These are representative sample records from Science.gov related to your search topic.
For comprehensive and current results, perform a real-time search at Science.gov.
1

Molecular Biology DNA: The Genetic Macromolecule  

E-print Network

Molecular Biology Lab 1 DNA: The Genetic Macromolecule (Bacterial Transformation) Although we have of the E.coli genome. The experiment will allow the student to indirectly observe the central dogma DNA molecule) to a bacterial strain of E. coli called HB101. The plasmid has been engineered

2

Persistence Length of DNA Macromolecule with Kinks  

E-print Network

The study of configurational parameters of deformed DNA is a relevant problem in research of such important biological process as double helix compactization in cell. The deformations accompanied with local disruptions of the regular macromolecule structure cause significant bending of the double helix, or kinks. In this paper an approach for Kratky-Porod model to calculate persistence length of DNA macromolecule with kinks is developed. The presented approach considers kinks of arbitrary configuration, including two basic types of kinks, type 1 - sharp kink caused by unstacking a single base pair step, and type 2 - intrinsic-induced kink that involves several base pairs. Within developed approach analytical expressions for persistence length, coil size and gyration radius of kinky double helix were obtained.

Simonov, Kyrylo

2014-01-01

3

Functionalized gold nanoparticles for the binding, stabilization, and delivery of therapeutic DNA, RNA, and other biological macromolecules  

PubMed Central

Nanotechnology has virtually exploded in the last few years with seemingly limitless opportunity across all segments of our society. If gene and RNA therapy are to ever realize their full potential, there is a great need for nanomaterials that can bind, stabilize, and deliver these macromolecular nucleic acids into human cells and tissues. Many researchers have turned to gold nanomaterials, as gold is thought to be relatively well tolerated in humans and provides an inert material upon which nucleic acids can attach. Here, we review the various strategies for associating macromolecular nucleic acids to the surface of gold nanoparticles (GNPs), the characterization chemistries involved, and the potential advantages of GNPs in terms of stabilization and delivery. PMID:24198471

DeLong, Robert K; Reynolds, Christopher M; Malcolm, Yaneika; Schaeffer, Ashley; Severs, Tiffany; Wanekaya, Adam

2010-01-01

4

DNA, RNA , and protein  

NSDL National Science Digital Library

Have you ever wondered why you look like your mother while your brother looks like your grandfather? Consult life's gigantic book of information! This resource contains an illustrated interactive explanation of RNA, DNA, and proteins. This resource is appropriate for all users as it provides useful background information to enhance STEM teaching and learning for all. Copyright 2005 EDC

E-Museum, Nobel

2001-01-01

5

Target Biological Structures: The Cell, Organelles, DNA and RNA  

NASA Astrophysics Data System (ADS)

Living organisms are self replicating molecular factories of staggering complexity [1]. As a result, we are often overwhelmed when trying to identify potential targets for therapeutics. Water, inorganic ions and a large array of relatively small organic molecules (e.g., sugars, vitamins and fatty acids) account for approximately 80% of living matter, with water being the most abundant. Macromolecules such as proteins, polysaccharides, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) constitute the rest. The majority of potential therapeutic targets are found within the cell. Small molecules which are vital for cellular function are imported into the cell by a variety of mechanisms but unlike smaller molecules, macromolecules are assembled within the cell itself. Drugs are usually designed to target cellular macromolecules, as they perform very specific roles in the metabolic processes.

van Holst, Marcelis; Grant, Maxine P.; Aldrich-Wright, Janice

6

Cationic amphiphilic macromolecule (CAM)-lipid complexes for efficient siRNA gene silencing.  

PubMed

The accumulated evidence has shown that lipids and polymers each have distinct advantages as carriers for siRNA delivery. Composite materials comprising both lipids and polymers may present improved properties that combine the advantage of each. Cationic amphiphilic macromolecules (CAMs) containing a hydrophobic alkylated mucic acid segment and a hydrophilic poly(ethylene glycol) (PEG) tail were non-covalently complexed with two lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), to serve as a siRNA delivery vehicle. By varying the weight ratio of CAM to lipid, cationic complexes with varying compositions were obtained in aqueous media and their properties evaluated. CAM-lipid complex sizes were relatively independent of composition, ranging from 100 to 200nm, and zeta potentials varied from 10 to 30mV. Transmission electron microscopy confirmed the spherical morphology of the complexes. The optimal N/P ratio was 50 as determined by electrophoretic mobility shift assay. The ability to achieve gene silencing was evaluated by anti-luciferase siRNA delivery to a U87-luciferase cell line. Several weight ratios of CAM-lipid complexes were found to have similar delivery efficiency compared to the gold standard, Lipofectamine. Isothermal titration calorimetry revealed that siRNA binds more tightly at pH=7.4 than pH=5 to CAM-lipid (1:10 w/w). Further intracellular trafficking studies monitored the siRNA escape from the endosomes at 24h following transfection of cells. The findings in the paper indicate that CAM-lipid complexes can serve as a novel and efficient siRNA delivery vehicle. PMID:24727076

Gu, Li; Nusblat, Leora M; Tishbi, Nasim; Noble, Sarah C; Pinson, Chaya M; Mintzer, Evan; Roth, Charles M; Uhrich, Kathryn E

2014-06-28

7

Delivery of siRNA and other macromolecules into skin and cells using a peptide enhancer.  

PubMed

Delivery of macromolecules into cells and tissues such as skin is a major challenge. This obstacle poses a particular challenge for the delivery of siRNA where cellular and tissue level transport barriers need to be overcome. siRNAs are potential therapeutics for various dermatological diseases including psoriasis, atopic dermatitis, and cancer; however, their utility is limited by their low absorption across the stratum corneum (SC) and into viable cells of skin. Here, we address this challenge using a peptide identified by phage display termed skin penetrating and cell entering (SPACE) peptide. In vitro studies indicated that the SPACE peptide, when conjugated to cargoes such as small molecules and proteins, was able to facilitate their penetration across the SC into epidermis and dermis. The peptide also exhibited increased penetration into various cells including keratinocytes, fibroblasts, and endothelial cells, likely through a macropinocytosis pathway. The ability of SPACE peptide to deliver siRNA was tested in vivo using two targets, interleukin-10 and GAPDH. Conjugation of the peptide to siRNA led to their enhanced absorption into skin and knockdown of corresponding protein targets. PMID:21903933

Hsu, Tracy; Mitragotri, Samir

2011-09-20

8

Specific DNA-RNA hybrid recognition by TAL effectors.  

PubMed

The transcription activator-like (TAL) effector targets specific host promoter through its central DNA-binding domain, which comprises multiple tandem repeats (TALE repeats). Recent structural analyses revealed that the TALE repeats form a superhelical structure that tracks along the forward strand of the DNA duplex. Here, we demonstrate that TALE repeats specifically recognize a DNA-RNA hybrid where the DNA strand determines the binding specificity. The crystal structure of a designed TALE in complex with the DNA-RNA hybrid was determined at a resolution of 2.5 Å. Although TALE repeats are in direct contact with only the DNA strand, the phosphodiester backbone of the RNA strand is inaccessible by macromolecules such as RNases. Consistent with this observation, sequence-specific recognition of an HIV-derived DNA-RNA hybrid by an engineered TALE efficiently blocked RNase H-mediated degradation of the RNA strand. Our study broadens the utility of TALE repeats and suggests potential applications in processes involving DNA replication and retroviral infections. PMID:23022487

Yin, Ping; Deng, Dong; Yan, Chuangye; Pan, Xiaojing; Xi, Jianzhong Jeff; Yan, Nieng; Shi, Yigong

2012-10-25

9

RNA-directed DNA methylation in Arabidopsis  

PubMed Central

In plants, double-stranded RNA that is processed to short RNAs ?21–24 nt in length can trigger two types of epigenetic gene silencing. Posttranscriptional gene silencing, which is related to RNA interference in animals and quelling in fungi, involves targeted elimination of homologous mRNA in the cytoplasm. RNA-directed DNA methylation involves de novo methylation of almost all cytosine residues within a region of RNA–DNA sequence identity. RNA-directed DNA methylation is presumed to be responsible for the methylation observed in protein coding regions of posttranscriptionally silenced genes. Moreover, a type of transcriptional gene silencing and de novo methylation of homologous promoters in trans can occur if a double-stranded RNA contains promoter sequences. Although RNA-directed DNA methylation has been described so far only in plants, there is increasing evidence that RNA can also target genome modifications in other organisms. To understand how RNA directs methylation to identical DNA sequences and how changes in chromatin configuration contribute to initiating or maintaining DNA methylation induced by RNA, a promoter double-stranded RNA-mediated transcriptional gene silencing system has been established in Arabidopsis. A genetic analysis of this system is helping to unravel the relationships among RNA signals, DNA methylation, and chromatin structure. PMID:12169664

Aufsatz, Werner; Mette, M. Florian; van der Winden, Johannes; Matzke, Antonius J. M.; Matzke, Marjori

2002-01-01

10

The relative flexibility of B-DNA and A-RNA duplexes: database analysis  

PubMed Central

An extensive analysis of structural databases is carried out to investigate the relative flexibility of B-DNA and A-RNA duplexes in crystal form. Our results show that the general anisotropic concept of flexibility is not very useful to compare the deformability of B-DNA and A-RNA duplexes, since the flexibility patterns of B-DNA and A-RNA are quite different. In other words, ‘flexibility’ is a dangerous word for describing macromolecules, unless it is clearly defined. A few soft essential movements explain most of the natural flexibility of A-RNA, whereas many are necessary for B-DNA. Essential movements occurring in naked B-DNAs are identical to those necessary to deform DNA in DNA–protein complexes, which suggest that evolution has designed DNA–protein complexes so that B-DNA is deformed according to its natural tendency. DNA is generally more flexible, but for some distortions A-RNA is easier to deform. Local stiffness constants obtained for naked B-DNAs and DNA complexes are very close, demonstrating that global distortions in DNA necessary for binding to proteins are the result of the addition of small concerted deformations at the base-pair level. Finally, it is worth noting that in general the picture of the relative deformability of A-RNA and DNA derived from database analysis agrees very well with that derived from state of the art molecular dynamics (MD) simulations. PMID:15562006

Pérez, Alberto; Noy, Agnes; Lankas, Filip; Luque, F. Javier; Orozco, Modesto

2004-01-01

11

Properties of Macromolecules  

NSDL National Science Digital Library

This lab activity from the Biotechnology Alliance for Suncoast Biology Educators explores different analytical tests that are used to detect the presence of specific macromolecule classes based on their properties. Students will also have a chance to measure their own body mass index by taking advantage of the bioelectrical impedance properties of body fat and an opportunity to extract their own DNA. The lesson includes background information on types of macromolecules, the materials needed, and the procedure.

Keirle, Matt

2012-07-10

12

A DNA enzyme that cleaves RNA  

NASA Technical Reports Server (NTRS)

BACKGROUND: Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identify a metal-dependent DNA enzyme using in vitro selection methodology. RESULTS: Beginning with a population of 10(14) DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb(2+)-dependent cleavage of a target ribonucleoside 3'-O-P bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 min-1. Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 min-1. This rate is about 10(5)-fold increased compared to the uncatalyzed reaction. CONCLUSIONS: Using in vitro selection techniques, we obtained a DNA enzyme that catalyzes the Pb(2+)-dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymes. We expect that other examples of DNA enzymes will soon be forthcoming.

Breaker, R. R.; Joyce, G. F.; Hoyce, G. F. (Principal Investigator)

1994-01-01

13

RNA Splicing Factors and RNA-Directed DNA Methylation  

PubMed Central

RNA-directed histone and/or DNA modification is a conserved mechanism for the establishment of epigenetic marks from yeasts and plants to mammals. The heterochromation formation in yeast is mediated by RNAi-directed silencing mechanism, while the establishment of DNA methylation in plants is through the RNA-directed DNA methylation (RdDM) pathway. Recently, splicing factors are reported to be involved in both RNAi-directed heterochromatin formation in yeast and the RdDM pathway in plants. In yeast, splicing factors may provide a platform for facilitating the siRNA generation through an interaction with RDRC and thereby affect the heterochromatin formation, whereas in plants, various splicing factors seem to act at different steps in the RdDM pathway. PMID:24833507

Huang, Chao-Feng; Zhu, Jian-Kang

2014-01-01

14

RNA Splicing Factors and RNA-Directed DNA Methylation.  

PubMed

RNA-directed histone and/or DNA modification is a conserved mechanism for the establishment of epigenetic marks from yeasts and plants to mammals. The heterochromation formation in yeast is mediated by RNAi-directed silencing mechanism, while the establishment of DNA methylation in plants is through the RNA-directed DNA methylation (RdDM) pathway. Recently, splicing factors are reported to be involved in both RNAi-directed heterochromatin formation in yeast and the RdDM pathway in plants. In yeast, splicing factors may provide a platform for facilitating the siRNA generation through an interaction with RDRC and thereby affect the heterochromatin formation, whereas in plants, various splicing factors seem to act at different steps in the RdDM pathway. PMID:24833507

Huang, Chao-Feng; Zhu, Jian-Kang

2014-01-01

15

Absolute cross section for low-energy-electron damage to condensed macromolecules: A case study of DNA  

PubMed Central

Cross sections (CSs) for the interaction of low-energy electrons (LEE) with condensed macromolecules are essential parameters for accurate modeling of radiation-induced molecular decomposition and chemical synthesis. Electron irradiation of dry nanometer-scale macromolecular solid films has often been employed to measure CSs and other quantitative parameters for LEE interactions. Since such films have thicknesses comparable with electron thermalization distances, energy deposition varies throughout the film. Moreover, charge accumulation occurring inside the films shields a proportion of the macromolecules from electron irradiation. Such effects complicate the quantitative comparison of the CSs obtained in films of different thicknesses and limit the applicability of such measurements. Here, we develop a simple mathematical model, termed the molecular survival model, that employs a CS for a particular damage process together with an attenuation length related to the total CS, to investigate how a measured CS might be expected to vary with experimental conditions. As a case study, we measure the absolute CS for the formation of DNA strand breaks (SBs) by electron irradiation at 10 and 100 eV of lyophilized plasmid DNA films with thicknesses between 10 and 30 nm. The measurements are shown to depend strongly on the thickness and charging condition of the nanometer-scale films. Such behaviors are in accord with the model and support its validity. Via this analysis, the CS obtained for SB damage is nearly independent of film thickness and charging effects. In principle, this model can be adapted to provide absolute CSs for electron-induced damage or reactions occurring in other molecular solids across a wider range of experimental conditions. PMID:23030950

Rezaee, Mohammad; Cloutier, Pierre; Bass, Andrew D.; Michaud, Marc; Hunting, Darel J.; Sanche, Leon

2013-01-01

16

Isolation of DNA and RNA.  

PubMed

Blood samples for most coagulation tests are collected into 3.8% trisodium citrate in a ratio of 1 part anticoagulant to 9 parts blood. Whole-blood samples for DNA isolation can be stored at -50°C and the DNA prepared at a later stage. A more convenient method requiring less freezer space is to store buffy coats-the interface between the red cells and the plasma that is seen following centrifugation of whole blood. This latter method also allows isolation of the plasma fraction. PMID:21340978

Perry, D J

1999-01-01

17

Isothermal amplified detection of DNA and RNA.  

PubMed

This review highlights various methods that can be used for a sensitive detection of nucleic acids without using thermal cycling procedures, as is done in PCR or LCR. Topics included are nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), loop-mediated amplification (LAMP), Invader assay, rolling circle amplification (RCA), signal mediated amplification of RNA technology (SMART), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), nicking endonuclease signal amplification (NESA) and nicking endonuclease assisted nanoparticle activation (NENNA), exonuclease-aided target recycling, Junction or Y-probes, split DNAZyme and deoxyribozyme amplification strategies, template-directed chemical reactions that lead to amplified signals, non-covalent DNA catalytic reactions, hybridization chain reactions (HCR) and detection via the self-assembly of DNA probes to give supramolecular structures. The majority of these isothermal amplification methods can detect DNA or RNA in complex biological matrices and have great potential for use at point-of-care. PMID:24643211

Yan, Lei; Zhou, Jie; Zheng, Yue; Gamson, Adam S; Roembke, Benjamin T; Nakayama, Shizuka; Sintim, Herman O

2014-05-01

18

Free-energy calculations for semi-flexible macromolecules: Applications to DNA knotting and looping  

PubMed Central

We present a method to obtain numerically accurate values of configurational free energies of semiflexible macromolecular systems, based on the technique of thermodynamic integration combined with normal-mode analysis of a reference system subject to harmonic constraints. Compared with previous free-energy calculations that depend on a reference state, our approach introduces two innovations, namely, the use of internal coordinates to constrain the reference states and the ability to freely select these reference states. As a consequence, it is possible to explore systems that undergo substantially larger fluctuations than those considered in previous calculations, including semiflexible biopolymers having arbitrary ratios of contour length L to persistence length P. To validate the method, high accuracy is demonstrated for free energies of prime DNA knots with L/P = 20 and L/P = 40, corresponding to DNA lengths of 3000 and 6000 base pairs, respectively. We then apply the method to study the free-energy landscape for a model of a synaptic nucleoprotein complex containing a pair of looped domains, revealing a bifurcation in the location of optimal synapse (crossover) sites. This transition is relevant to target-site selection by DNA-binding proteins that occupy multiple DNA sites separated by large linear distances along the genome, a problem that arises naturally in gene regulation, DNA recombination, and the action of type-II topoisomerases. PMID:25381542

Giovan, Stefan M.; Scharein, Robert G.; Hanke, Andreas

2014-01-01

19

Free-energy calculations for semi-flexible macromolecules: Applications to DNA knotting and looping  

E-print Network

We present a method to obtain numerically accurate values of configurational free energies of semiflexible macromolecular systems, based on the technique of thermodynamic integration combined with normal-mode analysis of a reference system subject to harmonic constraints. Compared with previous free-energy calculations that depend on a reference state, our approach introduces two innovations, namely the use of internal coordinates to constrain the reference states and the ability to freely select these reference states. As a consequence, it is possible to explore systems that undergo substantially larger fluctuations than those considered in previous calculations, including semiflexible biopolymers having arbitrary ratios of contour length L to persistence length P. To validate the method, high accuracy is demonstrated for free energies of prime DNA knots with L/P=20 and L/P=40, corresponding to DNA lengths of 3000 and 6000 base pairs, respectively. We then apply the method to study the free-energy landscape for a model of a synaptic nucleoprotein complex containing a pair of looped domains, revealing a bifurcation in the location of optimal synapse (crossover) sites. This transition is relevant to target-site selection by DNA-binding proteins that occupy multiple DNA sites separated by large linear distances along the genome, a problem that arises naturally in gene regulation, DNA recombination, and the action of type-II topoisomerases.

Stefan M. Giovan; Robert G. Scharein; Andreas Hanke; Stephen D. Levene

2014-10-24

20

Free-energy calculations for semi-flexible macromolecules: Applications to DNA knotting and looping.  

PubMed

We present a method to obtain numerically accurate values of configurational free energies of semiflexible macromolecular systems, based on the technique of thermodynamic integration combined with normal-mode analysis of a reference system subject to harmonic constraints. Compared with previous free-energy calculations that depend on a reference state, our approach introduces two innovations, namely, the use of internal coordinates to constrain the reference states and the ability to freely select these reference states. As a consequence, it is possible to explore systems that undergo substantially larger fluctuations than those considered in previous calculations, including semiflexible biopolymers having arbitrary ratios of contour length L to persistence length P. To validate the method, high accuracy is demonstrated for free energies of prime DNA knots with L/P = 20 and L/P = 40, corresponding to DNA lengths of 3000 and 6000 base pairs, respectively. We then apply the method to study the free-energy landscape for a model of a synaptic nucleoprotein complex containing a pair of looped domains, revealing a bifurcation in the location of optimal synapse (crossover) sites. This transition is relevant to target-site selection by DNA-binding proteins that occupy multiple DNA sites separated by large linear distances along the genome, a problem that arises naturally in gene regulation, DNA recombination, and the action of type-II topoisomerases. PMID:25381542

Giovan, Stefan M; Scharein, Robert G; Hanke, Andreas; Levene, Stephen D

2014-11-01

21

Splint ligation of RNA with T4 DNA ligase  

PubMed Central

Splint ligation of RNA, whereby specific RNA molecules are ligated together, can be carried out using T4 DNA ligase and a bridging DNA oligonucleotide complementary to the RNAs. This method takes advantage of the property of T4 DNA ligase to join RNA molecules when they are in an RNA:DNA hybrid. Splint ligation is a useful tool for the introduction of modified nucleotides into RNA molecules, insertion of a radiolabel into a specific position within an RNA and for the assembly of smaller synthetic RNAs into longer RNA molecules. Such modifications enable a wide range of experiments to be carried out with the modified RNA including structural studies, co-immunoprecipitations, and the ability to map sites of RNA:RNA and RNA:protein interactions. PMID:23065567

Kershaw, Christopher J.; O’Keefe, Raymond T.

2014-01-01

22

Pore forming polyalkylpyridinium salts from marine sponges versus synthetic lipofection systems: distinct tools for intracellular delivery of cDNA and siRNA  

Microsoft Academic Search

BACKGROUND: Haplosclerid marine sponges produce pore forming polyalkylpyridinium salts (poly-APS), which can be used to deliver macromolecules into cells. The aim of this study was to investigate the delivery of DNA, siRNA and lucifer yellow into cells mediated by poly-APS and its potential mechanisms as compared with other lipofection systems (lipofectamine and N4,N9-dioleoylspermine (LipoGen)). DNA condensation was evaluated and HEK

Debra McLaggan; Noppadon Adjimatera; Kristina Sep?i?; Marcel Jaspars; David J MacEwan; Ian S Blagbrough; Roderick H Scott

2006-01-01

23

Isolation of RNA and DNA fragments using diatomaceous earth  

Microsoft Academic Search

A series of simple and phenol-free silica-based protocols for isolating and cleaning RNA or DNA fragments from different sources were developed. Cytoplasmic RNA isolated from hybridoma cells by this method was used in RT-PCR. DNA fragments obtained using this protocol were suitable for further subcloning, gene transformation and DNA sequencing. © Rapid Science Ltd. 1998

Kai Koo; Peggy M. Foegeding; Harold E. Swaisgood

1998-01-01

24

DNA and RNA Quadruplex-Binding Proteins  

PubMed Central

Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc.), the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGG)n, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2) and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development. PMID:25268620

Brazda, Vaclav; Haronikova, Lucia; Liao, Jack C. C.; Fojta, Miroslav

2014-01-01

25

DNA and RNA quadruplex-binding proteins.  

PubMed

Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc.), the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGG)n, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2) and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development. PMID:25268620

Brázda, Václav; Hároníková, Lucia; Liao, Jack C C; Fojta, Miroslav

2014-01-01

26

Efficient isolation of total RNA from Clostridium without DNA contamination  

Microsoft Academic Search

Several molecular techniques require high-quality RNA, free from DNA. Various methods have been described to obtain RNA to be used in expression studies or as starting material in differential display-reverse transcriptase (dd-RT-PCR), for which high-quality RNA free from DNA is an essential requirement. In this report, we compare three different methods to isolate RNA from Gram-positive bacteria: (1) An acid–phenol

S. Nuyts; L. Van Mellaert; P. Lambin; J. Anné

2001-01-01

27

Polynucleotide 3'-terminal Phosphate Modifications by RNA and DNA Ligases.  

PubMed

RNA and DNA ligases catalyze the formation of a phosphodiester bond between the 5'-phosphate and 3'-hydroxyl ends of nucleic acids. In this work, we describe the ability of the thermophilic RNA ligase MthRnl from Methanobacterium thermoautotrophicum to recognize and modify the 3'-terminal phosphate of RNA and single-stranded DNA (ssDNA). This ligase can use an RNA 3'p substrate to generate an RNA 2',3'-cyclic phosphate or convert DNA3'p to ssDNA(3')pp(5')A. An RNA ligase from the Thermus scotoductus bacteriophage TS2126 and a predicted T4 Rnl1-like protein from Thermovibrio ammonificans, TVa, were also able to adenylate ssDNA 3'p. These modifications of RNA and DNA 3'-phosphates are similar to the activities of RtcA, an RNA 3'-phosphate cyclase. The initial step involves adenylation of the enzyme by ATP, which is then transferred to either RNA 3'p or DNA 3'p to generate the adenylated intermediate. For RNA (3')pp(5')A, the third step involves attack of the adjacent 2' hydroxyl to generate the RNA 2',3'-cyclic phosphate. These steps are analogous to those in classical 5' phosphate ligation. MthRnl and TS2126 RNA ligases were not able to modify a 3'p in nicked double-stranded DNA. However, T4 DNA ligase and RtcA can use 3'-phosphorylated nicks in double-stranded DNA to produce a 3'-adenylated product. These 3'-terminal phosphate-adenylated intermediates are substrates for deadenylation by yeast 5'Deadenylase. Our findings that classic ligases can duplicate the adenylation and phosphate cyclization activity of RtcA suggests that they have an essential role in metabolism of nucleic acids with 3'-terminal phosphates. PMID:25324547

Zhelkovsky, Alexander M; McReynolds, Larry A

2014-11-28

28

Transcript-RNA-templated DNA recombination and repair.  

PubMed

Homologous recombination is a molecular process that has multiple important roles in DNA metabolism, both for DNA repair and genetic variation in all forms of life. Generally, homologous recombination involves the exchange of genetic information between two identical or nearly identical DNA molecules; however, homologous recombination can also occur between RNA molecules, as shown for RNA viruses. Previous research showed that synthetic RNA oligonucleotides can act as templates for DNA double-strand break (DSB) repair in yeast and human cells, and artificial long RNA templates injected in ciliate cells can guide genomic rearrangements. Here we report that endogenous transcript RNA mediates homologous recombination with chromosomal DNA in yeast Saccharomyces cerevisiae. We developed a system to detect the events of homologous recombination initiated by transcript RNA following the repair of a chromosomal DSB occurring either in a homologous but remote locus, or in the same transcript-generating locus in reverse-transcription-defective yeast strains. We found that RNA-DNA recombination is blocked by ribonucleases H1 and H2. In the presence of H-type ribonucleases, DSB repair proceeds through a complementary DNA intermediate, whereas in their absence, it proceeds directly through RNA. The proximity of the transcript to its chromosomal DNA partner in the same locus facilitates Rad52-driven homologous recombination during DSB repair. We demonstrate that yeast and human Rad52 proteins efficiently catalyse annealing of RNA to a DSB-like DNA end in vitro. Our results reveal a novel mechanism of homologous recombination and DNA repair in which transcript RNA is used as a template for DSB repair. Thus, considering the abundance of RNA transcripts in cells, RNA may have a marked impact on genomic stability and plasticity. PMID:25186730

Keskin, Havva; Shen, Ying; Huang, Fei; Patel, Mikir; Yang, Taehwan; Ashley, Katie; Mazin, Alexander V; Storici, Francesca

2014-11-20

29

Unusual Thermal Stability of RNA/[RP-PS]-DNA/RNA Triplexes Containing a Homopurine DNA Strand  

PubMed Central

Homopurine deoxyribonucleoside phosphorothioates, as short as hexanucleotides and possessing all internucleotide linkages of RP configuration, form a triple helix with two RNA or 2?-OMe-RNA strands, with Watson-Crick and Hoogsteen complementarity. Melting temperature and fluorescence quenching experiments strongly suggest that the Hoogsteen RNA strand is parallel to the homopurine [RP-PS]-oligomer. Remarkably, these triplexes are thermally more stable than complexes formed by unmodified homopurine DNA molecules of the same sequence. The triplexes formed by phosphorothioate DNA dodecamers containing 4–6 dG residues are thermally stable at pH 7.4, although their stability increases significantly at pH 5.3. FTIR measurements suggest participation of the C2-carbonyl group of the pyrimidines in the stabilization of the triplex structure. Formation of triple-helix complexes with exogenously delivered PS-oligos may become useful for the reduction of RNA accessibility in vivo and, hence, selective suppression/inhibition of the translation process. PMID:17218459

Guga, Piotr; Boczkowska, Malgorzata; Janicka, Magdalena; Maciaszek, Anna; Kuberski, Slawomir; Stec, Wojciech J.

2007-01-01

30

DNA-RNA-Protein - Nobel Prize Educational Tutorial  

NSDL National Science Digital Library

The Nobel Prize in Physiology or Medicine 1959 was awarded jointly to Severo Ochoa and Arthur Kornberg "for their discovery of the mechanisms in the biological synthesis of ribonucleic acid and deoxyribonucleic acid." This tutorial goes through DNA replication, RNA transcription, RNA processing, mRNA transport, and protein translation. The tutorial has two levels: basic and advanced.

2009-01-01

31

Conserved tRNA gene cluster in starfish mitochondrial DNA  

Microsoft Academic Search

Partial sequencing of mtDNA from four long-diverged species of starfish reveals the existence of a conserved cluster of 13 tRNA genes, organized in a manner similar to that of the tRNA cluster of sea urchin mtDNA, but located at a position distant from the presumed replication origin. These findings suggest that a clustered organization of tRNA genes may have been

Howard T. Jacobs; Shuichi Asakawa; Takeyoshi Araki; Kin-ichiro Miura; Michael J. Smith; Kimitsuna Watanabe

1989-01-01

32

RADIA: RNA and DNA Integrated Analysis for Somatic Mutation Detection  

PubMed Central

The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA) to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis), a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual’s DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84%) and very high precision (98% and 99%) for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA. PMID:25405470

Radenbaugh, Amie J.; Ma, Singer; Ewing, Adam; Stuart, Joshua M.; Collisson, Eric A.; Zhu, Jingchun; Haussler, David

2014-01-01

33

Spatial and Temporal Variations in Bacterial Macromolecule Labeling with [methyl-H]Thymidine in a Hypertrophic Lake.  

PubMed

The incorporation of [methyl-H]thymidine into three macromolecular fractions, designated as DNA, RNA, and protein, by bacteria from Hartbeespoort Dam, South Africa, was measured over 1 year by acid-base hydrolysis procedures. Samples were collected at 10 m, which was at least 5 m beneath the euphotic zone. On four occasions, samples were concurrently collected at the surface. Approximately 80% of the label was incorporated into bacterial DNA in surface samples. At 10 m, total incorporation of label into bacterial macromolecules was correlated to bacterial utilization of glucose (r = 0.913, n = 13, P < 0.001). The labeling of DNA, which ranged between 0 and 78% of total macromolecule incorporation, was inversely related to glucose uptake (r = -0.823), total thymidine incorporation (r = -0.737), and euphotic zone algal production (r = -0.732, n = 13, P < 0.005). With decreased DNA labeling, increasing proportions of label were found in the RNA fraction and proteins. Enzymatic digestion followed by chromatographic separation of macromolecule fragments indicated that DNA and proteins were labeled while RNA was not. The RNA fraction may represent labeled lipids or other macromolecules or both. The data demonstrated a close coupling between phytoplankton production and heterotrophic bacterial activity in this hypertrophic lake but also confirmed the need for the routine extraction and purification of DNA during [methyl-H]thymidine studies of aquatic bacterial production. PMID:16347241

Robarts, R D; Wicks, R J; Sephton, L M

1986-12-01

34

Thermodynamic DNA-RNA hybridization software with applications to crispr RNA, an  

E-print Network

Thermodynamic DNA-RNA hybridization software with applications to crispr RNA, an acquired Repeats (crispr) and crispr-associated sequence (cas) proteins constitute a remarkable acquired, heritable (Lamarckian) immune system that is widespread in Bacteria and Archaea. RNA transcripts of crispr arrays, known

Clote, Peter

35

Drug binding to DNA x RNA hybrid structures.  

PubMed

The DNA x RNA hybrid duplexes are functionally important structures in gene expression that are underutilized as potential drug targets. Several tools are described here for the discovery and characterization of small molecules capable of the selective recognition of DNA x RNA hybrid structures. Competition dialysis and thermal denaturation of mixtures of polynucleotide structures can be used to identify small molecules that bind selectively to DNA x RNA hybrids. An assay that measures small molecule inhibition of RNase H can be used to measure a functional response to these ligands. PMID:19997877

Wheelhouse, Richard T; Chaires, Jonathan B

2010-01-01

36

RNA intrusions change DNA elastic properties and structure  

NASA Astrophysics Data System (ADS)

The units of RNA, termed ribonucleoside monophosphates (rNMPs), have been recently found as the most abundant defects present in DNA. Despite the relevance, it is largely unknown if and how rNMPs embedded in DNA can change the DNA structure and mechanical properties. Here, we report that rNMPs incorporated in DNA can change the elastic properties of DNA. Atomic force microscopy (AFM)-based single molecule elasticity measurements show that rNMP intrusions in short DNA duplexes can decrease - by 32% - or slightly increase the stretch modulus of DNA molecules for two sequences reported in this study. Molecular dynamics simulations and nuclear magnetic resonance spectroscopy identify a series of significant local structural alterations of DNA containing embedded rNMPs, especially at the rNMPs and nucleotide 3' to the rNMP sites. The demonstrated ability of rNMPs to locally alter DNA mechanical properties and structure may help in understanding how such intrusions impact DNA biological functions and find applications in structural DNA and RNA nanotechnology.The units of RNA, termed ribonucleoside monophosphates (rNMPs), have been recently found as the most abundant defects present in DNA. Despite the relevance, it is largely unknown if and how rNMPs embedded in DNA can change the DNA structure and mechanical properties. Here, we report that rNMPs incorporated in DNA can change the elastic properties of DNA. Atomic force microscopy (AFM)-based single molecule elasticity measurements show that rNMP intrusions in short DNA duplexes can decrease - by 32% - or slightly increase the stretch modulus of DNA molecules for two sequences reported in this study. Molecular dynamics simulations and nuclear magnetic resonance spectroscopy identify a series of significant local structural alterations of DNA containing embedded rNMPs, especially at the rNMPs and nucleotide 3' to the rNMP sites. The demonstrated ability of rNMPs to locally alter DNA mechanical properties and structure may help in understanding how such intrusions impact DNA biological functions and find applications in structural DNA and RNA nanotechnology. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr01794c

Chiu, Hsiang-Chih; Koh, Kyung Duk; Evich, Marina; Lesiak, Annie L.; Germann, Markus W.; Bongiorno, Angelo; Riedo, Elisa; Storici, Francesca

2014-08-01

37

Bridging the solution divide: comprehensive structural analyses of dynamic RNA, DNA, and protein assemblies by small angle X-ray scattering  

PubMed Central

Summary Small-Angle X-ray Scattering (SAXS) is changing how we perceive biological structures, because it reveals dynamic macromolecular conformations and assemblies in solution. SAXS information captures thermodynamic ensembles, enhances static structures detailed by high-resolution methods, uncovers commonalities among diverse macromolecules, and helps define biological mechanisms. SAXS-based experiments on RNA riboswitches and ribozymes and on DNA-protein complexes including DNA-PK and p53 discover flexibilities that better define structure-function relationships. Furthermore, SAXS results suggest conformational variation is a general functional feature of macromolecules. Thus, accurate structural analyses will require a comprehensive approach that assesses both flexibility, as seen by SAXS, and detail, as determined by X-ray crystallography and NMR. Here, we review recent SAXS computational tools, technologies, and applications to nucleic acids and related structures. PMID:20097063

Rambo, Robert P.; Tainer, John A.

2010-01-01

38

Biological Macromolecule Crystallization Database  

National Institute of Standards and Technology Data Gateway

SRD 21 Biological Macromolecule Crystallization Database (Web, free access)   The Biological Macromolecule Crystallization Database and NASA Archive for Protein Crystal Growth Data (BMCD) contains the conditions reported for the crystallization of proteins and nucleic acids used in X-ray structure determinations and archives the results of microgravity macromolecule crystallization studies.

39

a Hypothetical Pathway from the RNA to the DNA World  

NASA Astrophysics Data System (ADS)

If the DNA world was preceded by a RNA world as widely suggested a rational pathway should be discernable to link the two. This report uses as a starting point a membrane-enclosed ribozyme capable of polymerising itself and its counterpart copy. As molecular complexity increased, it is suggested that a consortia of the initial ribozyme polymerase and chaperone molecules formed a complex specifically for RNA replication. A mutation in one of several copy-genomes coding for these replication machines then led in step-wise fashion to a proto-ribosome that increasingly inserted specific amino acids instead of nucleotides into a growing RNA chain, the driving force being selection for improved or new function. Eventually the nucleotides would be entirely displaced in this proto-ribosome, after which the ribose-phosphate linkage would be replaced by peptide linkage. The final steps would be the formation of DNA from the RNA genomic material viareverse transcriptase, coupled with the evolution of enzymes for DNA polymerisation and transcription. At this point the original RNA-replicator machinery would be redundant and eliminated, the RNA genomic material would become mRNA and the present-day function of the ribosome would be fixed. In the scenario described a mechanism for the selection for l-amino acids becomes evident

Line, Martin A.

2005-08-01

40

1780 Macromolecules 1989, 22, 1780-1786 The Action of Interhelical Forces on the Organization of DNA  

E-print Network

of DNA Double Helices: Fluctuation-Enhanced Decay of Electrostatic Double-Layer and Hydration Forces Rudi-rangepotentials. At no point does the force vary in the way expected from traditional double-layer theory. Indeed separation, to a region of elec- trostatic double-layer interaction. The puzzle was that even here

Rau, Don C.

41

How can plant DNA viruses evade siRNA-directed DNA methylation and silencing?  

PubMed

Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes. PMID:23887650

Pooggin, Mikhail M

2013-01-01

42

How Can Plant DNA Viruses Evade siRNA-Directed DNA Methylation and Silencing?  

PubMed Central

Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes. PMID:23887650

Pooggin, Mikhail M.

2013-01-01

43

Molecular-Sized DNA or RNA Sequencing Machine  

Cancer.gov

Current high-throughput DNA sequencing methods suffer from several limitations. Many methods require multiple fluid handling steps, fixing of molecules on beads or a 2D surface, and provide very short read-lengths. Researchers at the National Cancer Institute's Gene Regulation and Chromosome Biology Laboratory offer a potential DNA or RNA sequencing device that drastically simplifies the process by combining all elements for sequence detection in a single molecule.

44

Molecular recognition of a DNA:RNA hybrid: Sub-nanomolar binding by a neomycinmethidium conjugate  

E-print Network

Ethidium bromide Intercalator Neomycin Aminoglycoside RNA DNA:RNA hybrid Telomerase a b s t r a c t A novel hypothesized that by conjugating neomycin with a specific DNA:RNA binding intercalator, ethidium bromide, highMolecular recognition of a DNA:RNA hybrid: Sub-nanomolar binding by a neomycin­methidium conjugate

Stuart, Steven J.

45

RNA-DNA Interactions and DNA Methylation in PostTranscriptional Gene Silencing  

Microsoft Academic Search

Post-transcriptional gene silencing (PTGS) is a homology-dependent process that reduces cytoplasmic RNA levels. In several experimental systems, there is also an association of PTGS with methylation of DNA. To investigate this associ- ation, we used plants carrying a transgene encoding the green fluorescent protein (GFP). Gene silencing was induced using potato virus X RNA vectors carrying parts of the coding

Al Jones; Andrew J. Hamilton; Olivier Voinnet; Carole L. Thomas; Andrew J. Maule; David C. Baulcombe

1999-01-01

46

DNA?RNA: What Do Students Think the Arrow Means?  

ERIC Educational Resources Information Center

The central dogma of molecular biology, a model that has remained intact for decades, describes the transfer of genetic information from DNA to protein though an RNA intermediate. While recent work has illustrated many exceptions to the central dogma, it is still a common model used to describe and study the relationship between genes and protein…

Wright, L. Kate; Fisk, J. Nick; Newman, Dina L.

2014-01-01

47

Isolation of an RNA-Directed RNA Polymerase Specific cDNA Clone from Tomato  

Microsoft Academic Search

A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP

Winfried Schiebel; Thierry Pélissier; Leonhard Riedel; Sabine Thalmeir; Rosemarie Schiebel; Dirk Kempe; Friedrich Lottspeich; Heinz L. Sänger; Michael Wassenegger; Worringer Weg

1998-01-01

48

Function of DNA polymerase I in RNA-primed synthesis of bacteriophage M-13 duplex DNA.  

PubMed Central

Cell-free extracts from Escherichia coli contain a DNA polymerase activity resistant to SH-blocking agents, which is capable of synthesizing complementary strand DNA on a circular M-13 DNA template by extension of RNA primers. This activity is considered to be identical with DNA polymerase I (or some altered form of this enzyme) since it is missing in extracts from po1A- cells. DNA synthesis in the presence of SH-blocking agents occurs at a reduced rate as compared to untreated controls and leads to the formation of DNA chains of defined size (0.4-0.5 genome's length). It is concluded that efficient M-13 duplex DNA synthesis requires the cooperation of both DNA polymerase I and III. PMID:1272793

Schneck, P K; Staudenbauer, W L; Hofschneider, P H

1976-01-01

49

Single-Molecule Electrical Random Resequencing of DNA and RNA  

NASA Astrophysics Data System (ADS)

Two paradigm shifts in DNA sequencing technologies--from bulk to single molecules and from optical to electrical detection--are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals. Here we report single-molecule electrical resequencing of DNA and RNA using a hybrid method of identifying single-base molecules via tunneling currents and random sequencing. Our method reads sequences of nine types of DNA oligomers. The complete sequence of 5'-UGAGGUA-3' from the let-7 microRNA family was also identified by creating a composite of overlapping fragment sequences, which was randomly determined using tunneling current conducted by single-base molecules as they passed between a pair of nanoelectrodes.

Ohshiro, Takahito; Matsubara, Kazuki; Tsutsui, Makusu; Furuhashi, Masayuki; Taniguchi, Masateru; Kawai, Tomoji

2012-07-01

50

A method to determine RNA and DNA oxidation simultaneously by HPLC-ECD: greater RNA than DNA oxidation in rat liver after doxorubicin administration.  

PubMed

We developed a novel method for the simultaneous extraction and analysis of total tissue RNA and DNA to quantify the RNA and DNA oxidation products 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine using HPLC coupled to electrochemical detection (HPLC-ECD). The protein denaturing agents guanidine thiocyanate and phenol/chloroform at neutral pH were found to be very efficient for the isolation of RNA and DNA from rat brain, liver and muscle. The method is very fast, allows extraction at 0 degrees C, gives high yields of pure RNA and DNA with low background oxidation levels, and also determines the RNA/DNA ratio. Experiments with isolated RNA and DNA exposed to the Fenton reagents H2O2/ascorbate/Fe3+ (or Cu2+) resulted in significantly greater RNA oxidation. The RNase inhibitor 2-mercaptoethanol, commonly used for RNA extraction, acted as a pro-oxidant during nucleic acid extraction, an effect attenuated by the inclusion of the metal chelator deferoxamine mesylate. In vivo, administration of doxorubicin (an oxidant generator) to Fisher-344 rats resulted in a significant increase in liver RNA oxidation, but no significantly increased DNA oxidation. This new method could be useful to assess oxidatively damaged RNA and DNA simultaneously, and our data show that RNA is more susceptible to oxidative stress than DNA in vivo and in vitro. PMID:16497170

Hofer, Tim; Seo, Arnold Y; Prudencio, Mercedes; Leeuwenburgh, Christiaan

2006-01-01

51

A DNA enzyme with Mg(2+)-Dependent RNA Phosphoesterase Activity  

NASA Technical Reports Server (NTRS)

Previously we demonstrated that DNA can act as an enzyme in the Pb(2+)-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of approx. 10(exp -4)/ min in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23 C. The Mg(2+) - dependent reaction is more difficult, with an uncatalyzed rate of approx. 10(exp -7)/ min under comparable conditions. Mg(2+) - dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg(2+) - dependent reaction. Results: We generated a population of greater than 10(exp 13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg(2+), Mn(2+), Zn(2+) or Pb(2+). Examination of individual clones from the Mg(2+) lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction.This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01/ min. The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions. Conclusions: We have generated a Mg(2+) - dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approx. 10(exp 5) - fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.

Breaker, Ronald R.; Joyce, Gerald F.

1995-01-01

52

DNA nanostructure-based ultrasensitive electrochemical microRNA biosensor.  

PubMed

MicroRNAs (miRNAs) are key regulators of a wide range of cellular processes, and have been identified as promising cancer biomarkers due to their stable presence in serum. As an surface-based electrochemical biosensors which offer great opportunities for low-cost, point-of-care tests (POCTs) of disease-associated miRNAs. Nevertheless, the sensitivity of miRNA sensors is often limited by mass transport and the surface crowding effect at the water-electrode interface. Here, we present a protocol as well as guidelines for ultrasensitive detection of miRNA with DNA nanostructure-based electrochemical miRNA biosensor. By employing the three-dimensional DNA nanostructure-based interfacial engineering approach, we can directly detect as few as attomolar (<1000 copies) miRNAs with high single-base discrimination ability. Since this ultrasensitive electrochemical miRNA sensor (EMRS) is highly reproducible and essentially free of prior target labeling and PCR amplification, it can conveniently and reliably analyze miRNA expression levels in clinical samples from esophageal squamous cell carcinoma (ESCC) patients. PMID:23911620

Wen, Yanli; Liu, Gang; Pei, Hao; Li, Lanying; Xu, Qin; Liang, Wen; Li, Yan; Xu, Li; Ren, Suzhen; Fan, Chunhai

2013-12-15

53

Synthesis and structural characterization of piperazino-modified DNA that favours hybridization towards DNA over RNA  

PubMed Central

We report the synthesis of two C4?-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4?-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilization to a maximum of 9°C per incorporation. Using fluorescence, ultraviolet and nuclear magnetic resonance (NMR) spectroscopy, we show that the stabilization is achieved by pyrene intercalation in the dsDNA duplex. The pyrene moiety is not restricted to one intercalation site but rather switches between multiple sites in intermediate exchange on the NMR timescale, resulting in broad lines in NMR spectra. We identified two intercalation sites with NOE data showing that the pyrene prefers to intercalate one base pair away from the modified nucleotide with its linker curled up in the minor groove. Both modifications are tolerated in DNA:RNA hybrids but leave their melting temperatures virtually unaffected. Fluorescence data indicate that the pyrene moiety is residing outside the helix. The available data suggest that the DNA discrimination is due to (i) the positive charge of the piperazino ring having a greater impact in the narrow and deep minor groove of a B-type dsDNA duplex than in the wide and shallow minor groove of an A-type DNA:RNA hybrid and (ii) the B-type dsDNA duplex allowing the pyrene to intercalate and bury its apolar surface. PMID:21062815

Skov, Joan; Bryld, Torsten; Lindegaard, Dorthe; Nielsen, Katrine E.; Højland, Torben; Wengel, Jesper; Petersen, Michael

2011-01-01

54

RNA's role in the cell, James WatsonSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Interviewee: James Watson DNAi Location:Code>Copying the code>players What does RNA do? After solving the structure of DNA, James Watson started working on RNA. He talks about what he thought of RNA and its function.

2008-10-06

55

Polyadenylated RNA complementary to repetitive DNA in mouse L-cells.  

PubMed

Complementary DNA, synthesized with L-cell polyadenylated RNA as template, renatured with total L-cell DNA to about 70%. About 30% complementary to unique sequence DNA and another 10 and 30% corresponded to sequences about 20- and 500-fold repetitive. Complementary DNA was fractionated after partial hybridization with total polyadenylated RNA to obtain preparations enriched or impoverished in complements of the most frequent polyadenylated RNA. Renaturation of these complementary DNA fractions with L-cell DNA revealed that most frequent RNAs are transcribed from repetitive DNA sequences, Complementary DNA, density labeled with bromodeoxyuridine, was fractionated by renaturation with L-cell DNA to yield fractions enriched in repetitive and unique sequence DNA. The denisty labeled complementary DNA was purified by equilibrium centrifiguation in an alkaline Cs2SO4 gradient. The complementary DNA representing mainly repetitive DNA sequences hybridized preferentially to frequent polyadenylated RNA. PMID:1168486

Ryffel, G U; McCarthy, B J

1975-04-01

56

Redefining regulation of DNA methylation by RNA interference  

PubMed Central

Epigenetic changes refer to heritable changes that may modulate gene expression without affecting DNA sequence. DNA methylation is one such heritable epigenetic change, which is causally associated with the transcription regulation of many genes in the mammalian genome. Altered DNA methylation has been implicated in a wide variety of human diseases including cancer. Understanding the regulation of DNA methylation is likely to improve the ability to diagnose and treat these diseases. With the advent of high-throughput RNA interference (RNAi) screens, answering epigenetic questions on a genomic scale is now possible. Two recent genome-wide RNAi screens have addressed the regulation of DNA methylation in cancer, leading to the identification of the regulators of epigenetic silencing by oncogenic RAS and how epigenetic silencing of the tumor suppressor RASSF1A is maintained. These RNAi screens have much wider applications, since similar screens can now be adapted to identify the mechanism of silencing of any human disease-associated gene that is epigenetically regulated. In this review, we discuss two recent genome-wide RNAi screens for epigenetic regulators and explore potential applications in understanding DNA methylation and gene expression regulation in mammalian cells. We also discuss some of the key unanswered questions in the field of DNA methylation and suggest genome-wide RNAi screens designed to answer them. PMID:20620207

Muthusamy, Viswanathan; Bosenberg, Marcus; Wajapeyee, Narendra

2013-01-01

57

Electron Attachment to DNA and RNA Nucleobases: An EOMCC Investigation  

E-print Network

We report a benchmark theoretical investigation of both adiabatic and vertical electron affinities of five DNA and RNA nucleobases: adenine, guanine, cytosine, thymine and uracil using state-of-the-art equation of motion coupled cluster (EOMCC) method. We have calculated the vertical electron affinity values of first five electron attached states of the DNA and RNA nucleobases and only the first electron attached state is found to be energetically accessible in gas phase. An analysis of the natural orbitals shows that the first electron attached states of uracil and thymine are valence-bound type and undergo significant structural changes on attachment of excess electron, which is reflected in the deviation of the adiabatic electron affinity from the vertical one. On the other hand, the first electron attached state of cytosine, adenine and guanine are dipole-bound type and their structure remain unaffected on attachment of an extra electron, which results in small deviation of adiabatic electron affinity fro...

Dutta, Chintya Kumar; Vaval, Nayana; Pal, Sourav

2014-01-01

58

DNA damage: RNA-binding proteins protect from near and far.  

PubMed

Recent work, including large-scale genetic and molecular analyses, identified RNA-binding proteins (RBPs) as major players in the prevention of genome instability. These studies show that RBPs prevent harmful RNA/DNA hybrids and are involved in the DNA damage response (DDR), from DNA repair to cell survival decisions. Indeed, specific RBPs allow the selective regulation of DDR genes at multiple post-transcriptional levels (from pre-mRNA splicing/polyadenylation to mRNA stability/translation) and are directly involved in DNA repair. These multiple activities are mediated by RBP binding to mRNAs, nascent transcripts, noncoding RNAs, and damaged DNA. Finally, because DNA damage modifies RBP localization and binding to different RNA/DNA molecules, we propose that upon DNA damage, RBPs coordinately regulate various aspects of both RNA and DNA metabolism. PMID:24534650

Dutertre, Martin; Lambert, Sarah; Carreira, Aura; Amor-Guéret, Mounira; Vagner, Stéphan

2014-03-01

59

Biochemical studies of the rat seminiferous epithelial wave : DNA and RNA syntheses and effects of adriamycin  

E-print Network

the effects of adriamycin on both DNA and RNA syntheses. Two distinct peaks of DNA synthe- sis were seenBiochemical studies of the rat seminiferous epithelial wave : DNA and RNA syntheses and effects to measure the rate of DNA synthesis in different segments of the seminiferous epithelial wave and to analyze

Paris-Sud XI, Université de

60

Did the Pre-RNA World Rest Upon DNA Molecules?  

