Mol Biol Cell, 2002 Sep, 13(9), 3192 - 202 The product of the survival of motor neuron (SMN) gene is a human telomerase-associated protein; Bachand F et al.; Telomerase is a ribonucleoprotein (RNP) complex that is minimally composed of a protein catalytic subunit, the telomerase reverse transcriptase (TERT), and an RNA component, the telomerase RNA . The survival of motor neuron (SMN) gene codes for a protein involved in the biogenesis of certain RNPs . Here, we report that SMN is a telomerase-associated protein . Using in vitro binding assays and immunoprecipitation experiments, we demonstrate an association between SMN and the telomerase RNP in vitro and in human cells . The specific immunopurification of SMN from human 293 cells copurified telomerase activity, suggesting that SMN associates with a subset of the functional telomerase holoenzyme . Our results also indicate that the human telomerase RNA and the human (h) TERT are not associated with Sm proteins, in contrast to Saccharomyces cerevisiae telomerase . Immunofluorescence analysis showed that hTERT does not specifically colocalize with wild-type SMN in gems or Cajal bodies . However, a dominant-negative mutant of SMN (SMNDeltaN27) previously characterized to elicit the cellular reorganization of small nuclear RNPs caused the accumulation of hTERT in specific SMNDeltaN27-induced cellular bodies . Furthermore, coexpression of SMNDeltaN27 and hTERT in rabbit reticulocyte lysates decreased the efficiency of human telomerase reconstitution in vitro . Our results establish SMN as a novel telomerase-associated protein that is likely to function in human telomerase biogenesis. Mol Biol Cell, 2002 Sep, 13(9), 3162 - 77 An essential subfamily of Drs2p-related P-type ATPases is required for protein trafficking between Golgi complex and endosomal/vacuolar system; Hua Z et al.; The Saccharomyces cerevisiae genome contains five genes encoding P-type ATPases that are potential aminophospholipid translocases (APTs): DRS2, NEO1, and three uncharacterized open reading frames that we have named DNF1, DNF2, and DNF3 for DRS2/NEO1 family . NEO1 is the only essential gene in APT family and seems to be functionally distinct from the DRS2/DNF genes . The drs2Delta dnf1Delta dnf2Delta dnf3Delta quadruple mutant is inviable, although any one member of this group can maintain viability, indicating that there is a substantial functional overlap between the encoded proteins . We have previously implicated Drs2p in clathrin function at the trans-Golgi network . In this study, we constructed strains carrying all possible viable combinations of null alleles from this group and analyzed them for defects in protein transport . The drs2Delta dnf1Delta mutant grows slowly, massively accumulates intracellular membranes, and exhibits a substantial defect in the transport of alkaline phosphatase to the vacuole . Transport of carboxypeptidase Y to the vacuole is also perturbed, but to a lesser extent . In addition, the dnf1Delta dnf2Delta dnf3Delta mutant exhibits a defect in recycling of GFP-Snc1p in the early endocytic-late secretory pathways . Drs2p and Dnf3p colocalize with the trans-Golgi network marker Kex2p, whereas Dnf1p and Dnf2p seem to localize to the plasma membrane and late exocytic or early endocytic membranes . We propose that eukaryotes express multiple APT subfamily members to facilitate protein transport in multiple pathways. Mol Biol Cell, 2002 Sep, 13(9), 3029 - 41 Distinct chromosome segregation roles for spindle checkpoint proteins; Warren CD et al.; The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensuring that anaphase is initiated only after kinetochore-microtubule associations of all sister chromatid pairs are complete . In this study, we find that known spindle checkpoint proteins do not contribute equally to chromosome segregation fidelity in Saccharomyces cerevisiae . Loss of Bub1 or Bub3 protein elicits the largest effect . Analysis of Bub1p reveals the presence of two molecular functions . An N-terminal 608-amino acid (nonkinase) portion of the protein supports robust checkpoint activity, and, as expected, contributes to chromosome segregation . A C-terminal kinase-encoding segment independently contributes to chromosome segregation through an unknown mechanism . Both molecular functions depend on association with Bub3p . A 156-amino acid fragment of Bub1p functions in Bub3p binding and in kinetochore localization by one-hybrid assay . An adjacent segment is required for Mad1p binding, detected by deletion analysis and coimmunoprecipitation . Finally, overexpression of wild-type BUB1 or MAD3 genes leads to chromosome instability . Analysis of this activity indicates that the Bub3p-binding domain of Bub1p contributes to this phenotype through disruption of checkpoint activity as well as through introduction of kinetochore or spindle damage. J Biol Chem, 2002 Nov 8, 277(45), 42875 - 80 Epub 2002 Sep 06. Removal of Pex3p is an important initial stage in selective peroxisome degradation in Hansenula polymorpha; Bellu AR et al.; Selective degradation of peroxisomes (macropexophagy) in Hansenula polymorpha involves the sequestration of individual organelles to be degraded by membranes prior to the fusion of this compartment with the vacuole and subsequent degradation of the whole organelle by vacuolar hydrolases . Here we show that Pex3p, a peroxisomal membrane protein essential for peroxisome biogenesis, escapes this autophagic process . Upon induction of macropexophagy, Pex3p is removed from the organelle tagged for degradation prior to its sequestration . Our data indicate that Pex3p degradation is essential to allow the initiation of the organellar degradation process . Also, in a specific peroxisome degradation-deficient (pdd) mutant in which sequestration still occurs but the vacuolar fusion event is disturbed, the turnover of Pex3p is still observed . Taken together, our data suggest that degradation of Pex3p is part of the initial degradation machinery of individual peroxisomes. Trends Cell Biol, 2002 Sep, 12(9), 404 - 6 Human Sir2 and the 'silencing' of p53 activity; Smith J; Members of the evolutionarily conserved silent information regulator 2 (Sir2) protein family are nicotinamide adenine dinucleotide (NAD(+))-dependent histone deacetylases . In yeast, the founding Sir2 protein is known to function in transcriptional silencing processes through the deacetylation of histones H3 and H4, thus setting up a repressive chromatin structure . Yeast and Caenorhabditis elegans Sir2 are also involved in regulating the life span of these organisms . Until recently, the function of mammalian Sir2 family members was completely unknown . However, several recent studies have now determined a remarkable function for the human SIRT1 protein, which is the closest human homolog of yeast Sir2 . SIRT1 specifically associates with the p53 tumor suppressor protein and deacetylates it, resulting in negative regulation of p53-mediated transcriptional activation . Importantly, p53 deacetylation by SIRT1 also prevents cellular senescence and apoptosis induced by DNA damage and stress. FEBS Lett, 2002 Sep 11, 527(1-3), 303 - 8 The regions of securin and cyclin B proteins recognized by the ubiquitination machinery are natively unfolded; Cox CJ et al.; The proteins securin and cyclin B are destroyed in mitosis by the ubiquitin/proteasome system . This destruction is important to mitotic progression . The N-terminal regions of these proteins contain the sequence features recognized by the ubiquitination system . We have demonstrated using circular dichroism and 1-D and 2-D nuclear magnetic resonance that these rather substantial regions are natively unfolded . Based on these findings, we propose a model that helps to explain previously enigmatic observations. Mutat Res, 2002 Sep, 512(1), 1 - 35 A survey of EPA/OPP and open literature on selected pesticide chemicals . III . Mutagenicity and carcinogenicity of benomyl and carbendazim; McCarroll NE et al.; The known aneuploidogens, benomyl and its metabolite, carbendazim (methyl 2-benzimidazole carbamate (MBC)), were selected for the third in a series of ongoing projects with selected pesticides . Mutagenicity and carcinogenicity data submitted to the US Environmental Protection Agency's (US EPA's) Office of Pesticide Programs (OPP) as part of the registration process are examined along with data from the open literature . Mutagenicity and carcinogenicity profiles are developed to provide a complete overview and to determine whether an association can be made between benomyl- and MBC-induced mouse liver tumors and aneuploidy . Since aneuploidogens are considered to indirectly affect DNA, the framework adopted by the Agency for evaluating any mode of action (MOA) for carcinogenesis is applied to the benomyl/MBC data.Both agents displayed consistent, positive results for aneuploidy induction but mostly negative results for gene mutations . Non-linear dose responses were seen both in vitro and in vivo for aneuploidy endpoints . No evidence was found suggesting that an alternative MOA other than aneuploidy may be operative . The data show that by 14 days of benomyl treatment, events associated with liver toxicity appear to set in motion the sequence of actions that leads to neoplasms . Genetic changes (as indicated by spindle impairment leading to missegregation of chromosomes, micronucleus induction and subsequent aneuploidy in bone marrow cells) can commence within 1-24h after dosing, well within the time frame for early key events . Critical steps associated with frank tumor formation in the mouse liver include hepatotoxicity, increased liver weights, cell proliferation, hypertrophy, and other steps involving hepatocellular alteration and eventual progression to neoplasms . The analysis, however, reveals weaknesses in the data base for both agents (i.e . no studies on mouse tubulin binding, no in vivo assays of aneuploidy on the target tissue (liver), and no clear data on cell proliferation relative to dose response and time dependency) . The deficiencies in defining the MOA for benomyl/MBC introduce uncertainties into the analysis; consequently, benomyl/MBC induction of aneuploidy cannot be definitively linked to mouse liver carcinogenicity at this time. Biochemistry, 2002 Sep 17, 41(37), 11218 - 25 RNA methyltransferases utilize two cysteine residues in the formation of 5-methylcytosine; King MY et al.; Proteins that have sequence homology with known RNA m(5)C methyltransferases contain two conserved cysteines, each of which lies within a sequence that bears similarity to a methyltransferase active site . Other enzymes that transfer a methyl group to carbon 5 of a pyrimidine nucleotide, such as the bacterial DNA m(5)C methyltransferases, utilize their single conserved cysteine residue to form a covalent Michael adduct with carbon 6 of the pyrimidine ring during catalysis . We present a model for the utilization of two cysteines in catalysis by RNA m(5)C methyltransferases . It is proposed that one thiol acts in a classical fashion by forming a covalent link to carbon 6 of the pyrimidine base, while the other cysteine assists breakdown of the covalent adduct . Therefore, alteration of the assisting cysteine is anticipated to stabilize the covalent enzyme-RNA intermediate . The model was conceived as a possible explanation for the effects of mutations that change the conserved cysteines in Nop2p, an apparent RNA m(5)C methyltransferase that is essential for ribosome assembly and yeast viability . Evidence for the predicted accumulation of protein-RNA complexes following mutation of the assisting cysteine has been obtained with Nop2p and a known tRNA m(5)C methyltransferase called Ncl1p (Trm4). Biochim Biophys Acta, 2002 Sep 10, 1555(1-3), 174 - 80 Is there a relationship between the supramolecular organization of the mitochondrial ATP synthase and the formation of cristae? Giraud MF, Paumard P, Soubannier V, Vaillier J, Arselin G, Salin B, Schaeffer J, Brethes D, di Rago JP, Velours J. Blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses of detergent mitochondrial extracts have provided evidence that the yeast ATP synthase could form dimers . Cross-linking experiments performed on a modified version of the i-subunit of this enzyme indicate the existence of such ATP synthase dimers in the yeast inner mitochondrial membrane . We also show that the first transmembrane segment of the eukaryotic b-subunit (bTM1), like the two supernumerary subunits e and g, is required for dimerization/oligomerization of ATP synthases . Unlike mitochondria of wild-type cells that display a well-developed cristae network, mitochondria of yeast cells devoid of subunits e, g, or bTM1 present morphological alterations with an abnormal proliferation of the inner mitochondrial membrane . From these observations, we postulate that an anomalous organization of the inner mitochondrial membrane occurs due to the absence of ATP synthase dimers/oligomers . We provide a model in which the mitochondrial ATP synthase is a key element in cristae morphogenesis. Biochim Biophys Acta, 2002 Sep 10, 1555(1-3), 101 - 5 Atp11p and Atp12p are chaperones for F(1)-ATPase biogenesis in mitochondria; Ackerman SH; The bioenergetic needs of aerobic cells are met principally through the action of the F(1)F(0) ATP synthase, which catalyzes ATP synthesis during oxidative phosphorylation . The catalytic unit of the enzyme (F(1)) is a multimeric protein of the subunit composition alpha(3)beta(3)(gamma)(delta) epsilon . Our work, which employs the yeast Saccharomyces cerevisiae as a model system for studies of mitochondrial function, has provided evidence that assembly of the mitochondrial alpha and beta subunits into the F(1) oligomer requires two molecular chaperone proteins called Atp11p and Atp12p . Comprehensive knowledge of Atp11p and Atp12p activities in mitochondria bears relevance to human physiology and disease as these chaperone actions are now known to exist in mitochondria of human cells. J Mol Biol, 2002 Aug 30, 321(5), 891 - 908 Unfolding of a leucine zipper is not a simple two-state transition; Dragan AI et al.; Temperature-induced unfolding of the leucine zipper, an alpha-helical, double-stranded, coiled-coil, was studied by circular dichroism spectroscopy, spectrofluorimetry