bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppression of Coatomer Mutants by a New Protein Family with COPI and COPII Binding Motifs inSaccharomyces cerevisiae
Authors: Sandmann Thomas , Herrmann Johannes M. , Dengjel Jörn , Schwarz Heinz , Spang Anne
Published: May 2003

Protein trafficking is achieved by a bidirectional vesicle flow between the various compartments of the eukaryotic cell. COPII coated vesicles mediate anterograde protein transport from the endoplasmic reticulum to the Golgi apparatus, whereas retrograde Golgi-to-endoplasmic reticulum vesicles use the COPI coat. Inactivation of COPI vesicle formation in conditional sec21 (γ-COP) mutants rapidly blocks transport of certain proteins along the early secretory pathway. We have identified the integral membrane protein Mst27p as a strong suppressor of sec21-3 and ret1-1 mutants. A C-terminal KKXX motif of Mst27p that allows direct binding to the COPI complex is crucial for its suppression ability. Mst27p and its homolog Yar033w (Mst28p) are part of the same complex. Both proteins contain cytoplasmic exposed C termini that have the ability to interact directly with COPI and COPII coat complexes. Site-specific mutations of the COPI binding domain abolished suppression of the sec21 mutants. Our results indicate that overexpression of MST27 provides an increased number of coat binding sites on membranes of the early secretory pathway and thereby promotes vesicle formation. As a consequence, the amount of cargo that can bind COPI might be important for the regulation of the vesicle flow in the early secretory pathway.
Possibility of Cytoplasmic pre-tRNA Splicing: the Yeast tRNA Splicing Endonuclease Mainly Localizes on the Mitochondria
Authors: Yoshihisa Tohru , Yunoki-Esaki Kaori , Ohshima Chie , Tanaka Nobuyuki , Endo Toshiya
Published: May 2003

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.
Directed Motion of Telomeres in the Formation of the Meiotic Bouquet Revealed by Time Course and Simulation Analysis
Authors: Carlton Peter M. , Cowan Carrie R. , Cande W. Zacheus
Published: May 2003

Chromosome movement is critical for homologous chromosome pairing during meiosis. A prominent and nearly universal meiotic chromosome reorganization is the formation of the bouquet, characterized by the close clustering of chromosome ends at the nuclear envelope. We have used a novel method of in vitro culture of rye anthers combined with fluorescent in situ hybridization (FISH) detection of telomeres to quantitatively study bouquet formation. The three-dimensional distribution of telomeres over time was used to obtain a quantitative profile of bouquet formation intermediates. The bouquet formed through a gradual, continuous tightening of telomeres over ∼6 h. To determine whether the motion of chromosomes was random or directed, we developed a computer simulation of bouquet formation to compare with our observations. We varied the diffusion rate of telomeres and the amount of directional bias in telomere movement. In our models, the bouquet was formed in a manner comparable to what we observed in cultured meiocytes only when the movement of telomeres was actively directed toward the bouquet site, whereas a wide range of diffusion rates were permitted. Directed motion, as opposed to random diffusion, was required to reproduce our observations, implying that an active process moves chromosomes to cause telomere clustering.
Differential Caveolin-1 Polarization in Endothelial Cells during Migration in Two and Three Dimensions
Authors: Parat Marie-Odile , Anand-Apte Bela , Fox Paul L.
Published: May 2003

Endothelial cell (EC) migration is a critical event during multiple physiological and pathological processes. ECs move in the plane of the endothelium to heal superficially injured blood vessels but migrate in three dimensions during angiogenesis. We herein investigate differences in these modes of movement focusing on caveolae and their defining protein caveolin-1. Using a novel approach for morphological analysis of transmigrating cells, we show that ECs exhibit a polarized distribution of caveolin-1 when traversing a filter pore. Strikingly, in these cells caveolin-1 seems to be released from caveolar structures in the cell rear and to relocalize at the cell front in a cytoplasmic form. In contrast, during planar movement caveolin-1 is concentrated at the rear of ECs, colocalizing with caveolae. The phosphorylatable Tyr14residue of caveolin-1 is required for polarization of the protein during transmigration but does not alter polarization during planar movement. Palmitoylation of caveolin-1 is not essential for redistribution of the protein during either mode of movement. Thus, ECs migrating in three dimensions uniquely exhibit dissociation of caveolin-1 from caveolae and phosphorylation-dependent relocalization to the cell front.
Kinetochore Protein Interactions and their Regulation by the Aurora Kinase Ipl1p
Authors: Shang Ching , Hazbun Tony R. , Cheeseman Iain M. , Aranda Jennifer , Fields Stanley , Drubin David G. , Barnes Georjana
Published: May 2003

