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1

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

2

Resolution enhancement in Raman spectra of biological macromolecules by Fourier deconvolution: applications to single-stranded DNA and RNA viruses1  

NASA Astrophysics Data System (ADS)

The application of a constrained, iterative Fourier deconvolution method to Raman spectra of viruses permits separation of overlapped vibrational bands assigned to viral protein and nucleic acid constituents. The intrinsically broad and extensively overlapped Raman lines, which cannot be resolved by instrumental methods, are sufficiently well separated in the deconvoluted spectra so that band areas may be measured with sufficient accuracy to allow conclusions about the secondary structures and hydrogen bonding interactions of viral molecular components. Deconvolution of Raman scattering in the region 1500-1750 cm -1 of filamentous bacteriophages resolves the contributions of(i)amide I modes ofalpha-helix and beta-strand conformations of the viral coat proteins, (ii) aromatic ring modes of tryptophan, tyrosine and phenylalanine side chains and (iii) purine ring modes of the coated DNA molecule. The results show that among six filamentous viruses the amount of alpha-helix in coat protein subunits decreases in the following order: Pfl (100%) > IKe (93%) > fd (92%) > Ifl (90%) > Pf3 (82%) > Xf (71%). In an application to tobacco mosaic virus (TMV), the complex band in the 800-850 cm -1 region is deconvoluted to resolve contributions from the encapsidated RNA genome at 813 and 822 cm -1, which indicate at least two distinct nucleoside conformers and a contribution from the tyrosine rings of protein subunits at 831 cm -1. Integrated intensity measurements suggest that at least two-thirds of the nucleosides do not contain the usual C3'-endo ring pucker and anti orientation of the glycosidic bond normally associated with nucleoside residues of single-stranded RNA.

Thomas, George J.

3

Molecular Biology DNA: The Genetic Macromolecule  

E-print Network

, turned on or off depending on the conditions of the cell. Bacteria normally take up DNA from to contain a 3 genes as well as signals that allow it to be replicated and expressed inside living cells begun our Molecular Biology course accepting that DNA is the genetic material of the cell, our first lab

4

Nonlinear electrophoretic focusing of DNA macromolecules.  

PubMed

A new approach to focusing DNA molecules in a nonuniform electric field based on nonlinear mobility (L. L. Frumin, S. E. Peltek, S. Bukshpan, V. V. Chasovskikh and G. V. Zilberstein, PhysChemComm, 2000, 11) is proposed. The focusing is carried out in an alternating nonuniform electric field, created by using a wedge gel with hyperbolic boundaries. Methods of the theory of analytical functions were used to demonstrate that the fractions separated electrophoretically in such a wedge retain their rectilinear shape. Solutions for the focusing points for the case of mere velocity cubic nonlinearity were obtained as well as for the square velocity nonlinearity, suggested in a number of modern approaches. Gel electrophoresis experiments supported the possibility of a pronounced nonlinear focusing of DNA molecules. This nonlinear separation technique presents encouraging prospects for preparative isolation of long DNA fragments and development of new separation methods for bacterial fingerprinting. PMID:11394292

Frumin, L L; Peltek, S E; Zilberstein, G V

2001-05-01

5

Triggering of RNA Interference with RNA-RNA, RNA-DNA, and DNA-RNA Nanoparticles.  

PubMed

Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use. PMID:25521794

Afonin, Kirill A; Viard, Mathias; Kagiampakis, Ioannis; Case, Christopher L; Dobrovolskaia, Marina A; Hofmann, Jen; Vrzak, Ashlee; Kireeva, Maria; Kasprzak, Wojciech K; KewalRamani, Vineet N; Shapiro, Bruce A

2015-01-27

6

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

7

Quantum Confinement in Hydrogen Bond of DNA and RNA  

E-print Network

The hydrogen bond is a fundamental ingredient to stabilize the DNA and RNA macromolecules. The main contribution of this work is to describe quantitatively this interaction as a consequence of the quantum confinement of the hydrogen. The results for the free and confined system are compared with experimental data. The formalism to compute the energy gap of the vibration motion used to identify the spectrum lines is the Variational Method allied to Supersymmetric Quantum Mechanics.

Santos, da Silva dos; Ricotta, Regina Maria

2015-01-01

8

Quantum Confinement in Hydrogen Bond of DNA and RNA  

E-print Network

The hydrogen bond is a fundamental ingredient to stabilize the DNA and RNA macromolecules. The main contribution of this work is to describe quantitatively this interaction as a consequence of the quantum confinement of the hydrogen. The results for the free and confined system are compared with experimental data. The formalism to compute the energy gap of the vibration motion used to identify the spectrum lines is the Variational Method allied to Supersymmetric Quantum Mechanics.

da Silva dos Santos; Elso Drigo Filho; Regina Maria Ricotta

2015-02-09

9

Inosine in DNA and RNA.  

PubMed

Deamination of the nucleobases in DNA and RNA is a result of spontaneous hydrolysis, endogenous or environmental factors as well as deaminase enzymes. Adenosine is deaminated to inosine which is miscoding and preferentially base pairs with cytosine. In the case of DNA, this is a premutagenic event that is counteracted by DNA repair enzymes specifically engaged in recognition and removal of inosine. However, in RNA, inosine is an essential modification introduced by specialized enzymes in a highly regulated manner to generate transcriptome diversity. Defect editing is seen in various human disease including cancer, viral infections and neurological and psychiatric disorders. Enzymes catalyzing the deaminase reaction are well characterized and recently an unexpected function of Endonuclease V in RNA processing was revealed. Whereas bacterial Endonuclease V enzymes are classified as DNA repair enzymes, it appears that the mammalian enzymes are involved in processing of inosine in RNA. This yields an interesting yet unexplored, link between DNA and RNA processing. Further work is needed to gain understanding of the impact of inosine in DNA and RNA under normal physiology and disease progression. PMID:25173738

Alseth, Ingrun; Dalhus, Bjørn; Bjørås, Magnar

2014-06-01

10

Diffraction in resonant electron scattering from helical macromolecules: A-and B-type DNA Laurent Caron* and Lon Sanche  

E-print Network

Diffraction in resonant electron scattering from helical macromolecules: A- and B-type DNA Laurent in B-type DNA. Diffraction patterns due to base-pair spacing are observed under all conditions. We discuss the role of electron diffraction in electron attachment to the bases leading to the formation

Simons, Jack

11

Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir  

Microsoft Academic Search

Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When (³H)thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. In an attempt to specifically label phytoplankton DNA, samples were incubated with (³H)adenine or ³²P{sub i} in

J. H. Paul; W. H. Jeffrey; J. P. Cannon

1990-01-01

12

RNA-Linked DNA Fragments In Vitro*  

PubMed Central

RNA-linked DNA fragments are intermediates in DNA replication in Escherichia coli cells made permeable to nucleoside triphosphates by treatment with toluene. Covalent linkage of a short RNA stretch to the 5? end of the DNA is proved by transfer of 32P from [?-32P]dNTP to ribonucleotides upon digestion with alkali or pancreatic RNase, and by a small decrease in the molecular size upon alkaline hydrolysis. The 32P transfer experiments reveal a unique structure...p(rPy)p(rA)p(rU or rC)p(dC)p... at the RNA-DNA junction. PMID:4567338

Sugino, Akio; Okazaki, Reiji

1973-01-01

13

RNA-Linked DNA Fragments in vitro  

Microsoft Academic Search

RNA-linked DNA fragments are intermediates in DNA replication in Escherichia coli cells made permeable to nucleoside triphosphates by treatment with toluene. Covalent linkage of a short RNA stretch to the 5' end of the DNA is proved by transfer of 32P from [alpha -32P]dNTP to ribonucleotides upon digestion with alkali or pancreatic RNase, and by a small decrease in the

Akio Sugino; Reiji Okazaki

1973-01-01

14

Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir.  

PubMed

Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When [3H]thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. The rate of production of labeled dissolved macromolecules was 3.1% the rate of cellular incorporation of [3H]thymidine, and the production of dissolved DNA represented 2.3% the rate of cellular DNA incorporation. Microautotrophic populations labeled with NaH[14C]CO3 produced dissolved RNA and protein at rates of 0.24 and 0.11 micrograms of C/liter per h, respectively, or 1.8% the total rate of carbon fixation, with no measurable dissolved DNA production. In an attempt to specifically label phytoplankton DNA, samples were incubated with [3H]adenine or 32Pi in the presence and absence of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Although DCMU inhibited 14C fixation by approximately 99%, this antimetabolite had only a slight effect on [3H]adenine incorporation and no effect on 32P incorporation into cellular macromolecules. Significant amounts of dissolved DNA were produced in both [3H]adenine and 32Pi incubations, but again DCMU had no effect on the production rates. These results indicate that actively growing populations of both phytoplankton and bacterioplankton produced dissolved RNA and protein, while only active bacterioplankton produced measurable quantities of dissolved DNA. Dead or senescent phytoplankton may have produced dissolved DNA, but would not be measured in the relatively short incubations used. These findings also indicate that [3H]adenine and 32Pi primarily labeled heterotrophic bacterioplankton and not phytoplankton in this environment. PMID:1704697

Paul, J H; Jeffrey, W H; Cannon, J P

1990-10-01

15

DNA repair meets the RNA world.  

PubMed

The ability to repair damaged DNA and to maintain genome stability is the utmost importance for the survival of any species. Hence, it is not surprising to find that DNA repair mechanisms are evolutionarily conserved and are expected to evolve to maintain the existence of species. In the last few years, there has been an exponential increase in the evidence linking RNA processing with DNA repair programs. For instance, the well-studied DNA base excision repair (BER) enzyme apurinic/apyrimidinic endonuclease 1 can cleave RNA molecules, regulate mRNA levels, and associate physically with proteins involved in RNA processing. It is now clear that not only the expression of noncoding RNAs are changed upon DNA damage, they can modulate the expression of genes involved in the genome stability programs. The five reviews in this Forum provide the up-to-date knowledge on DNA repair, with a focus on BER, and a perspective on how the two ancient biochemical pathways are linked. The contributions demonstrate the complexity of such interactions, but also pointed out the opportunities for new therapeutic interventions. Future in vivo studies on the link between DNA repair processes and RNA metabolism should contribute to our basic understanding of physiology, disease, and treatment strategies. PMID:24252191

Lee, Chow H

2014-02-01

16

A Computational Framework for Mechanical Response of Macromolecules: Application to the Salt Concentration Dependence of DNA Bendability  

PubMed Central

A computational framework is presented for studying the mechanical response of macromolecules. The method combines a continuum mechanics (CM) model for the mechanical properties of the macromolecule with a continuum electrostatic (CE) treatment of solvation. The molecules are represented by their shape and key physicochemical characteristics such as the distribution of materials properties and charge. As a test case, we apply the model to the effect of added salt on the bending of DNA. With a simple representation of DNA, the CM/CE framework using a Debye-Hückel model leads to results that are in good agreement with both analytical theories and recent experiments, including a modified Odijk-Skolnick-Fixman theory that takes the finite length of DNA into consideration. Calculations using a more sophisticated CE model (Poisson-Boltzmann), however, suffer from convergence problems, highlighting the importance of balancing numerical accuracy in the CM and CE models when dealing with very large systems, particularly those with a high degree of symmetry. PMID:19413960

Ma, Liang; Yethiraj, Arun; Chen, Xi; Cui, Qiang

2009-01-01

17

Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir  

SciTech Connect

Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When ({sup 3}H)thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. In an attempt to specifically label phytoplankton DNA, samples were incubated with ({sup 3}H)adenine or {sup 32}P{sub i} in the presence and absence of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Although DCMU inhibited {sup 14}C fixation by {approximately} 99%, this antimetabolite had only a slight effect on ({sup 3}H)adenine incorporation and no effect on {sup 32}P incorporation into cellular macromolecules. Significant amounts of dissolved DNA were produced in both ({sup 3}H)adenine and {sup 32}P{sub i} incubations, but again DCMU had no effect on the production rates. These results indicate that actively growing populations of both phytoplankton and bacterioplankton produced dissolved RNA and protein, while only active bacterioplankton produced measurable quantities of dissolved DNA. Dead or senescent phytoplankton may have produced dissolved DNA, but would not be measured in the relatively short incubations used. These findings also indicate that ({sup 3}H)adenine and {sup 32}P{sub i} primarily labeled heterotrophic bacterioplankton and not phytoplankton in this environment.

Paul, J.H.; Jeffrey, W.H.; Cannon, J.P. (Univ. of South Florida, St. Petersburg (USA))

1990-10-01

18

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

19

Opening dynamics of DNA and RNA Simona Cocco LDFC, Strasbourg  

E-print Network

Monasson ENS, Paris Barcelona, Nov 2001 #12;DNA P5ab P5aba RNA #12;The flow of genetic information DNA RNAOpening dynamics of DNA and RNA Simona Cocco LDFC, Strasbourg John F. Marko UIC, Chicago Rémi*(df/dt,N) Model #12;Opening dynamics, RNA P5ab f f Critical force fu Opening dynamics at the critical force t (fu

Cocco, Simona

20

RNA Silencing Genes Control de Novo DNA Methylation  

E-print Network

RNA Silencing Genes Control de Novo DNA Methylation Simon W.-L. Chan,1 Daniel Zilberman,1 Zhixin double mutants cannot initiate DNA methylation and silencing and therefore flow- er late (2). De novo DNA at the SUPERMAN locus, did not affect the initiation of FWA silencing. However, four mutants--rna dependent rna

Jacobsen, Steve

21

Interaction of zanamivir with DNA and RNA: Models for drug DNA and drug RNA bindings  

NASA Astrophysics Data System (ADS)

Zanamivir (ZAN) is the first of a new generation of influenza virus-specific drugs known as neuraminidase inhibitors, which acts by interfering with life cycles of influenza viruses A and B. It prevents the virus spreading infection to other cells by blocking the neuraminidase enzyme present on the surface of the virus. The aim of this study was to examine the stability and structural features of calf thymus DNA and yeast RNA complexes with zanamivir in aqueous solution, using constant DNA or RNA concentration (12.5 mM) and various zanamivir/polynucleotide ( P) ratios of 1/20, 1/10, 1/4, and 1/2. FTIR and UV-visible spectroscopy are used to determine the drug external binding modes, the binding constant and the stability of zanamivir-DNA and RNA complexes in aqueous solution. Structural analysis showed major interaction of zanamivir with G-C (major groove) and A-T (minor groove) base pairs and minor perturbations of the backbone PO 2 group with overall binding constants of Kzanamivir-DNA = 1.30 × 10 4 M -1 and Kzanamivir-RNA = 1.38 × 10 4 M -1. The drug interaction induces a partial B to A-DNA transition, while RNA remains in A-conformation.

Nafisi, Shohreh; Kahangi, Fatemeh Ghoreyshi; Azizi, Ebrahim; Zebarjad, Nader; Tajmir-Riahi, Heidar-Ali

2007-03-01

22

DNA-RNA transcription as an impact of viscosity  

NASA Astrophysics Data System (ADS)

The impact of viscosity on DNA dynamics is studied both analytically and numerically. It is assumed that the viscosity exists at the segments where DNA molecule is surrounded by RNA polymerase. We demonstrate that the frictional forces destroy the modulation of the incoming solitonic wave. We show that viscosity, crucial for demodulation, is essential for DNA-RNA transcription.

Zdravkovi?, Slobodan; Satari?, Miljko V.; Hadžievski, Ljup?o

2010-12-01

23

Repair of methyl lesions in DNA and RNA by oxidative demethylation.  

PubMed

It was established several decades ago that it is crucial for all organisms to repair their DNA to maintain genome integrity and numerous proteins are dedicated to this purpose. However, it is becoming increasingly clear that it is also important to prevent and repair lesions in the macromolecules encoded by the DNA, i.e. RNA and protein. Many neurological disorders such as Alzheimer's disease and Parkinson's disease are associated with the aggregation of defective, misfolded proteins, and several mechanisms exist to prevent such aggregation, both through direct protein repair and through the elimination and repair of faulty or damaged RNAs. A few years ago, it was discovered that the E. coli AlkB protein represented an iron and 2-oxoglutarate dependent oxygenase capable of repairing methyl lesions in DNA by a novel mechanism, termed oxidative demethylation. Furthermore, it was found that both human and bacterial AlkB proteins were able to demethylate lesions also in RNA, thus representing the first example of RNA repair. In the present review, recent findings on the AlkB mechanism, as well as on RNA damage in general, will be discussed. PMID:17175108

Falnes, P Ø; Klungland, A; Alseth, I

2007-04-14

24

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

25

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

26

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

NASA Astrophysics Data System (ADS)

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.

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

2014-11-01

27

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

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

2014-01-01

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

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

30

crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus  

PubMed Central

The Cas9-crRNA complex of the Streptococcus thermophilus DGCC7710 CRISPR3-Cas system functions as an RNA-guided endonuclease with crRNA-directed target sequence recognition and protein-mediated DNA cleavage. We show here that an additional RNA molecule, tracrRNA (trans-activating CRISPR RNA), co-purifies with the Cas9 protein isolated from the heterologous E. coli strain carrying the S. thermophilus DGCC7710 CRISPR3-Cas system. We provide experimental evidence that tracrRNA is required for Cas9-mediated DNA interference both in vitro and in vivo. We show that Cas9 specifically promotes duplex formation between the precursor crRNA (pre-crRNA) transcript and tracrRNA, in vitro. Furthermore, the housekeeping RNase III contributes to primary pre-crRNA-tracrRNA duplex cleavage for mature crRNA biogenesis. RNase III, however, is not required in the processing of a short pre-crRNA transcribed from a minimal CRISPR array containing a single spacer. Finally, we show that an in vitro-assembled ternary Cas9-crRNA-tracrRNA complex cleaves DNA. This study further specifies the molecular basis for crRNA-based re-programming of Cas9 to specifically cleave any target DNA sequence for precise genome surgery. The processes for crRNA maturation and effector complex assembly established here will contribute to the further development of the Cas9 re-programmable system for genome editing applications. PMID:23535272

Karvelis, Tautvydas; Gasiunas, Giedrius; Miksys, Algirdas; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus

2013-01-01

31

RNA-Linked Nascent DNA Fragments in Escherichia coli*  

PubMed Central

Nucleic acid that is extracted from E. coli labeled by a brief pulse of [3H]dT and depatured by treatment with heat, formamide, or formaldehyde bands in a region with a density higher than that of single-stranded E. coli DNA in a Cs2SO4 equilibrium density gradient. If treated with alkali or RNase, it then exhibits the density of single-stranded DNA. These results suggest the presence of a short strand of RNA covalently linked to the nascent DNA. Evidence for the presence of covalently linked RNA-DNA molecules is also obtained by pulse labeling with [3H]U. Analyses of nascent nucleic acids from cells pulse labeled for various times, and of the molecules with different sizes, support the hypothesis that the short DNA fragments are formed by extension of even shorter RNA chains, which are synthesized on the parental DNA strands and are removed before ligation of the DNA fragments. The synthesis of the RNA segment of the RNA-DNA molecule is much less sensitive to rifampicin than is the synthesis of bulk RNA. Images PMID:4558661

Sugino, Akio; Hirose, Susumu; Okazaki, Reiji

1972-01-01

32

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

33

Method for rapid base sequencing in DNA and RNA  

DOEpatents

A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed.

Jett, James H. (Los Alamos, NM); Keller, Richard A. (Los Alamos, NM); Martin, John C. (Los Alamos, NM); Moyzis, Robert K. (Los Alamos, NM); Ratliff, Robert L. (Los Alamos, NM); Shera, E. Brooks (Los Alamos, NM); Stewart, Carleton C. (Los Alamos, NM)

1990-01-01

34

Measuring RNA:DNA ratios in individual Acartia tonsa (Copepoda)  

Microsoft Academic Search

Acartia tonsa Dana is a dominant copepod in coastal waters and is therefore an important link in the food web between microplankton and\\u000a higher trophic levels. RNA:DNA ratios have been used to describe growth and nutritional condition of field-collected copepods\\u000a and to show strong correlation between RNA:DNA ratios and group egg production (EP). A method was developed using a sensitive,

Christa L. Speekmann; B. Scott Nunez; Edward J. Buskey

2007-01-01

35

Lipoplexes versus nanoparticles: pDNA/siRNA delivery.  

PubMed

Small interfering RNA (siRNA) has been widely used as potential therapeutic for treatment of various genetic disorders. However, rapid degradation, poor cellular uptake and limited stability in blood limit the effectiveness of the systemic delivery of siRNA. Therefore, an efficient delivery system is required to enhance its transfection and duration of therapeutics. In the present study, plasmid DNA (pEGFPN3) expressing green fluorescent protein (GFP) was used as a reporter gene. Chitosan nanoparticles/polyplexes and cationic liposomes/lipoplexes were developed and compared for their transfectivity and therapeutic activity in mammalian cell line (HEK 293). The nanoparticulates were first characterized by assessing the surface charge (zeta potential), size (dynamic light scattering) and morphology (transmission electron microscope) followed by evaluation for their DNA retardation ability, transfection efficiency and cytotoxicity on HEK 293 cell line. The chitosan nanoparticles/plasmid DNA (pDNA) complex and liposomes/pDNA complex were co-transfected with GFP-specific siRNA into HEK 293 cells and it was found that both are efficient delivery vehicles for siRNA transfection, resulting in ~57% and ~70% suppression of the targeted gene (GFP), respectively, as compared with the mock control (cells transfected with nanocarrier/pDNA complexes alone). This strong inhibition of GFP expression indicated that cationic liposomes are better than chitosan nanoparticles and can be used as an effective carrier of siRNA in mammalian cells. PMID:23537464

Khurana, Bharat; Goyal, Amit K; Budhiraja, Abhishek; Aora, Diasy; Vyas, Suresh P

2013-02-01

36

Analyses of DNA, RNA and Protein  

E-print Network

such as Deletions duplications Translocations Point mutations if they alter a restriction enzyme cutting site; Restriction endonucleases #12; Recombinant DNA technology #12;Cloning Vectors Plasmids Double stranded circular DNA origin of replication selectable marker (antibiotic resistance) 1 or more restriction

Dellaire, Graham

37

DNA Templated Synthesis (DTS) -Nature's effective molarity based approach-  

E-print Network

Translation DNA RNA Protein Replication mRNA tRNA Nucleic acid templated synthesis plays a important roleDNA Templated Synthesis (DTS) -Nature's effective molarity based approach- Organic Seminar 27th May of many reactants in one solution macromolecule- templated synthesis selective product formation one

Katsumoto, Shingo

38

Quantum-mechanical predictions of DNA and RNA ionization by energetic proton beams.  

PubMed

Among the numerous constituents of eukaryotic cells, the DNA macromolecule is considered as the most important critical target for radiation-induced damages. However, up to now ion-induced collisions on DNA components remain scarcely approached and theoretical support is still lacking for describing the main ionizing processes. In this context, we here report a theoretical description of the proton-induced ionization of the DNA and RNA bases as well as the sugar-phosphate backbone. Two different quantum-mechanical models are proposed: the first one based on a continuum distorted wave-eikonal initial state treatment and the second perturbative one developed within the first Born approximation with correct boundary conditions (CB1). Besides, the molecular structure information of the biological targets studied here was determined by ab initio calculations with the Gaussian 09 software at the restricted Hartree-Fock level of theory with geometry optimization. Doubly, singly differential and total ionization cross sections also provided by the two models were compared for a large range of incident and ejection energies and a very good agreement was observed for all the configurations investigated. Finally, in comparison with the rare experiment, we have noted a large underestimation of the total ionization cross sections of uracil impacted by 80 keV protons,whereas a very good agreement was shown with the recently reported ionization cross sections for protons on adenine, at both the differential and the total scale. PMID:22433314

Galassi, M E; Champion, C; Weck, P F; Rivarola, R D; Fojón, O; Hanssen, J

2012-04-01

39

Quantum-mechanical predictions of DNA and RNA ionization by energetic proton beams  

NASA Astrophysics Data System (ADS)

Among the numerous constituents of eukaryotic cells, the DNA macromolecule is considered as the most important critical target for radiation-induced damages. However, up to now ion-induced collisions on DNA components remain scarcely approached and theoretical support is still lacking for describing the main ionizing processes. In this context, we here report a theoretical description of the proton-induced ionization of the DNA and RNA bases as well as the sugar-phosphate backbone. Two different quantum-mechanical models are proposed: the first one based on a continuum distorted wave-eikonal initial state treatment and the second perturbative one developed within the first Born approximation with correct boundary conditions (CB1). Besides, the molecular structure information of the biological targets studied here was determined by ab initio calculations with the Gaussian 09 software at the restricted Hartree-Fock level of theory with geometry optimization. Doubly, singly differential and total ionization cross sections also provided by the two models were compared for a large range of incident and ejection energies and a very good agreement was observed for all the configurations investigated. Finally, in comparison with the rare experiment, we have noted a large underestimation of the total ionization cross sections of uracil impacted by 80 keV protons, whereas a very good agreement was shown with the recently reported ionization cross sections for protons on adenine, at both the differential and the total scale.

Galassi, M. E.; Champion, C.; Weck, P. F.; Rivarola, R. D.; Fojón, O.; Hanssen, J.

2012-04-01

40

RNA intrusions change DNA elastic properties and structure.  

PubMed

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. PMID:24992674

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

2014-09-01

41

Connections between RNA splicing and DNA intron mobility in yeast mitochondria: RNA maturase and DNA endonuclease switching experiments.  

PubMed Central

The intron-encoded proteins bI4 RNA maturase and aI4 DNA endonuclease can be faithfully expressed in yeast cytoplasm from engineered forms of their mitochondrial coding sequences. In this work we studied the relationships between these two activities associated with two homologous intron-encoded proteins: the bI4 RNA maturase encoded in the fourth intron of the cytochrome b gene and the aI4 DNA endonuclease (I-SceII) encoded in the fourth intron of the gene coding for the subunit I of cytochrome oxidase. Taking advantage of both the high recombinogenic properties of yeast and the similarities between the two genes, we constructed in vivo a family of hybrid genes carrying parts of both RNA maturase and DNA endonuclease coding sequences. The presence of a sequence coding for a mitochondrial targeting peptide upstream from these hybrid genes allowed us to study the properties of their translation products within the mitochondria in vivo. We thus could analyze the ability of the recombinant proteins to complement RNA maturase deficiencies in different strains. Many combinations of the two parental intronic sequences were found in the recombinants. Their structural and functional analysis revealed the following features. (i) The N-terminal half of the bI4 RNA maturase could be replaced in total by its equivalent from the aI4 DNA endonuclease without affecting the RNA maturase activity. In contrast, replacing the C-terminal half of the bI4 RNA maturase with its equivalent from the aI4 DNA endonuclease led to a very weak RNA maturase activity, indicating that this region is more differentiated and linked to the maturase activity. (ii) None of the hybrid proteins carrying an RNA maturase activity kept the DNA endonuclease activity, suggesting that the latter requires the integrity of the aI4 protein. These observations are interesting because the aI4 DNA endonuclease is known to promote the propagation, at the DNA level, of the aI4 intron, whereas the bI4 RNA maturase, which is required for the splicing of its coding intron, also controls the splicing process of the aI4 intron. We propose a scenario for the evolution of these intronic proteins that relies on a switch from DNA endonuclease to RNA maturase activity. Images PMID:1310149

Goguel, V; Delahodde, A; Jacq, C

1992-01-01

42

A Xenopus Zinc Finger Protein that Specifically Binds dsRNA and RNA-DNA Hybrids  

E-print Network

A Xenopus Zinc Finger Protein that Specifically Binds dsRNA and RNA-DNA Hybrids Patrick J. Finerty Building 533, University of Utah, Salt Lake City, UT 84112, USA Proteins containing C2H2 type zinc ®nger describe a novel zinc ®nger protein, dsRBP-ZFa, isolated by screening an ex- pression library with ds

Bass, Brenda L.

43

DNA and RNA unzipping using nanopore force spectroscopy  

NASA Astrophysics Data System (ADS)

DNA and RNA molecules can be electrophoreticaly threaded through nanoscale pores, such as the ˜1.5 nm alpha-Hemolysin. Information about their translocation dynamics is obtained by probing the ionic current flowing through the pore during their passage. We experimentally study the translocation process of unstructured and structured DNA molecules through a single nanopore. We find that the translocation process depends on DNA properties, such as its sequence and its direction of entry. With intense electrical field structured DNA and RNA can be unzipped in a controlled way, and the unzipping kinetics can be directly quantified. We study the unzipping kinetics of DNA and RNA molecules under a wide range of voltage gradients. We find that the unzipping kinetics is characterized by two limiting regimes: the strong field limit in which the system is unzipped in an irreversible process, and the weak field regime, in which it is in quasi equilibrium. Interestingly the unzipping kinetics of RNA molecules is very different from their DNA analogues. A theoretical model that accounts for our experimental results will be discussed.

Meller, Amit; Mathe, Jerome; Wanunu, Meni; Akabayov, Barak; Sagi, Irit

2006-03-01

44

RNA-DNA differences in human mitochondria restore ancestral form of 16S ribosomal RNA.  

PubMed

RNA transcripts are generally identical to the underlying DNA sequences. Nevertheless, RNA-DNA differences (RDDs) were found in the nuclear human genome and in plants and animals but not in human mitochondria. Here, by deep sequencing of human mitochondrial DNA (mtDNA) and RNA, we identified three RDD sites at mtDNA positions 295 (C-to-U), 13710 (A-to-U, A-to-G), and 2617 (A-to-U, A-to-G). Position 2617, within the 16S rRNA, harbored the most prevalent RDDs (>30% A-to-U and ?15% A-to-G of the reads in all tested samples). The 2617 RDDs appeared already at the precursor polycistrone mitochondrial transcript. By using traditional Sanger sequencing, we identified the A-to-U RDD in six different cell lines and representative primates (Gorilla gorilla, Pongo pigmaeus, and Macaca mulatta), suggesting conservation of the mechanism generating such RDD. Phylogenetic analysis of more than 1700 vertebrate mtDNA sequences supported a thymine as the primate ancestral allele at position 2617, suggesting that the 2617 RDD recapitulates the ancestral 16S rRNA. Modeling U or G (the RDDs) at position 2617 stabilized the large ribosomal subunit structure in contrast to destabilization by an A (the pre-RDDs). Hence, these mitochondrial RDDs are likely functional. PMID:23913925

Bar-Yaacov, Dan; Avital, Gal; Levin, Liron; Richards, Allison L; Hachen, Naomi; Rebolledo Jaramillo, Boris; Nekrutenko, Anton; Zarivach, Raz; Mishmar, Dan

2013-11-01

45

Effect of bleomycin on DNA, RNA, protein, chromatin and on cell transformation by oncogenic RNA viruses.  

PubMed

Bleomycin (BLM) exclusively affects thymidine-containing compounds such as DNA and polydeoxyribonucleotides by releasing free thymine and leaving aldehyde functions. Molecular morphology and base sequence of the DNA strongly influence BLM activity. High BLM concentrations, besides modifying DNA into oligothyminic or athyminic nucleic acids, cause strand scissions. Enzymatic DNA and RNA synthesis is strongly influenced by BLM. The inhibition in DNA-dependent DNA polymerase and DNA-dependent RNA polymerase assays is of the non-competitive type. Protein biosynthesis in in vitro systems is not affected by BLM even at high concentrations. BLM turns out to be a strong inhibitor of DNase I and of DNase II; the inhibition is of the competitive type. The enzymatic activities of nucleases using RNA as substrate (RNase A, RNase B, Rnase T1, venom phosphodiesterase I and spleen phosphodiesterase II) are not influenced by this antibiotic. The antibiotic reduces cell proliferation (L5178y mouse lymphoma cells) in vitro in low concentrations by cytostasis and at higher concentrations by cytotoxicity. In BLM-treated L5178y cells, DNA synthesis is strongly reduced, while RNA and protein synthesis are not affected. In vivo, using growing quail oviducts, cell proliferation and cytodifferentiation are markedly inhibited after BLM treatment. This is attributed to the observed inhibition of DNA synthesis. RNA and protein synthesis as well as gene expression are not influenced by BLM under the conditions used. The selective inhibition of DNA synthesis in vivo may be caused by the following mechanisms: (1) competition of BLM with RNA; (2) blocking of the accessibility of DNA in chromatin to BLM, and (3) dependence from the repair processes. BLM inhibits growth of sarcomas, induced by oncogenic RNA viruses in vivo; well-developed tumours show regression after BLM treatment. Transformation of chick embryo fibroblasts by oncogenic RNA viruses in vitro and growth of these viruses is blocked by BLM; the most sensitive period for BLM inhibition is the time during the first period (integration of viral genome into cellular genome?) after infection. PMID:63969

Müller, W E; Zahn, R K

1976-01-01

46

Properties of nicked and circular dumbbell RNA\\/DNA chimeric oligonucleotides containing antisense phosphodiester oligodeoxynucleotides  

Microsoft Academic Search

We have designed a new class of oligonucleotides, ‘dumbbell RNA\\/DNA chimeric phosphodiester oligonucleotides’, consisting of a sense RNA sequence and its complementary antisense DNA sequence, with two hairpin loop structures. The reaction of the Nicked (NDRDON) and Circular (CDRNON) dumbbell DNA\\/RNA chimeric oligonucleotides with RNase H gave the corresponding antisense phosphodiester oligodeoxynucleotide together with the sense RNA cleavage products. The

Hidefumi Yamakawa; Takayuki Abe; Takeshi Saito; Kazuyuki Takai; Naoki Yamamoto; Hiroshi Takaku

1998-01-01

47

Characterization of marine prokaryotic communities via DNA and RNA  

Microsoft Academic Search

We know very little about species distributions in prokaryotic marine plankton. Such information is very interesting in its own right, and ignorance of it is also beginning to hamper process studies, such as those on viral infection. New DNA- and RNA-based approaches avoid many prior limitations. Here we discuss four such applications: (1) cloning and sequencing of 16S rRNA genes

J. A. Fuhrman; S. H. Lee; Y. Masuchi; A. A. Davis; R. M. Wilcox

1994-01-01

48

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

49

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

50

Bifacial PNA complexation inhibits enzymatic access to DNA and RNA.  

PubMed

FULL STOP: Herein we report the effective in vitro inhibition of transcription, reverse-transcription and exonuclease function by formation of synthetic bPNA-nucleic acid triplex structures. Selective bPNA targeting of both DNA and RNA substrates suggests possible application of bPNAs as synthetic regulators of nucleic acid function. PMID:24259287

Xia, Xin; Piao, Xijun; Fredrick, Kurt; Bong, Dennis

2014-01-01

51

Simulations Using Random-Generated DNA and RNA Sequences  

ERIC Educational Resources Information Center

Using a very simple computer program written in BASIC, a very large number of random-generated DNA or RNA sequences are obtained. Students use these sequences to predict complementary sequences and translational products, evaluate base compositions, determine frequencies of particular triplet codons, and suggest possible secondary structures.…

Bryce, C. F. A.

1977-01-01

52

Dataflow diagram representation of biological process: DNA, RNA, and protein  

Microsoft Academic Search

In this paper, we introduce a new method of modeling tool for a biological process -central dogma. The data-flow diagram is used as a representation of the whole data input and output, which enables us to simulate, analyze, and manipulate (in the future) at our disposal. From DNA to protein via RNA is the one of most well-known biological process,

Joe W. Yeol; W. Samarrai; I. Barjis; Y. S. Ryu

2005-01-01

53

Photochemical Inactivation of DNA and RNA Viruses by Psoralen Derivatives  

Microsoft Academic Search

SUMMARY Western equine encephalitis virus, and RNA virus, and herpes simplex virus type I, a DNA virus, were efficiently inactivated in less than I rain by exposure to long- wave ultraviolet light (32o to 38o rim) in the presence &several psoralen derivatives. The psoralen photochemical reaction was chosen for study due to its known specificity for nucleic acids. Neither the

CARL VEITH HANSON; JOHN L. RIGGS; EDWIN H. LENNETTE

1978-01-01

54

Effects of hypoxanthine substitution on bleomycin-mediated DNA strand degradation in DNA-RNA hybrids.  

PubMed Central

We have reported on the differences in site-specific cleavage between DNA and DNA-RNA hybrids by various prototypic DNA cleavers (accompanying paper). In the case of bleomycin (BLM), degradation at 5'-GC-3'sites was suppressed relative to the same sequence in double-stranded DNA, while 5'-GT-3' damage remained constant. We now present results of our further investigation on the chemical and conformational factors that contribute to BLM-mediated DNA strand cleavage of DNA-RNA hybrids. Substitution of guanine by hypoxanthine on the RNA strand of hybrids resulted in a significant enhancement of 5'-GC-3' site damage on the DNA strand relative to double-stranded DNA, thus reversing the suppression noted at these sites. Additionally, 5'-AT-3' sites, which are damaged significantly more in the hybrid than in DNA, exhibit decreased product formation when hypoxanthine is present on the RNA strand of hybrids. However, when hypoxanthine is substituted for guanine on the DNA strand (a GC cleavage site becomes IC), 5'-IT-3' and 5'-IC-3' site cleavage is almost completely suppressed, whereas AT site cleavage is dramatically enhanced. The priority in metallobleomycin site-specific cleavage of hybrids changes with hypoxanthine substitution: the cleavage priority is AT > GT > GC in native hybrid; GC > GT > AT in hybrids substituted with hypoxanthine in the RNA strand; AT >> GT approximately GC in hybrids substituted with hypoxanthine in the DNA strand. The results of kinetic isotope effect studies on BLM cleavage are presented and, in most cases, the values are larger for the hypoxanthine-substituted hybrid. The results suggest that the 2-amino groups of guanine residues on both strands of the nucleic acid play an important role in modulation of the binding and cleavage specificity of BLM. PMID:9108170

Bansal, M; Stubbe, J; Kozarich, J W

1997-01-01

55

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

56

DNA/RNA Detection Using DNA-Templated Few-Atom Silver Nanoclusters  

PubMed Central

DNA-templated few-atom silver nanoclusters (DNA/Ag NCs) are a new class of organic/inorganic composite nanomaterials whose fluorescence emission can be tuned throughout the visible and near-IR range by simply programming the template sequences. Compared to organic dyes, DNA/Ag NCs can be brighter and more photostable. Compared to quantum dots, DNA/Ag NCs are smaller, less prone to blinking on long timescales, and do not have a toxic core. The preparation of DNA/Ag NCs is simple and there is no need to remove excess precursors as these precursors are non-fluorescent. Our recent discovery of the fluorogenic and color switching properties of DNA/Ag NCs have led to the invention of new molecular probes, termed NanoCluster Beacons (NCBs), for DNA detection, with the capability to differentiate single-nucleotide polymorphisms by emission colors. NCBs are inexpensive, easy to prepare, and compatible with commercial DNA synthesizers. Many other groups have also explored and taken advantage of the environment sensitivities of DNA/Ag NCs in creating new tools for DNA/RNA detection and single-nucleotide polymorphism identification. In this review, we summarize the recent trends in the use of DNA/Ag NCs for developing DNA/RNA sensors. PMID:25586126

Obliosca, Judy M.; Liu, Cong; Batson, Robert Austin; Babin, Mark C.; Werner, James H.; Yeh, Hsin-Chih

2013-01-01

57

Protein localization with flexible DNA or RNA.  