NASA Technical Reports Server (NTRS)

The isolation of a DNA sequence that catalyzes the ligation of oligodeoxynucleotides via the formation of 3' - 5' phosphodiester linkage significance in selection experiments has been reported. Ball recently used this to discuss the possibility that natural DNA molecules may have formed in the primitive Earth leading to the origin of life. As noted by Ferris and Usher, if metabolic pathways evolved backwards, it could be argued that the biosynthesis of 2-deoxyribose from ribose suggests that RNA came from DNA. As summarized elsewhere, there are several properties of deoxyribose which could be interpreted to support the possibility that DNA-like molecules arose prior to the RNA world. For example, 2-deoxyribose is slightly more soluble than ribose (which may have been an advantage in a drying pool scenario), may have been more reactive under possible prebiotic conditions (it forms a nucleoside approx. 150 times faster than ribose with the alternative base urazole at 25 C), while it decomposes in solution (approximately 2.6 times more slowly than ribose at 100 C). Other advantages of DNA over RNA are that it has one fewer chiral center, has a greater stability at the 8.2 pH value of the current oceans, and does not has the 2'5' and 3'5' ambiguity in polymerizations. Yet, there is strong molecular biological and biochemical evidence that RNA was featured in the biology well before the last common ancestor. The presence of sugar acids, including both ribo- and deoxysugar acids, in the 4.6 Ga old Murchison meteorite suggest that both may have been available in the primitive Earth, derived from the accretion of extraterrestrial sources and/or from endogenous processes involving formaldehyde and its derivatives. However, the abiotic synthesis of deoxyribose, ribose, and other sugars from glyceraldehyde and acetaldehyde under alkaline conditions is inefficient and unespecific. Although sugars are labile compounds, the role of cyanamide or borate minerals in the stabilization of the cyclic forms of ribose and other pentoses has recently been demonstrated. Nonetheless, the assumption either RNA or DNA was the first genetic material needs to be supplemented by laboratory models demonstrating that the prebiotic synthesis of activated beta-D-(deoxy)ribonucleotides and their polymers was feasible. As of today such evidence is lacking, and there is no convincing synthesis of any nucleotide, since all model experiments produce complex mixtures of products in which there is no preferential synthesis of chiral D-nucleotides. This strongly suggests that both DNA and RNA may have been preceded by pairing structures much simpler than extant nucleic acids. It is doubtful that DNA molecules, or indeed other (de0xy)ribofuranoid oligonucleotides formed the basis of these as yet undescribed pre-RNA worlds.

Lazcano, Antonio; Dworkin, Jason P.; Miller, Stanley L.

2004-01-01

61

The RNA World: Life Before DNA and Protein  

NASA Technical Reports Server (NTRS)

All of the life that is known, all organisms that exist on Earth today or are known to have existed on Earth in the past, are of the same life form: a life form based on DNA and protein. It does not necessarily have to be that way. Why not have two competing life forms on this planet? Why not have biology as we know it and some other biology that occupies its own distinct niche? Yet that is not how evolution has played out. From microbes living on the surface of antarctic ice to tube worms lying near the deep-sea hydrothermal vents, all known organisms on this planet are of the same biology. Looking at the single known biology on Earth, it is clear that this biology could not have simply sprung forth from the primordial soup. The biological system that is the basis for all known. life is far too complicated to have arisen spontaneously. This brings us to the notion that something else something simpler, must have preceded life based on DNA and protein. One suggestion that has gained considerable acceptance over the past decade is that DNA and protein-based life was preceded by RNA-based life in a period referred to as the 'RNA world'. Even an RNA-based life form would have been fairly complicated - not as complicated as our own DNA- and protein-based life form - but far too complicated, according to prevailing scientific thinking, to have arisen spontaneously from the primordial soup. Thus, it has been argued that something else must have preceded RNA-based life, or even that there was a succession of life forms leading from the primordial soup to RNA-based life. The experimental evidence to support this conjecture is not strong because, after all, the origin of life was a historical event that left no direct physical record. However, based on indirect evidence in both the geological record and the phylogenetic record of evolutionary history on earth, it is possible to reconstruct a rough picture of what life was like before DNA and protein.

Joyce, Gerald F.

1993-01-01

62

Detection Theory in Identification of RNA-DNA Sequence Differences Using RNA-Sequencing  

PubMed Central

Advances in sequencing technology have allowed for detailed analyses of the transcriptome at single-nucleotide resolution, facilitating the study of RNA editing or sequence differences between RNA and DNA genome-wide. In humans, two types of post-transcriptional RNA editing processes are known to occur: A-to-I deamination by ADAR and C-to-U deamination by APOBEC1. In addition to these sequence differences, researchers have reported the existence of all 12 types of RNA-DNA sequence differences (RDDs); however, the validity of these claims is debated, as many studies claim that technical artifacts account for the majority of these non-canonical sequence differences. In this study, we used a detection theory approach to evaluate the performance of RNA-Sequencing (RNA-Seq) and associated aligners in accurately identifying RNA-DNA sequence differences. By generating simulated RNA-Seq datasets containing RDDs, we assessed the effect of alignment artifacts and sequencing error on the sensitivity and false discovery rate of RDD detection. Overall, we found that even in the presence of sequencing errors, false negative and false discovery rates of RDD detection can be contained below 10% with relatively lenient thresholds. We also assessed the ability of various filters to target false positive RDDs and found them to be effective in discriminating between true and false positives. Lastly, we used the optimal thresholds we identified from our simulated analyses to identify RDDs in a human lymphoblastoid cell line. We found approximately 6,000 RDDs, the majority of which are A-to-G edits and likely to be mediated by ADAR. Moreover, we found the majority of non A-to-G RDDs to be associated with poorer alignments and conclude from these results that the evidence for widespread non-canonical RDDs in humans is weak. Overall, we found RNA-Seq to be a powerful technique for surveying RDDs genome-wide when coupled with the appropriate thresholds and filters. PMID:25396741

Toung, Jonathan M.; Lahens, Nicholas; Hogenesch, John B.; Grant, Gregory

2014-01-01

63

RNase H and multiple RNA biogenesis factors cooperate to prevent RNA-DNA hybrids from generating genome instability  

PubMed Central

Genome instability, a hallmark of cancer progression, is thought to arise through DNA double strand breaks (DSBs). Studies in yeast and mammalian cells have shown that DSBs and instability can occur through RNA-DNA hybrids generated by defects in RNA elongation and splicing. We report that in yeast hybrids naturally form at many loci in wild-type cells, likely due to transcriptional errors, but are removed by two evolutionarily conserved RNase H enzymes. Mutants defective in transcriptional repression, RNA export and RNA degradation show increased hybrid formation and associated genome instability. One mutant, sin3?, changes the genome profile of hybrids, enhancing formation at ribosomal DNA. Hybrids likely induce damage in G1, S and G2/M as assayed by Rad52 foci. In summary, RNA-DNA hybrids are a potent source for changing genome structure. By preventing their formation and accumulation, multiple RNA biogenesis factors and RNAse H act as guardians of the genome. PMID:22195970

Wahba, Lamia; Amon, Jeremy D.; Koshland, Douglas; Vuica-Ross, Milena

2012-01-01

64

DNA-DEPENDENT RNA SYNTHESIS IN CHLOROPLASTS OF EUGLENA GRACILIS  

E-print Network

been shown to be capable of incorporating amino acids into protein (2). This incorporation is inhibited by actinomycin D, a fact which suggests that the chloroplasts might be capable of DNAdependent RNA synthesis. Such RNA synthesis is also indicated in a recent report (6) showing inhibition of chlorophyll synthesis by actinomycin. In conjunction with our studies of chloroplast synthesis and replication in Euglena, we have investigated this phenomenon and present here evidence for DNA-dependent RNA synthesis in isolated chloroplasts of this organism. Chlorophyll formation in colorless Euglena cells is also reinvestigated with regard to the synthesis of RNA and DNA. Much evidence has been accumulating which indicates that the DNA of Euglena chloroplasts may have a genetic role in the inheritance of chloroplasts (5, 7). The ability of this DNA to direct RNA synthesis might add support for this contention. Euglena gradlis strain Z was grown heterotropically on defined medium in light or dark at room temperature (25°C) with constant agitation at moderate speed, as described previously (5). Effects of Actinomycin D and Mitomycin C on the Synthesis of Chlorophyll Dark-grown colorless cells in the log phase of growth were collected and transferred into fresh neutral medium, pH 6.8-7.2, containing actinomycin D (20 #g/ml) or mitomycin C (30 #g/ml) and allowed to become green under normal illumination. Aliquots of cells were taken at intervals of several hours. The number of cells was determined by haemocytometer and the chloro-phyll was determined by measuring the optical density at a wavelength of 665 m#. The results are shown in Table I. Although mltomycin C-treated cells do not undergo any further division, there is no inhibition cf chlorophyll formation. In fact, for a period of 38 hr they make almost twice the amount of chlorophyll per cell as compared with nontreated control~. Lctinomycin D, on the other hand, inhibited synthesis of chlorophyll soon after its introduction into the medium. After 38 hr in actinomycin D, the ceils appear to have less than 17 % of the amount of chlorophyll found in control cultures.

Vinod C. Shah; Harvard Lyman

65

DNA targeting specificity of RNA-guided Cas9 nucleases  

PubMed Central

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of singleguide RNAs (sgRNAs) to enable genome editing1–10. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses. PMID:23873081

Hsu, Patrick D; Scott, David A; Weinstein, Joshua A; Ran, F Ann; Konermann, Silvana; Agarwala, Vineeta; Li, Yinqing; Fine, Eli J; Wu, Xuebing; Shalem, Ophir; Cradick, Thomas J; Marraffini, Luciano A; Bao, Gang; Zhang, Feng

2014-01-01

66

Structural Basis for Telomerase Catalytic Subunit TERT Binding to RNA Template and Telomeric DNA  

SciTech Connect

Telomerase is a specialized DNA polymerase that extends the 3{prime} ends of eukaryotic linear chromosomes, a process required for genomic stability and cell viability. Here we present the crystal structure of the active Tribolium castaneum telomerase catalytic subunit, TERT, bound to an RNA-DNA hairpin designed to resemble the putative RNA-templating region and telomeric DNA. The RNA-DNA hybrid adopts a helical structure, docked in the interior cavity of the TERT ring. Contacts between the RNA template and motifs 2 and B{prime} position the solvent-accessible RNA bases close to the enzyme active site for nucleotide binding and selectivity. Nucleic acid binding induces rigid TERT conformational changes to form a tight catalytic complex. Overall, TERT-RNA template and TERT-telomeric DNA associations are remarkably similar to those observed for retroviral reverse transcriptases, suggesting common mechanistic aspects of DNA replication between the two families of enzymes.

Mitchell, M.; Gillis, A; Futahashi, M; Fujiwara, H; Skordalakes, E

2010-01-01

67

Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins  

E-print Network

Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins Brian that are resistant to cationic lipid-mediated siRNA transfection, results in potent siRNA delivery. In four of these five cell lines, siRNA transfected by 36 GFP suppresses target gene expression. We show that 36 GFP

Liu, David R.

68

The RNA experiment, 2D animationSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Sydney Brenner, Francois Jacob and Matt Meselson's experiment showed that RNA was a copy of the information in DNA. As a messenger, RNA transported the information from the nucleus to the protein-making machinery in the cell.

2008-10-06

69

Structure and assembly of the essential RNA ring component of a viral DNA packaging motor  

Microsoft Academic Search

Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage 29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 resolution,

Fang Ding; Changrui Lu; Wei Zhao; Kanagalaghatta R. Rajashankar; Dwight L. Anderson; Paul J. Jardine; Shelley Grimes; Ailong Ke

2011-01-01

70

C2?-Pyrene-functionalized Triazole-linked DNA: Universal DNA/RNA Hybridization Probes  

PubMed Central

Development of universal hybridization probes, i.e., oligonucleotides displaying identical affinity toward matched and mismatched DNA/RNA targets, has been a longstanding goal due to potential applications as degenerate PCR primers and microarray probes. The classic approach toward this end has been the use of ‘universal bases’ that either are based on aromatic base analogs without hydrogen-bonding capabilities or hydrogen-bonding purine derivatives. However, development of probes that enable truly ‘universal’ hybridization without compromising duplex thermostability has proven challenging. Here we have used the ‘click reaction’ to synthesize four C2?-pyrene-functionalized triazole-linked 2?-deoxyuridine phosphoramidites. We demonstrate that oligodeoxyribonucleotides modified with the corresponding monomers display: a) minimally decreased thermal affinity toward DNA/RNA complements relative to reference strands; b) highly robust universal hybridization characteristics (average differences in thermal denaturation temperatures of matched vs mismatched duplexes are < 1.5 °C); and c) exceptional affinity toward DNA targets containing abasic sites opposite of the modification site (?Tm up to +25 °C). The latter observation, along with results from absorption and fluorescence spectroscopy, indicates that the pyrene moiety is intercalating into the duplex whereby the opposing nucleotide is pushed into an extrahelical position. These properties render C2?-pyrene-functionalized triazole-linked DNA as promising universal hybridization probes for applications in nucleic acid chemistry and biotechnology. PMID:22087648

Sau, Sujay P.; Hrdlicka, Patrick J.

2011-01-01

71

Complex Interplay among DNA Modification, Noncoding RNA Expression and Protein-Coding RNA Expression in Salvia miltiorrhiza Chloroplast Genome  

PubMed Central

Salvia miltiorrhiza is one of the most widely used medicinal plants. As a first step to develop a chloroplast-based genetic engineering method for the over-production of active components from S. miltiorrhiza, we have analyzed the genome, transcriptome, and base modifications of the S. miltiorrhiza chloroplast. Total genomic DNA and RNA were extracted from fresh leaves and then subjected to strand-specific RNA-Seq and Single-Molecule Real-Time (SMRT) sequencing analyses. Mapping the RNA-Seq reads to the genome assembly allowed us to determine the relative expression levels of 80 protein-coding genes. In addition, we identified 19 polycistronic transcription units and 136 putative antisense and intergenic noncoding RNA (ncRNA) genes. Comparison of the abundance of protein-coding transcripts (cRNA) with and without overlapping antisense ncRNAs (asRNA) suggest that the presence of asRNA is associated with increased cRNA abundance (p<0.05). Using the SMRT Portal software (v1.3.2), 2687 potential DNA modification sites and two potential DNA modification motifs were predicted. The two motifs include a TATA box–like motif (CPGDMM1, “TATANNNATNA”), and an unknown motif (CPGDMM2 “WNYANTGAW”). Specifically, 35 of the 97 CPGDMM1 motifs (36.1%) and 91 of the 369 CPGDMM2 motifs (24.7%) were found to be significantly modified (p<0.01). Analysis of genes downstream of the CPGDMM1 motif revealed the significantly increased abundance of ncRNA genes that are less than 400 bp away from the significantly modified CPGDMM1motif (p<0.01). Taking together, the present study revealed a complex interplay among DNA modifications, ncRNA and cRNA expression in chloroplast genome. PMID:24914614

Chen, Haimei; Zhang, Jianhui; Yuan, George; Liu, Chang

2014-01-01

72

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9  

NASA Astrophysics Data System (ADS)

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

2014-03-01

73

The Roads to and from the RNA World  

NASA Technical Reports Server (NTRS)

The historical existence of the RNA world, in which early life used RNA for both genetic information and catalytic ability, is widely accepted. However, there has been little discussion of whether protein synthesis arose before DNA or what preceded the RNA world (i.e. the pre-RNA world). We outline arguments of what route life may have taken out of the RNA world: whether DNA or protein followed. The metabolic arguments favor the possibility that RNA genomes preceded the use of DNA as the informational macromolecule. However, the opposite can also be argued based on the enhanced stability, reactivity, and solubility of 2-deoxyribose as compared to ribose. The possibility that DNA may have come before RNA is discussed, although it is a less parsimonious explanation than DNA following RNA.

Dworkin, Jason P.; Lazcano, Antonio; Miller, Stanley L.

2003-01-01

74

Mechanism and manipulation of DNA:RNA hybrid G-quadruplex formation in transcription of G-rich DNA.  

PubMed

We recently reported that a DNA:RNA hybrid G-quadruplex (HQ) forms during transcription of DNA that bears two or more tandem guanine tracts (G-tract) on the nontemplate strand. Putative HQ-forming sequences are enriched in the nearby 1000 nt region right downstream of transcription start sites in the nontemplate strand of warm-blooded animals, and HQ regulates transcription under both in vitro and in vivo conditions. Therefore, knowledge of the mechanism of HQ formation is important for understanding the biological function of HQ as well as for manipulating gene expression by targeting HQ. In this work, we studied the mechanism of HQ formation using an in vitro T7 transcription model. We show that RNA synthesis initially produces an R-loop, a DNA:RNA heteroduplex formed by a nascent RNA transcript and the template DNA strand. In the following round of transcription, the RNA in the R-loop is displaced, releasing the RNA in single-stranded form (ssRNA). Then the G-tracts in the RNA can jointly form HQ with those in the nontemplate DNA strand. We demonstrate that the structural cascade R-loop ? ssRNA ? HQ offers opportunities to intercept HQ formation, which may provide a potential method to manipulate gene expression. PMID:24392825

Zhang, Jia-yu; Zheng, Ke-wei; Xiao, Shan; Hao, Yu-hua; Tan, Zheng

2014-01-29

75

RNA-directed DNA methylation induces transcriptional activation in plants  

PubMed Central

A class-C floral homeotic gene of Petunia, pMADS3, is specifically expressed in the stamen and carpels of developing flowers. We had previously reported the ect-pMADS3 phenomenon in which introduction of a part of the pMADS3 genomic sequence, including intron 2, induces ectopic expression of endogenous pMADS3. Unlike transcriptional or posttranscriptional gene silencing triggered by the introduction of homologous sequences, this observation is unique in that the gene expression is up-regulated. In this study, we demonstrated that the ect-pMADS3 phenomenon is due to transcriptional activation based on RNA-directed DNA methylation (RdDM) occurring in a particular CG in a putative cis-element in pMADS3 intron 2. The CG methylation was maintained over generations, along with pMADS3 ectopic expression, even in the absence of RNA triggers. These results demonstrate a previously undescribed transcriptional regulatory mechanism that could lead to the generation of a transcriptionally active epiallele, thereby contributing to plant evolution. Our results also reveal a putative negative cis-element for organ-specific transcriptional regulation of class-C floral homeotic genes, which could be difficult to identify by other approaches. PMID:19164525

Shibuya, Kenichi; Fukushima, Setsuko; Takatsuji, Hiroshi

2009-01-01

76

Modeling and time-dependent dynamics of processes of stimulated depolymerization, auto-repairing, degradation and radiation curing of DNA macromolecules and biopolymers at separated and combined actions of ionizing irradiation  

NASA Astrophysics Data System (ADS)

The time-dependent dynamics of the formation, relaxation and auto-repairing of double breaks of DNA macromolecules at the combined radiation action and non-radiation processes of degradation (e.g. by free radicals) were considered. The auto-repairing of DNA double breaks is connected with the peculiarities of long-range interaction of nucleotide charges, atoms and molecules in the intracellular milieu. The properties of intracellular liquid and the characteristics of force interaction between the end-pairs of nucleotides in the area of DNA break in response to radiation are changed. Each kind of radiation is characterized by a certain effectiveness of the double DNA break formation but simultaneously one creates the conditions for their liquidation. On the basis of the analysis and correlation of these processes the time-dependent theory for DNA degradation was created, including hormesis phenomenon, radiation antagonism, the validity of anomaly influence of low and large doses at sharp and chronic radiation and other effects. The qualitative and quantitative correspondences of the theory and experimental results of radiation biology were obtained.

Vysotskii, Vladimir I.; Pinchuk, Anatoliy O.; Kornilova, Alla A.; Samoylenko, Igor I.

2001-12-01

77

Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for genomic applications  

PubMed Central

Genomic technologies have revolutionized our understanding of complex Mendelian diseases and cancer. Solid tumors present several challenges for genomic analyses, such as tumor heterogeneity and tumor contamination with surrounding stroma and infiltrating lymphocytes. We developed a protocol to (i) select tissues of high cellular purity on the basis of histological analyses of immediately flanking sections and (ii) simultaneously extract genomic DNA (gDNA), messenger RNA (mRNA), noncoding RNA (ncRNA; enriched in microRNA (miRNA)) and protein from the same tissues. After tissue selection, about 12–16 extractions of DNA/RNA/protein can be obtained per day. Compared with other similar approaches, this fast and reliable methodology allowed us to identify mutations in tumors with remarkable sensitivity and to perform integrative analyses of whole-genome and exome data sets, DNA copy numbers (by single-nucleotide polymorphism (SNP) arrays), gene expression data (by transcriptome profiling and quantitative PCR (qPCR)) and protein levels (by western blotting and immunohistochemical analysis) from the same samples. Although we focused on renal cell carcinoma, this protocol may be adapted with minor changes to any human or animal tissue to obtain high-quality and high-yield nucleic acids and proteins. PMID:24136348

Pena-Llopis, Samuel; Brugarolas, James

2014-01-01

78

RNA:DNA Hybrids Initiate Quasi-Palindrome-Associated Mutations in Highly Transcribed Yeast DNA  

PubMed Central

RNase H enzymes promote genetic stability by degrading aberrant RNA?DNA hybrids and by removing ribonucleotide monophosphates (rNMPs) that are present in duplex DNA. Here, we report that loss of RNase H2 in yeast is associated with mutations that extend identity between the arms of imperfect inverted repeats (quasi-palindromes or QPs), a mutation type generally attributed to a template switch during DNA synthesis. QP events were detected using frameshift-reversion assays and were only observed under conditions of high transcription. In striking contrast to transcription-associated short deletions that also are detected by these assays, QP events do not require Top1 activity. QP mutation rates are strongly affected by the direction of DNA replication and, in contrast to their elevation in the absence of RNase H2, are reduced when RNase H1 is additionally eliminated. Finally, transcription-associated QP events are limited by components of the nucleotide excision repair pathway and are promoted by translesion synthesis DNA polymerases. We suggest that QP mutations reflect either a transcription-associated perturbation of Okazaki-fragment processing, or the use of a nascent transcript to resume replication following a transcription-replication conflict. PMID:24244191

Kim, Nayun; Cho, Jang-Eun; Li, Yue C.; Jinks-Robertson, Sue

2013-01-01

79

Involvement of putative SNF2 chromatin remodeling protein DRD1 in RNA-directed DNA methylation  

E-print Network

1 Involvement of putative SNF2 chromatin remodeling protein DRD1 in RNA-directed DNA methylation/SNF2-like proteins most similar to the RAD54/ATRX-subfamily. In drd1 mutants, RNA-induced non of centromeric and rDNA repeats is unaffected. Thus, unlike the SNF2-like proteins DDM1/Lsh1 [[6, 7

Kreil, David

80

In vivo label-free photoacoustic microscopy of cell nuclei by excitation of DNA and RNA  

E-print Network

In vivo label-free photoacoustic microscopy of cell nuclei by excitation of DNA and RNA Da-Kang Yao light excites unlabeled DNA and RNA in cell nuclei to produce photoacoustic waves. We applied UV of the histologically stained cell nuclei. Given intrinsic optical contrast and high spatial resolution, in vivo label-free

Wang, Lihong

81

RNA and DNA Puffs in Polytene Chromosomes of Rhynchosciara: Inhibition by Extirpation of Prothorax  

Microsoft Academic Search

In the giant polytene chromosomes, gene amplification is made visible by formation of DNA puffs, and gene transcription is made visible by formation of RNA puffs. Ligation of the anterior portion of the larva at the end of the fourth larval instar inhibited the formation of the DNA puffs that normally develop at this stage and caused regression of RNA

J. M. Amabis; Dulce Cabral

1970-01-01

82

A General Synthesis of Specifically Deuterated Nucleotides for Studies of DNA and RNA  

E-print Network

A General Synthesis of Specifically Deuterated Nucleotides for Studies of DNA and RNA Bingzi Chen by chemical synthesis and subsequently converted to ribonucleotides and deoxyribonucleotides via enzymatic nucleotides can be incorporated into DNA and RNA molecules to suppress nonessential proton resonances in NMR

Tullius, Thomas D.

83

Efficient transfection of DNA or shRNA vectors into neurons using magnetofection  

E-print Network

Efficient transfection of DNA or shRNA vectors into neurons using magnetofection Thomas Buerli1.445 Efficient and long-lasting transfection of primary neurons is an essential tool for addressing many and difficult to transfect with most methods. We provide a protocol for transfection of cDNA and RNA

Cossart, Rosa

84

Genome-Wide Profiling of Yeast DNA:RNA Hybrid Prone Sites with DRIP-Chip  

PubMed Central

DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013. PMID:24743342

Lu, Phoebe Y. T.; Luo, Zongli; Hamza, Akil; Kobor, Michael S.; Stirling, Peter C.; Hieter, Philip

2014-01-01

85

The Effect of Charge-Reversal Amphiphile Spacer Composition on DNA and siRNA Delivery  

E-print Network

The Effect of Charge-Reversal Amphiphile Spacer Composition on DNA and siRNA Delivery Xiao April 7, 2010 A series of charge-reversal amphiphiles with different spacers separating the headgroup from the hydrophobic chains are described for delivery of DNA and siRNA. Among them, the amphiphiles

86

A Pre-mRNA-Splicing Factor Is Required for RNA-Directed DNA Methylation in Arabidopsis  

PubMed Central

Cytosine DNA methylation is a stable epigenetic mark that is frequently associated with the silencing of genes and transposable elements (TEs). In Arabidopsis, the establishment of DNA methylation is through the RNA-directed DNA methylation (RdDM) pathway. Here, we report the identification and characterization of RDM16, a new factor in the RdDM pathway. Mutation of RDM16 reduced the DNA methylation levels and partially released the silencing of a reporter gene as well as some endogenous genomic loci in the DNA demethylase ros1-1 mutant background. The rdm16 mutant had morphological defects and was hypersensitive to salt stress and abscisic acid (ABA). Map-based cloning and complementation test led to the identification of RDM16, which encodes a pre-mRNA-splicing factor 3, a component of the U4/U6 snRNP. RNA-seq analysis showed that 308 intron retention events occurred in rdm16, confirming that RDM16 is involved in pre-mRNA splicing in planta. RNA-seq and mRNA expression analysis also revealed that the RDM16 mutation did not affect the pre-mRNA splicing of known RdDM genes, suggesting that RDM16 might be directly involved in RdDM. Small RNA expression analysis on loci showing RDM16-dependent DNA methylation suggested that unlike the previously reported putative splicing factor mutants, rdm16 did not affect small RNA levels; instead, the rdm16 mutation caused a decrease in the levels of Pol V transcripts. ChIP assays revealed that RDM16 was enriched at some Pol V target loci. Our results suggest that RDM16 regulates DNA methylation through influencing Pol V transcript levels. Finally, our genome-wide DNA methylation analysis indicated that RDM16 regulates the overall methylation of TEs and gene-surrounding regions, and preferentially targets Pol IV-dependent DNA methylation loci and the ROS1 target loci. Our work thus contributes to the understanding of RdDM and its interactions with active DNA demethylation. PMID:24068953

Huang, Chao-Feng; Miki, Daisuke; Tang, Kai; Zhou, Hao-Ran; Zheng, Zhimin; Chen, Wei; Ma, Ze-Yang; Yang, Lan; Zhang, Heng; Liu, Renyi; He, Xin-Jian; Zhu, Jian-Kang

2013-01-01

87

On-chip separation and analysis of RNA and DNA from single cells.  

PubMed

The simultaneous analysis of RNA and DNA of single cells remains a challenge as these species have very similar physical and biochemical properties and can cross-contaminate each other. Presented is an on-chip system that enables selective lysing of single living cells, extraction, focusing, and absolute quantification of cytoplasmic RNA mass and its physical separation from DNA in the nucleus using electrical lysing and isotachophoresis (ITP). This absolute quantitation is performed without enzymatic amplification in less than 5 min. The nucleus is preserved, and its DNA fluorescence signal can be measured independently. We demonstrate the technique using single mouse lymphocyte cells, for which we extracted an average of 14.1 pg of total RNA per cell. We also demonstrate correlation analysis between the absolute amount of RNA and relative amount of DNA, showing heterogeneity associated with cell cycles. The technique is compatible with fractionation of DNA and RNA and with downstream assays of each. PMID:24499009

Shintaku, Hirofumi; Nishikii, Hidekazu; Marshall, Lewis A; Kotera, Hidetoshi; Santiago, Juan G

2014-02-18

88

Molecular structure of r/GCG/d/TATACGC/ - A DNA-RNA hybrid helix joined to double helical DNA  

NASA Technical Reports Server (NTRS)

The molecule r(GCG)d(TATACGC) is self-complementary and forms two DNA-RNA hybrid segments surrounding a central region of double helical DNA; its molecular structure has been solved by X-ray analysis. All three parts of the molecule adopt a conformation which is close to that seen in the 11-fold RNA double helix. The conformation of the ribonucleotides is partly determined by water molecules bridging between the ribose O2' hydroxyl group and cytosine O2. The hybrid-DNA duplex junction contains no structural discontinuities. However, the central DNA TATA sequence has some structural irregularities.

Wang, A. H.-J.; Fujii, S.; Rich, A.; Van Boom, J. H.; Van Der Marel, G. A.; Van Boeckel, S. A. A.

1982-01-01

89

An Attempt to Detect siRNA-Mediated Genomic DNA Modification by Artificially Induced Mismatch siRNA in Arabidopsis  

PubMed Central

Although tremendous progress has been made in recent years in identifying molecular mechanisms of small interfering RNA (siRNA) functions in higher plants, the possibility of direct interaction between genomic DNA and siRNA remains an enigma. Such an interaction was proposed in the ‘RNA cache’ hypothesis, in which a mutant allele is restored based on template-directed gene conversion. To test this hypothesis, we generated transgenic Arabidopsis thaliana plants conditionally expressing a hairpin dsRNA construct of a mutated acetolactate synthase (mALS) gene coding sequence, which confers chlorsulfuron resistance, in the presence of dexamethasone (DEX). In the transgenic plants, suppression of the endogenous ALS mRNA expression as well as 21-nt mALS siRNA expression was detected after DEX treatment. After screening >100,000 progeny of the mALS siRNA-induced plants, no chlorsulfuron-resistant progeny were obtained. Further experiments using transgenic calli also showed that DEX-induced expression of mALS siRNA did not affect the number of chlorsulfuron-resistant calli. No trace of cytosine methylation of the genomic ALS region corresponding to the dsRNA region was observed in the DEX-treated calli. These results do not necessarily disprove the ‘RNA cache’ hypothesis, but indicate that an RNAi machinery for ALS mRNA suppression does not alter the ALS locus, either genetically or epigenetically. PMID:24278423

Miyagawa, Yosuke; Ogawa, Jun; Iwata, Yuji; Koizumi, Nozomu; Mishiba, Kei-ichiro

2013-01-01

90

In vitro transcription of herpes simplex virus ANG DNA by E-coli RNA polymerase.  

PubMed Central

HSV-1 ANG DNA and a defective genome of the same virus were transcribed with E. coli RNA polymerase under various salt conditions. The extent of transcription was assayed by hybridizing the cRNA to the Hind III, Hpa I and Hind II restriction fragments of the DNA templates using the blot technique of E. Southern. The transcripts proved to contain sequences homologous to all DNA fragments. A similar ratio of hybridized cRNA and the amount of fragment DNA was observed in all cases. The results suggest that both, the wt and the defective HSV ANG genome were completely transcribed. Images PMID:197492

Strauss, G P; Maichle, I B; Schatten, R; Kaerner, H C

1977-01-01

91

Production of infectious RNA transcripts from full-length cDNA clones representing two subgroups of peanut stunt virus strains: mapping satellite RNA support to RNA1  

Microsoft Academic Search

Full-length cDNA clones from which infectious trans- cripts could be generated were constructed from the genomic RNAs of two distinct strains of peanut stunt cucumovirus (PSV), PSV-ER and PSV-W. PSV- ER, a subgroup I strain, is known to support efficient replication of satellite RNA (satRNA) in infected plants, whereas PSV-W, a subgroup II strain, does not support satRNA replication. Although

Chung-Chi Hu; Margaret Sanger; Said A. Ghabrial

1998-01-01

92

Structure and assembly of the essential RNA ring component of a viral DNA packaging motor  

SciTech Connect

Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage {psi}29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 {angstrom} resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of {psi}29 DNA.

Ding, Fang; Lu, Changrui; Zhao, Wei; Rajashankar, Kanagalaghatta R.; Anderson, Dwight L.; Jardine, Paul J.; Grimes, Shelley; Ke, Ailong (Cornell); (UMM)

2011-07-25

93

A comparison of RNA with DNA in template-directed synthesis  

NASA Technical Reports Server (NTRS)

Nonenzymatic template-directed copying of RNA sequences rich in cytidylic acid using nucleoside 5'-(2-methylimidazol-1-yl phosphates) as substrates is substantially more efficient than the copying of corresponding DNA sequences. However, many sequences cannot be copied, and the prospect of replication in this system is remote, even for RNA. Surprisingly, wobble-pairing leads to much more efficient incorporation of G opposite U on RNA templates than of G opposite T on DNA templates.

Zielinski, M.; Kozlov, I. A.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

2000-01-01

94

Influences of DNA isolation and RNA contamination on carcinogen-DNA adduct analysis by 32P-postlabeling.  

PubMed

32P-Postlabeling is a widely applied assay for the analysis of carcinogen-DNA adducts. Optimization of most steps in this assay has been given attention, but influences of DNA isolation and DNA purity on adduct quantitation have not been investigated systematically. In this study, DNA was isolated from human lymphocytes exposed to benzo[a]pyrene (B[a]P, 10 microM) for 18 hr and from liver of rats i.p.-treated with B[a]P (10 mg/kg body weight) using two different DNA isolation methods: a phenol-extraction and a salting-out procedure. Subsequently, DNA was analysed by nuclease P1 (NP1) or butanol-enriched 32P-postlabeling. Influences of RNA contamination were studied by labeling RNA isolated from in vitro exposed lymphocytes. In the in vitro experiment, DNA adduct levels were significantly higher using the salting-out procedure (63.2 +/- 13.7 adducts per 10(8) nucleotides, n = 9) as compared with the phenol-extraction (14.3 +/- 0.8). RNA was approximately 4 times less efficiently labeled as compared to DNA. Nonetheless, RNA contamination of DNA samples may result in an overestimation of DNA adduct levels when butanol enrichment is used, because RNA adduct levels seemed to be substantially higher than DNA adduct levels in the same cells. DNA adduct analysis by nuclease P1 enrichment is probably less affected, since RNA adducts appeared to be NP1 sensitive. In vivo, three different adducts were found by NP1 enriched 32P-postlabeling in the liver of B[a]P-exposed rats. Again, DNA adduct levels were significantly higher using salting out as compared to phenol extraction for the adduct which comigrated with the BPDE-DNA adduct standard (adduct 1) and an unknown adduct (adduct 2). However, the results were the opposite for another B[a]P-derived DNA adduct (adduct 3). Our results suggest that differences in DNA isolation procedures as well as RNA contamination influence quantitative DNA adduct analysis by 32P-postlabeling. PMID:9882009

Godschalk, R W; Maas, L M; Kleinjans, J C; Van Schooten, F J

1998-01-01

95

Transcription: DNA codes for messenger RNA (mRNA), 3D animation with basic narrationSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

DNAi Location: Code>Copying the Code>putting it together>Transcription What you are about to see is DNA's most extraordinary secret, how a simple code is turned into flesh and blood. It begins with a bundle of factors (transcription factors)assembling at the start of a gene. A gene is simply a length of DNA instructions stretching away to the left. The assembled factors trigger the first phase of the process, reading off the information that will be needed to make the protein. Everything is ready to roll: three, two, one, GO! The blue molecule (RNA polymerase) racing along the DNA is reading the gene. It's unzipping the double helix, and copying one of the two strands. The yellow chain (messanger RNA or mRNA) snaking out of the top is a copy of the genetic message and it's made of a close chemical cousin of DNA called RNA. The building blocks to make the RNA enter through an intake hole. They are matched to the DNA - letter by letter - to copy the As, Cs, Ts and Gs of the gene. The only difference is that in the RNA copy, the letter T is replaced with a closely related building block known as \\"U\\". You are watching this process - called transcription - in real time. It's happening right now in almost every cell in your body.

2008-10-06

96

Co-transcriptional production of RNA-DNA hybrids for simultaneous release of multiple split functionalities  

PubMed Central

Control over the simultaneous delivery of different functionalities and their synchronized intracellular activation can greatly benefit the fields of RNA and DNA biomedical nanotechnologies and allow for the production of nanoparticles and various switching devices with controllable functions. We present a system of multiple split functionalities embedded in the cognate pairs of RNA–DNA hybrids which are programmed to recognize each other, re-associate and form a DNA duplex while also releasing the split RNA fragments which upon association regain their original functions. Simultaneous activation of three different functionalities (RNAi, Förster resonance energy transfer and RNA aptamer) confirmed by multiple in vitro and cell culture experiments prove the concept. To automate the design process, a novel computational tool that differentiates between the thermodynamic stabilities of RNA–RNA, RNA–DNA and DNA–DNA duplexes was developed. Moreover, here we demonstrate that besides being easily produced by annealing synthetic RNAs and DNAs, the individual hybrids carrying longer RNAs can be produced by RNA polymerase II-dependent transcription of single-stranded DNA templates. PMID:24194608

Afonin, Kirill A.; Desai, Ravi; Viard, Mathias; Kireeva, Maria L.; Bindewald, Eckart; Case, Christopher L.; Maciag, Anna E.; Kasprzak, Wojciech K.; Kim, Taejin; Sappe, Alison; Stepler, Marissa; KewalRamani, Vineet N.; Kashlev, Mikhail; Blumenthal, Robert; Shapiro, Bruce A.

2014-01-01

97

Tracking Fungal Community Responses to Maize Plants by DNA- and RNA-Based Pyrosequencing  

PubMed Central

We assessed soil fungal diversity and community structure at two sampling times (t1?=?47 days and t2?=?104 days of plant age) in pots associated with four maize cultivars, including two genetically modified (GM) cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA). The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most “active” fungi (as recovered via RNA). Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production). Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time. PMID:23875012

Kuramae, Eiko E.; Verbruggen, Erik; Hillekens, Remy; de Hollander, Mattias; Roling, Wilfred F. M.; van der Heijden, Marcel G. A.; Kowalchuk, George A.

2013-01-01

98

Interacting RNA polymerase motors on DNA track: effects of traffic congestion and intrinsic noise on RNA synthesis  

E-print Network

RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by a DNA. In many circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track; we refer to such collective movements of the RNAPs as RNAP traffic. Here we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechano-chemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the DNA track. We also introduce, and compute, two new measures of {\\it fluctuations} in the synthesis of RNA. Analyzing these fluctuations, we show how the level of intrinsic noise in mRNA synthesis dep...

Tripathi, Tripti

2007-01-01

99

RNA\\/DNA ratio and expression of 18S ribosomal RNA, actin and myosin heavy chain messenger RNAs in starved and fed larval Atlantic cod (Gadus morhua)  

Microsoft Academic Search

Levels of total RNA, total DNA, 18S ribosomal RNA (rRNA), poly(A) messenger RNA (mRNA), and two mRNAs coding for abundant\\u000a myofibrillar proteins were estimated in laboratory-reared Atlantic cod larvae (Gadusmorhua Linnaeus) under conditions of feeding and starvation. DNA probes specific for cod 18S rRNA, ?-actin mRNA and myosin heavy\\u000a chain mRNA were developed. In two experiments on newly hatched larvae

P. T. McNamara; E. M. Caldarone; L. J. Buckley

1999-01-01

100

Defects in purine nucleotide metabolism lead to substantial incorporation of xanthine and hypoxanthine into DNA and RNA  

E-print Network

Deamination of nucleobases in DNA and RNA results in the formation of xanthine (X), hypoxanthine (I), oxanine, and uracil, all of which are miscoding and mutagenic in DNA and can interfere with RNA editing and function. ...

Pang, Bo

101

Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction  

Microsoft Academic Search

Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase

D P Jackson; F A Lewis; G R Taylor; A W Boylston; P Quirke

1990-01-01

102

DNA, RNA, and Protein Extraction: The Past and The Present  

PubMed Central

Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination. PMID:20011662

Tan, Siun Chee; Yiap, Beow Chin

2009-01-01

103

RNA Interference by Single- and Double-stranded siRNA With a DNA Extension Containing a 3? Nuclease-resistant Mini-hairpin Structure  

PubMed Central

Selective gene silencing by RNA interference (RNAi) involves double-stranded small interfering RNA (ds siRNA) composed of single-stranded (ss) guide and passenger RNAs. siRNA is recognized and processed by Ago2 and C3PO, endonucleases of the RNA-induced silencing complex (RISC). RISC cleaves passenger RNA, exposing the guide RNA for base-pairing with its homologous mRNA target. Remarkably, the 3? end of passenger RNA can accommodate a DNA extension of 19-nucleotides without loss of RNAi function. This construct is termed passenger-3?-DNA/ds siRNA and includes a 3?-nuclease-resistant mini-hairpin structure. To test this novel modification further, we have now compared the following constructs: (I) guide-3?-DNA/ds siRNA, (II) passenger-3?-DNA/ds siRNA, (III) guide-3?-DNA/ss siRNA, and (IV) passenger-3?-DNA/ss siRNA. The RNAi target was SIRT1, a cancer-specific survival factor. Constructs I–III each induced selective knock-down of SIRT1 mRNA and protein in both noncancer and cancer cells, accompanied by apoptotic cell death in the cancer cells. Construct IV, which lacks the SIRT1 guide strand, had no effect. Importantly, the 3?-DNA mini-hairpin conferred nuclease resistance to constructs I and II. Resistance required the double-stranded RNA structure since single-stranded guide-3?-DNA/ss siRNA (construct III) was susceptible to serum nucleases with associated loss of RNAi activity. The potential applications of 3?-DNA/siRNA constructs are discussed. PMID:24399205

Allison, Simon J; Milner, Jo

2014-01-01

104

A novel method for constructing pathogen-regulated small RNA cDNA library.  

PubMed

Pathogen-responsive endogenous small non-coding RNAs regulate gene expression in relation to plant immune responses by serving as RNA silencing machinery. Decay caused by the bacterium, Erwinia carotovora subsp. carotovora (Ecc), often leads to soft rot disease in the plant Brassica campestris L. ssp. pekinensis (Bcp). To discover endogenous small RNA species in Bcp in response to Ecc infection, we developed a highly efficient approach for cloning pathogen-regulated small RNAs. A group of degenerate stem-loop reverse primers was designed to synthesize first single-stranded cDNA (sscDNA) and the sscDNA was then tailed with a poly(C) at its 3' end to create a forward priming site. A novel cDNA/RNA subtractive hybridization was performed to capture Ecc-regulated small RNAs and this subsequently allowed construction of small RNA cDNA libraries for sequencing. PMID:20515661

Sun, Chuan Bao; Du, Xian Ming; He, Yu Ke

2010-07-01

105

RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?  

PubMed

Tailor-made nucleases for precise genome modification, such as zinc finger or TALE nucleases, currently represent the state-of-the-art for genome editing. These nucleases combine a programmable protein module which guides the enzyme to the target site with a nuclease domain which cuts DNA at the addressed site. Reprogramming of these nucleases to cut genomes at specific locations requires major protein engineering efforts. RNA-guided DNA endonuclease Cas9 of the type II (clustered regularly interspaced short palindromic repeat) CRISPR-Cas system uses CRISPR RNA (crRNA) as a guide to locate the DNA target and the Cas9 protein to cut DNA. Easy programmability of the Cas9 endonuclease using customizable RNAs brings unprecedented flexibility and versatility for targeted genome modification. We highlight the potential of the Cas9 RNA-guided DNA endonuclease as a novel tool for genome surgery, and discuss possible constraints and future prospects. PMID:24095303

Gasiunas, Giedrius; Siksnys, Virginijus

2013-11-01

106

Pre-mRNA processing factors meet the DNA damage response  

PubMed Central

It is well-known that DNA-damaging agents induce genome instability, but only recently have we begun to appreciate that chromosomes are fragile per se and frequently subject to DNA breakage. DNA replication further magnifies such fragility, because it leads to accumulation of single-stranded DNA. Recent findings suggest that chromosome fragility is similarly increased during transcription. Transcripts produced by RNA polymerase II (RNAPII) are subject to multiple processing steps, including maturation of 5? and 3? ends and splicing, followed by transport to the cytoplasm. RNA maturation starts on nascent transcripts and is mediated by a number of diverse proteins and ribonucleoprotein particles some of which are recruited cotranscriptionally through interactions with the carboxy-terminal domain of RNAPII. This coupling is thought to maximize efficiency of pre-mRNA maturation and directly impacts the choice of alternative splice sites. Mounting evidence suggests that lack of coordination among different RNA maturation steps, by perturbing the interaction of nascent transcripts with the DNA template, has deleterious effects on genome stability. Thus, in the absence of proper surveillance mechanisms, transcription could be a major source of DNA damage in cancer. Recent high-throughput screenings in human cells and budding yeast have identified several factors implicated in RNA metabolism that are targets of DNA damage checkpoint kinases: ATM (ataxia telangiectasia mutated) and ATR (ATM-Rad3 related) (Tel1 and Mec1 in budding yeast, respectively). Moreover, inactivation of various RNA processing factors induces accumulation of ?H2AX foci, an early sign of DNA damage. Thus, a complex network is emerging that links DNA repair and RNA metabolism. In this review we provide a comprehensive overview of the role played by pre-mRNA processing factors in the cell response to DNA damage and in the maintenance of genome stability. PMID:23761808

Montecucco, Alessandra; Biamonti, Giuseppe

2013-01-01

107

Replication initiation and genome instability: a crossroads for DNA and RNA synthesis.  

PubMed

Nuclear DNA replication requires the concerted action of hundreds of proteins to efficiently unwind and duplicate the entire genome while also retaining epigenetic regulatory information. Initiation of DNA replication is tightly regulated, rapidly firing thousands of origins once the conditions to promote rapid and faithful replication are in place, and defects in replication initiation lead to proliferation defects, genome instability, and a range of developmental abnormalities. Interestingly, DNA replication in metazoans initiates in actively transcribed DNA, meaning that replication initiation occurs in DNA that is co-occupied with tens of thousands of poised and active RNA polymerase complexes. Active transcription can induce genome instability, particularly during DNA replication, as RNA polymerases can induce torsional stress, formation of secondary structures, and act as a physical barrier to other enzymes involved in DNA metabolism. Here we discuss the challenges facing mammalian DNA replication, their impact on genome instability, and the development of cancer. PMID:25238783

Barlow, Jacqueline H; Nussenzweig, André

2014-12-01

108

Inhibition of hepatitis B virus cccDNA by siRNA in transgenic mice.  

PubMed

The elimination of viral covalently closed circular DNA (cccDNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with siRNA is able to suppress viral cccDNA amplification using a HBV-transgenic mice model. The experimental results revealed that siRNAs can serve as efficient alternative anti-HBV agents, because they showed better inhibitory effect on viral replication and antigen expression in transgenic mice. More importantly, the siRNA markedly inhibited HBV cccDNA amplification. PMID:24569930

Li, Guiqiu; Jiang, Guotao; Lu, Juan; Chen, Shulan; Cui, Lanying; Jiao, Jundong; Wang, Yongchen

2014-07-01

109

Synthesis of Double-Stranded Complementary DNA from Poly(A)(+)mRNA.  

PubMed

The use of avian myeloblastosis virus reverse transcriptase (AMV RTase) to produce DNA copies of mRNA templates is a common and well-documented method (1-3). Briefly, the method involves synthesis of a complementary DNA strand to the mRNA from a short double-stranded region, usually provided by using an oligo(dT) primer on poly(A)(+)RNA. The enzyme does not always produce full length transcripts, but all the complementary strands are finished off with a short hairpin loop. This provides a ready-made primer for second strand synthesis, useful whether this is to be performed by more reverse transcriptase or by E. coli DNA polymerase 1 (pol 1). An idealized picture is shown in Fig. 1. Before the double-stranded cDNA (ds cDNA) copy can be cloned it is necessary to remove this hairpin loop using the single-strand specific nuclease S1. Fig. 1. Stages in the production of double-stranded cDNA from poly(A)(+)mRNA. The original RNA is represented by a solid line, while the cDNA is represented by a dashed line. Note that this diagram is not intended as an accurate representation of the enzymatic processes involved, but as a general guide to the principles of cDNA synthesis. PMID:21374188

McGookin, R

1985-01-01

110

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

PubMed

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. PMID:18834537

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

2008-01-01

111

Recognition of Chelerythrine to Human Telomeric DNA and RNA G-quadruplexes  

PubMed Central

A study on binding of antitumor chelerythrine to human telomeric DNA/RNA G-quadruplexes was performed by using DNA polymerase stop assay, UV-melting, ESI-TOF-MS, UV-Vis absorption spectrophotometry and fluorescent triazole orange displacement assay. Chelerythrine selectively binds to and stabilizes the K+-form hybrid-type human telomeric DNA G-quadruplex of biological significance, compared with the Na+-form antiparallel-type DNA G-quadruplex. ESI-TOF-MS study showed that chelerythrine possesses a binding strength for DNA G-quadruplex comparable to that of TMPyP4 tetrachloride. Both 1:1 and 2:1 stoichiometries were observed for chelerythrine's binding with DNA and RNA G-quadruplexes. The binding strength of chelerythrine with RNA G-quadruplex is stronger than that with DNA G-quadruplex. Fluorescent triazole orange displacement assay revealed that chelerythrine interacts with human telomeric RNA/DNA G-quadruplexes by the mode of end- stacking. The relative binding strength of chelerythrine for human telomeric RNA and DNA G-quadruplexes obtained from ESI-TOF-MS experiments are respectively 6.0- and 2.5-fold tighter than that with human telomeric double-stranded hairpin DNA. The binding selectivity of chelerythrine for the biologically significant K+-form human telomeric DNA G-quadruplex over the Na+-form analogue, and binding specificity for human telomeric RNA G-quadruplex established it as a promising candidate in the structure-based design and development of G-quadruplex specific ligands. PMID:25341562

Bai, Li-Ping; Hagihara, Masaki; Nakatani, Kazuhiko; Jiang, Zhi-Hong

2014-01-01

112

Recognition of Chelerythrine to Human Telomeric DNA and RNA G-quadruplexes  

NASA Astrophysics Data System (ADS)

A study on binding of antitumor chelerythrine to human telomeric DNA/RNA G-quadruplexes was performed by using DNA polymerase stop assay, UV-melting, ESI-TOF-MS, UV-Vis absorption spectrophotometry and fluorescent triazole orange displacement assay. Chelerythrine selectively binds to and stabilizes the K+-form hybrid-type human telomeric DNA G-quadruplex of biological significance, compared with the Na+-form antiparallel-type DNA G-quadruplex. ESI-TOF-MS study showed that chelerythrine possesses a binding strength for DNA G-quadruplex comparable to that of TMPyP4 tetrachloride. Both 1:1 and 2:1 stoichiometries were observed for chelerythrine's binding with DNA and RNA G-quadruplexes. The binding strength of chelerythrine with RNA G-quadruplex is stronger than that with DNA G-quadruplex. Fluorescent triazole orange displacement assay revealed that chelerythrine interacts with human telomeric RNA/DNA G-quadruplexes by the mode of end- stacking. The relative binding strength of chelerythrine for human telomeric RNA and DNA G-quadruplexes obtained from ESI-TOF-MS experiments are respectively 6.0- and 2.5-fold tighter than that with human telomeric double-stranded hairpin DNA. The binding selectivity of chelerythrine for the biologically significant K+-form human telomeric DNA G-quadruplex over the Na+-form analogue, and binding specificity for human telomeric RNA G-quadruplex established it as a promising candidate in the structure-based design and development of G-quadruplex specific ligands.

