M et al.; Through exhaustive two-hybrid screens using a budding yeast genomic library, and starting with the splicing factor and DEAH-box RNA helicase Prp22p as bait, we identified yeast Prp45p and Prp46p . We show that as well as interacting in two-hybrid screens, Prp45p and Prp46p interact with each other in vitro . We demonstrate that Prp45p and Prp46p are spliceosome associated throughout the splicing process and both are essential for pre-mRNA splicing . Under nonsplicing conditions they also associate in coprecipitation assays with low levels of the U2, U5, and U6 snRNAs that may indicate their presence in endogenous activated spliceosomes or in a postsplicing snRNP complex. RNA, 2003 Feb, 9(2), 239 - 51 The enhancer of decapping proteins, Edc1p and Edc2p, bind RNA and stimulate the activity of the decapping enzyme; Schwartz D et al.; A major pathway of eukaryotic mRNA turnover initiates with deadenylation, which allows a decapping reaction leading to 5'-3' exonucleolytic degradation . A key control point in this pathway is the decapping of the mRNA . Two proteins, Edc1 and Edc2, were genetically identified previously as enhancers of the decapping reaction . In this work, we demonstrate that Edc1p and Edc2p are RNA-binding proteins . In addition, recombinant Edc1p or Edc2p stimulates mRNA decapping in cell-free extracts or with purified decapping enzyme . These results suggest that Edc1p and Edc2p activate decapping directly by binding to the mRNA substrate and enhancing the activity of the decapping enzyme . Interestingly, edc1Delta strains show defects in utilization of glycerol as a carbon source and misregulation of several mRNAs in response to carbon-source changes . This identifies a critical role for decapping and Edc1p in alterations of gene expression in response to carbon-source changes. EMBO J, 2003 Feb 3, 22(3), 612 - 20 Molecular evidence for a positive role of Spt4 in transcription elongation; Rondon AG et al.; We have previously shown that yeast mutants of the THO complex have a defect in gene expression, observed as an impairment of lacZ transcription . Here we analyze the ability of mutants of different transcription elongation factors to transcribe lacZ . We found that spt4Delta, like THO mutants, impaired transcription of lacZ and of long and GC-rich DNA sequences fused to the GAL1 promoter . Using a newly developed in vitro transcription elongation assay, we show that Spt4 is required in elongation . There is a functional interaction between Spt4 and THO, detected by the lethality or strong gene expression defect and hyper-recombination phenotypes of double mutants in the W303 genetic background . Our results indicate that Spt4-Spt5 has a positive role in transcription elongation and suggest that Spt4-Spt5 and THO act at different steps during mRNA biogenesis. EMBO J, 2003 Feb 3, 22(3), 600 - 11 Chromatin exposes intrinsic differences in the transcriptional activities of estrogen receptors alpha and beta; Cheung E et al.; The biological actions of estrogens are mediated via two distinct intranuclear estrogen receptor (ER) proteins, ERalpha and ERbeta . We have used an in vitro chromatin assembly and transcription system to compare the transcriptional activities of the two ERs in the context of chromatin, the physiological template for transcription by RNA polymerase II . We find that under conditions where many biochemical activities of the receptors are similar (e.g . ligand binding, chromatin binding, chromatin remodeling and co-activator recruitment), liganded ERalpha is a much more potent transcriptional activator than ERbeta with chromatin templates, but not with naked DNA . This difference is attributable to the N-terminal A/B region of ERalpha, which contains a transferable activation function that facilitates transcription specifically with chromatin templates . Interestingly, chromatin selectively restricts ligand-dependent transcriptional activation by ERbeta under some conditions (e.g . with a closed chromatin architecture), while allowing it under other conditions (e.g . with an open chromatin architecture) . Collectively, our results define an important role for chromatin in determining signaling outcomes mediated by distinct subtypes of signal-transducing transcriptional activator proteins. EMBO J, 2003 Feb 3, 22(3), 362 - 71 Two distinct myosin light chain structures are induced by specific variations within the bound IQ motifs-functional implications; Terrak M et al.; IQ motifs are widespread in nature . Mlc1p is a calmodulin-like myosin light chain that binds to IQ motifs of a class V myosin, Myo2p, and an IQGAP-related protein, Iqg1p, playing a role in polarized growth and cytokinesis in Saccharomyces cerevisiae . The crystal structures of Mlc1p bound to IQ2 and IQ4 of Myo2p differ dramatically . When bound to IQ2, Mlc1p adopts a compact conformation in which both the N- and C-lobes interact with the IQ motif . However, in the complex with IQ4, the N-lobe no longer interacts with the IQ motif, resulting in an extended conformation of Mlc1p . The two light chain structures relate to two distinct subfamilies of IQ motifs, one of which does not interact with the N-lobes of calmodulin-like light chains . The correlation between light chain structure and IQ sequence is demonstrated further by sedimentation velocity analysis of complexes of Mlc1p with IQ motifs from Myo2p and Iqg1p . The resulting 'free' N-lobes of myosin light chains in the extended conformation could mediate the formation of ternary complexes during protein localization and/or partner recruitment. Cell, 2003 Jan 24, 112(2), 243 - 56 Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase; Orlicky S et al.; Cell cycle progression depends on precise elimination of cyclins and cyclin-dependent kinase (CDK) inhibitors by the ubiquitin system . Elimination of the CDK inhibitor Sic1 by the SCFCdc4 ubiquitin ligase at the onset of S phase requires phosphorylation of Sic1 on at least six of its nine Cdc4-phosphodegron (CPD) sites . A 2.7 A X-ray crystal structure of a Skp1-Cdc4 complex bound to a high-affinity CPD phosphopeptide from human cyclin E reveals a core CPD motif, Leu-Leu-pThr-Pro, bound to an eight-bladed WD40 propeller domain in Cdc4 . The low affinity of each CPD motif in Sic1 reflects structural discordance with one or more elements of the Cdc4 binding site . Reengineering of Cdc4 to reduce selection against Sic1 sequences allows ubiquitination of lower phosphorylated forms of Sic1 . These features account for the observed phosphorylation threshold in Sic1 recognition and suggest an equilibrium binding mode between a single receptor site in Cdc4 and multiple low-affinity CPD sites in Sic1. Cell, 2003 Jan 24, 112(2), 142 - 4 Ubiquitinating a phosphorylated Cdk inhibitor on the blades of the Cdc4 beta-propeller; Jackson PK; Substrate binding by the SCFCdc4 ubiquitin ligase is regulated by phosphorylation . In this issue of Cell, Orlicky et al . describe the crystal structure of the Cdc4 subunit bound to a high-affinity substrate phosphopeptide . This structure provides insights into the binding interaction and how a precise mechanism involving multiple regulatory phosphorylations may be mediated by a single binding site. Biochem J, 2003 May 1, 371(Pt 3), 823 - 30 Iron-sulphur cluster assembly in plants: distinct NFU proteins in mitochondria and plastids from Arabidopsis thaliana; Leon S et al.; Recent results are in favour of a role for NFU-like proteins in Fe-S cluster biogenesis . These polypeptides share a conserved CXXC motif in their NFU domain . In the present study, we have characterized Arabidopsis thaliana NFU1-5 genes . AtNFU proteins are separated into two classes . NFU4 and NFU5 are part of the mitochondrial type, presenting a structural organization similar to Saccharomyces cerevisiae Nfu1p . These proteins complement a Delta isu1 Delta nfu1 yeast mutant and NFU4 mitochondrial localization was confirmed by green fluorescent protein fusion analysis . AtNFU1-3 represent a new class of NFU proteins, unique to plants . These polypeptides are made of two NFU domains, the second having lost its CXXC motif . AtNFU1-3 proteins are more related to Synechocystis PCC6803 NFU-like proteins and are localized to plastids when fused with the green fluorescent protein . NFU2 and/or NFU3 were detected in leaf chloroplasts by immunoblotting . NFU1 and NFU2 are functional NFU capable of restoring the growth of a Delta isu1 Delta nfu1 yeast mutant, when addressed to yeast mitochondria . Furthermore, NFU2 recombinant protein is capable of binding a labile 2Fe-2S cluster in vitro . These results demonstrate the presence of distinct NFU proteins in Arabidopsis mitochondria and plastids . Such results suggest the existence of two different Fe-S assembly machineries in plant cells. Biol Chem, 2002 Dec, 383(12), 1895 - 905 Evidence for the involvement of the unique C-tail of S100A9 in the binding of arachidonic acid to the heterocomplex S100A8/A9; Sopalla C et al.; Protein complexes formed by S100A8 and S100A9 represent the only AA-binding capacity in the human neutrophilic cytosol and are involved in the intracellular arachidonic acid metabolism . The formation of S100A8/A9 protein complexes and the binding of calcium to the complexes are prerequisites for the specific binding of polyunsaturated fatty acids . The present study was undertaken to characterize the fatty acid binding site within the protein complex . Deletions at both termini and point mutations of different basic amino acids especially within the extended C-terminal tail of human S100A9 were introduced . The S100A9 mutant proteins were then analyzed with respect to protein-protein interaction (GST pull down-assay and yeast two-hybrid system) and functional properties (arachidonic acid and calcium binding) . The data give strong evidence that the unique C-tail of S100A9 containing the three consecutive histidine residues (His103-His105) represents the region to which the fatty acid carboxy-group is bound to the protein complex . The localization of the AA-binding site within the unique C-tail of S100A9 correlates with the fact that fatty acid binding has not yet been reported for other S100 proteins. J Virol, 2003 Feb, 77(4), 2369 - 76 Human cytomegalovirus infection leads to accumulation of geminin and inhibition of the licensing of cellular DNA replication; Biswas N et al.; Previous studies have shown that infection of G(0)-synchronized human fibroblasts by human cytomegalovirus (HCMV) results in a block to cellular DNA synthesis . In this study, we have examined the effect of viral infection on the formation of the host cell DNA prereplication complex (pre-RC) . We found that the Cdc6 protein level was significantly upregulated in the virus-infected cells and that there was a delay in the expression of the Mcm family of proteins . The loading of the Mcm proteins onto the DNA pre-RC complex also appeared to be defective in the virus-infected cells . This inhibition of DNA replication licensing was associated with the accumulation of geminin, a replication inhibitor . Cdt1, which participates in the loading of the Mcm proteins, was also downregulated and modified differentially in the infected cells . Early viral gene expression was sufficient for the virus-induced alteration of the pre-RC, and the immediate-early protein IE1 was not required . These studies show that the inhibition of replication licensing in HCMV-infected cells is one of the multiple pathways by which the virus dysregulates the host cell cycle. J Biol Chem, 2003 Apr 18, 278(16), 13712 - 8 Epub 2003 Jan 27. Location of the actual signal in the negatively charged leader sequence involved in the import into the mitochondrial matrix space; Mukhopadhyay A et al.; Proteins destined for the mitochondrial matrix space have leader sequences that are typically present at the most N-terminal end of the nuclear-encoded precursor protein . The leaders are rich in positive charges and usually deficient of negative charges . This observation led to the acid-chain hypothesis to explain how the leader sequences interact with negatively charged receptor proteins . Here we show using both chimeric leaders and one from isopropyl malate synthase that possesses a negative charge that the leader need not be at the very N terminus of the precursor . Experiments were performed with modified non-functioning leader sequences fused to either the native or a non-functioning leader of aldehyde dehydrogenase so that an internal leader sequence could exist . The internal leader is sufficient for the import of the modified precursor protein . It appears that this leader still needs to form an amphipathic helix just like the normal N-terminal leaders do . This internal leader could function even if the most N-terminal portion contained negative charges in the first 7-11 residues . If the first 11 residues were deleted from isopropyl malate synthase, the resulting protein was imported more successfully than the native protein . It appears that precursors that carry negatively charged leaders use an internal signal sequence to compensate for the non-functional segment at the most N-terminal portion of the protein. J Biol Chem, 2003 Apr 4, 278(14), 12513 - 21 Epub 2003 Jan 27. STAM and Hrs are subunits of a multivalent ubiquitin-binding complex on early endosomes; Bache KG et al.; STAM1 and STAM2, which have been identified as regulators of receptor signaling and trafficking, interact directly with Hrs, which mediates the endocytic sorting of ubiquitinated membrane proteins . The STAM proteins interact with the same coiled-coil domain that is involved in the targeting of Hrs to endosomes . In this work, we show that STAM1 and STAM2, as well as an endocytic regulator protein, Eps15, can be co-immunoprecipitated with Hrs both from membrane and cytosolic fractions and that recombinant Hrs, STAM1/STAM2, and Eps15 form a ternary complex . We find that overexpression of Hrs causes a strong recruitment of STAM2 to endosome membranes . Moreover, STAM2, like Hrs and Eps15, binds ubiquitin, and Hrs, STAM2, and Eps15 colocalize with ubiquitinated proteins in clathrin-containing endosomal microdomains . The localization of Hrs, STAM2, Eps15, and clathrin to endosome membranes is controlled by the AAA ATPase mVps4, which has been implicated in multivesicular body formation . Depletion of cellular Hrs by small interfering RNA results in a strongly reduced recruitment of STAM2 to endosome membranes and an impaired degradation of endocytosed epidermal growth factor receptors . We propose that Hrs, Eps15, and STAM proteins function in a multivalent complex that sorts ubiquitinated proteins into the multivesicular body pathway. Biochemistry, 2003 Feb 4, 42(4), 1053 - 61 Two of the five zinc fingers in the Zap1 transcription factor DNA binding domain dominate site-specific DNA binding; Evans-Galea MV et al.; The Zap1 transcriptional activator from Saccharomyces cerevisiae induces expression of a series of genes containing an 11 base pair conserved promoter element (ZRE) under conditions of zinc deficiency . This work shows that Zap1 uses four of its seven zinc finger domains to contact the ZRE and that two of these dominate the interaction by contacting the essential ACC-GGT ends . Two Zn finger domains (ZF1 and ZF2) do not contact DNA, and a third ZF3 may be more important for interfinger protein-protein interactions . Zn finger domains important for ZRE contact were identified from triple mutations in Zap1, changing three residues in the alpha helix in each finger known to be important for DNA contacts in Zn finger proteins . Replacement of -1, 3, and 6 helix residues in ZF4 and ZF7 reduced the affinity of Zap1 for the wild-type ZRE . In contrast, triple mutations within the intervening ZF5 and ZF6 domains had minimal effect . The data argue that fingers 4 and 7 contact the ACC-GGT ends while fingers 5 and 6 contact the 5 bp central ZRE sequence . This conclusion is corroborated by decreased Zap1 affinity for a ZRE DNA duplex containing mutations of the AC-GT ends of the ZRE, whereas transversion mutations within the central 5 bp of the ZRE had minimal effect on Zap1 binding affinity. Biochemistry, 2003 Feb 4, 42(4), 901 - 9 Differential helix stabilities and sites pre-organized for tertiary interactions revealed by monitoring local nucleotide flexibility in the bI5 group I intron RNA; Chamberlin SI et al.; The local environment at adenosine residues in the bI5 group I intron RNA was monitored as a function of Mg(2+) using both the traditional method of dimethyl sulfate (DMS) N1 methylation and a new approach, selective acylation of 2'-amine substituted nucleotides . These probes yield complementary structural information because N1 methylation reports accessibility at the base pairing face, whereas 2'-amine acylation scores overall residue flexibility . 2'-Amine acylation robustly detects RNA secondary structure and is sensitive to higher order interactions not monitored by DMS . Disruption of RNA structure due to the 2'-amine substitution is rare and can be compensated by stabilizing folding conditions . Peripheral helices that do not interact with other parts of the RNA are more stable than both base paired helices and tertiary interactions in the conserved catalytic core . The equilibrium state of the bI5 intron RNA, prior to assembly with its protein cofactor, thus features a relatively loosely packed core anchored by more stable external stem-loop structures . Adenosine residues in J4/5 and P9.0 form structures in which the nucleotide is constrained but the N1 position is accessible, consistent with pre-organization to form long-range interactions with the 5' and 3' splice sites. Nat Genet, 2003 Feb, 33(2), 145 - 53 Epub 2003 Jan 27. Ru2 and Ru encode mouse orthologs of the genes mutated in human Hermansky-Pudlak syndrome types 5 and 6; Zhang Q et al.; Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous disease involving abnormalities of melanosomes, platelet dense granules and lysosomes . Here we have used positional candidate and transgenic rescue approaches to identify the genes mutated in ruby-eye 2 and ruby-eye mice (ru2 and ru, respectively), two 'mimic' mouse models of HPS . We also show that these genes are orthologs of the genes mutated in individuals with HPS types 5 and 6, respectively, and that their protein products directly interact . Both genes are previously unknown and are found only in higher eukaryotes, and together represent a new class of genes that have evolved in higher organisms to govern the synthesis of highly specialized lysosome-related organelles. Nat Struct Biol, 2003 Mar, 10(3), 160 - 7 The structure and function of MCM from archaeal M . Thermoautotrophicum; Fletcher RJ et al.; Eukaryotic chromosomal DNA is licensed for replication precisely once in each cell cycle . The mini-chromosome maintenance (MCM) complex plays a role in this replication licensing . We have determined the structure of a fragment of MCM from Methanobacterium thermoautotrophicum (mtMCM), a model system for eukaryotic MCM . The structure reveals a novel dodecameric architecture with a remarkably long central channel . The channel surface has an unusually high positive charge and binds DNA . We also show that the structure of the N-terminal fragment is conserved for all MCMs proteins despite highly divergent sequences, suggesting a common architecture for a similar task: gripping/remodeling DNA and regulating MCM activity . An mtMCM mutant protein equivalent to a yeast MCM5 (CDC46) protein with the bob1 mutation at its N terminus has only subtle structural changes, suggesting a Cdc7-bypass mechanism by Bob1 in yeast . Yeast bypass experiments using MCM5 mutant proteins support the hypothesis for the bypass mechanism. Trends Genet, 2003 Feb, 19(2), 79 - 81 Life cycles of successful genes; Hoffmann R et al.; By exploring time-series data from MEDLINE abstracts, we observe that only a few genes have been quoted with increasing frequency during the past 25 years . This is probably the result of selective pressure by the scientific community . Over the years, this selection has produced an extreme power law distribution of the information available for individual genes . Interestingly, those genes that are successfully selected are not necessarily the most important genes to the cell . To stress the implication of this finding we show that there is no correlation between a gene's impact in the scientific literature and its centrality in protein-interaction networks. J Hepatol, 2003 Feb, 38(2), 148 - 55 Down-regulation of the Na+/taurocholate cotransporting polypeptide during pregnancy in the rat; Arrese M et al.; BACKGROUND: Experimental studies have shown decreased bile acid (BA) uptake and reduced excretion of cholephilic compounds in pregnant rodents . AIM: To assess the expression and function of the main BA importer, the Na(+)/taurocholate cotransporting polypeptide (Ntcp) in pregnant rats . METHODS: BA uptake and Ntcp expression were studied in control and timed-pregnant rats in late gestation . Ntcp protein, messenger RNA (mRNA) expression, and Ntcp tissue localization were determined by Northern blotting, Western analysis, and tissue immunofluorescence . The activity of three transactivators of the Ntcp promoter: hepatocyte nuclear factor 1-alpha (HNF1-alpha), nuclear receptor heterodimer retinoid X receptor:retinoid acid receptor (RXR:RAR) and signal transducer and activator of transcription 5 (Stat5) was assessed using gel electrophoretic mobility shift assays . RESULTS: A significantly reduced BA uptake and decreased Ntcp mRNA levels (-40%) and protein mass (-60%) was observed in pregnant rats . Nuclear extracts from pregnant rats showed a marked decrease of HNF1-alpha and RXR:RAR binding activities by -80 and -40% of basal activity, respectively . In contrast, binding activity of Stat-5 was increased by 50% in nuclear extracts from pregnant rats . CONCLUSIONS: Pregnancy is associated with reduced Ntcp expression and function in the rat . Our findings suggest that Ntcp down-regulation during pregnancy occurs primarily at the transcriptional level. DNA Repair (Amst), 2003 Mar 1, 2(3), 337 - 46 Isolation and genetic characterization of the Neurospora crassa REV1 and REV7 homologs: evidence for involvement in damage-induced mutagenesis; Sakai W et al.; In a previous paper, we reported that the Neurospora crassa upr-1 gene is a homolog of the yeast gene REV3, which encodes the catalytic subunit of DNA polymerase zeta (polzeta) . Characterization of the upr-1 mutant indicated that the UPR1 protein plays a role in DNA repair and mutagenesis . To help understand the mechanisms of mutagenic DNA repair in the N . crassa more extensively, we identified N . crassa homologs of yeast REV1 and REV7 and obtained mutants ncrev1 or ncrev7, which had similar phenotypes to the upr-1 mutant . Mutant carrying ncrev7 was more sensitive to UV and 4NQO, and slightly sensitive to MMS than the wild-type . The sensitivity to UV and MMS of the ncrev1 mutant was moderately higher than that of the wild-type, but the sensitivity to 4NQO of the mutant was similar to that of the wild-type . In reversion assay using testers with base substitution or frameshift mutation at the ad-3A locus, each of ncrev1 and ncrev7 mutants showed lower induced-mutability than the wild-type . Expression of ncrev1 and ncrev7 was found to be UV-inducible like the case of upr-1 . Genetic analyses showed that the ncrev7 was identical to mus-26, which belongs to the upr-1 epistasis group, and that the ncrev1 was a newly identified DNA repair gene and designated as mus-42 . Interestingly, all three mutants have a normal CPD photolyase gene, however, they showed a partial photoreactivation defect (PPD) phenotype, not completely defective but inefficient in photoreactivation . These results suggest that N . crassa REV homolog genes function in DNA repair and UV mutagenesis through the bypass of (6-4) photoproducts. J Mol Biol, 2003 Feb 7, 326(1), 65 - 