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Sequence Motifs (sequence + motif)
Kinds of Sequence Motifs Selected AbstractsFunctional properties of the protein disulfide oxidoreductase from the archaeon Pyrococcus furiosusFEBS JOURNAL, Issue 16 2004A member of a novel protein family related to protein disulfide-isomerase Protein disulfide oxidoreductases are ubiquitous redox enzymes that catalyse dithiol,disulfide exchange reactions with a CXXC sequence motif at their active site. A disulfide oxidoreductase, a highly thermostable protein, was isolated from Pyrococcus furiosus (PfPDO), which is characterized by two redox sites (CXXC) and an unusual molecular mass. Its 3D structure at high resolution suggests that it may be related to the multidomain protein disulfide-isomerase (PDI), which is currently known only in eukaryotes. This work focuses on the functional characterization of PfPDO as well as its relation to the eukaryotic PDIs. Assays of oxidative, reductive, and isomerase activities of PfPDO were performed, which revealed that the archaeal protein not only has oxidative and reductive activity, but also isomerase activity. On the basis of structural data, two single mutants (C35S and C146S) and a double mutant (C35S/C146S) of PfPDO were constructed and analyzed to elucidate the specific roles of the two redox sites. The results indicate that the CPYC site in the C-terminal half of the protein is fundamental to reductive/oxidative activity, whereas isomerase activity requires both active sites. In comparison with PDI, the ATPase activity was tested for PfPDO, which was found to be cation-dependent with a basic pH optimum and an optimum temperature of 90 °C. These results and an investigation on genomic sequence databases indicate that PfPDO may be an ancestor of the eukaryotic PDI and belongs to a novel protein disulfide oxidoreductase family. [source] Is Runx a linchpin for developmental signaling in metazoans?JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009James A. Coffman Abstract The Runt domain (Runx) is a 128 amino acid sequence motif that defines a metazoan family of sequence-specific DNA binding proteins, which appears to have originated in concert with the intercellular signaling systems that coordinate multicellular development in animals. In the model organisms where they have been studied (fruit fly, mouse, sea urchin, and nematode) Runx genes are essential for normal development, and in humans they are causally associated with a variety of cancers, manifesting both oncogenic and tumor suppressive attributes. During development Runx proteins support both cell proliferation and differentiation, and function in both transcriptional activation and repression. Runx function is thus context-dependent, with the context provided genetically by cis -regulatory sequence architecture and epigenetically by development. This context dependency makes it difficult to formulate reductionistic generalizations concerning Runx function in normal and carcinogenic development. However, a growing body of literature links Runx function to each of the major intercellular signaling systems in animals, suggesting that the general function of Runx transcription factors may be to potentiate and govern genomic responsiveness to developmental signaling. J. Cell. Biochem. 107: 194,202, 2009. © 2009 Wiley-Liss, Inc. [source] Regulation of GTP cyclohydrolase I gene transcription by basic region leucine zipper transcription factorsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2005Jude Al Sarraj Abstract Tetrahydrobiopterin is an essential cofactor for the phenylalanine, tyrosine and tryptophan hydroxylases, and the family of nitric oxide synthases. The initial and rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin is GTP cyclohydrolase I. The proximal promoter of the human GTP cyclohydrolase I gene contains the sequence motif 5,-TGACGCGA-3,, resembling a cAMP response element (CRE). The objective of this study was to analyze the regulation of GTP cyclohydrolase I gene transcription by basic region leucine zipper (bZIP) transcription factors. A constitutively active mutant of the cAMP response element binding (CREB) protein strongly stimulated GTP cyclohydrolase I promoter activity, indicating that the CRE in the context of the GTP cyclohydrolase I gene is functional. Likewise, GTP cyclohydrolase I promoter/luciferase gene transcription was stimulated following nuclear expression of the catalytic subunit of cAMP-dependent protein kinase. Constitutively active mutants of activating transcription factor 2 (ATF2) and c-Jun additionally stimulated GTP cyclohydrolase I promoter activity, but to a lesser extent than the constitutively active CREB mutant. The fact that stress-activated protein kinases target the GTP cyclohydrolase I gene was corroborated by expression experiments involving p38 and MEKK1 protein kinases. We conclude that signaling pathways involving either the cAMP-dependent protein kinase or stress-activated protein kinases converge to the GTP cyclohydrolase I gene. Hence, enzymatic reactions that require tetrahydrobiopterin as cofactor are therefore indirectly controlled by signaling cascades involving the signal-responsive transcription factors CREB, c-Jun, and ATF2. J. Cell. Biochem. © 2005 Wiley-Liss, Inc. [source] DNA aptamers developed against a soman derivative cross-react with the methylphosphonic acid core but not with flanking hydrophobic groupsJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2009John G. Bruno Abstract Twelve rounds of systematic evolution of ligands by exponential enrichment (SELEX) were conducted against a magnetic bead conjugate of the para -aminophenylpinacolylmethylphosphonate (PAPMP) derivative of the organophosphorus (OP) nerve agent soman (GD). The goal was to develop DNA aptamers that could scavenge GD in vivo, thereby reducing or eliminating the toxic effects of this dangerous compound. Aptamers were sequenced and screened in peroxidase-based colorimetric plate assays after rounds 8 and 12 of SELEX. The aptamer candidate sequences