Although there has been a recent explosion in the identification of budding yeast kinetochore components, the physical interactions that underlie kinetochore function remain obscure. To better understand how kinetochores attach to microtubules and how this attachment is regulated, we sought to characterize the interactions among kinetochore proteins, especially with respect to the microtubule-binding Dam1 complex. The Dam1 complex plays a crucial role in the chromosome-spindle attachment and is a key target for phospho-regulation of this attachment by the Aurora kinase Ipl1p. To identify protein–protein interactions involving the Dam1 complex, and the effects of Dam1p phosphorylation state on these physical interactions, we conducted both a genome-wide two-hybrid screen and a series of biochemical binding assays for Dam1p. A two-hybrid screen of a library of 6000 yeast open reading frames identified nine kinetochore proteins as Dam1p-interacting partners. From 113 in vitro binding reactions involving all nine subunits of the Dam1 complex and 32 kinetochore proteins, we found at least nine interactions within the Dam1 complex and 19 potential partners for the Dam1 complex. Strikingly, we found that the Dam1p–Ndc80p and Dam1p–Spc34p interactions were weakened by mutations mimicking phosphorylation at Ipl1p sites, allowing us to formulate a model for the effects of phosphoregulation on kinetochore function.
Nitric Oxide Impairs Normoxic Degradation of HIF-1α by Inhibition of Prolyl Hydroxylases
Authors: Metzen Eric , Zhou Jie , Jelkmann Wolfgang , Fandrey Joachim , Brüne Bernhard
Published: May 2003

Hypoxia inducible factor-1 (HIF-1) is the master regulator of metabolic adaptation to hypoxia. It is appreciated that HIF-1α accumulation is achieved under normoxic conditions by e.g., nitric oxide. We determined molecular mechanisms of HIF-1α accumulation under the impact of S-nitrosoglutathione (GSNO). In human embryonic kidney cells GSNO provoked nuclear accumulation of HIF-1α. This appeared unrelated to gene transcription and protein translation, thus pointing to inhibition of HIF-1α degradation. Indeed, GSNO as well as the hypoxia mimic CoCl2decreased ubiquitination of HIF-1α and GSNO-induced HIF-1α failed to coimmunoprecipitate with pVHL (von Hippel Lindau protein). Considering that HIF-1α-pVHL interactions require prolyl hydroxylation of HIF-1α, we went on to demonstrate inhibition of HIF-1α prolyl hydroxylases (PHDs) by GSNO. In vitro HIF-1α-pVHL interactions revealed that GSNO dose-dependently inhibits PHD activity but not the interaction of a synthetic peptide resembling the hydroxylated oxygen-dependent degradation domain of HIF-1α with pVHL. We conclude that GSNO-attenuated prolyl hydroxylase activity accounts for HIF-1α accumulation under conditions of NO formation during normoxia and that PHD activity is subject to regulation by NO.
The Kinesin-associated Protein UNC-76 Is Required for Axonal Transport in theDrosophilaNervous System
Authors: Gindhart Joseph G. , Chen Jinyun , Faulkner Melissa , Gandhi Rita , Doerner Karl , Wisniewski Tiffany , Nandlestadt Aline
Published: May 2003

Kinesin-I is essential for the transport of membrane-bound organelles in neural and nonneural cells. However, the means by which kinesin interacts with its intracellular cargoes, and the means by which kinesin–cargo interactions are regulated in response to cellular transport requirements are not fully understood. The C terminus of the Drosophila kinesin heavy chain (KHC) was used in a two-hybrid screen of a Drosophila cDNA library to identify proteins that bind specifically to the kinesin tail domain. UNC-76 is an evolutionarily conserved cytosolic protein that binds to the tail domain of KHC in two-hybrid and copurification assays, indicating that kinesin and UNC-76 form a stable complex in vivo. Loss of Drosophila Unc-76 function results in locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants, suggesting that UNC-76 is required for kinesin-dependent axonal transport. Unc-76 exhibits dosage-sensitive genetic relationships with Khc and Kinesin light chain mutations, further supporting the hypothesis that UNC-76 and kinesin-I work in a common transport pathway. Given the interaction of FEZ1, the mammalian homolog of UNC-76, with protein kinase Cζ, and the role of FEZ1 in axon outgrowth, we propose that UNC-76 helps integrate kinesin activity in response to transport requirements in axons.
Phosphorylation of the Autoinhibitory Domain of the Sso t-SNAREs Promotes Binding of the Vsm1 SNARE Regulator in Yeast
Authors: Marash Michael , Gerst Jeffrey E.
Published: May 2003

We have shown that protein kinase A phosphorylation of t-SNAREs inhibits SNARE assembly and suppresses endo- and exocytosis in yeast. Herein, we show that protein kinase A phosphorylation of the Sso exocytic t-SNAREs promotes the binding of Vsm1, a potential SNARE regulator identified previously in our laboratory. Phosphorylation of Sso increases its affinity for Vsm1 by more than fivefold in vitro and both phosphorylated Sso1, as well as Sso1 bearing an aspartate substitution at position 79, interact tightly with Vsm1. Vsm1 binding is dependent upon the NH2-terminal autoinhibitory domain of Sso, and constitutively “open” forms of the t-SNARE show a reduction in Vsm1 binding in vivo. The substitution of serine-79 in Sso1 with an alanine residue or the treatment of yeast with C2-ceramide, which results in the dephosphorylation of serine-79, both inhibit Vsm1 binding in vivo. Importantly, Vsm1 binding to Sso seems to preclude Sso binding to its partner t-SNARE, Sec9, and vice versa. This is consistent with the idea that Vsm1 is an inhibitor of SNARE assembly in yeast. Thus, one way by which phosphorylation inhibits SNARE assembly could be by regulating the association of inhibitory factors that control the ability of t-SNAREs to form complexes in vivo.
The Role of Subunit Assembly in Peripherin-2 Targeting to Rod Photoreceptor Disk Membranes and Retinitis Pigmentosa
Authors: Loewen Christopher J.R. , Moritz Orson L. , Tam Beatrice M. , Papermaster David S. , Molday Robert S.
Published: May 2003