76 Stepwise manipulation of DNA specificity in Flp recombinase: progressively adapting Flp to individual and combinatorial mutations in its target site; Voziyanov Y et al.; The Flp protein from Saccharomyces cerevisiae is one of the site-specific tyrosine family recombinases that are used widely in genomic engineering . As a first step towards mediating directed DNA rearrangements at non-native Flp recombination targets (mFRTs), we have evolved three separate groups of Flp variants that preferentially act on mFRTs containing substitutions at the first, seventh or both positions of the Flp-binding elements . The variants that recombine the double-mutant mFRT contain a subset of the mutations present in those that are active on the single-mutant mFRTs, plus additional mutations . Specificity for and discrimination between target sites, effected primarily by amino acid residues that contact DNA, can be modulated by those that do not interact with DNA or with a DNA-contacting residue . The degree of modulation can range from relaxed DNA specificity to almost completely altered specificity . Our results suggest that combined DNA shuffling and mutagenesis of libraries of Flp variants active on distinct mFRTs can yield variants that can recombine mFRTs containing combinations of the individual mutations. Curr Biol, 2003 Jan 21, 13(2), 173 - 7 Evolution of eukaryotic cell cycle regulation: stepwise addition of regulatory kinases and late advent of the CDKs; Krylov DM et al.; Protein kinases regulate a number of critical events in mitosis and meiosis . A study of the evolution of kinases involved in cell cycle control (CCC) might shed light on the evolution of the eukaryotic cell cycle . In particular, applying quantitative phylogenetic methods to key CCC kinases could provide information on the relative timing of gene duplication events . To investigate the evolution of CCC kinases, we constructed phylogenetic trees for the CDC28 family and performed statistical tests of the tree topology . This family includes the cyclin-dependent kinases (CDKs), which are key regulators of the eukaryotic cell cycle, as well as other CCC kinases . We found that CDKs and, in particular, the principal cell cycle regulator Cdc28p, branch off the phylogenetic tree at a late stage, after several other kinases involved in either mitosis or meiosis regulation . On the basis of this tree topology, it is proposed that, at early stages of evolution, the eukaryotic cell cycle was not controlled by CDKs and that only a subset of extant kinases, notably the DNA damage checkpoint kinase Chk1p, were in place . During subsequent evolution, a series of duplications of kinase genes occurred, gradually adding more kinases to the CCC system, the CDKs being among the last major additions. Biochem Soc Trans, 2003 Feb, 31(Pt 1), 266 - 9 Regulation of eukaryotic DNA replication at the initiation step; Pollok S et al.; The studies of cell growth and division have remained at the centre of biomedical research for more than 100 years . The combination of genetic, biochemical, molecular and cell biological techniques recently yielded a burst in what is known of the molecular control of cell growth processes . The initiation of DNA replication is crucial for the stability of the genetic information of a cell . Two factors, Cdc45p (cell division cycle 45p) and DNA polymerase alpha-primase, are necessary in this process . Depending on growth signals, Cdc45p is expressed as a late protein . New phosphorylation-specific antibodies specifically recognize the phosphorylated subunit, p68, of the four subunit DNA polymerase alpha-primase and show that the phosphorylated polypeptide is exclusively nuclear. Biochem Soc Trans, 2003 Feb, 31(Pt 1), 40 - 4 The complex matter of DNA double-strand break detection; Bradbury JM et al.; To maintain genomic stability, despite constant exposure to agents that damage DNA, eukaryotic cells have developed elaborate and highly conserved pathways of DNA damage sensing, signalling and repair . In this review, we concentrate mainly on what we know about DNA damage sensing with particular reference to Lcd1p, a yeast protein that functions early in DNA damage signalling, and MDC1 (mediator of DNA damage checkpoint 1), a recently identified human protein that may be involved in recruiting the MRE11 complex to radiation-induced nuclear foci . We describe a model for the DNA damage response in which factors are recruited sequentially to sites of DNA damage to form complexes that can amplify the original signal and propagate it to the multitude of response pathways necessary for genome stability. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Feb, 35(2), 113 - 6 The interactions of LIM protein KyoT with polycomb proteins; Li R et al.; LIM domain protein KyoT interacts with transcription factor RBP-J and modulates Notch signaling pathway . To study the function of KyoT, yeast two-hybrid was performed and proteins interacting with KyoT2 including Ring1 and hPc2 (human polycomb 2) that belong to PcG(polycomb group) were analyzed . The interactions between Ring1, hPc2, and KyoT2 were confirmed by changing vectors in two-hybrid analysis . Their interactions in vitro were confirmed by GST-pull down assay . Two-hybrid assay showed that the LIM domain of KyoT was responsible for interacting with the full-length Ring1 and hPc2 . Moreover, the LIM domain also interacted with the C-terminus of hPc2 . Interactions of KyoT2 with Ring1 and hPc2 suggested that PcG family might be involved in Notch signaling pathway. Neuroreport, 2003 Jan 20, 14(1), 25 - 30 A novel putative M9.2 isoform of V-ATPase expressed in the nervous system; Ueda T et al.; We have identified a cDNA encoding a novel putative neuron-specific isoform of vacuolar proton-translocating ATPase (V-ATPase), NM9.2, from rat and mouse . Sequence analysis revealed that NM9.2 conserved similar characteristic amino acid sequences with 60-70% identity to M9.2 previously isolated from V-ATPase in chromaffin granules . Using Northern blot analysis, NM9.2 mRNA was specifically detected in the brain, whereas M9.2 mRNA was widely expressed in various tissues . In situ hybridization showed that NM9.2 gene expression was restricted mainly to neuronal cells and consistent with that of the a1/Ac116 subunit of V-ATPase . NM9.2 is a putative neuronal isoform of the 9.2 kDa subunit in V-ATPase . Pharmacogenetics, 2003 Jan, 13(1), 29 - 38 A functional single-nucleotide polymorphism in the human cytidine deaminase gene contributing to ara-C sensitivity; Yue L et al.; To test the hypothesis that analyses of drug targets for polymorphism will help to establish gene-based information for the treatment of cancer patients, we investigated the functional single-nucleotide polymorphisms in the human cytidine deaminase (HDCA) gene . The cDNAs from 52 leukaemia/lymphoma samples and 169 control blood samples were direct-sequenced and analysed for the polymorphisms . Three different polymorphisms (A79C, G208A and T435C) were identified in the coding region of the HDCA gene and displayed allelic frequencies of 20.1%, 4.3% and 70.1%, respectively . No association with susceptibility to disease was observed . A novel polymorphism, G208A produced an alanine to threonine substitution (A70T) within the conserved catalytic domain . By introduction of the polymorphic HCDA genes into the yeast CDA-null mutants, the HCDA-70T showed 40% and 32% activity of prototype for cytidine and ara-C substrates, respectively (P < 0.01) . The ara-C IC50 value of the yeast transformants carrying HCDA-70T was 757 +/- 33 micromol and was significantly lower (P < 0.01) than that of prototype (941 +/- 58 micromol) . This study demonstrated a population characterized with 208A genotype for, which potentially leads one more sensitive to ara-C treatment than prototype . Accumulation of polymorphisms in the genes responsible for drug metabolism and determination of polymorphism-induced biological variations could provide the additional therapeutic strategies in risk-stratified protocols for the treatment of childhood malignancies. Mol Cell Proteomics, 2002 Dec, 1(12), 930 - 46 Deciphering networks of protein interactions at the nuclear pore complex; Allen NP et al.; The nuclear pore complex (NPC) gates the only known conduit for molecular exchange between the nucleus and cytoplasm of eukaryotic cells . Macromolecular transport across the NPC is mediated by nucleocytoplasmic shuttling receptors termed karyopherins (Kaps) . Kaps interact with NPC proteins (nucleoporins) that contain FG peptide repeats (FG Nups) and altogether carry hundreds of different cargoes across the NPC . Previously we described a biochemical strategy to identify proteins that interact with individual components of the nucleocytoplasmic transport machinery . We used bacterially expressed fusions of glutathione S-transferase with nucleoporins or karyopherins as bait to capture interacting proteins from yeast extracts . Forty-five distinct proteins were identified as binding to one or several FG Nups and Kaps . Most of the detected interactions were expected, such as Kap-Nup interactions, but others were unexpected, such as the interactions of the multisubunit Nup84p complex with several of the FG Nups . Also unexpected were the interactions of various FG Nups with the nucleoporins Nup2p and Nup133p, the Gsp1p-GTPase-activating protein Rna1p, and the mRNA-binding protein Pab1p . Here we resolve how these interactions occur . We show that Pab1p associates nonspecifically with immobilized baits via RNA . More interestingly, we demonstrate that the Nup84p complex contains Nup133p as a subunit and binds to the FG repeat regions of Nups directly via the Nup85p subunit . Binding of Nup85p to the GLFG region of Nup116p was quantified in vitro (K(D) = 1.5 micro M) and was confirmed in vivo using the yeast two-hybrid assay . We also demonstrate that Nup2p and Rna1p can be tethered directly to FG Nups via the importin Kap95p-Kap60p and the exportin Crm1p, respectively . We discuss possible roles of these novel interactions in the mechanisms of nucleocytoplasmic transport. J Biochem Mol Biol, 2003 Jan 31, 36(1), 110 - 9 Histone deacetylase in carcinogenesis and its inhibitors as anti-cancer agents; Kim DH et al.; The acetylation state of histone is reversibly regulated by histone acetyltransferase (HAT) and deacetylase (HDAC) . An imbalance of this reaction leads to an aberrant behavior of the cells in morphology, cell cycle, differentiation, and carcinogenesis . Recently, these key enzymes in the gene expression were cloned . They revealed a broad use of this modification, not only in histone, but also other proteins that involved transcription, nuclear transport, and cytoskeleton . These results suggest that HAT/HDAC takes charge of multiple-functions in the cell, not just the gene expression . HDAC is especially known to play an important role in carcinogenesis . The enzyme has been considered a target molecule for cancer therapy . The inhibition of HDAC activity by a specific inhibitor induces growth arrest, differentiation, and apoptosis of transformed or several cancer cells . Some of these inhibitors are in a clinical trial at phase I or phase II . The discovery and development of specific HDAC inhibitors are helpful for cancer therapy, and decipher the molecular mode of action for HDAC. Br J Haematol, 2003 Jan, 120(2), 271 - 3 A novel fusion variant of the MORF and CBP genes detected in therapy-related myelodysplastic syndrome with t(10;16)(q22;p13); Kojima K et al.; We report a case of therapy-related myelodysplastic syndrome (t-MDS) with t(10;16)(q22;p13), in which novel fusion transcripts of the MORF and CBP genes were detected . In one MORF-CBP fusion transcript, exon 15 of the MORF gene was fused in frame with exon 5 of the CBP gene . In a reciprocal CBP-MORF transcript, exon 4 of the CBP gene was fused in frame with exon 16 of the MORF gene . This is the first reported case of t-MDS associated with t(10;16), and provides molecular evidence that the novel MORF-CBP and/or CBP-MORF fusion protein(s) might play an important role in the development of t-MDS. Endocr Relat Cancer, 2002 Dec, 9(4), 221 - 6 Uncontrolled insulin secretion from a childhood pancreatic beta-cell adenoma is not due to the functional loss of ATP-sensitive potassium channels; Hussain K et al.; We report the case of an 8-year-old child who presented with severe hyperinsulinaemic hypoglycaemia due to a pancreatic islet cell adenoma . In vivo, there was no beneficial response to the hyperglycaemia-inducing agent diazoxide and as a consequence the child underwent a subtotal pancreatectomy . In vitro studies of adenomatous beta-cells revealed no operational defects in ATP-sensitive potassium channel activity and appropriate responses to diazoxide . In comparison with patients with focal adenomatous hyperplasia, genetic analysis of the isolated adenoma showed no loss of heterozygosity for chromosome 11p15 and expression of the cyclin-dependent kinase inhibitor p57(kip2) . This case illustrates that the excess insulin secretion from an infantile adenoma has an aetiology different from that observed in hyperinsulinism in infancy. Se Pu, 2002 Jul, 20(4), 364 - 6 {Analysis of mannoligosaccharides by liquid chromatography-electrospray ionization mass spectrometry}; Zhang YT et al.; Oligosaccharide characterization has been of utmost interest in various areas such as medicine, biochemistry, and food chemistry . These biologically relevant molecules are ideally suited for mass spectrometric investigation, because of the capability of this technique in offering structure and relative molecular mass information . Therefore, liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was applied to characterize the acetolysis of mannan from Saccharomyces cerevisiae . The electrospray using Na+ as adducts proved to be superior to the LC-MS for the determination of mannoligosaccharides . LC separation was accomplished by the use of NH2 column and the elution by acetonitrile-water (70:30, volume ratio) . The results showed that mannoligosaccharides side chain consisted of mannose, mannobiose, mannotriose and mannotetraose . The method developed is accurate, fast and convenient and can be used to characterize the relative molecular mass of the oligosaccharides. Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 880 - 5 Epub 2003 Jan 22. Quantitation of changes in protein phosphorylation: a simple method based on stable isotope labeling and mass spectrometry; Bonenfant D et al.; Reversible protein phosphorylation plays an important role in many cellular processes . However, a simple and reliable method to measure changes in the extent of phosphorylation is lacking . Here, we present a method to quantitate the changes in phosphorylation occurring in a protein in response to a stimulus . The method consists of three steps: (i) enzymatic digestion in H(2)16O or isotopically enriched H(2)18O to label individual pools of differentially phosphorylated proteins; (ii) affinity selection of phosphopeptides from the combined digests by immobilized metal-affinity chromatography; and (iii) dephosphorylation with alkaline phosphatase to allow for quantitation of changes of phosphorylation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry . We applied this strategy to the analysis of the yeast nitrogen permease reactivator protein kinase involved in the target of rapamycin signaling pathway . Alteration in the extent of phosphorylation at Ser-353 and Ser-357 could be easily assessed and quantitated both in wild-type yeast cells treated with rapamycin and in cells lacking the SIT4 phosphatase responsible for dephosphorylating nitrogen permease reactivator protein . The method described here is simple and allows quantitation of relative changes in the level of phosphorylation in signaling proteins, thus yielding information critical for understanding the regulation of complex protein phosphorylation cascades. Genome Biol . 2003;4(1):R6 . Epub 2003 Jan 06. Towards reconstruction of gene networks from expression data by supervised learning; Soinov LA et al.; BACKGROUND: Microarray experiments are generating datasets that can help in reconstructing gene networks . One of the most important problems in network reconstruction is finding, for each gene in the network, which genes can affect it and how . We use a supervised learning approach to address this question by building decision-tree-related classifiers, which predict gene expression from the expression data of other genes . RESULTS: We present algorithms that work for continuous expression levels and do not require a priori discretization . We apply our method to publicly available data for the budding yeast cell cycle . The obtained classifiers can be presented as simple rules defining gene interrelations . In most cases the extracted rules confirm the existing knowledge about cell-cycle gene expression, while hitherto unknown relationships can be treated as new hypotheses . CONCLUSIONS: All the relations between the considered genes are consistent with the facts reported in the literature . This indicates that the approach presented here is valid and that the resulting rules can be used as elements for building and explaining gene networks. J Bioenerg Biomembr, 2002 Oct, 34(5), 381 - 8 Copper chaperones for cytochrome c oxidase and human disease; Hamza I et al.; Biological processes in living cells are compartmentalized between lipid membranes . Integral membrane proteins often confer specific functions to these compartments and as such have a critical role in cellular metabolism and function . Cytochrome c oxidase is a macromolecular metalloprotein complex essential for the respiratory function of the cell . Elucidating the mechanisms of assembly of cytochrome c oxidase within the inner mitochondrial membrane represents a unique challenge for understanding metalloprotein biosynthesis . Elegant genetic experiments in yeast have defined several proteins required for copper delivery to cytochrome c oxidase . While the precise role of each of these proteins in copper incorporation remains unclear, recent studies have revealed that inherited mutations in two of these proteins can result in severe pathology in human infants in association with cytochrome c oxidase deficiency . Characterization of the molecular pathogenesis of these disorders offers new insights into the mechanisms of cellular copper metabolism and the role of these cytochrome c oxidase copper chaperones in human disease. J Bioenerg Biomembr, 2002 Oct, 34(5), 373 - 9 Copper chaperones: personal escorts for metal ions; Field LS et al.; Copper serves as the essential cofactor for a number of enzymes involved in redox chemistry and virtually all organisms must accumulate trace levels of copper in order to survive . However, this metal can also be toxic and a number of effective methods for sequestering and detoxifying copper prevent the metal from freely circulating inside a cell . Copper metalloenzymes are therefore faced with the challenge of acquiring their precious metal cofactor in the absence of available copper . To overcome this dilemma, all eukaryotic organisms have evolved with a family of intracellular copper binding proteins that help reserve a bioavailable pool of copper for the metalloenzymes, escort the metal to appropriate targets, and directly transfer the copper ion . These proteins have been collectively called "copper chaperones." The identification of such molecules has been made possible through molecular genetic studies in the bakers' yeast Saccharomyces cerevisiae . In this review, we highlight the findings that led to a new paradigm of intracellular trafficking of copper involving the action of copper chaperones . In particular, emphasis will be placed on the ATX1 and CCS copper chaperones that act to deliver copper to the secretory pathway and to Cu/Zn superoxide dismutase in the cytosol, respectively. Protein Sci, 2003 Feb, 12(2), 306 - 12 Insights into binding cooperativity of MATa1/MATalpha2 from the crystal structure of a MATa1 homeodomain-maltose binding protein chimera; Ke A et al.; The Yeast MATa1 and MATalpha2 are homeodomain proteins that bind DNA cooperatively to repress transcription of cell type specific genes . The DNA affinity and specificity of MATa1 in the absence of MATalpha2, however, is very low . MATa1 is converted to a higher affinity DNA-binding protein by its interaction with the C-terminal tail of MATalpha2 . To understand why MATa1 binds DNA weakly by itself, and how the MATalpha2 tail affects the affinity of MATa1 for DNA, we determined the crystal structure of a maltose-binding protein (MBP)-a1 chimera whose DNA binding behavior is similar to MATa1 . The overall MATa1 conformation in the MBP-a1 structure, which was determined in the absence of alpha2 and DNA, is similar to that in the a1/alpha2/DNA structure . The sole difference is in the C-terminal portion of the DNA recognition helix of MATa1, which is flexible in the present structure . However, these residues are not in a location likely to be affected by binding of the MATalpha2 tail . The results argue against conformational changes in a1 induced by the tail of MATalpha2, suggesting instead that the MATalpha2 tail energetically couples the DNA binding of MATalpha2 and MATa1. Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 934 - 9 Epub 2003 Jan 21. A single-stranded promoter for RNA polymerase III; Schroder O et al.; Single strands of DNA serve, in rare instances, as promoters for transcription; duplex DNA promoters with individual strands that also have a promoter capacityfunction have not been described . We show that the nontranscribed strand of the Saccharomyces cerevisiae U6 snRNA gene directs transcription initiation factor IIIB-requiring and accurately initiating transcription by RNA polymerase III . The nontranscribed strand promoter is much more extended than its duplex DNA counterpart, comprising the U6 gene TATA box, a downstream T(7) tract, and an upstream-lying segment . A requirement for placement of the 3' end of the transcribed (template) strand within the confines of the transcription bubble is seen as indicating that the nontranscribed strand provides a scaffold for RNA polymerase recruitment but is deficient at a subsequent step of transcription initiation factor IIIB's direct involvement in promoter opening. J Cell Sci, 2003 Feb 15, 116(Pt 4), 701 - 10 Krh1p and Krh2p act downstream of the Gpa2p G(alpha) subunit to negatively regulate haploid invasive growth; Batlle M et al.; The yeast G(alpha) subunit Gpa2p and its coupled receptor Gpr1p function in a signaling pathway that is required for the transition to pseudohyphal and invasive growth . A two-hybrid screen using a constitutively active allele of GPA2 identified the KRH1 gene as encoding a potential binding partner of Gpa2p . Strains containing deletions of KRH1 and its homolog KRH2 were hyper-invasive and displayed a high level of expression of FLO11, a gene involved in pseudohyphal and invasive growth . Therefore, KRH1 and KRH2 encode negative regulators of the invasive growth pathway . Cells containing krh1Delta krh2Delta mutations also displayed increased sensitivity to heat shock and decreased sporulation efficiency, indicating that Krh1p and Krh2p regulate multiple processes controlled by the cAMP/PKA pathway . The krh1Delta krh2Delta mutations suppressed the effect of a gpa2Delta mutation on FLO11 expression and eliminated the effect of a constitutively active GPA2 allele on induction of FLO11 and heat shock sensitivity, suggesting that Krh1p and Krh2p act downstream of Gpa2p . The Sch9p kinase was not required for the signal generated by deletion of KRH1 and KRH2; however, the cAMP-dependent kinase Tpk2p was required for generation of this signal . These results support a model in which activation of Gpa2p relieves the inhibition exerted by Krh1p and Krh2p on components of the cAMP/PKA signaling pathway. J Cell Biol, 2003 Jan 20, 160(2), 177 - 88 Epub 2003 Jan 21. HA95 and LAP2 beta mediate a novel chromatin-nuclear envelope interaction implicated in initiation of DNA replication; Martins S et al.; HA95 is a chromatin-associated protein that interfaces the nuclear envelope (NE) and chromatin . We report an interaction between HA95 and the inner nuclear membrane protein lamina-associated polypeptide (LAP) 2 beta, and a role of this association in initiation of DNA replication . Precipitation of GST-LAP2 beta fusion proteins and overlays of immobilized HA95 indicate that a first HA95-binding region lies within amino acids 137-242 of LAP2 beta . A second domain sufficient to bind HA95 colocalizes with the lamin B-binding domain of LAP2beta at residues 299-373 . HA95-LAP2 beta interaction is not required for NE formation . However, disruption of the association of HA95 with the NH2-terminal HA95-binding domain of LAP2 beta abolishes the initiation, but not elongation, of DNA replication in purified G1 phase nuclei incubated in S-phase extract . Inhibition of replication initiation correlates with proteasome-mediated proteolysis of Cdc6, a component of the prereplication complex . Rescue of Cdc6 degradation with proteasome inhibitors restores replication . We propose that an interaction of LAP2beta, or LAP2 proteins, with HA95 is involved in the control of initiation of DNA replication. J Cell Biol, 2003 Jan 20, 160(2), 157 - 63 Epub 2003 Jan 21. Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator; Fu L et al.; Newly synthesized proteins that do not fold correctly in the ER are targeted for ER-associated protein degradation (ERAD) through distinct sorting mechanisms; soluble ERAD substrates require ER-Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER . Retained transmembrane proteins are often sequestered into specialized ER subdomains, but the relevance of such sequestration to proteasomal degradation has not been explored . We used the yeast Saccharomyces cerevisiae and a model ERAD substrate, the cystic fibrosis transmembrane conductance regulator (CFTR), to explore whether CFTR is sequestered before degradation, to identify the molecular machinery regulating sequestration, and to analyze the relationship between sequestration and degradation . We report that CFTR is sequestered into ER subdomains containing the chaperone Kar2p, and that sequestration and CFTR degradation are disrupted in sec12ts strain (mutant in guanine-nucleotide exchange factor for Sar1p), sec13ts strain (mutant in the Sec13p component of COPII), and sec23ts strain (mutant in the Sec23p component of COPII) grown at restrictive temperature . The function of the Sar1p/COPII machinery in CFTR sequestration and degradation is independent of its role in ER-Golgi traffic . We propose that Sar1p/COPII-mediated sorting of CFTR into ER subdomains is essential for its entry into the proteasomal degradation pathway . These findings reveal a new aspect of the degradative mechanism, and suggest functional crosstalk between the secretory and the degradative pathways. Bioinformatics, 2003 Jan 22, 19(2), 212 - 8 Rank order metrics for quantifying the association of sequence features with gene regulation; Clarke ND et al.; MOTIVATION: Genome sequences and transcriptome analyses allow the correlation between gene regulation and DNA sequence features to be studied at the whole-genome level . To quantify these correlations, metrics are needed that can be applied to any sequence feature, regardless of its statistical distribution . It is also desirable for the metric values to be determined objectively, that is, without the use of subjective threshold values . RESULTS: We compare two metrics for quantifying the correlation of DNA sequence features with gene regulation . Each of the metrics is calculated from a rank-ordering of genes based on the value of the sequence feature of interest . The first metric is the area under the curve for a receiver operator characteristic plot (ROC AUC), a common way of summarizing the tradeoff between sensitivity and specificity for different values of a prediction criterion . We call the second metric the mean normalized conditional probability (MNCP) . The MNCP can be thought of as the predictive value of the sequence feature averaged over all regulated genes . The statistical significance (P-value) of each metric can be estimated from simulations . Importantly, the P-value of the MNCP metric is less dramatically affected by the presence of false positives among the set of co-regulated genes than is the ROC AUC . This is especially useful in analyzing gene sets identified by DNA microarray analysis because such data cannot distinguish direct regulation by transcription factor binding from indirect regulation . We demonstrate that these two metrics, taken together, are useful tools for defining the binding site representation and regulatory control regions that best explain the difference between genes that are regulated by a given transcription factor and those that are not . Applications to other gene features are also described . AVAILABILITY: A Python program for calculating the ROC AUC and MNCP metric values given input rank orders is available from ftp://ftp.bs.jhmi.edu/users/nclarke/MNCP/ Prostaglandins Leukot Essent Fatty Acids, 2003 Feb, 68(2), 123 - 33 Stearoyl-CoA desaturase, a short-lived protein of endoplasmic reticulum with multiple control mechanisms; Heinemann FS et al.; Stearoyl-CoA desaturase (SCD) is a short-lived, polytopic membrane-bound non-heme iron enzyme localized primarily in the endoplasmic reticulum . SCD is required for the biosynthesis of monounsaturated fatty acids, and plays a key role in hepatic synthesis of triglycerides and very-low-density lipoproteins . The intracellular concentration of SCD fluctuates in a wide range in response to complex and often competing hormonal and dietary factors . A combination of transcriptional regulation and rapid protein degradation produces transient elevations of SCD enzyme activity in response to physiologic demands . Dysregulation of SCD has been implicated in non-alcoholic fatty liver disease, hyperlipidemia, and obesity. Genome Biol . 2002;3(12):RESEARCH0071 . Epub 2002 Nov 20. Bayesian analysis of gene expression levels: statistical quantification of relative mRNA level across multiple strains or treatments; Townsend JP et al.; BACKGROUND: Methods of microarray analysis that suit experimentalists using the technology are vital . Many methodologies discard the quantitative results inherent in cDNA microarray comparisons or cannot be flexibly applied to multifactorial experimental design . Here we present a flexible, quantitative Bayesian framework . This framework can be used to analyze normalized microarray data acquired by any replicated experimental design in which any number of treatments, genotypes, or developmental states are studied using a continuous chain of comparisons . RESULTS: We apply this method to Saccharomyces cerevisiae microarray datasets on the transcriptional response to ethanol shock, to SNF2 and SWI1 deletion in rich and minimal media, and to wild-type and zap1 expression in media with high, medium, and low levels of zinc . The method is highly robust to missing data, and yields estimates of the magnitude of expression differences and experimental error variances on a per-gene basis . It reveals genes of interest that are differentially expressed at below the twofold level, genes with high 'fold-change' that are not statistically significantly different, and genes differentially regulated in quantitatively unanticipated ways . CONCLUSIONS: Anyone with replicated normalized cDNA microarray ratio datasets can use the freely available MacOS and Windows software, which yields increased biological insight by taking advantage of replication to discern important changes in expression level both above and below a twofold threshold . Not only does the method have utility at the moment, but also, within the Bayesian framework, there will be considerable opportunity for future development. Genome Biol . 2002;3(12):PREPRINT0013 . Epub 2002 Nov 21. The GRID: The General Repository for Interaction Datasets; Breitkreutz BJ et al.; We have developed a relational database, called the General Repository for Interaction Datasets (The GRID; to archive and display physical, genetic and functional interactions . The GRID displays data-rich interaction tables for any protein of interest, combines literature-derived and high throughput interaction datasets, and is readily accessible via the World Wide Web . Interactions parsed in the GRID can be viewed in graphical form with a versatile visualization tool called Osprey. Neuroscience, 2003, 116(1), 77 - 80 Methyl-CpG-binding protein 2 is localized in the postsynaptic compartment: an immunochemical study of subcellular fractions; Aber KM et al.; Methyl-CpG-binding protein 2 is a characteristic member of the methyl-CpG-binding protein family of transcription regulators . In conjunction with Sin3, MeCP2 recruits class I histone deacetylases to methyl-CpG regions to suppress transcription . Rett syndrome, a disorder characterized by mental retardation and autistic features, is associated in a majority of cases with mutations within the coding region of the MeCP2 gene . Considering that defective MeCP2 has mainly been related to Rett syndrome and other neurologic manifestations, we examined methyl-CpG-binding protein 2 cellular and subcellular compartmentalization in normal brain by immunochemical methods . Methyl-CpG-binding protein 2 immunoreactivity is present mainly in neurons; while the few immunostained glia show label confined to nuclei, many neurons also show slight perikaryal staining . Using well-characterized tissue fractions, we found that methyl-CpG-binding protein 2 but not Sin3 is found in both nuclear and postsynaptic compartments . This novel extranuclear localization is not unique to methyl-CpG-binding protein 2, since it has been previously reported for other transcription regulators such as c-Fos . These findings support the concept that methyl-CpG-binding protein 2 may link synaptic activity and transcriptional regulation in neurons . Biochem Biophys Res Commun, 2003 Jan 31, 301(1), 119 - 26 Dimerization of presenilin-1 in vivo: suggestion of novel regulatory mechanisms leading to higher order complexes; Hebert SS et al.; A growing body of evidence indicates that presenilins could exist and be active as oligomeric complexes . Using yeast two-hybrid and cell culture analysis, we provide evidence that presenilin-1 (PS1) may self-oligomerize giving rise to specific full-length/full-length homodimers . When expressed in N2A and HEK239T cultured cells, full-length PS1-wt and 5(')myc-PS1-wt form specific homodimers corresponding to twice their molecular weight . The Alzheimer's disease-associated PS1 mutations Y115H, M146L, L392V, deltaE10(PS1(1-289/320-467)), the gamma-secretase dominant negative mutant D257A, and the PS1 polymorphism mutant E318G do not affect their ability to self-oligomerize . Under non-denaturing conditions, endogenous PS1 forms specific homo-oligomers in human cultured cells . The results obtained herein suggest that PS1 associates intramolecularly to form higher order complexes, which may be needed for endoproteolytic cleavage and/or gamma-secretase-associated activity. Biochem Biophys Res Commun, 2003 Jan 31, 301(1), 92 - 7 Structural determinants of Rab and Rab Escort Protein interaction: Rab family motifs define a conserved binding surface; Pereira-Leal JB et al.; Rab proteins are a large family of monomeric GTPases with 60 members identified in the human genome . Rab GTPases require an isoprenyl modification to their C-terminus for membrane association and function in the regulation of vesicular trafficking pathways . This reaction is catalysed by Rab geranylgeranyl transferase, which recognises as protein substrate any given Rab in a 1:1 complex with Rab Escort Protein (REP) . REP is therefore able to bind many distinct Rab proteins but the molecular basis for this activity is still unclear . We recently identified conserved motifs in Rabs termed RabF motifs, which we proposed to mediate a conserved mode of interaction between Rabs and REPs . Here, we tested this hypothesis . We first used REP1 as a bait in the yeast two-hybrid system and isolated strictly full-length Rabs, suggesting that REP recognises multiple regions within and properly folded Rabs . We introduced point mutations in Rab3a as a model Rab and assessed the ability of the mutants to interact with REP using the yeast two-hybrid system and an in vitro prenylation assay . We identified several residues that affect REP:Rab binding in the RabF1, RabF3, and RabF4 regions (which include parts of the switch I and II regions), but not other RabF regions . These results support the hypothesis that Rabs bind REP via conserved RabF motifs and provide a molecular explanation for the preferential recognition of the GDP-bound conformation of Rab by REP. Biochim Biophys Acta, 2003 Jan 31, 1645(1), 22 - 9 Multiple sugar binding sites in alpha-glucosidase; Yao X et al.; Twenty-five analogs of D-glucose were examined as reversible inhibitors of yeast alpha-glucosidase (EC 3.2.1.20) . The K(i) values range from 0.38 mM for 6-deoxy-D-glucose (quinovose) to 1.0 M for D-lyxose at pH=6.3 (0.1 M NaCl, 25 degrees ) . All the monosaccharides and the three disaccharides (maltose, isomaltose and alpha,alpha-trehalose) were found to be linear competitive inhibitors with respect to alpha-p-nitrophenyl glucoside (pNPG) hydrolysis . Multiple inhibition analysis reveals that there are at least three monosaccharide binding sites on the enzyme . One of these can be occupied by glucose {K(i)=1.8(+/-0.1) mM}, one by D-galactose {K(i)=164(+/-11) mM} and one by D-mannose {K(i)=120(+/-9) mM} . The pH dependence for glucose binding closely follows that of V/K {pK(a1)=5.55(+/-0.15), pK(a2)=6.79(+/-0.15)}, but the binding of mannose does not . Although the glucose subsite can be occupied simultaneously with the mannose or galactose subsites in the enzyme-product complex, no transglucosylation can be detected between pNPG and either mannose or galactose . This suggests that neither of these nonglucose subsites can be occupied in a productive manner in the covalent glucosyl-enzyme intermediate. Mol Cell, 2003 Jan, 11(1), 267 - 74 Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter; Wood A et al.; Ubiquitination of histone H2B catalyzed by Rad6 is required for methylation of histone H3 by COMPASS . We identified Bre1 as the probable E3 for Rad6's role in transcription . Bre1 contains a C3HC4 (RING) finger and is present with Rad6 in a complex . The RING finger of Bre1 is required for ubiquitination of histone H2B, methylation of lysine 4 and 79 of H3 and for telomeric silencing . Chromatin immunoprecipitation experiments indicated that both Rad6 and Bre1 are recruited to a promoter . Bre1 is essential for this recruitment of Rad6 and is dedicated to the transcriptional pathway of Rad6 . These results suggest that Bre1 is the likely E3 enzyme that directs Rad6 to modify chromatin and ultimately to affect gene expression. Mol Cell, 2003 Jan, 11(1), 261 - 6 A conserved RING finger protein required for histone H2B monoubiquitination and cell size control; Hwang WW et al.; Monoubiquitination of histone H2B is required for methylation of histone H3 on lysine 4 (K4), a modification associated with active chromatin . The identity of the cognate ubiquitin ligase is unknown . We identify Bre1 as an evolutionarily conserved RING finger protein required in vivo for both H2B ubiquitination and H3 K4 methylation . The RING domain of Bre1 is essential for both of these modifications as is Lge1 (Large 1), a protein required for cell size control that copurifies with Bre1 . In cells lacking the euchromatin-associated histone variant H2A.Z, BRE1, RAD6, and LGE1 are each essential for cell viability, supporting redundant functions for H2B ubiquitination and H2A substitution in the formation of active chromatin . Notably, analysis of mutants demonstrates a function for Bre1/Lge1-dependent H2B monoubiquitination in the control of cell size. Mol Cell, 2003 Jan, 11(1), 237 - 48 Structural and dynamic functions establish chromatin domains; Ishii K et al.; Drosophila and mammalian proteins protect genes from heterochromatic repression in Saccharomyces cerevisiae by two different mechanisms . Factors termed genuine boundary activities (BAs) establish a structural, unidirectional bulwark against heterochromatin . In contrast, factors termed desilencing activities (DAs) act by the formation of a bidirectional, euchromatic island that blocks spreading of heterochromatin . The Drosophila boundary protein BEAF and, unexpectedly, the mammalian factor Sp1 exhibited a robust BA in yeast . In contrast, mammalian CTCF, Drosophila GAGA factor, yeast Gcn5p, and many mammalian transcription factors, although inactive as upregulators of nonsilenced genes, work as DAs . DAs but not BAs protect telomere-linked genes from silencing, presumably due to looping of telomeres and ensuing multidirectional silencing . The data demonstrate that "genetic autonomy" of chromatin domains is established by both passive and active mechanisms. Mol Cell, 2003 Jan, 11(1), 127 - 38 The Q motif: a newly identified motif in DEAD box helicases may regulate ATP binding and hydrolysis; Tanner NK et al.; SF1 and SF2 helicases have structurally conserved cores containing seven to eight distinctive motifs and variable amino- and carboxyl-terminal flanking sequences . We have discovered a motif upstream of motif I that is unique to and characteristic of the DEAD box family of RNA helicases . It consists of a 9 amino acid sequence containing an invariant glutamine . A conserved phenylalanine occurs 17 aa further upstream . Sequence alignments, site-specific mutagenesis, and ATPase assays show that this motif and the upstream phenylalanine are highly conserved, that they are essential for viability in the yeast Saccharomyces cerevisiae, and that they control ATP binding and hydrolysis in the yeast translation-initiation factor eIF4A . These results are consistent with computer studies of the solved crystal structures. Biol Reprod, 2003 Feb, 68(2), 383 - 9 Quantification of histone acetyltransferase and histone deacetylase transcripts during early bovine embryo development; McGraw S et al.; Mammalian oocytes are very unique cells with an unlimited developmental potential . These totipotent cells are able to remove existing gene-expression patterns and to impose new ones . However, genome reprogramming is still a mystery . Posttranslational modifications by acetylation of the N-termini portion of histones composing the nucleosome are involved in genome reprogramming . These modifications alter the higher-order chromatin structure to render the DNA accessible to the regulatory and transcriptional machinery . In the present study, we have investigated, to our knowledge for the first time, precise expression patterns of seven genes involved in chromatin structure throughout bovine embryo development . Oocytes harvested from bovine ovaries were used for in vitro production of germinal vesicle oocytes, metaphase II oocytes, 2- and 8-cell embryos, and blastocysts . Total RNA was extracted from pools (triplicates) of 20 oocytes or from embryos of each developmental stage . By means of quantitative reverse transcription-polymerase chain reaction using SYBR Green to detect double-stranded DNA, mRNA expression profiles for histone deacetylases (HDAC1, HDAC2, HDAC3, and HDAC7), histone acetyltransferases (GCN5 and HAT1), and histone H2A were established . Transcripts for all genes were detected at all stages from the oocyte to the blastocyst . The HDAC1, HDAC2 (class I HDAC), and HAT1 (type B HAT) revealed similar expression profiles . The HDAC3 (class I HDAC) tends to have an expression profile similar to those of HDAC1, HDAC2, and HAT1, whereas the HDAC7 (class II HDAC) and GCN5 (type A HAT) profiles were different from those three . These results indicate variable levels of histone deacetylases and histone acetyltransferases throughout embryonic development and may indicate the ones that are involved in somatic remodeling. Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 1010 - 5 Epub 2003 Jan 16. The C-terminal domain of myosin-like protein 1 (Mlp1p) is a docking site for heterogeneous nuclear ribonucleoproteins that are required for mRNA export; Green DM et al.; For mRNA to be transported from the nucleus to the cytoplasm, it must travel from the site of transcription through the nuclear interior to the nuclear pore . Studies in Saccharomyces cerevisiae have suggested a relationship between poly(A) RNA trafficking and myosin-like protein 1 (Mlp1p), a nuclear-pore associated protein that is homologous to the mammalian Tpr (translocated promoter region) protein {Kosova, B., Pante, N., Rollenhagen, C., Podtelejnikov, A., Mann, M., Aebi, U., and Hurt, E . (2000) J . Biol . Chem . 275, 343-350} . We identified a yeast two-hybrid interaction between the C-terminal globular domain of Mlp1p and Nab2p, a shuttling heterogeneous nuclear ribonucleoprotein that is required for mRNA export . Coimmunoprecipitation confirms that Nab2p also interacts with full-length Mlp1p and in vitro binding experiments show that Nab2p binds directly to the C-terminal domain of Mlp1p . In addition, our experiments reveal that the C-terminal domain of Mlp1p is both necessary and sufficient to cause accumulation of poly(A) RNA and Nab2p in the nucleus . We propose a model where Mlp1p acts as a checkpoint at the nuclear pore by interacting with export-competent ribonucleoprotein complexes through its C-terminal globular domain . This study identifies Nab2p as a heterogeneous nuclear ribonucleoprotein found in complex with Mlp1p and begins to delineate the path that mRNA travels from the chromatin to the nuclear pore. J Biol Chem, 2003 May 23, 278(21), 18833 - 41 Epub 2003 Jan 16. Regulation of p70 S6 kinase by complex formation between the Rac guanine nucleotide exchange factor (Rac-GEF) Tiam1 and the scaffold spinophilin; Buchsbaum RJ et al.; Tiam1 is a ubiquitous guanine nucleotide exchange factor (GEF) that activates the Rac GTPase . We have shown previously that the N terminus of Tiam1 contributes to the signaling specificity of its downstream target Rac via association with IB2, a scaffold that promotes Rac activation of a p38 kinase cascade . Here we show that the N terminus of Tiam1 can influence Rac signaling specificity in a different way by interaction with spinophilin, a scaffold that binds to p70 S6 kinase, another protein regulated by Rac . In particular, spinophilin binding promotes the plasma membrane localization of Tiam1 and enhances the ability of Tiam1 to activate p70 S6 kinase . In contrast, spinophilin binding suppresses the ability of Tiam to activate Pak1, a different Rac effector . Finally, a mutant spinophilin that cannot bind to Tiam1 suppresses serum-induced p70 S6 kinase activation in cells, suggesting that a Tiam1/spinophilin complex contributes to p70 S6 kinase regulation by extracellular signals . These findings add to a growing body of evidence supporting the concept that some Rac-GEFs not only activate Rac GTPases but also participate in the selection of Rac effector by binding to particular scaffolds that complex with components of specific Rac effector pathways. Vaccine, 2003 Jan 17, 21(5-6), 386 - 92 Construction of artificial virus-like particles exposing HIV epitopes, and the study of their immunogenic properties; Karpenko LI et al.; One of the major problems in the development of successful recombinant vaccines against human immunodeficiency virus (HIV) is that of correct identification of a safe and effective vaccine delivery system with which to induce protective immunity using soluble protein antigens . An original method for constructing artificial immunogens in the form of spherical particles with yeast dsRNA in the center and hybrid proteins exposing epitopes of an infectious agent on the surface is reported . The dsRNA and the proteins were linked with spermidine-polyglucin-glutathione conjugates . Particles exposing HIV-1 epitopes were constructed, and their immunogenicity tested. Biochim Biophys Acta, 2002 Dec 30, 1585(2-3), 193 - 201 Sphingosine kinase, sphingosine-1-phosphate, and apoptosis; Maceyka M et al.; The sphingolipid metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) play an important role in the regulation of cell proliferation, survival, and cell death . Cer and Sph usually inhibit proliferation and promote apoptosis, while the further metabolite S1P stimulates growth and suppresses apoptosis . Because these metabolites are interconvertible, it has been proposed that it is not the absolute amounts of these metabolites but rather their relative levels that determines cell fate . The relevance of this "sphingolipid rheostat" and its role in regulating cell fate has been borne out by work in many labs using many different cell types and experimental manipulations . A central finding of these studies is that Sph kinase (SphK), the enzyme that phosphorylates Sph to form S1P, is a critical regulator of the sphingolipid rheostat, as it not only produces the pro-growth, anti-apoptotic messenger S1P, but also decreases levels of pro-apoptotic Cer and Sph . Given the role of the sphingolipid rheostat in regulating growth and apoptosis, it is not surprising that sphingolipid metabolism is often found to be disregulated in cancer, a disease characterized by enhanced cell growth, diminished cell death, or both . Anticancer therapeutics targeting SphK are potentially clinically relevant . Indeed, inhibition of SphK has been shown to suppress gastric tumor growth {Cancer Res . 