and heat capacity scanning calorimetry . It is shown that this process does not represent a simple two-state transition, as previously believed, but consists of several stages . The first transition starts at the very beginning of heating from 0 degrees C and proceeds with significant heat absorption and decrease of ellipticity . This transition does not depend on the concentration of protein and is sensitive to modification of the N terminus; it is therefore associated with unfolding or fraying of this part of the leucine zipper . The second transition takes place at a considerably higher temperature; it is more pronounced than the first one and does not depend on the concentration of protein, i.e . it is unimolecular . This transition is sensitive to modification of both termini of the leucine zipper and affects the optical properties of a tryptophan residue placed in the central part of the zipper . It therefore involves the whole dimer but does not result in its dissociation, presumably being associated with some repacking of the coiled-coil . This second transition is followed at higher temperatures by the concentration-dependent cooperative unfolding/dissociation of the two strands . The enthalpy and entropy of the temperature-induced structural changes of the leucine zipper that take place before its cooperative unfolding/dissociation comprises almost 40% of the total enthalpy and entropy of unfolding of the completely folded coiled-coil, the state in which it appears to be below 0 degrees C . Comparison of the total enthalpy of leucine zipper unfolding with that of a single-stranded alpha-helix shows that their temperature-dependence correlates with the extent of intramolecular non-polar contacts and allows an assessment of the enthalpy of hydrogen bonding in alpha-helices, which appears to be about 3.3kJmol(-1) at 20 degrees C. Nat Struct Biol, 2002 Oct, 9(10), 764 - 70 Crystal structure of the intein homing endonuclease PI-SceI bound to its recognition sequence; Moure CM et al.; The first X-ray structures of an intein-DNA complex, that of the two-domain homing endonuclease PI-SceI bound to its 36-base pair DNA substrate, have been determined in the presence and absence of Ca(2+) . The DNA shows an asymmetric bending pattern, with a major 50 degree bend in the endonuclease domain and a minor 22 degree bend in the splicing domain region . Distortions of the DNA bound to the endonuclease domain cause the insertion of the two cleavage sites in the catalytic center . DNA binding induces changes in the protein conformation . The two overlapping non-identical active sites in the endonucleolytic center contain two Ca(+2) ions that coordinate to the catalytic Asp residues . Structure analysis indicates that the top strand may be cleaved first. Proc Natl Acad Sci U S A, 2002 Oct 1, 99(20), 12663 - 8 Epub 2002 Sep 06. The hDcp2 protein is a mammalian mRNA decapping enzyme; Wang Z et al.; Decapping of mRNA is a critical step in eukaryotic mRNA turnover, yet the proteins involved in this activity remain elusive in mammals . We identified the human Dcp2 protein (hDcp2) as an enzyme containing intrinsic decapping activity . hDcp2 specifically hydrolyzed methylated capped RNA to release m(7)GDP; however, it did not function on the cap structure alone . hDcp2 is therefore functionally distinct from the recently identified mammalian scavenger decapping enzyme, DcpS . hDcp2-mediated decapping required a functional Nudix (nucleotide diphosphate linked to an X moiety) pyrophosphatase motif as mutations in conserved amino acids within this motif disrupted the decapping activity . hDcp2 is detected exclusively in the cytoplasm and predominantly cosediments with polysomes . Consistent with the localization of hDcp2, endogenous Dcp2-like decapping activity was detected in polysomal fractions prepared from mammalian cells . Similar to decapping in yeast, the presence of the poly(A) tail was inhibitory to the endogenous decapping activity, yet unlike yeast, competition of cap-binding proteins by cap analog did not influence the efficiency of decapping . Therefore the mammalian homologue of the yeast Dcp2 protein is an mRNA decapping enzyme demonstrated to contain intrinsic decapping activity. Proc Natl Acad Sci U S A, 2002 Sep 17, 99(19), 12185 - 90 Epub 2002 Sep 06. Singularity in budding: a role for the evolutionarily conserved small GTPase Cdc42p; Caviston JP et al.; The budding yeast Saccharomyces cerevisiae initiates polarized growth or budding once per cell cycle at a specific time of the cell cycle and at a specific location on the cell surface . Little is known about the molecular nature of the temporal and spatial regulatory mechanisms . It is also unclear what factors, if any, among the numerous proteins required to make a bud are involved in the determination of budding frequency . Here we describe a class of cdc42 mutants that produce multiple buds at random locations on the cell surface within one nuclear cycle . The critical mutation responsible for this phenotype affects amino acid residue 60, which is located in a domain required for GTP binding and hydrolysis . This mutation bypasses the requirement for the essential guanine-nucleotide-exchange factor Cdc24p, suggesting that the alteration at residue 60 makes Cdc42p hyperactive, which was confirmed biochemically . This result also suggests that the only essential function of Cdc24p is to activate Cdc42p . Together, these data suggest that the temporal and spatial regulation of polarized growth converges at the level of Cdc42p and that the activity of Cdc42p determines the budding frequency. J Immunol, 2002 Sep 15, 169(6), 3069 - 75 Gene conversion-like sequence transfers between transgenic antibody V genes are independent of RAD54; D'Avirro N et al.; Homology-based Ig gene conversion is a major mechanism for Ab diversification in chickens and the Rad54 DNA repair protein plays an important role in this process . In mice, although gene conversion appears to be rare among endogenous Ig genes, Ab H chain transgenes undergo isotype switching and gene conversion-like sequence transfer processes that also appear to involve homologous recombination or gene conversion . Furthermore, homology-based DNA repair has been suggested to be important for somatic mutation of endogenous mouse Ig genes . To assess the role of Rad54 in these mouse B cell processes, we have analyzed H chain transgene isotype switching, sequence transfer, and somatic hypermutation in mice that lack RAD54 . We find that Rad54 is not required for either transgene switching or transgene hypermutation . Furthermore, even transgene sequence transfers that are known to require homology-based recombinations are Rad54 independent . These results indicate that mouse B cells must use factors for promoting homologous recombination that are distinct from the Rad54 proteins important in homology-based chicken Ab gene recombinations . Our findings also suggest that mouse H chain transgene sequence transfers might be more closely related to an error-prone homology-based somatic hypermutational mechanism than to the hyperconversion mechanism that operates in chicken B cells. Hum Mol Genet, 2002 Sep 15, 11(19), 2331 - 9 ALG12 mannosyltransferase defect in congenital disorder of glycosylation type lg; Grubenmann CE et al.; In the endoplasmic reticulum (ER) of eukaryotes, N-linked glycans are first assembled on the lipid carrier dolichyl pyrophosphate . The GlcNAc(2)Man(9)Glc(3) oligosaccharide is transferred to selected asparagine residues of nascent polypeptides . Defects along the biosynthetic pathway of N-glycans are associated with severe multisystemic syndromes called congenital disorders of glycosylation . Here, we describe a deficiency in the ALG12 ER alpha1,6-mannosyltransferase resulting in a novel type of glycosylation disorder . The severe disease was identified in a child presenting with psychomotor retardation, hypotonia, growth retardation, dysmorphic features and anorexia . In the patient's fibroblasts, the biosynthetic intermediate GlcNAc(2)Man(7) oligosaccharide was detected both on the lipid carrier dolichyl pyrophosphate and on newly synthesized glycoproteins, thus pointing to a defect in the dolichyl pyrophosphate-GlcNAc(2)Man(7)-dependent ALG12 alpha1,6 mannosyltransferase . Analysis of the ALG12 cDNA in the CDG patient revealed compound heterozygosity for two point mutations that resulted in the amino acid substitutions T67M and R146Q, respectively . The impact of these mutations on ALG12 protein function was investigated in the Saccharomyces cerevisiae alg12 glycosylation mutant by showing that the yeast ALG12 gene bearing the homologous mutations T61M and R161Q and the human mutant ALG12 cDNA alleles failed to normalize the growth defect phenotype of the alg12 yeast model, whereas expression of the normal ALG12 cDNA complemented the yeast mutation . The ALG12 mannosyltransferase defect defines a new type of congenital disorder of glycosylation, designated CDG-Ig. Bioinformatics, 2002 Sep, 18(9), 1194 - 206 Bayesian infinite mixture model based clustering of gene expression profiles; Medvedovic M et al.; MOTIVATION: The biologic significance of results obtained through cluster analyses of gene expression data generated in microarray experiments have been demonstrated in many studies . In this article we focus on the development of a clustering procedure based on the concept of Bayesian model-averaging and a precise statistical model of expression data . RESULTS: We developed a clustering procedure based on the Bayesian infinite mixture model and applied it to clustering gene expression profiles . Clusters of genes with similar expression patterns are identified from the posterior distribution of clusterings defined implicitly by the stochastic data-generation model . The posterior distribution of clusterings is estimated by a Gibbs sampler . We summarized the posterior distribution of clusterings by calculating posterior pairwise probabilities of co-expression and used the complete linkage principle to create clusters . This approach has several advantages over usual clustering procedures . The analysis allows for incorporation of a reasonable probabilistic model for generating data . The method does not require specifying the number of clusters and resulting optimal clustering is obtained by averaging over models with all possible numbers of clusters . Expression profiles that are not similar to any other profile are automatically detected, the method incorporates experimental replicates, and it can be extended to accommodate missing data . This approach represents a qualitative shift in the model-based cluster analysis of expression data because it allows for incorporation of uncertainties involved in the model selection in the final assessment of confidence in similarities of expression profiles . We also demonstrated the importance of incorporating the information on experimental variability into the clustering model . AVAILABILITY: The MS Windows(TM) based program implementing the Gibbs sampler and supplemental material is available at CONTACT: medvedm@email.uc.edu Bioinformatics, 2002 Sep, 18(9), 1176 - 83 Geometry of gene expression dynamics; Rifkin SA et al.; MOTIVATION: A gene expression trajectory moves through a high dimensional space where each axis represents the mRNA abundance of a different gene . Genome wide gene expression has a dynamic structure, especially in studies of development and temporal response . Both visualization and analyses of such data require an explicit attention to the temporal structure . RESULTS: Using three cell cycle trajectories from Saccharomyces cerevisiae to illustrate, we present several techniques which reveal the geometry of the data . We import phase-delay time plots from chaotic systems theory as a dynamic data visualization device and show how these plots capture important aspects of the trajectories . We construct an objective function to find an optimal two-dimensional projection of the cell cycle, demonstrate that the system returns to this plane after three different initial perturbations, and explore the conditions under which this geometric approach outperforms standard approaches such as singular value decomposition and Fourier analysis . Finally, we show how a geometric analysis can isolate distinct parts of the trajectories, in this case the initial perturbation versus the cell cycle . CONTACT: junhyong.kim@yale.edu Bioinformatics, 2002 Sep, 18(9), 1167 - 75 Identification of regulatory elements using a feature selection method; Keles S et al.; MOTIVATION: Many methods have been described to identify regulatory motifs in the transcription control regions of genes that exhibit similar patterns of gene expression across a variety of experimental conditions . Here we focus on a single experimental condition, and utilize gene expression data to identify sequence motifs associated with genes that are activated under this experimental condition . We use a linear model with two-way interactions to model gene expression as a function of sequence features (words) present in presumptive transcription control regions . The most relevant features are selected by a feature selection method called stepwise selection with monte carlo cross validation . We apply this method to a publicly available dataset of the yeast Saccharomyces cerevisiae, focussing on the 800 basepairs immediately upstream of each gene's translation start site (the upstream control region (UCR)) . RESULTS: We successfully identify regulatory motifs that are known to be active under the experimental conditions analyzed, and find additional significant sequences that may represent novel regulatory motifs . We also discuss a complementary method that utilizes gene expression data from a single microarray experiment and allows averaging over variety of experimental conditions as an alternative to motif finding methods that act on clusters of co-expressed genes . AVAILABILITY: The software is available upon request from the first author or may be downloaded from CONTACT: keles@stat.berkeley.edu Trends Endocrinol Metab, 2002 Oct, 13(8), 336 - 43 Lessons from constitutively active mutants of G protein-coupled receptors; Parnot C et al.; In the past decade, the concept