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See detailTargeting the DNA-binding activity of the human ERG transcription factor using new heterocyclic dithiophene diamidines
Nhili, Raja; Peixoto, Paul ULg; Depauw, Sabine et al

in Nucleic Acids Research (2012)

Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or ... [more ▼]

Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or translocated in leukaemia and prostate carcinoma. <br />In this work, we selected the di-(thiophene-phenylamidine) compound DB1255 as an ERG/DNA binding inhibitor using a screening test of synthetic inhibitors of the ERG/DNA interaction followed by electrophoretic mobility shift assays (EMSA) validation. Spectrometry, footprint and biosensor-surface plasmon resonance analyses of the DB1255/DNA interaction evidenced sequence selectivity and groove binding as dimer. Additional EMSA evidenced the precise DNA-binding sequence required for optimal DB1255/DNA binding and thus for an efficient ERG/DNA complex inhibition. We <br />further highlighted the structure activity relationshipsfrom comparison with derivatives. In cellulo luciferase assay confirmed this modulation both with the constructed optimal sequences and the Osteopontin promoter known to be regulated by ERG and which ERG-binding site was protected from DNaseI digestion on binding of DB1255. These data showed for the first time the ERG/DNA complex modulation, both in vitro and in cells, by a heterocyclic diamidine that specifically targets a portion of the ERG DNA recognition site. [less ▲]

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See detailLSD1 cooperates with CTIP2 to promote HIV-1 transcriptional silencing.
Le Douce, Valentin; Colin, Laurence; Redel, Laetitia et al

in Nucleic Acids Research (2012), 40(5), 1904-15

Microglial cells are the main HIV-1 targets in the central nervous system (CNS) and constitute an important reservoir of latently infected cells. Establishment and persistence of these reservoirs rely on ... [more ▼]

Microglial cells are the main HIV-1 targets in the central nervous system (CNS) and constitute an important reservoir of latently infected cells. Establishment and persistence of these reservoirs rely on the chromatin structure of the integrated proviruses. We have previously demonstrated that the cellular cofactor CTIP2 forces heterochromatin formation and HIV-1 gene silencing by recruiting HDAC and HMT activities at the integrated viral promoter. In the present work, we report that the histone demethylase LSD1 represses HIV-1 transcription and viral expression in a synergistic manner with CTIP2. We show that recruitment of LSD1 at the HIV-1 proximal promoter is associated with both H3K4me3 and H3K9me3 epigenetic marks. Finally, our data suggest that LSD1-induced H3K4 trimethylation is linked to hSET1 recruitment at the integrated provirus. [less ▲]

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See detailPhEVER: a database for the global exploration of virus–host evolutionary relationships
Palmeira, Leonor ULg; Penel, Simon; Lotteau, Vincent et al

in Nucleic Acids Research (2011)

Fast viral adaptation and the implication of this rapid evolution in the emergence of several new infectious diseases have turned this issue into a major challenge for various research domains. Indeed ... [more ▼]

Fast viral adaptation and the implication of this rapid evolution in the emergence of several new infectious diseases have turned this issue into a major challenge for various research domains. Indeed, viruses are involved in the development of a wide range of pathologies and understanding how viruses and host cells interact in the context of adaptation remains an open question. In order to provide insights into the complex interactions between viruses and their host organisms and namely in the acquisition of novel functions through exchanges of genetic material, we developed the PhEVER database. This database aims at providing accurate evolutionary and phylogenetic information to analyse the nature of virus–virus and virus–host lateral gene transfers. PhEVER (http://pbil.univ-lyon1.fr/databases/phever) is a unique database of homologous families both (i) between sequences from different viruses and (ii) between viral sequences and sequences from cellular organisms. PhEVER integrates extensive data from up-to-date completely sequenced genomes (2426 non-redundant viral genomes, 1007 non-redundant prokaryotic genomes, 43 eukaryotic genomes ranging from plants to vertebrates) and offers a clustering of proteins into homologous families containing at least one viral sequences, as well as alignments and phylogenies for each of these families. Public access to PhEVER is available through its webpage and through all dedicated ACNUC retrieval systems. [less ▲]

