Semin Cell Dev Biol, 2002 Apr, 13(2), 77 - 82 Membrane trafficking in cytokinesis; Xu H et al.; Until recently, two distinct types of cytokinesis were thought to be responsible for the division of plant and animal cells . Plant cells divide through the formation of a membrane plate between the daughter cells, while animal cells divide by the constriction of a cortical actin-based ring around the cell . However, accumulating evidence suggests that the two mechanisms may have more in common than previously thought . In this review we will focus on recent developments that raise the possibility of unexpected similarities between the final steps in cytokinesis in animal and plant cells. Arch Biochem Biophys, 2002 Aug 1, 404(1), 71 - 9 Glc8 is a glucose-repressible activator of Glc7 protein phosphatase-1; Nigavekar SS et al.; Regulation of Glc7 type 1 protein phosphatase stability and activity was studied in budding yeast . We found that the Glc7 protein has a half-life of over 180min, which is sufficient for several generations . Glc7 protein stability was constant during the cell cycle and in batch culture growth . Furthermore, deletion of regulatory subunit Gac1, Reg1, Reg2, Sds22, or Glc8 had no influence on Glc7 protein half-life . The activity of Glc7 assayed as okadaic acid-resistant phosphorylase phosphatase activity was constant during the cell cycle . Deletion of the aforementioned regulatory subunits revealed that only Glc8 deletion had a significant effect in reducing Glc7 activity . Glc7 activity was induced during stationary phase in a Glc8-dependent manner . In addition, extracellular glucose repressed the induction of Glc7 activity . These results are consistent with glucose repression of Glc8 expression and favor the role of Glc8 as a major Glc7 activator. Scand J Gastroenterol, 2002 Jun, 37(6), 692 - 8 Familial and sporadic inflammatory bowel disease: comparison of clinical features and serological markers in a genetically homogeneous population; Halme L et al.; BACKGROUND: The familial occurrence of inflammatory bowel disease (IBD) and the clinical features of familial and sporadic IBD in the genetically homogeneous Finnish population are evaluated . METHODS: 257 patients with Crohn disease (CD) and 436 with ulcerative colitis (UC) participated in the study . They were asked whether IBD was present (familial IBD) or absent (sporadic IBD) in their first-degree relatives . Data on the clinical course of the disease were collected from the patient records . Antibodies to Saccharomyces cerevisiae (ASCA) and anti-neutrophil cytoplasmic antibodies (ANCA) were determined from serum samples . RESULTS: Affected first-degree relatives were found in 15.6% of patients with CD and in 13.8% of patients with UC . In familial cases CD was more often located in the ileum (38% versus 21%) and less often in the ileocolon (35% versus 50%) (P< 0.05) than in sporadic cases . A greater percentage of CD patients than UC patients were smokers (47% versus 13%; P < 0.01) . An elevated level of IgA and/or IgG antibodies for ASCA was found more often in CD patients than in UC patients (59% versus 14%; P < 0.01), while pANCA were found more often in UC than in CD patients (48% versus 12%; P < 0.01) . The combination of pANCA-ASCA+ yielded a sensitivity, specificity and positive predictive value of 48%, 92% and 90%, respectively, for CD, and the combination of pANCA + ASCA- of 55%, 94% and 90%, respectively, for UC . CONCLUSIONS: The percentage of familial IBD cases in Finland is comparable to that reported elsewhere in Europe . No important clinical differences between patients with familial and sporadic forms of the disease were found . ASCA is associated with both familial and sporadic CD and pANCA with UC, but low sensitivity diminishes their value as a serological marker of IBD or as a differential diagnostic test between CD and UC. Protist, 2002 Jun, 153(2), 111 - 22 Polyadenylated transcripts containing random gene fragments are expressed in dinoflagellate mitochondria; Chaput H et al.; An AT-rich cDNA isolated from a Gonyaulax library was identified as a putative mitochondrial cytochrome oxidase subunit 3 (cox3) by sequence comparisons . Transcripts hybridizing to this cox3 probe are abundant, are found in poly(A) enriched RNA fractions and have a variable length when tested by Northern blot analysis . A PCR analysis of 21 cox3 clones, designed to measure the length of 5' and 3' ends separately, indicated that the greatest variability in length was found at the 3' end . The variable length of the 3' end may be due to polyadenylation at random sites or to a reduced stringency for polyadenylation signals . Eight cox3 cDNAs were completely sequenced, and were found to differ by up to thirteen bases in the regions of overlap . Curiously, the longest cDNA contained a 350 base pair fragment of a dinoflagellate cox1 gene and a 600 bp fragment of a cytochrome b (cob) gene in the region 3' to the cox3 sequence . Gene fragments or multiple copies of the cox3 and cob sequences may be abundant in the mitochondrial genome since Southern blots, using these sequences separately as probes, show multiple restriction fragments with several enzymes. Chem Commun (Camb), 2002 Jul 7, (13), 1428 - 9 A screening system for enantioselective enzymes based on differential cell growth; Reetz MT et al.; The esterase-catalyzed enantioselective hydrolysis of the fluoroacetate of pantolactone leads to fluoroacetic acid, a toxic compound which inhibits the growth of esterase-producing yeast; this forms the basis of an ee-assay. Hum Mutat, 2002 Aug, 20(2), 139 - 47 An amplification and ligation-based method to scan for unknown mutations in DNA; Zhang Y et al.; A new approach is presented for the sensitive and selective scanning for unknown DNA mutations, based on ligation-mediated PCR and the use of the glycosylases TDG and MutY . These two highly selective enzymes together can detect about 70% of commonly observed polymorphisms and mutations in human tumors . DNA is cross-hybridized to form mismatches at the positions of point mutations, de-phosphorylated to eliminate any pre-existing phosphorylated DNA ends, and then exposed to enzymatic treatment to remove mismatched thymidine (TDG) or adenine (MutY) . The resulting apurinic/apyrimidinic sites at the position of the mismatches are heat-converted to 5'-phosphate-containing strand breaks, the DNA is denatured, and an oligonucleotide is ligated at the position of the newly created 5'-phosphate-containing DNA ends . The ligated oligonucleotide then participates in a PCR reaction that amplifies exponentially only the mutation-containing fragments . Using this method, A-->G mutations in a p53 (TP53)-containing system, T-->G, G-->A, and C-->A, mutations in the Ku gene (XRCC5), and ATM, gene for a number of patient-derived genomic DNA samples have been successfully screened . This PCR-based assay is capable of detecting one mutated allele in 100 normal alleles and requires 5 to 100 ng of genomic DNA as starting material . The assay allows final visualization of the mutated fragments on a common ethidium gel or biotinylation and use in a capture format, potentially allowing the isolation of diverse mutated DNA fragments simultaneously . This versatile new approach should allow high throughput detection of DNA alterations and application in diverse areas of human mutation research . Nature, 2002 Jul 18, 418(6895), 348 - 52 Adenovirus oncoproteins inactivate the Mre11-Rad50-NBS1 DNA repair complex; Stracker TH et al.; In mammalian cells, a conserved multiprotein complex of Mre11, Rad50 and NBS1 (also known as nibrin and p95) is important for double-strand break repair, meiotic recombination and telomere maintenance . This complex forms nuclear foci and may be a sensor of double-strand breaks . In the absence of the early region E4, the double-stranded DNA genome of adenovirus is joined into concatemers too large to be packaged . We have investigated the cellular proteins involved in this concatemer formation and how they are inactivated by E4 products during a wild-type infection . Here we show that concatemerization requires functional Mre11 and NBS1, and that these proteins are found at foci adjacent to viral replication centres . Infection with wild-type virus results in both reorganization and degradation of members of the Mre11-Rad50-NBS1 complex . These activities are mediated by three viral oncoproteins that prevent concatemerization . This targeting of cellular proteins involved in genomic stability suggests a mechanism for 'hit-and-run' transformation observed for these viral oncoproteins. FEBS Lett, 2002 Jul 17, 523(1-3), 35 - 42 Identification of the first Rho-GEF inhibitor, TRIPalpha, which targets the RhoA-specific GEF domain of Trio; Schmidt S et al.; The Rho-guanine nucleotide exchange factors (Rho-GEFs) remodel the actin cytoskeleton via their Rho-GTPase targets and affect numerous physiological processes such as transformation and cell motility . They are therefore attractive targets to design specific inhibitors that may have therapeutic applications . Trio contains two Rho-GEF domains, GEFD1 and GEFD2, which activate the Rac and RhoA pathways, respectively . Here we have used a genetic screen in yeast to select in vivo peptides coupled to thioredoxin, called aptamers, that could inhibit GEFD2 activity . One aptamer, TRIAPalpha (TRio Inhibitory APtamer), specifically blocks GEFD2-exchange activity on RhoA in vitro . The corresponding peptide sequence, TRIPalpha, inhibits TrioGEFD2-mediated activation of RhoA in intact cells and specifically reverts the neurite retraction phenotype induced by TrioGEFD2 in PC12 cells . Thus TRIPalpha is the first Rho-GEF inhibitor isolated so far, and represents an important step in the design of inhibitors for the expanding family of Rho-GEFs. Curr Biol, 2002 Jun 25, 12(12), 973 - 82 The dual mechanism of separase regulation by securin; Hornig NC et al.; BACKGROUND: Sister chromatid separation and segregation at anaphase onset are triggered by cleavage of the chromosomal cohesin complex by the protease separase . Separase is regulated by its binding partner securin in two ways: securin is required to support separase activity in anaphase; and, at the same time, securin must be destroyed via ubiquitylation before separase becomes active . The molecular mechanisms underlying this dual regulation of separase by securin are unknown.RESULTS: We show that, in budding yeast, securin supports separase localization . Separase enters the nucleus independently of securin, but securin is required and sufficient to cause accumulation of separase in the nucleus, where its known cleavage targets reside . Securin also ensures that separase gains full proteolytic activity in anaphase . We also show that securin, while present, directly inhibits the proteolytic activity of separase . Securin prevents the binding of separase to its substrates . It also hinders the separase N terminus from interacting with and possibly inducing an activating conformational change at the protease active site 150 kDa downstream at the protein's C terminus.CONCLUSIONS: Securin inhibits the proteolytic activity of separase in a 2-fold manner . While inhibiting separase, securin is able to promote nuclear accumulation of separase and help separase to become fully activated after securin's own destruction at anaphase onset. Biochem Cell Biol, 2002, 80(3), 321 - 33 A feel for the template: zinc finger protein transcription factors and chromatin; Urnov FD; Transcription factors and chromatin collaborate in bringing the eukaryotic genome to life . An important, and poorly understood, aspect of this collaboration involves targeting the regulators to correct binding sites in vivo . An implicit and insufficiently tested assumption in the field has been that chromatin simply obstructs most sites and leaves only a few functionally relevant ones accessible . The major class of transcription factors in all metazoa, zinc finger proteins (ZFPs), can bind to chromatin in vitro (as clearly shown for Spl, GATA-1 and -4, and the nuclear hormone receptors, for example) . Data on the accessibility of DNA within heterochromatin to nonhistone regulators (E.A . Sekinger and D.S . Gross . 2001 . Mol . Cell 105: 403-414; C . Jolly et al . 2002 . J . Cell . Biol . 156: 775-781) and the ability of the basal transcription machinery to reside within highly condensed chromatin (most recently, R . Christova and T . Oelgeschlaeger . 2002 . Nat . Cell Biol . 4: 79-82) further weaken the argument that chromatin acts as an across-the-board deterrent to ZFP binding . These proteins, however, do not bind promiscuously in vivo, and recent data on human cells (C.E . Horak et al . 2002 . Proc . Natl . Acad . Sci . U.S.A . 99: 2924-2929) confirm earlier data on budding yeast (B . Ren et al . 