Peripherin-2 is a member of the tetraspanin family of membrane proteins that plays a critical role in photoreceptor outer segment disk morphogenesis. Mutations in peripherin-2 are responsible for various retinal degenerative diseases including autosomal dominant retinitis pigmentosa (ADRP). To identify determinants required for peripherin-2 targeting to disk membranes and elucidate mechanisms underlying ADRP, we have generated transgenic Xenopus tadpoles expressing wild-type and ADRP-linked peripherin-2 mutants as green fluorescent fusion proteins in rod photoreceptors. Wild-type peripherin-2 and P216L and C150S mutants, which assemble as tetramers, targeted to disk membranes as visualized by confocal and electron microscopy. In contrast the C214S and L185P mutants, which form homodimers, but not tetramers, were retained in the rod inner segment. Only the P216L disease mutant induced photoreceptor degeneration. These results indicate that tetramerization is required for peripherin-2 targeting and incorporation into disk membranes. Tetramerization-defective mutants cause ADRP through a deficiency in wild-type peripherin-2, whereas tetramerization-competent P216L peripherin-2 causes ADRP through a dominant negative effect, possibly arising from the introduction of a new oligosaccharide chain that destabilizes disks. Our results further indicate that a checkpoint between the photoreceptor inner and outer segments allows only correctly assembled peripherin-2 tetramers to be incorporated into nascent disk membranes.
Association of the Golgi UDP-Galactose Transporter with UDP-Galactose:Ceramide Galactosyltransferase Allows UDP-Galactose Import in the Endoplasmic Reticulum
Authors: Sprong Hein , Degroote Sophie , Nilsson Tommy , Kawakita Masao , Ishida Nobuhiro , van der Sluijs Peter , van Meer Gerrit
Published: May 2003

UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose:ceramide galactosyltransferase. It is not known how UDP-galactose is transported from the cytosol into the endoplasmic reticulum. We transfected ceramide galactosyltransferase cDNA into CHOlec8 cells, which have a defective UGT and no endogenous ceramide galactosyltransferase. Cotransfection with the human UGT1 greatly stimulated synthesis of lactosylceramide in the Golgi and of galactosylceramide in the endoplasmic reticulum. UDP-galactose was directly imported into the endoplasmic reticulum because transfection with UGT significantly enhanced synthesis of galactosylceramide in endoplasmic reticulum membranes. Subcellular fractionation and double label immunofluorescence microscopy showed that a sizeable fraction of ectopically expressed UGT and ceramide galactosyltransferase resided in the endoplasmic reticulum of CHOlec8 cells. The same was observed when UGT was expressed in human intestinal cells that have an endogenous ceramide galactosyltransferase. In contrast, in CHOlec8 singly transfected with UGT 1, the transporter localized exclusively to the Golgi complex. UGT and ceramide galactosyltransferase were entirely detergent soluble and form a complex because they could be coimmunoprecipitated. We conclude that the ceramide galactosyltransferase ensures a supply of UDP-galactose in the endoplasmic reticulum lumen by retaining UGT in a molecular complex.
A Monitor for Bud Emergence in the Yeast Morphogenesis Checkpoint
Authors: Theesfeld Chandra L. , Zyla Trevin R. , Bardes Elaine G.S. , Lew Daniel J.
Published: May 2003

Cell cycle transitions are subject to regulation by both external signals and internal checkpoints that monitor satisfactory progression of key cell cycle events. In budding yeast, the morphogenesis checkpoint arrests the cell cycle in response to perturbations that affect the actin cytoskeleton and bud formation. Herein, we identify a step in this checkpoint pathway that seems to be directly responsive to bud emergence. Activation of the kinase Hsl1p is dependent upon its recruitment to a cortical domain organized by the septins, a family of conserved filament-forming proteins. Under conditions that delayed or blocked bud emergence, Hsl1p recruitment to the septin cortex still took place, but hyperphosphorylation of Hsl1p and recruitment of the Hsl1p-binding protein Hsl7p to the septin cortex only occurred after bud emergence. At this time, the septin cortex spread to form a collar between mother and bud, and Hsl1p and Hsl7p were restricted to the bud side of the septin collar. We discuss models for translating cellular geometry (in this case, the emergence of a bud) into biochemical signals regulating cell proliferation.
Hypoxia Regulates Assembly of Cilia in Suppressors ofTetrahymenaLacking an Intraflagellar Transport Subunit Gene
Authors: Brown Jason M. , Fine Noah A. , Pandiyan Gautham , Thazhath Rupal , Gaertig Jacek
Published: May 2003