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See detaild(CGGTGGT) forms an octameric parallel G-quadruplex via stacking of unusual G(:C):G(:C):G(:C):G(:C) octads
Borbone, Nicola; Amato, Jussara; Oliviero, Giorgia et al

in Nucleic Acids Research (2011)

Among non-canonical DNA secondary structures, G-quadruplexes are currently widely studied because of their probable involvement in many pivotal biological roles, and for their potential use in ... [more ▼]

Among non-canonical DNA secondary structures, G-quadruplexes are currently widely studied because of their probable involvement in many pivotal biological roles, and for their potential use in nanotechnology. The overall quadruplex scaffold can exhibit several morphologies through intramolecular or intermolecular organization of G-rich oligodeoxyribonucleic acid strands. In particular, several G-rich strands can form higher order assemblies by multimerization between several G-quadruplex units. Here, we report on the identification of a novel dimerization pathway. Our Nuclear magnetic resonance, circular dichroism, UV, gel electrophoresis and mass spectrometry studies on the DNA sequence dCGGTGGT demonstrate that this sequence forms an octamer when annealed in presence of K+ or NH4+ ions, through the 5′-5′ stacking of two tetramolecular G-quadruplex subunits via unusual G(:C):G(:C):G(:C):G(:C) octads. [less ▲]

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See detailTetramolecular G-quadruplex formation pathways studied by electrospray mass spectrometry
Rosu, Frédéric ULg; Gabelica, Valérie ULg; Poncelet, Harmonie et al

in Nucleic Acids Research (2010)

Electrospray mass spectrometry was used to investigate the mechanism of tetramolecular G-quadruplex formation by the DNA oligonucleotide dTG5T, in ammonium acetate. The intermediates and products were ... [more ▼]

Electrospray mass spectrometry was used to investigate the mechanism of tetramolecular G-quadruplex formation by the DNA oligonucleotide dTG5T, in ammonium acetate. The intermediates and products were separated according to their mass (number of strands and inner cations) and quantified. The study of the temporal evolution of each species allows us to propose the following formation mechanism. (i) Monomers, dimers and trimers are present at equilibrium already in the absence of ammonium acetate. (ii) The addition of cations promotes the formation of tetramers and pentamers that incorporate ammonium ions and therefore presumably have stacked guanine quartets in their structure. (iii) The pentamers eventually disappear and tetramers become predominant. However, these tetramers do not have their four strands perfectly aligned to give five G-quartets: the structures contain one ammonium ion too few, and ion mobility spectrometry shows that their conformation is more extended. (iv) At 4°C, the rearrangement of the kinetically trapped tetramers with presumably slipped strand(s) into the perfect G-quadruplex structure is extremely slow (not complete after 4 months). We also show that the addition of methanol to the monomer solution significantly accelerates the cation-induced G-quadruplex assembly. [less ▲]

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See detailPatrocles: a database of polymorphic miRNA-mediated gene regulation in vertebrates
Hiard, Samuel ULg; Charlier, Carole ULg; Coppieters, Wouter ULg et al

in Nucleic Acids Research (2010), 38(Database), 640-651

The Patrocles database (http://www.patrocles.org/) compiles DNA sequence polymorphisms (DSPs) that are predicted to perturb miRNA-mediated gene regulation. Distinctive features include: (i) the coverage ... [more ▼]

The Patrocles database (http://www.patrocles.org/) compiles DNA sequence polymorphisms (DSPs) that are predicted to perturb miRNA-mediated gene regulation. Distinctive features include: (i) the coverage of seven vertebrate species in its present release, aiming for more when information becomes available, (ii) the coverage of the three compartments involved in the silencing process (i.e. targets, miRNA precursors and silencing machinery), (iii) contextual information that enables users to prioritize candidate ‘Patrocles DSPs’, including graphical information on miRNA-target coexpression and eQTL effect of genotype on target expression levels, (iv) the inclusion of Copy Number Variants and eQTL information that affect miRNA precursors as well as genes encoding components of the silencing machinery and (v) a tool (Patrocles finder) that allows the user to determine whether her favorite DSP may perturb miRNA-mediated gene regulation of custom target sequences. To support the biological relevance of Patrocles' content, we searched for signatures of selection acting on ‘Patrocles single nucleotide polymorphisms (pSNPs)’ in human and mice. As expected, we found a strong signature of purifying selection against not only SNPs that destroy conserved target sites but also against SNPs that create novel, illegitimate target sites, which is reminiscent of the Texel mutation in sheep. [less ▲]