2000 . Science (Washington, D.C.), 290: 2306-2309) that primary DNA sequence, i.e., density of binding sites per unit DNA length, is not the primary determinant of where a ZFP transcription factor will bind in vivo . This article reviews these data and uses ZFP transcription factors as a model system to compare in vitro binding to chromatin by transcription factors with their in vivo behavior in gene regulation . DNA binding domain structure, nonrandom nucleoprotein organization of chromatin at target promoters, and cooperativity of regulator action may all contribute to target site selection in vivo. J Biol Chem, 2002 Sep 20, 277(38), 35025 - 34 Epub 2002 Jul 16. A novel calcium-stimulated adenylyl cyclase from Trypanosoma cruzi, which interacts with the structural flagellar protein paraflagellar rod; D'Angelo MA et al.; Trypanosoma cruzi adenylyl cyclases are encoded by a large polymorphic gene family . Although several genes have been identified in this parasite, little is known about the properties and regulation of these enzymes . Here we report the cloning and characterization of TczAC, a novel member of T . cruzi adenylyl cyclase family . The TczAC gene is expressed in all of the parasite life forms and encodes a 1,313-amino acid protein that can complement a Saccharomyces cerevisiae mutant deficient in adenylyl cyclase activity . The recombinant enzyme expressed in yeasts is constitutively active, has a low affinity for ATP (K(m) = 406 microm), and requires a divalent cation for catalysis . TczAC is inhibited by Zn(2+) and the P-site inhibitor 2'-deoxyadenosine 3'-monophosphate, suggesting some level of conservation in the catalytic mechanism with mammalian adenylyl cyclases . It shows a dose-dependent stimulation by Ca(2+) which can be reversed by high concentrations of phenothiazinic calmodulin inhibitors . However, bovine calmodulin fails to stimulate the enzyme . Using a yeast two-hybrid screen it was found that TczAC interacts through its catalytic domain with the paraflagellar rod protein, a component of the flagellar structure . Furthermore, we demonstrate that TczAC can dimerize through the same domain . These results provide novel evidence of the possible localization and regulation of this protein. J Biol Chem, 2002 Sep 20, 277(38), 34959 - 66 Epub 2002 Jul 16. Interaction between the N-terminal and middle regions is essential for the in vivo function of HSP90 molecular chaperone; Matsumoto S et al.; At the primary structure level, the 90-kDa heat shock protein (HSP90) is composed of three regions: the N-terminal (Met(1)-Arg(400)), middle (Glu(401)-Lys(615)), and C-terminal (Asp(621)-Asp(732)) regions . In the present study, we investigated potential subregion structures of these three regions and their roles . Limited proteolysis revealed that the N-terminal region could be split into two fragments carrying residues Met(1) to Lys(281) (or Lys(283)) and Glu(282) (or Tyr(284)) to Arg(400) . The former is known to carry the ATP-binding domain . The fragments carrying the N-terminal two-thirds (Glu(401)-Lys(546)) and C-terminal one-third of the middle region were sufficient for the interactions with the N- and C-terminal regions, respectively . Yeast HSC82 that carried point mutations in the middle region causing deficient binding to the N-terminal region could not support the growth of HSP82-depleted cells at an elevated temperature . Taken together, our data show that the N-terminal and middle regions of the HSP90 family protein are structurally divided into two respective subregions . Moreover, the interaction between the N-terminal and middle regions is essential for the in vivo function of HSP90 in yeast. Curr Biol, 2002 Jul 9, 12(13), R458 - 60 Chromosome dynamics: new light on Aurora B kinase function; Shannon KB et al.; Aurora B family kinases play an essential role in chromosome segregation and cytokinesis . Recent work suggests that the kinase activity is required for bipolar chromosome orientation, kinetochore assembly, spindle checkpoint and microtubule dynamics . Aurora B also has additional functions in chromosome condensation and cohesion. Chem Commun (Camb), 2002 Mar 21, (6), 588 - 9 A C-terminal domain of the membrane copper pump Ctr1 exchanges copper(I) with the copper chaperone Atx1; Xiao Z et al.; A cloned C-terminal domain of the yeast high-affinity copper uptake pump Ctr1 exchanges Cu(I) rapidly with the yeast copper chaperone Atx1: 10(-2) < Kex < 10(+2). Gene, 2002 Jun 12, 292(1-2), 141 - 9 A system for deletion and complementation of Candida glabrata genes amenable to high-throughput application; Willins DA et al.; We describe a method for deleting or modifying genes from the pathogenic fungus Candida glabrata as well as a companion vector for complementation or ectopic expression experiments . A linear deletion fragment generated by polymerase chain reaction was used to replace a gene of interest with the C . glabrata gene encoding imidazoleglycerol-phosphate dehydratase (HIS3) . As test cases, the chromosomal loci of the C . glabrata genes encoding aminoimidazole ribonucleotide carboxylase (ADE2) and encoding isopropylmalate dehydrogenase (LEU2) were deleted . To facilitate application of the deletion technique to essential genes, we also constructed vectors to allow expression of a complementing copy of the wildtype gene under control of the copper-inducible C . glabrata metallothionein I (MT-1) promoter . One version of the vector carried the Saccharomyces cerevisiae centromere (CEN) and autonomously-replicating sequence (ARS) regions . The C . glabrata ADE2 and LEU2 genes and a transposon-derived neomycin/kanamycin resistance gene were successfully expressed from this vector, with expression of the ADE2 and LEU2 genes complementing the ADE2 and LEU2 deletion mutations, respectively . However, this vector showed regulated expression only for the ADE2 gene . A second version of the vector, which carried an additional C . glabrata CEN and ARS region for stable plasmid maintenance, did show regulated expression for the LEU2 and neomycin/kanamycin resistance genes . This deletion and expression system is potentially applicable to any C . glabrata gene and is amenable to high-throughput application . We anticipate that these tools will have broad utility in deletion or modification of specific C . glabrata genes . This approach is also applicable to other yeast fungi. Biochemistry, 2002 Jul 23, 41(29), 9070 - 8 Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase; Conway ME et al.; The human mitochondrial branched chain aminotransferase isoenzyme (hBCATm) must be stored in a reducing environment to remain active . Oxidation or labeling of hBCATm with sulfhydryl reagents results in enzyme inhibition . In this study, we investigated both the structural and biochemical basis for the sensitivity of hBCATm to these reagents . In its native form, hBCATm has two reactive cysteine residues which were identified as Cys315 and Cys318 using iodinated beta-(4-hydroxyphenyl)ethyl maleimide . These are located in the large domain of the homodimer, about 10 A from the active site . The crystal structures show evidence for a thiol-thiolate hydrogen bond between Cys315 and Cys318 . Under oxidizing conditions, these cysteine residues can reasonably form a disulfide bond because of the short distance between the sulfur atoms (3.09-3.46 A), requiring only a decrease of 1.1-1.5 A . In addition to Cys315 playing a structural role by anchoring Tyr173, which in the ketimine form increases access to the active site, our evidence indicates that these cysteine residues act as a redox switch in hBCATm . Electrospray ionization mass spectrometry analysis and UV-Vis spectroscopic studies of 5,5'-dithiobis(2-nitrobenzoic acid) labeled hBCATm showed that during labeling, an intrasubunit disulfide bond was formed in a significant portion of the protein . Furthermore, it was established that reaction of hBCATm with H2O2 abolished its activity and resulted in the formation of an intrasubunit disulfide bond between Cys315 and Cys318 . Addition of dithiothreitol completely reversed the oxidation and restored activity . Therefore, the results demonstrate that there is redox-linked regulation of hBCATm activity by a peroxide sensitive CXXC center . Future studies will determine if this center has an in vivo role in the regulation of branched chain amino acid metabolism. Mol Cell Proteomics, 2002 May, 1(5), 349 - 56 Protein interactions: two methods for assessment of the reliability of high throughput observations; Deane CM et al.; High throughput methods for detecting protein interactions require assessment of their accuracy . We present two forms of computational assessment . The first method is the expression profile reliability (EPR) index . The EPR index estimates the biologically relevant fraction of protein interactions detected in a high throughput screen . It does so by comparing the RNA expression profiles for the proteins whose interactions are found in the screen with expression profiles for known interacting and non-interacting pairs of proteins . The second form of assessment is the paralogous verification method (PVM) . This method judges an interaction likely if the putatively interacting pair has paralogs that also interact . In contrast to the EPR index, which evaluates datasets of interactions, PVM scores individual interactions . On a test set, PVM identifies correctly 40% of true interactions with a false positive rate of approximately 1% . EPR and PVM were applied to the Database of Interacting Proteins (DIP), a large and diverse collection of protein-protein interactions that contains over 8000 Saccharomyces cerevisiae pairwise protein interactions . Using these two methods, we estimate that approximately 50% of them are reliable, and with the aid of PVM we identify confidently 3003 of them . Web servers for both the PVM and EPR methods are available on the DIP website (dip.doe-mbi.ucla.edu/Services.cgi). Infect Immun, 2002 Aug, 70(8), 4510 - 22 Genes for glycosylphosphatidylinositol toxin biosynthesis in Plasmodium falciparum; Delorenzi M et al.; About 2.5 million people die of Plasmodium falciparum malaria every year . Fatalities are associated with systemic and organ-specific inflammation initiated by a parasite toxin . Recent studies show that glycosylphosphatidylinositol (GPI) functions as the dominant parasite toxin in the context of infection . GPIs also serve as membrane anchors for several of the most important surface antigens of parasite invasive stages . GPI anchoring is a complex posttranslational modification produced through the coordinated action of a multicomponent biosynthetic pathway . Here we present eight new genes of P . falciparum selected for encoding homologs of proteins essential for GPI synthesis: PIG-A, PIG-B, PIG-M, PIG-O, GPI1, GPI8, GAA-1, and DPM1 . We describe the experimentally verified mRNA and predicted amino acid sequences and in situ localization of the gene products to the parasite endoplasmic reticulum . Moreover, we show preliminary evidence for the PIG-L and PIG-C genes . The biosynthetic pathway of the malaria parasite GPI offers potential targets for drug development and may be useful for studying parasite cell biology and the molecular basis for the pathophysiology of parasitic diseases. Carcinogenesis, 2002 Jul, 23(7), 1139 - 48 Protocadherin LKC, a new candidate for a tumor suppressor of colon and liver cancers, its association with contact inhibition of cell proliferation; Okazaki N et al.; Protocadherins are a major subfamily of the cadherin superfamily, but little is known about their functions and intracellular signal transduction . We cloned a novel human protocadherin gene, containing seven EC domains, and identified functional aspects of this gene . The gene was predominantly expressed in liver, kidney and colon tissues, and was thus designated Protocadherin LKC . The expression of Protocadherin LKC is markedly reduced in cancers arising from these tissues at both transcriptional and protein levels . To investigate the effects of Protocadherin LKC expression in colon cancer, we introduced the gene into colon cancer cell line HCT116, which does not express this gene . Significantly, Protocadherin LKC expression induced contact inhibition of cell proliferation although it did not affect growth rate . When grown to post-confluence in monolayer cells cultures, Protocadherin LKC-expressing HCT116 no longer formed multiple cell layers and showed the typical paving stone morphology of normal epithelial cells . Furthermore, expression of Protocadherin LKC suppressed tumor formation of HCT116 cells in a nude mouse model . In addition, we identified a protein, hMAST205 (microtubule-associated serine/threonine kinase-205 kDa), which interacted with Protocadherin LKC; the interaction occurring between the PDZ domain of hMAST205 and C-terminal tail of Protocadherin LKC . Our results suggest that Protocadherin LKC, which directly binds PDZ protein, is a molecular switch for contact inhibition of epithelial cells in the liver, kidney and colon tissues. J Neurobiol, 2002 Jul, 52(1), 24 - 42 Regulation of neuronal excitability in Drosophila by constitutively active CaMKII; Park D et al.; The ability of calcium/calmodulin-dependent protein kinase II (CaMKII) to become calcium independent after autophosphorylation makes this enzyme a temporal marker of neuronal activity . Here we show that the calcium-independent form of CaMKII has unique effects on larval viability, locomotion, and neuronal excitability in Drosophila . Expression of constitutively active T287D, but not calcium-dependent T287A, mutant CaMKII in Drosophila neurons resulted in decreased viability, behavioral defects, and failure of action potential propagation . The actions of T287D may be mediated, at least in part, by increased potassium conductances . Expression of T287D CaMKII also stimulated an increase in the number of boutons at the larval neuromuscular junction, but did not affect the mechanics of release . This study defines a role for autophosphorylation of CaMKII in the regulation of multiple neuronal functions including the intrinsic properties of neurons . Plant Physiol, 2002 Jul, 129(3), 1232 - 40 Phosphite, an analog of phosphate, suppresses the coordinated expression of genes under phosphate starvation; Varadarajan DK et al.; Phosphate (Pi) and its analog phosphite (Phi) are acquired by plants via Pi transporters . Although the uptake and mobility of Phi and Pi are similar, there is no evidence suggesting that plants can utilize Phi as a sole source of phosphorus . Phi is also known to interfere with many of the Pi starvation responses in plants and yeast (Saccharomyces cerevisiae) . In this study, effects of Phi on plant growth and coordinated expression of genes induced by Pi starvation were analyzed . Phi suppressed many of the Pi starvation responses that are commonly observed in plants . Enhanced root growth and root to shoot ratio, a hallmark of Pi stress response, was strongly inhibited by Phi . The negative effects of Phi were not obvious in plants supplemented with Pi . The expression of Pi starvation-induced genes such as LePT1, LePT2, AtPT1, and AtPT2 (high-affinity Pi transporters); LePS2 (a novel acid phosphatase); LePS3 and TPSI1 (novel genes); and PAP1 (purple acid phosphatase) was suppressed by Phi in plants and cell cultures . Expression of luciferase reporter gene driven by the Pi starvation-induced AtPT2 promoter was also suppressed by Phi . These analyses showed that suppression of Pi starvation-induced genes is an early response to