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| J Mol Biol, 2002 Mar 15, 317(1), 159 - 67 Structure of POIA1, a homologous protein to the propeptide of subtilisin: implication for protein foldability and the function as an intramolecular chaperone; Sasakawa H et al.; Solution structure of POIA1 (Pleurotus ostreatus proteinase A inhibitor 1), which functions as an intramolecular chaperone and as an inhibitor to subtilisin, was determined . By making use of the fact that POIA1 is the only structured protein that shows homology to the propeptide of subtilisin, which is unstructured by itself, foldability of this protein was elucidated . It became clear that the evolutionarily conserved residues play two important roles, one for the maintenance of its own structure, and the other for the interaction with subtilisin . Structural softness and mutational tolerance contained in the POIA1 structure makes it an ideal material for designing a foldable protein . Genes Dev, 2002 Mar 15, 16(6), 729 - 42 C . elegans condensin promotes mitotic chromosome architecture, centromere organization, and sister chromatid segregation during mitosis and meiosis; Hagstrom KA et al.; Chromosome segregation and X-chromosome gene regulation in Caenorhabditis elegans share the component MIX-1, a mitotic protein that also represses X-linked genes during dosage compensation . MIX-1 achieves its dual roles through interactions with different protein partners . To repress gene expression, MIX-1 acts in an X-chromosome complex that resembles the mitotic condensin complex yet lacks chromosome segregation function . Here we show that MIX-1 interacts with a mitotic condensin subunit, SMC-4, to achieve chromosome segregation . The SMC-4/MIX-1 complex positively supercoils DNA in vitro and is required for mitotic chromosome structure and segregation in vivo . Thus, C . elegans has two condensin complexes, one conserved for mitosis and another specialized for gene regulation . SMC-4 and MIX-1 colocalize with centromere proteins on condensed mitotic chromosomes and are required for the restricted orientation of centromeres toward spindle poles . This cell cycle-dependent localization requires AIR-2/AuroraB kinase . Depletion of SMC-4/MIX-1 causes aberrant mitotic chromosome structure and segregation, but not dramatic decondensation at metaphase . Moreover, SMC-4/MIX-1 depletion disrupts sister chromatid segregation during meiosis II but not homologous chromosome segregation during meiosis I, although both processes require chromosome condensation . These results imply that condensin is not simply required for compaction, but plays a more complex role in chromosome architecture that is essential for mitotic and meiotic sister chromatid segregation. Genes Dev, 2002 Mar 15, 16(6), 687 - 92 Specific targeting and constitutive association of histone deacetylase complexes during transcriptional repression; Li J et al.; Specific recruitment of corepressor complexes containing histone deacetylases (HDAC) by transcription factors is believed to play an essential role in transcriptional repression . Recent studies indicate that repression by unliganded nuclear hormone receptors and by the Mad family of repressors requires distinct HDAC-containing corepressor complexes . In this work, we show that unliganded TR specifically recruits only the closely related N-CoR and SMRT-HDAC3 complexes, whereas the Mad1 recruits only the Sin3-HDAC1/2 complex . Significantly, both the Sin3 and Mi-2/NURD complexes also exhibit constitutive association with chromatin and contribute to chromatin deacetylation in a nontargeted fashion . These results suggest that HDAC complexes can contribute to gene repression by two distinct mechanisms as follows: (1) specific targeting by repressors and (2) constitutive association with chromatin. Biochemistry, 2002 Apr 2, 41(13), 4202 - 8 Determinants in the sequence specific binding of two plant transcription factors, CBF1 and NtERF2, to the DRE and GCC motifs; Hao D et al.; Arabidopsis ERF proteins such as DREB1, DREB2, and CBF1 bind to the dehydration-responsive element (DRE), which has the sequence TACCGACAT . Mutation analyses reveal that a central 5 bp CCGAC core of the DRE is the minimal sequence motif (designated as the DRE motif in this paper), to which the ERF domain fragment of CBF1 (CBF1-F) binds specifically with a binding K(d) at the nanomolar level . In contrast, the ERF domain fragment of the tobacco ERF2 (NtERF2-F) does not interact with the DRE motif, but restrictedly recognizes the sequence containing a minimal 6 bp GCCGCC motif (designated as the GCC motif in this paper) . However, CBF1-F binds to the GCC motif with a binding activity similar to its binding activity for the DRE motif . These in vitro binding variations were further demonstrated through reporter cotransformation assays, suggesting that the DRE and GCC motifs are two similar sequence motifs sharing a common core region of CCGNC with a discriminating guanine base at the 5'-end of the GCC motif . Binding analyses with the mutated ERF domain show that such a unique binding of NtERF2-F to the GCC motif can be altered by the substitution of A14 with valine in beta-strand 2 of its ERF domain, the mutant NtERF2-F, ERFav, acquiring a binding to the DRE motif with a K(d) comparable to that for CBF1-F binding to the DRE motif . This demonstrates that A14 is an important determinant of the NtERF2-F binding specificity . A possible mechanism of the binding specificity determination is discussed. DNA Seq, 2001 Dec, 12(5-6), 305 - 18 Genomic structure of mouse copper chaperone, COX17; Takahashi Y et al.; Coxl7p was first cloned as a cytoplasmic copper chaperone from yeast mutant and recent works suggested the existence of mammalian homologues . Previous report has shown that a gel filtration fraction of heart extract containing porcine Coxl7p peptide promoted the survival of NIH3T3 fibroblast cells . In the present study, we first cloned DNA fragments of the mouse COX17 gene . The mouse COX17 spans approximately 6kb and consists of three exons . It was mapped to the center of chromosome 16, using a radiation hybrid-mapping panel . The major transcription start site is 80 bp upstream of the ATG initiation codon as determined by rapid amplification of cDNA ends (5'-RACE) analysis . Two potential polyadenylation sites are 3233 and 3293 bp downstream of the termination codon, respectively . Transient transfection of reporter plasmids containing portions of the mouse COX17 5'-flanking region into AtT-20 and NIH3T3 cells allowed the localization of the essential promoter to a 0.8 kb region upstream of the transcription starting site . Furthermore, the transfected luciferase activity was much higher in AtT-20 than NIH3T3 . According to sequence analysis of the approximately 0.8kb 5'-flanking region, GC rich segments including consensus sequences for binding of the transcription factor Sp1, but no TATA/CAAT boxes, exist in the region of the transcription start site . Besides the GC box, binding sites for NRF-1 and 2 known as specific transcription factors for COX subunits are also localized around the transcription starting site. J Comput Chem, 2002 Jan 15, 23(1), 161 - 5 Is there a unique melting temperature for two-state proteins? Klimov DK, Thirumalai D. Thermal unfolding (or folding) in many proteins occurs in an apparent two-state manner, suggesting that only two states, unfolded and folded, are populated . At the melting temperature, Tm, the two states coexist . Using lattice models with side chains we show that individual residues become structured at temperatures that deviate from Tm, which implies that partially folded conformations make substantial contribution to thermodynamic properties of two-state proteins . We also find that the folding cooperativity for a given residue is linked to its accessible surface area . These results are consistent with the experiments on GCN4-like zipper peptide, which showed that local melting temperatures differ from Tm . Analysis of thermal unfolding of six proteins shows that deltaT/Tm approximately N(-1), where deltaT is the transition width and N is the number of residues . This scaling allows us to conclude that, when corrected for finite size effects, folding cooperativity can be captured using coarse grained models. Mol Cell Endocrinol, 2002 Feb 25, 188(1-2), 27 - 36 Polarized trafficking of thyrocyte proteins in MDCK cells; Zhang X et al.; Recent studies suggest striking similarities between polarized protein sorting in thyrocytes and MDCK epithelial cells, including apical trafficking of thyroglobulin (Tg), thyroid peroxidase, and aminopeptidase N; as well as basolateral targeting of heparan sulfate proteoglycans, thrombospondin 1 (TSP1), type 1 5'-deiodinase, sodium-potassium ATPase, and the thyrotropin receptor . In this report, we have firstly expressed in stably transfected MDCK II cells a range of truncation mutants lacking up to 78% of the C-terminus of TSP1; these studies indicate that the N-terminal region containing the heparin binding domain is sufficient for basolateral targeting of TSP1 . Secondly, we have stably transfected MDCK II cells with both Tg and sodium-iodide symporter (NIS) cDNAs, obtaining clones that simultaneously express both thyroid-specific proteins at the apical and basolateral cell surfaces, respectively . These studies represent promising early steps towards designing artificial thyrocytes by thyroid gene transfer into MDCK cells. FEBS Lett, 2002 Feb 20, 513(1), 124 - 8 Bromodomain: an acetyl-lysine binding domain; Zeng L et al.; Bromodomains, an extensive family of evolutionarily conserved protein modules originally found in proteins associated with chromatin and in nearly all nuclear histone acetyltransferases, have been recently discovered to function as acetyl-lysine binding domains . More recent structural studies of bromodomain/peptide ligand complexes have enriched our understanding of differences in ligand selectivity of bromodomains . These new findings demonstrate that bromodomain/acetyl-lysine recognition can serve as a pivotal mechanism for regulating protein-protein interactions in numerous cellular processes including chromatin remodeling and transcriptional activation, and reinforce the concept that functional diversity of a conserved protein modular structure is achieved by evolutionary changes of amino acid sequences in the ligand binding site. FEBS Lett, 2002 Feb 20, 513(1), 77 - 84 The phosphatidylinositol 3-phosphate-binding FYVE finger; Stenmark H et al.; The FYVE zinc finger domain is conserved from yeast (five proteins) to man (27 proteins) . It functions in the membrane recruitment of cytosolic proteins by binding to phosphatidylinositol 3-phosphate (PI3P), which is found mainly on endosomes . Here we review recent work that sheds light on the targeting of FYVE finger proteins to PI3P-containing membranes, and how these proteins serve to regulate multiple cellular functions. Biochem Pharmacol, 2002 Mar 1, 63(5), 865 - 9 Phentermine inhibition of recombinant human liver monoamine oxidases A and B; Nandigama RK et al.; Recent studies with rat tissue preparations have suggested that the anorectic drug phentermine inhibits serotonin degradation by inhibition of monoamine oxidase (MAO) A with a K(I) value of 85-88 microM, a potency suggested to be similar to that of other reversible MAO inhibitors (Ulus et al., Biochem Pharmacol 2000;59:1611-21) . Since there are known differences between rats and humans in substrate and inhibitor specificities of MAOs, the interactions of phentermine with recombinant human purified preparations of MAO A and MAO B were determined . Human MAO A was competitively inhibited by phentermine with a K(I) value of 498+/-60 microM, a value approximately 6-fold weaker than that observed for the rat enzyme . Phentermine was also observed to be a competitive inhibitor of recombinant human liver MAO B with a K(I) value of 375+/-42 microM, a value similar to that observed with the rat enzyme (310-416 microM) . In contrast to the behavior with rat tissue preparations, no slow time-dependent behavior was observed for phentermine inhibition of purified soluble human MAO preparations . Difference absorption spectral studies showed similar perturbations of the covalent FAD moieties of both human MAO A and MAO B, which suggests a similar mode of binding in both enzymes . These data suggest that phentermine inhibition of human MAO A (or of MAO B) is too weak to be of pharmacological relevance. RNA, 2002 Feb, 8(2), 252 - 60 Water counting: quantitating the hydration level of paramagnetic metal ions bound to nucleotides and nucleic acids; Hoogstraten CG et al.; Binding of divalent metal ions plays a key role in the structure and function of ribozymes and other RNAs . In turn, the energetics and kinetics of the specific binding process are dominated by the balance between the cost of dehydrating the aqueous ion and the energy gained from inner-sphere interactions with the macromolecule . In this work, we introduce the use of the pulsed EPR technique of 2H Electron Spin-Echo Envelope Modulation (ESEEM) to determine the hydration level of Mn2+ ions bound to nucleotides and nucleic acids . Mn2+ is an excellent structural and functional mimic for Mg2+, the most common divalent ion of physiological interest . Comparison of data in D2O and H2O, with aqueous Mn2+ as a reference standard, allows a robust and precise determination of the number of bound water molecules, and therefore the number of RNA-derived ligands . Examples of applications to the mononucleotide models MnGMP and MnATP, as well as to the paradigmatic RNA system tRNAPhe, are shown. J Genet, 2001 Apr, 80(1), 17 - 21 Accelerated molecular evolution of insect orthologues of ERG28/C14orf1: a link with ecdysteroid metabolism? Veitia RA, Hurst LD. We have analysed the evolution of ERG28/C14orf1, a gene coding for a protein involved in sterol biosynthesis . While primary sequence of the protein is well conserved in all organisms able to synthesize sterols de novo, strong divergence is noticed in insects, which are cholesterol auxotrophs . In spite of this virtual acceleration, our analysis suggests that the insect orthologues are evolving today at rates similar to those of the remaining members of the family . A plausible way to explain this acceleration and subsequent stabilization is that Erg28 plays a role in at least two different pathways . Discontinuation of the cholesterogenesis pathway in insects allowed the protein to evolve as much as the function in the other pathway was not compromised. Science, 2002 Mar 22, 295(5563), 2262 - 4 Exosome-mediated recognition and degradation of mRNAs lacking a termination codon; van Hoof A et al.; One role of messenger RNA (mRNA) degradation is to maintain the fidelity of gene expression by degrading aberrant transcripts . Recent results show that mRNAs without translation termination codons are unstable in eukaryotic cells . We used yeast mutants to demonstrate that these "nonstop" mRNAs are degraded by the exosome in a 3'-to-5' direction . The degradation of nonstop transcripts requires the exosome-associated protein Ski7p . Ski7p is closely related to the translation elongation factor EF1A and the translation termination factor eRF3 . This suggests that the recognition of nonstop mRNAs involves the binding of Ski7p to an empty aminoacyl-(RNA-binding) site (A site) on the ribosome, thereby bringing the exosome to a mRNA with a ribosome stalled near the 3' end . This system efficiently degrades mRNAs that are prematurely polyadenylated within the coding region and prevents their expression. Science, 2002 Mar 22, 295(5563), 2258 - 61 An mRNA surveillance mechanism that eliminates transcripts lacking termination codons; Frischmeyer PA et al.; Translation is an important mechanism to monitor the quality of messenger RNAs (mRNAs), as exemplified by the translation-dependent recognition and degradation of transcripts harboring premature termination codons (PTCs) by the nonsense-mediated mRNA decay (NMD) pathway . We demonstrate in yeast that mRNAs lacking all termination codons are as labile as nonsense transcripts . Decay of "nonstop" transcripts in yeast requires translation but is mechanistically distinguished from NMD and the major mRNA turnover pathway that requires deadenylation, decapping, and 5'-to-3' exonucleolytic decay . These data suggest that nonstop decay is initiated when the ribosome reaches the 3' terminus of the message . We demonstrate multiple physiologic sources of nonstop transcripts and conservation of their accelerated decay in mammalian cells . This process regulates the stability and expression of mRNAs that fail to signal translational termination. Protein Sci, 2002 Apr, 11(4), 841 - 51 Thermodynamic stability measurements on multimeric proteins using a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based method; Powell KD et al.; We recently reported on a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based technique, termed SUPREX, that removes several important limitations associated with measuring the thermodynamic stability of proteins . In contrast to conventional spectroscopy-based techniques for characterizing the equilibrium unfolding behavior of proteins, SUPREX is amenable to the thermodynamic analysis of both purified and unpurified proteins using mg to ng quantities of material . Here we report on the application of SUPREX to the analysis of multimeric protein systems . Included in this work are the SUPREX results we obtained in studies on six model multimeric proteins including the GCN4p1 dimer, the coil-V(a)L(d) trimer, the 4-oxalocrotonate tautomerase (4-OT) hexamer, the Trp repressor (TrpR) dimer, the Arc repressor (ArcR) dimer, and an ArcR mutant (the (DOA20)ArcR) dimer which contained two destabilizing mutations including an Asp to Ala mutation at position 20 and an amide to ester bond mutation between amino acid (aa) residues 19 and 20 . As part of the work described here, we present a new method for the analysis of SUPREX data that is generally applicable to both monomeric and multimeric protein systems . Our results on the model proteins in this study indicate that this new method can be used to determine folding free energies for proteins with the accuracy and precision of conventional spectroscopy-based methods. Mol Cell Biol, 2002 Apr, 22(8), 2743 - 50 Identification of mammalian Sds3 as an integral component of the Sin3/histone deacetylase corepressor complex; Alland L et al.; Silencing of gene transcription involves local chromatin modification achieved through the local recruitment of large multiprotein complexes containing histone deacetylase (HDAC) activity . The mammalian corepressors mSin3A and mSin3B have been shown to play a key role in this process by tethering HDACs 1 and 2 to promoter-bound transcription factors . Similar mechanisms appear to be operative in yeast, in which epistasis experiments have established that the mSin3 and HDAC orthologs (SIN3 and RPD3), along with a novel protein, SDS3, function in the same repressor pathway . Here, we report the identification of a component of the mSin3-HDAC complex that bears homology to yeast SDS3, physically associates with mSin3 proteins in vivo, represses transcription in a manner that is partially dependent on HDAC activity, and enables HDAC1 catalytic activity in vivo . That key physical and functional properties are also shared by yeast SDS3 underscores the central role of the Sin3-HDAC-Sds3 complex in eukaryotic cell biology, and the discovery of mSds3 in mammalian cells provides a new avenue for modulating the activity of this complex in human disease. Mol Cell Biol, 2002 Apr, 22(8), 2642 - 9 New insights into the pleiotropic drug resistance network from genome-wide characterization of the YRR1 transcription factor regulation system; Le Crom S et al.; Yrr1p is a recently described Zn(2)Cys(6) transcription factor involved in the pleiotropic drug resistance (PDR) phenomenon . It is controlled in a Pdr1p-dependent manner and is autoregulated . We describe here a new genome-wide approach to characterization of the set of genes directly regulated by Yrr1p . We found that the time-course production of an artificial chimera protein containing the DNA-binding domain of Yrr1p activated the 15 genes that are also up-regulated by a gain-of-function mutant of Yrr1p . Gel mobility shift assays showed that the promoters of the genes AZR1, FLR1, SNG1, YLL056C, YLR346C, and YPL088W interacted with Yrr1p . The putative consensus Yrr1p binding site deduced from these experiments, (T/A)CCG(C/T)(G/T)(G/T)(A/T)(A/T), is strikingly similar to the PDR element binding site sequence recognized by Pdr1p and Pdr3p . The minor differences between these sequences are consistent with Yrr1p and Pdr1p and Pdr3p having different sets of target genes . According to these data, some target genes are directly regulated by Pdr1p and Pdr3p or by Yrr1p, whereas some genes are indirectly regulated by the activation of Yrr1p . Some genes, such as YOR1, SNQ2, and FLR1, are clearly directly controlled by both classes of transcription factor, suggesting an important role for the corresponding membrane proteins. Mol Cell Biol, 2002 Apr, 22(8), 2564 - 74 Novel G-protein complex whose requirement is linked to the translational status of the cell; Carr-Schmid A et al.; G proteins, which bind and hydrolyze GTP, are involved in regulating a variety of critical cellular processes, including the process of protein synthesis . Many members of the subfamily of elongation factor class G proteins interact with the ribosome and function to regulate discrete steps during the process of protein synthesis . Despite sequence similarity to factors involved in translation, a role for the yeast Hbs1 protein has not been defined . In this work we have identified a genetic relationship between genes encoding components of the translational apparatus and HBS1 . HBS1, while not essential for viability, is important for efficient growth and protein synthesis under conditions of limiting translation initiation . The identification of an Hbs1p-interacting factor, Dom34p, which shares a similar genetic relationship with components of the translational apparatus, suggests that Hbs1p and Dom34p may function as part of a complex that facilitates gene expression . Dom34p contains an RNA binding motif present in several ribosomal proteins and factors that regulate translation of specific mRNAs . Thus, Hbs1p and Dom34p may function together to help directly or indirectly facilitate the expression either of specific mRNAs or under certain cellular conditions. Curr Biol, 2002 Mar 19, 12(6), R221 - 3 Mitotic exit: delaying the end without FEAR; Jensen S et al.; Activation of Cdc14 phosphatase, controlled by a signalling cascade known as the mitotic exit network, is the final switch that drives cells from mitosis into the next cell cycle . The recent discovery of a novel network that regulates early Cdc14 activation has revealed the unexpected existence of a two-step control of mitotic exit. Curr Biol, 2002 Mar 19, 12(6), R212 - 4 Exocytosis: the many masters of the exocyst; Lipschutz JH et al.; The exocyst is a conserved eight-subunit complex involved in the docking of exocytic vesicles . The exocyst has now been identified as an effector for five small GTPases, including Sec4, Rho1, Rho3, Cdc42 and, most recently, RalA. Cell, 2002 Feb 22, 108(4), 545 - 56 Gene-specific regulation by general translation factors; Dever TE; Protein synthesis is the ultimate step of gene expression and a key control point for regulation . In particular, it enables cells to rapidly manipulate protein production without new mRNA synthesis, processing, or export . Recent studies have enhanced our understanding of the translation initiation process and helped elucidate how modifications of the general translational machinery regulate gene-specific protein production. Cell, 2002 Feb 22, 108(4), 475 - 87 Cooperation between complexes that regulate chromatin structure and transcription; Narlikar GJ et al.; Chromatin structure creates barriers for each step in eukaryotic transcription . Here we discuss how the activities of two major classes of chromatin-modifying complexes, ATP-dependent remodeling complexes and HAT or HDAC complexes, might be coordinated to create a DNA template that is accessible to the general transcription apparatus. Adv Colloid Interface Sci, 2002 Feb 25, 96(1-3), 279 - 93 Viscosity of concentrated suspensions: influence of cluster formation; Starov V et al.; Dispersed particles can form clusters even at low concentrations . Colloidal and hydrodynamic forces are responsible for this phenomenon and these forces determine both structure and size of clusters . We assume that the viscosity of a concentrated suspension is completely determined by cluster size distribution, regardless if clusters form under the action of colloidal, hydrodynamic interactions or applied shear rates . Based on this assumption an equation, which describes dependency of viscosity on a concentration of dispersed particles taking into account cluster formation, is deduced . Under special restrictions the deduced dependency coincides with the well-known Dougherty-Krieger's equation except for a clear physical meaning of parameters entered . Our consideration shows that Dougherty-Krieger's equation has deeper physical background than it has been supposed earlier . Experimental verification of the suggested model shows a good agreement with the theory predictions and proves a presence of clusters even at low concentrations of dispersed particles. Pharmacogenomics J, 2001, 1(4), 288 - 92 CYP2C9*3 influences the metabolism and the drug-interaction of candesartan in vitro; Hanatani T et al.; Candesartan cilexetil is an angiotensin II receptor antagonist, and candesartan, its active metabolite, is metabolized by CYP2C9 . However, the effect of CYP2C9*3 on candesartan metabolism is not established . We characterized the kinetics of candesartan by CYP2C9*1/*1 and CYP2C9*1/*3 in human liver microsomes . The difference between the two was not significant . Subsequently, CYP2C9*1 and CYP2C9*3 (Leu359) were expressed in yeast, and the kinetics of candesartan were determined . The wild-type showed the lower Km (345 vs 439 microM; 3/4) and higher Vmax/Km (1/3) than the Leu359 variant . Also, we investigated potential interaction between candesartan and warfarin with both the wild-type and the Leu359 variant . Candesartan had no effect on S-warfarin 7-hydroxylation . In contrast, S-warfarin inhibited candesartan metabolism by the wild-type (K = 17microM) greater than by the Leu359 variant (Ki = 36 microM) . These findings suggest that CYP2C9*3 may change not only the metabolic activity but also the inhibitory susceptibility compared with CYP2C9*1. Mol Biol Cell, 2002 Mar, 13(3), 965 - 77 Dynein supports motility of endoplasmic reticulum in the fungus Ustilago maydis; Wedlich-Soldner R et al.; The endoplasmic reticulum (ER) of most vertebrate cells is spread out by kinesin-dependent transport along microtubules, whereas studies in Saccharomyces cerevisiae indicated that motility of fungal ER is an actin-based process . However, microtubules are of minor importance for organelle transport in yeast, but they are crucial for intracellular transport within numerous other fungi . Herein, we set out to elucidate the role of the tubulin cytoskeleton in ER organization and dynamics in the fungal pathogen Ustilago maydis . An ER-resident green fluorescent protein (GFP)-fusion protein localized to a peripheral network and the nuclear envelope . Tubules and patches within the network exhibited rapid dynein-driven motion along microtubules, whereas conventional kinesin did not participate in ER motility . Cortical ER organization was independent of microtubules or F-actin, but reformation of the network after experimental disruption was mediated by microtubules and dynein . In addition, a polar gradient of motile ER-GFP stained dots was detected that accumulated around the apical Golgi apparatus . Both the gradient and the Golgi apparatus were sensitive to brefeldin A or benomyl treatment, suggesting that the gradient represents microtubule-dependent vesicle trafficking between ER and Golgi . Our results demonstrate a role of cytoplasmic dynein and microtubules in motility, but not peripheral localization of the ER in U . maydis. Mol Biol Cell, 2002 Mar, 13(3), 880 - 91 Erv14p directs a transmembrane secretory protein into COPII-coated transport vesicles; Powers J et al.; Erv14p is a conserved integral membrane protein that traffics in COPII-coated vesicles and localizes to the early secretory pathway in yeast . Deletion of ERV14 causes a defect in polarized growth because Axl2p, a transmembrane secretory protein, accumulates in the endoplasmic reticulum and is not delivered to its site of function on the cell surface . Herein, we show that Erv14p is required for selection of Axl2p into COPII vesicles and for efficient formation of these vesicles . Erv14p binds to subunits of the COPII coat and binding depends on conserved residues in a cytoplasmically exposed loop domain of Erv14p . When mutations are introduced into this loop, an Erv14p-Axl2p complex accumulates in the endoplasmic reticulum, suggesting that Erv14p links Axl2p to the COPII coat . Based on these results and further genetic experiments, we propose Erv14p coordinates COPII vesicle formation with incorporation of specific secretory cargo. Mol Biol Cell, 2002 Mar, 13(3), 755 - 66 Checkpoint protein BubR1 acts synergistically with Mad2 to inhibit anaphase-promoting complex; Fang G; The spindle assembly checkpoint monitors the attachment of kinetochores to the mitotic spindle and the tension exerted on kinetochores by microtubules and delays the onset of anaphase until all the chromosomes are aligned at the metaphase plate . The target of the checkpoint control is the anaphase-promoting complex (APC)/cyclosome, a ubiquitin ligase whose activation by Cdc20 is required for separation of sister chromatids . In response to activation of the checkpoint, Mad2 binds to and inhibits Cdc20-APC . I show herein that in checkpoint-arrested cells, human Cdc20 forms two separate, inactive complexes, a lower affinity complex with Mad2 and a higher affinity complex with BubR1 . Purified BubR1 binds to recombinant Cdc20 and this interaction is direct . Binding of BubR1 to Cdc20 inhibits activation of APC and this inhibition is independent of its kinase activity . Quantitative analysis indicates that BubR1 is 12-fold more potent than Mad2 as an inhibitor of Cdc20 . Although at high protein concentrations BubR1 and Mad2 each is sufficient to inhibit Cdc20, BubR1 and Mad2 mutually promote each other's binding to Cdc20 and function synergistically at physiological concentrations to quantitatively inhibit Cdc20-APC . Thus, BubR1 and Mad2 act cooperatively to prevent premature separation of sister chromatids by directly inhibiting APC. Mol Biol Cell, 2002 Mar, 13(3), 739 - 54 Genetic ablation of phosphatidylinositol transfer protein function in murine embryonic stem cells; Alb JG Jr et al.; Phosphatidylinositol transfer proteins (PITPs) regulate the interface between signal transduction, membrane-trafficking, and lipid metabolic pathways in eukaryotic cells . The best characterized mammalian PITPs are PITP alpha and PITP beta, two highly homologous proteins that are encoded by distinct genes . Insights into PITP alpha and PITP beta function in mammalian systems have been gleaned exclusively from cell-free or permeabilized cell reconstitution and resolution studies . Herein, we report for the first time the use of genetic approaches to directly address the physiological functions of PITP alpha and PITP beta in murine cells . Contrary to expectations, we find that ablation of PITP alpha function in murine cells fails to compromise growth and has no significant consequence for bulk phospholipid metabolism . Moreover, the data show that PITP alpha does not play an obvious role in any of the cellular activities where it has been reconstituted as an essential stimulatory factor . These activities include protein trafficking through the constitutive secretory pathway, endocytic pathway function, biogenesis of mast cell dense core secretory granules, and the agonist-induced fusion of dense core secretory granules to the mast cell plasma membrane . Finally, the data demonstrate that PITP alpha-deficient cells not only retain their responsiveness to bulk growth factor stimulation but also retain their pluripotency . In contrast, we were unable to evict both PITP beta alleles from murine cells and show that PITP beta deficiency results in catastrophic failure early in murine embryonic development . We suggest that PITP beta is an essential housekeeping PITP in murine cells, whereas PITP alpha plays a far more specialized function in mammals than that indicated by in vitro systems that show PITP dependence. J Virol, 2002 Apr, 76(8), 3905 - 19 Long-distance base pairing in flock house virus RNA1 regulates subgenomic RNA3 synthesis and RNA2 replication; Lindenbach BD et al.; Replication of flock house virus (FHV) RNA1 and production of subgenomic RNA3 in the yeast Saccharomyces cerevisiae provide a useful tool for the dissection of FHV molecular biology and host-encoded functions involved in RNA replication . The replication template activity of RNA1 can be separated from its coding potential by supplying the RNA1-encoded replication factor protein A in trans . We constructed a trans-replication system in yeast to examine cis-acting elements in RNA1 that control RNA3 production, as well as RNA1 and RNA2 replication . Two cis elements controlling RNA3 production were found . A proximal subgenomic control element was located just upstream of the RNA3 start site (nucleotides {nt} 2282 to 2777) . A short distal element also controlling RNA3 production (distal subgenomic control element) was identified 1.5 kb upstream, at nt 1229 to 1239 . Base pairing between these distal and proximal elements was shown to be essential for RNA3 production by covariation analysis and in vivo selection of RNA3-expressing replicons from plasmid libraries containing random sequences in the distal element . Two distinct RNA1 replication elements (RE) were mapped within the 3' quarter of RNA1: the intRE (nt 2322 to 2501) and the 3'RE (nt 2735 to 3011) . The 3'RE significantly overlaps the RNA3 region in RNA1, and this information was applied to produce improved RNA3-based vectors for foreign-gene expression . In addition, replication of an RNA2 derivative was dependent on RNA1 templates capable of forming the long-distance interaction that controls RNA3 production. Biochem Biophys Res Commun, 2002 Mar 29, 292(2), 355 - 61 Exposure to power frequency magnetic fields suppresses X-ray-induced apoptosis transiently in Ku80-deficient xrs5 cells; Tian F et al.; In an attempt to determine whether exposure to extremely low frequency (ELF) electromagnetic fields can affect cells, Ku80-deficient cells (xrs5) and Ku80-proficient cells (CHO-K1) were exposed to ELF electromagnetic fields . Cell survival, and the levels of the apoptosis-related genes p21, p53, phospho-p53 (Ser(15)), caspase-3 and the anti-apoptosis gene bcl-2 were determined in xrs5 and CHO-K1 cells following exposure to ELF electromagnetic fields and X-rays . It was found that exposure of xrs5 and CHO-K1 cells to 60 Hz ELF electromagnetic fields had no effect on cell survival, cell cycle distribution and protein expression . Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields for 5 h after irradiation significantly inhibited G(1) cell cycle arrest induced by X-rays (1 Gy) and resulted in elevated bcl-2 expression . A significant decrease in the induction of p53, phospho-p53, caspase-3 and p21 proteins was observed in xrs5 cells when irradiation by X-rays (8 Gy) was followed by exposure to 5 mT ELF magnetic fields . Exposure of xrs5 cells to the ELF electromagnetic fields for 10 h following irradiation significantly decreased X-ray-induced apoptosis from about 1.7% to 0.7% . However, this effect was not found in CHO-K1 cells within 24 h of irradiation by X-rays alone and by X-rays combined with ELF electromagnetic fields . Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields following irradiation can affect cell cycle distribution and transiently suppress apoptosis by decreasing the levels of caspase-3, p21, p53 and phospho-p53 and by increasing bcl-2 expression . (c)2002 Elsevier Science (USA). Biotechniques, 2002 Mar, Suppl, 16 - 8, 20-4, 26-7 Bioinformatics of large-scale protein interaction networks; Schachter V; We survey recent techniques for construction and prediction of large-scale protein interaction networks, focusing on computational processing steps . Special emphasis is placed on critical assessment of data completeness and reliability of the various approaches . Once built, protein interaction networks can be used for functional annotation or to generate higher-level biological hypotheses on pathways. Plant Mol Biol, 2002 Mar, 48(4), 401 - 11 Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning, biochemical properties and developmentally regulated expression; Yamazaki M et al.; Two flavonoid glucosyltransferases, UDP-glucose:flavonoid 3-0-glucosyltransferase (3-GT) and UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT), are responsible for the glucosylation of anthocyani(di)ns to produce stable molecules in the anthocyanin biosynthetic pathway . The cDNAs encoding 3-GT and 5-GT were isolated from Petunia hybrida by hybridization screening with heterologous probes . The cDNA clones of 3-GT, PGT8, and 5-GT, PH1, encode putative polypeptides of 448 and 468 amino acids, respectively . A phylogenetic tree based on amino acid sequences of the family of glycosyltransferases from various plants shows that PGT8 belongs to the 3-GT subfamily and PH1 belongs to the 5-GT subfamily . The function of isolated cDNAs was identified by the catalytic activities for 3-GT and 5-GT exhibited by the recombinant proteins produced in yeast . The recombinant PGT8 protein could convert not only anthocyanidins but also flavonols into the corresponding 3-O-glucosides . In contrast, the recombinant PH1 protein exhibited a strict substrate specificity towards anthocyanidin 3-acylrutinoside, comparing with other 5-GTs from Perilla frutescens and Verbena hybrida, which showed broad substrate specificities towards several anthocyanidin 3-glucosides . The mRNA expression of both 3-GT and 5-GT increased in the early developmental stages of P . hybrida flower, reaching the maximum at the stage before flower opening . Southern blotting analysis of genomic DNA indicates that both 3-GT and 5-GT genes exist in two copies in P . hybrida, respectively . The results are discussed in relation to the molecular evolution of flavonoid glycosyltransferases. Acta Neuropathol (Berl), 2002 Apr, 103(4), 408 - 14 Epub 2002 Jan 09. Impairment of mitochondrial DNA repair enzymes against accumulation of 8-oxo-guanine in the spinal motor neurons of amyotrophic lateral sclerosis; Kikuchi H et al.; Oxidative stress plays an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS) . In the present study, we investigated the expression of two major human enzymes that prevent errors caused by 8-oxoguanine (8-oxoG), a mitochondrial form of 8-oxoG DNA glycosylase (hOGG1) and oxidized purine nucleoside triphosphatase (hMTH1) . We also investigated the relationship between their expression and the 8-oxoG accumulation observed in the large motor neurons of the lumbar spinal cord in seven cases of adult onset sporadic ALS, four cases of subarachnoid hemorrhage (SAH) and four control cases . 