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See detailSteady-state levels of imported tRNAs in Chlamydomonas mitochondria are correlated with both cytosolic and mitochondrial codon usages
Vinogradova, Elizaveta; Salinas, Thalia; Cognat, Valerie et al

in Nucleic Acids Research (2009), 37(5), 1521-1528

The mitochondrial genome of Chlamydomonas reinhardtii only encodes three expressed tRNA genes, thus most mitochondrial tRNAs are likely imported. The sharing of tRNAs between chloroplasts and mitochondria ... [more ▼]

The mitochondrial genome of Chlamydomonas reinhardtii only encodes three expressed tRNA genes, thus most mitochondrial tRNAs are likely imported. The sharing of tRNAs between chloroplasts and mitochondria has been speculated in this organism. We first demonstrate that no plastidial tRNA is present in mitochondria and that the mitochondrial translation mainly relies on the import of nucleus-encoded tRNA species. Then, using northern analysis, we show that the extent of mitochondrial localization for the 49 tRNA isoacceptor families encoded by the C. reinhardtii nuclear genome is highly variable. Until now the reasons for such variability were unknown. By comparing cytosolic and mitochondrial codon usage with the sub-cellular distribution of tRNAs, we provide unprecedented evidence that the steady-state level of a mitochondrial tRNA is linked not only to the frequency of the cognate codon in mitochondria but also to its frequency in the cytosol, then allowing optimal mitochondrial translation. [less ▲]

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See detailNovel method for high-throughput colony PCR screening in nanoliter-reactors.
Walser, Marcel; Pellaux, Rene; Meyer, Andreas et al

in Nucleic acids research (2009), 37(8), 57

We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a ... [more ▼]

We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20,000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100,000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies. [less ▲]

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See detailSelective recognition of pyrimidine–pyrimidine DNA mismatches by distance-constrained macrocyclic bis-intercalators
Bahr, Matthias; Gabelica, Valérie ULg; Granzhan, Anton et al

in Nucleic Acids Research (2008), 36

Binding of three macrocyclic bis-intercalators, derivatives of acridine and naphthalene, and two acyclic model compounds to mismatch-containing and matched duplex oligodeoxynucleotides was analyzed by ... [more ▼]

Binding of three macrocyclic bis-intercalators, derivatives of acridine and naphthalene, and two acyclic model compounds to mismatch-containing and matched duplex oligodeoxynucleotides was analyzed by thermal denaturation experiments, electrospray ionization mass spectrometry studies (ESI-MS) and fluorescent intercalator displacement (FID) titrations. The macrocyclic bis-intercalators bind to duplexes containing mismatched thymine bases with high selectivity over the fully matched ones, whereas the acyclic model compounds are much less selective and strongly bind to the matched DNA. Moreover, the results from thermal denaturation experiments are in very good agreement with the binding affinities obtained by ESI-MS and FID measurements. The FID results also demonstrate that the macrocyclic naphthalene derivative BisNP preferentially binds to pyrimidine– pyrimidine mismatches compared to all other possible base mismatches. This ligand also efficiently competes with a DNA enzyme (M.TaqI) for binding to a duplex with a TT-mismatch, as shown by competitive fluorescence titrations. Altogether, our results demonstrate that macrocyclic distance- constrained bis-intercalators are efficient and selective mismatch-binding ligands that can interfere with mismatch-binding enzymes. [less ▲]

