Modular structural elements in the replication origin region of Tetrahymena rDNA.

PubMed Central

Du, C; Sanzgiri, R P; Shaiu, W L; Choi, J K; Hou, Z; Benbow, R M; Dobbs, D L

1995-01-01

Computer analyses of the DNA replication origin region in the amplified rRNA genes of Tetrahymena thermophila identified a potential initiation zone in the 5'NTS [Dobbs, Shaiu and Benbow (1994), Nucleic Acids Res. 22, 2479-2489]. This region consists of a putative DNA unwinding element (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions. In this study, two mung bean nuclease-hypersensitive sites in super-coiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses. Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels. Restriction fragments containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the replication origin region with the nuclear matrix in vivo. The direct demonstration in a protozoan origin region of elements previously identified in Drosophila, chick and mammalian origin regions suggests that clusters of modular structural elements may be a conserved feature of eukaryotic chromosomal origins of replication. Images PMID:7784181

Exploring the read-write genome: mobile DNA and mammalian adaptation.

PubMed

Shapiro, James A

2017-02-01

The read-write genome idea predicts that mobile DNA elements will act in evolution to generate adaptive changes in organismal DNA. This prediction was examined in the context of mammalian adaptations involving regulatory non-coding RNAs, viviparous reproduction, early embryonic and stem cell development, the nervous system, and innate immunity. The evidence shows that mobile elements have played specific and sometimes major roles in mammalian adaptive evolution by generating regulatory sites in the DNA and providing interaction motifs in non-coding RNA. Endogenous retroviruses and retrotransposons have been the predominant mobile elements in mammalian adaptive evolution, with the notable exception of bats, where DNA transposons are the major agents of RW genome inscriptions. A few examples of independent but convergent exaptation of mobile DNA elements for similar regulatory rewiring functions are noted.

A Surrogate Approach to Study the Evolution of Noncoding DNA Elements That Organize Eukaryotic Genomes

PubMed Central

Vermaak, Danielle; Bayes, Joshua J.

2009-01-01

Comparative genomics provides a facile way to address issues of evolutionary constraint acting on different elements of the genome. However, several important DNA elements have not reaped the benefits of this new approach. Some have proved intractable to current day sequencing technology. These include centromeric and heterochromatic DNA, which are essential for chromosome segregation as well as gene regulation, but the highly repetitive nature of the DNA sequences in these regions make them difficult to assemble into longer contigs. Other sequences, like dosage compensation X chromosomal sites, origins of DNA replication, or heterochromatic sequences that encode piwi-associated RNAs, have proved difficult to study because they do not have recognizable DNA features that allow them to be described functionally or computationally. We have employed an alternate approach to the direct study of these DNA elements. By using proteins that specifically bind these noncoding DNAs as surrogates, we can indirectly assay the evolutionary constraints acting on these important DNA elements. We review the impact that such “surrogate strategies” have had on our understanding of the evolutionary constraints shaping centromeres, origins of DNA replication, and dosage compensation X chromosomal sites. These have begun to reveal that in contrast to the view that such structural DNA elements are either highly constrained (under purifying selection) or free to drift (under neutral evolution), some of them may instead be shaped by adaptive evolution and genetic conflicts (these are not mutually exclusive). These insights also help to explain why the same elements (e.g., centromeres and replication origins), which are so complex in some eukaryotic genomes, can be simple and well defined in other where similar conflicts do not exist. PMID:19635763

Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology

PubMed Central

Chappell, James; Jensen, Kirsten; Freemont, Paul S.

2013-01-01

A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. To address this, we have validated a completely in vitro approach for the characterization of DNA regulatory elements using Escherichia coli extract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performance in vivo for the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completely in vitro method to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. This in vitro approach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology. PMID:23371936

The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme.

PubMed Central

Burke, W D; Calalang, C C; Eickbush, T H

1987-01-01

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover. Images PMID:2439905

Concerted formation of macromolecular Suppressor–mutator transposition complexes

PubMed Central

Raina, Ramesh; Schläppi, Michael; Karunanandaa, Balasulojini; Elhofy, Adam; Fedoroff, Nina

1998-01-01

Transposition of the maize Suppressor–mutator (Spm) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA–DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein–protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range. PMID:9671711

Useful DNA polymorphisms are identified by snapback, a midrepetitive element in Tribolium castaneum.

PubMed

Stuart, J J; De Gortari, M J; Hall, P S; Maxwell, M E; Mocelin, G; Brown, S J; Muir, W M

1996-06-01

The red flour bettle, Tribolium castaneum, is both a pest of stored grain products and an important experimental organism. To improve its facility as a genetic model, we are developing DNA fingerprinting methods for this insect. A Tribolium DNA fragment, snapback-1 (SBI), identified among sequences that reassociate before a Cot of 0.03 mol.s/L, was found to produce a banding pattern in restriction endonuclease digested genomic DNA that is characteristic of a midrepetitive element. DNA fingerprints of individual beetles demonstrated that unvarying inherited DNA polymorphism is revealed, and that polymorphism is inherited in a dominant Mendelian fashion. Linkage between bands was minimal. The sequence of SBI was determined, and hybridization experiments indicated that SBI is a fragment of a larger midrepetitive element. Fingerprinting individuals with known inbreeding coefficients indicated that SBI loci have relatively high mutation rates. The possibility that SBI is a fragment of a transposable element is discussed.

Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic Cells.

PubMed

Zhao, Ming-Tao; Shao, Ning-Yi; Hu, Shijun; Ma, Ning; Srinivasan, Rajini; Jahanbani, Fereshteh; Lee, Jaecheol; Zhang, Sophia L; Snyder, Michael P; Wu, Joseph C

2017-11-10

Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is not well known how chromatin marks in regulatory DNA elements are modulated to establish cell type-specific gene expression in the human heart. We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression. We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing; n=84) in human endothelial cells (CD31 + CD144 + ), cardiac progenitor cells (Sca-1 + ), fibroblasts (DDR2 + ), and their respective induced pluripotent stem cells. We uncovered 2 classes of regulatory DNA elements: class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both class I and class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human induced pluripotent stem cells and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells and identified a few enhancers that could possibly regulate the cardiac-specific gene expression. Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering the epigenetic modulation of regulatory DNA elements that fine-tune spatiotemporal gene expression in human cardiac development and diseases. © 2017 American Heart Association, Inc.

Rat L (long interspersed repeated DNA) elements contain guanine-rich homopurine sequences that induce unpairing of contiguous duplex DNA.

PubMed Central

Usdin, K; Furano, A V

1988-01-01

The L family (long interspersed repeated DNA) of mobile genetic elements is a persistent feature of the mammalian genome. In rats, this family contains approximately equal to 40,000 members and accounts for approximately equal to 10% of the haploid genome. We demonstrate here that the guanine-rich homopurine stretches located at the right end of L-DNA induce oligonucleotide uptake by contiguous duplex DNA. The uptake is dependent on negative supercoiling and the length of the homopurine stretch and occurs even when the L-DNA homopurine stretches are introduced into a different DNA environment. The bound oligomer primes DNA synthesis when DNA polymerase and deoxyribonucleoside triphosphates are added, resulting in a faithful copy of the template to which the oligonucleotide had bound. The implications of this property of the L-DNA guanine-rich homopurine stretches in the amplification, recombination, and dispersal of L elements is discussed. Images PMID:2837766

Transcription factor trapping by RNA in gene regulatory elements.

PubMed

Sigova, Alla A; Abraham, Brian J; Ji, Xiong; Molinie, Benoit; Hannett, Nancy M; Guo, Yang Eric; Jangi, Mohini; Giallourakis, Cosmas C; Sharp, Phillip A; Young, Richard A

2015-11-20

Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs. Copyright © 2015, American Association for the Advancement of Science.

Genomic patterns associated with paternal/maternal distribution of transposable elements

NASA Astrophysics Data System (ADS)

Jurka, Jerzy

2003-03-01

Transposable elements (TEs) are specialized DNA or RNA fragments capable of surviving in intragenomic niches. They are commonly, perhaps unjustifiably referred to as "selfish" or "parasitic" elements. TEs can be divided in two major classes: retroelements and DNA transposons. The former include non-LTR retrotransposons and retrovirus-like elements, using reverse transriptase for their reproduction prior to integration into host DNA. The latter depend mostly on host DNA replication, with possible exception of rolling-circle transposons recently discovered by our team. I will review basic information on TEs, with emphasis on human Alu and L1 retroelements discussed in the context of genomic organization. TEs are non-randomly distributed in chromosomal DNA. In particular, human Alu elements tend to prefer GC-rich regions, whereas L1 accumulate in AT-rich regions. Current explanations of this phenomenon focus on the so called "target effects" and post-insertional selection. However, the proposed models appear to be unsatisfactory and alternative explanations invoking "channeling" to different chromosomal regions will be a major focus of my presentation. Transposable elements (TEs) can be expressed and integrated into host DNA in the male or female germlines, or both. Different models of expression and integration imply different proportions of TEs on sex chromosomes and autosomes. The density of recently retroposed human Alu elements is around three times higher on chromosome Y than on chromosome X, and over two times higher than the average density for all human autosomes. This implies Alu activity in paternal germlines. Analogous inter-chromosomal proportions for other repeat families should determine their compatibility with one of the three basic models describing the inheritance of TEs. Published evidence indicates that maternally and paternally imprinted genes roughly correspond to GC-rich and AT-rich DNA. This may explain the observed chromosomal distribution of Alu and L1 elements. Finally, paternal models of inheritance predict rapid accumulation of active TEs on chromosome Y. I will discuss potential implications of this phenomenon for evolution of chromosome Y and transposable elements.

Nucleosome core particles containing a poly(dA.dT) sequence element exhibit a locally distorted DNA structure.

PubMed

Bao, Yunhe; White, Cindy L; Luger, Karolin

2006-08-25

Poly(dA.dT) DNA sequence elements are thought to promote transcription by either excluding nucleosomes or by altering their structural or dynamic properties. Here, the stability and structure of a defined nucleosome core particle containing a 16 base-pair poly(dA.dT) element (A16 NCP) was investigated. The A16 NCP requires a significantly higher temperature for histone octamer sliding in vitro compared to comparable nucleosomes that do not contain a poly(dA.dT) element. Fluorescence resonance energy transfer showed that the interactions between the nucleosomal DNA ends and the histone octamer were destabilized in A16 NCP. The crystal structure of A16 NCP was determined to a resolution of 3.2 A. The overall structure was maintained except for local deviations in DNA conformation. These results are consistent with previous in vivo and in vitro observations that poly(dA.dT) elements cause only modest changes in DNA accessibility and modest increases in steady-state transcription levels.

Foldback intercoil DNA and the mechanism of DNA transposition.

PubMed

Kim, Byung-Dong

2014-09-01

Foldback intercoil (FBI) DNA is formed by the folding back at one point of a non-helical parallel track of double-stranded DNA at as sharp as 180° and the intertwining of two double helixes within each other's major groove to form an intercoil with a diameter of 2.2 nm. FBI DNA has been suggested to mediate intra-molecular homologous recombination of a deletion and inversion. Inter-molecular homologous recombination, known as site-specific insertion, on the other hand, is mediated by the direct perpendicular approach of the FBI DNA tip, as the attP site, onto the target DNA, as the attB site. Transposition of DNA transposons involves the pairing of terminal inverted repeats and 5-7-bp tandem target duplication. FBI DNA configuration effectively explains simple as well as replicative transposition, along with the involvement of an enhancer element. The majority of diverse retrotransposable elements that employ a target site duplication mechanism is also suggested to follow the FBI DNA-mediated perpendicular insertion of the paired intercoil ends by non-homologous end-joining, together with gap filling. A genome-wide perspective of transposable elements in light of FBI DNA is discussed.

Small RNA-Mediated trans-Nuclear and trans-Element Communications in Tetrahymena DNA Elimination.

PubMed

Noto, Tomoko; Mochizuki, Kazufumi

2018-06-18

Epigenetic inheritance of acquired traits is widespread among eukaryotes, but how and to what extent such information is transgenerationally inherited is still unclear. The patterns of programmed DNA elimination in ciliates are epigenetically and transgenerationally inherited, and it has been proposed that small RNAs, which shuttle between the germline and the soma, regulate this epigenetic inheritance. In this study, we test the existence and role of such small-RNA-mediated communication by epigenetically disturbing the pattern of DNA elimination in Tetrahymena. We show that the pattern of DNA elimination is, indeed, determined by the selective turnover of small RNAs, which is induced by the interaction between germline-derived small RNAs and the somatic genome. In addition, we show that DNA elimination of an element is regulated by small-RNA-mediated communication with other eliminated elements. By contrast, no evidence obtained thus far supports the notion that transfer of epigenetic information from the soma to the germline, if any, regulates DNA elimination. Our results indicate that small-RNA-mediated trans-nuclear and trans-element communication, in addition to unknown information in the germline genome, contributes to determining the pattern of DNA elimination. Copyright © 2018 Elsevier Ltd. All rights reserved.

Satellite DNA and Transposable Elements in Seabuckthorn (Hippophae rhamnoides), a Dioecious Plant with Small Y and Large X Chromosomes

PubMed Central

Puterova, Janka; Razumova, Olga; Martinek, Tomas; Alexandrov, Oleg; Divashuk, Mikhail; Kubat, Zdenek; Hobza, Roman; Karlov, Gennady

2017-01-01

Seabuckthorn (Hippophae rhamnoides) is a dioecious shrub commonly used in the pharmaceutical, cosmetic, and environmental industry as a source of oil, minerals and vitamins. In this study, we analyzed the transposable elements and satellites in its genome. We carried out Illumina DNA sequencing and reconstructed the main repetitive DNA sequences. For data analysis, we developed a new bioinformatics approach for advanced satellite DNA analysis and showed that about 25% of the genome consists of satellite DNA and about 24% is formed of transposable elements, dominated by Ty3/Gypsy and Ty1/Copia LTR retrotransposons. FISH mapping revealed X chromosome-accumulated, Y chromosome-specific or both sex chromosomes-accumulated satellites but most satellites were found on autosomes. Transposable elements were located mostly in the subtelomeres of all chromosomes. The 5S rDNA and 45S rDNA were localized on one autosomal locus each. Although we demonstrated the small size of the Y chromosome of the seabuckthorn and accumulated satellite DNA there, we were unable to estimate the age and extent of the Y chromosome degeneration. Analysis of dioecious relatives such as Shepherdia would shed more light on the evolution of these sex chromosomes. PMID:28057732

Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project.

PubMed

Gerstein, Mark B; Lu, Zhi John; Van Nostrand, Eric L; Cheng, Chao; Arshinoff, Bradley I; Liu, Tao; Yip, Kevin Y; Robilotto, Rebecca; Rechtsteiner, Andreas; Ikegami, Kohta; Alves, Pedro; Chateigner, Aurelien; Perry, Marc; Morris, Mitzi; Auerbach, Raymond K; Feng, Xin; Leng, Jing; Vielle, Anne; Niu, Wei; Rhrissorrakrai, Kahn; Agarwal, Ashish; Alexander, Roger P; Barber, Galt; Brdlik, Cathleen M; Brennan, Jennifer; Brouillet, Jeremy Jean; Carr, Adrian; Cheung, Ming-Sin; Clawson, Hiram; Contrino, Sergio; Dannenberg, Luke O; Dernburg, Abby F; Desai, Arshad; Dick, Lindsay; Dosé, Andréa C; Du, Jiang; Egelhofer, Thea; Ercan, Sevinc; Euskirchen, Ghia; Ewing, Brent; Feingold, Elise A; Gassmann, Reto; Good, Peter J; Green, Phil; Gullier, Francois; Gutwein, Michelle; Guyer, Mark S; Habegger, Lukas; Han, Ting; Henikoff, Jorja G; Henz, Stefan R; Hinrichs, Angie; Holster, Heather; Hyman, Tony; Iniguez, A Leo; Janette, Judith; Jensen, Morten; Kato, Masaomi; Kent, W James; Kephart, Ellen; Khivansara, Vishal; Khurana, Ekta; Kim, John K; Kolasinska-Zwierz, Paulina; Lai, Eric C; Latorre, Isabel; Leahey, Amber; Lewis, Suzanna; Lloyd, Paul; Lochovsky, Lucas; Lowdon, Rebecca F; Lubling, Yaniv; Lyne, Rachel; MacCoss, Michael; Mackowiak, Sebastian D; Mangone, Marco; McKay, Sheldon; Mecenas, Desirea; Merrihew, Gennifer; Miller, David M; Muroyama, Andrew; Murray, John I; Ooi, Siew-Loon; Pham, Hoang; Phippen, Taryn; Preston, Elicia A; Rajewsky, Nikolaus; Rätsch, Gunnar; Rosenbaum, Heidi; Rozowsky, Joel; Rutherford, Kim; Ruzanov, Peter; Sarov, Mihail; Sasidharan, Rajkumar; Sboner, Andrea; Scheid, Paul; Segal, Eran; Shin, Hyunjin; Shou, Chong; Slack, Frank J; Slightam, Cindie; Smith, Richard; Spencer, William C; Stinson, E O; Taing, Scott; Takasaki, Teruaki; Vafeados, Dionne; Voronina, Ksenia; Wang, Guilin; Washington, Nicole L; Whittle, Christina M; Wu, Beijing; Yan, Koon-Kiu; Zeller, Georg; Zha, Zheng; Zhong, Mei; Zhou, Xingliang; Ahringer, Julie; Strome, Susan; Gunsalus, Kristin C; Micklem, Gos; Liu, X Shirley; Reinke, Valerie; Kim, Stuart K; Hillier, LaDeana W; Henikoff, Steven; Piano, Fabio; Snyder, Michael; Stein, Lincoln; Lieb, Jason D; Waterston, Robert H

2010-12-24

We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor-binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor-binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.

Genome-wide colonization of gene regulatory elements by G4 DNA motifs

PubMed Central

Du, Zhuo; Zhao, Yiqiang; Li, Ning

2009-01-01

G-quadruplex (or G4 DNA), a stable four-stranded structure found in guanine-rich regions, is implicated in the transcriptional regulation of genes involved in growth and development. Previous studies on the role of G4 DNA in gene regulation mostly focused on genomic regions proximal to transcription start sites (TSSs). To gain a more comprehensive understanding of the regulatory role of G4 DNA, we examined the landscape of potential G4 DNA (PG4Ms) motifs in the human genome and found that G4 motifs, not restricted to those found in the TSS-proximal regions, are bias toward gene-associated regions. Significantly, analyses of G4 motifs in seven types of well-known gene regulatory elements revealed a constitutive enrichment pattern and the clusters of G4 motifs tend to be colocalized with regulatory elements. Considering our analysis from a genome evolutionary perspective, we found evidence that the occurrence and accumulation of certain progenitors and canonical G4 DNA motifs within regulatory regions were progressively favored by natural selection. Our results suggest that G4 DNA motifs are ‘colonized’ in regulatory regions, supporting a likely genome-wide role of G4 DNA in gene regulation. We hypothesize that G4 DNA is a regulatory apparatus situated in regulatory elements, acting as a molecular switch that can modulate the role of the host functional regions, by transition in DNA structure. PMID:19759215

Regulation of the yeast RAD2 gene: DNA damage-dependent induction correlates with protein binding to regulatory sequences and their deletion influences survival.

PubMed

Siede, W; Friedberg, E C

1992-03-01

In the yeast Saccharomyces cerevisiae the RAD2 gene is absolutely required for damage-specific incision of DNA during nucleotide excision repair and is inducible by DNA-damaging agents. In the present study we correlated sensitivity to killing by DNA-damaging agents with the deletion of previously defined specific promoter elements. Deletion of the element DRE2 increased the UV sensitivity of cells in both the G1/early S and S/G2 phases of the cell cycle as well as in stationary phase. On the other hand, increased UV sensitivity associated with deletion of the sequence-related element DRE1 was restricted to cells irradiated in G1/S. Specific binding of protein(s) to the promoter elements DRE1 and DRE2 was observed under non-inducing conditions using gel retardation assays. Exposure of cells to DNA-damaging agents resulted in increased protein binding that was dependent on de novo protein synthesis.

Spontaneous germline excision of Tol1, a DNA-based transposable element naturally occurring in the medaka fish genome.

PubMed

Watanabe, Kohei; Koga, Hajime; Nakamura, Kodai; Fujita, Akiko; Hattori, Akimasa; Matsuda, Masaru; Koga, Akihiko

2014-04-01

DNA-based transposable elements are ubiquitous constituents of eukaryotic genomes. Vertebrates are, however, exceptional in that most of their DNA-based elements appear to be inactivated. The Tol1 element of the medaka fish, Oryzias latipes, is one of the few elements for which copies containing an undamaged gene have been found. Spontaneous transposition of this element in somatic cells has previously been demonstrated, but there is only indirect evidence for its germline transposition. Here, we show direct evidence of spontaneous excision in the germline. Tyrosinase is the key enzyme in melanin biosynthesis. In an albino laboratory strain of medaka fish, which is homozygous for a mutant tyrosinase gene in which a Tol1 copy is inserted, we identified de novo reversion mutations related to melanin pigmentation. The gamete-based reversion rate was as high as 0.4%. The revertant fish carried the tyrosinase gene from which the Tol1 copy had been excised. We previously reported the germline transposition of Tol2, another DNA-based element that is thought to be a recent invader of the medaka fish genome. Tol1 is an ancient resident of the genome. Our results indicate that even an old element can contribute to genetic variation in the host genome as a natural mutator.

Trace elements are associated with urinary 8-hydroxy-2'-deoxyguanosine level: a case study of college students in Guangzhou, China.

PubMed

Lu, Shaoyou; Ren, Lu; Fang, Jianzhang; Ji, Jiajia; Liu, Guihua; Zhang, Jianqing; Zhang, Huimin; Luo, Ruorong; Lin, Kai; Fan, Ruifang

2016-05-01

Many trace heavy elements are carcinogenic and increase the incidence of cancer. However, a comprehensive study of the correlation between multiple trace elements and DNA oxidative damage is still lacking. The aim of this study is to investigate the relationships between the body burden of multiple trace elements and DNA oxidative stress in college students in Guangzhou, China. Seventeen trace elements in urine samples were determined by inductively coupled plasma-mass spectrometry (ICP-MS). Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of DNA oxidative stress, was also measured using liquid chromatography tandem mass spectrometer (LC-MS/MS). The concentrations of six essential elements including manganese (Mn), copper (Cu), nickel (Ni), selenium (Se), strontium (Sr), and molybdenum (Mo), and five non-essential elements including arsenic (As), cadmium (Cd), aluminum (Al), stibium (Sb), and thallium (Tl), were found to be significantly correlated with urinary 8-OHdG levels. Moreover, urinary levels of Ni, Se, Mo, As, Sr, and Tl were strongly significantly correlated with 8-OHdG (P < 0.01) concentration. Environmental exposure and dietary intake of these trace elements may play important roles in DNA oxidative damage in the population of Guangzhou, China.

Photoinduced Regeneration of an Aptamer-Based Electrochemical Sensor for Sensitively Detecting Adenosine Triphosphate.

PubMed

Zhang, Xiaoyu; Song, Chunxia; Yang, Ke; Hong, Wenwen; Lu, Ying; Yu, Ping; Mao, Lanqun

2018-04-17

Electrochemical aptasensors generally include three elements, that is, recognition element, signal-transformation element, and regeneration element. In this study, a new adenosine triphosphate (ATP) aptasensor is developed by combining three elements into one DNA oligonucleotide chain. In the DNA oligonucleotide chain, DNA aptamer is used as the recognition element, ferrocene group attached at the 3'-end of the aptamer is used as the signal-transformation element, and azobenzene moiety embedded into the DNA chain is used as the regeneration element. In addition to the similar analytical properties with the traditional ones, the aptasensor developed here is easily regenerated with UV-light irradiation. The current response recorded on the aptasensor increases with increasing the concentration of ATP in the incubation solution and is linear with the logarithm of ATP concentration in the range from 1 nM to 100 μM. The limit of detection is 0.5 nM (S/N = 3). The basal level of ATP in the rat brain cortex microdialysate is determined to be 21.33 ± 4.1 nM ( n = 3). After being challenged with ATP, the aptasensor could be readily regenerated by UV-light irradiation for more than seven cycles. The regeneration of the aptasensor is proposed to be regulated by conversing azobenzene from its trans to cis form under UV irradiation.

Movable Genetic Elements: Detection of Changes in Maize DNA at the Shrunken Locus Due to the Intervention of Ds Elements

DOE R&D Accomplishments Database

Burr, B.; Burr, F.A.

1980-05-28

This report describes our initial attempts at the molecular characterization of a maize controlling element. We have prepared a cDNA probe and used it to detect changes at a locus where Ds elements are found. Evidence of their presence are indicated by changes in the restriction patterns, but there is as yet no information on the physical nature of the controlling elements nor on the kinds of rearrangements they cause.

Satellite DNA and Transposable Elements in Seabuckthorn (Hippophae rhamnoides), a Dioecious Plant with Small Y and Large X Chromosomes.

PubMed

Puterova, Janka; Razumova, Olga; Martinek, Tomas; Alexandrov, Oleg; Divashuk, Mikhail; Kubat, Zdenek; Hobza, Roman; Karlov, Gennady; Kejnovsky, Eduard

2017-01-01

Seabuckthorn (Hippophae rhamnoides) is a dioecious shrub commonly used in the pharmaceutical, cosmetic, and environmental industry as a source of oil, minerals and vitamins. In this study, we analyzed the transposable elements and satellites in its genome. We carried out Illumina DNA sequencing and reconstructed the main repetitive DNA sequences. For data analysis, we developed a new bioinformatics approach for advanced satellite DNA analysis and showed that about 25% of the genome consists of satellite DNA and about 24% is formed of transposable elements, dominated by Ty3/Gypsy and Ty1/Copia LTR retrotransposons. FISH mapping revealed X chromosome-accumulated, Y chromosome-specific or both sex chromosomes-accumulated satellites but most satellites were found on autosomes. Transposable elements were located mostly in the subtelomeres of all chromosomes. The 5S rDNA and 45S rDNA were localized on one autosomal locus each. Although we demonstrated the small size of the Y chromosome of the seabuckthorn and accumulated satellite DNA there, we were unable to estimate the age and extent of the Y chromosome degeneration. Analysis of dioecious relatives such as Shepherdia would shed more light on the evolution of these sex chromosomes. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

RNA from the 5' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site.

PubMed

Christensen, Shawn M; Ye, Junqiang; Eickbush, Thomas H

2006-11-21

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.

The long (LINEs) and the short (SINEs) of it: altered methylation as a precursor to toxicity.

PubMed

Carnell, Ammie N; Goodman, Jay I

2003-10-01

Although once thought of as "junk" DNA, the importance of interspersed elements in the genome has become increasingly appreciated in recent years. In a broad sense these are collectively referred to as transposable elements, which encompass both transposons and retrotransposons. The latter include long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs). Expression of these elements leads to genetic instability. Therefore, it is important that they remain transcriptionally silenced, and DNA methylation plays a key role in this regard. A framework for understanding the possible interplay between altered DNA methylation, an epigenetic change, and mutational events is presented. A case is made as to how retrotransposable elements, specifically LINEs and SINEs, are likely to emerge as key players in furthering our understanding of mechanisms underlying a variety of toxicities, including carcinogenesis but not limited to this endpoint.

Conserved Elements Vaccine for HIV | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the National Cancer Institute (NCI) developed a DNA vaccine using conserved elements of HIV-1 Gag, administered in a prime-boost vaccination protocol. Two of the HIV Gag CE DNA vectors have been tested in a rhesus macaque model. Priming with the Gag CE vaccine and boosting with full length Gag DNA showed increased immune responses when compared to vaccination with Gag alone. Researchers seek licensing and/or co-development research collaborations for development this DNA vaccine.

Interactions between the R2R3-MYB Transcription Factor, AtMYB61, and Target DNA Binding Sites

PubMed Central

Prouse, Michael B.; Campbell, Malcolm M.

2013-01-01

Despite the prominent roles played by R2R3-MYB transcription factors in the regulation of plant gene expression, little is known about the details of how these proteins interact with their DNA targets. For example, while Arabidopsis thaliana R2R3-MYB protein AtMYB61 is known to alter transcript abundance of a specific set of target genes, little is known about the specific DNA sequences to which AtMYB61 binds. To address this gap in knowledge, DNA sequences bound by AtMYB61 were identified using cyclic amplification and selection of targets (CASTing). The DNA targets identified using this approach corresponded to AC elements, sequences enriched in adenosine and cytosine nucleotides. The preferred target sequence that bound with the greatest affinity to AtMYB61 recombinant protein was ACCTAC, the AC-I element. Mutational analyses based on the AC-I element showed that ACC nucleotides in the AC-I element served as the core recognition motif, critical for AtMYB61 binding. Molecular modelling predicted interactions between AtMYB61 amino acid residues and corresponding nucleotides in the DNA targets. The affinity between AtMYB61 and specific target DNA sequences did not correlate with AtMYB61-driven transcriptional activation with each of the target sequences. CASTing-selected motifs were found in the regulatory regions of genes previously shown to be regulated by AtMYB61. Taken together, these findings are consistent with the hypothesis that AtMYB61 regulates transcription from specific cis-acting AC elements in vivo. The results shed light on the specifics of DNA binding by an important family of plant-specific transcriptional regulators. PMID:23741471

Extended HSR/CARD domain mediates AIRE binding to DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maslovskaja, Julia, E-mail: julia.maslovskaja@ut.ee; Saare, Mario; Liiv, Ingrid

Autoimmune regulator (AIRE) activates the transcription of many genes in an unusual promiscuous and stochastic manner. The mechanism by which AIRE binds to the chromatin and DNA is not fully understood, and the regulatory elements that AIRE target genes possess are not delineated. In the current study, we demonstrate that AIRE activates the expression of transiently transfected luciferase reporters that lack defined promoter regions, as well as intron and poly(A) signal sequences. Our protein-DNA interaction experiments with mutated AIRE reveal that the intact homogeneously staining region/caspase recruitment domain (HSR/CARD) and amino acids R113 and K114 are key elements involved inmore » AIRE binding to DNA. - Highlights: • Promoter and mRNA processing elements are not important for AIRE to activate gene expression from reporter plasmids. • AIRE protein fragment aa 1–138 mediates direct binding to DNA. • Integrity of the HSR/CARD domain is needed for AIRE binding to DNA.« less

Interplay between DNA methylation, histone modification and chromatin remodeling in stem cells and during development.

PubMed

Ikegami, Kohta; Ohgane, Jun; Tanaka, Satoshi; Yagi, Shintaro; Shiota, Kunio

2009-01-01

Genes constitute only a small proportion of the mammalian genome, the majority of which is composed of non-genic repetitive elements including interspersed repeats and satellites. A unique feature of the mammalian genome is that there are numerous tissue-dependent, differentially methylated regions (T-DMRs) in the non-repetitive sequences, which include genes and their regulatory elements. The epigenetic status of T-DMRs varies from that of repetitive elements and constitutes the DNA methylation profile genome-wide. Since the DNA methylation profile is specific to each cell and tissue type, much like a fingerprint, it can be used as a means of identification. The formation of DNA methylation profiles is the basis for cell differentiation and development in mammals. The epigenetic status of each T-DMR is regulated by the interplay between DNA methyltransferases, histone modification enzymes, histone subtypes, non-histone nuclear proteins and non-coding RNAs. In this review, we will discuss how these epigenetic factors cooperate to establish cell- and tissue-specific DNA methylation profiles.

Whole DNA methylome profiling in mice exposed to secondhand smoke.

PubMed

Tommasi, Stella; Zheng, Albert; Yoon, Jae-In; Li, Arthur Xuejun; Wu, Xiwei; Besaratinia, Ahmad

2012-11-01

Aberration of DNA methylation is a prime epigenetic mechanism of carcinogenesis. Aberrant DNA methylation occurs frequently in lung cancer, with exposure to secondhand smoke (SHS) being an established risk factor. The causal role of SHS in the genesis of lung cancer, however, remains elusive. To investigate whether SHS can cause aberrant DNA methylation in vivo, we have constructed the whole DNA methylome in mice exposed to SHS for a duration of 4 mo, both after the termination of exposure and at ensuing intervals post-exposure (up to 10 mo). Our genome-wide and gene-specific profiling of DNA methylation in the lung of SHS-exposed mice revealed that all groups of SHS-exposed mice and controls share a similar pattern of DNA methylation. Furthermore, the methylation status of major repetitive DNA elements, including long-interspersed nuclear elements (LINE L1), intracisternal A particle long-terminal repeat retrotransposons (IAP-LTR), and short-interspersed nuclear elements (SINE B1), in the lung of all groups of SHS-exposed mice and controls remains comparable. The absence of locus-specific gain of DNA methylation and global loss of DNA methylation in the lung of SHS-exposed mice within a timeframe that precedes neoplastic-lesion formation underscore the challenges of lung cancer biomarker development. Identifying the initiating events that cause aberrant DNA methylation in lung carcinogenesis may help improve future strategies for prevention, early detection and treatment of this highly lethal disease.

FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin

PubMed Central

Giresi, Paul G.; Kim, Jonghwan; McDaniell, Ryan M.; Iyer, Vishwanath R.; Lieb, Jason D.

2007-01-01

DNA segments that actively regulate transcription in vivo are typically characterized by eviction of nucleosomes from chromatin and are experimentally identified by their hypersensitivity to nucleases. Here we demonstrate a simple procedure for the isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements). To perform FAIRE, chromatin is crosslinked with formaldehyde in vivo, sheared by sonication, and phenol-chloroform extracted. The DNA recovered in the aqueous phase is fluorescently labeled and hybridized to a DNA microarray. FAIRE performed in human cells strongly enriches DNA coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, and active promoters. Evidence for cell-type–specific patterns of FAIRE enrichment is also presented. FAIRE has utility as a positive selection for genomic regions associated with regulatory activity, including regions traditionally detected by nuclease hypersensitivity assays. PMID:17179217

Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements)

PubMed Central

Giresi, Paul G.; Lieb, Jason D.

2009-01-01

The binding of sequence-specific regulatory factors and the recruitment of chromatin remodeling activities cause nucleosomes to be evicted from chromatin in eukaryotic cells. Traditionally, these active sites have been identified experimentally through their sensitivity to nucleases. Here we describe the details of a simple procedure for the genome-wide isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements). We also provide protocols for different methods of detecting FAIRE-enriched DNA, including use of PCR, DNA microarrays, and next-generation sequencing. FAIRE works on all eukaryotic chromatin tested to date. To perform FAIRE, chromatin is crosslinked with formaldehyde, sheared by sonication, and phenol-chloroform extracted. Most genomic DNA is crosslinked to nucleosomes and is sequestered to the interphase, whereas DNA recovered in the aqueous phase corresponds to nucleosome-depleted regions of the genome. The isolated regions are largely coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, enhancers, insulators, and active promoters. Given its speed and simplicity, FAIRE has utility in establishing chromatin profiles of diverse cell types in health and disease, isolating DNA regulatory elements en masse for further characterization, and as a screening assay for the effects of small molecules on chromatin organization. PMID:19303047

Primary analysis of repeat elements of the Asian seabass (Lates calcarifer) transcriptome and genome

PubMed Central

Kuznetsova, Inna S.; Thevasagayam, Natascha M.; Sridatta, Prakki S. R.; Komissarov, Aleksey S.; Saju, Jolly M.; Ngoh, Si Y.; Jiang, Junhui; Shen, Xueyan; Orbán, László

2014-01-01

As part of our Asian seabass genome project, we are generating an inventory of repeat elements in the genome and transcriptome. The karyotype showed a diploid number of 2n = 24 chromosomes with a variable number of B-chromosomes. The transcriptome and genome of Asian seabass were searched for repetitive elements with experimental and bioinformatics tools. Six different types of repeats constituting 8–14% of the genome were characterized. Repetitive elements were clustered in the pericentromeric heterochromatin of all chromosomes, but some of them were preferentially accumulated in pretelomeric and pericentromeric regions of several chromosomes pairs and have chromosomes specific arrangement. From the dispersed class of fish-specific non-LTR retrotransposon elements Rex1 and MAUI-like repeats were analyzed. They were wide-spread both in the genome and transcriptome, accumulated on the pericentromeric and peritelomeric areas of all chromosomes. Every analyzed repeat was represented in the Asian seabass transcriptome, some showed differential expression between the gonads. The other group of repeats analyzed belongs to the rRNA multigene family. FISH signal for 5S rDNA was located on a single pair of chromosomes, whereas that for 18S rDNA was found on two pairs. A BAC-derived contig containing rDNA was sequenced and assembled into a scaffold containing incomplete fragments of 18S rDNA. Their assembly and chromosomal position revealed that this part of Asian seabass genome is extremely rich in repeats containing evolutionarily conserved and novel sequences. In summary, transcriptome assemblies and cDNA data are suitable for the identification of repetitive DNA from unknown genomes and for comparative investigation of conserved elements between teleosts and other vertebrates. PMID:25120555

Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens

PubMed Central

2018-01-01

Many implementations of pooled screens in mammalian cells rely on linking an element of interest to a barcode, with the latter subsequently quantitated by next generation sequencing. However, substantial uncoupling between these paired elements during lentiviral production has been reported, especially as the distance between elements increases. We detail that PCR amplification is another major source of uncoupling, and becomes more pronounced with increased amounts of DNA template molecules and PCR cycles. To lessen uncoupling in systems that use paired elements for detection, we recommend minimizing the distance between elements, using low and equal template DNA inputs for plasmid and genomic DNA during PCR, and minimizing the number of PCR cycles. We also present a vector design for conducting combinatorial CRISPR screens that enables accurate barcode-based detection with a single short sequencing read and minimal uncoupling. PMID:29799876

Whole DNA methylome profiling in mice exposed to secondhand smoke

PubMed Central

Tommasi, Stella; Zheng, Albert; Yoon, Jae-In; Li, Arthur Xuejun; Wu, Xiwei; Besaratinia, Ahmad

2012-01-01

Aberration of DNA methylation is a prime epigenetic mechanism of carcinogenesis. Aberrant DNA methylation occurs frequently in lung cancer, with exposure to secondhand smoke (SHS) being an established risk factor. The causal role of SHS in the genesis of lung cancer, however, remains elusive. To investigate whether SHS can cause aberrant DNA methylation in vivo, we have constructed the whole DNA methylome in mice exposed to SHS for a duration of 4 mo, both after the termination of exposure and at ensuing intervals post-exposure (up to 10 mo). Our genome-wide and gene-specific profiling of DNA methylation in the lung of SHS-exposed mice revealed that all groups of SHS-exposed mice and controls share a similar pattern of DNA methylation. Furthermore, the methylation status of major repetitive DNA elements, including long-interspersed nuclear elements (LINE L1), intracisternal A particle long-terminal repeat retrotransposons (IAP-LTR), and short-interspersed nuclear elements (SINE B1), in the lung of all groups of SHS-exposed mice and controls remains comparable. The absence of locus-specific gain of DNA methylation and global loss of DNA methylation in the lung of SHS-exposed mice within a timeframe that precedes neoplastic-lesion formation underscore the challenges of lung cancer biomarker development. Identifying the initiating events that cause aberrant DNA methylation in lung carcinogenesis may help improve future strategies for prevention, early detection and treatment of this highly lethal disease. PMID:23051858

Structure and conformational dynamics of scaffolded DNA origami nanoparticles

DTIC Science & Technology

2017-05-08

all-atom molecular dynamics and coarse-grained finite element modeling to DX-based nanoparticles to elucidate their fine-scale and global conforma... finite element (FE) modeling approach CanDo is also routinely used to predict the 3D equilibrium conformation of programmed DNA assemblies based on a...model with both experimental cryo-electron microscopy (cryo-EM) data and all-atom modeling. MATERIALS AND METHODS Lattice-free finite element model

DEPPDB - DNA electrostatic potential properties database. Electrostatic properties of genome DNA elements.

PubMed

Osypov, Alexander A; Krutinin, Gleb G; Krutinina, Eugenia A; Kamzolova, Svetlana G

2012-04-01

Electrostatic properties of genome DNA are important to its interactions with different proteins, in particular, related to transcription. DEPPDB - DNA Electrostatic Potential (and other Physical) Properties Database - provides information on the electrostatic and other physical properties of genome DNA combined with its sequence and annotation of biological and structural properties of genomes and their elements. Genomes are organized on taxonomical basis, supporting comparative and evolutionary studies. Currently, DEPPDB contains all completely sequenced bacterial, viral, mitochondrial, and plastids genomes according to the NCBI RefSeq, and some model eukaryotic genomes. Data for promoters, regulation sites, binding proteins, etc., are incorporated from established DBs and literature. The database is complemented by analytical tools. User sequences calculations are available. Case studies discovered electrostatics complementing DNA bending in E.coli plasmid BNT2 promoter functioning, possibly affecting host-environment metabolic switch. Transcription factors binding sites gravitate to high potential regions, confirming the electrostatics universal importance in protein-DNA interactions beyond the classical promoter-RNA polymerase recognition and regulation. Other genome elements, such as terminators, also show electrostatic peculiarities. Most intriguing are gene starts, exhibiting taxonomic correlations. The necessity of the genome electrostatic properties studies is discussed.

Promoter selection in human mitochondria involves binding of a transcription factor to orientation-independent upstream regulatory elements.

PubMed

Fisher, R P; Topper, J N; Clayton, D A

1987-07-17

Selective transcription of human mitochondrial DNA requires a transcription factor (mtTF) in addition to an essentially nonselective RNA polymerase. Partially purified mtTF is able to sequester promoter-containing DNA in preinitiation complexes in the absence of mitochondrial RNA polymerase, suggesting a DNA-binding mechanism for factor activity. Functional domains, required for positive transcriptional regulation by mtTF, are identified within both major promoters of human mtDNA through transcription of mutant promoter templates in a reconstituted in vitro system. These domains are essentially coextensive with DNA sequences protected from nuclease digestion by mtTF-binding. Comparison of the sequences of the two mtTF-responsive elements reveals significant homology only when one sequence is inverted; the binding sites are in opposite orientations with respect to the predominant direction of transcription. Thus mtTF may function bidirectionally, requiring additional protein-DNA interactions to dictate transcriptional polarity. The mtTF-responsive elements are arrayed as direct repeats, separated by approximately 80 bp within the displacement-loop region of human mitochondrial DNA; this arrangement may reflect duplication of an ancestral bidirectional promoter, giving rise to separate, unidirectional promoters for each strand.

A comprehensive list of cloned human DNA sequences

PubMed Central

Schmidtke, Jörg; Cooper, David N.

1987-01-01

A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:3575113

A comprehensive list of cloned human DNA sequences

PubMed Central

Schmidtke, Jörg; Cooper, David N.

1990-01-01

A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:2333227

A comprehensive list of cloned human DNA sequences

PubMed Central

Schmidtke, Jörg; Cooper, David N.

1988-01-01

A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:3368330

A comprehensive list of cloned human DNA sequences

PubMed Central

Schmidtke, Jörg; Cooper, David N.

1989-01-01

A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:2654889

Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells

PubMed Central

Shimizu, Akira; Nakatani, Yoko; Nakamura, Takako; Jinno-Oue, Atsushi; Ishikawa, Osamu; Boeke, Jef D.; Takeuchi, Yasuhiro; Hoshino, Hiroo

2014-01-01

The synthesis and subsequent genomic integration of DNA that is complementary to the genomes of non-retroviral RNA viruses are rarely observed. However, upon infection of various human cell lines and primary fibroblasts with the vesicular stomatitis virus (VSV), we detected DNA complementary to the VSV RNA. The VSV DNA was detected in the cytoplasm as single-stranded DNA fully complementary to the viral mRNA from the poly(A) region to the 7-methyl guanosine cap. The formation of this DNA was cell-dependent. Experimentally, we found that the transduction of cells that do not produce VSV DNA with the long interspersed nuclear element 1 and their infection with VSV could lead to the formation of VSV DNA. Viral DNA complementary to other RNA viruses was also detected in the respective infected human cells. Thus, the genetic information of the non-retroviral RNA virus genome can flow into the DNA of mammalian cells expressing LINE-1-like elements. PMID:24875540

Mycobacterium smegmatis strain for detection of Mycobacterium tuberculosis by PCR used as internal control for inhibition of amplification and for quantification of bacteria.

PubMed Central

Kolk, A H; Noordhoek, G T; de Leeuw, O; Kuijper, S; van Embden, J D

1994-01-01

For the detection of Mycobacterium tuberculosis by PCR, the IS6110 sequence was used. A modified target was constructed by insertion of 56 nucleotides in the IS6110 insertion element of Mycobacterium bovis BCG. This modified insertion sequence was integrated into the genome of Mycobacterium smegmatis, a mycobacterium species which does not contain the IS6110 element. When DNA from the modified M. smegmatis 1008 strain was amplified with IS6110-specific primers INS1 and INS2, a band of 301 bp was seen on agarose gel, whereas the PCR product of M. tuberculosis complex DNA was a 245-bp fragment with these primers. The addition of a small number of M. smegmatis 1008 cells to clinical samples before DNA purification enables the detection of problems which may be due to the loss of DNA in the isolation procedure or to the presence of inhibitors. The presence of inhibitors of the amplification reaction can be confirmed by the addition of M. smegmatis 1008 DNA after the DNA isolation procedure. Furthermore, competition between the different target DNAs of M. smegmatis 1008 DNA and M. tuberculosis complex DNA enables the estimation of the number of IS6110 elements in the clinical sample. Images PMID:8051267

VEZF1 Elements Mediate Protection from DNA Methylation

PubMed Central

Strogantsev, Ruslan; Gaszner, Miklos; Hair, Alan; Felsenfeld, Gary; West, Adam G.

2010-01-01

There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm β-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state. PMID:20062523

AP1 Keeps Chromatin Poised for Action | Center for Cancer Research

Cancer.gov

The human genome harbors gene-encoding DNA, the blueprint for building proteins that regulate cellular function. Embedded across the genome, in non-coding regions, are DNA elements to which regulatory factors bind. The interaction of regulatory factors with DNA at these sites modifies gene expression to modulate cell activity. In cells, DNA exists in a complex with proteins called chromatin that compacts the DNA in the nucleus, strongly restricting access to DNA sequences. As a result, regulatory factors only interact with a small subset of their potential binding elements in a given cell to regulate genes. How factors recognize and select sites in chromatin across the genome is not well understood -- but several discoveries in CCR’s Laboratory of Receptor Biology and Gene Expression (LRBGE) have shed light on the mechanisms that direct factors to DNA.

The twilight zone of cis element alignments.

PubMed

Sebastian, Alvaro; Contreras-Moreira, Bruno

2013-02-01

Sequence alignment of proteins and nucleic acids is a routine task in bioinformatics. Although the comparison of complete peptides, genes or genomes can be undertaken with a great variety of tools, the alignment of short DNA sequences and motifs entails pitfalls that have not been fully addressed yet. Here we confront the structural superposition of transcription factors with the sequence alignment of their recognized cis elements. Our goals are (i) to test TFcompare (http://floresta.eead.csic.es/tfcompare), a structural alignment method for protein-DNA complexes; (ii) to benchmark the pairwise alignment of regulatory elements; (iii) to define the confidence limits and the twilight zone of such alignments and (iv) to evaluate the relevance of these thresholds with elements obtained experimentally. We find that the structure of cis elements and protein-DNA interfaces is significantly more conserved than their sequence and measures how this correlates with alignment errors when only sequence information is considered. Our results confirm that DNA motifs in the form of matrices produce better alignments than individual sequences. Finally, we report that empirical and theoretically derived twilight thresholds are useful for estimating the natural plasticity of regulatory sequences, and hence for filtering out unreliable alignments.

The twilight zone of cis element alignments

PubMed Central

Sebastian, Alvaro; Contreras-Moreira, Bruno

2013-01-01

Sequence alignment of proteins and nucleic acids is a routine task in bioinformatics. Although the comparison of complete peptides, genes or genomes can be undertaken with a great variety of tools, the alignment of short DNA sequences and motifs entails pitfalls that have not been fully addressed yet. Here we confront the structural superposition of transcription factors with the sequence alignment of their recognized cis elements. Our goals are (i) to test TFcompare (http://floresta.eead.csic.es/tfcompare), a structural alignment method for protein–DNA complexes; (ii) to benchmark the pairwise alignment of regulatory elements; (iii) to define the confidence limits and the twilight zone of such alignments and (iv) to evaluate the relevance of these thresholds with elements obtained experimentally. We find that the structure of cis elements and protein–DNA interfaces is significantly more conserved than their sequence and measures how this correlates with alignment errors when only sequence information is considered. Our results confirm that DNA motifs in the form of matrices produce better alignments than individual sequences. Finally, we report that empirical and theoretically derived twilight thresholds are useful for estimating the natural plasticity of regulatory sequences, and hence for filtering out unreliable alignments. PMID:23268451

Evolutionary dynamics of selfish DNA explains the abundance distribution of genomic subsequences

PubMed Central

Sheinman, Michael; Ramisch, Anna; Massip, Florian; Arndt, Peter F.

2016-01-01

Since the sequencing of large genomes, many statistical features of their sequences have been found. One intriguing feature is that certain subsequences are much more abundant than others. In fact, abundances of subsequences of a given length are distributed with a scale-free power-law tail, resembling properties of human texts, such as Zipf’s law. Despite recent efforts, the understanding of this phenomenon is still lacking. Here we find that selfish DNA elements, such as those belonging to the Alu family of repeats, dominate the power-law tail. Interestingly, for the Alu elements the power-law exponent increases with the length of the considered subsequences. Motivated by these observations, we develop a model of selfish DNA expansion. The predictions of this model qualitatively and quantitatively agree with the empirical observations. This allows us to estimate parameters for the process of selfish DNA spreading in a genome during its evolution. The obtained results shed light on how evolution of selfish DNA elements shapes non-trivial statistical properties of genomes. PMID:27488939

DNA capture elements for rapid detection and identification of biological agents

NASA Astrophysics Data System (ADS)

Kiel, Johnathan L.; Parker, Jill E.; Holwitt, Eric A.; Vivekananda, Jeeva

2004-08-01

DNA capture elements (DCEs; aptamers) are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. Reporter molecules were attached to the finished sequences. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. These DCEs have demonstrated specificity and sensitivity equal to or better than antibody.

DNA methylation dynamics during early plant life.

PubMed

Bouyer, Daniel; Kramdi, Amira; Kassam, Mohamed; Heese, Maren; Schnittger, Arp; Roudier, François; Colot, Vincent

2017-09-25

Cytosine methylation is crucial for gene regulation and silencing of transposable elements in mammals and plants. While this epigenetic mark is extensively reprogrammed in the germline and early embryos of mammals, the extent to which DNA methylation is reset between generations in plants remains largely unknown. Using Arabidopsis as a model, we uncovered distinct DNA methylation dynamics over transposable element sequences during the early stages of plant development. Specifically, transposable elements and their relics show invariably high methylation at CG sites but increasing methylation at CHG and CHH sites. This non-CG methylation culminates in mature embryos, where it reaches saturation for a large fraction of methylated CHH sites, compared to the typical 10-20% methylation level observed in seedlings or adult plants. Moreover, the increase in CHH methylation during embryogenesis matches the hypomethylated state in the early endosperm. Finally, we show that interfering with the embryo-to-seedling transition results in the persistence of high CHH methylation levels after germination, specifically over sequences that are targeted by the RNA-directed DNA methylation (RdDM) machinery. Our findings indicate the absence of extensive resetting of DNA methylation patterns during early plant life and point instead to an important role of RdDM in reinforcing DNA methylation of transposable element sequences in every cell of the mature embryo. Furthermore, we provide evidence that this elevated RdDM activity is a specific property of embryogenesis.

Divergent genome evolution caused by regional variation in DNA gain and loss between human and mouse

PubMed Central

Kortschak, R. Daniel

2018-01-01

The forces driving the accumulation and removal of non-coding DNA and ultimately the evolution of genome size in complex organisms are intimately linked to genome structure and organisation. Our analysis provides a novel method for capturing the regional variation of lineage-specific DNA gain and loss events in their respective genomic contexts. To further understand this connection we used comparative genomics to identify genome-wide individual DNA gain and loss events in the human and mouse genomes. Focusing on the distribution of DNA gains and losses, relationships to important structural features and potential impact on biological processes, we found that in autosomes, DNA gains and losses both followed separate lineage-specific accumulation patterns. However, in both species chromosome X was particularly enriched for DNA gain, consistent with its high L1 retrotransposon content required for X inactivation. We found that DNA loss was associated with gene-rich open chromatin regions and DNA gain events with gene-poor closed chromatin regions. Additionally, we found that DNA loss events tended to be smaller than DNA gain events suggesting that they were able to accumulate in gene-rich open chromatin regions due to their reduced capacity to interrupt gene regulatory architecture. GO term enrichment showed that mouse loss hotspots were strongly enriched for terms related to developmental processes. However, these genes were also located in regions with a high density of conserved elements, suggesting that despite high levels of DNA loss, gene regulatory architecture remained conserved. This is consistent with a model in which DNA gain and loss results in turnover or “churning” in regulatory element dense regions of open chromatin, where interruption of regulatory elements is selected against. PMID:29677183

Turnover of R1 (Type I) and R2 (Type Ii) Retrotransposable Elements in the Ribosomal DNA of Drosophila Melanogaster

PubMed Central

Jakubczak, J. L.; Zenni, M. K.; Woodruff, R. C.; Eickbush, T. H.

1992-01-01

R1 and R2 are distantly related non-long terminal repeat retrotransposable elements each of which inserts into a specific site in the 28S rRNA genes of most insects. We have analyzed aspects of R1 and R2 abundance and sequence variation in 27 geographical isolates of Drosophila melanogaster. The fraction of 28S rRNA genes containing these elements varied greatly between strains, 17-67% for R1 elements and 2-28% for R2 elements. The total percentage of the rDNA repeats inserted ranged from 32 to 77%. The fraction of the rDNA repeats that contained both of these elements suggested that R1 and R2 exhibit neither an inhibition of nor preference for insertion into a 28S gene already containing the other type of element. Based on the conservation of restriction sites in the elements of all strains, and sequence analysis of individual elements from three strains, nucleotide divergence is very low for R1 and R2 elements within or between strains (<0.6%). This sequence uniformity is the expected result of the forces of concerted evolution (unequal crossovers and gene conversion) which act on the rRNA genes themselves. Evidence for the role of retrotransposition in the turnover of R1 and R2 was obtained by using naturally occurring 5' length polymorphisms of the elements as markers for independent transposition events. The pattern of these different length 5' truncations of R1 and R2 was found to be diverse and unique to most strains analyzed. Because recombination can only, with time, amplify or eliminate those length variants already present, the diversity found in each strain suggests that retrotransposition has played a critical role in maintaining these elements in the rDNA repeats of D. melanogaster. PMID:1317313

A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases.

PubMed

Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

2008-10-04

Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases.

A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases

PubMed Central

Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

2008-01-01

Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases. This article was reviewed by Eugene Koonin and Mark Ragan. PMID:18834537

Structure, replication efficiency and fragility of yeast ARS elements.

PubMed

Dhar, Manoj K; Sehgal, Shelly; Kaul, Sanjana

2012-05-01

DNA replication in eukaryotes initiates at specific sites known as origins of replication, or replicators. These replication origins occur throughout the genome, though the propensity of their occurrence depends on the type of organism. In eukaryotes, zones of initiation of replication spanning from about 100 to 50,000 base pairs have been reported. The characteristics of eukaryotic replication origins are best understood in the budding yeast Saccharomyces cerevisiae, where some autonomously replicating sequences, or ARS elements, confer origin activity. ARS elements are short DNA sequences of a few hundred base pairs, identified by their efficiency at initiating a replication event when cloned in a plasmid. ARS elements, although structurally diverse, maintain a basic structure composed of three domains, A, B and C. Domain A is comprised of a consensus sequence designated ACS (ARS consensus sequence), while the B domain has the DNA unwinding element and the C domain is important for DNA-protein interactions. Although there are ∼400 ARS elements in the yeast genome, not all of them are active origins of replication. Different groups within the genus Saccharomyces have ARS elements as components of replication origin. The present paper provides a comprehensive review of various aspects of ARSs, starting from their structural conservation to sequence thermodynamics. All significant and conserved functional sequence motifs within different types of ARS elements have been extensively described. Issues like silencing at ARSs, their inherent fragility and factors governing their replication efficiency have also been addressed. Progress in understanding crucial components associated with the replication machinery and timing at these ARS elements is discussed in the section entitled "The replicon revisited". Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Transcriptional activation of short interspersed elements by DNA-damaging agents.

PubMed

Rudin, C M; Thompson, C B

2001-01-01

Short interspersed elements (SINEs), typified by the human Alu repeat, are RNA polymerase III (pol III)-transcribed sequences that replicate within the genome through an RNA intermediate. Replication of SINEs has been extensive in mammalian evolution: an estimated 5% of the human genome consists of Alu repeats. The mechanisms regulating transcription, reverse transcription, and reinsertion of SINE elements in genomic DNA are poorly understood. Here we report that expression of murine SINE transcripts of both the B1 and B2 classes is strongly upregulated after prolonged exposure to cisplatin, etoposide, or gamma radiation. A similar induction of Alu transcripts in human cells occurs under these conditions. This induction is not due to a general upregulation of pol III activity in either species. Genotoxic treatment of murine cells containing an exogenous human Alu element induced Alu transcription. Concomitant with the increased expression of SINEs, an increase in cellular reverse transcriptase was observed after exposure to these same DNA-damaging agents. These findings suggest that genomic damage may be an important activator of SINEs, and that SINE mobility may contribute to secondary malignancy after exposure to DNA-damaging chemotherapy.

Functional characterization of piggyBat from the bat Myotis lucifugus unveils an active mammalian DNA transposon.

PubMed

Mitra, Rupak; Li, Xianghong; Kapusta, Aurélie; Mayhew, David; Mitra, Robi D; Feschotte, Cédric; Craig, Nancy L

2013-01-02

A revelation of the genomic age has been the contributions of the mobile DNA segments called transposable elements to chromosome structure, function, and evolution in virtually all organisms. Substantial fractions of vertebrate genomes derive from transposable elements, being dominated by retroelements that move via RNA intermediates. Although many of these elements have been inactivated by mutation, several active retroelements remain. Vertebrate genomes also contain substantial quantities and a high diversity of cut-and-paste DNA transposons, but no active representative of this class has been identified in mammals. Here we show that a cut-and-paste element called piggyBat, which has recently invaded the genome of the little brown bat (Myotis lucifugus) and is a member of the piggyBac superfamily, is active in its native form in transposition assays in bat and human cultured cells, as well as in the yeast Saccharomyces cerevisiae. Our study suggests that some DNA transposons are still actively shaping some mammalian genomes and reveals an unprecedented opportunity to study the mechanism, regulation, and genomic impact of cut-and-paste transposition in a natural mammalian host.

Complex structure of knob DNA on maize chromosome 9. Retrotransposon invasion into heterochromatin.

PubMed Central

Ananiev, E V; Phillips, R L; Rines, H W

1998-01-01

The recovery of maize (Zea mays L.) chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses enables us to analyze the structure and composition of specific regions, such as knobs, of individual maize chromosomes. A DNA hybridization blot panel of eight individual maize chromosome addition lines revealed that 180-bp repeats found in knobs are present in each of these maize chromosomes, but the copy number varies from approximately 100 to 25, 000. Cosmid clones with knob DNA segments were isolated from a genomic library of an oat-maize chromosome 9 addition line with the help of the 180-bp knob-associated repeated DNA sequence used as a probe. Cloned knob DNA segments revealed a complex organization in which blocks of tandemly arranged 180-bp repeating units are interrupted by insertions of other repeated DNA sequences, mostly represented by individual full size copies of retrotransposable elements. There is an obvious preference for the integration of retrotransposable elements into certain sites (hot spots) of the 180-bp repeat. Sequence microheterogeneity including point mutations and duplications was found in copies of 180-bp repeats. The 180-bp repeats within an array all had the same polarity. Restriction maps constructed for 23 cloned knob DNA fragments revealed the positions of polymorphic sites and sites of integration of insertion elements. Discovery of the interspersion of retrotransposable elements among blocks of tandem repeats in maize and some other organisms suggests that this pattern may be basic to heterochromatin organization for eukaryotes. PMID:9691055

SINE sequences detect DNA fingerprints in salmonid fishes.

PubMed

Spruell, P; Thorgaard, G H

1996-04-01

DNA probes homologous to two previously described salmonid short interspersed nuclear elements (SINEs) detected DNA fingerprint patterns in 14 species of salmonid fishes. The probes showed more homology to some species than to others and little homology to three nonsalmonid fishes. The DNA fingerprint patterns derived from the SINE probes are individual-specific and inherited in a Mendelian manner. Probes derived from different regions of the same SINE detect only partially overlapping banding patterns, reflecting a more complex SINE structure than has been previously reported. Like the human Alu sequence, the SINEs found in salmonids could provide useful genetic markers and primer sites for PCR-based techniques. These elements may be more desirable for some applications than traditional DNA fingerprinting probes that detect tandemly repeated arrays.

Detection of DNA polymerase λ activity during seed germination and enhancement after salinity stress and dehydration in the plumules of indica rice (Oryza sativa L.

PubMed

Sihi, Sayantani; Bakshi, Sankar; Sengupta, Dibyendu Narayan

2015-02-01

DNA polymerase λ (DNA pol λ) is the only reported X-family DNA polymerases in plants and has been shown to play a significant role in dry quiescent seeds, growth, development and nuclear DNA repair. cDNA for DNA pol λ has been reported in Arabidopsis and japonica rice cultivar and has been characterized from E. coli expressed protein, but very little is known about its activity at protein level in plants. The enzymatic activity of DNA pol λ was studied in dry, imbibed and during different germination stages of indica rice IR-8 (salt sensitive) by in-gel activity assay to determine its physiological role in important stages of growth and development. The upstream sequence was also analyzed using plantCARE database and was found to contain several cis-acting elements, including light responsive elements, dehydration responsive elements, Myb binding sites, etc. Hence, 4-day-old germinating seedlings of IR29, a salt-sensitive, but high yielding indica rice cultivar and Nonabokra, a salt-tolerant, but low yielding cultivar were treated with water (control) or 250 mM NaCl or 20% polyethyleneglycol-6000 for 4 and 8 h. The protein was analyzed by in vitro DNA pol λ activity assay, in-gel activity assay and Western blot analysis. DNA pol λ was not detected in dry seeds, but enhanced after imbibition and detectable from low level to high level during subsequent germination steps. Both salinity and dehydration stress led to the enhancement of the activity and protein level of DNA pol λ, as compared to control tissues. This is the first evidence of the salinity or dehydration stress induced enhancement of DNA pol λ activity in the plumules of rice (Oryza sativa L.) cultivars.

Evolutionary Dynamics of 5S rDNA and Recurrent Association of Transposable Elements in Electric Fish of the Family Gymnotidae (Gymnotiformes): The Case of Gymnotus mamiraua.

PubMed

da Silva, Maelin; Barbosa, Patricia; Artoni, Roberto F; Feldberg, Eliana

2016-01-01

Gymnotidae is a family of electric fish endemic to the Neotropics consisting of 2 genera: Electrophorus and Gymnotus. The genus Gymnotus is widely distributed and is found in all of the major Brazilian river systems. Physical and molecular mapping data for the ribosomal DNA (rDNA) in this genus are still scarce, with its chromosomal location known in only 11 species. As other species of Gymnotus with 2n = 54 chromosomes from the Paraná-Paraguay basin, G. mamiraua was found to have a large number of 5S rDNA sites. Isolation and cloning of the 5S rDNA sequences from G. mamiraua identified a fragment of a transposable element similar to the Tc1/mariner transposon associated with a non-transcribed spacer. Double fluorescence in situ hybridization analysis of this element and the 5S rDNA showed that they were colocalized on several chromosomes, in addition to acting as nonsyntenic markers on others. Our data show the association between these sequences and suggest that the Tc1 retrotransposon may be the agent that drives the spread of these 5S rDNA-like sequences in the G. mamiraua genome. © 2016 S. Karger AG, Basel.

Is junk DNA bunk? A critique of ENCODE.

PubMed

Doolittle, W Ford

2013-04-02

Do data from the Encyclopedia Of DNA Elements (ENCODE) project render the notion of junk DNA obsolete? Here, I review older arguments for junk grounded in the C-value paradox and propose a thought experiment to challenge ENCODE's ontology. Specifically, what would we expect for the number of functional elements (as ENCODE defines them) in genomes much larger than our own genome? If the number were to stay more or less constant, it would seem sensible to consider the rest of the DNA of larger genomes to be junk or, at least, assign it a different sort of role (structural rather than informational). If, however, the number of functional elements were to rise significantly with C-value then, (i) organisms with genomes larger than our genome are more complex phenotypically than we are, (ii) ENCODE's definition of functional element identifies many sites that would not be considered functional or phenotype-determining by standard uses in biology, or (iii) the same phenotypic functions are often determined in a more diffuse fashion in larger-genomed organisms. Good cases can be made for propositions ii and iii. A larger theoretical framework, embracing informational and structural roles for DNA, neutral as well as adaptive causes of complexity, and selection as a multilevel phenomenon, is needed.

Is junk DNA bunk? A critique of ENCODE

PubMed Central

Doolittle, W. Ford

2013-01-01

Do data from the Encyclopedia Of DNA Elements (ENCODE) project render the notion of junk DNA obsolete? Here, I review older arguments for junk grounded in the C-value paradox and propose a thought experiment to challenge ENCODE’s ontology. Specifically, what would we expect for the number of functional elements (as ENCODE defines them) in genomes much larger than our own genome? If the number were to stay more or less constant, it would seem sensible to consider the rest of the DNA of larger genomes to be junk or, at least, assign it a different sort of role (structural rather than informational). If, however, the number of functional elements were to rise significantly with C-value then, (i) organisms with genomes larger than our genome are more complex phenotypically than we are, (ii) ENCODE’s definition of functional element identifies many sites that would not be considered functional or phenotype-determining by standard uses in biology, or (iii) the same phenotypic functions are often determined in a more diffuse fashion in larger-genomed organisms. Good cases can be made for propositions ii and iii. A larger theoretical framework, embracing informational and structural roles for DNA, neutral as well as adaptive causes of complexity, and selection as a multilevel phenomenon, is needed. PMID:23479647

Satellite DNA Modulates Gene Expression in the Beetle Tribolium castaneum after Heat Stress

PubMed Central

Feliciello, Isidoro; Akrap, Ivana; Ugarković, Đurđica

2015-01-01

Non-coding repetitive DNAs have been proposed to perform a gene regulatory role, however for tandemly repeated satellite DNA no such role was defined until now. Here we provide the first evidence for a role of satellite DNA in the modulation of gene expression under specific environmental conditions. The major satellite DNA TCAST1 in the beetle Tribolium castaneum is preferentially located within pericentromeric heterochromatin but is also dispersed as single repeats or short arrays in the vicinity of protein-coding genes within euchromatin. Our results show enhanced suppression of activity of TCAST1-associated genes and slower recovery of their activity after long-term heat stress relative to the same genes without associated TCAST1 satellite DNA elements. The level of gene suppression is not influenced by the distance of TCAST1 elements from the associated genes up to 40 kb from the genes’ transcription start sites, but it does depend on the copy number of TCAST1 repeats within an element, being stronger for the higher number of copies. The enhanced gene suppression correlates with the enrichment of the repressive histone marks H3K9me2/3 at dispersed TCAST1 elements and their flanking regions as well as with increased expression of TCAST1 satellite DNA. The results reveal transient, RNAi based heterochromatin formation at dispersed TCAST1 repeats and their proximal regions as a mechanism responsible for enhanced silencing of TCAST1-associated genes. Differences in the pattern of distribution of TCAST1 elements contribute to gene expression diversity among T. castaneum strains after long-term heat stress and might have an impact on adaptation to different environmental conditions. PMID:26275223

Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications

PubMed Central

Hong, Ka Lok

2015-01-01

Molecular recognition elements (MREs) can be short sequences of single-stranded DNA, RNA, small peptides, or antibody fragments. They can bind to user-defined targets with high affinity and specificity. There has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the Gold and Szostak laboratories. A large number of target specific nucleic acids MREs and their applications are currently in the literature. This review first describes the general methodologies used in identifying single-stranded DNA (ssDNA) aptamers. It then summarizes advancements in the identification and biosensing application of ssDNA aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. Lastly, an overview of the basic principles of ssDNA aptamer-based biosensors is discussed. PMID:26199940

On-Chip Synthesis of Protein Microarrays from DNA Microarrays Via Coupled In Vitro Transcription and Translation for Surface Plasmon Resonance Imaging Biosensor Applications

PubMed Central

Seefeld, Ting H.; Halpern, Aaron R.; Corn, Robert M.

2012-01-01

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of messenger RNA (mRNA) in a surface RNA polymerase reaction; the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and anti-luciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications. PMID:22793370

Surveying DNA Elements within Functional Genes of Heterocyst-Forming Cyanobacteria

PubMed Central

Hilton, Jason A.; Meeks, John C.; Zehr, Jonathan P.

2016-01-01

Some cyanobacteria are capable of differentiating a variety of cell types in response to environmental factors. For instance, in low nitrogen conditions, some cyanobacteria form heterocysts, which are specialized for N2 fixation. Many heterocyst-forming cyanobacteria have DNA elements interrupting key N2 fixation genes, elements that are excised during heterocyst differentiation. While the mechanism for the excision of the element has been well-studied, many questions remain regarding the introduction of the elements into the cyanobacterial lineage and whether they have been retained ever since or have been lost and reintroduced. To examine the evolutionary relationships and possible function of DNA sequences that interrupt genes of heterocyst-forming cyanobacteria, we identified and compared 101 interruption element sequences within genes from 38 heterocyst-forming cyanobacterial genomes. The interruption element lengths ranged from about 1 kb (the minimum able to encode the recombinase responsible for element excision), up to nearly 1 Mb. The recombinase gene sequences served as genetic markers that were common across the interruption elements and were used to track element evolution. Elements were found that interrupted 22 different orthologs, only five of which had been previously observed to be interrupted by an element. Most of the newly identified interrupted orthologs encode proteins that have been shown to have heterocyst-specific activity. However, the presence of interruption elements within genes with no known role in N2 fixation, as well as in three non-heterocyst-forming cyanobacteria, indicates that the processes that trigger the excision of elements may not be limited to heterocyst development or that the elements move randomly within genomes. This comprehensive analysis provides the framework to study the history and behavior of these unique sequences, and offers new insight regarding the frequency and persistence of interruption elements in heterocyst-forming cyanobacteria. PMID:27206019

Surveying DNA Elements within Functional Genes of Heterocyst-Forming Cyanobacteria.