PubMed

Localization of activity is ubiquitous in life, and also within sub-cellular compartments. Localization provides potential advantages as different proteins involved in the same cellular process may supplement each other on a fast timescale. It might also prevent proteins from being active in other regions of the cell. However localization is at odds with the spreading of unbound molecules by diffusion. We model the cost and gain for specific enzyme activity using localization strategies based on binding to sites of intermediate specificity. While such bindings in themselves decrease the activity of the protein on its target site, they may increase protein activity if stochastic motion allows the acting protein to touch both the intermediate binding site and the specific site simultaneously. We discuss this strategy in view of recent suggestions on long non-coding RNA as a facilitator of localized activity of chromatin modifiers. PMID:22347995

Bernhardsson, Sebastian; Mitarai, Namiko; Sneppen, Kim

2012-01-01

58

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

59

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

60

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

61

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

62

Use of Laser Capture Microdissection for Analysis of Retinal mRNA/miRNA Expression and DNA Methylation  

PubMed Central

Laser capture microdissection (LCM) is a useful method to isolate specific cells or cell layers of interest from heterogeneous tissues, such as the retina. The collected cells can be used for DNA, RNA, or protein analysis. We have applied LCM technology to isolate cells from the outer nuclear, inner nuclear, and ganglion cell layers of the retina for mRNA and microRNA (miRNA) expression and epigenetic (DNA methylation) analysis. Here, we describe the methods we have employed for sample preparation, LCM-based isolation of retinal layers, RNA/DNA extraction, RNA quality check, microRNA analysis by quantitative PCR, and DNA methylation analysis by bisulfite sequencing. PMID:22688715

Hackler, Laszlo; Masuda, Tomohiro; Oliver, Verity F.; Merbs, Shannath L.; Zack, Donald J.

2014-01-01

63

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

64

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

65

A method for DNA and RNA co-extraction for use on forensic samples using the Promega DNA IQ™ system.  

PubMed

The use of messenger RNA profiling to identify the origin of biological samples (e.g. blood, semen and saliva) from crime scenes is now at the stage of being implemented into routine forensic casework. We report on the successful modification of the Promega DNA IQ™ system to enable co-extraction of DNA and RNA from the same sample without compromising the potential DNA profile. Using the protocol in our laboratory for extracting DNA using the DNA IQ™ system combined with the Zymo Research Mini RNA Isolation Kit™ II we demonstrate the simultaneous co-extraction of DNA and RNA from the same sample for routine DNA and mRNA profiling for the identification of both the individual and the biological stain. PMID:20457058

Bowden, Anna; Fleming, Rachel; Harbison, SallyAnn

2011-01-01

66

Spectrofluorometric determination of DNA and RNA with berberine  

NASA Astrophysics Data System (ADS)

On binding to nucleic acids, the dye berberine increases its fluorescence quantum efficiency by a factor of 25-30. Based on this, an easy, rapid and accurate method for the determination of nucleic acids was developed. Berberine is very like ethidium bromide (EB), but it is non-poisonous. Determination can be made at any pH between 4 and 10, where the native structure of DNA and RNA is not disrupted. The maximum emission is near 520 nm for excitation at 355 or 450 nm. This method has good sensitivity (0.01 ?g ml -1 of ctDNA), high selectivity and a wide linear range (0.05-14.0 ?g ml -1 of ctDNA).

Gong, Guo-Quan; Zong, Zhi-Xin; Song, Yu-Min

1999-08-01

67

Conflict RNA modification, host-parasite co-evolution, and the origins of DNA and DNA-binding proteins1.  

PubMed

Nearly 150 different enzymatically modified forms of the four canonical residues in RNA have been identified. For instance, enzymes of the ADAR (adenosine deaminase acting on RNA) family convert adenosine residues into inosine in cellular dsRNAs. Recent findings show that DNA endonuclease V enzymes have undergone an evolutionary transition from cleaving 3' to deoxyinosine in DNA and ssDNA to cleaving 3' to inosine in dsRNA and ssRNA in humans. Recent work on dsRNA-binding domains of ADARs and other proteins also shows that a degree of sequence specificity is achieved by direct readout in the minor groove. However, the level of sequence specificity observed is much less than that of DNA major groove-binding helix-turn-helix proteins. We suggest that the evolution of DNA-binding proteins following the RNA to DNA genome transition represents the major advantage that DNA genomes have over RNA genomes. We propose that a hypothetical RNA modification, a RRAR (ribose reductase acting on genomic dsRNA) produced the first stretches of DNA in RNA genomes. We discuss why this is the most satisfactory explanation for the origin of DNA. The evolution of this RNA modification and later steps to DNA genomes are likely to have been driven by cellular genome co-evolution with viruses and intragenomic parasites. RNA modifications continue to be involved in host-virus conflicts; in vertebrates, edited cellular dsRNAs with inosine-uracil base pairs appear to be recognized as self RNA and to suppress activation of innate immune sensors that detect viral dsRNA. PMID:25110019

McLaughlin, Paul J; Keegan, Liam P

2014-08-01

68

DNA targeting specificity of RNA-guided Cas9 nucleases.  

PubMed

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. 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

2013-09-01

69

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

PubMed Central

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 products. We investigated understanding of central dogma concepts and found that students are not primed to think about information when presented with the canonical figure of the central dogma. We also uncovered conceptual errors in student interpretation of the meaning of the transcription arrow in the central dogma representation; 36% of students (n = 128; all undergraduate levels) described transcription as a chemical conversion of DNA into RNA or suggested that RNA existed before the process of transcription began. Interviews confirm that students with weak conceptual understanding of information flow find inappropriate meaning in the canonical representation of central dogma. Therefore, we suggest that use of this representation during instruction can be counterproductive unless educators are explicit about the underlying meaning.

Fisk, J. Nick; Newman, Dina L.

2014-01-01

70

Labile Catalytic Packaging of DNA/siRNA: Control of Gold Nanoparticles “out” of DNA/siRNA Complexes  

PubMed Central

A novel approach was developed to efficiently package and deliver nucleic acids with low generation polypropylenimine (PPI) dendrimers by using Au nanoparticles as a “labile catalytic” packaging agent. The Au nanoparticles (Au NPs) helped low generation dendrimers to package nucleic acids into discrete nanoparticles but are not included in the final DNA/siRNA complexes. Therefore it becomes possible to eliminate the potential toxic problems associated with Au NPs by selectively removing the Au NPs from the resulting nucleic acid complexes before their delivery to targeted cells. This is a new concept in using inorganic engineered nanoparticles in nucleic acid packaging and delivery applications. Furthermore, compared to the siRNA nanostructures (mainly randomly aggregated nanofibers) fabricated by low generation dendrimer alone (Generation 3), the siRNA nanoparticles packaged using this novel approach (by Au NPs modified with G3 PPI) can be internalized by cancer cells and the delivered siRNAs can efficiently silence their target mRNA. The efficiency of mRNA silencing by this novel approach is even superior to higher generation dendrimers (Generation 5). PMID:20521827

Chen, Alex M.; Taratula, Oleh; Wei, Dongguang; Yen, Hsin-I; Thomas, Thresia; Thomas, T. J.; Minko, Tamara; He, Huixin

2010-01-01

71

Introduction Model Results Conclusions On the Origin of DNA Genomes in RNA World  

E-print Network

Introduction Model Results Conclusions On the Origin of DNA Genomes in RNA World Nobuto Takeuchi1 2011, Kyoto #12;Introduction Model Results Conclusions One of the fundamental properties of living. #12;Introduction Model Results Conclusions Proteins & DNA compared with RNA Proteins (vs. RNA

Utrecht, Universiteit

72

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

PubMed Central

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 293 and HtTA HeLa cells were used to investigate pore formation and intracellular delivery of cDNA, siRNA and lucifer yellow. Results Poly-APS and LipoGen were both found to be highly efficient DNA condensing agents. Fura-2 calcium imaging was used to measure calcium transients indicative of cell membrane pore forming activity. Calcium transients were evoked by poly-APS but not LipoGen and lipofectamine. The increases in intracellular calcium produced by poly-APS showed temperature sensitivity with greater responses being observed at 12°C compared to 21°C. Similarly, delivery of lucifer yellow into cells with poly-APS was enhanced at lower temperatures. Transfection with cDNA encoding for the expression enhanced green fluorescent protein was also evaluated at 12°C with poly-APS, lipofectamine and LipoGen. Intracellular delivery of siRNA was achieved with knockdown in beta-actin expression when lipofectamine and LipoGen were used as transfection reagents. However, intracellular delivery of siRNA was not achieved with poly-APS. Conclusion Poly-APS mediated pore formation is critical to its activity as a transfection reagent, but lipofection systems utilise distinct mechanisms to enable delivery of DNA and siRNA into cells. PMID:16412248

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

2006-01-01

73

Structures of HIV-1 RT-RNA/DNA ternary complexes with dATP and nevirapine reveal conformational flexibility of RNA/DNA: insights into requirements for RNase H cleavage  

PubMed Central

In synthesizing a double-stranded DNA from viral RNA, HIV-1 reverse transcriptase (RT) generates an RNA/DNA intermediate. RT also degrades the RNA strand and synthesizes the second DNA strand. The RNase H active site of RT functions as a nuclease to cleave the RNA strand; however, the structural basis for endonucleolytic cleavage of the RNA strand remains elusive. Here we report crystal structures of RT-RNA/DNA-dATP and RT-RNA/DNA-nevirapine (NVP) ternary complexes at 2.5 and 2.9 Å resolution, respectively. The polymerase region of RT-RNA/DNA-dATP complex resembles DNA/DNA ternary complexes apart from additional interactions of 2?-OH groups of the RNA strand. The conformation and binding of RNA/DNA deviates significantly after the seventh nucleotide versus a DNA/DNA substrate. Binding of NVP slides the RNA/DNA non-uniformly over RT, and the RNA strand moves closer to the RNase H active site. Two additional structures, one containing a gapped RNA and another a bulged RNA, reveal that conformational changes of an RNA/DNA and increased interactions with the RNase H domain, including the interaction of a 2?-OH with N474, help to position the RNA nearer to the active site. The structures and existing biochemical data suggest a nucleic acid conformation-induced mechanism for guiding cleavage of the RNA strand. PMID:24880687

Das, Kalyan; Martinez, Sergio E.; Bandwar, Rajiv P.; Arnold, Eddy

2014-01-01

74

Excited states of protonated DNA/RNA bases Matias Berdakin1  

E-print Network

1 Excited states of protonated DNA/RNA bases Matias Berdakin1 , Géraldine Féraud2 , Claude Dedonder of the excited states to the ground state in DNA/RNA bases is a necessary process to ensure the photostability of DNA and its rate is highly sensitive to the tautomeric form of the bases. Protonation of the bases

Boyer, Edmond

75

Protocol Cycle Time Optimization on a Microfluidic Cartridge for DNA\\/RNA Extraction Application  

Microsoft Academic Search

A disposable microfluidic cartridge for DNA\\/RNA extraction has been developed. Different reagents are used to bind and unbind DNA on the chip used in the cartridge. The extraction of DNA\\/RNA is based on a particular protocol, which involves the dispensing of different reagents at various flow rates. Dispensing of reagents is controlled by a needle valve which is integrated in

Michelle Chew; Ling Xie; CS Premachandran; Seung Wook Yoon

2007-01-01

76

Aprataxin resolves adenylated RNA-DNA junctions to maintain genome integrity.  

PubMed

Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions used by ATP-dependent DNA ligases. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5'-adenylated (5'-AMP) DNA lesions. Aprataxin (APTX) reverses DNA adenylation but the context for deadenylation repair is unclear. Here we examine the importance of APTX to RNase-H2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5' ends containing a ribose characteristic of RNase H2 incision. APTX efficiently repairs adenylated RNA-DNA, and acting in an RNA-DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure-function studies of human APTX-RNA-DNA-AMP-Zn complexes define a mechanism for detecting and reversing adenylation at RNA-DNA junctions. This involves A-form RNA binding, proper protein folding and conformational changes, all of which are affected by heritable APTX mutations in ataxia with oculomotor apraxia 1. Together, these results indicate that accumulation of adenylated RNA-DNA may contribute to neurological disease. PMID:24362567

Tumbale, Percy; Williams, Jessica S; Schellenberg, Matthew J; Kunkel, Thomas A; Williams, R Scott

2014-02-01

77

Aprataxin resolves adenylated RNA-DNA junctions to maintain genome integrity  

PubMed Central

Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions employed by ATP-dependent DNA ligases1,2. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5?-adenylated (5?-AMP) DNA lesions3–6 (Fig. 1a). Aprataxin (Aptx) reverses DNA-adenylation but the context for deadenylation repair is unclear. Here we examine the importance of Aptx to RNaseH2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5?-ends containing a ribose characteristic of RNaseH2 incision. Aptx efficiently repairs adenylated RNA-DNA, and acting in an RNA-DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure-function studies of human Aptx/RNA-DNA/AMP/Zn complexes define a mechanism for detecting and reversing adenylation at RNA-DNA junctions. This involves A-form RNA-binding, proper protein folding and conformational changes, all of which are impacted by heritable APTX mutations in Ataxia with Oculomotor Apraxia 1 (AOA1). Together, these results suggest that accumulation of adenylated RNA-DNA may contribute to neurological disease. PMID:24362567

Tumbale, Percy; Williams, Jessica S.; Schellenberg, Matthew J.; Kunkel, Thomas A.; Williams, R. Scott

2014-01-01

78

Structural mechanisms of RNA recognition: sequence-specific and non-specific RNA-binding proteins and the Cas9-RNA-DNA complex.  

PubMed

RNA-binding proteins play crucial roles in RNA processing and function as regulators of gene expression. Recent studies have defined the structural basis for RNA recognition by diverse RNA-binding motifs. While many RNA-binding proteins recognize RNA sequence non-specifically by associating with 5' or 3' RNA ends, sequence-specific recognition by RNA-binding proteins is typically achieved by combining multiple modular domains to form complex binding surfaces. In this review, we present examples of structures from different classes of RNA-binding proteins, identify the mechanisms utilized by them to target specific RNAs, and describe structural principles of how protein-protein interactions affect RNA recognition specificity. We also highlight the structural mechanism of sequence-dependent and -independent interactions in the Cas9-RNA-DNA complex. PMID:25432705

Ban, Ting; Zhu, Jian-Kang; Melcher, Karsten; Xu, H Eric

2014-11-29

79

A method for DNA and RNA co-extraction for use on forensic samples using the Promega DNA IQ™ system  

Microsoft Academic Search

The use of messenger RNA profiling to identify the origin of biological samples (e.g. blood, semen and saliva) from crime scenes is now at the stage of being implemented into routine forensic casework. We report on the successful modification of the Promega DNA IQ™ system to enable co-extraction of DNA and RNA from the same sample without compromising the potential

Anna Bowden; Rachel Fleming; SallyAnn Harbison

2011-01-01

80

Triplex DNA:RNA, 3'-to-5' inverted RNA and protein coding in mitochondrial genomes.  

PubMed

Triple-stranded DNA:RNA helices of unknown function in vertebrate mitochondria associate with replication and transcription. Antiparallel Hoogsteen pairings form triplexes at physiological conditions. Intermolecular antiparallel triplexes require inverted 3'-to-5' RNA polymerization, which was never observed. Three rare, long natural 3'-to-5' inverted GenBank RNAs from mice mitochondria suggest occasional inverted transcription, putatively coding for proteins. BLAST aligns 18 GenBank-stored proteins with hypothetical proteins translated from the 3'-to-5' inverted Mus musculus mitochondrial genome. Three are DNA-binding, five are membrane proteins. 25% of main frame codons contribute to their 3'-to-5' overlap coding. Properties of these codons match those of overlap coding protein genes, as compared to codons not expected involved in inverted coding: a) nucleotide contents at synonymous codon positions in mitochondrial genomes fit replicational deamination gradients (A->G and C->T), but digress from gradients when functioning as nonsynonymous positions in putative 3'-to-5' overlapping genes; b) bias against 'circular code' codons (codon groups creating unambiguity between frames), and favouring homogenous codons (AAA, CCC, GGG, TTT) characterize overlapping genes, including putative 3'-to-5' overlapping genes, as compared to nonoverlapping coding sequences from the same main frame gene. This signature correlates with digression from deamination gradients. Deamination and circular code tests confirm independently alignment-based predictions of overlapping 3'-to-5' protein coding genes. Results indicate varying expression for different 3'-to-5' overlapping genes. Inverted 3'-to-5' RNA is produced, perhaps by an unknown RNA polymerase (invertase) putatively coded by 3'-to-5' inverted RNA. PMID:23841652

Seligmann, Hervé

2013-09-01

81

Integrated RNA and DNA sequencing improves mutation detection in low purity tumors  

PubMed Central

Identifying somatic mutations is critical for cancer genome characterization and for prioritizing patient treatment. DNA whole exome sequencing (DNA-WES) is currently the most popular technology; however, this yields low sensitivity in low purity tumors. RNA sequencing (RNA-seq) covers the expressed exome with depth proportional to expression. We hypothesized that integrating DNA-WES and RNA-seq would enable superior mutation detection versus DNA-WES alone. We developed a first-of-its-kind method, called UNCeqR, that detects somatic mutations by integrating patient-matched RNA-seq and DNA-WES. In simulation, the integrated DNA and RNA model outperformed the DNA-WES only model. Validation by patient-matched whole genome sequencing demonstrated superior performance of the integrated model over DNA-WES only models, including a published method and published mutation profiles. Genome-wide mutational analysis of breast and lung cancer cohorts (n = 871) revealed remarkable tumor genomics properties. Low purity tumors experienced the largest gains in mutation detection by integrating RNA-seq and DNA-WES. RNA provided greater mutation signal than DNA in expressed mutations. Compared to earlier studies on this cohort, UNCeqR increased mutation rates of driver and therapeutically targeted genes (e.g. PIK3CA, ERBB2 and FGFR2). In summary, integrating RNA-seq with DNA-WES increases mutation detection performance, especially for low purity tumors. PMID:24970867

Wilkerson, Matthew D.; Cabanski, Christopher R.; Sun, Wei; Hoadley, Katherine A.; Walter, Vonn; Mose, Lisle E.; Troester, Melissa A.; Hammerman, Peter S.; Parker, Joel S.; Perou, Charles M.; Hayes, D. Neil

2014-01-01

82

Characterization of cloned cDNA sequences derived from Xenopus laevis poly A(+) oocyte RNA.  

PubMed Central

Double-stranded cDNA sequences were prepared from Xenopus laevis ovary poly A(+) RNA with AMV reverse transcriptase and nuclease S1. They were inserted into the plasmid pBR 322 after ligation with a Hind III linker and were cloned in E. coli strain X1776. Plasmid pools containing a cDNA insert were identified by Hind II restriction and hybridization of the DNA fragments with radiolabelled pBR 322 DNA. Hybridization of the positive pools with ovary RNA labelled in vitro led to the identification of cloned cDNA sequences which represent RNA species of high to intermediate abundance in the ovary. Positive clones were further challenged with in vitro labelled mitochondrial DNA and RNA from different developmental stages. One clone of mitochondrial origin has been detected. The hybridization characteristics of the cDNA sequences with the RNA probes from later developmental stages is discussed. Images PMID:6159593

Jacob, E

1980-01-01

83

Catalytic metal ions and enzymatic processing of DNA and RNA.  

PubMed

Conspectus Two-metal-ion-dependent nucleases cleave the phosphodiester bonds of nucleic acids via the two-metal-ion (2M) mechanism. Several high-resolution X-ray structures portraying the two-metal-aided catalytic site, together with mutagenesis and kinetics studies, have demonstrated a functional role of the ions for catalysis in numerous metallonucleases. Overall, the experimental data confirm the general mechanistic hypothesis for 2M-aided phosphoryl transfer originally reported by Steitz and Steitz ( Proc. Natl. Acad. Sci. U.S.A. 1993 , 90 ( 14 ), 6498 - 6502 ). This seminal paper proposed that one metal ion favors the formation of the nucleophile, while the nearby second metal ion facilitates leaving group departure during RNA hydrolysis. Both metals were suggested to stabilize the enzymatic transition state. Nevertheless, static X-ray structures alone cannot exhaustively unravel how the two ions execute their functional role along the enzymatic reaction during processing of DNA or RNA strands when moving from reactants to products, passing through metastable intermediates and high-energy transition states. In this Account, we discuss the role of multiscale molecular simulations in further disclosing mechanistic insights of 2M-aided catalysis for two prototypical enzymatic targets for drug discovery, namely, ribonuclease H (RNase H) and type II topoisomerase (topoII). In both examples, first-principles molecular simulations, integrated with structural data, emphasize a cooperative motion of the bimetal motif during catalysis. The coordinated motion of both ions is crucial for maintaining a flexible metal-centered structural architecture exquisitely tailored to accommodate the DNA or RNA sugar-phosphate backbone during phosphodiester bond cleavage. Furthermore, our analysis of RNase H and the N-terminal domain (PAN) of influenza polymerase shows that classical molecular dynamics simulations coupled with enhanced sampling techniques have contributed to describe the modulatory effect of metal ion concentration and metal uptake on the 2M mechanism and efficiency. These aspects all point to the emerging and intriguing role of additional adjacent ions potentially involved in the modulation of phosphoryl transfer reactions and enzymatic turnover in 2M-catalysis, as recently observed experimentally in polymerase ? and homing endonuclease I-DmoI. These computational results, integrated with experimental findings, describe and reinforce the nascent concept of a functional and cooperative dynamics of the catalytic metal ions during the 2M-dependent enzymatic processing of DNA and RNA. Encouraged by the insights provided by computational approaches, we foresee further experiments that will feature the functional and joint dynamics of the catalytic metal ions for nucleic acid processing. This could impact the de novo design of artificial metallonucleases and the rational design of potent metal-chelating inhibitors of pharmaceutically relevant enzymes. PMID:25590654

Palermo, Giulia; Cavalli, Andrea; Klein, Michael L; Alfonso-Prieto, Mercedes; Dal Peraro, Matteo; De Vivo, Marco

2015-02-17

84

RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries  

PubMed Central

Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3? and 5? ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1–249) and Rnl2(1–249)K227Q, for 3?-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5?-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/3?-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 5?-terminal 5-nt barcode extensions for a set of 20 barcoded 3? adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling. PMID:21775473

Hafner, Markus; Renwick, Neil; Brown, Miguel; Mihailovi?, Aleksandra; Holoch, Daniel; Lin, Carolina; Pena, John T.G.; Nusbaum, Jeffrey D.; Morozov, Pavel; Ludwig, Janos; Ojo, Tolulope; Luo, Shujun; Schroth, Gary; Tuschl, Thomas

2011-01-01

85

Double-stranded RNA adenosine deaminase binds Z-DNA in vitro.  

PubMed

A Z-DNA binding protein of 140,000 M(r) has been purified from chicken lungs by sedimentation through 40%(w/w) sucrose and Z-DNA affinity chromatography. Specificity of the protein for Z-DNA was confirmed by competition with polyd(CG) that had been stabilized in the Z-DNA conformer by chemical bromination and also with a supercoiled plasmid that contains a Z-DNA-forming insert. In addition to a Z-DNA binding site, the protein also has a separate binding site for double-stranded RNA. Peptide sequence of the protein shows that it has high similarity to the RNA editing enzyme double-stranded RNA adenosine deaminase (dsRAD), which deaminates adenosine in dsRNA to form inosine. The Z-DNA binding protein has this enzymatic activity, confirming its identity to dsRAD. Recombinant human dsRAD also binds to Z-DNA. Z-DNA is stabilized in a sequence-dependent manner by negative supercoiling, which occurs in actively transcribed genes upstream to RNA polymerase. It is proposed that Z-DNA links editing to transcription by localizing dsRAD to a particular region of a gene and thus determines the efficiency with which an RNA is edited. The presence of Z-DNA forming elements in many genes raises the possibility that RNA editing by dsRAD is far more prevalent than is currently thought. PMID:8643357

Herbert, A; Lowenhaupt, K; Spitzner, J; Rich, A

1995-01-01

86

RNA 3'-phosphate cyclase (RtcA) catalyzes ligase-like adenylylation of DNA and RNA 5'-monophosphate ends.  

PubMed

RNA 3'-phosphate cyclase (Rtc) enzymes are a widely distributed family that catalyze the synthesis of RNA 2',3'-cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of Rtc with ATP to form a covalent Rtc-(histidinyl-N)-AMP intermediate and release PP(i); transfer of AMP from Rtc to an RNA 3'-phosphate to form an RNA(3')pp(5')A intermediate; and attack by the terminal nucleoside O2' on the 3'-phosphate to form an RNA 2',3'-cyclic phosphate product and release AMP. The chemical transformations of the cyclase pathway resemble those of RNA and DNA ligases, with the key distinction being that ligases covalently adenylylate 5'-phosphate ends en route to phosphodiester synthesis. Here we show that the catalytic repertoire of RNA cyclase overlaps that of ligases. We report that Escherichia coli RtcA catalyzes adenylylation of 5'-phosphate ends of DNA or RNA strands to form AppDNA and AppRNA products. The polynucleotide 5' modification reaction requires the His(309) nucleophile, signifying that it proceeds through a covalent RtcA-AMP intermediate. We established this point directly by demonstrating transfer of [(32)P]AMP from RtcA to a pDNA strand. RtcA readily adenylylated the 5'-phosphate at a 5'-PO(4)/3'-OH nick in duplex DNA but was unable to covert the nicked DNA-adenylate to a sealed phosphodiester. Our findings raise the prospect that cyclization of RNA 3'-ends might not be the only biochemical pathway in which Rtc enzymes participate; we discuss scenarios in which the 5'-adenylyltransferase of RtcA might play a role. PMID:21098490

Chakravarty, Anupam K; Shuman, Stewart

2011-02-11

87

Role of DNA/RNA sensors and contribution to autoimmunity.  

PubMed

Innate immune detection and subsequent immune responses rely on the initial recognition of pathogen specific molecular motifs. Foreign nucleic acids are key structures recognised by the immune system, recognition of which occurs mainly through the use of nucleic acid receptors including members of the Toll-like receptors, AIM2-like receptors, RIG-I-like receptors and intracellular DNA receptors. While the immune system is critically important in protecting the host from infection, it is of utmost importance that it is tightly regulated, in order to prevent recognition of self-nucleic acids and the subsequent development of autoimmunity. Defects in the mechanisms regulating such pathways, for example mutations in endonucleases that clear DNA, altered expression of nucleic acid sensors and defects in negative regulators of these signalling pathways involved in RNA/DNA sensing, have all been implicated in promoting the generation of autoimmune responses. This evidence, as reviewed here, suggests that novel therapeutics targeting these sensors and their downstream pathways may be of use in the treatment of patients with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and primary Sjögren's syndrome. PMID:25193293

Smith, Siobhán; Jefferies, Caroline

2014-12-01

88

Fluorometric quantification of RNA and DNA in solutions containing both nucleic acids.  

PubMed

A fluorescence-based method for quantitative determination of RNA and DNA in probes containing both nucleic acids has been developed. The total concentration of nucleic acids is determined using SYBR Green II dye under conditions providing independent binding of the fluorophore with DNA and RNA. The concentration of DNA is specifically measured using the Hoechst 33258 dye and the RNA concentration is calculated from these data. The procedure allows for accurate determination of DNA concentration in the range 10-1000 ng/ml in the presence of 200-fold excess of RNA and determination of RNA concentrations in the range 10-1000 ng/ml in the presence of large excess of DNA. An absence of the treatment of mixed samples with RNase-free DNase I provides rapid, reproducible, and accurate RNA quantification. PMID:14705779

Morozkin, Evgeniy S; Laktionov, Pavel P; Rykova, Elena Y; Vlassov, Valentin V

2003-11-01

89

A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation  

PubMed Central

A prompt and efficient DNA damage response (DDR) eliminates the detrimental effects of DNA lesions in eukaryotic cells. Basic and preclinical studies suggest that the DDR is one of the primary anti-cancer barriers during tumorigenesis. The DDR involves a complex network of processes that detect and repair DNA damage, in which long non-coding RNAs (lncRNAs), a new class of regulatory RNAs, may play an important role. In the current study, we identified a novel lncRNA, lncRNA-JADE, that is induced after DNA damage in an ataxia-telangiectasia mutated (ATM)-dependent manner. LncRNA-JADE transcriptionally activates Jade1, a key component in the HBO1 (human acetylase binding to ORC1) histone acetylation complex. Consequently, lncRNA-JADE induces histone H4 acetylation in the DDR. Markedly higher levels of lncRNA-JADE were observed in human breast tumours in comparison with normal breast tissues. Knockdown of lncRNA-JADE significantly inhibited breast tumour growth in vivo. On the basis of these results, we propose that lncRNA-JADE is a key functional link that connects the DDR to histone H4 acetylation, and that dysregulation of lncRNA-JADE may contribute to breast tumorigenesis. PMID:24097061

Wan, Guohui; Hu, Xiaoxiao; Liu, Yunhua; Han, Cecil; Sood, Anil K; Calin, George A; Zhang, Xinna; Lu, Xiongbin

2013-01-01

90

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

91

Polyacid macromolecule primers  

DOEpatents

Hydrophilic polyacids are described, such as macromolecules of polyitaconic acid and polyacrylic acid, where such macromolecules have molecular weights >50,000 as primers between a polymeric top coating, such as polyurethane, and an oxidized aluminum or aluminum alloy. A near monolayer of primer is used in polymeric adhesive/oxidized aluminum adhered joint systems in 0.05% primer concentration to give superior results in standard peel tests. 2 figs.

Sugama, Toshifumi.

1989-12-26

92

Polyacid macromolecule primers  

DOEpatents

Hydrophylic polyacids, such as macromolecules of polyitaconic acid and polyacrylic acid, where such macromolecules have molecular weights >50,000 as primers between a polymeric top coating, such as polyurethane, and an oxidized aluminum or aluminum alloy. A near monolayer of primer is used in polymeric adhesive/oxidized aluminum adhered joint systems in 0.05% primer concentration to give superior results in standard peel tests.

Sugama, Toshifumi (Mastic Beach, NY)

1989-01-01

93

EBV Noncoding RNA Binds Nascent RNA to Drive Host PAX5 to Viral DNA.  

PubMed

EBER2 is an abundant nuclear noncoding RNA expressed by the Epstein-Barr virus (EBV). Probing its possible chromatin localization by CHART revealed EBER2's presence at the terminal repeats (TRs) of the latent EBV genome, overlapping previously identified binding sites for the B cell transcription factor PAX5. EBER2 interacts with PAX5 and is required for the localization of PAX5 to the TRs. EBER2 knockdown phenocopies PAX5 depletion in upregulating the expression of LMP2A/B and LMP1, genes nearest the TRs. Knockdown of EBER2 also decreases EBV lytic replication, underscoring the essential role of the TRs in viral replication. Recruitment of the EBER2-PAX5 complex is mediated by base-pairing between EBER2 and nascent transcripts from the TR locus. The interaction is evolutionarily conserved in the related primate herpesvirus CeHV15 despite great sequence divergence. Using base-pairing with nascent RNA to guide an interacting transcription factor to its DNA target site is a previously undescribed function for a trans-acting noncoding RNA. PMID:25662012

Lee, Nara; Moss, Walter N; Yario, Therese A; Steitz, Joan A

2015-02-12

94

Biopolymers Celebrates 50 Years of Nucleic Acids Research DNA AND RNA OVER HALF A CENTURY  

E-print Network

, which was quickly amended to include such non-canonical events as reverse transcription of RNA into DNAEditorial Biopolymers Celebrates 50 Years of Nucleic Acids Research DNA AND RNA OVER HALF A CENTURY of the DNA double helix by James Watson and Francis Crick 60 years ago, our grasp of the cen- tral role

Walter, Nils G.

95

Absolute molecular flux and angular distribution measurements to characterize DNA / RNA vapor jets  

E-print Network

1 Absolute molecular flux and angular distribution measurements to characterize DNA / RNA vapor.P.7955, Casablanca, Morocco Abstract Vapor jets of DNA and RNA bases (adenine, cytosine, thymine and viscous flow corresponding to Knudsen numbers in the range 0.1

Paris-Sud XI, Université de

96

Correlated measurements of DNA, RNA, and protein in individual cells by flow cytometry  

Microsoft Academic Search

A cytochemical method was developed to differentially stain cellular DNA, RNA, and proteins with fluorochromes Hoechst 33342, pyronin Y, and fluorescein isothiocyanate, respectively. The fluorescence intensities, reflecting the DNA, RNA, and protein content of individual cells, were measured in a flow cytometer after sequential excitation by three lasers tuned to different excitation wavelengths. The method offers rapid analysis of changes

H. A. Crissman; Z. Darzynkiewicz; R. A. Tobey; J. A. Steinkamp

1985-01-01

97

Fluorescence lifetime discrimination of cellular DNA and RNA using various intercalating dyes and flow cytometric analysis  

Microsoft Academic Search

Previously we showed that the fluorescence lifetimes of ethidium and propidium iodide were different when intercalated into cellular double-stranded DNA or RNA. Current studies of four ethidium derivatives showed that the lifetime difference of DNA and RNA bound dyes existed in all four compounds, and that the magnitude of the difference was dependent on the dye structure, the staining concentration,

H. H. Cui; Joseph G. Valdez; John A. Steinkamp; Harry A. Crissman

2001-01-01

98

Correlated Measurements of DNA, RNA, and Protein in Individual Cells by Flow Cytometry  

Microsoft Academic Search

A cytochemical method was developed to differentially stain cellular DNA, RNA, and proteins with fluorochromes Hoechst 33342, pyronin Y, and fluorescein isothiocyanate, respectively. The fluorescence intensities, reflecting the DNA, RNA, and protein content of individual cells, were measured in a flow cytometer after sequential excitation by three lasers tuned to different excitation wavelengths. The method offers rapid analysis of changes

Harry A. Crissman; Zbigniew Darzynkiewicz; Robert A. Tobey; John A. Steinkamp

1985-01-01

99

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

100

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

101

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

Peña-Llopis, Samuel; Brugarolas, James

2014-01-01

102

Initiation of simian virus 40 DNA replication in vitro: identification of RNA-primed nascent DNA chains.  

PubMed

Cell-free extracts of simian virus 40 (SV40)-infected CV-1 cells can initiate large tumor antigen dependent bidirectional replication in circular DNA molecules containing a functional SV40 origin of replication (ori). To determine whether or not DNA replication under these conditions involves RNA-primed DNA synthesis, replication was carried out in the presence of 5-mercuri-deoxycytidine triphosphate to label nascent DNA chains. Newly synthesized mercurated DNA was isolated by its affinity for thiol-agarose, and the 5'-ends of the isolated chains were radiolabeled to allow identification of RNA primers. At least 50% of the isolated chains contained 4 to 7 ribonucleotides covalently linked to their 5'-end; 80% of the oligoribonucleotides began with adenosine and 19% began with guanosine. About 60% of the nascent DNA chains annealed to the SV40 ori region, and about 80% of these chains were synthesized in the same direction as early mRNA. These results are consistent with the properties of SV40 DNA replication in vivo and support a model for initiation of SV40 DNA replication in which DNA primase initiates DNA synthesis on that strand of ori that encodes early mRNA. PMID:2444924

Taljanidisz, J; Decker, R S; Guo, Z S; DePamphilis, M L; Sarkar, N

1987-10-12

103

Initiation of simian virus 40 DNA replication in vitro: identification of RNA-primed nascent DNA chains.  

PubMed Central

Cell-free extracts of simian virus 40 (SV40)-infected CV-1 cells can initiate large tumor antigen dependent bidirectional replication in circular DNA molecules containing a functional SV40 origin of replication (ori). To determine whether or not DNA replication under these conditions involves RNA-primed DNA synthesis, replication was carried out in the presence of 5-mercuri-deoxycytidine triphosphate to label nascent DNA chains. Newly synthesized mercurated DNA was isolated by its affinity for thiol-agarose, and the 5'-ends of the isolated chains were radiolabeled to allow identification of RNA primers. At least 50% of the isolated chains contained 4 to 7 ribonucleotides covalently linked to their 5'-end; 80% of the oligoribonucleotides began with adenosine and 19% began with guanosine. About 60% of the nascent DNA chains annealed to the SV40 ori region, and about 80% of these chains were synthesized in the same direction as early mRNA. These results are consistent with the properties of SV40 DNA replication in vivo and support a model for initiation of SV40 DNA replication in which DNA primase initiates DNA synthesis on that strand of ori that encodes early mRNA. Images PMID:2444924

Taljanidisz, J; Decker, R S; Guo, Z S; DePamphilis, M L; Sarkar, N

1987-01-01

104

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

105

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

106

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

PubMed Central

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, 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 ?29 DNA. PMID:21471452

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

2011-01-01

107

Suppression of Hepatitis C Virus Genome Replication in Cells with RNA-Cleaving DNA Enzymes  

E-print Network

Suppression of Hepatitis C Virus Genome Replication in Cells with RNA-Cleaving DNA Enzymes, the hepatitis C virus nonstructural gene 3 (HCV NS3) RNA that encodes viral helicase and protease, from a pool. These selected DNAzyme and shRNA may be a viable therapeutic intervention to inhibit HCV replication in hepatic

Park, Jong-Sang

108

THE GREAT ESCAPE: Phloem Transport and Unloading of Macromolecules1.  

PubMed

The phloem of higher plants translocates a diverse range of macromolecules including proteins, RNAs, and pathogens. This review considers the origin and destination of such macromolecules. A survey of the literature reveals that the majority of phloem-mobile macromolecules are synthesized within companion cells and enter the sieve elements through the branched plasmodesmata that connect these cells. Examples of systemic macromolecules that originate outside the companion cell are rare and are restricted to viral and subviral pathogens and putative RNA gene-silencing signals, all of which involve a relay system in which the macromolecule is amplified in each successive cell along the pathway to companion cells. Evidence is presented that xenobiotic macromolecules may enter the sieve element by a default pathway as they do not possess the necessary signals for retention in the sieve element-companion cell complex. Several sink tissues possess plasmodesmata with a high-molecular-size exclusion limit, potentially allowing the nonspecific escape of a wide range of small (<50-kDa) macromolecules from the phloem. Larger macromolecules and systemic mRNAs appear to require facilitated transport through sink plasmodesmata. The fate of phloem-mobile macromolecules is considered in relation to current models of long-distance signaling in plants. PMID:15012195

Oparka, Karl J.; Cruz, Simon Santa

2000-06-01

109

Pulse Dipolar ESR of Doubly Labeled Mini TAR DNA and Its Annealing to Mini TAR RNA.  

