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The pioneering chromosome conformation capture (3C) method provides the opportunity to study chromosomal folding in the nucleus. It is based on formaldehyde cross-linking of living cells followed by enzyme digestion, intramolecular ligation and quantitative (Q)-PCR analysis. However, 3C requires prior knowledge of the bait and interacting sequence (termed interactor) rendering it less useful for genome-wide studies. As several recent reports document, this limitation has been overcome by exploiting a circular intermediate in a variant of the 3C method, termed 4C (for circular 3C). The strategic positioning of primers within the bait enables the identification of unknown interacting sequences, which form part of the circular DNA. Here, we describe a protocol for our 4C method, which produces a high-resolution interaction map potentially suitable for the analysis of cis-regulatory elements and for comparison with chromatin marks obtained by chromatin immunoprecipitation (ChIP) on chip at the sites of interaction. Following optimization of enzyme digestions and amplification conditions, the protocol can be completed in 2-3 weeks.  相似文献   

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Chromosome conformation capture (3C) is a powerful tool to study DNA looping. The procedure generates chimeric DNA templates after ligation of restriction enzyme fragments juxtaposed in vivo by looping. These unique ligation products (ULPs) are typically quantified by gel-based methods, which are practically inefficient. Taqman probes may be used, but are expensive. Cycle threshold (Ct) determined using SYBR Green, an inexpensive alternative, is hampered by non-specific products and/or background fluorescence, both due to high template/ULP ratio. SYBR Green melting curve analysis (MCA) is a well-known qualitative tool for assessing PCR specificity. Here we present for the first time MCA as a quantitative tool (qMCA) to compare template concentrations across different samples and apply it to 3C to assess looping among remote elements identified by STAT1 and IRF1 ChIP-chip at the interferon-γ responsive CIITA and SOCS1 loci. This rapid, inexpensive approach provided highly reproducible identification and quantification of ULPs over a significant linear range. Therefore, qMCA is a robust method to assess chromatin looping in vivo, and overcomes several drawbacks associated with other approaches. Our data suggest that basal and induced looping is a involving remote enhancers is a common mechanism at IFNγ-regulated targets.  相似文献   

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Quantitative analysis of chromosome conformation capture assays (3C-qPCR)   总被引:1,自引:0,他引:1  
Chromosome conformation capture (3C) technology is a pioneering methodology that allows in vivo genomic organization to be explored at a scale encompassing a few tens to a few hundred kilobase-pairs. Understanding the folding of the genome at this scale is particularly important in mammals where dispersed regulatory elements, like enhancers or insulators, are involved in gene regulation. 3C technology involves formaldehyde fixation of cells, followed by a polymerase chain reaction (PCR)-based analysis of the frequency with which pairs of selected DNA fragments are crosslinked in the population of cells. Accurate measurements of crosslinking frequencies require the best quantification techniques. We recently adapted the real-time TaqMan PCR technology to the analysis of 3C assays, resulting in a method that more accurately determines crosslinking frequencies than current semiquantitative 3C strategies that rely on measuring the intensity of ethidium bromide-stained PCR products separated by gel electrophoresis. Here, we provide a detailed protocol for this method, which we have named 3C-qPCR. Once preliminary controls and optimizations have been performed, the whole procedure (3C assays and quantitative analyses) can be completed in 7-9 days.  相似文献   

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Mammalian interphase chromosomes fold into a multitude of loops to fit the confines of cell nuclei, and looping is tightly linked to regulated function. Chromosome conformation capture (3C) technology has significantly advanced our understanding of this structure‐to‐function relationship. However, all 3C‐based methods rely on chemical cross‐linking to stabilize spatial interactions. This step remains a “black box” as regards the biases it may introduce, and some discrepancies between microscopy and 3C studies have now been reported. To address these concerns, we developed “i3C”, a novel approach for capturing spatial interactions without a need for cross‐linking. We apply i3C to intact nuclei of living cells and exploit native forces that stabilize chromatin folding. Using different cell types and loci, computational modeling, and a methylation‐based orthogonal validation method, “TALE‐iD”, we show that native interactions resemble cross‐linked ones, but display improved signal‐to‐noise ratios and are more focal on regulatory elements and CTCF sites, while strictly abiding to topologically associating domain restrictions.  相似文献   