addition of Phi . These data also provide evidence that Phi interferes with gene expression at the level of transcription . Synchronized suppression of multiple Pi starvation-induced genes by Phi points to its action on the early molecular events, probably signal transduction, in Pi starvation response. Plant Physiol, 2002 Jul, 129(3), 1207 - 15 Selective activation of the developmentally regulated Ha hsp17.6 G1 promoter by heat stress transcription factors; Rojas A et al.; Using two well-characterized heat stress transcription factors (Hsfs) from tomato (Lycopersicon peruvianum; LpHsfA1 and LpHsfA2), we analyzed the transcriptional activation of the Ha hsp17.6 G1 promoter in sunflower (Helianthus annuus) embryos . In this system, we observed transient promoter activation only with LpHsfA2 . In contrast, both factors were able to activate mutant versions of the promoter with improved consensus Hsf-binding sites . Exclusive activation by LpHsfA2 was also observed in yeast (Saccharomyces cerevisiae) without other Hsfs and with a minimal Cyc1 promoter fused to the Ha hsp17.6 G1 heat stress cis-element . Furthermore, the same promoter mutations reproduced the loss of activation selectivity, as observed in sunflower embryos . The results of in vitro binding experiments rule out differential DNA binding of the two factors as the explanation for the observed differential activation capacity . We conclude that the specific sequence of this heat stress cis-element is crucial for Hsf promoter selectivity, and that this selectivity could involve preferential transcriptional activation following DNA binding . In sunflower embryos, we also observed synergistic transcriptional activation by co-expression of LpHsfA1 and LpHsfA2 . Mutational analyses of the Ha hsp17.6 G1 promoter, combined with in vitro binding assays, suggest that mixed oligomers of the two factors may be involved in promoter activation . We discuss the relevance of our observations for mechanisms of developmental regulation of plant heat stress protein genes. Plant Physiol, 2002 Jul, 129(3), 1181 - 93 Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene; Hanaoka H et al.; Autophagy is an intracellular process for vacuolar bulk degradation of cytoplasmic components . The molecular machinery responsible for yeast and mammalian autophagy has recently begun to be elucidated at the cellular level, but the role that autophagy plays at the organismal level has yet to be determined . In this study, a genome-wide search revealed significant conservation between yeast and plant autophagy genes . Twenty-five plant genes that are homologous to 12 yeast genes essential for autophagy were discovered . We identified an Arabidopsis mutant carrying a T-DNA insertion within AtAPG9, which is the only ortholog of yeast Apg9 in Arabidopsis (atapg9-1) . AtAPG9 is transcribed in every wild-type organ tested but not in the atapg9-1 mutant . Under nitrogen or carbon-starvation conditions, chlorosis was observed earlier in atapg9-1 cotyledons and rosette leaves compared with wild-type plants . Furthermore, atapg9-1 exhibited a reduction in seed set when nitrogen starved . Even under nutrient growth conditions, bolting and natural leaf senescence were accelerated in atapg9-1 plants . Senescence-associated genes SEN1 and YSL4 were up-regulated in atapg9-1 before induction of senescence, unlike in wild type . All of these phenotypes were complemented by the expression of wild-type AtAPG9 in atapg9-1 plants . These results imply that autophagy is required for maintenance of the cellular viability under nutrient-limited conditions and for efficient nutrient use as a whole plant. J Biol Chem, 2002 Sep 27, 277(39), 36449 - 56 Epub 2002 Jul 11. The N-terminal domains of syntaxin 7 and vti1b form three-helix bundles that differ in their ability to regulate SNARE complex assembly; Antonin W et al.; The SNAREs syntaxin 7, syntaxin 8, vti1b, and endobrevin/VAMP8 function in the fusion of late endosomes . Although the core complex formed by these SNAREs is very similar to the neuronal SNARE complex, it differs from the neuronal complex in that three of the four SNAREs contain extended N-terminal regions of unknown structure and function . Here we show that the N-terminal regions of syntaxin 7, syntaxin 8, and vti1b contain well folded alpha-helical domains . Multidimensional NMR spectroscopy revealed that in syntaxin 7 and vti1b, the domains form three-helix bundles resembling those of syntaxin 1, Sso1p, and Vam3p . The three-helix bundle domain of vti1b is the first of its kind identified in a SNARE outside the syntaxin family . Only syntaxin 7 adopts a closed conformation, whereas in vti1b and syntaxin 8, the N-terminal domains do not interact with the adjacent SNARE motifs . Accordingly, the rate of SNARE complex assembly is retarded about 7-fold when syntaxin 7 contains its N-terminal domain, whereas the N-terminal domains of vti1b and syntaxin 8 have no influence on assembly kinetics . We conclude that three-helix bundles represent a common fold for SNARE N-terminal domains, not restricted to the syntaxin family . However, they differ in their ability to adopt closed conformations and thus to regulate the assembly of SNARE complexes. Bioorg Med Chem Lett, 2002 Aug 5, 12(15), 2011 - 4 Inhibition of Src kinase activity by 4-anilino-7-thienyl-3-quinolinecarbonitriles; Boschelli DH et al.; Based on a screening lead from a yeast-based assay to identify Src family kinase inhibitors, a series of 4-anilino-7-thienyl-3-quinolinecarbonitriles was prepared . When the thiophene ring was substituted with a water-solubilizing group in a 2,5-, 3,5- or 2,4-pattern, potent inhibition of Src kinase activity was observed. Ann Neurol, 2002 Jun, 51(6), 771 - 4 Human herpesvirus 6 encephalitis associated with hypersensitivity syndrome; Fujino Y et al.; Hypersensitivity syndrome, a serious systematic reaction to a limited number of drugs, is associated with the reactivation of human herpesvirus 6 . A 56-year-old man developed acute limbic encephalitis followed by multiple organ failure during the course of toxic dermatitis induced by aromatic anticonvulsants . The clinical features of skin eruptions, high fever, eosinophilia, and atypical lymphocytosis were compatible with drug hypersensitivity syndrome . The patient showed seroconversion for human herpesvirus 6, and polymerase chain reaction detected human herpesvirus 6 DNA in the cerebrospinal fluid . To our knowledge, this is the first report of human herpesvirus 6 encephalitis associated with hypersensitivity syndrome. Curr Genet, 2002 Jun, 41(3), 123 - 31 Epub 2002 Jun 19. Septins: a ring to part mother and daughter; Faty M et al.; The septins are well conserved GTPases found in animals and fungi . In yeast, they are required for the formation of 10-nm filaments, with which they co-localize at the bud neck . Therefore, septins have been proposed to be components of the neck filaments and to have polymerization properties . In support of this hypothesis, septin complexes purified from yeast and flies form filaments in vitro . However, recent studies have questioned the relevance of septin filament formation for septin function . Particularly, septin polymerization may not be required for their function in cytokinesis . New septin functions have also been recently uncovered: in budding yeast, the septin ring is required for the maintenance of cell polarity . It forms a cortical barrier that prevents lateral diffusion of membrane-associated proteins through the bud neck . Here, we review the most recent functional and biochemical data, to discuss whether there is a link between septin polymerization properties and septin function. J Biol Chem, 2002 Sep 13, 277(37), 34003 - 9 Epub 2002 Jul 10. Functional mapping of Bas2 . Identification of activation and Bas1-interaction domains; Hannum C et al.; The transcriptional activator protein Bas2 is required to express more than 20 genes in pathways for purine nucleotide and histidine biosynthesis, phosphate utilization, and the HO endonuclease by acting with co-regulator proteins Bas1, Pho4, and Swi5 . The role that Bas2 plays in transcriptional activation may be to unmask latent activation domains in the co-regulator and to promote ternary complex formation between Bas2, the co-regulator, and DNA . We show that Bas2 also contributes to transcriptional activation by providing an activation domain . We localize this domain in Bas2 to the C-terminal 156 amino acids using deletion analysis and fusion to a heterologous DNA binding domain . Additionally, we show that Bas2 makes direct contacts with Bas1 . This interaction is detected by co-immunoprecipitation and by two-hybrid analysis . We localize the interaction region to the central portion of Bas2, from amino acids 112 to 404. J Biol Chem, 2002 Sep 20, 277(38), 35688 - 95 Epub 2002 Jul 10. A conserved motif common to the histone acetyltransferase Esa1 and the histone deacetylase Rpd3; Adachi N et al.; Post-translational modification of histones enables dynamic regulation of chromatin structure in eukaryotes . Histone acetyltransferase (HAT) and histone deacetylase (HDAC) modify the N-terminal tails of histones by adding or removing acetyl groups to specific lysine residues . A particular pair of HAT (Esa1) and HDAC (Rpd3) is proposed to modify the same lysine residue in vitro and in vivo . Thus, HAT and HDAC might have similar structural and functional motifs . Here we show that HAT (Esa1 family) and HDAC (Rpd3 family) have similar amino acid stretches in the primary structures through evolution . We refer to this region as the "ER (Esa1-Rpd3) motif." In the tertiary structure of Esa1, the ER motif is located near the active center . In Rpd3, for which the tertiary structure remains unclear, we demonstrate that the ER motif contains the same secondary structure as found in Esa1 by circular dichroism analysis . We did alanine-scanning mutagenesis and found that the ER motif regions of Esa1 or Rpd3 are required for HAT activity of Esa1 or HDAC activity of Rpd3, respectively . Our discovery of the ER motif present in the pair of enzymes (HAT and HDAC) indicates that HAT and HDAC have common structural bases, although they catalyze the reaction with opposite functions. EMBO J, 2002 Jul 15, 21(14), 3620 - 31 How Tlg2p/syntaxin 16 'snares' Vps45; Dulubova I et al.; Soluble N-ethylmaleimide sensitive factor-attachment protein receptors (SNAREs) and Sec1p/Munc18-homologs (SM proteins) play key roles in intracellular membrane fusion . The SNAREs form tight four-helix bundles (core complexes) that bring the membranes together, but it is unclear how this activity is coupled to SM protein function . Studies of the yeast trans-Golgi network (TGN)/endosomal SNARE complex, which includes the syntaxin-like SNARE Tlg2p, have suggested that its assembly requires activation by binding of the SM protein Vps45p to the cytoplasmic region of Tlg2p folded into a closed conformation . Nuclear magnetic resonance and biochemical experiments now show that Tlg2p and Pep12p, a late- endosomal syntaxin that interacts functionally but not directly with Vps45p, have a domain structure characteristic of syntaxins but do not adopt a closed conformation . Tlg2p binds tightly to Vps45p via a short N-terminal peptide motif that is absent in Pep12p . The Tlg2p/Vps45p binding mode is shared by the mammalian syntaxin 16, confirming that it is a Tlg2p homolog, and resembles the mode of interaction between the SM protein Sly1p and the syntaxins Ufe1p and Sed5p . Thus, this mechanism represents the most widespread mode of coupling between syntaxins and SM proteins. Cell, 2002 Jun 28, 109(7), 835 - 48 Yph1p, an ORC-interacting protein: potential links between cell proliferation control, DNA replication, and ribosome biogenesis; Du YC et al.; Immunoprecipitation of the origin recognition complex (ORC) from yeast extracts identified Yph1p, an essential protein containing a BRCT domain . Two Yph1p complexes were characterized . Besides ORC, MCM proteins, cell-cycle regulatory proteins, checkpoint proteins, 60S ribosomal proteins, and preribosome particle proteins were found to be associated with Yph1p . Yph1p is predominantly nucleolar and is required for 60S ribosomal subunit biogenesis and possibly for translation on polysomes . Proliferating cells depleted of Yph1p arrest in G(1) or G(2), with no cells in S phase, or significantly delay S phase progression after release from a hydroxyurea arrest . Yph1p levels decline as cells commit to exit the cell cycle, and levels vary depending on energy source . Yph1p may link cell proliferation control to DNA replication, ribosome biogenesis, and translation on polysomes. Cancer Chemother Pharmacol, 2002 Jun, 49(6), 445 - 52 Epub 2002 Apr 20. Human colon cancer cells surviving high doses of cisplatin or oxaliplatin in vitro are not defective in DNA mismatch repair proteins; Sergent C et al.; PURPOSE: Alterations in the DNA mismatch repair (MMR) proteins have been associated with an increased resistance of many cancer cell lines to cisplatin . The aim of this work was to investigate whether defects in DNA MMR proteins are involved in the survival of human colorectal cancer cells in the presence of high concentrations of cisplatin and oxaliplatin, a diaminocyclohexane (DACH) platinum compound whose adducts are not recognized by the MMR system . METHODS: Six unselected human colon cancer cell lines (HT29, HCT15, HCT116, Caco2, SW480 and SW620) were treated with a single 3-h exposure to cisplatin or oxaliplatin at suprapharmacological concentrations, ranging from 50 to 200 microg/ml . The microsatellite stability and the expression of MMR proteins in the parental cell lines and in the drug-selected subpopulations were studied . RESULTS: Most cells underwent apoptosis in the days following the cisplatin or oxaliplatin treatment, but some colonies expanded 3 to 4 weeks after, suggesting the presence of innately resistant cells in the six parental cell lines . Microsatellite instability (MIN), which reflects genetic defects in the DNA MMR system, was detected only in the HCT116 parental cell line and its drug-selected counterparts, due to a known mutation in the hMLH1 gene . No acquired MIN was