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| J Exp Bot, 2001 Apr, 52(357), 663 - 8 Down-regulation of a ripening-related beta-galactosidase gene (TBG1) in transgenic tomato fruits; Carey AT et al.; Exo-galactanase/beta-galactosidase (EC 3.2.1.23) activity is thought to be responsible for the loss of galactosyl residues from the cell walls of ripening tomatoes . Transgenic tomato plants (Lycopersicon esculentum Mill cv . Ailsa Craig) with reduced exo-galactanase/beta-galactosidase mRNA were generated to test this hypothesis and to investigate the role of the enzyme in fruit softening . A previously identified tomato beta-galactosidase cDNA clone, TBG1, was used in the experiments . Heterologous expression of the clone in yeast demonstrated that TBG1 could release galactosyl residues from tomato cell wall galactans . Transgenic plants showed a reduction in TBG1 mRNA to 10% of normal levels in the ripening fruits . However, despite the reduction in message, total beta-galactosidase and exo-galactanase activities were unaffected . Furthermore, there was no apparent effect on levels of cell wall galactosyl residues when compared with the control . It was concluded that during the ripening of tomato fruits a family of beta-galactosidases capable of degrading cell wall galactans are active and down-regulation of TBG1 message to 10% was insufficient to alter the degree of galactan degradation. J Biol Chem, 2001 Aug 31, 276(35), 32642 - 7 Epub 2001 Jun 18. Thrombin receptor signaling to cytoskeleton requires Hsp90; Pai KS et al.; Thrombin is a serine protease that evokes various cellular responses involved in injury and repair of the nervous system through the activation of protease-activated receptor-1 (PAR-1) . Signals that modulate cell morphology precede most PAR-1 effects, but the initial signal transduction molecules are not known . Using the yeast two-hybrid system, we identified Hsp90, a chaperone with known signaling properties, as a binding partner of PAR-1 . The interaction was confirmed by glutathione S-transferase pull-down, overlay, and co-immunoprecipitation assays . Morphological assays in mouse astrocytes were carried out to evaluate the importance of Hsp90 during cytoskeletal signaling . Reducing Hsp90 levels by antisense treatment or disruption of the Hsp90.PAR-1 complex by the Hsp90-specific drug geldanamycin attenuated thrombin-mediated astrocyte shape changes . Furthermore, overexpression of the PAR-1 cytoplasmic tail abrogated thrombin-induced cytoskeletal changes in neuronal cells . Treatment with geldanamycin specifically inhibited activation of RhoA without affecting thrombin-mediated intracellular calcium release, revealing the regulation of a distinct signaling pathway by Hsp90 . Taken together, these studies demonstrate that Hsp90 may be essential for PAR-1-mediated signaling to the cytoskeleton. Curr Biol, 2001 Apr 3, 11(7), R253 - 6 Meiotic recombination: breaking the genome to save it; Lichten M; Recombination ensures the correct segregation of chromosomes to gametes during meiosis . Recent studies point to a universal mechanism for initiating meiotic recombination: the formation of double-strand DNA breaks by Spo11p. Curr Biol, 2001 Apr 3, 11(7), 508 - 13 Activation of the anaphase-promoting complex and degradation of cyclin B is not required for progression from Meiosis I to II in Xenopus oocytes; Taieb FE et al.; Sister chromatid separation and cyclin degradation in mitosis depend on the association of the anaphase-promoting complex (APC) with the Fizzy protein (Cdc20), leading to the metaphase/anaphase transition and exit from mitosis {1--3} . In Xenopus, after metaphase of the first meiotic division, only partial cyclin degradation occurs, and chromosome segregation during anaphase I proceeds without sister chromatid separation {4--7} . We investigated the role of xFizzy during meiosis using an antisense depletion approach . xFizzy accumulates to high levels in Meiosis I, and injection of antisense oligonucleotides to xFizzy blocks nearly all APC-mediated cyclin B degradation and Cdc2/cyclin B (MPF) inactivation between Meiosis I and II . However, even without APC activation, xFizzy-ablated oocytes progress to Meiosis II as shown by cyclin E synthesis, further accumulation of cyclin B, and evolution of the metaphase I spindle to a metaphase II spindle via a disc-shaped aggregate of microtubules known to follow anaphase I {8} . Inhibition of the MAPK pathway by U0126 in antisense-injected oocytes prevents cyclin B accumulation beyond the level that is present at metaphase I . Full synthesis and accumulation can be restored in the presence of U0126 by the expression of a constitutively active form of the MAPK target, p90(Rsk) . Thus, p90(Rsk) is sufficient not only to partially inhibit APC activity {7}, but also to stimulate cyclin B synthesis in Meiosis II. Curr Biol, 2001 Apr 3, 11(7), 482 - 8 CDC-42 controls early cell polarity and spindle orientation in C . elegans; Gotta M et al.; BACKGROUND: Generation of asymmetry in the one-cell embryo of C . elegans establishes the anterior--posterior axis (A-P), and is necessary for the proper identity of early blastomeres . Conserved PAR proteins are asymmetrically distributed and are required for the generation of this early asymmetry . The small G protein Cdc42 is a key regulator of polarity in other systems, and recently it has been shown to interact with the mammalian homolog of PAR-6 . The function of Cdc42 in C . elegans had not yet been investigated, however . RESULTS: Here, we show that C . elegans cdc-42 plays an essential role in the polarity of the one-cell embryo and the proper localization of PAR proteins . Inhibition of cdc-42 using RNA interference results in embryos with a phenotype that is nearly identical to par-3, par-6, and pkc-3 mutants, and asymmetric localization of these and other PAR proteins is lost . We further show that C . elegans CDC-42 physically interacts with PAR-6 in a yeast two-hybrid system, consistent with data on the interaction of human homologs . CONCLUSIONS: Our results show that CDC-42 acts in concert with the PAR proteins to control the polarity of the C . elegans embryo, and provide evidence that the interaction of CDC-42 and the PAR-3/PAR-6/PKC-3 complex has been evolutionarily conserved as a functional unit. FEBS Lett, 2001 Jun 8, 498(2-3), 150 - 6 Nuclear export of mRNA; Zenklusen D et al.; Export of mRNA through nuclear pore complexes (NPC) is preceded by multiple and well coordinated processing steps, resulting in the formation of an export competent ribonucleoprotein complex (mRNP) . Numerous factors involved in the translocation of the mRNP through the NPC and its release into the cytoplasm have been isolated mainly through genetic approaches in yeast, and putative functional homologues have been identified in metazoan systems . Understanding the mechanism of mRNA export relies, in part, on the functional characterization of these factors and the establishment of a complete network of molecular interactions . Here we summarize recent progress in the characterization of yeast and mammalian components implicated in the export of an mRNA from the nucleus to the cytoplasm. FEBS Lett, 2001 Jun 8, 498(2-3), 140 - 4 Transcriptomes, transcription activators and microarrays; Devaux F et al.; Gene-specific transcription activators are among the main factors which specifically shape the transcriptome profiles . It is tempting to take advantage of their properties to decipher the genome expression circuitry . The advent of microarray technology has offered fantastic opportunities to quickly analyze the expression profiles dictated by specific transcription factors . This review will first focus on the strategies which have been devised to control the activity of transcription factors and in the second part on the microarray experiments which addressed the role of these transcription factors in the genome-wide expression profile . This last part will mainly consider the case of the yeast Saccharomyces cerevisiae genome . All the collected data are available through the on-line database yTAFNET . yTAFNET is designed to help the characterization of connections between the different yeast regulatory networks. Exp Cell Res, 2001 Jul 1, 267(1), 126 - 34 hVPS41 is expressed in multiple isoforms and can associate with vesicles through a RING-H2 finger motif; McVey Ward D et al.; Vps41p, the protein encoded by the yeast gene VPS41, has been shown to mediate formation of AP-3 transport vesicles from the Golgi apparatus and to facilitate the docking and fusion of lysosomal vesicles . Although both of these activities involve transient association with membrane structures, the mechanisms that mediate those interactions have not been determined . Orthologues of VPS41 have been identified in humans, Drosophila, tomato, and Arabidopsis; the degree of sequence similarity among these genes suggests a highly conserved function . Here we provide evidence that hVps41, the human homologue of Vps41p, is expressed in two isoforms that differ in that one contains a C-terminal RING-H2 sequence motif . Transient expression analysis suggests that this RING-H2 domain is responsible for membrane association . This observation was further supported by the cytosolic localization of site-specific mutants . A truncated construct containing only the hVps41 RING-H2 domain was found to associate with a class of intracellular vesicles that originated from the Golgi and showed partial coincidence with the delta subunit of the adaptor protein complex-3 . Together with information from the homologous yeast system, these results suggest that hVps41 may also be involved in the formation and fusion of transport vesicles from the Golgi . J Biol Chem, 2001 Aug 17, 276(33), 30773 - 8 Epub 2001 Jun 15. Atp11p and Atp12p are assembly factors for the F(1)-ATPase in human mitochondria; Wang ZG et al.; Atp11p and Atp12p were first described as proteins required for assembly of the F(1) component of the mitochondrial ATP synthase in Saccharomyces cerevisiae (Ackerman, S . H., and Tzagoloff, A . (1990) Proc . Natl . Acad . Sci . U . S . A . 87, 4986-4990) . Here we report the isolation of the cDNAs and the characterization of the human genes for Atp11p and Atp12p and show that the human proteins function like their yeast counterparts . Human ATP11 spans 24 kilobase pairs in 9 exons and maps to 1p32.3-p33, while ATP12 contains > or =8 exons and localizes to 17p11.2 . Both genes are broadly conserved in eukaryotes and are expressed in a wide range of tissues, which suggests that Atp11p and Atp12p are essential housekeeping proteins of human cells . The information reported herein will be useful in the evaluation of patients with ascertained deficiencies in the ATP synthase, in which the underlying biochemical defect is unknown and may reside in a protein that influences the assembly of the enzyme. Gene, 2001 Jun 13, 271(1), 99 - 108 Cloning and characterization of two Arabidopsis genes that belong to the RAD21/REC8 family of chromosome cohesin proteins; Dong F et al.; Sister chromatid cohesion is required for proper chromosome segregation during cell division . One group of proteins that is essential for sister chromatid cohesion during mitosis and meiosis is the RAD21/REC8 family of cohesin proteins . Two cohesin proteins are found in yeast; one that functions mainly in mitosis while the other participates in meiosis . In contrast, only one cohesin gene appears to be present in Drosophila . In previous studies we identified an Arabidopsis cohesin protein that is required for meiosis . In this report we describe the isolation and characterization of two additional Arabidopsis cohesin genes . The structure of the genes suggests that they arose via a gene duplication event followed by extensive sequence evolution . Transcripts for the two genes are present throughout the plant and are highest in regions of active cell division, suggesting that the proteins may participate in chromosome cohesion during mitosis. Biochim Biophys Acta, 2001 Jun 15, 1526(3), 301 - 9 Study of the mode of action of endopolygalacturonase from Fusarium moniliforme; Bonnin E et al.; One endopolygalacturonase from Fusarium moniliforme was purified from the culture broth of a transformed strain of Saccharomyces cerevisiae . Its kinetic parameters and mode of action were studied on galacturonic acid oligomers and homogalacturonan . The dimer was not a substrate for the enzyme . The enzyme was shown to follow Michaelis-Menten behaviour towards the other substrates tested . Affinity and maximum rate of hydrolysis increased with increasing chain length, up to the hexamer or heptamer, for which V(max) was in the same range as with homogalacturonan . The enzyme was demonstrated to have a multi-chain attack mode of action and its active site included five subsites ranging from -3 to +2 . The final products of hydrolysis of homogalacturonan were the monomer and the dimer of galacturonic acid. Virus Res, 2001 Aug, 76(2), 183 - 9 The H1 double-stranded RNA genome of Ustilago maydis virus-H1 encodes a polyprotein that contains structural motifs for capsid polypeptide, papain-like protease, and RNA-dependent RNA polymerase; Kang J et al.; The Ustilago maydis viral (UmV) genome consists of three distinct size groups of double-stranded RNA (dsRNA) segments: H (heavy), M (medium), and L (light) . The H segments have been suggested to encode all essential viral proteins, but without any molecular evidences . As a preliminary step to understand viral genomic organization and the molecular mechanism governing gene expression in UmV, we determined the complete nucleotide sequence of the H1 dsRNA genome in P1 viral killer subtype . The H1 dsRNA genome (designated UmV-H1) contained a single open reading frame that encodes a polyprotein of 1820 residues, which is predicted to be autocatalytically processed by a viral papain-like protease to generate viral proteins . The amino-terminal region is the capsid polypeptide with a predicted molecular mass of 79.9 kDa . The carboxy-terminal region is the RNA-dependent RNA polymerase (RDRP) that has a high sequence homology to those of the totiviruses . The H2 dsRNA also encodes a distinct RDRP, suggesting that UmV is a complex virus system like the Saccharomyces cerevisiae viruses ScV-L1 and -La. EMBO J, 2001 Jun 15, 20(12), 3272 - 81 Role of the non-homologous DNA end joining pathway in the early steps of retroviral infection; Li L et al.; Early after infection, the retroviral RNA genome is reverse transcribed to generate a linear cDNA copy, then that copy is integrated into a chromosome of the host cell . We report that unintegrated viral cDNA is a substrate for the host cell non-homologous DNA end joining (NHEJ) pathway, which normally repairs cellular double-strand breaks by end ligation . NHEJ activity was found to be required for an end-ligation reaction that circularizes a portion of the unintegrated viral cDNA in infected cells . Consistent with this, the NHEJ proteins Ku70 and Ku80 were found to be bound to purified retroviral replication intermediates . Cells defective in NHEJ are known to undergo apoptosis in response to retroviral infection, a response that we show requires reverse transcription to form the cDNA genome but not subsequent integration . We propose that the double-strand ends present in unintegrated cDNA promote apoptosis, as is known to be the case for chromosomal double-strand breaks, and cDNA circularization removes the pro-apoptotic signal. EMBO J, 2001 Jun 15, 20(12), 3167 - 76 Cell signaling can direct either binary or graded transcriptional responses; Biggar SR et al.; Transcriptional control is generally thought to operate as a binary switch, a behavior that might explain observations such as monoallelic gene expression, stochastic phenotypic changes and bimodal gene activation kinetics . By measuring the activity of the single-copy GAL1 promoter in single cells, we found that changes in the activities of either the transcriptional activator, Gal4 (by simple recruitment with synthetic ligands), or the transcriptional repressor, Mig1, generated graded (non-binary) changes in gene expression that were proportional to signal intensity . However, in the context of the endogenous glucose-responsive signaling pathway, these transcription factors formed part of a binary transcriptional response . Genetic studies demonstrated that this binary response resulted from regulation of a second repressor, Gal80, whereas regulation of Mig1 by a distinct signaling pathway generated graded changes in GAL1 promoter activity . Surprisingly, isogenetic cells can respond to glucose with either binary or graded changes in gene expression, depending on growth conditions . Our studies demonstrate that a given promoter can adapt either binary or graded behavior, and identify the Mig1 and Gal80 genes as necessary for binary versus graded behavior of the Gal1 promoter. EMBO J, 2001 Jun 15, 20(12), 3145 - 55 Vac8p release from the SNARE complex and its palmitoylation are coupled and essential for vacuole fusion; Veit M et al.; Activated fatty acids stimulate budding and fusion in several cell-free assays for vesicular transport . This stimulation is thought to be due to protein palmitoylation, but relevant substrates have not yet been identified . We now report that Vac8p, a protein known to be required for vacuole inheritance, becomes palmitoylated when isolated yeast vacuoles are incubated under conditions that allow membrane fusion . Similar requirements for Vac8p palmitoylation and vacuole fusion, the inhibition of vacuole fusion by antibodies to Vac8p and the strongly reduced fusion of vacuoles lacking Vac8p suggest that palmitoylated Vac8p is essential for homotypic vacuole fusion . Strikingly, palmitoylation of Vac8p is blocked by the addition of antibodies to Sec18p (yeast NSF) only . Consistent with this, a portion of Vac8p is associated with the SNARE complex on vacuoles, which is lost during Sec18p- and ATP-dependent priming . During or after SNARE complex disassembly, palmitoylation occurs and anchors Vac8p to the vacuolar membrane . We propose that palmitoylation of Vac8p is regulated by the same machinery that controls membrane fusion. EMBO J, 2001 Jun 15, 20(12), 3124 - 31 Sec61p-independent degradation of the tail-anchored ER membrane protein Ubc6p; Walter J et al.; Tail-anchored proteins are distinct from other membrane proteins as they are thought to insert into the endoplasmic reticulum (ER) membrane independently of Sec61p translocation pores . These pores not only mediate import but are also assumed to catalyze export of proteins in a process called ER-associated protein degradation (ERAD) . In order to examine the Sec61p dependence of the export of tail-anchored proteins, we analyzed the degradation pathway of a tail-anchored ER membrane protein, the ubiquitin-conjugating enzyme 6 (Ubc6p) . In contrast to other ubiquitin conjugating enzymes (Ubcs), Ubc6p is naturally short-lived . Its proteolysis is mediated specifically by the unique Ubc6p tail region . Degradation further requires the activity of Cue1p-assembled Ubc7p, and its own catalytic site cysteine . However, it occurs independently of the other ERAD components Ubc1p, Hrd1p/Der3p, Hrd3p and Der1p . In contrast to other natural ERAD substrates, proteasomal mutants accumulate a membrane-bound degradation intermediate of Ubc6p . Most interestingly, mutations in SEC61 do not reduce the turnover of full-length Ubc6p nor cause a detectable accumulation of degradation intermediates . These data are in accordance with a model in which tail-anchored proteins can be extracted from membranes independently of Sec61p. Biochim Biophys Acta, 2001 May 31, 1536(2-3), 85 - 96 The unfolded protein response and Alzheimer's disease; Imaizumi K et al.; Disruption of calcium homeostasis, inhibition of protein glycosylation, and reduction of disulfide bonds provoke accumulation of unfolded protein in the endoplasmic reticulum (ER), and are therefore a type of 'ER stress' . Normal cells respond to ER stress by increasing transcription of genes encoding ER-resident chaperones such as GRP78/BiP, GRP94 and protein disulfide isomerase to facilitate protein folding . This induction system is termed the unfolded protein response . Familial Alzheimer's disease-linked presenilin-1 (PS1) mutation downregulates the unfolded protein response and leads to vulnerability to ER stress . The mechanisms by which mutant PS1 affects the ER stress response are attributed to the inhibited activation of ER stress transducers such as IRE1, PERK and ATF6. Biochim Biophys Acta, 2001 May 28, 1519(1-2), 70 - 7 Molecular cloning and functional characterization of two murine cDNAs which encode Ubc variants involved in DNA repair and mutagenesis; Franko J et al.; Ubiquitin-conjugating enzyme (Ubc) variants share structural similarity with Ubcs but lack the essential cysteine residue required to form a thioester bond with ubiquitin . Yeast Mms2 is a Ubc variant and plays an important role in error-free DNA postreplication repair to protect cells from killing by DNA damaging agents and mutagenesis . Ironically, one of two known Mms2 homologs, CROC1, has been linked to cell immortalization and tumorigenesis . To further investigate cellular roles played by mammalian Mms2 homologs, we report here the molecular cloning, tissue distribution and functional characterization of two mouse cDNAs encoding mMMS2 and mCROC1 . Unlike human CROC1, the mCROC1 gene does not encode two alternative transcripts in most tissues . Instead, nonoverlapping sequences were found in two distinct cDNA clones that together would constitute a full-length open reading frame homologous to CROC1B . Both mMMS2 and the C-terminal mCROC1 core domain are able to complement the yeast mms2 mutant functionally and are able to interact with Ubc13 in a yeast two-hybrid assay, indicating that they are true yeast Mms2 homologs and may play a similar role in DNA postreplication repair . We propose several hypotheses to reconcile the seemingly contradictory observations regarding roles of the two mammalian Mms2 homologs in tumorigenesis and carcinogenesis. Mutat Res, 2001 Jul 1, 478(1-2), 1 - 21 Recognition of cisplatin adducts by cellular proteins; Kartalou M et al.; Cisplatin is a widely used chemotherapeutic agent . It reacts with nucleophilic bases in DNA and forms 1,2-d(ApG), 1,2-d(GpG) and 1,3-d(GpTpG) intrastrand crosslinks, interstrand crosslinks and monofunctional adducts . The presence of these adducts in DNA is through to be responsible for the therapeutic efficacy of cisplatin . The exact signal transduction pathway that leads to cell cycle arrest and cell death following treatment with the drug is not known but cell death is believed to be mediated by the recognition of the adducts by cellular proteins . Here we describe the structural information available for cisplatin and related platinum adducts, the interactions of the adducts with cellular proteins and the implications of these interactions for cell survival. Mol Genet Genomics, 2001 May, 265(3), 497 - 507 Stability and inheritance of methylation states at PstI sites in Pisum; Knox MR et al.; The nuclear genome of pea is heavily methylated and the stability of this methylation pattern is unknown . In this study we investigated the stability of DNA methylation and its contribution to restriction fragment polymorphism as judged by AFLP fragment differences . To do this the results of a conventional AFLP analysis were compared with those obtained by a related procedure performed on pre-amplified DNA which provided a 5-methylcytosine-free DNA template (SDAFLP) . Genetic mapping in a recombinant inbred (RI) population showed that polymorphisms attributable to different methylation states of PstI sites were abundant, and generally appeared to be stably inherited, although occasional failures of the inheritance of methylation states have been found . Assessments of genetic diversity by AFLP and SDAFLP were in general agreement with each other and with the currently accepted phylogeny of Pisum, but within cultivated groups the number of differences appeared to be exaggerated by AFLP . The data suggest that epigenetic differences may have played a role in the domestication of pea. Curr Genet, 2001 Apr, 39(2), 77 - 82 Over-expression of the INO2 regulatory gene alters regulation of an INO1-lacZ reporter gene but does not affect regulation of INO1 expression; Miller LL et al.; The yeast INO2 gene encodes a transcriptional activator . Inositol and choline repress transcription of the INO2 gene, and its target genes . That is, INO2 transcription is auto-regulated . This observation prompted two separate investigations to determine if regulation of INO2 is required for regulation of its target genes . One study, using northern blot hybridization, showed that constitutive INO2 transcription did not affect regulation of the INO1 gene, while another study revealed that it severely dampened regulation of an INO1-lacZ gene . By repeating both assays from a single yeast strain we demonstrate that this disparity is due to the different reporter systems. Curr Genet, 2001 Apr, 39(2), 62 - 7 Ssb1 chaperone is a {PSI+} prion-curing factor; Chacinska A et al.; Yeast SUP7' or SUP11 nonsense suppressors have no phenotypic expression in strains deficient in the isopentenylation of A37 in tRNA . Here we show that such strains spontaneously produce cells with a nonsense suppressor phenotype which is related to the cytoplasmically inherited determinant and manifests all the key features of the {PSI+} prion . A screen of a multicopy yeast genomic library for genes that inactivate the {PSI+}-related suppressor phenotype resulted in the isolation of the SSB1 gene . Moreover, we demonstrate that multicopy plasmid encoding the Ssb1 chaperone cures cells of the {PSI+} prion. Proc Natl Acad Sci U S A, 2001 Jun 19, 98(13), 7325 - 30 Epub 2001 Jun 12. Protein kinase Cdc15 activates the Dbf2-Mob1 kinase complex; Mah AS et al.; Exit from mitosis in budding yeast requires inactivation of cyclin-dependent kinases through mechanisms triggered by the protein phosphatase Cdc14 . Cdc14 activity, in turn, is regulated by a group of proteins, the mitotic exit network (MEN), which includes Lte1, Tem1, Cdc5, Cdc15, Dbf2/Dbf20, and Mob1 . The direct biochemical interactions between the components of the MEN remain largely unresolved . Here, we investigate the mechanisms that underlie activation of the protein kinase Dbf2 . Dbf2 kinase activity depended on Tem1, Cdc15, and Mob1 in vivo . In vitro, recombinant protein kinase Cdc15 activated recombinant Dbf2, but only when Dbf2 was bound to Mob1 . Conserved phosphorylation sites Ser-374 and Thr-544 (present in the human, Caenorhabditis elegans, and Drosophila melanogaster relatives of Dbf2) were required for DBF2 function in vivo, and activation of Dbf2-Mob1 by Cdc15 in vitro . Although Cdc15 phosphorylated Dbf2, Dbf2-Mob1, and Dbf2(S374A/T544A)-Mob1, the pattern of phosphate incorporation into Dbf2 was substantially altered by either the S374A T544A mutations or omission of Mob1 . Thus, Cdc15 promotes the exit from mitosis by directly switching on the kinase activity of Dbf2 . We propose that Mob1 promotes this activation process by enabling Cdc15 to phosphorylate the critical Ser-374 and Thr-544 phosphoacceptor sites of Dbf2. Gene, 2001 May 30, 270(1-2), 31 - 40 Mouse fatty acid transport protein 4 (FATP4): characterization of the gene and functional assessment as a very long chain acyl-CoA synthetase; Herrmann T et al.; FATP4 (SLC27A4) is a member of the fatty acid transport protein (FATP) family, a group of evolutionarily conserved proteins that are involved in cellular uptake and metabolism of long and very long chain fatty acids . We cloned and characterized the murine FATP4 gene and its cDNA . From database analysis we identified the human FATP4 genomic sequence . The FATP4 gene was assigned to mouse chromosome 2 band B, syntenic to the region 9q34 encompassing the human gene . The open reading frame was determined to be 1929 bp in length, encoding a polypeptide of 643 amino acids . Within the coding region, the exon-intron structures of the murine FATP4 gene and its human counterpart are identical, revealing a high similarity to the FATP1 gene . The overall amino acid identity between the deduced murine and human FATP4 polypeptides is 92.2%, and between the murine FATP1 and FATP4 polypeptides is 60.3% . Northern analysis showed that FATP4 mRNA was expressed most abundantly in small intestine, brain, kidney, liver, skin and heart . Transfection of FATP4 cDNA into COS1 cells resulted in a 2-fold increase in palmitoyl-CoA synthetase (C16:0) and a 5-fold increase in lignoceroyl-CoA synthetase (C24:0) activity from membrane extracts, indicating that the FATP4 gene encodes an acyl-CoA synthetase with substrate specificity biased towards very long chain fatty acids. Mol Biol Cell, 2001 Jun, 12(6), 1671 - 85 Drosophila heterochromatin protein 1 (HP1)/origin recognition complex (ORC) protein is associated with HP1 and ORC and functions in heterochromatin-induced silencing; Shareef MM et al.; Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres . Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit . ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing . The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae . A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits . This complex appears to be poised to function in heterochromatin assembly later in embryonic development . Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein . This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro . The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes . Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit. Biotechnol Bioeng, 2001, 76(1), 86 - 90 Glutamate synthesis via photoreduction of NADP+ by photostable chlorophyllide coupled with polyethylene-glycol; Asada H et al.; Chlorophyllide a was coupled with alpha-(3-aminopropyl)-omega-methoxypoly(oxyethylene) (PEG-NH2) to form a PEG-chlorophyllide conjugate through an acid-amide bond . The conjugate catalyzed the reduction of methylviologen in the presence of 2-mercaptoethanol . It also catalyzed the photoreduction of NADP+ or NAD+ in the presence of ascorbate as an electron donor and ferredoxin-NADP+ reductase as the coupling enzyme . Utilizing the reducing power of NADPH generated by PEG-chlorophyllide conjugate under illumination, glutamate was synthesized from 2-oxoglutarate and NH4+ in the presence of glutamate dehydrogenase . PEG-chlorophyllide conjugate was quite stable toward light illumination compared with chlorophyll a . The increase in the molecular weight of PEG in the PEG-chlorophyllide conjugates was accompanied by the enhancement of photostability of the conjugate and also by the increased solubility in the aqueous solution . J Biol Chem, 2001 Aug 10, 276(32), 30483 - 9 Epub 2001 Jun 08. Distinct functional surface regions on ubiquitin; Sloper-Mould KE et al.; The characterized functions of the highly conserved polypeptide ubiquitin are to target proteins for proteasome degradation or endocytosis . The formation of a polyubiquitin chain of at least four units is required for efficient proteasome binding . By contrast, monoubiquitin serves as a signal for the endocytosis of plasma membrane proteins . We have defined surface residues that are important for ubiquitin's vital functions in Saccharomyces cerevisiae . Surprisingly, alanine scanning mutagenesis showed that only 16 of ubiquitin's 63 surface residues are essential for vegetative growth in yeast . Most of the essential residues localize to two hydrophobic clusters that participate in proteasome recognition and/or endocytosis . The others reside in or near the tail region, which is important for conjugation and deubiquitination . We also demonstrate that the essential residues comprise two distinct functional surfaces: residues surrounding Phe(4) are required for endocytosis, whereas residues surrounding Ile(44) are required for both endocytosis and proteasome degradation. J Biol Chem, 2001 Aug 3, 276(31), 29051 - 8 Epub 2001 Jun 08. Biochemical studies of Zmpste24-deficient mice; Leung GK et al.; Genetic studies in Saccharomyces cerevisiae identified two genes, STE24 and RCE1, involved in cleaving the three carboxyl-terminal amino acids from isoprenylated proteins that terminate with a CAAX sequence motif . Ste24p cleaves the carboxyl-terminal "-AAX" from the yeast mating pheromone a-factor, whereas Rce1p cleaves the -AAX from both a-factor and Ras2p . Ste24p also cleaves the amino terminus of a-factor . The mouse genome contains orthologues for both yeast RCE1 and STE24 . We previously demonstrated, with a gene-knockout experiment, that mouse Rce1 is essential for development and that Rce1 is entirely responsible for the carboxyl-terminal proteolytic processing of the mouse Ras proteins . In this study, we cloned mouse Zmpste24, the orthologue for yeast STE24 and showed that it could promote a-factor production when expressed in yeast . Then, to assess the importance of Zmpste24 in development, we generated Zmpste24-deficient mice . Unlike the Rce1 knockout mice, Zmpste24-deficient mice survived development and were fertile . Since no natural substrates for mammalian Zmpste24 have been identified, yeast a-factor was used as a surrogate substrate to investigate the biochemical activities in membranes from the cells and tissues of Zmpste24-deficient mice . We demonstrate that Zmpste24-deficient mouse membranes, like Ste24p-deficient yeast membranes, have diminished CAAX proteolytic activity and lack the ability to cleave the amino terminus of the a-factor precursor . Thus, both enzymatic activities of yeast Ste24p are conserved in mouse Zmpste24, but these enzymatic activities are not essential for mouse development or for fertility. J Biol Chem, 2001 Aug 10, 276(32), 29792 - 7 Epub 2001 Jun 13. The brain exocyst complex interacts with RalA in a GTP-dependent manner: identification of a novel mammalian Sec3 gene and a second Sec15 gene; Brymora A et al.; Ral is a small GTPase involved in critical cellular signaling pathways . The two isoforms, RalA and RalB, are widely distributed in different tissues, with RalA being enriched in brain . The best characterized RalA signaling pathways involve RalBP1 and phospholipase D . To investigate RalA signaling in neuronal cells we searched for RalA-binding proteins in brain . We found at least eight proteins that bound RalA in a GTP-dependent manner . Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified these as the components of the exocyst complex . The yeast exocyst is a regulator of polarized secretion, docking vesicles to regions of the plasma membrane involved in active exocytosis . We identified the human FLJ10893 protein as the mammalian homologue of the yeast exocyst protein Sec3p . The exocyst complex did not contain the previously identified exocyst component rSec15, but a new homologue of both yeast Sec15p and rSec15, called KIAA0919 . Western blots confirmed that two rat exocyst proteins, rSec6 and rSec8, bound active RalA in nerve terminals, as did RalBP1 . Phospholipase D bound RalA in a nucleotide-independent manner . This places the RalA signaling system in mammalian nerve terminals, where the exocyst may act as an effector for activated RalA in directing sites of exocytosis. Methods, 2001 Jul, 24(3), 218 - 29 The tandem affinity purification (TAP) method: a general procedure of protein complex purification; Puig O et al.; Identification of components present in biological complexes requires their purification to near homogeneity . Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases . We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level . Prior knowledge of complex composition or function is not required . The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest . Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications . Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented . The TAP method was initially developed in yeast but can be successfully adapted to various organisms . Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration . J Chromatogr A, 2001 May 11, 917(1-2), 55 - 61 Bioskin as an affinity matrix for the separation of glycoproteins; Vicente C et al.; Bioskin is a natural product produced by a mixed culture of Acetobacter xylinum, Saccharomyces cerevisiae and S . pombe cultured on media containing sucrose . It is of fibrillar nature able to retain some proteins, such as cytochrome c, by adsorption, and mainly composed of glucosamine and N-acetyl-D-glucosamine . This makes it possible that, at an adequate pH value, proteins charged as polyanionic molecules, such as catalase, can be retained by ionic adsorption using the positively charged amino groups of the matrix . In addition, bioskin can also be used as an affinity matrix to retain glycoproteins able to perform specific affinity reactions with the amino sugars of the matrix, such as invertase, fetuin or ovalbumin . Its possible use as a chromatographic support is discussed. Anal Chem, 2001 Jun 1, 73(11), 2578 - 86 Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses; Goshe MB et al.; A method has been developed that utilizes phosphoprotein isotope-coded affinity tags (PhIAT) that combines stable isotope and biotin labeling to enrich and quantitatively measure differences in the O-phosphorylation states of proteins . The PhIAT labeling approach involves hydroxide ion-mediated beta-elimination of the O-phosphate moiety and the addition of 1,2-ethanedithiol containing either four alkyl hydrogens (EDT-D0) or four alkyl deuteriums (EDT-D4) followed by biotinylation of the EDT-D0/D4 moiety using (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine . The PhIAT reagent, which contains the nucleophilic sulfhydryl and isotopic label covalently linked to a biotin moiety, was synthesized and has the potential utility to reduce the O-phosphorylation derivatization into a one-step process . The PhIAT labeling approach was initially demonstrated using the model phosphoprotein beta-casein . After proteolytic digestion, the PhIAT-labeled peptides were affinity isolated using immobilized avidin and analyzed using capillary reversed-phase liquid chromatography-mass spectrometry . PhIAT-labeled beta-casein peptides corresponding to peptides containing known sites of O-phosphorylation were isolated and identified . The PhIAT labeling method was also applied to a yeast protein extract . The PhIAT labeling technique provides a reliable method for making quantitative measurements of differences in the O-phosphorylation state of proteins. Inflammation, 2001 Jun, 25(3), 165 - 9 An intact cytoskeleton is required for prolonged respiratory burst activity during neutrophil phagocytosis; Granfeldt D et al.; The temporal relationship between phagocytosis and respiratory burst activity was investigated . Neutrophil uptake of yeast particles was synchronized and the kinetics of the oxidative burst was determined using an isoluminol/luminol amplified chemiluminescence system . The reactive oxygen species were mainly generated intracellularly (defined as the activity that remained in an luminol-enhanced system in the presence of superoxide dismutase and catalase) . Following phagocytosis, the intracellular response rapidly reached a level close to the maximum and the activity was almost constant for the first 10 to 15 min . The response then slowly declined . The presence of cytochalasin B, an inhibitor of actin polymerization, greatly reduced the respiratory burst activity, and this was true also when the inhibitor was added after completion of uptake of yeast particles . Our results thus show that there is a continuous production of oxygen metabolites long after phagocytosis is completed . There is also a requirement for an intact cytoskeleton for prolonged superoxide production inside the phagosome. Plant Physiol, 2001 Jun, 126(2), 910 - 20 Cloning and expression pattern of a gene encoding an alpha-xylosidase active against xyloglucan oligosaccharides from Arabidopsis; Sampedro J et al.; An alpha-xylosidase active against xyloglucan oligosaccharides was purified from cabbage (Brassica oleracea var . capitata) leaves . Two peptide sequences were obtained from this protein, the N-terminal and an internal one, and these were used to identify an Arabidopsis gene coding for an alpha-xylosidase that we propose to call AtXYL1 . It has been mapped to a region of chromosome I between markers at 100.44 and 107.48 cM . AtXYL1 comprised three exons and encoded a peptide that was 915 amino acids long, with a potential signal peptide of 22 amino acids and eight possible N-glycosylation sites . The protein encoded by AtXYL1 showed the signature regions of family 31 glycosyl hydrolases, which comprises not only alpha-xylosidases, but also alpha-glucosidases . The alpha-xylosidase activity is present in apoplastic extractions from Arabidopsis seedlings, as suggested by the deduced signal peptide . The first eight leaves from Arabidopsis plants were harvested to analyze alpha-xylosidase activity and AtXYL1 expression levels . Both increased from older to younger leaves, where xyloglucan turnover is expected to be higher . When this gene was introduced in a suitable expression vector and used to transform Saccharomyces cerevisiae, significantly higher alpha-xylosidase activity was detected in the yeast cells . alpha-Glucosidase activity was also increased in the transformed cells, although to a lesser extent . These results show that AtXYL1 encodes for an apoplastic alpha-xylosidase active against xyloglucan oligosaccharides that probably also has activity against p-nitrophenyl-alpha-D-glucoside. Plant Cell, 2001 Jun, 13(6), 1369 - 82 Nodule-specific regulation of phosphatidylinositol transfer protein expression in Lotus japonicus; Kapranov P et al.; Phosphatidylinositol transfer proteins (PITPs) modulate signal transduction pathways and membrane-trafficking functions in eukaryotes . Here, we describe the characterization of a gene family from Lotus japonicus that encodes a novel class of plant PITP-like proteins (LjPLPs) and that is regulated in an unusual nodule-specific manner . Members of this gene family were identified based on their nucleotide sequence homology with a previously described cDNA, LjNOD16, which encodes the L . japonicus late nodulin Nlj16 . Nlj16 or highly related amino acid sequences are shown to constitute C-terminal domains of LjPLPs and are suggested to function as specific plasma membrane targeting modules . The expression patterns of one member of this gene family (LjPLP-IV) revealed that LjNOD16 mRNA synthesis in nodules is the result of the transcriptional activity of a nodule-specific promoter located in an intron of the LjPLP-IV gene . This intron-borne bidirectional promoter also generates nodule-specific antisense transcripts derived from the N-terminal PITP domain coding region of the LjPLP-IV gene . We propose that Nlj16 protein synthesis and LjPLP-IV antisense transcript generation are components of an elaborate mechanism designed to control LjPLP synthesis and/or functioning in nodules. J Cell Biol, 2001 Jun 11, 153(6), 1151 - 60 Tom40, the pore-forming component of the protein-conducting TOM channel in the outer membrane of mitochondria; Ahting U et al.; Tom40 is the main component of the preprotein translocase of the outer membrane of mitochondria (TOM complex) . We have isolated Tom40 of Neurospora crassa by removing the receptor Tom22 and the small Tom components Tom6 and Tom7 from the purified TOM core complex . Tom40 is organized in a high molecular mass complex of approximately 350 kD . It forms a high conductance channel . Mitochondrial presequence peptides interact specifically with Tom40 reconstituted into planar lipid membranes and decrease the ion flow through the pores in a voltage-dependent manner . The secondary structure of Tom40 comprises approximately 31% beta-sheet, 22% alpha-helix, and 47% remaining structure as determined by circular dichroism measurements and Fourier transform infrared spectroscopy . Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation . They presumably represent an open pore with a diameter of approximately 2.5 nm, similar to the pores found in the TOM complex . Thus, Tom40 is the core element of the TOM translocase; it forms the protein-conducting channel in an oligomeric assembly. J Biol Chem, 2001 Aug 10, 276(32), 30002 - 10 Epub 2001 Jun 11. pEg2 aurora-A kinase, histone H3 phosphorylation, and chromosome assembly in Xenopus egg extract; Scrittori L et al.; In eukaryotes cell division is accompanied by phosphorylation of histone H3 at serine 10 . In this work we have studied the kinase activity responsible for this histone H3 modification by using cell-free extracts prepared from Xenopus eggs . We have found that the Xenopus aurora-A kinase pEg2, immunoprecipitated from the extract, is able to phosphorylate specifically histone H3 at serine 10 . The enzyme is incorporated into chromatin during in vitro chromosome assembly, and the kinetics of this incorporation parallels that of histone H3 phosphorylation . Recombinant pEg2 phosphorylates efficiently histone H3 at serine 10 in reconstituted nucleosomes and in sperm nuclei decondensed in heated extracts . These data identify pEg2 as a good candidate for mitotic histone H3 kinase . However, immunodepletion of pEg2 does not interfere with the chromosome assembly properties of the extract nor with the pattern of H3 phosphorylation, suggesting the existence of multiple kinases involved in this H3 modification in Xenopus eggs . This hypothesis is supported by in gel activity assay experiments using extracts from Saccharomyces cerevisiae. Mol Microbiol, 2001 Jun, 40(5), 1165 - 74 The Candida glabrata Amt1 copper-sensing transcription factor requires Swi/Snf and Gcn5 at a critical step in copper detoxification; Koch KA et al.; The yeast Candida glabrata rapidly autoactivates transcription of the AMT1 gene in response to potentially toxic copper levels through the copper-inducible binding of the Amt1 transcription factor to a metal response element (MRE) within a positioned nucleosome . Our previous studies have characterized the role of a 16 bp homopolymeric dA:dT DNA structural element in facilitating rapid Amt1 access to the AMT1 promoter nucleosomal MRE . In this study, we have used the genetically more facile yeast Saccharomyces cerevisiae to identify additional cellular factors that are important for promoting rapid autoactivation of the AMT1 gene in response to toxic copper levels . We demonstrate that the Swi/Snf nucleosome remodelling complex and the histone acetyltransferase Gcn5 are both essential for AMT1 gene autoregulation, and that the requirement for these chromatin remodelling factors is target gene specific . Chromatin accessibility measurements performed in vitro and in vivo indicate that part of the absolute requirement for these factors is derived from their involvement in facilitating nucleosomal access to the AMT1 promoter MRE . Additionally, these data implicate the involvement of Swi/Snf and Gcn5 at multiple levels of AMT1 gene autoregulation. Mol Microbiol, 2001 May, 40(4), 1009 - 19 RNA polymerase II and TBP occupy the repressed CYC1 promoter; Martens C et al.; Saccharomyces cerevisiae CYC1 gene expression has been studied in great detail with regard to the response to oxygen availability and carbon source . In the absence of oxygen and the presence of glucose, the CYC1 gene is completely repressed . Chromatin structure is thought to play an important role in CYC1 gene regulation, as nucleosome depletion results in 94-fold derepression . In addition, the CYC1 core promoter has been used extensively in hybrid constructs to study activation by heterologous transcription factors . Therefore, we set out to map the chromatin structure of the CYC1 promoter and determine its role in CYC1 gene regulation . We report here that the repressed CYC1 promoter contains no positioned nucleosomes over the core promoter . However, we did find TFIID and RNA polymerase II bound in a complex on the repressed promoter . These results indicate that recruitment of TFIID and RNA polymerase II are not rate-limiting steps in CYC1 activation. Biochemistry, 2001 Jun 19, 40(24), 7301 - 8 Role of phylogenetically conserved amino acids in folding of Na,K-ATPase; Jorgensen JR et al.; This paper focuses on the amino acid sequence 708-TGDGVNDSPALKK in pig kidney Na,K-ATPase as one of the best conserved among P-type ATPases . In Ca-ATPase this sequence forms a strand-loop-helix structure as part of a Rossman fold next to the phosphorylation site . Substitution of polar residues in the investigated sequence interfered with high-level accumulation of mutant protein . Mutant alpha1-subunit protein only accumulated in membranes from yeast cells grown at 15 degrees C whereas wild-type protein accumulated at both 15 and 35 degrees C . A systematic screen for the molecular mechanism behind lack of accumulation of mutant protein at 35 degrees C showed that transcription and translation were unaffected by the mutations . To demonstrate in vivo protein folding problems, an unfolded protein response reporter system was constructed in yeast . In this strain, only expression of mutant Na,K-ATPase alpha1-subunit caused induction of the unfolded protein response at 35 degrees C, indicating folding problems in the ER . Lowering the expression temperature to 15 degrees C prevented induction of the unfolded protein response after mutant protein expression, indicating correct folding at this temperature . At the permissive temperature mutant proteins were able to escape the endoplasmic reticulum quality control, reach the plasma membrane, and bind ouabain with high affinity . Since mutants in the 708-TGDGVNDSPALKK segment had a thermo inactivation profile identical to that of wild type, they were classified as temperature-sensitive synthesis mutants . The results indicate that this segment contributes side chains of importance for overall folding and maturation of Na,K-ATPase and all other P-type ATPases. J Mol Biol, 2001 Jun 15, 309(4), 845 - 53 The crystal structure of delta(3)-delta(2)-enoyl-CoA isomerase; Mursula AM et al.; The active-site geometry of the first crystal structure of a Delta(3)-Delta(2)-enoyl-coenzyme A (CoA) isomerase (the peroxisomal enzyme from the yeast Saccharomyces cerevisiae) shows that only one catalytic base, Glu158, is involved in shuttling the proton from the C2 carbon atom of the substrate, Delta(3)-enoyl-CoA, to the C4 atom of the product, Delta(2)-enoyl-CoA . Site-directed mutagenesis has been performed to confirm that this glutamate residue is essential for catalysis . This Delta(3)-Delta(2)-enoyl-CoA isomerase is a hexameric enzyme, consisting of six identical subunits . It belongs to the hydratase/isomerase superfamily of enzymes which catalyze a wide range of CoA-dependent reactions . The members of the hydratase/ isomerase superfamily have only a low level of sequence identity . Comparison of the crystal structure of the Delta(3)-Delta(2)-enoyl-CoA isomerase with the other structures of this superfamily shows only one region of large structural variability, which is in the second turn of the spiral fold and which is involved in defining the shape of the binding pocket . J Biol Chem, 2001 Aug 17, 276(33), 31321 - 31 Epub 2001 Jun 07. Mutational analysis of conserved residues in the GCN5 family of histone acetyltransferases; Langer MR et al.; GCN5 is a critical transcriptional co-activator and is the defining member of a large superfamily of N-acetyltransferases . GCN5 catalyzes the transfer of an acetyl group from acetyl-CoA to the epsilon-amino of lysine 14 within the core H3 histone protein . Previous biochemical analyses have indicated a fully ordered kinetic mechanism . Recent structural studies have implicated several conserved residues in catalysis and substrate binding . Here the roles of Glu-173, His-145, and Asp-214 in yeast GCN5 have been evaluated using site-directed mutagenesis, steady state and pre-steady state kinetics, pH analysis, isotope partitioning, and equilibrium binding studies . The results with wild type and E173Q, H145A, and D214A mutants are consistent with chemical catalysis being rate-determining in turnover . All mutants exhibited K(d) values (3.5-8.5 microm) for AcCoA that were similar to wild type enzyme, indicating no functional role for these residues in AcCoA binding . The E173Q mutant demonstrated a approximately 500-600-fold decreases in k(cat) and k(cat)/K(m),(H3), consistent with Glu-173 acting as the general base catalyst as proposed previously . No significant effect was observed on substrate binding steps . His-145 was identified as a residue in the peptide binding cleft that must be unprotonated (pK(a) = 5.8) for peptide binding and likely hydrogen-bonds to the Ser-10 hydroxyl of histone H3 . His-145 also contributes to lowering the pK(a) value (by 0.8 units) of general base Glu-173 through a water-mediated hydrogen bond to the carboxylate side chain . Analysis of D214A revealed an obligate protein isomerization step that occurs after AcCoA binding and permits efficient peptide binding . Asp-214 is part of a conformationally flexible loop that mediates the isomerization by stabilizing distinct conformers of the protein. Trends Endocrinol Metab, 2001 Jul, 12(5), 184 - 6 Molecular scaffold protein and cellular responses; Keenan SM et al.; Mitogen-activated kinases (MAPK) regulate many diverse cellular processes, including growth, differentiation and responses to stress . The organization of MAPKs through the use of scaffolding proteins is crucial for the selective activation of these kinases by different stimuli . Recent studies identify beta-arrestins as members of the family of MAPK scaffold proteins . beta-Arrestins not only shut off signaling by uncoupling G-protein-coupled receptors (GPCRs) from their heterotrimeric G proteins, but also contribute to the specificity of GPCRs signaling by recruiting and activating selective MAPKs. Phytochemistry, 2001 Jul, 57(5), 743 - 7 Antifungal polysulphides from Petiveria alliacea L; Benevides PJ et al.; Bioactivity-directed fractionation of the CH(2)Cl(2)/MeOH (2:1, v/v) extract of the roots of Petiveria alliacea, using mutant yeast strains of Saccharomyces cerevisiae and fungi Cladosporium cladosporioides and C . sphaerospermum led to the isolation of dipropyl disulphide (1), dibenzyl sulphide (2), dibenzyl disulphide (3), dibenzyl trisulphide (4), dibenzyl tetrasulphide (5), benzylhydroxymethyl sulphide (6) and di(benzyltrithio) methane (7) . Of these, 5-7 are new compounds and this is the first report of the natural occurrence of 2 and 3. Biochem Biophys Res Commun, 2001 Jun 15, 284(3), 757 - 62 A transgenic mouse expressing human CYP4B1 in the liver; Imaoka S et al.; The human CYP4B1 protein was expressed in the liver of a transgenic mouse line under the control of the promoter of the human apolipoprotein E (apo E) gene . Hepatic microsomes of transgenic mice catalyzed omega-hydroxylation of lauric acid and also activated 2-aminofluorene (2-AF), which is a typical substrate for CYP4B1, to mutagenic compounds detected by an umu gene expression assay . These activities observed in transgenic mouse were efficiently inhibited by CYP4B1 antibody . However, such inhibition was not observed in control mice . This is the first report to indicate catalytic activities of human CYP4B1 . For further characterization of human CYP4B1, a fusion protein of CYP4B1 and NADPH-P450 reductase was expressed in yeast cells . It was able to activate 2-AF and was also able to catalyze omega-hydroxylation of lauric acid . This transgenic mouse line and the recombinant fusion protein provide a useful tool to study human CYP4B1 and its relation to chemical toxicity and carcinogenesis . Biochemistry, 2001 Jun 5, 40(22), 6688 - 98 Mg2+-induced tRNA folding; Serebrov V et al.; Mg(2+)-induced folding of yeast tRNA(Phe) was examined at low ionic strength in steady-state and kinetic experiments . By using fluorescent labels attached to tRNA, four conformational transitions were revealed when the Mg(2+) concentration was gradually increased . The last two transitions were not accompanied by changes in the number of base pairs . The observed transitions were attributed to Mg(2+) binding to four distinct types of sites . The first two types are strong sites with K(diss) of 4 and 16 microM . The sites of the third and fourth types are weak with a K(diss) of 2 and 20 mM . Accordingly, the Mg(2+)-binding sites previously classified as "strong" and "weak" can be further subdivided into two subtypes each . Fluorescent transition I is likely to correspond to Mg(2+) binding to a unique strong site selective for Mg(2+); binding to this site causes only minor A(260) change . The transition at 2 mM Mg(2+) is accompanied by substantial conformational changes revealed by probing with ribonucleases T1 and V1 and likely enhances stacking of the tRNA bases . Fast and slow kinetic phases of tRNA refolding were observed . Time-resolved monitoring of Mg(2+) binding to tRNA suggested that the slow kinetic phase was caused by a misfolded tRNA structure formed in the absence of Mg(2+) . Our results suggest that, similarly to large RNAs, Mg(2+)-induced tRNA folding exhibits parallel folding pathways and the existence of kinetically trapped intermediates stabilized by Mg(2+) . A multistep scheme for Mg(2+)-induced tRNA folding is discussed. Nature, 2001 Jun 7, 411(6838), 713 - 6 Defects in mismatch repair promote telomerase-independent proliferation; Rizki A et al.; Mismatch repair has a central role in maintaining genomic stability by repairing DNA replication errors and inhibiting recombination between non-identical (homeologous) sequences . Defects in mismatch repair have been linked to certain human cancers, including hereditary non-polyposis colorectal cancer (HNPCC) and sporadic tumours . A crucial requirement for tumour cell proliferation is the maintenance of telomere length, and most tumours achieve this by reactivating telomerase . In both yeast and human cells, however, telomerase-independent telomere maintenance can occur as a result of recombination-dependent exchanges between often imperfectly matched telomeric sequences . Here we show that loss of mismatch-repair function promotes cellular proliferation in the absence of telomerase . Defects in mismatch repair, including mutations that correspond to the same amino-acid changes recovered from HNPCC tumours, enhance telomerase-independent survival in both Saccharomyces cerevisiae and a related budding yeast with a degree of telomere sequence homology that is similar to human telomeres . These results indicate that enhanced telomeric recombination in human cells with mismatch-repair defects may contribute to cell immortalization and hence tumorigenesis. J Biol Chem, 2001 Aug 10, 276(32), 30208 - 15 Epub 2001 Jun 06. p65-activated histone acetyltransferase activity is repressed by glucocorticoids: mifepristone fails to recruit HDAC2 to the p65-HAT complex; Ito K et al.; Glucocorticoids acting through their specific receptor can either enhance or repress gene transcription . Dexamethasone represses interleukin-1beta-stimulated histone acetylation and granulocyte-macrophage colony-stimulating factor expression through a combination of direct inhibition of p65-associated histone acetyltransferase (HAT) activity and by recruiting histone deacetylase 2 (HDAC2) to the p65-HAT complex . Here we show that mifepristone, a glucocorticoid receptor partial agonist, has no ability to induce gene expression but represses interleukin-1beta-stimulated histone acetylation and granulocyte-macrophage colony-stimulating factor release by 50% maximally . Mifepristone was able to inhibit p65-associated HAT activity to the same extent as dexamethasone but failed to inhibit the natural promoter to an equal extent due to an inability to recruit HDAC2 to the p65-associated HAT complex . These data suggest that the maximal repressive actions of glucocorticoids require recruitment of HDAC2 to a p65-HAT complex . These data also suggest that pharmacological manipulation of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases. J Biol Chem, 2001 Aug 3, 276(31), 29067 - 71 Epub 2001 Jun 06. Architecture of the human origin recognition complex; Dhar SK et al.; All the human homologs of the six subunits of Saccharomyces cerevisiae origin recognition complex have been reported so far . However, not much has been reported on the nature and the characteristics of the human origin recognition complex . In an attempt to purify recombinant human ORC from insect cells infected with baculoviruses expressing HsORC subunits, we found that human ORC2, -3, -4, and -5 form a core complex . HsORC1 and HsORC6 subunits did not enter into this core complex, suggesting that the interaction of these two subunits with the core ORC2-5 complex is extremely labile . We found that the C-terminal region of ORC2 interacts directly with the N-terminal region of ORC3 . The C-terminal region of ORC3 was, however, necessary to bring ORC4 and ORC5 into the core complex . A fragment containing the N-terminal 200 residues of ORC3 (ORC3N) competitively inhibited the ORC2-ORC3 interaction . Overexpression of this fragment in U2OS cells blocked the cells in G(1), providing the first evidence that a mammalian ORC subunit is important for the G(1)-S transition in mammalian cells. Bioinformatics, 2001 Jun, 17(6), 520 - 5 Missing value estimation methods for DNA microarrays; Troyanskaya O et al.; MOTIVATION: Gene expression microarray experiments can generate data sets with multiple missing expression values . Unfortunately, many algorithms for gene expression analysis require a complete matrix of gene array values as input . For example, methods such as hierarchical clustering and K-means clustering are not robust to missing data, and may lose effectiveness even with a few missing values . Methods for imputing missing data are needed, therefore, to minimize the effect of incomplete data sets on analyses, and to increase the range of data sets to which these algorithms can be applied . In this report, we investigate automated methods for estimating missing data . RESULTS: We present a comparative study of several methods for the estimation of missing values in gene microarray data . We implemented and evaluated three methods: a Singular Value Decomposition (SVD) based method (SVDimpute), weighted K-nearest neighbors (KNNimpute), and row average . We evaluated the methods using a variety of parameter settings and over different real data sets, and assessed the robustness of the imputation methods to the amount of missing data over the range of 1--20% missing values . We show that KNNimpute appears to provide a more robust and sensitive method for missing value estimation than SVDimpute, and both SVDimpute and KNNimpute surpass the commonly used row average method (as well as filling missing values with zeros) . We report results of the comparative experiments and provide recommendations and tools for accurate estimation of missing microarray data under a variety of conditions. Bioinformatics, 2001 Jun, 17(6), 495 - 508 Aligning gene expression time series with time warping algorithms; Aach J et al.; motivation: Increasingly, biological processes are being studied through time series of RNA expression data collected for large numbers of genes . Because common processes may unfold at varying rates in different experiments or individuals, methods are needed that will allow corresponding expression states in different time series to be mapped to one another . Results: We present implementations of time warping algorithms applicable to RNA and protein expression data and demonstrate their application to published yeast RNA expression time series . Programs executing two warping algorithms are described, a simple warping algorithm and an interpolative algorithm, along with programs that generate graphics that visually present alignment information . We show time warping to be superior to simple clustering at mapping corresponding time states . We document the impact of statistical measurement noise and sample size on the quality of time alignments, and present issues related to statistical assessment of alignment quality through alignment scores . We also discuss directions for algorithm improvement including development of multiple time series alignments and possible applications to causality searches and non-temporal processes ('concentration warping'). Annu Rev Biochem, 2001, 70, 677 - 701 Function, structure, and mechanism of intracellular copper trafficking proteins; Huffman DL et al.; Genetic, biochemical, and spectroscopic studies have established a new function for an intracellular protein, i.e., guiding and inserting a copper cofactor into the active site of a target enzyme . Studies of these new proteins have revealed a fundamental aspect of copper physiology, namely the vast overcapacity of the cytoplasm for copper sequestration . This finding framed the mechanistic, energetic, and structural aspects of intracellular copper trafficking proteins . One hallmark of the copper chaperones is the similarity of the protein fold between the chaperone and its target enzyme . The surface residues presented by each partner, however, are quite different, and some initial findings concerning the complementarity of these interfaces have led to mechanistic insights . The copper chaperones appear to lower the activation barrier for metal transfer into specific protein-binding sites . The manner in which they facilitate metal insertion appears to involve a docking of the metal donor and acceptor sites in close proximity to one another . Although the intimate mechanism is still open, it appears that a low activation barrier for metal transfer is achieved by a network of coordinate-covalent, electrostatic, and hydrogen bonding interactions in the vicinity of the metal-binding site itself. Annu Rev Biochem, 2001, 70, 475 - 501 Transcriptional coactivator complexes; Naar AM et al.; The last two decades have witnessed a tremendous expansion in our knowledge of the mechanisms employed by eukaryotic cells to control gene activity . A critical insight to transcriptional control mechanisms was provided by the discovery of coactivators, a diverse array of cellular factors that connect sequence-specific DNA binding activators to the general transcriptional machinery, or that help activators and the transcriptional apparatus to navigate through the constraints of chromatin . A number of coactivators have been isolated as large multifunctional complexes, and biochemical, genetic, molecular, and cellular strategies have all contributed to uncovering many of their components, activities, and modes of action . Coactivator functions can be broadly divide into two classes: (a) adaptors that direct activator recruitment of the transcriptional apparatus, (b) chromatin-remodeling or -modifying enzymes . Strikingly, several distinct coactivator complexes nonetheless share many subunits and appear to be assembled in a modular fashion . Such structural and functional modularity could provide the cell with building blocks from which to construct a versatile array of coactivator complexes according to its needs . The extent of functional interplay between these different activities in gene-specific transcriptional regulation is only now becoming apparent, and will remain an active area of research for years to come. Annu Rev Biochem, 2001, 70, 81 - 120 Histone acetyltransferases; Roth SY et al.; Transcriptional regulation in eukaryotes occurs within a chromatin setting and is strongly influenced by nucleosomal barriers imposed by histone proteins . Among the well-known covalent modifications of histones, the reversible acetylation of internal lysine residues in histone amino-terminal domains has long been positively linked to transcriptional activation . Recent biochemical and genetic studies have identified several large, multisubunit enzyme complexes responsible for bringing about the targeted acetylation of histones and other factors . This review discusses our current understanding of histone acetyltransferases (HATs) or acetyltransferases (ATs): their discovery, substrate specificity, catalytic mechanism, regulation, and functional links to transcription, as well as to other chromatin-modifying activities . Recent studies underscore unexpected connections to both cellular regulatory processes underlying normal development and differentiation, as well as abnormal processes that lead to oncogenesis . Although the functions of HATs and the mechanisms by which they are regulated are only beginning to be understood, these fundamental processes are likely to have far-reaching implications for human biology and disease. Bioseparation, 2000, 9(5), 269 - 76 Extraction of peptide tagged cutinase in detergent-based aqueous two-phase systems; Rodenbrock A et al.; Detergent-based aqueous two-phase systems have the advantage to require only one auxiliary chemical to induce phase separation above the cloud point . In a systematic study the efficiency of tryptophan-rich peptide tags was investigated to enhance the partitioning of an enzyme to the detergent-rich phase using cutinase as an example . Up to 90% enzyme activity could be extracted in a single step from whole broth of recombinant Saccharomyces cerevisiae expressing cutinase variants carrying a (WP)4 tag . In contrast, the extraction yield of wild type cutinase was 2-3% only . The detergent concentration and the temperature are the main parameters to optimize the extraction yield . Considering availability, extraction yields, and price the detergent Agrimul NRE 1205 served best for enzyme recovery. J Hum Genet, 2001, 46(6), 314 - 9 Identification of single-nucleotide polymorphisms (SNPs) of human N-acetyltransferase genes NAT1, NAT2, AANAT, ARD1 and L1CAM in the Japanese population; Sekine A et al.; By direct sequencing of regions of the human genome containing five genes belonging to the acetyltransferase family, arylamine N-acetyltransferase (NAT1), arylamine N-acetyltransferase (NAT2), arylalkylamine N-acetyltransferase (AANAT), L1 cell adhesion molecule (L1CAM), and the human homolog of Saccharomyces cerevisiae N-acetyltransferase ARD1, we identified 53 single-nucleotide polymorphisms (SNPs) and two insertion/ deletion polymorphisms in 48 healthy Japanese volunteers . NAT1 and NAT2 are so-called drug-metabolizing enzymes . In the NAT1 gene we found two SNPs and a 3-bp insertion/ deletion polymorphism that corresponded to the NAT1*3, *10, and *18A/*18B alleles reported in other populations . The frequencies of NAT1* alleles in our Japanese subjects were 52.6% for NAT1*4, 1.0% for NAT1*3, 40.6% for NAT1*10, 2.6% for NAT1*18A and 3.1% for NAT1*18B . In the NAT2 gene we found 32 SNPs and a 1-bp insertion/ deletion polymorphism; 6 SNPs within the coding region were reported previously and belonged to the slow acetylator group (NAT2*5, NAT2*6 and NAT2*7), and 2 of the 8 SNPs in the 5' flanking region were reported in the dbSNP of GenBank, but the remaining 24 SNPs and the insertion/deletion polymorphism were novel . The frequencies of NAT2* alleles in Japanese (51.3% for NAT2*4, 1.6% for *5B, 26.1% for *6A, 2.2% for *6B, 1.2% for *7A, 10.1% for *7B, 7.4% for *12A, and 1.1% for *13) were significantly different from those reported in Caucasian populations . In the AANAT gene we found 4 novel SNPs: 2 in the 5' flanking region, 1 in exon 4, and 1 in intron 3 . In the two genes belonging to the N-terminal N-acetyltransferase family, we identified 9 SNPs, 7 of them novel, for ARD1, and six novel SNPs for L1CAM . Variations at these loci may contribute to an understanding of the way in which different genotypes may affect the activities of human N-acetyltransferases, especially as regards the therapeutic efficacy of certain drugs and antibiotics. Proc Natl Acad Sci U S A, 2001 Jul 3, 98(14), 8133 - 8 Epub 2001 Jun 05. The role of haustoria in sugar supply during infection of broad bean by the rust fungus Uromyces fabae; Voegele RT et al.; Biotrophic plant pathogenic fungi differentiate specialized infection structures within the living cells of their host plants . These haustoria have been linked to nutrient uptake ever since their discovery . We have for the first time to our knowledge shown that the flow of sugars from the host Vicia faba to the rust fungus Uromyces fabae seems to occur largely through the haustorial complex . One of the most abundantly expressed genes in rust haustoria, the expression of which is negligible in other fungal structures, codes for a hexose transporter . Functional expression of the gene termed HXT1 in Saccharomyces cerevisiae and Xenopus laevis oocytes assigned a substrate specificity for D-glucose and D-fructose and indicated a proton symport mechanism . Abs against HXT1p exclusively labeled haustoria in immunofluorescence microscopy and the haustorial plasma membrane in electron microscopy . These results suggest that the fungus concentrates this transporter in haustoria to take advantage of a specialized compartment of the haustorial complex . The extrahaustorial matrix, delimited by the plasma membranes of both host and parasite, constitutes a newly formed apoplastic compartment with qualities distinct from those of the bulk apoplast . This organization might facilitate the competition of the parasite with natural sink organs of the host. Proc Natl Acad Sci U S A, 2001 Jun 19, 98(13), 7206 - 11 Epub 2001 Jun 05. Identification and characterization of a lysosomal transporter for small neutral amino acids; Sagne C et al.; In eukaryotic cells, lysosomes represent a major site for macromolecule degradation . Hydrolysis products are eventually exported from this acidic organelle into the cytosol through specific transporters . Impairment of this process at either the hydrolysis or the efflux step is responsible of several lysosomal storage diseases . However, most lysosomal transporters, although biochemically characterized, remain unknown at the molecular level . In this study, we report the molecular and functional characterization of a lysosomal amino acid transporter (LYAAT-1), remotely related to a family of H+-coupled plasma membrane and synaptic vesicle amino acid transporters . LYAAT-1 is expressed in most rat tissues, with highest levels in the brain where it is present in neurons . Upon overexpression in COS-7 cells, the recombinant protein mediates the accumulation of neutral amino acids, such as gamma-aminobutyric acid, l-alanine, and l-proline, through an H+/amino acid symport . Confocal microscopy on brain sections revealed that this transporter colocalizes with cathepsin D, an established lysosomal marker . LYAAT-1 thus appears as a lysosomal transporter that actively exports neutral amino acids from lysosomes by chemiosmotic coupling to the H+-ATPase of these organelles . Homology searching in eukaryotic genomes suggests that LYAAT-1 defines a subgroup of lysosomal transporters in the amino acid/auxin permease family. Mol Cell Biol, 2001 Jul, 21(13), 4311 - 20 Essential roles of Snf5p in Snf-Swi chromatin remodeling in vivo; Geng F et al.; Snf-Swi, the prototypical ATP-dependent nucleosome-remodeling complex, regulates transcription of a subset of yeast genes . With the exception of Snf2p, the ATPase subunit, the functions of the other components are unknown . We have investigated the role of the conserved Snf-Swi core subunit Snf5p through characterization of two conditional snf5 mutants . The mutants contain single amino acid alterations of invariant or conserved residues that abolish Snf-Swi-dependent transcription by distinct mechanisms . One mutation impairs Snf-Swi assembly and, consequently, its stable association with a target promoter . The other blocks a postrecruitment catalytic remodeling step . These findings suggest that Snf5p coordinates the assembly and nucleosome-remodeling activities of Snf-Swi. Mol Cell Biol, 2001 Jul, 21(13), 4276 - 91 In vivo action of the HRD ubiquitin ligase complex: mechanisms of endoplasmic reticulum quality control and sterol regulation; Gardner RG et al.; Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction . Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate . Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase . In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates . In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins . Using an in vivo cross-linking assay, we directly examined the association of the separate HRD complex components with various ERAD substrates . We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates . Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent "misfolded" state and a stable, tightly folded state . Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate. Mol Cell Biol, 2001 Jul, 21(13), 4246 - 55 Evidence of p53-dependent cross-talk between ribosome biogenesis and the cell cycle: effects of nucleolar protein Bop1 on G(1)/S transition; Pestov DG et al.; Bop1 is a novel nucleolar protein involved in rRNA processing and ribosome assembly . We have previously shown that expression of Bop1Delta, an amino-terminally truncated Bop1 that acts as a dominant negative mutant in mouse cells, results in inhibition of 28S and 5.8S rRNA formation and deficiency of newly synthesized 60S ribosomal subunits (Z . Strezoska, D . G . Pestov, and L . F . Lau, Mol . Cell . Biol . 20:5516-5528, 2000) . Perturbation of Bop1 activities by Bop1Delta also induces a powerful yet reversible cell cycle arrest in 3T3 fibroblasts . In the present study, we show that asynchronously growing cells are arrested by Bop1Delta in a highly concerted fashion in the G(1) phase . Kinase activities of the G(1)-specific Cdk2 and Cdk4 complexes were downregulated in cells expressing Bop1Delta, whereas levels of the Cdk inhibitors p21 and p27 were concomitantly increased . The cells also displayed lack of hyperphosphorylation of retinoblastoma protein (pRb) and decreased expression of cyclin A, indicating their inability to progress through the restriction point . Inactivation of functional p53 abrogated this Bop1Delta-induced cell cycle arrest but did not restore normal rRNA processing . These findings show that deficiencies in ribosome synthesis can be uncoupled from cell cycle arrest and reveal a new role for the p53 pathway as a mediator of the signaling link between ribosome biogenesis and the cell cycle . We propose that aberrant rRNA processing and/or ribosome biogenesis may cause "nucleolar stress," leading to cell cycle arrest in a p53-dependent manner. Mol Cell Biol, 2001 Jul, 21(13), 4233 - 45 New function of CDC13 in positive telomere length regulation; Meier B et al.; Two roles for the Saccharomyces cerevisiae Cdc13 protein at the telomere have previously been characterized: it recruits telomerase to the telomere and protects chromosome ends from degradation . In a synthetic lethality screen with YKU70, the 70-kDa subunit of the telomere-associated Yku heterodimer, we identified a new mutation in CDC13, cdc13-4, that points toward an additional regulatory function of CDC13 . Although CDC13 is an essential telomerase component in vivo, no replicative senescence can be observed in cdc13-4 cells . Telomeres of cdc13-4 mutants shorten for about 150 generations until they reach a stable level . Thus, in cdc13-4 mutants, telomerase seems to be inhibited at normal telomere length but fully active at short telomeres . Furthermore, chromosome end structure remains protected in cdc13-4 mutants . Progressive telomere shortening to a steady-state level has also been described for mutants of the positive telomere length regulator TEL1 . Strikingly, cdc13-4/tel1Delta double mutants display shorter telomeres than either single mutant after 125 generations and a significant amplification of Y' elements after 225 generations . Therefore CDC13, TEL1, and the Yku heterodimer seem to represent distinct pathways in telomere length maintenance . Whereas several CDC13 mutants have been reported to display elongated telomeres indicating that Cdc13p functions in negative telomere length control, we report a new mutation leading to shortened and eventually stable telomeres . Therefore we discuss a key role of CDC13 not only in telomerase recruitment but also in regulating telomerase access, which might be modulated by protein-protein interactions acting as inhibitors or activators of telomerase activity. J Biol Chem, 2001 Aug 31, 276(35), 32597 - 605 Epub 2001 Jun 04. The binding interaction of HMG-1 with the TATA-binding protein/TATA complex; Das D et al.; High mobility protein-1 (HMG-1) has been shown to regulate transcription by RNA polymerase II . In the context that it acts as a transcriptional repressor, it binds to the TATA-binding protein (TBP) to form the HMG-1/TBP/TATA complex, which is proposed to inhibit the assembly of the preinitiation complex . By using electrophoretic mobility shift assays, we show that the acidic C-terminal domain of HMG-1 and the N terminus of human TBP are the domains that are essential for the formation of a stable HMG-1/TBP/TATA complex . HMG-1 binding increases the affinity of TBP for the TATA element by 20-fold, which is reflected in a significant stimulation of the rate of TBP binding, with little effect on the dissociation rate constant . In support of the binding target of HMG-1 being the N terminus of hTBP, the N-terminal polypeptide of human TBP competes with and inhibits HMG-1/TBP/TATA complex formation . Deletion of segments of the N terminus of human TBP was used to map the region(s) where HMG-1 binds . These findings indicate that interaction of HMG-1 with the Q-tract (amino acids 55-95) in hTBP is primarily responsible for stable complex formation . In addition, HMG-1 and the monoclonal antibody, 1C2, specific to the Q-tract, compete for the same site . Furthermore, calf thymus HMG-1 forms a stable complex with the TBP/TATA complex that contains TBP from either human or Drosophila but not yeast . This is again consistent with the importance of the Q-tract for this stable interaction and shows that the interaction extends over many species but does not include yeast TBP. J Biol Chem, 2001 Aug 10, 276(32), 29628 - 31 Epub 2001 Jun 04. A multiprotein complex that interacts with RNA polymerase II elongator; Li Y et al.; A three-subunit Hap complex that interacts with the RNA polymerase II Elongator was isolated from yeast . Deletions of genes for two Hap subunits, HAP1 and HAP3, confer pGKL killer-insensitive and weak Elongator phenotypes . Preferential interaction of the Hap complex with free rather than RNA polymerase II-associated Elongator suggests a role in the regulation of Elongator activity. J Biol Chem, 2001 Jul 27, 276(30), 28541 - 5 Epub 2001 Jun 04. Golgi-localizing, gamma-adaptin ear homology domain, ADP-ribosylation factor-binding (GGA) proteins interact with acidic dileucine sequences within the cytoplasmic domains of sorting receptors through their Vps27p/Hrs/STAM (VHS) domains; Takatsu H et al.; GGA (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) proteins are potential effectors of ADP-ribosylation factors, are associated with the trans-Golgi network (TGN), and are involved in protein transport from this compartment . By yeast two-hybrid screening and subsequent two-hybrid and pull-down analyses, we have shown that GGA proteins, through their VHS (Vps27p/Hrs/STAM) domains, interact with acidic dileucine sequences found in the cytoplasmic domains of TGN-localized sorting receptors such as sortilin and mannose 6-phosphate receptor . A mutational analysis has revealed that a leucine pair and a cluster of acidic residues adjacent to the pair are mainly responsible for the interaction . A chimeric receptor with the sortilin cytoplasmic domain localizes to the TGN, whereas the chimeric receptor with a mutation at the leucine pair or the acidic cluster is mislocalized to punctate structures reminiscent of early endosomes . These results indicate that GGA proteins regulate the localization to or exit from the TGN of the sorting receptors. Genes Dev, 2001 Jun 1, 15(11), 1406 - 18 Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase; Gottlob K et al.; The serine/threonine kinase Akt/PKB is a major downstream effector of growth factor-mediated cell survival . Activated Akt, like Bcl-2 and Bcl-xL, prevents closure of a PT pore component, the voltage-dependent anion channel (VDAC); intracellular acidification; mitochondrial hyperpolarization; and the decline in oxidative phosphorylation that precedes cytochrome c release . However, unlike Bcl-2 and Bcl-xL, the ability of activated Akt to preserve mitochondrial integrity, and thereby inhibit apoptosis, requires glucose availability and is coupled to its metabolism . Hexokinases are known to bind to VDAC and directly couple intramitochondrial ATP synthesis to glucose metabolism . We provide evidence that such coupling serves as a downstream effector function for Akt . First, Akt increases mitochondria-associated hexokinase activity . Second, the antiapoptotic activity of Akt requires only the first committed step of glucose metabolism catalyzed