Inverted repeat (IR) sequences are DNA sequences that read the same from 5′ to 3′ in each strand. Some IRs can form cruciforms under the stress of negative supercoiling, and these IRs are widely found in genomes. However, their biological significance remains unclear. The aim of the current study is to explore this issue further. We constructed the first Escherichia coli genome-wide comprehensive map of IRs with cruciform-forming potential. Based on the map, we performed detailed and quantitative analyses. Here, we report that IRs with cruciform-forming potential are statistically enriched in the following five regions: the adjacent regions downstream of the stop codon-coding sites (referred to as the stop codons), on and around the positions corresponding to mRNA ends (referred to as the gene ends), ~ 20 to ~45 bp upstream of the start codon-coding sites (referred to as the start codons) within the 5′-UTR (untranslated region), ~ 25 to ~ 60 bp downstream of the start codons, and promoter regions. For the adjacent regions downstream of the stop codons and on and around the gene ends, most of the IRs with a repeat unit length of ≥ 8 bp and a spacer size of ≤ 8 bp were parts of the intrinsic terminators, regardless of the location, and presumably used for Rho-independent transcription termination. In contrast, fewer IRs were present in the small region preceding the start codons. In E. coli, IRs with cruciform-forming potential are actively placed or excluded in the regulatory regions for the initiation and termination of transcription and translation, indicating their deep involvement or influence in these processes.

Magnesium chloride and polyamines stabilize DNA and chromatin. Furthermore, they can induce nucleosome aggregation and chromatin condensation in vitro. To determine the effects of elevating the cation concentrations in the nucleus of a living cell, we microinjected various concentrations of mono-, di- and polyvalent cation solutions into the nuclei of mouse embryonic stem (ES) cells and traced their fates. Here, we show that an elevation of either MgCl2, spermidine or spermine concentration in the nucleus exerts a significant effect on mouse ES cells, and can differentiate a certain population of the cells into trophectoderm, a lineage that mouse ES cells do not normally generate, or endoderm. It is hypothesized that the cell differentiation was most probably caused by the condensation of chromatin including the Oct3/4 locus, which was induced by the elevated concentrations of these cations. (C) 2016 Elsevier Inc. All rights reserved.

Plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected mammalian cells, and such amplification generates chromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes (DMs). This method provides a novel, efficient, and rapid way to establish cells that stably produce high levels of recombinant proteins. However, because IR/MAR plasmids are amplified as repeats, they are frequently targeted by repeat-induced gene silencing (RIGS), which silences a variety of repeated sequences in transgenes and the genome. To address this problem, we developed a novel screening system using the IR/MAR plasmid to isolate human genome sequences that alleviate RIGS. The screen identified a 3,271 bp sequence (B-3-31) that elevated transgene expression without affecting the amplification process. Neither non-B structure (i.e., the inverted repeats or bending) nor known epigenetic modifier elements such as MARs, insulators, UCOEs, or STARs could explain the anti-silencing activity of B-3-31. Instead, the activity was distributed throughout the entire B-3-31 sequence, which was extremely A/T-rich and CpG-poor. Because B-3-31 effectively and reproducibly alleviated RIGS of repeated genes, it could be used to increase recombinant protein production.

Gene transfer agents (GTAs) are shaped like bacteriophage particles but have many properties that distinguish them from bacteriophages. GTAs play a role in horizontal gene transfer in nature and thus affect the evolution of prokaryotic genomes. In the course of studies on the extracellular production of designed RNAs using the marine bacterium Rhodovulum sulfidophilum, we found that this bacterium produces a GTA-like particle. The particle contains DNA fragments of 4.5. kb, which consist of randomly fragmented genomic DNA from the bacterium. This 4.5-kb DNA production was prevented while quorum sensing was inhibited. Direct observation of the particle by transmission electron microscopy revealed that the particle resembles a tailed phage and has a head diameter of about 40. nm and a tail length of about 60. nm. We also identified the structural genes for the GTA in the genome. Translated amino acid sequences and gene positions are closely related to those of the genes that encode the Rhodobacter capsulatus GTA. This is the first report of a GTA-like particle from the genus Rhodovulum. However, gene transfer activity of this particle has not yet been confirmed. The differences between this particle and other GTAs are discussed.