8-oxoG immunoreactivity increased in most large motor neurons in both the ALS and SAH cases . However, the large motor neurons in the control cases often lacked hOGG1 immunoreactivity, although some neurons expressed hOGG1 in either homogeneous or fine granular patterns . In SAH cases, most large motor neurons showed a fine granular pattern proportional to the increased 8-oxoG immunoreactivity . However, only half of the remaining motor neurons in ALS expressed hOGG1 in the fine granular pattern, and the rest did not show any immunoreactivity . In addition, small aggregates of hMTH1 in the nuclei of the anterior horn cells were present in several ALS cases . Our results indicate that the oxidative damage accumulates in the mitochondria of motor neurons in ALS, and that hOGG1 does not repair the damage efficiently, which may lead to a loss of motor neurons in ALS. Proc Natl Acad Sci U S A, 2002 Mar 19, 99(6), 3746 - 51 The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1; Zhao X et al.; Cell cycle checkpoints are evolutionarily conserved surveillance systems that protect genomic stability and prevent oncogenesis in mammals . One important target of checkpoint control is ribonucleotide reductase (RNR), which catalyzes the rate-limiting step in dNTP and DNA synthesis . In both yeast and humans, RNR is transcriptionally induced after DNA damage via Mec1/Rad53 (yeast) and ATM/CHK2 (human) checkpoint pathways . In addition, yeast checkpoint proteins Mec1 and Rad53 also regulate the RNR inhibitor Sml1 . After DNA damage or at S phase, Mec1 and Rad53 control the phosphorylation and concomitant degradation of Sml1 protein . This new layer of control contributes to the increased dNTP production likely necessary for DNA repair and replication; however, the molecular mechanism is unclear . Here we show that Dun1, a downstream kinase of Mec1/Rad53, genetically and physically interacts with Sml1 in vivo . The absence of Dun1 activity leads to the accumulation of Sml1 protein at S phase and after DNA damage . As a result, dun1Delta strains need more time to finish DNA replication, are defective in mitochondrial DNA propagation, and are sensitive to DNA-damaging agents . Moreover, phospho-Sml1 is absent or dramatically reduced in dun1Delta cells . Finally, Dun1 can phosphorylate Sml1 in vitro . These results suggest that Dun1 kinase function is the last step required in the Mec1/Rad53 cascade to remove Sml1 during S phase and after DNA damage. Proc Natl Acad Sci U S A, 2002 Mar 19, 99(6), 3517 - 22 Elongator is a histone H3 and H4 acetyltransferase important for normal histone acetylation levels in vivo; Winkler GS et al.; The elongating, hyperphosphorylated form of RNA polymerase II is associated with the Elongator complex, which has the histone acetyltransferase (HAT) Elp3 as a subunit . Here we show that, in contrast to the isolated Elp3 subunit, the activity of intact Elongator complex is directed specifically toward the amino-terminal tails of histone H3 and H4, and that Elongator can acetylate both core histones and nucleosomal substrates . The predominant acetylation sites are lysine-14 of histone H3 and lysine-8 of histone H4 . The three smallest Elongator subunits--Elp4, Elp5, and Elp6--are required for HAT activity, and Elongator binds to both naked and nucleosomal DNA . By using chromatin immunoprecipitation, we show that the levels of multiply acetylated histone H3 and H4 in chromatin are decreased in vivo in yeast cells lacking ELP3. Proc Natl Acad Sci U S A, 2002 Apr 2, 99(7), 4379 - 84 Epub 2002 Mar 19. Analysis of cell-cycle-specific gene expression in human cells as determined by microarrays and double-thymidine block synchronization; Shedden K et al.; Microarray analysis of gene expression patterns for thousands of human genes has led to the proposal that a large number of genes are expressed in a cell-cycle-specific manner . The identification of cyclically expressed genes was based on Affymetrix microarray analysis of gene expression after double-thymidine block synchronization . A statistical reanalysis of the original data leads to three principal findings . (i) Randomized data exhibit periodic patterns of similar or greater strength than the experimental data . This finding suggests that all apparent cyclicities in the expression measurements may arise from chance fluctuations . (ii) The presence of cyclicity and the timing of peak cyclicity in a given gene are not reproduced in two replicate experiments . This fact suggests there is an uncontrolled source of experimental variation that is stronger than the innate variation of gene expression in cells over time . (iii) The amplitude of peak expression in the second cycle is not consistently smaller than the corresponding amplitude in the first cycle . This finding places doubt on the assumption that the cells are actually synchronized . We propose that the microarray results do not support the proposal that there are numerous cell-cycle-specifically expressed genes in human cells. J Biol Chem, 2002 May 24, 277(21), 19049 - 55 Epub 2002 Mar 19. Cloning and characterization of a novel mammalian zinc transporter, zinc transporter 5, abundantly expressed in pancreatic beta cells; Kambe T et al.; Intracellular homeostasis for zinc is achieved through the coordinate regulation of specific transporters engaged in zinc influx, efflux, and intracellular compartmentalization . We have identified a novel mammalian zinc transporter, zinc transporter 5 (ZnT-5), by virtue of its similarity to ZRC1, a zinc transporter of Saccharomyces cerevisiae, a member of the cation diffusion facilitator family . Human ZnT-5 (hZnT-5) cDNA encodes a 765-amino acid protein with 15 predicted membrane-spanning domains . hZnT-5 was ubiquitously expressed in all tested human tissues and abundantly expressed in the pancreas . In the human pancreas, hZnT-5 was expressed abundantly in insulin-containing beta cells that contain zinc at the highest level in the body . The hZnT-5 immunoreactivity was found to be associated with secretory granules by electron microscopy . The hZnT-5-derived zinc transport activity was detected using the Golgi-enriched vesicles prepared from hZnT-5-induced HeLa/hZnT-5 cells in which exogenous hZnT-5 expression is inducible by the Tet-on gene regulation system . This activity was dependent on time, temperature, and concentration and was saturable . Moreover, zinc at a high concentration (10 mm) inhibited the growth of yeast expressing hZnT-5 . These results suggest that ZnT-5 plays an important role for transporting zinc into secretory granules in pancreatic beta cells. FEBS Lett, 2002 Feb 27, 513(2-3), 197 - 202 Cooperative activity of phospholipid-N-methyltransferases localized in different membranes; Janssen MJ et al.; The possibility that the phospholipid-N-methyltransferases from yeast are capable of acting upon a phospholipid substrate, localized in a different membrane than in which the enzymes reside ('trans-catalysis' hypothesis), was investigated using cho2 and opi3 gene disruptant strains, which are defective in phosphatidylethanolamine transferase (PEMT) and phospholipid methyltransferase (PLMT), respectively . When cell homogenates or microsomes of the two disruptant strains are mixed, the combined methyltransferase activity, measured as the incorporation of {(3)H}methyl label from S-adenosyl methionine, exceeds that expected based on the separate activities of PEMT and PLMT . The increased incorporation implies that monomethylphosphatidylethanolamine generated by PEMT becomes available for PLMT, as evidenced by increased synthesis of dimethylphosphatidylethanolamine and phosphatidylcholine . The kinetics of the cooperativity suggest a collision-based process, enabling either transport of substrate or 'trans-catalysis'. Int J Dev Biol, 2002 Jan, 46(1), 173 - 6 A screen for genes expressed in Drosophila imaginal discs; Gerlitz O et al.; The development of Drosophila imaginal discs serves as a model system to understand how genes determine the shape and size of an organ . The identification of genes involved in this process is an important step towards this goal . Here we describe a P-element based enhancer trap screen for genes expressed in the larval imaginal discs . Our aim was to establish a large collection of enhancer trap lines each showing expression of Gal4 in imaginal discs . To this end, we improved the well established P-element vector pGawB in order to obtain higher in vivo transposition frequencies . In addition we chose an F1-screening approach using UAS-GFP as a reporter gene . This system permits the efficient screening of larval and pupal stages of living animals and the detection of imaginal gene expression patterns through the transparent cuticle . The procedure has been optimized for high-throughput . 2'000 P-element insertions have been established which exhibit expression in imaginal discs. Nat Rev Cancer, 2001 Dec, 1(3), 194 - 202 Histone deacetylases and cancer: causes and therapies; Marks P et al.; Together, histone acetyltransferases and histone deacetylases (HDACs) determine the acetylation status of histones . This acetylation affects the regulation of gene expression, and inhibitors of HDACs have been found to cause growth arrest, differentiation and/or apoptosis of many tumours cells by altering the transcription of a small number of genes . HDAC inhibitors are proving to be an exciting therapeutic approach to cancer, but how do they exert this effect? Faraday Discuss, 2001, (120), 249 - 59; discussion 325-51 Spatio-temporal dynamics in glycolysis; Mair T et al.; During the glycolytic degradation of sugar in a thin layer of yeast extract, travelling waves of NADH and protons can be generated that carry a state of high enzymatic activity through the system . The controlled initiation of such waves with an activator of the enzyme phosphofructokinase (PFK) and the influence of various salts and co-factors on the propagation dynamics are investigated . Furthermore a first study of the dispersion of waves is presented . The experimental characterisation of this in vitro system contributes to unravelling the possible role of glycolysis for biological information processing . In this context, the provision of chemically available energy in the absence of compartmentation by glycolysis is of primary importance. Nat Cell Biol, 2002 Apr, 4(4), 317 - 22 Deregulated human Cdc14A phosphatase disrupts centrosome separation and chromosome segregation; Mailand N et al.; We show that human Cdc14A phosphatase interacts with interphase centrosomes, and that this interaction is independent of microtubules and Cdc14A phosphatase activity, but requires active nuclear export . Disrupting the nuclear export signal (NES) led to Cdc14A being localized in nucleoli, which in unperturbed cells selectively contain Cdc14B (ref . 1) . Conditional overproduction of Cdc14A, but not its phosphatase-dead or NES-deficient mutants, or Cdc14B, resulted in premature centrosome splitting and formation of supernumerary mitotic spindles . In contrast, downregulation of endogenous Cdc14A by short inhibitory RNA duplexes (siRNA) induced mitotic defects including impaired centrosome separation and failure to undergo productive cytokinesis . Consequently, both overexpression and downregulation of Cdc14A caused aberrant chromosome partitioning into daughter cells . These results indicate that Cdc14A is a physiological regulator of the centrosome duplication cycle, which, when disrupted, can lead to genomic instability in mammalian cells. Genetics, 2002 Mar, 160(3), 961 - 73 Identification and characterization of the genes encoding the core histones and histone variants of Neurospora crassa; Hays SM et al.; We have identified and characterized the complete complement of genes encoding the core histones of Neurospora crassa . In addition to the previously identified pair of genes that encode histones H3 and H4 (hH3 and hH4-1), we identified a second histone H4 gene (hH4-2), a divergently transcribed pair of genes that encode H2A and H2B (hH2A and hH2B), a homolog of the F/Z family of H2A variants (hH2Az), a homolog of the H3 variant CSE4 from Saccharomyces cerevisiae (hH3v), and a highly diverged H4 variant (hH4v) not described in other species . The hH4-1 and hH4-2 genes, which are 96% identical in their coding regions and encode identical proteins, were inactivated independently . Strains with inactivating mutations in either gene were phenotypically wild type, in terms of growth rates and fertility, but the double mutants were inviable . As expected, we were unable to isolate null alleles of hH2A, hH2B, or hH3 . The genomic arrangement of the histone and histone variant genes was determined . hH2Az and the hH3-hH4-1 gene pair are on LG IIR, with hH2Az centromere-proximal to hH3-hH4-1 and hH3 centromere-proximal to hH4-1 . hH3v and hH4-2 are on LG IIIR with hH3v centromere-proximal to hH4-2 . hH4v is on LG IVR and the hH2A-hH2B pair is located immediately right of the LG VII centromere, with hH2A centromere-proximal to hH2B . Except for the centromere-distal gene in the pairs, all of the histone genes are transcribed toward the centromere . Phylogenetic analysis of the N . crassa histone genes places them in the Euascomycota lineage . In contrast to the general case in eukaryotes, histone genes in euascomycetes are few in number and contain introns . This may be a reflection of the evolution of the RIP (repeat-induced point mutation) and MIP (methylation induced premeiotically) processes that detect sizable duplications and silence associated genes. Curr Mol Med, 2001 Sep, 1(4), 401 - 29 Histone acetylation/deacetylation and cancer: an "open" and "shut" case? Gray SG, Teh BT. DNA in eukaryotic cells is packaged into chromatin . The main packaging component of chromatin is the nucleosome, and this is composed of proteins known as histones . Histones can be reversibly modified in several ways, and the best characterized of these modifications is histone acetylation . This is a reversible modification, which is carried out by two families of enzymes, the histone acetyltransferases (HATs), and the histone deacetylases (HDACs) . These enzymes have important activities in many cellular processes including transcription, DNA replication and cell cycle progression . The mechanisms underlying tumor formation are multifaceted, and often involve mutations or alterations of genes involved with the regulation and control of the cell cycle or cell death . Because of their important roles in the regulation of such events, enzymes that affect histone acetylation status are increasingly being associated with tumors . This article describes some of the current knowledge about histone acetyltransferases and histone deacetylases, and how their multitudinal roles in cellular events may have important roles in tumorigensis. Folia Microbiol (Praha), 2001, 46(6), 559 - 63 Particulate 1,3-beta-D-glucan, carboxymethylglucan and sulfoethylglucan--influence of their oral or intraperitoneal administration on immunological respondence of mice; Mucksova J et al.; The effect of orally or intraperitoneally administered particulate 1,3-beta-D-glucan (PBG), carboxymethylglucan (CMG) or sulfoethylglucan (SEG), obtained from the culture filtrate of Saccharomyces cerevisiae, on the functions of murine peritoneal adherent cells (PC) (peroxidase activity, nitric oxide synthesis), on relative organ mass and on proliferation of splenocytes was determined . The modulating activities after parenteral and non-parenteral administration of these polysaccharides were compared . Significant enhancement of NO production was observed only after in vitro cultivation of PC in the presence of lipopolysaccharide (LPS) in groups of mice treated repeatedly orally with CMG, PBG and SEG at a dose of 50 mg/kg body mass . Peroxidase activity increased significantly after repeated oral administration of CMG and PBG at doses 150 and 50 mg/kg, SEG 150 mg/kg body mass . The peroxidase activity and NO synthesis in mice given a single intraperitoneal injection of glucans (15 mg/kg body mass) were slightly higher than those after oral administration . Neither a significant enhancement of relative organ mass nor enhancement of the proliferative response of splenocytes to in vitro added stimuli (LPS, phytohemagglutinin) after repeated oral or single intraperitoneal administration of beta-glucans was observed. J Biol Chem, 2002 May 24, 277(21), 18346 - 56 Epub 2002 Mar 15. The protein kinase/endoribonuclease IRE1alpha that signals the unfolded protein response has a luminal N-terminal ligand-independent dimerization domain; Liu CY et al.; In response to accumulation of unfolded proteins in the endoplasmic reticulum (ER), cells activate an intracellular signal transduction pathway called the unfolded protein response (UPR) . IRE and PERK are the two type-I ER transmembrane protein kinase receptors that signal the UPR . The N-terminal luminal domains (NLDs) of IRE1 and PERK sense ER stress conditions by a common mechanism and transmit the signal to regulate the cytoplasmic domains of these receptors . To provide an experimental system amenable to detailed biochemical and structural analysis to elucidate the mechanism of ER-transmembrane signaling mechanism mediated by the NLD, we overexpressed the soluble luminal domain of human IRE1alpha in COS-1 cells by transient DNA transfection . Here we report the expression, purification, and characterization of the soluble NLD . The biological function of the NLD was confirmed by its ability to associate with itself and to interact with both the membrane-bound full-length IRE1alpha receptor and the ER chaperone BiP . Functional and spectral studies suggested that the highly conserved N-linked glycosylation site is not required for proper protein folding and self-association . Interestingly, we demonstrated that the NLD forms stable dimers linked by intermolecular disulfide bridges . Our data support that the luminal domain represents a novel ligand-independent dimerization domain. EMBO Rep, 2002 Apr, 3(4), 329 - 34 Epub 2002 Mar 15. Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway; Mello JA et al.; The efficient assembly of newly replicated and repaired DNA into chromatin is essential for proper genome function . Based on genetic studies in Saccharomyces cerevisiae, the histone chaperone anti-silencing function 1 (Asf1) has been implicated in the DNA repair response . Here, the human homologs are shown to function synergistically with human CAF-1 to assemble nucleosomes during nucleotide excision repair in vitro . Furthermore, we demonstrate that hAsf1 proteins can interact directly with the p60 subunit of hCAF-1 . In contrast to hCAF-1 p60, the nuclear hAsf1 proteins are not significantly associated with chromatin in cells before or after the induction of DNA damage, nor specifically recruited to damaged DNA during repair in a bead-linked DNA assay . A model is proposed in which the synergism between hAsf1 and CAF-1 for nucleosome formation during DNA repair is achieved through a transient physical interaction allowing histone delivery from Asf1 to CAF-1. Genome Biol . 2002;3(3):RESEARCH0011 . Epub 2002 Feb 13. Vector algebra in the analysis of genome-wide expression data; Kuruvilla FG et al.; BACKGROUND: Data from thousands of transcription-profiling experiments in organisms ranging from yeast to humans are now publicly available . How best to analyze these data remains an important challenge . A variety of tools have been used for this purpose, including hierarchical clustering, self-organizing maps and principal components analysis . In particular, concepts from vector algebra have proven useful in the study of genome-wide expression data . RESULTS: Here we present a framework based on vector algebra for the analysis of transcription profiles that is geometrically intuitive and computationally efficient . Concepts in vector algebra such as angles, magnitudes, subspaces, singular value decomposition, bases and projections have natural and powerful interpretations in the analysis of microarray data . Angles in particular offer a rigorous method of defining 'similarity' and are useful in evaluating the claims of a microarray-based study . We present a sample analysis of cells treated with rapamycin, an immunosuppressant whose effects have been extensively studied with microarrays . In addition, the algebraic concept of a basis for a space affords the opportunity to simplify data analysis and uncover a limited number of expression vectors to span the transcriptional range of cell behavior . CONCLUSIONS: This framework represents a compact, powerful and scalable construction for analysis and computation . As the amount of microarray data in the public domain grows, these vector-based methods are relevant in determining statistical significance . These approaches are also well suited to extract biologically meaningful information in the analysis of signaling networks. Genome Biol . 2002;3(3):REPORTS4007 . Epub 2002 Feb 25. Genomics meets nanoscience: probing genes and the cell nucleus at 10-9 meters; Politz J et al.; A report on the Jackson Laboratory 'Genomics meets nanoscience' meeting, Bar Harbor, USA, 9-12 October 2001. Oncogene, 2002 Mar 7, 21(11), 1777 - 84 Cdc6 requires anchorage for its expression; Jinno S et al.; Fibroblasts need anchorage to extracellular matrix to transit from G1 to S phase, but no longer after oncogenic transformation . Here we report that Cdc6 protein essential for the activation of replication origins requires anchorage or oncogenic stimulation for its execution . Upon anchorage loss, Cdc6 expression is shut off both transcriptionally and post-transcriptionally in a rat fibroblast despite enforced activation of E2F-dependent promoters . However, stimulation of this cell with oncogenic growth factors suppresses this shutoff and concurrently activates Cdk2 and Cdk6/4, thereby overriding the anchorage requirement for the G1-S transition and consequently enabling cells to perform anchorage-independent S phase entry . Analysis with enforced expression of Cdc6 indicates that the G1 cyclin-dependent kinases and Cdc6 constitute major cell cycle targets for the restriction of the G1-S transition by anchorage loss. Eur J Biochem, 2002 Mar, 269(6), 1780 - 9 Characterization of RNA polymerase III transcription factor TFIIIC from the mulberry silkworm, Bombyx mori; Srinivasan L et al.; Fractionation of nuclear extracts from posterior silk glands of mulberry silkworm Bombyx mori, resolved the transcription factor TFIIIC into two components (designated here as TFIIIC and TFIIIC1) as in HeLa cell nuclear extracts . The reconstituted transcription of tRNA genes required the presence of both components . The affinity purified TFIIIC is a heteromeric complex comprising of five subunits ranging from 44 to 240 kDa . Of these, the 51-kDa subunit could be specifically crosslinked to the B box of tRNA1Gly . Purified swTFIIIC binds to the B box sequences with an affinity in the same range as of yTFIIIC or hTFIIIC2 . Although an histone acetyl transferase (HAT) activity was associated with the TFIIIC fractions during the initial stages of purification, the HAT activity, unlike the human TFIIIC preparations, was separated at the final DNA affinity step . The tRNA transcription from DNA template was independent of HAT activity but the repressed transcription from chromatin template could be partially restored by external supplementation of the dissociated HAT activity . This is the first report on the purification and characterization of TFIIIC from insect systems. Eur J Biochem, 2002 Mar, 269(6), 1734 - 45 The lipase/acyltransferase from Candida parapsilosis: molecular cloning and characterization of purified recombinant enzymes; Neugnot V et al.; Candida parapsilosis has been previously shown to produce a lipase (i.e . able to catalyze efficiently the hydrolysis of insoluble lipid esters such as triacylglycerols) that preferentially catalyses transfer reactions such as alcoholysis in the presence of suitable nucleophiles other than water, even in aqueous media with high (> 0.9) water thermodynamic activity . The present work describes the cloning and the overexpression of the gene coding for this enzyme . Two ORFs (CpLIP1 and CpLIP2) were isolated . The deduced 465-amino-acid protein sequences contained the consensus motif (G-X-S-X-G) which is conserved among lipolytic enzymes . Only one of the two deduced proteins (CpLIP2) contained peptide sequences obtained from the purified lipase/acyltransferase . Homology investigations showed that CpLIP2 has similarities principally with 11 lipases produced by C . albicans (42-61%) and the lipase A from Candida antarctica (31%) but not with the other lipases sequenced so far . Both CpLIP1 and CpLIP2 were expressed in Saccharomyces cerevisiae, but only CpLIP2 coded for an active protein . The substrate specificity and the catalytic behavior of purified recombinant CpLIP2, with or without a C-terminal histidine tag, were not changed compared to those of the native lipase. EMBO J, 1983, 2(1), 33 - 8 Conservation of RNA secondary structures in two intron families including mitochondrial-, chloroplast- and nuclear-encoded members; Michel F et al.; Two families of fungal mitochondrial introns that include all known sequences have been recognized . These families are now extended to incorporate a plant mitochondrial intron and several introns in chloroplast- and nuclear-encoded rRNA and tRNA precursors . Members of the same family share distinctive sequence stretches and a number of potential RNA secondary structures that would bring these stretches and the intron-exon junctions into relatively close proximity . Using several of these introns which have been extensively studied by either biochemical or genetic means, an attempt is made to integrate the available data into a common picture. Cell Biochem Biophys, 2001, 35(3), 289 - 301 Replication-related activities establish cohesion between sister chromatids; Wang Z et al.; Replicated sister chromatids are held together from their synthesis in S phase to their separation in anaphase . The process of sister chromatid cohesion is essential for the proper segregation of chromosomes in eukaryotic cells . Recent studies in Saccharomyces cerevisiae have advanced our understanding of how sister chromatid cohesion is established, maintained, and dissolved during the cell cycle . Historical observations have suggested that establishment of cohesion is roughly coincident with replication fork passage . Emerging evidence now indicates that replication fork components, such as PCNA, a novel DNA polymerase, Trf4p/Pol sigma (formerly Trf4p/Pol kappa), and a modified clamp-loader complex, actively participate in the process of the cohesion establishment . Here, we review the molecular events in the chromosome cycle with respect to cohesion . Failure of sister chromatid cohesion results in the aneuploidy characteristic of many birth defects and tumors in humans. J Biol Chem, 2002 May 24, 277(21), 18535 - 44 Epub 2002 Mar 13. Structural identification of 2'- and 3'-O-acetyl-ADP-ribose as novel metabolites derived from the Sir2 family of beta -NAD+-dependent histone/protein deacetylases; Jackson MD et al.; The Sir2 (silent information regulator 2) family of histone/protein deacetylases has been implicated in a wide range of biological activities, including gene silencing, life-span extension, and chromosomal stability . Their dependence on beta-NAD(+) for activity is unique among the known classes of histone/protein deacetylase . Sir2 enzymes have been shown to couple substrate deacetylation and beta-NAD(+) cleavage to the formation of O-acetyl-ADP-ribose, a newly described metabolite . To gain a better understanding of the catalytic mechanism and of the biological implications of producing this molecule, we have performed a detailed enzymatic and structural characterization of O-acetyl-ADP-ribose . Through the use of mass spectrometry, rapid quenching techniques, and NMR structural analyses, 2'-O-acetyl-ADP-ribose and 3'-O-acetyl-ADP-ribose were found to be the solution products produced by the Sir2 family of enzymes . Rapid quenching approaches under single-turnover conditions identified 2'-O-acetyl-ADP-ribose as the enzymatic product, whereas 3'-O-acetyl-ADP-ribose was formed by intramolecular transesterification after enzymatic release into bulk solvent, where 2'- and 3'-O-acetyl-ADP-ribose exist in equilibrium (48:52) . In addition to (1)H and (13)C chemical shift assignments for each regioisomer, heteronuclear multiple-bond correlation spectroscopy was used to assign unambiguously the position of the acetyl group . These findings are highly significant, because they differ from the previous conclusion, which suggested that 1'-O-acetyl-ADP-ribose was the solution product of the reaction . Possible mechanisms for the generation of 2'-O-acetyl-ADP-ribose are discussed. J Biol Chem, 2002 May 17, 277(20), 17531 - 7 Epub 2002 Mar 13. Specific modulation of Kex2/furin family proteases by potassium; Rockwell NC et al.; Kex2 protease is the prototype for a family of proteases responsible for endoproteolytic cleavage at multi-basic motifs in the eukaryotic secretory pathway . Here we demonstrate that potassium ion can act as a modulator of Kex2 activity with an apparent affinity of approximately 20 mm . Other monovalent cations (Li(+), Na(+), etc.) display similar effects, but affinities are all over 20-fold lower . Potassium ion binding stimulates turnover at physiologically relevant Lys-Arg cleavage sites but reduces turnover with at least one incorrect sequence . Furthermore, the mammalian Kex2 homolog furin displays similar effects . In contrast, the neuroendocrine homolog PC2 is inhibited by potassium ion with all substrates examined . The pre-steady-state behavior of Kex2 is also altered upon binding of potassium ion, with opposite effects on acylation and deacylation rates . These biochemical data indicate that potassium ion concentration may function as a regulator of processing protease specificity and activity in the eukaryotic secretory pathway, with such enzymes potentially encountering compartments high in potassium ion caused by the action of antiporters such as yeast NHX1 (VPS44) or the mammalian NHE7. Trends Biochem Sci, 2002 Mar, 27(3), 139 - 47 Conservation of amino acid transporters in fungi, plants and animals; Wipf D et al.; When comparing the transporters of three completely sequenced eukaryotic genomes--Saccharomyces cerevisiae, Arabidopsis thaliana and Homo sapiens--transporter types can be distinguished according to phylogeny, substrate spectrum, transport mechanism and cell specificity . The known amino acid transporters belong to five different superfamilies . Two preferentially Na(+)-coupled transporter superfamilies are not represented in the yeast and Arabidopsis genomes, whereas the other three groups, which often function as H(+)-coupled systems, have members in all investigated genomes . Additional superfamilies exist for organellar transport, including mitochondrial and plastidic carriers . When used in combination with phylogenetic analyses, functional comparison might aid our prediction of physiological functions for related but uncharacterized open reading frames. Trends Biochem Sci, 2002 Mar, 27(3), 115 - 7 A second catalytic domain in the Elp3 histone acetyltransferases: a candidate for histone demethylase activity? Chinenov Y. A new subfamily of two-domain histone acetyltransferases (HATs) related to Elp3 has been identified . In addition to a HAT domain in the C terminus, these proteins have an N-terminal domain similar to the catalytic domain of S-adenosylmethionine radical enzymes . Two-domain organization is preserved in evolution, suggesting that both enzymatic activities are functionally or mechanistically coupled and directed towards highly conserved substrates . The functional implications of this similarity and a possible role for Elp3-related proteins as histone demethylases are discussed. Curr Opin Genet Dev, 2002 Apr, 12(2), 198 - 209 Histone methylation in transcriptional control; Kouzarides T; Over the past year or so, methylation of histones has come to be recognised as a major player in the regulation of gene activity . This notion follows the discovery of lysine and arginine methyltransferases and proteins that recognise the methyl-lysine 'mark' on histones . Methylated histones have been implicated in heterochromatic repression, promoter regulation and the propagation of a repressed state via DNA methylation. Curr Opin Genet Dev, 2002 Apr, 12(2), 188 - 92 Breaking through to the other side: silencers and barriers; Dhillon N et al.; The establishment and restriction of transcriptionally inactive regions in the nucleus is mediated by silencer and barrier elements . Silencer-bound proteins recruit additional factors to establish the silenced domain during the S-phase of the cell cycle but, contrary to previous models, DNA replication is not a pre-requisite for the establishment . Characteristically, silenced domains contain hypoacetylated histones and recent data have identified residue-specific methylation of histone H3 as an additional signature that distinguishes active regions from inactive ones . Peaks of acetylated histones demarcate the boundaries between these regions and recruitment of HAT activities provides a mechanism to restrict the spread of heterochromatin. Curr Opin Genet Dev, 2002 Apr, 12(2), 178 - 87 Heterochromatin: new possibilities for the inheritance of structure; Grewal SI et al.; Significant portions of the eukaryotic genome are heterochromatic, made up largely of repetitious sequences and possessing a distinctive chromatin structure associated with gene silencing . New insights into the form of packaging, the associated histone modifications, and the associated nonhistone chromosomal proteins of heterochromatin have suggested a mechanism for providing an epigenetic mark that allows this distinctive chromatin structure to be maintained following replication and to spread within a given domain. Curr Opin Genet Dev, 2002 Apr, 12(2), 170 - 7 ChIPs of the beta-globin locus: unraveling gene regulation within an active domain; Bulger M et al.; Recent studies of beta-globin gene expression have concentrated on the analysis of factor binding and chromatin structure within the endogenous locus . These studies have more precisely defined the extent and nature of the active chromosomal domain and the elements that organize it . Surprisingly, the beta-globin locus control region (LCR), although critical for high-level gene expression, plays little role in the overall architecture of the active locus . Analysis of the effects of targeted deletion of the beta-globin LCR, along with emerging knowledge of the behavior of the erythroid transcription factor NF-E2, leads to a new perspective on factor binding and LCR function. Curr Opin Genet Dev, 2002 Apr, 12(2), 162 - 9 Histone H2A variants H2AX and H2AZ; Redon C et al.; Two of the nucleosomal histone families, H3 and H2A, have highly conserved variants with specialized functions . Recent studies have begun to elucidate the roles of two of the H2A variants, H2AX and H2AZ . H2AX is phosphorylated on a serine four residues from the carboxyl terminus in response to the introduction of DNA double-strand breaks, whether these breaks are a result of environmental insult, metabolic mistake, or programmed process . H2AZ appears to alter nucleosome stability, is partially redundant with nucleosome remodeling complexes, and is involved in transcriptional control. Curr Opin Genet Dev, 2002 Apr, 12(2), 149 - 55 Coactivators in transcription initiation: here are your orders; Featherstone M; Coactivators are diverse and multifunctional proteins that act downstream of DNA-binding activators to stimulate transcription . Recent studies elucidate the temporal sequence in which coactivators are recruited to target promoters, and how their enzymatic properties and molecular interactions culminate in transcriptional initiation. Curr Opin Genet Dev, 2002 Apr, 12(2), 142 - 8 Histone modifications in transcriptional regulation; Berger SL; Covalent modifications of the amino termini of the core histones in nucleosomes have important roles in gene regulation . Research in the past two years reveals these modifications to consist of phosphorylation, methylation and ubiquitination, in addition to the better-characterized acetylation . This multiplicity of modifications, and their occurrence in patterns and dependent sequences, argues persuasively for the existence of a histone code. Curr Opin Genet Dev, 2002 Apr, 12(2), 130 - 6 Deciphering gene expression regulatory networks; Wyrick JJ et al.; In the past year, great strides have been made in our understanding of the regulatory networks that control gene expression in the model eukaryote Saccharomyces cerevisiae . The development and use of a number of genomic tools, including genome-wide location and expression analysis, has fueled this progress . In addition, a variety of computational algorithms have been devised to mine genomic sequence for conserved regulatory motifs in co-regulated genes . The recent description of the genetic network controlling the cell cycle illustrates the tremendous potential of these approaches for deciphering gene expression regulatory networks in eukaryotic cells. J Pept Res, 2001 Nov, 58(5), 389 - 98 Solution structures of the N-terminal domain of histone H4; Bang E et al.; Histones, nuclear proteins that interact with DNA to form nucleosomes, are essential for both the regulation of transcription and the packaging of DNA within chromosomes . The N-terminal domain of histone H4 contains four acetylation sites at lysine residues and may play a separate role in chromatin structure from the remainder of the H4 chain . We performed circular dichroism and NMR characterization of both native (H4NTP) and acetylated (Ace-H4NTP) peptides containing N-terminal acetylation domain of histone H4 for various pH environments . Data from CD and NMR suggested that H4NTP exhibited a pH-dependent conformational change, whereas the Ace-H4NTP is insensitive to pH change . However, both peptides showed a defined structural form at acidic pH environments . The solution structure for Ace-H4NTP shows two structurally independent regions comprising residues of Leu10-Gly13 and Arg19-Leu22, demonstrating relatively well-defined turn-type structures . Our results suggest that N-terminal acetylated region of H4 prefers an extended backbone conformation at neutral pH, however, upon acetylation, the regions containing lysine residues induce structural transition, having defined structural form for its optimum function. EMBO J, 1983, 2(9), 1465 - 72 Map location of transcripts from Torulopsis glabrata mitochondrial DNA; Clark-Walker GD et al.; Unique transcripts for cytochrome b, ATPase subunits 6 and 9, cytochrome oxidase subunits 2 and 3 and S and L rRNA have been mapped by the S1 protection technique to the circular 19-kbp mitochondrial DNA (mtDNA) of the yeast Torulopsis glabrata . In contrast, a number of transcripts have been detected for the mosaic cytochrome oxidase subunit 1 gene with the largest being approximately 5000 nucleotides and the mature message having a length of 1760 nucleotides . Despite the presence in T . glabrata mtDNA of a sequence that hybridizes to the variant 1 gene of Saccharomyces cerevisiae mtDNA we have not detected a transcript of this region . Neither have we detected co-transcripts of adjacent genes in RNA from either glucose-repressed or derepressed cells . However, by comparison of RNA species from the two growth conditions, we have found that the ATPase subunit 6 transcript is lower in amount relative to other species in preparations from glucose-repressed cells . This information, together with the observation of separate transcripts and the knowledge that there are several species of mitochondrial RNA which can be capped by the guanylyl transferase catalysed addition of GMP, suggests that each of the genes investigated in the present study is separately transcribed. Curr Top Microbiol Immunol, 2002, 261, 165 - 77 Integration site selection by lentiviruses: biology and possible control; Bushman FD; Retroviruses integrate into naked DNA in a generally sequence nonspecific fashion, but closer study reveals a variety of forces that influence target site selection . Primary sequence of the target plays a small but detectable role . Proteins bound to target DNA can inhibit integration by blocking access of integration complexes or stimulate integration by distorting DNA . An important example of the latter is DNA distortion in nucleosomal DNA . In vivo integration has not yet been convincingly shown to be biased in favor of any identifiable sequence features, though this could still change in future studies . Many applications of retroviral vectors could be facilitated by targeting integration in vivo to predetermined sites . Towards this end, several groups have studied the properties of fusions of integrase proteins to sequence-specific DNA-binding domains . To date such studies establish that targeting can work well in reactions in vitro, but a variety of obstacles complicate applications in vivo . However, naturally occurring retrotransposons do carry out highly targeted integration using retrovirus-like integrase proteins, fueling