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All members of the human herpesvirus protease (HHV Pr) family are active as weakly associating dimers but inactive as monomers. A small-molecule allosteric inhibitor of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) traps the enzyme in an inactive monomeric state where the C-terminal helices are unfolded and the hydrophobic dimer interface is exposed. NMR titration studies demonstrate that the inhibitor binds to KSHV Pr monomers with low micromolar affinity. A 2.0-Å-resolution X-ray crystal structure of a C-terminal truncated KSHV Pr-inhibitor complex locates the binding pocket at the dimer interface and displays significant conformational perturbations at the active site, 15 Å from the allosteric site. NMR and CD data suggest that the small molecule inhibits human cytomegalovirus protease via a similar mechanism. As all HHV Prs are functionally and structurally homologous, the inhibitor represents a class of compounds that may be developed into broad-spectrum therapeutics that allosterically regulate enzymatic activity by disrupting protein-protein interactions.  相似文献   

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Summary In a vesicular-arbuscular mycorrhizal association a different texture in the cell wall of the host and fungus is revealed by using cryoultramicrotomy, that is a powerful tool in studying macromolecular arrangements. Using this technique the host wall displays fibrillar texture, whilst the fungal wall appears amorphous. The latter is not affected by the tested enzymatic digestions. On the contrary, it is labelled by ferritin linked to the wheat germ agglutinin, displaying the presence of N-acetylglucosamine. It is suggested that this sugar is present in single units or in short chains, whilst true chitin fibrils are not formed.  相似文献   

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Yi M  Yu Q 《Genetika》2002,38(11):1480-1484
Human sex chromosome-specific probes were hybridized to metaphase spreads of three fish species, Monopterus albus Zuiew, Danio rerio and Mastacembelus aculeatus Basilewsky, to reveal evolutionary conservation of sex chromosomal segments between distantly related species of vertebrates. The human X chromosomal paint disclosed 4, 8, and 6 conserved syntenic segments in the karyotypes of the three fish species respectively, which were scattered in several pairs of homologous chromosomes. But no conserved segment was identified in our experiments when the human Y chromosomal probes were used. The evolution of the X chromosome of vertebrates is discussed.  相似文献   

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Individual interphase chromosome domains revealed by in situ hybridization   总被引:15,自引:0,他引:15  
Summary The position and arrangement of individual chromosomes in interphase nuclei were examined in mouse-human cell hybrids by in situ hybridization of biotinylated human DNA probes. Intense and even labeling of human chromosomes with little background was observed when polyethylene glycol and Tween-20 were included in hybridization solutions. Human interphase chromosomes were separated from each other in the nucleus, and were confined to well localized domains. Hybrid cells with a single human chromosome showed a reproducible position of this chromosome in the nucleus. Some chromosomes appeared to have a characteristic folding pattern in interphase. Optical section as well as electron microscopy of labeled regions revealed the presence of 0.2 m wide fibers in each interphase domain, as well as adjacent, locally extended 500 nm fibers. Such fibers are consistent with previously proposed structural models of interphase chromosomes.  相似文献   

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The G-polymorphism of metaphase chromosomes of peripheral human lymphocytes and its inheritance in 32 families (268 persons) and 315 unrelated persons after G-staining has been studied. The site of Q-heterochromatine, its size and the length of secondary constrictions were accepted as morphological signs of Q-polymorphic variants of chromosomes. All the three chromosome signs are shown to be inherited according to codominant type and are characteristic features of each separate chromosome. No identical patterns of Q-polymorphic chromosome variants are found among all the persons studied, except the monozygotic twins. According to the data obtained, the question of individualization of each chromosome in the karyotype (3, 4, 13--15, 21, 22) and of each personal individuality in relation to Q-polymorphism is discussed.  相似文献   

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HAMP domains are widely abundant signaling modules. The putative mechanism of their function comprises switching between two distinct states. To unravel these conformational transitions, we apply site-directed spin labeling and time-resolved EPR spectroscopy to the phototactic receptor/transducer complex NpSRII/NpHtrII. We characterize the kinetic coupling of NpHtrII to NpSRII along with the activation period of the transducer and follow the transient conformational signal. The observed transient shift towards a more compact state of the HAMP domain upon light-activation agrees with structure-based calculations. It thereby validates the two modeled signaling states and integrates the domain’s dynamics into the current model.  相似文献   