Unlike plants with no carnivory, carnivorous plants seem to use S-like ribonucleases (RNases) as an enzyme for carnivory. Carnivorous plant-specific conserved amino acid residues are present at four positions around the conserved active site (CAS). The roles of these conserved amino acid residues in the enzymatic function were explored in the current study by preparing five recombinant variants of DA-I, the S-like RNase of Drosera adelae. The k(cat) and k(cat)/K-m values of the enzymes revealed that among the four variants with a single mutation, the serine to glycine mutation at position 111 most negatively influenced the enzymatic activity. The change in the bulkiness of the amino acid residue side-chain seemed to be the major cause of the above effect. Modeling of the three dimensional (3D) structures strongly suggested that the S to G mutation at 111 greatly altered the overall enzyme conformation. The conserved four amino acid residues are likely to function in keeping the two histidine residues, which are essential for the cleavage of RNA strands, and the CAS in the most functional enzymatic conformation. (C) 2015 Elsevier Inc. All rights reserved.

Although the S-like ribonucleases (RNases) share sequence homology with the S-RNases involved in the self-incompatibility mechanism in plants, they are not associated with this mechanism. They usually function in stress responses in non-carnivorous plants and in carnivory in carnivorous plants. In this study, we clarified the structures of the S-like RNases of Aldrovanda vesiculosa, Nepenthes bicalcarata and Sarracenia leucophylla, and compared them with those of other plants. At ten positions, amino acid residues are conserved or almost conserved only for carnivorous plants (six in total). In contrast, two positions are specific to non-carnivorous plants. A phylogenetic analysis revealed that the S-like RNases of the carnivorous plants form a group beyond the phylogenetic relationships of the plants. We also prepared and characterized recombinant S-like RNases of Dionaea muscipula, Cephalotus follicularis, A. vesiculosa, N. bicalcarata and S. leucophylla, and RNS1 of Arabidopsis thaliana. The recombinant carnivorous plant enzymes showed optimum activities at about pH 4.0. Generally, poly(C) was digested less efficiently than poly(A), poly(I) and poly(U). The kinetic parameters of the recombinant D. muscipula enzyme (DM-I) and A. thaliana enzyme RNS1 were similar. The k (cat)/K (m) of recombinant RNS1 was the highest among the enzymes, followed closely by that of recombinant DM-I. On the other hand, the k (cat)/K (m) of the recombinant S. leucophylla enzyme was the lowest, and was similar to 1/30 of that for recombinant RNS1. The magnitudes of the k (cat)/K (m) values or k (cat) values for carnivorous plant S-like RNases seem to correlate negatively with the dependency on symbionts for prey digestion.

Spatial positioning of genes in the cell nucleus plays an important role in the regulation of genomic functions. Evidence for changes in gene positioning associated with transcriptional activity has been reported. However, our understanding of this phenomenon is still quite limited. We examined how pluripotency genes and hepatocyte-specific genes behave during the differentiation of mouse embryonic stem (ES) cells into hepatocytes, by targeting the loci of the Klf4, Nanog, Oct4, Sox2, Cyp7a1, Pck1, Tat, and Tdo2 genes, and using three-dimensional fluorescence in situ hybridization analyses. We found that each gene has a distinctly inherent localization profile in the ES cell nucleus. During differentiation, the Klf4, Nanog, Oct4, Cyp7a1, Pck1, and Tat loci shifted toward the nuclear center, while the Sox2 and Tdo2 loci shifted toward the periphery. The Klf4, Nanog, Oct4, and Tdo2 seem to prefer the outer regions, rather than the inner regions, when they are active. We also found that the radial positioning of the focused genes in the hepatocyte cell nucleus was highly correlated with the local GC content and the gene density of the surrounding region, but not with gene activity.

Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory.

The structure of the nucleosome has been solved at atomic resolution, and the genome-wide nucleosome positions have been clarified for the budding yeast Saccharomyces cerevisiae. However, the genome-wide three-dimensional arrangement of nucleosomal arrays in the nucleus remains unclear. Several studies simulated overall interphase chromosome architectures by introducing the putative persistence length of the controversial 30-nm chromatin fibres into the modelling and using data-fitting approaches. However, the genome-folding mechanism still could not be linked with the chromosome shapes, to identify which structures or properties of chromatin fibres or DNA sequences determine the overall interphase chromosome architectures. Here we demonstrate that the paths of nucleosomal arrays and the chromatin architectures themselves are determined principally by the physical properties of genomic DNA and the nucleus size in yeast. We clarified the flexibilities and persistence lengths of all linker DNAs of the organism, deduced their spatial expanses and simulated the architectures of all 16 interphase chromosomes in the nucleus, at a resolution of beads-on-a-string chromatin fibre. For the average spatial distance between two given loci in a chromosome, the model predictions agreed well with all experimental data reported to date. These findings suggest a general mechanism underlying the folding of eukaryotic genomes into interphase chromosomes.