Bai, Li-Ping; Hagihara, Masaki; Nakatani, Kazuhiko; Jiang, Zhi-Hong

2014-10-01

113

Recognition of Chelerythrine to Human Telomeric DNA and RNA G-quadruplexes.  

PubMed

A study on binding of antitumor chelerythrine to human telomeric DNA/RNA G-quadruplexes was performed by using DNA polymerase stop assay, UV-melting, ESI-TOF-MS, UV-Vis absorption spectrophotometry and fluorescent triazole orange displacement assay. Chelerythrine selectively binds to and stabilizes the K(+)-form hybrid-type human telomeric DNA G-quadruplex of biological significance, compared with the Na(+)-form antiparallel-type DNA G-quadruplex. ESI-TOF-MS study showed that chelerythrine possesses a binding strength for DNA G-quadruplex comparable to that of TMPyP4 tetrachloride. Both 1:1 and 2:1 stoichiometries were observed for chelerythrine's binding with DNA and RNA G-quadruplexes. The binding strength of chelerythrine with RNA G-quadruplex is stronger than that with DNA G-quadruplex. Fluorescent triazole orange displacement assay revealed that chelerythrine interacts with human telomeric RNA/DNA G-quadruplexes by the mode of end- stacking. The relative binding strength of chelerythrine for human telomeric RNA and DNA G-quadruplexes obtained from ESI-TOF-MS experiments are respectively 6.0- and 2.5-fold tighter than that with human telomeric double-stranded hairpin DNA. The binding selectivity of chelerythrine for the biologically significant K(+)-form human telomeric DNA G-quadruplex over the Na(+)-form analogue, and binding specificity for human telomeric RNA G-quadruplex established it as a promising candidate in the structure-based design and development of G-quadruplex specific ligands. PMID:25341562

Bai, Li-Ping; Hagihara, Masaki; Nakatani, Kazuhiko; Jiang, Zhi-Hong

2014-01-01

114

Arginine-grafted biodegradable polymer: a versatile transfection reagent for both DNA and siRNA.  

PubMed

Effective delivery of DNA or siRNA into primary cells demands an efficient delivery system. However, the significant differences in physical and molecular characteristics of the two molecules generally necessitate distinct delivery systems or considerable differences in carrier formulation protocols for effective transfection. Arginine-grafted bioreducible poly (disulfide amine) (ABP) is a redox-sensitive, bioreducible, positively charged polymer which complexes with siRNA and DNA via charge interactions to form nanoplexes. ABP effectively mediates cytoplasmic delivery of both DNA and siRNA into multiple cell types, including primary cells like myoblast, human umbilical vein endothelial cells (HUVECs), and primary rat aorta vascular smooth muscle cells (SMCs) eliciting functional activity. In this chapter, we provide the detailed protocols for the synthesis of ABP as well as transfection of both siRNA and DNA using ABP. PMID:25030923

Beloor, Jagadish; Nam, Hye Yeong; Lee, Sang-Kyung; Kumar, Priti

2014-01-01

115

The structure, function and evolution of proteins that bind DNA and RNA.  

PubMed

Proteins that bind both DNA and RNA typify the ability of a single gene product to perform multiple functions. Such DNA- and RNA-binding proteins (DRBPs) have unique functional characteristics that stem from their specific structural features; these developed early in evolution and are widely conserved. Proteins that bind RNA have typically been considered as functionally distinct from proteins that bind DNA and studied independently. This practice is becoming outdated, in partly owing to the discovery of long non-coding RNAs (lncRNAs) that target DNA-binding proteins. Consequently, DRBPs were found to regulate many cellular processes, including transcription, translation, gene silencing, microRNA biogenesis and telomere maintenance. PMID:25269475

Hudson, William H; Ortlund, Eric A

2014-11-01

116

Isolation of DNA, RNA and protein from the starlet sea anemone Nematostella vectensis.  

PubMed

Among marine invertebrates, the starlet sea anemone Nematostella vectensis has emerged as an important laboratory model system. One advantage of working with this species relative to many other marine invertebrates is the ease of isolating relatively pure DNA, RNA and protein. Nematostella can be raised at high densities, under clean culture conditions, and it lacks integumentary or skeletal structures that can impede the recovery of DNA, RNA or protein. Here we describe methods used in our lab to isolate DNA, RNA and protein from Nematostella embryos, larvae and adults. The methods described here are less expensive than commercial kits and are more easily scalable to larger tissue amounts. Preparation of DNA can be completed in ?7 h, RNA preparation in ?1.5 h and protein preparation in ?1 h. PMID:23579778

Stefanik, Derek J; Wolenski, Francis S; Friedman, Lauren E; Gilmore, Thomas D; Finnerty, John R

2013-05-01

117

Targeted Delivery of siRNA-Generating DNA Nanocassettes Using Multifunctional Nanoparticles  

PubMed Central

Molecular therapy using a small interfering RNA (siRNA) has shown promise in the development of novel therapeutics. Various formulations have been used for in vivo delivery of siRNAs. However, the stability of short double-stranded RNA molecules in the blood and efficiency of siRNA delivery into target organs or tissues following systemic administration have been the major issues that limit applications of siRNA in human patients. In this study, multifunctional siRNA delivery nanoparticles are developed that combine imaging capability of nanoparticles with urokinase plasminogen activator receptor-targeted delivery of siRNA expressing DNA nanocassettes. This theranostic nanoparticle platform consists of a nanoparticle conjugated with targeting ligands and double-stranded DNA nanocassettes containing a U6 promoter and a shRNA gene for in vivo siRNA expression. Targeted delivery and gene silencing efficiency of firefly luciferase siRNA nanogenerators are demonstrated in tumor cells and in animal tumor models. Delivery of survivin siRNA expressing nanocassettes into tumor cells induces apoptotic cell death and sensitizes cells to chemotherapy drugs. The ability of expression of siRNAs from multiple nanocassettes conjugated to a single nanoparticle following receptor-mediated internalization should enhance the therapeutic effect of the siRNA-mediated cancer therapy. PMID:23292656

Cho, Y.-S.; Lee, G. Y.; Sajja, H. K.; Qian, W.; Cao, Z.; He, W.; Karna, P.; Chen, X.; Mao, H.; Wang, Y. A.; Yang, L.

2013-01-01

118

ADAR Proteins: Double-stranded RNA and Z-DNA Binding Domains  

PubMed Central

Adenosine deaminases acting on RNA (ADARs) catalyze adenosine to inosine editing within double-stranded RNA (dsRNA) substrates. Inosine is read as a guanine by most cellular processes and therefore these changes create codons for a different amino acid, stop codons or even a new splice-site allowing protein diversity generated from a single gene. We are reviewing here the current structural and molecular knowledge on RNA editing by the ADAR family of protein. We focus especially on two types of nucleic acid binding domains present in ADARs, namely the double-stranded RNA and Z-DNA binding domains. PMID:21728134

Barraud, Pierre; Allain, Frederic H.-T

2012-01-01

119

Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA  

PubMed Central

We present a novel Phi29 DNA polymerase application in RCA-based target RNA detection and analysis. The 3??5? RNase activity of Phi29 DNA polymerase converts target RNA into a primer and the polymerase uses this newly generated primer for RCA initiation. Therefore, using target RNA-primed RCA, padlock probes may be targeted to inner RNA sequences and their peculiarities can be analyzed directly. We demonstrate that the exoribonucleolytic activity of Phi29 DNA polymerase can be successfully applied in vitro and in situ. These findings expand the potential for detection and analysis of RNA sequences distanced from 3?-end. PMID:19244362

Lagunavicius, Arunas; Merkiene, Egle; Kiveryte, Zivile; Savaneviciute, Agne; Zimbaite-Ruskuliene, Vilma; Radzvilavicius, Tomas; Janulaitis, Arvydas

2009-01-01

120

Replication of Mengovirus I. Effect on Synthesis of Macromolecules by Host Cell  

PubMed Central

Plagemann, Peter G. W. (Western Reserve University, Cleveland, Ohio), and H. Earle Swim. Replication of mengovirus. I. Effect on synthesis of macromolecules by host cell. J. Bacteriol. 91:2317–2326. 1966.—The replication of mengovirus was studied in two strains of Novikoff (rat) hepatoma cells propagated in vitro. The replicative cycle in both strains required 6.5 to 7 hr. Infection resulted in a marked depression of ribonucleic acid (RNA) and protein synthesis by strain N1S1-63. Inhibition of RNA synthesis was reflected by a decrease in the deoxyribonucleic acid (DNA)-dependent RNA polymerase activity of isolated nuclei. Mengovirus had no effect on either protein or RNA synthesis or on the DNA-dependent RNA polymerase activity of a second strain, N1S1-67. The time course of viral-induced synthesis of RNA by cells was studied in cells treated with actinomycin D. It was first detectable between 2.5 and 3 hr after infection and continued until 6.5 to 7 hr. The formation of mature virus was estimated biochemically by measuring the amount of RNA synthesized as a result of viral infection which was resistant to degradation by ribonuclease in the presence of deoxycholate. Approximately 70% of the deoxycholate-ribonuclease-resistant RNA was located in mature virus, and the remainder was double-stranded. The formation of mature virus began about 45 min after viral-directed (actinomycin-resistant) synthesis of RNA was detectable in the cell, and only about 18 to 20% of the total RNA synthesized was incorporated into virus. Release of virus from cells began about 1 hr after maturation was first detectable. Release of virus from cells was accompanied by a loss of a large proportion of their cytoplasmic RNA and protein. PMID:4287585

Plagemann, Peter G. W.; Swim, H. Earle

1966-01-01

121

Structure of the RNA claw of the DNA packaging motor of bacteriophage ?29  

PubMed Central

Bacteriophage DNA packaging motors translocate their genomic DNA into viral heads, compacting it to near-crystalline density. The Bacillus subtilis phage ?29 has a unique ring of RNA (pRNA) that is an essential component of its motor, serving as a scaffold for the packaging ATPase. Previously, deletion of a three-base bulge (18-CCA-20) in the pRNA A-helix was shown to abolish packaging activity. Here, we solved the structure of this crucial bulge by nuclear magnetic resonance (NMR) using a 27mer RNA fragment containing the bulge (27b). The bulge actually involves five nucleotides (17-UCCA-20 and A100), as U17 and A100 are not base paired as predicted. Mutational analysis showed these newly identified bulge residues are important for DNA packaging. The bulge introduces a 33–35° bend in the helical axis, and inter-helical motion around this bend appears to be restricted. A model of the functional 120b pRNA was generated using a 27b NMR structure and the crystal structure of the 66b prohead-binding domain. Fitting this model into a cryo-EM map generated a pentameric pRNA structure; five helices projecting from the pRNA ring resemble an RNA claw. Biochemical analysis suggested that this shape is important for coordinated motor action required for DNA translocation. PMID:22879380

Harjes, Elena; Kitamura, Aya; Zhao, Wei; Morais, Marc C.; Jardine, Paul J.; Grimes, Shelley; Matsuo, Hiroshi

2012-01-01

122

RNA/DNA Ratios in the Estimation of Growth Stages of Oceanic Zooplankton Populations.  

National Technical Information Service (NTIS)

Current thought holds that the rate of protein synthesis is some function of the ribonucleic acid (RNA) concentration in growing animals. It is possible that measurements of the ratio of RNA to deoxyribonucleic acid (DNA) might provide an index of growth ...

D. E. Baugh

1974-01-01

123

ISOLATION AND CHARACTERIZATION OF EPIDERMAL DNA AND RNA FROM GUINEA PIG SKIN  

Microsoft Academic Search

DNA and RNA were isolated from mammalian epidermis in a relatively small scale procedure. The high purity and native state of the DNA isolated is reflected by its molar absorptivity E (P), its thermal hyperchromicity and its hyperchromicity upon DNase treatment and by its sedimentation profile as well as by its profile in a cesium chloride density gradient. The very

D. M. Kramer; M. A. Pathak; Ute Güngerich

1971-01-01

124

Transcription-coupled and DNA damage-dependent ubiquitination of RNA polymerase II in vitro  

Microsoft Academic Search

Transcription-coupled repair (TCR) is essential for the rapid, preferential removal of DNA damage in active genes. The large subunit of RNA polymerase (Pol) II is ubiquitinated in cells after UV-irradiation or cisplatin treatment, which induces DNA damage preferentially repaired by TCR. Several human mutations, such as Cockayne syndrome complementation groups A and B, are defective in TCR and incapable of

Keng-Boon Lee; Dong Wang; Stephen J. Lippard; Phillip A. Sharp

2002-01-01

125

New Concepts in Biochemistry Divalent Cations Stabilize Unstacked Conformations of DNA and RNA by  

E-print Network

for cations in DNA bending, DNA-protein recognition, base-flipping, RNA folding, and catalysis. We propose interactions (9). Experimental gas-phase en- thalpies for binding of monovalent cations to benzene range from Database (11). The interaction is generally dominated by electrostatics, but charge transfer, polarization

Williams, Loren

126

A novel cDNA/PCR strategy for efficient cloning of small amounts of undefined RNA.  

PubMed

In this report we present a strategy for generating a representative cDNA library from prohibitively low amounts of mRNA template. A defined DNA adapter, which carries an EcoRI site, is ligated to both ends of the products of a cDNA synthesis reaction. This allows low levels of cDNA to be amplified by a polymerase chain reaction. In studies with pg amounts of rabbit globin mRNA, the amplified cDNA product is shown to be full-length. Globin cDNA recombinants are positively identified in lambda gt10. The protocol should be widely applicable to mRNAs of low abundance, whose sequences have not been determined, and to limited samples from patients or animals. It may also be useful for generating representative libraries of low titer or variant viral sequences. PMID:2530138

Akowitz, A; Manuelidis, L

1989-09-30

127

PARP activation regulates the RNA-binding protein NONO in the DNA damage response to DNA double-strand breaks  

PubMed Central

After the generation of DNA double-strand breaks (DSBs), poly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins to be recruited and activated through its binding to the free DNA ends. Upon activation, PARP-1 uses NAD+ to generate large amounts of poly(ADP-ribose) (PAR), which facilitates the recruitment of DNA repair factors. Here, we identify the RNA-binding protein NONO, a partner protein of SFPQ, as a novel PAR-binding protein. The protein motif being primarily responsible for PAR-binding is the RNA recognition motif 1 (RRM1), which is also crucial for RNA-binding, highlighting a competition between RNA and PAR as they share the same binding site. Strikingly, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR, generated by activated PARP-1. Furthermore, we show that upon PAR-dependent recruitment, NONO stimulates nonhomologous end joining (NHEJ) and represses homologous recombination (HR) in vivo. Our results therefore place NONO after PARP activation in the context of DNA DSB repair pathway decision. Understanding the mechanism of action of proteins that act in the same pathway as PARP-1 is crucial to shed more light onto the effect of interference on PAR-mediated pathways with PARP inhibitors, which have already reached phase III clinical trials but are until date poorly understood. PMID:22941645

Krietsch, Jana; Caron, Marie-Christine; Gagne, Jean-Philippe; Ethier, Chantal; Vignard, Julien; Vincent, Michel; Rouleau, Michele; Hendzel, Michael J.; Poirier, Guy G.; Masson, Jean-Yves

2012-01-01

128

PARP activation regulates the RNA-binding protein NONO in the DNA damage response to DNA double-strand breaks.  

PubMed

After the generation of DNA double-strand breaks (DSBs), poly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins to be recruited and activated through its binding to the free DNA ends. Upon activation, PARP-1 uses NAD+ to generate large amounts of poly(ADP-ribose) (PAR), which facilitates the recruitment of DNA repair factors. Here, we identify the RNA-binding protein NONO, a partner protein of SFPQ, as a novel PAR-binding protein. The protein motif being primarily responsible for PAR-binding is the RNA recognition motif 1 (RRM1), which is also crucial for RNA-binding, highlighting a competition between RNA and PAR as they share the same binding site. Strikingly, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR, generated by activated PARP-1. Furthermore, we show that upon PAR-dependent recruitment, NONO stimulates nonhomologous end joining (NHEJ) and represses homologous recombination (HR) in vivo. Our results therefore place NONO after PARP activation in the context of DNA DSB repair pathway decision. Understanding the mechanism of action of proteins that act in the same pathway as PARP-1 is crucial to shed more light onto the effect of interference on PAR-mediated pathways with PARP inhibitors, which have already reached phase III clinical trials but are until date poorly understood. PMID:22941645

Krietsch, Jana; Caron, Marie-Christine; Gagné, Jean-Philippe; Ethier, Chantal; Vignard, Julien; Vincent, Michel; Rouleau, Michèle; Hendzel, Michael J; Poirier, Guy G; Masson, Jean-Yves

2012-11-01

129

Affinity Purification of DNA and RNA from Environmental Samples with Peptide Nucleic Acid Clamps  

Microsoft Academic Search

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sed- iment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO4, 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate (SDS)) a PNA clamp recovered significantly more

DARRELL P. CHANDLER; JENNIE R. STULTS; SHARON CEBULA; BEATRICE L. SCHUCK; DEREK W. WEAVER; KEVIN K. ANDERSON; MICHAEL EGHOLM; FRED J. BROCKMAN

2000-01-01

130

The Tree of Life's Macromolecules  

NSDL National Science Digital Library

Students start with images of living organisms, from bacteria to plants and animals. They "zoom" into cells and tissues to discover that they are made of different macromolecules. Students observe that these macromolecules are polymers. They zoom into polymers to find that some are made from almost identical monomers, while others, such as proteins, are made from a set of different monomers. They discover that all monomers making up biological macromolecules are composed of just a few types of chemical elements: C, H, O, N, P and S. Students will be able to:Identify typical molecular building blocks (monomers) that form biological macromolecules; determine the types of atoms that make up most biopolymers; reason about the uniformity and diversity at the atomic level of life's molecular building blocks.

Project, Molecular L.

131

Ribavirin inhibits DNA, RNA, and protein synthesis in PHA-stimulated human peripheral blood mononuclear cells: possible explanation for therapeutic efficacy in patients with chronic HCV infection.  

PubMed

The treatment of choice for patients infected chronically with HCV is the combination of IFN-alpha and ribavirin. Monotherapy with ribavirin leads to a clinical and histological improvement, but its exact mechanism of action is unknown. Therefore, the effect of ribavirin on synthesis of inflammatory cytokines and on apoptosis in stimulated peripheral blood mononuclear cells (PBMCs) was investigated. PBMCs were isolated from the blood of HCV infected patients and from healthy volunteers. The effect of ribavirin on IFN-gamma and IL-1beta release in the supernatant of unstimulated and phytohemagglutinin (PHA) stimulated PBMCs was investigated by enzyme linked immunosorbent assay (ELISA). The effect on total DNA, RNA, and protein synthesis was analyzed by measurement of 3H-thymidine, 3H-uridine and 3H-leucine incorporation into cellular macromolecules. Ribavirin led to a dose-dependent decrease of the IFN-gamma but an increase of IL-1beta release into the supernatant of PHA-stimulated PBMCs. At the same time, a dose-dependent decrease of total DNA, RNA, and protein synthesis in cultures of PHA-stimulated PBMCs was demonstrated. These effects could be compensated by the addition of equimolar amounts of guanosine. The rate of apoptotic CD45+ and CD14+ cells in PBMCs cultures increased in a dose-dependent manner. Our data suggest that ribavirin administration to chronically HCV-infected patients could lead to a decrease of the synthesis of proinflammatory cytokines (e.g., IFN-gamma) by an inhibition of total DNA-, RNA-, and protein-synthesis and by induction of apoptosis in the cells of the inflammatory infiltrate. Furthermore, ribavirin could influence the synthesis of viral particles in the hepatocytes. PMID:12436477

Meier, Volker; Bürger, Erik; Mihm, Sabine; Saile, Bernhard; Ramadori, Giuliano

2003-01-01

132

The chemical structure of DNA sequence signals for RNA transcription  

NASA Technical Reports Server (NTRS)

The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

George, D. G.; Dayhoff, M. O.

1982-01-01

133

Bacterial RNA:DNA hybrids are activators of the NLRP3 inflammasome.  

PubMed

Enterohemorrhagic Escherichia coli (EHEC) is an extracellular pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. The proinflammatory cytokine, interleukin-1?, has been linked to hemolytic uremic syndrome. Here we identify the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome as an essential mediator of EHEC-induced IL-1?. Whereas EHEC-specific virulence factors were dispensable for NLRP3 activation, bacterial nucleic acids such as RNA:DNA hybrids and RNA gained cytosolic access and mediated inflammasome-dependent responses. Consistent with a direct role for RNA:DNA hybrids in inflammasome activation, delivery of synthetic EHEC RNA:DNA hybrids into the cytosol triggered NLRP3-dependent responses, and introduction of RNase H, which degrades such hybrids, into infected cells specifically inhibited inflammasome activation. Notably, an E. coli rnhA mutant, which is incapable of producing RNase H and thus harbors increased levels of RNA:DNA hybrid, induced elevated levels of NLRP3-dependent caspase-1 activation and IL-1? maturation. Collectively, these findings identify RNA:DNA hybrids of bacterial origin as a unique microbial trigger of the NLRP3 inflammasome. PMID:24828532

Kailasan Vanaja, Sivapriya; Rathinam, Vijay A K; Atianand, Maninjay K; Kalantari, Parisa; Skehan, Brian; Fitzgerald, Katherine A; Leong, John M

2014-05-27

134

Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes  

SciTech Connect

Treatment of HeLa cells with a DNA topoisomerase I-specific inhibitor, camptothecin, results in rapid cessation of the synthesis of the 45S rRNA precursor. The inhibition of rRNA synthesis is reversible following drug removal and correlates with the presence of camptothecin-trapped topoisomerase I-DNA abortive complexes, which can be detected as topoisomerase I-linked DNA breaks upon lysis with sodium dodecyl sulfate. These breaks were found to be concentrated within the transcribed region of human rRNA genes. No such sites can be detected in the inactive human rRNA genes in mouse-human hybrid cells, suggesting a preferential association of topoisomerase I with actively transcribed genes. The distribution of RNA polymerase molecules along the transcription unit of human rRNA genes in camptothecin-treated HeLa cells, as assayed by nuclear run-on transcription, shows a graded decrease of the RNA polymerase density toward the 3' end of the transcription unit; the density is minimally affected near the 5' start of the transcription unit. These results suggest that DNA topoisomerase I is normally involved in the elongation step of transcription, especially when the transcripts are long, and that camptothecin interferes with this role.

Zhang, H.; Wang, J.C.; Liu, L.F.

1988-02-01

135

Effect of salts, solvents and buffer on miRNA detection using DNA silver nanocluster (DNA/AgNCs) probes  

NASA Astrophysics Data System (ADS)

MicroRNAs (miRNAs) are small regulatory RNAs (size ˜21 nt to ˜25 nt) which regulate a variety of important cellular events in plants, animals and single cell eukaryotes. Especially because of their use in diagnostics of human diseases, efforts have been directed towards the invention of a rapid, simple and sequence selective detection method for miRNAs. Recently, we reported an innovative method for the determination of miRNA levels using the red fluorescent properties of DNA/silver nanoclusters (DNA/AgNCs). Our method is based on monitoring the emission drop of a DNA/AgNCs probe in the presence of its specific target miRNA. Accordingly, the accuracy and efficiency of the method relies on the sensitivity of hybridization between the probe and target. To gain specific and robust hybridization between probe and target, we investigated a range of diverse salts, organic solvents, and buffer to optimize target sensing conditions. Under the newly adjusted conditions, the target sensitivity and the formation of emissive DNA/AgNCs probes were significantly improved. Also, fortification of the Tris-acetate buffer with inorganic salts or organic solvents improved the sensitivity of the DNA/AgNC probes. On the basis of these optimizations, the versatility of the DNA/AgNCs-based miRNA detection method can be expanded.

Shah, Pratik; Cho, Seok Keun; Waaben Thulstrup, Peter; Bhang, Yong-Joo; Ahn, Jong Cheol; Choi, Suk Won; Rørvig-Lund, Andreas; Yang, Seong Wook

2014-01-01

136

Oncolytic virus-mediated tumor radiosensitization in mice through DNA-PKcs-specific shRNA  

PubMed Central

One of the key issues in cancer radiotherapy research is to sensitize tumor cells to the cell killing effects of ionizing radiation while leaving normal tissues intact. One potential approach to achieve this is through tumor-specific targeting of DNA repair genes. In this study, we engineered a replication-deficient adenovirus encoding a mini shRNA gene targeted to the DNA-PKcs gene, which is involved in double strand break DNA repair, and evaluated its anti-tumor efficacy in combination with radiotherapy. Our shRNA-encoding adenovirus showed significant efficacy in down-regulating the levels of the DNA-PKcs protein that was accompanied by increased radiation sensitivity in the human HCT116 colon cancer cells. However, when delivered intratumorally to xenograft human tumors, minimal anti-tumor effects of the virus were seen either alone or in combination with radiation therapy, suggesting an inefficiency of the non-replicative adenovirus in delivering shRNA genes to the tumor mass. When a conditionally replicative adenovirus targeted to telomerase-positive tumor cells was used in conjunction with the DNA-PKcs-targeted shRNA-encoding non-replicative adenovirus, the efficiency of tumor-specific anti-DNA-PKcs shRNA gene expression was enhanced significantly. Most importantly, this enhanced shRNA expression led to significant anti-tumor efficacy of concurrently delivered radiation therapy. Our results suggest our shRNA-based DNA-PKcs- targeting approach in combination with tumor-targeting replicative adenovirus is a promising method to sensitize solid tumors to radiation therapy. PMID:22924158

Kon, Takashi; Zhang, Xiuwu; Huang, Qian; Yang, Zhonghui; Liu, Shanling; Yan, Bin; Li, Fang; Wang, He; Li, Chuan-Yuan

2012-01-01

137

The splicing machinery promotes RNA-directed DNA methylation and transcriptional silencing in Arabidopsis  

PubMed Central

DNA methylation in transposons and other DNA repeats is conserved in plants as well as in animals. In Arabidopsis thaliana, an RNA-directed DNA methylation (RdDM) pathway directs de novo DNA methylation. We performed a forward genetic screen for suppressors of the DNA demethylase mutant ros1 and identified a novel Zinc-finger and OCRE domain-containing Protein 1 (ZOP1) that promotes Pol IV-dependent siRNA accumulation, DNA methylation, and transcriptional silencing. Whole-genome methods disclosed the genome-wide effects of zop1 on Pol IV-dependent siRNA accumulation and DNA methylation, suggesting that ZOP1 has both RdDM-dependent and -independent roles in transcriptional silencing. We demonstrated that ZOP1 is a pre-mRNA splicing factor that associates with several typical components of the splicing machinery as well as with Pol II. Immunofluorescence assay revealed that ZOP1 overlaps with Cajal body and is partially colocalized with NRPE1 and DRM2. Moreover, we found that the other development-defective splicing mutants tested including mac3a3b, mos4, mos12 and mos14 show defects in RdDM and transcriptional silencing. We propose that the splicing machinery rather than specific splicing factors is involved in promoting RdDM and transcriptional silencing. PMID:23524848

Zhang, Cui-Jun; Zhou, Jin-Xing; Liu, Jun; Ma, Ze-Yang; Zhang, Su-Wei; Dou, Kun; Huang, Huan-Wei; Cai, Tao; Liu, Renyi; Zhu, Jian-Kang; He, Xin-Jian

2013-01-01

138

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development  

EPA Science Inventory

The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

139

RNA-Dependent DNA Polymerase Activity of RNA Tumor Viruses I. Directing Influence of DNA in the Reaction  

PubMed Central

The template requirements and deoxyribonucleic acid (DNA) products of the DNA polymerases isolated from Rauscher leukemia and avian myeloblastosis viruses have been examined. All DNA preparations or synthetic polydeoxynucleotides which are active as primers possess a duplex structure containing single-stranded regions with a 3?-hydroxyl terminus. Native DNA and fully single-stranded DNA are inactive; moreover, their activity is not enhanced by sonic oscillation or treatment with micrococcal nuclease, Neurospora nuclease, or low levels of deoxyribonuclease I. Poor DNA templates are activated by treatment with exonuclease III, large amounts of deoxyribonuclease I, or an endonuclease isolated from Rauscher viral preparations. In reactions primed with deoxyadenylate-deoxythymidylate copolymer, the product formed is covalently attached to primer strands, indicating that no new strands are initiated. DNA polymerase products formed with exonuclease III- or deoxyribonuclase I-treated DNA are duplex structures. Short single-stranded regions are completely filled in, whereas long single-stranded regions are only partly repaired. DNA preparations containing extensive single-stranded regions are poorly utilized as templates. PMID:4333538

Hurwitz, Jerard; Leis, Jonathan P.

1972-01-01

140

Lipid-mediated DNA and siRNA Transfection Efficiency Depends on Peptide Headgroup  

PubMed Central

A series of amphiphiles with differing cationic tri- and di- peptide headgroups, designed and synthesized based on lysine (K), ornithine (O), arginine (R), and glycine (G), have been characterized and evaluated for DNA and siRNA delivery. DNA-lipoplexes formed from the tri- and di- lipopeptides possessed lipid:nucleic acid charge ratios of 7:1 to 10:1, diameters of ~200 nm to 375 nm, zeta potentials of 23 mV to 41 mV, melting temperatures of 12 °C to 46 °C, and lamellar repeat periods of 6 nm to 8 nm. These lipid-DNA complexes formed supramolecular structures in which DNA is entrapped at the surface between multilamellar liposomal vesicles. Compared to their DNA counterparts, siRNA-lipoplexes formed slightly larger complexes (348 nm to 424 nm) and required higher charge ratios to form stable structures. Additionally, it was observed that lipids with multivalent, tripeptide headgroups (i.e., KGG, OGG, and RGG) were successful at transfecting DNA in vitro, whereas DNA transfection with the dipeptide lipids proved ineffective. Cellular uptake of DNA was more effective with the KGG compared to the KG lipopeptide. In siRNA knockdown experiments, both tri- and di- peptide lipids (i.e., RGG, GGG, KG, OG, RG, GG) showed some efficacy, but total cellular uptake of siRNA complexes was not indicative of knockdown outcomes and suggested that the intracellular fate of lipoplexes may be a factor. Overall, this lipopeptide study expands the library of efficient DNA transfection vectors available for use, introduces new vectors for siRNA delivery, and begins to address the structure-activity relationships which influence delivery and transfection efficacy. PMID:24391676

Zhang, Xiao-Xiang; LaManna, Caroline M.; Kohman, Richie E.; McIntosh, Thomas J.; Han, Xue; Grinstaff, Mark W.

2013-01-01

141

A pathogenic non-coding RNA induces changes in dynamic DNA methylation of ribosomal RNA genes in host plants.  

PubMed

Viroids are plant-pathogenic non-coding RNAs able to interfere with as yet poorly known host-regulatory pathways and to cause alterations recognized as diseases. The way in which these RNAs coerce the host to express symptoms remains to be totally deciphered. In recent years, diverse studies have proposed a close interplay between viroid-induced pathogenesis and RNA silencing, supporting the belief that viroid-derived small RNAs mediate the post-transcriptional cleavage of endogenous mRNAs by acting as elicitors of symptoms expression. Although the evidence supporting the role of viroid-derived small RNAs in pathogenesis is robust, the possibility that this phenomenon can be a more complex process, also involving viroid-induced alterations in plant gene expression at transcriptional levels, has been considered. Here we show that plants infected with the 'Hop stunt viroid' accumulate high levels of sRNAs derived from ribosomal transcripts. This effect was correlated with an increase in the transcription of ribosomal RNA (rRNA) precursors during infection. We observed that the transcriptional reactivation of rRNA genes correlates with a modification of DNA methylation in their promoter region and revealed that some rRNA genes are demethylated and transcriptionally reactivated during infection. This study reports a previously unknown mechanism associated with viroid (or any other pathogenic RNA) infection in plants providing new insights into aspects of host alterations induced by the viroid infectious cycle. PMID:24178032

Martinez, German; Castellano, Mayte; Tortosa, Maria; Pallas, Vicente; Gomez, Gustavo

2014-02-01

142

Insights into RNA/DNA hybrid recognition and processing by RNase H from the crystal structure of a non-specific enzyme-dsDNA complex  

SciTech Connect

Ribonuclease HI (RNase H) is a member of the nucleotidyl-transferase superfamily and endo-nucleolytically cleaves the RNA portion in RNA/DNA hybrids and removes RNA primers from Okazaki fragments. The enzyme also binds RNA and DNA duplexes but is unable to cleave either. Three-dimensional structures of bacterial and human RNase H catalytic domains bound to RNA/DNA hybrids have revealed the basis for substrate recognition and the mechanism of cleavage. In order to visualize the enzyme's interactions with duplex DNA and to establish the structural differences that afford tighter binding to RNA/DNA hybrids relative to dsDNA, we have determined the crystal structure of Bacillus halodurans RNase H in complex with the B-form DNA duplex [d(CGCGAATTCGCG)]2. The structure demonstrates that the inability of the enzyme to cleave DNA is due to the deviating curvature of the DNA strand relative to the substrate RNA strand and the absence of Mg{sup 2+} at the active site. A subset of amino acids engaged in contacts to RNA 2{prime}-hydroxyl groups in the substrate complex instead bind to bridging or non-bridging phosphodiester oxygens in the complex with dsDNA. Qualitative comparison of the enzyme's interactions with the substrate and inhibitor duplexes is consistent with the reduced binding affinity for the latter and sheds light on determinants of RNase H binding and cleavage specificity.

Pallan, Pradeep S.; Egli, Martin (Vanderbilt)

2009-06-17

143

RecFOR Proteins Target RecA Protein to a DNA Gap with Either DNA or RNA at the 5 Terminus  

E-print Network

: This work provides biochemical evidence for recognition by RecF of gaps created when DNA replication haltsRecFOR Proteins Target RecA Protein to a DNA Gap with Either DNA or RNA at the 5 Terminus proteins regulate assembly of RecA onto single-stranded DNA complexed with SSB protein. Results: Rec

Kowalczykowski, Stephen C.

144

Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil.  

PubMed

Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al?(SO?)? were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris-HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al?(SO?)? for treating rhizosphere soil of rice, tobacco, sugarcane, Rehmannia glutinosa, and Pseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses. PMID:24078111

Fang, Changxun; Xu, Tiecheng; Ye, Changliang; Huang, Likun; Wang, Qingshui; Lin, Wenxiong

2014-02-01

145

Affinity Purification of DNA and RNA from Environmental Samples with Peptide Nucleic Acid Clamps  

PubMed Central

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO4, 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO4, 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of ?100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10?21 M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective. PMID:10919804

Chandler, Darrell P.; Stults, Jennie R.; Cebula, Sharon; Schuck, Beatrice L.; Weaver, Derek W.; Anderson, Kevin K.; Egholm, Michael; Brockman, Fred J.

2000-01-01

146

Human papilloma virus, DNA methylation and microRNA expression in cervical cancer (Review)  

PubMed Central

Cancer is a complex disease caused by genetic and epigenetic abnormalities that affect gene expression. The progression from precursor lesions to invasive cervical cancer is influenced by persistent human papilloma virus (HPV) infection, which induces changes in the host genome and epigenome. Epigenetic alterations, such as aberrant miRNA expression and changes in DNA methylation status, favor the expression of oncogenes and the silencing of tumor-suppressor genes. Given that some miRNA genes can be regulated through epigenetic mechanisms, it has been proposed that alterations in the methylation status of miRNA promoters could be the driving mechanism behind their aberrant expression in cervical cancer. For these reasons, we assessed the relationship among HPV infection, cellular DNA methylation and miRNA expression. We conclude that alterations in the methylation status of protein-coding genes and various miRNA genes are influenced by HPV infection, the viral genotype, the physical state of the viral DNA, and viral oncogenic risk. Furthermore, HPV induces deregulation of miRNA expression, particularly at loci near fragile sites. This deregulation occurs through the E6 and E7 proteins, which target miRNA transcription factors such as p53. PMID:24737381

JIMÉNEZ-WENCES, HILDA; PERALTA-ZARAGOZA, OSCAR; FERNÁNDEZ-TILAPA, GLORIA

2014-01-01

147

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development  

Microsoft Academic Search

The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA\\u000a extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic\\u000a analysis of 761 DNA-based clone sequences showed that unclassified bacteria were the most abundant group, representing nearly\\u000a 62% of all DNA sequences analyzed.

Randy P. Revetta; Robin S. Matlib

2011-01-01

148

Archaeal DnaG contains a conserved N-terminal RNA-binding domain and enables tailing of rRNA by the exosome.  

PubMed

The archaeal exosome is a phosphorolytic 3'-5' exoribonuclease complex. In a reverse reaction it synthesizes A-rich RNA tails. Its RNA-binding cap comprises the eukaryotic orthologs Rrp4 and Csl4, and an archaea-specific subunit annotated as DnaG. In Sulfolobus solfataricus DnaG and Rrp4 but not Csl4 show preference for poly(rA). Archaeal DnaG contains N- and C-terminal domains (NTD and CTD) of unknown function flanking a TOPRIM domain. We found that the NT and TOPRIM domains have comparable, high conservation in all archaea, while the CTD conservation correlates with the presence of exosome. We show that the NTD is a novel RNA-binding domain with poly(rA)-preference cooperating with the TOPRIM domain in binding of RNA. Consistently, a fusion protein containing full-length Csl4 and NTD of DnaG led to enhanced degradation of A-rich RNA by the exosome. We also found that DnaG strongly binds native and in vitro transcribed rRNA and enables its polynucleotidylation by the exosome. Furthermore, rRNA-derived transcripts with heteropolymeric tails were degraded faster by the exosome than their non-tailed variants. Based on our data, we propose that archaeal DnaG is an RNA-binding protein, which, in the context of the exosome, is involved in targeting of stable RNA for degradation. PMID:25326320

Hou, Linlin; Klug, Gabriele; Evguenieva-Hackenberg, Elena

2014-11-10

149

Archaeal DnaG contains a conserved N-terminal RNA-binding domain and enables tailing of rRNA by the exosome  

PubMed Central

The archaeal exosome is a phosphorolytic 3?–5? exoribonuclease complex. In a reverse reaction it synthesizes A-rich RNA tails. Its RNA-binding cap comprises the eukaryotic orthologs Rrp4 and Csl4, and an archaea-specific subunit annotated as DnaG. In Sulfolobus solfataricus DnaG and Rrp4 but not Csl4 show preference for poly(rA). Archaeal DnaG contains N- and C-terminal domains (NTD and CTD) of unknown function flanking a TOPRIM domain. We found that the NT and TOPRIM domains have comparable, high conservation in all archaea, while the CTD conservation correlates with the presence of exosome. We show that the NTD is a novel RNA-binding domain with poly(rA)-preference cooperating with the TOPRIM domain in binding of RNA. Consistently, a fusion protein containing full-length Csl4 and NTD of DnaG led to enhanced degradation of A-rich RNA by the exosome. We also found that DnaG strongly binds native and in vitro transcribed rRNA and enables its polynucleotidylation by the exosome. Furthermore, rRNA-derived transcripts with heteropolymeric tails were degraded faster by the exosome than their non-tailed variants. Based on our data, we propose that archaeal DnaG is an RNA-binding protein, which, in the context of the exosome, is involved in targeting of stable RNA for degradation. PMID:25326320

Hou, Linlin; Klug, Gabriele; Evguenieva-Hackenberg, Elena

2014-01-01

150

A rolling circle amplification-based DNA machine for miRNA screening coupling catalytic hairpin assembly with DNAzyme formation.  

PubMed

A novel DNA nanomachine based on the linear rolling circle amplification strategy was designed for sensitive screening of microRNA (miRNA) at an ultralow concentration coupling catalytic hairpin assembly (CHA) with DNAzyme formation. PMID:24501741

Zhuang, Junyang; Lai, Wenqiang; Chen, Guonan; Tang, Dianping

2014-03-18

151

A program for the identification of tRNA-like structures in DNA sequence data.  

PubMed Central

A computer algorithm has been developed which identifies tRNA genes and tRNA-like structures in DNA sequences. The program searches the sequence string for specific base positions that correspond to the invariant and semi-invariant bases found in tRNAs. The tRNA nature of the sequence is confirmed by the presence of complementary base pairing at the tRNA's calculated 5' and 3' ends (which in situ constitutes the amino-acyl stem region). The program achieves greater than 96% accuracy when run against known tRNA sequences in the Genbank database. The program is modular and is readily modified to allow searching either a file or database. The program is written in "C" and operates on a D.E.C. Vax 750. The utility of the algorithm is demonstrated by the identification of a distinctive tRNA structure in an intron of a published bovine hemoglobin gene. PMID:3753778

Marvel, C C

1986-01-01

152

RNA:DNA hybrids are a novel molecular pattern sensed by TLR9.  

PubMed

The sensing of nucleic acids by receptors of the innate immune system is a key component of antimicrobial immunity. RNA:DNA hybrids, as essential intracellular replication intermediates generated during infection, could therefore represent a class of previously uncharacterised pathogen-associated molecular patterns sensed by pattern recognition receptors. Here we establish that RNA:DNA hybrids containing viral-derived sequences efficiently induce pro-inflammatory cytokine and antiviral type I interferon production in dendritic cells. We demonstrate that MyD88-dependent signalling is essential for this cytokine response and identify TLR9 as a specific sensor of RNA:DNA hybrids. Hybrids therefore represent a novel molecular pattern sensed by the innate immune system and so could play an important role in host response to viruses and the pathogenesis of autoimmune disease. PMID:24514026

Rigby, Rachel E; Webb, Lauren M; Mackenzie, Karen J; Li, Yue; Leitch, Andrea; Reijns, Martin A M; Lundie, Rachel J; Revuelta, Ailsa; Davidson, Donald J; Diebold, Sandra; Modis, Yorgo; MacDonald, Andrew S; Jackson, Andrew P

2014-03-18

153

The Chemical Synthesis of DNA/RNA: Our Gift to Science  

PubMed Central

It is a great privilege to contribute to the Reflections essays. In my particular case, this essay has allowed me to weave some of my major scientific contributions into a tapestry held together by what I have learned from three colleagues (Robert Letsinger, Gobind Khorana, and George Rathmann) who molded my career at every important junction. To these individuals, I remain eternally grateful, as they always led by example and showed many of us how to break new ground in both science and biotechnology. Relative to my scientific career, I have focused primarily on two related areas. The first is methodologies we developed for chemically synthesizing DNA and RNA. Synthetic DNA and RNA continue to be an essential research tool for biologists, biochemists, and molecular biologists. The second is developing new approaches for solving important biological problems using synthetic DNA, RNA, and their analogs. PMID:23223445

Caruthers, Marvin H.

2013-01-01

154

Preliminary Aspects Related to a DNA-RNA Simulator  

Microsoft Academic Search

Here we describe a project t hat i s being conduced at UNIVALI - Computer Science Center. Its main objective is to develop a software to simulate the DNA replication and translation processes in a user defined environment (temperature, molecule concentration, cell state, etc). Based in the ce llular automata technique, the simulator will present a bi-dimensional graphical view of

Rafael Luiz Cancian; Thomas Basten; André Oliveira de Souza

155

RNA-Cleaving DNA Enzymes with Altered Regio- or Enantioselectivity  

NASA Technical Reports Server (NTRS)

In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5' - phosphodiester following a wibonucleotide or a 3',5' -phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5' -phosphodiester exhibits a k(sub cat) of approx. 0.01/ min and catalytic efficiency, k(sub cat)/k(sub m) of approx. 10(exp 5)/ M min. The enzyme that cleaves an L-ribonudeotide is about 10-fold slower and has a catalytic efficiency of approx. 4 x 10(exp 5)/ M min. Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 C. In a comparison of each enzyme s activity with either its corresponding substrate that contains an unnatural ribonudeotide or a substrate that instead contains a standard ribonucleotide, the 2',5' -phosphodiester-deaving DNA enzyme exhibited a regioselectivity of 6000- fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 50-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.

Ordoukhanian, Phillip; Joyce, Gerald F.

2002-01-01

156

Macromolecules Relevant to Stone Formation  

NASA Astrophysics Data System (ADS)

Despite years of research, no single macromolecule in kidney calculi or in urine has yet been shown to fulfill a specific function in stone pathogenesis. In this paper we briefly review papers investigating the urinary excretion of individual macromolecules, their effects on calcium oxalate (CaOx) crystallization and attachment of crystals to renal epithelial cells, and the influence of lithogenic conditions on their renal expression in cultured cells and animal models. Using prothrombin fragment 1 (PTF1) and human serum albumin as examples, we show the types of patterns resulting from the binding of a fluorescently tagged protein to a specific CaOx monohydrate (COM) crystal face and its incorporation into the crystal structure. Molecular modeling is also used to illustrate how PTF1 can align with the atomic array on a COM crystal surface. We conclude that although many macromolecules are, by strict definition, relevant to stone formation, very few are probably truly influential.

Ryall, Rosemary L.; Cook, Alison F.; Thurgood, Lauren A.; Grover, Phulwinder K.

2007-04-01

157

Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing  

Microsoft Academic Search

Like other eukaryotes, plants use DICER-LIKE (DCL) proteins as the central enzymes of RNA silencing, which regulates gene expression and mediates defense against viruses. But why do plants like Arabidopsis express four DCLs, a diversity un- matched by other kingdoms? Here we show that two nuclear DNA viruses (geminivirus CaLCuV and pararetrovirus CaMV) and a cytoplasmic RNA tobamovirus ORMV are

Todd Blevins; Rajendran Rajeswaran; Padubidri V. Shivaprasad; Daria Beknazariants; Azeddine Si-Ammour; Hyun-Sook Park; Franck Vazquez; Dominique Robertson; Frederick Meins Jr; Thomas Hohn; Mikhail M. Pooggin

2006-01-01

158

DNA structural variation affects complex formation and promoter melting in ribosomal RNA transcription  

Microsoft Academic Search

Eukaryotic ribosomal RNA promoters exhibit an unusual conservation of non-canonical DNA structure (curvature, twist angle and duplex stability) despite a lack of primary sequence conservation. This raises the possibility that rRNA transcription factors might utilize structural anomalies in their sequence recognition process. We have analyzed in detail the interaction of the polymerase I transcription factor TIF-IB from Acanthmoeba castellanii with

M. Marilley; C. Radebaugh; G. Geiss; P. Laybourn; M. Paule

2002-01-01

159

DNA structure in human RNA polymerase II promoters1  

Microsoft Academic Search

this paper we present an analysis of a large set of human RNA polymerase II promoters with verylow sequence similarity. The sequences, which include both TATA-containing and TATA-less promoters,are aligned by hidden Markov models (HMMs). Using three different models of sequence-derived DNAbendability, the aligned promoters display a common structural profile with bendability being low in a regionupstream of the transcriptional

Anders Gorm Pedersen; Pierre Baldi; Yves Chauvin; Søren Brunak

1998-01-01

160

The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once.  

PubMed

To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by James Watson and Francis Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure twice these nucleases act as molecular level transformers that typically reshape the DNA and sometimes themselves to achieve extraordinary specificity and efficiency. PMID:24754999

Tsutakawa, Susan E; Lafrance-Vanasse, Julien; Tainer, John A

2014-07-01

161

The effect of as long-term Mars simulation on a microbial permafrost soil community and macromolecules such as DNA, polypeptides and cell wall components.  

NASA Astrophysics Data System (ADS)

Ten freeze-dried and homogenized samples of a 2300 years old Spitsbergen permafrost soil containing a complex microbial community were aseptically transferred to inert glass tubes and subjected to a 30 days Martian simulation experiment. During this period the samples received an UV dose equivalent to 80 Martian Sol. Data loggers in 4 out the ten samples monitored the temperature 0-2 mm below the surface of the sample. After removal from the simulation chamber, the samples were sliced in 1.5 to 6 mm thick horizons (H1, 0-1.5 mm; H2, 1.5-3 mm; H3, 3-6 mm; H4, 6-9 mm; H5, 9-15 mm; H6, 15-21 mm; H7, 21-27 mm and H8, 27-33 mm), resulting in 10 subsamples from each soil horizon. The subsamples from each horizon were pooled and used for the following investigations: 1. Determination of the bacterial number after staining with SYBR-gold, 2. Determination of the number of dead and living bacteria using the BacLight kit, 3. Determination of the total amount of extractable DNA, 4. Determination of the number of culturable aerobic and anaerobic bacteria, 5. Determination of the concentration of the total hydrolysable amino acids and D and L enantiomers, 6. Determination of the muramic acid contentration. The results of the experiments will be presented and discussed in our communication

Finster, K.; Hansen, A.; Liengaard, L.; Kristoffersen, T.; Mikkelsen, K.; Merrison, J.; Lomstein, B.

162

Nucleic acid determinants for selective deamination of DNA over RNA by activation-induced deaminase  

PubMed Central

Activation-induced deaminase (AID), a member of the larger AID/APOBEC family, is the key catalyst in initiating antibody somatic hypermutation and class-switch recombination. The DNA deamination model accounting for AID’s functional role posits that AID deaminates genomic deoxycytosine bases within the immunoglobulin locus, activating downstream repair pathways that result in antibody maturation. Although this model is well supported, the molecular basis for AID’s selectivity for DNA over RNA remains an open and pressing question, reflecting a broader need to elucidate how AID/APOBEC enzymes engage their substrates. To address these questions, we have synthesized a series of chimeric nucleic acid substrates and characterized their reactivity with AID. These chimeric substrates feature targeted variations at the 2?-position of nucleotide sugars, allowing us to interrogate the steric and conformational basis for nucleic acid selectivity. We demonstrate that modifications to the target nucleotide can significantly alter AID’s reactivity. Strikingly, within a substrate that is otherwise DNA, a single RNA-like 2?-hydroxyl substitution at the target cytosine is sufficient to compromise deamination. Alternatively, modifications that favor a DNA-like conformation (or sugar pucker) are compatible with deamination. AID’s closely related homolog APOBEC1 is similarly sensitive to RNA-like substitutions at the target cytosine. Inversely, with unreactive 2?-fluoro-RNA substrates, AID’s deaminase activity was rescued by introducing a trinucleotide DNA patch spanning the target cytosine and two nucleotides upstream. These data suggest a role for nucleotide sugar pucker in explaining the molecular basis for AID’s DNA selectivity and, more generally, suggest how other nucleic acid-modifying enzymes may distinguish DNA from RNA. PMID:23942124

Nabel, Christopher S.; Lee, Jae W.; Wang, Laura C.; Kohli, Rahul M.