by hexokinase . Finally, ectopic hexokinase expression mimics the ability of Akt to inhibit cytochrome c release and apoptosis . We therefore propose that Akt increases coupling of glucose metabolism to oxidative phosphorylation and regulates PT pore opening via the promotion of hexokinase-VDAC interaction at the outer mitochondrial membrane. Genes Dev, 2001 Jun 1, 15(11), 1361 - 72 Pds1 phosphorylation in response to DNA damage is essential for its DNA damage checkpoint function; Wang H et al.; In Saccharomyces cerevisiae, Pds1 is an anaphase inhibitor and plays an essential role in DNA damage and spindle checkpoint pathways . Pds1 is phosphorylated in response to DNA damage but not spindle disruption, indicating distinct mechanisms delaying anaphase entry . Phosphorylation of Pds1 is Mec1 and Chk1 dependent in vivo . Here, we show that Pds1 is phosphorylated at multiple sites in vivo in response to DNA damage by Chk1 . Mutation of the Chk1 phosphorylation sites on Pds1 abolished most of its DNA damage-inducible phosphorylation and its checkpoint function, whereas its anaphase inhibitor functions and spindle checkpoint functions remain intact . Loss of Pds1 phosphorylation correlates with APC-dependent Pds1 destruction in response to DNA damage . We also show that APC(Cdc20) is active in preanaphase arrested cells after DNA damage . This suggests that Pds1 is stabilized by phosphorylation in response to DNA damage, but APC(Cdc20) activity is not altered . Our results indicate that phosphorylation of Pds1 by Chk1 is the key function of Chk1 required to prevent anaphase entry. FEBS Lett, 2001 Jun 1, 498(1), 110 - 5 The predicted beta12-beta13 loop is important for inhibition of PP2Acalpha by the antitumor drug fostriecin; Evans DR et al.; The potential anticancer agent fostriecin (FOS) is a potent inhibitor of the protein Ser/Thr phosphatases PP2A and PP4 and a weaker inhibitor of PP1 . Random mutagenesis and automated screening in yeast identified residues in human PP2Acalpha important for inhibitory FOS binding . A C269S substitution in the predicted beta12-beta13 loop decreased the FOS sensitivity of intact cells and increased the IC(50) of PP2Acalpha by 10-fold in vitro . Changing PP2Acalpha Cys-269 to phenylalanine, the equivalent residue in PP1, and the Y267G and G270D substitutions caused a similar effect . The results provide information relevant to the design of novel protein Ser/Thr phosphatase inhibitory drugs. Mol Cell, 2001 May, 7(5), 1013 - 23 Evolutionarily conserved interaction between CstF-64 and PC4 links transcription, polyadenylation, and termination; Calvo O et al.; Tight connections exist between transcription and subsequent processing of mRNA precursors, and interactions between the transcription and polyadenylation machineries seem especially extensive . Using a yeast two-hybrid screen to identify factors that interact with the polyadenylation factor CstF-64, we uncovered an interaction with the transcriptional coactivator PC4 . Both human proteins have yeast homologs, Rna15p and Sub1p, respectively, and we show that these two proteins also interact . Given evidence that certain polyadenylation factors, including Rna15p, are necessary for termination in yeast, we show that deletion or overexpression of SUB1 suppresses or enhances, respectively, both growth and termination defects detected in an rna15 mutant strain . Our findings provide an additional, unexpected connection between transcription and polyadenylation and suggest that PC4/Sub1p, via its interaction with CstF-64/Rna15p, possesses an evolutionarily conserved antitermination activity. Mol Cell, 2001 May, 7(5), 981 - 91 The 19S regulatory particle of the proteasome is required for efficient transcription elongation by RNA polymerase II; Ferdous A et al.; It is generally thought that the primary or even sole activity of the 19S regulatory particle of the 26S proteasome is to facilitate the degradation of polyubiquitinated proteins by the 20S-core subunit . However, we present evidence that the 19S complex is required for efficient elongation of RNA polymerase II (RNAP II) in vitro and in vivo . First, yeast strains carrying alleles of SUG1 and SUG2, encoding 19S components, exhibit phenotypes indicative of elongation defects . Second, in vitro transcription is inhibited by antibodies raised against Sug1, or by heat-inactivating temperature-sensitive Sug1 mutants with restoration of elongation by addition of immunopurified 19S complex . Finally, Cdc68, a known elongation factor, coimmunoprecipitates with the 19S complex, indicating a physical interaction . Inhibition of the 20S proteolytic core of the proteasome has no effect on elongation . This work defines a nonproteolytic role for the 19S complex in RNAP II transcription. Cell, 2001 Jun 1, 105(5), 637 - 44 Immunity to K1 killer toxin: internal TOK1 blockade; Sesti F et al.; K1 killer strains of Saccharomyces cerevisiae harbor RNA viruses that mediate secretion of K1, a protein toxin that kills virus-free cells . Recently, external K1 toxin was shown to directly activate TOK1 channels in the plasma membranes of sensitive yeast cells, leading to excess potassium flux and cell death . Here, a mechanism by which killer cells resist their own toxin is shown: internal toxin inhibits TOK1 channels and suppresses activation by external toxin. Plant J, 2001 Apr, 26(2), 181 - 9 Arabidopsis IRT2 gene encodes a root-periphery iron transporter; Vert G et al.; Iron uptake from the soil is a tightly controlled process in plant roots, involving specialized transporters . One such transporter, IRT1, was identified in Arabidopsis thaliana and shown to function as a broad-range metal ion transporter in yeast . Here we report the cloning and characterization of the IRT2 cDNA, a member of the ZIP family of metal transporters, highly similar to IRT1 at the amino-acid level . IRT2 expression in yeast suppresses the growth defect of iron and zinc transport yeast mutants and enhances iron uptake and accumulation . However, unlike IRT1, IRT2 does not transport manganese or cadmium in yeast . IRT2 expression is detected only in roots of A . thaliana plants, and is upregulated by iron deficiency . By fusing the IRT2 promoter to the uidA reporter gene, we show that the IRT2 promoter is mainly active in the external cell layers of the root subapical zone, and therefore provide the first tissue localization of a plant metal transporter . Altogether, these data support a role for the IRT2 transporter in iron and zinc uptake from the soil in response to iron-limited conditions. Mol Reprod Dev, 2001 Jun, 59(2), 144 - 58 Expression of genes involved in mammalian meiosis during the transition from egg to embryo; Hwang SY et al.; The ooplasm of higher eukaryotes provides substances necessary for completing the last stages of meiosis and initiating the first mitotic division . These processes are firmly attuned to other events in the egg and newly formed embryo, such as switching from the use of maternal transcripts to the onset of zygotic transcription . In mammals little is known about the molecular mechanisms guiding this transition, largely due to the lack of information about genes expressed in the egg and early embryos . Studies of yeast mitosis have contributed much of what is known about the vertebrate cell cycle, and recent reports indicate that homologs of yeast DNA repair genes also function during mammalian gametogenesis . To examine whether this conservation can be expanded to include genes operative in oocyte meiosis, we performed a computer-based search for homologs of yeast genes that are induced during sporulation in C . elegans, Drosophila, and mammals . Results from this study suggest that yeast and higher eukaryotes share genes that coordinate the overall process of meiosis . However intriguing differences exist, reflecting the distinctive mechanisms governing the progression of meiosis in each organism . ESTs representing more than half of the mammalian homologs are present in mouse cDNA libraries that contains genes controlling the meiosis/mitosis transition . About 50% of these genes contain potential cis-elements for cytoplasmic polyadenylation in their 3'-UTR, suggesting the importance of controlled translation in the egg and zygote. Nat Cell Biol, 2001 Jun, 3(6), 531 - 7 Dynamics of the COPII coat with GTP and stable analogues; Antonny B et al.; We have developed an assay to monitor the assembly of the COPII coat onto liposomes in real time . We show that with Sar1pGTP bound to liposomes, a single round of assembly and disassembly of the COPII coat lasts a few seconds . The two large COPII complexes Sec23/24p and Sec13/31p bind almost instantaneously (in less than 1 s) to Sar1pGTP-doped liposomes . This binding is followed by a fast (less than 10 s) disassembly due to a 10-fold acceleration of the GTPase-activating protein activity of Sec23/24p by the Sec13/31p complex . Experiments with the phosphate analogue BeFx suggest that Sec23/24p provides residues directly involved in GTP hydrolysis on Sar1p. Blood, 2001 Jun 15, 97(12), 3763 - 7 Expression of the neuronal cyclin-dependent kinase 5 activator p35Nck5a in human monocytic cells is associated with differentiation; Chen F et al.; Although cyclin-dependent kinase 5 (Cdk5) is widely expressed in human tissues, its activator p35Nck5a is generally considered to be neuron specific . In addition to neuronal cells, active Cdk5 complexes have been reported in developing tissues, such as the embryonic muscle and ocular lens, and in human leukemia HL60 cells induced to differentiate by an exposure to 1,25-dihydroxyvitamin D(3); however, its activator in these cells has not been demonstrated . The results of this study indicate that p35Nck5a is associated with Cdk5 in monocytic differentiation of hematopoietic cells . Specifically, p35Nck5a is expressed in normal human monocytes and in leukemic cells induced to differentiate toward the monocytic lineage, but not in lymphocytes or cells induced to granulocytic differentiation by retinoic acid . It is present in a complex with Cdk5 that has protein kinase activity, and when ectopically expressed together with Cdk5 in undifferentiated HL60 cells, it induces the expression of CD14 and "nonspecific" esterase, markers of monocytic phenotype . These observations not only indicate a functional relationship between Cdk5 and p35Nck5a, but also support a role for this complex in monocytic differentiation . (Blood . 2001;97:3763-3767) Protein Expr Purif, 2001 Jun, 22(1), 1 - 10 Immunoaffinity purification and reconstitution of human alpha(2)-adrenergic receptor subtype C2 into phospholipid vesicles; Liitti S et al.; Large quantities of correctly folded, pure alpha(2)-adrenergic receptor protein are needed for structural analysis . We report here the first efficient method to purify human alpha(2)-adrenergic receptor subtype C2 to homogeneity from recombinant yeast Saccharomyces cerevisiae by one-step purification using a monoclonal antibody column (specific for alpha(2)C2) . We show that the adrenoceptor antagonist phentolamine stabilized the receptor during purification . We used a very effective chaotropic agent, NaSCN, to elute the receptor from the immunoaffinity column with an overall yield of 34% before reconstitution . Ligand binding of detergent-solubilized, immunoaffinity-purified receptors could not be demonstrated, but partial recovery of ligand binding activity was achieved when purified receptors were reconstituted into phospholipid vesicles . The reconstituted receptors still bound radioligand after storage on ice for 4 weeks . This purification procedure can be easily scaled-up and thus demonstrates the utility of a monoclonal antibody column and NaSCN elution to purify large quantities of G-protein-coupled receptors . Stroke, 2001 Jun, 32(6), 1401 - 7 Early decrease in dna repair proteins, Ku70 and Ku86, and subsequent DNA fragmentation after transient focal cerebral ischemia in mice; Kim GW et al.; BACKGROUND AND PURPOSE: Ku70 and Ku86, multifunctional DNA repair proteins, bind to broken DNA ends, including double-strand breaks, and trigger a DNA repair pathway . To investigate the involvement of these proteins in DNA fragmentation after ischemia/reperfusion, Ku protein expression was examined before and after transient focal cerebral ischemia (FCI) in mice . METHODS: Adult male CD-1 mice were subjected to 60 minutes of FCI by intraluminal suture blockade of the middle cerebral artery . Ku protein expression was studied by immunohistochemistry and Western blot analysis . DNA fragmentation was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) . The spatial relationship between Ku expression and DNA fragmentation was examined by double labeling with Ku and TUNEL after reperfusion . RESULTS: Immunohistochemistry showed constitutive expression of Ku proteins in control brains . The number of Ku-expressing cells was decreased in the entire middle cerebral artery territory as early as 4 hours after reperfusion and remained reduced until 24 hours . Western blot analyses confirmed the significant reduction of these proteins (59.4% and 57.7% reduction in optical density at 4 hours of reperfusion from the normal level of Ku70 and Ku86 bands, respectively; P<0.001) . DNA gel electrophoresis demonstrated DNA laddering 24 hours after reperfusion, but not at 4 hours . Double staining with Ku and TUNEL showed a concomitant loss of Ku immunoreactivity and TUNEL-positive staining . CONCLUSIONS: These results suggest that the early reduction of Ku proteins and the loss of defense against DNA damage may underlie the mechanism of DNA fragmentation after FCI. J Biol Chem, 2001 Aug 10, 276(32), 29782 - 91 Epub 2001 May 31. Marked stepwise differences within a common kinetic mechanism characterize TATA-binding protein interactions with two consensus promoters; Powell RM et al.; Binding of the TATA-binding protein (TBP) to promoter DNA bearing the TATA sequence is an obligatory initial step in RNA polymerase II transcription initiation . The interactions of Saccharomyces cerevisiae TBP with the E4 (TATATATA) and adenovirus major late (TATAAAAG) promoters have been modeled via global analysis of kinetic and thermodynamic data obtained using fluorescence resonance energy transfer . A linear two-intermediate kinetic mechanism describes the reaction of both of these consensus strong promoters with TBP . Qualitative features common to both interactions include tightly bound TBP-DNA complexes with similar solution geometries, simultaneous DNA binding and bending, and the presence of intermediate TBP-DNA conformers at high mole fraction throughout most of the reaction and at equilibrium . Despite very similar energetic changes overall, the stepwise entropic and enthalpic compensations along the two pathways differ markedly following the initial binding/bending event . Furthermore, TBP-E4 dissociation ensues from both replacement and displacement processes, in contrast to replacement alone for TBP-adenovirus major late promoter . A model is proposed that explicitly correlates these similarities and differences with the sequence-specific structural properties inherent to each promoter . This detailed mechanistic comparison of two strong promoters interacting with TBP provides a foundation for subsequent comparison between consensus and variant promoter sequences reacting with TBP. J Biol Chem, 2001 Aug 3, 276(31), 29268 - 74 Epub 2001 May 31. Proteomic analysis of nucleoporin interacting proteins; Allen NP et al.; The Saccharomyces cerevisiae nuclear pore complex is a supramolecular assembly of 30 nucleoporins that cooperatively facilitate nucleocytoplasmic transport . Thirteen nucleoporins that contain FG peptide repeats (FG Nups) are proposed to function as stepping stones in karyopherin-mediated transport pathways . Here, protein interactions that occur at individual FG Nups were sampled using immobilized nucleoporins and yeast extracts . We find that many proteins bind to FG Nups in highly reproducible patterns . Among 135 proteins identified by mass spectrometry, most were karyopherins and nucleoporins . The PSFG nucleoporin Nup42p and the GLFG nucleoporins Nup49p, Nup57p, Nup100p, and Nup116p exhibited generic interactions with karyopherins; each bound 6--10 different karyopherin betas, including importins as well as exportins . Unexpectedly, the same Nups also captured the hexameric Nup84p complex and Nup2p . In contrast, the FXFG nucleoporins Nup1p, Nup2p, and Nup60p were more selective and captured mostly the Kap95p.Kap60p heterodimer . When the concentration of Gsp1p-GTP was elevated in the extracts to mimic the nucleoplasmic environment, the patterns of interacting proteins changed; exportins exhibited enhanced binding to FG Nups, and importins exhibited reduced binding . The results demonstrate a global role for Gsp1p-GTP on karyopherin-nucleoporin interactions and provide a rudimentary map of the routes that karyopherins take as they cross the nuclear pore complex. EMBO J, 2001 Jun 1, 20(11), 2954 - 65 A subcomplex of three eIF3 subunits binds eIF1 and eIF5 and stimulates ribosome binding of mRNA and tRNA(i)Met; Phan L et al.; Yeast translation initiation factor 3 contains five core subunits (known as TIF32, PRT1, NIP1, TIF34 and TIF35) and a less tightly associated component known as HCR1 . We found that a stable subcomplex of His8-PRT1, NIP1 and TIF32 (PN2 subcomplex) could be affinity purified from a strain overexpressing these eIF3 subunits . eIF5, eIF1 and HCR1 co-purified with this subcomplex, but not with distinct His8-PRT1- TIF34-TIF35 (P45) or His8-PRT1-TIF32 (P2) sub complexes . His8-PRT1 and NIP1 did not form a stable binary subcomplex . These results provide in vivo evidence that TIF32 bridges PRT1 and NIP1, and that eIFs 1 and 5 bind to NIP1, in native eIF3 . Heat-treated prt1-1 extracts are defective for Met-tRNA(i)Met binding to 40S subunits, and we also observed defective 40S binding of mRNA, eIFs 1 and 5 and eIF3 itself in these extracts . We could rescue 40S binding of Met- tRNA(i)Met and mRNA, and translation of luciferase mRNA, in a prt1-1 extract almost as well with purified PN2 subcomplex as with five-subunit eIF3, whereas the P45 subcomplex was nearly inactive . Thus, several key functions of eIF3 can be carried out by the PRT1-TIF32-NIP1 subcomplex. EMBO J, 2001 Jun 1, 20(11), 2907 - 13 Engineered interphase chromosome loops guide intrachromosomal recombination; Kostriken R et al.; How large-scale topologies regulate interphase chromosome function remains an important question in eukaryotic cell biology . Looped structures are thought to modulate transcription by pairing promoters with distant control elements and to orchestrate intrachromosomal recombination events by pairing appropriate recombination partners . To explore the effects of chromosomal topology on intrachromosomal recombination, distinct loop geometries were engineered into chromosome III of the budding yeast Saccharomyces cerevisiae . These topologies were created by employing pairs of lac operator clusters to serve as pairing sites and a modified lac repressor to perform the role of a protein cross-bridge . The influence of these engineered loops on the selection of donor loci during mating-type switching was evaluated using novel genetic and molecular methods . These experiments demonstrate that engineered interphase chromosome loops are biologically active-capable of influencing the course of intrachromosomal recombination . They also provide insight into the mechanism of mating-type switching by revealing a causal relationship between defined chromosomal topologies and the choice of donor locus. EMBO J, 2001 Jun 1, 20(11), 2896 - 906 Silent repair accounts for cell cycle specificity in the signaling of oxidative DNA lesions; Leroy C et al.; Reactive oxygen species are the most important source of DNA lesions in aerobic organisms, but little is known about the activation of the DNA checkpoints in response to oxidative stress . We show that treatment of yeast cells with sublethal concentrations of hydrogen peroxide induces a Mec1-dependent phosphorylation of Rad53 and a Rad53-dependent cell cycle delay specifically during S phase . The lack of Rad53 phosphorylation after hydrogen peroxide treatment in the G1 and G2 phases is due to the silent repair of oxidative DNA lesions produced at these stages by the base excision repair (BER) pathway . Only the disruption of the BER pathway and the accumulation and/or treatment of DNA intermediates by alternative repair pathways reveal the existence of primary DNA lesions induced at all phases of the cell cycle by hydrogen peroxide . Our data illustrate both the concept of silent repair of DNA damage and the high sensitivity of S-phase cells to hydrogen peroxide. EMBO J, 2001 Jun 1, 20(11), 2823 - 34 The RNA polymerase III transcription initiation factor TFIIIB participates in two steps of promoter opening; Kassavetis GA et al.; Evidence for post-recruitment functions of yeast transcription factor (TF)IIIB in initiation of transcription was first provided by the properties of TFIIIB-RNA polymerase III-promoter complexes assembled with deletion mutants of its Brf and B" subunits that are transcriptionally inactive because they fail to open the promoter . The experiments presented here show that these defects can be repaired by unpairing short (3 or 5 bp) DNA segments spanning the transcription bubble of the open promoter complex . Analysis of this suppression phenomenon indicates that TFIIIB participates in two steps of promoter opening by RNA polymerase III that are comparable to the successive steps of promoter opening by bacterial RNA polymerase holoenzyme . B" deletions between amino acids 355 and 421 interfere with the initiating step of DNA strand separation at the upstream end of the transcription bubble . Removing an N-terminal domain of Brf interferes with downstream propagation of the transcription bubble to and beyond the transcriptional start site. EMBO J, 2001 Jun 1, 20(11), 2742 - 56 SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase; Farras R et al.; Arabidopsis Snf1-related protein kinases (SnRKs) are implicated in pleiotropic regulation of metabolic, hormonal and stress responses through their interaction with the kinase inhibitor PRL1 WD-protein . Here we show that SKP1/ASK1, a conserved SCF (Skp1-cullin-F-box) ubiquitin ligase subunit, which suppresses the skp1-4 mitotic defect in yeast, interacts with the PRL1-binding C-terminal domains of SnRKs . The same SnRK domains recruit an SKP1/ASK1-binding proteasomal protein, alpha4/PAD1, which enhances the formation of a trimeric SnRK complex with SKP1/ASK1 in vitro . By contrast, PRL1 reduces the interaction of SKP1/ASK1 with SnRKs . SKP1/ASK1 is co-immunoprecipitated with a cullin SCF subunit (AtCUL1) and an SnRK kinase, but not with PRL1 from Arabidopsis cell extracts . SKP1/ASK1, cullin and proteasomal alpha-subunits show nuclear co-localization in differentiated Arabidopsis cells, and are observed in association with mitotic spindles and phragmoplasts during cell division . Detection of SnRK in purified 26S proteasomes and co-purification of epitope- tagged SKP1/ASK1 with SnRK, cullin and proteasomal alpha-subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and alpha4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis. EMBO J, 2001 Jun 1, 20(11), 2702 - 14 The death substrate Gas2 binds m-calpain and increases susceptibility to p53-dependent apoptosis; Benetti R et al.; Gas2 is a caspase-3 substrate that plays a role in regulating microfilament and cell shape changes during apoptosis . Here we provide evidence that overexpression of Gas2 efficiently increases cell susceptibility to apoptosis following UV irradiation, etoposide and methyl methanesulfonate treatments, and that these effects are dependent on increased p53 stability and transcription activity . To investigate possible pathways linking Gas2 to p53, a yeast two-hybrid screen swas performed, indicating m-calpain as a strong Gas2- interacting protein . Moreover, we demonstrate that Gas2 physically interacts with m-calpain in vivo and that recombinant Gas2 inhibits calpain-dependent processing of p53 . Importantly, the Gas2 dominant-negative form (Gas2171-314) that binds calpain but is unable to inhibit its activity abrogates Gas2's ability to stabilize p53, to enhance p53 transcriptional activity and to induce p53-dependent apoptosis . Finally, we show that Gas2 is able to regulate the levels of p53 independently of Mdm2 status, suggesting that, like calpastatin, it may enhance p53 stability by inhibiting calpain activity. Mol Genet Metab, 2001 Jun, 73(2), 138 - 48 A novel inborn error in the ligand-binding domain of the vitamin D receptor causes hereditary vitamin D-resistant rickets; Malloy PJ et al.; Mutations in the vitamin D receptor (VDR) cause hereditary vitamin D-resistant rickets (HVDRR), an autosomal recessive disease resulting in target organ resistance to 1,25-dihydroxyvitamin D(3) {1,25(OH)(2)D(3)} . In this report, we describe the clinical case and molecular basis of HVDRR in an Asian boy exhibiting the typical clinical features of the disease including alopecia . Using cultured dermal fibroblasts from the patient, 1,25(OH)(2)D(3) resistance was demonstrated by a shift in the dose response required for 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) mRNA induction . Western blot showed that the cells express a normal size VDR but contained reduced levels of receptor compared to normal cells . At 24 degrees C, the affinity of the patient's VDR for {(3)H}1,25(OH)(2)D(3) was 50-fold lower than the VDR in normal fibroblasts . Sequence analysis identified a unique T to G missense mutation in exon 6 that changed phenylalanine to cysteine at amino acid 251 (F251C) . The recreated F251C mutant VDR showed reduced transactivation activity using a 24-hydroxylase promoter-luciferase reporter . Maximal transactivation activity exhibited by the WT VDR was not achieved by the mutant VDR even when the cells were treated with up to 10(-6) M 1,25(OH)(2)D(3) . However, the transactivation activity was partially rescued by addition of RXRalpha . In the yeast two-hybrid system and GST-pull-down assays, high concentrations of 1,25(OH)(2)D(3) were needed to promote F251C mutant VDR binding to RXRalpha, indicating defective heterodimerization . In conclusion, a novel mutation was identified in the VDR LBD that reduces VDR abundance and its affinity for 1,25(OH)(2)D(3) and interferes with RXRalpha heterodimerization resulting in the syndrome of HVDRR . J Biol Chem, 2001 Jul 27, 276(30), 28185 - 9 Epub 2001 May 30. Interaction between the noncatalytic region of Sid1p kinase and Cdc14p is required for full catalytic activity and localization of Sid1p; Guertin DA et al.; Sid1p is a group II p21-activated kinase/germinal center kinase family member that is part of a signaling network required for cytokinesis in fission yeast . Germinal center kinases are characterized by well conserved amino-terminal catalytic domains followed by less conserved carboxyl termini . The carboxyl termini among group I germinal center kinases are moderately conserved and thought to be regulatory regions . Little is known about the carboxyl termini of group II family members . Sid1p has been shown to bind the novel protein Cdc14p; however, the functional significance of this interaction is unknown . Here we report that the carboxyl terminus of Sid1p is an essential regulatory region . Our results indicate that this region contains the binding domain for Cdc14p, and this association is required for full Sid1p catalytic activity as well as intracellular localization . Furthermore, overexpression of the carboxyl terminus of Sid1p alone compromises the signaling of cytokinesis . We conclude that Cdc14p positively regulates the Sid1p kinase by binding the noncatalytic carboxyl-terminal region of the protein. J Biol Chem, 2001 Jul 27, 276(30), 28179 - 84 Epub 2001 May 30. The histone acetyltransferase, hGCN5, interacts with and acetylates the HIV transactivator, Tat; Col E et al.; Factor acetyltransferase activity associated with several histone acetyltransferases plays a key role in the control of transcription . Here we report that hGCN5, a well known histone acetyltransferase, specifically interacts with and acetylates the human immunodeficiency virus type 1 (HIV-1) transactivator protein, Tat . The interaction between Tat and hGCN5 is direct and involves the acetyltransferase and the bromodomain regions of hGCN5, as well as a limited region of Tat encompassing the cysteine-rich domain of the protein . Tat lysines 50 and 51, target of acetylation by p300/CBP, were also found to be acetylated by hGCN5 . The acetylation of these two lysines by p300/CBP has been recently shown to stimulate Tat transcriptional activity and accordingly, we have found that hGCN5 can considerably enhance Tat-dependent transcription of the HIV-1 long terminal repeat . These data highlight the importance of the acetylation of lysines 50 and 51 in the function of Tat, since different histone acetyltransferases involved in distinct signaling pathways, GCN5 and p300/CBP, converge to acetylate Tat on the same site. J Biol Chem, 2001 Jul 27, 276(30), 28171 - 8 Epub 2001 May 30. Reconstitution of acid secretion in digitonin-permeabilized rabbit gastric glands . Identification of cytosolic regulatory factors; Akagi K et al.; When isolated rabbit gastric glands were permeabilized with digitonin, they lost their ability to secrete acid, as monitored by {14C}aminopyrine accumulation, and they never recovered by supplement with cytosol prepared from gastric mucosa . However, the permeabilized glands elicited acid secretion when brain cytosol was supplemented . Fractionation of gastric cytosol by gel filtration revealed that the fraction at 30 kDa stimulated permeabilized glands by itself, whereas the 200-kDa fraction potently inhibited brain cytosol-stimulated acid secretion . Brain cytosol contained only the former stimulatory factor . With further gel filtration, the 30-kDa activator was separated into two components, 20 kDa (peak 1) and 1.8 kDa (peak 2), both of which are necessary for full activity . We purified peak 1 from bovine brain, and phosphatidylinositol transfer protein (PITP) was identified as the main component of the activity . The stimulating activity in brain and gastric mucosa correlated with the contents of PITP, and recombinant PITP mimicked the effect of peak 1, suggesting that PITP is one of the essential components in gastric acid secretion . When gastric glands were stimulated, the inhibitory activity, but not stimulatory activity, in the cytosol was increased . This suggests a regulatory mechanism such as stimulation translocates the inhibitory component from the secretory site on the membrane to cytosol . These results demonstrate a high degree of usefulness for our present model, the reconstituted digitonin-permeabilized gastric glands. J Biol Chem, 2001 Aug 10, 276(32), 30452 - 60 Epub 2001 May 29. Apg2p functions in autophagosome formation on the perivacuolar structure; Shintani T et al.; Autophagy is a degradative process in which cytoplasmic components are non-selectively sequestered by double-membrane structures, termed autophagosomes, and transported to the vacuole . We have identified and characterized a novel protein Apg2p essential for autophagy in yeast . Biochemical and fluorescence microscopic analyses indicate that Apg2p functions at the step of autophagosome formation . Apg2p localizes to some membranous structure distinct from any known organelle . Using fluorescent protein-tagged Apg2p, we showed that Apg2p localizes to a dot structure close to the vacuole, where Apg8p also exists, but not on autophagosomes unlike Apg8p . This punctate localization of Apg2p depends on the function of Apg1p kinase, phosphatidylinositol 3-kinase complex and Apg9p . Apg2p(G83E), encoded by an apg2-2 allele, shows a severely reduced activity of autophagy and a dispersed localization in the cytoplasm . Overexpression of the mutant Apg2p lessens the defect in autophagy . These results suggest that the dot structure is physiologically important . Apg2p and Apg8p are independently recruited to the structure but coordinately function there to form the autophagosome. J Biol Chem, 2001 Aug 3, 276(31), 29393 - 402 Epub 2001 May 29. Molecular characterization of mammalian homologues of class C Vps proteins that interact with syntaxin-7; Kim BY et al.; Vesicle-mediated protein sorting plays an important role in segregation of intracellular molecules into distinct organelles . Extensive genetic studies using yeast have identified more than 40 vacuolar protein sorting (VPS) genes involved in vesicle transport to vacuoles . However, their mammalian counterparts are not fully elucidated . In this study, we identified two human homologues of yeast Class C VPS genes, human VPS11 (hVPS11) and human VPS18 (hVPS18) . We also characterized the subcellular localization and interactions of the protein products not only from these genes but also from the other mammalian Class C VPS homologue genes, hVPS16 and rVPS33a . The protein products of hVPS11 (hVps11) and hVPS18 (hVps18) were ubiquitously expressed in peripheral tissues, suggesting that they have a fundamental role in cellular function . Indirect immunofluorescence microscopy revealed that the mammalian Class C Vps proteins are predominantly associated with late endosomes/lysosomes . Immunoprecipitation and gel filtration studies showed that the mammalian Class C Vps proteins constitute a large hetero-oligomeric complex that interacts with syntaxin-7 . These results indicate that like their yeast counterparts, mammalian Class C Vps proteins mediate vesicle trafficking steps in the endosome/lysosome pathway. J Mol Graph Model, 2001, 19(1), 3 - 12 NMR spin relaxation methods for characterization of disorder and folding in proteins; Bracken C; The flexibility and dynamics of proteins directly influence the processes of protein folding, recognition, and function . NMR spin relaxation methods are used to assess the dynamics and mobility of proteins, for fast ps and ns motions as well as slower microsecond and ms events . The degree of protein flexibility and disorder as well as the changes in protein flexibility can be assessed by NMR spin relaxation methods at individual residues within the protein . In addition to probing protein dynamics, the changes in the NMR-derived order parameters can be used to estimate the entropic contributions of order-disorder transitions . Furthermore, kinetic processes in the ms time regime may be directly investigated to extract the rates of conformational interconversion, ligand binding, and protein folding processes . We show how a variety of dynamical information can be obtained from NMR relaxation measurements . We present examples that illustrate the use of NMR spin relaxation analysis for investigation of folding and disorder in proteins. Nat Genet, 2001 Jun, 28(2), 119 - 20 A conserved sorting-associated protein is mutant in chorea-acanthocytosis; Rampoldi L et al.; Chorea-acanthocytosis (CHAC, MIM 200150) is an autosomal recessive neurodegenerative disorder characterized by the gradual onset of hyperkinetic movements and abnormal erythrocyte morphology (acanthocytosis) . Neurological findings closely resemble those observed in Huntington disease . We identified a gene in the CHAC critical region and found 16 different mutations in individuals with chorea-acanthocytosis . CHAC encodes an evolutionarily conserved protein that is probably involved in protein sorting. Mol Biochem Parasitol, 2001 May, 114(2), 183 - 95 Two classes of plant-like vacuolar-type H(+)-pyrophosphatases in malaria parasites; McIntosh MT et al.; In plants, cytosolic inorganic pyrophosphate (PP(i)) is hydrolyzed by energy-conserving vacuolar-type H(+)-pyrophosphatases (V-PPases) that harness the free energy of PP(i) hydrolysis to establish transmembrane H(+) gradients . Here we describe the identification and cloning of two genes, PfVP1 and PfVP2, from the malaria parasite Plasmodium falciparum . Inferred to encode type I (K(+)-dependent) and type II (K(+)-independent) V-PPases, respectively, PfVP1 and PfVP2 appeared more sequence divergent from each other than from their type I and type II counterparts in plants . The steady state levels of PfVP1 mRNA were high in comparison to PfVP2 mRNA throughout the erythrocytic phases of infection . Western analyses of trophozoite membranes using generic V-PPase antibodies (PAB(HK) and PAB(TK)) demonstrated appreciable amounts of a Mr 67000 polypeptide whose associated aminomethylenediphosphonate- (AMDP) inhibitable PPase activity was markedly stimulated by K(+) . Immunofluorescence microscopy of infected erythrocytes revealed PfVP antigen associated with both the parasite plasma membrane and punctate intracellular inclusions . Transient transfection of a PfVP1-GFP fusion further supported the localization of PfVP1 to the parasite plasma membrane . Based on these findings and the growth-retarding effects of AMDP, P . falciparum is concluded to possess both type I and type II V-PPases of which the former has the greatest potential for contributing to the establishment of H(+) gradients across the parasite plasma membrane under conditions of energy limitation. Mol Cell Endocrinol, 2001 May 25, 177(1-2), 13 - 8 Exiting the endoplasmic reticulum; Gorelick FS et al.; The movement of nascent proteins from sites of synthesis to final cellular or extracellular destinations involves their transport through a distinct series of vesicular compartments . Vesicle biogenesis is regulated by specific proteins and co-factors that control distinct steps including budding, transport, docking, and fusion with target membranes . Budding requires assembly of a coat protein complex on the membrane, membrane deformation and the subsequent cleavage of the nascent vesicle from donor membrane . Coat proteins may also mediate vesicle interactions with the cytoskeleton or insulate the vesicles from fusion with unwanted compartments . Three classes of cytoplasmic coats have been identified . (1) Clathrin, interacting with different adaptor proteins, participates in endocytosis, lysosome biogenesis and as yet unidentified vesicular transport processes that arise in the trans-Golgi region of cells {reviewed in (Kreis, T.E., Lowe, M., Pepperkok, R., 1995 . COPs regulating membrane traffic . Ann . Rev . Cell . Dev . Biol . 11, 677--706.)} . (2) The COPI coatomer is involved in retrograde traffic within the Golgi and from the cis-Golgi region to the endoplasmic reticulum (ER) . It may also participate in anterograde transport from the ER {reviewed in (Aridor, M., Balch, W.E., 1999 . Integration of endoplasmic reticulum signaling in health and disease . Nature 5, 745--751.)} . (3) COPII coats mediate anterograde transport of cargo out of the ER {Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M.F., Ravazazola, M., Amherdt, M., Schekman, R., 1994 . COPII: a membrane coat formed by sec proteins that drive vesicle budding from the endoplasmic reticulum . Cell 77, 895--907; Scales, S.J., Gomez, M., Kreis, T.E., 2000 . Coat proteins regulating membrane traffic . Int . Rev . Cytol . 195, 67--144.