51 (1991) 1613} and conversely, overexpression of SphK increases tumorigenicity {Curr . Biol . 10 (2000) 1527} . Moreover, S1P has also been shown to regulate angiogenesis, or new blood vessel formation {Cell 99 (1999) 301}, which is critical for tumor progression . Furthermore, there is intriguing new evidence that S1P can act in an autocrine and/or paracrine fashion {Science 291 (2001) 1800} to regulate blood vessel formation {J . Clin . Invest . 106 (2000) 951} . Thus, SphK may not only protect tumors from apoptosis, it may also increase their vascularization, further enhancing growth . The cytoprotective effects of SphK/S1P may also be important for clinical benefit, as S1P has been shown to protect oocytes from radiation-induced cell death in vivo {Nat . Med . 6 (2000) 1109} . Here we review the growing literature on the regulation of SphK and the role of SphK and its product, S1P, in apoptosis. DNA Repair (Amst), 2002 Nov 3, 1(11), 913 - 27 Functional crosstalk between hOgg1 and the helicase domain of Cockayne syndrome group B protein; Tuo J et al.; We have previously reported that the Cockayne syndrome group B gene product (CSB) contributes to base excision repair (BER) of 8-hydroxyguanine (8-OH-Gua) and the importance of motifs V and VI of the putative helicase domains of CSB in BER of 8-OH-Gua . To further elucidate the function of CSB in BER, we investigated its role in the pathway involving human 8-OH-Gua glycosylase/apurinic lyase (hOgg1) . Depletion of CSB protein with anti-CSB antibody reduced the 8-OH-Gua incision rate of wild type cell extracts but not of CSB null and motif VI mutant cell extracts, suggesting a direct contribution of CSB to the catalytic process of 8-OH-Gua incision and the importance of its motif VI in this pathway . Introduction of recombinant purified CSB partially complemented the depletion of CSB as shown by the recovery of the incision activity . This complementation could not fully recover the deficiency of the incision activity in WCE from CS-B null and mutant cell lines, suggesting that some additional factor(s) are necessary for the full activity . Electrophoretic mobility shift assays (EMSAs) showed a defect in binding of CSB null and motif VI mutant cell extracts to 8-OH-Gua-containing oligonucleotides . We detected less hOgg1 transcript and protein in the cell extracts from CS-B null and mutant cells, suggesting hOgg1 may be the missing component . Pull-down of hOgg1 by histidine-tagged CSB and co-localization of those two proteins after gamma-radiation indicated their co-existence in vivo, particularly under cellular stress . However, we did not detect any functional and physical interaction between purified CSB and hOgg1 by incision, gel shift and yeast two-hybrid assays, suggesting that even though hOgg1 and CSB might be in a common protein complex, they may not interact directly . We conclude that CSB functions in the catalysis of 8-OH-Gua BER and in the maintenance of efficient hOgg1 expression, and that motif VI of the putative helicase domain of CSB is crucial in these functions. DNA Repair (Amst), 2002 Dec 5, 1(12), 983 - 94 DNA structure dependent checkpoints as regulators of DNA repair; Carr AM; Checkpoint proteins were initially identified because their loss of function resulted in defects in cell cycle arrest in response to genotoxic treatments . Initially, the analysis of checkpoint pathways concentrated on their function as signal transducers and how the checkpoint signals were communicated to the core cell cycle machinery and transcriptional apparatus . Although some of the early genetic analysis indicated a complex relationship between DNA replication, DNA repair and the checkpoint pathways, it is only now becoming apparent that checkpoint proteins regulate multiple DNA repair and replication functions . Furthermore, recent data suggest that some checkpoint proteins may participate directly in DNA repair events . In this review I summarise the current models for DNA structure-dependent checkpoint activation and review the evidence linking checkpoint proteins both directly and indirectly to DNA repair . Dev Cell, 2003 Jan, 4(1), 8 - 9 In search of lipid translocases and their biological functions; Hoekstra D et al.; In plasma membranes, lipids distribute asymmetrically across the bilayer, a process that requires proteins . Recent work identified novel lipid translocators in yeast, and their activity was functionally correlated to endocytosis, thus boosting investigations on identity, mechanism, and function of lipid translocases. Cell Mol Life Sci, 2002 Nov, 59(11), 1819 - 32 Molecular aspects of membrane fission in the secretory pathway; Corda D et al.; Membrane fission is essential in various intracellular dissociative transport steps . The molecular mechanisms by which endocytic vesicles detach from the plasma membrane are being rapidly elucidated . Much less is known about the fission mechanisms operating at Golgi tubular networks; these include the Golgi transport and sorting stations, the trans-Golgi and cis-Golgi networks, where the geometry and physical properties of the membranes differ from those at the cell surface . Here we discuss the lipid and protein machineries that have so far been related to the fission process, with emphasis on those acting in the Golgi complex. Nature, 2003 Jan 16, 421(6920), 231 - 7 Systematic functional analysis of the Caenorhabditis elegans genome using RNAi; Kamath RS et al.; A principal challenge currently facing biologists is how to connect the complete DNA sequence of an organism to its development and behaviour . Large-scale targeted-deletions have been successful in defining gene functions in the single-celled yeast Saccharomyces cerevisiae, but comparable analyses have yet to be performed in an animal . Here we describe the use of RNA interference to inhibit the function of approximately 86% of the 19,427 predicted genes of C . elegans . We identified mutant phenotypes for 1,722 genes, about two-thirds of which were not previously associated with a phenotype . We find that genes of similar functions are clustered in distinct, multi-megabase regions of individual chromosomes; genes in these regions tend to share transcriptional profiles . Our resulting data set and reusable RNAi library of 16,757 bacterial clones will facilitate systematic analyses of the connections among gene sequence, chromosomal location and gene function in C . elegans. Plant Physiol, 2003 Jan, 131(1), 335 - 44 The pea gene NA encodes ent-kaurenoic acid oxidase; Davidson SE et al.; The gibberellin (GA)-deficient dwarf na mutant in pea (Pisum sativum) has severely reduced internode elongation, reduced root growth, and decreased leaflet size . However, the seeds develop normally . Two genes, PsKAO1 and PsKAO2, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated . Both PsKAO1 and PsKAO2 had ent-kaurenoic acid oxidase (KAO) activity, catalyzing the three steps of the GA biosynthetic pathway from ent-kaurenoic acid to GA(12) when expressed in yeast (Saccharomyces cerevisiae) . In addition to the intermediates ent-7alpha-hydroxykaurenoic acid and GA(12)-aldehyde, some additional products of the pea KAO activity were detected, including ent-6alpha,7alpha-dihydroxykaurenoic acid and 7beta-hydroxykaurenolide . The NA gene encodes PsKAO1, because in two independent mutant alleles, na-1 and na-2, PsKAO1 had altered sequences and the five-base deletion in PsKAO1 associated with the na-1 allele cosegregated with the dwarf na phenotype . PsKAO1 was expressed in the stem, apical bud, leaf, pod, and root, organs in which GA levels have previously been shown to be reduced in na plants . PsKAO2 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds of na plants. Plant Physiol, 2003 Jan, 131(1), 78 - 92 Regulatory networks in seeds integrating developmental, abscisic acid, sugar, and light signaling; Brocard-Gifford IM et al.; Progression through embryogenesis and the transition to germination is subject to regulation by many transcription factors, including those encoded by the Arabidopsis LEC1 (LEAFY COTYLEDON1), FUS3 (FUSCA3), and abscisic acid-insensitive (ABI) ABI3, ABI4, and ABI5 loci . To determine whether the ABI4, ABI5, LEC1, and FUS3 loci interact or act independently, we analyzed abi fus3 and abi lec1 double mutants . Our results show that both ABI4 and ABI5 interact genetically with both LEC1 and FUS3 in controlling pigment accumulation, suppression of vivipary, germination sensitivity to abscisic acid, gene expression during mid- and late embryogenesis, sugar metabolism, sensitivity to sugar, and etiolated growth . However, the relative strengths of the observed interactions vary among responses and may even be antagonistic . Furthermore, the interactions reveal cryptic effects of individual loci that are not detectable by analyses of single mutants . Despite these strong genetic interactions, but consistent with the disparities in peak expression of these loci, none of the ABI transcription factors appear to interact directly with either FUS3 or LEC1 in a yeast (Saccharomyces cerevisiae) two-hybrid assay system. Plant Physiol, 2003 Jan, 131(1), 70 - 7 AtSTP6, a new pollen-specific H+-monosaccharide symporter from Arabidopsis; Scholz-Starke J et al.; This paper describes the molecular, kinetic, and physiological characterization of AtSTP6, a new member of the Arabidopsis H(+)/monosaccharide transporter family . The AtSTP6 gene (At3g05960) is interrupted by two introns and encodes a protein of 507 amino acids containing 12 putative transmembrane helices . Expression in yeast (Saccharomyces cerevisiae) shows that AtSTP6 is a high-affinity (K(m) = 20 microM), broad-spectrum, and uncoupler-sensitive monosaccharide transporter that is targeted to the plasma membrane and that can complement a growth deficiency resulting from the disruption of most yeast hexose transporter genes . Analyses of AtSTP6-promoter::GUS plants and in situ hybridization experiments detected AtSTP6 expression only during the late stages of pollen development . A transposon-tagged Arabidopsis mutant was isolated and homozygous plants were analyzed for potential effects of the Atstp6 mutation on pollen viability, pollen germination, fertilization, and seed production . However, differences between wild-type and mutant plants could not be observed. Mol Biol Cell, 2003 Jan, 14(1), 26 - 39 A Bni4-Glc7 phosphatase complex that recruits chitin synthase to the site of bud emergence; Kozubowski L et al.; Bni4 is a scaffold protein in the yeast Saccharomyces cerevisiae that tethers chitin synthase III to the bud neck by interacting with septin neck filaments and with Chs4, a regulatory subunit of chitin synthase III . We show herein that Bni4 is also a limiting determinant for the targeting of the type 1 serine/threonine phosphatase (Glc7) to the bud neck . Yeast cells containing a Bni4 variant that fails to associate with Glc7 fail to tether Chs4 to the neck, due in part to the failure of Bni4(V831A/F833A) to localize properly . Conversely, the Glc7-129 mutant protein fails to bind Bni4 properly and glc7-129 mutants exhibit reduced levels of Bni4 at the bud neck . Bni4 is phosphorylated in a cell cycle-dependent manner and Bni4(V831A/F833A) is both hyperphosphorylated and mislocalized in vivo . Yeast cells lacking the protein kinase Hsl1 exhibit increased levels of Bni4-GFP at the bud neck . GFP-Chs4 does not accumulate at the incipient bud site in either a bni4::TRP1 or a bni4(V831A/F833A) mutant but does mobilize to the neck at cytokinesis . Together, these results indicate that the formation of the Bni4-Glc7 complex is required for localization to the site of bud emergence and for subsequent targeting of chitin synthase. Mol Cell Biol, 2003 Feb, 23(3), 1025 - 33 Interaction of histone acetylases and deacetylases in vivo; Yamagoe S et al.; Having opposing enzymatic activities, histone acetylases (HATs) and deacetylases affect chromatin and regulate transcription . The activities of the two enzymes are thought to be balanced in the cell by an unknown mechanism that may involve their direct interaction . Using fluorescence resonance energy transfer analysis, we demonstrated that the acetylase PCAF and histone deacetylase 1 (HDAC1) are in close spatial proximity in living cells, compatible with their physical interaction . In agreement, coimmunoprecipitation assays demonstrated that endogenous HDACs are associated with PCAF and another acetylase, GCN5, in HeLa cells . We found by glycerol gradient sedimentation analysis that HATs are integrated into a large multiprotein HDAC complex that is distinct from the previously described HDAC complexes containing mSin3A, Mi-2/NRD, or CoREST . This HDAC-HAT association is partly accounted for by a direct protein-protein interaction observed in vitro . The HDAC-HAT complex may play a role in establishing a dynamic equilibrium of the two enzymes in vivo. Mol Cell Biol, 2003 Feb, 23(3), 842 - 51 Nonsense-containing mRNAs that accumulate in the absence of a functional nonsense-mediated mRNA decay pathway are destabilized rapidly upon its restitution; Maderazo AB et al.; Nonsense-mediated mRNA decay (NMD) is a conserved proofreading mechanism that protects eukaryotic cells from the potentially deleterious effects of truncated proteins . Studies of Saccharomyces cerevisiae imply that NMD is a predominantly cytoplasmic decay pathway, while studies of mammalian systems suggest that decay of most substrate mRNAs may occur while they are still associated with the nucleus, possibly during a round of translation that occurs during their export to the cytoplasm . Complete entry of the latter mRNAs into the cytoplasm appears to render them immune to further NMD; i.e., they escape further susceptibility to this decay pathway . To determine if yeast cytoplasmic nonsense-containing mRNAs that evade decay are subsequently immune to NMD, we examined the consequences of placing each of the three UPF/NMD genes under the control of a galactose-inducible promoter . The decay kinetics of ADE2 and PGK1 nonsense-containing mRNAs were then analyzed when expression of UPF1, NMD2, or UPF3 was either repressed or subsequently induced . Results from these experiments demonstrated that activation of NMD caused rapid and immediate degradation of both substrate transcripts, with half-lives of both stable mRNA populations shortened to approximately 7 min . These findings make it unlikely that yeast nonsense-containing mRNAs can escape degradation by NMD and indicate that such mRNAs are available to this decay pathway at each round of translation. J Biol Chem, 2003 Apr 4, 278(14), 12356 - 60 Epub 2003 Jan 15. Structure and enzymatic properties of a stable complex of the human REV1 and REV7 proteins; Masuda Y et al.; With yeast Saccharomyces cerevisiae, results from a variety of genetic and biochemical investigations have demonstrated that the REV genes play a major role in induction of mutations through replication processes that directly copy the damaged DNA template during DNA replication . However, in higher eucaryotes functions of homologues are poorly understood and appear somewhat different from the yeast case . It has been suggested that human REV1 interacts with human REV7, this being specific to higher eucaryotes . Here we show that purified human REV1 and REV7 proteins form a heterodimer in solution, which is stable through intensive purification steps . Results from biochemical analysis of the transferase reactions of the REV1-REV7 complex demonstrated, in contrast to the case of yeast Rev3 whose polymerase activity is stimulated by assembly with yeast Rev7, that human REV7 did not influence the stability, substrate specificity, or kinetic parameters of the transferase reactions of REV1 protein . The possible role of human REV7 is discussed. J Biol Chem, 2003 Apr 4, 278(14), 12278 - 84 Epub 2003 Jan 14. Supplying copper to the cuproenzyme peptidylglycine alpha-amidating monooxygenase; El Meskini R et al.; We explored the role of known copper transporters and chaperones in delivering copper to peptidylglycine-alpha-hydroxylating monooxygenase (PHM), a copper-dependent enzyme that functions in the secretory pathway lumen . We examined the roles of yeast Ccc2, a P-type ATPase related to human ATP7A (Menkes disease protein) and ATP7B (Wilson disease protein), as well as yeast Atx1, a cytosolic copper chaperone . We expressed soluble PHMcc (catalytic core) in yeast using the yeast pre-pro-alpha-mating factor leader region to target the enzyme to the secretory pathway . Although the yeast genome encodes no PHM-like enzyme, PHMcc expressed in yeast is at least as active as PHMcc produced by mammalian cells . PHMcc partially co-migrated with a Golgi marker during subcellular fractionation and partially co-localized with Ccc2 based on immunofluorescence . To determine whether production of active PHM was dependent on copper trafficking pathways involving the CCC2 or ATX1 genes, we expressed PHMcc in wild-type, ccc2, and atx1 mutant yeast . Although ccc2 and atx1 mutant yeast produce normal levels of PHMcc protein, it lacks catalytic activity . Addition of exogenous copper yields fully active PHMcc . Similarly, production of active PHM in mouse fibroblasts is impaired in the presence of a mutant ATP7A gene . Although delivery of copper to lumenal cuproproteins like PAM involves ATP7A, lumenal chaperones may not be required. Biochem J, 2003 Apr 15, 371(Pt 2), 395 - 403 The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor-associated factors 1, 2, 3 and 5 and mediates nuclear factor-kappaB activation; Brown SA et al.; Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein . The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis) . In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein . Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif . We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory kappaBalpha phosphorylation and transcriptional activation of a nuclear factor-kappaB (NF-kappaB) enhancer/luciferase reporter construct . Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-kappaB activation, and this cellular response requires an intact TRAF binding site . These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-kappaB transcription factor signalling pathway. Biochem J, 2003 Mar 15, 370(Pt 3), 1077 - 85 Docking sites on mitogen-activated protein kinase (MAPK) kinases, MAPK phosphatases and the Elk-1 transcription factor compete for MAPK binding and are crucial for enzymic activity; Bardwell AJ et al.; Mitogen-activated protein kinase (MAPK) cascades control gene expression patterns in response to extracellular stimuli . MAPK/ERK (extracellular-signal-regulated kinase) kinases (MEKs) activate MAPKs by phosphorylating them; activated MAPKs, in turn, phosphorylate target transcription factors, and are deactivated by phosphatases . One mechanism for maintaining signal specificity and efficiency is the interaction of MAPKs with their substrates and regulators through high-affinity docking sites . In the present study, we show that peptides corresponding to the MAPK-docking sites of MEK1, MEK2, Ste7, Elk-1 and MAPK phosphatase (MKP)-2 potently inhibit MEK2 phosphorylation of ERK2, ERK2 phosphorylation of Elk-1, and MKP-1 dephosphorylation of ERK2 . Each peptide inhibited multiple reactions; for example, the MEK2 peptide inhibited not only MEK2, but also ERK2 and MKP-1 . In addition, these docking-site peptides inhibited MEK2-ERK2 binding . The MAPK-docking site of MEK1 also potently stimulated ERK2-mediated phosphorylation of a target site on the same peptide . Control peptides with mutations of conserved basic and hydrophobic residues of the MAPK-docking site consensus lacked biological activity . We conclude that MEKs, MKPs and the Elk-1 transcription factor compete for binding to the same region of ERK2 via protein-protein interactions that are crucial for kinase/phosphatase activity. Oncogene, 2003 Jan 16, 22(2), 246 - 55 Ras-mediated apoptosis of PC CL 3 rat thyroid cells induced by RET/PTC oncogenes; Castellone MD et al.; RET gene rearrangements, which generate chimeric RET/PTC oncogenes, are early events in the evolution of thyroid papillary carcinomas . Expression of RET/PTC oncogenes promotes neoplastic transformation of cultured thyroid cells and of thyroid glands in transgenic mice . Notwithstanding these oncogenic effects, we have found that the expression of two RET/PTC oncogenes (H4-RET and RFG-RET) induces apoptosis of rat thyroid PC CL 3 cells . Promotion of thyroid cell death depends on the kinase activity of RET/PTC and on the phosphorylation of a tyrosine residue (tyrosine 1062) that maps in the carboxy-terminus of the RET protein . Tyrosine 1062 is essential for RET/PTC-mediated activation of the Ras/ERK pathway . Inhibition of Ras/ERK by a dominant negative Ras or by the MEKI inhibitor, PD98059, obstructed RET/PTC-mediated apoptosis . We also show that signals transmitted by tyrosine 1062 mediate proapoptotic events like Bcl-2 down regulation and Bax upregulation, and that adoptive overexpression of Bcl-2 overcomes RET/PTC-induced apoptosis . Thus, gene rearrangements that generate RET/PTC oncogenes subvert RET function by converting it into a chronically active kinase that is constitutively phosphorylated on tyrosine 1062 . In turn, Y1062 phosphorylation transmits not only mitogenic but also proapoptotic signals to thyroid cells. Nucleic Acids Res, 2003 Jan 15, 31(2), 653 - 60 The phylogenetic diversity of eukaryotic transcription; Coulson RM et al.; Eukaryotic transcription is a highly regulated process involving interactions between large numbers of proteins . To analyse the phylogenetic distribution of the components of this process, six crown eukaryote group genomes were queried with a reference set of transcription-associated (TA) proteins . On average, one in 10 proteins encoded by these genomes were found to be homologous to sequences in the reference set . Analysis of families identified using an accurate sequence clustering algorithm and containing both TA proteins and eukaryotic sequences showed that in two-thirds of the families the homologues originate from a single kingdom . Furthermore, in