2013-01-01

163

Seasonal patterns of muscle RNA-DNA ratio and growth in black crappie, Pomoxis nigromaculatus  

Microsoft Academic Search

Synopsis  Black crappie (Pomoxis nigromaculatus) were collected weekly from a natural lake during the period mid-April to mid-September. The fish were weighed, state of\\u000a maturity determined and RNA-DNA ratio of white muscle was measured. Water temperature and primary production were measured\\u000a in the lake.\\u000a \\u000a RNA-DNA ratio declined during the spawning season, reaching a low in mid-May, then increased steadily during the

Terry A. Haines

1980-01-01

164

Conformations consistent with charge migration observed in DNA and RNA X-ray structures.  

PubMed

Electron holes are known to migrate along the DNA or RNA duplexes and to localize preferentially on successive guanines. The stationary point conformations of Gua pairs that can trap or let pass these holes have been characterized by quantum chemistry calculations. Here we show their recurrent occurrence in DNA and RNA X-ray structures, often in quadruplex conformations or in interaction with proteins, ligands or metal ions. These findings give support to the biological, possibly regulatory, roles of charge migration in cell functioning. PMID:21469755

Rooman, Marianne; Cauët, Emilie; Liévin, Jacques; Wintjens, René

2011-06-01

165

Using Amino-Labeled Nucleotide Probes for Simultaneous Single Molecule RNA-DNA FISH  

PubMed Central

Using amino-labeled oligonucleotide probes, we established a simple, robust and low-noise method for simultaneous detection of RNA and DNA by fluorescence in situ hybridization, a highly useful tool to study the large pool of long non-coding RNAs being identified in the current research. With probes either chemically or biologically synthesized, we demonstrate that the method can be applied to study a wide range of RNA and DNA targets at the single-cell and single-molecule level in cellular contexts. PMID:25226542

Wu, Jun; Shao, Fangwei; Zhang, Li-Feng

2014-01-01

166

Analysis of Cytokine mRNA and DNA: Detection and Quantitation by Competitive Polymerase Chain Reaction  

Microsoft Academic Search

The expression of two cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3), has been investigated in MLA-144 cells before and after induction with phorbol 12-myristate 13-acetate. We describe an adaptation of the polymerase chain reaction (PCR) for highly accurate quantitation of mRNA or DNA from a small number of cells. Aliquots of the PCR mixture containing cDNA copies of

Gary Gilliland; Steven Perrin; Kerry Blanchard; H. Franklin Bunn

1990-01-01

167

High-Fidelity Recognition of RNA: Solution Structure of a DNA:RNA Hybrid Duplex with a Molecular Cap.  

PubMed

Binding RNA targets, such as microRNAs, with high fidelity is challenging, particularly when the nucleobases to be bound are located at the terminus of the duplex between probe and target. Recently, a peptidyl chain terminating in a quinolone, called ogOA, was shown to act as a cap that enhances affinity and fidelity for RNAs, stabilizing duplexes with Watson-Crick pairing at their termini. Here we report the three-dimensional structure of an intramolecular complex between a DNA strand featuring the ogOA cap and an RNA segment, solved by NMR and restrained torsion angle molecular dynamics. The quinolone stacks on the terminal base pair of the hybrid duplex, positioned by the peptidyl chain, whose prolinol residue induces a sharp bend between the 5' terminus of the DNA chain and the glycine linked to the oxolinic acid residue. The structure explains why canonical base pairing is favored over hard-to-suppress mismatched base combinations, such as T:G and A:A, and helps to design improved high-fidelity probes for RNA. PMID:25318665

Gerlach, Claudia; Claasen, Birgit; Richert, Clemens

2014-11-24

168

INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis  

SciTech Connect

At least three pathways control maintenance of DNA cytosine methylation in Arabidopsis thaliana. However, the RNA-directed DNA methylation (RdDM) pathway is solely responsible for establishment of this silencing mark. We previously described INVOLVED IN DE NOVO 2 (IDN2) as being an RNA-binding RdDM component that is required for DNA methylation establishment. In this study, we describe the discovery of two partially redundant proteins that are paralogous to IDN2 and that form a stable complex with IDN2 in vivo. Null mutations in both genes, termed IDN2-LIKE 1 and IDN2-LIKE 2 (IDNL1 and IDNL2), result in a phenotype that mirrors, but does not further enhance, the idn2 mutant phenotype. Genetic analysis suggests that this complex acts in a step in the downstream portion of the RdDM pathway. We also have performed structural analysis showing that the IDN2 XS domain adopts an RNA recognition motif (RRM) fold. Finally, genome-wide DNA methylation and expression analysis confirms the placement of the IDN proteins in an RdDM pathway that affects DNA methylation and transcriptional control at many sites in the genome. Results from this study identify and describe two unique components of the RdDM machinery, adding to our understanding of DNA methylation control in the Arabidopsis genome.

Ausin, Israel; Greenberg, Maxim V.C.; Simanshu, Dhirendra K.; Hale, Christopher J.; Vashisht, Ajay A.; Simon, Stacey A.; Lee, Tzuu-fen; Feng, Suhua; Española, Sophia D.; Meyers, Blake C.; Wohlschlegel, James A.; Patel, Dinshaw J.; Jacobsen, Steven E. (UCLA); (MSKCC); (Delaware)

2012-10-23

169

Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing.  

PubMed

Like other eukaryotes, plants use DICER-LIKE (DCL) proteins as the central enzymes of RNA silencing, which regulates gene expression and mediates defense against viruses. But why do plants like Arabidopsis express four DCLs, a diversity unmatched by other kingdoms? Here we show that two nuclear DNA viruses (geminivirus CaLCuV and pararetrovirus CaMV) and a cytoplasmic RNA tobamovirus ORMV are differentially targeted by subsets of DCLs. DNA virus-derived small interfering RNAs (siRNAs) of specific size classes (21, 22 and 24 nt) are produced by all four DCLs, including DCL1, known to process microRNA precursors. Specifically, DCL1 generates 21 nt siRNAs from the CaMV leader region. In contrast, RNA virus infection is mainly affected by DCL4. While the four DCLs are partially redundant for CaLCuV-induced mRNA degradation, DCL4 in conjunction with RDR6 and HEN1 specifically facilitates extensive virus-induced silencing in new growth. Additionally, we show that CaMV infection impairs processing of endogenous RDR6-derived double-stranded RNA, while ORMV prevents HEN1-mediated methylation of small RNA duplexes, suggesting two novel viral strategies of silencing suppression. Our work highlights the complexity of virus interaction with host silencing pathways and suggests that DCL multiplicity helps mediate plant responses to diverse viral infections. PMID:17090584

Blevins, Todd; Rajeswaran, Rajendran; Shivaprasad, Padubidri V; Beknazariants, Daria; Si-Ammour, Azeddine; Park, Hyun-Sook; Vazquez, Franck; Robertson, Dominique; Meins, Frederick; Hohn, Thomas; Pooggin, Mikhail M

2006-01-01

170

Different modes of interaction by TIAR and HuR with target RNA and DNA  

PubMed Central

TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U–rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2?-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways. PMID:21233170

Kim, Henry S.; Wilce, Matthew C. J.; Yoga, Yano M. K.; Pendini, Nicole R.; Gunzburg, Menachem J.; Cowieson, Nathan P.; Wilson, Gerald M.; Williams, Bryan R. G.; Gorospe, Myriam; Wilce, Jacqueline A.

2011-01-01

171

Switch to raltegravir-based regimens and HIV DNA decrease in patients with suppressed HIV RNA  

PubMed Central

Introduction Raltegravir intensification is associated with an increase in 2-LTR episomal HIV DNA= circles, indicating a persistent low-level replication, in some individuals in ART with suppressed HIV RNA. We aimed at monitoring residual plasma HIV RNA and cellular HIV DNA in virologically suppressed patients switching to a raltegravir-based regimen. Materials and Methods Forty-six HIV-infected subjects on PI or NNRTI based-regimens, with plasma HIV RNA level <40 copies/mL for ?6 months and CD4 >200 cells/µL for ?12 months were enrolled. Thirty-four patients switched to raltegravir-based regimen (RASTA study group) and 12 continued a PI or NNRTI based-regimen (control group). Ultrasensitive HIV residual viremia and total PBMC HIV DNA were assessed at baseline (W0), 24 (W24) and 48 (W48) weeks. HIV RNA levels were determined by an ultrasensitive test derived from a commercial real time PCR (limit of detection 5 copies/ml). A real time PCR was used to quantify HIV DNA copy numbers in PBMCs. Results At W0, HIV DNA was detected in all patients while at W48 it was detectable in 82.3% of RASTA group vs 100% of controls (p=0.01). The difference between the average values of HIV DNA log10 copies/10°6 CD4 at W0 (median 3.11, IQR 2.70–3.45) and W48 (median 2.87, IQR 2.24–3.38) was statistically significant for RASTA group (p=0.035). Male gender (mean difference ?0.37 log10 copies/10°6 PBMC, p=0.023) and previous PI based-ART (mean difference +0.39 log10 copies/10°6 PBMC, p=0.036) were predictive of HIV DNA level at W0. After adjusting for previous PI based-ART, male gender was the only variable independently associated with HIV DNA size at W0 (mean difference ?0.326 log10 copies/10°6 PBMC, 95% CI ?0.641, ?0.011 p=0.043). Ultrasensitive HIV-1 RNA was detectable at W0 in 50% of RASTA group versus 66.7% of controls and at W48 in 32.4% versus 45.5%, respectively. No differences were found between HIV RNA levels at W0 and W48 within and between the two groups. Conclusions Switching to raltegravir-based regimens may be associated with a decrease of HIV reservoir, as measured by total PBMC HIV DNA. A larger sample size is required to confirm this finding.

Bianco, Claudia; Meini, Genny; Rossetti, Barbara; Lamonica, Silvia; Mondi, Annalisa; Belmonti, Simone; Fanti, Luri; Ciccarelli, Nicoletta; Di Giambenedetto, Simona; Zazzi, Maurizio; De Luca, Andrea

2014-01-01

172

Macromolecules in Undergraduate Physical Chemistry.  

ERIC Educational Resources Information Center

Suggests the topic of macromolecules and synthetic polymers be included in undergraduate courses. Two macromolecular systems (polyethylene in a state unperturbated by long-range interactions and a polypeptide undergoing a helix-coil transition) are described which are suitable for inclusion in the statistical mechanics section of physical…

Mattice, Wayne L.

1981-01-01

173

Selective Precipitation of RNA, Supercoiled Plasmid DNA, and Open?Circular Plasmid DNA with Different Cationic Surfactants  

Microsoft Academic Search

Precipitation of RNA and plasmid DNA in clarified lysate produced from a bacterial culture was studied using three cationic surfactants with a different number of alkyl groups in the hydrophobic tail and cationic head. All the precipitation mixtures contained 0.5 M of NaCl. The results obtained indicated that selective precipitation was achieved when different types of cationic surfactants were used. In

Panarat Tomanee; James T. Hsu

2006-01-01

174

Thermodynamics of Oligoarginines Binding to RNA and DNA David P. Mascotti and Timothy M. Lohman*  

E-print Network

Thermodynamics of Oligoarginines Binding to RNA and DNA David P. Mascotti and Timothy M. Lohman basis for the stabilities of these complexes requires information on the thermodynamics (thermodynamics) of well-defined model peptides to nucleic acids [for a review, see Lohman and Mascotti (1992a

Lohman, Timothy M.

175

Large scale chemical synthesis, purification and crystallization of RNA-DNA chimeras.  

PubMed Central

RNA-DNA chimeras, in which both DNA and RNA monomers are site-specifically substituted in the same strand, may be prepared only by chemical synthesis. Biochemical studies have revealed a number of surprising and subtle effects resulting from the insertion of either a ribonucleotide into a DNA strand or a deoxyribonucleotide into an RNA strand. The availability of large quantities of these chimeras allows for their crystallization and subsequent x-ray structure determination. We describe a flexible and efficient method for the large-scale preparation of these compounds, their purification, and their crystallization. The methodology is based on a combination of existing DNA phosphoramidite synthons and those recently introduced for the preparation of biochemically active RNA1. We demonstrate that these two different synthons are compatible, produce large quantities of nucleic acid needed for physical studies, and that high resolution diffraction quality crystals may be grown from these chimeras. Of the duplex chimeras synthesized and crystallized, [r(G)d(CGTATACGC)]2, [d(GCGT)r(A)d(TACGC)]2 and [r(GCG)d(TATACCC) + d(GGGTATACGC)] form A-helices and d(CG)r(CG)d(CG)]2 forms a left-handed Z-helix. Images PMID:1282704

Usman, N; Egli, M; Rich, A

1992-01-01

176

In vitro selection of optimal DNA substrates for T4 RNA ligase  

NASA Technical Reports Server (NTRS)

We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 RNA ligase. We find that the ensemble of selected sequences ligated about 10 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly, the majority of the selected sequences approximated a well-defined consensus sequence.

Harada, Kazuo; Orgel, Leslie E.

1993-01-01

177

RNA editing changes the lesion specificity for the DNA repair enzyme NEIL1  

E-print Network

RNA editing changes the lesion specificity for the DNA repair enzyme NEIL1 Jongchan Yeo, Rena A of the protein. The two forms of NEIL1 are shown here to have distinct enzymatic properties. The edited form for A to I editing in humans (ADAR1 and ADAR2) are expressed in tissues throughout the body, little is known

Beal, Peter A.

178

Recent Findings Concerning PAMAM Dendrimer Conjugates with Cyclodextrins as Carriers of DNA and RNA  

PubMed Central

We have evaluated the potential use of various polyamidoamine (PAMAM) dendrimer [dendrimer, generation (G) 2-4] conjugates with cyclodextrins (CyDs) as novel DNA and RNA carriers. Among the various dendrimer conjugates with CyDs, the dendrimer (G3) conjugate with ?-CyD having an average degree of substitution (DS) of 2.4 [?-CDE (G3, DS2)] displayed remarkable properties as DNA, shRNA and siRNA delivery carriers through the sensor function of ?-CDEs toward nucleic acid drugs, cell surface and endosomal membranes. In an attempt to develop cell-specific gene transfer carriers, we prepared sugar-appended ?-CDEs. Of the various sugar-appended ?-CDEs prepared, galactose- or mannose-appended ?-CDEs provided superior gene transfer activity to ?-CDE in various cells, but not cell-specific gene delivery ability. However, lactose-appended ?-CDE [Lac-?-CDE (G2)] was found to possess asialoglycoprotein receptor (AgpR)-mediated hepatocyte-selective gene transfer activity, both in vitro and in vivo. Most recently, we prepared folate-poly(ethylene glycol)-appended ?-CDE [Fol-P?C (G3)] and revealed that Fol-P?C (G3) imparted folate receptor (FR)-mediated cancer cell-selective gene transfer activity. Consequently, ?-CDEs bearing integrated, multifunctional molecules may possess the potential to be novel carriers for DNA, shRNA and siRNA. PMID:22454589

Arima, Hidetoshi; Motoyama, Keiichi

2009-01-01

179

INFLUENCE OF MACROMOLECULES ON CHEMICAL TRANSPORT  

EPA Science Inventory

Macromolecules in the pore fluid influence the mobility of hydrophobic compounds through soils. his study evaluated the significance of macromolecules in facilitating chemical transport under laboratory conditions. Partition coefficients between 14C-labeled hexachlorobenzene and ...

180

Protection by Anoxia of the Scrapie Agent and some DNA and RNA Viruses Irradiated as Dry Preparations  

Microsoft Academic Search

SUMMARY Freeze-dried preparations of the scrapie agent and of 5 strains of virus were used to determine the extent of protection afforded against ionizing radiation by anoxia. The five viruses tested were bacteriophages TI and T2 (DNA) and #2 (RNA); and the animal viruses herpes simplex (DNA) and yellow fever (RNA). Oxygen enhancement ratios (measured in terms of doses required

TIKVAH ALPER; D. A. Haig

1968-01-01

181

The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage.  

PubMed

Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks. PMID:25064736

Larsen, Dorthe H; Hari, Flurina; Clapperton, Julie A; Gwerder, Myriam; Gutsche, Katrin; Altmeyer, Matthias; Jungmichel, Stephanie; Toledo, Luis I; Fink, Daniel; Rask, Maj-Britt; Grøfte, Merete; Lukas, Claudia; Nielsen, Michael L; Smerdon, Stephen J; Lukas, Jiri; Stucki, Manuel

2014-08-01

182

Cloned single- and double-stranded DNA copies of potato spindle tuber viroid (PSTV) RNA and co-inoculated subgenomic DNA fragments are infectious.  

PubMed Central

A set of monomeric and oligomeric potato spindle tuber viroid (PSTV) specific DNA forms representing complete DNA copies of the circular PSTV RNA genome were constructed and cloned in plasmid pBR322 and bacteriophage M13. Both single- and double-stranded PSTV DNAs are capable of initiating viroid replication in mechanically inoculated tomato plants where it normally proceeds via the RNA-RNA pathway without DNA being involved. All dimeric and higher multimeric forms were infectious irrespective of their polarity in the case of single-stranded DNA and regardless of their orientation in the vector DNA in the case of double-stranded DNA. The vector-inserted monomeric PSTV DNA units were also found to be infectious but of low specific infectivity which was increased when these monomers had been excised. Even two subgenomic DNA fragments, representing together the 359 nucleotides of the PSTV RNA genome, initiated the synthesis of viroid RNA progeny when co-inoculated although each fragment by itself is non-infectious. These results are discussed with respect to the infectivity previously observed with certain cloned DNAs of conventional RNA and DNA viruses. Images Fig. 2. Fig. 3. PMID:6549294

Tabler, M; Sänger, H L

1984-01-01

183

RNA polymerase approaches its promoter without long-range sliding along DNA.  

PubMed

Sequence-specific DNA binding proteins must quickly bind target sequences, despite the enormously larger amount of nontarget DNA present in cells. RNA polymerases (or associated general transcription factors) are hypothesized to reach promoter sequences by facilitated diffusion (FD). In FD, a protein first binds to nontarget DNA and then reaches the target by a 1D sliding search. We tested whether Escherichia coli ?(54)RNA polymerase reaches a promoter by FD using the colocalization single-molecule spectroscopy (CoSMoS) multiwavelength fluorescence microscopy technique. Experiments directly compared the rates of initial polymerase binding to and dissociation from promoter and nonpromoter DNAs measured in the same sample under identical conditions. Binding to a nonpromoter DNA was much slower than binding to a promoter-containing DNA of the same length, indicating that the detected nonspecific binding events are not on the pathway to promoter binding. Truncating one of the DNA segments flanking the promoter to a length as short as 7 bp or lengthening it to ~3,000 bp did not alter the promoter-specific binding rate. These results exclude FD over distances corresponding to the length of the promoter or longer from playing any significant role in accelerating promoter search. Instead, the data support a direct binding mechanism, in which ?(54)RNA polymerase reaches the local vicinity of promoters by 3D diffusion through solution, and suggest that binding may be accelerated by atypical structural or dynamic features of promoter DNA. Direct binding explains how polymerase can quickly reach a promoter, despite occupancy of promoter-flanking DNA by bound proteins that would impede FD. PMID:23720315

Friedman, Larry J; Mumm, Jeffrey P; Gelles, Jeff

2013-06-11

184

First Principles Dynamics of Photoexcited DNA and RNA Bases  

SciTech Connect

The reaction dynamics of excited electronic states in nucleic acid bases is a key process in DNA photodamage. Recent ultrafast spectroscopy experiments have shown multi-component decays of excited uracil and thymine, tentatively assigned to nonadiabatic transitions involving multiple electronic states. Using both quantum chemistry and first principles quantum molecular dynamics methods we show that a true minimum on the bright S{sub 2} electronic state is responsible for the first step which occurs on a femtosecond timescale. Thus the observed femtosecond decay does not correspond to surface crossing as previously thought. We suggest that subsequent barrier crossing to the minimal energy S{sub 2}/S{sub 1} conical intersection is responsible for the picosecond decay.

Hudock, Hanneli R.; Levine, Benjamin G.; Thompson, Alexis L.; Martinez, Todd J. [Department of Chemistry, Beckman Institute, and Center for Biophysics and Computational Biology University of Illinois, Urbana, Illinois 61801 (United States)

2007-12-26

185

The effect of LNA nucleobases as enhancers for the binding of amiloride to an abasic site in DNA/DNA and DNA/RNA duplexes.  

PubMed

We report on a significant effect of locked nucleic acid (LNA) nucleobases on the binding of amiloride for abasic site (AP)-containing DNA duplexes. Fluorescence titration experiments showed that the binding affinity of amiloride for the target thymine (T) opposite an AP site significantly improves for the DNA duplexes possessing LNA nucleobases that flank the AP site, compared to the corresponding normal DNA duplexes. In particular, LNA flanking nucleobases on both 5'- and 3'-sides of the AP site are found to be effective for the enhancement of the binding affinity. From thermodynamic characterization of the amiloride binding, the loss in the binding entropy is remarkably reduced for the LNA-containing DNA duplexes, which is indeed responsible for the enhanced affinity of amiloride. Moreover, such an effect of LNA nucleobases was also observed for amiloride binding to DNA/RNA hybrid duplexes. PMID:25101634

Sato, Yusuke; Sato, Tetsushi; Sato, Takaya; Nishizawa, Seiichi; Teramae, Norio

2014-10-01

186

DNA-dependent RNA polymerase activity in silkmoth-wing epidermis after hormone treatment.  

PubMed

DNA-dependent RNA polymerase activity of wing epidermal tissue from the silkmoth, Antheraea polyphemus, has been studied after treatment of pupae with either molting hormone 20-hydroxyecdysone or 20-hydroxyecdysone and juvenile hormone. Enzyme activity has been measured both on endogenous template in isolated nuclei and on exogenous template after solubilization and correlated with transcriptional activity measured as the incorporation of [3H]uridine into RNA. Within 4 h of either hormonal regimen, increases in nuclear transcriptional activity for enzymes I and II are observed. Maximal nuclear activity for both enzyme classes was observed at 26 h. Solubilized enzyme activity, on the other hand, increased continuously up to 144 h. The increase in enzyme activity at 26 h, and probably earlier, is dependent on both RNA and protein synthesis, indicating that the increase is not a consequence of the activation of inactive molecules, but requires the synthesis of either new enzyme molecules or effector molecules. Application of 20-hydroxyecdysone + juvenile hormone does not significantly affect nuclear RNA polymerase activity, rates of RNA synthesis or even RNA content during the first 26 h. However, JH causes significant diminution in the rise of solubilized activity observed with 20-hydroxyecdysone. This reduction is not a consequence of diminished protein content. Therefore, the number of active RNA polymerase molecules appears not to directly correspond to the rate of RNA synthesis. PMID:7250486

Katula, K S; Gilbert, L I; Sridhara, S

1981-06-01

187

Conformational influence of the ribose 2'-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes  

NASA Technical Reports Server (NTRS)

We have crystallized three double-helical DNA-RNA chimeric duplexes and determined their structures by X-ray crystallography at resolutions between 2 and 2.25 A. The two self-complementary duplexes [r(G)d(CGTATACGC)]2 and [d(GCGT)r(A)d(TACGC)]2, as well as the Okazaki fragment d(GGGTATACGC).r(GCG)d(TATACCC), were found to adopt A-type conformations. The crystal structures are non-isomorphous, and the crystallographic environments for the three chimeras are different. A number of intramolecular interactions of the ribose 2'-hydroxyl groups contribute to the stabilization of the A-conformation. Hydrogen bonds between 2'-hydroxyls and 5'-oxygens or phosphate oxygens, in addition to the previously observed hydrogen bonds to 1'-oxygens of adjacent riboses and deoxyriboses, are observed in the DNA-RNA chimeric duplexes. The crystalline chimeric duplexes do not show a transition between the DNA A- and B-conformations. CD spectra suggest that the Okazaki fragment assumes an A-conformation in solution as well. In this molecule the three RNA residues may therefore lock the complete decamer in the A-conformation. Crystals of an all-DNA strand with the same sequence as the self-complementary chimeras show a morphology which is different from those of the chimera crystals. Moreover, the oligonucleotide does not match any of the sequence characteristics of DNAs usually adopting the A-conformation in the crystalline state (e.g., octamers with short alternating stretches of purines and pyrimidines). In DNA-RNA chimeric duplexes, it is therefore possible that a single RNA residue can drive the conformational equilibrium toward the A-conformation.

Egli, M.; Usman, N.; Rich, A.

1993-01-01

188

Accurate Quantification of microRNA via Single Strand Displacement Reaction on DNA Origami Motif  

PubMed Central

DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs. PMID:23990889

Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

2013-01-01

189

microRNA-152 Mediates DNMT1-Regulated DNA Methylation in the Estrogen Receptor ? Gene  

PubMed Central

Background Estrogen receptor ? (ER?) has been shown to protect against atherosclerosis. Methylation of the ER? gene can reduce ER? expression leading to a higher risk for cardiovascular disease. Recently, microRNAs have been found to regulate DNA methyltransferases (DNMTs) and thus control methylation status in several genes. We first searched for microRNAs involved in DNMT-associated DNA methylation in the ER? gene. We also tested whether statin and a traditional Chinese medicine (San-Huang-Xie-Xin-Tang, SHXXT) could exert a therapeutic effect on microRNA, DNMT and ER? methylation. Methodology/Principal Findings The ER? expression was decreased and ER? methylation was increased in LPS-treated human aortic smooth muscle cells (HASMCs) and the aorta from rats under a high-fat diet. microRNA-152 was found to be down regulated in the LPS-treated HASMCs. We validated that microRNA-152 can knock down DNMT1 in HASMCs leading to hypermethylation of the ER? gene. Statin had no effect on microRNA-152, DNMT1 or ER? expression. On the contrary, SHXXT could restore microRNA-152, decrease DNMT1 and increase ER? expression in both cellular and animal studies. Conclusions/Significance The present study showed that microRNA-152 decreases under the pro-atherosclerotic conditions. The reduced microRNA-152 can lose an inhibitory effect on DNA methyltransferase, which leads to hypermethylation of the ER? gene and a decrease of ER? level. Although statin can not reverse these cascade proatherosclerotic changes, the SHXXT shows a promising effect to inhibit this unwanted signaling pathway. PMID:22295098

Wang, Yung-Song; Chou, Wen-Wen; Chen, Ku-Chung; Cheng, Hsin-Yun; Lin, Ruey-Tay; Juo, Suh-Hang Hank

2012-01-01

190

An RNA-dependent DNA polymerase, different from the known viral reverse transcriptases, in the chicken system.  

PubMed Central

The properties of an RNA-dependent DNA polymerase (an RNA-dependent DNA nucleotidyltransferase), which occurs ubiquitously in the allantoic fluid of uninfected, leukosis-virus-free eggs, are described. It is shown that the enzyme can synthesize faithful transcripts from natural RNA (globin mRNA). By biochemical and immunological methods, the enzyme can be clearly distinguished from the reverse transcriptases of the known chicken RNA tumor viruses and therefore seems to be a member of a so far unknown class of chicken polymerases. PMID:61587

Bauer, G; Hofschneider, P H

1976-01-01

191

DNA, RNA, and protein synthesis in the cyanobacterium Synechocystis sp. PCC 6803 adapted to different salt concentrations  

Microsoft Academic Search

A salt shock of 684mm NaCl reduced RNA and DNA synthesis to about 30% of the control level inSynechocystis. DNA synthesis recovered to the initial level within 4 h, while for recovery of RNA synthesis about 8 h were necessary. In cells completely adapted to different salt concentrations (from 171 to 1026mm NaCl), a continuous decrease in the RNA content

Martin Hagemann; Sabine Fulda; Hendrik Schubert

1994-01-01

192

PATTERNS OF RNA SYNTHESIS IN T5-INFECTED CELLS I. AS STUDIED BY THE TECHNIQUE OF DNA-RNA HYBRIDIZATION-COMPETITION*  

PubMed Central

The RNA-labeling patterns obtained after T5 infection of Escherichia coli F agree with the patterns of protein labeling published by McCorquodale and Buchanan.1 Three distinct classes of RNA formed sequentially during the period of viral development can be recognized by the DNA-RNA hybridization-competition technique. Class I RNA is formed within 5 minutes after the beginning of viral metabolism and corresponds to the RNA synthesized in response to infection with the 8 per cent segment of T5 DNA. Protein synthesis directed by this 8 per cent segment is required in some capacity for the cessation of class I synthesis and the beginning of the synthesis of class II at 4 to 5 min after infection. Class III RNA synthesis begins between 9 and 12 minutes. Its appearance is prevented when chloramphenicol is added immediately after complete expression of class I functions. PMID:4916923

Moyer, Richard W.; Buchanan, John M.

1969-01-01

193

Crystal structure of RNase H3–substrate complex reveals parallel evolution of RNA/DNA hybrid recognition  

PubMed Central

RNases H participate in the replication and maintenance of genomic DNA. RNase H1 cleaves the RNA strand of RNA/DNA hybrids, and RNase H2 in addition hydrolyzes the RNA residue of RNA–DNA junctions. RNase H3 is structurally closely related to RNases H2, but its biochemical properties are similar to type 1 enzymes. Its unique N-terminal substrate-binding domain (N-domain) is related to TATA-binding protein. Here, we report the first crystal structure of RNase H3 in complex with its RNA/DNA substrate. Just like RNases H1, type 3 enzyme recognizes the 2?-OH groups of the RNA strand and detects the DNA strand by binding a phosphate group and inducing B-form conformation. Moreover, the N-domain recognizes RNA and DNA in a manner that is highly similar to the hybrid-binding domain of RNases H1. Our structure demonstrates a remarkable example of parallel evolution of the elements used in the specific recognition of RNA and DNA. PMID:25016521

Figiel, Ma?gorzata; Nowotny, Marcin

2014-01-01

194

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development  

EPA Science Inventory

We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

195

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster  

EPA Science Inventory

We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

196

Mutually exclusive binding of telomerase RNA and DNA by Ku alters telomerase recruitment model.  

PubMed

In Saccharomyces cerevisiae, the Ku heterodimer contributes to telomere maintenance as a component of telomeric chromatin and as an accessory subunit of telomerase. How Ku binding to double-stranded DNA (dsDNA) and to telomerase RNA (TLC1) promotes Ku's telomeric functions is incompletely understood. We demonstrate that deletions designed to constrict the DNA-binding ring of Ku80 disrupt nonhomologous end-joining (NHEJ), telomeric gene silencing, and telomere length maintenance, suggesting that these functions require Ku's DNA end-binding activity. Contrary to the current model, a mutant Ku with low affinity for dsDNA also loses affinity for TLC1 both in vitro and in vivo. Competition experiments reveal that wild-type Ku binds dsDNA and TLC1 mutually exclusively. Cells expressing the mutant Ku are deficient in nuclear accumulation of TLC1, as expected from the RNA-binding defect. These findings force reconsideration of the mechanisms by which Ku assists in recruiting telomerase to natural telomeres and broken chromosome ends. PAPERCLIP: PMID:22365814

Pfingsten, Jennifer S; Goodrich, Karen J; Taabazuing, Cornelius; Ouenzar, Faissal; Chartrand, Pascal; Cech, Thomas R

2012-03-01

197

Transcription-coupled and DNA damage-dependent ubiquitination of RNA polymerase II in vitro  

PubMed Central

Transcription-coupled repair (TCR) is essential for the rapid, preferential removal of DNA damage in active genes. The large subunit of RNA polymerase (Pol) II is ubiquitinated in cells after UV-irradiation or cisplatin treatment, which induces DNA damage preferentially repaired by TCR. Several human mutations, such as Cockayne syndrome complementation groups A and B, are defective in TCR and incapable of Pol II ubiquitination upon DNA damage. Here we demonstrate a correlation between ubiquitination of RNA Pol II and arrest of transcription in vitro. Ubiquitination of Pol II is significantly induced by ?-amanitin, an amatoxin that blocks Pol II elongation and causes its degradation in cells. Pol II undergoes similar ubiquitination on DNA containing cisplatin adducts that arrest transcription. Stimulation of ubiquitination requires the addition of template DNA and does not occur in the presence of an antibody to the general transcription factor TFIIB, indicating the transcription dependence of the reaction. We propose that components of the reaction recognize elongating Pol II–DNA complexes arrested by ?-amanitin or cisplatin lesions, triggering ubiquitination. PMID:11904382

Lee, Keng-Boon; Wang, Dong; Lippard, Stephen J.; Sharp, Phillip A.

2002-01-01

198

Transcription-coupled and DNA damage-dependent ubiquitination of RNA polymerase II in vitro.  

PubMed

Transcription-coupled repair (TCR) is essential for the rapid, preferential removal of DNA damage in active genes. The large subunit of RNA polymerase (Pol) II is ubiquitinated in cells after UV-irradiation or cisplatin treatment, which induces DNA damage preferentially repaired by TCR. Several human mutations, such as Cockayne syndrome complementation groups A and B, are defective in TCR and incapable of Pol II ubiquitination upon DNA damage. Here we demonstrate a correlation between ubiquitination of RNA Pol II and arrest of transcription in vitro. Ubiquitination of Pol II is significantly induced by alpha-amanitin, an amatoxin that blocks Pol II elongation and causes its degradation in cells. Pol II undergoes similar ubiquitination on DNA containing cisplatin adducts that arrest transcription. Stimulation of ubiquitination requires the addition of template DNA and does not occur in the presence of an antibody to the general transcription factor TFIIB, indicating the transcription dependence of the reaction. We propose that components of the reaction recognize elongating Pol II-DNA complexes arrested by alpha-amanitin or cisplatin lesions, triggering ubiquitination. PMID:11904382

Lee, Keng-Boon; Wang, Dong; Lippard, Stephen J; Sharp, Phillip A

2002-04-01

199

Quantification of pregenomic RNA and covalently closed circular DNA in hepatitis B virus-related hepatocellular carcinoma.  

PubMed

Pregenomic RNA (pgRNA) is generated from covalently closed circular DNA (cccDNA) and plays important roles in viral genome amplification and replication. Hepatic pgRNA and cccDNA expression levels indicate viral persistence and replication activity. This study was aimed to measure hepatic pgRNA and cccDNA expression levels in various states of hepatitis B virus (HBV) infection. Thirty-eight hepatocellular carcinoma (HCC) patients, including 14 positive for hepatitis B surface antigen (HBsAg) and 24 negative for HBsAg but positive for anti-hepatitis B core (anti-HBc) antibody, were enrolled in this study. In HBsAg-negative but anti-HBc-positive group, HBV-DNA was detected in 20 of 24 (83%) noncancerous liver tissues for at least two genomic regions based on polymerase chain reaction (PCR) analysis. pgRNA and cccDNA expression levels in occult HBV-infected patients were significantly lower than those in HBsAg-positive patients (P < 0.001). pgRNA and cccDNA in cancerous tissues were also detected without significant difference from those in noncancerous tissues. In conclusion, cccDNA and pgRNA are detected and represented HBV replication not only in noncancerous but also in cancerous liver tissues. In addition, the replication is shown in not only patients with HBsAg-positive but also occult HBV-infected patients, suggesting the contribution to HCC development. PMID:24455286

Bai, Fugui; Yano, Yoshihiko; Fukumoto, Takumi; Takebe, Atsushi; Tanaka, Motofumi; Kuramitsu, Kaori; Anggorowati, Nungki; Rinonce, Hanggoro Tri; Widasari, Dewiyani Indah; Saito, Masaya; Hirano, Hirotaka; Hayakumo, Takanobu; Seo, Yasushi; Azuma, Takeshi; Ku, Yonson; Hayashi, Yoshitake

2013-01-01

200

Double-stranded RNA under force and torque: Similarities to and striking differences from double-stranded DNA  

PubMed Central

RNA plays myriad roles in the transmission and regulation of genetic information that are fundamentally constrained by its mechanical properties, including the elasticity and conformational transitions of the double-stranded (dsRNA) form. Although double-stranded DNA (dsDNA) mechanics have been dissected with exquisite precision, much less is known about dsRNA. Here we present a comprehensive characterization of dsRNA under external forces and torques using magnetic tweezers. We find that dsRNA has a force–torque phase diagram similar to that of dsDNA, including plectoneme formation, melting of the double helix induced by torque, a highly overwound state termed “P-RNA,” and a highly underwound, left-handed state denoted “L-RNA.” Beyond these similarities, our experiments reveal two unexpected behaviors of dsRNA: Unlike dsDNA, dsRNA shortens upon overwinding, and its characteristic transition rate at the plectonemic buckling transition is two orders of magnitude slower than for dsDNA. Our results challenge current models of nucleic acid mechanics, provide a baseline for modeling RNAs in biological contexts, and pave the way for new classes of magnetic tweezers experiments to dissect the role of twist and torque for RNA–protein interactions at the single-molecule level. PMID:25313077

Lipfert, Jan; Skinner, Gary M.; Keegstra, Johannes M.; Hensgens, Toivo; Jager, Tessa; Dulin, David; Kober, Mariana; Yu, Zhongbo; Donkers, Serge P.; Chou, Fang-Chieh; Das, Rhiju; Dekker, Nynke H.

2014-01-01

201

Double-stranded RNA under force and torque: Similarities to and striking differences from double-stranded DNA.  

PubMed

RNA plays myriad roles in the transmission and regulation of genetic information that are fundamentally constrained by its mechanical properties, including the elasticity and conformational transitions of the double-stranded (dsRNA) form. Although double-stranded DNA (dsDNA) mechanics have been dissected with exquisite precision, much less is known about dsRNA. Here we present a comprehensive characterization of dsRNA under external forces and torques using magnetic tweezers. We find that dsRNA has a force-torque phase diagram similar to that of dsDNA, including plectoneme formation, melting of the double helix induced by torque, a highly overwound state termed "P-RNA," and a highly underwound, left-handed state denoted "L-RNA." Beyond these similarities, our experiments reveal two unexpected behaviors of dsRNA: Unlike dsDNA, dsRNA shortens upon overwinding, and its characteristic transition rate at the plectonemic buckling transition is two orders of magnitude slower than for dsDNA. Our results challenge current models of nucleic acid mechanics, provide a baseline for modeling RNAs in biological contexts, and pave the way for new classes of magnetic tweezers experiments to dissect the role of twist and torque for RNA-protein interactions at the single-molecule level. PMID:25313077

Lipfert, Jan; Skinner, Gary M; Keegstra, Johannes M; Hensgens, Toivo; Jager, Tessa; Dulin, David; Köber, Mariana; Yu, Zhongbo; Donkers, Serge P; Chou, Fang-Chieh; Das, Rhiju; Dekker, Nynke H

2014-10-28

202

Controlled cytoplasmic and nuclear localization of plasmid DNA and siRNA by differentially tailored polyethylenimine.  

PubMed

To maximize therapeutic effects, targeted delivery of nucleic acids (e.g., DNA and RNA) in their appropriate intracellular targets is highly desirable. In this study, primary amines of a model polymeric nonviral carrier, polyethylenimine (PEI), at two molecular weights (0.8 and 25 kDa) were differentially ketalized (i.e., 17-96%) in order to explore the possibility of precisely modulating intracellular localization of plasmid DNA- and siRNA-containing polyplexes. The size of the polyplexes revealed that the ketalization ratios of 35 to 70% were found to be the most efficient in condensing nucleic acids with the ketalized low molecular weight PEI (LMW PEI), while high molecular weight PEI (HMW PEI) ketalized at the ratio of 23% condensed nucleic acids most efficiently. Ketalization of LMW PEI (up to 70%) enhanced transfection; however, ketalization of HMW PEI reduced its transfection capability. On the contrary, HMW PEI ketalized at 23 and 37% ratios showed significant RNA interference, while LMW PEI could not successfully inhibit gene expression regardless of ketalization ratios. The results were explained by confocal microscopic studies demonstrating that ketalization ratios, molecular weights of ketalized PEI, and types of nucleic acids complexed in the polyplexes play crucial roles in intracellular localization of nucleic acids/ketalized PEI polyplexes and affect DNA transfection and RNA interference efficiencies. All ketalized PEI showed negligible cytotoxicity. This study implies a feasibility of selectively localizing nucleic acids in their intracellular targets by employing differentially tailored polymeric gene carriers. PMID:18992289

Shim, Min Suk; Kwon, Young Jik

2009-02-10

203

Cellular HIV-1 DNA load predicts HIV-RNA rebound and the outcome of highly active antiretroviral therapy  

E-print Network

Cellular HIV-1 DNA load predicts HIV-RNA rebound and the outcome of highly active antiretroviral HIV-1 DNA prior to highly active antiretroviral therapy (HAART) initiation predicts its outcome initiation were available. Cellular HIV-1 DNA quantification was performed by a molecular beacon-based real

204

Construction of rubella virus genome-length cDNA clones and synthesis of infectious RNA transcripts.  

PubMed Central

Plasmids containing a complete cDNA copy of the rubella virus (RUB) genomic RNA were constructed. Transfection into cell culture of genome-length RNA transcribed in vitro from one of these cDNA clones, Robo102, resulted in the production of virus which preserved the genetic and phenotypic characteristics of the parental virus from which the cDNA clone was derived. Prior to construction of the RUB genome-length cDNA clones, the 5'-terminal sequence of the RUB genomic RNA was determined to be 5'CAAUGG...3' following the cap structure. Analysis of the specific infectivity of RUB genomic RNA isolated from virions revealed that in Vero cells, the specific infectivity of RUB genomic RNA is roughly equivalent to that of Sindbis virus genomic RNA. In RUB virion RNA preparations, the subgenomic RNA was detected. It was demonstrated that subgenomic RNA was packaged into RUB virions; however, the presence of the subgenomic RNA was not essential for infectivity of the genomic RNA. Images PMID:8189494

Wang, C Y; Dominguez, G; Frey, T K

1994-01-01

205

Discovery of DNA Viruses in Wild-Caught Mosquitoes Using Small RNA High throughput Sequencing  

PubMed Central

Background Mosquito-borne infectious diseases pose a severe threat to public health in many areas of the world. Current methods for pathogen detection and surveillance are usually dependent on prior knowledge of the etiologic agents involved. Hence, efficient approaches are required for screening wild mosquito populations to detect known and unknown pathogens. Methodology/principal findings In this study, we explored the use of Next Generation Sequencing to identify viral agents in wild-caught mosquitoes. We extracted total RNA from different mosquito species from South China. Small 18–30 bp length RNA molecules were purified, reverse-transcribed into cDNA and sequenced using Illumina GAIIx instrumentation. Bioinformatic analyses to identify putative viral agents were conducted and the results confirmed by PCR. We identified a non-enveloped single-stranded DNA densovirus in the wild-caught Culex pipiens molestus mosquitoes. The majority of the viral transcripts (.>80% of the region) were covered by the small viral RNAs, with a few peaks of very high coverage obtained. The +/? strand sequence ratio of the small RNAs was approximately 7?1, indicating that the molecules were mainly derived from the viral RNA transcripts. The small viral RNAs overlapped, enabling contig assembly of the viral genome sequence. We identified some small RNAs in the reverse repeat regions of the viral 5?- and 3? -untranslated regions where no transcripts were expected. Conclusions/significance Our results demonstrate for the first time that high throughput sequencing of small RNA is feasible for identifying viral agents in wild-caught mosquitoes. Our results show that it is possible to detect DNA viruses by sequencing the small RNAs obtained from insects, although the underlying mechanism of small viral RNA biogenesis is unclear. Our data and those of other researchers show that high throughput small RNA sequencing can be used for pathogen surveillance in wild mosquito vectors. PMID:21949749

Ma, Maijuan; Huang, Yong; Gong, Zhengda; Zhuang, Lu; Li, Cun; Yang, Hong; Tong, Yigang; Liu, Wei; Cao, Wuchun

2011-01-01

206

DNA-dependent RNA polymerase detects hidden giant viruses in published databanks.  

PubMed

Environmental metagenomic studies show that there is a "dark matter," composed of sequences not linked to any known organism, as determined mainly using ribosomal DNA (rDNA) sequences, which therefore ignore giant viruses. DNA-dependent RNA polymerase (RNAP) genes are universal in microbes and conserved in giant viruses and may replace rDNA for identifying microbes. We found while reconstructing RNAP subunit 2 (RNAP2) phylogeny that a giant virus sequenced together with the genome of a large eukaryote, Hydra magnipapillata, has been overlooked. To explore the dark matter, we used viral RNAP2 and reconstructed putative ancestral RNAP2, which were significantly superior in detecting distant clades than current sequences, and we revealed two additional unknown mimiviruses, misclassified as an euryarchaeote and an oomycete plant pathogen, and detected unknown putative viral clades. We suggest using RNAP systematically to decipher the black matter and identify giant viruses. PMID:24929085

Sharma, Vikas; Colson, Philippe; Giorgi, Roch; Pontarotti, Pierre; Raoult, Didier

2014-07-01

207

Reprogramming DNA Methylation in Bovine Cells by Knocking Down DNA Methyltransferase-1 with RNA Interference  

E-print Network

Embryos derived by somatic cell nuclear transfer (SCNT) produce few pregnancies that result in a live, healthy offspring. This has largely been attributed to the aberrant reprogramming of the somatic cell DNA used for cloning. In order to improve...

Stroud, Todd

2010-01-20

208

Analysis of intermolecular base pair formation of prohead RNA of the phage ø29 DNA packaging motor using NMR spectroscopy  

Microsoft Academic Search

The bacteriophage ø29 DNA packaging motor that assembles on the precursor capsid (prohead) contains an essential 174-nt structural RNA (pRNA) that forms multimers. To determine the structural features of the CE- and D-loops believed to be involved in multimerization of pRNA, 35- and 19-nt RNA molecules containing the CE-loop or the D-loop, respectively, were produced and shown to form a

Aya Kitamura; Paul J. Jardine; Dwight L. Anderson; Shelley Grimes; Hiroshi Matsuo

2008-01-01

209

Different base per unit length ratios exist in single-stranded RNA and single-stranded DNA.  

PubMed Central

A significant difference was found to exist in the number of bases per unit length of single-stranded RNA as compared to single-stranded DNA when single-stranded RNA or DNA molecules of known nucleotide sequence were measured by electron microscopy using a cytochrome spreading technique. Using this technique, single-stranded RNA was found to have 17.5% more bases per unit of length than single-stranded DNA. These ratios were verified using four different denaturing conditions for the RNA: 80% formamide, 80% formamide plus glyoxal, 80% formamide/4M urea and 80% formamide/4M urea plus glyoxal. Molecules ranging in size from 1541 to 5386 nucleotides were examined and the number of bases per unit length was found to vary inversely with micrometer was consistent when RNA and DNA molecules of the same length were compared. Images PMID:6162153

Glass, J; Wertz, G W

1980-01-01

210

Strategy for Systematic Assembly of Large RNA and DNA Genomes: Transmissible Gastroenteritis Virus Model  

PubMed Central

A systematic method was developed to assemble functional full-length genomes of large RNA and DNA viruses. Coronaviruses contain the largest single-stranded positive-polarity RNA genome in nature. The ?30-kb genome, coupled with regions of genomic instability, has hindered the development of a full-length infectious cDNA construct. We have assembled a full-length infectious construct of transmissible gastroenteritis virus (TGEV), an important pathogen in swine. Using a novel approach, six adjoining cDNA subclones that span the entire TGEV genome were isolated. Each clone was engineered with unique flanking interconnecting junctions which determine a precise systematic assembly with only the adjacent cDNA subclones, resulting in an intact TGEV cDNA construct of ?28.5 kb in length. Transcripts derived from the full-length TGEV construct were infectious, and progeny virions were serially passaged in permissive host cells. Viral antigen production and subgenomic mRNA synthesis were evident during infection and throughout passage. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar plaque morphology in permissive host cells. Host range phenotypes of the molecularly cloned and wild-type viruses were similar in cells of swine and feline origin. The recombinant viruses were sequenced across the unique interconnecting junctions, conclusively demonstrating the marker mutations and restriction sites that were engineered into the component clones. Full-length infectious constructs of TGEV will permit the precise genetic modification of the coronavirus genome. The method that we have designed to generate an infectious cDNA construct of TGEV could theoretically be used to precisely reconstruct microbial or eukaryotic genomes approaching several million base pairs in length. PMID:11044104

Yount, Boyd; Curtis, Kristopher M.; Baric, Ralph S.

2000-01-01

211

An improved method for RNA isolation and cDNA library construction from immature seeds of Jatropha curcas L  

PubMed Central

Background RNA quality and quantity is sometimes unsuitable for cDNA library construction, from plant seeds rich in oil, polysaccharides and other secondary metabolites. Seeds of jatropha (Jatropha curcas L.) are rich in fatty acids/lipids, storage proteins, polysaccharides, and a number of other secondary metabolites that could either bind and/or co-precipitate with RNA, making it unsuitable for downstream applications. Existing RNA isolation methods and commercial kits often fail to deliver high-quality total RNA from immature jatropha seeds for poly(A)+ RNA purification and cDNA synthesis. Findings A protocol has been developed for isolating good quality total RNA from immature jatropha seeds, whereby a combination of the CTAB based RNA extraction method and a silica column of a commercial plant RNA extraction kit is used. The extraction time was reduced from two days to about 3 hours and the RNA was suitable for poly(A)+ RNA purification, cDNA synthesis, cDNA library construction, RT-PCR, and Northern hybridization. Based on sequence information from selected clones and amplified PCR product, the cDNA library seems to be a good source of full-length jatropha genes. The method was equally effective for isolating RNA from mustard and rice seeds. Conclusions This is a simple CTAB + silica column method to extract high quality RNA from oil rich immature jatropha seeds that is suitable for several downstream applications. This method takes less time for RNA extraction and is equally effective for other tissues where the quality and quantity of RNA is highly interfered by the presence of fatty acids, polysaccharides and polyphenols. PMID:20444276

2010-01-01

212

Small tandemly repeated DNA sequences of higher plants likely originate from a tRNA gene ancestor.  