of constitutive activity has profoundly modified our understanding of G protein-coupled-receptors (GPCRs) . Here, we review the contribution of constitutively active mutants (CAMs) to our understanding of three aspects of GPCR physiopathology: (1) GPCR activation is a complex mechanism involving both the release of inactive state conformational constraints, mimicked by most CAMs, and the creation of new interactions that stabilize the active state and are mimicked by a restricted set of CAMs; (2) GPCR phosphorylation, internalization and desensitization processes are activated by receptor conformations, which partly overlap those activating G protein; (3) natural CAMs, mostly affecting GPCRs of the endocrine system, are found in several hereditary and acquired diseases, including cancers . One major remaining question is how CAMs recapitulate the different structural modifications of the agonist-induced active conformation(s) of the wild-type receptor . This characterization is a prerequisite for further use of CAMs as ligand-free models of active GPCRs in structural, cellular and physiological studies. Dev Biol, 2002 Sep 1, 249(1), 140 - 55 Tubedown-1 in remodeling of the developing vitreal vasculature in vivo and regulation of capillary outgrowth in vitro; Paradis H et al.; Tubedown-1 (tbdn-1) is a mammalian homologue of the N-terminal acetyltransferase subunit NAT1 of Saccharomyces cerevisiae and copurifies with an acetyltransferase activity . Tbdn-1 expression in endothelial cells becomes downregulated during the formation of capillary-like structures in vitro and is regulated in vivo in a manner which suggests a functional role in dampening blood vessel development . Here we show that tbdn-1 is expressed highly in the vitreal vascular network (tunica vasculosa lentis and vasa hyaloidea propria) during the pruning and remodeling phases of this transient structure . The vitreal blood vessels of mice harboring a targeted inactivation of TGF-beta2 fail to remodel and abnormally accumulate, a phenomenon reminiscent of the ocular pathology resembling persistent fetal vasculature (PFV) in humans . Since suppression of normal tbdn-1 expression has been previously observed in retinal vessel proliferation, we analyzed vitreal vascular changes and tbdn-1 expression in TGF-beta2(-/-) eyes . The nuclei of vitreal vessel endothelial cells in TGF-beta2(-/-) eyes express proliferating cell nuclear antigen (PCNA) and exhibit increased levels of active (P42/44)mitogen-activated protein kinase (phospho-(P42/44)MAPK), characteristics consistent with proliferative endothelial cells . In contrast to normal vitreal vessels, collagen IV expression exhibited a disorganized pattern in the TGF-beta2(-/-) vitreal vessels, suggesting vessel disorganization and possibly a breakdown of vessel basal laminae . Moreover, vitreal vessels of TGF-beta2(-/-) mice lack expression of pericyte markers (CD13, alpha smooth muscle actin) and show ultrastructural changes consistent with pericyte degeneration . The accumulating vitreal blood vessels of TGF-beta2(-/-) mice, while maintaining expression of the endothelial marker von Willebrand Factor, show a significant decrease in the expression of tbdn-1 . We addressed the functional role of tbdn-1 in the regulation of vitreal blood vessels using an in vitro model of choroid-retina capillary outgrowth . Clones of the RF/6A fetal choroid-retina endothelial cell line showing suppression of tbdn-1 levels after overexpression of an antisense TBDN-1 cDNA display a significant increase in the formation of capillary-like structures in vitro compared with controls . These findings suggest that tbdn-1 inhibits capillary-like formation in vitro and may serve to dampen vitreal blood vessel formation preceding the regression of the vitreal vasculature during development . Our results also suggest that tbdn-1 may participate with TGF-beta2 in regulating normal development of the vitreal vasculature. Lipids, 2002 Jul, 37(7), 663 - 72 PC and PE synthesis: mixed micellar analysis of the cholinephosphotransferase and ethanolaminephosphotransferase activities of human choline/ethanolamine phosphotransferase 1 (CEPT1); Wright MM et al.; The human choline/ethanolamine phosphotransferase 1 (CEPT1) gene codes for a dual-specificity enzyme that catalyzes the de novo synthesis of the two major phospholipids through the transfer of a phosphobase from CDP-choline or CDP-ethanolamine to DAG to form PC and PE . We used an expression system devoid of endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities to assess the diradylglycerol specificity of CEPT1 . A mixed micellar assay was used to ensure that the diradylglycerols delivered were not affecting the membrane environment in which CEPT1 resides . The CEPT1 enzyme displayed an apparent Km of 36 microM for CDP-choline and 4.2 mol% for di-18:1 DAG with a Vmax of 14.3 nmol min(-1) mg(-1) . When CDP-ethanolamine was used as substrate, the apparent Km was 98 microM for CDP-ethanolamine and 4.3 mol% for di-18:1 DAG with a Vmax of 8.2 nmol min(-1) mg(-1) . The preferred diradylglycerol substrates used by CEPT1 with CDP-choline as the phosphobase donor were di-18:1 DAG, di-16:1 DAG, and 16:0/18:1 DAG . A major difference between previous emulsion-based assay results and the mixed micelle results was a complete inability to use 16:0(O)/2:0 as a substrate for the de novo synthesis of platelet-activating factor when the mixed micelle assay was used . When CDP-ethanolamine was used as the phosphobase donor, 16:0/18:1 DAG, di-18:1 DAG, and di-16:1 DAG were the preferred substrates . The mixed micelle assay also allowed the lipid activation of CEPT to be measured, and both the cholinephosphotransferase and ethanolaminephosphotransferase activities displayed the unusual property of product activation at 5 mol%, implying that specific lipid activation binding sites exist on CEPT1 . The protein kinase C inhibitor chelerythrine and the human DAG kinase inhibitor R59949 both inhibited CEPT1 activity with IC50 values of 40 microM. Cancer, 2002 Sep 15, 95(6), 1199 - 205 The expression pattern of Ku correlates with tumor radiosensitivity and disease free survival in patients with rectal carcinoma; Komuro Y et al.; BACKGROUND: Preoperative radiotherapy reduces the rate of local recurrence and improves the chance of survival in patients with resectable, advanced rectal carcinoma . However, because not all tumors respond similarly to radiation, sorting out suitable patients is required to irradiate tumors rationally . The authors examined the possible role of Ku protein in determining tumor radiosensitivity and disease free survival in patients with rectal carcinoma . METHODS: The authors studied 96 patients with advanced rectal carcinoma . In preradiation biopsy specimens of tumor samples, the number of cells that were stained positive for Ku protein was evaluated by immunohistochemistry . The expression pattern of Ku protein was examined for an association between tumor radiosensitivity (which was determined according to T classification downstaging, complete pathologic response, or Response Evaluation Criteria in Solid Tumors) and disease free survival . RESULTS: There was a high degree of correlation between the percentage of cells that expressed the 70-kDa Ku protein (Ku70) and the 86-kDa Ku protein (Ku86) in the tumor sections (correlation coefficient = 0.85; P < 0.001) . The expression pattern of Ku protein was correlated not only with tumor radiosensitivity but also with disease free survival . Pathologic TMN classification, histopathologic grade, and Ku70 expression were significant prognostic variables for disease free survival in a multivariate analysis (P = 0.0031, P = 0.030, and P = 0.023, respectively) . CONCLUSIONS: Ku70 and Ku86 raise the predictive possibility of tumor radiosensitivity . Ku may be a useful parameter for selecting patients with rectal carcinoma for preoperative radiotherapy . Am J Hum Genet, 2002 Oct, 71(4), 863 - 76 Epub 2002 Sep 05. GRACILE syndrome, a lethal metabolic disorder with iron overload, is caused by a point mutation in BCS1L; Visapaa I et al.; GRACILE (growth retardation, aminoaciduria, cholestasis, iron overload, lactacidosis, and early death) syndrome is a recessively inherited lethal disease characterized by fetal growth retardation, lactic acidosis, aminoaciduria, cholestasis, and abnormalities in iron metabolism . We previously localized the causative gene to a 1.5-cM region on chromosome 2q33-37 . In the present study, we report the molecular defect causing this metabolic disorder, by identifying a homozygous missense mutation that results in an S78G amino acid change in the BCS1L gene in Finnish patients with GRACILE syndrome, as well as five different mutations in three British infants . BCS1L, a mitochondrial inner-membrane protein, is a chaperone necessary for the assembly of mitochondrial respiratory chain complex III . Pulse-chase experiments performed in COS-1 cells indicated that the S78G amino acid change results in instability of the polypeptide, and yeast complementation studies revealed a functional defect in the mutated BCS1L protein . Four different mutations in the BCS1L gene have been reported elsewhere, in Turkish patients with a distinctly different phenotype . Interestingly, the British and Turkish patients had complex III deficiency, whereas in the Finnish patients with GRACILE syndrome complex III activity was within the normal range, implying that BCS1L has another cellular function that is uncharacterized but essential and is putatively involved in iron metabolism. Mol Cell Biol, 2002 Oct, 22(19), 6759 - 66 Molecular interaction and synergistic activation of a promoter by Six, Eya, and Dach proteins mediated through CREB binding protein; Ikeda K et al.; Drosophila sine oculis, eyes absent, and dachshund are essential for compound eye formation and form a gene network with direct protein interaction and genetic regulation . The vertebrate homologues of these genes, Six, Eya, and Dach, also form a similar genetic network during muscle formation . To elucidate the molecular mechanism underlying the network among Six, Eya, and Dach, we examined the molecular interactions among the encoded proteins . Eya interacted directly with Six but never with Dach . Dach transactivated a multimerized GAL4 reporter gene by coproduction of GAL4-Eya fusion proteins . Transactivation by Eya and Dach was repressed by overexpression of VP16 or E1A but not by E1A mutation, which is defective for CREB binding protein (CBP) binding . Recruitment of CBP to the immobilized chromatin DNA template was dependent on FLAG-Dach and GAL4-Eya3 . These results indicate that CBP is a mediator of the interaction between Eya and Dach . Contrary to our expectations, Dach binds to chromatin DNA by itself, not being tethered by GAL4-Eya3 . Dach also binds to naked DNA with lower affinity . The conserved DD1 domain is responsible for binding to DNA . Transactivation was also observed by coproduction of GAL4-Six, Eya, and Dach, indicating that Eya and Dach synergy is relevant when Eya is tethered to DNA through Six protein . Our results demonstrated that synergy is mediated through direct interaction of Six-Eya and through the interaction of Eya-Dach with CBP and explain the molecular basis for the genetic interactions among Six, Eya, and Dach . This work provides fundamental information on the role and the mechanism of action of this gene cassette in tissue differentiation and organogenesis. Mol Cell Biol, 2002 Oct, 22(19), 6750 - 8 Activation of the Bur1-Bur2 cyclin-dependent kinase complex by Cak1; Yao S et al.; Cyclin-dependent kinases (Cdks) were originally identified as regulators of eukaryotic cell cycle progression, but several Cdks were subsequently shown to perform important roles as transcriptional regulators . While the mechanisms regulating the Cdks involved in cell cycle progression are well documented, much less is known regarding how the Cdks that are involved in transcription are regulated . In Saccharomyces cerevisiae, Bur1 and Bur2 comprise a Cdk complex that is involved in transcriptional regulation, presumably mediated by its phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II . To investigate the regulation of Bur1 in vivo, we searched for high-copy-number suppressors of a bur1 temperature-sensitive mutation, identifying a single gene, CAK1 . Cak1 is known to activate two other Cdks in yeast by phosphorylating a threonine within their conserved T-loop domains . Bur1 also has the conserved threonine within its T loop and is therefore a potential direct target of Cak1 . Additional tests establish a direct functional interaction between Cak1 and the Bur1-Bur2 Cdk complex: Bur1 is phosphorylated in vivo, both the conserved Bur1 T-loop threonine and Cak1 are required for phosphorylation and Bur1 function in vivo, and recombinant Cak1 stimulates CTD kinase activity of the purified Bur1-Bur2 complex in vitro . Thus, both genetic and biochemical evidence demonstrate that Cak1 is a physiological regulator of the Bur1 kinase. Mol Cell Biol, 2002 Oct, 22(19), 6735 - 49 The Ccr4-not complex and yTAF1 (yTaf(II)130p/yTaf(II)145p) show physical and functional interactions; Deluen C et al.; The Saccharomyces cerevisiae Ccr4-Not complex is a global regulator of transcription that is thought to regulate TATA binding protein (TBP) function at certain promoters specifically . In this paper, we show interactions between the essential domain of Not1p, which interacts with Not4p and Not5p, and the N-terminal domain of yTAF1 . We isolated a temperature-sensitive nonsense allele of TAF1, taf1-4, which is synthetically lethal at the permissive temperature when combined with not4 and not5 mutants and which produces high levels of a C-terminally truncated yTAF1 derivative . Overexpression of C-terminally truncated yTAF1 is toxic in not4 or not5 mutants, whereas overexpression of full-length yTAF1 suppresses not4 . Furthermore, mutations in the autoinhibitory N-terminal TAND domain of yTAF1 suppress not5, and the overexpression of similar mutants does not suppress not4 . We find that, like Not5p, yTAF1 acts as a repressor of stress response element-dependent transcription . Finally, we have evidence for stress-regulated occupancy of promoter DNA by Not5p and for