only 15% of the fungal-specific clusters are the homologues present in both budding and fission yeast, as compared with the metazoan-specific clusters where 53% of the homologues originate from two or more species . Families whose members comprise general transcription factor or RNA polymerase subunits exhibit a low degree of taxon specificity, suggesting that the transcription initiation complex is highly conserved . This contrasts with transcriptional regulator families, that are primarily taxon-specific, indicating proteins controlling gene activation exhibit considerable sequence diversity across the eukaryotic domain. Nucleic Acids Res, 2003 Jan 15, 31(2), 619 - 28 Putative subunits of the maize origin of replication recognition complex ZmORC1-ZmORC5; Witmer X et al.; The finding in animal species of complexes homologous to the products of six Saccharomyces cerevisiae genes, origin of replication recognition complex (ORC), has suggested that ORC-related mechanisms have been conserved in all eukaryotes . In plants, however, the only cloned putative homologs of ORC subunits are the Arabidopsis ORC2 and the rice ORC1 . Homologs of other subunits of plant origin have not been cloned and characterized . A striking observation was the absence from the Arabidopsis genome of an obvious candidate gene-homolog of ORC4 . This fact raised compelling questions of whether plants, in general, and Arabidopsis, in particular, may have lost the ORC4 gene, whether ORC-homologous subunits function within a complex in plants, whether an ORC complex may form and function without an ORC4 subunit, whether a functional (but not sequence) protein homolog may have taken up the role of ORC4 in Arabidopsis, and whether lack of ORC4 is a plant feature, in general . Here, we report the first cloned and molecularly characterized five genes coding for the maize putative homologs of ORC subunits ZmORC1, ZmORC2, ZmORC3, ZmORC4 and ZmORC5 . Their expression profiles in tissues with different cell-dividing activities are compatible with a role in DNA replication . Based on the potential of ORC-homologous maize proteins to bind each other in yeast, we propose a model for their possible assembly within a maize ORC . The isolation and molecular characterization of an ORC4-homologous gene from maize argues that, in its evolution, Arabidopsis may have lost the homologous ORC4 gene. J Biol Chem, 2003 Mar 21, 278(12), 10322 - 7 Epub 2003 Jan 13. Glucose-mediated phosphorylation converts the transcription factor Rgt1 from a repressor to an activator; Mosley AL et al.; Glucose, the most abundant carbon and energy source, regulates the expression of genes required for its own efficient metabolism . In the yeast Saccharomyces cerevisiae, glucose induces the expression of the hexose transporter (HXT) genes by modulating the activity of the transcription factor Rgt1 that functions as a repressor when glucose is absent . However, in the presence of high concentrations of glucose, Rgt1 is converted from a repressor to an activator and is required for maximal induction of HXT1 gene expression . We report that Rgt1 binds to the HXT1 promoter only in the absence of glucose, suggesting that Rgt1 increases HXT1 gene expression at high levels of glucose by an indirect mechanism . It is likely that Rgt1 stimulates the expression of an activator of the HXT1 gene at high concentrations of glucose . In addition, we demonstrate that Rgt1 becomes hyperphosphorylated in response to high glucose levels and that this phosphorylation event is required for Rgt1 to activate transcription . Furthermore, Rgt1 lacks the glucose-mediated phosphorylation in the snf3 rgt2 and grr1 mutants, which are defective in glucose induction of HXT gene expression . In these mutants, Rgt1 behaves as a constitutive repressor independent of the carbon source . We conclude that phosphorylation of Rgt1 in response to glucose is required to abolish the Rgt1-mediated repression of the HXT genes and to convert Rgt1 from a transcriptional repressor to an activator. Gene, 2003 Jan 2, 302(1-2), 147 - 53 Diversity of mouse proton-translocating ATPase: presence of multiple isoforms of the C, d and G subunits; Sun-Wada GH et al.; Vacuolar-type proton-translocating ATPases (V-ATPases), multimeric proton pumps, are involved in a wide variety of physiological processes . For their diverse functions, V-ATPases utilize a specific subunit isoform(s) . Here, we reported the molecular cloning and characterization of three novel subunit isoforms, C2, d2 and G3, of mouse V-ATPase . These isoforms were expressed in a tissue-specific manner, in contrast to the ubiquitously expressed C1, d1 and G1 isoforms . C2 was expressed predominantly in lung and kidney, and d2 and G3 specifically in kidney . We introduced these isoforms into yeasts lacking the corresponding genes . Although the G3 and d2 did not rescue the vmaDelta phenotype, d1 and the two C isoforms functionally complemented the Deltavma6 and Deltavma5, respectively, indicating that they are bona fide subunits of V-ATPase. Gene, 2003 Jan 2, 302(1-2), 53 - 64 Evidence implicating Ku antigen as a structural factor in RNA polymerase II-mediated transcription; Bertinato J et al.; Ku antigen is an abundant nuclear protein with multiple functions that depend mainly on Ku's prolific and highly verstatile interactions with DNA . We have shown previously that the direct binding of Ku in vitro to negative regulatory element 1 (NRE1), a transcriptional regulatory element in the long terminal repeat of mouse mammary tumour virus, correlates with the regulation of viral transcription by Ku . In this study, we have sought to explore the interaction of Ku with NRE1 in vivo in yeast one-hybrid experiments . Unexpectedly, we observed that human Ku70 carrying a transcriptional activation domain from the yeast Gal4 protein induced transcription of yeast reporter genes pleiotrophically, independent of NRE1, promoter, reporter gene and chromosomal location . Ku80 with the same activation domain had no effect on transcription when expressed alone, but reconstituted activation when co-expressed with native human Ku70 . The requirements for transcriptional activation by Ku-Gal4 activation domain proteins correlated with previous descriptions of the requirements for DNA sequence-independent DNA binding by Ku, but were distinct from determinants for DNA-end binding by a truncated Ku heterodimer determined recently by crystallography . These results suggest a preferential targeting of Ku to transcriptionally active chromatin that indicate a possible function for Ku within the RNA polymerase II holoenzyme. Cell, 2002 Dec 13, 111(6), 791 - 802 Physical and functional interactions among basic chromosome organizational features govern early steps of meiotic chiasma formation; Blat Y et al.; Analysis of meiotic recombination by functional genomic approaches reveals prominent spatial and functional interactions among diverse organizational determinants . Recombination occurs between chromatin loop sequences; however, these sequences are spatially tethered to underlying chromosome axes via their recombinosomes . Meiotic chromosomal protein, Red1, localizes to chromosome axes; however, Red1 loading is modulated by R/G-bands isochores and thus by bulk chromatin state . Recombination is also modulated by isochore determinants: R-bands differentially favor double-strand break (DSB) formation but disfavor subsequent loading of meiotic RecA homolog, Dmc1 . Red1 promotes DSB formation in both R- and G-bands and then promotes Dmc1 loading, specifically counteracting disfavoring R-band effects . These complexities are discussed in the context of chiasma formation as a series of coordinated local changes at the DNA and chromosome-axis levels. Cell, 2003 Jan 10, 112(1), 41 - 50 Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70; Young JC et al.; The role of cytosolic factors in protein targeting to mitochondria is poorly understood . Here, we show that in mammals, the cytosolic chaperones Hsp90 and Hsp70 dock onto a specialized TPR domain in the import receptor Tom70 at the outer mitochondrial membrane . This interaction serves to deliver a set of preproteins to the receptor for subsequent membrane translocation dependent on the Hsp90 ATPase . Disruption of the chaperone/Tom70 recognition inhibits the import of these preproteins into mitochondria . In yeast, Hsp70 rather than Hsp90 is used in import, and Hsp70 docking is required for the formation of a productive preprotein/Tom70 complex . We outline a novel mechanism in which chaperones are recruited for a specific targeting event by a membrane-bound receptor. Cell, 2003 Jan 10, 112(1), 11 - 7 Regulating the regulators: lysine modifications make their mark; Freiman RN et al.; Decades of research have uncovered much of the molecular machinery responsible for establishing and maintaining proper gene transcription patterns in eukaryotes . Although the composition of this machinery is largely known, mechanisms regulating its activity by covalent modification are just coming into focus . Here, we review several cases of ubiquitination, sumoylation, and acetylation that link specific covalent modification of the transcriptional apparatus to their regulatory function . We propose that potential cascades of modifications serve as molecular rheostats that fine-tune the control of transcription in diverse organisms. Curr Biol, 2003 Jan 8, 13(1), R31 - 8 Periodic transcription: a cycle within a cycle; Breeden LL; Studies in model organisms indicate that one in every five genes may be subject to cell cycle regulated transcription . Moreover, a high proportion of periodically expressed genes have discrete roles in the cell division process, and their peaks of expression coincide with the interval during which they function . This periodic transcription is commonly regulated by transcription factors that are also periodically transcribed, and there is a growing number of examples where the transcription factors and their targets are conserved in yeast and mammalian cells . As such, it is worth considering why these regulatory circuits persist in such great number, how they are achieved and what role they may play in the cell cycle. Curr Biol, 2003 Jan 8, 13(1), 78 - 84 EGF regulation of PITP dynamics is blocked by inhibitors of phospholipase C and of the Ras-MAP kinase pathway; Larijani B et al.; Phosphatidylinositol transfer proteins (PITP) function in signal transduction and in membrane traffic . Studies aimed at elucidating the mechanism of action of PITP have yielded a singular theme; the activity of PITP stems from its ability to transfer phosphatidylinositol (PI) from its site of synthesis to sites of cellular activity and to stimulate the local synthesis of phosphorylated forms of PI . The participation of various phosphoinositides in EGF signal transduction and in the trafficking of the EGF receptors is well documented . Using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET) between EGFP-PITP proteins and fluorescently labeled phospholipids, we report that PITPalpha and PITPbeta can dynamically interact with PI or PC at the plasma membrane when stimulated with EGF . Additionally, PITPbeta is localized at the Golgi, and EGF stimulation resulted in enhanced FRET . Inhibitors of the PLC and the Ras/MAP kinase pathway were both able to inhibit the EGF-stimulated interaction of PITPalpha with PI at the plasma membrane . The mobility of PITP proteins was determined by using fluorescence recovery after photobleaching (FRAP), and EGF stimulation reduced the mobility at the plasma membrane . We conclude that the dynamic behavior of PITPalpha and PITPbeta in vivo is a regulated process involving multiple mechanisms. Curr Biol, 2003 Jan 8, 13(1), 68 - 72 A new role for the transcriptional corepressor SIN3; regulation of centromeres; Silverstein RA et al.; Centromeres play a vital role in maintaining the genomic stability of eukaryotes by coordinating the equal distribution of chromosomes to daughter cells during mitosis and meiosis . Fission yeast (S . pombe) centromeres consist of a 4-9 kb central core region and 30-100 kb of flanking inner (imr/B) and outer (otr/K) repeats . These sequences direct a laminar kinetochore structure similar to that of human centromeres . Centromeric heterochromatin is generally underacetylated . We have previously shown that inhibition of histone deacetylases (HDACs) caused hyperacetylation of centromeres and defective chromosome segregation . SIN3 is a HDAC corepressor that has the ability to mediate HDAC targeting in the repression of promoters . In this study, we have characterized S . pombe sin three corepressors (Pst1p and Pst2p) to investigate whether SIN3-HDAC is required in the regulation of centromeres . We show that only pst1-1 and not pst2Delta cells displayed anaphase defects and thiabendazole sensitivity . pst1-1 cells showed reduced centromeric silencing, increased histone acetylation in centromeric chromatin, and defective centromeric sister chromatid cohesion . The HDAC Clr6p and Pst1p coimmunoprecipitated, and Pst1p colocalized with centromeres, particularly in binucleate cells . These data are consistent with a model in which Pst1p-Clr6p temporally associate with centromeres to carry out the initial deacetylation necessary for subsequent steps in heterochromatin formation. Exp Physiol, 2003 Jan, 88(1), 57 - 64 Import of nuclear-encoded proteins into mitochondria; Stojanovski D et al.; The majority of mitochondrial proteins are encoded by nuclear genes, synthesized in the cytosol and subsequently imported into mitochondria through protein translocation machineries of the outer and inner membranes . In this review, we discuss the arrangement of the various translocation complexes and the function of individual import components . We also outline the various targeting pathways that preproteins can take in order to reach their appropriate sub-mitochondrial compartment. J Virol, 2003 Feb, 77(3), 2010 - 20 Importance of basic residues in binding of rous sarcoma virus nucleocapsid to the RNA packaging signal; Lee EG et al.; In the context of the Rous sarcoma virus Gag polyprotein, only the nucleocapsid (NC) domain is required to mediate the specificity of genomic RNA packaging . We have previously showed that the Saccharomyces cerevisiae three-hybrid system provides a rapid genetic assay to analyze the RNA and protein components of the avian retroviral RNA-Gag interactions necessary for specific encapsidation . In this study, using both site-directed mutagenesis and in vivo random screening in the yeast three-hybrid binding assay, we have examined the amino acids in NC required for genomic RNA binding . We found that we could delete either of the two Cys-His boxes without greatly abrogating either RNA binding or packaging, although the two Cys-His boxes are likely to be required for efficient viral assembly and release . In contrast, substitutions for the Zn-coordinating residues within the boxes did prevent RNA binding, suggesting changes in the overall conformation of the protein . In the basic region between the two Cys-His boxes, three positively charged residues, as well as basic residues flanking the two boxes, were necessary for both binding and packaging . Our results suggest that the stretches of positively charged residues within NC that need to be in a proper conformation appear to be responsible for selective recognition and binding to the packaging signal (Psi)-containing RNAs. J Med Genet, 2003 Jan, 40(1), 11 - 7 A third MRX family (MRX68) is the result of mutation in the long chain fatty acid-CoA ligase 4 (FACL4) gene: proposal of a rapid enzymatic assay for screening mentally retarded patients; Longo I et al.; BACKGROUND: The gene encoding fatty acid CoA ligase 4 (FACL4) is mutated in families with non-specific X linked mental retardation (MRX) and is responsible for cognitive impairment in the contiguous gene syndrome ATS-MR (Alport syndrome and mental retardation), mapped to Xq22.3 . This finding makes this gene a good candidate for other mental retardation disorders mapping in this region . METHODS: We have screened the FACL4 gene in eight families, two MRX and six syndromic X linked mental retardation (MRXS), mapping in a large interval encompassing Xq22.3 . RESULTS: We have found a missense mutation in MRX68 . The mutation (c.1001C>T in the brain isoform) cosegregates with the disease and changes a highly conserved proline into a leucine (p.P375L) in the first luciferase domain, which markedly reduces the enzymatic activity . Furthermore, all heterozygous females showed completely skewed X inactivation in blood leucocytes, as happens in all reported females with other FACL4 point mutations or deletions . CONCLUSIONS: Since the FACL4 gene is highly expressed in brain, where it encodes a brain specific isoform, and is located in hippocampal and cerebellar neurones, a role for this gene in cognitive processes can be expected . Here we report the third MRX family with a FACL4 mutation and describe the development of a rapid enzymatic assay on peripheral blood that we propose as a sensitive, robust, and efficient diagnostic tool in mentally retarded males. J Biol Chem, 2003 Mar 21, 278(12), 10328 - 33 Epub 2003 Jan 13. Sti1 is a non-competitive inhibitor of the Hsp90 ATPase . Binding prevents the N-terminal dimerization reaction during the atpase cycle; Richter K et al.; The molecular chaperone Hsp90 is known to be involved in the activation of key regulatory proteins such as kinases, steroid hormone receptors, and transcription factors in an ATP-dependent manner . During the chaperone cycle, Hsp90 has been found associated with the partner protein Hop/Sti1, which seems to be required for the progression of the cycle . However, little is known about its specific function . Here we have investigated the interaction of Sti1 from Saccharomyces cerevisiae with Hsp90 and its influence on the ATPase activity . We show that the inhibitory mechanism of Sti1 on the ATPase activity of Hsp90 is non-competitive . Sti1 binds to the N- and C-terminal part of Hsp90 and prevents the N-terminal dimerization reaction that is required for efficient ATP hydrolysis . The first 24 amino acids of Hsp90, a region shown previously to be important for the association of the N-terminal domains and stimulation of ATP hydrolysis, seems to be important for this interaction. BMC Bioinformatics . 2003 Jan 13;4(1):2. An automated method for finding molecular complexes in large protein interaction networks; Bader GD et al.; BACKGROUND: Recent advances in proteomics technologies such as two-hybrid, phage display and mass spectrometry have enabled us to create a detailed map of biomolecular interaction networks . Initial mapping efforts have already produced a wealth of data . As the size of the interaction set increases, databases and computational methods will be required to store, visualize and analyze the information in order to effectively aid in knowledge discovery . RESULTS: This paper describes a novel graph theoretic clustering algorithm, "Molecular Complex Detection" (MCODE), that detects densely connected regions in large protein-protein interaction networks that may represent molecular complexes . The method is based on vertex weighting by local neighborhood density and outward traversal from a locally dense seed protein to isolate the dense regions according to given parameters . The algorithm has the advantage over other graph clustering methods of having a directed mode that allows fine-tuning of clusters of interest without considering the rest of the network and allows examination of cluster interconnectivity, which is relevant for protein networks . Protein interaction and complex information from the yeast Saccharomyces cerevisiae was used for evaluation . CONCLUSION: Dense regions of protein interaction networks can be found, based solely on connectivity data, many of which correspond to known protein complexes . The algorithm is not affected by a known high rate of false positives in data from high-throughput interaction techniques . The program is available from ftp://ftp.mshri.on.ca/pub/BIND/Tools/MCODE. Biochemistry, 2003 Jan 21, 42(2), 535 - 42 Glycolipid intermembrane transfer is accelerated by HET-C2, a filamentous fungus gene product involved in the cell-cell incompatibility response; Mattjus P et al.; Among filamentous fungi capable of mycelial growth, het genes play crucial roles by regulating heterokaryon formation between different individuals . When fusion occurs between fungal mycelia that differ genetically at their het loci, the resulting heterokaryotic cells are quickly destroyed . It is unclear how het gene products of Podospora anserina trigger heterokaryon incompatibility . One unexplored possibility is that glycosphingolipids play a role because the het-c2 gene encodes a protein that displays 32% sequence identity and an additional 30% similarity to the mammalian glycolipid transfer protein . Here, P . anserina protoplasts containing wild-type het-c2 genes were shown to have greater glycosphingolipid transfer activity than protoplasts with disrupted het-c2 genes, a condition previously linked to altered cell compatibility following hyphal fusion . The observed glycolipid transfer activity could not be accounted for by nonspecific lipid transfer protein activity . Direct assessment showed that purified, recombinant HET-C2 accelerates the intermembrane transfer of glycolipid in vitro, but that the HET-C2 activity is mitigated much less by negatively charged membranes than the mammalian glycolipid transfer protein . The findings are discussed within the context of HET-C2 being a member of an emerging family of ancestral sphingolipid transfer proteins that play important roles in cell proliferation and accelerated death. Biochemistry, 2003 Jan 21, 42(2), 486 - 96 Mechanism of formin-induced nucleation of actin filaments; Pring M et al.; A fragment of the yeast formin Bni1 containing the FH1FH2 domains increases the rate of filament nucleation from pure G-actin {Pruyne et al . (2002) Science 297, 612-615} . To determine the mechanism of nucleation, we compared the G-actin dependence of Bni1FH1FH2-induced polymerization with theoretical models . The data best fit a model suggesting that Bni1FH1FH2 stabilizes an actin dimer . We also show that nucleation increases with the square root of the Bni1FH1FH2 concentration . We demonstrate that this relationship is expected for any such nucleator, independent of nucleus size . The proline-rich FH1 domain binds profilin, and deletion of this domain decreases the contribution of profilin-actin to the nucleation . A role for profilin binding to the FH1 domain in filament nucleation was supported by the inability of Bni1FH1FH2 to utilize a mutant profilin, H133S profilin, with defective binding to polyproline . Bni1FH1FH2 partially inhibits barbed-end elongation, and we find that the rate constants for both polymerization and depolymerization are decreased by approximately 50% . Bni1FH1FH2 has no effect on pointed-end kinetics or on the critical concentration . To investigate the domains of Bni1 required for these activities, the experiments were all duplicated with the FH2 domain alone . The FH2 domain is as effective as the FH1FH2 domains together in inhibiting barbed-end kinetics; it is less effective as a nucleator but the mechanism is again best fit by dimer stabilization. Biochemistry, 2003 Jan 21, 42(2), 450 - 7 Polycystin-2 interacts with troponin I, an angiogenesis inhibitor; Li Q et al.; Polycystin-2 (PC2), encoded by the PKD2 gene, is mutated in 10-15% of autosomal dominant polycystic kidney disease (ADPKD) patients . PC2 is a Ca(2+)-permeable nonselective cation channel and is present in kidney and many other organs . Likewise, PKD2-mutated patients and mice exhibit extrarenal abnormalities . In comparison with cysts in the kidney, liver, and pancreas, abnormalities in the heart, brain, and vascular vessels are less understood . In particular, roles of PC2 in muscle and endothelia remain largely unknown . In the present study, using a yeast two-hybrid screening, we discovered that the PC2 carboxyl terminal domain (D682-V968) interacts with the cardiac troponin I, an important regulatory component of the actin microfilament in cardiac muscle cells . This interaction was demonstrated by GST pull-down and microtiter binding assays . Dose-dependent binding between PC2 and troponin I followed a Michaelis-Menten relationship, indicating a 1:1 binding stoichiometry . The interacting domains were located to the R872-H927 segment of PC2 and the M1-V107 and K106-L158 segments of troponin I . Co-immunoprecipitation experiments demonstrated that the cardiac and two skeletal isoforms of troponin I were all associated with PC2, when coexpressed in mouse fibroblast NIH 3T3 cells and Xenopus oocytes . Furthermore, reciprocal co-immunoprecipitation verified the interaction between the native polycystin-2 and troponin I in human adult heart tissues . This study thus provides new evidence for a direct attachment of PC2 to the actin microfilament network, in addition to the recently identified association between PC2 and trypomyosin-1 . Troponin I functions as an inhibitory subunit of the troponin complex for calcium-dependent regulation of muscle contraction and as an inhibitor of angiogenesis seen in ADPKD . It is possible that altered interaction due to pathogenic polycystin-1 or -2 mutations can account for angiogenesis in ADPKD and may be corrected to some extent by exogenous troponin I. Biochemistry, 2003 Jan 21, 42(2), 293 - 301 Aromatic residues at the extracellular ends of transmembrane domains 5 and 6 promote ligand activation of the G protein-coupled alpha-factor receptor; Lin JC et al.; The alpha-factor receptor (STE2) stimulates a G protein signaling pathway that promotes mating of the yeast Saccharomyces cerevisiae . Previous random mutagenesis studies implicated residues in the regions near the extracellular ends of the transmembrane domains in ligand activation . In this study, systematic Cys scanning mutagenesis across the ends of transmembrane domains 5 and 6 identified two residues, Phe(204) and Tyr(266), that were important for receptor signaling . These residues play a specific role in responding to alpha-factor since the F204C and Y266C substituted receptors responded to an alternative agonist (novobiocin) . To better define the structure of this region, the Cys-substituted mutant receptors were assayed for reactivity with a thiol-specific probe that does not react with membrane-imbedded residues . A drop in reactivity coincided with residues likely to be buried in the membrane . Interestingly, both Phe(204) and Tyr(266) are located very near the interface region . However, these assays predict that Phe(204) is accessible at the surface of the receptor, consistent with the strong defect in binding alpha-factor caused by mutating this residue . In contrast, Tyr(266) was not accessible . This correlates with the ability of Y266C mutant receptors to bind alpha-factor and suggests that this residue is involved in the subsequent triggering of receptor activation . These results highlight the role of aromatic residues near the ends of the transmembrane segments in the alpha-factor receptor, and suggest that similar aromatic residues may play an important role in other G protein-coupled receptors. Zhongguo Zhong Yao Za Zhi, 2001 Feb, 26(2), 119 - 21 {Studies on the semi-biosynthesis of L-ephedrine (I) . Studies on the biosynthesis of L-phenyl acetyl carbinol}; Xu XY et al.; OBJECTIVE: The optimal biosynthesis of L-PAC by immobilized Sc-5 yeast cell was systematically studied in this paper . METHOD: Biosynthesis . RESULT AND CONCLUSION: The average of L-PAC yield produced by the immobilized Sc-5 yeast cell is > or = 2 g.L-1 and half life > or = 240 h. Nat Genet, 2003 Feb, 33(2), 155 - 61 Epub 2003 Jan 13. Genome-wide analysis of RNA-protein interactions illustrates specificity of the mRNA export machinery; Hieronymus H et al.; Nuclear export of mRNA is mediated by a complex machinery of RNA-binding proteins that recognizes and routes mRNAs through a messenger ribonucleoprotein (mRNP) network . The full spectrum of mRNA cargoes for any dedicated mRNA export factor is unknown . We identified the mRNAs that bind two conserved yeast mRNA export factors, Yra1 (refs . 1-5) and Mex67 (refs . 6,7), on a genome-wide scale and determined their level of binding . Yra1 and Mex67 bind approximately 1,000 and 1,150 mRNAs, respectively, corresponding to almost 20% of the yeast genome and roughly 36% of all transcriptional events each . The binding level of Yra1 targets is related to their transcriptional frequency, but that of Mex67 targets is not . Yra1-bound transcripts are enriched in mRNAs that are regulated by a number of transcription factors . Yra1- and Mex67-bound populations also show enrichment of mRNAs encoding distinct functional classes of proteins, some of which are regulated by these transcription factors . We determined that one such transcription factor, Abf1 (refs . 8-10), associates with Yra1 . These results indicate a previously unidentified specificity of mRNA export factors, which coordinates the export of transcriptionally co-regulated, functional classes of transcripts, perhaps through interactions with the transcriptional machinery. Genetics, 2002 Dec, 162(4), 1675 - 85 A Drosophila homologue of Sir2 modifies position-effect variegation but does not affect life span; Newman BL et al.; Control of chromosome structure is important in the regulation of gene expression, recombination, DNA repair, and chromosome stability . In a two-hybrid screen for proteins that interact with the Drosophila CREB-binding protein (dCBP), a known histone acetyltransferase and transcriptional coactivator, we identified the Drosophila homolog of a yeast chromatin regulator, Sir2 . In yeast, Sir2 silences genes via an intrinsic NAD(+)-dependent histone deacetylase activity . In addition, Sir2 promotes longevity in yeast and in Caenorhabditis elegans . In this report, we characterize the Drosophila Sir2 (dSir2) gene and its product and describe the generation of dSir2 amorphic alleles . We found that dSir2 expression is developmentally regulated and that dSir2 has an intrinsic NAD(+)-dependent histone deacetylase activity . The dSir2 mutants are viable, fertile, and recessive suppressors of position-effect variegation (PEV), indicating that, as in yeast, dSir2 is not an essential function for viability and is a regulator of heterochromatin formation and/or function . However, mutations in dSir2 do not shorten life span as predicted from studies in yeast and worms. Biophys J, 2003 Jan, 84(1), 124 - 35 The importance of thermodynamic equilibrium for high throughput gene expression arrays; Bhanot G et al.; We present an analysis of physical chemical constraints on the accuracy of DNA micro-arrays under equilibrium and nonequilibrium conditions . At the beginning of the article we describe an algorithm for choosing a probe set with high specificity for targeted genes under equilibrium conditions . The algorithm as well as existing methods is used to select probes from the full Saccharomyces cerevisiae genome, and these probe sets, along with a randomly selected set, are used to simulate array experiments and identify sources of error . Inasmuch as specificity and sensitivity are maximum at thermodynamic equilibrium, we are particularly interested in the factors that affect the approach to equilibrium . These are analyzed later in the article, where we develop and apply a rapidly executable method to simulate the kinetics of hybridization on a solid phase support . Although the difference between solution phase and solid phase hybridization is of little consequence for specificity and sensitivity when equilibrium is achieved, the kinetics of hybridization has a pronounced effect on both . We first use the model to estimate the effects of diffusion, crosshybridization, relaxation time, and target concentration on the hybridization kinetics, and then investigate the effects of the most important kinetic parameters on specificity . We find even when using probe sets that have high specificity at equilibrium that substantial crosshybridization is present under nonequilibrium conditions . Although those complexes that differ from perfect complementarity by more than a single base do not contribute to sources of error at equilibrium, they slow the approach to equilibrium dramatically and confound interpretation of the data when they dissociate on a time scale comparable to the time of the experiment . For the best probe set, our simulation shows that steady-state behavior is obtained in a relaxation time of approximately 12-15 h for experimental target concentrations approximately (10(-13) - 10(-14))M, but the time is greater for lower target concentrations in the range (10(-15)-10(-16))M . The result points to an asymmetry in the accuracy with which up- and downregulated genes are identified. J Chem Ecol, 2002 Nov, 28(11), 2141 - 68 Antifungal leaf-surface metabolites correlate with fungal abundance in sagebrush populations; Talley SM et al.; A central component in understanding plant-enemy interactions is to determine whether plant enemies, such as herbivores and pathogens, mediate the evolution of plant secondary metabolites . Using 26 populations of a broadly distributed plant species, sagebrush (Artemisia tridentata), we examined whether sagebrush populations in habitats with a greater prevalence of fungi contained antifungal secondary metabolites on leaf surfaces that were more active and diverse than sagebrush populations in habitats less favorable to fungi . Because moisture and temperature play a key role in the epidemiology of most plant-pathogen interactions, we also examined the relationship between the antifungal activity of secondary metabolites and the climate of a site . We evaluated the antifungal activity of sagebrush secondary metabolites against two fungi, a wild Penicillium sp . and a laboratory yeast, Saccharomyces cerevisiae, using a filter-paper disk assay and bioautography . Comparing the 26 sagebrush populations, we found that fungal abundance was a good predictor of both the activity (r2 = 0.36 for Saccharomyces, r2 = 0.37 for Penicillium) and number (r2 = 0.34 for Saccharomyces) of antifungal secondary metabolites . This suggests that selection imposed by fungal pathogens has led to more effective antifungal secondary metabolites . We found that the antifungal activity of sagebrush secondary metabolites was negatively related to average vapor pressure deficit of the habitat (r2 = 0.60 for Saccharomyces, r2 = 0.61 for Penicillium) . Differences in antifungal activity among populations were not due to the amount of secondary metabolites, but rather to qualitative differences in the composition of antifungal compounds . Although all populations in habitats with high fungal prevalence had secondary metabolites with high antifungal activity, different suites of compounds were responsible for this activity, suggesting independent outcomes of selection on plants by fungal pathogens . The location of antifungal secondary metabolites on the leaf surface is consistent with their putative defense role, and we found no evidence supporting other functions, such as protection from ultraviolet light or oxidation . That the antifungal activity of sagebrush secondary metabolites was similar for two different fungi provides support for broad antifungal defenses . The incidence and severity of fungal disease in the field (caused by Puccinia tanaceti) were similar in moist and dry habitats, possibly reflecting an equilibrium between plant defense and fungal attack, as sites with greater fungal abundance compensated with more effective secondary metabolites . The geographic correlation between fungal abundance and antifungal secondary metabolites of sagebrush, coupled with our other data showing heritable variation in these metabolites, suggests that pathogenic fungi have selected for antifungal secondary metabolites in sagebrush. J Biol Chem, 2003 Mar 14, 278(11), 8881 - 7 Epub 2003 Jan 08. Cytochrome oxidase assembly does not require catalytically active cytochrome C; Barrientos A et al.; Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen . COX assembly requires the coming together of nuclear- and mitochondrial-encoded subunits and the assistance of a large number of nuclear gene products acting at different stages of maturation of the enzyme . In Saccharomyces cerevisiae, expression of cytochrome c, encoded by CYC1 and CYC7, is required not only for electron transfer but also for COX assembly through a still unknown mechanism . We have attempted to distinguish between a functional and structural requirement of cytochrome c in COX assembly . A cyc1/cyc7 double null mutant strain was transformed with the cyc1-166 mutant gene (Schweingruber, M . E., Stewart, J . W., and Sherman, F . (1979) J . Biol . Chem . 254, 4132-4143) that expresses stable but catalytically inactive iso-1-cytochrome c . The COX content of the cyc1/cyc7 double mutant strain harboring non-functional iso-1-cytochrome c has been characterized spectrally, functionally, and immunochemically . The results of these studies demonstrate that cytochrome c plays a structural rather than functional role in assembly of cytochrome c oxidase . In addition to its requirement for COX assembly, cytochrome c also affects turnover of the enzyme . Mutants containing wild type apocytochrome c in mitochondria lack COX, suggesting that only the folded and mature protein is able to promote COX assembly. Blood, 2003 May 1, 101(9), 3699 - 707 Epub 2003 Jan 09. Iron transport by Nramp2/DMT1: pH regulation of transport by 2 histidines in transmembrane domain 6; Lam-Yuk-Tseung S et al.; Mutations at natural resistance-associated macrophage protein 1 (Nramp1) impair phagocyte function and cause susceptibility to infections while mutations at Nramp2 (divalent metal transporter 1 {DMT1}) affect iron homeostasis and cause severe microcytic anemia . Structure-function relationships in the Nramp superfamily were studied by mutagenesis, followed by functional characterization in yeast and in mammalian cells . These studies identify 3 negatively charged and highly conserved residues in transmembrane domains (TM) 1, 4, and 7 as essential for cation transport by Nramp2/DMT1 . The introduction of a charged residue (Gly185Arg) in TM4 found in the naturally occurring microcytic anemia mk (mouse) and Belgrade (rat) mutants is shown to cause a partial or complete loss of function in mammalian and yeast cells, respectively . A pair of mutation-sensitive and highly conserved histidines (His267, His272) was identified in TM6 . Surprisingly, inactive His267 and His272 mutants could be rescued by lowering the pH of the transport assay . This indicates that His267/His272 are not directly involved in metal binding but, rather, play an important role in pH regulation of metal transport by Nramp proteins. Blood, 2003 May 1, 101(9), 3687 - 9 Epub 2003 Jan 09. Hematopoietic progenitor kinase 1 (HPK1) negatively regulates prostaglandin E2-induced fos gene transcription; Sawasdikosol S et al.; Prostaglandin E(2) (PGE(2)) is the predominant eicosanoid product released by macrophages at the site of inflammation . Binding of PGE(2) to its cognate 7 transmembrane-spanning G protein-coupled receptors (GPCRs) activates signaling pathways, leading to the synthesis of the Fos transcription factor . Because the Ste20 serine/threonine protein kinase (S/TPK) is a critical signal transducer for the G protein-coupled pheromone receptor in Saccharomyces cerevisiae, we postulated that the PGE(2) GPCRs may activate one of the Ste20 mammalian orthologs . We demonstrate here that the catalytic activity of a hematopoietic cell-restricted, Ste20-related S/TPK, HPK1, is positively regulated by exposure to physiological concentrations of PGE(2) . Furthermore, ectopic expression studies implicated HPK1 as a negative regulator of PGE(2)-induced transcription of the fos gene . Our data suggest that PGE(2)-induced activation of HPK1 may represent a novel negative regulatory pathway capable of modulating PGE(2)-mediated gene transcription. Mol Cells, 2002 Dec 31, 14(3), 420 - 4 RACK-1, a receptor for activated C kinase, interacts with the transcription factor NFAT and represses its transactivation; Han J et al.; To isolate and characterize a novel protein that interacts with nuclear factor of activated T cells (NFAT) and potentially regulates its activity, we screened a Jurkat cDNA library by using the NFAT regulatory domain as bait in the yeast two-hybrid system . RACK-1, a receptor for activated protein kinase C and a homologue of the G-protein beta subunit, was identified as a NFAT-binding protein . Mammalian two hybrid tests in CV-1 cells and a coimmunoprecipitation assay confirmed protein-protein interaction between NFAT and RACK-1 . In addition, overexpression of RACK-1 specifically suppressed transcriptional activation derived by NFAT, but not by NF-kappaB . These results demonstrate RACK-1 as a potent negative modulator of NFAT activation and suggest a novel mechanism in NFAT regulation. Planta, 2003 Jan, 216(3), 377 - 86 Epub 2002 Oct 08. Molecular characterization of two plant BI-1 homologues which suppress Bax-induced apoptosis in human 293 cells; Bolduc N et al.; To date, few homologues of animal programmed cell death (PCD) regulators have been identified in plants . Among these is the plant Bax Inhibitor-1 (BI-1) protein, which possesses, like its human counterpart, the ability to suppress Bax-induced lethality in yeast cells . As the role of BI-1 in the regulation of plant PCD remains to be elucidated, we cloned BnBI-1 and NtBI-1 from cDNA libraries of oilseed rape ( Brassica napus L.) and tobacco ( Nicotiana tabacum L.) . The analysis of the deduced amino acid sequences of BnBI-1 and NtBI-1 indicated that these proteins share a relatively high level of identity with other plant BI-1 proteins (73-95%) as well as with animal BI-1 proteins (26-42%) . Comparative analysis with other available plant BI-1 proteins allowed the establishment of a structural model presenting seven transmembrane domains . Moreover, transient co-transfection of Bax with BnBI-1 or NtBI-1 in human embryonic kidney 293 cells revealed that both proteins can substantially inhibit apoptosis induced by Bax overexpression . Localization studies were also conducted using stable transformation of tobacco BY-2 cells and Saccharomyces cerevisiae, or transient expression in tobacco leaves, with the fusion protein BnBI-1GFP under control of the cauliflower mosaic virus 35S promoter . All transformants showed a fluorescence pattern of distribution typical of an endoplasmic reticulum (ER) protein . Results from differential permeabilization experiments in BY-2 cells expressing BnBI-1GFP also showed that the C-terminus is located on the cytosolic side of the ER . Taken altogether, our results suggest that BI-1 is evolutionarily conserved and could act as a key regulator of a death pathway common to plants and animals. Nucleic Acids Res, 2003 Jan 1, 31(1), 397 - 9 NLSdb: database of nuclear localization signals; Nair R et al.; NLSdb is a database of nuclear localization signals (NLSs) and of nuclear proteins . NLSs are short stretches of residues mediating transport of nuclear proteins into the nucleus . The database contains 114 experimentally determined NLSs that were obtained through an extensive literature search . Using 'in silico mutagenesis' this set was extended to 308 experimental and potential NLSs . This final set matched over 43% of all known nuclear proteins and matches no currently known non-nuclear protein . NLSdb contains over 6000 predicted nuclear proteins and their targeting signals from the PDB and SWISS-PROT/TrEMBL databases . The database also contains over 12 500 predicted nuclear proteins from six entirely sequenced eukaryotic proteomes (Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae) . NLS motifs often co-localize with DNA-binding regions . This observation was used to also annotate over 1500 DNA-binding proteins . NLSdb can be accessed via the web site: http://cubic.bioc.columbia.edu/db/NLSdb/. Nucleic Acids Res, 2003 Jan 1, 31(1), 325 - 7 The Mitochondrial Protein Import Machinery of Plants (MPIMP) database; Lister R et al.; The Mitochondrial Protein Import Machinery of Plants database (MPIMP) is an Internet-accessible database containing detailed information on the protein import apparatus of plant mitochondria . The Arabidopsis genome was searched for com-ponents of the mitochondrial protein import apparatus using components from the well-characterized model system of Saccharomyces cerevisiae . Twenty six homologues of 34 components could be found, encompassing the essential components for the general and carrier import pathways . The database is available through the Internet at http://millar3.biochem.uwa.edu.au/~lister/index.html. Nucleic Acids Res, 2003 Jan 1, 31(1), 207 - 11 The PEDANT genome database; Frishman D et al.; The PEDANT genome database provides exhaustive automatic analysis of genomic sequences by a large variety of established bioinformatics tools through a comprehensive Web-based user interface . One hundred and seventy seven completely sequenced and unfinished genomes have been processed so far, including large eukaryotic genomes (mouse, human) published recently . In this contribution, we describe the current status of the PEDANT database and novel analytical features added to the PEDANT server in 2002 . Those include: (i) integration with the BioRS data retrieval system which allows fast text queries, (ii) pre-computed sequence clusters in each complete genome, (iii) a comprehensive set of tools for genome comparison, including genome comparison tables and protein function prediction based on genomic context, and (iv) computation and visualization of protein-protein interaction (PPI) networks based on experimental data . The availability of functional and structural predictions for 650 000 genomic proteins in well organized form makes PEDANT a useful resource for both functional and structural genomics. J Biol Chem, 2003 Apr 4, 278(14), 11849 - 57 Epub 2003 Jan 07. Onset of carbon catabolite repression in Aspergillus nidulans . Parallel involvement of hexokinase and glucokinase in sugar signaling; Flipphi M et al.; The role of hexose phosphorylating enzymes in the signaling of carbon catabolite repression was investigated in the filamentous fungus Aspergillus nidulans . A d-fructose non-utilizing, hexokinase-deficient (hxkA1, formerly designated frA1) strain was utilized to obtain new mutants lacking either glucokinase (glkA4) or both hexose kinases (hxkA1/glkA4) . d-Glucose and d-fructose phosphorylation is completely abolished in the double mutant, which consequently cannot grow on either sugar . The glucokinase single mutant exhibits no nutritional deficiencies . Three repressible diagnostic systems, ethanol utilization (alcA and alcR genes), xylan degradation (xlnA), and acetate catabolism (facA), were analyzed in these hexose kinase mutants at the transcript level . Transcriptional repression by d-glucose is fully retained in the two single kinase mutants, whereas the hexokinase mutant is partially derepressed for d-fructose . Thus, hexokinase A and glucokinase A compensate each other for carbon catabolite repression by d-glucose in the single mutants . In contrast, both d-glucose and d-fructose repression are severely impaired for all three diagnostic systems in the double mutant . Unlike the situation in Saccharomyces cerevisiae, the hexose phosphorylating enzymes play parallel roles in glucose repression in A . nidulans. Xenobiotica, 2003 Jan, 33(1), 13 - 25 Role of specific cytochrome P450 enzymes in the N-oxidation of the antiarrhythmic agent mexiletine; Labbe L et al.; 1 . Mexiletine is extensively metabolized in man by C- and N-oxidation and the aim of the present study was to characterize major cytochrome P450 enzyme(s) involved in the formation of N-hydroxymexiletine . 