PubMed

Hilton, Jason A; Meeks, John C; Zehr, Jonathan P

2016-01-01

Some cyanobacteria are capable of differentiating a variety of cell types in response to environmental factors. For instance, in low nitrogen conditions, some cyanobacteria form heterocysts, which are specialized for N2 fixation. Many heterocyst-forming cyanobacteria have DNA elements interrupting key N2 fixation genes, elements that are excised during heterocyst differentiation. While the mechanism for the excision of the element has been well-studied, many questions remain regarding the introduction of the elements into the cyanobacterial lineage and whether they have been retained ever since or have been lost and reintroduced. To examine the evolutionary relationships and possible function of DNA sequences that interrupt genes of heterocyst-forming cyanobacteria, we identified and compared 101 interruption element sequences within genes from 38 heterocyst-forming cyanobacterial genomes. The interruption element lengths ranged from about 1 kb (the minimum able to encode the recombinase responsible for element excision), up to nearly 1 Mb. The recombinase gene sequences served as genetic markers that were common across the interruption elements and were used to track element evolution. Elements were found that interrupted 22 different orthologs, only five of which had been previously observed to be interrupted by an element. Most of the newly identified interrupted orthologs encode proteins that have been shown to have heterocyst-specific activity. However, the presence of interruption elements within genes with no known role in N2 fixation, as well as in three non-heterocyst-forming cyanobacteria, indicates that the processes that trigger the excision of elements may not be limited to heterocyst development or that the elements move randomly within genomes. This comprehensive analysis provides the framework to study the history and behavior of these unique sequences, and offers new insight regarding the frequency and persistence of interruption elements in heterocyst-forming cyanobacteria.

Nanoparticle-labeled DNA capture elements for detection and identification of biological agents

NASA Astrophysics Data System (ADS)

Kiel, Johnathan L.; Holwitt, Eric A.; Parker, Jill E.; Vivekananda, Jeevalatha; Franz, Veronica

2004-12-01

Aptamers, synthetic DNA capture elements (DCEs), can be made chemically or in genetically engineered bacteria. DNA capture elements are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare or terrorism agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. The probes can then be conjugated to Quantum Dots and super paramagnetic nanoparticles. The former provide intense, bleach-resistant fluorescent detection of bioagent and the latter provide a means to collect the bioagents with a magnet. The fluorescence can be detected in a flow cytometer, in a fluorescence plate reader, or with a fluorescence microscope. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. DCEs can easily distinguish Bacillus anthracis from its nearest relatives, Bacillus cereus and Bacillus thuringiensis. Development of a high through-put process is currently being investigated.

New insights into replication origin characteristics in metazoans

PubMed Central

Puy, Aurore; Rialle, Stéphanie; Kaplan, Noam; Segal, Eran

2012-01-01

We recently reported the identification and characterization of DNA replication origins (Oris) in metazoan cell lines. Here, we describe additional bioinformatic analyses showing that the previously identified GC-rich sequence elements form origin G-rich repeated elements (OGREs) that are present in 67% to 90% of the DNA replication origins from Drosophila to human cells, respectively. Our analyses also show that initiation of DNA synthesis takes place precisely at 160 bp (Drosophila) and 280 bp (mouse) from the OGRE. We also found that in most CpG islands, an OGRE is positioned in opposite orientation on each of the two DNA strands and detected two sites of initiation of DNA synthesis upstream or downstream of each OGRE. Conversely, Oris not associated with CpG islands have a single initiation site. OGRE density along chromosomes correlated with previously published replication timing data. Ori sequences centered on the OGRE are also predicted to have high intrinsic nucleosome occupancy. Finally, OGREs predict G-quadruplex structures at Oris that might be structural elements controlling the choice or activation of replication origins. PMID:22373526

The fission yeast CENP-B protein Abp1 prevents pervasive transcription of repetitive DNA elements.

PubMed

Daulny, Anne; Mejía-Ramírez, Eva; Reina, Oscar; Rosado-Lugo, Jesus; Aguilar-Arnal, Lorena; Auer, Herbert; Zaratiegui, Mikel; Azorin, Fernando

2016-10-01

It is well established that eukaryotic genomes are pervasively transcribed producing cryptic unstable transcripts (CUTs). However, the mechanisms regulating pervasive transcription are not well understood. Here, we report that the fission yeast CENP-B homolog Abp1 plays an important role in preventing pervasive transcription. We show that loss of abp1 results in the accumulation of CUTs, which are targeted for degradation by the exosome pathway. These CUTs originate from different types of genomic features, but the highest increase corresponds to Tf2 retrotransposons and rDNA repeats, where they map along the entire elements. In the absence of abp1, increased RNAPII-Ser5P occupancy is observed throughout the Tf2 coding region and, unexpectedly, RNAPII-Ser5P is enriched at rDNA repeats. Loss of abp1 also results in Tf2 derepression and increased nucleolus size. Altogether these results suggest that Abp1 prevents pervasive RNAPII transcription of repetitive DNA elements (i.e., Tf2 and rDNA repeats) from internal cryptic sites. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantification of transcription factor-DNA binding affinity in a living cell

PubMed Central

Belikov, Sergey; Berg, Otto G.; Wrange, Örjan

2016-01-01

The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [3H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element. PMID:26657626

Amplification of a specific repetitive DNA sequence for Trypanosoma rangeli identification and its potential application in epidemiological investigations.

PubMed

Vargas, N; Souto, R P; Carranza, J C; Vallejo, G A; Zingales, B

2000-11-01

Trypanosoma rangeli can infect humans as well as the same domestic and wild animals and triatomine vectors infected by Trypanosoma cruzi in Central and South America. This overlapping distribution complicates the epidemiology of American trypanosomiasis due to the cross-reactivity between T. rangeli and T. cruzi antigens and the presence of conserved DNA sequences in these parasites. We have isolated a T. rangeli-specific DNA repetitive element which is represented in approximately 103 copies per parasite genome and is distributed in several chromosomal bands. The 542-bp nucleotide sequence of this element, named P542, was determined and a PCR assay was standardized for its amplification. The sensitivity of the assay is high, allowing the detection of one tenth of the DNA content of a single parasite. The presence of the P542 element was confirmed in 11 T. rangeli isolates from mammalian hosts and insect vectors originating from several countries in Latin America. Negative amplification was observed with different T. cruzi strains and other trypanosomatids. The potential field application of the P542 PCR assay was investigated in simulated samples containing T. rangeli and/or T. cruzi and intestinal tract and feces of Rhodnius prolixus. Epidemiological studies were conducted in DNA preparations obtained from the digestive tracts of 12 Rhodnius colombiensis insects collected in a sylvatic area in Colombia. Positive amplification of the P542 element was obtained in 9/12 insects. We have also compared in the same samples the diagnostic performance of two PCR assays for the amplification of the variable domain of minicircle kinetoplast DNA (kDNA) and of the large subunit (LSU) of the ribosomal RNA gene of T. cruzi and T. rangeli. Data indicate that the kDNA PCR assay does not allow diagnosis of mixed infections in most insects. On the other hand, the PCR assay of the LSU RNA gene showed lower sensitivity in the detection of T. rangeli than the PCR assay of the P542 element. It is predicted that the use of sensitive detection techniques will indicate that the actual distribution of T. rangeli in America is wider than presumed. Copyright 2000 Academic Press.

Increased methylation of repetitive elements and DNA repair genes is associated with higher DNA oxidation in children in an urbanized, industrial environment.

PubMed

Alvarado-Cruz, Isabel; Sánchez-Guerra, Marco; Hernández-Cadena, Leticia; De Vizcaya-Ruiz, Andrea; Mugica, Violeta; Pelallo-Martínez, Nadia Azenet; Solís-Heredia, María de Jesús; Byun, Hyang-Min; Baccarelli, Andrea; Quintanilla-Vega, Betzabet

2017-01-01

DNA methylation in DNA repair genes participates in the DNA damage regulation. Particulate matter (PM), which has metals and polycyclic aromatic hydrocarbons (PAHs) adsorbed, among others has been linked to adverse health outcomes and may modify DNA methylation. To evaluate PM exposure impact on repetitive elements and gene-specific DNA methylation and DNA damage, we conducted a cross-sectional study in 150 schoolchildren (7-10 years old) from an urbanized, industrial area of the metropolitan area of Mexico City (MAMC), which frequently exhibits PM concentrations above safety standards. Methylation (5mC) of long interspersed nuclear element-1 (LINE1) and DNA repair gene (OGG1, APEX, and PARP1) was assessed by pyrosequencing in peripheral mononuclear cells, DNA damage by comet assay and DNA oxidation by 8-OHdG content. PAH and metal contents in PM 10 (≤10μm aerodynamic diameter) were determined by HPLC-MS and ICP-AES, respectively. Multiple regression analysis between DNA methylation, DNA damage, and PM 10 exposure showed that PM 10 was significantly associated with oxidative DNA damage; a 1% increase in 5mC at all CpG sites in PARP1 promoter was associated with a 35% increase in 8-OHdG, while a 1% increase at 1, 2, and 3 CpG sites resulted in 38, 9, and 56% increments, respectively. An increase of 10pg/m 3 in benzo[b]fluoranthene content of PM 10 was associated with a 6% increase in LINE1 methylation. Acenaphthene, indene [1,2,3-cd] pyrene, and pyrene concentrations correlated with higher dinucleotide methylation in OGG1, APEX and PARP1 genes, respectively. Vanadium concentration correlated with increased methylation at selected APEX and PARP1 CpG sites. DNA repair gene methylation was significantly correlated with DNA damage and with specific PM 10 -associated PAHs and Vanadium. Data suggest that exposure to PM and its components are associated with differences in DNA methylation of repair genes in children, which may contribute to DNA damage. Copyright © 2016 Elsevier B.V. All rights reserved.

The contribution of alu elements to mutagenic DNA double-strand break repair.

PubMed

Morales, Maria E; White, Travis B; Streva, Vincent A; DeFreece, Cecily B; Hedges, Dale J; Deininger, Prescott L

2015-03-01

Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.

LINE-1 Elements in Structural Variation and Disease

PubMed Central

Beck, Christine R.; Garcia-Perez, José Luis; Badge, Richard M.; Moran, John V.

2014-01-01

The completion of the human genome reference sequence ushered in a new era for the study and discovery of human transposable elements. It now is undeniable that transposable elements, historically dismissed as junk DNA, have had an instrumental role in sculpting the structure and function of our genomes. In particular, long interspersed element-1 (LINE-1 or L1) and short interspersed elements (SINEs) continue to affect our genome, and their movement can lead to sporadic cases of disease. Here, we briefly review the types of transposable elements present in the human genome and their mechanisms of mobility. We next highlight how advances in DNA sequencing and genomic technologies have enabled the discovery of novel retrotransposons in individual genomes. Finally, we discuss how L1-mediated retrotransposition events impact human genomes. PMID:21801021

The effect of inflammation-related lifestyle exposures and interactions with gene variants on long interspersed nuclear element-1 DNA methylation.

PubMed

Gogna, Priyanka; O'Sullivan, Dylan E; King, Will D

2018-06-11

To examine the relationship between inflammation-related lifestyle factors and long interspersed nuclear element-1 (LINE-1) DNA methylation, and test for interaction by gene variants involved in one-carbon metabolism. The study population consisted of 280 individuals undergoing colonoscopy screening. Multivariable linear regression was employed to examine associations of physical activity, BMI and NSAID use with LINE-1 DNA methylation and interactions with MTR and MTHFR gene variants. The highest quartile of physical activity compared with the lowest was associated with higher LINE-1 DNA methylation (p = 0.005). Long-term NSAID use and a normal BMI were associated with increased LINE-1 DNA methylation among individuals with the variant MTR allele (p = 0.02; p = 0.03). This study provides evidence that inflammation-related exposures may influence LINE-1 DNA methylation.

Sequence of retrovirus provirus resembles that of bacterial transposable elements

NASA Astrophysics Data System (ADS)

Shimotohno, Kunitada; Mizutani, Satoshi; Temin, Howard M.

1980-06-01

The nucleotide sequences of the terminal regions of an infectious integrated retrovirus cloned in the modified λ phage cloning vector Charon 4A have been elucidated. There is a 569-base pair direct repeat at both ends of the viral DNA. The cell-virus junctions at each end consist of a 5-base pair direct repeat of cell DNA next to a 3-base pair inverted repeat of viral DNA. This structure resembles that of a transposable element and is consistent with the protovirus hypothesis that retroviruses evolved from the cell genome.

Multivalent DNA-binding properties of the HMG-1 proteins.

PubMed Central

Maher, J F; Nathans, D

1996-01-01

HMG-I proteins are DNA-binding proteins thought to affect the formation and function of transcription complexes. Each protein contains three DNA-binding motifs, known as AT-hooks, that bind in the minor groove of AT tracts in DNA. Multiple AT-hooks within a polypeptide chain should contact multiple AT tracts, but the rules governing these interactions have not been defined. In this study, we demonstrate that high-affinity binding uses two or three appropriately spaced AT tracts as a single multivalent binding site. These principles have implications for binding to regulatory elements such as the interferon beta enhancer, TATA boxes, and serum response elements. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8692884

Control of DEMETER DNA demethylase gene transcription in male and female gamete companion cells in Arabidopsis thaliana.

PubMed

Park, Jin-Sup; Frost, Jennifer M; Park, Kyunghyuk; Ohr, Hyonhwa; Park, Guen Tae; Kim, Seohyun; Eom, Hyunjoo; Lee, Ilha; Brooks, Janie S; Fischer, Robert L; Choi, Yeonhee

2017-02-21

The DEMETER (DME) DNA glycosylase initiates active DNA demethylation via the base-excision repair pathway and is vital for reproduction in Arabidopsis thaliana DME-mediated DNA demethylation is preferentially targeted to small, AT-rich, and nucleosome-depleted euchromatic transposable elements, influencing expression of adjacent genes and leading to imprinting in the endosperm. In the female gametophyte, DME expression and subsequent genome-wide DNA demethylation are confined to the companion cell of the egg, the central cell. Here, we show that, in the male gametophyte, DME expression is limited to the companion cell of sperm, the vegetative cell, and to a narrow window of time: immediately after separation of the companion cell lineage from the germline. We define transcriptional regulatory elements of DME using reporter genes, showing that a small region, which surprisingly lies within the DME gene, controls its expression in male and female companion cells. DME expression from this minimal promoter is sufficient to rescue seed abortion and the aberrant DNA methylome associated with the null dme-2 mutation. Within this minimal promoter, we found short, conserved enhancer sequences necessary for the transcriptional activities of DME and combined predicted binding motifs with published transcription factor binding coordinates to produce a list of candidate upstream pathway members in the genetic circuitry controlling DNA demethylation in gamete companion cells. These data show how DNA demethylation is regulated to facilitate endosperm gene imprinting and potential transgenerational epigenetic regulation, without subjecting the germline to potentially deleterious transposable element demethylation.

Evidence of pervasive biologically functional secondary structures within the genomes of eukaryotic single-stranded DNA viruses.

PubMed

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y F; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie; Martin, Darren Patrick

2014-02-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.

Evidence of Pervasive Biologically Functional Secondary Structures within the Genomes of Eukaryotic Single-Stranded DNA Viruses

PubMed Central

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y. F.; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie

2014-01-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here. PMID:24284329

Hsmar1 Transposition Is Sensitive to the Topology of the Transposon Donor and the Target

PubMed Central

Claeys Bouuaert, Corentin; Chalmers, Ronald

2013-01-01

Hsmar1 is a member of the Tc1-mariner superfamily of DNA transposons. These elements mobilize within the genome of their host by a cut-and-paste mechanism. We have exploited the in vitro reaction provided by Hsmar1 to investigate the effect of DNA supercoiling on transposon integration. We found that the topology of both the transposon and the target affect integration. Relaxed transposons have an integration defect that can be partially restored in the presence of elevated levels of negatively supercoiled target DNA. Negatively supercoiled DNA is a better target than nicked or positively supercoiled DNA, suggesting that underwinding of the DNA helix promotes target interactions. Like other Tc1-mariner elements, Hsmar1 integrates into 5′-TA dinucleotides. The direct vicinity of the target TA provides little sequence specificity for target interactions. However, transposition within a plasmid substrate was not random and some TA dinucleotides were targeted preferentially. The distribution of intramolecular target sites was not affected by DNA topology. PMID:23341977

Structure of a bacterial RNA polymerase holoenzyme open promoter complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka

2015-09-08

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstreammore » of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.« less

Structure of a bacterial RNA polymerase holoenzyme open promoter complex

DOE PAGES

Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka; ...

2015-09-08

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstreammore » of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Additionally a RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σ A dissociation.« less

ICE Afe 1, an actively excising genetic element from the biomining bacterium Acidithiobacillus ferrooxidans.

PubMed

Bustamante, Paula; Covarrubias, Paulo C; Levicán, Gloria; Katz, Assaf; Tapia, Pablo; Holmes, David; Quatrini, Raquel; Orellana, Omar

2012-01-01

Integrative conjugative elements (ICEs) are self-transferred mobile genetic elements that contribute to horizontal gene transfer. An ICE (ICEAfe1) was identified in the genome of Acidithiobacillus ferrooxidans ATCC 23270. Excision of the element and expression of relevant genes under normal and DNA-damaging growth conditions was analyzed. Bioinformatic tools and DNA amplification methods were used to identify and to assess the excision and expression of genes related to the mobility of the element. Both basal and mitomycin C-inducible excision as well as expression and induction of the genes for integration/excision are demonstrated, suggesting that ICEAfe1 is an actively excising SOS-regulated mobile genetic element. The presence of a complete set of genes encoding self-transfer functions that are induced in response to DNA damage caused by mitomycin C additionally suggests that this element is capable of conjugative transfer to suitable recipient strains. Transfer of ICEAfe1 may provide selective advantages to other acidophiles in this ecological niche through dissemination of gene clusters expressing transfer RNAs, CRISPRs, and exopolysaccharide biosynthesis enzymes, probably by modification of translation efficiency, resistance to bacteriophage infection and biofilm formation, respectively. These data open novel avenues of research on conjugative transformation of biotechnologically relevant microorganisms recalcitrant to genetic manipulation. Copyright © 2013 S. Karger AG, Basel.

Cut-and-Paste Transposons in Fungi with Diverse Lifestyles

PubMed Central

Steczkiewicz, Kamil; Ginalski, Krzysztof

2017-01-01

Abstract Transposable elements (TEs) shape genomes via recombination and transposition, lead to chromosomal rearrangements, create new gene neighborhoods, and alter gene expression. They play key roles in adaptation either to symbiosis in Amanita genus or to pathogenicity in Pyrenophora tritici-repentis. Despite growing evidence of their importance, the abundance and distribution of mobile elements replicating in a “cut-and-paste” fashion is barely described so far. In order to improve our knowledge on this old and ubiquitous class of transposable elements, 1,730 fungal genomes were scanned using both de novo and homology-based approaches. DNA TEs have been identified across the whole data set and display uneven distribution from both DNA TE classification and fungal taxonomy perspectives. DNA TE content correlates with genome size, which confirms that many transposon families proliferate simultaneously. In contrast, it is independent from intron density, average gene distance and GC content. TE count is associated with species’ lifestyle and tends to be elevated in plant symbionts and decreased in animal parasites. Lastly, we found that fungi with both RIP and RNAi systems have more total DNA TE sequences but less elements retaining a functional transposase, what reflects stringent control over transposition. PMID:29228286

Strip biosensor for amplified detection of nerve growth factor-beta based on a molecular translator and catalytic DNA circuit.

PubMed

Liu, Jun; Lai, Ting; Mu, Kejie; Zhou, Zheng

2014-10-07

We have demonstrated a new visual detection approach based on a molecular translator and a catalytic DNA circuit for the detection of nerve growth factor-beta (NGF-β). In this assay, a molecular translator based on the binding-induced DNA strand-displacement reaction was employed to convert the input protein to an output DNA signal. The molecular translator is composed of a target recognition element and a signal output element. Target recognition is achieved by the binding of the anti-NGF-β antibody to the target protein. Polyclonal anti-NGF-β antibody is conjugated to DNA1 and DNA2. The antibody conjugated DNA1 is initially hybridized to DNA3 to form a stable DNA1/DNA3 duplex. In the presence of NGF-β, the binding of the same target protein brings DNA1 and DNA2 into close proximity, resulting in an increase in their local effective concentration. This process triggers the strand-displacement reaction between DNA2 and DNA3 and releases the output DNA3. The released DNA3 is further amplified by a catalytic DNA circuit. The product of the catalytic DNA circuit is detected by a strip biosensor. This proposed assay has high sensitivity and selectivity with a dynamic response ranging from 10 fM to 10 pM, and its detection limit is 10 fM of NGF-β. This work provides a sensitive, enzyme-free, and universal strategy for the detection of other proteins.

Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements.

PubMed

Redrejo-Rodríguez, Modesto; Ordóñez, Carlos D; Berjón-Otero, Mónica; Moreno-González, Juan; Aparicio-Maldonado, Cristian; Forterre, Patrick; Salas, Margarita; Krupovic, Mart

2017-11-07

Family B DNA polymerases (PolBs) play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB), that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Rapid Microarray Detection of DNA and Proteins in Microliter Volumes with SPR Imaging Measurements

PubMed Central

Seefeld, Ting Hu; Zhou, Wen-Juan; Corn, Robert M.

2011-01-01

A four chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 μL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The twelve element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm2 area; 45 nm thickness) each in individually addressable 0.5 μL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (single stranded DNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements were also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss was proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18,000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 seconds in the microliter volume format. PMID:21488682

In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against the Pesticide Fipronil and Sensitive Detection in River Water

PubMed Central

Sooter, Letha J.

2017-01-01

Fipronil is a commonly used insecticide that has been shown to have environmental and human health risks. The current standard methods of detection for fipronil and its metabolites, such as GC-MS, are time consuming and labor intensive. In this study, a variant of systematic evolution of ligands by exponential enrichment (SELEX), was utilized to identify the first single-stranded DNA (ssDNA) molecular recognition element (MRE) that binds to fipronil with high affinity (Kd = 48 ± 8 nM). The selected MRE displayed low cross binding activity on various environmentally relevant, structurally unrelated herbicides and pesticides, in addition to broad-spectrum binding activity on major metabolites of fipronil and a structurally similar pesticide in prepared river samples. Additionally, a proof-of-principle fluorescent detection assay was developed by using the selected ssDNA MRE as a signal-reporting element, with a limit of detection of 105 nM in a prepared river water sample. PMID:29283416

DNA extraction techniques compared for accurate detection of genetically modified organisms (GMOs) in maize food and feed products.

PubMed

Turkec, Aydin; Kazan, Hande; Karacanli, Burçin; Lucas, Stuart J

2015-08-01

In this paper, DNA extraction methods have been evaluated to detect the presence of genetically modified organisms (GMOs) in maize food and feed products commercialised in Turkey. All the extraction methods tested performed well for the majority of maize foods and feed products analysed. However, the highest DNA content was achieved by the Wizard, Genespin or the CTAB method, all of which produced optimal DNA yield and purity for different maize food and feed products. The samples were then screened for the presence of GM elements, along with certified reference materials. Of the food and feed samples, 8 % tested positive for the presence of one GM element (NOS terminator), of which half (4 % of the total) also contained a second element (the Cauliflower Mosaic Virus 35S promoter). The results obtained herein clearly demonstrate the presence of GM maize in the Turkish market, and that the Foodproof GMO Screening Kit provides reliable screening of maize food and feed products.

Tyrosine Recombinase Retrotransposons and Transposons.

PubMed

Poulter, Russell T M; Butler, Margi I

2015-04-01

Retrotransposons carrying tyrosine recombinases (YR) are widespread in eukaryotes. The first described tyrosine recombinase mobile element, DIRS1, is a retroelement from the slime mold Dictyostelium discoideum. The YR elements are bordered by terminal repeats related to their replication via free circular dsDNA intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. Recently a large number of YR retrotransposons have been described, including elements from fungi (mucorales and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish, amphibia and reptiles. YR retrotransposons can be divided into three major groups: the DIRS elements, PAT-like and the Ngaro elements. The three groups form distinct clades on phylogenetic trees based on alignments of reverse transcriptase/ribonuclease H (RT/RH) and YR sequences, and also having some structural distinctions. A group of eukaryote DNA transposons, cryptons, also carry tyrosine recombinases. These DNA transposons do not encode a reverse transcriptase. They have been detected in several pathogenic fungi and oomycetes. Sequence comparisons suggest that the crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon. This acquisition must have occurred at a very early point in the evolution of eukaryotes.

Specific interaction of mutant p53 with regions of matrix attachment region DNA elements (MARs) with a high potential for base-unpairing

PubMed Central

Will, Katrin; Warnecke, Gabriele; Wiesmüller, Lisa; Deppert, Wolfgang

1998-01-01

Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominant-oncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT “DNA-unwinding motif,” which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MAR-derived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GC-clamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA. PMID:9811860

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tully, D.B.; Cidlowski, J.A.

1989-03-07

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, ({sup 3}H)TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins priormore » to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. Activated ({sup 3}H)TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA. Stability of the complexes formed between GR and these three DNA fragments was strongly affected by even moderate alterations in either the salt concentration or the pH of the gradient buffer. Under all conditions tested, the complex formed with the MMTV LTR DNA fragment was more stable than the complexes formed with either of the pBR322 DNA fragments. Together these observations indicate that the formation of stable complexes between activated GR and isolated DNA fragments requires the presence of GRE consensus sequences in the DNA.« less

Integrated in silico and biological validation of the blocking effect of Cot-1 DNA on Microarray-CGH.

PubMed

Kang, Seung-Hui; Park, Chan Hee; Jeung, Hei Cheul; Kim, Ki-Yeol; Rha, Sun Young; Chung, Hyun Cheol

2007-06-01

In array-CGH, various factors may act as variables influencing the result of experiments. Among them, Cot-1 DNA, which has been used as a repetitive sequence-blocking agent, may become an artifact-inducing factor in BAC array-CGH. To identify the effect of Cot-1 DNA on Microarray-CGH experiments, Cot-1 DNA was labeled directly and Microarray-CGH experiments were performed. The results confirmed that probes which hybridized more completely with Cot-1 DNA had a higher sequence similarity to the Alu element. Further, in the sex-mismatched Microarray-CGH experiments, the variation and intensity in the fluorescent signal were reduced in the high intensity probe group in which probes were better hybridized with Cot-1 DNA. Otherwise, those of the low intensity probe group showed no alterations regardless of Cot-1 DNA. These results confirmed by in silico methods that Cot-1 DNA could block repetitive sequences in gDNA and probes. In addition, it was confirmed biologically that the blocking effect of Cot-1 DNA could be presented via its repetitive sequences, especially Alu elements. Thus, in contrast to BAC-array CGH, the use of Cot-1 DNA is advantageous in controlling experimental variation in Microarray-CGH.

Identification of a DNA Segment Exhibiting Rearrangement Modifying Effects upon Transgenic δ-deleting Elements

PubMed Central

Janowski, Karen M.; Ledbetter, Stephanie; Mayo, Matthew S.; Hockett, Richard D.

1997-01-01

Control of the rearrangement and expression of the T cell receptor α and δ chains is critical for determining T cell type. The process of δ deletion is a candidate mechanism for maintaining separation of the α and δ loci. Mice harboring a transgenic reporter δ deletion construct show α/β T cell lineage–specific use of the transgenic elements. A 48-basepair segment of DNA, termed HPS1A, when deleted from this reporter construct, loses tight lineage-specific rearrangement control of transgenic elements, with abundant rearrangements of transgenic δ-deleting elements now in γ/δ T cells. Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay. DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci. These data are consistent with δ deletion playing an important role in maintaining separate TCR α and δ loci. PMID:9207011

Mavericks, a novel class of giant transposable elements widespread in eukaryotes and related to DNA viruses.

PubMed

Pritham, Ellen J; Putliwala, Tasneem; Feschotte, Cédric

2007-04-01

We previously identified a group of atypical mobile elements designated Mavericks from the nematodes Caenorhabditis elegans and C. briggsae and the zebrafish Danio rerio. Here we present the results of comprehensive database searches of the genome sequences available, which reveal that Mavericks are widespread in invertebrates and non-mammalian vertebrates but show a patchy distribution in non-animal species, being present in the fungi Glomus intraradices and Phakopsora pachyrhizi and in several single-celled eukaryotes such as the ciliate Tetrahymena thermophila, the stramenopile Phytophthora infestans and the trichomonad Trichomonas vaginalis, but not detectable in plants. This distribution, together with comparative and phylogenetic analyses of Maverick-encoded proteins, is suggestive of an ancient origin of these elements in eukaryotes followed by lineage-specific losses and/or recurrent episodes of horizontal transmission. In addition, we report that Maverick elements have amplified recently to high copy numbers in T. vaginalis where they now occupy as much as 30% of the genome. Sequence analysis confirms that most Mavericks encode a retroviral-like integrase, but lack other open reading frames typically found in retroelements. Nevertheless, the length and conservation of the target site duplication created upon Maverick insertion (5- or 6-bp) is consistent with a role of the integrase-like protein in the integration of a double-stranded DNA transposition intermediate. Mavericks also display long terminal-inverted repeats but do not contain ORFs similar to proteins encoded by DNA transposons. Instead, Mavericks encode a conserved set of 5 to 9 genes (in addition to the integrase) that are predicted to encode proteins with homology to replication and packaging proteins of some bacteriophages and diverse eukaryotic double-stranded DNA viruses, including a DNA polymerase B homolog and putative capsid proteins. Based on these and other structural similarities, we speculate that Mavericks represent an evolutionary missing link between seemingly disparate invasive DNA elements that include bacteriophages, adenoviruses and eukaryotic linear plasmids.

Control of DEMETER DNA demethylase gene transcription in male and female gamete companion cells in Arabidopsis thaliana

PubMed Central

Park, Jin-Sup; Frost, Jennifer M.; Park, Kyunghyuk; Ohr, Hyonhwa; Park, Guen Tae; Kim, Seohyun; Eom, Hyunjoo; Lee, Ilha; Brooks, Janie S.; Fischer, Robert L.; Choi, Yeonhee

2017-01-01

The DEMETER (DME) DNA glycosylase initiates active DNA demethylation via the base-excision repair pathway and is vital for reproduction in Arabidopsis thaliana. DME-mediated DNA demethylation is preferentially targeted to small, AT-rich, and nucleosome-depleted euchromatic transposable elements, influencing expression of adjacent genes and leading to imprinting in the endosperm. In the female gametophyte, DME expression and subsequent genome-wide DNA demethylation are confined to the companion cell of the egg, the central cell. Here, we show that, in the male gametophyte, DME expression is limited to the companion cell of sperm, the vegetative cell, and to a narrow window of time: immediately after separation of the companion cell lineage from the germline. We define transcriptional regulatory elements of DME using reporter genes, showing that a small region, which surprisingly lies within the DME gene, controls its expression in male and female companion cells. DME expression from this minimal promoter is sufficient to rescue seed abortion and the aberrant DNA methylome associated with the null dme-2 mutation. Within this minimal promoter, we found short, conserved enhancer sequences necessary for the transcriptional activities of DME and combined predicted binding motifs with published transcription factor binding coordinates to produce a list of candidate upstream pathway members in the genetic circuitry controlling DNA demethylation in gamete companion cells. These data show how DNA demethylation is regulated to facilitate endosperm gene imprinting and potential transgenerational epigenetic regulation, without subjecting the germline to potentially deleterious transposable element demethylation. PMID:28130550

A methodology to annotate systems biology markup language models with the synthetic biology open language.

PubMed

Roehner, Nicholas; Myers, Chris J

2014-02-21

Recently, we have begun to witness the potential of synthetic biology, noted here in the form of bacteria and yeast that have been genetically engineered to produce biofuels, manufacture drug precursors, and even invade tumor cells. The success of these projects, however, has often failed in translation and application to new projects, a problem exacerbated by a lack of engineering standards that combine descriptions of the structure and function of DNA. To address this need, this paper describes a methodology to connect the systems biology markup language (SBML) to the synthetic biology open language (SBOL), existing standards that describe biochemical models and DNA components, respectively. Our methodology involves first annotating SBML model elements such as species and reactions with SBOL DNA components. A graph is then constructed from the model, with vertices corresponding to elements within the model and edges corresponding to the cause-and-effect relationships between these elements. Lastly, the graph is traversed to assemble the annotating DNA components into a composite DNA component, which is used to annotate the model itself and can be referenced by other composite models and DNA components. In this way, our methodology can be used to build up a hierarchical library of models annotated with DNA components. Such a library is a useful input to any future genetic technology mapping algorithm that would automate the process of composing DNA components to satisfy a behavioral specification. Our methodology for SBML-to-SBOL annotation is implemented in the latest version of our genetic design automation (GDA) software tool, iBioSim.

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach.

PubMed

Frimodt-Møller, Jakob; Charbon, Godefroid; Krogfelt, Karen A; Løbner-Olesen, Anders

2017-09-11

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Novel DNA Motif Binding Activity Observed In Vivo With an Estrogen Receptor α Mutant Mouse

PubMed Central

Li, Leping; Grimm, Sara A.; Winuthayanon, Wipawee; Hamilton, Katherine J.; Pockette, Brianna; Rubel, Cory A.; Pedersen, Lars C.; Fargo, David; Lanz, Rainer B.; DeMayo, Francesco J.; Schütz, Günther; Korach, Kenneth S.

2014-01-01

Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as “tethering.” Evidence for tethering is based on in vitro studies and a widely used “KIKO” mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the “EAAE” ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null–like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo. PMID:24713037

Evolution of double-stranded DNA viruses of eukaryotes: from bacteriophages to transposons to giant viruses

PubMed Central

Koonin, Eugene V; Krupovic, Mart; Yutin, Natalya

2015-01-01

Diverse eukaryotes including animals and protists are hosts to a broad variety of viruses with double-stranded (ds) DNA genomes, from the largest known viruses, such as pandoraviruses and mimiviruses, to tiny polyomaviruses. Recent comparative genomic analyses have revealed many evolutionary connections between dsDNA viruses of eukaryotes, bacteriophages, transposable elements, and linear DNA plasmids. These findings provide an evolutionary scenario that derives several major groups of eukaryotic dsDNA viruses, including the proposed order “Megavirales,” adenoviruses, and virophages from a group of large virus-like transposons known as Polintons (Mavericks). The Polintons have been recently shown to encode two capsid proteins, suggesting that these elements lead a dual lifestyle with both a transposon and a viral phase and should perhaps more appropriately be named polintoviruses. Here, we describe the recently identified evolutionary relationships between bacteriophages of the family Tectiviridae, polintoviruses, adenoviruses, virophages, large and giant DNA viruses of eukaryotes of the proposed order “Megavirales,” and linear mitochondrial and cytoplasmic plasmids. We outline an evolutionary scenario under which the polintoviruses were the first group of eukaryotic dsDNA viruses that evolved from bacteriophages and became the ancestors of most large DNA viruses of eukaryotes and a variety of other selfish elements. Distinct lines of origin are detectable only for herpesviruses (from a different bacteriophage root) and polyoma/papillomaviruses (from single-stranded DNA viruses and ultimately from plasmids). Phylogenomic analysis of giant viruses provides compelling evidence of their independent origins from smaller members of the putative order “Megavirales,” refuting the speculations on the evolution of these viruses from an extinct fourth domain of cellular life. PMID:25727355

Cryptic MCAT enhancer regulation in fibroblasts and smooth muscle cells. Suppression of TEF-1 mediated activation by the single-stranded DNA-binding proteins, Pur alpha, Pur beta, and MSY1.

PubMed

Carlini, Leslie E; Getz, Michael J; Strauch, Arthur R; Kelm, Robert J

2002-03-08

An asymmetric polypurine-polypyrimidine cis-element located in the 5' region of the mouse vascular smooth muscle alpha-actin gene serves as a binding site for multiple proteins with specific affinity for either single- or double-stranded DNA. Here, we test the hypothesis that single-stranded DNA-binding proteins are responsible for preventing a cryptic MCAT enhancer centered within this element from cooperating with a nearby serum response factor-interacting CArG motif to trans-activate the minimal promoter in fibroblasts and smooth muscle cells. DNA binding studies revealed that the core MCAT sequence mediates binding of transcription enhancer factor-1 to the double-stranded polypurine-polypyrimidine element while flanking nucleotides account for interaction of Pur alpha and Pur beta with the purine-rich strand and MSY1 with the complementary pyrimidine-rich strand. Mutations that selectively impaired high affinity single-stranded DNA binding by fibroblast or smooth muscle cell-derived Pur alpha, Pur beta, and MSY1 in vitro, released the cryptic MCAT enhancer from repression in transfected cells. Additional experiments indicated that Pur alpha, Pur beta, and MSY1 also interact specifically, albeit weakly, with double-stranded DNA and with transcription enhancer factor-1. These results are consistent with two plausible models of cryptic MCAT enhancer regulation by Pur alpha, Pur beta, and MSY1 involving either competitive single-stranded DNA binding or masking of MCAT-bound transcription enhancer factor-1.

A User's Guide to the Encyclopedia of DNA Elements (ENCODE)

PubMed Central

2011-01-01

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome. PMID:21526222

In vivo and in vitro characterization of σ70 constitutive promoters by real-time PCR and fluorescent measurements.

PubMed

Chappell, James; Freemont, Paul

2013-01-01

The characterization of DNA regulatory elements such as ribosome binding sites and transcriptional promoters is a fundamental aim of synthetic biology. Characterization of such DNA regulatory elements by monitoring the synthesis of fluorescent proteins is a commonly used technique to resolve the relative or absolute strengths. These measurements can be used in combination with mathematical models and computer simulation to rapidly assess performance of DNA regulatory elements both in isolation and in combination, to assist predictable and efficient engineering of complex novel biological devices and systems. Here we describe the construction and relative characterization of Escherichia coli (E. coli) σ(70) transcriptional promoters by monitoring the synthesis of green fluorescent protein (GFP) both in vivo in E. coli and in vitro in a E. coli cell-free transcription and translation reaction.

Genome-wide DNA methylation patterns in LSH mutant reveals de-repression of repeat elements and redundant epigenetic silencing pathways

PubMed Central

Yu, Weishi; McIntosh, Carl; Lister, Ryan; Zhu, Iris; Han, Yixing; Ren, Jianke; Landsman, David; Lee, Eunice; Briones, Victorino; Terashima, Minoru; Leighty, Robert; Ecker, Joseph R.

2014-01-01

Cytosine methylation is critical in mammalian development and plays a role in diverse biologic processes such as genomic imprinting, X chromosome inactivation, and silencing of repeat elements. Several factors regulate DNA methylation in early embryogenesis, but their precise role in the establishment of DNA methylation at a given site remains unclear. We have generated a comprehensive methylation map in fibroblasts derived from the murine DNA methylation mutant Hells−/− (helicase, lymphoid specific, also known as LSH). It has been previously shown that HELLS can influence de novo methylation of retroviral sequences and endogenous genes. Here, we describe that HELLS controls cytosine methylation in a nuclear compartment that is in part defined by lamin B1 attachment regions. Despite widespread loss of cytosine methylation at regulatory sequences, including promoter regions of protein-coding genes and noncoding RNA genes, overall relative transcript abundance levels in the absence of HELLS are similar to those in wild-type cells. A subset of promoter regions shows increases of the histone modification H3K27me3, suggesting redundancy of epigenetic silencing mechanisms. Furthermore, HELLS modulates CG methylation at all classes of repeat elements and is critical for repression of a subset of repeat elements. Overall, we provide a detailed analysis of gene expression changes in relation to DNA methylation alterations, which contributes to our understanding of the biological role of cytosine methylation. PMID:25170028

Repeated administration of CGP 46381, a gamma-aminobutyric acidB antagonist, and ethosuximide suppresses seizure-associated cyclic adenosine 3'5' monophosphate response element- and activator protein-1 DNA-binding activities in lethargic (lh/lh) mice.

PubMed

Ishige, K; Endo, H; Saito, H; Ito, Y

2001-01-19

To characterize seizure-associated increases in cerebral cortical and thalamic cyclic AMP responsive element (CRE)- and activator protein 1 (AP-1) DNA-binding activities in lethargic (lh/lh) mice, a genetic model of absence seizures, we examined the effects of ethosuximide and CGP 46381 on these DNA-binding activities. Repeated administration (twice a day for 5 days) of ethosuximide (200 mg/kg) or CGP 46381 (60 mg/kg) attenuated both seizure behavior and the increased DNA-binding activities, and was more effective than a single administration of these drugs. These treatments did not affect either normal behavior or basal DNA-binding activities in non-epileptic control (+/+) mice. Gel supershift assays revealed that the increased CRE-binding activity was attributable to activation of the binding activity of CREB, and that the c-Fos-c-Jun complex was a component of the increased AP-1 DNA-binding activity.

Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements.

PubMed

Guérin, Frédéric; Arnaiz, Olivier; Boggetto, Nicole; Denby Wilkes, Cyril; Meyer, Eric; Sperling, Linda; Duharcourt, Sandra

2017-04-26

DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome. We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.

What triggers differential DNA methylation of genes and TEs: contribution of body methylation?

PubMed

Inagaki, S; Kakutani, T

2012-01-01

Transposable elements (TEs) are epigenetically silenced with extensive DNA methylation. The silent epigenetic marks should, however, be excluded from active genes. By genetic approaches, we study mechanisms to remove the heterochromatin marks from transcribed genes. Based on our observations on control of TE transcription, we propose a possible trigger for the TE-specific accumulation of DNA methylation. A critical difference between TEs and genes could be their responses to the DNA methylation in the internal part of transcribed regions. When their internal region is methylated, genes are still transcribed, but TEs could be silenced, which may reflect the obligatory position of every critical cis-acting element within the TE itself. This initial difference of TEs and genes will be amplified by positive feedback loops to stabilize active or silent states. Thus, the mechanisms to accumulate heterochromatin marks within transcribed regions could provide a trigger to induce differential DNA methylation between genes and TEs.

pH-Driven Reversible Self-Assembly of Micron-Scale DNA Scaffolds.

PubMed

Green, Leopold N; Amodio, Alessia; Subramanian, Hari K K; Ricci, Francesco; Franco, Elisa

2017-12-13

Inspired by cytoskeletal scaffolds that sense and respond dynamically to environmental changes and chemical inputs with a unique capacity for reconfiguration, we propose a strategy that allows the dynamic and reversible control of the growth and breakage of micron-scale synthetic DNA structures upon pH changes. We do so by rationally designing a pH-responsive system composed of synthetic DNA strands that act as pH sensors, regulators, and structural elements. Sensor strands can dynamically respond to pH changes and route regulatory strands to direct the self-assembly of structural elements into tubular structures. This example represents the first demonstration of the reversible assembly and disassembly of micron-scale DNA scaffolds using an external chemical input other than DNA. The capacity to reversibly modulate nanostructure size may promote the development of smart devices for catalysis or drug-delivery applications.

In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax.

PubMed

Paca-Uccaralertkun, S; Zhao, L J; Adya, N; Cross, J V; Cullen, B R; Boros, I M; Giam, C Z

1994-01-01

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB. In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G- or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G- and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.

DNA is structured as a linear "jigsaw puzzle" in the genomes of Arabidopsis, rice, and budding yeast.

PubMed

Liu, Yun-Hua; Zhang, Meiping; Wu, Chengcang; Huang, James J; Zhang, Hong-Bin

2014-01-01

Knowledge of how a genome is structured and organized from its constituent elements is crucial to understanding its biology and evolution. Here, we report the genome structuring and organization pattern as revealed by systems analysis of the sequences of three model species, Arabidopsis, rice and yeast, at the whole-genome and chromosome levels. We found that all fundamental function elements (FFE) constituting the genomes, including genes (GEN), DNA transposable elements (DTE), retrotransposable elements (RTE), simple sequence repeats (SSR), and (or) low complexity repeats (LCR), are structured in a nonrandom and correlative manner, thus leading to a hypothesis that the DNA of the species is structured as a linear "jigsaw puzzle". Furthermore, we showed that different FFE differ in their importance in the formation and evolution of the DNA jigsaw puzzle structure between species. DTE and RTE play more important roles than GEN, LCR, and SSR in Arabidopsis, whereas GEN and RTE play more important roles than LCR, SSR, and DTE in rice. The genes having multiple recognized functions play more important roles than those having single functions. These results provide useful knowledge necessary for better understanding genome biology and evolution of the species and for effective molecular breeding of rice.

[Genome-scale sequence data processing and epigenetic analysis of DNA methylation].

PubMed

Wang, Ting-Zhang; Shan, Gao; Xu, Jian-Hong; Xue, Qing-Zhong

2013-06-01

A new approach recently developed for detecting cytosine DNA methylation (mC) and analyzing the genome-scale DNA methylation profiling, is called BS-Seq which is based on bisulfite conversion of genomic DNA combined with next-generation sequencing. The method can not only provide an insight into the difference of genome-scale DNA methylation among different organisms, but also reveal the conservation of DNA methylation in all contexts and nucleotide preference for different genomic regions, including genes, exons, and repetitive DNA sequences. It will be helpful to under-stand the epigenetic impacts of cytosine DNA methylation on the regulation of gene expression and maintaining silence of repetitive sequences, such as transposable elements. In this paper, we introduce the preprocessing steps of DNA methylation data, by which cytosine (C) and guanine (G) in the reference sequence are transferred to thymine (T) and adenine (A), and cytosine in reads is transferred to thymine, respectively. We also comprehensively review the main content of the DNA methylation analysis on the genomic scale: (1) the cytosine methylation under the context of different sequences; (2) the distribution of genomic methylcytosine; (3) DNA methylation context and the preference for the nucleotides; (4) DNA- protein interaction sites of DNA methylation; (5) degree of methylation of cytosine in the different structural elements of genes. DNA methylation analysis technique provides a powerful tool for the epigenome study in human and other species, and genes and environment interaction, and founds the theoretical basis for further development of disease diagnostics and therapeutics in human.

DNA topoisomerase 1α promotes transcriptional silencing of transposable elements through DNA methylation and histone lysine 9 dimethylation in Arabidopsis.

PubMed

Dinh, Thanh Theresa; Gao, Lei; Liu, Xigang; Li, Dongming; Li, Shengben; Zhao, Yuanyuan; O'Leary, Michael; Le, Brandon; Schmitz, Robert J; Manavella, Pablo A; Manavella, Pablo; Li, Shaofang; Weigel, Detlef; Pontes, Olga; Ecker, Joseph R; Chen, Xuemei

2014-07-01

RNA-directed DNA methylation (RdDM) and histone H3 lysine 9 dimethylation (H3K9me2) are related transcriptional silencing mechanisms that target transposable elements (TEs) and repeats to maintain genome stability in plants. RdDM is mediated by small and long noncoding RNAs produced by the plant-specific RNA polymerases Pol IV and Pol V, respectively. Through a chemical genetics screen with a luciferase-based DNA methylation reporter, LUCL, we found that camptothecin, a compound with anti-cancer properties that targets DNA topoisomerase 1α (TOP1α) was able to de-repress LUCL by reducing its DNA methylation and H3K9me2 levels. Further studies with Arabidopsis top1α mutants showed that TOP1α silences endogenous RdDM loci by facilitating the production of Pol V-dependent long non-coding RNAs, AGONAUTE4 recruitment and H3K9me2 deposition at TEs and repeats. This study assigned a new role in epigenetic silencing to an enzyme that affects DNA topology.

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.

PubMed

Guo, Tai Wei; Bartesaghi, Alberto; Yang, Hui; Falconieri, Veronica; Rao, Prashant; Merk, Alan; Eng, Edward T; Raczkowski, Ashleigh M; Fox, Tara; Earl, Lesley A; Patel, Dinshaw J; Subramaniam, Sriram

2017-10-05

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition. Published by Elsevier Inc.

Dead Element Replicating: Degenerate R2 Element Replication and rDNA Genomic Turnover in the Bacillus rossius Stick Insect (Insecta: Phasmida)

PubMed Central

Martoni, Francesco; Eickbush, Danna G.; Scavariello, Claudia; Luchetti, Andrea; Mantovani, Barbara

2015-01-01

R2 is an extensively investigated non-LTR retrotransposon that specifically inserts into the 28S rRNA gene sequences of a wide range of metazoans, disrupting its functionality. During R2 integration, first strand synthesis can be incomplete so that 5’ end deleted copies are occasionally inserted. While active R2 copies repopulate the locus by retrotransposing, the non-functional truncated elements should frequently be eliminated by molecular drive processes leading to the concerted evolution of the rDNA array(s). Although, multiple R2 lineages have been discovered in the genome of many animals, the rDNA of the stick insect Bacillus rossius exhibits a peculiar situation: it harbors both a canonical, functional R2 element (R2Brfun) as well as a full-length but degenerate element (R2Brdeg). An intensive sequencing survey in the present study reveals that all truncated variants in stick insects are present in multiple copies suggesting they were duplicated by unequal recombination. Sequencing results also demonstrate that all R2Brdeg copies are full-length, i. e. they have no associated 5' end deletions, and functional assays indicate they have lost the active ribozyme necessary for R2 RNA maturation. Although it cannot be completely ruled out, it seems unlikely that the degenerate elements replicate via reverse transcription, exploiting the R2Brfun element enzymatic machinery, but rather via genomic amplification of inserted 28S by unequal recombination. That inactive copies (both R2Brdeg or 5'-truncated elements) are not eliminated in a short term in stick insects contrasts with findings for the Drosophila R2, suggesting a widely different management of rDNA loci and a lower efficiency of the molecular drive while achieving the concerted evolution. PMID:25799008

Development and validation of an integrated DNA walking strategy to detect GMO expressing cry genes.

PubMed

Fraiture, Marie-Alice; Vandamme, Julie; Herman, Philippe; Roosens, Nancy H C

2018-06-27

Recently, an integrated DNA walking strategy has been proposed to prove the presence of GMO via the characterisation of sequences of interest, including their transgene flanking regions and the unnatural associations of elements in their transgenic cassettes. To this end, the p35S, tNOS and t35S pCAMBIA elements have been selected as key targets, allowing the coverage of most of GMO, EU authorized or not. In the present study, a bidirectional DNA walking method anchored on the CryAb/c genes is proposed with the aim to cover additional GMO and additional sequences of interest. The performance of the proposed bidirectional DNA walking method anchored on the CryAb/c genes has been evaluated in a first time for its feasibility using several GM events possessing these CryAb/c genes. Afterwards, its sensitivity has been investigated through low concentrations of targets (as low as 20 HGE). In addition, to illustrate its applicability, the entire workflow has been tested on a sample mimicking food/feed matrices analysed in GMO routine analysis. Given the successful assessment of its performance, the present bidirectional DNA walking method anchored on the CryAb/c genes can easily be implemented in GMO routine analysis by the enforcement laboratories and allows completing the entire DNA walking strategy in targeting an additional transgenic element frequently found in GMO.

Ectopic recombination between Ty elements in Saccharomyces cerevisiae is not induced by DNA damage.

PubMed

Parket, A; Kupiec, M

1992-10-01

Mitotic recombination is increased when cells are treated with a variety of physical and chemical agents that cause damage to their DNA. We show here, using Saccharomyces cerevisiae strains that carry marked Ty elements, that recombination between members of this family of retrotransposons is not increased by UV irradiation or by treatment with the radiomimetic drug methyl methanesulfonate. Both ectopic recombination and mutation events were elevated by these agents for non-Ty sequences in the same strain. We discuss possible mechanisms that can prevent the induction of recombination between Ty elements.

DNA transposons have colonized the genome of the giant virus Pandoravirus salinus.

PubMed

Sun, Cheng; Feschotte, Cédric; Wu, Zhiqiang; Mueller, Rachel Lockridge

2015-06-12

Transposable elements are mobile DNA sequences that are widely distributed in prokaryotic and eukaryotic genomes, where they represent a major force in genome evolution. However, transposable elements have rarely been documented in viruses, and their contribution to viral genome evolution remains largely unexplored. Pandoraviruses are recently described DNA viruses with genome sizes that exceed those of some prokaryotes, rivaling parasitic eukaryotes. These large genomes appear to include substantial noncoding intergenic spaces, which provide potential locations for transposable element insertions. However, no mobile genetic elements have yet been reported in pandoravirus genomes. Here, we report a family of miniature inverted-repeat transposable elements (MITEs) in the Pandoravirus salinus genome, representing the first description of a virus populated with a canonical transposable element family that proliferated by transposition within the viral genome. The MITE family, which we name Submariner, includes 30 copies with all the hallmarks of MITEs: short length, terminal inverted repeats, TA target site duplication, and no coding capacity. Submariner elements show signs of transposition and are undetectable in the genome of Pandoravirus dulcis, the closest known relative Pandoravirus salinus. We identified a DNA transposon related to Submariner in the genome of Acanthamoeba castellanii, a species thought to host pandoraviruses, which contains remnants of coding sequence for a Tc1/mariner transposase. These observations suggest that the Submariner MITEs of P. salinus belong to the widespread Tc1/mariner superfamily and may have been mobilized by an amoebozoan host. Ten of the 30 MITEs in the P. salinus genome are located within coding regions of predicted genes, while others are close to genes, suggesting that these transposons may have contributed to viral genetic novelty. Our discovery highlights the remarkable ability of DNA transposons to colonize and shape genomes from all domains of life, as well as giant viruses. Our findings continue to blur the division between viral and cellular genomes, adhering to the emerging view that the content, dynamics, and evolution of the genomes of giant viruses do not substantially differ from those of cellular organisms.

The site-specific ribosomal DNA insertion element R1Bm belongs to a class of non-long-terminal-repeat retrotransposons.

PubMed Central

Xiong, Y; Eickbush, T H

1988-01-01

Two types of insertion elements, R1 and R2 (previously called type I and type II), are known to interrupt the 28S ribosomal genes of several insect species. In the silkmoth, Bombyx mori, each element occupies approximately 10% of the estimated 240 ribosomal DNA units, while at most only a few copies are located outside the ribosomal DNA units. We present here the complete nucleotide sequence of an R1 insertion from B. mori (R1Bm). This 5.1-kilobase element contains two overlapping open reading frames (ORFs) which together occupy 88% of its length. ORF1 is 461 amino acids in length and exhibits characteristics of retroviral gag genes. ORF2 is 1,051 amino acids in length and contains homology to reverse transcriptase-like enzymes. The analysis of 3' and 5' ends of independent isolates from the ribosomal locus supports the suggestion that R1 is still functioning as a transposable element. The precise location of the element within the genome implies that its transposition must occur with remarkable insertion sequence specificity. Comparison of the deduced amino acid sequences from six retrotransposons, R1 and R2 of B. mori, I factor and F element of Drosophila melanogaster, L1 of Mus domesticus, and Ingi of Trypanosoma brucei, reveals a relatively high level of sequence homology in the reverse transcriptase region. Like R1, these elements lack long terminal repeats. We have therefore named this class of related elements the non-long-terminal-repeat (non-LTR) retrotransposons. Images PMID:2447482

Flavonoids and DNA Repair in Prostate Cancer

DTIC Science & Technology

2005-12-01

1-0114 TITLE: Flavonoids and DNA Repair in Prostate Cancer...SUBTITLE Flavonoids and DNA Repair in Prostate Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-04-1-0114 5c. PROGRAM ELEMENT NUMBER...test the hypothesis that natural products such as flavonoids are able to stimulate the repair of oxidative DNA damage. For this purpose LNCaP

AP1 Keeps Chromatin Poised for Action | Center for Cancer Research

Cancer.gov

The human genome harbors gene-encoding DNA, the blueprint for building proteins that regulate cellular function. Embedded across the genome, in non-coding regions, are DNA elements to which regulatory factors bind. The interaction of regulatory factors with DNA at these sites modifies gene expression to modulate cell activity. In cells, DNA exists in a complex with proteins

A cDNA from a mouse pancreatic beta cell encoding a putative transcription factor of the insulin gene.

PubMed Central

Walker, M D; Park, C W; Rosen, A; Aronheim, A

1990-01-01

Cell specific expression of the insulin gene is achieved through transcriptional mechanisms operating on multiple DNA sequence elements located in the 5' flanking region of the gene. Of particular importance in the rat insulin I gene are two closely similar 9 bp sequences (IEB1 and IEB2): mutation of either of these leads to 5-10 fold reduction in transcriptional activity. We have screened an expression cDNA library derived from mouse pancreatic endocrine beta cells with a radioactive DNA probe containing multiple copies of the IEB1 sequence. A cDNA clone (A1) isolated by this procedure encodes a protein which shows efficient binding to the IEB1 probe, but much weaker binding to either an unrelated DNA probe or to a probe bearing a single base pair insertion within the recognition sequence. DNA sequence analysis indicates a protein belonging to the helix-loop-helix family of DNA-binding proteins. The ability of the protein encoded by clone A1 to recognize a number of wild type and mutant DNA sequences correlates closely with the ability of each sequence element to support transcription in vivo in the context of the insulin 5' flanking DNA. We conclude that the isolated cDNA may encode a transcription factor that participates in control of insulin gene expression. Images PMID:2181401

Characterization of noncoding regulatory DNA in the human genome.

PubMed

Elkon, Ran; Agami, Reuven

2017-08-08

Genetic variants associated with common diseases are usually located in noncoding parts of the human genome. Delineation of the full repertoire of functional noncoding elements, together with efficient methods for probing their biological roles, is therefore of crucial importance. Over the past decade, DNA accessibility and various epigenetic modifications have been associated with regulatory functions. Mapping these features across the genome has enabled researchers to begin to document the full complement of putative regulatory elements. High-throughput reporter assays to probe the functions of regulatory regions have also been developed but these methods separate putative regulatory elements from the chromosome so that any effects of chromatin context and long-range regulatory interactions are lost. Definitive assignment of function(s) to putative cis-regulatory elements requires perturbation of these elements. Genome-editing technologies are now transforming our ability to perturb regulatory elements across entire genomes. Interpretation of high-throughput genetic screens that incorporate genome editors might enable the construction of an unbiased map of functional noncoding elements in the human genome.

The Human L1 Element Causes DNA Double-Strand Breaks in Breast Cancer

DTIC Science & Technology

2006-08-01

cancer is complex. However, defects in DNA repair genes in the double-strand break repair pathway are cancer predisposing. My lab has characterized...a new potentially important source of double-strand breaks (DSBs) in human cells and are interested in characterizing which DNA repair genes act on...this particular source of DNA damage. Selfish DNA accounts for 45% of the human genome. We have recently demonstrated that one particular selfish

[Molecular-genetic analysis of wheat (T. aestivum L.) genome with introgression of Ae. cylindrica Host genetic elements].

PubMed

Galaev, A V; Sivolap, Iu M

2005-01-01

Wheat-aegilops hybrid plants Triticum aestivum L. (2n = 42) x Aegilops cylindrica Host (2n = 28) were investigated with using microsatellite markers. In two BC1F9 lines some genome modifications connected with losing DNA fragments of initial variety or appearing of Aegilops genome elements were detected. In some investigated hybrids new amplicons lacking in parental plants were found. Substitution of wheat chromosomes for aegilops chromosomes was not revealed. Analysis of microsatellite loci in BC2F5 plants showed stable introgression of aegilops genetic elements into wheat; elimination of some transferred aegilops DNA fragments in the course of backcrossing; decreasing size of introgressive elements after backcrossing. Introgressive lines were classified according to genome changes.

Altruistic functions for selfish DNA.

PubMed

Faulkner, Geoffrey J; Carninci, Piero

2009-09-15

Mammalian genomes are comprised of 30-50% transposed elements (TEs). The vast majority of these TEs are truncated and mutated fragments of retrotransposons that are no longer capable of transposition. Although initially regarded as important factors in the evolution of gene regulatory networks, TEs are now commonly perceived as neutrally evolving and non-functional genomic elements. In a major development, recent works have strongly contradicted this "selfish DNA" or "junk DNA" dogma by demonstrating that TEs use a host of novel promoters to generate RNA on a massive scale across most eukaryotic cells. This transcription frequently functions to control the expression of protein-coding genes via alternative promoters, cis regulatory non protein-coding RNAs and the formation of double stranded short RNAs. If considered in sum, these findings challenge the designation of TEs as selfish and neutrally evolving genomic elements. Here, we will expand upon these themes and discuss challenges in establishing novel TE functions in vivo.

Specificity determinants for the abscisic acid response element.

PubMed

Sarkar, Aditya Kumar; Lahiri, Ansuman

2013-01-01

Abscisic acid (ABA) response elements (ABREs) are a group of cis-acting DNA elements that have been identified from promoter analysis of many ABA-regulated genes in plants. We are interested in understanding the mechanism of binding specificity between ABREs and a class of bZIP transcription factors known as ABRE binding factors (ABFs). In this work, we have modeled the homodimeric structure of the bZIP domain of ABRE binding factor 1 from Arabidopsis thaliana (AtABF1) and studied its interaction with ACGT core motif-containing ABRE sequences. We have also examined the variation in the stability of the protein-DNA complex upon mutating ABRE sequences using the protein design algorithm FoldX. The high throughput free energy calculations successfully predicted the ability of ABF1 to bind to alternative core motifs like GCGT or AAGT and also rationalized the role of the flanking sequences in determining the specificity of the protein-DNA interaction.

The MluI cell cycle box (MCB) motifs, but not damage-responsive elements (DREs), are responsible for the transcriptional induction of the rhp51+ gene in response to DNA replication stress.

PubMed

Sartagul, Wugangerile; Zhou, Xin; Yamada, Yuki; Ma, Ning; Tanaka, Katsunori; Furuyashiki, Tomoyuki; Ma, Yan

2014-01-01

DNA replication stress induces the transcriptional activation of rhp51+, a fission yeast recA homolog required for repair of DNA double strand breaks. However, the mechanism by which DNA replication stress activates rhp51+ transcription is not understood. The promoter region of rhp51+ contains two damage-responsive elements (DREs) and two MluI cell cycle box (MCB) motifs. Using luciferase reporter assays, we examined the role of these elements in rhp51+ transcription. The full-length rhp51+ promoter and a promoter fragment containing MCB motifs only, but not a fragment containing DREs, mediated transcriptional activation upon DNA replication stress. Removal of the MCB motifs from the rhp51+ promoter abolished the induction of rhp51+ transcription by DNA replication stress. Consistent with a role for MCB motifs in rhp51+ transcription activation, deletion of the MBF (MCB-binding factor) co-repressors Nrm1 and Yox1 precluded rhp51+ transcriptional induction in response to DNA replication stress. Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51+ transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways. Because MBF is critical for G1/S transcription, we examined how the cell cycle affected rhp51+ transcription. The transcription of rhp51+ and cdc18+, an MBF-dependent G1/S gene, peaked simultaneously in synchronized cdc25-22 cells. Furthermore, DNA replication stress maintained transcription of rhp51+ similarly to cdc18+. Collectively, these results suggest that MBF and its regulators mediate rhp51+ transcription in response to DNA replication stress, and underlie rhp51+ transcription at the G1/S transition.

Effects of Particulate Matter on Genomic DNA Methylation Content and iNOS Promoter Methylation

PubMed Central

Tarantini, Letizia; Bonzini, Matteo; Apostoli, Pietro; Pegoraro, Valeria; Bollati, Valentina; Marinelli, Barbara; Cantone, Laura; Rizzo, Giovanna; Hou, Lifang; Schwartz, Joel; Bertazzi, Pier Alberto; Baccarelli, Andrea

2009-01-01

Background Altered patterns of gene expression mediate the effects of particulate matter (PM) on human health, but mechanisms through which PM modifies gene expression are largely undetermined. Objectives We aimed at identifying short- and long-term effects of PM exposure on DNA methylation, a major genomic mechanism of gene expression control, in workers in an electric furnace steel plant with well-characterized exposure to PM with aerodynamic diameters < 10 μm (PM10). Methods We measured global genomic DNA methylation content estimated in Alu and long interspersed nuclear element-1 (LINE-1) repeated elements, and promoter DNA methylation of iNOS (inducible nitric oxide synthase), a gene suppressed by DNA methylation and induced by PM exposure in blood leukocytes. Quantitative DNA methylation analysis was performed through bisulfite PCR pyrosequencing on blood DNA obtained from 63 workers on the first day of a work week (baseline, after 2 days off work) and after 3 days of work (postexposure). Individual PM10 exposure was between 73.4 and 1,220 μg/m3. Results Global methylation content estimated in Alu and LINE-1 repeated elements did not show changes in postexposure measures compared with baseline. PM10 exposure levels were negatively associated with methylation in both Alu [β = −0.19 %5-methylcytosine (%5mC); p = 0.04] and LINE-1 [β = −0.34 %5mC; p = 0.04], likely reflecting long-term PM10 effects. iNOS promoter DNA methylation was significantly lower in postexposure blood samples compared with baseline (difference = −0.61 %5mC; p = 0.02). Conclusions We observed changes in global and gene specific methylation that should be further characterized in future investigations on the effects of PM. PMID:19270791

Thermodynamics of complex structures formed between single-stranded DNA oligomers and the KH domains of the far upstream element binding protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakraborty, Kaushik; Sinha, Sudipta Kumar; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in

The noncovalent interaction between protein and DNA is responsible for regulating the genetic activities in living organisms. The most critical issue in this problem is to understand the underlying driving force for the formation and stability of the complex. To address this issue, we have performed atomistic molecular dynamics simulations of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein (FBP) complexed with two single-stranded DNA (ss-DNA) oligomers in aqueous media. Attempts have been made to calculate the individual components of the net entropy change for the complexation process by adopting suitablemore » statistical mechanical approaches. Our calculations reveal that translational, rotational, and configurational entropy changes of the protein and the DNA components have unfavourable contributions for this protein-DNA association process and such entropy lost is compensated by the entropy gained due to the release of hydration layer water molecules. The free energy change corresponding to the association process has also been calculated using the Free Energy Perturbation (FEP) method. The free energy gain associated with the KH4–DNA complex formation has been found to be noticeably higher than that involving the formation of the KH3–DNA complex.« less

Transposon-like properties of the major, long repetitive sequence family in the genome of Physarum polycephalum

PubMed Central

Pearston, Douglas H.; Gordon, Mairi; Hardman, Norman

1985-01-01

A family of long, highly-repetitive sequences, referred to previously as `HpaII-repeats', dominates the genome of the eukaryotic slime mould Physarum polycephalum. These sequences are found exclusively in scrambled clusters. They account for about one-half of the total complement of repetitive DNA in Physarum, and represent the major sequence component found in hypermethylated, 20-50 kb segments of Physarum genomic DNA that fail to be cleaved using the restriction endonuclease HpaII. The structure of this abundant repetitive element was investigated by analysing cloned segments derived from the hypermethylated genomic DNA compartment. We show that the `HpaII-repeat' forms part of a larger repetitive DNA structure, ∼8.6 kb in length, with several structural features in common with recognised eukaryotic transposable genetic elements. Scrambled clusters of the sequence probably arise as a result of transposition-like events, during which the element preferentially recombines in either orientation with target sites located in other copies of the same repeated sequence. The target sites for transposition/recombination are not related in sequence but in all cases studied they are potentially capable of promoting the formation of small `cruciforms' or `Z-DNA' structures which might be recognised during the recombination process. ImagesFig. 3.Fig. 4. PMID:16453652

A model for genesis of transcription systems.

PubMed

Burton, Zachary F; Opron, Kristopher; Wei, Guowei; Geiger, James H

2016-01-01

Repeating sequences generated from RNA gene fusions/ligations dominate ancient life, indicating central importance of building structural complexity in evolving biological systems. A simple and coherent story of life on earth is told from tracking repeating motifs that generate α/β proteins, 2-double-Ψ-β-barrel (DPBB) type RNA polymerases (RNAPs), general transcription factors (GTFs), and promoters. A general rule that emerges is that biological complexity that arises through generation of repeats is often bounded by solubility and closure (i.e., to form a pseudo-dimer or a barrel). Because the first DNA genomes were replicated by DNA template-dependent RNA synthesis followed by RNA template-dependent DNA synthesis via reverse transcriptase, the first DNA replication origins were initially 2-DPBB type RNAP promoters. A simplifying model for evolution of promoters/replication origins via repetition of core promoter elements is proposed. The model can explain why Pribnow boxes in bacterial transcription (i.e., (-12)TATAATG(-6)) so closely resemble TATA boxes (i.e., (-31)TATAAAAG(-24)) in archaeal/eukaryotic transcription. The evolution of anchor DNA sequences in bacterial (i.e., (-35)TTGACA(-30)) and archaeal (BRE(up); BRE for TFB recognition element) promoters is potentially explained. The evolution of BRE(down) elements of archaeal promoters is potentially explained.

The Autographa californica Multiple Nucleopolyhedrovirus ac83 Gene Contains a cis-Acting Element That Is Essential for Nucleocapsid Assembly.

PubMed

Huang, Zhihong; Pan, Mengjia; Zhu, Silei; Zhang, Hao; Wu, Wenbi; Yuan, Meijin; Yang, Kai

2017-03-01

Baculoviridae is a family of insect-specific viruses that have a circular double-stranded DNA genome packaged within a rod-shaped capsid. The mechanism of baculovirus nucleocapsid assembly remains unclear. Previous studies have shown that deletion of the ac83 gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) blocks viral nucleocapsid assembly. Interestingly, the ac83 -encoded protein Ac83 is not a component of the nucleocapsid, implying a particular role for ac83 in nucleocapsid assembly that may be independent of its protein product. To examine this possibility, Ac83 synthesis was disrupted by insertion of a chloramphenicol resistance gene into its coding sequence or by deleting its promoter and translation start codon. Both mutants produced progeny viruses normally, indicating that the Ac83 protein is not required for nucleocapsid assembly. Subsequently, complementation assays showed that the production of progeny viruses required the presence of ac83 in the AcMNPV genome instead of its presence in trans Therefore, we reasoned that ac83 is involved in nucleocapsid assembly via an internal cis -acting element, which we named the nucleocapsid assembly-essential element (NAE). The NAE was identified to lie within nucleotides 1651 to 1850 of ac83 and had 8 conserved A/T-rich regions. Sequences homologous to the NAE were found only in alphabaculoviruses and have a conserved positional relationship with another essential cis -acting element that was recently identified. The identification of the NAE may help to connect the data of viral cis -acting elements and related proteins in the baculovirus nucleocapsid assembly, which is important for elucidating DNA-protein interaction events during this process. IMPORTANCE Virus nucleocapsid assembly usually requires specific cis -acting elements in the viral genome for various processes, such as the selection of the viral genome from the cellular nucleic acids, the cleavage of concatemeric viral genome replication intermediates, and the encapsidation of the viral genome into procapsids. In linear DNA viruses, such elements generally locate at the ends of the viral genome; however, most of these elements remain unidentified in circular DNA viruses (including baculovirus) due to their circular genomic conformation. Here, we identified a nucleocapsid assembly-essential element in the AcMNPV (the archetype of baculovirus) genome. This finding provides an important reference for studies of nucleocapsid assembly-related elements in baculoviruses and other circular DNA viruses. Moreover, as most of the previous studies of baculovirus nucleocapsid assembly have been focused on viral proteins, our study provides a novel entry point to investigate this mechanism via cis -acting elements in the viral genome. Copyright © 2017 American Society for Microbiology.

