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 cytopla