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Photosystem (PS) I is a large membrane protein complex vital for oxygenic photosynthesis, one of the most important biological processes on the planet. We present an "atomic" model of higher plant PSI, based on theoretical modeling using the recent 4.4 angstroms x-ray crystal structure of PSI from pea. Because of the lack of information on the amino acid side chains in the x-ray structural model and the high cofactor content in this system, novel modeling techniques were developed. Our model reveals some important structural features of plant PSI that were not visible in the crystal structure, and our model sheds light on the evolutionary relationship between plant and cyanobacterial PSI.  相似文献   

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Cells regulate functionally related genes cis- and trans-contacts in order to perform specific biological roles. To understand the cryptic spatial genomic contexts underlying these biological functions, we analyzed the gene association data from the gene ontology (GO) database and the genomic spatial organization data obtained by analysis of chromosome conformation capture (3C)-based data from the Sequence Read Archive, where GO and 3C-based data were used to measure functional similarity and spatial proximity, respectively, between genomic loci. In the human genome and the fission yeast genome, we observed that correlation between the two measures was statistically significant on a genome-wide scale. Specifically, it is also confirmed that the genomic spatial architecture is affected by functional similarity of genes by showing better correlation of functional similarities with spatial distances estimated by contact frequencies than those estimated by genomic distances for cis-contacts. Furthermore, we analyzed distances between the genomic segments sharing the same GO term using the two-sample t test, found that the genomic segments identified by various GO terms are spatially located closer than the average distance over statistically-valid contacts, and provided a list of the GO terms. The results suggested that genomic loci with similar biological functions are situated in close proximity to each other in the nuclear space by aggregating functionally related genes in a short spatial range.  相似文献   

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A variety of experimental evidence suggests that rapid, long-range propagation of conformational changes through the core of proteins plays a vital role in allosteric communication. Here, we describe a non-equilibrium molecular dynamics simulation method, anisotropic thermal diffusion (ATD), which allowed us to observe a dominant intramolecular signaling pathway in PSD-95, a member of the PDZ domain protein family. The observed pathway is in good accordance with a pathway previously inferred using a multiple sequence analysis of 276 PDZ domain proteins. In comparison with conventional solution molecular dynamics methods, the ATD method provides greatly enhanced signal-to-noise, allowing long-distance correlations to be observed clearly. The ATD method requires neither a large number of homologous proteins, nor extremely long simulation times to obtain a complete signaling pathway within a protein. Therefore, the ATD method should prove to be a powerful and general complement to experimental efforts to understand the physical basis of intramolecular signaling.  相似文献   

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Chromosomal evolution in carnivores has been revisited extensively using cross-species chromosome painting. Painting probes derived from flow-sorted chromosomes of the domestic dog, which has one of the most rearranged karyotypes in mammals and the highest dipoid number (2n=78) in carnivores, are a powerful tool in detecting both evolutionary intra- and inter-chromosomal rearrangements. However, only a few comparative maps have been established between dog and other non-Canidae species. Here, we extended cross-species painting with dog probes to seven more species representing six carnivore families: Eurasian lynx (Lynx lynx), the stone marten (Martes foina), the small Indian civet (Viverricula indica), the Asian palm civet (Paradoxurus hermaphrodites), Javan mongoose (Hepestes javanicas), the raccoon (Procyon lotor) and the giant panda (Ailuropoda melanoleuca). The numbers and positions of intra-chromosomal rearrangements were found to differ among these carnivore species. A comparative map between human and stone marten, and a map among the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), stone marten and human were also established to facilitate outgroup comparison and to integrate comparative maps between stone marten and other carnivores with such maps between human and other species. These comparative maps give further insight into genome evolution and karyotype phylogenetic relationships among carnivores, and will facilitate the transfer of gene mapping data from human, domestic dog and cat to other species.  相似文献   

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RNA aptamers are in vitro-selected binding domains that recognize their respective ligand with high affinity and specificity. They are characterized by complex three-dimensional conformations providing preformed binding pockets that undergo conformational changes upon ligand binding. Small molecule-binding aptamers have been exploited as synthetic riboswitches for conditional gene expression in various organisms. In the present study, double electron-electron resonance (DEER) spectroscopy combined with site-directed spin labeling was used to elucidate the conformational transition of a tetracycline aptamer upon ligand binding. Different sites were selected for post-synthetic introduction of either the (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate by reaction with a 4-thiouridine modified RNA or of 4-isocyanato-2,6-tetramethylpiperidyl-N-oxid spin label by reaction with 2'-aminouridine modified RNA. The results of the DEER experiments indicate the presence of a thermodynamic equilibrium between two aptamer conformations in the free state and capture of one conformation upon tetracycline binding.  相似文献   

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