PubMed Central

Several monomers (177 bp) of a tandemly arranged repetitive nuclear DNA sequence of Brassica oleracea have been cloned and sequenced. They share up to 95% homology between one another and up to 80% with other satellite DNA sequences of Cruciferae, suggesting a common ancestor. Both strands of these monomers show more than 50% homology with many tRNA genes; the best homologies have been obtained with Lys and His yeast mitochondrial tRNA genes (respectively 64% and 60%). These results suggest that small tandemly repeated DNA sequences of plants may have evolved from a tRNA gene ancestor. These tandem repeats have probably arisen via a process involving reverse transcription of polymerase III RNA intermediates, as is the case for interspersed DNA sequences of mammalians. A model is proposed to explain the formation of such small tandemly repeated DNA sequences. Images PMID:3774553

Benslimane, A A; Dron, M; Hartmann, C; Rode, A

1986-01-01

213

Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians  

PubMed Central

Background Identifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I (COI) has been proposed as universal marker for this purpose among animals. Results Herein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates.COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1–17%), with low degrees of pairwise haplotype divergence within populations (0–1%). Conclusion We strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem. PMID:15771783

Vences, Miguel; Thomas, Meike; van der Meijden, Arie; Chiari, Ylenia; Vieites, David R

2005-01-01

214

Structural Basis for DNA-Hairpin Promoter Recognition by the Bacteriophage N4 Virion RNA Polymerase  

SciTech Connect

Coliphage N4 virion-encapsidated RNA polymerase (vRNAP) is a member of the phage T7-like single-subunit RNA polymerase (RNAP) family. Its central domain (mini-vRNAP) contains all RNAP functions of the full-length vRNAP, which recognizes a 5 to 7 base pair stem and 3 nucleotide loop hairpin DNA promoter. Here, we report the X-ray crystal structures of mini-vRNAP bound to promoters. Mini-vRNAP uses four structural motifs to recognize DNA sequences at the hairpin loop and stem and to unwind DNA. Despite their low sequence similarity, three out of four motifs are shared with T7 RNAP that recognizes a double-stranded DNA promoter. The binary complex structure and results of engineered disulfide linkage experiments reveal that the plug and motif B loop, which block the access of template DNA to the active site in the apo-form mini-vRNAP, undergo a large-scale conformational change upon promoter binding, explaining the restricted promoter specificity that is critical for N4 phage early transcription.

Gleghorn, M.; Davydova, E; Rothman-Denes, L; Murakami, K

2008-01-01

215

The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis  

SciTech Connect

Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for {beta}-actin and GAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death.

Reich, Charles F. [Medical Research Service, 151G Durham VAMC, 508 Fulton Street, Durham, NC 27705 (United States); Division of Rheumatology and Immunology, Duke University Medical Center, Durham, NC 27705 (United States); Pisetsky, David S. [Medical Research Service, 151G Durham VAMC, 508 Fulton Street, Durham, NC 27705 (United States); Division of Rheumatology and Immunology, Duke University Medical Center, Durham, NC 27705 (United States)], E-mail: piset001@mc.duke.edu

2009-03-10

216

Bacterial and archaeal communities in long-term contaminated surface and subsurface soil evaluated through coextracted RNA and DNA.  

PubMed

Soil RNA and DNA were coextracted along a contamination gradient at a landfarming field with aged crude oil contamination to investigate pollution-dependent differences in 16S rRNA and rRNA gene pools. Microbial biomass correlated with nucleic acid yields as well as bacterial community change, indicating that the same factors controlled community size and structure. In surface soil, bacterial community evenness, estimated through length heterogeneity PCR (LH-PCR) fingerprinting, appeared higher for RNA-based than for DNA-based communities. The RNA-based community profiles resembled the DNA-based communities of soil with a lower contamination level. Cloning-based identification of bacterial hydrocarbon-degrading taxa in the RNA pool, representing the viable community with high protein synthesis potential, indicated that decontamination processes still continue. Analyses of archaea revealed that only Thaumarchaeota were present in the aerobic samples, whereas more diverse communities were found in the compacted subsurface soil with more crude oil. For subsurface bacteria, hydrocarbon concentration explained neither the community structure nor the difference between RNA-based and DNA-based communities. However, rRNA of bacterial taxa associated with syntrophic and sulphate-reducing alkane degradation was detected. Although the same prokaryotic taxa were identified in DNA and RNA, comparison of the two nucleic acid pools can aid in the assessment of past and future restoration success. PMID:24986450

Mikkonen, Anu; Santalahti, Minna; Lappi, Kaisa; Pulkkinen, Anni-Mari; Montonen, Leone; Suominen, Leena

2014-10-01

217

Molecular cloning and characterization of a complete DNA copy of potato spindle tuber viroid RNA.  

PubMed

Double-stranded cDNA has been synthesized from RNA of a severe strain of potato spindle tuber viroid using a synthetic oligodeoxyribonucleotide as a primer. Upon cloning in bacteriophage M13mp9, two recombinant phages were selected to construct a pBR322-derived plasmid containing a complete viroid DNA copy. Elucidation of the nucleotide sequence revealed four differences with the previously established sequence of another PSTV strain, three of which were base exchanges and one a deletion. PMID:6897677

van Wezenbeek, P; Vos, P; van Boom, J; van Kammen, A

1982-12-20

218

HYPOXIA-INDUCED GROWTH LIMITATION OF JUVENILE FISHES IN AN ESTUARINE NURSERY: ASSESSMENT OF SMALL-SCALE TEMPORAL DYNAMICS USING RNA:DNA  

EPA Science Inventory

The ratio of RNA to DNA (RNA:DNA) in white muscle tissue of juvenile summer flounder (Paralichthys dentatus) and weakfish (Cynoscion regalis) was used as a proxy for recent growth rate in an estuarine nursery. Variability in RNA:DNA was examined relative to temporal changes in te...

219

RNA-directed DNA methylation regulates parental genomic imprinting at several loci in Arabidopsis.  

PubMed

In mammals and plants, parental genomic imprinting restricts the expression of specific loci to one parental allele. Imprinting in mammals relies on sex-dependent de novo deposition of DNA methylation during gametogenesis but a comparable mechanism was not shown in plants. Rather, paternal silencing by the maintenance DNA methyltransferase 1 (MET1) and maternal activation by the DNA demethylase DEMETER (DME) cause maternal expression. However, genome-wide studies suggested other DNA methylation-dependent imprinting mechanisms. Here, we show that de novo RNA-directed DNA methylation (RdDM) regulates imprinting at specific loci expressed in endosperm. RdDM in somatic tissues is required to silence expression of the paternal allele. By contrast, the repression of RdDM in female gametes participates with or without DME requirement in the activation of the maternal allele. The contrasted activity of DNA methylation between male and female gametes appears sufficient to prime imprinted maternal expression. After fertilization, MET1 maintains differential expression between the parental alleles. RdDM depends on small interfering RNAs (siRNAs). The involvement of RdDM in imprinting supports the idea that sources of siRNAs such as transposons and de novo DNA methylation were recruited in a convergent manner in plants and mammals in the evolutionary process leading to selection of imprinted loci. PMID:23760956

Vu, Thiet Minh; Nakamura, Miyuki; Calarco, Joseph P; Susaki, Daichi; Lim, Pei Qi; Kinoshita, Tetsu; Higashiyama, Tetsuya; Martienssen, Robert A; Berger, Frédéric

2013-07-01

220

RNA-directed DNA methylation regulates parental genomic imprinting at several loci in Arabidopsis  

PubMed Central

In mammals and plants, parental genomic imprinting restricts the expression of specific loci to one parental allele. Imprinting in mammals relies on sex-dependent de novo deposition of DNA methylation during gametogenesis but a comparable mechanism was not shown in plants. Rather, paternal silencing by the maintenance DNA methyltransferase 1 (MET1) and maternal activation by the DNA demethylase DEMETER (DME) cause maternal expression. However, genome-wide studies suggested other DNA methylation-dependent imprinting mechanisms. Here, we show that de novo RNA-directed DNA methylation (RdDM) regulates imprinting at specific loci expressed in endosperm. RdDM in somatic tissues is required to silence expression of the paternal allele. By contrast, the repression of RdDM in female gametes participates with or without DME requirement in the activation of the maternal allele. The contrasted activity of DNA methylation between male and female gametes appears sufficient to prime imprinted maternal expression. After fertilization, MET1 maintains differential expression between the parental alleles. RdDM depends on small interfering RNAs (siRNAs). The involvement of RdDM in imprinting supports the idea that sources of siRNAs such as transposons and de novo DNA methylation were recruited in a convergent manner in plants and mammals in the evolutionary process leading to selection of imprinted loci. PMID:23760956

Vu, Thiet Minh; Nakamura, Miyuki; Calarco, Joseph P.; Susaki, Daichi; Lim, Pei Qi; Kinoshita, Tetsu; Higashiyama, Tetsuya; Martienssen, Robert A.; Berger, Frederic

2013-01-01

221

G4 Resolvase 1 Binds Both DNA and RNA Tetramolecular Quadruplex with High Affinity and Is the Major Source of Tetramolecular Quadruplex G4-DNA and G4-RNA Resolving Activity in HeLa Cell Lysates*  

PubMed Central

Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes. We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117–38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs. We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures. The apparent Kd values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 ± 6 and 77 ± 6pm, respectively, as measured by gel mobility shift assay. In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution. Down-regulation of G4R1/RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells. This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/RHAU in targeting in vivo RNA quadruplex structures. PMID:18842585

Creacy, Steven D.; Routh, Eric D.; Iwamoto, Fumiko; Nagamine, Yoshikuni; Akman, Steven A.; Vaughn, James P.

2008-01-01

222

G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates.  

PubMed

Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes. We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117-38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs. We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures. The apparent K(d) values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 +/- 6 and 77 +/- 6 Pm, respectively, as measured by gel mobility shift assay. In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution. Down-regulation of G4R1/RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells. This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/RHAU in targeting in vivo RNA quadruplex structures. PMID:18842585

Creacy, Steven D; Routh, Eric D; Iwamoto, Fumiko; Nagamine, Yoshikuni; Akman, Steven A; Vaughn, James P

2008-12-12

223

Lipidoid-coated iron oxide nanoparticles for efficient DNA and siRNA delivery.  

PubMed

The safe, targeted and effective delivery of gene therapeutics remains a significant barrier to their broad clinical application. Here we develop a magnetic nucleic acid delivery system composed of iron oxide nanoparticles and cationic lipid-like materials termed lipidoids. Coated nanoparticles are capable of delivering DNA and siRNA to cells in culture. The mean hydrodynamic size of these nanoparticles was systematically varied and optimized for delivery. While nanoparticles of different sizes showed similar siRNA delivery efficiency, nanoparticles of 50-100 nm displayed optimal DNA delivery activity. The application of an external magnetic field significantly enhanced the efficiency of nucleic acid delivery, with performance exceeding that of the commercially available lipid-based reagent, Lipofectamine 2000. The iron oxide nanoparticle delivery platform developed here offers the potential for magnetically guided targeting, as well as an opportunity to combine gene therapy with MRI imaging and magnetic hyperthermia. PMID:23394319

Jiang, Shan; Eltoukhy, Ahmed A; Love, Kevin T; Langer, Robert; Anderson, Daniel G

2013-03-13

224

DNA and RNA analyses in detection of genetic predisposition to cancer  

PubMed Central

During the past decade many new molecular methods for DNA and RNA analysis have emerged. The most popular thus far have been SSCP, HET, CMC, DGGE, RFLP or ASA, which have now been replaced by methods that are more cost effective and less time consuming. Real-time amplification techniques and particularly those with the capacity of multiplexing have become commonly used in laboratory practice. Novel screening methods enable the very rapid examination of large patients series. Use of liquid handling robotics applied to the isolation of DNA or RNA, the normalisation of sample concentration, and standardization of target amplification by PCR have also contributed to a reduced risk of sample contamination and have resulted in laboratory analysis being easier and faster. The aim of this study is the introduction of a few modern techniques, most commonly used in detection of genetic predisposition to cancer. PMID:23206658

2012-01-01

225

RNA:DNA ratio and other nucleic acid derived indices in marine ecology.  

PubMed

Some of most used indicators in marine ecology are nucleic acid-derived indices. They can be divided by target levels in three groups: 1) at the organism level as ecophysiologic indicators, indicators such as RNA:DNA ratios, DNA:dry weight and RNA:protein, 2) at the population level, indicators such as growth rate, starvation incidence or fisheries impact indicators, and 3) at the community level, indicators such as trophic interactions, exergy indices and prey identification. The nucleic acids derived indices, especially RNA:DNA ratio, have been applied with success as indicators of nutritional condition, well been and growth in marine organisms. They are also useful as indicators of natural or anthropogenic impacts in marine population and communities, such as upwelling or dredge fisheries, respectively. They can help in understanding important issues of marine ecology such as trophic interactions in marine environment, fish and invertebrate recruitment failure and biodiversity changes, without laborious work of counting, measuring and identification of small marine organisms. Besides the objective of integrate nucleic acid derived indices across levels of organization, the paper will also include a general characterization of most used nucleic acid derived indices in marine ecology and also advantages and limitations of them. We can conclude that using indicators, such RNA:DNA ratios and other nucleic acids derived indices concomitantly with organism and ecosystems measures of responses to climate change (distribution, abundance, activity, metabolic rate, survival) will allow for the development of more rigorous and realistic predictions of the effects of anthropogenic climate change on marine systems. PMID:19325815

Chícharo, Maria A; Chícharo, Luis

2008-08-01

226

Mutations affecting RNA polymerase I-stimulated exchange and rDNA recombination in yeast  

Microsoft Academic Search

HOT1 is a cis-acting recombination-stimulatory sequence isolated from the rDNA repeat unit of yeast. The ability of HOT1 to stimulate mitotic exchange appears to depend on its ability to promote high levels of RNA polymerase I transcription. A qualitative colony color sectoring assay was developed to screen for trans-acting mutations that alter the activity of HOT1. Both hypo-recombination and hyper-recombination

Yu-Huei Lin; Ralph L. Keil

1991-01-01

227

ARGONAUTE4 Control of Locus-Specific siRNA Accumulation and DNA and Histone Methylation  

Microsoft Academic Search

Proteins of the ARGONAUTE family are important in diverse posttranscriptional RNA-mediated gene-silencing systems as well as in transcriptional gene silencing in Drosophila and fission yeast and in programmed DNA elimination in Tetrahymena. We cloned ARGONAUTE4 (AGO4) from a screen for mutants that suppress silencing of the Arabidopsis SUPERMAN (SUP) gene. The ago4-1 mutant reactivated silent SUP alleles and decreased CpNpG

Daniel Zilberman; Xiaofeng Cao; Steven E. Jacobsen

2003-01-01

228

A comparison of methods for retrieval of DNA and RNA from FFPE tissues  

Microsoft Academic Search

Summary A variety of commercially available kits designed for the purpose of extracting DNA\\/RNA from formalin-fixed, paraffin-embedded (FFPE) samples were tested under controlled conditions. The kits were selected based on the method of extraction: Column-based, lysis only, solid-phase reverse binding, and magnetic based extraction. Kits were compared on the basis of resulting nucleic acid concentration, yield, protein and chemical contamination,

Sam Haldenby; Tom Burr; Stewart Martin; Andy Green; Cliff Murray; Jackie Rodgers

2008-01-01

229

ALS-associated mutation FUS-R521C causes DNA damage and RNA splicing defects  

PubMed Central

Autosomal dominant mutations of the RNA/DNA binding protein FUS are linked to familial amyotrophic lateral sclerosis (FALS); however, it is not clear how FUS mutations cause neurodegeneration. Using transgenic mice expressing a common FALS-associated FUS mutation (FUS-R521C mice), we found that mutant FUS proteins formed a stable complex with WT FUS proteins and interfered with the normal interactions between FUS and histone deacetylase 1 (HDAC1). Consequently, FUS-R521C mice exhibited evidence of DNA damage as well as profound dendritic and synaptic phenotypes in brain and spinal cord. To provide insights into these defects, we screened neural genes for nucleotide oxidation and identified brain-derived neurotrophic factor (Bdnf) as a target of FUS-R521C–associated DNA damage and RNA splicing defects in mice. Compared with WT FUS, mutant FUS-R521C proteins formed a more stable complex with Bdnf RNA in electrophoretic mobility shift assays. Stabilization of the FUS/Bdnf RNA complex contributed to Bdnf splicing defects and impaired BDNF signaling through receptor TrkB. Exogenous BDNF only partially restored dendrite phenotype in FUS-R521C neurons, suggesting that BDNF-independent mechanisms may contribute to the defects in these neurons. Indeed, RNA-seq analyses of FUS-R521C spinal cords revealed additional transcription and splicing defects in genes that regulate dendritic growth and synaptic functions. Together, our results provide insight into how gain-of-function FUS mutations affect critical neuronal functions. PMID:24509083

Qiu, Haiyan; Lee, Sebum; Shang, Yulei; Wang, Wen-Yuan; Au, Kin Fai; Kamiya, Sherry; Barmada, Sami J.; Finkbeiner, Steven; Lui, Hansen; Carlton, Caitlin E.; Tang, Amy A.; Oldham, Michael C.; Wang, Hejia; Shorter, James; Filiano, Anthony J.; Roberson, Erik D.; Tourtellotte, Warren G.; Chen, Bin; Tsai, Li-Huei; Huang, Eric J.

2014-01-01

230

Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning  

SciTech Connect

A {lambda}gt11 library constructed from poly(A{plus}) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first, screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3{prime}-untranslated region. A subsequent screen using a 5{prime} rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin CDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA.

Pierce, R.A.; Deak, S.B.; Stolle, C.A.; Boyd, C.D. (Univ. of Medicine and Dentistry of New Jersey, New Brunswick (USA))

1990-10-01

231

Plasmid DNA- and messenger RNA-based anti-cancer vaccination.  

PubMed

Tumor cells (over-) express specific antigens which allow them to be recognized and destroyed by the immune system. Triggering anti-tumor immunity in cancer patients by specific vaccination is foreseen as a safe and versatile method to control cancer. As a source of antigen, whole tumor cells, nucleic acids, proteins or derived peptides have been used. This review focuses on the utilization of vaccines based on plasmid DNA (pDNA) and messenger RNA (mRNA) coding for tumor associated antigens. Both vectors (pDNA and mRNA) are grouped under the designation "minimal nucleic acid vector" or MNAV. The current knowledge on anti-tumor vaccination based on MNAV-encoded tumor antigens, methods of delivery, principles of production and optimization is discussed. Furthermore, an up-to-date summary of published clinical trials using MNAV for the vaccination against solid tumors is given. Recent preclinical and early phase clinical trials demonstrate promising synergies between vaccination and other treatments such as chemotherapy or non-specific immune enhancement regimens. Combining optimized MNAV formulations and parallel adjuvant treatments could allow to turn MNAV-based vaccines into efficient anti-tumor immunotherapies in humans. PMID:18006079

Weide, Benjamin; Garbe, Claus; Rammensee, Hans-Georg; Pascolo, Steve

2008-01-15

232

Age-Dependent Accumulation of 8-Oxoguanine in the DNA and RNA in Various Rat Tissues  

PubMed Central

The relationship between the oxidative damage of nucleic acids and aging of animals was investigated by analyzing the nucleic acids derived from various tissue specimens of naturally aged Sprague-Dawley (SD) rats. For this purpose, we established an accurate and sensitive isotope-diluted LC-MS/MS method to determine the levels of 8-oxo-7,8-dihydro-2?-deoxyguanosine (8-oxo-dGsn) in DNA and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) in RNA. An age-dependent increase in oxidative DNA and RNA damage was observed in the various organs examined, including the brain, liver, kidneys, and testes. Similar increases in the 8-oxo-dGsn and 8-oxo-Gsn contents were observed in three parts of the brain, the hippocampus, cerebral cortex, and cerebellum, among which, the values for the hippocampus were always the highest. When the oxidized guanosine metabolites were quantified with urine, a similar age-dependent increase was observed for both 8-oxo-dGsn and 8-oxo-Gsn. However, unlike the results of nucleic acid samples derived from the tissues, the amount of 8-oxo-Gsn was significantly higher compared to that of 8-oxo-dGsn, probably reflecting the fact that RNA degradation occurs more frequently than DNA degradation. Our finding indicates that the amount of urinary 8-oxo-Gsn could be considered as a biomarker for the sensitive measurement of oxidative stress and aging. PMID:23738036

Nie, Ben; Gan, Wei; Shi, Fei; Hu, Guo-Xin; Chen, Lian-Guo; Hayakawa, Hiroshi; Sekiguchi, Mutsuo

2013-01-01

233

Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA  

PubMed Central

Background Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. Results We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. Conclusions A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities. PMID:25183040

2014-01-01

234

Elucidation of the Mechanism of Gene Silencing using Small Interferin RNA: DNA Hybrid Molecules  

SciTech Connect

The recent discovery that short hybrid RNA:DNA molecules (siHybrids) induce long-term silencing of gene expression in mammalian cells conflicts with the currently hypothesized mechanisms explaining the action of small, interfering RNA (siRNA). As a first step to elucidating the mechanism for this effect, we set out to quantify the delivery of siHybrids and determine their cellular localization in mammalian cells. We then tracked the segregation of the siHybrids into daughter cells after cell division. Markers for siHybrid delivery were shown to enter cells with and without the use of a transfection agent. Furthermore, delivery without transfection agent only occurred after a delay of 2-4 hours, suggesting a degradation process occurring in the cell culture media. Therefore, we studied the effects of nucleases and backbone modifications on the stability of siHybrids under cell culture conditions.

Dugan, L

2006-02-08

235

Activation of different split functionalities upon re-association of RNA-DNA hybrids  

PubMed Central

Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of the protein functions and improved detection sensitivity. Here we show a similar technique based on a pair of RNA-DNA hybrids that can be generally used for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept this work is mainly focused on activation of RNA interference; however the release of other functionalities (resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumors together with specific gene silencing. This split-functionality approach presents a new route in the development of “smart” nucleic acids based nanoparticles and switches for various biomedical applications. PMID:23542902

Afonin, Kirill A.; Viard, Mathias; Martins, Angelica N.; Lockett, Stephen J.; Maciag, Anna E.; Freed, Eric O.; Heldman, Eliahu; Jaeger, Luc; Blumenthal, Robert; Shapiro, Bruce A.

2013-01-01

236

Inhibition of Xenograft Tumor Growth by Gold Nanoparticle-DNA Oligonucleotide Conjugates-Assisted Delivery of BAX mRNA  

PubMed Central

Use of non-biological agents for mRNA delivery into living systems in order to induce heterologous expression of functional proteins may provide more advantages than the use of DNA and/or biological vectors for delivery. However, the low efficiency of mRNA delivery into live animals, using non-biological systems, has hampered the use of mRNA as a therapeutic molecule. Here, we show that gold nanoparticle-DNA oligonucleotide (AuNP-DNA) conjugates can serve as universal vehicles for more efficient delivery of mRNA into human cells, as well as into xenograft tumors generated in mice. Injections of BAX mRNA loaded on AuNP-DNA conjugates into xenograft tumors resulted in highly efficient mRNA delivery. The delivered mRNA directed the efficient production of biologically functional BAX protein, a pro-apoptotic factor, consequently inhibiting tumor growth. These results demonstrate that mRNA delivery by AuNP-DNA conjugates can serve as a new platform for the development of safe and efficient gene therapy. PMID:24073264

Won, Miae; Park, Mira; Bae, Jeehyeon; Lee, Kangseok

2013-01-01

237

Interplay of Structure, Hydration and Thermal Stability in Formacetal Modified Oligonucleotides: RNA May Tolerate Nonionic Modifications Better than DNA  

SciTech Connect

DNA and RNA oligonucleotides having formacetal internucleoside linkages between uridine and adenosine nucleosides have been prepared and studied using UV thermal melting, osmotic stress, and X-ray crystallography. Formacetal modifications have remarkably different effects on double helical RNA and DNAethe formacetal stabilizes the RNA helix by +0.7 C but destabilizes the DNA helix by -1.6 C per modification. The apparently hydrophobic formacetal has little effect on hydration of RNA but decreases the hydration of DNA, which suggests that at least part of the difference in thermal stability may be related to differences in hydration. A crystal structure of modified DNA shows that two isolated formacetal linkages fit almost perfectly in an A-type helix (decamer). Taken together, the data suggest that RNA may tolerate nonionic backbone modifications better than DNA. Overall, formacetal appears to be an excellent mimic of phosphate linkage in RNA and an interesting modification for potential applications in fundamental studies and RNA-based gene control strategies, such as RNA interference.

Kolarovic, A.; Schweizer, E; Greene, E; Gironda, M; Pallan, P; Egli, M; Rozners, E

2009-01-01

238

Rat ribosomal RNA gene can utilize primate RNA polymerase I transcription machinery: lack of absolute species specificity in rDNA transcription.  

PubMed

The transcriptional activity of rodent ribosomal RNA gene (rDNA) in the primate cell was examined in the light of reported species specificity of eukaryotic ribosomal RNA synthesis. The present study showed that rat rDNA can be transcribed in HeLa nuclear extract whereas mouse rDNA was not transcribed in the heterologous extract. Rat and mouse rDNA transcription factors were interchangeable with respect to efficiency and accuracy of transcription. The initiation of rat ribosomal gene transcription by RNA polymerase I occurred at the +1 site in the heterologous extract. Initiation of transcription at the correct site also occurred in vivo following transfection of cloned rat rDNA into the primate (COS-7) cells. These data indicate that rat ribosomal RNA gene can be expressed in the primate system in vitro and in vivo. The absolute lack of species specificity in rDNA transcription has been discussed based on the present data and other reports. PMID:8780707

Ghosh, A K; Niu, H; Jacob, S T

1996-08-23

239

Ultrasensitive detection of DNA and RNA based on enzyme-free click chemical ligation chain reaction on dispersed gold nanoparticles.  

PubMed

An ultrasensitive colorimetric DNA and RNA assay using a combination of enzyme-free click chemical ligation chain reaction (CCLCR) on dispersed gold nanoparticles (GNPs) and a magnetic separation process has been developed. The click chemical ligation between an azide-containing probe DNA-modified GNP and a dibenzocyclooctyne-containing probe biotinyl DNA occurred through hybridization with target DNA (RNA) to form the biotinyl-ligated GNPs (ligated products). Eventually, both the biotinyl-ligated GNPs and target DNA (RNA) were amplified exponentially using thermal cycling. After separation of the biotinyl-ligated GNPs using streptavidin-modified magnetic beads, the change in intensity of the surface plasmon band at 525 nm in the supernatants was observed by UV/vis measurement and was also evident visually. The CCLCR assay provides ultrasensitive detection (50 zM: several copies) of target DNA that is comparable to PCR-based approaches. Note that target RNA could also be detected with similar sensitivity without the need for reverse transcription to the corresponding cDNA. The amplification efficiency of the CCLCR assay was as high as 82% due to the pseudohomogeneous reaction behavior of CCLCR on dispersed GNPs. In addition, the CCLCR assay was able to discriminate differences in single-base mismatches and to specifically detect target DNA and target RNA from the cell lysate. PMID:25256209

Kato, Daiki; Oishi, Motoi

2014-10-28

240

DNA and RNA polymerase activity in a Moniliophthora perniciosa mitochondrial plasmid and self-defense against oxidative stress.  

PubMed

Moniliophthora perniciosa (Stahel) Aime and Phillips-Mora is a hemibiotrophic basidiomycete (Agaricales, Tricholomataceae) that causes witches' broom disease in cocoa (Theobroma cacao L.). This pathogen carries a stable integrated invertron-type linear plasmid in its mitochondrial genome that encodes viral-like DNA and RNA polymerases related to fungal senescence and longevity. After culturing the fungus and obtaining its various stages of development in triplicate, we carried out total RNA extraction and subsequent complementary DNA synthesis. To analyze DNA and RNA polymerase expression levels, we performed real-time reverse transcriptase polymerase chain reaction for various fungal phases of development. Our results showed that DNA and RNA polymerase gene expression in the primordium phase of M. perniciosa is related to a potential defense mechanism against T. cacao oxidative attack. PMID:23913377

Andrade, B S; Villela-Dias, C; Gomes, D S; Micheli, F; Góes-Neto, A

2013-01-01

241

An Undergraduate Investigation into the 10-23 DNA Enzyme that Cleaves RNA: DNA Can Cut It in the Biochemistry Laboratory  

ERIC Educational Resources Information Center

A low-cost biochemistry experiment is described that demonstrates current techniques in the use of catalytic DNA molecules and introduces a nonradioactive, nonfluorescent, inexpensive, fast, and safe method for monitoring these nucleic acid reactions. The laboratory involves the exploration of the 10-23 DNA enzyme as it cleaves a specific RNA

Flynn-Charlebois, Amber; Burns, Jamie; Chapelliquen, Stephanie; Sanmartino, Holly

2011-01-01

242

The Complete Mitochondrial DNA Sequence of the Basal Hexapod Tetrodontophora bielanensis: Evidence for Heteroplasmy and tRNA Translocations  

Microsoft Academic Search

We present the complete 15,455-nt mitochondrial DNA sequence of the springtail Tetrodontophora bielanensis (Arthropoda, Hexapoda, Collembola). The gene content is typical of most metazoans, with 13 protein-coding genes (PCGs), 2 genes encoding for ribosomal RNA subunits, and 22 tRNA genes. The nucleotide sequence shows the well-known A1T bias typical of insect mtDNA; its A1T content is lower (72.7%) than that

Francesco Nardi; Antonio Carapelli; Pietro Paolo Fanciulli; Romano Dallai; Francesco Frati; Biol Evol

243

Selective nucleic acid removal via exclusion (SNARE): capturing mRNA and DNA from a single sample.  

PubMed

The path from gene (DNA) to gene product (RNA or protein) is the foundation of genotype giving rise to phenotype. Comparison of genomic analyses (DNA) with paired transcriptomic studies (mRNA) is critical to evaluating the pathogenic processes that give rise to human disease. The ability to analyze both DNA and mRNA from the same sample is not only important for biologic interrogation but also to minimize variance (e.g., sample loss) unrelated to the biology. Existing methods for RNA and DNA purification from a single sample are typically time-consuming and labor intensive or require large sample sizes to split for separate RNA and DNA extraction procedures. Thus, there is a need for more efficient and cost-effective methods to purify both RNA and DNA from a single sample. To address this need, we have developed a technique, termed SNARE (Selective Nucleic Acid Removal via Exclusion), that uses pinned oil interfaces to simultaneous purify mRNA and DNA from a single sample. A unique advantage of SNARE is the elimination of dilutive wash and centrifugation processes that are fundamental to conventional methods where sample is typically discarded. This minimizes loss and maximizes recovery by allowing nondilutive reinterrogation of the sample. We demonstrate that SNARE is more sensitive than commercially available kits, robustly and repeatably achieving mRNA and DNA purification from extremely low numbers of cells for downstream analyses. In addition to sensitivity, SNARE is fast, easy to use, and cost-effective and requires no laboratory infrastructure or hazardous chemicals. We demonstrate the clinical utility of the SNARE with prostate cancer circulating tumor cells to demonstrate its ability to perform both genomic and transcriptomic interrogation on rare cell populations that would be difficult to achieve with any current method. PMID:24016179

Strotman, Lindsay; O'Connell, Rachel; Casavant, Benjamin P; Berry, Scott M; Sperger, Jamie M; Lang, Joshua M; Beebe, David J

2013-10-15

244

Exploring the recovery and detection of messenger RNA and DNA from enhanced fingermarks in blood.  

PubMed

Often in the examination of bloodstained fingermarks discussion occurs around whether to prioritise the fingerprint evidence or focus on the biological evidence. Collecting a sample for genetic profiling may result in the loss of ridge detail that could have been used for fingerprint comparison. Fingermark enhancement and recovery methods along with sample collection methods could also compromise downstream genetic analysis. Previous forensic casework has highlighted circumstances where, after enhancement had been performed, it would have been extremely valuable to both identify the body fluid and generate a DNA profile from the same sample. We enhanced depletion series of fingermarks made in blood, using single treatments consisting of aqueous amido black, methanol-based amido black, acid yellow and leucocrystal violet, and exposure to long wave UV light. We then extracted the DNA and RNA for profiling, to assess the recovery and detection of genetic material from the enhanced fingermarks. We have shown that genetic profiling of bloodstained fingermarks can be successful after chemical enhancement; however it may still be necessary to prioritise evidence types in certain circumstances. From our results it appears that even with visible bloodstained fingermarks, leucocrystal violet can reduce the effectiveness of subsequent messenger RNA profiling. Aqueous amido black and acid yellow also have adverse effects on messenger RNA profiling of depleted fingermarks with low levels of cellular material. These results help with forensic decision-making by expanding knowledge of the extent of the detrimental effects of blood-enhancement reagents on both DNA profiling and body fluid identification using messenger RNA profiling. PMID:24796948

Fox, A; Gittos, M; Harbison, S A; Fleming, R; Wivell, R

2014-05-01

245

Strong inverse correlation between microRNA-125b and human papillomavirus DNA in productive infection.  

PubMed

Infection by the human papillomavirus (HPV) is a cause of cervical intraepithelial neoplasia (CIN) and cancer. microRNA (miRNA) in situ analysis of the transformation zone epithelia, the site of initial cervical HPV infection, showed that miRNAs let-7c, -99a, 26a, and 125b were the most abundantly expressed. In situ testing of CIN 1 showed a dramatic reduction in miR-125b expression in the koilocytes, the cytologic marker of productive HPV infection. A marked reduction in miR-125b was likewise observed in the HPV-infected cells of the condyloma acuminatum, verruca vulgaris, and epidermodysplasia verruciformis. Reverse transcriptase in situ polymerase chain reaction (PCR) showed that the pre-miRNA 125b was present in the koilocyte, suggesting direct inactivation of the mature miRNA. HEK cells transfected with only the antimiR-125b showed perinuclear halos equivalent to HPV-infected koilocytes. NIH 3T3 cells transfected with the HPV 16 full-length genome and mimetic miR-125b showed a marked reduction in viral DNA and protein synthesis by quantitative PCR and in situ-based analyses, respectively (P=0.002). Alternatively, cotransfection with anti-miR-125b and HPV 16 markedly increased HPV DNA (P=0.002). Sequence analyses showed strong homology between L2 of different HPV genotypes and miR-125b. Transfection with HPV 16 L2 resulted in a marked reduction in miR-125b levels in the NIH 3T3 cells. HPV L2-induced inactivation of miR-125b is associated with the classic cytologic changes of the koilocyte, and the exogenous application of mimetic miR-125b markedly inhibits HPV DNA synthesis. PMID:20736742

Nuovo, Gerard J; Wu, Xin; Volinia, Stefano; Yan, Fengting; di Leva, Gianpiero; Chin, Nena; Nicol, Alcina F; Jiang, Jinmai; Otterson, Gregory; Schmittgen, Thomas D; Croce, Carlo

2010-09-01

246

A DNA Damage Response System Associated with the phosphoCTD of Elongating RNA Polymerase II  

PubMed Central

RNA polymerase II translocates across much of the genome and since it can be blocked by many kinds of DNA lesions, detects DNA damage proficiently; it thereby contributes to DNA repair and to normal levels of DNA damage resistance. However, the components and mechanisms that respond to polymerase blockage are largely unknown, except in the case of UV-induced damage that is corrected by nucleotide excision repair. Because elongating RNAPII carries with it numerous proteins that bind to its hyperphosphorylated CTD, we tested for effects of interfering with this binding. We find that expressing a decoy CTD-carrying protein in the nucleus, but not in the cytoplasm, leads to reduced DNA damage resistance. Likewise, inducing aberrant phosphorylation of the CTD, by deleting CTK1, reduces damage resistance and also alters rates of homologous recombination-mediated repair. In line with these results, extant data sets reveal a remarkable, highly significant overlap between phosphoCTD-associating protein genes and DNA damage-resistance genes. For one well-known phosphoCTD-associating protein, the histone methyltransferase Set2, we demonstrate a role in DNA damage resistance, and we show that this role requires the phosphoCTD binding ability of Set2; surprisingly, Set2’s role in damage resistance does not depend on its catalytic activity. To explain all of these observations, we posit the existence of a CTD-Associated DNA damage Response (CAR) system, organized around the phosphoCTD of elongating RNAPII and comprising a subset of phosphoCTD-associating proteins. PMID:23613755

Winsor, Tiffany Sabin; Bartkowiak, Bartlomiej; Bennett, Craig B.; Greenleaf, Arno L.

2013-01-01

247

Pisum sativum p68 DEAD-box protein is ATP-dependent RNA helicase and unique bipolar DNA helicase.  

PubMed

DEAD-box helicases play essential role in DNA and RNA metabolism such as replication, repair, recombination, transcription, translation, ribosome biogenesis and splicing which regulate plant growth and development. The presence of helicases in the stress-induced ORFs identified by cDNA microarray indicates that helicases might be playing an important role in stabilizing growth in plants under stress. p68 DEAD-box helicase has been identified and characterized from animal systems but the properties and functions of plant p68 are poorly understood. In this study, the identification, purification and characterization of recombinant p68 from Pisum sativum (Psp68) is presented. Psp68 possesses all the characteristic motifs like DEAD-box ATP-binding and helicase C terminal motifs and is structurally similar to human p68 homologue. Psp68 exhibits ATPase activity in the presence of both DNA and RNA and it binds to DNA as well as RNA. It contains the characteristic RNA helicase activity. Interestingly Psp68 also shows the unique DNA helicase activity, which is bipolar in nature (unwinds DNA in both the 5'-3' and 3'-5' directions). The Km values of Psp68 for ATPase are 0.5126 and 0.9142 mM in the presence of DNA and RNA, respectively. The Km values of Psp68 are 1.6129 and 1.14 nM for DNA helicase and RNA helicase, respectively. The unique properties of Psp68 suggest that it could be a multifunctional protein involved in different aspect of DNA and RNA metabolism. This discovery should make an important contribution to better understanding of nucleic acids metabolism plants. PMID:24908423

Tuteja, Narendra; Tarique, Mohammed; Banu, Mst Sufara Akhter; Ahmad, Moaz; Tuteja, Renu

2014-08-01

248

The Relative Reactivity of Deoxyribose and Ribose: Did DNA Come Before RNA?  

NASA Technical Reports Server (NTRS)

If it is assumed that there was a precursor to the ribose-phosphate backbone of RNA in the preRNA world (such as peptide nucleic acid), then the entry of various sugars into the genetic material may be related to the stability and non-enzymatic reactivity of the aldose. The rate of decomposition of 2-deoxyribose has been determined to be 1/3 that of ribose. In addition we have measured the amount of free aldehyde by H-1 and C-13 NMR and find that it has approximately 0.15% free aldehyde compared to 0.05% for ribose at 25 C. This suggests that deoxyribose would be significantly more reactive with early bases in the absence of enzymes. This is confirmed by urazole and deoxyribose reacting to form the deoxynucleoside 45 times faster as 25 C than urazole reacts with ribose to form the Ribonucleoside. Urazole is a potential precursor of uracil and is a plausible prebiotic compound which reacts with aldoses to form nucleosides. Thus the non-enzymatic reactivity of deoxyribose would favor its early use over ribose until enzymes could change the relative reactivities. Most of the reasons that RNA is presumed to have come before DNA are extrapolations back from contemporary metabolism (e.g. the abundance of ribose based coenzymes, the biosynthesis of histidine, deoxyribonucleotides are synthesized from ribonucleotides, etc.). It is very difficult to reconstruct biochemical pathways much before the last common ancestor, and it is even more difficult to do more than guess at the biochemistry of very early self-replicating systems. Thus we believe that these reasons are not compelling and that the non-enzymatic chemistry may be more important than enzymatic pathways for constructing the earliest of biochemical pathways. While the RNA world has been discussed at great length, there has not been an exploration of the transition out of the RNA world. We have constructed many possible schemes of genetic takeover events from preRNA to modern DNA, RNA, protein system which could generate the RNA metabolic fossils we see today.

Dworkin, Jason P.; Miller, Stanley L.

1995-01-01

249

De novo reconstruction of consensus master genomes of plant RNA and DNA viruses from siRNAs.  

PubMed

Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense. PMID:24523907

Seguin, Jonathan; Rajeswaran, Rajendran; Malpica-López, Nachelli; Martin, Robert R; Kasschau, Kristin; Dolja, Valerian V; Otten, Patricia; Farinelli, Laurent; Pooggin, Mikhail M

2014-01-01

250

Using a commercial DNA extraction kit to obtain RNA for RT-PCR from starchy rice endosperm.  

PubMed

The extraction of RNA from a starchy plant material, such as many common food grains, is difficult, and especially so from the mature endosperm of rice. Most commercial RNA kits are not suitable for starchy materials. Traditional RNA extraction procedures, in addition to being laborious and time consuming, leave hazardous organic wastes that result in expensive disposal costs. Interestingly, the numerous commercial DNA isolation kits now available often include directions for eliminating co-isolated RNA. This indicated an approach to obtain the generally unwanted RNA by-product by treating the total extraction product to intentionally retain RNA. A method was developed by which a two-step DNase procedure was applied to the product of the Cartagen Food DNA extraction kit that eliminated the DNA but left the co-extracted RNA. This modified procedure was compared with several other commercial and standard methods that are promoted as being able to work under high polysaccharide conditions. Successful extraction was determined by the production and amplification of cDNA by RT-PCR of actin. Extraction was successful from milled rice, as well as from cornmeal and wheat flour. The modification provides an RNA extraction method that is quick, easy, and inexpensive, and also eliminates the production of hazardous wastes. PMID:18228540

Belefant-Miller, Helen; Ledbetter, Cindy; Bennett, Selester

2008-03-01

251

Global Analysis of mRNA Decay in Halobacterium salinarum NRC1 at Single-Gene Resolution Using DNA Microarrays  

Microsoft Academic Search

RNA degradation is an important factor in the regulation of gene expression. It allows organisms to quickly respond to changing environmental conditions by adapting the expression of individual genes. The stability of individual mRNAs within an organism varies considerably, contributing to differential amounts of proteins ex- pressed. In this study we used DNA microarrays to analyze mRNA degradation in exponentially

Sonja Hundt; Alexander Zaigler; Christian Lange; Jorg Soppa; Gabriele Klug

2007-01-01

252

An adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U deamination of DNA  

E-print Network

An adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U deamination of DNA Mary Anne T (received for review February 24, 2007) Adenosine-to-inosine editing in the anticodon of tRNAs is essential for viability. Enzymes mediating tRNA adenosine deamination in bacteria and yeast contain cytidine deaminase

Papavasiliou, F. Nina

253

Mutations affecting RNA polymerase I-stimulated exchange and rDNA recombination in yeast  

SciTech Connect

HOT1 is a cis-acting recombination-stimulatory sequence isolated from the rDNA repeat unit of yeast. The ability of HOT1 to stimulate mitotic exchange appears to depend on its ability to promote high levels of RNA polymerase I transcription. A qualitative colony color sectoring assay was developed to screen for trans-acting mutations that alter the activity of HOT1. Both hypo-recombination and hyper-recombination mutants were isolated. Genetic analysis of seven HOT1 recombination mutants (hrm) that decrease HOT1 activity shows that they behave as recessive nuclear mutations and belong to five linkage groups. Three of these mutations, hrm1, hrm2, and hrm3, also decrease rDNA exchange but do not alter recombination in the absence of HOT1. Another mutation, hrm4, decreases HOT1-stimulated recombination but does not affect rDNA recombination or exchange in the absence of HOT1. Two new alleles of RAD52 were also isolated using this screen. With regard to HOT1 activity, rad52 is epistatic to all four hrm mutations indicating that the products of the HRM genes and of RAD52 mediate steps in the same recombination pathway. Finding mutations that decrease both the activity of HOT1 and exchange in the rDNA supports the hypothesis that HOT1 plays a role in rDNA recombination.

Lin, Y.H.; Keil, R.L. (Milton S. Hershey Medical Center, PA (USA))

1991-01-01

254

Electrolyzed-reduced water protects against oxidative damage to DNA, RNA, and protein.  

PubMed

The generation of reactive oxygen species is thought to cause extensive oxidative damage to various biomolecules such as DNA, RNA, and protein. In this study, the preventive, suppressive, and protective effects of in vitro supplementation with electrolyzed-reduced water on H2O2-induced DNA damage in human lymphocytes were examined using a comet assay. Pretreatment, cotreatment, and posttreatment with electrolyzed-reduced water enhanced human lymphocyte resistance to the DNA strand breaks induced by H2O2 in vitro. Moreover, electrolyzed-reduced water was much more effective than diethylpyrocarbonate-treated water in preventing total RNA degradation at 4 and 25 degrees C. In addition, electrolyzed-reduced water completely prevented the oxidative cleavage of horseradish peroxidase, as determined using sodium dodecyl sulfate-polyacrylamide gels. Enhancement of the antioxidant activity of ascorbic acid dissolved in electrolyzed-reduced water was about threefold that of ascorbic acid dissolved in nonelectrolyzed deionized water, as measured by a xanthine-xanthine oxidase superoxide scavenging assay system, suggesting an inhibitory effect of electrolyzedreduced water on the oxidation of ascorbic acid. PMID:17159237

Lee, Mi Young; Kim, Yoon Kyoung; Ryoo, Kun Kul; Lee, Yoon Bae; Park, Eun Ju

2006-11-01

255

Protozoan ALKBH8 Oxygenases Display both DNA Repair and tRNA Modification Activities  

PubMed Central

The ALKBH family of Fe(II) and 2-oxoglutarate dependent oxygenases comprises enzymes that display sequence homology to AlkB from E. coli, a DNA repair enzyme that uses an oxidative mechanism to dealkylate methyl and etheno adducts on the nucleobases. Humans have nine different ALKBH proteins, ALKBH1–8 and FTO. Mammalian and plant ALKBH8 are tRNA hydroxylases targeting 5-methoxycarbonylmethyl-modified uridine (mcm5U) at the wobble position of tRNAGly(UCC). In contrast, the genomes of some bacteria encode a protein with strong sequence homology to ALKBH8, and robust DNA repair activity was previously demonstrated for one such protein. To further explore this apparent functional duality of the ALKBH8 proteins, we have here enzymatically characterized a panel of such proteins, originating from bacteria, protozoa and mimivirus. All the enzymes showed DNA repair activity in vitro, but, interestingly, two protozoan ALKBH8s also catalyzed wobble uridine modification of tRNA, thus displaying a dual in vitro activity. Also, we found the modification status of tRNAGly(UCC) to be unaltered in an ALKBH8 deficient mutant of Agrobacterium tumefaciens, indicating that bacterial ALKBH8s have a function different from that of their eukaryotic counterparts. The present study provides new insights on the function and evolution of the ALKBH8 family of proteins. PMID:24914785

Zdzalik, Daria; Vagb?, Cathrine B.; Kirpekar, Finn; Davydova, Erna; Puscian, Alicja; Maciejewska, Agnieszka M.; Krokan, Hans E.; Klungland, Arne; Tudek, Barbara; van den Born, Erwin; Falnes, Pal ?.

2014-01-01

256

Recovery of an arenavirus entirely from RNA polymerase I/II-driven cDNA  

PubMed Central

The prototypic arenavirus lymphocytic choriomeningitis virus has been a primary workhorse of viral immunologists for almost a century, and it has served as an important model for studying basic principles of arenavirus molecular biology. Its negative-stranded bisegmented RNA genome has, however, posed a major obstacle to attempts at manipulating the infectious virus by reverse genetic techniques. Here, we report the recovery of infectious lymphocytic choriomeningitis virus (the immunosuppressive strain clone 13) entirely from cDNA. Intracellular transcription of the short and the long viral genome segment from polymerase (pol) I-driven vectors and coexpression of the minimal viral-transacting factors NP and L from pol II-driven plasmids resulted in the efficient formation of infectious virus with genetic tags in both genome segments. The cDNA-derived viruses behaved identically to wild-type virus in both cell culture and infected mice. Importantly, they caused a chronic infection and suppressed the adaptive immune response to an unrelated third-party virus. This technology provides an important basis for investigating viral determinants of persistent infection and immunosuppression. In addition, our findings demonstrate that pol I/II-based vector systems may represent an efficient alternative strategy for the recovery of cytoplasmic negative-strand RNA viruses from cDNA. PMID:16537369

Flatz, Lukas; Bergthaler, Andreas; de la Torre, Juan Carlos; Pinschewer, Daniel D.

2006-01-01

257

Recovery of an arenavirus entirely from RNA polymerase I/II-driven cDNA.  

PubMed

The prototypic arenavirus lymphocytic choriomeningitis virus has been a primary workhorse of viral immunologists for almost a century, and it has served as an important model for studying basic principles of arenavirus molecular biology. Its negative-stranded bisegmented RNA genome has, however, posed a major obstacle to attempts at manipulating the infectious virus by reverse genetic techniques. Here, we report the recovery of infectious lymphocytic choriomeningitis virus (the immunosuppressive strain clone 13) entirely from cDNA. Intracellular transcription of the short and the long viral genome segment from polymerase (pol) I-driven vectors and coexpression of the minimal viral-transacting factors NP and L from pol II-driven plasmids resulted in the efficient formation of infectious virus with genetic tags in both genome segments. The cDNA-derived viruses behaved identically to wild-type virus in both cell culture and infected mice. Importantly, they caused a chronic infection and suppressed the adaptive immune response to an unrelated third-party virus. This technology provides an important basis for investigating viral determinants of persistent infection and immunosuppression. In addition, our findings demonstrate that pol I/II-based vector systems may represent an efficient alternative strategy for the recovery of cytoplasmic negative-strand RNA viruses from cDNA. PMID:16537369

Flatz, Lukas; Bergthaler, Andreas; de la Torre, Juan Carlos; Pinschewer, Daniel D

2006-03-21

258

Short hairpin RNA induces methylation of hepatitis B virus covalently closed circular DNA in human hepatoma cells.  

PubMed

Small interfering RNAs not only modulate gene expression at a post-transcriptional level, but also induce transcriptional gene silencing by RNA interference-mediated heterochromatin formation and RNA-directed DNA methylation (RdDM). However, although established in plants, there have been controversies whether RdDM operates in mammals. Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) serves as a template for viral RNA transcription, and transcriptional activity of HBV cccDNA is regulated by methylation in patients with chronic HBV infection. In this study, we stably expressed short hairpin RNA (shRNA) against HBV in human hepatoma cells to determine whether shRNA induces methylation of HBV cccDNA. HepAD38 cells which permit replication of HBV under control of tetracycline-responsive promoter were transduced with lentiviral vectors which encode sh-1580, a shRNA against the hepatitis B viral protein HBx. Bisulfite sequencing PCR analysis revealed that sh-1580 induced CpG methylations at a higher rate compared to control (31.3% vs. 12.8%, p<0.05). The sh-1580-induced CpG methylation was localized near the target sequence of sh-1580 in more than a half of the clones. Methylation-induced transcriptional suppression was confirmed by in vitro transcription assay. These results confirm the feasibility of RdDM of HBV cccDNA in human cells. Lentiviral vector-mediated transfer of shRNA may be used as a tool for novel transcriptional modulation by epigenetic modification of HBV cccDNA. PMID:23727428

Park, Hyun Kyung; Min, Bo Young; Kim, Nam Young; Jang, Eun Sun; Shin, Cheol Min; Park, Young Soo; Hwang, Jin-Hyeok; Jeong, Sook-Hyang; Kim, Nayoung; Lee, Dong Ho; Kim, Jin-Wook

2013-06-28

259

Human origin recognition complex binds preferentially to G-quadruplex-preferable RNA and single-stranded DNA.  