} . The COPII coat is required for budding from the ER and ER to Golgi trafficking . Further, COPII proteins also participate in cargo selection and concentrate some nascent proteins in the budding vesicle . Recent studies have shown that human disease may result from mutations that affect proteins in COPII vesicles. Adv Drug Deliv Rev, 2001 Jul 2, 49(1-2), 199 - 215 RNA delivery into mitochondria; Entelis NS et al.; Mitochondria, though containing their own genome, import the vast majority of their macromolecular components from the cytoplasm . If the mechanisms of pre-protein import are well understood, the import of nuclear-coded RNAs into mitochondria was investigated to a much lesser extent . This targeting, if not universal, is widely spread among species . The origin and the mechanisms of RNA import seem to differ from one system to another and striking differences are observed even in closely related species . We describe data concerning the various experimental systems of studying RNA import with emphasis on the model of the yeast Saccharomyces cerevisiae, which was studied in our laboratory . We compare various requirements of RNA import into mitochondria in different species and demonstrate that this pathway can be transferred from yeast to human cells, in which tRNAs normally are not imported . We speculate on the possibility to use RNA import for biomedical purposes. J Biomed Inform, 2001 Feb, 34(1), 15 - 27 Extracting knowledge from dynamics in gene expression; Reis BY et al.; Most investigations of coordinated gene expression have focused on identifying correlated expression patterns between genes by examining their normalized static expression levels . In this study, we focus on the dynamics of gene expression by seeking to identify correlated patterns of changes in genetic expression level . In doing so, we build upon methods developed in clinical informatics to detect temporal trends of laboratory and other clinical data . We construct relevance networks from Saccharomyces cerevisiae gene-expression dynamics data and find genes with related functional annotations grouped together . While some of these associations are also found using a standard expression level analysis, many are identified exclusively through the dynamic analysis . These results strongly suggest that the analysis of gene expression dynamics is a necessary and important tool for studying regulatory and other functional relationships among genes . The source code developed for this investigation is freely available to all non-commercial investigators by contacting the authors. Dev Dyn, 2001 Jun, 221(2), 231 - 7 Zebrafish E-cadherin: expression during early embryogenesis and regulation during brain development; Babb SG et al.; Zebrafish E-cadherin (cdh1) cell adhesion molecule cDNAs were cloned . We investigated spatial and temporal expression of cdh1 during early embryogenesis . Expression was observed in blastomeres, the anterior mesoderm during gastrulation, and developing epithelial structures . In the developing nervous system, cdh1 was detected at the pharyngula stage (24 hpf) in the midbrain-hindbrain boundary (MHB) . Developmental regulation of MHB formation involves wnt1 and pax2.1 . wnt1 expression preceded cdh1 expression during MHB formation, and cdh1 expression in the MHB was dependent on normal development of this structure . J Biol Chem, 2001 Jun 1, 276(22), 18992 - 8 Epub 2001 Mar 16. Protein phosphatases regulate DNA-dependent protein kinase activity; Douglas P et al.; DNA-dependent protein kinase (DNA-PK) is a complex of DNA-PK catalytic subunit (DNA-PKcs) and the DNA end-binding Ku70/Ku80 heterodimer . DNA-PK is required for DNA double strand break repair by the process of nonhomologous end joining . Nonhomologous end joining is a major mechanism for the repair of DNA double strand breaks in mammalian cells . As such, DNA-PK plays essential roles in the cellular response to ionizing radiation and in V(D)J recombination . In vitro, DNA-PK undergoes phosphorylation of all three protein subunits (DNA-PK catalytic subunit, Ku70 and Ku80) and phosphorylation correlates with inactivation of the serine/threonine protein kinase activity of DNA-PK . Here we show that phosphorylation-induced loss of the protein kinase activity of DNA-PK is restored by the addition of the purified catalytic subunit of either protein phosphatase 1 or protein phosphatase 2A (PP2A) and that this reactivation is blocked by the potent protein phosphatase inhibitor, microcystin . We also show that treating human lymphoblastoid cells with either okadaic acid or fostriecin, at PP2A-selective concentrations, causes a 50-60% decrease in DNA-PK protein kinase activity, although the protein phosphatase 1 activity in these cells was unaffected . In vivo phosphorylation of DNA-PKcs, Ku70, and Ku80 was observed when cells were labeled with {(32)P}inorganic phosphate in the presence of the protein phosphatase inhibitor, okadaic acid . Together, our data suggest that reversible protein phosphorylation is an important mechanism for the regulation of DNA-PK protein kinase activity and that the protein phosphatase responsible for reactivation in vivo is a PP2A-like enzyme. J Biol Chem, 2001 Aug 3, 276(31), 29299 - 306 Epub 2001 May 25. Lung Krüppel-like factor contains an autoinhibitory domain that regulates its transcriptional activation by binding WWP1, an E3 ubiquitin ligase; Conkright MD et al.; Lung Kruppel-like factor (LKLF/Kruppel-like factor 2), a member of the Kruppel-like factor family of transcription factors, is expressed predominantly in the lungs, with low levels of expression in other organs such as heart, spleen, skeletal muscle, and testis . LKLF is essential during pulmonary development and single-positive T-cell development and is indispensable during mouse embryogenesis . In this study, we performed a series of experiments to define the activation domain of LKLF as a means to further advance the understanding of the molecular mechanisms underlying transcriptional regulation by this transcription factor . Using deletion analysis, it is shown that LKLF contains a transcriptional activation domain as well as a strong autoinhibitory subdomain . The inhibitory subdomain is able to independently suppress transcriptional activation of other strong activators such as viral protein 16, VP16 . This occurs either when the inhibitory domain is fused directly to VP16 or when the inhibitory domain is independently bound to DNA by GAL4 DNA-binding domain independent of the VP16 activator . Overexpression of the LKLF autoinhibitory domain alone potentiates transactivation by wild type LKLF, suggesting that the inhibitory domain binds a cofactor that prevents LKLF from transactivating . A yeast-two hybrid screen identified WWP1, an E3 ubiquitin ligase that binds specifically to the LKLF inhibitory domain but not to other transcription factors . In mammalian cells, WWP1 functions as a cofactor by binding LKLF and suppressing transactivation . These data demonstrate that LKLF contains multiple domains that either potentiate or inhibit the ability of this factor to function as an activator of transcription; moreover, regulation of LKLF transactivation is attenuated by an E3 ubiquitin ligase, WWP1. J Biol Chem, 2001 Aug 3, 276(31), 28814 - 8 Epub 2001 May 25. Protein matrix and dielectric effect in cytochrome c; Blouin C et al.; The effect of the protein matrix on the standard potential of a buried redox center has been investigated by using a selection of mutants and chemical derivatives in Saccharomyces cerevisiae cytochrome c isoform 1 . Assuming only local structural perturbation and no alteration of the iron-ligation chemistry, Delta E(m)(0)' can be regarded as a measure of the difference in polypeptide solvation of the heme charge, which reflects the dielectric properties of the protein . The evaluation of an apparent dielectric constant (U(exp)/U(theo)) yields variable, and sometimes even negative, values if U(exp) = Delta G(0)redox . However, some consistent result are observed if U(exp) = Delta H(0)redox, with a measured epsilon(Delta Delta)(H)(redox) = 19 +/- 6 . The variability is thus attributed to an entropic factor (epsilon(Delta Delta)(S)(redox)) that is investigated using a series of substitutions of Asn(52) and/or Tyr(67) . In double mutants Y67F/N52I Y67F/N52V, where most of the hydrogen bond network in the heme crevice is eliminated, Delta S(redox) compares to the wild type . This indicates that a fully consistent hydrogen bond network has a similar polarizability as an apolar matrix . We therefore argue that the variability in net dielectric susceptibility arises from conformational polarizability, a factor that is not a function of atomic properties and coordinates and is therefore hard to predict using conventional physical relationships. J Biol Chem, 2001 Aug 31, 276(35), 32427 - 36 Epub 2001 May 25. The hemidesmosomal protein bullous pemphigoid antigen 1 and the integrin beta 4 subunit bind to ERBIN . Molecular cloning of multiple alternative splice variants of ERBIN and analysis of their tissue expression; Favre B et al.; The bullous pemphigoid antigen 1 (eBPAG1) is a constituent of hemidesmosomes (HDs), cell-substrate adhesion complexes in stratified epithelia . Although its COOH terminus interacts with intermediate filaments, its NH(2) terminus is important for its recruitment into HDs . To identify proteins that interact with the NH(2) terminus of human eBPAG1, we performed a yeast two-hybrid screen, which uncovered a protein belonging to the LAP/LERP (for LRR and PDZ domain) protein family with 16 NH(2)-terminal leucine-rich repeats and a COOH-terminal PDZ domain . The gene for this LAP/LERP protein comprises at least 26 exons located on the long arm of chromosome 5 . In most human tissues, several transcripts were detected differing in the coding region situated upstream of or within the PDZ domain . One of the encoded variants was found to correspond to the recently described protein ERBIN . In yeast and in vitro binding experiments, ERBIN was shown to interact not only with eBPAG1 but also with the COOH-terminal region of the cytoplasmic domain of the integrin beta4 subunit, another component of HDs . Antibodies raised against the COOH terminus showed that ERBIN is expressed in keratinocytes . In transfected epithelial cells the protein, however, was not localized in HDs but was either diffusely distributed over the cytoplasm or concentrated at the basolateral plasma membrane . Because ERBIN had been shown previously to interact with the transmembrane tyrosine kinase receptor Erb-B2, which in turn associates with the integrin beta4 subunit, we suggest that ERBIN provides a link between HD assembly and Erb-B2 receptor signaling. Acta Crystallogr D Biol Crystallogr, 2001 Jun, 57(Pt 6), 867 - 9 Epub 2001 May 25. Optimization of Met8p crystals through protein-storage buffer manipulation; Schubert HL et al.; Sirohaem, the prosthetic group of assimilatory sulfite and nitrite reductases, is a modified tetrapyrrole that belongs to the same fraternity of metallo-prosthetic groups as haem, chlorophyll, cobalamin and coenzyme F430 {Warren & Scott (1990), Trends Biochem Sci . 15, 486-491} . In Saccharomyces cerevisiae, the last step in the biosynthesis of sirohaem involves Met8p, a bifunctional enzyme responsible for both the NAD(+)-dependent dehydrogenation of the corrin ring and ferrochelation . Optimization of the protein storage buffer according to the results of crystallization trials resulted in a more monodisperse protein solution . Crystals were grown that diffracted to 2.1 A. J Biol Chem, 2001 Jul 27, 276(30), 27975 - 80 Epub 2001 May 24. Combinations of protein-disulfide isomerase domains show that there is little correlation between isomerase activity and wild-type growth; Xiao R et al.; Protein-disulfide isomerase (PDI) has five domains: a, b, b', a' and c, all of which except c have a thioredoxin fold . A single catalytic domain (a or a') is effective in catalyzing oxidation of a reduced protein but not isomerization of disulfides (Darby, N . J., and Creighton, T . E . (1995) Biochemistry 34, 11725-11735) . To examine the structural basis for this oxidase and isomerase activity of PDI, shuffled domain mutants were generated using a method that should be generally applicable to multidomain proteins . Domains a and a' along with constructs ab, aa', aba', ab'a' display low disulfide isomerase activity, but all show significant reactivity with mammalian thioredoxin reductase, suggesting that the structure is not seriously compromised . The only domain order that retains significant isomerase activity has the b' domain coupled to the N terminus of the a' domain . This b'a'c has 38% of the isomerase activity of wild-type PDI, equivalent to the activity of full-length PDI with one of the active sites inactivated by mutation (Walker, K . W., Lyles, M . M., and Gilbert, H . F . (1996) Biochemistry 35, 1972-1980) . Individual a and a' domains, despite their very low isomerase activities in vitro, support wild-type growth of a pdi1Delta Saccharomyces cerevisiae strain yeast . Thus, most of the PDI structure is dispensable for its essential function in yeast, and high-level isomerase activity appears not required for viability or rapid growth. Appl Environ Microbiol, 2001 Jun, 67(6), 2610 - 6 Cloning of a phenol oxidase gene from Acremonium murorum and its expression in Aspergillus awamori; Gouka RJ et al.; Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions . We isolated a phenol oxidase from the fungus Acremonium murorum var . murorum that was capable of decolorizing plant chromophores (such as anthocyanins) . This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores . We expressed an A . murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore . The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids . The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography . The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60 degrees C) and is fully stable for at least 1 h at 60 degrees C under alkaline conditions . These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning. J Nat Prod, 2001 May, 64(5), 640 - 2 Onnamide F: a new nematocide from a southern Australian marine sponge, Trachycladus laevispirulifer; Vuong D et al.; A southern Australian marine sponge, Trachycladus laevispirulifer, has yielded a potent new nematocide with antifungal activity which has been identified as onnamide F (1) . The structure for 1 was assigned by detailed spectroscopic analysis and chemical conversion to the methyl ester 2 . Onnamide F contains a common structural motif previously described in a number of natural products exhibiting interesting pharmacological activities, including the insect chemical defense agent pederin (3), and the sponge metabolites the onnamides, mycalamides, and theopederins. Biochem Biophys Res Commun, 2001 Jun 1, 284(1), 194 - 202 Ray38p, a homolog of a purine motif triple-helical DNA-binding protein, Stm1p, is a ribosome-associated protein and dissociated from ribosomes prior to the induction of cycloheximide resistance in Candida maltosa; Takaku H et al.; Cycloheximide (CYH) resistance in Candida maltosa is dependent on the induction of a ribosomal protein, Q-type L41, the 56th residue of which is glutamine, not proline as in ordinary P-type L41 . We found that a 38-kDa protein in a wild-type C . maltosa ribosomal fraction became undetectable upon CYH treatment but detectable again with the establishment of CYH resistance by the induction of Q-type L41 . We cloned a gene coding for this protein and named it RAY38 (ribosome-associated protein of yeast) . Ray38p is a homolog of a purine motif triple-helical DNA-binding protein, Stm1p, and has a putative RNA-binding motif RGG . The ribosome-associated Ray38p was phosphorylated at serine and threonine residues, and Ray38p that was dissociated from ribosome by CYH treatment was highly phosphorylated in threonine residues . A ray38 null mutant recovered faster from CYH-caused growth stasis than the wild-type strain, suggesting that the dissociation of Ray38p from ribosome facilitates the induction of CYH resistance in C . maltosa . Biochem Biophys Res Commun, 2001 Jun 1, 284(1), 133 - 41 Structure and expression of the Arabidopsis thaliana homeobox gene Athb-12; Lee YH et al.; We have isolated the Arabidopsis thaliana homeobox gene Athb-12, determined its structure and activation domain, demonstrated that its promoter is inducible in response to abscisic acid (ABA) treatment, and characterized the cellular distribution of its transcripts . The single intron of the gene interrupted the leucine-zipper domain region . The 5' regulatory region of Athb-12 can drive beta-glucuronidase (GUS) expression in tobacco transgenic plants . Athb-12 gene expression was further examined using in situ hybridization to determine the cellular distribution of Athb-12 transcripts during ABA induction . A complex pattern of Athb-12 expression was observed, often associated with regions of developing vascular tissues . Analysis of chimeras constructed from Athb-12 and the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that the activation domain of Athb-12 lies in the C-terminal region (amino acids 180 to 235) . Taken together, our data suggest that Athb-12 is a transcriptional activator important in regulating certain developmental processes as well as in the plant's response to water stress involving ABA-mediated gene expression . Nat Struct Biol, 2001 Jun, 8(6), 515 - 20 Visualizing induced fit in early assembly of the human signal recognition particle; Rose MA et al.; Assembly of almost all ribonucleoprotein complexes involves induced fit in the RNA and, thus, formation of one or more intermediate states . In assembly of the human signal recognition particle (SRP), we show that SRP19 binding to SRP RNA involves obligatory intermediates . An apparent discrepancy exists between the ratio of dissociation and association rate constants, determined in a partitioning experiment, and the equilibrium binding constant; this kinetic signature reflects formation of a stable intermediate in assembly of the ribonucleoprotein complex . Assembly intermediates were observed directly by time-resolved footprinting . SRP19 binds rapidly to SRP RNA to form an initial labile, but structurally specific, encounter complex involving both helices III and IV . Two subsequent steps of structural consolidation yield the native RNA-protein interface . SRP19 binding stabilizes helix IV in the region recognized by SRP54, consistent with protein-protein cooperativity mediated in part by mutual recognition of similar RNA structures . This mechanism illustrates principles general to ribonucleoprotein assembly reactions that rely on recruitment of architectural RNA binding proteins. J Biol Chem, 2001 Jul 27, 276(30), 28620 - 7 Epub 2001 May 23. The regulation of catalytic activity of the menkes copper-translocating P-type ATPase . Role of high affinity copper-binding sites; Voskoboinik I et al.; The Menkes protein is a transmembrane copper translocating P-type ATPase . Mutations in the Menkes gene that affect the function of the Menkes protein may cause Menkes disease in humans, which is associated with severe systemic copper deficiency . The catalytic mechanism of the Menkes protein, including the formation of transient acylphosphate, is poorly understood . We transfected and overexpressed wild-type and targeted mutant Menkes protein in yeast and investigated its transient acyl phosphorylation . We demonstrated that the Menkes protein is transiently phosphorylated by ATP in a copper-specific and copper-dependent manner and appears to undergo conformational changes in accordance with the classical P-type ATPase model . Our data suggest that the catalytic cycle of the Menkes protein begins with the binding of copper to high affinity binding sites in the transmembrane channel, followed by ATP binding and transient phosphorylation . We propose that putative copper-binding sites at the N-terminal domain of the Menkes protein are important as sensors of low concentrations of copper but are not essential for the overall catalytic activity. Reproduction, 2001 Jun, 121(6), 873 - 80 Extracellular domain of YWK-II, a human sperm transmembrane protein, interacts with rat Müllerian-inhibiting substance; Tian XY et al.; The YWK-II protein in human spermatozoa is structurally related to the betaA4-amyloid precursor protein of Alzheimer disease and has high similarity with amyloid precusor homologues . Antibodies to the YWK-II protein agglutinate human spermatozoa and may be a potential cause of infertility . In the present study, a yeast two-hybrid system (MATCHMAKER Two-Hybrid System 2; Clontech, Palo Alto, CA) was used to screen a rat ovary cDNA library for potential ligands capable of interacting with the YWK-II component . Mullerian-inhibiting substance was found to interact with the extracellular domain of YWK-II protein . The interaction was confirmed by a binding experiment in vitro and surface plasmon resonance assays . The recombinant Mullerian-inhibiting substance can significantly increase the viability and longevity of human spermatozoa after 5 and 22 h of incubation, presumably through binding the YWK-II component on the sperm membrane . The results of this study indicate that the YWK-II sperm membrane protein may function as a receptor for Mullerian-inhibiting substance. Thromb Haemost, 2001 May, 85(5), 837 - 44 The NFY transcription factor mediates induction of the von Willebrand factor promoter by irradiation; Bertagna A et al.; Ionizing irradiation in patients is proposed to cause thrombus formation . An increase in von Willebrand factor secretion in response to irradiation is a major contributing factor to thrombus formation . We have previously reported that the increased VWF secretion in response to irradiation is mediated at the transcriptional level . The VWF core promoter fragment (sequences -90 to +22) was shown to contain the necessary cis-acting element(s) to mediate the irradiation response of the VWF gene . Here we report that a CCAAT element in the VWF promoter is the cis-acting element necessary for irradiation induction and that the NFY transcription factor interacts with this element . These analyses demonstrate that inhibition of NFY's interaction with the CCAAT element abolishes the irradiation induction of the VWF promoter . These results provide a novel role for NFY and add this factor to the small list of irradiation-responsive transcription factors . Coimmunoprecipitation experiments demonstrated that NFY is associated with the histone acetylase P/CAF in vivo and that irradiation resulted in an increased association of NFY with coactivator P/CAF . We propose that irradiation induction of the VWF promoter involves a mechanism resulting in increased recruitment of the coactivator P/CAF to the promoter via the NFY transcription factor. Proc Natl Acad Sci U S A, 2001 Jun 5, 98(12), 6611 - 6 Epub 2001 May 22. O-linkage of N-acetylglucosamine to Sp1 activation domain inhibits its transcriptional capability; Yang X et al.; The posttranslational modification of eukaryotic intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides is essential for cell viability, yet its precise functional roles are largely unknown . O-GlcNAc transferase utilizes UDP-GlcNAc, the end product of hexosamine biosynthesis, to catalyze this modification . The availability of UDP-GlcNAc correlates with glycosylation levels of intracellular proteins as well as with transcriptional levels of some genes . Meanwhile, transcription factors and RNA polymerase II can be modified by O-GlcNAc . A linkage between transcription factor O-GlcNAcylation and transcriptional regulation therefore has been postulated . Here, we show that O-GlcNAcylation of a chimeric transcriptional activator containing the second activation domain of Sp1 decreases its transcriptional activity both in an in vitro transcription system and in living cells, which is in concert with our observation that O-GlcNAcylation of Sp1 activation domain blocks its in vitro and in vivo interactions with other Sp1 molecules and TATA-binding protein-associated factor II 110 . Furthermore, overexpression of O-GlcNAc transferase specifically inhibits transcriptional activation by native Sp1 in cells . Thus, our studies provide direct evidence that O-GlcNAcylation of transcription factors is involved in transcriptional regulation. Cell, 2001 May 18, 105(4), 499 - 509 Maturation and intranuclear transport of pre-ribosomes requires Noc proteins; Milkereit P et al.; How pre-ribosomes temporally and spatially mature during intranuclear biogenesis is not known . Here, we report three nucleolar proteins, Noc1p to Noc3p, that are required for ribosome maturation and transport . They can be isolated in two distinct complexes: Noc1p/Noc2p associates with 90S and 66S pre-ribosomes and is enriched in the nucleolus, and Noc2p/Noc3p associates with 66S pre-ribosomes and is mainly nucleoplasmic . Mutation of each Noc protein impairs intranuclear transport of 60S subunits at different stages and inhibits pre-rRNA processing . Overexpression of a conserved domain common to Noc1p and Noc3p is dominant-negative for cell growth, with a defect in nuclear 60S subunit transport, but no inhibition of pre-rRNA processing . We propose that the dynamic interaction of Noc proteins is crucial for intranuclear movement of ribosomal precursor particles, and, thereby represent a prerequisite for proper maturation. Biochemistry, 2001 May 29, 40(21), 6361 - 70 18O-exchange evidence that mutations of arginine in a signature sequence for P-type pumps affect inorganic phosphate binding; Farley RA et al.; We have proposed a model for part of the catalytic site of P-type pumps in which arginine in a signature sequence functions like lysine in P-loop-containing enzymes that catalyze adenosine 5'-triphosphate hydrolysis {Smirnova, I . N., Kasho, V . N., and Faller, L . D . (1998) FEBS Lett . 431, 309-314} . The model originated with evidence from site-directed mutagenesis that aspartic acid in the DPPR sequence of Na,K-ATPase binds Mg(2+) {Farley, R . A., et al . (1997) Biochemistry 36, 941-951} . It was developed by assuming that the catalytic domain of P-type pumps evolved from enzymes that catalyze phosphoryl group transfer . The functions of the positively charged amino group in P-loops are to bind substrate and to facilitate nucleophilic attack upon phosphorus by polarizing the gamma-phosphorus-oxygen bond . To test the prediction that the positively charged guanidinium group of R596 in human alpha(1) Na,K-ATPase participates in phosphoryl group transfer, the charge was progressively decreased by site-directed mutagenesis . Mutants R596K, -Q, -T, -M, -A, -G, and -E were expressed in yeast membranes, and their ability to catalyze phosphorylation with inorganic phosphate was evaluated by following (18)O exchange . R596K, in which the positive charge is retained, resembled the wild type . Substitution of a negative charge (R596E) resulted in complete loss of activity . The remaining mutants with uncharged side chains had both lowered affinity for inorganic phosphate and altered phosphate isotopomer distributions, consistent with increased phosphate-off rate constants compared to that of the wild type . Therefore, mutations of R596 strengthen our hypothesis that the oppositely charged side chains of the DPPR peptide in Na,K-ATPase form a quaternary complex with magnesium phosphate. Biochemistry, 2001 May 29, 40(21), 6352 - 60 Buried polar residues in coiled-coil interfaces; Akey DL et al.; Coiled coils, estimated to constitute 3-5% of the encoded residues in most genomes, are characterized by a heptad repeat, (abcdefg)(n), where the buried a and d positions form the interface between multiple alpha-helices . Although generally hydrophobic, a substantial fraction ( approximately 20%) of these a- and d-position residues are polar or charged . We constructed variants of the well-characterized coiled coil GCN4-p1 with a single polar residue (Asn, Gln, Ser, or Thr) at either an a or a d position . The stability and oligomeric specificity of each variant were measured, and crystal structures of coiled-coil trimers with threonine or serine at either an a or a d position were determined . The structures show how single polar residues in the interface affect not only local packing, but also overall coiled-coil geometry as seen by changes in the Crick supercoil parameters and core cavity volumes. Biochemistry, 2001 May 29, 40(21), 6227 - 32 Role of enzyme-ribofuranosyl contacts in the ground state and transition state for orotidine 5'-phosphate decarboxylase: a role for substrate destabilization? Miller BG, Butterfoss GL, Short SA, Wolfenden R. The crystal structure of yeast orotidine 5'-monophosphate decarboxylase (ODCase) complexed with the inhibitor 6-hydroxyuridine 5'-phosphate (BMP) reveals the presence of a series of strong interactions between enzyme residues and functional groups of this ligand . Enzyme contacts with the phosphoribofuranosyl moiety of orotidine 5'-phosphate (OMP) have been shown to contribute at least 16.6 kcal/mol of intrinsic binding free energy to the stabilization of the transition state for the reaction catalyzed by yeast ODCase . In addition to these enzyme-ligand contacts, active site residues contributed by both subunits of the dimeric enzyme are positioned to form hydrogen bonds with the 2'- and 3'-OH groups of the ligand's ribosyl moiety . These involve Thr-100 of one subunit and Asp-37 of the opposite subunit, respectively . To evaluate the contributions of these ribofuranosyl contacts to ground state and transition state stabilization, Thr-100 and Asp-37 were each mutated to alanine . Elimination of the enzyme's capacity to contact individual ribosyl OH groups reduced the k(cat)/K(m) value of the T100A enzyme by 60-fold and that of the D37A enzyme by 300-fold . Removal of the 2'-OH group from the substrate OMP decreased the binding affinity by less than a factor of 10, but decreased k(cat) by more that 2 orders of magnitude . Upon removal of the complementary hydroxymethyl group from the enzyme, little further reduction in k(cat)/K(m) for 2'-deoxyOMP was observed . To assess the contribution made by contacts involving both ribosyl hydroxyl groups at once, the ability of the D37A mutant enzyme to decarboxylate 2'-deoxyOMP was measured . The value of k(cat)/K(m) for this enzyme-substrate pair was 170 M(-1) s(-1), representing a decrease of more than 7.6 kcal/mol of binding free energy in the transition state . To the extent that electrostatic repulsion in the ground state can be tested by these simple alterations, the results do not lend obvious support to the view that electrostatic destabilization in the ground state enzyme-substrate complex plays a major role in catalysis. J Mol Biol, 2001 Jun 1, 309(2), 465 - 76 Large scale expression, purification and 2D crystallization of recombinant plant plasma membrane H+-ATPase; Jahn T et al.; P-type ATPases convert chemical energy into electrochemical gradients that are used to energize secondary active transport . Analysis of the structure and function of P-type ATPases has been limited by the lack of active recombinant ATPases in quantities suitable for crystallographic studies aiming at solving their three-dimensional structure . We have expressed Arabidopsis thaliana plasma membrane H+-ATPase isoform AHA2, equipped with a His(6)-tag, in the yeast Saccharomyces cerevisiae . The H+-ATPase could be purified both in the presence and in the absence of regulatory 14-3-3 protein depending on the presence of the diterpene fusicoccin which specifically induces formation of the H+-ATPase/14-3-3 protein complex . Amino acid analysis of the purified complex suggested a stoichiometry of two 14-3-3 proteins per H+-ATPase polypeptide . The purified H(+)-ATPase readily formed two-dimensional crystals following reconstitution into lipid vesicles . Electron cryo-microscopy of the crystals yielded a projection map at approximately 8 A resolution, the p22(1)2(1) symmetry of which suggests a dimeric protein complex . Three distinct regions of density of approximately equal size are apparent and may reflect different domains in individual molecules of AHA2 . J Biol Chem, 2001 Jul 27, 276(30), 27936 - 43 Epub 2001 May 21. In vitro assembly and recognition of Lys-63 polyubiquitin chains; Hofmann RM et al.; Polyubiquitin chains assembled through lysine 48 (Lys-48) of ubiquitin act as a signal for substrate proteolysis by 26 S proteasomes, whereas chains assembled through Lys-63 play a mechanistically undefined role in post-replicative DNA repair . We showed previously that the products of the UBC13 and MMS2 genes function in error-free post-replicative DNA repair in the yeast Saccharomyces cerevisiae and form a complex that assembles Lys-63-linked polyubiquitin chains in vitro . Here we confirm that the Mms2.Ubc13 complex functions as a high affinity heterodimer in the chain assembly reaction in vitro and report the results of a kinetic characterization of the polyubiquitin chain assembly reaction . To test whether a Lys-63-linked polyubiquitin chain can signal degradation, we conjugated Lys-63-linked tetra-ubiquitin to a model substrate of 26 S proteasomes . Although the noncanonical chain effectively signaled substrate degradation, the results of new genetic epistasis studies agree with previous genetic data in suggesting that the proteolytic activity of proteasomes is not required for error-free post-replicative repair. Int Immunol, 2001 Jun, 13(6), 791 - 7 Role of DNA-dependent protein kinase in recognition of radiation-induced DNA damage in human peripheral blood mononuclear cells; Frasca D et al.; The DNA-dependent protein kinase (DNA-PK) complex plays a crucial role in radiation-induced DNA damage recognition . The complex includes the ku heterodimer, which comprises ku 70 and ku 80 subunits, that binds DNA termini of breaks without sequence specificity, and the catalytic subunit DNA-PKCS: The activation of the DNA-PK complex was studied in X-irradiated peripheral blood mononuclear cells (PBMC) from subjects of different ages . Radiation-induced changes in the DNA-binding activity of the ku heterodimer, and in the concentrations of ku 70, ku 80, DNA-PKcs and phosphorylated ku 80 were determined in nuclear and cytoplasmic extracts . DNA-binding activity was increased by irradiation only in the nuclear extract of PBMC from young, but not from elderly subjects, whereas it was found unchanged in cytoplasmic extracts regardless of age . The radiation-induced activation of the DNA-PK complex may result from the increased concentrations of ku 80 and DNA-PKcs in the cytoplasm of PBMC from young, but not from elderly subjects, leading to a higher concentration of phosphorylated ku 80 which readily migrates to the nucleus where, after dimerization with ku 70, binds to DNA breaks . These findings suggest major steps involved in DNA-PK activation, and the intracellular and molecular changes that may account for the age-dependent impairment of DNA repair capacity in irradiated mammalian cells. Curr Biol, 2001 Apr 17, 11(8), R326 - 9 Nuclear migration: cortical anchors for cytoplasmic dynein; Bloom K; Nuclear migration in yeast provides a model system for studying how a cell polarizes the actin and microtubule cytoskeletons toward sites of cell growth . Recent findings indicate that cortical anchors are necessary for directing microtubule-based processes. Gene, 2001 May 2, 268(1-2), 1 - 7 The BAH domain, polybromo and the RSC chromatin remodelling complex; Goodwin GH et al.; The BAH (Bromo-adjacent homology) domain is a domain first identified in the vertebrate polybromo protein, a protein present in a large nuclear complex . Polybromo has two BAH domains, six bromodomains and an HMG-box . The BAH domain has been identified in a number of proteins involved in gene transcription and repression and is likely to be involved in protein-protein interactions . Polybromo resembles two related proteins in yeast, the Rsc1 and Rsc2 proteins, both having a BAH domain and two bromodomains as well as a DNA binding motif, the AT -hook . The Rsc1 and 2 proteins are components of the RSC (remodelling the structure of chromatin) complex and are required for transcriptional control . In this paper we review recent data on the function of the BAH and bromodomains in relation to polybromo and the Rsc proteins. Biochem J, 2001 Jun 1, 356(Pt 2), 589 - 94 Proline residues in two tightly coupled helices of the sulphate transporter, SHST1, are important for sulphate transport; Shelden MC et al.; The sulphate transporter SHST1, from Stylosanthes hamata, features three tightly coupled transmembrane helices which include proline residues that are conserved in most related transporters . We used site-directed mutagenesis and expression of the mutant transporters in yeast to test whether these proline residues are important for function . Four proline residues were replaced by both alanine and leucine . Only one of these proline residues, Pro-144, was essential for sulphate transport . However, mutation of either Pro-133 or Pro-160 resulted in a severe decrease in sulphate transport activity; this was due more to a decrease in transport activity than to a decrease in the amount of mutant SHST1 in the plasma membrane . These results suggest that all three proline residues are important for transport, and that the conformation of the three tightly coupled helices may play a critical role in sulphate transport . We also show that SHST1 undergoes a post-translational modification that is required for trafficking to the plasma membrane. Arch Biochem Biophys, 2001 Apr 15, 388(2), 293 - 8 Functional identification of a delta8-sphingolipid desaturase from Borago officinalis; Sperling P et al.; The similarities between delta12- and delta5-fatty acyl desaturase sequences were used to construct degenerate primers for PCR experiments with cDNA transcribed from mRNA of developing borage seeds . Screening of a borage seed cDNA library with an amplified DNA fragment resulted in the isolation of a full-length cDNA corresponding to a deduced open-reading frame of 446 amino acids . The protein showed high similarity to plant delta8-sphingolipid desaturases as well as to the delta6-fatty acyl desaturase from Borago officinalis . The sequence is characterized by the presence of a N-terminal cytochrome b5 domain . Expression of this open-reading frame in Saccharomyces cerevisiae resulted in the formation of delta8-trans/cis-phytosphingenines not present in wild-type cells, as shown by HPLC analysis of sphingoid bases as their dinitrophenyl derivatives . GLC-MS analysis of the methylated di-O-trimethylsilyl ether derivatives confirmed the presence of delta8-stereoisomers of C18- and C20-phytosphingenine . Furthermore, Northern blotting showed that the gene encoding a stereo-unselective delta8-sphingolipid desaturase is primarily expressed in young borage leaves. Int Immunopharmacol, 2001 Mar, 1(3), 539 - 50 Immunostimulant oxidized beta-glucan conjugates; Cross GG et al.; beta-Glucans are polysaccharides that act as nonspecific immune system stimulants . However, many beta-Glucans are sparingly soluble in water . This work describes an oxidative procedure, which solubilizes the beta-Glucan from Saccharomyces cerevisiae and maintains its immunostimulatory properties . Furthermore, the carboxylates at the site of oxidation allow for the conjugation of small molecule immunostimulants . Both the parent oxidized beta-glucan and its conjugates with O-beta-alanyl-5-{6-(N,N'-dimethylamino)purin-9-yl}pentanol stimulate cytotoxic T-lymphocytes (CTLs), B cells and macrophages . In addition, they both stimulate natural killer (NK) cells, a property which the small molecule purine does not possess. J Neurosci, 2001 May 15, 21(10), 3383 - 91 Increased histone acetyltransferase and lysine acetyltransferase activity and biphasic activation of the ERK/RSK cascade in insular cortex during novel taste learning; Swank MW et al.; Changes in gene expression are thought to be involved in neuronal plasticity associated with learning and memory . Although acetylation of lysine residues on histones by histone acetyltransferases (HAT) is an obligatory component of transcription, HAT activity has been largely ignored in studies of the nervous system . We developed a new model for studying novel taste learning using novel solid food presentation to nondeprived animals . Using this behavioral paradigm, we investigated short- and long-term regulation of lysine acetyltransferase activity and the ERK/mitogen-activated protein kinase (MAPK)/RSK cascade in insular cortex, a CNS region known to be crucial for the formation of novel taste memories . We observed that novel taste learning elicited biphasic (acute and long-lasting) activation of two distinct lysine acetyltransferase activities along with the ERK/MAPK cascade in insular cortex . In vitro studies revealed that the ERK cascade could regulate the lysine acetylation of a 42 kDa lysine acetyltransferase substrate, suggesting a causal relationship between ERK activation and lysine acetyltransferase activity in insular cortex . Overall, our studies reveal an unanticipated long-lasting activation of insular cortex signal transduction cascades in novel taste learning . Furthermore, our studies suggest the hypothesis that acute and long-term ERK activation and lysine-histone acetyltransferase activation may play a role in regulating gene expression in single-trial learning and long-term memory formation. Mol Genet Genomics, 2001 Apr, 265(2), 215 - 24 Cloning and expression analysis of NhL1, a gene encoding an extracellular lipase from the fungal pea pathogen Nectria haematococca MP VI (Fusarium solani f . sp . pisi) that is expressed in planta; Eddine AN et al.; The filamentous fungus Nectria haematococca (anamorph Fusarium solani f . sp . pisi) resides in soil, and attacks pea seedlings in the area of the underground epicotyl and upper tap root, causing foot rot disease . We detected lipase activity during in vitro growth of N . haematococca . Subsequently, a lipase gene was cloned and functionally characterised by heterologous expression in Saccharomyces cerevisiae . The full-length cDNA of 1152 bp was cloned using a 3' RACE-PCR approach coupled with cDNA library screening . The genomic clone, comprising an ORF of 999 bp interrupted by two introns of 56 and 64 bp, was isolated from a newly constructed lambda phage library . Analysis of the deduced protein sequence revealed the presence of a typical signal peptide at the N-terminus, and of the three conserved amino acids forming the active site of lipases . The lipase of N . haematococca has a low degree of similarity to the lipases from Humicola lanuginosa (37.2%), Rhizomucor miehei (21.6%), Rhizopus delemar (23.1%), Rhizopus niveus (25.9%), and to mono- and diacylglycerol lipase from Penicillium camembertii (30.8%), and very high similarity (94.6%) to a lipase from Fusarium heterosporum . The lipase from N . haematococca shows maximal activity at 37 degrees C and pH 8.0 . Based on Southern analysis, the lipase clone represents a single-copy gene in N . haematococca . Expression analysis was performed by RT-PCR . In vitro, the lipase gene shows a low basal expression, but is highly inducible by lipase substrates, and repressed by glucose . During plant infection, transcripts of this fungal lipase gene were detected 4, 8, and 10 days after infection. Acta Pharmacol Sin, 2000 Jul, 21(7), 641 - 5 Thrombin stimulates MMP-9 mRNA expression through AP-1 pathway in human mesangial cells; Liu WH et al.; AIM: To investigate the thrombin mediated induction of gelatinase B (MMP-9) in mesangial cells (MC) and the underlying role of activator protein-1 (AP-1) . METHODS: Cultured human mesangial cells were exposed to thrombin in the presence or absence of hirudin, curcumin, and c-fos antisense or sense oligonucleotides . Northern hybridization was employed to assess MMP-9 mRNA expression, and electrophoretic mobility shift assay (EMSA) for AP-1 DNA binding activity . RESULTS: The levels of MMP-9 mRNA in the cell treated with different doses of thrombin (500, 1500, and 4500 u/L, respectively) were 1.1, 3.3, and 4.8 times higher than that in the control, respectively . There was also an increase in AP-1 binding activity (3.5, 5.9, and 7.1 fold than that of the control) in accordance with MMP-9 mRNA levels in the presence of thrombin . Hirudin, curcumin, and c-fos antisense oligonucleotides could block thrombin-induced expression of MMP-9 mRNA as well as AP-1 binding activity . CONCLUSION: Thrombin is a potent stimulator of MMP-9 gene expression in human mesangial cells, and the underlying intracellular events are mediated, at least partly, by AP-1 pathway. Oncogene, 2001 Apr 19, 20(17), 2091 - 100 Sensitivity of wild type and mutant ras alleles to Ras specific exchange factors: Identification of factor specific requirements; Nielsen KH et al.; We have investigated the productive interaction between the four mammalian Ras proteins (H-, N-, KA- and KB-Ras) and their activators, the mammalian exchange factors mSos1, GRF1 and GRP, by using a modified Saccharomyces cerevisiae whose growth is dependent on activation of a mammalian Ras protein by its activator . All four mammalian Ras proteins were activated with similar efficiencies by the individual exchange factors . The H-Ras mutant V103E, which is competent for membrane localization, nucleotide binding, intrinsic and stimulated GTPase activity as well as intrinsic exchange, was defective for activation by all factors tested, suggesting that the integrity of this residue is necessary for catalyzed exchange . However, when other H-Ras mutants were studied, some distinct sensitivities to the exchange factors were observed . GRP-mediated, but not mSos1-mediated, exchange was blocked in additional mutants, suggesting different structural requirements for GRP . Analysis of Ras-mediated gene activation in murine fibroblasts confirmed these results. Oncogene, 2001 Apr 12, 20(16), 1990 - 9 Ku antigen is required to relieve G2 arrest caused by inhibition of DNA topoisomerase II activity by the bisdioxopiperazine ICRF-193; Munoz P et al.; Ku antigen is necessary for DNA double-strand break (DSB) repair through its ability to bind DNA ends with high affinity and to recruit the catalytic subunit of DNA-PK to the DSBs . Ku-deficient cells are hypersensitive to agents causing DSBs in DNA but also to the DNA topoisomerase II (topo II) inhibitor ICRF-193, which does not induce DSBs . This suggests a new role of Ku antigen, that is independent of DSB repair by DNA-PK . Here we characterize the basis for the hypersensitivity of Ku-deficient cells to ICRF-193 . Chromosome condensation and segregation, which are dependent on topo II, but also the catalytic activity of topo II in late S-G2 were inhibited to a comparable extent when ICRF-193 was applied to Ku-deficient cells or wild-type cells . However, mutant cells arrested in G2 by ICRF-193 treatment were unable to progress into M phase upon drug removal, although drug-trapped topo II complexes were removed from DNA and the two isoforms of topo II recovered their catalytic activity as in wild-type cells . The reversibility of G2 arrest was recovered by complementation of mutant cells with a human Ku86 cDNA . Notably, chromosome condensation was abnormal in Ku-deficient cells after suppression of the G2 arrest by caffeine, even in the absence of ICRF-193 . These results reflect the involvement of Ku-antigen in the cellular response to topo II inhibition, more particularly in relieving G2 arrest caused by topo II inhibition in late S/G2 and the subsequent recovery of chromosome condensation. Mol Biol Cell, 2001 May, 12(5), 1381 - 92 Role of nuclear pools of aminoacyl-tRNA synthetases in tRNA nuclear export; Azad AK et al.; Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 20001) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases . We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p . By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein . Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location . The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol . Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths . Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths . No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export. Mol Biol Cell, 2001 May, 12(5), 1189 - 98 Structural requirements of Tom40 for assembly into preexisting TOM complexes of mitochondria; Rapaport D et al.; Tom40 is the major subunit of the translocase of the outer mitochondrial membrane (the TOM complex) . To study the assembly pathway of Tom40, we have followed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 protein . Upon import into isolated mitochondria, Tom40 precursor proteins lacking the first 20 or the first 40 amino acid residues were assembled as the wild-type protein . In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested at an intermediate stage of assembly . We constructed mutant versions of Tom40 affecting this region and transformed the genes into a sheltered heterokaryon containing a tom40 null nucleus . Homokaryotic strains expressing the mutant Tom40 proteins had growth rate defects and were deficient in their ability to form conidia . Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobility and a tendency to lose Tom40 subunits from the complex . Thus, both in vitro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex . Finally, we found that TOM complexes in the mitochondrial outer membrane were capable of exchanging subunits in vitro . A model is proposed for the integration of Tom40 subunits into the TOM complex. Mol Cell Biol, 2001 Jun, 21(12), 4075 - 88 A Pcl-like cyclin of Aspergillus nidulans is transcriptionally activated by developmental regulators and is involved in sporulation; Schier N et al.; The filamentous fungus Aspergillus nidulans reproduces asexually through the formation of spores on a multicellular aerial structure, called a conidiophore . A key regulator of asexual development is the TFIIIA-type zinc finger containing transcriptional activator Bristle (BRLA) . Besides BRLA, the transcription factor ABAA, which is located downstream of BRLA in the developmental regulation cascade, is necessary to direct later gene expression during sporulation . We isolated a new developmental mutant and identified a leaky brlA mutation and the mutated Saccharomyces cerevisiae cyclin homologue pclA, both contributing to the developmental phenotype of the mutant . pclA was found to be 10-fold transcriptionally upregulated during conidiation, and a pclA deletion strain was reduced three- to fivefold in production of conidia . Expression of pclA was strongly induced by ectopic expression of brlA or abaA under conidiation-suppressing conditions, indicating a direct role for brlA and abaA in pclA regulation . PCLA is homologous to yeast Pcl cyclins, which interact with the Pho85 cyclin-dependent kinase . Although interaction with a PSTAIRE kinase was shown in vivo, PCLA function during sporulation was independent of the A . nidulans Pho85 homologue PHOA . Besides the developmental regulation, pclA expression was cell cycle dependent with peak transcript levels in S phase . Our findings suggest a role for PCLA in mediating cell cycle events during late stages of sporulation. Mol Cell Biol, 2001 Jun, 21(12), 3913 - 25 Characterization of mec1 kinase-deficient mutants and of new hypomorphic mec1 alleles impairing subsets of the DNA damage response pathway; Paciotti V et al.; DNA damage checkpoints lead to the inhibition of cell cycle progression following DNA damage . The Saccharomyces cerevisiae Mec1 checkpoint protein, a phosphatidylinositol kinase-related protein, is required for transient cell cycle arrest in response to DNA damage or DNA replication defects . We show that mec1 kinase-deficient (mec1kd) mutants are indistinguishable from mec1Delta cells, indicating that the Mec1 conserved kinase domain is required for all known Mec1 functions, including cell viability and proper DNA damage response . Mec1kd variants maintain the ability to physically interact with both Ddc2 and wild-type Mec1 and cause dominant checkpoint defects when overproduced in MEC1 cells, impairing the ability of cells to slow down S phase entry and progression after DNA damage in G(1) or during S phase . Conversely, an excess of Mec1kd in MEC1 cells does not abrogate the G(2)/M checkpoint, suggesting that Mec1 functions required for response to aberrant DNA structures during specific cell cycle stages can be separable . In agreement with this hypothesis, we describe two new hypomorphic mec1 mutants that are completely defective in the G(1)/S and intra-S DNA damage checkpoints but properly delay nuclear division after UV irradiation in G(2) . The finding that these mutants, although indistinguishable from mec1Delta cells with respect to the ability to replicate a damaged DNA template, do not lose viability after UV light and methyl methanesulfonate treatment suggests that checkpoint impairments do not necessarily result in hypersensitivity to DNA-damaging agents. Mol Cell Biol, 2001 Jun, 21(12), 3888 - 900 The neural RNA-binding protein Musashi1 translationally regulates mammalian numb gene expression by interacting with its mRNA; Imai T et al.; Musashi1 (Msi1) is an RNA-binding protein that is highly expressed in neural progenitor cells, including neural stem cells . In this study, the RNA-binding sequences for Msi1 were determined by in vitro selection using a pool of degenerate 50-mer sequences . All of the selected RNA species contained repeats of (G/A)U(n)AGU (n = 1 to 3) sequences which were essential for Msi1 binding . These consensus elements were identified in some neural mRNAs . One of these, mammalian numb (m-numb), which encodes a membrane-associated antagonist of Notch signaling, is a likely target of Msi1 . Msi1 protein binds in vitro-transcribed m-numb RNA in its 3'-untranslated region (UTR) and binds endogenous m-numb mRNA in vivo, as shown by affinity precipitation followed by reverse transcription-PCR . Furthermore, adenovirus-induced Msi1 expression resulted in the down-regulation of endogenous m-Numb protein expression . Reporter assays using a chimeric mRNA that combined luciferase and the 3'-UTR of m-numb demonstrated that Msi1 decreased the reporter activity without altering the reporter mRNA level . Thus, our results suggested that Msi1 could regulate the expression of its target gene at the translational level . Furthermore, we found that Notch signaling activity was increased by Msi1 expression in connection with the posttranscriptional down-regulation of the m-numb gene. J Neurochem, 2001 May, 77(4), 1097 - 107 Interaction of phosphorylated tyrosine hydroxylase with 14-3-3 proteins: evidence for a phosphoserine 40-dependent association; Kleppe R et al.; Tyrosine hydroxylase (TH) has been reported to require binding of 14-3-3 proteins for optimal activation by phosphorylation . We examined the effects of phosphorylation at Ser19, Ser31 and Ser40 of bovine TH and human TH isoforms on their binding to the 14-3-3 proteins BMH1/BMH2, as well as 14-3-3 zeta and a mixture of sheep brain 14-3-3 proteins . Phosphorylation of Ser31 did not result in 14-3-3 binding, however, phosphorylation of TH on Ser40 increased its affinity towards the yeast 14-3-3 isoforms BMH1/BMH2 and sheep brain 14-3-3, but not for 14-3-3 zeta . On phosphorylation of both Ser19 and Ser40, binding to the 14-3-3 zeta isoform also occurred, and the binding affinity to BMH1 and sheep brain 14-3-3 increased . Both phosphoserine-specific antibodies directed against the 10 amino acids surrounding Ser19 or Ser40 of TH, and the phosphorylated peptides themselves, inhibited the association between phosphorylated TH and 14-3-3 proteins . This was also found when heparin was added, or after proteolytic removal of the N-terminal 37 amino acids of Ser40-phosphorylated TH . Binding of BMH1 to phosphorylated TH decreased the rate of dephosphorylation by protein phosphatase 2A, but no significant change in enzymatic activity was observed in the presence of BMH1 . These findings further support a role for 14-3-3 proteins in the regulation of catecholamine biosynthesis and demonstrate isoform specificity for both TH and 14-3-3 proteins. Mol Microbiol, 2001 May, 40(3), 719 - 30 A homologue of the transcriptional repressor Ssn6p antagonizes cAMP signalling in Ustilago maydis; Loubradou G et al.; In Ustilago maydis, cAMP signalling is crucial for successful infection of maize plants . Strains are non-pathogenic if mutated in any of the currently identified components of this signalling pathway, such as the alpha-subunit of a heterotrimeric G protein Gpa3, the adenylate cyclase Uac1 and the regulatory and catalytic subunit of protein kinase A Ubc1 and Adr1 respectively . Deletion of gpa3, uac1 or adr1 triggers filamentous growth, and certain point mutations in gpa3 and ubc1 that mimic a high cAMP level display a glossy colony phenotype . Screening an autonomously replicating U . maydis library in such a background (gpa3Q206L), we identified sql1 as a suppressor of the glossy colony phenotype . Interestingly, only alleles carrying C-terminal truncations of Sql1 were able to complement the mutant phenotype, suggesting a gain-of-function by these variants . Sql1 is a functional homologue of the yeast transcriptional repressor Ssn6p and contains 10 tetratricopeptide repeats (TPRs), of which the first six are important for suppressor function . Truncated sql1 alleles that suppress the glossy colony phenotype of gpa3Q206L strains induce filamentous growth when introduced in wild type . Filamentation of these strains is reversed in the presence of cAMP . We present a model in which Sql1 is part of an evolutionary conserved Sql1-Tup1 transcriptional repressor complex that antagonizes cAMP signalling by repressing cAMP-regulated genes. J Biol Chem, 2001 Jul 13, 276(28), 26090 - 8 Epub 2001 May 17. Alternative mechanisms of transcriptional activation by Rap1p; Idrissi FZ et al.; Single Rap1p DNA-binding sites are poor activators of transcription of yeast minimal promoters, even when fully occupied in vivo . This low efficiency is due to two independent repression mechanisms as follows: one that requires the presence of histones, and one that requires Hrs1p, a component of the RNA polymerase II mediator complex . Both repression mechanisms were greatly reduced for constructs with tandemly arranged sites . In these constructs, UASrpg sequences (ACACCCATACATTT) activated better than telomere-like sequences (ACACCCACACACCC) in an orientation-dependent manner . Both mutations in the SWI/SNF complex and a deletion of amino acids 597--629 of Rap1p (Tox domain) decreased synergistic effects of contiguous telomeric sites . Conversely, deletion of amino acids 700--798 of Rap1p (Sil domain) made UASrpg and telomeric sites functionally indistinguishable . We propose that the Sil domain masks the main transactivation domain of Rap1p in Rap1p-telomere complexes, where the Tox domain behaves as a secondary activation domain, probably by interacting with chromatin-remodeling complexes . Rap1p DNA-binding sites in ribosomal protein gene promoters are mainly UASrpg-like; their replacement by telomeric sequences in one of these promoters (RPS17B) decreased transcription by two-thirds . The functional differences between UASrpgs and telomeric sequences may thus contribute to the differential expression of Rap1p-regulated promoters in vivo. Glycobiology, 2001 Apr, 11(4), 321 - 33 Large-scale isolation of dolichol-linked oligosaccharides with homogeneous oligosaccharide structures: determination of steady-state dolichol-linked oligosaccharide compositions; Kelleher DJ et al.; The dolichol-linked oligosaccharide donor (Glc(3)Man(9)GlcNAc(2)-PP-Dol) for N-linked glycosylation of proteins is assembled in a series of reactions that initiate on the cytoplasmic face of the rough endoplasmic reticulum and terminate within the lumen . The biochemical analysis of the oligosaccharyltransferase and the glycosyltransferases that mediate assembly of dolichol-linked oligosaccharides (OS-PP-Dol) has been hindered by the lack of structurally homogeneous substrate preparations . We have developed an improved method for the preparative-scale isolation of dolichol-linked oligosaccharides from vertebrate tissues and yeast cells . Preparations that were highly enriched in either Glc(3)Man(9)GlcNAc(2)-PP-Dol or Man(9)GlcNAc(2)-PP-Dol were obtained from porcine pancreas and a Man(5)GlcNAc(2)-PP-Dol preparation was obtained from an alg3 yeast culture . Chromatography of the OS-PP-Dol preparations on an aminopropyl silica column was used to obtain dolichol-linked oligosaccharides with defined structures . A single chromatography step could achieve near-baseline resolution of dolichol-linked oligosaccharides that differed by one sugar residue . A sensitive oligosaccharyltransferase endpoint assay was used to determine the concentration and composition of the OS-PP-Dol preparations . Typical yields of Glc(3)Man(9)GlcNAc(2)-PP-Dol, Man(9)GlcNAc(2)-PP-Dol, and Man(5)GlcNAc(2)-PP-Dol ranged between 5 and 15 nmol per chromatographic run . The homogeneity of these preparations ranged between 85 and 98% with respect to oligosaccharide composition . Purification of dolichol-linked oligosaccharides from cultures of alg mutant yeast strains provides a general method to obtain authentic OS-PP-Dol assembly intermediates of high purity . The analytical methods described here can be used to accurately evaluate the steady-state dolichol-linked oligosaccharide compositions of wild-type and mutant cell lines. Cancer Res, 2001 May 15, 61(10), 4136 - 42 Dynamic, site-specific interaction of hypoxia-inducible factor-1alpha with the von Hippel-Lindau tumor suppressor protein; Yu F et al.; Hypoxia-inducible factor (HIF)-1alpha is a transcription factor that plays a critical role in regulating genes involved in erythropoiesis and angiogenesis . Recent evidence indicates that the von Hippel-Lindau tumor suppressor protein (VHL) is part of a ubiquitin ligase complex that promotes the degradation of HIF-1alpha under normoxic conditions . Under hypoxic conditions, HIF-1alpha is markedly stabilized . A critical issue in understanding the hypoxic response is the identification of hypoxia-regulated steps . We show here that hypoxia and cobalt treatment modulate the capacity of a HIF-1alpha fragment comprising residues 531-652 to coimmunoprecipitate with VHL . Hypoxia and cobalt both significantly diminish the interaction, and furthermore, normoxia treatment after hypoxia rapidly normalizes it . This HIF-1alpha fragment confers hypoxia and cobalt inducibility on a heterologous protein . Significantly, contained within this fragment is a short 27-residue sequence that behaves identically in all respects noted above . Finally, evidence is provided to show that cobalt and hypoxia both induce a posttranslational modification (or loss of one) in HIF-1alpha that affects its binding to VHL . We propose that dynamic, site-specific interaction of HIF-1alpha with VHL provides one mechanism by which HIF-1alpha can be regulated. Cancer Res, 2001 May 15, 61(10), 3886 - 93 Radiation-induced genomic rearrangements formed by nonhomologous end-joining of DNA double-strand breaks; Rothkamm K et al.; Two major pathways for repairing DNA double-strand breaks (DSBs) have been identified in mammalian cells, nonhomologous end-joining (NHEJ) and homologous recombination (HR) . Inactivation of NHEJ is known to lead to an elevated level of spontaneous and radiation-induced chromosomal rearrangements associated with an increased risk of tumorigenesis . This has raised the idea of a caretaker role for NHEJ . It is, however, not known whether NHEJ itself can also cause rearrangements . To investigate, on the DNA level, the influence of a defect in NHEJ on the formation of genomic rearrangements, we applied an assay based on Southern hybridization that allows the identification and quantification of incorrectly rejoined DSB ends produced by ionizing radiation . After 80 Gy of X-irradiation at a high dose rate (23 Gy/min), wild-type cells repaired 50% of the induced DSBs within 24 h by incorrect rejoining . This frequency of DSB misrejoining is considerably reduced in NHEJ-deficient cells . Low-dose-rate experiments, in which the cells were exposed to 80 Gy over a period of 14 days under repair conditions, led to no detectable misrejoining in wild-type cells but revealed a misrejoining frequency of 10% in NHEJ-deficient cells . This shows that in situations of separated breaks, NHEJ deficiency leads to genomic rearrangements, in agreement with chromosomal studies . However, if multiple DSBs coincide, even wild-type cells form genomic rearrangements frequently . These repair events are absent in Ku80-, DNA-PKcs-, and DNA ligase IV-deficient cells but are present in RAD54(-/-) cells . This strongly suggests that NHEJ has, in addition to its caretaker role, also the potential to effect genomic rearrangements . We propose that it serves as an efficient pathway for rejoining correct break ends in situations of separated breaks but generates genomic rearrangements if DSBs are close in time and space. Life Sci, 2001 Apr 6, 68(19-20), 2301 - 8 Receptor-independent activators of heterotrimeric G-proteins; Cismowski MJ et al.; Heterotrimeric G-protein signalling systems are primarily activated via cell surface receptors possessing the seven membrane span motif . Several observations suggest the existence of other modes of input to such signalling systems either downstream of effectors or at the level of G-proteins themselves . Using a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae, we identified three proteins, AGS1-3 (for Activators of G-protein Signalling), that activated heterotrimeric G-protein signalling pathways in the absence of a typical receptor . AGS1 defines a distinct member of the super family of ras related proteins . AGS2 is identical to mouse Tctex1, a protein that exists as a light chain component of the cytoplasmic motor protein dynein and subserves as yet undefined functions in cell signalling pathways . AGS3 possesses a series of tetratrico repeat motifs and a series of four amino acid repeats termed G-protein regulatory motifs . The GPR motifs are found in a number of proteins that interact with and regulate Galpha . Although each AGS protein activates G-protein signaling, they do so by different mechanisms within the context of the G-protein activation/deactivation cycle . AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signalling pathways. Electrophoresis, 2001 Apr, 22(6), 990 - 8 Quantitative distance information on protein-DNA complexes determined in polyacrylamide gels by fluorescence resonance energy transfer; Lorenz M et al.; In polyacrylamide gels, we have quantitatively determined Forster transfer (fluorescense resonance energy transfer, FRET) between two fluorescent dyes attached to DNA in protein-DNA complexes . The donor-dye fluorescein labeled to DNA retains its free mobility in the polyacrylamide gel, however, its fluorescence properties change . The different quantum yield of fluorescein in the gel is found to be independent of the gel concentration and can thus be quantitatively taken into account by a reduced Forster distance R0 of 46 A compared to 50 A in solution . We have determined global structural properties of two proteins binding to double-labeled DNA using a novel gel-based fluorescence resonance energy transfer assay . In polyacrylamide gels we have analyzed the binding of integration host factor (IHF) and the high mobility group protein NHP6a to their substrate DNA . The measured Forster transfer efficiency allows us to deduce information on the overall shape and the DNA bending angle in the complex. J Biol Chem, 2001 Jul 13, 276(28), 25974 - 81 Epub 2001 May 16. Multiple roles for Rsp5p-dependent ubiquitination at the internalization step of endocytosis; Dunn R et al.; Ubiquitination of integral plasma membrane proteins triggers their rapid internalization into the endocytic pathway . The yeast ubiquitin ligase Rsp5p, a homologue of mammalian Nedd4 and Itch, is required for the ubiquitination and subsequent internalization of multiple plasma membrane proteins, including the alpha-factor receptor (Ste2p) . Here we demonstrate that Rsp5p plays multiple roles at the internalization step of endocytosis . Temperature-sensitive rsp5 mutant cells were defective in the internalization of alpha-factor by a Ste2p-ubiquitin chimera, a receptor that does not require post-translational ubiquitination . Similarly, a modified version of Ste2p bearing a NPFXD linear peptide sequence as its only internalization signal was not internalized in rsp5 cells . Internalization of these variant receptors was dependent on the catalytic cysteine residue of Rsp5p and on ubiquitin-conjugating enzymes that bind Rsp5p . Thus, a Rsp5p-dependent ubiquitination event is required for internalization mediated by ubiquitin-dependent and -independent endocytosis signals . Constitutive Ste2p-ubiquitin internalization and fluid-phase endocytosis also required active ubiquitination machinery, including Rsp5p . These observations indicate that Rsp5p-dependent ubiquitination of a trans-acting protein component of the endocytosis machinery is required for the internalization step of endocytosis. J Biol Chem, 2001 Jul 27, 276(30), 27778 - 86 Epub 2001 May 16. Functional characterization of recombinant chloroplast signal recognition particle; Groves MR et al.; The signal recognition particle (SRP) is a ubiquitous system for the targeting of membrane and secreted proteins . The chloroplast SRP (cpSRP) is unique among SRPs in that it possesses no RNA and is functional in post-translational as well as co-translational targeting . We have expressed and purified the two components of the Arabidopsis thaliana chloroplast signal recognition particle (cpSRP) involved in post-translational transport: cpSRP54 and the chloroplast-specific protein, cpSRP43 . Recombinant cpSRP supports the efficient in vitro insertion of pea preLhcb1 into isolated thylakoid membranes . Recombinant cpSRP is a stable heterodimer with a molecular mass of approximately 100 kDa as determined by analytical ultracentrifugation, gel filtration analysis, and dynamic light scattering . The interactions of the components of the recombinant heterodimer and pea preLhcb1 were probed using an immobilized peptide library (pepscan) approach . These data confirm two previously reported interactions with the L18 region and the third transmembrane helix of Lhcb1 and suggest that the interface of the cpSRP43 and cpSRP54 proteins is involved in substrate binding . Additionally, cpSRP components are shown to recognize peptides from the cleavable, N-terminal chloroplast transit peptide of preLhcb1 . The interaction of cpSRP43 with cpSRP54 was probed in a similar experiment with a peptide library representing cpSPR54 . The C terminus of cpSRP54 is essential for the formation of the stable cpSRP complex and cpSPR43 interacts with distinct regions of the M domain of cpSRP54. Biochem Soc Trans, 2001 May, 29(Pt 2), 316 - 20 Post-transcriptional regulation of rat carnitine octanoyltransferase; Hegardt FG et al.; Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which can lead to the synthesis of two proteins . The occurrence of the three COT transcripts in rat has been found in all tissues examined and does not depend on sex, fat feeding, peroxisome proliferators or hyperinsulinaemia . Rat COT exon 2 contains a putative exonic splicing enhancer (ESE) sequence . Mutation of this ESE (GAAGAAG) to AAAAAAA decreased trans-splicing in vitro, from which it is deduced that this ESE sequence is partly responsible for the formation of the three transcripts . The protein encoded by cis-spliced mRNA of rat COT is inhibited by malonyl-CoA and etomoxir . cDNA species encoding full-length wild-type COT and one double mutant COT were expressed in Saccharomyces cerevisiae . The recombinant enzymes showed full activity towards both substrates, carnitine and decanoyl-CoA . The activity of the doubly mutated H131A/H340A enzyme was similar to that of the rat peroxisomal enzyme but was completely insensitive to malonyl-CoA and etomoxir . These results indicate that the histidine residues His-131 and His-340 are the sites responsible for the interaction of these two inhibitors, which inhibit COT by interacting with the same sites. Biochem Soc Trans, 2001 May, 29(Pt 2), 187 - 91 Eukaryotic mutagenesis and translesion replication dependent on DNA polymerase zeta and Rev1 protein; Lawrence CW et al.; Translesion replication is a mechanism that employs specialized DNA polymerases for promoting continued nascent strand extension at forks blocked by the presence of unrepaired DNA damage . In Saccharomyces cerevisiae at least, this process contributes only modestly to the ability of cells to tolerate DNA damage, but is a major source of DNA-damage-induced substitutions and frameshifts, and of spontaneous mutations . Translesion replication past many types of DNA damage in yeast depends on the activities of DNA polymerase zeta (pol zeta) and Rev1p . Pol zeta is found in most, but not all, eukaryotes investigated, whereas Rev1p appears to be universal . Genes encoding these enzymes are found in humans, and appear to perform functions similar to those in yeast. Biochem Soc Trans, 2001 May, 29(Pt 2), 128 - 35 Cytochrome P450 (CYP) mutants and substrate-specificity alterations: segment-directed mutagenesis applied to human CYP1A1; Urban P et al.; Cytochrome P450 (CYP) enzymes represent a large superfamily that displays extraordinarily diverse substrate specificities . After a concise review about CYPs of the CYP1A subfamily, which plays a crucial role in procarcinogen activation, this paper presents segment-directed mutagenesis . This approach generates a library of random combinatorial mutants limited to a precise region of human CYP1A1, namely amino acids 204-214 in which nine positions differ between CYP1A1 and CYP1A2 . The resulting mutants present all combinations possible among these nine positions shifting mutated residues to their CYP1A2 counterpart . The mutants were cloned and expressed in an engineered Saccharomyces cerevisiae strain that has a microsomal oxido-reduction environment optimized for CYPs . This procedure resulted in yeast transformants that express a library of mutant CYP1A1 . A subset of transformants were chosen at random, assayed for a typical CYP1A1 activity and the plasmidic DNA of functional clones was rescued and sequenced . In this approach, no preconceived idea is made as to which combination of amino acid residues controls substrate selectivity . The functional mutants were analysed further for alteration of substrate specificity with a series of heterocyclic and polycyclic aromatic hydrocarbons . Some of the implications of these analyses are discussed for the role of this region in substrate specificity, since it corresponds to a putative loop and is not part of one of the CYP substrate-recognition sites. J Invertebr Pathol, 2001 Apr, 77(3), 180 - 5 Comparative study of in vivo and in vitro phagocytosis including germicidal capacity in Odontaster validus (Koehler, 1906) at 0 degree C; Silva JR et al.; The phagocytosis and germicidal capacity of Saccharomyces cerevisiae by phagocytic amoebocytes (PA) of the Antarctic starfish Odontaster validus were studied in vivo (after incubation periods of 1, 2, and 4 h) and in vitro (after incubation periods of 1, 2, 4, 8, and 12 h) at 0 degree C . The total number of PA and the phagocytic capacity (PC), phagocytic index (PI), and germicidal capacity (GC) of the PA were calculated . Results showed significant variability of the total PA number in different animals . There was a significant increase in PC and no significant differences in PI and GC for different in vitro incubation times . In vivo, experiments showed no significant difference of PC and PI, but there was a significant increase in GC as incubation periods increased . Comparison between in vitro and in vivo results revealed that PI and PC were significantly higher in vitro and that GC was significantly higher in vivo . The present study shows for the first time the phagocytosis and GC of an Antarctic invertebrate in vivo at low temperature (0 degree C), and the results are comparing with the available literature for echinoderms . Mol Cells, 2001 Apr 30, 11(2), 263 - 6 An improved strategy for a genetic assay for site-specific proteolysis; Kang H et al.; We have previously reported a genetic assay that is suitable for the study of substrate specificity of a protease in vivo, and herein present a simplified version of the method . In this procedure, expressed in Saccharomyces cerevisiae by using the constitutive alcohol dehydrogenase promoter is a fusion protein in which a transcription factor is linked to the intracellular domain of an integral membrane protein by a protease substrate sequence . Following this, a protease is expressed by using the inducible GAL promoter in the same yeast cells . The cleavage of the substrate sequence by the specific protease results in the release of the transcription factor and subsequent activation of reporter genes in nucleus . Since the expression of a protease is strictly under the control of the inducible GAL promoter, false substrate sequences that are cleaved by endogenous yeast proteases can be easily recognized and eliminated from further characterization . This suggests that the modified strategy provides an efficient tool for the analysis of substrate sequences of a protease in vivo. Cancer Radiother, 2001 Apr, 5(2), 109 - 29 {Molecular mechanisms controlling the cell cycle: fundamental aspects and implications for oncology}; Viallard JF et al.; INTRODUCTION: Comprehension of cell cycle regulation mechanisms has progressed very quickly these past few years and regulators of the cell cycle have gained widespread importance in cancer . This review first summarizes major advances in the understanding of the control of cell cycle mechanisms . Examples of how this control is altered in tumoral cells are then described . CURRENT KNOWLEDGE AND KEY POINTS: The typical mammalian cell cycle consists of four distinct phases occurring in a well-defined order, each of which should be completed successfully before the next begins . Progression of eukaryotic cells through major cell cycle transitions is mediated by sequential assembly and activation of a family of serine-threonine protein kinases, the cyclin dependent kinases (CDK) . The timing of their activation is determined by their post-translational modifications (phosphorylations/dephosphorylations), and by the association of a protein called cyclin, which is the regulatory subunit of the kinase complex . The cyclin family is divided into two main classes . The 'G1 cyclins' include cyclins C, D1-3, and E, and their accumulation is rate-limiting for progression from the G1 to S phase . The 'mitotic or G2 cyclins', which include cyclin A and cyclin B, are involved in the control of G2/M transition and mitosis . The cyclins bind to and activate the CDK, which leads to phosphorylation (and then inhibition) of the tumor suppressor protein, pRb . pRb controls commitment to progress from the G1 to S phase, at least in part by repressing the activity of the E2F transcription factors known to promote cell proliferation . Both the D-type cyclins and their partner kinases CDK4/6 have proto-oncogenic properties, and their activity is carefully regulated at multiple levels including negative control by two families of CDK inhibitors . While members of the INK4 family (p16INK4A, p15INK4B, p18INK4C, p19INK4D) interact specifically with CDK4 and CDK6, the CIP/KIP inhibitors p21CIP1/WAF1, p27KIP1 and p57KIP2 inhibit a broader spectrum of CDK . The interplay between p16INK4A, cyclin D/CDK, and pRb/E2F together constitute a functional unit collectively known as the 'pRb pathway' . Each of the major components of this mechanism may become deregulated in cancer, and accumulating evidence points to the 'pRb pathway' as a candidate obligatory target in multistep oncogenesis of possibly all human tumor types . FUTURE PROSPECTS AND PROJECTS: Major advances in the understanding of cell cycle regulation mechanisms provided a better knowledge of the molecular interactions involved in human cancer . This progress has led to the promotion of new therapeutic agents presently in clinical trials or under development . Moreover, the components of the cell cycle are probably involved in other non-cancerous diseases and their role must be defined. Biotechniques, 2001 May, 30(5), 1134 - 8, 1140 Stable luciferase reporter cell lines for signal transduction pathway readout using GAL4 fusion transactivators; Hexdall L et al.; While GAL4 fusion activators have been widely used for dissecting signal transduction pathways in transient assays, there has been surprisingly little reported on utilizing cell lines with stably integrated fusion activators . To avoid problems with the efficiency and reproducibility inherent to transient transfection, we describe here the generation and characterization of HeLa reporter cell lines, which contain a stably integrated luciferase gene responsive to stably integrated and constitutively expressed GAL4-CREB or GAL4-Elk1 fusion activators . These cell lines exhibited extremely low basal luciferase expression but robust response to various extracellular stimuli or the expression of signaling molecules that resulted in elevated MAP kinase or PKA activities . This integrated two-component reporter system allows one to focus specifically on particular signaling pathway endpoints and the altered transactivation activity of either Elk1 or CREB . With the procedures described here, many novel cell-based assays can be developed by generating new reporter cell lines with medically important but difficult-to-transfect cell types, and by using different reporter genes or different fusion transactivator genes. J Hum Genet, 2001, 46(5), 251 - 9 Fragile XE-associated familial mental retardation protein 2 (FMR2) acts as a potent transcription activator; Hillman MA et al.; Expansion of the FRAXE CCG repeat to a full mutation is associated with methylation and transcriptional silencing of the FMR2 gene, and as a consequence, mild-to-borderline mental retardation . FMR2 is a member of a family of four proteins, AF4, LAF4, FMR2, and AF5q31 . The proteins associated with this family localize to the cell nucleus . Various regions of FMR2, and each of the other members of the protein family, were cloned and analyzed for transcription activation in yeast and mammalian cells . In both yeast and mammalian cells, FMR2 showed strong transcription activation . AF4 activation potential was several-fold lower . Interestingly, isoforms of both FMR2 and LAF4 lacking exon 3 activated transcription better than the larger isoforms containing exon 3 . Compared with the other members of the family, activation by FMR2 was the strongest . Our results show that FMR2 is a potent transcription activator and that its function is conserved . Elucidation of the function of the FMR2 protein as a transcription activator may place FMR2 within the molecular signalling pathways involved in nonspecific X-linked mental retardation (MRX). Bioorg Med Chem Lett, 2001 May 7, 11(9), 1137 - 9 Glycosidase inhibition by cyclic sulfonium compounds; Yuasa H et al.; Inhibitory activities of various cyclic sulfonium compounds including salacinol against several glycosidases were studied and some compounds showed significant inhibition . The sulfonium ion structure was found to be essential for the inhibitory activity . Specific inhibition of salacinol toward rice alpha-glucosidase was ascribed to the tether arm. Proc Natl Acad Sci U S A, 2001 May 22, 98(11), 6267 - 72 Epub 2001 May 15. BimD/SPO76 is at the interface of cell cycle progression, chromosome morphogenesis, and recombination; van Heemst D et al.; BIMD of Aspergillus nidulans belongs to a highly conserved protein family implicated, in filamentous fungi, in sister-chromatid cohesion and DNA repair . We show here that BIMD is chromosome associated at all stages, except from late prophase through anaphase, during mitosis and meiosis, and is involved in several aspects of both programs . First, bimD(+) function must be executed during S through M . Second, in bimD6 germlings, mitotic nuclear divisions and overall cellular program occur more rapidly than in wild type . Thus, BIMD, an abundant chromosomal protein, is a negative regulator of normal cell cycle progression . Third, bimD6 reduces the level of mitotic interhomolog recombination but does not alter the ratio between crossover and noncrossover outcomes . Moreover, bimD6 is normal for intrachromosomal recombination . Therefore, BIMD is probably not involved in the enzymology of recombinational repair per se . Finally, during meiosis, staining of the Sordaria ortholog Spo76p delineates robust chromosomal axes, whereas BIMD stains all chromatin . SPO76 and bimD are functional homologs with respect to their roles in mitotic chromosome metabolism but not in meiosis . We propose that BIMD exerts its diverse influences on cell cycle progression as well as chromosome morphogenesis and recombination by modulating chromosome structure. J Cell Biol, 2001 May 14, 153(4), 745 - 62 Biogenesis of the signal recognition particle (SRP) involves import of SRP proteins into the nucleolus, assembly with the SRP-RNA, and Xpo1p-mediated export; Grosshans H et al.; The signal recognition particle (SRP) targets nascent secretory proteins to the ER, but how and where the SRP assembles is largely unknown . Here we analyze the biogenesis of yeast SRP, which consists of an RNA molecule (scR1) and six proteins, by localizing all its components . Although scR1 is cytoplasmic in wild-type cells, nuclear localization was observed in cells lacking any one of the four SRP "core proteins" Srp14p, Srp21p, Srp68p, or Srp72p . Consistently, a major nucleolar pool was detected for these proteins . Sec65p, on the other hand, was found in both the nucleoplasm and the nucleolus, whereas Srp54p was predominantly cytoplasmic . Import of the core proteins into the nucleolus requires the ribosomal protein import receptors Pse1p and Kap123p/Yrb4p, which might, thus, constitute a nucleolar import pathway . Nuclear export of scR1 is mediated by the nuclear export signal receptor Xpo1p, is distinct from mRNA transport, and requires, as evidenced by the nucleolar accumulation of scR1 in a dis3/rrp44 exosome component mutant, an intact scR1 3' end . A subset of nucleoporins, including Nsp1p and Nup159p (Rat7p), are also necessary for efficient translocation of scR1 from the nucleus to the cytoplasm . We propose that assembly of the SRP requires import of all SRP core proteins into the nucleolus, where they assemble into a pre-SRP with scR1 . This particle can then be targeted to the nuclear pores and is subsequently exported to the cytoplasm in an Xpo1p-dependent way. J Cell Biol, 2001 May 14, 153(4), 709 - 24 A link between the synthesis of nucleoporins and the biogenesis of the nuclear envelope; Marelli M et al.; The nuclear pore complex (NPC) is a multicomponent structure containing a subset of proteins that bind nuclear transport factors or karyopherins and mediate their movement across the nuclear envelope . By altering the expression of a single nucleoporin gene, NUP53, we showed that the overproduction of Nup53p altered nuclear transport and had a profound effect on the structure of the nuclear membrane . Strikingly, conventional and immunoelectron microscopy analysis revealed that excess Nup53p entered the nucleus and associated with the nuclear membrane . Here, Nup53p induced the formation of intranuclear, tubular membranes that later formed flattened, double membrane lamellae structurally similar to the nuclear envelope . Like the nuclear envelope, the intranuclear double membrane lamellae enclosed a defined cisterna that was interrupted by pores but, unlike the nuclear envelope pores, they lacked NPCs . Consistent with this observation, we detected only two NPC proteins, the pore membrane proteins Pom152p and Ndc1p, in association with these membrane structures . Thus, these pores likely represent an intermediate in NPC assembly . We also demonstrated that the targeting of excess Nup53p to the NPC and its specific association with intranuclear membranes were dependent on the