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See detailDirect inhibition of the DNA-binding activity of POU transcription factors Pit-1 and Brn-3 by selective binding of a phenyl-furan-benzimidazole dication.
Peixoto, Paul ULg; Liu, Yang; Depauw, Sabine et al

in Nucleic Acids Research (2008), 36(10), 3341-53

The development of small molecules to control gene expression could be the spearhead of future-targeted therapeutic approaches in multiple pathologies. Among heterocyclic dications developed with this aim ... [more ▼]

The development of small molecules to control gene expression could be the spearhead of future-targeted therapeutic approaches in multiple pathologies. Among heterocyclic dications developed with this aim, a phenyl-furan-benzimidazole dication DB293 binds AT-rich sites as a monomer and 5'-ATGA sequence as a stacked dimer, both in the minor groove. Here, we used a protein/DNA array approach to evaluate the ability of DB293 to specifically inhibit transcription factors DNA-binding in a single-step, competitive mode. DB293 inhibits two POU-domain transcription factors Pit-1 and Brn-3 but not IRF-1, despite the presence of an ATGA and AT-rich sites within all three consensus sequences. EMSA, DNase I footprinting and surface-plasmon-resonance experiments determined the precise binding site, affinity and stoichiometry of DB293 interaction to the consensus targets. Binding of DB293 occurred as a cooperative dimer on the ATGA part of Brn-3 site but as two monomers on AT-rich sites of IRF-1 sequence. For Pit-1 site, ATGA or AT-rich mutated sequences identified the contribution of both sites for DB293 recognition. In conclusion, DB293 is a strong inhibitor of two POU-domain transcription factors through a cooperative binding to ATGA. These findings are the first to show that heterocyclic dications can inhibit major groove transcription factors and they open the door to the control of transcription factors activity by those compounds. [less ▲]

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See detailNORINE: a database of nonribosomal peptides.
Caboche, Segolene; Pupin, Maude; Leclere, Valerie et al

in Nucleic acids research (2008), 36(Database issue), 326-31

Norine is the first database entirely dedicated to nonribosomal peptides (NRPs). In bacteria and fungi, in addition to the traditional ribosomal proteic biosynthesis, an alternative ribosome-independent ... [more ▼]

Norine is the first database entirely dedicated to nonribosomal peptides (NRPs). In bacteria and fungi, in addition to the traditional ribosomal proteic biosynthesis, an alternative ribosome-independent pathway called NRP synthesis allows peptide production. It is performed by huge protein complexes called nonribosomal peptide synthetases (NRPSs). The molecules synthesized by NRPS contain a high proportion of nonproteogenic amino acids. The primary structure of these peptides is not always linear but often more complex and may contain cycles and branchings. In recent years, NRPs attracted a lot of attention because of their biological activities and pharmacological properties (antibiotic, immunosuppressor, antitumor, etc.). However, few computational resources and tools dedicated to those peptides have been available so far. Norine is focused on NRPs and contains more than 700 entries. The database is freely accessible at http://bioinfo.lifl.fr/norine/. It provides a complete computational tool for systematic study of NRPs in numerous species, and as such, should permit to obtain a better knowledge of these metabolic products and underlying biological mechanisms, and ultimately to contribute to the redesigning of natural products in order to obtain new bioactive compounds for drug discovery. [less ▲]

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See detailConformational and thermodynamic changes of the repressor/DNA operator complex upon monomerization shed new light an regulation mechanisms of bacterial resistance against beta-lactam antibiotics
Boudet, J.; Duval, V.; Van Melckebeke, H. et al

in Nucleic Acids Research (2007), 35(13), 4384-4395

In absence of beta-lactam antibiotics, Blal and Mecl homodimeric repressors negatively control the expression of genes involved in P-lactam resistance in Bacillus licheniformis and in Staphylococcus ... [more ▼]

In absence of beta-lactam antibiotics, Blal and Mecl homodimeric repressors negatively control the expression of genes involved in P-lactam resistance in Bacillus licheniformis and in Staphylococcus aureus. Subsequently to P-lactam presence, Blal/Mecl is inactivated by a single-point proteolysis that separates its N-terminal DNA-binding domain to its C-terminal domain responsible for its dimerization. Concomitantly to this proteolysis, the truncated repressor acquires a low affinity for its DNA target that explains the expression of the structural gene for resistance. To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1, of blaP (1/20P(1)blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner. Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator-repressor dimer complex, leading to a 300 rotation of the monomer with respect to a central axis extended across the DNA. These results open new insights for the repression and induction mechanisms of bacterial resistance to beta-lactams. [less ▲]

