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The composition and orientation of the house mouse satellite DNA sequences (minor, major, TLC) were investigated by a FISH and CO-FISH approach in 11 taxa belonging to three clades of the subgenus Mus. Using a phylogenetic framework, our results highlighted two distribution patterns. The TLC satellite, the most recently discovered satellite, was present in all clades but varied quantitatively among species. This distribution supported its appearance in the ancestor of the subgenus followed by independent evolution in species of each clade. In contrast, the minor and major satellites occurred in only two clades of the subgenus indicating the simultaneous and recent amplification of these sequences. In addition, although qualitative differences in the composition and orientation of the satellite sequences were observed among the taxa, none of the features studied were unique to the house mouse and could account for the extensive chromosomal plasticity evidenced in Mus musculus domesticus.

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AIMS: A new assay, much more rapid and efficient than the existing standardized tests, is introduced for the evaluation of bactericidal activity of chemical disinfectants and antiseptics under simulated practical conditions of use. METHODS AND RESULTS: The bactericidal activity of biocides was quantified using a novel semi-automated assay based on the European Norm (EN) standard suspension tests but determining bacterial cell viability by intracellular adenosine tri-phosphate (ATP) content quantification instead of traditional culture-based microbiological techniques. The new test was validated by comparison to the standard suspension tests EN 1276 and EN 13727. During the validation, the linearity of the ATP detection system, limit of detection, specificity, sensitivity, relative accuracy and precision (repeatability and reproducibility) were determined. CONCLUSIONS: The validation study showed that the new assay evaluates the activity of biocides as well as the EN standard suspension tests, but it allows a large number of test conditions to be efficiently analysed. SIGNIFICANCE AND IMPACT OF THE STUDY: The new test can therefore be applied to accurately establish the lowest active concentration (MBCs) of disinfectants or antiseptics under simulated practical conditions of use and to compare the susceptibility of a large number of strains and conditions via inactivation curves. This is not possible in any reasonably practicable way with the EN standards considering the time and cost required for each determination.

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An oligonucleotide was circularized around double-stranded DNA thanks to triple helix formation. Short oligonucleotides are known to be able to form DNA triple helices by binding into the DNA major groove at an oligopurine.oligopyrimidine sequence. After sequence-specific recognition of a double-stranded DNA target through triple helix formation, the ends of the triplex-forming oligonucleotide were joined through the action of T4 DNA ligase, thus creating a circular DNA molecule catenated to the plasmid containing the target sequence. The labeling of the double-stranded DNA sequence has been carried out without any chemical or enzymatic modification of this sequence. These "padlock" oligonucleotides provide a tool to attach a noncovalent tag in an irreversible way to supercoiled plasmid or other double-stranded DNAs. Such a complex may find applications in the development of new techniques for duplex DNA detection or plasmid delivery methods for gene therapy.

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DNA triple helices offer new perspectives toward oligonucleotide-directed gene regulation. However, the poor stability of some of these structures might limit their use under physiological conditions. Specific ligands can intercalate into DNA triple helices and stabilize them. Molecular modeling and thermal denaturation experiments suggest that benzo[f]pyrido[3, 4-b]quinoxaline derivatives intercalate into triple helices by stacking preferentially with the Hoogsteen-paired bases. Based on this model, it was predicted that a benzo[f]quino[3,4-b]quinoxaline derivative, which possesses an additional aromatic ring, could engage additional stacking interactions with the pyrimidine strand of the Watson-Crick double helix upon binding of this pentacyclic ligand to a triplex structure. This compound was synthesized. Thermal denaturation experiments and inhibition of restriction enzyme cleavage show that this new compound can indeed stabilize triple helices with great efficiency and specificity and/or induce triple helix formation under physiological conditions.

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A 16-base pair oligo(purine)-oligo(pyrimidine) sequence present in the coding region of two HIV 1 proviral genes (pol and nef) was chosen as a target for triplex-forming oligonucleotides in in vitro transcription assays. Inhibition of transcription elongation was observed with triplex-forming oligonucleotide-acridine conjugates (Acr-15-TCG:5'-Acr-T4CT4G6-3' and Acr-9-TC:5'-Acr-T4CT4-3' where C is 5-methylcytosine) under conditions where the unsubstituted oligomers did not exhibit any inhibitory effect. Both SP6 bacteriophage RNA polymerase and eukaryotic RNA polymerase II were physically blocked by such a triplex barrier. The polymerase arrest is caused by the triple-helical complex involving the hydrogen-bonded oligonucleotide stabilized by the intercalated moiety and not solely by the acridine molecule specifically intercalated at the duplex-triplex junction. The stability of the triple-helical complex formed by the 15-mer containing thymines, cytosine, and guanines (15-TCG) and involving the formation of six contiguous C.GxG base triplets was strongly enhanced in the presence of a benzopyridoindole derivative (BePI), which intercalates in triplex structures. This improvement of the binding affinity led to an increased inhibition of transcription elongation. The present results demonstrate the necessity to use triplex-forming oligonucleotides with high binding affinity and a long residence time on their double-stranded target to efficiently inhibit transcription elongation. These data provide a rational basis for the optimization and the development of triplex-forming oligonucleotides as transcriptional blockers, even when they are targeted to the transcribed portion of a gene, downstream of the transcription initiation site.