PubMed

Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3'- and 5'-termini of the 27-base mini TAR DNA relocated to opposite ends of a 27 bp RNA-DNA duplex with 76.5 Å between labels, a distance which was consistent with the distance between the two labels in a thermally annealed 27-bp TAR DNA-TAR RNA duplex. Different sets of double labels initially located 26-27 Å apart in the mini TAR DNA upper stem, appropriately altered their interlabel distance to ?35 Å when a 27 bp TAR DNA-TAR RNA duplex formed, where the formation was caused either through NCp7-induced annealing or by thermal annealing. In summary, clear structural evidence was obtained for the fraying and destabilization brought on by NCp7 in its biochemical function as an annealing agent and for the detailed structural change from stem-loop to duplex RNA-DNA when complementary RNA was present. PMID:25692594

Sun, Yan; Borbat, Peter P; Grigoryants, Vladimir M; Myers, William K; Freed, Jack H; Scholes, Charles P

2015-02-17

110

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; Röling, Wilfred F. M.; van der Heijden, Marcel G. A.; Kowalchuk, George A.

2013-01-01

111

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

112

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

113

An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference.  

PubMed

CRISPR-Cas systems provide a small RNA-based mechanism to defend against invasive genetic elements in archaea and bacteria. To investigate the in vivo mechanism of RNA interference by two type III-B systems (Cmr-? and Cmr-?) in Sulfolobus islandicus, a genetic assay was developed using plasmids carrying an artificial mini-CRISPR (AC) locus with a single spacer. After pAC plasmids were introduced into different strains, Northern analyses confirmed that mature crRNAs were produced from the plasmid-borne CRISPR loci, which then guided gene silencing to target gene expression. Spacer mutagenesis identified a trinucleotide sequence in the 3'-region of crRNA that was crucial for RNA interference. Studying mutants lacking Cmr-? or Cmr-? system showed that each Cmr complex exhibited RNA interference. Strikingly, these analyses further revealed that the two Cmr systems displayed distinctive interference features. Whereas Cmr-? complexes targeted transcripts and could be recycled in RNA cleavage, Cmr-? complexes probably targeted nascent RNA transcripts and remained associated with the substrate. Moreover, Cmr-? exhibited much stronger RNA cleavage activity than Cmr-?. Since we previously showed that S. islandicus Cmr-? mediated transcription-dependent DNA interference, the Cmr-? constitutes the first CRISPR system exhibiting dual targeting of RNA and DNA. PMID:25505143

Peng, Wenfang; Feng, Mingxia; Feng, Xu; Liang, Yun Xiang; She, Qunxin

2015-01-01

114

An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference  

PubMed Central

CRISPR-Cas systems provide a small RNA-based mechanism to defend against invasive genetic elements in archaea and bacteria. To investigate the in vivo mechanism of RNA interference by two type III-B systems (Cmr-? and Cmr-?) in Sulfolobus islandicus, a genetic assay was developed using plasmids carrying an artificial mini-CRISPR (AC) locus with a single spacer. After pAC plasmids were introduced into different strains, Northern analyses confirmed that mature crRNAs were produced from the plasmid-borne CRISPR loci, which then guided gene silencing to target gene expression. Spacer mutagenesis identified a trinucleotide sequence in the 3?-region of crRNA that was crucial for RNA interference. Studying mutants lacking Cmr-? or Cmr-? system showed that each Cmr complex exhibited RNA interference. Strikingly, these analyses further revealed that the two Cmr systems displayed distinctive interference features. Whereas Cmr-? complexes targeted transcripts and could be recycled in RNA cleavage, Cmr-? complexes probably targeted nascent RNA transcripts and remained associated with the substrate. Moreover, Cmr-? exhibited much stronger RNA cleavage activity than Cmr-?. Since we previously showed that S. islandicus Cmr-? mediated transcription-dependent DNA interference, the Cmr-? constitutes the first CRISPR system exhibiting dual targeting of RNA and DNA. PMID:25505143

Peng, Wenfang; Feng, Mingxia; Feng, Xu; Liang, Yun Xiang; She, Qunxin

2015-01-01

115

Cytosolic RNA:DNA hybrids activate the cGAS-STING axis.  

PubMed

Intracellular recognition of non-self and also self-nucleic acids can result in the initiation of potent pro-inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2'-5'), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP-1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS-STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA. PMID:25425575

Mankan, Arun K; Schmidt, Tobias; Chauhan, Dhruv; Goldeck, Marion; Höning, Klara; Gaidt, Moritz; Kubarenko, Andrew V; Andreeva, Liudmila; Hopfner, Karl-Peter; Hornung, Veit

2014-12-17

116

Small RNAs tackle large viruses: RNA interference-based antiviral defense against DNA viruses in insects.  

PubMed

The antiviral RNA interference (RNAi) pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNA) that guide the recognition and cleavage of complementary viral target RNAs. In RNA virus infections, viral replication intermediates, dsRNA genomes or viral structured RNAs have been implicated as Dicer-2 substrates. In a recent publication, we demonstrated that a double-stranded DNA virus, Invertebrate iridescent virus 6, is a target of the Drosophila RNAi machinery, and we proposed that overlapping converging transcripts base pair to form the dsRNA substrates for vsiRNA biogenesis. Here, we discuss the role of RNAi in antiviral defense to DNA viruses in Drosophila and other invertebrate model systems. PMID:23974177

Bronkhorst, Alfred W; Miesen, Pascal; van Rij, Ronald P

2013-01-01

117

Small RNAs tackle large viruses: RNA interference-based antiviral defense against DNA viruses in insects  

PubMed Central

The antiviral RNA interference (RNAi) pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNA) that guide the recognition and cleavage of complementary viral target RNAs. In RNA virus infections, viral replication intermediates, dsRNA genomes or viral structured RNAs have been implicated as Dicer-2 substrates. In a recent publication, we demonstrated that a double-stranded DNA virus, Invertebrate iridescent virus 6, is a target of the Drosophila RNAi machinery, and we proposed that overlapping converging transcripts base pair to form the dsRNA substrates for vsiRNA biogenesis. Here, we discuss the role of RNAi in antiviral defense to DNA viruses in Drosophila and other invertebrate model systems. PMID:23974177

Bronkhorst, Alfred W; Miesen, Pascal; van Rij, Ronald P

2013-01-01

118

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

119

Analysis of macromolecules, ligands and macromolecule-ligand complexes  

DOEpatents

A method for determining atomic level structures of macromolecule-ligand complexes through high-resolution powder diffraction analysis and a method for providing suitable microcrystalline powder for diffraction analysis are provided. In one embodiment, powder diffraction data is collected from samples of polycrystalline macromolecule and macromolecule-ligand complex and the refined structure of the macromolecule is used as an approximate model for a combined Rietveld and stereochemical restraint refinement of the macromolecule-ligand complex. A difference Fourier map is calculated and the ligand position and points of interaction between the atoms of the macromolecule and the atoms of the ligand can be deduced and visualized. A suitable polycrystalline sample of macromolecule-ligand complex can be produced by physically agitating a mixture of lyophilized macromolecule, ligand and a solvent.

Von Dreele, Robert B. (Los Alamos, NM)

2008-12-23

120

Correlated measurements of DNA, RNA, and protein in individual cells by flow cytometry  

SciTech Connect

A cytochemical method was developed to differentially stain cellular DNA, RNA, and proteins with fluorochromes Hoechst 33342, pyronin Y, and fluorescein isothiocyanate, respectively. The fluorescence intensities, reflecting the DNA, RNA, and protein content of individual cells, were measured in a flow cytometer after sequential excitation by three lasers tuned to different excitation wavelengths. The method offers rapid analysis of changes in the cellular content of RNA and protein as well as in the RNA-protein, RNA-DNA, and protein-DNA ratios in relation to cell cycle position for large cell populations. An analysis of cycling cell populations (exponentially growing CHO cultures) and noncycling CHO cells arrested in the G1 phase by growth in isoleucine-free medium demonstrated the potential of the technique. 18 references, 2 figures, 1 table.

Crissman, H.A.; Darzynkiewicz, Z.; Tobey, R.A.; Steinkamp, J.A.

1985-06-14

121

Possible involvement of DNA-linked RNA in the initiation of Bacillus subtilis chromosome replication.  

PubMed Central

After thermal denaturation, an in vivo-labeled RNA was found in a temperature-sensitive initiation mutant of Bacillus subtilis (dna-37) associated with high-molecular-weight DNA. This RNA could be clearly distinguished from other RNA species by different techniques of separation, such as Sepharose 2B filtration, chromatography on nitrocellulose, and equilibrium centrifugation in density gradient. It was obtained even when HCHO was present during denaturation and chilling of nucleic acids and was still detected after a second denaturation as well as after incubation with proteinase K. Properties of the complex were not altered by prior treatment with RNase H. A control experiment using two samples of the complex treated either with pancreatic DNase or with pancreatic RNase, denatured together and centrifuged in the same density gradient, showed that no artifactual associations occur between the DNA and the RNA components of the complex. These results demonstrate that the DNA and RNA in the complex are associated by neither hydrogen bonds nor proteins, but are indicative of a DNA-RNA covalent linkage. In addition, during synchronous replication after a previous period at a nonpermissive temperature, DNA-linked RNA synthesis took place at specific times which coincided with the appearance of rifampin resistance of the first and the second replication cycles. A possible involvement of this RNA in the initiation of chromosome replication is discussed. PMID:6172420

Henckes, G; Vannier, F; Buu, A; Seror-Laurent, S J

1982-01-01

122

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

123

Fast Fenton Footprinting: A Laboratory-Based Method for the Time-Resolved Analysis of DNA, RNA and Proteins  

SciTech Connect

'Footprinting' describes assays in which ligand binding or structure formation protects polymers such as nucleic acids and proteins from either cleavage or modification; footprinting allows the accessibility of individual residues to be mapped in solution. Equilibrium and time-dependent footprinting links site-specific structural information with thermodynamic and kinetic transitions. The hydroxyl radical ({center_dot}OH) is a particularly valuable footprinting probe by virtue of it being among the most reactive of chemical oxidants; it reports the solvent accessibility of reactive sites on macromolecules with as fine as a single residue resolution. A novel method of millisecond time-resolved {center_dot}OH footprinting has been developed based on the Fenton reaction, Fe(II) + H{sub 2}O{sub 2} {yields} Fe(III) + {center_dot}OH + OH{sup -}. This method can be implemented in laboratories using widely available three-syringe quench flow mixers and inexpensive reagents to study local changes in the solvent accessibility of DNA, RNA and proteins associated with their biological function.

Shcherbakova,I.; Mitra, S.; Beer, R.; Brenowitz, M.

2006-01-01

124

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

125

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

PubMed

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

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

2014-01-01

126

A guanine-linked end-effect is a sensitive reporter of charge flow through DNA and RNA double helices  

Microsoft Academic Search

The property of charge (electron hole) flow in DNA duplexes has been the subject of intensive study. RNA–DNA heteroduplexes have also been investigated; however, little information exists on the conductive properties of purely RNA duplexes. In investigating the relative conductive properties of a three molecule DNA–DNA duplex design, using piperidine and aniline to break strands at modified bases, we observed

Lucien Junior Bergeron; Kaushiki Sen; Dipankar Sen

2008-01-01

127

A novel rat genomic simple repeat DNA with RNA-homology shows triplex (H-DNA)-like structure and tissue-specific RNA expression.  

PubMed

Mammalian genome contains a wide variety of repetitive DNA sequences of relatively unknown function. We report a novel 227 bp simple repeat DNA (3.3 DNA) with a d{(GA)7A(AG)7} dinucleotide mirror repeat from the rat (Rattus norvegicus) genome. 3.3 DNA showed 75-85% homology with several eukaryotic mRNAs due to (GA/CU)n dinucleotide repeats by nBlast search and a dispersed distribution in the rat genome by Southern blot hybridization with [32P]3.3 DNA. The d{(GA)7A(AG)7} mirror repeat formed a triplex (H-DNA)-like structure in vitro. Two large RNAs of 9.1 and 7.5 kb were detected by [32P]3.3 DNA in rat brain by Northern blot hybridization indicating expression of such simple sequence repeats at RNA level in vivo. Further, several cDNAs were isolated from a rat cDNA library by [32P]3.3 DNA probe. Three such cDNAs showed tissue-specific RNA expression in rat. pRT 4.1 cDNA showed strong expression of a 2.39 kb RNA in brain and spleen, pRT 5.5 cDNA showed strong expression of a 2.8 kb RNA in brain and a 3.9 kb RNA in lungs, and pRT 11.4 cDNA showed weak expression of a 2.4 kb RNA in lungs. Thus, genomic simple sequence repeats containing d(GA/CT)n dinucleotides are transcriptionally expressed and regulated in rat tissues. Such d(GA/CT)n dinucleotide repeats may form structural elements (e.g., triplex) which may be sites for functional regulation of genomic coding sequences as well as RNAs. This may be a general function of such transcriptionally active simple sequence repeats widely dispersed in mammalian genome. PMID:15629459

Dey, Indranil; Rath, Pramod C

2005-02-01

128

Simultaneous analysis of micro-RNA and DNA for determining the body fluid origin of DNA profiles.  

PubMed

Micro-RNAs (miRNAs) can be specifically expressed in forensically relevant body fluids such as blood or saliva. The aim of the study was to develop a simultaneous extraction and analysis protocol that allows for the acquisition of a DNA profile and the identity of the body fluid using a single process. DNA and micro-RNA were extracted from blood and saliva before undergoing a cDNA synthesis step by using stem-loop reverse transcription PCR. The resulting extracts containing DNA and cDNA synthesized from body fluid-specific miRNA markers then underwent standard STR analysis using a modified ABI AmpF?STR(®) NGM SElect™ kit. In all samples, a full DNA profile was obtained along with additional peaks corresponding to the miRNA marker targeted. In all cases, blood samples profiled exhibited a peak indicating the presence of the blood-specific miRNA marker and the saliva sample profiled exhibited a peak indicating the presence of the saliva-specific miRNA marker. PMID:23683171

van der Meer, Donny; Uchimoto, Mari L; Williams, Graham

2013-07-01

129

Analysis of the cellular DNA and RNA content in acute leukemias by flow cytometry  

Microsoft Academic Search

Summary In the present study bone marrow samples from 573 patients with newly diagnosed acute myeloid (AML) and lymphoblastic or undifferentiated leukemias (ALL\\/AUL), were analysed for their cellular DNA und DNA\\/RNA content, respectively, by means of flow cytometry. From 237 patients with AML 35.4% revealed aneuploid DNA stemlines. While no relation of DNA aneuploidy with other pretherapeutic parameters, including FAB

W. Hiddemann; B. Wiirmann; D. Messerer; R. Springefeld; Th. Büchner

1990-01-01

130

Tandem Duplication of D-Loop and Ribosomal RNA Sequences in Lizard Mitochondrial DNA  

Microsoft Academic Search

Some Cnemidophorus exsanguis have mitochondrial DNA's (mtDNA's) that are 22.2 kilobases (kb) in size, whereas most have mtDNA's of 17.4 kb. Restriction site mapping, DNA transfer hybridization experiments, and electron microscopy show that the size increment stems from the tandem duplication of a 4.8-kb region that includes regulatory sequences and transfer and ribosomal RNA genes. This observation is notable in

Craig Moritz; Wesley M. Brown

1986-01-01

131

A Course on Macromolecules.  

ERIC Educational Resources Information Center

Describes a senior-level course that: (1) focuses on the structure and reactions of macromolecules; (2) treats industrial polymers in a unified way; and (3) uses analysis of conformation and conformational statistics as a unifying approach. Also discusses course topics, including polysaccharides, proteins, nucleic acids, and others. (JN)

Horta, Arturo

1985-01-01

132

Transmission of viral RNA and DNA to maize kernels by vascular puncture inoculation.  

PubMed

Vascular puncture inoculation (VPI) is an effective technique for transmission of maize viruses without using arthropods or other biological vectors. It involves using a jeweler's engraving tool to push minuten pins through a droplet of virus inoculum toward the major vascular bundle in the scutellum of germinating kernels. Here, VPI is shown to be useful for introducing RNA and DNA viral genomes into maize. Maize dwarf mosaic potyvirus (MDMV) virions, MDMV genomic RNA, foxtail mosaic potexvirus (FoMV) genomic RNA and maize streak geminivirus (MSV) DNA were introduced into kernels by VPI, and infection rates determined. At high concentrations, both MDMV virion and genomic RNA preparations produced 100% infection of susceptible maize. However, MDMV genomic RNA was transmitted with about 100-fold lower efficiency than virions. FoMV genomic RNA and MSV DNA were transmitted at lower efficiency than the MDMV RNA, and the highest transmission rates were about 50%. Ribonuclease A pretreatment prevented genomic MDMV and FoMV RNA transmission, but not MDMV virion transmission indicating the viral RNA was the infectious entity. Proteinase K (ProK) pretreatment reduced transmission of MDMV RNA suggesting that integrity of the viral genomic protein bound covalently to the viral RNA may be important for efficient transmission. PMID:11576640

Redinbaugh, M G; Louie, R; Ngwira, P; Edema, R; Gordon, D T; Bisaro, D M

2001-11-01

133

The Midblastula Transition Defines the Onset of Y RNA-Dependent DNA Replication in Xenopus laevis ?  

PubMed Central

Noncoding Y RNAs are essential for the initiation of chromosomal DNA replication in mammalian cell extracts, but their role in this process during early vertebrate development is unknown. Here, we use antisense morpholino nucleotides (MOs) to investigate Y RNA function in Xenopus laevis and zebrafish embryos. We show that embryos in which Y RNA function is inhibited by MOs develop normally until the midblastula transition (MBT) but then fail to replicate their DNA and die before gastrulation. Consistent with this observation, Y RNA function is not required for DNA replication in Xenopus egg extracts but is required for replication in a post-MBT cell line. Y RNAs do not bind chromatin in karyomeres before MBT, but they associate with interphase nuclei after MBT in an origin recognition complex (ORC)-dependent manner. Y RNA-specific MOs inhibit the association of Y RNAs with ORC, Cdt1, and HMGA1a proteins, suggesting that these molecular associations are essential for Y RNA function in DNA replication. The MBT is thus a transition point between Y RNA-independent and Y RNA-dependent control of vertebrate DNA replication. Our data suggest that in vertebrates Y RNAs function as a developmentally regulated layer of control over the evolutionarily conserved eukaryotic DNA replication machinery. PMID:21791613

Collart, Clara; Christov, Christo P.; Smith, James C.; Krude, Torsten

2011-01-01

134

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

135

Single molecule photobleaching (SMPB) technology for counting of RNA, DNA, protein and other molecules in nanoparticles and biological complexes by TIRF instrumentation.  

PubMed

Direct counting of biomolecules within biological complexes or nanomachines is demanding. Single molecule counting using optical microscopy is challenging due to the diffraction limit. The single molecule photobleaching (SMPB) technology for direct counting developed by our team (Shu et al., 2007 [18]; Zhang et al., 2007 [19]) offers a simple and straightforward method to determine the stoichiometry of molecules or subunits within biocomplexes or nanomachines at nanometer scales. Stoichiometry is determined by real-time observation of the number of descending steps resulted from the photobleaching of individual fluorophore. This technology has now been used extensively for single molecule counting of protein, RNA, and other macromolecules in a variety of complexes or nanostructures. Here, we elucidate the SMPB technology, using the counting of RNA molecules within a bacteriophage phi29 DNA-packaging biomotor as an example. The method described here can be applied to the single molecule counting of other molecules in other systems. The construction of a concise, simple and economical single molecule total internal reflection fluorescence (TIRF) microscope combining prism-type and objective-type TIRF is described. The imaging system contains a deep-cooled sensitive EMCCD camera with single fluorophore detection sensitivity, a laser combiner for simultaneous dual-color excitation, and a Dual-View™ imager to split the multiple outcome signals to different detector channels based on their wavelengths. Methodology of the single molecule photobleaching assay used to elucidate the stoichiometry of RNA on phi29 DNA packaging motor and the mechanism of protein/RNA interaction are described. Different methods for single fluorophore labeling of RNA molecules are reviewed. The process of statistical modeling to reveal the true copy number of the biomolecules based on binomial distribution is also described. PMID:24440482

Zhang, Hui; Guo, Peixuan

2014-05-15

136

A Computational Model for Predicting Experimental RNA and DNA Nearest-Neighbor Free Energy Rankings  

PubMed Central

Hydrogen-bonding, intra-strand base-stacking, and inter-strand base-stacking energies were calculated for RNA and DNA dimers at the MP2(full)/6-311G** level of theory. Standard A-form RNA and B-form DNA geometries from average fiber diffraction data were employed for all base monomer and dimer geometries, and all dimer binding energies were obtained via single-point calculations. The effects of water solvation were considered using the PCM model. The resulting dimer binding energies were used to calculate the 10 unique RNA and 10 unique DNA computational nearest-neighbor energies, and the ranking of these computational nearest neighbor energies are in excellent agreement with the ranking of the experimental nearest neighbor free energies. These results dispel the notion that average fiber diffraction geometries are insufficient for calculating RNA and DNA stacking energies. PMID:21619071

Johnson, Charles A.; Bloomingdale, Richard J.; Ponnusamy, Vikram E.; Tillinghast, Conor A.; Znosko, Brent M.; Lewis, Michael

2011-01-01

137

SPERM RNA AMPLIFICATION FOR GENE EXPRESSION PROFILING BY DNA MICROARRAY TECHNOLOGY  

EPA Science Inventory

Sperm RNA Amplification for Gene Expression Profiling by DNA Microarray Technology Hongzu Ren, Kary E. Thompson, Judith E. Schmid and David J. Dix, Reproductive Toxicology Division, NHEERL, Office of Research and Development, US Environmental Protection Agency, Research Triang...

138

Very Few RNA and DNA Sequence Differences in the Human Transcriptome  

E-print Network

,2 *. , Jean-Francois Gout1. , Matthew W. Hahn1,2 1 Department of Biology, Indiana University, Bloomington than originally proposed. Citation: Schrider DR, Gout J-F, Hahn MW (2011) Very Few RNA and DNA Sequence

Hahn, Matthew

139

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

140

DNA and RNA Synthesis in Animal Cells in Culture--Methods for Use in Schools  

ERIC Educational Resources Information Center

Describes the experimental procedures used for detecting DNA and RNA synthesis in xenopus cells by autoradiography. The method described is suitable for senior high school laboratory classes or biology projects, if supervised by a teacher qualified to handle radioisotopes. (JR)

Godsell, P. M.; Balls, M.

1973-01-01

141

UV light-induced DNA lesions cause dissociation of yeast RNA polymerases-I and establishment of a specialized chromatin structure at rRNA genes.  

PubMed

The cytotoxicity of UV light-induced DNA lesions results from their interference with transcription and replication. DNA lesions arrest elongating RNA polymerases, an event that triggers transcription-coupled nucleotide excision repair. Since arrested RNA polymerases reduce the accessibility of repair factors to DNA lesions, they might be displaced. The fate of arrested RNA polymerases-II at DNA lesions has been extensively studied, yielding partially contradictory results. Considerably less is known about RNA polymerases-I that transcribe nucleosomes-depleted rRNA genes at very high rate. To investigate the fate of arrested RNA polymerases-I at DNA lesions, chromatin-immunoprecipitation, electron microscopy, transcription run-on, psoralen-cross-linking and chromatin-endogenous cleavage were employed. We found that RNA polymerases-I density increased at the 5'-end of the gene, likely due to continued transcription initiation followed by elongation and pausing/release at the first DNA lesion. Most RNA polymerases-I dissociated downstream of the first DNA lesion, concomitant with chromatin closing that resulted from deposition of nucleosomes. Although nucleosomes were deposited, the high mobility group-box Hmo1 (component of actively transcribed rRNA genes) remained associated. After repair of DNA lesions, Hmo1 containing chromatin might help to restore transcription elongation and reopening of rRNA genes chromatin. PMID:24097442

Tremblay, Maxime; Charton, Romain; Wittner, Manuel; Levasseur, Geneviève; Griesenbeck, Joachim; Conconi, Antonio

2014-01-01

142

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

PubMed Central

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

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

2008-01-01

143

IDN2 has a role downstream of siRNA formation in RNA-directed DNA methylation  

PubMed Central

In plants, a particular class of short interfering (si)RNAs can serve as a signal to induce cytosine methylation at homologous genomic regions. If the targeted DNA has promoter function, this RNA-directed DNA methylation (RdDM) can result in transcriptional gene silencing (TGS). RNA-directed transcriptional gene silencing (RdTGS) of transgenes provides a versatile system for the study of epigenetic gene regulation. We used transcription of a nopaline synthase promoter (ProNOS)-inverted repeat (IR) to provide a RNA signal that triggers de novo cytosine methylation and TGS of a homologous ProNOS copy in trans. Utilizing a ProNOS-NPTII reporter gene showing high sensitivity to silencing in this two component system, a forward genetic screen for EMS-induced no rna-directed transcriptional silencing (nrd) mutations was performed in Arabidopsis thaliana. Three nrd mutant lines were found to contain one novel loss-of-function allele of idn2/rdm12 and two of nrpd2a/nrpe2a. IDN2/RDM12 encodes a XH/XS domain protein that is able to bind double-stranded RNA with 5? overhangs, while NRPD2a/NRPE2a encodes the common second-largest subunit of the plant specific DNA-dependent RNA polymerases IV and V involved in silencing processes. Both idn2/rdm12 and nrpd2a/nrpe2a release target transgene expression and reduce CHH context methylation at transgenic as well as endogenous RdDM target regions to similar extents. Nevertheless, accumulation of IR-derived siRNA is not affected, allowing us to present a refined model for the pathway of RdDM and RdTGS that positions function of IDN2 downstream of siRNA formation and points to an important role for its XH domain. PMID:22810086

Finke, Andreas; Kuhlmann, Markus; Florian Mette, Michael

2012-01-01

144

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

145

CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli.  

PubMed

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5' handle (8 nt) and a 3' handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5' handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA. PMID:25488810

Beloglazova, Natalia; Kuznedelov, Konstantin; Flick, Robert; Datsenko, Kirill A; Brown, Greg; Popovic, Ana; Lemak, Sofia; Semenova, Ekaterina; Severinov, Konstantin; Yakunin, Alexander F

2015-01-01

146

CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli  

PubMed Central

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5? handle (8 nt) and a 3? handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5? handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA. PMID:25488810

Beloglazova, Natalia; Kuznedelov, Konstantin; Flick, Robert; Datsenko, Kirill A.; Brown, Greg; Popovic, Ana; Lemak, Sofia; Semenova, Ekaterina; Severinov, Konstantin; Yakunin, Alexander F.

2015-01-01

147

Normal and perturbed Chinese hamster ovary cells: correlation of DNA, RNA, and protein content by flow cytometry  

Microsoft Academic Search

Quantitative, correlated determinations of DNA, RNA, and protein, as well as RNA to DNA and RNA to protein ratios, were performed on three-color stained cells using a multiwavelength-excitation flow cytometer. DNA-bound Hoechst 33342 (blue), protein- fluorescein isothiocyanate {green), and RNA-bound pyronin Y (red) fluorescence measure- ments were correlated as each stained cell intersected three spatially separated laser beams. The analytical

H. A. Crissman; Z. DARZYNKIEWlCZ; R. A. TOBEY; J. A. STEINKAMP

1985-01-01

148

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, Frédéric H.-T

2012-01-01

149

Calculated cross sections for electron elastic and inelastic scattering from DNA and RNA bases  

NASA Astrophysics Data System (ADS)

Electron scattering cross sections from DNA/RNA bases are calculated in the 10 eV 10 keV energy range by combining screening-corrected aditivity-rule (SCAR) with ab-initio quasifree inelastic atomic potential. Similar calculations compare very favourably with available experimental data for benzene, C6F6 and tetrahydrofuran molecules. For DNA/RNA bases, where no experimental data is available, present results are compared with other theoretical values.

Blanco, F.; García, G.

2007-01-01

150

Method for rapid base sequencing in DNA and RNA with two base labeling  

DOEpatents

Method for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand.

Jett, James H. (Los Alamos, NM); Keller, Richard A. (Los Alamos, NM); Martin, John C. (Los Alamos, NM); Posner, Richard G. (Los Alamos, NM); Marrone, Babetta L. (Los Alamos, NM); Hammond, Mark L. (Los Alamos, NM); Simpson, Daniel J. (Los Alamos, NM)

1995-01-01

151

Method for rapid base sequencing in DNA and RNA with two base labeling  

DOEpatents

A method is described for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand. 4 figures.

Jett, J.H.; Keller, R.A.; Martin, J.C.; Posner, R.G.; Marrone, B.L.; Hammond, M.L.; Simpson, D.J.

1995-04-11

152

Combined RNA/DNA fluorescence in situ hybridization on whole-mount Drosophila ovaries.  

PubMed

DNA FISH (fluorescent in situ hybridization) analysis reveals the chromosomal location of the gene of interest. RNA in situ hybridization is used to examine the amounts and cell location of transcripts. This method is commonly used to describe the localization of processed transcripts in different tissues or cell lines. Gene activation studies are often aimed at determining the mechanism of this activation (transcriptional or posttranscriptional). Elucidation of the mechanism of piRNA-mediated silencing of genomic repeats is at the cutting edge of small RNA research. The RNA/DNA FISH technique is a powerful method for assessing transcriptional changes at any particular genomic locus. Colocalization of the RNA and DNA FISH signals allows a determination of the accumulation of nascent transcripts at the transcribed genomic locus. This would be suggest that this gene is activated at the transcriptional (or co-transcriptional) level. Moreover, this method allows for the identification of transcriptional derepression of a distinct copy (copies) among a genomic repeat family. Here, a RNA/DNA FISH protocol is presented for the simultaneous detection of RNA and DNA in situ on whole-mount Drosophila ovaries using tyramide signal amplification. With subsequent immunostaining of chromatin components, this protocol can be easily extended for studying the interdependence between chromatin changes at genomic loci and their transcriptional activity. PMID:24178564

Shpiz, Sergey; Lavrov, Sergey; Kalmykova, Alla

2014-01-01

153

Using a commercial DNA extraction kit to obtain RNA from mature rice kernels  

Technology Transfer Automated Retrieval System (TEKTRAN)

Few RNA extraction protocols or commercial kits work well with the starchy endosperm of cereal grains. Standard RNA extraction protocols are time consuming, use large amounts of expensive chemicals, and leave behind hazardous wastes. However, there are numerous commercial DNA extraction kits that ...

154

[Breast cancer diagnostics based on extracellular DNA and RNA circulating in blood].  

PubMed

Extracellular DNA and RNA were extracted from blood plasma and cell surface-bound fractions of patients with breast tumors and healthy controls. Frequency of RASSF1A, Cyclin D2 and RARbeta2 methylation was detected using methylation-specific PCR in the extracellular DNA, extracted from plasma and cell-surface bound fractions of patient blood. Methylation of at least one of these genes was found in plasma of 13% patients with benign breast fibroadenoma and in 60% of breast cancer patients. Using cell-surface bound DNA as a substrate for PCR have lead to increase of gene methylation detection frequency up to 87% in fibroadenoma and 95% in breast cancer patients without false positive controls. GAPDH, RASSF8, Ki-67 RNA and 18S RNA were quantified using RT-qPCR of the extracellular RNA circulating in blood of patients with breast tumors and healthy controls. The main part of the extracellular RNA was shown to be cell-surface bound. Results show a higher amount of RASSF8, Ki-67 RNA and 18S RNA in plasma and cell-bound fraction of patients with breast cancer compared with patients with benign tumors and healthy controls. The data indicate that the specific RNA quantification in blood plasma is valuable for discrimination between cancer and benign tumors, which can be detected with high sensitivity using analysis of methylated RASSF1A, Cyclin D2 and RARbeta2 genes in extracellular circulating DNA. PMID:18421914

Rykova, E Iu; Skvortsova, T E; Hoffmann, A L; Tamkovich, S N; Starikov, A V; Bryzgunova, O E; Permiakova, V I; Warnecke, J M; Sczakiel, G; Vlasov, V V; Laktionov, P P

2008-01-01

155

DNA-, rRNA- and mRNA-based stable isotope probing of aerobic methanotrophs in lake sediment.  

PubMed

A stable isotope probing (SIP) approach was used to study aerobic methane-oxidizing bacteria (methanotrophs) in lake sediment. Oligotrophic Lake Stechlin was chosen because it has a permanently oxic sediment surface. 16S rRNA and the pmoA gene, which encodes a subunit of the methane monooxygenase enzyme, were analysed following the incubation of sediment with (13) CH(4) and the separation of (13) C-labelled DNA and RNA from unlabelled nucleic acids. The incubation with (13) CH(4) was performed over a 4-day time-course and the pmoA genes and transcripts became progressively labelled such that approximately 70% of the pmoA genes and 80% of the transcripts were labelled at 96 h. The labelling of pmoA mRNA was quicker than pmoA genes, demonstrating that mRNA-SIP is more sensitive than DNA-SIP; however, the general rate of pmoA transcript labelling was comparable to that of the pmoA genes, indicating that the incorporation of (13) C into ribonucleic acids of methanotrophs was a gradual process. Labelling of Betaproteobacteria was clearly seen in analyses of 16S rRNA by DNA-SIP and not by RNA-SIP, suggesting that cross-feeding of the (13) C was primarily detected by DNA-SIP. In general, we show that the combination of SIP approaches provided valuable information about the activity and growth of the methanotrophic populations and the cross-feeding of methanotroph metabolites by other microorganisms. PMID:21261798

Dumont, Marc G; Pommerenke, Bianca; Casper, Peter; Conrad, Ralf

2011-05-01

156

A General Strategy for Effector-mediated Control of RNA-cleaving Ribozymes and DNA Enzymes  

Microsoft Academic Search

A novel and general approach is described for generating versions of RNA-cleaving ribozymes (RNA enzymes) and DNAzymes (DNA enzymes), whose catalytic activity can be controlled by the binding of activator molecules. Variants of the RNA-cleaving 10–23 DNAzyme and 8–17 DNAzyme were created, whose catalysis was activated by up to ?35-fold by the binding of the effector adenosine. The design of

Dennis Y. Wang; Beatrice H. Y. Lai; Dipankar Sen

2002-01-01

157

Nucleocytoplasmic transport of macromolecules.  

PubMed Central

Nucleocytoplasmic transport is a complex process that consists of the movement of numerous macromolecules back and forth across the nuclear envelope. All macromolecules that move in and out of the nucleus do so via nuclear pore complexes that form large proteinaceous channels in the nuclear envelope. In addition to nuclear pores, nuclear transport of macromolecules requires a number of soluble factors that are found both in the cytoplasm and in the nucleus. A combination of biochemical, genetic, and cell biological approaches have been used to identify and characterize the various components of the nuclear transport machinery. Recent studies have shown that both import to and export from the nucleus are mediated by signals found within the transport substrates. Several studies have demonstrated that these signals are recognized by soluble factors that target these substrates to the nuclear pore. Once substrates have been directed to the pore, most transport events depend on a cycle of GTP hydrolysis mediated by the small Ras-like GTPase, Ran, as well as other proteins that regulate the guanine nucleotide-bound state of Ran. Many of the essential factors have been identified, and the challenge that remains is to determine the exact mechanism by which transport occurs. This review attempts to present an integrated view of our current understanding of nuclear transport while highlighting the contributions that have been made through studies with genetic organisms such as the budding yeast, Saccharomyces cerevisiae. PMID:9184010

Corbett, A H; Silver, P A

1997-01-01

158

DNA and RNA content analysis by flow cytometry in the pathobiologic assessment of bone tumors  

Microsoft Academic Search

Studies of simultaneous DNA and RNA contents by flow cytometry in hematologic and some solid neoplasms have been shown to provide information that may be useful in pathobiological evaluation of these neoplasms. We contend that similar analysis may be equally valuable in assessing bone tumors. Our data revealed significant statistical differences in DNA ploidy and proliferative fraction between benign and

Adel K. El-Naggar; Kenneth Hurr; Z. Nora Tu; Kim Teague; Kevin A. Raymond; Alberto G. Ayala; John Murray

1995-01-01

159

Evaluation of branched DNA signal amplification for the detection of hepatitis C virus RNA.  

PubMed

There is an increasing need for a practical assay to measure HCV RNA to assess the viral burden in chronic hepatitis C virus (HCV) infection as viral load relates to transmission and therapeutic response. This study evaluates branched DNA (bDNA) signal amplification, a technique that avoids many of the pitfalls of polymerase chain reaction (PCR). The bDNA assay uses a microtitre well format and a series of capture, target and amplification probes that bind RNA to the well and then successively bind oligonucleotides to the RNA and branched DNA molecules to the oligonucleotides. Enzyme-labelled probes are bound to the arms of the bDNA and light output from a chemiluminescent substrate is directly proportional to the amount of starting HCV RNA. Appropriate standards provide direct quantitation. Whereas PCR amplifies the HCV genome, bDNA amplifies the hybridization signal. In testing a standardized, coded panel, bDNA showed 100% specificity and detected five of six sera proven to transmit hepatitis C to the chimpanzee; PCR detected all six infectious sera. Serial samples were measured in two acute and five chronic cases of transfusion-associated hepatitis and in three commercial seroconversion panels. In acute cases, 10(7)-10(8) molecular equivalents per ml (eq per ml) of HCV RNA were detected prior to peak alanine aminotransferase (ALT) activity and then rapidly declined to non-detectable levels. Similar levels of HCV RNA were observed early in the course of two patients who progressed to chronic hepatitis; the chronic course was characterized by diminished, fluctuating and sometimes non-detectable levels of HCV RNA. In two chronic cases, HCV RNA was not detected, or only transiently detected by bDNA, but was present when assayed by PCR. In one chronic case, the periodicity of HCV RNA levels closely paralleled the fluctuations of ALT suggesting a relationship between viral replication and subsequent hepatocellular injury. In testing 50 blood donors whose anti-HCV reactivity was confirmed by a recombinant immunoblot assay (RIBA), HCV RNA was detected by bDNA in 41 (81%), while PCR was positive in 45 (90%); the overall concordance between bDNA and PCR in 100 anti-HCV enzyme immunoassays (EIA) reactive donor samples was 96%.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7493306

Alter, H J; Sanchez-Pescador, R; Urdea, M S; Wilber, J C; Lagier, R J; Di Bisceglie, A M; Shih, J W; Neuwald, P D

1995-01-01

160

The diversity of Malassezia yeasts confirmed by rRNA sequence and nuclear DNA comparisons  

Microsoft Academic Search

One hundred and fourMalassezia strains (52 isolated from humans and 52 from animals) were compared using large subunit (LSU) ribosomal RNA sequence similarity and nuclear DNA complementarity. Eight groups of strains were recognized as genetically distinct species. Each taxon was confirmed by a homogeneous mole % GC and percentages of DNA\\/DNA reassociations higher than 85%. The non-lipid-dependentMalassezia yeasts were maintained

Jacques Guillot; Eveline Guého

1995-01-01

161

Evaluation of Commercial Kits for Dual Extraction of DNA and RNA from Human Body Fluids(,) (.)  