observed in the other cisplatin-selected sublines derived from the HT29, HCT15, Caco2, SW480 or SW620 parental cells . In the same way, Western blot analysis showed that expression of the DNA MMR proteins hMLH1, hPMS1, hPMS2, hMSH2 and hMSH6 did not differ between the parental and the drug-surviving cells . CONCLUSIONS: These results indicate that high-level resistance of human colon cancer cells to high doses of cisplatin and oxaliplatin does not seem to be related to acquired defects in the DNA MMR proteins. Proc Natl Acad Sci U S A, 2002 Jul 23, 99(15), 9668 - 73 Epub 2002 Jul 09. Molecular dynamic study of orotidine-5'-monophosphate decarboxylase in ground state and in intermediate state: a role of the 203-218 loop dynamics; Hur S et al.; Molecular dynamics simulations have been used to derive the structures of ground (orotidine-5'-monophosphate decarboxylase x orotidine 5'-monophosphate; ODC x OMP) and intermediate (ODC x intermediate; ODC x I(-)) states in the ODC-catalyzed decarboxylation of OMP . For comparison, a molecular dynamics simulation of the conformers of OMP dissolved in water was also studied . This structural information is unavailable from present crystal structures . The electrostatic network in the active site around the carboxylate moiety of OMP exhibits remarkable stability . The conformation of enzyme-bound OMP is very similar to the conformation of OMP in water . Thus, the proposed Circe effect mechanism for ODC catalysis is unlikely . Comparison of ground state and intermediate state structures shows that on decarboxylation C6 takes the position of the carboxylate O8 . This significant movement of the ligand is accompanied by a placement of the C6 carbanion in the vicinity of the protonated Lys-93 and is enforced by a change of the 203-218 loop from an unstructured form to an ordered beta-hairpin . Previously proposed mechanisms involving protonation at O2, O4, or C5 have in common internal stabilization of the anionic intermediate by conjugation with positive charge on the pyrimidine ring . These mechanisms are not supported because there are no proton sources near O2, O4, and C5 . We propose that the stabilization of intermediate ODC x I(-) is achieved by movement of the carbanion toward the external cation Lys-93 on decarboxylation and organization of the 203-218 loop . Because the intermediate and transition state are energetically similar, stabilization of the former decreases the free energy content of the latter. Mol Biochem Parasitol, 2002 Jul, 122(2), 189 - 200 Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi; Takashima E et al.; Trypanosoma cruzi, a protozoan causing Chagas' disease, excretes a considerable amount of succinate even though it uses the TCA cycle and the aerobic respiratory chain . For this reason, it was believed that unknown metabolic pathways participate in succinate production in this parasite . In the present study, we examined the molecular properties of dihydroorotate dehydrogenase (DHOD), the fourth enzyme of de novo pyrimidine biosynthetic pathway, as a soluble fumarate reductase (FRD) because our sequence analysis of pyr genes cluster showed that the amino acid sequence of T . cruzi DHOD is quite similar to that of type 1A DHOD of Saccharomyces cerevisiae, an enzyme that uses fumarate as an electron acceptor and produces succinate . Biochemical analyses of the cytosolic enzyme purified from the parasite and of the recombinant enzyme revealed that T . cruzi DHOD has methylviologen-fumarate reductase (MV-FRD) activity . In addition, T . cruzi DHOD was found to catalyze electron transfer from dihydroorotate to fumarate by a ping-pong Bi-Bi mechanism . The recombinant enzyme contained FMN as a prosthetic group . Dynamic light scattering analysis indicated that T . cruzi DHOD is a homodimer . These results clearly indicated that the cytosolic MV-FRD is attributable to T . cruzi DHOD . The DHOD may play an important role in succinate/fumarate metabolism as well as de novo pyrimidine biosynthesis in T . cruzi. Neurochem Int, 2002 Oct, 41(4), 251 - 9 Relationship between protein phosphatase type-2C activity and induction of apoptosis in cultured neuronal cells; Klumpp S et al.; The cellular composition and concentration of fatty acids are crucial for proliferation and survival . We recently showed stimulation of protein phosphatase type-2C (PP2C) by unsaturated fatty acids . Here, we describe that treatment of cultured chick neurons with 100 microM oleic acid for 24h increased the percentage of damaged neurons to 61+/-9% compared with 25+/-4% in controls . Oleic acid-induced cell death showed features of apoptosis such as chromatin condensation, shrinkage of the nucleus, DNA fragmentation and caspase-3 activation . Extensive studies with a variety of fatty acids revealed a striking correlation between activation of PP2C and induction of apoptosis . Lipophilicity, oxidizability, and an acidic group were required for both effects . In addition, activation of PP2C and induction of apoptosis could discriminate between cis- and trans-conformation of the fatty acids . The results are in favor of PP2C playing an important, yet unidentified role in apoptosis. Neurochem Int, 2002 Oct, 41(4), 229 - 36 Alteration of phosphatidylinositol transfer protein during global brain ischemia-reperfusion in gerbils; Chalimoniuk M et al.; Phosphatidylinositol transfer proteins (PI-TPs) are responsible for the transport of phosphatidylinositol and other phospholipids . Moreover, these proteins are involved in vesicle transport and in the function of cytoskeleton . Our previous data indicated that brain ischemia affected phosphoinositides metabolism and the level of lipid derived second messengers . In this study, the effect of ischemia-reperfusion injury on the level of PI-TPs and of the role of NMDA receptor stimulation on the alteration of these proteins was investigated during reperfusion after 5 min of forebrain ischemia in gerbils . Some groups of animals were injected intraperitoneally with MK-801, an antagonist of NMDA receptor 30 min before ischemia . The levels of both PI-TP isoforms alpha+beta and separately the alpha-isoform were determined in cytosol and membrane fraction from brain cortex and hippocampus using Western blot analysis . In the cytosolic fractions, the concentration of both isoforms of PI-TP was 2 times higher when compared to the membrane fraction . In brain cortex, PI-TP alpha isoform consist about 32-44% but in hippocampus 72-82% of both isoforms (PI-TP alpha+beta) in cytosolic and membrane fraction respectively . Ischemia-reperfusion had no effect on PI-TPs in brain cortex . However, in hippocampus after 5 min ischemia and during whole reperfusion time up till 7 days the level of PI-TP alpha+beta and PI-TP alpha was significantly higher by about 20-55%, respectively when compared to control . MK-801 eliminated ischemia-reperfusion evoked alteration of PI-TPs . To confirm the role of NMDA receptor in PI-TP alteration additional experiments were carried out on PC-12 cells in culture . The results indicated that activation of NMDA receptor enhances significantly the level of PI-TP alpha . The competitive antagonist of NMDA receptor inhibited this effect . These results indicated that activation of NMDA receptor is connected with PI-TPs alteration and plays an important role in modulation of PI-TPs during ischemia-reperfusion injury that may have important physiopathological consequence. J Biol Chem, 2002 Aug 30, 277(35), 31834 - 41 Epub 2002 Jun 24. Phosphorylation-dependent interaction between the splicing factors SAP155 and NIPP1; Boudrez A et al.; NIPP1 is a ubiquitously expressed nuclear protein that functions both as a regulator of protein Ser/Thr phosphatase-1 and as a splicing factor . The N-terminal part of NIPP1 consists of a phosphothreonine-interacting Forkhead-associated (FHA) domain . We show here that the FHA domain of NIPP1 interacts in vitro and in vivo with a TP dipeptide-rich fragment of the splicing factor SAP155/SF3b(155), a component of the U2 small nuclear ribonucleoprotein particle . The NIPP1-SAP155 interaction was entirely dependent on the phosphorylation of specific TP motifs in SAP155 . Mutagenesis and competition studies revealed that various phosphorylated TP motifs competed for binding to the same site in the FHA domain . The SAP155 kinases in cell lysates were blocked by the Ca(2+) chelator EGTA and by the cyclin-dependent protein kinase inhibitor roscovitine . The phosphorylation level of SAP155 was dramatically increased during mitosis, and accordingly the activity of SAP155 kinases was augmented in mitotic lysates . We discuss how the interaction between NIPP1 and SAP155 could contribute to spliceosome (dis)assembly and the catalytic steps of splicing. J Cell Biol, 2002 Jul 8, 158(1), 91 - 101 Epub 2002 Jul 08. An HRD/DER-independent ER quality control mechanism involves Rsp5p-dependent ubiquitination and ER-Golgi transport; Haynes CM et al.; We have identified a new pathway of ER-associated degradation in Saccharomyces cerevisiae that functions separately from the HRD/DER pathway comprised of Hrd1p, Hrd3p, Der1p, and Ubc7p . This pathway, termed Hrd1p independent-proteolysis (HIP), is capable of recognizing and degrading both lumenal (CPY* and PrA*), and integral membrane proteins (Sec61-2p) that misfold in the ER . CPY* overexpression likely saturates the HRD/DER pathway and activates the HIP pathway, so the slowed degradation kinetics of CPY* in a hrd1 Delta strain is restored to a wild-type rate when CPY* is overexpressed . Substrates of HIP require vesicular trafficking between the ER and Golgi apparatus before degradation by the ubiquitin-proteasome system . Ubiquitination of HIP substrates does not involve the HRD/DER pathway ubiquitin ligase Hrd1p, but instead uses another ubiquitin ligase, Rsp5p . HIP is regulated by the unfolded protein response as Ire1p is necessary for the degradation of CPY* when overexpressed, but not when CPY* is expressed at normal levels . Both the HIP and HRD/DER pathways contribute to the degradation of CPY*, and only by eliminating both is CPY* degradation completely blocked. Prog Nucleic Acid Res Mol Biol, 2002, 71, 69 - 90 AMP- and stress-activated protein kinases: key regulators of glucose-dependent gene transcription in mammalian cells? Leclerc I, da Silva Xavier G, Rutter GA. This article will discuss the role of two classes of serine/threonine protein kinases in the regulation of gene transcription in mammals . The first is AMP-activated protein kinase (AMPK), which is responsive to changes in the intracellular energy status . The second is the 'stress-activated" family of protein kinases, members of the mitogen-activated protein (MAP) kinase superfamily, whose regulation by a number of extracellular agents (including osmotic stresses, cytokines, and heat) is less well understood . Interest in these enzymes has grown in the past few years due to mounting evidence (both pharmacological and genetic) which has implicated them in the regulation of a number genes important in mammalian metabolism. Prog Nucleic Acid Res Mol Biol, 2002, 71, 493 - 511 Sphingosine kinases: a novel family of lipid kinases; Liu H et al.; Sphingosine kinase (SPHK) catalyzes the formation of sphingosine-1-phosphate (S11) . S1P plays an important role in regulation of a variety of biological processes through intracellular and extracellular actions . S1P has recently been shown to be the ligand for the EDG-1 family of G-protein-coupled receptors . To date, seven cloned SPHKs have been reported with confirmed SPHK activity, including human, mouse, yeast, and plant . A computer search of various databases suggests that a new SPHK family is emerging . The cloning and manipulation of SPHK genes will no doubt provide us with important information about the functions of S1P in a wide range of organisms. Nat Biotechnol, 2002 Aug, 20(8), 835 - 9 Epub 2002 Jul 08. An algorithm for finding protein-DNA binding sites with applications to chromatin-immunoprecipitation microarray experiments; Liu XS et al.; Chromatin immunoprecipitation followed by cDNA microarray hybridization (ChIP-array) has become a popular procedure for studying genome-wide protein-DNA interactions and transcription regulation . However, it can only map the probable protein-DNA interaction loci within 1-2 kilobases resolution . To pinpoint interaction sites down to the base-pair level, we introduce a computational method, Motif Discovery scan (MDscan), that examines the ChIP-array-selected sequences and searches for DNA sequence motifs representing the protein-DNA interaction sites . MDscan combines the advantages of two widely adopted motif search strategies, word enumeration and position-specific weight matrix updating, and incorporates the ChIP-array ranking information to accelerate searches and enhance their success rates . MDscan correctly identified all the experimentally verified motifs from published ChIP-array experiments in yeast (STE12, GAL4, RAP1, SCB, MCB, MCM1, SFF, and SWI5), and predicted two motif patterns for the differential binding of Rap1 protein in telomere regions . In our studies, the method was faster and more accurate than several established motif-finding algorithms . MDscan can be used to find DNA motifs not only in ChIP-array experiments but also in other experiments in which a subgroup of the sequences can be inferred to contain relatively abundant motif sites . The MDscan web server can be accessed at http://BioProspector.stanford.edu/MDscan/. Mol Cell Biol, 2002 Aug, 22(15), 5395 - 404 Regulation of the transcription factor Gcn4 by Pho85 cyclin PCL5; Shemer R et al.; The yeast transcription factor Gcn4 is regulated by amino acid starvation at the levels of both protein synthesis and stability . Gcn4 degradation depends on the ubiquitination complex SCF(CDC4) and requires phosphorylation by the cyclin-dependent kinase Pho85 . Here, we show that Pcl5 is the Pho85 cyclin specifically required for Gcn4 degradation . PCL5 is itself induced by Gcn4 at the level of transcription . However, even when PCL5 is constitutively overexpressed, Pho85-associated Gcn4 phosphorylation activity is reduced in starved cells and Gcn4 degradation is