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Takashi Ohyama - One of the best experts on this subject based on the ideXlab platform.
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nuclear localization of reporter genes activated by Curved DNA
Journal of Bioscience and Bioengineering, 2012Co-Authors: Koji Udagawa, Hajime Kimura, Hideyuki Tanabe, Takashi OhyamaAbstract:Curved DNA structures with a left-handed superhelical conformation can activate eukaryotic transcription. Mechanistically, these structures favor binding to histone cores and can function as a docking site for sliding nucleosomes. Thus, promoters with this kind of Curved DNA can adopt a more open structure, facilitating transcription initiation. However, whether the Curved DNA segment can affect localization of a reporter gene is an open question. Localization of a gene in the nucleus often plays an important role in its expression and this phenomenon may also have a Curved DNA-dependent mechanism. We examined this issue in transient and stable assay systems using a 180-bp synthetic Curved DNA with a left-handed superhelical conformation. The results clearly showed that Curved DNA of this kind does not have a property to deliver reporter constructs to nuclear positions that are preferable for transcription. We also identify the spatial location to which electroporation delivers a reporter plasmid in the nucleus.
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Competence of an artificial bent DNA as a transcriptional activator in mouse ES cells
Molecular Biology Reports, 2011Co-Authors: Jun Ichi Tanase, Jun-ichi Nishikawa, Koji Udagawa, Tasuku Mitani, Takashi OhyamaAbstract:Curved DNA structures with a left-handed superhelical conformation can activate eukaryotic transcription. However, their potency in transgene activation in embryonic stem (ES) cells has not been examined. T20 is an artificial Curved DNA of 180 bp that serves as a transcriptional activator. We investigated the effect of T20 on transcription in mouse ES cell lines or hepatocytes differentiated from them. We established 10 sets of cell lines each harboring a single copy of the reporter construct. Each set comprised a T20-harboring cell line and a T20-less control cell line. Analyses showed that in ES cells and in hepatocytes originating from these cells, T20 both activated and repressed transcription in a manner that was dependent on the locus of reporter. The present and previous studies strongly suggest that in cells that have a strict gene regulation system, transcriptional activation by T20 occurs only in a transcriptionally active locus in the genome.
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highly efficient chromatin transcription induced by superhelically Curved DNA segments the underlying mechanism revealed by a yeast system
Biochemistry, 2010Co-Authors: Jun Ichi Tanase, Jun-ichi Nishikawa, Nobuyuki Morohashi, Masashi Fujita, Mitsuhiro Shimizu, Takashi OhyamaAbstract:Superhelically Curved DNA structures can strongly activate transcription in mammalian cells. However, the mechanism underlying the activation has not been clarified. We investigated this mechanism ...
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the location of the left handedly Curved DNA sequence affects exogenous DNA expression in vivo
Archives of Biochemistry and Biophysics, 2007Co-Authors: H Kamiya, Takashi Ohyama, S Fukunaga, H HarashimaAbstract:The intranuclear disposition of a plasmid is extremely important for transgene expression. The effects of a left-handedly Curved sequence with high histone affinity on plasmid expression were examined in vivo. A naked luciferase-plasmid was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. The location of the left-handedly Curved sequence determined the transgene expression, without affecting the amount of intranuclear exogenous DNA. The plasmid containing the Curved sequence at the location that results in the exposure of the TATA box out of the nucleosome core showed the highest expression. These results suggest that sequences with high histone affinity could control transgene expression from plasmids in vivo.
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regulation of chromatin structure by Curved DNA how activator binding sites become accessible
2007Co-Authors: Takashi OhyamaAbstract:A single somatic cell of humans contains DNA fibers of a total length of approximately 2 m, which are compacted, without entanglement, into the nucleus of approximately 1×10−5 m in diameter. To greater or lesser degrees, all organisms compact their DNA. Biologically important DNA regions, such as the origins of DNA replication, regulatory regions of transcription, and recombination loci, must all be compacted. The tightly constrained DNA, however, presents the appropriate environment for replication, transcription, and recombination to take place.
Takeshi Mizuno - One of the best experts on this subject based on the ideXlab platform.