PubMed

STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body-fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR-Duet(™) kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand-alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification. PMID:25284026

Schweighardt, Andrew J; Tate, Courtney M; Scott, Kristina A; Harper, Kathryn A; Robertson, James M

2014-10-01

162

From face to interface recognition: a differential geometric approach to distinguish DNA from RNA binding surfaces  

PubMed Central

Protein nucleic acid interactions play a critical role in all steps of the gene expression pathway. Nucleic acid (NA) binding proteins interact with their partners, DNA or RNA, via distinct regions on their surface that are characterized by an ensemble of chemical, physical and geometrical properties. In this study, we introduce a novel methodology based on differential geometry, commonly used in face recognition, to characterize and predict NA binding surfaces on proteins. Applying the method on experimentally solved three-dimensional structures of proteins we successfully classify double-stranded DNA (dsDNA) from single-stranded RNA (ssRNA) binding proteins, with 83% accuracy. We show that the method is insensitive to conformational changes that occur upon binding and can be applicable for de novo protein-function prediction. Remarkably, when concentrating on the zinc finger motif, we distinguish successfully between RNA and DNA binding interfaces possessing the same binding motif even within the same protein, as demonstrated for the RNA polymerase transcription-factor, TFIIIA. In conclusion, we present a novel methodology to characterize protein surfaces, which can accurately tell apart dsDNA from an ssRNA binding interfaces. The strength of our method in recognizing fine-tuned differences on NA binding interfaces make it applicable for many other molecular recognition problems, with potential implications for drug design. PMID:21693557

Shazman, Shula; Elber, Gershon; Mandel-Gutfreund, Yael

2011-01-01

163

Telomeric RNA-DNA hybrids affect telomere-length dynamics and senescence.  

PubMed

Although telomeres are heterochromatic, they are transcribed into noncoding telomeric repeat-containing RNA (TERRA). Here we show that RNA-DNA hybrids form at telomeres and are removed by RNase H enzymes in the budding yeast, Saccharomyces cerevisiae. In recombination-competent telomerase mutants, telomeric RNA-DNA hybrids promote recombination-mediated elongation events that delay the onset of cellular senescence. Reduction of TERRA and telomeric RNA-DNA-hybrid levels diminishes rates of recombination-mediated telomere elongation in cis. Overexpression of RNase H decreases telomere recombination rates and accelerates senescence in recombination-competent but not recombination-deficient cells. In contrast, in the absence of both telomerase and homologous recombination, accumulation of telomeric RNA-DNA hybrids leads to telomere loss and accelerated rates of cellular senescence. Therefore, the regulation of TERRA transcription and telomeric RNA-DNA-hybrid formation are important determinants of both telomere-length dynamics and proliferative potential after the inactivation of telomerase. PMID:24013207

Balk, Bettina; Maicher, André; Dees, Martina; Klermund, Julia; Luke-Glaser, Sarah; Bender, Katharina; Luke, Brian

2013-10-01

164

Simultaneous Extraction from Bacterioplankton of Total RNA and DNA Suitable for Quantitative Structure and Function Analyses  

PubMed Central

The aim of this study was to develop a protocol for the simultaneous extraction from bacterioplankton of RNA and DNA suitable for quantitative molecular analysis. By using a combined mechanical and chemical extraction method, the highest RNA and DNA yield was obtained with sodium lauryl sarcosinate-phenol or DivoLab-phenol as the extraction mix. The efficiency of extraction of nucleic acids was comparatively high and varied only moderately in gram-negative bacterial isolates and bacterioplankton (RNA, 52 to 66%; DNA, 43 to 61%); significant amounts of nucleic acids were also obtained for a gram-positive bacterial isolate (RNA, 20 to 30%; DNA, 20 to 25%). Reverse transcription-PCR and PCR amplification products of fragments of 16S rRNA and its genes were obtained from all isolates and communities, indicating that the extracted nucleic acids were intact and pure enough for community structure analyses. By using single-strand conformation polymorphism of fragments of 16S rRNA and its gene, community fingerprints were obtained from pond bacterioplankton. mRNA transcripts encoding fragments of the enzyme nitrite reductase gene (nir gene) could be detected in a pond water sample, indicating that the extraction method is also suitable for studying gene expression. The extraction method presented yields nucleic acids that can be used to perform structural and functional studies of bacterioplankton communities from a single sample. PMID:11872453

Weinbauer, Markus G.; Fritz, Ingo; Wenderoth, Dirk F.; Höfle, Manfred G.

2002-01-01

165

Development of multiplex PCR for simultaneous detection of six swine DNA and RNA viruses.  

PubMed

Uniplex and multiplex reverse transcription-polymerase chain reaction (RT-PCR) and PCR protocols were developed and evaluated subsequently for its effectiveness in detecting simultaneously single and mixed infections in swine. Specific primers for three DNA viruses and three RNA viruses, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV) were used for testing procedure. A single nucleic acid extraction protocol was adopted for the simultaneous extraction of both RNA and DNA viruses. The multiplex PCR consisted with two-step procedure which included reverse transcription of RNA virus and multiplex PCR of viral cDNA and DNA. The multiplex PCR assay was shown to be sensitive detecting at least 450pg of viral genomic DNA or RNA from a mixture of six viruses in a reaction. The assay was also highly specific in detecting one or more of the same viruses in various combinations in specimens. Thirty clinical samples and aborted fetuses collected from 4- to 12-week-old piglets were detected among 39 samples tested by both uniplex and multiplex PCR, showing highly identification. Because of the sensitivity and specificity, the multiplex PCR is a useful approach for clinical diagnosis of mixed infections of DNA and RNA viruses in swine. PMID:22575688

Xu, Xin-Gang; Chen, Guang-Da; Huang, Yong; Ding, Li; Li, Zhao-Cai; Chang, Ching-Dong; Wang, Chi-Young; Tong, De-Wen; Liu, Hung-Jen

2012-07-01

166

DOMAINS REARRANGED METHYLTRANSFERASE3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis.  

PubMed

DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DOMAINS REARRANGED METHYLASE 2 (DRM2) controls RNA-directed DNA methylation in a pathway that also involves the plant-specific RNA Polymerase V (Pol V). Additionally, the Arabidopsis genome encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase, DRM3. Here, we show that DRM3 has moderate effects on global DNA methylation and small RNA abundance and that DRM3 physically interacts with Pol V. In Arabidopsis drm3 mutants, we observe a lower level of Pol V-dependent noncoding RNA transcripts even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts. This work sheds further light on the mechanism by which long noncoding RNAs facilitate RNA-directed DNA methylation. PMID:25561521

Zhong, Xuehua; Hale, Christopher J; Nguyen, Minh; Ausin, Israel; Groth, Martin; Hetzel, Jonathan; Vashisht, Ajay A; Henderson, Ian R; Wohlschlegel, James A; Jacobsen, Steven E

2015-01-20

167

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 ...

168

Structural Basis of Transcription Initiation: An RNA Polymerase Holoenzyme-DNA Complex  

NASA Astrophysics Data System (ADS)

The crystal structure of Thermus aquaticus RNA polymerase holoenzyme (?2??'??A) complexed with a fork-junction promoter DNA fragment has been determined by fitting high-resolution x-ray structures of individual components into a 6.5-angstrom resolution map. The DNA lies across one face of the holoenzyme, completely outside the RNA polymerase active site channel. All sequence-specific contacts with core promoter elements are mediated by the ? subunit. A universally conserved tryptophan is ideally positioned to stack on the exposed face of the base pair at the upstream edge of the transcription bubble. Universally conserved basic residues of the ? subunit provide critical contacts with the DNA phosphate backbone and play a role in directing the melted DNA template strand into the RNA polymerase active site. The structure explains how holoenzyme recognizes promoters containing variably spaced -10 and -35 elements and provides the basis for models of the closed and open promoter complexes.

Murakami, Katsuhiko S.; Masuda, Shoko; Campbell, Elizabeth A.; Muzzin, Oriana; Darst, Seth A.

2002-05-01

169

The regulation of microRNA expression by DNA methylation in hepatocellular carcinoma.  

PubMed

Emerging evidence indicates that microRNAs (miRNAs) are often dysregulated and play a fundamental role in hepatocellular carcinoma (HCC). However, the mechanism underlying miRNA dysregulation is still elusive. In the present study, we adopted an integrated analysis strategy combining data from genome-wide methylated DNA immunoprecipitation chip and miRNA expression microarray to study the regulation of DNA methylation on miRNA expression in HCC. We first characterized 864 differentially methylated regions (DMRs) located in 236 miRNA regions between cancerous and normal hepatocytes in HCC. We observed that the occurrence of miRNA DNA hypomethylation was more common than its hypermethylation while miRNA DNA hypermethylation was usually found in CpG islands. Then through correlation analysis between miRNA methylation and expression data, we identified 10 dysregulated miRNAs under the potential regulation of DNA methylation in HCC. Five of them (miR-148a, miR-375, miR-195, miR-497 and miR-378) were in hypermethylation and down-regulation status, while another five (miR-106b, miR-25, miR-93, miR-23a and miR-27a) were in hypomethylation and up-regulation status in HCC. Bioinformatics analysis showed that miR-148a may form a negative feedback loop with its targets DNMT1 and DNMT3B and the expression of the miR-195/497 cluster may be affected not only by their hypermethylated promoter region but also by their hypermethylated transcription factors NEUROG2 and DDIT3. Conclusion: our preliminary data and bioinformatics analysis suggest that DNA methylation plays an important and complex role in the regulation of miRNA expression in HCC, which may provide insights into the pathogenesis of HCC and thus may be used for diagnosis and intervention. PMID:25424171

He, Xing-Xing; Kuang, Shu-Zhen; Liao, Jia-Zhi; Xu, Chuan-Rui; Chang, Ying; Wu, Yu-Liang; Gong, Jing; Tian, De-An; Guo, An-Yuan; Lin, Ju-Sheng

2014-11-26

170

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

171

Measuring the differential stoichiometry and energetics of ligand binding to macromolecules by single-molecule force spectroscopy: an extended theory.  

PubMed

Many chemical techniques exist for measuring the stoichiometry of ligand binding to a macromolecule; however, these techniques are often specific to certain ligands or require the presumption of specific binding models. Here, we further develop a previously reported, general, thermodynamic method for extracting the change in number of ligands bound to a macromolecule as that macromolecule undergoes a conformational transition driven by mechanical stretching, for example, by magnetic tweezers or optical trapping. We extend the theory of this method to consider systems with many ligands, experiments conducted in different thermodynamic ensembles (e.g., constant-force, constant-extension), and experiments in which the system is not at equilibrium. Further, we show that analysis of the same single-molecule mechanical manipulation data yields a measure of the differential free energy of stabilization due to ligand binding, that is, the free energy contribution by which ligand binding favors one conformation of the macromolecule over another. We interpret an existing data set measuring ion binding to RNA and DNA in terms of this free energy. PMID:25621932

Jacobson, David R; Saleh, Omar A

2015-02-01

172

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

173

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

174

Simultaneous detection of RNA and DNA targets based on multiplex isothermal amplification.  

PubMed

The detection of pathogenic microorganisms present in food, feed, plant, and other samples is important for providing safe food as well as for preventing the spread of microbes. The genome of pathogens is made of DNA or RNA, therefore a multiplex diagnostics tool would ideally be able to amplify and detect both RNA and DNA targets in parallel. With this goal we have developed an isothermal nucleic acid sequence based amplification [NASBA] implemented microarray analysis (NAIMA) procedure, suitable for the simultaneous multiplex amplification of RNA and DNA targets, coupled with the detection on ArrayTubes. The method is demonstrated to be very sensitive and specific for the detection of two economically important quarantine plant pathogens of potato, the potato spindle tuber viroid (RNA target) and Ralstonia solanacearum (DNA target). Because of its isothermal amplification and simple detection equipment, the method is also applicable for on-site analyses. NAIMA can be used in any domain where there is the need to detect RNA and DNA targets simultaneously. PMID:24625323

Dobnik, David; Morisset, Dany; Lenar?i?, Rok; Ravnikar, Maja

2014-04-01

175

Codelivery of DNA and siRNA via Arginine-Rich PEI-Based Polyplexes.  

PubMed

In this study, we formulated polyplexes with different compositions for codelivery of DNA and small-interfering RNA (siRNA). Since DNA and siRNA have distinctive and complementary morphological characteristics (DNA is long and winding and siRNA is short and rigid), we hypothesized that their codelivery using polyplex would enhance each other's transfection. To test this hypothesis, cationic polymer branched polyethylenimine (bPEI) as a standard transfecting agent and its derivative arginine-rich oligopeptide-grafted bPEI modified with polyethylene glycol (P(SiDAAr)5P3), synthesized in our laboratory, were used as carriers for transfection. Polyplexes at different nucleic acid to polymer weight ratios were characterized for transfection in breast cancer sensitive (MCF-7) and resistant (MCF-7/Adr) cell lines. Gene silencing effect of polyplexes was determined in MDA-MB-231-luc-D3H2LN cell line. The results demonstrated that the polyplexes formed with derivative P(SiDAAr)5P3 show significantly lower toxicity compared to polyplexes formed using bPEI. Further, codelivery resulted in 20-fold higher DNA transfection and 2-fold higher siRNA transfection as compared to the respective single nucleotide delivery. DNA transfection was ?100-fold lower in resistant MCF-7/Adr cells than in sensitive MCF-7 cells. Confocal imaging and flow cytometry data demonstrated that enhanced transfection does not solely depend on DNA's cellular uptake, suggesting that other mechanisms contribute to increased transfection. DNA-co-siRNA delivery could be a promising therapeutic approach to achieve synergistic effects because it can simultaneously target and interfere with multiple regulatory levels in a cell to halt and reverse disease progression. PMID:25591125

Lu, Shan; Morris, Viola B; Labhasetwar, Vinod

2015-02-01

176

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

177

Reduction of DNA contamination in RNA samples for reverse transcription-polymerase chain reaction using selective precipitation by compaction agents.  

PubMed

An important problem in measurement of messenger RNA (mRNA) levels by reverse transcription-polymerase chain reaction (RT-PCR) is DNA contamination, which can produce artifactually increased mRNA concentration. Current methods to eliminate contaminating DNA can compromise the integrity of the RNA, are time-consuming, and/or are hazardous. We present a rapid, nuclease-free, and cost-effective method of eliminating contaminating DNA in RNA samples using selective precipitation by compaction agents. Compaction agents are cationic molecules that bind to double-stranded nucleic acids, driven by electrostatic interactions and steric complementarity. The effectiveness and DNA selectivity of six compaction agents were investigated: trivalent spermidine, Triquat A, and Triquat 7; tetravalent spermine and Quatro-quat; and hexavalent Quatro-diquat. Effectiveness was measured initially by supernatant UV absorbance after precipitation of salmon sperm DNA. Effectiveness and selectivity were then investigated using differences in RT-PCR C(t) values with synthetic mixtures of human genomic DNA and total RNA and with total RNA isolated from cells. With 500 microM spermidine or Triquat A, the supernatant DNA could not be detected up to 40 cycles of PCR (C(t)12.6), whereas the C(t) for the mRNA was increased by only five cycles. Therefore, spermidine and Triquat A each show strong DNA selectivity and could be used to eliminate contaminating DNA in measurements of mRNA. PMID:18831957

Añez-Lingerfelt, Mariaclara; Fox, George E; Willson, Richard C

2009-01-01

178

Estimation of gene expression in heterocysts of Anabaena variabilis by using DNA-RNA hybridization.  

PubMed Central

In the filamentous cyanobacterium Anabaena variabilis, specialized cells called heterocysts occur in a regular pattern along the filament and are the sites of nitrogen fixation. We used two different types of DNA-excess RNA hybridization techniques to estimate the number of genes expressed in recently differentiated, mature heterocysts. In the first, RNA and DNA were incubated in a phosphate buffer at 60 degrees C, and the hybrids were separated from the unhybridized material by hydroxylapatite chromatography. In the second, the nucleic acids were incubated at 50 degrees C in a buffer containing 50% formamide, and the fraction of DNA in duplexes was assayed by S1 nuclease digestion. Both techniques revealed that approximately 65% of the A. variabilis genome was expressed in vegetative cells and 45% of the genome was expressed in heterocysts. Two experiments were conducted to estimate the number of heterocyst-specific mRNA transcripts. In one, hybridization of heterocyst RNA to a null DNA probe (DNA not transcribed in vegetative cells) revealed that heterocyst-specific transcripts were encoded by 25% of the DNA sense strand, representing approximately 1,000 genes (assuming each to be 1,500 nucleotides in length). The second approach, in which total cell DNA was hybridized to a mixture of heterocyst and vegetative cell RNA, indicated that 14.7% of the DNA sense strand, or about 600 genes, was transcribed exclusively in the heterocyst. The remaining 900 to 1,300 transcripts present in the heterocyst appeared to be constitutively produced in both vegetative cells and heterocysts. The heterocyst-specific transcripts were present in abundant copies in the cell, while transcripts that occurred in both cell types were present at much lower frequency. PMID:2427500

Lynn, M E; Bantle, J A; Ownby, J D

1986-01-01

179

RNA-Linked Nascent DNA Fragments in Escherichia coli  

Microsoft Academic Search

Nucleic acid that is extracted from E. coli labeled by a brief pulse of [3H]dT and denatured by treatment with heat, formamide, or formaldehyde bands in a region with a density higher than that of single-stranded E. coli DNA in a Cs2SO4 equilibrium density gradient. If treated with alkali or RNase, it then exhibits the density of single-stranded DNA. These

Akio Sugino; Susumu Hirose; Reiji Okazaki

1972-01-01

180

Mitochondrial Ribosomal RNA (rRNA) Methyltransferase Family Members Are Positioned to Modify Nascent rRNA in Foci near the Mitochondrial DNA Nucleoid*  

PubMed Central

We have identified RNMTL1, MRM1, and MRM2 (FtsJ2) as members of the RNA methyltransferase family that may be responsible for the three known 2?-O-ribose modifications of the 16 S rRNA core of the large mitochondrial ribosome subunit. These proteins are confined to foci located in the vicinity of mtDNA nucleoids. They show distinct patterns of association with mtDNA nucleoids and/or mitochondrial ribosomes in cell fractionation studies. We focused on the role of the least studied protein in this set, RNMTL1, to show that this protein interacts with the large ribosomal subunit as well as with a series of non-ribosomal proteins that may be involved in coupling of the rate of rRNA transcription and ribosome assembly in mitochondria. siRNA-directed silencing of RNMTL1 resulted in a significant inhibition of translation on mitochondrial ribosomes. Our results are consistent with a role for RNMTL1 in methylation of G1370 of human 16 S rRNA. PMID:24036117

Lee, Ken-Wing; Okot-Kotber, Cynthia; LaComb, Joseph F.; Bogenhagen, Daniel F.

2013-01-01

181

Effect of seven days of spaceflight on hindlimb muscle protein, RNA and DNA in adult rats  

NASA Technical Reports Server (NTRS)

Effects of seven days of spaceflight on skeletal muscle (soleus, gastrocnemius, EDL) content of protein, RNA and DNA were determined in adult rats. Whereas total protein contents were reduced in parallel with muscle weights, myofibrillar protein appeared to be more affected. There were no significant changes in absolute DNA contents, but a significant (P less than 0.05) increase in DNA concentration (microgram/milligram) in soleus muscles from flight rats. Absolute RNA contents were significantly (P less than 0.025) decreased in the soleus and gastrocnemius muscles of flight rats, with RNA concentrations reduced 15-30 percent. These results agree with previous ground-based observations on the suspended rat with unloaded hindlimbs and support continued use of this model.

Steffen, J. M.; Musacchia, X. J.

1985-01-01

182

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

PubMed Central

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

2014-01-01

183

A conserved transcriptional regulator is required for RNA-directed DNA methylation and plant development  

PubMed Central

RNA-directed DNA methylation (RdDM) is a conserved mechanism for epigenetic silencing of transposons and other repetitive elements. We report that the rdm4 (RNA-directed DNA Methylation4) mutation not only impairs RdDM, but also causes pleiotropic developmental defects in Arabidopsis. Both RNA polymerase II (Pol II)- and Pol V-dependent transcripts are affected in the rdm4 mutant. RDM4 encodes a novel protein that is conserved from yeast to humans and interacts with Pol II and Pol V in plants. Our results suggest that RDM4 functions in epigenetic regulation and plant development by serving as a transcriptional regulator for RNA Pol V and Pol II, respectively. PMID:19903758

He, Xin-Jian; Hsu, Yi-Feng; Zhu, Shihua; Liu, Hai-Liang; Pontes, Olga; Zhu, Jianhua; Cui, Xinping; Wang, Co-Shine; Zhu, Jian-Kang

2009-01-01

184

A FRET-based DNA nano-tweezer technique for the imaging analysis of specific mRNA.  

PubMed

A DNA nano-tweezer (DNA-NT) structure-based target mRNA detection probe, which uses fluorescence resonance energy transfer (FRET) as a detection signal and works as a single molecule, has been developed. This FRET-paired fluorescent dye-modified DNA-NT, self-assembled from three single-stranded DNAs, alters its structure from open to closed states and produces a FRET signal in response to in vitro transcripts of Hes-1 mRNA. Our results showed that the FRET-based DNA-NT detected both GLUT1 mRNA as a pre-fixed target mRNA model and Hes-1 mRNA as a model expressed inside a living cell. These results confirm the feasibility of using the FRET-based DNA-NT for imaging analysis of target mRNA. PMID:25529369

Funabashi, Hisakage; Shigeto, Hajime; Nakatsuka, Keisuke; Kuroda, Akio

2015-02-01

185

When you can’t trust the DNA: RNA editing changes transcript sequences  

Microsoft Academic Search

RNA editing describes targeted sequence alterations in RNAs so that the transcript sequences differ from their DNA template.\\u000a Since the original discovery of RNA editing in trypanosomes nearly 25 years ago more than a dozen such processes of nucleotide\\u000a insertions, deletions, and exchanges have been identified in evolutionarily widely separated groups of the living world including\\u000a plants, animals, fungi, protists, bacteria,

Volker Knoop

2011-01-01

186

The Fitness Effects of Random Mutations in Single-Stranded DNA and RNA Bacteriophages  

Microsoft Academic Search

Mutational fitness effects can be measured with relatively high accuracy in viruses due to their small genome size, which facilitates full-length sequencing and genetic manipulation. Previous work has shown that animal and plant RNA viruses are very sensitive to mutation. Here, we characterize mutational fitness effects in single-stranded (ss) DNA and ssRNA bacterial viruses. First, we performed a mutation-accumulation experiment

Pilar Domingo-Calap; José M. Cuevas; Rafael Sanjuán

2009-01-01

187

How do RNA sequence, DNA sequence, and chromatin properties regulate splicing?  

PubMed Central

Recent genome-wide studies have revealed a remarkable correspondence between nucleosome positions and exon-intron boundaries, and several studies have implicated specific histone modifications in regulating alternative splicing. In addition, recent progress in cracking the ‘splicing code’ shows that sequence motifs carried on the nascent RNA molecule itself are sufficient to accurately predict tissue-specific alternative splicing patterns. Together, these studies shed light on the complex interplay between RNA sequence, DNA sequence, and chromatin properties in regulating splicing. PMID:21173847

2010-01-01

188

Fabrication of Stable and RNase-Resistant RNA Nanoparticles Active in Gearing the Nanomotors for Viral DNA-Packaging  

PubMed Central

Both DNA and RNA can serve as powerful building blocks for bottom-up fabrication of nanostructures. A pioneering concept proposed by Ned Seeman 30 years ago has led to an explosion of knowledge in DNA nanotechnology. RNA can be manipulated with simplicity characteristic of DNA, while possessing noncanonical base-pairing, versatile function and catalytic activity similar to proteins. However, standing in awe of the sensitivity of RNA to RNase degradation has made many scientists flinch away from RNA nanotechnology. Here we report the construction of stable RNA nanoparticles resistant to RNase digestion. The chemically modified RNA retained its property for correct folding in dimer formation, appropriate structure in procapsid binding, and biological activity in gearing phi29 nanomotor to package viral DNA and producing infectious viral particles. Our results demonstrate that it is practical to produce RNase resistant, biologically active and stable RNA for application in nanotechnology. PMID:21155596

Liu, Jing; Guo, Songchuan; Cinier, Mathieu; Shu, Yi; Chen, Chaoping; Shen, Guanxin; Guo, Peixuan

2010-01-01

189

Effects of long DNA folding and small RNA stem–loop in thermophoresis  

PubMed Central

In thermophoresis, with the fluid at rest, suspensions move along a gradient of temperature. In an aqueous solution, a PEG polymer suspension is depleted from the hot region and builds a concentration gradient. In this gradient, DNA polymers of different sizes can be separated. In this work the effect of the polymer structure for genomic DNA and small RNA is studied. For genome-size DNA, individual single T4 DNA is visualized and tracked in a PEG solution under a temperature gradient built by infrared laser focusing. We find that T4 DNA follows steps of depletion, ring-like localization, and accumulation patterns as the PEG volume fraction is increased. Furthermore, a coil–globule transition for DNA is observed for a large enough PEG volume fraction. This drastically affects the localization position of T4 DNA. In a similar experiment, with small RNA such as ribozymes we find that the stem–loop folding of such polymers has important consequences. The RNA polymers having a long and rigid stem accumulate, whereas a polymer with stem length less than 4 base pairs shows depletion. Such measurements emphasize the crucial contribution of the double-stranded parts of RNA for thermal separation and selection under a temperature gradient. Because huge temperature gradients are present around hydrothermal vents in the deep ocean seafloor, this process might be relevant, at the origin of life, in an RNA world hypothesis. Ribozymes could be selected from a pool of random sequences depending on the length of their stems. PMID:23071341

Maeda, Yusuke T.; Tlusty, Tsvi; Libchaber, Albert

2012-01-01

190

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

PubMed

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

2013-08-27

191

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

192

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

193

Improved protocol for the simultaneous extraction and column-based separation of DNA and RNA from different soils.  

PubMed

We developed an improved protocol, allowing the simultaneous extraction of DNA and RNA from soil using phenol-chloroform with subsequent column-based separation of DNA and RNA (PCS). We compared this new approach with the well established protocol published by Griffiths et al. (2000), where DNA and RNA are separated by selective enzymatic digestions and two commercial kits used for DNA or RNA extraction, respectively, using four different agricultural soils. We compared yield and purity of the nucleic acids as well as abundance and diversity profiles of the soil bacterial communities targeting the nosZ gene via quantitative real-time PCR and terminal restriction fragment length polymorphism on DNA and RNA level. The newly developed protocol provided purer nucleic acid extracts compared to the used kit-based protocols. All protocols were suitable for DNA- and RNA-based gene quantification, however high variations between replicates were obtained for RNA samples using the original Griffiths protocol. Diversity patterns of nosZ were highly influenced by the extraction protocol used both on the DNA and RNA level. Finally, our data showed that the new protocol allows a simultaneous and reproducible extraction and separation of DNA and RNA, which were suitable for reliable analyses of gene and transcript copy numbers and diversity pattern. PMID:21256887

Töwe, Stefanie; Wallisch, Stefanie; Bannert, Andrea; Fischer, Doreen; Hai, Brigitte; Haesler, Felix; Kleineidam, Kristina; Schloter, Michael

2011-03-01

194

Stabilizing RNA by the sonochemical formation of RNA nanospheres.  

PubMed

Biological macromolecules, including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom-up fabrication of nanodevices. Unlike DNA, RNA is a more versatile molecule whose range in the cell is from 21 to thousands of nucleotides and is usually folded into stem and loop structures. RNA is unique in nanoscale fabrication due to its diversity in size, function, and structure. Because gene expression analysis is becoming a clinical reality and there is a need to collect RNA in minute amounts from clinical samples, keeping the RNA intact is a growing challenge. RNA samples are notoriously difficult to handle because of their highly labile nature and tendency to degrade even under controlled RNase-free conditions and maintenance in the cold. Silencing the RNA that induces the RNA interference is viewed as the next generation of therapeutics. The stabilization and delivery of RNA to cells are the major concerns in making siRNAs usable drugs. For the first time, ultrasonic waves are shown to convert native RNA molecules to RNA nanospheres. The creation of the nanobubbles is performed by a one-step reaction. The RNA nanospheres are stable at room temperature for at least one month. Additionally, the nanospheres can be inserted into mammalian cancer cells (U2OS). This research achieves: 1) a solution to RNA storage; and 2) a way to convert RNA molecules to RNA particles. RNA nanosphere formation is a reversible process, and by using denaturing conditions, the RNA can be refolded into intact molecules. PMID:21456085

Shimanovich, Ulyana; Volkov, Vadim; Eliaz, Dror; Aizer, Adva; Michaeli, Shulamit; Gedanken, Aharon

2011-04-18

195

Oxidative DNA lesions as blocks to in vitro transcription by phage 17 RNA polymerase  

SciTech Connect

In recent years, a link between the transcriptional state of damaged DNA and the rate at which it is repaired has been demonstrated in both prokaryotes and eukaryotes. DNA containing bulky adducts, cross-links, and UV damage processed by nucleotide excision repair is repaired at a higher rate when it is actively transcribed. For these damages, evidence exists that an RNA polymerase molecule, stalled opposite a lesion, works as a signal to initiate repair, thus linking the two processes. However, no conclusive demonstration exists between base excision repair processing and transcription. Accordingly, we have examined the ability of several oxidative DNA lesions to block in vitro transcription by phage T7 RNA polymerase. Previous and ongoing work in this laboratory suggests that the effect that these lesions have on DNA polymerases is greatly influenced by the sequence context in which they are found. Future work will examine if sequence context regulates the role of these lesions as blocks to transcription.

Hatahet, Z.; Purmal, A.A.; Wallace, S.S. [Univ. of Vermont, Burlington, VT (United States)

1994-12-31

196

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

197

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

198

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

199

The Escherichia coli tRNA-guanine transglycosylase can recognize and modify DNA.  

PubMed

tRNA-guanine transglycosylase (TGT) catalyzes the exchange of queuine (or a precursor) for guanine 34 in tRNA. The minimal RNA recognition motif for TGT has been found to involve a UGU sequence in the anticodon loop of the queuine-cognate tRNAs. Recent studies have shown that the enzyme is capable of recognizing the UGU sequence in alternative contexts (Kung, F. L., Nonekowski, S., and Garcia, G. A. (2000) RNA 6, 233-244) and have investigated the role of the first U of the UGU sequence in tRNA recognition by TGT (Nonekowski, S. T., and Garcia, G. A. (2001) RNA 7, 1432-1441). The TGT reaction involves the breakage and re-formation of a glycosidic bond. To rule out a potential chemical mechanism involving the 2'-hydroxyl at position 34, we synthesized and evaluated an RNA minihelix with 2'-deoxy-G at 34. The high level of activity exhibited by this analogue indicates that the 2'-hydroxyl of G(34) is not required for catalysis. Furthermore, we find that TGT can recognize analogues composed entirely of DNA, but only when 2'-deoxyuridines replace the thymidines in the DNA. The requirement for uridine bases for recognition is perhaps not surprising given the UGU recognition motif for TGT. However, it is not clear if the uracil requirement is due to specific recognition by TGT or due to the effect of uracils on the conformation of the oligonucleotide. PMID:11751936

Nonekowski, Susanne T; Kung, Fan-Lu; Garcia, George A

2002-03-01

200

A sequential co-extraction method for DNA, RNA and protein recovery from soil for future system-based approaches.  

PubMed

A co-extraction protocol that sequentially isolates core biopolymer fractions (DNA, RNA, protein) from edaphic microbial communities is presented. In order to confirm compatibility with downstream analyses, bacterial T-RFLP profiles were generated from the DNA- and RNA-derived fractions of an arid-based soil, with metaproteomics undertaken on the corresponding protein fraction. PMID:24929037

Gunnigle, Eoin; Ramond, Jean-Baptiste; Frossard, Aline; Seeley, Mary; Cowan, Don

2014-08-01

201

Ratios between Contents of DNA, RNA and Protein in Different Microorganisms as a Function of Maximal Growth Rate  

Microsoft Academic Search

THE growth medium determines the macromolecular composition as well as the growth rate (number of doublings per hour) of micro-organisms in the steady state of growth. For a given micro-organism the ratios of RNA : protein and RNA : DNA are linear functions of the growth rate, whereas the ratio of DNA : protein is virtually independent of the rate

Vagn Leick

1968-01-01

202

In vitro selection from combinatorial nucleic acid libraries has provided new RNA and DNA molecules that have catalytic  

E-print Network

257 In vitro selection from combinatorial nucleic acid libraries has provided new RNA and DNA of nucleic acid molecules. The future application of in vitro selected RNA and DNA catalysts in bioorganic-state analog Introduction Combinatorial nucleic-acid libraries have found increasing use for the isolation

Weiblen, George D

203

Efficient extraction of viral DNA and viral RNA by the Chemagic viral DNA/RNA kit allows sensitive detection of cytomegalovirus, hepatitis B virus, and hepatitis G virus by PCR.  

PubMed

The Chemagic Viral DNA/RNA kit was evaluated for extraction of cytomegalovirus (CMV), hepatitis B virus (HBV), and hepatitis G virus (HGV) by using the QIAamp DNA Blood Mini kit and the QIAamp Viral RNA Mini kit as reference protocols. The extraction efficiencies of the different kits for CMV DNA and HBV DNA were not distinguishable, but the extraction efficiency for HGV RNA was better with the Chemagen protocol. All clinical specimens tested HBV DNA- or HGV RNA-positive after QIAGEN protocols for extraction were confirmed by using the Chemagen protocol. The Chemagen kit failed to confirm one of 75 CMV DNA-positive specimens. Thus, a new competitive extraction method using a technology with a high potential for automation is available. PMID:14605182

Kleines, Michael; Schellenberg, Kirsten; Ritter, Klaus

2003-11-01

204

A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA  

PubMed Central

Background The transcribed sequences of a cell, the transcriptome, represent the trans-acting fraction of the genetic information, yet eukaryotic cDNA libraries are typically made from only the poly-adenylated fraction. The non-coding or translated but non-polyadenylated RNAs are therefore not represented. The goal of this study was to develop a method that would more completely represent the transcriptome in a useful format, avoiding over-representation of some of the abundant, but low-complexity non-translated transcripts. Results We developed a combination of self-subtraction and directional cloning procedures for this purpose. Libraries were prepared from partially degraded (hydrolyzed) total RNA from three different species. A restriction endonuclease site was added to the 3' end during first-strand synthesis using a directional random-priming technique. The abundant non-polyadenylated rRNA and tRNA sequences were largely removed by using self-subtraction to equalize the representation of the various RNA species. Sequencing random clones from the libraries showed that 87% of clones were in the forward orientation with respect to known or predicted transcripts. 70% matched identified or predicted translated RNAs in the sequence databases. Abundant mRNAs were less frequent in the self-subtracted libraries compared to a non-subtracted mRNA library. 3% of the sequences were from known or hypothesized ncRNA loci, including five matches to miRNA loci. Conclusion We describe a simple method for making high-quality, directional, random-primed, cDNA libraries from small amounts of degraded total RNA. This technique is advantageous in situations where a cDNA library with complete but equalized representation of transcribed sequences, whether polyadenylated or not, is desired. PMID:17925018

Davis, Claytus; Barvish, Zeev; Gitelman, Inna

2007-01-01

205

Simple and Safe Method for Simultaneous Isolation of Microbial RNA and DNA from Problematic Populations?  

PubMed Central

We describe a novel, rapid, and safe method for extracting RNA and DNA from refractory microbes, which avoids the use of phenol or chloroform. It has been used successfully to isolate high-quality nucleic acids from pure cultures and environmental populations known to resist widely used extraction protocols. PMID:18791011

McIlroy, Simon; Porter, Kate; Seviour, Robert J.; Tillett, Daniel

2008-01-01

206

Aminoglycoside-Nucleic Acid Interactions: Remarkable Stabilization of DNA and RNA Triple Helices by Neomycin  

E-print Network

Aminoglycoside-Nucleic Acid Interactions: Remarkable Stabilization of DNA and RNA Triple Helices by Neomycin Dev P. Arya,* R. Lane Coffee, Jr., Bert Willis, and Anna I. Abramovitch Contribution from. H. Prog. Nucleic Acid Res. Mol. Biol.. 1998, 59, 55-94. (7) Thuong, N. T.; Helene, C. Angew. Chem

Stuart, Steven J.

207

Correction of the Mutation Responsible for Sickle Cell Anemia by an RNA-DNA Oligonucleotide  

Microsoft Academic Search

A chimeric oligonucleotide composed of DNA and modified RNA residues was used to direct correction of the mutation in the hemoglobin beta^S allele. After introduction of the chimeric molecule into lymphoblastoid cells homozygous for the beta^S mutation, there was a detectable level of gene conversion of the mutant allele to the normal sequence. The efficient and specific conversion directed by

Allyson Cole-Strauss; Kyonggeun Yoon; Yufei Xiang; Bruce C. Byrne; Michael C. Rice; Jeff Gryn; William K. Holloman; Eric B. Kmiec

1996-01-01

208

Comparison of commercial systems for extraction of nucleic acids from DNA/RNA respiratory pathogens.  