We cloned a Tetrahymena thermophila gene, IFT52, encoding a homolog of the Chlamydomonas intraflagellar transport protein, IFT52. Disruption of IFT52 led to loss of cilia and incomplete cytokinesis, a phenotype indistinguishable from that of mutants lacking kinesin-II, a known ciliary assembly transporter. The cytokinesis failures seem to result from lack of cell movement rather than from direct involvement of ciliary assembly pathway components in cytokinesis. Spontaneous partial suppressors of the IFT52 null mutants occurred, which assembled cilia at high cell density and resorbed cilia at low cell density. The stimulating effect of high cell density on cilia formation is based on the creation of pericellular hypoxia. Thus, at least under certain conditions, ciliary assembly is affected by an extracellular signal and the Ift52p function may be integrated into signaling pathways that regulate ciliogenesis.
Subcellular Localization of LGN During Mitosis: Evidence for Its Cortical Localization in Mitotic Cell Culture Systems and Its Requirement for Normal Cell Cycle Progression
Authors: Kaushik Rachna , Yu Fengwei , Chia William , Yang Xiaohang , Bahri Sami
Published: May 2003

Mammalian LGN/AGS3 proteins and their Drosophila Pins orthologue are cytoplasmic regulators of G-protein signaling. In Drosophila, Pins localizes to the lateral cortex of polarized epithelial cells and to the apical cortex of neuroblasts where it plays important roles in their asymmetric division. Using overexpression studies in different cell line systems, we demonstrate here that, like Drosophila Pins, LGN can exhibit enriched localization at the cell cortex, depending on the cell cycle and the culture system used. We find that in WISH, PC12, and NRK but not COS cells, LGN is largely directed to the cell cortex during mitosis. Overexpression of truncated protein domains further identified the Gα-binding C-terminal portion of LGN as a sufficient domain for cortical localization in cell culture. In mitotic COS cells that normally do not exhibit cortical LGN localization, LGN is redirected to the cell cortex upon overexpression of Gα subunits of heterotrimeric G-proteins. The results also show that the cortical localization of LGN is dependent on microfilaments and that interfering with LGN function in cultured cell lines causes early disruption to cell cycle progression.
NF2deficiency promotes tumorigenesis and metastasis by destabilizing adherens junctions
Authors: Lallemand Dominique , Curto Marcello , Saotome Ichiko , Giovannini Marco , McClatchey Andrea I.
Published: May 2003

Mutation of theNeurofibromatosis 2(NF2) tumor suppressor gene leads to cancer development in humans and mice. Recent studies suggest thatNf2loss also contributes to tumor metastasis. TheNf2-encoded protein, merlin, is related to the ERM (ezrin,radixin, andmoesin) family of membrane:cytoskeleton-associated proteins. However, the cellular mechanism whereby merlin controls cell proliferation from this location is not known. Here we show that the major cellular consequence ofNf2deficiency in primary cells is an inability to undergo contact-dependent growth arrest and to form stable cadherin-containing cell:cell junctions. Merlin colocalizes and interacts with adherens junction (AJ) components in confluent wild-type cells, suggesting that the lack of AJs and contact-dependent growth arrest inNf2−/−cells directly results from the absence of merlin at sites of cell:cell contact. Our studies indicate that merlin functions as a tumor and metastasis suppressor by controlling cadherin-mediated cell:cell contact.
GINS, a novel multiprotein complex required for chromosomal DNA replication in budding yeast
Authors: Takayama Yuko , Kamimura Yoichiro , Okawa Mariko , Muramatsu Sachiko , Sugino Akio , Araki Hiroyuki
Published: May 2003

Eukaryotic chromosomal DNA replication requires a two-step assembly of replication proteins on origins; formation of the prereplicative complex (pre-RC) in late M and G1 phases of the cell cycle, and assembly of other replication proteins in S phase to load DNA polymerases to initiate DNA synthesis. In budding yeast, assembly of Dpb11 and the Sld3–Cdc45 complex on the pre-RC at origins is required for loading DNA polymerases. Here we describe a novel replication complex, GINS (Go,Ichi,Nii, andSan; five, one, two, and three in Japanese), in budding yeast, consisting of Sld5, Psf1 (partner ofSldfive1), Psf2, and Psf3 proteins, all of which are highly conserved in eukaryotic cells. Since the conditional mutations of Sld5 and Psf1 confer defect of DNA replication under nonpermissive conditions, GINS is suggested to function for chromosomal DNA replication. Consistently, in S phase, GINS associates first with replication origins and then with neighboring sequences. Without GINS, neither Dpb11 nor Cdc45 associates properly with chromatin DNA. Conversely, without Dpb11 or Sld3, GINS does not associate with origins. Moreover, genetic and two-hybrid interactions suggest that GINS interacts with Sld3 and Dpb11. Therefore, Dpb11, Sld3, Cdc45, and GINS assemble in a mutually dependent manner on replication origins to initiate DNA synthesis.
Genomic binding by theDrosophilaMyc, Max, Mad/Mnt transcription factor network
Authors: Orian Amir , van Steensel Bas , Delrow Jeffrey , Bussemaker Harmen J. , Li Ling , Sawado Tomoyuki , Williams Eleanor , Loo Lenora W.M. , Cowley Shaun M. , Yost Cynthia , Pierce Sarah , Edgar Bruce A. , Parkhurst Susan M. , Eisenman Robert N.
Published: May 2003

