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