As solid as a rock-comparison of CE- and MPS-based analyses of the petrosal bone as a source of DNA for forensic identification of challenging cranial bones.

PubMed

Kulstein, Galina; Hadrys, Thorsten; Wiegand, Peter

2018-01-01

Short tandem repeat (STR) typing from skeletal remains can be a difficult task. Dependent on the environmental conditions of the provenance of the bones, DNA can be degraded and STR typing inhibited. Generally, dense and compact bones are known to preserve DNA better. Several studies already proved that femora and teeth have high DNA typing success rates. Unfortunately, these elements are not present in all cases involving skeletal remains. Processing partial or singular skeletal elements, it is favorable to select bone areas where DNA preservation is comparably higher. Especially, cranial bones are often accidentally discovered during criminal investigations. The cranial bone is composed of multiple parts. In this examination, we evaluated the potential of the petrous bone for human identification of skeletal remains in forensic case work. Material from different sections of eight unknown cranial bones and-where available-additionally other skeletal elements, collected at the DNA department of the Institute of Legal Medicine in Ulm, Germany, from 2010 to 2017, were processed with an optimized DNA extraction and STR typing strategy. The results highlight that STR typing from the petrous bones leads to reportable profiles in all individuals, even in cases where the analysis of the parietal bone failed. Moreover, the comparison of capillary electrophorese (CE) typing to massively parallel sequencing (MPS) analysis shows that MPS has the potential to analyze degraded human remains and is even capable to provide additional information about phenotype and ancestry of unknown individuals.

Germ line insertion of mtDNA at the breakpoint junction of a reciprocal constitutional translocation.

PubMed

Willett-Brozick, J E; Savul, S A; Richey, L E; Baysal, B E

2001-08-01

Constitutional chromosomal translocations are relatively common causes of human morbidity, yet the DNA double-strand break (DSB) repair mechanisms that generate them are incompletely understood. We cloned, sequenced and analyzed the breakpoint junctions of a familial constitutional reciprocal translocation t(9;11)(p24;q23). Within the 10-kb region flanking the breakpoints, chromosome 11 had 25% repeat elements, whereas chromosome 9 had 98% repeats, 95% of which were L1-type LINE elements. The breakpoints occurred within an L1-type repeat element at 9p24 and at the 3'-end of an Alu sequence at 11q23. At the breakpoint junction of derivative chromosome 9, we discovered an unusually large 41-bp insertion, which showed 100% identity to 12S mitochondrial DNA (mtDNA) between nucleotides 896 and 936 of the mtDNA sequence. Analysis of the human genome failed to show the preexistence of the inserted sequence at normal chromosomes 9 and 11 breakpoint junctions or elsewhere in the genome, strongly suggesting that the insertion was derived from human mtDNA and captured into the junction during the DSB repair process. To our knowledge, these findings represent the first observation of spontaneous germ line insertion of modern human mtDNA sequences and suggest that DSB repair may play a role in inter-organellar gene transfer in vivo. Our findings also provide evidence for a previously unrecognized insertional mechanism in human, by which non-mobile extra-chromosomal fragments can be inserted into the genome at DSB repair junctions.

Differential DNA methylation at conserved non-genic elements and evidence for transgenerational inheritance following developmental exposure to mono(2-ethylhexyl) phthalate and 5-azacytidine in zebrafish.

PubMed

Kamstra, Jorke H; Sales, Liana Bastos; Aleström, Peter; Legler, Juliette

2017-01-01

Exposure to environmental stressors during development may lead to latent and transgenerational adverse health effects. To understand the role of DNA methylation in these effects, we used zebrafish as a vertebrate model to investigate heritable changes in DNA methylation following chemical-induced stress during early development. We exposed zebrafish embryos to non-embryotoxic concentrations of the biologically active phthalate metabolite mono(2-ethylhexyl) phthalate (MEHP, 30 µM) and the DNA methyltransferase 1 inhibitor 5-azacytidine (5AC, 10 µM). Direct, latent and transgenerational effects on DNA methylation were assessed using global, genome-wide and locus-specific DNA methylation analyses. Following direct exposure in zebrafish embryos from 0 to 6 days post-fertilization, genome-wide analysis revealed a multitude of differentially methylated regions, strongly enriched at conserved non-genic elements for both compounds. Pathways involved in adipogenesis were enriched with the putative obesogenic compound MEHP. Exposure to 5AC resulted in enrichment of pathways involved in embryonic development and transgenerational effects on larval body length. Locus-specific methylation analysis of 10 differentially methylated sites revealed six of these loci differentially methylated in sperm sampled from adult zebrafish exposed during development to 5AC, and in first and second generation larvae. With MEHP, consistent changes were found at 2 specific loci in first and second generation larvae. Our results suggest a functional role for DNA methylation on cis-regulatory conserved elements following developmental exposure to compounds. Effects on these regions are potentially transferred to subsequent generations.

Changes in DNA methylation induced by multi-walled carbon nanotube exposure in the workplace.

PubMed

Ghosh, Manosij; Öner, Deniz; Poels, Katrien; Tabish, Ali M; Vlaanderen, Jelle; Pronk, Anjoeka; Kuijpers, Eelco; Lan, Qing; Vermeulen, Roel; Bekaert, Bram; Hoet, Peter Hm; Godderis, Lode

This study was designed to assess the epigenetic alterations in blood cells, induced by occupational exposure to multi-wall carbon nanotubes (MWCNT). The study population comprised of MWCNT-exposed workers (n=24) and unexposed controls (n=43) from the same workplace. We measured global DNA methylation/hydroxymethylation levels on the 5th cytosine residues using a validated liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. Sequence-specific methylation of LINE1 retrotransposable element 1 (L1RE1) elements, and promoter regions of functionally important genes associated with epigenetic regulation [DNA methyltransferase-1 (DNMT1) and histone deacetylase 4 (HDAC4)], DNA damage/repair and cell cycle pathways [nuclear protein, coactivator of histone transcription/ATM serine/threonine kinase (NPAT/ATM)], and a potential transforming growth factor beta (TGF-β) repressor [SKI proto-oncogene (SKI)] were studied using bisulfite pyrosequencing. Analysis of global DNA methylation levels and hydroxymethylation did not reveal significant difference between the MWCNT-exposed and control groups. No significant changes in Cytosine-phosphate-Guanine (CpG) site methylation were observed for the LINE1 (L1RE1) elements. Further analysis of gene-specific DNA methylation showed a significant change in methylation for DNMT1, ATM, SKI, and HDAC4 promoter CpGs in MWCNT-exposed workers. Since DNA methylation plays an important role in silencing/regulation of the genes, and many of these genes have been associated with occupational and smoking-induced diseases and cancer (risk), aberrant methylation of these genes might have a potential effect in MWCNT-exposed workers.

Interaction of the alpha-subunit of Escherichia coli RNA polymerase with DNA: rigid body nature of the protein-DNA contact.

PubMed

Heyduk, E; Baichoo, N; Heyduk, T

2001-11-30

The alpha-subunit of Escherichia coli RNA polymerase plays an important role in the activity of many promoters by providing a direct protein-DNA contact with a specific sequence (UP element) located upstream of the core promoter sequence. To obtain insight into the nature of thermodynamic forces involved in the formation of this protein-DNA contact, the binding of the alpha-subunit of E. coli RNA polymerase to a fluorochrome-labeled DNA fragment containing the rrnB P1 promoter UP element sequence was quantitatively studied using fluorescence polarization. The alpha dimer and DNA formed a 1:1 complex in solution. Complex formation at 25 degrees C was enthalpy-driven, the binding was accompanied by a net release of 1-2 ions, and no significant specific ion effects were observed. The van't Hoff plot of temperature dependence of binding was linear suggesting that the heat capacity change (Deltac(p)) was close to zero. Protein footprinting with hydroxyradicals showed that the protein did not change its conformation upon protein-DNA contact formation. No conformational changes in the DNA molecule were detected by CD spectroscopy upon protein-DNA complex formation. The thermodynamic characteristics of the binding together with the lack of significant conformational changes in the protein and in the DNA suggested that the alpha-subunit formed a rigid body-like contact with the DNA in which a tight complementary recognition interface between alpha-subunit and DNA was not formed.

Restless 5S: the re-arrangement(s) and evolution of the nuclear ribosomal DNA in land plants.

PubMed

Wicke, Susann; Costa, Andrea; Muñoz, Jesùs; Quandt, Dietmar

2011-11-01

Among eukaryotes two types of nuclear ribosomal DNA (nrDNA) organization have been observed. Either all components, i.e. the small ribosomal subunit, 5.8S, large ribosomal subunit, and 5S occur tandemly arranged or the 5S rDNA forms a separate cluster of its own. Generalizations based on data derived from just a few model organisms have led to a superimposition of structural and evolutionary traits to the entire plant kingdom asserting that plants generally possess separate arrays. This study reveals that plant nrDNA organization into separate arrays is not a distinctive feature, but rather assignable almost solely to seed plants. We show that early diverging land plants and presumably streptophyte algae share a co-localization of all rRNA genes within one repeat unit. This raises the possibility that the state of rDNA gene co-localization had occurred in their common ancestor. Separate rDNA arrays were identified for all basal seed plants and water ferns, implying at least two independent 5S rDNA transposition events during land plant evolution. Screening for 5S derived Cassandra transposable elements which might have played a role during the transposition events, indicated that this retrotransposon is absent in early diverging vascular plants including early fern lineages. Thus, Cassandra can be rejected as a primary mechanism for 5S rDNA transposition in water ferns. However, the evolution of Cassandra and other eukaryotic 5S derived elements might have been a side effect of the 5S rDNA cluster formation. Structural analysis of the intergenic spacers of the ribosomal clusters revealed that transposition events partially affect spacer regions and suggests a slightly different transcription regulation of 5S rDNA in early land plants. 5S rDNA upstream regulatory elements are highly divergent or absent from the LSU-5S spacers of most early divergent land plant lineages. Several putative scenarios and mechanisms involved in the concerted relocation of hundreds of 5S rRNA gene copies are discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

A novel species-specific tandem repeat DNA family from Sinapis arvensis: detection of telomere-like sequences.

PubMed

Kapila, R; Das, S; Srivastava, P S; Lakshmikumaran, M

1996-08-01

DNA sequences representing a tandemly repeated DNA family of the Sinapis arvensis genome were cloned and characterized. The 700-bp tandem repeat family is represented by two clones, pSA35 and pSA52, which are 697 and 709 bp in length, respectively. Dot matrix analysis of the sequences indicates the presence of repeated elements within each monomeric unit. Sequence analysis of the repetitive region of clones pSA35 and pSA52 shows that there are several copies of a 7-bp repeat element organized in tandem. The consensus sequence of this repeat element is 5'-TTTAGGG-3'. These elements are highly mutated and the difference in length between the two clones is due to different copy numbers of these elements. The repetitive region of clone pSA35 has 26 copies of the element TTTAGGG, whereas clone pSA52 has 28 copies. The repetitive region in both clones is flanked on either side by inverted repeats that may be footprints of a transposition event. Sequence comparison indicates that the element TTTAGGG is identical to telomeric repeats present in Arabidopsis, maize, tomato, and other plants. However, Bal31 digestion kinetics indicates non-telomeric localization of the 700-bp tandem repeats. The clones represent a novel repeat family as (i) they contain telomere-like motifs as subrepeats within each unit; and (ii) they do not hybridize to related crucifers and are species-specific in nature.

Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakraborty, Kaushik; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in

2015-07-28

Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging themore » ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.« less

A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells.

PubMed

Kang, Shin-Young; Kim, Yeon-Gu; Kang, Seunghee; Lee, Hong Weon; Lee, Eun Gyo

2016-05-01

Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Billions of basepairs of recently expanded, repetitive sequences are eliminated from the somatic genome during copepod development.

PubMed

Sun, Cheng; Wyngaard, Grace; Walton, D Brian; Wichman, Holly A; Mueller, Rachel Lockridge

2014-03-11

Chromatin diminution is the programmed deletion of DNA from presomatic cell or nuclear lineages during development, producing single organisms that contain two different nuclear genomes. Phylogenetically diverse taxa undergo chromatin diminution--some ciliates, nematodes, copepods, and vertebrates. In cyclopoid copepods, chromatin diminution occurs in taxa with massively expanded germline genomes; depending on species, germline genome sizes range from 15 - 75 Gb, 12-74 Gb of which are lost from pre-somatic cell lineages at germline--soma differentiation. This is more than an order of magnitude more sequence than is lost from other taxa. To date, the sequences excised from copepods have not been analyzed using large-scale genomic datasets, and the processes underlying germline genomic gigantism in this clade, as well as the functional significance of chromatin diminution, have remained unknown. Here, we used high-throughput genomic sequencing and qPCR to characterize the germline and somatic genomes of Mesocyclops edax, a freshwater cyclopoid copepod with a germline genome of ~15 Gb and a somatic genome of ~3 Gb. We show that most of the excised DNA consists of repetitive sequences that are either 1) verifiable transposable elements (TEs), or 2) non-simple repeats of likely TE origin. Repeat elements in both genomes are skewed towards younger (i.e. less divergent) elements. Excised DNA is a non-random sample of the germline repeat element landscape; younger elements, and high frequency DNA transposons and LINEs, are disproportionately eliminated from the somatic genome. Our results suggest that germline genome expansion in M. edax reflects explosive repeat element proliferation, and that billions of base pairs of such repeats are deleted from the somatic genome every generation. Thus, we hypothesize that chromatin diminution is a mechanism that controls repeat element load, and that this load can evolve to be divergent between tissue types within single organisms.

Billions of basepairs of recently expanded, repetitive sequences are eliminated from the somatic genome during copepod development

PubMed Central

2014-01-01

Background Chromatin diminution is the programmed deletion of DNA from presomatic cell or nuclear lineages during development, producing single organisms that contain two different nuclear genomes. Phylogenetically diverse taxa undergo chromatin diminution — some ciliates, nematodes, copepods, and vertebrates. In cyclopoid copepods, chromatin diminution occurs in taxa with massively expanded germline genomes; depending on species, germline genome sizes range from 15 – 75 Gb, 12–74 Gb of which are lost from pre-somatic cell lineages at germline – soma differentiation. This is more than an order of magnitude more sequence than is lost from other taxa. To date, the sequences excised from copepods have not been analyzed using large-scale genomic datasets, and the processes underlying germline genomic gigantism in this clade, as well as the functional significance of chromatin diminution, have remained unknown. Results Here, we used high-throughput genomic sequencing and qPCR to characterize the germline and somatic genomes of Mesocyclops edax, a freshwater cyclopoid copepod with a germline genome of ~15 Gb and a somatic genome of ~3 Gb. We show that most of the excised DNA consists of repetitive sequences that are either 1) verifiable transposable elements (TEs), or 2) non-simple repeats of likely TE origin. Repeat elements in both genomes are skewed towards younger (i.e. less divergent) elements. Excised DNA is a non-random sample of the germline repeat element landscape; younger elements, and high frequency DNA transposons and LINEs, are disproportionately eliminated from the somatic genome. Conclusions Our results suggest that germline genome expansion in M. edax reflects explosive repeat element proliferation, and that billions of base pairs of such repeats are deleted from the somatic genome every generation. Thus, we hypothesize that chromatin diminution is a mechanism that controls repeat element load, and that this load can evolve to be divergent between tissue types within single organisms. PMID:24618421

Ribosomal DNA Organization Before and After Magnification in Drosophila melanogaster

PubMed Central

Bianciardi, Alessio; Boschi, Manuela; Swanson, Ellen E.; Belloni, Massimo; Robbins, Leonard G.

2012-01-01

In all eukaryotes, the ribosomal RNA genes are stably inherited redundant elements. In Drosophila melanogaster, the presence of a Ybb− chromosome in males, or the maternal presence of the Ribosomal exchange (Rex) element, induces magnification: a heritable increase of rDNA copy number. To date, several alternative classes of mechanisms have been proposed for magnification: in situ replication or extra-chromosomal replication, either of which might act on short or extended strings of rDNA units, or unequal sister chromatid exchange. To eliminate some of these hypotheses, none of which has been clearly proven, we examined molecular-variant composition and compared genetic maps of the rDNA in the bb2 mutant and in some magnified bb+ alleles. The genetic markers used are molecular-length variants of IGS sequences and of R1 and R2 mobile elements present in many 28S sequences. Direct comparison of PCR products does not reveal any particularly intensified electrophoretic bands in magnified alleles compared to the nonmagnified bb2 allele. Hence, the increase of rDNA copy number is diluted among multiple variants. We can therefore reject mechanisms of magnification based on multiple rounds of replication of short strings. Moreover, we find no changes of marker order when pre- and postmagnification maps are compared. Thus, we can further restrict the possible mechanisms to two: replication in situ of an extended string of rDNA units or unequal exchange between sister chromatids. PMID:22505623

A role for circadian evening elements in cold-regulated gene expression in Arabidopsis.

PubMed

Mikkelsen, Michael D; Thomashow, Michael F

2009-10-01

The plant transcriptome is dramatically altered in response to low temperature. The cis-acting DNA regulatory elements and trans-acting factors that regulate the majority of cold-regulated genes are unknown. Previous bioinformatic analysis has indicated that the promoters of cold-induced genes are enriched in the Evening Element (EE), AAAATATCT, a DNA regulatory element that has a role in circadian-regulated gene expression. Here we tested the role of EE and EE-like (EEL) elements in cold-induced expression of two Arabidopsis genes, CONSTANS-like 1 (COL1; At5g54470) and a gene encoding a 27-kDa protein of unknown function that we designated COLD-REGULATED GENE 27 (COR27; At5g42900). Mutational analysis indicated that the EE/EEL elements were required for cold induction of COL1 and COR27, and that their action was amplified through coupling with ABA response element (ABRE)-like (ABREL) motifs. An artificial promoter consisting solely of four EE motifs interspersed with three ABREL motifs was sufficient to impart cold-induced gene expression. Both COL1 and COR27 were found to be regulated by the circadian clock at warm growth temperatures and cold-induction of COR27 was gated by the clock. These results suggest that cold- and clock-regulated gene expression are integrated through regulatory proteins that bind to EE and EEL elements supported by transcription factors acting at ABREL sequences. Bioinformatic analysis indicated that the coupling of EE and EEL motifs with ABREL motifs is highly enriched in cold-induced genes and thus may constitute a DNA regulatory element pair with a significant role in configuring the low-temperature transcriptome.

Scanning sequences after Gibbs sampling to find multiple occurrences of functional elements

PubMed Central

Tharakaraman, Kannan; Mariño-Ramírez, Leonardo; Sheetlin, Sergey L; Landsman, David; Spouge, John L

2006-01-01

Background Many DNA regulatory elements occur as multiple instances within a target promoter. Gibbs sampling programs for finding DNA regulatory elements de novo can be prohibitively slow in locating all instances of such an element in a sequence set. Results We describe an improvement to the A-GLAM computer program, which predicts regulatory elements within DNA sequences with Gibbs sampling. The improvement adds an optional "scanning step" after Gibbs sampling. Gibbs sampling produces a position specific scoring matrix (PSSM). The new scanning step resembles an iterative PSI-BLAST search based on the PSSM. First, it assigns an "individual score" to each subsequence of appropriate length within the input sequences using the initial PSSM. Second, it computes an E-value from each individual score, to assess the agreement between the corresponding subsequence and the PSSM. Third, it permits subsequences with E-values falling below a threshold to contribute to the underlying PSSM, which is then updated using the Bayesian calculus. A-GLAM iterates its scanning step to convergence, at which point no new subsequences contribute to the PSSM. After convergence, A-GLAM reports predicted regulatory elements within each sequence in order of increasing E-values, so users have a statistical evaluation of the predicted elements in a convenient presentation. Thus, although the Gibbs sampling step in A-GLAM finds at most one regulatory element per input sequence, the scanning step can now rapidly locate further instances of the element in each sequence. Conclusion Datasets from experiments determining the binding sites of transcription factors were used to evaluate the improvement to A-GLAM. Typically, the datasets included several sequences containing multiple instances of a regulatory motif. The improvements to A-GLAM permitted it to predict the multiple instances. PMID:16961919

Applications of the rep-PCR DNA fingerprinting technique to study microbial diversity, ecology and evolution.

PubMed

Ishii, Satoshi; Sadowsky, Michael J

2009-04-01

A large number of repetitive DNA sequences are found in multiple sites in the genomes of numerous bacteria, archaea and eukarya. While the functions of many of these repetitive sequence elements are unknown, they have proven to be useful as the basis of several powerful tools for use in molecular diagnostics, medical microbiology, epidemiological analyses and environmental microbiology. The repetitive sequence-based PCR or rep-PCR DNA fingerprint technique uses primers targeting several of these repetitive elements and PCR to generate unique DNA profiles or 'fingerprints' of individual microbial strains. Although this technique has been extensively used to examine diversity among variety of prokaryotic microorganisms, rep-PCR DNA fingerprinting can also be applied to microbial ecology and microbial evolution studies since it has the power to distinguish microbes at the strain or isolate level. Recent advancement in rep-PCR methodology has resulted in increased accuracy, reproducibility and throughput. In this minireview, we summarize recent improvements in rep-PCR DNA fingerprinting methodology, and discuss its applications to address fundamentally important questions in microbial ecology and evolution.

Tetris Is a Foldback Transposon that Provided the Building Blocks for an Emerging Satellite DNA of Drosophila virilis

PubMed Central

Dias, Guilherme B.; Svartman, Marta; Delprat, Alejandra; Ruiz, Alfredo; Kuhn, Gustavo C.S.

2014-01-01

Transposable elements (TEs) and satellite DNAs (satDNAs) are abundant components of most eukaryotic genomes studied so far and their impact on evolution has been the focus of several studies. A number of studies linked TEs with satDNAs, but the nature of their evolutionary relationships remains unclear. During in silico analyses of the Drosophila virilis assembled genome, we found a novel DNA transposon we named Tetris based on its modular structure and diversity of rearranged forms. We aimed to characterize Tetris and investigate its role in generating satDNAs. Data mining and sequence analysis showed that Tetris is apparently nonautonomous, with a structure similar to foldback elements, and present in D. virilis and D. americana. Herein, we show that Tetris shares the final portions of its terminal inverted repeats (TIRs) with DAIBAM, a previously described miniature inverted transposable element implicated in the generation of chromosome inversions. Both elements are likely to be mobilized by the same autonomous TE. Tetris TIRs contain approximately 220-bp internal tandem repeats that we have named TIR-220. We also found TIR-220 repeats making up longer (kb-size) satDNA-like arrays. Using bioinformatic, phylogenetic and cytogenomic tools, we demonstrated that Tetris has contributed to shaping the genomes of D. virilis and D. americana, providing internal tandem repeats that served as building blocks for the amplification of satDNA arrays. The β-heterochromatic genomic environment seemed to have favored such amplification. Our results imply for the first time a role for foldback elements in generating satDNAs. PMID:24858539

Atomically precise arrays of fluorescent silver clusters: a modular approach for metal cluster photonics on DNA nanostructures.

PubMed

Copp, Stacy M; Schultz, Danielle E; Swasey, Steven; Gwinn, Elisabeth G

2015-03-24

The remarkable precision that DNA scaffolds provide for arraying nanoscale optical elements enables optical phenomena that arise from interactions of metal nanoparticles, dye molecules, and quantum dots placed at nanoscale separations. However, control of ensemble optical properties has been limited by the difficulty of achieving uniform particle sizes and shapes. Ligand-stabilized metal clusters offer a route to atomically precise arrays that combine desirable attributes of both metals and molecules. Exploiting the unique advantages of the cluster regime requires techniques to realize controlled nanoscale placement of select cluster structures. Here we show that atomically monodisperse arrays of fluorescent, DNA-stabilized silver clusters can be realized on a prototypical scaffold, a DNA nanotube, with attachment sites separated by <10 nm. Cluster attachment is mediated by designed DNA linkers that enable isolation of specific clusters prior to assembly on nanotubes and preserve cluster structure and spectral purity after assembly. The modularity of this approach generalizes to silver clusters of diverse sizes and DNA scaffolds of many types. Thus, these silver cluster nano-optical elements, which themselves have colors selected by their particular DNA templating oligomer, bring unique dimensions of control and flexibility to the rapidly expanding field of nano-optics.

Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System.

PubMed

Cooper, Lauren A; Stringer, Anne M; Wade, Joseph T

2018-04-17

In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo , for the type I-E system of Escherichia coli Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5' end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing. IMPORTANCE Many bacterial and archaeal species encode CRISPR-Cas immunity systems that protect against invasion by foreign DNA. In the Escherichia coli CRISPR-Cas system, a protein complex, Cascade, binds 61-nucleotide (nt) CRISPR RNAs (crRNAs). The Cascade complex is directed to invading DNA molecules through base pairing between the crRNA and target DNA. This leads to recruitment of the Cas3 nuclease, which destroys the invading DNA molecule and promotes acquisition of new immunity elements. We made the first in vivo measurements of Cascade binding to DNA targets. Thus, we show that Cascade binding to DNA is highly promiscuous; endogenous E. coli crRNAs can direct Cascade binding to >100 chromosomal locations. In contrast, we show that targeted degradation and acquisition of new immunity elements require highly specific association of Cascade with DNA, limiting CRISPR-Cas function to the appropriate targets. Copyright © 2018 Cooper et al.

A Short Interspersed Nuclear Element (SINE)-Based Real-Time PCR Approach to Detect and Quantify Porcine Component in Meat Products.

PubMed

Zhang, Chi; Fang, Xin; Qiu, Haopu; Li, Ning

2015-01-01

Real-time PCR amplification of mitochondria gene could not be used for DNA quantification, and that of single copy DNA did not allow an ideal sensitivity. Moreover, cross-reactions among similar species were commonly observed in the published methods amplifying repetitive sequence, which hindered their further application. The purpose of this study was to establish a short interspersed nuclear element (SINE)-based real-time PCR approach having high specificity for species detection that could be used in DNA quantification. After massive screening of candidate Sus scrofa SINEs, one optimal combination of primers and probe was selected, which had no cross-reaction with other common meat species. LOD of the method was 44 fg DNA/reaction. Further, quantification tests showed this approach was practical in DNA estimation without tissue variance. Thus, this study provided a new tool for qualitative detection of porcine component, which could be promising in the QC of meat products.

Modulating the DNA polymerase β reaction equilibrium to dissect the reverse reaction

PubMed Central

Shock, David D.; Freudenthal, Bret D.; Beard, William A.; Wilson, Samuel H.

2017-01-01

DNA polymerases catalyze efficient and high fidelity DNA synthesis. While this reaction favors nucleotide incorporation, polymerases also catalyze a reverse reaction, pyrophosphorolysis, removing the DNA primer terminus and generating deoxynucleoside triphosphates. Since pyrophosphorolysis can influence polymerase fidelity and sensitivity to chain-terminating nucleosides, we analyzed pyrophosphorolysis with human DNA polymerase β and found the reaction to be inefficient. The lack of a thio-elemental effect indicated that it was limited by a non-chemical step. Utilizing a pyrophosphate analog, where the bridging oxygen is replaced with an imido-group (PNP), increased the rate of the reverse reaction and displayed a large thio-elemental effect indicating that chemistry was now rate determining. Time-lapse crystallography with PNP captured structures consistent with a chemical equilibrium that favored the reverse reaction. These results highlight the importance of the bridging atom between the β- and γ-phosphates of the incoming nucleotide in reaction chemistry, enzyme conformational changes, and overall reaction equilibrium. PMID:28759020

Structure of p73 DNA-binding domain tetramer modulates p73 transactivation

PubMed Central

Ethayathulla, Abdul S.; Tse, Pui-Wah; Monti, Paola; Nguyen, Sonha; Inga, Alberto; Fronza, Gilberto; Viadiu, Hector

2012-01-01

The transcription factor p73 triggers developmental pathways and overlaps stress-induced p53 transcriptional pathways. How p53-family response elements determine and regulate transcriptional specificity remains an unsolved problem. In this work, we have determined the first crystal structures of p73 DNA-binding domain tetramer bound to response elements with spacers of different length. The structure and function of the adaptable tetramer are determined by the distance between two half-sites. The structures with zero and one base-pair spacers show compact p73 DNA-binding domain tetramers with large tetramerization interfaces; a two base-pair spacer results in DNA unwinding and a smaller tetramerization interface, whereas a four base-pair spacer hinders tetramerization. Functionally, p73 is more sensitive to spacer length than p53, with one base-pair spacer reducing 90% of transactivation activity and longer spacers reducing transactivation to basal levels. Our results establish the quaternary structure of the p73 DNA-binding domain required as a scaffold to promote transactivation. PMID:22474346

DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses

PubMed Central

Wicker, Thomas; Yu, Yeisoo; Haberer, Georg; Mayer, Klaus F. X.; Marri, Pradeep Reddy; Rounsley, Steve; Chen, Mingsheng; Zuccolo, Andrea; Panaud, Olivier; Wing, Rod A.; Roffler, Stefan

2016-01-01

DNA (class 2) transposons are mobile genetic elements which move within their ‘host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind. PMID:27599761

Active Site Sharing and Subterminal Hairpin Recognition in a New Class of DNA Transposases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ronning, Donald R.; Guynet, Catherine; Ton-Hoang, Bao

2010-07-20

Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg{sup 2+} and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalentmore » intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.« less

Deletion endpoint allele-specificity in the developmentally regulated elimination of an internal sequence (IES) in Paramecium.

PubMed Central

Dubrana, K; Le Mouël, A; Amar, L

1997-01-01

Ciliated protozoa undergo thousands of site-specific DNA deletion events during the programmed development of micronuclear genomes to macronuclear genomes. Two deletion elements, W1 and W2, were identified in the Paramecium primaurelia wild-type 156 strain. Here, we report the characterization of both elements in wild-type strain 168 and show that they display variant deletion patterns when compared with those of strain 156. The W1 ( 168 ) element is defective for deletion. The W2 ( 168 ) element is excised utilizing two alternative boundaries on one side, both are different from the boundary utilized to excise the W2156 element. By crossing the 156 and 168 strains, we demonstrate that the definition of all deletion endpoints are each controlled by cis -acting determinant(s) rather than by strain-specific trans-acting factor(s). Sequence comparison of all deleted DNA segments indicates that the 5'-TA-3'terminal sequence is strictly required at their ends. Furthermore the identity of the first eight base pairs of these ends to a previously established consensus sequence correlates with the frequency of the corresponding deletion events. Our data implies the existence of an adaptive convergent evolution of these Paramecium deleted DNA segment end sequences. PMID:9171098

A novel non prophage(-like) gene-intervening element within gerE that is reconstituted during sporulation in Bacillus cereus ATCC10987.

PubMed

Abe, Kimihiro; Shimizu, Shin-Ya; Tsuda, Shuhei; Sato, Tsutomu

2017-09-12

Gene rearrangement is a widely-shared phenomenon in spore forming bacteria, in which prophage(-like) elements interrupting sporulation-specific genes are excised from the host genome to reconstitute the intact gene. Here, we report a novel class of gene-intervening elements, named gin, inserted in the 225 bp gerE-coding region of the B. cereus ATCC10987 genome, which generates a sporulation-specific rearrangement. gin has no phage-related genes and possesses three site-specific recombinase genes; girA, girB, and girC. We demonstrated that the gerE rearrangement occurs at the middle stage of sporulation, in which site-specific DNA recombination took place within the 9 bp consensus sequence flanking the disrupted gerE segments. Deletion analysis of gin uncovered that GirC and an additional factor, GirX, are responsible for gerE reconstitution. Involvement of GirC and GirX in DNA recombination was confirmed by an in vitro recombination assay. These results broaden the definition of the sporulation-specific gene rearrangement phenomenon: gene-intervening elements are not limited to phage DNA but may include non-viral genetic elements that carry a developmentally-regulated site-specific recombination system.

Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modified organisms.

PubMed

Dinon, Andréia Z; Prins, Theo W; van Dijk, Jeroen P; Arisi, Ana Carolina M; Scholtens, Ingrid M J; Kok, Esther J

2011-05-01

Primers and probes were developed for the element-specific detection of cry1A.105 and cry2Ab2 genes, based on their DNA sequence as present in GM maize MON89034. Cry genes are present in many genetically modified (GM) plants and they are important targets for developing GMO element-specific detection methods. Element-specific methods can be of use to screen for the presence of GMOs in food and feed supply chains. Moreover, a combination of GMO elements may indicate the potential presence of unapproved GMOs (UGMs). Primer-probe combinations were evaluated in terms of specificity, efficiency and limit of detection. Except for specificity, the complete experiment was performed in 9 PCR runs, on 9 different days and by testing 8 DNA concentrations. The results showed a high specificity and efficiency for cry1A.105 and cry2Ab2 detection. The limit of detection was between 0.05 and 0.01 ng DNA per PCR reaction for both assays. These data confirm the applicability of these new primer-probe combinations for element detection that can contribute to the screening for GM and UGM crops in food and feed samples.

Identification and chromosome mapping of repetitive elements in the Astyanax scabripinnis (Teleostei: Characidae) species complex.

PubMed

Barbosa, Patrícia; de Oliveira, Luiz Antonio; Pucci, Marcela Baer; Santos, Mateus Henrique; Moreira-Filho, Orlando; Vicari, Marcelo Ricardo; Nogaroto, Viviane; de Almeida, Mara Cristina; Artoni, Roberto Ferreira

2015-02-01

Most part of the eukaryotic genome is composed of repeated sequences or multiple copies of DNA, which were considered as "junk DNA", and may be associated to the heterochromatin. In this study, three populations of Astyanax aff. scabripinnis from Brazilian rivers of Guaratinguetá and Pindamonhangaba (São Paulo) and a population from Maringá (Paraná) were analyzed concerning the localization of the nucleolar organizer regions (Ag-NORs), the As51 satellite DNA, the 18S ribosomal DNA (rDNA), and the 5S rDNA. Repeated sequences were also isolated and identified by the Cot - 1 method, which indicated similarity (90%) with the LINE UnaL2 retrotransposon. The fluorescence in situ hybridization (FISH) showed the retrotransposon dispersed and more concentrated markers in centromeric and telomeric chromosomal regions. These sequences were co-localized and interspaced with 18S and 5S rDNA and As51, confirmed by fiber-FISH essay. The B chromosome found in these populations pointed to a conspicuous hybridization with LINE probe, which is also co-located in As51 sequences. The NORs were active at unique sites of a homologous pair in the three populations. There were no evidences that transposable elements and repetitive DNA had influence in the transcriptional regulation of ribosomal genes in our analyses.

Integrated DNA walking system to characterize a broad spectrum of GMOs in food/feed matrices.

PubMed

Fraiture, Marie-Alice; Herman, Philippe; Lefèvre, Loic; Taverniers, Isabel; De Loose, Marc; Deforce, Dieter; Roosens, Nancy H

2015-08-14

In order to provide a system fully integrated with qPCR screening, usually used in GMO routine analysis, as well as being able to detect, characterize and identify a broad spectrum of GMOs in food/feed matrices, two bidirectional DNA walking methods targeting p35S or tNOS, the most common transgenic elements found in GM crops, were developed. These newly developed DNA walking methods are completing the previously implemented DNA walking method targeting the t35S pCAMBIA element. Food/feed matrices containing transgenic crops (Bt rice or MON863 maize) were analysed using the integrated DNA walking system. First, the newly developed DNA walking methods, anchored on the sequences used for the p35S or tNOS qPCR screening, were tested on Bt rice that contains these two transgenic elements. Second, the methods were assessed on a maize sample containing a low amount of the GM MON863 event, representing a more complex matrix in terms of genome size and sensitivity. Finally, to illustrate its applicability in GMO routine analysis by enforcement laboratories, the entire workflow of the integrated strategy, including qPCR screening to detect the potential presence of GMOs and the subsequent DNA walking methods to characterize and identify the detected GMOs, was applied on a GeMMA Scheme Proficiency Test matrix. Via the characterization of the transgene flanking region between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette, the presence of GMOs was properly confirmed or infirmed in all tested samples. Due to their simple procedure and their short time-frame to get results, the developed DNA walking methods proposed here can be easily implemented in GMO routine analysis by the enforcement laboratories. In providing crucial information about the transgene flanking regions and/or the transgenic cassettes, this DNA walking strategy is a key molecular tool to prove the presence of GMOs in any given food/feed matrix.

Global Quantitative Modeling of Chromatin Factor Interactions

PubMed Central

Zhou, Jian; Troyanskaya, Olga G.

2014-01-01

Chromatin is the driver of gene regulation, yet understanding the molecular interactions underlying chromatin factor combinatorial patterns (or the “chromatin codes”) remains a fundamental challenge in chromatin biology. Here we developed a global modeling framework that leverages chromatin profiling data to produce a systems-level view of the macromolecular complex of chromatin. Our model ultilizes maximum entropy modeling with regularization-based structure learning to statistically dissect dependencies between chromatin factors and produce an accurate probability distribution of chromatin code. Our unsupervised quantitative model, trained on genome-wide chromatin profiles of 73 histone marks and chromatin proteins from modENCODE, enabled making various data-driven inferences about chromatin profiles and interactions. We provided a highly accurate predictor of chromatin factor pairwise interactions validated by known experimental evidence, and for the first time enabled higher-order interaction prediction. Our predictions can thus help guide future experimental studies. The model can also serve as an inference engine for predicting unknown chromatin profiles — we demonstrated that with this approach we can leverage data from well-characterized cell types to help understand less-studied cell type or conditions. PMID:24675896

RNAi drives nonreciprocal translocations at eroding chromosome ends to establish telomere-free linear chromosomes.

PubMed

Begnis, Martina; Apte, Manasi S; Masuda, Hirohisa; Jain, Devanshi; Wheeler, David Lee; Cooper, Julia Promisel

2018-04-01

The identification of telomerase-negative HAATI (heterochromatin amplification-mediated and telomerase-independent) cells, in which telomeres are superseded by nontelomeric heterochromatin tracts, challenged the idea that canonical telomeres are essential for chromosome linearity and raised crucial questions as to how such tracts translocate to eroding chromosome ends and confer end protection. Here we show that HAATI arises when telomere loss triggers a newly recognized illegitimate translocation pathway that requires RNAi factors. While RNAi is necessary for the translocation events that mobilize ribosomal DNA (rDNA) tracts to all chromosome ends (forming "HAATI rDNA " chromosomes), it is dispensable for HAATI rDNA maintenance. Surprisingly, Dicer (Dcr1) plays a separate, RNAi-independent role in preventing formation of the rare HAATI subtype in which a different repetitive element (the subtelomeric element) replaces telomeres. Using genetics and fusions between shelterin components and rDNA-binding proteins, we mapped the mechanism by which rDNA loci engage crucial end protection factors-despite the absence of telomere repeats-and secure end protection. Sequence analysis of HAATI rDNA genomes allowed us to propose RNA and DNA polymerase template-switching models for the mechanism of RNAi-triggered rDNA translocations. Collectively, our results reveal unforeseen roles for noncoding RNAs (ncRNAs) in assembling a telomere-free chromosome end protection device. © 2018 Begnis et al.; Published by Cold Spring Harbor Laboratory Press.

Photoabsorption Study of Bacillus megaterium, DNA and Related Biological Materials in the Phosphorus K-edge Region

NASA Technical Reports Server (NTRS)

Frigo, Sean P.; McNulty, Ian; Richmond, Robert C.; Ehret, Charles F.

2002-01-01

We have measured the x-ray transmission spectra of several biologically related samples in the phosphorus K-edge absorption region. These include elemental red phosphorus, hydrated sodium phosphate (Na3PO4.12H2O), deoxyribonucleic acid (DNA), adenosinetriphosphate (ATP), diolylphosphatidyl choline (DOPC), and Bacillus megaterium spores. Elemental red phosphorus essentially displays an edge-jump. All other spectra are similar in form and energy position. Each spectrum for these substances is dominated by a narrower, more intense first peak and a broader but less intense second peak. The corresponding K-edge absorption thresholds are shifted towards higher energy relative to that for elemental red phosphorus, as expected for increasing degrees of phosphorus oxidation. The B. megaterium spectrum has aspects common to both the phosphate and DNA spectra and is therefore interpreted as a composite of spectra arising from DNA/RNA and phosphates within the spore. The B. megaterium spore spectrum provides needed information for resonant radiation damage studies in the phosphorus K-edge absorption region by identifying candidate photoexcitations. In addition,the absorption spectra will be useful in macromolecular crystallography studies employing anomalous dispersion effects at the phosphorus K-edge.

Defining functional DNA elements in the human genome

PubMed Central

Kellis, Manolis; Wold, Barbara; Snyder, Michael P.; Bernstein, Bradley E.; Kundaje, Anshul; Marinov, Georgi K.; Ward, Lucas D.; Birney, Ewan; Crawford, Gregory E.; Dekker, Job; Dunham, Ian; Elnitski, Laura L.; Farnham, Peggy J.; Feingold, Elise A.; Gerstein, Mark; Giddings, Morgan C.; Gilbert, David M.; Gingeras, Thomas R.; Green, Eric D.; Guigo, Roderic; Hubbard, Tim; Kent, Jim; Lieb, Jason D.; Myers, Richard M.; Pazin, Michael J.; Ren, Bing; Stamatoyannopoulos, John A.; Weng, Zhiping; White, Kevin P.; Hardison, Ross C.

2014-01-01

With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease. PMID:24753594

Prenatal maternal immune activation causes epigenetic differences in adolescent mouse brain

PubMed Central

Basil, P; Li, Q; Dempster, E L; Mill, J; Sham, P-C; Wong, C C Y; McAlonan, G M

2014-01-01

Epigenetic processes such as DNA methylation have been implicated in the pathophysiology of neurodevelopmental disorders including schizophrenia and autism. Epigenetic changes can be induced by environmental exposures such as inflammation. Here we tested the hypothesis that prenatal inflammation, a recognized risk factor for schizophrenia and related neurodevelopmental conditions, alters DNA methylation in key brain regions linked to schizophrenia, namely the dopamine rich striatum and endocrine regulatory centre, the hypothalamus. DNA methylation across highly repetitive elements (long interspersed element 1 (LINE1) and intracisternal A-particles (IAPs)) were used to proxy global DNA methylation. We also investigated the Mecp2 gene because it regulates transcription of LINE1 and has a known association with neurodevelopmental disorders. Brain tissue was harvested from 6 week old offspring of mice exposed to the viral analog PolyI:C or saline on gestation day 9. We used Sequenom EpiTYPER assay to quantitatively analyze differences in DNA methylation at IAPs, LINE1 elements and the promoter region of Mecp2. In the hypothalamus, prenatal exposure to PolyI:C caused significant global DNA hypomethylation (t=2.44, P=0.019, PolyI:C mean 69.67%, saline mean 70.19%), especially in females, and significant hypomethylation of the promoter region of Mecp2, (t=3.32, P=0.002; PolyI:C mean 26.57%, saline mean 34.63%). IAP methylation was unaltered. DNA methylation in the striatum was not significantly altered. This study provides the first experimental evidence that exposure to inflammation during prenatal life is associated with epigenetic changes, including Mecp2 promoter hypomethylation. This suggests that environmental and genetic risk factors associated with neurodevelopmental disorders may act upon similar pathways. This is important because epigenetic changes are potentially modifiable and their investigation may open new avenues for treatment. PMID:25180573

E2F mediates induction of the Sp1-controlled promoter of the human DNA polymerase ɛ B-subunit gene POLE2

PubMed Central

Huang, Deqi; Jokela, Maarit; Tuusa, Jussi; Skog, Sven; Poikonen, Kari; Syväoja, Juhani E.

2001-01-01

The B-subunits of replicative DNA polymerases from Archaea to humans belong to the same protein family, suggesting that they share a common fundamental function. We report here the gene structure for the B-subunit of human DNA polymerase ɛ (POLE2), whose expression and transcriptional regulation is typical for replication proteins with some unique features. The 75 bp core promoter region, located within exon 1, contains an Sp1 element that is a critical determinant of promoter activity as shown by the luciferase reporter, electrophoretic mobility shift and DNase I footprinting assays. Two overlapping E2F elements adjacent to the Sp1 element are essential for full promoter activity and serum response. Binding sites for E2F1 and NF-1 reside immediately downstream from the core promoter region. Our results suggest that human POLE2 is regulated by two E2F–pocket protein complexes, one associated with Sp1 and the other with NF-1. So far, only one replicative DNA polymerase B-subunit gene promoter, POLA2 encoding the B-subunit of DNA polymerase α, has been characterized. Mitogenic activation of the POLE2 promoter by an E2F-mediated mechanism resembles that of POLA2, but the regulation of basal promoter activity is different between these two genes. PMID:11433027

Associations between genetic variation in one-carbon metabolism and LINE-1 DNA methylation in histologically normal breast tissues

USDA-ARS?s Scientific Manuscript database

Genome-wide DNA hypomethylation is an early event in the carcinogenic process. Percent methylation of long interspersed nucleotide element-1 (LINE-1) is a biomarker of genome-wide methylation and is a potential biomarker for breast cancer. Understanding factors associated with percent LINE-1 DNA met...

The DL1 repeats in the genome of Diphyllobothrium latum.

PubMed

Usmanova, Nadezhda M; Kazakov, Vasiliy I

2010-07-01

Diphyllobothrium latum is a widespread intestinal parasite, which has a great clinical relevance, but there are no sequences of its nuclear genome. In this paper, a repetitive element in the D. latum genome is firstly described. The adult D. latum was obtained in the result of expulsion from intestinum of a patient suffering from diphyllobothriasis. Genomic DNA was isolated from several proglottids of this individual. PstI restriction products of D. latum genomic DNA were sequenced. Polymerase chain reaction (PCR) amplification of these products using genomic DNA and selected primers was carried out. Thereby a cluster of a repetitive element, called DL1, was discovered. For precise identification of a beginning and an end of the repeat, a product of PCR amplification of D. latum genomic DNA with one specific primer was sequenced. In discussion, several evidences that DL1 repeat is a member of the SINE family of retroposons were adduced.

Genome-wide DNA methylation map of human neutrophils reveals widespread inter-individual epigenetic variation

PubMed Central

Chatterjee, Aniruddha; Stockwell, Peter A.; Rodger, Euan J.; Duncan, Elizabeth J.; Parry, Matthew F.; Weeks, Robert J.; Morison, Ian M.

2015-01-01

The extent of variation in DNA methylation patterns in healthy individuals is not yet well documented. Identification of inter-individual epigenetic variation is important for understanding phenotypic variation and disease susceptibility. Using neutrophils from a cohort of healthy individuals, we generated base-resolution DNA methylation maps to document inter-individual epigenetic variation. We identified 12851 autosomal inter-individual variably methylated fragments (iVMFs). Gene promoters were the least variable, whereas gene body and upstream regions showed higher variation in DNA methylation. The iVMFs were relatively enriched in repetitive elements compared to non-iVMFs, and were associated with genome regulation and chromatin function elements. Further, variably methylated genes were disproportionately associated with regulation of transcription, responsive function and signal transduction pathways. Transcriptome analysis indicates that iVMF methylation at differentially expressed exons has a positive correlation and local effect on the inclusion of that exon in the mRNA transcript. PMID:26612583

DNA sequence analysis of ARS elements from chromosome III of Saccharomyces cerevisiae: identification of a new conserved sequence.

PubMed Central

Palzkill, T G; Oliver, S G; Newlon, C S

1986-01-01

Four fragments of Saccharomyces cerevisiae chromosome III DNA which carry ARS elements have been sequenced. Each fragment contains multiple copies of sequences that have at least 10 out of 11 bases of homology to a previously reported 11 bp core consensus sequence. A survey of these new ARS sequences and previously reported sequences revealed the presence of an additional 11 bp conserved element located on the 3' side of the T-rich strand of the core consensus. Subcloning analysis as well as deletion and transposon insertion mutagenesis of ARS fragments support a role for 3' conserved sequence in promoting ARS activity. PMID:3529036

cDNA cloning, genomic organization and expression analysis during somatic embryogenesis of the translationally controlled tumor protein (TCTP) gene from Japanese larch (Larix leptolepis).

PubMed

Zhang, Li-Feng; Li, Wan-Feng; Han, Su-Ying; Yang, Wen-Hua; Qi, Li-Wang

2013-10-15

A full-length cDNA and genomic sequences of a translationally controlled tumor protein (TCTP) gene were isolated from Japanese larch (Larix leptolepis) and designated LaTCTP. The length of the cDNA was 1, 043 bp and contained a 504 bp open reading frame that encodes a predicted protein of 167 amino acids, characterized by two signature sequences of the TCTP protein family. Analysis of the LaTCTP gene structure indicated four introns and five exons, and it is the largest of all currently known TCTP genes in plants. The 5'-flanking promoter region of LaTCTP was cloned using an improved TAIL-PCR technique. In this region we identified many important potential cis-acting elements, such as a Box-W1 (fungal elicitor responsive element), a CAT-box (cis-acting regulatory element related to meristem expression), a CGTCA-motif (cis-acting regulatory element involved in MeJA-responsiveness), a GT1-motif (light responsive element), a Skn-1-motif (cis-acting regulatory element required for endosperm expression) and a TGA-element (auxin-responsive element), suggesting that expression of LaTCTP is highly regulated. Expression analysis demonstrated ubiquitous localization of LaTCTP mRNA in the roots, stems and needles, high mRNA levels in the embryonal-suspensor mass (ESM), browning embryogenic cultures and mature somatic embryos, and low levels of mRNA at day five during somatic embryogenesis. We suggest that LaTCTP might participate in the regulation of somatic embryo development. These results provide a theoretical basis for understanding the molecular regulatory mechanism of LaTCTP and lay the foundation for artificial regulation of somatic embryogenesis. © 2013.

Lactase non-persistence is directed by DNA variation-dependent epigenetic aging

PubMed Central

Labrie, Viviane; Buske, Orion J; Oh, Edward; Jeremian, Richie; Ptak, Carolyn; Gasiūnas, Giedrius; Maleckas, Almantas; Petereit, Rūta; Žvirbliene, Aida; Adamonis, Kęstutis; Kriukienė, Edita; Koncevičius, Karolis; Gordevičius, Juozas; Nair, Akhil; Zhang, Aiping; Ebrahimi, Sasha; Oh, Gabriel; Šikšnys, Virginijus; Kupčinskas, Limas; Brudno, Michael; Petronis, Arturas

2016-01-01

Inability to digest lactose due to lactase non-persistence is a common trait in adult mammals, with the exception of certain human populations that exhibit lactase persistence. It is not clear how the lactase gene can be dramatically downregulated with age in most individuals, but remains active in some. We performed a comprehensive epigenetic study of the human and mouse intestine using chromosome-wide DNA modification profiling and targeted bisulfite sequencing. Epigenetically-controlled regulatory elements were found to account for the differences in lactase mRNA levels between individuals, intestinal cell types and species. The importance of these regulatory elements in modulating lactase mRNA levels was confirmed by CRISPR-Cas9-induced deletions. Genetic factors contribute to epigenetic changes occurring with age at the regulatory elements, as lactase persistence- and non-persistence-DNA haplotypes demonstrated markedly different epigenetic aging. Thus, genetic factors facilitate a gradual accumulation of epigenetic changes with age to affect phenotypic outcome. PMID:27159559

Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster

PubMed Central

Simoni, Alekos; Siniscalchi, Carla; Chan, Yuk-Sang; Huen, David S.; Russell, Steven; Windbichler, Nikolai; Crisanti, Andrea

2014-01-01

Selfish genes are DNA elements that increase their rate of genetic transmission at the expense of other genes in the genome and can therefore quickly spread within a population. It has been suggested that selfish elements could be exploited to modify the genome of entire populations for medical and ecological applications. Here we report that transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) can be engineered into site-specific synthetic selfish elements (SSEs) and demonstrate their transmission of up to 70% in the Drosophila germline. We show here that SSEs can spread via DNA break-induced homologous recombination, a process known as ‘homing’ similar to that observed for homing endonuclease genes (HEGs), despite their fundamentally different modes of DNA binding and cleavage. We observed that TALEN and ZFN have a reduced capability of secondary homing compared to HEG as their repetitive structure had a negative effect on their genetic stability. The modular architecture of ZFNs and TALENs allows for the rapid design of novel SSEs against specific genomic sequences making them potentially suitable for the genetic engineering of wild-type populations of animals and plants, in applications such as gene replacement or population suppression of pest species. PMID:24803674

Tetris is a foldback transposon that provided the building blocks for an emerging satellite DNA of Drosophila virilis.

PubMed

Dias, Guilherme B; Svartman, Marta; Delprat, Alejandra; Ruiz, Alfredo; Kuhn, Gustavo C S

2014-05-24

Transposable elements (TEs) and satellite DNAs (satDNAs) are abundant components of most eukaryotic genomes studied so far and their impact on evolution has been the focus of several studies. A number of studies linked TEs with satDNAs, but the nature of their evolutionary relationships remains unclear. During in silico analyses of the Drosophila virilis assembled genome, we found a novel DNA transposon we named Tetris based on its modular structure and diversity of rearranged forms. We aimed to characterize Tetris and investigate its role in generating satDNAs. Data mining and sequence analysis showed that Tetris is apparently nonautonomous, with a structure similar to foldback elements, and present in D. virilis and D. americana. Herein, we show that Tetris shares the final portions of its terminal inverted repeats (TIRs) with DAIBAM, a previously described miniature inverted transposable element implicated in the generation of chromosome inversions. Both elements are likely to be mobilized by the same autonomous TE. Tetris TIRs contain approximately 220-bp internal tandem repeats that we have named TIR-220. We also found TIR-220 repeats making up longer (kb-size) satDNA-like arrays. Using bioinformatic, phylogenetic and cytogenomic tools, we demonstrated that Tetris has contributed to shaping the genomes of D. virilis and D. americana, providing internal tandem repeats that served as building blocks for the amplification of satDNA arrays. The β-heterochromatic genomic environment seemed to have favored such amplification. Our results imply for the first time a role for foldback elements in generating satDNAs. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

A unique mitigator sequence determines the species specificity of the major late promoter in adenovirus type 12 DNA.

PubMed Central

Zock, C; Iselt, A; Doerfler, W

1993-01-01

Human adenovirus type 12 (Ad12) cannot replicate in hamster cells, whereas human cells are permissive for Ad12. Ad12 DNA replication and late-gene and virus-associated RNA expression are blocked in hamster cells. Early Ad12 genes are transcribed, and the viral DNA can be integrated into the host genome. Ad12 DNA replication and late-gene transcription can be complemented in hamster cells by E1 functions of Ad2 or Ad5, for which hamster cells are fully permissive (for a review, see W. Doerfler, Adv. Virus Res. 39:89-128, 1991). We have previously demonstrated that a 33-nucleotide mitigator sequence, which is located in the downstream region of the major late promoter (MLP) of Ad12 DNA, is responsible for the inactivity of the Ad12 MLP in hamster cells (C. Zock and W. Doerfler, EMBO J. 9:1615-1623, 1990). A similar negative regulator has not been found in the MLP of Ad2 DNA. We have now studied the mechanism of action of this mitigator element. The results of nuclear run-on experiments document the absence of MLP transcripts in the nuclei of Ad12-infected BHK21 hamster cells. Surprisingly, the mitigator element cannot elicit its function in in vitro transcription experiments with nuclear extracts from both hamster BHK21 and human HeLa cells. Intact nuclear topology and/or tightly bound nuclear elements that cannot be eluted in nuclear extracts are somehow required for recognition of the Ad12 mitigator. Electrophoretic mobility shift assays have not revealed significant differences in the binding of proteins from human HeLa or hamster BHK21 cells to the mitigator sequence in the MLP of Ad12 DNA or to the corresponding sequence in Ad2 DNA. We have converted the sequence of the mitigator in the MLP of Ad12 DNA to the equivalent sequence in the MLP of Ad2 DNA by site-directed mutagenesis. This construct was not active in hamster cells. When the Ad12 mitigator, on the other hand, was inserted into the Ad2 MLP, the latter's function in hamster cells was not compromised. Deletions in the 5' upstream region of the Ad12 MLP have provided evidence for the existence of additional sequences that codetermine the deficiency of the Ad12 MLP in hamster cells. The amphifunctional YY1 protein from HeLa cells can bind specifically to the mitigator and to upstream elements of the MLP of Ad12 DNA.(ABSTRACT TRUNCATED AT 400 WORDS) Images PMID:8419643

Mechanisms of radiation-induced gene responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woloschak, G.E.; Paunesku, T.

1996-10-01

In the process of identifying genes differentially expressed in cells exposed ultraviolet radiation, we have identified a transcript having a 26-bp region that is highly conserved in a variety of species including Bacillus circulans, yeast, pumpkin, Drosophila, mouse, and man. When the 5` region (flanking region or UTR) of a gene, the sequence is predominantly in +/+ orientation with respect to the coding DNA strand; while in the coding region and the 3` region (UTR), the sequence is most frequently in the +/-orientation with respect to the coding DNA strand. In two genes, the element is split into two parts;more » however, in most cases, it is found only once but with a minimum of 11 consecutive nucleotides precisely depicting the original sequence. The element is found in a large number of different genes with diverse functions (from human ras p21 to B. circulans chitonase). Gel shift assays demonstrated the presence of a protein in HeLa cell extracts that binds to the sense and antisense single-stranded consensus oligomers, as well as to the double- stranded oligonucleotide. When double-stranded oligomer was used, the size shift demonstrated as additional protein-oligomer complex larger than the one bound to either sense or antisense single-stranded consensus oligomers alone. It is speculated either that this element binds to protein(s) important in maintaining DNA is a single-stranded orientation for transcription or, alternatively that this element is important in the transcription-coupled DNA repair process.« less

Co-located hAT transposable element and 5S rDNA in an interstitial telomeric sequence suggest the formation of Robertsonian fusion in armored catfish.

PubMed

Glugoski, Larissa; Giuliano-Caetano, Lucia; Moreira-Filho, Orlando; Vicari, Marcelo R; Nogaroto, Viviane

2018-04-15

Co-located 5S rDNA genes and interstitial telomeric sites (ITS) revealed the involvement of multiple 5S rDNA clusters in chromosome rearrangements of Loricariidae. Interstitial (TTAGGG)n vestiges, in addition to telomeric sites, can coincide with locations of chromosomal rearrangements, and they are considered to be hotspots for chromosome breaks. This study aimed the molecular characterization of 5S rDNA in two Rineloricaria latirostris populations and examination of roles of 5S rDNA in breakpoint sites and its in situ localization. Rineloricaria latirostris from Brazil's Das Pedras river (2n = 46 chromosomes) presented five pairs identified using a 5S rDNA probe, in addition to a pair bearing a co-located ITS/5S rDNA. Rineloricaria latirostris from the Piumhi river (2n = 48 chromosomes) revealed two pairs containing 5S rDNA, without ITS. A 702-bp amplified sequence, using 5S rDNA primers, revealed an insertion of the hAT transposable element (TE), referred to as a degenerate 5S rDNA. Double-FISH (fluorescence in situ hybridization) demonstrated co-localization of 5S rDNA/degenerate 5S rDNA, 5S rDNA/hAT and ITS/5S rDNA from the Das Pedras river population. Piumhi river isolates possessed only 5S rDNA sites. We suggest that the degenerate 5S rDNA was generated by unequal crossing over, which was driven by invasion of hAT, establishing a breakpoint region susceptible to chromosome breakage, non-homologous recombination and Robertsonian (Rb) fusion. Furthermore, the presence of clusters of 5S rDNA at fusion points in other armored catfish species suggests its re-use and that these regions represent hotspots for evolutionary rearrangements within Loricariidae genomes. Copyright © 2018 Elsevier B.V. All rights reserved.

The Dynamic Mu Transpososome: MuB activation prevents disintegration

PubMed Central

Lemberg, Kathryn M.; Schweidenback, Caterina T. H.; Baker, Tania A.

2007-01-01

Summary DNA transposases use a single active center to sequentially cleave the transposable element DNA and join this DNA to a target site. Recombination requires controlled conformational changes within the transposase to ensure that these chemically distinct steps occur at the right time and place, and that the reaction proceeds in the net forward direction. Mu transposition is catalyzed by a stable complex of MuA transposase bound to paired Mu DNA ends (a transpososome). We find that Mu transpososomes efficiently catalyze disintegration when recombination on one end of the Mu DNA is blocked. The MuB activator protein controls the integration vs. disintegration equilibrium. When MuB is present, disintegration occurs slowly and transpososomes that have disintegrated catalyze subsequent rounds of recombination. In the absence of MuB, disintegration goes to completion. These results together with experiments mapping the MuA-MuB contacts during DNA joining suggest that MuB controls progression of recombination by specifically stabilizing a concerted transition to the ‘joining’ configuration of MuA. Thus, we propose that MuB's interaction with the transpososome actively promotes coupled joining of both ends of the element DNA into the same target site and thus may provide a mechanism to antagonize formation of single-end transposition products. PMID:17988683

Different domains of the murine RNA polymerase I-specific termination factor mTTF-I serve distinct functions in transcription termination.

PubMed

Evers, R; Smid, A; Rudloff, U; Lottspeich, F; Grummt, I

1995-03-15

Termination of mouse ribosomal gene transcription by RNA polymerase I (Pol I) requires the specific interaction of a DNA binding protein, mTTF-I, with an 18 bp sequence element located downstream of the rRNA coding region. Here we describe the molecular cloning and functional characterization of the cDNA encoding this transcription termination factor. Recombinant mTTF-I binds specifically to the murine terminator elements and terminates Pol I transcription in a reconstituted in vitro system. Deletion analysis has defined a modular structure of mTTF-I comprising a dispensable N-terminal half, a large C-terminal DNA binding region and an internal domain which is required for transcription termination. Significantly, the C-terminal region of mTTF-I reveals striking homology to the DNA binding domains of the proto-oncogene c-Myb and the yeast transcription factor Reb1p. Site-directed mutagenesis of one of the tryptophan residues that is conserved in the homology region of c-Myb, Reb1p and mTTF-I abolishes specific DNA binding, a finding which underscores the functional relevance of these residues in DNA-protein interactions.

Different domains of the murine RNA polymerase I-specific termination factor mTTF-I serve distinct functions in transcription termination.

PubMed Central

Evers, R; Smid, A; Rudloff, U; Lottspeich, F; Grummt, I

1995-01-01

Termination of mouse ribosomal gene transcription by RNA polymerase I (Pol I) requires the specific interaction of a DNA binding protein, mTTF-I, with an 18 bp sequence element located downstream of the rRNA coding region. Here we describe the molecular cloning and functional characterization of the cDNA encoding this transcription termination factor. Recombinant mTTF-I binds specifically to the murine terminator elements and terminates Pol I transcription in a reconstituted in vitro system. Deletion analysis has defined a modular structure of mTTF-I comprising a dispensable N-terminal half, a large C-terminal DNA binding region and an internal domain which is required for transcription termination. Significantly, the C-terminal region of mTTF-I reveals striking homology to the DNA binding domains of the proto-oncogene c-Myb and the yeast transcription factor Reb1p. Site-directed mutagenesis of one of the tryptophan residues that is conserved in the homology region of c-Myb, Reb1p and mTTF-I abolishes specific DNA binding, a finding which underscores the functional relevance of these residues in DNA-protein interactions. Images PMID:7720715

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

PubMed

NandyMazumdar, Monali; Nedialkov, Yuri; Svetlov, Dmitri; Sevostyanova, Anastasia; Belogurov, Georgiy A; Artsimovitch, Irina

2016-12-27

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

DNA-nanostructure-assembly by sequential spotting

PubMed Central

2011-01-01

Background The ability to create nanostructures with biomolecules is one of the key elements in nanobiotechnology. One of the problems is the expensive and mostly custom made equipment which is needed for their development. We intended to reduce material costs and aimed at miniaturization of the necessary tools that are essential for nanofabrication. Thus we combined the capabilities of molecular ink lithography with DNA-self-assembling capabilities to arrange DNA in an independent array which allows addressing molecules in nanoscale dimensions. Results For the construction of DNA based nanostructures a method is presented that allows an arrangement of DNA strands in such a way that they can form a grid that only depends on the spotted pattern of the anchor molecules. An atomic force microscope (AFM) has been used for molecular ink lithography to generate small spots. The sequential spotting process allows the immobilization of several different functional biomolecules with a single AFM-tip. This grid which delivers specific addresses for the prepared DNA-strand serves as a two-dimensional anchor to arrange the sequence according to the pattern. Once the DNA-nanoarray has been formed, it can be functionalized by PNA (peptide nucleic acid) to incorporate advanced structures. Conclusions The production of DNA-nanoarrays is a promising task for nanobiotechnology. The described method allows convenient and low cost preparation of nanoarrays. PNA can be used for complex functionalization purposes as well as a structural element. PMID:22099392

Neurotoxic Doses of Chronic Methamphetamine Trigger Retrotransposition of the Identifier Element in Rat Dorsal Dentate Gyrus

PubMed Central

Moszczynska, Anna; Burghardt, Kyle J.; Yu, Dongyue

2017-01-01

Short interspersed elements (SINEs) are typically silenced by DNA hypermethylation in somatic cells, but can retrotranspose in proliferating cells during adult neurogenesis. Hypomethylation caused by disease pathology or genotoxic stress leads to genomic instability of SINEs. The goal of the present investigation was to determine whether neurotoxic doses of binge or chronic methamphetamine (METH) trigger retrotransposition of the identifier (ID) element, a member of the rat SINE family, in the dentate gyrus genomic DNA. Adult male Sprague-Dawley rats were treated with saline or high doses of binge or chronic METH and sacrificed at three different time points thereafter. DNA methylation analysis, immunohistochemistry and next-generation sequencing (NGS) were performed on the dorsal dentate gyrus samples. Binge METH triggered hypomethylation, while chronic METH triggered hypermethylation of the CpG-2 site. Both METH regimens were associated with increased intensities in poly(A)-binding protein 1 (PABP1, a SINE regulatory protein)-like immunohistochemical staining in the dentate gyrus. The amplification of several ID element sequences was significantly higher in the chronic METH group than in the control group a week after METH, and they mapped to genes coding for proteins regulating cell growth and proliferation, transcription, protein function as well as for a variety of transporters. The results suggest that chronic METH induces ID element retrotransposition in the dorsal dentate gyrus and may affect hippocampal neurogenesis. PMID:28272323

Cytoplasmic transfer of heritable elements other than mtDNA from SAMP1 mice into mouse tumor cells suppresses their ability to form tumors in C57BL6 mice.

PubMed

Shimizu, Akinori; Tani, Haruna; Takibuchi, Gaku; Ishikawa, Kaori; Sakurazawa, Ryota; Inoue, Takafumi; Hashimoto, Tetsuo; Nakada, Kazuto; Takenaga, Keizo; Hayashi, Jun-Ichi

2017-11-04

In a previous study, we generated transmitochondrial P29mtSAMP1 cybrids, which had nuclear DNA from the C57BL6 (referred to as B6) mouse strain-derived P29 tumor cells and mitochondrial DNA (mtDNA) exogenously-transferred from the allogeneic strain SAMP1. Because P29mtSAMP1 cybrids did not form tumors in syngeneic B6 mice, we proposed that allogeneic SAMP1 mtDNA suppressed tumor formation of P29mtSAMP1 cybrids. To test this hypothesis, current study generated P29mt(sp)B6 cybrids carrying all genomes (nuclear DNA and mtDNA) from syngeneic B6 mice by eliminating SAMP1 mtDNA from P29mtSAMP1 cybrids and reintroducing B6 mtDNA. However, the P29mt(sp)B6 cybrids did not form tumors in B6 mice, even though they had no SAMP1 mtDNA, suggesting that SAMP1 mtDNA is not involved in tumor suppression. Then, we examined another possibility of whether SAMP1 mtDNA fragments potentially integrated into the nuclear DNA of P29mtSAMP1 cybrids are responsible for tumor suppression. We generated P29 H (sp)B6 cybrids by eliminating nuclear DNA from P29mt(sp)B6 cybrids and reintroducing nuclear DNA with no integrated SAMP1 mtDNA fragment from mtDNA-less P29 cells resistant to hygromycin in selection medium containing hygromycin. However, the P29 H (sp)B6 cybrids did not form tumors in B6 mice, even though they carried neither SAMP1 mtDNA nor nuclear DNA with integrated SAMP1 mtDNA fragments. Moreover, overproduction of reactive oxygen species (ROS) and bacterial infection were not involved in tumor suppression. These observations suggest that tumor suppression was caused not by mtDNA with polymorphic mutations or infection of cytozoic bacteria but by hypothetical heritable cytoplasmic elements other than mtDNA from SAMP1 mice. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

A new family of satellite DNA sequences as a major component of centromeric heterochromatin in owls (Strigiformes).

PubMed

Yamada, Kazuhiko; Nishida-Umehara, Chizuko; Matsuda, Yoichi

2004-03-01

We isolated a new family of satellite DNA sequences from HaeIII- and EcoRI-digested genomic DNA of the Blakiston's fish owl ( Ketupa blakistoni). The repetitive sequences were organized in tandem arrays of the 174 bp element, and localized to the centromeric regions of all macrochromosomes, including the Z and W chromosomes, and microchromosomes. This hybridization pattern was consistent with the distribution of C-band-positive centromeric heterochromatin, and the satellite DNA sequences occupied 10% of the total genome as a major component of centromeric heterochromatin. The sequences were homogenized between macro- and microchromosomes in this species, and therefore intraspecific divergence of the nucleotide sequences was low. The 174 bp element cross-hybridized to the genomic DNA of six other Strigidae species, but not to that of the Tytonidae, suggesting that the satellite DNA sequences are conserved in the same family but fairly divergent between the different families in the Strigiformes. Secondly, the centromeric satellite DNAs were cloned from eight Strigidae species, and the nucleotide sequences of 41 monomer fragments were compared within and between species. Molecular phylogenetic relationships of the nucleotide sequences were highly correlated with both the taxonomy based on morphological traits and the phylogenetic tree constructed by DNA-DNA hybridization. These results suggest that the satellite DNA sequence has evolved by concerted evolution in the Strigidae and that it is a good taxonomic and phylogenetic marker to examine genetic diversity between Strigiformes species.

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

PubMed

Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

2017-05-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

High copy number of highly similar mariner-like transposons in planarian (Platyhelminthe): evidence for a trans-phyla horizontal transfer.

PubMed

Garcia-Fernàndez, J; Bayascas-Ramírez, J R; Marfany, G; Muñoz-Mármol, A M; Casali, A; Baguñà, J; Saló, E

1995-05-01

Several DNA sequences similar to the mariner element were isolated and characterized in the platyhelminthe Dugesia (Girardia) tigrina. They were 1,288 bp long, flanked by two 32 bp-inverted repeats, and contained a single 339 amino acid open-reading frame (ORF) encoding the transposase. The number of copies of this element is approximately 8,000 per haploid genome, constituting a member of the middle-repetitive DNA of Dugesia tigrina. Sequence analysis of several elements showed a high percentage of conservation between the different copies. Most of them presented an intact ORF and the standard signals of actively expressed genes, which suggests that some of them are or have recently been functional transposons. The high degree of similarity shared with other mariner elements from some arthropods, together with the fact that this element is undetectable in other planarian species, strongly suggests a case of horizontal transfer between these two distant phyla.