Remarkable progress has been made in genome science during the past decade, but understanding of genomes of eukaryotes is far from complete. We have created DNA flexibility maps of the human, mouse, fruit fly, and nematode chromosomes. The maps revealed that all of these chromosomes have markedly flexible DNA regions (We named them SPIKEs). SPIKEs occur more frequently in the human chromosomes than in the mouse, fruit fly, and nematode chromosomes. Markedly rigid DNA regions (rSPIKEs) are also present in these chromosomes. The ratio of the number of SPIKEs to the total number of SPIKEs and rSPIKEs correlated positively with evolutionary stage among the organisms. Repetitive DNA sequences with flexible and rigid properties contribute to the formation of SPIKEs and rSPIKEs respectively. However, non-repetitive flexible and rigid sequences appear to play a major role in SPIKE and rSPIKE formation respectively. They might be involved in the genome-folding mechanism of eukaryotes.

DNA methylation in eukaryotes occurs on the cytosine bases in CG, CHG, and CHH (where H indicates non-G nucleotides) contexts and provides an important epigenetic mark in various biological processes. However, the structural and physical properties of methylated DNA are poorly understood. Using nondenaturing polyacrylamide gel electrophoresis, we performed a systematic study of the influence of DNA methylation on the conformation and physical properties of DNA for all CG, CHG, and CHH contexts. In the CG context, methylated multimers of the CG/CG-containing unit fragment migrated in gels slightly faster than their unmethylated counterparts. In the CHG context, both homo- and hemimethylation caused retarded migration of multimers of the CAG/CTG-containing fragment. In the CHH context, methylation caused or enhanced retarded migration of the multimers of CAA/TTG-, CAT/ATG-, CAC/GTG-, CTA/TAG-, or CTT/AAG-containing fragments. These results suggest that methylation increases DNA rigidity in the CG context and introduces distortions into several CHG and CHH sequences. More interestingly, we found that nearly all of the methylation repertoires in the CHG context and 98% of those in the CHH context in human embryonic stem cells were species that undergo conformational changes upon methylation. Similarly, most of the methylation repertoires in the Arabidopsis CHG and CHH contexts were sequences with methylation-induced distortion. We hypothesize that the methylation-induced properties or conformational changes in DNA may facilitate nucleosome formation, which provides the essential mechanism for alterations of chromatin density. © 2013 American Chemical Society.

Self-assembly is the autonomous organization of constituents into higher order structures or assemblages and is a fundamental mechanism in biological systems. There has been an unfounded idea that self-assembly may be used in the sensing and pairing of homologous chromosomes or chromatin, including meiotic chromosome pairing, polytene chromosome formation in Diptera and transvection. Recent studies proved that double-stranded DNA molecules have a sequence-sensing property and can self-assemble, which may play a role in the above phenomena. However, to explain these processes in terms of self-assembly, it first must be proved that nucleosomes retain a DNA sequence-sensing property and can self-assemble. Here, using atomic force microscopy (AFM)-based analyses and a quantitative interaction assay, we show that nucleosomes with identical DNA sequences preferentially associate with each other in the presence of Mg2+ ions. Using Xenopus borealis 5S rDNA nucleosome-positioning sequence and 601 and 603 sequences, homomeric or heteromeric octa-or tetranucleosomes were reconstituted in vitro and induced to form weak intracondensates by MgCl2. AFM clearly showed that DNA sequence-based selective association occurs between nucleosomes with identical DNA sequences. Selective association was also detected between mononucleosomes. We propose that nucleosome self-assembly and DNA self-assembly constitute the mechanism underlying sensing and pairing of homologous chromosomes or chromatin.

The intranuclear disposition of plasmid DNA is extremely important for transgene expression. The interactions between the plasmid DNA and the histone proteins are one of the keys for controlling the disposition. In this study, the effects of a left-handedly curved sequence (20-40 repeated A center dot T tracts) on transgene expression from a plasmid were examined in vivo. A naked luciferase plasmid with the curved sequence was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. Interestingly, transgene expression was markedly increased by the addition of the curved sequence. An analysis of the nucleosome positions near the left-handedly curved sequence suggested that the sequence functions as an acceptor of the histone core and allows nucleosome sliding, resulting in transcriptional activation. These results suggested that the designed curved DNA sequences could control transgene expression from plasmid DNAs in vivo.

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. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.

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.

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 in yeast cells, using 108, 180, and 252 bp synthetic curved DNA segments. Even in the presence of nucleosomes, these DNAs activated transcription front it UAS-deleted. CYC1 promoter that is silenced in the presence of nucleosomes. The fold-activations of transcription by these segments, relative to the transcription on the control that lacked Such segments, were 51.4, 63.4, and 56.4, respectively. The superhelically curved DNA structures favored nucleosome formation. However, the translational positions of the nucleosomes were dynamic. The high mobility of the nucleosomes on the superhelically curved DNA Structures seemed to influence the mobility of the nucleosomes formed oil the promoter and eventually enhanced the access to the center region or one TATA sequence. Functioning as a dock for the historic core and allowing nucleosome sliding seem to be the mechanisms underlying the transcriptional activation by superhelically curved DNA Structures in chromatin. The present study provides important clues for designing and constructing artificial chromatin modulators, as a tool for chromatin engineering.