exhibiting the highest affinity for the GD derivative from round 8 also reappeared in several clones from round 12. Each of the highest affinity PAPMP-binding aptamers also bound methylphosphonic acid (MPA). In addition, the aptamer with the highest overall affinity for PAPMP carried a sequence motif (TTTAGT) thought to bind MPA based on previously published data (J. Fluoresc 18: 867,876, 2008). This sequence motif was found in several other relatively high affinity PAPMP aptamer candidates as well. In studies with the nerve agent GD, pre-incubation of a large molar excess of aptamer candidates failed to protect human butyrylcholinesterase (BuChE) from inhibition. With the aid of three-dimensional molecular modeling of the GD derivative it appears that a hydrophilic cleft sandwiched between the pinacolyl group and the p -aminophenyl ring might channel nucleotide interactions to the phosphonate portion of the immobilized GD derivative. However, bona fide GD free in solution may be repulsed by the negative phosphate backbone of aptamers and rotate its phosphonate and fluorine moieties away from the aptamer to avoid being bound. Future attempts to develop aptamers to GD might benefit from immobilizing the pinacolyl group of bona fide GD to enhance exposure of the phosphonate and fluorine to the random DNA library. Copyright © 2008 John Wiley & Sons, Ltd. [source] Amino acids involved in conformational dynamics and G protein coupling of an odorant receptor: targeting gain-of-function mutationJOURNAL OF NEUROCHEMISTRY, Issue 5 2008Aya Kato Abstract Thousands of different odorants are recognized and discriminated by odorant receptors (ORs) in the guanine nucleotide-binding protein (G protein)-coupled seven-transmembrane receptor family. Odorant-bound ORs stimulate Gs-type G proteins, G,olf, which in turn activates cAMP-mediated signaling pathway in olfactory sensory neurons. To better understand the molecular basis for OR activation and G protein coupling, we analyzed the effects of a series of site-directed mutations of mouse ORs, on function. Mutations of conserved amino acid residues in an intracellular loop or the C-terminus resulted in loss of activity without impairing ligand-binding activity, indicating that these residues are involved in G,s/olf coupling. Moreover, mutation of the serine in KAFSTC, the OR-specific sequence motif, resulted in a dramatic increase in odorant responsiveness, suggesting that the motif is involved in a conformational change of the receptor that regulates G protein coupling efficiency. Our results provide insights into how ORs switch from an inactive to an active state, as well as where and how activated ORs interact with G proteins. [source] The changing faces of Streptococcus antigen I/II polypeptide family adhesinsMOLECULAR MICROBIOLOGY, Issue 2 2010L. Jeannine Brady Summary Streptococcus mutans antigen I/II (AgI/II) protein was one of the first cell wall-anchored adhesins identified in Gram-positive bacteria. It mediates attachment of S. mutans to tooth surfaces and has been a focus for immunization studies against dental caries. The AgI/II family polypeptides recognize salivary glycoproteins, and are also involved in biofilm formation, platelet aggregation, tissue invasion and immune modulation. The genes encoding AgI/II family polypeptides are found among Streptococcus species indigenous to the human mouth, as well as in Streptococcus pyogenes, S. agalactiae and S. suis. Evidence of functionalities for different regions of the AgI/II proteins has emerged. A sequence motif within the C-terminal portion of Streptococcus gordonii SspB (AgI/II) is bound by Porphyromonas gingivalis, thus promoting oral colonization by this anaerobic pathogen. The significance of other epitopes is now clearer following resolution of regional crystal structures. A new picture emerges of the central V (variable) region, predicted to contain a carbohydrate-binding trench, being projected from the cell surface by a stalk formed by an unusual association between an N-terminal ,-helix and a C-terminal polyproline helix. This presentation mode might be important in determining functional conformations of other Gram-positive surface proteins that have adhesin domains flanked by ,-helical and proline-rich regions. [source] Identification and characterization of TSAP, a novel gene specifically expressed in testis during spermatogenesisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2007Li Bin Abstract Through in silico screens, we have identified many previously uncharacterized genes that display similar expression patterns as the mouse Dazl gene, a germ line-specific marker. Here, we report the identification and characterization of one of these novel genes. TSAP gene encodes a protein with 350 amino acids and contains five ankyrin repeats and a PEST sequence motif. Furthermore, we have generated an anti-TSAP antibody and have used three different approaches (RT-PCR, in situ hybridization, and immunohistochemistry) to investigate the expression profiles of TSAP mRNAs and proteins. TSAP is specifically expressed in testis, but not in other tissues such as ovary. Within the testis, TSAP is detected 10 days after birth and is mainly expressed in spermatocytes (ST) and later stage of germ cells, but not in spermatogonia (SG) or sertoli cells. Therefore, TSAP protein likely plays a role in spermatogenesis. Mol. Reprod. Dev. 74: 1141,1148, 2007. © 2007 Wiley-Liss, Inc. [source] Sequence-structure analysis of FAD-containing proteinsPROTEIN SCIENCE, Issue 9 2001Orly Dym We have analyzed structure-sequence relationships in 32 families of flavin adenine dinucleotide (FAD)-binding proteins, to prepare for genomic-scale analyses of this family. Four different FAD-family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p -cresol methylhydroxylase (PCMH) were previously defined, and a family represented by pyruvate oxidase (PO) is newly defined. For each of the families, several conserved sequence motifs have been characterized. Several newly recognized sequence motifs are reported here for the