Not5p-dependent regulation of yTAF1 association with promoter DNA . Taken together with our finding that Not1p copurifies with glutathione S-transferase-yTaf1 in large complexes, these results provide the first molecular evidence that the Ccr4-Not complex might interact with yTAF1 to regulate its association at promoters, a function that might in turn regulate the autoinhibitory N-terminal domain of yTAF1. Mol Cell Biol, 2002 Oct, 22(19), 6669 - 80 Isolation and characterization of new proliferating cell nuclear antigen (POL30) mutator mutants that are defective in DNA mismatch repair; Lau PJ et al.; A number of studies have suggested a role for proliferating cell nuclear antigen (PCNA) in DNA mismatch repair (MMR) . However, the majority of mutations in the POL30 gene encoding PCNA that cause MMR defects also cause replication and other repair defects that contribute to the increased mutation rate caused by these mutations . Here, 20 new pol30 mutants were identified and screened for MMR and other defects, resulting in the identification of two mutations, pol30-201 and pol30-204, that appear to cause MMR defects but little if any other defects . The pol30-204 mutation altered an amino acid (C81R) in the monomer-monomer interface region and resulted in a partial general MMR defect and a defect in MSH2-MSH6 binding in vitro . The pol30-201 mutation altered an amino acid (C22Y) located on the surface of the PCNA trimer that slides over the DNA but did not cause a defect in MSH2-MSH6 binding in vitro . The pol30-201 mutation caused an intermediate mutator phenotype . However, the pol30-201 mutation caused almost a complete defect in the repair of AC and GT mispairs and only a small defect in the repair of a "+T" insertion, an effect similar to that caused by an msh6Delta mutation, indicating that pol30-201 primarily effects MSH6-dependent MMR . The chromosomal double mutant msh3-FF>AA msh6-FF>AA eliminating the conserved FF residues of the PCNA interacting motif of these proteins caused a small (<10%) defect in MMR but showed synergistic interactions with mutations in POL30, indicating that the FF>AA substitution may not eliminate PCNA interactions in vivo . These results indicate that the interaction between PCNA and MMR proteins is more complex than was previously appreciated. Mol Cell Biol, 2002 Oct, 22(19), 6663 - 8 Purified box C/D snoRNPs are able to reproduce site-specific 2'-O-methylation of target RNA in vitro; Galardi S et al.; Small nucleolar RNAs (snoRNAs) are associated in ribonucleoprotein particles localized to the nucleolus (snoRNPs) . Most of the members of the box C/D family function in directing site-specific 2'-O-methylation of substrate RNAs . Although the selection of the target nucleotide requires the antisense element and the conserved box D or D' of the snoRNA, the methyltransferase activity is supposed to reside in one of the protein components . Through protein tagging of a snoRNP-specific factor, we purified to homogeneity box C/D snoRNPs from the yeast Saccharomyces cerevisiae . Mass spectrometric analysis demonstrated the presence of Nop1p, Nop58p, Nop56p, and Snu13p as integral components of the particle . We show that purified snoRNPs are able to reproduce the site-specific methylation pattern on target RNA and that the predicted S-adenosyl-L-methionine-binding region of Nop1p is responsible for the catalytic activity. J Biol Chem, 2002 Nov 8, 277(45), 43288 - 300 Epub 2002 Sep 04. Barrier to autointegration factor interacts with the cone-rod homeobox and represses its transactivation function; Wang X et al.; Crx (cone-rod homeobox) is a homeodomain transcription factor implicated in regulating the expression of photoreceptor and pineal genes . To identify proteins that interact with Crx in the retina, we carried out a yeast two-hybrid screen of a retinal cDNA library . One of the identified clones encodes Baf (barrier to autointegration factor), which was previously shown to have a role in mitosis and retroviral integration . Additional biochemical assays provided supporting evidence for a Baf-Crx interaction . The Baf protein is detectable in all nuclear layers of the mouse retina, including the photoreceptors and the bipolar cells where Crx is expressed . Transient transfection assays with a rhodopsin-luciferase reporter in HEK293 cells demonstrate that overexpression of Baf represses Crx-mediated transactivation, suggesting that Baf acts as a negative regulator of Crx . Consistent with this role for Baf, an E80A mutation of CRX associated with cone-rod dystrophy has a higher than normal transactivation potency but a reduced interaction with Baf . Although our studies did not identify a causative Baf mutation in retinopathies, we suggest that Baf may contribute to the phenotype of a photoreceptor degenerative disease by modifying the activity of Crx . In view of the ubiquitous expression of Baf, we hypothesize that it may play a role in regulating tissue- or cell type-specific gene expression by interacting with homeodomain transcription factors. J Mol Biol, 2002 Sep 6, 322(1), 27 - 39 Characterization of mutations in NOT2 indicates that it plays an important role in maintaining the integrity of the CCR4-NOT complex; Russell P et al.; The NOT2 protein is a component of the CCR4-NOT complex that plays multiple roles in the regulation of mRNA production in the yeast Saccharomyces cerevisiae . We have identified four novel not2 mutations and have characterized these and two previously described alleles as to the means by which they affect CCR4-NOT function . While two of the not2 alleles, not2-4 (carrying a G31R alteration) and not2::L9P, resulted in severe growth defects and caused a not phenotype at the HIS3 locus, these phenotypes appear to arise from partially different effects . The not2::L9P mutation resulted in complete loss of the 1.9x10(6)Da (1.9MDa) CCR4-NOT complex, and the not2::L9P protein displayed increased ability to associate with the NOT5 protein . In contrast, the not2-4 allele destabilized the CCR4-NOT complex to a lesser extent and had no effect on NOT5 association with NOT2 . Instead, as previously reported, it displayed defective interactions with ADA2, a component of the SAGA complex . The not2::R165G also abrogated NOT2 ability to interact with ADA2 but had little effect on the integrity of the CCR4-NOT complex . Alterations in NOT2 contacts to ADA2, therefore, do not necessarily result in effects on the CCR4-NOT complex nor result in severe growth defects . We also observed that the four NOT2 N-terminal mutations affected NOT5 association with the CCR4-NOT complexes, suggesting that it is the N terminus of NOT2 that contacts and stabilizes NOT5 interactions . These results indicate that it is the loss of the integrity of the CCR4-NOT complex which leads to severe not2 phenotypes and that the NOT2 contacts to ADA2 play a lesser role in NOT2 function. Oncogene, 2002 Sep 12, 21(41), 6377 - 81 Ku affects the ATM-dependent S phase checkpoint following ionizing radiation; Zhou XY et al.; Following exposure to genotoxic stress, proliferating cells actively slow down DNA replication through an S phase checkpoint to provide time for repair . The ATM-dependent pathway plays an important role in the S phase checkpoint response following ionizing irradiation . We report that there is a stronger S phase checkpoint response in irradiated Ku80(-/-) cells as compared with their wild-type counterparts, which has no relationship to DNA-dependent protein kinase (DNA-PK) activity but correlates with a higher ATM activity and with more ATM bound to chromatin DNA in such cells . Wortmannin, a nonspecific inhibitor of ATM, not only reduces the higher activity of ATM kinase, but also abolishes the stronger S phase checkpoint response in Ku80(-/-) cells . Furthermore, a specific ATM antisense oligonucleotide abolishes the stronger S checkpoint response in Ku80(-/-) cells and renders these cells practically indistinguishable from Ku80(+/+) cells for this endpoint . These results in aggregate indicate that the stronger S checkpoint in irradiated Ku80(-/-) cells is due to the higher ATM kinase activity. Microbiology, 2002 Sep, 148(Pt 9), 2811 - 7 Compensatory expression of five chitin synthase genes, a response to stress stimuli, in Wangiella (Exophiala) dermatitidis, a melanized fungal pathogen of humans; Wang Q et al.; Numerous chitin synthase structural (CHS) genes have been identified in fungi, and usually there are several CHS genes per species . Compensatory expression of one CHS gene in response to defects in other CHS genes has not been reported . Five chitin synthase structural (WdCHS) genes have been identified in the melanized human pathogen Wangiella dermatitidis: WdCHS1, WdCHS2, WdCHS3, WdCHS4 and WdCHS5 . This study showed that increased WdCHS expression existed as a compensatory mechanism in response to stress induced by chitin synthase gene disruptions, or by exposure of the wild-type or two temperature-sensitive morphological mutants, for short or long periods, to 37 degrees C . In general, the compensatory responses varied with each WdCHS gene, and in accordance with the hypothesized functions of the chitin synthase (WdChsp) encoded . It is suggested that these compensatory responses indicate that WdCHS gene transcription in W . dermatitidis functions as part of a cell-wall integrity pathway in a manner similar to that recently described for Saccharomyces cerevisiae. J Biol Chem, 2002 Nov 8, 277(45), 42686 - 93 Epub 2002 Sep 03. Functional characterization of the 11 non-ATPase subunit proteins in the trypanosome 19 S proteasomal regulatory complex; Li Z et al.; The ubiquitin-proteasome pathway is responsible for selective degradation of short-lived and dysfunctional proteins in eukaryotes . The recently demonstrated presence of a functional 26 S proteasome in Trypanosoma brucei led to the identification and isolation of genes encoding all 11 non-ATPase (Rpn) subunit proteins in the trypanosome 19 S regulatory complex . Using the technique of RNA interference, expression of individual RPN genes was disrupted in the procyclic form of T . brucei, resulting, in each case, in intracellular accumulation of polyubiquitinated protein, cell arrest at the G2/M phase, and eventual cell death . With the exception of Rpn10, depletion of individual Rpn proteins disrupted also trypanosome 19 S complex formation, with the complex virtually depleted in the cell lysate . This functional and structural essentiality of 10 of the 11 Rpn proteins in T . brucei differs significantly from that observed in other organisms . When Rpn10 was deficient in trypanosomes, a 19 S complex without Rpn10 was still formed, whereas cell growth was arrested . This structural dispensability but functional indispensability of Rpn10 may constitute another unique aspect of the proteasomes in T . brucei. J Biol Chem, 2002 Nov 15, 277(46), 44244 - 51 Epub 2002 Sep 03. Glucose down-regulates Per1 and Per2 mRNA levels and induces circadian gene expression in cultured Rat-1 fibroblasts; Hirota T et al.; In mammals, peripheral circadian clocks are present in most tissues, but little is known about how these clocks are synchronized with the ambient 24-h cycles . By using rat-1 fibroblasts, a model cell system of the peripheral clock, we found that an exchange of the culture medium triggered circadian gene expression that was preceded by slow down-regulation of Per1 and Per2 mRNA levels . This profile contrasts to the immediate up-regulation of these genes often observed for clock resetting . The screening of factor(s) responsible for the down-regulation revealed glucose as a key component triggering the circadian rhythm . The requirement of both glucose metabolism and RNA/protein synthesis for the down-regulation suggests the involvement of gene(s) immediately up-regulated by glucose metabolism . An analysis with high density oligonucleotide microarrays identified >100 glucose-regulated genes . We found among others immediately up-regulated genes encoding transcriptional regulators TIEG1, VDUP1, and HES1, in addition to cooperatively regulated genes that are associated with cholesterol biosynthesis and cell cycle . The immediate up-regulation of Tieg1 and Vdup1 expression was dependent on glucose metabolism but not on protein synthesis, suggesting that the transcriptional regulators mediate the glucose-induced down-regulation of Per1 and Per2 expression . These results illustrate a novel mode of peripheral clock resetting by external glucose, a major food metabolite. Biochim Biophys Acta, 2002 Sep 13, 1577(2), 308 - 24 RNA polymerase II conducts a symphony of pre-mRNA processing activities; Howe KJ; RNA polymerase II (RNAP II) and its associated factors interact with a diverse collection of nuclear proteins during the course of precursor messenger RNA synthesis . This growing list of known contacts provides compelling evidence for the existence of large multifunctional complexes, a.k.a . transcriptosomes, within which the biosynthesis of mature mRNAs is coordinated . Recent studies have demonstrated that the unique carboxy-terminal domain (CTD) of the largest subunit of RNAP II plays an important role in recruiting many of these activities to the transcriptional machinery . Throughout the transcription cycle the CTD undergoes a variety of covalent and structural modifications which can, in turn, modulate the interactions and functions of processing factors during transcription initiation, elongation and termination . New evidence suggests that the possibility that interaction of some of these processing factors with the polymerase can affect its elongation rate . Besides the CTD, proteins involved in pre-mRNA processing can interact with general transcription factors (GTFs) and transcriptional activators, which associate with polymerase at promoters . This suggests a mechanism for the recruitment of specific processing activities to different transcription units . This harmonic integration of transcriptional and post-transcriptional activities, many of which once were considered to be functionally isolated within the cell, supports a general model for the coordination of gene expression by RNAP II within the nucleus. Biochim Biophys Acta, 2002 Sep 13, 1577(2), 276 - 86 Transcript elongation on a nucleoprotein