Using FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) to isolate active regulatory DNA

PubMed Central

Simon, Jeremy M.; Giresi, Paul G.; Davis, Ian J.; Lieb, Jason D.

2013-01-01

Eviction or destabilization of nucleosomes from chromatin is a hallmark of functional regulatory elements of the eukaryotic genome. Historically identified by nuclease hypersensitivity, these regulatory elements are typically bound by transcription factors or other regulatory proteins. FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) is an alternative approach to identify these genomic regions and has proven successful in a multitude of eukaryotic cell and tissue types. Cells or dissociated tissues are crosslinked briefly with formaldehyde, lysed, and sonicated. Sheared chromatin is subjected to phenol-chloroform extraction and the isolated DNA, typically encompassing 1–3% of the human genome, is purified. We provide guidelines for quantitative analysis by PCR, microarrays, or next-generation sequencing. Regulatory elements enriched by FAIRE display high concordance with those identified by nuclease hypersensitivity or ChIP, and the entire procedure can be completed in three days. FAIRE exhibits low technical variability, which allows its use in large-scale studies of chromatin from normal or diseased tissues. PMID:22262007

Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus.

PubMed

Lakshmikumaran, M S; D'Ambrosio, E; Laimins, L A; Lin, D T; Furano, A V

1985-09-01

The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this.

Mechanism of DNA-binding enhancement by the human T-cell leukaemia virus transactivator Tax.

PubMed

Baranger, A M; Palmer, C R; Hamm, M K; Giebler, H A; Brauweiler, A; Nyborg, J K; Schepartz, A

1995-08-17

Tax protein activates transcription of the human T-cell leukaemia virus type I (HTLV-I) genome through three imperfect cyclic AMP-responsive element (CRE) target sites located within the viral promoter. Previous work has shown that Tax interacts with the bZIP element of proteins that bind the CRE target site to promote peptide dimerization, suggesting an association between Tax and bZIP coiled coil. Here we show that the site of interaction with Tax is not the coiled coil, but the basic segment. This interaction increases the stability of the GCN4 bZIP dimer by 1.7 kcal mol-1 and the DNA affinity of the dimer by 1.9 kcal mol-1. The differential effect of Tax on several bZip-DNA complexes that differ in peptide sequence or DNA conformation suggests a model for Tax action based on stabilization of a distinct DNA-bound protein structure. This model may explain how Tax interacts with transcription factors of considerable sequence diversity to alter patterns of gene expression.

Characterization of "cis"-regulatory elements ("c"RE) associated with mammary gland function

USDA-ARS?s Scientific Manuscript database

The Bos taurus genome assembly has propelled dairy science into a new era; still, most of the information encoded in the genome has not yet been decoded. The human Encyclopedia of DNA Elements (ENCODE) project has spearheaded the identification and annotation of functional genomic elements in the hu...

Transposable genetic elements in Spirulina and potential applications for genetic engineering

NASA Astrophysics Data System (ADS)

Hiroyuki, Kojima; Qin, Song; Thankappan, Ajith Kumar; Yoshikazu, Kawata; Shin-Ichi, Yano

1998-03-01

Transposable elements in cyanobacteria are briefly reviewed. Evidence is presented to show that transposable elements in Spirulina platensis is actually reflected on the phenotype change, i e., helical to straight filaments. Transposition intermediates of DNA were isolated from the extrachromosome and the transposition was related to helical variations in Spirulina. Uses of transposable elements for microalgal recombination are discussed based on the transposition mechanism.

Mobile DNA in cancer. Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes.

PubMed

Tubio, Jose M C; Li, Yilong; Ju, Young Seok; Martincorena, Inigo; Cooke, Susanna L; Tojo, Marta; Gundem, Gunes; Pipinikas, Christodoulos P; Zamora, Jorge; Raine, Keiran; Menzies, Andrew; Roman-Garcia, Pablo; Fullam, Anthony; Gerstung, Moritz; Shlien, Adam; Tarpey, Patrick S; Papaemmanuil, Elli; Knappskog, Stian; Van Loo, Peter; Ramakrishna, Manasa; Davies, Helen R; Marshall, John; Wedge, David C; Teague, Jon W; Butler, Adam P; Nik-Zainal, Serena; Alexandrov, Ludmil; Behjati, Sam; Yates, Lucy R; Bolli, Niccolo; Mudie, Laura; Hardy, Claire; Martin, Sancha; McLaren, Stuart; O'Meara, Sarah; Anderson, Elizabeth; Maddison, Mark; Gamble, Stephen; Foster, Christopher; Warren, Anne Y; Whitaker, Hayley; Brewer, Daniel; Eeles, Rosalind; Cooper, Colin; Neal, David; Lynch, Andy G; Visakorpi, Tapio; Isaacs, William B; Veer, Laura Van't; Caldas, Carlos; Desmedt, Christine; Sotiriou, Christos; Aparicio, Sam; Foekens, John A; Eyfjörd, Jórunn Erla; Lakhani, Sunil R; Thomas, Gilles; Myklebost, Ola; Span, Paul N; Børresen-Dale, Anne-Lise; Richardson, Andrea L; Van de Vijver, Marc; Vincent-Salomon, Anne; Van den Eynden, Gert G; Flanagan, Adrienne M; Futreal, P Andrew; Janes, Sam M; Bova, G Steven; Stratton, Michael R; McDermott, Ultan; Campbell, Peter J

2014-08-01

Long interspersed nuclear element-1 (L1) retrotransposons are mobile repetitive elements that are abundant in the human genome. L1 elements propagate through RNA intermediates. In the germ line, neighboring, nonrepetitive sequences are occasionally mobilized by the L1 machinery, a process called 3' transduction. Because 3' transductions are potentially mutagenic, we explored the extent to which they occur somatically during tumorigenesis. Studying cancer genomes from 244 patients, we found that tumors from 53% of the patients had somatic retrotranspositions, of which 24% were 3' transductions. Fingerprinting of donor L1s revealed that a handful of source L1 elements in a tumor can spawn from tens to hundreds of 3' transductions, which can themselves seed further retrotranspositions. The activity of individual L1 elements fluctuated during tumor evolution and correlated with L1 promoter hypomethylation. The 3' transductions disseminated genes, exons, and regulatory elements to new locations, most often to heterochromatic regions of the genome. Copyright © 2014, American Association for the Advancement of Science.

Powering the programmed nanostructure and function of gold nanoparticles with catenated DNA machines

NASA Astrophysics Data System (ADS)

Elbaz, Johann; Cecconello, Alessandro; Fan, Zhiyuan; Govorov, Alexander O.; Willner, Itamar

2013-06-01

DNA nanotechnology is a rapidly developing research area in nanoscience. It includes the development of DNA machines, tailoring of DNA nanostructures, application of DNA nanostructures for computing, and more. Different DNA machines were reported in the past and DNA-guided assembly of nanoparticles represents an active research effort in DNA nanotechnology. Several DNA-dictated nanoparticle structures were reported, including a tetrahedron, a triangle or linear nanoengineered nanoparticle structures; however, the programmed, dynamic reversible switching of nanoparticle structures and, particularly, the dictated switchable functions emerging from the nanostructures, are missing elements in DNA nanotechnology. Here we introduce DNA catenane systems (interlocked DNA rings) as molecular DNA machines for the programmed, reversible and switchable arrangement of different-sized gold nanoparticles. We further demonstrate that the machine-powered gold nanoparticle structures reveal unique emerging switchable spectroscopic features, such as plasmonic coupling or surface-enhanced fluorescence.

Optimization of hCFTR Lung Expression in Mice Using DNA Nanoparticles

PubMed Central

Padegimas, Linas; Kowalczyk, Tomasz H; Adams, Sam; Gedeon, Chris R; Oette, Sharon M; Dines, Karla; Hyatt, Susannah L; Sesenoglu-Laird, Ozge; Tyr, Olena; Moen, Robert C; Cooper, Mark J

2012-01-01

Efficient and prolonged human cystic fibrosis transmembrane conductance regulator (hCFTR) expression is a major goal for cystic fibrosis (CF) lung therapy. A hCFTR expression plasmid was optimized as a payload for compacted DNA nanoparticles formulated with polyethylene glycol (PEG)-substituted 30-mer lysine peptides. A codon-optimized and CpG-reduced hCFTR synthetic gene (CO-CFTR) was placed in a polyubiquitin C expression plasmid. Compared to hCFTR complementary DNA (cDNA), CO-CFTR produced a ninefold increased level of hCFTR protein in transfected HEK293 cells and, when compacted as DNA nanoparticles, produced a similar improvement in lung mRNA expression in Balb/c and fatty acid binding protein promoter (FABP) CF mice, although expression duration was transient. Various vector modifications were tested to extend duration of CO-CFTR expression. A novel prolonged expression (PE) element derived from the bovine growth hormone (BGH) gene 3′ flanking sequence produced prolonged expression of CO-CFTR mRNA at biologically relevant levels. A time course study in the mouse lung revealed that CO-CFTR mRNA did not change significantly, with CO-CFTR/mCFTR geometric mean ratios of 94% on day 2, 71% on day 14, 53% on day 30, and 14% on day 59. Prolonged CO-CFTR expression is dependent on the orientation of the PE element and its transcription, is not specific to the UbC promoter, and is less dependent on other vector backbone elements. PMID:21952168

Bovine papilloma virus contains an activator of gene expression at the distal end of the early transcription unit.

PubMed Central

Lusky, M; Berg, L; Weiher, H; Botchan, M

1983-01-01

Bovine papilloma virus (BPV) contains a cis-acting DNA element which can enhance transcription of distal promoters. Utilizing both direct and indirect transient transfection assays, we showed that a 59-base-pair DNA sequence from the BPV genome could activate the simian virus 40 promoter from distances exceeding 2.5 kilobases and in an orientation-independent manner. In contrast to the promoter 5'-proximal localization of other known viral activators, this element was located immediately 3' to the early polyadenylation signal in the BPV genome. Deletion of these sequences from the BPV genome inactivated the transforming ability of BPV recombinant plasmids. Orientation-independent reinsertion of this 59-base-pair sequence, or alternatively of activator DNA sequences from simian virus 40 or polyoma virus, restored the transforming activity of the BPV recombinant plasmids. Furthermore, the stable transformation frequency of the herpes simplex virus type 1 thymidine kinase gene was enhanced when linked to restriction fragments of BPV DNA which included the defined activator element. This enhancement was orientation independent with respect to the thymidine kinase promoter. The enhancement also appeared to be unrelated to the establishment of the recombinant plasmids as episomes, since in transformed cells these sequences are found linked to high-molecular-weight DNA. We propose that the enhancement of stable transformation frequencies and the activation of transcription units are in this case alternate manifestations of the same biochemical events. Images PMID:6308425

An Integrated Encyclopedia of DNA Elements in the Human Genome

PubMed Central

2012-01-01

Summary The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure, and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall the project provides new insights into the organization and regulation of our genes and genome, and an expansive resource of functional annotations for biomedical research. PMID:22955616

Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter.

PubMed

Wang, Xiaohong; Liu, Haibin; Ge, Hui; Ajiro, Masahiko; Sharma, Nishi R; Meyers, Craig; Morozov, Pavel; Tuschl, Thomas; Klar, Amar; Court, Donald; Zheng, Zhi-Ming

2017-05-30

The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P 811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis -acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors. IMPORTANCE It has been known for decades that the activity of viral late promoters is associated with viral DNA replication among almost all DNA viruses. However, the mechanism of how DNA replication activates the viral late promoter and what components of the replication machinery are involved remain largely unknown. In this study, we characterized the P 811 promoter region of HPV18 and demonstrated that its activation depends on the orientation of DNA replication. Using single-stranded oligonucleotides targeting the replication fork on either leading or lagging strands, we showed that viral lagging-strand replication activates the promoter. We also identified a transcriptional repressor element located upstream of the promoter transcription start site which interacts with cellular proteins hnRNP D0B and hnRNP A/B and modulates the late promoter activity. This is the first report on how DNA replication activates a viral late promoter. Copyright © 2017 Wang et al.

Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity

PubMed Central

Song, Lingyun; Zhang, Zhancheng; Grasfeder, Linda L.; Boyle, Alan P.; Giresi, Paul G.; Lee, Bum-Kyu; Sheffield, Nathan C.; Gräf, Stefan; Huss, Mikael; Keefe, Damian; Liu, Zheng; London, Darin; McDaniell, Ryan M.; Shibata, Yoichiro; Showers, Kimberly A.; Simon, Jeremy M.; Vales, Teresa; Wang, Tianyuan; Winter, Deborah; Zhang, Zhuzhu; Clarke, Neil D.; Birney, Ewan; Iyer, Vishwanath R.; Crawford, Gregory E.; Lieb, Jason D.; Furey, Terrence S.

2011-01-01

The human body contains thousands of unique cell types, each with specialized functions. Cell identity is governed in large part by gene transcription programs, which are determined by regulatory elements encoded in DNA. To identify regulatory elements active in seven cell lines representative of diverse human cell types, we used DNase-seq and FAIRE-seq (Formaldehyde Assisted Isolation of Regulatory Elements) to map “open chromatin.” Over 870,000 DNaseI or FAIRE sites, which correspond tightly to nucleosome-depleted regions, were identified across the seven cell lines, covering nearly 9% of the genome. The combination of DNaseI and FAIRE is more effective than either assay alone in identifying likely regulatory elements, as judged by coincidence with transcription factor binding locations determined in the same cells. Open chromatin common to all seven cell types tended to be at or near transcription start sites and to be coincident with CTCF binding sites, while open chromatin sites found in only one cell type were typically located away from transcription start sites and contained DNA motifs recognized by regulators of cell-type identity. We show that open chromatin regions bound by CTCF are potent insulators. We identified clusters of open regulatory elements (COREs) that were physically near each other and whose appearance was coordinated among one or more cell types. Gene expression and RNA Pol II binding data support the hypothesis that COREs control gene activity required for the maintenance of cell-type identity. This publicly available atlas of regulatory elements may prove valuable in identifying noncoding DNA sequence variants that are causally linked to human disease. PMID:21750106

Sequence variations in RepMP2/3 and RepMP4 elements reveal intragenomic homologous DNA recombination events in Mycoplasma pneumoniae.

PubMed

Spuesens, Emiel B M; Oduber, Minoushka; Hoogenboezem, Theo; Sluijter, Marcel; Hartwig, Nico G; van Rossum, Annemarie M C; Vink, Cornelis

2009-07-01

The gene encoding major adhesin protein P1 of Mycoplasma pneumoniae, MPN141, contains two DNA sequence stretches, designated RepMP2/3 and RepMP4, which display variation among strains. This variation allows strains to be differentiated into two major P1 genotypes (1 and 2) and several variants. Interestingly, multiple versions of the RepMP2/3 and RepMP4 elements exist at other sites within the bacterial genome. Because these versions are closely related in sequence, but not identical, it has been hypothesized that they have the capacity to recombine with their counterparts within MPN141, and thereby serve as a source of sequence variation of the P1 protein. In order to determine the variation within the RepMP2/3 and RepMP4 elements, both within the bacterial genome and among strains, we analysed the DNA sequences of all RepMP2/3 and RepMP4 elements within the genomes of 23 M. pneumoniae strains. Our data demonstrate that: (i) recombination is likely to have occurred between two RepMP2/3 elements in four of the strains, and (ii) all previously described P1 genotypes can be explained by inter-RepMP recombination events. Moreover, the difference between the two major P1 genotypes was reflected in all RepMP elements, such that subtype 1 and 2 strains can be differentiated on the basis of sequence variation in each RepMP element. This implies that subtype 1 and subtype 2 strains represent evolutionarily diverged strain lineages. Finally, a classification scheme is proposed in which the P1 genotype of M. pneumoniae isolates can be described in a sequence-based, universal fashion.

Epigenetic regulation of transcription and possible functions of mammalian short interspersed elements, SINEs.

PubMed

Ichiyanagi, Kenji

2013-01-01

Short interspersed elements (SINEs) are a class of retrotransposons, which amplify their copy numbers in their host genomes by retrotransposition. More than a million copies of SINEs are present in a mammalian genome, constituting over 10% of the total genomic sequence. In contrast to the other two classes of retrotransposons, long interspersed elements (LINEs) and long terminal repeat (LTR) elements, SINEs are transcribed by RNA polymerase III. However, like LINEs and LTR elements, the SINE transcription is likely regulated by epigenetic mechanisms such as DNA methylation, at least for human Alu and mouse B1. Whereas SINEs and other transposable elements have long been thought as selfish or junk DNA, recent studies have revealed that they play functional roles at their genomic locations, for example, as distal enhancers, chromatin boundaries and binding sites of many transcription factors. These activities imply that SINE retrotransposition has shaped the regulatory network and chromatin landscape of their hosts. Whereas it is thought that the epigenetic mechanisms were originated as a host defense system against proliferation of parasitic elements, this review discusses a possibility that the same mechanisms are also used to regulate the SINE-derived functions.

[Structure and function of eukaryotic nuclear DNA-dependent RNA polymerase I].

PubMed

Shematorova, E K; Shpakovskiĭ, G V

2002-01-01

In the eukaryotic cell, normal protein biosynthesis is sustained by several million ribosomes, which contain rRNA as an essential component. The high-molecular-weight precursor of large and 5.8S rRNAs is synthesized by DNA-dependent RNA polymerase I (Pol I) in the nucleolus. Data on DNA regulatory elements, protein factors involved in rDNA transcription by Pol I, subunit composition of Pol I, and on the interactions and possible functions of individual subunits are summarized.

Transitional circuitry for studying the properties of DNA

NASA Astrophysics Data System (ADS)

Trubochkina, N.

2018-01-01

The article is devoted to a new view of the structure of DNA as an intellectual scheme possessing the properties of logic and memory. The theory of transient circuitry, developed by the author for optimal computer circuits, revealed an amazing structural similarity between mathematical models of transition silicon elements and logic and memory circuits of solid state transient circuitry and atomic models of parts of DNA.

The Interplay Between Estrogen and Replication Origins in Breast Cancer DNA Amplification

DTIC Science & Technology

2013-09-01

Replication Origins in Breast Cancer DNA Amplification PRINCIPAL INVESTIGATOR: Cinzia Casella CONTRACTING ORGANIZATION: Brown...Interplay Between Estrogen and Replication Origins in Breast Cancer DNA Amplification 5b. GRANT NUMBER W81XWH-11-1-0599 5c. PROGRAM ELEMENT NUMBER 6... amplification and oncogenes activation in breast cancer cells? This project aims to understand the role of estrogen in inducing re-replication, thus

Molecular beacon-based real-time PCR method for detection of porcine DNA in gelatin and gelatin capsules.

PubMed

Mohamad, Nurhidayatul Asma; Mustafa, Shuhaimi; Khairil Mokhtar, Nur Fadhilah; El Sheikha, Aly Farag

2018-03-05

The pharmaceutical industry has boosted gelatin consumption worldwide. This is supported by the availability of cost-effective gelatin production from porcine by-products. However, cross-contamination of gelatin materials, where porcine gelatin was unintentionally included in the other animal sources of gelatin, has caused significant concerns about halal authenticity. The real-time polymerase chain reaction (PCR) has enabled a highly specific and sensitive animal species detection method in various food products. Hence, such a technique was employed in the present study to detect and quantify porcine DNA in gelatin using a molecular beacon probe, with differences in performance between mitochondrial (cytochrome b gene) and chromosomal DNA-(MPRE42 repetitive element) based porcine-specific PCR assays being compared. A higher sensitivity was observed in chromosomal DNA (MPRE-PCR assay), where this assay allows the detection of gelatin DNA at amounts as as low as 1 pg, whereas mitochondrial DNA (CBH-PCR assay) can only detect at levels down to 10 pg of gelatin DNA. When an analysis with commercial gelatin and gelatin capsule samples was conducted, the same result was observed, with a significantly more sensitive detection being provided by the repetitive element of chromosomal DNA. The present study has established highly sensitive DNA-based porcine detection systems derived from chromosomal DNA that are feasible for highly processed products such as gelatin and gelatin capsules containing a minute amount of DNA. This sensitive detection method can also be implemented to assist the halal authentication process of various food products available on the market. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

The Evolution of Tyrosine-Recombinase Elements in Nematoda

PubMed Central

Szitenberg, Amir; Koutsovoulos, Georgios; Blaxter, Mark L.; Lunt, David H.

2014-01-01

Transposable elements can be categorised into DNA and RNA elements based on their mechanism of transposition. Tyrosine recombinase elements (YREs) are relatively rare and poorly understood, despite sharing characteristics with both DNA and RNA elements. Previously, the Nematoda have been reported to have a substantially different diversity of YREs compared to other animal phyla: the Dirs1-like YRE retrotransposon was encountered in most animal phyla but not in Nematoda, and a unique Pat1-like YRE retrotransposon has only been recorded from Nematoda. We explored the diversity of YREs in Nematoda by sampling broadly across the phylum and including 34 genomes representing the three classes within Nematoda. We developed a method to isolate and classify YREs based on both feature organization and phylogenetic relationships in an open and reproducible workflow. We also ensured that our phylogenetic approach to YRE classification identified truncated and degenerate elements, informatively increasing the number of elements sampled. We identified Dirs1-like elements (thought to be absent from Nematoda) in the nematode classes Enoplia and Dorylaimia indicating that nematode model species do not adequately represent the diversity of transposable elements in the phylum. Nematode Pat1-like elements were found to be a derived form of another Pat1-like element that is present more widely in animals. Several sequence features used widely for the classification of YREs were found to be homoplasious, highlighting the need for a phylogenetically-based classification scheme. Nematode model species do not represent the diversity of transposable elements in the phylum. PMID:25197791

The evolution of tyrosine-recombinase elements in Nematoda.

PubMed

Szitenberg, Amir; Koutsovoulos, Georgios; Blaxter, Mark L; Lunt, David H

2014-01-01

Transposable elements can be categorised into DNA and RNA elements based on their mechanism of transposition. Tyrosine recombinase elements (YREs) are relatively rare and poorly understood, despite sharing characteristics with both DNA and RNA elements. Previously, the Nematoda have been reported to have a substantially different diversity of YREs compared to other animal phyla: the Dirs1-like YRE retrotransposon was encountered in most animal phyla but not in Nematoda, and a unique Pat1-like YRE retrotransposon has only been recorded from Nematoda. We explored the diversity of YREs in Nematoda by sampling broadly across the phylum and including 34 genomes representing the three classes within Nematoda. We developed a method to isolate and classify YREs based on both feature organization and phylogenetic relationships in an open and reproducible workflow. We also ensured that our phylogenetic approach to YRE classification identified truncated and degenerate elements, informatively increasing the number of elements sampled. We identified Dirs1-like elements (thought to be absent from Nematoda) in the nematode classes Enoplia and Dorylaimia indicating that nematode model species do not adequately represent the diversity of transposable elements in the phylum. Nematode Pat1-like elements were found to be a derived form of another Pat1-like element that is present more widely in animals. Several sequence features used widely for the classification of YREs were found to be homoplasious, highlighting the need for a phylogenetically-based classification scheme. Nematode model species do not represent the diversity of transposable elements in the phylum.

Two CGTCA motifs and a GHF1/Pit1 binding site mediate cAMP-dependent protein kinase A regulation of human growth hormone gene expression in rat anterior pituitary GC cells.

PubMed

Shepard, A R; Zhang, W; Eberhardt, N L

1994-01-21

We established the cis-acting elements which mediate cAMP responsiveness of the human growth hormone (hGH) gene in transiently transfected rat anterior pituitary tumor GC cells. Analysis of the intact hGH gene or hGH 5'-flanking DNA (5'-FR) coupled to the hGh cDNA or chloramphenicol acetyltransferase or luciferase genes, indicated that cAMP primarily stimulated hGH promoter activity. Cotransfection of a protein kinase A inhibitory protein cDNA demonstrated that the cAMP response was mediated by protein kinase A. Mutational analysis of the hGH promoter identified two core cAMP response element motifs (CGTCA) located at nucleotides -187/-183 (distal cAMP response element; dCRE) and -99/-95 (proximal cAMP response element; pCRE) and a pituitary-specific transcription factor (GHF1/Pit1) binding site at nucleotides -123/-112 (dGHF1) which were required for cAMP responsiveness. GHF1 was not a limiting factor, since overexpression of GHF1 in cotransfections increased basal but not forskolin induction levels. Gel shift analyses indicated that similar, ubiquitous, thermostable protein(s) specifically bound the pCRE and dCRE motifs. The CGTCA motif-binding factors were cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1)-related, since the DNA-protein complex was competed by unlabeled CREB consensus oligonucleotide, specifically supershifted by antisera to CREB and ATF-1 but not ATF-2, and was bound by purified CREB with the same relative binding affinity (pCRE < dCRE < CREB) and mobility as the GC nuclear extract. UV cross-linking and Southwestern blot analyses revealed multiple DNA-protein interactions of which approximately 100- and approximately 45-kDa proteins were predominant; the approximately 45-kDa protein may represent CREB. These results indicate that CREB/ATF-1-related factors act coordinately with the cell-specific factor GHF1 to mediate cAMP-dependent regulation of hGH-1 gene transcription in anterior pituitary somatotrophs.

The SIDER2 elements, interspersed repeated sequences that populate the Leishmania genomes, constitute subfamilies showing chromosomal proximity relationship.

PubMed

Requena, Jose M; Folgueira, Cristina; López, Manuel C; Thomas, M Carmen

2008-06-02

Protozoan parasites of the genus Leishmania are causative agents of a diverse spectrum of human diseases collectively known as leishmaniasis. These eukaryotic pathogens that diverged early from the main eukaryotic lineage possess a number of unusual genomic, molecular and biochemical features. The completion of the genome projects for three Leishmania species has generated invaluable information enabling a direct analysis of genome structure and organization. By using DNA macroarrays, made with Leishmania infantum genomic clones and hybridized with total DNA from the parasite, we identified a clone containing a repeated sequence. An analysis of the recently completed genome sequence of L. infantum, using this repeated sequence as bait, led to the identification of a new class of repeated elements that are interspersed along the different L. infantum chromosomes. These elements turned out to be homologues of SIDER2 sequences, which were recently identified in the Leishmania major genome; thus, we adopted this nomenclature for the Leishmania elements described herein. Since SIDER2 elements are very heterogeneous in sequence, their precise identification is rather laborious. We have characterized 54 LiSIDER2 elements in chromosome 32 and 27 ones in chromosome 20. The mean size for these elements is 550 bp and their sequence is G+C rich (mean value of 66.5%). On the basis of sequence similarity, these elements can be grouped in subfamilies that show a remarkable relationship of proximity, i.e. SIDER2s of a given subfamily locate close in a chromosomal region without intercalating elements. For comparative purposes, we have identified the SIDER2 elements existing in L. major and Leishmania braziliensis chromosomes 32. While SIDER2 elements are highly conserved both in number and location between L. infantum and L. major, no such conservation exists when comparing with SIDER2s in L. braziliensis chromosome 32. SIDER2 elements constitute a relevant piece in the Leishmania genome organization. Sequence characteristics, genomic distribution and evolutionarily conservation of SIDER2s are suggestive of relevant functions for these elements in Leishmania. Apart from a proved involvement in post-transcriptional mechanisms of gene regulation, SIDER2 elements could be involved in DNA amplification processes and, perhaps, in chromosome segregation as centromeric sequences.

Promoter-proximal rDNA terminator augments initiation by preventing disruption of the stable transcription complex caused by polymerase read-in

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henderson, S.L.; Ryan, K.; Sollner-Webb, B.

1989-02-01

We have examined the mechanism by which transcriptional initiation at the mouse rDNA promoter is augmented by the RNA polymerase I terminator element that resides just upstream of it. Using templates in which terminator elements are instead positioned at the opposite side of the plasmid rather than proximal to the promoter, or conditions where transcription is terminated elsewhere in the plasmid by UV-induced lesions, we show that the terminator's stimulatory effect is not position dependent. Mouse terminator elements therefore do not stimulate via the previously postulated 'read-through enhancement' model in which terminated polymerases are handed off to an adjacent promotermore » in a concerted reaction. The position independence and orientation dependence of the terminator also makes it unlikely that the terminator functions as a promoter element or as an enhancer. Instead, terminators serve to augment initiation by preventing polymerases from reading completely around the plasmid and through the promoter from upstream, an event which we show interferes with subsequent rounds of initiation. Notably, this transcriptional interference arises because polymerase passage across a promoter disrupts the otherwise stable transcription complex, specifically releasing the bound transcription factor D. These liberated D molecules can then bind to other templates and activate their expression. The rDNA transcriptional interference is not due to a steric impediment to the binding of new polymerase molecules, and it does not similarly liberate the initiation-competent polymerase (factor C). These studies have also convincingly demonstrated that multiple rounds of transcription are obtained from rDNA template molecules in vitro.« less

Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stark, M.; Naiman, T.; Canaani, D.

1989-08-15

In a previous work, an immortal xeroderma pigmentosum cell line belonging to complementation group C was complemented to a UV-resistant phenotype by transfection with a human cDNA clone library. We now report that the primary transformants selected for UV-resistance also acquired normal levels of DNA repair. This was assessed both by measurement of UV-induced ({sup 3}H)thymidine incorporation and by equilibrium sedimentation analysis of repair-DNA synthesis. Therefore, the transduced DNA element which confers normal UV-resistance also corrects the excision repair defect of the xeroderma pigmentosum group C cell line.

Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster.

PubMed

Simoni, Alekos; Siniscalchi, Carla; Chan, Yuk-Sang; Huen, David S; Russell, Steven; Windbichler, Nikolai; Crisanti, Andrea

2014-06-01

Selfish genes are DNA elements that increase their rate of genetic transmission at the expense of other genes in the genome and can therefore quickly spread within a population. It has been suggested that selfish elements could be exploited to modify the genome of entire populations for medical and ecological applications. Here we report that transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) can be engineered into site-specific synthetic selfish elements (SSEs) and demonstrate their transmission of up to 70% in the Drosophila germline. We show here that SSEs can spread via DNA break-induced homologous recombination, a process known as 'homing' similar to that observed for homing endonuclease genes (HEGs), despite their fundamentally different modes of DNA binding and cleavage. We observed that TALEN and ZFN have a reduced capability of secondary homing compared to HEG as their repetitive structure had a negative effect on their genetic stability. The modular architecture of ZFNs and TALENs allows for the rapid design of novel SSEs against specific genomic sequences making them potentially suitable for the genetic engineering of wild-type populations of animals and plants, in applications such as gene replacement or population suppression of pest species. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Distribution of Unlinked Transpositions of a Ds Element from a T-DNA Locus on Tomato Chromosome 4

PubMed Central

Briza, J.; Carroll, B. J.; Klimyuk, V. I.; Thomas, C. M.; Jones, D. A.; Jones, JDG.

1995-01-01

In maize, receptor sites for unlinked transpositions of Activator (Ac) elements are not distributed randomly. To test whether the same is true in tomato, the receptor sites for a Dissociation (Ds) element derived from Ac, were mapped for 26 transpositions unlinked to a donor T-DNA locus on chromosome 4. Four independent transposed Dss mapped to sites on chromosome 4 genetically unlinked to the donor T-DNA, consistent with a preference for transposition to unlinked sites on the same chromosome as opposed to sites on other chromosomes. There was little preference among the nondonor chromosomes, except perhaps for chromosome 2, which carried seven transposed Dss, but these could not be proven to be independent. However, these data, when combined with those from other studies in tomato examining the distribution of transposed Acs or Dss among nondonor chromosomes, suggest there may be absolute preferences for transposition irrespective of the chromosomal location of the donor site. If true, transposition to nondonor chromosomes in tomato would differ from that in maize, where the preference seems to be determined by the spatial arrangement of chromosomes in the interphase nucleus. The tomato lines carrying Ds elements at known locations are available for targeted transposon tagging experiments. PMID:8536985

Evolution of Genome Size and Complexity in Pinus

PubMed Central

Morse, Alison M.; Peterson, Daniel G.; Islam-Faridi, M. Nurul; Smith, Katherine E.; Magbanua, Zenaida; Garcia, Saul A.; Kubisiak, Thomas L.; Amerson, Henry V.; Carlson, John E.; Nelson, C. Dana; Davis, John M.

2009-01-01

Background Genome evolution in the gymnosperm lineage of seed plants has given rise to many of the most complex and largest plant genomes, however the elements involved are poorly understood. Methodology/Principal Findings Gymny is a previously undescribed retrotransposon family in Pinus that is related to Athila elements in Arabidopsis. Gymny elements are dispersed throughout the modern Pinus genome and occupy a physical space at least the size of the Arabidopsis thaliana genome. In contrast to previously described retroelements in Pinus, the Gymny family was amplified or introduced after the divergence of pine and spruce (Picea). If retrotransposon expansions are responsible for genome size differences within the Pinaceae, as they are in angiosperms, then they have yet to be identified. In contrast, molecular divergence of Gymny retrotransposons together with other families of retrotransposons can account for the large genome complexity of pines along with protein-coding genic DNA, as revealed by massively parallel DNA sequence analysis of Cot fractionated genomic DNA. Conclusions/Significance Most of the enormous genome complexity of pines can be explained by divergence of retrotransposons, however the elements responsible for genome size variation are yet to be identified. Genomic resources for Pinus including those reported here should assist in further defining whether and how the roles of retrotransposons differ in the evolution of angiosperm and gymnosperm genomes. PMID:19194510

AzaHx, a novel fluorescent, DNA minor groove and G·C recognition element: Synthesis and DNA binding properties of a p-anisyl-4-aza-benzimidazole-pyrrole-imidazole (azaHx-PI) polyamide.

PubMed

Satam, Vijay; Babu, Balaji; Patil, Pravin; Brien, Kimberly A; Olson, Kevin; Savagian, Mia; Lee, Megan; Mepham, Andrew; Jobe, Laura Beth; Bingham, John P; Pett, Luke; Wang, Shuo; Ferrara, Maddi; Bruce, Chrystal D; Wilson, W David; Lee, Moses; Hartley, John A; Kiakos, Konstantinos

2015-09-01

The design, synthesis, and DNA binding properties of azaHx-PI or p-anisyl-4-aza-benzimidazole-pyrrole-imidazole (5) are described. AzaHx, 2-(p-anisyl)-4-aza-benzimidazole-5-carboxamide, is a novel, fluorescent DNA recognition element, derived from Hoechst 33258 to recognize G·C base pairs. Supported by theoretical data, the results from DNase I footprinting, CD, ΔT(M), and SPR studies provided evidence that an azaHx/IP pairing, formed from antiparallel stacking of two azaHx-PI molecules in a side-by-side manner in the minor groove, selectively recognized a C-G doublet. AzaHx-PI was found to target 5'-ACGCGT-3', the Mlu1 Cell Cycle Box (MCB) promoter sequence with specificity and significant affinity (K(eq) 4.0±0.2×10(7) M(-1)). Copyright © 2015 Elsevier Ltd. All rights reserved.

A SHORT SEQUENCE IMMEDIATELY UPSTREAM OF THE INTERNAL REPEAT ELEMENTS IS CRITICAL FOR KSHV LANA MEDIATED DNA REPLICATION AND IMPACTS EPISOME PERSISTENCE

PubMed Central

León Vázquez, Erika De; Juillard, Franceline; Rosner, Bernard; Kaye, Kenneth M.

2013-01-01

Kaposi’s sarcoma-associated herpesvirus LANA (1162 residues) mediates episomal persistence of viral genomes during latency. LANA mediates viral DNA replication and segregates episomes to daughter nuclei. A 59 residue deletion immediately upstream of the internal repeat elements rendered LANA highly deficient for DNA replication and modestly deficient for the ability to segregate episomes, while smaller deletions did not. The 59 amino acid deletion reduced LANA episome persistence by ~14-fold, while sequentially smaller deletions resulted in ~3-fold, or no deficiency. Three distinct LANA regions reorganized heterochromatin, one of which contains the deleted sequence, but the deletion did not abolish LANA’s ability to alter chromatin. Therefore, this work identifies a short internal LANA sequence that is critical for DNA replication, has modest effects on episome segregation, and substantially impacts episome persistence; this region may exert its effects through an interacting host cell protein(s). PMID:24314665

Nanopore sequencing technology: a new route for the fast detection of unauthorized GMO.

PubMed

Fraiture, Marie-Alice; Saltykova, Assia; Hoffman, Stefan; Winand, Raf; Deforce, Dieter; Vanneste, Kevin; De Keersmaecker, Sigrid C J; Roosens, Nancy H C

2018-05-21

In order to strengthen the current genetically modified organism (GMO) detection system for unauthorized GMO, we have recently developed a new workflow based on DNA walking to amplify unknown sequences surrounding a known DNA region. This DNA walking is performed on transgenic elements, commonly found in GMO, that were earlier detected by real-time PCR (qPCR) screening. Previously, we have demonstrated the ability of this approach to detect unauthorized GMO via the identification of unique transgene flanking regions and the unnatural associations of elements from the transgenic cassette. In the present study, we investigate the feasibility to integrate the described workflow with the MinION Next-Generation-Sequencing (NGS). The MinION sequencing platform can provide long read-lengths and deal with heterogenic DNA libraries, allowing for rapid and efficient delivery of sequences of interest. In addition, the ability of this NGS platform to characterize unauthorized and unknown GMO without any a priori knowledge has been assessed.

The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

PubMed Central

Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

2009-01-01

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

Quercetin-Iron Complex: Synthesis, Characterization, Antioxidant, DNA Binding, DNA Cleavage, and Antibacterial Activity Studies.

PubMed

Raza, Aun; Xu, Xiuquan; Xia, Li; Xia, Changkun; Tang, Jian; Ouyang, Zhen

2016-11-01

Quercetin-iron (II) complex was synthesized and characterized by elemental analysis, ultraviolet-visible spectrophotometry, fourier transform infrared spectroscopy, mass spectrometry, proton nuclear magnetic resonance spectroscopy, thermogravimetry and differential scanning calorimetry, scanning electron micrography and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal:ligand) of the complex. Antioxidant study of the quercetin and its metal complex against 2, 2-di-phenyl-1-picryl hydrazyl radical showed that the complex has much more radical scavenging activity than free quercetin. The interaction of quercetin-iron (II) complex with DNA was determined using ultraviolet visible spectra, fluorescence spectra and agarose gel electrophoresis. The results showed that quercetin-iron (II) complex can intercalate moderately with DNA, quench a strong intercalator ethidium bromide and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form to nicked circular form and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was an oxidative cleavage pathway. These results revealed the potential nuclease activity of complex to cleave DNA. In addition, antibacterial activity of complex on E.coli and S. aureus was also investigated. The results showed that complex has higher antibacterial activity than ligand.

Spy: a new group of eukaryotic DNA transposons without target site duplications.

PubMed

Han, Min-Jin; Xu, Hong-En; Zhang, Hua-Hao; Feschotte, Cédric; Zhang, Ze

2014-06-24

Class 2 or DNA transposons populate the genomes of most eukaryotes and like other mobile genetic elements have a profound impact on genome evolution. Most DNA transposons belong to the cut-and-paste types, which are relatively simple elements characterized by terminal-inverted repeats (TIRs) flanking a single gene encoding a transposase. All eukaryotic cut-and-paste transposons so far described are also characterized by target site duplications (TSDs) of host DNA generated upon chromosomal insertion. Here, we report a new group of evolutionarily related DNA transposons called Spy, which also include TIRs and DDE motif-containing transposase but surprisingly do not create TSDs upon insertion. Instead, Spy transposons appear to transpose precisely between 5'-AAA and TTT-3' host nucleotides, without duplication or modification of the AAATTT target sites. Spy transposons were identified in the genomes of diverse invertebrate species based on transposase homology searches and structure-based approaches. Phylogenetic analyses indicate that Spy transposases are distantly related to IS5, ISL2EU, and PIF/Harbinger transposases. However, Spy transposons are distinct from these and other DNA transposon superfamilies by their lack of TSD and their target site preference. Our findings expand the known diversity of DNA transposons and reveal a new group of eukaryotic DDE transposases with unusual catalytic properties. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schellenberg, Matthew J; Appel, C Denise; Adhikari, Sanjay

The topoisomerase II (topo II) DNA incision-and-ligation cycle can be poisoned (for example following treatment with cancer chemotherapeutics) to generate cytotoxic DNA double-strand breaks (DSBs) with topo II covalently conjugated to DNA. Tyrosyl-DNA phosphodiesterase 2 (Tdp2) protects genomic integrity by reversing 5'-phosphotyrosyl–linked topo II–DNA adducts. Here, X-ray structures of mouse Tdp2–DNA complexes reveal that Tdp2 β–2-helix–β DNA damage–binding 'grasp', helical 'cap' and DNA lesion–binding elements fuse to form an elongated protein-DNA conjugate substrate-interaction groove. The Tdp2 DNA-binding surface is highly tailored for engagement of 5'-adducted single-stranded DNA ends and restricts nonspecific endonucleolytic or exonucleolytic processing. Structural, mutational and functional analysesmore » support a single–metal ion catalytic mechanism for the exonuclease-endonuclease-phosphatase (EEP) nuclease superfamily and establish a molecular framework for targeted small-molecule blockade of Tdp2-mediated resistance to anticancer topoisomerase drugs.« less

DNA Fingerprinting of Lactobacillus crispatus Strain CTV-05 by Repetitive Element Sequence-Based PCR Analysis in a Pilot Study of Vaginal Colonization

PubMed Central

Antonio, May A. D.; Hillier, Sharon L.

2003-01-01

Lactobacillus crispatus is one of the predominant hydrogen peroxide (H2O2)-producing species found in the vagina and is under development as a probiotic for the treatment of bacterial vaginosis. In this study, we assessed whether DNA fingerprinting by repetitive element sequence-based PCR (rep-PCR) can be used to distinguish the capsule strain of L. crispatus (CTV-05) from other endogenous strains as well as other species of vaginal lactobacilli. Vaginal and rectal lactobacilli were identified to the species level by using whole-chromosome probe DNA hybridization. The DNAs from L. crispatus, L. jensenii, L. gasseri, and an as-yet-unnamed H2O2-negative Lactobacillus species designated 1086V were subjected to rep-PCR. The results of gel electrophoresis and ethidium bromide staining of the DNA fingerprints obtained were compared. L. crispatus CTV-05 had a unique DNA fingerprint compared to all other lactobacilli. DNA fingerprints for 27 production lots of L. crispatus sampled from 1994 through 2001 were identical to that of the original strain isolated in 1993, suggesting strain stability. In a pilot study of nine women, this DNA fingerprinting method distinguished CTV-05 from other endogenous vaginal lactobacilli prior to and after vaginal capsule use. rep-PCR DNA fingerprinting is useful for strain typing and for evaluating longitudinal loss or acquisition of vaginal lactobacilli used as probiotics. PMID:12734221

Evolution in the block: common elements of 5S rDNA organization and evolutionary patterns in distant fish genera.

PubMed

Campo, Daniel; García-Vázquez, Eva

2012-01-01

The 5S rDNA is organized in the genome as tandemly repeated copies of a structural unit composed of a coding sequence plus a nontranscribed spacer (NTS). The coding region is highly conserved in the evolution, whereas the NTS vary in both length and sequence. It has been proposed that 5S rRNA genes are members of a gene family that have arisen through concerted evolution. In this study, we describe the molecular organization and evolution of the 5S rDNA in the genera Lepidorhombus and Scophthalmus (Scophthalmidae) and compared it with already known 5S rDNA of the very different genera Merluccius (Merluccidae) and Salmo (Salmoninae), to identify common structural elements or patterns for understanding 5S rDNA evolution in fish. High intra- and interspecific diversity within the 5S rDNA family in all the genera can be explained by a combination of duplications, deletions, and transposition events. Sequence blocks with high similarity in all the 5S rDNA members across species were identified for the four studied genera, with evidences of intense gene conversion within noncoding regions. We propose a model to explain the evolution of the 5S rDNA, in which the evolutionary units are blocks of nucleotides rather than the entire sequences or single nucleotides. This model implies a "two-speed" evolution: slow within blocks (homogenized by recombination) and fast within the gene family (diversified by duplications and deletions).

Presynaptic Filament Dynamics in Homologous Recombination and DNA Repair

PubMed Central

Liu, Jie; Ehmsen, Kirk T.; Heyer, Wolf-Dietrich; Morrical, Scott W.

2014-01-01

Homologous Recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments—helical filaments of a recombinase enzyme bound to single-stranded DNA. Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we review the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments: some intrinsic such as recombinase ATP binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examine dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examine the biochemical properties of recombination proteins from four model systems (T4 phage, E. coli, S. cerevisiae, and H. sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We propose that the presynaptic filament has evolved to rely on multiple external factors for increased multi-level regulation of HR processes in genomes with greater structural and sequence complexity. PMID:21599536

In vitro selection of single-stranded DNA molecular recognition elements against S. aureus alpha toxin and sensitive detection in human serum.

PubMed

Hong, Ka L; Battistella, Luisa; Salva, Alysia D; Williams, Ryan M; Sooter, Letha J

2015-01-27

Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX) variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE) targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (Kd) of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA) was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved.

The Dfam database of repetitive DNA families.

PubMed

Hubley, Robert; Finn, Robert D; Clements, Jody; Eddy, Sean R; Jones, Thomas A; Bao, Weidong; Smit, Arian F A; Wheeler, Travis J

2016-01-04

Repetitive DNA, especially that due to transposable elements (TEs), makes up a large fraction of many genomes. Dfam is an open access database of families of repetitive DNA elements, in which each family is represented by a multiple sequence alignment and a profile hidden Markov model (HMM). The initial release of Dfam, featured in the 2013 NAR Database Issue, contained 1143 families of repetitive elements found in humans, and was used to produce more than 100 Mb of additional annotation of TE-derived regions in the human genome, with improved speed. Here, we describe recent advances, most notably expansion to 4150 total families including a comprehensive set of known repeat families from four new organisms (mouse, zebrafish, fly and nematode). We describe improvements to coverage, and to our methods for identifying and reducing false annotation. We also describe updates to the website interface. The Dfam website has moved to http://dfam.org. Seed alignments, profile HMMs, hit lists and other underlying data are available for download. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

A deep-branching clade of retrovirus-like retrotransposons in bdelloid rotifers

PubMed Central

Gladyshev, Eugene A.; Meselson, Matthew; Arkhipova, Irina R.

2007-01-01

Rotifers of class Bdelloidea, a group of aquatic invertebrates in which males and meiosis have never been documented, are also unusual in their lack of multicopy LINE-like and gypsy-like retrotransposons, groups inhabiting the genomes of nearly all other metazoans. Bdelloids do contain numerous DNA transposons, both intact and decayed, and domesticated Penelope-like retroelements Athena, concentrated at telomeric regions. Here we describe two LTR retrotransposons, each found at low copy number in a different bdelloid species, which define a clade different from previously known clades of LTR retrotransposons. Like bdelloid DNA transposons and Athena, these elements have been found preferentially in telomeric regions. Unlike bdelloid DNA transposons, many of which are decayed, the newly described elements, named Vesta and Juno, inhabiting the genomes of Philodina roseola and Adineta vaga, respectively, appear to be intact and to represent recent insertions, possibly from an exogenous source. We describe the retrovirus-like structure of the new elements, containing gag, pol, and env-like open reading frames, and discuss their possible origins, transmission, and behavior in bdelloid genomes. PMID:17129685

Population genetics and molecular evolution of DNA sequences in transposable elements. I. A simulation framework.

PubMed

Kijima, T E; Innan, Hideki

2013-11-01

A population genetic simulation framework is developed to understand the behavior and molecular evolution of DNA sequences of transposable elements. Our model incorporates random transposition and excision of transposable element (TE) copies, two modes of selection against TEs, and degeneration of transpositional activity by point mutations. We first investigated the relationships between the behavior of the copy number of TEs and these parameters. Our results show that when selection is weak, the genome can maintain a relatively large number of TEs, but most of them are less active. In contrast, with strong selection, the genome can maintain only a limited number of TEs but the proportion of active copies is large. In such a case, there could be substantial fluctuations of the copy number over generations. We also explored how DNA sequences of TEs evolve through the simulations. In general, active copies form clusters around the original sequence, while less active copies have long branches specific to themselves, exhibiting a star-shaped phylogeny. It is demonstrated that the phylogeny of TE sequences could be informative to understand the dynamics of TE evolution.

Homing endonucleases from mobile group I introns: discovery to genome engineering

PubMed Central

2014-01-01

Homing endonucleases are highly specific DNA cleaving enzymes that are encoded within genomes of all forms of microbial life including phage and eukaryotic organelles. These proteins drive the mobility and persistence of their own reading frames. The genes that encode homing endonucleases are often embedded within self-splicing elements such as group I introns, group II introns and inteins. This combination of molecular functions is mutually advantageous: the endonuclease activity allows surrounding introns and inteins to act as invasive DNA elements, while the splicing activity allows the endonuclease gene to invade a coding sequence without disrupting its product. Crystallographic analyses of representatives from all known homing endonuclease families have illustrated both their mechanisms of action and their evolutionary relationships to a wide range of host proteins. Several homing endonucleases have been completely redesigned and used for a variety of genome engineering applications. Recent efforts to augment homing endonucleases with auxiliary DNA recognition elements and/or nucleic acid processing factors has further accelerated their use for applications that demand exceptionally high specificity and activity. PMID:24589358

Transposon-mediated epigenetic regulation contributes to phenotypic diversity and environmental adaptation in rice.

PubMed

Song, Xianwei; Cao, Xiaofeng

2017-04-01

Transposable elements (TEs) have long been regarded as 'selfish DNA', and are generally silenced by epigenetic mechanisms. However, work in the past decade has identified positive roles for TEs in generating genomic novelty and diversity in plants. In particular, recent studies suggested that TE-induced epigenetic alterations and modification of gene expression contribute to phenotypic variation and adaptation to geography or stress. These findings have led many to regard TEs, not as junk DNA, but as sources of control elements and genomic diversity. As a staple food crop and model system for genomic research on monocot plants, rice (Oryza sativa) has a modest-sized genome that harbors massive numbers of DNA transposons (class II transposable elements) scattered across the genome, which may make TE regulation of genes more prevalent. In this review, we summarize recent progress in research on the functions of rice TEs in modulating gene expression and creating new genes. We also examine the contributions of TEs to phenotypic diversity and adaptation to environmental conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Seedling lethality in Nicotiana plumbaginifolia conferred by Ds transposable element insertion into a plant-specific gene.

PubMed

Majira, Amel; Domin, Monique; Grandjean, Olivier; Gofron, Krystyna; Houba-Hérin, Nicole

2002-10-01

A seedling lethal mutant of Nicotiana plumbaginifolia (sdl-1) was isolated by transposon tagging using a maize Dissociation (Ds) element. The insertion mutation was produced by direct co-transformation of protoplasts with two plasmids: one containing Ds and a second with an Ac transposase gene. sdl-1 seedlings exhibit several phenotypes: swollen organs, short hypocotyls in light and dark conditions, and enlarged and multinucleated cells, that altogether suggest cell growth defects. Mutant cells are able to proliferate under in vitro culture conditions. Genomic DNA sequences bordering the transposon were used to recover cDNA from the normal allele. Complementation of the mutant phenotype with the cDNA confirmed that the transposon had caused the mutation. The Ds element was inserted into the first exon of the open reading frame and the homozygous mutant lacked detectable transcript. Phenocopies of the mutant were obtained by an antisense approach. SDL-1 encodes a novel protein found in several plant genomes but apparently missingfrom animal and fungal genomes; the protein is highly conserved and has a potential plastid targeting motif.

Polyvalent Proteins, a Pervasive Theme in the Intergenomic Biological Conflicts of Bacteriophages and Conjugative Elements

PubMed Central

Iyer, Lakshminarayan M.; Burroughs, A. Maxwell; Anand, Swadha; de Souza, Robson F.

2017-01-01

ABSTRACT Intense biological conflicts between prokaryotic genomes and their genomic parasites have resulted in an arms race in terms of the molecular “weaponry” deployed on both sides. Using a recursive computational approach, we uncovered a remarkable class of multidomain proteins with 2 to 15 domains in the same polypeptide deployed by viruses and plasmids in such conflicts. Domain architectures and genomic contexts indicate that they are part of a widespread conflict strategy involving proteins injected into the host cell along with parasite DNA during the earliest phase of infection. Their unique feature is the combination of domains with highly disparate biochemical activities in the same polypeptide; accordingly, we term them polyvalent proteins. Of the 131 domains in polyvalent proteins, a large fraction are enzymatic domains predicted to modify proteins, target nucleic acids, alter nucleotide signaling/metabolism, and attack peptidoglycan or cytoskeletal components. They further contain nucleic acid-binding domains, virion structural domains, and 40 novel uncharacterized domains. Analysis of their architectural network reveals both pervasive common themes and specialized strategies for conjugative elements and plasmids or (pro)phages. The themes include likely processing of multidomain polypeptides by zincin-like metallopeptidases and mechanisms to counter restriction or CRISPR/Cas systems and jump-start transcription or replication. DNA-binding domains acquired by eukaryotes from such systems have been reused in XPC/RAD4-dependent DNA repair and mitochondrial genome replication in kinetoplastids. Characterization of the novel domains discovered here, such as RNases and peptidases, are likely to aid in the development of new reagents and elucidation of the spread of antibiotic resistance. IMPORTANCE This is the first report of the widespread presence of large proteins, termed polyvalent proteins, predicted to be transmitted by genomic parasites such as conjugative elements, plasmids, and phages during the initial phase of infection along with their DNA. They are typified by the presence of multiple domains with disparate activities combined in the same protein. While some of these domains are predicted to assist the invasive element in replication, transcription, or protection of their DNA, several are likely to target various host defense systems or modify the host to favor the parasite's life cycle. Notably, DNA-binding domains from these systems have been transferred to eukaryotes, where they have been incorporated into DNA repair and mitochondrial genome replication systems. PMID:28559295

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex

PubMed Central

Ganaie, Safder S.; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve

2017-01-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases. PMID:28459842

Characterization of various promoter regions of the human DNA helicase-encoding genes and identification of duplicated ets (GGAA) motifs as an essential transcription regulatory element.

PubMed

Uchiumi, Fumiaki; Watanabe, Takeshi; Tanuma, Sei-ichi

2010-05-15

DNA helicases are important in the regulation of DNA transaction and thereby various cellular functions. In this study, we developed a cost-effective multiple DNA transfection assay with DEAE-dextran reagent and analyzed the promoter activities of the human DNA helicases. The 5'-flanking regions of the human DNA helicase-encoding genes were isolated and subcloned into luciferase (Luc) expression plasmids. They were coated onto 96-well plate and used for co-transfection with a renilla-Luc expression vector into various cells, and dual-Luc assays were performed. The profiles of promoter activities were dependent on cell lines used. Among these human DNA helicase genes, XPB, RecQL5, and RTEL promoters were activated during TPA-induced HL-60 cell differentiation. Interestingly, duplicated ets (GGAA) elements are commonly located around the transcription start sites of these genes. The duplicated GGAA motifs are also found in the promoters of DNA replication/repair synthesis factor genes including PARG, ATR, TERC, and Rb1. Mutation analyses suggested that the duplicated GGAA-motifs are necessary for the basal promoter activity in various cells and some of them positively respond to TPA in HL-60 cells. TPA-induced response of 44-bp in the RTEL promoter was attenuated by co-transfection of the PU.1 expression vector. These findings suggest that the duplicated ets motifs regulate DNA-repair associated gene expressions during macrophage-like differentiation of HL-60 cells. Copyright 2010 Elsevier Inc. All rights reserved.

Reconstructing the complex evolutionary history of mobile plasmids in red algal genomes

PubMed Central

Lee, JunMo; Kim, Kyeong Mi; Yang, Eun Chan; Miller, Kathy Ann; Boo, Sung Min; Bhattacharya, Debashish; Yoon, Hwan Su

2016-01-01

The integration of foreign DNA into algal and plant plastid genomes is a rare event, with only a few known examples of horizontal gene transfer (HGT). Plasmids, which are well-studied drivers of HGT in prokaryotes, have been reported previously in red algae (Rhodophyta). However, the distribution of these mobile DNA elements and their sites of integration into the plastid (ptDNA), mitochondrial (mtDNA), and nuclear genomes of Rhodophyta remain unknown. Here we reconstructed the complex evolutionary history of plasmid-derived DNAs in red algae. Comparative analysis of 21 rhodophyte ptDNAs, including new genome data for 5 species, turned up 22 plasmid-derived open reading frames (ORFs) that showed syntenic and copy number variation among species, but were conserved within different individuals in three lineages. Several plasmid-derived homologs were found not only in ptDNA but also in mtDNA and in the nuclear genome of green plants, stramenopiles, and rhizarians. Phylogenetic and plasmid-derived ORF analyses showed that the majority of plasmid DNAs originated within red algae, whereas others were derived from cyanobacteria, other bacteria, and viruses. Our results elucidate the evolution of plasmid DNAs in red algae and suggest that they spread as parasitic genetic elements. This hypothesis is consistent with their sporadic distribution within Rhodophyta. PMID:27030297

Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing

PubMed Central

Yang, Christine; McLeod, Andrea J.; Cotton, Allison M.; de Leeuw, Charles N.; Laprise, Stéphanie; Banks, Kathleen G.; Simpson, Elizabeth M.; Brown, Carolyn J.

2012-01-01

Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome. PMID:23023002

Perspective on the pipeline of drugs being developed with modulation of DNA damage as a target.

PubMed

Plummer, Ruth

2010-09-15

Inhibitors of various elements of the DNA repair pathways have entered clinical development or are in late preclinical stages of drug development. It was initially considered that agents targeting DNA repair would act to overcome tumor resistance to chemotherapy and radiotherapy. More recent data have shown that targeting DNA repair pathways can be effective in selected tumors via a synthetically lethal route, with single agent activity having been shown with poly-ADP ribose polymerase (PARP) inhibitors. An increased understanding of the biology and interaction of the DNA repair pathways also means that rational combination of DNA repair inhibitors may also give great benefit in the clinic. ©2010 AACR.

Engineering the Structure and Properties of DNA-Nanoparticle Superstructures Using Polyvalent Counterions.

PubMed

Chou, Leo Y T; Song, Fayi; Chan, Warren C W

2016-04-06

DNA assembly of nanoparticles is a powerful approach to control their properties and prototype new materials. However, the structure and properties of DNA-assembled nanoparticles are labile and sensitive to interactions with counterions, which vary with processing and application environment. Here we show that substituting polyamines in place of elemental counterions significantly enhanced the structural rigidity and plasmonic properties of DNA-assembled metal nanoparticles. These effects arose from the ability of polyamines to condense DNA and cross-link DNA-coated nanoparticles. We further used polyamine wrapped DNA nanostructures as structural templates to seed the growth of polymer multilayers via layer-by-layer assembly, and controlled the degree of DNA condensation, plasmon coupling efficiency, and material responsiveness to environmental stimuli by varying polyelectrolyte composition. These results highlight counterion engineering as a versatile strategy to tailor the properties of DNA-nanoparticle assemblies for various applications, and should be applicable to other classes of DNA nanostructures.

Spread of X-chromosome inactivation into autosomal sequences: role for DNA elements, chromatin features and chromosomal domains

PubMed Central

Cotton, Allison M.; Chen, Chih-Yu; Lam, Lucia L.; Wasserman, Wyeth W.; Kobor, Michael S.; Brown, Carolyn J.

2014-01-01

X-chromosome inactivation results in dosage equivalence between the X chromosome in males and females; however, over 15% of human X-linked genes escape silencing and these genes are enriched on the evolutionarily younger short arm of the X chromosome. The spread of inactivation onto translocated autosomal material allows the study of inactivation without the confounding evolutionary history of the X chromosome. The heterogeneity and reduced extent of silencing on autosomes are evidence for the importance of DNA elements underlying the spread of silencing. We have assessed DNA methylation in six unbalanced X-autosome translocations using the Illumina Infinium HumanMethylation450 array. Two to 42% of translocated autosomal genes showed this mark of silencing, with the highest degree of inactivation observed for trisomic autosomal regions. Generally, the extent of silencing was greatest close to the translocation breakpoint; however, silencing was detected well over 100 kb into the autosomal DNA. Alu elements were found to be enriched at autosomal genes that escaped from inactivation while L1s were enriched at subject genes. In cells without the translocation, there was enrichment of heterochromatic features such as EZH2 and H3K27me3 for those genes that become silenced when translocated, suggesting that underlying chromatin structure predisposes genes towards silencing. Additionally, the analysis of topological domains indicated physical clustering of autosomal genes of common inactivation status. Overall, our analysis indicated a complex interaction between DNA sequence, chromatin features and the three-dimensional structure of the chromosome. PMID:24158853

DNA detection on ultrahigh-density optical fiber-based nanoarrays.

PubMed

Tam, Jenny M; Song, Linan; Walt, David R

2009-04-15

Nanoarrays for DNA detection were fabricated on etched nanofiber bundles based on recently developed techniques for microscale arrays. Two different-sized nanoarrays were created: one with 700 nm feature sizes and a 1 microm center-to-center pitch (approximately 1x10(6) array elements/mm(2)) and one with 300 nm feature sizes and a 500 nm center-to-center pitch (4.6x10(6) array elements/mm(2)). A random, multiplexed array composed of oligonucleotide-functionalized nanospheres was constructed and used for parallel detection and analysis of fluorescently labeled DNA targets. We have used these arrays to detect a variety of target sequences including Bacillus thuringiensis kurstaki and vaccina virus sequences, two potential biowarfare agents, as well as interleukin-2 sequences, an immune system modulator that has been used for the diagnosis of HIV.

Identification of structural variation in mouse genomes.

PubMed

Keane, Thomas M; Wong, Kim; Adams, David J; Flint, Jonathan; Reymond, Alexandre; Yalcin, Binnaz

2014-01-01

Structural variation is variation in structure of DNA regions affecting DNA sequence length and/or orientation. It generally includes deletions, insertions, copy-number gains, inversions, and transposable elements. Traditionally, the identification of structural variation in genomes has been challenging. However, with the recent advances in high-throughput DNA sequencing and paired-end mapping (PEM) methods, the ability to identify structural variation and their respective association to human diseases has improved considerably. In this review, we describe our current knowledge of structural variation in the mouse, one of the prime model systems for studying human diseases and mammalian biology. We further present the evolutionary implications of structural variation on transposable elements. We conclude with future directions on the study of structural variation in mouse genomes that will increase our understanding of molecular architecture and functional consequences of structural variation.

Preferential Nucleosome Assembly at DNA Triplet Repeats from the Myotonic Dystrophy Gene

NASA Astrophysics Data System (ADS)

Wang, Yuh-Hwa; Amirhaeri, Sorour; Kang, Seongman; Wells, Robert D.; Griffith, Jack D.

1994-07-01

The expansion of CTG repeats in DNA occurs in or near genes involved in several human diseases, including myotonic dystrophy and Huntington's disease. Nucleosomes, the basic structural element of chromosomes, consist of 146 base pairs of DNA coiled about an octamer of histone proteins and mediate general transcriptional repression. Electron microscopy was used to examine in vitro the nucleosome assembly of DNA containing repeating CTG triplets. The efficiency of nucleosome formation increased with expanded triplet blocks, suggesting that such blocks may repress transcription through the creation of stable nucleosomes.

Structure Prediction and Analysis of DNA Transposon and LINE Retrotransposon Proteins*

PubMed Central

Abrusán, György; Zhang, Yang; Szilágyi, András

2013-01-01

Despite the considerable amount of research on transposable elements, no large-scale structural analyses of the TE proteome have been performed so far. We predicted the structures of hundreds of proteins from a representative set of DNA and LINE transposable elements and used the obtained structural data to provide the first general structural characterization of TE proteins and to estimate the frequency of TE domestication and horizontal transfer events. We show that 1) ORF1 and Gag proteins of retrotransposons contain high amounts of structural disorder; thus, despite their very low conservation, the presence of disordered regions and probably their chaperone function is conserved. 2) The distribution of SCOP classes in DNA transposons and LINEs indicates that the proteins of DNA transposons are more ancient, containing folds that already existed when the first cellular organisms appeared. 3) DNA transposon proteins have lower contact order than randomly selected reference proteins, indicating rapid folding, most likely to avoid protein aggregation. 4) Structure-based searches for TE homologs indicate that the overall frequency of TE domestication events is low, whereas we found a relatively high number of cases where horizontal transfer, frequently involving parasites, is the most likely explanation for the observed homology. PMID:23530042

DNA nanotechnology for nanophotonic applications.

PubMed

Samanta, Anirban; Banerjee, Saswata; Liu, Yan

2015-02-14

DNA nanotechnology has touched the epitome of miniaturization by integrating various nanometer size particles with nanometer precision. This enticing bottom-up approach has employed small DNA tiles, large multi-dimensional polymeric structures or more recently DNA origami to organize nanoparticles of different inorganic materials, small organic molecules or macro-biomolecules like proteins, and RNAs into fascinating patterns that are difficult to achieve by other conventional methods. Here, we are especially interested in the self-assembly of nanomaterials that are potentially attractive elements in the burgeoning field of nanophotonics. These materials include plasmonic nanoparticles, quantum dots, fluorescent organic dyes, etc. DNA based self-assembly allows excellent control over distance, orientation and stoichiometry of these nano-elements that helps to engineer intelligent systems that can potentially pave the path for future technology. Many outstanding structures have been fabricated that are capable of fine tuning optical properties, such as fluorescence intensity and lifetime modulation, enhancement of Raman scattering and emergence of circular dichroism responses. Within the limited scope of this review we have tried to give a glimpse of the development of this still nascent but highly promising field to its current status as well as the existing challenges before us.

The structure of the regulatory region of the rat L1 (L1Rn, long interspersed repeated) DNA family of transposable elements.

PubMed Central

Furano, A V; Robb, S M; Robb, F T

1988-01-01

Here we report the DNA structure of the left 1.5 kb of two newly isolated full length members of the rat L1 DNA family (L1Rn, long interspersed repeated DNA). In contrast to earlier isolated rat L1 members, both of these contain promoter-like regions that are most likely full length. In addition, the promoter-like region of both members has undergone a partial tandem duplication. A second internal region of the left end of one of the reported members is also tandemly duplicated. The propensity of the left end of rat L1 elements to undergo this form of genetic rearrangement, as well as other structural features revealed by the present work, is discussed in light of the fact that during evolution the otherwise conserved mammalian L1 DNA families have each acquired completely different promoter-like regions. In an accompanying paper [Nur, I., Pascale, E., and Furano, A. V. (1988) Nucleic Acids Res. 16, submitted], we report that one of the rat promoter-like regions can function as a promoter in rat cells when fused to the Escherichia coli chloramphenicol acyltransferase gene. PMID:2845369

Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus.

PubMed Central

Lakshmikumaran, M S; D'Ambrosio, E; Laimins, L A; Lin, D T; Furano, A V

1985-01-01

The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this. Images PMID:3016521

piRNA pathway targets active LINE1 elements to establish the repressive H3K9me3 mark in germ cells

PubMed Central

Pezic, Dubravka; Manakov, Sergei A.; Sachidanandam, Ravi; Aravin, Alexei A.

2014-01-01

Transposable elements (TEs) occupy a large fraction of metazoan genomes and pose a constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small noncoding piwi-interacting RNAs (piRNAs) recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments. PMID:24939875

Improved results of LINE-1 methylation analysis in formalin-fixed, paraffin-embedded tissues with the application of a heating step during the DNA extraction process.

PubMed

Wen, Xianyu; Jeong, Seorin; Kim, Younghoon; Bae, Jeong Mo; Cho, Nam Yun; Kim, Jung Ho; Kang, Gyeong Hoon

2017-01-01

Formalin-fixed, paraffin-embedded (FFPE) tissues are important resources for profiling DNA methylation changes and for studying a variety of diseases. However, formalin fixation introduces inter-strand crosslinking, which might cause incomplete bisulfite conversion of unmethylated cytosines, which might lead to falsely elevated measurements of methylation levels in pyrosequencing assays. Long interspersed nucleotide element-1 (LINE-1) is a major constituent of repetitive transposable DNA elements, and its methylation is referred to correlates with global DNA methylation. To identify whether formalin fixation might impact the measured values of methylation in LINE-1 repetitive elements and whether prolonged heat-induced denaturation of DNA might reduce the artificial increases in measured values caused by formalin fixation, we analyzed paired fresh-frozen (FF) and FFPE xenograft tissue samples for their methylation levels in LINE-1 using a pyrosequencing assay. To further confirm the effect of a heating step in the measurement of LINE-1 or single gene methylation levels, we analyzed FFPE tissue samples of gastric cancer and colorectal cancer for their methylation status in LINE-1 and eight single genes, respectively. Formalin fixation led to an increase in the measured values of LINE-1 methylation regardless of the duration of fixation. Prolonged heating of the DNA at 95 °C for 30 min before bisulfite conversion was found (1) to decrease the discrepancy in the measured values between the paired FF and FFPE tissue samples, (2) to decrease the standard deviation of the measured value of LINE-1 methylation levels in FFPE tissue samples of gastric cancer, and (3) to improve the performance in the measurement of single gene methylation levels in FFPE tissue samples of colorectal cancer. Formalin fixation leads to artificial increases in the measured values of LINE-1 methylation, and the application of prolonged heating of DNA samples decreases the discrepancy in the measured values of LINE-1 methylation between paired FF and FFPE tissue samples. The application of prolonged heating of DNA samples improves bisulfite conversion-based measurement of LINE-1 or single gene methylation levels in FFPE tissue samples.

Optimal Ancient DNA Yields from the Inner Ear Part of the Human Petrous Bone.