decreased . Under these conditions, the Pcl5 protein disappears because of rapid constitutive turnover . We suggest that, by virtue of its constitutive metabolic instability, Pcl5 may be a sensor of cellular protein biosynthetic capacity . The fact that PCL5 is transcriptionally induced in the presence of Gcn4 suggests that it is part of a homeostatic mechanism that reduces Gcn4 levels upon recovery from starvation. Genes Dev, 2002 Jul 1, 16(13), 1672 - 81 Spo13 regulates cohesin cleavage; Lee BH et al.; A key aspect of meiotic chromosome segregation is that cohesin, the protein complex that holds sister chromatids together, dissociates from chromosome arms during meiosis I and from centromeric regions during meiosis II . The budding yeast protein Spo13 plays a key role in preventing centromeric cohesin from being lost during meiosis I . We have determined the molecular basis for the metaphase arrest obtained when SPO13 is overexpressed during the mitotic cell cycle . Overexpression of SPO13 inhibits anaphase onset by at least two mechanisms . First, Spo13 causes a transient delay in degradation of the anaphase inhibitor Pds1 . Second, Spo13 inhibits cleavage of the cohesin subunit Scc1/Mcd1 or its meiosis-specific homolog, Rec8, by the separase Esp1 . The finding that Spo13 did not prevent cleavage of another Esp1 substrate, Slk19, suggests that overexpression of SPO13 is sufficient to prevent cohesin cleavage by protecting specific substrates from separase activity. Genes Dev, 2002 Jul 1, 16(13), 1659 - 71 Spo13 protects meiotic cohesin at centromeres in meiosis I; Shonn MA et al.; In the absence of Spo13, budding yeast cells complete a single meiotic division during which sister chromatids often separate . We investigated the function of Spo13 by following chromosomes tagged with green fluorescent protein . The occurrence of a single division in spo13Delta homozygous diploids depends on the spindle checkpoint . Eliminating the checkpoint accelerates meiosis I in spo13Delta cells and allows them to undergo two divisions in which sister chromatids often separate in meiosis I and segregate randomly in meiosis II . Overexpression of Spo13 and the meiosis-specific cohesin Rec8 in mitotic cells prevents separation of sister chromatids despite destruction of Pds1 and activation of Esp1 . This phenotype depends on the combined overexpression of both proteins and mimics one aspect of meiosis I chromosome behavior . Overexpressing the mitotic cohesin, Scc1/Mcd1, does not substitute for Rec8, suggesting that the combined actions of Spo13 and Rec8 are important for preventing sister centromere separation in meiosis I. EMBO Rep, 2002 Jul, 3(7), 636 - 40 Functional involvement of a novel Nedd4-like ubiquitin ligase on retrovirus budding; Yasuda J et al.; In this study, we have identified a novel Nedd4-like ubiquitin ligase, BUL1, as the host factor involved in budding of type D retrovirus Mason-Pfizer monkey virus (M-PMV) . Overexpression of BUL1 enhanced virus particle release, while a BUL1 mutant in which a W to G substitution was introduced into a WW domain, W791G, lost the ability to bind to the viral Gag protein and abolished its ability to mediate virus budding . In addition, a fragment of BUL1 containing only the WW domains inhibited virus budding in a dominant negative manner . These results, together with previous findings, indicate that the M-PMV Gag L domain interacts with the BUL1 WW domain and that this interaction is essential for virus budding . Our observations provide new insights into the mechanism of virus budding, and could be useful in establishing new antiviral strategies targeted at progeny virus release from a host cell. Breast Cancer Res, 2002, 4(4), 145 - 7 Epub 2002 Jun 07. Cyclins in breast cancer: too much of a good thing; Enders GH; Cyclin E, a key mediator of entry into the cell division cycle, is expressed abundantly in many breast cancers . However, amplification of the cognate gene is observed rarely, leaving the responsible mechanism(s) and its importance in tumorigenesis in doubt . In a recent report, Steve Reed's lab demonstrates that hCdc4/Fbw7 targets cyclin E for ubiquitin-mediated proteolysis and is mutant in a breast cancer cell line with high cyclin E levels . Independent work demonstrates that a Drosophila hCdc4 homologue constrains cyclin E expression in vivo . These results suggest that lesions in protein degradation pathways may contribute to cyclin E deregulation in breast cancer. Mol Microbiol, 2002 Jul, 45(1), 219 - 31 Regulation of cell separation in the dimorphic fungus Ustilago maydis; Weinzierl G et al.; During its haploid phase the dimorphic fungus Ustilago maydis grows vegetatively by budding . We have identified two genes, don1 and don3, which control the separation of mother and daughter cells . Mutant cells form tree-like clusters in liquid culture and grow as ring-like (donut-shaped) colonies on solid medium . In wild-type U . maydis cells, two distinct septa are formed during cytokinesis and delimit a fragmentation zone . Cells defective for either don1 or don3 display only a single septum and fail to complete cell separation . don1 encodes a guanine nucleotide exchange factor (GEF) of the Dbl family specific for Rho/Rac GTPases . Don3 belongs to the germinal-centre-kinase (GC) subfamily of Ste20-like protein kinases . We have isolated the U . maydis homologues of the small GTP binding proteins Rho2, Rho3, Rac1 and Cdc42 . Out of these, only Cdc42 interacts specifically with Don1 and Don3 in the yeast two-hybrid system . We propose that Don1 and Don3 regulate the initiation of the secondary septum, which is required for proper cell separation. J Virol, 2002 Aug, 76(15), 7868 - 73 Analysis of natural variants of the human immunodeficiency virus type 1 gag-pol frameshift stem-loop structure; Telenti A et al.; Human immunodeficiency virus type 1 uses ribosomal frameshifting for translation of the Gag-Pol polyprotein . Frameshift activities are thought to be tightly regulated . Analysis of gag p1 sequences from 270 plasma virions identified in 64% of the samples the occurrence of polymorphism that could lead to changes in thermodynamic stability of the stem-loop . Expression in Saccharomyces cerevisiae of p1-beta-galactosidase fusion proteins from 10 representative natural stem-loop variants and three laboratory mutant constructs (predicted the thermodynamic stability {Delta G degrees} ranging from -23.0 to -4.3 kcal/mol) identified a reduction in frameshift activity of 13 to 67% compared with constructs with the wild-type stem-loop (Delta G degrees, -23.5 kcal/mol) . Viruses carrying stem-loops associated with greater than 60% reductions in frameshift activity presented profound defects in viral replication . In contrast, viruses with stem-loop structures associated with 16 to 42% reductions in frameshift efficiency displayed no significant viral replication deficit. Genome Res, 2002 Jul, 12(7), 1121 - 6 Pathway Processor: a tool for integrating whole-genome expression results into metabolic networks; Grosu P et al.; We have developed a new tool to visualize expression data on metabolic pathways and to evaluate which metabolic pathways are most affected by transcriptional changes in whole-genome expression experiments . Using the Fisher Exact Test, the method scores biochemical pathways according to the probability that as many or more genes in a pathway would be significantly altered in a given experiment by chance alone . This method has been validated on diauxic shift experiments and reproduces well known effects of carbon source on yeast metabolism . The analysis is implemented with Pathway Analyzer, one of the tools of Pathway Processor, a new statistical package for the analysis of whole-genome expression data . Results from multiple experiments can be compared, reducing the analysis from the full set of individual genes to a limited number of pathways of interest . The pathways are visualized with OpenDX, an open-source visualization software package, and the relationship between genes in the pathways can be examined in detail using Expression Mapper, the second program of the package . This program features a graphical output displaying differences in expression on metabolic charts of the biochemical pathways to which the open reading frames are assigned. J Biol Chem, 2002 Aug 23, 277(34), 30421 - 4 Epub 2002 Jul 03. Disruptor of telomeric silencing-1 is a chromatin-specific histone H3 methyltransferase; Lacoste N et al.; Yeast disruptor of telomeric silencing-1 (DOT1) is involved in gene silencing and in the pachytene checkpoint during meiotic cell cycle . Here we show that the Dot1 protein possesses intrinsic histone methyltransferase (HMT) activity . When compared with Rmt1, another putative yeast HMT, Dot1 shows very distinct substrate specificity . While Rmt1 methylates histone H4, Dot1 targets histone H3 . In contrast to Rmt1, which can only modify free histones, Dot1 activity is specific to nucleosomal substrates . This was also confirmed using native chromatin purified from yeast cells . We also demonstrate that, like its mammalian homolog PRMT1, Rmt1 specifically dimethylates an arginine residue at position 3 of histone H4 N-terminal tail . In surprising contrast, methylation by Dot1 occurs in the globular domain of nucleosomal histone H3 . Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis suggests that H3 lysine 79 is trimethylated by Dot1 . The intrinsic nucleosomal histone H3 methyltransferase activity of Dot1 is certainly a key aspect of its function in gene silencing at telomeres, most likely by directly modulating chromatin structure and Sir protein localization . In agreement with a role in regulating localization of histone deacetylase complexes like SIR, an increase of bulk histone acetylation is detected in dot1- cells. Cancer Res, 2002 Jul 1, 62(13), 3773 - 81 A common DNA-binding site for SZF1 and the BRCA1-associated zinc finger protein, ZBRK1; Peng H et al.; More than 220 Kruppel-associated box-zinc finger protein (KRAB-ZFP) genes are encoded in the human genome . KRAB-ZFPs function as transcriptionalrepressors by binding DNA through their tandem zinc finger motifs.Gene silencing is mediated by the highly conserved KRAB domain, which recruits histone deacetylase complexes, histone methylases, and heterochromatin proteins . However, little is known of the biological programs regulated by KRAB-ZFPs, in large part because of the difficulty in identifying DNA-binding sites recognized by long arrays of zinc fingers . In an attempt to identify the natural target genes for a KRAB-ZFP, we chose SZF1, a hematopoietic progenitor-restricted, KRAB-ZFP that contains only four C(2)H(2) zinc finger motifs . Using recombinant SZF1 protein and a PCR-based binding site selection strategy, we identified a 15-bp consensus DNA sequence recognized by SZF1 . Remarkably, this sequence is similar to the core DNA-binding site described recently for ZBRK1, a KRAB-ZFP that binds to BRCA1 and is involved in coordinating the cellular DNA damage response . The SZF1 and ZBRK1 proteins bind to both the experimentally derived SZF1 site and the canonical ZBRK1 site . The KRAB domain from SZF1 bound directly to the KAP-1 corepressor and displayed intrinsic silencing activity . Moreover, full-length SZF1 repressed a promoter containing ZBRK1 recognition sequences . Thus, SZF1 and ZBRK1 may regulate a common set of target genes in vivo. Mol Cell Proteomics, 2002 Jan, 1(1), 19 - 29 Quantitative protein profiling using two-dimensional gel electrophoresis, isotope-coded affinity tag labeling, and mass spectrometry; Smolka M et al.; Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples . We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry . The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel . By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates . The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research. Mol Cell Proteomics, 2002 Mar, 1(3), 204 - 12 Deciphering protein complexes and protein interaction networks by tandem affinity purification and mass spectrometry: analytical perspective; Shevchenko A et al.; We employed a combination of tandem affinity purification and mass spectrometry for deciphering protein complexes and the protein interaction network in budding yeast . 53 genes were epitope-tagged, and their interaction partners were isolated by two-step immunoaffinity chromatography from whole cell lysates . 