template; Hartzog GA et al.; Chromatin forms a general, repeating barrier to elongation of transcripts by eukaryotic RNA polymerases . Recent studies of nucleosome structure and histone modifications reveal a set of likely mechanisms for control of elongation through chromatin . Genetic and biochemical studies of transcription have identified a set of accessory factors for transcript elongation by RNA polymerase II (Pol II) that appear to function in the context of chromatin . The C-terminal repeated domain (CTD) of Pol II may also play a role in regulating elongation through chromatin. Biochim Biophys Acta, 2002 Sep 13, 1577(2), 261 - 275 Regulation of transcription elongation by phosphorylation; Kobor MS et al.; The synthesis of mRNA by RNA polymerase II (RNAPII) is a multistep process that is regulated by different mechanisms . One important aspect of transcriptional regulation is phosphorylation of components of the transcription apparatus . The phosphorylation state of RNAPII carboxy-terminal domain (CTD) is controlled by a variety of protein kinases and at least one protein phosphatase . We discuss emerging genetic and biochemical evidence that points to a role of these factors not only in transcription initiation but also in elongation and possibly termination . In addition, we review phosphorylation events involving some of the general transcription factors (GTFs) and other regulatory proteins . As an interesting example, we describe the modulation of transcription associated kinases and phosphatase by the HIV Tat protein . We focus on bringing together recent findings and propose a revised model for the RNAPII phosphorylation cycle. Biochem Pharmacol, 2002 Sep, 64(5-6), 935 - 42 Oxidative stress, transcription factors and chromatin remodelling in lung inflammation; Rahman I; Oxidative stress has been implicated in the pathogenesis of several inflammatory lung disorders . Oxidants and inflammatory mediators such as tumour necrosis factor-alpha (TNF-alpha) activate transcription factors such as nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) leading to the expression of pro-inflammatory genes . The expression of many genes, including those encoding pro-inflammatory mediators involves the remodelling of the chromatin structure provided by histone proteins . Histone acetylation causes the unwinding of chromatin structure therefore allowing transcription factor access to promoter sites . Nuclear histone acetylation is a reversible process, and is regulated by a group of acetyltransferases (HATs) which promote acetylation, and deacetylases (HDACs) which promote deacetylation . In addition, several co-activators, transcription factors and nuclear proteins also have histone acetyltransferase activity . Both TNF-alpha and the oxidant, hydrogen peroxide (H2O2) alter histone acetylation/deacetylation, and the activation of NF-kappaB and AP-1, leading to the release of the pro-inflammatory cytokine interleukin-8 (IL-8) in human alveolar epithelial cells (A549) . Pharmacological inhibition of HDAC leads to the increased HAT activity, AP-1 and NF-kappaB activation, and IL-8 release by H2O2 or TNF-alpha treatments . This suggests that the remodelling of chromatin by histone acetylation plays a role in the oxidant-mediated pro-inflammatory responses in the lungs. Insect Biochem Mol Biol, 2002 Sep, 32(9), 961 - 6 Acyl-CoA Z9- and Z10-desaturase genes from a New Zealand leafroller moth species, Planotortrix octo; Hao G et al.; Two cDNAs encoding acyl-CoA Z9-desaturase from the fat body and Z10-desaturase from the pheromone gland of the greenhead leafroller moth, Planotortrix octo, were obtained by RACE PCR . The Z9-desaturase (Pocto-Z9) cDNA spans 2291 nt with an ORF encoding a 352 amino-acid protein, which has 65% identity to Trichoplusia ni Delta 9 desaturase (Tni-Z9) . The Z10-desaturase (Pocto-Z10) cDNA spans 2777 nt with an ORF encoding a protein with 356 amino acids . Pocto-Z10 shows lower identity to Pocto-Z9 and Tni-Z9 (48 and 46%, respectively) and relatively higher identity to the Delta 11 desaturases of T . ni and Helicoverpa zea (57 and 56%, respectively) . The ORFs of these two P . octo cDNAs were constructed into an expression vector, YEpOLEX, that complemented the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient ole1 strain of Saccharomyces cerevisiae . Expression of Pocto-Z9 produced a 5:2 ratio of Z9-16 and Z9-18 acids, with minor amounts (<4%) of Z9-14, Z9-15, and Z9-17 acids . Pocto-Z10 was successfully expressed in the YEpOLEX system when complemented with Z11-18:Me, and the major desaturase product proved to be Z10-16:Acid . The results confirm the regio- and stereo-selectivity of this unusual Delta 10 desaturase. Proteins, 2002 Oct 1, 49(1), 125 - 34 Understanding protein structure-function relationships in Family 47 alpha-1,2-mannosidases through computational docking of ligands; Mulakala C et al.; Family 47 alpha-1,2-mannosidases are crucial enzymes involved in N-glycan maturation in the endoplasmic reticula and Golgi apparati of eukaryotic cells . High-resolution crystal structures of the human and yeast endoplasmic reticulum alpha-1,2-mannosidases have been recently determined, the former complexed with the inhibitors 1-deoxymannojirimycin and kifunensine, both of which bind in its active site in the unusual 1C4 conformation . However, unambiguous identification of the catalytic proton donor and nucleophile involved in glycoside bond hydrolysis was not possible from this structural information . In this work, alpha-D-galactose, alpha-D-glucose, and alpha-D-mannose were computationally docked in the active site in the energetically stable 4C1 conformation as well as in the 1C4 conformation to compare their interaction energetics . From these docked structures, a model for substrate and conformer selectivity based on the dimensions of the active site was proposed . Alpha-D-galactopyranosyl-(1-->2)-alpha-D-mannopyranose, alpha-D-glucopyranosyl-(1-->2)-alpha-D-mannopyranose, and alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranose were also docked into the active site with their nonreducing-end residues in the 1C4 and E4 (representing the transition state) conformations . Based on the docked structure of alpha-D-mannopyranosyl-E4-(1-->2)-alpha-D-mannopyranose, the catalytic acid and base are Glu132 and Glu435, respectively . Yeast, 2002 Sep 15, 19(12), 1075 - 86 Genetic interactions link ARF1, YPT31/32 and TRS130; Zhang CJ et al.; A genetic screen for synthetic lethal interactions with arf1(-) identified a recessive mutation in TRS130, one of 10 components in the trafficking protein particle (TRAPP) complex (Sacher et al., 2000) . As TRS130 is an essential gene, the synthetic lethal allele (trs130-101) is a novel one that requires ARF1 for viability . This allele was found to exhibit no defects in secretory function, i.e . processing of carboxypeptidase Y or invertase . YPT31 and YPT32 were identified in a subsequent screen as high-copy suppressors of arf1(-)trs130-101 . Increasing the gene dosage of YPT31/32 also suppressed lethality resulting from deletion of TRS130 or TRS120 but not three other essential TRAPP subunit-encoding genes . Although unable to suppress defects in several alleles of ARF1, increasing the gene dosage of YPT31/32 suppressed the cold sensitivity of gcs1(-), an Arf GTPase-activating protein (GAP) . Thus, these genetic interactions provide initial evidence for linkage of Arf and TRAPP signalling and for Ypt31/32 proteins functioning downstream of both components in the TRAPP complex and of Arf signalling via the Gcs1 Arf GAP . Yeast, 2002 Sep 15, 19(12), 1015 - 27 Characterization of the putative maltose transporters encoded by YDL247w and YJR160c; Day RE et al.; The maltose permease family of Saccharomyces cerevisiae comprises five proteins, three of which are characterized, MAL31, MAL61 and AGT1 and two putative permeases, YDL247w (MPH2) and YJR160c (MPH3) . The two uncharacterized permeases share 100% identity and have 75% identity with MAL31 and MAL61 and 55% identity with AGT1 . Characterization of the genes YDL247w and YJR160c confirmed that they encode alpha-glucoside permeases capable of transporting maltose, maltotriose, alpha-methylglucoside and turanose . Analysis of the promoter regions identified regulatory elements, binding sites for the transcriptional activator, Malx3p and the inhibitory protein, Mig1p . Further analysis of the flanking sequences located blocks of identity covering five open reading frames, indicating that this region was involved in chromosomal block duplication . The members of the maltose permease family are proteins that have strongly overlapping but nevertheless distinct functions, which is a selective advantage for yeast, as it reflects successful adaptation to the variety of environmental conditions to which the yeast cells are exposed; such adaptability is very important in an industrial context . Bioessays, 2002 Sep, 24(9), 785 - 8 Non-stop decay--a new mRNA surveillance pathway; Vasudevan S et al.; Gene expression is an inherently complex process and errors often occur during the transcription and processing of mRNAs . Several surveillance mechanisms have evolved to check the fidelity at each step of mRNA manufacture . Two recent reports describe the identification of a novel pathway in eukaryotes that recognizes and degrades mRNAs that lack a stop codon . The non-stop decay mechanism releases ribosomes stalled at the 3' end of a mRNA and stimulates the exosome to rapidly degrade the transcript . Bioessays, 2002 Sep, 24(9), 780 - 4 When machines get stuck--obstructed RNA polymerase II: displacement, degradation or suicide; van den Boom V et al.; The severe hereditary progeroid disorder Cockayne syndrome is a consequence of a defective transcription-coupled repair (TCR) pathway . This special mode of DNA repair aids a RNA polymerase that is stalled by a DNA lesion in the template and ensures efficient DNA repair to permit resumption of transcription and prevent cell death . Although some key players in TCR, such as the Cockayne syndrome A (CSA) and B (CSB) proteins have been identified, the exact molecular mechanism still remains illusive . A recent report provides new unexpected insights into TCR in yeast . The identification and characterisation of a novel protein co-purifying with the yeast homologue of CSB (Rad26) imposes reassessment of our current understanding of TCR in yeast . What about humans? J Pathol, 2002 Sep, 198(1), 121 - 30 Heterogeneous expression of Ku70 in human tissues is associated with morphological and functional alterations of the nucleus; Choi EK et al.; Ku70 is a subunit of DNA-protein kinase complex and involved in diverse intranuclear events including the repair of double-stranded DNA breaks . Ku70 is rich in the interphase nucleus of cultured cells . In human tissues, however, the distribution of Ku70 has not yet been systematically examined . To characterize the difference of Ku70 distribution between cells of human tissues and cultured cells, the expression of Ku70 was examined in various normal and neoplastic human tissues by immunohistochemistry and immunoblot . In addition, the role of Ku70 in the cellular response against ionizing radiation (IR) was analysed in fibroblasts after exposure to 5 Gy IR and apoptotic indices were examined in Ku70-overexpressed fibroblasts from an ataxia telangiectasia patient and in normal fibroblasts, before and after irradiation . In contrast to cultured cells, Ku70 was not detected in some interphase cells of human tissues and was distributed heterogeneously, even in the same nucleus . Ku70 expression was strikingly low in terminally differentiated cells such as neutrophils, eosinophils, glomerular capillary endothelial cells and fibroblasts, and was absent in spermatids . In spermatocytes, Ku70 was tightly integrated with chromosome filaments, unlike other somatic cells under mitosis . After exposure to IR, Ku70 expression was not increased in ataxia telangiectasia fibroblasts, but was significantly increased in normal fibroblasts . Most of the increased Ku70 was of soluble nuclear protein fraction . Furthermore, overexpression of Ku70 increased radiation resistance both in ataxia telangiectasia fibroblasts and normal fibroblasts . The presented data indicate that the distribution of Ku70 in cells of human tissues is closely associated with the cell cycle, cellular differentiation, nuclear shape and the process of repair of DNA damage caused by IR . J Pathol, 2002 Sep, 198(1), 55 - 9 Distinct promoter hypermethylation of p16INK4a, CDH1, and RAR-beta in intestinal, diffuse-adherent, and diffuse-scattered type gastric carcinomas; Oue N et al.; Hypermethylation of CpG islands in gene promoters is associated with silencing of various tumour suppressor genes . Recent studies of colorectal and gastric carcinomas have defined a CpG island methylator phenotype (CIMP), which involves the targeting of multiple genes by promoter hypermethylation . In this study, methylation-specific polymerase chain reaction (PCR) was performed to study methylation of CpG islands in the promoters of the p16(INK4a), cadherin 1 (CDH1), and retinoic acid receptor-beta (RAR-beta) genes in 45 gastric carcinomas and to investigate whether CDH1 and RAR-beta promoter hypermethylation is associated with CIMP-positive gastric carcinoma . CpG island hypermethylation of the p16(INK4a), CDH1, and RAR-beta promoters was detected in 12 (27%), 26 (58%), and 24 (53%) of the 45 gastric carcinomas, respectively . Hypermethylation of the p16(INK4a) promoter was more common in intestinal type than in diffuse type gastric carcinomas (p = 0.0023; Fisher's exact test) and was inversely associated with p53 mutations (p = 0.0225; Fisher's exact test) . However, CDH1 and RAR-beta promoter hypermethylation was observed more frequently in diffuse-scattered type gastric carcinoma than in other types (intestinal and diffuse-adherent types) (p = 0.0175 and p = 0.0335, respectively; Fisher's exact test) and was not associated with p53 mutation status . Moreover, hypermethylation of the CDH1 and RAR-beta promoters occurred concordantly (p < 0.0001; Fisher's exact test) . These results suggest that at least two types of promoter methylation status are involved in the development of the intestinal (p16(INK4a) promoter hypermethylation) and diffuse-scattered types (CDH1 and RAR-beta promoter hypermethylation) of gastric carcinoma . J Cell Physiol, 2002 Oct, 193(1), 120 - 31 Titin-cap associates with, and regulates secretion of, Myostatin; Nicholas G et al.; Myostatin, a secreted growth factor, is a key negative regulator of skeletal muscle growth . To identify modifiers of Myostatin function, we screened for Myostatin interacting proteins . Using a yeast two-hybrid screen, we identified Titin-cap (T-cap) protein as interacting with Myostatin . T-cap is a sarcomeric protein that binds to the N-terminal domain of Titin and is a substrate of the titin kinase . Mammalian two-hybrid studies, in vitro binding assays and protein truncations in the yeast two-hybrid system verified the specific interaction between processed mature Myostatin and full-length T-cap . Analysis of protein-protein interaction using surface plasmon resonance (Biacore, Uppsala, Sweden) kinetics revealed a high affinity between Myostatin and T-cap with a KD of 40 nM . When T-cap was stably overexpressed in C(2)C(12) myoblasts, the rate of cell proliferation was significantly increased . Western analyses showed that production and processing of Myostatin were not altered in cells overexpressing T-cap, but an increase in the retention of mature Myostatin indicated that T-cap may block Myostatin secretion . Bioassay for Myostatin confirmed that conditioned media from myoblasts overexpressing T-cap contained lower levels of Myostatin . Given that Myostatin negatively regulates myoblast proliferation, the increase in proliferation observed in myoblasts overexpressing T-cap could thus be due to reduced Myostatin secretion . These results suggest that T-cap, by interacting with Myostatin, controls Myostatin secretion in myogenic precursor cells without affecting the processing step of precursor Myostatin . J Virol, 2002 Oct, 76(19), 9856 - 67 Flock house virus RNA polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion; Miller DJ et al.; Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses . Replication complexes of flock house virus (FHV), the best-studied alphanodavirus, are located on outer mitochondrial membranes in infected Drosophila melanogaster cells and are associated with the formation of membrane-bound spherules, similar to structures found for many other positive-strand RNA viruses . To further study FHV replication complex formation, we investigated the subcellular localization, membrane association, and membrane topology of protein A, the FHV RNA-dependent RNA polymerase, in the yeast Saccharomyces cerevisiae, a host able to support full FHV RNA replication and virion formation . Confocal immunofluorescence revealed that protein A localized to mitochondria in yeast, as in Drosophila cells, and that this mitochondrial localization was independent of viral RNA synthesis . Nycodenz gradient flotation and dissociation assays showed that protein A behaved as an integral membrane protein, a finding consistent with a predicted N-proximal transmembrane domain . Protease digestion and selective permeabilization after differential epitope tagging demonstrated that protein A was inserted into the outer mitochondrial membrane with the N terminus in the inner membrane space or matrix and that the C terminus was exposed to the cytoplasm . Flotation and immunofluorescence studies with deletion mutants indicated that the N-proximal region of protein A was important for both membrane association and mitochondrial localization . Gain-of-function studies with green fluorescent protein fusions demonstrated that the N-terminal 46 amino acids of protein A were sufficient for mitochondrial localization and membrane insertion . We conclude that protein A targets and anchors FHV RNA replication complexes to outer mitochondrial membranes, in part through an N-proximal mitochondrial localization signal and transmembrane domain. FEBS Lett, 2002 Aug 28, 526(1-3), 142 - 6 PIAS3 (protein inhibitor of activated STAT-3) modulates the transcriptional activation mediated by the nuclear receptor coactivator TIF2; Jimenez-Lara AM et al.; PIAS3, a member of the protein inhibitor of activated STAT family, was found to interact in vivo and in vitro with TIF2, a previously described coactivator for nuclear receptors . The interaction is mediated by two distinct non-contiguous regions of TIF2 . We found that TIF2-PIAS3 interaction occurs through a unique domain of PIAS3, very rich in acidic residues and conserved throughout the PIAS family . PIAS3 modulates the ability of TIF2 to mediate ligand-enhanced transcription activation positively or negatively, for different steroid receptors . Taken together, our results indicate a potential role of PIAS3 as transcriptional modulator of TIF2-mediated signalling. FEBS Lett, 2002 Aug 28, 526(1-3), 71 - 6 Ccz1p/Aut11p/Cvt16p is essential for autophagy and the cvt pathway; Meiling-Wesse K et al.; In a reverse genetics screen, we here identify Ccz1p as an essential component of the cvt pathway and autophagy . Ccz1p is identical with the so far unknown Cvt16p . GFP-Aut7p, a specific cargo of autophagosomes, accumulates in ccz1 Delta cells at punctate, vesicular structures in the cytosol, suggesting a block in the autophagic pathway prior to vacuolar fusion of autophagosomes . Proteinase protection experiments using hypotonically lysed ccz1 Delta spheroplasts demonstrate that proaminopeptidase I, another specific cargo of autophagy and the cvt pathway, is trapped inside membrane-enclosed vesicles . Taken together our findings are compatible with a function of Ccz1p in vacuolar fusion of cvt vesicles and autophagosomes. Biochem Biophys Res Commun, 2002 Sep 6, 296(5), 1164 - 70 Mammalian Apg12p, but not the Apg12p.Apg5p conjugate, facilitates LC3 processing; Tanida I et al.; A dynamic membrane rearrangement occurs in cells during autophagy to form autophagosomes . In this dynamic process, two ubiquitin-like modifications, Apg12p-conjugation and LC3-modification, are essential for the formation of autophagosomes . Apg7p and Apg10p catalyze the conjugation of Apg12p to Apg5p . The same Apg7p and Apg3p catalyze the processing of LC3 to a membrane-bound form, LC3-II . In this paper, we investigated whether Apg12p has an influence on the second LC3-modification system . A cross-linking experiment revealed that Apg3p interacts with the endogenous Apg12p.Apg5p conjugate . However, Apg3p itself interacts with free Apg12p more preferentially than the Apg12p.Apg5p conjugate, when free Apg12p exists . When Apg12p was overexpressed, LC3 processing was significantly enhanced in the presence of Apg7p . In contrast, when the Apg12p.Apg5p conjugate itself was accumulated by the overexpression of Apg12p and Apg5p, LC3 processing was dominantly inhibited, even in the presence of Apg7p . These results indicate that both Apg12p and the Apg12p.Apg5p conjugate are regulatory factors for LC3 processing. Biochem Biophys Res Commun, 2002 Sep 6, 296(5), 1104 - 11 VCY2 protein interacts with the HECT domain of ubiquitin-protein ligase E3A; Wong EY et al.; VCY2 locates in the AZFc region on chromosome Yq and is frequently deleted in infertile men with severe oligozoospermia or azoospermia . VCY2 is a testis-specific protein with unknown function . This study was to identify the protein that interacts with VCY2 . We used the full-length VCY2 as bait to screen the human testis cDNA library using yeast two-hybrid approach . We identified a number of positive-interacting clones that encode ubiquitin-protein ligase E3A (UBE3A) . UBE3A contains a HECT domain that binds VCY2 . The specificity of the interaction was confirmed by co-immunoprecipitation and yeast mating . Northern blot analyses revealed two UBE3A transcripts 1.4 and 2kb that were abundantly expressed in human testis . We also showed that both VCY2 and UBE3A mRNAs were expressed in ejaculated human spermatozoa, indicating that both genes localize in the germ cell compartment . These data suggest that UBE3A ubiquitination may be required for VCY2 function. J Agric Food Chem, 2002 Sep 11, 50(19), 5496 - 502 Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by human cytochrome P450-dependent monooxygenase systems; Inouye K et al.; Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by monooxygenase systems dependent on 12 forms of human cytochrome P450 (CYP) was examined with the recombinant yeast microsomes containing each of the human CYP . The metabolites of PCDDs were analyzed by HPLC and GC-MS . Remarkable metabolism by the multiple CYP forms was observed toward dibenzo-p-dioxin (DD) and mono-, di-, and trichloroDDs . The metabolism contained multiple reactions such as hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, and hydroxylation with elimination of a chloride substituent . Although major CYPs in human liver such as CYP2C8, CYP2C9, and CYP3A4 showed no significant metabolism toward the PCDDs, CYP1A1 and CYP1A2 showed high catalytic activity toward DD and mono-, di-, and trichloroDDs . The kinetic parameters K(m)(app) and V(max) of the CYP1A1-dependent 8-hydroxylation activity of 2,3,7-trichloro-DD (2,3,7-triCDD) were estimated to be 0.30 microM and 51 (mol/min/mol of P450), respectively, suggesting that 2,3,7-triCDD was a good substrate for CYP1A1 . However, none of the CYPs showed any detectable activity {<0.01 mol/min/mol of P450)} toward 2,3,7,8-tetraCDD . Substrate-induced absorption spectrum and inhibition studies indicated that CYP1A1 could bind 2,3,7,8-tetraCDD with considerably high affinity . It was strongly suggested that the long half-life (7.1 years) of 2,3,7,8-tetraCDD in humans was due to an extremely low activity of CYPs toward 2,3,7,8-tetraCDD in addition to its chemical stability. Mech Dev, 2002 Sep, 117(1-2), 235 - 41 Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines; Chae J et al.; Analysis of gene function in vertebrates is facilitated by gain-of-function studies, such as injection of synthetic mRNA in amphibian embryos . This approach is hampered by lack of spatial and temporal control of expression of the introduced gene product . An additional level of control is obtained by nuclear-transfer-mediated transgenesis, but functional analyses are complicated by variability and background abnormalities in primary transgenic embryos . The GAL4/UAS system permits establishment of stable lines and elimination of nuclear-transfer-associated abnormalities, through generation of separate UAS-'effector' and GAL4 'transactivator' transgenic lines . When the GAL4 DNA-binding domain is combined with a steroid hormone ligand-binding domain, this system allows full temporal regulation of transgene expression by introduction of an exogenous steroid analogue, the progesterone antagonist RU486 . We show here that by crossing stable Xenopus tropicalis transgenic lines, one bearing a UAS-enhanced cyan fluorescent protein (ECFP) reporter construct, and the other with a GAL4-progesterone receptor fusion driven by a retina-specific promoter, reporter expression in the resulting embryos can be induced with RU486 in a tissue-specific manner . These results suggest that the inducible binary system, in which the target gene expression can be controlled in a stage- and tissue-specific pattern, should be readily applicable for gene function studies at all stages of development . J Biol Chem, 2002 Nov 1, 277(44), 41744 - 9 Epub 2002 Aug 28. Differential sensitivity between Fks1p and Fks2p against a novel beta -1,3-glucan synthase inhibitor, aerothricin3 {corrected}; Kondoh O et al.; Fks1p and Fks2p are catalytic subunits of beta-1,3-glucan synthase, which synthesize beta-1,3-glucan, a main component of the cell wall in Saccharomyces cerevisiae . Although Fks1p and Fks2p are highly homologous, sharing 88.1% identity, it has been shown that Fks2p is more sensitive than Fks1p to one of echinocandin derivatives, which inhibits beta-1,3-glucan synthase activity . Here we show a similar differential sensitivity between Fks1p and Fks2p to a novel beta-1,3-glucan synthase inhibitor, aerothricin3 {corrected} . To investigate the molecular mechanism of this differential sensitivity, we constructed a series of chimeric genes of FKSs and examined their sensitivity to aerothricin3 {corrected} . As a result, it was shown that a region around the fourth extracellular domain of Fks2p, containing 10 different amino acid residues from those of Fks1p, provided Fks1p aerothricin3 {corrected} sensitivity when the region was replaced with a corresponding region of Fks1p . In order to identify essential amino acid residues responsible for the sensitivity, each of the 10 non-conserved amino acids of Fks1p was substituted into the corresponding amino acid of Fks2p by site-directed mutagenesis . Surprisingly, only one amino acid substitution of Fks1p (K1336I) conferred Fks1p hypersensitivity to aerothricin3 {corrected} . On the other hand, reverse substitution of the corresponding amino acid of Fks2p (I1355K) resulted in loss of hypersensitivity to aerothricin3 {corrected} . These results suggest that the 1355th isoleucine of Fks2p plays a key role in aerothricin3 {corrected} sensitivity. Anal Biochem, 2002 Aug 15, 307(2), 273 - 9 An efficient assay for dolichyl phosphate-mannose: protein O-mannosyltransferase; Hendershot LL et al.; A novel method for quantifying the reaction product from dolichyl phosphoryl mannose:polypeptide mannosyltransferase (protein mannosyl transferase; PMT), was developed . The assay quantifies the amount of radioactivity incorporated into the acceptor peptide YNPTSV from dolichyl phosphoryl {3H}mannose (Dol-P-Man) . A novel delivery system, large unilamellar vesicles (LUV), composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), is used to keep the poorly soluble donor substrate, Dol-P-Man, in solution . The use of LUV allows generation of truly reproducible data and, as an additional benefit, also results in a more than 10 times increase in transfer efficiency . In contrast to the solvent extraction procedures commonly used in previously described PMT assays, the assay reaction product is separated from the radioactive donor substrate on C(18) cartridges . The use of C(18) cartridges allows generation of reproducible data with a low, consistent background and also produces a significant