PubMed

Pinhasi, Ron; Fernandes, Daniel; Sirak, Kendra; Novak, Mario; Connell, Sarah; Alpaslan-Roodenberg, Songül; Gerritsen, Fokke; Moiseyev, Vyacheslav; Gromov, Andrey; Raczky, Pál; Anders, Alexandra; Pietrusewsky, Michael; Rollefson, Gary; Jovanovic, Marija; Trinhhoang, Hiep; Bar-Oz, Guy; Oxenham, Marc; Matsumura, Hirofumi; Hofreiter, Michael

2015-01-01

The invention and development of next or second generation sequencing methods has resulted in a dramatic transformation of ancient DNA research and allowed shotgun sequencing of entire genomes from fossil specimens. However, although there are exceptions, most fossil specimens contain only low (~ 1% or less) percentages of endogenous DNA. The only skeletal element for which a systematically higher endogenous DNA content compared to other skeletal elements has been shown is the petrous part of the temporal bone. In this study we investigate whether (a) different parts of the petrous bone of archaeological human specimens give different percentages of endogenous DNA yields, (b) there are significant differences in average DNA read lengths, damage patterns and total DNA concentration, and (c) it is possible to obtain endogenous ancient DNA from petrous bones from hot environments. We carried out intra-petrous comparisons for ten petrous bones from specimens from Holocene archaeological contexts across Eurasia dated between 10,000-1,800 calibrated years before present (cal. BP). We obtained shotgun DNA sequences from three distinct areas within the petrous: a spongy part of trabecular bone (part A), the dense part of cortical bone encircling the osseous inner ear, or otic capsule (part B), and the dense part within the otic capsule (part C). Our results confirm that dense bone parts of the petrous bone can provide high endogenous aDNA yields and indicate that endogenous DNA fractions for part C can exceed those obtained for part B by up to 65-fold and those from part A by up to 177-fold, while total endogenous DNA concentrations are up to 126-fold and 109-fold higher for these comparisons. Our results also show that while endogenous yields from part C were lower than 1% for samples from hot (both arid and humid) parts, the DNA damage patterns indicate that at least some of the reads originate from ancient DNA molecules, potentially enabling ancient DNA analyses of samples from hot regions that are otherwise not amenable to ancient DNA analyses.

Nucleases Encoded by Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter jejuni

USDA-ARS?s Scientific Manuscript database

The species Campylobacter jejuni displays huge genetic diversity, and is naturally competent for DNA uptake. Nevertheless, not every strain is able to acquire foreign DNA since nonnaturally transformable strains do exist. Previously we showed that many nonnaturally transformable C. jejuni strains ex...

Insights into the strategies used by related group II introns to adapt successfully for the colonisation of a bacterial genome

PubMed Central

Martínez-Rodríguez, Laura; García-Rodríguez, Fernando M; Molina-Sánchez, María Dolores; Toro, Nicolás; Martínez-Abarca, Francisco

2014-01-01

Group II introns are self-splicing RNAs and site-specific mobile retroelements found in bacterial and organellar genomes. The group II intron RmInt1 is present at high copy number in Sinorhizobium meliloti species, and has a multifunctional intron-encoded protein (IEP) with reverse transcriptase/maturase activities, but lacking the DNA-binding and endonuclease domains. We characterized two RmInt1-related group II introns RmInt2 from S. meliloti strain GR4 and Sr.md.I1 from S. medicae strain WSM419 in terms of splicing and mobility activities. We used both wild-type and engineered intron-donor constructs based on ribozyme ΔORF-coding sequence derivatives, and we determined the DNA target requirements for RmInt2, the element most distantly related to RmInt1. The excision and mobility patterns of intron-donor constructs expressing different combinations of IEP and intron RNA provided experimental evidence for the co-operation of IEPs and intron RNAs from related elements in intron splicing and, in some cases, in intron homing. We were also able to identify the DNA target regions recognized by these IEPs lacking the DNA endonuclease domain. Our results provide new insight into the versatility of related group II introns and the possible co-operation between these elements to facilitate the colonization of bacterial genomes. PMID:25482895

Insights into the strategies used by related group II introns to adapt successfully for the colonisation of a bacterial genome.

PubMed

Martínez-Rodríguez, Laura; García-Rodríguez, Fernando M; Molina-Sánchez, María Dolores; Toro, Nicolás; Martínez-Abarca, Francisco

2014-01-01

Group II introns are self-splicing RNAs and site-specific mobile retroelements found in bacterial and organellar genomes. The group II intron RmInt1 is present at high copy number in Sinorhizobium meliloti species, and has a multifunctional intron-encoded protein (IEP) with reverse transcriptase/maturase activities, but lacking the DNA-binding and endonuclease domains. We characterized two RmInt1-related group II introns RmInt2 from S. meliloti strain GR4 and Sr.md.I1 from S. medicae strain WSM419 in terms of splicing and mobility activities. We used both wild-type and engineered intron-donor constructs based on ribozyme ΔORF-coding sequence derivatives, and we determined the DNA target requirements for RmInt2, the element most distantly related to RmInt1. The excision and mobility patterns of intron-donor constructs expressing different combinations of IEP and intron RNA provided experimental evidence for the co-operation of IEPs and intron RNAs from related elements in intron splicing and, in some cases, in intron homing. We were also able to identify the DNA target regions recognized by these IEPs lacking the DNA endonuclease domain. Our results provide new insight into the versatility of related group II introns and the possible co-operation between these elements to facilitate the colonization of bacterial genomes.

Mutations on the DNA Binding Surface of TBP Discriminate between Yeast TATA and TATA-Less Gene Transcription

PubMed Central

Kamenova, Ivanka; Warfield, Linda

2014-01-01

Most RNA polymerase (Pol) II promoters lack a TATA element, yet nearly all Pol II transcription requires TATA binding protein (TBP). While the TBP-TATA interaction is critical for transcription at TATA-containing promoters, it has been unclear whether TBP sequence-specific DNA contacts are required for transcription at TATA-less genes. Transcription factor IID (TFIID), the TBP-containing coactivator that functions at most TATA-less genes, recognizes short sequence-specific promoter elements in metazoans, but analogous promoter elements have not been identified in Saccharomyces cerevisiae. We generated a set of mutations in the yeast TBP DNA binding surface and found that most support growth of yeast. Both in vivo and in vitro, many of these mutations are specifically defective for transcription of two TATA-containing genes with only minor defects in transcription of two TATA-less, TFIID-dependent genes. TBP binds several TATA-less promoters with apparent high affinity, but our results suggest that this binding is not important for transcription activity. Our results are consistent with the model that sequence-specific TBP-DNA contacts are not important at yeast TATA-less genes and suggest that other general transcription factors or coactivator subunits are responsible for recognition of TATA-less promoters. Our results also explain why yeast TBP derivatives defective for TATA binding appear defective in activated transcription. PMID:24865972

Mutations on the DNA binding surface of TBP discriminate between yeast TATA and TATA-less gene transcription.

PubMed

Kamenova, Ivanka; Warfield, Linda; Hahn, Steven

2014-08-01

Most RNA polymerase (Pol) II promoters lack a TATA element, yet nearly all Pol II transcription requires TATA binding protein (TBP). While the TBP-TATA interaction is critical for transcription at TATA-containing promoters, it has been unclear whether TBP sequence-specific DNA contacts are required for transcription at TATA-less genes. Transcription factor IID (TFIID), the TBP-containing coactivator that functions at most TATA-less genes, recognizes short sequence-specific promoter elements in metazoans, but analogous promoter elements have not been identified in Saccharomyces cerevisiae. We generated a set of mutations in the yeast TBP DNA binding surface and found that most support growth of yeast. Both in vivo and in vitro, many of these mutations are specifically defective for transcription of two TATA-containing genes with only minor defects in transcription of two TATA-less, TFIID-dependent genes. TBP binds several TATA-less promoters with apparent high affinity, but our results suggest that this binding is not important for transcription activity. Our results are consistent with the model that sequence-specific TBP-DNA contacts are not important at yeast TATA-less genes and suggest that other general transcription factors or coactivator subunits are responsible for recognition of TATA-less promoters. Our results also explain why yeast TBP derivatives defective for TATA binding appear defective in activated transcription. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A HLA class I cis-regulatory element whose activity can be modulated by hormones.

PubMed

Sim, B C; Hui, K M

1994-12-01

To elucidate the basis of the down-regulation in major histocompatibility complex (MHC) class I gene expression and to identify possible DNA-binding regulatory elements that have the potential to interact with class I MHC genes, we have studied the transcriptional regulation of class I HLA genes in human breast carcinoma cells. A 9 base pair (bp) negative cis-regulatory element (NRE) has been identified using band-shift assays employing DNA sequences derived from the 5'-flanking region of HLA class I genes. This 9-bp element, GTCATGGCG, located within exon I of the HLA class I gene, can potently inhibit the expression of a heterologous thymidine kinase (TK) gene promoter and the HLA enhancer element. Furthermore, this regulatory element can exert its suppressive function in either the sense or anti-sense orientation. More interestingly, NRE can suppress dexamethasone-mediated gene activation in the context of the reported glucocorticoid-responsive element (GRE) in MCF-7 cells but has no influence on the estrogen-mediated transcriptional activation of MCF-7 cells in the context of the reported estrogen-responsive element (ERE). Furthermore, the presence of such a regulatory element within the HLA class I gene whose activity can be modulated by hormones correlates well with our observation that the level of HLA class I gene expression can be down-regulated by hormones in human breast carcinoma cells. Such interactions between negative regulatory elements and specific hormone trans-activators are novel and suggest a versatile form of transcriptional control.

FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology.

PubMed

Hernanz-Koers, Miguel; Gandía, Mónica; Garrigues, Sandra; Manzanares, Paloma; Yenush, Lynne; Orzaez, Diego; Marcos, Jose F

2018-07-01

Current challenges in the study and biotechnological exploitation of filamentous fungi are the optimization of DNA cloning and fungal genetic transformation beyond model fungi, the open exchange of ready-to-use and standardized genetic elements among the research community, and the availability of universal synthetic biology tools and rules. The GoldenBraid (GB) cloning framework is a Golden Gate-based DNA cloning system developed for plant synthetic biology through Agrobacterium tumefaciens-mediated genetic transformation (ATMT). In this study, we develop reagents for the adaptation of GB version 3.0 from plants to filamentous fungi through: (i) the expansion of the GB toolbox with the domestication of fungal-specific genetic elements; (ii) the design of fungal-specific GB structures; and (iii) the ATMT and gene disruption of the plant pathogen Penicillium digitatum as a proof of concept. Genetic elements domesticated into the GB entry vector pUPD2 include promoters, positive and negative selection markers and terminators. Interestingly, some GB elements can be directly exchanged between plants and fungi, as demonstrated with the marker hph for Hyg R or the fluorescent protein reporter YFP. The iterative modular assembly of elements generates an endless number of diverse transcriptional units and other higher order combinations in the pDGB3α/pDGB3Ω destination vectors. Furthermore, the original plant GB syntax was adapted here to incorporate specific GB structures for gene disruption through homologous recombination and dual selection. We therefore have successfully adapted the GB technology for the ATMT of fungi. We propose the name of FungalBraid (FB) for this new branch of the GB technology that provides open, exchangeable and collaborative resources to the fungal research community. Copyright © 2018 Elsevier Inc. All rights reserved.

DNA methylation aberrancies as a guide for surveillance and treatment of human cancers

PubMed Central

Liang, Gangning; Weisenberger, Daniel J.

2017-01-01

ABSTRACT DNA methylation aberrancies are hallmarks of human cancers and are characterized by global DNA hypomethylation of repetitive elements and non-CpG rich regions concomitant with locus-specific DNA hypermethylation. DNA methylation changes may result in altered gene expression profiles, most notably the silencing of tumor suppressors, microRNAs, endogenous retorviruses and tumor antigens due to promoter DNA hypermethylation, as well as oncogene upregulation due to gene-body DNA hypermethylation. Here, we review DNA methylation aberrancies in human cancers, their use in cancer surveillance and the interplay between DNA methylation and histone modifications in gene regulation. We also summarize DNA methylation inhibitors and their therapeutic effects in cancer treatment. In this context, we describe the integration of DNA methylation inhibitors with conventional chemotherapies, DNA repair inhibitors and immune-based therapies, to bring the epigenome closer to its normal state and increase sensitivity to other therapeutic agents to improve patient outcome and survival. PMID:28358281

Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA.

PubMed

Pineda, Gina M; Montgomery, Anne H; Thompson, Robyn; Indest, Brooke; Carroll, Marion; Sinha, Sudhir K

2014-11-01

There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition. However, a system which can also assess the extent of degradation in a forensic sample will be a useful tool for DNA analysts. Possessing this information prior to genotyping will allow an analyst to more informatively make downstream decisions for the successful typing of a forensic sample without unnecessarily consuming DNA extract. Real-time PCR provides a reliable method for determining the amount and quality of amplifiable DNA in a biological sample. Alu are Short Interspersed Elements (SINE), approximately 300bp insertions which are distributed throughout the human genome in large copy number. The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human genome, which minimizes the individual specific variation possible when using a multi-copy target quantification system. This study utilizes two independent retrotransposon genomic targets to obtain quantification of an 80bp "short" DNA fragment and a 207bp "long" DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™. The ratio of the two quantitation values provides a "Degradation Index", or a qualitative measure of a sample's extent of degradation. The Degradation Index was found to be predictive of the observed loss of STR markers and alleles as degradation increases. Use of a synthetic target as an internal positive control (IPC) provides an additional assessment for the presence of PCR inhibitors in the test sample. In conclusion, a DNA based qualitative/quantitative/inhibition assessment system that accurately predicts the status of a biological sample, will be a valuable tool for deciding which DNA test kit to utilize and how much target DNA to use, when processing compromised forensic samples for DNA testing. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Evaluating differential nuclear DNA yield rates and osteocyte numbers among human bone tissue types: A synchrotron radiation micro-CT approach.

PubMed

Andronowski, Janna M; Mundorff, Amy Z; Pratt, Isaac V; Davoren, Jon M; Cooper, David M L

2017-05-01

Molecular human identification has conventionally focused on DNA sampling from dense, weight-bearing cortical bone tissue, typically from femora or tibiae. A comparison of skeletal elements from three contemporary individuals demonstrated that elements with high quantities of cancellous bone yielded nuclear DNA at the highest rates, suggesting that preferentially sampling cortical bone may be suboptimal (Mundorff & Davoren, 2014). Despite these findings, the reason for the differential DNA yields between cortical and cancellous bone tissues remains unknown. The primary goal of this work is to ascertain whether differences in bone microstructure can be used to explain differential nuclear DNA yield among bone tissue types observed by Mundorff and Davoren (2014), with a focus on osteocytes and the three-dimensional (3D) quantification of their associated lacunae. Osteocytes and other bone cells are recognized to house DNA in bone tissue, thus examining the density of their lacunae may explain why nuclear DNA yield rates differ among bone tissue types. Lacunae were visualized and quantified using synchrotron radiation-based micro-Computed Tomographic imaging (SR micro-CT). Volumes of interest (VOIs) from cortical and cancellous bone tissues (n=129) were comparatively analyzed from the three skeletons sampled for Mundorff and Davoren's (2014) study. Analyses tested the primary hypothesis that the abundance and density of osteocytes (inferred from their lacunar spaces) vary between cortical and cancellous bone tissue types. Results demonstrated that osteocyte lacunar abundance and density vary between cortical and cancellous bone tissue types, with cortical bone VOIs containing a higher lacunar abundance and density. We found that the osteocyte lacunar density values are independent of nuclear DNA yield, suggesting an alternative explanation for the higher nuclear DNA yields from bones with greater quantities of cancellous bone tissue. The use of SR micro-CT allowed for a scale of analysis that revealed a high range of variation in lacunar abundance in both tissue types. Moreover, high-resolution SR micro-CT imaging revealed potential soft tissue remnants within marrow spaces not visible macroscopically. It is hypothesized that soft tissue remnants observed among the trabeculae of skeletal elements with high quantities of cancellous bone tissue are responsible for the high nuclear DNA yields. These findings have significant implications for bone-sample selection for nuclear DNA analysis in a forensic context when skeletal remains are recovered from the ground surface. Copyright © 2017 Elsevier B.V. All rights reserved.

Transposable elements in fish chromosomes: a study in the marine cobia species.

PubMed

Costa, G W W F; Cioffi, M B; Bertollo, L A C; Molina, W F

2013-01-01

Rachycentron canadum, a unique representative of the Rachycentridae family, has been the subject of considerable biotechnological interest due to its potential use in marine fish farming. This species has undergone extensive research concerning the location of genes and multigene families on its chromosomes. Although most of the genome of some organisms is composed of repeated DNA sequences, aspects of the origin and dispersion of these elements are still largely unknown. The physical mapping of repetitive sequences on the chromosomes of R. canadum proved to be relevant for evolutionary and applied purposes. Therefore, here, we present the mapping by fluorescence in situ hybridization of the transposable element (TE) Tol2, the non-LTR retrotransposons Rex1 and Rex3, together with the 18S and 5S rRNA genes in the chromosome of this species. The Tol2 TE, belonging to the family of hAT transposons, is homogeneously distributed in the euchromatic regions of the chromosomes but with huge colocalization with the 18S rDNA sites. The hybridization signals for Rex1 and Rex3 revealed a semi-arbitrary distribution pattern, presenting differentiated dispersion in euchromatic and heterochromatic regions. Rex1 elements are associated preferentially in heterochromatic regions, while Rex3 shows a scarce distribution in the euchromatic regions of the chromosomes. The colocalization of TEs with 18S and 5S rDNA revealed complex chromosomal regions of repetitive sequences. In addition, the nonpreferential distribution of Rex1 and Rex3 in all heterochromatic regions, as well as the preferential distribution of the Tol2 transposon associated with 18S rDNA sequences, reveals a distinct pattern of organization of TEs in the genome of this species. A heterogeneous chromosomal colonization of TEs may confer different evolutionary rates to the heterochromatic regions of this species.

Translocation and gross deletion breakpoints in human inherited disease and cancer II: Potential involvement of repetitive sequence elements in secondary structure formation between DNA ends.

PubMed

Chuzhanova, Nadia; Abeysinghe, Shaun S; Krawczak, Michael; Cooper, David N

2003-09-01

Translocations and gross deletions are responsible for a significant proportion of both cancer and inherited disease. Although such gene rearrangements are nonuniformly distributed in the human genome, the underlying mutational mechanisms remain unclear. We have studied the potential involvement of various types of repetitive sequence elements in the formation of secondary structure intermediates between the single-stranded DNA ends that recombine during rearrangements. Complexity analysis was used to assess the potential of these ends to form secondary structures, the maximum decrease in complexity consequent to a gross rearrangement being used as an indicator of the type of repeat and the specific DNA ends involved. A total of 175 pairs of deletion/translocation breakpoint junction sequences available from the Gross Rearrangement Breakpoint Database [GRaBD; www.uwcm.ac.uk/uwcm/mg/grabd/grabd.html] were analyzed. Potential secondary structure was noted between the 5' flanking sequence of the first breakpoint and the 3' flanking sequence of the second breakpoint in 49% of rearrangements and between the 5' flanking sequence of the second breakpoint and the 3' flanking sequence of the first breakpoint in 36% of rearrangements. Inverted repeats, inversions of inverted repeats, and symmetric elements were found in association with gross rearrangements at approximately the same frequency. However, inverted repeats and inversions of inverted repeats accounted for the vast majority (83%) of deletions plus small insertions, symmetric elements for one-half of all antigen receptor-mediated translocations, while direct repeats appear only to be involved in mediating simple deletions. These findings extend our understanding of illegitimate recombination by highlighting the importance of secondary structure formation between single-stranded DNA ends at breakpoint junctions. Copyright 2003 Wiley-Liss, Inc.

Repetitive sequences: the hidden diversity of heterochromatin in prochilodontid fish

PubMed Central

Terencio, Maria L.; Schneider, Carlos H.; Gross, Maria C.; do Carmo, Edson Junior; Nogaroto, Viviane; de Almeida, Mara Cristina; Artoni, Roberto Ferreira; Vicari, Marcelo R.; Feldberg, Eliana

2015-01-01

Abstract The structure and organization of repetitive elements in fish genomes are still relatively poorly understood, although most of these elements are believed to be located in heterochromatic regions. Repetitive elements are considered essential in evolutionary processes as hotspots for mutations and chromosomal rearrangements, among other functions – thus providing new genomic alternatives and regulatory sites for gene expression. The present study sought to characterize repetitive DNA sequences in the genomes of Semaprochilodus insignis (Jardine & Schomburgk, 1841) and Semaprochilodus taeniurus (Valenciennes, 1817) and identify regions of conserved syntenic blocks in this genome fraction of three species of Prochilodontidae (Semaprochilodus insignis, Semaprochilodus taeniurus, and Prochilodus lineatus (Valenciennes, 1836) by cross-FISH using Cot-1 DNA (renaturation kinetics) probes. We found that the repetitive fractions of the genomes of Semaprochilodus insignis and Semaprochilodus taeniurus have significant amounts of conserved syntenic blocks in hybridization sites, but with low degrees of similarity between them and the genome of Prochilodus lineatus, especially in relation to B chromosomes. The cloning and sequencing of the repetitive genomic elements of Semaprochilodus insignis and Semaprochilodus taeniurus using Cot-1 DNA identified 48 fragments that displayed high similarity with repetitive sequences deposited in public DNA databases and classified as microsatellites, transposons, and retrotransposons. The repetitive fractions of the Semaprochilodus insignis and Semaprochilodus taeniurus genomes exhibited high degrees of conserved syntenic blocks in terms of both the structures and locations of hybridization sites, but a low degree of similarity with the syntenic blocks of the Prochilodus lineatus genome. Future comparative analyses of other prochilodontidae species will be needed to advance our understanding of the organization and evolution of the genomes in this group of fish. PMID:26752156

Rotifer rDNA-specific R9 retrotransposable elements generate an exceptionally long target site duplication upon insertion.

PubMed

Gladyshev, Eugene A; Arkhipova, Irina R

2009-12-15

Ribosomal DNA genes in many eukaryotes contain insertions of non-LTR retrotransposable elements belonging to the R2 clade. These elements persist in the host genomes by inserting site-specifically into multicopy target sites, thereby avoiding random disruption of single-copy host genes. Here we describe R9 retrotransposons from the R2 clade in the 28S RNA genes of bdelloid rotifers, small freshwater invertebrate animals best known for their long-term asexuality and for their ability to survive repeated cycles of desiccation and rehydration. While the structural organization of R9 elements is highly similar to that of other members of the R2 clade, they are characterized by two distinct features: site-specific insertion into a previously unreported target sequence within the 28S gene, and an unusually long target site duplication of 126 bp. We discuss the implications of these findings in the context of bdelloid genome organization and the mechanisms of target-primed reverse transcription.

Accelerated Evolution in Distinctive Species Reveals Candidate Elements for Clinically Relevant Traits, Including Mutation and Cancer Resistance.

PubMed

Ferris, Elliott; Abegglen, Lisa M; Schiffman, Joshua D; Gregg, Christopher

2018-03-06

The identity of most functional elements in the mammalian genome and the phenotypes they impact are unclear. Here, we perform a genome-wide comparative analysis of patterns of accelerated evolution in species with highly distinctive traits to discover candidate functional elements for clinically important phenotypes. We identify accelerated regions (ARs) in the elephant, hibernating bat, orca, dolphin, naked mole rat, and thirteen-lined ground squirrel lineages in mammalian conserved regions, uncovering ∼33,000 elements that bind hundreds of different regulatory proteins in humans and mice. ARs in the elephant, the largest land mammal, are uniquely enriched near elephant DNA damage response genes. The genomic hotspot for elephant ARs is the E3 ligase subunit of the Fanconi anemia complex, a master regulator of DNA repair. Additionally, ARs in the six species are associated with specific human clinical phenotypes that have apparent concordance with overt traits in each species. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Heterochromatin and molecular characterization of DsmarMITE transposable element in the beetle Dichotomius schiffleri (Coleoptera: Scarabaeidae).

PubMed

Xavier, Crislaine; Cabral-de-Mello, Diogo Cavalcanti; de Moura, Rita Cássia

2014-12-01

Cytogenetic studies of the Neotropical beetle genus Dichotomius (Scarabaeinae, Coleoptera) have shown dynamism for centromeric constitutive heterochromatin sequences. In the present work we studied the chromosomes and isolated repetitive sequences of Dichotomius schiffleri aiming to contribute to the understanding of coleopteran genome/chromosomal organization. Dichotomius schiffleri presented a conserved karyotype and heterochromatin distribution in comparison to other species of the genus with 2n = 18, biarmed chromosomes, and pericentromeric C-positive blocks. Similarly to heterochromatin distributional patterns, the highly and moderately repetitive DNA fraction (C 0 t-1 DNA) was detected in pericentromeric areas, contrasting with the euchromatic mapping of an isolated TE (named DsmarMITE). After structural analyses, the DsmarMITE was classified as a non-autonomous element of the type miniature inverted-repeat transposable element (MITE) with terminal inverted repeats similar to Mariner elements of insects from different orders. The euchromatic distribution for DsmarMITE indicates that it does not play a part in the dynamics of constitutive heterochromatin sequences.

Repetitive sequence analysis and karyotyping reveals centromere-associated DNA sequences in radish (Raphanus sativus L.).

PubMed

He, Qunyan; Cai, Zexi; Hu, Tianhua; Liu, Huijun; Bao, Chonglai; Mao, Weihai; Jin, Weiwei

2015-04-18

Radish (Raphanus sativus L., 2n = 2x = 18) is a major root vegetable crop especially in eastern Asia. Radish root contains various nutritions which play an important role in strengthening immunity. Repetitive elements are primary components of the genomic sequence and the most important factors in genome size variations in higher eukaryotes. To date, studies about repetitive elements of radish are still limited. To better understand genome structure of radish, we undertook a study to evaluate the proportion of repetitive elements and their distribution in radish. We conducted genome-wide characterization of repetitive elements in radish with low coverage genome sequencing followed by similarity-based cluster analysis. Results showed that about 31% of the genome was composed of repetitive sequences. Satellite repeats were the most dominating elements of the genome. The distribution pattern of three satellite repeat sequences (CL1, CL25, and CL43) on radish chromosomes was characterized using fluorescence in situ hybridization (FISH). CL1 was predominantly located at the centromeric region of all chromosomes, CL25 located at the subtelomeric region, and CL43 was a telomeric satellite. FISH signals of two satellite repeats, CL1 and CL25, together with 5S rDNA and 45S rDNA, provide useful cytogenetic markers to identify each individual somatic metaphase chromosome. The centromere-specific histone H3 (CENH3) has been used as a marker to identify centromere DNA sequences. One putative CENH3 (RsCENH3) was characterized and cloned from radish. Its deduced amino acid sequence shares high similarities to those of the CENH3s in Brassica species. An antibody against B. rapa CENH3, specifically stained radish centromeres. Immunostaining and chromatin immunoprecipitation (ChIP) tests with anti-BrCENH3 antibody demonstrated that both the centromere-specific retrotransposon (CR-Radish) and satellite repeat (CL1) are directly associated with RsCENH3 in radish. Proportions of repetitive elements in radish were estimated and satellite repeats were the most dominating elements. Fine karyotyping analysis was established which allow us to easily identify each individual somatic metaphase chromosome. Immunofluorescence- and ChIP-based assays demonstrated the functional significance of satellite and centromere-specific retrotransposon at centromeres. Our study provides a valuable basis for future genomic studies in radish.

MD simulations of papillomavirus DNA-E2 protein complexes hints at a protein structural code for DNA deformation.

PubMed

Falconi, M; Oteri, F; Eliseo, T; Cicero, D O; Desideri, A

2008-08-01

The structural dynamics of the DNA binding domains of the human papillomavirus strain 16 and the bovine papillomavirus strain 1, complexed with their DNA targets, has been investigated by modeling, molecular dynamics simulations, and nuclear magnetic resonance analysis. The simulations underline different dynamical features of the protein scaffolds and a different mechanical interaction of the two proteins with DNA. The two protein structures, although very similar, show differences in the relative mobility of secondary structure elements. Protein structural analyses, principal component analysis, and geometrical and energetic DNA analyses indicate that the two transcription factors utilize a different strategy in DNA recognition and deformation. Results show that the protein indirect DNA readout is not only addressable to the DNA molecule flexibility but it is finely tuned by the mechanical and dynamical properties of the protein scaffold involved in the interaction.

Engineering bacteria to solve the Burnt Pancake Problem

PubMed Central

Haynes, Karmella A; Broderick, Marian L; Brown, Adam D; Butner, Trevor L; Dickson, James O; Harden, W Lance; Heard, Lane H; Jessen, Eric L; Malloy, Kelly J; Ogden, Brad J; Rosemond, Sabriya; Simpson, Samantha; Zwack, Erin; Campbell, A Malcolm; Eckdahl, Todd T; Heyer, Laurie J; Poet, Jeffrey L

2008-01-01

Background We investigated the possibility of executing DNA-based computation in living cells by engineering Escherichia coli to address a classic mathematical puzzle called the Burnt Pancake Problem (BPP). The BPP is solved by sorting a stack of distinct objects (pancakes) into proper order and orientation using the minimum number of manipulations. Each manipulation reverses the order and orientation of one or more adjacent objects in the stack. We have designed a system that uses site-specific DNA recombination to mediate inversions of genetic elements that represent pancakes within plasmid DNA. Results Inversions (or "flips") of the DNA fragment pancakes are driven by the Salmonella typhimurium Hin/hix DNA recombinase system that we reconstituted as a collection of modular genetic elements for use in E. coli. Our system sorts DNA segments by inversions to produce different permutations of a promoter and a tetracycline resistance coding region; E. coli cells become antibiotic resistant when the segments are properly sorted. Hin recombinase can mediate all possible inversion operations on adjacent flippable DNA fragments. Mathematical modeling predicts that the system reaches equilibrium after very few flips, where equal numbers of permutations are randomly sorted and unsorted. Semiquantitative PCR analysis of in vivo flipping suggests that inversion products accumulate on a time scale of hours or days rather than minutes. Conclusion The Hin/hix system is a proof-of-concept demonstration of in vivo computation with the potential to be scaled up to accommodate larger and more challenging problems. Hin/hix may provide a flexible new tool for manipulating transgenic DNA in vivo. PMID:18492232

pH-Dependent DNA Distortion and Repression of Gene Expression by Pectobacterium atrosepticum PecS.

PubMed

Deochand, Dinesh K; Meariman, Jacob K; Grove, Anne

2016-07-15

Transcriptional activity is exquisitely sensitive to changes in promoter DNA topology. Transcription factors may therefore control gene activity by modulating the relative positioning of -10 and -35 promoter elements. The plant pathogen Pectobacterium atrosepticum, which causes soft rot in potatoes, must alter gene expression patterns to ensure growth in planta. In the related soft-rot enterobacterium Dickeya dadantii, PecS functions as a master regulator of virulence gene expression. Here, we report that P. atrosepticum PecS controls gene activity by altering promoter DNA topology in response to pH. While PecS binds the pecS promoter with high affinity regardless of pH, it induces significant DNA distortion only at neutral pH, the pH at which the pecS promoter is repressed in vivo. At pH ∼8, DNA distortions are attenuated, and PecS no longer represses the pecS promoter. A specific histidine (H142) located in a crevice between the dimerization- and DNA-binding regions is required for pH-dependent changes in DNA distortion and repression of gene activity, and mutation of this histidine renders the mutant protein incapable of repressing the pecS promoter. We propose that protonated PecS induces a DNA conformation at neutral pH in which -10 and -35 promoter elements are suboptimally positioned for RNA polymerase binding; on deprotonation of PecS, binding is no longer associated with significant changes in DNA conformation, allowing gene expression. We suggest that this mode of gene regulation leads to differential expression of the PecS regulon in response to alkalinization of the plant apoplast.

Architecture of the bacteriophage T4 activator MotA/promoter DNA interaction during sigma appropriation.

PubMed

Hsieh, Meng-Lun; James, Tamara D; Knipling, Leslie; Waddell, M Brett; White, Stephen; Hinton, Deborah M

2013-09-20

Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called σ appropriation, the T4 co-activator AsiA structurally remodels the σ(70) subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of σ(70) and binds to a DNA promoter element, the MotA box. Structures for the N-terminal (NTD) and C-terminal (CTD) domains of MotA are available, but no structure exists for MotA with or without DNA. We report the first molecular map of the MotA/DNA interaction within the σ-appropriated complex, which we obtained by using the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). We conjugated surface-exposed, single cysteines in MotA with FeBABE and performed cleavage reactions in the context of stable transcription complexes. The DNA cleavage sites were analyzed using ICM Molsoft software and three-dimensional physical models of MotA(NTD), MotA(CTD), and the DNA to investigate shape complementarity between the protein and the DNA and to position MotA on the DNA. We found that the unusual "double wing" motif present within MotA(CTD) resides in the major groove of the MotA box. In addition, we have used surface plasmon resonance to show that MotA alone is in a very dynamic equilibrium with the MotA element. Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses.

Sequence-Level Mechanisms of Human Epigenome Evolution

PubMed Central

Prendergast, James G.D.; Chambers, Emily V.; Semple, Colin A.M.

2014-01-01

DNA methylation and chromatin states play key roles in development and disease. However, the extent of recent evolutionary divergence in the human epigenome and the influential factors that have shaped it are poorly understood. To determine the links between genome sequence and human epigenome evolution, we examined the divergence of DNA methylation and chromatin states following segmental duplication events in the human lineage. Chromatin and DNA methylation states were found to have been generally well conserved following a duplication event, with the evolution of the epigenome largely uncoupled from the total number of genetic changes in the surrounding DNA sequence. However, the epigenome at tissue-specific, distal regulatory regions was observed to be unusually prone to diverge following duplication, with particular sequence differences, altering known sequence motifs, found to be associated with divergence in patterns of DNA methylation and chromatin. Alu elements were found to have played a particularly prominent role in shaping human epigenome evolution, and we show that human-specific AluY insertion events are strongly linked to the evolution of the DNA methylation landscape and gene expression levels, including at key neurological genes in the human brain. Studying paralogous regions within the same sample enables the study of the links between genome and epigenome evolution while controlling for biological and technical variation. We show DNA methylation and chromatin divergence between duplicated regions are linked to the divergence of particular genetic motifs, with Alu elements having played a disproportionate role in the evolution of the epigenome in the human lineage. PMID:24966180

Flavin Charge Transfer Transitions Assist DNA Photolyase Electron Transfer

NASA Astrophysics Data System (ADS)

Skourtis, Spiros S.; Prytkova, Tatiana; Beratan, David N.

2007-12-01

This contribution describes molecular dynamics, semi-empirical and ab-initio studies of the primary photo-induced electron transfer reaction in DNA photolyase. DNA photolyases are FADH--containing proteins that repair UV-damaged DNA by photo-induced electron transfer. A DNA photolyase recognizes and binds to cyclobutatne pyrimidine dimer lesions of DNA. The protein repairs a bound lesion by transferring an electron to the lesion from FADH-, upon photo-excitation of FADH- with 350-450 nm light. We compute the lowest singlet excited states of FADH- in DNA photolyase using INDO/S configuration interaction, time-dependent density-functional, and time-dependent Hartree-Fock methods. The calculations identify the lowest singlet excited state of FADH- that is populated after photo-excitation and that acts as the electron donor. For this donor state we compute conformationally-averaged tunneling matrix elements to empty electron-acceptor states of a thymine dimer bound to photolyase. The conformational averaging involves different FADH--thymine dimer confromations obtained from molecular dynamics simulations of the solvated protein with a thymine dimer docked in its active site. The tunneling matrix element computations use INDO/S-level Green's function, energy splitting, and Generalized Mulliken-Hush methods. These calculations indicate that photo-excitation of FADH- causes a π→π* charge-transfer transition that shifts electron density to the side of the flavin isoalloxazine ring that is adjacent to the docked thymine dimer. This shift in electron density enhances the FADH--to-dimer electronic coupling, thus inducing rapid electron transfer.

copia-like retrotransposons are ubiquitous among plants.

PubMed Central

Voytas, D F; Cummings, M P; Koniczny, A; Ausubel, F M; Rodermel, S R

1992-01-01

Transposable genetic elements are assumed to be a feature of all eukaryotic genomes. Their identification, however, has largely been haphazard, limited principally to organisms subjected to molecular or genetic scrutiny. We assessed the phylogenetic distribution of copia-like retrotransposons, a class of transposable element that proliferates by reverse transcription, using a polymerase chain reaction assay designed to detect copia-like element reverse transcriptase sequences. copia-like retrotransposons were identified in 64 plant species as well as the photosynthetic protist Volvox carteri. The plant species included representatives from 9 of 10 plant divisions, including bryophytes, lycopods, ferns, gymnosperms, and angiosperms. DNA sequence analysis of 29 cloned PCR products and of a maize retrotransposon cDNA confirmed the identity of these sequences as copia-like reverse transcriptase sequences, thereby demonstrating that this class of retrotransposons is a ubiquitous component of plant genomes. Images PMID:1379734

Repetitive DNA loci and their modulation by the non-canonical nucleic acid structures R-loops and G-quadruplexes

PubMed Central

Hall, Amanda C.; Ostrowski, Lauren A.; Mekhail, Karim

2017-01-01

ABSTRACT Cells have evolved intricate mechanisms to maintain genome stability despite allowing mutational changes to drive evolutionary adaptation. Repetitive DNA sequences, which represent the bulk of most genomes, are a major threat to genome stability often driving chromosome rearrangements and disease. The major source of repetitive DNA sequences and thus the most vulnerable constituents of the genome are the rDNA (rDNA) repeats, telomeres, and transposable elements. Maintaining the stability of these loci is critical to overall cellular fitness and lifespan. Therefore, cells have evolved mechanisms to regulate rDNA copy number, telomere length and transposon activity, as well as DNA repair at these loci. In addition, non-canonical structure-forming DNA motifs can also modulate the function of these repetitive DNA loci by impacting their transcription, replication, and stability. Here, we discuss key mechanisms that maintain rDNA repeats, telomeres, and transposons in yeast and human before highlighting emerging roles for non-canonical DNA structures at these repetitive loci. PMID:28406751

DNA sequence requirements for the accurate transcription of a protein-coding plastid gene in a plastid in vitro system from mustard (Sinapis alba L.)

PubMed Central

Link, Gerhard

1984-01-01

A nuclease-treated plastid extract from mustard (Sinapis alba L.) allows efficient transcription of cloned plastid DNA templates. In this in vitro system, the major runoff transcript of the truncated gene for the 32 000 mol. wt. photosystem II protein was accurately initiated from a site close to or identical with the in vivo start site. By using plasmids with deletions in the 5'-flanking region of this gene as templates, a DNA region required for efficient and selective initiation was detected ˜28-35 nucleotides upstream of the transcription start site. This region contains the sequence element TTGACA, which matches the consensus sequence for prokaryotic `−35' promoter elements. In the absence of this region, a region ˜13-27 nucleotides upstream of the start site still enables a basic level of specific transcription. This second region contains the sequence element TATATAA, which matches the consensus sequence for the `TATA' box of genes transcribed by RNA polymerase II (or B). The region between the `TATA'-like element and the transcription start site is not sufficient but may be required for specific transcription of the plastid gene. This latter region contains the sequence element TATACT, which resembles the prokaryotic `−10' (Pribnow) box. Based on the structural and transcriptional features of the 5' upstream region, a `promoter switch' mechanism is proposed, which may account for the developmentally regulated expression of this plastid gene. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Figure 5. PMID:16453540

Fragile X-related element 2 methylation analysis may provide a suitable option for inclusion of fragile X syndrome and/or sex chromosome aneuploidy into newborn screening: a technical validation study.

PubMed

Inaba, Yoshimi; Herlihy, Amy S; Schwartz, Charles E; Skinner, Cindy; Bui, Quang M; Cobb, Joanna; Shi, Elva Z; Francis, David; Arvaj, Alison; Amor, David J; Pope, Kate; Wotton, Tiffany; Cohen, Jonathan; Hewitt, Jacqueline K; Hagerman, Randi J; Metcalfe, Sylvia A; Hopper, John L; Loesch, Danuta Z; Slater, Howard R; Godler, David E

2013-04-01

We show that a novel fragile X-related epigenetic element 2 FMR1 methylation test can be used along with a test for sex-determining region Y (SRY) to provide the option of combined fragile X syndrome and sex chromosome aneuploidy newborn screening. Fragile X-related epigenetic element 2, SRY, and FMR1 CGG repeat analyses were performed on blood and saliva DNA, and in adult and newborn blood spots. The cohort consisted of 159 controls (CGG <40), 187 premutation (CGG 56-170), and 242 full-mutation (CGG ~200-2,000) males and females, 106 sex chromosome aneuploidy individuals, and 151 cytogenetically normal controls. At the 0.435 threshold, fragile X-related epigenetic element 2 analysis in males was robust on both blood DNA and newborn blood spots, with specificity and sensitivity of ~100% for full-mutation genotype. In females, the specificity was 99%, whereas half of full-mutation females were above the 0.435 threshold in both blood DNA and newborn blood spots. Furthermore, at this threshold, the test could not differentiate individuals with Klinefelter syndrome from female controls without using the SRY marker. When combined with SRY analysis, the test was consistent with most results for sex chromosome aneuploidies from karyotyping. Setting specific thresholds for fragile X-related epigenetic element 2 analysis and including the SRY marker provides the option to either include or exclude detection of sex chromosome aneuploidies as part of fragile X syndrome newborn screening.

Species-Specific Elements in the Large T-Antigen J Domain Are Required for Cellular Transformation and DNA Replication by Simian Virus 40

PubMed Central

Sullivan, Christopher S.; Tremblay, James D.; Fewell, Sheara W.; Lewis, John A.; Brodsky, Jeffrey L.; Pipas, James M.

2000-01-01

The J domain of simian virus 40 (SV40) large T antigen is required for efficient DNA replication and transformation. Despite previous reports demonstrating the promiscuity of J domains in heterologous systems, results presented here show the requirement for specific J-domain sequences in SV40 large-T-antigen-mediated activities. In particular, chimeric-T-antigen constructs in which the SV40 T-antigen J domain was replaced with that from the yeast Ydj1p or Escherichia coli DnaJ proteins failed to replicate in BSC40 cells and did not transform REF52 cells. However, T antigen containing the JC virus J domain was functional in these assays, although it was less efficient than the wild type. The inability of some large-T-antigen chimeras to promote DNA replication and elicit cellular transformation was not due to a failure to interact with hsc70, since a nonfunctional chimera, containing the DnaJ J domain, bound hsc70. However, this nonfunctional chimeric T antigen was reduced in its ability to stimulate hsc70 ATPase activity and unable to liberate E2F from p130, indicating that transcriptional activation of factors required for cell growth and DNA replication may be compromised. Our data suggest that the T-antigen J domain harbors species-specific elements required for viral activities in vivo. PMID:10891510

Plasticity of DNA methylation and gene expression under zinc deficiency in Arabidopsis roots.

PubMed

Chen, Xiaochao; Schönberger, Brigitte; Menz, Jochen; Ludewig, Uwe

2018-05-25

DNA methylation is a heritable chromatin modification that maintains chromosome stability, regulates transposon silencing and appears to be involved in gene expression in response to environmental conditions. Environmental stress alters DNA methylation patterns that are correlated with gene expression differences. Here, genome-wide differential DNA-methylation was identified upon prolonged Zn deficiency, leading to hypo- and hyper-methylated chromosomal regions. Preferential CpG methylation changes occurred in gene promoters and gene bodies, but did not overlap with transcriptional start sites. Methylation changes were also prominent in transposable elements. By contrast, non-CG methylation differences were exclusively found in promoters of protein coding genes and in transposable elements. Strongly Zn deficiency-induced genes and their promoters were mostly non-methylated, irrespective of Zn supply. Differential DNA methylation in the CpG and CHG, but not in the CHH context, was found close to a few up-regulated Zn-deficiency genes. However, the transcriptional Zn-deficiency response in roots appeared little correlated with associated DNA methylation changes in promoters or gene bodies. Furthermore, under Zn deficiency, developmental defects were identified in an Arabidopsis mutant lacking non-CpG methylation. The root methylome thus responds specifically to a micro-nutrient deficiency and is important for efficient Zn utilization at low availability, but the relationship of differential methylation and differentially expressed genes is surprisingly poor.

Recent Advancements in DNA Damage-Transcription Crosstalk and High-Resolution Mapping of DNA Breaks.

PubMed

Vitelli, Valerio; Galbiati, Alessandro; Iannelli, Fabio; Pessina, Fabio; Sharma, Sheetal; d'Adda di Fagagna, Fabrizio

2017-08-31

Until recently, DNA damage arising from physiological DNA metabolism was considered a detrimental by-product for cells. However, an increasing amount of evidence has shown that DNA damage could have a positive role in transcription activation. In particular, DNA damage has been detected in transcriptional elements following different stimuli. These physiological DNA breaks are thought to be instrumental for the correct expression of genomic loci through different mechanisms. In this regard, although a plethora of methods are available to precisely map transcribed regions and transcription start sites, commonly used techniques for mapping DNA breaks lack sufficient resolution and sensitivity to draw a robust correlation between DNA damage generation and transcription. Recently, however, several methods have been developed to map DNA damage at single-nucleotide resolution, thus providing a new set of tools to correlate DNA damage and transcription. Here, we review how DNA damage can positively regulate transcription initiation, the current techniques for mapping DNA breaks at high resolution, and how these techniques can benefit future studies of DNA damage and transcription.

Detection and characterization of miniature inverted-repeat transposable elements in “Candidatus Liberibacter asiaticus”

USDA-ARS?s Scientific Manuscript database

Miniature inverted-repeat transposable elements (MITEs) are non-autonomous transposons (devoid a transposase gene, tps) involving insertion/deletion of genomic DNA in bacterial genomes influencing gene functions. No transposon has yet been reported in “Candidatus Liberibacter asiaticus”, an alpha-pr...

Transposable elements and G-quadruplexes.

PubMed

Kejnovsky, Eduard; Tokan, Viktor; Lexa, Matej

2015-09-01

A significant part of eukaryotic genomes is formed by transposable elements (TEs) containing not only genes but also regulatory sequences. Some of the regulatory sequences located within TEs can form secondary structures like hairpins or three-stranded (triplex DNA) and four-stranded (quadruplex DNA) conformations. This review focuses on recent evidence showing that G-quadruplex-forming sequences in particular are often present in specific parts of TEs in plants and humans. We discuss the potential role of these structures in the TE life cycle as well as the impact of G-quadruplexes on replication, transcription, translation, chromatin status, and recombination. The aim of this review is to emphasize that TEs may serve as vehicles for the genomic spread of G-quadruplexes. These non-canonical DNA structures and their conformational switches may constitute another regulatory system that, together with small and long non-coding RNA molecules and proteins, contribute to the complex cellular network resulting in the large diversity of eukaryotes.

Fabrication of microfluidic integrated biosensor

NASA Astrophysics Data System (ADS)

Adam, Tijjani; Dhahi, Th S.; Mohammed, Mohammed; Hashim, U.; Noriman, N. Z.; Dahham, Omar S.

2017-09-01

An event of miniaturizing for sensor systems to carry out biological diagnostics are gaining wade spread acceptance. The system may contain several different sensor units for the detection of specific analyte, the analyte to be detected might be any kind of biological molecules (DNA, mRNA or proteins) or chemical substances. In most cases, the detection is based on receptor-ligand binding like DNA hybridization or antibody-antigen interaction, achieving this on a nanostructure. DNA or protein must be attached to certain locations within the structure. Critical for this is to have a robust binding chemistry to the surface in the microstructure. Here we successfully designed and fabricated microfluidics element for passive fluid delivery into polysilicon Nanowire sensing domain, we further demonstrated a very simple and effective way of integrating the two devices to give full functionalities of laboratory on a single chip. The sensing element was successfully surface modified and tested on real biomedical clinical sample for evaluation and validation.

Molecular Cloning and Analysis of a DNA Repetitive Element from the Mouse Genome

ERIC Educational Resources Information Center

Geisinger, Adriana; Cossio, Gabriela; Wettstein, Rodolfo

2006-01-01

We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry…

Targeted gene insertion for molecular medicine.

PubMed

Voigt, Katrin; Izsvák, Zsuzsanna; Ivics, Zoltán

2008-11-01

Genomic insertion of a functional gene together with suitable transcriptional regulatory elements is often required for long-term therapeutical benefit in gene therapy for several genetic diseases. A variety of integrating vectors for gene delivery exist. Some of them exhibit random genomic integration, whereas others have integration preferences based on attributes of the targeted site, such as primary DNA sequence and physical structure of the DNA, or through tethering to certain DNA sequences by host-encoded cellular factors. Uncontrolled genomic insertion bears the risk of the transgene being silenced due to chromosomal position effects, and can lead to genotoxic effects due to mutagenesis of cellular genes. None of the vector systems currently used in either preclinical experiments or clinical trials displays sufficient preferences for target DNA sequences that would ensure appropriate and reliable expression of the transgene and simultaneously prevent hazardous side effects. We review in this paper the advantages and disadvantages of both viral and non-viral gene delivery technologies, discuss mechanisms of target site selection of integrating genetic elements (viruses and transposons), and suggest distinct molecular strategies for targeted gene delivery.

Cyclosporin A and FK-506 both affect DNA binding of regulatory nuclear proteins to the human interleukin-2 promoter.

PubMed

Baumann, G; Geisse, S; Sullivan, M

1991-03-01

The structurally unrelated immunosuppressive drugs cyclosporin A (Sandimmun) and FK-506 both interfere with the process of T-cell proliferation by blocking the transcription of the T-cell growth factor interleukin-2 (IL-2). Here we demonstrate that the transcriptional activation of this gene requires the binding of regulatory nuclear proteins to a promoter element with sequence similarity to the consensus binding site for NF-kappa B-related transcription factors. We present evidence that the binding by regulatory nuclear proteins to the kappa B element of the IL-2 promoter is affected negatively by cyclosporin A and FK-506 at concentrations paralleling their immunosuppressive activity in vivo. The decrease in DNA-protein complex formation induced by the immunosuppressive drugs correlates with a decrease in IL-2 production. FK-506 is 10 to 100 times more potent than cyclosporin A in its ability to inhibit sequence-specific DNA binding and IL-2 production. Our findings suggest that the actions of both drugs converge at the level of DNA-protein interaction.

Systematic prediction of control proteins and their DNA binding sites

PubMed Central

Sorokin, Valeriy; Severinov, Konstantin; Gelfand, Mikhail S.

2009-01-01

We present here the results of a systematic bioinformatics analysis of control (C) proteins, a class of DNA-binding regulators that control time-delayed transcription of their own genes as well as restriction endonuclease genes in many type II restriction-modification systems. More than 290 C protein homologs were identified and DNA-binding sites for ∼70% of new and previously known C proteins were predicted by a combination of phylogenetic footprinting and motif searches in DNA upstream of C protein genes. Additional analysis revealed that a large proportion of C protein genes are translated from leaderless RNA, which may contribute to time-delayed nature of genetic switches operated by these proteins. Analysis of genetic contexts of newly identified C protein genes revealed that they are not exclusively associated with restriction-modification genes; numerous instances of associations with genes originating from mobile genetic elements were observed. These instances might be vestiges of ancient horizontal transfers and indicate that during evolution ancestral restriction-modification system genes were the sites of mobile elements insertions. PMID:19056824

Libraries of Synthetic TALE-Activated Promoters: Methods and Applications.

PubMed

Schreiber, T; Tissier, A

2016-01-01

The discovery of proteins with programmable DNA-binding specificities triggered a whole array of applications in synthetic biology, including genome editing, regulation of transcription, and epigenetic modifications. Among those, transcription activator-like effectors (TALEs) due to their natural function as transcription regulators, are especially well-suited for the development of orthogonal systems for the control of gene expression. We describe here the construction and testing of libraries of synthetic TALE-activated promoters which are under the control of a single TALE with a given DNA-binding specificity. These libraries consist of a fixed DNA-binding element for the TALE, a TATA box, and variable sequences of 19 bases upstream and 43 bases downstream of the DNA-binding element. These libraries were cloned using a Golden Gate cloning strategy making them usable as standard parts in a modular cloning system. The broad range of promoter activities detected and the versatility of these promoter libraries make them valuable tools for applications in the fine-tuning of expression in metabolic engineering projects or in the design and implementation of regulatory circuits. © 2016 Elsevier Inc. All rights reserved.

Recovery and separation of rare earth elements using columns loaded with DNA-filter hybrid.

PubMed

Takahashi, Yoshio; Kondo, Kazuhiro; Miyaji, Asami; Umeo, Miyuki; Honma, Tetsuo; Asaoka, Satoshi

2012-01-01

Given that the supply of several rare earth elements (REEs) is sometimes limited, recycling REEs used in various advanced materials, such as Nd magnets, is important for realizing efficient use of REE resources. In the present work, the feasibility of using DNA for REE recovery and separation was examined, along with the identification of the binding site of REEs in DNA. In particular, a DNA-cellulose filter paper hybrid was prepared so that DNA-based materials can be used for the separation of REEs using columns loaded with DNA. N,N'-Disuccinimidyl was used as a cross-linker reagent for the fixation of DNA onto a fibrous cellulose filter. The results showed that (i) the DNA-filter hybrid has a sufficiently high affinity to adsorb REEs; (ii) the adsorption capacity was 0.182 mg/g for Nd; and (iii) the affinity of REEs for DNA was stronger for REEs with larger atomic numbers. The difference of the affinity among REEs in the third result was compared with the adsorption patterns of REEs discussed in the literature. The comparison suggests that phosphate in the DNA-filter paper hybrid was responsible for REE adsorption onto the hybrid. The results were supported by the Nd, Dy, and Lu L(III)-edge EXAFS; the REE-P shell was identified for the second neighboring atom, showing the importance of the phosphate site as REE binding sites. The difference in the affinity among REEs suggest that group separation of REEs (such as La, Ce, (Pr and Nd), (Ho, Dy, and Er), (Tb and Gd), (Sm, Eu), Tm, Yb, and Lu) is possible, although complete isolation of each REE from a solution containing all REEs may be difficult. For practical applications, Nd and Fe(III) were successfully separated from a synthetic solution of Nd magnet waste using columns loaded with the DNA-filter hybrid.

Inducible in vivo DNA footprints define sequences necessary for UV light activation of the parsley chalcone synthase gene.

PubMed Central

Schulze-Lefert, P; Dangl, J L; Becker-André, M; Hahlbrock, K; Schulz, W

1989-01-01

We began characterization of the protein--DNA interactions necessary for UV light induced transcriptional activation of the gene encoding chalcone synthase (CHS), a key plant defense enzyme. Three light dependent in vivo footprints appear on a 90 bp stretch of the CHS promoter with a time course correlated with the onset of CHS transcription. We define a minimal light responsive promoter by functional analysis of truncated CHS promoter fusions with a reporter gene in transient expression experiments in parsley protoplasts. Two of the three footprinted sequence 'boxes' reside within the minimal promoter. Replacement of 10 bp within either of these 'boxes' leads to complete loss of light responsiveness. We conclude that these sequences define the necessary cis elements of the minimal CHS promoter's light responsive element. One of the functionally defined 'boxes' is homologous to an element implicated in regulation of genes involved in photosynthesis. These data represent the first example in a plant defense gene of an induced change in protein--DNA contacts necessary for transcriptional activation. Also, our data argue strongly that divergent light induced biosynthetic pathways share common regulatory units. Images PMID:2566481

Radiation-Induced Epigenetic Alterations after Low and High LET Irradiations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aypar, Umut; Morgan, William F.; Baulch, Janet E.

Epigenetics, including DNA methylation and microRNA (miRNA) expression, could be the missing link in understanding the delayed, non-targeted effects of radiation including radiationinduced genomic instability (RIGI). This study tests the hypothesis that irradiation induces epigenetic aberrations, which could eventually lead to RIGI, and that the epigenetic aberrations induced by low linear energy transfer (LET) irradiation are different than those induced by high LET irradiations. GM10115 cells were irradiated with low LET x-rays and high LET iron (Fe) ions and evaluated for DNA damage, cell survival and chromosomal instability. The cells were also evaluated for specific locus methylation of nuclear factor-kappamore » B (NFκB), tumor suppressor in lung cancer 1 (TSLC1) and cadherin 1 (CDH1) gene promoter regions, long interspersed nuclear element 1 (LINE-1) and Alu repeat element methylation, CpG and non-CpG global methylation and miRNA expression levels. Irradiated cells showed increased micronucleus induction and cell killing immediately following exposure, but were chromosomally stable at delayed times post-irradiation. At this same delayed time, alterations in repeat element and global DNA methylation and miRNA expression were observed. Analyses of DNA methylation predominantly showed hypomethylation, however hypermethylation was also observed. MiRNA shown to be altered in expression level after x-ray irradiation are involved in chromatin remodeling and DNA methylation. Different and higher incidence of epigenetic changes were observed after exposure to low LET x-rays than high LET Fe ions even though Fe ions elicited more chromosomal damage and cell killing. This study also shows that the irradiated cells acquire epigenetic changes even though they are chromosomally stable suggesting that epigenetic aberrations may arise in the cell without initiating RIGI.« less

UV-induced DNA damage is an intermediate step in UV-induced expression of human immunodeficiency virus type 1, collagenase, c-fos, and metallothionein.

PubMed Central

Stein, B; Rahmsdorf, H J; Steffen, A; Litfin, M; Herrlich, P

1989-01-01

UV irradiation of human and murine cells enhances the transcription of several genes. Here we report on the primary target of relevant UV absorption, on pathways leading to gene activation, and on the elements receiving the UV-induced signal in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, in the gene coding for collagenase, and in the cellular oncogene fos. In order to induce the expression of genes. UV radiation needs to be absorbed by DNA and to cause DNA damage of the kind that cannot be repaired by cells from patients with xeroderma pigmentosum group A. UV-induced activation of the three genes is mediated by the major enhancer elements (located between nucleotide positions -105 and -79 of HIV-1, between positions -72 and -65 of the collagenase gene, and between positions -320 and -299 of fos). These elements share no apparent sequence motif and bind different trans-acting proteins; a member of the NF kappa B family binds to the HIV-1 enhancer, the heterodimer of Jun and Fos (AP-1) binds to the collagenase enhancer, and the serum response factors p67 and p62 bind to fos. DNA-binding activities of the factors recognizing the HIV-1 and collagenase enhancers are augmented in extracts from UV-treated cells. The increase in activity is due to posttranslational modification. While AP-1 resides in the nucleus and must be modulated there, NF kappa B is activated in the cytoplasm, indicating the existence of a cytoplasmic signal transduction pathway triggered by UV-induced DNA damage. In addition to activation, new synthesis of AP-1 is induced by UV radiation. Images PMID:2557547

The genomes of many yam species contain transcriptionally active endogenous geminiviral sequences that may be functionally expressed

PubMed Central

Filloux, Denis; Murrell, Sasha; Koohapitagtam, Maneerat; Golden, Michael; Julian, Charlotte; Galzi, Serge; Uzest, Marilyne; Rodier-Goud, Marguerite; D’Hont, Angélique; Vernerey, Marie Stephanie; Wilkin, Paul; Peterschmitt, Michel; Winter, Stephan; Murrell, Ben; Martin, Darren P.; Roumagnac, Philippe

2015-01-01

Endogenous viral sequences are essentially ‘fossil records’ that can sometimes reveal the genomic features of long extinct virus species. Although numerous known instances exist of single-stranded DNA (ssDNA) genomes becoming stably integrated within the genomes of bacteria and animals, there remain very few examples of such integration events in plants. The best studied of these events are those which yielded the geminivirus-related DNA elements found within the nuclear genomes of various Nicotiana species. Although other ssDNA virus-like sequences are included within the draft genomes of various plant species, it is not entirely certain that these are not contaminants. The Nicotiana geminivirus-related DNA elements therefore remain the only definitively proven instances of endogenous plant ssDNA virus sequences. Here, we characterize two new classes of endogenous plant virus sequence that are also apparently derived from ancient geminiviruses in the genus Begomovirus. These two endogenous geminivirus-like elements (EGV1 and EGV2) are present in the Dioscorea spp. of the Enantiophyllum clade. We used fluorescence in situ hybridization to confirm that the EGV1 sequences are integrated in the D. alata genome and showed that one or two ancestral EGV sequences likely became integrated more than 1.4 million years ago during or before the diversification of the Asian and African Enantiophyllum Dioscorea spp. Unexpectedly, we found evidence of natural selection actively favouring the maintenance of EGV-expressed replication-associated protein (Rep) amino acid sequences, which clearly indicates that functional EGV Rep proteins were probably expressed for prolonged periods following endogenization. Further, the detection in D. alata of EGV gene transcripts, small 21–24 nt RNAs that are apparently derived from these transcripts, and expressed Rep proteins, provides evidence that some EGV genes are possibly still functionally expressed in at least some of the Enantiophyllum clade species. PMID:27774276

DNA nanotechnology-enabled biosensors.

PubMed

Chao, Jie; Zhu, Dan; Zhang, Yinan; Wang, Lianhui; Fan, Chunhai

2016-02-15

Biosensors employ biological molecules to recognize the target and utilize output elements which can translate the biorecognition event into electrical, optical or mass-sensitive signals to determine the quantities of the target. DNA-based biosensors, as a sub-field to biosensor, utilize DNA strands with short oligonucleotides as probes for target recognition. Although DNA-based biosensors have offered a promising alternative for fast, simple and cheap detection of target molecules, there still exist key challenges including poor stability and reproducibility that hinder their competition with the current gold standard for DNA assays. By exploiting the self-recognition properties of DNA molecules, researchers have dedicated to make versatile DNA nanostructures in a highly rigid, controllable and functionalized manner, which offers unprecedented opportunities for developing DNA-based biosensors. In this review, we will briefly introduce the recent advances on design and fabrication of static and dynamic DNA nanostructures, and summarize their applications for fabrication and functionalization of DNA-based biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

Recent Insights into the Control of Human Papillomavirus (HPV) Genome Stability, Loss, and Degradation.

PubMed

Fisher, Chris

2015-01-01

Most human papillomavirus (HPV) antiviral strategies have focused upon inhibiting viral DNA replication, but it is increasingly apparent that viral DNA levels can be chemically controlled by approaches that promote its instability. HPVs and other DNA viruses have a tenuous relationship with their hosts. They must replicate and hide from the DNA damage response (DDR) and innate immune systems, which serve to protect cells from foreign or "non-self" DNA, and yet they draft these same systems to support their life cycles. DNA binding antiviral agents promoting massive viral DNA instability and elimination are reviewed. Mechanistic studies of these agents have identified genetic antiviral enhancers and repressors, antiviral sensitizers, and host cell elements that protect and stabilize HPV genomes. Viral DNA degradation appears to be an important means of controlling HPV DNA levels in some cases, but the underlying mechanisms remain poorly understood. These findings may prove useful not only for understanding viral DNA persistence but also in devising future antiviral strategies.

Graphene oxide-DNA based sensors.

PubMed

Gao, Li; Lian, Chaoqun; Zhou, Yang; Yan, Lirong; Li, Qin; Zhang, Chunxia; Chen, Liang; Chen, Keping

2014-10-15

Since graphene oxide (GO) is readily available and exhibits exceptional optical, electrical, mechanical and chemical properties, it has attracted increasing interests for use in GO-DNA based sensors. This paper reviews the advances in GO-DNA based sensors using DNA as recognition elements. In solution, GO is as an excellent acceptor of fluorescence resonance energy transfer (FRET) to quench the fluorescence in dye labeled DNA sequences. This review discusses the emerging GO-DNA based sensors related to FRET for use in the detection of DNA, proteins, metal ions, cysteine (Cys), and others. The application of the electrochemical GO-DNA based sensors is also summarized because GO possesses exceptional electrochemical properties. The detection mechanisms and the advantages of GO are also revealed and discussed. GO-DNA based sensors perform well at low cost, and high sensitivity, and provide low detection limits. Additionally, GO-DNA based sensors should appear in the near future as scientists explore their usefulness and properties. Finally, future perspectives and possible challenges in this area are outlined. Copyright © 2014 Elsevier B.V. All rights reserved.

Structural Basis for Sequence-specific DNA Recognition by an Arabidopsis WRKY Transcription Factor*

PubMed Central

Yamasaki, Kazuhiko; Kigawa, Takanori; Watanabe, Satoru; Inoue, Makoto; Yamasaki, Tomoko; Seki, Motoaki; Shinozaki, Kazuo; Yokoyama, Shigeyuki

2012-01-01

The WRKY family transcription factors regulate plant-specific reactions that are mostly related to biotic and abiotic stresses. They share the WRKY domain, which recognizes a DNA element (TTGAC(C/T)) termed the W-box, in target genes. Here, we determined the solution structure of the C-terminal WRKY domain of Arabidopsis WRKY4 in complex with the W-box DNA by NMR. A four-stranded β-sheet enters the major groove of DNA in an atypical mode termed the β-wedge, where the sheet is nearly perpendicular to the DNA helical axis. Residues in the conserved WRKYGQK motif contact DNA bases mainly through extensive apolar contacts with thymine methyl groups. The importance of these contacts was verified by substituting the relevant T bases with U and by surface plasmon resonance analyses of DNA binding. PMID:22219184

FARME DB: a functional antibiotic resistance element database

PubMed Central

Wallace, James C.; Port, Jesse A.; Smith, Marissa N.; Faustman, Elaine M.

2017-01-01

Antibiotic resistance (AR) is a major global public health threat but few resources exist that catalog AR genes outside of a clinical context. Current AR sequence databases are assembled almost exclusively from genomic sequences derived from clinical bacterial isolates and thus do not include many microbial sequences derived from environmental samples that confer resistance in functional metagenomic studies. These environmental metagenomic sequences often show little or no similarity to AR sequences from clinical isolates using standard classification criteria. In addition, existing AR databases provide no information about flanking sequences containing regulatory or mobile genetic elements. To help address this issue, we created an annotated database of DNA and protein sequences derived exclusively from environmental metagenomic sequences showing AR in laboratory experiments. Our Functional Antibiotic Resistant Metagenomic Element (FARME) database is a compilation of publically available DNA sequences and predicted protein sequences conferring AR as well as regulatory elements, mobile genetic elements and predicted proteins flanking antibiotic resistant genes. FARME is the first database to focus on functional metagenomic AR gene elements and provides a resource to better understand AR in the 99% of bacteria which cannot be cultured and the relationship between environmental AR sequences and antibiotic resistant genes derived from cultured isolates. Database URL: http://staff.washington.edu/jwallace/farme PMID:28077567

An updated version of NPIDB includes new classifications of DNA–protein complexes and their families

PubMed Central

Zanegina, Olga; Kirsanov, Dmitriy; Baulin, Eugene; Karyagina, Anna; Alexeevski, Andrei; Spirin, Sergey

2016-01-01

The recent upgrade of nucleic acid–protein interaction database (NPIDB, http://npidb.belozersky.msu.ru/) includes a newly elaborated classification of complexes of protein domains with double-stranded DNA and a classification of families of related complexes. Our classifications are based on contacting structural elements of both DNA: the major groove, the minor groove and the backbone; and protein: helices, beta-strands and unstructured segments. We took into account both hydrogen bonds and hydrophobic interaction. The analyzed material contains 1942 structures of protein domains from 748 PDB entries. We have identified 97 interaction modes of individual protein domain–DNA complexes and 17 DNA–protein interaction classes of protein domain families. We analyzed the sources of diversity of DNA–protein interaction modes in different complexes of one protein domain family. The observed interaction mode is sometimes influenced by artifacts of crystallization or diversity in secondary structure assignment. The interaction classes of domain families are more stable and thus possess more biological sense than a classification of single complexes. Integration of the classification into NPIDB allows the user to browse the database according to the interacting structural elements of DNA and protein molecules. For each family, we present average DNA shape parameters in contact zones with domains of the family. PMID:26656949

Alpha3, a transposable element that promotes host sexual reproduction.

PubMed

Barsoum, Emad; Martinez, Paula; Aström, Stefan U

2010-01-01

Theoretical models predict that selfish DNA elements require host sex to persist in a population. Therefore, a transposon that induces sex would strongly favor its own spread. We demonstrate that a protein homologous to transposases, called alpha3, was essential for mating type switch in Kluyveromyces lactis. Mutational analysis showed that amino acids conserved among transposases were essential for its function. During switching, sequences in the 5' and 3' flanking regions of the alpha3 gene were joined, forming a DNA circle, showing that alpha3 mobilized from the genome. The sequences encompassing the alpha3 gene circle junctions in the mating type alpha (MATalpha) locus were essential for switching from MATalpha to MATa, suggesting that alpha3 mobilization was a coupled event. Switching also required a DNA-binding protein, Mating type switch 1 (Mts1), whose binding sites in MATalpha were important. Expression of Mts1 was repressed in MATa/MATalpha diploids and by nutrients, limiting switching to haploids in low-nutrient conditions. A hairpin-capped DNA double-strand break (DSB) was observed in the MATa locus in mre11 mutant strains, indicating that mating type switch was induced by MAT-specific DSBs. This study provides empirical evidence for selfish DNA promoting host sexual reproduction by mediating mating type switch.

Paramecium tetraurelia chromatin assembly factor-1-like protein PtCAF-1 is involved in RNA-mediated control of DNA elimination.

PubMed

Ignarski, Michael; Singh, Aditi; Swart, Estienne C; Arambasic, Miroslav; Sandoval, Pamela Y; Nowacki, Mariusz

2014-10-29

Genome-wide DNA remodelling in the ciliate Paramecium is ensured by RNA-mediated trans-nuclear crosstalk between the germline and the somatic genomes during sexual development. The rearrangements include elimination of transposable elements, minisatellites and tens of thousands non-coding elements called internally eliminated sequences (IESs). The trans-nuclear genome comparison process employs a distinct class of germline small RNAs (scnRNAs) that are compared against the parental somatic genome to select the germline-specific subset of scnRNAs that subsequently target DNA elimination in the progeny genome. Only a handful of proteins involved in this process have been identified so far and the mechanism of DNA targeting is unknown. Here we describe chromatin assembly factor-1-like protein (PtCAF-1), which we show is required for the survival of sexual progeny and localizes first in the parental and later in the newly developing macronucleus. Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs. PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development. We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells. Our results demonstrate the importance of PtCAF-1 for the epigenetic trans-nuclear cross-talk mechanism. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

WordCluster: detecting clusters of DNA words and genomic elements

PubMed Central

2011-01-01

Background Many k-mers (or DNA words) and genomic elements are known to be spatially clustered in the genome. Well established examples are the genes, TFBSs, CpG dinucleotides, microRNA genes and ultra-conserved non-coding regions. Currently, no algorithm exists to find these clusters in a statistically comprehensible way. The detection of clustering often relies on densities and sliding-window approaches or arbitrarily chosen distance thresholds. Results We introduce here an algorithm to detect clusters of DNA words (k-mers), or any other genomic element, based on the distance between consecutive copies and an assigned statistical significance. We implemented the method into a web server connected to a MySQL backend, which also determines the co-localization with gene annotations. We demonstrate the usefulness of this approach by detecting the clusters of CAG/CTG (cytosine contexts that can be methylated in undifferentiated cells), showing that the degree of methylation vary drastically between inside and outside of the clusters. As another example, we used WordCluster to search for statistically significant clusters of olfactory receptor (OR) genes in the human genome. Conclusions WordCluster seems to predict biological meaningful clusters of DNA words (k-mers) and genomic entities. The implementation of the method into a web server is available at http://bioinfo2.ugr.es/wordCluster/wordCluster.php including additional features like the detection of co-localization with gene regions or the annotation enrichment tool for functional analysis of overlapped genes. PMID:21261981

An Ancient Transkingdom Horizontal Transfer of Penelope-Like Retroelements from Arthropods to Conifers

PubMed Central

Lin, Xuan; Faridi, Nurul; Casola, Claudio

2016-01-01

Comparative genomics analyses empowered by the wealth of sequenced genomes have revealed numerous instances of horizontal DNA transfers between distantly related species. In eukaryotes, repetitive DNA sequences known as transposable elements (TEs) are especially prone to move across species boundaries. Such horizontal transposon transfers, or HTTs, are relatively common within major eukaryotic kingdoms, including animals, plants, and fungi, while rarely occurring across these kingdoms. Here, we describe the first case of HTT from animals to plants, involving TEs known as Penelope-like elements, or PLEs, a group of retrotransposons closely related to eukaryotic telomerases. Using a combination of in situ hybridization on chromosomes, polymerase chain reaction experiments, and computational analyses we show that the predominant PLE lineage, EN(+)PLEs, is highly diversified in loblolly pine and other conifers, but appears to be absent in other gymnosperms. Phylogenetic analyses of both protein and DNA sequences reveal that conifers EN(+)PLEs, or Dryads, form a monophyletic group clustering within a clade of primarily arthropod elements. Additionally, no EN(+)PLEs were detected in 1,928 genome assemblies from 1,029 nonmetazoan and nonconifer genomes from 14 major eukaryotic lineages. These findings indicate that Dryads emerged following an ancient horizontal transfer of EN(+)PLEs from arthropods to a common ancestor of conifers approximately 340 Ma. This represents one of the oldest known interspecific transmissions of TEs, and the most conspicuous case of DNA transfer between animals and plants. PMID:27190138

Preferential inclusion of extrachromosomal genetic elements in yeast meiotic spores.