PO, GR, and PCMH families. Each FAD fold can be uniquely identified by the presence of distinctive conserved sequence motifs. We also analyzed cofactor properties, some of which are conserved within a family fold while others display variability. Among the conserved properties is cofactor directionality: in some FAD-structural families, the adenine ring of the FAD points toward the FAD-binding domain, whereas in others the isoalloxazine ring points toward this domain. In contrast, the FAD conformation and orientation are conserved in some families while in others it displays some variability. Nevertheless, there are clear correlations among the FAD-family fold, the shape of the pocket, and the FAD conformation. Our general findings are as follows: (a) no single protein ,pharmacophore' exists for binding FAD; (b) in every FAD-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif, suggesting that pyrophosphate binding is a significant component of molecular recognition; and (c) sequence motifs can identify proteins that bind phosphate-containing ligands. [source] Open-and-shut cases in coiled-coil assembly: ,-sheets and ,-cylindersPROTEIN SCIENCE, Issue 3 2001John Walshaw Abstract The coiled coil is a ubiquitous protein-folding motif. It generally is accepted that coiled coils are characterized by sequence patterns known as heptad repeats. Such patterns direct the formation and assembly of amphipathic ,-helices, the hydrophobic faces of which interface in a specific manner first proposed by Crick and termed "knobs-into-holes packing". We developed software, socket, to recognize this packing in protein structures. As expected, in a trawl of the protein data bank, we found examples of canonical coiled coils with a single contiguous heptad repeat. In addition, we identified structures with multiple, overlapping heptad repeats. This observation extends Crick's original postulate: Multiple, offset heptad repeats help explain assemblies with more than two helices. Indeed, we have found that the sequence offset of the multiple heptad repeats is related to the coiled-coil oligomer state. Here we focus on one particular sequence motif in which two heptad repeats are offset by two residues. This offset sets up two hydrophobic faces separated by ,150°,160° around the ,-helix. In turn, two different combinations of these faces are possible. Either similar or opposite faces can interface, which leads to open or closed multihelix assemblies. Accordingly, we refer to these two forms as ,-sheets and ,-cylinders. We illustrate these structures with our own predictions and by reference to natural variants on these designs that have recently come to light. [source] Distribution of corticotropin-releasing hormone in the developing zebrafish brainTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 4 2007Gayathri Chandrasekar Abstract Corticotropin-releasing hormone (CRH) plays a central role in the physiological regulation of the hypothalamus-pituitary-adrenal/interrenal axis mediating endocrine, behavioral, autonomic, and immune responses to stress. Despite the wealth of knowledge about the physiological roles of CRH, the genetic mechanisms by which CRH neurons arise during development are poorly understood. As a first step toward analyzing the molecular and genetic pathways involved in CRH lineage specification, we describe the developmental distribution of CRH neurons in the embryonic zebrafish, a model organism for functional genomics and developmental biology. We searched available zebrafish expressed sequence tag (EST) databases for CRH-like sequences and identified one EST that contained the complete zebrafish CRH open reading frame (ORF). The CRH precursor sequence contained a signal peptide, the CRH peptide, and a cryptic peptide with a conserved sequence motif. RT-PCR analysis showed crh expression in a wide range of adult tissues as well as during embryonic and larval stages. By whole-mount in situ hybridization histochemistry, discrete crh -expressing cell clusters were found in different parts of the embryonic zebrafish brain, including telencephalon, preoptic region, hypothalamus, posterior tuberculum, thalamus, epiphysis, midbrain tegmentum, and rostral hindbrain and in the neural retina. The localization of crh mRNA within the preoptic region is consistent with the central role of CRH in the teleost stress response through activation of the hypothalamic-pituitary-interrenal axis. The widespread distribution of CRH-synthesizing cells outside the preoptic region suggests additional functions of CRH in the embryonic zebrafish brain. J. Comp. Neurol. 505:337,351, 2007. © 2007 Wiley-Liss, Inc. [source] Identification and characterization of a new conserved motif within the presequence of proteins targeted into complex diatom plastidsTHE PLANT JOURNAL, Issue 2 2005Oliver Kilian Summary Several groups of algae evolved by secondary endocytobiosis, which is defined as the uptake of a eukaryotic alga into a eukaryotic host cell and the subsequent transformation of the endosymbiont into an organelle. Due to this explicit evolutionary history such algae possess plastids that are surrounded by either three or four membranes. Protein targeting into plastids of these organisms depends on N-terminal bipartite presequences consisting of a signal and a transit peptide domain. This suggests that different protein targeting systems may have been combined during establishment of secondary endocytobiosis to enable the transport of proteins into the plastids. Here we demonstrate the presence of an apparently new type of transport into diatom plastids. We analyzed protein targeting into the plastids of diatoms and identified a conserved amino acid sequence motif within plastid preprotein targeting sequences. We expressed several diatom plastid presequence:GFP fusion proteins with or without modifications within that motif in the diatom Phaeodactylum tricornutum and found that a single conserved phenylalanine is crucial for protein transport into the diatom plastids in vivo, thus indicating the presence of a so far