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Triplex-forming oligophosphoramidates containing thymines and cytosines or 5-methyl cytosines (5' T4CT4C6T 3') bind strongly to a 16 basepair oligopurine.oligopyrimidine sequence of HIV proviral DNA even at neutral pH. These triple-helical complexes formed with oligonucleotide analogues with N3'-->P5' phosphoramidate linkages are remarkably stable compared to oligonucleotides with natural phosphodiester linkages. In transcription assays the (T,C)-containing phosphoramidate oligomers induce an efficient arrest of both bacteriophage and eukaryotic transcriptional machineries under conditions where the isosequential phosphodiesters have no inhibitory effect. In both cases the RNA polymerase (SP6, T7 or Pol II) is physically blocked by the non-covalent triplex and RNA synthesis is stopped at the triplex site. However the eukaryotic transcription machinery is blocked more efficiently (at submicromolar concentration) than the bacteriophage polymerases. The analysis of the 3'-ends of the truncated transcripts provides evidence for differences in the termination patterns induced by the triplex barrier for the bacteriophage and the eukaryotic systems. This in vitro comparative study provides the basis for the rational design of strong transcriptional inhibitors. The efficient in vitro inhibition obtained using the phosphoramidate oligomers in the eukaryotic transcription assay makes them good candidates for the development of sequence-specific antigene agents.

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We have examined the effects of benzopyridoindole derivatives on triple helices with antiparallel third strands. Absorption spectroscopy, footprinting, and gel retardation experiments demonstrate that a benzopyridoindole derivative (BePI) is able to induce formation of a triple helix with an antiparallel (G, T)-containing third strand, which does not form in the absence of this ligand. This triple-helical complex is very stable with a half-dissociation temperature as high as 51 degrees C, and its formation is pH independent. Antiparallel oligonucleotides containing thymine and guanine bind strongly to double-helical DNA under physiological conditions in the presence of only 0.5 microM BePI. Formation of a BePI-stabilized triple helix strongly inhibits cleavage of the target duplex by DNase I.

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Oligonucleotide analogs with N3'-->P5' phosphoramidate linkages bind to the major groove of double-helical DNA at specific oligopurine.oligopyrimidine sequences. These triple-helical complexes are much more stable than those formed by oligonucleotides with natural phosphodiester linkages. Oligonucleotide phosphoramidates containing thymine and cytosine or thymine, cytosine, and guanine bind strongly to the polypurine tract of human immunodeficiency virus proviral DNA under physiological conditions. Site-specific cleavage by the Dra I restriction enzyme at the 5' end of the polypurine sequence was inhibited by triplex formation. A eukaryotic transcription assay was used to investigate the effect of oligophosphoramidate binding to the polypurine tract sequence on transcription of the type 1 human immunodeficiency virus nef gene under the control of a cytomegalovirus promoter. An efficient arrest of RNA polymerase II was observed at the specific triplex site at submicromolar concentrations.

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INTRODUCTION: Based on molecular modeling studies, a model has been proposed for intercalation of triple-helix-specific ligands (benzopyridoindole (BPI) derivatives) into triple helices, in which the intercalating compounds interact mainly with the Hoogsteen-paired strands of the triple helix. We set out to test this model experimentally using DNA duplexes capable of forming parallel Hoogsteen base-paired structures. RESULTS: We have investigated the possible formation of a parallel DNA structure involving Hoogsteen hydrogen bonds by thermal denaturation, FTIR spectroscopy and gel-shift experiments. We show that BPI derivatives bind to Hoogsteen base-paired duplexes and stabilize them. The compounds induce a reorganization from a non-perfectly matched antiparallel Watson- Crick duplex into a perfectly matched parallel Hoogsteen-paired duplex. CONCLUSIONS: These results suggest that preferential intercalation of BPI derivatives in triple helices is due to their ability to interact specifically with the Hoogsteen-paired bases. The results are consistent with a model proposed on the basis of molecular modeling studies using energy minimization, and they open a new field of investigations regarding the biological relevance of Hoogsteen base-pairing.

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UV-absorption spectrophotometry and molecular modeling have been used to study the influence of the chemical nature of sugars (ribose or deoxyribose) on triple helix stability. For the Pyrimidine.purine* Pyrimidine motif, all eight combinations were tested with each of the three strands composed of either DNA or RNA. The chemical nature of sugars has a dramatic influence on triple helix stability. For each double helix composition, a more stable triple helix was formed when the third strand was RNA rather than DNA. No stable triple helix was detected when the polypurine sequence was made of RNA with a third strand made of DNA. Energy minimization studies using the JUMNA program suggested that interactions between the 2'-hydroxyl group of the third strand and the phosphates of the polypurine strand play an important role in determining the relative stabilities of triple-helical structures in which the polypyrimidine third strand is oriented parallel to the polypurine sequence. These interactions are not allowed when the third strand adopts an antiparallel orientation with respect to the target polypurine sequence, as observed when the third strand contains G and A or G and T/U. We show by footprinting and gel retardation experiments that an oligoribonucleotide containing G and A or G and U fails to bind double helical DNA, while the corresponding DNA oligomers form stable triple-helical complexes.

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Pyrimidine oligoribonucleotides bind to the major groove of double-helical DNA at homopurine.homopyrimidine sequences. They recognize Watson-Crick base pairs by forming T.A x U and C.G x C base triplets via Hoogsteen hydrogen bonding. The stability of these triple helices is much higher than that of triple helices formed by oligodeoxyribonucleotides as shown by an increase of the temperature at which half-dissociation of the third strand occurs. When the 2'-hydroxyl group of ribose moieties is replaced by 2'-O-methyl substituent, triple helix stability is further increased.

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