PubMed

Brewer, B J; Fangman, W L

1980-09-01

During meiosis and sporulation in the yeast Saccharomyces cerevisiae, extrachromosomal traits are efficiently transmitted to haploid spores. Although the pattern of inheritance of chromosomal traits reflects the mechanism of regular chromosomal segregation in meiosis, it is not known what processes are reflected by the efficient inheritance of extrachromosomal traits. Because extrachromosomal genetic elements in yeast are present in multiple copies, perpetuation of an extrachromosomal trait could occur by the passive envelopment of a subset of copies or by an active sequestering of all or a subset of copies within the four spores. We show that only subsets of the four extrachromosomal nucleic acids commonly found in yeast are transmitted through meiosis--55% of mitochondrial DNA copies, 82% of the 2-micron DNA plasmids, and about 70% of the L and M double-stranded RNAs. However, electron micrographs of serial sections through yeast asci indicate that the four spore enclose only 30% of the total ascus material. Thus these extrachromosomal elements are preferentially included within the spores, indicating that their inheritance is not a random process. Transmission of mitochondrial DNA can be accounted for by the observed enclosure of 52% of the mitochondrial volume within the spores. The high transmission frequencies of the double-stranded RNAs (which exist as virus-like particles in the cytoplasm) and 2-micron DNA must indicate that either these nucleic acids are actively recruited from the cytoplasm by some mechanism or they are associated in some way with the nucleus during meiosis.

Origins and evolution of viruses of eukaryotes: The ultimate modularity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koonin, Eugene V., E-mail: koonin@ncbi.nlm.nih.gov; Dolja, Valerian V., E-mail: doljav@science.oregonstate.edu; Krupovic, Mart, E-mail: krupovic@pasteur.fr

2015-05-15

Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes of viruses are dramatically different between prokaryotes and eukaryotes. In prokaryotes, the great majority of viruses possess double-stranded (ds) DNA genomes, with a substantial minority of single-stranded (ss) DNA viruses and only limited presence of RNA viruses. In contrast, in eukaryotes, RNA viruses account for the majority of the virome diversity although ssDNA and dsDNA viruses are common as well. Phylogenomic analysis yields tangiblemore » clues for the origins of major classes of eukaryotic viruses and in particular their likely roots in prokaryotes. Specifically, the ancestral genome of positive-strand RNA viruses of eukaryotes might have been assembled de novo from genes derived from prokaryotic retroelements and bacteria although a primordial origin of this class of viruses cannot be ruled out. Different groups of double-stranded RNA viruses derive either from dsRNA bacteriophages or from positive-strand RNA viruses. The eukaryotic ssDNA viruses apparently evolved via a fusion of genes from prokaryotic rolling circle-replicating plasmids and positive-strand RNA viruses. Different families of eukaryotic dsDNA viruses appear to have originated from specific groups of bacteriophages on at least two independent occasions. Polintons, the largest known eukaryotic transposons, predicted to also form virus particles, most likely, were the evolutionary intermediates between bacterial tectiviruses and several groups of eukaryotic dsDNA viruses including the proposed order “Megavirales” that unites diverse families of large and giant viruses. Strikingly, evolution of all classes of eukaryotic viruses appears to have involved fusion between structural and replicative gene modules derived from different sources along with additional acquisitions of diverse genes. - Highlights: • Eukaryotic virome dramatically differs from the viromes of bacteria and archaea. • Eukaryotic virome is dominated by RNA viruses and retroelements. • All classes of eukaryotic viruses evolved by gene module exchange. • Prokaryotic ancestry is traceable for core gene modules of most eukaryotic viruses. • Evolutionary histories of viruses and transposable elements are tightly linked.« less

DNA N(6)-methyladenine: a new epigenetic mark in eukaryotes?

PubMed

Luo, Guan-Zheng; Blanco, Mario Andres; Greer, Eric Lieberman; He, Chuan; Shi, Yang

2015-12-01

DNA N(6)-adenine methylation (N(6)-methyladenine; 6mA) in prokaryotes functions primarily in the host defence system. The prevalence and significance of this modification in eukaryotes had been unclear until recently. Here, we discuss recent publications documenting the presence of 6mA in Chlamydomonas reinhardtii, Drosophila melanogaster and Caenorhabditis elegans; consider possible roles for this DNA modification in regulating transcription, the activity of transposable elements and transgenerational epigenetic inheritance; and propose 6mA as a new epigenetic mark in eukaryotes.

Construction and Nonclinical Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine

DTIC Science & Technology

2012-12-12

immunogenic hantavirus M gene-based DNA vaccines against the HFRS hantaviruses , we ini- tiated preclinical testing of these vaccines, delivered using a...Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR (S) 5d. PROJECT...Vaccination with pWRG/ PUU-M(s2) protected hamsters against infection with PUUV but not against infection by related HFRS-associated hantaviruses

Comparing anterior and posterior Hox complex formation reveals guidelines for predicting cis-regulatory elements

PubMed Central

Uhl, Juli D.; Cook, Tiffany A.; Gebelein, Brian

2010-01-01

Hox transcription factors specify numerous cell fates along the anterior-posterior axis by regulating the expression of downstream target genes. While expression analysis has uncovered large numbers of de-regulated genes in cells with altered Hox activity, determining which are direct versus indirect targets has remained a significant challenge. Here, we characterize the DNA binding activity of Hox transcription factor complexes on eight experimentally verified cis-regulatory elements. Hox factors regulate the activity of each element by forming protein complexes with two cofactor proteins, Extradenticle (Exd) and Homothorax (Hth). Using comparative DNA binding assays, we found that a number of flexible arrangements of Hox, Exd, and Hth binding sites mediate cooperative transcription factor complexes. Moreover, analysis of a Distal-less regulatory element (DMXR) that is repressed by abdominal Hox factors revealed that suboptimal binding sites can be combined to form high affinity transcription complexes. Lastly, we determined that the anterior Hox factors are more dependent upon Exd and Hth for complex formation than posterior Hox factors. Based upon these findings, we suggest a general set of guidelines to serve as a basis for designing bioinformatics algorithms aimed at identifying Hox regulatory elements using the wealth of recently sequenced genomes. PMID:20398649

Detecting authorized and unauthorized genetically modified organisms containing vip3A by real-time PCR and next-generation sequencing.

PubMed

Liang, Chanjuan; van Dijk, Jeroen P; Scholtens, Ingrid M J; Staats, Martijn; Prins, Theo W; Voorhuijzen, Marleen M; da Silva, Andrea M; Arisi, Ana Carolina Maisonnave; den Dunnen, Johan T; Kok, Esther J

2014-04-01

The growing number of biotech crops with novel genetic elements increasingly complicates the detection of genetically modified organisms (GMOs) in food and feed samples using conventional screening methods. Unauthorized GMOs (UGMOs) in food and feed are currently identified through combining GMO element screening with sequencing the DNA flanking these elements. In this study, a specific and sensitive qPCR assay was developed for vip3A element detection based on the vip3Aa20 coding sequences of the recently marketed MIR162 maize and COT102 cotton. Furthermore, SiteFinding-PCR in combination with Sanger, Illumina or Pacific BioSciences (PacBio) sequencing was performed targeting the flanking DNA of the vip3Aa20 element in MIR162. De novo assembly and Basic Local Alignment Search Tool searches were used to mimic UGMO identification. PacBio data resulted in relatively long contigs in the upstream (1,326 nucleotides (nt); 95 % identity) and downstream (1,135 nt; 92 % identity) regions, whereas Illumina data resulted in two smaller contigs of 858 and 1,038 nt with higher sequence identity (>99 % identity). Both approaches outperformed Sanger sequencing, underlining the potential for next-generation sequencing in UGMO identification.

Analytical applications of aptamers

NASA Astrophysics Data System (ADS)

Tombelli, S.; Minunni, M.; Mascini, M.

2007-05-01

Aptamers are single stranded DNA or RNA ligands which can be selected for different targets starting from a library of molecules containing randomly created sequences. Aptamers have been selected to bind very different targets, from proteins to small organic dyes. Aptamers are proposed as alternatives to antibodies as biorecognition elements in analytical devices with ever increasing frequency. This in order to satisfy the demand for quick, cheap, simple and highly reproducible analytical devices, especially for protein detection in the medical field or for the detection of smaller molecules in environmental and food analysis. In our recent experience, DNA and RNA aptamers, specific for three different proteins (Tat, IgE and thrombin), have been exploited as bio-recognition elements to develop specific biosensors (aptasensors). These recognition elements have been coupled to piezoelectric quartz crystals and surface plasmon resonance (SPR) devices as transducers where the aptamers have been immobilized on the gold surface of the crystals electrodes or on SPR chips, respectively.

Colonization of heterochromatic genes by transposable elements in Drosophila.

PubMed

Dimitri, Patrizio; Junakovic, Nikolaj; Arcà, Bruno

2003-04-01

As a further step toward understanding transposable element-host genome interactions, we investigated the molecular anatomy of introns from five heterochromatic and 22 euchromatic protein-coding genes of Drosophila melanogaster. A total of 79 kb of intronic sequences from heterochromatic genes and 355 kb of intronic sequences from euchromatic genes have been used in Blast searches against Drosophila transposable elements (TEs). The results show that TE-homologous sequences belonging to 19 different families represent about 50% of intronic DNA from heterochromatic genes. In contrast, only 0.1% of the euchromatic intron DNA exhibits homology to known TEs. Intraspecific and interspecific size polymorphisms of introns were found, which are likely to be associated with changes in TE-related sequences. Together, the enrichment in TEs and the apparent dynamic state of heterochromatic introns suggest that TEs contribute significantly to the evolution of genes located in heterochromatin.

Repetitive elements dynamics in cell identity programming, maintenance and disease.

PubMed

Bodega, Beatrice; Orlando, Valerio

2014-12-01

The days of 'junk DNA' seem to be over. The rapid progress of genomics technologies has been unveiling unexpected mechanisms by which repetitive DNA and in particular transposable elements (TEs) have evolved, becoming key issues in understanding genome structure and function. Indeed, rather than 'parasites', recent findings strongly suggest that TEs may have a positive function by contributing to tissue specific transcriptional programs, in particular as enhancer-like elements and/or modules for regulation of higher order chromatin structure. Further, it appears that during development and aging genomes experience several waves of TEs activation, and this contributes to individual genome shaping during lifetime. Interestingly, TEs activity is major target of epigenomic regulation. These findings are shedding new light on the genome-phenotype relationship and set the premises to help to explain complex disease manifestation, as consequence of TEs activity deregulation. Copyright © 2014. Published by Elsevier Ltd.

Discrimination of Single Base Pair Differences Among Individual DNA Molecules Using a Nanopore

NASA Technical Reports Server (NTRS)

Vercoutere, Wenonah; DeGuzman, Veronica

2003-01-01

The protein toxin alpha-hemolysin form nanometer scale channels across lipid membranes. Our lab uses a single channel in an artificial lipid bilayer in a patch clamp device to capture and examine individual DNA molecules. This nanopore detector used with a support vector machine (SVM) can analyze DNA hairpin molecules on the millisecond time scale. We distinguish duplex stem length, base pair mismatches, loop length, and single base pair differences. The residual current fluxes also reveal structural molecular dynamics elements. DNA end-fraying (terminal base pair dissociation) can be observed as near full blockades, or spikes, in current. This technique can be used to investigate other biological processes dependent on DNA end-fraying, such as the processing of HIV DNA by HIV integrase.

Understanding the relationship between DNA methylation and histone lysine methylation☆

PubMed Central

Rose, Nathan R.; Klose, Robert J.

2014-01-01

DNA methylation acts as an epigenetic modification in vertebrate DNA. Recently it has become clear that the DNA and histone lysine methylation systems are highly interrelated and rely mechanistically on each other for normal chromatin function in vivo. Here we examine some of the functional links between these systems, with a particular focus on several recent discoveries suggesting how lysine methylation may help to target DNA methylation during development, and vice versa. In addition, the emerging role of non-methylated DNA found in CpG islands in defining histone lysine methylation profiles at gene regulatory elements will be discussed in the context of gene regulation. This article is part of a Special Issue entitled: Methylation: A Multifaceted Modification — looking at transcription and beyond. PMID:24560929

Genome-wide analysis of salinity-stress induced DNA methylation alterations in cotton (Gossypium hirsutum L.) using the Me-DIP sequencing technology.

PubMed

Lu, X K; Shu, N; Wang, J J; Chen, X G; Wang, D L; Wang, S; Fan, W L; Guo, X N; Guo, L X; Ye, W W

2017-06-29

Cytosine DNA methylation is a significant form of DNA modification closely associated with gene expression in eukaryotes, fungi, animals, and plants. Although the reference genomes of cotton (Gossypium hirsutum L.) have been publically available, the salinity-stress-induced DNA methylome alterations in cotton are not well understood. Here, we constructed a map of genome-wide DNA methylation characteristics of cotton leaves under salt stress using the methylated DNA immunoprecipitation sequencing method. The results showed that the methylation reads on chromosome 9 were most comparable with those on the other chromosomes, but the greatest changes occurred on chromosome 8 under salt stress. The DNA methylation pattern analysis indicated that a relatively higher methylation density was found in the upstream2k and downstream2k elements of the CDS region and CG-islands. Almost 94% of the reads belonged to LTR-gspsy and LTR-copia, and the number of methylation reads in LTR-gypsy was four times greater than that in LTR-copia in both control and stressed samples. The analysis of differentially methylated regions (DMRs) showed that the gene elements upstream2k, intron, and downstream2k were hypomethylated, but the CDS regions were hypermethylated. The GO (Gene Ontology) analysis suggested that the methylated genes were most enriched in cellular processes, metabolic processes, cell parts and catalytic activities, which might be closely correlated with response to NaCl stress. In this study, we completed a genomic DNA methylation profile and conducted a DMR analysis under salt stress, which provided valuable information for the better understanding of epigenetics in response to salt stress in cotton.

Loop-mediated isothermal amplification (LAMP): early detection of Toxoplasma gondii infection in mice.

PubMed

Kong, Qing-Ming; Lu, Shao-Hong; Tong, Qun-Bo; Lou, Di; Chen, Rui; Zheng, Bin; Kumagai, Takashi; Wen, Li-Yong; Ohta, Nobuo; Zhou, Xiao-Nong

2012-01-03

Toxoplasmosis is a widespread zoonotic parasitic disease that occurs in both animals and humans. Traditional molecular assays are often difficult to perform, especially for the early diagnosis of Toxoplasma gondii infections. Here, we established a novel loop-mediated isothermal amplification targeting the 529 bp repeat element (529 bp-LAMP) to detect T. gondii DNA in blood samples of experimental mice infected with tachyzoites of the RH strain. The assay was performed with Bst DNA polymerase at 65°C for 1 h. The detection limit of the 529 bp-LAMP assay was as low as 0.6 fg of T. gondii DNA. The sensitivity of this assay was 100 and 1000 fold higher than that of the LAMP targeting B1 gene (B1-LAMP) and nested PCR targeting 529 bp repeat element (529 bp-nested PCR), respectively. The specificity of the 529 bp-LAMP assay was determined using the DNA samples of Trypanosoma evansi, Plasmodium falciparum, Paragonimus westermani, Schistosoma japonicum, Fasciola hepatica and Angiostrongylus cantonensis. No cross-reactivity with the DNA of any parasites was found. The assay was able to detect T. gondii DNA in all mouse blood samples at one day post infection (dpi). We report the following findings: (i) The detection limit of the 529 bp-LAMP assay is 0.6 fg of T. gondii DNA; (ii) The assay does not involve any cross-reactivity with the DNA of other parasites; (iii) This is the first report on the application of the LAMP assay for early diagnosis of toxoplasmosis in blood samples from experimentally infected mice. Due to its simplicity, sensitivity and cost-effectiveness for common use, we suggest that this assay should be used as an early diagnostic tool for health control of toxoplasmosis.

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in

The Nucleotide Excision Repair Pathway Limits L1 Retrotransposition

PubMed Central

Servant, Geraldine; Streva, Vincent A.; Derbes, Rebecca S.; Wijetunge, Madushani I.; Neeland, Marc; White, Travis B.; Belancio, Victoria P.; Roy-Engel, Astrid M.; Deininger, Prescott L.

2017-01-01

Long interspersed elements 1 (L1) are active mobile elements that constitute almost 17% of the human genome. They amplify through a “copy-and-paste” mechanism termed retrotransposition, and de novo insertions related to these elements have been reported to cause 0.2% of genetic diseases. Our previous data demonstrated that the endonuclease complex ERCC1-XPF, which cleaves a 3′ DNA flap structure, limits L1 retrotransposition. Although the ERCC1-XPF endonuclease participates in several different DNA repair pathways, such as single-strand annealing, or in telomere maintenance, its recruitment to DNA lesions is best characterized in the nucleotide excision repair (NER) pathway. To determine if the NER pathway prevents the insertion of retroelements in the genome, we monitored the retrotransposition efficiencies of engineered L1 elements in NER-deficient cells and in their complemented versions. Core proteins of the NER pathway, XPD and XPA, and the lesion binding protein, XPC, are involved in limiting L1 retrotransposition. In addition, sequence analysis of recovered de novo L1 inserts and their genomic locations in NER-deficient cells demonstrated the presence of abnormally large duplications at the site of insertion, suggesting that NER proteins may also play a role in the normal L1 insertion process. Here, we propose new functions for the NER pathway in the maintenance of genome integrity: limitation of insertional mutations caused by retrotransposons and the prevention of potentially mutagenic large genomic duplications at the site of retrotransposon insertion events. PMID:28049704

Early demethylation of non-CpG, CpC-rich, elements in the myogenin 5′-flanking region

PubMed Central

Fuso, Andrea; Ferraguti, Giampiero; Grandoni, Francesco; Ruggeri, Raffaella; Scarpa, Sigfrido; Strom, Roberto

2010-01-01

The dynamic changes and structural patterns of DNA methylation of genes without CpG islands are poorly characterized. The relevance of CpG to the non-CpG methylation equilibrium in transcriptional repression is unknown. In this work, we analyzed the DNA methylation pattern of the 5′-flanking of the myogenin gene, a positive regulator of muscle differentiation with no CpG island and low CpG density, in both C2C12 muscle satellite cells and embryonic muscle. Embryonic brain was studied as a non-expressing tissue. High levels of both CpG and non-CpG methylation were observed in non-expressing experimental conditions. Both CpG and non-CpG methylation rapidly dropped during muscle differentiation and myogenin transcriptional activation with active demethylation dynamics. Non-CpG demethylation occurred more rapidly than CpG demethylation. Demethylation spread from initially highly methylated short CpC-rich elements to a virtually unmethylated status. These short elements have a high CpC content and density, share some motifs and largely coincide with putative recognition sequences of some differentiation-related transcription factors. Our findings point to a dynamically controlled equilibrium between CpG and non-CpG active demethylation in the transcriptional control of tissue-specific genes. The short CpC-rich elements are new structural features of the methylation machinery, whose functions may include priming the complete demethylation of a transcriptionally crucial DNA region. PMID:20935518

DNA-directed mutations. Leading and lagging strand specificity

NASA Technical Reports Server (NTRS)

Sinden, R. R.; Hashem, V. I.; Rosche, W. A.

1999-01-01

The fidelity of replication has evolved to reproduce B-form DNA accurately, while allowing a low frequency of mutation. The fidelity of replication can be compromised, however, by defined order sequence DNA (dosDNA) that can adopt unusual or non B-DNA conformations. These alternative DNA conformations, including hairpins, cruciforms, triplex DNAs, and slipped-strand structures, may affect enzyme-template interactions that potentially lead to mutations. To analyze the effect of dosDNA elements on spontaneous mutagenesis, various mutational inserts containing inverted repeats or direct repeats were cloned in a plasmid containing a unidirectional origin of replication and a selectable marker for the mutation. This system allows for analysis of mutational events that are specific for the leading or lagging strands during DNA replication in Escherichia coli. Deletions between direct repeats, involving misalignment stabilized by DNA secondary structure, occurred preferentially on the lagging strand. Intermolecular strand switch events, correcting quasipalindromes to perfect inverted repeats, occurred preferentially during replication of the leading strand.

Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia.

PubMed

Pal, Arnab; Srivastava, Tapasya; Sharma, Manish K; Mehndiratta, Mohit; Das, Prerna; Sinha, Subrata; Chattopadhyay, Parthaprasad

2010-11-01

Hypoxia is an integral part of tumorigenesis and contributes extensively to the neoplastic phenotype including drug resistance and genomic instability. It has also been reported that hypoxia results in global demethylation. Because a majority of the cytosine-phosphate-guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability. U87MG glioblastoma cells were cultured in 0.1% O₂ for 6 weeks and compared with cells cultured in 21% O₂ for the same duration. Real-time PCR analysis showed a significant increase in SINE and reverse transcriptase coding long interspersed nuclear element (LINE) transcripts during hypoxia. Sequencing of bisulphite treated DNA as well as the Combined Bisulfite Restriction Analysis (COBRA) assay showed that the SINE loci studied underwent significant hypomethylation though there was patchy hypermethylation at a few sites. The inter-alu PCR profile of DNA from cells cultured under 6-week hypoxia, its 4-week revert back to normoxia and 6-week normoxia showed several changes in the band pattern indicating increased alu mediated genomic alteration. Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia. This might be a cause of genetic heterogeneity in tumours especially in variegated hypoxic environment and lead to a development of foci of more aggressive tumour cells. © 2009 The Authors Journal compilation © 2010 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Conversion at large intergenic regions of mitochondrial DNA in Saccharomyces cerevisiae.

PubMed

Skelly, P J; Clark-Walker, G D

1990-04-01

Saccharomyces cerevisiae mitochondrial DNA deletion mutants have been used to examine whether base-biased intergenic regions of the genome influence mitochondrial biogenesis. One strain (delta 5.0) lacks a 5-kilobase (kb) segment extending from the proline tRNA gene to the small rRNA gene that includes ori1, while a second strain (delta 3.7) is missing a 3.7-kb region between the genes for ATPase subunit 6 and glutamic acid tRNA that encompasses ori7 plus ori2. Growth of these strains on both fermentable and nonfermentable substrates does not differ from growth of the wild-type strain, indicating that the deletable regions of the genome do not play a direct role in the expression of mitochondrial genes. Examination of whether the 5- or 3.7-kb regions influence mitochondrial DNA transmission was undertaken by crossing strains and examining mitochondrial genotypes in zygotic colonies. In a cross between strain delta 5.0, harboring three active ori elements (ori2, ori3, and ori5), and strain delta 3.7, containing only two active ori elements (ori3 and ori5), there is a preferential recovery of the genome containing two active ori elements (37% of progeny) over that containing three active elements (20%). This unexpected result, suggesting that active ori elements do not influence transmission of respiratory-competent genomes, is interpreted to reflect a preferential conversion of the delta 5.0 genome to the wild type (41% of progeny). Supporting evidence for conversion over biased transmission is shown by preferential recovery of a nonparental genome in the progeny of a heterozygous cross in which both parental molecules can be identified by size polymorphisms.

Elements in the transcriptional regulatory region flanking herpes simplex virus type 1 oriS stimulate origin function.

PubMed

Wong, S W; Schaffer, P A

1991-05-01

Like other DNA-containing viruses, the three origins of herpes simplex virus type 1 (HSV-1) DNA replication are flanked by sequences containing transcriptional regulatory elements. In a transient plasmid replication assay, deletion of sequences comprising the transcriptional regulatory elements of ICP4 and ICP22/47, which flank oriS, resulted in a greater than 80-fold decrease in origin function compared with a plasmid, pOS-822, which retains these sequences. In an effort to identify specific cis-acting elements responsible for this effect, we conducted systematic deletion analysis of the flanking region with plasmid pOS-822 and tested the resulting mutant plasmids for origin function. Stimulation by cis-acting elements was shown to be both distance and orientation dependent, as changes in either parameter resulted in a decrease in oriS function. Additional evidence for the stimulatory effect of flanking sequences on origin function was demonstrated by replacement of these sequences with the cytomegalovirus immediate-early promoter, resulting in nearly wild-type levels of oriS function. In competition experiments, cotransfection of cells with the test plasmid, pOS-822, and increasing molar concentrations of a competitor plasmid which contained the ICP4 and ICP22/47 transcriptional regulatory regions but lacked core origin sequences resulted in a significant reduction in the replication efficiency of pOS-822, demonstrating that factors which bind specifically to the oriS-flanking sequences are likely involved as auxiliary proteins in oriS function. Together, these studies demonstrate that trans-acting factors and the sites to which they bind play a critical role in the efficiency of HSV-1 DNA replication from oriS in transient-replication assays.

Conversion at large intergenic regions of mitochondrial DNA in Saccharomyces cerevisiae.

PubMed Central

Skelly, P J; Clark-Walker, G D

1990-01-01

Saccharomyces cerevisiae mitochondrial DNA deletion mutants have been used to examine whether base-biased intergenic regions of the genome influence mitochondrial biogenesis. One strain (delta 5.0) lacks a 5-kilobase (kb) segment extending from the proline tRNA gene to the small rRNA gene that includes ori1, while a second strain (delta 3.7) is missing a 3.7-kb region between the genes for ATPase subunit 6 and glutamic acid tRNA that encompasses ori7 plus ori2. Growth of these strains on both fermentable and nonfermentable substrates does not differ from growth of the wild-type strain, indicating that the deletable regions of the genome do not play a direct role in the expression of mitochondrial genes. Examination of whether the 5- or 3.7-kb regions influence mitochondrial DNA transmission was undertaken by crossing strains and examining mitochondrial genotypes in zygotic colonies. In a cross between strain delta 5.0, harboring three active ori elements (ori2, ori3, and ori5), and strain delta 3.7, containing only two active ori elements (ori3 and ori5), there is a preferential recovery of the genome containing two active ori elements (37% of progeny) over that containing three active elements (20%). This unexpected result, suggesting that active ori elements do not influence transmission of respiratory-competent genomes, is interpreted to reflect a preferential conversion of the delta 5.0 genome to the wild type (41% of progeny). Supporting evidence for conversion over biased transmission is shown by preferential recovery of a nonparental genome in the progeny of a heterozygous cross in which both parental molecules can be identified by size polymorphisms. Images PMID:2181277

Estrogen receptor accessory proteins augment receptor-DNA interaction and DNA bending.

PubMed

Landel, C C; Potthoff, S J; Nardulli, A M; Kushner, P J; Greene, G L

1997-01-01

Increasing evidence suggests that accessory proteins play an important role in the ability of the estrogen receptor (ER) and other nuclear hormone receptors to modulate transcription when bound to cis-acting hormone response elements in target genes. We have previously shown that four proteins, hsp70, protein disulfide isomerase (PDI) and two unknown proteins (p48 and p45), copurify with ER that has been isolated by site-specific DNA chromatography (BERE) and influence the interaction of ER with DNA in vitro. To better define the nature of these effects, we used filter binding and electrophoretic mobility shift assays to study the ability of these proteins to alter the kinetics of ER-DNA interaction and to influence the ability of ER to bend DNA when bound to an estrogen response element (ERE). The results of both assays indicate that ERE-purified ER, with its four associated proteins (hsp70, PDI, p48, p45), has a greater ability to bind to the vitellogenin A2 ERE than ER purified by estradiol-Sepharose chromatography in the absence (ESeph) or presence (EATP) of ATP, in which p48, p45 (ESeph) and hsp70 (EATP) are removed. Surprisingly, the rates of association and dissociation of ER and ERE were essentially the same for all three mixtures, suggesting that one or more ER-associated proteins, especially p45 and p48, may be required for ER to attain maximum DNA binding activity. In addition, circular permutation and phasing analyses demonstrated that the same ER-associated proteins produced higher order ER-DNA complexes that significantly increased the magnitude of DNA distortion, but did not alter the direction of the ER-induced bend of ERE-containing DNA fragments, which was toward the major groove of the DNA helix. These results suggest that p45 and/or p48 and possibly hsp70, play an important role both in the specific DNA binding and bending activities of ER and thus contribute to the overall stimulation of transcription in target genes that contain cis-acting EREs.

DNA methylation differences in exposed workers and nearby residents of the Ma Ta Phut industrial estate, Rayong, Thailand

PubMed Central

Peluso, Marco; Bollati, Valentina; Munnia, Armelle; Srivatanakul, Petcharin; Jedpiyawongse, Adisorn; Sangrajrang, Suleeporn; Piro, Sara; Ceppi, Marcello; Bertazzi, Pier Alberto; Boffetta, Paolo; Baccarelli, Andrea A

2012-01-01

Background Adverse biological effects from airborne pollutants are a primary environmental concern in highly industrialized areas. Recent studies linked air pollution exposures with altered blood Deoxyribo-nucleic acid (DNA) methylation, but effects from industrial sources and underlying biological mechanisms are still largely unexplored. Methods The Ma Ta Phut industrial estate (MIE) in Rayong, Thailand hosts one of the largest steel, oil refinery and petrochemical complexes in south-eastern Asia. We measured a panel of blood DNA methylation markers previously associated with air pollution exposures, including repeated elements [long interspersed nuclear element-1 (LINE-1) and Alu] and genes [p53, hypermethylated-in-cancer-1 (HIC1), p16 and interleukin-6 (IL-6)], in 67 MIE workers, 65 Ma Ta Phut residents and 45 rural controls. To evaluate the role of DNA damage and oxidation, we correlated DNA methylation measures with bulky DNA and 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) adducts. Results In covariate-adjusted models, MIE workers, compared with rural residents, showed lower LINE-1 (74.8% vs 78.0%; P < 0.001), p53 (8.0% vs 15.7%; P < 0.001) and IL-6 methylation (39.2% vs 45.0%; P = 0.027) and higher HIC1 methylation (22.2% vs 15.3%, P < 0.001). For all four markers, Ma Ta Phut residents exhibited methylation levels intermediate between MIE workers and rural controls (LINE-1, 75.7%, P < 0.001; p53, 9.0%, P < 0.001; IL-6, 39.8%, P = 0.041; HIC1, 17.8%, P = 0.05; all P-values vs rural controls). Bulky DNA adducts showed negative correlation with p53 methylation (P = 0.01). M1dG showed negative correlations with LINE-1 (P = 0.003) and IL-6 methylation (P = 0.05). Conclusions Our findings indicate that industrial exposures may induce alterations of DNA methylation patterns detectable in blood leucocyte DNA. Correlation of DNA adducts with DNA hypomethylation suggests potential mediation by DNA damage. PMID:23064502

Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Hymenoptera): structure, organization, and retrotransposable elements

PubMed Central

Gillespie, J J; Johnston, J S; Cannone, J J; Gutell, R R

2006-01-01

As an accompanying manuscript to the release of the honey bee genome, we report the entire sequence of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) ribosomal RNA (rRNA)-encoding gene sequences (rDNA) and related internally and externally transcribed spacer regions of Apis mellifera (Insecta: Hymenoptera: Apocrita). Additionally, we predict secondary structures for the mature rRNA molecules based on comparative sequence analyses with other arthropod taxa and reference to recently published crystal structures of the ribosome. In general, the structures of honey bee rRNAs are in agreement with previously predicted rRNA models from other arthropods in core regions of the rRNA, with little additional expansion in non-conserved regions. Our multiple sequence alignments are made available on several public databases and provide a preliminary establishment of a global structural model of all rRNAs from the insects. Additionally, we provide conserved stretches of sequences flanking the rDNA cistrons that comprise the externally transcribed spacer regions (ETS) and part of the intergenic spacer region (IGS), including several repetitive motifs. Finally, we report the occurrence of retrotransposition in the nuclear large subunit rDNA, as R2 elements are present in the usual insertion points found in other arthropods. Interestingly, functional R1 elements usually present in the genomes of insects were not detected in the honey bee rRNA genes. The reverse transcriptase products of the R2 elements are deduced from their putative open reading frames and structurally aligned with those from another hymenopteran insect, the jewel wasp Nasonia (Pteromalidae). Stretches of conserved amino acids shared between Apis and Nasonia are illustrated and serve as potential sites for primer design, as target amplicons within these R2 elements may serve as novel phylogenetic markers for Hymenoptera. Given the impending completion of the sequencing of the Nasonia genome, we expect our report eventually to shed light on the evolution of the hymenopteran genome within higher insects, particularly regarding the relative maintenance of conserved rDNA genes, related variable spacer regions and retrotransposable elements. PMID:17069639

UV-vis spectroscopy and dynamic light scattering study of gold nanorods aggregation.

PubMed

Kanjanawarut, Roejarek; Yuan, Bo; XiaoDi, Su

2013-08-01

Gold nanorods (AuNRs) were used as spectroscopic sensing elements to detect specific DNA sequences with a single-base mismatch sensitivity. The assay was based on the observation that the stabilizing repulsive forces between CTA(+)-coated AuNRs can be removed by citrate ions, which causes aggregation among AuNRs; whereas nucleic acids of different structures[ i.e., peptide nucleic acid (PNA), single-stranded DNA (ssDNA), PNA-DNA complex, and double-stranded DNA (dsDNA)] can retard the aggregation. Moreover, the dsDNA PNA-DNA duplexes provide larger retardation than that by unhybridized ssDNA and PNA probe. This assay can differentiate single-base mismatched targets with base substitution at different locations (center and end) with AuNRs of a larger aspect ratio. Besides ultraviolet-visable spectroscopy measurement of particle assembly-induced plasmonic coupling that in turn provides a spectroscopic detection of the specific DNA, dynamic light scattering and transmission electron microscope (TEM) were used to measure smaller degree of aggregation that can reveal sodium citrate- and dsDNA-AuNRs interactions in fine detail.

Trichomonas vaginalis ribosomal RNA: identification and characterisation of the transcription promoter and terminator sequences.

PubMed

Franco, Bernardo; Hernández, Roberto; López-Villaseñor, Imelda

2012-09-01

Trichomonas vaginalis is a parasitic protozoan of both medical and biological relevance. Transcriptional studies in this organism have focused mainly on type II pol promoters, whereas the elements necessary for transcription by polI or polIII have not been investigated. Here, with the aid of a transient transcription system, we characterised the rDNA intergenic region, defining both the promoter and the terminator sequences required for transcription. We defined the promoter as a compact region of approximately 180 bp. We also identified a potential upstream control element (UCE) that was located 80 bp upstream of the transcription start point (TSP). A transcription termination element was identified within a 34 bp region that was located immediately downstream of the 28S coding sequence. The function of this element depends upon polarity and the presence of both a stretch of uridine residues (U's) and a hairpin structure in the transcript. Our observations provide a strong basis for the study of DNA recognition by the polI transcriptional machinery in this early divergent organism. Copyright © 2012 Elsevier B.V. All rights reserved.

Rapid Mitochondrial Genome Evolution through Invasion of Mobile Elements in Two Closely Related Species of Arbuscular Mycorrhizal Fungi

PubMed Central

Beaudet, Denis; Nadimi, Maryam; Iffis, Bachir; Hijri, Mohamed

2013-01-01

Arbuscular mycorrhizal fungi (AMF) are common and important plant symbionts. They have coenocytic hyphae and form multinucleated spores. The nuclear genome of AMF is polymorphic and its organization is not well understood, which makes the development of reliable molecular markers challenging. In stark contrast, their mitochondrial genome (mtDNA) is homogeneous. To assess the intra- and inter-specific mitochondrial variability in closely related Glomus species, we performed 454 sequencing on total genomic DNA of Glomus sp. isolate DAOM-229456 and we compared its mtDNA with two G. irregulare isolates. We found that the mtDNA of Glomus sp. is homogeneous, identical in gene order and, with respect to the sequences of coding regions, almost identical to G. irregulare. However, certain genomic regions vary substantially, due to insertions/deletions of elements such as introns, mitochondrial plasmid-like DNA polymerase genes and mobile open reading frames. We found no evidence of mitochondrial or cytoplasmic plasmids in Glomus species, and mobile ORFs in Glomus are responsible for the formation of four gene hybrids in atp6, atp9, cox2, and nad3, which are most probably the result of horizontal gene transfer and are expressed at the mRNA level. We found evidence for substantial sequence variation in defined regions of mtDNA, even among closely related isolates with otherwise identical coding gene sequences. This variation makes it possible to design reliable intra- and inter-specific markers. PMID:23637766

Rapid mitochondrial genome evolution through invasion of mobile elements in two closely related species of arbuscular mycorrhizal fungi.

PubMed

Beaudet, Denis; Nadimi, Maryam; Iffis, Bachir; Hijri, Mohamed

2013-01-01

Arbuscular mycorrhizal fungi (AMF) are common and important plant symbionts. They have coenocytic hyphae and form multinucleated spores. The nuclear genome of AMF is polymorphic and its organization is not well understood, which makes the development of reliable molecular markers challenging. In stark contrast, their mitochondrial genome (mtDNA) is homogeneous. To assess the intra- and inter-specific mitochondrial variability in closely related Glomus species, we performed 454 sequencing on total genomic DNA of Glomus sp. isolate DAOM-229456 and we compared its mtDNA with two G. irregulare isolates. We found that the mtDNA of Glomus sp. is homogeneous, identical in gene order and, with respect to the sequences of coding regions, almost identical to G. irregulare. However, certain genomic regions vary substantially, due to insertions/deletions of elements such as introns, mitochondrial plasmid-like DNA polymerase genes and mobile open reading frames. We found no evidence of mitochondrial or cytoplasmic plasmids in Glomus species, and mobile ORFs in Glomus are responsible for the formation of four gene hybrids in atp6, atp9, cox2, and nad3, which are most probably the result of horizontal gene transfer and are expressed at the mRNA level. We found evidence for substantial sequence variation in defined regions of mtDNA, even among closely related isolates with otherwise identical coding gene sequences. This variation makes it possible to design reliable intra- and inter-specific markers.

GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology1[C][W][OA

PubMed Central

Sarrion-Perdigones, Alejandro; Vazquez-Vilar, Marta; Palací, Jorge; Castelijns, Bas; Forment, Javier; Ziarsolo, Peio; Blanca, José; Granell, Antonio; Orzaez, Diego

2013-01-01

Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an ever-growing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects. PMID:23669743

GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology.

PubMed

Sarrion-Perdigones, Alejandro; Vazquez-Vilar, Marta; Palací, Jorge; Castelijns, Bas; Forment, Javier; Ziarsolo, Peio; Blanca, José; Granell, Antonio; Orzaez, Diego

2013-07-01

Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an evergrowing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects.

A genomic island integrated into recA of Vibrio cholerae contains a divergent recA and provides multi-pathway protection from DNA damage.

PubMed

Rapa, Rita A; Islam, Atiqul; Monahan, Leigh G; Mutreja, Ankur; Thomson, Nicholas; Charles, Ian G; Stokes, Harold W; Labbate, Maurizio

2015-04-01

Lateral gene transfer (LGT) has been crucial in the evolution of the cholera pathogen, Vibrio cholerae. The two major virulence factors are present on two different mobile genetic elements, a bacteriophage containing the cholera toxin genes and a genomic island (GI) containing the intestinal adhesin genes. Non-toxigenic V. cholerae in the aquatic environment are a major source of novel DNA that allows the pathogen to morph via LGT. In this study, we report a novel GI from a non-toxigenic V. cholerae strain containing multiple genes involved in DNA repair including the recombination repair gene recA that is 23% divergent from the indigenous recA and genes involved in the translesion synthesis pathway. This is the first report of a GI containing the critical gene recA and the first report of a GI that targets insertion into a specific site within recA. We show that possession of the island in Escherichia coli is protective against DNA damage induced by UV-irradiation and DNA targeting antibiotics. This study highlights the importance of genetic elements such as GIs in the evolution of V. cholerae and emphasizes the importance of environmental strains as a source of novel DNA that can influence the pathogenicity of toxigenic strains. © 2014 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter

PubMed Central

Wang, Xiaohong; Liu, Haibin; Ge, Hui; Ajiro, Masahiko; Sharma, Nishi R.; Meyers, Craig; Morozov, Pavel; Tuschl, Thomas; Klar, Amar; Court, Donald

2017-01-01

ABSTRACT The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis-acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors. PMID:28559488

Annotation, submission and screening of repetitive elements in Repbase: RepbaseSubmitter and Censor.

PubMed

Kohany, Oleksiy; Gentles, Andrew J; Hankus, Lukasz; Jurka, Jerzy

2006-10-25

Repbase is a reference database of eukaryotic repetitive DNA, which includes prototypic sequences of repeats and basic information described in annotations. Updating and maintenance of the database requires specialized tools, which we have created and made available for use with Repbase, and which may be useful as a template for other curated databases. We describe the software tools RepbaseSubmitter and Censor, which are designed to facilitate updating and screening the content of Repbase. RepbaseSubmitter is a java-based interface for formatting and annotating Repbase entries. It eliminates many common formatting errors, and automates actions such as calculation of sequence lengths and composition, thus facilitating curation of Repbase sequences. In addition, it has several features for predicting protein coding regions in sequences; searching and including Pubmed references in Repbase entries; and searching the NCBI taxonomy database for correct inclusion of species information and taxonomic position. Censor is a tool to rapidly identify repetitive elements by comparison to known repeats. It uses WU-BLAST for speed and sensitivity, and can conduct DNA-DNA, DNA-protein, or translated DNA-translated DNA searches of genomic sequence. Defragmented output includes a map of repeats present in the query sequence, with the options to report masked query sequence(s), repeat sequences found in the query, and alignments. Censor and RepbaseSubmitter are available as both web-based services and downloadable versions. They can be found at http://www.girinst.org/repbase/submission.html (RepbaseSubmitter) and http://www.girinst.org/censor/index.php (Censor).

Programming a topologically constrained DNA nanostructure into a sensor

NASA Astrophysics Data System (ADS)

Liu, Meng; Zhang, Qiang; Li, Zhongping; Gu, Jimmy; Brennan, John D.; Li, Yingfu

2016-06-01

Many rationally engineered DNA nanostructures use mechanically interlocked topologies to connect individual DNA components, and their physical connectivity is achieved through the formation of a strong linking duplex. The existence of such a structural element also poses a significant topological constraint on functions of component rings. Herein, we hypothesize and confirm that DNA catenanes with a strong linking duplex prevent component rings from acting as the template for rolling circle amplification (RCA). However, by using an RNA-containing DNA [2] catenane with a strong linking duplex, we show that a stimuli-responsive RNA-cleaving DNAzyme can linearize one component ring, and thus enable RCA, producing an ultra-sensitive biosensing system. As an example, a DNA catenane biosensor is engineered to detect the model bacterial pathogen Escherichia coli through binding of a secreted protein, with a detection limit of 10 cells ml-1, thus establishing a new platform for further applications of mechanically interlocked DNA nanostructures.

Programming a topologically constrained DNA nanostructure into a sensor

PubMed Central

Liu, Meng; Zhang, Qiang; Li, Zhongping; Gu, Jimmy; Brennan, John D.; Li, Yingfu

2016-01-01

Many rationally engineered DNA nanostructures use mechanically interlocked topologies to connect individual DNA components, and their physical connectivity is achieved through the formation of a strong linking duplex. The existence of such a structural element also poses a significant topological constraint on functions of component rings. Herein, we hypothesize and confirm that DNA catenanes with a strong linking duplex prevent component rings from acting as the template for rolling circle amplification (RCA). However, by using an RNA-containing DNA [2] catenane with a strong linking duplex, we show that a stimuli-responsive RNA-cleaving DNAzyme can linearize one component ring, and thus enable RCA, producing an ultra-sensitive biosensing system. As an example, a DNA catenane biosensor is engineered to detect the model bacterial pathogen Escherichia coli through binding of a secreted protein, with a detection limit of 10 cells ml−1, thus establishing a new platform for further applications of mechanically interlocked DNA nanostructures. PMID:27337657

Tetrahelical monomolecular architecture of DNA: a new building block for nanotechnology.

PubMed

Kankia, Besik

2014-06-12

DNA nanotechnology typically relies on Watson-Crick base pairing as both a recognition and structural element. This limits structural versatility and introduces errors during self-assembly of DNA. Guanine (G) quartet motifs show promise as an alternative to DNA duplexes, but the synthesis of long, precisely defined molecules is a significant challenge. Here we demonstrate a continuous tetrahelical DNA architecture capable of programmed self-assembly. We report that the homopolymer consisting of (G3T)3G3 monomeric units has the capability to fold into a monomolecular DNA tetrahelix with unprecedented speed and stability. For instance, in the presence of 1 mM K(+) ions the dimer, (G3T)2, folds readily and melts above 100 °C. These findings have the potential to revolutionize DNA nanotechnology by introducing fast and error-free self-assembly of long and extraordinarily stable molecules.

Stereochemical analysis of the functional significance of the conserved inverted CCAAT and TATA elements in the rat bone sialoprotein gene promoter.

PubMed

Su, Ming; Lee, Daniel; Ganss, Bernhard; Sodek, Jaro

2006-04-14

Basal transcription of the bone sialoprotein gene is mediated by highly conserved inverted CCAAT (ICE; ATTGG) and TATA elements (TTTATA) separated by precisely 21 nucleotides. Here we studied the importance of the relative position and orientation of the CCAAT and TATA elements in the proximal promoter by measuring the transcriptional activity of a series of mutated reporter constructs in transient transfection assays. Whereas inverting the TTTATA (wild type) to a TATAAA (consensus TATA) sequence increased transcription slightly, transcription was reduced when the flanking dinucleotides were also inverted. In contrast, reversing the ATTGG (wild type; ICE) to a CCAAT (RICE) sequence caused a marked reduction in transcription, whereas both transcription and NF-Y binding were progressively increased with the simultaneous inversion of flanking nucleotides (f-RICE-f). Reducing the distance between the ICE and TATA elements produced cyclical changes in transcriptional activity that correlated with progressive alterations in the relative positions of the CCAAT and TATA elements on the face of the DNA helix. Minimal transcription was observed after 5 nucleotides were deleted (equivalent to approximately one half turn of the helix), whereas transcription was fully restored after deleting 10 nucleotides (approximately one full turn of the DNA helix), transcriptional activity being progressively lost with deletions beyond 10 nucleotides. In comparison, when deletions were made with the ICE in the reversed (f-RICE-f) orientation transcriptional activity was progressively lost with no recovery. These results show that, although transcription can still occur when the CCAAT box is reversed and/or displaced relative to the TATA box, the activity is dependent upon the flexibility of the intervening DNA helix needed to align the NF-Y complex on the CCAAT box with preinitiation complex proteins that bind to the TATA box. Thus, the precise location and orientation of the CCAAT element is necessary for optimizing basal transcription of the bone sialoprotein gene.

Genetic variant modifies the effect of N3 PUFAs on DNA methylation of IL6 in the Genetics of Lipid Lowering Drugs and Diet Network study

USDA-ARS?s Scientific Manuscript database

N3 polyunsaturated fatty acids (N3 PUFAs) ameliorate inflammation status with specific regulation on interleukin-6 (IL6) expression. However, the molecular mechanism for this regulation is unclear. Using both cell lines data from Encyclopedia of DNA Elements (ENCODE) consortium and population data f...

An ancient trans-kingdom horizontal transfer of Penelope -like retroelements from arthropods to conifers

Treesearch

Xuan Lin; Nurul Faridi; Claudio Casola

2016-01-01

Comparative genomics analyses empowered by the wealth of sequenced genomes have revealed numerous instances of horizontal DNA transfers between distantly related species. In  eukaryotes, repetitive DNA sequences known as transposable elements (TEs) are especially prone to  move across species boundaries. Such horizontal transposon transfers, or HTTs, are relatively  ...

Deep Investigation of Arabidopsis thaliana Junk DNA Reveals a Continuum between Repetitive Elements and Genomic Dark Matter

PubMed Central

Maumus, Florian; Quesneville, Hadi

2014-01-01

Eukaryotic genomes contain highly variable amounts of DNA with no apparent function. This so-called junk DNA is composed of two components: repeated and repeat-derived sequences (together referred to as the repeatome), and non-annotated sequences also known as genomic dark matter. Because of their high duplication rates as compared to other genomic features, transposable elements are predominant contributors to the repeatome and the products of their decay is thought to be a major source of genomic dark matter. Determining the origin and composition of junk DNA is thus important to help understanding genome evolution as well as host biology. In this study, we have used a combination of tools enabling to show that the repeatome from the small and reducing A. thaliana genome is significantly larger than previously thought. Furthermore, we present the concepts and results from a series of innovative approaches suggesting that a significant amount of the A. thaliana dark matter is of repetitive origin. As a tentative standard for the community, we propose a deep compendium annotation of the A. thaliana repeatome that may help addressing farther genome evolution as well as transcriptional and epigenetic regulation in this model plant. PMID:24709859

[Molecular variability in the commom shrew Sorex araneus L. from European Russia and Siberia inferred from the length polymorphism of DNA regions flanked by short interspersed elements (Inter-SINE PCR) and the relationships between the Moscow and Seliger chromosome races].

PubMed

Bannikova, A A; Bulatova, N Sh; Kramerov, D A

2006-06-01

Genetic exchange among chromosomal races of the common shrew Sorex araneus and the problem of reproductive barriers have been extensively studied by means of such molecular markers as mtDNA, microsatellites, and allozymes. In the present study, the interpopulation and interracial polymorphism in the common shrew was derived, using fingerprints generated by amplified DNA regions flanked by short interspersed repeats (SINEs)-interSINE PCR (IS-PCR). We used primers, complementary to consensus sequences of two short retroposons: mammalian element MIR and the SOR element from the genome of Sorex araneus. Genetic differentiation among eleven populations of the common shrew from eight chromosome races was estimated. The NP and MJ analyses, as well as multidimensional scaling showed that all samples examined grouped into two main clusters, corresponding to European Russia and Siberia. The bootstrap support of the European Russia cluster in the NJ and MP analyses was respectively 76 and 61%. The bootstrap index for the Siberian cluster was 100% in both analyses; the Tomsk race, included into this cluster, was separated with the bootstrap support of NJ/MP 92/95%.

The high mobility group protein 1 enhances binding of the estrogen receptor DNA binding domain to the estrogen response element.

PubMed

Romine, L E; Wood, J R; Lamia, L A; Prendergast, P; Edwards, D P; Nardulli, A M

1998-05-01

We have examined the ability of the high-mobility group protein 1 (HMG1) to alter binding of the estrogen receptor DNA-binding domain (DBD) to the estrogen response element (ERE). HMG1 dramatically enhanced binding of purified, bacterially expressed DBD to the consensus vitellogenin A2 ERE in a dose-dependent manner. The ability of HMG1 to stabilize the DBD-ERE complex resulted in part from a decrease in the dissociation rate of the DBD from the ERE. Antibody supershift experiments demonstrated that HMG1 was also capable of forming a ternary complex with the ERE-bound DBD in the presence of HMG1-specific antibody. HMG1 did not substantially affect DBD-ERE contacts as assessed by methylation interference assays, nor did it alter the ability of the DBD to induce distortion in ERE-containing DNA fragments. Because HMG1 dramatically enhanced estrogen receptor DBD binding to the ERE, and the DBD is the most highly conserved region among the nuclear receptor superfamily members, HMG1 may function to enhance binding of other nuclear receptors to their respective response elements and act in concert with coactivator proteins to regulate expression of hormone-responsive genes.

qPMS9: An Efficient Algorithm for Quorum Planted Motif Search

NASA Astrophysics Data System (ADS)

Nicolae, Marius; Rajasekaran, Sanguthevar

2015-01-01

Discovering patterns in biological sequences is a crucial problem. For example, the identification of patterns in DNA sequences has resulted in the determination of open reading frames, identification of gene promoter elements, intron/exon splicing sites, and SH RNAs, location of RNA degradation signals, identification of alternative splicing sites, etc. In protein sequences, patterns have led to domain identification, location of protease cleavage sites, identification of signal peptides, protein interactions, determination of protein degradation elements, identification of protein trafficking elements, discovery of short functional motifs, etc. In this paper we focus on the identification of an important class of patterns, namely, motifs. We study the (l, d) motif search problem or Planted Motif Search (PMS). PMS receives as input n strings and two integers l and d. It returns all sequences M of length l that occur in each input string, where each occurrence differs from M in at most d positions. Another formulation is quorum PMS (qPMS), where the motif appears in at least q% of the strings. We introduce qPMS9, a parallel exact qPMS algorithm that offers significant runtime improvements on DNA and protein datasets. qPMS9 solves the challenging DNA (l, d)-instances (28, 12) and (30, 13). The source code is available at https://code.google.com/p/qpms9/.

A novel cold-inducible zinc finger protein from soybean, SCOF-1, enhances cold tolerance in transgenic plants.

PubMed

Kim, J C; Lee, S H; Cheong, Y H; Yoo, C M; Lee, S I; Chun, H J; Yun, D J; Hong, J C; Lee, S Y; Lim, C O; Cho, M J

2001-02-01

Cold stress on plants induces changes in the transcription of cold response genes. A cDNA clone encoding C2H2-type zinc finger protein, SCOF-1, was isolated from soybean. The transcription of SCOF-1 is specifically induced by low temperature and abscisic acid (ABA) but not by dehydration or high salinity. Constitutive overexpression of SCOF-1 induced cold-regulated (COR) gene expression and enhanced cold tolerance of non-acclimated transgenic Arabidopsis and tobacco plants. SCOF-1 localized to the nucleus but did not bind directly to either C-repeat/dehydration (CRT/DRE) or ABA responsive element (ABRE), cis-acting DNA regulatory elements present in COR gene promoters. However, SCOF-1 greatly enhanced the DNA binding activity of SGBF-1, a soybean G-box binding bZIP transcription factor, to ABRE in vitro. SCOF-1 also interacted with SGBF-1 in a yeast two-hybrid system. The SGBF-1 transactivated the beta-glucuronidase reporter gene driven by the ABRE element in Arabidopsis leaf protoplasts. Furthermore, the SCOF-1 enhanced ABRE-dependent gene expression mediated by SGBF-1. These results suggest that SCOF-1 may function as a positive regulator of COR gene expression mediated by ABRE via protein-protein interaction, which in turn enhances cold tolerance of plants.

Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance

PubMed Central

Jeyapalan, J N; Noor, D A Mohamed; Lee, S-H; Tan, C L; Appleby, V A; Kilday, J P; Palmer, R D; Schwalbe, E C; Clifford, S C; Walker, D A; Murray, M J; Coleman, N; Nicholson, J C; Scotting, P J

2011-01-01

Background: Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes. Methods: A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays. Results: Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a ‘methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype. Conclusion: Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy. PMID:21712824

Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance.

PubMed

Jeyapalan, J N; Noor, D A Mohamed; Lee, S-H; Tan, C L; Appleby, V A; Kilday, J P; Palmer, R D; Schwalbe, E C; Clifford, S C; Walker, D A; Murray, M J; Coleman, N; Nicholson, J C; Scotting, P J

2011-08-09

Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes. A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays. Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a 'methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype. Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy.

A conserved MCM single-stranded DNA binding element is essential for replication initiation.

PubMed

Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B; Bell, Stephen P; Enemark, Eric J

2014-04-01

The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001.

A conserved MCM single-stranded DNA binding element is essential for replication initiation

PubMed Central

Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B; Bell, Stephen P; Enemark, Eric J

2014-01-01

The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001 PMID:24692448

UV-Vis Spectroscopy and Dynamic Light Scattering Study of Gold Nanorods Aggregation

PubMed Central

Kanjanawarut, Roejarek; Yuan, Bo

2013-01-01

Gold nanorods (AuNRs) were used as spectroscopic sensing elements to detect specific DNA sequences with a single-base mismatch sensitivity. The assay was based on the observation that the stabilizing repulsive forces between CTA+-coated AuNRs can be removed by citrate ions, which causes aggregation among AuNRs; whereas nucleic acids of different structures[ i.e., peptide nucleic acid (PNA), single-stranded DNA (ssDNA), PNA-DNA complex, and double-stranded DNA (dsDNA)] can retard the aggregation. Moreover, the dsDNA PNA-DNA duplexes provide larger retardation than that by unhybridized ssDNA and PNA probe. This assay can differentiate single-base mismatched targets with base substitution at different locations (center and end) with AuNRs of a larger aspect ratio. Besides ultraviolet–visable spectroscopy measurement of particle assembly-induced plasmonic coupling that in turn provides a spectroscopic detection of the specific DNA, dynamic light scattering and transmission electron microscope (TEM) were used to measure smaller degree of aggregation that can reveal sodium citrate– and dsDNA–AuNRs interactions in fine detail. PMID:23902360

Palindromic repetitive DNA elements with coding potential in Methanocaldococcus jannaschii.

PubMed

Suyama, Mikita; Lathe, Warren C; Bork, Peer

2005-10-10

We have identified 141 novel palindromic repetitive elements in the genome of euryarchaeon Methanocaldococcus jannaschii. The total length of these elements is 14.3kb, which corresponds to 0.9% of the total genomic sequence and 6.3% of all extragenic regions. The elements can be divided into three groups (MJRE1-3) based on the sequence similarity. The low sequence identity within each of the groups suggests rather old origin of these elements in M. jannaschii. Three MJRE2 elements were located within the protein coding regions without disrupting the coding potential of the host genes, indicating that insertion of repeats might be a widespread mechanism to enhance sequence diversity in coding regions.

Transposable Elements in Human Cancer: Causes and Consequences of Deregulation.

PubMed

Anwar, Sumadi Lukman; Wulaningsih, Wahyu; Lehmann, Ulrich

2017-05-04

Transposable elements (TEs) comprise nearly half of the human genome and play an essential role in the maintenance of genomic stability, chromosomal architecture, and transcriptional regulation. TEs are repetitive sequences consisting of RNA transposons, DNA transposons, and endogenous retroviruses that can invade the human genome with a substantial contribution in human evolution and genomic diversity. TEs are therefore firmly regulated from early embryonic development and during the entire course of human life by epigenetic mechanisms, in particular DNA methylation and histone modifications. The deregulation of TEs has been reported in some developmental diseases, as well as for different types of human cancers. To date, the role of TEs, the mechanisms underlying TE reactivation, and the interplay with DNA methylation in human cancers remain largely unexplained. We reviewed the loss of epigenetic regulation and subsequent genomic instability, chromosomal aberrations, transcriptional deregulation, oncogenic activation, and aberrations of non-coding RNAs as the potential mechanisms underlying TE deregulation in human cancers.

Transposable Elements in Human Cancer: Causes and Consequences of Deregulation

PubMed Central

Anwar, Sumadi Lukman; Wulaningsih, Wahyu; Lehmann, Ulrich

2017-01-01

Transposable elements (TEs) comprise nearly half of the human genome and play an essential role in the maintenance of genomic stability, chromosomal architecture, and transcriptional regulation. TEs are repetitive sequences consisting of RNA transposons, DNA transposons, and endogenous retroviruses that can invade the human genome with a substantial contribution in human evolution and genomic diversity. TEs are therefore firmly regulated from early embryonic development and during the entire course of human life by epigenetic mechanisms, in particular DNA methylation and histone modifications. The deregulation of TEs has been reported in some developmental diseases, as well as for different types of human cancers. To date, the role of TEs, the mechanisms underlying TE reactivation, and the interplay with DNA methylation in human cancers remain largely unexplained. We reviewed the loss of epigenetic regulation and subsequent genomic instability, chromosomal aberrations, transcriptional deregulation, oncogenic activation, and aberrations of non-coding RNAs as the potential mechanisms underlying TE deregulation in human cancers. PMID:28471386

Analysis of mutations in oral poliovirus vaccine by hybridization with generic oligonucleotide microchips.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Proudnikov, D.; Kirillov, E.; Chumakov, K.

2000-01-01

This paper describes use of a new technology of hybridization with a micro-array of immobilized oligonucleotides for detection and quantification of neurovirulent mutants in Oral Poliovirus Vaccine (OPV). We used a micro-array consisting of three-dimensional gel-elements containing all possible hexamers (total of 4096 probes). Hybridization of fluorescently labelled viral cDNA samples with such microchips resulted in a pattern of spots that was registered and quantified by a computer-linked CCD camera, so that the sequence of the original cDNA could be deduced. The method could reliably identify single point mutations, since each of them affected fluorescence intensity of 12 micro-array elements.more » Micro-array hybridization of DNA mixtures with varying contents of point mutants demonstrated that the method can detect as little as 10% of revertants in a population of vaccine virus. This new technology should be useful for quality control of live viral vaccines, as well as for other applications requiring identification and quantification of point mutations.« less

Functional consequences of inducible genetic elements from the p53 SOS response in a mammalian organ system.

PubMed

Guthrie, O'neil W

2017-10-01

In response to DNA damage from ultraviolet (UV) radiation, bacteria deploy the SOS response in order to limit cell death. This bacterial SOS response is characterized by an increase in the recA gene that transactivates expression of multiple DNA repair genes. The current series of experiments demonstrate that a mammalian organ system (the cochlea) that is not evolutionarily conditioned to UV radiation can elicit SOS responses that are reminiscent of that of bacteria. This mammalian SOS response is characterized by an increase in the p53 gene with activation of multiple DNA repair genes that harbor p53 response elements in their promoters. Furthermore, the experimental results provide support for the notion of a convergent trigger paradox, where independent SOS triggers facilitate disparate physiologic sequelae (loss vs. recovery of function). Therefore, it is proposed that the mammalian SOS response is multifunctional and manipulation of this endogenous response could be exploited in future biomedical interventions. Copyright © 2017 Elsevier Inc. All rights reserved.

Uniformity of nucleosome preservation pattern in Mammalian sperm and its connection to repetitive DNA elements.

PubMed

Samans, Birgit; Yang, Yang; Krebs, Stefan; Sarode, Gaurav Vilas; Blum, Helmut; Reichenbach, Myriam; Wolf, Eckhard; Steger, Klaus; Dansranjavin, Temuujin; Schagdarsurengin, Undraga

2014-07-14

Nucleosome-to-protamine exchange during mammalian spermiogenesis is essential for compaction and protection of paternal DNA. It is interesting that, depending on the species, 1% to 15% of nucleosomes are retained, but the generalizability and biological function of this retention are unknown. Here, we show concordantly in human and bovine that nucleosomes remained in sperm chromatin predominantly within distal intergenic regions and introns and associated with centromere repeats and retrotransposons (LINE1 and SINEs). In contrast, nucleosome depletion concerned particularly exons, 5'-UTR, 3'-UTR, TSS, and TTS and was associated with simple and low-complexity repeats. Overlap of human and bovine genes exhibiting nucleosome preservation in the promoter and gene body revealed a significant enrichment of signal transduction and RNA- and protein-processing factors. Our study demonstrates the genome-wide uniformity of the nucleosome preservation pattern in mammalian sperm and its connection to repetitive DNA elements and suggests a function in preimplantation processes for paternally derived nucleosomes. Copyright © 2014 Elsevier Inc. All rights reserved.

Insertion of an SVA element, a nonautonomous retrotransposon, in PMS2 intron 7 as a novel cause of Lynch syndrome.

PubMed

van der Klift, Heleen M; Tops, Carli M; Hes, Frederik J; Devilee, Peter; Wijnen, Juul T

2012-07-01

Heterozygous germline mutations in the mismatch repair gene PMS2 predispose carriers for Lynch syndrome, an autosomal dominant predisposition to cancer. Here, we present a LINE-1-mediated retrotranspositional insertion in PMS2 as a novel mutation type for Lynch syndrome. This insertion, detected with Southern blot analysis in the genomic DNA of the patient, is characterized as a 2.2 kb long 5' truncated SVA_F element. The insertion is not detectable by current diagnostic testing limited to MLPA and direct Sanger sequencing on genomic DNA. The molecular nature of this insertion could only be resolved in RNA from cultured lymphocytes in which nonsense-mediated RNA decay was inhibited. Our report illustrates the technical problems encountered in the detection of this mutation type. Especially large heterozygous insertions will remain unnoticed because of preferential amplification of the smaller wild-type allele in genomic DNA, and are probably underreported in the mutation spectra of autosomal dominant disorders. © 2012 Wiley Periodicals, Inc.

Characterizing the stress/defense transcriptome of Arabidopsis

PubMed Central

Mahalingam, Ramamurthy; Gomez-Buitrago, AnaMaria; Eckardt, Nancy; Shah, Nigam; Guevara-Garcia, Angel; Day, Philip; Raina, Ramesh; Fedoroff, Nina V

2003-01-01

Background To understand the gene networks that underlie plant stress and defense responses, it is necessary to identify and characterize the genes that respond both initially and as the physiological response to the stress or pathogen develops. We used PCR-based suppression subtractive hybridization to identify Arabidopsis genes that are differentially expressed in response to ozone, bacterial and oomycete pathogens and the signaling molecules salicylic acid (SA) and jasmonic acid. Results We identified a total of 1,058 differentially expressed genes from eight stress cDNA libraries. Digital northern analysis revealed that 55% of the stress-inducible genes are rarely transcribed in unstressed plants and 17% of them were not previously represented in Arabidopsis expressed sequence tag databases. More than two-thirds of the genes in the stress cDNA collection have not been identified in previous studies as stress/defense response genes. Several stress-responsive cis-elements showed a statistically significant over-representation in the promoters of the genes in the stress cDNA collection. These include W- and G-boxes, the SA-inducible element, the abscisic acid response element and the TGA motif. Conclusions The stress cDNA collection comprises a broad repertoire of stress-responsive genes encoding proteins that are involved in both the initial and subsequent stages of the physiological response to abiotic stress and pathogens. This set of stress-, pathogen- and hormone-modulated genes is an important resource for understanding the genetic interactions underlying stress signaling and responses and may contribute to the characterization of the stress transcriptome through the construction of standardized specialized arrays. PMID:12620105

Small gene family encoding an eggshell (chorion) protein of the human parasite Schistosoma mansoni

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bobek, L.A.; Rekosh, D.M.; Lo Verde, P.T.

1988-08-01

The authors isolated six independent genomic clones encoding schistosome chorion or eggshell proteins from a Schistosoma mansoni genomic library. A linkage map of five of the clones spanning 35 kilobase pairs (kbp) of the S. mansoni genome was constructed. The region contained two eggshell protein genes closely linked, separated by 7.5 kbp of intergenic DNA. The two genes of the cluster were arranged in the same orientation, that is, they were transcribed from the same strand. The sixth clone probably represents a third copy of the eggshell gene that is not contained within the 35-kbp region. The 5- end ofmore » the mRNA transcribed from these genes was defined by primer extension directly off the RNA. The ATCAT cap site sequence was homologous to a silkmoth chorion PuTCATT cap site sequence, where Pu indicates any purine. DNA sequence analysis showed that there were no introns in these genes. The DNA sequences of the three genes were very homologous to each other and to a cDNA clone, pSMf61-46, differing only in three or four nucleotices. A multiple TATA box was located at positions -23 to -31, and a CAAAT sequence was located at -52 upstream of the eggshell transcription unit. Comparison of sequences in regions further upstream with silkmoth and Drosophila sequences revealed very short elements that were shared. One such element, TCACGT, recently shown to be an essential cis-regulatory element for silkmoth chorion gene promoter function, was found at a similar position in all three organisms.« less

Mitochondrial genome rearrangements in glomus species triggered by homologous recombination between distinct mtDNA haplotypes.

PubMed

Beaudet, Denis; Terrat, Yves; Halary, Sébastien; de la Providencia, Ivan Enrique; Hijri, Mohamed

2013-01-01

Comparative mitochondrial genomics of arbuscular mycorrhizal fungi (AMF) provide new avenues to overcome long-lasting obstacles that have hampered studies aimed at understanding the community structure, diversity, and evolution of these multinucleated and genetically polymorphic organisms.AMF mitochondrial (mt) genomes are homogeneous within isolates, and their intergenic regions harbor numerous mobile elements that have rapidly diverged, including homing endonuclease genes, small inverted repeats, and plasmid-related DNA polymerase genes (dpo), making them suitable targets for the development of reliable strain-specific markers. However, these elements may also lead to genome rearrangements through homologous recombination, although this has never previously been reported in this group of obligate symbiotic fungi. To investigate whether such rearrangements are present and caused by mobile elements in AMF, the mitochondrial genomes from two Glomeraceae members (i.e., Glomus cerebriforme and Glomus sp.) with substantial mtDNA synteny divergence,were sequenced and compared with available glomeromycotan mitochondrial genomes. We used an extensive nucleotide/protein similarity network-based approach to investigated podiversity in AMF as well as in other organisms for which sequences are publicly available. We provide strong evidence of dpo-induced inter-haplotype recombination, leading to a reshuffled mitochondrial genome in Glomus sp. These findings raise questions as to whether AMF single spore cultivations artificially underestimate mtDNA genetic diversity.We assessed potential dpo dispersal mechanisms in AMF and inferred a robust phylogenetic relationship with plant mitochondrial plasmids. Along with other indirect evidence, our analyses indicate that members of the Glomeromycota phylum are potential donors of mitochondrial plasmids to plants.