PubMed

This study compared six automated nucleic acid extraction systems and one manual kit for their ability to recover nucleic acids from human nasal wash specimens spiked with five respiratory pathogens, representing Gram-positive bacteria (Streptococcus pyogenes), Gram-negative bacteria (Legionella pneumophila), DNA viruses (adenovirus), segmented RNA viruses (human influenza virus A), and non-segmented RNA viruses (respiratory syncytial virus). The robots and kit evaluated represent major commercially available methods that are capable of simultaneous extraction of DNA and RNA from respiratory specimens, and included platforms based on magnetic-bead technology (KingFisher mL, Biorobot EZ1, easyMAG, KingFisher Flex, and MagNA Pure Compact) or glass fiber filter technology (Biorobot MDX and the manual kit Allprep). All methods yielded extracts free of cross-contamination and RT-PCR inhibition. All automated systems recovered L. pneumophila and adenovirus DNA equivalently. However, the MagNA Pure protocol demonstrated more than 4-fold higher DNA recovery from the S. pyogenes than other methods. The KingFisher mL and easyMAG protocols provided 1- to 3-log wider linearity and extracted 3- to 4-fold more RNA from the human influenza virus and respiratory syncytial virus. These findings suggest that systems differed in nucleic acid recovery, reproducibility, and linearity in a pathogen specific manner. PMID:21034773

Yang, Genyan; Erdman, Dean E; Kodani, Maja; Kools, John; Bowen, Michael D; Fields, Barry S

2011-01-01

209

Detection of RNA Polymerase II Promoters and Polyadenylation Sites in Human DNA Sequence  

Microsoft Academic Search

Detection of RNA polymerase II promoters and polyadenylation sites helps to locate gene boundaries and can enhance accurate gene recognition and modeling in genomic DNA sequence. We describe a system which can be used to detect polyadenylation sites and thus delineate the 3? boundary of a gene, and discuss improvements to a system first described in Matis et al. (1995)

Sherri Matis; Ying Xu; Manesh J. Shah; Xiaojun Guan; J. Ralph Einstein; Richard J. Mural; Edward C. Uberbacher

1996-01-01

210

Introduction Parasites Traveling Waves Ecosystem DNA Evolution of RNA-like Replicators  

E-print Network

Introduction Parasites Traveling Waves Ecosystem DNA Evolution of RNA-like Replicators --Roles of Parasites-- Nobuto Takeuchi National Center for Biotechnology Information National Library of MedicineD defense of Folkert K. de Boer Utrecht University May 15, 2012 #12;Introduction Parasites Traveling Waves

Utrecht, Universiteit

211

RNA, DNA, and Cell Surface Characteristics of Lesional and Nonlesional Psoriatic Skin  

Microsoft Academic Search

We have measured the RNA and DNA content and examined cell surface characteristics of human epidermal cells derived from normal skin, and lesional and nonlesional areas of psoriatic skin prior to and following treatment on a modified Goeckerman protocol. Our results show that cells from active psoriatic lesions contain greater numbers of basal keratinocytes when compared with either nonlesional skin

Lisa Staiano-Coico; Alice B. Gottlieb; Lance Barazani; D. Martin Carter

1987-01-01

212

Indirect read-out of the promoter DNA by RNA polymerase in the closed complex  

PubMed Central

Transcription is initiated when RNA polymerase recognizes the duplex promoter DNA in the closed complex. Due to its transient nature, the closed complex has not been well characterized. How the initial promoter recognition occurs may offer important clues to regulation of transcription initiation. In this article, we have carried out single-base pair substitution experiments on two Escherichia coli promoters belonging to two different classes, the ?35 and the extended ?10, under conditions which stabilize the closed complex. Single-base pair substitution experiments indicate modest base-specific effects on the stability of the closed complex of both promoters. Mutations of base pairs in the ?10 region affect the closed complexes of two promoters differently, suggesting different modes of interaction of the RNA polymerase and the promoter in the two closed complexes. Two residues on ?70 which have been suggested to play important role in promoter recognition, Q437 and R436, were mutated and found to have different effects on the closed-complex stability. DNA circular dichroism (CD) and FRET suggest that the promoter DNA in the closed complex is distorted. Modeling suggests two different orientations of the recognition helix of the RNA polymerase in the closed complex. We propose that the RNA polymerase recognizes the sequence dependent conformation of the promoter DNA in the closed complex. PMID:23118489

Debnath, Subrata; Roy, Neeladri Sekhar; Bera, Indrani; Ghoshal, Nanda; Roy, Siddhartha

2013-01-01

213

Short Hairpin RNA Suppression of Thymidylate Synthase Produces DNA Mismatches and Results in Excellent Radiosensitization  

SciTech Connect

Purpose: To determine the effect of short hairpin ribonucleic acid (shRNA)-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur. Methods and Materials: shRNA suppression of TS was compared with 5-fluoro-2 Prime -deoxyuridine (FdUrd) inactivation of TS with or without ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured by clonogenic assay. Cell cycle effects were measured by flow cytometry. The effects of FdUrd or shRNA suppression of TS on dNTP deoxynucleotide triphosphate imbalances and consequent nucleotide misincorporations into deoxyribonucleic acid (DNA) were analyzed by high-pressure liquid chromatography and as pSP189 plasmid mutations, respectively. Results: TS shRNA produced profound ({>=}90%) and prolonged ({>=}8 days) suppression of TS in HCT116 and HT29 cells, whereas FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs deoxynucleotide triphosphates compared with FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, although its effects were not as long-lasting as those of FdUrd. Both treatments resulted in phosphorylation of Chk1. TS shRNA alone was less cytotoxic than FdUrd but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio: TS shRNA, 1.5-1.7; FdUrd, 1.4-1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations. Conclusions: TS shRNA produced less cytotoxicity than FdUrd but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but it only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches after TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP thymidine triphosphate and consequent DNA mismatches underlying radiosensitization. Importantly, shRNA suppression of TS avoids FP-mediated TS elevation and its negative prognostic role. These studies support the further exploration of TS suppression as a novel radiosensitizing strategy.

Flanagan, Sheryl A., E-mail: sflan@umich.edu [Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Cooper, Kristin S. [Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)] [Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Mannava, Sudha; Nikiforov, Mikhail A. [Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York (United States)] [Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York (United States); Shewach, Donna S. [Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)] [Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)

2012-12-01

214

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

215

Simplified methods for the construction of RNA and DNA virus infectious clones.  

PubMed

Infectious virus clones are one of the most powerful tools in plant pathology, molecular biology, and biotechnology. The construction of infectious clones of RNA and DNA viruses, however, usually requires laborious cloning and subcloning steps. In addition, instability of the RNA virus genome is frequently reported after its introduction into the vector and transference to Escherichia coli. These difficulties hamper the cloning procedures, making it tedious and cumbersome. This chapter describes two protocols for a simple construction of infectious viruses, an RNA virus, the tobamovirus Pepper mild mottle virus, and a DNA virus, a bipartite begomovirus. For this purpose, the strategy of overlap-extension PCR was used for the construction of infectious tobamovirus clone and of rolling circle amplification (RCA) for the construction of a dimeric form of the begomovirus clone. PMID:25287508

Nagata, Tatsuya; Inoue-Nagata, Alice Kazuko

2015-01-01

216

Inhibition of Hepatitis B virus cccDNA replication by siRNA  

SciTech Connect

The development of an effective therapy for Hepatitis B virus (HBV) infection is still a challenge. Progress in RNA interference (RNAi) has shed slight on developing a new anti-HBV strategy. Here, we present a series of experiments showing a significant reduction in HBV transcripts and replication intermediates in HepG2.2.15 cells by vector-based siRNA targeted nuclear localization signal (NLS) region. More importantly, we showed that siRNA1 markedly inhibited HBV covalently closed circular DNA (cccDNA) replication. Our results indicated that HBV NLS may serve as a novel RNAi target to combat HBV infection, which can enhance anti-HBV efficacy and overcome the drawbacks of current therapies.

Li Guiqiu [Department of Microbiology, Harbin Medical University, Harbin, Heilongjiang Province (China); Gu Hongxi [Department of Microbiology, Harbin Medical University, Harbin, Heilongjiang Province (China)]. E-mail: hxgu2432@163.com; Li Di [Department of Microbiology, Harbin Medical University, Harbin, Heilongjiang Province (China); Xu Weizhen [Department of Microbiology, Harbin Medical University, Harbin, Heilongjiang Province (China)

2007-04-06

217

DNA versus RNA-based denaturing gradient gel electrophoresis profiles of a bacterial community during replenishment after soil fumigation  

Microsoft Academic Search

We compared the responsiveness and sensitivity to soil fumigation of DNA- and RNA-based analyses of a bacterial community. We first established an improved RNA extraction method using DNA as an adsorption competitor, because it is extremely difficult to extract nucleic acids from clay-rich volcanic ash soil (Andisol), which adsorbs nucleic acids. This novel method facilitated RNA extraction from 500mg of

Yuko Takada Hoshino; Naoyuki Matsumoto

2007-01-01

218

Removal of RNA impurities by tangential flow filtration in an RNase-free plasmid DNA purification process  

Microsoft Academic Search

Addition of animal-derived ribonuclease A to degrade RNA impurities is not recommended in the manufacture of pharmaceutical-grade plasmid DNA. Tangential flow filtration (TFF) takes advantage of the significant size difference between RNA and plasmid DNA to remove RNA in the permeate while plasmid remains in the retentate, in an RNase-free plasmid purification process. Operating conditions including transmembrane pressure, membrane pore

Alex Eon-Duval; Robert H MacDuff; Carol A Fisher; Mark J Harris; Chris Brook

2003-01-01

219

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

220

PfAlbas constitute a new eukaryotic DNA/RNA-binding protein family in malaria parasites.  

PubMed

In Plasmodium falciparum, perinuclear subtelomeric chromatin conveys monoallelic expression of virulence genes. However, proteins that directly bind to chromosome ends are poorly described. Here we identify a novel DNA/RNA-binding protein family that bears homology to the archaeal protein Alba (Acetylation lowers binding affinity). We isolated three of the four PfAlba paralogs as part of a molecular complex that is associated with the P. falciparum-specific TARE6 (Telomere-Associated Repetitive Elements 6) subtelomeric region and showed in electromobility shift assays (EMSAs) that the PfAlbas bind to TARE6 repeats. In early blood stages, the PfAlba proteins were enriched at the nuclear periphery and partially co-localized with PfSir2, a TARE6-associated histone deacetylase linked to the process of antigenic variation. The nuclear location changed at the onset of parasite proliferation (trophozoite-schizont), where the PfAlba proteins were also detectable in the cytoplasm in a punctate pattern. Using single-stranded RNA (ssRNA) probes in EMSAs, we found that PfAlbas bind to ssRNA, albeit with different binding preferences. We demonstrate for the first time in eukaryotes that Alba-like proteins bind to both DNA and RNA and that their intracellular location is developmentally regulated. Discovery of the PfAlbas may provide a link between the previously described subtelomeric non-coding RNA and the regulation of antigenic variation. PMID:22167473

Chêne, Arnaud; Vembar, Shruthi S; Rivière, Loïc; Lopez-Rubio, José Juan; Claes, Aurelie; Siegel, T Nicolai; Sakamoto, Hiroshi; Scheidig-Benatar, Christine; Hernandez-Rivas, Rosaura; Scherf, Artur

2012-04-01

221

Peptide and peptide nucleic acid syntheses using a DNA/RNA synthesizer.  

PubMed

The use of an ABI 394 DNA/RNA synthesizer for peptide and peptide nucleic acid (PNA) syntheses is described. No additional physical part or software is needed for the application. A commercially available large DNA synthesis column was used, and only about half of its volume was filled with resin when the resin was fully swollen. With additional space in the top portion of the column, agitation of reaction mixture was achieved by bubbling argon from the bottom without losing solution. Removing solutions from column was achieved by flushing argon from top to bottom. Two peptide and two PNA sequences were synthesized. Good yields were obtained in all the cases. The method is easy to follow by researchers who are familiar with DNA/RNA synthesizer. © 2014 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 487-493, 2014. PMID:25298082

Pokharel, Durga; Fueangfung, Suntara; Zhang, Mingcui; Fang, Shiyue

2014-11-01

222

Femtosecond fluorescence studies of DNA/RNA constituents  

NASA Astrophysics Data System (ADS)

In this overview, femtosecond fluorescence studies of various DNA constituents are presented, ranging from the monomeric chromophores to different model helices. In order to interpret the experimental results in terms of fundamental processes on the molecular scale they are discussed in the light of recent theoretical calculations. The ultrafast fluorescence decay observed for the monomers is explained by the involvement of highly efficient conical intersections (CI) between the first singlet excited state and the ground state. For the model helices, the picture is more complex, but fluorescence anisotropy data reveal collective effects.

Gustavsson, T.; Banyasz, A.; Improta, R.; Markovitsi, D.

2011-01-01

223

A 3.(ET743)-DNA complex that both resembles an RNA-DNA hybrid and mimicks zinc finger-induced DNA structural distortions.  

PubMed

The antitumor ecteinascidin ET743 has been shown to inhibit the transcriptional activation of a number of genes at nanomolar concentrations. Cell sensitivity to subnanomolar concentrations of the drug has also been shown to specifically depend on the transcription-coupled nucleotide excision repair system. ET743 is known to bind covalently to the minor groove of a DNA double helix in regions comprising selected sets of three consecutive base pairs. Following alkylation of a central guanine, the minor groove is widened and the DNA is bent toward the major groove. We have previously shown that in the resulting adduct the DNA triplet containing the covalently modified guanine bears a strong resemblance to a DNA triplet recognized by a C(2)H(2) zinc finger. We now expand this earlier finding and use simulation methods to show that head-to-tail binding of three ET743 molecules to three adjacent optimal binding sites stabilizes a DNA structure whose conformation is intermediate between A- and B-form DNA. Furthermore, despite the increase in roll at the sites of covalent attachment, no net curvature is apparent in this complex due to cancellation of the localized bends over virtually one turn of the helix. Both observations are in good analogy to findings in zinc finger-DNA complexes. Triplets are virtually superimposable both directly and upon shifting the register one base pair. In this latter case, the central guanine in a triplet alkylated by ET743 corresponds to the third nucleic base in the triplet recognized by a zinc finger of transcription factors such as EGR1 or Sp-1. The DNA conformation found in the ET743-DNA complex is also strongly reminiscent of an RNA-DNA hybrid, as found in the RNA polymerase II elongation complex. The possible biological implications of these findings in relation to the antitumor action of ET743 are discussed. PMID:11831898

Marco, Esther; García-Nieto, Raquel; Mendieta, Jesús; Manzanares, Ignacio; Cuevas, Carmen; Gago, Federico

2002-02-14

224

Reduced mRNA Expression of the DNA Demethylase, MBD2, in Human Colorectal and Stomach Cancers  

Microsoft Academic Search

A study was performed to evaluate the significance of aberrations of the newly identified DNA demethylase, MBD2, in human carcinogenesis. Levels of expression of DNA demethylase mRNA were examined by reverse transcription followed by real-time quantitative detection of the PCR products in 32 samples of colorectal cancer tissue, 24 stomach cancers, and the corresponding noncancerous mucosae. DNA demethylase mRNA levels

Yae Kanai; Saori Ushijima; Yukihiro Nakanishi; Setsuo Hirohashi

1999-01-01

225

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.

Molecular Literacy Project

226

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 ...

227

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 ...

228

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

229

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; Köber, Mariana; Yu, Zhongbo; Donkers, Serge P.; Chou, Fang-Chieh; Das, Rhiju; Dekker, Nynke H.

2014-01-01

230

Transcription of T7 DNA containing modified nucleotides by bacteriophage T7 specific RNA polymerase.  

PubMed

The interaction of bacteriophage T7 specific RNA polymerase with its cognate promoter sites has been probed by selectively replacing bases in one T7 promoter site with base analogs. Base analogs such as 2,6-diaminopurine or hypoxanthine, which alter residues appearing in the minor groove of the DNA helix, prevent utilization of the promoter by T7 RNA polymerase. These analogs do not affect transcription which starts outside of the modified region. In contrast, base analogs that have alterations that appear in the major groove of the DNA helix, such as uracil, 5-bromouracil, 5-methylcytosine, 5-hydroxymethylcytosine, and [5-HgSR]pyrimidines, do not prevent utilization of the promoter. The deoxyribonucleoside analog 5'-imino-5'-deoxythymidine, an alteration appearing in the deoxyribose-phosphodiester backbone of the DNA helix, does not prevent promoter recognition. Haemophilus aegyptius restriction endonuclease III, which cleaves DNA at the sequence 5'GGCC3', does not act at sites in which the guanine residues in one of the two DNA strands have been substituted with hypoxanthine. This implicates the guanine amino group in the minor groove of the DNA helix as a possible recognition point for this restriction endonuclease. PMID:353045

Stahl, S J; Chamberlin, M J

1978-07-25

231

SRA/SET domain-containing proteins link RNA polymerase V occupancy to DNA methylation  

PubMed Central

RNA-directed DNA methylation (RdDM) in Arabidopsis thaliana depends on the upstream synthesis of 24-nucleotide small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV)1,2 and downstream synthesis of non-coding transcripts by Pol V. Pol V transcripts are thought to interact with siRNAs which then recruit DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to methylate DNA3-7. The SU(VAR)3-9 homologs SUVH2 and SUVH9 act in this downstream step but the mechanism of their action is unknown8,9. Here we show that genome-wide Pol V association with chromatin redundantly requires, SUVH2 and SUVH9. Although SUVH2 and SUVH9 resemble histone methyltransferases a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S-adenosyl methionine (SAM) binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins8. SUVH2 and SUVH9 both contain SET- and RING-ASSOCIATED (SRA) domains capable of binding methylated DNA8, suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of DNA METHYLTRANSFERASE 1 (MET1) causes loss of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. Furthermore, tethering SUVH2 with a zinc finger to an unmethylated site is sufficient to recruit Pol V and establish DNA methylation and gene silencing. These results suggest that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of plant genomes for epigenetic silencing. PMID:24463519

Hale, Christopher J.; Bischof, Sylvain; Feng, Suhua; Chodavarapu, Ramakrishna K.; Zhong, Xuehua; Marson, Giuseppe; Pellegrini, Matteo; Segal, David J.; Patel, Dinshaw J.; Jacobsen, Steven E.

2014-01-01

232

Peculiar feature of the organization of rRNA genes of the Chlorella chloroplast DNA.  

PubMed Central

The organization of a cloned rRNA gene cluster from Chlorella ellipsoidea chloroplast DNA (cpDNA) has been analyzed. Southern hybridization experiments with labelled chloroplast rRNAs as probes revealed an extraordinarily large size of the 16S-23S rRNA spacer region, ca. 4.8 kbp, almost twice as large as those of most higher plants. The nucleotide sequence determined on this region has shown that: (1) The tRNAIle gene locating in this region is similar to those of higher plant chloroplasts, blue-green algae and E. coli but does not contain any introns in contrast to higher plant chloroplasts. (2) The tRNAAla gene is absent from this region. (3) There are four open reading frames (ORFs) coding for 55, 102, 107 and 110 amino acids, respectively. (4) A few sets of unique sequence were found repeatedly in this region. (5) The 23S rRNA gene is coded on the opposite strand in the reverse order. This arrangement of the 16S-23S rRNA region of Chlorella cpDNA is quite different from any of those reported so far for various organisms. Images PMID:3714498

Yamada, T; Shimaji, M

1986-01-01

233

A Begomovirus DNA?-Encoded Protein Binds DNA, Functions as a Suppressor of RNA Silencing, and Targets the Cell Nucleus  

PubMed Central

Our previous results demonstrated that the DNA? satellite (Y10?) associated with Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) is essential for induction of leaf curl symptoms in plants and that transgenic expression of its ?C1 gene in Nicotiana plants induces virus-like symptoms. In the present study, in vitro DNA binding activity of the ?C1 proteins of Y10? and DNA? (Y35?) found in the Tobacco curly shoot virus Y35 isolate (TbCSV-Y35) were studied following their expression as six-His fusion proteins in Escherichia coli. Electrophoretic mobility shift assays and UV cross-linking experiments revealed that ?C1 proteins could bind both single-stranded and double-stranded DNA without size or sequence specificity. Suppression of green fluorescent protein (GFP) transgene silencing was observed with the new leaves of GFP-expressing Nicotiana benthamiana plants coinoculated by TYLCCNV-Y10 plus Y10? or by TbCSV-Y35 plus Y35?. In a patch agroinfiltration assay, the transiently expressed ?C1 gene of Y10? or Y35? was able to suppress host RNA silencing activities and permitted the accumulation of high levels of GFP mRNA in the infiltrated leaf patches of GFP transgenic N. benthamiana plants. The ?C1 protein of Y10? accumulated primarily in the nuclei of plant and insect cells when fused with ?-glucuronidase or GFP and immunogold labeling showed that the ?C1 protein is present in the nuclei of infected N. benthamiana plants. A mutant version of Y10? carrying the mutations within the putative nuclear localization sequence of the Y10 ?C1 protein failed to induce disease symptoms, suppress RNA silencing, or accumulate in the nucleus, suggesting that nuclear localization of the ?C1 protein is a key requirement for symptom induction and silencing suppression. PMID:16051868

Cui, Xiaofeng; Li, Guixin; Wang, Daowen; Hu, Dongwei; Zhou, Xueping

2005-01-01

234

The effects of upstream DNA on open complex formation by Escherichia coli RNA polymerase  

PubMed Central

Binding of activators to upstream DNA sequences regulates transcription initiation by affecting the stability of the initial RNA polymerase (RNAP)–promoter complex and/or the rate of subsequent conformational changes required to form the open complex (RPO). Here we observe that the presence of nonspecific upstream DNA profoundly affects an early step in formation of the transcription bubble. Kinetic studies with the ?PR promoter and Escherichia coli RNAP reveal that the presence of DNA upstream of base pair -47 greatly increases the rate of forming RPO, without significantly affecting its rate of dissociation. We find that this increase is largely due to an acceleration of the rate-limiting step (isomerization) in RPO formation, a step that occurs after polymerase binds. Footprinting experiments reveal striking structural differences downstream of the transcription start site (+1) in the first kinetically significant intermediate when upstream DNA is present. On the template strand, the DNase I downstream boundary of this early intermediate is +20 when upstream DNA is present but is shortened by approximately two helical turns when upstream DNA beyond -47 is removed. KMnO4 footprinting reveals an identical initiation bubble (-11 to +2), but unusual reactivity of template strand upstream cytosines (-12, -14, and -15) on the truncated promoter. Based on this work, we propose that early wrapping interactions between upstream DNA and the polymerase exterior strongly affect the events that control entry and subsequent unwinding of the DNA start site in the jaws of polymerase. PMID:15626761

Davis, Caroline A.; Capp, Michael W.; Record, M. Thomas; Saecker, Ruth M.

2005-01-01

235

SETX sumoylation: A link between DNA damage and RNA surveillance disrupted in AOA2.  

PubMed

Senataxin (SETX) is a putative RNA:DNA helicase that is mutated in two distinct juvenile neurological disorders, AOA2 and ALS4. SETX is involved in the response to oxidative stress and is suggested to resolve R loops formed at transcription termination sites or at sites of collisions between the transcription and replication machineries. R loops are hybrids between RNA and DNA that are believed to lead to DNA damage and genomic instability. We discovered that Rrp45, a core component of the exosome, is a SETX-interacting protein and that the interaction depends on modification of SETX by sumoylation. Importantly, we showed that AOA2 but not ALS4 mutations prevented both SETX sumoylation and the Rrp45 interaction. We also found that upon replication stress induction, SETX and Rrp45 co-localize in nuclear foci that constitute sites of R-loop formation generated by transcription and replication machinery collisions. We suggest that SETX links transcription, DNA damage and RNA surveillance, and discuss here how this link can be relevant to AOA2 disease. PMID:25054092

Richard, Patricia; Manley, James L

2014-01-01

236

Fluorescence lifetime discrimination of cellular DNA and RNA using various intercalating dyes and flow cytometric analysis  

NASA Astrophysics Data System (ADS)

Previously we showed that the fluorescence lifetimes of ethidium and propidium iodide were different when intercalated into cellular double-stranded DNA or RNA. Current studies of four ethidium derivatives showed that the lifetime difference of DNA and RNA bound dyes existed in all four compounds, and that the magnitude of the difference was dependent on the dye structure, the staining concentration, and the conditions in which flow cytometry (FCM) analysis of stained cells was performed. The fluorescence lifetime of both DNA and RNA bound fluorochromes was reduced with increasing dye concentration; however the level of decrease varied in different dyes. Further analysis of stained cellular DNA in dye-free solution, which favors only high affinity binding, led to elevated fluorescence lifetime values compared to lifetime values obtained from analysis in the dye solution under equilibrium binding conditions. The changes of fluorescence lifetime value reflect the difference in dye structure and distinct interaction between the dyes and nucleic acids. The staining and analysis variables used in these studies may potentially provide additional information on differences in nucleic acid conformation and function in cell populations.

Cui, H. H.; Valdez, Joseph G.; Steinkamp, John A.; Crissman, Harry A.

2001-05-01

237

Diel variation of the RNA/DNA ratios in Crassostrea angulata (Lamarck) and Ruditapes decussatus (Linnaeus 1758) (Mollusca: Bivalvia).  

PubMed

The aim of this study was to investigate the effect of time of day on RNA/DNA ratios among fed and starved Crassostrea angulata and Ruditapes decussatus juveniles. Sampling to investigate the day and night condition of juveniles was carried out for 48 h. A highly sensitive method for nucleic acid quantification was applied to bivalves. The results suggest that there is some variation in nucleic acid quantities with the time of the day. For the two species analysed, the RNA/DNA ratio was particularly high during the night and was higher in the fed animals. The results seem to indicate that there is some endogenous rhythm in the production of RNA. If there are diel changes in RNA/DNA ratios, it follows that average RNA/DNA ratios can be unrepresentative if there is any day or night bias in sampling. PMID:11325380

Chícharo, L M.Z.; Chícharo, M A.; Alves, F; Amaral, A; Pereira, A; Regala, J

2001-04-30

238

Site-specific DICER and DROSHA RNA products control the DNA damage response  

PubMed Central

Non-coding RNAs (ncRNAs) are involved in an increasing number of cellular events1. Some ncRNAs are processed by DICER and DROSHA ribonucleases to give rise to small double-stranded RNAs involved in RNA interference (RNAi)2. The DNA-damage response (DDR) is a signaling pathway that originates from the DNA lesion and arrests cell proliferation3. So far, DICER or DROSHA RNA products have not been reported to control DDR activation. Here we show that DICER and DROSHA, but not downstream elements of the RNAi pathway, are necessary to activate DDR upon oncogene-induced genotoxic stress and exogenous DNA damage, as studied also by DDR foci formation in mammalian cells and zebrafish and by checkpoint assays. DDR foci are sensitive to RNase A treatment, and DICER- and DROSHA-dependent RNA products are required to restore DDR foci in treated cells. Through RNA deep sequencing and studies of DDR activation at an inducible unique DNA double-strand break (DSB), we demonstrate that DDR foci formation requires site-specific DICER- and DROSHA-dependent small RNAs, named DDRNAs, which act in a MRE11-RAD50-NBS1 (MRN) complex-dependent manner. Chemically synthesized or in vitro-generated by DICER cleavage, DDRNAs are sufficient to restore DDR in RNase A-treated cells, also in the absence of other cellular RNAs. Our results describe an unanticipated direct role of a novel class of ncRNAs in the control of DDR activation at sites of DNA damage. PMID:22722852

Francia, Sofia; Michelini, Flavia; Saxena, Alka; Tang, Dave; de Hoon, Michiel; Anelli, Viviana; Mione, Marina; Carninci, Piero; d’Adda di Fagagna, Fabrizio

2012-01-01

239

DNA/RNA Hybrid Primer Mediated Poly(A) Tag Library Construction for Illumina Sequencing.  

PubMed

Alternation polyadenylation is widespread in eukaryotes, and has demonstrated roles in gene expression regulation. Owing to deep DNA sequencing technologies, global analyses of alternation polyadenylation and their functions have become possible. We present a method to generate poly(A) tags libraries for high-throughput sequencing (PAT-seq). This protocol targets the junction of the 3'-UTR and poly(A) tail of a transcript so it can be positively identified as a poly(A) site. Upon Zinc-mediated limited digestion of total RNA, RNA fragments with poly(A) tail are then isolated and 5'-end repaired. A DNA/RNA hybrid adaptor is ligated to the 5' end as an anchor. Then the library is generated by reverse transcription with oligo(dT)-adapter followed by PCR amplification. Such a custom poly(A) tags library can be generated from any source poly(A) containing RNA and good for both single- or paired-end sequencing in any Illumina sequencing platforms. This new method has been applied to investigate mRNA polyadenylation in Arabidopsis. PMID:25487213

Liu, Man; Wu, Xiaohui; Li, Qingshun Quinn

2015-01-01

240

Flexibility of the DNA enhances promoter affinity of Escherichia coli RNA polymerase.  

PubMed Central

Two types of mechanisms are discussed for the formation of active protein-DNA complexes: contacts with specific bases and interaction via specific DNA structures within the cognate DNA. We have studied the effect of a single nucleoside deletion on the interaction of Escherichia coli RNA polymerase with a strong promoter. This study reveals three patterns of interaction which can be attributed to different sites of the promoter, (i) direct base contact with the template strand in the '-35 region' (the 'recognition domain'), (ii) a DNA structure dependent interaction in the '-10 region' (the 'melting domain'), and (iii) an interaction which is based on a defined spatial relationship between the two domains of a promoter, namely the 'recognition domain' and the 'melting domain'. Images PMID:1868834

Werel, W; Schickor, P; Heumann, H

1991-01-01

241

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

242

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

243

Highly stable triple helix formation by homopyrimidine (l)-acyclic threoninol nucleic acids with single stranded DNA and RNA.  

PubMed

Acyclic (l)-threoninol nucleic acid (aTNA) containing thymine, cytosine and adenine nucleobases were synthesized and shown to form surprisingly stable triplexes with complementary single stranded homopurine DNA or RNA targets. The triplex structures consist of two (l)-aTNA strands and one DNA or RNA, and these triplexes are significantly stronger than the corresponding DNA or RNA duplexes as shown in competition experiments. As a unique property the (l)-aTNAs exclusively form triplex structures with DNA and RNA and no duplex structures are observed by gel electrophoresis. The results were compared to the known enantiomer (d)-aTNA, which forms much weaker triplexes depending upon temperature and time. It was demonstrated that (l)-aTNA triplexes are able to stop primer extension on a DNA template, showing the potential of (l)-aTNA for antisense applications. PMID:25564220

Kumar, Vipin; Kesavan, Venkitasamy; Gothelf, Kurt V

2015-02-10

244

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

245

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

246

Blind Predictions of DNA and RNA Tweezers Experiments with Force and Torque  

PubMed Central

Single-molecule tweezers measurements of double-stranded nucleic acids (dsDNA and dsRNA) provide unprecedented opportunities to dissect how these fundamental molecules respond to forces and torques analogous to those applied by topoisomerases, viral capsids, and other biological partners. However, tweezers data are still most commonly interpreted post facto in the framework of simple analytical models. Testing falsifiable predictions of state-of-the-art nucleic acid models would be more illuminating but has not been performed. Here we describe a blind challenge in which numerical predictions of nucleic acid mechanical properties were compared to experimental data obtained recently for dsRNA under applied force and torque. The predictions were enabled by the HelixMC package, first presented in this paper. HelixMC advances crystallography-derived base-pair level models (BPLMs) to simulate kilobase-length dsDNAs and dsRNAs under external forces and torques, including their global linking numbers. These calculations recovered the experimental bending persistence length of dsRNA within the error of the simulations and accurately predicted that dsRNA's “spring-like” conformation would give a two-fold decrease of stretch modulus relative to dsDNA. Further blind predictions of helix torsional properties, however, exposed inaccuracies in current BPLM theory, including three-fold discrepancies in torsional persistence length at the high force limit and the incorrect sign of dsRNA link-extension (twist-stretch) coupling. Beyond these experiments, HelixMC predicted that ‘nucleosome-excluding’ poly(A)/poly(T) is at least two-fold stiffer than random-sequence dsDNA in bending, stretching, and torsional behaviors; Z-DNA to be at least three-fold stiffer than random-sequence dsDNA, with a near-zero link-extension coupling; and non-negligible effects from base pair step correlations. We propose that experimentally testing these predictions should be powerful next steps for understanding the flexibility of dsDNA and dsRNA in sequence contexts and under mechanical stresses relevant to their biology. PMID:25102226

Chou, Fang-Chieh; Lipfert, Jan; Das, Rhiju

2014-01-01

247

Intronic Non-CG DNA hydroxymethylation and alternative mRNA splicing in honey bees  

PubMed Central

Background Previous whole-genome shotgun bisulfite sequencing experiments showed that DNA cytosine methylation in the honey bee (Apis mellifera) is almost exclusively at CG dinucleotides in exons. However, the most commonly used method, bisulfite sequencing, cannot distinguish 5-methylcytosine from 5-hydroxymethylcytosine, an oxidized form of 5-methylcytosine that is catalyzed by the TET family of dioxygenases. Furthermore, some analysis software programs under-represent non-CG DNA methylation and hydryoxymethylation for a variety of reasons. Therefore, we used an unbiased analysis of bisulfite sequencing data combined with molecular and bioinformatics approaches to distinguish 5-methylcytosine from 5-hydroxymethylcytosine. By doing this, we have performed the first whole genome analyses of DNA modifications at non-CG sites in honey bees and correlated the effects of these DNA modifications on gene expression and alternative mRNA splicing. Results We confirmed, using unbiased analyses of whole-genome shotgun bisulfite sequencing (BS-seq) data, with both new data and published data, the previous finding that CG DNA methylation is enriched in exons in honey bees. However, we also found evidence that cytosine methylation and hydroxymethylation at non-CG sites is enriched in introns. Using antibodies against 5-hydroxmethylcytosine, we confirmed that DNA hydroxymethylation at non-CG sites is enriched in introns. Additionally, using a new technique, Pvu-seq (which employs the enzyme PvuRts1l to digest DNA at 5-hydroxymethylcytosine sites followed by next-generation DNA sequencing), we further confirmed that hydroxymethylation is enriched in introns at non-CG sites. Conclusions Cytosine hydroxymethylation at non-CG sites might have more functional significance than previously appreciated, and in honey bees these modifications might be related to the regulation of alternative mRNA splicing by defining the locations of the introns. PMID:24079845

2013-01-01

248

A Z-DNA binding domain present in the human editing enzyme, double-stranded RNA adenosine?deaminase  

PubMed Central

Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Z?) that binds specifically to the left-handed Z-DNA conformation with high affinity (KD = 4 nM). As formation of Z-DNA in vivo occurs 5? to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. Analysis of sequences related to Z? has allowed identification of motifs common to this class of nucleic acid binding domain. PMID:9237992

Herbert, Alan; Alfken, Jens; Kim, Yang-Gyun; Mian, I. Saira; Nishikura, Kazuko; Rich, Alexander

1997-01-01

249

RNA and DNA association to zwitterionic and charged monolayers at the air-liquid interface.  

PubMed

The objective of this work is to establish under which conditions short RNA molecules (similar to miRNA) associate with zwitterionic phospholipids and how this differs from the association with cationic surfactants. We study how the base pairing (i.e., single stranded versus double stranded nucleic acids) and the length of the nucleic acid and the charge of the lipid/surfactant monolayer affect the association behavior. For this purpose, we study the adsorption of nucleic acids to monolayers composed of dipalmitoyl phosphatidylcholine (DPPC) or dioctadecyl-dimethyl-ammoniumbromide (DODAB) using the surface film balance, neutron reflectometry, and fluorescence microscopy. The monolayer studies with the surface film balance suggested that short single-stranded ssRNA associates with liquid expanded zwitterionic phospholipid monolayers, whereas less or no association is detected for double-stranded dsRNA and dsDNA. In order to quantify the interaction and to determine the location of the nucleic acid in the lipid/surfactant monolayer we performed neutron reflectometry measurements. It was shown that ssRNA adsorbs to and penetrates the liquid expanded monolayers, whereas there is no penetration of nucleic acids into the liquid condensed monolayer. No adsorption was detected for dsDNA to zwitterionic monolayers. On the basis of these results, we propose that the association is driven by the hydrophobic interactions between the exposed hydrophobic bases of the ssRNA and the hydrocarbon chains of the phospholipids. The addition of ssRNA also influences domain formation in the DPPC monolayer, leading to fractal-like interconnected domains. The experimental results are discussed in terms of the implication for biological processes and new leads for applications in medicine and biotechnology. PMID:22624628

Michanek, Agnes; Yanez, Marianna; Wacklin, Hanna; Hughes, Arwel; Nylander, Tommy; Sparr, Emma

2012-06-26

250

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

251

Y-box binding protein from Schistosoma mansoni: interaction with DNA and RNA.  

PubMed

A Schistosoma mansoni homologue of the human Y-box binding protein (SMYB1), as well as truncated proteins containing its N-terminal Cold Shock Domain (CSD) or its C-terminal domain (TAIL) were cloned into the p-MAL-c2 expression vector and produced in Escherichia coli. In order to characterize the interactions of these proteins to an inverted CCAAT motif present in a number of gene promoters, their binding to DNA was measured by Electrophoretic Mobility Shift Assays. SMYB1 bound to single- and double-stranded DNA containing the CCAAT motif and could bind also to RNA. The truncated CSD and TAIL domain proteins bound to dsDNA and RNA, but exhibited distinct binding patterns. Protein-DNA interaction was also investigated in vivo, using the Yeast One-Hybrid System. The plasmid constructs were GSTTRI, a DNA fragment composed of three copies of the CCAAT motif of the S. mansoni glutathione S-transferase gene promoter and four oligonucleotides spanning different regions of the S. mansoni p14 gene promoter. None of the yeast clones transformed with the above plasmids was able to grow in selective medium or to activate the transcription of the HIS3 reporter gene, suggesting that SMYB1 could not interact with these promoters in vivo. PMID:12467973

Valadão, A F; Fantappie, M R; LoVerde, P T; Pena, S D J; Rumjanek, F D; Franco, G R

2002-01-01

252

Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles  

SciTech Connect

RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie; Weinberg, Marc S. [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa)] [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa); Arbuthnot, Patrick, E-mail: Patrick.Arbuthnot@wits.ac.za [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa)] [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa)

2009-11-20

253

Structural basis for DNA-hairpin promoter recognition by the bacteriophage N4 virion RNA polymerase  

PubMed Central

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 five- to seven-base pair stem and three-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. PMID:19061645

Gleghorn, Michael L.; Davydova, Elena K.; Rothman-Denes, Lucia B.; Murakami, Katsuhiko S.

2008-01-01

254

Stopped in its tracks: the RNA polymerase molecular motor as a robust sensor of DNA damage.  

PubMed

DNA repair is often a complex, multi-component, multi-step process; this makes detailed kinetic analysis of the different steps of repair a challenging task using standard biochemical methods. At the same time, single-molecule methods are well-suited for extracting kinetic information despite time-averaging due to diffusion of biochemical components and stochasticity of chemical reaction steps. Here we discuss recent experiments using DNA nanomanipulation in a magnetic trap to study the initiation of transcription-coupled repair in a model bacterial system comprising the canonical Escherichia coli RNA polymerase and the Mfd translocase which specifically binds to it. These experiments provide kinetic insight into the reaction process, helping to explain how Mfd discriminates between transcribing RNAP and stalled RNAP. They also identify a reliably long-lived intermediate containing Mfd translocase and, potentially, RNA polymerase. This intermediate presumably serves as a platform for assembly of downstream repair components UvrAB(C). PMID:24685770

Howan, K; Monnet, J; Fan, J; Strick, T R

2014-08-01

255

RNA-DNA differences are rarer in proto-oncogenes than in tumor suppressor genes  

PubMed Central

It has long been assumed that DNA sequences and corresponding RNA transcripts are almost identical; a recent discovery, however, revealed widespread RNA-DNA differences (RDDs), which represent a largely unexplored aspect of human genome variation. It has been speculated that RDDs can affect disease susceptibility and manifestations; however, almost nothing is known about how RDDs are related to disease. Here, we show that RDDs are rarer in proto-oncogenes than in tumor suppressor genes; the number of RDDs in coding exons, but not in 3?UTR and 5?UTR, is significantly lower in the former than the latter, and this trend is especially pronounced in non-synonymous RDDs, i.e., those cause amino acid changes. A potential mechanism is that, unlike proto-oncogenes, the requirement of tumor suppressor genes to have both alleles affected to cause tumor ‘buffers' these genes to tolerate more RDDs. PMID:22355757

Gao, Feng; Lin, Yan; Zhang, Randy Ren

2012-01-01

256

RNA:DNA Ratio and Other Nucleic Acid Derived Indices in Marine Ecology  

PubMed Central

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 Alexandra; Chícharo, Luis

2008-01-01

257

Ab initio determination of the ionization potentials of DNA and RNA nucleobases  

NASA Astrophysics Data System (ADS)

Quantum chemical high level ab initio coupled-cluster and multiconfigurational perturbation methods have been used to compute vertical and adiabatic ionization potentials of the five canonical DNA and RNA nucleobases: uracil, thymine, cytosine, adenine, and guanine. Several states of their cations have been also calculated. The present results represent a systematic compendium of these magnitudes, establishing theoretical reference values at a level not reported before, calibrating computational strategies, and guiding the assignment of the features in the experimental photoelectron spectra.