unknown new type of targeting signal. We also provide experimental data about the minimal requirements of a diatom plastid targeting presequence and demonstrate that the signal peptides of plastid preproteins and of endoplasmic reticulum-targeted preproteins in diatoms are functionally equivalent. Furthermore we show that treatment of the cells with Brefeldin A arrests protein transport into the diatom plastids suggesting that a vesicular transport step within the plastid membranes may occur. [source] HLA polymorphisms in African Americans with idiopathic inflammatory myopathy: Allelic profiles distinguish patients with different clinical phenotypes and myositis autoantibodiesARTHRITIS & RHEUMATISM, Issue 11 2006Terrance P. O'Hanlon Objective To investigate possible associations of HLA polymorphisms with idiopathic inflammatory myopathy (IIM) in African Americans, and to compare this with HLA associations in European American IIM patients with IIM. Methods Molecular genetic analyses of HLA,A, B, Cw, DRB1, and DQA1 polymorphisms were performed in a large population of African American patients with IIM (n = 262) in whom the major clinical and autoantibody subgroups were represented. These data were compared with similar information previously obtained from European American patients with IIM (n = 571). Results In contrast to European American patients with IIM, African American patients with IIM, in particular those with polymyositis, had no strong disease associations with HLA alleles of the 8.1 ancestral haplotype; however, African Americans with dermatomyositis or with anti,Jo-1 autoantibodies shared the risk factor HLA,DRB1*0301 with European Americans. We detected novel HLA risk factors in African American patients with myositis overlap (DRB1*08) and in African American patients producing anti,signal recognition particle (DQA1*0102) and anti,Mi-2 autoantibodies (DRB1*0302). DRB1*0302 and the European American,, anti,Mi-2,associated risk factor DRB1*0701 were found to share a 4,amino-acid sequence motif, which was predicted by comparative homology analyses to have identical 3-dimensional orientations within the peptide-binding groove. Conclusion These data demonstrate that North American IIM patients from different ethnic groups have both shared and distinct immunogenetic susceptibility factors, depending on the clinical phenotype. These findings, obtained from the largest cohort of North American minority patients with IIM studied to date, add additional support to the hypothesis that the myositis syndromes comprise multiple, distinct disease entities, perhaps arising from divergent pathogenic mechanisms and/or different gene,environment interactions. [source] Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of Mrs2, a magnesium ion transporter from yeast inner mitochondrial membraneACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Muhammad Bashir Khan Mrs2 transporters are distantly related to the major bacterial Mg2+ transporter CorA and to Alr1, which is found in the plasma membranes of lower eukaryotes. Common features of all Mrs2 proteins are the presence of an N-terminal soluble domain followed by two adjacent transmembrane helices (TM1 and TM2) near the C-terminus and of the highly conserved F/Y-G-M-N sequence motif at the end of TM1. The inner mitochondrial domain of the Mrs2 from Saccharomyces cerevisae was overexpressed, purified and crystallized in two different crystal forms corresponding to an orthorhombic and a hexagonal space group. The crystals diffracted X-rays to 1.83 and 4.16,Å resolution, respectively. Matthews volume calculations suggested the presence of one molecule per asymmetric unit in the orthorhombic crystal form and of five or six molecules per asymmetric unit in the hexagonal crystal form. The phase problem was solved for the orthorhombic form by a single-wavelength anomalous dispersion experiment exploiting the sulfur anomalous signal. [source] DNA sequence motifs conserved in endocrine promoters are essential for Pax4 expressionDEVELOPMENTAL DYNAMICS, Issue 4 2003Christopher Brink Abstract The paired box transcription factor Pax4 is required for maturation of insulin-producing ,- and somatostatin secreting ,-cells in the murine pancreas. It starts to be expressed in pancreatic precursors and later is restricted to ,- and ,-cells to finally be switched off after birth. A 0.9-kb genomic DNA fragment has been shown to mediate the Pax4 expression pattern. Transcription factors Pdx1 and NeuroD bind to this fragment at A2- and E1-sequence motifs. In this study, we downscale the size of this fragment to 409 bp. Another genomic fragment of 254 bp is still able to mediate the specificity, but not the strength of Pax4 expression. Deletion of the A2 and E1 elements results in loss or weakening of reporter gene expression. Because A2 and E1 elements are conserved in numerous pancreatic promoters, they might play a general role in regulating endocrine gene expression. Developmental Dynamics 228:617,622, 2003. © 2003 Wiley-Liss, Inc. [source] Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domainsFEBS JOURNAL, Issue 2 2007Anne R. Karow RNA helicases mediate structural rearrangements of RNA or RNA,protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis,Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication. [source] Chromatin structure of the bovine Cyp19 promoter 1.1FEBS JOURNAL, Issue 5 2001DNA hypomethylation correlate with placental expression, DNaseI hypersensitive sites Expression of the Cyp19 gene, encoding aromatase cytochrome P450, is driven by several tissue-specific promoters. The underlying mechanisms of this complex regulation have not yet been elucidated in detail. In the present report we investigate a possible link between chromatin structure and tissue-specific regulation of the bovine Cyp19 gene. We analysed the DNA methylation status and mapped DNaseI hypersensitive sites in the region encompassing the Cyp19 promoter 1.1 (P1.1) which controls Cyp19 expression in the bovine placenta. We show that P1.1 is hypomethylated in placental