long-term hopes for targeting with retroviral integrases as well. Plant Physiol, 2002 Mar, 128(3), 833 - 43 Interaction of the Arabidopsis E2F and DP proteins confers their concomitant nuclear translocation and transactivation; Kosugi S et al.; E2F transcription factors are required for the progression and arrest of the cell cycle in animals . Like animals, plants have evolved to conserve the E2F family . The Arabidopsis genome encodes E2F and DP proteins that share a high similarity with the animal E2F and DP families . Here, we show that Arabidopsis E2F and DP proteins are not predominantly localized to the nucleus in analyses with green fluorescent protein, and that the complete nuclear localization of some members is driven by the co-expression of their specific partner proteins . Both AtE2F1 and AtE2F3 were translocated to the nucleus and transactivate an E2F reporter gene when co-expressed with DPa but not DPb . In contrast, AtE2F2 was inactive for both nuclear translocation and transactivation even when Dpa or DPb was co-expressed . Because the DNA binding activities of the three E2Fs are equally stimulated by the interaction with DPa or DPb in vitro, the observed transactivation of AtE2F1 and AtE2F3 is DPa specific and nuclear import dependent . A green fluorescent protein fusion with an AtE2F3 mutant, in which a conserved nuclear export signal-like sequence in the dimerization domain was deleted, was localized to the nucleus . Thus, the concomitant nuclear translocation seems to be conferred by the DPa interaction to release an activity that inhibits an intrinsic nuclear import activity of AtE2Fs . Furthermore, the nuclear translocation of AtE2F3 stimulated by DPa was abolished by the deletion of the N-terminal region of AtE2F3, which is conserved among all the E2F proteins identified in plants to date . Replacement of the N-terminal region of AtE2F3 with a canonical nuclear localization signal only partially mimicked the effect of the DPa co-expression, demonstrating the function of plant E2F distinct from that observed for animal E2Fs . These observations suggest that the function of plant E2F and DP proteins is primarily controlled by their nuclear localization mediated by the interaction with specific partner proteins. Gene, 2002 Feb 6, 284(1-2), 161 - 8 Cloning of rat thymic stromal lymphopoietin receptor (TSLPR) and characterization of genomic structure of murine Tslpr gene; Blagoev B et al.; Thymic stromal derived lymphopoietin receptor (TSLPR) is a novel receptor subunit that is related in sequence to the interleukin (IL)-2 receptor common gamma chain . TSLPR forms a heterodimeric complex with the IL-7 receptor alpha chain to form the receptor for thymic stromal derived lymphopoietin, a cytokine involved in B- and T-cell function . We have cloned the TSLP receptor from rat and find that the WSXWX motif commonly found in extracellular domains of cytokine receptors is conserved as a W(T/S)XV(T/A) motif among TSLP receptors from mouse, rat and human . As in the mouse, TSLP receptor is widely expressed in rats suggesting that TSLPR may have roles in signaling outside the hematopoietic system . A zooblot analysis revealed that TSLPR is expressed in all vertebrate species examined . The absence of TSLPR in Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans genomes is similar to the expression of several other cytokine receptors that have been characterized thus far . We have also characterized the genomic structure of the murine Tslpr gene which shows that in addition to primary sequence homology, it shares a common genomic organization of coding exons with the murine IL-2 receptor common gamma chain (Il2rg) . Use of an alternative splice acceptor site leads to two alternatively spliced transcript variants of murine TSLPR, both of which are functional receptors . Finally, using linkage analysis, we mapped the murine Tslpr gene to mouse chromosome 5 between the Ecm2 and Pxn genes. Biochem Biophys Res Commun, 2002 Mar 22, 292(1), 256 - 62 Murine Apg12p has a substrate preference for murine Apg7p over three Apg8p homologs; Tanida I et al.; Apg7p is a unique E1 enzyme which is essential for both the Apg12p- and Apg8p-modification systems, and plays indispensable roles in yeast autophagy . A cDNA encoding murine Apg7p homolog (mApg7p) was isolated from a mouse brain cDNA library . The predicted amino acid sequence of the clone shows a significant homology to human Apg7p and yeast Apg7p . Murine Apg12p as well as the three mammalian Apg8p homologs co-immunoprecipitate with mApg7p . Site-directed mutagenesis revealed that an active-site cysteine within mApg7p is Cys(567), indicating that mApg7p is an authentic E1 enzyme for murine Apg12p and mammalian Apg8p homologs . The mutagenesis study also revealed that Apg12p has a substrate preference for mApg7p over the three Apg8p homologs, suggesting that the Apg12p conjugation by Apg7p occurs preferentially in mammalian cells compared with the modification of the three Apg8p homologs . We also report here on the ubiquitous expression of human APG7 mRNA in human adult and fetal tissues and of rat Apg7p in adult tissues . (C)2002 Elsevier Science (USA). Nat Genet, 2002 Apr, 30(4), 436 - 40 Epub 2002 Mar 11. FACL4, encoding fatty acid-CoA ligase 4, is mutated in nonspecific X-linked mental retardation; Meloni I et al.; X-linked mental retardation (XLMR) is an inherited condition that causes failure to develop cognitive abilities, owing to mutations in a gene on the X chromosome . The latest XLMR update lists up to 136 conditions leading to 'syndromic', or 'specific', mental retardation (MRXS) and 66 entries leading to 'nonspecific' mental retardation (MRX) . For 9 of the 66 MRX entries, the causative gene has been identified . Our recent discovery of the contiguous gene deletion syndrome ATS-MR (previously known as Alport syndrome, mental retardation, midface hypoplasia, elliptocytosis, OMIM #300194), characterized by Alport syndrome (ATS) and mental retardation (MR), indicated Xq22.3 as a region containing one mental retardation gene . Comparing the extent of deletion between individuals with ATS-MR and individuals with ATS alone allowed us to define a critical region for mental retardation of approximately 380 kb, containing four genes . Here we report the identification of two point mutations, one missense and one splice-site change, in the gene FACL4 in two families with nonspecific mental retardation . Analysis of enzymatic activity in lymphoblastoid cell lines from affected individuals of both families revealed low levels compared with normal cells, indicating that both mutations are null mutations . All carrier females with either point mutations or genomic deletions in FACL4 showed a completely skewed X-inactivation, suggesting that the gene influences survival advantage . FACL4 is the first gene shown to be involved in nonspecific mental retardation and fatty-acid metabolism. J Cell Biol, 2002 Mar 18, 156(6), 1015 - 28 Epub 2002 Mar 11. Osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p; Bonangelino CJ et al.; Phosphatidylinositol 3,5-bisphosphate (PtdIns{3,5}P(2)) was first identified as a non-abundant phospholipid whose levels increase in response to osmotic stress . In yeast, Fab1p catalyzes formation of PtdIns(3,5)P(2) via phosphorylation of PtdIns(3)P . We have identified Vac14p, a novel vacuolar protein that regulates PtdIns(3,5)P(2) synthesis by modulating Fab1p activity in both the absence and presence of osmotic stress . We find that PtdIns(3)P levels are also elevated in response to osmotic stress, yet, only the elevation of PtdIns(3,5)P(2) levels are regulated by Vac14p . Under basal conditions the levels of PtdIns(3,5)P(2) are 18-28-fold lower than the levels of PtdIns(3)P, PtdIns(4)P, and PtdIns(4,5)P(2) . After a 10 min exposure to hyperosmotic stress the levels of PtdIns(3,5)P(2) rise 20-fold, bringing it to a cellular concentration that is similar to the other phosphoinositides . This suggests that PtdIns(3,5)P(2) plays a major role in osmotic stress, perhaps via regulation of vacuolar volume . In fact, during hyperosmotic stress the vacuole morphology of wild-type cells changes dramatically, to smaller, more highly fragmented vacuoles, whereas mutants unable to synthesize PtdIns(3,5)P(2) continue to maintain a single large vacuole . These findings demonstrate that Vac14p regulates the levels of PtdIns(3,5)P(2) and provide insight into why PtdIns(3,5)P(2) levels rise in response to osmotic stress. J Biol Chem, 2002 May 24, 277(21), 18489 - 93 Epub 2002 Mar 11. A quality control pathway that down-regulates aberrant T-cell receptor (TCR) transcripts by a mechanism requiring UPF2 and translation; Wang J et al.; Nonsense-mediated decay (NMD) is an RNA surveillance pathway that degrades mRNAs containing premature termination codons (PTC) . T-cell receptor (TCR) and immunoglobulin (Ig) transcripts, which are encoded by genes that very frequently acquire PTCs during lymphoid ontogeny, are down-regulated much more dramatically in response to PTCs than are other known transcripts . Another feature unique to TCR, Ig, and a subset of other mRNAs is that they are down-regulated in response to nonsense codons in the nuclear fraction of cells . This is paradoxical, as the only well recognized entity that recognizes nonsense codons is the cytoplasmic translation apparatus . Therefore, we investigated whether translation is responsible for this nuclear-associated mechanism . We found that the down-regulation of TCR-beta transcripts in response to nonsense codons requires several features of translation, including an initiator ATG and the ability to scan . We also found that optimal down-regulation depends on a Kozak consensus sequence surrounding the initiator ATG and that it can be initiated by an internal ribosome entry site, neither of which has been demonstrated before for any other PTC-bearing mRNA . At least a portion of this down-regulatory response is mediated by the NMD pathway as antisense hUPF2 transcripts increased the levels of PTC-bearing TCR-beta transcripts in the nuclear fraction of cells . We conclude that a hUPF2-dependent RNA surveillance pathway with translation-like features operating in the nuclear fraction of cells prevents the expression of potentially deleterious truncated proteins encoded by non-productively rearranged TCR genes. EMBO J, 2002 Mar 15, 21(6), 1414 - 26 CCR4, a 3'-5' poly(A) RNA and ssDNA exonuclease, is the catalytic component of the cytoplasmic deadenylase; Chen J et al.; The CCR4-NOT complex from Saccharomyces cerevisiae is a general transcriptional regulatory complex . The proteins of this complex are involved in several aspects of mRNA metabolism, including transcription initiation and elongation and mRNA degradation . The evolutionarily conserved CCR4 protein, which is part of the cytoplasmic deadenylase, contains a C-terminal domain that displays homology to an Mg2+-dependent DNase/phosphatase family of proteins . We have analyzed the putative enzymatic properties of CCR4 and have found that it contains both RNA and single-stranded DNA 3'-5' exonuclease activities . CCR4 displays a preference for RNA and for 3' poly(A) substrates, implicating it as the catalytic component of the cytoplasmic deadenylase . Mutations in the key, conserved catalytic residues in the CCR4 exonuclease domain abolished both its in vitro activities and its in vivo functions . Importantly, CCR4 was active as a monomer and remained active in the absence of CAF1, which links CCR4 to the remainder of the CCR4-NOT complex components . These results establish that CCR4 and most probably other members of a widely distributed CCR4-like family of proteins constitute a novel class of RNA-DNA exonucleases . The various regulatory effects of the CCR4-NOT complex on gene expression may be executed in part through these CCR4 exonuclease activities. EMBO J, 2002 Mar 15, 21(6), 1406 - 13 GAL4 directs nucleosome sliding induced by NURF; Kang JG et al.; The Drosophila nucleosome remodeling factor (NURF) is an imitation switch (ISWI)-containing chromatin remodeling complex that can catalyze nucleosome repositioning at promoter regions to regulate access by the transcription machinery . Mononucleosomes reconstituted in vitro by salt dialysis adopt an ensemble of translational positions on DNA templates . NURF induces bi-directional 'sliding' of these nucleosomes to a subset of preferred positions . Here we show that mononucleosome sliding catalyzed by NURF bears similarity to nucleosome movement induced by elevated temperature . Moreover, we demonstrate that the GAL4 DNA-binding domain can extend NURF-induced nucleosome movement on a GAL4-E4 promoter, expanding the stretch of histone-free DNA at GAL4 recognition sites . The direction of NURF-induced nucleosome movement can be significantly modulated by asymmetric placement of tandem GAL4 sites relative to the nucleosome core particle . As such, sequence-specific, transcription factor-directed nucleosome sliding is likely to have substantial influence on promoter activation. EMBO J, 2002 Mar 15, 21(6), 1379 - 88 Promoter-specific functions of CIITA and the MHC class II enhanceosome in transcriptional activation; Masternak K et al.; Transcription of the major histocompatibility complex class II family of genes is regulated by conserved promoter elements and two gene-specific trans-activators, RFX and CIITA . RFX binds DNA and nucleates the assembly of an enhanceosome, which recruits CIITA through protein--protein interactions . Transcriptional activation is a complex, multi-step process involving chromatin modification and recruitment of the transcription apparatus . To examine the roles of the enhanceosome and CIITA in these processes, we analysed the level of promoter-associated hyperacetylated histones H3 and H4, TBP, TFIIB and RNA poly merase II in cells lacking RFX or CIITA . We compared four genes co-regulated by RFX and CIITA (HLA-DRA, HLA-DPB, HLA-DMB and Ii) and found that the enhanceosome and CIITA make variable, promoter-dependent contributions to histone acetylation and transcription apparatus recruitment . CIITA is generally implicated at multiple levels of the activation process, while the enhanceosome contributes in a CIITA-independent manner only at certain promoters . Our results support the general notion that the impact of a particular activator on transcription in vivo may vary depending on the promoter and the chromatin context. EMBO J, 2002 Mar 15, 21(6), 1267 - 79 The ANGUSTIFOLIA gene of Arabidopsis, a plant CtBP gene, regulates leaf-cell expansion, the arrangement of cortical microtubules in leaf cells and expression of a gene involved in cell-wall formation; Kim GT et al.; We previously showed that the ANGUSTIFOLIA (AN) gene regulates the width of leaves of Arabidopsis thaliana, by controlling the polar elongation of leaf cells . In the present study, we found that the abnormal arrangement of cortical microtubules (MTs) in an leaf cells appeared to account entirely for the abnormal shape of the cells . It suggested that the AN gene might regulate the polarity of cell growth by controlling the arrangement of cortical MTs . We cloned the AN gene using a map-based strategy and identified it as the first member of the CtBP family to be found in plants . Wild-type AN cDNA reversed the narrow-leaved phenotype and the abnormal arrangement of cortical MTs of the an-1 mutation . In the animal kingdom, CtBPs self-associate and act as co-repressors of transcription . The AN protein can also self-associate in the yeast two-hybrid system . Furthermore, microarray analysis suggested that the AN gene might regulate the expression of certain genes, e.g . the gene involved in formation of cell walls, MERI5 . A discussion of the molecular mechanisms involved in the leaf shape regulation is presented based on our observations. Dev Comp Immunol, 2002 May, 26(4), 345 - 54 Phagocyte spreading and phagocytosis in the compound ascidian Botryllus schlosseri: evidence for an integrin-like, RGD-dependent recognition mechanism; Ballarin L et al.; The involvement of integrins in phagocyte spreading and phagocytosis was investigated in the compound ascidian Botryllus schlosseri . The number of spreading cells was significantly reduced when adhesion occurred in the presence of the tetrapeptide Arg--Gly--Asp--Ser (RGDS), but not of Arg--Gly--Glu--Ser (RGES) indicating the involvement of RGD-mediated adhesion mechanisms in phagocyte spreading . The significant decrease of the fraction of spreading cells in the presence of Botryllus blood plasma suggests the presence of RGD-containing molecules in the blood of our species . The increase in the same index when blood plasma-coated slides as well as fibrinogen- and fibronectin-coated coverslips were used, fits with the above hypothesis . Adhesion in the presence of RGDS leads to a consistent alteration of the actin cytoskeleton, in agreement with the known role of integrin adhesion in microfilament organization . Phagocytosis was greatly reduced by RGDS in the incubation medium, but not by RGES, and was significantly increased by coating yeast cells with fibronectin or blood plasma . Both spreading and phagocytic capability were severely inhibited by wortmannin, suggesting the importance of phosphatidylinositol-3-kinase in integrin-mediated signal transduction in ascidians. Biochemistry, 2002 Mar 19, 41(11), 3605 - 12 Mechanism of domain closure of Sec7 domains and role in BFA sensitivity; Renault L et al.; Activation of small G proteins of the Arf family is initiated by guanine nucleotide exchange factors whose catalytic Sec7 domain stimulates the dissociation of the tightly bound GDP nucleotide . The exchange reaction involves distinct sequential steps that can be trapped by the noncompetitive inhibitor brefeldin A, by mutation of an invariant catalytic glutamate, or by removal of guanine nucleotides . Arf-GDP retains most characteristics of its GDP-bound form at the initial low-affinity Arf-GDP-Sec7 step . It then undergoes large conformational changes toward its GTP-bound form at the next step, and eventually dissociates GDP to form a nucleotide-free high-affinity Arf-Sec7 complex at the last step . Thus, Arf proteins evolve through different conformations that must be accommodated by Sec7 domains in the course of the reaction . Here the contribution of the flexibility of Sec7 domains to the exchange reaction was investigated with the crystal structure of the unbound Sec7 domain of yeast Gea2 . Comparison with Gea2 in complex with nucleotide-free Arf1 Delta 17 {Goldberg, J . (1998) Cell 95, 237-248} reveals that Arf induces closure of the two subdomains that form the sides of its active site . Several residues that determine sensitivity to brefeldin A are involved in interdomain and local movements, pointing to the importance of the flexibility of Sec7 domains for the inhibition mechanism . Altogether, this suggests a model for the initial steps of the exchange reaction where Arf docks onto the C-terminal domain of the Sec7 domain before closure of the N-terminal domain positions the catalytic glutamate to complete the reaction. Nat Struct Biol, 2002 Apr, 9(4), 301 - 7 Protein unfolding by the mitochondrial membrane potential; Huang S et al.; Mitochondria can unfold importing precursor proteins by unraveling them from their N-termini . However, how this unraveling is induced is not known . Two candidates for the unfolding activity are the electrical potential across the inner mitochondrial membrane and mitochondrial Hsp70 in the matrix . Here, we propose that many precursors are unfolded by the electrical potential acting directly on positively charged amino acid side chains in the targeting sequences . Only precursor proteins with targeting sequences that are long enough to reach the matrix at the initial interaction with the import machinery are unfolded by mitochondrial Hsp70, and this unfolding occurs even in the absence of a membrane potential. J Biol Chem, 2002 May 17, 277(20), 17743 - 50 Epub 2002 Mar 08. Prp43 is an essential RNA-dependent ATPase required for release of lariat-intron from the spliceosome; Martin A et al.; The essential Saccharomyces cerevisiae PRP43 gene encodes a 767-amino acid protein of the DEXH-box family . Prp43 has been implicated in spliceosome disassembly (Arenas, J . E., and Abelson, J . N . (1997) Proc . Natl . Acad . Sci . U . S . A . 94, 11798-11802) . Here we show that purified recombinant Prp43 is an RNA-dependent ATPase . Alanine mutations at conserved residues within motifs I ((119)GSGKT(123)), II ((215)DEAH(218)) and VI ((423)QRAGRAGR(430)) that diminished ATPase activity in vitro were lethal in vivo, indicating that ATP hydrolysis is necessary for the biological function of Prp43 . Overexpression of lethal, ATPase-defective mutants in a wild-type strain resulted in dominant-negative growth inhibition . The ATPase-defective mutant T123A interfered in trans with the in vitro splicing function of wild-type Prp43 . T123A did not affect the chemical steps of splicing or the release of mature mRNA from the spliceosome, but it blocked the release of the excised lariat-intron from the spliceosome . We show that the lariat-intron is not accessible to debranching by purified Dbr1 when it is held in the T123A-arrested splicing complex . Our results define a new ATP-dependent step of splicing that is catalyzed by Prp43. J Biol Chem, 2002 May 10, 277(19), 17125 - 38 Epub 2002 Mar 08. CD40 induces interleukin-6 gene transcription in dendritic cells: regulation by TRAF2, AP-1, NF-kappa B, AND CBF1; Mann J et al.; CD40-induced activation of cytokine gene expression in dendritic cells (DC) is an important process in the initiation of primary immune responses . We have determined the intracellular signaling events that lead to CD40 ligation-induced activation of interleukin-6 (IL-6) gene transcription in a murine DC line, FSDC, that is phenotypically representative of bone marrow-derived DC . IL-6 reverse transcriptase-PCR and promoter assays established the responsiveness of FSDC to anti-CD40 ligation . Further promoter assays showed that the transcription factors NF-kappaB and AP-1 are downstream transcriptional mediators of CD40-induced IL-6 gene expression . Anti-CD40 treatment of FSDC stimulated increased expression of specific NF-kappaB (p50:p65) and AP-1 (c-Jun, JunB, JunD, and c-Fos) DNA-protein complexes . Overexpression of an IkappaB-alpha super-repressor or a dominant negative JunD resulted in a strong inhibition of CD40-inducible IL-6 promoter activity supporting a role for both transcription factors . Upstream signal transduction events were studied by transfection of wild type and mutant human CD40 expression constructs into FSDC followed by stimulation with an anti-human CD40 antibody . These experiments revealed that anti-CD40 stimulation of NF-kappaB and IL-6 gene transcription requires specific amino acid residues in the cytoplasmic region of CD40 involved in the recruitment of TRAF2 . Induction of IL-6 mRNA by anti-CD40 treatment was found to be a transient event (24 h) and was followed by a diminution of IL-6 transcript to levels below those found in unstimulated cells . This loss of IL-6 expression was associated with reduced p50:p65 NF-kappaB DNA binding and elevated binding of CBF1 to a site overlapping the NF-kappaB site . Overexpression of CBF1 resulted in a profound inhibition of basal and anti-CD40-induced IL-6 promoter activities indicating that prolonged induction of CBF1 may contribute to the transient nature of the IL-6 response . The physiological relevance of these molecular events to DC function is discussed. J Invest Dermatol, 2001 Dec, 117(6), 1505 - 11 Transcriptional repression of the microphthalmia gene in melanoma cells correlates with the unresponsiveness of target genes to ectopic microphthalmia-associated transcription factor; Vachtenheim J et al.; In the melanocyte, expression of genes required for pigment formation is mediated by the microphthalmia transcription factor, which is also critical for the development and survival of normal melanocytes during embryogenesis . Here we show that the expression of the melanocyte-specific isoform of microphthalmia transcription factor is lost in a subset of human melanoma cell lines, accompanied by the repression of tyrosinase and tyrosinase-related proteins 1 and 2, the three transcriptional target genes for microphthalmia . After the forced expression of microphthalmia transcription factor in melanoma cells where the expression of endogenous microphthalmia gene was found to be extinguished, no restoration of the melanogenic phenotype occurred and the transcription of the three microphthalmia transcription factor target genes remained silent . The transcription activation domain of microphthalmia transcription factor, tested as a GAL-MITF fusion protein, remained fully functional in these cells, however, and ectopic microphthalmia transcription factor localized normally to the nucleus and bound to the tyrosinase initiator E-box in gel retardation assays . Thus, the block of differentiation in microphthalmia-transcription-factor-negative melanomas extended the transcriptional repression of the microphthalmia transcription factor gene alone, and endogenous promoters in these melanoma cells became no longer responsive to microphthalmia transcription factor when this was substituted exogenously . The data presented suggest that a specific nuclear context is required for the transcriptional activation of the melanocyte markers by the microphthalmia transcription factor in malignant melanocytes and this specificity is lost concomitantly with the transcriptional repression of microphthalmia transcription factor. Mol Cell, 2001 Dec, 8(6), 1155 - 6 Mitotic exit: closing the gap; Pintard L et al.; Completion of mitosis is triggered by the activation of the Ras-like GTP-binding protein Tem1p . In the November 30, 2001 issue of Cell, Hu et al . suggest that Tem1p activation is achieved by inhibition of its two-component GAP Bub2p/Bfa1p via phosphorylation of Bfa1p by the Polo kinase Cdc5p . Interestingly, activation of spindle checkpoints inhibits Bfa1p phosphorylation, suggesting that these signaling pathways prevent mitotic exit by maintaining the GAP activity of Bub2p/Bfa1p. Science, 2002 Mar 8, 295(5561), 1901 - 4 Coordination of PIC assembly and chromatin remodeling during differentiation-induced gene activation; Soutoglou E et al.; We analyzed the ordered recruitment of factors to the human alpha1 antitrypsin promoter around the initial activation of the gene during enterocyte differentiation . We found that a complete preinitiation complex, including phosphorylated RNA pol II, was assembled at the promoter long before transcriptional activation . The histone acetyltransferases CBP and P/CAF were recruited subsequently, but local histone hyperacetylation was delayed . After transient recruitment of the human Brahma homolog hBrm, remodeling of the neighboring nucleosome coincided with transcription initiation . The results suggest that, at this promoter, chromatin reconfiguration is a defining step of the initiation process, acting after the assembly of the Pol II machinery. Plant Cell, 2002 Feb, 14(2), 465 - 77 The putative plasma membrane Na(+)/H(+) antiporter SOS1 controls long-distance Na(+) transport in plants; Shi H et al.; The salt tolerance locus SOS1 from Arabidopsis has been shown to encode a putative plasma membrane Na(+)/H(+) antiporter . In this study, we examined the tissue-specific pattern of gene expression as well as the Na(+) transport activity and subcellular localization of SOS1 . When expressed in a yeast mutant deficient in endogenous Na(+) transporters, SOS1 was able to reduce Na(+) accumulation and improve salt tolerance of the mutant cells . Confocal imaging of a SOS1-green fluorescent protein fusion protein in transgenic Arabidopsis plants indicated that SOS1 is localized in the plasma membrane . Analysis of SOS1 promoter-beta-glucuronidase transgenic Arabidopsis plants revealed preferential expression of SOS1 in epidermal cells at the root tip and in parenchyma cells at the xylem/symplast boundary of roots, stems, and leaves . Under mild salt stress (25 mM NaCl), sos1 mutant shoot accumulated less Na(+) than did the wild-type shoot . However, under severe salt stress (100 mM NaCl), sos1 mutant plants accumulated more Na(+) than did the wild type . There also was greater Na(+) content in the xylem sap of sos1 mutant plants exposed to 100 mM NaCl . These results suggest that SOS1 is critical for controlling long-distance Na(+) transport from root to shoot . We present a model in which SOS1 functions in retrieving Na(+) from the xylem stream under severe salt stress, whereas under mild salt stress it may function in loading Na(+) into the xylem. Nucleic Acids Res, 2002 Mar 15, 30(6), 1306 - 15 The CUP1 upstream repeated element renders CUP1 promoter activation insensitive to mutations in the RNA polymerase II transcription complex; Badi L et al.; Activation of transcription in eukaryotes requires the concerted action of numerous components of the RNA polymerase II transcriptional apparatus . The degree of dependence on many of these components varies from gene to gene and it is still largely unknown how the requirement for any particular component is determined at any given gene . We show that removal of Gal11 from the yeast transcription complex can affect activation from the CUP1 UAS in a manner dependent on its genomic context . Our results indicate a novel function for the CUP1 upstream repeated element (CURE) located upstream of the CUP1 UAS at the naturally multimerized CUP1 locus . The presence of CURE endowed the CUP1 UAS with a reduced susceptibility to the effects of deleting Gal11 . Similar results were obtained with the Srb/mediator subunit Srb5 . Restoration of activation from the CUP1 promoter to wild-type levels by the CURE correlated with changes in the accessibility of local chromatin to nucleases . The CURE sequence may serve to protect the stress-inducible CUP1 UAS-promoter elements against reduced activation that may result from crippled transcription complexes under stress conditions. Mol Cell Biol, 2002 Apr, 22(7), 2159 - 69 Involvement of mouse Rev3 in tolerance of endogenous and exogenous DNA damage; Van Sloun PP et al.; The Rev3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta that is implicated in mutagenic translesion synthesis of damaged DNA . To investigate the function of its mouse homologue, we have generated mouse embryonic stem cells and mice carrying a targeted disruption of Rev3 . Although some strain-dependent variation was observed, Rev3(-/-) embryos died around midgestation, displaying retarded growth in the absence of consistent developmental abnormalities . Rev3(-/-) cell lines could not be established, indicating a cell-autonomous requirement of Rev3 for long-term viability . Histochemical analysis of Rev3(-/-) embryos did not reveal aberrant replication or cellular proliferation but demonstrated massive apoptosis in all embryonic lineages . Although increased levels of p53 are detected in Rev3(-/-) embryos, the embryonic phenotype was not rescued by the absence of p53 . A significant increase in double-stranded DNA breaks as well as chromatid and chromosome aberrations was observed in cells from Rev3(-/-) embryos . The inner cell mass of cultured Rev3(-/-) blastocysts dies of a delayed apoptotic response after exposure to a low dose of N-acetoxy-2-acetylaminofluorene . These combined data are compatible with a model in which, in the absence of polymerase zeta, double-stranded DNA breaks accumulate at sites of unreplicated DNA damage, eliciting a p53-independent apoptotic response . Together, these data are consistent with involvement of polymerase zeta in translesion synthesis of endogenously and exogenously induced DNA lesions. Mol Cell Biol, 2002 Apr, 22(7), 1971 - 80 Ctr9, Rtf1, and Leo1 are components of the Paf1/RNA polymerase II complex; Mueller CL et al.; The Saccharomyces cerevisiae Paf1-RNA polymerase II (Pol II) complex is biochemically and functionally distinct from the Srb-mediator form of Pol II holoenzyme and is required for full expression of a subset of genes . In this work we have used tandem affinity purification tags to isolate the Paf1 complex and mass spectrometry to identify additional components . We have established that Ctr9, Rtf1, and Leo1 are factors that associate with Paf1, Cdc73, and Pol II, but not with the Srb-mediator . Deletion of either PAF1 or CTR9 leads to similar severe pleiotropic phenotypes, which are unaltered when the two mutations are combined . In contrast, we found that deletion of LEO1 or RTF1 leads to few obvious phenotypes, although mutation of RTF1 suppresses mutations in TATA-binding protein, alters transcriptional start sites, and affects elongation . Remarkably, deletion of LEO1 or RTF1 suppresses many paf1Delta phenotypes . In particular, an rtf1Delta paf1Delta double mutant grew faster, was less temperature sensitive, and was more resistant to caffeine and hydroxyurea than a paf1Delta single mutant . In addition, expression of the G(1) cyclin CLN1, reduced nearly threefold in paf1Delta, is restored to wild-type levels in the rtf1Delta paf1Delta double mutant . We suggest that lack of Paf1 results in a defective complex and a block in transcription, which is relieved by removal of Leo1 or Rtf1. Mol Cell Biol, 2002 Apr, 22(7), 1961 - 70 The PHD type zinc finger is an integral part of the CBP acetyltransferase domain; Kalkhoven E et al.; Histone acetyltransferases (HATs) such as CBP and p300 are regarded as key regulators of RNA polymerase II-mediated transcription, but the critical structural features of their HAT modules remain ill defined . The HAT domains of CBP and p300 are characterized by the presence of a highly conserved putative plant homeodomain (PHD) (C4HC3) type zinc finger, which is part of the functionally uncharacterized cysteine-histidine-rich region 2 (CH2) . Here we show that this region conforms to the PHD type zinc finger consensus and that it is essential for in vitro acetylation of core histones and the basal transcription factor TFIIE34 as well as for CBP autoacetylation . PHD finger mutations also reduced the transcriptional activity of the full-length CBP protein when tested on transfected reporter genes . Importantly, similar results were obtained on integrated reporters, which reflect a more natural chromatinized state . Taken together, our results indicate that the PHD finger forms an integral part of the enzymatic core of the HAT domain of CBP. J Virol, 2002 Apr, 76(7), 3240 - 7 Ty5 gag mutations increase retrotransposition and suggest a role for hydrogen bonding in the function of the nucleocapsid zinc finger; Gao X et al.; The Ty5 retrotransposon of Saccharomyces paradoxus transposes in Saccharomyces cerevisiae at frequencies 1,000-fold lower than do the native Ty1 elements . The low transposition activity of Ty5 could be due to differences in cellular environments between these yeast species or to naturally occurring mutations in Ty5 . By screening of a Ty5 mutant library, two single mutants (D252N and Y68C) were each found to increase transposition approximately sixfold . When combined, transposition increased 36-fold, implying that the two mutations act independently . Neither mutation affected Ty5 protein synthesis, processing, cDNA recombination, or target site choice . However, cDNA levels in both single mutants and the double mutant were significantly higher than in the wild type . The D252N mutation resides in the zinc finger of nucleocapsid and increases the potential for hydrogen bonding with nucleic acids . We generated other mutations that increase the hydrogen bonding potential (i.e., D252R and D252K) and found that they similarly increased transposition . This suggests that hydrogen bonding within the zinc finger motif is important for cDNA production and builds upon previous studies implicating basic amino acids flanking the zinc finger as important for zinc finger function . Although NCp zinc fingers differ from the zinc finger motifs of cellular enzymes, the requirement for efficient hydrogen bonding is likely universal. J Cell Sci, 2002 Mar 15, 115(Pt 6), 1259 - 71 Identification of septin-interacting proteins and characterization of the Smt3/SUMO-conjugation system in Drosophila; Shih HP et al.; The septins are a family of proteins involved in cytokinesis and other aspects of cell-cortex organization . In a two-hybrid screen designed to identify septin-interacting proteins in Drosophila, we isolated several genes, including homologues (Dmuba2 and Dmubc9) of yeast UBA2 and UBC9 . Yeast Uba2p and Ubc9p are involved in the activation and conjugation, respectively, of the ubiquitin-like protein Smt3p/SUMO, which becomes conjugated to a variety of proteins through this pathway . Uba2p functions together with a second protein, Aos1p . We also cloned and characterized the Drosophila homologues of AOS1 (Dmaos1) and SMT3 (Dmsmt3) . Our biochemical data suggest that DmUba2/DmAos1 and DmUbc9 indeed act as activating and conjugating enzymes for DmSmt3, implying that this protein-conjugation pathway is well conserved in DROSOPHILA: Immunofluorescence studies showed that DmUba2 shuttles between the embryonic cortex and nuclei during the syncytial blastoderm stage . In older embryos, DmUba2 and DmSmt3 are both concentrated in the nuclei during interphase but dispersed throughout the cells during mitosis, with DmSmt3 also enriched on the chromosomes during mitosis . These data suggest that DmSmt3 could modify target proteins both inside and outside the nuclei . We did not observe any concentration of DmUba2 at sites where the septins are concentrated, and we could not detect DmSmt3 modification of the three Drosophila septins tested . However, we did observe DmSmt3 localization to the midbody during cytokinesis both in tissue-culture cells and in embryonic mitotic domains, suggesting that DmSmt3 modification of septins and/or other midzone proteins occurs during cytokinesis in Drosophila. J Biol Chem, 2002 May 24, 277(21), 18881 - 90 Epub 2002 Mar 07. Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels; Anderson RM et al.; Yeast deprived of nutrients exhibit a marked life span extension that requires the activity of the NAD(+)-dependent histone deacetylase, Sir2p . Here we show that increased dosage of NPT1, encoding a nicotinate phosphoribosyltransferase critical for the NAD(+) salvage pathway, increases Sir2-dependent silencing, stabilizes the rDNA locus, and extends yeast replicative life span by up to 60% . Both NPT1 and SIR2 provide resistance against heat shock, demonstrating that these genes act in a more general manner to promote cell survival . We show that Npt1 and a previously uncharacterized salvage pathway enzyme, Nma2, are both concentrated in the nucleus, indicating that a significant amount of NAD(+) is regenerated in this organelle . Additional copies of the salvage pathway genes, PNC1, NMA1, and NMA2, increase telomeric and rDNA silencing, implying that multiple steps affect the rate of the pathway . Although SIR2-dependent processes are enhanced by additional NPT1, steady-state NAD(+) levels and NAD(+)/NADH ratios remain unaltered . This finding suggests that yeast life span extension may be facilitated by an increase in the availability of NAD(+) to Sir2, although not through a simple increase in steady-state levels . We propose a model in which increased flux through the NAD(+) salvage pathway is responsible for the Sir2-dependent extension of life span. J Mol Biol, 2002 Mar 1, 316(4), 955 - 68 Implications for the ubiquitination reaction of the anaphase-promoting complex from the crystal structure of the Doc1/Apc10 subunit; Au SW et al.; The anaphase-promoting complex (APC) is a multi-subunit E3 protein ubiquitin ligase that is responsible for the metaphase to anaphase transition and the exit from mitosis . One of the subunits of the APC that is required for its ubiquitination activity is Doc1/Apc10, a protein composed of a Doc1 homology domain that has been identified in a number of diverse putative E3 ubiquitin ligases . Here, we present the crystal structure of Saccharomyces cerevisiae Doc1/Apc10 at 2.2A resolution . The Doc1 homology domain forms a beta-sandwich structure that is related in architecture to the galactose-binding domain of galactose oxidase, the coagulation factor C2 domain and a domain of XRCC1 . Residues that are invariant amongst Doc1/Apc10 sequences, including a temperature-sensitive mitotic arrest mutant, map to a beta-sheet region of the molecule, whose counterpart in galactose oxidase, the coagulation factor C2 domains and XRCC1, mediate bio-molecular interactions . This finding suggests the identification of the functionally important and conserved region of Doc1/Apc10 and, since invariant residues of Doc1/Apc10 colocalise with conserved residues of other Doc1 homology domains, we propose that the Doc1 homology domains perform common ubiquitination functions in the APC and other E3 ubiquitin ligases . J Mol Biol, 2002 Mar 1, 316(4), 919 - 29 Protein-splicing reaction via a thiazolidine intermediate: crystal structure of the VMA1-derived endonuclease bearing the N and C-terminal propeptides; Mizutani R et al.; Protein splicing excises an internal intein segment from a protein precursor precisely, and concomitantly ligates flanking N and C-extein polypeptides at the respective sides of the precursor . Here, a series of precursor recombinants bearing 11 N-extein and ten C-extein residues is prepared for the intein of the Saccharomyces cerevisiae VMA1-derived homing endonuclease referred to as VDE and as PI-SceI . The recombinant with replacements of C284S, H362N, N737S, and C738S is chosen as a spliceable precursor model and is then subjected to a 2.1A resolution crystallographic analysis . The crystal structure shows that the introduced extein polypeptides are located in the vicinity of the splicing site, and that each of their peptide bonds is in the trans conformation . The S284 O(gamma) atom located at a distance of 3.1A from the G283 C atom in the N-terminal junction suggests that a nucleophilic attack of the C284 S(gamma) atom on the G283 C atom forms a tetrahedral intermediate containing a five-membered thiazolidine ring . The tetrahedral intermediate is supposedly resolved into a thioester acyl group upon the cleavage of the linkage between the G283 C and C284 N atoms, and this thioester acyl formation completes the initial steps of Nright arrowS acyl shift at the junction between the N-extein and intein . The S738 O(gamma) atom in the C-terminal junction is placed in close proximity to the S284 O(gamma) atom at a distance of 3.6A, and is well suited for another nucleophilic attack on the resultant thioester acyl group that is then subjected to the transesterification in the next step . The reaction steps proposed for the acyl shift are driven entirely by protonation and deprotonation, in which proton ingress and egress is balanced within the splicing site . Planta, 2002 Mar, 214(5), 703 - 7 Epub 2001 Nov 21. Spatial distribution of the 26S proteasome in meristematic tissues and primordia of rice (Oryza sativa L.); Yanagawa Y et al.; The 26S proteasome is known to play central roles in the growth of many eukaryotes . However, little is known regarding its distribution in higher plants . Here, we report the spatial distribution pattern of Rpn3 (a regulatory PA700 subunit) and C2 (a subunit of the 20S proteasome) in rice ( Oryza sativa L.) seedlings as determined by in situ hybridization . The transcripts were abundantly co-expressed in the apical and marginal meristems of shoots and roots . Interestingly, these transcripts also accumulated in the leaf and ligule primordia of the shoot apex . Our results suggest that the 26S proteasome is spatially distributed among various tissues and may be involved not only in cell division but also in organ formation in higher plants. Naunyn Schmiedebergs Arch Pharmacol, 2002 Mar, 365(3), 231 - 41 Epub 2002 Jan 17. G-protein betagamma-subunits contribute to the coupling specificity of the beta2-adrenergic receptor to G(s); Kuhn B et al.; Receptors and heterotrimeric G-proteins interact with a high degree of specificity, the molecular basis of which is only partially understood . In the present study, we analyzed the influence of different G-protein betagamma-subunits on the coupling of the beta2-adrenergic receptor to G(s) . Sf9-cells were infected with baculoviruses coding for the beta2-adrenergic receptor, alpha(s,Short) or alpha(s,Long), and various beta- and gamma-subunits . The ability of different beta- and gamma-subunits to correctly dimerize was assessed by limited proteolysis of proteins expressed in Sf9-cells and additionally by analysis of beta/gamma-interaction in the yeast two-hybrid system . Agonist-induced GTPgammaS-binding to alpha(s,Short)beta(1)gamma-trimers was significantly higher than to alpha(s,Short)beta2gamma-combinations, when gamma4, gamma5, or gamma7 were co-expressed . Because beta(5) did not support coupling of the beta(2)-adrenergic receptor to G(s), the 87 C-terminal amino acids of Gbeta(5) assumed to encompass the beta-subunit interface with the receptor were substituted by the corresponding sequence of beta(1) . Whereas this beta(5)/beta(1)-chimera did not promote GTPgammaS-binding to alpha(s), histamine H(1)-receptor-dependent GTPgammaS-binding to alpha(q) was supported by this chimeric beta-subunit and by wild-type beta(5) . Our findings argue that the betagamma-subunit composition contributes directly to the specificity of beta(2)-adrenergic receptor-mediated G(s)-activation. EMBO Rep, 2002 Mar, 3(3), 242 - 7 Modulation of ISWI function by site-specific histone acetylation; Corona DF et al.; Mutations in Drosophila ISWI, a member of the SWI2/SNF2 family of chromatin remodeling ATPases, alter the global architecture of the male X chromosome . The transcription of genes on this chromosome is increased 2-fold relative to females due to dosage compensation, a process involving the acetylation of histone H4 at lysine 16 (H4K16) . Here we show that blocking H4K16 acetylation suppresses the X chromosome defects resulting from loss of ISWI function in males . In contrast, the forced acetylation of H4K16 in ISWI mutant females causes X chromosome defects indistinguishable from those seen in ISWI mutant males . Increased expression of MOF, the histone acetyltransferase that acetylates H4K16, strongly enhances phenotypes resulting from the partial loss of ISWI function . Peptide competition assays revealed that H4K16 acetylation reduces the ability of ISWI to interact productively with its substrate . These findings suggest that H4K16 acetylation directly counteracts chromatin compaction mediated by the ISWI ATPase. EMBO Rep, 2002 Mar, 3(3), 224 - 9 Histone acetylation: a switch between repressive and permissive chromatin . Second in review series on chromatin dynamics; Eberharter A et al.; The organization of eukaryotic chromatin has a major impact on all nuclear processes involving DNA substrates . Gene expression is affected by the positioning of individual nucleosomes relative to regulatory sequence elements, by the folding of the nucleosomal fiber into higher-order structures and by the compartmentalization of functional domains within the nucleus . Because site-specific acetylation of nucleosomal histones influences all three aspects of chromatin organization, it is central to the switch between permissive and repressive chromatin structure . The targeting of enzymes that modulate the histone acetylation status of chromatin, in synergy with the effects mediated by other chromatin remodeling factors, is central to gene regulation. Exp Hematol, 2002 Mar, 30(3), 212 - 20 Translocation of Ku86/Ku70 to the multiple myeloma cell membrane: functional implications; Tai YT et al.; OBJECTIVE: Since the central hallmarks of human multiple myeloma (MM) are abnormalities in immunoglobulin (Ig) gene rearrangement, IgH class switching, and DNA damage repair, and since Ku86 and Ku70 proteins are central to these processes, aberrant Ku function may play a role in MM pathogenesis . Our prior studies demonstrated a 69-kDa Ku86 variant in freshly isolated patient MM cells that confers sensitivity to DNA damage . We also showed that Ku86 on the cell surface of CD40-activated MM cells mediates homotypic tumor cell adhesion, as well as heterotypic adhesion to bone marrow stromal cells . We here define the mechanism and functional significance of CD40-induced Ku translocation from the cytoplasm to the cell membrane in MM cells vs normal B cells . MATERIALS AND METHODS: We examined Ku86 and Ku70 translocation following CD40 activation in human MM cells vs normal tonsillar B lymphocytes . We then identified the functional sequelae of membrane Ku86 and Ku70 expression on CD40-activated human MM cells . RESULTS: CD40 activation induces translocation of both Ku86 and Ku70 to the cell surface of MM cells, but not normal tonsillar B cells . Moreover, CD40 activation triggers Ku association with CD40 only in CD40-activated MM cells . Finally, CD40-activated MM cells adhere to fibronectin and are protected against apoptosis triggered by irradiation or doxorubicin; conversely, antibodies to Ku both inhibit tumor cell binding and restore sensitivity to these agents . CONCLUSION: These results demonstrate functional significance of Ku translocation to the cell membrane of CD40-activated human MM cells . Therefore, targeting Ku86 and Ku70, with blocking peptides for example, might serve as a novel treatment strategy in human MM. Curr Biol, 2002 Mar 5, 12(5), R166 - 7 Golgi complex: biogenesis de novo? Lowe M. Whether Golgi biogenesis occurs by self-assembly or around a pre-existing template is currently a matter of debate . Recent studies have shown that Golgi structural proteins are more dynamic than previously thought, suggesting that self-assembly of the Golgi complex may be possible. Curr Biol, 2002 Mar 5, 12(5), 397 - 402 Oxygen metabolism causes chromosome breaks and is associated with the neuronal apoptosis observed in DNA double-strand break repair mutants; Karanjawala ZE et al.; Cells deficient in a major DNA double-strand break repair pathway (nonhomologous DNA end joining {NHEJ}) have increased spontaneous chromosome breaks; however, the source of these chromosome breaks has remained undefined . Here, we show that the observed spontaneous chromosome breaks are partially suppressed by reducing the cellular oxygen tension . Conversely, elevating the level of reactive oxygen species by overexpressing the antioxidant enzyme superoxide dismutase 1 (SOD1), in a transgenic mouse, increases chromosome breakage . The effect of SOD1 can also be modulated by cellular oxygen tension . The elevated chromosome breakage correlates histologically with a significant increase in the amount of neuronal cell death in Ku86(-/-) SOD1 transgenic embryos over that seen in Ku86(-/-) embryos . Therefore, oxygen metabolism is a major source of the genomic instability observed in NHEJ-deficient cells and, presumably, in all cells. Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 2666 - 71 Mot1 activates and represses transcription by direct, ATPase-dependent mechanisms; Dasgupta A et al.; Mot1 is an essential yeast Snf2/Swi2-related ATPase that exerts both positive and negative effects on gene expression . In vitro, Mot1 can disrupt TATA-binding protein-DNA complexes in an ATP-dependent reaction . This activity can explain Mot1-mediated transcriptional repression, but how Mot1 activates transcription is unknown . We demonstrate that, remarkably, Mot1 is localized in vivo to promoters for both Mot1-repressed and Mot1-activated genes . Moreover, Mot1 ATPase activity is required for both activation and repression of gene activity . These findings suggest a novel function for the Mot1 ATPase at activated genes, perhaps involving ATP-driven reorganization of the preinitiation complex . Mot1 regulates the expression of approximately 3% of yeast genes in cells grown in rich medium . Most of these genes are repressed by Mot1, consistent with Mot1's ATP-dependent TATA-binding protein-DNA dissociating activity . Additionally, approximately 77% of the Mot1-repressed genes are involved in the diauxic shift, stress response, mating, or sporulation . The gene sets controlled by NC2 and Srb10 are strongly correlated with the Mot1-controlled set, suggesting that these factors cooperate in transcriptional control on a global scale. Chem Biol, 2002 Feb, 9(2), 245 - 51 Transcriptional effects of the potent enediyne anti-cancer agent Calicheamicin gamma(I)(1); Watanabe CM et al.; We have investigated the mode of action of calicheamicin in living cells by using oligonucleotide microarrays to monitor its effects on gene expression across the entire yeast genome . Transcriptional effects were observed as early as 2 min into drug exposure . Among these effects were the upregulation of two nuclear proteins encoding a Y'-helicase (a subtelomerically encoded protein whose function is to maintain telomeres) and a suppressor of rpc10 and rpb40 mutations (both rpc10 and rpb40 encode RNA polymerase subunits) . With longer calicheamicin exposure, genes involved in chromatin arrangement, DNA repair and/or oxidative damage, DNA synthesis and cell cycle checkpoint control as well as other nuclear proteins were all differentially expressed . Additionally, ribosomal proteins and a variety of metabolic, biosynthetic, and stress response genes were also altered in their expression. Lancet, 2002 Feb 23, 359(9307), 667 - 71 Activation markers of coagulation and fibrinolysis in twins: heritability of the prethrombotic state; Ariens RA et al.; BACKGROUND: Activation markers of coagulation and fibrinolysis are increased in individuals at risk of coronary-artery disease and other thrombotic disorders--a condition defined as the prethrombotic state . We aimed to find out the extent to which the prethrombotic state is determined by genetic factors . METHODS: We analysed concentrations of prothrombin, prothrombin fragment 1+2, thrombin-antithrombin complex, crosslinked fibrin degradation product D-dimer, and thrombin-activatable fibrinolysis inhibitor by ELISA in 118 monozygotic and 112 dizygotic unselected female twins aged 21-73 years from the St Thomas' UK Adult Twin Registry . We used quantitative genetic-model fitting to estimate heritability . FINDINGS: We found significant heritabilities in concentrations of the activation markers in plasma . Genetic factors contributed 45, 40, and 65% of the variation in concentrations of fragment 1+2, thrombin-antithrombin complex, and D-dimer, respectively . Age was important only in fragment 1+2 concentrations, in which it accounted for 12% of the variation . The remaining variation could be attributed to unique environmental factors . Variation in concentrations of precursor prothrombin in plasma was determined by 57% heritability, and that of zymogen thrombin-activatable fibrinolysis inhibitor showed a very strong genetic component (82%) . INTERPRETATION: The activation mechanisms of the coagulation and fibrinolytic systems, and therefore the prethrombotic state, are controlled to a substantial degree by genetic factors . Genes influencing activation of haemostasis are likely to be an important component of the overall thrombotic tendency in the general population. Biochimie, 2001 Nov-Dec, 83(11-12), 1029 - 39 Chromatin structure and dynamics: functional implications; Morales V et al.; In eucaryotes, DNA packaging into nucleosomes and its organization in a chromatin fiber generate constraints for all processes involving DNA, such as DNA-replication, -repair, -recombination, and -transcription . Transient changes in chromatin structure allow overcoming these constraints with different requirements in regions where processes described above are initiated . Mechanisms involved in chromatin dynamics are complex . Multiprotein complexes which can contain histone-acetyltransferase, -deacetylase, -methyltransferase or -kinase activities are targeted by regulatory factors to precise regions of the genome . These enzymes have been shown to modify histone-tails within specific nucleosomes . Post-translational modifications of histone-tails constitute a code that is thought to contribute to the nucleosome or to the chromatin fiber remodeling, either directly, or through the recruitment of other proteins . Other multiprotein complexes, such as ATP-dependent remodeling complexes, play an essential role in chromatin fiber dynamics allowing nucleosome sliding and redistribution on the DNA . We will focus here on the chromatin structure and its consequences for DNA damaging, replication, repair, and transcription and we will discuss the mechanisms of chromatin remodeling. Biochimie, 2001 Nov-Dec, 83(11-12), 1003 - 8 Cloning of the PpMSH-2 cDNA of Physcomitrella patens, a moss in which gene targeting by homologous recombination occurs at high frequency; Brun F et al.; In the moss Physcomitrella patens integrative transformants from homologous recombination are obtained at an efficiency comparable to that found for yeast . This property, unique in the plant kingdom, allows the knockout of specific genes . It also makes the moss a convenient model to study the regulation of homologous recombination in plants . We used degenerate oligonucleotides designed from AtMSH2 from Arabidopsis thaliana and other known MutS homologues to isolate the P . patens MSH2 (PpMSH2) cDNA . The deduced sequence of the PpMSH2 protein is respectively 60.8% and 59.6% identical to the maize and A . thaliana MSH2 . Phylogenic studies show that PpMSH2 is closely related to the group of plant MSH2 proteins . Southern analysis reveals that the gene exists as a single copy in the P . patens genome. Dev Cell, 2002 Mar, 2(3), 307 - 17 The Rab GTPase Ypt1p and tethering factors couple protein sorting at the ER to vesicle targeting to the Golgi apparatus; Morsomme P et al.; GPI-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event can be reproduced in vitro . When extracts from a uso1 mutant were used, the sorting of GPI-anchored proteins from other secretory proteins was defective . Complementation with purified Uso1p restored sorting . The Rab GTPase Ypt1p and the tethering factors Sec34p and Sec35p, but not Bet3p, a member of the TRAPP complex, were also required for protein sorting upon ER exit . Therefore, the Ypt1p tethering complex couples protein sorting in the ER to vesicle targeting to the Golgi apparatus . Sorting of GPI-anchored proteins from other secretory proteins was also observed in vivo . The sorting defect observed in vitro with uso1 and ypt1 mutants was reproduced in vivo. Dev Cell, 2002 Mar, 2(3), 295 - 305 Sly1 binds to Golgi and ER syntaxins via a conserved N-terminal peptide motif; Yamaguchi T et al.; Sec1/munc18-like proteins (SM proteins) and SNARE complexes are probably universally required for membrane fusion . However, the molecular mechanism by which they interact has only been defined for synaptic vesicle fusion where munc18 binds to syntaxin in a closed conformation that is incompatible with SNARE complex assembly . We now show that Sly1, an SM protein involved in Golgi and ER fusion, binds to a short, evolutionarily conserved N-terminal peptide of Sed5p and Ufe1p in yeast and of syntaxins 5 and 18 in vertebrates . In these syntaxins, the Sly1 binding peptide is upstream of a separate, autonomously folded N-terminal domain . These data suggest a potentially general mechanism by which SM proteins could interact with peptides in target proteins independent of core complex assembly and suggest that munc18 binding to syntaxin is an exception. Breast Cancer Res, 2002, 4(2), 70 - 6 Epub 2002 Feb 12. The p53 pathway in breast cancer; Gasco M et al.; p53 mutation remains the most common genetic change identified in human neoplasia . In breast cancer, p53 mutation is associated with more aggressive disease and worse overall survival . The frequency of mutation in p53 is, however, lower in breast cancer than in other solid tumours . Changes, both genetic and epigenetic, have been identified in regulators of p53 activity and in some downstream transcriptional targets of p53 in breast cancers that express wild-type p53 . Molecular pathological analysis of the structure and expression of constituents of the p53 pathway is likely to have value in diagnosis, in prognostic assessment and, ultimately, in treatment of breast cancer. Biochem J, 2002 Mar 15, 362(Pt 3), 749 - 54 A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect; Abdullaev ZKh et al.; A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed . To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal alpha-helix or position 72 were obtained . It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H(2)O(2) production by the initial segment of the respiratory chain of intact rat heart mitochondria . The major role in the difference between the horse and yeast proteins was shown to be played by the amino acid residue in position 4 (glutamate in horse, and lysine in yeast; horse protein numbering) . A mutant of the yeast cytochrome c containing K4E and some other "horse" modifications in the N-terminal alpha-helix, proved to be (i) much more active in electron transfer and antioxidant activity than the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c, inactive in caspase stimulation, even if added in 400-fold excess compared with the horse protein . Thus this mutant seems to be a good candidate for knock-in studies of the role of cytochrome c-mediated apoptosis, in contrast with the horse K72R, K72G, K72L and K72A mutant cytochromes that at low concentrations were less active in apoptosis than the wild-type, but were quite active when the concentrations were increased by a factor of 2-12. Genes Dev, 2002 Mar 1, 16(5), 608 - 19 Calcineurin-dependent regulation of Crz1p nuclear export requires Msn5p and a conserved calcineurin docking site; Boustany LM et al.; Calcineurin, a conserved Ca(2+)/calmodulin-regulated protein phosphatase, plays a crucial role in Ca(2+) signaling in a wide variety of cell types . In Saccharomyces cerevisiae, calcineurin positively regulates transcription in response to stress by dephosphorylating the transcription factor Crz1p/Tcn1p . Dephosphorylation promotes Crz1p nuclear localization in part by increasing the efficiency of its nuclear import . In this work, we show that calcineurin-dependent dephosphorylation of Crz1p also down-regulates its nuclear export . Using a genetic approach, we identify Msn5p as the exportin for Crz1p . In addition, we define the Crz1p nuclear export signal (NES) and show that it interacts with Msn5p in a phosphorylation-dependent manner . This indicates that calcineurin regulates Crz1p nuclear export by dephosphorylating and inactivating its NES . Finally, we define a motif in Crz1p, PIISIQ, similar to the PxIxIT docking site for calcineurin on the mammalian transcription factor NFAT, that mediates the in vivo interaction between calcineurin and Crz1p and is required for calcineurin-dependent regulation of Crz1p nuclear export and activity . Therefore, in yeast as in mammals, a docking site is required to target calcineurin to its substrate such that it can dephosphorylate it efficiently. Hum Mol Genet, 2002 Mar 1, 11(5), 599 - 604 A frequent mild mutation in ALG6 may exacerbate the clinical severity of patients with congenital disorder of glycosylation Ia (CDG-Ia) caused by phosphomannomutase deficiency; Westphal V et al.; Single nucleotide polymorphisms occur throughout the human genome . A gene that causes one of the congenital disorders of glycosylation (CDG) has a mutation (911T-->C ) that changes a phenylalanine to serine at position 304 (F304S) of the alpha 1,3 glucosyl transferase . We show that this change reduces the ability of the gene product to rescue defective glycosylation of an alg6-deficient strain of Saccharomyces cerevisiae during rapid growth . This finding suggested that the mutation might affect glycosylation in humans . We therefore compared the frequency of this variant in 301 controls and in 101 CDG patients who carry known mutations in other genes involved in CDG, i.e . PMM2 (CDG-Ia; 91 patients) and MPI (CDG-Ib; 10 patients) . The variant allele frequency is identical in both CDG patients (0.30) and controls (0.28) . Importantly, the F304S genotype frequency in 55 CDG-Ia patients classified as mild/moderate (n = 28), or severe (n = 27) was significantly higher in severely affected patients (0.41) than in mild/moderately affected patients (0.21) . Mortality (n = 9) was higher when F304S was present (n = 6) . Severely affected patients with the PMM2 mutations F119L/R141H (n = 22) carry the F304S mutation more often (0.36) than mildly affected patients (0.18, n = 11) with this mutation . Clinical severity of mildly affected sibs with the same PMM2 mutations did not correlate with F304S genotype . Thus, the presence of the F304S allele may exacerbate the clinical outcome, especially in severely affected CDG patients . We speculate that this type of variant may be implicated in other multi-factorial disorders that involve N-glycosylation. Genome Res, 2002 Mar, 12(3), 379 - 90 The human ribosomal protein genes: sequencing and comparative analysis of 73 genes; Yoshihama M et al.; The ribosome, as a catalyst for protein synthesis, is universal and essential for all organisms . Here we describe the structure of the genes encoding human ribosomal proteins (RPs) and compare this class of genes among several eukaryotes . Using genomic and full-length cDNA sequences, we characterized 73 RP genes and found that (1) transcription starts at a C residue within a characteristic oligopyrimidine tract; (2) the promoter region is GC rich, but often has a TATA box or similar sequence element; (3) the genes are small (4.4 kb), but have as many as 5.6 exons on average; (4) the initiator ATG is in the first or second exon and is within plus minus 5 bp of the first intron boundaries in about half of cases; and (5) 5'- and 3'-UTRs are significantly smaller (42 bp and 56 bp, respectively) than the genome average . Comparison of RP genes from humans, Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae revealed the coding sequences to be highly conserved (63% homology on average), although gene size and the number of exons vary . The positions of the introns are also conserved among these species as follows: 44% of human introns are present at the same position in either D . melanogaster or C . elegans, suggesting RP genes are highly suitable for studying the evolution of introns. Plant J, 2002 Mar, 29(5), 531 - 43 Double mutation cpSRP43--/cpSRP54-- is necessary to abolish the cpSRP pathway required for thylakoid targeting of the light-harvesting chlorophyll proteins; Hutin C et al.; Biochemical and genetic studies have established that the light-harvesting chlorophyll proteins (LHCPs) of the photosystems use the cpSRP (chloroplast signal recognition particle) pathway for their targeting to thylakoids . Previous analyses of single cpSRP mutants, chaos and ffc, deficient in cpSRP43 and cpSRP54, respectively, have revealed that half of the LHCPs are still integrated into the thylakoid membranes . Surprisingly, the effects of both mutations are additive in the double mutant ffc/chaos described here . This mutant has pale yellow leaves at all stages of growth and drastically reduced levels of all the LHCPs except Lhcb 4 . Although the chloroplasts have a normal shape, the thylakoid structure is affected by the mutation, probably as a consequence of reduction of all the LHCPs . ELIPs (early light-inducible proteins), nuclear-encoded proteins related to the LHCP family and inducible by light stress, were also drastically reduced in the double mutant . However, proteins targeted by other chloroplastic targeting pathways (DeltapH, Sec and spontaneous pathways) accumulated to similar levels in the wild-type and the double mutant . Therefore, the near total loss of LHCPs and ELIPs in the double mutant suggests that cpSRP is the predominant, if not exclusive, targeting pathway for these proteins . Phenotypic analysis of the double mutant, compared to the single mutants, suggests that the cpSRP subunits cpSRP43 and cpSRP54 contribute to antenna targeting in an independent but additive way. J Chromatogr A, 2002 Feb 8, 946(1-2), 141 - 55 Cutinase-peptide fusions in thermoseparating aqueous two-phase systems . Prediction of partitioning and enhanced tag efficiency by detergent addition; Nilsson A et al.; It is of increasing importance to develop efficient purification methods for recombinant proteins where the number of steps can be minimised . The aim has been to establish a method for predicting the partitioning of the wild-type target protein in an aqueous two-phase system, and with this as basis, develop fusion tags and optimise the phase system for enhanced partitioning of the target protein . The surface of the lipolytic enzyme cutinase from Fusarium solani pisi was investigated with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP) . The accessible surface areas for the different amino acid residues were used together with peptide partitioning data to calculate the partition coefficient for the protein . The separation system was composed of a thermoseparating random copolymer of ethylene oxide and propylene oxide . Breox PAG 50A 1000, as top phase forming polymer and a hydroxypropyl starch polymer, Reppal PES 200, as bottom phase polymer . The calculated partition coefficient for the wild-type protein (K= 1.0) agreed reasonably well with the experimentally determined value (K=0.85) . Genetic engineering was used to construct fusion proteins expressed in Saccharomyces cerevisiae based on cutinase and peptide tags containing tryptophan, to enhance the partitioning in aqueous two-phase systems . The partitioning of the cutinase constructs could qualitatively be predicted from peptide partitioning data, i.e . the trends in partitioning could be predicted . A spacer peptide introduced between protein and tag increased the partitioning of the protein towards the ethylene oxide-propylene oxide (EOPO) copolymer top phase . The aqueous two-phase system was modified by addition of detergent to increase the partitioning of the cutinase variants towards the EOPO copolymer phase . Triton and a series of C12En detergents selectively increased the partitioning of cutinase constructs with (WP)4-based tags up to 14 times compared to wild-type cutinase . The protein partition could almost quantitatively be predicted from the peptide partition data. Yeast, 2002 Mar 15, 19(4), 373 - 92 Proliferation of the endoplasmic reticulum occurs normally in cells that lack a functional unfolded protein response; Larson LL et al.; Increased expression of certain ER membrane proteins leads to biogenesis of novel ER membrane arrays . These structures provide models in which to explore the mechanisms by which cells control the size and organization of organelles in response to changing physiological demands . In yeast, elevated levels of HMG-CoA reductase induce ER arrays known as karmellae . Cox and co-workers (1997) discovered that karmellae assembly is toxic to ire1 mutants . These mutants are unable to initiate the unfolded protein response, which enables cells to adjust levels of ER chaperones in response to stresses . We sought to determine whether the karmellae-dependent death of ire1 mutants was due to karmellae assembly or to increased levels of HMG-CoA reductase activity . Unexpectedly, we found that ire1 cells could assemble normal levels of karmellae that were structurally identical to those of wild-type cells . In addition, karmellae assembly did not itself induce the unfolded protein response . Certain ire1 strains produced significant numbers of transformants that were unable to utilize galactose as sole carbon source . These results suggest that the karmellae-dependent death of certain ire1 strains may simply reflect their inability to grow on galactose . Eur J Immunol, 2002 Mar, 32(3), 701 - 9 Different types of V(D)J recombination and end-joining defects in DNA double-strand break repair mutant mammalian cells; Verkaik NS et al.; The end-joining pathway of DNA double-strand break (DSB) repair is necessary for proper V(D)J recombination and repair of DSB caused by ionizing radiation . This DNA repair pathway can either use short stretches of (micro)homology near the DNA ends or use no homology at all (direct end-joining) . We designed assays to determine the relative efficiencies of these (sub)pathways of DNA end-joining . In one version, a DNA substrate is linearized in such a way that joining on a particular microhomology creates a novel restriction enzyme recognition site . In the other one, the DSB is made by the RAG1 and RAG2 proteins . After PCR amplification of the junctions, the different end-joining modes can be discriminated by restriction enzyme digestion . We show that inactivation of the 'classic' end-joining factors (Ku80, DNA-PK(CS), ligase IV and XRCC4) results in a dramatic increase of microhomology-directed joining of the linear substrate, but very little decrease in overall joining efficiency . V(D)J recombination, on the other hand, is severely impaired, but also shows a dramatic shift towards microhomology use . Interestingly, two interstrand cross-linker-sensitive cell lines showed decreased microhomology-directed end-joining, but without an effect on V(D)J recombination . These results suggest that direct end-joining and microhomology-directed end-joining constitute genetically distinct DSB repair pathways. Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 2732 - 7 Epub 2002 Feb 26. Analysis of the AAA sensor-2 motif in the C-terminal ATPase domain of Hsp104 with a site-specific fluorescent probe of nucleotide binding; Hattendorf DA et al.; Hsp104 from Saccharomyces cerevisiae is a hexameric protein with two AAA ATPase domains (N- and C-terminal nucleotide-binding domains NBD1 and NBD2, respectively) per monomer . Our previous analysis of the Hsp104 ATP hydrolysis cycle revealed that NBD1 and NBD2 have very different catalytic properties, but each shows positive cooperativity in hydrolysis . There is also communication between the two domains, in that ATP hydrolysis at NBD1 depends on the nucleotide that is bound to NBD2 . Here, we extend our understanding of the Hsp104 ATP hydrolysis cycle through mutagenesis of the AAA sensor-2 motif in NBD2 . To do so, we took advantage of the lack of tryptophan residues in Hsp104 to place a single tryptophan in the C-terminal domain (Y819W) . The Y819W substitution has no significant effects on folding stability of the C-terminal domain or on ATP hydrolysis by NBD1 or NBD2 . The fluorescence of this tryptophan changes in response to ATP and ADP binding, allowing the K(d) and Hill coefficient to be determined for each nucleotide . By using this site-specific probe of binding, we analyze the effect of mutating the conserved arginine residue in the sensor-2 motif in Hsp104 NBD2 . An R826M mutation causes nearly equal decreases in affinity of NBD2 for both ATP and ADP, indicating that at this site, the sensor-2 provides binding energy, but does not act to sense the difference between these nucleotides . In addition, the rate of ATP hydrolysis at NBD1 is decreased by the R826M mutation, providing further evidence for interdomain communication in the Hsp104 ATP hydrolysis cycle. J Gerontol A Biol Sci Med Sci, 2002 Mar, 57(3), B93 - 8 Histone acetyltransferase activities of cAMP-regulated enhancer-binding protein and p300 in tissues of fetal, young, and old mice; Li Q et al.; CBP, a protein that binds to cyclic adenosine monophosphate-regulated enhancer-binding protein, and homologue protein, p300, have histone acetyltransferase (HAT) activity and are important in gene transcription, although their physiological functions in vivo remain to be further elucidated . By using immunoprecipitation and HAT activity assay we have found that p300 and CBP have similar tissue patterns of HAT activities, with the highest level in the brain, a relatively high level in the lung, spleen, and heart, an intermediate level in testes and muscle, and a lower level in liver and kidney; that HAT activities of p300 and CBP are relatively stable with advancing age in most examined tissues, but in liver, muscle, and testes, the activities are attenuated with aging; and that HAT activities of p300 and CBP are high in the brain and liver of E14 fetal and newborn mice . These data suggest that the HAT activities of p300 and CBP are important for gene transcription involved in tissue-specific expression, aging, and developing processes. EMBO J, 2002 Mar 1, 21(5), 1148 - 57 Protein 61K, encoded by a gene (PRPF31) linked to autosomal dominant retinitis pigmentosa, is required for U4/U6*U5 tri-snRNP formation and pre-mRNA splicing; Makarova OV et al.; In each round of nuclear pre-mRNA splicing, the U4/U6*U5 tri-snRNP must be assembled from U4/U6 and U5 snRNPs, a reaction that is at present poorly understood . We have characterized a 61 kDa protein (61K) found in human U4/U6*U5 tri-snRNPs, which is homologous to yeast Prp31p, and show that it is required for this step . Immunodepletion of protein 61K from HeLa nuclear extracts inhibits tri-snRNP formation and subsequent spliceosome assembly and pre-mRNA splicing . Significantly, complementation with recombinant 61K protein restores each of these steps . Protein 61K is operationally defined as U4/U6 snRNP-specific as it remains bound to this particle at salt concentrations where the tri-snRNP dissociates . However, as shown by two-hybrid analysis and biochemical assays, protein 61K also interacts specifically with the U5 snRNP-associated 102K protein, indicating that it physically tethers U4/U6 to the U5 snRNP to yield the tri-snRNP . Interestingly, protein 61K is encoded by a gene (PRPF31) that has been shown to be linked to autosomal dominant retinitis pigmentosa . Thus, our studies suggest that disruptions in tri-snRNP formation and function resulting from mutations in the 61K protein may contribute to the manifestation of this disease. EMBO J, 2002 Mar 1, 21(5), 1101 - 11 Hyperacetylation of chromatin at the ADH2 promoter allows Adr1 to bind in repressed conditions; Verdone L et al.; We report that in vivo increased acetylation of the repressed Saccharomyces cerevisiae ADH2 promoter chromatin, as obtained by disrupting the genes for the two deacetylases HDA1 and RPD3, destabilizes the structure of the TATA box-containing nucleosome . This acetylation-dependent chromatin remodeling is not sufficient to allow the binding of the TATA box-binding protein, but facilitates the recruitment of the transcriptional activator Adr1 and induces faster kinetics of mRNA accumulation when the cells are shifted to derepressing conditions. EMBO J, 2002 Mar 1, 21(5), 942 - 53 The Tim9p-Tim10p complex binds to the transmembrane domains of the ADP/ATP carrier; Curran SP et al.; The soluble Tim9p-Tim10p (Tim, translocase of inner membrane) complex of the mitochondrial intermembrane space mediates the import of the carrier proteins and is a component of the TIM22 import system . The mechanism by which the Tim9p-Tim10p complex assembles and binds the carriers is not well understood, but previous studies have proposed that the conserved cysteine residues in the 'twin CX3C' motif coordinate zinc and potentially generate a zinc-finger-like structure that binds to the matrix loops of the carrier proteins . Here we have purified the native and recombinant Tim9p-Tim10p complex, and show that both complexes resemble each other and consist of three Tim9p and three Tim10p . Results from inductively coupled plasma--mass spectrometry studies failed to detect zinc in the Tim9p-Tim10p complex . Instead, the cysteine residues seemingly formed disulfide linkages . The Tim9p-Tim10p complex bound specifically to the transmembrane domains of the ADP/ATP carrier, but had no affinity for Tim23p, an inner membrane protein that is inserted via the TIM22 complex . The chaperone-like Tim9p-Tim10p complex thus may prevent aggregation of the unfolded carrier proteins in the aqueous intermembrane space. EMBO J, 2002 Mar 1, 21(5), 920 - 9 The Ppz protein phosphatases are key regulators of K+ and pH homeostasis: implications for salt tolerance, cell wall integrity and cell cycle progression; Yenush L et al.; The yeast Ppz protein phosphatases and the Hal3p inhibitory subunit are important determinants of salt tolerance, cell wall integrity and cell cycle progression . We present several lines of evidence showing that these disparate phenotypes are connected by the fact that Ppz regulates K+ transport . First, salt tolerance, cell wall integrity and cell cycle phenotypes of Ppz mutants are dependent on the Trk K+ transporters . Secondly, Ppz mutants exhibit altered activity of the Trk system, as measured by rubidium uptake . Thirdly, Ppz mutants exhibit altered intracellular K+ and pH, as expected from H+ efflux providing electrical balance