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See detailGuanines are a quartet's best friend: impact of base substitutions on the kinetics and stability of tetramolecular quadruplexes
Gros, Julien; Rosu, Frédéric ULg; Amrane, Samir et al

in Nucleic Acids Research (2007), 35(9), 3064-3075

Parallel tetramolecular quadruplexes may be formed with short oligodeoxynucleotides bearing a block of three or more guanines. We analyze the properties of sequence variants of parallel quadruplexes in ... [more ▼]

Parallel tetramolecular quadruplexes may be formed with short oligodeoxynucleotides bearing a block of three or more guanines. We analyze the properties of sequence variants of parallel quadruplexes in which each guanine of the central block was systematically substituted with a different base. Twelve types of substitutions were assessed in more than 100 different sequences. We conducted a comparative kinetic analysis of all tetramers. Electrospray mass spectrometry was used to count the number of inner cations, which is an indicator of the number of effective tetrads. In general, the presence of a single substitution has a strong deleterious impact on quadruplex stability, resulting in reduced quadruplex lifetime/ thermal stability and in decreased association rate constants. We demonstrate extremely large differences in the association rate constants of these quadruplexes depending on modification position and type. These results demonstrate that most guanine substitutions are deleterious to tetramolecular quadruplex structure. Despite the presence of well- defined non- guanine base quartets in a number of NMR and X- ray structures, our data suggest that most non- guanine quartets do not participate favorably in structural stability, and that these quartets are formed only by virtue of the docking platform provided by neighboring G- quartets. Two notable exceptions were found with 8- bromoguanine ( X) and 6- methyl- isoxanthopterin ( P) substitutions, which accelerate quadruplex formation by a factor of 10 when present at the 50 end. The thermodynamic and kinetic data compiled here are highly valuable for the design of DNA quadruplex assemblies with tunable association/ dissociation properties. [less ▲]

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See detailMolecular characterization of geminivirus-derived small RNAs in different plant species.
Akbergenov, Rashid; Si-Ammour, Azeddine; Blevins, Todd et al

in Nucleic acids research (2006), 34(2), 462-71

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs-the hallmarks of silencing-associated with Cabbage leaf curl begomovirus in Arabidopsis and ... [more ▼]

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs-the hallmarks of silencing-associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5' end phosphorylated, as shown by phosphatase treatments, and methylated at the 3'-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to beta-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interactions. [less ▲]

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See detailExtending the classification of bacterial transcription factors beyond the helix-turn-helix motif as an alternative approach to discover new cis/trans relationships
Rigali, Sébastien ULg; Schlicht, M.; Hoskisson, P. et al

in Nucleic Acids Research (2004), 32(11), 3418-3426

Transcription factors (TFs) of bacterial helix-turn-helix superfamilies exhibit different effector-binding domains (EBDs) fused to a DNA-binding domain with a common feature. In a previous study of the ... [more ▼]

Transcription factors (TFs) of bacterial helix-turn-helix superfamilies exhibit different effector-binding domains (EBDs) fused to a DNA-binding domain with a common feature. In a previous study of the GntR superfamily, we demonstrated that classifying members into subfamilies according to the EBD heterogeneity highlighted unsuspected and accurate TF-binding site signatures. In this work, we present how such in silico analysis can provide prediction tools to discover new cis/trans relationships. The TF-binding site consensus of the HutC/GntR subfamily was used to (i) predict target sites within the Streptomyces coelicolor genome, (ii) discover a new HutC/GntR regulon and (iii) discover its specific TF. By scanning the S.coelicolor genome we identified a presumed new HutC regulon that comprises genes of the phosphotransferase system (PTS) specific for the uptake of N-acetylglucosamine (PTSNag). A weight matrix was derived from the compilation of the predicted cis-acting elements upstream of each gene of the presumed regulon. Under the assumption that TFs are often subject to autoregulation, we used this matrix to scan the upstream region of the 24 HutC-like members of S.coelicolor. orf SCO5231 (dasR) was selected as the best candidate according to the high score of a 16 bp sequence identified in its upstream region. Our prediction that DasR regulates the PTSNag regulon was confirmed by in vivo and in vitro experiments. In conclusion, our in silico approach permitted to highlight the specific TF of a regulon out of the 673 orfs annotated as 'regulatory proteins' within the genome of S.coelicolor. [less ▲]