cotyledons (foetal layer) whereas it is methylated in placental caruncles (maternal layer), testis and corpus luteum. Furthermore, two placenta-specific DNaseI hypersensitive sites, HS1 and HS2, were observed within P1.1. Both DNA hypomethylation and the presence of DNaseI hypersensitive sites correlate with transcriptional activity of P1.1. Sequence analysis of hypersensitive sites revealed potential cis -regulatory elements, an E-box in HS1 and a trophoblast-specific element-like sequence in HS2. It could be demonstrated by electrophoretic mobility shift assays that both sequence motifs are specific targets for placenta-derived nuclear factors. In conclusion, observed tissue-specific differences of the chromatin structure which correlate with tissue-specific promoter activity suggest that chromatin might be an important regulator of aromatase expression in cattle. [source] Cloning of the guanylate kinase homologues AGK-1 and AGK-2 from Arabidopsis thaliana and characterization of AGK-1FEBS JOURNAL, Issue 2 2000Vinod Kumar Guanylate kinase is an essential enzyme for nucleotide metabolism, phosphorylating GMP to GDP or dGMP to dGDP. The low molecular mass cytosolic forms of guanylate kinase are implicated primarily in the regulation of the supply of guanine nucleotides to cell signalling pathways. The high molecular mass and membrane-associated forms of guanylate kinase homologues, notably found in neuronal tissues, are assigned roles in cell junction organization and transmembrane regulation. Here, we describe the first plant guanylate kinase-encoding genes, AGK1 and AGK2, from Arabidopsis thaliana. The nucleotide sequences of their genomic and cDNA clones predict proteins that carry N-terminal and C-terminal extensions of the guanylate kinase-like domain. The amino acid sequences of this domain share 46,52% identity with guanylate kinases from yeast, Escherichia coli, human, mouse and Caenorhabditis elegans. Arabidopsis guanylate kinases (AGKs) exhibit a high degree of conservation of active site residues and sequence motifs in common with other nucleoside monophosphate kinases, which suggests overall structural similarity of the plant proteins. Although bacterially expressed AGK-1 is enzymatically much less active than yeast guanylate kinase, its kinase domain is shown to complement yeast GUK1 recessive lethal mutations. AGKs are expressed ubiquitously in plant tissues with highest transcriptional activity detected in roots. The identification of AGKs provides new perspectives for understanding the role of guanylate kinases in plant cell signalling pathways. [source] Identification of a novel REV1-interacting motif necessary for DNA polymerase , functionGENES TO CELLS, Issue 2 2009Eiji Ohashi When a replicative DNA polymerase (Pol) is stalled by damaged DNA, a "polymerase switch" recruits specialized translesion synthesis (TLS) DNA polymerase(s) to sites of damage. Mammalian cells have several TLS DNA polymerases, including the four Y-family enzymes (Pol,, Pol,, Pol, and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Pol,, Pol,, and Pol, and therefore appears to play a central role during TLS in vivo. Here we have investigated the molecular basis for interactions between REV1 and Pol,. We have identified novel REV1-interacting regions (RIRs) present in Pol,, Pol, and Pol,. Within the RIRs, the presence of two consecutive phenylalanines (FF) is essential for REV1-binding. The consensus sequence for REV1-binding is denoted by x-x-x-F-F-y-y-y-y (x, no specific residue and y, no specific residue but not proline). Our results identify structural requirements that are necessary for FF-flanking residues to confer interactions with REV1. A Pol, mutant lacking REV1-binding activity did not complement the genotoxin-sensitivity of Polk -null mouse embryonic fibroblast cells, thereby demonstrating that the REV1-interaction is essential for Pol, function in vivo. [source] The Versatility of Helicobacter pylori CagA Effector Protein Functions: The Master Key HypothesisHELICOBACTER, Issue 3 2010Steffen Backert Abstract Several bacterial pathogens inject virulence proteins into host target cells that are substrates of eukaryotic tyrosine kinases. One of the key examples is the Helicobacter pylori CagA effector protein which is translocated by a type-IV secretion system. Injected CagA becomes tyrosine-phosphorylated on EPIYA sequence motifs by Src and Abl family kinases. CagA then binds to and activates/inactivates multiple signaling proteins in a phosphorylation-dependent and phosphorylation-independent manner. A recent proteomic screen systematically identified eukaryotic binding partners of the EPIYA phosphorylation sites of CagA and similar sites in other bacterial effectors by high-resolution mass spectrometry. Individual phosphorylation sites recruited a surprisingly high number of interaction partners suggesting that each phosphorylation site can interfere with many downstream pathways. We now count 20 reported cellular binding partners of CagA, which represents the highest quantitiy among all yet known virulence-associated effector proteins in the microbial world. This complexity generates a highly remarkable and puzzling scenario. In addition, the first crystal structure of CagA provided us with new information on the function of this important virulence determinant. Here we review the recent advances in characterizing the multiple binding signaling activities of CagA. Injected CagA can act as a ,master key' that evolved the ability to highjack multiple host cell signalling cascades, which include the induction of membrane dynamics, actin-cytoskeletal rearrangements and the disruption of cell-to-cell junctions as well as proliferative, pro-inflammatory and anti-apoptotic nuclear responses. The discovery that different pathogens use this common strategy to subvert host cell functions suggests that more examples will emerge soon. [source] Complete ascertainment of intragenic copy number mutations (CNMs) in the CFTR gene and its implications for CNM formation at other autosomal loci,HUMAN MUTATION, Issue 