reduction in the time and labor needed for the product workup . In a reaction mixture consisting of 100 microg POPC LUV, 9 x 10(5)cpm (approximately 15 pmol) Dol-P-Man, 100 nmol YNPTSV, and aproximately 4 microg of crude yeast microsomal extract, time-dependent formation of glycosylated product obeys Michaelis-Menten-type kinetics throughout the course of the reaction-until exhaustion of the donor substrate . The linear initial rates of the reaction allowed calculation of an apparent K(m) of 1mM, for the acceptor peptide YNPTSV . Variations in detergent concentration in the assay influence transfer efficiency, possibly through interference with the LUV-based donor substrate delivery system . Hence detergent concentrations should be kept constant. Virology, 2002 Aug 15, 300(1), 100 - 8 Effect of Ku80 depletion on the preintegrative steps of HIV-1 replication in human cells; Jeanson L et al.; To gain new insights regarding the role of Ku, the DNA-PK DNA-binding component, during lentiviral DNA integration, we have investigated the HIV-1 replication in Ku80-depleted human cells . CEM4fx cells underexpressing the Ku80 factor were selected after transduction by a retroviral vector expressing the Ku80 full-length antisense sequence . De novo infection experiment with NL4.3 HIV-1 strain led to the observation that the viral replication was delayed in the Ku80-depleted cells . Early events of the replicative cycle, including nuclear import of the viral DNA, were not affected . In contrast, the formation of the 2-LTR circles was impaired, thus demonstrating the implication of Ku in HIV-1 DNA circularization, for the first time in human cells . Furthermore, the detection of integrated proviruses by an Alu-LTR-nested PCR amplification method was affected in cells underexpressing Ku80 . These results suggest that this factor may also be involved in the mechanisms leading to the stable establishment of HIV-1 provirus. Virology, 2002 Aug 15, 300(1), 60 - 70 New targets for antivirals: the ribosomal A-site and the factors that interact with it; Goss Kinzy T et al.; Many viruses use programmed -1 ribosomal frameshifting to ensure the correct ratio of viral structural to enzymatic proteins . Alteration of frameshift efficiencies changes these ratios, in turn inhibiting viral particle assembly and virus propagation . Previous studies determined that anisomycin, a peptidyl transferase inhibitor, specifically inhibited -1 frameshifting and the ability of yeast cells to propagate the L-A and M(1) dsRNA viruses (J . D . Dinman, M . J . Ruiz-Echevarria, K . Czaplinski, and S . W . Peltz, 1997, Proc . Natl . Acad . Sci . USA 94, 6606-6611) . Here we show that preussin, a pyrollidine that is structurally similar to anisomycin (R . E . Schwartz, J . Liesch, O . Hensens, L . Zitano, S . Honeycutt, G . Garrity, R . A . Fromtling, J . Onishi, and R . Monaghan, 1988 . J . Antibiot . (Tokyo) 41, 1774--1779), also inhibits -1 programmed ribosomal frameshifting and virus propagation by acting at the same site or through the same mechanism as anisomycin . Since anisomycin is known to assert its effect at the ribosomal A-site, we undertook a pharmacogenetic analysis of mutants of trans-acting eukaryotic elongation factors (eEFs) that function at this region of the ribosome . Among mutants of eEF1A, a correlation is observed between resistance/susceptibility profiles to preussin and anisomycin, and these in turn correlate with programmed -1 ribosomal frameshifting efficiencies and killer virus phenotypes . Among mutants of eEF2, the extent of resistance to preussin correlates with resistance to sordarin, an eEF2 inhibitor . These results suggest that structural features associated with the ribosomal A-site and with the trans-acting factors that interact with it may present a new set of molecular targets for the rational design of antiviral compounds. J Biol Chem, 2002 Nov 8, 277(45), 42572 - 8 Epub 2002 Aug 27. Acute activation of de novo sphingolipid biosynthesis upon heat shock causes an accumulation of ceramide and subsequent dephosphorylation of SR proteins; Jenkins GM et al.; Recent studies are beginning to implicate sphingolipids in the heat stress response . In the yeast Saccharomyces cerevisiae, heat stress has been shown to activate de novo biosynthesis of sphingolipids, whereas in mammalian cells the sphingolipid ceramide has been implicated in the heat shock responses . In the current study, we found an increase in the ceramide mass of Molt-4 cells in response to heat shock, corroborating findings in HL-60 cells . Increased ceramide was determined to be from de novo biosynthesis by two major lines of evidence . First, the accumulation of ceramide was dependent upon the activities of both ceramide synthase and serine palmitoyltransferase . Second, pulse labeling studies demonstrated increased production of ceramide through the de novo biosynthetic pathway . Significantly, the de novo sphingolipid biosynthetic pathway was acutely induced upon heat shock, which resulted in a 2-fold increased flux in newly made ceramides within 1-2 min of exposure to 42.5 degrees C . Functionally, heat shock induced the dephosphorylation of the SR proteins, and this effect was demonstrated to be dependent upon the accumulation of de novo-produced ceramides . Thus, these studies disclose an evolutionary conserved activation of the de novo pathway in response to heat shock . Moreover, SR dephosphorylation is emerging as a specific downstream target of accumulation of newly made ceramides in mammalian cells. J Biol Chem, 2002 Nov 1, 277(44), 41706 - 14 Epub 2002 Aug 27. Mcm3 is polyubiquitinated during mitosis before establishment of the pre-replication complex; Cheng IH et al.; To ensure fidelity in genome duplication, eukaryotes restrict DNA synthesis to once every cell division by a cascade of regulated steps . Central to this cascade is the periodic assembly of the hexameric MCM2-7 complex at replication origins . However, in Saccharomyces cerevisiae, only a fraction of each MCM protein is able to assemble into hexamers and associate with replication origins during M phase, suggesting that MCM complex assembly and recruitment may be regulated post-translationally . Here we show that a small fraction of Mcm3p is polyubiquitinated at the onset of MCM complex assembly . Reducing the rate of ubiquitination by uba1-165, a suppressor of mcm3-10, restored the interaction of Mcm3-10p with subunits of the MCM complex and its recruitment to the replication origin . Possible roles for ubiquitinated Mcm3p in the assembly of the MCM complex at replication origins are discussed. J Biol Chem, 2002 Nov 1, 277(44), 42259 - 67 Epub 2002 Aug 27. CpG-binding protein is a nuclear matrix- and euchromatin-associated protein localized to nuclear speckles containing human trithorax . Identification of nuclear matrix targeting signals; Lee JH et al.; CpG-binding protein (CGBP) binds unmethylated CpG dinucleotides and is essential for mammalian development . CGBP exhibits a punctate nuclear localization correlated with 4,6-diamidino-2-phenylindole light regions and is excluded from metaphase chromosomes . The distribution of CGBP is distinct from the heterochromatin-associated proteins MBD1, methyl-CpG-binding protein 2, and HP1alpha . Some CGBP-containing nuclear speckles co-localize with splicing factor SC-35 and actively transcribed regions of the genome, whereas most CGBP co-localizes with acetylated histones, indicating that CGBP is localized to active chromatin . CGBP contains two nuclear localization signals that are insufficient to direct punctate subnuclear distribution . Instead, localization of CGBP to nuclear speckles requires signals within the acidic, basic, and coiled-coil domains . CGBP associates with the nuclear matrix, and fragments of CGBP that fail to associate with the nuclear matrix fail to localize to nuclear speckles and exhibit reduced transcriptional activation activity . Mutated versions of CGBP that lack DNA binding activity exhibit a normal nuclear distribution, suggesting that CGBP accumulates at nuclear speckles as a result of protein/protein interactions . Importantly, the subcellular distribution of CGBP is identical to human trithorax, suggesting that these proteins may be components of a multimeric complex analogous to the histone-methylating Set1 complex of Saccharomyces cerevisiae that contains CGBP and trithorax homologues. J Biol Chem, 2002 Nov 1, 277(44), 41736 - 43 Epub 2002 Aug 27. General regulatory factors (GRFs) as genome partitioners; Fourel G et al.; Insulators are sequences that uncouple adjacent chromosome domains . Here we have shown that Saccharomyces cerevisiae Rap1p and Abf1p proteins are endowed with a potent insulating capacity . Insulating domains in Rap1p coincide with previously described transcription activation domains, whereas four adjacent subdomains spanning the whole of the Abf1p C terminus (440-731) were found to display autonomous insulating capacity . That both Rap1p and Abf1p silencing domains either contain or largely overlap with an insulating domain suggests that insulation conveys some undefined chromosome organization capacity that also contributes a function in silencing . Together with Reb1p and Tbf1p, previously involved in the activity of Saccharomyces cerevisiae subtelomeric insulators, insulating potential emerges as a supplementary common property of General Regulatory Factors (GRFs) . Thus GRFs, which bind to sites scattered throughout the genome within promoters, would not only play a key role in regulating gene expression but also partition the genome in functionally independent domains. Appl Environ Microbiol, 2002 Sep, 68(9), 4233 - 9 Cyclization reaction catalyzed by glycogen debranching enzyme (EC 2.4.1.25/EC 3.2.1.33) and its potential for cycloamylose production; Yanase M et al.; Glycogen debranching enzyme (GDE) has 4-alpha-glucanotransferase and amylo-1,6-glucosidase activities in the single polypeptide chain . We analyzed the detailed action profile of GDE from Saccharomyces cerevisiae on amylose and tested whether GDE catalyzes cyclization of amylose . GDE treatment resulted in a rapid reduction of absorbance of iodine-amylose complex and the accumulation of a product that was resistant to an exo-amylase (glucoamylase {GA}) but was degraded by an endo-type alpha-amylase to glucose and maltose . These results indicated that GDE catalyzed cyclization of amylose to produce cyclic alpha-1,4 glucan (cycloamylose) . The formation of cycloamylose was confirmed by high-performance anion-exchange chromatography, and the size was shown to range from a degree of polymerization of 11 to a degree of polymerization around 50 . The minimum size and the size distribution of cycloamylose were different from those of cycloamylose produced by other 4-alpha-glucanotransferases . GDE also efficiently produced cycloamylose even from the branched glucan substrate, starch, demonstrating its potential for industrial production of cycloamylose. Biochem Biophys Res Commun, 2002 Aug 30, 296(4), 813 - 9 Identification of ubiquitin-like protein-binding subunits of the 26S proteasome; Saeki Y et al.; Ubiquitin-like proteins Rad23 and Dsk2 have recently been shown to be capable of binding both polyubiquitin chains and the 26S proteasome . The ubiquitin-like domains (Ubls) of Rad23 and Dsk2 are indispensable for their interaction with the 26S proteasome, but the proteasome subunits capable of binding the Ubl have not been identified . Here, we report that the Ubls of both Rad23 and Dsk2 can bind with the 19S regulatory particle (RP) of the 26S proteasome in vivo and in vitro . A competition assay using the respective Ubls of Rad23 and Dsk2 revealed that they bind to the RP in a competitive manner . The base subcomplex of the RP was found to have the ability to bind the Ubl . By cross-linking experiments, Rpn1 and Rpn2 were identified as Ubl-binding subunits . Taken together, the results suggest that the Rpn1 and Rpn2 in the base subcomplex form the receptor for the ubiquitin-like protein. Int J Biochem Cell Biol, 2002 Nov, 34(11), 1491 - 5 LAG1 puts the focus on ceramide signaling; Jazwinski SM et al.; Longevity-assurance gene 1 (LAG 1) is a yeast longevity gene . Homologues of the Lag 1 protein can be found throughout phylogeny, although sequence similarity is very limited . The Lag 1 protein is located in the endoplasmic reticulum (ER), where it helps to accelerate the transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi . This function of Lag 1 p results from its participation in ceramide synthesis . Thus, Lag 1 p and its homologues are likely to play a role in ceramide signaling, which affects growth, proliferation, stress resistance, and apoptosis . This provides a wide range of physiologic processes through which Lag 1 p may impinge upon life span. Int J Biochem Cell Biol, 2002 Nov, 34(11), 1475 - 90 Senescence and epigenetic dysregulation in cancer; Neumeister P et al.; Mammalian cells have a finite proliferative lifespan, at the end of which they are unable to enter S phase in response to mitogenic stimuli . They undergo morphological changes and synthesize an altered repertoire of cell type-specific proteins . This non-proliferative state is termed replicative senescence and is regarded as a major tumor suppressor mechanism . The ability to overcome senescence and obtain a limitless replicative potential is called immortalization, and considered to be one of the prerequisites of cancer formation . While senescence mainly represents a genetically governed process, epigenetic changes in cancer have received increasing attention as an alternative mechanism for mediating gene expression changes in transformed cells . DNA methylation of promoter-containing CpG islands has emerged as an epigenetic mechanism of silencing tumor suppressor genes . New insights are being gained into the mechanisms causing aberrant methylation in cancer and evidence suggests that aging is accompanied by accumulation of cells with aberrant CpG island methylation . Aberrant methylation may contribute to many of the physiological and pathological changes associated with aging including tumor development . Finally, we describe how genes involved in promoting longevity might inhibit pathways promoting tumorigenesis. Int J Biochem