The Myc/Max/Mad transcription factor network is critically involved in cell behavior; however, there is relatively little information on its genomic binding sites. We have employed the DamID method to carry out global genomic mapping of theDrosophilaMyc, Max, and Mad/Mnt proteins. Each protein was tethered toEscherichia coliDNA adenine-methyltransferase (Dam) permitting methylation proximal to in vivo binding sites inKccells. Microarray analyses of methylated DNA fragments reveals binding to multiple loci on all majorDrosophilachromosomes. This approach also reveals dynamic interactions among network members as we find that increased levels of dMax influence the extent of dMyc, but not dMnt, binding. Computer analysis using the REDUCE algorithm demonstrates that binding regions correlate with the presence of E-boxes, CG repeats, and other sequence motifs. The surprisingly large number of directly bound loci (∼15% of coding regions) suggests that the network interacts widely with the genome. Furthermore, we employ microarray expression analysis to demonstrate that hundreds of DamID-binding loci correspond to genes whose expression is directly regulated by dMyc in larvae. These results suggest that a fundamental aspect of Max network function involves widespread binding and regulation of gene expression.
Genomic targets of the human c-Myc protein
Authors: Fernandez Paula C. , Frank Scott R. , Wang Luquan , Schroeder Marianne , Liu Suxing , Greene Jonathan , Cocito Andrea , Amati Bruno
Published: May 2003

The transcription factor Myc is induced by mitogenic signals and regulates downstream cellular responses. If overexpressed, Myc promotes malignant transformation. Myc modulates expression of diverse genes in experimental systems, but few are proven direct targets. Here, we present a large-scale screen for genomic Myc-binding sites in live human cells. We used bioinformatics to select consensus DNA elements (CACGTG or E-boxes) situated in the 5′ regulatory region of genes and measured Myc binding to those sequences in vivo by quantitative chromatin immunoprecipitation. Strikingly, most promoter-associated E-boxes showed selective recovery with Myc, unlike non-E-box promoters or E-boxes in bulk genomic DNA. Promoter E-boxes were distributed in two groups bound by Myc at distinct frequencies. The high-affinity group included an estimated 11% of all cellular loci, was highly conserved among different cells, and was bound independently of Myc expression levels. Overexpressed Myc associated at increased frequency with low-affinity targets and, at extreme levels, also with other sequences, suggesting that some binding was not sequence-specific. The strongest DNA-sequence parameter defining high-affinity targets was the location of E-boxes within CpG islands, correlating with an open, preacetylated state of chromatin. Myc further enhanced histone acetylation, with or without accompanying induction of mRNA expression. Our findings point to a high regulatory and biological diversity among Myc-target genes.
A novel ring-like complex ofXenopusproteins essential for the initiation of DNA replication
Authors: Kubota Yumiko , Takase Youhei , Komori Yasunori , Hashimoto Yoshitami , Arata Toshiaki , Kamimura Yoichiro , Araki Hiroyuki , Takisawa Haruhiko
Published: May 2003

We have identifiedXenopushomologs of the budding yeast Sld5 and its three interacting proteins. These form a novel complex essential for the initiation of DNA replication inXenopusegg extracts. The complex binds to chromatin in a manner dependent on replication licensing and S-phase CDK. The chromatin binding of the complex and that of Cdc45 are mutually dependent and both bindings requireXenopusCut5, the yeast homolog of which interacts with Sld5. On replicating chromatin the complex interacts with Cdc45 and MCM, putative components of replication machinery. Electron microscopy further reveals that the complex has a ring-like structure. These results suggest that the complex plays an essential role in the elongation stage of DNA replication as well as the initiation stage.
Latent ClpX-recognition signals ensure LexA destruction after DNA damage
Authors: Neher Saskia B. , Flynn Julia M. , Sauer Robert T. , Baker Tania A.
Published: May 2003

The DNA-damage response genes in bacteria are up-regulated when LexA repressor undergoes autocatalytic cleavage stimulated by activated RecA protein. Intact LexA is stable to intracellular degradation but its auto-cleavage fragments are degraded rapidly. Here, both fragments of LexA are shown to be substrates for the ClpXP protease. ClpXP recognizes these fragments using sequence motifs that flank the auto-cleavage site but are dormant in intact LexA. Furthermore, ClpXP degradation of the LexA-DNA-binding fragment is important to cell survival after DNA damage. These results demonstrate how one protein-processing event can activate latent protease recognition signals, triggering a cascade of protein turnover in response to environmental stress.
Cop9/signalosome subunits and Pcu4 regulate ribonucleotide reductase by both checkpoint-dependent and -independent mechanisms
Authors: Liu Cong , Powell Kelly A. , Mundt Kirsten , Wu LeJung , Carr Antony M. , Caspari Thomas
Published: May 2003