4 2010Sylvia Quemener Abstract Over the last 20 years since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, more than 1,600 different putatively pathological CFTR mutations have been identified. Until now, however, copy number mutations (CNMs) involving the CFTR gene have not been methodically analyzed, resulting almost certainly in the underascertainment of CFTR gene duplications compared with deletions. Here, high-resolution array comparative genomic hybridization (averaging one interrogating probe every 95,bp) was used to analyze the entire length of the CFTR gene (189,kb) in 233 cystic fibrosis chromosomes lacking conventional mutations. We succeeded in identifying five duplication CNMs that would otherwise have been refractory to analysis. Based upon findings from this and other studies, we propose that deletion and duplication CNMs in the human autosomal genome are likely to be generated in the proportion of approximately 2,3:1. We further postulate that intragenic gene duplication CNMs in other disease loci may have been routinely underascertained. Finally, our analysis of ±20,bp flanking each of the 40 CFTR breakpoints characterized at the DNA sequence level provide support for the emerging concept that non-B DNA conformations in combination with specific sequence motifs predispose to both recurring and nonrecurring genomic rearrangements. Hum Mutat 31:1,8, 2010. © 2010 Wiley-Liss, Inc. [source] Gene conversion causing human inherited disease: Evidence for involvement of non-B-DNA-forming sequences and recombination-promoting motifs in DNA breakage and repair,HUMAN MUTATION, Issue 8 2009Nadia Chuzhanova Abstract A variety of DNA sequence motifs including inverted repeats, minisatellites, and the , recombination hotspot, have been reported in association with gene conversion in human genes causing inherited disease. However, no methodical statistically based analysis has been performed to formalize these observations. We have performed an in silico analysis of the DNA sequence tracts involved in 27 nonoverlapping gene conversion events in 19 different genes reported in the context of inherited disease. We found that gene conversion events tend to occur within (C+G)- and CpG-rich regions and that sequences with the potential to form non-B-DNA structures, and which may be involved in the generation of double-strand breaks that could, in turn, serve to promote gene conversion, occur disproportionately within maximal converted tracts and/or short flanking regions. Maximal converted tracts were also found to be enriched (P<0.01) in a truncated version of the ,-element (a TGGTGG motif), immunoglobulin heavy chain class switch repeats, translin target sites and several novel motifs including (or overlapping) the classical meiotic recombination hotspot, CCTCCCCT. Finally, gene conversions tend to occur in genomic regions that have the potential to fold into stable hairpin conformations. These findings support the concept that recombination-inducing motifs, in association with alternative DNA conformations, can promote recombination in the human genome. Hum Mutat 30:1,10, 2009. © 2009 Wiley-Liss, Inc. [source] cDNA sequence, mRNA expression and genomic DNA of trypsinogen from the Indianmeal moth, Plodia interpunctellaINSECT MOLECULAR BIOLOGY, Issue 1 2000Y. C. Zhu Abstract Trypsin-like enzymes are major insect gut enzymes that digest dietary proteins and proteolytically activate insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). Resistance to Bt in a strain of the Indianmeal moth, Plodia interpunctella, was linked to the absence of a major trypsin-like proteinase (Oppert et al., 1997). In this study, trypsin-like proteinases, cDNA sequences, mRNA expression levels and genomic DNAs from Bt-susceptible and -resistant strains of the Indianmeal moth were compared. Proteinase activity blots of gut extracts indicated that the susceptible strain had two major trypsin-like proteinases, whereas the resistant strain had only one. Several trypsinogen-like cDNA clones were isolated and sequenced from cDNA libraries of both strains using a probe deduced from a conserved sequence for a serine proteinase active site. cDNAs of 852 nucleotides from the susceptible strain and 848 nucleotides from the resistant strain contained an open reading frame of 783 nucleotides which encoded a 261-amino acid trypsinogen-like protein. There was a single silent nucleotide difference between the two cDNAs in the open reading frame and the predicted amino acid sequence from the cDNA clones was most similar to sequences of trypsin-like proteinases from the spruce budworm, Choristoneura fumiferana, and the tobacco hornworm, Manduca sexta. The encoded protein included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Northern blotting analysis showed no major difference between the two strains in mRNA expression in fourth-instar larvae, indicating that transcription was similar in the strains. Southern blotting analysis revealed that the restriction sites for the trypsinogen genes from the susceptible and resistant strains were different. Based on an enzyme size comparison, the cDNA isolated in this study corresponded to the gene for the smaller of two trypsin-like proteinases, which is found in both the Bt-susceptible and -resistant strains of the Indianmeal moth. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF064525 for the RC688 strain and AF064526 for HD198). [source] Mammalian Phosphatidylinositol 4-KinasesIUBMB LIFE, Issue 2 2003Ludwig M. G. Heilmeyer Jr. Abstract Three phosphatidylinositol 4-kinase isoforms, PI4K 230, 92 and 55 have been cloned and sequenced allowing a much wider characterization than the previously employed enzymological typing into type II and III enzymes. PI4K 230 and 92 contain a highly conserved catalytic core, PI4K55 one with a much lower degree of similarity. Candidate kinase motifs, deduced from the protein kinase super family, are absolutely conserved in all isoforms. Kinase activities are described based on their sensitivity and reactivity towards wortmannin, phenylarsine oxide (PAO) and 5,-p-fluorosulfonylbenzoyladenosine (FSBA). Localization of all isoforms in the cell is reported. All enzymes contain nuclear localization and export sequence motifs (NLS and NES) leading to the expectation that they can be transferred to the nucleus. PI4K230 has been found in the nucleolus, PI4K92 in the nucleus, additionally further broadening the function of these enzymes. In the cytoplasm of neuronal cells, PI4K230 is distributed evenly on membranes that are ultra structurally cisterns of the rough endoplasmatic reticulum, outer membranes of mitochondria, multivesicular bodies, and are in close vicinity of synaptic contacts. PI4K92 is functionally characterized as a key enzyme regulating Golgi disintegration/reorganization during mitosis probably via phosphorylation by cyclin-dependent kinases on well-defined sites. PI4K55 is involved in the production of second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3) at the plasma membrane, moreover, in the endocytotic pathway in the cytoplasm. [source] Correlation between translation efficiency and outcome of combination therapy in chronic hepatitis C genotype 3JOURNAL OF VIRAL HEPATITIS, Issue 2 2006A. Yasmeen Summary., Combination therapy with interferon- , (IFN- ,) and ribavirin (RBV) in chronic hepatitis C demonstrates the best responses against hepatitis C virus (HCV) of genotype 3. Still, it has proven to be ineffective in 20,30% of patients infected with this genotype. In the present study, we analysed the translation efficiency mediated by the internal ribosome entry site (IRES) region in HCV genotype 3 genomes isolated from sustained responders (SR) and non-responders (NR), assuming that this may influence the outcome of treatment. Pretreatment isolates of genotype 3 from 22 individuals (15 SR, seven NR) were selected for such analyses. The IRES region [nucleotide (nt) 1,407] was cloned into a dual luciferase vector and IRES activity assessed following transfection into various cell lines. Low relative translation efficiency was observed for IRES elements derived from SR patients, whereas those of NR patients showed significantly greater translation efficiency (29.7 ± 13 vs 69.4 ± 22; P < 0.01). Subsequently, the effect of IFN- , plus RBV on IRES-driven translation in vitro was determined. A greater suppressive effect was observed on IRES activity isolated from seven SR patients, when compared with seven NR patients. In conclusion, IRES efficiency in vitro correlated with treatment response for HCV genotype 3. Further studies are warranted to investigate whether IRES efficiency in vitro, or sequence motifs associated with IRES efficiency, will be worthwhile to explore as prognostic tools for other HCV genotypes in the treatment of chronic HCV infection. [source] RNA-dependent RNA polymerase activity encoded by GB virus-B non-structural protein 5BJOURNAL OF VIRAL HEPATITIS, Issue 5 2000Zhong Phylogenetic analysis and polyprotein organization comparison have shown that GB virus-B (GBV-B) is closely related to hepatitis C virus (HCV). In this study, the coding region for GBV-B non-structural protein 5B (NS5B) was isolated by reverse transcription,polymerase chain reaction (RT,PCR) from pooled serum of GBV-B-infected tamarins. Expression of soluble GBV-B NS5B protein in Escherichia coli was achieved by removal of a 19-amino acid hydrophobic domain at the C-terminus of the protein. The truncated GBV-B NS5B (NS5B,CT19) was purified to homogeneity and shown to possess an RNA-dependent RNA polymerase (RdRp) activity in both gel-based and scintillation proximity assays. NS5B,CT19 required the divalent cation Mn2+ for enzymatic activity, at an optimal concentration of 15 m M. Interestingly, Mg2+, at concentrations up to 20 m M, did not support the GBV-B NS5B activity. This differs from HCV NS5B where both Mn2+ and Mg2+ can support RdRp activity. Zn2+ was found to inhibit the activity of GBV-B NS5B, with a 50% inhibitory concentration (IC50) of 5,10 ,M. Higher concentrations of monovalent salts (NaCl or KCl > 100 m M) and glycerol (> 3%) were also inhibitory. NS5B,CT19 was able to bind to RNA homopolymers, but utilized most efficiently poly(C), the one with the lowest binding affinity for RNA synthesis. Mutational analysis of GBV-B NS5B demonstrated the importance of several conserved sequence motifs for enzymatic activity. Based on sequence homology (, 37% identity and 52% similarity) between GBV-B and HCV NS5B proteins, the active GBV-B RdRp provides a good surrogate assay system for HCV polymerase studies. [source] Discrimination of Arcobacter butzleri isolates by polymerase chain reaction-mediated DNA fingerprintingLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2002H.I. Atabay Aims:,The objective of this study was to subtype Arcobacter butzleri isolates using RAPD-PCR. Methods and Results:,Thirty-five A. butzleri isolates obtained from chicken carcasses were examined. PCR-mediated DNA fingerprinting technique with primers of the variable sequence motifs was used to detect polymorphism within the isolates. Eleven distinct DNA profiles were obtained as follows: Of the 35 strains, 10 as profile 4; seven as profile 1; five as profile 3; three as profiles 2 and 9; two as profile 10; one as profiles 5, 6, 7, 8 and 11. Conclusions:,Chicken carcasses sold in markets were found to be contaminated with several different strains of A. butzleri. RAPD-PCR technique was found to be a useful technique for distinguishing A. butzleri isolates. Significance and Impact of the Study:,The presence of several different A. butzleri strains on chicken carcasses may indicate multiple sources of contamination. The epidemiological role of A. butzleri in human and animal diseases should be investigated further. [source] Evolution and structural organisation of mitochondrial DNA control region of myiasis-causing fliesMEDICAL AND VETERINARY ENTOMOLOGY, Issue 1 2000A. C. Lessinger Summary This study reports the molecular characterization of the mtDNA control region (called the A + T-rich region in insects) of five dipteran species which cause