38 baits pulled down a total of 220 interaction partners, which are members of 19 functionally distinct protein complexes . We identified four proteins shared between complexes of different functionality thus charting segments of a protein interaction network . Concordance with the results of genome-wide two-hybrid screening was poor (14% of identified interactors overlapped) suggesting that the two approaches may provide complementary views on physical interactions within the proteome. J Exp Bot, 2002 Jul, 53(374), 1677 - 82 Cloning an iron-regulated metal transporter from rice; Bughio N et al.; Rice cDNA and genomic libraries were screened in order to clone an Fe(II) transporter gene . A cDNA clone highly homologous to the Arabidopsis Fe(II) transporter gene IRT1 was isolated from Fe-deficient rice roots . The cDNA clone was named OsIRT1 . A genomic clone corresponding to the cDNA was also obtained, sequenced and analysed . When expressed in yeast cells, OsIRT1 cDNA reversed the growth defects of the yeast iron-uptake mutant . Northern blot analysis revealed that OsIRT1 mRNA was predominantly expressed in roots and was induced by Fe- and Cu-deficiency . This suggests that OsIRT1 is a functional metal transporter for iron, and is actively engaged in Fe uptake from soils, especially under limiting conditions. Ann N Y Acad Sci, 2002 Jun, 963, 221 - 8 Human type 1 estrogen sulfotransferase: catecholestrogen metabolism and potential involvement in cancer promotion; Faucher F et al.; Using purified human type 1 estrogen sulfotransferase (hEST1), we show that the best substrate for this enzyme is 2-hydroxy-catecholestrogen . The enzyme also catalyzes the transformation of 4-hydroxy-estrogens and 16-hydroxy-estrogens, but with a lower affinity . We also present evidence to indicate that estrogen sulfotransferase may play a role in processes other than the detoxification and elimination of steroids . Indeed, hEST1 may also be involved in the production of stable precursors for local steroid biosynthesis or in the activation of promutagenic estrogen metabolites into carcinogens. Gene, 2002 May 29, 291(1-2), 95 - 104 Identification of putative interaction partners for the Xenopus Polycomb-group protein Xeed; Showell C et al.; The extra sex combs (esc) gene of Drosophila and its mammalian homologue embryonic ectoderm development (eed) play pivotal roles in establishing Polycomb-group (Pc-G) mediated transcriptional silencing of regulatory genes during early development . We have carried out a two-hybrid screen in yeast to identify maternally expressed proteins that interact directly with the product of the Xenopus eed homologue, Xeed . Xeed-interacting proteins that were recovered in this screen included a maternal Xenopus histone deacetylase (HDACm), the Xeed protein itself, and a Xenopus homologue of Enhancer of zeste (XEZ) - a second member of the Pc-G that is closely related by sequence similarity to histone methyltransferases . We have also identified a novel interaction between Xeed and a component of the Xenopus basal transcription machinery, TAF(II)32 . We show for the first time that each of these proteins interacts with the Xeed polypeptide, both in the yeast two-hybrid assay and in vitro using purified recombinant proteins . XEZ, HDACm and TAF(II)32 mRNAs are all strongly co-expressed with Xeed mRNA in the fertilized egg, further suggesting that their encoded proteins could interact with Xeed during early embryonic development . Our observations support a multi-step model for the onset of transcriptional silencing in which Xeed binds to and inhibits the function of the transcription initiation complex and also recruits proteins that mediate the acquisition by associated chromatin of epigenetically heritable, post-translational modifications. FEBS Lett, 2002 Jul 3, 522(1-3), 177 - 82 Association of HSP70 with endonucleases allows the expression of otherwise silent mutations; Mizumura H et al.; A subpopulation of the 70 kDa heat shock protein (HSP70) found within the mitochondria of Saccharomyces cerevisiae functions as a stable binding partner of the endonuclease SceI . We have previously found that the SceI endonuclease monomer recognizes and cleaves a unique, 26 bp sequence in vitro . Dimerization with HSP70 changes the specificity of SceI, allowing it to cleave at multiple sequences . This study shows that SuvI, an ortholog of SceI isolated from a different yeast strain, contains two amino acid substitutions, yet it shows the same uni-site specificity in its monomeric form . Binding of HSP70 to the SuvI monomer confers multi-site specificity that is different from that exhibited by the HSP70/SceI heterodimer . Mutation of single residues of SceI to the corresponding residue in SuvI provides enzymes with specificities intermediate between SceI and SuvI when complexed with HSP70 . These results suggest that HSP70 interaction with certain endonucleases allows the expression of otherwise silent mutations in them, causing a change in enzyme cleavage specificity. J Mol Biol, 2002 Jul 19, 320(4), 697 - 704 Interference between the PHA-4 and PEB-1 transcription factors in formation of the Caenorhabditis elegans pharynx; Kalb JM et al.; PHA-4 is a forkhead/winged helix transcription factor that acts as an organ identity factor in the development of the Caenorhabditis elegans pharynx . PEB-1 is a novel DNA-binding protein also involved in pharyngeal morphogenesis . PHA-4 and PEB-1 bind at overlapping sites on the C183 sequence element that controls pharynx-specific expression of the C . elegans myo-2 gene . It has been suggested that PHA-4 and PEB-1 act cooperatively on the C183 sequence . In this study, we test this model and assess the C183-dependent transcriptional activity of PHA-4 and PEB-1, both individually and in combination . We show that PHA-4 and PEB-1 are both modest transcriptional activators in yeast but that co-expression of the two factors does not result in significantly increased expression of a C183-regulated reporter gene . Electrophoretic mobility-shift assays provide no evidence for the formation of a PHA-4/PEB-1 complex in vitro but rather show that PHA-4 and PEB-1 cannot bind C183 simultaneously . As we have reported previously, ectopic expression of PHA-4 in C . elegans causes ectopic expression of a C183-regulated reporter gene . We show that ectopic expression of PEB-1 cannot cause ectopic expression of the same reporter but rather ectopic PEB-1 inhibits reporter gene activation by PHA-4 . Overall, our results do not support a model in which PHA-4 and PEB-1 synergize in vivo but rather support a model in which PEB-1 may negatively modulate PHA-4's ability to activate transcription through C183 during formation of the C . elegans pharynx . (c) 2002 Elsevier Science Ltd. Am J Gastroenterol, 2002 Jun, 97(6), 1458 - 62 Serological markers for prediction of response to anti-tumor necrosis factor treatment in Crohn's disease; Esters N et al.; OBJECTIVES: The use of monoclonal anti-tumor necrosis factor (TNF) antibodies (infliximab, Remicade) is a new therapeutic approach for severe refractory luminal or fistulizing, Crohn's disease (CD) . However, up to 30% of patients do not respond to this treatment . So far, no parameters predictive of response to anti-TNF have been identified . Our aim was to determine whether serological markers ASCA (anti-Saccharomyces cerevisiae antibodies) or pANCA (perinuclear antineutrophil cytoplasmic antibodies) could identify Crohn's patients likely to benefit from anti-TNF therapy . METHODS: Serum samples of 279 CD patients were analyzed for ASCA and pANCA before anti-TNF therapy . A blinded physician determined clinical response at week 4 (refractory luminal CD) or week 10 (fistulizing CD) after the first infusion of infliximab (5 mg/kg) . RESULTS: Overall, there was no relationship between ASCA or pANCA and response to therapy . However, lower response rates were observed for patients with refractory intestinal disease carrying the pANCA+/ASCA- combination, although this lacked significance (p = 0.067) . CONCLUSIONS: In this cohort of infliximab-treated patients, neither ASCA nor pANCA could predict response to treatment . However, the combination pANCA+/ASCA- might warrant further investigation for its value in predicting nonresponse in patients with refractory luminal disease. J Biol Chem, 2002 Sep 13, 277(37), 33720 - 6 Epub 2002 Jul 01. The C2 domain of phosphatidylserine decarboxylase 2 is not required for catalysis but is essential for in vivo function; Kitamura H et al.; Phosphatidylserine decarboxylase 2 (Psd2p) is currently being used to study lipid trafficking processes in intact and permeabilized yeast cells . The Psd2p contains a C2 homology domain and a putative Golgi retention/localization (GR) domain . C2 domains play important functions in membrane binding and docking reactions involving phospholipids and proteins . We constructed a C2 domain deletion variant (C2Delta) and a GR deletion variant (GRDelta) of Psd2p and examined their effects on in vivo function and catalysis . Immunoblotting confirmed that the predicted immature and mature forms of Psd2(C2Delta)p, Psd2(GRDelta)p, and wild type Psd2p were produced in vivo and that the proteins localized normally . Enzymology revealed that the Psd2(C2Delta)p and Psd2(GRDelta)p were catalytically active and could readily be expressed at levels 10-fold higher than endogenous Psd2p . Both Psd2p and Psd2(GRDelta)p expression complemented the growth defect of psd1Deltapsd2Delta strains and resulted in normal aminoglycerophospholipid metabolism . In contrast, the Psd2(C2Delta)p failed to complement psd1Deltapsd2Delta strains, and {(3)H}serine labeling revealed a severe defect in the formation of PtdEtn in both intact and permeabilized cells, indicative of disruption of lipid trafficking . These findings identify an essential, non-catalytic function of the C2 domain of Psd2p and raise the possibility that it plays a direct role in membrane docking and/or PtdSer transport to the enzyme. J Biol Chem, 2002 Sep 20, 277(38), 35650 - 6 Epub 2002 Jul 01. Convergence of the target of rapamycin and the Snf1 protein kinase pathways in the regulation of the subcellular localization of Msn2, a transcriptional activator of STRE (Stress Response Element)-regulated genes; Mayordomo I et al.; The subcellular localization of Msn2, a transcriptional activator of STRE (stress response element)-regulated genes, is modulated by carbon source availability . In cells growing in glucose, Msn2 is located mainly in the cytosol, whereas in carbon source-starved cells, Msn2 is located largely inside the nucleus . However, in cells lacking Reg1 (the regulatory subunit of the Reg1/Glc7 protein phosphatase complex), the regulation of subcellular distribution is absent, Msn2 being constitutively present in the cytosol . The localization defect in these mutants is specific for carbon starvation stress, and it is because of the presence of an abnormally active Snf1 protein kinase that inhibits the nuclear localization of Msn2 upon carbon starvation . Active Snf1 kinase is also able to avoid the effects of rapamycin, a drug that by inhibiting the TOR kinase pathway leads to a nuclear localization of Msn2 in wild type cells . Therefore, active Snf1 and the TOR kinase pathway may affect similar cytosolic steps in the regulation of the subcellular localization of Msn2. Genome Biol . 2002;3(6):REVIEWS1016 . Epub 2002 May 24. Surveying genome replication; Kearsey S; Two recent studies have added microarrays to the toolkit used to analyze the origins of replication in yeast chromosomes, providing a fuller picture of how genomic DNA replication is organized. Genome Biol . 2002;3(6):RESEARCH0027 . Epub 2002 May 14. ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins; Hjelmqvist L et al.; BACKGROUND: Annotations of completely sequenced genomes reveal that nearly half of the genes identified are of unknown function, and that some belong to uncharacterized gene families . To help resolve such issues, information can be obtained from the comparative analysis of homologous genes in model organisms . RESULTS: While characterizing genes from the retinitis pigmentosa locus RP26 at 2q31-q33, we have identified a new gene, ORMDL1, that belongs to a novel gene family comprising three genes in humans (ORMDL1, ORMDL2 and ORMDL3), and homologs in yeast, microsporidia, plants, Drosophila, urochordates and vertebrates . The human genes are expressed ubiquitously in adult and fetal tissues . The Drosophila ORMDL homolog is also expressed throughout embryonic and larval stages, particularly in ectodermally derived tissues . The ORMDL genes encode transmembrane proteins anchored in the endoplasmic reticulum (ER) . Double knockout of the two Saccharomyces cerevisiae homologs leads to decreased growth rate and greater sensitivity to tunicamycin and dithiothreitol . Yeast mutants can be rescued by human ORMDL homologs . CONCLUSIONS: From protein sequence comparisons we have defined a novel gene family, not previously recognized because of the absence of a characterized functional signature . The sequence conservation of this family from yeast to vertebrates, the maintenance of duplicate copies in different lineages, the ubiquitous pattern of expression in human and Drosophila, the partial functional redundancy of the yeast homologs and phenotypic rescue by the human homologs, strongly support functional conservation . Subcellular localization and the response of yeast mutants to specific agents point to the involvement of ORMDL in protein folding in the ER. J Pept Sci, 2002 Jun, 8(6), 235 - 40 A novel peptide with ribonuclease and translation-inhibitory activities from fruiting bodies of the oyster mushroom Pleurotus ostreatus; Ye XY et al.; From the fresh fruiting bodies of the oyster mushroom a peptide with a molecular weight of 9 kDa and demonstrating a novel N-terminal sequence GPCYLVAFYESSGRR was isolated . The isolation procedure involved ion exchange chromatography on CM-Sepharose and Mono S . The peptide was adsorbed on both types of chromatographic media . The peptide demonstrated a ribonuclease activity of 650 U/mg toward yeast transfer RNA . It inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 15 nM. J Neurogenet, 2001, 15(3-4), 205 - 19 The power law distribution for walking-time intervals correlates with the ellipsoid-body in Drosophila; Martin J et al.; The temporal properties of a variety of behavioral traits obey power law distributions, a property often referred to as fractal . We recently showed that the temporal pattern of locomotor activity of the fruitfly Drosophila melanogaster follows this distribution . Although an increasing number of such fractal patterns are being discovered, the brain areas and neuronal networks responsible remain unknown . In this study, we show that specifically blocking synapses established by neurons of the Drosophila ellipsoid-body, a substructure of the central complex in the brain, leads to a loss of the fractal properties in the temporal pattern . We conclude that the temporal fractal pattern of locomotor activity is regulated in the ellipsoid-body. J Neurogenet, 2001, 15(3-4), 193 - 204 Rnai: a new technology in the post-genomic sequencing era; Ueda R; The complete genome sequences and huge numbers of predicted gene sequences from many complex organisms are available . Reverse-genetic analyses of these organisms will now be necessary to understand what all these genes are doing and how their functions interact . RNAi technology is very useful in this regard for studying gene function, not only in these model organisms, but also in the organisms previously considered not to be amenable to genetic analysis . This treatment reviews the discovery of RNAi, advances in the study of the mechanisms by which this material impinges on gene-product expressions, and technical improvements that will allow RNAi to be applied to a wide variety of organisms. Invest Ophthalmol Vis Sci, 