during K+ uptake . Our unifying picture of Ppz phenotypes contends that activation of Trk by decreased Ppz activity results in plasma membrane depolarization (reducing uptake of toxic cations), increased intracellular K+ and turgor (compromising cell integrity), and increased intracellular pH (augmenting the expression of pH-regulated genes and facilitating alpha-factor recovery) . In addition to providing a coherent explanation for all Ppz-dependent phenotypes, our results provide evidence for a causal relationship between intracellular cation homeostasis and a potential cell cycle checkpoint. J Mol Biol, 2002 Feb 22, 316(3), 657 - 66 Protein translocase of the outer mitochondrial membrane: role of import receptors in the structural organization of the TOM complex; Model K et al.; The mitochondrial outer membrane contains a multi-subunit machinery responsible for the specific recognition and translocation of precursor proteins . This translocase of the outer membrane (TOM) consists of three receptor proteins, Tom20, Tom22 and Tom70, the channel protein Tom40, and several small Tom proteins . Single-particle electron microscopy analysis of the Neurospora TOM complex has led to different views with two or three stain-filled centers resembling channels . Based on biochemical and electron microscopy studies of the TOM complex isolated from yeast mitochondria, we have discovered the molecular reason for the different number of channel-like structures . The TOM complex from wild-type yeast contains up to three stain-filled centers, while from a mutant yeast selectively lacking Tom20, the TOM complex particles contain only two channel-like structures . From mutant mitochondria lacking Tom22, native electrophoresis separates an approximately 80 kDa subcomplex that consists of Tom40 only and is functional for accumulation of a precursor protein . We conclude that while Tom40 forms the import channels, the two receptors Tom22 and Tom20 are required for the organization of Tom40 dimers into larger TOM structures . J Infect Dis, 2002 Mar 1, 185(5), 657 - 64 Epub 2002 Feb 14. Merozoite surface protein 3 and protection against malaria in Aotus nancymai monkeys; Hisaeda H et al.; A blood-stage vaccine based on Plasmodium falciparum merozoite surface protein 3 (MSP3) was tested for efficacy in a primate model . Aotus nancymai monkeys were vaccinated with yeast-expressed MSP3 before a lethal challenge with Plasmodium falciparum parasites . Five of 7 control monkeys had acute infections and required treatment to control parasitemia . Only 1 of 7 monkeys vaccinated with MSP3 required this treatment . The efficacy of the MSP3 vaccination appeared to be comparable to that of MSP1(42), a leading asexual vaccine candidate, in response to which 2 monkeys experienced acute infections . In the MSP3-vaccinated group, protection correlated with prechallenge titers of antibody to MSP3 . In the MSP1 and control groups, protection correlated with antibody to MSP3 raised by challenge infection. Mol Cell Biol, 2002 Mar, 22(6), 1936 - 46 Binding of the RING polycomb proteins to specific target genes in complex with the grainyhead-like family of developmental transcription factors; Tuckfield A et al.; The Polycomb group (PcG) of proteins represses homeotic gene expression through the assembly of multiprotein complexes on key regulatory elements . The mechanisms mediating complex assembly have remained enigmatic since most PcG proteins fail to bind DNA . We now demonstrate that the human PcG protein dinG interacts with CP2, a mammalian member of the grainyhead-like family of transcription factors, in vitro and in vivo . The functional consequence of this interaction is repression of CP2-dependent transcription . The CP2-dinG interaction is conserved in evolution with the Drosophila factor grainyhead binding to dring, the fly homologue of dinG . Electrophoretic mobility shift assays demonstrate that the grh-dring complex forms on regulatory elements of genes whose expression is repressed by grh but not on elements where grh plays an activator role . These observations reveal a novel mechanism by which PcG proteins may be anchored to specific regulatory elements in developmental genes. Mol Cell Biol, 2002 Mar, 22(6), 1615 - 25 Transcription activator interactions with multiple SWI/SNF subunits; Neely KE et al.; We have previously shown that the yeast SWI/SNF complex stimulates in vitro transcription from chromatin templates in an ATP-dependent manner . SWI/SNF function in this regard requires the presence of an activator with which it can interact directly, linking activator recruitment of SWI/SNF to transcriptional stimulation . In this study, we determine the SWI/SNF subunits that mediate its interaction with activators . Using a photo-cross-linking label transfer strategy, we show that the Snf5, Swi1, and Swi2/Snf2 subunits are contacted by the yeast acidic activators, Gcn4 and Hap4, in the context of the intact native SWI/SNF complex . In addition, we show that the same three subunits can interact individually with acidic activation domains, indicating that each subunit contributes to binding activators . Furthermore, mutations that reduce the activation potential of these activators also diminish its interaction with each of these SWI/SNF subunits . Thus, three distinct subunits of the SWI/SNF complex contribute to its interactions with activation domains. J Cell Biol, 2002 Mar 4, 156(5), 805 - 15 Epub 2002 Feb 25. In vivo dissection of the chromosome condensation machinery: reversibility of condensation distinguishes contributions of condensin and cohesin; Lavoie BD et al.; The machinery mediating chromosome condensation is poorly understood . To begin to dissect the in vivo function(s) of individual components, we monitored mitotic chromosome structure in mutants of condensin, cohesin, histone H3, and topoisomerase II (topo II) . In budding yeast, both condensation establishment and maintenance require all of the condensin subunits, but not topo II activity or phospho-histone H3 . Structural maintenance of chromosome (SMC) protein 2, as well as each of the three non-SMC proteins (Ycg1p, Ycs4p, and Brn1p), was required for chromatin binding of the condensin complex in vivo . Using reversible condensin alleles, we show that chromosome condensation does not involve an irreversible modification of condensin or chromosomes . Finally, we provide the first evidence of a mechanistic link between condensin and cohesin function . A model discussing the functional interplay between cohesin and condensin is presented. J Biol Chem, 2002 May 3, 277(18), 15613 - 20 Epub 2002 Feb 25. Desaturation and hydroxylation . Residues 148 and 324 of Arabidopsis FAD2, in addition to substrate chain length, exert a major influence in partitioning of catalytic specificity; Broadwater JA et al.; Exchanging the identity of amino acids at four key locations within the Arabidopsis thaliana oleate desaturase (FAD2) and the Lesquerella fendleri hydroxylase/desaturase (LFAH) was shown to influence partitioning between desaturation and hydroxylation (Broun, P., Shanklin, J., Whittle, E., and Somerville, C . (1998) Science 282, 1315-1317) . We report that four analogous substitutions in the FAD2 sequence by their equivalents from the castor oleate hydroxylase result in hydroxy fatty acid accumulation in A . thaliana to the same levels as for the wild-type castor hydroxylase . We also describe the relative contribution of these substitutions, both individually and in combination, by analyzing the products resulting from their expression in A . thaliana and/or Saccharomyces cerevisiae . Yeast expression showed that M324V, a change reachable by a single point mutation, altered the product distribution approximately 49-fold, and that residue 148 is also a predominant determinant of reaction outcome . Comparison of residues at position 148 of FAD2, LFAH, and the Ricinus oleate hydroxylase prompted us to rationally engineer LFAH-N149I, a variant with approximately 1.9-fold increase in hydroxylation specificity compared with that of wild-type LFAH . Control experiments showed that the wild-type Arabidopsis thaliana FAD2 desaturase has inherent, low level, hydroxylation activity . Further, fatty acid desaturases from different kingdoms and with different regiospecificities exhibit similar intrinsic hydroxylase activity, underscoring fundamental mechanistic similarities between desaturation and hydroxylation . For LFAH mutants the hydroxylation:desaturation ratio is 5-9-fold higher for 18-carbon versus 16-carbon substrates, supporting our hypothesis that substrate positioning in the active site plays a key role in the partitioning of catalytic specificity. J Biol Chem, 2002 May 3, 277(18), 15766 - 72 Epub 2002 Feb 25. Pheromone-dependent ubiquitination of the mitogen-activated protein kinase kinase Ste7; Wang Y et al.; Many cell signaling pathways are regulated by phosphorylation, ubiquitination, and degradation of constituent proteins . As with phosphorylation, protein ubiquitination can be reversed, through the action of ubiquitin-specific processing proteases (UBPs) . Here we have analyzed 15 UBP disruption mutants in the yeast Saccharomyces cerevisiae and identified one (ubp3 Delta) that acts specifically in the pheromone response pathway . Upon pheromone stimulation, ubp3 Delta mutants accumulate unconjugated polyubiquitin chains as well as polyubiquitinated forms of the mitogen-activated protein kinase kinase Ste7 . The ubp3 Delta mutants exhibit a potentiated response to pheromone, as measured by in vivo MAP kinase activity, transcriptional induction, and cell cycle arrest . Signaling is likewise enhanced upon direct activation of Ste4 (G protein beta subunit) and Ste11 (Ste7 kinase) but not the downstream transcription factor Ste12 . These findings reveal a mechanism by which pheromone-triggered ubiquitination of Ste7 can modulate the pheromone response in vivo. J Biol Chem, 2002 May 3, 277(18), 15629 - 37 Epub 2002 Feb 25. Salt-inducible kinase represses cAMP-dependent protein kinase-mediated activation of human cholesterol side chain cleavage cytochrome P450 promoter through the CREB basic leucine zipper domain; Doi J et al.; Salt-inducible kinase (SIK), one of the serine/threonine protein kinases, was transiently expressed in Y1 cells during the early phase of the ACTH/cAMP-dependent protein kinase (PKA)-mediated signal transduction . The overexpression of SIK(N), the SIK's N-terminal kinase domain, repressed the expression of the side chain cleavage cytochrome P450 (CYP11A) gene . To elucidate the mechanism of the repression by SIK, several CYP11A promoter constructs were tested for the promoter activities in the presence of PKA and/or SIK(N) . A cAMP-response element (CRE)-like sequence present in the promoter was shown to be responsible not only for the PKA-mediated promoter activation but also for the SIK(N)-mediated repression . When the Gal4 DNA binding domain-linked full-length CRE-binding protein (CREB) construct was cotransfected with Gal4 reporter gene, SIK(N) repressed the PKA-induced reporter gene expression . However, SIK(N) could not repress the PKA-induced reporter activity conferred by Gal4 DNA binding domain-linked basic leucine zipper (bZIP)-less CREB or bZIP-disrupted CREB . On the other hand, SIK(N) could repress the kinase-inducible domain-disrupted CREB-dependent reporter gene expression in the presence of PKA . The in vitro kinase reaction studies showed that SIK(N) could not phosphorylate CREB, and PKA failed to phosphorylate SIK(N) . Taken together, these results suggest that SIK(N), cooperating with PKA, may act on the CREB's bZIP domain and repress the CREB-mediated transcriptional activation of the CYP11A gene. Mol Cell, 2002 Feb, 9(2), 363 - 73 Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signal-gated channel; Kovermann P et al.; The protein insertion complex of the mitochondrial inner membrane is crucial for import of the numerous multitopic membrane proteins with internal targeting signals . Little is known about the molecular mechanism of this complex, including whether it forms a real channel or merely acts as scaffold for protein insertion . We report the unexpected observation that Tim22 is the only essential membrane-integrated subunit of the complex . Reconstituted Tim22 forms a hydrophilic, high-conductance channel with distinct opening states and pore diameters . The channel is voltage-activated and specifically responds to an internal targeting signal, but not to presequences . Thus, a protein insertion complex can combine three essential functions, signal recognition, channel formation, and energy transduction, in one central component. Mol Cell, 2002 Feb, 9(2), 341 - 51 Ssf1p prevents premature processing of an early pre-60S ribosomal particle; Fatica A et al.; Ssf1p and Ssf2p are two nearly identical and functionally redundant nucleolar proteins . In the absence of Ssf1p and Ssf2p, the 27SA(2) pre-rRNA was prematurely cleaved, inhibiting synthesis of the 27SB and 7S pre-rRNAs and the 5.8S and 25S rRNA components of the large ribosomal subunit . On sucrose gradients, Ssf1p sedimented with pre-60S ribosomal particles . The 27SA(2), 27SA(3), and 27SB pre-rRNAs were copurified with tagged Ssf1p, as were 23 large subunit ribosomal proteins and 21 other proteins implicated in ribosome biogenesis . These included four Brix family proteins, Ssf1p, Rpf1p, Rpf2p, and Brx1p, indicating that the entire family functions in ribosome synthesis . This complex is distinct from recently reported pre-60S complexes in RNA and protein composition . We describe a multistep pathway of 60S preribosome maturation. Mol Cell, 2002 Feb, 9(2), 233 - 40 Xenopus Mcm10 binds to origins of DNA replication after Mcm2-7 and stimulates origin binding of Cdc45; Wohlschlegel JA et al.; Current models suggest that the replication initiation factor Mcm10 is required for association of Mcm2-7 with origins of replication to generate the prereplicative complex (pre-RC) . Here we report that Xenopus Mcm10 (XMcm10) is not required for origin binding of XMcm2-7 . Instead, the chromatin binding of XMcm10 at the onset of DNA replication requires chromatin-bound XMcm2-7, and it is independent of Cdk2 and Cdc7 . In the absence of XMcm10, XCdc45 binding, XRPA binding, and initiation-dependent plasmid supercoiling are blocked . Therefore, XMcm10 performs its function after pre-RC assembly and before origin unwinding . As one of the earliest known pre-RC activation steps, chromatin binding of XMcm10 is an attractive target for regulation by cell cycle checkpoints. Mol Cell, 2002 Feb, 9(2), 207 - 9 The FEAR factor; Saunders WS; The decision to exit from mitosis is a fateful one, and Amon and colleagues have shown that it involves both an early and a late component . The FEAR network initiates the exit machinery, while the previously described MEN pathway maintains it. Curr Biol, 2002 Feb 19, 12(4), 323 - 8 Eco1 is a novel acetyltransferase that can acetylate proteins involved in cohesion; Ivanov D et al.; Cohesion between sister chromatids is established during S phase and maintained through G2 phase until it is resolved in anaphase (for review, see {1-3}) . In Saccharomyces cerevisiae, a complex consisting of Scc1, Smc1, Smc3, and Scc3 proteins, called "cohesin," mediates the connection between sister chromatids . The evolutionary conserved yeast protein Eco1 is required for establishment of sister chromatid cohesion during S phase but not for its further maintenance during G2 or M phases or for loading the cohesin complex onto DNA . We address the molecular functions of Eco1 with sensitive sequence analytic techniques, including hidden Markov model domain fragment searches . We found a two-domain architecture with an N-terminal C2H2 Zn finger-like domain and an approximately 150 residue C-terminal domain with an apparent acetyl coenzyme A binding motif . Biochemical tests confirm that Eco1 has the acetyltransferase activity in vitro . In vitro Eco1 acetylates itself and components of the cohesin complex but not histones . Thus, the establishment of cohesion between sister chromatids appears to be regulated, directly or indirectly, by a specific acetyltransferase. Curr Biol, 2002 Feb 19, 12(4), 317 - 21 The p21-activated kinase PAK is negatively regulated by POPX1 and POPX2, a pair of serine/threonine phosphatases of the PP2C family; Koh CG et al.; The Rho GTPases are involved in many signaling pathways and cellular functions, including the organization of the actin cytoskeleton, regulation of transcription, cell motility, and cell division . The p21 (Cdc42/Rac)-activated kinase PAK mediates a number of biological effects downstream of these Rho GTPases (reviewed by {1}) . The phosphorylation state of mammalian PAK is highly regulated: upon binding of GTPases, PAK is potently activated by autophosphorylation at multiple sites, although the mechanisms of PAK downregulation are not known . We now report two PP2C-like serine/threonine phosphatases (POPX1 and POPX2) that efficiently inactivate PAK . POPX1 was isolated as a binding partner for the PAK interacting guanine nucleotide exchange factor PIX . The dephosphorylating activity of POPX correlates with an ability to block the in vivo effects of active PAK . Consonant with these effects on PAK, POPX can also inhibit actin stress fiber breakdown and morphological changes driven by active Cdc42(V12) . The association of the POPX phosphatases with PAK complexes may allow PAK to cycle rapidly between active and inactive states; it represents a unique regulatory component of the signaling pathways of the PAK kinase family. Biochemistry, 2002 Mar 5, 41(9), 3254 - 61 Single replacement constructs of all hydroxyl, basic, and acidic amino acids identify new function and structure-sensitive regions of the mitochondrial phosphate transport protein; Wohlrab H et al.; The phosphate transport protein (PTP) catalyzes the proton cotransport of phosphate into the mitochondrial matrix . It functions as a homodimer, and thus residues of the phosphate and proton pores are somewhat scattered throughout the primary sequence . With 71 new single mutation per subunit PTPs, all its hydroxyl, basic, and acidic residues have now been replaced to identify these essential residues . We assayed the initial rate of pH gradient-dependent unidirectional phosphate transport activity and the liposome incorporation efficiency (LIE) of these mutants . Single mutations of Thr79, Tyr83, Lys90, Tyr94, and Lys98 inactivate transport . The spacings between these residues imply that they are located along the same face of transmembrane (TM) helix B, requiring an extension of its current model C-terminal domain by 10 residues . This extension superimposes very well onto the shorter bovine PTP helix B, leaving a 15-residue hydrophobic extension of the yeast helix B N-terminus . This is similar to the helix D and F regions of the yeast PTP . Only one transport-inhibiting mutation is located within loops: Ser158Thr in the matrix loop between helices C and D . All other transport-inhibiting mutations are located within the TM helices . Mutations that yield LIEs of <6% are all, except for four, within helices . The four exceptions are Tyr12Ala near the PTP N-terminus and Arg159Ala, Glu163Gln, and Glu164Gln in the loop between helices C and D . The PTP C-terminal segment beyond Thr214 at the N-terminus of helix E has 11 mutations with LIEs >20% and none with LIE <6% . Mutations with LIEs >20% are located near the ends of all the TM helices except TM helix D . Only a few mutations alter PTP structure (LIE) and also affect PTP transport activity . A novel observation is that Ser4Ala blocks the formation of PTP bacterial inclusion bodies. Biochemistry, 2002 Mar 5, 41(9), 3147 - 55 Gal4-VP16 and Gal4-AH increase the orientational and axial specificity of TATA box recognition by TATA box binding protein; Kays AR et al.; Previous work has shown that binding of the TATA box binding protein (TBP) to the TATA box is a rate-limiting step during pre-initiation complex (PIC) formation . Although the transcription of eukaryotic genes normally proceeds in one direction, studies in solution have shown that TBP lacks the information necessary to orient itself on the TATA box . Instead, yeast TBP binds TATA-containing promoters in two orientations that are related by a 180 rotation about TBP's pseudo-2-fold symmetry axis . Recruitment of PIC components by gene-specific activators is considered a primary mechanism of transcriptional enhancement . Here we ask whether activators might function, at least in part, by increasing the fraction of PICs assembled with TBP bound in the orientation necessary for transcription . We use DNA affinity cleavage and a TBP-phenanthroline-copper conjugate to monitor the orientation of TBP in the presence of the well-studied activators Gal4-VP16 and Gal4-AH . In the absence of a transcriptional activator, only 51% of the TBP x TATA box complexes were bound in the orientation necessary for the initiation of transcription . However, in the presence of saturating Gal4-VP16, 87% of the TBP bound to the TATA box was oriented correctly at equilibrium . This increase in orientational specificity corresponds to a free energy difference (Delta Delta G(obs)) of 1.1 kcal x mol(-1) and was accompanied by a dramatic increase in axial specificity, reminiscent of the effects of transcription factors TFIIB and TFIIA reported previously . Gal4-AH also enhanced the orientational and axial specificity of the TBP x TATA complex, although to a lesser extent . We suggest that these effects on specificity represent a variation of recruitment, since they require direct interactions between the activator and a PIC component but only increase the effective concentration of the correctly oriented PIC component . These findings add to increasing evidence that recruitment may encompass a broad range of mechanisms. Nucleic Acids Res, 2002 Mar 1, 30(5), 1198 - 204 Processing of nucleopeptides mimicking the topoisomerase I-DNA covalent complex by tyrosyl-DNA phosphodiesterase; Debethune L et al.; Tyrosyl-DNA phosphodiesterase-1 (Tdp1) is the only known enzyme to remove tyrosine from complexes in which the amino acid is linked to the 3'-end of DNA fragments . Such complexes can be produced following DNA processing by topoisomerase I, and recent studies in yeast have demonstrated the importance of TDP1 for cell survival following topoisomerase I-mediated DNA damage . In the present study, we used synthetic oligodeoxynucleotide-peptide conjugates (nucleopeptides) and recombinant yeast Tdp1 to investigate the molecular determinants for Tdp1 activity . We find that Tdp1 can process nucleopeptides with up to 13 amino acid residues but is poorly active with a 70 kDa fragment of topoisomerase I covalently linked to a suicide DNA substrate . Furthermore, Tdp1 was more effective with nucleopeptides with one to four amino acids than 15 amino acids . Tdp1 was also more effective with nucleopeptides containing 15 nt than with homolog nucleopeptides containing 4 nt . These results suggest that DNA binding contributes to the activity of Tdp1 and that Tdp1 would be most effective after topoisomerase I has been proteolyzed in vivo. Nucleic Acids Res, 2002 Mar 1, 30(5), 1103 - 13 SGS1 is a multicopy suppressor of srs2: functional overlap between DNA helicases; Mankouri HW et al.; Sgs1 is a member of the RecQ family of DNA helicases, which have been implicated in genomic stability, cancer and ageing . Srs2 is another DNA helicase that shares several phenotypic features with Sgs1 and double sgs1srs2 mutants have a severe synthetic growth phenotype . This suggests that there may be functional overlap between these two DNA helicases . Consistent with this idea, we found the srs2Delta mutant to have a similar genotoxin sensitivity profile and replicative lifespan to the sgs1Delta mutant . In order to directly test if Sgs1 and Srs2 are functionally interchangeable, the ability of high-copy SGS1 and SRS2 plasmids to complement the srs2Delta and sgs1Delta mutants was assessed . We report here that SGS1 is a multicopy suppressor of the methyl methanesulphonate (MMS) and hydroxyurea sensitivity of the srs2Delta mutant, whereas SRS2 overexpression had no complementing ability in the sgs1Delta mutant . Domains of Sgs1 directly required for processing MMS-induced DNA damage, most notably the helicase domain, are also required for complementation of the srs2Delta mutant . Although SGS1 overexpression was unable to rescue the shortened mean replicative lifespan of the srs2Delta mutant, maximum lifespan was significantly increased by multicopy SGS1 . We conclude that Sgs1 is able to partially compensate for the loss of Srs2. Nucleic Acids Res, 2002 Mar 1, 30(5), 1083 - 90 A question of size: the eukaryotic proteome and the problems in defining it; Harrison PM et al.; We discuss the problems in defining the extent of the proteomes for completely sequenced eukaryotic organisms (i.e . the total number of protein-coding sequences), focusing on yeast, worm, fly and human . (i) Six years after completion of its genome sequence, the true size of the yeast proteome is still not defined . New small genes are still being discovered, and a large number of existing annotations are being called into question, with these questionable ORFs (qORFs) comprising up to one-fifth of the 'current' proteome . We discuss these in the context of an ideal genome-annotation strategy that considers the proteome as a rigorously defined subset of all possible coding sequences ('the orfome') . (ii) Despite the greater apparent complexity of the fly (more cells, more complex physiology, longer lifespan), the nematode worm appears to have more genes . To explain this, we compare the annotated proteomes of worm and fly, relating to both genome-annotation and genome evolution issues . (iii) The unexpectedly small size of the gene complement estimated for the complete human genome provoked much public debate about the nature of biological complexity . However, in the first instance, for the human genome, the relationship between gene number and proteome size is far from simple . We survey the current estimates for the numbers of human genes and, from this, we estimate a range for the size of the human proteome . The determination of this is substantially hampered by the unknown extent of the cohort of pseudogenes ('dead' genes), in combination with the prevalence of alternative splicing . (Further information relating to yeast is available at http://genecensus.org/yeast/orfome) Mol Biol Evol, 2002 Mar, 19(3), 239 - 46 Adaptation for horizontal transfer in a homing endonuclease; Koufopanou V et al.; Selfish genes of no function other than self-propagation are susceptible to degeneration if they become fixed in a population, and regular transfer to new species may be the only means for their long-term persistence . To test this idea we surveyed 24 species of yeast for VDE, a nuclear, intein-associated homing endonuclease gene (HEG) originally discovered in Saccharomyces cerevisiae . Phylogenetic analyses show that horizontal transmission has been a regular occurrence in its evolutionary history . Moreover, VDE appears to be specifically adapted for horizontal transmission . Its 31-bp recognition sequence is an unusually well-conserved region in an unusually well-conserved gene . In addition, the nine nucleotide sites most critical for homing are also unusually well conserved . Such adaptation for horizontal transmission presumably arose as a consequence of selection, both among HEGs at different locations in the genome and among variants at the same location . The frequency of horizontal transmission must therefore be a key feature constraining the distribution and abundance of these genes. J Virol, 2002 Mar, 76(6), 2804 - 16 Ty3 integrase is required for initiation of reverse transcription; Nymark-McMahon MH et al.; The integrase (IN) encoded by the Saccharomyces cerevisiae retrovirus-like element Ty3 has features found in retrovirus IN proteins including the catalytic triad, an amino-terminal zinc-binding motif, and a nuclear localization sequence . Mutations in the amino- and carboxyl-terminal domains of Ty3 IN cause reduced accumulation of full-length cDNA in the viruslike particles . We show that the reduction in cDNA is accompanied by reduced amounts of early intermediates such as minus-strand, strong-stop DNA . Expression of a capsid (CA)-IN fusion protein (CA-IN) complemented catalytic site and nuclear localization mutants, but not DNA mutants . However, expression of a fusion of CA, reverse transcriptase (RT), and IN (CA-RT-IN) complemented transposition of catalytic site and nuclear localization signal mutants, increased the amount of cDNA in some of the mutants, and complemented transposition of several mutants to low frequencies . Expression of a CA-RT-IN protein with a Ty3 IN catalytic site mutation did not complement transposition of either a Ty3 catalytic site mutant or a nuclear localization mutant but did increase the amount of cDNA in several mutants and complement at least one of the cDNA mutants for transposition . These in vivo data support a model in which independent IN domains can contribute to reverse transcription and integration . We conclude that during reverse transcription, the Ty3 IN domain interacts closely with the polymerase domain and may even constitute a domain within a heterodimeric RT . These studies also suggest that during integration the IN catalytic site and at least portions of the IN carboxyl-terminal domain act in cis. J Cell Sci, 2002 Feb 1, 115(Pt 3), 655 - 66 Vid22p, a novel plasma membrane protein, is required for the fructose-1,6-bisphosphatase degradation pathway; Brown CR et al.; Fructose-1,6-bisphosphatase (FBPase), an important enzyme in the gluconeogenic pathway in Saccharomyces cerevisiae, is expressed when cells are grown in media containing a poor carbon source . Following glucose replenishment, FBPase is targeted from the cytosol to intermediate Vid (vacuole import and degradation) vesicles and then to the vacuole for degradation . Recently, several vid mutants that are unable to degrade FBPase in response to glucose were identified . Here, we present VID22, a novel gene involved in FBPase degradation . VID22 encodes a glycosylated integral membrane protein that localizes to the plasma membrane . Newly synthesized Vid22p was found in the cytoplasm and then targeted to the plasma membrane independent of the classical secretory pathway . A null mutation of VID22 failed to degrade FBPase following a glucose shift and accumulated FBPase in the cytosol . Furthermore, the majority of FBPase remained in a proteinase K sensitive compartment in the Deltavid22 mutant, implying that VID22 is involved in FBPase transport from the cytosol to Vid vesicles . By contrast, starvation-induced autophagy and peroxisome degradation were not impaired in the Deltavid22 mutant . This strain also exhibited the proper processing of carboxypeptidase Y and aminopeptidase I in the vacuole . Therefore, Vid22p appears to play a specific role in the FBPase trafficking pathway. J Biol Chem, 2002 May 10, 277(19), 16383 - 90 Epub 2002 Feb 22. Differential role of the proline-rich domain of nuclear factor 1-C splice variants in DNA binding and transactivation; Prado F et al.; We have addressed the functional significance of the existence of several natural splice variants of NF1-C* differing in their COOH-terminal proline-rich transactivation domain (PRD) by studying their specific DNA binding and transactivation in the yeast Saccharomyces cerevisiae . These parameters yielded the intrinsic transactivation potential (ITP), defined as the activation observed with equal amounts of DNA bound protein . Exchange of 83 amino acids at the COOH-terminal end of the PRD by 16 unrelated amino acids, as found in NF1-C2, and splicing out the central region of the PRD, as found in NF1-C7, enhanced DNA binding in vivo and in vitro . However, the ITP of the splice variants NF1-C2 and NF1-C7 was found to be similar to that of the intact NF1-C1 . Additional mutations showed that the ITP of NF1-C requires the synergistic action of the PRD and a novel domain encoded in exons 5 and 6 . Intriguingly the carboxyl-terminal domain-like motif encoded in exons 9/10 is not essential for transactivation of a reporter with a single NF1 site but is required for activation of a reporter with six NF1 sites in tandem . Our results imply that differential splicing is used to regulate transcription by generating variants with different DNA binding affinities but similar ITPs. Genetics, 2002 Feb, 160(2), 799 - 803 Caenorhabditis elegans unc-37/groucho interacts genetically with components of the transcriptional mediator complex; Zhang H et al.; Groucho functions as a general corepressor by modulating chromatin structure and has a widespread role in many developmental processes . Here we show that Groucho may also interact with the basal transcriptional machinery . Mutations in Caenorhabditis elegans groucho interact with mutations in components of the transcriptional Mediator complex, resulting in synthetic lethality and loss of male sensory neurons. J Biotechnol, 2002 Apr 11, 94(3), 299 - 308 Single-cell viability assessment with a novel spectro-imaging system; Matsuoka H et al.; Single-cell viability assessment by means of plural dye probes require the spectral and temporal analysis of microscopic images of the test cells . To meet this requirement, we have developed a simple and compact spectro-imaging system using an image slicer and a grism . The image slicer was made of a bundle of 100 optical fibers . The field of view is divided into 10 x 10 sections . The spectral data of each section could be recorded every 5 s in the range from 400 to 800 nm at 5 nm resolution . The viability changes of yeast or tobacco single-cells were measured with this system . Using BY-2 cells, for example, the response to a chemical stress of saponin was measured by means of two fluorescent probes . The spectral-spatial-temporal data of fluorescein and DNA bound ethidium bromide provided us with useful information about the dynamic change of cell membrane permeability from which the cell viability was assessed. Nature, 2002 Feb 21, 415(6874), 929 - 33 A Rad26-Def1 complex coordinates repair and RNA pol II proteolysis in response to DNA damage; Woudstra EC et al.; Eukaryotic cells use multiple, highly conserved mechanisms to contend with ultraviolet-light-induced DNA damage . One important response mechanism is transcription-coupled repair (TCR), during which DNA lesions in the transcribed strand of an active gene are repaired much faster than in the genome overall . In mammalian cells, defective TCR gives rise to the severe human disorder Cockayne's syndrome (CS) . The best-studied CS gene, CSB, codes for a Swi/Snf-like DNA-dependent ATPase, whose yeast homologue is called Rad26 (ref . 