myiasis: Cochliomyia hominivorax Coquerel, Cochliomyia macellaria Fabricius, Chrysomya megacephala Fabricius, Lucilia eximia Wiedemann (Diptera: Calliphoridae) and Dermatobia hominis Linnaeus Jr (Diptera: Oestridae). The control region in these species varies in length from 1000 to 1600 bp. Two structural domains with specific evolutionary patterns were identified. These were (1) conserved sequence blocks containing primary sequence motifs, including dinucleotide pyrimidine-purine series and long T-stretches, located at the 5, end adjacent to the tRNAIle gene and (2) a hypervariable domain at the 3, end characterized by increased nucleotide divergence and size variation. A high frequency of A,T transversions at nucleotide substitution level indicated directional mutation pressure. The phylogenetic usefulness of the insect control region is discussed. [source] A novel member of the glycosyltransferase family, ,3Gn-T2, highly downregulated in invasive human bladder transitional cell carcinomasMOLECULAR CARCINOGENESIS, Issue 2 2001Irina Gromova Abstract Differential display reverse transcription (DDRT),polymerase chain reaction (PCR) was used to compare the transcriptomes of invasive and noninvasive fresh human bladder transitional cell carcinomas. A differentially expressed novel gene sharing structural similarity with the human ,3-galactosyltransferase family, ,-1,3- N -acetylglucosaminyltransferase-T2 (,3Gn-T2), was identified. The full-length ,3Gn-T2 cDNA, containing a complete open reading frame of 1193 bp, was cloned and sequenced. ,3Gn-T2 exhibited 29,41% homology to the multigene ,3-galactosyltransferase family. Expression of the full-length ,3Gn-T2 cDNA in an in vitro coupled transcription/translation assay yielded a primary translation product with an apparent Mr of 46 kDa, which is in agreement with the predicted 397-amino-acid protein encoded by ,3Gn-T2. Multiple peptide alignment showed several sequence motifs corresponding to putative catalytic domains that are conserved throughout all members of the ,3-galactosyltransferase family, namely, a type II transmembrane domain, a conserved DxD motif, an N -glycosylation site, and five conserved cysteins. By RT-PCR strong downregulation of ,3Gn-T2 expression was noted in invasive human bladder transitional cell carcinomas (16 fresh biopsy samples: grade III, T2,T4) compared with their noninvasive counterparts (15 fresh biopsies: grade II, Ta), suggesting that ,3Gn-T2 may be involved in cancer progression. © 2001 Wiley-Liss, Inc. [source] Characterization of HMW-PBPs from the rod-shaped actinomycete Corynebacterium glutamicum: peptidoglycan synthesis in cells lacking actin-like cytoskeletal structuresMOLECULAR MICROBIOLOGY, Issue 3 2007Noelia Valbuena Summary Analysis of the complete genome sequence of Corynebacterium glutamicum indicated that, in addition to ftsI, there are eight proteins with sequence motifs that are strongly conserved in penicillin binding proteins (PBPs): four genes that code for high-molecular-weight (HMW)-PBPs (PBP1a, PBP1b, PBP2a and PBP2b), two genes encoding low-molecular-weight PBPs (PBP4 and PBP4b) and two probable ,-lactamases (PBP5 and PBP6). Here, the function of the four HMW-PBPs in C. glutamicum was investigated using a combination of genetic knockouts, enhanced green fluorescent protein 2 (EGFP2) fusions and penicillin staining of membrane preparations. The four HMW-PBPs were expressed in a growing culture of C. glutamicum, but none of four pbp genes was individually essential for the growth of the bacterium, and only the simultaneous disruption of both pbp1b and pbp2b was lethal. The fused EGFP2,PBP proteins were functional in vivo, which allowed correct determination of their cellular localization. EGFP2 fusions to PBP1a, PBP1b and PBP2b localized at the poles and at the septum, whereas EGFP2,PBP2a was predominantly found at the septum. Cefsulodin treatment specifically delocalized PBP1a and PBP1b (class A HMW-PBPs), whereas mecillinam caused the specific delocalization of PBP2b and PBP2a (class B HMW-PBPs). The results provide new insight into the mechanisms involved in the synthesis of the cell wall in this bacterial species, which lacks a known actin-like cytoskeletal structure. [source] The TEA/ATTS transcription factor CaTec1p regulates hyphal development and virulence in Candida albicansMOLECULAR MICROBIOLOGY, Issue 3 2000Anja Schweizer The temporal and spatial expression of stage-specific genes during morphological development of fungi and higher eukaryotes is controlled by transcription factors. In this study, we report the cloning and functional analysis of the Candida albicans TEC1 (CaTEC1) gene, a new member of the TEA/ATTS family of transcription factors that regulates C. albicans virulence. The promoters of the type 4, 5 and 6 proteinase isogenes (SAP4,6) contain repetitive TEA/ATTS consensus sequence motifs. This finding suggests a possible role for a homologue of Saccharomyces cerevisiae TEC1 during the activation of proteinase gene expression in C. albicans. CaTEC1 is predominantly expressed in the hyphal form of C. albicans. In vitro, serum-induced hyphal formation as well as evasion from M, after phagocytosis is suppressed in catec1/catec1 mutant cells. Furthermore, expression of the proteinase isogenes SAP4,6 is no longer inducible in these mutant cells. The deletion of the CaTEC1 gene attenuates virulence of C. albicans in a systemic model of murine candidiasis, although both mutant and revertant cells that were prepared from infected tissues or the vaginal mucosa grew in a hyphal morphology in vivo. CaTEC1 complements the pseudohyphal and invasive growth defect of haploid and diploid S. cerevisiae tec1/tec1 mutant cells and strongly activates the promoter of FLO11, a gene required for pseudohyphal growth. This study provides the first evidence pointing to an essential role for a member of the TEA/ATTS transcription factor family that had so far only been ascribed to function during development as a virulence regulator in microbial pathogenesis. [source] | |||||||||||||||||||||||||||||||