The intranuclear disposition of plasmid DNA is highly important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on transgene expression were examined in COS-7 cells with two kinds of carriers (Lipofectamine Plus and TransIT-LT1). Three plasmids containing the curved sequence at different positions were transfected. The transgene expression was affected by the position of the left-handedly curved sequence, and the sequence at appropriate locations enhanced the expression from plasmid DNAs. However, the position effects on the expression differed from those obtained by electroporation of the same plasmid DNAs in a naked form. In addition, the degree of expression enhancement seemed to depend on the carriers. These results suggest that the left-handedly curved sequence with high histone affinity could increase the transgene expression from a plasmid delivered with carriers. (C) 2009 Elsevier B.V. All rights reserved.

Certain actin-related proteins (Arps) of budding yeast are localized in the nucleus, and have essential roles as stoichiometric components of histone acetyltransferase (HAT) and chromatin remodeling complexes. On the other hand, identification of vertebrate nuclear Arps and their functional analyses are just beginning. We show that human Arp5 (hArp5) proteins are localized in the nucleus, and that arp5 Delta yeast cells are partially complemented by hArp5. Thus, hArp5 is a novel member of the nuclear Arps of vertebrates, which possess evolutionarily conserved functions from yeast to humans. We show here that hArp5 shuttles between the nucleus and the cytoplasm. Furthermore, after the induction of DNA double strand breaks (DSB), cell growth and the accumulation of phosphorylated histone H2AX (gamma-H2AX) are impaired by hArp5 depletion. Association of hArp5 with the hIno80 chromatin remodeling enzyme and decrease of chromatinbound hIno80 by hArp5-depletion indicate that hArp5 may have a role in the recruitment of the hINO80 complex to chromatin. Overexpression of hArp5 and hIno80 enhanced gamma-H2AX accumulation. These observations suggest that hArp5 is involved in the process of DSB repair through the regulation of the chromatin remodelling machinery. (C) 2008 Elsevier Inc. All rights reserved.

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. (c) 2007 Elsevier Inc. All rights reserved.

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.

Some proteins have the property of self-assembly, known to be an important mechanism in constructing supramolecular architectures for cellular functions. However, as yet, the ability of double-stranded (ds) DNA molecules to self-assemble has not been established. Here we report that dsDNA molecules also have a property of self-assembly in aqueous solutions containing physiological concentrations of Mg2+. We show that DNA molecules preferentially interact with molecules with an identical sequence and length even in a solution composed of heterogeneous DNA species. Curved DNA and DNA with an unusual conformation and property also exhibit this phenomenon, indicating that it is not specific to usual B-form DNA. Atomic force microscopy (AFM) directly reveals the assembled DNA molecules formed at concentrations of 10 nM but rarely at 1 nM. The self-assembly is concentration-dependent. We suggest that the attractive force causing DNA self-assembly may function in biological processes such as folding of repetitive DNA, recombination between homologous sequences, and synapsis in meiosis.

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.

The conformation and mechanical properties of DNA provide additional structural and functional dimensions to chromatin organization and gene expression. The reason why transcriptional regulatory regions often contain curved DNA structures has been roughly understood They seem to construct chromatin structures that leave target elements exposed, permitting activators to recognize them and bind. Concerning the mechanical properties of DNA, a surprisingfinding was recently made for eukaryotic class II gene promoters. They have common mechanical properties irrespective of the promoter type. These properties are suggested to function as general markers in promoter recognition by transcription factors. Here, we discuss genetic information carried in DNA conformation and properties.

Bent DNAs are known to migrate slower than ordinary DNA in non-denaturing polyacrylamide gel electrophoresis. In contrast, several satellite DNAs have been shown to migrate fast. The structural property that causes the fast migration, however, is not clarified so far on molecular basis. We have investigated the structural property of a satellite DNA, which contains consecutive purine sequences and migrates faster in gel, by CD spectroscopy. Partial formation of an A-form-like structure has been suggested. Reduction in DNA length due to the formation of the A-form-like structure may be responsible for the fast migration. The pronounced rigidity of DNA may also contribute to the behavior.