PubMed

Origin recognition complex (ORC), consisting of six subunits ORC1-6, is known to bind to replication origins and function in the initiation of DNA replication in eukaryotic cells. In contrast to the fact that Saccharomyces cerevisiae ORC recognizes the replication origin in a sequence-specific manner, metazoan ORC has not exhibited strict sequence-specificity for DNA binding. Here we report that human ORC binds preferentially to G-quadruplex (G4)-preferable G-rich RNA or single-stranded DNA (ssDNA). We mapped the G-rich RNA-binding domain in the ORC1 subunit, in a region adjacent to its ATPase domain. This domain itself has an ability to preferentially recognize G4-preferable sequences of ssDNA. Furthermore, we found, by structure modeling, that the G-rich RNA-binding domain is similar to the N-terminal portion of AdoMet_MTase domain of mammalian DNA methyltransferase 1. Therefore, in contrast with the binding to double-stranded DNA, human ORC has an apparent sequence preference with respect to its RNA/ssDNA binding. Interestingly, this specificity coincides with the common signature present in most of the human replication origins. We expect that our findings provide new insights into the regulations of function and chromatin binding of metazoan ORCs. PMID:24003239

Hoshina, Shoko; Yura, Kei; Teranishi, Honami; Kiyasu, Noriko; Tominaga, Ayumi; Kadoma, Haruka; Nakatsuka, Ayaka; Kunichika, Tomoko; Obuse, Chikashi; Waga, Shou

2013-10-18

260

Antimicrobial Activity Spectrum, cDNA Cloning, and mRNA Expression of a Newly Isolated Member of the  

E-print Network

Antimicrobial Activity Spectrum, cDNA Cloning, and mRNA Expression of a Newly Isolated Member, named cecropin A, was purified to homogeneity and fully characterized after cDNA cloning. The 34-residue for serious debilitating human diseases such as malaria, lymphatic filari- asis, and numerous arboviruses

Lowenberger, Carl

261

Detection of Avian leukosis virus genome by a nested polymerase chain reaction using DNA and RNA from dried feather shafts.  

PubMed

The nested polymerase chain reaction (nPCR) using frozen feather pulp is useful for detecting fowl glioma-inducing virus (FGV), which belongs to the Avian leukosis virus family, and it has recently been suggested that FGV has spread to ornamental chickens kept in Japanese zoological gardens. In the current study, the practicality of using DNA and RNA from dried feather shafts as PCR samples was examined to establish a simple method for tissue preservation. Feather shafts were collected from 7 FGV-positive chickens and stored at room temperature for 30 days. DNA and RNA were extracted from these dried materials. All DNA and complementary DNA (cDNA) prepared from the RNA showed positive results for chicken beta-actin and FGV, respectively. Screening for FGV was performed on Japanese fowls kept in zoological garden N. Of the feather shafts collected from 57 birds, 1 sample tested positive for FGV according to PCR of DNA and cDNA samples from the dried feather shafts. This positive bird originated from zoological garden A and had brain lesions suggestive of fowl glioma. The results suggest that DNA and RNA from dried feather shafts can be used in nPCR to detect the FGV genome. PMID:19564502

Hatai, Hitoshi; Ochiai, Kenji; Umemura, Takashi

2009-07-01

262

Human Origin Recognition Complex Binds Preferentially to G-quadruplex-preferable RNA and Single-stranded DNA*  

PubMed Central

Origin recognition complex (ORC), consisting of six subunits ORC1–6, is known to bind to replication origins and function in the initiation of DNA replication in eukaryotic cells. In contrast to the fact that Saccharomyces cerevisiae ORC recognizes the replication origin in a sequence-specific manner, metazoan ORC has not exhibited strict sequence-specificity for DNA binding. Here we report that human ORC binds preferentially to G-quadruplex (G4)-preferable G-rich RNA or single-stranded DNA (ssDNA). We mapped the G-rich RNA-binding domain in the ORC1 subunit, in a region adjacent to its ATPase domain. This domain itself has an ability to preferentially recognize G4-preferable sequences of ssDNA. Furthermore, we found, by structure modeling, that the G-rich RNA-binding domain is similar to the N-terminal portion of AdoMet_MTase domain of mammalian DNA methyltransferase 1. Therefore, in contrast with the binding to double-stranded DNA, human ORC has an apparent sequence preference with respect to its RNA/ssDNA binding. Interestingly, this specificity coincides with the common signature present in most of the human replication origins. We expect that our findings provide new insights into the regulations of function and chromatin binding of metazoan ORCs. PMID:24003239

Hoshina, Shoko; Yura, Kei; Teranishi, Honami; Kiyasu, Noriko; Tominaga, Ayumi; Kadoma, Haruka; Nakatsuka, Ayaka; Kunichika, Tomoko; Obuse, Chikashi; Waga, Shou

2013-01-01

263

Why does TNA cross-pair more strongly with RNA than with DNA? An answer from X-ray analysis  

SciTech Connect

L-{alpha}-threofuranosyl (3' {yields} 2') nucleic acid (TNA) residues adopt a C4'-exo pucker when incorporated into an A- (left) or a B-form DNA duplex (right). The resulting intranucleotide P {hor_ellipsis} P distance in TNA is very similar to that in RNA (represented by a C3'-endo puckered adenosine residue; green). The structural data explain earlier observations that TNA hydridizes more stably with RNA than with DNA and that RNA constitutes the better template for ligating TNA fragments.

Pallan, P.S.; Wilds, C.J.; Wawrzak, Z.; Krishnamurthy, R.; Eschenmoser, A.; Egli, M. (Vanderbilt); (Scripps); (NWU); (ConU)

2010-03-08

264

Metal chelate affinity precipitation of RNA and purification of plasmid DNA  

NASA Technical Reports Server (NTRS)

The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.

Balan, Sindhu; Murphy, Jason; Galaev, Igor; Kumar, Ashok; Fox, George E.; Mattiasson, Bo; Willson, Richard C.

2003-01-01

265

DNA-RNA chimera indicates the flexibility of the backbone influences the encapsulation of fluorescent AgNC emitters.  

PubMed

Many DNA scaffolds efficiently encapsulate highly emissive silver nanoclusters (AgNCs). The secondary structures and the arrangement of sequences of DNA scaffolds are important factors by which the specific features of AgNCs emitters can be determined. By introducing DNA-RNA chimera scaffolds, we here explore another factor - the flexibility of the backbone of nucleic acid-templates - in creating highly fluorescent AgNC emitters. PMID:25247813

Shah, Pratik; Thulstrup, Peter W; Cho, Seok Keun; Bjerrum, Morten Jannik; Yang, Seong Wook

2014-10-01

266

Combined DNA-RNA fluorescent in situ hybridization (FISH) to study X chromosome inactivation in differentiated female mouse embryonic stem cells.  

PubMed

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected. PMID:24961515

Barakat, Tahsin Stefan; Gribnau, Joost

2014-01-01

267

A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site  

PubMed Central

Human mitochondrial DNA contains a distinctive guanine-rich motif denoted conserved sequence block II (CSB II) that stops RNA transcription, producing prematurely terminated transcripts to prime mitochondrial DNA replication. Recently, we reported a general phenomenon that DNA:RNA hybrid G-quadruplexes (HQs) readily form during transcription when the non-template DNA strand is guanine-rich and such HQs in turn regulate transcription. In this work, we show that transcription of mitochondrial DNA leads to the formation of a stable HQ or alternatively an unstable intramolecular DNA G-quadruplex (DQ) at the CSB II. The HQ is the dominant species and contributes to the majority of the premature transcription termination. Manipulating the stability of the DQ has little effect on the termination even in the absence of HQ; however, abolishing the formation of HQs by preventing the participation of either DNA or RNA abolishes the vast majority of the termination. These results demonstrate that the type of G-quadruplexes (HQ or DQ) is a crucial determinant in directing the transcription termination at the CSB II and suggest a potential functionality of the co-transcriptionally formed HQ in DNA replication initiation. They also suggest that the competition/conversion between an HQ and a DQ may regulate the function of a G-quadruplex-forming sequence. PMID:25140009

Zheng, Ke-wei; Wu, Ren-yi; He, Yi-de; Xiao, Shan; Zhang, Jia-yu; Liu, Jia-quan; Hao, Yu-hua; Tan, Zheng

2014-01-01

268

A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site.  

PubMed

Human mitochondrial DNA contains a distinctive guanine-rich motif denoted conserved sequence block II (CSB II) that stops RNA transcription, producing prematurely terminated transcripts to prime mitochondrial DNA replication. Recently, we reported a general phenomenon that DNA:RNA hybrid G-quadruplexes (HQs) readily form during transcription when the non-template DNA strand is guanine-rich and such HQs in turn regulate transcription. In this work, we show that transcription of mitochondrial DNA leads to the formation of a stable HQ or alternatively an unstable intramolecular DNA G-quadruplex (DQ) at the CSB II. The HQ is the dominant species and contributes to the majority of the premature transcription termination. Manipulating the stability of the DQ has little effect on the termination even in the absence of HQ; however, abolishing the formation of HQs by preventing the participation of either DNA or RNA abolishes the vast majority of the termination. These results demonstrate that the type of G-quadruplexes (HQ or DQ) is a crucial determinant in directing the transcription termination at the CSB II and suggest a potential functionality of the co-transcriptionally formed HQ in DNA replication initiation. They also suggest that the competition/conversion between an HQ and a DQ may regulate the function of a G-quadruplex-forming sequence. PMID:25140009

Zheng, Ke-wei; Wu, Ren-yi; He, Yi-de; Xiao, Shan; Zhang, Jia-yu; Liu, Jia-quan; Hao, Yu-hua; Tan, Zheng

2014-01-01

269

Nonenzymatic synthesis of RNA and DNA oligomers on hexitol nucleic acid templates: the importance of the A structure  

NASA Technical Reports Server (NTRS)

Hexitol nucleic acid (HNA) is an analogue of DNA containing the standard nucleoside bases, but with a phosphorylated 1,5-anhydrohexitol backbone. HNA oligomers form duplexes having the nucleic acid A structure with complementary DNA or RNA oligomers. The HNA decacytidylate oligomer is an efficient template for the oligomerization of the 5'-phosphoroimidazolides of guanosine or deoxyguanosine. Comparison of the oligomerization efficiencies on HNA, RNA, and DNA decacytidylate templates under various conditions suggests strongly that only nucleic acid double helices with the A structure support efficient template-directed synthesis when 5'-phosphoroimidazolides of nucleosides are used as substrates.

Kozlov, I. A.; Politis, P. K.; Van Aerschot, A.; Busson, R.; Herdewijn, P.; Orgel, L. E.; Bada, J. L. (Principal Investigator); Dolan, M. (Principal Investigator)

1999-01-01

270

24-Hour Rhythms of DNA Methylation and Their Relation with Rhythms of RNA Expression in the Human Dorsolateral Prefrontal Cortex  

PubMed Central

Circadian rhythms modulate the biology of many human tissues, including brain tissues, and are driven by a near 24-hour transcriptional feedback loop. These rhythms are paralleled by 24-hour rhythms of large portions of the transcriptome. The role of dynamic DNA methylation in influencing these rhythms is uncertain. While recent work in Neurospora suggests that dynamic site-specific circadian rhythms of DNA methylation may play a role in modulating the fungal molecular clock, such rhythms and their relationship to RNA expression have not, to our knowledge, been elucidated in mammalian tissues, including human brain tissues. We hypothesized that 24-hour rhythms of DNA methylation exist in the human brain, and play a role in driving 24-hour rhythms of RNA expression. We analyzed DNA methylation levels in post-mortem human dorsolateral prefrontal cortex samples from 738 subjects. We assessed for 24-hour rhythmicity of 420,132 DNA methylation sites throughout the genome by considering methylation levels as a function of clock time of death and parameterizing these data using cosine functions. We determined global statistical significance by permutation. We then related rhythms of DNA methylation with rhythms of RNA expression determined by RNA sequencing. We found evidence of significant 24-hour rhythmicity of DNA methylation. Regions near transcription start sites were enriched for high-amplitude rhythmic DNA methylation sites, which were in turn time locked to 24-hour rhythms of RNA expression of nearby genes, with the nadir of methylation preceding peak transcript expression by 1–3 hours. Weak ante-mortem rest-activity rhythms were associated with lower amplitude DNA methylation rhythms as were older age and the presence of Alzheimer's disease. These findings support the hypothesis that 24-hour rhythms of DNA methylation, particularly near transcription start sites, may play a role in driving 24-hour rhythms of gene expression in the human dorsolateral prefrontal cortex, and may be affected by age and Alzheimer's disease. PMID:25375876

Lim, Andrew S. P.; Srivastava, Gyan P.; Yu, Lei; Chibnik, Lori B.; Xu, Jishu; Buchman, Aron S.; Schneider, Julie A.; Myers, Amanda J.; Bennett, David A.; De Jager, Philip L.

2014-01-01

271

24-hour rhythms of DNA methylation and their relation with rhythms of RNA expression in the human dorsolateral prefrontal cortex.  

PubMed

Circadian rhythms modulate the biology of many human tissues, including brain tissues, and are driven by a near 24-hour transcriptional feedback loop. These rhythms are paralleled by 24-hour rhythms of large portions of the transcriptome. The role of dynamic DNA methylation in influencing these rhythms is uncertain. While recent work in Neurospora suggests that dynamic site-specific circadian rhythms of DNA methylation may play a role in modulating the fungal molecular clock, such rhythms and their relationship to RNA expression have not, to our knowledge, been elucidated in mammalian tissues, including human brain tissues. We hypothesized that 24-hour rhythms of DNA methylation exist in the human brain, and play a role in driving 24-hour rhythms of RNA expression. We analyzed DNA methylation levels in post-mortem human dorsolateral prefrontal cortex samples from 738 subjects. We assessed for 24-hour rhythmicity of 420,132 DNA methylation sites throughout the genome by considering methylation levels as a function of clock time of death and parameterizing these data using cosine functions. We determined global statistical significance by permutation. We then related rhythms of DNA methylation with rhythms of RNA expression determined by RNA sequencing. We found evidence of significant 24-hour rhythmicity of DNA methylation. Regions near transcription start sites were enriched for high-amplitude rhythmic DNA methylation sites, which were in turn time locked to 24-hour rhythms of RNA expression of nearby genes, with the nadir of methylation preceding peak transcript expression by 1-3 hours. Weak ante-mortem rest-activity rhythms were associated with lower amplitude DNA methylation rhythms as were older age and the presence of Alzheimer's disease. These findings support the hypothesis that 24-hour rhythms of DNA methylation, particularly near transcription start sites, may play a role in driving 24-hour rhythms of gene expression in the human dorsolateral prefrontal cortex, and may be affected by age and Alzheimer's disease. PMID:25375876

Lim, Andrew S P; Srivastava, Gyan P; Yu, Lei; Chibnik, Lori B; Xu, Jishu; Buchman, Aron S; Schneider, Julie A; Myers, Amanda J; Bennett, David A; De Jager, Philip L

2014-11-01

272

H-NS Can Facilitate Specific DNA-binding by RNA Polymerase in AT-rich Gene Regulatory Regions  

PubMed Central

Extremely AT-rich DNA sequences present a challenging template for specific recognition by RNA polymerase. In bacteria, this is because the promoter ?10 hexamer, the major DNA element recognised by RNA polymerase, is itself AT-rich. We show that Histone-like Nucleoid Structuring (H-NS) protein can facilitate correct recognition of a promoter by RNA polymerase in AT-rich gene regulatory regions. Thus, at the Escherichia coli ehxCABD operon, RNA polymerase is unable to distinguish between the promoter ?10 element and similar overlapping sequences. This problem is resolved in native nucleoprotein because the overlapping sequences are masked by H-NS. Our work provides mechanistic insight into nucleoprotein structure and its effect on protein-DNA interactions in prokaryotic cells. PMID:23818873

Singh, Shivani S.; Grainger, David C.

2013-01-01

273

Transcription in methanogens. Evidence for specific in vitro transcription of the purified DNA-dependent RNA polymerase of Methanococcus thermolithotrophicus.  

PubMed

The purification of the DNA-dependent RNA polymerase of Methanococcus thermolithotrophicus is described. As the first step of purification the endogenous template was removed from the enzyme by hydrophobic interaction chromatography. The purified enzyme consists of seven components with different molecular masses. Transcription studies on T7 DNA and the recombinant plasmid pMV15, containing rRNA genes of Methanococcus vannielii, revealed that only the methanogen DNA is transcribed specifically, indicating a principal structural difference between archaebacterial and eubacterial promoters. This could be shown both by analysis of ternary transcription complexes and Southern hybridization. The site of initiation was found within a restriction fragment harbouring the first 390 nucleotides of the sequence coding for mature 16S rRNA and 1100 base pairs of upstream sequences. The specific initiation on this fragment strongly suggests that the enzyme can start in vitro transcription from the promoter(s) of rRNA synthesis. PMID:3996411

Thomm, M; Stetter, K O

1985-06-01

274

Impacts of Pretranscriptional DNA Methylation, Transcriptional Transcription Factor, and Posttranscriptional microRNA Regulations on Protein Evolutionary Rate  

PubMed Central

Gene expression is largely regulated by DNA methylation, transcription factor (TF), and microRNA (miRNA) before, during, and after transcription, respectively. Although the evolutionary effects of TF/miRNA regulations have been widely studied, evolutionary analysis of simultaneously accounting for DNA methylation, TF, and miRNA regulations and whether promoter methylation and gene body (coding regions) methylation have different effects on the rate of gene evolution remain uninvestigated. Here, we compared human–macaque and human–mouse protein evolutionary rates against experimentally determined single base-resolution DNA methylation data, revealing that promoter methylation level is positively correlated with protein evolutionary rates but negatively correlated with TF/miRNA regulations, whereas the opposite was observed for gene body methylation level. Our results showed that the relative importance of these regulatory factors in determining the rate of mammalian protein evolution is as follows: Promoter methylation ? miRNA regulation > gene body methylation > TF regulation, and further indicated that promoter methylation and miRNA regulation have a significant dependent effect on protein evolutionary rates. Although the mechanisms underlying cooperation between DNA methylation and TFs/miRNAs in gene regulation remain unclear, our study helps to not only illuminate the impact of these regulatory factors on mammalian protein evolution but also their intricate interaction within gene regulatory networks. PMID:24923326

Chuang, Trees-Juen; Chiang, Tai-Wei

2014-01-01

275

DNA damage triggers SAF-A and RNA biogenesis factors exclusion from chromatin coupled to R-loops removal  

PubMed Central

We previously identified the heterogeneous ribonucleoprotein SAF-A/hnRNP U as a substrate for DNA-PK, a protein kinase involved in DNA damage response (DDR). Using laser micro-irradiation in human cells, we report here that SAF-A exhibits a two-phase dynamics at sites of DNA damage, with a rapid and transient recruitment followed by a prolonged exclusion. SAF-A recruitment corresponds to its binding to Poly(ADP-ribose) while its exclusion is dependent on the activity of ATM, ATR and DNA-PK and reflects the dissociation from chromatin of SAF-A associated with ongoing transcription. Having established that SAF-A RNA-binding domain recapitulates SAF-A dynamics, we show that this domain is part of a complex comprising several mRNA biogenesis proteins of which at least two, FUS/TLS and TAFII68/TAF15, exhibit similar biphasic dynamics at sites of damage. Using an original reporter for live imaging of DNA:RNA hybrids (R-loops), we show a transient transcription-dependent accumulation of R-loops at sites of DNA damage that is prolonged upon inhibition of RNA biogenesis factors exclusion. We propose that a new component of the DDR is an active anti-R-loop mechanism operating at damaged transcribed sites which includes the exclusion of mRNA biogenesis factors such as SAF-A, FUS and TAF15. PMID:25030905

Britton, Sébastien; Dernoncourt, Emma; Delteil, Christine; Froment, Carine; Schiltz, Odile; Salles, Bernard; Frit, Philippe; Calsou, Patrick

2014-01-01

276

Simplified Identification of mRNA or DNA in Whole Cells  

NASA Technical Reports Server (NTRS)

A recently invented method of detecting a selected messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA) sequence offers two important advantages over prior such methods: it is simpler and can be implemented by means of compact equipment. The simplification and miniaturization achieved by this invention are such that this method is suitable for use outside laboratories, in field settings in which space and power supplies may be limited. The present method is based partly on hybridization of nucleic acid, which is a powerful technique for detection of specific complementary nucleic acid sequences and is increasingly being used for detection of changes in gene expression in microarrays containing thousands of gene probes.

Almeida, Eduardo; Kadambi, Geeta

2007-01-01

277

Continuous cell electroporation for efficient DNA and siRNA delivery based on laminar microfluidic chips.  

PubMed

Electroporation is a high-efficiency and low-toxicity physical gene transfer method. Traditional electroporation is limited to only low volume cell samples. Here we present a continuous cell electroporation method based on commonly used microfluidic chip fabrication technology. Using easily fabricated PDMS microfluidic chip, syringe pumps, and pulse generator, we show efficient delivery of both DNA and siRNA into different cell lines. We describe the protocol of chip fabrication, apparatus setup, and cell electroporation assay. Typically, the fabrication of the devices takes 1 or 2 days and the continuous electroporation assay takes 1 h. PMID:24510815

Wei, Zewen; Li, Zhihong

2014-01-01

278

Ab initio determination of the electron affinities of DNA and RNA nucleobases  

NASA Astrophysics Data System (ADS)

High-level quantum-chemical ab initio coupled-cluster and multiconfigurational perturbation methods have been used to compute the vertical and adiabatic electron affinities of the five canonical DNA and RNA nucleobases: uracil, thymine, cytosine, adenine, and guanine. The present results aim for the accurate determination of the intrinsic electron acceptor properties of the isolated nucleic acid bases as described by their electron affinities, establishing an overall set of theoretical reference values at a level not reported before and helping to rule out less reliable theoretical and experimental data and to calibrate theoretical strategies.

Roca-Sanjuán, Daniel; Merchán, Manuela; Serrano-Andrés, Luis; Rubio, Mercedes

2008-09-01

279

RNA Initiation with Dinucleoside Monophosphates during Transcription of Bacteriophage T4 DNA with RNA Polymerase of Escherichia coli  

PubMed Central

The effects of dinucleoside monophosphates on the transcription of phage T4 DNA by E. coli RNA polymerase have been examined at various concentrations of the sigma subunit and extremely low concentration of ribonucleoside triphosphate. The following conclusions were reached: (i) Labeled specific dinucleoside monophosphates are incorporated as chain initiators. (ii) When the ratio of sigma factor to core enzyme is small, there is a general stimulation by most 5?-guanosyl dinucleoside monophosphates. (iii) When the ratio is increased or holoenzyme is present, ApU, CpA, UpA, and GpU are the most effective stimulators. (iv) At high concentrations of sigma factor, only certain adenosine-containing dinucleoside monophosphates (ApU, CpA, UpA, and ApA) stimulate the reaction. (v) Competition hybridization studies indicate that the RNAs stimulated by dinucleoside monophosphates (ApU, CpA, UpA, and GpU) are of the T4 “early” type. (vi) Studies involving both combinations of stimulatory dinucleoside monophosphates and competitive effects of these compounds on chain initiation by ATP and GTP suggest that the stimulatory dinucleoside monophosphates act as chain initiators and may recognize part of a continuous sequence in a promoter region. Studies based on the incorporation of 3H-labeled stimulatory dinucleoside monophosphates support the above conclusions. PMID:4568732

Hoffman, David J.; Niyogi, Salil K.

1973-01-01

280

Structural mimicry in transcription regulation of human RNA polymerase II by the DNA helicase RECQL5  

PubMed Central

RECQL5 is a member of the highly conserved RecQ family of DNA helicases involved in DNA repair. RECQL5 interacts with RNA polymerase II (Pol II) and inhibits transcription of protein–coding genes by an unknown mechanism. We show that RECQL5 contacts the Rpb1 jaw domain of Pol II at a site that overlaps with the binding site for the transcription elongation factor TFIIS. Our cryo–electron microscopy structure of elongating Pol II arrested in complex with RECQL5 shows that the RECQL5 helicase domain is positioned to sterically block elongation. The crystal structure of the RECQL5 KIX domain reveals similarities with TFIIS, and binding of RECQL5 to Pol II interferes with the ability of TFIIS to promote transcriptional read–through in vitro. Together, our findings reveal a dual mode of transcriptional repression by RECQL5 that includes structural mimicry of the Pol II–TFIIS interaction. PMID:23748380

Kassube, Susanne A.; Jinek, Martin; Fang, Jie; Tsutakawa, Susan; Nogales, Eva

2013-01-01

281

Oxidative Stress Diverts tRNA Synthetase to Nucleus for Protection against DNA Damage.  

PubMed

Tyrosyl-tRNA synthetase (TyrRS) is known for its essential aminoacylation function in protein synthesis. Here we report a function for TyrRS in DNA damage protection. We found that oxidative stress, which often downregulates protein synthesis, induces TyrRS to rapidly translocate from the cytosol to the nucleus. We also found that angiogenin mediates or potentiates this stress-induced translocalization. The nuclear-localized TyrRS activates transcription factor E2F1 to upregulate the expression of DNA damage repair genes such as BRCA1 and RAD51. The activation is achieved through direct interaction of TyrRS with TRIM28 to sequester this vertebrate-specific epigenetic repressor and its associated HDAC1 from deacetylating and suppressing E2F1. Remarkably, overexpression of TyrRS strongly protects against UV-induced DNA double-strand breaks in zebrafish, whereas restricting TyrRS nuclear entry completely abolishes the protection. Therefore, oxidative stress triggers an essential cytoplasmic enzyme used for protein synthesis to translocate to the nucleus to protect against DNA damage. PMID:25284223

Wei, Na; Shi, Yi; Truong, Lan N; Fisch, Kathleen M; Xu, Tao; Gardiner, Elisabeth; Fu, Guangsen; Hsu, Yun-Shiuan Olivia; Kishi, Shuji; Su, Andrew I; Wu, Xiaohua; Yang, Xiang-Lei

2014-10-23

282

DNA sequence-directed nucleosome reconstitution on 5S RNA genes of Xenopus laevis.  

PubMed Central

Nucleosomes were reconstituted in vitro with several singly end-labeled restriction fragments derived from a cloned somatic-type 5S RNA gene of Xenopus laevis and purified nucleosome core particles from Xenopus cultured cells or chicken erythrocytes. Nucleosome locations were determined by digestion of the reconstitutes with exonuclease III and DNase I and were the same for all fragments investigated, extending from 20 base pairs (bp) within the 5S gene to 80 bp beyond the 3' end of the gene. Both core particles and crude nuclear extracts gave equivalent results, suggesting that no factors other than the core histones are responsible for recognition of DNA sequence during reconstitution. The histone octamer and the 5S gene-specific transcription factor TFIIIA both bind to the same region and face of 5S DNA, and nucleosome reconstitution on the 5S gene excluded binding of TFIIIA. The helical repeat of somatic-type 5S DNA in solution was measured by the band shift method and was 10.5 to 10.6 bp per turn over the region of the TFIIIA-binding site. The difference in helical repeat between DNA in solution and on the surface of the nucleosome (10.0-bp spacing between DNase I cutting sites) may explain the linking number paradox. Images PMID:3600640

Gottesfeld, J M

1987-01-01

283

Unlocked nucleic acids: implications of increased conformational flexibility for RNA/DNA triplex formation.  

PubMed

Unlocked nucleic acids (UNAs) have been introduced at specific positions in short model DNA hairpins and RNA/DNA triplexes for the first time. UNA residues destabilize the hairpins and decrease triplex thermodynamic stability or suppress triplex formation for most of the evaluated structures. Nevertheless, the incorporation of UNA residues at certain positions of dsDNA was found to be energetically favourable or at least did not affect triplex stability. Notably, the most thermodynamically stable UNA-modified triplexes exhibited improved stability at both acidic and physiological pH. The specificity of the interactions between the triplex-forming oligonucleotide and dsDNA was characterized using EMSA for the most thermodynamically stable structures, and triplex dissociation constants were determined. One of the modified triplexes exhibited an improved Kd in comparison with the unmodified triplex. CD and thermal difference spectra indicated that UNA residues do not alter the overall structure of the most thermodynamically stable triplexes. In addition, incubation of the modified oligonucleotides with human serum indicated that the UNAs demonstrate the potential to improve the biological stability of nucleic acids. PMID:25226286

Kotkowiak, Weronika; Kotkowiak, Micha?; Kierzek, Ryszard; Pasternak, Anna

2014-12-01

284

HPV mRNA Is More Specific than HPV DNA in Triage of Women with Minor Cervical Lesions  

PubMed Central

Background In Norway, repeat cytology and HPV testing comprise delayed triage of women with minor cytological lesions. The objective of this study was to evaluate HPV DNA and HPV mRNA testing in triage of women with an ASC-US/LSIL diagnosis. Materials and Methods We used repeat cytology, HPV DNA testing (Cobas 4800) and HPV mRNA testing (PreTect HPV-Proofer) to follow up 311 women aged 25–69 years with ASC-US/LSIL index cytology. Results Of 311 women scheduled for secondary screening, 30 women (9.6%) had ASC-H/HSIL cytology at triage and 281 women (90.4%) had ASC-US/LSIL or normal cytology. The HPV DNA test was positive in 92 (32.7%) of 281 instances, and 37 (13.2%) were mRNA positive. Of the 132 women with repeated ASC-US/LSIL, we received biopsies from 97.0% (65/67) of the DNA-positive and 92.9% (26/28) of the mRNA-positive cases. The positive predictive values for CIN2+ were 21.5% (14/65) for DNA positive and 34.6% (9/26) for mRNA positive (ns). The odds ratio for being referred to colposcopy in DNA-positive cases were 2.8 times (95% CI: 1.8–4.6) higher that of mRNA-positive cases. Compared to the mRNA test, the DNA test detected four more cases of CIN2 and one case of CIN3. Conclusions The higher positivity rate of the DNA test in triage leads to higher referral rate for colposcopy and biopsy, and subsequent additional follow-up of negative biopsies. By following mRNA-negative women who had ASC-US/LSIL at triage with cytology, the additional cases of CIN2+ gained in DNA screening can be discovered. Our study indicates that in triage of repeated ASC-US/LSIL, HPV mRNA testing is more specific and is more relevant in clinical use than an HPV DNA test. PMID:25405981

Sørbye, Sveinung Wergeland; Fismen, Silje; Gutteberg, Tore Jarl; Mortensen, Elin Synnøve; Skjeldestad, Finn Egil

2014-01-01

285

Accurate quantification of hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-DNA technology.  

PubMed Central

In studies monitoring disease progression and therapeutic response, it is essential that the method used for hepatitis C virus (HCV) quantification not be influenced by genotypic variability. The branched DNA assay provides a reliable method for the quantification of HCV RNA. A modified set of oligonucleotide probes for the branched DNA assay was developed to enhance the efficiency of binding to genotypic variants of HCV. The improved branched DNA assay (HCV RNA 2.0) yielded highly reproducible quantification of hepatitis C virus RNA and displayed a nearly 600-fold dynamic range in quantification up to 120 Meq of HCV RNA per ml. The quantification limit was set at 0.2 Meg of HCV RNA per ml to ensure a specificity of > or = 95%. With this lowered quantification limit and the enhanced hybridization of the probes, the HCV RNA 2.0 assay exhibited a high level of sensitivity (96%) and was virtually unaffected by the genotypic variability of HCV. The HCV RNA 2.0 assay may be a useful tool for following HCV RNA levels throughout the course of disease, selecting patients for therapy, and evaluating therapeutic response. PMID:8815105

Detmer, J; Lagier, R; Flynn, J; Zayati, C; Kolberg, J; Collins, M; Urdea, M; Sanchez-Pescador, R

1996-01-01

286

TFIIH-mediated nucleotide excision repair and initiation of mRNA transcription in an optimized cell-free DNA repair and RNA transcription assay  

Microsoft Academic Search

In mammalian cells, mRNA transcription is initiated with the aid of transcription initiation factors. Of these, TFIIH has also been shown to play an essential role in nucleotide excision repair (NER), which is a versatile biochemical pathway that corrects a broad range of DNA damage. Since the dual role of TFIIH is conserved among eukaryotes, including yeast and mammalian cells,

Masahiko S. Satoh; Philip C. Hanawalt

1996-01-01

287

Genome-Wide Distribution of RNA-DNA Hybrids Identifies RNase H Targets in tRNA Genes, Retrotransposons and Mitochondria  

PubMed Central

During transcription, the nascent RNA can invade the DNA template, forming extended RNA-DNA duplexes (R-loops). Here we employ ChIP-seq in strains expressing or lacking RNase H to map targets of RNase H activity throughout the budding yeast genome. In wild-type strains, R-loops were readily detected over the 35S rDNA region, transcribed by Pol I, and over the 5S rDNA, transcribed by Pol III. In strains lacking RNase H activity, R-loops were elevated over other Pol III genes, notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5?-flanking regions of tRNA genes. Unexpectedly, R-loops were also associated with mitochondrial genes in the absence of RNase H1, but not of RNase H2. Finally, R-loops were detected on actively transcribed protein-coding genes in the wild-type, particularly over the second exon of spliced ribosomal protein genes. PMID:25357144

El Hage, Aziz; Webb, Shaun; Kerr, Alastair; Tollervey, David

2014-01-01

288

The Origins of the RNA World  

PubMed Central

The general notion of an “RNA World” is that, in the early development of life on the Earth, genetic continuity was assured by the replication of RNA and genetically encoded proteins were not involved as catalysts. There is now strong evidence indicating that an RNA World did indeed exist before DNA- and protein-based life. However, arguments regarding whether life on Earth began with RNA are more tenuous. It might be imagined that all of the components of RNA were available in some prebiotic pool, and that these components assembled into replicating, evolving polynucleotides without the prior existence of any evolved macromolecules. A thorough consideration of this “RNA-first” view of the origin of life must reconcile concerns regarding the intractable mixtures that are obtained in experiments designed to simulate the chemistry of the primitive Earth. Perhaps these concerns will eventually be resolved, and recent experimental findings provide some reason for optimism. However, the problem of the origin of the RNA World is far from being solved, and it is fruitful to consider the alternative possibility that RNA was preceded by some other replicating, evolving molecule, just as DNA and proteins were preceded by RNA. PMID:20739415

Robertson, Michael P; Joyce, Gerald F

2012-01-01

289

Structural analysis of monomeric retroviral reverse transcriptase in complex with an RNA/DNA hybrid  

PubMed Central

A key step in proliferation of retroviruses is the conversion of their RNA genome to double-stranded DNA, a process catalysed by multifunctional reverse transcriptases (RTs). Dimeric and monomeric RTs have been described, the latter exemplified by the enzyme of Moloney murine leukaemia virus. However, structural information is lacking that describes the substrate binding mechanism for a monomeric RT. We report here the first crystal structure of a complex between an RNA/DNA hybrid substrate and polymerase-connection fragment of the single-subunit RT from xenotropic murine leukaemia virus-related virus, a close relative of Moloney murine leukaemia virus. A comparison with p66/p51 human immunodeficiency virus-1 RT shows that substrate binding around the polymerase active site is conserved but differs in the thumb and connection subdomains. Small-angle X-ray scattering was used to model full-length xenotropic murine leukaemia virus-related virus RT, demonstrating that its mobile RNase H domain becomes ordered in the presence of a substrate—a key difference between monomeric and dimeric RTs. PMID:23382176

Nowak, El?bieta; Potrzebowski, Wojciech; Konarev, Petr V.; Rausch, Jason W.; Bona, Marion K.; Svergun, Dmitri I.; Bujnicki, Janusz M.; Le Grice, Stuart F. J.; Nowotny, Marcin

2013-01-01

290

ICR Noncoding RNA Expression Controls Imprinting and DNA Replication at the Dlk1-Dio3 Domain.  

PubMed

Imprinted genes play essential roles in development, and their allelic expression is mediated by imprinting control regions (ICRs). The Dlk1-Dio3 locus is among the few imprinted domains controlled by a paternally methylated ICR. The unmethylated maternal copy activates imprinted expression early in development through an unknown mechanism. We find that in mouse embryonic stem cells (ESCs) and in blastocysts, this function is linked to maternal, bidirectional expression of noncoding RNAs (ncRNAs) from the ICR. Disruption of ICR ncRNA expression in ESCs affected gene expression in cis, led to acquisition of aberrant histone and DNA methylation, delayed replication timing along the domain on the maternal chromosome, and changed its subnuclear localization. The epigenetic alterations persisted during differentiation and affected the neurogenic potential of the stem cells. Our data indicate that monoallelic expression at an ICR of enhancer RNA-like ncRNAs controls imprinted gene expression, epigenetic maintenance processes, and DNA replication in embryonic cells. PMID:25263792

Kota, Satya K; Llères, David; Bouschet, Tristan; Hirasawa, Ryutaro; Marchand, Alice; Begon-Pescia, Christina; Sanli, Ildem; Arnaud, Philippe; Journot, Laurent; Girardot, Michael; Feil, Robert

2014-10-13

291

A novel microRNA assay with optical detection and enzyme-free DNA circuits  

NASA Astrophysics Data System (ADS)

MicroRNAs (miRNAs) participate in the significant processes of life course, can be used as quantificational biomarkers for cellular level researches and related diseases. Conventional methods of miRNAs' quantitative detection are obsessed with low sensitivity, time and labour consuming. Otherwise, the emerging miRNAs detection approaches are mostly exposed to the expensive equipment demands and the professional operation, remains at the stage of laboratory and concept demonstration phase. Herein, we designed a novel miRNAs detection platform that based on enzyme-free DNA circuits and electrochemical luminescence (ECL). MicroRNA21 was chosen as the ideal analyte of this platform. The whole process consists of enzyme-free DNA circuits and ECL signal giving-out steps, achieves advantages of operating in constant temperature condition, without the participation of the enzyme, preferable sensitivity and specificity. Through this approach, the sensitivity achieved at 10pM. It is indicated that this miRNAs detection platform possesses potentials to be an innovation of miRNA detection technologies in routine tests. Benefits of the high penetration of ECL in well-equipped medical establishment, this approach could greatly lessen the obstacles in process of popularization and possess excellent prospects of practical application.

Liao, Yuhui; Zhou, Xiaoming

2014-09-01

292

Identification of active denitrifiers in rice paddy soil by DNA- and RNA-based analyses.  

PubMed

Denitrification occurs markedly in rice paddy fields; however, few microbes that are actively involved in denitrification in these environments have been identified. In this study, we used a laboratory soil microcosm system in which denitrification activity was enhanced. DNA and RNA were extracted from soil at six time points after enhancing denitrification activity, and quantitative PCR and clone library analyses were performed targeting the 16S rRNA gene and denitrification functional genes (nirS, nirK and nosZ) to clarify which microbes are actively involved in denitrification in rice paddy soil. Based on the quantitative PCR results, transcription levels of the functional genes agreed with the denitrification activity, although gene abundance did not change at the DNA level. Diverse denitrifiers were detected in clone library analysis, but comparative analysis suggested that only some of the putative denitrifiers, especially those belonging to the orders Neisseriales, Rhodocyclales and Burkholderiales, were actively involved in denitrification in rice paddy soil. PMID:22972387

Yoshida, Megumi; Ishii, Satoshi; Fujii, Daichi; Otsuka, Shigeto; Senoo, Keishi

2012-01-01

293

Identification of Active Denitrifiers in Rice Paddy Soil by DNA- and RNA-Based Analyses  

PubMed Central

Denitrification occurs markedly in rice paddy fields; however, few microbes that are actively involved in denitrification in these environments have been identified. In this study, we used a laboratory soil microcosm system in which denitrification activity was enhanced. DNA and RNA were extracted from soil at six time points after enhancing denitrification activity, and quantitative PCR and clone library analyses were performed targeting the 16S rRNA gene and denitrification functional genes (nirS, nirK and nosZ) to clarify which microbes are actively involved in denitrification in rice paddy soil. Based on the quantitative PCR results, transcription levels of the functional genes agreed with the denitrification activity, although gene abundance did not change at the DNA level. Diverse denitrifiers were detected in clone library analysis, but comparative analysis suggested that only some of the putative denitrifiers, especially those belonging to the orders Neisseriales, Rhodocyclales and Burkholderiales, were actively involved in denitrification in rice paddy soil. PMID:22972387

Yoshida, Megumi; Ishii, Satoshi; Fujii, Daichi; Otsuka, Shigeto; Senoo, Keishi

2012-01-01

294

Structural analysis of monomeric retroviral reverse transcriptase in complex with an RNA/DNA hybrid.  

PubMed

A key step in proliferation of retroviruses is the conversion of their RNA genome to double-stranded DNA, a process catalysed by multifunctional reverse transcriptases (RTs). Dimeric and monomeric RTs have been described, the latter exemplified by the enzyme of Moloney murine leukaemia virus. However, structural information is lacking that describes the substrate binding mechanism for a monomeric RT. We report here the first crystal structure of a complex between an RNA/DNA hybrid substrate and polymerase-connection fragment of the single-subunit RT from xenotropic murine leukaemia virus-related virus, a close relative of Moloney murine leukaemia virus. A comparison with p66/p51 human immunodeficiency virus-1 RT shows that substrate binding around the polymerase active site is conserved but differs in the thumb and connection subdomains. Small-angle X-ray scattering was used to model full-length xenotropic murine leukaemia virus-related virus RT, demonstrating that its mobile RNase H domain becomes ordered in the presence of a substrate-a key difference between monomeric and dimeric RTs. PMID:23382176

Nowak, Elzbieta; Potrzebowski, Wojciech; Konarev, Petr V; Rausch, Jason W; Bona, Marion K; Svergun, Dmitri I; Bujnicki, Janusz M; Le Grice, Stuart F J; Nowotny, Marcin

2013-04-01

295

Integrative DNA, RNA, and protein evidence connects TREML4 to coronary artery calcification.  

PubMed

Coronary artery calcification (CAC) is a heritable and definitive morphologic marker of atherosclerosis that strongly predicts risk for future cardiovascular events. To search for genes involved in CAC, we used an integrative transcriptomic, genomic, and protein expression strategy by using next-generation DNA sequencing in the discovery phase with follow-up studies using traditional molecular biology and histopathology techniques. RNA sequencing of peripheral blood from a discovery set of CAC cases and controls was used to identify dysregulated genes, which were validated by ClinSeq and Framingham Heart Study data. Only a single gene, TREML4, was upregulated in CAC cases in both studies. Further examination showed that rs2803496 was a TREML4 cis-eQTL and that the minor allele at this locus conferred up to a 6.5-fold increased relative risk of CAC. We characterized human TREML4 and demonstrated by immunohistochemical techniques that it is localized in macrophages surrounding the necrotic core of coronary plaques complicated by calcification (but not in arteries with less advanced disease). Finally, we determined by von Kossa staining that TREML4 colocalizes with areas of microcalcification within coronary plaques. Overall, we present integrative RNA, DNA, and protein evidence implicating TREML4 in coronary artery calcification. Our findings connect multimodal genomics data with a commonly used clinical marker of cardiovascular disease. PMID:24975946

Sen, Shurjo K; Boelte, Kimberly C; Barb, Jennifer J; Joehanes, Roby; Zhao, XiaoQing; Cheng, Qi; Adams, Lila; Teer, Jamie K; Accame, David S; Chowdhury, Soma; Singh, Larry N; Kavousi, Maryam; Peyser, Patricia A; Quigley, Laura; Priel, Debra Long; Lau, Karen; Kuhns, Douglas B; Yoshimura, Teizo; Johnson, Andrew D; Hwang, Shih-Jen; Chen, Marcus Y; Arai, Andrew E; Green, Eric D; Mullikin, James C; Kolodgie, Frank D; O'Donnell, Christopher J; Virmani, Renu; Munson, Peter J; McVicar, Daniel W; Biesecker, Leslie G

2014-07-01

296

A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response  

PubMed Central

Invertebrate RNA viruses are targets of the host RNA interference (RNAi) pathway, which limits virus infection by degrading viral RNA substrates. Several insect RNA viruses encode suppressor proteins to counteract this antiviral response. We recently demonstrated that the dsDNA virus Invertebrate iridescent virus 6 (IIV-6) induces an RNAi response in Drosophila. Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R. Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs). We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex. Finally, we show that 340R is able to rescue a Flock House virus replicon that lacks its viral suppressor of RNAi. Together, our findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response. PMID:25274730

Bronkhorst, Alfred W.; van Cleef, Koen W.R.; Venselaar, Hanka; van Rij, Ronald P.

2014-01-01

297

Development of a Novel Self-Enclosed Sample Preparation Device for DNA/RNA Isolation in Space  

NASA Technical Reports Server (NTRS)

Modern biology techniques present potentials for a wide range of molecular, cellular, and biochemistry applications in space, including detection of infectious pathogens and environmental contaminations, monitoring of drug-resistant microbial and dangerous mutations, identification of new phenotypes of microbial and new life species. However, one of the major technological blockades in enabling these technologies in space is a lack of devices for sample preparation in the space environment. To overcome such an obstacle, we constructed a prototype of a DNA/RNA isolation device based on our novel designs documented in the NASA New Technology Reporting System (MSC-24811-1/3-1). This device is self-enclosed and pipette free, purposely designed for use in the absence of gravity. Our design can also be modified easily for preparing samples in space for other applications, such as flowcytometry, immunostaining, cell separation, sample purification and separation according to its size and charges, sample chemical labeling, and sample purification. The prototype of our DNA/RNA isolation device was tested for efficiencies of DNA and RNA isolation from various cell types for PCR analysis. The purity and integrity of purified DNA and RNA were determined as well. Results showed that our developed DNA/RNA isolation device offers similar efficiency and quality in comparison to the samples prepared using the standard protocol in the laboratory.

Zhang, Ye; Mehta, Satish K.; Pensinger, Stuart J.; Pickering, Karen D.

2011-01-01

298

Characterization of Damage to Bacteria and Bio-macromolecules Caused by (V)UV Radiation and Particles Generated by a Microscale Atmospheric Pressure Plasma Jet  

NASA Astrophysics Data System (ADS)

Atmospheric pressure plasma jets effectively inactivate bacteria on ­surfaces including infected tissues. This is due to the combined effects of (V)UV radiation, reactive oxygen and nitrogen species, ions, and high electric fields. A well-characterized microscale atmospheric pressure plasma jet (?-APPJ) operated with He/O2 gas mixture has been modified so that (V)UV radiation and heavy reactive particles (mainly O3 molecules and O atoms) emitted from the plasma source can be separated effectively. The separation is achieved by an additional lateral He flow, which diverts the heavy particles from the jet axis. The new jet geometry is called X-Jet. Separation of different plasma components allows studying their effects on living cells and bio-macromolecules separately. First, the effectiveness of the separation of different plasma components was demonstrated by treatment of monolayers of vegetative Bacillus subtilis cells. To characterize effects on nucleic acids, dried plasmid DNA and total cellular RNA were treated with the separated plasma components. Dried bovine serum albumin was used to study etching effects of (V)UV radiation and heavy particles on proteins. We found that heavy particles emitted from the X-Jet kill vegetative cells more effectively than the (V)UV radiation from this type of plasma source. All bio-macromolecules investigated, DNA, RNA, and proteins, are affected by plasma treatment. DNA exposed to the (V)UV-channel of the jet seems to be prone to thymine dimer formation not only in vitro but also in vivo as indicated by induction of the photolyase in Escherichia coli, while DNA strand breaks occur under both jet channels. Heavy particles seem more effective in degrading RNA and in etching protein in vitro.

Lackmann, Jan-Wilm; Schneider, Simon; Narberhaus, Franz; Benedikt, Jan; Bandow, Julia E.

299

Transduction of RNA-directed DNA methylation signals to repressive histone marks in Arabidopsis thaliana  

PubMed Central

RNA-directed modification of histones is essential for the maintenance of heterochromatin in higher eukaryotes. In plants, cytosine methylation is an additional factor regulating inactive chromatin, but the mechanisms regulating the coexistence of cytosine methylation and repressive histone modification remain obscure. In this study, we analysed the mechanism of gene silencing mediated by MORPHEUS' MOLECULE1 (MOM1) of Arabidopsis thaliana. Transcript profiling revealed that the majority of up-regulated loci in mom1 carry sequences related to transposons and homologous to the 24-nt siRNAs accumulated in wild-type plants that are the hallmarks of RNA-directed DNA methylation (RdDM). Analysis of a single-copy gene, SUPPRESSOR OF drm1 drm2 cmt3 (SDC), revealed that mom1 activates SDC with concomitant reduction of di-methylated histone H3 lysine 9 (H3K9me2) at the tandem repeats in the promoter region without changes in siRNA accumulation and cytosine methylation. The reduction of H3K9me2 is not observed in regions flanking the tandem repeats. The results suggest that MOM1 transduces RdDM signals to repressive histone modification in the core region of RdDM. PMID:20010696

Numa, Hisataka; Kim, Jong-Myong; Matsui, Akihiro; Kurihara, Yukio; Morosawa, Taeko; Ishida, Junko; Mochizuki, Yoshiki; Kimura, Hiroshi; Shinozaki, Kazuo; Toyoda, Tetsuro; Seki, Motoaki; Yoshikawa, Manabu; Habu, Yoshiki

2010-01-01

300

MicroRNA-31 suppresses medulloblastoma cell growth by inhibiting DNA replication through minichromosome maintenance 2  

PubMed Central

Medulloblastoma is an aggressive childhood brain tumor with poor prognosis. Recent studies indicate that dys-regulation of microRNA expression plays important roles in tumorigenesis. By comparing microRNA levels between mouse medulloblastoma and normal cerebellar tissues, we identified a set of down-regulated microRNAs including miR-31. Here, we show that the genomic region surrounding human miR-31 at 9p21.3 is frequently deleted in many solid tumor cell lines, and reintroducing miR-31 into DAOY cells, a line of human medulloblastoma cells devoid of miR-31, strongly suppresses cell growth, causes cell cycle arrest at the G1/S boundary, and inhibits colony formation in vitro and xenograft tumorigenesis in nude mice. Global gene expression profiling of mouse medulloblastomas and bioinformatics analyses of microRNA targets suggest that minichromosome maintenance complex component 2 (MCM2) is a likely target gene of miR-31 in suppressing cell growth. We demonstrate that miR-31 inhibits MCM2 expression via its 3'-untranslated region, that knockdown of MCM2 in DAOY cells leads to a degree of growth inhibition comparable to that by miR-31 restoration, and that overexpression of miR-31 reduces the chromatin loading of MCM2 at the point of G1/S transition. Taken together, these data indicate that miR-31 suppresses medulloblastoma tumorigenesis by negatively regulating DNA replication via MCM2. PMID:24970811

Zhang, Ziyu; Li, Sanen; Huang, Huijie; Yu, Ting-ting; Cao, Xiumei; Cheng, Steven Y.

2014-01-01

301

Artificial riboswitches for gene expression and replication control of DNA and RNA viruses.  