Mitochondrial Genome Rearrangements in Glomus Species Triggered by Homologous Recombination between Distinct mtDNA Haplotypes

PubMed Central

Beaudet, Denis; Terrat, Yves; Halary, Sébastien; de la Providencia, Ivan Enrique; Hijri, Mohamed

2013-01-01

Comparative mitochondrial genomics of arbuscular mycorrhizal fungi (AMF) provide new avenues to overcome long-lasting obstacles that have hampered studies aimed at understanding the community structure, diversity, and evolution of these multinucleated and genetically polymorphic organisms. AMF mitochondrial (mt) genomes are homogeneous within isolates, and their intergenic regions harbor numerous mobile elements that have rapidly diverged, including homing endonuclease genes, small inverted repeats, and plasmid-related DNA polymerase genes (dpo), making them suitable targets for the development of reliable strain-specific markers. However, these elements may also lead to genome rearrangements through homologous recombination, although this has never previously been reported in this group of obligate symbiotic fungi. To investigate whether such rearrangements are present and caused by mobile elements in AMF, the mitochondrial genomes from two Glomeraceae members (i.e., Glomus cerebriforme and Glomus sp.) with substantial mtDNA synteny divergence, were sequenced and compared with available glomeromycotan mitochondrial genomes. We used an extensive nucleotide/protein similarity network-based approach to investigate dpo diversity in AMF as well as in other organisms for which sequences are publicly available. We provide strong evidence of dpo-induced inter-haplotype recombination, leading to a reshuffled mitochondrial genome in Glomus sp. These findings raise questions as to whether AMF single spore cultivations artificially underestimate mtDNA genetic diversity. We assessed potential dpo dispersal mechanisms in AMF and inferred a robust phylogenetic relationship with plant mitochondrial plasmids. Along with other indirect evidence, our analyses indicate that members of the Glomeromycota phylum are potential donors of mitochondrial plasmids to plants. PMID:23925788

Endogenous sex hormone exposure and repetitive element DNA methylation in healthy postmenopausal women.

PubMed

Boyne, Devon J; Friedenreich, Christine M; McIntyre, John B; Stanczyk, Frank Z; Courneya, Kerry S; King, Will D

2017-12-01

Epigenetic mechanisms may help to explain the complex and heterogeneous relation between sex hormones and cancer. Few studies have investigated the effects of sex hormones on epigenetic markers related to cancer risk such as levels of methylation within repetitive DNA elements. Our objective was to describe the association between endogenous sex hormone exposure and levels of LINE-1 and Alu methylation in healthy postmenopausal women. We nested a cross-sectional study within the Alberta Physical Activity and Breast Cancer Prevention Trial (2003-2006). Study participants consisted of healthy postmenopausal women who had never been diagnosed with cancer (n = 289). Sex hormone exposures included serum concentrations of estradiol, estrone, testosterone, androstenedione, and sex hormone-binding globulin. We estimated the participants' lifetime number of menstrual cycles (LNMC) as a proxy for cumulative exposure to ovarian sex hormones. Buffy coat samples were assessed for DNA methylation. Linear regression was used to model the associations of interest and to control for confounding. Both estradiol and estrone had a significant positive dose-response association with LINE-1 methylation. LNMC was associated with both LINE-1 and Alu methylation. Specifically, LNMC had a non-linear "U-shaped" association with LINE-1 methylation regardless of folate intake and a negative linear association with Alu methylation, but only amongst low folate consumers. Androgen exposure was not associated with either outcome. Current and cumulative estrogen exposure was associated with repetitive element DNA methylation in a group of healthy postmenopausal women. LINE-1 and Alu methylation may be epigenetic mechanisms through which estrogen exposure impacts cancer risk.

The human haptoglobin gene promoter: interleukin-6-responsive elements interact with a DNA-binding protein induced by interleukin-6.

PubMed Central

Oliviero, S; Cortese, R

1989-01-01

Transcription of the human haptoglobin (Hp) gene is induced by interleukin-6 (IL-6) in the human hepatoma cell line Hep3B. Cis-acting elements responsible for this response are localized within the first 186 bp of the 5'-flanking region. Site-specific mutants of the Hp promoter fused to the chloramphenicol acetyl transferase (CAT) gene were analysed by transient transfection into uninduced and IL-6-treated Hep3B cells. We identified three regions, A, B and C, defined by mutation, which are important for the IL-6 response. Band shift experiments using nuclear extracts from untreated or IL-6-treated cells revealed the presence of IL-6-inducible DNA binding activities when DNA fragments containing the A or the C sequences were used. Competition experiments showed that both sequences bind to the same nuclear factors. Polymers of oligonucleotides containing either the A or the C regions confer IL-6 responsiveness to a truncated SV40 promoter. The B region forms several complexes with specific DNA-binding proteins different from those which bind to the A and C region. The B region complexes are identical in nuclear extracts from IL-6-treated and untreated cells. While important for IL-6 induction in the context of the haptoglobin promoter, the B site does not confer IL-6 inducibility to the SV40 promoter. Our results indicate that the IL-6 response of the haptoglobin promoter is dependent on the presence of multiple, partly redundant, cis-acting elements. Images PMID:2787245

Modeling Interactions between Transposable Elements and the Plant Epigenetic Response: A Surprising Reliance on Element Retention.

PubMed

Roessler, Kyria; Bousios, Alexandros; Meca, Esteban; Gaut, Brandon S

2018-03-01

Transposable elements (TEs) compose the majority of angiosperm DNA. Plants counteract TE activity by silencing them epigenetically. One form of epigenetic silencing requires 21-22 nt small interfering RNAs that act to degrade TE mRNA and may also trigger DNA methylation. DNA methylation is reinforced by a second mechanism, the RNA-dependent DNA methylation (RdDM) pathway. RdDM relies on 24 nt small interfering RNAs and ultimately establishes TEs in a quiescent state. These host factors interact at a systems level, but there have been no system level analyses of their interactions. Here, we define a deterministic model that represents the propagation of active TEs, aspects of the host response and the accumulation of silenced TEs. We describe general properties of the model and also fit it to biological data in order to explore two questions. The first is why two overlapping pathways are maintained, given that both are likely energetically expensive. Under our model, RdDM silenced TEs effectively even when the initiation of silencing was weak. This relationship implies that only a small amount of RNAi is needed to initiate TE silencing, but reinforcement by RdDM is necessary to efficiently counter TE propagation. Second, we investigated the reliance of the host response on rates of TE deletion. The model predicted that low levels of deletion lead to few active TEs, suggesting that silencing is most efficient when methylated TEs are retained in the genome, thereby providing one explanation for the large size of plant genomes.

Transposable elements at the center of the crossroads between embryogenesis, embryonic stem cells, reprogramming, and long non-coding RNAs.

PubMed

Hutchins, Andrew Paul; Pei, Duanqing

Transposable elements (TEs) are mobile genomic sequences of DNA capable of autonomous and non-autonomous duplication. TEs have been highly successful, and nearly half of the human genome now consists of various families of TEs. Originally thought to be non-functional, these elements have been co-opted by animal genomes to perform a variety of physiological functions ranging from TE-derived proteins acting directly in normal biological functions, to innovations in transcription factor logic and influence on epigenetic control of gene expression. During embryonic development, when the genome is epigenetically reprogrammed and DNA-demethylated, TEs are released from repression and show embryonic stage-specific expression, and in human and mouse embryos, intact TE-derived endogenous viral particles can even be detected. A similar process occurs during the reprogramming of somatic cells to pluripotent cells: When the somatic DNA is demethylated, TEs are released from repression. In embryonic stem cells (ESCs), where DNA is hypomethylated, an elaborate system of epigenetic control is employed to suppress TEs, a system that often overlaps with normal epigenetic control of ESC gene expression. Finally, many long non-coding RNAs (lncRNAs) involved in normal ESC function and those assisting or impairing reprogramming contain multiple TEs in their RNA. These TEs may act as regulatory units to recruit RNA-binding proteins and epigenetic modifiers. This review covers how TEs are interlinked with the epigenetic machinery and lncRNAs, and how these links influence each other to modulate aspects of ESCs, embryogenesis, and somatic cell reprogramming.

Programmable DNA switches and their applications.

PubMed

Harroun, Scott G; Prévost-Tremblay, Carl; Lauzon, Dominic; Desrosiers, Arnaud; Wang, Xiaomeng; Pedro, Liliana; Vallée-Bélisle, Alexis

2018-03-08

DNA switches are ideally suited for numerous nanotechnological applications, and increasing efforts are being directed toward their engineering. In this review, we discuss how to engineer these switches starting from the selection of a specific DNA-based recognition element, to its adaptation and optimisation into a switch, with applications ranging from sensing to drug delivery, smart materials, molecular transporters, logic gates and others. We provide many examples showcasing their high programmability and recent advances towards their real life applications. We conclude with a short perspective on this exciting emerging field.

Dysregulation of RNA Interference in Breast Cancer

DTIC Science & Technology

2007-07-01

of miRNA complementary elements in 3-UTR of target mRNAs, the concentration in the seed (6–8 bp) of continuous Watson - Crick base pairing in the 5...treated the transfected cells with the anticancer drug topotecan (TPT) that is known to inhibit DNA topoisomerase I and cause DNA damage (Tanizawa et...as DNA damage caused by TPT, can increase the inhibitory effect mediated by R el at iv e C el l G ro w th R el at iv e C el l G ro w th Negative

DNA-Templated Pd Conductive Metallic Nanowires

NASA Astrophysics Data System (ADS)

Nguyen, K.; Monteverde, M.; Lyonnais, S.; Campidelli, S.; Bourgoin, J.-Ph.; Filoramo, A.

2008-10-01

Because of its unique recognition properties, its size and the sub-nanometric resolution, DNA is of particular interest for positioning and organizing nanomaterials. However, in DNA-directed nanoelectronic it can be envisioned to use DNA not only as a positioning scaffold, but also as a support for the conducting element. To ensure this function a metallization process is necessary and among the various DNA metallization methods the Pd based ones are of particular interest for carbon nanotube transistor connections. In this field, the major drawback of the existing methods is the fast kinetics of the process which lead to a stochastic growth. Here, we present a novel approach to DNA Pd metalization where the DNA molecule is previously deposited on a dry substrate in a typical nanodevice configuration. In our approach the progressive growth of nanowires is achieved by the slow and selective precipitation of PdO, followed by a subsequent reduction step. Thanks to this strategy we fabricated homogeneous, continuous and conductive Pd nanowires on the DNA scaffolds of very thin diameter (20-25 nm).

Transcription initiation complex structures elucidate DNA opening.

PubMed

Plaschka, C; Hantsche, M; Dienemann, C; Burzinski, C; Plitzko, J; Cramer, P

2016-05-19

Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts.

Pico-level DNA sensing by hetero-polymetalate, Na10{Dy2W10O30(µ-S)6}·80H2O, cluster

NASA Astrophysics Data System (ADS)

Dutta, Taposhree; Ganguly, Jhuma; Sarkar, Sabyasachi

2018-04-01

The polyoxometalate dysprosium cluster, (Dy-S-W POM) , Na10[Dy2W10O30(µ-S)6]·80H2O, shows remarkable dsDNA denaturation property. In the presence of 0.22 µmol of this Dy-S-W POM, the melting temperature (Tm) of calf-thymus (CT) dsDNA is decreased to 62.35 °C. Dy-S-W POM shows bleaching of methylene blue (MB). Addition of CT-DNA in the MB bleached solution of Dy-S-W POM apparently intercalates MB. Such trapped MB by CT-DNA responds to its re-oxidation by elemental sulfur formed in the bleaching process involving Dy-S-W POM. This reduction-oxidation property of MB with Dy-S-W POM led to the detection of pico (13.20 pmol) level of DNA even by naked eye, which will be helpful for rapid trace DNA detection in forensic science and DNA-related diagnostics, complimenting time-consuming sophisticated methodology.

Programmable motion of DNA origami mechanisms.

PubMed

Marras, Alexander E; Zhou, Lifeng; Su, Hai-Jun; Castro, Carlos E

2015-01-20

DNA origami enables the precise fabrication of nanoscale geometries. We demonstrate an approach to engineer complex and reversible motion of nanoscale DNA origami machine elements. We first design, fabricate, and characterize the mechanical behavior of flexible DNA origami rotational and linear joints that integrate stiff double-stranded DNA components and flexible single-stranded DNA components to constrain motion along a single degree of freedom and demonstrate the ability to tune the flexibility and range of motion. Multiple joints with simple 1D motion were then integrated into higher order mechanisms. One mechanism is a crank-slider that couples rotational and linear motion, and the other is a Bennett linkage that moves between a compacted bundle and an expanded frame configuration with a constrained 3D motion path. Finally, we demonstrate distributed actuation of the linkage using DNA input strands to achieve reversible conformational changes of the entire structure on ∼ minute timescales. Our results demonstrate programmable motion of 2D and 3D DNA origami mechanisms constructed following a macroscopic machine design approach.

Purification of functionalized DNA origami nanostructures.

PubMed

Shaw, Alan; Benson, Erik; Högberg, Björn

2015-05-26

The high programmability of DNA origami has provided tools for precise manipulation of matter at the nanoscale. This manipulation of matter opens up the possibility to arrange functional elements for a diverse range of applications that utilize the nanometer precision provided by these structures. However, the realization of functionalized DNA origami still suffers from imperfect production methods, in particular in the purification step, where excess material is separated from the desired functionalized DNA origami. In this article we demonstrate and optimize two purification methods that have not previously been applied to DNA origami. In addition, we provide a systematic study comparing the purification efficacy of these and five other commonly used purification methods. Three types of functionalized DNA origami were used as model systems in this study. DNA origami was patterned with either small molecules, antibodies, or larger proteins. With the results of our work we aim to provide a guideline in quality fabrication of various types of functionalized DNA origami and to provide a route for scalable production of these promising tools.

Programmable motion of DNA origami mechanisms

PubMed Central

Marras, Alexander E.; Zhou, Lifeng; Su, Hai-Jun; Castro, Carlos E.

2015-01-01

DNA origami enables the precise fabrication of nanoscale geometries. We demonstrate an approach to engineer complex and reversible motion of nanoscale DNA origami machine elements. We first design, fabricate, and characterize the mechanical behavior of flexible DNA origami rotational and linear joints that integrate stiff double-stranded DNA components and flexible single-stranded DNA components to constrain motion along a single degree of freedom and demonstrate the ability to tune the flexibility and range of motion. Multiple joints with simple 1D motion were then integrated into higher order mechanisms. One mechanism is a crank–slider that couples rotational and linear motion, and the other is a Bennett linkage that moves between a compacted bundle and an expanded frame configuration with a constrained 3D motion path. Finally, we demonstrate distributed actuation of the linkage using DNA input strands to achieve reversible conformational changes of the entire structure on ∼minute timescales. Our results demonstrate programmable motion of 2D and 3D DNA origami mechanisms constructed following a macroscopic machine design approach. PMID:25561550

[Bacteriophage λ: electrostatic properties of the genome and its elements].

PubMed

Krutinina, G G; Krutinin, E A; Kamzolova, S G; Osypov, A A

2015-01-01

Bacteriophage λ is a classical model object in molecular biology, but little is still known on the physical properties of its DNA and regulatory elements. A study was made of the electrostatic properties of phage λ DNA and regulatory elements. A global electrostatic potential distribution along the phage genome was found to be nonuniform with main regulatory elements being located in a limited region with a high potential. The RNA polymerase binding frequency on the linearized phage chromosome directly correlates with its local potential. Strong promoters of the phage and its host Escherichia coli have distinct electrostatic upstream elements, which differ in nucleotide sequence. Attachment and recombination sites of phage λ and its host have a higher potential, which possibly facilitates their recognition by integrase. Phage λ and host Rho-independent terminators have a symmetrical M-shaped potential profile, which only slightly depends on the annotated terminator palindrome length, and occur in a region with a substantially higher potential, which may cause polymerase retention, facilitating the formation of a terminator hairpin in RNA. It was concluded that virtually all elements of phage λ genome have potential distribution specifics, which are related to their structural properties and may play a role in their biological function. The global potential distribution along the phage genome reflects the architecture of the regulation of its transcription and integration in the host genome.

The Foldback-like element Galileo belongs to the P superfamily of DNA transposons and is widespread within the Drosophila genus.

PubMed

Marzo, Mar; Puig, Marta; Ruiz, Alfredo

2008-02-26

Galileo is the only transposable element (TE) known to have generated natural chromosomal inversions in the genus Drosophila. It was discovered in Drosophila buzzatii and classified as a Foldback-like element because of its long, internally repetitive, terminal inverted repeats (TIRs) and lack of coding capacity. Here, we characterized a seemingly complete copy of Galileo from the D. buzzatii genome. It is 5,406 bp long, possesses 1,229-bp TIRs, and encodes a 912-aa transposase similar to those of the Drosophila melanogaster 1360 (Hoppel) and P elements. We also searched the recently available genome sequences of 12 Drosophila species for elements similar to Dbuz\\Galileo by using bioinformatic tools. Galileo was found in six species (ananassae, willistoni, peudoobscura, persimilis, virilis, and mojavensis) from the two main lineages within the Drosophila genus. Our observations place Galileo within the P superfamily of cut-and-paste transposons and extend considerably its phylogenetic distribution. The interspecific distribution of Galileo indicates an ancient presence in the genus, but the phylogenetic tree built with the transposase amino acid sequences contrasts significantly with that of the species, indicating lineage sorting and/or horizontal transfer events. Our results also suggest that Foldback-like elements such as Galileo may evolve from DNA-based transposon ancestors by loss of the transposase gene and disproportionate elongation of TIRs.

The Foldback-like element Galileo belongs to the P superfamily of DNA transposons and is widespread within the Drosophila genus

PubMed Central

Marzo, Mar; Puig, Marta; Ruiz, Alfredo

2008-01-01

Galileo is the only transposable element (TE) known to have generated natural chromosomal inversions in the genus Drosophila. It was discovered in Drosophila buzzatii and classified as a Foldback-like element because of its long, internally repetitive, terminal inverted repeats (TIRs) and lack of coding capacity. Here, we characterized a seemingly complete copy of Galileo from the D. buzzatii genome. It is 5,406 bp long, possesses 1,229-bp TIRs, and encodes a 912-aa transposase similar to those of the Drosophila melanogaster 1360 (Hoppel) and P elements. We also searched the recently available genome sequences of 12 Drosophila species for elements similar to Dbuz\\Galileo by using bioinformatic tools. Galileo was found in six species (ananassae, willistoni, peudoobscura, persimilis, virilis, and mojavensis) from the two main lineages within the Drosophila genus. Our observations place Galileo within the P superfamily of cut-and-paste transposons and extend considerably its phylogenetic distribution. The interspecific distribution of Galileo indicates an ancient presence in the genus, but the phylogenetic tree built with the transposase amino acid sequences contrasts significantly with that of the species, indicating lineage sorting and/or horizontal transfer events. Our results also suggest that Foldback-like elements such as Galileo may evolve from DNA-based transposon ancestors by loss of the transposase gene and disproportionate elongation of TIRs. PMID:18287066

Spacing requirements for interactions between the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase and the cAMP receptor protein.

PubMed Central

Lloyd, G S; Busby, S J; Savery, N J

1998-01-01

During transcription initiation at bacterial promoters, the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) can interact with DNA-sequence elements (known as UP elements) and with activator proteins. We have constructed a series of semi-synthetic promoters carrying both an UP element and a consensus DNA-binding site for the Escherichia coli cAMP receptor protein (CRP; a factor that activates transcription by making direct contacts with alphaCTD). At these promoters, the UP element was located at a variety of distances upstream of the CRP-binding site, which was fixed at position -41.5 bp upstream of the transcript start. At some positions, the UP element caused enhanced promoter activity whereas, at other positions, it had very little effect. In no case was the CRP-dependence of the promoter relieved. DNase I and hydroxyl-radical footprinting were used to study ternary RNA polymerase-CRP-promoter complexes formed at two of the most active of these promoters, and co-operativity between the binding of CRP and purified alpha subunits was studied. The footprints show that alphaCTD binds to the UP element as it is displaced upstream but that this displacement does not prevent alphaCTD from being contacted by CRP. Models to account for this are discussed. PMID:9461538

APOBEC3G Inhibits HIV-1 RNA Elongation by Inactivating the Viral Trans-Activation Response Element

PubMed Central

Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

2014-01-01

Deamination of cytidine residues in viral DNA (vDNA) is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient HIV-1 replication. dC to dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here we demonstrate that A3G provides an additional layer of defence against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. Finally, we show that free ssDNA termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3′+5′ ends is sufficient for A3G deamination, identifying A3G as an efficient mutator, and that deamination of (−)SSDNA results in an early block of HIV-1 transcription. PMID:24859335

APOBEC3G inhibits HIV-1 RNA elongation by inactivating the viral trans-activation response element.

PubMed

Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

2014-07-29

Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mutations at Several Loci Cause Increased Expression of Ribonucleotide Reductase in Escherichia coli

PubMed Central

Feeney, Morgan Anne; Ke, Na

2012-01-01

Production of deoxyribonucleotides for DNA synthesis is an essential and tightly regulated process. The class Ia ribonucleotide reductase (RNR), the product of the nrdAB genes, is required for aerobic growth of Escherichia coli. In catalyzing the reduction of ribonucleotides, two of the cysteines of RNR become oxidized, forming a disulfide bond. To regenerate active RNR, the cell uses thioredoxins and glutaredoxins to reduce the disulfide bond. Strains that lack thioredoxins 1 and 2 and glutaredoxin 1 do not grow because RNR remains in its oxidized, inactive form. However, suppressor mutations that lead to RNR overproduction allow glutaredoxin 3 to reduce sufficient RNR for growth of these mutant strains. We previously described suppressor mutations in the dnaA and dnaN genes that had such effects. Here we report the isolation of new mutations that lead to increased levels of RNR. These include mutations that were not known to influence production of RNR previously, such as a mutation in the hda gene and insertions in the nrdAB promoter region of insertion elements IS1 and IS5. Bioinformatic analysis raises the possibility that IS element insertion in this region represents an adaptive mechanism in nrdAB regulation in E. coli and closely related species. We also characterize mutations altering different amino acids in DnaA and DnaN from those isolated before. PMID:22247510

Metal based biologically active compounds: Design, synthesis, DNA binding and antidiabetic activity of 6-methyl-3-formyl chromone derived hydrazones and their metal (II) complexes.

PubMed

Philip, Jessica Elizabeth; Shahid, Muhammad; Prathapachandra Kurup, M R; Velayudhan, Mohanan Puzhavoorparambil

2017-10-01

Two chromone hydrazone ligands HL 1 and HL 2 were synthesized and characterized by elemental analyses, IR, 1 H NMR & 13 C NMR, electronic absorption and mass spectra. The reactions of the chromone hydrazones with transition metals such as Ni, Cu, and Zn (II) salts of acetate afforded mononuclear metal complexes. Characterization and structure elucidation of the prepared chromone hydrazone metal (II) complexes were done by elemental, IR, electronic, EPR spectra and thermo gravimetric analyses as well as conductivity and magnetic susceptibility measurements. The spectroscopic data showed that the ligand acts as a mono basic bidentate with coordination sites are azomethine nitrogen and hydrazonic oxygen, and they exhibited distorted geometry. The biological studies involved antidiabetic activity i.e. enzyme inhibition of α-amylase and α-glucosidase, Calf Thymus - DNA (CT-DNA) interaction and molecular docking. Potential capacity of synthesized compounds to inhibit the α-amylase and α-glucosidase activity was assayed whereas DNA interaction studies were carried out with the help UV-Vis absorption titration and viscosity method. The docking studies of chromone hydrazones show that they are minor groove binders. Complexes were found to be good DNA - intercalates. Chromone hydrazones and its transition metal complexes have shown comparable antidiabetic activity with a standard drug acarbose. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of DNA type on response of DNA biosensor for carcinogens

NASA Astrophysics Data System (ADS)

Sani, Nor Diyana bt. Md.; Heng, Lee Yook; Surif, Salmijah; Lazim, Azwani Mat

2013-11-01

Carcinogens are cancer causing chemicals that can bind to DNA and cause damage to the DNA. These chemicals are available everywhere including in water, air, soil and food. Therefore, a sensor that can detect the presence of these chemicals will be a very useful tool. Since carcinogens bind to DNA, DNA can be used as the biological element in a biosensor. This study has utilized different types of DNA in a biosensor for carcinogen detection. The DNAs include double stranded calf thymus DNA, single stranded calf thymus DNA and guanine rich single stranded DNA. The modified SPE was exposed to a carcinogen followed by interaction with methylene blue which acts as the electroactive indicator. The SPE was then analysed using differential pulse voltammetry (DPV). Optimization studies were conducted for MB concentration and accumulation time, DNA concentration, as well as effect of buffer concentration, buffer pH and ionic strength. The performance of the biosensor was tested on a group 1 carcinogen, formaldehyde. The results indicated that the usage of guanine rich single stranded DNA also gives higher response as carcinogens prefer to bind with guanine compared to other bases.

A novel image encryption algorithm based on the chaotic system and DNA computing

NASA Astrophysics Data System (ADS)

Chai, Xiuli; Gan, Zhihua; Lu, Yang; Chen, Yiran; Han, Daojun

A novel image encryption algorithm using the chaotic system and deoxyribonucleic acid (DNA) computing is presented. Different from the traditional encryption methods, the permutation and diffusion of our method are manipulated on the 3D DNA matrix. Firstly, a 3D DNA matrix is obtained through bit plane splitting, bit plane recombination, DNA encoding of the plain image. Secondly, 3D DNA level permutation based on position sequence group (3DDNALPBPSG) is introduced, and chaotic sequences generated from the chaotic system are employed to permutate the positions of the elements of the 3D DNA matrix. Thirdly, 3D DNA level diffusion (3DDNALD) is given, the confused 3D DNA matrix is split into sub-blocks, and XOR operation by block is manipulated to the sub-DNA matrix and the key DNA matrix from the chaotic system. At last, by decoding the diffused DNA matrix, we get the cipher image. SHA 256 hash of the plain image is employed to calculate the initial values of the chaotic system to avoid chosen plaintext attack. Experimental results and security analyses show that our scheme is secure against several known attacks, and it can effectively protect the security of the images.

Origins and evolution of viruses of eukaryotes: The ultimate modularity

PubMed Central

Koonin, Eugene V.; Dolja, Valerian V.; Krupovic, Mart

2018-01-01

Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes of viruses are dramatically different between prokaryotes and eukaryotes. In prokaryotes, the great majority of viruses possess double-stranded (ds) DNA genomes, with a substantial minority of single-stranded (ss) DNA viruses and only limited presence of RNA viruses. In contrast, in eukaryotes, RNA viruses account for the majority of the virome diversity although ssDNA and dsDNA viruses are common as well. Phylogenomic analysis yields tangible clues for the origins of major classes of eukaryotic viruses and in particular their likely roots in prokaryotes. Specifically, the ancestral genome of positive-strand RNA viruses of eukaryotes might have been assembled de novo from genes derived from prokaryotic retroelements and bacteria although a primordial origin of this class of viruses cannot be ruled out. Different groups of double-stranded RNA viruses derive either from dsRNA bacteriophages or from positive-strand RNA viruses. The eukaryotic ssDNA viruses apparently evolved via a fusion of genes from prokaryotic rolling circle-replicating plasmids and positive-strand RNA viruses. Different families of eukaryotic dsDNA viruses appear to have originated from specific groups of bacteriophages on at least two independent occasions. Polintons, the largest known eukaryotic transposons, predicted to also form virus particles, most likely, were the evolutionary intermediates between bacterial tectiviruses and several groups of eukaryotic dsDNA viruses including the proposed order “Megavirales” that unites diverse families of large and giant viruses. Strikingly, evolution of all classes of eukaryotic viruses appears to have involved fusion between structural and replicative gene modules derived from different sources along with additional acquisitions of diverse genes. PMID:25771806

In silico modeling of epigenetic-induced changes in photoreceptor cis-regulatory elements.

PubMed

Hossain, Reafa A; Dunham, Nicholas R; Enke, Raymond A; Berndsen, Christopher E

2018-01-01

DNA methylation is a well-characterized epigenetic repressor of mRNA transcription in many plant and vertebrate systems. However, the mechanism of this repression is not fully understood. The process of transcription is controlled by proteins that regulate recruitment and activity of RNA polymerase by binding to specific cis-regulatory sequences. Cone-rod homeobox (CRX) is a well-characterized mammalian transcription factor that controls photoreceptor cell-specific gene expression. Although much is known about the functions and DNA binding specificity of CRX, little is known about how DNA methylation modulates CRX binding affinity to genomic cis-regulatory elements. We used bisulfite pyrosequencing of human ocular tissues to measure DNA methylation levels of the regulatory regions of RHO , PDE6B, PAX6 , and LINE1 retrotransposon repeats. To describe the molecular mechanism of repression, we used molecular modeling to illustrate the effect of DNA methylation on human RHO regulatory sequences. In this study, we demonstrate an inverse correlation between DNA methylation in regulatory regions adjacent to the human RHO and PDE6B genes and their subsequent transcription in human ocular tissues. Docking of CRX to the DNA models shows that CRX interacts with the grooves of these sequences, suggesting changes in groove structure could regulate binding. Molecular dynamics simulations of the RHO promoter and enhancer regions show changes in the flexibility and groove width upon epigenetic modification. Models also demonstrate changes in the local dynamics of CRX binding sites within RHO regulatory sequences which may account for the repression of CRX-dependent transcription. Collectively, these data demonstrate epigenetic regulation of CRX binding sites in human retinal tissue and provide insight into the mechanism of this mode of epigenetic regulation to be tested in future experiments.

Accurate Prediction of Inducible Transcription Factor Binding Intensities In Vivo

PubMed Central

Siepel, Adam; Lis, John T.

2012-01-01

DNA sequence and local chromatin landscape act jointly to determine transcription factor (TF) binding intensity profiles. To disentangle these influences, we developed an experimental approach, called protein/DNA binding followed by high-throughput sequencing (PB–seq), that allows the binding energy landscape to be characterized genome-wide in the absence of chromatin. We applied our methods to the Drosophila Heat Shock Factor (HSF), which inducibly binds a target DNA sequence element (HSE) following heat shock stress. PB–seq involves incubating sheared naked genomic DNA with recombinant HSF, partitioning the HSF–bound and HSF–free DNA, and then detecting HSF–bound DNA by high-throughput sequencing. We compared PB–seq binding profiles with ones observed in vivo by ChIP–seq and developed statistical models to predict the observed departures from idealized binding patterns based on covariates describing the local chromatin environment. We found that DNase I hypersensitivity and tetra-acetylation of H4 were the most influential covariates in predicting changes in HSF binding affinity. We also investigated the extent to which DNA accessibility, as measured by digital DNase I footprinting data, could be predicted from MNase–seq data and the ChIP–chip profiles for many histone modifications and TFs, and found GAGA element associated factor (GAF), tetra-acetylation of H4, and H4K16 acetylation to be the most predictive covariates. Lastly, we generated an unbiased model of HSF binding sequences, which revealed distinct biophysical properties of the HSF/HSE interaction and a previously unrecognized substructure within the HSE. These findings provide new insights into the interplay between the genomic sequence and the chromatin landscape in determining transcription factor binding intensity. PMID:22479205

Evolutionary conservation of regulatory elements in vertebrate HOX gene clusters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santini, Simona; Boore, Jeffrey L.; Meyer, Axel

2003-12-31

Due to their high degree of conservation, comparisons of DNA sequences among evolutionarily distantly-related genomes permit to identify functional regions in noncoding DNA. Hox genes are optimal candidate sequences for comparative genome analyses, because they are extremely conserved in vertebrates and occur in clusters. We aligned (Pipmaker) the nucleotide sequences of HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human and mouse (over 500 million years of evolutionary distance). We identified several highly conserved intergenic sequences, likely to be important in gene regulation. Only a few of these putative regulatory elements have been previously described as being involvedmore » in the regulation of Hox genes, while several others are new elements that might have regulatory functions. The majority of these newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac). The conserved intergenic regions located between the most rostrally expressed genes in the developing embryo are longer and better retained through evolution. We document that presumed regulatory sequences are retained differentially in either A or A clusters resulting from a genome duplication in the fish lineage. This observation supports both the hypothesis that the conserved elements are involved in gene regulation and the Duplication-Deletion-Complementation model.« less

Localization of Action of the Is50-Encoded Transposase Protein

PubMed Central

Phadnis, Suhas H.; Sasakawa, Chihiro; Berg, Douglas E.

1986-01-01

The movement of the bacterial insertion sequence IS50 and of composite elements containing direct terminal repeats of IS50 involves the two ends of IS50, designated O (outside) and I (inside), which are weakly matched in DNA sequence, and an IS50 encoded protein, transposase, which recognizes the O and I ends and acts preferentially in cis. Previous data had suggested that, initially, transposase interacts preferentially with the O end sequence and then, in a second step, with either an O or an I end. To better understand the cis action of transposase and how IS50 ends are selected, we generated a series of composite transposons which contain direct repeats of IS50 elements. In each transposon, one IS50 element encoded transposase (tnp +), and the other contained a null (tnp-) allele. In each of the five sets of composite transposons studied, the transposon for which the tnp+ IS50 element contained its O end was more active than a complementary transposon for which the tnp - IS50 element contained its O end. This pattern of O end use suggests models in which the cis action of transposase and its choice of ends is determined by protein tracking along DNA molecules. PMID:3007274

Structural analysis of the rDNA intergenic spacer of Brassica nigra: evolutionary divergence of the spacers of the three diploid Brassica species.

PubMed

Bhatia, S; Singh Negi, M; Lakshmikumaran, M

1996-11-01

EcoRI restriction of the B. nigra rDNA recombinants, isolated from a lambda genomic library, showed that the 3.9-kb fragment corresponded to the Intergenic Spacer (IGS), which was sequenced and found to be 3,928 bp in size. Sequence and dot-matrix analyses showed that the organization of the B. nigra rDNA IGS was typical of most rDNA spacers, consisting of a central repetitive region and flanking unique sequences on either side. The repetitive region was composed of two repeat families-RF 'A' and RF 'B.' The B. nigra RF 'A' consisted of a tandem array of three full-length copies of a 106-bp sequence element. RF 'B' was composed of 66 tandemly repeated elements. Each 'B' element was only 21-bp in size and this is the smallest repeat unit identified in plant rDNA to date. The putative transcription initiation site (TIS) was identified as nucleotide position 3,110. Based on the sequence analysis it was suggested that the present organization of the repeat families was generated by successive cycles of deletions and amplifications and was being maintained by homogenization processes such as gene conversion and crossing-over.A detailed comparison of the rDNA IGS sequences of the three diploid Brassica species-namely, B. nigra, B. campestris, and B. oleracea-was carried out. First, comparisons revealed that B. campestris and B. oleracea were close to each other as the repeat families in both showed high sequence homology between each other. Second, the repeat elements in both the species were organized in an interspersed manner. Third, a 52-bp sequence, present just downstream of the repeats in B. campestris, was found to be identical to the B. oleracea repeats, thereby suggesting a common progenitor. On the other hand, in B. nigra no interspersion pattern of organization of repeats was observed. Further, the B. nigra RF 'A' was identified as distinct from the repeat families of B. campestris and B. oleracea. Based on this analysis, it was suggested that during speciation B. campestris and B. oleracea evolved in one lineage whereas B. nigra diverged into a separate lineage. The comparative analysis of the IGS helped in identifying not only conserved ancestral sequence motifs of possible functional significance such as promoters and enhancers, but also sequences which showed variation between the three diploid species and were therefore identified as species-specific sequences.

Polintons: a hotbed of eukaryotic virus, transposon and plasmid evolution

PubMed Central

Krupovic, Mart; Koonin, Eugene V.

2018-01-01

Polintons (also known as Mavericks) are large DNA transposons that are widespread in the genomes of eukaryotes. We have recently shown that Polintons encode virus capsid proteins, which suggests that these transposons might form virions, at least under some conditions. In this Opinion article, we delineate the evolutionary relationships among bacterial tectiviruses, Polintons, adenoviruses, virophages, large and giant DNA viruses of eukaryotes of the proposed order ‘Megavirales’, and linear mitochondrial and cytoplasmic plasmids. We hypothesize that Polintons were the first group of eukaryotic double-stranded DNA viruses to evolve from bacteriophages and that they gave rise to most large DNA viruses of eukaryotes and various other selfish genetic elements. PMID:25534808

Transposon facilitated DNA sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berg, D.E.; Berg, C.M.; Huang, H.V.

1990-01-01

The purpose of this research is to investigate and develop methods that exploit the power of bacterial transposable elements for large scale DNA sequencing: Our premise is that the use of transposons to put primer binding sites randomly in target DNAs should provide access to all portions of large DNA fragments, without the inefficiencies of methods involving random subcloning and attendant repetitive sequencing, or of sequential synthesis of many oligonucleotide primers that are used to match systematically along a DNA molecule. Two unrelated bacterial transposons, Tn5 and {gamma}{delta}, are being used because they have both proven useful for molecular analyses,more » and because they differ sufficiently in mechanism and specificity of transposition to merit parallel development.« less

Exact solution of a model DNA-inversion genetic switch with orientational control.

PubMed

Visco, Paolo; Allen, Rosalind J; Evans, Martin R

2008-09-12

DNA inversion is an important mechanism by which bacteria and bacteriophage switch reversibly between phenotypic states. In such switches, the orientation of a short DNA element is flipped by a site-specific recombinase enzyme. We propose a simple model for a DNA-inversion switch in which recombinase production is dependent on the switch state (orientational control). Our model is inspired by the fim switch in E. coli. We present an exact analytical solution of the chemical master equation for the model switch, as well as stochastic simulations. Orientational control causes the switch to deviate from Poissonian behavior: the distribution of times in the on state shows a peak and successive flip times are correlated.

Control of transcriptional pausing by biased thermal fluctuations on repetitive genomic sequences

PubMed Central

Imashimizu, Masahiko; Afek, Ariel; Takahashi, Hiroki; Lubkowska, Lucyna; Lukatsky, David B.

2016-01-01

In the process of transcription elongation, RNA polymerase (RNAP) pauses at highly nonrandom positions across genomic DNA, broadly regulating transcription; however, molecular mechanisms responsible for the recognition of such pausing positions remain poorly understood. Here, using a combination of statistical mechanical modeling and high-throughput sequencing and biochemical data, we evaluate the effect of thermal fluctuations on the regulation of RNAP pausing. We demonstrate that diffusive backtracking of RNAP, which is biased by repetitive DNA sequence elements, causes transcriptional pausing. This effect stems from the increased microscopic heterogeneity of an elongation complex, and thus is entropy-dominated. This report shows a linkage between repetitive sequence elements encoded in the genome and regulation of RNAP pausing driven by thermal fluctuations. PMID:27830653

Use of a Drosophila Genome-Wide Conserved Sequence Database to Identify Functionally Related cis-Regulatory Enhancers

PubMed Central

Brody, Thomas; Yavatkar, Amarendra S; Kuzin, Alexander; Kundu, Mukta; Tyson, Leonard J; Ross, Jermaine; Lin, Tzu-Yang; Lee, Chi-Hon; Awasaki, Takeshi; Lee, Tzumin; Odenwald, Ward F

2012-01-01

Background: Phylogenetic footprinting has revealed that cis-regulatory enhancers consist of conserved DNA sequence clusters (CSCs). Currently, there is no systematic approach for enhancer discovery and analysis that takes full-advantage of the sequence information within enhancer CSCs. Results: We have generated a Drosophila genome-wide database of conserved DNA consisting of >100,000 CSCs derived from EvoPrints spanning over 90% of the genome. cis-Decoder database search and alignment algorithms enable the discovery of functionally related enhancers. The program first identifies conserved repeat elements within an input enhancer and then searches the database for CSCs that score highly against the input CSC. Scoring is based on shared repeats as well as uniquely shared matches, and includes measures of the balance of shared elements, a diagnostic that has proven to be useful in predicting cis-regulatory function. To demonstrate the utility of these tools, a temporally-restricted CNS neuroblast enhancer was used to identify other functionally related enhancers and analyze their structural organization. Conclusions: cis-Decoder reveals that co-regulating enhancers consist of combinations of overlapping shared sequence elements, providing insights into the mode of integration of multiple regulating transcription factors. The database and accompanying algorithms should prove useful in the discovery and analysis of enhancers involved in any developmental process. Developmental Dynamics 241:169–189, 2012. © 2011 Wiley Periodicals, Inc. Key findings A genome-wide catalog of Drosophila conserved DNA sequence clusters. cis-Decoder discovers functionally related enhancers. Functionally related enhancers share balanced sequence element copy numbers. Many enhancers function during multiple phases of development. PMID:22174086

Friends-enemies: endogenous retroviruses are major transcriptional regulators of human DNA

NASA Astrophysics Data System (ADS)

Buzdin, Anton A.; Prassolov, Vladimir; Garazha, Andrew V.

2017-06-01

Endogenous retroviruses are mobile genetic elements hardly distinguishable from infectious, or “exogenous”, retroviruses at the time of insertion in the host DNA. Human endogenous retroviruses (HERVs) are not rare. They gave rise to multiple families of closely related mobile elements that occupy 8% of the human genome. Together, they shape genomic regulatory landscape by providing at least 320,000 human transcription factor binding sites (TFBS) located on 110,000 individual HERV elements. The HERVs host as many as 155,000 mapped DNaseI hypersensitivity sites, which denote loci active in the regulation of gene expression or chromatin structure. The contemporary view of the HERVs evolutionary dynamics suggests that at the early stages after insertion, the HERV is treated by the host cells as a foreign genetic element, and is likely to be suppressed by the targeted methylation and mutations. However, at the later stages, when significant number of mutations has been already accumulated and when the retroviral genes are broken, the regulatory potential of a HERV may be released and recruited to modify the genomic balance of transcription factor binding sites. This process goes together with further accumulation and selection of mutations, which reshape the regulatory landscape of the human DNA. However, developmental reprogramming, stress or pathological conditions like cancer, inflammation and infectious diseases, can remove the blocks limiting expression and HERV-mediated host gene regulation. This, in turn, can dramatically alter the gene expression equilibrium and shift it to a newer state, thus further amplifying instability and exacerbating the stressful situation.

Dynamics and biological relevance of DNA demethylation in Arabidopsis antibacterial defense.

PubMed

Yu, Agnès; Lepère, Gersende; Jay, Florence; Wang, Jingyu; Bapaume, Laure; Wang, Yu; Abraham, Anne-Laure; Penterman, Jon; Fischer, Robert L; Voinnet, Olivier; Navarro, Lionel

2013-02-05

DNA methylation is an epigenetic mark that silences transposable elements (TEs) and repeats. Whereas the establishment and maintenance of DNA methylation are relatively well understood, little is known about their dynamics and biological relevance in plant and animal innate immunity. Here, we show that some TEs are demethylated and transcriptionally reactivated during antibacterial defense in Arabidopsis. This effect is correlated with the down-regulation of key transcriptional gene silencing factors and is partly dependent on an active demethylation process. DNA demethylation restricts multiplication and vascular propagation of the bacterial pathogen Pseudomonas syringae in leaves and, accordingly, some immune-response genes, containing repeats in their promoter regions, are negatively regulated by DNA methylation. This study provides evidence that DNA demethylation is part of a plant-induced immune response, potentially acting to prime transcriptional activation of some defense genes linked to TEs/repeats.

Protein Detection via Direct Enzymatic Amplification of Short DNA Aptamers

PubMed Central

Fischer, Nicholas O.; Tarasow, Theodore M.; Tok, Jeffrey B.-H.

2008-01-01

Aptamers are single-stranded nucleic acids that fold into defined tertiary structures to bind target molecules with high specificities and affinities. DNA aptamers have garnered much interest as recognition elements for biodetection and diagnostic applications due to their small size, ease of discovery and synthesis, and chemical and thermal stability. Herein, we describe the design and application of a short DNA molecule capable of both protein target binding and amplifiable bioreadout processes. As both recognition and readout capabilities are incorporated into a single DNA molecule, tedious conjugation procedures required for protein-DNA hybrids can be omitted. The DNA aptamer is designed to be amplified directly by either the polymerase chain reaction (PCR) or rolling circle amplification (RCA) processes, taking advantage of real-time amplification monitoring techniques for target detection. A combination of both RCA and PCR provides a wide protein target dynamic range (1 μM to 10 pM). PMID:17980857

Current trends in electrochemical sensing and biosensing of DNA methylation.

PubMed

Krejcova, Ludmila; Richtera, Lukas; Hynek, David; Labuda, Jan; Adam, Vojtech

2017-11-15

DNA methylation plays an important role in physiological and pathological processes. Several genetic diseases and most malignancies tend to be associated with aberrant DNA methylation. Among other analytical methods, electrochemical approaches have been successfully employed for characterisation of DNA methylation patterns that are essential for the diagnosis and treatment of particular diseases. This article discusses current trends in the electrochemical sensing and biosensing of DNA methylation. Particularly, it provides an overview of applied electrode materials, electrode modifications and biorecognition elements applications with an emphasis on strategies that form the core DNA methylation detection approaches. The three main strategies as (i) bisulfite treatment, (ii) cleavage by restriction endonucleases, and (iii) immuno/affinity reaction were described in greater detail. Additionally, the availability of the reviewed platforms for early cancer diagnosis and the approval of methylation inhibitors for anticancer therapy were discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

A label-free DNA hairpin biosensor for colorimetric detection of target with suitable functional DNA partners.

PubMed

Nie, Ji; Zhang, De-Wen; Tie, Cai; Zhou, Ying-Lin; Zhang, Xin-Xiang

2013-11-15

The combination of aptamer and peroxidase-mimicking DNAzyme within a hairpin structure can form a functional DNA probe. The activities of both aptamer (as biorecognition element) and DNAzyme (as signal amplification element) are blocked via base pairing in the hairpin structure. The presence of target triggers the opening of the hairpin to form target/aptamer complex and releases G-quadruplex sequence which can generate amplified colorimetric signals. In this work, we elaborated a universal and simple procedure to design an efficient and sensitive hairpin probe with suitable functional DNA partners. A fill-in-the-blank process was developed for sequence design, and two key points including the pretreatment of the hairpin probe and the selection of suitable signal transducer sequence were proved to enhance the detection sensitivity. Cocaine was chosen as a model target for a proof of concept. A series of hairpins with different numbers of base pairs in the stem region were prepared. Hairpin-C10 with ten base pairs was screened out and a lowest detectable cocaine concentration of 5 μM by colorimetry was obtained. The proposed functional DNA hairpin showed good selectivity and satisfactory analysis in spiked biologic fluid. The whole "mix-and-measure" detection based on DNA hairpin without the need of immobilization and labeling was indicated to be time and labor saving. The strategy has potential to be transplanted into more smart hairpins toward other targets for general application in bioanalytical chemistry. Copyright © 2013 Elsevier B.V. All rights reserved.

In Vivo Control of CpG and Non-CpG DNA Methylation by DNA Methyltransferases

PubMed Central

Arand, Julia; Spieler, David; Karius, Tommy; Branco, Miguel R.; Meilinger, Daniela; Meissner, Alexander; Jenuwein, Thomas; Xu, Guoliang; Leonhardt, Heinrich; Wolf, Verena; Walter, Jörn

2012-01-01

The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position–, cell type–, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs. PMID:22761581

Effects of age, sex, and persistent organic pollutants on DNA methylation in children

PubMed Central

Huen, Karen; Yousefi, Paul; Bradman, Asa; Yan, Liying; Harley, Kim G.; Kogut, Katherine; Eskenazi, Brenda; Holland, Nina

2015-01-01

Epigenetic changes such as DNA methylation may be a molecular mechanism through which environmental exposures affect health. Methylation of Alu and long interspersed nucleotide elements (LINE-1) is a well-established measure of DNA methylation often used in epidemiologic studies. Yet, few studies have examined the effects of host factors on LINE-1 and Alu methylation in children. We characterized the relationship of age, sex, and prenatal exposure to persistent organic pollutants (POPs), dichlorodiphenyl trichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE), and polybrominated diphenyl ethers (PBDEs), with DNA methylation in a birth cohort of Mexican-American children participating in the CHAMACOS study. We measured Alu and LINE-1 methylation by pyrosequencing bisulfite-treated DNA isolated from whole blood samples collected from newborns and 9-year old children (n=358). POPs were measured in maternal serum during late pregnancy. Levels of DNA methylation were lower in 9-year olds compared to newborns and were higher in boys compared to girls. Higher prenatal DDT/E exposure was associated with lower Alu methylation at birth, particularly after adjusting for cell type composition (p=0.02 for o,p′ -DDT). Associations of POPs with LINE-1 methylation were only identified after examining the co-exposure of DDT/E with PBDEs simultaneously. Our data suggest that repeat element methylation can be an informative marker of epigenetic differences by age and sex and that prenatal exposure to POPs may be linked to hypomethylation in fetal blood. Accounting for co-exposure to different types of chemicals and adjusting for blood cell types may increase sensitivity of epigenetic analyses for epidemiological studies. PMID:24375655

On the roles of repetitive DNA elements in the context of a unified genomic-epigenetic system.

PubMed

von Sternberg, Richard

2002-12-01

Repetitive DNA sequences comprise a substantial portion of most eukaryotic and some prokaryotic chromosomes. Despite nearly forty years of research, the functions of various sequence families as a whole and their monomer units remain largely unknown. The inability to map specific functional roles onto many repetitive DNA elements (REs), coupled with the taxon-specificity of sequence families, have led many to speculate that these genomic components are "selfish" replicators generating genomic "junk." The purpose of this paper is to critically examine the selfishness, evolutionary effects, and functionality of REs. First, a brief overview of the range of ideas pertaining to RE function is presented. Second, the argument is presented that the selfish DNA "hypothesis" is actually a narrative scheme, that it serves to protect neo-Darwinian assumptions from criticism, and that this story is untestable and therefore not a hypothesis. Third, attempts to synthesize the selfish DNA concept with complex systems models of the genome and RE functionality are critiqued. Fourth, the supposed connection between RE-induced mutations and macroevolutionary events are stated to be at variance with empirical evidence and theoretical considerations. Hypotheses that base phylogenetic transitions in repetitive sequence changes thus remain speculative. Fifth and finally, the case is made for viewing REs as integrally functional components of chromosomes, genomes, and cells. It is argued throughout that a new conceptual framework is needed for understanding the roles of repetitive DNA in genomic/epigenetic systems, and that neo-Darwinian "narratives" have been the primary obstacle to elucidating the effects of these enigmatic components of chromosomes.

Genomic Heat Shock Element Sequences Drive Cooperative Human Heat Shock Factor 1 DNA Binding and Selectivity*

PubMed Central

Jaeger, Alex M.; Makley, Leah N.; Gestwicki, Jason E.; Thiele, Dennis J.

2014-01-01

The heat shock transcription factor 1 (HSF1) activates expression of a variety of genes involved in cell survival, including protein chaperones, the protein degradation machinery, anti-apoptotic proteins, and transcription factors. Although HSF1 activation has been linked to amelioration of neurodegenerative disease, cancer cells exhibit a dependence on HSF1 for survival. Indeed, HSF1 drives a program of gene expression in cancer cells that is distinct from that activated in response to proteotoxic stress, and HSF1 DNA binding activity is elevated in cycling cells as compared with arrested cells. Active HSF1 homotrimerizes and binds to a DNA sequence consisting of inverted repeats of the pentameric sequence nGAAn, known as heat shock elements (HSEs). Recent comprehensive ChIP-seq experiments demonstrated that the architecture of HSEs is very diverse in the human genome, with deviations from the consensus sequence in the spacing, orientation, and extent of HSE repeats that could influence HSF1 DNA binding efficacy and the kinetics and magnitude of target gene expression. To understand the mechanisms that dictate binding specificity, HSF1 was purified as either a monomer or trimer and used to evaluate DNA-binding site preferences in vitro using fluorescence polarization and thermal denaturation profiling. These results were compared with quantitative chromatin immunoprecipitation assays in vivo. We demonstrate a role for specific orientations of extended HSE sequences in driving preferential HSF1 DNA binding to target loci in vivo. These studies provide a biochemical basis for understanding differential HSF1 target gene recognition and transcription in neurodegenerative disease and in cancer. PMID:25204655

Role of transposon-derived small RNAs in the interplay between genomes and parasitic DNA in rice.

PubMed

Nosaka, Misuzu; Itoh, Jun-Ichi; Nagato, Yasuo; Ono, Akemi; Ishiwata, Aiko; Sato, Yutaka

2012-09-01

RNA silencing is a defense system against "genomic parasites" such as transposable elements (TE), which are potentially harmful to host genomes. In plants, transcripts from TEs induce production of double-stranded RNAs (dsRNAs) and are processed into small RNAs (small interfering RNAs, siRNAs) that suppress TEs by RNA-directed DNA methylation. Thus, the majority of TEs are epigenetically silenced. On the other hand, most of the eukaryotic genome is composed of TEs and their remnants, suggesting that TEs have evolved countermeasures against host-mediated silencing. Under some circumstances, TEs can become active and increase in copy number. Knowledge is accumulating on the mechanisms of TE silencing by the host; however, the mechanisms by which TEs counteract silencing are poorly understood. Here, we show that a class of TEs in rice produces a microRNA (miRNA) to suppress host silencing. Members of the microRNA820 (miR820) gene family are located within CACTA DNA transposons in rice and target a de novo DNA methyltransferase gene, OsDRM2, one of the components of epigenetic silencing. We confirmed that miR820 negatively regulates the expression of OsDRM2. In addition, we found that expression levels of various TEs are increased quite sensitively in response to decreased OsDRM2 expression and DNA methylation at TE loci. Furthermore, we found that the nucleotide sequence of miR820 and its recognition site within the target gene in some Oryza species have co-evolved to maintain their base-pairing ability. The co-evolution of these sequences provides evidence for the functionality of this regulation. Our results demonstrate how parasitic elements in the genome escape the host's defense machinery. Furthermore, our analysis of the regulation of OsDRM2 by miR820 sheds light on the action of transposon-derived small RNAs, not only as a defense mechanism for host genomes but also as a regulator of interactions between hosts and their parasitic elements.

Stress induced gene expression drives transient DNA methylation changes at adjacent repetitive elements.

PubMed

Secco, David; Wang, Chuang; Shou, Huixia; Schultz, Matthew D; Chiarenza, Serge; Nussaume, Laurent; Ecker, Joseph R; Whelan, James; Lister, Ryan

2015-07-21

Cytosine DNA methylation (mC) is a genome modification that can regulate the expression of coding and non-coding genetic elements. However, little is known about the involvement of mC in response to environmental cues. Using whole genome bisulfite sequencing to assess the spatio-temporal dynamics of mC in rice grown under phosphate starvation and recovery conditions, we identified widespread phosphate starvation-induced changes in mC, preferentially localized in transposable elements (TEs) close to highly induced genes. These changes in mC occurred after changes in nearby gene transcription, were mostly DCL3a-independent, and could partially be propagated through mitosis, however no evidence of meiotic transmission was observed. Similar analyses performed in Arabidopsis revealed a very limited effect of phosphate starvation on mC, suggesting a species-specific mechanism. Overall, this suggests that TEs in proximity to environmentally induced genes are silenced via hypermethylation, and establishes the temporal hierarchy of transcriptional and epigenomic changes in response to stress.

Identification and functional characterization of BTas transactivator as a DNA-binding protein.

PubMed

Tan, Juan; Hao, Peng; Jia, Rui; Yang, Wei; Liu, Ruichang; Wang, Jinzhong; Xi, Zhen; Geng, Yunqi; Qiao, Wentao

2010-09-30

The genome of bovine foamy virus (BFV) encodes a transcriptional transactivator, namely BTas, that remarkably enhances gene expression by binding to the viral long-terminal repeat promoter (LTR) and internal promoter (IP). In this report, we characterized the functional domains of BFV BTas. BTas contains two major functional domains: the N-terminal DNA-binding domain (residues 1-133) and the C-terminal activation domain (residues 198-249). The complete BTas responsive regions were mapped to the positions -380/-140 of LTR and 9205/9276 of IP. Four BTas responsive elements were identified at the positions -368/-346, -327/-307, -306/-285 and -186/-165 of the BFV LTR, and one element was identified at the position 9243/9264 of the BFV IP. Unlike other foamy viruses, the five BTas responsive elements in BFV shared obvious sequence homology. These data suggest that among the complex retroviruses, BFV appears to have a unique transactivation mechanism. Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed Central

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-01-01

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions. Images PMID:2556266

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-12-20

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions.

The bornavirus-derived human protein EBLN1 promotes efficient cell cycle transit, microtubule organisation and genome stability.

PubMed

Myers, Katie N; Barone, Giancarlo; Ganesh, Anil; Staples, Christopher J; Howard, Anna E; Beveridge, Ryan D; Maslen, Sarah; Skehel, J Mark; Collis, Spencer J

2016-10-14

It was recently discovered that vertebrate genomes contain multiple endogenised nucleotide sequences derived from the non-retroviral RNA bornavirus. Strikingly, some of these elements have been evolutionary maintained as open reading frames in host genomes for over 40 million years, suggesting that some endogenised bornavirus-derived elements (EBL) might encode functional proteins. EBLN1 is one such element established through endogenisation of the bornavirus N gene (BDV N). Here, we functionally characterise human EBLN1 as a novel regulator of genome stability. Cells depleted of human EBLN1 accumulate DNA damage both under non-stressed conditions and following exogenously induced DNA damage. EBLN1-depleted cells also exhibit cell cycle abnormalities and defects in microtubule organisation as well as premature centrosome splitting, which we attribute in part, to improper localisation of the nuclear envelope protein TPR. Our data therefore reveal that human EBLN1 possesses important cellular functions within human cells, and suggest that other EBLs present within vertebrate genomes may also possess important cellular functions.

Isolation, characterization and expression analysis of the BABY BOOM (BBM) gene from Larix kaempferi × L. olgensis during adventitious rooting.

PubMed

Li, Kui-Peng; Sun, Xiao-Mei; Han, Hua; Zhang, Shou-Gong

2014-11-10

The full-length cDNA and genomic sequences of the BABY BOOM (BBM) gene, designated LkBBM, were isolated from Larix kaempferi × Larix olgensis. The 3324 bp cDNA was cloned and its open reading frame (ORF) consists of 2370 nucleotides. The deduced 789 amino acid protein contains two AP2 domains and a BBM specific motif. Four conserved motifs between BBM and PLT were identified, which may be conducive to the similar function of BBM and PLT. The three dimensional (3D) structure of LkBBM was predicted and β-sheets in the AP2-R2 domain of LkBBM might recognize the specific base pairs in the major groove. Analysis of the LkBBM gene structure indicates that the gene has eight introns and nine exons. In the 5'-flanking promoter region of LkBBM, many important potential cis-acting elements were identified, such as the TATABOX5 element (a functional TATA element), ROOTMOTIFTAPOX1 element (element of root specificity), AUXREPSIAA4 element (element involved in auxin responsiveness and gene expression in root meristem), MYB1AT element (element involved in MYB recognition), ARR1AT element (element involved in cytokinin responsiveness), GARE1OSREP1 element (element involved in gibberellin responsiveness) and PYRIMIDINEBOXHVEPB1 element (element involved in abscisic acid responsiveness), which all suggested that the expression of LkBBM is highly regulated. Compared with gene expression levels in the stem, stem tip and leaf, LkBBM shows a specific expression in the root, which indicates that LkBBM plays a key role in regulating the development and growth of root in larch. In the processing of larch adventitious root formation, LkBBM started to express on the eighth day after rooting treatment and its transcript level increased continuously afterwards. According to the gene characteristics, LkBBM is proposed as a molecular marker for root primordia of larch, and the initial period of LkBBM expression may be the formation period of root primordia in the processing of adventitious rooting of larch. Copyright © 2014 Elsevier B.V. All rights reserved.

A Helitron-like Transposon Superfamily from Lepidoptera Disrupts (GAAA)n Microsatellites and is Responsible for Flanking Sequence Similarity within a Microsatellite Family

USDA-ARS?s Scientific Manuscript database

Transposable elements (TEs) are mobile DNA regions that alter host genome structure and gene expression. A novel 588 bp non-autonomous high copy number TE in the Ostrinia nubilalis genome has features in common with miniature inverted-repeat transposable elements (MITEs): high A+T content (62.3%),...

SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

PubMed Central

Varshney, Dhaval; Vavrova-Anderson, Jana; Oler, Andrew J.; Cowling, Victoria H.; Cairns, Bradley R.; White, Robert J.

2015-01-01

Short interspersed nuclear elements (SINEs), such as Alu, spread by retrotransposition, which requires their transcripts to be copied into DNA and then inserted into new chromosomal sites. This can lead to genetic damage through insertional mutagenesis and chromosomal rearrangements between non-allelic SINEs at distinct loci. SINE DNA is heavily methylated and this was thought to suppress its accessibility and transcription, thereby protecting against retrotransposition. Here we provide several lines of evidence that methylated SINE DNA is occupied by RNA polymerase III, including the use of high-throughput bisulphite sequencing of ChIP DNA. We find that loss of DNA methylation has little effect on accessibility of SINEs to transcription machinery or their expression in vivo. In contrast, a histone methyltransferase inhibitor selectively promotes SINE expression and occupancy by RNA polymerase III. The data suggest that methylation of histones rather than DNA plays a dominant role in suppressing SINE transcription. PMID:25798578

Chromatin-Specific Regulation of Mammalian rDNA Transcription by Clustered TTF-I Binding Sites

PubMed Central

Diermeier, Sarah D.; Németh, Attila; Rehli, Michael; Grummt, Ingrid; Längst, Gernot

2013-01-01

Enhancers and promoters often contain multiple binding sites for the same transcription factor, suggesting that homotypic clustering of binding sites may serve a role in transcription regulation. Here we show that clustering of binding sites for the transcription termination factor TTF-I downstream of the pre-rRNA coding region specifies transcription termination, increases the efficiency of transcription initiation and affects the three-dimensional structure of rRNA genes. On chromatin templates, but not on free rDNA, clustered binding sites promote cooperative binding of TTF-I, loading TTF-I to the downstream terminators before it binds to the rDNA promoter. Interaction of TTF-I with target sites upstream and downstream of the rDNA transcription unit connects these distal DNA elements by forming a chromatin loop between the rDNA promoter and the terminators. The results imply that clustered binding sites increase the binding affinity of transcription factors in chromatin, thus influencing the timing and strength of DNA-dependent processes. PMID:24068958

Modulating nanoparticle superlattice structure using proteins with tunable bond distributions

DOE PAGES

McMillan, Janet R.; Brodin, Jeffrey D.; Millan, Jaime A.; ...

2017-01-25

Here, we investigate the use of proteins with tunable DNA modification distributions to modulate nanoparticle superlattice structure. Using Beta-galactosidase (βgal) as a model system, we have employed the orthogonal chemical reactivities of surface amines and thiols to synthesize protein-DNA conjugates with 36 evenly distributed or 8 specifically positioned oligonucleotides. When assembled into crystalline superlattices with AuNPs, we find that the distribution of DNA modifications modulates the favored structure: βgal with uniformly distributed DNA bonding elements results in body-centered cubic crystals, whereas DNA functionalization of cysteines results in AB 2 packing. We probe the role of protein oligonucleotide number and conjugatemore » size on this observation, which revealed the importance of oligonucleotide distribution and number in this observed assembly behavior. These results indicate that proteins with defined DNA-modification patterns are powerful tools to control the nanoparticle superlattices architecture, and establish the importance of oligonucleotide distribution in the assembly behavior of protein-DNA conjugates.« less

Specific interaction of the nonstructural protein NS1 of minute virus of mice (MVM) with [ACCA](2) motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication.

PubMed

Willwand, Kurt; Moroianu, Adela; Hörlein, Rita; Stremmel, Wolfgang; Rommelaere, Jean

2002-07-01

The linear single-stranded DNA genome of minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate DNA. Amplification of viral RF DNA requires the structural transition of the right-end palindrome from a linear duplex into a double-hairpin structure, which serves for the repriming of unidirectional DNA synthesis. This conformational transition was found previously to be induced by the MVM nonstructural protein NS1. Elimination of the cognate NS1-binding sites, [ACCA](2), from the central region of the right-end palindrome next to the axis of symmetry was shown to markedly reduce the efficiency of hairpin-primed DNA replication, as measured in a reconstituted in vitro replication system. Thus, [ACCA](2) sequence motifs are essential as NS1-binding elements in the context of the structural transition of the right-end MVM palindrome.

Isolation and characterization of active LINE and SINEs from the eel.

PubMed

Kajikawa, Masaki; Ichiyanagi, Kenji; Tanaka, Nozomu; Okada, Norihiro

2005-03-01

Long interspersed elements (LINEs) and short interspersed elements (SINEs) are retrotransposons. These elements can mobilize by the "copy-and-paste" mechanism, in which their own RNA is reverse-transcribed into complementary DNA (cDNA). LINEs and SINEs not only are components of eukaryotic genomes but also drivers of genomic evolution. Thus, studies of the amplification mechanism of LINEs and SINEs are important for understanding eukaryotic genome evolution. Here we report the characterization of one LINE family (UnaL2) and two SINE families (UnaSINE1 and UnaSINE2) from the eel (Anguilla japonica) genome. UnaL2 is approximately 3.6 kilobases (kb) and encodes only one open reading frame (ORF). UnaL2 belongs to the stringent type--thought to be a major group of LINEs--and can mobilize in HeLa cells. We also show that UnaL2 and the two UnaSINEs have similar 3' tails, and that both UnaSINE1 and UnaSINE2 can be mobilized by UnaL2 in HeLa cells. These elements are thus useful for delineating the amplification mechanism of stringent type LINEs as well as that of SINEs.

Identification of Regulatory DNA Elements Using Genome-wide Mapping of DNase I Hypersensitive Sites during Tomato Fruit Development.

PubMed

Qiu, Zhengkun; Li, Ren; Zhang, Shuaibin; Wang, Ketao; Xu, Meng; Li, Jiayang; Du, Yongchen; Yu, Hong; Cui, Xia

2016-08-01

Development and ripening of tomato fruit are precisely controlled by transcriptional regulation, which depends on the orchestrated accessibility of regulatory proteins to promoters and other cis-regulatory DNA elements. This accessibility and its effect on gene expression play a major role in defining the developmental process. To understand the regulatory mechanism and functional elements modulating morphological and anatomical changes during fruit development, we generated genome-wide high-resolution maps of DNase I hypersensitive sites (DHSs) from the fruit tissues of the tomato cultivar "Moneymaker" at 20 days post anthesis as well as break stage. By exploring variation of DHSs across fruit development stages, we pinpointed the most likely hypersensitive sites related to development-specific genes. By detecting binding motifs on DHSs of these development-specific genes or genes in the ascorbic acid biosynthetic pathway, we revealed the common regulatory elements contributing to coordinating gene transcription of plant ripening and specialized metabolic pathways. Our results contribute to a better understanding of the regulatory dynamics of genes involved in tomato fruit development and ripening. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Formation and loss of large, unstable tandem arrays of the piggyBac transposable element in the yellow fever mosquito, Aedes aegypti.

PubMed

Adelman, Zach N; Jasinskiene, Nijole; Vally, K J M; Peek, Corrie; Travanty, Emily A; Olson, Ken E; Brown, Susan E; Stephens, Janice L; Knudson, Dennis L; Coates, Craig J; James, Anthony A

2004-10-01

The Class II transposable element, piggyBac, was used to transform the yellow fever mosquito, Aedes aegypti. In two transformed lines only 15-30% of progeny inherited the transgene, with these individuals displaying mosaic expression of the EGFP marker gene. Southern analyses, gene amplification of genomic DNA, and plasmid rescue experiments provided evidence that these lines contained a high copy number of piggyBac transformation constructs and that much of this DNA consisted of both donor and helper plasmids. A detailed analysis of one line showed that the majority of piggyBac sequences were unit-length donor or helper plasmids arranged in a large tandem array that could be lost en masse in a single generation. Despite the presence of a transposase source and many intact donor elements, no conservative (cut and paste) transposition of piggyBac was observed in these lines. These results reveal one possible outcome of uncontrolled and/or unexpected recombination in this mosquito, and support the conclusion that further investigation is necessary before transposable elements such as piggyBac can be used as genetic drive mechanisms to move pathogen-resistance genes into mosquito populations.

The cis decoy against the estrogen response element suppresses breast cancer cells via target disrupting c-fos not mitogen-activated protein kinase activity.

PubMed

Wang, Li Hua; Yang, Xiao Yi; Zhang, Xiaohu; Mihalic, Kelly; Xiao, Weihua; Farrar, William L

2003-05-01

Breast cancer, the most common malignancy in women, has been demonstrated to be associated with the steroid hormone estrogen and its receptor (ER), a ligand-activated transcription factor. Therefore, we developed a phosphorothiolate cis-element decoy against the estrogen response element (ERE decoy) to target disruption of ER DNA binding and transcriptional activity. Here, we showed that the ERE decoy potently ablated the 17beta-estrogen-inducible cell proliferation and induced apoptosis of human breast carcinoma cells by functionally affecting expression of c-fos gene and AP-1 luciferase gene reporter activity. Specificity of the decoy was demonstrated by its ability to directly block ER binding to a cis-element probe and transactivation. Moreover, the decoy failed to inhibit ER-mediated mitogen-activated protein kinase signaling pathways and cell growth of ER-negative breast cancer cells. Taken together, these data suggest that estrogen-mediated cell growth of breast cancer cells can be preferentially restricted via targeted disruption of ER at the level of DNA binding by a novel and specific decoy strategy applied to steroid nuclear receptors.

Enzymatically Controlled Vacancies in Nanoparticle Crystals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barnaby, Stacey N.; Ross, Michael B.; Thaner, Ryan V.

In atomic systems, the mixing of metals results in distinct phase behavior that depends on the identity and bonding characteristics of the atoms. In nanoscale systems, the use of oligonucleotides as programmable “bonds” that link nanoparticle “atoms” into superlattices allows for the decoupling of atom identity and bonding. While much research in atomic systems is dedicated to understanding different phase behavior of mixed metals, it is not well understood on the nanoscale how changes in the nanoscale “bond” affect the phase behavior of nanoparticle crystals. In this work, the identity of the atom is kept the same but the chemicalmore » nature of the bond is altered, which is not possible in atomic systems, through the use of DNA and RNA bonding elements. These building blocks assemble into single crystal nanoparticle superlattices with mixed DNA and RNA bonding elements throughout. The nanoparticle crystals can be dynamically changed through the selective and enzymatic hydrolysis of the RNA bonding elements, resulting in superlattices that retain their crystalline structure and habit, while incorporating up to 35% random vacancies generated from the nanoparticles removed. Therefore, the bonding elements of nanoparticle crystals can be enzymatically and selectively addressed without affecting the nature of the atom.« less

Current strategies for mobilome research.