Carnivorous plants usually grow in nutrient-deficient habitats, and thus they partly depend on insects for nitrogen and phosphate needed for amino acid and nucleotide synthesis. We report that a sticky digestive liquid from a sundew, Drosera adelae, contains an abundant amount of an S-like ribonuclease (RNase) that shows high amino acid-sequence similarity to S-like RNases induced by phosphate starvation or wounding in normal plants. By giving leaves an RNase "coat", D. adelae seems to achieve two requirements simultaneously to adapt itself to its specific surroundings: it obtains phosphates from insects, and defends itself against pathogen attack. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A recent study revealed that TATA boxes and initiator sequences have a common anomalous mechanical property, i.e. they comprise distinctive flexible and rigid sequences when compared with the other parts of the promoter region. In the present study, using the flexibility parameters from two different models, we calculated the average flexibility profiles of 1004 human promoters that do not contain canonical promoter elements, such as a TATA box, initiator (Inr) sequence, downstream promoter element or a GC box, and those of 382 human promoters that contain the GC box only. Here, we show that they have a common characteristic mechanical property that is strikingly similar to those of the TATA box-containing or Inr-containing promoters. Their most interesting feature is that the TATA- or Inr-corresponding region lies in the several nucleotides around the transcription start site. We have also found that a dinucleotide step from -1 to +1 (transcription start site) has a slight tendency to adopt CA that is known to be flexible. We also demonstrate that certain synthetic DNA fragments designed to mimic the average mechanical property of these 1386 promoters can drive transcription. This distinctive mechanical property may be the hallmark of a promoter.

We improved the aluminum borate whisker-mediated method of DNA delivery into rice callus (Oryza saliva L., cv. Notohikari). The following factors were examined: amount of whiskers, the kind of apparatus for agitation, the type of whiskers, duration of agitation, and agitation speed. Twenty callused scutellar tissues were agitated in a 1.5 ml microtube containing aluminum borate whiskers (ABW), liquid medium, and the plasmid pAct1-F carrying the beta-glucuronidase (GUS) gene. After the agitation, the scutellar tissues were subjected to transient GUS expression assay, which visualizes GUS-expressing cells as blue spots. Efficiencies of DNA delivery were evaluated as the number of blue spots resulting from the assay. In the present study, we succeeded in improving the efficiency of DNA delivery by changing the apparatus for agitation from rotary (Vortex Genie 2) to multidirectional (MT-360), and the type of ABW from Alborex Y to an aminosilane-coated Alborex (Alborex YS3A).

In spite of the abundant data on DNA sequence, the mechanical aspects of promoter DNA remain poorly understood. We classified 1871 human and 196 mouse RNA polymerase II promoters and investigated average flexibility profiles of the human promoters containing either a TATA box or an initiator (Inr) sequence only. Here, we show that TATA boxes and Inr sequences have a common anomalous mechanical property: they are comprised of distinctively flexible and rigid sequences, compared with the other parts of the promoter region. The +2 position in the Inr consensus sequence does not favor adenine to keep the high flexibility and thus this position is more accurately represented as 'T, G, C>A'. Additionally, it was also found that DNA region upstream of TATA box or Inr sequence is more rigid than region downstream of each element. These properties may function as a marker for recognition by TATA-binding protein and Inr-binding protein.

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.

A unique DNA curvature, the CIT, has been found in the 5'-upstream region of the psbA2 gene, which exhibits basal, light-responsive and circadian rhythmic transcription, in a unicellular photosynthetic cyanobacterium, Microcystis aeruginosa K-81. In this study, we report the universality of curvatures found in 5'-upstream regions in the psbA family and the function of the curvature in gene expression. Intrinsic curvatures were identified within 1000 bp upstream from the psbA genes in another cyanobacterium, a red alga and in plants (monocot and dicot). Mutagenized curvatures were constructed and confirmed to have disrupted architecture by gel electrophoresis and atomic force microscopy. Relatively small amounts but light-responsive transcripts of psbA2 were observed in cyanobacterial transformants harboring the mutagenized curvature under light/dark and light/high-light conditions. This shows that the curvature is important for basal transcription. In vitro primer extension and DNA mobility shift assay revealed that factors which might bind to the region upstream from the bending center contribute to the effective basal transcription of psbA2.

The electrophoretic mobility shift assay (EMSA) or simply the "gel shift assay" is one of the most sensitive methods for studying the ability of a protein to bind to DNA. EMSAs are also widely used to investigate protein- or sequence-dependent DNA bending. Here we report that electrophoresis using physiological concentrations of Mg2+ can cause a mobility shift of restriction fragments in nondenaturing polyacrylamide gels as the overhangs form stable base pairs. This phenomenon was observed at even 37 degreesC. The retardation was, however, more pronounced at low temperatures, where a three-nucleotide overhang 5'-GAC also caused a mobility shift. The stability of the pairing was generally high when the overhangs of four nucleotides display high GC content, while the mobility shift caused by 5'-AATT was greater than those caused by 5'-GATC, 5'-TCGA, and 5'-CTAG. This observation should be taken into account to avoid misinterpretation of the data when the EMSA, especially the circular permutation gel mobility shift assay, is performed using a running buffer that contains Mg2+ ions. The stable adhesion between short overhangs may present an important basis for genome stability and many genetic processes occurring in living cells.