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an escherichia coli Curved DNA binding protein whose expression is affected by the stationary phase specific sigma factor sigma s
Molecular Genetics and Genomics, 1995Co-Authors: Minoru Kakeda, Hisami Yamada, Chiharu Ueguchi, Takeshi MizunoAbstract:FromEscherichia coli, a DNA-binding protein that preferentially recognizes a Curved DNA sequence was isolated and shown to correspond to one that has recently been reported as a binding protein for the replication origin of theE. coli chromosome, named Rob. Here, arob promoter-lacZ transcriptional fusion was constructed on the chromosome, and used to demonstrate that the expression ofrob is notably enhanced at the onset of stationary phase in Luria-broth and also under certain growth conditions in a minimal medium, such as glucose- and phosphate-starvation medium. It was further shown that this growth condition-dependent expression ofrob is notably reduced in a null mutant for the stationary phase-specific sigma subunit of RNA polymerase, σs, although σs-independent expression ofrob was significant during the logarithmic growth phase. Furthermore therob null mutant was found to exhibit, as compared with the wild-type, an altered profile of protein synthesis, particularly at the very late stationary phase.
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a fission yeast gene encoding a protein that preferentially associates with Curved DNA
Yeast, 1994Co-Authors: Hisami Yamada, Hideki Mori, Hiroyuki Momoi, Yoshiyuki Nakagawa, Chiharu Ueguchi, Takeshi MizunoAbstract:We searched for fission yeast (Schizosaccharomyces pombe) proteins that preferentially bind to a synthetic Curved DNA sequence, by means of a DNA-binding gel shift assay in the presence of an excess amount of a non-Curved DNA sequence as a competitor. We identified such a protein in S. pombe. The protein, thus purified, has an apparent molecular weight of 42,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was suggested that this protein (42 K-protein) recognizes and binds to a Curved DNA structure in a given nucleotide sequence, although it also binds to a non-Curved DNA sequence with lower affinity. As its putative coding sequence, a 1.9-kilobase genomic DNA from S. pombe was cloned and sequenced. Sequencing of a cDNA clone also revealed the existence of an open reading frame, with no intron, encoding a 381-amino-acid protein with a calculated molecular mass, 41,597. This protein appears to be located in the nucleus. The predicted protein sequence revealed that the 42 K-protein exhibits no significant similarity to any other known proteins, except to a hypothetical protein of Caenorhabditis elegans.
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importance of stereospecific positioning of the upstream cis acting DNA element containing a Curved DNA structure for the functioning of the escherichia coli pro v promoter
Bioscience Biotechnology and Biochemistry, 1994Co-Authors: Ken-ichi Tanaka, Chiharu Ueguchi, Takeshi MizunoAbstract:The mechanism by which the Escherichia coli pro V promoter is activated more than 100-fold in response to the medium osmolarity, without the help of any known trans-acting activators, is not yet fully understood. In this context, it has recently begun to be realized that structural features, not the primary sequences, of cis-acting DNA elements may be important for transcriptional regulation in prokaryotes. From this point of view, in this study the pro V promoter was characterized by constructing a series of spacer-insertion mutants in a pro V-lac Z fusion on the chromosome. Here it was found that the upstream cis-acting sequence must be positioned stereospecifically with respect to the principal − 35 and −10 regions for the pro V promoter to be fully activated. In this regard, it was suggested that an overall DNA structure, particularly DNA curvature, is an important cis-acting parameter for activation of the pro V promoter.
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molecular analysis of the escherichia coli hns gene encoding a DNA binding protein which preferentially recognizes Curved DNA sequences
Molecular Genetics and Genomics, 1991Co-Authors: Hisami Yamada, Ken-ichi Tanaka, Takayuki Yoshida, Chihiro Sasakawa, Takeshi MizunoAbstract:We previously demonstrated that the E. coli protein, H-NS (or H1a), encoded by the gene hns (or osmZ or bglY) preferentially recognizes Curved DNA sequences in vitro. In order to gain further insight into the complex function of H-NS and the significance of DNA curvature, we constructed a structurally defined hns deletion mutant on the E. coli chromosome. The hns deletion mutant thus obtained showed a variety of phenotypes previously for other lesions in hns. It was further demonstrated that, in this hns deletion background, numerous E. coli cellular proteins were either strongly expressed or remarkably repressed, as compared to their expression levels in wild-type cells.