2 . Incubations with genetically engineered microsomes indicated that the formation rate of N-hydroxymexiletine was highest in the presence of microsomes expressing high levels of either CYP1A2 or CYP2E1 and the formation of N-hydroxymexiletine by human liver microsomes was inhibited about 40% by antibodies directed against CYP1A1/1A2 or CYP2E1 . Additional incubations demonstrated that formation of N-hydroxymexiletine was decreased 47 and 51% by furafylline, 40 microm and 120 microm, respectively, and decreased 55 and 67% by alpha-naphthoflavone, 1 microm and 3 microm, respectively (all p < 0.05 versus control) . 3 . The formation rate of N-hydroxymexiletine in human liver microsomes was highly correlated with CYP2B6 (RS-mexiletine, r = 0.7827; R-(-)-enantiomer, r = 0.7034; S-(+)-enantiomer, r = 0.7495), CYP2E1 (S-(+)-enantiomer, r = 0.7057) and CYP1A2 (RS-mexiletine, r = 0.5334; S-(+)-enantiomer, r = 0.6035) . 4 . In conclusion, we have demonstrated that CYP1A2 is a major human cytochrome P450 enzyme involved in the formation of N-hydroxymexiletine . However, other cytochrome P450 enzymes (CYP2E1 and CYP2B6) also appear to play a role in the N-oxidation of this drug. Mol Microbiol, 2003 Jan, 47(2), 411 - 30 Molecular characterization, function and regulation of ammonium transporters (Amt) and ammonium-metabolizing enzymes (GS, NADP-GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum; Javelle A et al.; External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structures of the ectomycorrhizal symbiosis . Here, we have cloned and characterized Hebeloma cylindrosporum AMT1, GLNA and GDHA genes, which encode a third ammonium transporter, a glutamine synthetase and an NADP-dependent glutamate dehydrogenase respectively . Amt1 can fully restore the pseudohyphal growth defect of a Saccharomyces cerevisiae mep2 mutant, and this is the first evidence that a heterologous member of the Mep/Amt family complements this dimorphic change defect . Dixon plots of the inhibition of methylamine uptake by ammonium indicate that Amt1 has a much higher affinity than the two previously characterized members (Amt2 and Amt3) of the Amt/Mep family in H . cylindrosporum . We also identified the intracellular nitrogen pool(s) responsible for the modulation of expression of AMT1, AMT2, AMT3, GDHA and GLNA . In response to exogenously supplied ammonium or glutamine, AMT1, AMT2 and GDHA were downregulated and, therefore, these genes are subjected to nitrogen repression in H . cylindrosporum . Exogenously supplied nitrate failed to induce a downregulation of the five mRNAs after transfer of mycelia from a N-starved condition . Our results demonstrate that glutamine is the main effector for AMT1 and AMT2 repression, whereas GDHA repression is controlled by intracellular ammonium, independently of the intracellular glutamine or glutamate concentration . Ammonium transport activity may be controlled by intracellular NH4+ . AMT3 and GLNA are highly expressed but not highly regulated . A model for ammonium assimilation in H . cylindrosporum is presented. Mol Microbiol, 2003 Jan, 47(2), 373 - 81 Lithium and valproate decrease the membrane phosphatidylinositol/phosphatidylcholine ratio; Ding D et al.; Lithium and valproate, two structurally different anti-bipolar drugs, cause decreased intracellular inositol in the yeast Saccharomyces cerevisiae and an in-crease in expression of a structural (INO1) and a regulatory (INO2) gene for phospholipid synthesis that responds to inositol depletion (Vaden, D., Ding, D., Peterson, B., and Greenberg, M.L., 2001, J Biol Chem 276: 15466-15471) . We report here that both drugs decrease the relative rate of membrane phosphatidylinositol synthesis and, to a lesser but still significant degree, the steady state relative phosphatidylinositol composition . In addition, both drugs increase the rate of phosphatidylcholine (PC) synthesis . Finally, valproate, but not lithium, increases expression of phosphatidylcholine pathway genes CHO1 and OPI3 . The overall effect on membrane phospholipid composition is a reduction in the phosphatidylinositol/phosphatidylcholine ratio by both drugs . Because maintenance of the appropriate phosphatidylinositol/phosphatidylcholine ratio is required for secretory vesicle formation, a decrease in this ratio may have far-reaching implications for understanding the therapeutic mechanisms of action of these drugs. Curr Opin Cell Biol, 2003 Feb, 15(1), 6 - 13 Impact of genome-wide functional analyses on cell biology research; Martin AC et al.; The availability of complete genome sequences for a variety of organisms, coupled with novel approaches that allow evaluation of the functions of thousands of genes in parallel, have the potential to greatly impact on cell biology research . Functional genomic approaches in Saccharomyces cerevisiae are beginning to make significant contributions to the understanding of complex biological systems. BMC Plant Biol . 2003 Jan 07;3(1):1. Expression pattern of a nuclear encoded mitochondrial arginine-ornithine translocator gene from Arabidopsis; Catoni E et al.; BACKGROUND: Arginine and citrulline serve as nitrogen storage forms, but are also involved in biosynthetic and catabolic pathways . Metabolism of arginine, citrulline and ornithine is distributed between mitochondria and cytosol . For the shuttle of intermediates between cytosol and mitochondria transporters present on the inner mitochondrial membrane are required . Yeast contains a mitochondrial translocator for ornithine and arginine, Ort1p/Arg11p . Ort1p/Arg11p is a member of the mitochondrial carrier family (MCF) essential for ornithine export from mitochondria . The yeast arg11 mutant, which is deficient in Ort1p/Arg11p grows poorly on media lacking arginine . RESULTS: High-level expression of a nuclear encoded Arabidopsis thaliana homolog (AtmBAC2) of Ort1p/Arg11p was able to suppress the growth deficiency of arg11 . RT-PCR analysis demonstrated expression of AtmBAC2 in all tissues with highest levels in flowers . Promoter-GUS fusions showed preferential expression in flowers, i.e . pollen, in the vasculature of siliques and in aborted seeds . Variable expression was observed in leaf vasculature . Induction of the promoter was not observed during the first two weeks in seedlings grown on media containing NH4NO3, arginine or ornithine as sole nitrogen sources . CONCLUSION: AtmBAC2 was isolated as a mitochondrial transporter for arginine in Arabidopsis . The absence of expression in developing seeds and in cotyledons of seedlings indicates that other transporters are responsible for storage and mobilization of arginine in seeds. Plant Mol Biol, 2002 Dec, 50(6), 949 - 70 Protein-protein interactions required during translation; Gallie DR; Protein synthesis requires the involvement of numerous accessory factors that assist the ribosome in translation initiation, elongation, and termination . Extensive protein-protein and protein-RNA interactions are required to bring together the accessory factors, tRNAs, ribosomes, and mRNA into a productive complex and these interactions undergo dynamic alterations during each step of the translation initiation process . Initiation represents the most complex aspect of translation, requiring more accessory proteins, called initiation factors, than either elongation or termination . Not surprisingly, initiation is most often the rate-limiting step of translation and, as such, most (but not all) examples of translational regulation involve the regulation of protein-protein or protein-RNA interactions of the initiation complex . In this review, we focus on those interactions required for efficient translation initiation and how such interactions are regulated by developmental or environmental signals. Plant Mol Biol, 2002 Dec, 50(6), 925 - 47 Multi-protein complexes in eukaryotic gene transcription; Martinez E; Specific transcription initiation by RNA polymerase II at eukaryotic protein-coding genes involves the cooperative assembly at the core promoter of more than 40 distinct proteins--with a total mass of over 2 MDa--including RNA polymerase II itself and general/basal transcription initiation factors, to form a stable pre-initiation complex (PIC) . In vivo, PIC assembly is a major point of regulation by sequence-specific transcription regulators (activators and repressors) and is hindered by the packaging of promoter DNA into nucleosomes and higher order chromatin structures . Genetic and biochemical studies have recently identified a variety of transcription cofactors/co-regulators (coactivators and corepressors) that interact with sequence-specific regulators and/or various components of the general/basal transcription machinery and are essential for regulated transcription . An emerging view from these studies is that regulators must target two types of transcription cofactors: chromatin-modifying/remodeling cofactors and general cofactors that associate with and/or influence the activities of components of the general/basal transcription machinery . The recent biochemical identification and characterization of many different chromatin-modifying and general transcription cofactors has revealed their often complex multi-subunit nature and a previously unsuspected level of structural and functional redundancy . Another emerging theme is the multi-functional nature of chromatin-modifying cofactor complexes that appear to couple gene-specific transcription to other cellular processes. J Fr Ophtalmol, 2002 Nov, 25(9), 890 - 6 {Fungal keratitis at the Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts: retrospective study of 19 cases}; Rondeau N et al.; PURPOSE: To report the spectrum of fungal keratitis at the Centre Hospitalier National d'Ophtalmologie XV-XX, Paris . METHODS: We reviewed 19 cases of fungal keratitis from January 1993 to January 2001 . We evaluated the different risk factors, fungal identification, topical and systemic antifungal therapy, surgical treatment and outcome . RESULTS: Nineteen patients were included, 7 women and 12 men, with visual acuity ranging from 9/10 to no perception of light . The mean age was 56.2 years . Patients were hospitalized for an average stay of 16.3 days and all received a diagnostic and therapeutic scraping and 16 received a local antifungal treatment . The most common risk factors were topical steroid treatment (42.1%), corneal graft (31.6%), trauma or foreign body (31.6%) . The mean delay between the first signs and fungal keratitis diagnosis was 14 days . Yeast as Candida parapsilosis and albicans were the most frequently isolated fungi (58%), followed by Aspergillus sp . (21%) and Fusarium sp . (21%) . The most commonly used topical treatment was amphotericin B, and itraconazole was used as systemic treatment . Five patients had evisceration, 6 had penetrating keratoplasty and 5 retained leukoma . CONCLUSION: Candida was the most frequently isolated fungi and topical steroid treatment the main risk factor . The prognosis is relatively poor (26% of lost vision) because of a delay in diagnosis and other previous ocular pathology or surgery. J Cell Biol, 2003 Jan 6, 160(1), 25 - 39 Epub 2003 Jan 06. Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway; Goshima G et al.; Kinetochores are the chromosomal sites for spindle interaction and play a vital role for chromosome segregation . The composition of kinetochore proteins and their cellular roles are, however, poorly understood in higher eukaryotes . We identified a novel kinetochore protein family conserved from yeast to human that is essential for equal chromosome segregation . The human homologue hMis12 of yeast spMis12/scMtw1 retains conserved sequence features and locates at the kinetochore region indistinguishable from CENP-A, a centromeric histone variant . RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores . Further, the metaphase spindle length is abnormally extended . Spindle checkpoint protein hMad2 temporally localizes at kinetochores at early mitotic stages after RNAi . The RNAi deficiency of CENP-A leads to a similar mitotic phenotype, but the kinetochore signals of other kinetochore proteins, hMis6 and CENP-C, are greatly diminished . RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest . Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores. RNA, 2002 Dec, 8(12), 1502 - 14 Naf1 p is a box H/ACA snoRNP assembly factor; Fatica A et al.; Box H/ACA small nucleolar ribonucleoprotein particles (snoRNPs) contain four essential proteins, Cbf5p, Gar1p, Nhp2p, and Nop10p, each of which, with the exception of Gar1p, is required for box H/ACA snoRNA accumulation . Database searches identified a novel essential protein, which we termed Naf1p, with a region of homology to the RNA-binding domain of Gar1p and other features in common with hnRNP-like proteins . Naf1p is localized to the nucleus and is not a stable component of the H/ACA snoRNPs, but it is required for the accumulation of all box H/ACA snoRNAs . This requirement is not at the level of snoRNA transcription initiation or termination . Naf1 p shows in vitro RNA-binding activity and also binds directly to Cbf5p and Nhp2p . Naf1p was shown to bind to the CTD in vivo in a two-hybrid assay, and the phosphorylated CTD, but not the nonphosphorylated CTD, was shown to precipitate tagged Naf1p from a cell lysate . We propose that Naf1 p is recruited to the CTD of RNA polymerase II and binds to nascent box H/ACA snoRNAs promoting snoRNP assembly. RNA, 2002 Dec, 8(12), 1489 - 501 The human LSm1-7 proteins colocalize with the mRNA-degrading enzymes Dcp1/2 and Xrnl in distinct cytoplasmic foci; Ingelfinger D et al.; Sm and Sm-like (LSm) proteins form heptameric complexes that are involved in various steps of RNA metabolism . In yeast, the Lsm1-7 complex functions in mRNA degradation and is associated with several enzymes of this pathway, while the complex LSm2-8, the composition of which largely overlaps with that of LSm1-7, has a role in pre-mRNA splicing . A human gene encoding an LSm1 homolog has been identified, but its role in mRNA degradation has yet to be elucidated . We performed subcellular localization studies and found hLSm1 predominantly in the cytoplasm . However, it is not distributed evenly; rather, it is highly enriched in small, discrete foci . The endogenous hLSm4 is similarly localized, as are the overexpressed proteins hLSm1-7, but not hLSm8 . The foci also contain two key factors in mRNA degradation, namely the decapping enzyme hDcp1/2 and the exonuclease hXrn1 . Moreover, coexpression of wild-type and mutant LSm proteins, as well as fluorescence resonance energy transfer (FRET) studies, indicate that the mammalian proteins hLSm1-7 form a complex similar to the one found in yeast, and that complex formation is required for enrichment of the proteins in the cytoplasmic foci . Therefore, the foci contain a partially or fully assembled machinery for the degradation of mRNA. J Biol Chem, 2003 Mar 14, 278(11), 9212 - 8 Epub 2003 Jan 03. Rad54p is a chromatin remodeling enzyme required for heteroduplex DNA joint formation with chromatin; Jaskelioff M et al.; In eukaryotic cells, the repair of DNA double-strand breaks by homologous recombination requires a RecA-like recombinase, Rad51p, and a Swi2p/Snf2p-like ATPase, Rad54p . Here we find that yeast Rad51p and Rad54p support robust homologous pairing between single-stranded DNA and a chromatin donor . In contrast, bacterial RecA is incapable of catalyzing homologous pairing with a chromatin donor . We also show that Rad54p possesses many of the biochemical properties of bona fide ATP-dependent chromatin-remodeling enzymes, such as ySWI/SNF . Rad54p can enhance the accessibility of DNA within nucleosomal arrays, but it does not seem to disrupt nucleosome positioning . Taken together, our results indicate that Rad54p is a chromatin-remodeling enzyme that promotes homologous DNA pairing events within the context of chromatin. EMBO J, 2003 Jan 15, 22(2), 281 - 91 MAP kinase phosphorylation-dependent activation of Elk-1 leads to activation of the co-activator p300; Li QJ et al.; CBP/p300 recruitment to enhancer-bound complexes is a key determinant in promoter activation by many transcription factors . We present a novel mechanism of activating such complexes and show that pre-assembled Elk-1-p300 complexes become activated following Elk-1 phosphorylation by changes in Elk-1-p300 interactions rather than recruitment . It is known that Elk-1 binds to promoter in the absence of stimuli . However, it is unclear how activation of Elk-1 by mitogen-acivated protein kinase (MAPK)-mediated phosphorylation leads to targeted gene transactivation . We show that Elk-1 can interact with p300 in vitro and in vivo in the absence of a stimulus through the Elk-1 C-terminus and the p300 N-terminus . Phosphorylation on Ser383 and Ser389 of Elk-1 by MAPK enhances this basal binding but, most importantly, Elk-1 exhibits new interactions with p300 . These interaction changes render a strong histone acetyltransferase activity in the Elk-1-associated complex that could play a critical role in chromatin remodeling and gene activation . The pre-assembly mechanism may greatly accelerate transcription activation, which is important in regulation of expression of immediate-early response genes, in particular those involved in stress responses. EMBO J, 2003 Jan 15, 22(2), 193 - 204 Domains of eIF1A that mediate binding to eIF2, eIF3 and eIF5B and promote ternary complex recruitment in vivo; Olsen DS et al.; Translation initiation factor 1A (eIF1A) is predicted to bind in the decoding site of the 40S ribosome and has been implicated in recruitment of the eIF2-GTP-Met-tRNA i Met ternary complex (TC) and ribosomal scanning . We show that the unstructured C-terminus of eIF1A interacts with the C-terminus of eIF5B, a factor that stimulates 40S-60S subunit joining, and removal of this domain of eIF1A diminishes translation initiation in vivo . These findings support the idea that eIF1A-eIF5B association is instrumental in releasing eIF1A from the ribosome after subunit joining . A larger C-terminal truncation that removes a 3(10) helix in eIF1A deregulates GCN4 translation in a manner suppressed by overexpressing TC, implicating eIF1A in TC binding to 40S ribosomes in vivo . The unstructured N-terminus of eIF1A interacts with eIF2 and eIF3 and is required at low temperatures for a step following TC recruitment . We propose a modular organization for eIF1A wherein a core ribosome-binding domain is flanked by flexible segments that mediate interactions with other factors involved in recruitment of TC and release of eIF1A at subunit joining. Genes Dev, 2003 Jan 1, 17(1), 101 - 14 The highly conserved Ndc80 complex is required for kinetochore assembly, chromosome congression, and spindle checkpoint activity; McCleland ML et al.; We show that the Xenopus homologs of Ndc80/Tid3/HEC1 (xNdc80) and Nuf2/MPP1/Him-10 (xNuf2) proteins physically interact in a 190-kD complex that associates with the outer kinetochore from prometaphase through anaphase . Injecting function-blocking antibodies to either xNdc80 or xNuf2 into XTC cells caused premature exit from mitosis without detectable chromosome congression or anaphase movements . Injected cells did not arrest in response to microtubule drugs, showing that the complex is required for the spindle checkpoint . Kinetochores assembled in Xenopus extracts after immunodepletion of the complex did not contain xRod, xZw10, xP150 glued (Dynactin), xMad1, xMad2, xBub1, and xBub3, demonstrating that the xNdc80 complex is required for functional kinetochore assembly . In contrast, function-blocking antibodies did not affect the localization of other kinetochore proteins when added to extracts containing previously assembled kinetochores . These extracts with intact kinetochores were deficient in checkpoint signaling, suggesting that the Ndc80 complex participates in the spindle checkpoint . We also demonstrate that the spindle checkpoint can arrest budding yeast cells lacking Ndc80 or Nuf2, whereas yeast lacking both proteins fail to arrest in mitosis . Systematic deletion of yeast kinetochore genes suggests that the Ndc80 complex has a unique role in spindle checkpoint signaling . We propose that the Ndc80 complex has conserved roles in kinetochore assembly, chromosome congression, and spindle checkpoint signaling. Genes Dev, 2003 Jan 1, 17(1), 43 - 8 Phosphorylation of histone H3 during transcriptional activation depends on promoter structure; Labrador M et al.; Covalent modifications of histone N-terminal tails are required for the proper assembly and activation of the general transcription factors at promoters . Here, we analyze histone acetylation and phosphorylation in Drosophila transgenes activated by the yeast Gal4 transcriptional activator in the context of different promoters . We show that, independent of the promoter, transcription does not correlate with acetylation of either H3-Lys 14 or H4-Lys 8 . Histone H3 associated with the DNA of Gal4-induced transcribing transgenes driven by the Drosophila Hsp70 promoter is hyperphosphorylated at Ser 10 during transcription . Surprisingly, histone H3 at Gal4-induced transgenes driven by the P element Transposase promoter is not hyperphosphorylated . The data suggest that transcription occurs without acetylated H4 and H3 in both transgenes in Drosophila polytene chromosomes . Instead, phosphorylation of H3 is linked to transcription and can be modulated by the structure of the promoter. Science, 2003 Feb 14, 299(5609), 1061 - 4 Epub 2003 Jan 02. Rewiring MAP kinase pathways using alternative scaffold assembly mechanisms; Park SH et al.; How scaffold proteins control information flow in signaling pathways is poorly understood: Do they simply tether components, or do they precisely orient and activate them? We found that the yeast mitogen-activated protein (MAP) kinase scaffold Ste5 is tolerant to major stereochemical perturbations; heterologous protein interactions could functionally replace native kinase recruitment interactions, indicating that simple tethering is largely sufficient for scaffold-mediated signaling . Moreover, by engineering a scaffold that tethers a unique kinase set, we could create a synthetic MAP kinase pathway with non-natural input-output properties . These findings demonstrate that scaffolds are highly flexible organizing factors that can facilitate pathway evolution and engineering. J Biol Chem, 2003 Mar 14, 278(11), 8897 - 903 Epub 2003 Jan 02. The Set2 histone methyltransferase functions through the phosphorylated carboxyl-terminal domain of RNA polymerase II; Li B et al.; The histone methyltransferase Set2, which specifically methylates lysine 36 of histone H3, has been shown to repress transcription upon tethering to a heterologous promoter . However, the mechanism of targeting and the consequence of Set2-dependent methylation have yet to be demonstrated . We sought to identify the protein components associated with Set2 to gain some insights into the in vivo function of this protein . Mass spectrometry analysis of the Set2 complex, purified using a tandem affinity method, revealed that RNA polymerase II (pol II) is associated with Set2 . Immunoblotting and immunoprecipitation using antibodies against subunits of pol II confirmed that the phosphorylated form of pol II is indeed an integral part of the Set2 complex . Gst-Set2 preferentially binds to CTD synthetic peptides phosphorylated at serine 2, and to a lesser extent, serine 5 phosphorylated peptides, but has no affinity for unphosphorylated CTD, suggesting that Set2 associates with the elongating form of the pol II . Furthermore, we show that set2Delta ppr2Delta double mutants (PPR2 encodes TFIIS, a transcription elongation factor) are synthetically hypersensitive to 6-azauracil, and that deletions in the