2002 Jul, 43(7), 2326 - 33 Interaction of GABA receptor/channel rho(1) and gamma(2) subunit; Ekema GM et al.; PURPOSE: To determine whether protein-protein and functional interactions can occur between gamma-aminobutyric acid (GABA)(A) receptor/channels gamma(2) subunit and the retina-specific GABA(C) rho(1) subunit . METHODS: Protein-protein interaction was characterized by immunocoprecipitation of these subunits in brain and spinal cord with anti-gamma(2) subunit antibody and by Western blot analysis with anti-rho(1) subunit antibody . The rho(1) and gamma(2) subunits were detected in the adult rat brain and spinal cord lysates that had been previously precipitated with the specific antibodies against the rho(1) and gamma(2) subunits, respectively . A two-microelectrode voltage clamp was used to measure GABA-induced currents in oocytes . In addition, a yeast two-hybrid system was used to detect the interactions of these subunits in vivo . RESULTS: Based on yeast transformed with the N-terminal fragment of the gamma(2) subunit (gamma(2)-N'), the N-terminal fragment of the rho(1) subunit (rho(1)-N'), and the full-length rho(1) subunit, the protein-protein interaction of the GABA(A) gamma(2) subunit and the GABA(C) rho(1) subunit was found in yeast grown in triple-dropout medium (deficient in Leu, Trp, and His) and expressing the LacZ reporter gene . Interaction of the rho(1) and gamma(2) subunits was investigated by functional studies in which gamma(2) (gamma(2)-N' with 837 bp) and rho(1) cRNAs were coinjected in Xenopus oocytes . In studies of the functional interaction, after injection of the gamma(2) subunit mutant cRNA containing a N-terminal fragment, GABA-induced rho(1) originated currents declined to 16% of the control level of homooligomeric rho(1) current . This inhibitory effect of coexpressing gamma(2) subunit mutants with rho(1) subunit on the rho(1)-originated current in oocytes was dose dependent . In addition, coexpression of the GABA rho(1) and gamma(2) subunits in oocytes altered pharmacologic properties of the homooligomeric receptor/channel formed by rho(1) or gamma(2) subunits . Further evidence was provided by results obtained with specific antibodies showing that the rho(1) subunit was coimmunoprecipitated with the gamma(2) subunit from the retina, brain, and spinal cord . CONCLUSIONS: The results indicate that protein-protein and functional interactions can occur between the GABA(A) gamma(2) subunit and the GABA(C) rho(1) subunit . Therefore, the functional role of GABA receptor/channels in the brain, retina, and spinal cord is more diversified because of the possible assembly between the GABA(A) gamma(2) subunit and GABA(C) rho(1) subunit. J Biol Chem, 2002 Sep 13, 277(37), 34489 - 98 Epub 2002 Jun 28. Structural basis for the NAD-dependent deacetylase mechanism of Sir2; Chang JH et al.; The NAD-dependent histone/protein deacetylase activity of Sir2 (silent information regulator 2) accounts for its diverse biological roles including gene silencing, DNA damage repair, cell cycle regulation, and life span extension . We provide crystallographic evidence that 2'-O-acetyl ADP-ribose is the reaction product that is formed at the active site of Sir2 from the 2.6-A co-crystal structure of 2'-O-acetyl-ADP-ribose and Sir2 from Archaeoglobus fulgidus . In addition, we show that His-116 and Phe-159 play critical roles in the catalysis and substrate recognition . The conserved Ser-24 and Asp-101 contribute to the stability for NAD binding rather than being directly involved in the catalysis . The crystal structures of wild type and mutant derivatives of Sir2, in conjunction with biochemical analyses of the mutants, provide novel insights into the reaction mechanism of Sir2-mediated deacetylation. Development, 2002 Jul, 129(14), 3325 - 34 A common translational control mechanism functions in axial patterning and neuroendocrine signaling in Drosophila; Clark IE et al.; Translational repression of maternal nanos (nos) mRNA by a cis-acting Translational Control Element (TCE) in the nos 3'UTR is critical for anterior-posterior patterning of the Drosophila embryo . We show, through ectopic expression experiments, that the nos TCE is capable of repressing gene expression at later stages of development in neuronal cells that regulate the molting cycle . Our results predict additional targets of TCE-mediated repression within the nervous system . They also suggest that mechanisms that regulate maternal mRNAs, like TCE-mediated repression, may function more widely during development to spatially or temporally control gene expression. Plant Mol Biol, 2002 Jul, 49(5), 503 - 14 KAP-2, a protein that binds to the H-box in a bean chalcone synthase promoter, is a novel plant transcription factor with sequence identity to the large subunit of human Ku autoantigen; Lindsay WP et al.; The KAP-2 protein that binds to the H-box (CCTACC) element in the bean CHS15 chalcone synthase promoter was purified, and internal peptide sequence used to design primers leading to the cloning of KAP-2 from bean (Phaseolus vulgaris) and barrel medic (Medicago truncatula) . KAP-2 shares sequence similarity to the large subunit of mammalian Ku autoantigen, a protein proposed to be involved in control of DNA recombination and transcription . KAP-2 sequences were present in genomic DNA from a range of legumes, and a related protein is found in Arabidopsis thaliana . Recombinant KAP-2 expressed in insect cells showed the same binding specificity for the CHS15 H-box as the protein purified from bean cell extracts . In vitro transcription assays confirmed that KAP-2 stimulates transcription from a promoter harboring the H-box cis element. Plant Mol Biol, 2002 Jul, 49(5), 483 - 90 Regulation of the high-affinity ammonium transporter (BnAMT1;2) in the leaves of Brassica napus by nitrogen status; Pearson JN et al.; Substantial concentrations of NH4+ are found in the apoplast of the leaves of Brassica napus . Physiological studies on isolated mesophyll protoplasts with 15NH4+ revealed the presence of a high-affinity ammonium transporter that shared physiological similarity to the high-affinity NH4+ transporters in Arabidopsis thaliana (AtAMT1;3) . PCR techniques were used to isolate a full-length clone of a B . napus homologue of AMT1 from shoot mRNA which showed 97% similarity to AtAMT1;3 . The full-length cDNA when cloned into the yeast expression vector pFL61 was able to complement a yeast mutant unable to grow on media with NH4+ as the sole nitrogen source . Regulatory studies with detached leaves revealed a stimulation of both NH4+ uptake and expression of mRNA when the leaves were supplied with increasing concentrations of NH4+ . Withdrawal of NH4+ supply for up to 96 h had little effect on mRNA expression or NH4+ uptake; however, plants grown continuously at high NH4+ levels exhibited decreased mRNA expression . BnAMT1;2 mRNA expression was highest when NH4+ was supplied directly to the leaf and lowest when either glutamine or glutamate was supplied to the leaves, which directly paralleled chloroplastic glutamine synthetase (GS2) activity in the same leaves . These results provide tentative evidence that BnAMT1;2 may be regulated by similar mechanisms to GS2 in leaves. Mol Endocrinol, 2002 Jul, 16(7), 1482 - 91 Direct interactions between corepressors and coactivators permit the integration of nuclear receptor-mediated repression and activation; Li X et al.; The unliganded thyroid hormone receptor beta (TRbeta) represses the basal transcriptional activity of target genes, in part through interactions with the nuclear receptor corepressor (N-CoR) . In this study we have identified a rather unexpected interaction between N-CoR and the nuclear receptor coactivator ACTR . We have demonstrated in vitro and in intact cells that N-CoR directly associates with ACTR and that the interaction surfaces on N-CoR and ACTR are distinct from those required for TR binding . The significance of this finding was demonstrated by showing that N-CoR facilitates an interaction between unliganded-TRbeta and ACTR . One possible consequence of the formation of the trimeric complex of N-CoR/ACTR/unliganded-TR is that N-CoR may raise the local concentration of ACTR at target gene promoters . In support of this hypothesis it was demonstrated that the presence of N-CoR can enhance TRbeta-mediated transcriptional activation . It is proposed, therefore, that TRbeta- mediated activation and repression are integrally linked in a manner that is not predicted by the current models of nuclear receptor action. J Biol Chem, 2002 Aug 23, 277(34), 30413 - 6 Epub 2002 Jun 27. Broad requirement for the mediator subunit RGR-1 for transcription in the Caenorhabditis elegans embryo; Shim EY et al.; The Mediator-related transcription co-factors integrate positive and negative inputs and recruit and activate the RNA polymerase II complex . To understand the role of Mediator during transcription, it is important to identify Mediator subunits that are essential for its functions . In the yeast Mediator, the conserved component Rgr1 is associated with multiple subunits that are required for specific activation or repression events . Yeast rgr1 is essential for viability, for certain repression mechanisms, and for activation of heat shock genes, but it is not known whether rgr1 is generally important for transcription . Here we have performed the first analysis of rgr-1 function in a metazoan . We found that in the developing Caenorhabditis elegans embryo rgr-1 is broadly required for transcription and for phosphorylation of both Ser-2 and Ser-5 of the RNA polymerase II C-terminal domain repeat . We conclude that RGR-1 fulfills a critical Mediator function that is broadly essential for metazoan mRNA transcription and that RGR-1 may be required at an early recruitment or initiation step. J Nat Prod, 2002 Jun, 65(6), 938 - 41 Convolutindole A and convolutamine H, new nematocidal brominated alkaloids from the marine bryozoan Amathia convoluta; Narkowicz CK et al.; Nematocidal activity of an extract of the marine bryozoan Amathia convoluta, collected from Tasmania's east coast, was ascribed to two novel tribrominated alkaloids: convolutamine H (2) and convolutindole A (5), an indole possessing the unusual N-methoxy moiety . The structures were established by spectroscopic techniques. J Nat Prod, 2002 Jun, 65(6), 932 - 4 Novel diterpene lactones from Suregada multiflora; Jahan IA et al.; Two new diterpene lactones, suregadolides A (1) and B (2), were isolated from a dichloromethane extract of Suregada multiflora bark . These compounds possess a novel skeleton, which contains a cyclopropane ring bridging C-3 and C-4 of the abietane skeleton . The structures were established on the basis of one- and two-dimensional NMR and other spectroscopic studies . Compound 1 showed moderate inhibitory activity in a mutant yeast strain bioassay. Nucleic Acids Res, 2002 Jul 1, 30(13), 2862 - 70 Effects of double-strand break repair proteins on vertebrate telomere structure; Wei C et al.; Although telomeres are not recognized as double-strand breaks (DSBs), some DSB repair proteins are present at telomeres and are required for telomere maintenance . To learn more about the telomeric function of proteins from the homologous recombination (HR) and non-homologous end joining pathways (NHEJ), we have screened a panel of chicken DT40 knockout cell lines for changes in telomere structure . In contrast to what has been observed in Ku-deficient mice, we found that Ku70 disruption did not result in telomere-telomere fusions and had no effect on telomere length or the structure of the telomeric G-strand overhang . G-overhang length was increased by Rad51 disruption but unchanged by disruption of DNA-PKcs, Mre11, Rad52, Rad54, XRCC2 or XRCC3 . The effect of Rad51 depletion was unexpected because gross alterations in telomere structure have not been detected in yeast HR mutants . Thus, our results indicate that Rad51 has a previously undiscovered function at vertebrate telomeres . They also indicate that Mre11 is not required to generate G-overhangs . Although Mre11 has been implicated in overhang generation, overhang structure had not previously been examined in Mre11-deficient cells . Overall our findings indicate that there are significant species-specific differences in the telomeric function of DSB repair proteins. Proc Natl Acad Sci U S A, 2002 Jul 9, 99(14), 9145 - 9 Epub 2002 Jun 26. Distinct domains of splicing factor Prp8 mediate different aspects of spliceosome activation; Kuhn AN et al.; Prp8 is the largest and most highly conserved protein in the spliceosome yet its mechanism of function is poorly understood . Our previous studies implicate Prp8 in control of spliceosome activation for the first catalytic step of splicing, because substitutions in five distinct regions (a-e) of Prp8 suppress a cold-sensitive block to activation caused by a mutation in U4 RNA . Catalytic activation of the spliceosome is thought to require unwinding of the U1 RNA/5' splice site and U4/U6 RNA helices by the Prp28 and Prp44/Brr2 DExD/H-box helicases, respectively . Here we show that mutations in regions a, d, and e of Prp8 exhibit allele-specific genetic interactions with mutations in Prp28, Prp44/Brr2, and U6 RNA, respectively . These results indicate that Prp8 coordinates multiple processes in spliceosome activation and enable an initial correlation of Prp8 structure and function . Furthermore, additional genetic interactions with U4-cs1 support a two-state model for this RNA conformational switch and implicate another splicing factor, Prp31, in Prp8-mediated spliceosome activation. J Biol Chem, 2002 Sep 6, 277(36), 33049 - 57 Epub 2002 Jun 26. MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts; Edwards MC et al.; The MCM2-7 complex is believed to function as the eukaryotic replicative DNA helicase . It is recruited to chromatin by the origin recognition complex (ORC), Cdc6, and Cdt1, and it is activated at the G(1)/S transition by Cdc45 and the protein kinases Cdc7 and Cdk2 . Paradoxically, the number of chromatin-bound MCM complexes greatly exceeds the number of bound ORC complexes . To understand how the high MCM2-7:ORC ratio comes about, we