PubMed

Aptazymes are small, ligand-dependent self-cleaving ribozymes that function independently of transcription factors and can be customized for induction by various small molecules. Here, we introduce these artificial riboswitches for regulation of DNA and RNA viruses. We hypothesize that they represent universally applicable tools for studying viral gene functions and for applications as a safety switch for oncolytic and live vaccine viruses. Our study shows that the insertion of artificial aptazymes into the adenoviral immediate early gene E1A enables small-molecule-triggered, dose-dependent inhibition of gene expression. Aptazyme-mediated shutdown of E1A expression translates into inhibition of adenoviral genome replication, infectious particle production, and cytotoxicity/oncolysis. These results provide proof of concept for the aptazyme approach for effective control of biological outcomes in eukaryotic systems, specifically in virus infections. Importantly, we also demonstrate aptazyme-dependent regulation of measles virus fusion protein expression, translating into potent reduction of progeny infectivity and virus spread. This not only establishes functionality of aptazymes in fully cytoplasmic genetic systems, but also implicates general feasibility of this strategy for application in viruses with either DNA or RNA genomes. Our study implies that gene regulation by artificial riboswitches may be an appealing alternative to Tet- and other protein-dependent gene regulation systems, based on their small size, RNA-intrinsic mode of action, and flexibility of the inducing molecule. Future applications range from gene analysis in basic research to medicine, for example as a safety switch for new generations of efficiency-enhanced oncolytic viruses. PMID:24449891

Ketzer, Patrick; Kaufmann, Johanna K; Engelhardt, Sarah; Bossow, Sascha; von Kalle, Christof; Hartig, Jörg S; Ungerechts, Guy; Nettelbeck, Dirk M

2014-02-01

302

Bleomycin mediated degradation of DNA-RNA hybrids does not involve C-1' chemistry.  

PubMed Central

Incubation of Fe(II) bleomycin and O2 with a number of 'A'-like DNA-RNA hybrid homopolymers at 4 atm O2 results in formation of base propenal and base in a ratio of approximately 1.0:1.0. This ratio differs dramatically from the corresponding ratio of approximately 10:1.0 observed when activated BLM degrades 'B'-like DNA homopolymers. Experiments were undertaken to determine if the shift to enhanced base production observed in the A-like hybrids is the result of C-1' chemistry in addition to the C-4' chemistry normally observed with B-like DNA under identical conditions. Increased accessibility of the 1'-hydrogen might be anticipated due to widening of the minor groove in the A-like conformers. Experiments using poly([1'-3H]dA) poly(rU) and poly([U-14C]dA) poly(rU) indicated that neither 3H2O nor deoxyribonolactone accompanied adenine release. In addition, studies using poly([4'-2H]dA) poly(rU) and poly([1'-2H]dA) poly(rU) unambiguously establish that the altered base to base propenal ratio is not the result of C-1' chemistry, but a direct consequence of C-4' chemistry. PMID:1380696

Absalon, M J; Krishnamoorthy, C R; McGall, G; Kozarich, J W; Stubbe, J

1992-01-01

303

Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivo protein production in Bacillus megaterium.  

PubMed

Gene "7" of Escherichia coli phage K1E was proposed to encode a novel DNA-dependent RNA polymerase (RNAP). The corresponding protein was produced recombinantly, purified to apparent homogeneity via affinity chromatography, and successfully employed for in vitro RNA synthesis. Optimal assay conditions (pH 8, 37 degrees C, 10 mM magnesium chloride and 1.3 mM spermidine) were established. The corresponding promoter regions were identified on the phage genome and summarized in a sequence logo. Surprisingly, next to K1E promoters, the SP6 promoter was also recognized efficiently in vitro by K1E RNAP, while the T7 RNAP promoter was not recognized at all. Based on these results, a system for high-yield in vitro RNA synthesis using K1E RNAP was established. The template plasmid is a pUC18 derivative, which enables blue/white screening for positive cloning of the target DNA. Production of more than 5 microg of purified RNA per microgram plasmid DNA was achieved. Finally, in vivo protein production systems for Bacillus megaterium were established based on K1E and SP6 phage RNAP transcription. Up to 61.4 mg g (CDW) (-1) (K1E RNAP) of the reporter protein Gfp was produced in shaking flask cultures of B. megaterium. PMID:20596705

Stammen, Simon; Schuller, Franziska; Dietrich, Sylvia; Gamer, Martin; Biedendieck, Rebekka; Jahn, Dieter

2010-09-01

304

Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability  

PubMed Central

Summary Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through the assembly of multiple protein complexes involved in DNA repair, cell-cycle arrest, and transcriptional regulation. Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery. In response to DNA damage, this complex regulates pre-mRNA splicing of a number of genes involved in DNA damage signaling and repair, thereby promoting the stability of these transcripts/proteins. Further, we show that abrogation of this complex results in sensitivity to DNA damage, defective DNA repair, and genomic instability. Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types. These data suggest that regulation of splicing by the BRCA1-mRNA splicing complex plays an important role in the cellular response to DNA damage. PMID:24746700

Savage, Kienan I.; Gorski, Julia J.; Barros, Eliana M.; Irwin, Gareth W.; Manti, Lorenzo; Powell, Alexander J.; Pellagatti, Andrea; Lukashchuk, Natalia; McCance, Dennis J.; McCluggage, W. Glenn; Schettino, Giuseppe; Salto-Tellez, Manuel; Boultwood, Jacqueline; Richard, Derek J.; McDade, Simon S.; Harkin, D. Paul

2014-01-01

305

A major role for Tau in neuronal DNA and RNA protection in vivo under physiological and hyperthermic conditions  

PubMed Central

Nucleic acid protection is a substantial challenge for neurons, which are continuously exposed to oxidative stress in the brain. Neurons require powerful mechanisms to protect DNA and RNA integrity and ensure their functionality and longevity. Beside its well known role in microtubule dynamics, we recently discovered that Tau is also a key nuclear player in the protection of neuronal genomic DNA integrity under reactive oxygen species (ROS)-inducing heat stress (HS) conditions in primary neuronal cultures. In this report, we analyzed the capacity of Tau to protect neuronal DNA integrity in vivo in adult mice under physiological and HS conditions. We designed an in vivo mouse model of hyperthermia/HS to induce a transient increase in ROS production in the brain. Comet and Terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assays demonstrated that Tau protected genomic DNA in adult cortical and hippocampal neurons in vivo under physiological conditions in wild-type (WT) and Tau-deficient (KO-Tau) mice. HS increased DNA breaks in KO-Tau neurons. Notably, KO-Tau hippocampal neurons in the CA1 subfield restored DNA integrity after HS more weakly than the dentate gyrus (DG) neurons. The formation of phosphorylated histone H2AX foci, a double-strand break marker, was observed in KO-Tau neurons only after HS, indicating that Tau deletion did not trigger similar DNA damage under physiological or HS conditions. Moreover, genomic DNA and cytoplasmic and nuclear RNA integrity were altered under HS in hippocampal neurons exhibiting Tau deficiency, which suggests that Tau also modulates RNA metabolism. Our results suggest that Tau alterations lead to a loss of its nucleic acid safeguarding functions and participate in the accumulation of DNA and RNA oxidative damage observed in the Alzheimer’s disease (AD) brain. PMID:24672431

Violet, Marie; Delattre, Lucie; Tardivel, Meryem; Sultan, Audrey; Chauderlier, Alban; Caillierez, Raphaelle; Talahari, Smail; Nesslany, Fabrice; Lefebvre, Bruno; Bonnefoy, Eliette; Buee, Luc; Galas, Marie-Christine

2014-01-01

306

A DNA-dependent RNA synthesis by wheat-germ RNA polymerase II insensitive to the fungal toxin alpha-amanitin.  

PubMed Central

Wheat-germ RNA polymerase II is able to catalyse a DNA-dependent reaction of RNA synthesis in the presence of a high concentration (1 mg/ml) of the fungal toxin alpha-amanitin. This anomalous reaction is specifically directed by single-stranded or double-stranded homopolymer templates, such as poly(dC) or poly(dC).poly(dG), and occurs in the presence of either Mn2+ or Mg2+ as the bivalent metal cofactor. In contrast, the transcription of other synthetic templates, such as poly(dT), poly(dA).poly(dT) or poly[d(A-T)] is completely abolished in the presence of 1 microgram of alpha-amanitin/ml, in agreement with well-established biochemical properties of class II RNA polymerases. Size analysis of reaction products resulting from transcription of (dC)n templates of defined lengths suggests that polymerization of RNA chains proceeds through a slippage mechanism. The fact that alpha-amanitin does not impede this synthetic reaction implies that the amatoxin interferes with the translocation of wheat-germ RNA polymerase II along the DNA template. Images Fig. 3. Fig. 4. PMID:1379042

Job, C; Shire, D; Sure, V; Job, D

1992-01-01

307

A DNA-dependent RNA synthesis by wheat-germ RNA polymerase II insensitive to the fungal toxin alpha-amanitin.  

PubMed

Wheat-germ RNA polymerase II is able to catalyse a DNA-dependent reaction of RNA synthesis in the presence of a high concentration (1 mg/ml) of the fungal toxin alpha-amanitin. This anomalous reaction is specifically directed by single-stranded or double-stranded homopolymer templates, such as poly(dC) or poly(dC).poly(dG), and occurs in the presence of either Mn2+ or Mg2+ as the bivalent metal cofactor. In contrast, the transcription of other synthetic templates, such as poly(dT), poly(dA).poly(dT) or poly[d(A-T)] is completely abolished in the presence of 1 microgram of alpha-amanitin/ml, in agreement with well-established biochemical properties of class II RNA polymerases. Size analysis of reaction products resulting from transcription of (dC)n templates of defined lengths suggests that polymerization of RNA chains proceeds through a slippage mechanism. The fact that alpha-amanitin does not impede this synthetic reaction implies that the amatoxin interferes with the translocation of wheat-germ RNA polymerase II along the DNA template. PMID:1379042

Job, C; Shire, D; Sure, V; Job, D

1992-07-01

308

Size separation of macromolecules during spreading.  

PubMed

Spreading of homogeneous mixtures of bottle-brush and linear macromolecules of poly(n-butylacrylate) on a solid substrate has been monitored on the molecular scale by atomic force microscopy. Despite the nearly identical chemical composition and similar molecular weight, brush-like macromolecules move markedly slower than linear chains. Moreover, smaller bottle-brushes have been shown to flow faster than the larger bottle-brushes, resulting in fractionation of the macromolecules along the spreading direction. This behavior was explained by the difference in sliding friction coefficient between the bottle-brush macromolecules and linear chains with the substrate. A theoretical model of molecular size separation is in a good agreement with experimental data. PMID:20839779

Barrett, Michael J; Sun, Frank C; Nese, Alper; Matyjaszewski, Krzysztof; Carrillo, Jan-Michael Y; Dobrynin, Andrey V; Sheiko, Sergei S

2010-10-01

309

Nanomechanics of cartilage extracellular matrix macromolecules  

E-print Network

In this thesis, the shear and self-adhesion nanomechanical properties between opposing cartilage aggrecan macromolecules were probed. In addition, nanoscale dynamic oscillatory mechanical properties of cartilage and its ...

Han, Lin, Ph. D. Massachusetts Institute of Technology

2007-01-01

310

Putative DNA/RNA helicase Schlafen-11 (SLFN11) sensitizes cancer cells to DNA-damaging agents  

PubMed Central

DNA-damaging agents (DDAs) constitute the backbone of treatment for most human tumors. Here we used the National Cancer Institute Antitumor Cell Line Panel (the NCI-60) to identify predictors of cancer cell response to topoisomerase I (Top1) inhibitors, a widely used class of DDAs. We assessed the NCI-60 transcriptome using Affymetrix Human Exon 1.0 ST microarrays and correlated the in vitro activity of four Top1 inhibitors with gene expression in the 60 cell lines. A single gene, Schlafen-11 (SLFN11), showed an extremely significant positive correlation with the response not only to Top1 inhibitors, but also to Top2 inhibitors, alkylating agents, and DNA synthesis inhibitors. Using cells with endogenously high and low SLFN11 expression and siRNA-mediated silencing, we show that SLFN11 is causative in determining cell death and cell cycle arrest in response to DDAs in cancer cells from different tissues of origin. We next analyzed SLFN11 expression in ovarian and colorectal cancers and normal corresponding tissues from The Cancer Genome Atlas database and observed that SLFN11 has a wide expression range. We also observed that high SLFN11 expression independently predicts overall survival in a group of ovarian cancer patients treated with cisplatin-containing regimens. We conclude that SLFN11 expression is causally associated with the activity of DDAs in cancer cells, has a broad expression range in colon and ovarian adenocarcinomas, and may behave as a biomarker for prediction of response to DDAs in the clinical setting. PMID:22927417

Zoppoli, Gabriele; Regairaz, Marie; Leo, Elisabetta; Reinhold, William C.; Varma, Sudhir; Ballestrero, Alberto; Doroshow, James H.; Pommier, Yves

2012-01-01

311

In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells.  

PubMed

Orf, which is caused by orf virus (ORFV), is distributed worldwide and is endemic in most sheep- and/or goat-raising countries. RNA interference (RNAi) pathways have emerged as important regulators of virus-host cell interactions. In this study, the specific effect of RNAi on the replication of ORFV was explored. The application of RNA interference (RNAi) inhibited the replication of ORFV in cell culture by targeting the ORF025 gene of ORFV, which encodes the viral polymerase. Three small interfering RNA (siRNA) (named siRNA704, siRNA1017 and siRNA1388) were prepared by in vitro transcription. The siRNAs were evaluated for antiviral activity against the ORFV Jilin isolate by the observation of cytopathic effects (CPE), virus titration, and real-time PCR. After 48 h of infection, siRNA704, siRNA1017 and siRNA1388 reduced virus titers by 59- to 199-fold and reduced the level of viral replication by 73-89 %. These results suggest that these three siRNAs can efficiently inhibit ORFV genome replication and infectious virus production. RNAi targeting of the DNA polymerase gene is therefore potentially useful for studying the replication of ORFV and may have potential therapeutic applications. PMID:24178308

Wang, Gaili; He, Wenqi; Song, Deguang; Li, Jida; Bao, Yingfu; Lu, Rongguang; Bi, Jingying; Zhao, Kui; Gao, Feng

2014-05-01

312

Ovalbumin Gene: Evidence for a Leader Sequence in mRNA and DNA Sequences at the Exon-Intron Boundaries  

Microsoft Academic Search

Selected regions of cloned EcoRI fragments of the chicken ovalbumin gene have been sequenced. The positions where the sequences coding for ovalbumin mRNA (ov-mRNA) are interrupted in the genome have been determined, and a previously unreported interruption in the DNA sequences coding for the 5' nontranslated region of the messenger has been discovered. Because directly repeated sequences are found at

R. Breathnach; C. Benoist; K. O'Hare; F. Gannon; P. Chambon

1978-01-01

313

Nine duplicated tRNA genes on the plastid-like DNA of the malaria parasite Plasmodium falciparum.  

PubMed

A major feature of the plastid-like circular DNA of Plasmodium falciparum is an inverted repeat comprising duplicated genes for rRNA (rrn) and tRNA (trn). We have identified nine putative trn genes in each arm of the repeat on the basis of their potential clover-leaf structures and conserved residues. Northern blots indicate that these trn genes are expressed. PMID:8039718

Gardner, M J; Preiser, P; Rangachari, K; Moore, D; Feagin, J E; Williamson, D H; Wilson, R J

1994-07-01

314

HPLC of biological macromolecules: The first decade  

Microsoft Academic Search

Summary  During the past decade, HPLC has developed into a powerful new technique for the analysis of complex mixtures of biological\\u000a macromolecules. Through the use of microparticulate supports of vastly improved mechanican strength, superior stationary phase\\u000a chemistry, and advanced instrumentation, it is now possible to separate biological macromolecules more than 10 times faster\\u000a and with greater resolution than in the classical

F. E. Regnier

1987-01-01

315

Electrochemical Branched-DNA Assay for Polymerase Chain Reaction-Free Detection and Quantification of Oncogenes in Messenger RNA  

SciTech Connect

We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcript in the population of messenger RNA (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify targets signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-napthyl-phosphate. The specificity and sensitivity of assay enabled direct detection of target transcript in as little as 4.6 ng mRNA without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcript in total mRNA population. The approach thus provides a simple, sensitive, accurate and quantitative tool alternate to the RQ-PCR for early disease diagnosis.

Lee, Ai Cheng; Dai, Ziyu; Chen, Baowei; Wu, Hong; Wang, Jun; Zhang, Aiguo; Zhang, Lurong; Lim, Tit-Meng; Lin, Yuehe

2008-12-01

316

Macromolecule-Assisted de novo Protein Folding  

PubMed Central

In the processes of protein synthesis and folding, newly synthesized polypeptides are tightly connected to the macromolecules, such as ribosomes, lipid bilayers, or cotranslationally folded domains in multidomain proteins, representing a hallmark of de novo protein folding environments in vivo. Such linkage effects on the aggregation of endogenous polypeptides have been largely neglected, although all these macromolecules have been known to effectively and robustly solubilize their linked heterologous proteins in fusion or display technology. Thus, their roles in the aggregation of linked endogenous polypeptides need to be elucidated and incorporated into the mechanisms of de novo protein folding in vivo. In the classic hydrophobic interaction-based stabilizing mechanism underlying the molecular chaperone-assisted protein folding, it has been assumed that the macromolecules connected through a simple linkage without hydrophobic interactions and conformational changes would make no effect on the aggregation of their linked polypeptide chains. However, an increasing line of evidence indicates that the intrinsic properties of soluble macromolecules, especially their surface charges and excluded volume, could be important and universal factors for stabilizing their linked polypeptides against aggregation. Taken together, these macromolecules could act as folding helpers by keeping their linked nascent chains in a folding-competent state. The folding assistance provided by these macromolecules in the linkage context would give new insights into de novo protein folding inside the cell. PMID:22949867

Choi, Seong Il; Son, Ahyun; Lim, Keo-Heun; Jeong, Hotcherl; Seong, Baik L.

2012-01-01

317

PCR primers MAGE A3 mRNA cross exon boundaries allowing cDNA amplification identified genomic DNA. Try avoid placing primer sequences known psuedogene homologues. Primer Blast web site select your primers gene http:  

EPA Pesticide Factsheets

Search instead for PCR primers MAGE A3 mRNA cross exon boundaries allowing cDNA amplification identified genomic DNA. Try avoid placing primer sequences known psuedogene homologues. Primer Blast web site select your primers gene http: ?

318

Cisplatin-DNA adducts are molecular decoys for the ribosomal RNA transcription factor hUBF (human upstream binding factor).  

PubMed

The toxicity of DNA-damaging agents is widely believed to result from the formation of lesions that block polymerases or disrupt the integrity of the genome. A mechanism heretofore not addressed is that DNA damage may titrate essential DNA-binding proteins away from their natural sites of action. This report shows that the ribosomal RNA (rRNA) transcription factor hUBF (human upstream binding factor) binds with striking affinity (Kd(app) approximately 60 pM) to the intrastrand cis-[Pt(NH3)2](2+-d(GpG) crosslink formed by the anticancer drug cis-diamminedichloroplatinum(II) (cisplatin). When protein blots of human cell extracts are probed with cisplatin-modified DNA, 97- and 94-kDa proteins are detected, consistent with the known sites of hUBF species. A similar analysis of blots containing in vitro translated hUBF confirmed that the protein binds cisplatin adducts with high specificity. By contrast, DNA adducts of the clinically ineffective trans isomer of cisplatin, trans-diamminedichloroplatinum(II), are not recognized by hUBF. DNase I inhibition patterns of hUBF bound to a 100-base-pair DNA fragment containing a centrally located cis-[Pt(NH3)2](2+)-d(GpG) crosslink reveal specific protein-DNA interactions in a 14-base-pair region flanking the adduct. The affinity of hUBF for the rRNA promoter is similar (Kd(app) approximately 18 pM) to that measured for the cisplatin adduct. In addition, we observe that the hUBF-promoter interaction is highly sensitive to the antagonistic effects of cisplatin-DNA adducts. These results suggest that a cisplatin-mediated transcription-factor-hijacking mechanisms could disrupt rRNA synthesis, which is stimulated in proliferating cells. PMID:8202546

Treiber, D K; Zhai, X; Jantzen, H M; Essigmann, J M

1994-06-01

319

Simultaneous isolation of DNA, RNA, and protein from Medicago truncatula L.  

PubMed

We describe a method for the simultaneous extraction of proteins and nucleic acids from Medicago truncatula tissues. Using a modified TRIzol reagent method, we developed a simple and an effective way to simultaneously extract proteins and nucleic acids from a single sample. We verified that this method does not affect the quality or quantitation of the isolated DNA and RNA. Furthermore, we used 2-DE to compare M. truncatula leaf, stem, and root samples processed using this new method with two commonly used methods: phenol extraction/methanol-ammonium acetate precipitation and trichloroacetic acid/acetone precipitation. The results showed that our method was superior to the other methods, based on 2-DE patterns. We also demonstrated that our protocol is compatible with proteomic analysis, as 10 out of 14 selected proteins isolated by the method were identified by MALDI-TOF-MS/MS. The protocol described can be used with sample preparation protocols for proteomic, transcriptomic, and genomic studies. PMID:21254131

Xiong, Junbo; Yang, Qingchuan; Kang, Junmei; Sun, Yan; Zhang, Tiejun; Margaret, Gruber; Ding, Wang

2011-01-01

320

Experimental and ab initio study of the photofragmentation of DNA and RNA sugars  

SciTech Connect

The photoelectron-photoion-photoion coincidence method is used to measure the photodissociation of doubly charged D-ribose (C{sub 5}H{sub 10}O{sub 5}), the RNA sugar molecules, and 2-deoxy-D-ribose (C{sub 5}H{sub 10}O{sub 4}), the DNA sugar molecules, following normal Auger decay after initial C 1s and O 1s core ionizations. The fragment identification is facilitated by measuring isotopically labeled D-ribose, such as D-ribose deuterated at C(1), and with {sup 13}C at the C(5) position. Ab initio quantum chemistry calculations are used to gain further insight into the abundant appearance of the CHO{sup +} fragment.

Ha, D. T. [Department of Physics and Astronomy, University of Turku (Finland); Graduate School of Materials Research, Turku (Finland); Huels, M. A. [Department of Nuclear Medicine and Radiobiology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec (Canada); Huttula, M.; Urpelainen, S. [Department of Physics, University of Oulu (Finland); Kukk, E. [Department of Physics and Astronomy, University of Turku (Finland); Turku University Centre for Materials and Surfaces (MatSurf), Turku (Finland)

2011-09-15

321

On the aromatic character of the heterocyclic bases of DNA and RNA.  

PubMed

Studies based on ab initio optimized geometries (at B3LYP/6-311+G** and MP2/6-311+G** levels) and on experimental structures retrieved from the Cambridge Structural Database (CSD) reveal that the nucleobases constituting DNA and RNA differ significantly in their aromatic character, as shown by the geometry-based index of aromaticity HOMA that ranges from 0.466 for thymine to 0.917 for adenine, based on B3LYP/6-311+G** calculations, and 0.495-0.926, respectively, if based on the MP2/6-311+G** level. Aromaticity of the bases decreases markedly with an increase of the number of double-bond C=X (X = N, O) substituents at the rings. H-bonds involving C=O groups in Watson-Crick pairs cause an increase of the aromatic character of the rings. PMID:14575493

Cyra?ski, Micha? K; Gilski, Miros?aw; Jaskólski, Mariusz; Krygowski, Tadeusz Marek

2003-10-31

322

Renewable pencil electrodes for highly sensitive stripping potentiometric measurements of DNA and RNA.  

PubMed

Renewable graphite pencil electrodes are demonstrated to be excellent materials for adsorptive stripping measurements of trace nucleic acids. While displaying an attractive stripping performance, comparable to that of conventional carbon paste electrodes, the pencil electrode offers a convenient (mechanical) renewal, with each stripping potentiogram recorded at a fresh surface. Various pencil lead materials and lengths have been examined and experimental variables of the pretreatment and measurement procedures have been explored and optimized. The extremely low detection limits (e.g., 3 micrograms l-1 tRNA with 10 min accumulation) are coupled to a good surface-to-surface reproducibility (RSD of 6.4% for 14 repetitive measurements of 1 mg l-1 ssDNA). PMID:10885061

Wang, J; Kawde, A N; Sahlin, E

2000-01-01

323

Oxidative demethylation of DNA and RNA mediated by non-heme iron-dependent dioxygenases.  

PubMed

DNA/RNA methylation can be generated by methyltransferases and thus plays a critical role in regulating cellular processes; alternatively, nucleic acid methylation can be produced by methylation agents and is cytotoxic/mutagenic if left unrepaired. Oxidative demethylation mediated by non-heme iron-dependent dioxygenases is an efficient way to reverse either the cellular roles of regulatory methylation or the cytotoxic/mutagenic effects of methylation damage. In this Focus Review we summarize recent advances in the study of nucleic acid dioxygenases exemplified by the TET and AlkB family proteins, with an emphasis on chemical insights from the recent literature. Comparison of the chemical mechanisms of these dioxygenases revealed that differences in the mechanism also contribute significantly to their distinct biological functions. PMID:24909658

Lu, Lining; Zhu, Chenxu; Xia, Bo; Yi, Chengqi

2014-08-01

324

QIP, a protein that converts duplex siRNA into single strands, is required for meiotic silencing by unpaired DNA.  

PubMed

RNA interference (RNAi) depends on the production of small RNA to regulate gene expression in eukaryotes. Two RNAi systems exist to control repetitive selfish elements in Neurospora crassa. Quelling targets transgenes during vegetative growth, whereas meiotic silencing by unpaired DNA (MSUD) silences unpaired genes during meiosis. The two mechanisms require common RNAi proteins, such as RNA-directed RNA polymerases, Dicers, and Argonaute slicers. We have previously demonstrated that, while Quelling depends on the redundant dicer activity of DCL-1 and DCL-2, only DCL-1 is required for MSUD. Here, we show that QDE-2-interacting protein (QIP), an exonuclease that is important for the production of single-stranded siRNA during Quelling, is also required for MSUD. QIP is crucial for sexual development and is shown to colocalize with other MSUD proteins in the perinuclear region. PMID:20551436

Xiao, Hua; Alexander, William G; Hammond, Thomas M; Boone, Erin C; Perdue, Tony D; Pukkila, Patricia J; Shiu, Patrick K T

2010-09-01

325

Component H of the DNA-Dependent RNA Polymerases of Archaea is Homologous to a Subunit Shared by the Three Eucaryal Nuclear RNA Polymerases  

Microsoft Academic Search

The gene encoding component H of the DNA-dependent RNA polymerase (RNAP, EC 2.7.7.6) of Sulfolobus acidocaldarius has been identified by comparison of the amino acid sequence with the derived amino acid sequence of an open reading frame (ORF88) in the RNAP operon. Corresponding genes were identified in Halobacterium halobium and were cloned and sequenced from Thermococcus celer and Methanococcus vannielii.

Hans-Peter Klenk; Peter Palm; Friedrich Lottspeich; Wolfram Zillig

1992-01-01

326

Soluble Interleukin-6 Receptor-Mediated Innate Immune Response to DNA and RNA Viruses  

PubMed Central

The interleukin-6 (IL-6) receptor, which exists as membrane-bound and soluble forms, plays critical roles in the immune response. The soluble IL-6 receptor (sIL6R) has been identified as a potential therapeutic target for preventing coronary heart disease. However, little is known about the role of this receptor during viral infection. In this study, we show that sIL6R, but not IL-6, is induced by viral infection via the cyclooxygenase-2 pathway. Interestingly, sIL6R, but not IL-6, exhibited extensive antiviral activity against DNA and RNA viruses, including hepatitis B virus, influenza virus, human enterovirus 71, and vesicular stomatitis virus. No synergistic effects on antiviral action were observed by combining sIL6R and IL-6. Furthermore, sIL6R mediated antiviral action via the p28 pathway and induced alpha interferon (IFN-?) by promoting the nuclear translocation of IFN regulatory factor 3 (IRF3) and NF-?B, which led to the activation of downstream IFN effectors, including 2?,5?-oligoadenylate synthetase (OAS), double-stranded RNA-dependent protein kinase (PKR), and myxovirus resistance protein (Mx). Thus, our results demonstrate that sIL6R, but not IL-6, plays an important role in the host antiviral response. PMID:23946454

Wang, Qing; Chen, Xueyuan; Feng, Jian; Cao, Yanhua; Song, Yu; Wang, Hui; Zhu, Chengliang; Liu, Shi

2013-01-01

327

A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection  

PubMed Central

Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), are completely restricted in their replication after entry into Lepidopteran cells. This restriction is overcome when cells are co-infected with vaccinia virus (VACV), a vertebrate DNA virus. Using RNAi screening, we show that Lepidopteran RNAi, Nuclear Factor-?B, and ubiquitin-proteasome pathways restrict RNA virus infection. Surprisingly, a highly conserved, uncharacterized VACV protein, A51R, can partially overcome this virus restriction. We show that A51R is also critical for VACV replication in vertebrate cells and for pathogenesis in mice. Interestingly, A51R colocalizes with, and stabilizes, host microtubules and also associates with ubiquitin. We show that A51R promotes viral protein stability, possibly by preventing ubiquitin-dependent targeting of viral proteins for destruction. Importantly, our studies reveal exciting new opportunities to study virus-host interactions in experimentally-tractable Lepidopteran systems. DOI: http://dx.doi.org/10.7554/eLife.02910.001 PMID:24966209

Gammon, Don B; Duraffour, Sophie; Rozelle, Daniel K; Hehnly, Heidi; Sharma, Rita; Sparks, Michael E; West, Cara C; Chen, Ying; Moresco, James J; Andrei, Graciela; Connor, John H; Conte, Darryl; Gundersen-Rindal, Dawn E; Marshall, William L; Yates, John R; Silverman, Neal; Mello, Craig C

2014-01-01

328

Evidence for and Localization of Vegetative Viral DNA Replication by Autoradiographic Detection of RNA?DNA Hybrids in Sections of Tumors Induced by Shope Papilloma Virus  

PubMed Central

The occurrence and localization of vegetative viral DNA replication was studied in sections of tumors induced by the rabbit Shope papilloma virus, in cottontail and domestic rabbit papillomas, in primary domestic rabbit carcinoma, and in transplantable VX2 carcinoma, by in situ hybridization of radioactive RNA complementary to viral DNA. Vegetative viral DNA replication and viral protein synthesis were compared by means of cytological hybridization and immunofluorescence techniques on adjacent frozen sections. Vegetative viral DNA replication is completely repressed in the proliferating cellular layers of these tumors, which suggests a provirus state of the viral genome, as in other cells transformed by oncogenic DNA viruses. Vegetative viral DNA replication is induced, after initiation of the keratinization, in cells of cottonail rabbit papillomas, where it is usually followed by viral protein synthesis; this illustrates the influence of the physiological state of the host cell on the control of viral functions. Vegetative viral DNA replication is deteced only in a few cells of domestic rabbit papillomas, at the end of the keratinization process; this observation provides indirect evidence that the DNA synthesis specifically induced in these tumors after the onset of keratinization reflects mostly the induction of cellular DNA synthesis. Images PMID:4331563

Orth, Gerard; Jeanteur, Philippe; Croissant, Odile

1971-01-01

329

Alignment of Synaptic Vesicle Macromolecules with the Macromolecules in Active Zone Material that Direct Vesicle Docking  

PubMed Central

Synaptic vesicles dock at active zones on the presynaptic plasma membrane of a neuron’s axon terminals as a precondition for fusing with the membrane and releasing their neurotransmitter to mediate synaptic impulse transmission. Typically, docked vesicles are next to aggregates of plasma membrane-bound macromolecules called active zone material (AZM). Electron tomography on tissue sections from fixed and stained axon terminals of active and resting frog neuromuscular junctions has led to the conclusion that undocked vesicles are directed to and held at the docking sites by the successive formation of stable connections between vesicle membrane proteins and proteins in different classes of AZM macromolecules. Using the same nanometer scale 3D imaging technology on appropriately stained frog neuromuscular junctions, we found that ?10% of a vesicle’s luminal volume is occupied by a radial assembly of elongate macromolecules attached by narrow projections, nubs, to the vesicle membrane at ?25 sites. The assembly’s chiral, bilateral shape is nearly the same vesicle to vesicle, and nubs, at their sites of connection to the vesicle membrane, are linked to macromolecules that span the membrane. For docked vesicles, the orientation of the assembly’s shape relative to the AZM and the presynaptic membrane is the same vesicle to vesicle, whereas for undocked vesicles it is not. The connection sites of most nubs on the membrane of docked vesicles are paired with the connection sites of the different classes of AZM macromolecules that regulate docking, and the membrane spanning macromolecules linked to these nubs are also attached to the AZM macromolecules. We conclude that the luminal assembly of macromolecules anchors in a particular arrangement vesicle membrane macromolecules, which contain the proteins that connect the vesicles to AZM macromolecules during docking. Undocked vesicles must move in a way that aligns this arrangement with the AZM macromolecules for docking to proceed. PMID:23894473

Xu, Jing; Jung, Jae Hoon; Marshall, Robert M.; McMahan, Uel J.

2013-01-01

330

An integrative analysis of DNA methylation and RNA-Seq data for human heart, kidney and liver  

PubMed Central

Background Many groups, including our own, have proposed the use of DNA methylation profiles as biomarkers for various disease states. While much research has been done identifying DNA methylation signatures in cancer vs. normal etc., we still lack sufficient knowledge of the role that differential methylation plays during normal cellular differentiation and tissue specification. We also need thorough, genome level studies to determine the meaning of methylation of individual CpG dinucleotides in terms of gene expression. Results In this study, we have used (insert statistical method here) to compile unique DNA methylation signatures from normal human heart, lung, and kidney using the Illumina Infinium 27 K methylation arraysand compared those to gene expression by RNA sequencing. We have identified unique signatures of global DNA methylation for human heart, kidney and liver, and showed that DNA methylation data can be used to correctly classify various tissues. It indicates that DNA methylation reflects tissue specificity and may play an important role in tissue differentiation. The integrative analysis of methylation and RNA-Seq data showed that gene methylation and its transcriptional levels were comprehensively correlated. The location of methylation markers in terms of distance to transcription start site and CpG island showed no effects on the regulation of gene expression by DNA methylation in normal tissues. Conclusions This study showed that an integrative analysis of methylation array and RNA-Seq data can be utilized to discover the global regulation of gene expression by DNA methylation and suggests that DNA methylation plays an important role in normal tissue differentiation via modulation of gene expression. PMID:22784623

2011-01-01

331

Physical Localization and DNA Methylation of 45S rRNA Gene Loci in Jatropha curcas L.  

PubMed Central

In eukaryotes, 45S rRNA genes are arranged in tandem arrays of repeat units, and not all copies are transcribed during mitosis. DNA methylation is considered to be an epigenetic marker for rDNA activation. Here, we established a clear and accurate karyogram for Jatropha curcas L. The chromosomal formula was found to be 2n?=?2x?=?22?=?12m+10sm. We found that the 45S rDNA loci were located at the termini of chromosomes 7 and 9 in J. curcas. The distribution of 45S rDNA has no significant difference in J. curcas from different sources. Based on the hybridization signal patterns, there were two forms of rDNA - dispersed and condensed. The dispersed type of signals appeared during interphase and prophase, while the condensed types appeared during different stages of mitosis. DNA methylation analysis showed that when 45S rDNA stronger signals were dispersed and connected to the nucleolus, DNA methylation levels were lower at interphase and prophase. However, when the 45S rDNA loci were condensed, especially during metaphase, they showed different forms of DNA methylation. PMID:24386362

Gong, Zhiyun; Xue, Chao; Zhang, Mingliang; Guo, Rui; Zhou, Yong; Shi, Guoxin

2013-01-01

332

Detection of bovine viral diarrhea virus genome in leukocytes from persistently infected cattle by RNA-cDNA hybridization.  

PubMed Central

A bovine viral diarrhea virus (BVDV) cDNA library was constructed. One cloned complementary DNA sequence was used as a probe to detect BVDV RNA by hybridization in infected cell cultures and in mononuclear leukocytes from persistently infected cattle by dot blot and in situ hybridization. The cDNA probe hybridized with all cytopathic and noncytopathic BVDV isolates tested. The hybridization results were consistent with results obtained using conventional subculturing and immunofluorescent staining methods and by inoculation of seronegative test cattle. Images Fig. 1. Fig. 2. Fig. 3. PMID:2162729

Jensen, J; Aiken, J; Schultz, R D

1990-01-01

333

An atypical component of RNA-directed DNA methylation machinery has both DNA methylation-dependent and -independent roles in locus-specific transcriptional gene silencing  

PubMed Central

RNA-directed DNA methylation (RdDM) is an important de novo DNA methylation pathway in plants. RdDM mediates the transcriptional silencing of many endogenous genomic loci, most of which are transposon related. A forward genetics screen identified DTF1 (DNA-binding transcription factor 1) as a new component for RdDM in Arabidopsis. Loss-of-function mutations in DTF1 release the transcriptional silencing of RdDM target loci and reduce the accumulation of 24-nt small interfering RNAs (siRNAs) from some of the targets. Interestingly, in the dtf1 mutant plants, the release of transcriptional gene silencing at solo-LTR is accompanied by decreased siRNA accumulation but not by reduced DNA methylation. These results suggest that DTF1 is an atypical component of RdDM and has both DNA methylation-dependent and -independent roles in transcriptional gene silencing. We suggest that besides DNA methylation, siRNAs may cause some other uncharacterized epigenetic modifications that lead to transcriptional gene silencing. PMID:22064704

Liu, Jun; Bai, Ge; Zhang, Cuijun; Chen, Wei; Zhou, Jinxing; Zhang, Suwei; Chen, Qing; Deng, Xin; He, Xin-Jian; Zhu, Jian-Kang

2011-01-01

334

In planta engineering of viral RNA replicons: Efficient assembly by recombination of DNA modules delivered by Agrobacterium  

Microsoft Academic Search

We have developed an efficient, versatile, and user-friendly viral engineering and expression system that is based on in planta assembly of functional viral vectors from separate pro-vector modules. With this new system, instead of supplying a plant cell with a complete viral vector as a mature viral particle, an RNA or a linear DNA molecule, we use agrobacteria to deliver

Sylvestre Marillonnet; Anatoli Giritch; Mario Gils; Romy Kandzia; Victor Klimyuk; Yuri Gleba

2004-01-01

335

Sequence analysis of 16S rRNA from mycoplasmas by direct solid-phase DNA sequencing.  

PubMed Central

Automated solid-phase DNA sequencing was used for determination of partial 16S ribosomal DNA sequences of mycoplasmas. The sequence information was used to establish phylogenetic relationships of 11 different mycoplasmas whose 16S rRNA sequences had not been determined earlier. A biotinylated fragment corresponding to positions 344 to 939 in the Escherichia coli sequence was generated by PCR. The PCR product was immobilized onto streptavidin-coated paramagnetic beads, and direct sequencing was performed in both directions. One previously unclassified avian mycoplasma was found to belong to the Mycoplasma lipophilum cluster of the hominis group. Microheterogeneities were discovered in the rRNA operons of Mycoplasma mycoides subsp. mycoides (SC type), confirming the existence of two different rRNA operons. The 16S rRNA sequence of M. mycoides subsp. capri was identical to that of M. mycoides subsp. mycoides (type SC), except that no microheterogeneities were revealed. Furthermore, automated solid-phase DNA sequencing was used to identify a mycoplasmal contamination of a cell culture as Mycoplasma hyorhinis, which proved to be very difficult by conventional methods. The results suggest that the direct solid-phase DNA sequencing procedure is a powerful tool for identification of mycoplasmas and is also useful in taxonomic studies. Images PMID:7521158

Pettersson, B; Johansson, K E; Uhlen, M

1994-01-01

336

A New Three-Dimensional Educational Model Kit for Building DNA and RNA Molecules: Development and Evaluation  

ERIC Educational Resources Information Center

International specialized literature focused on research in biology education is sadly scarce, especially regarding biochemical and molecular aspects. In this light, researchers from this Centre for Structural Molecular Biotechnology developed and evaluated a three-dimensional educational model named "Building Life Molecules DNA and RNA." The…

Beltramini, Leila Maria; Araujo, Ana Paula Ulian; de Oliveira, Tales Henrique Goncalves; dos Santos Abel, Luciano Douglas; da Silva, Aparecido Rodrigues; dos Santos, Neusa Fernandes

2006-01-01

337

Catalog of mRNA expression patterns for DNA methylating and demethylating genes in developing mouse lower urinary tract.  

PubMed

The mouse prostate develops from a component of the lower urinary tract (LUT) known as the urogenital sinus (UGS). This process requires androgens and signaling between mesenchyme and epithelium. Little is known about DNA methylation during prostate development, including which factors are expressed, whether their expression changes over time, and if DNA methylation contributes to androgen signaling or influences signaling between mesenchyme and epithelium. We used in situ hybridization to evaluate the spatial and temporal expression pattern of mRNAs which encode proteins responsible for establishing, maintaining or remodeling DNA methylation. These include DNA methyltransferases, DNA deaminases, DNA glycosylases, base excision repair and mismatch repair pathway members. The mRNA expression patterns were compared between male and female LUT prior to prostatic bud formation (14.5 days post coitus (dpc)), during prostatic bud formation (17.5 dpc) and during prostatic branching morphogenesis (postnatal day (P) 5). We found dramatic changes in the patterns of these mRNAs over the course of prostate development and identified examples of sexually dimorphic mRNA expression. Future investigation into how DNA methylation patterns are established, maintained and remodeled during the course of embryonic prostatic bud formation may provide insight into prostate morphogenesis and disease. PMID:23920106

Keil, Kimberly P; Altmann, Helene M; Mehta, Vatsal; Abler, Lisa L; Elton, Erik A; Vezina, Chad M

2013-12-01

338

Evaluation of novel carbon nano-tube particles in the bacterial and viral DNA and RNA extraction from the clinical samples  

Microsoft Academic Search

Molecular techniques have become the most im- portant methods of detecting bacterial and viral pathogens. However, current genomic extraction methods are currently limited in term of automation. In this study, carbon nano-tube was used as the vector to trap DNA and RNA molecules. The capability of carbon nano-tube to trap DNA and RNA was evaluated using samples (TB and HBV

Nguyen KC; Vo DXA; Hoang HN; Ho LTT; Pham HV

2010-01-01

339

Self assembly of poly(o-methoxy aniline) with RNA and RNA/DNA hybrids: physical properties and conformational change of poly(o-methoxy aniline).  

PubMed

Biomolecular hybrids of a conducting polymer [poly(o-methoxy aniline) (POMA)] and RNA are prepared at the three different compositions by mixing aqueous solutions of diethyl, 2-hydroxy ethyl, ammonium salt of RNA (type IX from Torula Yeast) and POMA (ES, emeraldine salt; doping level [Cl]/[N]=0.52). A slow increase of pH up to 30 h of aging occurs in the mixture till it levels up. The TEM micrographs indicate a fibrillar network structure in all the hybrid compositions (POMA: RNA=1:3, 1:1, 3:1, by weight). In the complexes three types of supramolecular interactions, viz. (i) electrostatic, (ii) H-bonding and (iii) pi-pi interactions, are evident from the FTIR spectroscopy. The CD spectra indicate a small distortion of A-RNA conformation towards its B form during the hybrid formation. Time and temperature dependent UV-vis spectral studies indicate a slow red shift of the pi-band to polaron band transition peak (lambda(max)) for the uncoiling of the POMA (P) chain on the RNA (R) surface. The repulsive interaction between the radical cations of POMA (ES) absorbed on the RNA surface is attributed to the conformational change causing the uncoiling of POMA chain. UV-vis spectral study indicates that the uncoiling and attachment of POMA on RNA surface is much faster than that on DNA (D). In POMA-RNA-DNA (PRD) hybrid solutions slower red shift of lambda(max) indicates more disordered array of the phosphate groups than that in PR and PD systems. The conductivity values of the PR hybrids (10(-)(6) S/cm(-1)) are three orders higher than that of RNA, rendering the PR hybrids to be useful for fabricating good biosensors. In the PRD hybrids conductivity decreases by two orders than those of PR and PD hybrids suggesting a disorder arrangement of POMA chains in the PRD hybrids. The I-V characteristic curves of the PR and PRD hybrids indicate a semiconducting nature of the hybrids. PMID:19482408

Routh, Parimal; Mukherjee, Pratap; Dawn, Arnab; Nandi, Arun K

2009-08-01

340

Missing Genes, Multiple ORFs, and C-to-U Type RNA Editing in Acrasis kona (Heterolobosea, Excavata) Mitochondrial DNA  

PubMed Central

Discoba (Excavata) is an ancient group of eukaryotes with great morphological and ecological diversity. Unlike the other major divisions of Discoba (Jakobida and Euglenozoa), little is known about the mitochondrial DNAs (mtDNAs) of Heterolobosea. We have assembled a complete mtDNA genome from the aggregating heterolobosean amoeba, Acrasis kona, which consists of a single circular highly AT-rich (83.3%) molecule of 51.5 kb. Unexpectedly, A. kona mtDNA is missing roughly 40% of the protein-coding genes and nearly half of the transfer RNAs found in the only other sequenced heterolobosean mtDNAs, those of Naegleria spp. Instead, over a quarter of A. kona mtDNA consists of novel open reading frames. Eleven of the 16 protein-coding genes missing from A. kona mtDNA were identified in its nuclear DNA and polyA RNA, and phylogenetic analyses indicate that at least 10 of these 11 putative nuclear-encoded mitochondrial (NcMt) proteins arose by direct transfer from the mitochondrion. Acrasis kona mtDNA also employs C-to-U type RNA editing, and 12 homologs of DYW-type pentatricopeptide repeat (PPR) proteins implicated in plant organellar RNA editing are found in A. kona nuclear DNA. A mapping of mitochondrial gene content onto a consensus phylogeny reveals a sporadic pattern of relative stasis and rampant gene loss in Discoba. Rampant loss occurred independently in the unique common lineage leading to Heterolobosea + Tsukubamonadida and later in the unique lineage leading to Acrasis. Meanwhile, mtDNA gene content appears to be remarkably stable in the Acrasis sister lineage leading to Naegleria and in their distant relatives Jakobida. PMID:25146648

Fu, Cheng-Jie; Sheikh, Sanea; Miao, Wei; Andersson, Siv G.E.; Baldauf, Sandra L.

2014-01-01

341

Phylogeny and taxonomy of mesophilic Methanococcus spp. and comparison of rRNA, DNA hybridization, and phenotypic methods.  

PubMed

The phylogeny and taxonomy of the mesophilic methane-producing archaea of the order Methanococcales were examined by DNA relatedness, 16S rRNA sequence analysis, cellular protein patterns, and phenotypic methods. The mesophilic species Methanococcus maripaludis, Methanococcus vannielii, Methanococcus voltaei, and "Methanococcus aeolicus" formed a deep group with 5 to 30% DNA relatedness and 92 to 96% 16S rRNA sequence similarity. Twenty-two additional isolates and Methanococcus deltae were similar to the type strain of either M. voltaei or M. maripaludis. Two isolates, strains A2 and A3, exhibited 37% DNA relatedness and 99.2% 16S rRNA sequence similarity to M. voltaei PS(T) (T = type strain). In the absence of phenotypic differences, these organisms were assigned to M. voltaei. Similarly, four autotrophic isolates, strains C5, C6, C7, and C8, exhibited 54 to 69% DNA relatedness and 99.2% 16S rRNA sequence similarity to M. maripaludis JJT and were assigned to M. Maripaludis. While these isolates were sufficiently genetically diverse to justify classification in novel species, few differences were apparent in the phenotypic properties available for measurement. Thus, the phenotypic properties of these lithotrophic archaea were highly conserved and poor indicators of genetic diversity. Partial sequencing of about 200 bases of both the 16S and 23S rRNAs of the isolates demonstrated allelic diversity within methanococcal species. This allelic diversity did not correlate with diversity measured by DNA relatedness, cellular protein pattern, and other methods. Similarly, antisera to whole cells of the type strains did not cross-react strongly to whole cells of strains that were genetically similar, and serological cross-reactivity was not a useful taxonomic method for methanococci. Lastly, on the basis of the results of 16S rRNA sequence analyses and biochemical data, the ancestor of the mesophilic methanococci may have been an autotrophic thermophile. PMID:8782682

Keswani, J; Orkand, S; Premachandran, U; Mandelco, L; Franklin, M J; Whitman, W B

1996-07-01

342

Seasonality of rDNA- and rRNA-derived archaeal communities and methanogenic potential in a boreal mire.  

PubMed

Methane (CH(4)) emissions from boreal wetlands show considerable seasonal variation, including small winter emissions. We addressed the seasonality of CH(4)-producing microbes by comparing archaeal communities and the rates and temperature response of CH(4) production in a boreal fen at three key phases of growing season and in winter. Archaeal community analysis by terminal restriction fragment length polymorphism and cloning of 16S ribosomal DNA and reverse-transcribed RNA revealed slight community shifts with season. The main archaeal groups remained the same throughout the year and were Methanosarcinaceae, Rice cluster II and Methanomicrobiales-associated Fen cluster. These methanogens and the crenarchaeal groups 1.1c and 1.3 were detected from DNA and RNA, but the family Methanosaetaceae was detected only from RNA. Differences between DNA- and RNA-based results suggested higher stability of DNA-derived communities and better representation of the active CH(4) producers in RNA. Methane production potential, measured as formation of CH(4) in anoxic laboratory incubations, showed prominent seasonality. The potential was strikingly highest in winter, possibly due to accumulation of methanogenic substrates, and maximal CH(4) production was observed at ca. 30 degrees C. Archaeal community size, determined by quantitative PCR, remained similar from winter to summer. Low production potential in late summer after a water level draw-down suggested diminished activity due to oxygen exposure. Our results indicated that archaeal community composition and size in the boreal fen varied only slightly despite the large fluctuations of methanogenic potential. Detection of mRNA of the methanogenic mcrA gene confirmed activity of methanogens in winter, accounting for previously reported winter CH(4) emissions. PMID:18650929

Juottonen, Heli; Tuittila, Eeva-Stiina; Juutinen, Sari; Fritze, Hannu; Yrjälä, Kim

2008-11-01

343

Cytonuclear conflict in interpopulation hybrids: the role of RNA polymerase in mtDNA transcription and replication.  

PubMed

Organismal fitness requires functional integration of nuclear and mitochondrial genomes. Structural and regulatory elements coevolve within lineages and several studies have found that interpopulation hybridization disrupts mitonuclear interactions. Because mitochondrial RNA polymerase (mtRPOL) plays key roles in both mitochondrial DNA (mtDNA) replication and transcription, the interaction between mtRPOL and coevolved regulatory sites in the mtDNA may be central to mitonuclear integration. Here, we generate interpopulation hybrids between divergent populations of the copepod Tigriopus californicus to obtain lines having different combinations of mtRPOL and mtDNA. Lines were scored for mtDNA copy number and ATP6 (mtDNA) gene expression. We find that there is a genotype-dependent negative association between mitochondrial transcriptional response and mtDNA copy number. We argue that an observed increase in mtDNA copy number and reduced mtDNA transcription in hybrids reflects the regulatory role of mtRPOL; depending on the mitonuclear genotype, hybridization may disrupt the normal balance between transcription and replication of the mitochondrial genome. PMID:20070459

Ellison, C K; Burton, R S

2010-03-01

344

DNA sequence-based "bar codes" for tracking the origins of expressed sequence tags from a maize cDNA library constructed using multiple mRNA sources.  