karyopherin Kap121p and the nucleoporin Nup170p . At the nuclear envelope, the abilities of Nup53p to associate with the membrane and drive membrane proliferation were dependent on a COOH-terminal segment containing a potential amphipathic alpha-helix . The implications of these results with regards to the biogenesis of the nuclear envelope are discussed. J Cell Biol, 2001 May 14, 153(4), 649 - 62 Components of a ubiquitin ligase complex specify polyubiquitination and intracellular trafficking of the general amino acid permease; Helliwell SB et al.; Gap1p, the general amino acid permease of Saccharomyces cerevisiae, is regulated by intracellular sorting decisions that occur in either Golgi or endosomal compartments . Depending on nitrogen source, Gap1p is transported to the plasma membrane, where it functions for amino acid uptake, or to the vacuole, where it is degraded . We found that overexpression of Bul1p or Bul2p, two nonessential components of the Rsp5p E3-ubiquitin ligase complex, causes Gap1p to be sorted to the vacuole regardless of nitrogen source . The double mutant bul1Delta bul2Delta has the inverse phenotype, causing Gap1p to be delivered to the plasma membrane more efficiently than in wild-type cells . In addition, bul1Delta bul2Delta can reverse the effect of lst4Delta, a mutation that normally prevents Gap1p from reaching the plasma membrane . Evaluation of Gap1p ubiquitination revealed a prominent polyubiquitinated species that was greatly diminished in a bul1Delta bul2Delta mutant . Both a rsp5-1 mutant and a COOH-terminal truncation of Gap1p behave as bul1Delta bul2Delta, causing constitutive delivery of Gap1p to the plasma membrane and decreasing Gap1p polyubiquitination . These results indicate that Bul1p and Bul2p, together with Rsp5p, generate a polyubiquitin signal on Gap1p that specifies its intracellular targeting to the vacuole. J Biol Chem, 2001 Jul 13, 276(28), 26526 - 33 Epub 2001 May 14. Interaction of gamma 1-syntrophin with diacylglycerol kinase-zeta . Regulation of nuclear localization by PDZ interactions; Hogan A et al.; Syntrophins are modular adapter proteins that link ion channels and signaling proteins to dystrophin and its homologues . A yeast two-hybrid screen of a human brain cDNA library using the PDZ domain of gamma 1- syntrophin, a recently identified brain-specific isoform, yielded overlapping clones encoding the C terminus of diacylglycerol kinase-zeta (DGK-zeta), an enzyme that converts diacylglycerol into phosphatidic acid . In biochemical assays, the C terminus of DGK-zeta, which contains a consensus PDZ-binding motif, was found to be necessary and sufficient for association with gamma 1-syntrophin . When coexpressed in HeLa cells, DGK-zeta and gamma 1-syntrophin formed a stable complex that partitioned between the cytoplasm and nucleus . DGK-zeta translocates from the cytosol to the nucleus, a process negatively regulated by protein kinase C phosphorylation . We found that DGK-zeta recruits gamma 1-syntrophin into the nucleus and that the PDZ-binding motif is required . Disrupting the interaction altered the intracellular localization of both proteins; DGK-zeta accumulated in the nucleus, whereas gamma 1-syntrophin remained in the cytoplasm . The level of endogenous syntrophins in the nucleus of HeLa cells also reflected the amount of nuclear DGK-zeta . In the brain, DGK-zeta and gamma 1-syntrophin were colocalized in cell bodies and dendrites of cerebellar Purkinjie neurons and other neuronal cell types, suggesting that their interaction is physiologically relevant . Moreover, coimmunoprecipitation and pull-down experiments from brain extracts and cells suggest that DGK-zeta, gamma 1-syntrophin, and dystrophin form a ternary complex . Collectively, our results suggest that gamma 1-syntrophin participates in regulating the subcellular localization of DGK-zeta to ensure correct termination of diacylglycerol signaling. Biochem Biophys Res Commun, 2001 Apr 13, 282(4), 916 - 20 The deubiquitinating enzyme Dot4p is involved in regulating nutrient uptake; Kahana A; In yeast, several membrane-bound nutrient transporters have been shown to be regulated by the covalent attachment of ubiquitin, a signal for internalization and degradation . The yeast gene DOT4 encodes one of a family of enzymes which remove ubiquitin from proteins to which the peptide has been attached . Mutations in DOT4 cause a growth defect that is particularly severe when combined with mutations in nutrient biosynthetic enzymes (1) . These results suggest that nutrient transport or utilization may be compromised in dot4 mutants . We now report that preventing the down-regulation by endocytosis of membrane proteins partially suppressed the dot4Delta growth defect . We also show that the activity of the amino acid permease Gap1p is reduced in DOT4 mutants . This correlates with a reduction in Gap1 protein level, while GAP1 mRNA levels remains unchanged . We conclude that Dot4p is involved in posttranscriptionally regulating Gap1p, and possibly other transporters as well . Int J Cancer, 2001 Jun 15, 92(6), 784 - 9 Induction of histone acetylation in mouse erythroleukemia cells by some organosulfur compounds including allyl isothiocyanate; Lea MA et al.; In previous studies we observed that some allyl sulfides can cause increased acetylation of histones and differentiation in DS19 mouse erythroleukemia cells . In the present work we observed increased acetylation of histones with allyl isothiocyanate and butanethiol but not with butyl sulfide or butyl disulfide . Increased acetylation of histones was established by change in electrophoretic mobility, incorporation of {3H}acetate or immunoblotting . Histone deacetylase in nuclei of DS19 cells was inhibited 74% by 0.5 mM allyl mercaptan and 43% by 0.5 mM butanethiol but was not significantly affected by 0.5 mM allyl isothiocyanate . There was some degree of reversibility in the effect of allyl isothiocyanate when the cells were incubated for 15 hr in fresh medium . The data suggested that allyl isothiocyanate may stimulate histone acetylation rather than inhibit histone deacetylation . Addition of allyl isothiocyanate, however, had very little or no additional effect on the induction of histone acetylation caused by trichostatin A . Histone acetyltransferase activity determined in cell homogenates was not increased by preincubation of cells with allyl isothiocyanate or inclusion of allyl isothiocyanate in the assay medium . It was concluded that treatment of mouse erythroleukemia cells with allyl isothiocyanate can cause increased acetylation of histones but the mechanism for this effect requires further elucidation . J Biol Chem, 2001 May 18, 276(20), 16944 - 51 Epub 2001 Feb 28. Gcn2 mediates Gcn4 activation in response to glucose stimulation or UV radiation not via GCN4 translation; Marbach I et al.; In mammalian cells transcription factors of the AP-1 family are activated by either stress signals such as UV radiation, or mitogenic signals such as growth factors . Here we show that a similar situation exists in the yeast Saccharomyces cerevisiae . The AP-1 transcriptional activator Gcn4, known to be activated by stress signals such as UV radiation and amino acids starvation, is also induced by growth stimulation such as glucose . We show that glucose-dependent Gcn4 activation is mediated through the Ras/cAMP pathway . This pathway is also responsible for UV-dependent Gcn4 activation but is not involved in Gcn4 activation by amino acid starvation . Thus, the unusual phenomenon of activation of mitogenic pathways and AP-1 factors by contradictory stimuli through Ras is conserved from yeast to mammals . We also show that activation of Gcn4 by glucose and UV requires Gcn2 activity . However, in contrast to its role in amino acid starvation, Gcn2 does not increase eIF2alpha phosphorylation or translation of GCN4 mRNA in response to glucose or UV . These findings suggest a novel mechanism of action for Gcn2 . The finding that Gcn4 is activated in response to glucose via the Ras/cAMP pathway suggests that this cascade coordinates glucose metabolism with amino acids and purine biosynthesis and thereby ensures availability of both energy and essential building blocks for continuation of the cell cycle. EMBO J, 2001 May 15, 20(10), 2536 - 44 Dnmt3a binds deacetylases and is recruited by a sequence-specific repressor to silence transcription; Fuks F et al.; The Dnmt3a DNA methyltransferase is essential for mammalian development and is responsible for the generation of genomic methylation patterns, which lead to transcriptional silencing . Here, we show that Dnmt3a associates with RP58, a DNA-binding transcriptional repressor protein found at transcriptionally silent heterochromatin . Dnmt3a acts as a co-repressor for RP58 in a manner that does not require its de novo methyltransferase activity . Like other characterized co-repressors, Dnmt3a associates with the histone deacetylase HDAC1 using its ATRX-homology domain . This domain of Dnmt3a represents an independent transcriptional repressor domain whose silencing functions require HDAC activity . These results identify Dnmt3a as a co-repressor protein carrying deacetylase activity and show that Dnmt3a can be targeted to specific regulatory foci via its association with DNA-binding transcription factors. EMBO J, 2001 May 15, 20(10), 2528 - 35 Positive feedback in eukaryotic gene networks: cell differentiation by graded to binary response conversion; Becskei A et al.; Feedback is a ubiquitous control mechanism of gene networks . Here, we have used positive feedback to construct a synthetic eukaryotic gene switch in Saccharomyces cerevisiae . Within this system, a continuous gradient of constitutively expressed transcriptional activator is translated into a cell phenotype switch when the activator is expressed autocatalytically . This finding is consistent with a mathematical model whose analysis shows that continuous input parameters are converted into a bimodal probability distribution by positive feedback, and that this resembles analog-digital conversion . The autocatalytic switch is a robust property in eukaryotic gene expression . Although the behavior of individual cells within a population is random, the proportion of the cell population displaying either low or high expression states can be regulated . These results have implications for understanding the graded and probabilistic mechanisms of enhancer action and cell differentiation. EMBO J, 2001 May 15, 20(10), 2435 - 42 The role of Sis1 in the maintenance of the {RNQ+} prion; Sondheimer N et al.; Yeast prions are inherited through proteins that exist in alternate, self-perpetuating conformational states . The mechanisms by which these states arise and are maintained are still poorly defined . Here we demonstrate for the first time that Sis1, a member of the Hsp40 chaperone family, plays a critical role in the maintenance of a prion . The prion {RNQ+} is formed by Rnq1, which is present in the same physical complex as Sis1, but only when Rnq1 is in the prion state . The G/F domain of Sis1 is dispensable for rapid growth on rich medium, but is required for {RNQ+} maintenance, distinguishing essential regions of Sis1 from those needed for prion interaction . A specific Sis1 deletion mutant altered the physical aggregation pattern of Rnq1 without curing the prion . This variant state propagated in a heritable fashion after wild-type Sis1 function was restored, indicating that multiple physical states are compatible with prion maintenance and that changes in chaperone activity can create prion variants . Using a prion chimera we demonstrate that the prion-determinant domain of Rnq1 is genetically sufficient for control by Sis1. Clin Cancer Res, 2001 May, 7(5), 1287 - 92 Significant correlation between interleukin 10 expression and vascularization through angiopoietin/TIE2 networks in non-small cell lung cancer; Hatanaka H et al.; The expression of interleukin 10 (IL-10) is correlated with clinical prognosis in non-small cell lung cancer {NSCLC (H . Hatanaka et al., ANN: ONCOL:, 11: 815--819, 2000)} . However, the effects of IL-10 expression on vascularization in NSCLC are not apparent . We examined the gene expression of IL-10/IL-10 receptor and various angiogenic/angioinhibitory factors in 95 NSCLC samples to determine the correlation between IL-10 production and vascularization . Vascular endothelial growth factor, angiopoietin {Ang (Ang-1 and Ang-2)}, thrombospondin, brain-specific angiogenesis inhibitor 1, vascular endothelial growth factor receptors (KDR and flt-1), and Ang receptor (TIE2) gene expression were evaluated by reverse transcription-PCR . The cellular localization of these factors and vascularity in the cancer stroma were examined immunohistochemically . Seventy-eight (82.1%) and 93 (97.9%) of these 95 NSCLCs were positive for IL-10 and IL-10 receptor, respectively . Ang-1, Ang-2, and TIE2 gene expression was seen in 76 (97.4%), 73 (93.6%), and 78 (100%) of 78 IL-10-positive NSCLCs, respectively, and was significantly correlated with IL-10 gene expression (P < 0.0088, <0.0008, and 0.0305, respectively; Fisher's exact method) . The localizations of Ang-1, Ang-2, and TIE2 were confirmed within tumor cells immunohistochemically . Vascular number and measurement area were significantly higher in the IL-10-positive NSCLCs (33.500 +/- 9.299/microm(2) and 4.742 +/- 1.287%) as compared with IL-10-negative NSCLCs (10.611 +/- 2.839/microm(2) and 0.718 +/- 0.331%; Mann-Whitney U test, P = 0.0039) . The IL-10 expression did not show any significant correlation with the expression of other factors . These results suggested that tumor-produced IL-10 promotes stromal vascularization through expression of Ang-1, Ang-2, and TIE2. Traffic, 2001 May, 2(5), 321 - 35 Evidence for prebudding arrest of ER export in animal cell mitosis and its role in generating Golgi partitioning intermediates; Prescott AR et al.; During mitosis the interconnected Golgi complex of animal cells breaks down to produce both finely dispersed elements and discrete vesiculotubular structures . The endoplasmic reticulum (ER) plays a controversial role in generating these partitioning intermediates and here we highlight the importance of mitotic ER export arrest in this process . We show that experimental inhibition of ER export (by microinjecting dominant negative Sar1 mutant proteins) is sufficient to induce and maintain transformation of Golgi cisternae to vesiculotubular remnants during interphase and telophase, respectively . We also show that buds on the ER, ER exit sites and COPII vesicles are markedly depleted in mitotic cells and COPII components Sec23p, Sec24p, Sec13p and Sec31p redistribute into the cytosol, indicating ER export is inhibited at an early stage . Finally, we find a markedly uneven distribution of Golgi residents over residual exit sites of metaphase cells, consistent with tubulovesicular Golgi remnants arising by fragmentation rather than redistribution via the ER . Together, these results suggest selective recycling of Golgi residents, combined with prebudding cessation of ER export, induces transformation of Golgi cisternae to vesiculotubular remnants in mitotic cells . The vesiculotubular Golgi remnants, containing populations of slow or nonrecycling Golgi components, arise by fragmentation of a depleted Golgi ribbon independently from the ER. Mol Genet Metab, 2001 May, 73(1), 71 - 6 Functional analysis of novel mutations in a congenital disorder of glycosylation Ia patient with mixed Asian ancestry; Westphal V et al.; Congenital disorders of glycosylation (CDG) are caused by autosomal recessive mutations in genes affecting N-glycan biosynthesis . Mutations in the PMM2 gene, which encodes the enzyme phosphomannomutase (mannose 6-phosphate <--> mannose 1-phosphate), give rise to the most common form: CDG-Ia . These patients typically present with dysmorphic features and neurological abnormalities, cerebellar hypoplasia, ataxia, hypotonia, and coagulopathy, in addition to feeding problems . However, the clinical symptoms vary greatly . The great majority of known CDG-Ia patients are of European descent where the most common mutant alleles originated . This ethnic bias can also be explained by lack of global awareness of the disorder . Here we report an Asian patient with prominent systemic features that we diagnosed with CDG-Ia resulting from two new mutations in the PMM2 gene (310C --> G resulting in L104V and an intronic mutation IVS1-1G --> A) . The latter mutation seems to result in lower mRNA levels, and the L104V has been functionally analyzed in a yeast expression system together with known mutations . The Filipino and Cambodian origins of the parents show that CDG-Ia mutations occur in these ethnic groups as well as in Caucasians . Mol Genet Metab, 2001 May, 73(1), 46 - 54 Molecular and enzymatic characterization of a unique carnitine palmitoyltransferase 1A mutation in the Hutterite community; Prip-Buus C et al.; Hepatic carnitine palmitoyltransferase 1 (CPT1A) deficiency is a rare disorder of mitochondrial fatty acid oxidation inherited as an autosomal recessive trait . Symptomatology comprises attacks of hypoketotic hypoglycemia with risk of sudden death or neurological sequelae . Only one CPT1A mutation has been reported so far . Identification of the disease-causing mutations allows both insights into the structure-function relationships of CPT1A and management of the patients and their relatives . The molecular analysis of CPT1A deficiency in a large Hutterite kindred illustrates this point . Both cDNA and genomic DNA analysis demonstrate that the affected patients are homozygous for a 2129G>A mutation predicting a G710E substitution . Studies in fibroblasts from one patient as well as heterologous expression of the mutagenized CPT1A in yeast show that the G710E mutation alters neither mitochondrial targeting nor stability of the CPT1A protein . By contrast, kinetic studies conclusively establish that the mutant CPT1A is totally inactive, indicating that the G710E mutation dramatically impairs the catalytic function of CPT1A . Finally, due to a strongly suspected founder effect for the origin of CPT1A deficiency in this Hutterite kindred, identification of this disease-causing mutation allows the setup of a targeted DNA-based newborn screening in this at-risk population . J Mol Biol, 2001 May 11, 308(4), 609 - 22 A comprehensive characterization of a group IB intron and its encoded maturase reveals that protein-assisted splicing requires an almost intact intron RNA; Geese WJ et al.; The group I intron (AnCOB) of the mitochondrial apocytochrome b gene from Aspergillus nidulans encodes a bi-functional maturase protein that is also a DNA endonuclease . Although the AnCOB intron self-splices, the encoded maturase protein greatly facilitates splicing, in part, by stabilizing RNA tertiary structure . To determine their role in self-splicing and in protein-assisted splicing, several peripheral RNA sub-domains in the 313 nucleotide intron were deleted (P2, P9, P9.1) or truncated (P5ab, P6a) . The sequence in two helices (P2 and P9) was also inverted . Except for P9, the deleted regions are not highly conserved among group I introns and are often dispensable for catalytic activity . Nevertheless, despite the very tight binding of AnCOB RNA to the maturase and the high activity of the bimolecular complex (the rate of 5' splice-site cleavage was >20 min(-1) with guanosine as the cofactor), the intron was surprisingly sensitive to these modifications . Several mutations inactivated splicing completely and virtually all impaired splicing to varying degrees . Mutants containing comparatively small deletions in various regions of the intron significantly decreased binding affinity (generally >10(4)-fold), indicating that none of the domains that remained constitutes the primary recognition site of the maturase . The data argue that tight binding requires tertiary interactions that can be maintained by only a relatively intact intron RNA, and that the binding mechanism of the maturase differs from those of two other well-characterized group I intron splicing factors, CYT-18 and Cpb2 . A model is proposed in which the protein promotes widespread cooperative folding of an RNA lacking extensive initial tertiary structure . RNA, 2001 May, 7(5), 753 - 64 Defects in the mRNA export factors Rat7p, Gle1p, Mex67p, and Rat8p cause hyperadenylation during 3'-end formation of nascent transcripts; Hilleren P et al.; The biosynthesis and function of eukaryotic mRNAs requires a series of events including nuclear polyadenylation, transport to the cytoplasm, translation, and ultimately mRNA degradation . To identify the interrelationships between these events, we examined the synthesis and fate of mRNAs in several strains defective in mRNA export . Strains carrying lesions in RAT7, GLE1, MEX67, and RAT8, produce nascent transcripts carrying poly(A) tails roughly 30 residues longer than the nascent poly(A) tails observed in wild type . In the rat7-1, rat8-2, and mex67-5 strains, the hyperadenylated transcripts undergo a novel form of deadenylation to chase into a population with normal poly(A) tail lengths, which cofractionate with polysomes, undergo nonsense-mediated decay, and are degraded by the normal cytoplasmic decay machinery . This suggests a relationship between the mechanism of processing to a normal poly(A) tail length and the ability of these transcripts to proceed in their metabolism . These observations provide further support for the view that mRNA 3'-end formation and mRNA export are mechanistically coupled events. RNA, 2001 May, 7(5), 682 - 91 Crystal structure of a model branchpoint-U2 snRNA duplex containing bulged adenosines; Berglund JA et al.; Bulged nucleotides play a variety of important roles in RNA structure and function, frequently forming tertiary interactions and sometimes even participating in RNA catalysis . In pre-mRNA splicing, the U2 snRNA base pairs with the intron branchpoint sequence (BPS) to form a short RNA duplex that contains a bulged adenosine that ultimately serves as the nucleophile that attacks the 5' splice site . We have determined a 2.18-A resolution crystal structure of a self-complementary RNA designed to mimic the highly conserved yeast (Saccharomyces cerevisiae) branchpoint sequence (5'-UACUAACGUAGUA with the BPS italicized and the branchsite adenosine underlined) base paired with its complementary sequence from U2 snRNA . The structure shows a nearly ideal A-form helix from which two unpaired adenosines flip out . Although the adenosine adjacent to the branchsite adenosine is the one bulged out in the structure described here, either of these adenosines can serve as the nucleophile in mammalian but not in yeast pre-mRNA splicing . In addition, the packing of the bulged RNA helices within the crystal reveals a novel RNA tertiary interaction in which three RNA helices interact through bulged adenosines in the absence of any divalent metal ions. Infect Immun, 2001 Jun, 69(6), 3933 - 8 Normal human fibroblasts express pattern recognition receptors for fungal (1-->3)-beta-D-glucans; Kougias P et al.; Fungal cell wall glucans nonspecifically stimulate various aspects of innate immunity . Glucans are thought to mediate their effects via interaction with membrane receptors on macrophages, neutrophils, and NK cells . There have been no reports of glucan receptors on nonimmune cells . We investigated the binding of a water-soluble glucan in primary cultures of normal human dermal fibroblasts (NHDF) . Membranes from NHDF exhibited saturable binding with an apparent dissociation constant (K(D)) of 8.9 +/- 1.9 microg of protein per ml and a maximum binding of 100 +/- 8 resonance units . Competition studies demonstrated the presence of at least two glucan binding sites on NHDF . Glucan phosphate competed for all binding sites, with a K(D) of 5.6 microM (95% confidence interval {CI}, 3.0 to 11 microM), while laminarin competed for 69% +/- 6% of binding sites, with a K(D) of 3.7 microM (95% CI, 1.9 to 7.3 microM) . Glucan (1 microg/ml) stimulated fibroblast NF-kappaB nuclear binding activity and interleukin 6 (IL-6) gene expression in a time-dependent manner . NF-kappaB was activated at 4, 8, and 12 h, while IL-6 mRNA levels were increased by 48% at 8 h . This is the first report of pattern recognition receptors for glucan on human fibroblasts and the first demonstration of glucan binding sites on cells other than leukocytes . It also provides the first evidence that glucans can directly modulate the functional activity of NHDF . These results provide new insights into the mechanisms by which the host recognizes and responds to fungal (1-->3)-beta-D-glucans and suggests that the response to glucans may not be confined to cells of the immune system. Mech Ageing Dev, 2001 Jul 15, 122(9), 921 - 44 Unwinding the molecular basis of the Werner syndrome; Shen J et al.; Werner syndrome (WS) is an autosomal recessive disease manifested by the premature onset of age-related phenotypes, including diseases such as atherosclerosis and cancer . This mimicry of normal aging with the possible exception of central nervous system manifestations has made it a focus of recent molecular studies on the pathophysiology of aging . In culture, cells obtained from patients with WS are genetically unstable, characterized by an increased frequency of nonclonal translocations and extensive DNA deletions . The WS gene product (WRN) is a DNA helicase belonging to the RecQ family, but is unique within this family in that it also contains an exonuclease activity . In addition to unwinding double-stranded DNA, WRN helicase is able to resolve aberrant DNA structures such as G4 tetraplexes, triplexes and 4-way junctions . Concordant with this structure-specificity, WRN exonuclease preferentially hydrolyzes alternative DNA that contains bubbles, extra-helical loops, 3-way junctions or 4-way junctions . WRN has been shown to bind to and/or functionally interact with other proteins, including replication protein A (RPA), proliferating cell nuclear antigen (PCNA), DNA topoisomerase I, Ku 86/70, DNA polymerase delta and p53 . Each of these interacting proteins is involved in DNA transactions including those that resolve alternative DNA structures or repair DNA damage . The biochemical activities of WRN and the functions of WRN associated proteins suggest that in vivo WRN resolves DNA topological or structural aberrations that either occur during DNA metabolic processes such as recombination, replication and repair, or are the outcome of DNA damage. Cell, 2001 May 4, 105(3), 403 - 14 Silenced chromatin is permissive to activator binding and PIC recruitment; Sekinger EA et al.; Chromatin is thought to repress transcription by limiting access of the DNA to transcription factors . Using a yeast heat shock gene flanked by mating-type silencers as a model system, we find that repressive, SIR-generated heterochromatin is permissive to the constitutive binding of an activator, HSF, and two components of the preinitiation complex (PIC), TBP and Pol II . These factors cohabitate the promoter with Sir silencing proteins and deacetylated nucleosomal histones . The heterochromatic HMRa1 promoter is also occupied by TBP and Pol II, suggesting that SIR regulates gene expression not by restricting factor access to DNA but rather by blocking a step downstream of PIC recruitment . Interestingly, activation of silent promoter chromatin occurs in the absence of histone displacement and without change in histone acetylation state. Biol Chem, 2001 Mar, 382(3), 443 - 8 Effects of cysteine to serine substitutions in the two inter-chain disulfide bonds of insulin; Guo ZY et al.; Using site-directed mutagenesis we deleted the two inter-chain disulfide bonds of insulin, separately or both, by substitution of the cysteine residues with serine . Deletion of A20-B19 or both of the two inter-chain disulfide bonds resulted in the complete loss of secretion of the mutant single-chain porcine insulin precursor (PIP) from Saccharomyces cerevisiae cells . Removal of the A7-B7 disulfide bond resulted in a large reduction of secretion, but we could obtain the mutant for analysis of its biological and some physico-chemical properties . The A7-B7 disulfide bond deleted insulin mutant retained only 0.1% receptor-binding activity compared with porcine insulin, and its in vivo biological potency measured by mouse convulsion assay was also very low . We also studied some physico-chemical properties of the mutant using circular dichroism, native polyacrylamide gel electrophoresis and reversed-phase HPLC, which revealed some structural changes of the mutant peptides compared to native insulin . The present study shows that the two inter-chain disulfide bonds are important for efficient in vivo folding/secretion of PIP from yeast, especially the A20-B19 disulfide bond, and that the A7-B7 disulfide bond is crucial for maintaining the native conformation and biological activity of insulin. J Biol Chem, 2001 Jul 20, 276(29), 26947 - 54 Epub 2001 May 09. Multiple mechanisms regulate subcellular localization of human CDC6; Delmolino LM et al.; CDC6 is a protein essential for DNA replication, the expression and abundance of which are cell cycle-regulated in Saccharomyces cerevisiae . We have demonstrated previously that the subcellular localization of the human CDC6 homolog, HsCDC6, is cell cycle-dependent: nuclear during G(1) phase and cytoplasmic during S phase . Here we demonstrate that endogenous HsCDC6 is phosphorylated during the G(1)/S transition . The N-terminal region contains putative cyclin-dependent kinase phosphorylation sites adjoining nuclear localization sequences (NLSs) and a cyclin-docking motif, whereas the C-terminal region contains a nuclear export signal (NES) . In addition, we show that the observed regulated subcellular localization depends on phosphorylation status, NLS, and NES . When the four putative substrate sites (serines 45, 54, 74, and 106) for cyclin-dependent kinases are mutated to alanines, the resulting HsCDC6A4 protein is localized predominantly to the nucleus . This localization depends upon two functional NLSs, because expression of HsCDC6 containing mutations in the two putative NLSs results in predominantly cytoplasmic distribution . Furthermore, mutation of the four serines to phosphate-mimicking aspartates (HsCDC6D4) results in strictly cytoplasmic localization . This cytoplasmic localization depends upon the C-terminal NES . Together these results demonstrate that HsCDC6 is phosphorylated at the G(1)/S phase of the cell cycle and that the phosphorylation status determines the subcellular localization. Immunogenetics, 2001 Mar, 53(2), 87 - 94 PAProC: a prediction algorithm for proteasomal cleavages available on the WWW; Nussbaum AK et al.; The first version of PAProC (Prediction Algorithm for Proteasomal Cleavages) is now available to the general public . PAProC is a prediction tool for cleavages by human and yeast proteasomes, based on experimental cleavage data . It will be particularly useful for immunologists working on antigen processing and the prediction of major histocompatibility complex class I molecule (MHC I) ligands and cytotoxic T-lymphocyte (CTL) epitopes . Likewise, in cases in which proteasomal protein degradation has been indicated in disease, PAProC can be used to assess the general cleavability of disease-linked proteins . On its web site , background information and hyperlinks are provided for the user (e.g., to SYFPEITHI, the database for the prediction of MHC I ligands). RNA, 2001 Apr, 7(4), 565 - 75 An essential protein-binding domain of nuclear RNase P RNA; Ziehler WA et al.; Eukaryotic RNase P and RNase MRP are endoribonucleases composed of RNA and protein subunits . The RNA subunits of each enzyme share substantial secondary structural features, and most of the protein subunits are shared between the two . One of the conserved RNA subdomains, designated P3, has previously been shown to be required for nucleolar localization . Phylogenetic sequence analysis suggests that the P3 domain interacts with one of the proteins common to RNase P and RNase MRP, a conclusion strengthened by an earlier observation that the essential domain can be interchanged between the two enzymes . To examine possible functions of the P3 domain, four conserved nucleotides in the P3 domain of Saccharomyces cerevisiae RNase P RNA (RPR1) were randomized to create a library of all possible sequence combinations at those positions . Selection of functional genes in vivo identified permissible variations, and viable clones that caused yeast to exhibit conditional growth phenotypes were tested for defects in RNase P RNA and tRNA biosynthesis . Under nonpermissive conditions, the mutants had reduced maturation of the RPR1 RNA precursor, an expected phenotype in cases where RNase P holoenzyme assembly is defective . This loss of RPR1 RNA maturation coincided, as expected, with a loss of pre-tRNA maturation characteristic of RNase P defects . To test whether mutations at the conserved positions inhibited interactions with a particular protein, specific binding of the individual protein subunits to the RNA subunit was tested in yeast using the three-hybrid system . Pop1p, the largest subunit shared by RNases P and MRP, bound specifically to RPR1 RNA and the isolated P3 domain, and this binding was eliminated by mutations at the conserved P3 residues . These results indicate that Pop1p interacts with the P3 domain common to RNases P and MRP, and that this interaction is critical in the maturation of RNase P holoenzyme. Biotechnol Bioeng, 2001 Jun 20, 73(6), 465 - 75 Purification of recombinant cutinase by extraction in an aqueous two-phase system facilitated by a fatty acid substrate; Fernandes S et al.; Purification of recombinant wild-type cutinase from the culture supernatant of Saccharomyces cerevisiae by extraction in aqueous two-phase system was investigated . The partition of the enzyme in a polyethylene glycol (PEG)-potassium phosphate system to the top phase was increased with lower molecular weight PEG . Enzyme partition in a 20% PEG/15% phosphate two-phase system was studied in the presence of detergents, fatty acids, and alcohols, respectively . Addition of 0.5% (w/w) butyrate increased the partition coefficient from 17 to 135 and the purification factor from 10 to 23 . The effect of butyrate was also confirmed by using the countercurrent mode of extraction . Recovery of cutinase from the top phase was achieved by a secondary extraction into a new salt phase at a lower pH or a lower temperature . A specific interaction of butyrate to the active site of the enzyme was demonstrated by fluorescence spectroscopy . Size exclusion chromatography showed the cutinase-butyrate complex to be over two times the size of the free enzyme . J Biol Chem, 2001 Jul 13, 276(28), 25856 - 61 Epub 2001 May 08. Modular structure of the TIM23 preprotein translocase of mitochondria; Milisav I et al.; The TIM23 complex mediates import into mitochondria of nuclear encoded preproteins with a matrix-targeting signal . It is composed of the integral membrane proteins Tim17 and Tim23 and the peripheral membrane protein Tim44, which recruits mitochondrial Hsp70 to the sites of protein import . We have analyzed the functions of these constituents using a combined genetic and biochemical approach . Depletion of either Tim17 or Tim23 led to loss of import competence of mitochondria and to a reduction in the number of preprotein-conducting channels . Upon depletion of Tim44, mitochondria also lost their ability to import proteins but maintained normal numbers of import channels . In the absence of Tim44 precursor protein was specifically recognized . The presequence was translocated in a Delta psi-dependent manner across the inner membrane and cleaved by matrix-processing peptidase . However, the preprotein did not move further into the matrix but rather underwent retrograde sliding out of the TIM23 complex . Thus, the TIM23 complex is composed of functionally independent modules . Tim17 and Tim23 are necessary for initiating translocation, whereas Tim44 and mitochondrial Hsp70 are indispensable for complete transport of preproteins and for unfolding of folded domains of preproteins. J Bacteriol, 2001 Jun, 183(11), 3447 - 57 The CDC42 homolog of the dimorphic fungus Penicillium marneffei is required for correct cell polarization during growth but not development; Boyce KJ et al.; The opportunistic human pathogenic fungus Penicillium marneffei is dimorphic and is thereby capable of growth either as filamentous multinucleate hyphae or as uninucleate yeast cells which divide by fission . The dimorphic switch is temperature dependent and requires regulated changes in morphology and cell shape . Cdc42p is a Rho family GTPase which in Saccharomyces cerevisiae is required for changes in polarized growth during mating and pseudohyphal development . Cdc42p homologs in higher organisms are also associated with changes in cell shape and polarity . We have cloned a highly conserved CDC42 homolog from P . marneffei named cflA . By the generation of dominant-negative and dominant-activated cflA transformants, we have shown that CflA initiates polarized growth and extension of the germ tube and subsequently maintains polarized growth in the vegetative mycelium . CflA is also required for polarization and determination of correct cell shape during yeast-like growth, and active CflA is required for the separation of yeast cells . However, correct cflA function is not required for dimorphic switching and does not appear to play a role during the generation of specialized structures during asexual development . In contrast, heterologous expression of cflA alleles in Aspergillus nidulans prevented conidiation. Trends Biochem Sci, 2001 May, 26(5), 310 - 7 Glucose-sensing mechanisms in eukaryotic cells; Rolland F et al.; Glucose not only serves as a nutrient but also exerts many hormone-like regulatory effects in a wide variety of eukaryotic cell types . Recently, interest in identifying general mechanisms and principles used to sense the presence of glucose has significantly increased and promising advances have been made: in yeast, the first proteins with an apparently specific function in glucose detection have been discovered; in plant cells, there is increasing evidence for a diverse array of glucose-induced signalling mechanisms; and in mammals, glucose-sensing phenomena have turned out to be much more widespread than just in the well-known example of pancreatic beta cells. Curr Opin Cell Biol, 2001 Jun, 13(3), 363 - 75 Nuclear pores and nuclear assembly; Vasu SK et al.; Communication between the nucleus and cytoplasm occurs through large macromolecular structures, the nuclear pores . Quantitative scanning transmission electron microscopy has estimated the mass of a nuclear pore to be 60 million Daltons in yeast and 120 million Daltons in vertebrates . The past two years were noteworthy in that they saw: 1) the purification of both the yeast and vertebrate nuclear pores, 2) the initial description of routes through the pore for specific transport receptors, 3) glimpses of intranuclear organization imposed by the nuclear pores and envelope and 4) the revelation of new and pivotal roles for the small GTPase Ran not only in nuclear import but in spindle assembly and nuclear membrane fusion. Curr Opin Cell Biol, 2001 Jun, 13(3), 281 - 9 Turning telomeres off and on; Dubrana K et al.; We envision multiple steps in telomere maintenance, based largely on genetic data from budding yeast . First, the telomere must unfold or open itself such that the free end is accessible to the appropriate enzymatic machinery . Second, telomerase must be recruited, together with the DNA replication machinery that synthesizes the C-rich strand . The processivity of telomerase is regulated both by a length-sensing feedback mechanism and by second-strand synthesis . Finally, the telosome refolds into a protective end structure . If telomerase is nonfunctional, recombination may occur once telomeres are open . Multiple pathways regulate these different steps, producing a highly dynamic chromosomal cap. FEBS Lett, 2001 May 4, 496(1), 31 - 5 Bromodomain factor 1 (Bdf1) protein interacts with histones; Pamblanco M et al.; Using a yeast two-hybrid assay we detected an interaction between the N-terminal region of histone H4 (amino acids 1--59) and a fragment of the bromodomain factor 1 protein (Bdf1p) (amino acids 304--571) that includes one of the two bromodomains of this protein . No interaction was observed using fragments of histone H4 sequence smaller than the first 59 amino acids . Recombinant Bdf1p (rBdf1p) demonstrates binding affinity for histones H4 and H3 but not H2A and H2B in vitro . Moreover, rBdf1p is able to bind histones H3 and H4 having different degrees of acetylation . Finally, we have not detected histone acetyltransferase activity associated with Bdf1p. Hepatology, 2001 May, 33(5), 1206 - 16 Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C-dependent mechanisms in cholestatic rat liver; Beuers U et al.; Ursodeoxycholic acid (UDCA) exerts anticholestatic effects by undefined mechanisms . Previous work suggested that UDCA stimulates biliary exocytosis via Ca(++)- and protein kinase C (PKC)-dependent mechanisms . Therefore, the effect of taurine-conjugated UDCA (TUDCA) was studied in the experimental model of taurolithocholic acid (TLCA)-induced cholestasis on bile flow, hepatobiliary exocytosis, distribution of PKC isoforms, and density of the apical conjugate export pump, Mrp2, in canalicular membranes . Isolated perfused rat livers were preloaded with horseradish peroxidase (HRP), a marker of vesicular exocytosis, and were perfused with bile acids or dimethylsulfoxide (control) only . PKC isoform distribution and membrane density of Mrp2 were studied using immunoblotting and immunoelectron-microscopic techniques . Biliary secretion of the Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione (GS-DNP), was studied in the presence or absence of the PKC inhibitor, bisindolylmaleimide I (BIM-I; 1 micromol/L) . TLCA (10 micromol/L) impaired bile flow by 51%; biliary secretion of HRP and GS-DNP by 46% and 95%, respectively; membrane binding of the Ca(++)-sensitive alpha-isoform of PKC by 32%; and density of Mrp2 in the canalicular membrane by 79% . TUDCA (25 micromol/L) reversed the effects of TLCA on bile flow, secretion of HRP and GS-DNP, and distribution of alpha-PKC . TUDCA reduced membrane binding of epsilon-PKC and increased Mrp2 density 4-fold in canalicular