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

2006-08-01

258

Continuous-flow DNA and RNA amplification chip combined with laser-induced fluorescence detection  

Microsoft Academic Search

DNA and RNA amplification, through polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR) are essential steps in most procedures of nucleic acid analysis. Microfabricated devices (chips) for continuous-flow PCR and\\/or RT-PCR provide very rapid thermal energy transfer and cycling compared to conventional PCR systems. However, time-consuming slab gel electrophoresis has been used for analysis of the amplified fragments

Pierre J Obeid; Theodore K Christopoulos

2003-01-01

259

Effects of trace elements and pesticides on dephosphorylation of RNA and DNA added to soils  

SciTech Connect

This study was carried out to assess the effects of 14 trace elements, 12 herbicides, and two fungicides on dephosphorylation of yeast ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) added to soils (Xerollic Calciorthids and Typic Haploxeralfs). The cumulative amount of ortho phosphate (Pi) released from nucleic acids increased linearly with time of incubation (up to 72 h), decreased with profile depth, and was highly influenced by soil pH. When trace elements were applied and compared by using 2.5 mmol kg/sup -1/ of soil, the average inhibition in dephosphorylation of RNA and DNA in two soils ranged from 17% with Co(II) to 52% with Cu(II). The most effective inhibitors of nucleic acid dephosphorylation were Ag(I), Cu(I), Cd(II), Cu(II), Mn(II), Ni(II), and Pb(II) (avg inhibition greater than or equal to 35%). Other elements that inhibited dephosphorylation of RNA and DNA added to soils included Ba(II), Co(II), Hg(II), Zn(II), Ti(IV), V(IV), and W(VI). When the pesticides were compared by using 5 mg of active ingredient kg/sup -1/ of soil, the average inhibition in nucleic acid dephosphorylation ranged from 14% with butylate to 39% with chloramben. The most effective inhibitors (> 25%) were atrazine, naptalam, chloramben, dicamba, trifluralin, and maneb. Other pesticides that inhibited RNA and DNA dephosphorylation in soils included cyanazine, 2,4-D, dinitroamine, EPTC plus R-25788, alachlor, paraquat, butylate, and captan.

Frankenberger, W.T. Jr.; Johanson, J.B.; Lund L.J.

1986-01-01

260

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

261

Ion distributions around left- and right-handed DNA and RNA duplexes: a comparative study  

PubMed Central

The ion atmosphere around nucleic acids is an integral part of their solvated structure. However, detailed aspects of the ionic distribution are difficult to probe experimentally, and comparative studies for different structures of the same sequence are almost non-existent. Here, we have used large-scale molecular dynamics simulations to perform a comparative study of the ion distribution around (5?-CGCGCGCGCGCG-3?)2 dodecamers in solution in B-DNA, A-RNA, Z-DNA and Z-RNA forms. The CG sequence is very sensitive to ionic strength and it allows the comparison with the rare but important left-handed forms. The ions investigated include Na+, K+ and Mg2 +, with various concentrations of their chloride salts. Our results quantitatively describe the characteristics of the ionic distributions for different structures at varying ionic strengths, tracing these differences to nucleic acid structure and ion type. Several binding pockets with rather long ion residence times are described, both for the monovalent ions and for the hexahydrated Mg[(H2O)6]2+ ion. The conformations of these binding pockets include direct binding through desolvated ion bridges in the GpC steps in B-DNA and A-RNA; direct binding to backbone oxygens; binding of Mg[(H2O)6]2+ to distant phosphates, resulting in acute bending of A-RNA; tight ‘ion traps’ in Z-RNA between C-O2 and the C-O2? atoms in GpC steps; and others. PMID:25428372

Pan, Feng; Roland, Christopher; Sagui, Celeste

2014-01-01

262

Ion distributions around left- and right-handed DNA and RNA duplexes: a comparative study.  

PubMed

The ion atmosphere around nucleic acids is an integral part of their solvated structure. However, detailed aspects of the ionic distribution are difficult to probe experimentally, and comparative studies for different structures of the same sequence are almost non-existent. Here, we have used large-scale molecular dynamics simulations to perform a comparative study of the ion distribution around (5'-CGCGCGCGCGCG-3')2 dodecamers in solution in B-DNA, A-RNA, Z-DNA and Z-RNA forms. The CG sequence is very sensitive to ionic strength and it allows the comparison with the rare but important left-handed forms. The ions investigated include Na(+), K(+) and Mg(2 +), with various concentrations of their chloride salts. Our results quantitatively describe the characteristics of the ionic distributions for different structures at varying ionic strengths, tracing these differences to nucleic acid structure and ion type. Several binding pockets with rather long ion residence times are described, both for the monovalent ions and for the hexahydrated Mg[(H2O)6](2+) ion. The conformations of these binding pockets include direct binding through desolvated ion bridges in the GpC steps in B-DNA and A-RNA; direct binding to backbone oxygens; binding of Mg[(H2O)6](2+) to distant phosphates, resulting in acute bending of A-RNA; tight 'ion traps' in Z-RNA between C-O2 and the C-O2' atoms in GpC steps; and others. PMID:25428372

Pan, Feng; Roland, Christopher; Sagui, Celeste

2014-12-16

263

Vaccinia virus encodes four putative DNA and/or RNA helicases distantly related to each other.  

PubMed

Computer-assisted analysis of the amino acid sequences of vaccinia virus proteins containing the purine NTP-binding pattern revealed the seven motifs typical of the DNA (RNA) helicase superfamily II in the proteins I8R and A18R. Together with the previously described putative helicases D6R and D11L, the number of putative helicases of this superfamily encoded by the genome of vaccinia virus now reaches four. Aside from the helicase motifs, the sequences of I8R and A18R showed no strong similarity to each other, nor to D6R and D11L. Statistically significant similarity was demonstrated between I8R and the putative RNA helicases involved in pre-mRNA splicing in yeast, PRP2, PRP16 and PRP22, whereas A18R appeared to be related to the putative helicases encoded by the human DNA repair gene ERCC3 and the D10 gene of bacteriophage T5. These findings suggest that I8R may be an RNA helicase. Based on the known properties of the virion NTPases of vaccinia virus, it is possible that the I8R protein may be identical to the previously characterized virion NTPase II. A18R is likely to possess DNA and/or RNA helicase activity. Circumstantial evidence suggests that this activity might be involved in melting duplex structures in late mRNAs. The possibility of independent acquisition of the putative helicases I8R, A18R and a common progenitor to D6R and D11L by an ancestral poxvirus is discussed. PMID:1321883

Koonin, E V; Senkevich, T G

1992-04-01

264

Thermodynamics and kinetics of DNA, RNA, and hybrid oligonucleotide double-strand formation  

SciTech Connect

The double strands formed by the RNA, DNA, and RNA.DNA hybrid oligonucleotides rCA/sub 5/G + rCU/sub 5/G, dCA/sub 5/G + dCt/sub 5/G and rCA/sub 5/G + dCT/sub 5/G were studied in order to determine the differences in the stability and dynamics of RNA and DNA. The thermodynamics of double-strand formation were determined by measuring the absorbance vs temperature at 260 nm for different strand concentrations. The deoxyribo-oligonucleotides were found to be more stable, due to a more favorable enthalpy, than the ribo-oligonucleotides. The double strands were found to aggregate, and the extent of aggregation was determined by analytical untracentrifugation. It was found that the double strands formed dangling ends by breaking the terminal C.G base pairs and forming all of the internal A.U base pairs. The kinetics of double-strand formation of these oligonucleotides were studied using temperature-jump kinetic techniques. The greater stability of these deoxyribo-oligonucleotides relative to the ribo-oligonucleotides was found to be due to both a faster recombination rate and a slower dissociation rate for the deoxyribo-oligonucleotides. The hybrid oligonucleotide kinetic properties were similar to the ribo-oligonucleotides. The intercalation of ethidium ion into these oligonucleotide double strands was measured by monitoring the absorbance vs temperature at two wavelengths, 260 and 283 nm.

Nelson, J.W.

1982-09-01

265

Macrocyclic Pyridyl Polyoxazoles: Selective RNA and DNA G-Quadruplex Ligands as Antitumor Agents  

PubMed Central

The synthesis of a series of 24-membered pyridine-containing polyoxazole macrocycles is described. Seventeen new macrocycles were evaluated for cytotoxic activity against RPMI 8402, KB-3, and KB-3 cell lines that overexpress the efflux transporters MDR1 (KBV-1) and BCRP (KBH5.0). Macrocycles in which the pyridyl-polyoxazole moiety is linked by a 1,3-bis(aminomethyl)phenyl group with a 5-(2-aminoethyl)-18, or a 5-(2-dimethylaminoethyl)-substituent 19, displayed the greatest cytotoxic potency. These compounds exhibit exquisite selectivity for stabilizing G-quadruplex DNA with no stabilization of duplex DNA or RNA. Compound 19 stabilizes quadruplex mRNA that encodes the cell-cycle checkpoint protein kinase Aurora A to a greater extent than the quadruplex DNA of a human telomeric sequence. These data may suggest a prominent role for G-quadruplex ligands interacting with mRNA being associated with the biological activity of macrocyclic polyoxazoles. Compound 19 has significant in vivo anticancer activity against a human breast cancer xenograft (MDA-MB-435) in athymic nude mice. PMID:20359224

Rzuczek, Suzanne G.; Pilch, Daniel S.; Liu, Angela; Liu, Leroy; LaVoie, Edmond J.; Rice, Joseph E.

2010-01-01

266

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

NASA Astrophysics Data System (ADS)

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 protein functions and improved detection sensitivity. Here we report a similar technique based on a pair of RNA-DNA hybrids that can be used generally 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 mainly focuses on the activation of RNA interference. However, the release of other functionalities (such as resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumours together with specific gene silencing. This split-functionality approach presents a new route in the development of `smart' nucleic acid-based nanoparticles and switches for various biomedical applications.

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.

2013-04-01

267

Expression of a cDNA encoding a functional 241-kilodalton vesicular stomatitis virus RNA polymerase.  

PubMed Central

The large gene, L, of vesicular stomatitis virus (VSV), which codes for the multifunctional RNA-dependent RNA polymerase, was assembled from five overlapping cDNA clones. The sequence of the 6.4-kilobase gene of the final construct was identical to the consensus sequence reported earlier. The gene was inserted into the simian virus 40 transient expression vector pJC119. Antibodies directed against synthetic peptides corresponding to the amino and carboxyl termini of the L protein were raised in rabbits. Both antibodies specifically immunostained the cytoplasm of COS cells that had been transfected with the vector DNA. The expressed L protein was immunoprecipitated from cell extracts and it was identical in size to the L protein of the virion (241 kilodaltons). Most importantly, COS cells that expressed the recombinant L protein transcribed, replicated, and consequently complemented and rescued temperature-sensitive RNA polymerase mutants of VSV at the nonpermissive temperature. The kinetics of virus release were similar to those of a wild-type VSV infection. We conclude that the recombinant RNA polymerase protein L is indistinguishable in its size and its functions from the VSV polymerase. Images PMID:2999788

Schubert, M; Harmison, G G; Richardson, C D; Meier, E

1985-01-01

268

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

269

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

270

Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens  

PubMed Central

Background Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. Principal Findings For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. Significance We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies. PMID:22514653

Liu, Christina; Lin, Juan; Ye, Kenny; Kim, Ryung; Hazan, Rachel; Rohan, Thomas; Fineberg, Susan; Loudig, Olivier

2012-01-01

271

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

272

Initiation and termination of DNA replication in human rRNA genes.  

PubMed Central

We have used the multicopy human rRNA genes as a model system to study replication initiation and termination in mammalian chromosomes. Enrichment for replicating molecules was achieved by isolating S-phase enriched populations of cells by centrifugal elutriation, purification of DNA associated with the nuclear matrix, and a chromatographic procedure that enriches for molecules containing single-stranded regions, a characteristic of replication forks. Two-dimensional agarose gel electrophoresis techniques were used to demonstrate that replication appears to initiate at multiple sites throughout most of the 31-kb nontranscribed spacer (NTS) of human ribosomal DNA but not within the 13-kb transcription unit or adjacent regulatory elements. Although initiation events were detected throughout the majority of the NTS, some regions may initiate more frequently than others. Termination of replication, the convergence of opposing replication forks, was found throughout the ribosomal DNA repeat units, and, in some repeats, specifically at the junction of the 3' end of the transcription unit and the NTS. This site-specific termination of replication is the result of pausing of replication forks near the sites of transcription termination. The naturally occurring multicopy rRNA gene family offers a unique system to study mammalian DNA replication without the use of chemical synchronization agents. Images PMID:8413256

Little, R D; Platt, T H; Schildkraut, C L

1993-01-01

273

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

274

Chitosan graft-(PEI-?-cyclodextrin) copolymers and their supramolecular PEGylation for DNA and siRNA delivery  

Microsoft Academic Search

Two water-soluble chitosan-graft-(polyethylenimine-?-cyclodextrin) (CPC) cationic copolymers were synthesized via reductive amination between oxidized chitosan (CTS) and low molecular weight polyethylenimine-modified ?-cyclodextrin (?-CD-PEI). The two polycations, termed as CPC1 and CPC2, were characterized by proton nuclear magnetic resonance spectroscopy, gel permeation chromatography, and elemental analysis. These polycations exhibited good ability to condense both plasmid DNA (pDNA) and small interfering RNA (siRNA)

Yuan Ping; Chengde Liu; Zhongxing Zhang; Kerh Li Liu; Jianhai Chen; Jun Li

2011-01-01

275

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

276

Proteolytic footprinting of transcription factor TFIIIA reveals different tightly binding sites for 5S RNA and 5S DNA.  

PubMed Central

Transcription factor IIIA (TFIIIA) employs an array of nine N-terminal zinc fingers to bind specifically to both 5S RNA and 5S DNA. The binding of TFIIIA to 5S RNA and 5S DNA was studied by using a protease footprinting technique. Brief treatment of free or bound TFIIA with trypsin or chymotrypsin generated fragments which were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fragments retaining the N terminus of TFIIA were identified by immunoblotting with an antibody directed against the N terminus of TFIIIA. Proteolytic footprinting of TFIIIA complexed with 5S DNA derivatives reinforced other evidence that the three N-terminal zinc fingers of TFIIIA bind most tightly to 5S DNA. Proteolytic footprinting of TFIIIA in reconstituted 7S ribonucleoprotein particles revealed different patterns of trypsin sensitivity for TFIIIA bound to oocyte versus somatic 5S RNA. Trypsin cleaved TFIIIA between zinc fingers 3 and 4 more readily when the protein was bound to somatic 5S RNA than when it was bound to oocyte 5S RNA. A tryptic fragment of TFIIIA containing zinc fingers 4 through 7 remained tightly associated with somatic 5S RNA. Zinc fingers 4 through 7 may represent a tightly binding site for 5S RNA in the same sense that fingers 1 through 3 represent a tightly binding site for 5S DNA. Images PMID:7689146

Bogenhagen, D F

1993-01-01

277

Ionic strength-dependent persistence lengths of single-stranded RNA and DNA  

PubMed Central

Dynamic RNA molecules carry out essential processes in the cell including translation and splicing. Base-pair interactions stabilize RNA into relatively rigid structures, while flexible non-base-paired regions allow RNA to undergo conformational changes required for function. To advance our understanding of RNA folding and dynamics it is critical to know the flexibility of these un-base-paired regions and how it depends on counterions. Yet, information about nucleic acid polymer properties is mainly derived from studies of ssDNA. Here we measure the persistence lengths (lp) of ssRNA. We observe valence and ionic strength-dependent differences in lp in a direct comparison between 40-mers of deoxythymidylate (dT40) and uridylate (rU40) measured using the powerful combination of SAXS and smFRET. We also show that nucleic acid flexibility is influenced by local environment (an adjoining double helix). Our results illustrate the complex interplay between conformation and ion environment that modulates nucleic acid function in vivo. PMID:22203973

Chen, Huimin; Meisburger, Steve P.; Pabit, Suzette A.; Sutton, Julie L.; Webb, Watt W.; Pollack, Lois

2012-01-01

278

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

E-print Network

III KNOCKDOWN OF DNMT1 IN BOVINE SOMATIC CELLS ......... 26 Materials and Methods????????????????.. 28 Bovine Fetal Cell Isolation and Culture ................................. 28 Transient Transfection... structural states that an RNAi molecule can assume when introduced. One method for introducing the RNAi pathway is by transfection of synthetic double stranded siRNA. These molecules tend to have a shorter half-life once introduced into the host cell...

Stroud, Todd

2010-01-20

279

Strong Inverse Correlation Between MicroRNA-125b and Human Papillomavirus DNA in Productive Infection  

PubMed Central

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

2014-01-01

280

Retrotransposon Ty1 integration targets specifically positioned asymmetric nucleosomal DNA segments in tRNA hotspots.  

PubMed

The Saccharomyces cerevisiae genome contains about 35 copies of dispersed retrotransposons called Ty1 elements. Ty1 elements target regions upstream of tRNA genes and other Pol III-transcribed genes when retrotransposing to new sites. We used deep sequencing of Ty1-flanking sequence amplicons to characterize Ty1 integration. Surprisingly, some insertions were found in mitochondrial DNA sequences, presumably reflecting insertion into mitochondrial DNA segments that had migrated to the nucleus. The overwhelming majority of insertions were associated with the 5' regions of Pol III transcribed genes; alignment of Ty1 insertion sites revealed a strong sequence motif centered on but extending beyond the target site duplication. A strong sequence-independent preference for nucleosomal integration sites was observed, in distinction to the preferences of the Hermes DNA transposon engineered to jump in yeast and the Tf1 retrotransposon of Schizosaccharomyces pombe, both of which prefer nucleosome free regions. Remarkably, an exquisitely specific relationship between Ty1 integration and nucleosomal position was revealed by alignment of hotspot Ty1 insertion position regions to peak nucleosome positions, geographically implicating nucleosomal DNA segments at specific positions on the nucleosome lateral surface as targets, near the "bottom" of the nucleosome. The specificity is observed in the three tRNA 5'-proximal nucleosomes, with insertion frequency dropping off sharply 5' of the tRNA gene. The sites are disposed asymmetrically on the nucleosome relative to its dyad axis, ruling out several simple molecular models for Ty1 targeting, and instead suggesting association with a dynamic or directional process such as nucleosome remodeling associated with these regions. PMID:22219510

Mularoni, Loris; Zhou, Yulian; Bowen, Tyson; Gangadharan, Sunil; Wheelan, Sarah J; Boeke, Jef D

2012-04-01

281

RNA and DNA binding of inert oligonuclear ruthenium(ii) complexes in live eukaryotic cells.  

PubMed

Confocal microscopy was used to study the intracellular localisation of a series of inert polypyridylruthenium(ii) complexes with three eukaryotic cells lines - baby hamster kidney (BHK), human embryonic kidney (HEK-293) and liver carcinoma (Hep-G2). Co-staining experiments with the DNA-selective dye DAPI demonstrated that the di-, tri- and tetra-nuclear polypyridylruthenium(ii) complexes that are linked by the bis[4(4'-methyl-2,2'-bipyridyl)]-1,12-dodecane bridging ligand ("bb12") showed a high degree of selectivity for the nucleus of the eukaryotic cells. Additional co-localisation experiments with the general nucleic acid stain SYTO 9 indicated that the ruthenium complexes showed a considerable preference for the RNA-rich nucleolus, rather than chromosomal DNA. No significant differences were observed in the intracellular localisation between the ?? and ?? enantiomers of the dinuclear complex. Cytotoxicity assays carried out over 72 hours indicated that the ruthenium complexes, particularly the tri- and tetra-nuclear species, were significantly toxic to the eukaryotic cells. However, when the activity of the least cytotoxic compound (the ?? enantiomer of the dinuclear species) was determined over a 24 hour period, the results indicated that the ruthenium complex was approximately a 100-fold less toxic to liver and kidney cells than to Gram positive bacteria. Circular dichroism (CD) spectroscopy was used to examine the effect of the ?? and ?? enantiomers of the dinuclear complex on the solution conformations of RNA and DNA. The CD experiments indicated that the RNA maintained the A-type conformation, and the DNA the B-type structure, upon binding by the ruthenium complexes. PMID:25333883

Li, Xin; Gorle, Anil K; Ainsworth, Tracy D; Heimann, Kirsten; Woodward, Clifford E; Grant Collins, J; Richard Keene, F

2014-10-21

282

RNA\\/protein and RNA\\/DNA ratios determined by flow cytometry and their relationship to growth limitation of selected planktonic algae in culture  

Microsoft Academic Search

RNA\\/protein and RNA\\/DNA ratios correlate closely to the growth rate for many organisms. However, this has not yet been shown for planktonic algae and could be a basis for a new approach to determine in situ growth rates. Therefore, cultures of Stephanodiscus minutulus, Cyclostephanos invisitatus (Bacillariophyceae), Chlamydomonas sp., a eukaryotic pico-alga, (Chlorophyceae) and Rhodomonas minuta (Cryptophyceae) were grown under limitation

Andreas Nicklisch; Christian E. W. Steinberg

2009-01-01

283

cDNA-RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG  

Microsoft Academic Search

Identifying genes that are differentially expressed by Mycobacterium bovis BCG after phagocytosis by macrophages will facilitate the understanding of the molecular mechanisms of host cell-intracellular pathogen interactions. To identify such genes a cDNA-total RNA subtractive hybridization strategy has been used that circumvents the problems both of limited availability of bacterial RNA from models of infection and the high rRNA backgrounds

Ming-Shi Li; Irene M. Monahan; Simon J. Waddell; Joseph A. Mangan; Steve L. Martin; Martin J. Everett; Philip D. Butcher

284

Typing of coagulase-negative staphylococci by southern hybridization of chromosomal DNA fingerprints using a ribosomal RNA probe  

Microsoft Academic Search

Ribotyping consists of restriction endonuclease fingerprinting of ribosomal RNA (rRNA) genes visualized by Southern hybridization with an rRNA probe. This method was developed and compared with restriction endonuclease fingerprinting of chromosomal DNA for typing coagulase-negative staphylococci. Twenty-five American Type Culture Collection reference type strains and 53 clinical isolates were typed. Both methods clearly distinguished all 15 species of coagulase-negative staphylococci

H. Bialkowska-Hobrzanska; V. Harry; D. Jaskot; O. Hammerberg

1990-01-01

285

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

286

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

287

Reduction of DNA contamination in RNA samples for reverse transcription-polymerase chain reaction using selective precipitation by compaction agents.  

E-print Network

Reduction of DNA contamination in RNA samples for reverse transcription-polymerase chain reaction of Chemical and Biomolecular Engineering, 4800 Calhoun Road, S222 Engineering Building 1, University by reverse transcription-polymerase chain reaction (RT-PCR) is DNA contamination, which can produce

Fox, George

288

Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods.  

PubMed

To better characterize lentiviral vector supernatants, we compared three methods of titer assessment. These titer methods include assessment of vector RNA sequences in supernatants, DNA sequences in transduced cells, and vector expression in transduced cells (using a vector which expressed the green fluorescence protein, GFP). For analysis of RNA and DNA, we developed a real-time PCR method for detecting the lentiviral packaging sequence and used this methodology to quantitate the number of vector sequences. Vector expression was assessed by flow cytometric analysis for GFP. As functional titers (DNA and GFP expression titers) are dependent on transduction efficiency, we calculated the titer of a lentiviral vector, RRL-CMV-GFP, after transduction of 293, HeLa, or Mus dunni cells. Genomic DNA was extracted at 4 and 14 days after transduction and the number of vector DNA molecules was determined against a plasmid standard. Of the three cell lines tested, 293 cells provided the highest rate of transduction (PCR estimated DNA titer for RRL-CMV-GFP vector was 2.52 +/- 0.25 x 10(6) molecules/ml at 14 days, and 2.31 +/- 0.15 x 10(6) molecules/ml at 4 days). When titer was calculated based on GFP expression, the highest titer was also obtained on 293 cells (0.26 +/- 0.04 x 10(6) TU/ml at 14 days, and 0.24 +/- 0.03 +/- 10(6) TU/ml at 4 days). The titers obtained by GFP expression assay were approximately one log lower than those obtained by DNA analysis suggesting that variability in vector expression may underestimate titer. Measurement of RNA titers directly from vector supernatants against a plasmid standard indicated that the RNA titers are substantially higher than the DNA (approximately 10(3)-fold) and GFP titers (approximately 10(4)-fold). To show that the lentiviral probe and primers could be used for titering a variety of lentiviral vectors, we have also used the real-time PCR method to determine the DNA titers of two other HIV1 derived vectors, RRL-PGK-GFP (6.1 +/- 1.4 x 10(5) molecules/ml), and SMPU-RRE-BN (1.26 +/- 0.2 x 10(6) molecules/ml). We conclude that of the three methods tested, titers assessed by DNA analysis of transduced cells provide the most reliable estimate of functional titers as these are least likely to be influenced by factors, such as defective interfering particles and vector expression levels. The real-time PCR method described offers a reproducible method for lentiviral titering and can be applied to a wide variety of vectors, regardless of transgene. PMID:12170379

Sastry, L; Johnson, T; Hobson, M J; Smucker, B; Cornetta, K

2002-09-01

289

A mammalian microRNA cluster controls DNA methylation and telomere recombination via Rbl2-dependent regulation of DNA methyltransferases  

PubMed Central

Dicer initiates RNA interference by generating small RNAs involved in various silencing pathways. Dicer participates in centromeric silencing, but its role in the epigenetic regulation of other chromatin domains has not been explored. Here we show that Dicer1 deficiency in Mus musculus leads to decreased DNA methylation, concomitant with increased telomere recombination and telomere elongation. These DNA-methylation defects correlate with decreased expression of Dnmt1, Dnmt3a and Dnmt3b DNA methyltransferases (Dnmts), and methylation levels can be recovered by their overexpression. We identify the retinoblastoma-like 2 protein (Rbl2) as responsible for decreased Dnmt expression in Dicer1-null cells, suggesting the existence of Dicer-dependent small RNAs that target Rbl2. We identify the miR-290 cluster as being downregulated in Dicer1-deficient cells and show that it silences Rbl2, thereby controlling Dnmt expression. These results identify a pathway by which miR-290 directly regulates Rbl2-dependent Dnmt expression, indirectly affecting telomere-length homeostasis. PMID:18311151

Benetti, Roberta; Gonzalo, Susana; Jaco, Isabel; Muñoz, Purificación; Gonzalez, Susana; Schoeftner, Stefan; Murchison, Elizabeth; Andl, Thomas; Chen, Taiping; Klatt, Peter; Li, En; Serrano, Manuel; Millar, Sarah; Hannon, Gregory; Blasco, Maria A

2010-01-01

290

Cloning, expression, and complementation test of the RNA lariat debranching enzyme cDNA from mouse.  

PubMed

The RNA lariat debranching enzyme of mouse (mDBR1) was cloned by screening a NIH/3T3 cDNA library. The sequence of full-length mDBR1 cDNA contained a single 515 amino acid open reading frame of 58 kDa protein. Comparison of the amino acid sequence of mDBR1 to other DBR proteins showed 40%, 44%, 43%, 42%, and 80% identity to Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, and human debranching enzymes, respectively. The mDBR1 cDNA was shown to be functional in an interspecies specific complementation experiment, and an in vitro debranching enzyme assay. Mouse DBR1 could complement the intron accumulation phenotype of a S. cerevisiae dbrl null mutant strain. However, the level of complementation depended on the copy number of the mDBR1 cDNA. The integration of the mDBR1 cDNA in the chromosome of S. pombe also complemented both intron accumulation and slow growth phenotypes of the S. pombe dbr1 knock-out mutant strain. PMID:11355701

Kim, H C; Kim, G M; Yang, J M; Ki, J W

2001-04-30

291

LNA units present in the (2'-OMe)-RNA strand stabilize parallel duplexes (2'-OMe)-RNA/[All-RP-PS]-DNA and parallel triplexes (2'-OMe)-RNA/[All-RP-PS]-DNA/RNA. An improved tool for the inhibition of reverse transcription.  

PubMed

Homopurine phosphorothioate analogs of DNA, possessing all phosphorus atoms of RP configuration ([All-RP-PS]-DNA), when interact with appropriate complementary RNA or (2'-OMe)-RNA templates, form parallel triplexes or parallel duplexes of very high thermodynamic stability. The present results show that T-LNA or 5-Me-C-LNA units introduced into the parallel Hoogsteen-paired (2'-OMe)-RNA strands (up to four units in the oligomers of 9 or 12 nt in length) stabilize these parallel complexes. At neutral pH, dodecameric parallel duplexes have Tm values of 62-68 °C, which are by 4-10 °C higher than Tm for the reference duplex (with no LNA units present), while for the corresponding triplexes, Tm values exceeded 85 °C. For nonameric parallel duplexes, melting temperatures of 38-62 °C were found and (2'-OMe)-RNA oligomers containing 5-Me-C-LNA units stabilized the complexes more efficiently than the T-LNA containing congeners. In both series the stability of the parallel complexes increased with an increasing number of LNA units present. The same trend was observed in experiments of reverse transcription RNA?DNA (using AMV RT reverse transcriptase) where the formation of parallel triplexes (consisting of an RNA template, [All-RP-PS]-DNA nonamer and Hoogsteen-paired (2'-OMe)-RNA strands containing the LNA units) led to the efficient inhibition of the process. Under the best conditions checked (four 5-Me-C-LNA units, three-fold excess over the RNA template) the inhibition was 94% effective, compared to 71% inhibition observed in the reference system with the Hoogsteen-paired (2'-OMe)-RNA strand carrying no LNA units. This kind of complexation may "arrest" harmful RNA oligomers (e.g., viral RNA or mRNA of unwanted proteins) and, beneficially, exclude them from enzymatic processes, otherwise leading to viral or genetic diseases. PMID:25564351

Maciaszek, Anna; Krakowiak, Agnieszka; Janicka, Magdalena; Tomaszewska-Antczak, Agnieszka; Sobczak, Milena; Miko?ajczyk, Barbara; Guga, Piotr

2015-02-10

292

Micelle-like Nanoparticles as Carriers for DNA and siRNA.  

PubMed

Gene therapy represents a potential efficient approach of disease prevention and therapy. However, due to their poor in vivo stability, gene molecules need to be associated with delivery systems to overcome extracellular and intracellular barriers and allow access to the site of action. Cationic polymeric nanoparticles are popular carriers for small interfering RNA (siRNA) and DNA-based therapeutics for which efficient and safe delivery are important factors that need to be optimized. Micelle-like nanoparticles (MNP) (half micelles, half polymeric nanoparticles) can overcome some of the disadvantages of such cationic carriers by unifying in one single carrier the best of both delivery systems. In this review, we will discuss how the unique properties of MNP including self-assembly, condensation and protection of nucleic acids, improved cell association and gene transfection, and low toxicity may contribute to the successful application of siRNA- and DNA-based therapeutics into the clinic. Recent developments of MNP involving the addition of stimulus-sensitive functions to respond specifically to pathological or externally applied "triggers" (e.g., temperature, pH or enzymatic catalysis, light, or magnetic fields) will be discussed. Finally, we will overview the use of MNP as two-in-one carriers for the simultaneous delivery of different agents (small molecules, imaging agents) and nucleic acid combinations. PMID:25557580

Navarro, Gemma; Pan, Jiayi; Torchilin, Vladimir P

2015-02-01

293

Lipid-mediated delivery of RNA is more efficient than delivery of DNA in non-dividing cells  

PubMed Central

The design of appropriate gene delivery systems is essential for the successful application of gene therapy to clinical medicine. Cationic lipid-mediated delivery is a viable alternative to viral vector-mediated gene delivery in applications where transient gene expression is desirable. However, cationic lipid-mediated delivery of DNA to post-mitotic cells such as neurons is often reported to be of low efficiency, due to the presumed inability of the DNA to translocate to the nucleus. Lipid-mediated delivery of RNA is an attractive alternative to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary. Here we report a comparative investigation of cationic lipid-mediated delivery of RNA versus DNA vectors encoding the reporter gene green fluorescent protein (GFP) in Chinese Hamster Ovary (CHO) and NIH3T3 cells following chemical inhibition of proliferation, and in primary mixed neuronal cell cultures. Using optimized formulations and transfection procedures, we assess gene expression by flow cytometry to specifically address some of the advantages and disadvantages of lipid-mediated RNA and DNA gene transfer. Despite inhibition of cell proliferation, over 45% of CHO cells express GFP after lipid-mediated transfection with RNA vectors. Transfection efficiency of DNA encoding GFP in proliferation-inhibited CHO cells was less than 5%. Detectable expression after RNA transfection occurs at least 3 hours earlier than after DNA transfection, but DNA transfection eventually produces a mean level of per cell GFP expression (as assayed by flow cytometry) that is higher than after RNA transfection. Transfection of proliferation-inhibited NIH3T3 cells and primary mixed neuronal cultures produced similar results, with RNA encoded GFP expression in 2 to 4 times the number of cells as after DNA encoded GFP expression. These results demonstrate the increased efficiency of RNA transfection relative to DNA transfection in non-dividing cells. We used firefly luciferase encoded by RNA and DNA vectors to investigate the time course of gene expression after delivery of RNA or DNA to primary neuronal cortical cells. Delivery of mRNA resulted in rapid onset (within 1 hour) of luciferase expression after transfection, a peak in expression 5– 7 hours after transfection, and a return to baseline within 12 hours after transfection. After DNA delivery significant luciferase activity did not appear until 7 hours after transfection, but peak luciferase expression was always at least one order of magnitude higher than after RNA delivery. The peak expression after luciferase-expressing DNA delivery occurred 36–48 hours after transfection and remained at a significant level for at least one week before dropping to baseline. This observation is consistent with our in-vivo delivery results, which are shown as well. RNA delivery may therefore be more suitable for short-term transient gene expression due to rapid onset, shorter duration of expression and greater efficiency, particularly in non-dividing cells. Higher mean levels of expression per cell obtained following DNA delivery and the longer duration of expression confirm a continuing role for DNA gene delivery in clinical applications that require longer term transient gene expression. PMID:20080162

Zou, S; Scarfo, K; Nantz, M H; Hecker, J G

2010-01-01

294

Enzymatic Amplification of DNA/RNA Hybrid Molecular Beacon Signaling in Nucleic Acid Detection  

PubMed Central

A rapid assay operable under isothermal or non-isothermal conditions is described wherein the sensitivity of a typical molecular beacon (MB) system is improved by utilizing thermostable RNase H to enzymatically cleave an MB comprised of a DNA stem and RNA loop (R/D-MB). Upon hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7x above background) due to an opening of the probe and concomitant reduction in the Förster resonance energy transfer efficiency. Addition of thermostable RNase H resulted in the cleavage of the RNA loop which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9x above background), resulting in a ~2–2.8 fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time PCR reactions by measuring enhancement of donor fluorescence upon R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB-RNase H scheme can be applied to a broad range of nucleic acid amplification methods. PMID:23000602

Jacroux, Thomas; Rieck, Daniel C.; Cui, Rong; Ouyang, Yexin; Dong, Wen-Ji

2012-01-01

295

Hydration effects on the duplex stability of phosphoramidate DNA-RNA oligomers.  

PubMed Central

Recent studies on uniformly modified oligonucleotides containing 3'-NHP(O)(O-)O-5'internucleoside linkages (3'amidate) and alternatively modified oligonucleotides containing 3'-O(O-)(O)PNH-5'internucleoside linkages (5'amidate) have shown that 3'amidate duplexes, formed with DNA or RNA complementary strands, are more stable in water than those of the corresponding phosphodiesters. In contrast, 5'amidates do not form duplexes at all. There is no steric reason that the 5'amidate duplex should not form. We demonstrate that these differences arise from differential solvation of the sugar-phosphate backbones. By molecular dynamics calculations on models of 10mer single-stranded DNA and double-stranded DNA-RNA molecules, both with and without the phosphoramidate backbone modifications, we show that the single-stranded 3'amidate and 5'amidate backbones are equally well solvated, but the 5'amidate backbone is not adequately solvated in an A-form duplex. These results are supported by quantum chemical free energy of solvation calculations which show that the 3'amidate backbone is favored relative to the 5'amidate backbone. PMID:9016634

Barsky, D; Colvin, M E; Zon, G; Gryaznov, S M

1997-01-01

296

Tandem repeats upstream of the Arabidopsis endogene SDC recruit non-CG DNA methylation and initiate siRNA spreading  

Microsoft Academic Search

Plants use siRNAs to target cytosine DNA methylation to both symmetrical CG and nonsymmetrical (CHG and CHH) sequence contexts. DNA methylation and siRNA clusters most frequently overlap with transposons in the Arabidopsis thaliana genome. However, a significant number of protein-coding genes also show promoter DNA methylation, and this can be used to silence their expression. Loss of the majority of

Ian R. Henderson; Steven E. Jacobsen

2008-01-01

297

RNA.  

ERIC Educational Resources Information Center

Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

Darnell, James E., Jr.

1985-01-01

298

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

299

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

300

Ultrastable pRNA hexameric ring gearing hexameric phi29 DNA-packaging motor by revolving without rotating and coiling  

PubMed Central

Biomotors have previously been classified into two categories: linear and rotational motors. It has long been popularly believed that viral DNA packaging motors are rotation motors. We have recently found that the DNA-packaging motor of bacteriophage phi29 uses a third mechanism: revolution without rotation. phi29 motor consists of three-coaxial rings of hexameric RNA, a hexameric ATPase, and a dodecameric channel. The motor uses six ATP to revolve one helical turn of dsDNA around the hexameric ring of ATPase gp16. Each dodecameric segment tilts at a 30°-angle and runs anti-parallel to the dsDNA helix to facilitate translation in one direction. The negatively charged phosphate backbone interacts with four positively charged lysine rings, resulting in four steps of transition. This review will discuss how the novel pRNA meets motor requirements for translocation concerning structure, stoichiometry, and thermostability; how pRNA studies have led to the generation of the concept of RNA nanotechnology; and how pRNA is fabricated into nanoparticles to deliver siRNA, miRNA, and ribozymes to cancer and virus-infected cells. PMID:23683853

Schwartz, Chad; Guo, Peixuan

2013-01-01

301

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

302

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

303

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

304

Human growth hormone DNA sequence and mRNA structure: possible alternative splicing.  

PubMed Central

We have determined the complete sequence of the human growth hormone (hGH) gene and the position of the mature 5' end of the hGH mRNA within the sequence. Comparison of this sequence with that of a cloned hGH cDNA shows that the gene is interrupted by four intervening sequences. S1 mapping shows that one of these intervening sequences has two different 3' splice sites. These alternate splicing pathways generate hGH peptides of different sizes which are found in normal pituitaries. Comparison of sequences near the 5' end of the hGH mRNA with a similar region of the alpha subunit of the human glycoprotein hormones reveals an unexpected region of homology between these otherwise unrelated peptide hormones. Images PMID:6269091

DeNoto, F M; Moore, D D; Goodman, H M

1981-01-01

305

Chimeric DNA-RNA hammerhead ribozyme targeting transforming growth factor-beta 1 mRNA inhibits neointima formation in rat carotid artery after balloon injury.  