PubMed

Jørgensen, Tue S; Kiil, Anne S; Hansen, Martin A; Sørensen, Søren J; Hansen, Lars H

2014-01-01

Mobile genetic elements (MGEs) are pivotal for bacterial evolution and adaptation, allowing shuffling of genes even between distantly related bacterial species. The study of these elements is biologically interesting as the mode of genetic propagation is kaleidoscopic and important, as MGEs are the main vehicles of the increasing bacterial antibiotic resistance that causes thousands of human deaths each year. The study of MGEs has previously focused on plasmids from individual isolates, but the revolution in sequencing technology has allowed the study of mobile genomic elements of entire communities using metagenomic approaches. The problem in using metagenomic sequencing for the study of MGEs is that plasmids and other mobile elements only comprise a small fraction of the total genetic content that are difficult to separate from chromosomal DNA based on sequence alone. The distinction between plasmid and chromosome is important as the mobility and regulation of genes largely depend on their genetic context. Several different approaches have been proposed that specifically enrich plasmid DNA from community samples. Here, we review recent approaches used to study entire plasmid pools from complex environments, and point out possible future developments for and pitfalls of these approaches. Further, we discuss the use of the PacBio long-read sequencing technology for MGE discovery.

The targeting expression of the vascular endothelial growth factor gene in endothelial cells regulated by HRE.ppET-1.

PubMed

Zheng, Xiangrong; Zhang, Shangshang; Yang, Yujia; Wang, Xia; Zhong, Le; Yu, Xiaohe

2008-11-01

The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can deliver genes to target organs or cells selectively and efficiently with minimal toxicity. Here, we show that by using the HRE.ppET-1 regulatory element, we were able to restrict expression of the transgene of vascular endothelial growth factor (VEGF) to endothelial cells exclusively in hypoxic conditions. Eukaryotic expression vectors such as pEGFP-HRE.ppET-1, pcDNA3.1-VEGF+Pa, pcDNA3.1-ppET-1+ EGF+Pa, and pcDNA3.1-HRE.ppET-1+VEGF+Pa were constructed by using a series of nuclear molecule handling methods like PCR, enzyme digestion. The recombinant vectors were transfected into HUVEC cells and HL7702 cells by the lipofectin method. GFP expression was observed with a fluorescence microscope to validate the specificity of expression in endothelial cells under the regulation of HRE.ppET-1 element. Cobalt chloride (final concentration 100 mumol/L) was added to the medium to mimic hypoxia in vitro. After transfection of vectors, the expression of VEGF mRNA was detected by RT-PCR, and the expression of VEGF was detected by Western blotting and ELISA methods under normoxia and hypoxia, respectively. The cell proliferation rate was detected by the MTT test. The expression of GFP revealed that the exterior gene was transcripted effectively in endothelial cells regulated by the HRE.ppET-1 element, while the expression of GFP was very weak in nonendothelial cells. The results of RT-PCR, Western blotting and ELISA showed that VEGF gene expression in the pcDNA3.1-HRE.ppET-1+VEGF+Pa group and in the pcDNA3.1-ppET-1+VEGF+Pa group was higher in hypoxia than it was in normoxia (P<0.05). The MTT test showed that the proliferation rate of HUVEC transfected with HPVA under hypoxia exceeded that of the control group. We conclude that the HRE.ppET-1 element was expressed specifically in endothelial cells, and can increase the expression of VEGF in hypoxia and stimulate proliferation of endothelial cells. Taking advantage of these facts could greatly improve the efficiency of gene therapy. The vector would be valuable for various gene transfer studies targeting endothelial cells.

Characterization of three active transposable elements recently inserted in three independent DFR-A alleles and one high-copy DNA transposon isolated from the Pink allele of the ANS gene in onion (Allium cepa L.).

PubMed

Kim, Sunggil; Park, Jee Young; Yang, Tae-Jin

2015-06-01

Intact retrotransposon and DNA transposons inserted in a single gene were characterized in onions (Allium cepa) and their transcription and copy numbers were estimated in this study. While analyzing diverse onion germplasm, large insertions in the DFR-A gene encoding dihydroflavonol 4-reductase (DFR) involved in the anthocyanin biosynthesis pathway were found in two accessions. A 5,070-bp long terminal repeat (LTR) retrotransposon inserted in the active DFR-A (R4) allele was identified from one of the large insertions and designated AcCOPIA1. An intact ORF encoded typical domains of copia-like LTR retrotransposons. However, AcCOPIA1 contained atypical 'TG' and 'TA' dinucleotides at the ends of the LTRs. A 4,615-bp DNA transposon was identified in the other large insertion. This DNA transposon, designated AcCACTA1, contained an ORF coding for a transposase showing homology with the CACTA superfamily transposable elements (TEs). Another 5,073-bp DNA transposon was identified from the DFR-A (TRN) allele. This DNA transposon, designated AchAT1, belonged to the hAT superfamily with short 4-bp terminal inverted repeats (TIRs). Finally, a 6,258-bp non-autonomous DNA transposon, designated AcPINK, was identified in the ANS-p allele encoding anthocyanidin synthase, the next downstream enzyme to DFR in the anthocyanin biosynthesis pathway. AcPINK also possessed very short 3-bp TIRs. Active transcription of AcCOPIA1, AcCACTA1, and AchAT1 was observed through RNA-Seq analysis and RT-PCR. The copy numbers of AcPINK estimated by mapping the genomic DNA reads produced by NextSeq 500 were predominantly high compared with the other TEs. A series of evidence indicated that these TEs might have transposed in these onion genes very recently, providing a stepping stone for elucidation of enormously large-sized onion genome structure.

Fine organization of genomic regions tagged to the 5S rDNA locus of the bread wheat 5B chromosome.

PubMed

Sergeeva, Ekaterina M; Shcherban, Andrey B; Adonina, Irina G; Nesterov, Michail A; Beletsky, Alexey V; Rakitin, Andrey L; Mardanov, Andrey V; Ravin, Nikolai V; Salina, Elena A

2017-11-14

The multigene family encoding the 5S rRNA, one of the most important structurally-functional part of the large ribosomal subunit, is an obligate component of all eukaryotic genomes. 5S rDNA has long been a favored target for cytological and phylogenetic studies due to the inherent peculiarities of its structural organization, such as the tandem arrays of repetitive units and their high interspecific divergence. The complex polyploid nature of the genome of bread wheat, Triticum aestivum, and the technically difficult task of sequencing clusters of tandem repeats mean that the detailed organization of extended genomic regions containing 5S rRNA genes remains unclear. This is despite the recent progress made in wheat genomic sequencing. Using pyrosequencing of BAC clones, in this work we studied the organization of two distinct 5S rDNA-tagged regions of the 5BS chromosome of bread wheat. Three BAC-clones containing 5S rDNA were identified in the 5BS chromosome-specific BAC-library of Triticum aestivum. Using the results of pyrosequencing and assembling, we obtained six 5S rDNA- containing contigs with a total length of 140,417 bp, and two sets (pools) of individual 5S rDNA sequences belonging to separate, but closely located genomic regions on the 5BS chromosome. Both regions are characterized by the presence of approximately 70-80 copies of 5S rDNA, however, they are completely different in their structural organization. The first region contained highly diverged short-type 5S rDNA units that were disrupted by multiple insertions of transposable elements. The second region contained the more conserved long-type 5S rDNA, organized as a single tandem array. FISH using probes specific to both 5S rDNA unit types showed differences in the distribution and intensity of signals on the chromosomes of polyploid wheat species and their diploid progenitors. A detailed structural organization of two closely located 5S rDNA-tagged genomic regions on the 5BS chromosome of bread wheat has been established. These two regions differ in the organization of both 5S rDNA and the neighboring sequences comprised of transposable elements, implying different modes of evolution for these regions.

Development and Synthesis of DNA-Encoded Benzimidazole Library.

PubMed

Ding, Yun; Chai, Jing; Centrella, Paolo A; Gondo, Chenaimwoyo; DeLorey, Jennifer L; Clark, Matthew A

2018-04-25

Encoded library technology (ELT) is an effective approach to the discovery of novel small-molecule ligands for biological targets. A key factor for the success of the technology is the chemical diversity of the libraries. Here we report the development of DNA-conjugated benzimidazoles. Using 4-fluoro-3-nitrobenzoic acid as a key synthon, we synthesized a 320 million-member DNA-encoded benzimidazole library using Fmoc-protected amino acids, amines and aldehydes as diversity elements. Affinity selection of the library led to the discovery of a novel, potent and specific antagonist of the NK3 receptor.

A Locus Encoding Variable Defense Systems against Invading DNA Identified in Streptococcus suis

PubMed Central

Okura, Masatoshi; Nozawa, Takashi; Watanabe, Takayasu; Murase, Kazunori; Nakagawa, Ichiro; Takamatsu, Daisuke; Osaki, Makoto; Sekizaki, Tsutomu; Gottschalk, Marcelo; Hamada, Shigeyuki

2017-01-01

Streptococcus suis, an important zoonotic pathogen, is known to have an open pan-genome and to develop a competent state. In S. suis, limited genetic lineages are suggested to be associated with zoonosis. However, little is known about the evolution of diversified lineages and their respective phenotypic or ecological characteristics. In this study, we performed comparative genome analyses of S. suis, with a focus on the competence genes, mobile genetic elements, and genetic elements related to various defense systems against exogenous DNAs (defense elements) that are associated with gene gain/loss/exchange mediated by horizontal DNA movements and their restrictions. Our genome analyses revealed a conserved competence-inducing peptide type (pherotype) of the competence system and large-scale genome rearrangements in certain clusters based on the genome phylogeny of 58 S. suis strains. Moreover, the profiles of the defense elements were similar or identical to each other among the strains belonging to the same genomic clusters. Our findings suggest that these genetic characteristics of each cluster might exert specific effects on the phenotypic or ecological differences between the clusters. We also found certain loci that shift several types of defense elements in S. suis. Of note, one of these loci is a previously unrecognized variable region in bacteria, at which strains of distinct clusters code for different and various defense elements. This locus might represent a novel defense mechanism that has evolved through an arms race between bacteria and invading DNAs, mediated by mobile genetic elements and genetic competence. PMID:28379509

Effects of a Transposable Element Insertion on Alcohol Dehydrogenase Expression in Drosophila Melanogaster

PubMed Central

Dunn, R. C.; Laurie, C. C.

1995-01-01

Variation in the DNA sequence and level of alcohol dehydrogenase (Adh) gene expression in Drosophila melanogaster have been studied to determine what types of DNA polymorphisms contribute to phenotypic variation in natural populations. The Adh gene, like many others, shows a high level of variability in both DNA sequence and quantitative level of expression. A number of transposable element insertions occur in the Adh region and one of these, a copia insertion in the 5' flanking region, is associated with unusually low Adh expression. To determine whether this insertion (called RI42) causes the low expression level, the insertion was excised from the cloned RI42 Adh gene and the effect was assessed by P-element transformation. Removal of this insertion causes a threefold increase in the level of ADH, clearly showing that it contributes to the naturally occurring variation in expression at this locus. Removal of all but one LTR also causes a threefold increase, indicating that the mechanism is not a simple sequence disruption. Furthermore, this copia insertion, which is located between the two Adh promoters and their upstream enhancer sequences, has differential effects on the levels of proximal and distal transcripts. Finally, a test for the possible modifying effects of two suppressor loci, su(w(a)) and su(f), on this insertional mutation was negative, in contrast to a previous report in the literature. PMID:7498745

Robust k-mer frequency estimation using gapped k-mers

PubMed Central

Ghandi, Mahmoud; Mohammad-Noori, Morteza

2013-01-01

Oligomers of fixed length, k, commonly known as k-mers, are often used as fundamental elements in the description of DNA sequence features of diverse biological function, or as intermediate elements in the constuction of more complex descriptors of sequence features such as position weight matrices. k-mers are very useful as general sequence features because they constitute a complete and unbiased feature set, and do not require parameterization based on incomplete knowledge of biological mechanisms. However, a fundamental limitation in the use of k-mers as sequence features is that as k is increased, larger spatial correlations in DNA sequence elements can be described, but the frequency of observing any specific k-mer becomes very small, and rapidly approaches a sparse matrix of binary counts. Thus any statistical learning approach using k-mers will be susceptible to noisy estimation of k-mer frequencies once k becomes large. Because all molecular DNA interactions have limited spatial extent, gapped k-mers often carry the relevant biological signal. Here we use gapped k-mer counts to more robustly estimate the ungapped k-mer frequencies, by deriving an equation for the minimum norm estimate of k-mer frequencies given an observed set of gapped k-mer frequencies. We demonstrate that this approach provides a more accurate estimate of the k-mer frequencies in real biological sequences using a sample of CTCF binding sites in the human genome. PMID:23861010

Robust k-mer frequency estimation using gapped k-mers.

PubMed

Ghandi, Mahmoud; Mohammad-Noori, Morteza; Beer, Michael A

2014-08-01

Oligomers of fixed length, k, commonly known as k-mers, are often used as fundamental elements in the description of DNA sequence features of diverse biological function, or as intermediate elements in the constuction of more complex descriptors of sequence features such as position weight matrices. k-mers are very useful as general sequence features because they constitute a complete and unbiased feature set, and do not require parameterization based on incomplete knowledge of biological mechanisms. However, a fundamental limitation in the use of k-mers as sequence features is that as k is increased, larger spatial correlations in DNA sequence elements can be described, but the frequency of observing any specific k-mer becomes very small, and rapidly approaches a sparse matrix of binary counts. Thus any statistical learning approach using k-mers will be susceptible to noisy estimation of k-mer frequencies once k becomes large. Because all molecular DNA interactions have limited spatial extent, gapped k-mers often carry the relevant biological signal. Here we use gapped k-mer counts to more robustly estimate the ungapped k-mer frequencies, by deriving an equation for the minimum norm estimate of k-mer frequencies given an observed set of gapped k-mer frequencies. We demonstrate that this approach provides a more accurate estimate of the k-mer frequencies in real biological sequences using a sample of CTCF binding sites in the human genome.

Transcriptional activation of human mu-opioid receptor gene by insulin-like growth factor-I in neuronal cells is modulated by the transcription factor REST.

PubMed

Bedini, Andrea; Baiula, Monica; Spampinato, Santi

2008-06-01

The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. We investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I up-regulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 signaling pathway and this transcription factor, binding to the signal transducer and activator of transcription-1/3 DNA element located in the promoter, increases OPRM1 transcription. We propose that a reduction in REST is a critical switch enabling IGF-I to up-regulate hMOPr. These findings help clarify how hMOPr expression is regulated in neuronal cells.

Evolutionary genomics of miniature inverted-repeat transposable elements (MITEs) in Brassica.

PubMed

Nouroz, Faisal; Noreen, Shumaila; Heslop-Harrison, J S

2015-12-01

Miniature inverted-repeat transposable elements (MITEs) are truncated derivatives of autonomous DNA transposons, and are dispersed abundantly in most eukaryotic genomes. We aimed to characterize various MITEs families in Brassica in terms of their presence, sequence characteristics and evolutionary activity. Dot plot analyses involving comparison of homoeologous bacterial artificial chromosome (BAC) sequences allowed identification of 15 novel families of mobile MITEs. Of which, 5 were Stowaway-like with TA Target Site Duplications (TSDs), 4 Tourist-like with TAA/TTA TSDs, 5 Mutator-like with 9-10 bp TSDs and 1 novel MITE (BoXMITE1) flanked by 3 bp TSDs. Our data suggested that there are about 30,000 MITE-related sequences in Brassica rapa and B. oleracea genomes. In situ hybridization showed one abundant family was dispersed in the A-genome, while another was located near 45S rDNA sites. PCR analysis using primers flanking sequences of MITE elements detected MITE insertion polymorphisms between and within the three Brassica (AA, BB, CC) genomes, with many insertions being specific to single genomes and others showing evidence of more recent evolutionary insertions. Our BAC sequence comparison strategy enables identification of evolutionarily active MITEs with no prior knowledge of MITE sequences. The details of MITE families reported in Brassica enable their identification, characterization and annotation. Insertion polymorphisms of MITEs and their transposition activity indicated important mechanism of genome evolution and diversification. MITE families derived from known Mariner, Harbinger and Mutator DNA transposons were discovered, as well as some novel structures. The identification of Brassica MITEs will have broad applications in Brassica genomics, breeding, hybridization and phylogeny through their use as DNA markers.

Estimates of electronic coupling for excess electron transfer in DNA

NASA Astrophysics Data System (ADS)

Voityuk, Alexander A.

2005-07-01

Electronic coupling Vda is one of the key parameters that determine the rate of charge transfer through DNA. While there have been several computational studies of Vda for hole transfer, estimates of electronic couplings for excess electron transfer (ET) in DNA remain unavailable. In the paper, an efficient strategy is established for calculating the ET matrix elements between base pairs in a π stack. Two approaches are considered. First, we employ the diabatic-state (DS) method in which donor and acceptor are represented with radical anions of the canonical base pairs adenine-thymine (AT) and guanine-cytosine (GC). In this approach, similar values of Vda are obtained with the standard 6-31G* and extended 6-31++G** basis sets. Second, the electronic couplings are derived from lowest unoccupied molecular orbitals (LUMOs) of neutral systems by using the generalized Mulliken-Hush or fragment charge methods. Because the radical-anion states of AT and GC are well reproduced by LUMOs of the neutral base pairs calculated without diffuse functions, the estimated values of Vda are in good agreement with the couplings obtained for radical-anion states using the DS method. However, when the calculation of a neutral stack is carried out with diffuse functions, LUMOs of the system exhibit the dipole-bound character and cannot be used for estimating electronic couplings. Our calculations suggest that the ET matrix elements Vda for models containing intrastrand thymine and cytosine bases are essentially larger than the couplings in complexes with interstrand pyrimidine bases. The matrix elements for excess electron transfer are found to be considerably smaller than the corresponding values for hole transfer and to be very responsive to structural changes in a DNA stack.

An Ancient Transkingdom Horizontal Transfer of Penelope-Like Retroelements from Arthropods to Conifers.

PubMed

Lin, Xuan; Faridi, Nurul; Casola, Claudio

2016-05-02

Comparative genomics analyses empowered by the wealth of sequenced genomes have revealed numerous instances of horizontal DNA transfers between distantly related species. In eukaryotes, repetitive DNA sequences known as transposable elements (TEs) are especially prone to move across species boundaries. Such horizontal transposon transfers, or HTTs, are relatively common within major eukaryotic kingdoms, including animals, plants, and fungi, while rarely occurring across these kingdoms. Here, we describe the first case of HTT from animals to plants, involving TEs known as Penelope-like elements, or PLEs, a group of retrotransposons closely related to eukaryotic telomerases. Using a combination of in situ hybridization on chromosomes, polymerase chain reaction experiments, and computational analyses we show that the predominant PLE lineage, EN(+)PLEs, is highly diversified in loblolly pine and other conifers, but appears to be absent in other gymnosperms. Phylogenetic analyses of both protein and DNA sequences reveal that conifers EN(+)PLEs, or Dryads, form a monophyletic group clustering within a clade of primarily arthropod elements. Additionally, no EN(+)PLEs were detected in 1,928 genome assemblies from 1,029 nonmetazoan and nonconifer genomes from 14 major eukaryotic lineages. These findings indicate that Dryads emerged following an ancient horizontal transfer of EN(+)PLEs from arthropods to a common ancestor of conifers approximately 340 Ma. This represents one of the oldest known interspecific transmissions of TEs, and the most conspicuous case of DNA transfer between animals and plants. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2016. This work is written by US Government employees and is in the public domain in the US.

NF-κB– and AP-1–Mediated DNA Looping Regulates Osteopontin Transcription in Endotoxin-Stimulated Murine Macrophages

PubMed Central

Zhao, Wei; Wang, Lijuan; Zhang, Meng; Wang, Peng; Zhang, Lei; Yuan, Chao; Qi, Jianni; Qiao, Yu; Kuo, Paul C.; Gao, Chengjiang

2013-01-01

Osteopontin (OPN) is expressed by various immune cells and modulates both innate and adaptive immune responses. However, the molecular mechanisms that control opn gene expression, especially at the chromatin level, remain largely unknown. We have previously demonstrated many specific cis- and trans-regulatory elements that determine the extent of endotoxin (LPS)-mediated induction of OPN synthesis in murine macrophages. In the present study, we confirm that NF-κB also plays an important role in the setting of LPS-stimulated OPN expression through binding to a distal regulatory element. Importantly, we demonstrate that LPS stimulates chromosomal loops in the OPN promoter between NF-κB binding site and AP-1 binding site using chromosome conformation capture technology. The crucial role of NF-κB and AP-1 in LPS-stimulated DNA looping was confirmed, as small interfering RNA knock-down of NF-κB p65 and AP-1 c-Jun exhibited decreased levels of DNA looping. Furthermore, we demonstrate that p300 can form a complex with NF-κB and AP-1 and is involved in DNA looping and LPS-induced OPN expression. Therefore, we have identified an essential mechanism to remodel the local chromatin structures and spatial conformations to regulate LPS-induced OPN expression. PMID:21257959

Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts

PubMed Central

Hurst, Gregory D.D; Jiggins, Francis M

2005-01-01

Mitochondrial DNA (mtDNA) has been a marker of choice for reconstructing historical patterns of population demography, admixture, biogeography and speciation. However, it has recently been suggested that the pervasive nature of direct and indirect selection on this molecule renders any conclusion derived from it ambiguous. We review here the evidence for indirect selection on mtDNA in arthropods arising from linkage disequilibrium with maternally inherited symbionts. We note first that these symbionts are very common in arthropods and then review studies that reveal the extent to which they shape mtDNA evolution. mtDNA diversity patterns are compatible with neutral expectations for an uninfected population in only 2 of 19 cases. The remaining 17 studies revealed cases of symbiont-driven reduction in mtDNA diversity, symbiont-driven increases in diversity, symbiont-driven changes in mtDNA variation over space and symbiont-associated paraphyly of mtDNA. We therefore conclude that these elements often confound the inference of an organism's evolutionary history from mtDNA data and that mtDNA on its own is an unsuitable marker for the study of recent historical events in arthropods. We also discuss the impact of these studies on the current programme of taxonomy based on DNA bar-coding. PMID:16048766

Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms.

PubMed

Martins, Margarida; Uppuluri, Priya; Thomas, Derek P; Cleary, Ian A; Henriques, Mariana; Lopez-Ribot, José L; Oliveira, Rosário

2010-05-01

DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.

Structural rearrangements in the mitochondrial genome of Drosophila melanogaster induced by elevated levels of the replicative DNA helicase

PubMed Central

Ciesielski, Grzegorz L; Nadalutti, Cristina A; Oliveira, Marcos T; Griffith, Jack D; Kaguni, Laurie S

2018-01-01

Abstract Pathological conditions impairing functions of mitochondria often lead to compensatory upregulation of the mitochondrial DNA (mtDNA) replisome machinery, and the replicative DNA helicase appears to be a key factor in regulating mtDNA copy number. Moreover, mtDNA helicase mutations have been associated with structural rearrangements of the mitochondrial genome. To evaluate the effects of elevated levels of the mtDNA helicase on the integrity and replication of the mitochondrial genome, we overexpressed the helicase in Drosophila melanogaster Schneider cells and analyzed the mtDNA by two-dimensional neutral agarose gel electrophoresis and electron microscopy. We found that elevation of mtDNA helicase levels increases the quantity of replication intermediates and alleviates pausing at the replication slow zones. Though we did not observe a concomitant alteration in mtDNA copy number, we observed deletions specific to the segment of repeated elements in the immediate vicinity of the origin of replication, and an accumulation of species characteristic of replication fork stalling. We also found elevated levels of RNA that are retained in the replication intermediates. Together, our results suggest that upregulation of mtDNA helicase promotes the process of mtDNA replication but also results in genome destabilization. PMID:29432582

A selective and label-free strategy for rapid screening of telomere-binding Ligands via fluorescence regulation of DNA/silver nanocluster

NASA Astrophysics Data System (ADS)

Cheng, Rui; Xu, Jing; Zhang, Xiafei; Shi, Zhilu; Zhang, Qi; Jin, Yan

2017-03-01

Herein, the conformational switch of G-rich oligonucleotide (GDNA) demonstrated the obvious functional switch of GDNA which was found to significantly affect the fluorescence of the in-situ synthesized DNA/silver nanocluster (DNA-AgNC) in homogeneous solution. We envisioned that the allosteric interaction between GDNA and DNA-AgNC would be possible to be used for screening telomere-binding ligands. A unimolecular probe (12C5TG) is ingeniously designed consisting of three contiguous DNA elements: G-rich telomeric DNA (GDNA) as molecular recognition sequence, T-rich DNA as linker and C-rich DNA as template of DNA-AgNC. The quantum yield and stability of 12C5TG-AgNC is greatly improved because the nearby deoxyguanosines tended to protect DNA/AgNC against oxidation. However, in the presence of ligands, the formation of G-quadruplex obviously quenched the fluorescence of DNA-AgNC. By taking full advantage of intramolecular allosteric effect, telomere-binding ligands were selectively and label-free screened by using deoxyguanines and G-quadruplex as natural fluorescence enhancer and quencher of DNA-AgNC respectively. Therefore, the functional switching of G-rich structure offers a cost-effective, facile and reliable way to screen drugs, which holds a great potential in bioanalysis as well.

An Evolutionary Classification of Genomic Function

PubMed Central

Graur, Dan; Zheng, Yichen; Azevedo, Ricardo B.R.

2015-01-01

The pronouncements of the ENCODE Project Consortium regarding “junk DNA” exposed the need for an evolutionary classification of genomic elements according to their selected-effect function. In the classification scheme presented here, we divide the genome into “functional DNA,” that is, DNA sequences that have a selected-effect function, and “rubbish DNA,” that is, sequences that do not. Functional DNA is further subdivided into “literal DNA” and “indifferent DNA.” In literal DNA, the order of nucleotides is under selection; in indifferent DNA, only the presence or absence of the sequence is under selection. Rubbish DNA is further subdivided into “junk DNA” and “garbage DNA.” Junk DNA neither contributes to nor detracts from the fitness of the organism and, hence, evolves under selective neutrality. Garbage DNA, on the other hand, decreases the fitness of its carriers. Garbage DNA exists in the genome only because natural selection is neither omnipotent nor instantaneous. Each of these four functional categories can be 1) transcribed and translated, 2) transcribed but not translated, or 3) not transcribed. The affiliation of a DNA segment to a particular functional category may change during evolution: Functional DNA may become junk DNA, junk DNA may become garbage DNA, rubbish DNA may become functional DNA, and so on; however, determining the functionality or nonfunctionality of a genomic sequence must be based on its present status rather than on its potential to change (or not to change) in the future. Changes in functional affiliation are divided into pseudogenes, Lazarus DNA, zombie DNA, and Jekyll-to-Hyde DNA. PMID:25635041

[Structural organization of 5S ribosomal DNA of Rosa rugosa].

PubMed

Tynkevych, Iu O; Volkov, R A

2014-01-01

In order to clarify molecular organization of the genomic region encoding 5S rRNA in diploid species Rosa rugosa several 5S rDNA repeated units were cloned and sequenced. Analysis of the obtained sequences revealed that only one length variant of 5S rDNA repeated units, which contains intact promoter elements in the intergenic spacer region (IGS) and appears to be transcriptionally active is present in the genome. Additionally, a limited number of 5S rDNA pseudogenes lacking a portion of coding sequence and the complete IGS was detected. A high level of sequence similarity (from 93.7 to 97.5%) between the IGS of major 5S rDNA variants of East Asian R. rugosa and North American R. nitida was found indicating comparatively recent divergence of these species.

Allosteric transcriptional regulation via changes in the overall topology of the core promoter

DOE PAGES

Philips, Steven J.; Canalizo-Hernandez, Monica; Yildirim, Ilyas; ...

2015-08-21

Many transcriptional activators act at a distance from core promoter elements and work by recruiting RNA polymerase through protein-protein interactions. We show here how the prokaryotic regulatory protein CueR both represses and activates transcription by differentially modulating local DNA structure within the promoter. Structural studies reveal that the repressor state slightly bends the promoter DNA, precluding optimal RNA polymerase-promoter recognition. Upon binding a metal ion in the allosteric site, CueR switches into an activator conformation. It maintains all protein-DNA contacts but introduces torsional stresses that kink and undertwist the promoter, stabilizing an A-DNA-like conformation. Finally, these factors switch on andmore » off transcription by exerting dynamic control of DNA stereochemistry, reshaping the core promoter and making it a better or worse substrate for polymerase.« less

Comparison of the gentamicin resistance transposon Tn5281 with regions encoding gentamicin resistance in Enterococcus faecalis isolates from diverse geographic locations.

PubMed Central

Hodel-Christian, S L; Murray, B E

1992-01-01

The genetic determinant encoding gentamicin resistance (Gmr) on the beta-lactamase encoding plasmid pBEM10 of Enterococcus faecalis HH22 is carried on a transposon, termed Tn5281, that is highly related to the staphylococcal Gmr transposons Tn4001 found in Australian isolates of Staphylococcus aureus and Tn4031 found in United States isolates of Staphylococcus epidermidis. We have now studied plasmid DNA from Gmr strains of E. faecalis isolated from diverse geographical locations (Houston, Pennsylvania, Thailand, and Chile) by using restriction endonuclease analysis and DNA-DNA hybridization to determine whether other Gmr E. faecalis carry Tn5281 or a similar type of element. We also compared these enterococci to several United States isolates of Staphylococcus aureus with nonmobile Gmr determinants. Three E. faecalis isolates (from Houston and Chile) carried Tn5281-like elements, whereas two isolates (from Houston and Pennsylvania) had restriction endonuclease and DNA-DNA hybridization patterns more similar to those of the Tn4001-IS257 hybrid found in the nonmobile Gmr determinants in United States isolates of S. aureus. A strain from Thailand had a third pattern unrelated to either Tn5281 or the nonmobile Gmr determinants present in United States isolates of S. aureus. Our results demonstrate that there is both similarity and diversity between the Gmr determinant of strains of E. faecalis isolated in diverse geographic locations. Images PMID:1332593

HDA6 directly interacts with DNA methyltransferase MET1 and maintains transposable element silencing in Arabidopsis.

PubMed

Liu, Xuncheng; Yu, Chun-Wei; Duan, Jun; Luo, Ming; Wang, Koching; Tian, Gang; Cui, Yuhai; Wu, Keqiang

2012-01-01

The molecular mechanism of how the histone deacetylase HDA6 participates in maintaining transposable element (TE) silencing in Arabidopsis (Arabidopsis thaliana) is not yet defined. In this study, we show that a subset of TEs was transcriptionally reactivated and that TE reactivation was associated with elevated histone H3 and H4 acetylation as well as increased H3K4Me3 and H3K4Me2 in hda6 mutants. Decreased DNA methylation of the TEs was also detected in hda6 mutants, suggesting that HDA6 silences the TEs by regulating histone acetylation and methylation as well as the DNA methylation status of the TEs. Similarly, transcripts of some of these TEs were also increased in the methyltransferase1 (met1) mutant, with decreased DNA methylation. Furthermore, H4 acetylation, H3K4Me3, H3K4Me2, and H3K36Me2 were enriched at the coregulated TEs in the met1 and hda6 met1 mutants. Protein-protein interaction analysis indicated that HDA6 physically interacts with MET1 in vitro and in vivo, and further deletion analysis demonstrated that the carboxyl-terminal region of HDA6 and the bromo-adjacent homology domain of MET1 were responsible for the interaction. These results suggested that HDA6 and MET1 interact directly and act together to silence TEs by modulating DNA methylation, histone acetylation, and histone methylation status.

DNA AND THE FINE STRUCTURE OF SYNAPTIC CHROMOSOMES IN THE DOMESTIC ROOSTER (GALLUS DOMESTICUS)

PubMed Central

Coleman, James R.; Moses, Montrose J.

1964-01-01

The indium trichloride method of Watson and Aldridge (38) for staining nucleic acids for electron microscopy was employed to study the relationship of DNA to the structure of the synaptinemal complex in meiotic prophase chromosomes of the domestic rooster. The selectivity of the method was demonstrated in untreated and DNase-digested testis material by comparing the distribution of indium staining in the electron microscope to Feulgen staining and ultraviolet absorption in thicker sections seen with the light microscope. Following staining by indium, DNA was found mainly in the microfibril component of the synaptinemal complex. When DNA was known to have been removed from aldehyde-fixed material by digestion with DNase, indium stainability was also lost. However, staining of the digested material with non-selective heavy metal techniques demonstrated the presence of material other than DNA in the microfibrils and showed that little alteration in appearance of the chromosome resulted from DNA removal. The two dense lateral axial elements of the synaptinemal complex, but not the central one to any extent, also contained DNA, together with non-DNA material. PMID:14228519

Role for a region of helically unstable DNA within the Epstein-Barr virus latent cycle origin of DNA replication oriP in origin function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Polonskaya, Zhanna; Benham, Craig J.; Hearing, Janet

The minimal replicator of the Epstein-Barr virus (EBV) latent cycle origin of DNA replication oriP is composed of two binding sites for the Epstein-Barr virus nuclear antigen-1 (EBNA-1) and flanking inverted repeats that bind the telomere repeat binding factor TRF2. Although not required for minimal replicator activity, additional binding sites for EBNA-1 and TRF2 and one or more auxiliary elements located to the right of the EBNA-1/TRF2 sites are required for the efficient replication of oriP plasmids. Another region of oriP that is predicted to be destabilized by DNA supercoiling is shown here to be an important functional component ofmore » oriP. The ability of DNA fragments of unrelated sequence and possessing supercoiled-induced DNA duplex destabilized (SIDD) structures, but not fragments characterized by helically stable DNA, to substitute for this component of oriP demonstrates a role for the SIDD region in the initiation of oriP-plasmid DNA replication.« less

Architecture and DNA Recognition Elements of the Fanconi Anemia FANCM-FAAP24 Complex

PubMed Central

Coulthard, Rachel; Deans, Andrew J.; Swuec, Paolo; Bowles, Maureen; Costa, Alessandro; West, Stephen C.; McDonald, Neil Q.

2013-01-01

Summary Fanconi anemia (FA) is a disorder associated with a failure in DNA repair. FANCM (defective in FA complementation group M) and its partner FAAP24 target other FA proteins to sites of DNA damage. FANCM-FAAP24 is related to XPF/MUS81 endonucleases but lacks endonucleolytic activity. We report a structure of an FANCM C-terminal fragment (FANCMCTD) bound to FAAP24 and DNA. This S-shaped structure reveals the FANCM (HhH)2 domain is buried, whereas the FAAP24 (HhH)2 domain engages DNA. We identify a second DNA contact and a metal center within the FANCM pseudo-nuclease domain and demonstrate that mutations in either region impair double-stranded DNA binding in vitro and FANCM-FAAP24 function in vivo. We show the FANCM translocase domain lies in proximity to FANCMCTD by electron microscopy and that binding fork DNA structures stimulate its ATPase activity. This suggests a tracking model for FANCM-FAAP24 until an encounter with a stalled replication fork triggers ATPase-mediated fork remodeling. PMID:23932590

Design of copper DNA intercalators with leishmanicidal activity.

PubMed

Navarro, Maribel; Cisneros-Fajardo, Efrén José; Sierralta, Aníbal; Fernández-Mestre, Mercedes; Silva, Pedro; Arrieche, Dwight; Marchán, Edgar

2003-04-01

The complexes [Cu(dppz)(NO(3))]NO(3) (1), [Cu(dppz)(2)(NO(3))]NO(3) (2), [Cu(dpq)(NO(3))]NO(3) (3), and [Cu(dpq)(2)(NO(3))]NO(3) (4) were synthesized and characterized by elemental analysis, FAB-mass spectrometry, EPR, UV, and IR spectroscopies, and molar conductivity. DNA interaction studies showed that intercalation is an important way of interacting with DNA for these complexes. The biological activity of these copper complexes was evaluated on Leishmania braziliensis promastigotes, and the results showed leishmanicidal activity. Preliminary ultrastructural studies with the most active complex (2) at 1 h revealed parasite swelling and binucleated cells. This finding suggests that the leishmanicidal activity of the copper complexes could be associated with their interaction with the parasitic DNA.

Computational Micromodel for Epigenetic Mechanisms

PubMed Central

Raghavan, Karthika; Ruskin, Heather J.; Perrin, Dimitri; Goasmat, Francois; Burns, John

2010-01-01

Characterization of the epigenetic profile of humans since the initial breakthrough on the human genome project has strongly established the key role of histone modifications and DNA methylation. These dynamic elements interact to determine the normal level of expression or methylation status of the constituent genes in the genome. Recently, considerable evidence has been put forward to demonstrate that environmental stress implicitly alters epigenetic patterns causing imbalance that can lead to cancer initiation. This chain of consequences has motivated attempts to computationally model the influence of histone modification and DNA methylation in gene expression and investigate their intrinsic interdependency. In this paper, we explore the relation between DNA methylation and transcription and characterize in detail the histone modifications for specific DNA methylation levels using a stochastic approach. PMID:21152421

A DNA sequence element that advances replication origin activation time in Saccharomyces cerevisiae.

PubMed

Pohl, Thomas J; Kolor, Katherine; Fangman, Walton L; Brewer, Bonita J; Raghuraman, M K

2013-11-06

Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.

Alternative DNA structure formation in the mutagenic human c-MYC promoter

PubMed Central

del Mundo, Imee Marie A.; Zewail-Foote, Maha; Kerwin, Sean M.

2017-01-01

Abstract Mutation ‘hotspot’ regions in the genome are susceptible to genetic instability, implicating them in diseases. These hotspots are not random and often co-localize with DNA sequences potentially capable of adopting alternative DNA structures (non-B DNA, e.g. H-DNA and G4-DNA), which have been identified as endogenous sources of genomic instability. There are regions that contain overlapping sequences that may form more than one non-B DNA structure. The extent to which one structure impacts the formation/stability of another, within the sequence, is not fully understood. To address this issue, we investigated the folding preferences of oligonucleotides from a chromosomal breakpoint hotspot in the human c-MYC oncogene containing both potential G4-forming and H-DNA-forming elements. We characterized the structures formed in the presence of G4-DNA-stabilizing K+ ions or H-DNA-stabilizing Mg2+ ions using multiple techniques. We found that under conditions favorable for H-DNA formation, a stable intramolecular triplex DNA structure predominated; whereas, under K+-rich, G4-DNA-forming conditions, a plurality of unfolded and folded species were present. Thus, within a limited region containing sequences with the potential to adopt multiple structures, only one structure predominates under a given condition. The predominance of H-DNA implicates this structure in the instability associated with the human c-MYC oncogene. PMID:28334873

Molecular genotyping of Colletotrichum species based on arbitrarily primed PCR, A + T-Rich DNA, and nuclear DNA analyses

USGS Publications Warehouse

Freeman, S.; Pham, M.; Rodriguez, R.J.

1993-01-01

Molecular genotyping of Colletotrichum species based on arbitrarily primed PCR, A + T-rich DNA, and nuclear DNA analyses. Experimental Mycology 17, 309-322. Isolates of Colletotrichum were grouped into 10 separate species based on arbitrarily primed PCR (ap-PCR), A + T-rich DNA (AT-DNA) and nuclear DNA banding patterns. In general, the grouping of Colletotrichum isolates by these molecular approaches corresponded to that done by classical taxonomic identification, however, some exceptions were observed. PCR amplification of genomic DNA using four different primers allowed for reliable differentiation between isolates of the 10 species. HaeIII digestion patterns of AT-DNA also distinguished between species of Colletotrichum by generating species-specific band patterns. In addition, hybridization of the repetitive DNA element (GcpR1) to genomic DNA identified a unique set of Pst 1-digested nuclear DNA fragments in each of the 10 species of Colletotrichum tested. Multiple isolates of C. acutatum, C. coccodes, C. fragariae, C. lindemuthianum, C. magna, C. orbiculare, C. graminicola from maize, and C. graminicola from sorghum showed 86-100% intraspecies similarity based on ap-PCR and AT-DNA analyses. Interspecies similarity determined by ap-PCR and AT-DNA analyses varied between 0 and 33%. Three distinct banding patterns were detected in isolates of C. gloeosporioides from strawberry. Similarly, three different banding patterns were observed among isolates of C. musae from diseased banana.

Behavior of restriction–modification systems as selfish mobile elements and their impact on genome evolution

PubMed Central

Kobayashi, Ichizo

2001-01-01

Restriction–modification (RM) systems are composed of genes that encode a restriction enzyme and a modification methylase. RM systems sometimes behave as discrete units of life, like viruses and transposons. RM complexes attack invading DNA that has not been properly modified and thus may serve as a tool of defense for bacterial cells. However, any threat to their maintenance, such as a challenge by a competing genetic element (an incompatible plasmid or an allelic homologous stretch of DNA, for example) can lead to cell death through restriction breakage in the genome. This post-segregational or post-disturbance cell killing may provide the RM complexes (and any DNA linked with them) with a competitive advantage. There is evidence that they have undergone extensive horizontal transfer between genomes, as inferred from their sequence homology, codon usage bias and GC content difference. They are often linked with mobile genetic elements such as plasmids, viruses, transposons and integrons. The comparison of closely related bacterial genomes also suggests that, at times, RM genes themselves behave as mobile elements and cause genome rearrangements. Indeed some bacterial genomes that survived post-disturbance attack by an RM gene complex in the laboratory have experienced genome rearrangements. The avoidance of some restriction sites by bacterial genomes may result from selection by past restriction attacks. Both bacteriophages and bacteria also appear to use homologous recombination to cope with the selfish behavior of RM systems. RM systems compete with each other in several ways. One is competition for recognition sequences in post-segregational killing. Another is super-infection exclusion, that is, the killing of the cell carrying an RM system when it is infected with another RM system of the same regulatory specificity but of a different sequence specificity. The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and function of RM enzymes. PMID:11557807

Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution.

PubMed

Kobayashi, I

2001-09-15

Restriction-modification (RM) systems are composed of genes that encode a restriction enzyme and a modification methylase. RM systems sometimes behave as discrete units of life, like viruses and transposons. RM complexes attack invading DNA that has not been properly modified and thus may serve as a tool of defense for bacterial cells. However, any threat to their maintenance, such as a challenge by a competing genetic element (an incompatible plasmid or an allelic homologous stretch of DNA, for example) can lead to cell death through restriction breakage in the genome. This post-segregational or post-disturbance cell killing may provide the RM complexes (and any DNA linked with them) with a competitive advantage. There is evidence that they have undergone extensive horizontal transfer between genomes, as inferred from their sequence homology, codon usage bias and GC content difference. They are often linked with mobile genetic elements such as plasmids, viruses, transposons and integrons. The comparison of closely related bacterial genomes also suggests that, at times, RM genes themselves behave as mobile elements and cause genome rearrangements. Indeed some bacterial genomes that survived post-disturbance attack by an RM gene complex in the laboratory have experienced genome rearrangements. The avoidance of some restriction sites by bacterial genomes may result from selection by past restriction attacks. Both bacteriophages and bacteria also appear to use homologous recombination to cope with the selfish behavior of RM systems. RM systems compete with each other in several ways. One is competition for recognition sequences in post-segregational killing. Another is super-infection exclusion, that is, the killing of the cell carrying an RM system when it is infected with another RM system of the same regulatory specificity but of a different sequence specificity. The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and function of RM enzymes.

The RY/Sph element mediates transcriptional repression of maturation genes from late maturation to early seedling growth.

PubMed

Guerriero, Gea; Martin, Nathalie; Golovko, Anna; Sundström, Jens F; Rask, Lars; Ezcurra, Ines

2009-11-01

In orthodox seeds, the transcriptional activator ABI3 regulates two major stages in embryo maturation: a mid-maturation (MAT) stage leading to accumulation of storage compounds, and a late maturation (LEA) stage leading to quiescence and desiccation tolerance. Our aim was to elucidate mechanisms for transcriptional shutdown of MAT genes during late maturation, to better understand phase transition between MAT and LEA stages. Using transgenic and transient approaches in Nicotiana, we examined activities of two ABI3-dependent reporter genes driven by multimeric RY and abscisic acid response elements (ABREs) from a Brassica napus napin gene, termed RY and ABRE, where the RY reporter requires ABI3 DNA binding. Expression of RY peaks during mid-maturation and drops during late maturation, mimicking the MAT gene program, and in Arabidopsis thaliana RY elements are over-represented in MAT, but not in LEA, genes. The ABI3 transactivation of RY is inhibited by staurosporine, by a PP2C phosphatase, and by a repressor of maturation genes, VAL1/HSI2. The RY element mediates repression of MAT genes, and we propose that transcriptional shutdown of the MAT program during late maturation involves inhibition of ABI3 DNA binding by dephosphorylation. Later, during seedling growth, VAL1/HSI2 family repressors silence MAT genes by binding RY elements.

Mobilization of a plant transposon by expression of the transposon-encoded anti-silencing factor.

PubMed

Fu, Yu; Kawabe, Akira; Etcheverry, Mathilde; Ito, Tasuku; Toyoda, Atsushi; Fujiyama, Asao; Colot, Vincent; Tarutani, Yoshiaki; Kakutani, Tetsuji

2013-08-28

Transposable elements (TEs) have a major impact on genome evolution, but they are potentially deleterious, and most of them are silenced by epigenetic mechanisms, such as DNA methylation. Here, we report the characterization of a TE encoding an activity to counteract epigenetic silencing by the host. In Arabidopsis thaliana, we identified a mobile copy of the Mutator-like element (MULE) with degenerated terminal inverted repeats (TIRs). This TE, named Hiun (Hi), is silent in wild-type plants, but it transposes when DNA methylation is abolished. When a Hi transgene was introduced into the wild-type background, it induced excision of the endogenous Hi copy, suggesting that Hi is the autonomously mobile copy. In addition, the transgene induced loss of DNA methylation and transcriptional activation of the endogenous Hi. Most importantly, the trans-activation of Hi depends on a Hi-encoded protein different from the conserved transposase. Proteins related to this anti-silencing factor, which we named VANC, are widespread in the non-TIR MULEs and may have contributed to the recent success of these TEs in natural Arabidopsis populations.

The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure.

PubMed

Valle-García, David; Griffiths, Lyra M; Dyer, Michael A; Bernstein, Emily; Recillas-Targa, Félix

2014-01-01

The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of α-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of ATRX isoform 2 coding sequence in two different plasmids derived from a single source. We demonstrate that these insertion events are common and there is an insertion hotspot within the ATRX cDNA. Such IS10 insertions produce a truncated form of ATRX, which significantly compromises its nuclear localization. In turn, we describe ways to prevent IS10 insertion during propagation and cloning of ATRX-containing vectors, including optimal growth conditions, bacterial strains, and suggested sequencing strategies. Finally, we have generated an insertion-free plasmid that is available to the community for expression studies of ATRX.

Lineage-specific genomics: Frequent birth and death in the human genome: The human genome contains many lineage-specific elements created by both sequence and functional turnover.

PubMed

Young, Robert S

2016-07-01

Frequent evolutionary birth and death events have created a large quantity of biologically important, lineage-specific DNA within mammalian genomes. The birth and death of DNA sequences is so frequent that the total number of these insertions and deletions in the human population remains unknown, although there are differences between these groups, e.g. transposable elements contribute predominantly to sequence insertion. Functional turnover - where the activity of a locus is specific to one lineage, but the underlying DNA remains conserved - can also drive birth and death. However, this does not appear to be a major driver of divergent transcriptional regulation. Both sequence and functional turnover have contributed to the birth and death of thousands of functional promoters in the human and mouse genomes. These findings reveal the pervasive nature of evolutionary birth and death and suggest that lineage-specific regions may play an important but previously underappreciated role in human biology and disease. © 2016 The Authors BioEssays Published by WILEY Periodicals, Inc.

BPF-1, a pathogen-induced DNA-binding protein involved in the plant defense response.

PubMed

da Costa e Silva, O; Klein, L; Schmelzer, E; Trezzini, G F; Hahlbrock, K

1993-07-01

The mechanisms by which plants restrict the growth of pathogens include transient activation of numerous defense-related genes. Box P is a putative cis-acting element of a distinct group of such genes, including those encoding the enzyme phenylalanine ammonialyase (PAL). A DNA-binding activity to Box P was identified in nuclear extracts from cultured parsley cells and a cDNA encoding the protein BPF-1 (Box P-binding Factor) partially characterized. BPF-1 binds to this element with specificity similar to that of the binding activity in nuclear extracts. BPF-1 mRNA accumulates rapidly in elicitor-treated parsley cells and around fungal infection sites on parsley leaves. This accumulation is, at least partly, due to a rapid and transient increase in the transcription rate of BPF-1. Moreover, tight correlation between the relative amounts of BPF-1 and PAL mRNAs was observed in different organs of a parsley plant. These results are consistent with the hypothesis that BPF-1 is involved in disease resistance by modulating plant defense gene expression.

A purified transcription factor (TIF-IB) binds to essential sequences of the mouse rDNA promoter.

PubMed Central

Clos, J; Buttgereit, D; Grummt, I

1986-01-01

A transcription factor that is specific for mouse rDNA has been partially purified from Ehrlich ascites cells. This factor [designated transcription initiation factor (TIF)-IB] is required for accurate in vitro synthesis of mouse rRNA in addition to RNA polymerase I and another regulatory factor, TIF-IA. TIF-IB activity is present in extracts both from growing and nongrowing cells in comparable amounts. Prebinding competition experiments with wild-type and mutant templates suggest that TIF-IB interacts with the core control element of the rDNA promoter, which is located immediately upstream of the initiation site. The specific binding of TIF-IB to the RNA polymerase I promoter is demonstrated by exonuclease III protection experiments. The 3' border of the sequences protected by TIF-IB is shown to be on the coding strand at position -21 and on the noncoding strand at position -7. The results suggest that direct binding of TIF-IB to sequences in the core promoter element is the mechanism by which this factor imparts promoter selectivity to RNA polymerase I. Images PMID:3456157

Structural diversity and dynamics of genomic replication origins in Schizosaccharomyces pombe

PubMed Central

Cotobal, Cristina; Segurado, Mónica; Antequera, Francisco

2010-01-01

DNA replication origins (ORI) in Schizosaccharomyces pombe colocalize with adenine and thymine (A+T)-rich regions, and earlier analyses have established a size from 0.5 to over 3 kb for a DNA fragment to drive replication in plasmid assays. We have asked what are the requirements for ORI function in the chromosomal context. By designing artificial ORIs, we have found that A+T-rich fragments as short as 100 bp without homology to S. pombe DNA are able to initiate replication in the genome. On the other hand, functional dissection of endogenous ORIs has revealed that some of them span a few kilobases and include several modules that may be as short as 25–30 contiguous A+Ts capable of initiating replication from ectopic chromosome positions. The search for elements with these characteristics across the genome has uncovered an earlier unnoticed class of low-efficiency ORIs that fire late during S phase. These results indicate that ORI specification and dynamics varies widely in S. pombe, ranging from very short elements to large regions reminiscent of replication initiation zones in mammals. PMID:20094030

A high-throughput method for GMO multi-detection using a microfluidic dynamic array.

PubMed

Brod, Fábio Cristiano Angonesi; van Dijk, Jeroen P; Voorhuijzen, Marleen M; Dinon, Andréia Zilio; Guimarães, Luis Henrique S; Scholtens, Ingrid M J; Arisi, Ana Carolina Maisonnave; Kok, Esther J

2014-02-01

The ever-increasing production of genetically modified crops generates a demand for high-throughput DNA-based methods for the enforcement of genetically modified organisms (GMO) labelling requirements. The application of standard real-time PCR will become increasingly costly with the growth of the number of GMOs that is potentially present in an individual sample. The present work presents the results of an innovative approach in genetically modified crops analysis by DNA based methods, which is the use of a microfluidic dynamic array as a high throughput multi-detection system. In order to evaluate the system, six test samples with an increasing degree of complexity were prepared, preamplified and subsequently analysed in the Fluidigm system. Twenty-eight assays targeting different DNA elements, GM events and species-specific reference genes were used in the experiment. The large majority of the assays tested presented expected results. The power of low level detection was assessed and elements present at concentrations as low as 0.06 % were successfully detected. The approach proposed in this work presents the Fluidigm system as a suitable and promising platform for GMO multi-detection.

Cytogenotoxicity of sewage sludge leachate before and after calcium oxide-based solidification in human lymphocytes.

PubMed

Gajski, Goran; Oreščanin, Višnja; Garaj-Vrhovac, Vera

2011-07-01

Present study aimed to establish the chemical composition of sewage sludge leachate before/after calcium oxide-based solidification using energy dispersive X-ray fluorescence (EDXRF). The other aim was to determine leachate effects on human lymphocyte and DNA integrity in vitro using a battery of bioassays (DNA diffusion assay, micronucleus test and comet assay) to determine effects of those complex mixtures of elements on cell and DNA integrity. EDXRF showed that nickel concentration in the leachate of untreated sludge was 18.5 times higher than the upper permissible limit for inert waste landfills. Other elements were kept below the permissible values. After sludge solidification, leachate concentrations of Cr, Mn, Fe, Ni, Cu, Zn, and Pb dropped 1.6, 2.7, 37, 5.9, 3.2, 7.8, and 2.6 times, respectively. Untreated sludge leachate was cytogenotoxic to lymphocytes, and may lead to adverse effects on the exposed human populations, but calcium oxide-based solidification reduced these effects in significant manner. Copyright © 2011 Elsevier Inc. All rights reserved.

High-efficiency transformation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence, ARS2.

PubMed Central

Yang, V W; Marks, J A; Davis, B P; Jeffries, T W

1994-01-01

This paper describes the first high-efficiency transformation system for the xylose-fermenting yeast Pichia stipitis. The system includes integrating and autonomously replicating plasmids based on the gene for orotidine-5'-phosphate decarboxylase (URA3) and an autonomous replicating sequence (ARS) element (ARS2) isolated from P. stipitis CBS 6054. Ura- auxotrophs were obtained by selecting for resistance to 5-fluoroorotic acid and were identified as ura3 mutants by transformation with P. stipitis URA3. P. stipitis URA3 was cloned by its homology to Saccharomyces cerevisiae URA3, with which it is 69% identical in the coding region. P. stipitis ARS elements were cloned functionally through plasmid rescue. These sequences confer autonomous replication when cloned into vectors bearing the P. stipitis URA3 gene. P. stipitis ARS2 has features similar to those of the consensus ARS of S. cerevisiae and other ARS elements. Circular plasmids bearing the P. stipitis URA3 gene with various amounts of flanking sequences produced 600 to 8,600 Ura+ transformants per micrograms of DNA by electroporation. Most transformants obtained with circular vectors arose without integration of vector sequences. One vector yielded 5,200 to 12,500 Ura+ transformants per micrograms of DNA after it was linearized at various restriction enzyme sites within the P. stipitis URA3 insert. Transformants arising from linearized vectors produced stable integrants, and integration events were site specific for the genomic ura3 in 20% of the transformants examined. Plasmids bearing the P. stipitis URA3 gene and ARS2 element produced more than 30,000 transformants per micrograms of plasmid DNA. Autonomously replicating plasmids were stable for at least 50 generations in selection medium and were present at an average of 10 copies per nucleus. Images PMID:7811063

The impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells.

PubMed

Sharma, Nynne; Hollensen, Anne Kruse; Bak, Rasmus O; Staunstrup, Nicklas Heine; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

2012-01-01

DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5'-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and Tol2 transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells.

The Impact of cHS4 Insulators on DNA Transposon Vector Mobilization and Silencing in Retinal Pigment Epithelium Cells

PubMed Central

Sharma, Nynne; Hollensen, Anne Kruse; Bak, Rasmus O.; Staunstrup, Nicklas Heine; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

2012-01-01

DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5′-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and Tol2 transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells. PMID:23110238

Maternal phthalate exposure during pregnancy is associated with DNA methylation of LINE-1 and Alu repetitive elements in Mexican-American children

PubMed Central

Huen, Karen; Calafat, Antonia M.; Bradman, Asa; Yousefi, Paul; Eskenazi, Brenda; Holland, Nina

2016-01-01

Phthalates are frequently used in personal care products and plasticizers and phthalate exposure is ubiquitous in the US population. Exposure to phthalates during critical periods in utero has been associated with a variety of adverse health outcomes but the biological mechanisms linking these exposures with disease are not well characterized. In this study, we examined the relationship of in utero phthalate exposure with repetitive element DNA methylation, an epigenetic marker of genome instability, in children from the longitudinal birth cohort CHAMACOS. Methylation of Alu and long interspersed nucleotide elements (LINE-1) was determined using pyrosequencing of bisulfite-treated DNA isolated from whole blood samples collected from newborns and 9 year old children (n=355). Concentrations of eleven phthalate metabolites were measured in urine collected from pregnant mothers at 13 and 26 weeks gestation. We found a consistent inverse association between prenatal concentrations of monoethyl phthalate, the most frequently detected urinary metabolite, with cord blood methylation of Alu repeats (β(95%CI):−0.14(−0.28,0.00) and −0.16(−0.31,−0.02)) for early and late pregnancy, respectively, and a similar but weaker association with LINE-1 methylation. Additionally, increases in urinary concentrations of di-(2-ethylhexyl) phthalate metabolites during late pregnancy were associated with lower levels of methylation of Alu repeats in 9 year old blood (significant p-values ranged from 0.003 to 0.03). Our findings suggest that prenatal exposure to some phthalates may influence differences in repetitive element methylation, highlighting epigenetics as a plausible biological mechanism through which phthalates may affect health. PMID:27019040

Analysis of Two Cosmid Clones from Chromosome 4 of Drosophila melanogaster Reveals Two New Genes Amid an Unusual Arrangement of Repeated Sequences

PubMed Central

Locke, John; Podemski, Lynn; Roy, Ken; Pilgrim, David; Hodgetts, Ross

1999-01-01

Chromosome 4 from Drosophila melanogaster has several unusual features that distinguish it from the other chromosomes. These include a diffuse appearance in salivary gland polytene chromosomes, an absence of recombination, and the variegated expression of P-element transgenes. As part of a larger project to understand these properties, we are assembling a physical map of this chromosome. Here we report the sequence of two cosmids representing ∼5% of the polytenized region. Both cosmid clones contain numerous repeated DNA sequences, as identified by cross hybridization with labeled genomic DNA, BLAST searches, and dot matrix analysis, which are positioned between and within the transcribed sequences. The repetitive sequences include three copies of the mobile element Hoppel, one copy of the mobile element HB, and 18 DINE repeats. DINE is a novel, short repeated sequence dispersed throughout both cosmid sequences. One cosmid includes the previously described cubitus interruptus (ci) gene and two new genes: that a gene with a predicted amino acid sequence similar to ribosomal protein S3a which is consistent with the Minute(4)101 locus thought to be in the region, and a novel member of the protein family that includes plexin and met–hepatocyte growth factor receptor. The other cosmid contains only the two short 5′-most exons from the zinc-finger-homolog-2 (zfh-2) gene. This is the first extensive sequence analysis of noncoding DNA from chromosome 4. The distribution of the various repeats suggests its organization is similar to the β-heterochromatic regions near the base of the major chromosome arms. Such a pattern may account for the diffuse banding of the polytene chromosome 4 and the variegation of many P-element transgenes on the chromosome. PMID:10022978

[Construction of a general AAV vector regulated by minimal and artificial hypoxic-responsive element].

PubMed

Nie, Xiao-wei; Sun, Li-jun; Hao, Yue-wen; Yang, Guang-xiao; Wang, Quan-ying

2011-03-01

To synthesize the minimal and artificial HRE, and to insert it into the anterior extremity of CMV promoter of a AAV plasmid, and then to construct the AAV regulated by hypoxic-responsive element which was introduced into 293 cell by method of Ca3(PO4)2 using three plasmids. Thus obtaining the adenoassociated virus vector regulated by hypoxic-responsive element was possibly used for gene therapy in ischemia angiocardiopathy and cerebrovascular disease. Artificially synthesize the 36 bp nucleotide sequences of four connection in series HIF-binding sites A/GCGTG(4×HBS)and a 35 bp nucleotide sequences spacing inserted into anterior extremity of CMV promoter TATA Box, then amplified by PCR. The cDNA fragment was confirmed to be right by DNA sequencing. Molecular biology routine method was used to construct a AAV vector regulated by minimal hypoxic-responsive element after the normal CMV promoter in AAV vector was replaced by the CMV promoter included minimal hypoxic-responsive element. Then, NT4-6His-PR39 fusogenic peptide was inserted into MCS of the plasmid, the recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells, in which we can also investigate the expression of 6×His using immunochemistry in hypoxia environment. Artificial HRE was inserted into anterior extremity of CMV promoter and there was a correct spacing between the HRE and the TATA-box. The DNA sequencing and restriction enzyme digestion results indicated that the AAV regulated by hypoxic-responsive element was successfully constructed. Compared to the control group, the expressions of 6×His was significantly increased in the experimental groups in hypoxia environment, which confirmed that the AAV effectually regulated by the minimal HRE was inserted into anterior extremity of CMV promoter. The HRE is inserted into anterior extremity of CMV promoter to lack incision enzyme recognition site by PCR. And eukaryotic expression vector regulated by hypoxic-responsive is constructed. The AAV effectually regulated by the minimal HRE inserted into anterior extremity of CMV promoter. The vector is successfully constructed and it has important theoretical and practical value in the synteresis and therapy of ischemia angiocardiopathy and cerebrovascular disease.

The mitochondrial genome of the gymnosperm Cycas taitungensis contains a novel family of short interspersed elements, Bpu sequences, and abundant RNA editing sites.

PubMed

Chaw, Shu-Miaw; Shih, Arthur Chun-Chieh; Wang, Daryi; Wu, Yu-Wei; Liu, Shu-Mei; Chou, The-Yuan

2008-03-01

The mtDNA of Cycas taitungensis is a circular molecule of 414,903 bp, making it 2- to 6-fold larger than the known mtDNAs of charophytes and bryophytes, but similar to the average of 7 elucidated angiosperm mtDNAs. It is characterized by abundant RNA editing sites (1,084), more than twice the number found in the angiosperm mtDNAs. The A + T content of Cycas mtDNA is 53.1%, the lowest among known land plants. About 5% of the Cycas mtDNA is composed of a novel family of mobile elements, which we designated as "Bpu sequences." They share a consensus sequence of 36 bp with 2 terminal direct repeats (AAGG) and a recognition site for the Bpu 10I restriction endonuclease (CCTGAAGC). Comparison of the Cycas mtDNA with other plant mtDNAs revealed many new insights into the biology and evolution of land plant mtDNAs. For example, the noncoding sequences in mtDNAs have drastically expanded as land plants have evolved, with abrupt increases appearing in the bryophytes, and then in the seed plants. As a result, the genomic organizations of seed plant mtDNAs are much less compact than in other plants. Also, the Cycas mtDNA appears to have been exempted from the frequent gene loss observed in angiosperm mtDNAs. Similar to the angiosperms, the 3 Cycas genes nad1, nad2, and nad5 are disrupted by 5 group II intron squences, which have brought the genes into trans-splicing arrangements. The evolutionary origin and invasion/duplication mechanism of the Bpu sequences in Cycas mtDNA are hypothesized and discussed.

Unusual Properties of Regulatory DNA from the Drosophila Engrailed Gene: Three ``pairing-Sensitive'' Sites within a 1.6-Kb Region

PubMed Central

Kassis, J. A.

1994-01-01

We have previously shown that a 2-kb fragment of engrailed DNA can suppress expression of a linked marker gene, white, in the P element vector CaSpeR. This suppression is dependent on the presence of two copies of engrailed DNA-containing P elements (P[en]) in proximity in the Drosophila genome (either in cis or in trans). In this study, the 2-kb fragment was dissected and found to contain three fragments of DNA which could mediate white suppression [called ``pairing-sensitive sites'' (PS)]. A PS site was also identified in regulatory DNA from the Drosophila escargot gene. The eye colors of six different P[en] insertions in the escargot gene suggest an interaction between P[en]-encoded and genome-encoded PS sites. I hypothesize that white gene expression from P[en] is repressed by the formation of a protein complex which is initiated at the engrailed PS sites and also requires interactions with flanking genomic DNA. Genes were sought which influence the function of PS sites. Mutations in some Polycomb and trithorax group genes were found to affect the eye color from some P[en] insertion sites. However, different mutations affected expression from different P[en] insertion sites and no one mutation was found to affect expression from all P[en] insertion sites examined. These results suggest that white expression from P[en] is not directly regulated by members of the Polycomb and trithorax group genes, but in some cases can be influenced by them. I propose that engrailed PS sites normally act to promote interactions between distantly located engrailed regulatory sites and the engrailed promoter. PMID:8005412

Thermodynamic and structural insights into CSL-DNA complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Friedmann, David R.; Kovall, Rhett A.

The Notch pathway is an intercellular signaling mechanism that plays important roles in cell fates decisions throughout the developing and adult organism. Extracellular complexation of Notch receptors with ligands ultimately results in changes in gene expression, which is regulated by the nuclear effector of the pathway, CSL (C-promoter binding factor 1 (CBF-1), suppressor of hairless (Su(H)), lin-12 and glp-1 (Lag-1)). CSL is a DNA binding protein that is involved in both repression and activation of transcription from genes that are responsive to Notch signaling. One well-characterized Notch target gene is hairy and enhancer of split-1 (HES-1), which is regulated bymore » a promoter element consisting of two CSL binding sites oriented in a head-to-head arrangement. Although previous studies have identified in vivo and consensus binding sites for CSL, and crystal structures of these complexes have been determined, to date, a quantitative description of the energetics that underlie CSL-DNA binding is unknown. Here, we provide a thermodynamic and structural analysis of the interaction between CSL and the two individual sites that comprise the HES-1 promoter element. Our comprehensive studies that analyze binding as a function of temperature, salt, and pH reveal moderate, but distinct, differences in the affinities of CSL for the two HES-1 binding sites. Similarly, our structural results indicate that overall CSL binds both DNA sites in a similar manner; however, minor changes are observed in both the conformation of CSL and DNA. Taken together, our results provide a quantitative and biophysical basis for understanding how CSL interacts with DNA sites in vivo.« less

Surface-enhanced Raman spectroscopy for the detection of pathogenic DNA and protein in foods

NASA Astrophysics Data System (ADS)

Chowdhury, Mustafa H.; Atkinson, Brad; Good, Theresa; Cote, Gerard L.

2003-07-01

Traditional Raman spectroscopy while extremely sensitive to structure and conformation, is an ineffective tool for the detection of bioanalytes at the sub milimolar level. Surface Enhanced Raman Spectroscopy (SERS) is a technique developed more recently that has been used with applaudable success to enhance the Raman cross-section of a molecule by factors of 106 to 1014. This technique can be exploited in a nanoscale biosensor for the detection of pathogenic proteins and DNA in foods by using a biorecognition molecule to bring a target analyte in close proximity to the mental surface. This is expected to produce a SERS signal of the target analyte, thus making it possible to easily discriminate between the target analyte and possible confounders. In order for the sensor to be effective, the Raman spectra of the target analyte would have to be distinct from that of the biorecognition molecule, as both would be in close proximity to the metal surface and thus be subjected to the SERS effect. In our preliminary studies we have successfully used citrate reduced silver colloidal particles to obtain unique SERS spectra of α-helical and β-sheet bovine serum albumin (BSA) that served as models of an α helical antiobiody (biorecognition element) and a β-sheet target protein (pathogenic prion). In addition, the unique SERS spectra of double stranded and single stranded DNA were also obtained where the single stranded DNA served as the model for the biorecognition element and the double stranded DNA served as themodel for the DNA probe/target hybrid. This provides a confirmation of the feasibility of the method which opens opportunities for potentially wide spread applications in the detection of food pathogens, biowarefare agents, andother bio-analytes.

Antibiotic Resistance Genes in the Bacteriophage DNA Fraction of Environmental Samples

PubMed Central

Colomer-Lluch, Marta; Jofre, Juan; Muniesa, Maite

2011-01-01

Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, β-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to β-lactam antibiotics is conferred by β-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to β-lactam antibiotics, namely two β-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment. PMID:21390233

Theoretical rate constants of super-exchange hole transfer and thermally induced hopping in DNA.

PubMed

Shimazaki, Tomomi; Asai, Yoshihiro; Yamashita, Koichi

2005-01-27

Recently, the electronic properties of DNA have been extensively studied, because its conductivity is important not only to the study of fundamental biological problems, but also in the development of molecular-sized electronics and biosensors. We have studied theoretically the reorganization energies, the activation energies, the electronic coupling matrix elements, and the rate constants of hole transfer in B-form double-helix DNA in water. To accommodate the effects of DNA nuclear motions, a subset of reaction coordinates for hole transfer was extracted from classical molecular dynamics (MD) trajectories of DNA in water and then used for ab initio quantum chemical calculations of electron coupling constants based on the generalized Mulliken-Hush model. A molecular mechanics (MM) method was used to determine the nuclear Franck-Condon factor. The rate constants for two types of mechanisms of hole transfer-the thermally induced hopping (TIH) and the super-exchange mechanisms-were determined based on Marcus theory. We found that the calculated matrix elements are strongly dependent on the conformations of the nucleobase pairs of hole-transferable DNA and extend over a wide range of values for the "rise" base-step parameter but cluster around a particular value for the "twist" parameter. The calculated activation energies are in good agreement with experimental results. Whereas the rate constant for the TIH mechanism is not dependent on the number of A-T nucleobase pairs that act as a bridge, the rate constant for the super-exchange process rapidly decreases when the length of the bridge increases. These characteristic trends in the calculated rate constants effectively reproduce those in the experimental data of Giese et al. [Nature 2001, 412, 318]. The calculated rate constants were also compared with the experimental results of Lewis et al. [Nature 2000, 406, 51].

Role of UME6 in transcriptional regulation of a DNA repair gene in Saccharomyces cerevisiae.

PubMed

Sweet, D H; Jang, Y K; Sancar, G B

1997-11-01

In Saccharomyces cerevisiae UV radiation and a variety of chemical DNA-damaging agents induce the transcription of specific genes, including several involved in DNA repair. One of the best characterized of these genes is PHR1, which encodes the apoenzyme for DNA photolyase. Basal-level and damage-induced expression of PHR1 require an upstream activation sequence, UAS(PHR1), which has homology with DRC elements found upstream of at least 19 other DNA repair and DNA metabolism genes in yeast. Here we report the identification of the UME6 gene of S. cerevisiae as a regulator of UAS(PHR1) activity. Multiple copies of UME6 stimulate expression from UAS(PHR1) and the intact PHR1 gene. Surprisingly, the effect of deletion of UME6 is growth phase dependent. In wild-type cells PHR1 is induced in late exponential phase, concomitant with the initiation of glycogen accumulation that precedes the diauxic shift. Deletion of UME6 abolishes this induction, decreases the steady-state concentration of photolyase molecules and PHR1 mRNA, and increases the UV sensitivity of a rad2 mutant. Despite the fact that UAS(PHR1) does not contain the URS1 sequence, which has been previously implicated in UME6-mediated transcriptional regulation, we find that Ume6p binds to UAS(PHR1) with an affinity and a specificity similar to those seen for a URS1 site. Similar binding is also seen for DRC elements from RAD2, RAD7, and RAD53, suggesting that UME6 contributes to the regulated expression of a subset of damage-responsive genes in yeast.