membranes of cholestatic hepatocytes . BIM-I inhibited the effect of TUDCA on GS-DNP secretion in cholestatic livers by 49% without affecting secretion in controls . In conclusion, TUDCA may enhance the secretory capacity of cholestatic hepatocytes by stimulation of exocytosis and insertion of transport proteins into apical membranes via PKC-dependent mechanisms. Hepatology, 2001 May, 33(5), 1036 - 42 Human REG I gene is up-regulated in intrahepatic cholangiocarcinoma and its precursor lesions; Harada K et al.; The Reg I gene (regenerating gene) and its product (Reg protein) are a regenerating and/or proliferating factor(s) of pancreatic islet cells . The ectopic expression of REG Ialpha was shown in colorectal carcinomas, suggesting that REG Ialpha is related to their carcinogenesis . In this study, we examined the expression of REG I in intrahepatic cholangiocarcinoma (ICC) and its precursor lesion (biliary dysplasia) . By polymerase chain reaction and in situ hybridization (ISH) studies using a total of 16 fresh liver specimens, REG Ialpha mRNA was demonstrated in 6 of 11 (55%) ICC cases, but in 0 of 5 (0%) normal livers . Immunohistochemistry for REG I protein was performed in 100 formalin-fixed, paraffin-embedded sections obtained from the 18 cases of ICC alone, 45 hepatolithiasis with ICC (n = 19) or biliary dysplasia (n = 26), 21 hepatolithiasis alone (all with hyperplasia), and 16 normal livers . In ICC, the expression of REG I protein was significantly dependent on the histologic differentiation; 12 of 13 (92%) cases in papillary and well-differentiated, 6 of 16 (38%) cases in moderately differentiated, and 0 of 8 (0%) cases in poorly differentiated types . Moreover, in the lesions of hyperplasia, low-grade dysplasia, and high-grade dysplasia in hepatolithiasis, REG I protein was expressed in 4 of 21 (19%), 7 of 12 (58%), and 13 of 14 (93%) cases, respectively . In normal liver, intrahepatic bile ducts were constantly negative for REG I protein . These findings suggest that neoexpression of REG I is a good marker for biliary mucosa at risk for development of ICC, and also that REG I plays a role in the early stages of biliary carcinogenesis, probably via a cell-proliferative effect. J Neurogenet, 2000 Dec, 14(4), 227 - 43,271 The effects of ectopic white and transformer expression on Drosophila courtship behavior; An X et al.; The sex determining genes of Drosophila males and females function to establish the potential for sex-specific behaviors . Previous studies suggest that ectopic GAL4-directed misexpression of the female-specific isoform of the sex-determining gene transformer (tra) in specific sub-domains of an otherwise male brain can lead to bisexual courtship behavior, thus identifying brain domains that may mediate sex-specific behavior . However, expression of mini-white, the marker gene used in both P{GAL4} and P{UAS(G)} constructs, also induces males to court other males, questioning whether GAL4-mediated tra expression alone can induce bisexual behavior . Here we demonstrate the consequences of inducing mutations in the mini-white genes within P{GAL4} and P{UAS(G)} constructs to generate flies in which a white mutant phenotype is revealed . In these mini-white mutant strains, P{GAL4}-mediated transformer expression alone is both sufficient and necessary to generate bisexual behavior . In addition, using RT-PCR, we reveal the presence of female transcripts of doublesex and fruitless in the brains of otherwise male (XY) flies exhibiting P{GAL4}-directed tra-expression, demonstrating that P{GAL4}-directed tra is functional at the molecular level . We conclude that P{GAL4}-directed misexpression of tra is responsible for the bisexual behavior previously described and that this is mediated via sex-specific splicing of dsx and fru . Our results support the validity of such strategies for identifying regions of the fly brain that underlie sex-specific behaviors. Proteins, 2001 Jun 1, 43(4), 382 - 94 Theoretical evidence of a salt bridge disruption as the initiating process for the alpha1d-adrenergic receptor activation: a molecular dynamics and docking study; Carrieri A et al.; This study reports the building of the three-dimensional structure of the rat alpha1d-adrenergic receptor through a topology approach based on the structure of the rhodopsin receptor from cryoelectron microscopy . The validity and reliability of the receptor model were assessed through exhaustive molecular dynamics and docking studies . Some interesting ligand-receptor interactions were identified along with significant differences between the binding mode of agonists and antagonists . The importance of the disruption of a salt bridge as a possible initial event leading to receptor activation is discussed on the basis of data from mutagenesis and molecular dynamics studies . Science, 2001 May 4, 292(5518), 929 - 34 Integrated genomic and proteomic analyses of a systematically perturbed metabolic network; Ideker T et al.; We demonstrate an integrated approach to build, test, and refine a model of a cellular pathway, in which perturbations to critical pathway components are analyzed using DNA microarrays, quantitative proteomics, and databases of known physical interactions . Using this approach, we identify 997 messenger RNAs responding to 20 systematic perturbations of the yeast galactose-utilization pathway, provide evidence that approximately 15 of 289 detected proteins are regulated posttranscriptionally, and identify explicit physical interactions governing the cellular response to each perturbation . We refine the model through further iterations of perturbation and global measurements, suggesting hypotheses about the regulation of galactose utilization and physical interactions between this and a variety of other metabolic pathways. Plant Cell, 2001 May, 13(5), 1231 - 44 The maize golden2 gene defines a novel class of transcriptional regulators in plants; Rossini L et al.; In the C4 plant maize, three photosynthetic cell types differentiate: C4 bundle sheath, C4 mesophyll, and C3 mesophyll cells . C3 mesophyll cells represent the ground state, whereas C4 bundle sheath and C4 mesophyll cells are specialized cells that differentiate in response to light-induced positional signals . The Golden2 (G2) gene regulates plastid biogenesis in all photosynthetic cells during the C3 stages of development . However, G2 function is specifically committed to the differentiation of bundle sheath cell chloroplasts in C4 leaf blades . In this article, we report the isolation of G2-like (Glk) genes from maize and rice, providing evidence for a family of Glk genes in plants . The expression profiles of the rice Glk genes suggest that these genes may act redundantly to promote photosynthetic development in this C3 species . In maize, G2 and ZmGlk1 transcripts accumulate primarily in C4 bundle sheath and C4 mesophyll cells, respectively, suggesting a specific role for each gene in C4 differentiation . We show that G2 and ZmGLK1 both can transactivate reporter gene transcription and dimerize in yeast, which supports the idea that these proteins act as transcriptional regulators of cell-type differentiation processes. Plant Cell, 2001 May, 13(5), 1205 - 19 Hyperosmotic stress induces the rapid phosphorylation of a soybean phosphatidylinositol transfer protein homolog through activation of the protein kinases SPK1 and SPK2; Monks DE et al.; Although phosphatidylinositol transfer proteins (PITPs) are known to serve critical functions in regulating a varied array of signal transduction processes in animals and yeast, the discovery of a similar class of proteins in plants occurred only recently . Here, we report the participation of Ssh1p, a soybean PITP-like protein, in the early events of osmosensory signal transduction in plants, a function not attributed previously to animal or yeast PITPs . Exposure of plant tissues to hyperosmotic stress led to the rapid phosphorylation of Ssh1p, a modification that decreased its ability to associate with membranes . An osmotic stress-activated Ssh1p kinase activity was detected in several plant species by presenting recombinant Ssh1p as a substrate in in-gel kinase assays . Elements of a similar osmosensory signaling pathway also were conserved in yeast, an observation that facilitated the identification of soybean protein kinases SPK1 and SPK2 as stress-activated Ssh1p kinases . This study reveals the activation of SPK1 and/or SPK2 and the subsequent phosphorylation of Ssh1p as two early successive events in a hyperosmotic stress-induced signaling cascade in plants . Furthermore, Ssh1p is shown to enhance the activities of a plant phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase, an observation that suggests that the ultimate function of Ssh1p in cellular signaling is to alter the plant's capacity to synthesize phosphoinositides during periods of hyperosmotic stress. Comput Methods Programs Biomed, 2001 Jun, 65(3), 163 - 74 An integrated approach for genome-wide gene expression analysis; Hu YJ; Since efficient and relatively cheap methods were developed for determining biosequences, a lot of biosequence data has been generated . As the main problem in molecular biology is the analysis of the data instead of the data acquisition, part of the study of computational biology is to extract all kinds of meaningful information from the sequences . Computer-assisted methods have become very important in analyzing biosequence data . However, most of the current computer-assisted methods are limited to finding motifs . Genes can be regulated in many ways, including combinations of regulatory elements . This research is aimed at developing a new integrated system for genome-wide gene expression analysis . This research begins with a new motif-finding method, using a new objective function combining multiple well defined components and an improved stochastic iterative sampling strategy . Combinatorial motif analysis is accomplished by constructive induction that analyzes potential motif combinations . We then apply standard inductive learning algorithms to generate hypotheses for different gene behaviors . A genome-wide gene expression analysis demonstrated the value of this novel integrated system. Bioelectrochemistry, 2001 Mar, 53(2), 175 - 81 In situ infrared spectroelectrochemical studies on adsorption and oxidation of nucleic acids at glassy carbon electrode; Wang Z et al.; The adsorption and oxidation of yeast RNA and herring sperm DNA (HS DNA) at glass carbon (GC) electrode are studied by differential pulse voltammetry (DPV) and in situ FTIR spectroelectrochemistry . Two oxidation peaks of yeast RNA are obtained by DPV, whose peak potentials shift negatively with increasing pH . The peak currents decrease gradually in successive scans and no corresponding reduction peaks occur, thus indicating that the oxidation process of yeast RNA is completely irreversible . The IR bands in the 1200-1800 cm(-1) range, attributed to the stretching and ring vibrations of nucleic acid bases, show the main spectral changes when the potential is shifted positively, which gives evidence that the oxidation process takes place in the base residues . The oxidation process of HS DNA is similar to that of yeast RNA . The results both from DPV and in situ FTIR spectroelectrochemistry confirm that the guanine and adenine residues can be oxidized at the electrode surface, which is consistent with the oxidation mechanism of nucleic acids proposed previously. Am J Pathol, 2001 May, 158(5), 1859 - 66 Mapping the binding domain of immunoglobulin light chains for Tamm-Horsfall protein; Ying WZ et al.; Cast nephropathy, or myeloma kidney, is a potentially reversible cause of chronic renal failure . In this condition, filtered light chains bind to a common site on Tamm-Horsfall protein (THP), which is produced by cells of the thick ascending limb of the loop of HENLE: Subsequent aggregation of these proteins produces casts that obstruct tubule fluid flow and results in renal failure . In the present study, we used the yeast two-hybrid system to determine the site of interaction of light chains with THP . The third complementarity-determining region (CDR3) of both kappa and lambda light chains interacted with THP . These findings were confirmed in a series of competition studies using a synthetic peptide that corresponded to the CDR3 region and purified THP and light chains . Variations in the CDR3 sequence of the light chain affected binding . Thus, the current studies increase our understanding of the process of cast formation and provide an opportunity to develop strategies that may inhibit this interaction and prevent the clinical manifestations of myeloma kidney. Mutat Res, 2001 May 9, 476(1-2), 29 - 42 Identification of a deletion hotspot on distal mouse chromosome 4 by YAC fingerprinting; Potter T et al.; Using repetitive elements as probes, genomic DNA fingerprints of four randomly selected yeast artificial chromosome (YAC) clones (two human and two mouse-derived YAC) were analyzed to determine the mutation level following X-ray exposure . Because the repetitive probes were derived from the mammalian host DNA, most of the fingerprint bands originated from the artificial chromosomes and not from the yeast genome . For none of the YAC clones was the mutation frequency elevated following X-ray exposure . However, for one mouse-derived YAC, the mutation level was unusually high (7%; 42 mutants of 607 clones analyzed), whereas for the other three YACs, the mutation level was nearly 0% . Surprisingly, 40 of the 42 mutations were deletions occurring only at three of the 20 mouse specific fingerprint bands . One of the frequently deleted fragments was cloned, sequenced and mapped to distal mouse chromosome 4, which has been repeatedly reported to be the most unstable region of the whole mouse genome, associated with various tumors . Deletion mapping of six YAC mutants revealed this fragment to be completely deleted in four YACs . In the other two mutants, recombination occurred within the fragment, in each case initiated at the same LINE-1 element . In conclusion, the presented YAC fingerprint is a useful tool for detecting and characterizing unstable regions in mammalian genomes. Biochim Biophys Acta, 2001 Apr 23, 1538(2-3), 305 - 12 Ku protein in human T and B lymphocytes: full length functional form and signs of degradation; Sallmyr A et al.; DNA-dependent protein kinase (DNA-PK) has been shown to take part in cell cycle regulatory signal transduction and in the repair of X-ray-induced DNA double-strand breaks . Functional DNA-PK is furthermore needed for the generation of antigen specificity during lymphocyte maturation . The Ku86 subunit of DNA-PK has been reported to exist in human B lymphocytes in a truncated form capable of binding to broken DNA but lacking the ability to activate the kinase function of DNA-PK . In the present work the Ku70 and Ku86 dimer proteins in T and B lymphocytes from human blood donors were analysed by immunoblotting and were observed apparently to be of full length . Also, nuclear protein extracted from B and non-B lymphocytes displayed DNA-dependent kinase activity . However, a minor fraction of Ku86 in lymphocytes was observed to be truncated with a molecular mass of approx . 70 kDa. Int J Parasitol, 2001 May 15, 31(7), 728 - 37 Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease; Cornelissen JB et al.; A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F . hepatica in sheep and cattle . The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F . hepatica flukes in Saccharomyces cerevisiae . Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F . hepatica and six-seven other parasites . The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively . In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera . In experimentally infected cattle and sheep, the first detection of F . hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F . hepatica . In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F . hepatica infection . Detection of Fasciola gigantica infections was similar to detection of F . hepatica . The first detection occurred at week 5 and signals persisted for at least 20 weeks . All sera from naturally F . hepatica infected sheep were seropositive in the cathepsin L-like ELISA . The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts . For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2% . The 'apparent' specificity under these conditions was calculated to be 75.3% . In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas . The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis. Mol Cell, 2001 Apr, 7(4), 753 - 65 Bonus, a Drosophila homolog of TIF1 proteins, interacts with nuclear receptors and can inhibit betaFTZ-F1-dependent transcription; Beckstead R et al.; The Drosophila bonus (bon) gene encodes a homolog of the vertebrate TIF1 transcriptional cofactors . bon is required for male viability, molting, and numerous events in metamorphosis including leg elongation, bristle development, and pigmentation . Most of these processes are associated with genes that have been implicated in the ecdysone pathway, a nuclear hormone receptor pathway required throughout Drosophila development . Bon is associated with sites on the polytene chromosomes and can interact with numerous Drosophila nuclear receptor proteins . Bon binds via an LxxLL motif to the AF-2 activation domain present in the ligand binding domain of betaFTZ-F1 and behaves as a transcriptional inhibitor in vivo. Mol Cell, 2001 Apr, 7(4), 741 - 51 A Rsc3/Rsc30 zinc cluster dimer reveals novel roles for the chromatin remodeler RSC in gene expression and cell cycle control; Angus-Hill ML et al.; Chromatin remodeling complexes perform central roles in transcriptional regulation . Here, we identify Rsc3 and Rsc30 as novel components of the essential yeast remodeler RSC complex . Rsc3 and Rsc30 function requires their zinc cluster domain, a known site-specific DNA binding motif . RSC3 is essential, and rsc3 Ts- mutants display a G2/M cell cycle arrest involving the spindle assembly checkpoint pathway, whereas rsc30Delta mutants are viable and osmosensitive . Rsc3 and Rsc30 interact functionally and also physically as a stable Rsc3/Rsc30 heteromeric complex . However, DNA microarray analysis with rsc3 or rsc30 mutants reveals different effects on the expression levels of ribosomal protein genes and cell wall genes . We propose that Rsc3 and Rsc30 interact physically but have different roles in targeting or regulating RSC. Mol Cell, 2001 Apr, 7(4), 715 - 27 The large subunit of replication factor C promotes cell survival after DNA damage in an LxCxE motif- and Rb-dependent manner; Pennaneach V et al.; Retinoblastoma (Rb) protein promotes cell survival after DNA damage . We show here that the LxCxE binding site in Rb mediates both cell survival and cell-cycle arrest after DNA damage . Replication factor C (RF-C) complex plays an important role in DNA replication . We describe a novel function of the large subunit of RF-C in promoting cell survival after DNA damage . RF-Cp145 contains an LxCxE motif, and mutation of this motif abolishes the protective effect of RF-Cp145 . The inability of wild-type RF-Cp145 to promote cell survival in Rb-null cells is rescued by Rb but not by Rb mutants defective in binding LxCxE proteins . RF-C thus enhances cell survival after DNA damage in an Rb-dependent manner. Cell, 2001 Apr 20, 105(2), 269 - 79 Crystal structure of a SIR2 homolog-NAD complex; Min J et al.; The SIR2 protein family comprises a novel class of nicotinamide-adenine dinucleotide (NAD)-dependent protein deacetylases that function in transcriptional silencing, DNA repair, and life-span extension in Saccharomyces cerevisiae . Two crystal structures of a SIR2 homolog from Archaeoglobus fulgidus complexed with NAD have been determined at 2.1 A and 2.4 A resolutions . The structures reveal that the protein consists of a large domain having a Rossmann fold and a small domain containing a three-stranded zinc ribbon motif . NAD is bound in a pocket between the two domains . A distinct mode of NAD binding and an unusual configuration of the zinc ribbon motif are observed . The structures also provide important insights into the catalytic mechanism of NAD-dependent protein deacetylation by this family of enzymes. Biochem J, 2001 May 15, 356(Pt 1), 171 - 80 Transcriptional regulation of the KEL gene and Kell protein expression in erythroid and non-erythroid cells; Camara-Clayette V et al.; The Kell blood-group antigen was originally reported to be a protein expressed in erythroid tissue only . Transcriptional analysis of the KEL promoter activity in human erythroleukaemia K562 and epithelial HeLa cells by electrophoretic mobility-shift and supershift assays, chloramphenicol acetyltransferase assays, co-transfection studies and site-directed mutagenesis provided the following results: (i) the KEL promoter exhibits a strong transcriptional activity in K562 cells and, unexpectedly, a basal non-erythroid activity in HeLa cells, (ii) up-regulation of the 5' distal promoter activity occurs only in the erythroid context, and (iii) two motifs localized in the exon 1 region, which bind the Sp1/Sp3 and the human GATA-1/Ku70/80 factors, were required for down-regulation of the promoter activity, but inhibition of the promoter activity by the repressing factors in HeLa cells was incomplete . KEL expression in HeLa cells was performed further by primer-extension analysis, which revealed the presence of a low amount of Kell transcript correlating with basal expression of the Kell protein in these cells, as shown by immunopurification and Western-blot analysis . DNA sequencing of the transcript revealed a sequence identical to that obtained from erythroid tissue . In human tissues, KEL expression was investigated by dot-blot analysis and revealed high levels of Kell mRNAs, particularly in brain tissues, testis and lymphoid tissues . Moreover, most tissues analysed exhibited low levels of Kell transcripts . The Kell protein was also detected by immunohistochemistry in the Sertoli cells of the testis and in lymphoid tissues like spleen and tonsil, specifically localized in the follicular dendritic cells . Altogether, the results indicated that KEL expression is not restricted to erythroid tissue. Dev Biol, 2001 May 15, 233(2), 380 - 93 Dominant Drop mutants are gain-of-function alleles of the muscle segment homeobox gene (msh) whose overexpression leads to the arrest of eye development; Mozer BA; Dominant Drop (Dr) mutations are nearly eyeless and have additional recessive phenotypes including lethality and patterning defects in eye and sensory bristles due to cis-regulatory lesions in the cell cycle regulator string (stg) . Genetic analysis demonstrates that the dominant small eye phenotype is the result of separate gain-of-function mutations in the closely linked muscle segment homeobox (msh) gene, encoding a homeodomain transcription factor required for patterning of muscle and nervous system . Reversion of the Dr(Mio) allele was coincident with the generation of lethal loss-of-function mutations in msh in cis, suggesting that the dominant eye phenotype is the result of ectopic expression . Molecular genetic analysis revealed that two dominant Dr alleles contain lesions upstream of the msh transcription start site . In the Dr(Mio) mutant, a 3S18 retrotransposon insertion is the target of second-site mutations (P-element insertions or deletions) which suppress the dominant eye phenotype following reversion . The pattern of 3S18 expression and the absence of msh in eye imaginal discs suggest that transcriptional activation of the msh promoter accounts for ectopic expression . Dr dominant mutations arrest eye development by blocking the progression of the morphogenetic furrow leading to photoreceptor cell loss via apoptosis . Gal4-mediated ubiquitous expression of msh in third-instar larvae was sufficient to arrest the morphogenetic furrow in the eye imaginal disc and resulted in lethality prior to eclosion . Dominant mutations in the human msx2 gene, one of the vertebrate homologs of msh, are associated with craniosynostosis, a disease affecting cranial development . The Dr mutations are the first example of gain-of-function mutations in the msh/msx gene family identified in a genetically tractible model organism and may serve as a useful tool to identify additional genes that regulate this class of homeodomain proteins . Trends Genet, 2001 May, 17(5), 237 - 9 Birth and death of duplicated genes in completely sequenced eukaryotes; Wagner A; Gene and genome duplications are commonly regarded as being of major evolutionary significance . But how often does gene duplication occur? And, once duplicated, what are the fates of duplicated genes? How do they contribute to evolution? In a recent article, Lynch and Conery analyze divergence between duplicate genes from six eukaryotic genomes . They estimate the rate of gene duplication, the rate of gene loss after duplication and the strength of selection experienced by duplicate genes . They conclude that although the rate of gene duplications is high, so is the rate of gene loss, and they argue that gene duplications could be a major factor in speciation. Genetics, 2001 May, 158(1), 133 - 43 Rap1p requires Gcr1p and Gcr2p homodimers to activate ribosomal protein and glycolytic genes, respectively; Deminoff SJ et al.; Efficient transcription of ribosomal protein (RP) and glycolytic genes requires the Rap1p/Gcr1p regulatory complex . A third factor, Gcr2p, is required for only the glycolytic (specialized) mode of transcriptional activation . It is recruited to the complex by Gcr1p and likely mediates a change in the phosphorylation state and/or conformation of the latter . We show here that leucine zipper motifs in Gcr1p and Gcr2p (1LZ and 2LZ) are each specific to one of the two activation mechanisms-mutations in 1LZ and 2LZ impair transcription of RP and glycolytic genes, respectively . Although neither class of mutations causes more than a mild growth defect, simultaneous impairment of 1LZ and 2LZ results in a severe synthetic defect and a reduction in the expression of both sets of genes . Intracistronic complementation by point mutations in the charged e and g positions confirmed that Gcr1p/Gcr1p and Gcr2p/Gcr2p homodimers are the forms required for the different roles of the activator complex . Direct heterodimerization between 1LZ and 2LZ apparently does not occur . Dichotomous Rap1p activation and its striking requirement for distinct homodimeric subunits give cells the capacity to switch between coordinated and uncoupled RP and glycolytic gene regulation. RNA, 2001 Mar, 7(3), 382 - 94 The requirement for eukaryotic initiation factor 4A (elF4A) in translation is in direct proportion to the degree of mRNA 5' secondary structure; Svitkin YV et al.; Eukaryotic initiation factor (elF) 4A functions as a subunit of the initiation factor complex elF4F, which mediates the binding of mRNA to the ribosome . elF4A possesses ATPase and RNA helicase activities and is the prototype for a large family of putative RNA helicases (the DEAD box family) . It is thought that the function of elF4A during translation initiation is to unwind the mRNA secondary structure in the 5' UTR to facilitate ribosome binding . However, the evidence to support this hypothesis is rather indirect, and it was reported that elF4A is also required for the translation of mRNAs possessing minimal 5' UTR secondary structure . Were this hypothesis correct, the requirement for elF4A should correlate with the degree of mRNA secondary structure . To test this hypothesis, the effect of a dominant-negative mutant of mammalian elF4A on translation of mRNAs with various degrees of secondary structure was studied in vitro . Here, we show that mRNAs containing stable secondary structure in the 5' untranslated region are more susceptible to inhibition by the elF4A mutant . The mutant protein also strongly inhibits translation from several picornavirus internal ribosome entry sites (IRES), although to different extents . UV crosslinking of elF4F subunits and elF4B to the mRNA cap structure is dramatically reduced by the elF4A mutant and RNA secondary structure . Finally, the elF4A mutant forms a more stable complex with elF4G, as compared to the wild-type elF4A, thus explaining the mechanism by which substoichiometric amounts of mutant elF4A inhibit translation. RNA, 2001 Mar, 7(3), 361 - 73 Basis for regulated RNA cleavage by functional analysis of RNase L and Ire1p; Dong B et al.; RNase L and Ire1p are members of a superfamily of regulated endoribonucleases that play essential roles in mediating diverse types of cellular stress responses . 2'-5' oligoadenylates, produced in response to interferon treatment and viral double-stranded RNA, are necessary to activate RNase L . In contrast, unfolded proteins in the endoplasmic reticulum activate Ire1p, a transmembrane serine/threonine kinase and endoribonuclease . To probe their similarities and differences, molecular properties of wild-type and mutant forms of human RNase L and yeast Ire1p were compared . Surprisingly, RNase L and Ire1p showed mutually exclusive RNA substrate specificity and partially overlapping but not identical requirements for phylogenetically conserved amino acid residues in their nuclease domains . A functional model for RNase L was generated based on the comparative analysis with Ire1p that assigns novel roles for ankyrin repeats and kinase-like domains. Mol Plant Microbe Interact, 2001 May, 14(5), 618 - 28 Isolation and characterization of a symbiosis-regulated ras from the ectomycorrhizal fungus Laccaria bicolor; Sundaram S et al.; Ectomycorrhizae formed by the symbiotic interaction between ectomycorrhizal fungi and plant roots play a key role in maintaining and improving the health of a wide range of plants . Mycorrhizal initiation, development, and functional maintenance involve morphological changes that are mediated by activation and suppression of several fungal and plant genes . We identified a gene, Lbras, in the ectomycorrhizal fungus Laccaria bicolor that belongs to the ras family of genes, which has been shown in other systems to be associated with signaling pathways controlling cell growth and proliferation . The Lbras cDNA complemented ras2 function in Saccharomyces cerevisiae and had the ability to transform mammalian cells . Expression of Lbras, present as a single copy in the genome, was dependent upon interaction with host roots . Northern analysis showed that expression was detectable in L bicolor 48 h after interaction as well as in the established mycorrhizal tissue . Phylogenetic analysis with other Ras proteins showed that Lbras is related most closely to Aras of Aspergillus nidulans. Nat Rev Genet, 2001 May, 2(5), 333 - 41 Yesterday's polyploids and the mystery of diploidization; Wolfe KH; Thirty years after Susumu Ohno proposed that vertebrate genomes are degenerate polyploids, the extent to which genome duplication contributed to the evolution of the vertebrate genome, if at all, is still uncertain . Sequence-level studies on model organisms whose genomes show clearer evidence of ancient polyploidy are invaluable because they indicate what the evolutionary products of genome duplication can look like . The greatest mystery is the molecular basis of diploidization, the evolutionary process by which a polyploid genome turns into a diploid one. Proc Natl Acad Sci U S A, 2001 May 8, 98(10), 5538 - 43 Epub 2001 May 01. Homologous-pairing activity of the human DNA-repair proteins Xrcc3.Rad51C; Kurumizaka H et al.; The human Xrcc3 protein is involved in the repair of damaged DNA through homologous recombination, in which homologous pairing is a key step . The Rad51 protein is believed to be the only protein factor that promotes homologous pairing in recombinational DNA repair in mitotic cells . In the brain, however, Rad51 expression is extremely low, whereas XRCC3, a human homologue of Saccharomyces cerevisiae RAD57 that activates the Rad51-dependent homologous pairing with the yeast Rad55 protein, is expressed . In this study, a two-hybrid analysis conducted with the use of a human brain cDNA library revealed that the major Xrcc3-interacting protein is a Rad51 paralog, Rad51C/Rad51L2 . The purified Xrcc3.Rad51C complex, which shows apparent 1:1 stoichiometry, was found to catalyze the homologous pairing . Although the activity is reduced, the Rad51C protein alone also catalyzed homologous pairing, suggesting that Rad51C is a catalytic subunit for homologous pairing . The DNA-binding activity of Xrcc3.Rad51C was drastically decreased in the absence of Xrcc3, indicating that Xrcc3 is important for the DNA binding of Xrcc3.Rad51C . Electron microscopic observations revealed that Xrcc3.Rad51C and Rad51C formed similar filamentous structures with circular single-stranded DNA. Hum Reprod, 2001 May, 16(5), 1030 - 6 Assessment of oestrogenic potency of chemicals used as growth promoter by in-vitro methods; Le Guevel R et al.; Three in-vitro bioassays were used to compare the oestrogenic potency of chemicals used as growth promoter in beef cattle in certain non-European Union countries (17beta-oestradiol, alpha-zearalanol, testosterone, trenbolone, trenbolone acetate, melengestrol acetate) or found as food contaminant such as the mycotoxin zearalenone and some of their metabolites (17alpha-oestradiol, oestrone, 17alpha-epitestosterone, 19-nortestosterone, androstendione, zearalanone, alpha-zearalanol, beta-zearalanol, alpha-zearalenol, beta-zearalenol) . The strong oestrogens 17alpha-ethinyl oestradiol and diethylstilboestrol were used as standards . The first bioassay was based on the activation of a reporter gene by oestrogens in recombinant yeast expressing human or rainbow trout oestrogen receptor . In the second bioassay, the vitellogenin gene induction of rainbow trout hepatocyte cultures was used as a biomarker for the exposure to oestrogens . The third bioassay was based on the alkaline phosphatase gene induction by oestrogens in the human endometrial Ishikawa cell line . The assessment of oestrogenic potency of these chemicals clearly demonstrates the strong oestrogenicity of the mycotoxin zearalenone and its metabolites and particularly alpha-zearalenol which was as potent as ethinyl oestradiol and diethylstilboestrol in the human endometrial Ishikawa cell line. Hum Mol Genet, 2001 May 1, 10(10), 1071 - 6 Defect of histone acetyltransferase activity of the nuclear transcriptional coactivator CBP in Rubinstein-Taybi syndrome; Murata T et al.; CREB-binding protein (CBP) is a transcriptional coactivator that has intrinsic histone acetyltransferase (HAT) activity . CBP is the causative gene of Rubinstein-Taybi syndrome (RTS) . To investigate the relationships between CBP HAT activity and RTS, we analyzed 16 RTS patients . A microdeletion was identified in one patient by fluorescent in situ hybridization analysis . Heteroallelic mutations were identified in five patients by reverse transcriptase-polymerase chain reaction-single-strand conformation polymorphism analysis and sequencing . These included a 2 bp deletion between nucleotides 4319 and 4320, an 11 bp deletion between nucleotides 4898 and 4908, a 14 bp insertion (CCTCGGTCCTGCAC) between nucleotides 5212 and 5213, a 2 bp deletion between nucleotides 5222 and 5223, and a missense mutation from guanine (G) to cytosine (C) at nucleotide 4951 that changed codon 1378 from CGG (arginine) to CCG (proline) . The identical missense mutation was introduced into the recombinant mouse CBP . It abolished the HAT activity of CBP and the ability of CBP to transactivate cyclic AMP-response element binding protein (CREB), in HAT assays and in microinjection experiments, respectively . These results suggest that the loss of the HAT activity of CBP may cause RTS, as the first example of a defect of HAT activity in a human disease . Our findings raise the possibility that treatment of RTS patients with histone deacetylase inhibitors might have beneficial effects. Genes Dev, 2001 May 1, 15(9), 1104 - 14 Subcellular localization of the Snf1 kinase is regulated by specific beta subunits and a novel glucose signaling mechanism; Vincent O et al.; The Snf1/AMP-activated protein kinase family has broad roles in transcriptional, metabolic, and developmental regulation in response to stress . In Saccharomyces cerevisiae, Snf1 is required for the response to glucose limitation . Snf1 kinase complexes contain the alpha (catalytic) subunit Snf1, one of the three related beta subunits Gal83, Sip1, or Sip2, and the gamma subunit Snf4 . We present evidence that the beta subunits regulate the subcellular localization of the Snf1 kinase . Green fluorescent protein fusions to Gal83, Sip1, and Sip2 show different patterns of localization to the nucleus, vacuole, and/or cytoplasm . We show that Gal83 directs Snf1 to the nucleus in a glucose-regulated manner . We further identify a novel signaling pathway that controls this nuclear localization in response to glucose phosphorylation . This pathway is distinct from the glucose signaling pathway that inhibits Snf1 kinase activity and responds not only to glucose but also to galactose and sucrose . Such independent regulation of the localization and the activity of the Snf1 kinase, combined with the distinct localization of kinases containing different beta subunits, affords versatility in regulating physiological responses. Genes Dev, 2001 May 1, 15(9), 1078 - 92 Negative regulation of Gcn4 and Msn2 transcription factors by Srb10 cyclin-dependent kinase; Chi Y et al.; The budding yeast transcriptional activator Gcn4 is rapidly degraded in an SCF(Cdc4)-dependent manner in vivo . Upon fractionation of yeast extracts to identify factors that mediate Gcn4 ubiquitination, we found that Srb10 phosphorylates Gcn4 and thereby marks it for recognition by SCF(Cdc4) ubiquitin ligase . Srb10 is a physiological regulator of Gcn4 stability because both phosphorylation and turnover of Gcn4 are diminished in srb10 mutants . Gcn4 is almost completely stabilized in srb10Delta pho85Delta cells, or upon mutation of all Srb10 phosphorylation sites within Gcn4, suggesting that the Pho85 and Srb10 cyclin-dependent kinases (CDKs) conspire to limit the accumulation of Gcn4 . The multistress response transcriptional regulator Msn2 is also a substrate for Srb10 and is hyperphosphorylated in an Srb10-dependent manner upon heat-stress-induced translocation into the nucleus . Whereas Msn2 is cytoplasmic in resting wild-type cells, its nuclear exclusion is partially compromised in srb10 mutant cells . Srb10 has been shown to repress a subset of genes in vivo, and has been proposed to inhibit transcription via phosphorylation of the C-terminal domain of RNA polymerase II . We propose that Srb10 also inhibits gene expression by promoting the rapid degradation or nuclear export of specific transcription factors . Simultaneous down-regulation of both transcriptional regulatory proteins and RNA polymerase may enhance the potency and specificity of transcriptional inhibition by Srb10. Genes Dev, 2001 May 1, 15(9), 1061 - 6 Asf1 links Rad53 to control of chromatin assembly; Hu F et al.; Yeast defective in the checkpoint kinase Rad53 fail to recover from transient DNA replication blocks and synthesize intact chromosomes . The effectors of Rad53 relevant to this recovery process are unknown . Here we report that overproduction of the chromatin assembly factor Asf1 can suppress the Ts phenotype of mrc1rad53 double mutants and the HU sensitivity of rad53 mutants . Eliminating silencing also suppresses this lethality, further implicating chromatin structure in checkpoint function . We find that Asf1 and Rad53 exist in a dynamic complex that dissociates in response to replication blocks and DNA damage . Thus, checkpoint pathways directly regulate chromatin assembly to promote survival in response to DNA damage and replication blocks. Genes Dev, 2001 May 1, 15(9), 1055 - 60 RAD51-independent break-induced replication to repair a broken chromosome depends on a distant enhancer site; Malkova A et al.; Without the RAD51 strand exchange protein, Saccharomyces cerevisiae cannot repair a double-strand break (DSB) by gene conversion . However, cells can repair DSBs by recombination-dependent, break-induced replication (BIR) . RAD51-independent BIR is initiated more than 13 kb from the DSB . Repair depends on a 200-bp sequence adjacent to ARS310, located approximately 34 kb centromere-proximal to the DSB, but does not depend on the origin activity of ARS310 . We conclude that the ability of a recombination-induced replication fork to copy > 130 kb to the end of the chromosome depends on a special site that enhances assembly of a processive repair replication fork. Genes Dev, 2001 May 1, 15(9), 1045 - 50 Transcriptional activation: risky business; Tansey WP; Transcriptional regulation is all about getting RNA polymerase to the right place on the gene at the right time and making sure that it is competent to conduct transcription . Traditional views of this process place most of their emphasis on the events that precede initiation of transcription . We imagine a promoter-bound transcriptional activator (or collection of activators) recruiting components of the basal transcriptional machinery to the DNA, eventually leading to the recruitment of RNA polymerase II and the onset of gene transcription . Although these events play a crucial role in regulating gene expression, they are only half the story . Correct regulation of transcription requires that polymerase not only initiates when and where it should, but that it stops initiating when no longer appropriate . But how are the signals from transcriptional activators, telling RNA polymerase to fire, terminated? Is this process governed by chance, with activators simply falling off the promoter at a certain frequency? Or is there some more direct mechanism, whereby activators are aggressively limited from uncontrolled promoter activation? A new article by suggests the latter may be true, and provides a mechanism for how a component of the basal transcription machinery can mark the activators it has encountered, sentencing them to an early death or banishing them from the nucleus . The ability of the basal transcriptional apparatus to mark activators provides an efficient way to limit activator function and ensures that continuing transcription initiation at a promoter is coupled to the continuing synthesis and activation of transcriptional activators. EMBO J, 2001 May 1, 20(9), 2326 - 37 Multiple roles for the