DNA with a curved trajectory of its helix axis is called bent DNA, or curved DNA. Interestingly, biologically important DNA regions often contain this structure, irrespective of the origin of DNA. In the last decade, considerable progress has been made in clarifying one role of bent DNA in prokaryotic transcription and its mechanism of action. However, the role of bent DNA in eukaryotic transcription remains unclear. Our recent study raises the possibility that bent DNA is implicated in the "functional packaging" of transcriptional regulatory regions into chromatin. In this article, I review recent progress in bent DNA research in eukaryotic transcription, and summarize the history of bent DNA research and several subjects relevant to this theme. Finally, I propose a hypothesis that bent DNA structures that mimic a negative supercoil, or have a right-handed superhelical writhe, organize local chromatin infrastructure to help the very first interaction between cis-acting DNA elements and activators that trigger transcription. (C) 2001 John Wiley & Sons, Inc.

A bent DNA library was constructed from human genomic DNA, from which a new clone belonging to the human LINE-1 sequence family was isolated and characterized. This clone, with a length of 378 base pairs and termed HBC-1 (human bent clone-1), contained an intrinsically occurring curved DNA structure. By permutation analysis, the center of curvature of this fragment was mapped onto the nucleotide position 886 from the 5' terminus of the complete LINE-1 sequence. Reporter plasmids, which contain HBC-1, were effectively integrated into human chromosome, indicating that the bent DNA structure provides a preferential donor site for the integration of human LINE-1 sequences. The present finding may provide an explanation as to why some inactivated LINE-1 sequences on human chromosomes carry the deletion at their 5' termini. (C) 2001 Elsevier Science B.V. All rights reserved.

Eukaryotic promoters often contain a bent DNA structure, suggesting that this structure plays some role in transcription. To reveal the role, we need more information on the promoters that contain or flank a bent DNA structure. In this study, we collected such promoters by the following approach: we first isolated human genomic DNA fragments that contained at least one bent DNA structure, then shotgun cloned them into a promoter trap vector, screened DNA fragments that functioned as a promoter, and finally found the promoters of interest by determining the bent DNA locus and the region expressing promoter activity. From 1187 recombinant plasmids, we isolated 51 that showed promoter activity. Structural and functional analyses of randomly selected 10 clones with inserts of 548-913 bp demonstrated 11 sequences that could drive transcription. Unexpectedly, all of these clones met our purpose: i.e., each segment that showed a promoter activity (67-179 bp) was very close to the bent DNA structure (spanning about 150 bp in all clones), and in some cases overlapped it. More interestingly, these bent DNA structures all had a superhelical writhe. We propose a hypothesis that in the bent-DNA-containing eukaryotic promoters, bent DNA organizes local chromatin infrastructure appropriately for transcription initiation.

We have cloned a human genomic DNA fragment that contains a complex bent DNA structure and a LINE-1 (L1) sequence. The bent DNA structure spans from 70 to -440 relative to the transcription start site of L1, and contains a left-handed sup erhelical structure from about 70 to -200. When we changed the rotational orientation of the bent DNA relative to the L1 promoter by inserting double-stranded oligonucleotides between positions -3 and -4, L1 promoter activity was profoundly affected Inserts of 5 and 16 by altered the rotational position by about 180°, and stimulated transcription 2- to 3-fold Insertions of 11 and 21 base pairs, which altered the rotational orientati on minimally, had much less effect. We present here the first experimental evidence that upstream DNA architecture influences the machinery employed in L1 transcription.

During investigation of the molecular mechanism of large DNA fragmentation in apoptosis, we found that one of our clones, which were derived from 30kb DNA fragments generated from heat-treated human cells, mached perfectly with many sequences in E. coli genomic DNA, E. coli cDNA, a Neiserria plasmid, and several human genomic DNAs and cDNAs. All had exactly the same 153bp sequence. In fact, 1147bp DNA, containing the 153bp sequence, was apparently shared between human chromosomes 12, 13, and 21 and E. coli. This DNA was identified as E. coli insertion element 5. We need to pay carefull attention to such contaminations in genome data bases.

We reported previously that DNA bend sites appear in the human beta-globin locus at an average distance of 680 bp. The relative locations of the sites were conserved among the five active beta-like globin genes and one pseudogene. Here, we mapped the sites in the beta-like globin genes from various species and examined their conservation. The locations of the bend sites in the bovine, rabbit and chicken beta-globin genes mapped here showed marked conservation in their locations relative to the cap site and showed similar locations to the previously mapped sites in the human beta- and mouse beta(maj)-globin genes. Further analysis of the first bend sites from the cap site (B-1 sites) indicated that they contained tracts of adenines and thymines longer than or equal to two bases. This sequence feature contributed mostly to the curvature profiles revealed by gel assays and/or by computer-based TRIF analysis. TRIF analysis indicated that most of the B-1 sites showed right-handed superhelical twists accompanied by left handed twists. This was confirmed by the effect of ethidium bromide on the superhelical twists in the assays.