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Systematic characterization of Curved DNA segments randomly cloned from Escherichia coli and their functional significance
Molecular and General Genetics MGG, 1991Co-Authors: Ken-ichi Tanaka, Shuji Muramatsu, Hisami Yamada, Takeshi MizunoAbstract:In addition to the set of Curved DNA segments isolated previously from Escherichia coli , another set of Curved DNA segments has now been isolated. To gain an insight into the functional significance of these Curved DNA sequences, systematic analyses were carried out, which included not only mapping of the precise locations of the segments on the E. coli chromosome but also clarification of the gene organization in the chromosomal regions surrounding the Curved DNA sequences. It was demonstrated that most of the Curved DNA sequences, which have been characterized so far, appear to be located immediately upstream of the coding sequences of adjacent genes. It was also demonstrated that an E. coli histone-like protein, named H-NS (or H1a), exhibits a strong affinity for naturally occurring Curved DNA sequences in regions upstream promoters.
Jun-ichi Nishikawa - One of the best experts on this subject based on the ideXlab platform.
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Competence of an artificial bent DNA as a transcriptional activator in mouse ES cells
Molecular Biology Reports, 2011Co-Authors: Jun Ichi Tanase, Jun-ichi Nishikawa, Koji Udagawa, Tasuku Mitani, Takashi OhyamaAbstract:Curved DNA structures with a left-handed superhelical conformation can activate eukaryotic transcription. However, their potency in transgene activation in embryonic stem (ES) cells has not been examined. T20 is an artificial Curved DNA of 180 bp that serves as a transcriptional activator. We investigated the effect of T20 on transcription in mouse ES cell lines or hepatocytes differentiated from them. We established 10 sets of cell lines each harboring a single copy of the reporter construct. Each set comprised a T20-harboring cell line and a T20-less control cell line. Analyses showed that in ES cells and in hepatocytes originating from these cells, T20 both activated and repressed transcription in a manner that was dependent on the locus of reporter. The present and previous studies strongly suggest that in cells that have a strict gene regulation system, transcriptional activation by T20 occurs only in a transcriptionally active locus in the genome.
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highly efficient chromatin transcription induced by superhelically Curved DNA segments the underlying mechanism revealed by a yeast system
Biochemistry, 2010Co-Authors: Jun Ichi Tanase, Jun-ichi Nishikawa, Nobuyuki Morohashi, Masashi Fujita, Mitsuhiro Shimizu, Takashi OhyamaAbstract:Superhelically Curved DNA structures can strongly activate transcription in mammalian cells. However, the mechanism underlying the activation has not been clarified. We investigated this mechanism ...
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a designed Curved DNA segment that is a remarkable activator of eukaryotic transcription
FEBS Journal, 2006Co-Authors: Noriyuki Sumida, Jun-ichi Nishikawa, Miho Amano, Haruka Kishi, Takayo Furuya, Haruyuki Sonobe, Takashi OhyamaAbstract:To identify artificial DNA segments that can stably express transgenes in the genome of host cells, we built a series of Curved DNA segments that mimic a left-handed superhelical structure. Curved DNA segments of 288 bp (T32) and 180 bp (T20) were able to activate transcription from the herpes simplex virus thymidine kinase (tk) promoter by approximately 150-fold and 70-fold, respectively, compared to a control in a transient transfection assay in COS-7 cells. The T20 segment was also able to activate transcription from the human adenovirus type 2 E1A promoter with an 18-fold increase in the same assay system, and also activated transcription from the tk promoter on episomes in COS-7 cells. We also established five HeLa cell lines with genomes containing T20 upstream of the transgene promoter and control cell lines with T20 deleted from the transgene locus. Interestingly, T20 was found to activate transcription in all the stable transformants, irrespective of the locus. This suggests that the T20 segment may allow stable expression of transgenes, which is of importance in many fields, and may also be useful for the construction of nonviral vectors for gene therapy.
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Left-handedly Curved DNA introduced into episomes can activate transcription in a TATA box-dependent manner
Journal of Advanced Science, 2004Co-Authors: Jun-ichi Nishikawa, Yoshiro Fukue, Takashi OhyamaAbstract:Using a transient transfection assay system, we have recently demonstrated that Curved DNA that mimics a negative supercoil can activate transcription by influencing chromatin structure. To know whether this phenomenon is general, we investigated the effect of the DNA curvature on transcription in an episomal assay system. It was confirmed that the left-handedly Curved DNA can activate transcription even in the episomal system. Furthermore, it was found that conversion of the TATA sequence in the reporter constructs from the original non-canonical sequence to the canonical one considerably elevated transcription from the control template and, as the result, diminished the difference in promoter activity between the control reporter and the reporters containing the left-handedly Curved DNA, indicating that transcriptional activation by the DNA curvature is more evident when the TATA box is not optimized.