The signalosome is implicated in regulating cullin-dependent ubiquitin ligases. We find that two signalosome subunits, Csn1 and Csn2, are required to regulate ribonucleotide reductase (RNR) through the degradation of a small protein, Spd1, that acts to anchor the small RNR subunit in the nucleus. Spd1 destruction correlates with the nuclear export of the small RNR subunit, which, in turn, correlates with a requirement for RNR in replication and repair. Spd1 degradation is promoted by two separate CSN-dependent mechanisms. During unperturbed S phase, Spd1 degradation is independent of checkpoint proteins. In irradiated G2 cells, Spd1 degradation requires the DNA damage checkpoint. The signalosome copurifies with Pcu4 (cullin 4). Pcu4, Csn1, and Csn2 promote the degradation of Spd1, identifying a new function for the signalosome as a regulator of Pcu4-containing E3 ubiquitin ligase.
Molecular analysis of theLATERAL SUPPRESSORgene inArabidopsisreveals a conserved control mechanism for axillary meristem formation
Authors: Greb Thomas , Clarenz Oliver , Schäfer Elisabeth , Müller Dörte , Herrero Rubén , Schmitz Gregor , Theres Klaus
Published: May 2003

In seed plants, shoot branching is initiated by the formation of new meristems in the axils of leaves, which subsequently develop into new axes of growth. This study describes the genetic control of axillary meristem formation by theLATERAL SUPPRESSOR(LAS) gene inArabidopsis thaliana. lasmutants show a novel phenotype that is characterized by the inability to form lateral shoots during vegetative development. The analysis shows that axillary meristem formation is differently regulated during different phases of development. During reproductive development, axillary meristems initiate in close proximity to the shoot apical meristem and do not requireLASfunction. In contrast, during the vegetative phase, axillary meristems initiate at a distance to the SAM and requireLASfunction. This control mechanism is conserved between the distantly related species tomato andArabidopsis. Monitoring the patterns ofLASandSHOOT MERISTEMLESStranscript accumulation allowed us to identify early steps in the development of leaf axil identity, which seem to be a prerequisite for axillary meristem initiation. Other regulators of shoot branching, likeREVOLUTAandAUXIN RESISTANT 1, act downstream ofLAS. The results are discussed in the context of the “detached meristem” and the “de novo formation” concepts of axillary meristem formation.
Wear properties of punch with sheared edge
Authors: Yilbas B.S. , Abdul Aleem B.J. , Zainullabdeen S.
Published: May 2003

Metal forming through punching finds wide interest in industry. The punch life and the end product quality depend on the punch and workpiece properties as well as punching conditions. Recently, TiN coating of the punch surface has reported to improve considerably the tool life and the end product quality. This is because of the high hardness and low friction coefficient of TiN coating. In the present study, the wear properties of sheared edges punch is investigated through SEM micrograph and EDS analysis. Mild steel is selected as workpiece material while the punch material is cold‐work tool steel (A2). The punch surface is coated with TiN (PVD) and an uniform coat thickness is assured. It is found that the wear mechanism is governed by adhesive and abrasive wear through shearing. The TiN coating protects the punch surface in the initial cycles of the punching process. As the punching cycle progresses, local defects in TiN coating are observed, i.e. coating is locally worn away.
Evaluating the real profile length in turning of carbon steels
Authors: Petropoulos G. , Pandazaras C.
Published: May 2003

The real profile length constitutes a hybrid parameter in surface metrology which is rather neglected in literature, albeit it affects or controls sometimes surface functional properties like corrosion resistance, adhesibility, wear, lubrication etc. Real profile length values are assessed here in turning of different carbon steels under a wide range of cutting conditions variation leading to a variety of relevant textures. Useful quantitative information for evaluation and modeling of the turning process is provided, as well as correlation is sought with other topography parameters and the statistical distributions over the turned surfaces are determined. Also, possibilities of turned profiles discrimination are discussed.
Life after graduation as a “very uncomfortable world”: an Australian case study
Authors: Perrone Lisa , Vickers Margaret H.
Published: May 2003

Few studies have addressed the experiences of a graduate’s transition from university to the world of work. Understanding how graduates react and respond to this journey will provide universities with the ability to prepare and equip students for the road ahead. The purpose of this particular Australian case study was to extend understanding of the experience of making the transition from university to work, and to identify questions for further study. Data were collected using semi‐structured interviews, which were transcribed and analysed for clusters of common themes. Four themes emerged from the case study: “an uncertain feeling”, “inflated expectations”, “the work experience paradox” and “a low time”. It is hoped that understanding this graduate’s experiences will assist higher education institutions, recruiting companies and students to better understand and prepare for this significant life passage.
Social deprivation and educational underachievement: lessons from London
Authors: Cooper Mark , Lloyd‐Reason Lester , Wall Stuart
Published: May 2003

A study by the OECD in 2001 indicated that the UK had one of the strongest links between social deprivation and educational underachievement. This article uses original analysis to report a close correlation over the period 1997‐2001 between educational achievement in the London boroughs and various indicators of the extent of social deprivation in those boroughs. When the data are further disaggregated in terms of “inner” and “outer” London locations of those boroughs the so‐called “cycle of deprivation” hypothesis is supported still more strongly. The article goes on to discuss the implications of these results for broader policy issues such as central government use of the “standards fund” to target finance to the more deprived schools and the recently announced government decision to appoint a commissioner to improve standards in London schools.
Student placement – structure, skills and e‐support
Authors: Neill Neville T. , Mulholland Gwyneth E.
Published: May 2003