C-terminal domain of eIF5 in translation initiation complex assembly and GTPase activation; Asano K et al.; eIF5 stimulates the GTPase activity of eIF2 bound to Met-tRNA(i)(Met), and its C-terminal domain (eIF5-CTD) bridges interaction between eIF2 and eIF3/eIF1 in a multifactor complex containing Met-tRNA(i)(Met) . The tif5-7A mutation in eIF5-CTD, which destabilizes the multifactor complex in vivo, reduced the binding of Met-tRNA(i)(Met) and mRNA to 40S subunits in vitro . Interestingly, eIF5-CTD bound simultaneously to the eIF4G subunit of the cap-binding complex and the NIP1 subunit of eIF3 . These interactions may enhance association of eIF4G with eIF3 to promote mRNA binding to the ribosome . In vivo, tif5-7A eliminated eIF5 as a stable component of the pre-initiation complex and led to accumulation of 48S complexes containing eIF2; thus, conversion of 48S to 80S complexes is the rate-limiting defect in this mutant . We propose that eIF5-CTD stimulates binding of Met-tRNA(i)(Met) and mRNA to 40S subunits through interactions with eIF2, eIF3 and eIF4G; however, its most important function is to anchor eIF5 to other components of the 48S complex in a manner required to couple GTP hydrolysis to AUG recognition during the scanning phase of initiation. EMBO J, 2001 May 1, 20(9), 2180 - 90 The sortilin cytoplasmic tail conveys Golgi-endosome transport and binds the VHS domain of the GGA2 sorting protein; Nielsen MS et al.; Sortilin belongs to a growing family of multiligand type-1 receptors with homology to the yeast receptor Vps10p . Based on structural features and sortilin's intracellular predominance, we have proposed it to be a sorting receptor for ligands in the synthetic pathway as well as on the cell membrane . To test this hypothesis we examine here the cellular trafficking of chimeric receptors containing constructs of the sortilin tail . We report that sorting signals conforming to YXX and dileucine motifs mediate rapid endocytosis of sortilin chimeras, which subsequently travel to the trans-Golgi network, showing little or no recycling . Furthermore, we found that cation-independent mannose 6-phosphate receptor (MPR300)-sortilin chimeras, expressed in mannose 6-phosphate receptor knockout cells, were almost as efficient as MPR300 itself for transport of newly synthesized beta-hexosaminidase and beta-glucuronidase to lysosomes, and established that the sortilin tail contains potent signals for Golgi-endosome sorting . Finally, we provide evidence suggesting that sortilin is the first example of a mammalian receptor targeted by the recently described GGA family of cytosolic sorting proteins, which condition the Vps10p-mediated sorting of yeast carboxypeptidase Y. EMBO J, 2001 May 1, 20(9), 2160 - 70 Modulation of integrin signal transduction by ILKAP, a protein phosphatase 2C associating with the integrin-linked kinase, ILK1; Leung-Hagesteijn C et al.; ILKAP, a protein serine/threonine (S/T) phosphatase of the PP2C family, was isolated in a yeast two-hybrid screen baited with integrin-linked kinase, ILK1 . Association of ILK1 and ILKAP was independent of the catalytic activity of either partner, as assayed in co-precipitation and two-hybrid experiments . Condi tional expression of ILKAP in HEK 293 cells resulted in selective inhibition of ECM- and growth factor-stimulated ILK1 activity, but did not inhibit Raf-1 kinase activity . A catalytic mutant of ILKAP, H154D, did not inhibit ILK1 kinase activity . Two cellular targets of ILK1, glycogen synthase kinase 3 beta (GSK3beta) and protein kinase B (PKB)/AKT, were differentially affected by ILKAP-mediated inhibition of ILK1 . Catalytically active, but not mutant ILKAP, strongly inhibited insulin-like growth factor-1-stimulated GSK3beta phosphorylation on Ser9, but did not affect phosphorylation of PKB on Ser473, suggesting that ILKAP selectively affects ILK-mediated GSK3beta signalling . Consistent with this, active, but not H154D mutant or the related PP2Calpha, selectively inhibited transactivation of a Tcf/Lef reporter gene, TOPFlash, in 293 cells . We propose that ILKAP regulates ILK1 activity, targeting ILK1 signalling of Wnt pathway components via modulation of GSK3beta phosphorylation. J Cell Biol, 2001 Apr 30, 153(3), 627 - 34 Activation of the Arp2/3 complex by the actin filament binding protein Abp1p; Goode BL et al.; The actin-related protein (Arp) 2/3 complex plays a central role in assembly of actin networks . Because distinct actin-based structures mediate diverse processes, many proteins are likely to make spatially and temporally regulated interactions with the Arp2/3 complex . We have isolated a new activator, Abp1p, which associates tightly with the yeast Arp2/3 complex . Abp1p contains two acidic sequences (DDW) similar to those found in SCAR/WASp proteins . We demonstrate that mutation of these sequences abolishes Arp2/3 complex activation in vitro . Genetic studies indicate that this activity is important for Abp1p functions in vivo . In contrast to SCAR/WASp proteins, Abp1p binds specifically to actin filaments, not monomers . Actin filament binding is mediated by the ADF/cofilin homology (ADF-H) domain of Abp1p and is required for Arp2/3 complex activation in vitro . We demonstrate that Abp1p recruits Arp2/3 complex to the sides of filaments, suggesting a novel mechanism of activation . Studies in yeast and mammalian cells indicate that Abp1p is involved functionally in endocytosis . Based on these results, we speculate that Abp1p may link Arp2/3-mediated actin assembly to a specific step in endocytosis. Bioinformatics, 2001 May, 17(5), 405 - 14 Analysis of temporal gene expression profiles: clustering by simulated annealing and determining the optimal number of clusters; Lukashin AV et al.; MOTIVATION: Cluster analysis of genome-wide expression data from DNA microarray hybridization studies has proved to be a useful tool for identifying biologically relevant groupings of genes and samples . In the present paper, we focus on several important issues related to clustering algorithms that have not yet been fully studied . RESULTS: We describe a simple and robust algorithm for the clustering of temporal gene expression profiles that is based on the simulated annealing procedure . In general, this algorithm guarantees to eventually find the globally optimal distribution of genes over clusters . We introduce an iterative scheme that serves to evaluate quantitatively the optimal number of clusters for each specific data set . The scheme is based on standard approaches used in regular statistical tests . The basic idea is to organize the search of the optimal number of clusters simultaneously with the optimization of the distribution of genes over clusters . The efficiency of the proposed algorithm has been evaluated by means of a reverse engineering experiment, that is, a situation in which the correct distribution of genes over clusters is known a priori . The employment of this statistically rigorous test has shown that our algorithm places greater than 90% genes into correct clusters . Finally, the algorithm has been tested on real gene expression data (expression changes during yeast cell cycle) for which the fundamental patterns of gene expression and the assignment of genes to clusters are well understood from numerous previous studies. Yi Chuan Xue Bao, 2001, 28(4), 379 - 84 {Establishment of suc2 signal sequence trap system}; Sun Q et al.; The secretion of proteins depends on the signal peptide located to the N-terminal of the protein precursor . We established a genetic system in yeast to screen cDNA library for the signal peptide encoding sequences . To do it, we mutated genomic suc2 gene (encoding yeast invertase) of EGY48 by one-step gene disruption method, and got yeast cell lines without invertase expression (EGY48-delta suc) . To get vector for library screening, we inserted suc2 gene encoding mature peptide of invertase downstream to yeast promoter P-ADH1, and multiple cloning sites for insertion of library is between suc2 and P-ADH1 . EGY48-delta suc transformed with the vector can grow on the medium with glucose as carbon source, but not on the medium with raffinose . Signal peptide of suc2 and alpha chain of human interleukin-2 was fused in frame to suc2 gene, then the two resulting vectors were transformed into EGY48-delta suc, all the transformants can grow in the medium with either raffinose or glucose as carbon source . Hence, the system established here can discern cDNA encoding signal peptide from the one not encoding signal peptide. Biochemistry, 2001 Feb 20, 40(7), 2080 - 6 Mitochondrial phosphate transport protein . Reversions of inhibitory conservative mutations identify four helices and a nonhelix protein segment with transmembrane interactions and Asp39, Glu137, and Ser158 as nonessential for transport; Phelps A et al.; The mitochondrial phosphate transport protein (PTP) has six (A--F) transmembrane (TM) helices per subunit of functional homodimer with all mutations referring to the subunit of the homodimer . In earlier studies, conservative replacements of several residues located either at the matrix end (Asp39/helix A, Glu137/helix C, Asp236/helix E) or at the membrane center (His32/helix A, Glu136/helix C) of TM helices yielded inactive single mutation PTPs . Some of these residues were suggested to act as phosphate ligands or as part of the proton cotransport path . We now show that the mutation Ser158Thr, not part of a TM helix but located near the center of the matrix loop (Ile141--Ser171) between TM helices C and D, inactivates PTP and is thus also functionally relevant . On the other side of the membrane, the single mutation Glu192Asp at the intermembrane space end of TM helix D yields a PTP with 33% wild-type activity . We constructed double mutants by adding this mutation to the six transport-inactivating mutations . Transport was detected only in those with Asp39Asn, Glu137Gln, or Ser158Thr . We conclude that TM helix D can interact with TM helices A and C and matrix loop Ile141--Ser171 and that Asp39, Glu137, and Ser158 are not essential for phosphate transport . Since our results are consistent with residues present in all 12 functionally identified members of the mitochondrial transport protein (MTP) family, they lead to a general rule that specifies MTP residue types at 7 separate locations . The conformations of all the double mutation PTPs (except that with the matrix loop Ser158Thr) are significantly different from those of the single mutation PTPs, as indicated by their very low liposome incorporation efficiency and their requirement for less detergent (Triton X-100) to stay in solution . These dramatic conformational differences also suggest an interaction between TM helices D and E . The results are discussed in terms of TM helix movements and changes in the PTP monomer/dimer ratio. Biochemistry, 2001 Feb 27, 40(8), 2448 - 54 Kinetic properties of chitinase-1 from the fungal pathogen Coccidioides immitis; Fukamizo T et al.; The endochitinase from Coccidioides immitis (CiX1) is a member of the class 18 chitinase family . Here we show the enzyme functions by a retaining catalytic mechanism; that is, the beta-conformation of the chitin substrate linkages is preserved after hydrolysis . The pattern of cleavage of N-acetyglucosamine (GlcNAc) oligosaccharide substrates has been determined . (GlcNAc)6 is predominantly cleaved into (GlcNAc)2 and (GlcNAc)4, where the (GlcNAc)2 group arises from the nonreducing end of the substrate and is formed as the beta-anomer . With time, transglycosylation occurs, generating (GlcNAc)8 from the product dimer and fresh hexamer . Similar patterns are seen for the cleavage of (GlcNAc)5 and (GlcNAc)4 where dimers cleaved from the nonreducing end reflect the most common binding and hydrolysis pattern . Intrinsic fluorescence measurements suggest the dissociation constant for (GlcNAc)4 is 50 microM . Synthetic substrates with fluorescent leaving groups exhibit complicated profiles in the relationship between initial velocity and substrate concentration, making it difficult to obtain the values of kinetic constants . An improved theoretical analysis of the time-course of (GlcNAc)6 degradation allows the unitary free energy of binding of the individual subsites of the enzyme to be estimated . The free energy values obtained are consistent with the dissociation constant obtained by fluorescence measurements, and generate a model of substrate interaction that can be tested against the crystal structure of the enzyme. Biochemistry, 2001 Feb 27, 40(8), 2433 - 8 In vivo recognition of an RNA aptamer by its transcription factor target; Cassiday LA et al.; In vitro-selected RNA aptamers are potential inhibitors of disease-related macromolecules . Our laboratory previously isolated an RNA aptamer that specifically binds to the human transcription factor NF-kappaB . This RNA aptamer competitively inhibits DNA binding by NF-kappaB in vitro . In the study presented here, this aptamer was tested for binding to the p50 homodimer form of NF-kappaB (p50(2)) in eukaryotic cells using a yeast three-hybrid system . We show that the alpha-p50 RNA aptamer selectively binds recombinant p50(2) expressed in yeast, demonstrating in vivo recognition of an in vitro-selected RNA aptamer by its protein target . This result suggests that RNA decoys might be used to inhibit the function of DNA-binding proteins in vivo. Biochemistry, 2001 Feb 13, 40(6), 1624 - 34 Analysis of etoposide binding to subdomains of human DNA topoisomerase II alpha in the absence of DNA; Leroy D et al.; Epipodophyllotoxins are effective anti-tumor drugs that inhibit eukaryotic DNA topoisomerase II by trapping the enzyme in a covalent complex with DNA . We show that both the recombinant N-terminal ATPase domain and the B'A' core domain of human topoisomerase IIalpha (htopoIIalpha) bind radiolabeled etoposide specifically, even in the absence of DNA . The addition of ATP impairs etoposide binding to the holoenzyme and the N-terminal domain, but not to the core domain . To see if this interference resembles that between novobiocin and ATP in the bacterial GyrB subunit, we modeled the structure of the N-terminal domain of htopoIIalpha and performed molecular docking analysis with etoposide . Mutagenesis of critical amino acids, predicted to stabilize the drug within the N-terminal domain, reveals a less efficient binding of etoposide to the mutated proteins as monitored by direct drug binding assays, although the binding of ATP is not affected. Methods, 2001 May, 24(1), 29 - 34 Protein recruitment systems for the analysis of protein +/- protein interactions; Aronheim A; The yeast Saccharomyces cerevisiae serves as an excellent genetic tool for the analysis of protein +/- protein interactions . The most common system, used to date, is the two-hybrid system . Although proven very powerful, the two-hybrid system exhibits several inherent problems and limitations . Recently, two alternative systems have been described that take advantage of the fact that localization of signal transduction effectors to the inner leaflet of the plasma membrane is absolutely necessary for yeast viability . These effectors can either be the Ras guanyl nucleotide exchange factor or Ras itself . The yeast strain used in both systems is a temperature-sensitive mutant in the yeast Ras guanyl nucleotide exchange factor, CDC25 . Membrane localization of these effectors is achieved via protein +/- protein interaction . Each system can be used to test interaction between known protein pairs, as well as for isolation of novel protein interactions . Described here are the scientific and technical steps to be considered for both protein recruitment systems . Am J Hum Genet, 2001 Jun, 68(6), 1398 - 407 Epub 2001 Apr 26. A recurrent RNA-splicing mutation in the SEDL gene causes X-linked spondyloepiphyseal dysplasia tarda; Tiller GE et al.; Spondyloepiphyseal dysplasia tarda (SEDL) is a genetically heterogeneous disorder characterized by mild-to-moderate short stature and early-onset osteoarthritis . Both autosomal and X-linked forms have been described . Elsewhere, we have reported the identification of the gene for the X-linked recessive form, which maps to Xp22.2 . We now report characterization of an exon-skipping mutation (IVS3+5G-->A at the intron 3 splice-donor site) in two unrelated families with SEDL . Using reverse transcriptase (RT)-PCR, we demonstrated that the mutation resulted in elimination of the first 31 codons of the open reading frame . The mutation was not detected in 120 control X chromosomes . Articular cartilage from an adult who had SEDL and carried this mutation contained chondrocytes with abundant Golgi complexes and dilated rough endoplasmic reticulum (ER) . RT-PCR experiments using mouse/human cell hybrids revealed that the SEDL gene escapes X inactivation . Homologues of the SEDL gene include a transcribed retropseudogene on chromosome 19, as well as expressed genes in mouse, rat, Drosophila melanogaster Caenorhabditis elegans, and Saccharomyces cerevisiae . The latter homologue, p20, has a putative role in vesicular transport from ER to Golgi complex . These data suggest that SEDL mutations may perturb an intracellular pathway that is important for cartilage homeostasis. Cancer Res, 2001 May 1, 61(9), 3682 - 8 Intersection of interferon and hypoxia signal transduction pathways in nitric oxide-induced tumor apoptosis; Tendler DS et al.; Activated macrophages play a central role in antitumor immunity . However, the stimuli that activate macrophages to kill tumor cells are not completely understood . Because the center of solid tumors can be hypoxic, we hypothesized that hypoxia may be an important signal in activating macrophages to kill tumor cells . Hypoxia stimulates IFN-primed macrophages to express the inducible nitric oxide synthase (NOS2) and to synthesize nitric oxide (NO) . We show that this synergy between IFN and hypoxia is mediated by the direct interaction of the hypoxia inducible factor-1 (HIF-1) and IFN regulatory factor-1 (IRF-1), which are both required for the hypoxic transcription of NOS2 . This interaction between HIF-1 and IRF-1 may explain the mechanism by which macrophages infiltrating into tumors are activated to express NOS2 and to produce NO, a mediator of tumor apoptosis. Biophys J, 2001 May, 80(5), 2248 - 61 Hydration state of single cytochrome c monolayers on soft interfaces via neutron interferometry; Kneller LR et al.; Yeast cytochrome c (YCC) can be covalently tethered to, and thereby vectorially oriented on, the soft surface of a mixed endgroup (e.g., -CH3/-SH = 6:1, or -OH/-SH = 6:1) organic self-assembled monolayer (SAM) chemisorbed on the surface of a silicon substrate utilizing a disulfide linkage between its unique surface cysteine residue and a thiol endgroup . Neutron reflectivities from such monolayers of YCC on Fe/Si or Fe/Au/Si multilayer substrates with H2O versus D2O hydrating the protein monolayer at 88% relative humidity for the nonpolar SAM (-CH3/-SH = 6:1 mixed endgroups) surface and 81% for the uncharged-polar SAM (-OH/-SH = 6:1mixed endgroups) surface were collected on the NG1 reflectometer at NIST . These data were analyzed using a new interferometric phasing method employing the neutron scattering contrast between the Si and Fe layers in a single reference multilayer structure and a constrained refinement approach utilizing the finite extent of the gradient of the profile structures for the systems . This provided the water distribution profiles for the two tethered protein monolayers consistent with their electron density profile determined previously via x-ray interferometry (Chupa et al., 1994). J Nat Prod, 2001 Apr, 64(4), 536 - 9 Two bioactive saponins from Albizia subdimidiata from the Suriname rainforest; Abdel-Kader M et al.; Bioassay-guided fractionation of a methanol extract of Albizia subdimidiata using the engineered yeast strains 1138, 1140, 1353, and Sc7 of Saccharomyces cerevisiae as the bioassay tool resulted in the isolation of the two active saponins 1 and 2; one of these, albiziatrioside A (1), is described for the first time . The structures of 1 and 2 were established on the basis of HRMS, 1D and 2D NMR spectral data, and GC--MS analysis of the sugar units . Both isolated compounds showed significant cytotoxicity against the A2780 cell line. Sheng Li Xue Bao, 1998 Aug, 50(4), 385 - 91 The changes of AP-1 DNA binding activity and components in hippocampus of seizure-sensitive rat induced by kainate; Li DD et al.; Seizure-sensitive Fisher 344 rats were prepared 7 d after seizure episodes induced by a single subcutaneous administration of convulsive dose (7.5 mg/kg) of kainic acid . The activator protein-1 (AP-1) DNA binding activity and its components in the hippocampus of such rats were examined by Gel shift, Super-shift and Western blot assay . Gel shift assay showed that the basal level of AP-1 DNA binding activity was significantly higher than that of control rat hippocampus . Super-shift showed that only Fra and Jun D antibodies could super-shift AP-1 complex to a higher position . It suggested that the AP-1 complex was composed of Fra and Jun D in hippocampus of the seizure-sensitive rats . Western blot analysis further confirmed that Jun D proteins contained components of 43, 39 and 28 kDa . When secondary seizure stimulations were given to seizure-sensitive rats, the AP-1 DNA binding activity was attenuated and the components of AP-1 proteins changed transiently . In light of our results and those of literature, it appears that all the mentioned changes may play an important role in induction of long-lasting enhancement in seizure susceptibility. Nat Struct Biol, 2001 May, 8(5), 417 - 22 UBA domains of DNA damage-inducible proteins interact with ubiquitin; Bertolaet BL et al.; Rad23 is a highly conserved protein involved in nucleotide excision repair (NER) that associates with the proteasome via its N-terminus . Its C-terminal ubiquitin-associated (UBA) domain is evolutionarily conserved from yeast to humans . However, the cellular function of UBA domains is not completely understood . Recently, RAD23 and DDI1, both DNA damage-inducible genes encoding proteins with UBA domains, were implicated genetically in Pds1-dependent mitotic control in yeast . The UBA domains of RAD23 and DDI1 are required for these interactions . Timely degradation of Pds1 via the ubiquitin/proteasome pathway allows anaphase onset and is crucial for chromosome maintenance . Here, we show that Rad23 and Ddi1 interact directly with ubiquitin and that this interaction is dependent on their UBA domains, providing a possible mechanism for UBA-dependent cell cycle control . Moreover, we show that a hydrophobic surface on the UBA domain, which from structural work had been predicted to be a protein-protein interaction interface, is indeed required for ubiquitin binding . By demonstrating that UBA domains interact with ubiquitin, we have provided the first indication of a cellular function for the UBA domain. J Biol Chem, 2001 Jul 13, 276(28), 26666 - 73 Epub 2001 Apr 25. Assembly of the human origin recognition complex; Vashee S et al.; The six-subunit origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae . Yeast ORC binds specifically to origins of replication and serves as a platform for the assembly of additional initiation factors, such as Cdc6 and the Mcm proteins . Human homologues of all six ORC subunits have been identified by sequence similarity to their yeast counterparts, but little is known about the biochemical characteristics of human ORC (HsORC) . We have extracted HsORC from HeLa cell chromatin and probed its subunit composition using specific antibodies . The endogenous HsORC, identified in these experiments, contained homologues of Orc1-Orc5 but lacked a putative homologue of Orc6 . By expressing HsORC subunits in insect cells using the baculovirus system, we were able to identify a complex containing all six subunits . To explore the subunit-subunit interactions that are required for the assembly of HsORC, we carried out extensive co-immunoprecipitation experiments with recombinant ORC subunits expressed in different combinations . These studies revealed the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4, HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6 . HsOrc5 did not form stable binary complexes with any other HsORC subunit but interacted with sub-complexes containing any two of subunits HsOrc2, HsOrc3, or HsOrc4 . Complex formation by HsOrc1 required the presence of HsOrc2, HsOrc3, HsOrc4, and HsOrc5 subunits . These results suggest that the subunits HsOrc2, HsOrc3, and HsOrc4 form a core upon which the ordered assembly of HsOrc5 and HsOrc1 takes place . The characterization of HsORC should facilitate the identification of human origins of DNA replication. FEBS Lett, 2001 Apr 20, 495(1-2), 87 - 93 Molecular characterization of human UDP-glucuronic acid/UDP-N-acetylgalactosamine transporter, a novel nucleotide sugar transporter with dual substrate specificity; Muraoka M et al.; A novel human nucleotide sugar transporter (NST) which transports both UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylgalactosamine (UDP-GalNAc) has been identified, cloned and characterized . The strategy for the identification of the novel NST involved a search of the expressed sequence tags database for genes related to the human UDP-galactose transporter-related isozyme 1, followed by heterologous expression of a candidate gene (hUGTrel7) in Saccharomyces cerevisiae and biochemical analyses . Significantly more UDP-GlcA and UDP-GalNAc were translocated from the reaction medium into the lumen of microsomes prepared from the hUGTrel7-expressing yeast cells than into the control microsomes from cells not expressing hUGTrel7 . The possibility that this transporter participates in glucuronidation and/or chondroitin sulfate biosynthesis is discussed. Bioseparation, 2000, 9(4), 231 - 8 Scale-up of recombinant cutinase recovery by whole broth extraction with PEG-phosphate aqueous two-phase; Costa MJ et al.; A whole broth extraction using an aqueous two-phase system (ATPS) composed by 5% (w/w) PEG 3350 and 15% (w/w) phosphate was used for the scale-up extraction and isolation of a recombinant Fusarium solani pisi cutinase, an extracellular mutant enzyme expressed in Saccharomyces cerevisiae, containing a fusion peptide (WP)4 . The experiments were carried out at three different scales (10 ml, 1 l and 30 l) . Mixing time and stirrer speed were evaluated at lab scale (1 l) with two different system compositions . Stirrer speed between 400 and 800 rpm and mixing time between 2 and 5 min led to the highest recoveries of cutinase . In all cases, inclusive of pilot scale (30 l), the equilibrium was reached after a few minutes . The performance of ATPS was reproducible within the scale range of 0.010-30 l and provided a standard deviation of the yield lower than 8%, leading to (i) a partition coefficient over 50, (ii) a yield over 95% and (iii) a concentration factor over 5 . The fusion of the peptide (WP)4 to the cutinase protein enabled a 400 increase of the partition coefficient relative to the wild-type strain. Anal Chem, 2001 Apr 1, 73(7), 1610 - 3 Attomole-level protein fingerprinting based on intrinsic peptide fluorescence; Okerberg E et al.; Protein identification has relied heavily on proteolytic analysis, but current techniques are often slow and generally consume large quantities of valuable protein sample . We report the development of a rapid, ultralow volume protein analysis strategy based on tryptic digestion within the tip of a 1.5-microm capillary channel followed by separation of the proteolytic fragments using capillary electrophoresis (CE) . Two-photon excitation is used to probe the intrinsic fluorescence of peptide fragments through "deep-UV" excitation of aromatic amino acid residues at the outlet of the CE channel . Detection limits using this technique are 0.7, 2.4, and 23 amol for the aromatic amino acids tryptophan, tyrosine, and phenylalanine, respectively . In these studies, we demonstrate the capacity to differentiate bovine and yeast cytochrome c variants using less than 15 amol of protein through tryptic fingerprinting . Moreover, the detection of a single amino acid substitution between bovine and canine cytochrome c illustrates the sensitivity of this approach to minor differences in protein sequence . The 2-pL sample volume required for this on-column tryptic digestion is, to our knowledge, the smallest yet reported for a proteolytic assay. Proc Natl Acad Sci U S A, 2001 Apr 24, 98(9), 4961 - 5 Controlling small guanine-nucleotide-exchange factor function through cytoplasmic RNA intramers; Mayer G et al.; ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the control of diverse biological functions . Two main classes of positive regulatory elements for ARF have been discovered so far: the large Sec7/Gea and the small cytohesin/ARNO families, respectively . These proteins harbor guanine-nucleotide-exchange factor (GEF) activity exerted by the common Sec7 domain . The availability of a specific inhibitor, the fungal metabolite brefeldin A, has enabled documentation of the involvement of the large GEFs in vesicle transport . However, because of the lack of such tools, the biological roles of the small GEFs have remained controversial . Here, we have selected a series of RNA aptamers that specifically recognize the Sec7 domain of cytohesin 1 . Some aptamers inhibit guanine-nucleotide exchange on ARF1, thereby preventing ARF activation in vitro . Among them, aptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the GEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs . The inhibitory effect demonstrated in vitro clearly is observed as well in vivo, based on the finding that M69 produces similar results as a dominant-negative, GEF-deficient mutant of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces stimulated adhesion to intercellular adhesion molecule-1 and results in a dramatic reorganization of F-actin distribution . These highly specific cellular effects suggest that the ARF-GEF activity of cytohesin 1 plays an important role in cytoskeletal remodeling events of lymphoid cells. Int J Obes Relat Metab Disord, 2001 Apr, 25(4), 467 - 71 The association between the val/ala-55 polymorphism of the uncoupling protein 2 gene and exercise efficiency; Buemann B et al.; BACKGROUND: Energy expenditure may partly be determined by genetic variations in uncoupling proteins . We have previously found an increased physical activity but a similar 24-h energy expenditure (EE) in subjects with the val/val-55 UCP2 genotype compared to those with the ala/ala genotype which indicates that the val-55 allele is statistically associated with a higher metabolic efficiency . DESIGN: EE during bicycling was determined by indirect calorimetry at three different loads (30, 40 and 60% of VO2max in eight subjects with the val/val-55 genotype (35+/-6 y weight=76.8+/-13.6 kg, VO2max=2.79+/-0.71 l/min) and eight subjects with the ala/ala-55 genotype (37+/-3 y, weight=78.3+/-16.5 kg, VO2max=2.66+/-0.41 l/min) . RESULTS: Incremental exercise efficiency across the three different work levels was higher in the val/val (25.3%, c.i . 24.2-26.4%) than in the ala/ala (23.6%, c.i . 22.5-24.7%) genotype P<0.05 . Gross exercise efficiency at 40% VO2max was higher in the val/val (15.3+/-0.6%) than in the ala/ala (13.5+/-0.4%) group . CONCLUSION: As the val/ala-55 polymorphism is located in a domain of the protein without any known function, the different exercise efficiency between the two genotypes most likely reflects a linkage disequilibrium with a functionally significant polymorphism in UCP2 or in the neighbouring UCP3 gene . The study suggests that variations in the UCP genes may affect not only basal metabolic rate but also influence energy costs of exercise. Plant J, 2001 Mar, 25(6), 617 - 26 CKS1At overexpression in Arabidopsis thaliana inhibits growth by reducing meristem size and inhibiting cell-cycle progression; De Veylder L et al.; The SUC1/CKS1 proteins associate with cyclin-dependent kinases (CDKs) and play an essential role in the regulation of the cell cycle . Recently, an Arabidopsis thaliana SUC1/CKS1 homologous gene, designated CKS1At, has been cloned . Here, overexpression of CKS1At in Arabidopsis is shown to reduce leaf size and root growth rates . Reduced root growth resulted primarily from an increase of the cell-cycle duration and a shortening of the meristem . Endoreduplication was unaffected . The increased cell-cycle duration was associated with an equal extension of both the G1 and G2 phases . This inhibition was due to the binding of CDK subunits with CDKs . The reduced growth rates in response to altered cell-cycle gene expression demonstrates a direct dependence of plant growth rates on cell-cycle regulation. Biochemistry, 2001 May 1, 40(17), 5243 - 8 Role of intrahelical arginine residues in functional properties of uncoupling protein (UCP1); Echtay KS et al.; The functional role of the four intrahelical arginines in uncoupling protein (UCP1) from brown adipose tissue were studied in mutants where they were replaced by noncharged residues . Wild-type and mutant UCP1 were expressed in Saccharomyces cerevisiae . As measured in isolated UCP1, nucleotide binding was largely lost in mutants of R83, R182, and R276 occurring in three repeated domains and common to mitochondrial carrier family, whereas mutation of the UCP typical R91 shows normal binding capacity but > 20-fold lower binding affinity and a near loss of pH dependency of binding . In reconstituted UCP1, fatty acid dependent H(+) transport is retained in all four mutants, but inhibition by nucleotide changes according to the binding ability of UCP1 . Cl(-) transport is inhibited only by mutations of arginines in the first domain (R83 and R91) . Also in isolated mitochondria H(+) transport and respiration with all four mutants is similar to wt, and inhibition by GDP is found only in R91T . The three "regular" arginines are suggested to influence the nucleotide binding site indirectly via a charge network and the "extra" R91 directly via an ion bond with the previously characterised pH sensor E190 . The mutants were also used to assess intrahelical control of UCP1 . In the yeast cells expressing UCP1, the aerobic growth could be reduced by fatty acid addition only with the nucleotide insensitive mutants . This demonstrates an intracellular control of UCP1 by nucleotides and fatty acids. Curr Genet, 2001 Feb, 39(1), 10 - 5 Characterization of the amyR gene encoding a transcriptional activator for the amylase genes in Aspergillus nidulans; Tani S et al.; The Aspergillus nidulans amyR gene and its cDNA were cloned and sequenced . The genomic gene comprised 2,092 bp, interrupted by two short introns, and encoded a cys-6 zinc transcriptional activator (AMYR) of 662 amino acid residues with a calculated molecular mass of 72,862 Da . Disruption of the amyR gene caused defects in the utilization of maltose and starch and abolished expression of the taaG2 gene encoding A . oryzae Taka-amylase A, which is inducibly and abundantly expressed in the wild-type A . nidulans . Expression of the amyR gene was under the control of the carbon catabolite repressor, CREA . The growth defect of the malA1 mutant on maltose was complemented by the amyR gene; and the amyR gene derived from the mutant possessed a single mutation, from A to T, at position 1,483, resulting in a substitution of His478 to Leu . These results indicate that the amyR gene is identical to the genetically defined malA gene . AMYR possessed five domains (Zn and MH1-MH4) homologous to Mal63p, a transcriptional activator for the genes involved in maltose utilization in Saccharomyces cerevisiae . The His478 to Leu substitution lay within the MH3 domain, corresponding to the negative regulatory domain of Mal63p which relieves the inhibitory effect on the activation function in response to maltose. Protein Sci, 2001 May, 10(5), 1005 - 14 Genomic-scale comparison of sequence- and structure-based methods of function prediction: does structure provide additional insight? Fetrow JS, Siew N, Di Gennaro JA, Martinez-Yamout M, Dyson HJ, Skolnick J. A function annotation method using the sequence-to-structure-to-function paradigm is applied to the identification of all disulfide oxidoreductases in the Saccharomyces cerevisiae genome . The method identifies 27 sequences as potential disulfide oxidoreductases . All previously known thioredoxins, glutaredoxins, and disulfide isomerases are correctly identified . Three of the 27 predictions are probable false-positives . Three novel predictions, which subsequently have been experimentally validated, are presented . Two additional novel predictions suggest a disulfide oxidoreductase regulatory mechanism for two subunits (OST3 and OST6) of the yeast oligosaccharyltransferase complex . Based on homology, this prediction can be extended to a potential tumor suppressor gene, N33, in humans, whose biochemical function was not previously known . Attempts to obtain a folded, active N33 construct to test the prediction were unsuccessful . The results show that structure prediction coupled with biochemically relevant structural motifs is a powerful method for the function annotation of genome sequences and can provide more detailed, robust predictions than function prediction methods that rely on sequence comparison alone. J Biol Chem, 2001 Jun 29, 276(26), 23296 - 303 Epub 2001 Apr 20. Degradation of endoplasmic reticulum (ER) quality control substrates requires transport between the ER and Golgi; Caldwell SR et al.; Endoplasmic reticulum (ER) quality control (ERQC) components retain and degrade misfolded proteins, and our results have found that the degradation of the soluble ERQC substrates CPY* and PrA* but not membrane spanning ERQC substrates requires transport between the ER and Golgi . Stabilization of these misfolded soluble proteins was seen in cells lacking Erv29p, a probable Golgi localized protein that cycles through the ER by means of a di-lysine ER retrieval motif (KKKIY) . Cells lacking Erv29p also displayed severely retarded ER exit kinetics for a subset of correctly folded proteins . We suggest that Erv29p is likely involved in cargo loading of a subset of proteins, including soluble misfolded proteins, into vesicles for ER exit . The stabilization of soluble ERQC substrates in both erv29Delta cells and sec mutants blocked in either ER exit (sec12) or vesicle delivery to the Golgi (sec18) suggests that ER-Golgi transport is required for ERQC and reveals a new aspect of the degradative mechanism. Genes Dev, 2001 Apr 15, 15(8), 1031 - 40 PIE-1 is a bifunctional protein that regulates maternal and zygotic gene expression in the embryonic germ line of Caenorhabditis elegans; Tenenhaus C et al.; The CCCH zinc finger protein PIE-1 is an essential regulator of germ cell fate that segregates with the germ lineage during the first cleavages of the Caenorhabditis elegans embryo . We have shown previously that one function of PIE-1 is to inhibit mRNA transcription . Here we show that PIE-1 has a second function in germ cells; it is required for efficient expression of the maternally encoded Nanos homolog NOS-2 . This second function is genetically separable from PIE-1's inhibitory effect on transcription . A mutation in PIE-1's second CCCH finger reduces NOS-2 expression without affecting transcriptional repression and causes primordial germ cells to stray away from the somatic gonad, occasionally exiting the embryo entirely . Our results indicate that PIE-1 promotes germ cell fate by two independent mechanisms as follows: (1) inhibition of transcription, which blocks zygotic programs that drive somatic development, and (2) activation of protein expression from nos-2 and possibly other maternal RNAs, which promotes primordial germ cell development. Genes Dev, 2001 Apr 15, 15(8), 1007 - 20 Recruitment of the transcriptional machinery through GAL11P: structure and interactions of the GAL4 dimerization domain; Hidalgo P et al.; The GAL4 dimerization domain (GAL4-dd) is a powerful transcriptional activator when tethered to DNA in a cell bearing a mutant of the GAL11 protein, named GAL11P . GAL11P (like GAL11) is a component of the RNA-polymerase II holoenzyme . Nuclear magnetic resonance (NMR) studies of GAL4-dd revealed an elongated dimer structure with C(2) symmetry containing three helices that mediate dimerization via coiled-coil contacts . The two loops between the three coiled coils form mobile bulges causing a variation of twist angles between the helix pairs . Chemical shift perturbation analysis mapped the GAL11P-binding site to the C-terminal helix alpha3 and the loop between alpha1 and alpha2 . One GAL11P monomer binds to one GAL4-dd dimer rendering the dimer asymmetric and implying an extreme negative cooperativity mechanism . Alanine-scanning mutagenesis of GAL4-dd showed that the NMR-derived GAL11P-binding face is crucial for the novel transcriptional activating function of the GAL4-dd on GAL11P interaction . The binding of GAL4 to GAL11P, although an artificial interaction, represents a unique structural motif for an activating region capable of binding to a single target to effect gene expression. Genes Dev, 2001 Apr 15, 15(8), 955 - 67 Conversion of a gene-specific repressor to a regional silencer; Rusche LN et al.; In Saccharomyces cerevisiae, gene silencing at the HMR and HML loci is normally dependent on Sir2p, Sir3p, and Sir4p, which are structural components of silenced chromatin . Sir2p is a NAD+-dependent histone deacetylase required for silencing . Silencing can be restored in cells lacking Sir proteins by a dominant mutation in SUM1, which normally acts as a mitotic repressor of meiotic genes . This study found that mutant Sum1-1p, but not wild-type Sum1p, associated directly with HM loci . The origin recognition complex (ORC) was required for Sum1-1p-mediated silencing, and mutations in ORC genes reduced association of Sum1-1p with the HM loci . Sum1-1p-mediated silencing also depended on HST1, a paralog of SIR2 . Both Sum1-1p and wild-type Sum1p interacted with Hst1p in coimmunoprecipitation experiments . Therefore, the SUM1-1 mutation did not change the affinity of Sum1p for Hst1p, but rather relocalized Sum1p to the HM loci . Sum1-1-Hst1p action led to hypoacetylation of the nucleosomes at HM loci . Thus, Sum1-1p and Hst1p could substitute for Sir proteins to achieve silencing through formation of a compositionally distinct type of heterochromatin.
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