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left handedly Curved DNA regulates accessibility to cis DNA elements in chromatin
Nucleic Acids Research, 2003Co-Authors: Jun-ichi Nishikawa, Yoshiro Fukue, Miho Amano, Shigeo Tanaka, Haruka Kishi, Yoshiko Hirota, Kinya Yoda, Takashi OhyamaAbstract:There is little information on chromatin structure that allows access of trans-acting transcription factors. Logically, the target DNA elements become accessible by either exposing themselves towards the environment on the surface of the nucleosome, or making the regulatory region free of the nucleosome. Here, we demonstrate that Curved DNA that mimics a negative supercoil can play both roles in the promoter region. By constructing 35 reporter plasmids and using in vivo assay systems, we scrutinized the relationships between upstream DNA geometry, nucleosome positioning and promoter activity. When the left-handedly Curved DNA was linked to the herpes simplex virus thymidine kinase (HSV tk) promoter at a specific rotational phase and distance, the Curved DNA attracted the nucleosome and the TATA box was thereby left in the linker DNA with its minor groove facing outwards, which led to the activation of transcription. Neither planar curving, nor right-handedly Curved DNA nor straight DNA had this effect. Our results seem to provide a clue for solving the problem of why Curved DNA is often located near transcriptional control regions.
Michael Famulok - One of the best experts on this subject based on the ideXlab platform.
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Design, assembly, characterization, and operation of double-stranded interlocked DNA nanostructures
Nature Protocols, 2019Co-Authors: Julián Valero, Marko Škugor, Michael W. Haydell, Mathias Centola, Yinzhou Ma, Daniel Keppner, Ze Yu, Michael FamulokAbstract:Mechanically interlocked DNA nanostructures are useful as flexible entities for operating DNA-based nanomachines. Interlocked structures made of double-stranded (ds) DNA components can be constructed by irreversibly threading them through one another to mechanically link them. The interlocked components thus remain bound to one another while still permitting large-amplitude motion about the mechanical bond. The construction of interlocked dsDNA architectures is challenging because it usually involves the synthesis and modification of small dsDNA nanocircles of various sizes, dependent on intrinsically Curved DNA. Here we describe the design, generation, purification, and characterization of interlocked dsDNA structures such as catenanes, rotaxanes, and daisy-chain rotaxanes (DCRs). Their construction requires precise control of threading and hybridization of the interlocking components at each step during the assembly process. The protocol details the characterization of these nanostructures with gel electrophoresis and atomic force microscopy (AFM), including acquisition of high-resolution AFM images obtained in intermittent contact mode in liquid. Additional functionality can be conferred on the DNA architectures by incorporating proteins, molecular switches such as photo-switchable azobenzene derivatives, or fluorophores for studying their mechanical behavior by fluorescence quenching or fluorescent resonance energy transfer experiments. These modified interlocked DNA architectures provide access to more complex mechanical devices and nanomachines that can perform a variety of desired functions and operations. The assembly of catenanes can be completed in 2 d, and that of rotaxanes in 3 d. Addition of azobenzene functionality, fluorophores, anchor groups, or the site-specific linkage of proteins to the nanostructure can extend the time line.This protocol describes the detailed procedures for design, assembly, and characterization of different types of double-stranded DNA nanostructures, as well as a number of downstream applications.
Nancy C Stellwagen - One of the best experts on this subject based on the ideXlab platform.
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monovalent cation binding by Curved DNA molecules containing variable numbers of a tracts
Biophysical Journal, 2008Co-Authors: Nancy C StellwagenAbstract:Monovalent cation binding by DNA A-tracts, runs of four or more contiguous adenine or thymine residues, has been determined for two Curved ∼200 basepair (bp) restriction fragments, one taken from the M13 origin of replication and the other from the VP1 gene of SV40. These two fragments have previously been shown to contain stable, centrally located bends of 44° and 46°, respectively, located within ∼60 bp “curvature modules” containing four or five irregularly spaced A-tracts. Transient electric birefringence measurements of these two fragments, sequence variants containing reduced numbers of A-tracts in the SV40 curvature module or changes in the residues flanking the A-tracts in the M13 curvature module, have been combined with the free solution electrophoretic mobilities of the same fragments using known equations to estimate the effective charge of each fragment. The effective charge is reduced, on average, by one-third charge for each A-tract in the curvature module, suggesting that each A-tract binds a monovalent cation approximately one-third of the time. Monovalent cation binding to two or more A-tracts is required to observe significant curvature of the DNA helix axis.