PubMed

To enhance gene discovery, expressed sequence tag (EST) projects often make use of cDNA libraries produced using diverse mixtures of mRNAs. As such, expression data are lost because the origins of the resulting ESTs cannot be determined. Alternatively, multiple libraries can be prepared, each from a more restricted source of mRNAs. Although this approach allows the origins of ESTs to be determined, it requires the production of multiple libraries. A hybrid approach is reported here. A cDNA library was prepared using 21 different pools of maize (Zea mays) mRNAs. DNA sequence "bar codes" were added during first-strand cDNA synthesis to uniquely identify the mRNA source pool from which individual cDNAs were derived. Using a decoding algorithm that included error correction, it was possible to identify the source mRNA pool of more than 97% of the ESTs. The frequency at which a bar code is represented in an EST contig should be proportional to the abundance of the corresponding mRNA in the source pool. Consistent with this, all ESTs derived from several genes (zein and adh1) that are known to be exclusively expressed in kernels or preferentially expressed under anaerobic conditions, respectively, were exclusively tagged with bar codes associated with mRNA pools prepared from kernel and anaerobically treated seedlings, respectively. Hence, by allowing for the retention of expression data, the bar coding of cDNA libraries can enhance the value of EST projects. PMID:14555776

Qiu, Fang; Guo, Ling; Wen, Tsui-Jung; Liu, Feng; Ashlock, Daniel A; Schnable, Patrick S

2003-10-01

345

Use of RNA:DNA ratios to evaluate the condition and growth of the copepod Calanus sinicus in the Southern Yellow Sea  

NASA Astrophysics Data System (ADS)

Calanus sinicus, a dominant calanoid copepod in the Yellow Sea, is an important link in the food web between phytoplankton and higher trophic levels. Its populations typically start to develop in later winter with a maximum of individuals in early summer. To study the correlation between changes in the abundance of this species and changes in food resources and the physical environment, RNA and DNA concentrations and egg production rates (EPR) were measured, and RNA:DNA ratios were calculated as indices of growth and nutritional conditions of copepods collected in the Yellow Sea from February to July. We observed pronounced seasonal and spatial variations of RNA concentrations and resulting RNA:DNA ratios. There was a positive correlation between the EPR and RNA:DNA ratios. The copepods collected in March and April, when phytoplankton were more abundant, had high RNA:DNA ratios, and contained more RNA than copepods collected during the other months. There was no significant correlation between the growth indices (RNA:DNA ratios and EPR) and chlorophyll-a concentrations (Chl a) or temperature at large temporal and spatial scales. We tracked the development of two phytoplankton blooms in April, which were dominated in turn by diatoms and dinoflagellates. We observed high concentrations of RNA and a high RNA:DNA ratio at both bloom sites during the respective blooms. During the diatom bloom, the RNA:DNA ratios in copepods increased at the onset of the bloom and decreased thereafter. In addition, we observed a positive correlation (P<0.001) between RNA-based indices and Chl a. Our results suggest that food availability plays a more important role than temperature in controlling the growth of C. sinicus in the field. Thus, the spring phytoplankton blooms in the Yellow Sea are important regulators of copepod abundance.

Ning, Juan; Li, Chaolun; Yang, Guang; Wan, Aiyong; Sun, Song

2013-12-01

346

Changes in macromolecule syntheses under special consideration of UNA and the energy providing system in imbibing embryos of different aged agrostemma githago L. Seeds  

Microsoft Academic Search

Changes in macromolecule syntheses, especially RNA synthesis, and the energy providing system were investigated in seeds ofAgrostemma githago aged for different periods. In embryos of aged seeds all macromolecule syntheses start later and reach a lower level than\\u000a young ones. It was found that the synthesis of rRNA in embryos of aged seeds is reduced whereas the synthesis of poly

R. Kosanke; D. Bernhardt; K. H. KÖhler; Birgit Voigt

1990-01-01

347

Fast-SAXS-pro: A unified approach to computing SAXS profiles of DNA, RNA, protein, and their complexes  

NASA Astrophysics Data System (ADS)

A generalized method, termed Fast-SAXS-pro, for computing small angle x-ray scattering (SAXS) profiles of proteins, nucleic acids, and their complexes is presented. First, effective coarse-grained structure factors of DNA nucleotides are derived using a simplified two-particle-per-nucleotide representation. Second, SAXS data of a 18-bp double-stranded DNA are measured and used for the calibration of the scattering contribution from excess electron density in the DNA solvation layer. Additional test on a 25-bp DNA duplex validates this SAXS computational method and suggests that DNA has a different contribution from its hydration surface to the total scattering compared to RNA and protein. To account for such a difference, a sigmoidal function is implemented for the treatment of non-uniform electron density across the surface of a protein/nucleic-acid complex. This treatment allows differential scattering from the solvation layer surrounding protein/nucleic-acid complexes. Finally, the applications of this Fast-SAXS-pro method are demonstrated for protein/DNA and protein/RNA complexes.

Ravikumar, Krishnakumar M.; Huang, Wei; Yang, Sichun

2013-01-01

348

Cell type-specific induction of O 6 -alkylguanine-DNA alkyltransferase mRNA expression in rat liver during regeneration, inflammation and preneoplasia  

Microsoft Academic Search

Purpose and Methods: To investigate the potential role of an aberrant cellular DNA repair in target cells during malignant transformation we studied cell type-specific mRNA expression of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (O6-AGT) in normal and regenerating rat liver, chronic hepatitis and preneoplastic liver lesions by in situ hybridization and semiautomatic image analysis. Results: O6-AGT mRNA expression was found

Volker Vielhauer; Marina Sarafoff; Peter Gais; Hartmut M. Rabes

2001-01-01

349

Overcoming RNA inhibition in the fluorescent polymerase chain reaction assay to enhance detection of bovine DNA in cattle feeds.  

PubMed

The practice of incorporating mammalian protein in ruminant feeds was banned in the United States in 1997 as a measure to avoid transmission of bovine spongiform encephalopathy (BSE). A sensitive means of identifying the banned additives in feeds would be by detection of species-specific DNA using the polymerase chain reaction (PCR). However, problems may arise in the PCR due to the presence of inhibitory substances. Using human DNA as an internal PCR control, inhibitory substances were evident in the DNA extraction products of cattle feeds. The results of heating experiments excluded enzymes as a cause of inhibition, and spectrophotometric calculations suggested the possibility of RNA contamination. Co-electrophoresis of untreated and RNAse digested extracts confirmed the presence of RNA in the undigested product. Seven cattle feeds were spiked with predetermined amounts of bovine meat and bone meal (BMBM). The DNA extracted products were treated with RNAse and the bovine specific mitochondrial DNA (B-mtDNA) was amplified by PCR. The minimum level of detection of B-mtDNA was influenced by RNAse treatment and feed composition. RNAse treatment decreased false-negative results overall by 75%. False-negative results were decreased 100% in the higher BMBM concentrations and 50% in the lower BMBM concentrations. Also, each cattle feed was spiked to attain a 2% wt/wt concentration with each swine, fish, sheep, or poultry product, or cattle dried blood. Amplification of B-mtDNA occurred only with the cattle dried blood and only in three feeds in which B-mtDNA was detected at the only level tested (2%). A commercial immunochromotographic assay (Neogen) detected the spiked BMBM in only one of the seven feeds and only at the upper concentration (1%). PMID:15992269

Sawyer, Mary; Rensen, Gabriel; Smith, Wayne; Yee, Melanie; Wong, Alice; Osburn, Bennie; Cullor, James

2004-01-01

350

A novel thermostable polymerase for RNA and DNA loop-mediated isothermal amplification (LAMP).  

PubMed

Meeting the goal of providing point of care (POC) tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol) is a thermostable viral enzyme that enables true POC use in clinics or in the field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP) for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations. Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst) and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular detection of pathogens. PMID:25136338

Chander, Yogesh; Koelbl, Jim; Puckett, Jamie; Moser, Michael J; Klingele, Audrey J; Liles, Mark R; Carrias, Abel; Mead, David A; Schoenfeld, Thomas W

2014-01-01

351

Mitochondria of cold hardy insects: responses to cold and hypoxia assessed at enzymatic, mRNA and DNA levels.  

PubMed

Winter survival for larvae of goldenrod gall insects, the freeze-avoiding Epiblema scudderiana, and the freeze tolerant, Eurosta solidaginis, includes entry into diapause (a torpid state of arrested development) and expression of a variety of cryoprotective adaptations. Diapause and cold winter temperatures, as well as freezing in E. solidaginis, all strongly reduce the need for mitochondrial activity. To evaluate the responses of mitochondria to these conditions, we assessed the maximal activity of cytochrome c oxidase (COX), transcript levels of COX subunit 1 (encoded on the mitochondrial genome), mitochondrial 12S rRNA levels and mitochondrial DNA content. COX activity decreased over the winter months in both species to levels that were about one-third of September values. COX activity also dropped significantly in E. scudderiana in response to cold acclimation (4,-4,-20 degrees C) or hypoxia exposure. COX activity was less sensitive to these stresses in E. solidaginis but rose by approximately 50% when larvae were thawed after freezing. COX 1 mRNA transcripts and 12S rRNA levels were unchanged over the winter months in E. scudderiana, as was COX 1 DNA content; this indicates that changes in COX enzymatic activity are likely mediated mainly by post-translational modification. However, both COX transcript and 12S rRNA levels decreased in response to hypoxia exposure in both species, whereas COX DNA did not, which indicates that transcription of the mitochondrial genome is sensitive to oxygen levels. PMID:18252250

McMullen, David C; Storey, Kenneth B

2008-03-01

352

Degradation of DNA damage-independently stalled RNA polymerase II is independent of the E3 ligase Elc1  

PubMed Central

Transcription elongation is a highly dynamic and discontinuous process, which includes frequent pausing of RNA polymerase II (RNAPII). RNAPII complexes that stall persistently on a gene during transcription elongation block transcription and thus have to be removed. It has been proposed that the cellular pathway for removal of these DNA damage-independently stalled RNAPII complexes is similar or identical to the removal of RNAPII complexes stalled due to DNA damage. Here, we show that—consistent with previous data—DNA damage-independent stalling causes polyubiquitylation and proteasome-mediated degradation of Rpb1, the largest subunit of RNAPII, using Saccharomyces cerevisiae as model system. Moreover, recruitment of the proteasome to RNAPII and transcribed genes is increased when transcription elongation is impaired indicating that Rpb1 degradation takes place at the gene. Importantly, in contrast to the DNA damage-dependent pathway Rpb1 degradation of DNA damage-independently stalled RNAPII is independent of the E3 ligase Elc1. In addition, deubiquitylation of RNAPII is also independent of the Elc1-antagonizing deubiquitylase Ubp3. Thus, the pathway for degradation of DNA damage-independently stalled RNAPII is overlapping yet distinct from the previously described pathway for degradation of RNAPII stalled due to DNA damage. Taken together, we provide the first evidence that the cell discriminates between DNA damage-dependently and -independently stalled RNAPII. PMID:25120264

Karakasili, Eleni; Burkert-Kautzsch, Cornelia; Kieser, Anja; Sträßer, Katja

2014-01-01

353

Characterizing Flexible and Instrinsically Unstructured Biological Macromolecules by SAS using the Porod-Debye Law  

PubMed Central

Unstructured proteins, RNA or DNA components provide functionally important flexibility that is key to many macromolecular assemblies throughout cell biology. As objective, quantitative experimental measures of flexibility and disorder in solution are limited, small angle scattering (SAS), and in particular small angle X-ray scattering (SAXS), provides a critical technology to assess macromolecular flexibility as well as shape and assembly. Here, we consider the Porod-Debye law as a powerful tool for detecting biopolymer flexibility in SAS experiments. We show that the Porod-Debye region fundamentally describes the nature of the scattering intensity decay, which captures information needed for distinguishing between folded and flexible particles. Particularly for comparative SAS experiments, application of the law, as described here, can distinguish between discrete conformational changes and localized flexibility relevant to molecular recognition and interaction networks. This approach aids insightful analyses of fully and partly flexible macromolecules that is more robust and conclusive than traditional Kratky analyses. Furthermore, we demonstrate for prototypic SAXS data that the ability to calculate particle density by the Porod-Debye criteria, as shown here, provides an objective quality assurance parameter that may prove of general use for SAXS modeling and validation. PMID:21509745

Rambo, Robert P.; Tainer, John A.

2011-01-01

354

Effective inhibition of infectious bursal disease virus replication in vitro by DNA vector-based RNA interference.  

PubMed

Infectious bursal disease (IBD) leads to considerable economic losses for the poultry industry by inducing severe immunosuppression and high mortality in chickens. The objective of this study was to determine if RNA interference (RNAi) could be utilized to inhibit IBDV replication in vitro. We selected 3 short interfering RNA (siRNA) sequences (siVP1(618), siVP1(1,115), and siVP1(2,571)) based on conserved regions in the vp1 gene of the infectious bursal disease virus (IBDV). When the Vero cells were transfected with siRNA, synthesized via in vitro transcription, and then infected with IBDV, siVP1(2,571) was discovered to be the most effective site for inhibiting IBDV replication. For long-term expression of siRNA and due to its suitability for large-scale preparation, the mouse U6 promoter was amplified using primers designed according to the siVP1(2,571) sequence. The resulting products were then subcloned into pEGFP-C1 to construct the shRNA expression vector pEC2571-shRNA. The shRNA-transfected Vero cells were then infected with IBDV. As compared to the control, the inhibitory rate in the pEC2,571-shRNA-transfected group was 87.4%. Indirect immunofluorescence and real-time polymerase chain reaction (PCR) confirmed that VP1 expression decreased at both the protein and RNA levels as compared to that in the controls. The results presented here indicate that DNA vector-based RNAi could effectively inhibit IBDV replication in vitro. PMID:18378010

Gao, Yulong; Liu, Wei; Gao, Honglei; Qi, Xiaole; Lin, Huan; Wang, Xiaomei; Shen, Rongxian

2008-08-01

355

Induction of amphiregulin by p53 promotes apoptosis via control of microRNA biogenesis in response to DNA damage.  

PubMed

Upon DNA damage, tumor suppressor p53 determines cell fate by repairing DNA lesions to survive or by inducing apoptosis to eliminate damaged cells. The decision is based on its posttranslational modifications. Especially, p53 phosphorylation at Ser46 exerts apoptotic cell death. However, little is known about the precise mechanism of p53 phosphorylation on the induction of apoptosis. Here, we show that amphiregulin (AREG) is identified for a direct target of Ser46 phosphorylation via the comprehensive expression analyses. Ser46-phosphorylated p53 selectively binds to the promoter region of AREG gene, indicating that the p53 modification changes target genes by altering its binding affinity to the promoter. Although AREG belongs to a family of the epidermal growth factor, it also emerges in the nucleus under DNA damage. To clarify nuclear function of AREG, we analyze AREG-binding proteins by mass spectrometry. AREG interacts with DEAD-box RNA helicase p68 (DDX5). Intriguingly, AREG regulates precursor microRNA processing (i.e., miR-15a) with DDX5 to reduce the expression of antiapoptotic protein Bcl-2. These findings collectively support a mechanism in which the induction of AREG by Ser46-phosphorylated p53 is required for the microRNA biogenesis in the apoptotic response to DNA damage. PMID:24379358

Taira, Naoe; Yamaguchi, Tomoko; Kimura, Junko; Lu, Zheng-Guang; Fukuda, Shinji; Higashiyama, Shigeki; Ono, Masaya; Yoshida, Kiyotsugu

2014-01-14

356

Coilin participates in the suppression of RNA polymerase I in response to cisplatin-induced DNA damage  

PubMed Central

Coilin is a nuclear phosphoprotein that concentrates within Cajal bodies (CBs) and impacts small nuclear ribonucleoprotein (snRNP) biogenesis. Cisplatin and ?-irradiation, which cause distinct types of DNA damage, both trigger the nucleolar accumulation of coilin, and this temporally coincides with the repression of RNA polymerase I (Pol I) activity. Knockdown of endogenous coilin partially overrides the Pol I transcriptional arrest caused by cisplatin, while both ectopically expressed and exogenous coilin accumulate in the nucleolus and suppress rRNA synthesis. In support of this mechanism, we demonstrate that both cisplatin and ?-irradiation induce the colocalization of coilin with RPA-194 (the largest subunit of Pol I), and we further show that coilin can specifically interact with RPA-194 and the key regulator of Pol I activity, upstream binding factor (UBF). Using chromatin immunoprecipitation analysis, we provide evidence that coilin modulates the association of Pol I with ribosomal DNA. Collectively, our data suggest that coilin acts to repress Pol I activity in response to cisplatin-induced DNA damage. Our findings identify a novel and unexpected function for coilin, independent of its role in snRNP biogenesis, establishing a new link between the DNA damage response and the inhibition of rRNA synthesis. PMID:21289084

Gilder, Andrew S.; Do, Phi M.; Carrero, Zunamys I; Cosman, Angela M.; Broome, Hanna J.; Velma, Venkatramreddy; Martinez, Luis A.; Hebert, Michael D.

2011-01-01

357

Use of synthetic oligodeoxyribonucleotides and primed cDNA as probes for pea (Pisum sativum L.) RNA and genomic DNA sequences  

Microsoft Academic Search

Two oligonucleotide sequences were synthesised by a solid-phase phosphotriester method. One of these sequences, A was a copy of part of a characterised cDNA clone encoding the basic subunit of legumin, a seed storage protein of Pisum sativum L. (garden pea); the other sequence B was predicted to be complementary to the 5' region of legumin mRNA on the basis

I. Marta Evans; John A. Gatehouse; Grantley W. Lycett; Donald Boulter

1984-01-01

358

High variety of known and new RNA and DNA viruses of diverse origins in untreated sewage.  

PubMed

Deep sequencing of untreated sewage provides an opportunity to monitor enteric infections in large populations and for high-throughput viral discovery. A metagenomics analysis of purified viral particles in untreated sewage from the United States (San Francisco, CA), Nigeria (Maiduguri), Thailand (Bangkok), and Nepal (Kathmandu) revealed sequences related to 29 eukaryotic viral families infecting vertebrates, invertebrates, and plants (BLASTx E score, <10(-4)), including known pathogens (>90% protein identities) in numerous viral families infecting humans (Adenoviridae, Astroviridae, Caliciviridae, Hepeviridae, Parvoviridae, Picornaviridae, Picobirnaviridae, and Reoviridae), plants (Alphaflexiviridae, Betaflexiviridae, Partitiviridae, Sobemovirus, Secoviridae, Tombusviridae, Tymoviridae, Virgaviridae), and insects (Dicistroviridae, Nodaviridae, and Parvoviridae). The full and partial genomes of a novel kobuvirus, salivirus, and sapovirus are described. A novel astrovirus (casa astrovirus) basal to those infecting mammals and birds, potentially representing a third astrovirus genus, was partially characterized. Potential new genera and families of viruses distantly related to members of the single-stranded RNA picorna-like virus superfamily were genetically characterized and named Picalivirus, Secalivirus, Hepelivirus, Nedicistrovirus, Cadicistrovirus, and Niflavirus. Phylogenetic analysis placed these highly divergent genomes near the root of the picorna-like virus superfamily, with possible vertebrate, plant, or arthropod hosts inferred from nucleotide composition analysis. Circular DNA genomes distantly related to the plant-infecting Geminiviridae family were named Baminivirus, Nimivirus, and Niminivirus. These results highlight the utility of analyzing sewage to monitor shedding of viral pathogens and the high viral diversity found in this common pollutant and provide genetic information to facilitate future studies of these newly characterized viruses. PMID:22933275

Ng, Terry Fei Fan; Marine, Rachel; Wang, Chunlin; Simmonds, Peter; Kapusinszky, Beatrix; Bodhidatta, Ladaporn; Oderinde, Bamidele Soji; Wommack, K Eric; Delwart, Eric

2012-11-01

359

SAD-2 is required for meiotic silencing by unpaired DNA and perinuclear localization of SAD-1 RNA-directed RNA polymerase  

PubMed Central

A gene unpaired during the meiotic homolog pairing stage in Neurospora generates a sequence-specific signal that silences the expression of all copies of that gene. This process is called Meiotic Silencing by Unpaired DNA (MSUD). Previously, we have shown that SAD-1, an RNA-directed RNA polymerase (RdRP), is required for MSUD. We isolated a second gene involved in this process, sad-2. Mutated Sad-2 RIP alleles, like those of Sad-1, are dominant and suppress MSUD. Crosses homozygous for Sad-2 are blocked at meiotic prophase. SAD-2 colocalizes with SAD-1 in the perinuclear region, where small interfering RNAs have been shown to reside in mammalian cells. A functional sad-2+ gene is necessary for SAD-1 localization, but the converse is not true. The data suggest that SAD-2 may function to recruit SAD-1 to the perinuclear region, and that the proper localization of SAD-1 is important for its activity. PMID:16461906

Shiu, Patrick K. T.; Zickler, Denise; Raju, Namboori B.; Ruprich-Robert, Gwenael; Metzenberg, Robert L.

2006-01-01

360

Quantitative model of R-loop forming structures reveals a novel level of RNA-DNA interactome complexity  

PubMed Central

R-loop is the structure co-transcriptionally formed between nascent RNA transcript and DNA template, leaving the non-transcribed DNA strand unpaired. This structure can be involved in the hyper-mutation and dsDNA breaks in mammalian immunoglobulin (Ig) genes, oncogenes and neurodegenerative disease related genes. R-loops have not been studied at the genome scale yet. To identify the R-loops, we developed a computational algorithm and mapped R-loop forming sequences (RLFS) onto 66?803 sequences defined by UCSC as ‘known’ genes. We found that ?59% of these transcribed sequences contain at least one RLFS. We created R-loopDB (http://rloop.bii.a-star.edu.sg/), the database that collects all RLFS identified within over half of the human genes and links to the UCSC Genome Browser for information integration and visualisation across a variety of bioinformatics sources. We found that many oncogenes and tumour suppressors (e.g. Tp53, BRCA1, BRCA2, Kras and Ptprd) and neurodegenerative diseases related genes (e.g. ATM, Park2, Ptprd and GLDC) could be prone to significant R-loop formation. Our findings suggest that R-loops provide a novel level of RNA–DNA interactome complexity, playing key roles in gene expression controls, mutagenesis, recombination process, chromosomal rearrangement, alternative splicing, DNA-editing and epigenetic modifications. RLFSs could be used as a novel source of prospective therapeutic targets. PMID:22121227

Wongsurawat, Thidathip; Jenjaroenpun, Piroon; Kwoh, Chee Keong; Kuznetsov, Vladimir

2012-01-01

361

Advantages and Limitations of Ribosomal RNA PCR and DNA Sequencing for Identification of Bacteria in Cardiac Valves of Danish Patients  

PubMed Central

Studies on the value of culture-independent molecular identification of bacteria in cardiac valves are mostly restricted to comparing agreement of identification to what is obtained by culture to the number of identified bacteria in culture-negative cases. However, evaluation of the usefulness of direct molecular identification should also address weaknesses, their relevance in the given setting, and possible improvements. In this study cardiac valves from 56 Danish patients referred for surgery for infective endocarditis were analysed by microscopy and culture as well as by PCR targeting part of the bacterial 16S rRNA gene followed by DNA sequencing of the PCR product. PCR and DNA sequencing identified significant bacteria in 49 samples from 43 patients, including five out of 13 culture-negative cases. No rare, exotic, or intracellular bacteria were identified. There was a general agreement between bacterial identity obtained by ribosomal PCR and DNA sequencing from the valves and bacterial isolates from blood culture. However, DNA sequencing of the 16S rRNA gene did not discriminate well among non-haemolytic streptococci, especially within the Streptococcus mitis group. Ribosomal PCR with subsequent DNA sequencing is an efficient and reliable method of identifying the cause of IE, but exact species identification of some of the most common causes, i.e. non-haemolytic streptococci, may be improved with other molecular methods. PMID:24403979

Kemp, Michael; Bangsborg, Jette; Kjerulf, Anne; Schmidt, Thomas Andersen; Christensen, John; Irmukhamedov, Akhmadjon 6; Bruun, Niels Eske; Dargis, Rimtas; Andresen, Keld; Christensen, Jens J?rgen

2013-01-01

362

Peach (Prunus persica) endopolygalacturonase cDNA isolation and mRNA analysis in melting and nonmelting peach cultivars.  

PubMed Central

Two distinct partial cDNAs, PRF1 and PRF3, similar in sequence to previously described polygalacturonases, were amplified from ripe peach (Prunus persica L. Batsch cv Flavorcrest) fruit cDNA by the polymerase chain reaction. PRF1-related RNA was present in fruit from early ripening at levels not detected by northern analysis. PRF3-related RNA was readily detectable in ripe fruit by northern analysis. PRF3 was used to isolate a cDNA with a complete open reading frame, PRF5, from a lambda ZAP II cDNA library prepared from poly(A)+ RNA of ripe peach fruit. PRF5 coded for a predicted protein of 393 amino acids with a molecular mass of 41,500 D. The derived amino acid sequence of PRF5 included a putative leader sequence of 23 amino acids, followed by a sequence that matched the N terminus of endopolygalacturonase protein purified from ripe peach fruit. By northern analysis, PRF3-related RNA was undetectable in firm, unripe Flavorcrest fruit. It appeared at low levels as a 1.7-kb transcript in fruit that had begun to ripen and soften and was very abundant in ripe fruit that had undergone the "melting" stage of softening. The marked increase in PRF3-related RNA levels took place over a period of less than 2 d at 20 degrees C and coincided with the climacteric peak in ethylene evolution. Levels of 1-aminocyclopropane-1-carboxylate oxidase-related RNA increased during ripening at a much earlier stage than levels of PRF3-related RNA. Lower levels of 1.7-kb RNA transcript were detected by PRF3 in ripe fruit of the melting cultivar Fragar, which are firmer than Flavorcrest fruit. In ripe fruit of the nonmelting cultivar Carolyn, PRF3 detected a 1.45-kb RNA transcript that was present at low levels. Transcripts of a peach polygalacturonase-related genomic sequence were not detected in ripening fruit. PMID:8029352

Lester, D R; Speirs, J; Orr, G; Brady, C J

1994-01-01

363

Efficient targeted pDNA/siRNA delivery with folate-low-molecular-weight polyethyleneimine-modified pullulan as non-viral carrier.  

PubMed

Folate receptor (FR)-mediated gene/short interfering RNA (siRNA) targeting shows advantage for the delivery of gene/siRNA into specific FR-overexpressing cancer cells. In this study, the non-targeted gene vector P-PEI was synthesized by grafting low-molecular-weight (1kDa) branched polyethyleneimine (PEI) to succinylated pullulan, and the targeted gene vector P-PEI-FA was synthesized by coupling the carboxyl of folate (FA) to the amino of PEI. Gel electrophoresis retardation assay demonstrated that both P-PEI and P-PEI-FA can efficiently wrap pDNA and siRNA with electrostatic interaction at N/P ratios higher than 1.56 and can protect pDNA from degradation by DNase I and serum. Compared with PEI/pDNA, P-PEI/pDNA and P-PEI-FA/pDNA showed lower cytotoxicity against different cells. Under serum-containing conditions, compared with Lipofamine 2000/DNA and Lipofamine2000/siRNA, P-PEI-FA/DNA at N/P ratio of 6.25 displayed higher gene transfection efficiency, whereas P-PEI-FA/siRNA at N/P ratio of 12.5 demonstrated better enhanced gene silencing effect. P-PEI-FA/siRNA can also deliver FAM-labeled siRNA to endosomes and escape. Moreover, the gene transfection and silencing effects of P-PEI-FA were higher than those of P-PEI, and were dependent on the dose of FA in FR(+) HeLa cells. Thus, P-PEI-FA can assist DNA or siRNA targeting to FR-overexpressing cells, and the uptake pathway of P-PEI-FA/siRNA was FR-mediated endocytosis. These results indicate that P-PEI-FA is a potential candidate for safe and targeted gene delivery applications. PMID:24268238

Wang, Jingyun; Dou, Bairui; Bao, Yongming

2014-01-01

364

RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee  

PubMed Central

Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing. PMID:23852726

Li-Byarlay, Hongmei; Li, Yang; Stroud, Hume; Feng, Suhua; Newman, Thomas C.; Kaneda, Megan; Hou, Kirk K.; Worley, Kim C.; Elsik, Christine G.; Wickline, Samuel A.; Jacobsen, Steven E.; Ma, Jian; Robinson, Gene E.

2013-01-01

365

Nonempirically Tuned Range-Separated DFT Accurately Predicts Both Fundamental and Excitation Gaps in DNA and RNA Nucleobases  

PubMed Central

Using a nonempirically tuned range-separated DFT approach, we study both the quasiparticle properties (HOMO–LUMO fundamental gaps) and excitation energies of DNA and RNA nucleobases (adenine, thymine, cytosine, guanine, and uracil). Our calculations demonstrate that a physically motivated, first-principles tuned DFT approach accurately reproduces results from both experimental benchmarks and more computationally intensive techniques such as many-body GW theory. Furthermore, in the same set of nucleobases, we show that the nonempirical range-separated procedure also leads to significantly improved results for excitation energies compared to conventional DFT methods. The present results emphasize the importance of a nonempirically tuned range-separation approach for accurately predicting both fundamental and excitation gaps in DNA and RNA nucleobases. PMID:22904693

2012-01-01

366

Non-Empirically Tuned Range-Separated DFT Accurately Predicts Both Fundamental and Excitation Gaps in DNA and RNA Nucleobases  

E-print Network

Using a non-empirically tuned range-separated DFT approach, we study both the quasiparticle properties (HOMO-LUMO fundamental gaps) and excitation energies of DNA and RNA nucleobases (adenine, thymine, cytosine, guanine, and uracil). Our calculations demonstrate that a physically-motivated, first-principles tuned DFT approach accurately reproduces results from both experimental benchmarks and more computationally intensive techniques such as many-body GW theory. Furthermore, in the same set of nucleobases, we show that the non-empirical range-separated procedure also leads to significantly improved results for excitation energies compared to conventional DFT methods. The present results emphasize the importance of a non-empirically tuned range-separation approach for accurately predicting both fundamental and excitation gaps in DNA and RNA nucleobases.

Foster, Michael E; 10.1021/ct300420f

2012-01-01

367

Nonempirically Tuned Range-Separated DFT Accurately Predicts Both Fundamental and Excitation Gaps in DNA and RNA Nucleobases.  

PubMed

Using a nonempirically tuned range-separated DFT approach, we study both the quasiparticle properties (HOMO-LUMO fundamental gaps) and excitation energies of DNA and RNA nucleobases (adenine, thymine, cytosine, guanine, and uracil). Our calculations demonstrate that a physically motivated, first-principles tuned DFT approach accurately reproduces results from both experimental benchmarks and more computationally intensive techniques such as many-body GW theory. Furthermore, in the same set of nucleobases, we show that the nonempirical range-separated procedure also leads to significantly improved results for excitation energies compared to conventional DFT methods. The present results emphasize the importance of a nonempirically tuned range-separation approach for accurately predicting both fundamental and excitation gaps in DNA and RNA nucleobases. PMID:22904693

Foster, Michael E; Wong, Bryan M

2012-08-14

368

Recovery of Infectious Virus by Transfection of In Vitro-Generated RNA from Tulane Calicivirus cDNA?  

PubMed Central

Tulane virus (TV) is a newly reported calicivirus that was isolated from stool samples of captive rhesus macaques from the Tulane National Primate Research Center (TNPRC). The virus has been cultivated successfully in LLC-MK2 rhesus monkey kidney cells. Its complete genomic sequence suggests that TV represents a new genus and is evolutionarily more closely related to Norovirus than to any other genus of Caliciviridae. In this study, we demonstrated that RNA transcripts made in vitro from the full-length genomic cDNA of TV were infectious upon transfection into permissive LLC-MK2 cells. The recombinant virus exhibited plaque morphologies and growth kinetics similar to those of the wild-type virus in this cell line. Capping was required for TV RNA infectivity. Although a subgenomic RNA has been detected in TV-transfected cells, a separate subgenomic RNA transcript was not required for the initial transfection to establish the replication. Transfection of truncated RNA lacking open reading frame 2 (ORF2) and ORF3 or TV-norovirus chimeric RNA resulted in abortive replication without the production of infectious progeny viruses, indicating that both ORFs are essential for the replication of TV. A heterologous insertion at the 5? end of the genome also hampered viral replication, suggesting that an authentic 5? end of the genome is critical for replication. The availability of the complete genomic sequence and the reverse genetics system described herein make TV a valuable model for studying calicivirus pathogenesis and replication. PMID:18787011

Wei, Chao; Farkas, Tibor; Sestak, Karol; Jiang, Xi

2008-01-01

369

Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma  

Microsoft Academic Search

Background  RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream\\u000a applications. Extraction of high quality nucleic acids is difficult from neuronal cells and brain tissues as they are particularly\\u000a rich in lipids. In addition, most common RNA extraction methods are phenol-based, resulting in RNA that may be incompatible\\u000a with downstream applications

Lucélia Tavares; Paula M Alves; Ricardo B Ferreira; Claudia N Santos

2011-01-01

370

Effects of trace metals on growth of yellow perch ( Perca flavescens ) as measured by RNA-DNA ratios  

Microsoft Academic Search

Synopsis Relationships between sublethal concentrations of cadmium and zinc in natural water and metal uptake by and growth of fish were investigated. RNA-DNA ratios and weight gain were used to assess seasonal growth differences between yellow perch populations from contaminated and control sites. Whole-body concentrations of cadmium and zinc in young-of-the-year perch (Perca flavescens) were significantly different between sites. Measurable

Paul K. Kearns; Gary J. Atchison

1979-01-01

371

Repression of mRNA for the PLK cell cycle gene after DNA damage requires BRCA1.  

PubMed

DNA damage activates the G2 cell cycle checkpoint to allow time for DNA repair before mitotic entry. The mechanism involves inhibition of the enzymatic activity for polo-like kinase 1 (Plk1), rendering Cdc25C with a basal phosphatase activity that is insufficient for converting Cdc2 to the fully active G2/M transition kinase. We found that cell cycle arrest at the G2/M boundary after ionizing radiation (IR) of breast carcinoma cells may involve repression of the gene for Plk1, PLK, mediated by the tumor-suppressor protein BRCA1. The p53-defective MT-1 cell line had an apparent accumulation of G2/M phase cells 12 h after irradiation. This response was preceded by a transient downregulation of PLK mRNA expression with a barely detectable level 6 h after exposure to IR but recovered after 12 h. A significantly lower fraction of irradiated BRCA1(-/-) HCC1937 cells arrested in the G2/M phase after 12 h, and the transient response of PLK mRNA was also considerably impaired. After reconstitution of wild-type BRCA1 in the HCC1937 cells however, downregulation of PLK mRNA as well as Plk1 protein expression after IR was restored. Moreover, the suppression of PLK mRNA expression 6 h after irradiation was completely abolished by the specific CHEK1 kinase inhibitor UCN-01, further indicating that the effector mechanism of DNA damage on PLK signals through BRCA1 and its downstream CHEK1. Our observations provide new information about the diversity of regulatory mechanisms governed by BRCA1 in DNA damage checkpoint control. PMID:14654792

Ree, Anne Hansen; Bratland, Ase; Nome, Ragnhild V; Stokke, Trond; Fodstad, Øystein

2003-12-01

372

Coordinating responses to iron and oxygen stress with DNA and mRNA promoters: The ferritin story  

Microsoft Academic Search

Combinations of DNA antioxidant response element and mRNA iron responsive element regulate ferritin expression in animals\\u000a in response to oxidant and iron stress, or normal developmental signals. Ferritins are protein nanocages, found in animals,\\u000a plants, bacteria, and archaea, that convert iron and oxygen to ferric oxy biominerals in the protein central cavity; the mineral\\u000a traps potentially toxic reactants and concentrates

Elizabeth C. Theil

2007-01-01

373

Effects of downregulating DNA methyltransferase 1 transcript by RNA interference on DNA methylation status of the satellite I region and in vitro development of bovine somatic cell nuclear transfer embryos.  

PubMed

For the successful production of cloned animals by somatic cell nuclear transfer (NT), the epigenetic status of the differentiated donor cell is reversed to an embryonic totipotent status. However, in NT embryos, this process is aberrant, with genomic hypermethylation consistently observed. Here, we investigated the effects of silencing DNA methyltransferase 1 (DNMT1) mRNA by small interfering RNA (siRNA) on the DNA methylation status of the satellite I region and in vitro development of bovine NT embryos. First, the levels of DNMT1 expression were analyzed at 0, 24, 48, 72, 120 and 192 h after in vitro culture. Real-time PCR and western blotting analyses detected a significant decrease in DNMT1 mRNA in the siRNA-injected NT (siRNA-NT) group up to 72 h after in vitro culture. Next, the levels of DNA methylation of the satellite I region were analyzed at several time points after in vitro culture. The level of DNA methylation detected in siRNA-NT embryos was significantly less than those in NT embryos throughout in vitro development. Moreover, the developmental rate of embryos to blastocysts in the siRNA-NT group was significantly higher than that of NT embryos. Our data suggest that knockdown of DNMT1 mRNA in NT embryos can induce DNA demethylation, which may enhance reprogramming efficiency. PMID:21343670

Yamanaka, Ken-ichi; Sakatani, Miki; Kubota, Kaiyu; Balboula, Ahmed Zaky; Sawai, Ken; Takahashi, Masashi

2011-06-01

374

Messenger RNA, Matthew MeselsonSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Interviewee: Matt Meselson DNAi Location:Code>Copying the code>players Messenger RNA experiment Matt Meselson also had a hand in Sydney Brenner's RNA experiment. He talks about the experiment and how they waited for James Watson's group to finished their RNA work before publishing.

2008-10-06

375

Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB.  

PubMed

Nucleic acid damage by environmental and endogenous alkylation reagents creates lesions that are both mutagenic and cytotoxic, with the latter effect accounting for their widespread use in clinical cancer chemotherapy. Escherichia coli AlkB and the homologous human proteins ABH2 and ABH3 (refs 5, 7) promiscuously repair DNA and RNA bases damaged by S(N)2 alkylation reagents, which attach hydrocarbons to endocyclic ring nitrogen atoms (N1 of adenine and guanine and N3 of thymine and cytosine). Although the role of AlkB in DNA repair has long been established based on phenotypic studies, its exact biochemical activity was only elucidated recently after sequence profile analysis revealed it to be a member of the Fe-oxoglutarate-dependent dioxygenase superfamily. These enzymes use an Fe(II) cofactor and 2-oxoglutarate co-substrate to oxidize organic substrates. AlkB hydroxylates an alkylated nucleotide base to produce an unstable product that releases an aldehyde to regenerate the unmodified base. Here we have determined crystal structures of substrate and product complexes of E. coli AlkB at resolutions from 1.8 to 2.3 A. Whereas the Fe-2-oxoglutarate dioxygenase core matches that in other superfamily members, a unique subdomain holds a methylated trinucleotide substrate into the active site through contacts to the polynucleotide backbone. Amide hydrogen exchange studies and crystallographic analyses suggest that this substrate-binding 'lid' is conformationally flexible, which may enable docking of diverse alkylated nucleotide substrates in optimal catalytic geometry. Different crystal structures show open and closed states of a tunnel putatively gating O2 diffusion into the active site. Exposing crystals of the anaerobic Michaelis complex to air yields slow but substantial oxidation of 2-oxoglutarate that is inefficiently coupled to nucleotide oxidation. These observations suggest that protein dynamics modulate redox chemistry and that a hypothesized migration of the reactive oxy-ferryl ligand on the catalytic Fe ion may be impeded when the protein is constrained in the crystal lattice. PMID:16482161

Yu, Bomina; Edstrom, William C; Benach, Jordi; Hamuro, Yoshitomo; Weber, Patricia C; Gibney, Brian R; Hunt, John F

2006-02-16

376

Generation of marker-free transgenic plants concurrently resistant to a DNA geminivirus and a RNA tospovirus.  

PubMed

Global threats of ssDNA geminivirus and ss(-)RNA tospovirus on crops necessitate the development of transgenic resistance. Here, we constructed a two-T DNA vector carrying a hairpin of the intergenic region (IGR) of Ageratum yellow vein virus (AYVV), residing in an intron inserted in an untranslatable nucleocapsid protein (NP) fragment of Melon yellow spot virus (MYSV). Transgenic tobacco lines highly resistant to AYVV and MYSV were generated. Accumulation of 24-nt siRNA, higher methylation levels on the IGR promoters of the transgene, and suppression of IGR promoter activity of invading AYVV indicate that AYVV resistance is mediated by transcriptional gene silencing. Lack of NP transcript and accumulation of corresponding siRNAs indicate that MYSV resistance is mediated through post-transcriptional gene silencing. Marker-free progenies with concurrent resistance to both AYVV and MYSV, stably inherited as dominant nuclear traits, were obtained. Hence, we provide a novel way for concurrent control of noxious DNA and RNA viruses with less biosafety concerns. PMID:25030413

Yang, Ching-Fu; Chen, Kuan-Chun; Cheng, Ying-Hui; Raja, Joseph A J; Huang, Ya-Ling; Chien, Wan-Chu; Yeh, Shyi-Dong

2014-01-01

377

5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D 19F NMR spectroscopy  

PubMed Central

19F NMR spectroscopy has proved to be a valuable tool to monitor functionally important conformational transitions of nucleic acids. Here, we present a systematic investigation on the application of 5-fluoro pyrimidines to probe DNA and RNA secondary structures. Oligonucleotides with the propensity to adapt secondary structure equilibria were chosen as model systems and analyzed by 1D 19F and 1H NMR spectroscopy. A comparison with the unmodified analogs revealed that the equilibrium characteristics of the bistable DNA and RNA oligonucleotides were hardly affected upon fluorine substitution at C5 of pyrimidines. This observation was in accordance with UV spectroscopic melting experiments which demonstrated that single 5-fluoro substitutions in double helices lead to comparable thermodynamic stabilities. Thus, 5-fluoro pyrimidine labeling of DNA and RNA can be reliably applied for NMR based nucleic acid secondary structure evaluation. Furthermore, we developed a facile synthetic route towards 5-fluoro cytidine phosphoramidites that enables their convenient site-specific incorporation into oligonucleotides by solid-phase synthesis. PMID:19843610

Puffer, Barbara; Kreutz, Christoph; Rieder, Ulrike; Ebert, Marc-Olivier; Konrat, Robert; Micura, Ronald

2009-01-01

378

Multiplexed microRNA detection using lanthanide-labeled DNA probes and laser ablation inductively coupled plasma mass spectrometry.  

PubMed

In the past decade, microRNAs (miRNAs) have drawn increasing attention due to their role in regulation of gene expression. Especially, their potential as biomarkers in disease diagnostics has motivated miRNA research, including the development of simple, accurate, and sensitive detection methods. The narrow size range of miRNAs (20-24 nucleotides) combined with the chemical properties of conventional reporter tags has hampered the development of multiplexed miRNA assays. In this study, we have used lanthanide-labeled DNA probes for the detection of miRNAs on membranes using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). Three miRNAs from Arabidopsis thaliana were analyzed simultaneously with high specificity, and the sensitivity of the method was comparable to radioactive detection (low femtomol range). The perspective of the developed method is highly multiplexed and quantitative miRNA analysis with high specificity and sensitivity. PMID:24945747

de Bang, Thomas Christian; Shah, Pratik; Cho, Seok Keun; Yang, Seong Wook; Husted, Søren

2014-07-15

379

A rat brain mRNA encoding a transcriptional activator homologous to the DNA binding domain of retroviral integrases.  

PubMed Central

We have isolated a rat cDNA, named FE65, hybridizing to an mRNA of about 2,300 nucleotides present in rat brain, undetectable in rat liver and very poorly represented in other tissues. An mRNA of the same size is present in human neuroblastoma cells and is absent from other human cell lines. The FE65 cDNA contains an open reading frame (ORF) coding for a polypeptide of 499 amino acids in which 143 residues can be aligned with the DNA binding domain of the integrases encoded by mammalian immunodeficiency viruses. The remaining part of the FE65 ORF is not homologous with the correspondent regions of the integrases; the first 206 residues of the FE65 ORF show numerous negative charges and a short sequence not dispensable for the function of the transactivating acidic domain of the jun family transcriptional factors. A plasmid which expresses FE65 amino acids 1-232 fused to the yeast GAL4 DNA binding domain was co-transfected with a plasmid containing five GAL4 binding sites upstream of a minimal Adenovirus promoter controlling the expression of the CAT gene. This experiment showed that the fused protein GAL4-FE65 is able to obtain a 30-40 fold increase of the CAT gene expression compared to the expression observed in the presence of the GAL4 DNA binding domain alone. Two types of FE65 mRNA are present in rat brain, differing only for six nucleotides. We demonstrate that this is the consequence of a neuron-specific alternative splicing of a six-nucleotide miniexon, which is also present in the human genome, in an intron/exon context very similar to that of the rat FE65 gene. Images PMID:1923810

Duilio, A; Zambrano, N; Mogavero, A R; Ammendola, R; Cimino, F; Russo, T

1991-01-01

380

Genome-wide DNA Methylation Profiles and Their Relationships with mRNA and the microRNA Transcriptome in Bovine Muscle Tissue (Bos taurine).  

PubMed

DNA methylation is a key epigenetic modification in mammals and plays important roles in muscle development. We sampled longissimus dorsi muscle (LDM) from a well-known elite native breed of Chinese Qinchuan cattle living within the same environment but displaying distinct skeletal muscle at the fetal and adult stages. We generated and provided a genome-wide landscape of DNA methylomes and their relationship with mRNA and miRNA for fetal and adult muscle studies. Integration analysis revealed a total of 77 and 1,054 negatively correlated genes with methylation in the promoter and gene body regions, respectively, in both the fetal and adult bovine libraries. Furthermore, we identified expression patterns of high-read genes that exhibit a negative correlation between methylation and expression from nine different tissues at multiple developmental stages of bovine muscle-related tissue or organs. In addition, we validated the MeDIP-Seq results by bisulfite sequencing PCR (BSP) in some of the differentially methylated promoters. Together, these results provide valuable data for future biomedical research and genomic and epigenomic studies of bovine skeletal muscle that may help uncover the molecular basis underlying economically valuable traits in cattle. This comprehensive map also provides a solid basis for exploring the epigenetic mechanisms of muscle growth and development. PMID:25306978

Huang, Yong-Zhen; Sun, Jia-Jie; Zhang, Liang-Zhi; Li, Cong-Jun; Womack, James E; Li, Zhuan-Jian; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

2014-01-01

381

Genome-wide DNA Methylation Profiles and Their Relationships with mRNA and the microRNA Transcriptome in Bovine Muscle Tissue (Bos taurine)  

PubMed Central

DNA methylation is a key epigenetic modification in mammals and plays important roles in muscle development. We sampled longissimus dorsi muscle (LDM) from a well-known elite native breed of Chinese Qinchuan cattle living within the same environment but displaying distinct skeletal muscle at the fetal and adult stages. We generated and provided a genome-wide landscape of DNA methylomes and their relationship with mRNA and miRNA for fetal and adult muscle studies. Integration analysis revealed a total of 77 and 1,054 negatively correlated genes with methylation in the promoter and gene body regions, respectively, in both the fetal and adult bovine libraries. Furthermore, we identified expression patterns of high-read genes that exhibit a negative correlation between methylation and expression from nine different tissues at multiple developmental stages of bovine muscle-related tissue or organs. In addition, we validated the MeDIP-Seq results by bisulfite sequencing PCR (BSP) in some of the differentially methylated promoters. Together, these results provide valuable data for future biomedical research and genomic and epigenomic studies of bovine skeletal muscle that may help uncover the molecular basis underlying economically valuable traits in cattle. This comprehensive map also provides a solid basis for exploring the epigenetic mechanisms of muscle growth and development. PMID:25306978

Huang, Yong-Zhen; Sun, Jia-Jie; Zhang, Liang-Zhi; Li, Cong-Jun; Womack, James E.; Li, Zhuan-Jian; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

2014-01-01

382

Study of the genetic organisation of a plant viral RNA genome by in vitro expression of a full-length DNA copy.  

PubMed

The genetic approach for elucidating functions encoded by RNA plant viruses has been hampered by the lack of methods to select desired mutants following random mutagenesis. An alternative might be to copy RNA genomes into DNA and use methods for site-directed mutagenesis to modify specific regions of the DNA copy. Transcription of the DNA copy will subsequently produce viral RNA with desired mutations. We have constructed a full-length DNA copy of the smaller of the two cowpea mosaic virus (CPMV) RNAs, referred to as M RNA. The DNA copy was positioned downstream from the promoter of bacteriophage SP6 and using SP6 RNA polymerase, this copy and two derivatives of it containing a specific deletion and insertion, respectively, have been transcribed into RNA molecules which are efficiently translated in rabbit reticulocyte lysates. The results obtained show that the subsequent in vitro transcription and translation of DNA copies may be a powerful tool to unravel the genetic properties of viral RNA genomes. PMID:6549293

Vos, P; Verver, J; van Wezenbeek, P; van Kammen, A; Goldbach, R

1984-12-20

383

Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors  

PubMed Central

Background The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer. Results We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein. Conclusions Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer. PMID:24257477

2013-01-01

384

Composition and Dynamics of Bacterial Communities of a Drinking Water Supply System as Assessed by RNA- and DNA-Based 16S rRNA Gene Fingerprinting  

PubMed Central

Bacterial community dynamics of a whole drinking water supply system (DWSS) were studied from source to tap. Raw water for this DWSS is provided by two reservoirs with different water characteristics in the Harz mountains of Northern Germany. Samples were taken after different steps of treatment of raw water (i.e., flocculation, sand filtration, and chlorination) and at different points along the supply system to the tap. RNA and DNA were extracted from the sampled water. The 16S rRNA or its genes were partially amplified by reverse transcription-PCR or PCR and analyzed by single-strand conformation polymorphism community fingerprints. The bacterial community structures of the raw water samples from the two reservoirs were very different, but no major changes of these structures occurred after flocculation and sand filtration. Chlorination of the processed raw water strongly affected bacterial community structure, as reflected by the RNA-based fingerprints. This effect was less pronounced for the DNA-based fingerprints. After chlorination, the bacterial community remained rather constant from the storage containers to the tap. Furthermore, the community structure of the tap water did not change substantially for several months. Community composition was assessed by sequencing of abundant bands and phylogenetic analysis of the sequences obtained. The taxonomic compositions of the bacterial communities from both reservoirs were very different at the species level due to their different limnologies. On the other hand, major taxonomic groups, well known to occur in freshwater, such as Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes, were found in both reservoirs. Significant differences in the detection of the major groups were observed between DNA-based and RNA-based fingerprints irrespective of the reservoir. Chlorination of the drinking water seemed to promote growth of nitrifying bacteria. Detailed analysis of the community dynamics of the whole DWSS revealed a significant influence of both source waters on the overall composition of the drinking water microflora and demonstrated the relevance of the raw water microflora for the drinking water microflora provided to the end user. PMID:16517632

Eichler, Stefan; Christen, Richard; Holtje, Claudia; Westphal, Petra; Botel, Julia; Brettar, Ingrid; Mehling, Arndt; Hofle, Manfred G.

2006-01-01