CTD reduce in vivo levels of H3 lysine 36 methylation . Collectively, these results suggest that Set2 is involved in regulating transcription elongation through its direct contact with pol II. Nat Cell Biol, 2003 Jan, 5(1), 28 - 37 Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis; Shirane M et al.; The mitochondrial localization of the membrane proteins Bcl-2 and Bcl-x(L) is essential for their anti-apoptotic function . Here we show that mitochondrial FK506-binding protein 38 (FKBP38), unlike FKBP12, binds to and inhibits calcineurin in the absence of the immunosuppressant FK506, suggesting that FKBP38 is an inherent inhibitor of this phosphatase . FKBP38 is associated with Bcl-2 and Bcl-x(L) in immunoprecipitation assays and colocalizes with these proteins in mitochondria; in addition, the expression of FKBP38 mutant proteins induces a marked redistribution of Bcl-2 and Bcl-x(L) . Overexpression of FKBP38 blocks apoptosis, whereas functional inhibition of this protein by a dominant-negative mutant or by RNA interference promotes apoptosis . Thus, FKBP38 might function to inhibit apoptosis by anchoring Bcl-2 and Bcl-x(L) to mitochondria. Proc Natl Acad Sci U S A, 2003 Jan 7, 100(1), 283 - 8 Epub 2002 Dec 30. Interaction of "readthrough" acetylcholinesterase with RACK1 and PKCbeta II correlates with intensified fear-induced conflict behavior; Birikh KR et al.; Behavioral reactions to stress are altered in numerous psychiatric and neurodegenerative syndromes, but the corresponding molecular processes and signal transduction pathways are yet unknown . Here, we report that, in mice, the stress-induced splice variant of acetylcholinesterase, AChE-R, interacts intraneuronally with the scaffold protein RACK1 and through it, with its target, protein kinase CbetaII (PKCbetaII), which is known to be involved in fear conditioning . In stress-responsive brain regions of normal FVBN mice, the mild stress of i.p . injection increased AChE and PKCbetaII levels in a manner suppressible by antisense prevention of AChE-R accumulation . Injection stress also prolonged conflict between escape and hiding in the emergence into an open field test . Moreover, transgenic FVBN mice overexpressing AChE-R displayed prolonged delay to emerge into another field (fear-induced behavioral inhibition), associated with chronically intensified neuronal colabeling of RACK1 and PKCbetaII in stress-responsive brain regions . These findings are consistent with the hypothesis that stress-associated changes in cholinergic gene expression regulate neuronal PKCbetaII functioning, promoting fear-induced conflict behavior after stress. Proc Natl Acad Sci U S A, 2003 Jan 7, 100(1), 50 - 5 Epub 2002 Dec 30. Optimal alignment for enzymatic proton transfer: structure of the Michaelis complex of triosephosphate isomerase at 1.2-A resolution; Jogl G et al.; In enzyme catalysis, where exquisitely positioned functionality is the sine qua non, atomic coordinates for a Michaelis complex can provide powerful insights into activation of the substrate . We focus here on the initial proton transfer of the isomerization reaction catalyzed by triosephosphate isomerase and present the crystal structure of its Michaelis complex with the substrate dihydroxyacetone phosphate at near-atomic resolution . The active site is highly compact, with unusually short and bifurcated hydrogen bonds for both catalytic Glu-165 and His-95 residues . The carboxylate oxygen of the catalytic base Glu-165 is positioned in an unprecedented close interaction with the ketone and the alpha-hydroxy carbons of the substrate (C em leader O approximately 3.0 A), which is optimal for the proton transfer involving these centers . The electrophile that polarizes the substrate, His-95, has close contacts to the substrate's O1 and O2 (N em leader O < or = 3.0 and 2.6 A, respectively) . The substrate is conformationally relaxed in the Michaelis complex: the phosphate group is out of the plane of the ketone group, and the hydroxy and ketone oxygen atoms are not in the cisoid configuration . The epsilon ammonium group of the electrophilic Lys-12 is within hydrogen-bonding distance of the substrate's ketone oxygen, the bridging oxygen, and a terminal phosphate's oxygen, suggesting a role for this residue in both catalysis and in controlling the flexibility of active-site loop. Mol Cell Biol, 2003 Jan, 23(2), 699 - 707 Autoregulation in the biosynthesis of ribosomes; Zhao Y et al.; The synthesis of ribosomes in Saccharomyces cerevisiae consumes a prodigious amount of the cell's resources and, consequently, is tightly regulated . The rate of ribosome synthesis responds not only to nutritional cues but also to signals dependent on other macromolecular pathways of the cell, e.g., a defect in the secretory pathway leads to severe repression of transcription of both rRNA and ribosomal protein genes . A search for mutants that interrupted this repression revealed, surprisingly, that inactivation of RPL1B, one of a pair of genes encoding the 60S ribosomal protein L1, almost completely blocked the repression of rRNA and ribosomal protein gene transcription that usually follows a defect in the secretory pathway . Further experiments showed that almost any mutation leading to a defect in 60S subunit synthesis had the same effect, whereas mutations affecting 40S subunit synthesis did not . Although one might suspect that this effect would be due to a decrease in the initiation of translation or to the presence of half-mers, i.e., polyribosomes awaiting a 60S subunit, our data show that this is not the case . Rather, a variety of experiments suggest that some aspect of the production of defective 60S particles or, more likely, their breakdown suppresses the signal generated by a defect in the secretory pathway that represses ribosome synthesis. Mol Cell Biol, 2003 Jan, 23(2), 629 - 35 The tor pathway regulates gene expression by linking nutrient sensing to histone acetylation; Rohde JR et al.; The Tor pathway mediates cell growth in response to nutrient availability, in part by inducing ribosomal protein (RP) gene expression via an unknown mechanism . Expression of RP genes coincides with recruitment of the Esa1 histone acetylase to RP gene promoters . We show that inhibition of Tor with rapamycin releases Esa1 from RP gene promoters and leads to histone H4 deacetylation without affecting promoter occupancy by Rap1 and Abf1 . Genetic and biochemical evidence identifies Rpd3 as the major histone deacetylase responsible for reversing histone H4 acetylation at RP gene promoters in response to Tor inhibition by rapamycin or nutrient limitation . Our results illustrate that the Tor pathway links nutrient sensing with histone acetylation to control RP gene expression and cell growth. Mol Cell Biol, 2003 Jan, 23(2), 607 - 19 The inv(16) fusion protein associates with corepressors via a smooth muscle myosin heavy-chain domain; Durst KL et al.; Inversion(16) is one of the most frequent chromosomal translocations found in acute myeloid leukemia (AML), occurring in over 8% of AML cases . This translocation results in a protein product that fuses the first 165 amino acids of core binding factor beta to the coiled-coil region of a smooth muscle myosin heavy chain (CBFbeta/SMMHC) . CBFbeta interacts with AML1 to form a heterodimer that binds DNA; this interaction increases the affinity of AML1 for DNA . The CBFbeta/SMMHC fusion protein cooperates with AML1 to repress the transcription of AML1-regulated genes . We show that CBFbeta/SMMHC contains a repression domain in the C-terminal 163 amino acids of the SMMHC region that is required for inv(16)-mediated transcriptional repression . This minimal repression domain is sufficient for the association of CBFbeta/SMMHC with the mSin3A corepressor . In addition, the inv(16) fusion protein specifically associates with histone deacetylase 8 (HDAC8) . inv(16)-mediated repression is sensitive to HDAC inhibitors . We propose a model whereby the inv(16) fusion protein associates with AML1 to convert AML1 into a constitutive transcriptional repressor. Mol Cell Biol, 2003 Jan, 23(2), 534 - 42 Binding dynamics of structural nucleoporins govern nuclear pore complex permeability and may mediate channel gating; Shulga N et al.; The nuclear pore complex (NPC) is a permeable sieve that can dilate to facilitate the bidirectional translocation of a wide size range of receptor-cargo complexes . The binding of receptors to FG nucleoporin docking sites triggers channel gating by an unknown mechanism . Previously, we used deoxyglucose and chilling treatments to implicate Nup170p and Nup188p in the control of NPC sieving in Saccharomyces cerevisiae . Here, we report that aliphatic alcohols increase the permeability of wild-type and nup170Delta NPCs . In conjunction with increases in permeability, aliphatic alcohols, deoxyglucose, and chilling trigger the reversible dissociation of several nucleoporins from nup170Delta NPCs . These results are consistent with the hypothesis that NPC gating occurs when molecular latches composed of FG repeats and structural nucleoporins dissociate. Mol Cell Biol, 2003 Jan, 23(2), 474 - 81 Enhanced genomic instability and defective postreplication repair in RAD18 knockout mouse embryonic stem cells; Tateishi S et al.; In lower eukaryotes, Rad18 plays a crucial role in postreplication repair . Previously, we isolated a human homologue of RAD18 (hRAD18) and showed that human cells overexpressing hRad18 protein with a mutation in the RING finger motif are defective in postreplication repair . Here, we report the construction of RAD18-knockout mouse embryonic stem cells by gene targeting . These cells had almost the same growth rate as wild-type cells and manifested phenotypes similar to those of human cells expressing mutant Rad18 protein: hypersensitivity to multiple DNA damaging agents and a defect in postreplication repair . Mutation was not induced in the knockout cells with any higher frequencies than in wild-type cells, as shown by ouabain resistance . In the knockout cells, spontaneous sister chromatid exchange (SCE) occurred with twice the frequency observed in normal cells . After mild DNA damage, SCE was threefold higher in the knockout cells, while no increase was observed in normal cells . Stable transformation efficiencies were approximately 20-fold higher in knockout cells, and gene targeting occurred with approximately 40-fold-higher frequency than in wild-type cells at the Oct3/4 locus . These results indicate that dysfunction of Rad18 greatly increases both the frequency of homologous as well as illegitimate recombination, and that RAD18 contributes to maintenance of genomic stability through postreplication repair. J Biol Chem, 2003 Mar 28, 278(13), 10957 - 62 Epub 2002 Dec 30. Orientation of 1,3-bisphosphoglycerate analogs bound to phosphoglycerate kinase; Jakeman DL et al.; We have previously reported dissociation constants for a range of bisphosphonate analogs of 1,3-bisphospho-D-glyceric acid binding to yeast phosphoglycerate kinase . Data for the unsymmetrical analogs were difficult to interpret because it was not clear in which of the two possible orientations these ligands bound . Here we report a novel NMR method for quantifying orientation preference based on relaxation effects induced by titration with CrADP, which is applied to these ligands . It is shown that all ligands can bind in both orientations but that the driving force for the orientational preference is to put the alpha,alpha-difluoromethanephosphonate group in the "basic patch" (nontransferable phosphate) position . The relevance to the design of phosphoglycerate kinase inhibitors is discussed. DNA Repair (Amst), 2002 Sep 4, 1(9), 719 - 29 Evidence for a direct association of hMRE11 with the human mismatch repair protein hMLH1; Her C et al.; In both mitotic and meiotic processes, cellular surveillance of the integrity of genetic information transmission from parental cells to their subsequent generations is carried out by a network of proteins primarily involved in cell-cycle regulation, DNA replication, DNA repair, and chromosome segregation . Within this context, the mammalian MRE11 represents an essential multifunctional protein that promotes repair of DNA double-strand breaks and plays a role in the signaling of DNA damage response . Mutations in human hMRE11 gene could contribute to the rare "AT-like" disorder . However, at present time the functional roles of hMRE11 in these cellular processes are elusive . In the current study, we provide evidence that hMRE11 interacts physically with the mismatch repair protein hMLH1 through yeast two-hybrid analysis . In addition, we show that recombinant hMRE11 and hMLH1 proteins interact when these two proteins are coexpressed in bacterial cells, and both proteins can be co-immunoprecipitated from human cell extracts . Furthermore, hMRE11 and hMLH1 display similar expression patterns when examined with a human normal/tumor DNA array . Together, these data suggest that hMRE11 and hMLH1 might act in a co-operative fashion during DNA damage detection, signaling, and repair. DNA Repair (Amst), 2002 Feb 28, 1(2), 137 - 42 Arsenic-induced Mre11 phosphorylation is cell cycle-dependent and defective in NBS cells; Yuan SS et al.; Cancer-prone diseases ataxia-telangiectasia (AT), Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD) are defective in the repair of DNA double-stranded break (DSB) . On the other hand, arsenic (As) has been reported to cause DSB and to be involved in the occurrence of skin, lung and bladder cancers . To dissect the repair mechanism of As-induced DSB, wild type, AT and NBS cells were treated with sodium arsenite to study the complex formation and post-translational modification of Rad50/NBS1/Mre11 repair proteins . Our results showed that Mre11 went through cell cycle-dependent phosphorylation upon sodium arsenite treatment and this post-translational modification required NBS1 but not ATM . Defective As-induced Mre11 phosphorylation was rescued by reconstitution with full length NBS1 in NBS cells . Although As-induced Mre11 phosphorylation was not required for Rad50/NBS1/Mre11 complex formation, it might be required for the formation of Rad50/NBS1/Mre11 nuclear foci upon DNA damage. DNA Repair (Amst), 2002 May 30, 1(5), 379 - 90 Translation of hSNM1 is mediated by an internal ribosome entry site that upregulates expression during mitosis; Zhang X et al.; SNM1 is involved in the repair of DNA interstrand cross-links (ICLs) in Saccharomyces cerevisiae and possibly in human cells, although relatively little is known about its biochemical function . The hSNM1 contains a long 5' untranslated region (5'UTR) predicted to fold into a complex secondary structure, and which contains numerous short open reading frames (ORFs) . We show here using bicistronic constructs that human SNM1 mRNA contains an internal ribosome entry site (IRES) that generally suppresses translation, except during mitosis where translation is upregulated . These results suggest that hSNM1 may have a mitotic function possibly involved in response to DNA interstrand cross-linking agents. DNA Repair (Amst), 2002 Jun 21, 1(6), 425 - 35 Cellular roles of DNA polymerase zeta and Rev1 protein; Lawrence CW; The majority of both spontaneous and DNA damage-induced mutations in eukaryotes result from replication processes in which DNA polymerase zeta (Polzeta) and Rev1 protein (Rev1p) play major roles . Understanding these roles is likely to provide information relevant to the origin of genetic diseases, such as cancer, and may provide new insights for their prevention . DNA Polzeta also appears to be involved in the somatic hypermutability that occurs during development of the immune response . The results from a variety of genetic and enzymological investigations have started to delineate the cellular roles of these enzymes, but a number of important issues have not yet been resolved and much remains to be learned . Questions concerning the possible existence of other subunits to these enzymes, of their possible association with one another or with other proteins, of the nature of their enzymatic activities and of the relative roles played by these and other DNA polymerases in the bypass of different kinds of DNA damage, require further investigation . Finally, very little is known about the way these enzymes are regulated and brought into play when needed. Bioessays, 2003 Jan, 25(1), 58 - 65 The viral control of cellular acetylation signaling; Caron C et al.; It is becoming clear that the post-translational modification of histone and non-histone proteins by acetylation is part of an important cellular signaling process controlling a wide variety of functions in both the nucleus and the cytoplasm . Recent investigations designate this signaling pathway as one of the primary targets of viral proteins after infection . Indeed, specific viral proteins have acquired the capacity to interact with cellular acetyltransferases (HATs) and deacetylases (HDACs) and consequently to disrupt normal acetylation signaling pathways, thereby affecting viral and cellular gene expression . Here we review the targeting of cellular HATs and HDACs by viral proteins and highlight different strategies adopted by viruses to control cellular acetylation signaling and to accomplish their life cycle . Comput Methods Programs Biomed, 2003 Feb, 70(2), 107 - 19 Partitioning large-sample microarray-based gene expression profiles using principal components analysis; Peterson LE; Principal components analysis (PCA) is useful for reproducing the total variation among hundreds or thousands of continuously-scaled variables with a much smaller number of unobservable variables called 'latent factors' . The CLUSFAVOR computer program was used to implement PCA for identifying groups of genes with similar expression profiles from a large number of genes used on DNA microarrays . This paper describes the principal components solution to the factor model of the correlation matrix R, calculation of eigenvalues and eigenvectors of R, extraction of factors, and calculation of factor loadings and identification of genes with similar loading patterns to construct groups of genes with similar expression profiles . With regard to extraction of factors, it was found that more than 90% of the total variance in input data could be accounted for by extracting factors whose eigenvalues exceed unity . Bipolar factors containing strong positive and negative loadings can also be used for identifying two unique groups of genes, since expression profiles of genes that load positive are unlike expression profiles of genes that load negative on the same factor . While PCA does not provide the absolute answer to a multidimensional problem, it nevertheless can provide a heuristic with which natural groupings of genes with similar expression profiles can be assembled . While cluster analysis essentially generates a single dendogram (tree branch) containing every gene in the input data, PCA can be used to assemble gene expression profiles that strongly correlate with the latent factors accounting for a majority of total variance . Example results for CLUSFAVOR computer program runs are provided. Biochem Biophys Res Commun, 2003 Jan 17, 300(3), 694 - 8 Identification of residues which regulate activity of the STE20-related kinase hMINK; Lim J et al.; Activity of the STE20-related kinase hMINK was investigated . hMINK was expressed widely, though not ubiquitously, in human tissues; highest levels being found in haematopoietic tissues but also in brain, placenta, and lung . Mutagenesis revealed that T(191) and Y(193) in the substrate recognition loop of the catalytic domain were critical for kinase activity against exogenous substrates and autophosphorylation . A mutation on T(187) showed reduced enzymatic activity against exogenous substrates but retained autophosphorylation activity . Phosphorylation was confirmed by the use of a phospho-specific T(187) antibody . hMINK activated the JNK signal transduction pathway and optimal JNK activation occurred when the C-terminus was deleted . In addition, overexpression of the C-terminal domain devoid of kinase activity also resulted in significant activation of the JNK pathway . These data suggest that hMINK requires an activation step that dissociates the C terminal, thereby freeing the catalytic domain to interact with substrates . Models for receptor-mediated activation of hMINK are discussed. Cell, 2002 Dec 27, 111(7), 1003 - 14 RNA polymerase I propagates unidirectional spreading of rDNA silent chromatin; Buck SW et al.; The ribosomal DNA (rDNA) tandem array in Saccharomyces cerevisiae induces transcriptional silencing of RNA polymerase II-transcribed genes . This SIR2-dependent form of repression (rDNA silencing) also functions to limit rDNA recombination and is involved in life span control . In this report, we demonstrate that rDNA silencing spreads into the centromere-proximal unique sequence located downstream of RNA polymerase I (Pol I) transcription, but fails to enter the upstream telomere-proximal sequences . The spreading of silencing correlates with SIR2-dependent histone H3 and H4 deacetylation and can be extended by SIR2 overexpression . Surprisingly, rDNA silencing required transcription by RNA polymerase I and the direction of spreading was controlled by the direction of Pol I transcription. Biosci Biotechnol Biochem, 2002 Nov, 66(11), 2429 - 36 Compilation and characterization of a novel WNK family of protein kinases in Arabiodpsis thaliana with reference to circadian rhythms; Nakamichi N et al.; The complete genome sequence of Arabidopsis thaliana revealed that this higher plant has a tremendous number of protein kinases . We recently isolated a novel type of protein kinase, named AtWNK1, which shows an in vitro ability to phosphorylate the APRR3 member of the APRR1/TOC1 quintet that has been implicated in a mechanism underlying circadian rhythms in Arabidopsis . We here address two issues, one general and one specific, as to this novel protein kinase . We first asked the general question of how many WNK family members are present in this higher plant, then whether or not other members are also relevant to circadian rhythms . The results of our analyses showed that Arabidopsis has at least 9 members of the WNK1 family of protein kinases (designated here as WNK1 to WNK9), the structural design of which is clearly distinct from those of other known protein kinases, such as receptor-like kinases and mitogen-activated protein kinases . They were examined with special reference to the circadian-related APRR1/TOC1 quintet . It was found that not only the transcription of the WNK1 gene, but also those of three other members (WNK2, WNK4, and WNK6) are under the control of circadian rhythms . These results suggested that certain members of the WNK family of protein kinases might play roles in a mechanism that generates circadian rhythms in Arabidopsis. Arch Biochem Biophys, 2003 Jan 15, 409(2), 243 - 50 Identification and characterization of a sucrose transporter isolated from the developing cotyledons of soybean; Aldape MJ et al.; Reduced carbon produced in mature leaves is distributed throughout plants in the form of sucrose . Sucrose transporter proteins (SUT) play a crucial role in transporting sucrose . We isolated a cDNA encoding a sucrose transporter, GmSUT1, which is expressed in the developing cotyledons of soybean (Glycine max) . {14C}sucrose uptake assays demonstrate that GmSUT1 has a K(m) of 5.6mM and a V(max) of 5.8 nmol sucrose min(-1)(mg cells)(-1), which are similar to those of the low-affinity-high-capacity sucrose transporter family . GmSUT1 protein accumulates gradually during cotyledon development, correlating with increasing sucrose levels in the maturing cotyledons . Collectively, these data suggest that GmSUT1 plays an active role in the movement of sucrose into the developing seeds.
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