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Curved DNA molecules migrate anomalously slowly in polyacrylamide gels even at zero gel concentration
Electrophoresis, 2006Co-Authors: Nancy C StellwagenAbstract:The electrophoretic mobilities of Curved and normal DNA molecules of the same size have been measured in polyacrylamide gels containing various acrylamide concentrations and cross-linker ratios. Ferguson plots were constructed to extrapolate the observed mobilities to zero gel concentration. The DNA samples were two 147-bp restriction fragments, called 12A and 12B, obtained from the MspI digestion of plasmid pBR322, and head-to-tail multimers of each fragment. Fragment 12A is stably Curved and migrates anomalously slowly in polyacrylamide gels; fragment 12B has the conformation of normal DNA and migrates with normal electrophoretic mobilities. The extrapolated mobilities of the Curved fragment 12A and its multimers at zero gel concentration are lower than the extrapolated mobilities of the normal fragment 12B and its multimers. The free solution mobility of the Curved fragment 12A, measured by CE, is also lower than that of the normal fragment 12B. The combined results indicate that the extrapolated mobilities observed for Curved DNA molecules at zero polyacrylamide gel concentration reflect the intrinsic differences in their free solution mobilities.
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Curved DNA molecules migrate anomalously slowly in free solution
Nucleic Acids Research, 2005Co-Authors: Earle Stellwagen, Nancy C StellwagenAbstract:The electrophoretic mobility of a Curved DNA restriction fragment taken from the VP1 gene in the SV40 minichromosome has been measured in polyacrylamide gels and free solution, using capillary electrophoresis. The 199 bp restriction fragment has an apparent bend angle of 46 ± 2° located at SV40 sequence position 1922 ± 2 bp [Lu Y.J., Weers B.D. and Stellwagen N. C. (2005) Biophys. J., 88, 1191–1206]. The ‘curvature module’ surrounding the apparent bend center contains five unevenly spaced A- and T-tracts, which are responsible for the observed curvature. The parent 199 bp fragment and sequence mutants containing at least one A-tract in the curvature module migrate anomalously slowly in free solution, as well as in polyacrylamide gels. Hence, the anomalously slow mobilities observed for Curved DNA molecules in polyacrylamide gels are due in part to their anomalously slow mobilities in free solution. Analysis of the gel and free solution mobility decrements indicates that each A- or T-tract contributes independently, but not equally, to the curvature of the 199 bp fragment and its A-tract mutants. The relative contribution of each A- or T-tract to the observed curvature depends on its spacing with respect to the first A-tract in the curvature module.
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conformational isomers of Curved DNA molecules can be observed by polyacrylamide gel electrophoresis
Electrophoresis, 2000Co-Authors: Nancy C StellwagenAbstract:Dimers, trimers and higher multimers of two 147-base pair restriction fragments called 12 A and 12B, obtained from the MspI digest of plasmid pBR322, migrate as sharp bands in agarose and dilute polyacrylamide gels, indicating that they are homogeneous in molecular weight. However, the electrophoretic bands corresponding to multimers of the Curved fragment 12A are split into sharp sub-bands in more concentrated polyacrylamide gels. The relative intensities and spacing of the sub-bands depend on the number of monomers in the multimer, the pH of the buffer, and the presence or absence of divalent cations in the solution. Since band splitting is not observed for the normal 12B multimers under any gel-running conditions, the sub-bands observed for multimers of the Curved fragment 12A must be attributed to conformational isomers which are in slow exchange on the electrophoretic time scale. Band splitting is also observed for multimers of a Curved DNA fragment containing the kinetoplast bending locus and for plasmid pUC19 linearized by digestion with certain restriction enzymes. Plasmid pUC19 contains two nearly equidistant regions of intrinsic curvature (Strutz, K., Stellwagen, N. C., Electrophoresis 1996, 17, 989-995). Hence, DNA molecules containing two or more regions of curvature exist as discrete subpopulations of conformational isomers which can be observed as separate bands migrating in polyacrylamide gels.