PubMed

We designed and synthesized a chimeric DNA-RNA hammerhead ribozyme targeting transforming growth factor (TGF)-beta 1 mRNA and found that this ribozyme effectively and specifically inhibited growth of vascular smooth muscle cells. We examined the effects of the chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta 1 mRNA on neointima formation and investigated the underlying mechanism to develop a possible gene therapy for coronary artery restenosis after percutaneous transluminal coronary angioplasty. Expression of mRNAs encoding TGF-beta 1, p27kip1, and connective tissue growth factor (CTGF) in carotid artery increased after balloon injury. Fluorescein-isothiocyanate (FITC)-labeled ribozyme was taken up into the midlayer smooth muscle of the injured carotid artery. Both 2 and 5 mg of ribozyme reduced neointima formation by 65% compared to that of controls. Ribozyme markedly decreased expression of TGF-beta 1 mRNA and protein in injured vessel. Mismatch ribozyme had no effect on expression of TGF-beta 1 mRNA protein in injured vessel. Ribozyme markedly decreased expression of fibronectin, p27kip1, and CTGF mRNAs in injured vessel, whereas a mismatch ribozyme had no effect on these mRNAs. These findings indicate that the chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta 1 mRNA inhibits neointima formation in rat carotid artery after balloon injury with suppression of TGF-beta 1 and inhibition of extracellular matrix and CTGF. In conclusion, the hammerhead ribozyme against TGF-beta 1 may have promise as a therapy for coronary artery restenosis after percutaneous transluminal coronary angioplasty. PMID:14729108

Ando, Hideyuki; Fukuda, Noboru; Kotani, Motoko; Yokoyama, Shin ichiro; Kunimoto, Satoshi; Matsumoto, Koichi; Saito, Satoshi; Kanmatsuse, Katsuo; Mugishima, Hideo

2004-01-12

306

piRNA-823 contributes to tumorigenesis by regulating de novo DNA methylation and angiogenesis in multiple myeloma.  

PubMed

Aberrant DNA hypermethylation contributes to myelomagenesis by silencing tumor-suppressor genes. Recently, a few reports have suggested that a novel class of small non-coding RNAs, called Piwi-interacting RNAs (piRNAs), may be involved in the epigenetic regulation of cancer. In this study, for the first time we provided evidence that the expression of piRNA-823 was upregulated in multiple myeloma (MM) patients and cell lines, and positively correlated with clinical stage. Silencing piRNA-823 in MM cells induced deregulation of cell cycle regulators and apoptosis-related proteins expression, accompanied by inhibition of tumorigenicity in vitro and in vivo. Moreover, piRNA-823 was directly relevant to de novo DNA methyltransferases, DNMT3A and 3B, in primary CD138(+) MM cells. The inhibited expression of piRNA-823 in MM cells resulted in marked reduction of DNMT3A and 3B at both mRNA and protein levels, which in turn led to decrease in global DNA methylation and reexpression of methylation-silenced tumor suppressor, p16(INK4A). In addition, piRNA-823 abrogation in MM cells induced reduction of vascular endothelial growth factor secretion, with consequent decreased proangiogenic activity. Altogether, these data support an oncogenic role of piRNA-823 in the biology of MM, providing a rational for the development of piRNA-targeted therapeutic strategies in MM. PMID:24732595

Yan, H; Wu, Q-L; Sun, C-Y; Ai, L-S; Deng, J; Zhang, L; Chen, L; Chu, Z-B; Tang, B; Wang, K; Wu, X-F; Xu, J; Hu, Y

2015-01-01

307

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

308

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

309

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

310

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

311

Higher-Level Snake Phylogeny Inferred from Mitochondrial DNA Sequences of 12s rRNA and 16s rRNA Genes  

E-print Network

Higher-Level Snake Phylogeny Inferred from Mitochondrial DNA Sequences of 12s rRNA and 16s r sequenced to determine the phylogenetic relationships among the major clades of snakes. Thirty-six species families of lizards. Snakes were found to constitute a monophyletic group (confidence probability [CP] = 96

Hedges, Blair

312

A novel approach on fluid dispensing for a DNA/RNA extraction chip package  

NASA Astrophysics Data System (ADS)

Micro fluidic package with integrated reservoirs has been developed for DNA /RNA extraction application. A membrane based pump which consists of a reservoir to store reagents and a pin valve to control the fluid is developed to dispense the reagents into the chip. A programmable external actuator is fabricated to dispense the fluid from the membrane pump into the DNA chip. An elastic and high elongation thin rubber membrane is used to seal the membrane pump and at the same time prevent actuator from mixing with different reagents in the micro fluidic package. Break displacement during actuation of membrane pump sealing material is studied with different ratios of PDMS and other types of rubber materials. The fluid flow from the reservoir to the chip is controlled by a pin valve which is activated during the external actuation. A CFD simulation is performed to study the pumping action dusting the external actuation and is validated with experimental results.

Xie, Ling; Premachandran, C. S.; Chew, Michelle; Yao, Qiang; Xu, Diao; Pinjala, D.

2008-02-01

313

Cloning of cDNA to rat mammary-gland fatty acid synthase mRNA. Evidence for the expression of two mRNA species during lactation.  

PubMed Central

A cDNA library was constructed in the expression vector lambda gt11, by synthesizing cDNA from size-selected poly(A) RNA from lactating rat mammary gland, using random hexanucleotide primers. Using this library we identified two recombinants which, on addition of a lac z inducer, produced proteins recognized by affinity-purified anti-fatty-acid synthase antibody, and which, therefore, contained fatty acid synthase coding sequences. The inserts were subcloned, were shown to be between 500 and 600 base pairs in size, and to cross-hybridize. The cloned DNA was then used in Northern hybridizations with mRNA isolated at various stages throughout lactation. Two mRNA species were identified of approx. 9.7 and 10.4 kilobases, which increased and decreased in parallel during lactation, reaching a peak at 12-13 days. Both mRNA species disappeared rapidly if the pups were removed prematurely. This study provides evidence that, during hormonal induction in lactation, regulation of the level of fatty acid synthase protein can be accounted for by variation in the level of mRNA. Images Fig. 1. Fig. 2. Fig. 3. PMID:3342031

Braddock, M; Hardie, D G

1988-01-01

314

Purification, Characterization, and Comparison of the DNA Polymerases from Two Primate RNA Tumor Viruses  

PubMed Central

The DNA polymerase from the Mason-Pfizer monkey virus (M-PMV), an RNA tumor virus not typical type-C or type-B, has been purified a thousand-fold over the original crude viral suspension. This purified enzyme is compared to a similarly purified DNA polymerase from the primate woolly monkey virus, a type-C virus. The two enzymes have similar template specificities but differ in their requirements for optimum activity. Both DNA polymerases have a pH optimum of 7.3 in Tris buffer. M-PMV enzyme has maximum activity with 5 mM Mg2+ and 40 mM potassium chloride, whereas the woolly monkey virus optima are 100 mM potassium chloride with 0.8 mM Mn2+. The apparent molecular weight of the M-PMV enzyme is approximately 110,000, whereas the woolly monkey virus polymerase is approximately 70,000. The biochemical properties of these two enzymes were also compared to a similarly purified enzyme from a type-C virus from a lower mammal (Rauscher murine leukemia virus). The results show that more similarity exists between the DNA polymerases from viruses of the same type (type-C), than between the polymerases from viruses of different types but from closely related species. Images PMID:4127027

Abrell, John W.; Gallo, Robert C.

1973-01-01

315

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

316

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

317

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

PubMed

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

2014-10-29

318

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

319

A collaborative European exercise on mRNA-based body fluid/skin typing and interpretation of DNA and RNA results.  

PubMed

The European Forensic Genetics Network of Excellence (EUROFORGEN-NoE) undertook a collaborative project on mRNA-based body fluid/skin typing and the interpretation of the resulting RNA and DNA data. Although both body fluids and skin are composed of a variety of cell types with different functions and gene expression profiles, we refer to the procedure as 'cell type inference'. Nine laboratories participated in the project and used a 20-marker multiplex to analyse samples that were centrally prepared and thoroughly tested prior to shipment. Specimens of increasing complexity were assessed that ranged from reference PCR products, cDNAs of indicated or unnamed cell type source(s), to challenging mock casework stains. From this specimen set, information on the overall sensitivity and specificity of the various markers was obtained. In addition, the reliability of a scoring system for inference of cell types was assessed. This scoring system builds on replicate RNA analyses and the ratio observed/possible peaks for each cell type [1]. The results of the exercise support the usefulness of this scoring system. When interpreting the data obtained from the analysis of the mock casework stains, the participating laboratories were asked to integrate the DNA and RNA results and associate donor and cell type where possible. A large variation for the integrated interpretations of the DNA and RNA data was obtained including correct interpretations. We infer that with expertise in analysing RNA profiles, clear guidelines for data interpretation and awareness regarding potential pitfalls in associating donors and cell types, mRNA-based cell type inference can be implemented for forensic casework. PMID:24552886

van den Berge, M; Carracedo, A; Gomes, I; Graham, E A M; Haas, C; Hjort, B; Hoff-Olsen, P; Maroñas, O; Mevåg, B; Morling, N; Niederstätter, H; Parson, W; Schneider, P M; Court, D Syndercombe; Vidaki, A; Sijen, T

2014-05-01

320

Site selection and structure of DNA-linked RNA primers synthesized by the primosome in phage phi X174 DNA replication in vitro.  

PubMed

Synthesis of a complementary strand on the circular viral (+)-DNA of phage phiX174, coated with single-stranded DNA binding protein, is primed by the synthesis of an oligonucleotide by the primosome. Processive primosome movement on the lagging strand with the replication fork was proposed as a model for the discontinuous portion of Escherichia coli chromosome replication (Arai, K. and Kornberg, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 69-73; Arai, K., Low, R. L., and Kornberg, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 707-711). RNA primers covalently bound to the 5'-end of a DNA chain are heterogeneous with respect to both size and nucleotide composition. The chain length of the DNA-linked RNA primers is shorter than a decanucleotide, predominantly ranging from 1 to 9 residues. The primers start with adenylate followed mainly by a purine nucleotide (Pu) at the second position suggesting that pppA-Pu is a preferred initiation sequence. The inner sequences are more heterogeneous and no consensus or preferred sequence was found beyond the third position. The size distribution of the primer is influenced by the relative concentration of ribo- and deoxyribonucleoside triphosphates; the proportion of mononucleotide (riboadenylate) primer increases upon decreasing the relative ribonucleoside triphosphate concentration. Mapping of the transition sites from RNA to DNA on HaeIII endonuclease fragments suggest they are distributed randomly and occur frequently on the phiX174 genome. These results suggest that the selection of RNA priming sites is affected by primase at the preferred sequence 3'-T-pyrimidine nucleotide-5' on the template within the DNA domain generated by the dnaB protein. These properties of RNA priming have important implications for site selection by the primosome on the lagging strand at the replication fork of the E. coli chromosome. PMID:6195163

Ogawa, T; Arai, K; Okazaki, T

1983-11-10

321

Mass Isotopomer Analysis of Nucleosides Isolated from RNA and DNA Using GC/MS.  

PubMed

Nucleosides are biosynthesized from metabolites that are at key nodes of intermediary metabolism. Therefore, (13)C labeling patterns in nucleosides from ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) in suitably designed isotopic tracer studies provide information on metabolic flux distributions of proliferating cells. Here, we present a gas chromatography (GC)-mass spectrometry (MS)-based approach that permits one to exploit that potential. In order to elucidate positional isotopomers of nucleosides from RNA and DNA, we screened the fragmentation spectra of their trimethylsilyl derivatives. We identified the molecular ion moieties retained in the respective fragment ions, focusing particularly on the carbon backbone. Nucleosides fragmented at the N-glycosidic bond provide nucleobase and/or ribose or 2'-deoxyribose fragment ions and fragments thereof. Nucleoside fragments composed of the nucleobase plus some carbons of the ribose ring were also observed. In total, we unequivocally assigned 31 fragments. The mass-isotopic distribution of the assigned fragments provides valuable information for later (13)C metabolic flux analysis as indicated by a labeling experiment applying [1-(13)C]glucose in a yeast culture. PMID:25458249

Miranda-Santos, Ines; Gramacho, Silvia; Pineiro, Marta; Martinez-Gomez, Karla; Fritz, Michel; Hollemeyer, Klaus; Salvador, Armindo; Heinzle, Elmar

2015-01-01

322

Integrative DNA, RNA, and Protein Evidence Connects TREML4 to Coronary Artery Calcification  

PubMed Central

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.

2014-01-01

323

Automatic on-chip RNA-DNA hybridization assay with integrated phase change microvalves  

NASA Astrophysics Data System (ADS)

An RNA-DNA hybridization assay microfluidic chip integrated with electrothermally actuated phase change microvalves for detecting pathogenic bacteria is presented in this paper. In order to realize the sequential loading and washing processes required in such an assay, gravity-based pressure-driven flow and phase-change microvalves were used in the microfluidic chip. Paraffin wax was used as the phase change material in the valves and thin film heaters were used to electrothermally actuate microvalves. Light absorption measured by a photodetector to determine the concentrations of the samples. The automatic control of the complete assay was implemented by a self-coded LabVIEW program. To examine the performance of this chip, Salmonella was used as a sample pathogen. Significantly, reduction in reagent/sample consumption (up to 20 folds) was achieved by this on-chip assay, compared with using the commercial test kit following the same protocol in conventional labs. The experimental results show that the quantitative detection can be obtained in approximately 26 min, and the detection limit is as low as 103 CFU ml-1. This RNA-DNA hybridization assay microfluidic chip shows an excellent potential in the development of a portable device for point-of-testing applications.

Weng, Xuan; Jiang, Hai; Wang, Junsheng; Chen, Shu; Cao, Honghe; Li, Dongqing

2012-07-01

324

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

325

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

326

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

327

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

328

Absolute molecular flux and angular distribution measurements to characterize DNA/RNA vapor jets  

NASA Astrophysics Data System (ADS)

Vapor jets of DNA and RNA bases (adenine, cytosine, thymine, and uracil) from an oven with a capillary exit have been studied in the intermediate regime between molecular and viscous flow corresponding to Knudsen numbers in the range 0.1 < K n < 10. The temperature control method ensured stationary flow. Assuming the Knudsen hypothesis, the pressure of sublimated molecules in the oven was determined as a function of temperature and the transmission probability of the capillary (Clausing factor). Thus it was possible to relate the oven temperature and pressure to the total flux through the capillary, determined by measuring the total mass of DNA/RNA base molecules condensed on a cold surface intersecting the jet. The angular distribution of molecules in the jet has been also studied experimentally using an optical interference method. The measured profiles are in good agreement with Troïtskii's [Sov. Phys. JETP 7 (1962) 353] analytical law for (cos ?) 3/2 angular dependence in the intermediate regime with error functions associated with the mean free path between intermolecular collisions.

Tabet, J.; Eden, S.; Feil, S.; Abdoul-Carime, H.; Farizon, B.; Farizon, M.; Ouaskit, S.; Märk, T. D.

2010-08-01

329

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

330

DNA/RNA binding and anticancer/antimicrobial activities of polymer-copper(II) complexes  

NASA Astrophysics Data System (ADS)

Water soluble polymer-copper(II) complexes with various degrees of coordination in the polymer chain were synthesized and characterized by elemental analysis, IR, UV-visible and EPR spectra. The DNA/RNA binding behavior of these polymer-copper(II) complexes was examined by UV-visible absorption, emission and circular dichroism spectroscopic methods, and cyclic voltammetry techniques. The binding of the polymer-copper(II) complexes with DNA/RNA was mainly through intercalation but some amount of electrostatic interaction was also observed. This binding capacity increased with the degree of coordination of the complexes. The polymer-copper(II) complex having the highest degree of coordination was subjected to analysis of cytotoxic and antimicrobial properties. The cytotoxicity study indicated that the polymer-copper(II) complexes affected the viability of MCF-7 mammary carcinoma cells, and the cells responded to the treatment with mostly through apoptosis although a few cells succumbed to necrosis. The antimicrobial screening showed activity against some human pathogens.

Lakshmipraba, Jagadeesan; Arunachalam, Sankaralingam; Riyasdeen, Anvarbatcha; Dhivya, Rajakumar; Vignesh, Sivanandham; Akbarsha, Mohammad Abdulkader; James, Rathinam Arthur

2013-05-01

331

Adleman DNA ([14], [15])  

E-print Network

­ 1 Adleman DNA " " PCR PCR DNA DNA RNA " " " " 1 #12;PCR DNA 2 1. ([10]) DNA DNA DNA RNA RNA GFP RNA RNA RNA RNA GFP 2. DNA S S+ ([14], [15]) S+ ([17]) n (m ) S+ O(m6 n6 ) 2 #12;3. Abstract Rewriting System on Multisets, ARMS P53 DNA P53 4 " ()self-reinforcement reaction)" ARMS P Systems P Systems 4

Hagiya, Masami

332

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples  

PubMed Central

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios < 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios > 1.53. However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at ?280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 < ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1). PMID:23407542

Patterson, Jennifer; Mura, Cameron

2013-01-01

333

Polyamidoamine Dendrimer Conjugates with Cyclodextrins as Novel Carriers for DNA, shRNA and siRNA  

PubMed Central

Gene, short hairpin RNA (shRNA) and small interfering RNA (siRNA) delivery can be particularly used for the treatment of diseases by the entry of genetic materials mammalian cells either to express new proteins or to suppress the expression of proteins, respectively. Polyamidoamine (PAMAM) StarburstTM dendrimers are used as non-viral vectors (carriers) for gene, shRNA and siRNA delivery. Recently, multifunctional PAMAM dendrimers can be used for the wide range of biomedical applications including intracellular delivery of genes and nucleic acid drugs. In this context, this review paper provides the recent findings on PAMAM dendrimer conjugates with cyclodextrins (CyDs) for gene, shRNA and siRNA delivery. PMID:24300184

Arima, Hidetoshi; Motoyama, Keiichi; Higashi, Taishi

2012-01-01

334

Catalytic Activity of a Binary Informational Macromolecule  

NASA Technical Reports Server (NTRS)

RNA molecules are thought to have played a prominent role in the early history of life on Earth based on their ability both to encode genetic information and to exhibit catalytic function. The modern genetic alphabet relies on two sets of complementary base pairs to store genetic information. However, due to the chemical instability of cytosine, which readily deaminates to uracil, a primitive genetic system composed of the bases A, U, G and C may have been difficult to establish. It has been suggested that the first genetic material instead contained only a single base-pairing unie'. Here we show that binary informational macromolecules, containing only two different nucleotide subunits, can act as catalysts. In vitro evolution was used to obtain ligase ribozymes composed of only 2,6-diaminopurine and uracil nucleotides, which catalyze the template-directed joining of two RNA molecules, one bearing a 5'-triphosphate and the other a 3'-hydroxyl. The active conformation of the fastest isolated ribozyme had a catalytic rate that was about 36,000-fold faster than the uncatalyzed rate of reaction. This ribozyme is specific for the formation of biologically relevant 3',5'-phosphodiester linkages.

Reader, John S.; Joyce, Gerald F.

2003-01-01

335

Rapid and Sensitive Colorimetric Method for Visualizing Biotin-Labeled DNA Probes Hybridized to DNA or RNA Immobilized on Nitrocellulose: Bio-Blots  

Microsoft Academic Search

Biotin-labeled DNA probes, prepared by nicktranslation in the presence of biotinylated analogs of TTP, are hybridized to DNA or RNA immobilized on nitrocellulose filters. After removal of residual probe, the filters are incubated for 2--5 min with a preformed complex made with avidin-DH (or streptavidin) and biotinylated polymers of intestinal alkaline phosphatase. The filters are then incubated with a mixture

Jeffry J. Leary; David J. Brigati; David C. Ward

1983-01-01

336

DNA/RNA Helicase Gene Mutations in a Form of Juvenile Amyotrophic Lateral Sclerosis (ALS4)  

PubMed Central

Juvenile amyotrophic lateral sclerosis (ALS4) is a rare autosomal dominant form of juvenile amyotrophic lateral sclerosis (ALS) characterized by distal muscle weakness and atrophy, normal sensation, and pyramidal signs. Individuals affected with ALS4 usually have an onset of symptoms at age <25 years, a slow rate of progression, and a normal life span. The ALS4 locus maps to a 1.7-Mb interval on chromosome 9q34 flanked by D9S64 and D9S1198. To identify the molecular basis of ALS4, we tested 19 genes within the ALS4 interval and detected missense mutations (T3I, L389S, and R2136H) in the Senataxin gene (SETX). The SETX gene encodes a novel 302.8-kD protein. Although its function remains unknown, SETX contains a DNA/RNA helicase domain with strong homology to human RENT1 and IGHMBP2, two genes encoding proteins known to have roles in RNA processing. These observations of ALS4 suggest that mutations in SETX may cause neuronal degeneration through dysfunction of the helicase activity or other steps in RNA processing. PMID:15106121

Chen, Ying-Zhang; Bennett, Craig L.; Huynh, Huy M.; Blair, Ian P.; Puls, Imke; Irobi, Joy; Dierick, Ines; Abel, Annette; Kennerson, Marina L.; Rabin, Bruce A.; Nicholson, Garth A.; Auer-Grumbach, Michaela; Wagner, Klaus; De Jonghe, Peter; Griffin, John W.; Fischbeck, Kenneth H.; Timmerman, Vincent; Cornblath, David R.; Chance, Phillip F.

2004-01-01

337

DNA/RNA helicase gene mutations in a form of juvenile amyotrophic lateral sclerosis (ALS4).  

PubMed

Juvenile amyotrophic lateral sclerosis (ALS4) is a rare autosomal dominant form of juvenile amyotrophic lateral sclerosis (ALS) characterized by distal muscle weakness and atrophy, normal sensation, and pyramidal signs. Individuals affected with ALS4 usually have an onset of symptoms at age <25 years, a slow rate of progression, and a normal life span. The ALS4 locus maps to a 1.7-Mb interval on chromosome 9q34 flanked by D9S64 and D9S1198. To identify the molecular basis of ALS4, we tested 19 genes within the ALS4 interval and detected missense mutations (T3I, L389S, and R2136H) in the Senataxin gene (SETX). The SETX gene encodes a novel 302.8-kD protein. Although its function remains unknown, SETX contains a DNA/RNA helicase domain with strong homology to human RENT1 and IGHMBP2, two genes encoding proteins known to have roles in RNA processing. These observations of ALS4 suggest that mutations in SETX may cause neuronal degeneration through dysfunction of the helicase activity or other steps in RNA processing. PMID:15106121

Chen, Ying-Zhang; Bennett, Craig L; Huynh, Huy M; Blair, Ian P; Puls, Imke; Irobi, Joy; Dierick, Ines; Abel, Annette; Kennerson, Marina L; Rabin, Bruce A; Nicholson, Garth A; Auer-Grumbach, Michaela; Wagner, Klaus; De Jonghe, Peter; Griffin, John W; Fischbeck, Kenneth H; Timmerman, Vincent; Cornblath, David R; Chance, Phillip F

2004-06-01

338

Bacteriophage N4 virion RNA polymerase interaction with its promoter DNA hairpin  

PubMed Central

Bacteriophage N4 minivirion RNA polymerase (mini-vRNAP), the RNA polymerase (RNAP) domain of vRNAP, is a member of the T7-like RNAP family. Mini-vRNAP recognizes promoters that comprise conserved sequences and a 3-base loop–5-base pair (bp) stem DNA hairpin structure on single-stranded templates. Here, we defined the DNA structural and sequence requirements for mini-vRNAP promoter recognition. Mini-vRNAP binds a 20-nucleotide (nt) N4 P2 promoter deoxyoligonucleotide with high affinity (Kd = 2 nM) to form a salt-resistant complex. We show that mini-vRNAP interacts specifically with the central base of the hairpin loop (?11G) and a base at the stem (?8G) and that the guanine 6-keto and 7-imino groups at both positions are essential for binding and complex salt resistance. The major determinant (?11G), which must be presented to mini-vRNAP in the context of a hairpin loop, appears to interact with mini-vRNAP Trp-129. This interaction requires template single-strandedness at positions ?2 and ?1. Contacts with the promoter are disrupted when the RNA product becomes 11–12 nt long. This detailed description of vRNAP interaction with its promoter hairpin provides insights into RNAP–promoter interactions and explains how the injected vRNAP, which is present in one or two copies, recognizes its promoters on a single copy of the injected genome. PMID:17438270

Davydova, Elena K.; Santangelo, Thomas J.; Rothman-Denes, Lucia B.

2007-01-01

339

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

340

Structural biology. Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target.  

PubMed

In prokaryotes, RNA derived from type I and type III CRISPR loci direct large ribonucleoprotein complexes to destroy invading bacteriophage and plasmids. In Escherichia coli, this 405-kilodalton complex is called Cascade. We report the crystal structure of Cascade bound to a single-stranded DNA (ssDNA) target at a resolution of 3.03 angstroms. The structure reveals that the CRISPR RNA and target strands do not form a double helix but instead adopt an underwound ribbon-like structure. This noncanonical structure is facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid and is stabilized by the highly interlocked organization of protein subunits. These studies provide insight into both the assembly and the activity of this complex and suggest a mechanism to enforce fidelity of target binding. PMID:25123481

Mulepati, Sabin; Héroux, Annie; Bailey, Scott

2014-09-19

341

16S rRNA sequences of uncultivated hot spring cyanobacterial mat inhabitants retrieved as randomly primed cDNA  

SciTech Connect

Cloning and analysis of cDNAs synthesized from rRNAs is one approach to assess the species composition of natural microbial communities. In some earlier attempts to synthesize cDNA from 16S rRNA (16S rcDNA) from the Octopus Spring cyanobacterial mat, a dominance of short 16S rcDNAs was observed, which appear to have originated only from certain organisms. Priming of cDNA synthesis from small ribosomal subunit RNA with random deoxyhexanucleotides can retrieve longer sequences, more suitable for phylogenetic analysis. Here we report the retrieval of 16S rRNA sequences form three formerly uncultured community members. One sequence type, which was retrieved three times from a total of five sequences analyzed, can be placed in the cyanobacterial phylum. A second sequence type is related to 16S rRNAs from green nonsulfur bacteria. The third sequence type may represent a novel phylogenetic type.

Weller, R.; Ward, D.M. (Montana State Univ., Bozeman (United States)); Weller, J.W. (Univ. of Montana, Missoula (United States))

1991-04-01

342

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; Buée, Luc; Galas, Marie-Christine

2014-01-01

343

Construction and identification of a cDNA clone for human type II procollagen mRNA.  

PubMed Central

Double-stranded cDNA was constructed for poly(A)-containing RNA isolated from foetal human articular cartilage known to contain small amounts of pro alpha 1 (II) collagen mRNA. A 585 base pair PstI-EcoRI cDNA fragment was isolated and cloned into plasmid pBR322. A resulting recombinant plasmid pHCAR1 was shown to hybridize specifically to a 5.4 kilobase mRNA in cartilage but not in calvarial RNA. Definite identification of clone pHCAR1 was based on sequence analysis; marked homology with the corresponding chick gene and complete agreement with the human gene sequences available were observed. Images Fig. 1. PMID:3840017

Elima, K; Mäkelä, J K; Vuorio, T; Kauppinen, S; Knowles, J; Vuorio, E

1985-01-01

344

Characterization of cDNA encoding the human tRNA-guanine transglycosylase (TGT) catalytic subunit.  

PubMed

Queuosine (Q) is a 7-deazaguanosine found in the first position of the anticodon of tRNAs that recognize NAU and NAC codons (Tyr, Asn, Asp and His). Eukaryotes synthesize Q by the base-for-base exchange of queuine (Q base) for guanine in the unmodified tRNA, a reaction catalyzed by TGT. A search of the human EST database for sequences with significant homology to the well studied TGT from Escherichia coli identified several candidates for full-length (1.3-1.4 kb) cDNA clones. Three candidate cDNA clones, available from IMAGE Consortium, LLNL, (Lennon et al., 1996, Genomics 33, 151-152) were obtained: IMAGE Clone Id Nos. 611146, 1422928, and 72154. Here we report the complete sequences of these clones. IMAGE:72154 contains an ORF encoding a 44 kDa polypeptide with high homology to bacterial TGTs and was subcloned into the mammalian expression vector pMAMneo-Cat. When this construct was transfected into the TGT-negative cell line, GC(3)/c1 (Gündüz et al., 1992, Biochim. Biophys. Acta 1139, 229-238), it restored the ability of the cells to form Q-containing tRNA. This TGT cDNA sequence is encoded in human chromosome 19 clone CTC-539A10 (GenBank accession no. AC011475), enabling determination of the exon-intron boundaries for the TGT gene. The sequence of IMAGE:611146 is 5'-truncated by 76 bp compared to that from IMAGE:72154 and, except for two differences in the 3'-non-coding region, the remainder of the sequence is identical to that of IMAGE:72154. IMAGE:1422928 is a 1390 bp chimera: the 5'-portion, bp 1-708, is identical to a genomic DNA sequence from chromosome 15 (GenBank accession no. AC067805, bp 148976-149683); the 3'-end, bp 726-1390, is identical to the 3'-end of the TGT cDNA sequence from IMAGE:611146. PMID:11255023

Deshpande, K L; Katze, J R

2001-03-01

345

Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions.  

PubMed

In human cells, the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5') next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5'-templating base, indicating that it derives from nontemplated synthesis according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. Thus, the translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, translesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions. PMID:25605892

Walmacq, Celine; Wang, Lanfeng; Chong, Jenny; Scibelli, Kathleen; Lubkowska, Lucyna; Gnatt, Averell; Brooks, Philip J; Wang, Dong; Kashlev, Mikhail

2015-02-01

346

Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions  

PubMed Central

In human cells, the oxidative DNA lesion 8,5?-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5?) next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5?-templating base, indicating that it derives from nontemplated synthesis according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. Thus, the translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, translesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions. PMID:25605892

Walmacq, Celine; Wang, Lanfeng; Chong, Jenny; Scibelli, Kathleen; Lubkowska, Lucyna; Gnatt, Averell; Brooks, Philip J.; Wang, Dong; Kashlev, Mikhail

2015-01-01

347

Repressor activity of the RpoS/?S-dependent RNA polymerase requires DNA binding.  

PubMed

The RpoS/?(S) sigma subunit of RNA polymerase (RNAP) activates transcription of stationary phase genes in many Gram-negative bacteria and controls adaptive functions, including stress resistance, biofilm formation and virulence. In this study, we address an important but poorly understood aspect of ?(S)-dependent control, that of a repressor. Negative regulation by ?(S) has been proposed to result largely from competition between ?(S) and other ? factors for binding to a limited amount of core RNAP (E). To assess whether ?(S) binding to E alone results in significant downregulation of gene expression by other ? factors, we characterized an rpoS mutant of Salmonella enterica serovar Typhimurium producing a ?(S) protein proficient for E?(S) complex formation but deficient in promoter DNA binding. Genome expression profiling and physiological assays revealed that this mutant was defective for negative regulation, indicating that gene repression by ?(S) requires its binding to DNA. Although the mechanisms of repression by ?(S) are likely specific to individual genes and environmental conditions, the study of transcription downregulation of the succinate dehydrogenase operon suggests that ? competition at the promoter DNA level plays an important role in gene repression by E?(S). PMID:25578965

Lévi-Meyrueis, Corinne; Monteil, Véronique; Sismeiro, Odile; Dillies, Marie-Agnès; Kolb, Annie; Monot, Marc; Dupuy, Bruno; Duarte, Sara Serradas; Jagla, Bernd; Coppée, Jean-Yves; Beraud, Mélanie; Norel, Françoise

2015-02-18

348

Structural Basis of the RNase H1 Activity on Stereo Regular Borano Phosphonate DNA/RNA Hybrids  

PubMed Central

Numerous DNA chemistries have been explored to improve oligodeoxynucleotide (ODN) based RNA targeting. The majority of the modifications render the ODN/RNA target insensitive to RNase H1. Borano phosphonate ODN’s are among the few modifications that are tolerated by RNase H1. To understand the effect of the stereochemistry of the BH3 modification on the nucleic acid structure and RNase H1 enzyme activity we have investigated two DNA/RNA hybrids containing either a RP or SP BH3 modification by NMR spectroscopy. TM studies show that the stability of either RP or SP modified DNA/RNA hybrids are essentially identical (313.8 K) and similar to an unmodified control (312.9 K). The similarity is also reflected in the imino proton spectra. In order to characterize such similar structures, a large number of NMR restraints (including dipolar couplings and backbone torsion angles) were used to determine structural features important for RNase H1 activity. The final NMR structures exhibit excellent agreement with the data (total RX values < 6.0) with helical properties between those of an A and B helix. Subtle backbone variations are observed in the DNA near the modification, while the RNA strands are relatively unperturbed. In case of the SP modification, for which more perturbations are recorded, a slightly narrower minor groove is also obtained. Unique NOE base contacts localize the SP -BH3 group in the major groove while the RP -BH3 group points away from the DNA. However, this creates a potential clash of the RP -BH3 groups with important RNase H1 residues in a complex while the SP -BH3 groups could be tolerated. We therefore predict that based on our NMR structures a fully RP BH3 DNA/RNA hybrid would not be a substrate for RNase H1. PMID:21443203

Johnson, Christopher N; Spring, Alexander M.; Shaw, Barbara R.; Germann, Markus W.

2011-01-01

349

DNA and RNA virus species are inhibited by xanthates, a class of antiviral compounds with unique properties.  

PubMed Central

Various DNA and RNA virus species are inhibited by xanthate compounds at concentrations that leave the mitotic activity of uninfected cells unimpaired. The concentration of tricyclodecan -9-yl- xanthogenate that reduces the yield of herpes simplex virus types 1 and 2 by 50% is between 4.5 and 33 microM. The replication of DNA viruses such as simian virus 40 can be blocked at the DNA and RNA level both early and late after infection. The xanthates are not incorporated into nucleic acids. Episomal bovine papilloma virus DNA replication and transcription are also inhibited in transformed cells. The treated cells revert to the normal phenotype by acquisition of contact inhibition and a flat morphology. Images PMID:6328507

Sauer, G; Amtmann, E; Melber, K; Knapp, A; Müller, K; Hummel, K; Scherm, A

1984-01-01

350

Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells.  

PubMed

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) is a well-known marker of oxidative stress. We report a mechanistic analysis of several pathways by which 8-oxodG is converted to nucleotide triphosphates and incorporated into both DNA and RNA. Exposure of MCF-7 cells to [(14)C]8-oxodG combined with specific inhibitors of several nucleotide salvage enzymes followed with accelerator mass spectrometry provided precise quantitation of the resulting radiocarbon-labeled species. Concentrations of exogenously dosed nucleobase in RNA reached one per 10(6) nucleotides, 5-6-fold higher than the maximum observed in DNA. Radiocarbon incorporation into DNA and RNA was abrogated by Immucillin H, an inhibitor of human purine nucleoside phosphorylase (PNP). Inhibition of ribonucleotide reductase (RR) decreased the radiocarbon content of the DNA, but not in RNA, indicating an important role for RR in the formation of 8-oxodG-derived deoxyribonucleotides. Inhibition of deoxycytidine kinase had little effect on radiocarbon incorporation in DNA, which is in contrast to the known ability of mammalian cells to phosphorylate dG. Our data indicate that PNP and RR enable nucleotide salvage of 8-oxodG in MCF-7 cells, a previously unrecognized mechanism that may contribute to mutagenesis and carcinogenesis. PMID:18025045

Mundt, Janna M; Hah, Sang Soo; Sumbad, Rhoda A; Schramm, Vern; Henderson, Paul T

2008-01-01

351

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

352

Measurement of human prolactin messenger RNA in decidual tissues using complementary DNA probe cloned in M13mp9 bacteriophage.  

PubMed

A human prolactin (PRL) cDNA clone was digested with restriction enzyme Pst I and the resultant fragments were cloned into bacteriophage M13mp9. Single stranded recombinant DNAs having a coding strand of the PRL cDNA were selected by hybridization with 125I-labeled PRL mRNA obtained from human prolactinoma tissue. One of the single stranded recombinant DNAs was purified by agarose gel electrophoresis and labeled with 125I to a specific activity of 1.4 X 10(8) cpm per microgram of DNA. The probe could be successfully used in RNA dot hybridization. Analysis of poly (A) RNAs from prolactinoma, liver and placental tissues revealed that this probe was specific to PRL mRNA sequence. Hybridization of poly (A) RNA from decidual tissue to this probe revealed the presence of PRL mRNA sequence. However, PRL mRNA in decidua was at least 20,000 times less than that in pituitary prolactinoma. PMID:3007638

Suganuma, N; Seo, H; Narita, O; Tomoda, Y; Matsui, N

1986-02-01

353

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.

354

A light-activated metal complex targets both DNA and RNA in a fluorescent in vitro transcription and translation assay.  

PubMed

A coupled in vitro transcription and translation (IVTT) assay that uses GFP as a fluorescent reporter allowed the potency of a light-activated cytotoxic ruthenium agent to be quantified. The compound inhibits the function of both DNA and mRNA only upon light activation. The IVTT functional assay provides estimates of potency that are consistent with cellular cytotoxicity values, in marked contrast to the values obtained from traditional DNA-damage assays. PMID:24482049

Heidary, David K; Glazer, Edith C

2014-03-01

355

Following the Assembly of RNA Polymerase-DNA Complexes in Aqueous Solutions with the Scanning Force Microscope  

Microsoft Academic Search

The capability of the scanning force microscope (SFM) to image molecules in aqueous buffers has opened the exciting possibility of following processes of molecular assembly in real time and in near-physiological environments. This capability is demonstrated in this paper by following the assembly process of RNA polymerase-DNA complexes. DNA fragments deposited on mica and imaged in Hepes\\/MgCl_2 are shown before

Martin Guthold; Magdalena Bezanilla; Dorothy A. Erie; Bethany Jenkins; Helen G. Hansma; Carlos Bustamante

1994-01-01

356

Rabies RNA synthesis, detected with cDNA probes, as a marker for virus transport in the rat nervous system.  

PubMed

The kinetics of viral RNA synthesis in different parts of the rat brain, infected with fixed or street rabies virus strains, is correlated with their anatomical neuronal connections with the masseter muscles, using hybridization with rabies cDNA probes. Viral RNA synthesis is first detected in the brain-stem and in the pons where the direct anatomical projection of the masseter muscle nervous arborization into the sensory and motor nuclei is located, through the trigeminus nerve. Rabies RNA detection is delayed in the other regions of the rat brain depending on the time course of virus transport from the trigeminal nuclei through multiple nervous connections. PMID:7681151

Ermine, A; Ceccaldi, P E; Masson, G; Tsiang, H

1993-02-01

357

Galloflavin prevents the binding of lactate dehydrogenase A to single stranded DNA and inhibits RNA synthesis in cultured cells.  