The concept of a student spending part of their undergraduate course in industry or commerce is not new. It is remarkable, however, to note how little literature exists on how best to manage, evaluate and support such a period of placement. At the University of Ulster approximately 1,400 students per year undertake paid placement, generally spending one year in a range of settings throughout Ireland, Great Britain or further afield. Within the Faculty of Informatics each of the nine degree programmes incorporates a compulsory placement year, as does the largest of the Higher National Diploma courses. This paper discusses why work experience should be incorporated within undergraduate courses, how placement is managed within Informatics, reports on how students have perceived the placement experience and outlines how the entire process will soon be supported by a Web portal for use by students, staff and employers alike.
Mentoring for high ability high school students
Authors: Earl Irving S. , Moore Dennis W. , Hamilton Richard J.
Published: May 2003

Examines the effects of a three‐year mentoring programme on the academic achievement of high ability year 13 students at a New Zealand high school. The programme’s purpose was to improve the academic results in the university bursary examination. The study covered a period of six years. The programme matched each selected student with a staff member in a flexible mentoring arrangement which focused on generic skills such as study skills, goal setting and time management. Protégés and mentors felt that the programme was enjoyable and successful. Protégés felt that they gained from the skills they were taught. Statistical analysis indicates that the programme did not have a measurable effect on the academic achievement of the mentored students. Reasons for this and implications for programmes of this kind are discussed.
Online project based learning in innovation management
Authors: O’Sullivan David
Published: May 2003

Learning pedagogies increasingly involve a synthesis between traditional lectures and the use of case studies and project based group work. The Internet provides new opportunities to this learning approach. Internet applications can be developed for project based assignments, which are cheap and easy to deploy. They offer students interactive assignments and the opportunity to benchmark with other assignments online. Teachers have opportunities to review assignments online and offer project groups online advice and assessment. This paper presents a pedagogical approach to teaching innovation management that uses both traditional and online learning and assessment techniques.
Devolving HR responsibility to the line
Authors: Whittaker Susan , Marchington Mick
Published: May 2003

Investigates the devolution of human resources (HR) responsibilities to the line within a large food manufacturing company. A total of 13 senior/board level line managers took part in this study. Records questionnaire and interview data with reference to line manager perceptions of their involvement in HR activities. Of interest is the finding that the line managers claimed to be satisfied with the HR responsibilities that have been devolved to them and are keen to take on activities that relate explicitly to the development of their team. Most line managers report working closely with their HR counterparts and see the configuration moving towards a partnership. The line managers’ main concern is that a lack of support from HR during the delivery of the service can detract from the overall effectiveness. They also note that junior level line managers are likely to feel less supported by HR and comment that it is merely their high level position that drives the HR‐line partnership in their situations. Suggests that this research will act as a first step in exploring the relationship between varying levels of line managers and their HR counterparts in order to ascertain whether the higher your status in the organisation, the better your relationship is with HR.
Line manager involvement in HRM: an inside view
Authors: Renwick Douglas
Published: May 2003

Although line managers have always been involved in managing human resources (HR), it is within human resource management (HRM) that their involvement has been placed centre‐stage as a core element of an HR approach. This article reports findings from 40 interviews with line managers on their experiences in handling HR work that has been devolved to them, from a study of three different UK work organisations. The study finds that significant organisational benefits and costs exist from involving the line in HR work. The article concludes that participation of both line and HR managers in HRM needs to be re‐assessed, as line involvement in HRM is a problematic initiative for organisations to adopt.
New management practices and enterprise training in Australia
Authors: Smith Andrew , Oczkowski Eddie , Noble Charles , Macklin Robert
Published: May 2003

The widespread implementation of new management practices (NMPs) in industrialised countries has had a significant impact on employee training. Examines five NMPs: the learning organisation; total quality management; lean production/high performance work organisations; teamworking; and business process re‐engineering. Focuses on the relationship between organisational change and training at the enterprise level. The research identified important findings in six key areas: small business; the use of the vocational education and training system; the importance of the individual; the nature of training; the importance of behavioural skills; and organisational change. The study confirmed that workplace change is a major driver of improved training provision in enterprises. It showed unambiguously that most NMPs are associated with higher levels of training. The integration of training with business strategy was found to be the most important factor in driving training across a wide range of training activities and appears to lead to an across the board boost to enterprise training in all its forms.
Developing added value skills within an academic programme through work‐based learning
Authors: Falconer Stewart , Pettigrew Malcolm
Published: May 2003

This paper examines the extent to which the softer or transferable skills that are apparently sought by firms are developed through work‐based learning. The study is a pilot study, as it is restricted to the experience of students and graduates of a single degree programme at Napier University, Edinburgh. However, the results indicate a positive contribution of work‐based learning to the development of the transferable skills of those involved in the programme. The paper concludes that these tentative positive results drawn from the pilot study need to be tested across a broader sample of students and graduates from a wide range of programmes and institutions. This would enable an effective evaluation of the role of work‐based learning in skilling the firm.
Anticipating employers’ skills needs: the case for intervention
Authors: Ellis Simon P.
Published: May 2003