examined the binding of these proteins to immobilized linear DNA fragments in Xenopus egg extracts . The minimum length of DNA required to recruit ORC and MCM2-7 was approximately 80 bp, and the MCM2-7:ORC ratio on this fragment was approximately 1:1 . With longer DNA fragments, the MCM2-7:ORC ratio increased dramatically, indicating that MCM complexes normally become distributed over a large region of DNA surrounding ORC . Only a small subset of the chromatin-bound MCM2-7 complexes recruited Cdc45 at the onset of DNA replication, and unlike Cdc45, MCM2-7 was not limiting for DNA replication . However, all the chromatin-bound MCM complexes may be functional, because they were phosphorylated in a Cdc7-dependent fashion, and because they could be induced to support Cdk2-dependent Cdc45 loading . The data suggest that in Xenopus egg extracts, origins of replication contain multiple, distributed, initiation-competent MCM2-7 complexes. Mol Cell, 2002 Jun, 9(6), 1307 - 17 Hog1 kinase converts the Sko1-Cyc8-Tup1 repressor complex into an activator that recruits SAGA and SWI/SNF in response to osmotic stress; Proft M et al.; The yeast ATF/CREB repressor Sko1(Acr1) regulates genes that are induced upon hyperosmotic stress by recruiting the Cyc8(Ssn6)-Tup1 corepressor complex to target promoters . During hyperosmotic stress, Hog1 MAP kinase associates with target promoters, phosphorylates Sko1, and converts Sko1 into a transcriptional activator . Unexpectedly, Tup1 remains bound to target promoters during osmotic stress . Sko1, Hog1, and Tup1 are all important for recruitment of SAGA histone acetylase and SWI/SNF nucleosome-remodeling complexes to osmotic-inducible promoters, and both complexes are important for activation upon osmotic stress . Thus, osmotic induction involves a switch of Sko1-Cyc8-Tup1 from a repressing to an activating state in a process that is triggered by Hog1 phosphorylation . Cyc8-Tup1 is not simply a corepressor but is also involved in recruiting SWI/SNF and SAGA during the transcriptional induction process. Mol Cell, 2002 Jun, 9(6), 1297 - 305 Cti6, a PHD domain protein, bridges the Cyc8-Tup1 corepressor and the SAGA coactivator to overcome repression at GAL1; Papamichos-Chronakis M et al.; The yeast Cyc8 and Tup1 proteins form a corepressor complex that, when tethered to DNA, turns off transcription . Release of the Cyc8-Tup1 corepressor from a promoter has been considered as a prerequisite for subsequent transcriptional activation . Contrasting this, we demonstrate that Cyc8-Tup1 is continuously associated with target promoters under both repressive and inducing conditions . At the GAL1 promoter, Cyc8-Tup1 facilitates recruitment of SAGA (Spt-Ada-Gcn5-acetyltranferase) via Cti6, a PHD domain protein that physically links the Cyc8-Tup1 and SAGA complexes . Lack of functional corepressor renders GAL1 transcription largely independent of specific SAGA subunits . Thus, corepressor's release is not the mechanism of derepression; instead, it is the coactivator complex that alleviates Cyc8-Tup1-mediated repression under induction conditions. Mol Cell, 2002 Jun, 9(6), 1285 - 96 Processing of 3'-extended read-through transcripts by the exosome can generate functional mRNAs; Torchet C et al.; Strains carrying rna14.1 and rna15.2 mutations are defective in pre-mRNA 3' cleavage, polyadenylation, and transcription termination . Long extended read-through transcripts generated in rna14.1 and rna15.2 strains are greatly stabilized by depletion of Rrp41p, a core component of the exosome complex or the RNA helicase Dob1p/Mtr4p . The absence of the nuclear-specific exosome component, Rrp6p, from the rna14.1 strain gave a very different phenotype . Short polyadenylated pre-mRNAs were strongly stabilized, and these were functional for translation . Production of these mRNAs was suppressed by depletion of Rrp41p, indicating that they are the products of exosome processing followed by uncoupled polyadenylation . The balance between complete degradation of 3'-unprocessed pre-mRNAs and their processing to functional mRNAs is regulated, with degradation favored on glucose media. Mol Cell, 2002 Jun, 9(6), 1154 - 6 A mark in the core: silence no more! Varga-Weisz PD, Dalgaard JZ. The histone modification repertoire has recently been expanded . Dot1p is a new type of methyltransferase that methylates lysine 79 in the histone H3 core only in its nucleosomal context and has a possible role in marking open chromatin regions. Cell, 2002 May 17, 109(4), 447 - 58 Drosophila Sir2 is required for heterochromatic silencing and by euchromatic Hairy/E(Spl) bHLH repressors in segmentation and sex determination; Rosenberg MI et al.; Yeast SIR2 is a NAD+-dependent histone deacetylase required for heterochromatic silencing at telomeres, rDNA, and mating-type loci . We find that the Drosophila homolog of Sir2 (dSir2) also encodes deacetylase activity and is required for heterochromatic silencing, but unlike ySir2, is not required for silencing at telomeres . We show that dSir2 interacts genetically and physically with members of the Hairy/Deadpan/E(Spl) family of bHLH euchromatic repressors, key regulators of Drosophila development . dSir2 is an essential gene whose loss of function results in both segmentation defects and skewed sex ratios, associated with reduced activities of the Hairy and Deadpan bHLH repressors . These results indicate that Sir2 in higher organisms plays an essential role in both euchromatic repression and heterochromatic silencing. Syst Appl Microbiol, 2002 Apr, 25(1), 153 - 61 Study of the first hours of microvinification by the use of osmotic stress-response genes as probes; Perez-Torrado R et al.; When yeast cells are inoculated into grape must for vinification they find stress conditions because of osmolarity, which is due to very high sugar concentration, and pH lower than 4 . In this work an analysis of the expression of three osmotic stress induced genes (GPD1, HSP12 and HSP104) under microvinification conditions is shown as a way to probe those stress situations and the regulatory mechanisms that control them . The results indicate that during the first hours of microvinification there is an increase in the GPDI mRNA levels with a maximum about one hour after inoculation, and a decrease in the amount of HSP12 and HSP104 mRNAs, although with differences between them . The RNA steady-state levels of all the genes considered, and in some cases the microvinification progress are significantly affected by the composition of the must (pH, nature of the osmotic agent and carbon source) . These results point out the importance of the control of these parameters and the yeast molecular response during the first hours of vinification for an accurate winemaking process. Br J Cancer, 2002 May 20, 86(10), 1592 - 6 Investigations on a clinically and functionally unusual and novel germline p53 mutation; Rutherford J et al.; This report describes an individual with a rare choroid plexus papilloma in adulthood (age 29) after earlier having an osteosarcoma (age 22) . The results from this study, and others, suggest that it may be advisable to consider the possibility of a germline p53 mutation in adults presenting with choroid plexus tumours . In the current study automated DNA sequencing of genomic DNA detected a novel germline 7 base pair insertion in exon 5 of the p53 gene in this patient . The alteration in frame would produce amino acid substitutions beginning with alanine to glycine at position 161 and a stop codon at position 182 in the mutated protein . Surprisingly two assays of p53 function gave apparently wild-type results on peripheral blood lymphocytes from this individual . These results led us to carry out more detailed functional tests on the mutant protein . The mutant allele was expressed either at very low levels or not at all in phytohaemagglutinin stimulated lymphocytes . Further, the mutant protein was completely non-functional in terms of its ability to transactivate a series of p53-responsive genes (p21(WAF1), bax, PIG3), to transrepress a target gene and to inhibit colony growth in transfected Saos-2 cells . However, surprisingly, data from irradiated peripheral blood lymphocytes and transfected Saos-2 cells, suggested that this truncated, mutant protein retains significant ability to induce apoptosis . com Proc Natl Acad Sci U S A, 2002 Jun 25, 99(13), 8548 - 53 Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein; Peng G et al.; Galactose-inducible genes (GAL genes) in yeast Saccharomyces cerevisiae are efficiently transcribed only when the sequence-specific transcription activator Gal4p is activated . Activation of Gal4p requires the interaction between the Gal4p inhibitory protein Gal80p and the galactokinase paralog, Gal3p . It has been proposed that Gal3p binds to a Gal80p-Gal4p complex in the nucleus to activate Gal4p . Here, we present evidence that the Gal3p-Gal80p interaction occurs in the cytoplasm, and concurrently, Gal80p is removed from Gal4p at the GAL gene promoter . We also show that GAL gene expression can be activated by heterologous protein-protein interaction in the cytoplasm that is independent of galactose and Gal3p function . These results indicate that galactose-triggered Gal3p-Gal80p association in the cytoplasm activates Gal4p in the nucleus. J Biol Chem, 2002 Aug 30, 277(35), 31857 - 62 Epub 2002 Jun 25. The synthesis of inositol hexakisphosphate . Characterization of human inositol 1,3,4,5,6-pentakisphosphate 2-kinase; Verbsky JW et al.; The enzyme(s) responsible for the production of inositol hexakisphosphate (InsP(6)) in vertebrate cells are unknown . In fungal cells, a 2-kinase designated Ipk1 is responsible for synthesis of InsP(6) by phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) . Based on limited conserved sequence motifs among five Ipk1 proteins from different fungal species, we have identified a human genomic DNA sequence on chromosome 9 that encodes human inositol 1,3,4,5,6-pentakisphosphate 2-kinase (InsP(5) 2-kinase) . Recombinant human enzyme was produced in Sf21 cells, purified, and shown to catalyze the synthesis of InsP(6) or phytic acid in vitro . The recombinant protein converted 31 nmol of InsP(5) to InsP(6)/min/mg of protein (V(max)) . The Michaelis-Menten constant for InsP(5) was 0.4 microM and for ATP was 21 microM . Saccharomyces cerevisiae lacking IPK1 do not produce InsP(6) and show lethality in combination with a gle1 mutant allele . Here we show that expression of the human InsP(5) 2-kinase in a yeast ipk1 null strain restored the synthesis of InsP(6) and rescued the gle1-2 ipk1-4 lethal phenotype . Northern analysis on human tissues showed expression of the human InsP(5) 2-kinase mRNA predominantly in brain, heart, placenta, and testis . The isolation of the gene responsible for InsP(6) synthesis in mammalian cells will allow for further studies of the InsP(6) signaling functions. J Biol Chem, 2002 Aug 30, 277(35), 31988 - 93 Epub 2002 Jun 25. Pctaire1 interacts with p35 and is a novel substrate for Cdk5/p35; Cheng K et al.; Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that plays important roles during central nervous system development . Cdk5 kinase activity depends on its regulatory partners, p35 or p39, which are prominently expressed in the central nervous system . We have previously demonstrated the involvement of Cdk5 in the regulation of acetylcholine receptor expression at the neuromuscular junction, suggesting a novel functional role of Cdk5 at the synapse . Here we report the identification of Pctaire1, a member of the Cdk-related kinase family, as a p35-interacting protein in muscle . Binding of Pctaire1 to p35 can be demonstrated by in vitro binding assay and co-immunoprecipitation experiments . Pctaire1 is associated with p35 in cultured myotubes and skeletal muscle, and is concentrated at the neuromuscular junction . Furthermore, Pctaire1 can be phosphorylated by the Cdk5/p25 complex, and serine 95 is the major phosphorylation site . In brain and muscle of Cdk5 null mice, Pctaire1 activity is significantly reduced . Moreover, Pctaire1 activity is increased following preincubation with brain extracts and phosphorylation by the Cdk5/p25 complex . Taken together, our findings demonstrate that Pctaire1 interacts with p35, both in vitro and in vivo, and that phosphorylation of Pctaire1 by Cdk5 enhances its kinase activity. Biochim Biophys Acta, 2002 Jul 19, 1576(3), 316 - 23 Molecular and functional characterization of a second copy of the aflatoxin regulatory gene, aflR-2, from Aspergillus parasiticus; Cary JW et al.; The genes required for the synthesis of aflatoxin (AF) in Aspergillus flavus and Aspergillus parasiticus have been shown to be clustered on a chromosome in these fungi . Transcription of most of these genes is dependent upon the activity of the aflR gene, also present on the gene cluster, which encodes a zinc binuclear cluster DNA-binding protein . While many strains of A . parasiticus have only one copy of aflR (aflR-1), many others contain a second copy of this gene (aflR-2) which resides on a duplicated region of the aflatoxin gene cluster . Targeted disruption of aflR-1 generated a number of non-aflatoxin producing transformants of A . parasiticus SU-1 which still harbored a wild-type aflR-2 gene . Southern and Northern hybridization analyses and ELISA assays demonstrated that aflR-1 had been successfully inactivated in strain AFS10 . DNA sequence analysis showed that aflR-2 was capable of encoding a deduced 47 kDa protein . Northern and RT-PCR analysis of RNA from a toxin producing strain indicated that aflR-2 was transcribed at extremely low levels compared to aflR-1 . RT-PCR analysis of RNA from AFS10 demonstrated that mRNAs of aflatoxin pathway genes were not processed to their mature forms . Functional analysis of aflr-2 protein in a yeast system showed that it was not activating transcription. Amino Acids, 2002, 22(3), 245 - 57 Molecular and biochemical analysis of the enzymes of cysteine biosynthesis in the plant Arabidopsis thaliana; Hell R et al.; Among the amino acids produced by plants cysteine plays a special role as a mediator between assimilatory sulfate reduction and provision of reduced sulfur f