PubMed

Lactate dehydrogenase A (LDH-A) binds single stranded DNA (ssDNA) and stimulates cell transcription. Binding is prevented by NADH, suggesting that the coenzyme site is involved in the interaction LDH-A/ssDNA. We recently identified an inhibitor of LDH-A enzymatic activity (Galloflavin, GF) which occupies the NADH site. In the experiments reported here we studied whether GF can also hinder the binding of LDH-A to ssDNA and investigated its effects on RNA synthesis in cultured cells. Using a filter binding assay we observed that 4 ?M GF inhibited the binding of human LDH-A to a single stranded [(3)H]DNA sample by 50%. After only 0.5-1h, 50-100 ?M GF inhibited RNA synthesis in SW620 cells maintained in a medium in which galactose substituted glucose. In these culture conditions, SW620 cells did not produce lactic acid and effects caused by the inhibition of the enzymatic activity of LDH-A could be excluded. Novel LDH-A inhibitors which hinder aerobic glycolysis of cancer cells are at present actively searched. Our results suggest that: (i) inhibitors which bind the NADH site can exert their antiproliferative activity not only by blocking aerobic glycolysis but also by causing an inhibition of RNA synthesis independent from the effect on glycolysis; (ii) GF can be a useful tool to study the biological role of LDH-A binding to ssDNA. PMID:23237800

Fiume, Luigi; Vettraino, Marina; Carnicelli, Domenica; Arfilli, Valentina; Di Stefano, Giuseppina; Brigotti, Maurizio

2013-01-11

358

RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.  

PubMed

Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability. PMID:25527408

Manfrini, Nicola; Trovesi, Camilla; Wery, Maxime; Martina, Marina; Cesena, Daniele; Descrimes, Marc; Morillon, Antonin; d'Adda di Fagagna, Fabrizio; Longhese, Maria Pia

2015-02-01

359

HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double-stranded RNA  

PubMed Central

To analyze relationships between RNA signals, DNA methylation and chromatin modifications, we performed a genetic screen to recover Arabidopsis mutants defective in RNA-directed transcriptional silencing and methylation of a nopaline synthase promoter–neomycinphosphotransferase II (NOSpro– NPTII) target gene. Mutants were identified by screening for recovery of kanamycin resistance in the presence of an unlinked silencing complex encoding NOSpro double-stranded RNA. One mutant, rts1 (RNA-mediated transcriptional silencing), displayed moderate recovery of NPTII gene expression and partial loss of methylation in the target NOSpro, predominantly at symmetrical C(N)Gs. The RTS1 gene was isolated by positional cloning and found to encode a putative histone deacetylase, HDA6. The more substantial decrease in methylation of symmetrical compared with asymmetrical cytosines in rts1 mutants suggests that HDA6 is dispensable for RNA-directed de novo methylation, which results in intermediate methylation of cytosines in all sequence contexts, but is necessary for reinforcing primarily C(N)G methylation induced by RNA. Because CG methylation in centromeric and rDNA repeats was not reduced in rts1 mutants, HDA6 might be specialized for the RNA- directed pathway of genome modification. PMID:12486004

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

2002-01-01

360

Formation of DNA:RNA Hybrid G-Quadruplex in Bacterial Cells and Its Dominance over the Intramolecular DNA G-Quadruplex in Mediating Transcription Termination.  

PubMed

DNA with four guanine tracts can fold into G-quadruplexes that are targets of transcription regulation. We recently found that hybrid DNA:RNA G-quadruplexes (HQs) can form during in?vitro transcription. However, it is unclear whether they can form in cells. Evidence is presented supporting their formation in plasmids in bacterial cells. The formation of the HQs is indicated by a unique pattern of prematurely terminated transcripts under two conditions where the RNA transcripts do or do not participate in G-quadruplex assembly and further supported by a number of chemical and biochemical analysis. HQs dominate over the intramolecular DNA G-quadruplexes (DQ) in mediating the transcription termination when both structures are able to form. These findings provide the first evidence of HQ formation in cells and suggest that the competition/conversion between HQ and DQ may regulate transcription and serve as drug target in pharmaceutical applications. PMID:25613367

Wu, Ren-Yi; Zheng, Ke-Wei; Zhang, Jia-Yu; Hao, Yu-Hua; Tan, Zheng

2015-02-16

361

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

362

Structure and function of dioxygenases in histone demethylation and DNA/RNA demethylation  

PubMed Central

Iron(II) and 2-oxoglutarate (2OG)-dependent dioxygenases involved in histone and DNA/RNA demethylation convert the cosubstrate 2OG and oxygen to succinate and carbon dioxide, resulting in hydroxylation of the methyl group of the substrates and subsequent demethylation. Recent evidence has shown that these 2OG dioxygenases play vital roles in a variety of biological processes, including transcriptional regulation and gene expression. In this review, the structure and function of these dioxygenases in histone and nucleic acid demethylation will be discussed. Given the important roles of these 2OG dioxygenases, detailed analysis and comparison of the 2OG dioxygenases will guide the design of target-specific small-molecule chemical probes and inhibitors. PMID:25485134

Dong, Cheng; Zhang, Heng; Xu, Chao; Arrowsmith, Cheryl H.; Min, Jinrong

2014-01-01

363

A Sequence-Specific Interaction between the Saccharomyces cerevisiae rRNA Gene Repeats and a Locus Encoding an RNA Polymerase I Subunit Affects Ribosomal DNA Stability.  

PubMed

The spatial organization of eukaryotic genomes is linked to their functions. However, how individual features of the global spatial structure contribute to nuclear function remains largely unknown. We previously identified a high-frequency interchromosomal interaction within the Saccharomyces cerevisiae genome that occurs between the intergenic spacer of the ribosomal DNA (rDNA) repeats and the intergenic sequence between the locus encoding the second largest RNA polymerase I subunit and a lysine tRNA gene [i.e., RPA135-tK(CUU)P]. Here, we used quantitative chromosome conformation capture in combination with replacement mapping to identify a 75-bp sequence within the RPA135-tK(CUU)P intergenic region that is involved in the interaction. We demonstrate that the RPA135-IGS1 interaction is dependent on the rDNA copy number and the Msn2 protein. Surprisingly, we found that the interaction does not govern RPA135 transcription. Instead, replacement of a 605-bp region within the RPA135-tK(CUU)P intergenic region results in a reduction in the RPA135-IGS1 interaction level and fluctuations in rDNA copy number. We conclude that the chromosomal interaction that occurs between the RPA135-tK(CUU)P and rDNA IGS1 loci stabilizes rDNA repeat number and contributes to the maintenance of nucleolar stability. Our results provide evidence that the DNA loci involved in chromosomal interactions are composite elements, sections of which function in stabilizing the interaction or mediating a functional outcome. PMID:25421713

Cahyani, Inswasti; Cridge, Andrew G; Engelke, David R; Ganley, Austen R D; O'Sullivan, Justin M

2015-02-01

364

A Systematic Analysis on DNA Methylation and the Expression of Both mRNA and microRNA in Bladder Cancer  

PubMed Central

Background DNA methylation aberration and microRNA (miRNA) deregulation have been observed in many types of cancers. A systematic study of methylome and transcriptome in bladder urothelial carcinoma has never been reported. Methodology/Principal Findings The DNA methylation was profiled by modified methylation-specific digital karyotyping (MMSDK) and the expression of mRNAs and miRNAs was analyzed by digital gene expression (DGE) sequencing in tumors and matched normal adjacent tissues obtained from 9 bladder urothelial carcinoma patients. We found that a set of significantly enriched pathways disrupted in bladder urothelial carcinoma primarily related to “neurogenesis” and “cell differentiation” by integrated analysis of -omics data. Furthermore, we identified an intriguing collection of cancer-related genes that were deregulated at the levels of DNA methylation and mRNA expression, and we validated several of these genes (HIC1, SLIT2, RASAL1, and KRT17) by Bisulfite Sequencing PCR and Reverse Transcription qPCR in a panel of 33 bladder cancer samples. Conclusions/Significance We characterized the profiles between methylome and transcriptome in bladder urothelial carcinoma, identified a set of significantly enriched key pathways, and screened four aberrantly methylated and expressed genes. Conclusively, our findings shed light on a new avenue for basic bladder cancer research. PMID:22140553

Zhou, Liang; Chen, Jiahao; Luo, Huijuan; Sun, Jihua; Wu, Song; Han, Yonghua; Yin, Guangliang; Chen, Maoshan; Han, Zujing; Li, Xianxin; Huang, Yi; Zhang, Weixing; Zhou, Fangjian; Chen, Tong; Fa, Pingping; Wang, Yong; Sun, Liang; Leng, Huimin; Sun, Fenghao; Liu, Yuchen; Ye, Mingzhi; Yang, Huanming; Cai, Zhiming; Gui, Yaoting; Zhang, Xiuqing

2011-01-01

365

MicroRNA-182-5p targets a network of genes involved in DNA repair  

PubMed Central

MicroRNAs are noncoding regulators of gene expression, which act by repressing protein translation and/or degrading mRNA. Many have been shown to drive tumorigenesis in cancer, but functional studies to understand their mode of action are typically limited to single-target genes. In this study, we use synthetic biotinylated miRNA to pull down endogenous targets of miR-182-5p. We identified more than 1000 genes as potential targets of miR-182-5p, most of which have a known function in pathways underlying tumor biology. Specifically, functional enrichment analysis identified components of both the DNA damage response pathway and cell cycle to be highly represented in this target cohort. Experimental validation confirmed that miR-182-5p-mediated disruption of the homologous recombination (HR) pathway is a consequence of its ability to target multiple components in that pathway. Although there is a strong enrichment for the cell cycle ontology, we do not see primary proliferative defects as a consequence of miR-182-5p overexpression. We highlight targets that could be responsible for miR-182-5p-mediated disruption of other biological processes attributed in the literature so far. Finally, we show that miR-182-5p is highly expressed in a panel of human breast cancer samples, highlighting its role as a potential oncomir in breast cancer. PMID:23249749

Krishnan, Keerthana; Steptoe, Anita L.; Martin, Hilary C.; Wani, Shivangi; Nones, Katia; Waddell, Nic; Mariasegaram, Mythily; Simpson, Peter T.; Lakhani, Sunil R.; Gabrielli, Brian; Vlassov, Alexander; Cloonan, Nicole; Grimmond, Sean M.

2013-01-01

366

Transient expression of DNA and RNA in parasitic helminths by using particle bombardment.  

PubMed

Parasitic helminths (worms belonging to several metazoan phyla) cause considerable morbidity and mortality in humans. They are an important veterinary problem, and they result in significant economic losses in animal grazing and agriculture. Experimental studies on parasitic helminths have been limited by a lack of parasite cell lines and methods for molecular genetic analyses. We evaluated particle bombardment (biolistics) as a strategy to introduce and express nucleic acids in these multicellular parasites. By using embryos of the parasitic nematode Ascaris as a model, we developed methods to introduce and express both DNA and RNA during several stages of Ascaris embryogenesis. Biolistic transfection will facilitate experimental strategies in Ascaris embryos complementing other biochemical tools available (e.g., in vitro whole-cell embryo extracts for transcription, RNA processing, and translation). Transfection experiments with adult schistosomes further suggest that the biolistic strategy should be applicable to a variety of other parasitic helminths. The development of these methods provides molecular genetic tools to study gene expression and the biology of a variety of types and developmental stages of important helminth parasites. PMID:10411936

Davis, R E; Parra, A; LoVerde, P T; Ribeiro, E; Glorioso, G; Hodgson, S

1999-07-20

367

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

368

Impacts of copepods on marine seston, and resulting effects on Calanus finmarchicus RNA:DNA ratios in mesocosm experiments  

Microsoft Academic Search

We investigated the impact of copepods on the seston community in a mesocosm set-up, and assessed how the changes in food quantity, quality and size affected the condition of the grazers, by measuring the RNA:DNA ratios in different developmental stages of Calanus finmarchicus. Manipulated copepod densities did not affect the particulate carbon concentration in the mesocosms. On the other hand,

C. Becker; D. Brepohl; H. Feuchtmayr; E. Zöllner; F. Sommer; C. Clemmesen; U. Sommer; M. Boersma

2005-01-01

369

The day/night switch of the circadian clock of synechococcus elongatus and hydrogen bonds of dna and rna  

E-print Network

are the same for isolated A:U and A:T base pairs. Replacing uridine residues in RNA with 5-methyl uridine and substituting deoxythymidines in DNA with deoxyuridines do not statistically shift empirical 2h?13C2 values. Thus, we show experimentally...

Kim, Yong-Ick

2009-05-15

370

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; Uhlén, M

1994-01-01

371

Coilin participates in the suppression of RNA polymerase I in response to cisplatin-induced DNA damage  

Microsoft Academic Search

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. Knock- down of endogenous coilin partially overrides the Pol I transcriptional

Andrew S. Gilder; Phi M. Do; Zunamys I Carrero; Angela M. Cosman; Hanna J. Broome; Venkatramreddy Velma; Luis A. Martinez; Michael D. Hebert; Karsten Weis

2011-01-01

372

Levels and size complexity of DNA polymerase beta mRNA in rat regenerating liver and other organs.  

PubMed

A cDNA probe encoding DNA polymerase beta (beta-pol) was used to study the level and size complexity of beta-pol mRNA in regenerating rat liver and other rat tissues. An almost 2-fold increase in beta-pol mRNA was observed 18-24 h after partial hepatectomy. In most adult rat tissues (liver, heart, kidney, stomach, spleen, thymus, lung and brain) the abundance of beta-pol mRNA was low. In contrast, young brain and testes exhibited beta-pol mRNA levels 5- and 15-times higher, respectively. The observed changes in the level of beta-pol mRNA in regenerating rat liver and in developing brain are correlated with reported changes in DNA polymerase beta activity. Four different (4.0, 2.5, 2.2, 1.4 kb) transcripts hybridizing to beta-pol probe were found in all tissues examined. The 4.0 kb transcript was dominant for young and adult brain, whereas the 1.4 kb transcript was dominant for testes. The significance of these transcripts is discussed. PMID:2736248

Nowak, R; Siedlecki, J A; Kaczmarek, L; Zmudzka, B Z; Wilson, S H

1989-07-01

373

MACROMOLECULES FACILITATE THE TRANSPORT OF TRACE ORGANICS  

EPA Science Inventory

Macromolecules in the pore fluid of a soil may influence the mobility of hydrophobic compounds by their partitioning to the macromolecule, which moves with, or even faster than, the water. The mobility is described mathematically by a chemical transport model. The significance of...

374

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

375

Missing genes, multiple ORFs, and C-to-U type RNA editing in Acrasis kona (Heterolobosea, Excavata) mitochondrial DNA.  

PubMed

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

2014-09-01

376

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

377

Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing  

PubMed Central

Background and objective The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction procedures is important when comparing studies that use different methods. Identifying the method that best portrays communities is also desirable. Accordingly, the aim of this study was to evaluate bias introduced by different DNA extraction procedures on oral microbiome profiles. Design Four DNA extraction methods were tested on mock communities consisting of seven representative oral bacteria. Additionally, supragingival plaque samples were collected from seven individuals and divided equally to test two commonly used DNA extraction procedures. Amplicon libraries of the 16S rRNA gene were generated and sequenced via 454-pyrosequencing. Results Evaluation of mock communities revealed that DNA yield and bacterial species representation varied with DNA extraction methods. Despite producing the lowest yield of DNA, a method that included bead beating was the only protocol capable of detecting all seven species in the mock community. Comparison of the performance of two commonly used methods (crude lysis and a chemical/enzymatic lysis+column-based DNA isolation) on plaque samples showed no effect of extraction protocols on taxa prevalence but global community structure and relative abundance of individual taxa were affected. At the phylum level, the latter method improved the recovery of Actinobacteria, Bacteroidetes, and Spirochaetes over crude lysis. Conclusion DNA extraction distorts microbial profiles in simulated and clinical oral samples, reinforcing the importance of careful selection of a DNA extraction protocol to improve species recovery and facilitate data comparison across oral microbiology studies. PMID:24778776

Abusleme, Loreto; Hong, Bo-Young; Dupuy, Amanda K.; Strausbaugh, Linda D.; Diaz, Patricia I.

2014-01-01

378

Improving the specificity and efficacy of CRISPR/CAS9 and gRNA through target specific DNA reporter.  

PubMed

Genomic engineering by the guide RNA (gRNA)-directed CRISPR/CAS9 is rapidly becoming a method of choice for various biological systems. However, pressing concerns remain regarding its off-target activities and wide variations in efficacies. While next generation sequencing (NGS) has been primarily used to evaluate the efficacies and off-target activities of gRNAs, it only detects the imperfectly repaired double strand DNA breaks (DSB) by the error-prone non-homologous end joining (NHEJ) mechanism and may not faithfully represent the DSB activities because the efficiency of NHEJ-mediated repair varies depending on the local chromatin environment. Here we describe a reporter system for unbiased detection and comparison of DSB activities that promises to improve the chance of success in genomic engineering and to facilitate large-scale screening of CAS9 activities and gRNA libraries. Additionally, we demonstrated that the tolerances to mismatches between a gRNA and the corresponding target DNA can occur at any position of the gRNA, and depend on both specific gRNA sequences and CAS9 constructs used. PMID:25193712

Zhang, Jian-Hua; Pandey, Mritunjay; Kahler, John F; Loshakov, Anna; Harris, Benjamin; Dagur, Pradeep K; Mo, Yin-Yuan; Simonds, William F

2014-11-10

379

Interferon Antagonist NSs of La Crosse Virus Triggers a DNA Damage Response-like Degradation of Transcribing RNA Polymerase II*  

PubMed Central

La Crosse encephalitis virus (LACV) is a mosquito-borne member of the negative-strand RNA virus family Bunyaviridae. We have previously shown that the virulence factor NSs of LACV is an efficient inhibitor of the antiviral type I interferon system. A recombinant virus unable to express NSs (rLACVdelNSs) strongly induced interferon transcription, whereas the corresponding wt virus (rLACV) suppressed it. Here, we show that interferon induction by rLACVdelNSs mainly occurs through the signaling pathway leading from the pattern recognition receptor RIG-I to the transcription factor IRF-3. NSs expressed by rLACV, however, acts downstream of IRF-3 by specifically blocking RNA polymerase II-dependent transcription. Further investigations revealed that NSs induces proteasomal degradation of the mammalian RNA polymerase II subunit RPB1. NSs thereby selectively targets RPB1 molecules of elongating RNA polymerase II complexes, the so-called IIo form. This phenotype has similarities to the cellular DNA damage response, and NSs was indeed found to transactivate the DNA damage response gene pak6. Moreover, NSs expressed by rLACV boosted serine 139 phosphorylation of histone H2A.X, one of the earliest cellular reactions to damaged DNA. However, other DNA damage response markers such as up-regulation and serine 15 phosphorylation of p53 or serine 1524 phosphorylation of BRCA1 were not triggered by LACV infection. Collectively, our data indicate that the strong suppression of interferon induction by LACV NSs is based on a shutdown of RNA polymerase II transcription and that NSs achieves this by exploiting parts of the cellular DNA damage response pathway to degrade IIo-borne RPB1 subunits. PMID:21118815

Verbruggen, Paul; Ruf, Marius; Blakqori, Gjon; Överby, Anna K.; Heidemann, Martin; Eick, Dirk; Weber, Friedemann

2011-01-01

380

Role of a non-natural beta-C-nucleotide unit in DNA as a template for DNA and RNA syntheses and as a substrate for nucleolytic digestion.  

PubMed

A non-natural beta-C-nucleoside bearing a 3,4-dibenzyloxyphenyl group as a nucleobase (X) was synthesized and incorporated into a 34-mer oligomer with the sequence 5'-dTTTTTAAAAAAXATATAGCAGCGACATGTCACCG-3'. This synthetic oligonucleotide was examined for template activity in the enzymatic syntheses of DNA by the Klenow fragments of Escherichia coli DNA polymerase I and the recombinant DNA polymerase I, and in the synthesis of RNA by the E. coli RNA polymerase core enzyme. As a result, the template-directed polymerization of both DNA and RNA was precisely terminated at the position of X. The X-containing oligonucleotide was also tested for digestion by an exonuclease, Exo III nuclease (Exo III), and an endonuclease, Mung Bean nuclease (MB). The results indicate that the artificial nucleobase X acts as a terminator for digestion by Exo III, whereas the site X becomes susceptible to digestion by MB. These findings provide a useful tool for the size control of products in the synthesis and degradation of nucleic acids. PMID:13678792

Aketani, Saeko; Tanaka, Kentaro; Yamamoto, Kaneyoshi; Ishihama, Akira; Cao, Honghua; Tengeiji, Atsushi; Shionoya, Mitsuhiko

2003-09-01

381

DNA methylation and mRNA and microRNA expression of SLE CD4+ T cells correlate with disease phenotype.  

PubMed

Systemic lupus erythematosus (SLE) is an autoimmune disease well known for its clinical heterogeneity, and its etiology secondary to a cross-talk involving genetic predisposition and environmental stimuli. Although genome-wide analysis has contributed greatly to our understanding of the genetic basis of SLE, there is increasing evidence for a role of epigenetics. Indeed, recent data have demonstrated that in patients with SLE, there are striking alterations of DNA methylation, histone modifications, and deregulated microRNA expression, the sum of which contribute to over-expression of select autoimmune-related genes and loss of tolerance. To address this issue at the level of clinical phenotype, we performed DNA methylation, mRNA and microRNA expression screening using high-throughput sequencing of purified CD4+ T cells from patients with SLE, compared to age and sex matched controls. In particular, we studied 42 patients with SLE and divided this group into three clinical phenotypes: a) the presence of skin lesions without signs of systemic pathology; b) skin lesions but also chronic renal pathology; and c) skin lesions, chronic renal pathology and polyarticular disease. Interestingly, and as expected, sequencing data revealed changes in DNA methylation in SLE compared to controls. However, and more importantly, although there were common methylation changes found in all groups of SLE compared to controls, there was specific DNA methylation changes that correlated with clinical phenotype. These included changes in the novel key target genes NLRP2, CD300LB and S1PR3, as well as changes in the critical pathways, including the adherens junction and leukocyte transendothelial migration. We also noted that a significant proportion of genes undergoing DNA methylation changes were inversely correlated with gene expression and that miRNA screening revealed the existence of subsets with changes in expression. Integrated analysis of this data highlights specific sets of miRNAs controlled by DNA methylation, and genes that are altered by methylation and targeted by miRNAs. In conclusion, our findings suggest select epigenetic mechanisms that contribute to clinical phenotypes and further shed light on a new venue for basic SLE research. PMID:25091625

Zhao, Ming; Liu, Siyang; Luo, Shuangyan; Wu, Honglong; Tang, Meini; Cheng, Wenjing; Zhang, Qing; Zhang, Peng; Yu, Xinhai; Xia, Yudong; Yi, Na; Gao, Fei; Wang, Li; Yung, Susan; Chan, Tak Mao; Sawalha, Amr H; Richardson, Bruce; Gershwin, M Eric; Li, Ning; Lu, Qianjin

2014-11-01

382

The ability to form homodimers is essential for RDM1 to function in RNA-directed DNA methylation.  

PubMed

RDM1 (RNA-DIRECTED DNA METHYLATION1) is a small plant-specific protein required for RNA-directed DNA methylation (RdDM). RDM1 interacts with RNA polymerase II (Pol II), ARGONAUTE4 (AGO4), and the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) and binds to methylated single stranded DNA. As the only protein identified so far that interacts directly with DRM2, RDM1 plays a pivotal role in the RdDM mechanism by linking the de novo DNA methyltransferase activity to AGO4, which binds short interfering RNAs (siRNAs) that presumably base-pair with Pol II or Pol V scaffold transcripts synthesized at target loci. RDM1 also acts together with the chromatin remodeler DEFECTIVE IN RNA-DIRECTED DNA METHYLATION1 (DRD1) and the structural-maintenance-of-chromosomes solo hinge protein DEFECTIVE IN MERISTEM SILENCING3 (DMS3) to form the DDR complex, which facilitates synthesis of Pol V scaffold transcripts. The manner in which RDM1 acts in both the DDR complex and as a factor bridging DRM2 and AGO4 remains unclear. RDM1 contains no known protein domains but a prior structural analysis suggested distinct regions that create a hydrophobic pocket and promote homodimer formation, respectively. We have tested several mutated forms of RDM1 altered in the predicted pocket and dimerization regions for their ability to complement defects in RdDM and transcriptional gene silencing, support synthesis of Pol V transcripts, form homodimers, and interact with DMS3. Our results indicate that the ability to form homodimers is essential for RDM1 to function fully in the RdDM pathway and may be particularly important during the de novo methylation step. PMID:24498436

Sasaki, Taku; Lorkovi?, Zdravko J; Liang, Shih-Chieh; Matzke, Antonius J M; Matzke, Marjori

2014-01-01

383

Circular dichroism of biological macromolecules.  

PubMed

Circular dichroism, the unequal absorption of right and left circularly polarized light, is a manifestation of optical activity in the vicinity of absorption bands. To the experimental scientist interested in the conformation of macromolecules and in the sensitive response of optical activity to conformational alteration, it offers a relatively new and powerful means of understanding the environment of chromophoric residues. As a tool in the elucidation of electronic spectra, it should be useful to the theoretical scientist in identifying weakly allowed absorption bands as well as in providing rotational parameters which can be compared with the developing theory of optical activity. I have stressed application of circular dichroism, to experimental aspects of protein and nucleic acid conformation in solution. Much is still uncertain in particular quantitative details. However, even these early results shed new light and yield new information on the conformation of these molecules. PMID:5332570

Beychok, S

1966-12-01

384

LRP130, a single-stranded DNA/RNA-binding protein, localizes at the outer nuclear and endoplasmic reticulum membrane, and interacts with mRNA in vivo.  

PubMed

LRP130 (also known as a LRPPRC) is an RNA and single-stranded DNA-binding protein, and recently identified as a candidate gene responsible for the Leigh syndrome, a French-Canadian type cytochrome c oxidase deficiency. However, the biological function of LRP130 still remains largely unresolved. In the present study, we found that the C-terminal half of the mouse LRP130 located within a 120 amino acid sequence (a.a. 845-964) binds to synthetic RNA homopolymers, poly(G), poly(U), and poly(C), as well as r(CUGCC)(6). Assessment of the subcellular localization indicated both nuclear/endoplasmic reticulum (ER) and mitochondrial fractions to be positive. To further analyze the subcellular localization of LRP130, a nuclear/ER fraction was fractionated into the nucleoplasm (NP) and nuclear envelope (NE)/ER, and the latter was further separated into outer nuclear membrane (ONM)/ER and inner nuclear membrane (INM) by treatment with Triton X-100. LRP130 was detectable in all three fractions, and the distribution pattern was in good accordance with that known for ONM/ER proteins. Interestingly, immunostaining of HeLa cells demonstrated nuclear rim staining of LRP130, specifically at the outside of the NE and also at ER, and association of LRP130 with poly(A)(+) RNA was restricted only to the ONM/ER fraction. Overexpression of full-length mouse LRP130 fused with EGFP resulted in nuclear accumulation of poly(A)(+) RNA in HeLa cells. Taking all these results together, it is suggested that LRP130, a novel type of RNA-binding protein, associates with mRNA/mRNP complexes at the outside of NE and ER, and plays a role in control of mRNA metabolisms. PMID:15081402

Tsuchiya, Naoto; Fukuda, Hirokazu; Nakashima, Katsuhiko; Nagao, Minako; Sugimura, Takashi; Nakagama, Hitoshi

2004-05-01

385

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

386

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

387

Expression of mRNA for DNA methyltransferases and methyl-CpG–binding proteins and DNA methylation status on CpG islands and pericentromeric satellite regions during human hepatocarcinogenesis  

Microsoft Academic Search

To evaluate the significance of alterations in DNA methylation during human hepatocarcinogenesis, we examined levels of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and the DNA methylation status in 67 hepatocellular carcinomas (HCCs). The average level of mRNA for DNMT1 and DNMT3a was significantly higher in noncancerous liver tissues showing chronic hepatitis or cirrhosis than in histologically normal liver tissues,

Yoshimasa Saito; Yae Kanai; Michiie Sakamoto; Hidetsugu Saito; Hiromasa Ishii; Setsuo Hirohashi

2001-01-01

388

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

389

Sequential serum hepatitis C viral RNA levels longitudinally assessed by branched DNA signal amplification.  

PubMed

The aim of this study was to determine the stability of viral load over an extended period in patients with chronic hepatitis C virus (HCV). Sequential serum specimens collected from fourteen non-alcoholic adult patients with chronic HCV between 1990 and 1997 were tested retrospectively for HCV RNA levels by branched DNA assay (Quantiplex HCV RNA 2.0 [Chiron Diagnostics, Emeryville, CA]). A minimum of three serum samples was obtained at various intervals from each patient. None of the patients received antiviral therapy. Liver biopsies, available for 10 of 14 patients, showed mild or moderate hepatitis in seven and cirrhosis in three (one developed cirrhosis during follow-up). RIBA strip immunoassay showed that 7, 3, and 4 patients had viral genotypes 1, 2, and 3, respectively. The follow-up time averaged 5.3 years (range, 3.7 to 6.6 years). Eight patients (57.2%) showed increased viral levels from baseline to follow-up, the remaining six patients (42.8%) showed decreased viral levels. The three cirrhotic patients had the highest viral levels over time. The mean change was a 0.29-fold decrease (median, +1.14 [corrected]; range, -17.49 to +7.32). A less than twofold change in either direction was demonstrated for six patients (42.8%), and a less than threefold change was demonstrated for 10 patients (71.4%). Variation from baseline to last follow-up as calculated by log determination showed that the viremic load varied less than one log10 in all but one individual. These results show that viral load remains relatively stable over prolonged periods in most untreated patients with chronic hepatitis C. PMID:9828238

Gordon, S C; Dailey, P J; Silverman, A L; Khan, B A; Kodali, V P; Wilber, J C

1998-12-01

390

Induction of amphiregulin by p53 promotes apoptosis via control of microRNA biogenesis in response to DNA damage  

PubMed Central

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

2014-01-01

391

Comparison of biotinylated DNA and RNA probes for rapid detection of varicella-zoster virus genome by in situ hybridization.  

PubMed Central

We describe a general method for the production of nonisotopic DNA and RNA probes for the detection of the varicella-zoster virus (VZV) genome by in situ hybridization. VZV DNA was extracted from purified viral nucleocapsids, cleaved with restriction enzyme (RE) BamHI, and cloned into plasmid pBR322 by the standard vector insert procedure. We cloned over 85% of the VZV genome and obtained 18 recombinants. Plasmids containing the B, F, G, H, and J fragments of VZV DNA were labeled by the nick translation method with biotin-11-dUTP as the dTTP analog. Additionally, the B fragment was cleaved with RE AvaI, subcloned into the plasmid pGEM-4 transcription vector, and subsequently linearized with REs PstI and EcoRI. RNA was transcribed with T7 or SP6 polymerase, with a substitution of allylamine-UTP as the UTP analog, and labeled with epsilon-caproylamidobiotin-N-hydroxysuccinimide ester. The DNA and RNA probes were used under full-stringency conditions for in situ hybridization with alkaline phosphatase as the detector and 5-bromo-4-chloro-3-indolyl phosphate-Nitro Blue Tetrazolium as the substrate. When tested under comparable conditions, the RNA probe was slightly more sensitive than was the DNA probe: both probes showed homology only with VZV-infected cells and clinical tissues and not with the other herpesviruses. Probes prepared from variable regions of the genome (fragments F and J) performed as well as did those from conserved regions (fragments B. G. and H). Biotinylated probes have distinct advantages over isotopic probes and retain their full potency for more than 2 years when stored properly. Images PMID:1645371

Forghani, B; Yu, G J; Hurst, J W

1991-01-01

392

RNA:DNA ratios of Baltic Sea herring larvae and copepods in embayment and open sea habitats  

NASA Astrophysics Data System (ADS)

Elucidation of important nursery habitats for young fish can aid in the management and assessment of fish stocks. Herring ( Clupea harengus) in the Baltic Sea primarily spawn in coastal areas, but larvae are also present in off-shore, open sea areas. To investigate if sheltered coastal habitats provide a better growth environment for larval herring, we compared short-term growth (as indexed by whole body RNA:DNA ratios) of larval herring from three habitat types of the northwest Baltic proper (sheltered inner bay, exposed outer bay, and open sea). In addition, we compared individual RNA content of adult female Eurytemora affinis (a common Baltic copepod) among these different habitats. High RNA levels in these copepods indicate high production of nauplii, which are important food for larval herring. Both RNA:DNA ratios of larval herring and RNA content of E. affinis were significantly greater in embayment habitats, suggesting that the sheltered coastal areas are high quality nursery habitats for young Baltic herring.

Höök, Tomas O.; Gorokhova, Elena; Hansson, Sture

2008-01-01

393

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

394

A novel thermostable polymerase for RNA and DNA loop-mediated isothermal amplification (LAMP)  

PubMed Central

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

395

Effective DNA\\/RNA CoExtraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens  

Microsoft Academic Search

BackgroundRetrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter

Adam Kotorashvili; Andrew Ramnauth; Christina Liu; Juan Lin; Kenny Ye; Ryung Kim; Rachel Hazan; Thomas Rohan; Susan Fineberg; Olivier Loudig

2012-01-01

396

Two novel pathogenic mitochondrial DNA mutations affecting organelle number and protein synthesis. Is the tRNA(Leu(UUR)) gene an etiologic hot spot?  

PubMed Central

We identified two patients with pathogenic single nucleotide changes in two different mitochondrial tRNA genes: the first mutation in the tRNA(Asn) gene, and the ninth known mutation in the tRNA(Leu(UUR)) gene. The mutation in tRNA(Asn) was associated with isolated ophthalmoplegia, whereas the mutation in tRNA(Leu(UUR)) caused a neurological syndrome resembling MERRF (myoclonus epilepsy and ragged-red fibers) plus optic neuropathy, retinopathy, and diabetes. Both mutations were heteroplasmic, with higher percentages of mutant mtDNA in affected tissues, and undetectable levels in maternal relatives. Analysis of single muscle fibers indicated that morphological and biochemical alterations appeared only when the proportions of mutant mtDNA exceeded 90% of the total cellular mtDNA pool. The high incidence of mutations in the tRNA(Leu(UUR)) gene suggests that this region is an "etiologic hot spot" in mitochondrial disease. Images PMID:8254046

Moraes, C T; Ciacci, F; Bonilla, E; Jansen, C; Hirano, M; Rao, N; Lovelace, R E; Rowland, L P; Schon, E A; DiMauro, S

1993-01-01

397

Ultrasensitive detection of mRNA extracted from cancerous cells achieved by DNA rotaxane-based cross-rolling circle amplification.  

PubMed

An ultrasensitive and highly selective method for polymerase chain reaction-free (PCR-free) messenger RNA (mRNA) expression profiling is developed through a novel cross-rolling circle amplification (C-RCA) process based on DNA-rotaxane nanostructures. Two species of DNA pseudorotaxane (DPR) superstructures (DPR-I and DPR-II) are assembled by threading a linear DNA rod through a double-stranded DNA (dsDNA) ring containing two single-stranded gaps. In this assay, cDNA that is specific for ?-actin (ACTB) mRNA is taken as a model analyte. Upon the introduction of the target cDNA, the cDNA and the biotin-modified primer are hybridized to the single-stranded regions of the DNA rod and the gap-ring, respectively. As a result, the DPR-I dethreads into free DNA macrocycle and a dumbbell-shaped DNA nanostructure. In the presence of DNA polymerase/dNTPs, two release-DNA on the DPR-I are replaced by polymerase with strand-displacement activity, which can act as the input of the DPR-II to trigger the dethreading of DPR-II and the RCA reaction, releasing another two specified release-DNA strands those in turn serve as the "mimic cDNA" for DPR-I. The C-RCA reaction then proceeds autonomously. To overcome the high background induced by hemin itself, the biotinylated rolling circle products are captured by streptavidin-coated MNPs, achieving a detection limit as low as 0.1 zmol cDNA. The assay also exhibits an excellent selectivity due to its unique DNA nanostructure fabricated through base pairing hybridization. The ACTB mRNA expression in mammary cancer cells (MCF-7) is successfully detected. PMID:23148205

Bi, Sai; Cui, Yangyang; Li, Li

2013-01-01

398

HIV-1 Reverse Transcriptase Can Simultaneously Engage its DNA/RNA Substrate at both the DNA Polymerase and RNase H Active Sites: Implications for RNase H Inhibition  

PubMed Central

Reverse transcriptase (RT) of the human immunodeficiency virus (HIV) possesses DNA polymerase and ribonuclease (RNase) H activities. Although the nucleic acid binding cleft separating these domains can accommodate structurally-diverse duplexes, it is currently unknown whether regular DNA/RNA hybrids can simultaneously contact both active sites. In this study we demonstrate that ligands capable of trapping the 3’-end of the primer at the polymerase active site affect specificity of RNase H cleavage without altering the efficiency of the reaction. Experiments under single turnover conditions reveal that complexes with a bound nucleotide substrate show specific RNase H cleavage at template position -18, while complexes with the pyrophosphate analogue foscarnet show a specific cut at position -19. This pattern is indicative for post- and pre-translocated conformations. The data are inconsistent with models postulating that the substrate toggles between both active sites, such that the primer 3’-terminus is disengaged from the polymerase active site when the template is in contact with the RNase H active site. In contrast, our findings provide strong evidence to suggest that the nucleic acid substrate can engage both active sites at the same time. As a consequence, the bound and intact DNA/RNA hybrid can restrict access of RNase H active site inhibitors. We have mapped the binding site of the recently discovered inhibitor ?-thujaplicinol between the RNase H active site and Y501 of the RNase H primer grip and show that the inhibitor is unable to bind to a pre-formed RT-DNA/RNA complex. In conclusion, the bound nucleic acid substrate, and in turn, active DNA synthesis can represent an obstacle to RNase H inhibition with compounds that bind to the RNase H active site. PMID:19289131

Beilhartz, Greg L.; Wendeler, Michaela; Baichoo, Noel; Rausch, Jason; Le Grice, Stuart; Götte, Matthias

2009-01-01

399

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 Jørgen

2013-01-01

400

Chitosan-plasmid DNA nanoparticles encoding small hairpin RNA targeting MMP-3 and -13 to inhibit the expression of dedifferentiation related genes in expanded chondrocytes.  

PubMed

Overexpression of matrix metalloproteinase (MMP)-3 and -13 can lead to the dedifferentiation of expanded chondrocytes. After implanting dedifferentiated cells for cartilage defect repair, graft failure may occur. Short hairpin RNA (shRNA) is a powerful genetic tool to reduce the expression of target genes. This study investigated the effects of chitosan-plasmid DNA (pDNA) nanoparticles encoding shRNA targeting MMP-3 and -13 on the dedifferentiation of expanded chondrocytes. The objective was to optimize the parameters of chitosan-pDNA formulation for achieving higher efficiency of pDNA delivery and gene silencing. The chitosan-pDNA nanoparticles were prepared using a complex coacervation process. Then the characteristics including size, shape, stab