This paper demonstrates the need to anticipate employers’ future skills requirements, and shows how London Skills Forecasting Unit is addressing this problem. The paper first suggests that employers require advice and labour market intelligence in order to determine their skill requirements effectively, and to ensure their future competitiveness. It then shows that educational and training institutions also need to anticipate employers’ requirements, and so supply side institutions also need to anticipate employers’ skills demand. Finally, some theoretical evidence for skills forecasting is presented together with some practical developments of forecasting techniques undertaken by the Unit.
An evaluation of state‐subsidized, firm‐based training
Authors: Van Horn Carl E. , Fichtner Aaron R.
Published: May 2003

State‐subsidized, firm‐based training is an increasingly popular strategy used by state governments in the USA to encourage the economic growth of firms by upgrading the skills of employees. In 1999, 47 of the 50 states provided cash‐assistance totalling $600 million to employers to assist them in upgrading the skills of current or new employees. A comprehensive two‐year evaluation of one of the larger programs in the USA, the State of New Jersey's Workforce Development Partnership Program, concludes that such programs can help businesses remain competitive, contribute to the state's tax base and offer expanded employment opportunities for firm employees. As governments and private companies throughout the world search for the most effective ways to encourage economic growth, state‐subsidized, firm‐based training programs can form an important element of successful strategies for human and economic development.
Trust in managerial relationships
Authors: Atkinson Sally , Butcher David
Published: May 2003

Despite significant theoretical work over the past decade, the phenomenon of trust and the process of its development in managerial relationships remain elusive in theory and practice. This paper revisits theories that frame trust development in order to explore the development of trust in the specific social context of managerial relationships. Managerial relationships are often characterised by politics and the pursuit of hidden agendas and self‐interest. Competing perspectives and personal motivations can conspire to render even the most innocent of acts subject to scrutiny and suspicion. In these senses, high levels of trust are not commensurate with ideal conditions for managerial effectiveness. Examines the realistic possibilities for trust development set in the context of managerial relationships, and in the process of this analysis, creates a set of propositions that could inform further theory development and empirical investigation of the area.
Parental attitudes towards pocket money, trait competitiveness and occupational stress
Authors: Kirkcaldy Bruce , Furnham Adrian , Martin Terence
Published: May 2003

Several hundred German parents completed a questionnaire to assess their attitudes towards pocket money and economic socialisation. In addition trait competitiveness and occupational stress were measured. Demographic variables were less predictive of competitiveness compared to psychological/attitudinal factors. The more competitive oriented parents displayed a distinct monetary attitude profile: they were less liberal, more structured and budget‐oriented. They used money significantly more as a reinforcer for educational purposes, e.g. educational or scholarly success, and as an instrument to teach autonomy. Subjectively perceived occupational stress was determined by diverse socio‐demographic variables, although the stress‐demographic relationship was moderated by gender. Older fathers and men from a poor social‐economic background (as children) tended to show greater job‐related stress. Conversely, mothers from “superior” SES, with more siblings, and fewer children of their own, reported more occupational stress.
The good society: the Illuminated, Karl Marx and Adam Smith
Authors: Conway O'Brien John
Published: May 2003

Examines the history of the Illuminated, a secret society founded by Adam Weishaupt in Bavaria, on 1 May 1776, which aimed to overthrow civil and religious institutions with the claim that the ends justify the means. Looks at Karl Marx and the links with the Illuminated's doctrine and also compares the Illuminated's ideas with the teaching of Smith. Concludes that although the Illuminated may have been activated by the most altruistic of motives, their search for the good society was doomed from the start.
Impact of institutional and socioeconomic changes on marital relationship
Authors: Hsing Yu
Published: May 2003

Based on the GARCH model, the divorce rate in Taiwan is found to be negatively affected by the fertility rate and male earnings and positively influenced by the male unemployment rate, female earnings, female education, and institutional changes providing married women more protection in child custody, child support and alimony and property rights in divorce. The conventional OLS regression method yields poor statistical outcomes.
Fuzziness and funds allocation in portfolio optimization
Authors: Allen Jack , Bhattacharya Sukanto , Smarandache Florentin
Published: May 2003

Each individual investor is different, with different financial goals, levels of risk tolerance and personal preferences. From the point of view of investment management, these characteristics are often defined as objectives and constraints. Objectives can be the type of return being sought, while constraints include factors such as time horizon, how liquid the investor is, any personal tax situation and how risk is handled. It is really a balancing act between risk and return with each investor having unique requirements, as well as a unique financial outlook – essentially a constrained utility maximization objective. To analyze how well a customer fits into a particular investor class, one investment house has even designed a structured questionnaire with about 24 questions that each has to be answered with values from 1 to 5. The questions range from personal background to what the customer expects from an investment. A fuzzy logic system has been designed for the evaluation of the answers to the above questions. The notion of fuzziness with respect to funds allocation is investigated.