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See detailPathbase: a database of mutant mouse pathology
Schofield, P. N.; Bard, J. B. L.; Booth, C. et al

in Nucleic Acids Research (2004), 32(Sp. Iss. SI), 512-515

Pathbase is a database that stores images of the abnormal histology associated with spontaneous and induced mutations of both embryonic and adult mice including those produced by transgenesis, targeted ... [more ▼]

Pathbase is a database that stores images of the abnormal histology associated with spontaneous and induced mutations of both embryonic and adult mice including those produced by transgenesis, targeted multagenesis and chemical mutagenesis. Images of normal mouse histology and strain-dependent background lesions are also available. The database and the images are publicly accessible (http://www.pathbase.net) and linked by anatomical site, gene and other identifiers to relevant databases; there are also facilities for public comment and record annotation. The database is structured around a novel ontology of mouse disorders (MPATH) and provides high-resolution downloadable images of normal and diseased tissues that are searchable through orthogonal ontologies for pathology, developmental stage, anatomy and gene attributes (GO terms), together with controlled vocabularies for type of genetic manipulation or mutation, genotype and free text annotation for mouse strain and additional attributes. The database is actively curated and data records assessed by pathologists in the Pathbase Consortium before publication. The database interface is designed to have optimal browser and platform compatibility and to interact directly with other web-based mouse genetic resources. [less ▲]

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See detailCellular Uptake Of Antennapedia Penetratin Peptides Is A Two-Step Process In Which Phase Transfer Precedes A Tryptophan-Dependent Translocation
Dom, G.; Shaw-Jackson, C.; Matis, C. et al

in Nucleic Acids Research (2003), 31(2), 556-61

Several homeodomains and homeodomain-containing proteins enter live cells through a receptor- and energy-independent mechanism. Translocation through biological membranes is conferred by the third alpha ... [more ▼]

Several homeodomains and homeodomain-containing proteins enter live cells through a receptor- and energy-independent mechanism. Translocation through biological membranes is conferred by the third alpha-helix of the homeodomain, also known as Penetratin. Biophysical studies demonstrate that entry of Penetratin into cells requires its binding to surface lipids but that binding and translocation are differentially affected by modifications of some physico-chemical properties of the peptide, like helical amphipathicity or net charge. This suggests that the plasma membrane lipid composition affects the internalization of Penetratin and that internalization requires both lipid binding and other specific properties. Using a phase transfer assay, it is shown that negatively charged lipids promote the transfer of Penetratin from a hydrophilic into a hydrophobic environment, probably through charge neutralization. Accordingly, transfer into a hydrophobic milieu can also be obtained in the absence of negatively charged lipids, by the addition of DNA oligonucleotides. Strikingly, phase transfer by charge neutralization was also observed with a variant peptide of same charge and hydrophobicity in which the tryptophan at position 6 was replaced by a phenylalanine. However, Penetratin, but not its mutant version, is internalized by live cells. This underscores that charge neutralization and phase transfer represent only a first step in the internalization process and that further crossing of a biological membrane necessitates the critical tryptophan residue at position 6. [less ▲]

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See detailDetermination of affinity, stoichiometry and sequence selectivity of minor groove binder complexes with double-stranded oligodeoxynucleotides by electrospray ionization mass spectrometry
Rosu, Frédéric ULg; Gabelica, Valérie ULg; Houssier, Claude ULg et al

in Nucleic Acids Research (2002), 30(16), 82

Electrospray mass spectrometry was evaluated regarding the reliability of the determination of the stoichiometries and equilibrium association constants from single spectra. Complexes between minor groove ... [more ▼]