Two fragments that could function as replicational cue elements were isolated from a genomic DNA digest of Aspergillus oryzae on the basis of abnormal behavior in polyacrylamide gel electrophoresis. The vector used in this study contained a scaffold-associated region of the Drosophila melanogaster ftz gene to provide nuclear retention. Neither fragment contained a yeast ARS consensus sequence or an eukaryotic topoisomerase II binding sequence. One of the fragments showed sequence homology with the mitochondrial replication origin of Candida utilis and a portion of mitochondrial DNA of Aspergillus nidulans. This plasmid carrying the cue fragment could also replicate in HeLa and NIH3T3 cells.

Recombinant plasmids carrying a highly curved DNA structure are sometimes unstable in Escherichia coli. In order to know the underlying mechanism, several plasmids carrying one or two highly bent DNA segment(s) from the human adenovirus type 2 (Ad2) enhancer and/or origin region of phage lambda replication were systematically constructed and propagated in E. coli. The highly bent DNA segments disturbed the action of DNA topoisomerases: i.e. they were shown to be able to produce an anomalously wide spectrum of linking number topoisomers that tails toward lower supercoiling with a little of the DNA actually positively supercoiled. Furthermore, bent DNA caused multimeric plasmid formation. The linking number topoisomers and multimers seemed to be intermediate topological states of the bent DNA-containing plasmids that would lead to the deletion occurring in them. The nucleotide sequence of a deletion product of a bent DNA-containing plasmid showed that the putative source of the severe topological constraint was entirely eliminated from the plasmid.

The rpoD1 gene in the unicellular cyanobacterium Microcystis aeruginosa K-81 encodes a principal sigma factor of RNA polymerase and is transcribed under light and dark conditions to produce multiple monocistronic transcripts. In the 5'-upstream region from rpoD1 Promoter 2, which has a sequence of Escherichia coli type, we found a sequence-directed DNA curvature with an AT-rich sequence. Insertions of 2 to 21 base pairs introduced into the curved center changed a gross geometry of the original curved DNA structure. The rpoD1 promoter activities assayed in vivo by using transcriptional lacZ fusions were correlated with the change in the gross geometry in not only a cyanobacterium but also E. coli. In addition, RNA polymerase binding to the rpoD1 promoter region and the efficiency of the mRNA synthesis from the rpoD1 Promoter 2 were also affected in vitro by the change in the geometry. These results suggest that the tertiary structure of the curved DNA is important for the rpoD1 transcription. The deletion of the center region of the curvature resulted in a considerable reduction of the transcription from Promoter 2 in the cyanobacterium. This report demonstrates that a curved DNA plays a significant role in transcription in cyanobacteria, and that this functional curvature is located in the 5'-upstream region from the rpoD gene, which encodes a principal sigma factor in eubacteria.

An in vivo gene transfer technique for living mouse testes was used to develop a novel transient expression assay system for transcriptional regulatory elements of spermatogenic specific genes. The combination of DNA injection into seminiferous tubules and subsequent in vivo electroporation resulted in an efficient and convenient assay system for gene expression during spermatogenesis. The transfer of the firefly luciferase reporting gene driven by the Protamine-1 (Prm-1) enhancer region revealed a significant increase in the activity of the reporter enzyme. Histochemical studies of the transfer of the lacZ gene driven by the Prm-1 enhancer showed specific lacZ expression only in haploid spermatid cells in adult testes, corresponding with the expression pattern of endogenous Prm-1. We were able to detect long-lasting transgene expression in the transfected spermatogenic cells. A group of spermatogenic differentiating cells maintained the transfected lacZ expression after more than 2 mo of transfection, suggesting that spermatogenic stem cells and/or spermatogonia could also incorporate foreign DNA and that the transgene could be transmitted to the progenitor cells derived from a transfected proliferating germ cell.

Intrinsic DNA curvature is speculated to be a common feature of all satellite DNA sequences and may aid in the tight winding of DNA in constitutive heterochromatin. Several satellite DNAs, however, show unusually rapid migration in non-denaturing polyacrylamide gels, which is just the opposite behavior of that shown by curved DNA structures. Employing bovine satellite I DNA monomer, we attempted to understand the molecular mechanism of 'rapid migration'. The phenomenon of rapid migration was temperature-dependent and to a small extent polyacrylamide-concentration-dependent. Physiological or near-physiological concentrations of Mg2+ and Ca2+ ions bent the rapid migrating DNA segment, Predominance of purine-purine base stacking over purine-pyrimidine in nucleotide sequence was strongly indicated to be the cause of the rapid migration, Furthermore, they seemed to be implicated in the formation of induced DNA bend. We also found that the satellite I monomer contains an intrinsic DNA curvature as do many other satellites, Heretofore, the rapid migration property has concealed the presence of curvature.