Cell Biol, 2002 Nov, 34(11), 1355 - 71 Copper homeostasis and aging in the fungal model system Podospora anserina: differential expression of PaCtr3 encoding a copper transporter; Borghouts C et al.; Lifespan extension of Podospora anserina mutant grisea is caused by a loss-of-function mutation in the nuclear gene Grisea . This gene encodes the copper regulated transcription factor GRISEA recently shown to be involved in the expression of PaSod2 encoding the mitochondrial manganese superoxide dismutase . Here we report the identification and characterization of a second target gene . This gene, PaCtr3, encodes a functional homologue of the Saccharomyces cerevisiae high affinity copper permease yCTR3 . PaCtr3 is not expressed in the grisea mutant confirming the assumption that the extension of lifespan is primarily caused by cellular copper limitation and a switch from a cytochrome oxidase (COX)-dependent to and alternative oxidase (AOX)-dependent respiration . Transcript levels of PaCtr3 and PaSod2 respond to copper, iron, manganese and zinc . Transcription of PaCtr3 was found to be down-regulated during senescence of wild-type cultures suggesting that the intracellular copper concentration is raised in old cultures . A two hybrid analysis suggested that GRISEA acts as a homodimer . In accordance, an inverted repeat was identified as a putative binding sequence in the promoter region of PaCtr3 and of PaSod2 . Finally, the expression of PaCtr3 in transformants of the grisea mutant led to lifespan shortening . This effect correlates with the activity of the copper-dependent COX demonstrating a strong link between copper-uptake, respiration and lifespan. Eur J Biochem, 2002 Sep, 269(17), 4202 - 11 Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials; Saloheimo M et al.; Plant cell wall proteins called expansins are thought to disrupt hydrogen bonding between cell wall polysaccharides without hydrolyzing them . We describe here a novel gene with sequence similarity to plant expansins, isolated from the cellulolytic fungus Trichoderma reesei . The protein named swollenin has an N-terminal fungal type cellulose binding domain connected by a linker region to the expansin-like domain . The protein also contains regions similar to mammalian fibronectin type III repeats, found for the first time in a fungal protein . The swollenin gene is regulated in a largely similar manner as the T . reesei cellulase genes . The biological role of SWOI was studied by disrupting the swo1 gene from T . reesei . The disruption had no apparent effect on the growth rate on glucose or on different cellulosic carbon sources . Non-stringent Southern hybridization of Trichoderma genomic DNA with swo1 showed the presence of other swollenin-like genes, which could substitute for the loss of SWOI in the disruptant . The swollenin gene was expressed in yeast and Aspergillus niger var . awamori . Activity assays on cotton fibers and filter paper were performed with concentrated SWOI-containing yeast supernatant that disrupted the structure of the cotton fibers without detectable formation of reducing sugars . It also weakened filter paper as assayed by an extensometer . The SWOI protein was purified from A . niger var . awamori culture supernatant and used in an activity assay with Valonia cell walls . It disrupted the structure of the cell walls without producing detectable amounts of reducing sugars. J Biomed Inform, 2001 Dec, 34(6), 396 - 405 Comparing the similarity of time-series gene expression using signal processing metrics; Butte AJ et al.; Many algorithms have been used to cluster genes measured by microarray across a time series . Instead of clustering, our goal was to compare all pairs of genes to determine whether there was evidence of a phase shift between them . We describe a technique where gene expression is treated as a discrete time-invariant signal, allowing the use of digital signal-processing tools, including power spectral density, coherence, and transfer gain and phase shift . We used these on a public RNA expression set of 2467 genes measured every 7 min for 119 min and found 18 putative associations . Two of these were known in the biomedical literature and may have been missed using correlation coefficients . Digital signal processing tools can be embedded and enhance existing clustering algorithms. Nat Cell Biol, 2002 Sep, 4(9), 725 - 30 Proteasome subunit Rpn1 binds ubiquitin-like protein domains; Elsasser S et al.; The yeast protein Rad23 belongs to a diverse family of proteins that contain an amino-terminal ubiquitin-like (UBL) domain . This domain mediates the binding of Rad23 to proteasomes, which in turn promotes DNA repair and modulates protein degradation, possibly by delivering ubiquitinylated cargo to proteasomes . Here we show that Rad23 binds proteasomes by directly interacting with the base subcomplex of the regulatory particle of the proteasome . A component of the base, Rpn1, specifically recognizes the UBL domain of Rad23 through its leucine-rich-repeat-like (LRR-like) domain . A second UBL protein, Dsk2, competes with Rad23 for proteasome binding, which suggests that the LRR-like domain of Rpn1 may participate in the recognition of several ligands of the proteasome . We propose that the LRR domain of Rpn1 may be positioned in the base to allow the cargo proteins carried by Rad23 to be presented to the proteasomal ATPases for unfolding . We also report that, contrary to expectation, the base subunit Rpn10 does not mediate the binding of UBL proteins to the proteasome in yeast, although it can apparently contribute to the binding of ubiquitin chains by intact proteasomes. Mol Endocrinol, 2002 Sep, 16(9), 1999 - 2007 Ubc6p and ubc7p are required for normal and substrate-induced endoplasmic reticulum-associated degradation of the human selenoprotein type 2 iodothyronine monodeiodinase; Botero D et al.; The type 2 monodeiodinase (D2) is an endoplasmic reticulum-resident membrane selenoprotein responsible for catalyzing the first step in thyroid hormone action, T(4) deiodination to T(3) . Its short half-life is due to ubiquitination and proteolysis by proteasomes, a mechanism that is accelerated by D2 interaction with T(4) . To identify proteins involved in D2 ubiquitination, a FLAG-tagged selenocystine133-to-Cys mutation of the human D2 (CysD2) was created and expressed in Saccharomyces cerevisiae using the GAL1 gene promoter . CysD2 activity was detected in the microsomes, indistinguishable from transiently expressed CysD2 in vertebrate cells . Treatment with 100 mg/ml cycloheximide or 30 micro M T(4) caused rapid loss of CysD2 (t(1/2) = approximately 30 min) . Clasto-lactacystin beta-lactone not only increased galactose-inducible CysD2 but also stabilized CysD2 in the presence of cycloheximide or T(4) . Immunoprecipitation with anti-FLAG antibody combined with Western analysis with antiubiquitin revealed that CysD2 is heavily ubiquitinated . Expression of CysD2 in yeast strains that lack the ubiquitin conjugases Ubc6p or Ubc7p stabilized CysD2 half-life by markedly reducing CysD2 ubiquitination, whereas no difference was detected in Ubc1p-deficient mutants . Similarly, expression of CysD2 in UBC6 and UBC7 mutants also impaired the substrate-induced loss of CysD2 activity and protein . In conclusion, Ubc6p and Ubc7p are required for normal and substrate-induced ubiquitination and proteolysis of D2. EMBO J, 2002 Sep 2, 21(17), 4730 - 40 Recombination-dependent mtDNA partitioning: in vivo role of Mhr1p to promote pairing of homologous DNA; Ling F et al.; Yeast mhr1-1 was isolated as a defective mutation in mitochondrial DNA (mtDNA) recombination . About half of mhr1-1 cells lose mtDNA during growth at a higher temperature . Here, we show that mhr1-1 exhibits a defect in the partitioning of nascent mtDNA into buds and is a base-substitution mutation in MHR1 encoding a mitochondrial matrix protein . We found that the Mhr1 protein (Mhr1p) has activity to pair single-stranded DNA and homologous double-stranded DNA to form heteroduplex joints in vitro, and that mhr1-1 causes the loss of this activity, indicating its role in homologous mtDNA recombination . While the majority of the mtDNA in the mother cells consists of head-to-tail concatemers, more than half of the mtDNA in the buds exists as genome-sized monomers . The mhr1-1 deltacce1 double mutant cells do not maintain any mtDNA, indicating the strict dependence of mtDNA maintenance on recombination functions . These results suggest a mechanism for mtDNA inheritance similar to that operating in the replication and packaging of phage DNA. EMBO J, 2002 Sep 2, 21(17), 4699 - 708 The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases; Liu H et al.; We recently demonstrated that the major decapping activity in mammalian cells involves DcpS, a scavenger pyrophosphatase that hydrolyzes the residual cap structure following 3' to 5' decay of an mRNA . The association of DcpS with 3' to 5' exonuclease exosome components suggests that these two activities are linked and there is a coupled exonucleolytic decay-dependent decapping pathway . We purified DcpS from mammalian cells and identified the cDNA encoding a novel 40 kDa protein possessing DcpS activity . Consistent with purified DcpS, the recombinant protein specifically hydrolyzed methylated cap analog but did not hydrolyze unmethylated cap analog nor did it function on intact capped RNA . Sequence alignments of DcpS from different organisms revealed the presence of a conserved hexapeptide, containing a histidine triad (HIT) sequence with three histidines separated by hydrophobic residues . Mutagenesis analysis revealed that the central histidine within the DcpS HIT motif is critical for decapping activity and defines the HIT motif as a new mRNA decapping domain, making DcpS the first member of the HIT family of proteins with a defined biological function. EMBO J, 2002 Sep 2, 21(17), 4600 - 11 Structure and function of the BAH-containing domain of Orc1p in epigenetic silencing; Zhang Z et al.; The N-terminal domain of the largest subunit of the Saccharomyces cerevisiae origin recognition complex (Orc1p) functions in transcriptional silencing and contains a bromo-adjacent homology (BAH) domain found in some chromatin-associated proteins including Sir3p . The 2.2 A crystal structure of the N-terminal domain of Orc1p revealed a BAH core and a non-conserved helical sub-domain . Mutational analyses demonstrated that the helical sub-domain was necessary and sufficient to bind Sir1p, and critical for targeting Sir1p primarily to the cis-acting E silencers at the HMR and HML silent chromatin domains . In the absence of the BAH domain, approximately 14-20% of cells in a population were silenced at the HML locus . Moreover, the distributions of the Sir2p, Sir3p and Sir4p proteins, while normal, were at levels lower than found in wild-type cells . Thus, in the absence of the Orc1p BAH domain, HML resembled silencing of genes adjacent to telomeres . These data are consistent with the view that the Orc1p-Sir1p interaction at the E silencers ensures stable inheritance of pre-established Sir2p, Sir3p and Sir4p complexes at the silent mating type loci. EMBO J, 2002 Sep 2, 21(17), 4500 - 10 Timing of APC/C substrate degradation is determined by fzy/fzr specificity of destruction boxes; Zur A et al.; The anaphase promoting complex/cyclosome (APC/C), activated by fzy and fzr, degrades cell cycle proteins that carry RXXL or KEN destruction boxes (d-boxes) . APC/C substrates regulate sequential events and must be degraded in the correct order during mitosis and G(1) . We studied how d-boxes determine APC/C(fzy)/APC/C(fzr) specificity and degradation timing . Cyclin B1 has an RXXL box and is degraded by both APC/C(fzy) and APC/C(fzr); fzy has a KEN box and is degraded by APC/C(fzr) only . We characterized the degradation of substrates with swapped d-boxes . Cyclin B1 with KEN was degraded by APC/C(fzr) only . Fzy with RXXL could be degraded by APC/C(fzy) and APC/C(fzr) . Interestingly, APC/C(fzy)- but not APC/C(fzr)-specific degradation is highly dependent on the location of RXXL . We studied degradation of tagged substrates in real time and observed that APC/C(fzr) is activated in early G(1) . These observations demonstrate how d-box specificities of APC/C(fzy) and APC/C(fzr), and the successive activation of APC/C by fzy and fzr, establish the temporal degradation pattern . Our observations can explain further why some endogenous RXXL substrates are degraded by APC/C(fzy), while others are restricted to APC/C(fzr). EMBO J, 2002 Sep 2, 21(17), 4491 - 9 Suppression of the STK15 oncogenic activity requires a transactivation-independent p53 function; Chen SS et al.; Using a transactivation-defective p53 derivative as bait, STK15, a centrosome-associated oncogenic serine/threonine kinase, was isolated as a p53 partner . The p53-STK15 interaction was confirmed further by co-immunoprecipitation and GST pull-down studies . In co-transfection experiments, p53 suppressed STK15-induced centrosome amplification and cellular transformation in a transactivation-independent manner . The suppression of STK15 oncogenic activity by p53 might be explained in part by the finding that p53 inhibited STK15 kinase activity via direct interaction with the latter's Aurora box . Taken together, these findings revealed a novel mechanism for the tumor suppressor function of p53. J Biol Chem, 2002 Nov 22, 277(47), 45226 - 34 Epub 2002 Aug 26. Identification and characterization of a cDNA encoding a dolichyl pyrophosphate phosphatase located in the endoplasmic reticulum of mammalian cells; Rush JS et al.; The CWH8 gene in Saccharomyces cerevisiae has been shown recently (Fernandez, F., Rush, J . S., Toke, D . A., Han, G., Quinn, J . E., Carman, G . M., Choi, J.-Y., Voelker, D . R., Aebi, M., and Waechter, C . J . (2001) J . Biol . Chem . 276, 41455-41464) to encode a dolichyl pyrophosphate (Dol-P-P) phosphatase associated with crude microsomal fractions . Mutations in CWH8 result in the accumulation of Dol-P-P, deficiency in lipid intermediate synthesis, defective protein N-glycosylation, and a reduced growth rate . A cDNA (DOLPP1, GenBank accession number AB030189) from mouse brain encoding a homologue of the yeast CWH8 gene is now shown to complement th