Electrospray mass spectrometry was evaluated regarding the reliability of the determination of the stoichiometries and equilibrium association constants from single spectra. Complexes between minor groove binders (Hoechst 33258, Hoechst 33342, DAPI, netropsin and berenil) and 12mer oligonucleotide duplexes with a central sequence (A/T)(4) flanked by G/C base pairs were chosen as model systems. To validate the electrospray ionization mass spectrometry (ESI-MS) method, comparisons were made with circular dichroism and fluorescence spectroscopy measurements. ESI-MS allowed the detection of minor (2 drug + DNA) species for Hoechst 33258, Hoechst 33342, DAPI and berenil with duplex d(GGGG(A/T)(4)GGGG). d(CCCC(A/T)(4)CCCC), which were undetectable with the other techniques. Assuming that the duplexes and the complexes have the same electrospray response factors, the equilbrium association constants of the 1:1 and 2:1 complexes were determined by ESI-MS, and the values show a good quantitative agreement with fluorescence determined constants for Hoechst 33258 and Hoechst 33342. It is also shown that ESI-MS can quickly give reliable information on the A/T sequence selectivity of a drug: the signal of a complex is directly related to the affinity of the drug for that particular duplex. The potential of ESI-MS as a qualitative and quantitative affinity screening method is emphasized. [less ▲]

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See detailDNA-binding mechanism of the Escherichia coli Ada O(6)-alkylguanine-DNA alkyltransferase.
Verdemato, P. E.; Brannigan, J. A.; Damblon, Christian ULg et al

in Nucleic Acids Research (2000), 28(19), 3710-8

The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O:(6)-alkylguanine lesions in DNA. Structural and ... [more ▼]

The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O:(6)-alkylguanine lesions in DNA. Structural and thermodynamic studies were carried out to obtain a model of the DNA-binding process. Nuclear magnetic resonance (NMR) studies map the DNA-binding site to helix 5, and a loop region (residues 151-160) which form the recognition helix and the 'wing' of a helix-turn-wing motif, respectively. The NMR data also suggest the absence of a large conformational change in the protein upon binding to DNA. Hence, an O:(6)-methylguanine (O:(6)meG) lesion would be inaccessible to active site nucleophile Cys146 if the modified base remained stacked within the DNA duplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator protein, and the accepted base-flipping mechanism, to construct a model of how Ada-C binds to DNA in a productive manner. To complement the structural studies, thermodynamic data were obtained which demonstrate that binding to unmethylated DNA was entropically driven, whilst the demethylation reaction provoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain. [less ▲]

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See detailDNA bending by the silencer protein NeP1 is modulated by TR and RXR.
Arnold, R.; Burcin, M.; Kaiser, Bruno ULg et al

in Nucleic Acids Research (1996), 24(14), 2640-7

NeP1 binds to the F1 silencer element of the chicken lysozyme gene and, in the presence of TR, v-ERBA or RAR, synergistically represses transcriptional activity. This repression involves a silencing ... [more ▼]

NeP1 binds to the F1 silencer element of the chicken lysozyme gene and, in the presence of TR, v-ERBA or RAR, synergistically represses transcriptional activity. This repression involves a silencing mechanism acting independently of the relative promoter position. Here we show that NeP1 alone can induce a significant directed bend on DNA. The chicken homologue of human NeP1, CTCF, shows identical binding and bending properties. In contrast, the isolated DNA binding domain of CTCF efficiently binds DNA, but fails to confer bending. Similarly, the TR-RXR hetero- or homodimer, binding adjacent to NeP1 at the F2 sequence, do not show significant DNA bending. The binding of the T3 ligand to TR changes neither the magnitude nor the direction of the NeP1 induced bend. However, when all factors are bound simultaneously as a quaternary complex, the TR-RXR heterodimer changes the location of the bend center, the flexure angle and the bending direction. [less ▲]

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