The upstream half of the human adenovirus type 2 enhancer adopts a curved DNA structure. Most of the enhancer elements are within the curvature, suggesting that this unusual structure is linked to enhancer function. To verify this experimentally, I constructed in vitro transcription assay systems which could distinguish any effects generated by conformational changes in a DNA template, The curved DNA conformation in the enhancer clearly affected the extent of the stimulation of the E1A gene transcription: assays using the wildtype DNA template showed that the moderately curved enhancer was superior to the highly curved enhancer in transcriptional stimulation. In additional experiments, the enhancer region was substituted with a curved DNA derived from the bacteriophage lambda origin of replication, Assays using this mutant revealed that this curved segment could also act as an enhancer when it had the proper conformation. Consequently, DNA conformation may play a general role in transcriptional stimulation.

This work investigates the effect on transcription of superhelical writhe located in the region immediately upstream of the -35 consensus sequence of Escherichia coli promoters. A set of double-stranded oligonucleotides, each with an unique DNA configuration, were designed, synthesized and substituted into an area of naturally occurring right-handed superhelical curvature immediately. upstream of the beta-lactamase promoter in plasmid pUC19. All the mutants showed reduced promoter activities in E. coli cells. However, rightward superhelical writhe clearly facilitated transcription when compared with the effect produced by a straight DNA segment. Leftward writhe greatly repressed transcription. A plane curve hewed an intermediate effect. This phenomenon was due not only to the difference in the ability of the segment to drive an open complex formation, but also to the difference in the binding affinity of RNA polymerase to the promoter. The positive effect of rightward writhe was also observed in vivo for the promoter of the tetracycline resistance gene of pBR322. The sense and extent of superhelical writhe of a DNA curvature seem to determine its influence on promoter activity. (C) 1995 Academic Press Limited

A new vector, pATO, was constructed for rapid cloning and analysis of eukaryotic promoters. When a recombinant pATO, carrying a promoter sequence in its multiple cloning site, was introduced into COS cells, Thy-1.2 protein was produced on the cell surface, and was easily identified by an fluorescein-conjugated anti-Thy-1.2 antibody. The intensity of the fluorescence reflected the strength of the inserted promoter. Since pATO could replicate efficiently in COS cells, the recombinant plasmids recovered from a single COS cell were sufficient to transform Escherichia coli cells. This plasmid is applicable for the rapid and labor saving cloning of promoter elements.

It has been known that transfer RNA assumes a functional conformation in the presence of certain metal ions, most commonly magnesium ions. Several Mg²⁺-binding sites of high affinity have been revealed by X-ray crystallographic analyses of yeast tRNA^Phe. On the other hand, ATP, a naturally occurring high-energy compound and which is one of the substrates in aminoacylation of tRNA, is known to have ability to chelate Mg²⁺. We therefore examined the possibility that the conformation of tRNA is regulated by the quantitative balance between Mg²⁺ and ATP, and consequently biological activity of tRNA is modulated as well. Structural transition of tRNA was monitored by measuring the melting profiles of yeast tRNA^Tyr in the presence of Mg²⁺ and/or ATP at various [ATP]/[Mg²⁺] ratios. Tyrosine-accepting activity of yeast tRNA^Tyr was also measured under the similar solution conditions. These results clearly indicated that conformation of tRNA and its aminoacylation capacity can be easily modulated by changing the relative concentration ratio of Mg²⁺ and ATP.

The region preceding the beta-lactamase promoter of Escherichia coli plasmid pUC19 has a curved DNA (bent DNA) structure. The center of the curvature was revealed to exist around nucleotide position 2580 of the plasmid, which is just beside RNA polymerase binding region. It was indicated that the identified region is curved even at 60-degrees-C. The gross geometry of the curvature was altered by inserting synthetic double-stranded oligonucleotides between positions 2585 and 2586. Effect of the alteration on strength of the promoter was not detected in vitro. However, in vivo analyses showed that the promoter strength is apparently dependent, in part, on the gross geometry of the curvature. Insertions of 4 and 16 bp, both of which altered the gross geometry of the curvature greatly, caused considerable reductions of in vivo level of beta-lactamase mRNA. In vivo, overall three-dimensional structure of the region covering the promoter and the curvature seems to play some significant role in transcription of the gene.