4) . Here we identify a yeast protein, termed Def1, which forms a complex with Rad26 in chromatin . The phenotypes of cells lacking DEF1 are consistent with a role for this factor in the DNA damage response, but Def1 is not required for TCR . Rather, def1 cells are compromised for transcript elongation, and are unable to degrade RNA polymerase II (RNAPII) in response to DNA damage . Our data suggest that RNAPII stalled at a DNA lesion triggers a coordinated rescue mechanism that requires the Rad26-Def1 complex, and that Def1 enables ubiquitination and proteolysis of RNAPII when the lesion cannot be rapidly removed by Rad26-promoted DNA repair. J Immunol, 2002 Mar 1, 168(5), 2348 - 55 Transient association of Ku with nuclear substrates characterized using fluorescence photobleaching; Rodgers W et al.; The autoantigen Ku, composed of subunits Ku70 and Ku86, is necessary for repair of DNA double-strand breaks by nonhomologous end joining . Similarly, Ku participates in repair of DNA double-strand breaks that occur during V(D)J recombination, and it is therefore required for the development of B and T lymphocytes . Although previous studies have identified the DNA-binding activities of Ku, little is known concerning its dynamics, such as the mobility of Ku in the nucleus and its rate of association with substrates . To address this question, fluorescence photobleaching experiments were performed using HeLa cells and B cells expressing a green fluorescent protein (GFP) fusion construct of either Ku70 or Ku86 . The results show that Ku moves rapidly throughout the nucleus even following irradiation of the cells . However, the rate of diffusion of Ku was approximately 100-fold slower than that predicted from its size . Association of Ku-GFP with a filamentous nuclear structure was also evident, and nuclear extraction experiments suggest that this represents nuclear matrix . A central domain of Ku70 containing its DNA-binding and heterodimerization regions and its nuclear localization signal shows that this alone is sufficient for the observed mobility of Ku70-GFP and its association with nuclear matrix . These data suggest the mobility of Ku is characterized by a transient, high flux association with nuclear substrates that includes both DNA and the nuclear matrix and may represent a mechanism for repair of double-strand breaks using the nuclear matrix as a scaffold. Trends Cell Biol, 2002 Mar, 12(3), 104 - 7 A phosphorylation-driven ubiquitination switch for cell-cycle control; Harper JW; Cellular changes in state can be dictated by complex all-or-nothing switches built from ultrasensitive protein kinase cascades, positive-feedback loops and other mechanisms . Recent work has established that phosphorylation-driven protein destruction through the SCF ubiquitin-ligase pathway can also occur in a switch-like manner . In this context, multiple phosphorylation events are used to set a threshold for substrate targeting, thereby providing a framework for understanding the inter-relationship between protein phosphorylation and ubiquitin-mediated proteolysis. Protein Expr Purif, 2002 Mar, 24(2), 268 - 73 Two-step purification of mitochondrial Hsp70, Ssc1p, using Mge1(His)(6) immobilized on Ni-agarose; Weiss C et al.; The most abundant mitochondrial homolog of Hsp70, Ssc1p, is involved in the import and folding of mitochondrial proteins . We have developed an easy and efficient method for purifying Ssc1p . Following a first step of anion exchange at pH 6.6, a column of Mge1(His)(6) immobilized on Ni(2+)-agarose provides an efficient second dimension that results in highly purified protein . The strong and specific interaction between Ssc1p and its cofactor protein, Mge1, ensures that primarily functional protein is isolated . Ssc1p purified by this method hydrolyzed ATP with a turnover rate of 0.3/min . The ATP hydrolysis was enhanced slightly by Mge1, about 5 times by Mdj1, and 12 times by both cofactors together . The CD spectrum of Ssc1p had a pattern and temperature dependence similar to those shown for other hsp70 homologs, with a midpoint of the major transition at approximately 70 degrees C . J Cell Physiol, 2002 Mar, 190(3), 287 - 96 Cdc7 kinase complex: a key regulator in the initiation of DNA replication; Masai H et al.; DNA replication results from the action of a staged set of highly regulated processes . Among the stages of DNA replication, initiation is the key point at which all the G1 regulatory signals culminate . Cdc7 kinase is the critical regulator for the ultimate firing of the origins of initiation . Cdc7, originally identified in budding yeast and later in higher eukaryotes, forms a complex with a Dbf4-related regulatory subunit to generate an active kinase . Genetic evidence in mammals demonstrates essential roles for Cdc7 in mammalian DNA replication . Mini-chromosome maintenance protein (MCM) is the major physiological target of Cdc7 . Genetic studies in yeasts indicate additional roles of Cdc7 in meiosis, checkpoint responses, maintenance of chromosome structures, and repair . The interplay between Cdc7 and Cdk, another kinase essential for the S phase, is also discussed . Int J Cancer, 2002 Feb 20, 97(6), 726 - 31 TCL-1, MTCP-1 and TML-1 gene expression profile in non-leukemic clonal proliferations associated with ataxia-telangiectasia; Chun HH et al.; We analyzed the role of 4 genes, TCL-1, MTCP-1, TML-1 and ATM, in the early pathogenesis of T cell leukemia, with particular interest in the characteristics of long-standing non-leukemic clonal proliferations in ataxia-telangiectasia (A-T) patients . Five patients were studied: 4 patients had A-T (2 of whom had non-leukemic clonal proliferations {ATCP}), 1 had B cell lymphoma and 1 had T-ALL; a fifth patient with T-PLL did not have A-T . We measured the levels of expression for TCL-1, MTCP-1 and TML-1 . TCL-1, not expressed in unstimulated mature T cells, was upregulated in the peripheral blood leukocytes (PBL) of the 2 A-T patients with ATCP . It was also expressed in the malignant cells of the A-T patient with B cell lymphoma and the T-PLL cells of the patient without A-T . In the same cells, MTCP-1 type A was expressed equally in all 5 patients, as well as in the controls; MTCP-1 type B transcripts were not observed . TML-1, also not expressed in unstimulated T cells, was expressed in the PBL of one A-T patient with ATCP and in the leukemic cells of the non-A-T T-PLL patient . These expression patterns were compared to cellular immunophenotypes . The non-leukemic clonal T cell populations had the characteristics of immature T cells . We conclude that TCL-1 and TML-1 play a role in cell proliferation and survival but are not pivotal genes in the progression to malignancy, even when the ATM gene is mutated . Additional genetic alterations must occur to initiate tumorigenesis . J Biol Chem, 2002 May 3, 277(18), 15452 - 8 Epub 2002 Feb 20. Characterization of dominant-negative mutants of the DEAH-box splicing factors Prp22 and Prp16; Schneider S et al.; Saccharomyces cerevisiae Prp22 and Prp16 are RNA-dependent ATPases required for pre-mRNA splicing . Both proteins are members of the DEXH-box family of nucleic acid-dependent NTPases . Prior mutational analysis of Prp22 and Prp16 identified residues within conserved motifs I (GXGKT), II (DEAH), and VI (QRXGRXGR) that are required for their biological activity . Nonfunctional Prp22 and Prp16 mutants exerted a dominant negative effect on cell growth . Here we show that overexpression of lethal Prp22 mutants leads to accumulation of unspliced pre-mRNAs and excised introns in vivo . The biochemical basis for the lethality and inhibition of splicing in vivo was determined by purifying and characterizing recombinant mutant proteins . The lethal Prp22 mutants D603A and E604A in motif II and Q804A and R808A in motif VI were defective for ATP hydrolysis and mRNA release from the spliceosome, but were active in promoting step 2 transesterification . Lethal Prp16 mutants G378A and K379A in motif I; D473A and E474A in motif II; and Q685A, G688A, R689A, and R692A in motif VI were defective for ATP hydrolysis and step 2 transesterification chemistry . The ATPase-defective mutants of Prp16 and Prp22 bound to spliceosomes in vitro and blocked the function of the respective wild-type proteins in trans . Comparing the mutational effects in Prp16 and Prp22 highlights common as well as distinct structural requirements for the ATP-dependent steps in pre-mRNA splicing. J Biol Chem, 2002 May 3, 277(18), 15426 - 31 Epub 2002 Feb 20. Suppression of androgen receptor transactivation by Pyk2 via interaction and phosphorylation of the ARA55 coregulator; Wang X et al.; The proline-rich tyrosine kinase 2 (Pyk2) was first identified as a key kinase linked to the MAP kinase and JNK signaling pathways that play important roles in cell growth and adhesion . The linkage between Pyk2 and the androgen receptor (AR), an important transcription factor in prostate cancer progression, however, remains unclear . Here we report that using the full-length androgen receptor-associated protein, ARA55, coregulator as bait, we were able to isolate an ARA55-interacting protein, Pyk2, and demonstrated that Pyk2 could repress AR transactivation via inactivation of ARA55 . This inactivation may result from the direct phosphorylation of ARA55 by Pyk2 at tyrosine 43, impairing the coactivator activity of ARA55 and/or sequestering ARA55 to reduce its interaction with AR . Our finding that Pyk2 can indirectly modulate AR function via interaction and/or phosphorylation of ARA55 not only expands the role of Pyk2 in AR-mediated prostate cancer growth but also strengthens the role of ARA55 as an AR coregulator. Eur J Biochem, 2002 Feb, 269(4), 1060 - 6 Regulation of stress-activated protein kinase signaling pathways by protein phosphatases; Tamura S et al.; Stress-activated protein kinase (SAPK) signaling plays essential roles in eliciting adequate cellular responses to stresses and proinflammatory cytokines . SAPK pathways are composed of three successive protein kinase reactions . The phosphorylation of SAPK signaling components on Ser/Thr or Thr/Tyr residues suggests the involvement of various protein phosphatases in the negative regulation of these systems . Accumulating evidence indicates that three families of protein phosphatases, namely the Ser/Thr phosphatases, the Tyr phosphatases and the dual specificity Ser/Thr/Tyr phosphatases regulate these pathways, each mediating a distinct function . Differences in substrate specificities and regulatory mechanisms for these phosphatases form the molecular basis for the complex regulation of SAPK signaling . Here we describe the properties of the protein phosphatases responsible for the regulation of SAPK signaling pathways. Biochem Biophys Res Commun, 2002 Mar 1, 291(3), 535 - 41 Structural domains of vault proteins: a role for the coiled coil domain in vault assembly; van Zon A et al.; Vaults consist of multiple copies of three proteins (MVP, VPARP, and TEP1) and several untranslated RNAs . The function of vaults is unknown but the typical and evolutionary conserved structure indicates a role in intracellular transport . Although all vault components have been identified and characterized, not much is known about vault protein assembly . In this study we identified and analyzed structural domains involved in vault assembly with emphasis on protein-protein interactions . Using a yeast two-hybrid system, we demonstrate within MVP an intramolecular binding site and show that MVP molecules interact with each other via their coiled coil domain . We show that purified MVP is able to bind calcium, most likely at calcium-binding EF-hands . No interactions could be detected between TEP1 and other vault proteins . However, the N-terminal half of MVP binds to a specific domain in the C-terminus of VPARP . Furthermore, VPARP contains amino acid stretches mediating intramolecular binding . (c)2002 Elsevier Science (USA). Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 3105 - 10 Epub 2002 Feb 19. Noninvasive quantitative imaging of protein-protein interactions in living subjects; Ray P et al.; We are developing methods to image molecular and cellular events in living subjects . In this study, we validate imaging of protein-protein interactions in living mice by using bioluminescent optical imaging . We use the well studied yeast two-hybrid system adapted for mammalian cells and modify it to be inducible . We employ the NF-kappaB promoter to drive expression of two fusion proteins (VP16-MyoD and GAL4-ID) . We modulate the NF-kappaB promoter through tumor necrosis factor alpha . Firefly luciferase reporter gene expression is driven by the interaction of MyoD and ID through a transcriptional activation strategy . We demonstrate the ability to detect this induced protein-protein interaction in cell culture and image this induced interaction in living mice by using transiently transfected cells . The current approach will be a valuable and potentially generalizable tool to noninvasively and quantitatively image protein-protein interactions in living subjects . The approaches validated should have important implications for the study of protein-protein interactions in cells maintained in their natural in vivo environment as well as for the in vivo evaluation of new pharmaceuticals targeted to modulate protein-protein interactions. Mol Biol Cell, 2002 Feb, 13(2), 646 - 55 CSN1 N-terminal-dependent activity is required for Arabidopsis development but not for Rub1/Nedd8 deconjugation of cullins: a structure-function study of CSN1 subunit of COP9 signalosome; Wang X et al.; The COP9 signalosome (CSN) is a multifunctional protein complex essential for arabidopsis development . One of its functions is to promote Rub1/Nedd8 deconjugation from the cullin subunit of the Skp1-cullin-F-box ubiquitin ligase . Little is known about the specific role of its eight subunits in deneddylation or any of the physiological functions of CSN . In the absence of CSN1 (the fus6 mutant), arabidopsis CSN complex cannot assemble, which destabilizes multiple CSN subunits and contributes, together with the loss of CSN1, to the phenotype of fus6 . To distinguish CSN1-specific functions, we attempted to rescue the complex formation with deletion or point-mutation forms of CSN1 expressed as transgenes in fus6 . We show that the central domain of CSN1 is critical for complex assembly, whereas the C-terminal domain has a supporting role . By expressing the C231 fragment, which contains the structural information but lacks the presumed functional domain located at the N terminus, we have rescued the complex formation and restored the Rub1/Nedd8 deconjugation activity on cullins (fus6/C231) . Nonetheless, fus6/C231 exhibits pleiotropic phenotype, including photomorphogenic defects and growth arrest at seedling stage . We conclude that CSN1 N-terminal domain is not required for the Rub1/Nedd8 deconjugation activity of cullins, but contributes to a significant aspect of CSN functions that are essential for plant development. Mol Biol Cell, 2002 Feb, 13(2), 435 - 44 Essential role of MCM proteins in premeiotic DNA replication; Lindner K et al.; A critical event in eukaryotic DNA replication involves association of minichromosome maintenance (MCM2-7) proteins with origins, to form prereplicative complexes (pre-RCs) that are competent for initiation . The ability of mutants defective in MCM2-7 function to complete meiosis had suggested that pre-RC components could be irrelevant to premeiotic S phase . We show here that MCM2-7 proteins bind to chromatin in fission yeast cells preparing for meiosis and during premeiotic S phase in a manner suggesting they in fact are required for DNA replication in the meiotic cycle . This is confirmed by analysis of a degron mcm4 mutant, which cannot carry out premeiotic DNA replication . Later in meiosis, Mcm4 chromatin association is blocked between meiotic nuclear divisions, presumably accounting for the absence of a second round of DNA replication . Together, these results emphasize similarity between replication mechanisms in mitotic and meiotic cell cycles. Mol Biol Cell, 2002 Feb, 13(2), 412 - 24 The eukaryotic two-component histidine kinase Sln1p regulates OCH1 via the transcription factor, Skn7p; Li S et al.; The yeast "two-component" osmotic stress phosphorelay consists of the histidine kinase, Sln1p, the phosphorelay intermediate, Ypd1p and two response regulators, Ssk1p and Skn7p, whose activities are regulated by phosphorylation of a conserved aspartyl residue in the receiver domain . Dephospho-Ssk1p leads to activation of the hyper-osmotic response (HOG) pathway, whereas phospho-Skn7p presumably leads to activation of hypo-osmotic response genes . The multifunctional Skn7 protein is important in oxidative as well as osmotic stress; however, the Skn7p receiver domain aspartate that is the phosphoacceptor in the SLN1 pathway is dispensable for oxidative stress . Like many well-characterized bacterial response regulators, Skn7p is a transcription factor . In this report we investigate the role of Skn7p in osmotic response gene activation . Our studies reveal that the Skn7p HSF-like DNA binding domain interacts with a cis-acting element identified upstream of OCH1 that is distinct from the previously defined HSE-like Skn7p binding site . Our data support a model in which Skn7p receiver domain phosphorylation affects transcriptional activation rather than DNA binding to this class of DNA binding site. J Biol Chem, 2002 May 3, 277(18), 15486 - 98 Epub 2002 Feb 19. An easily dissociated 26 S proteasome catalyzes an essential ubiquitin-mediated protein degradation pathway in Trypanosoma brucei; Li Z et al.; The 26 S proteasome, a complex between the 20 S proteasome and 19 S regulatory units, catalyzes ATP-dependent degradation of unfolded and ubiquitinated proteins in eukaryotes . We have identified previously 20 S and activated 20 S proteasomes in Trypanosoma brucei, but not 26 S proteasome . However, the presence of 26 S proteasome in T . brucei was suggested by the hydrolysis of casein by cell lysate, a process that requires ATP but is inhibited by lactacystin, and the lactacystin-sensitive turnover of ubiquitinated proteins in the intact cells . T . brucei cDNAs encoding the six proteasome ATPase homologues (Rpt) were cloned and expressed . Five of the six T . brucei Rpt cDNAs, except for Rpt2, were capable of functionally complementing the corresponding rpt deletion mutants of Saccharomyces cerevisiae . Immunoblots showed the presence in T . brucei lysate of the Rpt proteins, which co-fractionated with the yeast 19 S proteasome complex by gel filtration and localized in the 19 S fraction of a glycerol gradient . All the Rpt and putative 19 S non-ATPase (Rpn) proteins were co-immunoprecipitated from T . brucei lysate by individual anti-Rpt antibodies . Treatment of T . brucei cells with a chemical cross-linker resulted in co-immunoprecipitation of 20 S proteasome with all the Rpt and Rpn proteins that sedimented in a glycerol gradient to the position of 26 S proteasome . These data demonstrate the presence of 26 S proteasome in T . brucei cells, which apparently dissociate into 19 S and 20 S complexes upon cell lysis . RNA interference to block selectively the expression of proteasome 20 S core and Rpt subunits resulted in significant accumulation of ubiquitinated proteins accompanied by cessation of cell growth . Expression of yeast RPT2 gene in T . brucei Rpt2-deficient cells could not rescue the lethal phenotype, thus confirming the incompatibility between the two Rpt2s . The T . brucei 11 S regulator (PA26)-deficient RNA interference cells grew normally, suggesting the dispensability of activated 20 S proteasome in T . brucei. Biochim Biophys Acta, 2002 Feb 15, 1559(2), 171 - 8 Inhibition of Mrp2- and Ycf1p-mediated transport by reducing agents: evidence for GSH transport on rat Mrp2; Rebbeor JF et al.; Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions . Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles . Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity . Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles . Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and beta-mercaptoethanol, with concentrations of 10 mM inhibiting by approximately 40% . DTT's inhibition of DNP-SG transport was noncompetitive . In contrast, ATP-dependent transport of {(3)H}taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT . DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p . As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected . These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport . When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism. Cell, 2002 Feb 8, 108(3), 357 - 69 Vacuole fusion at a ring of vertex docking sites leaves membrane fragments within the organelle; Wang L et al.; Three membrane microdomains can be identified on docked vacuoles: "outside" membrane, not in contact with other vacuoles, "boundary" membrane that contacts adjacent vacuoles, and "vertices," where boundary and outside membrane meet . In living cells and in vitro, vacuole fusion occurs at vertices rather than from a central pore expanding radially . Vertex fusion leaves boundary membrane within the fused organelle and is an unexpected pathway for the formation of intralumenal membranes . Proteins that regulate docking and fusion (Vac8p, the GTPase Ypt7p, its HOPS/Vps-C effector complex, the t-SNARE Vam3p, and protein phosphatase 1) accumulate at these vertices during docking . Their vertex enrichment requires cis-SNARE complex disassembly and is thus part of the normal fusion pathway. Cell, 2002 Feb 8, 108(3), 317 - 29 Evidence that the Ipl1-Sli15 (Aurora kinase-INCENP) complex promotes chromosome bi-orientation by altering kinetochore-spindle pole connections; Tanaka TU et al.; How sister kinetochores attach to microtubules from opposite spindle poles during mitosis (bi-orientation) remains poorly understood . In yeast, the ortholog of the Aurora B-INCENP protein kinase complex (Ipl1-Sli15) may have a role in this crucial process, because it is necessary to prevent attachment of sister kinetochores to microtubules from the same spindle pole . We investigated IPL1 function in cells that cannot replicate their chromosomes but nevertheless duplicate their spindle pole bodies (SPBs) . Kinetochores detach from old SPBs and reattach to old and new SPBs with equal frequency in IPL1+ cells, but remain attached to old SPBs in ipl1 mutants . This raises the possibility that Ipl1-Sli15 facilitates bi-orientation by promoting turnover of kinetochore-SPB connections until traction of sister kinetochores toward opposite spindle poles creates tension in the surrounding chromatin. Trends Biochem Sci, 2002 Feb, 27(2), 87 - 93 Pho85 and signaling environmental conditions; Carroll AS et al.; Through its association with a family of ten cyclins, the Pho85 cyclin-dependent kinase is involved in several signal transduction pathways in the yeast Saccharomyces cerevisiae . The responses mediated by Pho85 include cell-cycle progression and metabolism of nutrients such as phosphate and carbon sources . Although these responses require the phosphorylation of different substrates, and have different mechanistic consequences as a result of this phosphorylation, all appear to be involved in responses to changes in environmental conditions . Few of the activating signals or regulated targets have been unambiguously identified, but the kinase activity of Pho85 appears to inform the cell that the current environment is satisfactory. FEBS Lett, 2002 Feb 13, 512(1-3), 173 - 9 Mai1p is essential for maturation of proaminopeptidase I but not for autophagy; Barth H et al.; We here identify Mai1p, a homologue of the autophagy protein Aut10p, as a novel component essential for proaminopeptidase I (proAPI) maturation under non-starvation conditions . In mai1Delta cells mature vacuolar proteinases are detectable and vacuolar acidification is normal . In mai1Delta cells autophagy occurs, though at a somewhat reduced level . This is indicated by proAPI maturation during starvation and accumulation of autophagic bodies during starvation with phenylmethylsulfonyl fluoride . Homozygous diploid mai1Delta cells sporulate, but with a slightly reduced frequency . Biologically active Ha-tagged Mai1p, chromosomally expressed under its native promoter, is at least in part peripherally membrane-associated . In indirect immunofluorescence it localizes to the vacuolar membrane or structures nearby . In some cells Ha-tagged Mai1p appears concentrated at regions adjacent to the nucleus. FEBS Lett, 2002 Feb 13, 512(1-3), 91 - 4 Serine (threonine) phosphatase(s) acting on cAMP-dependent phosphoproteins in mammalian mitochondria; Signorile A et al.; Immunochemical and functional evidence showing the existence in the inner membrane and matrix fraction of mammalian mitochondria of serine/threonine phosphatases acting on cAMP-dependent phosphoproteins is presented . Mg(2+)-dependent Ca(2+)-inhibitable PP2C phosphatase, associated to the inner membrane, dephosphorylates the 18 kDa (NDUFS4 gene) of complex I. Biochemistry, 2002 Feb 26, 41(8), 2774 - 85 Transcription activation by ultrabithorax Ib protein requires a predicted alpha-helical region; Tan XX et al.; Characterization of their transcription activation domains is critical to understanding functional specificity within the Hox family of proteins . However, few Hox activation domains have been identified and none characterized in detail . In this study, promotor-reporter assays in yeast and Drosophila S2 cell culture were used to refine the boundaries of the activation domain of the Drosophila Hox protein Ultrabithorax (Ubx) and to identify critical elements within this domain . We found that residues 159-242 were sufficient for 50% function, and full transactivation capacity was achieved with inclusion of additional N-terminal sequences . Activation domain sequence and placement relative to the homeodomain differ between Ubx and other Hox proteins, consistent with the possibility that diverse activation mechanisms contribute to functional distinctions in vivo . The essential residues 159-242 in the UbxIb activation domain are predicted to contain a beta-sheet segment followed by an alpha-helix . This putative alpha-helical region was established to be necessary, but not sufficient, for transcriptional activation . Disruption of the helix by proline substitutions abolished activation function, while alteration of side chains presented on the surface of this putative helix with alanine or lysine mutations had no significant effect on activity . Collectively, these data indicate that this secondary structural element is a key component in forming an effective activation domain in the UbxIb protein . Interestingly, the alpha-helix critical for transcriptional activation is found only for Ubx orthologs from flies and not other species . The mutant Ubx proteins generated in this study have potential applications in deciphering Hox functions in vivo. J Mol Biol, 2002 Feb 15, 316(2), 247 - 56 Engineered improvements in DNA-binding function of the MATa1 homeodomain reveal structural changes involved in combinatorial control; Hart B et al.; We have engineered enhanced DNA-binding function into the a1 homeodomain by making changes in a loop distant from the DNA-binding surface . Comparison of the free and bound a1 structures suggested a mechanism linking van der Waals stacking changes in this loop to the ordering of a final turn in the DNA-binding helix of a1 . Inspection of the protein sequence revealed striking differences in amino acid identity at positions 24 and 25 compared to related homeodomain proteins . These positions lie in the loop connecting helix-1 and helix-2, which is involved in heterodimerization with the alpha 2 protein . A series of single and double amino acid substitutions (a1-Q24R, a1-S25Y, a1-S25F and a1-Q24R/S25Y) were engineered, expressed and purified for biochemical and biophysical study . Calorimetric measurements and HSQC NMR spectra confirm that the engineered variants are folded and are equally or more stable than the wild-type a1 homeodomain . NMR analysis of a1-Q24R/S25Y demonstrates that the DNA recognition helix (helix-3) is extended by at least one turn as a result of the changes in the loop connecting helix-1 and helix-2 . As shown by EMSA, the engineered variants bind DNA with enhanced affinity (16-fold) in the absence of the alpha 2 cofactor and the variant alpha 2/a1 heterodimers bind cognate DNA with specificity and affinity reflective of the enhanced a1 binding affinity . Importantly, in vivo assays demonstrate that the a1-Q24R/S25Y protein binds with fivefold greater affinity than wild-type a1 and is able to partially suppress defects in repression by alpha 2 mutants . As a result of these studies, we show how subtle differences in residues at a surface distant from the functional site code for a conformational switch that allows the a1 homeodomain to become active in DNA binding in association with its cofactor alpha 2. Oncogene, 2002 Jan 21, 21(4), 532 - 40 Protection of mammalian telomeres; de Lange T; Telomeres allow cells to distinguish natural chromosome ends from damaged DNA . When telomere function is disrupted, a potentially lethal DNA damage response can ensue, DNA repair activities threaten the integrity of chromosome ends, and extensive genome instability can arise . It is not clear exactly how the structure of telomere ends differs from sites of DNA damage and how telomeres protect chromosome ends from DNA repair activities . What are the defining structural features of telomeres and through which mechanisms do they ensure chromosome end protection? What is the molecular basis of the telomeric cap and how does it act to sequester the chromosome end? Here I discuss data gathered in the last few years, suggesting that the protection of human chromosome ends primarily depends on the telomeric protein TRF2 and that telomere capping involves the formation of a higher order structure, the telomeric loop or t-loop. J Biol Chem, 2002 Apr 26, 277(17), 14688 - 94 Epub 2002 Feb 15. Phosphorylation positively regulates DNA binding of the carbon catabolite repressor Cre1 of Hypocrea jecorina (Trichoderma reesei); Cziferszky A et al.; Cre1 of the ascomycete Hypocrea jecorina is a Cys(2)His(2) zinc finger DNA-binding protein functioning as regulator for carbon catabolite repression . It represents the functional equivalent of yeast Mig1, known to be negatively regulated by the Snf1-kinase at the nuclear import level . We demonstrate that Cre1 is also a phosphoprotein, and identify Ser(241) within an acidic protein region as phosphorylation target . In contrast to Mig1 phosphorylation is required for DNA binding of Cre1 . A S241E mutation mimics phosphorylation, whereas a S241A mutant protein shows phosphorylation-independent DNA binding activity, suggesting that phosphorylation is required to release Cre1 from an inactive conformation involving unphosphorylated Ser(241) . Retransformation of a H . jecorina cre1-non functional mutant with Cre1-S241A leads to permanent carbon catabolite repression in cellobiohydrolase I expression . Contrary to Mig1, the amino acid sequence surrounding Ser(241) (HSNDEDD) suggests that phosphorylation may occur by a casein kinase II-like protein . This is supported by a mutation of E244V leading to loss of phosphorylation, loss of DNA binding, and gain of carbon catabolite derepression . Our results imply that the regulation of carbon catabolite repression at the level of DNA binding strongly differs between Saccharomyces cerevisiae and H . jecorina. Genes Dev, 2002 Feb 15, 16(4), 503 - 17 fzr-1 and lin-35/Rb function redundantly to control cell proliferation in C . elegans as revealed by a nonbiased synthetic screen; Fay DS et al.; We report here a synthetic-lethal screen in Caenorhabditis elegans that overcomes a number of obstacles associated with the analysis of functionally redundant genes . Using this approach, we have identified mutations that synthetically interact with lin-35/Rb, a SynMuv gene and the sole member of the Rb/pocket protein family in C . elegans . Unlike the original SynMuv screens, our approach is completely nonbiased and can theoretically be applied to any situation in which a mutation fails to produce a detectable phenotype . From this screen we have identified fzr-1, a gene that synthetically interacts with lin-35 to produce global defects in cell proliferation control . fzr-1 encodes the C . elegans homolog of Cdh1/Hct1/FZR, a gene product shown in other systems to regulate the APC cyclosome . We have also uncovered genetic interactions between fzr-1 and a subset of class B SynMuv genes, and between lin-35 and the putative SCF regulator lin-23 . We propose that lin-35, fzr-1, and lin-23 function redundantly to control cell cycle progression through the regulation of cyclin levels. Antimicrob Agents Chemother, 2002 Mar, 46(3), 615 - 24 Molecular evaluation of the plasma membrane proton pump from Aspergillus fumigatus; Burghoorn HP et al.; The gene encoding the plasma membrane proton pump (H+ -ATPase) of Aspergillus fumigatus, PMA1, was characterized from A . fumigatus strain NIH 5233 and clinical isolate H11-20 . An open reading frame of 3,109 nucleotides with two introns near the N terminus predicts a protein consisting of 989 amino acids with a molecular mass of approximately 108 kDa . The predicted A . fumigatus enzyme is 89 and 51% identical to H+ - ATPases of Aspergillus nidulans and Saccharomyces cerevisiae, respectively . The A . fumigatus PMA1 is a typical member of the P-type ATPase family that contains 10 predicted transmembrane segments and conserved sequence motifs TGES, CSDKTGT, MLTGD, and GDGVN within the catalytic region . The enzyme represents 2% of the total plasma membrane protein, and it is characteristically inhibited by orthovanadate, with a 50% inhibitory concentration of approximately 1.8 microM . H+ -ATPases from Aspergillus spp . contain a highly acidic insertion region of 60 amino acids between transmembrane segments 2 and 3, which was confirmed for the membrane-assembled enzyme with a peptide-derived antibody . An increasing A . fumigatus PMA1 copy number confers enhanced growth in low-pH medium, consistent with its role as a proton pump . These results provide support for the development of the A . fumigatus H+ -ATPase as a potential drug discovery target. J Steroid Biochem Mol Biol, 2001 Dec, 79(1-5), 11 - 7 Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17beta-estradiol or a peroxisome proliferator; Toda K et al.; We generated aromatase gene knockout mice (ArKO mice) by targeting disruption of Cyp19, which encodes an enzyme responsible for conversion of androgens to estrogens . We found that ArKO males developed hepatic steatosis spontaneously with aging, indicating that the function of Cyp19 is required to maintain constitutive lipid metabolism in male mice . Plasma lipoprotein analysis using a gel permeation chromatography revealed that high density lipoprotein (HDL)-cholesterol levels were slightly higher in ArKO males than in wild-type males, whereas no other obvious alternations in the profiles were detected . Nevertheless, analysis of lipoprotein compositions by SDS-polyacrylamide gel electrophoresis demonstrated apparent reduction in the amounts of apolipoprotein E, functioning in receptor-mediated clearance of lipoproteins in the liver, in the IDL/LDL fraction of ArKO males as compared with that of wild-type males . Biochemical analysis on the ArKO livers revealed suppression of mRNA expression and activity of enzymes involved in fatty acid beta-oxidation . The impairment was reversed to the wild-type levels by treatment with 17beta-estradiol or bezafibrate, the latter is a synthetic peroxisome proliferator . These findings indicated a pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in fatty acid beta-oxidation and in maintaining lipid homeostasis. Mech Dev, 2002 Mar, 112(1-2), 9 - 14 A quantitative analysis of the kinetics of Gal4 activator and effector gene expression in the zebrafish; Scheer N et al.; Using a temperature-inducible hsp70:Gal4 activator and UAS:myc-notch1a-intra as effector, we determined quantitatively the kinetics of expression of both transgenes and analysed the effects of varying their expressivity on several phenotypic traits in the developing zebrafish . hsp70:Gal4 is transcribed within 15 min after temperature-mediated induction, but Gal4 RNA decays rapidly . The Gal4 protein was found to be quite stable, as functional Gal4, which was detectable 1.5 h after heat shock (HS), persisted for at least 13 h . myc-notch1a-intra RNA is expressed approximately 1.5 h after HS, but unlike the Gal4 RNA, it was found to be very stable; it continues to accumulate during the succeeding 17 h after HS . Fully penetrant phenotypic effects are obtained after a relatively long activator induction with a 30-min HS. Expert Opin Biol Ther, 2002 Feb, 2(2), 211 - 8 Becaplermin: recombinant platelet derived growth factor, a new treatment for healing diabetic foot ulcers; Nagai MK et al.; Chronic or non-healing lower extremity ulcerations in diabetics are a significant cause of morbidity and mortality, and account for a large proportion of the financial burden related to the care of diabetics . Lower extremity ulcerations develop primarily as a consequence of neuropathy and the goal in addressing any wound is to re-establish tissue integrity as soon as possible . The healing of wounds is a complex procedure involving multiple growth factors, some of which have multiple effects on different cell types, in particular, platelet derived growth factor (PDGF) is a prominent agent, active in all stages of the healing process . Becaplermin (0.01% Regranex gel) is a homodimeric protein produced by recombinant DNA technology through the insertion of the gene for the B chain PDGF into the yeast Saccharomyces cerevisiae . The biological activity of becaplermin is similar to that of indigenous PDGF-BB, specifically, the promotion of chemotactic recruitment and the proliferation of cells involved in wound repair . Becaplermin has undergone extensive animal and human studies, demonstrating that it is highly effective as an adjunctive measure for the healing of ulcerations in the feet of diabetics when used in conjunction with standard wound healing practices . Specifically these practices include the provision of a moist environment free of debris and necrotic tissue, control of infection and optimal weight displacement from the affected area . Becaplermin is safe and easy to use, being applied once-daily and at present, becaplermin is the only growth factor licensed for use in wound healing. Fungal Genet Biol, 2002 Mar, 35(2), 115 - 34 Comparative analysis of sequences expressed during the liquid-cultured mycelia and fruit body stages of Pleurotus ostreatus; Lee SH et al.; To characterize genes involved in fruit body development, two complementary DNA (cDNA) libraries were constructed from RNA isolated from liquid-cultured mycelia and fruit bodies of Pleurotus ostreatus . Using single-pass sequencing of cDNA clones, 952 and 1069 expressed sequence tags (ESTs) were generated from liquid-cultured mycelia and fruit body cDNA library, respectively . A BLASTX search revealed that 390 of the liquid-cultured mycelia ESTs (41%) and 531 of the fruit body ESTs (50%) showed significant similarity to protein sequences described in the nonredudant database (E values < or =1 x 10(-5)) . When liquid-cultured mycelia and fruit body ESTs were compared by the SeqMan II program, among the total of 2021 ESTs, 1256 ESTs were unigenes, and 66 unigenes (5.3%) were commonly expressed during both stages . The functional catalogs of the ESTs were made by comparison with functionally identified Saccharomyces cerevisiae genes . Liquid-cultured mycelium ESTs were compared with fruit body ESTs and changes of the expressed genes during fruit body development were analyzed. Biotechniques, 2002 Feb, 32(2), 356 - 8, 360, 362, 364 Analysis of microliter volumes of dye-labeled nucleic acids; Nagai T et al.; We describe methodology useful for determining the fluorescent activity of small volumes (e.g., 1 microL) of dye-labeled cDNAs using microabsorbance spectroscopy . A direct relationship was found between the average fluorescent hybridization signal from an array and the fluorescent activity of the cDNA used in the hybridization . The microabsorbance method makes possible the screening of small-volume samples before hybridization, allowing for a more efficient use of available microarrays . The standardization of the concentration of RNA samples contained in 1 microL before reverse transcription is also described. Environ Toxicol, 2002 Feb, 17(1), 14 - 23 Structure-activity relationships for gene activation oestrogenicity: evaluation of a diverse set of aromatic chemicals; Schultz TW et al.; Structure-activity relationships for oestrogenicity were developed based on 120 aromatic chemicals evaluated in the Saccharomyces cerevisiae-based Lac-Z reporter assay . Relative gene activation was compared to 17 beta-estradiol and varied over eight orders of magnitude . Analysis of the data compared to 17 beta-estradiol identified three structural criteria that were related to xenoestrogen activity and potency: (1) the hydrogen-bonding ability of the phenolic ring mimicking the A-ring, (2) a hydrophobic centre similar in size and shape to the B- and C-rings, and (3) a hydrogen-bond donor mimicking the 17 beta-hydroxyl moiety of the D-ring, especially with an oxygen-to-oxygen distance similar to that between the 3- and 17 beta-hydroxyl groups of 17 beta-estradiol . Binding data were segregated into activity clusters including strong, moderate, weak, and detectable gene expression, and those compounds that were inactive . The hydrogen-bonding ability of hydroxy group in the 3-position on 17 beta-estradiol was observed to be essential for gene activation . Compounds with a 4-hydroxyl substituted benzene ring and a hydrophobic moiety of size and shape equivalent to the B-ring of 17 beta-estradiol were generally observed to be weakly active compounds . Moderately active compounds have a 4-hydroxyl substituted benzene ring with a hydrophobic moiety equivalent in size and shape to the B- and C-ring of 17 beta-estradiol, or have a high hydrogen-bond donor capacity owing to the presence of halogens on a nonphenolic ring . Strongly active compounds, similar to 4,4'-diethylethylene bisphenol (DES), possess the same hydrophobic ring structure as described for moderately active compounds and an additional hydroxyl group with an oxygen-to-oxygen distance close to that exhibited by the 3- and 17-hydroxyl groups of 17 beta-estradiol. Science, 2002 Mar 15, 295(5562), 2073 - 6 Epub 2002 Feb 14. The RAR1 interactor SGT1, an essential component of R gene-triggered disease resistance; Azevedo C et al.; Plant disease resistance (R) genes trigger innate immune responses upon pathogen attack . RAR1 is an early convergence point in a signaling pathway engaged by multiple R genes . Here, we show that RAR1 interacts with plant orthologs of the yeast protein SGT1, an essential regulator in the cell cycle . Silencing the barley gene Sgt1 reveals its role in R gene-triggered, Rar1-dependent disease resistance . SGT1 associates with SKP1 and CUL1, subunits of the SCF (Skp1-Cullin-F-box) ubiquitin ligase complex . Furthermore, the RAR1-SGT1 complex also interacts with two COP9 signalosome components . The interactions among RAR1, SGT1, SCF, and signalosome subunits indicate a link between disease resistance and ubiquitination. J Exp Bot, 2002 Mar, 53(368), 439 - 45 Modulation of cyanoalanine synthase and O-acetylserine (thiol) lyases A and B activity by beta-substituted alanyl and anion inhibitors; Warrilow AG et al.; The reaction mechanisms of three enzymes belonging to a single gene family are compared: a cyanoalanine synthase and two isoforms of O-acetylserine (thiol) lyase (O-ASTL) isolated from spinach (Spinacea oleracea L . cv . Medina) . O-ASTL represents a major regulatory point in the S-assimilatory pathway, and the related cyanoalanine synthase, which is specific to the mitochondrial compartment, has evolved an independent function of cyanide detoxification . All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions although with different efficiencies, and which may be explained by a single amino acid substitution in the substrate-binding pocket of the enzyme . Substituted alanine and nucleophillic inhibitors caused predominantly non-competitive inhibition, indicating binding to both E- and F-forms of the enzyme in a bi-bi ping-pong kinetic model . Michaelis-Menten kinetics were observed when the alanyl substrate was varied in the presence and absence of inhibitors . The use of alanyl inhibitors has shown that the alanyl half-cycle of both the cysteine synthesis and cyanoalanine synthesis reactions of cyanoalanine synthase and O-acetylserine (thiol) lyases are similar . This is in contrast to the results observed with nucleophillic inhibitors, which have shown that the mechanisms of anion binding and processing differ between cyanoalanine synthase and O-ASTLs. J Biol Chem, 2002 Apr 26, 277(17), 14629 - 34 Epub 2002 Feb 14. Functional overlap between retinitis pigmentosa 2 protein and the tubulin-specific chaperone cofactor C; Bartolini F et al.; Mutations in the X-linked retinitis pigmentosa 2 gene cause progressive degeneration of photoreceptor cells . The retinitis pigmentosa 2 protein (RP2) is similar in sequence to the tubulin-specific chaperone cofactor C . Together with cofactors D and E, cofactor C stimulates the GTPase activity of native tubulin, a reaction regulated by ADP-ribosylation factor-like 2 protein . Here we show that in the presence of cofactor D, RP2 protein also stimulates the GTPase activity of tubulin . We find that this function is abolished by mutation in an arginine residue that is conserved in both cofactor C and RP2 . Notably, mutations that alter this arginine codon cause familial retinitis pigmentosa . Our data imply that this residue acts as an "arginine finger" to trigger the tubulin GTPase activity and suggest that loss of this function in RP2 contributes to retinal degeneration . We also show that in Saccharomyces cerevisiae, both cofactor C and RP2 partially complement the microtubule phenotype resulting from deletion of the cofactor C homolog, demonstrating their functional overlap in vivo . Finally, we find that RP2 interacts with GTP-bound ADP ribosylation factor-like 3 protein, providing a link between RP2 and several retinal-specific proteins, mutations in which also cause retinitis pigmentosa. EMBO J, 2002 Feb 15, 21(4), 615 - 21 Role of the ubiquitin-selective CDC48(UFD1/NPL4 )chaperone (segregase) in ERAD of OLE1 and other substrates; Braun S et al.; The OLE pathway of yeast regulates the abundance of the ER-bound enzyme Delta-9 fatty acid desaturase OLE1, thereby controlling unsaturated fatty acid pools and membrane fluidity . Previously, we showed that this pathway is exquisitely regulated by the ubiquitin/proteasome system . Activation of the pathway involves proteasomal processing of a membrane-bound transcription factor and the subsequent mobilization of the cleaved, ubiquitylated transcription factor from its partner molecule by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone-like enzyme . Here we report that the OLE1 protein itself is naturally short-lived and is degraded by ubiquitin/proteasome-dependent ER-associated degradation (ERAD) . We found that CDC48(UFD1/NPL4) plays a second role in the OLE pathway by mediating ERAD of OLE1 . Intriguingly, other ERAD substrates also require CDC48(UFD1/NPL4) for degradation, indicating that this enzyme is a novel, constitutive component of the ERAD machinery . We propose that CDC48(UFD1/NPL4) functions as a segregase that liberates ubiquitylated proteins from non-modified partners. J Interferon Cytokine Res, 2002 Jan, 22(1), 59 - 71 On the role of IRF in host defense; Barnes B et al.; Transcription factors of the interferon (IFN) regulatory factor (IRF) family have been shown to play an essential role in the regulated expression of type I IFN genes, IFN-stimulated genes (ISG), and other cytokines and chemokines . Three members of the IRF family, IRF-3, IRF-5, and IRF-7, have been identified as acting as direct transducers of virus-mediated signaling . In infected cells, these factors are activated by phosphorylation on the serine residues, transported to the nucleus, where they bind to the promoters of IFNA and IFNB genes and tether histone transacetylases to the transcription complex enhanceosome . IFNB and IFNA subtypes are expressed at different levels in infected cells . The ratio between the relative levels of IRF-3 and IRF-7 was shown to play an essential role in the inducible expression of type I IFN genes, whereas IRF-3 alone is sufficient for expression of the IFNB gene . IRF-5 was identified recently as another inducer of IFNA genes, which has two unique properties: (1) its activation is virus specific, and (2) the profile of IFNA genes induced by IRF-5 is distinct from that induced by IRF-7 . Several viruses target functions of IRF to eliminate the early inflammatory response . Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of four genes with homology to cellular IRF . Three of these vIRF were shown to inhibit induction of IFN genes and ISG in infected cells and function as dominant negative mutants of cellular IRF . The unique properties of previously uncharacterized vIRF-2 and vIRF-3 are discussed. Plant J, 2002 Feb, 29(4), 475 - 86 Selenate-resistant mutants of Arabidopsis thaliana identify Sultr1;2, a sulfate transporter required for efficient transport of sulfate into roots; Shibagaki N et al.; To investigate how plants acquire and assimilate sulfur from their environment, we isolated and characterized two mutants of Arabidopsis thaliana deficient in sulfate transport . The mutants are resistant to selenate, a toxic analogue of sulfate . They are allelic to each other and to the previously isolated sel1 (selenate-resistant) mutants, and have been designated sel1-8 and sel1-9 . Root elongation in these mutants is less sensitive to selenate than in wild-type plants . Sulfate uptake into the roots is impaired in the mutants under both sulfur-sufficient and sulfur-deficient conditions, but transport of sulfate to the shoot is not affected . The sel1 mutants contain lesions in the sulfate transporter gene Sultr1;2 located on the lower arm of chromosome 1 . The sel1-1, sel1-3 and sel1-8 mutants contain point mutations in the coding sequences of Sultr1;2, while the sel1-9 mutant has a T-DNA insertion in the Sultr1;2 promoter . The Sultr1;2 cDNA derived from wild-type plants is able to complement Saccharomyces cerevisiae mutants defective in sulfate transport, but the Sultr1;2 cDNA from sel1-8 is not . The Sultr1;2 gene is expressed mainly in roots, and accumulation of transcripts increases during sulfate deprivation . Examination of transgenic plants containing the Sultr1;2 promoter fused to the GUS-reporter gene indicates that Sultr1;2 is expressed mainly in the root cortex, the root tip and lateral roots . Weaker expression of the reporter gene was observed in hydathodes, guard cells and auxiliary buds of leaves, and in anthers and the basal parts of flowers . The results indicate that Sultr1;2 is primarily involved in importing sulfate from the environment into the root. Pflugers Arch, 2001, 443 Suppl 1, S92 - 6 Epub 2001 Jul 13. Regulation of the CFTR channel by phosphorylation; Dahan D et al.; Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are regulated tightly by protein kinases and phosphatases . The regulatory domain of CFTR has about 20 potential sites for phosphorylation by protein kinases A (PKA) and C (PKC) . The reason for this large number of sites is not known, however their conservation from fish to humans implies that they play important roles in vivo . PKA is an important activator, and its stimulation of CFTR is enhanced by PKC via mechanisms which are not fully understood . The physiological stimuli of CFTR are not known for some epithelia, and it appears likely that other serine/threonine and even tyrosine kinases also regulate CFTR in particular tissues . Phosphatases that deactivate CFTR have yet to be identified definitively at the molecular level, however CFTR is regulated by a membrane-bound form of protein phosphatase-2C (PP2C) in several cell types . Patch-clamp studies of channel rundown, co-immunoprecipitation, chemical cross-linking studies, and pull-down assays all indicate that CFTR and PP2C are closely associated within a stable regulatory complex . Understanding the regulation of CFTR by PP2C is a priority due to its potential as a target for pharmacotherapies in the treatment of cystic fibrosis. Cell Mol Biol (Noisy-le-grand), 2001 Nov, 47(7), 1149 - 62 The novel BLM3 gene encodes a protein that protects against lethal effects of oxidative damage; Febres DE et al.; Mutational alteration of the BLM3 gene in Saccharomyces cerevisiae confers hypersensitivities to lethal effects of ionizing radiation, anticancer bleomycins and structurally-related phleomycins . Bleomycin is used clinically in the treatment of many types of cancers, including Kaposi's sarcoma . The BLM3 gene was cloned from a genomic library by complementing the drug hypersensitivities conferred by the codominant blm3-1 mutation . The nucleotide sequence of BLM3 encodes a predicted integral protein of 1804 amino acids with seven to ten potential transmembrane domains and additional motifs . The blm3 null mutation was created by gene replacement, and found not to be essential for growth in the absence of the bleomycin-phleomycin antibiotics . Sequence analyses suggest the Blm3p could be a potential member of the major facilitator superfamily (MFS) of permeases . Northern dot blot analyses using a human RNA master tissue blot containing RNA from fifty different fetal and adult tissues revealed sequence homology in adult tissues to BLM3, but no sequence homology in fetal tissues . The function of the Blm3p is presently unknown . We propose several functions for the Blm3p in protecting cells against oxidative agents, including roles in detoxification, transport and defending against DNA damage. J Gen Virol, 2002 Mar, 83(Pt 3), 517 - 24 Analysis of DNA binding by the adenovirus type 5 E1A oncoprotein; Avvakumov N et al.; Adenovirus type 5 E1A proteins interact with cellular regulators of transcription to reprogram gene expression in the infected or transformed cell . Although E1A also interacts with DNA directly in vitro, it is not clear how this relates to its function in vivo . The N-terminal conserved regions 1, 2 and 3 and the C-terminal portions of E1A were prepared as purified recombinant proteins and analyses showed that only the C-terminal region bound DNA in vitro . Deletion of E1A amino acids 201-220 inhibited binding and a minimal fragment encompassing amino acids 201-218 of E1A was sufficient for binding single- and double-stranded DNA . This portion of E1A also bound the cation-exchange resins cellulose phosphate and carboxymethyl Sepharose . As this region contains six basic amino acids, in vitro binding of E1A to DNA probably results from an ionic interaction with the phosphodiester backbone of DNA . Studies in Saccharomyces cerevisiae have shown that expression of a strong transcriptional activation domain fused to a DNA-binding domain can inhibit growth . Although fusion of the C-terminal region of E1A to a strong transcriptional activation domain inhibited growth when expressed in yeast, this was not mediated by the DNA-binding domain identified in vitro . These data suggest that E1A does not bind DNA in vivo. Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 2240 - 5 Epub 2002 Feb 12. WASp verprolin homology, cofilin homology, and acidic region domain-mediated actin polymerization is required for T cell development; Zhang J et al.; All members of the Wiskott-Aldrich syndrome protein (WASp) family contain a carboxyl-terminal verprolin homology, cofilin homology, and acidic region (VCA) domain that binds and activates the Arp2/3 complex, thereby linking these proteins to the induction of actin polymerization . Although the VCA domain imbues WASp and other WASp family members with the capacity to modulate cytoskeletal organization, little is known about the impact of this domain activity on lymphoid cell function . Here we demonstrate that T cell-restricted expression of VCA domain-deleted WASp (WASpdeltaVCA) in WAS(-/-) mice engenders a severe early block in T lymphopoiesis associated with impaired T cell antigen receptor alphabeta expression and a consequent failure to generate single-positive CD4(+) and CD8(+) T cells . These latter defects, which are not observed in WAS(-/-) mice, are associated with impaired induction of cellular actin polymerization and a failure in the terminal differentiation of double-negative thymocytes . These findings indicate that WASp family proteins play an essential role in modulating the signaling events required for early thymocyte development and reveal their capacity to subserve this role to depend on VCA domain-mediated actin polymerization. Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 1990 - 5 Epub 2002 Feb 12. Arsenite-induced Cdc25C degradation is through the KEN-box and ubiquitin-proteasome pathway; Chen F et al.; Arsenite is a known human carcinogen that induces tumorigenesis through either a genotoxic or an epigenetic mechanism . In this study, the effect of arsenite on cell cycle regulation and the mechanisms that contribute to this effect were investigated . Treatment of the cells with arsenite suppressed cell proliferation and reduced cell viability in a dose- or time-dependent manner . Analysis of cell cycle profile and cell cycle regulatory proteins indicated that arsenite arrested the cell cycle at G(2)/M phase, partially through induction of cell division cycle 25 (Cdc25) isoform C (Cdc25C) degradation via ubiquitin-proteasome pathways . Mutation of the putative KEN box within the region 151 to 157 of human Cdc25C or treatment of the cells with a peptide competitor encompassing the KEN box partially inhibited arsenite-induced ubiquitination of Cdc25C . Thus, these results indicate that the regulated ubiquitination of Cdc25C may be involved in the arsenite-induced proteolytic down-regulation of Cdc25C activity in the G(2)/M phase of the cell cycle and suggest a link between cell cycle and the carcinogenic effects of arsenite. Plant Physiol, 2002 Feb, 128(2), 615 - 24 Transgenic production of epoxy fatty acids by expression of a cytochrome P450 enzyme from Euphorbia lagascae seed; Cahoon EB et al.; Seed oils of a number of Asteraceae and Euphorbiaceae species are enriched in 12-epoxyoctadeca-cis-9-enoic acid (vernolic acid), an unusual 18-carbon Delta(12)-epoxy fatty acid with potential industrial value . It has been previously demonstrated that the epoxy group of vernolic acid is synthesized by the activity of a Delta(12)-oleic acid desaturase-like enzyme in seeds of the Asteraceae Crepis palaestina and Vernonia galamensis . In contrast, results from metabolic studies have suggested the involvement of a cytochrome P450 enzyme in vernolic acid synthesis in seeds of the Euphorbiaceae species Euphorbia lagascae . To clarify the biosynthetic origin of vernolic acid in E . lagascae seed, an expressed sequence tag analysis was conducted . Among 1,006 randomly sequenced cDNAs from developing E . lagascae seeds, two identical expressed sequence tags were identified that encode a cytochrome P450 enzyme classified as CYP726A1 . Consistent with the seed-specific occurrence of vernolic acid in E . lagascae, mRNA corresponding to the CYP726A1 gene was abundant in developing seeds, but was not detected in leaves . In addition, expression of the E . lagascae CYP726A1 cDNA in Saccharomyces cerevisiae was accompanied by production of vernolic acid in cultures supplied with linoleic acid and an epoxy fatty acid tentatively identified as 12-epoxyoctadeca-9,15-dienoic acid (12-epoxy-18:2Delta(9,15)) in cultures supplied with alpha-linolenic acid . Consistent with this, expression of CYP726A1 in transgenic tobacco (Nicotiana tabacum) callus or somatic soybean (Glycine max) embryos resulted in the accumulation of vernolic acid and 12-epoxy-18:2Delta(9,15) . Overall, these results conclusively demonstrate that Asteraceae species and the Euphorbiaceae E . lagascae have evolved structurally unrelated enzymes to generate the Delta(12)-epoxy group of vernolic acid. Nucleic Acids Res, 2002 Feb 15, 30(4), 1029 - 37 Functional and physical interactions between components of the Prp19p-associated complex; Chen CH et al.; The Prp19p-associated complex is essential for the yeast pre-mRNA splicing reaction . The complex consists of at least eight protein components, but is not tightly associated with spliceosomal snRNAs . By a combination of genetic and biochemical methods we previously identified four components of this complex, Ntc25p, Ntc85p, Ntc30p and Ntc20p, all of them being novel splicing factors . We have now identified three other components of the complex, Ntc90p, Ntc77p and Ntc31p . These three proteins were also associated with the spliceosome during the splicing reaction in the same manner as Prp19p, concurrently with or immediately after dissociation of U4 snRNA . Two-hybrid analysis revealed that none of these proteins interacted with Prp19p or Ntc25p, but all interacted with Ntc85p . An interaction network between the identified components of the Prp19p-associated complex is demonstrated . Biochemical analysis revealed that Ntc90p, Ntc31p, Ntc30p and Ntc20p form a subcomplex, which, through interacting with Ntc85p and Ntc77p, can associate with Prp19p and Ntc25p to form the Prp19p-associated complex . Genetic analysis suggests that Ntc31p, Ntc30p and Ntc20p may play roles in modulating the function of Ntc90p. Chem Biol, 2002 Jan, 9(1), 49 - 61 Synthesis of calcineurin-resistant derivatives of FK506 and selection of compensatory receptors; Clemons PA et al.; We used olefin metathesis to synthesize C40 derivatives of FK506 and measured their ability, when complexed to FKBP12, to inhibit calcineurin's phosphatase activity . We identified modular dimerization domains (CABs) containing segments of the calcineurin A and B polypeptides . These CABs respond to FK506 both when overexpressed in mammalian cells and in yeast or mammalian three-hybrid assays . Using chemical genetic selection, we identified compensatory mutant CABs that respond to a calcineurin-resistant FK506 derivative at concentrations well below the response threshold for CABs containing only wild-type calcineurin sequence . These reagents provide a small molecule-protein combination orthogonal to existing dimerizer systems and may be used with existing systems to increase the complexity of induced-proximity experiments . This new use of the "bump-hole" strategy protects target cells from complications arising from the inhibition of endogenous calcineurin. Biochemistry, 2002 Feb 19, 41(7), 2184 - 90 Insertion of a reversible redox switch into a rare-cutting DNA endonuclease; Posey KL et al.; Target sites for homing endonucleases occur infrequently in complex genomes . As a consequence, these enzymes can be used in mammalian systems to introduce double-strand breaks at recognition sites inserted within defined loci to study DNA repair by homologous and nonhomologous recombination . Using homing endonucleases for gene targeting in vivo would be more feasible if temporal or spatial regulation of their enzymatic activity were possible . Here, we show that the DNA cleavage activity of the yeast PI-SceI homing endonuclease can be turned on and off using a redox switch . Two cysteine pairs (Cys-64/Cys-344 and Cys-67/Cys-365) were separately inserted into flexible DNA binding loop(s) to create disulfide bonds that lock the endonuclease into a nonproductive conformation . The cleavage activities of the reduced Cys-64/Cys-344 and Cys-67/Cys-365 variants are similar or slightly lower than that of the control protein, but the activities of the proteins in the oxidized state are decreased more than 30-fold . Modulating the activity of the proteins is easily accomplished by adding or removing the reducing agent . We show that defects in DNA binding account for the decreased DNA cleavage activities of the proteins containing disulfide bonds . Interestingly, the Cys-67/Cys-365 variant toggles between two different DNA binding conformations under reducing and oxidizing conditions, which may permit the identification of structural differences between the two states . These studies demonstrate that homing endonuclease activity can be controlled using a molecular switch. Biochemistry, 2002 Feb 19, 41(7), 2177 - 83 Structure-based design of a leucine zipper protein with new DNA contacting region; Morii T et al.; We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains the amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4 and have compared its DNA binding with GCN4 . The monomeric motif folds into a stable domain and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide . The addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4 . Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon DNA binding . CD spectral and thermodynamic studies indicate that the DNA-contacting region is folded in the presence or absence of DNA and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA . These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of basic leucine zipper proteins. Biochemistry, 2002 Feb 19, 41(7), 2120 - 9 Contribution of hydrogen bonding to protein stability estimated from isotope effects; Shi Z et al.; An unresolved issue in structural biology concerns the relative contribution of H bonds to protein stability . We use the small molecules 4-acetamidobenzoic acid and N-acetylanthranilic acid as model compounds to relate the energetic contribution from hydrogen bonds (H bonds) to the deuterium/hydrogen amide isotope effect . N-Acetylanthranilic acid models carbonyl-amide H bonds formed during protein folding; 4-acetamidobenzoic acid models the unfolded state in which the amide H bonds to water . NMR is used to measure shifts in the pK(a) of the ionizable carboxyl group when the amides of the compounds are either protonated or deuterated . From the pK(a) shift, we obtain a quantitative scale factor: SF = partial partial differential(DeltaG(HB))/partial partial differential(RT ln Phi), where DeltaG(HB) is the change in free energy of an H bond upon isotope substitution and Phi is the fractionation factor . Isotope effect data also are reported for a small globular protein, lambda repressor, using the "C(m) experiment" . The protein's isotope effect, which reports on the shape of the energy well, is converted to H-bonding free energy by applying the scale factor . We estimate that amide-related H bonds (amide-carbonyl and amide-water) contribute favorably to protein stability by approximately 30-50 kcal/mol in lambda repressor, GCN4 coiled coil, and cytochrome c but unfavorably by approximately 6 kcal/mol in ubiquitin . The results indicate that H-bond strength varies from one protein to another and presumably at different sites within the same protein. Proteomics, 2002 Feb, 2(2), 157 - 63 Stable isotope labelling in vivo as an aid to protein identification in peptide mass fingerprinting; Pratt JM et al.; Peptide mass fingerprinting (PMF) is a powerful technique for identification of proteins derived from in-gel digests by virtue of their matrix-assisted laser desorption/ionization-time of flight mass spectra . However, there are circumstances where the under-representation of peptides in the mass spectrum and the complexity of the source proteome mean that PMF is inadequate as an identification tool . In this paper, we show that identification is substantially enhanced by inclusion of composition data for a single amino acid . Labelling in vivo with a stable isotope labelled amino acid (in this paper, decadeuterated leucine) identifies the number of such amino acids in each digest fragment, and show a considerable gain in the ability of PMF to identify the parent protein . The method is tolerant to the extent of labelling, and as such, may be applicable to a range of single cell systems. Mol Cell Biol, 2002 Mar, 22(5), 1307 - 16 BAF53 forms distinct nuclear complexes and functions as a critical c-Myc-interacting nuclear cofactor for oncogenic transformation; Park J et al.; The c-Myc oncoprotein functions as a transcription factor that can transform normal cells into tumor cells, as well as playing a direct role in normal cell proliferation . The c-Myc protein transactivates cellular promoters by recruiting nuclear cofactors to chromosomal sites through an N-terminal transactivation domain . We have previously reported the identification and functional characterization of four different c-Myc cofactors: TRRAP, hGCN5, TIP49, and TIP48 . Here we present the identification and characterization of the actin-related protein BAF53 as a c-Myc-interacting nuclear cofactor that forms distinct nuclear complexes . In addition to the human SWI/SNF-related BAF complex, BAF53 forms a complex with TIP49 and TIP48 and a separate biochemically distinct complex containing TRRAP and a histone acetyltransferase which does not contain TIP60 . Using deletion mutants of BAF53, we show that BAF53 is critical for c-Myc oncogenic activity . Our results indicate that BAF53 plays a functional role in c-Myc-interacting nuclear complexes. Mol Cell Biol, 2002 Mar, 22(5), 1298 - 306 Set2 is a nucleosomal histone H3-selective methyltransferase that mediates transcriptional repression; Strahl BD et al.; Recent studies of histone methylation have yielded fundamental new insights pertaining to the role of this modification in gene activation as well as in gene silencing . While a number of methylation sites are known to occur on histones, only limited information exists regarding the relevant enzymes that mediate these methylation events . We thus sought to identify native histone methyltransferase (HMT) activities from Saccharomyces cerevisiae . Here, we describe the biochemical purification and characterization of Set2, a novel HMT that is site-specific for lysine 36 (Lys36) of the H3 tail . Using an antiserum directed against Lys36 methylation in H3, we show that Set2, via its SET domain, is responsible for methylation at this site in vivo . Tethering of Set2 to a heterologous promoter reveals that Set2 represses transcription, and part of this repression is mediated through the HMT activity of the SET domain . These results suggest that Set2 and methylation at H3 Lys36 play a role in the repression of gene transcription. Mol Cell Biol, 2002 Mar, 22(5), 1288 - 97 Kin28 is found within TFIIH and a Kin28-Ccl1-Tfb3 trimer complex with differential sensitivities to T-loop phosphorylation; Keogh MC et al.; Basal transcription factor TFIIH phosphorylates the RNA polymerase II (RNApII) carboxy-terminal domain (CTD) within the transcription initiation complex . The catalytic kinase subunit of TFIIH is a member of the cyclin-dependent kinase (Cdk) family, designated Kin28 in Saccharomyces cerevisiae and Cdk7 in higher eukaryotes . Together with TFIIH subunits cyclin H and Mat1, Cdk7 kinase is also found in a trimer complex known as Cdk activating kinase (CAK) . A yeast trimer complex has not previously been identified, although a Kin28-Ccl1 dimer called TFIIK has been isolated as a breakdown product of TFIIH . Here we show that a trimeric complex of Kin28-Ccl1-Tfb3 exists in yeast extracts . Several Kin28 point mutants that are defective in CTD phosphorylation were created . Consistent with earlier studies, these mutants have no transcriptional defect in vitro . Like other Cdks, Kin28 is activated by phosphorylation on T162 of the T loop . Kin28 T162 mutants have no growth defects alone but do demonstrate synthetic phenotypes when combined with mutant versions of the cyclin partner, Ccl1 . Surprisingly, these phosphorylation site mutants appear to destabilize the association of the cyclin subunit within the context of TFIIH but not within the trimer complex. J Cell Biol, 2002 Feb 18, 156(4), 643 - 51 Epub 2002 Feb 11. PEX11 promotes peroxisome division independently of peroxisome metabolism; Li X et al.; The PEX11 peroxisomal membrane proteins are the only factors known to promote peroxisome division in multiple species . It has been proposed that PEX11 proteins have a direct role in peroxisomal fatty acid oxidation, and that they only affect peroxisome abundance indirectly . Here we show that PEX11 proteins are unique in their ability to promote peroxisome division, and that PEX11 overexpression promotes peroxisome division in the absence of peroxisomal metabolic activity . We also observed that mouse cells lacking PEX11beta display reduced peroxisome abundance, even in the absence of peroxisomal metabolic substrates, and that PEX11beta(-/-) mice are partially deficient in two distinct peroxisomal metabolic pathways, ether lipid synthesis and very long chain fatty acid oxidation . Based on these and other observations, we propose that PEX11 proteins act directly in peroxisome division, and that their loss has indirect effects on peroxisome metabolism. Curr Opin Struct Biol, 2002 Feb, 12(1), 115 - 22 DNA double-strand break repair from head to tail; Hopfner KP et al.; DNA double-strand break repair is a complex process that requires multiple enzymatic and structural activities to rejoin or repair the broken DNA ends using one of several repair pathways . These enzymatic and structural activities include end detection, end processing and alignment of DNA ends . Recent structural and functional studies of the DNA double-strand break repair factors Mre11/Rad50, Ku70/80 and Xrcc4 show how these enzymes combine and assemble both enzymatic and structural activities in DNA double-strand break repair. Biosens Bioelectron, 2002 Mar, 17(3), 173 - 9 A novel ISFET-type biosensor based on P450 monooxygenases; Hara M et al.; We made a biosensor based on ion-sensitive field effect transistor (ISFET) using P450 monooxygenase . ISFETs are electrical devices and have been used as pH sensors . We used genetically engineered P450 monooxygenase for our research because of its high enzymatic activity . The fusion enzyme between rat CYP1A1P450 monooxygenase and yeast NADPH-cytochrome P450 oxidoreductase was expressed in yeast Saccharomyces cerevisiae strain AH22 . Yeast microsomal membranes were immobilized in an agarose layer on the ISFET . o-Deethylation of 7-ethoxycoumarin to 7-hydroxycoumarin was catalyzed by the enzyme in the presence of nicotinamide adenine dinucleotide phosphate reduced form (NADPH) . Formation of 7-hydroxycoumarin from 7-ethoxycoumarin was also measured by fluorescence . The difference of the voltage between the ISFET device and control device without enzymes showed a voltage increase along with the enzymatic reaction of P450 monooxygenases, and this voltage increase in the device was inhibited by addition of MnCl(2), an inhibitor of P450 monooxygenase . There was a positive correlation between the voltage increase in the ISFET device and the fluorescence intensity . This is the first electrochemical biosensing using P450 monooxygenases immobilized on the ISFET, and is applicable to the sensing of chlorophenol compounds. Curr Biol, 2002 Feb 5, 12(3), R105 - 7 Checkpoints: how to flag up double-strand breaks; Caspari T et al.; How checkpoint pathways recognise double-strand breaks has long been a mystery . Recent studies have found that two distinct checkpoint protein complexes associate independently with chromatin at the sites of DNA damage . Why do two distinct mechanisms recognise strand lesions, and what does this tell us about the checkpoint pathways? Bioinformatics, 2002 Jan, 18(1), 51 - 60 Linear modes of gene expression determined by independent component analysis; Liebermeister W; MOTIVATION: The expression of genes is controlled by specific combinations of cellular variables . We applied Independent Component Analysis (ICA) to gene expression data, deriving a linear model based on hidden variables, which we term 'expression modes' . The expression of each gene is a linear function of the expression modes, where, according to the ICA model, the linear influences of different modes show a minimal statistical dependence, and their distributions deviate sharply from the normal distribution . RESULTS: Studying cell cycle-related gene expression in yeast, we found that the dominant expression modes could be related to distinct biological functions, such as phases of the cell cycle or the mating response . Analysis of human lymphocytes revealed modes that were related to characteristic differences between cell types . With both data sets, the linear influences of the dominant modes showed distributions with large tails, indicating the existence of specifically up- and downregulated target genes . The expression modes and their influences can be used to visualize the samples and genes in low-dimensional spaces . A projection to expression modes helps to highlight particular biological functions, to reduce noise, and to compress the data in a biologically sensible way. Toxicology, 2002 Feb 28, 171(2-3), 137 - 46 Influence of redox-active compounds and PXR-activators on human MRP1 and MRP2 gene expression; Kauffmann HM et al.; In the present study, we investigated the inducibility of the drug conjugate transporter genes MRP1 and MRP2 by redox-active compounds such as tertiary butylated hydroquinone (tBHQ) and quercetin and by chemicals known to activate the pregnane X receptor (PXR) such as rifampicin and clotrimazol and by the metalloid compound arsenite . The human MRP2 gene was found to be inducible in HepG2 cells by rifampicin, clotrimazol, arsenite and tBHQ . As MRP1 expression is extremely low in HepG2 cells, its inducibility was studied in MCF-7 cells . However, only tBHQ and quercetin acted as inducers, but not the other compounds investigated . Reporter gene assays demonstrated that proximal promoter regions of the genes contribute to the induction by tBHQ, quercetin (MRP1) and clotrimazol (MRP2) . However, the deletion of binding sites supposed to mediate the induction process (a PXR-binding element-like sequence for the clotrimazol effect and an ARE (antioxidative response element) for the tBHQ/quercetin effect) did not result in a significant decrease in the induction factor indicating that other parts of the promoter are probably involved in the induction process . In summary, expression of both genes can be up-regulated by redox-active compounds, while the other compounds tested induced only MRP2 but not MRP1 expression. Arterioscler Thromb Vasc Biol, 2002 Feb 1, 22(2), 256 - 62 Prostacyclin synthase gene transfer modulates cyclooxygenase-2-derived prostanoid synthesis and inhibits neointimal formation in rat balloon-injured arteries; Yamada M et al.; Previous studies have shown that prostacyclin (PGI(2)) synthase (PCS) gene transfer inhibits neointimal formation in balloon-injured arteries . However, the role of each cyclooxygenase (COX) isoform in this healing mechanism remains unknown . We hypothesized that overexpression of PCS may modulate COX-2-mediated prostaglandin (PG) metabolism . That is to say, excessive PGH(2) derived from COX-2 after balloon injury may be converted into PGI(2) rather than PGE(2) or thromboxane (TX) A(2) by overexpressed PCS . We examined the expression of COX isoforms and evaluated the role of COX-2 with regard to the effects of PCS gene transfer by using 4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide (JTE-522), a selective COX-2 inhibitor . Rats were divided into 4 groups in conjunction with PCS gene transfer and JTE-522 treatment . The PCS gene (30 microg) was transfected into rat balloon-injured arteries by a lipotransfection method . JTE-522 (30 mg/kg per day) was administered for 14 days after balloon injury . Immunohistochemical analysis demonstrated marked COX-2 expression on the neointima . PCS gene transfer markedly inhibited neointimal formation, but JTE-522 reversed this beneficial effect . PCS gene transfer augmented PGI(2) production and decreased PGE(2) production without affecting TXA(2) production, but JTE-522 inhibited this increase in PGI(2) production . In conclusion, PCS gene transfer modulated COX-2-mediated prostanoid synthesis and inhibited neointimal formation after balloon injury. Dev Cell, 2002 Feb, 2(2), 195 - 206 The anaphase-promoting complex and separin are required for embryonic anterior-posterior axis formation; Rappleye CA et al.; Polarization of the one-cell C . elegans embryo establishes the animal's anterior-posterior (a-p) axis . We have identified reduction-of-function anaphase-promoting complex (APC) mutations that eliminate a-p polarity . We also demonstrate that the APC activator cdc20 is required for polarity . The APC excludes PAR-3 from the posterior cortex, allowing PAR-2 to accumulate there . The APC is also required for tight cortical association and posterior movement of the paternal pronucleus and its associated centrosome . Depletion of the protease separin, a downstream target of the APC, causes similar pronuclear and a-p polarity defects . We propose that the APC/separin pathway promotes close association of the centrosome with the cortex, which in turn excludes PAR-3 from the posterior pole early in a-p axis formation. Dev Cell, 2002 Feb, 2(2), 143 - 51 The utility of prions; Osherovich LZ et al.; Infectious, self-propagating protein aggregates (prions) as well as structurally related amyloid fibrils have traditionally been associated with neurodegenerative diseases in mammals . However, recent work in fungi indicates that prions are not simply aberrations of protein folding, but are in fact widespread, conserved, and in certain cases, apparently beneficial . Analysis of prion behavior in yeast has led to insights into the mechanisms of prion appearance and propagation as well as the effect of prions on cellular physiology and perhaps evolution . The prion-forming proteins of Saccharomyces cerevisiae are members of a larger class of Gln/Asn-rich proteins that is abundantly represented in the genomes of higher eukaryotes, raising the prospect of genetically programmed prion-like behavior in other organisms. Cell, 2002 Jan 25, 108(2), 207 - 20 Separase, polo kinase, the kinetochore protein Slk19, and Spo12 function in a network that controls Cdc14 localization during early anaphase; Stegmeier F et al.; In budding yeast, the phosphatase Cdc14, a key regulator of exit from mitosis, is released from its inhibitor Cfi1/Net1 in the nucleolus during anaphase . A signaling cascade, known as the mitotic exit network (MEN), controls this release . We have identified a regulatory network, the FEAR (Cdc fourteen early anaphase release) network that promotes Cdc14 release from the nucleolus during early anaphase . The FEAR network is comprised of the polo kinase Cdc5, the separase Esp1, the kinetochore-associated protein Slk19, and Spo12 . We also show that the FEAR network initiates Cdc14 release from Cfi1/Net1 during early anaphase, and MEN maintains Cdc14 in the released state during late anaphase . We propose that one function of Cdc14 released by the FEAR network is to stimulate MEN activity. Cell, 2002 Jan 25, 108(2), 183 - 93 The Mre11 complex is required for repair of hairpin-capped double-strand breaks and prevention of chromosome rearrangements; Lobachev KS et al.; Inverted repeats (IRs) that can form a hairpin or cruciform structure are common in the human genome and may be sources of instability . An IR involving the human Alu sequence (Alu-IR) has been studied as a model of such structures in yeast . We found that an Alu-IR is a mitotic recombination hotspot requiring MRE11/RAD50/XRS2 and SAE2 . Using a newly developed approach for mapping rare double-strand breaks (DSBs), we established that induction of recombination results from breaks that are terminated by hairpins . Failure of the mre11, rad50, xrs2, and sae2 mutants to process the hairpins blocks recombinational repair of the DSBs and leads to generation of chromosome inverted duplications . Our results suggest an additional role for the Mre11 complex in maintaining genome stability. Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1461 - 6 Complementary whole-genome technologies reveal the cellular response to proteasome inhibition by PS-341; Fleming JA et al.; Although the biochemical targets of most drugs are known, the biological consequences of their actions are typically less well understood . In this study, we have used two whole-genome technologies in Saccharomyces cerevisiae to determine the cellular impact of the proteasome inhibitor PS-341 . By combining population genomics, the screening of a comprehensive panel of bar-coded mutant strains, and transcript profiling, we have identified the genes and pathways most affected by proteasome inhibition . Many of these function in regulated protein degradation or a subset of mitotic activities . In addition, we identified Rpn4p as the transcription factor most responsible for the cell's ability to compensate for proteasome inhibition . Used together, these complementary technologies provide a general and powerful means to elucidate the cellular ramifications of drug treatment. Cancer Res, 2002 Feb 1, 62(3), 703 - 7 Molecular cloning and functional analysis of a novel cadmium-responsive proto-oncogene; Joseph P et al.; The molecular mechanisms potentially responsible for cell transformation and tumorigenesis induced by cadmium, a human carcinogen, were investigated by differential gene expression analysis of BALB/c-3T3 cells transformed with cadmium chloride (CdCl(2)) . Differential display analysis of gene expression revealed consistent overexpression of mouse translation initiation factor 3 (TIF3; GenBank accession number AF271072) in the cells transformed with CdCl(2) when compared with nontransformed cells . The predicted protein encoded by TIF3 cDNA exhibited 99% similarity to human eukaryotic initiation factor 3 p36 protein . A M(r) 36,000 protein was detected in cells transfected with an expression vector containing TIF3 cDNA . Transfection of NIH3T3 cells with an expression vector containing TIF3 cDNA resulted in overexpression of the encoded protein, and this was associated with cell transformation, as evidenced by the appearance of transformed foci exhibiting anchorage-independent growth on soft agar and tumorigenic potential in nude mice . Expression of the antisense RNA against TIF3 mRNA resulted in significant reversal of oncogenic potential of the CdCl(2)-transformed BALB/c-3T3 cells . Taken together, these findings demonstrate for the first time that the cell transformation and tumorigenesis induced by CdCl(2) are due, at least in part, to the overexpression of TIF3, a novel cadmium-responsive proto-oncogene. Biochem Biophys Res Commun, 2002 Feb 15, 291(1), 41 - 7 WICH, a novel verprolin homology domain-containing protein that functions cooperatively with N-WASP in actin-microspike formation; Kato M et al.; We describe a novel protein that contains a verprolin-homology (V) region, through which several actin-regulating proteins, including Wiskott-Aldrich syndrome protein (WASP) family members, bind directly to actin . The amino acid sequence is homologous to the sequences of WASP-interacting protein (WIP) and CR16, both of which associate with WASP and/or N-WASP, and thus these three proteins constitute a new protein family . We named the protein WICH (WIP- and CR16-homologous protein) . WICH associates strongly with N-WASP but only weakly with WASP via its C-terminal WASP-interacting (W) region . Ectopic expression of WICH induces actin-microspike formation through cooperation with N-WASP . In addition, expression of the W fragment of WICH suppresses microspike formation induced by N-WASP, indicating an essential role for WICH in N-WASP-induced microspike formation . (c)2002 Elsevier Science (USA). J Clin Invest, 2002 Feb, 109(3), 357 - 62 Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy; Arad M et al.; Mutations in PRKAG2, the gene for the gamma 2 regulatory subunit of AMP-activated protein kinase, cause cardiac hypertrophy and electrophysiologic abnormalities, particularly preexcitation (Wolff-Parkinson-White syndrome) and atrioventricular conduction block . To understand the mechanisms by which PRKAG2 defects cause disease, we defined novel mutations, characterized the associated cardiac histopathology, and studied the consequences of introducing these mutations into the yeast homologue of PRKAG2, Snf4 . Although the cardiac pathology caused by PRKAG2 mutations Arg302Gln, Thr400Asn, and Asn488Ile include myocyte enlargement and minimal interstitial fibrosis, these mutations were not associated with myocyte and myofibrillar disarray, the pathognomonic features of hypertrophic cardiomyopathy caused by sarcomere protein mutations . Instead PRKAG2 mutations caused pronounced vacuole formation within myocytes . Several lines of evidence indicated these vacuoles were filled with glycogen-associated granules . Analyses of the effects of human PRKAG2 mutations on Snf1/Snf4 kinase function demonstrated constitutive activity, which could foster glycogen accumulation . Taken together, our data indicate that PRKAG2 mutations do not cause hypertrophic cardiomyopathy but rather lead to a novel myocardial metabolic storage disease, in which hypertrophy, ventricular pre-excitation and conduction system defects coexist. Genome Res, 2002 Feb, 12(2), 309 - 15 Estimating coarse gene network structure from large-scale gene perturbation data; Wagner A; Large scale gene perturbation experiments generate information about the number of genes whose activity is directly or indirectly affected by a gene perturbation . From this information, one can numerically estimate coarse structural network features such as the total number of direct regulatory interactions and the number of isolated subnetworks in a transcriptional regulation network . Applied to the results of a large-scale gene knockout experiment in the yeast Saccharomyces cerevisiae, the results suggest that the yeast transcriptional regulatory network is very sparse, containing no more direct regulatory interactions than genes . The network comprises >100 independent subnetworks. Free Radic Biol Med, 2002 Feb 1, 32(3), 278 - 88 Regulation of mitochondrial thioredoxin peroxidase I expression by two different pathways: one dependent on cAMP and the other on heme; Monteiro G et al.; Mitochondrial isoform of thioredoxin peroxidase (mTPx I) is an antioxidant protein recently described in Saccharomyces cerevisiae . Here we characterized pathways that lead to mTPx I induction in two situations: growth in media containing low glucose concentrations and treatment with peroxides . The induction of mTPx I by growth on low glucose concentrations was dependent on cAMP and on the transcription factors Msn2p/Msn4p as demonstrated by northern blot experiments using yeast strains with deletion of MSN2 and MSN4 genes and also using a strain permeable to cAMP . mTPx I expression was also induced by peroxides in a time- and dose-dependent manner and varied with the carbon source present in the media . Deletion of HAP1 or inhibition of heme synthesis abolished induction of mTPx I by H(2)O(2) on cells which were grown in media containing glucose, indicating that Hap1p is involved in the regulation of this process . mTPx I was induced by H(2)O(2) on glycerol/ethanol-containing media, but we could not associate any transcription factor with this phenomenon . Finally, mTPx I also induced by t-butyl hydroperoxide in a Hap1p-independent manner . In conclusion, mTPx I expression is under a complex regulatory network, which involves, at least, two signaling pathways: one sensing the carbon source (which is signalized by cAMP) and the other sensing the intracellular redox state (which is signalized by heme). Mol Cell Biochem, 2001 Nov, 227(1-2), 119 - 27 Distinctive features of plant protein kinase CK2; Riera M et al.; In plants, protein kinase CK2 is involved in different processes that control many aspects of metabolism and development . In mammals and yeast the enzyme is a heterotetramer composed of two types of subunits . During years the subunit composition of the maize protein kinase CK2 enzyme has been a source of controversy . We have recently characterized the maize holoenzyme subunits . Our results show that multiple catalytic and regulatory subunits are expressed in maize and are able to specifically interact with other alpha and beta subunits suggesting a high level of heterogeneity in the typical heterotetrameric structure . Here, we summarize data available on plant CK2 enzymes, in order to clarify the distinctive features and functions of plant protein kinase CK2. Plant Cell, 2002 Jan, 14(1), 149 - 63 Arabidopsis CAP regulates the actin cytoskeleton necessary for plant cell elongation and division; Barrero RA et al.; An Arabidopsis cDNA (AtCAP1) that encodes a predicted protein of 476 amino acids highly homologous with the yeast cyclase-associated protein (CAP) was isolated . Expression of AtCAP1 in the budding yeast CAP mutant was able to rescue defects such as abnormal cell morphology and random budding pattern . The C-terminal domain, 158 amino acids of AtCAP1 possessing in vitro actin binding activity, was needed for the regulation of cytoskeleton-related defects of yeast . Transgenic plants overexpressing AtCAP1 under the regulation of a glucocorticoid-inducible promoter showed different levels of AtCAP1 accumulation related to the extent of growth abnormalities, in particular size reduction of leaves as well as petioles . Morphological alterations in leaves were attributable to decreased cell size and cell number in both epidermal and mesophyll cells . Tobacco suspension-cultured cells (Bright Yellow 2) overexpressing AtCAP1 exhibited defects in actin filaments and were unable to undergo mitosis . Furthermore, an immunoprecipitation experiment suggested that AtCAP1 interacted with actin in vivo . Therefore, AtCAP1 may play a functional role in actin cytoskeleton networking that is essential for proper cell elongation and division. Annu Rev Physiol, 2002, 64, 129 - 52 G proteins and pheromone signaling; Dohlman HG; All cells have the capacity to respond to chemical and sensory stimuli . Central to many such signaling pathways is the heterotrimeric G protein, which transmits a signal from cell surface receptors to intracellular effectors . Recent studies using the yeast Saccharomyces cerevisiae have produced important advances in our understanding of G protein activation and inactivation . This review focuses on the mechanisms by which G proteins transmit a signal from peptide pheromone receptors to the mating response in yeast and how mechanisms elucidated in yeast can provide insights to signaling events in more complex organisms. Genes Dev, 2002 Feb 1, 16(3), 363 - 76 A role for Ddc1 in signaling meiotic double-strand breaks at the pachytene checkpoint; Hong EJ et al.; The pachytene checkpoint prevents meiotic cell cycle progression in response to unrepaired recombination intermediates . We show that Ddc1 is required for the pachytene checkpoint in Saccharomyces cerevisiae . During meiotic prophase, Ddc1 localizes to chromosomes and becomes phosphorylated; these events depend on the formation and processing of double-strand breaks (DSBs) . Ddc1 colocalizes with Rad51, a DSB-repair protein, indicating that Ddc1 associates with sites of DSB repair . The Rad24 checkpoint protein interacts with Ddc1 and with recombination proteins (Sae1, Sae2, Rad57, and Msh5) in the two-hybrid protein system, suggesting that Rad24 also functions at DSB sites . Ddc1 phosphorylation and localization depend on Rad24 and Mec3, consistent with the hypothesis that Rad24 loads the Ddc1/Mec3/Rad17 complex onto chromosomes . Phosphorylation of Ddc1 depends on the meiosis-specific kinase Mek1 . In turn, Ddc1 promotes the stable association of Mek1 with chromosomes and is required for Mek1-dependent phosphorylation of the meiotic chromosomal protein Red1 . Ddc1 therefore appears to operate in a positive feedback loop that promotes Mek1 function. Biol Pharm Bull, 2002 Jan, 25(1), 48 - 52 Interaction of phytoestrogens with estrogen receptors alpha and beta (II); Morito K et al.; We investigated the estrogenic activities of isoflavone derivatives in competition binding assays with human estrogen receptor (hER) alpha or hER beta protein, and in a gene expression assay using a yeast system . Coumestrol binds as strongly as 17beta-estradiol to both hERs . Biochanin A, 5-OMe-genistein, formononetin, and tectorigenin bind well to hER beta, but significant binding to hER alpha is only observed with 5-OMe-genistein, formononetin and tectorigenin . The binding of 7-OMe-genistein and irisolidone is poor to both receptors . Among the glucosides, sissotorin binds both receptors and the binding is stronger than genistin . Coumestrol induces transcription as strongly as genistein . Tectorigenin also induces transcription with both hERs . Though biochanin A, 5-OMe-genistein, 7-OMe-genistein, irisolidone and formononetin slightly induce transcription with hER beta, they act as antagonists in the induction of transcription by 17beta-estradiol . The results show that methylation or glucosidation of isoflavones generally inhibits their phytoestrogenic activities. Biol Pharm Bull, 2002 Jan, 25(1), 145 - 6 Estrogenic activities of chemicals in diesel exhaust particles; Mori Y et al.; In a previous study, we focused on estrogenic activity of the hexane extract of diesel exhaust particles (DEP) . The extract of hexane was first fractionated to acidic, phenolic and neutral portions according to their chemical properties, of which the neutral fraction was fractionated by column chromatography on silica gel . The chemical structures of compounds in these fractions were then analyzed . It was found that the neutral fraction of the hexane extract of DEP contains dibenzothiophene derivatives, one of which, 4,6-dimethyldibenzothiophene, possesses estrogenic activity. Science, 2002 Feb 1, 295(5556), 848 - 51 Partitioning of the matrix fraction of the Golgi apparatus during mitosis in animal cells; Seemann J et al.; The Golgi apparatus is partitioned during mitosis in animal cells by a process of fragmentation, dispersal, and reassembly in each daughter cell . We fractionated the Golgi apparatus in vivo using the drug brefeldin A or a dominant-negative mutant of the Sar1p protein . After these treatments, Golgi enzymes moved back to the endoplasmic reticulum, leaving behind a matrix of Golgi structural proteins . Under these conditions, cells still entered and exited mitosis normally, and their Golgi matrix partitioned in a manner very similar to that of the complete organelle . Thus, the matrix may be the partitioning unit of the Golgi apparatus and may carry the Golgi enzyme-containing membranes into the daughter cells. EMBO J, 2002 Feb 1, 21(3), 355 - 64 Identification of a ubiquitin-protein ligase subunit within the CCR4-NOT transcription repressor complex; Albert TK et al.; The RING finger protein CNOT4 is a component of the CCR4-NOT complex . This complex is implicated in repression of RNA polymerase II transcription . Here we demonstrate that CNOT4 functions as a ubiquitin-protein ligase (E3) . We show that the unique C4C4 RING domain of CNOT4 interacts with a subset of ubiquitin-conjugating enzymes (E2s) . Using NMR spectroscopy, we detail the interaction of CNOT4 with UbcH5B and characterize RING residues that are critical for this interaction . CNOT4 acts as a potent E3 ligase in vitro . Mutations that destabilize the E2-E3 interface abolish this activity . Based on these results, we present a model of how E3 ligase function within the CCR4-NOT complex relates to transcriptional regulation. EMBO J, 2002 Feb 1, 21(3), 259 - 69 The Vtc proteins in vacuole fusion: coupling NSF activity to V(0) trans-complex formation; Muller O et al.; The fusion of cellular membranes comprises several steps; membrane attachment requires priming of SNAREs and tethering factors by Sec18p/NSF (N-ethylmaleimide sensitive factor) and LMA1 . This leads to trans-SNARE pairing, i.e . formation of SNARE complexes between apposed membranes . The yeast vacuole system has revealed two subsequent molecular events: trans-complex formation of V-ATPase proteolipid sectors (V(0)) and release of LMA1 from the membrane . We have now identified a hetero-oligomeric membrane integral complex of vacuolar transporter chaperone (Vtc) proteins integrating these events . The Vtc complex associates with the R-SNARE Nyv1p and with V(0) . Subunits Vtc1p and Vtc4p control the initial steps of fusion . They are required for Sec18p/NSF activity in SNARE priming, membrane binding of LMA1 and V(0) trans-complex formation . In contrast, subunit Vtc3p is required for the latest step, LMA1 release, but dispensible for all preceding steps, including V(0) trans-complex formation . This suggests that Vtc3p might act close to or at fusion pore opening . We propose that Vtc proteins may couple ATP-dependent NSF activity to a subset of V(0) sectors in order to activate them for V(0) trans-complex formation and/or control fusion pore opening. EMBO J, 2002 Feb 1, 21(3), 221 - 30 The ATP synthase is involved in generating mitochondrial cristae morphology; Paumard P et al.; The inner membrane of the mitochondrion folds inwards, forming the cristae . This folding allows a greater amount of membrane to be packed into the mitochondrion . The data in this study demonstrate that subunits e and g of the mitochondrial ATP synthase are involved in generating mitochondrial cristae morphology . These two subunits are non-essential components of ATP synthase and are required for the dimerization and oligomerization of ATP synthase . Mitochondria of yeast cells deficient in either subunits e or g were found to have numerous digitations and onion-like structures that correspond to an uncontrolled biogenesis and/or folding of the inner mitochondrial membrane . The present data show that there is a link between dimerization of the mitochondrial ATP synthase and cristae morphology . A model is proposed of the assembly of ATP synthase dimers, taking into account the oligomerization of the yeast enzyme and earlier data on the ultrastructure of mitochondrial cristae, which suggests that the association of ATP synthase dimers is involved in the control of the biogenesis of the inner mitochondrial membrane. Appl Environ Microbiol, 2002 Feb, 68(2), 532 - 8 Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue; Zhang Y et al.; Neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl . We found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil . To understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection . os-2 encodes a putative mitogen-activated protein (MAP) kinase homologous to HOG1 and can complement the osmosensitive phenotype of a Saccharomyces cerevisiae hog1 mutant . We sequenced three os-2 alleles and found that all of them were null with either frameshift or nonsense point mutations . An os-2 gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides . Conversely, os-2 mutants transformed with the wild-type os-2 gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides . Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in os-2 mutants . Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst . These results suggest that the hyperosmotic stress response pathway of N . crassa is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive os-2 MAP kinase pathway. Biotechnol Prog, 2002 Jan-Feb, 18(1), 116 - 23 Recovery of recombinant cutinase using detergent foam; Fernandes S et al.; Foam generated by vigorous stirring of a nonionic detergent, Triton X-114, was used for the recovery of recombinant cutinase expressed by Saccharomyces cerevisiae . The enzyme with a hydrophobic fusion tag, (Trp-Pro)(4), was recovered with a higher yield as compared to the wild-type cutinase, indicating the involvement of hydrophobic interactions in protein isolation with the foam . The influence of various factors including volume, dilution, pH, different additives, and cell concentration in the medium on enzyme recovery was investigated . Interaction of the enzyme with detergent was monitored using fluorescence spectroscopy . No significant changes in protein conformation after the isolation procedure were observed using circular dichroism. Nat Biotechnol, 2002 Feb, 20(2), 163 - 70 De novo peptide sequencing and quantitative profiling of complex protein mixtures using mass-coded abundance tagging; Cagney G et al.; Proteomic studies require efficient, robust, and practical methods of characterizing proteins present in biological samples . Here we describe an integrated strategy for systematic proteome analysis based on differential guanidination of C-terminal lysine residues on tryptic peptides followed by capillary liquid chromatography-electrospray tandem mass spectrometry . The approach, termed mass-coded abundance tagging (MCAT), facilitates the automated, large-scale, and comprehensive de novo determination of peptide sequence and relative quantitation of proteins in biological samples in a single analysis . MCAT offers marked advantages as compared with previously described methods and is simple, economic, and effective when applied to complex proteomic mixtures . MCAT is used to identify proteins, including polymorphic variants, from complex mixtures and measure variation in protein levels from diverse cell types. J Biol Chem, 2002 Mar 29, 277(13), 10756 - 9 Epub 2002 Jan 30. Binding of inositol hexakisphosphate (IP6) to Ku but not to DNA-PKcs; Ma Y et al.; The nonhomologous DNA end joining (NHEJ) pathway is responsible for repairing a major fraction of double strand DNA breaks in somatic cells of all multicellular eukaryotes . As an indispensable protein in the NHEJ pathway, Ku has been hypothesized to be the first protein to bind at the DNA ends generated at a double strand break being repaired by this pathway . When bound to a DNA end, Ku improves the affinity of another DNA end-binding protein, DNA-PK(cs), to that end . The Ku.DNA-PK(cs) complex is often termed the DNA-PK holoenzyme . It was recently shown that myo-inositol hexakisphosphate (IP(6)) stimulates the joining of complementary DNA ends in a cell free system . Moreover, the binding data suggested that IP(6) bound to DNA-PK(cs) (not to Ku) . Here we clearly show that, in fact, IP(6) associates not with DNA-PK(cs), but rather with Ku . Furthermore, the binding of DNA ends and IP(6) to Ku are independent of each other . The possible relationship between inositol phosphate metabolism and DNA repair is discussed in light of these findings. Folia Histochem Cytobiol, 2001, 39 Suppl 2, 91 - 2 Characterization of a novel protein inhibitor of protein kinases specific to acidic ribosomal proteins; Pilecki M et al.; The ribosomal stalk composed of acidic P1/P2 proteins and protein P0 is involved directly in the interaction of the elongation factors and mRNAs with the ribosome during protein synthesis . All P proteins are found to be phosphorylated in eucaryotic organisms . In Saccharomyces cerevisiae five different cAMP-independent protein kinases phosphorylating P proteins have been identified and characterized . In contrast to many other protein kinases, relatively little is known about inhibitors of these enzymes . A new protein inhibitor of protein kinases has been purified and characterized . It is a small (18.5 kDa) and acidic (pI = 4.2) protein with high inhibitory potency for PK60S and CK 2 . The inhibitor is competitive with respect to protein substrates with Ki values in the range of approximately 6.5 microM for PK60S and approximately 22 microM for CK 2. Int J Hyperthermia, 2002 Jan-Feb, 18(1), 40 - 9 Some general regularities of synergistic interaction of hyperthermia with various physical and chemical inactivating agents; Petin VG et al.; Various cellular systems have been analysed for synergistic interactions of the simultaneous action of hyperthermia with physical agents including ionizing radiation, ultraviolet light, and ultrasound, and with chemical agents including thio-TEPA and cis-DDP . Some general rules of synergistic interaction were revealed . First, for every constant rate or intensity of exposure to physical factors or concentration of chemical agents, synergy can be observed only within a certain temperature range that is different for various cellular systems . Secondly, within this range, there is a specific temperature that maximizes the synergistic effect . Any deviation of temperature from the optimal one results in a reduction in synergy . Thirdly, the rate of exposure to physical agents or the concentration of chemical agents strongly influences the synergy; i.e . as the dose rate or concentration is reduced, the temperature for maximum synergism decreases . Possible interpretations of the mechanisms responsible for these relationships of synergy are discussed. Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1206 - 11 Epub 2002 Jan 29. The regulatory role for the ERCC3 helicase of general transcription factor TFIIH during promoter escape in transcriptional activation; Fukuda A et al.; Eukaryotic transcriptional activators have been proposed to function, for the most part, by promoting the assembly of preinitiation complex through the recruitment of the RNA polymerase II transcriptional machinery to the promoter . Previous studies have shown that transcriptional activation is critically dependent on transcription factor IIH (TFIIH), which functions during promoter opening and promoter escape, the steps following preinitiation complex assembly . Here we have analyzed the role of TFIIH in transcriptional activation and show that the excision repair cross-complementing (ERCC) 3 helicase activity of TFIIH plays a regulatory role to stimulate promoter escape in activated transcription . The stimulatory effect of the ERCC3 helicase is observed until approximately 10-nt RNA is synthesized, and the helicase seems to act throughout the entire course of promoter escape . Analyses of the early phase of transcription show that a majority of the initiated complexes abort transcription and fail to escape the promoter; however, the proportion of productive complexes that escape the promoter apparently increases in response to activation . Our results establish that promoter escape is an important regulatory step stimulated by the ERCC3 helicase activity in response to activation and reveal a possible mechanism of transcriptional synergy. Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1241 - 6 Epub 2002 Jan 29. Human Elongator facilitates RNA polymerase II transcription through chromatin; Kim JH et al.; A human Elongator complex was purified from HeLa cells and found to be composed of three polypeptides . Human Elongator contains histone acetyltransferase activity with specificity to histone H3 and, to a much lesser extent, to histone H4 . Although many reports have suggested a role for the yeast Elongator in transcription elongation through chromatin templates, no direct evidence supporting this function exists . In the present study, we demonstrate that the human Elongator facilitates transcription by RNA polymerase II in a chromatin- and acetyl-CoA-dependent manner . The complex was found to directly interact with RNA polymerase II but failed to interact with other factors that facilitated RNA polymerase II to traverse through nucleosomes . From our results, we postulate that different mechanisms operate to ensure efficient transcription by RNA polymerase II on chromatin templates. Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1253 - 8 Epub 2002 Jan 29. Probing protein conformational changes in living cells by using designer binding proteins: application to the estrogen receptor; Koide A et al.; A challenge in understanding the mechanism of protein function in biology is to establish the correlation between functional form in the intracellular environment and high-resolution structures obtained with in vitro techniques . Here we present a strategy to probe conformational changes of proteins inside cells . Our method involves: (i) engineering binding proteins to different conformations of a target protein, and (ii) using them to sense changes in the surface property of the target in cells . We probed ligand-induced conformational changes of the estrogen receptor alpha (ER alpha) ligand-binding domain (LBD) . By using yeast two-hybrid techniques, we first performed combinatorial library screening of "monobodies" (small antibody mimics using the scaffold of a fibronectin type III domain) for clones that bind to ER alpha and then characterized their interactions with ER alpha in the nucleus, the native environment of ER alpha, in the presence of various ligands . A library using a highly flexible loop yielded monobodies that specifically recognize a particular ligand complex of ER alpha, and the pattern of monobody specificity was consistent with the structural differences found in known crystal structures of ER alpha-LBD . A more restrained loop library yielded clones that bind both agonist- and antagonist-bound ER alpha . Furthermore, we found that a deletion of the ER alpha F domain that is C-terminally adjacent to the LBD increased the crossreactivity of monobodies to the apo-ER alpha-LBD, suggesting a dynamic nature of the ER alpha-LBD conformation and a role of the F domain in restraining the LBD in an inactive conformation. Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1182 - 7 Epub 2002 Jan 29. Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex; Gao ZH et al.; Wnt signaling plays a key role in cell proliferation and development . Recently, casein kinase I (CKI) and protein phosphatase 2A (PP2A) have emerged as positive and negative regulators of the Wnt pathway, respectively . However, it is not clear how these two enzymes with opposing functions regulate Wnt signaling . Here we show that both CKI delta and CKI epsilon interacted directly with Dvl-1, and that CKI phosphorylated multiple components of the Wnt-regulated beta-catenin degradation complex in vitro, including Dvl-1, adenomatous polyposis coli (APC), axin, and beta-catenin . Comparison of peptide maps from in vivo and in vitro phosphorylated beta-catenin and axin suggests that CKI phosphorylates these proteins in vivo as well . CKI abrogated beta-catenin degradation in Xenopus egg extracts . Notably, CKI decreased, whereas inhibition of CKI increased, the association of PP2A with the beta-catenin degradation complex in vitro . Additionally, inhibition of CKI in vivo stabilized the beta-catenin degradation complex, suggesting that CKI actively destabilizes the complex in vivo . The ability of CKI to induce secondary body axes in Xenopus embryos was reduced by the B56 regulatory subunit of PP2A, and kinase-dead CKI epsilon acted synergistically with B56 in inhibiting Wnt signaling . The data suggest that CKI phosphorylates and destabilizes the beta-catenin degradation complex, likely through the dissociation of PP2A, providing a mechanism by which CKI stabilizes beta-catenin and propagates the Wnt signal. Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1377 - 82 Epub 2002 Jan 29. Targeted gene expression in transgenic Xenopus using the binary Gal4-UAS system; Hartley KO et al.; The transgenic technique in Xenopus allows one to misexpress genes in a temporally and spatially controlled manner . However, this system suffers from two experimental limitations . First, the restriction enzyme-mediated integration procedure relies on chromosomal damage, resulting in a percentage of embryos failing to develop normally . Second, every transgenic embryo has unique sites of integration and unique transgene copy number, resulting in variable transgene expression levels and variable phenotypes . For these reasons, we have adapted the Gal4-UAS method for targeted gene expression to Xenopus . This technique relies on the generation of transgenic lines that carry "activator" or "effector" constructs . Activator lines express the yeast transcription factor, Gal4, under the control of a desired promoter, whereas effector lines contain DNA-binding motifs for Gal4-(UAS) linked to the gene of interest . We show that on intercrossing of these lines, the effector gene is transcribed in the temporal and spatial manner of the activator's promoter . Furthermore, we use the Gal4-UAS system to misexpress Xvent-2, a transcriptional target of bone morphogenetic protein 4 (BMP4) signaling during early embryogenesis . Embryos inheriting both the Gal4 activator and Xvent-2 effector transgenes display a consistent microcephalic phenotype . Finally, we exploit this system to characterize the neural and mesodermal defects obtained from early misexpression of Xvent-2 . These results emphasize the potential of this system for the controlled analyses of gene function in Xenopus. Mol Endocrinol, 2002 Feb, 16(2), 315 - 30 The activating enzyme of NEDD8 inhibits steroid receptor function; Fan M et al.; Coregulator proteins, coactivators and corepressors, have a profound influence on steroid receptor activity and play a role in regulating receptor levels . To identify novel coregulators of nuclear receptors, we used the ligand-binding and hinge region of ERalpha as bait in a yeast two-hybrid screen of a cDNA library derived from rat uterine luminal epithelium . We report the cloning and characterization of a cDNA encoding a protein homologous to yeast and human ubiquitin-activating enzyme 3 (Uba3), the catalytic subunit of the activating enzyme of the ubiquitin-like NEDD8 (neural precursor cellexpressed developmentally down-regulated) conjugation pathway (known as neddylation) . Sequence analysis revealed that Uba3 contains multiple nuclear receptor (NR)-interacting motifs (NR boxes), which are known to mediate interactions between coregulatory proteins and ligand-activated NRs . Yeast two-hybrid and glutathione-S-transferase pull-down assays demonstrated that Uba3 directly interacts with ligand-occupied ERalpha and ERbeta . Transient transfection of Uba3 in mammalian cells inhibited ER-mediated transactivation in a time-dependent fashion; Uba3 had no effect on the initial events of transcriptional activation by liganded ER, but it blocked the progressive increase in target gene expression during continuous stimulation . Uba3 also inhibited transactivation by AR and PR in mammalian cells but had no effect on a steroid receptor-independent transactivation pathway . An enzymatically silent form of Uba3 did not inhibit ER-induced transcription, and a Uba3-binding fragment of amyloid precursor protein-binding protein, the other subunit of the NEDD8-activating enzyme, partially overcame Uba3-mediated inhibition, demonstrating that the neddylation activity of Uba3 is required for its inhibition of steroid receptor transactivation . Thus, Uba3 inhibits transcription induced by steroid hormone receptors through a novel mechanism that involves the neddylation pathway . Understanding the mechanisms controlling hormone responsiveness of target tissues, such as the uterus and mammary gland, may lead to novel insights of therapeutic intervention. J Leukoc Biol, 2002 Feb, 71(2), 195 - 204 Myosin-V colocalizes with MHC class II in blood mononuclear cells and is up-regulated by T-lymphocyte activation; Bizario JC et al.; Myosin-V is involved in organelle and vesicle trafficking in Saccharomyces cerevisiae and in other eukaryotic cells from yeast to human . In the present study, we determined by FACS that the major subpopulations of the peripheral blood mononuclear cells express myosin-V with similar fluorescence intensity . Confocal microscopy showed intense labeling for myosin-V at the centrosomal region and a punctate staining throughout the cytoplasm, frequently associated with the central microtubule arrays and the actin-rich cortex . Some degree of overlap with an endolysosomal marker and dynein light-chain 8 k was found at the cell center . Striking colocalization was observed with the major histocompatibility complex (MHC) class II molecules near the cell surface . Treatment with phytohemagglutinin, which induces T-lymphocyte activation, associated with MHC class II expression, increased the levels of myosin-V protein and mRNA for the three members of class V myosins . These data suggest that class V myosins might be involved in relevant functions in the immune response. J Am Chem Soc, 2002 Feb 6, 124(5), 834 - 42 Structural analysis of metal ion ligation to nucleotides and nucleic acids using pulsed EPR spectroscopy; Hoogstraten CG et al.; Metal ions play key structural and functional roles in many nucleic acid systems, particularly as required cofactors for many catalytic RNA molecules (ribozymes) . We apply the pulsed EPR technologies of electron spin-echo envelope modulation and electron spin-echo-electron nuclear double resonance to the structural analysis of the paramagnetic metal ion Mn(II) bound to nucleotides and nucleic acids . We demonstrate that pulsed EPR, supplemented with specific isotope labeling, can characterize ligation to nucleotide base nitrogens, outer-sphere interactions with phosphate groups, distances to sites of specific (2)H atom labels, and the hydration level of the metal ion . These techniques allow a comprehensive structural analysis of the mononucleotide model system MnGMP . Spectra of phenylalanine-specific transfer RNA from budding yeast and of the hammerhead ribozyme demonstrate the applicability of the methods to larger, structured RNA systems . This suite of experiments opens the way to detailed structural characterization of specifically bound metal ions in a variety of ribozymes and other nucleic acids of biological interest. Anal Chem, 2001 Nov 15, 73(22), 5395 - 402 Chemical cleavage at aspartyl residues for protein identification; Li A et al.; An alternative method to enzymatic digestion for protein identification by mass spectrometry has been developed that is based on chemical cleavage by formic acid . This method was tested on gel-purified apomyoglobin and BSA, as well as unknown proteins that cofractionate with Tyl-virus-like particles from Saccharomyces cerevisiae . Cleavage at aspartyl residues was found to be efficient and specific, and this specificity of cleavage lent itself easily to database searches . Parallel digestions using trypsin were also performed . The formic acid cleavage method generated comparable or better results than tryptic digestion for protein identification. Yeast, 2002 Feb, 19(3), 203 - 14 Phenotypic analysis of gene deletant strains for sensitivity to oxidative stress; Higgins VJ et al.; Ascertaining the impact of inhibitors on the growth phenotype of yeast mutants can be useful in elucidating the function of genes within the cell . Microtitre plates and robotics have been used to screen over 600 deletions from EUROSCARF, constructed in an FY1679 strain background, for sensitivity to various oxidants . These included the inorganic hydroperoxide, H(2)O(2), an organic peroxide (cumene hydroperoxide) and a lipid hydroperoxide (linoleic acid hydroperoxide) . These produce within the cell several different reactive oxygen species that can cause damage to DNA, proteins and lipids . Approximately 14% of deletants displayed sensitivity to at least one of the oxidants and there was also a distribution of deletants that showed sensitivity to all or different combinations of the oxidants . Deletants included genes encoding proteins involved in stress responses, heavy metal homeostasis and putative cell wall proteins . Although global mechanisms have been identified that provide general stress responses, these results imply that there are also distinct mechanisms involved in the protection of the cell against specific damage caused by different oxidants . Further analysis of these genes may reveal unknown mechanisms protecting the cell against reactive oxygen species . Gene, 2002 Jan 9, 282(1-2), 33 - 41 ORF-FINDER: a vector for high-throughput gene identification; Rombel IT et al.; We have developed a simple and efficient system (ORF-FINDER) for selecting open reading frames (ORFs) from randomly fragmented genomic DNA fragments . The ORF-FINDER vectors are plasmids that contain a translational start site out of frame with respect to the gene for green fluorescent protein (GFP) . Insertion of DNA fragments that bring the initiating ATG in frame with GFP and that contain no stop codons (that is, ORFs) results in the expression of ORF-GFP fusion proteins . In addition, we have developed software (GeneWorks and GenomeAnalyzer) to predict the optimal insert size for maximizing the number of gene-coding ORFs and minimizing unintentionally selected non-coding ORFs . To demonstrate the feasibility of using the ORF-FINDER system to screen genomes for ORFs, we cloned yeast genomic DNA and succeeded in enriching for ORFs by 25-fold . Furthermore, we have shown that the vector can effectively isolate ORFs from the more complex genomes of eukaryotic parasites . We envision that ORF-FINDER will have several applications including genome sequencing projects, gene building from oligonucleotides and construction of expression libraries enriched for ORFs. Psychiatry Res, 2001 Dec 31, 105(3), 273 - 8 Susceptibility genes for a trait measure of attention deficit hyperactivity disorder: a pilot study in a non-clinical sample of twins; Payton A et al.; Attention deficit hyperactivity disorder (ADHD) is a highly heritable disorder, and molecular genetic studies are underway, with most researchers focusing on identifying susceptibility genes in clinical samples with ADHD . An alternative approach is to search for quantitative trait loci underlying the trait measure of ADHD in non-clinical samples . Positive findings of association of the dopamine transporter DAT1 480 bp allele (allele 10) and the DRD4 7 repeat allele with clinical ADHD have been previously reported . In this pilot study, we examined these polymorphisms in a selected population-based sample of twins (50 high scoring pairs, 42 low scoring pairs) . There was a trend for an increase in the frequency of the dopamine receptor DRD4 7 repeat allele in the high-scoring concordant monozygotic twins (odds ratio=1.4) . Although this result was not statistically significant, the frequency of the 7 repeat allele was similar to that reported for our clinic sample of ADHD patients drawn from the same geographical area . There was a non-significant trend for an increased frequency of the DAT1 allele 10 (odds ratio=1.3) . These results suggest that a molecular genetic study based on a questionnaire-derived measure of ADHD in a non-clinical sample is feasible and the results appear to be comparable with those from studies of clinical cases . However, sample size and power are key issues to consider when using this approach.
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